WO2020127498A1 - Compounds for use in the treatment of diseases associated with altered autophagy, altered shh transduction, and/or neurodegenerative diseases - Google Patents

Compounds for use in the treatment of diseases associated with altered autophagy, altered shh transduction, and/or neurodegenerative diseases Download PDF

Info

Publication number
WO2020127498A1
WO2020127498A1 PCT/EP2019/085971 EP2019085971W WO2020127498A1 WO 2020127498 A1 WO2020127498 A1 WO 2020127498A1 EP 2019085971 W EP2019085971 W EP 2019085971W WO 2020127498 A1 WO2020127498 A1 WO 2020127498A1
Authority
WO
WIPO (PCT)
Prior art keywords
gigaxonin
atg16l1
interaction
disease
autophagy
Prior art date
Application number
PCT/EP2019/085971
Other languages
French (fr)
Inventor
Pascale BOMONT
Aurora SCRIVO
Yoan ARRIBAT
Original Assignee
Institut National De La Sante Et De La Recherche Medicale (Inserm)
Université De Montpellier
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institut National De La Sante Et De La Recherche Medicale (Inserm), Université De Montpellier filed Critical Institut National De La Sante Et De La Recherche Medicale (Inserm)
Publication of WO2020127498A1 publication Critical patent/WO2020127498A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Definitions

  • the present invention relates to the prevention and/or the treatment of neurodegenerative diseases, and/or diseases associated with altered autophagy.
  • the present invention also relates to the prevention and/or the treatment of diseases associated with altered Sonic Hedgehog (Shh) transduction.
  • Shh Sonic Hedgehog
  • the invention relies upon the discovery that modulation of the expression of Gigaxonin regulates the fine-tuning of the production of autophagosomes, notably through the modulation of the Gigaxonin/ATG16Ll interaction, and the control of the steady-level of ATG16L1.
  • the invention also relies upon the identification of Gigaxonin as the first E3 ligase that positively controls the initiation of Sonic Hedgehog (Shh) transduction, ensuring that Shh responses in spinal cord and muscles are conveyed into efficient motor activity.
  • Sonic Hedgehog Sonic Hedgehog
  • PTCH Patched
  • Autophagy is an essential degradative pathway that delivers cytoplasmic components to lysosomes for degradation. Evolutionarily conserved, this complex machinery is activated to recycle a wide range of substrates in normal conditions and to promote the degradation of damaged components (dysfunctional organelles, protein aggregates) in diseases. Therefore, alteration of autophagy perturbs cellular homeostasis and important physiological processes, and is associated with various pathological conditions, including cancer and neurodegenerative diseases (Mizushima and Komatsu (Cell. 2011 Nov 11;147(4):728-41); Schneider and Cuervo (Curr Opin Genet Dev. 2014 Jun;26: 16-23); Menzies et al. (Nat Rev Neurosci. 2015 Jun;16(6):345-57)).
  • Macroautophagy (hereafter referred to as autophagy) is characterised by the nucleation of a double-membrane fragment (phagophore) around the material to be degraded, which elongates to form a complete autophagosome and subsequently fuses to a lysosome.
  • phagophore double-membrane fragment
  • the mechanisms driving membrane expansion are key elements in autophagy.
  • the molecular determinants of membrane elongation are complex and involve two highly conserved ubiquitin-like (UBL) conjugation systems, ATG12 and LC3 (the mammalian homolog of the yeast ATG8). Structurally related to ubiquitin, ATG12 and LC3 are transferred by El- and E2-like enzymes to their final substrates.
  • the covalent conjugation of ATG12 to ATG5 generates the E3 ligase activity necessary for the last step of ATG8/LC3 conjugation to phosphatidylethanolamine (PtdEth) on the nascent membranes.
  • PtdEth phosphatidylethanolamine
  • ATG16L1 is a key determinant of autophagy elongation. Indeed, ATG16L1 interacts with the conjugate ATG12-ATG5 to form a multimeric structure and triggers the binding of the complex to the membrane. Through the subsequent interaction of ATG12 with LC3-conjugated- ATG3, ATG16L1 specifies the site of LC3 lipidation onto nascent membranes.
  • ATG16L1 alterations in ATG16L1, either using genetic mutants or the overexpressed protein, all result in impaired localization of ATG12-ATG5 to the phagophore and failure in ATG8/LC3 lipidation onto the membranes, leading to inhibition of autophagosome formation.
  • forced localization of ATG16L1 to the plasma membrane has been shown to be sufficient to promote ectopic LC3 lipidation at the cell surface.
  • the biological importance of ATG16L1 was further evidenced in vivo , where A I ’ G I6 I ⁇ mice, defective in autophagosome formation, did not survive neonatal starvation and died within 1 day of delivery.
  • miRNAs microRNAS
  • ATG7 and ATG16L1 have been proved to attenuate status epilepticus-induced brain injuries (Gan et al.; Sci Rep. 2017 Aug 31;7(1): 10270).
  • miR-410 has been evidenced to target ATG16L1 and subsequently to markedly inhibit autophagy in osteosarcoma cells, leading to, upon exposure to anticancer drugs, a protective increased autophagy and a better chemosensitivity in cancer cells. (Chen et al.; Mol Med Rep. 2017 Mar;15(3): 1326-1334).
  • a disease or a stage in the disease progression
  • a disease wherein autophagy is inhibited may benefit from a modulator that restores autophagy to a physiological level.
  • Sonic Hedgehog assigns neuronal and muscle fate, acting in a graded manner to pattern the dorso-ventral axis of the neural tube and the muscles.
  • Dysregulation of Shh signalling causes a wide range of human diseases, including congenital malformations of the central nervous system, of the axial skeleton and limbs, cancers and malignancies in children and adults. Therefore, understanding the Shh pathway is essential for both basic developmental biology and to explore therapeutic intervention for human diseases.
  • compounds that suitably modulate Shh signalling in disease cells/tissues especially neurodegenerative diseases, such as those associated with altered autophagy and Shh signaling.
  • the invention relates to a modulator of the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ATG16Ll interaction and/or of the functionality of the Gigaxonin/ ATG16L1 interaction for use as a medicament.
  • the invention relates to a polypeptide expressed by a nucleic acid comprising a nucleic acid sequence having at least 75% identity with SEQ ID NO. 1; or
  • polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2; for use as a medicament.
  • the diseases which are particularly considered by the invention include those associated with altered autophagy and/or altered Shh transduction and/or neurodegenerative diseases.
  • the invention relates to a polypeptide expressed by a nucleic acid comprising a nucleic acid sequence having at least 75% identity with SEQ ID NO. 1; or
  • a polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy.
  • the invention relates to a nucleic acid coding for such a polypeptide; for use as a medicament.
  • the invention relates to a nucleic acid coding for such a polypeptide; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
  • Another aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising:
  • Such a pharmaceutical composition may thus comprise a nucleic acid having at least 75% identity with SEQ ID NO. 1; or a polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2.
  • the invention relates to a pharmaceutical composition according to the present disclosure, for use in the prevention and/or the treatment of a disease associated with altered autophagy.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a nucleic acid having at least 75% identity with SEQ ID NO. 1; or
  • the invention also relates to a pharmaceutical composition according to the present disclosure, for use in the prevention and/or the treatment of a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
  • a further aspect of the invention relates to a method for screening a modulator of the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ATG16Ll interaction and/or of the functionality of the Gigaxonin/ ATG16L1 interaction, comprising the steps of:
  • Another aspect of the invention also relates to a method for assessing the efficiency of a candidate treatment of a disease associated with altered autophagy in an individual in need thereof, comprising the steps of:
  • step (d) determining a difference between the level measured in step (a) and the level measured in step (c).
  • a still further aspect of the invention concerns a method to activate autophagy in a target cell, comprising a step of increasing the level of expression, stability of Gigaxonin, decreasing its degradation and/or increasing activity of Gigaxonin and/or Gigaxonin/ ATG16L1 interaction to reach physiological functions of ATG16L1.
  • a still further aspect of the invention concerns a method to inhibit autophagy in a target cell, comprising a step of decreasing the level of expression, stability of Gigaxonin, increasing its degradation and/or decreasing the activity of Gigaxonin and/or Gigaxonin/ ATG16L1 interaction to decrease physiological functions of ATG16L1.
  • the invention in another aspect, relates to a method for the fine-tuned activation or inhibition of autophagy in a target cell comprising the step of modulating the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ATG16Ll interaction and/or of the functionality of the Gigaxonin/ATG16Ll interaction.
  • the invention relates to the use of gigaxonin and/or any derivatives (cells/constructs/animal model/methods%) to serve as a template to screen existent and/or novel drugs acting on the autophagy pathway.
  • the invention relates to a method for the fine-tuned activation or inhibition of Shh transduction in a target cell comprising the step of modulating the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/PTCH interaction and/or of the functionality of the Gigaxonin/PTCH interaction.
  • the invention relates to the use of gigaxonin and/or derivatives (cells/constructs/animal model/methods...) to serve as a template to screen existent and/or drugs acting on the Shh pathway.
  • FIG. 1 Immunoblotting evidencing the interaction of Gigaxonin with ATG16L1. Reverse Immunoprecipitation experiment was performed in COS-7 cells transfected with Ch-ATG16 and Flag-Gig and treated with MG132. Gigaxonin and ATG16L1 complexes were recovered using anti-flag (IP Flag) and anti-cherry (IP Cherry) antibodies and revealed by immunoblotting with cherry/Flag antibodies. Immunoprecipitation with anti-IgG antibodies (IP IgG) served as negative control.
  • IP Flag anti-flag
  • IP Cherry anti-cherry
  • FIG. 1 Schematic representation of the mouse ATG16L1 (ATG16L1-F) and its deletion constructs. N-terminal domain (ATG5 binding domain), central coiled-coil domain (CCD domain) and C-terminal WD40 repeats domain are indicated in white, gray stripes and black, respectively.
  • FIG. 3 Immunoblotting demonstrating the interaction of Gigaxonin with the WD40 repeats domain of ATG16L1.
  • the domain of interaction of ATG16L1 with Gigaxonin was evidenced by immunoprecipitation.
  • Full length and deletion constructs of Flag-ATG16 were transfected in COS-7 cells with Ch-Gig in presence of MG132. Please note that the Full-length ATG16L1 band (black star in the input panel) is enriched in pull down condition (in the IP Flag panel).
  • Immunoprecipitation using anti-Flag antibodies evidenced Gigaxonin in the immunocomplexes formed by the Full length-, C-, and DN- ATG16L1 proteins.
  • Negative controls include immunoprecipitation of the Ch-Gig + Flag- ATG16L1-F cell lysate with anti-IgG antibodies (IP IgG).
  • FIG. 4 Schematic representation of the human Gigaxonin (Gig-F) with its N-terminal BTB domain (stripes) and C-terminal Kelch repeats domain (black) and the BTB (Gig-AN) and Kelch (Gig-AC) deletion constructs.
  • FIG. 6 Immunoblotting showing that Gigaxonin induces the degradation of ectopic ATG16L1, through the proteasome and the lysosomal pathways
  • COS-7 cells co- were transfected with Flag-Gig and Cherry-ATG16L1 (double transfection), in basal condition (-), or treated with Mgl32 (+Mgl32) or Bafilomycin A1 (+Baf).
  • Figure 7. Immunoblotting demonstrating the Gigaxonin-induced degradation of endogenous ATG16L1, through the proteasome and lysosomal pathways. Endogenous ATG16L1 protein is greatly diminished in cells transfected with Flag-Gig, and partially restored under MG132 (+Mgl32) or Bafilomycin A1 (+Baf) treatment.
  • FIG. 8 Immunoblotting demonstrating the in vivo ubiquitination of ATG16L1 by Gigaxonin.
  • COS-7 cells were transfected with Ch-ATG16 alone, or in combination with His-Ubiquitin construct (His-Ub) in the presence or absence of siRNA against endogenous Gigaxonin (siRNA) or the mismatch counterpart (ms), and treated with MG132.
  • the pull down of ubiquitinated ATG16L1 is performed in denaturing conditions using nickel agarose beads.
  • FIG. 9 Immunoblotting showing the defect in autophagosome production caused by Gigaxonin depletion in neurons. Wild type and GANG cortical neurons were cultured for 4 div under basal condition, or with different treatments to evaluate autophagy activity. Starvation was induced with EBSS, autophagosome-lysosome fusion was blocked with Bafilomycin A1 (Baf). Immunoblotting of cortical neurons shows the lipidation of LC3 under different conditions, in control (WT) and mutant (GANG) neurons.
  • FIG. 10 Quantification of the lipidation of LC3 showing the defect in autophagosome production caused by Gigaxonin depletion.
  • Wild type (WT) and GANG cortical neurons are treated as in Figure 9.
  • Quantification of the lipidation of LC3 was measured with the LC3-II to tubulin ratio, and expressed as a fold increase over the control level in the basal condition.
  • the defect in autophagy in GAN ⁇ / ⁇ neurons, evidenced by the decreased LC3 lipidation in EBBS+Baf 6h condition was further defined as a decreased production of autophagosomes, by comparing the EBBS+Baf ratio at two different times (2h and 6h of treatment).
  • WT and mutant values are represented in black and grey circles, respectively.
  • n 3 independent experiments and values are means ⁇ SD. Differences between WT and mutant values are only significant for the (EBSS+Baf6h) condition **** EO.OOOl using 2Way ANOVA test (Bonferroni post-hoc test).
  • FIG. 12 Quantification showing the defectiveness of GAN ⁇ / ⁇ neurons in autophagosome-lysosome fusion.
  • Co-staining of LC3 with lysotracker evidenced decreased fusion between autophagic structures and lysosomes in GAN ⁇ / ⁇ neurons.
  • the decrease in the colocalisation of LC3 with the lysotracker was determined by the Pearson Coefficient.
  • FIG. 13 Immunoblotting showing the rescue of autophagy by Gigaxonin in GAN / neurons. Immunoblots for ATG16L1 were performed on 4div control and mutant neurons, after transduction with GFP-mock or Flag-Gigaxonin lentivirus. Both normal and aggregated ATG16L1 were cleared upon expression of Gigaxonin in control and GANG neurons, respectively.
  • FIG. 14 Quantification of neurons demonstrating the progressive degeneration of GANG neurons.
  • Doublecortine (Dbx) and MAP2 neurons were identified in E15.5 cortices prepared from wild type (WT) and GANG mice after 3 hours, 5, 15 and 20 div (days in vitro).
  • the neurodegeneration of GANG neurons was evidenced from 15 div onwards, as measured by the decreased proportion of neurons (as expressed by a MAP2 to DAPI ratio), relatively to the 3h time points (Dbx to Dapi ratio).
  • WT and mutant cells are represented in black and grey circles, respectively.
  • n 3 independent experiments with triplicate measures (161-821 DAPI positive cells counted per measure). Individual measures and means ⁇ SEM are represented; **E ⁇ 0.01 and *P ⁇ 0.05 values at 15 and 20 div, respectively using the 2way ANOVA test (Bonferroni post-hoc test).
  • FIG. 15 A Schematic model of Gigaxonin control of autophagosome synthesis.
  • Figure 15 A Gigaxonin controls the steady-state level of ATG16L1, by interacting with its C-terminal WD40 domain and promoting its K48 poly-ubiquitination and clearance by the proteasome and the autophagy pathways.
  • ATG16L1 binds to the ATG12-ATG5 elongation conjugate and targets it to the nascent autophagic membrane, through its interaction with WIPI2.
  • WIPI2 the E3 ligase activity of the ATG12-ATG5- ATG16 complex lipidates LC3 onto the membranes, allowing the elongation of the phagophore.
  • the mature autophagosome sequesters cytosolic material, including p62 bound cargo, which are degraded upon fusion to the lysosome.
  • the Gigaxonin-E3 ligase promotes autophagosome production and ensures a normal autophagic flux within cells.
  • autophagosome biogenesis occurs at the neurite tip but also in the soma of primary neurons.
  • Gigaxonin depletion ( Figure 15B) in primary neurons induces aggregation of ATG16L1, without impairing the formation of the ATG12-ATG5 conjugate.
  • Gigaxonin depletion alters autophagosome synthesis and causes an abnormal accumulation of the main autophagy receptor p62, hence impairing the autophagic flux.
  • ATG16L1 and P62 accumulation are localized within the soma, opening interesting perspectives on the study of autophagy associationalisation within neurons, and on its role in human neuropathy, as exemplified in GAN.
  • FIG. 16 Model of action of the Gigaxonin-E3 ligase in the initiation of Shh signalling.
  • A In an OFF state, prior to Shh activation, receiving cells silence the signalisation cascade through the inhibitory effect of the Ptch receptor on the effector Smo.
  • the cleaved active Shh N form is released and addressed to progenitor cells.
  • Gigaxonin acts as an initiator of Shh signalling by degrading Shh-bound Ptch receptor, hence allowing the derepression of the signal transducer Smo.
  • Gigaxonin an E3 ligase adaptor which is mutated in a fatale neurodegenerative disease called Giant Axonal Neuropathy (GAN) (Kulenbaumer, Timmerman & Bomont, Genereview (2016)), as being the first regulator of ATG16L1.
  • GAN Giant Axonal Neuropathy
  • Gigaxonin interacts with the WD40 domain of ATG16L1 to drive its ubiquitination and subsequent degradation. Accumulation of ATG16L1, as a result of Gigaxonin depletion, alters early events of LC3 lipidation onto nascent phagophore and diminishes fusion to the lysosome and degradation of the autophagy receptor p62.
  • the inventors have demonstrated that, as in human, repression of Gigaxonin in zebrafish leads to a loss of motor neurons in the spinal cord, severe axonal defects and the abolishment of locomotion, phenotypes all reversed upon co-injection of the human Gig transcripts.
  • gigaxonin induces the production of motoneurons in the spinal chord, in a manner which is compatible with restoration of normal motility.
  • injection of a nucleic acid coding for gigaxonin i.e. a human messenger RNA coding for gigaxonin
  • restores morphological defects and motility as evidence in zebrafish larvae
  • restores axonal growth of primary motor neurons and induces a production of secondary motor neurons, as exemplified herein.
  • the Gigaxonin-E3 ligase is the 1 st regulator of the Shh pathway, which acts in the receiving tissues to initiate Shh activity in a spatial and temporally regulated manner.
  • the Gigaxonin regulatory mechanism which was identified is evolutionary conserved, is required for both neuron and muscle patterning in vivo, and is necessary for movement.
  • Gigaxonin controls Shh signaling.
  • Shh regulates the initial steps of Shh induction, which is sufficient to specify neuronal and muscle fate in vertebrates.
  • physiological evidence is provided, showing that the Gigaxonin-E3 ligase is a key regulator of Shh activation, by controlling the degradation of the Ptch receptor in a Shh-dependent manner.
  • the positive control of Gigaxonin on the pathway is Shh-dependent, as revealed independently in the Gig-depleted zebrafish, in a cellular system using a Shh activity reporter assay, and in human patient cells.
  • Gigaxonin interacts with Ptch and mediates its degradation in a Shh- dependent manner, hence identifying Gigaxonin as the 1 st regulator of the initiation of Shh signaling.
  • the findings obtained in the gan zebrafish model mimic the motor dysfunctions found in patients, hence providing the first hints into the pathophysiological mechanisms in GAN, and supporting a developmental origin in the pathogenesis of GAN.
  • This notion is further endorsed by the functional rescue of the developmental deficits in the gan zebrafish by the human Gigaxonin, and the evidence of an impairment of Shh signaling in patient cells.
  • the invention relates to a modulator of the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ ATG16L1 interaction and/or of the functionality of the Gigaxonin/ ATG16L1 interaction for use as a medicament.
  • the invention in another complementary aspect, relates to a modulator of the expression, stability, degradation and/or activity of the Gigaxonin/PTCH interaction and/or of the functionality of the Gigaxonin/PTCH interaction, for use as a medicament.
  • such a modulator may be in the form of a polypeptide having at least 75% identity with gigaxonin, or a nucleic acid (such as a messenger RNA) coding for such a polypeptide.
  • the invention relates to a polypeptide expressed by a nucleic acid comprising a nucleic acid sequence having at least 75% identity with SEQ ID NO. 1; or
  • polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2; for use as a medicament.
  • the invention relates to a polypeptide expressed by a nucleic acid comprising a nucleic acid sequence having at least 80% identity with SEQ ID NO. 1; or - a polypeptide comprising a polypeptide sequence having at least 80% identity with SEQ ID NO. 2; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
  • a neurodegenerative disease such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
  • the invention relates to a polypeptide expressed by a nucleic acid comprising a nucleic acid sequence having at least 85% identity with SEQ ID NO. 1; or
  • a polypeptide comprising a polypeptide sequence having at least 85% identity with SEQ ID NO. 2; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
  • the invention relates to a polypeptide expressed by a nucleic acid comprising a nucleic acid sequence having at least 90% identity with SEQ ID NO. 1; or
  • a polypeptide comprising a polypeptide sequence having at least 90% identity with SEQ ID NO. 2; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
  • the invention relates to a polypeptide expressed by a nucleic acid comprising a nucleic acid sequence having at least 95% identity with SEQ ID NO. 1; or
  • a polypeptide comprising a polypeptide sequence having at least 95% identity with SEQ ID NO. 2; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
  • the invention relates to a nucleic acid coding for such a polypeptide; for use as a medicament.
  • the invention relates to a nucleic acid comprising a nucleic acid sequence having at least 75% identity with SEQ ID NO. 1; or a nucleic acid coding for a polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2; for use as a medicament.
  • the invention relates to a nucleic acid comprising a nucleic acid sequence having at least 80% identity with SEQ ID NO. 1; or a nucleic acid coding for a polypeptide comprising a polypeptide sequence having at least 80% identity with SEQ ID NO. 2; for use as a medicament.
  • the invention relates to a nucleic acid comprising a nucleic acid sequence having at least 85% identity with SEQ ID NO. 1; or a nucleic acid coding for a polypeptide comprising a polypeptide sequence having at least 85% identity with SEQ ID NO. 2; for use as a medicament.
  • the invention relates to a nucleic acid comprising a nucleic acid sequence having at least 90% identity with SEQ ID NO. 1; or a nucleic acid coding for a polypeptide comprising a polypeptide sequence having at least 90% identity with SEQ ID NO. 2; for use as a medicament.
  • the invention relates to a nucleic acid comprising a nucleic acid sequence having at least 95% identity with SEQ ID NO. 1; or a nucleic acid coding for a polypeptide comprising a polypeptide sequence having at least 95% identity with SEQ ID NO. 2; for use as a medicament.
  • the invention relates to a nucleic acid coding for such a polypeptide; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
  • the invention relates to a nucleic acid comprising a nucleic acid sequence having at least 75% identity with SEQ ID NO. 1; or a nucleic acid coding for a polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
  • a neurodegenerative disease such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
  • the invention relates to a nucleic acid comprising a nucleic acid sequence having at least 80% identity with SEQ ID NO. 1; or a nucleic acid coding for a polypeptide comprising a polypeptide sequence having at least 80% identity with SEQ ID NO. 2; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
  • a neurodegenerative disease such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
  • the invention relates to a nucleic acid comprising a nucleic acid sequence having at least 85% identity with SEQ ID NO. 1; or a nucleic acid coding for a polypeptide comprising a polypeptide sequence having at least 85% identity with SEQ ID NO. 2; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
  • a neurodegenerative disease such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
  • the invention relates to a nucleic acid comprising a nucleic acid sequence having at least 90% identity with SEQ ID NO. 1; or a nucleic acid coding for a polypeptide comprising a polypeptide sequence having at least 90% identity with SEQ ID NO. 2; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
  • a neurodegenerative disease such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
  • the invention relates to a nucleic acid comprising a nucleic acid sequence having at least 95% identity with SEQ ID NO. 1; or a nucleic acid coding for a polypeptide comprising a polypeptide seqeuence having at least 95% identity with SEQ ID NO. 2; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
  • a neurodegenerative disease such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
  • the modulator of the expression and/or the activity of Gigaxonin, of the Gigaxonin/ATG16Ll interaction and/or of the Gigaxonin/ATG16Ll functionality, as disclosed herein, may be for use for the preparation of a medicament, in particular a medicament intended to treat a disease associated with altered autophagy.
  • the modulator of the expression and/or the activity of Gigaxonin, of the Gigaxonin/PTCH interaction and/or of the Gigaxonin/PTCH functionality, as disclosed herein, may be for use for the preparation of a medicament, in particular a medicament intended to treat a disease associated with altered Shh transduction.
  • the human Gigaxonin polypeptide refers to a 597 amino acid residues protein, also known as Kelch-Like Family Member 16, Kelch-Like Protein 16, KLHL16 and GAN1, which Genbank nucleic acid access is AF291673.1 (SEQ ID NO. 1) and which protein access number is AAG35311.1 (SEQ ID NO. 2), as initially published in Bomont et al, Nat Genet 2000.
  • nucleic acid N in positions 4022 and 4633 of SEQ ID NO. l corresponds to any one of nucleic acids A, C, G or T.
  • the human ATG16L1 protein refers to a 624 amino acid residues, also known as Autophagy Related 16 Like 1, APG16L, ATG16 Autophagy Related 16-Like 1, ATG16 Autophagy Related 16-Like, APG16 Autophagy 16-Like, ATG16 Autophagy Related 16-Like 1, Autophagy -Related Protein 16-1, WD Repeat Domain 30, APGl 6-Like 1, APG16L Beta, ATG16A, ATG16L, IBD10 and WDR30, which Genbank nucleic acid access is NM_001363742.1 (SEQ ID NO. 3) and which protein access number is NP_001350671.1 (SEQ IN NO. 4).
  • the human Ptch receptor (PTCH) protein refers to the patched homolog 1 (PTCH1) and the patched homolog 2 (PTCH2) protein, which are transmembrane receptors of the patched gene family.
  • the PTCH1 Genbank nucleic acid access is Gene ID 5727.
  • the PTCH2 Genbank nucleic acid access is Gene ID 8643.
  • isoform polypeptides of PTCH1 protein patched homolog 1 isoform L (NP_000255.2); protein patched homolog 1 isoform M (NP_001077071.1); protein patched homolog 1 isoform L' (NP_001077072.1); protein patched homolog 1 isoform S (NP_001077073.1); protein patched homolog 1 isoform S
  • isoform polypeptides of PTCH2 protein patched homolog 2 isoform 2 (NP_001159764.1); protein patched homolog 2 isoform 1: NP 003729.3
  • autophagy is intended to refer to the well documented cellular process that mediates the breakdown and the recycling of intracellular components by the means of fusion between autophagosome and lysosomes.
  • autophagy and macroautophagy are intended to be equivalent and substitutable with one another.
  • the expression“altered autophagy” is intended to refer to a level of autophagy which is either increased or decreased as compared to the physiological level of autophagy.
  • autophagy may be assessed by conventional methods and protocols described in the state of the art.
  • autophagy and autophagy flux may be assessed in normal (physiological) conditions and conditions enhancing autophagy and/or blocking autophagosome-lysosome fusion: by quantifying LC3 lipidation; quantifying LC3 puncta formation; quantification of autophagy maturation with pH dependent LC3 -fluorescent probes, quantifying degradation of p62 and other cargos; quantifying lysosomal fusion; visualizing the formation of the double membrane autophagosomes; using techniques ranging from photonic microscopy and electron microscopy, and various immunoblotting techniques.
  • Sonic Hedgehog (Shh) transduction is intended to refer to the well-documented Sonic hedehog signaling pathway, which triggers the activation of the canonical and non-canonical Shh pathway.
  • the activation of Shh reflects Shh transduction and it can be also assessed by conventional methods and protocols described in the state of the art.
  • Shh activation or repression can be assessed by several assays, including but not restricted to the measures of i) the level of expression of Shh-responsive target genes (NKc ⁇ .I, PTCH, Gli.7) using in situ hybridization, and/or immunofluorescence and/or western-blotting ii) Shh signaling activity using cellular and/or plasmid reporter (such as Shh-Light 2 cells, 7Gli::GFP plasmid....), iii) Smothened localization to the cilium, iv) the cilium length, and any other methods developed to measure Shh activity (ratio of active and non active from of Ci/Gli .).
  • Shh activity ratio of active and non active from of Ci/Gli .
  • the term“altered Sonic Hedgehog (Shh) transduction” is intended to refer to a level of Shh transduction which is either increased or decreased as compared to the physiological level of Shh transduction.
  • the modulator for use according to the invention is for the prevention and/or the treatment of a disease associated with altered autophagy.
  • the modulator for use according to the invention is for the prevention and/or the treatment of a neurodegenerative disease.
  • the modulator for use according to the invention is for the prevention and/or the treatment of a disease associated with altered Shh signaling.
  • the modulator for use according to the invention for the prevention and/or treatment of a neurodegenerative disease is a nucleic acid having at least 75% identity with SEQ ID NO. 1; or
  • polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2.
  • the modulator for use according to the invention for the prevention and/or treatment of a neurodegenerative disease is a nucleic acid having at least 80% identity with SEQ ID NO. 1; or
  • polypeptide comprising a polypeptide sequence having at least 80% identity with SEQ ID NO. 2.
  • the modulator for use according to the invention for the prevention and/or treatment of a neurodegenerative disease is a nucleic acid having at least 85% identity with SEQ ID NO. 1; or
  • polypeptide comprising a polypeptide sequence having at least 85% identity with SEQ ID NO. 2.
  • the modulator for use according to the invention for the prevention and/or treatment of a neurodegenerative disease is a nucleic acid having at least 90% identity with SEQ ID NO. 1; or
  • polypeptide comprising a polypeptide sequence having at least 90% identity with SEQ ID NO. 2.
  • the modulator for use according to the invention for the prevention and/or treatment of a neurodegenerative disease is a nucleic acid having at least 95% identity with SEQ ID NO. 1; or
  • polypeptide comprising a polypeptide sequence having at least 95% identity with SEQ ID NO. 2.
  • prevention is intended to refer to a reduction of the risk of occurrence of a disease associated with altered autophagy.
  • the term“treatment” is intended to refer to the effects achieved following the administration of a modulator according to the invention in an individual suffering from a disease, such as one associated with altered autophagy, and resulting in the partial or total alleviating of the symptoms linked to said disease.
  • the invention further relates to a method for the prevention and/or the treatment of a disease associated with altered autophagy in an individual in need thereof, comprising the step of administering to the said individual a modulator according to the invention.
  • the invention further relates to a method for the prevention and/or the treatment of a disease associated with altered autophagy in an individual in need thereof, comprising the step of administering to the said individual an efficient amount of a modulator according to the invention.
  • an“individual in need thereof’ is intended to refer to any human or non-human animal suffering or susceptible to suffer from a disease associated with altered autophagy.
  • said individual may be a mammal, and more preferably an animal of economic importance, such as a sheep, swine, cattle, goat, dog, cat, horse, poultry, mouse and a rat. More preferably, an individual according to the invention may be a human.
  • the individual is a zebrafish, which may represent a valuable model for disease and drug screening.
  • the disease in particular associated with altered autophagy is a genetic disease.
  • the disease in particular associated with altered autophagy is an acquired disease.
  • the disease in particular associated with altered autophagy is selected in a group consisting of a cancer, an immune disease, an infectious disease, a metabolic disease, a cardiovascular disease, a (cardio)myopathy, a lysosomal disease, spinal cord injury and trauma, a neurodegenerative disease and a pulmonary disease.
  • the cancer is selected in a non-limitative group comprising a bladder cancer, a blastoma, a bone cancer, a brain cancer, a breast cancer, a cancer of the central nervous system, a cancer of the cervix, a cancer of the upper aero digestive tract, a carcinoma, a colorectal cancer, an endometrial cancer, a germ cell cancer, a glioblastoma, a Hodgkin lymphoma, a kidney cancer, a laryngeal cancer, a leukaemia, a liver cancer, a lung cancer, a melanoma, a myeloma, a nephroblastoma (Wilms tumor), a neuroblastoma, a non-Hodgkin lymphoma, a non-small cell lung cancer, an oesophageal cancer, an osteosarcoma, an ovarian cancer, a pancreatic cancer, a pleural cancer, a
  • the immune disease is selected in a group consisting of Achalasia, Addison’s disease, Adult Still's disease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome, Autoimmune angioedema, Autoimmune dysautonomia, Autoimmune encephalomyelitis, Autoimmune hepatitis, Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune orchitis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmune urticaria, Axonal & neuronal neuropathy (AMAN), Balo disease, Behcet’s disease, Benign mucosal pemphigoid, Bullous pemphigoid, Castleman disease (CD), Celiac
  • the infectious disease may be a microbial infection, in particular a bacterial infection or a viral infection.
  • the infectious disease selected in a non-limiting group comprising Anaplasmosis; Anthrax; Babesiosis; Botulism; Brucellosis; Burkholderia mallei infection (glanders); Burkholderia pseudomallei infection (melioidosis); Campylobacteriosis; Carbapenem-resistant Enterobacteriaceae infection (CRE); Chancroid; Chikungunya infection; Chlamydia infection; Ciguatera; Clostridium difficile infection; Clostridium perfringens infection (Epsilon Toxin); Coccidioidomycosis fungal infection (Valley fever); Creutzfeldt- Jacob Disease, transmissible spongioform (CJD); Cryptosporidiosis; Cyclosporiasis; Dengue Fever; Diphtheria; E.
  • Anaplasmosis Anaplasmosis
  • Anthrax Bacesiosis
  • Botulism Brucellosis
  • Coli infection Eastern Equine Encephalitis (EEE); Ebola Hemorrhagic Fever (Ebola); Ehrlichiosis; Arboviral or parainfectious encephalitis; Non-polio enterovirus infection; D68 enterovirus infection, (EV-D68); Giardiasis; Gonococcal infection (Gonorrhea); Granuloma inguinale; Type B Haemophilus Influenza disease, (Hib or H-flu); Hantavirus pulmonary syndrome (HPS); Hemolytic uremic syndrome (HUS); Hepatitis A (Hep A); Hepatitis B (Hep B); Hepatitis C (Hep C); Hepatitis D (Hep D); Hepatitis E (Hep E); Herpes; Herpes zoster, zoster VZV (Shingles); Histoplasmosis; Human Immunodeficiency Virus/ AIDS (HIV/AIDS); Human Papillomarivus (HP
  • the bacterial infection may encompass an infection by a bacterium of the genus Bacillus , such as the species Bacillus anthracis and Bacillus cereus Bartonella , such as the species Bartonella henselae and Bartonella Quintana ; Bordetella , such as the species Bordetella pertussis ; Borrelia, such as the species Borrelia burgdorferi , Borrelia garinii, Borrelia afzelii , Borrelia recurrently Brucella , such as the specied Brucella abortus , Brucella canis, Brucella melitensis , Brucella suis; Campylobacter , such as the species Campylobacter jejuni ; Chlamydia , such as the species Chlamydia pneumoniae , Chlamydia trachomatis ; Chlamydophila , such as the species Chlamydophila
  • the cardiovascular disease may be selected in a non- limitative group comprising a coronary artery disease, a heart attack, an abnormal heart rhythm (arrhythmia), a heart failure, a heart valve disease, a congenital heart disease, a heart muscle disease (cardiomyopathy), a pericardial disease, an aorta disease, a Marfan syndrome and a vascular disease.
  • a coronary artery disease a heart attack, an abnormal heart rhythm (arrhythmia), a heart failure, a heart valve disease, a congenital heart disease, a heart muscle disease (cardiomyopathy), a pericardial disease, an aorta disease, a Marfan syndrome and a vascular disease.
  • the metabolic disease encompasses acute pancreatic, diabetes, a lysosomal storage disease, obesity and Paget’s disease.
  • lysosomal storage diseases are described, e.g., in Settembre et al. (Autophagy. 2008 Jan;4(l): 113-4).
  • the myopathy is selected in a group consisting of cardiomyopathies, Bethlem myopathy, Danon’s disease, Dilated cardiomyopathy (DCM), Pompe’s disease, limb girdle muscular dystrophy type 2B, Miyoshi disease, sporadic inclusion body myositis, Ullrich congenital muscular dystrophy (UCMD) and X-Linked Myopathy with Excessive Autophagy (XMEA).
  • cardiomyopathies Bethlem myopathy, Danon’s disease, Dilated cardiomyopathy (DCM), Pompe’s disease, limb girdle muscular dystrophy type 2B, Miyoshi disease, sporadic inclusion body myositis, Ullrich congenital muscular dystrophy (UCMD) and X-Linked Myopathy with Excessive Autophagy (XMEA).
  • the neurodegenerative disease is selected in a non- limitative group comprising Alexander’s disease, Ataxia telangiectasia, Alper’s disease, Alzheimer's disease (AD), Amyotrophic Lateral Sclerosis (ALS), Batten disease, Beta- propeller protein-associated neurodegeneration (BPAN), Canavan disease, Cockayne disease, Charcot-Marie-Tooth disease (CMT), Cortico-basal degeneration (CBD), Epilepsy, Friedreich's ataxia, Fronto-temporal dementia (FTD), Gerstmann-Straussler- Scheinker disease (GSS), Giant Axonal Neuropathy (GAN), Guam-ALS syndrome, HIV- associated disease, Huntington's disease (HD), Lafora disease, Leigh's disease, Lewy body disease, Machado-Joseph disease, Neurodegeneration due to stroke, Neurodegeneration with brain iron accumulation (NBIA), Pallido-ponto-nigral degeneration (PPND), Pallido- ni
  • the neurodegenerative diseases which are particularly considered by the invention include neurodegenerative diseases associated with altered autophagy, and/or those associated with altered motor activity, and infantile neurological diseases.
  • a selection of neurodegenerative diseases which is particularly considered, in the sense of the invention, includes neurodegenerative diseases which are characterized by a loss of motor neurons, including Amyotrophic Lateral Sclerosis (ALS), Spinal muscular atrophy (SMA), and Charcot-Marie-Tooth disease (CMT).
  • ALS Amyotrophic Lateral Sclerosis
  • SMA Spinal muscular atrophy
  • CMT Charcot-Marie-Tooth disease
  • the neurodegenerative disease may in particular be Giant Axonal Neuropathy (GAN), as exemplified.
  • GAN Giant Axonal Neuropathy
  • the neurodegenerative disease may be distinct from Giant Axonal Neuropathy (GAN).
  • the lysosomal disease may be selected in a non- limitative group comprising an alpha-mannosidosis, an aspartylglucosaminuria, a Batten disease, a beta-mannosidosis, a cystinosis, a Danon disease, a Fabry disease, a Farber disease, a fucosidosis, a galactosialidosis, a Gaucher disease, a gangliosidosis (GM1 gangliosidosis and GM2-gangliosidosis AB variant), a Krabbe disease, a metachromatic leukodystrophy, a mucopolysaccharidose disorder, a mucolipidosis, a multiple sulfatase deficiency, a Niemann-Pick disease, a Pompe disease (glycogen storage disease), a pycnodysostosis, a Sandhoff disease, a Schindler disease, a Salla disease (
  • the neurodegenerative disease is selected in a group consisting of Alzheimer’s disease, Amyotrophic Lateral Sclerosis (ALS), Giant Axonal Neuropathy (GAN), Huntington’s disease, Parkinson’s disease, Spinal Muscular Atrophy and Transmissible spongiform encephalopathies.
  • ALS Amyotrophic Lateral Sclerosis
  • GAN Giant Axonal Neuropathy
  • Parkinson Parkinson’s disease
  • Spinal Muscular Atrophy Transmissible spongiform encephalopathies.
  • the pulmonary disease is cystic fibrosis.
  • the disease associated with altered autophagy is a disease characterized by a cellular accumulation or depletion, or mislocalization of ATG16L1.
  • the disease is associated with altered Shh transduction.
  • diseases associated with altered Shh transduction are known in the Art and further defined in Bale et al.;“ Hedgehog Signaling and Human Disease”; Annu. Rev. Genomics Hum. Genet; 3:47-65; 2002).
  • a disease may be associated both to an altered autophagy and to an altered Shh transduction in a patient.
  • the disease associated with altered Shh transduction is a disease characterized by a cellular accumulation or depletion, or mislocalization of PTCH.
  • the disease associated with altered Shh transduction is a neurodevelopmental or neurodegenetative disease, a congenital malformation of the central nervous system, of the axial skeleton and limbs, cancers and malignancies, including malignancies in children and adults.
  • the disease associated with altered Shh transduction is selected from a list consisting of: Huntington’s disease, Alzheimer’s disease, holoprosencephaly, Grieg cephalopolysyndactly, Pallister- Hall syndrome, Postaxial polydactyly type 3, VATER association (or “VACTERL Syndrome”), Smith-Lemli-Opitz syndrome, Gorlin syndrome, Sporadic basal-cell carcinoma, Sporadic medulloblastoma, Glioblastoma, Joubert syndrome, microphtalmia, holoproencephaly, epilepsy.
  • the disease associated with altered Shh transduction is basal cell carcinoma.
  • the modulator for use according to the present invention is selected in a group comprising Gigaxonin, an activator of Gigaxonin expression, an inhibitor of Gigaxonin expression, an activator of Gigaxonin stability, an inhibitor of Gigaxonin stability, an activator of Gigaxonin degradation, an inhibitor of Gigaxonin degradation, an activator of Gigaxonin activity and an inhibitor of Gigaxonin activity, and is preferably Gigaxonin.
  • the modulator is selected in a group consisting of a polypeptide and a nucleic acid.
  • the nucleic acid is a deoxyribonucleic acid or a ribonucleic acid.
  • the modulator when it is a nucleic acid, it may be selected from a complementary DNA, a plasmid DNA, or a messenger RNA.
  • “Gigaxonin” is intended to refer to either the polypeptide itself or the polypeptide as expressed by the GAN gene, i.e. the gene encoding the Gigaxonin polypeptide.
  • the Gigaxonin is:
  • nucleic acid comprising a nucleic acid (sequence) having at least 75% identity with SEQ ID NO. 1;
  • polypeptide comprising a polypeptide (sequence) having at least 75% identity with SEQ ID NO. 2.
  • the Gigaxonin is:
  • nucleic acid comprising a nucleic acid sequence having at least 80% identity with SEQ ID NO. 1;
  • polypeptide comprising a polypeptide sequence having at least 80% identity with SEQ ID NO. 2
  • the Gigaxonin is:
  • nucleic acid comprising a nucleic acid seqeunce having at least 85% identity with SEQ ID NO. 1;
  • polypeptide comprising a polypeptide sequence having at least 85% identity with SEQ ID NO. 2
  • the Gigaxonin is:
  • nucleic acid comprising a nucleic acid sequence having at least 90% identity with SEQ ID NO. 1;
  • polypeptide comprising a polypeptide sequence having at least 90% identity with SEQ ID NO. 2
  • the Gigaxonin is:
  • nucleic acid comprising a nucleic acid sequence having at least 95% identity with SEQ ID NO. 1;
  • the Gigaxonin may be provided as a nucleic acid encoding the Gigaxonin polypeptide, for example, as in the form of a plasmid or a nucleic acid vector that will be expressed and lead to the production of Gigaxonin when the said nucleic acid or the said plasmid or the said nucleic acid vector is introduced into a cell or bacteria, i.e. when a cell is transfected or transformed with the said nucleic acid or with the said plasmid or nucleic acid vector.
  • the nucleic acid encoding the Gigaxonin polypeptide comprises a nucleic acid having at least 75% identity with SEQ ID NO. 1.
  • the Gigaxonin comprises a polypeptide having at least 75% identity with SEQ ID NO. 2.
  • the expression “at least 75% identity” encompasses 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% identity.
  • the nucleic acid encoding the Gigaxonin polypeptide has at least 80% identity with SEQ ID NO. 1.
  • the nucleic acid encoding the Gigaxonin polypeptide has at least 85% identity with SEQ ID NO. 1.
  • the nucleic acid encoding the Gigaxonin polypeptide has at least 90% identity with SEQ ID NO. 1.
  • the nucleic acid encoding the Gigaxonin polypeptide has at least 95% identity with SEQ ID NO. 1.
  • the“percentage identity” between two sequences of nucleic acids or proteins means the percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after optimal alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly along their length.
  • the comparison of sequences is traditionally carried out by comparing the sequences after having optimally aligned them, said comparison being able to be conducted by segment or by using an“alignment window”.
  • Optimal alignment of the sequences for comparison can be carried out, in addition to comparison by hand, by means of the local homology algorithm of Smith and Waterman (1981), by means of the local homology algorithm of Neddleman and Wunsch (1970), by means of the similarity search method of Pearson and Lipman (1988) or by means of computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by the comparison software BLAST NR or BLAST P).
  • the percentage identity between two sequences is determined by comparing the two optimally-aligned sequences in which the sequence to compare can have additions or deletions compared to the reference sequence for optimal alignment between the two sequences. Percentage identity is calculated by determining the number of positions at which the nucleotide or amino acid residue is identical between the two sequences, preferably between the two complete sequences, dividing the number of identical positions by the total number of positions in the alignment window and multiplying the result by 100 to obtain the percentage identity between the two sequences.
  • the nucleic acid encoding the Gigaxonin polypeptide comprises a nucleic acid of SEQ ID NO. 1. In some other embodiments, the nucleic acid encoding the Gigaxonin polypeptide consists of a nucleic acid of SEQ ID NO. 1
  • the Gigaxonin polypeptide comprises a polypeptide of SEQ ID NO. 2. In some other embodiments, the Gigaxonin polypeptide consists of a polypeptide of SEQ ID NO. 2.
  • vectorization of a nucleic acid encoding the Gigaxonin polypeptide may be performed by the mean of conventional viral vector particles well known in the art.
  • viral vector particles encompass, e.g., baculovirus, retroviral vector particles, lentiviral vector particles, adenoviral vector particles and adeno-associated viral vector particles.
  • the Gigaxonin may be provided as a purified and/or recombinant polypeptide.
  • recombinant polypeptide can be produced in eucaryotic cells, such as insect cells.
  • Gigaxonin in order to implement the invention may be adapted, e.g., from Sambrook and Green (2012; Molecular Cloning: A Laboratory Manual (Fourth Edition); Cold Spring Harbor Laboratory Press).
  • an activator of Gigaxonin expression is intended to refer to a compound able to increase, at least in part, the physiological cellular content of the Gigaxonin polypeptide.
  • an inhibitor of Gigaxonin expression is intended to refer to a compound causing a decrease of the cellular content of the Gigaxonin polypeptide.
  • Non-limitative examples of inhibitors of Gigaxonin expression encompass siRNAs, miRNAs, piRNAs that specifically bind to the Gigaxonin encoding nucleic acid or its corresponding mRNA, or alternatively, to a regulator of Gigaxonin expression.
  • the inhibitor of Gigaxonin expression is a siRNA, a miRNA, or a piRNA that binds to a RNA complementary to a nucleic acid having at least 75% identity with SEQ ID NO. 1.
  • Non-limitative examples of activators of Gigaxonin expression encompass siRNAs, miRNAs, piRNAs that specifically bind to a negative regulator of Gigaxonin expression, and/or transcription factors and co-activators that bind to the Gigaxonin promotor to mediate transcription of the GAN gene.
  • the term “complementary” is intended to mean that a first nucleic acid is complementary to a second nucleic acid when these nucleic acids have the base on each position which is the complementary (i.e. A to T, C to G) and in the reverse order.
  • the complementary sequence to TTAC is GTAA. If one strand of the double-stranded DNA is considered the sense strand, then the other strand, considered the antisense strand, will have the complementary sequence to the sense strand.
  • the siRNAs or the miRNAs according to the invention bind to RNA complementary to a nucleic acid comprising a nucleic acid (sequence) of SEQ ID NO. 1. In some other embodiments, the siRNAs or the miRNAs according to the invention bind to RNA complementary to a nucleic acid of SEQ ID NO.
  • the term“Gigaxonin stability” is intended to refer to the equilibrium reached between the synthesis and the degradation of the Gigaxonin polypeptide.
  • an activator of Gigaxonin stability encompasses a compound which promotes the synthesis of Gigaxonin and/or decreases or reduces the degradation of the Gigaxonin polypeptide.
  • an inhibitor of Gigaxonin stability encompasses a compound which decreases or reduces the synthesis of Gigaxonin and/or promotes or increases the degradation of the Gigaxonin polypeptide.
  • E3-Ligases deubiquitination enzymes (DUB), kinases, phosphatases and other enzymes which may cause post-translational modifications (PTM) of Gigaxonin: such as acetylation, amidation, hydroxylation, methylation, N-glycosylation, O-glycosylation, phosphorylation, sulfation, sumoylation, fumoylation, lipidation, neddylation, and ubiquitination.
  • DAB deubiquitination enzymes
  • kinases kinases
  • phosphatases phosphatases
  • PTM post-translational modifications
  • an activator of Gigaxonin activity is intended to refer to a compound able to increase, at least in part, the ability of the Gigaxonin polypeptide to promote its physiological role in the cell.
  • an inhibitor of Gigaxonin activity is intended to refer to a compound able to decrease, at least in part, the ability of the Gigaxonin polypeptide to promote its physiological role in the cell.
  • a suitable inhibitor of Gigaxonin activity may be an antibody, an aptamer specifically binding to Gigaxonin and altering its interaction with ATG16L1, or altering the subsequent ubiquitination of ATG16 in presence of an intact ATG16/Gig complex.
  • a suitable inhibitor of Gigaxonin activity may be an antibody, an aptamer specifically binding to Gigaxonin and altering its interaction with PTCH, or altering the subsequent ubiquitination of PTCH in presence of an intact PTCH/Gig complex.
  • the inhibitor of Gigaxonin activity is an antibody or an aptamer that binds to a polypeptide having at least 75% identity with SEQ ID NO. 2.
  • the antibody or aptamer according to the invention binds to a polypeptide of SEQ ID NO. 2.
  • a suitable antibody may encompass IgA, IgD, IgE, IgG and IgM immunoglobulin.
  • Suitable antibodies may be monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, humanized antibodies and optimized antibodies, for example antibodies with modified glycosylation and antibodies having a variant Fc region having optimized binding affinity with one or more Fc receptors.
  • Chimeric antibodies contain naturally occurring variable region (light chain and heavy chain) derived from an antibody from a given first species which is fused with the constant regions of the light chain and of the heavy chain derived from an antibody of a second species, distinct from the first species.
  • Antibodies suitable for the instant invention can be prepared using genetic recombination techniques. Chimeric or humanized antibodies can be prepared using standard methods described in the state of the art.
  • a modulator of the Gigaxonin/ATG16Ll or Gigaxonin/PTCH interaction is intended to encompass any compound which either increases or decreases the ability of Gigaxonin and ATG16L1/PTCH to interact in physiological conditions.
  • a modulator of the Gigaxonin/ATG16Ll interaction or Gigaxonin/PTCH interaction may encompass an antibody, a chemical, a cofactor and an enzyme, which modifies either protein to impair its ability to interact with the other one, in particular a phoshorylase and kinase, or an E3 ligase or a Deubiquitinating enzyme (DUB).
  • a modulator of the functionality of the Gigaxonin/ATG16Ll interaction or of the Gigaxonin/PTCH interaction may encompass an antibody, a chemical, a cofactor and an enzyme, which modifies either protein to impair its ability to interact with the other one, in particular a phoshorylase and kinase, or an E3 ligase or a Deubiquitinating enzyme (DUB).
  • a modulator of the functionality of the Gigaxonin/ATG16Ll interaction is intended to encompass any compound which either increases or decreases the physiological function of the Gigaxonin/ATG16Ll complex.
  • Gigaxonin/ATG16Ll interaction may encompass a compound that alters the ubiquitination or any other Post Translational Modifications of ATG16L1 or Gigaxonin.
  • a modulator of the functionality of the Gigaxonin/PTCH interaction is intended to encompass any compound which either increases or decreases the physiological function of the Gigaxonin/PTCH complex.
  • Gigaxonin/PTCH interaction may encompass a compound that alters the ubiquitination or any other Post Translational Modifications of PTCH or Gigaxonin.
  • the level of abundance of Gigaxonin the level of abundance of Gigaxonin
  • Gigaxonin/ ATG16L interaction the Gigaxonin/PTCH interaction, the functionality of the Gigaxonin/ ATG16L1 interaction, and the functionality of the Gigaxonin/PTCH interaction may be measured by any suitable technique, such as, e.g. Western Blot, Co- immunoprecipitation, ELISA, FRET (Fluorescence Resonance Energy Transfer), Bimolecular Fluorescence Complementation (BiFC), Proximity Ligation Assay (PL A), ubiquitination (or ubiquitin-like) assay, phosphorylation and kinase assay, pull-down assay, Crosslinking protein interaction analysis, Label transfer protein interaction analysis.
  • any suitable technique such as, e.g. Western Blot, Co- immunoprecipitation, ELISA, FRET (Fluorescence Resonance Energy Transfer), Bimolecular Fluorescence Complementation (BiFC), Proximity Ligation Assay (PL A), ubiquitination
  • a modulator of the functionality of the Gigaxonin/PTCH interaction is intended to encompass any compound which either increases or decreases the physiological function of the Gigaxonin/PTCH complex.
  • a modulator of the functionality of the Gigaxonin/PTCH interaction may encompass a compound that alters the ubiquitination or any other Post Translational Modifications of PTCH or Gigaxonin. • Delivery particles
  • Another aspect of the invention further relates to a delivery particle comprising a modulator of Gigaxonin, the Gigaxonin/ATG16Ll interaction, the Gigaxonin/PTCH interaction and/or functionality as disclosed herein.
  • the invention relates to a delivery particle comprising a polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2, or a nucleic acid coding for such a polypeptide; such as a nucleic acid having at least 75% identity with SEQ ID NO.l.
  • the delivery particle may be in the form of a lipoplex or a lipid nanocapsule, comprising cationic lipids; a lipid nano-emulsion; a solid lipid nanoparticle; a peptide based particle; a polymer based particle, in particular comprising natural and/or synthetic polymers.
  • a polymer based particle may comprise a synthetic polymer, in particular, a polyethylene glycol (PEG), a polyethylene imine (PEI), a dendrimer, a poly (DL- Lactide) (PLA), a poly(DL-Lactide-co-glycoside) (PLGA), a polymethacrylate and a polyphosphoesters.
  • the delivery may further comprise at its surface one or more targeting ligands suitable for specifically addressing said particle to a targeted cell.
  • a polymer based particle may comprise a protein, in particular an antibody or a fragment thereof; a peptide; a mono-saccharide, an oligo saccharide or a polysaccharide, in particular chitosan; a hormone; a vitamin; a ligand of a cellular receptor.
  • the delivery particles according to the invention may be introduced in one or more target cells by the means of suitable methods known in the art, such as methods used for transfecting cells, which include electroporation, osmotic choc, sonoporation, cell squeezing and the like.
  • the delivery particle may comprise a viral vector, in particular an adenovirus, an adeno-associated virus (AAV), an alphavirus, a herpesvirus, a lentivirus, a non-integrative lentivirus, a retrovirus, vaccinia virus or a bacculovirus.
  • a viral vector in particular an adenovirus, an adeno-associated virus (AAV), an alphavirus, a herpesvirus, a lentivirus, a non-integrative lentivirus, a retrovirus, vaccinia virus or a bacculovirus.
  • composition and uses thereof may be performed by following any suitable method known in the state of the art. • Pharmaceutical composition and uses thereof
  • the invention also pertains to a pharmaceutical composition
  • a pharmaceutical composition comprising:
  • the invention also pertains to a pharmaceutical composition
  • a pharmaceutical composition comprising:
  • a modulator may be a polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2, or a nucleic acid coding for such a polypeptide.
  • Such a pharmaceutical composition may thus comprise a nucleic acid having at least 75% identity with SEQ ID NO. 1; or a polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2.
  • such a pharmaceutical composition may thus comprise a nucleic acid having at least 80% identity with SEQ ID NO. 1; or a polypeptide comprising a polypeptide sequence having at least 80% identity with SEQ ID NO. 2.
  • such a pharmaceutical composition may thus comprise a nucleic acid having at least 85% identity with SEQ ID NO. 1; or a polypeptide comprising a polypeptide sequence having at least 85% identity with SEQ ID NO. 2.
  • such a pharmaceutical composition may thus comprise a nucleic acid having at least 90% identity with SEQ ID NO. 1; or a polypeptide comprising a polypeptide sequence having at least 90% identity with SEQ ID NO. 2.
  • such a pharmaceutical composition may thus comprise a nucleic acid having at least 95% identity with SEQ ID NO. 1; or a polypeptide comprising a polypeptide sequence having at least 95% identity with SEQ ID NO. 2.
  • the invention relates to a pharmaceutical composition, as defined above, comprising a nucleic acid having at least 75% identity with
  • the invention also relates to a pharmaceutical composition according to the present disclosure, for use in the prevention and/or the treatment of a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
  • compositions suitable to implement the disclosed invention may be obtained by following the routine and common principles, methods and techniques disclosed in the state of the art.
  • a suitable pharmaceutically acceptable vehicle according to the invention may include any conventional solvent, dispersion medium, filler, solid carrier, aqueous solution, coating, antibacterial and antifungal agent, isotonic and absorption delaying agent, the like and a mixture thereof.
  • suitable pharmaceutically acceptable vehicles may include, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and a mixture thereof.
  • pharmaceutically acceptable vehicles may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the cells.
  • the pharmaceutical composition according to the present invention is for use in the prevention and/or the treatment of a disease associated with altered autophagy.
  • modulator and/or the pharmaceutical composition, as disclosed herein may be administered to an individual in need thereof by any route, i.e. by an oral administration, a topical administration or a parenteral administration, e.g., by injection, including a sub-cutaneous administration, a venous administration, an arterial administration, in intra-muscular administration, an intra-ocular administration and an intra-auricular administration.
  • the administration of the modulator and/or the pharmaceutical composition, as encompassed herein, by injection may be directly performed in the target tissue of interest, in particular in order to avoid spreading of the said product.
  • Suitable modes of administration may also employ pulmonary formulations, suppositories, and transdermal applications.
  • an oral formulation according to the invention includes usual excipients, such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • an effective amount of said modulator is administered to said individual in need thereof.
  • an“effective amount” refers to the amount of said compound that alone stimulates the desired outcome, i.e. alleviates or eradicates the symptoms of the encompassed disease.
  • the effective amount of the modulator and/or the pharmaceutical composition to be administered may be determined by a physician or any trained and authorized person skilled in the art and can be suitably adapted within the time course of the treatment.
  • the effective amount to be administered may depend upon a variety of parameters, including the material selected for administration, whether the administration is in single or multiple doses, and the individual’s parameters including age, physical conditions, size, weight, gender, and the severity of the disease to be treated.
  • an effective amount of the modulator and/or the pharmaceutical composition according to the instant invention may comprise from about 0.001 mg to about 3000 mg, per dosage unit, preferably from about 0.05 mg to about 100 mg, per dosage unit.
  • from about 0.001 mg to about 3000 mg includes, from about 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1100
  • 1850 mg 1900 mg, 1950 mg, 2000 mg, 2100 mg, 2150 mg, 2200 mg, 2250 mg, 2300 mg,
  • the modulator and/or the pharmaceutical composition according to the instant invention may be administered at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day.
  • an effective amount of the nucleic acid encoding the Gigaxonin polypeptide may comprise from about 1 ng to about 1 mg, per dosage unit, preferably from about 50 ng to about 100 pg, per dosage unit.
  • from about 1 ng to about 1 mg includes, about 2 ng, 3 ng, 4 ng, 5 ng, 6 ng, 7 ng, 8 ng, 9 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 150 ng, 200 ng, 250 ng, 300 ng, 350 ng, 400 ng, 450 ng, 500 ng, 550 ng, 600 ng, 650 ng, 700 ng, 750 ng, 800 ng, 850 ng, 900 ng, 950 ng,
  • the nucleic acid encoding the Gigaxonin polypeptide or the nucleic acid vector may be administered at dosage levels sufficient to deliver from about 0.01 ng/kg to about 10 pg/kg, from about 0.1 ng/kg to about 5 pg/kg, preferably from about 1 ng/kg to about 1 pg/kg of subject body weight per day.
  • the pharmaceutically composition may further comprise one or more additional active agent, in particular selected in a group comprising an antimicrobial compound and an anticancer compound.
  • the antimicrobial compound is an antibiotic selected in a group comprising a penicillin, in particular penicillin and amoxicillin; a carbapenem, in particular imipenem; a cephalosporin, in particular cephalexin; an aminoglycoside, in particular gentamicin and tobramycin; a tetracycline, in particular tetracycline and doxycycline; a macrolide, in particular erythromycin and clarithromycin; a quinolone, in particular ciprofloxacin and levofloxacin; and a sulphonamide, in particular sulfamethizole and sulfamethoxazole.
  • a penicillin in particular penicillin and amoxicillin
  • a carbapenem in particular imipenem
  • a cephalosporin in particular cephalexin
  • an aminoglycoside in particular gentamicin and tobramycin
  • a tetracycline in particular tetra
  • the antimicrobial compound is an antiviral agent selected in a non-limiting group comprising a neuraminidase inhibitor; a nucleoside analogue of guanine; a nucleoside analogue of thymidine; a nucleotide reverse transcriptase inhibitor; and a protease inhibitor.
  • the anti-cancer compound may be selected in a group comprising an alkylating agent, a purine analogue, a pyrimidine analogue, an anthracycline, bleomycin, mytomycin, an inhibitor of topo-isom erase 1, an inhibitor of topo-isom erase 2, a taxan, a monoclonal antibody, a cytokine, an inhibitor of a protein kinase, and the like.
  • the methods disclosed herein may be achieved in vitro , in vivo or ex vivo , preferably, in vitro or ex vivo.
  • the system may be a sample obtained from an individual, such as, without limitation, one or more cells of the central nervous system, one or more embryonic cells, one or more epithelial cells, one or more germ cells, one or more hematopoietic progenitor cells, one or more hematopoietic stem cells, one or more induced Pluripotent Stem Cells (iPSC), one or more muscular cells, one or more progenitor cells, one or more stem cells, and a mixture thereof.
  • an individual such as, without limitation, one or more cells of the central nervous system, one or more embryonic cells, one or more epithelial cells, one or more germ cells, one or more hematopoietic progenitor cells, one or more hematopoietic stem cells, one or more induced Pluripotent Stem Cells (iPSC), one or more muscular cells, one or more progenitor cells, one or more stem cells, and a mixture thereof.
  • iPSC induced Pluripotent Stem
  • the sample may originate from a tissue selected in a group comprising a connective tissue, an epithelial tissue, a muscle tissue and a nervous tissue.
  • the sample may originate an organ selected in a group comprising a bladder, a bone, a brain, a breast, a central nervous system, a cervix, a colon, an endometrium, a kidney, a larynx, a liver, a lung, an oesophagus, an ovarian, a pancreas, a pleura, a prostate, a rectum, a retina, a salivary gland, a skin, a small intestine, a soft tissue, a stomach, a testis, a thyroid, an uterus, a vagina.
  • the invention relates to a method for screening a modulator of the expression stability and/or activity of Gigaxonin, of the Gigaxonin/ATG16Ll interaction and/or of the functionality of the Gigaxonin/ATG16Ll interaction, comprising the steps of:
  • step (d) determining a difference between the level measured in step (c) and a corresponding reference level obtained in the absence of the said candidate modulator.
  • determining a significant difference is indicative of the ability of the candidate modulator to efficiently modulate Gigaxonin, the interaction between Gigaxonin and ATG16L1 and the functionality of the complex.
  • the system provided in step (a) comprises Gigaxonin and ATG16L1 in conditions to form a complex.
  • the suitable conditions for the interaction between Gigaxonin and ATG16L1 to occur may be determined by a skilled artisan in the art. These conditions are non-limited to parameters such as temperature, pH, O2 content, salinity, within the said system.
  • the invention also relates to a method for screening a modulator of the expression stability and/or activity of Gigaxonin, of the Gigaxonin/PTCH interaction and/or of the functionality of the Gigaxonin/PTCH interaction, comprising the steps of
  • step (d) determining a difference between the level measured in step (c) and a corresponding reference level obtained in the absence of the said candidate modulator.
  • determining a significant difference is indicative of the ability of the candidate modulator to efficiently modulate Gigaxonin, the interaction between Gigaxonin and PTCH and the functionality of the complex.
  • the invention thus also relates to a method aimed at using Gigaxonin and/or ATG16L1 and/or PTCH (including biological samples such as cells comprising or expressing them) for developing readout tests and methodologies to screen existent and/or novel drugs to modulate autophagy activity and Shh transduction, and associated diseases.
  • Gigaxonin and/or ATG16L1 and/or PTCH including biological samples such as cells comprising or expressing them
  • the above-mentioned methods are thus applicable to methods for screening a candidate treatment for treating and/or preventing a disease associated with altered autophagy, or with altered Shh transduction, and/or neurodegenerative diseases.
  • the expression “significant difference” is intended to refer to a level of interaction measured in step (c) which is at least 10% or more, for example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% higher or lower than the reference level measured in step (a).
  • a “significant difference” refers to a level of interaction measured in step (c) which is at least 1.2 fold, 1.4 fold, 1.6 fold, 1.8 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold, 10 fold, 50 fold, 100 fold, 500 fold higher or lower than the reference level measured in step (a).
  • the method may further comprise a step (c’) comprising a step of measuring the effect on the steady state level and distribution of ATG16L1 and Gigaxonin.
  • the method may further comprise a step (c’) comprising a step of measuring the effect on the steady state level and distribution of PTCH and Gigaxonin.
  • step (d) comprises a step of determining a difference between the level measured in step (d) and a reference level obtained in the absence of the said candidate modulator.
  • a method for assessing the efficiency of a candidate treatment of a disease may include a step of providing an individual with a candidate treatment.
  • the invention may also pertain to a method for assessing the efficiency of a candidate treatment of a disease associated with altered autophagy in an individual in need thereof, comprising the steps of:
  • the invention relates to a method for assessing the efficiency of a candidate treatment of a disease associated with altered Shh transduction in an individual in need thereof, comprising the steps of:
  • step (d) determining a difference between the level measured in step (a) and the level measured in step (c).
  • the sample of step (a) may comprise, without limitation, one or more cells of the central nervous system, one or more embryonic cells, one or more epithelial cells, one or more germ cells, one or more hematopoietic progenitor cells, one or more hematopoietic stem cells, one or more induced Pluripotent Stem Cells (iPSC), one or more muscular cells, one or more progenitor cells, one or more stem cells, and a mixture thereof.
  • iPSC induced Pluripotent Stem Cells
  • the sample of step (a) may originate from a tissue selected in a group comprising a connective tissue, an epithelial tissue, a muscle tissue and a nervous tissue.
  • the sample of step (a) may originate an organ selected in a group comprising a bladder, a bone, a brain, a breast, a central nervous system, a cervix, a colon, an endometrium, a kidney, a larynx, a liver, a lung, an oesophagus, an ovarian, a pancreas, a pleura, a prostate, a rectum, a retina, a salivary gland, a skin, a small intestine, a soft tissue, a stomach, a testis, a thyroid, an uterus, a vagina.
  • a group comprising a bladder, a bone, a brain, a breast, a central nervous system, a cervix, a colon, an endometrium, a kidney, a larynx, a liver, a lung, an oesophagus, an ovarian, a pancreas, a pleura,
  • determining a significant difference is indicative of the ability of the candidate treatment to efficiently treat the individual in need thereof.
  • a method for assessing the efficiency of a candidate treatment of a disease associated with altered autophagy in an individual in need thereof comprising the steps of: - (a) measuring the level of the Gigaxonin/ATG16Ll interaction and/or the Gigaxonin/ATG16Ll functionality in a sample from said individual in need thereof, in suitable conditions for the interaction to occur;
  • step (f) determining a difference between (i) the levels measured in step (a) and (d), and (ii) the levels measured in step (b) and (e).
  • the invention further relates to a method to activate autophagy in a target cell, comprising a step of increasing the level of expression, stability, degradation and/or activity of Gigaxonin to reach physiological functions of ATG16L1.
  • the activation of autophagy is performed in conditions of ATG16L1 -dependant inhibition and/or aggregation
  • the said method is achieved by a reduction of the aggregation of ATG16L1 and/or the reduction of the stability and/or the expression of ATG16L1, or alternatively by an increase of the degradation of ATG16L1 in the target cell.
  • the expression “to reach physiological functions of ATG16L1” is intended to refer to a physiological level of ATG16L1 upon which ATG16L1 exerts its normal function in a target cell. In other words, this expression is intended to mean that the ATG16L1 has reached a balance between its synthesis and its degradation in order to perform its physiological role in autophagy.
  • the invention further relates to a method to activate, induce or restore Sonic Hedgehog (Shh) transduction in a target cell, comprising a step of increasing the level of expression, stability, degradation and/or activity of Gigaxonin to reach physiological functions of PTCH.
  • the increasing of the level of expression of Gigaxonin comprises the step of providing the target cell with (i) a nucleic acid encoding a Gigaxonin polypeptide or (ii) a Gigaxonin polypeptide.
  • the increasing of the level of expression of Gigaxonin comprises the step of providing the target cell with (i) a nucleic acid encoding a Gigaxonin polypeptide or (ii) a Gigaxonin polypeptide, or (iii) siRNAs or (iv) miRNAs or (v) piRNAs that specifically bind to a negative regulator of Gigaxonin expression or (vi) transcription factors and/or co-activators that bind to the Gigaxonin promoter to mediate transcription of the GAN gene.
  • the increasing of the level of expression of Gigaxonin comprises the step of providing the target cell with compounds able to increase Gigaxonin expression, stability or to impair its degradation.
  • the invention further relates to a method for the fine-tuned activation or inhibition of autophagy in a target cell comprising the step of modulating the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ ATG16L1 interaction and/or of the functionality of the Gigaxonin/ATG16Ll interaction.
  • the invention further relates to a method for the fine-tuned activation or inhibition of Sonic Hedgehog (Shh) transduction in a target cell comprising the step of modulating the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/PTCH interaction and/or of the functionality of the Gigaxonin/PTCH interaction.
  • Sonic Hedgehog Sonic Hedgehog
  • the invention may also pertain to a method for inducing autophagy in an individual in need thereof, comprising the step of administering to the said individual an efficient amount of Gigaxonin or its gene, of an activator of Gigaxonin expression, of an activator of Gigaxonin stability or an activator of Gigaxonin activity, or a molecule able to reduce Gigaxonin degradation.
  • the invention may also pertain to a method for inhibiting autophagy in an individual in need thereof, comprising the step of administering to the said individual an efficient amount of Gigaxonin, of an inhibitor of Gigaxonin expression, of an inhibitor of Gigaxonin stability or an inhibitor of Gigaxonin activity or an activator of Gigaxonin degradation. It is understood that inhibition of Gigaxonin steady level/activity inhibits autophagy, and that below a certain threshold, administration of Gigaxonin may induce autophagy, whereas above a certain threshold, administration of Gigaxonin may inhibit autophagy.
  • the above-mentioned threshold may be determined by a physician or any trained and authorized person skilled in the art.
  • the invention also relates to a method for assessing the efficiency of the treatment of a disease associated with altered autophagy in an individual in need thereof, comprising the steps of:
  • step (d) determining a difference between the level measured in step (a) and the level measured in step (c).
  • determining a significant difference is indicative of the ability of the candidate treatment to efficiently treat the individual in need thereof.
  • the present disclosure also relates to a method for assessing a post- translational modification (PTM) of ATG16L1, in response to the modulation of the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ ATG16L1 interaction and/or of the functionality of the Gigaxonin/ ATG16L1 interaction, comprising the steps of:
  • PTM post- translational modification
  • the PTM may be selected in a non limited group comprising acetylation, amidation, hydroxylation, methylation, N- glycosylation, O-glycosylation, phosphorylation, sulfation, sumoylation, fumoylation, lipidation, neddylation, and ubiquitination of ATG16L1
  • the PTM status of the ATG16L1 polypeptide may be determined accordingly with the standard methods known in the state of the art, i.e.
  • the instant disclosure further relates to a method for assessing the activity of the autophagy pathway, in response to the modulation of ATG16L1 by Gigaxonin, comprising the steps of:
  • step (c) monitoring the autophagy in the cellular system at step (b).
  • the said method may be implemented by any suitable method mentioned above.
  • a“cellular system” encompasses any collection of cells (i.e. primary culture cells or cell lines), especially any collection of human cells, which include cells originating from various organs or tissue of a human individual, or derived from a human individual (iPS cells), such as a healthy human individual and a human individual affected with a disease, including an individual affected with cancer.
  • cells i.e. primary culture cells or cell lines
  • human cells which include cells originating from various organs or tissue of a human individual, or derived from a human individual (iPS cells), such as a healthy human individual and a human individual affected with a disease, including an individual affected with cancer.
  • the present disclosure also concerns a method for blocking autophagy in conditions wherein autophagy is increased, regardless of the steps involved in the autophagy pathway.
  • the said method comprises a step of decreasing the Gigaxonin expression and/or stability.
  • the said method comprises a step of increasing the Gigaxonin expression and/or stability.
  • the said method comprises a step of increasing
  • the said method comprises a step of decreasing
  • the said method comprises a step of decreasing Gigaxonin/ATG16Ll interaction.
  • the inventors believe that decreasing Gigaxonin/ATG16Ll interaction would exert a dominant negative effect on autophagy, through the increased abundance of ATG16L1.
  • COS-7 cells (clone CRL-1651 from ATCC) and HEK293T cells (ATCC CRL- 11268) were maintained in DMEM medium (THERMOFISHER®) containing 10% foetal bovine serum (THERMOFISHER®) and 1% Penicillin/Streptomycin. Cells were tested negative for mycoplasma. Cortical neurons were obtained from El 5.5 old embryos from the GAN / model (Ganay et al.; Mol Neurodegene 12, 25 (2011)) and control littermates. Animal were treated in accordance with the European Union guide for the care and the use of animals in research (2010/63/UE).
  • brains cortices were dissected in HBSS (THERMOFISHER®) supplemented with 0.44 % glucose; enzymatically dissociated in HAMF 10/0.025% trypsin (THERMOFISHER®); mechanically dissociated in neurobasal medium/2% foetal bovine serum; and concentrated after centrifugation at 470xg on BSA cushions (SIGMA®).
  • Neurons were resuspended in Neurobasal Medium (THERMOFISHER®), supplemented with 1% Sodium pyruvate (THERMOFISHER®), 2% B27-Supplement (THERMOFISHER®) and 1% Penicillin/Streptomycin (THERMOFISHER®).
  • LC3-II/tubulin intensity was performed by the Image Lab software (http://www.bio-rad.com/fr-fr/product/image-lab-software).
  • the level of LC3 lipidation was determined by the relative LC3-II/tubulin ratio.
  • the LC3 flux was determined after comparison of the basal condition with serum deprivation condition (EBSS), with or without lysosome inhibitor (Bafilomycin Al).
  • EBSS serum deprivation condition
  • Bafilomycin Al lysosome inhibitor
  • the autophagosome synthesis was measured as the differences of LC3 lipidation at two different times of EBBS+Baf, at 2h and 6h. All values were expressed relatively to the LC3-Q/tubulin ratio in wild type neurons in basal condition (fixed to 1).
  • LC3 puncta formation cortical neurons were fixed as described in the immunostaining section. LC3 fluorescence intensity of individual cell was quantified by ImageJ software (https://imagej.nih.gov/ij/).
  • P62 aggregation in primary neurons was visualised by immunofluorescence, using rabbit anti-p62 (1 : 1000, PM045, MBL®) and mouse anti-MAP2 (1 : 1000, M4403, SIGMA®), in basal condition and EBSS, Baf and EBSS+Baf conditions. Lysosomal fusion was assessed by two independent analyses, by immunofluorescence on cortical neurons. The co-localisation of LC3 dots with lysosomes (lysotracker, 75 nM, L7528, THERMOFISHER®) was quantified in individual neurons by the ImageJ software (https://imagej.nih.gov/ij/) with the Pearson coefficient. The fusion of p62 with lysosomes was assessed by immunofluorescence, in conditions that either promote fusion (EBSS) or inhibit autolysosome formation (Baf and EBSS+Baf).
  • EBSS promote fusion
  • Baf and EBSS+Baf autolys
  • the lentiviral vector pLEX-MCS-FLAG-Gigaxonin, pLEX-MCS-GFP, p- VSVG and pAX2 were gifts from R.D. Goldman/P. Opal (J Clin Invest 123, 1964-1975 (2013)).
  • Lentiviral particles were produced after cotransfection of the pLEX-MCS-FLAG- Gigaxonin or pLEX-MCS-GFP plasmids, together with the helper plasmid p-VSYG and pAX2 into HEK293T cells. The supernatant was collected from the cells two days later and the virus was concentrated by ultracentrifugation.
  • the human Cherry-ATG16L1 plasmid was generated by reverse transcription (Superscript III Kit, INVITROGEN®) from HeLa cells mRNA. cDNA was amplified using primers flanked by ATTB1/ATTB2 sequences, and subcloned into gateway vectors. Flag-ATG16L1 deletion constructs are disclosed in Fujita et al. (Mol Biol Cell 19, 2092- 2100 (2008)). The human Full-length and deletion Gigaxonin cDNAs are disclosed in Bomont and Koenig & Cleveland et al. (Hum Mol Genet 12, 813-822 (2003), Hum Mol Genet. 2009 Apr 15; 18(8): 1384-94).
  • Gateway plasmids were pcDNA-Cherry-N or pCi-3xFlag-N.
  • Bimolecular Fluorescence Complementation (BiFC) assay ATG16L1 and Gigaxonin cDNA were cloned in Gateway BiFC vectors, in fusion with the N-terminal region (YFPN: l-154aa), and the C-terminal region (YFPC: 155-239aa) of the YFP protein, respectively.
  • the primers used are the following (ATTB sequences in lower cases): and are common across gateway vectors (ATTB sequences in lower cases):
  • siRNAs Dharmacon of the following antisense sequences: siRNA-5’ AUAACAUAAAUACUGGCUC 3’ (SEQ ID NO. 13) with the mismatch counterpart ms antisense sequence -5’ AUAAAAUAAAUACGGGCUC 3’ (SEQ ID NO. 14).
  • mice anti-Cherry (1 : 1000, ab 125096 ABCAM®
  • mouse anti-Flag (1 : 1000, F3165, SIGMA®
  • mouse FITC anti-Flag (1 : 1000, F4049, SIGMA®
  • mouse anti-GAPDH (1 :2000, AM4300, AMBION®)
  • mouse anti-a tubulin (1 :200, clone DM la, CALBIOCHEM®
  • rabbit anti-HA (1 :2000, H6908, SIGMA®
  • mouse anti-HA (1 : 1000, #26183, THERMOSCIENTIFIC®
  • rabbit anti- doublecortin Dbx (1 : 1000, abl8723, ABCAM®
  • mouse anti-MAP2 (1 : 1000, M4403, SIGMA®
  • rabbit anti-ATG16Ll (1 : 1000, PM040, MBL
  • mouse anti-ATG5 (1 : 100 immunostaining, 1 : 1000 immunoblotting, M153-3, MBL®
  • HRP-secondary antibodies were from the following sources: goat anti-rabbit (1 :5000, #31460, Thermofisher), goat anti-mouse (1 :5000, #31430, Thermofisher), rat anti-mouse IgG (1: 1000, abl31368, Abeam).
  • Fluorescent-labeled secondary antibodies used for immunoblotting were: donkey anti-mouse IRDye 800 CW (1 : 15000, #926-32212, Eurobio), donkey anti-rabbit IRDye 680 RD (1 : 15000, #926-68073, Eurobio).
  • Alexa 488, Alexa 594 and Alexa 647-conjugated secondary antibodies were from Jackson Labs.
  • Lysotracker dye (Molecular probes, L7528, THERMOFISHER®), used to stain lysosome, was applied for 30 minutes at 50 nM concentration. Fluorescence pictures were taken with confocal laser scanning microscope model LSM700 (CARL ZEISS®). 1.7- Co-immunoprecipitation
  • Protein extracts were prepared from transiently transfected COS cells as described in Cleveland et al. (Hum Mol Genet 18, 1384-1394 (2009)) and in the presence of the proteasome inhibitor MG132 (1748/5, TOCRIS BIOSCIENCE®). Epitope-tagged proteins were immunoprecipitated with anti-Flag and anti-Cherry antibodies as following. Negative controls correspond to Normal Mouse IgG (SC2025, Santa Cruz). Antibodies, linked with 50 m ⁇ Dynabeads Protein G (10004D, THERMOFISHER®) (3 hours, at room temperature in 500 m ⁇ PBS tween 0.02%), were incubated with 500 mg of proteins for 2 hours at room temperature in 500 m ⁇ PBS tween 0.02%.
  • Dynabeads Protein G 10004D, THERMOFISHER®
  • Proteins-Antibody-bead complexes were washed with PBS tween 0.02%, re-suspended in Laemmli IX solution and eluted at 70°C for 10 min. The totality of the sample was loaded on SDS Page gel, while inputs correspond to 50pg of initial protein lysates.
  • siRNAs siRNAs (siRNA) (SEQ ID NO. 13) or mismatch siRNAs (ms) (SEQ ID NO. 14) against the endogenous Gigaxonin were transfected the day before, using Dharmafect 2 reagent, and accordingly to the manufacturer’s instructions (DHARMACON®). Protein extraction and ubiquitin pull down were performed under denaturing conditions, to examine only the covalent binding of ubiquitin onto ATG16L1.
  • solution I supplemented with 10 mM B-mercaptoethanol and 0.1% Triton X-100
  • solution II 8 M urea, 0.1M Na 2 HP0 4 /NaH 2 P0 4 , 0.01 M Tris-HCl, pH 8.0
  • solution III 8 M urea, 0.1M Na 2 HP0 4 /NaH 2 P0 4 , 0.01 M Tris-HCl, pH 6.3
  • Elution was performed under agitation for 20 min within 80 m ⁇ of 200 mM imidazole, 0.15 M Tris-HCl pH 6.7, 30% glycerol, 0.72 M B- mercaptoethanol, 5% SDS. For each sample, equal quantity of the elution was analysed, accordingly to the determination of protein concentration in the respective input samples.
  • Proximity ligation assay was performed according to the manufacturer’s instructions (Duolink In Situ-Fluorescence, SIGMA®). COS cells were transiently transfected with different combinations of plasmids: Ha-ubiquitin with ⁇ Cherry -ATG16 and ⁇ Flag-Gigaxonin. Fixation, blocking and primary antibody incubation were performed as described in the immunofluorescence section.
  • the PLA probes a-mouse minus and a- rabbit plus were diluted and mixed in ratio 1 :5 (except for two negative controls where only one probe was added) in the Duolink in Situ Buffer for 20 minutes at room temperature, and subsequently applied to the cells for 1 hour at 37 °C.
  • Gigaxonin was previously proposed as a possible partner of ATG16L1, in a study reconstructing the autophagy interaction network. To determine whether this interaction occurs with biological significance, cellular assays were combined for constructs bearing the Cherry-ATG16L1 (Ch-ATG16L1) and Flag-tagged Gigaxonin (Flag-Gig). Strikingly, immunofluorescence of COS cells expressing both constructs revealed that ATG16L1 was degraded upon Gigaxonin expression. Restoring ATG16L1 content using the proteasome inhibitor MG132, or focusing on the residual ATG16L1, evidenced a co-localization between Gigaxonin and ATG16L1.
  • ATG16L1 is composed of three main structural domains: an N-terminal binding fragment followed by a central self-oligomerization coiled-coil domain (CCD) and a C- terminal WD40 domain ( Figure 2). To determine which domain of ATG16L1 interacts with Gigaxonin, co-immunoprecipitation with Flag-tagged ATG16L1 deletion constructs was performed ( Figure 3).
  • Ch-Gig was identified in immuno-complexes with the full- length ATG16L1 (F), the ATG16L1-C (C) and the ATG 16L 1 -DN (DN ) proteins, and not the other deletion products, demonstrating a specific interaction of Gigaxonin with the C- terminal WD40 repeats domain of ATG16L1.
  • ch-ATG16Ll was pulled down with truncated portions of Gigaxonin to reveal that both BTB and Kelch repeats domains, or alternatively a common domain lying in the linker domain can interact with ATG16L1 ( Figures 4 and 5). 2 2- Gigaxonin mediates ubiquitin dependent degradation of ATG16L1
  • Gigaxonin greatly increases the proximity between ubiquitin/poly-ubiquitin chains and the endogenous ATG16L1, hence supporting an activity of Gigaxonin in the poly- ubiquitination of endogenous ATG16L1.
  • Gigaxonin In light of the important role of Gigaxonin in controlling ATG16L1 degradation, the effect and the significance of this regulation in a physiological context was investigated. For that purpose, a cellular model deficient for Gigaxonin was developed, from the knock-out mouse depleted in the GAN gene, which causes a fatal and recessive neurodegenerative disorder called Giant Axonal Neuropathy in human. In agreement with the above results, ATG16L1 is controlled by Gigaxonin in primary neuronal cells. Examination of MAP2 positive control cells revealed a spatial distribution/biogenesis of the endogenous ATG16L1 over time, which was mostly located in the soma at 4div (days in vitro ) and within neurites at 15div.
  • GAN / neurons exhibited large perinuclear aggregates of ATG16L1 within the soma at 4div, with a pronounced increase in abundance.
  • the ATG16L1 aggregates persisted but were not distributed in neurites, as in wild type cells.
  • the ATG16L1 distribution and abundance were compared in the presence of MG132.
  • ATG16L1 was mostly found in neurites at 4div, similarly to older neurons, which suggests a constitutive and active degradation of ATG16L1 locally within neurites in early developmental stages.
  • ATG16L1 is essential in the elongation phase of autophagy. Indeed, it forms a ternary complex with the preformed ATG12-ATG5 conjugate, which constitutes the E3 ligase promoting the LC3 conjugation to the nascent autophagic membrane.
  • ATG16L1 determines the site of LC3 lipidation and therefore primes the elongation of the autophagosomes.
  • p62 is the main selective autophagy receptor and is conventionally used as a marker of effective autophagic degradation.
  • p62 accumulated only in Bafilomycin Al treatment, as a result of the impairment of the degradation of autophagosome content by lysosomal enzymes.
  • p62 was shown to accumulate in all conditions in GAN / neurons, hence demonstrating a defect in basal autophagy when Gigaxonin is absent.
  • p62 most probably decorates phagophores, which failed to elongate and therefore provoked p62 accumulation.
  • Gig morphants exhibited an aberrant growth of pMN axons reminiscent of Shh mutants, including an absence or significant shortening of the caudal (CaP) primary axons and misguided axons with ectopic ventral projections.
  • CaP caudal
  • 48hpf morphants evidenced by LightSheet microscopy revealed additional phenotypes.
  • morphants exhibited abnormal protrusions of axons from the spinal cord, and apparent absence of the MiP and RoP motor axons, leading overall to a profound alteration of the structure of the spinal tracts and an increased spacing between the two motor columns.
  • Gig morphants exhibit U-shaped somites with an absence of horizontal myoseptum, and less dense and wavy myofibers very similarly to Shh inactivation. This effect, further reproduced using Mo exl-2 for Gigaxonin was detected as early as 28hpf, concomitantly to the abnormalities of pMN axonal pathfmding, which suggests a dual effect of Gigaxonin in neuronal and muscle development.
  • Shh signalling is crucial to specify neuronal identity and for somitogenesis. Genetic or pharmacological ablation of the Shh pathways in zebrafish has been shown to abolish motor neuron specification in the spinal cord and to generate U-shaped somites. The striking similarities with our morphants indicate a potential role of Gigaxonin in regulating Shh signalling in zebrafish.
  • a first validation towards a downregulation of Shh activity in GAN was provided by the pronounced reduction in the expression of the Shh responsive target Nkx6.1 gene in the spinal cord of Gig morphants.
  • Treated wild type embryos exhibited both an aberrant somitogenesis with U- shaped structures and a total loss of secondary Motor Neurons (sMN), and also resulted in a pinwheel swimming behavior in 80% of the embryos upon mechanical stimulation at 72hpf.
  • sMN secondary Motor Neurons
  • the elevation of Shh signalling in Gig morphants suppressed the muscle deficits, as shown by the restoration of V-shape somites and denser myofibers.
  • the restoration of MN differentiation was not so robust when the drug was administered during sMN specification, as revealed by the partial rescue of zn8 staining in Gig morphants.
  • the restoration of sMN differentiation in morphants was more efficient when purmorphamine was applied earlier, during the wave of pMN birth, indicating a possible control of the Shh cascade by Gigaxonin from 8hpf.
  • Shh pathway relies on the presence of cilium for signal transduction
  • downstream targets of Shh have also been shown to increase ciliary length by the regulation of the actin cytoskeleton (Bershteyn et ah, 2010) possibly in a negative feedback loop to attenuate the signalling (Fleet et ah, 2016).
  • the control human fibroblasts exposed to Shh increase the cilia length by 29%, the ciliary length of the independent GAN fibroblasts was not altered upon Shh addition, hence showing their inability to respond to the Shh activation.
  • the ATG16L1 protein orchestrates the two UBL systems required for membrane elongation during autophagosome formation, but its control and dynamics are unknown.
  • the Gigaxonin-E3 ligase as the first regulator of ATG16L1 turn-over, essential in ensuring ATG16L1 functions in phagophore expansion (Figure 15).
  • Gigaxonin interacts with the C-terminal WD40 repeats domain of ATG16L1 to promote its ubiquitination and degradation by the proteasome and the autophagy pathways.
  • Gigaxonin depletion induces a massive aggregation of ATG16L1 in primary neurons and inhibits autophagy flux by impairing phagophore elongation.
  • ATG16L1 bundles within GAN / neurons were mostly present in a perinuclear position in the soma, and while not totally absent, rarely seen within neurites.
  • Autophagosome biogenesis has been shown to be spatially regulated along the axons; it occurs distally in the neurite tip and the autophagosome matures and fuse to the lysosome while undergoing a retrograde transport towards the soma. Still, some autophagosome formation was observed in the soma, showing specificity in their maturation, localisation and dynamics (Figure 15).
  • Gigaxonin While several functions of axonal autophagy have been identified, including axonal homeostasis and presynaptic functions, nothing is known about its role in the soma. Thus, Gigaxonin will contribute to deciphering the meaning of the high degree of associationalisation of autophagy within the neuron, and its function in cell survival. Indeed, Gigaxonin loss-of-function causes widespread neuronal death throughout the nervous system in Giant Axonal Neuropathy patients, and a progressive degeneration of GAN cortical neurons.
  • GAN represents one of the few disease-associated genes, which encode for proteins that are either autophagic core components (WIPI4, ATG16L1) or their regulator (Gigaxonin), causing respectively encephalopathy, Crohn’s disease and Giant Axonal Neuropathy.
  • GAN is an infantile neurodegenerative disease characterised by a loss of neurons both in the spinal cord and the brain, however, its mechanisms are not understood.
  • Our study provides the first evidences of a link between GAN and Shh signaling, and identifies in the zebrafish, neurodevelopmental defects as a cause of the cellular and physiological markers of the pathology.
  • GAN adds up to the emerging concept whereby neurodevelopmental deficits disrupt homestasis and generate vulnerability that further evolve in adult stages towards clinical manifestations, as exemplified in Huntington’s disease. While this suggests that the GAN pathology has a neurodevelopmental component in human, the roles played by the Shh pathway in the adult nervous system could also be of relevance in the disease progression.
  • Atgl6L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy. Mol Biol Cell. 2008 May; 19(5):2092-100. doi: 10.1091/mbc.E07-12-1257.
  • ATG16L1 is associated with decreased risk of brain metastasis in patients with non-small cell lung cancer. Autophagy, 2017;13(6): 1053-1063. doi: 10.1080/15548627.2017.1308997.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Neurosurgery (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Neurology (AREA)
  • General Chemical & Material Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Food Science & Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biotechnology (AREA)
  • Hospice & Palliative Care (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Psychiatry (AREA)
  • Toxicology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Marine Sciences & Fisheries (AREA)

Abstract

The present invention relates to the prevention and/or the treatment of a disease associated with altered autophagy and/or Shh transduction, such as a cancer, an immune disease, an infectious disease, a metabolic disease, a cardiovascular disease, a (cardio)myopathy, heart failure, a lysosomal disease, spinal cord injury and trauma, a neurodegenerative disease and a pulmonary disease. The inventors showed that Gigaxonin promotes the fine-tuning of autophagy, by the means of its interaction with ATG16L1. In particular, cells depleted of Gigaxonin (GAN -/- neurons) exhibit ATG16L1 aggregation and a reduced capacity to produce autophagosomes, whereas overexpression of Gigaxonin rescues ATG16L1 aggregation and restores autophagy flux in GAN -/- neurons. The inventors also demonstrate that Gigaxonin is a key positive regulator of the Shh pathway, enabling receiving cells to convey signaling into motility. In particular, Gigaxonin depletion inhibits Shh transduction in the gan zebrafish model and patient cells, through a mechanism involving the interaction and degradation of the PTCH receptor. This pinpoints to an evolutionary-conserved and reversible mechanism that controls the entry point of Shh signalling and to the developmental origin of GAN. Thus, the present invention relates to a modulator of the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ATG16L1 or PTCH interaction and/or of the functionality of the Gigaxonin/ATG16L1 or PTCH interaction, for the prevention and/or the treatment of a disease associated with altered autophagy, Shh signaling and/or neurodegenerative disease.

Description

COMPOUNDS FOR USE IN THE TREATMENT OF DISEASES
ASSOCIATED WITH ALTERED AUTOPHAGY ALTERED SHH TRANSDUCTION AND/OR NEURODEGENERATIVE DISEASES
FTFUD OF nil INVENTION
The present invention relates to the prevention and/or the treatment of neurodegenerative diseases, and/or diseases associated with altered autophagy.
The present invention also relates to the prevention and/or the treatment of diseases associated with altered Sonic Hedgehog (Shh) transduction.
More particularly, the invention relies upon the discovery that modulation of the expression of Gigaxonin regulates the fine-tuning of the production of autophagosomes, notably through the modulation of the Gigaxonin/ATG16Ll interaction, and the control of the steady-level of ATG16L1. The invention also relies upon the identification of Gigaxonin as the first E3 ligase that positively controls the initiation of Sonic Hedgehog (Shh) transduction, ensuring that Shh responses in spinal cord and muscles are conveyed into efficient motor activity. An interaction between Gigaxonin and the Patched (PTCH) receptor is also reported.
BACKGROUND OF THF INVENTION
Autophagy is an essential degradative pathway that delivers cytoplasmic components to lysosomes for degradation. Evolutionarily conserved, this complex machinery is activated to recycle a wide range of substrates in normal conditions and to promote the degradation of damaged components (dysfunctional organelles, protein aggregates) in diseases. Therefore, alteration of autophagy perturbs cellular homeostasis and important physiological processes, and is associated with various pathological conditions, including cancer and neurodegenerative diseases (Mizushima and Komatsu (Cell. 2011 Nov 11;147(4):728-41); Schneider and Cuervo (Curr Opin Genet Dev. 2014 Jun;26: 16-23); Menzies et al. (Nat Rev Neurosci. 2015 Jun;16(6):345-57)).
Macroautophagy (hereafter referred to as autophagy) is characterised by the nucleation of a double-membrane fragment (phagophore) around the material to be degraded, which elongates to form a complete autophagosome and subsequently fuses to a lysosome.
The mechanisms driving membrane expansion are key elements in autophagy. The molecular determinants of membrane elongation are complex and involve two highly conserved ubiquitin-like (UBL) conjugation systems, ATG12 and LC3 (the mammalian homolog of the yeast ATG8). Structurally related to ubiquitin, ATG12 and LC3 are transferred by El- and E2-like enzymes to their final substrates. The covalent conjugation of ATG12 to ATG5 generates the E3 ligase activity necessary for the last step of ATG8/LC3 conjugation to phosphatidylethanolamine (PtdEth) on the nascent membranes.
Orchestrating this cascade at the site of the nascent phagophore, ATG16L1 is a key determinant of autophagy elongation. Indeed, ATG16L1 interacts with the conjugate ATG12-ATG5 to form a multimeric structure and triggers the binding of the complex to the membrane. Through the subsequent interaction of ATG12 with LC3-conjugated- ATG3, ATG16L1 specifies the site of LC3 lipidation onto nascent membranes. Several studies in yeast and mammalian cells have shown that alterations in ATG16L1, either using genetic mutants or the overexpressed protein, all result in impaired localization of ATG12-ATG5 to the phagophore and failure in ATG8/LC3 lipidation onto the membranes, leading to inhibition of autophagosome formation. Likewise, forced localization of ATG16L1 to the plasma membrane has been shown to be sufficient to promote ectopic LC3 lipidation at the cell surface. The biological importance of ATG16L1 was further evidenced in vivo , where A I’G I6 I~ mice, defective in autophagosome formation, did not survive neonatal starvation and died within 1 day of delivery. Thus, regulation of the scaffold ATG16L1 protein constitutes not only a fundamental question to apprehend the complex dynamics of autophagic activity but also represents a substantial target for therapy to activate autophagy in disease. Post-translational modifications (PTMs) of ATG proteins are essential in modulating their activity. While more than 300 PTMs of autophagic proteins have been characterized, very little is known about ATG16L1, and only Ser278 phosphorylation has been evidenced in acute intestinal inflammation.
An increasing body of evidence accumulated over the years is pointing to the alterations of autophagy in a growing number of various diseases, such as neurodegenerative diseases, cancer, metabolic diseases, infectious diseases, autoimmune diseases, to name a few. For an illustration of these evidences, one can refer to, e.g., Mizushima and Komatsu (Cell. 2011 Nov 11;147(4):728-41), Schneider and Cuervo (Curr Opin Genet Dev. 2014 Jun;26: 16-23), Menzies et al. (Nat Rev Neurosci. 2015 Jun;16(6):345-57), Lippai and Szatmari (Cell Biol Toxicol. 2017 Apr;33(2): 145-168), Vomero et al. (Front Immunol. 2018 Jul 18;9: 1577), Scrivo et al. (Lancet Neurol. 2018 Sep;17(9):802-815).
It has recently emerged from these discoveries that targeting key components involved in the autophagy process may influence the level/activity of autophagy in diseased cells and tissues and may provide suitable therapeutic approaches.
Illustratively, targeting ATG7 and ATG16L1 with microRNAS (miRNAs), has been proved to attenuate status epilepticus-induced brain injuries (Gan et al.; Sci Rep. 2017 Aug 31;7(1): 10270). Similarly, miR-410 has been evidenced to target ATG16L1 and subsequently to markedly inhibit autophagy in osteosarcoma cells, leading to, upon exposure to anticancer drugs, a protective increased autophagy and a better chemosensitivity in cancer cells. (Chen et al.; Mol Med Rep. 2017 Mar;15(3): 1326-1334). Very interestingly, a genetic variant of the ATG16L1 (Thr300Ala, which enhances its degradation) has been linked, in patients suffering from Non-small cell lung cancer to a decreased risk of brain metastasis (Li et al., Autophagy, 2017).
Due to the prominent role of alterations of the autophagy pathway in numerous diseases, there is a need to identify compounds that suitably modulate autophagy in disease cells/tissues. Notably, a disease (or a stage in the disease progression) wherein autophagy is enhanced may be cured/ameliorated by a modulator that inhibits autophagy, whereas a disease wherein autophagy is inhibited may benefit from a modulator that restores autophagy to a physiological level.
Besides, in vertebrates, Sonic Hedgehog (Shh) assigns neuronal and muscle fate, acting in a graded manner to pattern the dorso-ventral axis of the neural tube and the muscles. Dysregulation of Shh signalling causes a wide range of human diseases, including congenital malformations of the central nervous system, of the axial skeleton and limbs, cancers and malignancies in children and adults. Therefore, understanding the Shh pathway is essential for both basic developmental biology and to explore therapeutic intervention for human diseases. Thus, there is also a need to identify compounds that suitably modulate Shh signalling in disease cells/tissues; especially neurodegenerative diseases, such as those associated with altered autophagy and Shh signaling. SUMMARY OF THE INVENTION
In a first aspect, the invention relates to a modulator of the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ATG16Ll interaction and/or of the functionality of the Gigaxonin/ ATG16L1 interaction for use as a medicament.
In particular, the invention relates to a polypeptide expressed by a nucleic acid comprising a nucleic acid sequence having at least 75% identity with SEQ ID NO. 1; or
- a polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2; for use as a medicament.
Most notably, the diseases which are particularly considered by the invention include those associated with altered autophagy and/or altered Shh transduction and/or neurodegenerative diseases.
More particularly, the invention relates to a polypeptide expressed by a nucleic acid comprising a nucleic acid sequence having at least 75% identity with SEQ ID NO. 1; or
- a polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy.
Alternatively, the invention relates to a nucleic acid coding for such a polypeptide; for use as a medicament.
According to said alternative embodiment, the invention relates to a nucleic acid coding for such a polypeptide; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
Another aspect of the invention relates to a pharmaceutical composition comprising:
- (i) a modulator of the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ ATG16L1 interaction and/or of the functionality of the Gigaxonin/ ATG16L1 interaction, and
- (ii) a pharmaceutically acceptable vehicle. Such a pharmaceutical composition may thus comprise a nucleic acid having at least 75% identity with SEQ ID NO. 1; or a polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2.
In a further aspect, the invention relates to a pharmaceutical composition according to the present disclosure, for use in the prevention and/or the treatment of a disease associated with altered autophagy.
In a further aspect, the invention relates to a pharmaceutical composition comprising a nucleic acid having at least 75% identity with SEQ ID NO. 1; or
- a polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2; for use in the prevention and/or the treatment of a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity. Hence, the invention also relates to a pharmaceutical composition according to the present disclosure, for use in the prevention and/or the treatment of a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
A further aspect of the invention relates to a method for screening a modulator of the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ATG16Ll interaction and/or of the functionality of the Gigaxonin/ ATG16L1 interaction, comprising the steps of:
- (a) providing a system comprising Gigaxonin and ATG16L1 in suitable conditions for the production of the corresponding proteins and for the interaction between Gigaxonin and ATG16L1 to occur;
- (b) providing to the said system a candidate modulator of the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ ATG16L1 interaction and/or of the functionality of the Gigaxonin/ATG16Ll interaction;
- (c) measuring the level of expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ ATG16L1 interaction and/or of the functionality of the Gigaxonin/ ATG16L1 interaction in the system at step (b);
- (d) determining a difference between the level measured in step (c) and a corresponding reference level obtained in the absence of the said candidate modulator. Another aspect of the invention also relates to a method for assessing the efficiency of a candidate treatment of a disease associated with altered autophagy in an individual in need thereof, comprising the steps of:
- (a) measuring the level of Gigaxonin, ATG16L1, the Gigaxonin/ATG16Ll interaction and/or the functionality of the Gigaxonin/ ATG16L1 interaction in a sample from said individual in need thereof, in suitable conditions for the interaction to occur;
- (b) providing the said individual with a candidate treatment;
- (c) measuring the level of Gigaxonin, ATG16L1, the Gigaxonin/ ATG16L1 interaction and/or the functionality of the Gigaxonin/ ATG16L1 interaction in a sample obtained at step (b);
- (d) determining a difference between the level measured in step (a) and the level measured in step (c).
A still further aspect of the invention concerns a method to activate autophagy in a target cell, comprising a step of increasing the level of expression, stability of Gigaxonin, decreasing its degradation and/or increasing activity of Gigaxonin and/or Gigaxonin/ ATG16L1 interaction to reach physiological functions of ATG16L1.
A still further aspect of the invention concerns a method to inhibit autophagy in a target cell, comprising a step of decreasing the level of expression, stability of Gigaxonin, increasing its degradation and/or decreasing the activity of Gigaxonin and/or Gigaxonin/ ATG16L1 interaction to decrease physiological functions of ATG16L1.
In another aspect, the invention relates to a method for the fine-tuned activation or inhibition of autophagy in a target cell comprising the step of modulating the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ATG16Ll interaction and/or of the functionality of the Gigaxonin/ATG16Ll interaction.
In another aspect, the invention relates to the use of gigaxonin and/or any derivatives (cells/constructs/animal model/methods...) to serve as a template to screen existent and/or novel drugs acting on the autophagy pathway.
In another aspect, the invention relates to a method for the fine-tuned activation or inhibition of Shh transduction in a target cell comprising the step of modulating the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/PTCH interaction and/or of the functionality of the Gigaxonin/PTCH interaction. In another aspect, the invention relates to the use of gigaxonin and/or derivatives (cells/constructs/animal model/methods...) to serve as a template to screen existent and/or drugs acting on the Shh pathway.
LEGENDS OF THE FTGTTRES
Figure 1. Immunoblotting evidencing the interaction of Gigaxonin with ATG16L1. Reverse Immunoprecipitation experiment was performed in COS-7 cells transfected with Ch-ATG16 and Flag-Gig and treated with MG132. Gigaxonin and ATG16L1 complexes were recovered using anti-flag (IP Flag) and anti-cherry (IP Cherry) antibodies and revealed by immunoblotting with cherry/Flag antibodies. Immunoprecipitation with anti-IgG antibodies (IP IgG) served as negative control.
Figure 2. Schematic representation of the mouse ATG16L1 (ATG16L1-F) and its deletion constructs. N-terminal domain (ATG5 binding domain), central coiled-coil domain (CCD domain) and C-terminal WD40 repeats domain are indicated in white, gray stripes and black, respectively.
Figure 3. Immunoblotting demonstrating the interaction of Gigaxonin with the WD40 repeats domain of ATG16L1. The domain of interaction of ATG16L1 with Gigaxonin was evidenced by immunoprecipitation. Full length and deletion constructs of Flag-ATG16 were transfected in COS-7 cells with Ch-Gig in presence of MG132. Please note that the Full-length ATG16L1 band (black star in the input panel) is enriched in pull down condition (in the IP Flag panel). Immunoprecipitation using anti-Flag antibodies evidenced Gigaxonin in the immunocomplexes formed by the Full length-, C-, and DN- ATG16L1 proteins. Negative controls include immunoprecipitation of the Ch-Gig + Flag- ATG16L1-F cell lysate with anti-IgG antibodies (IP IgG).
Figure 4. Schematic representation of the human Gigaxonin (Gig-F) with its N-terminal BTB domain (stripes) and C-terminal Kelch repeats domain (black) and the BTB (Gig-AN) and Kelch (Gig-AC) deletion constructs.
Figure 5. Immunoblotting of the immunoprecipitation of Flag-Gig complexes identified Ch-ATG16 with both deletion constructs of Gigaxonin,
Figure 6. Immunoblotting showing that Gigaxonin induces the degradation of ectopic ATG16L1, through the proteasome and the lysosomal pathways COS-7 cells co- were transfected with Flag-Gig and Cherry-ATG16L1 (double transfection), in basal condition (-), or treated with Mgl32 (+Mgl32) or Bafilomycin A1 (+Baf). Figure 7. Immunoblotting demonstrating the Gigaxonin-induced degradation of endogenous ATG16L1, through the proteasome and lysosomal pathways. Endogenous ATG16L1 protein is greatly diminished in cells transfected with Flag-Gig, and partially restored under MG132 (+Mgl32) or Bafilomycin A1 (+Baf) treatment.
Figure 8. Immunoblotting demonstrating the in vivo ubiquitination of ATG16L1 by Gigaxonin. COS-7 cells were transfected with Ch-ATG16 alone, or in combination with His-Ubiquitin construct (His-Ub) in the presence or absence of siRNA against endogenous Gigaxonin (siRNA) or the mismatch counterpart (ms), and treated with MG132. The pull down of ubiquitinated ATG16L1 is performed in denaturing conditions using nickel agarose beads.
Figure 9. Immunoblotting showing the defect in autophagosome production caused by Gigaxonin depletion in neurons. Wild type and GANG cortical neurons were cultured for 4 div under basal condition, or with different treatments to evaluate autophagy activity. Starvation was induced with EBSS, autophagosome-lysosome fusion was blocked with Bafilomycin A1 (Baf). Immunoblotting of cortical neurons shows the lipidation of LC3 under different conditions, in control (WT) and mutant (GANG) neurons.
Figure 10. Quantification of the lipidation of LC3 showing the defect in autophagosome production caused by Gigaxonin depletion. Wild type (WT) and GANG cortical neurons are treated as in Figure 9. Quantification of the lipidation of LC3 was measured with the LC3-II to tubulin ratio, and expressed as a fold increase over the control level in the basal condition. The defect in autophagy in GAN~/~ neurons, evidenced by the decreased LC3 lipidation in EBBS+Baf 6h condition was further defined as a decreased production of autophagosomes, by comparing the EBBS+Baf ratio at two different times (2h and 6h of treatment). WT and mutant values are represented in black and grey circles, respectively. n= 3 independent experiments and values are means ± SD. Differences between WT and mutant values are only significant for the (EBSS+Baf6h) condition **** EO.OOOl using 2Way ANOVA test (Bonferroni post-hoc test).
Figure 11. Quantification of LC3 dot fluorescence showing the defect in autophagosome production caused by Gigaxonin depletion. Wild type (WT) and GANG cortical neurons are treated as in Figure 9. The quantification of LC3 dots was obtained by measuring the fluorescence intensity of LC3 staining in EBSS+Baf 6h condition (n=37 for WT cells represented in black circles, n= 43 cells for mutant cells represented in grey circles, from three independent experiments). Individual measures and medians with interquartile range are represented; statistical significance was obtained using the two- tailed Mann Whitney test with a ** <0.01 value.
Figure 12. Quantification showing the defectiveness of GAN~/~ neurons in autophagosome-lysosome fusion. Co-staining of LC3 with lysotracker evidenced decreased fusion between autophagic structures and lysosomes in GAN~/~ neurons. The decrease in the colocalisation of LC3 with the lysotracker was determined by the Pearson Coefficient. N=27 for wild type cells represented in black circles, and n=22 for mutant cells represented in grey circles, from three independent experiments; individual measures and medians with interquartile range are represented; statistical significance was obtained with a ****/><o 0001 value with the two-tailed Mann Whitney test.
Figure 13. Immunoblotting showing the rescue of autophagy by Gigaxonin in GAN / neurons. Immunoblots for ATG16L1 were performed on 4div control and mutant neurons, after transduction with GFP-mock or Flag-Gigaxonin lentivirus. Both normal and aggregated ATG16L1 were cleared upon expression of Gigaxonin in control and GANG neurons, respectively.
Figure 14. Quantification of neurons demonstrating the progressive degeneration of GANG neurons. Doublecortine (Dbx) and MAP2 neurons were identified in E15.5 cortices prepared from wild type (WT) and GANG mice after 3 hours, 5, 15 and 20 div (days in vitro). The neurodegeneration of GANG neurons was evidenced from 15 div onwards, as measured by the decreased proportion of neurons (as expressed by a MAP2 to DAPI ratio), relatively to the 3h time points (Dbx to Dapi ratio). WT and mutant cells are represented in black and grey circles, respectively. n=3 independent experiments with triplicate measures (161-821 DAPI positive cells counted per measure). Individual measures and means ± SEM are represented; **E<0.01 and *P< 0.05 values at 15 and 20 div, respectively using the 2way ANOVA test (Bonferroni post-hoc test).
Figure 15. Schematic model of Gigaxonin control of autophagosome synthesis. Figure 15 A: Gigaxonin controls the steady-state level of ATG16L1, by interacting with its C-terminal WD40 domain and promoting its K48 poly-ubiquitination and clearance by the proteasome and the autophagy pathways. ATG16L1 binds to the ATG12-ATG5 elongation conjugate and targets it to the nascent autophagic membrane, through its interaction with WIPI2. Thus, the E3 ligase activity of the ATG12-ATG5- ATG16 complex lipidates LC3 onto the membranes, allowing the elongation of the phagophore. The mature autophagosome sequesters cytosolic material, including p62 bound cargo, which are degraded upon fusion to the lysosome. Thus, by controlling ATG16L1 steady state, the Gigaxonin-E3 ligase promotes autophagosome production and ensures a normal autophagic flux within cells. In a physiological context, autophagosome biogenesis occurs at the neurite tip but also in the soma of primary neurons. Gigaxonin depletion (Figure 15B) in primary neurons induces aggregation of ATG16L1, without impairing the formation of the ATG12-ATG5 conjugate. The inventors propose that the impairment in LC3 lipidation observed in GAN7 neurons is due to a defect in anchoring ATG12-ATG5 to the autophagic membranes. Overall, Gigaxonin depletion alters autophagosome synthesis and causes an abnormal accumulation of the main autophagy receptor p62, hence impairing the autophagic flux. ATG16L1 and P62 accumulation are localized within the soma, opening interesting perspectives on the study of autophagy compartimentalisation within neurons, and on its role in human neuropathy, as exemplified in GAN.
Figure 16. Model of action of the Gigaxonin-E3 ligase in the initiation of Shh signalling. (A) In an OFF state, prior to Shh activation, receiving cells silence the signalisation cascade through the inhibitory effect of the Ptch receptor on the effector Smo. Upon Shh production, the cleaved active ShhN form is released and addressed to progenitor cells. Gigaxonin acts as an initiator of Shh signalling by degrading Shh-bound Ptch receptor, hence allowing the derepression of the signal transducer Smo. In absence of Gigaxonin, receiving tissues are unable to interpret Shh signalling, due to the constitutive repression of Smo induced by the accumulation of Shh-bound Ptch receptor. (B) In zebrafish, Gigaxonin depletion induces massive neuronal defects, which encompass aberrant axonal outgrowth of CaP pMN (blue), absence of MiP (green) and RoP (red) axons, and defective specification of sMN. Concomitantly, somitogenesis is altered, and generate U-shape somites and alteration of myofiber architecture. Collectively, the Gigaxonin-mediated inhibition of Shh activity impairs MN specification and somitogenesis during development, hampering neuromuscular junction formation and leading to an impairment of locomotion PET ATT, ED DESCRIPTION OF TU I INVENTION
The inventors have identified Gigaxonin, an E3 ligase adaptor which is mutated in a fatale neurodegenerative disease called Giant Axonal Neuropathy (GAN) (Kulenbaumer, Timmerman & Bomont, Genereview (2018)), as being the first regulator of ATG16L1. Gigaxonin interacts with the WD40 domain of ATG16L1 to drive its ubiquitination and subsequent degradation. Accumulation of ATG16L1, as a result of Gigaxonin depletion, alters early events of LC3 lipidation onto nascent phagophore and diminishes fusion to the lysosome and degradation of the autophagy receptor p62. The inventors demonstrated that Gigaxonin depletion inhibits autophagosome synthesis, which is rescued upon reintroduction of the E3-ligase. Altogether, these data unveil the regulatory mechanism that drives the dynamics of autophagosome formation by ATG16L1, and position Gigaxonin as a significant therapeutic target to modulate autophagy activity in disease.
Also, the inventors have demonstrated that, as in human, repression of Gigaxonin in zebrafish leads to a loss of motor neurons in the spinal cord, severe axonal defects and the abolishment of locomotion, phenotypes all reversed upon co-injection of the human Gig transcripts.
Surprisingly, it has been found that gigaxonin induces the production of motoneurons in the spinal chord, in a manner which is compatible with restoration of normal motility. In particular, it has been found that injection of a nucleic acid coding for gigaxonin (i.e. a human messenger RNA coding for gigaxonin) restores morphological defects and motility (as evidence in zebrafish larvae), and restores axonal growth of primary motor neurons, and induces a production of secondary motor neurons, as exemplified herein.
Without wishing to be bound by the theory, they also suggest that the Gigaxonin-E3 ligase is the 1st regulator of the Shh pathway, which acts in the receiving tissues to initiate Shh activity in a spatial and temporally regulated manner. The Gigaxonin regulatory mechanism which was identified is evolutionary conserved, is required for both neuron and muscle patterning in vivo, and is necessary for movement.
Indeed, it is shown herein that Gigaxonin controls Shh signaling. In particular, Shh regulates the initial steps of Shh induction, which is sufficient to specify neuronal and muscle fate in vertebrates. Using the zebrafish as a model system, physiological evidence is provided, showing that the Gigaxonin-E3 ligase is a key regulator of Shh activation, by controlling the degradation of the Ptch receptor in a Shh-dependent manner.
The deficits of the Gigaxonin-null embryos reproduce numerous animal models of Shh inhibition, and are restored upon Shh activation. The positive control of Gigaxonin on the pathway is Shh-dependent, as revealed independently in the Gig-depleted zebrafish, in a cellular system using a Shh activity reporter assay, and in human patient cells. Furthermore, Gigaxonin interacts with Ptch and mediates its degradation in a Shh- dependent manner, hence identifying Gigaxonin as the 1 st regulator of the initiation of Shh signaling. Notably, the findings obtained in the gan zebrafish model mimic the motor dysfunctions found in patients, hence providing the first hints into the pathophysiological mechanisms in GAN, and supporting a developmental origin in the pathogenesis of GAN. This notion is further endorsed by the functional rescue of the developmental deficits in the gan zebrafish by the human Gigaxonin, and the evidence of an impairment of Shh signaling in patient cells.
In a first aspect, the invention relates to a modulator of the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ ATG16L1 interaction and/or of the functionality of the Gigaxonin/ ATG16L1 interaction for use as a medicament.
In another complementary aspect, the invention relates to a modulator of the expression, stability, degradation and/or activity of the Gigaxonin/PTCH interaction and/or of the functionality of the Gigaxonin/PTCH interaction, for use as a medicament.
In particular, such a modulator may be in the form of a polypeptide having at least 75% identity with gigaxonin, or a nucleic acid (such as a messenger RNA) coding for such a polypeptide.
In particular, the invention relates to a polypeptide expressed by a nucleic acid comprising a nucleic acid sequence having at least 75% identity with SEQ ID NO. 1; or
- a polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2; for use as a medicament.
More particularly, the invention relates to a polypeptide expressed by a nucleic acid comprising a nucleic acid sequence having at least 80% identity with SEQ ID NO. 1; or - a polypeptide comprising a polypeptide sequence having at least 80% identity with SEQ ID NO. 2; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
More particularly, the invention relates to a polypeptide expressed by a nucleic acid comprising a nucleic acid sequence having at least 85% identity with SEQ ID NO. 1; or
- a polypeptide comprising a polypeptide sequence having at least 85% identity with SEQ ID NO. 2; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
More particularly, the invention relates to a polypeptide expressed by a nucleic acid comprising a nucleic acid sequence having at least 90% identity with SEQ ID NO. 1; or
- a polypeptide comprising a polypeptide sequence having at least 90% identity with SEQ ID NO. 2; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
More particularly, the invention relates to a polypeptide expressed by a nucleic acid comprising a nucleic acid sequence having at least 95% identity with SEQ ID NO. 1; or
- a polypeptide comprising a polypeptide sequence having at least 95% identity with SEQ ID NO. 2; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
Alternatively, the invention relates to a nucleic acid coding for such a polypeptide; for use as a medicament.
In particular, the invention relates to a nucleic acid comprising a nucleic acid sequence having at least 75% identity with SEQ ID NO. 1; or a nucleic acid coding for a polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2; for use as a medicament. In particular, the invention relates to a nucleic acid comprising a nucleic acid sequence having at least 80% identity with SEQ ID NO. 1; or a nucleic acid coding for a polypeptide comprising a polypeptide sequence having at least 80% identity with SEQ ID NO. 2; for use as a medicament.
In particular, the invention relates to a nucleic acid comprising a nucleic acid sequence having at least 85% identity with SEQ ID NO. 1; or a nucleic acid coding for a polypeptide comprising a polypeptide sequence having at least 85% identity with SEQ ID NO. 2; for use as a medicament.
In particular, the invention relates to a nucleic acid comprising a nucleic acid sequence having at least 90% identity with SEQ ID NO. 1; or a nucleic acid coding for a polypeptide comprising a polypeptide sequence having at least 90% identity with SEQ ID NO. 2; for use as a medicament.
In particular, the invention relates to a nucleic acid comprising a nucleic acid sequence having at least 95% identity with SEQ ID NO. 1; or a nucleic acid coding for a polypeptide comprising a polypeptide sequence having at least 95% identity with SEQ ID NO. 2; for use as a medicament.
According to said alternative embodiment, the invention relates to a nucleic acid coding for such a polypeptide; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
In particular, the invention relates to a nucleic acid comprising a nucleic acid sequence having at least 75% identity with SEQ ID NO. 1; or a nucleic acid coding for a polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
In particular, the invention relates to a nucleic acid comprising a nucleic acid sequence having at least 80% identity with SEQ ID NO. 1; or a nucleic acid coding for a polypeptide comprising a polypeptide sequence having at least 80% identity with SEQ ID NO. 2; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
In particular, the invention relates to a nucleic acid comprising a nucleic acid sequence having at least 85% identity with SEQ ID NO. 1; or a nucleic acid coding for a polypeptide comprising a polypeptide sequence having at least 85% identity with SEQ ID NO. 2; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
In particular, the invention relates to a nucleic acid comprising a nucleic acid sequence having at least 90% identity with SEQ ID NO. 1; or a nucleic acid coding for a polypeptide comprising a polypeptide sequence having at least 90% identity with SEQ ID NO. 2; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
In particular, the invention relates to a nucleic acid comprising a nucleic acid sequence having at least 95% identity with SEQ ID NO. 1; or a nucleic acid coding for a polypeptide comprising a polypeptide seqeuence having at least 95% identity with SEQ ID NO. 2; for use in a method for treating or preventing a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
In some embodiments, the modulator of the expression and/or the activity of Gigaxonin, of the Gigaxonin/ATG16Ll interaction and/or of the Gigaxonin/ATG16Ll functionality, as disclosed herein, may be for use for the preparation of a medicament, in particular a medicament intended to treat a disease associated with altered autophagy.
In some embodiments, the modulator of the expression and/or the activity of Gigaxonin, of the Gigaxonin/PTCH interaction and/or of the Gigaxonin/PTCH functionality, as disclosed herein, may be for use for the preparation of a medicament, in particular a medicament intended to treat a disease associated with altered Shh transduction.
Within the scope of the present invention, the human Gigaxonin polypeptide refers to a 597 amino acid residues protein, also known as Kelch-Like Family Member 16, Kelch-Like Protein 16, KLHL16 and GAN1, which Genbank nucleic acid access is AF291673.1 (SEQ ID NO. 1) and which protein access number is AAG35311.1 (SEQ ID NO. 2), as initially published in Bomont et al, Nat Genet 2000. SEQ ID NO. 1:
CAGGCACGTCCCGGGGGCTCCAGCTTCTGCTCAGAGCGCGGAGAGC CGGGCCGGGCGGGCGCGCGCGCAGGACTCGGGCCGCTCGAGGGGTCCGGCCG GACGGTGTCGGGAGCCGGACCCGTCGGCAGAGGAGCGGGCGCCGCGATGGCT GAGGGCAGTGCCGTGTCTGACCCTCAGCACGCCGCGCGTCTGCTGCGAGCGCT CAGCTCTTTCCGCGAGGAGTCTCGCTTCTGCGACGCGCACCTGGTCCTCGACGG GGAGGAGATCCCGGTGCAGAAGAACATCCTGGCGGCGGCCAGCCCGTACATC AGGACAAAGTTAAACTATAATCCTCCAAAAGATGATGGATCAACTTATAAGAT TGAACTTGAAGGGATATCGGTAATGGTTATGAGAGAGATCCTGGATTACATCT T C AGT GGGC AGATC AGGCT AAATGAAGAT AC AATCC A AGATGTTGTT C AGGC A GCTGACCTGCTGCTACTGACGGACCTTAAAACCCTGTGCTGTGAGTTTTTGGAA GGCTGCATTGCTGCTGAGAACTGTATTGGTATCCGTGACTTTGCACTACATTAC TGCCTCCATCACGTTCATTACCTTGCCACAGAATACCTGGAGACTCATTTCCGA GACGTCAGCAGCACGGAAGAATTCTTAGAGCTGAGTCCTCAAAAGCTTAAAGA AGT GATTTCTCTT GAGAAGTT AAACGTTGGC AAT GAAAGAT AT GTCTTTGA AG CAGTAATTCGATGGATAGCACATGATACAGAAATAAGAAAGGTCCACATGAA GGATGTTATGTCAGCTCTGTGGGTTTCAGGGTTGGACTCCAGTTATTTACGGGA ACAGATGCTGAATGAACCATTAGTACGAGAAATTGTCAAAGAGTGTAGCAATA TACCGCTCAGCCAGCCGCAGCAAGGGGAGGCGATGCTGGCCAACTTCAAACCC CGGGGCTACTCTGAGTGCATCGTGACTGTTGGTGGAGAAGAGAGAGTTTCACG GAAACCCACAGCAGCGATGCGATGCATGTGCCCTCTCTATGACCCTAACAGGC AGCTTTGGATCGAACTGGCCCCTTTAAGCATGCCGAGAATTAACCATGGAGTT CTCTCAGCAGAAGGATTTTTGTTTGTATTCGGGGGCCAAGATGAAAATAAGCA GACTCTT AGCTC AGGAGAAAAGT AT GATCC AGAT GC AAAT AC AT GGAC AGC AT TGCCACCTATGAACGAGGCAAGACATAACTTCGGAATTGTGGAGATAGATGGG AT GCTGT AC ATTTTGGGAGGAGAGGATGGTGAAAAGGAGCTGATTTCC AT GGA GTGTTACGATATTTATTCTAAAACCTGGACAAAGCAACCTGATTTGACCATGGT C AGAAAGATCGGCTGCT AT GC AGCT AT GAAAAAGAAA ATCT ACGCC AT GGGT G GAGGCTCCTATGGAAAGCTTTTTGAGTCTGTAGAGTGTTATGATCCCAGGACCC AGC AGT GGAC T GC CAT AT GTCC ACT A A A AGAGAGGAGGTTT GGAGC GGT GGC C TGTGGAGTTGCTATGGAGCTGTATGTGTTTGGGGGAGTCCGAAGTCGTGAGGA CGCCCAGGGTAGCGAGATGGTAACTTGCAAGTCCGAGTTCTACCATGATGAGT TTAAAAGGTGGATCTATCTTAACGACCAGAATTTATGCATCCCCGCCAGTTCCT
CTTTTGTTTATGGAGCTGTACCTATAGGAGCCAGTATTTATGTTATTGGAGATC
TTGATACAGGTACCAATTACGACTACGTGCGTGAGTTTAAAAGAAGCACAGGA
ACCTGGCACCACACTAAACCACTCCTTCCATCCGACCTTCGCCGTACAGGATGT
GCAGCCTTACGCATTGCGAATTGCAAGCTTTTCCGCCTGCAGCTTCAGCAAGGC
TTATTCCGTATTCGTGTTCATTCCCCTTGAGGAGGAAGCAGAGCAGAGTGCGA
GATCCTGACCC AAGAGC ACC AT A AC AT AGCTCCGA AAGGGAGAGC AGAGAT G
GC AGCTGAAACTC ACTCTGT GCTGGGCTTT GGT AT GGT AACTCTTT GGT GGTTT
TATGATGCTTACAAACTTGAGCTTTAGCTCTTGTTTGGGAGAACACGTAACTGT
TGAAAAACTACCTGGGAGGAGTGAGTTCCTCCAGTTAAATGTGGCTGTAGATG
TTGGAGGCT AGGGAGGCT AGT A A AT AT C A A A AGGA A A AGGGAGT GGGA ATT G
CTATCATGTAAAATATCAAAGTTAAAATACTAAGGTGCATTTTCCCTGAAGGG
AACTC ATGTCTGACTGCTGT ATTC AAAT ACGT AGCTTT GGT AAC AAAC AAAAT C
CGTATATGCAAATCAACATATCCAAACATGCCAAGACTGCTTTTCCACTGCACT
TGGAAGGATATATTATGCCTAACCCTGCCCAACAAATTAAGGTTTGTGCCTAA
AATGTTAGATTGGACTGTATGCCAGTTAGTCTCCATTTATTCCTAGT ACTCTGT
CCTAAGAATCTTTTTAAAACTATATCATGATGAATTGAAATGAAGATAAAATT
GCTCTTTTGTAACTTTATCTTAGTAATGTAAAGATTCAGTAAATTGATGAGTCA
GGTTGCAGCCCTCATGTGAACTGAAAGAAGTTGCTCGCTTCTGTGTTGACTTAG
ATCAAGACACGTCACGCATCCTTTCTGGGGTAGTACCTGTGGAGCCGGGAAGG
GTCTCCTGCAGTGCCATTCTGCCTTCTCAATGAGCAAAACCATTTTCTAAGTAT
GAGGAT ATT AGT GAGT AGG AGATTTT AT A A A AG A A AG ACC T GAGT C AG AC A A
ATAATAAAGGTCTGCTGTGGCTAAAAAAGCAAAAACAAAAAAGCTATCCAGA
CAAAATACATAGGGATCAGAAACTTTTTCATATAACCTGTCTGAAAGCACACA
AAGTCTTGTTTACTACTGTTTAGGGTTTGAAAACTTGTCTTAGAATGCGAATCT
GT GTT AAGAATCT AGTCTTTGT AAT AGAAGTTTCCC AGT GGC ATTC ACT AGT GA
GAGTTTT AGAC AA ATT AT GTT ACGAGT AAAGTTTGGCTGGT AGAAT AAACT AC
CTCAACTTAATTTCATTCTGTTCATAGTAGAGCAAATTAATCTTTTCCTCCTTCC
CTCCTTTCCTATCTCTCATTCTCACCTCAACCCACTTTACCCCACTTCTCATTGG
GGGAAAAATGTCTTTCGTTCGTGGAACGTATCTTGTAAGATATTTTGTTTTCCA
CTTGAATTACACCACCCTAGTGTGAAATCGGGGACTTCAAAGTGCTTGATTCCT
TCCTAAATTGAATTTTATGCAATGTCAAATTTCTAATCAATTTAATATACAGTA CTTCATTTTTAATTGTTTTCTAAAAAAAAGGTTTTTTTTTTCCTTTTGGAATATG
GCTGTAAGAAGCAGCATTTTGTCTTATTAACACATGTATAAAGGTATCCTTTGG
TTTTAAGTCGAGAACAGGAATTTCTTCTAGAAACTTCCTGTATGGAACGTTTAT
ATGCAATTAACATGCATATGAAAAACATCACTTACGTTCTCCCACCCATAGAC
ATAAAAAAGGGTGACCTGTGATTTTTTTTCTTATACCTGCTGGAAGTCAGTACA
AT GCGT C AAAAGTT GCTGAAAGAT GCTGTGCCT ATGGGGTTCT AGAGAGT GTT
GAGTGTGGTACATTAACTCTCCATTTTAGCCAGAAGATACCTGATAAGAGAAG
GTGTAAGGTTTTTATCTTATATGCCAGAGGCACCAGGCCAGATGTGCCGCACA
GTCATGTAATTCAATCATGTTTAATATGTCAGTCAAAACCAAATTCAGAATTGT
TTTGTGTACCAGTCAGCTGTTGTCTGCACATTTTCGTAGTGACAGTGTGGAGAT
CTTTTTAATGAAAGCAAACTATGTGGCCTCTGCAAAGAAAGGAAGATTTTTCTT
TTTACAACTAGATATTAGTTTTAGAGGAAGGAAATAGCTGAAAAACTAAATTT
GCTTTGGTGAAATGTCCTGTACAGANCAGTTCCTTGGCATTCAGCAGCTGTAAT
TGGGGAACATTAAAACAGTAACTGACATCCAGTTAAAGCCACGATCGTCAGCA
ATTCTCCTTTTTTAATTTCTGATATTTAAAGTTTTTTTTCCAGTCTACACCAGGC
CTCTCCAAGGAGACAGTTCATTATTTAGGAGTGAATGTGTTCCTCTTGCAATAT
TATCAGTACCTGCATGACTTGGTAAATTCATTTTATAAAAATAGTGTTTTTTTTT
TTTAATTTCAGTTCATTGACTCTATAACTGCAGAAATTAGATAATGTTTTATAA
AATAAATTTGCCACATAATATGGGATGCAATAACCAACAAAGCTGCTAAGTGC
CAAACTGTTATTTTACTATATATAAATATTAAAATATTGTGTTGAAGTATAGGG
ATGTATTTAATTTTACTATGCTCCAACATTAATCATGACTCTTTTGTAAATTACA
GTTATTTCAGTATTGTAAAATAAATGTTGACTCATTTCATGCAGGTTTGTGTTTT
AACATTCCACTTATGCCTTGAAACATCATTTCTTGATAACTTTTTGATACTTCTT
TCTT GAT AAGGC ACTTTT ACC AGGT GTGT GTTGGA ATT GNT GGCTC AGGAT AGG
TCTTAAATTTTAAATACAAATATCTTTGGG
According to the rules of the International Union of Pure and Applied Chemistry (UP AC), the nucleic acid N in positions 4022 and 4633 of SEQ ID NO. l corresponds to any one of nucleic acids A, C, G or T. SEQ ID NO. 2:
MAEGS AV SDPQHAARLLRAL S SFREESRF CD AHL VLDGEEIP V QKNIL A AASPYIRTKLNYNPPKDDGSTYKIELEGISVMVMREILDYIFSGQIRLNEDTIQDVV QAADLLLLTDLKTLCCEFLEGCIAAENCIGIRDFALHYCLHHVHYLATEYLETHFR D V S STEEFLEL SPQKLKE VISLEKLNV GNERYVFE AVIRWIAHDTEIRKVHIVIKD VM S ALW VSGLD S S YLREQMLNEPL VREIVKEC SNIPL SQPQQGEAML ANFKPRGY SEC I VT V GGEERV SRKPT AAMRCMCPL YDPNRQL WIEL APL SMPRINHGVL S AEGFLF VF GGQDENKQTL S SGEK YDPD ANTWT ALPPMNEARHNF GIVEIDGML YILGGEDG EKELISMECYDIYSKTWTKQPDLTMVRKIGCYAAMKKKIYAMGGGSYGKLFESV ECYDPRTQQWTAICPLKERRFGAVACGVAMELYVFGGVRSREDAQGSEMVTCKS EF YHDEFKRWIYLNDQNLCIP AS S SF VY GAVPIGASIYVIGDLDTGTNYD YVREFK RSTGTWHHTKPLLP SDLRRT GC AALRI ANCKLFRLQLQQGLFRIRVHSP
Within the scope of the present invention, the human ATG16L1 protein refers to a 624 amino acid residues, also known as Autophagy Related 16 Like 1, APG16L, ATG16 Autophagy Related 16-Like 1, ATG16 Autophagy Related 16-Like, APG16 Autophagy 16-Like, ATG16 Autophagy Related 16-Like 1, Autophagy -Related Protein 16-1, WD Repeat Domain 30, APGl 6-Like 1, APG16L Beta, ATG16A, ATG16L, IBD10 and WDR30, which Genbank nucleic acid access is NM_001363742.1 (SEQ ID NO. 3) and which protein access number is NP_001350671.1 (SEQ IN NO. 4).
SEP ID NO.3:
ACTAGCGAGCGCCCTGCGTAGGCACCGGCTCCTGAGCCCGTGCTTC GGGTGAGGGGGCGGGTCTTCCGGCCCTCTCGAAAATCATTTCCGGCATGAGCC GGAAGACCGTCCCGGATGGCCTCGGGGACTGCCAGTGTGTGGAGGTGAGCTCC GGGATTGCCGGCATTCCCGCTTCTGCTGGTTGCTTCATGCTGCAGGCTGCGGCC GT C AGCCCTCGCTCGC ATT GGT GGCGCTGAGGT GCCGGGGC AGC AAGT GAC AT GTCGTCGGGCCTCCGCGCCGCTGACTTCCCCCGCTGGAAGCGCCACATCTCGG AGCAACTGAGGCGCCGGGACCGGCTGCAGAGACAGGCGTTCGAGGAGATCAT CCTGCAGTATAACAAATTGCTGGAAAAGTCAGATCTTCATTCAGTGTTGGCCC AGAAACTACAGGCTGAAAAGCATGACGTACCAAACAGGCACGAGATAAGTCC CGGAC AT GATGGC AC AT GGAAT GAC AAT C AGCT AC AAGAAAT GGCCC AACTG AGGATTAAGCACCAAGAGGAACTGACTGAATTACACAAGAAACGTGGGGAGT
T AGCTC AACTGGTGATT GACCTGAAT AACC AAATGC AGCGGAAGGAC AGGGA
GATGCAGATGAATGAAGCAAAAATTGCAGAATGTTTGCAGACTATCTCTGACC
TGGAGACGGAGTGCCTAGACCTGCGCACTAAGCTTTGTGACCTTGAAAGAGCC
AACCAGACCCTGAAGGATGAATATGATGCCCTGCAGATCACTTTTACTGCCTT
GGAGGGAA AACTGAGGAAAACT ACGGA AGAGAACC AGGAGCTGGTC ACC AGA
TGGATGGCTGAGAAAGCCCAGGAAGCCAATCGGCTTAATGCAGAGAATGAAA
AAGACTCCAGGAGGCGGCAAGCCCGGCTGCAGAAAGAGCTTGCAGAAGCAGC
AAAGGA ACCTCT ACC AGTCGAAC AGGATGAT GAC ATT GAGGT C ATTGT GGAT G
AAACTTCTGATCACACAGAAGAGACCTCTCCTGTGCGAGCCATCAGCAGAGCA
GCCACTAAGCGACTCTCGCAGCCTGCTGGAGGCCTTCTGGATTCTATCACTAAT
ATCTTTGGTCTGTCCGAGTCTCCCCTTTTGGGACATCATTCTTCTTCTGATGCTG
CCAGGAGACGCTCTGTCTCTTCCTTCCCAGTCCCCCAGGACAATGTGGATACTC
ATCCTGGTTCTGGTAAAGAAGTGAGGGTACCAGCTACTGCCTTGTGTGTCTTCG
ATGCACATGATGGGGAAGTCAACGCTGTGCAGTTCAGTCCAGGTTCCCGGTTA
CTGGCC ACTGGAGGC AT GGACCGC AGGGTT AAGCTTT GGGAAGT ATTTGGAGA
AAAATGTGAGTTCAAGGGTTCCCTATCTGGCAGTAATGCAGGAATTACAAGCA
TTGAATTTGATAGTGCTGGATCTTACCTCTTAGCAGCTTCAAATGATTTTGCAA
GCCGAATCTGGACTGTGGATGATTATCGATTACGGCACACACTCACGGGACAC
AGTGGGAAAGTGCTGTCTGCTAAGTTCCTGCTGGACAATGCGCGGATTGTCTC
AGGAAGTCACGACCGGACTCTCAAACTCTGGGATCTACGCAGCAAAGTCTGCA
T AAAGAC AGT GTTT GC AGGATCC AGTT GC AAT GAT ATT GTCTGC AC AGAGC AA
TGTGTAATGAGTGGACATTTTGACAAGAAAATTCGTTTCTGGGACATTCGATCA
GAGAGCATAGTTCGAGAGATGGAGCTGTTGGGAAAGATTACTGCCCTGGACTT
AAACCCAGAAAGGACTGAGCTCCTGAGCTGCTCCCGTGATGACTTGCTAAAAG
TTATTGATCTCCGAACAAATGCTATCAAGCAGACATTCAGTGCACCTGGGTTCA
AGTGCGGCTCTGACTGGACCAGAGTTGTCTTCAGCCCTGATGGCAGTTACGTG
GCGGC AGGCTCTGCTGAGGGCTCTCTGT AT ATCTGGAGTGT GCTC AC AGGGAA
AGT GGAAAAGGTTCTTTC AAAGC AGC AC AGCTC ATCC AT C AAT GCGGT GGCGT
GGTCGCCCTCTGGCTCGCACGTTGTCAGTGTGGACAAAGGATGCAAAGCTGTG
CTGTGGGCACAGTACTGACGGGGCTCTCAGGGCTGGGAGGACCCCAGTGCCCT
CCTCAGAAGAAGCACATGGGCTCCTGCAGCCCTGTCCTGGCAGGTGATGTGCT GGGTATAGCATGGACCTCCCAGAGAAGCTCAAGCTATGTGGCACTGTAGCTTT
GCCGTGAATGGGATTTCTGAAGATTTGACTGAGGTCTCTCTTGGCCTGGAAGA
ATAACACTGAAAAAACCTGACGCTGCGGTCACTTAGCAGAGGCTCAGGTTCTT
GCCTTGGGAAACACTACTAGCTCTGACCTTCCATACCTCACTTGGGGGAGCAC
AGGGCCCCGCTGGGCCTCCTCACCAACGGCAGTGCCAAAATCAGCCCCCACAT
CAAGGTGGTGTTCTCTGTGCTTTCTCTCGTCCTTCCAAAGTCGGTTCTGGCCTA
ACGCATGTCCCAACACCTTGGGTTCATTTGCCCGGTGAACTCACTTTAAGCATT
GGATTAACGGAAACTCCCGAACTACAGACCCCTCCCTGGTGGGTTGCATGAAT
GTGTCTCATTACTGCTGAAATGTCCTCACATCTCTTTCACTGTTCTTCAGAGCTT
TCTGGCTCTCTTTCCCCCACAAAATTCGACATATTTAAAAATCTCCGTGTGGCT
TTAAAAAATGGTTTTTTGTTTTTTTGTTTTTTTGAGGTGGGAGAGGATGTGTGA
AAATCTTTTCCAGGGAAATGGGTTCGCTGCAGAGGTAAGGATGTGTTCCTGTA
TCGATCTGCAGACACCCAGAAGGTGGGTGCACACTGCATGCTTGGGGGTGCCA
AGGGATTCGAGACCTCCAACATACTTGTCTGAAGGTGGTGATTCTGGCCATGG
CCCCTCTGCCAAGCCTGTGTGCGATGCCCTTGGTGCTTTAGTGCAAGAAGCCTA
GGCTCAGAAGCACAGCAGCGCCATCTTTCCGTTTCAGGGGTTGTGATGAAGGC
CAAGGAAAAACATTTATCTTTACTATTTTACCTACGTATAAAGTTTTAGTTCAT
TGGGTGTGCGAAACACCCTTTTTATCACTTTTAAATTTGCACTTTATTTTTTTTC
TTCCATGCTTGTTCTCTGGACATTTGGGGATGTGAGTGTTAGAGCTGGTGAGAG
AGGAGTCAGGTGGCCTTCCCACCGATGGTCCTGGCCTCCACCTGCCCTCTCTTC
CCTGCCTGATCACCGCTTTCCAATTTGCCCTTCAGAGAACTTAAGTCAAGGAGA
GTTGAAATTCACAGGCCAGGGCACATCTTTTATTTATTTCATTATGTTGGCCAA
CAGAACTTGATTGTAAATAATAATAAAGAAATCTGTTATATACTTTTCAAACTC
CA
SEQ ID NO. 4:
MSSGLRAADFPRWKRHISEQLRRRDRLQRQAFEEIILQYNKLLEKSDLH SVLAQKLQAEKHDVPNRHEISPGHDGTWNDNQLQEMAQLRIKHQEELTELHKKR GELAQLVIDLNNQMQRKDREMQMNEAKIAECLQTISDLETECLDLRTKLCDLERA NQTLKDE YD ALQITF T ALEGKLRKTTEEN QEL VTRWM AEK AQE ANRLN AENEKD SRRRQ ARLQKEL AE A AKEPLP VEQDDDIE VI VDET SDHTEET SP VRAI SRA ATKRL S QP AGGLLD SITNIF GL SESPLLGHHS S SD A ARRRS V S SFP VPQDNVDTHPGSGKEVR VPATALCVFDAHDGEVNAVQFSPGSRLLATGGMDRRVKLWEVFGEKCEFKGSLS
GSNAGITSIEFDSAGSYLLAASNDFASRIWTVDDYRLRHTLTGHSGKVLSAKFLLD
NARIVSGSHDRTLKLWDLRSKVCIKTVFAGSSCNDIVCTEQCVMSGHFDKKIRFW
DIRSESIVREMELLGKITALDLNPERTELLSCSRDDLLKVIDLRTNAIKQTFSAPGFK
CGSDWTRVVFSPDGSYVAAGSAEGSLYIWSVLTGKVEKVLSKQHSSSINAVAWSP
S GSH V V S VDKGCK A VL W AQ Y
Within the scope of the invention, the human Ptch receptor (PTCH) protein refers to the patched homolog 1 (PTCH1) and the patched homolog 2 (PTCH2) protein, which are transmembrane receptors of the patched gene family.
For PTCH1, the PTCH1 Genbank nucleic acid access is Gene ID 5727. For PTCH2, the PTCH2 Genbank nucleic acid access is Gene ID 8643.
For reference, there are at least nine known isoform polypeptides of PTCH1 : protein patched homolog 1 isoform L (NP_000255.2); protein patched homolog 1 isoform M (NP_001077071.1); protein patched homolog 1 isoform L' (NP_001077072.1); protein patched homolog 1 isoform S (NP_001077073.1); protein patched homolog 1 isoform S
(NP_001077074.1); protein patched homolog 1 isoform S (NP_001077075.1); protein patched homolog 1 isoform S (NP_001077076.1); protein patched homolog 1 isoform 8
(NP_001341847.1); protein patched homolog 1 isoform 9 (NP_001341848.1).
For reference, there are at least two known isoform polypeptides of PTCH2: protein patched homolog 2 isoform 2 (NP_001159764.1); protein patched homolog 2 isoform 1: NP 003729.3
Within the scope of the invention, the term“autophagy” is intended to refer to the well documented cellular process that mediates the breakdown and the recycling of intracellular components by the means of fusion between autophagosome and lysosomes. For the purpose of the invention, the terms “autophagy” and “macroautophagy” are intended to be equivalent and substitutable with one another.
Within the scope of the instant invention, the expression“altered autophagy” is intended to refer to a level of autophagy which is either increased or decreased as compared to the physiological level of autophagy.
It is needless to mention that autophagy may be assessed by conventional methods and protocols described in the state of the art. Illustratively, autophagy and autophagy flux may be assessed in normal (physiological) conditions and conditions enhancing autophagy and/or blocking autophagosome-lysosome fusion: by quantifying LC3 lipidation; quantifying LC3 puncta formation; quantification of autophagy maturation with pH dependent LC3 -fluorescent probes, quantifying degradation of p62 and other cargos; quantifying lysosomal fusion; visualizing the formation of the double membrane autophagosomes; using techniques ranging from photonic microscopy and electron microscopy, and various immunoblotting techniques.
Within the scope of the invention, the term “Sonic Hedgehog (Shh) transduction” is intended to refer to the well-documented Sonic hedehog signaling pathway, which triggers the activation of the canonical and non-canonical Shh pathway. The activation of Shh reflects Shh transduction and it can be also assessed by conventional methods and protocols described in the state of the art. Illustratively, Shh activation or repression can be assessed by several assays, including but not restricted to the measures of i) the level of expression of Shh-responsive target genes (NKcό.I, PTCH, Gli....) using in situ hybridization, and/or immunofluorescence and/or western-blotting ii) Shh signaling activity using cellular and/or plasmid reporter (such as Shh-Light 2 cells, 7Gli::GFP plasmid....), iii) Smothened localization to the cilium, iv) the cilium length, and any other methods developed to measure Shh activity (ratio of active and non active from of Ci/Gli ....).
Within the scope of the invention, the term“altered Sonic Hedgehog (Shh) transduction” is intended to refer to a level of Shh transduction which is either increased or decreased as compared to the physiological level of Shh transduction.
• Therapeutic uses
In some embodiments, the modulator for use according to the invention is for the prevention and/or the treatment of a disease associated with altered autophagy.
In some embodiments, the modulator for use according to the invention is for the prevention and/or the treatment of a neurodegenerative disease.
In some embodiments, the modulator for use according to the invention is for the prevention and/or the treatment of a disease associated with altered Shh signaling. In some embodiments, the modulator for use according to the invention for the prevention and/or treatment of a neurodegenerative disease is a nucleic acid having at least 75% identity with SEQ ID NO. 1; or
- a polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2.
In some particular embodiments, the modulator for use according to the invention for the prevention and/or treatment of a neurodegenerative disease is a nucleic acid having at least 80% identity with SEQ ID NO. 1; or
- a polypeptide comprising a polypeptide sequence having at least 80% identity with SEQ ID NO. 2.
In some particular embodiments, the modulator for use according to the invention for the prevention and/or treatment of a neurodegenerative disease is a nucleic acid having at least 85% identity with SEQ ID NO. 1; or
- a polypeptide comprising a polypeptide sequence having at least 85% identity with SEQ ID NO. 2.
In some particular embodiments, the modulator for use according to the invention for the prevention and/or treatment of a neurodegenerative disease is a nucleic acid having at least 90% identity with SEQ ID NO. 1; or
- a polypeptide comprising a polypeptide sequence having at least 90% identity with SEQ ID NO. 2.
In some particular embodiments, the modulator for use according to the invention for the prevention and/or treatment of a neurodegenerative disease is a nucleic acid having at least 95% identity with SEQ ID NO. 1; or
- a polypeptide comprising a polypeptide sequence having at least 95% identity with SEQ ID NO. 2.
Within the scope of the instant invention, the term“prevention” is intended to refer to a reduction of the risk of occurrence of a disease associated with altered autophagy.
Within the scope of the instant invention, the term“treatment” is intended to refer to the effects achieved following the administration of a modulator according to the invention in an individual suffering from a disease, such as one associated with altered autophagy, and resulting in the partial or total alleviating of the symptoms linked to said disease. The invention further relates to a method for the prevention and/or the treatment of a disease associated with altered autophagy in an individual in need thereof, comprising the step of administering to the said individual a modulator according to the invention.
The invention further relates to a method for the prevention and/or the treatment of a disease associated with altered autophagy in an individual in need thereof, comprising the step of administering to the said individual an efficient amount of a modulator according to the invention.
Within the scope of the invention, the expression an“individual in need thereof’ is intended to refer to any human or non-human animal suffering or susceptible to suffer from a disease associated with altered autophagy. Preferably, said individual may be a mammal, and more preferably an animal of economic importance, such as a sheep, swine, cattle, goat, dog, cat, horse, poultry, mouse and a rat. More preferably, an individual according to the invention may be a human.
In some embodiment, the individual is a zebrafish, which may represent a valuable model for disease and drug screening.
It is known in the state of the art that alteration of autophagy, i.e. either increased or decreased autophagy, has been linked to a variety of human diseases. One can refer to Mizushima and Komatsu (Cell. 2011 Nov 11;147(4):728-41), Schneider and Cuervo (Curr Opin Genet Dev. 2014 Jun;26: 16-23), Menzies et al. (Nat Rev Neurosci. 2015 Jun;16(6):345-57), Lippai and Szatmari (Cell Biol Toxicol. 2017 Apr;33(2): 145- 168), Vomero et al. (Front Immunol. 2018 Jul 18;9: 1577).
It has been acknowledged by the medical community that depending on the nature of the disease, as well as the stage of the disease, one may want to either increase or decrease the functioning of the autophagy process in order to provide an adapted treatment to the individual in need thereof. For example, in early stage of cancer, enhancing the production of autophagosomes may benefit the organism as to combat the cancerous cells. By contrast, in late stage of some cancers, decreasing the autophagy may benefit the organism as to stop the progression of the tumor.
Therefore, a trained physician and/or clinician is able to determine the most suitable treatment depending of the information gathered from the individual and the nature and the stage of the disease for which a treatment is needed. In some embodiments, the disease (in particular associated with altered autophagy) is a genetic disease.
Alternatively, the disease (in particular associated with altered autophagy) is an acquired disease.
In some embodiments, the disease (in particular associated with altered autophagy) is selected in a group consisting of a cancer, an immune disease, an infectious disease, a metabolic disease, a cardiovascular disease, a (cardio)myopathy, a lysosomal disease, spinal cord injury and trauma, a neurodegenerative disease and a pulmonary disease.
In some embodiments, the cancer is selected in a non-limitative group comprising a bladder cancer, a blastoma, a bone cancer, a brain cancer, a breast cancer, a cancer of the central nervous system, a cancer of the cervix, a cancer of the upper aero digestive tract, a carcinoma, a colorectal cancer, an endometrial cancer, a germ cell cancer, a glioblastoma, a Hodgkin lymphoma, a kidney cancer, a laryngeal cancer, a leukaemia, a liver cancer, a lung cancer, a melanoma, a myeloma, a nephroblastoma (Wilms tumor), a neuroblastoma, a non-Hodgkin lymphoma, a non-small cell lung cancer, an oesophageal cancer, an osteosarcoma, an ovarian cancer, a pancreatic cancer, a pleural cancer, a prostate cancer, a retinoblastoma, a sarcoma, a skin cancer (including a melanoma), a small intestine cancer, a soft tissue sarcoma, a stomach cancer, a testicular cancer and a thyroid cancer.
In certain embodiments, the immune disease is selected in a group consisting of Achalasia, Addison’s disease, Adult Still's disease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome, Autoimmune angioedema, Autoimmune dysautonomia, Autoimmune encephalomyelitis, Autoimmune hepatitis, Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune orchitis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmune urticaria, Axonal & neuronal neuropathy (AMAN), Balo disease, Behcet’s disease, Benign mucosal pemphigoid, Bullous pemphigoid, Castleman disease (CD), Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss Syndrome (CSS) or Eosinophilic Granulomatosis (EGPA), Cicatricial pemphigoid, Cogan’s syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn’s disease, Dermatitis herpetiformis, Dermatomyositis, Devic’s disease (neuromyelitis optica), Discoid lupus, Dressier’ s syndrome, Endometriosis, Eosinophilic esophagitis (EoE), Eosinophilic fasciitis, Erythema nodosum, Essential mixed cryoglobulinemia, Evans syndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture’s syndrome, Granulomatosis with Polyangiitis, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura (HSP), Herpes gestationis or pemphigoid gestationis (PG), Hidradenitis Suppurativa (HS) (Acne Inversa), Hypogammalglobulinemia, IgA Nephropathy, IgG4-related sclerosing disease, Immune thrombocytopenic purpura (ITP), Inclusion body myositis (IBM), Interstitial cystitis (IC), Juvenile arthritis, Juvenile diabetes (Type 1 diabetes), Juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus, Lyme disease chronic, Meniere’s disease, Microscopic polyangiitis (MPA), Mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, Multifocal Motor Neuropathy (MMN) or MMNCB, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neonatal Lupus, Neuromyelitis optica, Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism (PR), PANDAS, Paraneoplastic cerebellar degeneration (PCD), Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Pars planitis (peripheral uveitis), Parsonnage-Turner syndrome,, Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia (PA), POEMS syndrome, Polyarteritis nodosa, Polyglandular syndromes type I, II, III, Polymyalgia rheumatica, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Primary biliary cirrhosis, Primary sclerosing cholangitis, Progesterone dermatitis, Psoriasis, Psoriatic arthritis, Pure red cell aplasia (PRCA), Pyoderma gangrenosum, Raynaud’s phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy, Relapsing polychondritis, Restless legs syndrome (RLS), Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, Sjogren’s syndrome, Sperm & testicular autoimmunity, Spinocerebellar ataxia, Stiff person syndrome (SPS), Subacute bacterial endocarditis (SBE), Susac’s syndrome, Sympathetic ophthalmia (SO), Takayasu’s arteritis, Temporal arteritis/Giant cell arteritis, Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), Transverse myelitis, Type 1 diabetes, Ulcerative colitis (UC), Undifferentiated connective tissue disease (UCTD), Uveitis, Vasculitis, Vitiligo, Vogt-Koyanagi-Harada Disease and Wegener’s granulomatosis.
In certain embodiments, the infectious disease may be a microbial infection, in particular a bacterial infection or a viral infection.
In certain embodiments, the infectious disease selected in a non-limiting group comprising Anaplasmosis; Anthrax; Babesiosis; Botulism; Brucellosis; Burkholderia mallei infection (glanders); Burkholderia pseudomallei infection (melioidosis); Campylobacteriosis; Carbapenem-resistant Enterobacteriaceae infection (CRE); Chancroid; Chikungunya infection; Chlamydia infection; Ciguatera; Clostridium difficile infection; Clostridium perfringens infection (Epsilon Toxin); Coccidioidomycosis fungal infection (Valley fever); Creutzfeldt- Jacob Disease, transmissible spongioform (CJD); Cryptosporidiosis; Cyclosporiasis; Dengue Fever; Diphtheria; E. Coli infection; Eastern Equine Encephalitis (EEE); Ebola Hemorrhagic Fever (Ebola); Ehrlichiosis; Arboviral or parainfectious encephalitis; Non-polio enterovirus infection; D68 enterovirus infection, (EV-D68); Giardiasis; Gonococcal infection (Gonorrhea); Granuloma inguinale; Type B Haemophilus Influenza disease, (Hib or H-flu); Hantavirus pulmonary syndrome (HPS); Hemolytic uremic syndrome (HUS); Hepatitis A (Hep A); Hepatitis B (Hep B); Hepatitis C (Hep C); Hepatitis D (Hep D); Hepatitis E (Hep E); Herpes; Herpes zoster, zoster VZV (Shingles); Histoplasmosis; Human Immunodeficiency Virus/ AIDS (HIV/AIDS); Human Papillomarivus (HPV); Influenza (Flu); Lead poisoning; Legionellosis (Legionnaires Disease); Leprosy (Hansens Disease); Leptospirosis; Listeriosis; Lyme Disease; Lymphogranuloma venereum infection (LVG); Malaria; Measles; Viral meningitis; Meningococcal disease; Middle East respiratory syndrome coronavirus (MERS-CoV); Mumps; Norovirus; Paralytic shellfish poisoning; Pediculosis (lice, head and body lice); Pelvic inflammatory disease (PID); Pertussis; Bubonic, septicemic or pneumonic plague,; Pneumococcal disease; Poliomyelitis (Polio); Psittacosis; Pthiriasis (crabs; pubic lice infestation); Pustular rash diseases (small pox, monkeypox, cowpox); Q-Fever; Rabies; Ricin poisoning; Rickettsiosis (Rocky Mountain Spotted Fever); Rubella, including congenital rubella (German Measles); Salmonellosis gastroenteritis infection; Scabies infestation; Scombroid; Severe acute respiratory syndrome (SARS); Shigellosis gastroenteritis infection; Smallpox; Methicillin-resistant Staphyloccal infection (MRSA); Staphylococcal food poisoning; Vancomycin intermediate Staphylococcal infection (VISA); Vancomycin resistant Staphylococcal infection (VRSA); Streptococcal disease, Group A; Streptococcal disease, Group B; Streptococcal toxic-shock syndrome (STSS); Primary, secondary, early latent, late latent or congenital syphilis; Tetanus infection (Lock Jaw); Trichonosis; Tuberculosis (TB); Latent tuberculosis (LTBI); Tularemia (rabbit fever); Typhoid fever, Group D; Typhus; Vaginosis; Varicella (chickenpox); Vibrio cholerae infection (Cholera); Vibriosis (Vibrio); Viral hemorrhagic fever (Ebola, Lassa, Marburg); West Nile virus infection; Yellow Fever; Yersenia infection and Zika virus infection.
In particular embodiments, the bacterial infection may encompass an infection by a bacterium of the genus Bacillus , such as the species Bacillus anthracis and Bacillus cereus Bartonella , such as the species Bartonella henselae and Bartonella Quintana ; Bordetella , such as the species Bordetella pertussis ; Borrelia, such as the species Borrelia burgdorferi , Borrelia garinii, Borrelia afzelii , Borrelia recurrently Brucella , such as the specied Brucella abortus , Brucella canis, Brucella melitensis , Brucella suis; Campylobacter , such as the species Campylobacter jejuni ; Chlamydia , such as the species Chlamydia pneumoniae , Chlamydia trachomatis ; Chlamydophila , such as the species Chlamydophila psittacy Clostridium , such as the species Clostridium botulinum , Clostridium difficile , Clostridium perfringens , Clostridium tetany ('or yne bacterium, such as the species Corynebacterium diphtheria ; Enterococcus , such as the species Enterococcus faecalis , Enterococcus faecium ; Escherichia , such as the species Escherichia coly Francisella , such as the species Francisella tularensis ; Haemophilus , such as the species Haemophilus influenza ; Helicobacter , such as the species Helicobacter pylori ; Legionella , such as the species Legionella pneumophila ; Leptospira , such as the species Leptospira interrogans , Leptospira santarosai, Leptospira weilii , Leptospira noguchiy Listeria , such as the species Listeria monocytogenes ; Mycobacterium , such as the species Mycobacterium leprae , Mycobacterium tuberculosis , Mycobacterium ulcerany Mycoplasma , such as the species Mycoplasma pneumoniae ; Neisseria , such as the species Neisseria gonorrhoeae , Neisseria meningitides ; Pseudomonas , such as the species Pseudomonas aeruginosa; Rickettsia , such as the species Rickettsia rickettsia ; Salmonella , such as the species Salmonella typhi , Salmonella typhimurium ; Shigella , such as the species Shigella sonnet, Staphylococcus , such as the species Staphylococcus aureus , Staphylococcus epidermidis , Staphylococcus saprophyticus ; Streptococcus , such as the species Streptococcus agalactiae , Streptococcus pneumoniae , Streptococcus pyogenes ; Treponema , such as the species Treponema pallidum ; Ureaplasma , such as the species Ureaplasma urealyticum Vibrio , such as the species Vibrio cholerae Yersinia , such as the species Yersinia pestis, Yersinia enterocolitica , Yersinia pseudotuberculosis.
In certain embodiments, the cardiovascular disease may be selected in a non- limitative group comprising a coronary artery disease, a heart attack, an abnormal heart rhythm (arrhythmia), a heart failure, a heart valve disease, a congenital heart disease, a heart muscle disease (cardiomyopathy), a pericardial disease, an aorta disease, a Marfan syndrome and a vascular disease.
In certain embodiments, the metabolic disease encompasses acute pancreatic, diabetes, a lysosomal storage disease, obesity and Paget’s disease.
The lysosomal storage diseases are described, e.g., in Settembre et al. (Autophagy. 2008 Jan;4(l): 113-4).
In certain embodiments, the myopathy is selected in a group consisting of cardiomyopathies, Bethlem myopathy, Danon’s disease, Dilated cardiomyopathy (DCM), Pompe’s disease, limb girdle muscular dystrophy type 2B, Miyoshi disease, sporadic inclusion body myositis, Ullrich congenital muscular dystrophy (UCMD) and X-Linked Myopathy with Excessive Autophagy (XMEA).
In certain embodiments, the neurodegenerative disease is selected in a non- limitative group comprising Alexander’s disease, Ataxia telangiectasia, Alper’s disease, Alzheimer's disease (AD), Amyotrophic Lateral Sclerosis (ALS), Batten disease, Beta- propeller protein-associated neurodegeneration (BPAN), Canavan disease, Cockayne disease, Charcot-Marie-Tooth disease (CMT), Cortico-basal degeneration (CBD), Epilepsy, Friedreich's ataxia, Fronto-temporal dementia (FTD), Gerstmann-Straussler- Scheinker disease (GSS), Giant Axonal Neuropathy (GAN), Guam-ALS syndrome, HIV- associated disease, Huntington's disease (HD), Lafora disease, Leigh's disease, Lewy body disease, Machado-Joseph disease, Neurodegeneration due to stroke, Neurodegeneration with brain iron accumulation (NBIA), Pallido-ponto-nigral degeneration (PPND), Pallido- nigro-luysian degeneration (PNLD), Parkinson's disease (PD), Pelizaeus-Merzbacher disease, Pick’s disease, Primary lateral sclerosis, Progressive supranuclear palsy (PSP), Refsum’s disease, Sandhoff disease, Schilder’s disease, Spinal muscular atrophy (SMA), Spino Cerebellar Ataxia (SCA) Static encephalopathy of childhood with neurodegeneration in adulthood (SENDA), Striatonigral degeneration, Transmissible spongiform encephalopathies (Prion diseases), Zellweger disease.
The neurodegenerative diseases which are particularly considered by the invention include neurodegenerative diseases associated with altered autophagy, and/or those associated with altered motor activity, and infantile neurological diseases.
For instance, a selection of neurodegenerative diseases which is particularly considered, in the sense of the invention, includes neurodegenerative diseases which are characterized by a loss of motor neurons, including Amyotrophic Lateral Sclerosis (ALS), Spinal muscular atrophy (SMA), and Charcot-Marie-Tooth disease (CMT).
Also, the neurodegenerative disease may in particular be Giant Axonal Neuropathy (GAN), as exemplified. Alternatively, the neurodegenerative disease may be distinct from Giant Axonal Neuropathy (GAN).
In some embodiments, the lysosomal disease may be selected in a non- limitative group comprising an alpha-mannosidosis, an aspartylglucosaminuria, a Batten disease, a beta-mannosidosis, a cystinosis, a Danon disease, a Fabry disease, a Farber disease, a fucosidosis, a galactosialidosis, a Gaucher disease, a gangliosidosis (GM1 gangliosidosis and GM2-gangliosidosis AB variant), a Krabbe disease, a metachromatic leukodystrophy, a mucopolysaccharidose disorder, a mucolipidosis, a multiple sulfatase deficiency, a Niemann-Pick disease, a Pompe disease (glycogen storage disease), a pycnodysostosis, a Sandhoff disease, a Schindler disease, a Salla disease (sialic acid storage disease), a Tay-Sachs disease and a Wolman disease.
In certain embodiments, the neurodegenerative disease is selected in a group consisting of Alzheimer’s disease, Amyotrophic Lateral Sclerosis (ALS), Giant Axonal Neuropathy (GAN), Huntington’s disease, Parkinson’s disease, Spinal Muscular Atrophy and Transmissible spongiform encephalopathies.
In certain embodiments, the pulmonary disease is cystic fibrosis.
In certain embodiments, the disease associated with altered autophagy is a disease characterized by a cellular accumulation or depletion, or mislocalization of ATG16L1.
In certain embodiments, the disease is associated with altered Shh transduction. In a general manner, diseases associated with altered Shh transduction, are known in the Art and further defined in Bale et al.;“ Hedgehog Signaling and Human Disease”; Annu. Rev. Genomics Hum. Genet; 3:47-65; 2002).
It will be understood herein that a disease may be associated both to an altered autophagy and to an altered Shh transduction in a patient.
In certain embodiments, the disease associated with altered Shh transduction is a disease characterized by a cellular accumulation or depletion, or mislocalization of PTCH.
In certain embodiments, the disease associated with altered Shh transduction is a neurodevelopmental or neurodegenetative disease, a congenital malformation of the central nervous system, of the axial skeleton and limbs, cancers and malignancies, including malignancies in children and adults. In certain embodiments, the disease associated with altered Shh transduction is selected from a list consisting of: Huntington’s disease, Alzheimer’s disease, holoprosencephaly, Grieg cephalopolysyndactly, Pallister- Hall syndrome, Postaxial polydactyly type 3, VATER association (or “VACTERL Syndrome”), Smith-Lemli-Opitz syndrome, Gorlin syndrome, Sporadic basal-cell carcinoma, Sporadic medulloblastoma, Glioblastoma, Joubert syndrome, microphtalmia, holoproencephaly, epilepsy. In certain embodiments, the disease associated with altered Shh transduction is basal cell carcinoma.
• Modulator of the expression, stability , degradation and/or activity of Gisaxonin .
In certain embodiments, the modulator for use according to the present invention is selected in a group comprising Gigaxonin, an activator of Gigaxonin expression, an inhibitor of Gigaxonin expression, an activator of Gigaxonin stability, an inhibitor of Gigaxonin stability, an activator of Gigaxonin degradation, an inhibitor of Gigaxonin degradation, an activator of Gigaxonin activity and an inhibitor of Gigaxonin activity, and is preferably Gigaxonin.
In some embodiments, the modulator is selected in a group consisting of a polypeptide and a nucleic acid.
In some embodiments, the nucleic acid is a deoxyribonucleic acid or a ribonucleic acid. For instance, when the modulator is a nucleic acid, it may be selected from a complementary DNA, a plasmid DNA, or a messenger RNA.
* Gisaxonin
Within the scope of the present invention,“Gigaxonin” is intended to refer to either the polypeptide itself or the polypeptide as expressed by the GAN gene, i.e. the gene encoding the Gigaxonin polypeptide.
In some embodiments, the Gigaxonin is:
- a polypeptide expressed by a nucleic acid comprising a nucleic acid (sequence) having at least 75% identity with SEQ ID NO. 1; or
- a polypeptide comprising a polypeptide (sequence) having at least 75% identity with SEQ ID NO. 2.
In some particular embodiments, the Gigaxonin is:
- a polypeptide expressed by a nucleic acid comprising a nucleic acid sequence having at least 80% identity with SEQ ID NO. 1; or
- a polypeptide comprising a polypeptide sequence having at least 80% identity with SEQ ID NO. 2
In some particular embodiments, the Gigaxonin is:
- a polypeptide expressed by a nucleic acid comprising a nucleic acid seqeunce having at least 85% identity with SEQ ID NO. 1; or
- a polypeptide comprising a polypeptide sequence having at least 85% identity with SEQ ID NO. 2
In some particular embodiments, the Gigaxonin is:
- a polypeptide expressed by a nucleic acid comprising a nucleic acid sequence having at least 90% identity with SEQ ID NO. 1; or
- a polypeptide comprising a polypeptide sequence having at least 90% identity with SEQ ID NO. 2
In some particular embodiments, the Gigaxonin is:
- a polypeptide expressed by a nucleic acid comprising a nucleic acid sequence having at least 95% identity with SEQ ID NO. 1; or
- a polypeptide comprising a polypeptide sequence having at least 95% identity with SEQ ID NO. 2 In some embodiments, the Gigaxonin may be provided as a nucleic acid encoding the Gigaxonin polypeptide, for example, as in the form of a plasmid or a nucleic acid vector that will be expressed and lead to the production of Gigaxonin when the said nucleic acid or the said plasmid or the said nucleic acid vector is introduced into a cell or bacteria, i.e. when a cell is transfected or transformed with the said nucleic acid or with the said plasmid or nucleic acid vector.
In some embodiments, the nucleic acid encoding the Gigaxonin polypeptide comprises a nucleic acid having at least 75% identity with SEQ ID NO. 1.
In some embodiments, the Gigaxonin comprises a polypeptide having at least 75% identity with SEQ ID NO. 2.
Within the scope of the present invention, the expression “at least 75% identity” encompasses 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% identity.
Hence, in some embodiments, the nucleic acid encoding the Gigaxonin polypeptide has at least 80% identity with SEQ ID NO. 1.
Hence, in some embodiments, the nucleic acid encoding the Gigaxonin polypeptide has at least 85% identity with SEQ ID NO. 1.
Hence, in some embodiments, the nucleic acid encoding the Gigaxonin polypeptide has at least 90% identity with SEQ ID NO. 1.
Hence, in some embodiments, the nucleic acid encoding the Gigaxonin polypeptide has at least 95% identity with SEQ ID NO. 1.
Within the scope of the present invention, the“percentage identity” between two sequences of nucleic acids or proteins means the percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after optimal alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly along their length. The comparison of sequences is traditionally carried out by comparing the sequences after having optimally aligned them, said comparison being able to be conducted by segment or by using an“alignment window”. Optimal alignment of the sequences for comparison can be carried out, in addition to comparison by hand, by means of the local homology algorithm of Smith and Waterman (1981), by means of the local homology algorithm of Neddleman and Wunsch (1970), by means of the similarity search method of Pearson and Lipman (1988) or by means of computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by the comparison software BLAST NR or BLAST P).
The percentage identity between two sequences is determined by comparing the two optimally-aligned sequences in which the sequence to compare can have additions or deletions compared to the reference sequence for optimal alignment between the two sequences. Percentage identity is calculated by determining the number of positions at which the nucleotide or amino acid residue is identical between the two sequences, preferably between the two complete sequences, dividing the number of identical positions by the total number of positions in the alignment window and multiplying the result by 100 to obtain the percentage identity between the two sequences.
In some particular embodiments, the nucleic acid encoding the Gigaxonin polypeptide comprises a nucleic acid of SEQ ID NO. 1. In some other embodiments, the nucleic acid encoding the Gigaxonin polypeptide consists of a nucleic acid of SEQ ID NO. 1
In some particular embodiments, the Gigaxonin polypeptide comprises a polypeptide of SEQ ID NO. 2. In some other embodiments, the Gigaxonin polypeptide consists of a polypeptide of SEQ ID NO. 2.
Illustratively, vectorization of a nucleic acid encoding the Gigaxonin polypeptide may be performed by the mean of conventional viral vector particles well known in the art. These viral vector particles encompass, e.g., baculovirus, retroviral vector particles, lentiviral vector particles, adenoviral vector particles and adeno-associated viral vector particles.
In some embodiments, the Gigaxonin may be provided as a purified and/or recombinant polypeptide. For instance, such recombinant polypeptide can be produced in eucaryotic cells, such as insect cells.
In practice, suitable techniques and protocols for the preparation of Gigaxonin in order to implement the invention may be adapted, e.g., from Sambrook and Green (2012; Molecular Cloning: A Laboratory Manual (Fourth Edition); Cold Spring Harbor Laboratory Press).
* Activators and inhibitors of the Gisaxonin expression
Within the scope of the present invention “an activator of Gigaxonin expression” is intended to refer to a compound able to increase, at least in part, the physiological cellular content of the Gigaxonin polypeptide.
Within the scope of the present invention “an inhibitor of Gigaxonin expression” is intended to refer to a compound causing a decrease of the cellular content of the Gigaxonin polypeptide.
Non-limitative examples of inhibitors of Gigaxonin expression encompass siRNAs, miRNAs, piRNAs that specifically bind to the Gigaxonin encoding nucleic acid or its corresponding mRNA, or alternatively, to a regulator of Gigaxonin expression.
In some embodiments, the inhibitor of Gigaxonin expression is a siRNA, a miRNA, or a piRNA that binds to a RNA complementary to a nucleic acid having at least 75% identity with SEQ ID NO. 1.
Non-limitative examples of activators of Gigaxonin expression encompass siRNAs, miRNAs, piRNAs that specifically bind to a negative regulator of Gigaxonin expression, and/or transcription factors and co-activators that bind to the Gigaxonin promotor to mediate transcription of the GAN gene.
Within the scope of the present invention, the term “complementary” is intended to mean that a first nucleic acid is complementary to a second nucleic acid when these nucleic acids have the base on each position which is the complementary (i.e. A to T, C to G) and in the reverse order. For example, the complementary sequence to TTAC is GTAA. If one strand of the double-stranded DNA is considered the sense strand, then the other strand, considered the antisense strand, will have the complementary sequence to the sense strand.
In some particular embodiments, the siRNAs or the miRNAs according to the invention bind to RNA complementary to a nucleic acid comprising a nucleic acid (sequence) of SEQ ID NO. 1. In some other embodiments, the siRNAs or the miRNAs according to the invention bind to RNA complementary to a nucleic acid of SEQ ID NO.
1 * Activators and inhibitors of the Gisaxonin stability
Within the scope of the present invention, the term“Gigaxonin stability” is intended to refer to the equilibrium reached between the synthesis and the degradation of the Gigaxonin polypeptide.
In some embodiments,“an activator of Gigaxonin stability” encompasses a compound which promotes the synthesis of Gigaxonin and/or decreases or reduces the degradation of the Gigaxonin polypeptide. For example, mention may be made of inactivation of Gigaxonin repressor, and/or transcription factors and/or translation initiation factors for the GAN gene.
In some embodiments,“an inhibitor of Gigaxonin stability” encompasses a compound which decreases or reduces the synthesis of Gigaxonin and/or promotes or increases the degradation of the Gigaxonin polypeptide. For example, mention may be made of E3-Ligases, deubiquitination enzymes (DUB), kinases, phosphatases and other enzymes which may cause post-translational modifications (PTM) of Gigaxonin: such as acetylation, amidation, hydroxylation, methylation, N-glycosylation, O-glycosylation, phosphorylation, sulfation, sumoylation, fumoylation, lipidation, neddylation, and ubiquitination.
Different chaperones may also take part in promoting Gigaxonin stability.
* Activators and inhibitors of the Gisaxonin activity
Within the scope of the present invention“an activator of Gigaxonin activity” is intended to refer to a compound able to increase, at least in part, the ability of the Gigaxonin polypeptide to promote its physiological role in the cell.
Within the scope of the present invention“an inhibitor of Gigaxonin activity” is intended to refer to a compound able to decrease, at least in part, the ability of the Gigaxonin polypeptide to promote its physiological role in the cell.
Illustratively, a suitable inhibitor of Gigaxonin activity may be an antibody, an aptamer specifically binding to Gigaxonin and altering its interaction with ATG16L1, or altering the subsequent ubiquitination of ATG16 in presence of an intact ATG16/Gig complex.
Illustratively, a suitable inhibitor of Gigaxonin activity may be an antibody, an aptamer specifically binding to Gigaxonin and altering its interaction with PTCH, or altering the subsequent ubiquitination of PTCH in presence of an intact PTCH/Gig complex.
In some embodiments, the inhibitor of Gigaxonin activity is an antibody or an aptamer that binds to a polypeptide having at least 75% identity with SEQ ID NO. 2.
In some particular embodiments, the antibody or aptamer according to the invention binds to a polypeptide of SEQ ID NO. 2.
A suitable antibody (or immunoglobulin) may encompass IgA, IgD, IgE, IgG and IgM immunoglobulin.
Suitable antibodies may be monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, humanized antibodies and optimized antibodies, for example antibodies with modified glycosylation and antibodies having a variant Fc region having optimized binding affinity with one or more Fc receptors.
Chimeric antibodies contain naturally occurring variable region (light chain and heavy chain) derived from an antibody from a given first species which is fused with the constant regions of the light chain and of the heavy chain derived from an antibody of a second species, distinct from the first species.
Antibodies suitable for the instant invention can be prepared using genetic recombination techniques. Chimeric or humanized antibodies can be prepared using standard methods described in the state of the art.
• Modulator of the Gigaxonin/ATG16Ll interaction or of the Gigaxonin/PTCH interaction
Within the scope of the instant invention, a modulator of the Gigaxonin/ATG16Ll or Gigaxonin/PTCH interaction is intended to encompass any compound which either increases or decreases the ability of Gigaxonin and ATG16L1/PTCH to interact in physiological conditions.
In some embodiments, a modulator of the Gigaxonin/ATG16Ll interaction or Gigaxonin/PTCH interaction may encompass an antibody, a chemical, a cofactor and an enzyme, which modifies either protein to impair its ability to interact with the other one, in particular a phoshorylase and kinase, or an E3 ligase or a Deubiquitinating enzyme (DUB). • Modulator of the functionality of the Gigaxonin/ATG16Ll interaction or of the Gigaxonin/PTCH interaction.
Within the scope of the instant invention, a modulator of the functionality of the Gigaxonin/ATG16Ll interaction is intended to encompass any compound which either increases or decreases the physiological function of the Gigaxonin/ATG16Ll complex.
In some embodiments, a modulator of the functionality of the
Gigaxonin/ATG16Ll interaction may encompass a compound that alters the ubiquitination or any other Post Translational Modifications of ATG16L1 or Gigaxonin.
Within the scope of the instant invention, a modulator of the functionality of the Gigaxonin/PTCH interaction is intended to encompass any compound which either increases or decreases the physiological function of the Gigaxonin/PTCH complex.
In some embodiments, a modulator of the functionality of the
Gigaxonin/PTCH interaction may encompass a compound that alters the ubiquitination or any other Post Translational Modifications of PTCH or Gigaxonin.
In some embodiments, the level of abundance of Gigaxonin, the
Gigaxonin/ ATG16L interaction, the Gigaxonin/PTCH interaction, the functionality of the Gigaxonin/ ATG16L1 interaction, and the functionality of the Gigaxonin/PTCH interaction may be measured by any suitable technique, such as, e.g. Western Blot, Co- immunoprecipitation, ELISA, FRET (Fluorescence Resonance Energy Transfer), Bimolecular Fluorescence Complementation (BiFC), Proximity Ligation Assay (PL A), ubiquitination (or ubiquitin-like) assay, phosphorylation and kinase assay, pull-down assay, Crosslinking protein interaction analysis, Label transfer protein interaction analysis.
Within the scope of the instant invention, a modulator of the functionality of the Gigaxonin/PTCH interaction is intended to encompass any compound which either increases or decreases the physiological function of the Gigaxonin/PTCH complex.
In some embodiments, a modulator of the functionality of the Gigaxonin/PTCH interaction may encompass a compound that alters the ubiquitination or any other Post Translational Modifications of PTCH or Gigaxonin. • Delivery particles
Another aspect of the invention further relates to a delivery particle comprising a modulator of Gigaxonin, the Gigaxonin/ATG16Ll interaction, the Gigaxonin/PTCH interaction and/or functionality as disclosed herein.
In particular, the invention relates to a delivery particle comprising a polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2, or a nucleic acid coding for such a polypeptide; such as a nucleic acid having at least 75% identity with SEQ ID NO.l.
In certain embodiments, the delivery particle may be in the form of a lipoplex or a lipid nanocapsule, comprising cationic lipids; a lipid nano-emulsion; a solid lipid nanoparticle; a peptide based particle; a polymer based particle, in particular comprising natural and/or synthetic polymers.
In some embodiments, a polymer based particle may comprise a synthetic polymer, in particular, a polyethylene glycol (PEG), a polyethylene imine (PEI), a dendrimer, a poly (DL- Lactide) (PLA), a poly(DL-Lactide-co-glycoside) (PLGA), a polymethacrylate and a polyphosphoesters.
In some embodiments, the delivery may further comprise at its surface one or more targeting ligands suitable for specifically addressing said particle to a targeted cell.
In some embodiments, a polymer based particle may comprise a protein, in particular an antibody or a fragment thereof; a peptide; a mono-saccharide, an oligo saccharide or a polysaccharide, in particular chitosan; a hormone; a vitamin; a ligand of a cellular receptor.
In some embodiments, the delivery particles according to the invention may be introduced in one or more target cells by the means of suitable methods known in the art, such as methods used for transfecting cells, which include electroporation, osmotic choc, sonoporation, cell squeezing and the like.
In some embodiments, the delivery particle may comprise a viral vector, in particular an adenovirus, an adeno-associated virus (AAV), an alphavirus, a herpesvirus, a lentivirus, a non-integrative lentivirus, a retrovirus, vaccinia virus or a bacculovirus.
The preparation of a delivery particle according to the instant invention may be performed by following any suitable method known in the state of the art. • Pharmaceutical composition and uses thereof
The invention also pertains to a pharmaceutical composition comprising:
- (i) a modulator of the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ATG16Ll interaction and/or of the functionality of the Gigaxonin/ATG16Ll interaction, and
- (ii) a pharmaceutically acceptable vehicle.
The invention also pertains to a pharmaceutical composition comprising:
- (i) a modulator of the expression, stability, degradation and/or activity of the Gigaxonin/PTCH interaction and/or of the functionality of the Gigaxonin/PTCH interaction, and
- (ii) a pharmaceutically acceptable vehicle.
In particular, as defined above, a modulator may be a polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2, or a nucleic acid coding for such a polypeptide.
Such a pharmaceutical composition may thus comprise a nucleic acid having at least 75% identity with SEQ ID NO. 1; or a polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2.
Hence, such a pharmaceutical composition may thus comprise a nucleic acid having at least 80% identity with SEQ ID NO. 1; or a polypeptide comprising a polypeptide sequence having at least 80% identity with SEQ ID NO. 2.
Hence, such a pharmaceutical composition may thus comprise a nucleic acid having at least 85% identity with SEQ ID NO. 1; or a polypeptide comprising a polypeptide sequence having at least 85% identity with SEQ ID NO. 2.
Hence, such a pharmaceutical composition may thus comprise a nucleic acid having at least 90% identity with SEQ ID NO. 1; or a polypeptide comprising a polypeptide sequence having at least 90% identity with SEQ ID NO. 2.
Hence, such a pharmaceutical composition may thus comprise a nucleic acid having at least 95% identity with SEQ ID NO. 1; or a polypeptide comprising a polypeptide sequence having at least 95% identity with SEQ ID NO. 2. In a further particular aspect, the invention relates to a pharmaceutical composition, as defined above, comprising a nucleic acid having at least 75% identity with
SEQ ID NO. 1; or
- a polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2; for use in the prevention and/or the treatment of a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity. Hence, the invention also relates to a pharmaceutical composition according to the present disclosure, for use in the prevention and/or the treatment of a neurodegenerative disease, such as a neurodegenerative disease associated with altered autophagy or altered motor activity.
The formulations of pharmaceutical compositions suitable to implement the disclosed invention may be obtained by following the routine and common principles, methods and techniques disclosed in the state of the art.
In some embodiments, a suitable pharmaceutically acceptable vehicle according to the invention may include any conventional solvent, dispersion medium, filler, solid carrier, aqueous solution, coating, antibacterial and antifungal agent, isotonic and absorption delaying agent, the like and a mixture thereof.
In certain embodiments, suitable pharmaceutically acceptable vehicles may include, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and a mixture thereof.
In some embodiments, pharmaceutically acceptable vehicles may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the cells.
In some embodiments, the pharmaceutical composition according to the present invention is for use in the prevention and/or the treatment of a disease associated with altered autophagy.
In some embodiments, modulator and/or the pharmaceutical composition, as disclosed herein, may be administered to an individual in need thereof by any route, i.e. by an oral administration, a topical administration or a parenteral administration, e.g., by injection, including a sub-cutaneous administration, a venous administration, an arterial administration, in intra-muscular administration, an intra-ocular administration and an intra-auricular administration. In certain embodiments, the administration of the modulator and/or the pharmaceutical composition, as encompassed herein, by injection may be directly performed in the target tissue of interest, in particular in order to avoid spreading of the said product.
Other suitable modes of administration may also employ pulmonary formulations, suppositories, and transdermal applications.
In some embodiments, an oral formulation according to the invention includes usual excipients, such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
In some embodiments, an effective amount of said modulator is administered to said individual in need thereof.
Within the scope of the instant invention, an“effective amount” refers to the amount of said compound that alone stimulates the desired outcome, i.e. alleviates or eradicates the symptoms of the encompassed disease.
It is within the routine and the common knowledge of a skilled artisan to determine the effective amount of the modulator and/or the pharmaceutical composition encompassed by the invention, in order to observe the desired outcome.
Within the scope of the instant invention, the effective amount of the modulator and/or the pharmaceutical composition to be administered may be determined by a physician or any trained and authorized person skilled in the art and can be suitably adapted within the time course of the treatment.
In certain embodiments, the effective amount to be administered may depend upon a variety of parameters, including the material selected for administration, whether the administration is in single or multiple doses, and the individual’s parameters including age, physical conditions, size, weight, gender, and the severity of the disease to be treated.
In certain embodiments, an effective amount of the modulator and/or the pharmaceutical composition according to the instant invention may comprise from about 0.001 mg to about 3000 mg, per dosage unit, preferably from about 0.05 mg to about 100 mg, per dosage unit.
Within the scope of the instant invention, from about 0.001 mg to about 3000 mg includes, from about 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1100 mg, 1150 mg, 1200 mg, 1250 mg, 1300 mg, 1350 mg,
1400 mg, 1450 mg, 1500 mg, 1550 mg, 1600 mg, 1650 mg, 1700 mg, 1750 mg, 1800 mg,
1850 mg, 1900 mg, 1950 mg, 2000 mg, 2100 mg, 2150 mg, 2200 mg, 2250 mg, 2300 mg,
2350 mg, 2400 mg, 2450 mg, 2500 mg, 2550 mg, 2600 mg, 2650 mg, 2700 mg, 2750 mg,
2800 mg, 2850 mg, 2900 mg and 2950 mg, per dosage unit.
In certain embodiments, the modulator and/or the pharmaceutical composition according to the instant invention may be administered at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day.
In other embodiments, an effective amount of the nucleic acid encoding the Gigaxonin polypeptide may comprise from about 1 ng to about 1 mg, per dosage unit, preferably from about 50 ng to about 100 pg, per dosage unit.
Within the scope of the instant invention, from about 1 ng to about 1 mg includes, about 2 ng, 3 ng, 4 ng, 5 ng, 6 ng, 7 ng, 8 ng, 9 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 150 ng, 200 ng, 250 ng, 300 ng, 350 ng, 400 ng, 450 ng, 500 ng, 550 ng, 600 ng, 650 ng, 700 ng, 750 ng, 800 ng, 850 ng, 900 ng, 950 ng,
! m& 2 m& 3 m& 4 m& 5 m& 6 m& 7 m& 8 kg, 9 kg, 10 m& 20 gg, 30 m& 40 gg, 50 gg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg, 150 pg, 200 pg, 250 pg, 300 pg, 350 pg, 400 pg, 450 pg, 500 pg, 550 pg, 600 pg, 650 pg, 700 pg, 750 pg, 800 pg, 850 pg, 900 pg and 950 pg per dosage unit.
In certain embodiments, the nucleic acid encoding the Gigaxonin polypeptide or the nucleic acid vector may be administered at dosage levels sufficient to deliver from about 0.01 ng/kg to about 10 pg/kg, from about 0.1 ng/kg to about 5 pg/kg, preferably from about 1 ng/kg to about 1 pg/kg of subject body weight per day. In some embodiments, the pharmaceutically composition may further comprise one or more additional active agent, in particular selected in a group comprising an antimicrobial compound and an anticancer compound.
In certain embodiments, the antimicrobial compound is an antibiotic selected in a group comprising a penicillin, in particular penicillin and amoxicillin; a carbapenem, in particular imipenem; a cephalosporin, in particular cephalexin; an aminoglycoside, in particular gentamicin and tobramycin; a tetracycline, in particular tetracycline and doxycycline; a macrolide, in particular erythromycin and clarithromycin; a quinolone, in particular ciprofloxacin and levofloxacin; and a sulphonamide, in particular sulfamethizole and sulfamethoxazole.
In certain embodiments, the antimicrobial compound is an antiviral agent selected in a non-limiting group comprising a neuraminidase inhibitor; a nucleoside analogue of guanine; a nucleoside analogue of thymidine; a nucleotide reverse transcriptase inhibitor; and a protease inhibitor.
In certain embodiments, the anti-cancer compound may be selected in a group comprising an alkylating agent, a purine analogue, a pyrimidine analogue, an anthracycline, bleomycin, mytomycin, an inhibitor of topo-isom erase 1, an inhibitor of topo-isom erase 2, a taxan, a monoclonal antibody, a cytokine, an inhibitor of a protein kinase, and the like.
• Miscellaneous methods
The methods disclosed herein may be achieved in vitro , in vivo or ex vivo , preferably, in vitro or ex vivo.
The system may be a sample obtained from an individual, such as, without limitation, one or more cells of the central nervous system, one or more embryonic cells, one or more epithelial cells, one or more germ cells, one or more hematopoietic progenitor cells, one or more hematopoietic stem cells, one or more induced Pluripotent Stem Cells (iPSC), one or more muscular cells, one or more progenitor cells, one or more stem cells, and a mixture thereof.
In some embodiments, the sample may originate from a tissue selected in a group comprising a connective tissue, an epithelial tissue, a muscle tissue and a nervous tissue. In some embodiments, the sample may originate an organ selected in a group comprising a bladder, a bone, a brain, a breast, a central nervous system, a cervix, a colon, an endometrium, a kidney, a larynx, a liver, a lung, an oesophagus, an ovarian, a pancreas, a pleura, a prostate, a rectum, a retina, a salivary gland, a skin, a small intestine, a soft tissue, a stomach, a testis, a thyroid, an uterus, a vagina.
Thus, the invention relates to a method for screening a modulator of the expression stability and/or activity of Gigaxonin, of the Gigaxonin/ATG16Ll interaction and/or of the functionality of the Gigaxonin/ATG16Ll interaction, comprising the steps of:
- (a) providing a system comprising Gigaxonin and ATG16L1 in suitable conditions for the production of the corresponding proteins and for the interaction between Gigaxonin and ATG16L1 to occur;
- (b) providing to the said system a candidate modulator of the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ ATG16L1 interaction and/or of the functionality of the Gigaxonin/ ATG16L1 interaction;
- (c) measuring the level of expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ ATG16L1 interaction and/or of the functionality of the Gigaxonin/ ATG16L1 interaction in the system at step (b);
- (d) determining a difference between the level measured in step (c) and a corresponding reference level obtained in the absence of the said candidate modulator.
In some embodiments, determining a significant difference is indicative of the ability of the candidate modulator to efficiently modulate Gigaxonin, the interaction between Gigaxonin and ATG16L1 and the functionality of the complex.
It is understood that the system provided in step (a) comprises Gigaxonin and ATG16L1 in conditions to form a complex. The suitable conditions for the interaction between Gigaxonin and ATG16L1 to occur may be determined by a skilled artisan in the art. These conditions are non-limited to parameters such as temperature, pH, O2 content, salinity, within the said system. The invention also relates to a method for screening a modulator of the expression stability and/or activity of Gigaxonin, of the Gigaxonin/PTCH interaction and/or of the functionality of the Gigaxonin/PTCH interaction, comprising the steps of
- (a) providing a system comprising Gigaxonin and PTCH in suitable conditions for the production of the corresponding proteins and for the interaction between Gigaxonin and PTCH to occur;
- (b) providing to the said system a candidate modulator of the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/PTCH interaction and/or of the functionality of the Gigaxonin/PTCH interaction;
- (c) measuring the level of expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/PTCH interaction and/or of the functionality of the Gigaxonin/PTCH interaction in the system at step (b);
- (d) determining a difference between the level measured in step (c) and a corresponding reference level obtained in the absence of the said candidate modulator.
In some embodiments, determining a significant difference is indicative of the ability of the candidate modulator to efficiently modulate Gigaxonin, the interaction between Gigaxonin and PTCH and the functionality of the complex.
The invention thus also relates to a method aimed at using Gigaxonin and/or ATG16L1 and/or PTCH (including biological samples such as cells comprising or expressing them) for developing readout tests and methodologies to screen existent and/or novel drugs to modulate autophagy activity and Shh transduction, and associated diseases.
The above-mentioned methods are thus applicable to methods for screening a candidate treatment for treating and/or preventing a disease associated with altered autophagy, or with altered Shh transduction, and/or neurodegenerative diseases.
Within the scope of the instant invention, the expression “significant difference” is intended to refer to a level of interaction measured in step (c) which is at least 10% or more, for example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% higher or lower than the reference level measured in step (a).
In certain embodiments, a “significant difference” refers to a level of interaction measured in step (c) which is at least 1.2 fold, 1.4 fold, 1.6 fold, 1.8 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold, 10 fold, 50 fold, 100 fold, 500 fold higher or lower than the reference level measured in step (a).
In some embodiments, the method may further comprise a step (c’) comprising a step of measuring the effect on the steady state level and distribution of ATG16L1 and Gigaxonin.
In some embodiments, the method may further comprise a step (c’) comprising a step of measuring the effect on the steady state level and distribution of PTCH and Gigaxonin.
In said embodiment, the step (d) comprises a step of determining a difference between the level measured in step (d) and a reference level obtained in the absence of the said candidate modulator.
According to an alternative embodiment, a method for assessing the efficiency of a candidate treatment of a disease may include a step of providing an individual with a candidate treatment.
Hence, the invention may also pertain to a method for assessing the efficiency of a candidate treatment of a disease associated with altered autophagy in an individual in need thereof, comprising the steps of:
- (a) measuring the level and distribution of Gigaxonin, ATG16L1, the Gigaxonin/ ATG16L1 interaction and/or the functionality of the Gigaxonin/ ATG16L1 interaction in a sample from said individual in need thereof, in suitable conditions for the interaction to occur;
- (b) providing the said individual with a candidate treatment;
- (c) measuring the level and distribution of Gigaxonin, ATG16L1, the Gigaxonin/ ATG16L1 interaction and/or the functionality of the Gigaxonin/ATG16Ll interaction in a sample obtained at step (b);
- (d) determining a difference between the level measured in step (a) and the level measured in step (c). In a further aspect, the invention relates to a method for assessing the efficiency of a candidate treatment of a disease associated with altered Shh transduction in an individual in need thereof, comprising the steps of:
- (a) measuring the level of Gigaxonin, PTCH, the Gigaxonin/PTCH interaction and/or the functionality of the Gigaxonin/PTCH interaction in a sample from said individual in need thereof, in suitable conditions for the interaction to occur;
- (b) providing the said individual with a candidate treatment;
- (c) measuring the level of Gigaxonin, PTCH, the Gigaxonin/PTCH interaction and/or the functionality of the Gigaxonin/PTCH interaction in a sample obtained at step
(b);
- (d) determining a difference between the level measured in step (a) and the level measured in step (c).
In certain embodiments, the sample of step (a) may comprise, without limitation, one or more cells of the central nervous system, one or more embryonic cells, one or more epithelial cells, one or more germ cells, one or more hematopoietic progenitor cells, one or more hematopoietic stem cells, one or more induced Pluripotent Stem Cells (iPSC), one or more muscular cells, one or more progenitor cells, one or more stem cells, and a mixture thereof.
In some embodiments, the sample of step (a) may originate from a tissue selected in a group comprising a connective tissue, an epithelial tissue, a muscle tissue and a nervous tissue.
In some embodiments, the sample of step (a) may originate an organ selected in a group comprising a bladder, a bone, a brain, a breast, a central nervous system, a cervix, a colon, an endometrium, a kidney, a larynx, a liver, a lung, an oesophagus, an ovarian, a pancreas, a pleura, a prostate, a rectum, a retina, a salivary gland, a skin, a small intestine, a soft tissue, a stomach, a testis, a thyroid, an uterus, a vagina.
In some embodiments, determining a significant difference is indicative of the ability of the candidate treatment to efficiently treat the individual in need thereof.
In another aspect, is also disclosed herein a method for assessing the efficiency of a candidate treatment of a disease associated with altered autophagy in an individual in need thereof, comprising the steps of: - (a) measuring the level of the Gigaxonin/ATG16Ll interaction and/or the Gigaxonin/ATG16Ll functionality in a sample from said individual in need thereof, in suitable conditions for the interaction to occur;
- (b) measuring the steady state levels and distribution of Gigaxonin and
ATG16L1
- (c) providing the said individual with a candidate treatment;
- (d) measuring the level of the Gigaxonin/ATG16Ll interaction and functionality in a sample obtained at step (c);
- (e) measuring the steady levels of Gigaxonin and ATG16L1 in a sample obtained at step (c);
- (f) determining a difference between (i) the levels measured in step (a) and (d), and (ii) the levels measured in step (b) and (e).
The invention further relates to a method to activate autophagy in a target cell, comprising a step of increasing the level of expression, stability, degradation and/or activity of Gigaxonin to reach physiological functions of ATG16L1.
In some embodiments, the activation of autophagy is performed in conditions of ATG16L1 -dependant inhibition and/or aggregation
In some embodiments, the said method is achieved by a reduction of the aggregation of ATG16L1 and/or the reduction of the stability and/or the expression of ATG16L1, or alternatively by an increase of the degradation of ATG16L1 in the target cell.
Within the scope of the instant invention, the expression “to reach physiological functions of ATG16L1” is intended to refer to a physiological level of ATG16L1 upon which ATG16L1 exerts its normal function in a target cell. In other words, this expression is intended to mean that the ATG16L1 has reached a balance between its synthesis and its degradation in order to perform its physiological role in autophagy.
The invention further relates to a method to activate, induce or restore Sonic Hedgehog (Shh) transduction in a target cell, comprising a step of increasing the level of expression, stability, degradation and/or activity of Gigaxonin to reach physiological functions of PTCH. In some embodiments, the increasing of the level of expression of Gigaxonin comprises the step of providing the target cell with (i) a nucleic acid encoding a Gigaxonin polypeptide or (ii) a Gigaxonin polypeptide.
In particular, the increasing of the level of expression of Gigaxonin comprises the step of providing the target cell with (i) a nucleic acid encoding a Gigaxonin polypeptide or (ii) a Gigaxonin polypeptide, or (iii) siRNAs or (iv) miRNAs or (v) piRNAs that specifically bind to a negative regulator of Gigaxonin expression or (vi) transcription factors and/or co-activators that bind to the Gigaxonin promoter to mediate transcription of the GAN gene.
In some embodiments, the increasing of the level of expression of Gigaxonin comprises the step of providing the target cell with compounds able to increase Gigaxonin expression, stability or to impair its degradation.
The invention further relates to a method for the fine-tuned activation or inhibition of autophagy in a target cell comprising the step of modulating the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ ATG16L1 interaction and/or of the functionality of the Gigaxonin/ATG16Ll interaction.
The invention further relates to a method for the fine-tuned activation or inhibition of Sonic Hedgehog (Shh) transduction in a target cell comprising the step of modulating the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/PTCH interaction and/or of the functionality of the Gigaxonin/PTCH interaction.
The invention may also pertain to a method for inducing autophagy in an individual in need thereof, comprising the step of administering to the said individual an efficient amount of Gigaxonin or its gene, of an activator of Gigaxonin expression, of an activator of Gigaxonin stability or an activator of Gigaxonin activity, or a molecule able to reduce Gigaxonin degradation.
The invention may also pertain to a method for inhibiting autophagy in an individual in need thereof, comprising the step of administering to the said individual an efficient amount of Gigaxonin, of an inhibitor of Gigaxonin expression, of an inhibitor of Gigaxonin stability or an inhibitor of Gigaxonin activity or an activator of Gigaxonin degradation. It is understood that inhibition of Gigaxonin steady level/activity inhibits autophagy, and that below a certain threshold, administration of Gigaxonin may induce autophagy, whereas above a certain threshold, administration of Gigaxonin may inhibit autophagy.
The above-mentioned threshold may be determined by a physician or any trained and authorized person skilled in the art.
The invention also relates to a method for assessing the efficiency of the treatment of a disease associated with altered autophagy in an individual in need thereof, comprising the steps of:
- (a) measuring the level of autophagy in a sample from said individual in need thereof, in suitable conditions for autophagy to occur;
- (b) providing the said individual with a candidate treatment;
- (c) measuring the level of autophagy of the sample of step (b);
- (d) determining a difference between the level measured in step (a) and the level measured in step (c).
In some embodiments, determining a significant difference is indicative of the ability of the candidate treatment to efficiently treat the individual in need thereof.
The present disclosure also relates to a method for assessing a post- translational modification (PTM) of ATG16L1, in response to the modulation of the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ ATG16L1 interaction and/or of the functionality of the Gigaxonin/ ATG16L1 interaction, comprising the steps of:
- (a) determining the PTM status of ATG16L1 in a system comprising the ATG16L1 polypeptide;
- (b) providing the said system with a candidate modulator of the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ ATG16L1 interaction and/or of the functionality of the Gigaxonin/ ATG16L1 interaction;
- (c) determining the PTM status of ATG16L1 in the system at step (b).
Within the scope of the instant invention, the PTM may be selected in a non limited group comprising acetylation, amidation, hydroxylation, methylation, N- glycosylation, O-glycosylation, phosphorylation, sulfation, sumoylation, fumoylation, lipidation, neddylation, and ubiquitination of ATG16L1 The PTM status of the ATG16L1 polypeptide may be determined accordingly with the standard methods known in the state of the art, i.e. measuring the level of acetylation, amidation, hydroxylation, methylation, N-glycosylation, O-glycosylation, phosphorylation, sulfation, sumoylation, fumoylation, lipidation, neddylation and ubiquitination of ATG16L1
The instant disclosure further relates to a method for assessing the activity of the autophagy pathway, in response to the modulation of ATG16L1 by Gigaxonin, comprising the steps of:
(a) monitoring the autophagy flux and maturation in a suitable cellular system;
- (b) providing the said cellular system with a candidate modulator of the expression, stability and/or activity of Gigaxonin, of the Gigaxonin/ATG16Ll interaction and/or of the functionality of the Gigaxonin/ ATG16L1 interaction;
(c) monitoring the autophagy in the cellular system at step (b).
The said method may be implemented by any suitable method mentioned above.
As used herein, a“cellular system” encompasses any collection of cells (i.e. primary culture cells or cell lines), especially any collection of human cells, which include cells originating from various organs or tissue of a human individual, or derived from a human individual (iPS cells), such as a healthy human individual and a human individual affected with a disease, including an individual affected with cancer.
The present disclosure also concerns a method for blocking autophagy in conditions wherein autophagy is increased, regardless of the steps involved in the autophagy pathway.
In some embodiment, the said method comprises a step of decreasing the Gigaxonin expression and/or stability.
In some embodiment, the said method comprises a step of increasing the Gigaxonin expression and/or stability.
In some embodiment, the said method comprises a step of increasing
Gigaxonin turn-over, preferably increasing Gigaxonin degradation.
In some embodiment, the said method comprises a step of decreasing
Gigaxonin turn-over, preferably decreasing Gigaxonin degradation. In some embodiments, the said method comprises a step of decreasing Gigaxonin/ATG16Ll interaction. The inventors believe that decreasing Gigaxonin/ATG16Ll interaction would exert a dominant negative effect on autophagy, through the increased abundance of ATG16L1.
The invention is further illustrated by the non -limitative examples below.
EXAMPLES
1- METHODS
1.1- Cell culture and transient transfection
COS-7 cells (clone CRL-1651 from ATCC) and HEK293T cells (ATCC CRL- 11268) were maintained in DMEM medium (THERMOFISHER®) containing 10% foetal bovine serum (THERMOFISHER®) and 1% Penicillin/Streptomycin. Cells were tested negative for mycoplasma. Cortical neurons were obtained from El 5.5 old embryos from the GAN / model (Ganay et al.; Mol Neurodegene 12, 25 (2011)) and control littermates. Animal were treated in accordance with the European Union guide for the care and the use of animals in research (2010/63/UE). Briefly, brains cortices were dissected in HBSS (THERMOFISHER®) supplemented with 0.44 % glucose; enzymatically dissociated in HAMF 10/0.025% trypsin (THERMOFISHER®); mechanically dissociated in neurobasal medium/2% foetal bovine serum; and concentrated after centrifugation at 470xg on BSA cushions (SIGMA®). Neurons were resuspended in Neurobasal Medium (THERMOFISHER®), supplemented with 1% Sodium pyruvate (THERMOFISHER®), 2% B27-Supplement (THERMOFISHER®) and 1% Penicillin/Streptomycin (THERMOFISHER®). Cells were plated on 14 mm-diameter coverslips (for immunostaining), or in 35mm petri dish (for immunoblot), previously coated with 3pg/ml of poly-D-ornithine (SIGMA®) and 2pg/ml of laminin (SIGMA®), at a density of 1-2 xlO4 cells or 0.75-1 x 106 cells, respectively. Transfection of plasmids was performed using Lipofectamin 2000 (INVITROGEN®) according to the manufacturer’s instructions. For induction of starvation-mediated autophagy, DMEM medium was replaced with EBSS (THERMOFISHER®, SH30029.02) for 2h or 6h, as indicated. Lysosomal and proteasome inhibition was achieved by 200 nM Bafilomycin Al (SIGMA®, B1793) and 20mM MG132 (TOCRIS BIOSCIENCE®) for 12 hours. 1.2- Autophagic measurements
Autophagy activity and flux was determined using complementary approaches: quantification of i) LC3 lipidation, ii) LC3 puncta formation, iii) p62 aggregation, iv) lysosomal fusion. For the quantification of LC3 lipidation, protein extracts were prepared as described in the western blot section. LC3-II and tubulin control were detected by rabbit anti-LC3B (1 : 1000, L7543, SIGMA®) and mouse anti-a tubulin (1 : 1000, clone DMla, CALBIOCHEM®), respectively. Quantification of LC3-II/tubulin intensity was performed by the Image Lab software (http://www.bio-rad.com/fr-fr/product/image-lab-software). The level of LC3 lipidation was determined by the relative LC3-II/tubulin ratio. The LC3 flux was determined after comparison of the basal condition with serum deprivation condition (EBSS), with or without lysosome inhibitor (Bafilomycin Al). The autophagosome synthesis was measured as the differences of LC3 lipidation at two different times of EBBS+Baf, at 2h and 6h. All values were expressed relatively to the LC3-Q/tubulin ratio in wild type neurons in basal condition (fixed to 1).
For the LC3 puncta formation, cortical neurons were fixed as described in the immunostaining section. LC3 fluorescence intensity of individual cell was quantified by ImageJ software (https://imagej.nih.gov/ij/).
P62 aggregation in primary neurons was visualised by immunofluorescence, using rabbit anti-p62 (1 : 1000, PM045, MBL®) and mouse anti-MAP2 (1 : 1000, M4403, SIGMA®), in basal condition and EBSS, Baf and EBSS+Baf conditions. Lysosomal fusion was assessed by two independent analyses, by immunofluorescence on cortical neurons. The co-localisation of LC3 dots with lysosomes (lysotracker, 75 nM, L7528, THERMOFISHER®) was quantified in individual neurons by the ImageJ software (https://imagej.nih.gov/ij/) with the Pearson coefficient. The fusion of p62 with lysosomes was assessed by immunofluorescence, in conditions that either promote fusion (EBSS) or inhibit autolysosome formation (Baf and EBSS+Baf).
1 3- Lentiviral infection
The lentiviral vector pLEX-MCS-FLAG-Gigaxonin, pLEX-MCS-GFP, p- VSVG and pAX2 were gifts from R.D. Goldman/P. Opal (J Clin Invest 123, 1964-1975 (2013)). Lentiviral particles were produced after cotransfection of the pLEX-MCS-FLAG- Gigaxonin or pLEX-MCS-GFP plasmids, together with the helper plasmid p-VSYG and pAX2 into HEK293T cells. The supernatant was collected from the cells two days later and the virus was concentrated by ultracentrifugation. Primary cortical neurons from GAN mouse and control littermates were infected with at 2 days in vitro (div) with MOCK- GFP and Flag-Gigaxonin lentivirus. Medium was changed 6-8 hours after transduction and cells were examined at 4 div.
1.4- Plasmids and siRNA
The human Cherry-ATG16L1 plasmid was generated by reverse transcription (Superscript III Kit, INVITROGEN®) from HeLa cells mRNA. cDNA was amplified using primers flanked by ATTB1/ATTB2 sequences, and subcloned into gateway vectors. Flag-ATG16L1 deletion constructs are disclosed in Fujita et al. (Mol Biol Cell 19, 2092- 2100 (2008)). The human Full-length and deletion Gigaxonin cDNAs are disclosed in Bomont and Koenig & Cleveland et al. (Hum Mol Genet 12, 813-822 (2003), Hum Mol Genet. 2009 Apr 15; 18(8): 1384-94). Final gateway plasmids were pcDNA-Cherry-N or pCi-3xFlag-N. For the Bimolecular Fluorescence Complementation (BiFC) assay, ATG16L1 and Gigaxonin cDNA were cloned in Gateway BiFC vectors, in fusion with the N-terminal region (YFPN: l-154aa), and the C-terminal region (YFPC: 155-239aa) of the YFP protein, respectively.
The primers used are the following (ATTB sequences in lower cases): and are common across gateway vectors (ATTB sequences in lower cases):
ATG16L1 Forward
ggggacaagtttgtacaaaaaagcaggcttcATGTCGTCGGGCCTCCGCGCC (SEQ ID
NO. 5),
ATG16L1 Reverse
ggggaccactttgtacaagaaagctgggttGTACTGTGCCCACAGCACAGC (SEQ ID
NO. 6) ;
Gigaxonin Forward (full length)
ggggacaagtttgtacaaaaaagcaggcttcATGGCTGAGGGCAGTGCCGTG (SEQ ID
NO. 7), Gigaxonin Reverse (full length)
ggggaccactttgtacaagaaagctgggttAGGGGAATGAACACGAATACG (SEQ ID
NO. 8);
DN Gigaxonin Forward
ggggacaagtttgtacaaaaaagcaggcttcGCACTACATTACTGCCTCCAT (SEQ ID
NO. 9),
DN Gigaxonin Reverse
ggggaccactttgtacaagaaagctgggttAGGGGAATGAACACGAATACG (SEQ ID
NO. 10);
AC Gigaxonin Forward
ggggacaagtttgtacaaaaaagcaggcttcATGGCTGAGGGCAGTGCCGTG (SEQ ID
NO. 11),
AC Gigaxonin Reverse
ggggaccactttgtacaagaaagctgggttAGCTGACATAACATCCTTCAT (SEQ ID
NO. 12)
Silencing of Gigaxonin was performed using siRNAs (Dharmacon) of the following antisense sequences: siRNA-5’ AUAACAUAAAUACUGGCUC 3’ (SEQ ID NO. 13) with the mismatch counterpart ms antisense sequence -5’ AUAAAAUAAAUACGGGCUC 3’ (SEQ ID NO. 14).
1.5- Western Blotting and immunostaining
Cells were lysed in a solution containing 50mM Tris pFI 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA and a cocktail of protease inhibitors Cleveland et al. (Hum Mol Genet 18, 1384-1394 (2009)). Proteins were separated in 10-15% SDS-PAGE gels and transferred to nitro-cellulose membrane (hybond C-Extra, AMERSHAM BIOSCIENCES®). After blocking with 5% non-fat milk in PBS-0.05% Tween, membranes were incubated with primary and HRP-secondary antibodies and the signals detected by Chemiluminescent substrate HRP (THERMOFISHER®, MILLIPORE®), using a conventional developer or a digital detection system (ChemiDoc, Biorad). Alternatively, fluorescent-labeled secondary antibodies were used and detection was performed thanks to the Odyssey® CLx imaging system (LI-COR). Uncropped scans of immunoblots are supplied in the Supplemental Information section. For immunofluorescence, cells were fixed with 4% Paraformaldehyde (PFA) for 15 minutes. Blocking and permeabilisation were performed in PBS/0. l%Triton, 4% Bovine Serum Albumin (SIGMA®), 4% Donkey Serum (SIGMA®) for 1 hour at room temperature. Subsequently primary antibodies, diluted in blocking buffer were incubated over night at 4°C. Following washes with PBS/0. l%Triton, secondary antibodies was incubated for 1 hour at room temperature, and mounted in Mowiol solution (SIGMA®, 81381).
1.6- Antibodies and reagent
Primary antibodies were from following sources: mouse anti-Cherry (1 : 1000, ab 125096 ABCAM®), mouse anti-Flag (1 : 1000, F3165, SIGMA®), mouse FITC anti-Flag (1 : 1000, F4049, SIGMA®), mouse anti-GAPDH (1 :2000, AM4300, AMBION®), mouse anti-a tubulin (1 :200, clone DM la, CALBIOCHEM®), rabbit anti-HA (1 :2000, H6908, SIGMA®), mouse anti-HA (1 : 1000, #26183, THERMOSCIENTIFIC®), rabbit anti- doublecortin Dbx (1 : 1000, abl8723, ABCAM®), mouse anti-MAP2 (1 : 1000, M4403, SIGMA®), rabbit anti-ATG16Ll (1 : 1000, PM040, MBL), mouse anti-ATG5 (1 : 100 immunostaining, 1 : 1000 immunoblotting, M153-3, MBL®), rabbit anti-LC3B (1 : 1000, L7543, SIGMA®), rabbit anti-p62 (1 : 1000, PM045, MBL®), rabbit anti-Gigaxonin (1 : 1000, sab4200104, SIGMA®), rabbit anti-K48 chain specific (1 : 1000, Apu2, MILLIPORE®), mouse anti-i chains (1 :500, FK1, ENZO®). For the immunoblotting, HRP-secondary antibodies were from the following sources: goat anti-rabbit (1 :5000, #31460, Thermofisher), goat anti-mouse (1 :5000, #31430, Thermofisher), rat anti-mouse IgG (1: 1000, abl31368, Abeam). Fluorescent-labeled secondary antibodies used for immunoblotting were: donkey anti-mouse IRDye 800 CW (1 : 15000, #926-32212, Eurobio), donkey anti-rabbit IRDye 680 RD (1 : 15000, #926-68073, Eurobio). For immunofluorescence, Alexa 488, Alexa 594 and Alexa 647-conjugated secondary antibodies were from Jackson Labs. Lysotracker dye (Molecular probes, L7528, THERMOFISHER®), used to stain lysosome, was applied for 30 minutes at 50 nM concentration. Fluorescence pictures were taken with confocal laser scanning microscope model LSM700 (CARL ZEISS®). 1.7- Co-immunoprecipitation
Protein extracts were prepared from transiently transfected COS cells as described in Cleveland et al. (Hum Mol Genet 18, 1384-1394 (2009)) and in the presence of the proteasome inhibitor MG132 (1748/5, TOCRIS BIOSCIENCE®). Epitope-tagged proteins were immunoprecipitated with anti-Flag and anti-Cherry antibodies as following. Negative controls correspond to Normal Mouse IgG (SC2025, Santa Cruz). Antibodies, linked with 50 mΐ Dynabeads Protein G (10004D, THERMOFISHER®) (3 hours, at room temperature in 500 mΐ PBS tween 0.02%), were incubated with 500 mg of proteins for 2 hours at room temperature in 500 mΐ PBS tween 0.02%. Proteins-Antibody-bead complexes were washed with PBS tween 0.02%, re-suspended in Laemmli IX solution and eluted at 70°C for 10 min. The totality of the sample was loaded on SDS Page gel, while inputs correspond to 50pg of initial protein lysates.
1.8- Ubiquitination assay
In vivo ubiquitination assay of ATG16L1 was performed in COS cells, upon transfection of Cherry-ATG16L1 ± His-ubiquitin constructs and Mgl32 treatment. Whenever mentioned, siRNAs (siRNA) (SEQ ID NO. 13) or mismatch siRNAs (ms) (SEQ ID NO. 14) against the endogenous Gigaxonin were transfected the day before, using Dharmafect 2 reagent, and accordingly to the manufacturer’s instructions (DHARMACON®). Protein extraction and ubiquitin pull down were performed under denaturing conditions, to examine only the covalent binding of ubiquitin onto ATG16L1. Briefly, cells were recovered, washed in PBS IX, and subsequently divided for a direct lysis i) in a 2x laemmli buffer for the input samples and ii) in guanidium buffer for the nickel agarose pull down i) Input samples were further lysed mechanically through a 23 G syringe, boiled at 95 °C and protein concentration was quantified using a BCA kit (PIERCE®), prior to the addition of 0.5M B-mercaptoethanol. ii) Sample lysis and binding of ubiquitinated proteins was performed simultaneously in solution I (6M guanidium-HCl, 0.1M Na2HP04/NaH2P04, 0.01 M Tris-HCl, pH 8.0) supplemented with 5mM imidazole and 10 mM B-mercaptoethanol, and with 50 mΐ of Ni2+-NTA-agarose beads, with overnight agitation at 4°C. Subsequently, different denaturing washes were performed with: solution I supplemented with 10 mM B-mercaptoethanol and 0.1% Triton X-100, solution II (8 M urea, 0.1M Na2HP04/NaH2P04, 0.01 M Tris-HCl, pH 8.0) supplemented with 10 mM B- mercaptoethanol and 0.1% Triton X-100, and solution III (8 M urea, 0.1M Na2HP04/NaH2P04, 0.01 M Tris-HCl, pH 6.3) supplemented with 10 mM B- mercaptoethanol and 0.1% Triton X-100. Elution was performed under agitation for 20 min within 80 mΐ of 200 mM imidazole, 0.15 M Tris-HCl pH 6.7, 30% glycerol, 0.72 M B- mercaptoethanol, 5% SDS. For each sample, equal quantity of the elution was analysed, accordingly to the determination of protein concentration in the respective input samples.
1.9- Proximity ligation assay
Proximity ligation assay was performed according to the manufacturer’s instructions (Duolink In Situ-Fluorescence, SIGMA®). COS cells were transiently transfected with different combinations of plasmids: Ha-ubiquitin with ± Cherry -ATG16 and ± Flag-Gigaxonin. Fixation, blocking and primary antibody incubation were performed as described in the immunofluorescence section. The PLA probes a-mouse minus and a- rabbit plus were diluted and mixed in ratio 1 :5 (except for two negative controls where only one probe was added) in the Duolink in Situ Buffer for 20 minutes at room temperature, and subsequently applied to the cells for 1 hour at 37 °C. After washing with the PLA wash buffer, ligation between the two PLA probes was achieved in the Ligation- Ligase solution, applied for 30 minutes at 37°C. The phase of amplification was performed using the Amplification-Polymerase solution green for 100 minutes at 37°C.
1.10- Statistics analysis
The statistical significance of the difference between experimental groups was determined by the two-tailed 2way ANOVA test (2 quantitative variables: genotype and treatment/time; 1 qualitative factor) with Bonferroni post-hoc test (Figures 10 and 14) or by the Mann Whitney test (Figures 11 and 12), after an assessment of the normality of distribution of the data. Accordingly, either means or medians are represented in the figures, and individual values are shown. The sample sizes (n) are indicated in each figure. Individual measures were normalised to internal controls (tubulin for Figure 9) or background intensity (Figures 11 and 12). In addition, GAN values were normalised to control values in every quantification. As presented in the figures, the differences between experimental groups are significant for * P<0.05; ** P<0.01, *** P<0.001 and **** PO.OOOl. 2- RESULTS
2 1- Gigaxonin interacts with the WD40 domain of ATG16L1
Gigaxonin was previously proposed as a possible partner of ATG16L1, in a study reconstructing the autophagy interaction network. To determine whether this interaction occurs with biological significance, cellular assays were combined for constructs bearing the Cherry-ATG16L1 (Ch-ATG16L1) and Flag-tagged Gigaxonin (Flag-Gig). Strikingly, immunofluorescence of COS cells expressing both constructs revealed that ATG16L1 was degraded upon Gigaxonin expression. Restoring ATG16L1 content using the proteasome inhibitor MG132, or focusing on the residual ATG16L1, evidenced a co-localization between Gigaxonin and ATG16L1.
In order to demonstrate the physical interaction between ATG16L1 and Gigaxonin in COS cells, reverse immunoprecipitation experiments were performed, in which ATG16L1 was stabilized with proteasome inhibitor (Figure 1). To further confirm their direct interaction, a Bimolecular Fluorescence Complementation (BiFC) assay was performed, which relies upon the reconstitution of a fluorescent reporter protein in live cells, as the result of the physical proximity of its complementary fragments upon interaction of the proteins fused to the fragments. This live assay revealed a specific interaction between Gigaxonin and ATG16L1, which was promoted by proteasome inhibition. ATG16L1 is composed of three main structural domains: an N-terminal binding fragment followed by a central self-oligomerization coiled-coil domain (CCD) and a C- terminal WD40 domain (Figure 2). To determine which domain of ATG16L1 interacts with Gigaxonin, co-immunoprecipitation with Flag-tagged ATG16L1 deletion constructs was performed (Figure 3). Ch-Gig was identified in immuno-complexes with the full- length ATG16L1 (F), the ATG16L1-C (C) and the ATG 16L 1 -DN (DN ) proteins, and not the other deletion products, demonstrating a specific interaction of Gigaxonin with the C- terminal WD40 repeats domain of ATG16L1. Similarly, ch-ATG16Ll was pulled down with truncated portions of Gigaxonin to reveal that both BTB and Kelch repeats domains, or alternatively a common domain lying in the linker domain can interact with ATG16L1 (Figures 4 and 5). 2 2- Gigaxonin mediates ubiquitin dependent degradation of ATG16L1
Considering the physical interaction between Gigaxonin and ATG16L1 and the substantial effect in ATG16L1 clearance (see section 2.1 above), the role of the Gigaxonin- E3 ligase in ATG16L1 turn-over was further examined. To overcome possible bias due to transfection, Gigaxonin function in overexpressed ATG16L1 (Cherry-ATG16L1) and on the endogenous ATG16L1 was assessed. While Cherry-ATG16L1 was completely degraded by ectopic Gigaxonin, the inhibition of the proteasome (by MG132) or lysosomal proteolysis (using Bafilomycin Al) restored to some extend Cherry-ATG16L1 levels, as revealed by immunofluorescence and immunoblotting (Figure 6). Importantly, the role of Gigaxonin in mediating the degradation of the endogenous ATG16L1 was confirmed, through the proteasome and autophagy routes (Figure 7). Considering the role of Gigaxonin as a substrate adaptor of Cul3-E3 ubiquitin ligases, we next adressed whether the degradation of ATG16L1 by Gigaxonin is mediated by its ubiquitination activity. Toward this aim, an in vivo ubiquitination assay for ATG16L1 was conducted, and the effect of Gigaxonin was determined by depleting the endogenous Gigaxonin protein. Considering the ability of the WD40 domain of ATG16L1 to transiently bind ubiquitin, we performed the ubiquitin pull down experiments under denaturing conditions. Thus, the covalent ubiquitination of the Cherry-ATG16L1 protein was assessed after transfection with a His-ubiquitin construct in presence or absence of Gigaxonin siRNAs, and the subsequent pull down of ubiquitinated proteins with nickel agarose beads (Figure 8). This experiment demonstrates that while Gigaxonin is not the only E3 ligase to promote ATG16L1 covalent ubiquitination in COS cells, its repression diminishes this PTM, hence providing the direct evidence of the role of Gigaxonin in promoting ATG16L1 ubiquitination. To further confirm this result, colocalisation experiments and Proximity Ligation Assay (PLA) were combined. First, a cell-based analysis for in vivo ubiquitination was performed, by co-transfecting HA-Ubiquitin (Ha-Ub) with Ch-ATG16 ± Flag-Gig. This analysis revealed that while a small proportion of ATG16L1 was ubiquitin positive when expressed alone, all residual ATG16L1 was co-labelled with ubiquitin upon Gigaxonin expression, hence confirming the Gigaxonin-dependent ubiquitination of ATG16L1. Moreover, staining with anti-K48-ubiquitin antibodies revealed a complete colocalisation of this Ub chain with the residual ATG16L1 upon Gigaxonin expression, thereby suggesting that Gigaxonin induces the specific elongation of K48-type ubiquitin chain onto ectopic ATG16L1. Furthermore, a Proximity Ligation Assay (PLA) for ubiquitin and ATG16L1 was performed. Combining a wide range of negative and positive controls, it was further confirmed the interaction between ATG16L1 (exogenous and endogenous) and Gigaxonin, and demonstrated the specificity of the PLA assay. This analysis demonstrated that while a minor pool of ectopic ATG16L1 is decorated with ubiquitin, Gigaxonin dramatically increases this colocalisation. Indeed, it was showed that Gigaxonin enhanced the fluorescence intensity, as a result of an increased proximity between ubiquitin and ATG16L1. Furthermore, the comparison between the PLA and ATG16L1 pattern revealed that while only a sub-population of ATG16L1 is ubiquitinated, the presence of Gigaxonin decorated all ATG16L1 with ubiquitin, hence supporting the dramatic effect of Gigaxonin on ATG16L1 clearance. Importantly, it was also confirmed that Gigaxonin greatly increases the proximity between ubiquitin/poly-ubiquitin chains and the endogenous ATG16L1, hence supporting an activity of Gigaxonin in the poly- ubiquitination of endogenous ATG16L1.
Altogether, our data combine to evidence that the turn-over of ATG16L1 is controlled by Gigaxonin, through its K48-poly-ubiquitination and degradation by the proteasome and autophagy.
2.3- Gigaxonin depletion induces ATG16L1 aggregation in neurons
In light of the important role of Gigaxonin in controlling ATG16L1 degradation, the effect and the significance of this regulation in a physiological context was investigated. For that purpose, a cellular model deficient for Gigaxonin was developed, from the knock-out mouse depleted in the GAN gene, which causes a fatal and recessive neurodegenerative disorder called Giant Axonal Neuropathy in human. In agreement with the above results, ATG16L1 is controlled by Gigaxonin in primary neuronal cells. Examination of MAP2 positive control cells revealed a spatial distribution/biogenesis of the endogenous ATG16L1 over time, which was mostly located in the soma at 4div (days in vitro ) and within neurites at 15div. On the contrary, GAN / neurons exhibited large perinuclear aggregates of ATG16L1 within the soma at 4div, with a pronounced increase in abundance. At 15div, the ATG16L1 aggregates persisted but were not distributed in neurites, as in wild type cells. To assess for the contribution of the proteasome in the impaired degradation of ATG16L1 upon Gigaxonin depletion, the ATG16L1 distribution and abundance were compared in the presence of MG132. Interestingly, in treated controls cells, ATG16L1 was mostly found in neurites at 4div, similarly to older neurons, which suggests a constitutive and active degradation of ATG16L1 locally within neurites in early developmental stages. Strikingly, the GAN / treated neurons were able to respond to some extent to the proteasome inhibition in neurites, but did not exhibit overt exacerbation of ATG16L1 aggregates in the soma, at any time point. Collectively, these data demonstrate that Gigaxonin may constitute the main or possibly the unique E3 ligase for ATG16L1 in the soma. Moreover, proteasome inhibition was not effective in generating ATG16L1 aggregates in the soma of wild type neurons, which emphasizes the potent role of Gigaxonin in degrading ATG16L1 through several routes as demonstrated in this study.
2.4- Gigaxonin alters the elongation complex but not ATG12-ATG5
ATG16L1 is essential in the elongation phase of autophagy. Indeed, it forms a ternary complex with the preformed ATG12-ATG5 conjugate, which constitutes the E3 ligase promoting the LC3 conjugation to the nascent autophagic membrane. Crucial for the recruitment of ATG12-ATG5 to the phagophore, ATG16L1 determines the site of LC3 lipidation and therefore primes the elongation of the autophagosomes. Considering the above results on the ubiquitin-dependent degradation of ATG16L1 by Gigaxonin (see sections 2.1 to 2.3 above) and its aggregation in absence of Gigaxonin, the question was addressed whether the ATG12-ATG5 elongation conjugate was altered in Gigaxonin- depleted neurons. Firstly, Gigaxonin-overexpression did not alter the formation of the ATG12-ATG5 conjugates, nor induce their aggregation in GAN / neurons. These findings further support the specificity of Gigaxonin action on ATG16L1 and recapitulate previous studies showing that neither ATG16L1 overexpression nor repression alters ATG12-ATG5 conjugation. Extremely challenging to evaluate with endogenous proteins in primary neurons, it could not clearly be evidenced that accumulation of ATG16L1 precludes the docking of the elongation conjugate to the membranes, as shown with the decrease of GFP-ATG5 dots upon ATG16L1 modulation. Still, it was observed that LC3 dots were less frequently decorated by endogenous ATG5 in GANG neurons, in comparison to control cells. Altogether, these data pinpoints a unique role of Gigaxonin in controlling the autophagy elongation complex, by degrading ATG16L1. 2.5- GAN neurons show defects in autophagosome synthesis
To directly demonstrate the role of Gigaxonin in the early steps of autophagosome maturation, the different phases of the autophagy pathway in GAN / neurons were investigated. For that purpose, the responsiveness of control and mutant cells was compared to an inducer of autophagy (EBSS) and to a drug blocking the autophagosome-lysosome fusion (Bafilomycin Al). First, the quantification of LC3 lipidation (Figures 9 and 10) and intensity (Figure 11) was measured to ascertain the number of autophagosomes. This analysis revealed that, as control cells, GAN / neurons were able to produce autophagic vesicules in basal condition and upon serum deprivation (Basal and EBBS conditions in Figures 9 and 10). Nevertheless, conditions in which autophagy induction were combined to a blocking of autophagosome-lysosome fusion evidenced a severe alteration of autophagosome synthesis in absence of Gigaxonin. Indeed, control cells were able to further increase autophagosome number when Bafilomycin Al was applied for 6h, while GAN / neurons did not (EBSS+ Baf 6h in Figures 9, 10 and 11). The decrease in the net production of autophagosome synthesis was further confirmed by the inability of Gigaxonin-null neurons to promote LC3 lipidation between 2h and 6h in EBBS and Bafilomycine A condition (EBSS+Baf 6h and EBSS+ Baf 2h in Figures 9 and 10). This analysis was further complemented by examining p62, which is the main selective autophagy receptor and is conventionally used as a marker of effective autophagic degradation. In control cells, p62 accumulated only in Bafilomycin Al treatment, as a result of the impairment of the degradation of autophagosome content by lysosomal enzymes. On the contrary, p62 was shown to accumulate in all conditions in GAN / neurons, hence demonstrating a defect in basal autophagy when Gigaxonin is absent. Considering the findings of an impairment of autophagosome synthesis in GAN / neurons (see Figures 9, 10 and 11), p62 most probably decorates phagophores, which failed to elongate and therefore provoked p62 accumulation. To verify this hypothesis the late stage of autophagosome maturation was examined, and it was observed that, contrary to control neurons, neither LC3 positive membranes (Figure 12) nor p62 aggregates co localized efficiently with lysosomes in basal condition. In addition, in conditions which favour the fusion as a result of autophagy induction in control cells, GAN / neurons were still unable to fuse their p62 structures to lysosomes, hence demonstrating a severe blocking of the maturation of autophagic structures upon Gigaxonin depletion.
Collectively, these data evidence that Gigaxonin depletion alters early steps of autophagy, by impairing autophagosome formation.
2.6- Gigaxonin overexpression restores autophagy in GAN neurons
To confirm that Gigaxonin controls autophagosome production through the regulation of ATG16L1 turn-over in a physiological context, a rescue experiment was performed in GAN / neurons. Using a lentiviral approach, it was observed that Gigaxonin overexpression but not the mock counterpart was able to reduce endogenous levels of ATG16L1 in control cells, further corroborating the control of its abundance by Gigaxonin in primary neurons (Figure 13). Robustly, Gigaxonin totally cleared ATG16L1 bundles in GAN / neurons. It was next confirmed that this rescue on ATG16L1 may be conveyed into a restoration of autophagy flux in GAN / neurons, by demonstrating a restoration of p62 degradation. Thus, these data confirm the action of the Gigaxonin-E3 ligase in ATG16L1 turn-over in primary neurons, but also support the notion of a restoration of autophagosome maturation upon Gigaxonin expression.
Considering that loss of Gigaxonin causes a neurodegenerative disorder, our results suggest that autophagosome membrane elongation is likely to be impaired in patients. While this cannot be assessed in humans, we have generated a neuronal model for GAN to mimick Gigaxonin loss-of-function as a very valuable biological system in determining the contribution of autophagy in the disease. Interestingly, we revealed a progressive neurodegeneration in GAN~/~ cortical neurons at late stage (15div, Figure 14), subsequent to apparition of the autophagy deficits (5div). Thus, targeting ATG16L1 may represent an exciting therapeutic avenue for GAN, but also for other conditions with alterations in the initial steps of autophagy activation, for which targeted approaches are needed to reduce deleterious side effects.
2.7- Gigaxonin depletion causes severe morphological abnormalities
To investigate the function of Gigaxonin in zebrafish development, we conducted first a transient repression approach to enable a dose-response analysis. Thus, we abrogated GAN pre-mRNA splicing with anti-sense morpholino (Mo) oligonucleotides, at the acceptor splice site of exon 3. The effective disruption of splicing was confirmed by RT-PCR in morpholino-injected embryos (called Gig morphants) from lOhpf (hours post fertilization) and from a dose of 0.25 pmol of GAN oligonucleotides. Importantly, increasing doses of morpholino were compared to identify 0.25 pmol as a non-toxic dose that did not perturb the global development and the number of somites at 24hpf, as compared to non-injected embryos.
Injection of GAN morpholino, and not the 5-bp mismatch control morpholino induced significant abnormalities from 48 hpf, as revealed by hematoxylin/eosin staining of whole embryos. Morphants exhibited penetrant and strong morphological phenotypes, including shortened body length, absent yolk extension, pronounced head and eye atrophies and heart defect. Importantly, the developmental deficits of Gig morphants were rescued by co-injection of the human Gig mRNA, hence providing the evidence of the specificity of the morpholino and the functional conservation of Gigaxonin in zebrafish and human. Additionally, we also disrupted Gig mRNA processing with another Mo, targeting the acceptor splice site of exon 2 (Mo ex 1-2), and obtained similar morphological deficits.
2.8- Gigaxonin depletion impairs motility in zebrafish
In regards to the neuronal and muscle expression of Gigaxonin during development, we investigated the motor performances of the Gig morphants, upon touch stimulation and by monitoring their spontaneous motility. This analysis revealed a marked reduction of motility in Gig-depleted animals. The touch response assay, performed at 72hpf revealed swimming abnormalities in 72.4 % of the morphants, with a circular swim (referred to as looping ), circular swim around the axis of the head (referred to as pinwheel) or absence of motion (referred as motion less). To confirm these results and evaluate pure motor capacities, we monitored the spontaneous locomotion of 5dpf (day post-fertilization) old larva. The spontaneous motility of Gig morphants was considerably impaired. While 80% of the morphants did not move, the remaining moved significantly slower and over shorter period than did control larvae.
The specificity of the motility defects for Gigaxonin was demonstrated by the rescue upon co-injection of the human Gig mRNA. 2 9- Gigaxonin is required for the specification of secondary motor neuron
To explore the role of Gigaxonin in sustaining motility in zebrafish, we analysed the two consecutive waves of motor neuron birth in the spinal cord: the primary Motor Neurons (pMN) and secondary Motor Neurons (sMN). Analysis of different key developmental stages using the islet marker revealed a normal proportion of pMN at 20hpf, but a massive reduction of sMN at 36hpf in morphants compared with the control group. The similarities with zebrafish mutants carrying mutations in the Shh pathway prompted us to test whether the decrease in MN number might result from an impaired cell specification in the Gig morphants. For that purpose, we labelled MN progenitors with the Shh target gene Nkx6.1. While the number of Nkx6.1 positive progenitor cells was not notably affected in morphants at 20hpf, Nkx6.1 expression was markedly reduced prior to sMN differentiation from 28hpf onwards.
These data indicate that Gigaxonin depletion inhibits sMN specification through a decrease of Nkx6.1 expression in progenitor cells, as would an inhibition of Shh signalling do during the second wave of MN birth. As a result, the structure of the spinal cord was severely impaired in older embryos, with decreased motor neurons as revealed by ultrastructural examination at 72hpf.
Collectively, our data demonstrate a role of Gigaxonin in controlling the differentiation of secondary motor neurons in the zebrafish spinal cord.
2 10- Gigaxonin controls the axonal pathfinding of primary motor neurons
The absence of sMN in morphants was further confirmed by immunostaining using the specific neuronal cell surface marker Zn8. Thus, zn8+ cells were completely absent in morphants at 56hpf, time of completion of sMN axonogenesis in control embryos. Next, we determined whether Gig-depleted pMN exhibit any defects in axonal pathfmding. At 56hpf, repression of Gigaxonin resulted in a wide range of axonal defects. Concomitant to an increased arborisation of primary motor axons, Gig morphants exhibited an aberrant growth of pMN axons reminiscent of Shh mutants, including an absence or significant shortening of the caudal (CaP) primary axons and misguided axons with ectopic ventral projections. Interestingly, the three-dimensional view of 48hpf morphants, evidenced by LightSheet microscopy revealed additional phenotypes. Indeed, morphants exhibited abnormal protrusions of axons from the spinal cord, and apparent absence of the MiP and RoP motor axons, leading overall to a profound alteration of the structure of the spinal tracts and an increased spacing between the two motor columns.
Importantly, the axonal deficits and the absence of sMN were reproduced by targeting an independent region of the z -Gig mRNA (Mo exl-2), and rescued upon co-injection of the human Gig mRNA, hence demonstrating the specificity of Gig depletion.
2.11- Gigaxonin promotes muscle innervation and somitogenesis
The severity of the defects in both primary motor neurons (pMN) and secondary motor neurons (sMN), together with the locomotion disabilities in morphants prompted us to investigate the integrity of the neuro-muscular junction and muscles. Visualization of acetylcholine receptors using a-bungarotoxin staining revealed a total absence of synapses along the axons at 48hpf, as compared to control embryos. We further analysed the muscle integrity to reveal profound structural abnormalities of muscles trunk somites in Gig morphants at 48hpf. Unlike control zebrafish, which have V-shaped somites and well-organized myofibers, Gig morphants exhibit U-shaped somites with an absence of horizontal myoseptum, and less dense and wavy myofibers very similarly to Shh inactivation. This effect, further reproduced using Mo exl-2 for Gigaxonin was detected as early as 28hpf, concomitantly to the abnormalities of pMN axonal pathfmding, which suggests a dual effect of Gigaxonin in neuronal and muscle development.
Ultrastructural examination of morphant muscles at later stage further revealed a massive alteration of myofiber structure, characterized by a marked shrinking of myofibers, an apparent denser content and an invasion of conjunctive tissue. While control embryos presented a regular hexagonal arrangement of the thick filaments with intercalating thin filaments, morphants exhibited a disorganization of sarcomeres, with a pronounced disparity in the spacing and distribution of the myosin and actin filaments.
2.12- Shh activation restores neuronal specification and somitogenesis deficits in Gigaxonin depleted zebrafish.
Shh signalling is crucial to specify neuronal identity and for somitogenesis. Genetic or pharmacological ablation of the Shh pathways in zebrafish has been shown to abolish motor neuron specification in the spinal cord and to generate U-shaped somites. The striking similarities with our morphants indicate a potential role of Gigaxonin in regulating Shh signalling in zebrafish. A first validation towards a downregulation of Shh activity in GAN was provided by the pronounced reduction in the expression of the Shh responsive target Nkx6.1 gene in the spinal cord of Gig morphants. To further expand on these results, we modulated Shh signalling in zebrafish, to either inhibit or activate it, using cyclopamine and purmorphamine, respectively.
We demonstrated that cyclopamine administration from 14hpf to 28hpf (hours post fertilization) in wild type embryos reproduced fully the phenotype induced by Gigaxonin depletion.
Treated wild type embryos exhibited both an aberrant somitogenesis with U- shaped structures and a total loss of secondary Motor Neurons (sMN), and also resulted in a pinwheel swimming behavior in 80% of the embryos upon mechanical stimulation at 72hpf. Conversely, the elevation of Shh signalling in Gig morphants suppressed the muscle deficits, as shown by the restoration of V-shape somites and denser myofibers. The restoration of MN differentiation was not so robust when the drug was administered during sMN specification, as revealed by the partial rescue of zn8 staining in Gig morphants. Remarkably, the restoration of sMN differentiation in morphants was more efficient when purmorphamine was applied earlier, during the wave of pMN birth, indicating a possible control of the Shh cascade by Gigaxonin from 8hpf.
The decreased abundance of the Shh target Nkx6.1 gene in morphants, together with the similarities of alterations between our and Shh defective models, indicate that Gigaxonin functions as a positive modulator of Shh signalling in zebrafish, to promote both neuronal and muscle development.
2 13- Gigaxonin acts as a positive regulator of the Shh signalling.
To directly demonstrate that Gigaxonin acts on the Shh pathway, we studied the activity of the Shh pathway in three independent biological systems, where Gigaxonin expression was ablated.
Firstly, we analysed Shh signalling in the GAN morphants and the GANdel/del line, by performing an in situ hybridisation for the expression pattern of ptch2 , a direct target of Shh signalling and a well-established indicator of the Shh pathway activation. In the spinal cords of zebrafish embryos at 32hpf, ptch2 displays a ventral-high dorsal-low pattern of expression. In conditions where Gigaxonin expression was reduced, either through MO knock-down or genetic ablation, the expression of ptch2 in the spinal cord was dramatically decreased, hence evidencing an inhibition of Shh signalling in absence of Gigaxonin.
Secondly, we knocked down Gigaxonin with RNAi in Shh-Light2 cells, an NIH-3T3 cell line that is stably expressing a Gli-dependent luciferase reporter and becomes activated upon Shh stimulation. In this study, we demonstrate that while Shh alone increased the activity of the luciferase by 19-fold, the decrease in Gigaxonin levels diminished this effect by 22%. Importantly, this effect is only significant in the presence of the morphogen, and not in basal condition, which indicate an effect specifically upon activation of the Shh signalling.
Thirdly, we analysed primary fibroblasts derived from GAN patients, which represent a well-established cellular model for the human pathology (Bomont and Koenig, 2003; Cleveland et ah, 2009). We selected independent primary fibroblasts, carrying different mutation types representative of the pathology: large deletion (GANAexllM 1), and a missense mutation (GANA49E) for which Gigaxonin was shown to be dramatically reduced (Boizot et ah, 2014). Conveniently, human primary fibroblasts present primary cilium, the antenna for Shh signalling in vertebrates, which can be easily detected. Not only Shh pathway relies on the presence of cilium for signal transduction, downstream targets of Shh have also been shown to increase ciliary length by the regulation of the actin cytoskeleton (Bershteyn et ah, 2010) possibly in a negative feedback loop to attenuate the signalling (Fleet et ah, 2016). We assessed the ability of primary fibroblasts to respond to Shh signalling, by comparing the effects of Shh induction on cilium length between control and mutant samples. Interestingly, whilst the control human fibroblasts exposed to Shh increase the cilia length by 29%, the ciliary length of the independent GAN fibroblasts was not altered upon Shh addition, hence showing their inability to respond to the Shh activation.
Altogether, we demonstrate that the Shh pathway fails to activate properly in absence of Gigaxonin in zebrafish, mouse and human systems, hence providing substantial evidence for a critical role of Gigaxonin in promoting Shh induction. 2.14- Gigaxonin acts together with Shh to degrade the receptor Ptch and initiate the signaling cascade.
To gain insight into the molecular mechanism by which the Gigaxonin-E3 ligase positively controls the Shh signaling, we directly addressed whether Gigaxonin can act together with Shh to target Ptch for degradation, and therefore initiate the Shh pathway.
Using both loss- and gain-of-function methodologies, we investigated the levels of endogenous Ptch in the presence or absence of Shh. Overexpressing Gigaxonin, with or without Shh, provided evidence that endogenous Ptch could only be efficiently degraded when both Gigaxonin and Shh were added to the system. Conversely, reduction of the levels of endogenous Gigaxonin with RNA interference revealed an increase in the abundance of endogenous Ptch, which was exacerbated when Shh was present.
From these results, we hypothesized that Gigaxonin might interact with Ptch. Indeed, using a co-immunoprecipitation (co-IP) assay in Light2 cells, we detected the presence of endogenous Ptch in the Gig complex. We confirmed these results by overexpressing both Cherry-Ptch and Flag-Gig in COS cells and performing co-IP in both directions. Interestingly, we observed multiple bands with Cherry-Ptch in Gigaxonin immunocomplex, resembling the laddering characteristic of ubiquitination. We confirmed this by co-labeling the Cherry-Ptch signal with anti-Ubiquitin antibody exhibiting specific Lysine-48 linkage antibody, hence identifying poly-ubiquitinated Ptch in Gigaxonin complex. The comparison with Ptch immunocomplex was also informative. Beyond the confirmation of the interaction of both proteins, the absence of laddering of Ptch and the weaker pull down of Gigaxonin revealed that while Gigaxonin does not interact with the entire pool of Ptch within the cell, Gigaxonin complex is significantly enriched in ubiquitinated Ptch.
Collectively, our data demonstrate that Gigaxonin acts positively on the Shh pathway, through the interaction with the Ptch receptor and a Shh-dependent targeting for degradation.
3- DISCUSSION
The ATG16L1 protein orchestrates the two UBL systems required for membrane elongation during autophagosome formation, but its control and dynamics are unknown. In this study, we identify the Gigaxonin-E3 ligase as the first regulator of ATG16L1 turn-over, essential in ensuring ATG16L1 functions in phagophore expansion (Figure 15). First, Gigaxonin interacts with the C-terminal WD40 repeats domain of ATG16L1 to promote its ubiquitination and degradation by the proteasome and the autophagy pathways. Second, Gigaxonin depletion induces a massive aggregation of ATG16L1 in primary neurons and inhibits autophagy flux by impairing phagophore elongation. These data suggest a pivotal role of the Gigaxonin-E3 ligase in controlling autophagosome formation, hence ensuring the fine-tuning of the activation of autophagy (Figure 15).
Up to now, no E3 ligase was shown to act on membrane elongation and no ubiquitin PTM has been identified for ATG16L1. Only one polymorphic variant of ATG16L1, associated to the inflammatory Crohn’s disease, has shown an increased sensitivity to caspase-3 -mediated processing, which can be modulated by phosphorylation during inflammation. The regulation of ATG16L1 by Gigaxonin is highly robust, as revealed by the complete ubiquitination and clearance of ATG16L1, and the absence of increased ATG16L1 aggregation upon proteasome inhibition in GAN / neuronal cells.
It emerges from the data obtained herein that an impairment of autophagosome production is observed in absence of Gigaxonin, which causes defective autophagic degradation with decreased fusion to lysosomes and p62 accumulation. The specificity and efficacy of Gigaxonin was further corroborated in a rescue experiment showing not only a clearance of ATG16L1 upon Gigaxonin overexpression in control and mutant neurons but also a restoration of p62 degradation in GAN cells. Strikingly, the phenotypes are similar to the effects caused by the alterations of ATG16L1 and WIPI2 (a protein which allows ATG16L1 localisation to the Ptdlns3P generated on membranes upon autophagy initiation). Indeed, autophagosome formation was impaired upon WIPI2 depletion and ATG16L1 overexpression, but also ATG16L1 depletion. It is likely that the level of lipidated LC3 that was observed in basal condition in GAN / cells reflects an accumulation of incomplete autophagosomes. Indeed, a similar LC3-II increase was already reported and was associated with the presence of phagophores and open autophagosomes in ATG16L1 / MEF cells on electron micrographs. Thus, it is here revealed the mechanism of regulation on ATG16L1 that controls the early steps of phagophore elongation. In absence of Gigaxonin, ATG16L1 accumulates and would, as previously reported, exert a dominant effect on the localisation of ATG12-ATG5 to the membranes, without affecting the formation of the conjugate.
The localisation of the ATG16L1 bundles within GAN / neurons was intriguing. Indeed, ATG16L1 bundles are mostly present in a perinuclear position in the soma, and while not totally absent, rarely seen within neurites. Autophagosome biogenesis has been shown to be spatially regulated along the axons; it occurs distally in the neurite tip and the autophagosome matures and fuse to the lysosome while undergoing a retrograde transport towards the soma. Still, some autophagosome formation was observed in the soma, showing specificity in their maturation, localisation and dynamics (Figure 15). While several functions of axonal autophagy have been identified, including axonal homeostasis and presynaptic functions, nothing is known about its role in the soma. Thus, Gigaxonin will contribute to deciphering the meaning of the high degree of compartimentalisation of autophagy within the neuron, and its function in cell survival. Indeed, Gigaxonin loss-of-function causes widespread neuronal death throughout the nervous system in Giant Axonal Neuropathy patients, and a progressive degeneration of GAN cortical neurons.
Neurodegeneration is one of the many different afflictions that autophagy impairment can cause to human. Indeed, autophagy is altered in a wide number of conditions, including cancer, myopathies, immune and neurodegenerative diseases. While most of the mutated genes are not autophagic components per se, a growing body of evidence shows that they play additional roles in regulating autophagy. Among them, GAN represents one of the few disease-associated genes, which encode for proteins that are either autophagic core components (WIPI4, ATG16L1) or their regulator (Gigaxonin), causing respectively encephalopathy, Crohn’s disease and Giant Axonal Neuropathy. The data provided herein show that Gigaxonin acts by poly-ubiquitinating ATG16L1 with K48 chains, which leads to its degradation, but do not exclude other (non)ubiquitin dependent control by Gigaxonin, which could regulate ATG16L1 trafficking within neurons. This further adds to the emerging concept of cross-talk between the Ubiquitin Proteasome System and the autophagy pathway. These data pinpoints the first E3 ligase to date that controls the membrane elongation step, hence unveiling a molecular switch to fine-tune the activation of autophagy. In this study, we further combined physiological evidences, cellular assays and biochemical data to demonstrate that the Gigaxonin-E3 ligase is a positive regulator of the Shh pathway. Furthemore, we showed that Gigaxonin interacts with endogenous Ptch and that its degradation is potentiated by the presence of Shh, hence placing Gigaxonin as the first E3 ligase enabling the receiving cells to interpret the morphogen signal. Indeed, the E3 ligases Itchy and Smurf act on Ptch to control the basal turnover of the receptor in absence of the morphogen (44), or unbound Ptch as a mechanism to turn-off Shh signaling, respectively. Controlling the degradation of the Ptch receptor, the upstream components of the cascade, and in a Shh dependant manner, we propose Gigaxonin-E3 ligase as a key molecular switch to the initiation of the signal transduction mediated by Shh. At an organism level, this Gig-mediated regulatory mechanism is essential to specify both neuronal and muscle patterning, to sustain locomotor activity in vivo.
In this study, we also show that Gigaxonin function is evolutionary conserved in vertebrates. Indeed, we demonstrate that, as in human, repression of Gigaxonin in zebrafish leads to a loss of motor neurons in the spinal cord, severe axonal defects and the abolishment of locomotion, phenotypes all reversed upon co-injection of the human Gig transcripts.
GAN is an infantile neurodegenerative disease characterised by a loss of neurons both in the spinal cord and the brain, however, its mechanisms are not understood. Our study provides the first evidences of a link between GAN and Shh signaling, and identifies in the zebrafish, neurodevelopmental defects as a cause of the cellular and physiological markers of the pathology. Thus, GAN adds up to the emerging concept whereby neurodevelopmental deficits disrupt homestasis and generate vulnerability that further evolve in adult stages towards clinical manifestations, as exemplified in Huntington’s disease. While this suggests that the GAN pathology has a neurodevelopmental component in human, the roles played by the Shh pathway in the adult nervous system could also be of relevance in the disease progression.
In light of the therapeutic benefits obtained by modulating the Shh pathway in several neurodegenerative diseases, our study may also offer a specific target for therapeutic intervention aimed at reactivating neurogenesis in disease; and hence for the treatment or prevention of neurodegenerative diseases. While this study provides important insights into the control of the Shh pathway by Gigaxonin within the motor system, it is possible to extend this to other modalities, within and outside the nervous system. Indeed, Gigaxonin is enriched in the nervous system and during prenatal stages, but its expression is ubiquitous. Accordingly, Shh signaling has been implicated in the development of multiple non-neuronal tissues, including skin, hair, mammary gland, stomach and kidney, all of which are reported to be affected in GAN patients.
In conclusion, this study opens an exciting new avenue for a role of the Shh pathway in adulthood. Indeed, in the mammalian adult brain, Shh signaling has emerged as an important neuromodulator through different mechanisms, including the proliferation of postnatal neural stem cells and fate specification.
LISTING OF SEQUENCES
Figure imgf000077_0001
REFERENCES
- Bale. Hedgehog Signaling and Human Disease. Annu. Rev. Genomics Hum. Genet. 2002. 3:47-65.
- Bomont P, Cavalier L, Blondeau F, Ben Hamida C, Belal S, Tazir M, Demir E, Topaloglu H, Korinthenberg R, Tiiysiiz B, Landrieu P, Hentati F, Koenig M. The gene encoding gigaxonin, a new member of the cytoskeletal BTB/kelch repeat family, is mutated in giant axonal neuropathy. Nat Genet. 2000 Nov; 26(3):370-4.
- Bomont P, Koenig M. Intermediate filament aggregation in fibroblasts of giant axonal neuropathy patients is aggravated in non dividing cells and by microtubule destabilization. Hum Mol Genet. 2003 Apr 15; 12(8): 813-22.
- Chen R, Li X, He B, Hu W. MicroRNA-410 regulates autophagy -related gene ATG16L1 expression and enhances chemosensitivity via autophagy inhibition in osteosarcoma. Mol Med Rep. 2017 Mar;15(3): 1326-1334. doi: 10.3892/mmr.2017.6149.
- Chen, X., Morales- Alcala, C.C., Riobo-Del Galdo, N.A., 2018. Autophagic Flux Is Regulated by Interaction Between the C-terminal Domain of PATCHED 1 and ATG101. Mol. Cancer Res. MCR 16, 909-919. https://doi.org/10.1158/1541-7786.MCR-17-0597
- Cleveland DW, Yamanaka K, Bomont P. Gigaxonin controls vimentin organization through a tubulin chaperone-independent pathway. Hum Mol Genet. 2009 Apr 15; 18(8): 1384-94. doi: 10.1093/hmg/ddp044.
- Fujita N, Itoh T, Omori H, Fukuda M, Noda T, Yoshimori T. The Atgl6L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy. Mol Biol Cell. 2008 May; 19(5):2092-100. doi: 10.1091/mbc.E07-12-1257.
- Gan J, Cai Q, Qu Y, Zhao F, Wan C, Luo R, Mu D. miR-96 attenuates status epilepticus-induced brain injury by directly targeting Atg7 and Atgl6Ll. Sci Rep. 2017 Aug 31 ;7(1): 10270. doi: 10.1038/s41598-017-10619-0.
- Ganay T, Boizot A., Burrer R, Chauvin JP, Bomont P. Sensory-motor deficits and neurofilament disorganization in gigaxonin-null mice. Mol Neurodegene (2011) 12, 25.
- Kuhlenbaumer G, Timmerman V, Bomont P. Giant Axonal Neuropathy. GeneReviews [internet] 2003 Jan 9, Seattle (WA), University of Washington, Seattle, 1993-2018. - Lippai M, Szatmari Z. Autophagy-from molecular mechanisms to clinical relevance. Cell Biol Toxicol. 2017 Apr;33(2): 145-168. doi: 10.1007/sl0565-016-9374-5.
- Li QX, Zhou X, Huang TT, Tang Y, Liu B, Peng P, Sun L, Wang YH, Yuan XL. The Thr300Ala variant of ATG16L1 is associated with decreased risk of brain metastasis in patients with non-small cell lung cancer. Autophagy, 2017;13(6): 1053-1063. doi: 10.1080/15548627.2017.1308997.
- Mahammad S, Murthy SN, Didonna A, Grin B, Israeli E, Perrot R, Bomont P, Julien JP, Kuczmarski E, Opal P, Goldman RD. Giant axonal neuropathy-associated gigaxonin mutations impair intermediate filament protein degradation. J Clin Invest. 2013 May; 123(5): 1964-75. doi: 10.1172/JCI66387.
- Menzies FM, Fleming A, Rubinsztein DC. Compromised autophagy and neurodegenerative diseases. Nat Rev Neurosci. 2015 Jun;16(6):345-57. doi: 10.1038/nm3961.
- Mizushima N, Komatsu M. Autophagy: renovation of cells and tissues. Cell. 2011 Nov 11;147(4):728-41. doi: 10.1016/j cell.2011.10.026.
- Sambrook and Green. Molecular Cloning: A Laboratory Manual (Fourth Edition); 2012; Cold Spring Harbor Laboratory Press)
- Schneider JL, Cuervo AM. Autophagy and human disease: emerging themes. Curr Opin Genet Dev. 2014 Jun;26: 16-23. doi: 10.1016/j.gde.2014.04.003.
- Scrivo A, Bourdenx M, Pampliega O, Cuervo AM. Selective autophagy as a potential therapeutic target for neurodegenerative disorders. Lancet Neurol. 2018 Sep;17(9):802- 815.
- Settembre C, Fraldi A, Rubinsztein DC, Ballabio A. Lysosomal storage diseases as disorders of autophagy. Autophagy. 2008 Jan;4(l): 113-4.
- Vomero M, Barbati C, Colasanti T, Perricone C, Novelli L, Ceccarelli F, Spinelli FR, Di Franco M, Conti F, Valesini G, Alessandri C. Autophagy and Rheumatoid Arthritis: Current Knowledges and Future Perspectives. Front Immunol. 2018 Jul 18;9: 1577. doi: 10.3389/fimmu.2018.01577.

Claims

1. A modulator of the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ATG16Ll interaction and/or of the functionality of the Gigaxonin/ATG16Ll interaction for use as a medicament.
2. The modulator for use according to claim 1, in a method for the prevention and/or the treatment of a disease associated with altered autophagy.
3. The modulator for use according to claim 1, in a method for the prevention and/or the treatment of a disease associated with altered Sonic Hedgehog (Shh) transduction.
4. The modulator for use according to claim 2, wherein the disease associated with altered autophagy is selected in a group consisting of a cancer, an immune disease, an infectious disease, a metabolic disease, a cardiovascular disease, a (cardio)myopathy, a lysosomal disease, spinal cord injury and trauma, a neurodegenerative disease and a pulmonary disease.
5. The modulator for use according to any one of claims 1 to 4, wherein said modulator is selected in a group comprising Gigaxonin, an activator of Gigaxonin expression, an inhibitor of Gigaxonin expression, an activator of Gigaxonin stability, an inhibitor of Gigaxonin stability, an activator of Gigaxonin degradation, an inhibitor of Gigaxonin degradation, an activator of Gigaxonin activity and an inhibitor of Gigaxonin activity, and is preferably Gigaxonin.
6. The modulator for use according to any one of claims 1 to 5, wherein Gigaxonin is:
- a polypeptide expressed by a nucleic acid comprising a nucleic acid sequence having at least 75% identity with SEQ ID NO. 1; or
- a polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2
7. The modulator for use according to any one of claims 1 to 6, wherein the inhibitor of Gigaxonin expression is a siRNA, a miRNA or a piRNA that binds to a RNA complementary to a nucleic acid having at least 75% identity with SEQ ID NO. 1.
8. The modulator for use according to any one of claims 1 to 7, wherein the inhibitor of Gigaxonin activity is an antibody or an aptamer that binds to a polypeptide having at least 75% identity with SEQ ID NO. 2.
9. The modulator for use according to claim 8, wherein the antibody or aptamer binds to a polypeptide of SEQ ID NO. 2.
10. A pharmaceutical composition comprising:
- (i) a modulator of the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ ATG16L1 interaction and/or of the functionality of the Gigaxonin/ ATG16L1 interaction, and
- (ii) a pharmaceutically acceptable vehicle.
11. A pharmaceutical composition according to claim 10, for use in a method for the prevention and/or the treatment of a disease associated with altered autophagy.
12. A method for screening a modulator of the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ ATG16L1 interaction and/or of the functionality of the Gigaxonin/ ATG16L1 interaction, comprising the steps of:
- (a) providing a system comprising Gigaxonin and ATG16L1 in suitable conditions for the production of the corresponding proteins and for the interaction between Gigaxonin and ATG16L1 to occur;
- (b) providing to the said system a candidate modulator of the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ ATG16L1 interaction and/or of the functionality of the Gigaxonin/ ATG16L1 interaction;
- (c) measuring the level of expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ ATG16L1 interaction and/or functionality of the Gigaxonin/ ATG16L1 interaction in the system at step (b);
- (d) determining a difference between the level measured in step (c) and a corresponding reference level obtained in the absence of the said candidate modulator.
13. A method for assessing the efficiency of a candidate treatment of a disease associated with altered autophagy in an individual in need thereof, comprising the steps of:
- (a) measuring the level of Gigaxonin, ATG16L1, the Gigaxonin/ ATG16L1 interaction and/or the functionality of the Gigaxonin/ ATG16L1 interaction in a sample from said individual in need thereof, in suitable conditions for the interaction to occur;
- (b) providing the said individual with a candidate treatment; - (c) measuring the level of Gigaxonin, ATG16L1, the Gigaxonin/ATG16Ll interaction and/or the functionality of the Gigaxonin/ ATG16L1 interaction in a sample obtained at step (b);
- (d) determining a difference between the level measured in step (a) and the level measured in step (c).
14. A method to activate autophagy in a target cell, comprising a step of increasing the level of expression, stability of Gigaxonin, decreasing its degradation and/or increasing activity of Gigaxonin and/or Gigaxonin/ ATG16L1 interaction to reach physiological functions of ATG16L1.
15. The method according to claim 14, wherein increasing the level of expression of Gigaxonin comprises the step of providing the target cell with (i) a nucleic acid encoding a Gigaxonin polypeptide or (ii) a Gigaxonin polypeptide, or (iii) siRNAs or (iv) miRNAs or (v) piRNAs that specifically bind to a negative regulator of Gigaxonin expression or (vi) transcription factors and/or co-activators that bind to the Gigaxonin promoter to mediate transcription of the GAN gene.
16. A method to inhibit autophagy in a target cell, comprising a step of decreasing the level of expression, stability of Gigaxonin, increasing its degradation and/or decreasing activity of Gigaxonin and/or Gigaxonin/ ATG16L1 interaction to decrease physiological functions of ATG16L1.
17. A method for the fine-tuned activation or inhibition of autophagy in a target cell comprising the step of modulating the expression, stability, degradation and/or activity of Gigaxonin, of the Gigaxonin/ ATG16L1 interaction and/or of the functionality of the Gigaxonin/ ATG16L1 interaction.
18. A polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2; or a nucleic acid coding for said polypeptide; for use as a medicament.
19. The polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2; or a nucleic acid coding for said polypeptide; for use according to the preceding claim, in a method for treating and/or preventing a neurodegenerative disease.
20. The polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2; or a nucleic acid coding for said polypeptide; for use according to the preceding claim, in a method for treating and/or preventing a neurodegenerative disease associated with altered motor activity.
21. The polypeptide comprising a polypeptide sequence having at least 75% identity with SEQ ID NO. 2; or a nucleic acid coding for said polypeptide; for use according to the claim 19, in a method for treating and/or preventing a neurodegenerative disease associated with altered autophagy or altered Sonic Hedgehog (Shh) transduction.
22. A pharmaceutical composition comprising a polypeptide or a nucleic acid according to claim 18.
23. The pharmaceutical composition according to claim 22; for use in a method for treating and/or preventing a neurodegenerative disease.
24. The pharmaceutical composition according to claim 23; for use in a method for treating and/or preventing a neurodegenerative disease associated with altered motor activity.
25. The pharmaceutical composition according to claim 23; for use in a method for treating and/or preventing a neurodegenerative disease associated with altered autophagy.
26. The pharmaceutical composition according to claim 23; for use in a method for treating and/or preventing a neurodegenerative disease associated with altered Sonic Hedgehog (Shh) transduction.
PCT/EP2019/085971 2018-12-18 2019-12-18 Compounds for use in the treatment of diseases associated with altered autophagy, altered shh transduction, and/or neurodegenerative diseases WO2020127498A1 (en)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
EP18306732.1 2018-12-18
EP18306732 2018-12-18
EP19305189 2019-02-15
EP19305189.3 2019-02-15
EP19193467 2019-08-23
EP19193467.8 2019-08-23

Publications (1)

Publication Number Publication Date
WO2020127498A1 true WO2020127498A1 (en) 2020-06-25

Family

ID=69061354

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2019/085971 WO2020127498A1 (en) 2018-12-18 2019-12-18 Compounds for use in the treatment of diseases associated with altered autophagy, altered shh transduction, and/or neurodegenerative diseases

Country Status (1)

Country Link
WO (1) WO2020127498A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112226434A (en) * 2020-09-24 2021-01-15 中山大学附属第六医院 Application of siRNA for inhibiting GAN gene expression

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013063309A1 (en) * 2011-10-25 2013-05-02 Indiana University Research & Technology Corporation Gigaxonin fusion protein and methods for treating giant axonal neuropathy

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013063309A1 (en) * 2011-10-25 2013-05-02 Indiana University Research & Technology Corporation Gigaxonin fusion protein and methods for treating giant axonal neuropathy

Non-Patent Citations (36)

* Cited by examiner, † Cited by third party
Title
"Genbank", Database accession no. NM_001363742.1
"METHODS IN ENZYMOLOGY", vol. 569, 1 January 2016, ACADEMIC PRESS, US, ISSN: 0076-6879, article PASCALE BOMONT ET AL: "Degradation of the Intermediate Filament Family by Gigaxonin", pages: 215 - 231, XP055593809, DOI: 10.1016/bs.mie.2015.07.009 *
ALEXIA BOIZOT ET AL: "The instability of the BTB-KELCH protein Gigaxonin causes Giant Axonal Neuropathy and constitutes a new penetrant and specific diagnostic test", ACTA NEUROPATHOLOGICA COMMUNICATIONS, BIOMED CENTRAL LTD, LONDON, UK, vol. 2, no. 1, 24 April 2014 (2014-04-24), pages 47, XP021185448, ISSN: 2051-5960, DOI: 10.1186/2051-5960-2-47 *
ANDREW THORBURN: "Autophagy and disease", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 293, no. 15, 30 November 2017 (2017-11-30), US, pages 5425 - 5430, XP055594031, ISSN: 0021-9258, DOI: 10.1074/jbc.R117.810739 *
AURORA SCRIVO ET AL: "Gigaxonin E3 ligase governs ATG16L1 turnover to control autophagosome production", NATURE COMMUNICATIONS, vol. 10, no. 1, 15 February 2019 (2019-02-15), XP055593792, DOI: 10.1038/s41467-019-08331-w *
BALE ET AL.: "Hedgehog Signaling and Human Disease", ANNU. REV. GENOMICS HUM. GENET, vol. 3, 2002, pages 47 - 65
BALE: "Hedgehog Signaling and Human Disease", ANNU. REV. GENOMICS HUM. GENET., vol. 3, 2002, pages 47 - 65
BOMONT ET AL., NAT GENET, 2000
BOMONT PCAVALIER LBLONDEAU FBEN HAMIDA CBELAL STAZIR MDEMIR ETOPALOGLU HKORINTHENBERG RTIIYSIIZ B: "The gene encoding gigaxonin, a new member of the cytoskeletal BTB/kelch repeat family, is mutated in giant axonal neuropathy", NAT GENET., vol. 26, no. 3, November 2000 (2000-11-01), pages 370 - 4, XP001156497, DOI: 10.1038/81701
BOMONT PKOENIG M: "Intermediate filament aggregation in fibroblasts of giant axonal neuropathy patients is aggravated in non dividing cells and by microtubule destabilization", HUM MOL GENET., vol. 12, no. 8, 15 April 2003 (2003-04-15), pages 813 - 22, XP055166900, DOI: 10.1093/hmg/ddg092
BOMONTKOENIGCLEVELAND ET AL., HUM MOL GENET, vol. 12, 2003, pages 813 - 822
CHEN RLI XHE BHU W: "MicroRNA-410 regulates autophagy-related gene ATG16L1 expression and enhances chemosensitivity via autophagy inhibition in osteosarcoma", MOL MED REP., vol. 15, no. 3, March 2017 (2017-03-01), pages 1326 - 1334
CHEN, X.MORALES-ALCALA, C.C.RIOBO-DEL GALDO, N.A.: "Autophagic Flux Is Regulated by Interaction Between the C-terminal Domain of PATCHED 1 and ATG101", MOL. CANCER RES. MCR, vol. 16, 2018, pages 909 - 919, Retrieved from the Internet <URL:https://doi.org/10.1158/1541-7786.MCR-17-0597>
CLEVELAND DWYAMANAKA KBOMONT P: "Gigaxonin controls vimentin organization through a tubulin chaperone-independent pathway", HUM MOL GENET., vol. 18, no. 8, 15 April 2009 (2009-04-15), pages 1384 - 94, XP055166905, DOI: 10.1093/hmg/ddp044
CLEVELAND ET AL., HUM MOL GENET, vol. 18, 2009, pages 1384 - 1394
FUJITA ET AL., MOL BIOL CELL, vol. 19, 2008, pages 2092 - 2100
FUJITA NITOH TOMORI HFUKUDA MNODA TYOSHIMORI T: "The Atgl6L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy", MOL BIOL CELL, vol. 19, no. 5, May 2008 (2008-05-01), pages 2092 - 100
GAN JCAI QQU YZHAO FWAN CLUO RMU D: "miR-96 attenuates status epilepticus-induced brain injury by directly targeting Atg7 and Atgl6Ll", SCI REP., vol. 7, no. 1, 31 August 2017 (2017-08-31), pages 10270
GANAY TBOIZOT A.BURRER RCHAUVIN JPBOMONT P: "Sensory-motor deficits and neurofilament disorganization in gigaxonin-null mice", MOL NEURODEGENE, vol. 12, 2011, pages 25
JASON LOWERY ET AL: "Abnormal intermediate filament organization alters mitochondrial motility in giant axonal neuropathy fibroblasts", MOLECULAR BIOLOGY OF THE CELL, vol. 27, no. 4, 15 February 2016 (2016-02-15), US, pages 608 - 616, XP055593802, ISSN: 1059-1524, DOI: 10.1091/mbc.E15-09-0627 *
KUHLENBAUMER GTIMMERMAN VBOMONT P: "GeneReviews", 9 January 2003, UNIVERSITY OF WASHINGTON, article "Giant Axonal Neuropathy"
KULENBAUMERTIMMERMANBOMONT, GENEREVIEW, 2018
LI QXZHOU XHUANG TTTANG YLIU BPENG PSUN LWANG YHYUAN XL: "The Thr300Ala variant of ATG16L1 is associated with decreased risk of brain metastasis in patients with non-small cell lung cancer", AUTOPHAGY, vol. 13, no. 6, 2017, pages 1053 - 1063
LIPPAI MSZATMARI Z: "Autophagy-from molecular mechanisms to clinical relevance", CELL BIOL TOXICOL., vol. 33, no. 2, April 2017 (2017-04-01), pages 145 - 168, XP036184974, DOI: 10.1007/s10565-016-9374-5
MAHAMMAD SMURTHY SNDIDONNA AGRIN BISRAELI EPERROT RBOMONT PJULIEN JPKUCZMARSKI EOPAL P: "Giant axonal neuropathy-associated gigaxonin mutations impair intermediate filament protein degradation", J CLIN INVEST., vol. 123, no. 5, May 2013 (2013-05-01), pages 1964 - 75, XP055166507, DOI: 10.1172/JCI66387
MENZIES FMFLEMING ARUBINSZTEIN DC: "Compromised autophagy and neurodegenerative diseases", NAT REV NEUROSCI., vol. 16, no. 6, June 2015 (2015-06-01), pages 345 - 57
MIZUSHIMA NKOMATSU M: "Autophagy: renovation of cells and tissues", CELL, vol. 147, no. 4, 11 November 2011 (2011-11-11), pages 728 - 41, XP028109261, DOI: 10.1016/j.cell.2011.10.026
R.D. GOLDMANP. OPAL, J CLIN INVEST, vol. 123, 2013, pages 1964 - 1975
SAMBROOKGREEN: "Molecular Cloning: A Laboratory Manual", 2012, COLD SPRING HARBOR LABORATORY PRESS
SCHNEIDER JLCUERVO AM: "Autophagy and human disease: emerging themes", CURR OPIN GENET DEV., vol. 26, June 2014 (2014-06-01), pages 16 - 23
SCRIVO ABOURDENX MPAMPLIEGA OCUERVO AM: "Selective autophagy as a potential therapeutic target for neurodegenerative disorders", LANCET NEUROL., vol. 17, no. 9, September 2018 (2018-09-01), pages 802 - 815, XP085443991, DOI: 10.1016/S1474-4422(18)30238-2
SETTEMBRE CFRALDI ARUBINSZTEIN DCBALLABIO A: "Lysosomal storage diseases as disorders of autophagy", AUTOPHAGY, vol. 4, no. l, January 2008 (2008-01-01), pages 113 - 4, XP009159089, DOI: 10.4161/auto.5227
VOMERO MBARBATI CCOLASANTI TPERRICONE CNOVELLI LCECCARELLI FSPINELLI FRDI FRANCO MCONTI FVALESINI G: "Autophagy and Rheumatoid Arthritis: Current Knowledges and Future Perspectives", FRONT IMMUNOL., vol. 9, 18 July 2018 (2018-07-18), pages 1577
YAN LI ET AL: "Mir223 restrains autophagy and promotes CNS inflammation by targeting ATG16L1", AUTOPHAGY, vol. 15, no. 3, 22 September 2018 (2018-09-22), US, pages 478 - 492, XP055593828, ISSN: 1554-8627, DOI: 10.1080/15548627.2018.1522467 *
YOAN ARRIBAT ET AL: "Sonic Hedgehog repression underlies gigaxonin mutation-induced motor deficits in giant axonal neuropathy", JOURNAL OF CLINICAL INVESTIGATION, vol. 129, no. 12, 28 October 2019 (2019-10-28), GB, pages 5312 - 5326, XP055673966, ISSN: 0021-9738, DOI: 10.1172/JCI129788 *
ZILI ZHAI ET AL: "Human autophagy gene ATG16L1 is post-transcriptionally regulated by MIR142-3p", AUTOPHAGY, vol. 10, no. 3, 6 January 2014 (2014-01-06), US, pages 468 - 479, XP055593827, ISSN: 1554-8627, DOI: 10.4161/auto.27553 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112226434A (en) * 2020-09-24 2021-01-15 中山大学附属第六医院 Application of siRNA for inhibiting GAN gene expression
CN112226434B (en) * 2020-09-24 2023-01-13 中山大学附属第六医院 Application of siRNA for inhibiting GAN gene expression

Similar Documents

Publication Publication Date Title
Mishra et al. Functional role of the interaction between polysialic acid and extracellular histone H1
Lee et al. Enhanced Ca2+-dependent glutamate release from astrocytes of the BACHD Huntington's disease mouse model
Löw et al. Netrin-1 is a novel myelin-associated inhibitor to axon growth
Groeneweg et al. Gephyrin: a key regulatory protein of inhibitory synapses and beyond
Ivanova et al. Endo-lysosomal dysfunction in human proximal tubular epithelial cells deficient for lysosomal cystine transporter cystinosin
Puzzo et al. Tau is not necessary for amyloid-β–induced synaptic and memory impairments
Casellas‐Díaz et al. Mfn2 localization in the ER is necessary for its bioenergetic function and neuritic development
Banks et al. Muscle structure influences utrophin expression in mdx mice
JP7225115B2 (en) Compositions and methods for treating lysosomal storage diseases and lysosomal storage disorders
Hu et al. Lentivirus-mediated PGC-1α overexpression protects against traumatic spinal cord injury in rats
Duerson et al. Detergent‐insoluble EAAC1/EAAT3 aberrantly accumulates in hippocampal neurons of Alzheimer's disease patients
Young et al. Dystonin/Bpag1 is a necessary endoplasmic reticulum/nuclear envelope protein in sensory neurons
US9988626B2 (en) Neurocalcin delta inhibitors and therapeutic and non-therapeutic uses thereof
EP2327412A1 (en) Treatment of nerve injuries
Moharir et al. Identification of a splice variant of optineurin which is defective in autophagy and phosphorylation
Kwon et al. Nanogel-mediated delivery of oncomodulin secreted from regeneration-associated macrophages promotes sensory axon regeneration in the spinal cord
WO2020127498A1 (en) Compounds for use in the treatment of diseases associated with altered autophagy, altered shh transduction, and/or neurodegenerative diseases
Janßen et al. A pivotal role of nonmuscle myosin II during microglial activation
Li et al. Binding of pro-prion to filamin A: by design or an unfortunate blunder
JP2019182873A (en) Compositions and methods for treating and preventing pancreatitis, renal injury and renal cancer
US11439658B2 (en) Compositions and methods for suppressing MSUT2
Lazarowski et al. Multidrug-resistance (MDR) proteins develops refractory epilepsy phenotype: clinical and experimental evidences
WO2021201116A1 (en) Method for screening compound having lysosome-damage-repairing activity
US20050106152A1 (en) Preventives/remedies for cancer
US20140242609A1 (en) Diagnosis and treatment of kidney stones, methods and compositions therefor

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19829140

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19829140

Country of ref document: EP

Kind code of ref document: A1