WO2023242844A1 - Method and therapeutic agent for treatment of disease or disorder associated with impaired firing rate and/or mitochondrial calcium homeostasis - Google Patents
Method and therapeutic agent for treatment of disease or disorder associated with impaired firing rate and/or mitochondrial calcium homeostasis Download PDFInfo
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- WO2023242844A1 WO2023242844A1 PCT/IL2023/050612 IL2023050612W WO2023242844A1 WO 2023242844 A1 WO2023242844 A1 WO 2023242844A1 IL 2023050612 W IL2023050612 W IL 2023050612W WO 2023242844 A1 WO2023242844 A1 WO 2023242844A1
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Definitions
- Neural circuits are composed of a large number of dynamic elements at various levels of organization. The operation of a neuronal circuit depends on the interaction between the intrinsic properties of the individual neurons and the synaptic interactions that connect them into functional ensembles. While some aspects of synaptic and spiking activity are dynamic, others show remarkable stability over long time periods (Chambers et al., 2017). Despite a large variability in synaptic and intrinsic parameters, firing rate distributions and their mean firing rate (MFR) are maintained at a specific set-point value during ongoing spontaneous activity. MFRs are typically restored even in the presence of large perturbations to activity rates and patterns.
- MFR mean firing rate
- MFR homeostasis can be achieved by a wide repertoire of homeostatic processes, including adjustments of synaptic strength, intrinsic excitability, and excitation-to-inhibition balance (Davis and Muller, 2015; Turrigiano, 2011). Dysregulation of homeostatic plasticity has been proposed to drive synaptic and cognitive deficits in distinct brain disorders, including neurodevelopmental disorders (Kavalali and Monteggia, 2020) and neurodegenerative disorders like Alzheimer’s disease (Frere and Slutsky, 2018).
- cytoCa 2+ Intracellular somatic cytosolic [Ca 2+ ] (cytoCa 2+ ) has been proposed to serve as a proxy of spiking activity because of their tight coupling and is therefore modeled as a feedback control signal (O’Leary et al., 2014). According to these models, deviations from a specific target cytoCa 2+ induce changes in effector proteins that result in renormalization of firing properties to a set point value.
- cytoCa 2+ in excitatory neurons returns to set-point value following sensory deprivation in vivo (Barnes et al., 2015) and following neuronal inactivity ex vivo (Slomowitz et al., 2015).
- IGF1R Insulin-like growth factor- 1 receptor
- a method for treatment of a disease or disorder associated with impaired MFR and/or mitoCa 2+ homeostasis e.g., a neurodevelopmental disorder such as Phelan-McDermid syndrome, Rett syndrome and autism, or neurodegenerative disease or disorder such as Alzheimer’s disease and Parkinson’s disease, in an individual in need thereof, said method comprising administering to said individual a therapeutically effective amount of a therapeutic agent selected from:
- mitochondria targeted-insulin-like growth factor- 1 receptor mitoIGFIR
- the therapeutic agent administered is a nucleic acid molecule encoding for the expression of either mitoIGFIR or a mitochondria targeted- portion thereof; or MCU optionally together with an additional subunit of the MCU complex.
- Nucleic acid molecules as referred to herein, each independently may be a cDNA or RNA, e.g., a cDNA-based plasmid, or a viral vector selected from, e.g., retrovirus, adenovirus, adeno-associated virus, poxvirus, alphavirus, herpes virus, and lentivirus.
- a viral vector selected from, e.g., retrovirus, adenovirus, adeno-associated virus, poxvirus, alphavirus, herpes virus, and lentivirus.
- a pharmaceutical composition comprising a therapeutic agent as defined above, encapsulated, e.g., within a capsule made of a phospholipid and/or a polymer, or within a viral- or viral-like-envelope, and a pharmaceutically acceptable carrier and/or excipient.
- the therapeutic agent and pharmaceutical composition disclosed herein are useful in the treatment of diseases or disorders associated with impaired MFR and/or mitoCa 2+ homeostasis, e.g., neurodevelopmental disorders such as Phelan-McDermid syndrome, Rett syndrome and autism, or neurodegenerative diseases or disorders such as Alzheimer’s disease and Parkinson’s disease.
- diseases or disorders associated with impaired MFR and/or mitoCa 2+ homeostasis e.g., neurodevelopmental disorders such as Phelan-McDermid syndrome, Rett syndrome and autism, or neurodegenerative diseases or disorders such as Alzheimer’s disease and Parkinson’s disease.
- a therapeutic agent as defined above encapsulated, e.g., within a capsule made of a phospholipid and/or a polymer, or within a viral- or viral-like-envelope, for use in the treatment of a disease or disorder associated with impaired MFR and/or mitoCa 2+ homeostasis.
- FIGs. 3A-3G show that lack of intrinsic excitability and postsynaptic homeostatic adaptations in IGF1R-KO neurons.
- (3B) Representative traces of Ctrl neurons before (left) and after (right) Bac2d.
- 3D Representative traces of IGF1R-KO neurons before (left) and after (right) Bac2d.
- 3E Representative mEPSC recordings of neurons from each group (scale bars: 1 s, 20 pA).
- Figs. 4A-4F show that IGF1R deletion decreases somatic mitoCa 2+ and cytoCa 2+ - to-mitoCa 2+ coupling evoked by spike bursts.
- Figs. 5A-5G show that IGF1R is present in brain mitochondria and is colocalized with MCUc within neurons.
- 5A Western blot (WB) analysis of mouse brain lysates and purified mitochondria (free mitoch.). The mitochondria are strongly enriched in the marker protein Cox4-1. The bands demonstrate the presence of the IGF1R protein in or on mitochondria.
- 5B Samples were treated with trypsin to cleave all proteins from the outer membranes of the organelles in the samples. Remarkably, a prominent IGF1R band is still visible in mitochondria after this treatment, suggesting that a significant proportion of IGF1R is present within mitochondria. The experiments are shown in triplicate, to indicate experimental reproducibility.
- (5E’) Enlarged views of the delineated areas alongside the filled arrows depict examples of colabeling of IGF1R and the MCUc proteins. Scale bars: 5 pm.
- (5F) Square regions of interest (ROIs) were obtained for all MCUc spots, both in the MCUc and in the IGF1R channels. The ROIs were then overlaid, which provides a visual indication of the presence of IGF1R in relation to MCUc spots. The arrows in the images point to the ROI centers, where the MCUc spots are located, and where an enrichment of IGF1R is also observed.
- IGF 1 -bound IGFIRs in neuronal mitochondria were detected, co-localized with mitochondrial calcium uniporter (MCU) and other members (subunits) of the MCU complex (MCUc).
- MCU mitochondrial calcium uniporter
- MCUc mitochondrial calcium uniporter
- Deletion of IGFIRs did not alter spike-to-cytoCa 2+ coupling, but weakened burst-to-mitoCa 2+ coupling by downregulating transcription of several MCUc members.
- a pronounced increase in the fraction of spike bursts was detected in the initial phase of the perturbation, this change in spike pattern was lost in IGFIR-deficient neurons.
- mitochondria targeted-insulin-like growth factor- 1 receptor (mitoIGFIR) agonist refers to any agent capable of agonizing the mitochondrial IGF1 receptor.
- an agent may be, e.g., a protein or peptide, as well as a small molecule, but it is preferably a short protein or a peptide.
- said mitoIGFIR agonist is IGF1, more specifically a human IGF1 (SEQ ID NO: 1).
- such a variant protein or peptide has an amino acid sequence that is at least 85%, preferably 90%, and most preferably 95%, 99%, or more, identical to the amino acid sequence of IGF1 from which it is derived, or said fragment thereof.
- Particular fragments of IGF1 or analogues thereof for use as mitoIGFIR agonists are peptides comprising the sequence glycine -proline-glutamate (GPE tripeptide) or an analog thereof such as Gly-l-methylPro-Glu, as the amino -terminal thereof.
- IGF1 fragment consists of the sequence Gly-Pro-Glu
- IGF1 analogue consists of the sequence Gly-l-methylPro-Glu (glycyl-alpha-methyl-L-prolyl-L- glutamic acid; (2S)-2- ⁇ [(2S)-l-(2-aminoacetyl)-2-methylpyrrolidine-2-carbonyl]amino ⁇ pentanedioic acid; trofinetide).
- Preferred mitoIGFIR agonists for use according to the present invention are IGF1, the tripeptide GPE, and trofinetide.
- Non-limiting examples of non-natural amino acids include citrulline (Cit), diaminopropionic acid (Dap), diaminobutyric acid (Dab), ornithine (Om), aminoadipic acid, P-alanine, 1 -naphthylalanine, 3-(l-naphthyl)alanine, 3- (2-naphthyl)alanine, y-aminobutiric acid (GABA), 3 -(aminomethyl) benzoic acid, p- ethynyl-phenylalanine, m-ethynyl-phenylalanine, p-chlorophenylalanine (4ClPhe), p- bromophenylalanine, p-iodopheny lalanine, -accty Ipheny lalanine, p-azidopheny lalanine, p- propargly-oxy-phenylalanine, ind
- peptide bond or "amide bond” as used herein refers to the covalent bond -C(O)NH- formed between two molecules, e.g., two amino acids, when a carboxyl group of one of the molecules reacts with an amino group of the other molecule, causing the release of a water molecule.
- nucleic acid molecule refers to nucleic acid, DNA or RNA, that comprises noncoding or coding sequences. Coding sequences are necessary for the production of a polypeptide or protein precursor. A polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence, as long as the desired protein activity is retained. Noncoding sequences refer to nucleic acid molecules which do not code for a polypeptide or protein precursor, and may include regulatory elements such as transcription factor binding sites, poly(A) sites, restriction endonuclease sites, stop codons and/or promoter sequences.
- a “nucleic acid”, as used herein, is a covalently linked sequence of nucleotides in which the 3' position of the pentose of one nucleotide is joined by a phosphodiester group to the 5' position of the pentose of the next, and in which the nucleotide residues (bases) are linked in specific sequence, i.e., a linear order of nucleotides.
- a “polynucleotide”, as used herein, is a nucleic acid containing a sequence that is greater than about 100 nucleotides in length.
- Nucleic acid molecules are said to have a “5 '-terminus” (5' end) and a “3 '-terminus” (3' end) because nucleic acid phosphodiester linkages occur to the 5' carbon and 3' carbon of the pentose ring of the substituent mononucleotides.
- the end of a polynucleotide at which a new linkage would be to a 5' carbon is its 5' terminal nucleotide; and the end of a polynucleotide at which a new linkage would be to a 3' carbon is its 3' terminal nucleotide.
- a terminal nucleotide, as used herein, is the nucleotide at the end position of the 3'- or 5'- terminus.
- DNA molecules are said to have “5' ends” and “3' ends” because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotides referred to as the “5' end” if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the “3' end” if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring.
- a nucleic acid sequence even if internal to a larger oligonucleotide or polynucleotide, also may be said to have 5' and 3' ends.
- discrete elements are referred to as being “upstream” or 5' of the “downstream” or 3' elements. This terminology reflects the fact that transcription proceeds in a 5' to 3' fashion along the DNA strand.
- promoter and enhancer elements that direct transcription of a linked gene are generally located 5' or upstream of the coding region.
- enhancer elements can exert their effect even when located 3' of the promoter element and the coding region.
- the therapeutic agent administered according to the method disclosed is a nucleic acid molecule encoding for the expression of either mitoIGFIR or a mitochondria targeted -portion thereof; or MCU optionally together with an additional subunit of the MCU complex.
- the nucleic acid molecule encoding for the expression of either mitoIGFIR or a mitochondria targeted-portion thereof is an expression vector comprising a sequence encoding IGF1R (SEQ ID NO: 2) or a portion thereof, fused to a sequence encoding a MTS.
- said expression vector comprises a sequence encoding IGF1R (SEQ ID NO: 2) fused to a sequence encoding a MTS.
- said expression vector comprises a sequence encoding a portion of IGF1R fused to a sequence encoding a MTS.
- said expression vector comprises a sequence encoding IGF1R (SEQ ID NO: 2) or a portion thereof, fused to both a sequence encoding CD8 (SEQ ID NO: 3) and a sequence encoding a MTS.
- the fusion to a sequence encoding a MTS is necessary so as to direct, i.e., target, the IGF1R encoded or said portion thereof to the mitochondria.
- mitochondria-targeting sequence refers to a short peptide, e.g., about 15-70 amino acids long, bearing positively charged basic residues, that directs the transport of a protein to the mitochondria, i.e., targets said protein to the mitochondria.
- Such a sequences is usually located at the N-terminal of a given protein, consisting of an alternating pattern of hydrophobic and positively charged residues that form an amphipathic helix.
- mitochondrial-targeting sequences include, without being limited to, 4mt (SEQ ID No: 4) as well as the sequence disclosed in Faria et al., 2021, Hurt et al., 1985 (SEQ ID NO: 5), and the sequence disclosed in Obita et al., 2003 (SEQ ID No: 6).
- the nucleic acid molecule encoding for the expression of MCU optionally together with said additional subunit of the MCU complex is an expression vector comprising a sequence encoding MCU and optionally a sequence encoding said additional subunit of the MCU complex.
- said expression vector comprises a sequence encoding MCU (SEQ ID NO: 7).
- said expression vector comprises a sequence encoding MCU (SEQ ID NO: 7) and a sequence encoding MICU 1 (SEQ ID NO:8).
- said expression vector comprises a sequence encoding MCU (SEQ ID NO: 7) and a sequence encoding MICU3 (SEQ ID NO:9).
- said expression vector comprises a sequence encoding MCU (SEQ ID NO:7), a sequence encoding MICU1 (SEQ ID NO:8), and a sequence encoding MICU3 (SEQ ID NO:9).
- a nucleic acid molecule encoding for the expression of MCU optionally together with an additional subunit of the MCU complex as referred to hereinabove does not have to comprise a sequence encoding a MTS, as both the sequence encoding MCU as well as each one of the sequences encoding the other MCU complex subunits each comprises an inherent sequence aimed at targeting the MCU complex subunit encoded to the mitochondria.
- the therapeutic agent administered according to the method disclosed herein is an expression vector comprising a sequence encoding IGF1R or a portion thereof, fused to a sequence encoding a MTS; or an expression vector comprising a sequence encoding MCU optionally together with an additional subunit of the MCU complex.
- said sequence according to any one of the embodiments above, each independently is a complementary DNA (cDNA) or RNA.
- said sequence each independently is a cDNA; and said expression vector is a plasmid, e.g., an adeno-associated virus (AAV) plasmid such as the AAV2-CBAP plasmid exemplified herein (this plasmid has all other default components of an AAV plasmid as defined, e.g., in the Adeno- Associated Virus Guide of Addgene (https://www.addgene.org/guides/aav/), wherein the features distinguish this plasmid are the promoter (CBAP) and the serotype (2)).
- AAV adeno-associated virus
- said expression vector each independently is a viral vector selected from, e.g., retrovirus, adenovirus, adeno-associated virus, poxvirus, alphavirus, herpes virus, and lentivirus.
- Gene therapy methods and methods of delivering genes to subjects are well known, and description thereof may be found, e.g., in WO 2013/142114, WO 2014/059029, WO 2014/059031, WO 2014/093622, WO 2014/127198, US 2014/0100265, and US 7,977,049, each of which is incorporated herein by reference in its entirety.
- AAV adeno-associated virus
- the present invention provides gene therapy methods, such as combinational gene therapy methods, to provide or regulate one or more endogenous proteins.
- gene therapy methods are aimed at treating or preventing a disease or disorder associated with impaired MFR and/or mitoCa 2+ homeostasis, by restoration of MFR and/or mitoCa 2+ homeostasis.
- the invention provides the identification of genes related to diseases and disorders associated with impaired MFR and/or mitoCa 2+ homeostasis, and further the regulation of such genes either by increasing a protein related to a gene or decreasing a protein related to a gene.
- the invention provides gene therapy methods for increasing mitoCa 2+ influx by introducing a nucleic acid encoding the functional protein which is expressed within a cell. Generally, such an expression results in the increased mitoCa 2+ influx by direct or indirect regulation of MCU complex activity.
- mitoCa 2+ influx is increased by mitoIGFIR or MCU complex subunits.
- mitoCa 2+ influx is increased by a mitoIGFIR agonist, e.g., human IGF1 (SEQ ID NO: 1) or a fragment, analogue or variant thereof capable of agonizing IGF1R.
- the present invention provides gene therapy methods for the regulation of one or more or a plurality of genes or their associated functional proteins in a method of treating or preventing diseases or disorders associated with impaired MFR and/or mitoCa 2+ homeostasis.
- the gene therapy can be based on, e.g., one or more of a nucleic acid or gene which overexpress a functional protein or a mutant form thereof; expression of a functional protein which regulates another target gene/protein; expression of polynucleotides, such as inhibitory RNA, to regulate expression of a target gene; and expression of gene editing systems that modify in situ the target gene.
- a nucleic acid can be a "synthetic nucleotide sequence", i.e., a nucleotide sequence which does not occur as such in nature, but was rather designed, engineered and/or constructed by human intervention.
- the method of the present invention is aimed at treating a disease or disorder associated with impaired MFR and/or mitoCa 2+ homeostasis, by restoration of MFR and/or mitoCa 2+ homeostasis, and the therapeutic agent administered thus has to be delivered into the cells, more particularly into the neuronal cells.
- said therapeutic agent is encapsulated within a capsule capable of delivering said therapeutic agent into the cell, more specifically, integrating with the cell membrane, following which it is opened and consequently release its content, i.e., the therapeutic agent, within/into the cell.
- a capsule may be made of, e.g., a phospholipid and/or a polymer; or may be a viral- or viral-like-envelope.
- the therapeutic agent administered is encapsulated within a phospholipid-based capsule.
- phospholipids include, without being limited to, a lecithin such as egg or soybean lecithin, or a derivative thereof, e.g., a lecithin having polyethylene glycol (PEG) chains; a phosphatidylcholine such as egg phosphatidylcholine; a hydrogenated phospho tidy Icholine; a lysophosphatidylcholine; dipalmitoylphosphatidylcholine; distearoylphosphatidylcholine; dimyristoylphosphatidylcholine; dilauroylphosphatidylcholine; a glycerophospholipid such as phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylinositol, phosphati
- PEG polyethylene glycol
- the therapeutic agent administered is encapsulated within a phospholipid-based capsule as defined above, wherein said phospholipid is admixed with one or more, e.g., two, three or four, nonpho sphorous- containing molecules each independently is a fatty amine, a fatty acid, a fatty acid amide, an ester of a fatty acid, cholesterol, a cholesterol ester, a diacylglycerol, or a glycerol ester.
- Non-limiting examples of suitable nonphosphorous-containing molecules include fatty amines such as octylamine, laurylamine, N-tetradecylamine, hexadecylamine, stearylamine, oleylamine, tallowamine, hydrogenated tallowamine, and cocoamine; fatty acids; fatty acid amides; esters of fatty acid such as isopropyl myristate, hexadecyl stearate, and cetyl palmitate; cholesterol; cholesterol esters; diacylglycerols; or glycerol esters such as glycerol ricinoleate.
- fatty amines such as octylamine, laurylamine, N-tetradecylamine, hexadecylamine, stearylamine, oleylamine, tallowamine, hydrogenated tallowamine, and cocoamine
- fatty acids such as isopropyl myristate,
- the therapeutic agent administered is encapsulated within a phospholipid-based capsule as defined above, wherein said phospholipid is admixed with one or more, e.g., two, three or four, PEGylated phospholipids.
- the method disclosed herein is aimed at treatment of a disease or disorder associated with, i.e., characterized by, impaired MFR and/or mitoCa 2+ homeostasis.
- diseases or disorders include, without being limited to, neurodevelopmental disorders, as well as neurodeg enerative diseases or disorders.
- Particular neurodevelopmental disorders that may be treated by the method of the present invention include, without limiting, Phelan-McDermid syndrome, Rett syndrome, attention- deficit/hyperactivity disorder (ADHD), autism, developmental language disorder (DLD), learning disability, intellectual disability (mental retardation), and an impairment in vision and hearing; and particular neurodeg enerative diseases or disorders that may be treated by said method include, without being limited to, Alzheimer’s disease and Parkinson’s disease.
- the present invention provides a therapeutic agent as referred to above, i.e., a therapeutic agent selected from:
- said nucleic acid molecule encoding for the expression of either mitoIGFIR or a mitochondria targeted-portion thereof is an expression vector comprising a sequence encoding IGF1R or a portion thereof, fused to a sequence encoding a MTS.
- said expression vector comprises a sequence encoding IGF1R (SEQ ID NO: 2) fused to a sequence encoding a MTS (e.g., SEQ ID NOs: 4-6).
- said expression vector comprises a sequence encoding a portion of IGF1R fused to a sequence encoding a MTS (e.g., SEQ ID NOs: 4-6).
- said expression vector comprises a sequence encoding IGF1R (SEQ ID NO: 2) or a portion thereof, fused to both a sequence encoding CD8 (SEQ ID NO: 3) and a sequence encoding a MTS (e.g., SEQ ID NOs: 4-6).
- said nucleic acid molecule encoding for the expression of MCU optionally together with said additional subunit of the MCU complex is an expression vector comprising a sequence encoding MCU and optionally a sequence encoding said additional subunit of the MCU complex.
- said expression vector comprises a sequence encoding MCU (SEQ ID NO: 7).
- said expression vector comprises a sequence encoding MCU (SEQ ID NO: 7) and a sequence encoding MICU1 (SEQ ID NO: 8).
- said expression vector comprises a sequence encoding MCU (SEQ ID NO: 7) and a sequence encoding MICU3 (SEQ ID NO: 9).
- said expression vector comprises a sequence encoding MCU (SEQ ID NO: 7), a sequence encoding MICU1 (SEQ ID NO:8), and a sequence encoding MICU3 (SEQ ID NO:9).
- a therapeutic agent as disclosed herein which is an expression vector according to any one of the embodiments above, may be a cDNA or RNA.
- said sequence each independently is a cDNA; and said expression vector is a plasmid, e.g., an adeno-associated virus plasmid.
- said expression vector each independently is a viral vector selected from, e.g., retrovirus, adenovirus, adeno-associated virus, poxvirus, alphavirus, herpes virus, and lentivirus.
- the therapeutic agent disclosed is encapsulated within a capsule capable of delivering said therapeutic agent into a cell, more specifically integrating with the cell membrane, following which it is opened and consequently release said therapeutic agent within/into the cell.
- a capsule capable of delivering said therapeutic agent into a cell, more specifically integrating with the cell membrane, following which it is opened and consequently release said therapeutic agent within/into the cell.
- the content of the capsule is injected into the cell.
- a capsule may be made of, e.g., a phospholipid and/or a polymer each as defined in any one of the embodiments above; or may be a viral- or viral-like-envelope.
- the present invention provides a pharmaceutical composition
- a pharmaceutical composition comprising a therapeutic agent (also referred to herein as an "active agent”) according to any one of the embodiments above, encapsulated, e.g., within a capsule made of a phospholipid and/or a polymer, or within a viral- or viral-like-envelope, and a pharmaceutically acceptable carrier and/or excipient.
- a therapeutic agent also referred to herein as an "active agent”
- encapsulated e.g., within a capsule made of a phospholipid and/or a polymer, or within a viral- or viral-like-envelope, and a pharmaceutically acceptable carrier and/or excipient.
- compositions should meet sterility, pyrogenicity, and general safety and purity standards as required by, e.g., the U.S. Food and Drug Administration (FDA), or the European Medicines Agency (EMA).
- FDA U.S. Food and Drug Administration
- EMA European Medicines Agency
- compositions provided by the present invention may be prepared by conventional techniques known in the art, e.g., as described in Remington: The Science and Practice of Pharmacy, 19 th Ed., 1995.
- the compositions may be prepared, e.g., by uniformly and intimately bringing the active agent, i.e., said therapeutic agent, into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.
- compositions according to the present invention may be formulated for both enteral administration, e.g., oral or rectal administration; and parenteral administration, e.g., sublingual, sublabial, intravenous, intraarterial, intrathecal, intramuscular, intraperitoneal, intracerebroventricularl, subcutaneous, topical, nasal, or ophthalmic (e.g., as eye drops) administration.
- parenteral administration e.g., sublingual, sublabial, intravenous, intraarterial, intrathecal, intramuscular, intraperitoneal, intracerebroventricularl, subcutaneous, topical, nasal, or ophthalmic (e.g., as eye drops) administration.
- the composition may further be formulated for inhalation.
- Useful dosage forms of the pharmaceutical compositions include orally disintegrating systems including, but not limited to, solid, semi-solid and liquid systems including disintegrating or dissolving tablets, soft or hard capsules, gels, fast dispersing dosage forms, controlled dispersing dosage forms, caplets, films, wafers, ovules, granules, buccal/mucoadhesive patches, powders, freeze dried (lyophilized) wafers, chewable tablets which disintegrate with saliva in the buccal/mouth cavity and combinations thereof.
- Useful films include, but are not limited to, single layer stand-alone films and dry multiple layer stand-alone films.
- the pharmaceutical compositions are formulated for oral administration, and are in the form of matrix tablets wherein the release of the active agent(s) is controlled by having said active agent(s) diffuse through a gel formed after the swelling of a hydrophilic polymer brought into contact with dissolving liquid (in vitro) or gastrointestinal fluid (in vivo).
- a hydrophilic polymer brought into contact with dissolving liquid (in vitro) or gastrointestinal fluid (in vivo).
- Many polymers have been described as capable of forming such gel, e.g., derivatives of cellulose, in particular the cellulose ethers such as hydroxypropyl cellulose, hydroxymethyl cellulose, methylcellulose or methyl hydroxypropyl cellulose, and among the different commercial grades of these ethers are those showing fairly high viscosity.
- the tablets are formulated as bi- or multi-layer tablets, made up of two or more distinct layers of granulation compressed together with the individual layers lying one on top of another, with each separate layer containing the same of different active agent.
- Bilayer tablets have the appearance of a sandwich since the edge of each layer or zone is exposed.
- compositions for oral administration might be formulated so as to inhibit the release of one or more of the active agents in the stomach, i.e., delay the release of said active agent(s) until at least a portion of the dosage form has traversed the stomach, in order to avoid the acidity of the gastric contents from hydrolyzing the active agent(s).
- Particular such compositions are those wherein the active agent(s) is coated by a pH- dependent enteric-coating polymer.
- pH-dependent enteric-coating polymer examples include, without being limited to, Eudragit® S (poly(methacrylicacid, methylmethacrylate), 1:2), Eudragit® L 55 (poly (methacrylicacid, ethylacrylate), 1:1), Kollicoat® (poly(methacrylicacid, ethylacrylate), 1:1), hydroxypropyl methylcellulose phthalate (HPMCP), alginates, carboxymethylcellulose, and combinations thereof.
- the pH-dependent enteric-coating polymer may be present in the composition in an amount from about 10% to about 95% by weight of the entire composition.
- Another contemplated formulation is depot systems, based on biodegradable polymers. As the polymer degrades, the active agent(s) is slowly released.
- the most common class of biodegradable polymers is the hydrolytically labile polyesters prepared from lactic acid, glycolic acid, or combinations of these two molecules. Polymers prepared from these individual monomers include poly (D,L-lactide) (PLA), poly (glycolide) (PGA), and the copolymer poly (D,L-lactide-co-glycolide) (PLG).
- compositions formulated for parenteral administration may be in the form of a sterile, optionally injectable, aqueous or oleaginous suspension, which may be formulated according to the known art using suitable dispersing, wetting or suspending agents.
- the sterile injectable preparation may also be an injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent.
- Acceptable vehicles and solvents include, without limiting, water, Ringer's solution, polyethylene glycol (PEG), 2-hydroxypropyl-P-cyclodextrin (HPCD), a surfactant such as Tween-80, and isotonic sodium chloride solution.
- PEG polyethylene glycol
- HPCD 2-hydroxypropyl-P-cyclodextrin
- surfactant such as Tween-80
- isotonic sodium chloride solution isotonic sodium chloride solution.
- the therapeutic agent and pharmaceutical composition disclosed herein are useful in the treatment of diseases or disorders associated with impaired MFR and/or mitoCa 2+ homeostasis as defined above.
- the present invention relates to a therapeutic agent according to any one of the embodiments above, encapsulated, e.g., within a capsule made of a phospholipid and/or a polymer, or within a viral- or viral-like-envelope, for use in the treatment of a disease or disorder associated with impaired MFR and/or mitoCa 2+ homeostasis.
- the present invention relates to a therapeutic agent according to any one of the embodiments above, encapsulated, e.g., within a capsule made of a phospholipid and/or a polymer, or within a viral- or viral-like-envelope, for use in the preparation of a medicament for the treatment of a disease or disorder associated with impaired MFR and/or mitoCa 2+ homeostasis.
- HBSS HBSS supplemented with 13 mM MgSO 4 and 0.5 mg/mL DNase by titration with fire -polished pipettes of decreasing diameter. Sedimentation of cells was accomplished by centrifugation at 1000 ref for 10 min at 4°C. After the removal of the supernatant, cells were re-suspended with plating medium (MEM supplemented with 10% FBS, 32.7 mM glucose, 25 mg/mL insulin, 2 mM Glutamax, 0.1 mg/mL transferrin, 0.1% SMI) and then plated on matrigel- coated glass coverslips, glass-bottom 24-wells or MEA plates.
- plating medium MEM supplemented with 10% FBS, 32.7 mM glucose, 25 mg/mL insulin, 2 mM Glutamax, 0.1 mg/mL transferrin, 0.1% SMI
- AAV-hSynl-jRGECO1a was prepared by inserting jRGECOla (pGP-CMV-NES-jRGECOla, Addgene plasmid # 61563) into AAV2-hSynI- AT1.03 NL (gift from Daniel Gitler), replacing AT 1.03 NL.
- jRGECOla pGP-CMV-NES-jRGECOla
- Addgene plasmid # 61563 AAV2-hSynI- AT1.03 NL (gift from Daniel Gitler), replacing AT 1.03 NL.
- AAV2-CaMII2a- 4mtGCaMP8m 4mt fragment (synthesized by GenScript) was in frame fused to jGCaMP8m (Addgene #162372) and cloned into Addgene plasmid #51086, replacing GCaMP6s-p2A- nls_dTomato.
- Raw data was collected at 10 kHz, with a hardware high-pass filter of 1 Hz and an upper cut off of 3.3 kHz for the MEA2100- system and 3.5 kHz for the MEA2100- minisystem.
- MCS data analyzer software offline, raw data were filtered by a Butterworth 2 nd order high-pass filter at 200 Hz. Spikes were then detected by a fixed threshold of 6 SD. To reduce processing and analysis time, each hour was represented by 20 min of recording, which was previously shown to reliably represent the MFR of the full hour (Slomowitz et al., 2015).
- Plexon offline- sorter V3 Plexon inc. USA
- bursts were defined as 2 or more spikes at a minimum of 20 Hz based on the code we previously published (Slomowitz et al., 2015). Our previous analysis shows that the results are robust over a wide range of burst parameters (Slomowitz et al., 2015).
- Electrophysiological data were analyzed using pClamp (Molecular Devices LLC, USA) and MiniAnalysis (Synaptosoft, Decatur, Georgia, USA) for mEPSC.
- pClamp Molecular Devices LLC, USA
- MiniAnalysis Synaptosoft, Decatur, Georgia, USA
- 90 first events were taken from each neuron to prevent overrepresentation of high-frequency neurons.
- Synaptic blockers were used to prevent recurrent activity from stimulation (in pM): 10 CNQX, 50 AP-5. Cultures were infected with AAVl/2-hSyn- jRGECOla and AAVl/2-CaMKIIa-4mt-GCaMP8m, so that only excitatory neurons expressed both Ca +2 sensors. Only cells with mitochondrial response to a 10-stimuli burst at 50 Hz were considered. Infection with AAV1/2-CB AP-Cre-Cerulean or AAV1/2-CBAP- Cerulean was verified with a 440 nm laser.
- the coverslips were mounted using Mowiol (Merck Millipore, Kenilworth, NJ, USA).
- the following primary antibodies were used: MICU1 rabbit polyclonal (1:100; Sigma), MICU2 rabbit polyclonal (1:50; Sigma), MICU3 rabbit polyclonal (1:150; Sigma), MCU rabbit polyclonal (1:100; Sigma), TOM20 mouse monoclonal (1:100; Sigma), and IGFIRb mouse monoclonal (1:100; Invitrogen, 194Q13).
- the applied secondary antibodies were anti-mouse STAR580 and anti-rabbit STAR635P purchased from Abberior GmbH, Gottingen, Germany.
- Epifluorescence images were obtained by means of an 1X83 inverted microscope (Olympus). STED imaging was captured using a STED Abberior microscope, Gottingen, Germany. Excitation lines of 640 nm and 561 nm were adopted for exciting Star653P (MCUc subunits) and Star580 (TOM20 and IGF1R). For STED excitation, pulsed lasers were set at 640 nm and 580 nm. STED depletion was implemented via 775 nm depletion laser and the images were acquired at 20 nm pixel size.
- Co ver slips were embedded with Duolink® In Situ Mounting Medium with DAPI and sealed to avoid leaking. Imaging was performed in the confocal mode using an Abberior microscope (Gottingen, Germany). The CoraLite®488 fluorophore was excited using a 488 nm laser line and the PLA fluorophore was excited using a 561 nm laser line. Images were acquired in 40x40 pm size with a pixel size of 40x40 nm and lOps pixel dwell time.
- Brain and free mitochondria purification Brain and free mitochondria purification. Brain lysates and free mitochondria were isolated as previously described (Sims and Anderson, 2008) form 5-month-old C57BL/6Rj with minor modifications. Briefly, upon rapid brain extraction, the forebrain was dissected, cleaned from meninges and washed in isolation buffer, to remove blood. Buffers were prepared exactly as described in (Sims and Anderson, 2008). The tissue was then homogenized with method A (using a dunce homogenizer) with a two-step approach, as detailed in (Sims and Anderson, 2008). The homogenate obtained was used for the "total brain lysate" samples.
- method A using a dunce homogenizer
- Membranes were incubated with the respective primary antibodies diluted in the blocking buffer overnight at 4°C. Membranes were rinsed for 30 min in TBS-Tween20 and incubated for 1 h at room temperature with the corresponding secondary LICOR antibodies (see antibody list for details) and finally washed in TBS-Tween20 for 30 min. As a control for loading reproducibility, the blots were probed with an anti-GADPH antibody and revealed with the respective secondary antibody. After secondary antibody incubation, the fluorescent images of the nitrocellulose membranes were acquired with a LICOR Odyssey CLX-2088 imaging system.
- Donor (mseCFP) was excited with a 440 nm laser, and emission was measured at [460-500] nm before (I DA ) and after (I D ) photobleaching. Bleaching was accomplished using a 514 nm laser. Images of acceptor were taken before and after bleaching at [530-600] nm, to assess bleaching level: neurons with less than 85% reduction in fluorescence were excluded. FRET efficiency was calculated as [I DA / I D ].
- FM-based imaging Activity-dependent FM1-43 (10 pM) styryl dye was used to estimate basal synaptic vesicle recycling and short-term plasticity using protocols described previously (Abramov et al. , 2009). Briefly, APs in neurons were initiated by field stimulation during dye loading, and the terminals, after undergoing vesicle exocytosis coupled to endocytosis, were stained by FM1-43 10 pM FM1-43 has been present 5 sec before and 20 sec after the electrical stimulation. During FM loading and unloading, kynurenic acid (0.5 mM) was added to Tyrode solution to prevent recurrent activity through blockage of excitatory postsynaptic responses during loading and unloading.
- kynurenic acid 0.5 mM
- IGF1R deficiency limits homeostatic compensation of mean firing rate and pattern to inactivity
- Adeno- associated virus was used under the general promoter CBAP (AAVl/2-CBAP-Cre- Cerulean), creating IGF1R knockout (IGF1R-KO) networks. It is envisioned that the promoter could be different - here a general promoter was used, but in other embodiments more specific promoters are envisioned, e.g., hSynl - for neurons, CaMKIIa - for excitatory neurons, hDlx - for inhibitory neurons, GFAP - for astrocytes, etc. For control (Ctrl) experiments, IGFlR fl/fl cultures were infected with AAV1/2-CB AP-Cerulean.
- somatic cytoCa 2+ is also homeostatically regulated, and whether IGF1R is necessary for this process.
- Continuous imaging of somatic cytoCa 2+ during spontaneous spiking activity in excitatory hippocampal neurons was conducted at baseline, and following 2 days of baclofen perturbation.
- the average amplitude, frequency, and Ca 2+ influx index (a product of average amplitude x frequency) of cytosolic events were quantified for Ctrl and IGF1R-KO neurons.
- 2 days of baclofen did not affect cytoCa 2+ event amplitudes, rate, and subsequently the Ca 2+ influx index (not shown).
- IGF1 is the principal ligand of the IGF1R (Hakuno and Takahashi, 2018). To test if it is the necessary signal for IGF1R to enable MFR homeostasis, IGF1 was knocked-down using an AAV carrying a small-hairpin RNA against it (shlGFl). shlGFl mimicked the effect of IGF1R-KO, showing a limited compensatory MFR response to baclofen in comparison to a control vector carrying a scrambled sequence (shScr, not shown). These results indicate that MFR homeostatic response is mediated by IGF 1 -activated IGFIRs.
- MFR homeostasis is achieved by intrinsic and synaptic adaptations that act in a negative-feedback manner to counteract disturbances to ongoing activity (Turrigiano, 2011).
- IGF1R intrinsic and synaptic adaptations that act in a negative-feedback manner to counteract disturbances to ongoing activity
- several parameters of intrinsic excitability and synaptic strength in excitatory neurons were measured using whole-cell patch clamp.
- Action potentials were elicited by injecting somatic currents ranging from zero to 600 pA (F-I curves) in the presence of postsynaptic receptor blockers in Ctrl (Figs.
- presynaptic vesicle recycling was quantified directly using FM-based method (Slutsky et al., 2004). As baclofen-induced increase in mEPSC frequency is associated with an increase in synaptic release probability (Slomowitz et al., 2015), FM1-43 was used to test if homeostatic increase in synaptic release probability remains intact in IGFIR-KOs.
- MCUc is the primary Ca 2+ source into mitochondria (De Stefani et al., 2016)
- IGF1R deletion the reduction in mitoCa 2+ by IGF1R deletion could result from downregulation of the MCUc subunits.
- the mRNA expression levels of MCU and the Ca 2+ -sensing subunits MICU1, MICU2 and MICU3 were measured.
- IGF1R deletion caused a downregulation in the expression level of MCU and the brainspecific (Patron et al., 2019) MICU3 subunits, while it did not affect the expression levels of MICU1 and MICU2 subunits (Fig. 6A). It was therefore concluded that IGF1R affects MCU and MICU3 expression at the transcriptional level.
- IGFIRs in upward MFR homeostasis are two-fold: 1) to enable an increase in the fraction of spike bursts during the induction phase of homeostatic plasticity; 2) to maintain burst-to-mitoCa 2+ coupling. Either reduction in secretion of IGF1 or in IGF1R expression level may decrease MCUc activation and thus impair the induction of intrinsic and postsynaptic homeostatic plasticity.
- mitoIGFIRs in IGF1R-KO neurons rescued all 3 components: baclofen-induced increase in the fraction of spike bursts, burst-to-mitoCa 2+ coupling and upward MFR homeostasis.
- mitoIGFIR signaling emerges as a critical element in the induction of the integrated MFR response by regulating spike pattern and burst-to-mitoCa 2+ coupling (Fig. 8).
- IG1-R does not alter presynaptic homeostatic plasticity, other homeostatic sensors, such as the endoplasmic reticulum (ER) Ca 2+ sensor MCTP may drive presynaptic homeostasis (Gene et al., 2017).
- Amyloid -beta as a positive endogenous regulator of release probability at hippocampal synapses. Nat. Neurosci., 2009, 12, 1567-1576
- MCTP is an ER-resident calcium sensor that stabilizes synaptic transmission and homeostatic plasticity.
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Abstract
The present invention provides a therapeutic agent selected from (a) a mitochondria targeted-insulin-like growth factor- 1 receptor (mitoIGFIR) agonist; and (b) a nucleic acid molecule encoding for the expression of mitoIGFIR or a portion thereof, or mitochondrial calcium uniporter, encapsulated for delivering into a cell, and a method for treatment of a disease or disorder associated with impaired firing rate and/or mitochondrial calcium homeostasis by administration of said therapeutic agent.
Description
METHOD AND THERAPEUTIC AGENT FOR TREATMENT OF DISEASE OR DISORDER ASSOCIATED WITH IMPAIRED FIRING RATE AND/OR MITOCHONDRIAL CALCIUM HOMEOSTASIS
[0001] The project leading to the present application has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. 724866).
TECHNICAL FIELD
[0002] The present invention provides a therapeutic agent selected from (a) a mitochondria targeted-insulin-like growth factor- 1 receptor (mitoIGFIR) agonist; and (b) a nucleic acid molecule encoding for the expression of mitoIGFIR or a portion thereof, or mitochondrial calcium uniporter (MCU), encapsulated for delivering into a cell, and a method for treatment of a disease or disorder associated with impaired firing rate and/or mitochondrial calcium homeostasis by administration of said therapeutic agent.
BACKGROUND ART
[0003] Neural circuits are composed of a large number of dynamic elements at various levels of organization. The operation of a neuronal circuit depends on the interaction between the intrinsic properties of the individual neurons and the synaptic interactions that connect them into functional ensembles. While some aspects of synaptic and spiking activity are dynamic, others show remarkable stability over long time periods (Chambers et al., 2017). Despite a large variability in synaptic and intrinsic parameters, firing rate distributions and their mean firing rate (MFR) are maintained at a specific set-point value during ongoing spontaneous activity. MFRs are typically restored even in the presence of large perturbations to activity rates and patterns. The renormalization of MFRs to a set-point value has been observed in response to activity-dependent perturbations in cortical and hippocampal networks grown ex vivo (Turrigiano et al., 1998; Burrone et al., 2002; Slomowitz et al., 2015; Vertkin et al., 2015; Styr et al., 2019) and to sensory deprivation in VI cortex in vivo (Hengen et al., 2016; Hengen et al., 2013). MFR homeostasis can be achieved by a wide repertoire of homeostatic processes, including adjustments of synaptic strength, intrinsic excitability, and excitation-to-inhibition balance (Davis and Muller, 2015; Turrigiano, 2011). Dysregulation of homeostatic plasticity has been proposed to drive
synaptic and cognitive deficits in distinct brain disorders, including neurodevelopmental disorders (Kavalali and Monteggia, 2020) and neurodegenerative disorders like Alzheimer’s disease (Frere and Slutsky, 2018).
[0004] Despite significant progress in our understanding of neuronal homeostasis, how firing rates are sensed and how this information is converted into feedback signaling remain largely unknown. Intracellular somatic cytosolic [Ca2+] (cytoCa2+) has been proposed to serve as a proxy of spiking activity because of their tight coupling and is therefore modeled as a feedback control signal (O’Leary et al., 2014). According to these models, deviations from a specific target cytoCa2+ induce changes in effector proteins that result in renormalization of firing properties to a set point value. Indeed, cytoCa2+ in excitatory neurons returns to set-point value following sensory deprivation in vivo (Barnes et al., 2015) and following neuronal inactivity ex vivo (Slomowitz et al., 2015).
[0005] Insulin-like growth factor- 1 receptor (IGF1R) signaling is a well-known, evolutionary-conserved pathway regulating brain development (Fernandez and Torres- Aleman, 2012) and aging (Kenyon et al., 1993). In the central nervous system, IGF1/IGF1R signaling is critical for experience-dependent synaptic and neuronal plasticity in sensory cortices (Mardinly et al., 2016; Tropea et al., 2006; Maya-Vetencourt et al., 2012), adult neurogenesis (Trejo et al., 2001; Chaker et al., 2016), synaptic vesicle release (Prister et al., 2019), and neuronal excitability (Prister et al., 2019; Maglio et al., 2021).
[0006] Katsenelson et al. (2021) provides the first evidence that IGF1R is necessary for MFR and mitoCa2+ homeostasis, and discloses super-resolution images suggesting that IGF1R is present in the mitochondria. Said reference shows that deletion of IGF1R limits firing rate homeostasis in response to inactivity, without altering the baseline firing rate distribution. Disruption of both synaptic and intrinsic homeostatic plasticity contributes to deficient firing rate homeostatic response. As specifically shown, a fraction of IGFIRs was localized in mitochondria with the mitochondrial calcium uniporter complex (MCUc). IGF1R deletion suppressed spike burst-evoked mitoCa2+ by weakening mitochondria-to- cytosol Ca2+ coupling; and MCUc overexpression in IGFIR-deficient neurons rescued the deficits in spike-to-mitoCa2+ coupling and firing rate homeostasis.
SUMMARY OF INVENTION
[0007] In one aspect, disclosed herein is a method for treatment of a disease or disorder associated with impaired MFR and/or mitoCa2+ homeostasis, e.g., a neurodevelopmental
disorder such as Phelan-McDermid syndrome, Rett syndrome and autism, or neurodegenerative disease or disorder such as Alzheimer’s disease and Parkinson’s disease, in an individual in need thereof, said method comprising administering to said individual a therapeutically effective amount of a therapeutic agent selected from:
(i) a mitochondria targeted-insulin-like growth factor- 1 receptor (mitoIGFIR) agonist; and
(ii) a nucleic acid molecule encoding for the expression of either mitoIGFIR or a mitochondria targeted-portion thereof; or mitochondrial calcium uniporter (MCU) optionally together with an additional subunit of the MCU complex, wherein said therapeutic agent is encapsulated, e.g., within a capsule made of a phospholipid and/or a polymer, or within a viral- or viral-like-envelope, for enabling delivering said therapeutic agent into a cell, thereby restoring MFR and/or mitoCa2+ homeostasis.
[0008] In certain embodiments, the therapeutic agent administered is a mitoIGFIR agonist. Said mitoIGFIR agonist may be, e.g., IGF1, a fragment thereof, or an analogue thereof. Particular fragments of IGF1 and analogues thereof include fragments of IGF1 comprising the sequence glycine -proline-glutamate (GPE tripeptide) or an analog thereof as the amino-terminal thereof, e.g., the peptide consisting of the sequence GPE or Gly-1- methylPro-Glu (trofinetide).
[0009] In other embodiments, the therapeutic agent administered is a nucleic acid molecule encoding for the expression of either mitoIGFIR or a mitochondria targeted- portion thereof; or MCU optionally together with an additional subunit of the MCU complex. Said nucleic acid molecule encoding for the expression of mitoIGFIR or a mitochondria targeted-portion thereof may be, e.g., an expression vector comprising a sequence encoding IGF1R or a portion thereof, fused to a sequence encoding a mitochondrial-targeting sequence (MTS); and said nucleic acid molecule encoding for the expression of MCU optionally together with an additional subunit of the MCU complex may be, e.g., an expression vector comprising a sequence encoding MCU optionally together with said additional subunit of the MCU complex, e.g., MICU1 and/or MICU3. Nucleic acid molecules as referred to herein, each independently may be a cDNA or RNA, e.g., a cDNA-based plasmid, or a viral vector selected from, e.g., retrovirus, adenovirus, adeno-associated virus, poxvirus, alphavirus, herpes virus, and lentivirus.
[0010] In another aspect, disclosed herein is a therapeutic agent selected from:
(i) a mitoIGFIR agonist; and
(ii) a nucleic acid molecule encoding for the expression of either mitoIGFIR or a mitochondria targeted-portion thereof; or MCU optionally together with an additional subunit of the MCU complex, wherein said therapeutic agent is encapsulated, e.g., within a capsule made of a phospholipid and/or a polymer, or within a viral- or viral-like-envelope, for enabling delivering said therapeutic agent into a cell.
[0011] In a further aspect, disclosed herein is a pharmaceutical composition comprising a therapeutic agent as defined above, encapsulated, e.g., within a capsule made of a phospholipid and/or a polymer, or within a viral- or viral-like-envelope, and a pharmaceutically acceptable carrier and/or excipient.
[0012] The therapeutic agent and pharmaceutical composition disclosed herein are useful in the treatment of diseases or disorders associated with impaired MFR and/or mitoCa2+ homeostasis, e.g., neurodevelopmental disorders such as Phelan-McDermid syndrome, Rett syndrome and autism, or neurodegenerative diseases or disorders such as Alzheimer’s disease and Parkinson’s disease.
[0013] In yet another aspect, disclosed herein is a therapeutic agent as defined above, encapsulated, e.g., within a capsule made of a phospholipid and/or a polymer, or within a viral- or viral-like-envelope, for use in the treatment of a disease or disorder associated with impaired MFR and/or mitoCa2+ homeostasis.
BRIEF DESCRIPTION OF DRAWINGS
[0014] Figs. 1A-1F show that IGF1R deletion limits MFR homeostasis at the network level. (1A) Raster plots of the same neurons during baseline (Bsl, left), acute application of 10 pM baclofen (Bac, center), and following two days of Bac (Bac2d, right) in Ctrl (Ctrl) cultures. (IB) A typical MFR homeostatic response to chronic inactivity induced by 10 pM baclofen in Ctrl cultures, showing renormalization to the baseline network MFR (6 experiments, 535 units). (1C) Summary of full MFR recovery following two days of Bac in Ctrl networks (P=0.68, each point represents individual experiment, same data as IB). (ID) Raster plots of the same neurons during baseline (left), acute application of Bac (center), and following Bac2d (right) in IGF1R-KO culture. (IE) MFR homeostatic response was limited by IGF1R-KO (6 experiments, 371 units). (IF) Summary of partial recovery of MFRs following Bac2d in IGF1R-KO networks (P=O.O313, each point represents individual
experiment, same data as IE). Wilcoxon test (1C and IF). Error bars indicate SEM. ns, not significant, *P<0.05.
[0015] Figs. 2A-2K show that deletion of IGF1R limits firing rate and pattern homeostasis, without affecting their basal metrics. (2A-2C) Effect of Bac2d on 535 single units in Ctrl cultures. (2A) Firing rate distributions were unchanged following Bac2d (P=0.23). (2B) Changes in MFR per neuron (line indicates no change). (2C) MFRs were unchanged following Bac2d (P=0.79). (2D-2F) Effect of Bac2d on 371 single units in IGF1R- KO cultures. (2D) Firing rate distributions were left-shifted following Bac2d. (2E) Changes in MFR per neuron (line indicates no change). (2F) MFRs were reduced following Bac2d. (0.60+0.05 Hz for IGF1R-KO, 0.35+0.05 Hz for KO+Bac). (2G and 2H) Fraction of spikes participating in bursts following Bac2d in Ctrl and IGFIR-KOs (n=6 experiments, 535 units in Ctrl; n=6 experiments, 371 units in IGF1R-KO). (2I-2K) IGF1R-KO does not affect basal firing properties (same data as in 2A-2F). No difference was found in MFR distributions between Ctrl and IGF1R-KO neurons (21, P=0.1), in fraction of spikes participating in bursts (2J, P=0.31, 0.23+0.01 for Ctrl, 0.24+0.01 for IGF1R-KO) and in the intraburst spike frequency (2K, P=0.32, 185.1+4.1 Hz for Ctrl, 188.4+4.7 Hz, for IGF1R-KO). Wilcoxon test (2C and 2F), Kolmogorov-Smirnov test (2A, 2D and 21), Mann- Whitney U test (2J and 2K), Two-way ANOVA with Sidak’s multiple comparison test (2H). Error bars indicate SEM. ns, not significant, *P<0.05, ****P<0.0001.
[0016] Figs. 3A-3G show that lack of intrinsic excitability and postsynaptic homeostatic adaptations in IGF1R-KO neurons. (3A) Increased firing of Ctrl neurons in response to depolarizing currents following Bac2d (n=29 for Ctrl, n=24 for Ctrl+Bac). (3B) Representative traces of Ctrl neurons before (left) and after (right) Bac2d. (3C) No change (P=0.56) in firing of IGF1R-KO neurons in response to depolarizing currents following Bac2d (n=26 for IGF1R-KO, n=26 for KO+Bac). (3D) Representative traces of IGF1R-KO neurons before (left) and after (right) Bac2d. (3E) Representative mEPSC recordings of neurons from each group (scale bars: 1 s, 20 pA). (3F) Cumulative distribution of mEPSC amplitudes in Ctrl was skewed toward larger amplitudes following Bac2d (n=2,061 events from 23 Ctrl neurons and n=l,887 events from 21 Ctrl+Bac neurons). Inset: mean of amplitudes is increased following Bac2d (16.20+0.64 pA, n=23 for Ctrl; 19.37+0.89 pA, n=21 for KO+Bac). (3G) Cumulative distribution of mEPSC amplitudes in IGF1R-KO did not change following Bac2d (P=0.87, n=l,860 events from 21 IGF1R-KO neurons, 1,890 events from 21 KO+Bac neurons). Inset: mean of mEPSC amplitudes did not change
(P=0.87) following Bac2d (18.92+1.15 pA, n=21 for IGF1R-K0; 19.17+1.12 pA, n=21 for KO+Bac. Two-way ANOVA mixed-effects analysis (3A and 3C), Kolmogorov-Smirnov and Mann-Whitney U tests (3F and 3G). Error bars indicate SEM. ns, not significant, **P<0.01, ***P<0.001.
[0017] Figs. 4A-4F show that IGF1R deletion decreases somatic mitoCa2+ and cytoCa2+- to-mitoCa2+ coupling evoked by spike bursts. (4A) An excitatory neuron expressing the cytosolic calcium sensor jRGCECO la and mitochondrial calcium sensor GCaMP8m. Scale bar: 10 pm. (4B) Mean (dark lines) and SEM of cytoCa2+ traces for a single spike and spike bursts composed of 3, 5 and 10 stimuli at 50 Hz. Scale bars: 0.5 AF/F, 2 s (4C) Mean (dark lines) and SEM of mitoCa2+ traces for a single spike and spike bursts composed of 3, 5 and 10 stimuli at 50 Hz. Scale bars: 0.5 AF/F, 20 s (4D) CytoCa2+ responses were not changed (P=0.42) by deletion of IGF1R (n=40-42 for Ctrl, n=32-37 for IGF1R-KO). (4E) MitoCa2+ transients evoked by spike bursts were reduced in IGF1R-KO neurons (n=40-42 for Ctrl, n=3237 for IGF1R-KO). (4F) CytoCa2+-to-mitoCa2+ coupling was reduced in IGF1R-KO (n=165 events for Ctrl, n=142 events for IGF1R-KO). Squares are mean of cytoCa2+ events from 4D and mean of mitoCa2+ events from 4E. Two-way ANOVA mixed effects analysis (4D and 4E), Least square exponential regression. Extra sum-of-squares F test (4F). Error bars indicate SEM. ns, not significant, ***P<0.001, ****P<0.0001.
[0018] Figs. 5A-5G show that IGF1R is present in brain mitochondria and is colocalized with MCUc within neurons. (5A) Western blot (WB) analysis of mouse brain lysates and purified mitochondria (free mitoch.). The mitochondria are strongly enriched in the marker protein Cox4-1. The bands demonstrate the presence of the IGF1R protein in or on mitochondria. (5B) Samples were treated with trypsin to cleave all proteins from the outer membranes of the organelles in the samples. Remarkably, a prominent IGF1R band is still visible in mitochondria after this treatment, suggesting that a significant proportion of IGF1R is present within mitochondria. The experiments are shown in triplicate, to indicate experimental reproducibility. (5C) Hippocampal cultured neurons were immunostained for TOM20, as a mitochondrial marker (magenta), in combination with antibodies against four different subunits of the MCUc. From left to right: MICU1, MICU2, MICU3, and MCU. Scale bars: 50 pm. The images were taken with an epifluorescence microscope. (5D) Cultured neurons were immunostained as above, to test the colocalization of IGF1R (magenta) and the respective MCUc subunits. Scale bars: 50 pm. (5E) Representative STED images of neural cell bodies immunostained for IGF1R (magenta) and MCUc subunits. (5E’)
Enlarged views of the delineated areas alongside the filled arrows depict examples of colabeling of IGF1R and the MCUc proteins. Scale bars: 5 pm. (5F) Square regions of interest (ROIs) were obtained for all MCUc spots, both in the MCUc and in the IGF1R channels. The ROIs were then overlaid, which provides a visual indication of the presence of IGF1R in relation to MCUc spots. The arrows in the images point to the ROI centers, where the MCUc spots are located, and where an enrichment of IGF1R is also observed. To analyze these images, line scan profiles of intensity for IGF1R (magenta) and the four MCUc subunits were performed across the ROIs, and are shown as means ± SEM from 8 samples for each MCUc subunit. A small, but significant enrichment of IGF1R near MCUc spots can be observed for all samples (Arrows indicate the IGF1R intensity in each graph. Kruskal- Wallis tests followed by a post hoc Tukey test, corrected for multiple comparisons; P<0.001 for colocalization with MCU, MICU1 and MICU2; P<0.05 for MICU3). (5G) The cultured neurons were immunostained for IGF1R and the mitochondrial marker TOM20, to estimate the proportion of IGF1R that colocalizes with mitochondria. STED images strongly suggest a partial colocalization in the soma (some colocalized molecules are shown by the arrows). Scale bars, 50 pm and 5 pm, respectively. The quantification shows the percentage of IGF1R spots found within the space delimited by the TOM20 staining (i.e., found on mitochondria), which averages to ~30%. The box shows the median and the quartiles, while the whiskers show the range of data (n=5 independent experiments, with 5-7 cells imaged for each experiment).
[0019] Figs. 6A-6J show that over-expression of mitoIGFIR or MCUc rescues mitoCa2+ and MFR homeostasis in the absence of IGF1R. (6A) mRNA expression of MCUc proteins in IGF1R-KO cultures relative to Ctrl: The mRNA of MCU and MICU3 was reduced in IGF1R-KO culture (0.815+0.045 for MCU; 0.649+0.046 for MICU3), and did not change in MICU1 and MICU2 (0.954+0.064, P=0.925 for MICU1; 1.070+0.054, P=0.832 for MICU2, n=15 for IGF1R-KO; n=13 for Ctrl. Samples from 3 experiments for MCU and MICU3, samples from 2 experiments (n=9) for MICU 1 and MICU2). (6B) Mean (dark line) and SEM of mitoCa2+ event traces in response to 10 stimuli at 50 Hz shown in C. (6C) MitoCa2+ is reduced by IGF1R deletion, and is rescued by over-expression of MCUc in IGF1R-KO neurons (P>0.99, 1.25+0.07, n=49 for Ctrl; 0.93+0.06, n=52 for IGF1R-KO, 1.33+0.08, n=42 for KO+MCUc). (6D) Impaired MFR response to chronic inactivity induced by 10 pM baclofen in IG1R-KO cultures expressing mCherry, normalized to the baseline MFR (broken line, 6 experiments, 291 channels). Restoration of a normal MFR homeostatic response in
IGFIR-KO+MCUc, normalized to the baseline MFR (continuous line, 5 experiments, 252 channels). (6E) Summary (same data as 6D and 61) shows restoration of MFR recovery following Bac2d by MCUc or mitoIGFIR in IGF1R-K0 (93.1+5.9% of baseline for IGF1R- KO+MCUc and 93.9+13.9% for IGFIR-KO+mitoIGFIR, compared to 36.1+8.0% for IGFIR-KO+mCherry). Each circle is the mean of the last four time -points of each experiment in 6D and 61. (6F) Representative confocal images of a hippocampal neuron expressing mitoIGFIR (4mtIGFlR-mTagBFP2, top left), mito-mCherry (2mt-mCherry, top right) and the merged image of both markers showing overlapping expression (bottom). Arrows show examples of mitoIGFIR and mito-mCherry colocalization. Scale bar: 5 pm. (6G) Mean (dark line) and SEM of mitoCa2+ event traces in (6F) scale bars: 20 s, 0.5 AF/F. (6H) Expression of mitoIGFIR in IGF1R-KO neurons increased mitoCa2+ in response to 10 stimuli at 50 Hz (0.72+0.07, n=27 for IGF1R-KO, 1.24+0.07, n=26 for KO+mitoIGFIR). (61) Normal MFR homeostatic response in IGFIR-KO+mitoIGFIR, normalized to the baseline MFR (5 experiments, 458 channels). (6J) Fraction of spikes participating in bursts following Bac2d in IGFIR-KOs expressing mitoIGFIR (n=4 experiments, 421 units). Two- way ANOVA with Sidak’s multiple comparison test (6A), Kruskal-Wallis test with Dunn’s correction for multiple comparisons (6B and 6E), Mann- Whitney U test (6G). Error bars indicate SEM. ns, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
[0020] Figs. 7A-7D show impairment of MFR homeostasis and mitochondrial calcium in Shank3-InsG3680 mutant neurons and restoration of mitochondrial calcium by mitochondrial IGF1R. (7A) mitCa2+ in response to 10 stimuli given at 50Hz is reduced in Shank3-InsG3680 excitatory neurons (WT n=49, Shank3-InsG3680 n=32, Shank3+mitoIGFlR n=36, Shank3+mitoCD8-IGFlTR n=21). (7B) mean traces of data presented in 7A. (7C) MFR homeostasis in response to baclofen is impaired in Shank3- InsG3680 networks (7 experiments, 728 channels). (7D) Summary of MFR recovery following two days of Bac in Shank3-InsG3680 networks (each point represents the mean of 4h of the respective epochs in individual experiments, same data as 7C). Kruskal-Wallis test with Dunn’s correction for multiple comparisons (7A), Wilcoxon test (7D). Error bars indicate SEM. ns, not significant, *P<0.05, **P<0.01, ****P<0.0001.
[0021] Fig. 8 shows a model for IGF1R signaling in firing rate homeostasis. Top: diagram of MFR homeostatic plasticity using the framework of control theory. Titles in blue describe processes that require functional IGF1R. The processes are numbered according to the bottom scheme. Bottom: proposed model for the induction phase of upward MFR
homeostasis. (1) GABABR activation causes an acute decrease in MFR. (2) IGF1R is required for an increase in the fraction of spike bursts in response to the perturbation, leading to an increase in cytoCa2+ and subsequent (3) activation of MCUc. (4) IGF1R also maintains the coupling of mitochondria-to-cytosol Ca2+ coupling. (5) As a result, IGF1R enables the induction of intrinsic and postsynaptic homeostatic plasticity that underlies MFR recovery to a set-point level.
DETAILED DESCRIPTION
[0022] Since mitochondria are involved in neuronal Ca2+ homeostasis (De Stefani et al., 2016), it has been hypothesized that IGF1R is necessary for the integrated homeostatic response to activity perturbations. To test this hypothesis, the question of how IGF1R affects spike-to-Ca2+ transfer functions, quantitatively describing the coupling between spikes and the compartment-specific Ca2+ events they evoke, was examined. The coupling was measured at two different Ca2+ compartments (cytosol and mitochondria) in soma of excitatory hippocampal neurons, and the role of spike-to-cytoCa2+ and spike-to-mitoCa2+ in the MFR homeostatic response to inactivity at the network level was examined. The results shown herein demonstrate that somatic mitochondria selectively uptake Ca2+ evoked by spike bursts, but not by single spikes.
[0023] Utilizing super-resolution imaging and biochemistry, a population of IGF 1 -bound IGFIRs in neuronal mitochondria were detected, co-localized with mitochondrial calcium uniporter (MCU) and other members (subunits) of the MCU complex (MCUc). Deletion of IGFIRs did not alter spike-to-cytoCa2+ coupling, but weakened burst-to-mitoCa2+ coupling by downregulating transcription of several MCUc members. Moreover, while a pronounced increase in the fraction of spike bursts was detected in the initial phase of the perturbation, this change in spike pattern was lost in IGFIR-deficient neurons. Reduction in burst fraction and in burst-to-mitoCa2+ coupling resulted in impaired MFR compensation in response to inactivity. Overexpression of either MCUc or mitochondria-targeted IGF1R (mitoIGFIR) in IGF1R knockout (IGF1R-KO) neurons was sufficient to rescue both mitoCa2+ and MFR homeostatic response. These results provide direct evidence for the critical role of IGFIR/MCUc signaling in upward MFR homeostasis at the population level in hippocampal networks.
[0024] As shown in the Experimental section herein, IGFIRs are a critical component of the neuronal MFR homeostasis machinery, since without it, compensatory processes are
disrupted, and activity fails to renormalize in response to perturbation. As further shown, a sub-population of IGFIRs is localized to mitochondria, where it regulates mitoCa2+ uptake; and expression of mitoIGFIR in IGF1R deficient neural networks is sufficient to restore mitoCa2+ and MFR homeostasis.
[0025] Several neurodevelopmental disorders, such as Phelan-McDermid syndrome (Kolevzon et al., 2022; Kolevzon et al., 2014) and Rett syndrome (Vahdatpour et al., 2016) were shown to have a partial relief of symptoms by administration IGF1 analogues, with the latter having a treatment based on trofinetide ((2S)-2-{ [(2S)-l-(2-aminoacetyl)-2- methylpyrrolidine-2-carbonyl] amino Jpentanedioic acid; a synthetic analog of the aminoterminal tripeptide of IGF-1; Daybue™, formulated as oral solution) recently approved by the FDA. Based on the findings above, it was hypothesized that disorders involving failure of MFR and mitoCa2+ homeostasis may in fact be treated by mitoIGFIR expression. More specifically, it was hypothesized that by manipulating the activation of the newly discovered population of mitoIGFIR we might achieve better results than with administration of IGF1 analogues, which not only achieve limited results but have deleterious side-effects, due to its systemic application. Indeed, by using neural cell cultures from PMS model mice (Zhou et al., 2016), we were able to show that MFR and mitoCa2+ homeostasis were impaired in these neurons, and that mitoIGFIRs were able to restore mitoCa2+ back to healthy levels. As further shown, by using a shorter, constitutively activate mitochondrial IGF1R (mitoCD8- IGF1R, Carboni et al. 2005; the fusion to CD8 was in order to make IGF1R constitutively active, i.e., always active regardless of whether it is bound to a ligand or not), we were able to get similar recovery of mitoCa2+, making the ectopic protein shorter, more compatible with in-vivo injections.
[0026] In summary, as shown herein using biochemical assays, IGF1R is indeed present in brain mitochondria, and expression of IGF1R specifically in the mitochondria (mitoIGFIR) when total IGF1R is absent (IGF1R-KO) is sufficient for restoring mitoCa2+ and MFR homeostasis. These results demonstrate, for the first time, the importance of the newly discovered mitoIGFIR for MFR and mitoCa2+ homeostasis.
[0027] Given the evidence for the therapeutic potential of IGF1 in neurodevelopmental disorders, and the new findings discussed above, it is postulated that targeting mitoIGFIRs sub-population by either gene-therapy or specific pharmacological agents will provide a better treatment for relevant neurodevelopmental disorders, with a broader effect on patients, while minimalizing systemic side effects.
[0028] In one aspect, the present invention thus relates to a method for treatment of a disease or disorder associated with impaired MFR and/or mitoCa2+ homeostasis in an individual in need thereof, said method comprising administering to said individual a therapeutically effective amount of a therapeutic agent selected from:
(i) a mitoIGFIR agonist; and
(ii) a nucleic acid molecule encoding for the expression of either mitoIGFIR or a mitochondria targeted-portion thereof; or MCU optionally together with an additional subunit of the MCU complex, wherein said therapeutic agent is encapsulated for enabling delivering said therapeutic agent into a cell, thereby restoring MFR and/or mitoCa2+ homeostasis.
[0029] In certain embodiments, the therapeutic agent administered according to the method disclosed is a mitoIGFIR agonist.
[0030] The term “mitochondria targeted-insulin-like growth factor- 1 receptor (mitoIGFIR) agonist” as used herein refers to any agent capable of agonizing the mitochondrial IGF1 receptor. Such an agent may be, e.g., a protein or peptide, as well as a small molecule, but it is preferably a short protein or a peptide. In certain embodiments, said mitoIGFIR agonist is IGF1, more specifically a human IGF1 (SEQ ID NO: 1). In other embodiments, said mitoIGFIR agonist is a fragment of IGF1, i.e., a peptide consisting of a partial sequence of the amino acid sequence of IGF 1, capable of agonizing IGF1R. In further embodiments, said mitoIGFIR agonist is an analogue or variant of either IGF1 or said fragment, i.e., a peptide based on the amino acid sequence of either IGF1 or said fragment, in which at least one of the amino acids has been substituted, replaced by an alternative amino acid, or deleted, or to which at least one amino acid has been added (at any position along the sequence). Preferably, such a variant protein or peptide has an amino acid sequence that is at least 85%, preferably 90%, and most preferably 95%, 99%, or more, identical to the amino acid sequence of IGF1 from which it is derived, or said fragment thereof. Particular fragments of IGF1 or analogues thereof for use as mitoIGFIR agonists are peptides comprising the sequence glycine -proline-glutamate (GPE tripeptide) or an analog thereof such as Gly-l-methylPro-Glu, as the amino -terminal thereof. A more particular such IGF1 fragment consists of the sequence Gly-Pro-Glu, and a more particular such IGF1 analogue consists of the sequence Gly-l-methylPro-Glu (glycyl-alpha-methyl-L-prolyl-L- glutamic acid; (2S)-2-{ [(2S)-l-(2-aminoacetyl)-2-methylpyrrolidine-2-carbonyl]amino}
pentanedioic acid; trofinetide). Preferred mitoIGFIR agonists for use according to the present invention are IGF1, the tripeptide GPE, and trofinetide.
[0031] The term “comprising” as used herein with respect to an amino acid sequence means that said amino acid sequence consists, either exclusively or not, of a particular sequence referred to. For example, an amino acid sequence comprising the tetrapeptide MQEP may consist exclusively of said tetrapeptide, or of an amino acid sequence including said tetrapeptide as a sub-sequence thereof (e.g., as the amino or carboxy terminal of said sequence, or at any non-terminal position of the sequence). The term “consisting” as used herein with respect to an amino acid sequence means that said amino acid sequence consists exclusively of a particular sequence referred to.
[0032] The term "amino acid" as used herein refers to an organic compound comprising both amine and carboxylic acid functional groups, which may be either natural or nonnatural, and occur in both L and D isomeric forms. The twenty-two amino acids naturally occurring in proteins are aspartic acid (Asp), tyrosine (Tyr), leucine (Leu), tryptophan (Trp), arginine (Arg), valine (Vai), glutamic acid (Glu), methionine (Met), phenylalanine (Phe), serine (Ser), alanine (Ala), glutamine (Gin), glycine (Gly), proline (Pro), threonine (Thr), asparagine (Asn), lysine (Lys), histidine (His), isoleucine (He), cysteine (Cys), selenocysteine (Sec), and pyrrolysine (Pyl). Non-limiting examples of non-natural amino acids include citrulline (Cit), diaminopropionic acid (Dap), diaminobutyric acid (Dab), ornithine (Om), aminoadipic acid, P-alanine, 1 -naphthylalanine, 3-(l-naphthyl)alanine, 3- (2-naphthyl)alanine, y-aminobutiric acid (GABA), 3 -(aminomethyl) benzoic acid, p- ethynyl-phenylalanine, m-ethynyl-phenylalanine, p-chlorophenylalanine (4ClPhe), p- bromophenylalanine, p-iodopheny lalanine, -accty Ipheny lalanine, p-azidopheny lalanine, p- propargly-oxy-phenylalanine, indanylglycine (Igl), (benzyl)cysteine, norleucine (Nle), azidonorleucine, 6-ethynyl-tryptophan, 5-ethynyl-tryptophan, 3-(6-chloroindolyl)alanine, 3- (6-bromoindolyl)alanine, 3-(5-bromoindolyl)alanine, azidohomoalanine, a-aminocaprylic acid, O-methyl-L-tyrosine, N-acetylgalactosamine-a-threonine, N-acetylgalactosamine-a- serine, and 1 -methylproline.
[0033] The term "amino acid residue" as used herein refers to a residue of an amino acid after removal of hydrogen atom from an amino group thereof, e.g., its a- amino group or side chain amino group when present, and -OH group from a carboxyl group thereof, e.g., its a- carboxyl group or side chain carboxyl group when present.
[0034] The term "peptide" refers to a short chain of amino acid monomers (residues), e.g., a chain consisting of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more amino acid residues, linked by peptide (amide) bonds. The term "peptide bond" or "amide bond" as used herein refers to the covalent bond -C(O)NH- formed between two molecules, e.g., two amino acids, when a carboxyl group of one of the molecules reacts with an amino group of the other molecule, causing the release of a water molecule.
[0035] The term “nucleic acid molecule” or “nucleic acid sequence”, used herein interchangeably, refers to nucleic acid, DNA or RNA, that comprises noncoding or coding sequences. Coding sequences are necessary for the production of a polypeptide or protein precursor. A polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence, as long as the desired protein activity is retained. Noncoding sequences refer to nucleic acid molecules which do not code for a polypeptide or protein precursor, and may include regulatory elements such as transcription factor binding sites, poly(A) sites, restriction endonuclease sites, stop codons and/or promoter sequences.
[0036] A “nucleic acid”, as used herein, is a covalently linked sequence of nucleotides in which the 3' position of the pentose of one nucleotide is joined by a phosphodiester group to the 5' position of the pentose of the next, and in which the nucleotide residues (bases) are linked in specific sequence, i.e., a linear order of nucleotides. A “polynucleotide”, as used herein, is a nucleic acid containing a sequence that is greater than about 100 nucleotides in length. An “oligonucleotide” or “primer”, as used herein, is a short polynucleotide or a portion of a polynucleotide. An oligonucleotide typically contains a sequence of about two to about one hundred bases.
[0037] Nucleic acid molecules are said to have a “5 '-terminus” (5' end) and a “3 '-terminus” (3' end) because nucleic acid phosphodiester linkages occur to the 5' carbon and 3' carbon of the pentose ring of the substituent mononucleotides. The end of a polynucleotide at which a new linkage would be to a 5' carbon is its 5' terminal nucleotide; and the end of a polynucleotide at which a new linkage would be to a 3' carbon is its 3' terminal nucleotide. A terminal nucleotide, as used herein, is the nucleotide at the end position of the 3'- or 5'- terminus.
[0038] DNA molecules are said to have “5' ends” and “3' ends” because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotides referred to as the “5' end”
if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the “3' end” if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring.
[0039] As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide or polynucleotide, also may be said to have 5' and 3' ends. In either a linear or circular DNA molecule, discrete elements are referred to as being “upstream” or 5' of the “downstream” or 3' elements. This terminology reflects the fact that transcription proceeds in a 5' to 3' fashion along the DNA strand. Typically, promoter and enhancer elements that direct transcription of a linked gene (e.g., open reading frame or coding region) are generally located 5' or upstream of the coding region. However, enhancer elements can exert their effect even when located 3' of the promoter element and the coding region. Transcription termination and polyadenylation signals are located 3' or downstream of the coding region. [0040] In certain embodiments, the therapeutic agent administered according to the method disclosed is a nucleic acid molecule encoding for the expression of either mitoIGFIR or a mitochondria targeted -portion thereof; or MCU optionally together with an additional subunit of the MCU complex.
[0041] In certain particular embodiments, the nucleic acid molecule encoding for the expression of either mitoIGFIR or a mitochondria targeted-portion thereof is an expression vector comprising a sequence encoding IGF1R (SEQ ID NO: 2) or a portion thereof, fused to a sequence encoding a MTS. In certain more particular embodiments, said expression vector comprises a sequence encoding IGF1R (SEQ ID NO: 2) fused to a sequence encoding a MTS. In other more particular embodiments, said expression vector comprises a sequence encoding a portion of IGF1R fused to a sequence encoding a MTS. In further more particular embodiments, said expression vector comprises a sequence encoding IGF1R (SEQ ID NO: 2) or a portion thereof, fused to both a sequence encoding CD8 (SEQ ID NO: 3) and a sequence encoding a MTS. The fusion to a sequence encoding a MTS is necessary so as to direct, i.e., target, the IGF1R encoded or said portion thereof to the mitochondria.
[0042] The term “mitochondrial-targeting sequence (MTS)” as used herein refers to a short peptide, e.g., about 15-70 amino acids long, bearing positively charged basic residues, that directs the transport of a protein to the mitochondria, i.e., targets said protein to the mitochondria. Such a sequences is usually located at the N-terminal of a given protein, consisting of an alternating pattern of hydrophobic and positively charged residues that form an amphipathic helix. Examples of mitochondrial-targeting sequences include, without
being limited to, 4mt (SEQ ID No: 4) as well as the sequence disclosed in Faria et al., 2021, Hurt et al., 1985 (SEQ ID NO: 5), and the sequence disclosed in Obita et al., 2003 (SEQ ID No: 6).
[0043] In other particular embodiments, the nucleic acid molecule encoding for the expression of MCU optionally together with said additional subunit of the MCU complex is an expression vector comprising a sequence encoding MCU and optionally a sequence encoding said additional subunit of the MCU complex. In certain more particular embodiments, said expression vector comprises a sequence encoding MCU (SEQ ID NO: 7). In other more particular embodiments, said expression vector comprises a sequence encoding MCU (SEQ ID NO: 7) and a sequence encoding MICU 1 (SEQ ID NO:8). In further more particular embodiments, said expression vector comprises a sequence encoding MCU (SEQ ID NO: 7) and a sequence encoding MICU3 (SEQ ID NO:9). In yet other more particular embodiments, said expression vector comprises a sequence encoding MCU (SEQ ID NO:7), a sequence encoding MICU1 (SEQ ID NO:8), and a sequence encoding MICU3 (SEQ ID NO:9). In contrast to expression vectors comprising a sequence encoding IGF1R or a portion thereof, wherein said sequence has to be fused to a sequence encoding a MTS so as to target the protein encoded to the mitochondria, a nucleic acid molecule encoding for the expression of MCU optionally together with an additional subunit of the MCU complex as referred to hereinabove does not have to comprise a sequence encoding a MTS, as both the sequence encoding MCU as well as each one of the sequences encoding the other MCU complex subunits each comprises an inherent sequence aimed at targeting the MCU complex subunit encoded to the mitochondria.
[0044] The term “expression vector” as used herein refers to a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. The vector is engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is the efficient production of protein, and this may be achieved by the production of a significant amount of stable messenger RNA, which can then be translated into protein. The expression of a protein may be tightly controlled, and the protein is only produced in significant quantity when necessary through the use of an inducer, in some systems however the protein may be expressed constitutively.
[0045] In certain embodiments, the therapeutic agent administered according to the method disclosed herein is an expression vector comprising a sequence encoding IGF1R or a portion thereof, fused to a sequence encoding a MTS; or an expression vector comprising a sequence encoding MCU optionally together with an additional subunit of the MCU complex. In certain particular such embodiments, said sequence, according to any one of the embodiments above, each independently is a complementary DNA (cDNA) or RNA. In other particular such embodiments, said sequence, according to any one of the embodiments above, each independently is a cDNA; and said expression vector is a plasmid, e.g., an adeno-associated virus (AAV) plasmid such as the AAV2-CBAP plasmid exemplified herein (this plasmid has all other default components of an AAV plasmid as defined, e.g., in the Adeno- Associated Virus Guide of Addgene (https://www.addgene.org/guides/aav/), wherein the features distinguish this plasmid are the promoter (CBAP) and the serotype (2)). In further particular such embodiments, said expression vector, according to any one of the embodiments above, each independently is a viral vector selected from, e.g., retrovirus, adenovirus, adeno-associated virus, poxvirus, alphavirus, herpes virus, and lentivirus.
[0046] Gene therapy methods and methods of delivering genes to subjects, e.g., using adeno-associated virus (AAV), are well known, and description thereof may be found, e.g., in WO 2013/142114, WO 2014/059029, WO 2014/059031, WO 2014/093622, WO 2014/127198, US 2014/0100265, and US 7,977,049, each of which is incorporated herein by reference in its entirety.
[0047] In certain embodiments, the present invention provides gene therapy methods, such as combinational gene therapy methods, to provide or regulate one or more endogenous proteins. Such gene therapy methods are aimed at treating or preventing a disease or disorder associated with impaired MFR and/or mitoCa2+ homeostasis, by restoration of MFR and/or mitoCa2+ homeostasis. According to some embodiments, the invention provides the identification of genes related to diseases and disorders associated with impaired MFR and/or mitoCa2+ homeostasis, and further the regulation of such genes either by increasing a protein related to a gene or decreasing a protein related to a gene.
[0048] In certain embodiments, the invention provides gene therapy methods for increasing mitoCa2+ influx by introducing a nucleic acid encoding the functional protein which is expressed within a cell. Generally, such an expression results in the increased mitoCa2+ influx by direct or indirect regulation of MCU complex activity. In specific embodiments, mitoCa2+ influx is increased by mitoIGFIR or MCU complex subunits. In
alternative or added embodiments, mitoCa2+ influx is increased by a mitoIGFIR agonist, e.g., human IGF1 (SEQ ID NO: 1) or a fragment, analogue or variant thereof capable of agonizing IGF1R.
[0049] In certain embodiments, the invention provides gene therapy methods using genetic constructs targeting cells in a human, and the delivery of such genetic constructs using methods such as, but not limited to, liposomes, synthetic or naturally occurring polymers, coated or non-coated nanoparticles, biolistic (“biological ballistics”) particles, laser mediate transfection (optoporation or phototransfection), etc.
[0050] Accordingly, the present invention provides gene therapy methods for the regulation of one or more or a plurality of genes or their associated functional proteins in a method of treating or preventing diseases or disorders associated with impaired MFR and/or mitoCa2+ homeostasis. The gene therapy can be based on, e.g., one or more of a nucleic acid or gene which overexpress a functional protein or a mutant form thereof; expression of a functional protein which regulates another target gene/protein; expression of polynucleotides, such as inhibitory RNA, to regulate expression of a target gene; and expression of gene editing systems that modify in situ the target gene. Such a nucleic acid can be a "synthetic nucleotide sequence", i.e., a nucleotide sequence which does not occur as such in nature, but was rather designed, engineered and/or constructed by human intervention.
[0051] The method of the present invention is aimed at treating a disease or disorder associated with impaired MFR and/or mitoCa2+ homeostasis, by restoration of MFR and/or mitoCa2+ homeostasis, and the therapeutic agent administered thus has to be delivered into the cells, more particularly into the neuronal cells. To achieve this goal, said therapeutic agent is encapsulated within a capsule capable of delivering said therapeutic agent into the cell, more specifically, integrating with the cell membrane, following which it is opened and consequently release its content, i.e., the therapeutic agent, within/into the cell. Alternatively, after binding to the cell membrane, the content of the capsule is injected into the cell. Such a capsule may be made of, e.g., a phospholipid and/or a polymer; or may be a viral- or viral-like-envelope.
[0052] In certain embodiments, the therapeutic agent administered is encapsulated within a phospholipid-based capsule. Examples of phospholipids include, without being limited to, a lecithin such as egg or soybean lecithin, or a derivative thereof, e.g., a lecithin having polyethylene glycol (PEG) chains; a phosphatidylcholine such as egg phosphatidylcholine;
a hydrogenated phospho tidy Icholine; a lysophosphatidylcholine; dipalmitoylphosphatidylcholine; distearoylphosphatidylcholine; dimyristoylphosphatidylcholine; dilauroylphosphatidylcholine; a glycerophospholipid such as phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol phosphate, phosphatidylinositol bisphosphate and phosphatidylinositol triphosphate; sphingomyelin; cardiolipin; a phosphatidic acid; a glycolipid such as a glyceroglycolipid, e.g., a galactolipid and a sulfolipid, a glycosphingolipid, e.g., a cerebroside (a glucocerebroside and a galactocerebroside), and a glycosylphosphatidylinositol; a plasmalogen; a phospho sphingolipid such as a ceramide phosphorylcholine, a ceramide phosphorylglycerol, and a ceramide phosphorylethanolamine; or a mixture thereof.
[0053] In certain particular embodiments, the therapeutic agent administered is encapsulated within a phospholipid-based capsule as defined above, wherein said phospholipid is a commercially available product such as Phospholipon® 50, i.e., a soybean lecithin comprising about 45% phosphatidylcholine and about 10 to about 18% phosphatidylethanolamine; Phospholipon® 75, i.e., a soybean lecithin comprising about 75% phosphatidylcholine; Phospholipon® 85G or Phospholipon® 90G, essentially consisting of soybean lecithins and phospholipids; Phospholipon® 80H or Phospholipon® 90H, essentially consisting of hydrogenated soybean lecithins and phospholipids; Phospholipon® E25, Phospholipon® E35 or Phospholipon® E, essentially consisting of egg yolk lecithins and phospholipids; and Phospholipon® LPC20, Phospholipon® LPC25 or Phospholipon® LPC65, essentially consisting of partially hydrolyzed soybean lecithins (all of Lipoid).
[0054] In other particular embodiments, the therapeutic agent administered is encapsulated within a phospholipid-based capsule as defined above, wherein said phospholipid is admixed with one or more, e.g., two, three or four, nonpho sphorous- containing molecules each independently is a fatty amine, a fatty acid, a fatty acid amide, an ester of a fatty acid, cholesterol, a cholesterol ester, a diacylglycerol, or a glycerol ester. Non-limiting examples of suitable nonphosphorous-containing molecules include fatty amines such as octylamine, laurylamine, N-tetradecylamine, hexadecylamine, stearylamine, oleylamine, tallowamine, hydrogenated tallowamine, and cocoamine; fatty acids; fatty acid amides; esters of fatty acid such as isopropyl myristate, hexadecyl stearate, and cetyl palmitate; cholesterol; cholesterol esters; diacylglycerols; or glycerol esters such as glycerol ricinoleate.
[0055] In further particular embodiments, the therapeutic agent administered is encapsulated within a phospholipid-based capsule as defined above, wherein said phospholipid is admixed with one or more, e.g., two, three or four, PEGylated phospholipids. Examples of suitable PEGylated phospholipids include, without being limited to, PEGylated dipalmitoyl phosphatidylethanolamine (DPPE-PEG), PEGylated palmitoyloleoyl phosphatidylethanolamine (POPE-PEG), PEGylated dioleoyl phosphatidylethanolamine (DOPE-PEG) and PEGylated distearoyl phosphatidylethanolamine (DSPE-PEG), preferably 1 ,2-distearoyl- sn-glycero-3 -phosphoethanolamine-N- [polyethyleneglycol 2000] (PEG- DSPE-2000).
[0056] In other embodiments, the therapeutic agent administered is encapsulated within a polymer-based capsule. Non-limiting examples of such polymers include polyethylene gly col-pho sphatidylethanolamine (PEG-PE).
[0057] In further embodiments, the therapeutic agent administered is encapsulated within a viral- or viral-like-capsule (envelope). Such therapeutic agents are those consisting of a nucleic acid molecule as referred to above, which are expression vectors comprising a sequence encoding either IGF1R or a portion thereof, fused to a sequence encoding a MTS; or MCU optionally together with an additional subunit of the MCU complex. As stated above, said expression vector may be a viral vector selected from, e.g., retrovirus, adenovirus, adeno-associated virus, poxvirus, alphavirus, herpes virus, and lentivirus, and the capsule encapsulating said therapeutic agent is in fact the viral envelope. Alternatively, said nucleic acid molecule is a cDNA; said expression vector is a plasmid; and said plasmid is encapsulated within a capsid (viral-like capsule) or a capsule as defined above.
[0058] The method disclosed herein is aimed at treatment of a disease or disorder associated with, i.e., characterized by, impaired MFR and/or mitoCa2+ homeostasis. Examples of such diseases or disorders include, without being limited to, neurodevelopmental disorders, as well as neurodeg enerative diseases or disorders. Particular neurodevelopmental disorders that may be treated by the method of the present invention include, without limiting, Phelan-McDermid syndrome, Rett syndrome, attention- deficit/hyperactivity disorder (ADHD), autism, developmental language disorder (DLD), learning disability, intellectual disability (mental retardation), and an impairment in vision and hearing; and particular neurodeg enerative diseases or disorders that may be treated by said method include, without being limited to, Alzheimer’s disease and Parkinson’s disease.
[0059] In another aspect, the present invention provides a therapeutic agent as referred to above, i.e., a therapeutic agent selected from:
(i) a mitoIGFIR agonist; and
(ii) a nucleic acid molecule encoding for the expression of either mitoIGFIR or a mitochondria targeted-portion thereof; or MCU optionally together with an additional subunit of the MCU complex, wherein said therapeutic agent is encapsulated for enabling delivering said therapeutic agent into a cell, more particularly into a neuronal cell.
[0060] In certain embodiments, the therapeutic agent disclosed is a mitoIGFIR agonist. In certain particular such embodiments, said mitoIGFIR agonist is IGF1, more specifically a human IGF1 (SEQ ID NO: 1). In other particular such embodiments, said mitoIGFIR agonist is a fragment of IGF1, i.e., a peptide consisting of a partial sequence of the amino acid sequence of IGF 1, capable of agonizing IGF1R. In further particular such embodiments, said mitoIGFIR agonist is an analogue or variant of either IGF1 or said fragment, i.e., a peptide based on the amino acid sequence of either IGF1 or said fragment in which at least one of the amino acids has been substituted, replaced by an alternative amino acid, or deleted, or to which at least one amino acid has been added (at any position along the sequence). Particular fragments of IGF1 or analogues thereof referred to herein are peptides comprising the GPE tripeptide or an analog thereof such as Gly-l-methylPro-Glu, as the amino-terminal thereof. A more particular such IGF1 fragment consists of the sequence Gly-Pro-Glu, and a more particular such IGF1 analogue is trofinetide. Preferred mitoIGFIR agonists referred to herein are IGF1, the tripeptide GPE, and trofinetide.
[0061] In certain embodiments, the therapeutic agent disclosed is a nucleic acid molecule encoding for the expression of either mitoIGFIR or a mitochondria targeted-portion thereof; or MCU optionally together with an additional subunit of the MCU complex.
[0062] In certain particular embodiments, said nucleic acid molecule encoding for the expression of either mitoIGFIR or a mitochondria targeted-portion thereof is an expression vector comprising a sequence encoding IGF1R or a portion thereof, fused to a sequence encoding a MTS. In some more particular embodiments, said expression vector comprises a sequence encoding IGF1R (SEQ ID NO: 2) fused to a sequence encoding a MTS (e.g., SEQ ID NOs: 4-6). In other more particular embodiments, said expression vector comprises a sequence encoding a portion of IGF1R fused to a sequence encoding a MTS (e.g., SEQ ID NOs: 4-6). In further more particular embodiments, said expression vector comprises a
sequence encoding IGF1R (SEQ ID NO: 2) or a portion thereof, fused to both a sequence encoding CD8 (SEQ ID NO: 3) and a sequence encoding a MTS (e.g., SEQ ID NOs: 4-6).
[0063] In other particular embodiments, said nucleic acid molecule encoding for the expression of MCU optionally together with said additional subunit of the MCU complex is an expression vector comprising a sequence encoding MCU and optionally a sequence encoding said additional subunit of the MCU complex. In some more particular embodiments, said expression vector comprises a sequence encoding MCU (SEQ ID NO: 7). In other more particular embodiments, said expression vector comprises a sequence encoding MCU (SEQ ID NO: 7) and a sequence encoding MICU1 (SEQ ID NO: 8). In further more particular embodiments, said expression vector comprises a sequence encoding MCU (SEQ ID NO: 7) and a sequence encoding MICU3 (SEQ ID NO: 9). In yet other more particular embodiments, said expression vector comprises a sequence encoding MCU (SEQ ID NO: 7), a sequence encoding MICU1 (SEQ ID NO:8), and a sequence encoding MICU3 (SEQ ID NO:9).
[0064] A therapeutic agent as disclosed herein, which is an expression vector according to any one of the embodiments above, may be a cDNA or RNA. In certain particular such embodiments, said sequence each independently is a cDNA; and said expression vector is a plasmid, e.g., an adeno-associated virus plasmid. In other particular such embodiments, said expression vector each independently is a viral vector selected from, e.g., retrovirus, adenovirus, adeno-associated virus, poxvirus, alphavirus, herpes virus, and lentivirus.
[0065] According to the present invention, the therapeutic agent disclosed is encapsulated within a capsule capable of delivering said therapeutic agent into a cell, more specifically integrating with the cell membrane, following which it is opened and consequently release said therapeutic agent within/into the cell. Alternatively, after binding to the cell membrane,
the content of the capsule is injected into the cell. Such a capsule may be made of, e.g., a phospholipid and/or a polymer each as defined in any one of the embodiments above; or may be a viral- or viral-like-envelope.
[0066] In a further aspect, the present invention provides a pharmaceutical composition comprising a therapeutic agent (also referred to herein as an "active agent”) according to any one of the embodiments above, encapsulated, e.g., within a capsule made of a phospholipid and/or a polymer, or within a viral- or viral-like-envelope, and a pharmaceutically acceptable carrier and/or excipient.
[0067] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" as used herein interchangeably refers to any non-active ingredient such as a solvent, dispersion medium, preservative, antioxidant, coating, isotonic and absorption delaying agent, and the like, that is compatible with pharmaceutical administration, and does not produce an adverse, allergic, or other untoward reaction when administered to a mammal or human as appropriate. For human administration, compositions should meet sterility, pyrogenicity, and general safety and purity standards as required by, e.g., the U.S. Food and Drug Administration (FDA), or the European Medicines Agency (EMA).
[0068] The compositions provided by the present invention may be prepared by conventional techniques known in the art, e.g., as described in Remington: The Science and Practice of Pharmacy, 19th Ed., 1995. In particular, the compositions may be prepared, e.g., by uniformly and intimately bringing the active agent, i.e., said therapeutic agent, into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.
[0069] Pharmaceutical compositions according to the present invention may be formulated for both enteral administration, e.g., oral or rectal administration; and parenteral administration, e.g., sublingual, sublabial, intravenous, intraarterial, intrathecal, intramuscular, intraperitoneal, intracerebroventricularl, subcutaneous, topical, nasal, or ophthalmic (e.g., as eye drops) administration. The composition may further be formulated for inhalation.
[0070] Pharmaceutical compositions formulated for oral administration may be in the form of a liquid, e.g., a solution in an edible solvent such as ethanol, tincture, syrup, or elixir; a semi-solid; or a solid such as tablets, caplets, pills, troches, lozenges, dispersible powder or granules, hard or soft capsules, and sachets. In some particular embodiments, the pharmaceutical composition is in the form of a bi- or multilayer tablet, in which each one of
the layers comprises the active agents, and the layers are optionally separated by an intermediate, inactive layer, e.g., a layer comprising one or more disintegrants.
[0071] Useful dosage forms of the pharmaceutical compositions include orally disintegrating systems including, but not limited to, solid, semi-solid and liquid systems including disintegrating or dissolving tablets, soft or hard capsules, gels, fast dispersing dosage forms, controlled dispersing dosage forms, caplets, films, wafers, ovules, granules, buccal/mucoadhesive patches, powders, freeze dried (lyophilized) wafers, chewable tablets which disintegrate with saliva in the buccal/mouth cavity and combinations thereof. Useful films include, but are not limited to, single layer stand-alone films and dry multiple layer stand-alone films.
[0072] In certain embodiments, the pharmaceutical compositions are formulated for oral administration, and are in the form of matrix tablets wherein the release of the active agent(s) is controlled by having said active agent(s) diffuse through a gel formed after the swelling of a hydrophilic polymer brought into contact with dissolving liquid (in vitro) or gastrointestinal fluid (in vivo). Many polymers have been described as capable of forming such gel, e.g., derivatives of cellulose, in particular the cellulose ethers such as hydroxypropyl cellulose, hydroxymethyl cellulose, methylcellulose or methyl hydroxypropyl cellulose, and among the different commercial grades of these ethers are those showing fairly high viscosity. In other embodiments, the tablets are formulated as bi- or multi-layer tablets, made up of two or more distinct layers of granulation compressed together with the individual layers lying one on top of another, with each separate layer containing the same of different active agent. Bilayer tablets have the appearance of a sandwich since the edge of each layer or zone is exposed.
[0073] Pharmaceutical compositions for oral administration might be formulated so as to inhibit the release of one or more of the active agents in the stomach, i.e., delay the release of said active agent(s) until at least a portion of the dosage form has traversed the stomach, in order to avoid the acidity of the gastric contents from hydrolyzing the active agent(s). Particular such compositions are those wherein the active agent(s) is coated by a pH- dependent enteric-coating polymer. Examples of pH-dependent enteric-coating polymer include, without being limited to, Eudragit® S (poly(methacrylicacid, methylmethacrylate), 1:2), Eudragit® L 55 (poly (methacrylicacid, ethylacrylate), 1:1), Kollicoat® (poly(methacrylicacid, ethylacrylate), 1:1), hydroxypropyl methylcellulose phthalate (HPMCP), alginates, carboxymethylcellulose, and combinations thereof. The pH-dependent
enteric-coating polymer may be present in the composition in an amount from about 10% to about 95% by weight of the entire composition.
[0074] Another contemplated formulation is depot systems, based on biodegradable polymers. As the polymer degrades, the active agent(s) is slowly released. The most common class of biodegradable polymers is the hydrolytically labile polyesters prepared from lactic acid, glycolic acid, or combinations of these two molecules. Polymers prepared from these individual monomers include poly (D,L-lactide) (PLA), poly (glycolide) (PGA), and the copolymer poly (D,L-lactide-co-glycolide) (PLG).
[0075] Pharmaceutical compositions for oral administration may further comprise one or more agents selected from sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. In addition, said compositions may comprise one or more pharmaceutically acceptable excipients. For example, a tablet may comprise at least one filler, e.g., lactose, ethylcellulose, microcrystalline cellulose, silicified microcrystalline cellulose; at least one disintegrant, e.g., cross-linked polyvinylpyrrolidinone; at least one binder, e.g., polyvinylpyridone, hydroxypropylmethyl cellulose; at least one surfactant, e.g., sodium laurylsulfate; at least one glidant, e.g., colloidal silicon dioxide; and at least one lubricant, e.g., magnesium stearate.
[0076] Pharmaceutical compositions formulated for parenteral administration, i.e., for administration elsewhere in the body than the mouth and alimentary canal, may be in the form of a sterile, optionally injectable, aqueous or oleaginous suspension, which may be formulated according to the known art using suitable dispersing, wetting or suspending agents. The sterile injectable preparation may also be an injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Acceptable vehicles and solvents that may be employed include, without limiting, water, Ringer's solution, polyethylene glycol (PEG), 2-hydroxypropyl-P-cyclodextrin (HPCD), a surfactant such as Tween-80, and isotonic sodium chloride solution.
[0077] The therapeutic agent and pharmaceutical composition disclosed herein are useful in the treatment of diseases or disorders associated with impaired MFR and/or mitoCa2+ homeostasis as defined above.
[0078] In yet another aspect, the present invention relates to a therapeutic agent according to any one of the embodiments above, encapsulated, e.g., within a capsule made of a
phospholipid and/or a polymer, or within a viral- or viral-like-envelope, for use in the treatment of a disease or disorder associated with impaired MFR and/or mitoCa2+ homeostasis.
[0079] In still another aspect, the present invention relates to a therapeutic agent according to any one of the embodiments above, encapsulated, e.g., within a capsule made of a phospholipid and/or a polymer, or within a viral- or viral-like-envelope, for use in the preparation of a medicament for the treatment of a disease or disorder associated with impaired MFR and/or mitoCa2+ homeostasis.
[0080] The invention will now be illustrated by the following non limiting Examples.
EXAMPLES
Study 1. IGF1 receptor regulates upward firing rate homeostasis via the mitochondrial calcium uniporter
Methods
[0081] Primary hippocampal culture preparation. Hippocampi were dissected from (Pozzi et al., 2013) pups (both sexes) at PO-lin ice cold Leibovitz L-15 medium.
Tissue was washed 3 times with Hank’s balance salt solution (HBSS). Chemical dissociation of cells was done using digestion solution (137 mM NaCl, 5 mM KC1, 7 mM Na2HPO4, 25 mM HEPES, 2 mg/mL trypsin, 0.5 mg/mL DNase) for 10 min in an incubator. Solution was replaced by HBSS supplemented with 20% fetal bovine serum (FBS) to inactivate trypsin and once again with only HBSS. Cells were then mechanically dissociated in HBSS supplemented with 13 mM MgSO4 and 0.5 mg/mL DNase by titration with fire -polished pipettes of decreasing diameter. Sedimentation of cells was accomplished by centrifugation at 1000 ref for 10 min at 4°C. After the removal of the supernatant, cells were re-suspended with plating medium (MEM supplemented with 10% FBS, 32.7 mM glucose, 25 mg/mL insulin, 2 mM Glutamax, 0.1 mg/mL transferrin, 0.1% SMI) and then plated on matrigel- coated glass coverslips, glass-bottom 24-wells or MEA plates. Half of the serum medium was replaced with feeding medium the day after (MEM supplemented with 32.7 mM glucose, 2 mM Glutamax, 3 pM ARA-C, 0.1 mg/mL transferrin, 2% SMI). Afterwards, half of the medium was replaced with fresh feeding medium every 3-4 days for three additional times. For all electrophysiology and live cell imaging, cultures were infected at 5-6 days in- vitro (DIV) with Cre+ or Cre- AAVs, shScr or shlGFl AAVs, and with MCUc (MCU,
MICU1 and MICU3) or mtIGFIR on DIV 8&9 (MCU and MICU1 on day 8, and MICU3 on the next day). The experiments were performed in cultures after 14-21 DIV.
[0082] Plasmids. For CBAP-Cre-P2a-Cerulean and CBAP-P2a-Cerulean, cDNA encoding for Cre was obtained from K.Villa. AAV2-CBAP plasmid was obtained from Daniel Gitler (BGU, Israel). Cre-P2a-Cerulean or P2a-Cerulean were fused in frame by PCR and cloned into AAV2-CBAP. AAV-hSynl-jRGECO1a was prepared by inserting jRGECOla (pGP-CMV-NES-jRGECOla, Addgene plasmid # 61563) into AAV2-hSynI- AT1.03 NL (gift from Daniel Gitler), replacing AT 1.03 NL. For AAV2-CaMII2a- 4mtGCaMP8m, 4mt fragment (synthesized by GenScript) was in frame fused to jGCaMP8m (Addgene #162372) and cloned into Addgene plasmid #51086, replacing GCaMP6s-p2A- nls_dTomato. Construction of AAV-hSynI-2mt-mCherry was described in (Styr et al., 2019). Mouse MCU (NM_001033259.4) and MICU1 (NM_144822.3) were synthesized by GenScript. Mouse MICU3 (NM_030110.2) was synthesized by Genwiz. P2a-mCherry was inserted into AAV2-CBAP, then MCU, MICU1 or MICU3 were cloned in frame with P2a- mCherry. For shRNA-mediated knockdown of mouse IGF1 or control vector, the whole cassette U6-shIGFl-hSyn-mCherry from pLL3.7 (Gazit et al., 2016) or U6-shScrambled- hSyn-mCherry were cloned into AAV2-hSynl, replacing the pre-existing promoter. For 4mtIGFlR-mTagBFP2: cDNA encoding to IGF1R was in frame fused to 4mt (synthesized by GenScript) and mTagBFP2 (p773, ETH, Switzerland) by PCR. The obtained 4mtIGFlR mTagBFP2 was inserted into AAV2-CBAP plasmid, giving AAV2-CBAP-4mtIGFlR- mTagBFP2 (called mitoIGFIR).
Electrophysiology
[0083] ME A data acquisition and analysis. Cells were grown on MEA plates [Multi Channel Systems (MCS), 120MEA200/30iR-Ti] containing 120 titanium nitride (TiN) electrodes with 4 internal reference electrodes. Each electrode’s diameter is 30 pm and they are spaced on a 12x12 grid (24 spaces in the 4 comers did not contain electrodes), spaced 200 pm apart. Data were recorded by either a MEA2100-System (MCS) with a chamber that maintained 37°C and 5% CO2, or a MEA1200-minisystem (MCS) that was constantly placed inside an incubator. Raw data was collected at 10 kHz, with a hardware high-pass filter of 1 Hz and an upper cut off of 3.3 kHz for the MEA2100- system and 3.5 kHz for the MEA2100- minisystem.
[0084] Using MCS data analyzer software offline, raw data were filtered by a Butterworth 2nd order high-pass filter at 200 Hz. Spikes were then detected by a fixed threshold of 6 SD. To reduce processing and analysis time, each hour was represented by 20 min of recording, which was previously shown to reliably represent the MFR of the full hour (Slomowitz et al., 2015). For spike sorting, Plexon offline- sorter V3 (Plexon inc. USA) was used. Principle component analysis was carried out on a 2-D or 3-D space. Distinct clusters were manually selected, and an automatic template sorting was done on the rest. Clusters’ stability throughout the recording was manually inspected. Only clusters that fulfilled the following requirements were considered units and used for the analysis: (1) No spikes in refractory periods. (2) The clusters were well defined during the entire experiment. (3) There were no sudden jumps in cluster location on PC axes. The rest of the analysis was carried out using a custom- written MATLAB (Mathworks) script as described in Slomowitz et al., 2015. For detection of bursts at the single-unit level, bursts were defined as 2 or more spikes at a minimum of 20 Hz based on the code we previously published (Slomowitz et al., 2015). Our previous analysis shows that the results are robust over a wide range of burst parameters (Slomowitz et al., 2015).
[0085] Patch clamp whole-cell recordings and analysis. Experiments were performed at room temperature in a RC-26G recording chamber (Warner instrument LCC, USA) on the stage of FV300 inverted confocal microscope (Olympus, Japan) using a Multiclamp 700B amplifier and a Digidata 1440A digitizer (Molecular devices, LLC, USA). All experiments were carried out using extracellular Tyrode solution containing (in mM): NaCl, 145; KC1, 3; glucose, 15; HEPES, 10; MgCh, 1.2; CaCh, 1.2; pH adjusted to 7.4 with NaOH, and intracellular solution containing (in mM): K-gluconate 120; KC1 10; HEPESs 10; Na- phosphocreatine 10; ATP-Na2 4; GTP-Na 0.3; MgCh 0.5. Intracellular solution was supplemented with 20 pM Alexa fluor 488 for dendritic spines imaging. For intrinsic excitability, synaptic blockers were used (in pM): 10 CNQX, 50 AP-5, and 10 gabazine. For mEPSCs recordings, tetrodotoxin (1 pM), AP-5 (50 pM), and gabazine (10 pM) were added to Tyrode’s solution. Intrinsic excitability protocols: small DC current was injected in current-clamp mode to maintain membrane potential at -65 mV. For LF curve: positive currents from 40 to 600 pA were injected in 40 pA increments for 500 ms. Input resistance (Rin) was measured by calculating the slope of the voltage change in response to increasing current injections from -80 to +20 mV in 20 mV increments. For single AP measurements,
2 ms currents were injected at 40 pA increments. For mEPSCs recordings, neurons were voltage-clamped at -65 mV. Neurons were excluded from the analysis if no dendritic spines were observed, serial resistance was > 15 MQ, serial resistance changed by >20% during recording, or if Rin was < 75 MQ. Signals were recorded at 10 kHz, and low-pass filtered with Bessel filter 2 kHz. Electrophysiological data were analyzed using pClamp (Molecular Devices LLC, USA) and MiniAnalysis (Synaptosoft, Decatur, Georgia, USA) for mEPSC. For distributions of mEPSCs, 90 first events were taken from each neuron to prevent overrepresentation of high-frequency neurons.
Confocal live cell imaging
[0086] Evoked cytosolic and mitochondrial calcium imaging in neuronal soma. Experiments were performed on a FV-1000 (Olympus, Japan) system using a QR/RC- 47FSLP chamber with a TC-324C temperature controller (Warner Instruments LLC, USA) at 33-34°C. In-chamber temperature stability was verified between coverslips with a Newtron TM-5005 thermometer. Coverslips were placed in Tyrode’s solution that contained (in mM): NaCl, 145; KC1, 3; glucose, 15; HEPES, 10; MgCl2, 1.2; CaCl2, 1.2; pH adjusted to 7.4 with NaOH at 34°C. Synaptic blockers were used to prevent recurrent activity from stimulation (in pM): 10 CNQX, 50 AP-5. Cultures were infected with AAVl/2-hSyn- jRGECOla and AAVl/2-CaMKIIa-4mt-GCaMP8m, so that only excitatory neurons expressed both Ca+2 sensors. Only cells with mitochondrial response to a 10-stimuli burst at 50 Hz were considered. Infection with AAV1/2-CB AP-Cre-Cerulean or AAV1/2-CBAP- Cerulean was verified with a 440 nm laser. Imaging was done with a 60x lens and xl.5 digital magnification at -12.5 frames per second with 488 nm and 561 nm lasers, and emission spectra of 505-540 nm and 575-675 nm for GCaMP8m and jRGECOla, respectively. Field stimulation was given using a SIU-102 stimulation unit (Warner Instruments LLC, USA) connected to an Axon Digidata 1440A digitizer (Molecular devices, LLC, USA). Each imaged neuron was stimulated by a single stimulus and bursts of 3, 5 and 10 stimuli at 50 Hz. Some neurons had more than one clear response to 3, 5 or 10 stimuli and thus excluded from analysis. Analysis was done using ImageJ.
[0087] Spontaneous cytosolic calcium in neuronal soma. Experiments were performed on a FV-1000 (Olympus, Japan) system. Cultures were plated on #0 glass bottom 24 well (Cellvis, USA) and imaged in a heated (34-35°C) Stage-Top Incubator System TC, connected to a CU-501 temperature controller and a humidifier delivering 5% CO2 air (Live
Cell Instruments, Republic of Korea). Imaging parameters were the same as for the evoked imaging. Each neuron was recorded 3 times for 3 min, with a 3-min interval to prevent bleaching and phototoxicity. Analysis was done using ImageJ (for extraction of florescence intensity over time) and a custom- written MATLAB script (for dF/F calculation and event identification and quantification).
[0088] STED microscopy . Immuno staining was carried out based on standard protocols. Cultured hippocampal neurons (prepared as described in Jahne el al., 2021) were fixed with 4% PFA in PBS for 30 min, followed by quenching with 100 mM glycine in PBS for 15 min, before being permeabilized and blocked using a blocking buffer prepared with 2% BS A (Sigma, 9048-46-8) in PBS added to 0.1% TritonX-100 (Merck, Kenilworth, NJ, USA). Next, the cells were incubated overnight with primary antibodies diluted in blocking reagent, at 4°C. The neurons were then incubated with the appropriate secondary antibody (1:400) for 60 min at room temperature. The coverslips were mounted using Mowiol (Merck Millipore, Kenilworth, NJ, USA). The following primary antibodies were used: MICU1 rabbit polyclonal (1:100; Sigma), MICU2 rabbit polyclonal (1:50; Sigma), MICU3 rabbit polyclonal (1:150; Sigma), MCU rabbit polyclonal (1:100; Sigma), TOM20 mouse monoclonal (1:100; Sigma), and IGFIRb mouse monoclonal (1:100; Invitrogen, 194Q13). The applied secondary antibodies were anti-mouse STAR580 and anti-rabbit STAR635P purchased from Abberior GmbH, Gottingen, Germany.
[0089] Epifluorescence images were obtained by means of an 1X83 inverted microscope (Olympus). STED imaging was captured using a STED Abberior microscope, Gottingen, Germany. Excitation lines of 640 nm and 561 nm were adopted for exciting Star653P (MCUc subunits) and Star580 (TOM20 and IGF1R). For STED excitation, pulsed lasers were set at 640 nm and 580 nm. STED depletion was implemented via 775 nm depletion laser and the images were acquired at 20 nm pixel size.
[0090] Proximity ligation assay (PLA). Primary hippocampal neurons were cultured and fixed as described above. PLA was carried out using Duolink™ In Situ Orange Starterkit Mouse/Rabbit (DUO92102-1KT, Merck) according to the Duolink® PLA Fluorescence Protocol. Primary hippocampal neurons were immunostained with anti-mouse IGF1RP (1:100, Invitrogen, 194Q13) and anti-rabbit IGF1 (1:100, abeam, ab9572) primary antibodies. After the PLA reaction, neurons were immunostained with the anti-rabbit Tom20 polyclonal antibody directly conjugated to CoraLite®488 (1:100, CL488-11802,
Proteintech). Co ver slips were embedded with Duolink® In Situ Mounting Medium with DAPI and sealed to avoid leaking. Imaging was performed in the confocal mode using an Abberior microscope (Gottingen, Germany). The CoraLite®488 fluorophore was excited using a 488 nm laser line and the PLA fluorophore was excited using a 561 nm laser line. Images were acquired in 40x40 pm size with a pixel size of 40x40 nm and lOps pixel dwell time.
[0091] Brain and free mitochondria purification. Brain lysates and free mitochondria were isolated as previously described (Sims and Anderson, 2008) form 5-month-old C57BL/6Rj with minor modifications. Briefly, upon rapid brain extraction, the forebrain was dissected, cleaned from meninges and washed in isolation buffer, to remove blood. Buffers were prepared exactly as described in (Sims and Anderson, 2008). The tissue was then homogenized with method A (using a dunce homogenizer) with a two-step approach, as detailed in (Sims and Anderson, 2008). The homogenate obtained was used for the "total brain lysate" samples. After the two centrifugations at 1,300 g at 4°C, the supernatants were further centrifuged at 21,000 g at 4°C for 10 min while preparing the Percoll (GE Healthcare, cat. no. 17089101) gradients. The pellet was resuspended in 15% Percoll solution at 4°C by gentle pipetting. This resuspended crude mitochondrial fraction was layered gently on the Percoll gradient and centrifuged at 30,700 g at 4°C for 5 min. After centrifugation, the lower band that contains the highly enriched mitochondrial fraction at the interface between the 23% and 40% Percoll layers (termed band 3) was collected, further concentrated by a 16,700g 10 min centrifugation at 4°C. These mitochondria were named "free mitochondria" and were used for further analyses.
[0092] Western blotting. Protein concentration was measured with the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, 23225) following the manufacturer’s guidelines. Samples of identical protein concentrations were run in parallel gels containing 10% or 20% polyacrylamide (Roth A124.2), and bands were separated with SDS-PAGE. Gels were then transferred to nitrocellulose blotting membranes (Amersham Protran Premium 10600003) for 2 hours at 1.25 A. Membranes were blocked in 5% skim milk powder (Sucofin) for Ih at RT in TBS-Tween20 buffer (20 mM Tris; 150 mM NaCl; 0.1% Tween 20). Membranes were incubated with the respective primary antibodies diluted in the blocking buffer overnight at 4°C. Membranes were rinsed for 30 min in TBS-Tween20 and incubated for 1 h at room temperature with the corresponding secondary LICOR antibodies
(see antibody list for details) and finally washed in TBS-Tween20 for 30 min. As a control for loading reproducibility, the blots were probed with an anti-GADPH antibody and revealed with the respective secondary antibody. After secondary antibody incubation, the fluorescent images of the nitrocellulose membranes were acquired with a LICOR Odyssey CLX-2088 imaging system. To cleave all proteins from the outer membranes of the organelles in the brain lysates and the free mitochondria, the samples were divided into 2 groups: one treated with trypsin (+ trypsin; Thermofisher Gibco 25300-054) and an untreated group (- trypsin). Primary antibodies: mouse anti-IGFIRb antibody (1:500; Invitrogen, 194Q13); mouse anti-GAPDH (1:5000; Proteintech 60004-1-Ig), mouse anti-Cox4-l (1:500; custom-made, identifier PRAB1522, a generous gift from Dr. Sven Dennerlein (University Medical Center Gottingen, Germany) characterized in (Richter-Dennerlein el al., 2016). Secondary antibodies: donkey anti-mouse IgG antibody conjugated to IRDye 800 CW (1:5000; Li-Cor 926-32212); donkey anti -rabbit IgG antibody conjugated to IRDye 680 RD (1:5000; Li-Cor 925-68072).
[0093] Real-time PCR. RNA was extracted using the TRI Reagent, according to the manufacturer’s instructions (Sigma- Aldrich). Equal amount of mRNA was reverse- transcribed to cDNA with Superscript III reverse transcriptase (Invitrogen, cat. No: 18080- 051). Real-time qPCR was performed with Sybr mix (Applied Biosystems). Reactions were run in triplicate in a StepOnePlus real-time PCR system (Applied Biosystems). mRNA abundance was calculated by means of the comparative cycle threshold (Ct) method following the manufacturer’s guidelines. mRNA expression level was normalized to the average expression of B2m and Polr2a.
[0094] AAV Vector Production. AAV vector production was carried out in 293T cells. Cells were transfected 24 h after seeding with helper plasmids encoding AAV rep, cap and plasmid for the rAAV cassette expressing the relevant DNA. Cells were harvested 72 h after transfection, cells pellet was resuspended in lysis solution (150 mM NaCl, 50 mM Tris-HCl, pH 8.5), ImL of lysis buffer per 150 mm dish. Cells were lysed by a few freeze-thaw cycles. The obtained crude lysate was treated with 50 U benzonase (Sigma, E1014) per 1 mL of lysate at 37°C for 1.5 h to degrade genomic DNA. Cell debris were pelleted by centrifugation at 3000g for 15 min 4°C. Supernatant contains crude virus was filtered through a 0.45 um filter and stored at 4°C.
[0095] Western Blot. Primary hippocampal cultures were infected on DIV5-6 by AAVs encoding cre-P2a-mCherry sequences (+Cre) or P2a-mCherry sequence (-Cre). Lysates of equal number of cells (~500k) were separated by SDS-PAGE, transferred to nitrocellulose, blocked with 5% skim milk powder (Difco # 232100) and incubated ON with anti-IGFIR antibody (#3027 CST), followed by anti-rabbit-HRP secondary antibody (#11-035-044 Jackson ImmunoResearch). To normalize the signal, the blot was re-probed with anti-actin antibody (Sigma), followed by anti-mouse-HRP secondary antibody (#115-035-146 Jackson ImmunoResearch).
[0096] Knockout validation at the level of genomic DNA . Primary hippocampal cultures were infected on DIV5-6 by AAVs encoding cre-P2a-mCherry sequences (+Cre) or P2a- mCherry sequence (-Cre). Cells were collected and lysed, and DNA was purified using the MasterPure™ DNA&RNA Complete purification kit according to the company’s manual (Cat. 19 MC85200 epicentre). DNA was probed with primers for the excised and not excised version of the floxed exon3 of the IGF1R gene with the following primers:
[0097] FRET efficiency imaging. Primary hippocampal cultures were infected on DIV5- 6 by AAV1/2 encoding Cre-P2a-mCherry sequences or P2a-mCherry sequence. Experiments were performed on a FV-1000 (Olympus, Japan) system at room temperature. Some coverslips were placed in Tyrode’s solution that contained (in mM): NaCl, 145: KC1, 3: glucose, 15: HEPES, 10: MgCh, 1.2; CaCh, 1.2; pH adjusted to 7.4 with NaOH. Others were placed in their own media, in a heated (34-35°C) Stage-Top Incubator System TC,
connected to a CU-501 temperature controller and a humidifier delivering 5% CO2 air (Live Cell Instruments, Republic of Korea). The results were similar for both conditions so they were pooled. Infection of neurons with AAVl/2-CBAP-Cre-mCherry or AAV1/2-CBAP- mCherry was verified with a 561 nm laser, images of neuronal soma were taken before and after acceptor (cpmVen) photobleaching. Donor (mseCFP) was excited with a 440 nm laser, and emission was measured at [460-500] nm before (IDA) and after (ID) photobleaching. Bleaching was accomplished using a 514 nm laser. Images of acceptor were taken before and after bleaching at [530-600] nm, to assess bleaching level: neurons with less than 85% reduction in fluorescence were excluded. FRET efficiency was calculated as [IDA/ ID].
[0098] FM-based imaging. Activity-dependent FM1-43 (10 pM) styryl dye was used to estimate basal synaptic vesicle recycling and short-term plasticity using protocols described previously (Abramov et al. , 2009). Briefly, APs in neurons were initiated by field stimulation during dye loading, and the terminals, after undergoing vesicle exocytosis coupled to endocytosis, were stained by FM1-43 10 pM FM1-43 has been present 5 sec before and 20 sec after the electrical stimulation. During FM loading and unloading, kynurenic acid (0.5 mM) was added to Tyrode solution to prevent recurrent activity through blockage of excitatory postsynaptic responses during loading and unloading. After dye loading, external dye was washed away in Ca2+-free solution containing ADVASEP-7 (0.1 mM; Sigma). To confirm that the fluorescent spots corresponded to release sites, we evoked 600 APs @ 5 Hz during the unloading step to obtain release of dye-filled vesicles. The total amount of releasable fluorescence at each bouton (DF) was calculated from the difference between fluorescence after loading and after unloading (DF = Fioading - Funioading). The total presynaptic strength during low-frequency stimulation (loading of 30 APs @ 1 Hz) has been calculated as Ssingie = DF " D, whereas D is the number of FM-(+) terminals per FM image. To determine the total presynaptic strength during burst patterns (Sburst), 30 APs have been delivered by bursts consisting of 5 APs @ 50 Hz, inter-burst-intervals of 5 s. To determine the sign and magnitude of short-term plasticity, we calculated the Sburst / Ssingie ratio over the same image area. Image analysis was carried out using ImageJ.
[0099] Statistical analysis. Experiments were repeated at least three times (three independent cultures) for each group. Data are shown as mean ± standard error of the mean (SEM). All statistical analyses were carried out using GraphPad Prism 8.0 (GraphPad
software, USA), and the specific tests of each figure, along with their P-values and total number of cells/repetitions per experimental group (n/N) are specified in the figure legends.
Results
IGF1R deficiency limits homeostatic compensation of mean firing rate and pattern to inactivity
[00100] To explore the role of mitoCa2+ in MFR homeostasis, we first tested whether IGF1R is necessary for the homeostatic regulation of firing rate distributions and their means. For this, multi-electrode arrays (MEAs) were used for long-term recordings of spiking activity at the same cultured hippocampal neurons during baseline and throughout two days of activity perturbation. The GAB AB receptor agonist baclofen (Bac) was used as a persistent inhibitory perturbation (Slomowitz et al., 2015). For conditional deletion of IGFIRs,
mice with a viral delivery of Cre-recombinase were used. Adeno- associated virus (AAV) was used under the general promoter CBAP (AAVl/2-CBAP-Cre- Cerulean), creating IGF1R knockout (IGF1R-KO) networks. It is envisioned that the promoter could be different - here a general promoter was used, but in other embodiments more specific promoters are envisioned, e.g., hSynl - for neurons, CaMKIIa - for excitatory neurons, hDlx - for inhibitory neurons, GFAP - for astrocytes, etc. For control (Ctrl) experiments, IGFlRfl/fl cultures were infected with AAV1/2-CB AP-Cerulean.
[00101] As expected from our previous work (Slomowitz et al., 2015; Styr et al., 2019), the Ctrl networks displayed a pronounced suppression of activity by baclofen (10 pM) that was restored to network’s baseline MFR after a period of two days (Figs. 1A-1C). Conversely, the average firing rates of IGF1R-KO networks exhibited only partial (-45%) recovery subsequent to baclofen application (Figs. 1D-1F). Moreover, distributions of Ctrl single-unit firing rates were indistinguishable before, and two days after, baclofen application (Fig. 2A). While effects of 2 day baclofen were variable per single unit in Ctrl conditions (Fig. 2B), on average per-unit MFR was not different following baclofen application (Fig. 2C). In contrast, MFR distribution was left-shifted following baclofen application in IGF1R-KO neurons (Fig. 2D) due to incomplete single-unit recovery (Figs. 2E-2F).
[00102] In addition to changes in mean rate, temporal spike pattern was also affected by baclofen, confirming our early study (Slomowitz et al., 2015). Namely, in Ctrl networks, the fraction of spikes participating in single-unit bursts (Figs. 2G-2H) and burst frequency normalized to MFR showed an acute decrease and subsequent increase beyond the baseline
level, which gradually declined during the perturbation (not shown). In contrast, IGF1R- KOs showed lower fraction of spikes participating in bursts at any given moment of time: baclofen caused an acute decrease in the fraction of spikes in bursts that recovered to the baseline during the first day of the perturbation and eventually declined below the baseline during the second day of the perturbation (Figs. 2G-2H). These experiments point towards IGF1R as a necessary component of homeostatic regulation of firing rate and pattern. Notably, IGF1R deletion did not affect baseline firing rates (Fig. 21) and patterns (Figs. 2J- 2K), suggesting that IGF1R is not necessary for the maintenance of the baseline spontaneous network activity, but for the homeostatic response to chronic inactivity.
[00103] Next, it was investigated whether somatic cytoCa2+ is also homeostatically regulated, and whether IGF1R is necessary for this process. Continuous imaging of somatic cytoCa2+ during spontaneous spiking activity in excitatory hippocampal neurons was conducted at baseline, and following 2 days of baclofen perturbation. The average amplitude, frequency, and Ca2+ influx index (a product of average amplitude x frequency) of cytosolic events were quantified for Ctrl and IGF1R-KO neurons. In Ctrl, 2 days of baclofen did not affect cytoCa2+ event amplitudes, rate, and subsequently the Ca2+ influx index (not shown). In IGF1R-KO neurons, the amplitude of cytoCa2+ events remained unaltered following the perturbation, but their frequency was diminished, resulting in lower Ca2+ influx index following 2 days of the perturbation (not shown). These results are in line with the electrophysiological data showing limited recovery of both MFR and burst rate (Figs. 1E- 1F). Moreover, these data further support somatic cytoCa2+ as a regulated variable maintained by a homeostatic system and demonstrate that IGF1R deletion impairs this regulation.
[00104] IGF1 is the principal ligand of the IGF1R (Hakuno and Takahashi, 2018). To test if it is the necessary signal for IGF1R to enable MFR homeostasis, IGF1 was knocked-down using an AAV carrying a small-hairpin RNA against it (shlGFl). shlGFl mimicked the effect of IGF1R-KO, showing a limited compensatory MFR response to baclofen in comparison to a control vector carrying a scrambled sequence (shScr, not shown). These results indicate that MFR homeostatic response is mediated by IGF 1 -activated IGFIRs.
[00105] To test if IGF1R is necessary for MFR renormalization to a set-point value for bidirectional changes in activity, chronic hyperactivity in IGF1R-KO neurons was induced by enhancing glutamate spillover by inhibiting glutamate transporters (Asztely et al., 1997). Application of 10 pM TBOA (threo-P-benzyloxyaspartate), a competitive glutamate
transporter antagonist (Christie and Jahr, 2006), induced a transient increase in MFR that was gradually renormalized during the following 2 days to the set-point value (not shown). Taken together, these results indicate that IGF1R is essential for upward but not for downward homeostatic restoration of MFRs.
IGF1R deletion blocks postsynaptic and intrinsic homeostatic plasticity
[00106] MFR homeostasis is achieved by intrinsic and synaptic adaptations that act in a negative-feedback manner to counteract disturbances to ongoing activity (Turrigiano, 2011). To address the question of whether IGF1R is necessary for a single but crucial compensatory mechanism, or is it an up-stream regulator of distinct compensatory mechanisms, several parameters of intrinsic excitability and synaptic strength in excitatory neurons were measured using whole-cell patch clamp. First, we tested whether intrinsic excitability is changed after 2 days of baclofen. Action potentials were elicited by injecting somatic currents ranging from zero to 600 pA (F-I curves) in the presence of postsynaptic receptor blockers in Ctrl (Figs. 3A-3B) and IGF1R-KO (Figs. 3C-3D) excitatory neurons. In Ctrl, 2 days of baclofen elicited an increase in firing rate in response to current injections (Figs. 3A- 3B) and augmented the maximal firing frequency (not shown). However, this plasticity of intrinsic excitability was lost in IGF1R-KO neurons, as reflected by the lack of change in the F-I curve (Figs. 3C-3D) and the maximal firing frequency (not shown) in response to 2 days of baclofen. Input resistance, spike threshold voltage, spike amplitude and half-width were unaltered by 2 days of baclofen in either Ctrl or IGF1R-KO groups (not shown), but the KO group exhibited increased input resistance and decreased action potential amplitude (not shown). These changes, however, did not result in changes of spontaneous firing (Fig.
21).
[00107] Next, we tested whether IGF1R deletion alters presynaptic and postsynaptic adaptations to inactivity. Indeed, in Ctrl neurons, 2 days of baclofen elicited a marked increase in the amplitude of miniature excitatory postsynaptic currents (mEPSCs) (Figs. 3E- 3F), whereas in IGF1R-KO neurons this homeostatic postsynaptic plasticity was lost, reflected by the lack of baclofen effect on the mEPSC amplitude (Figs. 3E-3G). On the other hand, the mEPSC frequency was increased in both groups (not shown). To test if IGF1R preserves the integrity of presynaptic compensatory mechanisms, presynaptic vesicle recycling was quantified directly using FM-based method (Slutsky et al., 2004). As baclofen-induced increase in mEPSC frequency is associated with an increase in synaptic
release probability (Slomowitz et al., 2015), FM1-43 was used to test if homeostatic increase in synaptic release probability remains intact in IGFIR-KOs. In response to 2 days of baclofen perturbation, both Ctrl and IGFIR-KOs exhibited increased presynaptic strength evoked by low-frequency stimulation and decreased short-term synaptic facilitation evoked by high-frequency spike bursts (not shown), indicating an increase in release probability. These results suggest that IGF1R is dispensable for the presynaptic homeostatic plasticity. However, the presynaptic compensation is insufficient for MFR renormalization. Taken together, these results demonstrate that deletion of IGFIRs impaired both postsynaptic and intrinsic homeostatic mechanisms that may contribute to the failure of MFR homeostatic recovery at the network level.
IGF1R deletion suppresses spike-to-mitoCa2+ coupling
[00108] How changes in firing rates and patterns are translated into physiological error signals within neurons remains unknown. Somatic cytoCa2+ has long been assumed to be a regulated variable through which neurons sense changes in spiking activity, and by doing so are able to correct deviations from MFR set points (O’Leary et al., 2014). Mitochondria are known to uptake Ca2+ upon neuronal activation (Kann and Kovacs, 2007), and thus may serve as a sensor of activity -induced changes in cytoCa2+ (Ruggiero et al., 2021).
[00109] As our early work found that IGFIRs modulate mitoCa2+ at the presynaptic compartment (Gazit et al., 2016), we asked how do IGFIRs regulate activity -dependent cytoCa2+ and mitoCa2+ in neuronal soma. To address this question, spike-to-cytoCa2+ and cyto-to-mitoCa2+ coupling for different patterns of spikes in Ctrl vs. IGF1R-KO excitatory neurons was measured. Simultaneous dual-color imaging of cytosolic and mitochondrial Ca2+ dynamics were conducted by using jRGECOla and 4mt-GCaMP8m, respectively (Fig. 4A). To identify excitatory neurons, 4mt-GCaMP8m under the CaMKIIa promoter were used. Neurons were stimulated by a single spike and spike bursts comprising of 3, 5 and 10 spikes at 50 Hz, while spontaneous firing was blocked. Under these conditions, spike-to- cytoCa2+ coupling did not differ between Ctrl and IGF1R-KO neurons (Figs. 4B, 4D). Conversely, IGF1R deletion caused a significant reduction in spike-to-mitoCa2+ coupling (Figs. 4C, 4E). Importantly, mitoCa2+ transients were activated only by spike bursts, but not by single spikes. Hence, IGF1R deletion diminished Ca2+ uptake by mitochondria evoked by spike bursts only. These changes resulted in weaker cytoCa2+-to-mitoCa2+ coupling, as seen by the right-shift of their transfer function (Fig. 4F).
[00110] Given the central role of mitochondria in ATP production, we tested whether IGF1R deletion suppressed ATP homeostasis. The cytosolic FRET sensor ATeam (Imamura et al., 2009) was used to estimate somatic ATP levels in Ctrl and IGF1R-KO hippocampal neurons. The results show no difference in FRET efficiency by IGF1R deletion during spontaneous activity (not shown). Taken together, these data suggest that IGF1R-KO impairs somatic Ca2+ uptake by mitochondria, without affecting cytoCa2+ and ATP levels. The decreased mitoCa2+ transients elicited by burst-induced cytoCa2+ events, ultimately lead to a weakening of cytoCa2+-to-mitoCa2+ coupling.
IGF1R is present in neuronal mitochondria
[00111] The mechanism used by IGF1R to regulate the mitoCa2+ influx does not appear to be straightforward, when one considers that IGF1R is a canonical plasma membrane receptor. One solution to this problem would be the presence of IGF1R in mitochondria, where the MCUc is located. This was tested by biochemical fractionation and superresolution imaging approaches (Fig. 5). Using an antibody against the P subunit of IGF1R, the unmodified IGF1R P subunit (~95 kD), which was predominant in the total lysate of mouse brain, and an additional - 100-kD band that was enriched in the mitochondrial fraction (Fig. 5A) were detected. This second band was stable when the lysates were treated with trypsin, implying that a fraction of IGF1R is found in the inner mitochondrial space, and is not simply attached to the outer mitochondria membrane (Fig. 5B).
[00112] Next, we tested whether IGF1R is co-localized in mitochondria with MCUc, using 2-color stimulated emission depletion (STED) microscopy (Figs. 5C-5D). This revealed a partial colocalization of IGF1R and the four MCUc components - the pore-forming subunit MCU, as well as regulatory subunits MICU1, MICU2 and MICU3 (Figs. 5E-5E’). Quantitative analysis indicated that the colocalization is statistically significant, with the IGF1R signal in the MCUc spots being significantly higher than elsewhere (Fig. 5F). On average, -30% of IGFIRs in soma are localized with the bona fide mitochondrial marker TOM20 (Fig. 5G).
[00113] Finally, we tested if mitochondrial IGF1R binds IGF1 using a proximity ligation assay (PLA). The PLA allows visualization of the interaction between two endogenous proteins in fixed neurons strictly dependent on the simultaneous recognition of the target by two antibodies (see Methods). About 40% of IGFIRs bound to IGF1 were localized to the
mitochondria (not shown). Taken together, these results suggest that IGF 1 -bound IGF1R is present in the mitochondria, where it co-localizes significantly with MCUc complexes.
Rescue of mitoCa2+ and upward MFR homeostatic response by mitoIGFIR / MCUc in IGF1R-KO neurons
[00114] Given that MCUc is the primary Ca2+ source into mitochondria (De Stefani et al., 2016), it was tested whether the reduction in mitoCa2+by IGF1R deletion could result from downregulation of the MCUc subunits. For this test, the mRNA expression levels of MCU and the Ca2+-sensing subunits MICU1, MICU2 and MICU3 were measured. As found, IGF1R deletion caused a downregulation in the expression level of MCU and the brainspecific (Patron et al., 2019) MICU3 subunits, while it did not affect the expression levels of MICU1 and MICU2 subunits (Fig. 6A). It was therefore concluded that IGF1R affects MCU and MICU3 expression at the transcriptional level.
[00115] To directly test whether reduction in mitoCa2+ is the primary cause of impaired MFR homeostasis in IGFIR-KOs, we tested whether ectopic overexpression of MCUc may rescue mitoCa2+ and MFR homeostasis. As found, overexpression of MCU, together with its regulatory subunits (MICU1 and MICU3), led to an increase in mitoCa2+ transients evoked by spike bursts in IGFIR-KOs, rescuing it to control level (Figs. 6B-6C). Then, we tested if the rescue of mitoCa2+ in IGF1R-KO neurons is sufficient to restore MFR homeostasis in response to inactivity. While MFR recovered only to 36% of the baseline in IGF1R-KO networks overexpressing mCherry, overexpression of MCUc markedly increased the compensatory response of IGF1R-KO networks to 93% of the baseline (Figs. 6D-6E). As MCUc rescues both mitoCa2+ and MFR homeostasis in response to inactivity, these results strongly suggest that IGF1R deletion impairs MFR homeostatic recovery from inactivity by reducing mitoCa2+ uptake during spike bursts.
[00116] Finally, we examined whether restoring IGF1R exclusively in the mitochondria is sufficient for functional recovery of mitoCa2+ dynamics and MFR homeostasis. To localize IGF1R to mitochondria, the IGF1R sequence was fused at the N-terminus to a tandem of four repeats of the Cox-8 mitochondria-targeting peptide (AAV-CBAP-4mtIGFlR- mTagBFP2, called mitoIGFIR). Mito-IGFIRs were colocalized with mitochondrial marker mito-mCherry in neurons (Fig. 6F). Expression of mitoIGFIR in IGF1R-KO neurons increased mitoCa2+ to Ctrl levels (Figs. 6G-6H) and rescued MFR homeostasis (Figs. 61, 6E). Furthermore, mitoIGFIR expression rescued baclofen-induced change in spike pattern
in IGFIR-KOs; the fraction of spikes participating in single-unit bursts was increased above the baseline during the initial phase of the perturbation and gradually decreased to the baseline (Fig. 6J), similarly to control conditions (Fig. 2G). Thus, a sub-population of IGF1R, mitoIGFIR, is sufficient for the proper functioning of both processes - mitoCa2+ and MFR homeostasis.
Rescue of mitoCa2+ and upward MFR homeostatic response by mitoIGFIR / MCUc in IGF1R-KO neurons
[00117] While mitochondrial dysfunctions have been implicated in ASD704, the relationship between Shank3 and mitoCa2+ has remained unexplored. To address this question, we used a Shank3-InsG3680 mouse model. We tested if the InsG3680 mutation causes deficits in mitoCa2+ and MFR homeostatic responses in hippocampal neuronal networks. Our results demonstrate that excitatory hippocampal neurons of Shank3-InsG3680 mutant mice show a profound decrease in mitoCa2+ evoked by spike bursts in excitatory hippocampal neurons (Figs. 7A-7B) and impaired MFR homeostatic response to inactivity (Figs. 7C-7D).
[00118] To test whether mitochondrial targeted IGF1R can rescue mioCa2+ in Shank3- InsG3680 mutant neurons, we expressed mitoIGFIRs in Shank3-InsG3680 mutant neurons. Our results show that mitoIGFIR fully restores mitoCa2+ influx in Shank3-InsG3680 excitatory neurons (Figs. 7A-7B). These results demonstrate that targeting of IGFIRs to mitochondria can rescue cellular deficits induced by Shank3 mutations.
Discussion
[00119] Long-term stability of ongoing spiking dynamics is crucial for neural circuits’ functions (Abbott and Nelson, 2000; Turrigiano and Nelson, 2004; Davis, 2006; O’Leary, 2018; Marder, 2011). Although a large number of fine-tuned parameters regulating synaptic and intrinsic membrane properties can generate similar firing properties (Prinz et al., 2004; Marder and Goaillard, 2006), the cellular and molecular design underlying MFR homeostasis in central neural networks remains largely unknown. Here, we identify a novel role of IGFIRs, known regulators of brain development, proteostasis (Cohen et al., 2009; Cohen et al., 2006) and lifespan (Kenyon et al., 1993; Holzenberger et al., 2003), in homeostasis of neural network activity. Our results provide converging evidence on the necessity of evolutionary-conserved IGF1R signaling in the stabilization of firing rate distributions at the population level in hippocampal networks. These results are important for several reasons. First, they demonstrate that IGFIRs are dispensable in regulating MFR
set points during spontaneous neuronal activity, but are critical for the homeostatic compensation of MFR to inactivity. Second, they reveal a role of IGF1R in regulating temporal Ca2+ filtering via MCUc of neuronal mitochondria during periods of spike bursts. Third, they reveal that a subpopulation of IGFIRs is present in mitochondria and colocalized with MCUc. Fourth, they show that either mitoIGFIR or MCUc can restore upward MFR homeostasis in IGFIR-deficient networks. Finally, they point to a critical role of mitochondria in regulating the integrated homeostatic response at the network level.
[00120] Mitochondria as high pass filters in central neurons . The results shown herein indicate that neurons are extremely unreliable at transferring information encoded by single spikes to somatic mitochondria. The coupling of mitochondria-to-cytosolic Ca2+ is nonlinear, showing almost complete uncoupling during periods of low-frequency, single spikes. However, spike bursts, known to play an important role in synaptic plasticity and information processing (Lisman, 1997), are reliably signaled to mitochondria by activating mitoCa2+ influx via MCUc. Thus, neuronal mitochondria can be viewed as filters that transmit bursts, but filter out single spikes. Our results demonstrate that these filter properties are regulated by IGFIRs. We identified a subpopulation of IGFIRs that localizes in mitochondria and regulates mitoCa2+ entry in hippocampal neurons. While previous work identified several other members of receptor tyrosine kinase family that can translocate to mitochondria, such as EGFR (Demory et al., 2009; Che et al., 2015) and ErbB2R (Ding et al., 2012). Similarly to EGF (Che et al., 2015), IGF1 may induce internalization and translocation of the IGF1R from the plasma membrane (i.e., the membrane that defines the cell) to the mitochondria (Fig. 8) (alternatively, it cannot be excluded that IGF1R is moved to the mitochondria directly after translation). Irrespective of the precise mechanism, our results point to mitoIGFIR as a key regulator of mitoCa2+ influx. Although our conclusions are based on cultured neurons, a recent in vivo study in the cortex of awake mice showed a positive relationship between mitoCa2+ and cytoCa2+ peak amplitudes, wherein large cytoCa2+ events are likely to be evoked by spike bursts (Lin et al., 2019). Therefore, unreliable spike-to-mitoCa2+ coupling during single spikes appears to be a universal property of somatic mitochondria in central neurons. Given that the duration of mitoCa2+ events is ~8 times longer than cytoCa2+, mitochondria perform amplification and high-pass filtering of burst-evoked cytoCa2+ signals. Therefore, MCUc does not only sense cytoCa2+ to control the threshold and gain as has been previously proposed (Csordas et al., 2013), but also
controls information content transferred from cytoplasmic membrane potential to mitochondria.
[00121] MCUc as a homeostatic sensor. What is the role of IGF1R/ MCUc signaling in upward MFR homeostasis? Here, we propose that MCUc is a homeostatic sensor that drives expression of intrinsic and postsynaptic homeostatic plasticity, and eventually MFR homeostatic response (Fig. 8). MCUc senses the changes in cytoCa2+ by Ca2+-binding EF- hand-containing regulatory subunits MICU (Marchi and Pinton, 2014). At the resting level of cytoCa2+, MICU1-MICU2 and MICU1-MICU3 heterodimers prevent ion conduction through the MCU channel, and they permit it when Ca2+ levels rise (Kamer and Mootha, 2015). Our results indicate that the rise of cytoCa2+ evoked by spike bursts is sufficient to activate MCUc in neuronal soma. Our current and previous (Slomowitz et al., 2015) results demonstrate that GABABR-mediated suppression of MFR is associated with a change in temporal spike pattern: the fraction of spike participating in bursts is increased during the initial phase of the perturbation. These changes in the temporal spike structure promote a more efficient activation of MCUc during the induction phase of homeostatic plasticity. The function of IGFIRs in upward MFR homeostasis is two-fold: 1) to enable an increase in the fraction of spike bursts during the induction phase of homeostatic plasticity; 2) to maintain burst-to-mitoCa2+ coupling. Either reduction in secretion of IGF1 or in IGF1R expression level may decrease MCUc activation and thus impair the induction of intrinsic and postsynaptic homeostatic plasticity. Importantly, expression of mitoIGFIRs in IGF1R-KO neurons rescued all 3 components: baclofen-induced increase in the fraction of spike bursts, burst-to-mitoCa2+ coupling and upward MFR homeostasis. Thus, mitoIGFIR signaling emerges as a critical element in the induction of the integrated MFR response by regulating spike pattern and burst-to-mitoCa2+ coupling (Fig. 8). As IG1-R does not alter presynaptic homeostatic plasticity, other homeostatic sensors, such as the endoplasmic reticulum (ER) Ca2+ sensor MCTP may drive presynaptic homeostasis (Gene et al., 2017).
[00122] Spike bursts are known to induce frequency- and spike-timing dependent plasticity (Paulsen and Sejnowski, 2000). Moreover, recent findings demonstrate that spike bursts evoke pronounced mitoCa2+ transients in soma and apical dendrites (Staler et al., 2022) and mitoCa2+ is implicated in the induction of long-term potentiation (Divakaruni et al., 2018). What are the mechanisms that separate the functions of burst-induced mitoCa2+ in Hebbian vs. homeostatic plasticity? One possibility is that different sources of Ca2+ entry to the
cytosol are coupled to different intracellular signaling pathways. While Ca2+ entry trough NMDARs is critical for induction of Hebbian-like synaptic plasticity (Malenka et al., 2004), Ca2+ entry through L-type (O’Leary et al., 2010) and T-type (Schaukowitch et al., 2017) voltage-gated calcium channels has been implicated in homeostatic plasticity. Moreover, the impact of N-type calcium channels on transcription is proposed to depend on Ca2+ buffering via mitochondria and ER (Wheeler et al., 2012). Thus, IGFIR-depedent mitoCa2+ may regulate MFR homeostasis by tuning spike bursts - transcription coupling.
[00123] IGF1R and MFR Homeostasis. The network’s ability to yield the same output despite different molecular compositions, called degeneracy, is proposed to be a ubiquitous biological property at all levels of organization (Edelman and Gaily, 2001). However, homeostatic regulation may fail when one of the core homeostatic machinery components becomes dysfunctional (Frere and Slutsky, 2018). Here, we show that IGF1R deficiency limits upward firing rate homeostasis by suppressing mitoCa2+-to-cytoCa2+ coupling via MCUc. Thus, in addition to cytoCa2+, mitoCa2+-to-cytoCa2+ coupling may play a crucial role in MFR homeostasis.
[00124] It has been recently demonstrated that the loss of Shank3, implicated in autism spectrum disorders, impairs upward MFR homeostasis and ocular dominance plasticity in the Vim cortex (Tatavarty et al., 2020). Interestingly, injection of IGF1 prevents effects of monocular deprivation on ocular dominance plasticity in the V 1 cortex (Tropea et al. , 2006). Moreover, IGF1 restored deficits in excitatory synaptic transmission in neurons with reduced Shank3 expression from 22ql3 deletion syndrome patients (Shcheglovitov et al., 2013). However, whether deficiency in mitoCa2+ evoked by spike bursts also limits upward MFR homeostasis in visual cortex and hippocampus in vivo is unknown. We hypothesize that maintaining a delicate balance of IGF1R signaling is critical for normal brain functioning. On the one hand, some brain disorders are associated with down-regulation of IGF1. For example, Mecp2 mutant mice, a model of Rett syndrome, exhibit decreased levels of serum IGF1 (Castro et al., 2014). A treatment of Mecp2 mutant mice with systemic IGF1 restored Rett syndrome-like symptoms, including synaptic and cognitive deficits (Castro et al., 2014; Tropea et al., 2009). As Mecp2 deletion impairs homeostatic synaptic scaling (Qiu et al., 2012) and excitation-to-inhibition balance (Dani et al., 2005), it would be important to test if IGF1 restores the symptoms by rescuing homeostatic failures. On the other hand, a decrease in IGF1R signaling protects from amyloid-P-mediated pathology as well as from
synaptic, neuronal and cognitive deficits in Alzheimer’s disease mouse models (Gazit et al., 2016; Gontier et al., 2015). Moreover, inhibition of MCU decreases mitoCa2+ overload in cortical neurons of Alzheimer’s disease model mice (Calvo-Rodriguez and Bacskai, 2020). Reduced mitoIGFIR/ MCUc signaling may be neuroprotective by suppressing disease- associated hyperactivity of hippocampal synapses (Gazit et al., 2016) and by restricting upward MFR homeostasis.
REFERENCES
Abbott, L.F.; Nelson, S.B., Synaptic plasticity: Taming the beast. Nat. Neurosci., 2000, 3 (suppl.), 1178-1183
Abramov, E.; et al., Amyloid -beta as a positive endogenous regulator of release probability at hippocampal synapses. Nat. Neurosci., 2009, 12, 1567-1576
Asztely, F.; Erdemli, G.; Kullmann, D.M., Extrasynaptic glutamate spillover in the hippocampus: Dependence on temperature and the role of active glutamate uptake. Neuron, 1997, 18, 281-293
Barnes, S.J.; et al., Subnetwork- specific homeostatic plasticity in mouse visual cortex in vivo. Neuron, 2015, 86, 1290-1303
Burrone, J.; O’Byrne, M.; Murthy, V.N., Multiple forms of synaptic plasticity triggered by selective suppression of activity in individual neurons. Nature, 2002, 420, 414- 418
Calvo-Rodriguez, M.; Bacskai, B.J., Mitochondria and calcium in Alzheimer’s disease: From cell signaling to neuronal cell death. Trends Neurosci., 2020, 44, 136-151
Carboni, J.M.; Lee, A.V.; Hadsell, D.L.; Rowley, B.R.; Lee, F.Y.: Bol, D.K.; Camuso, A.E.; Gottardis, M.: Greer, A.F.; Ho, C.P.: Hurlburt, W.: Li, A.; Saulnier. M.; Velaparthi, U.; Wang, C.; Wen, M.L.; Westhouse, R.A.: Wittman, M.; Zimmermann, K.: Rupnow, B.A.; Wong, T.W., Tumor development by transgenic expression of a constitutively active insulin-like growth factor 1 receptor. Cancer Res., 2005, 65(9), 3781 - 3787
Castro, J.; et al., Functional recovery with recombinant human IGF1 treatment in a mouse model of Rett Syndrome. Proc. Natl. Acad. Sci. U.S.A., 2014, 111, 9941-9946
Chaker, Z.; et al., Hypothalamic neurogenesis persists in the aging brain and is controlled by energy-sensing IGF-I pathway. Neurobiol. Aging, 2016, 41, 64-72
Chambers, A.R.; Rumpel, S., A stable brain from unstable components: Emerging concepts and implications for neural computation. Neuroscience, 2017, 357, 172-184
Che, T.-F.; et al., Mitochondrial translocation of EGFR regulates mitochondria dynamics and promotes metastasis in NSCLC. Oncotarget, 2015, 6, 37349-37366
Christie, J.M.; Jahr, C.E., Multivesicular release at Schaffer collateral-CAl hippocampal synapses. J. Neurosci., 2006, 26, 210-216
Cohen, E.; Bieschke, J.; Perciavalle, R.M.; Kelly, J.W.; Dillin, A., Opposing activities protect against age-onset proteotoxicity. Science, 2006, 313, 1604-1610
Cohen, E.; et al., Reduced IGF-1 signaling delays age- associated proteotoxicity in mice. Cell, 2009, 139, 1157-1169
Csordas, G.; et al., MICU1 controls both the threshold and cooperative activation of the mitochondrial Ca2+ uniporter. Cell Metab., 2013, 17, 976-987
Dani, V.S.; et al., Reduced cortical activity due to a shift in the balance between excitation and inhibition in a mouse model of Rett syndrome. Proc. Natl. Acad. Sci. U.S.A., 2005, 102, 12560-12565
Davis, G.W., Homeostatic control of neural activity: From phenomenology to molecular design. Anna. Rev. Neurosci., 2006, 29, 307-323
Davis, G.W.; Muller, M., Homeostatic control of presynaptic neurotransmitter release. Annu. Rev. Physiol., 2015, 77, 251-270
Demory, M.L.; et al., Epidermal growth factor receptor translocation to the mitochondria: Regulation and effect. J. Biol. Chem., 2009, 284, 36592-36604
De Stefani, D.; Rizzuto, R.; Pozzan, T., Enjoy the trip: Calcium in mitochondria back and forth. Annu. Rev. Biochem., 2016, 85, 161-192
Ding, T.; et al., Receptor tyrosine kinase ErbB2 translocates into mitochondria and regulates cellular metabolism. Nat. Commun., 2012, 3, 1271
Divakaruni, S.S.; et al., Long-term potentiation requires a rapid burst of dendritic mitochondrial fission during induction. Neuron, 2018, 100, 860-875. e7
Edelman, G.M.; Gaily, J. A., Degeneracy and complexity in biological systems. Proc. Natl. Acad. Sci. U.S.A., 2001, 98, 13763-13768
Faria, R.; Vives, E.; Boisguerin, P.; Sousa, A.; Costa, D., Development of peptide- based nanoparticles for mitochondrial plasmid DNA delivery. Polymers, 2021, 13, 1836
Fernandez, A.M.; Torres-Aleman, I., The many faces of insulin-like peptide signalling in the brain. Nat. Rev. Neurosci., 2012, 13, 225-239
Frere, S.; Slutsky, I., Alzheimer’s disease: From firing instability to homeostasis network collapse. Neuron, 2018, 97, 32-58
Gazit, N.; et al., IGF-1 receptor differentially regulates spontaneous and evoked transmission via mitochondria at hippocampal synapses. Neuron, 2016, 89, 583-597
Gene, O.; et al., MCTP is an ER-resident calcium sensor that stabilizes synaptic transmission and homeostatic plasticity. ELife, 2017, 6, e22904
Gontier, G.; George, C.; Chaker, Z.; Holzenberger, M.; Aid, S., Blocking IGF signaling in adult neurons alleviates Alzheimer’s disease pathology through amyloid-P clearance. J. Neurosci., 2015, 35, 11500-11513
Hakuno, F.; Takahashi, S.-L, IGF1 receptor signaling pathways. J. Mol. Endocrinol., 2018, 61, T69-T86
Hengen, K.B.; Lambo, M.E.; Van Hooser, S.D.; Katz, D.B.; Turrigiano, G.G., Firing rate homeostasis in visual cortex of freely behaving rodents. Neuron, 2013, 80, 335-342
Hengen, K.B.; Torrado Pacheco, A.; McGregor, J.N.; Van Hooser, S.D.; Turrigiano, G.G., Neuronal firing rate homeostasis is inhibited by sleep and promoted by wake. Cell, 2016, 165, 180-191
Holzenberger, M.; etal., IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice. Nature, 2003, 421, 182-187
Hurt, E.C., Pesold-Hurt, B., Suda, K., Oppliger, W .; Schatz, G., The first twelve amino acids (less than half of the pre-sequence) of an imported mitochondrial protein can direct mouse cytosolic dihydrofolate reductase into the yeast mitochondrial matrix. The EMBO Journal, 1985, 4, 2061-2068
Imamura, H.; et al., Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators. Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 15651-15656
Jahne, S.; et al., Presynaptic activity and protein turnover are correlated at the singlesynapse level. Cell Reports, 2021, 34(11), 108841
Kamer, K.J.; Mootha, V.K., The molecular era of the mitochondrial calcium uniporter. Nat. Rev. Mol. Cell Biol., 2015, 16, 545-553
Kann, O.; Kovacs, R., Mitochondria and neuronal activity. Am. J. Physiol. Cell. Physiol., 2007, 292, C641-C657
Katsenelson, M.; Shapira, I.; Abbas, E.; Styr, B.; Aid, S.; Holzenberger, M.; Rizzoli, S.; Slutsky, I., IGF1 receptor regulates upward firing rate homeostasis via the mitochondrial calcium uniporter. bioRxiv, 2021 (December 12)
Kavalali, E.T.; Monteggia, L.M., Targeting homeostatic synaptic plasticity for treatment of mood disorders. Neuron, 2020, 106, 715-726
Kenyon, C.; Chang, J.; Gensch, E.; Rudner, A.; R. Tabtiang, A C. elegans mutant that lives twice as long as wild type. Nature, 1993, 366, 461-464
Kolevzon, A. ; et al. , A pilot controlled trial of insulin-like growth factor- 1 in children with Phelan-McDermid syndrome. Molecular Autism, 2014, 5(1), 54
Kolevzon, A.; et al., Clinical trial of insulin-like growth factor- 1 in Phelan- McDermid syndrome. Molecular Autism, 2022, 13(1), 17
Lin, Y.; et al., Brain activity regulates loose coupling between mitochondrial and cytosolic Ca2+ transients. Nat. Commun., 2019, 10, 5277
Lisman, J.E., Bursts as a unit of neural information: Making unreliable synapses reliable. Trends Neurosci., 1997, 20, 38-43
Maglio, L.E.; et al., IGF-1 facilitates extinction of conditioned fear. ELife, 2021, 10, e67267
Malenka, R.C.; Bear, M.F.; LTP and LTD: An embarrassment of riches. Neuron, 2004, 44, 5-21
Marchi, S.; Pinton, P., The mitochondrial calcium uniporter complex: Molecular components, structure and physiopathological implications. J. Physiol., 2014, 592, 829-839 Marder, E.; Goaillard, J.M., Variability, compensation and homeostasis in neuron and network function. Nat. Rev. Neurosci., 2006, 7, 563-574
Marder, E., Variability, compensation, and modulation in neurons and circuits. Proc. Natl. Acad. Sci. U.S.A., 2011, 108 (Suppl. 3), 15542-15548
Mardinly, A.R.; et al., Sensory experience regulates cortical inhibition by inducing IGF1 in VIP neurons. Nature, 2016, 531, 371-375
Maya-Vetencourt, J.F.; et al., IGF-1 restores visual cortex plasticity in adult life by reducing local GABA levels. Neural Plast., 2012, 250421
Obita, T.; Muto, T.; Endo, T.; Kohda, D., Peptide library approach with a disulfide tether to refine the Tom20 recognition motif in mitochondrial presequences. J. Mol. Biol., 2003, 328 (2), 495-504
O’Leary, T.; van Rossum, M.C.W.; Wyllie, D.J.A., Homeostasis of intrinsic excitability in hippocampal neurones: Dynamics and mechanism of the response to chronic depolarization. J. Physiol., 2010, 588, 157-170
O’Leary, T.; Williams, A.H.; Franci, A.; Marder, E., Cell types, network homeostasis, and pathological compensation from a biologically plausible ion channel expression model. Neuron, 2014, 82, 809-821
O’Leary, T., Homeostasis, failure of homeostasis and degenerate ion channel regulation. Curr. Opin. Physiol., 2018, 2, 129-138
Patron, M.; Granatiero, V.; Espino, J.; Rizzuto, R.; De Stefani, D., MICU3 is a tissuespecific enhancer of mitochondrial calcium uptake. Cell Death Differ., 2019, 26, 179-195
Paulsen, O.; Sejnowski, T.J., Natural patterns of activity and long-term synaptic plasticity. Curr. Opin. Neurobiol., 2000, 10, 172-179
Pozzi, D.; et al., REST/NRSF-mediated intrinsic homeostasis protects neuronal networks from hyperexcitability. EMBO J., 2013, 32, 2994-3007
Prinz, A.A.; Bucher, D.; Marder, E., Similar network activity from disparate circuit parameters. Nat. Neurosci., 2004, 7, 1345-1352
Prister, A.; et al., Dopamine neuron-derived IGF-1 controls dopamine neuron firing, skill learning, and exploration. Proc. Natl. Acad. Sci. U.S.A., 2019, 116, 3817-3826
Qiu, Z.; et al., The Rett syndrome protein MeCP2 regulates synaptic scaling. J. Neurosci., 2012, 32, 989-994
Richter-Dennerlein, R.; et al., Mitochondrial protein synthesis adapts to influx of nuclear-encoded protein. Cell, 2016, 167(2), 471-483.e410
Ruggiero, A.; Katsenelson, M.; Slutsky, I., Mitochondria: New players in homeostatic regulation of firing rate set points. Trends Neurosci., 2021, 44, 605-618
Schaukowitch, K.; et al., An intrinsic transcriptional program underlying synaptic scaling during activity suppression. Cell Rep., 2017, 18, 1512-1526
Shcheglovitov, A.; et al., SHANK3 and IGF1 restore synaptic deficits in neurons from 22ql3 deletion syndrome patients. Nature, 2013, 503, 267-271
Sims, N.R.; Anderson, M.F., Isolation of mitochondria from rat brain using Percoll density gradient centrifugation. Nat. Protoc., 2008, 3, 1228-1239
Slomowitz E.; et al., Interplay between population firing stability and single neuron dynamics in hippocampal networks. ELife, 2015, 4, e04378
Slutsky, I.; Sadeghpour, S.; Li, B.; Liu, G., Enhancement of synaptic plasticity through chronically reduced Ca2+ flux during uncorrelated activity. Neuron, 2004, 44, 835- 849
Staler, O.; et al., Frequency- and spike-timing-dependent mitochondrial Ca2+ signaling regulates the metabolic rate and synaptic efficacy in cortical neurons. ELife, 2022, ll, e74606
Styr, B.; et al., Mitochondrial regulation of the hippocampal firing rate set point and seizure susceptibility. Neuron, 2019, 102, 1009- 1024. e8
Tatavarty, V.; et al., Autism-associated Shank3 is essential for homeostatic compensation in rodent VI. Neuron, 2020, 106, 769-777. e4
Trejo, J.L.; Carro, E.; Torres-Aleman, I., Circulating insulin-like growth factor I mediates exerciseinduced increases in the number of new neurons in the adult hippocampus. J. Neurosci., 2001, 21, 1628-1634
Tropea, D.; et al., Gene expression changes and molecular pathways mediating activity-dependent plasticity in visual cortex. Nat. Neurosci., 2006, 9, 660-668
Tropea, D.; etal., Partial reversal of Rett Syndrome-like symptoms in MeCP2 mutant mice. Proc. Natl. Acad. Sci. U.S.A., 2009, 106, 2029-2034
Turrigiano, G.G.; Leslie, J.R.; Desai, N.S.; Rutherford, L.C.; Nelson, S.B., Activitydependent scaling of quantal amplitude in neocortical neurons. Nature, 1998, 391, 892-896
Turrigiano, G.G.; Nelson, S.B., Homeostatic plasticity in the developing nervous system. Nat. Rev. Neurosci., 2004, 5, 97-107
Turrigiano, G., Too many cooks? Intrinsic and synaptic homeostatic mechanisms in cortical circuit refinement. Annu. Rev. Neurosci., 2011, 34, 89-103
Vahdatpour, C.; Dyer, A.H.; Tropea, D., Insulin-like growth factor 1 and related compounds in the treatment of childhood-onset neurodevelopmental disorders. Frontiers in neuroscience, 2016, 10, 450
Vertkin, I.; et al., GAB AB receptor deficiency causes failure of neuronal homeostasis in hippocampal networks. Proc. Natl. Acad. Sci. U.S.A., 2015, 112, E3291-E3299
Wheeler, D.G.; et al., Cavl and Cav2 channels engage distinct modes of Ca2+ signaling to control CREB -dependent gene expression. Cell, 2012, 149, 1112-1124
Zhou, Y.; et al., Mice with Shank3 mutations associated with ASD and schizophrenia display both shared and distinct defects. Neuron, 2016, 89(1), 147-162
Claims
1. A method for treatment of a disease or disorder associated with impaired mean firing rate (MFR) and/or mitochondrial calcium (mitoCa2+) homeostasis in an individual in need thereof, said method comprising administering to said individual a therapeutically effective amount of a therapeutic agent selected from:
(i) a mitochondria targeted-insulin-like growth factor- 1 receptor (mitoIGFIR) agonist; and
(ii) a nucleic acid molecule encoding for the expression of either mitoIGFIR or a mitochondria targeted -portion thereof; or mitochondrial calcium uniporter (MCU) optionally together with an additional subunit of the MCU complex, wherein said therapeutic agent is encapsulated for enabling delivering said therapeutic agent into a cell, thereby restoring MFR and/or mitoCa2+ homeostasis.
2. The method of claim 1, wherein said mitoIGFIR agonist is insulin-like growth factor- 1 (IGF1), a fragment thereof, or an analogue thereof.
3. The method of claim 2, wherein said mitoIGFIR agonist is IGF1 (SEQ ID NO: 1).
4. The method of claim 2, wherein said mitoIGFIR agonist is (i) a fragment of IGF1 comprising the sequence glycine-proline-glutamate (GPE tripeptide) as the amino-terminal thereof; or (ii) an analog of IGF1 comprising the sequence Gly-l-methylPro-Glu as the amino-terminal thereof.
5. The method of claim 4, wherein said fragment consists of the sequence GPE, or said analog consists of the sequence Gly-l-methylPro-Glu (glycyl-alpha-methyl-L-prolyl-L- glutamic acid; trofinetide; (2S)-2-{ [(2S)-l-(2-aminoacetyl)-2-methylpyrrolidine-2- carbonyl] amino } pen tanedioic acid) .
6. The method of claim 1, wherein: said nucleic acid molecule encoding for the expression of mitoIGFIR or said mitochondria targeted-portion thereof is an expression vector comprising a sequence encoding IGF1R or a portion thereof, fused to a sequence encoding a mitochondrial-targeting sequence (MTS); and said nucleic acid molecule encoding for the expression of MCU optionally together with said additional subunit of the MCU complex is an expression vector comprising a
sequence encoding MCU optionally together with said additional subunit of the MCU complex.
7. The method of claim 6, wherein said expression vector comprises a sequence encoding IGF1R (SEQ ID NO: 2) fused to a sequence encoding said MTS.
8. The method of claim 6, wherein said expression vector comprises a sequence encoding IGF1R (SEQ ID NO: 2) fused to both a sequence encoding CD8 (SEQ ID NO: 3) and a sequence encoding said MTS.
9. The method of any one of claims 6-8, wherein said MTS is selected from SEQ ID NOs: 4-6.
10. The method of claim 6, wherein said expression vector comprises a sequence encoding MCU (SEQ ID NO: 7).
11. The method of claim 6, wherein said expression vector comprises a sequence encoding MCU (SEQ ID NO: 7) together with a sequence encoding MICU1 (SEQ ID NO: 8) and/or a sequence encoding MICU3 (SEQ ID NO: 9).
12. The method of any one of claims 6-11, wherein said sequence each independently is a cDNA or RNA.
13. The method of any one of claims 6-12, wherein said sequence each independently is a cDNA, and said expression vector is a plasmid.
14. The method of any one of claims 6-12, wherein said expression vector is a viral vector selected from retrovirus, adenovirus, adeno-associated virus, poxvirus, alphavirus, herpes virus, and lentivirus.
15. The method of any one of claims 1-14, wherein said therapeutic agent is encapsulated within a capsule made of a phospholipid and/or a polymer, or within a viral- or viral-like- envelope.
16. The method of claim 15, wherein said phospholipid is a lecithin or a PEGylated derivative thereof, a phosphatidylcholine, a hydrogenated phosphotidylcholine, a lysophosphatidylcholine; dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine,
dimyristoylphosphatidylcholine, dilauroylphosphatidylcholine, a glycerophospholipid; sphingomyelin; cardiolipin, a phosphatidic acid, a glycolipid, a plasmalogen, a phosphosphingolipid, or a mixture thereof.
17. The method of claim 16, wherein said lecithin is egg lecithin, soybean lecithin, or a
PEGylated derivative thereof; said phosphatidylcholine is egg phosphatidylcholin; said glycerophospholipid is phosphatidylglycerol, phosphatidylserine, pho sphatidy lethanolamine , ly sopho sphatidy lethanolamine, pho sphatidy lino sitol, phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, or phosphatidylinositol triphosphate; said glycolipid is glyceroglycolipid, a glycosphingolipid, or a glycosylphosphatidylinositol; or said phospho sphingolipid is a ceramide phosphorylcholine, a ceramide phosphorylglycerol, or a ceramide phosphorylethanolamine.
18. The method of claim 17, wherein said glyceroglycolipid is a galactolipid, or a sulfolipid; or said glyco sphingolipid is a cerebroside (a glucocerebroside and a galactocerebroside).
19. The method of any one of claims 15-18, wherein said phospholipid is admixed with one or more nonpho sphorous -containing molecules each independently is a fatty amine, a fatty acid, a fatty acid amide, an ester of a fatty acid, cholesterol, a cholesterol ester, a diacylglycerol, or a glycerol ester.
20. The method of claim 19, wherein said fatty amine is octylamine, laurylamine, N- tetradecylamine, hexadecylamine, stearylamine, oleylamine, tallowamine, hydrogenated tallowamine, or cocoamine; said ester of a fatty acid is isopropyl myristate, hexadecyl stearate, or cetyl palmitate; or said glycerol ester is glycerol ricinoleate.
21. The method of any one of claims 15-20, wherein said phospholipid is admixed with one or more PEGylated phospholipids.
22. The method of claim 21, wherein said PEGylated phospholipid is PEGylated dipalmitoyl phosphatidylethanolamine (DPPE-PEG), PEGylated palmitoyloleoyl phosphatidylethanolamine (POPE-PEG), PEGylated dioleoyl phosphatidylethanolamine (DOPE-PEG), or PEGylated distearoyl phosphatidylethanolamine (DSPE-PEG), preferably
l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[polyethyleneglycol 2000] (DSPE- PEG-2000).
23. The method of any one of claims 15-22, wherein said polymer comprises polyethylene gly col-pho sphatidylethanolamine (PEG-PE).
24. The method of any one of claims 1-23, wherein said disease or disorder associated with impaired MFR and/or mitoCa2+ homeostasis is a neurodevelopmental disorder or neurodegenerative disease or disorder.
25. The method of claim 24, wherein said neurodevelopmental disorder is selected from Phelan-McDermid syndrome (PMS), Rett syndrome, attention-deficit/hyperactivity disorder (ADHD), autism, developmental language disorder (DLD), learning disability, intellectual disability (mental retardation), motor disorder, neurogenetic disorder, conduct disorder, cerebral palsy, and an impairment in vision and hearing; and said neurodegenerative disease or disorder is Alzheimer’s disease or Parkinson’s disease.
26. A therapeutic agent selected from:
(i) a mitochondria targeted-insulin-like growth factor- 1 receptor (mitoIGFIR) agonist; and
(ii) a nucleic acid molecule encoding for the expression of either mitoIGFIR or a mitochondria targeted -portion thereof; or mitochondrial calcium uniporter (MCU) optionally together with an additional subunit of the MCU complex, wherein said therapeutic agent is encapsulated for enabling delivering said therapeutic agent into a cell.
27. The therapeutic agent of claim 26, wherein said mitoIGFIR agonist is insulin-like growth factor- 1 (IGF1), a fragment thereof, or an analogue thereof.
28. The therapeutic agent of claim 27, wherein said mitoIGFIR agonist is IGF1 (SEQ ID NO: 1).
29. The therapeutic agent of claim 27, wherein said mitoIGFIR agonist is (i) a fragment of IGF1 comprising the sequence glycine-proline-glutamate (GPE tripeptide) as the amino terminal thereof; or (ii) an analog of IGF 1 comprising the sequence Gly-l-methylPro-Glu as the amino-terminal thereof.
30. The method of claim 29, wherein said fragment consists of the sequence GPE, or said analog consists of the sequence Gly-l-methylPro-Glu (trofinetide; (2S)-2-{ [(2S)-l-(2- aminoacetyl)-2-methylpyrrolidine-2-carbonyl]amino}pentanedioic acid).
31. The therapeutic agent of claim 26, wherein: said nucleic acid molecule encoding for the expression of mitoIGFIR or said mitochondria targeted-portion thereof is an expression vector comprising a sequence encoding IGF1R or a portion thereof, fused to a sequence encoding a mitochondrial-targeting sequence (MTS); and said nucleic acid molecule encoding for the expression of MCU optionally together with said additional subunit of the MCU complex is an expression vector comprising a sequence encoding MCU optionally together with said additional subunit of the MCU complex.
32. The therapeutic agent of claim 31, wherein said expression vector comprises a sequence encoding IGF1R (SEQ ID NO: 2) fused to a sequence encoding said MTS.
33. The therapeutic agent of claim 31, wherein said expression vector comprises a sequence encoding a portion of IGF1R (SEQ ID NO: 2) fused to both a sequence encoding CD8 (SEQ ID NO: 3) and a sequence encoding said MTS.
34. The therapeutic agent of any one of claims 31-33, wherein said MTS is selected from SEQ ID NOs: 4-6.
35. The therapeutic agent of claim 31, wherein said expression vector comprises a sequence encoding MCU (SEQ ID NO: 7).
36. The therapeutic agent of claim 31, wherein said expression vector comprises a sequence encoding MCU (SEQ ID NO: 7) together with a sequence encoding MICU1 (SEQ ID NO: 8) and/or a sequence encoding MICU3 (SEQ ID NO: 9).
37. The therapeutic agent of any one of claims 31-36, wherein said sequence each independently is a cDNA or RNA.
38. The therapeutic agent of any one of claims 31-37, wherein said sequence each independently is a cDNA, and said expression vector is a plasmid.
39. The therapeutic agent of any one of claims 31-37, wherein said expression vector is a viral vector selected from retrovirus, adenovirus, adeno-associated virus, poxvirus, alphavirus, herpes virus, and lentivirus.
40. The therapeutic agent of any one of claims 26-39, wherein said therapeutic agent is encapsulated within a capsule made of a phospholipid and/or a polymer, or within a viral- or viral-like-envelope.
41. A pharmaceutical composition comprising a therapeutic agent according to any one of claims 26-40, and a pharmaceutically acceptable carrier.
42. The therapeutic agent of any one of claims 26-40, or pharmaceutical composition according to claim 41, for use in the treatment of a disease or disorder associated with impaired mean firing rate (MFR) and/or mitochondrial calcium (mitoCa2+) homeostasis.
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000074724A2 (en) * | 1999-06-03 | 2000-12-14 | Bioinnovation Limited | Conjugates comprising cytokines and nucleic acids for treating proliferating cells |
WO2007071962A1 (en) * | 2005-12-23 | 2007-06-28 | Medical Research Council | Polypeptide targeting to mitochondria |
-
2023
- 2023-06-14 WO PCT/IL2023/050612 patent/WO2023242844A1/en unknown
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000074724A2 (en) * | 1999-06-03 | 2000-12-14 | Bioinnovation Limited | Conjugates comprising cytokines and nucleic acids for treating proliferating cells |
WO2007071962A1 (en) * | 2005-12-23 | 2007-06-28 | Medical Research Council | Polypeptide targeting to mitochondria |
Non-Patent Citations (3)
Title |
---|
CHO YOUNG-LAI, HUR SUNG-MO, KIM JI-YOON, KIM JI-HEE, LEE DONG-KEON, CHOE JONGEON, WON MOO-HO, HA KWON-SOO, JEOUNG DOOIL, HAN SANGH: "Specific Activation of Insulin-like Growth Factor-1 Receptor by Ginsenoside Rg5 Promotes Angiogenesis and Vasorelaxation", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 290, no. 1, 1 January 2015 (2015-01-01), US , pages 467 - 477, XP093117285, ISSN: 0021-9258, DOI: 10.1074/jbc.M114.603142 * |
DEVINE MICHAEL J., KITTLER JOSEF T.: "Mitochondria at the neuronal presynapse in health and disease", NATURE REVIEWS. NEUROSCIENCE, NATURE PUBLISHING GROUP, GB, vol. 19, no. 2, 1 February 2018 (2018-02-01), GB , pages 63 - 80, XP093117283, ISSN: 1471-003X, DOI: 10.1038/nrn.2017.170 * |
RUGGIERO ANTONELLA; KATSENELSON MAXIM; SLUTSKY INNA: "Mitochondria: new players in homeostatic regulation of firing rate set points", TRENDS IN NEUROSCIENCES, ELSEVIER, AMSTERDAM., NL, vol. 44, no. 8, 14 April 2021 (2021-04-14), NL , pages 605 - 618, XP086706807, ISSN: 0166-2236, DOI: 10.1016/j.tins.2021.03.002 * |
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