WO2012076555A1 - Autophagy enhancing compounds, peptides and peptidomimetic compounds for use in the treatment of neuronal diseases - Google Patents

Autophagy enhancing compounds, peptides and peptidomimetic compounds for use in the treatment of neuronal diseases Download PDF

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WO2012076555A1
WO2012076555A1 PCT/EP2011/071980 EP2011071980W WO2012076555A1 WO 2012076555 A1 WO2012076555 A1 WO 2012076555A1 EP 2011071980 W EP2011071980 W EP 2011071980W WO 2012076555 A1 WO2012076555 A1 WO 2012076555A1
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compound
autophagy
hcb
gfp
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Marcello D'amelio
Marco Molinari
Maria Teresa Viscomi
Francesco Cecconi
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Fondazione Santa Lucia
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • 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

Definitions

  • the present invention relates to autophagy enhancing compounds, peptides and peptidomimetic compounds for use in the treatment of neuronal diseases.
  • autophagy a catabolic process in which misfolded or aggregated proteins, lipids, and organelles are engulfed by double- membrane vesicles, called autophagosomes (APs), and degraded after fusing with lysosomes.
  • APs autophagosomes
  • autophagy leads to the accumulation of protein aggregates and damaged organelles in neurons, mediating the pathogenesis of neurodegenerative diseases. This evidence implicates the activation of autophagy as a therapeutic strategy in several chronic neurodegenerative disorders, such as Alzheimer's disease, Huntington's disease and Parkinson's disease. Yet, increased autophagy has been recently associated with more severe axonal degeneration in a model of optic nerve damage.
  • axonal damage affects the parent neuronal cell bodies in regions that are remote but functionally connected to the primary lesion.
  • These degenerative phenomena are termed "remote damage” and have been studied by several groups. Remote changes are sustained by many factors, including etiology, excitotoxicity, inflammation, and oxidative stress, for which various therapeutic approaches have been proposed.
  • remote degeneration is critical for the overall clinical profile in many acute CNS pathologies.
  • the present invention satisfies the above said needs by providing compounds or compositions according to claim 1.
  • peptidomimetic compound refers to a synthetic molecule that resembles in structure and steric conformation that of the peptide after which has been designed such that it can mimic its function either enhancing it or inhibiting it.
  • these pept idomimetic compounds are assembled with modified amino acids and/or organic molecules that have the same 3D conformation of the L-amino acid but are not recognized by cellular and extra-cellular proteases.
  • a similar approach has been recently validated by a synthetic molecule mimicking the structure of a MyD88 inhibitory peptide.
  • this peptidomimet ic compound (ST2825) maintained the same activity and specificity of inhibition displayed by the original MyD88 inhibitory peptide, proving the feasibility of this approach (Loiarro et al . , J. Leukocyte Biology 2007) .
  • AMBRAl refers to the protein disclosed in Fimia G.M et al . Nature 447 (7148), 1121-1125 (2007) (accession number ABI74670 GI : 114432124) .
  • mTOR refers to the protein designated mammalian target of rapamycin.
  • Figure 1 shows a schematic of the hemicerebellectomy (HCb) model. Due to the crossed input-output organization of the cerebellar connections, unilateral lesion of a cerebellar hemisphere induces axonal lesions and subsequent degeneration of the contralateral (experimental side) inferior olive (10) and pontine nuclei (Pn) , with sparing of the 10 and Pn on the ipsilateral side (control side) .
  • DCN deep cerebellar nuclei; icp: inferior cerebellar peduncle.
  • Figure 2 shows that acute cerebellar lesion activates autophagy in precerebellar neurons.
  • A Double-labeled confocal images of GFP-LC3 (green) and Dapi (blue) of neurons in pontine nuclei of CTRL-GFP-LC3 (CTRL) and HCb-GFP-LC3 mice at 4 days (HCb-4d) .
  • B Number of GFP-LC3-positive neurons (percentage of NeuN- positive neurons expressing GFP-LC3 punctate staining) in control (CTRL) and at various time points after HCb in inferior olive and pontine nuclei.
  • (C) GFP-LC3 dots per neuron in both inferior olive and pontine nuclei from CTRL-GFP-LC3 and HCb-GFP-LC3 mice at 4 days (mean ⁇ s.d.; ***p ⁇ 0.001 versus CTRL). The number of GFP-LC3 dots per neuron was determined for a minimum of five inferior olive and pontine nuclei sections /mice . Quantification was done on 150 cells per group (n 5 mice per group) .
  • D-E Electron photomicrographs of inferior olive neurons in CTRL (D) and HCb mice at 4 days (E) .
  • D' and E' enlargements of insets D and E, respectively.
  • Figure 3 shows that Acute cerebellar lesion upregulates key proteins involved in autophagosomes formation.
  • Beclin 1, p62, and LC3 immunoblots and densitometric graphs in CTRL and HCb mice in inferior olive (A) and pontine nuclei (B) at various time points (mean ⁇ s.d.; n 5) .
  • the upstream marker Beclin 1 is significantly increased already at HCb-24h compared with control values (**P ⁇ 0.01) .
  • the downstream markers p62 and LC3-I/LC3-II conversion are reduced and increased, respectively, compared with control values (**P ⁇ 0.01; ***p ⁇ 0.001).
  • Figure 4 shows the effects of the lysosomal inhibitor chloroquine (CQ) on autophagy activity.
  • CQ chloroquine
  • C Number of GFPLC3 punctae in inferior olive and pontine neurons from CTRL-GFP-LC3 and HCb-GFP-LC3 mice injected with saline (CTRL+sal and HCb+sal) or CQ (CTRL+CQ and HCb+CQ) . Note the significant effects of CQ treatment in HCb mice (compare HCb+sal and HCb+CQ; ***p ⁇ 0.001).
  • Figure 5 shows that mitochondrial cytochrome-c release precedes activation of autophagy and neuronal death.
  • A Representative immunoblots and densitometric graphs of the cytosolic levels of cytochrome-c in inferior olive and pontine nuclei at various time points after HCb. Note that in both nuclei the levels of cytosolic cytochrome-c is significantly increased already at HCb-12h and it tended to augment at the following time points considered. **P ⁇ 0.001; ***P ⁇ 0.0001 (versus HCb-12h) .
  • Figure 6 shows genetic and pharmacological manipulation of autophagy indexes.
  • A Histograms of the number GFP-LC3 dots per neuron in inferior olive and pontine nuclei in CTRL-GFP-LC3+sal , CTRLGFP-LC3+rap, CTRL- GFP-LC3-Becnl +/" , HCb-GFP-LC3+sal , HCb-GFP-LC3+rap and HCb- GFPLC3-Becnl +/" at various time points after HCb.
  • Figure 7 shows that enhancement of autophagy reduces neuronal death after brain lesion.
  • A Representative immunoblots and densitometric graphs of the levels of cytosolic cytochrome-c in inferior olive and pontine nuclei in CTRL+sal, CTRL+rap, CTRL-Becnl +/ ⁇ , HCb+sal, HCb+rap and HCb-Becnl + ⁇ at various time points after HCb. Note that, in both nuclei, the release of cytochrome-c is increased in HCb-Becnl + ⁇ when compared with HCb+sal, and conversely the cytochrome-c release results decreased in HCb+rap.
  • Figure 8 shows that rapamycin does not influence autophagy in Becnl + ⁇ mice.
  • A Histograms of the number GFP-LC3 punctae per neuron in inferior olive and pontine nuclei in CTRL-GFP-LC3-Becnl +/ ⁇ +sal, CTRL-GFP-LC3-Becnl +/" +rap, HCb-GFP-LC3-Becnl +/ ⁇ +sal, and HCb-GFP-LC3-Becnl +/" +rap mice.
  • Figure 9 shows a table listing mice strains, survival time, lesion, treatments, and procedures in the different experimental groups .
  • an autophagy enhancing compound for use in the treatment of neuronal diseases.
  • the autophagy enhancing compound can be associated to any pharmaceutically acceptable carrier and be provided in a pharmaceutical composition.
  • the neuronal disease can be in particular a central nervous system disease. More specifically, it can be a chronic neurodegenerative disease or an acute focal lesion.
  • the chronic neurodegenerative disease is advantageously Alzheimer's disease, Parkinson's disease, Huntington's disease and Batten's disease.
  • the acute focal lesion is advantageously stroke, spinal or brain trauma, and multiple sclerosis.
  • the compound or composition preferably increases the autophagic clearance of damaged mithocondria .
  • the compound or composition reduces the release of cytochrome-c in cells.
  • the compound or composition preferably induces mammalian target of rapamycin (mTOR) inactivation .
  • mTOR mammalian target of rapamycin
  • Preferred compounds according to the invention are selected from the group consisting of rapamycin, CCI-779, Glc, Glc-6-P, Torinl, perhexiline, niclosamide, rottlerin, lithium, L-690,330, carbamazepine , sodium valproate, verapamil, loperamide, amiodarone, nimodipine, nitrendipine, niguldipine, pimozide, calpastatin, calpeptin, clonidine, rilmenidine, 2 ' , 5 ' -dideoxyadenosine, NF449, minoxidil, penitrem A, fluspirilene, trifluoperazine, trehalose, SMER10, SMERl 8 , SMER28, and SMER analogs.
  • the compound is preferably rapamycin.
  • the Ambra 1 protein has been identified as a crucial factor in regulating autophagy in vertebrates.
  • Peptides or peptidomimetic compounds related to the Ambra 1 protein may be used in the treatment of the above said neuronal diseases.
  • these peptides comprise a sequence having SEQ ID NO : 1 , SEQ ID NO : 2 or SEQ ID NO : 3. More preferably these peptides have SEQ ID NO : 1 , SEQ ID NO : 2 or SEQ ID NO : 3.
  • Peptidomimetic compounds of said peptides are advantageously used.
  • HCb hemicerebellectomy
  • autophagy is reported to protect neurons from remote degeneration after CNS focal damage and pharmacological enhancement of autophagy is shown to increase cell survival and promotes functional recovery.
  • HCb Autophagy is activated in axotomized neurons.
  • GFP-LC3 ubiquitously expressed LC3-con ugated green fluorescent protein
  • Figure 9 The purpose of HCb is to remove half of the vermis and one cerebellar hemisphere, including the deep cerebellar nuclei, while sparing the vestibular nuclei and all surrounding structures (Fig. 1) . Due to the crossed input-output cerebellar organization, HCb damages the axons of all neurons of the contralateral (experimental side, Fig.
  • Beclin 1 is an evolutionarily conserved protein that promotes autophagy by participating in the nucleation of APs formation. Thus, Beclin 1 protein levels were measured by Western blot (Wb) . Twenty-four hours after HCb, Beclin 1 protein levels increased compared with CTRL, declining slightly at 4 days (Fig. 3A) .
  • microtubule-associated light chain 3 As regards microtubule-associated light chain 3 (LC3), during autophagy, it is proteolyzed, conjugated to phosphatidyl-ethanolamine, and incorporated into structures that become APs . Conversion from nonlipidated LC3 (LC3-I) to lipidated LC3 (LC3-II), detected as a mobility shift by Wb 27, was observed at 2 and 4 days after HCb, differing significantly from CTRL (Fig. 3A) .
  • p62/SQSTMl was evaluated.
  • p62 is degraded by autophagy and links ubiquitinated proteins to the autophagic machinery to effect their degradation in lysosomes. Because p62 accumulates when autophagy is inhibited and because decreased levels of p62 can be observed when autophagy is induced, p62 is considered to be a marker of autophagy. Thus, p62 levels were measured by Wb . Two days after HCb, p62 levels declined compared with CTRL; this decrease became more prominent after 4 days (Fig. 3A) .
  • CQ increased the conversion of LC3-I to LC3 II as demonstrated by Western blotting analysis (Fig. 4A) and by counting of the number of GFP-LC3 punctae per neuron (Fig. 4B-C) .
  • CQ significantly affected autophagy indices.
  • LC3-II was significantly upregulated compared to HCb+sal (Fig. 4A) .
  • the increase in LC3-II was paralleled by rise in the density of GFP-LC3 punctae per neuron (Fig. 4B-C) , although the number of neurons with GFP-LC3 punctae did not change (Fig. 4D) .
  • Cytochrome-c (cytc) is released by the mitochondria in response to proapoptotic stimuli and is considered an early marker of apoptosis.
  • cytc Cytochrome-c
  • the kinetics of mitochondrial cyt-c release and the activation of autophagy after HCb were analyzed.
  • mitochondrial-cytosol ic fractionation cyt-c was demonstrated to be released into the cytosol starting from 12 h after HCb (Fig. 5A) .
  • Cyt-c release from mitochondria is a crucial step in the activation of apoptotic machinery, and caspase-3 activation is considered an irreversible commitment of the cell toward apoptosis.
  • caspase-3 activation is considered an irreversible commitment of the cell toward apoptosis.
  • the apoptotic pathway downstream of cyt-c release was investigated to assess the fate of axotomized neurons.
  • caspase-3 activity did not differ significantly from control.
  • Caspase-3 activity differences were detected 2 days after HCb and increased significantly 4 days after the lesion (Fig. 5D) .
  • TUNEL TdT- mediated dUTP-biotin nick end-labeling
  • the kinetic data indicate that autophagy, in acute brain damage, occurs after cyt-c release and before caspase-3 activation and subsequent DNA fragmentation.
  • rapamicyn Activation of autophagy by rapamicyn is neuroprotective .
  • HCb-GFP-LC3-Becnl + ⁇ mice had fewer GFP-LC3- dots per neuron in 10 and Pn compared with HCb-GFP-LC3+sal mice (Fig. 6A) .
  • This autophagy reduction was confirmed by analysis of LC3 and p62 by Wb.
  • HCb-GFP-LC3-Becnl +/" mice LC3-II/LC3-I conversion decreased and p62 levels rose compared with HCb-GFP-LC3+sal mice (Fig. 6B) .
  • Rapamycin treatment as expected, induced mTOR inactivation as shown by the reduction in the phosphorylation of downstream substrates of mTOR, namely p70S6K and S6RP (compare to HCb+sal vs HCb+rap mice in Fig. S3) . Furthermore, treatment with rapamycin (HCb-GFP-LC3+rap mice) increased all autophagy markers compared with HCb-GFP-LC3+sal mice at all time points (Fig 6A-B) .
  • HCb- GFP-LC3-Becnl + ⁇ experienced increases in cyt-c release (Fig. 7A) , higher neuronal death (Fig. 7B) , and worse functional recovery, as shown by the Neurological Severity Score (NSS - Fig. 7C) .
  • Becnl + ⁇ mice were treated with rapamycin to evaluate whether this treatment was able to enhance or to fail autophagy activation and neuronal survival in mice with impaired autophagy responses ( Figure 9) .
  • autophagy is a protective mechanism that attempts to counteract axotomy induced degeneration but, at least for precerebellar neurons, it is incapable of preserving neuronal survival. In line with reduced cellular damage, better survival and better recovery is achieved when autophagy is pharmacologically enhanced.
  • HCb model A large percentage of CNS pathologies present acute focal brain lesions and their consequences have been studied in various animal models.
  • the HCb model is well established, in which neuronal degeneration can be analyzed remotely from the primary site of damage, where effects of the primary lesion can confound the study of degeneration. These remote phenomena can be used to exploit therapeutic approaches, because they are active long after the primary damage has subsided and influence postlesion impairments.
  • the time and location in which autophagy is activated are critical to the understanding of its relationships with apoptosis. These data, which are consistent with those of previous studies implicate cyt-c release as an early event of the neuronal reaction to axonal damage.
  • the strict kinetics of the activation of autophagy and the reduction in cyt-c release after enhanced autophagy suggest that cyt-c release triggers the activation of autophagy .
  • Cyt-c release from the mitochondria is the most logical target of the neuroprotective effect of autophagy.
  • Rapamycin protects against neurodegeneration, an effect that is linked presumably to enhanced autophagic clearance of damaged mithocondria .
  • rapamycin treatment was associated with reduced cyt-c release and greater neuronal survival.
  • HCbBecnl + ⁇ mice - with reduced autophagic response - showed inefficient autophagy-dependent clearance of mitochondria, which was associated with both poor neuronal survival and functional recovery.
  • HCbBecnl + ⁇ mice implicates autophagy mechanisms as the chief target of rapamycin-associated neuroprotective effects.
  • APs clearance was prevented by intracerebroventricularly injection of chloroquine to determine whether APs accumulation after HCb is due to an increase in their formation or reduced degradation. Based on the morphological and biochemical indices of autophagy activity, after HCb, APs were actively formed and efficiently cleared, demonstrating that autophagic flux was not impaired.
  • mice Animals and surgery. Experiments were performed using male adult mice (Figure 9) (weight 20-25 g) .
  • C57BL6 mice (10-12 weeks of age) were obtained from Harlan Italy, and GFP-LC3 transgenic mice were obtained from Dr. N. Mizushima.
  • the GFP-LC3 mice contain a transgene in which LC3 is fused to GFP and a CAG promoter.
  • Homozygous and heterozygous Beclinl mice were obtained from Dr. B. Levine. Further, the Beclinl homozygous and heterozygous mice were crossed to GFP-LC3 transgenic mice ( Figure 9) .
  • mice were deeply anesthetized by i.p. injections of xylazine (10 mg/ml) and tiletamine e zolazepam (50 mg/ml) and positioned in a stereotaxic apparatus. An incision was made in the skin on the skull, and the occipital bone was drilled and removed. Subsequently, the dura was incised to expose the cerebellum, and the right cerebellar hemisphere was removed by suction. The wound was sutured, and the animals were returned to their cages. For the CTRL group, surgery was interrupted after the dura lesion was made, and after suturing, the animals were returned to their cages.
  • CTRL group surgery was interrupted after the dura lesion was made, and after suturing, the animals were returned to their cages.
  • the experimental groups are detailed in Figure 9.
  • Rapamycin (Rap; Alexis Biochemicals , 380-004-M001 ) was dissolved in DMSO (25 mg/ml) and injected once daily (2 mg/Kg; i.p.) .
  • Chloroquine diphosphate-salt Chlor; Sigma, C6628 was dissolved in sterile saline and injected intracerebroventricular ly (i.e. v.; 25 mg/Kg) 60 min before hemicerebellectomy .48 Treatments in the different experimental groups are shown in detail in Figure 9.
  • Neurological evaluation Neurological impairment was evaluated by the Neurological Severity Score (NSS) (18) .
  • the Neurologic Severity Score (NSS) is a composite of motor, sensory, reflex, and balance tests, in which for each test, one point is awarded for the inability to perform or for the lack of a tested reflex, and zero points are awarded for success.
  • An NSS of 18 indicates severe injury, whereas a score of zero signifies healthy, uninjured rats.
  • the NSS was evaluated at 1, 2, and 4 days after damage by an investigator who was blinded to the experimental groups .
  • mice anti-neuronal nuclei Neuronal nuclei
  • GFAP rabbit anti-Glial fibrillary acidic protein
  • mouse anti-OX-42 (1:200; Serotec, #MCA275G. All primary antibody solutions were prepared in PB and 0.3% Triton X- 100 and incubated overnight. Each incubation step was followed by three, 5-min rinses in PB .
  • cytochrome-c cytochrome-c
  • Sections were incubated with the following primary antibody goat anti-Cyt-c (1:500; Santa Cruz Biotechnology, #sc-8385). Afterwards, sections were incubated 2h at RT with a Alexa Fluor 555 donkey anti-goat IgG (1:200; Molecular Probes, #21432).
  • Sections were examined under a confocal laser scanning microscope (ZEISS, LSM700; Germany) equipped with four laser lines: violet diode emitting at 405 nm for DAPI, argon emitting at 488 nm and helium/neon emitting at 543 nm and 633 nm. Plates were generated adjusting the brightness and contrast of digital images (Corel Draw, 9) .
  • Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TU EL) assay was performed on 15 ⁇ fresh frozen coronal sections at the level of 10 and Pn by using the ApopTag Fluorescein In Situ Apoptosis Detection Kit from Millipore (#S7110) . The assay was carried out according to manufacturer's instructions. After being washed in PBS, the sections were incubated overnight with a solution of PBS+0.4% Triton-X-100 containing mouse-anti NeuN (Millipore, MAB-377), DAPI (Sigma #D9542) counter-stained and coverslipped with Fluoromount (Sigma, #F4680) .
  • Fluorometric assay of caspase-3 activity The left 10 and Pn were punched. The tissues were homogenized separately in lysis assay buffer (100 mM Hepes pH 7.4, 0.1% Chaps, 1 mM EDTA, 10 mM DTT, 1 mM PMSF) and lysed by subsequent freezing in liquid N2 and thawing at 37°C three times. After centrifugation at 11, 500 x g for 5 min, the protein concentration of resulting supernatant was quantified, and proteins were incubated at 37°C in lysis assay buffer containing 50 ⁇ Ac-DEVDAMC (BD Pharmingen, #556449) . The fluorescence was measured at an excitation and emission wavelength of 380 nm and 460 nm, respectively. For further details see ref.
  • Precerebellar nuclei (10 and Pn separately) were homogenized in Buffer A (320 mM sucrose, 1 mM EDTA, 50 mM Tris-HCl pH7.4, 1 mM DTT, 1 mM PMSF, with protease inhibitor cocktail - Sigma, #P8340) by 30 strokes with a glass Pyrex micro homogenizer. The homogenate was centrifuged at 1,000 ⁇ g for 10 min and the resulting supernatant was centrifuged at 10, 000 x g for 20 min to obtain the mitochondrial pellet and the supernatant.
  • Buffer A 320 mM sucrose, 1 mM EDTA, 50 mM Tris-HCl pH7.4, 1 mM DTT, 1 mM PMSF, with protease inhibitor cocktail - Sigma, #P8340
  • the mitochondria-containing pellet was washed three times with Buffer B (250 mM sucrose, 1 mM EGTA, 10 mM TrisHCl pH7.4) by centrifugation for 10 min at 10, 000 x g. The supernatant was centrifuged at 100,000 ⁇ g for 1 hr to generate the cytosolic fraction .
  • Buffer B 250 mM sucrose, 1 mM EGTA, 10 mM TrisHCl pH7.4
  • Precerebellar nuclei (10 and Pn separately) were homogenized in lysis buffer (320 mM sucrose, 10% Glycerol, 50 mM NaCl, 50 mM Tris-HCl pH 7.5, 1% Triton X-100, 1 mM PMSF, with protease inhibitor cocktail (Sigma, #P8340), incubated on ice for 30 min and centrifuged at 13,000 x g for 20 min. The total protein content of resulting supernatant was determined. Proteins were applied to SDS-PAGE and electroblotted on a PVDF membrane. Immunoblott ing analysis was performed using a chemiluminescence detection kit.
  • Hawthorne, NY, USA was configured to interface an Olympus BX 50 microscope with a motorized stage and a HV-C20 Hitachi colour digital camera with a Pentium II PC workstation.
  • a three-dimensional optical dissector counting probe (x, y, z dimension of 30 x 30 x 10 ⁇ respectively) was applied.
  • the two nuclei were outlined using the 4x objective, while the lOOx oil immersion objective was used for marking the neuronal cells.
  • Total 10 and Pn cell number was estimated according to the formula given below:
  • N ⁇ Q x 1/ssf x 1/asf x 1/tsf.
  • ⁇ Q represents the total number of neurons counted in all optically sampled fields of the 10 and Pn
  • ssf is the section sampling fraction
  • asf is the area sampling fraction
  • tsf is the thickness sampling fraction
  • Electron microscopy Animals were transcardially perfused with a calcium-free Ringer's variant (pH 7.3), followed by 2% freshly depolymerised paraformaldehyde and 1% glutaraldehyde in 0.12 M phosphate buffer (PB) , pH 7.4. Brains were dissected out and cut on a vibratome in the coronal plane, to obtain 100 ⁇ thick sections which were collected in PB, pH 7.4. Slices were post-fixed in 1% osmium tetroxide in PB for 1 hr at 4 °C, in the dark, then gradually dehydrated in ethanol . All the steps of the above procedure were carried out at 4 °C.
  • PB phosphate buffer
  • Sections were infiltrated with graded mixtures of propylene oxide and Epon812 (TAAB, Reading) , then flat-embedded in the same resin, allowing specimens to polymerize at 60 °C, for 3 days. Selected areas, namely 10 and Pn, were then remounted on Epon blanks and sectioned by a Reichert Ultracut S ultramicrotome (Leica Microsystems), to obtain ultrathin sections (60-70 ⁇ ) which were collected on nickel grids. These were briefly contrasted with 1% uranyl acetate and observed in a Philips CM120 electron microscope, equipped with a Philips Megaview III videocamera. Images were electronically captured by AnalySys 2.0 software and composed in an Adobe Photoshop CS3 format.
  • Cecconi F Levine B. The role of autophagy in mammalian development: cell makeover rather than cell death. Dev Cell 2008; 15:344-57.
  • Fimia GM Piacentini M. Regulation of autophagy in mammals and its interplay with apoptosis. Cell Mol Life Sci 2010; 67:1581-88.
  • Kihara A Kabeya Y, Ohsumi Y, et al . Beclin- phosphatidylinositol 3-kinase complex functions at the trans-Golgi network. EMBO Rep 2001; 2:303-25.
  • Block F Dihne M, Loos M. Inflammation in areas of remote changes following focal brain lesion. Prog Neurobiol 2005; 75:342-65.

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Abstract

The present invention relates to autophagy enhancing compounds or a pharmaceutical compositions comprising these compounds for use in the treatment of neuronal diseases, in particular acute focal brain lesions.

Description

AUTOPHAGY ENHANCING COMPOUNDS, PEPTIDES AND PEPTIDOMIMETIC COMPOUNDS FOR USE IN THE TREATMENT OF NEURONAL DISEASES
TECHNICAL FIELD
The present invention relates to autophagy enhancing compounds, peptides and peptidomimetic compounds for use in the treatment of neuronal diseases.
STATE OF THE ART
Under normal conditions, cells maintain a balance between synthesis, degradation and recycling of cellular components. One key homeostatic mechanism is autophagy, a catabolic process in which misfolded or aggregated proteins, lipids, and organelles are engulfed by double- membrane vesicles, called autophagosomes (APs), and degraded after fusing with lysosomes.
Stress induces cell death primarily through two pathways: immediate/necrotic and delayed/apoptotic . In the latter, autophagy mechanisms are activated, although their function in life-and-death decisions is unknown. Thus, the links between autophagy and apoptosis are critical.
Although autophagy has been known for more than five decades, its importance in the central nervous system (CNS) homeostasis has only recently been demonstrated. Within the CNS, despite recent advances in the understanding of its molecular mechanisms and biological functions, the physiological and pathological functions of autophagy are largely unknown.
The absence of autophagy leads to the accumulation of protein aggregates and damaged organelles in neurons, mediating the pathogenesis of neurodegenerative diseases. This evidence implicates the activation of autophagy as a therapeutic strategy in several chronic neurodegenerative disorders, such as Alzheimer's disease, Huntington's disease and Parkinson's disease. Yet, increased autophagy has been recently associated with more severe axonal degeneration in a model of optic nerve damage.
At present consensus is gathering on the idea that autophagy is an important element in the pathogenesis of chronic diseases. Conversely, the function of autophagy in acute brain damage remains unknown.
Functional impairments after acute brain damage are associated with axonal lesions in common pathologies, such as multiple sclerosis, stroke, and spinal and brain trauma. In addition to interrupting the flow of information, axonal damage affects the parent neuronal cell bodies in regions that are remote but functionally connected to the primary lesion. These degenerative phenomena are termed "remote damage" and have been studied by several groups. Remote changes are sustained by many factors, including etiology, excitotoxicity, inflammation, and oxidative stress, for which various therapeutic approaches have been proposed. There is a consensus that mechanisms of remote degeneration are critical for the overall clinical profile in many acute CNS pathologies. Technical problem
Both chronic neurodegenerative disorders and acute neuronal damage trauma are a leading cause of morbidity and disability, they are dramatically debilitating and eventually result in death. Moreover, the management of patients affected by these diseases represents a very heavy burden for public health costs. Unfortunately, few therapeutic compounds are known for the effective treatment of the same. At present there is no satisfactory cure for most of these pathologies and their treatment is still scarcely effective. The need is therefore felt to find medicaments for the treatment of these disorders.
Summary of the invention
It is the object of the present invention to provide medicaments for use in the treatment of these disorders.
The present invention satisfies the above said needs by providing compounds or compositions according to claim 1.
Definitions
As used herein, the term "peptidomimetic compound" refers to a synthetic molecule that resembles in structure and steric conformation that of the peptide after which has been designed such that it can mimic its function either enhancing it or inhibiting it. In particular, these pept idomimetic compounds are assembled with modified amino acids and/or organic molecules that have the same 3D conformation of the L-amino acid but are not recognized by cellular and extra-cellular proteases. A similar approach has been recently validated by a synthetic molecule mimicking the structure of a MyD88 inhibitory peptide. Remarkably, this peptidomimet ic compound (ST2825) maintained the same activity and specificity of inhibition displayed by the original MyD88 inhibitory peptide, proving the feasibility of this approach (Loiarro et al . , J. Leukocyte Biology 2007) .
As used herein, the term "AMBRAl" refers to the protein disclosed in Fimia G.M et al . Nature 447 (7148), 1121-1125 (2007) (accession number ABI74670 GI : 114432124) .
As used herein, the term "mTOR" refers to the protein designated mammalian target of rapamycin.
Brief description of the drawings
The present invention will now be described with reference to the accompanying drawings, wherein:
Figure 1 shows a schematic of the hemicerebellectomy (HCb) model. Due to the crossed input-output organization of the cerebellar connections, unilateral lesion of a cerebellar hemisphere induces axonal lesions and subsequent degeneration of the contralateral (experimental side) inferior olive (10) and pontine nuclei (Pn) , with sparing of the 10 and Pn on the ipsilateral side (control side) . DCN: deep cerebellar nuclei; icp: inferior cerebellar peduncle.
Figure 2 shows that acute cerebellar lesion activates autophagy in precerebellar neurons. (A) Double-labeled confocal images of GFP-LC3 (green) and Dapi (blue) of neurons in pontine nuclei of CTRL-GFP-LC3 (CTRL) and HCb-GFP-LC3 mice at 4 days (HCb-4d) . (B) Number of GFP-LC3-positive neurons (percentage of NeuN- positive neurons expressing GFP-LC3 punctate staining) in control (CTRL) and at various time points after HCb in inferior olive and pontine nuclei. (C) GFP-LC3 dots per neuron in both inferior olive and pontine nuclei from CTRL-GFP-LC3 and HCb-GFP-LC3 mice at 4 days (mean ± s.d.; ***p < 0.001 versus CTRL). The number of GFP-LC3 dots per neuron was determined for a minimum of five inferior olive and pontine nuclei sections /mice . Quantification was done on 150 cells per group (n=5 mice per group) . (D-E) Electron photomicrographs of inferior olive neurons in CTRL (D) and HCb mice at 4 days (E) . D' and E': enlargements of insets D and E, respectively. Note the preserved mitochondria in control (D-D') and the altered nuclear envelopes (N) , abnormal mitochondrial cristae (#), and autophagosomes (**double- membrane surrounding a mitochondrion), in HCb-4d mice (Ε-Ε') . Scale bars: A= 8 pm; D-E= 1 pm; D'-E'= 200 nm.
Figure 3 shows that Acute cerebellar lesion upregulates key proteins involved in autophagosomes formation. Beclin 1, p62, and LC3 immunoblots and densitometric graphs in CTRL and HCb mice in inferior olive (A) and pontine nuclei (B) at various time points (mean ± s.d.; n=5) . Note that in the inferior olive and pontine nuclei the upstream marker Beclin 1 is significantly increased already at HCb-24h compared with control values (**P< 0.01) . By contrast, starting from 2d after lesion, the downstream markers p62 and LC3-I/LC3-II conversion are reduced and increased, respectively, compared with control values (**P< 0.01; ***p< 0.001). One-way ANOVA followed by Bonferroni multiple comparison test .
Figure 4 shows the effects of the lysosomal inhibitor chloroquine (CQ) on autophagy activity. (A) LC3 immunoblots and densitometric graphs in CTRL+sal, CTRL+CQ, HCb+sal, and HCb+CQ mice of the inferior olive and pontine nuclei 24 hours after CQ intracerebroventricular injection (mean ± s.d.; n=5) . Note that compared with CTRL+sal, CQ treatment in CTRL mice led to an slightly increase of LC3- II (*P< 0.05). CQ treatment in HCb mice led to a significant increase in and LC3-II (**P< 0.01) both in inferior olive and pontine nuclei (compare HCb+sal vs HCb+CQ) . One-way ANOVA, followed by Bonferroni multiple comparison test. (B) Double-labeled confocal images of GFP-LC3 (green) plus DAPI (blue) staining of neurons in the pontine nuclei of CTRL-GFP-LC3 mice intracerebroventricularly injected with saline (CTRL+sal) or CQ (CTRL+CQ) and in HCb-GFP-LC3 mice at 24 hours after injection with saline (HCb+sal 24h) or CQ (HCb+CQ 24h) . (C) Number of GFPLC3 punctae in inferior olive and pontine neurons from CTRL-GFP-LC3 and HCb-GFP-LC3 mice injected with saline (CTRL+sal and HCb+sal) or CQ (CTRL+CQ and HCb+CQ) . Note the significant effects of CQ treatment in HCb mice (compare HCb+sal and HCb+CQ; ***p < 0.001). (D) Number of GFP-LC3-positive neurons (percentage of NeuN- positive neurons with GFP-LC3 punctate staining) in CTRL- GFP-LC3+sal (CTRL+sal), CTRL-GFP-LC3+CQ (CTRL+CQ), HCb- GFP-LC3+sal (HCb+sal), and HCb-GFP-LC3+CQ (HCb+CQ) mice. Note that CQ treatment in HCb mice does not modify the number of neurons with GFP-LC3 punctate staining (compare HCb+sal and HCb+CQ). Scale bar: B= 20 μηι
Figure 5 shows that mitochondrial cytochrome-c release precedes activation of autophagy and neuronal death. (A) Representative immunoblots and densitometric graphs of the cytosolic levels of cytochrome-c in inferior olive and pontine nuclei at various time points after HCb. Note that in both nuclei the levels of cytosolic cytochrome-c is significantly increased already at HCb-12h and it tended to augment at the following time points considered. **P<0.001; ***P<0.0001 (versus HCb-12h) . (B) Time course of percentage of neurons (NeuN positive) presenting cytochrome-c-release and GFP-LC3 dots in inferior olive and pontine nuclei in CTRL and after HCb at various time points. Data are expressed as mean ± s.d. (n=5/group) . **P<0.001; ***P<0.0001 (versus HCb-12h) . (C) Time course of percentage of cytochrome-c-releasing neurons expressing GFP-LC3 dots in inferior olive and pontine nuclei in CTRL and after HCb. Data are expressed as mean ± s.d. (n=5/group) . ***P<0.0001 (versus CTRL). (D) Time course of caspase-3 activity in inferior olive and pontine nuclei in CTRL and after HCb. (E) Time course of stereological counts of NeuN- and TUNEL-positive neurons in inferior olive and pontine nuclei in CTRL and after HCb. Data are expressed as mean±s.d. (n=5/group) . One-way ANOVA followed by Bonferroni multiple comparison test. **P<0.001; ***P<0.0001 (versus CTRL).
Figure 6 shows genetic and pharmacological manipulation of autophagy indexes. (A) Histograms of the number GFP-LC3 dots per neuron in inferior olive and pontine nuclei in CTRL-GFP-LC3+sal , CTRLGFP-LC3+rap, CTRL- GFP-LC3-Becnl+/", HCb-GFP-LC3+sal , HCb-GFP-LC3+rap and HCb- GFPLC3-Becnl+/" at various time points after HCb. GFP-LC3 dots counting was performed as described in Material and Methods. Data are expressed as mean+s .d. (n=150/group) . **P<0.001; ***P<0.0001. Two-way ANOVA followed by Bonferroni multiple comparison test. (B) p62 and LC3 immunoblots and densitometric graphs in inferior olive and pontine nuclei in HCb+sal, HCb+rap and HCb-Becnl+ ~ mice at various time points after HCb . Data are expressed as mean+s.d. (n=5/group) . *P<0.05; **P<0.001; ***P<0.0001. Two-way ANOVA followed by Bonferroni multiple comparison test .
Figure 7 shows that enhancement of autophagy reduces neuronal death after brain lesion. (A) Representative immunoblots and densitometric graphs of the levels of cytosolic cytochrome-c in inferior olive and pontine nuclei in CTRL+sal, CTRL+rap, CTRL-Becnl+/~, HCb+sal, HCb+rap and HCb-Becnl+ ~ at various time points after HCb. Note that, in both nuclei, the release of cytochrome-c is increased in HCb-Becnl+ ~ when compared with HCb+sal, and conversely the cytochrome-c release results decreased in HCb+rap. *P<0.01; **P<0.001; ***P<0.0001. (B) Histograms of stereological neuronal counts in CTRL-GFP-LC3+sal , CTRL-GFP-LC3+rap, CTRL-GFP-LC3-Becnl+/", HCb-GFP-LC3+sal , HCb-GFP-LC3+rap and HCb-GFP-LC3-Becnl+/" at various time points after HCb. Cell counting of surviving NeuN positive neurons was performed as described in Material and Methods. Data are expressed as mean+s.d. (n=5/group) . (C) Time course of neurological recovery (NSS) . Data are expressed as mean+s.d. (n=5/group) . *P<0.01; **P<0.001; ***P<0.0001. Two-way ANOVA followed by Bonferroni multiple comparison test.
Figure 8 shows that rapamycin does not influence autophagy in Becnl+ ~ mice. (A) Histograms of the number GFP-LC3 punctae per neuron in inferior olive and pontine nuclei in CTRL-GFP-LC3-Becnl+/~+sal, CTRL-GFP-LC3-Becnl+/" +rap, HCb-GFP-LC3-Becnl+/~+sal, and HCb-GFP-LC3-Becnl+/"+rap mice. (B) p62 and LC3 immunoblots and densitometric graphs in inferior olive and pontine nuclei in CTRL- Becnl+ ~+sal , CTRL-Becnl+/~+rap, HCb-Becnl+/"+sal , and HCb-Becnl+/"+rap mice 4 days after HCb . Data are expressed as mean+s .d. (n=5/group) . (C) Histograms of stereological neuronal counts in CTRL-GFP-LC3-Becnl+/-+sal , CTRL-GFP-LC3-Becnl+/" +rap, HCb-GFP-LC3-Becnl+/~+sal, and HCbGFP-LC3-Becnl+/"+rap mice. Data are expressed as mean+s .d. (n=5/group) . (D) Time course of neurological recovery (NSS) . Data are expressed as mean+s .d. (n=5/group) . One-way ANOVA, followed by Bonferroni multiple comparison test.
Figure 9 shows a table listing mice strains, survival time, lesion, treatments, and procedures in the different experimental groups .
Detailed description of the invention
According to the present invention an autophagy enhancing compound is provided for use in the treatment of neuronal diseases. The autophagy enhancing compound can be associated to any pharmaceutically acceptable carrier and be provided in a pharmaceutical composition.
The neuronal disease can be in particular a central nervous system disease. More specifically, it can be a chronic neurodegenerative disease or an acute focal lesion. The chronic neurodegenerative disease is advantageously Alzheimer's disease, Parkinson's disease, Huntington's disease and Batten's disease. The acute focal lesion is advantageously stroke, spinal or brain trauma, and multiple sclerosis.
The compound or composition preferably increases the autophagic clearance of damaged mithocondria . Advantageously, the compound or composition reduces the release of cytochrome-c in cells.
The compound or composition preferably induces mammalian target of rapamycin (mTOR) inactivation .
Preferred compounds according to the invention are selected from the group consisting of rapamycin, CCI-779, Glc, Glc-6-P, Torinl, perhexiline, niclosamide, rottlerin, lithium, L-690,330, carbamazepine , sodium valproate, verapamil, loperamide, amiodarone, nimodipine, nitrendipine, niguldipine, pimozide, calpastatin, calpeptin, clonidine, rilmenidine, 2 ' , 5 ' -dideoxyadenosine, NF449, minoxidil, penitrem A, fluspirilene, trifluoperazine, trehalose, SMER10, SMERl 8 , SMER28, and SMER analogs. The compound is preferably rapamycin. Moreover, the Ambra 1 protein has been identified as a crucial factor in regulating autophagy in vertebrates. Peptides or peptidomimetic compounds related to the Ambra 1 protein may be used in the treatment of the above said neuronal diseases. Preferably, these peptides comprise a sequence having SEQ ID NO : 1 , SEQ ID NO : 2 or SEQ ID NO : 3. More preferably these peptides have SEQ ID NO : 1 , SEQ ID NO : 2 or SEQ ID NO : 3. Peptidomimetic compounds of said peptides are advantageously used.
In this study, the function of autophagy and its kinetics during apoptosis was examined in an in vivo model of acute focal CNS lesion, focusing on remote changes that are induced by hemicerebellectomy (HCb) . HCb is a unique experimental paradigm in which axonal damage-induced neuronal death mechanisms can be studied. In this model, neuronal degeneration is induced by target deprivation and axonal damage of contralateral neurons of the inferior olive (10) and pontine nuclei (Pn) . Thus, degeneration occurs in regions that are remote but functionally connected to the site of the lesion.
Herein, autophagy is reported to protect neurons from remote degeneration after CNS focal damage and pharmacological enhancement of autophagy is shown to increase cell survival and promotes functional recovery. Experimental
Autophagy is activated in axotomized neurons. To examine the induction of autophagy after focal brain damage, HCb was performed in transgenic mice that ubiquitously expressed LC3-con ugated green fluorescent protein (GFP-LC3) (Figure 9) . The purpose of HCb is to remove half of the vermis and one cerebellar hemisphere, including the deep cerebellar nuclei, while sparing the vestibular nuclei and all surrounding structures (Fig. 1) . Due to the crossed input-output cerebellar organization, HCb damages the axons of all neurons of the contralateral (experimental side, Fig. 1) inferior olive (10) and pontine nuclei (Pn) , sparing the same structures on the side that is ipsilateral to the cerebellar lesion (control side, Fig. 1) . In GFP-LC3 mice after HCb, autophagic activity was monitored by changes in GFP-LC3 signal, wherein activation was reflected by the appearance of GFP- LC3 punctate dots, representing enhanced conversion of LC3-I (cytosolic) to LC-3II (lipidated) . This conversion is concomitant with GFP-LC3 recruitment to APs. Thus, changes in GFP-LC3 signal in axotomized neurons in the chief precerebellar stations — the 10 and Pn — reflect autophagy status after the development of focal brain les ions .
Under physiological conditions ( CTRL-GFP-LC3 mice), the GFP-LC3 signal was low and diffuse in the cytoplasm of 10 and Pn neurons (Fig. 2A; CTRL) . Conversely, punctate GFP-LC3 expression was observed in axotomized 10 and Pn neurons in HCb-GFP-LC3 mice (Fig. 2A; HCb-4d) .
In time course quantitative analyses of the autophagy level, it was observed that starting from 24 h, the number of neurons with GFP-LC3 punctae, suggestive of APs formation, increased gradually (Fig. 2B) , as did the number of GFP-LC3 punctae per neuron (Fig. 2C) .
To determine the morphological signatures of autophagy in axotomized neurons, the 10 and Pn of control and HCb mice were examined by electron microscopy. No neuronal autophagosomal vacuoles were observed in CTRL- GFP-LC3 mice (Fig, 2D, D'; CTRL), but in HCb-GFP-LC3 mice, autophagosomal vacuoles, secondary lysosomes, double- membrane structures, and multilamellar bodies were detected in the 10 and Pn neurons (Fig, 2E, E'; HCb-4d) . Double-membraned structures that contained mitochondria, suggestive of mitochondrial degradation by autophagy, were also observed (Fig, E'; HCb-4d) .
The activation of autophagy after HCb was confirmed by analysis of the expression of proteins (Beclin 1, LC3, and p62) that regulate the formation of APs.
Beclin 1 is an evolutionarily conserved protein that promotes autophagy by participating in the nucleation of APs formation. Thus, Beclin 1 protein levels were measured by Western blot (Wb) . Twenty-four hours after HCb, Beclin 1 protein levels increased compared with CTRL, declining slightly at 4 days (Fig. 3A) .
As regards microtubule-associated light chain 3 (LC3), during autophagy, it is proteolyzed, conjugated to phosphatidyl-ethanolamine, and incorporated into structures that become APs . Conversion from nonlipidated LC3 (LC3-I) to lipidated LC3 (LC3-II), detected as a mobility shift by Wb 27, was observed at 2 and 4 days after HCb, differing significantly from CTRL (Fig. 3A) .
Moreover, the expression of p62/SQSTMl (p62) was evaluated. p62 is degraded by autophagy and links ubiquitinated proteins to the autophagic machinery to effect their degradation in lysosomes. Because p62 accumulates when autophagy is inhibited and because decreased levels of p62 can be observed when autophagy is induced, p62 is considered to be a marker of autophagy. Thus, p62 levels were measured by Wb . Two days after HCb, p62 levels declined compared with CTRL; this decrease became more prominent after 4 days (Fig. 3A) .
The morphological and biochemical data demonstrated an increase in APs after axonal damage. However, because APs fuse with and are degraded by lysososmes, the accumulation of APs after HCb can be attributed to increased APs formation or decreased lysososmal degradation. To distinguish between mechanisms, Control and HCb mice were intracerebroventricularly injected with the lysosomal inhibitor chloroquine (CQ) or saline (sal) (Figure 9) . Twenty-four hours after injection, LC3-II protein levels, the number of GFP-LC3 punctae per neuron, and the number of neurons with GFP-LC3 punctae were measured (Fig. 4A-D) .
In control mice, compared with saline treatment, CQ increased the conversion of LC3-I to LC3 II as demonstrated by Western blotting analysis (Fig. 4A) and by counting of the number of GFP-LC3 punctae per neuron (Fig. 4B-C) . After HCb, CQ significantly affected autophagy indices. In HCb+CQ, LC3-II was significantly upregulated compared to HCb+sal (Fig. 4A) . The increase in LC3-II was paralleled by rise in the density of GFP-LC3 punctae per neuron (Fig. 4B-C) , although the number of neurons with GFP-LC3 punctae did not change (Fig. 4D) . These data suggest that HCb induces APs formation, not their degradation .
Mitochondrial damage precedes neuronal autophagy.
Axotomyinduced remote cell death was recently demonstrated to be associated with mitochondrial-dependent apoptosis. Cytochrome-c (cytc) is released by the mitochondria in response to proapoptotic stimuli and is considered an early marker of apoptosis. To examine the interplay between apoptosis and autophagy, the kinetics of mitochondrial cyt-c release and the activation of autophagy after HCb were analyzed. By means of mitochondrial-cytosol ic fractionation, cyt-c was demonstrated to be released into the cytosol starting from 12 h after HCb (Fig. 5A) . The purity of mitochondrial fractionation was confirmed using immunoblot analysis for the presence of marker proteins (MnSOD as mitochondrial marker, actin and GAPDH as cytosolic markers, Fig. SI) . Accordingly to Wb analyses, morphological data proved that the percentage of neurons displaying cytosolic cyt-c release increased starting from 12 h after lesion (Fig. 5B) .
Notably, the kinetics of the two markers — cyt-c and GFP-LC3 — was observed to differ. Mitochondrial cyt-c was released as early as 12 h after HCb (Fig. 5B) , but GFP- LC3-dotted neurons, indicative of autophagy, were detected at 24 h after lesion (Fig. 5B) . Starting from this latter time point, the majority of cyt-c-releas ing neurons also exhibited GFP-LC3 punctae (Fig. 5C) .
Cyt-c release from mitochondria is a crucial step in the activation of apoptotic machinery, and caspase-3 activation is considered an irreversible commitment of the cell toward apoptosis. Thus, the apoptotic pathway downstream of cyt-c release was investigated to assess the fate of axotomized neurons. At early time points (12 h and 24 h) , caspase-3 activity did not differ significantly from control. Caspase-3 activity differences were detected 2 days after HCb and increased significantly 4 days after the lesion (Fig. 5D) .
Further, apoptotic death was confirmed by TdT- mediated dUTP-biotin nick end-labeling (TUNEL) DNA fragmentation analysis. No TUNEL-positivities were detected among 10 or Pn neurons of control animals (Fig. S2A) . By contrast, extensive TUNEL-positive neuronal staining was observed in the 10 and Pn of lesioned animals 2 and 4 days after HCb (Fig. S2B) .
At early time points (12 h and 24 h after HCb), the number of TUNEL-positive neurons did not differ significantly from control (data not shown) . From day 2 after damage, the number of TUNELpositive neurons in the 10 and Pn was significantly higher versus the control (Fig. 5E) .
The kinetic data indicate that autophagy, in acute brain damage, occurs after cyt-c release and before caspase-3 activation and subsequent DNA fragmentation.
Activation of autophagy by rapamicyn is neuroprotective .
To determine the contribution of the activation of autophagy to neuronal degeneration in acute brain damage, autophagy was manipulated using pharmacological and genetic approaches. Control and HCb animals were treated with saline or rapamycin (Figure 9), which enhances autophagy by inactivating mTOR. Beclin 1 heterozygous mice (Becnl ) , which show impaired autophagic responses, and Beclin 1 wild type mice (Becnl+ +) , which display normal autophagic responses, were also used by crossing them with GFP-LC3 mice (Figure 9) .
After HCb, autophagy markers, functional recovery, cyt-c release, and neuronal survival were measured in Becnl+/" and Becnl+/+ GFP-LC3 and in GFP-LC3 mice treated with rapamycin or saline. Compared with HCb-GFP-LC3+sal mice, at each time point, HCb-GFP-LC3-Becnl+ ~ animals showed reduced autophagic activation. No differences between HCb-GFP-LC3+sal and HCb-GFP-LC3- Becnl+/+ were observed (data not shown) . Notably, HCb-GFP-LC3-Becnl+ ~ mice had fewer GFP-LC3- dots per neuron in 10 and Pn compared with HCb-GFP-LC3+sal mice (Fig. 6A) . This autophagy reduction was confirmed by analysis of LC3 and p62 by Wb. In HCb-GFP-LC3-Becnl+/" mice, LC3-II/LC3-I conversion decreased and p62 levels rose compared with HCb-GFP-LC3+sal mice (Fig. 6B) . Rapamycin treatment, as expected, induced mTOR inactivation as shown by the reduction in the phosphorylation of downstream substrates of mTOR, namely p70S6K and S6RP (compare to HCb+sal vs HCb+rap mice in Fig. S3) . Furthermore, treatment with rapamycin (HCb-GFP-LC3+rap mice) increased all autophagy markers compared with HCb-GFP-LC3+sal mice at all time points (Fig 6A-B) . In HCb-GFP-LC3+rap mice, an increase of the number of LC3-GFP dots per neuron and of LC3-II/LC3-I conversion (and a decrease in p62 expression) was observed compared with HCb-GFP-LC3+sal mice (Fig. 6B).
Moreover, compared with HCb-GFP-LC3+sal animals, HCb- GFP-LC3-Becnl+ ~ experienced increases in cyt-c release (Fig. 7A) , higher neuronal death (Fig. 7B) , and worse functional recovery, as shown by the Neurological Severity Score (NSS - Fig. 7C) .
Notably, the increase in autophagy activation after rapamycin treatment was associated with a significant reduction in cyt-c release (Fig. 7A) , higher neuronal survival (Fig. 7B) , and greater functional recovery (NSS; Fig. 7C) , consistent with the neuroprotective role of autophagy in remote degeneration after acute brain damage.
Further, to exclude possible autophagy-independent effects, Becnl+ ~ mice were treated with rapamycin to evaluate whether this treatment was able to enhance or to fail autophagy activation and neuronal survival in mice with impaired autophagy responses (Figure 9) .
After 4 days of treatment, autophagy and the recovery indices were evaluated. In Becnl+ ~ mice, compared with saline treatment, rapamycin did not affect autophagy both in CTRL and HCb mice. Quantitative analysis of LC3/GFP punctae per neuron, Wb of LC3, neuronal survival and functional recovery as measured by NSS were unresponsive of rapamycin treatment (compare CTRL-Becnl+ ~+sal vs CTRL- Becnl+/"+rap and HCb-GFP-LC3-Becnl+/"+sal vs HCb-GFP-LC3- Becnl+/"+rap in Fig. 8A-D) .
These results demonstrate that, in contrast to what was observed in wild-type mice, rapamycin does not increase autophagy or cell survival in HCb-Becnl+ ~ mice, supporting a model in which the neuroprotective role of rapamycin depends on its ability to enhance autophagy.
Herein, it is demonstrated that acute brain lesions activate autophagy in axotomized neurons and that rapamycin-enhanced autophagy reduces neuronal death supporting functional recovery.
Further, the cascade of events that link the early stages of mitochondrial dysfunction to cell death, are described, identifying the time frame in which neuronal autophagy is active. These results support the idea that autophagy machinery is activated in response to mitochondria sufferance. When damaged mitochondria release cytochrome-c (cyt-c) into the cytosol, autophagy is activated possibly to engulf the suffering mitochondria thus neutralizing pro-apoptotic factors release. It can be hypothesized that reduced activation of autophagy reduces the clearance of damaged mitochondria thus allowing continuous cyt-c release into the cytosol leading to a further activation of caspase-3 and ultimately cell death. By enhancing autophagy, it is possible to support functional recovery, and a reduction in autophagic activation is associated with increased cell death and impaired functional recovery. This model shows that autophagy is a protective mechanism that attempts to counteract axotomy induced degeneration but, at least for precerebellar neurons, it is incapable of preserving neuronal survival. In line with reduced cellular damage, better survival and better recovery is achieved when autophagy is pharmacologically enhanced.
Many autophagy studies have been performed in recent years, and a consensus on the parameters that should be used to examine autophagy has been reached. In this study, the activation of autophagy was analyzed morphologically, ultrastructurally, and biochemically in strict compliance with the "Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes".
A large percentage of CNS pathologies present acute focal brain lesions and their consequences have been studied in various animal models. The HCb model is well established, in which neuronal degeneration can be analyzed remotely from the primary site of damage, where effects of the primary lesion can confound the study of degeneration. These remote phenomena can be used to exploit therapeutic approaches, because they are active long after the primary damage has subsided and influence postlesion impairments. The time and location in which autophagy is activated are critical to the understanding of its relationships with apoptosis. These data, which are consistent with those of previous studies implicate cyt-c release as an early event of the neuronal reaction to axonal damage. The strict kinetics of the activation of autophagy and the reduction in cyt-c release after enhanced autophagy suggest that cyt-c release triggers the activation of autophagy .
Cyt-c release from the mitochondria is the most logical target of the neuroprotective effect of autophagy. These data on rapamycin and Becnl+ ~ mice support this interpretation. Rapamycin protects against neurodegeneration, an effect that is linked presumably to enhanced autophagic clearance of damaged mithocondria . Similarly, in this model, rapamycin treatment was associated with reduced cyt-c release and greater neuronal survival. Further, by histological and molecular analysis, HCbBecnl+ ~ mice - with reduced autophagic response - showed inefficient autophagy-dependent clearance of mitochondria, which was associated with both poor neuronal survival and functional recovery.
Moreover, the lack of efficacy of rapamycin in mice with impaired autophagic response, as HCbBecnl+ ~ mice, implicates autophagy mechanisms as the chief target of rapamycin-associated neuroprotective effects. Moreover, in this study, APs clearance was prevented by intracerebroventricularly injection of chloroquine to determine whether APs accumulation after HCb is due to an increase in their formation or reduced degradation. Based on the morphological and biochemical indices of autophagy activity, after HCb, APs were actively formed and efficiently cleared, demonstrating that autophagic flux was not impaired.
A growing body of evidence suggests that alteration or dysfunction of autophagy has been implicated in several neurodegenerative disorders, including Alzheimer's disease and Parkinson's diseases.
Accelerating the autophagic removal of toxic accumulation is proposed to be a therapeutic strategy for neurodegenerative disorders. These data clearly demonstrate that autophagy not only is a long-term homeostatic process but also intervenes in the early stages of neuronal damage. Acute pathologies, such as stroke and brain trauma, would benefit tremendously if early autophagy mechanisms are targeted therapeutically.
Material and Methods
Animals and surgery. Experiments were performed using male adult mice (Figure 9) (weight 20-25 g) . C57BL6 mice (10-12 weeks of age) were obtained from Harlan Italy, and GFP-LC3 transgenic mice were obtained from Dr. N. Mizushima. The GFP-LC3 mice contain a transgene in which LC3 is fused to GFP and a CAG promoter. Homozygous and heterozygous Beclinl mice were obtained from Dr. B. Levine. Further, the Beclinl homozygous and heterozygous mice were crossed to GFP-LC3 transgenic mice (Figure 9) .
The experimental protocol was approved by the Italian
Ministry of Health per the guidelines of the European Communities Council Directive, November 24, 1986 ( 86 /609 /EEC ) , for the care and use of laboratory animals. All efforts were made to minimize the number of animals used and their suffering. Cerebellar lesions were induced by performing a right hemicerebel lectomy (HCb) .
For surgical procedures, the mice were deeply anesthetized by i.p. injections of xylazine (10 mg/ml) and tiletamine e zolazepam (50 mg/ml) and positioned in a stereotaxic apparatus. An incision was made in the skin on the skull, and the occipital bone was drilled and removed. Subsequently, the dura was incised to expose the cerebellum, and the right cerebellar hemisphere was removed by suction. The wound was sutured, and the animals were returned to their cages. For the CTRL group, surgery was interrupted after the dura lesion was made, and after suturing, the animals were returned to their cages. The experimental groups are detailed in Figure 9.
Drug treatment. Rapamycin (Rap; Alexis Biochemicals , 380-004-M001 ) was dissolved in DMSO (25 mg/ml) and injected once daily (2 mg/Kg; i.p.) . Chloroquine diphosphate-salt (Chlor; Sigma, C6628) was dissolved in sterile saline and injected intracerebroventricular ly (i.e. v.; 25 mg/Kg) 60 min before hemicerebellectomy .48 Treatments in the different experimental groups are shown in detail in Figure 9.
Neurological evaluation. Neurological impairment was evaluated by the Neurological Severity Score (NSS) (18) . The Neurologic Severity Score (NSS) is a composite of motor, sensory, reflex, and balance tests, in which for each test, one point is awarded for the inability to perform or for the lack of a tested reflex, and zero points are awarded for success. An NSS of 18 indicates severe injury, whereas a score of zero signifies healthy, uninjured rats. The NSS was evaluated at 1, 2, and 4 days after damage by an investigator who was blinded to the experimental groups .
Histology and immunohistochemistry. Control and lesioned animals, after various postlesional survival times (6h, 12h, 24h, 2d and 4d) , were perfused transcardially with 50 ml of saline followed by 50 ml of 4% paraformaldehyde in phosphate buffer (PB; 0.1 M; pH 7.4) under renewed general anesthesia induced by i.p. injections of sodium pentobarbital (60 mg/kg) . Each brain was removed fromthe skull, post-fixed in the same fixative for 2 h and then transferred to 30% sucrose in PB at 4 °C until it sank. Brainstem and cerebellum were cut into using a freezing microtome and collected in PB .
The sections were Nissl-stained to assess the number of surviving neurons . Finally, sections were mounted on chrome alum-coated slides, air dried, dehydrated with ethanol, cleared in xylene, and coverslipped. Bright field images were taken using a light microscope (Zeiss, Axioskop 2) equipped with a digital camera (Nikon, Coolpix 990) .
To assess autophagy activation, one series of sections was incubated overnight with a cocktail of primary antibodies, including mouse anti-neuronal nuclei (NeuN; 1:200; Millipore, MAB-377), rabbit anti-Glial fibrillary acidic protein (GFAP; 1:500; Dako, #Z0334) and mouse anti-OX-42 (1:200; Serotec, #MCA275G). All primary antibody solutions were prepared in PB and 0.3% Triton X- 100 and incubated overnight. Each incubation step was followed by three, 5-min rinses in PB . Afterwards, sections were incubated 2h at RT with a cocktail of secondary antibodies, including Alexa Fluor 555 donkey anti-rabbit IgG (1:200; Molecular Probes, #A31572) and Alexa Fluor 647 donkey anti-mouse IgG (1:200; Molecular Probes, #31571) .
For each animal, a series of sections were processed to investigate the effects of lesion and treatment on the release of cytochrome-c (Cyt-c) . Sections were incubated with the following primary antibody goat anti-Cyt-c (1:500; Santa Cruz Biotechnology, #sc-8385). Afterwards, sections were incubated 2h at RT with a Alexa Fluor 555 donkey anti-goat IgG (1:200; Molecular Probes, #21432).
Sections were examined under a confocal laser scanning microscope (ZEISS, LSM700; Germany) equipped with four laser lines: violet diode emitting at 405 nm for DAPI, argon emitting at 488 nm and helium/neon emitting at 543 nm and 633 nm. Plates were generated adjusting the brightness and contrast of digital images (Corel Draw, 9) .
Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TU EL) assay. TUNEL assay was performed on 15 μηι fresh frozen coronal sections at the level of 10 and Pn by using the ApopTag Fluorescein In Situ Apoptosis Detection Kit from Millipore (#S7110) . The assay was carried out according to manufacturer's instructions. After being washed in PBS, the sections were incubated overnight with a solution of PBS+0.4% Triton-X-100 containing mouse-anti NeuN (Millipore, MAB-377), DAPI (Sigma #D9542) counter-stained and coverslipped with Fluoromount (Sigma, #F4680) .
Fluorometric assay of caspase-3 activity. The left 10 and Pn were punched. The tissues were homogenized separately in lysis assay buffer (100 mM Hepes pH 7.4, 0.1% Chaps, 1 mM EDTA, 10 mM DTT, 1 mM PMSF) and lysed by subsequent freezing in liquid N2 and thawing at 37°C three times. After centrifugation at 11, 500 x g for 5 min, the protein concentration of resulting supernatant was quantified, and proteins were incubated at 37°C in lysis assay buffer containing 50 μΜ Ac-DEVDAMC (BD Pharmingen, #556449) . The fluorescence was measured at an excitation and emission wavelength of 380 nm and 460 nm, respectively. For further details see ref.
Mitochondrial and cytosolic fraction. Precerebellar nuclei (10 and Pn separately) were homogenized in Buffer A (320 mM sucrose, 1 mM EDTA, 50 mM Tris-HCl pH7.4, 1 mM DTT, 1 mM PMSF, with protease inhibitor cocktail - Sigma, #P8340) by 30 strokes with a glass Pyrex micro homogenizer. The homogenate was centrifuged at 1,000 χ g for 10 min and the resulting supernatant was centrifuged at 10, 000 x g for 20 min to obtain the mitochondrial pellet and the supernatant. The mitochondria-containing pellet was washed three times with Buffer B (250 mM sucrose, 1 mM EGTA, 10 mM TrisHCl pH7.4) by centrifugation for 10 min at 10, 000 x g. The supernatant was centrifuged at 100,000 χ g for 1 hr to generate the cytosolic fraction .
Protein isolation and Western blot. Precerebellar nuclei (10 and Pn separately) were homogenized in lysis buffer (320 mM sucrose, 10% Glycerol, 50 mM NaCl, 50 mM Tris-HCl pH 7.5, 1% Triton X-100, 1 mM PMSF, with protease inhibitor cocktail (Sigma, #P8340), incubated on ice for 30 min and centrifuged at 13,000 x g for 20 min. The total protein content of resulting supernatant was determined. Proteins were applied to SDS-PAGE and electroblotted on a PVDF membrane. Immunoblott ing analysis was performed using a chemiluminescence detection kit. The relative levels of immunoreactivity were determined by densitometry using the software ImageQuant 5.0. Samples were incubated with the following primary antibodies: rabbit polyclonal anti-p62 (1:1000; MBL International, #PM045); mouse monoclonal anti-LC3 (1:250; Nanotools, Teningen, #0260-100); mouse monoclonal anti-Beclin 1 (1:1000; BD Transduction Laboratories, #612113); mouse monoclonal antiCytochrome-c , (1:1000; BD Pharmingen, #556433); rabbit polyclonal anti- MnSOD (1:1000; Stressgen, # SOD-110); mouse monoclonal anti-phospho-p70 S6 Kinase (Thr389) (1A5) (1:1000; Cell Signaling #9206); mouse monoclonal anti-S6 Ribosomal Protein (54D2) (1:1000; Cell Signaling #2317); rabbit polyclonal anti-Ribosomal Protein S6 (pSpS235/236 ) phosphospecif ic antibody (1:1000; Invitrogen #44-922G) ; mouse monoclonal anti- GAPDH (1:1000; Calbiochem #CB1001); rabbit polyclonal antiB-actin (1:10,000; Sigma, #A5441) .
Quantitative and statistical analysis. Qualitative and quantitative observations were limited to the inferior olive (10) and pontine nuclei (Pn) of the experimental side; i.e., projecting to the lesioned hemicerebellum (Fig. 1) . Using the Stereo Investigator System (MicroBrightField Europe e.K., Magdeburg, Germany) an optical fractionator stereological design was applied to obtain unbiased estimates of total NeuN+, LC3+ and TUNEL+ neurons of 10 and Pn . A stack of MAC 5000 controller modules (Ludl Electronic Products, Ltd. Hawthorne, NY, USA) was configured to interface an Olympus BX 50 microscope with a motorized stage and a HV-C20 Hitachi colour digital camera with a Pentium II PC workstation. A three-dimensional optical dissector counting probe (x, y, z dimension of 30 x 30 x 10 μηι respectively) was applied. The two nuclei were outlined using the 4x objective, while the lOOx oil immersion objective was used for marking the neuronal cells. Total 10 and Pn cell number was estimated according to the formula given below:
N = ∑Q x 1/ssf x 1/asf x 1/tsf.
where ∑Q represents the total number of neurons counted in all optically sampled fields of the 10 and Pn, ssf is the section sampling fraction, asf is the area sampling fraction and tsf is the thickness sampling fraction.
To assess the number of 10 and Pn neurons presenting cyt-c release and GFP-LC3 dots, quantitative analyses were performed off-line on confocal images acquired through the 20x objective at the 0.07 zoom factor. For the 10, all labeled cells in a square box (200 μηι wide x 200 pm long), randomly positioned in five regularly spaced sections, were counted. For the Pn, quantitative data were obtained by adopting a different sampling strategy. Three digital square frames (200 x 200 μηι) were placed at a regular distance to sample the entire medio-lateral extent of the Pn .
All quantitative analyses were conducted blind to the animal's experimental group assignment.
All values were expressed as mean ± SD. Differences between means were analyzed by one- or two-way analysis of variance (ANOVA) followed by Bonferroni post hoc multiple comparison (Prism-5 software GraphPAD Software for Science, San Diego, CA) . Differences were considered significant at P < 0.05.
Morphometric analysis of GFP-LC3-positive punctae. In the various experimental groups, GFPLC3 punctae in the neuronal cell body were analyzed with a 63X oil immersion objective, and photographs were taken on a confocal laser scanning microscope (ZEISS, LSM700; Germany) . Cells were classified as neurons with vesicular structures or neurons with no punctae (diffuse GFP-LC3 only) . Only cells with detectable punctate GFP-LC3 staining (in the soma, axon, or both) were counted. GFP-LC3 punctae were counted manually in 150 cells per group (n=5 mice per group) . The GFP-LC3 punctae cell ratio was the mean number of GFP-LC3 punctae in individual cells, normalized to the total number of neurons. All quantitative analyses were performed blind to the animal's experimental group.
Electron microscopy. Animals were transcardially perfused with a calcium-free Ringer's variant (pH 7.3), followed by 2% freshly depolymerised paraformaldehyde and 1% glutaraldehyde in 0.12 M phosphate buffer (PB) , pH 7.4. Brains were dissected out and cut on a vibratome in the coronal plane, to obtain 100 μηι thick sections which were collected in PB, pH 7.4. Slices were post-fixed in 1% osmium tetroxide in PB for 1 hr at 4 °C, in the dark, then gradually dehydrated in ethanol . All the steps of the above procedure were carried out at 4 °C. Sections were infiltrated with graded mixtures of propylene oxide and Epon812 (TAAB, Reading) , then flat-embedded in the same resin, allowing specimens to polymerize at 60 °C, for 3 days. Selected areas, namely 10 and Pn, were then remounted on Epon blanks and sectioned by a Reichert Ultracut S ultramicrotome (Leica Microsystems), to obtain ultrathin sections (60-70 μηι) which were collected on nickel grids. These were briefly contrasted with 1% uranyl acetate and observed in a Philips CM120 electron microscope, equipped with a Philips Megaview III videocamera. Images were electronically captured by AnalySys 2.0 software and composed in an Adobe Photoshop CS3 format. REFERENCES
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Claims

1. An autophagy enhancing compound or a pharmaceutical composition comprising said compound, for use in the treatment of neuronal diseases.
2. The compound or composition according to claim 1, wherein the neuronal disease is a central nervous system disease .
3. The compound or composition according to claim 1 or 2, wherein the disease is a chronic neurodegenerative disease or an acute focal lesion.
4. The compound or composition according to claim 3, wherein the chronic neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease and Batten's disease.
5. The compound or composition according to claim 3, wherein the acute focal lesion is selected from the group consisting of stroke, spinal or brain trauma, and multiple sclerosis .
6. The compound or composition according to any of the preceding claims, wherein the compound increases the autophagic clearance of damaged mithocondria.
7. The compound or composition according to any of claims 1 to 5, wherein the compound reduces the release of cytochrome-c in cells.
8. The compound or composition according to any of claims 1 to 5, wherein the compound induces mammalian target of rapamycin (mTOR) inactivation .
9. The compound or composition according to any of claims 1 to 5, wherein the compound is selected from the group consisting of rapamycin, CCI-779, Glc, Glc-6-P, Torinl, perhexiline, niclosamide, rottlerin, lithium, L- 690,330, carbamazepine , sodium valproate, verapamil, loperamide, amiodarone, nimodipine, nitrendipine, niguldipine, pimozide, calpastatin, calpeptin, clonidine, rilmenidine, 2 ', 5 ' -dideoxyadenos ine , NF449, minoxidil, penitrem A, fluspirilene, trifluoperazine, trehalose, SMER10, SMER18, SMER28, and SMER analogs.
10. The compound or composition according to claim 9, wherein the compound is rapamycin.
11. The compound or composition according to any of claims 1 to 5, wherein the compound is a peptide comprising a sequence having SEQ ID NO : 1 , SEQ ID NO : 2 or SEQ ID NO : 3 or a peptidomimetic compound of said peptides.
12. The compound or composition according to claim
11, wherein the compound is a peptide of SEQ ID NO : 1 , SEQ ID NO: 2 or SEQ ID NO : 3.
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