Methods for promoting survival and / or function of motor neurons, and related agents, uses, and methods

Abstract

Provided are methods for promoting survival and / or function of amyotrophic lateral sclerosis (ALS) or ALS-like motor neurons, comprising contacting the motor neurons with an agent capable of reducing mitochondrial protein acetylation, particularly an agent selected from deacetylase activators, such as nicotinamide (NAM) and 7-hydroxy-3-(4'-methoxyphenyl)coumarin (C12), or an acetyltransferase inhibitor, such as GCN5L1 siRNA of SEQ ID NO: 1. Also provided are related agents, oligonucleotides, uses, and methods for identifying the agents.

Description

[Technical field]

[0001] The present disclosure relates generally to methods of promoting survival and / or function of motor neurons, such as amyotrophic lateral sclerosis (ALS) or ALS-like motor neurons, and related agents, uses, and methods. [Background technology]

[0002] Motor neuron diseases are a group of neurodegenerative disorders that affect motor neurons, among which amyotrophic lateral sclerosis (ALS) is an old-onset progressive neurodegenerative disorder that affects both upper and lower motor neurons (MNs).

[0003] In ALS, loss of MNs leads to denervation of skeletal muscles, resulting in muscle stiffness and atrophy, affecting patients' speech, swallowing, walking, and breathing. Disease progression is rapid and persistent, ultimately leading to death. Only two drugs, riluzole and edaravone, are FDA-approved for the treatment of ALS, but their efficacy is mild. Riluzole was found to be effective in patients with bulbar ALS, but not in subjects with limb ALS, which accounts for the majority of ALS patients. Riluzole extends life expectancy by an average of three months, whereas edaravone was only effective in a small proportion of ALS patients. Despite extensive research, an effective treatment has yet to be found, at least in part due to a poor understanding of the pathogenesis of ALS.

[0004] The majority of ALS cases (up to 90%) are sporadic, in which the cause of the disease is largely unknown. The remaining ALS patients have a familial form of the disease, where mutations in genes such as SOD1, C9ORF72, and TDP43 are the most common. Despite genetic differences, the clinical symptoms of sporadic and familial ALS patients are indistinguishable, suggesting a possible confluence of pathogenic mechanisms. However, common pathogenic nodes in both sporadic and familial ALS that could open up novel ALS therapeutic approaches for both types of ALS remain to be elucidated.

[0005] There is therefore a need to provide alternative methods for promoting the survival and / or function of motor neurons in motor neuron diseases such as amyotrophic lateral sclerosis (ALS), as well as related agents, uses and methods. Summary of the Invention

[0006] In one aspect, a method of promoting survival and / or function of amyotrophic lateral sclerosis (ALS) or ALS-like motor neurons is provided, the method comprising contacting the motor neurons with an agent capable of reducing acetylation of a mitochondrial protein.

[0007] In one embodiment, the agent is selected from the group consisting of a deacetylase activator, an acetyltransferase inhibitor, and combinations thereof.

[0008] In one embodiment, the deacetylase activator comprises a SIRT3 (sirtuin 3) activator.

[0009] In one embodiment, the SIRT3 activator is selected from the group consisting of a small molecule, NAD (nicotinamide adenine dinucleotide) or a precursor thereof, and combinations thereof.

[0010] In one embodiment, the small molecule is selected from the group consisting of 7-hydroxy-3-(4'-methoxyphenyl)coumarin (C12), honokiol, dihydromyricetin (DHM), and derivatives, analogs, and combinations thereof.

[0011] In one embodiment, the NAD precursor is selected from the group consisting of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (Nam), nicotinamide mononucleotide (NMN) nicotinamide riboside (NR), nicotinic acid, and derivatives, analogs, and combinations thereof.

[0012] In one embodiment, the acetyltransferase inhibitor comprises a GCN5L1 (GCN5 (general control of amino acid synthesis 5)-like 1) inhibitor.

[0013] In one embodiment, the GCN5L1 inhibitor comprises an oligonucleotide.

[0014] In one embodiment, the oligonucleotide is selected from the group consisting of an antisense oligonucleotide (ASO), a gapmer, a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), a guide RNA (gRNA), a single guide RNA (sgRNA), and combinations thereof.

[0015] In one embodiment, the oligonucleotide comprises a) a sequence complementary to the coding sequence (CDS) of the BLOC1S1 gene or a portion thereof, or SEQ ID NO:3 or a portion thereof, or SEQ ID NO:4 or a portion thereof, or b) a sequence sharing at least about 75% sequence identity with the sequence in a).

[0016] In one embodiment, the oligonucleotide comprises a sequence that shares at least about 75% sequence identity with SEQ ID NO:1, or a sequence that differs from SEQ ID NO:1 by about 1 nucleotide, about 2 nucleotides, about 3 nucleotides, about 4 nucleotides, or about 5 nucleotides.

[0017] In one aspect, there is provided an agent capable of reducing acetylation of a mitochondrial protein for use in the treatment of ALS or an ALS-like disease.

[0018] In one embodiment, the agent is selected from the group consisting of a deacetylase activator, an acetyltransferase inhibitor, and combinations thereof.

[0019] In one embodiment, the deacetylase activator comprises a SIRT3 activator, optionally the SIRT3 activator is selected from the group consisting of a small molecule, NAD or a precursor thereof, and combinations thereof, optionally the small molecule is selected from the group consisting of C12, honokiol, dihydromyricetin (DHM), and derivatives, analogs, and combinations thereof, and optionally the NAD precursor is selected from the group consisting of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (Nam), nicotinamide mononucleotide (NMN) nicotinamide riboside (NR), nicotinic acid, and derivatives, analogs, and combinations thereof.

[0020] In one embodiment, the acetyltransferase inhibitor comprises a GCN5L1 inhibitor, optionally the GCN5L1 inhibitor comprises an oligonucleotide, optionally the oligonucleotide is selected from the group consisting of an antisense oligonucleotide (ASO), a gapmer, a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), a guide RNA (gRNA), a single guide RNA (sgRNA), and combinations thereof, optionally the oligonucleotide comprises a) a sequence complementary to the CDS of the BLOC1S1 gene or a portion thereof, or SEQ ID NO: 3 or a portion thereof, or SEQ ID NO: 4 or a portion thereof, or a sequence sharing at least about 75% sequence identity with the sequence of a), optionally the oligonucleotide comprises a sequence sharing at least about 75% sequence identity with SEQ ID NO: 1, or a sequence that differs from SEQ ID NO: 1 by about 1 nucleotide, about 2 nucleotides, about 3 nucleotides, about 4 nucleotides, or about 5 nucleotides.

[0021] In one aspect, there is provided a use of an agent in the manufacture of a medicament for treating ALS or an ALS-like disorder.

[0022] In one aspect, a method of treating ALS or an ALS-like disease in a subject is provided, the method comprising administering to the subject an agent.

[0023] In one aspect, oligonucleotides sharing at least about 75% sequence identity with SEQ ID NO:1, or sequences that differ from SEQ ID NO:1 by about 1 nucleotide, about 2 nucleotides, about 3 nucleotides, about 4 nucleotides, or about 5 nucleotides, are provided.

[0024] In one aspect, an oligonucleotide for use in therapy is provided.

[0025] In one aspect, there is a method of identifying an agent for treating ALS or an ALS-like disease, comprising contacting an ALS or ALS-like motor neuron with a candidate agent and determining whether acetylation of a mitochondrial protein in said motor neuron is reduced following contact, wherein if acetylation of a mitochondrial protein in said motor neuron is reduced following contact, concluding that the candidate agent is an agent for treating ALS or an ALS-like disease, and if acetylation of a mitochondrial protein in said motor neuron is not reduced following contact, concluding that the candidate agent is not an agent for treating ALS or an ALS-like disease, and optionally wherein the agent for treating ALS or an ALS-like disease so identified comprises an agent or oligonucleotide as described herein.

[0026] definition The terms "treatment," "treat," and "therapy," and their equivalents, as used herein, refer to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent, slow down (attenuate), or reverse medical conditions, including, but not limited to, diseases (such as degenerative diseases and / or motor neuron diseases), symptoms, and disorders. Medical conditions also include the body's response to a disease or disorder, such as inflammation. Those in need of such treatment include those who are predisposed to a medical condition or those in whom a medical condition is desired to be prevented, as well as those who already have a medical condition.

[0027] As used herein, the term "therapeutically effective amount" refers to an amount of active agent that can prevent, reverse, or at least slow down (attenuate) medical conditions such as motor neuron disease, autoimmune disease, inflammation, and cancer. Dosage and administration of the agents, compounds, compositions, and formulations of the present disclosure can be determined by those skilled in the art of clinical pharmacology or pharmacokinetics. See, for example, Mordenti and Rescigno, (1992) Pharmaceutical Research. 9:17-25; Morenti et al., (1991) Pharmaceutical Research. 8:1351-1359; and Mordenti and Chappell, "The use of interspecies scaling in toxicokinetics" in Toxicokinetics and New Drug Development, Yacobi et al. (eds) (Pergamon Press: NY, 1989), pp. 42-96. The effective amount of the active agent of the present disclosure to be used therapeutically depends, for example, on the therapeutic purpose, the route of administration, and the condition of the subject. Accordingly, it may be necessary for the therapist to titrate the dosage or modify the route of administration as required to obtain the optimal therapeutic effect.

[0028] The term "subject" as used herein includes patients and non-patients. The term "patient" refers to an individual who is afflicted with or likely to be afflicted with a medical condition, such as a motor neuron disease, while "non-patient" refers to an individual who is not afflicted with and likely not afflicted with a medical condition. "Non-patient" includes healthy individuals, non-diseased individuals, and / or individuals who do not have a medical condition. The term "subject" includes humans and animals. Animals include mice and the like. "Rodent" refers to any mammal of the murine family, such as mice, rats, and the like.

[0029] The term "micro" as used herein should be interpreted broadly to include dimensions from about 1 micrometer to about 1000 micrometers.

[0030] The term "nano" as used herein should be interpreted broadly to include dimensions less than about 1000 nm.

[0031] The term "particle" as used herein broadly refers to a discrete entity or body. Particles as described herein may include organic particles, inorganic particles, or biological particles. Particles as used herein may also be macroparticles formed by the aggregation of multiple subparticles or fragments of smaller objects. Particles of the present disclosure may be spherical, substantially spherical, or non-spherical, such as irregularly shaped particles or ellipsoidal particles. The term "size", when used to refer to a particle, broadly refers to the largest dimension of the particle. For example, if the particle is substantially spherical, the term "size" may refer to the diameter of the particle, or if the particle is substantially non-spherical, the term "size" may refer to the largest length of the particle.

[0032] The terms "coupled" or "connected," as used herein, unless otherwise specified, are intended to encompass both a direct connection or a connection through one or more intermediary means.

[0033] The term "associated" as used herein when referring to two elements refers to a broad relationship between the two elements. The relationship includes, but is not limited to, a physical, chemical, or biological relationship. For example, when element A is associated with element B, elements A and B may be directly or indirectly bound to each other, element A may contain element B, or vice versa.

[0034] The term "adjacent" as used herein when referring to two elements refers to the close proximity of one element to another, which may be elements that are in contact with each other, but may also include elements that are separated by one or more additional elements disposed between them.

[0035] The term "and / or", e.g., "X and / or Y", should be understood to mean either "X and Y" or "X or Y" and should be considered to provide explicit support for both meanings or either meaning.

[0036] Further, in the description herein, the term "substantially", whenever used, is understood to include but is not limited to "entirely" or "completely", etc. Furthermore, terms such as "comprising", "comprise", etc., whenever used, are intended to be open-ended descriptive language in that they broadly include the elements / components listed after such term, in addition to other components not expressly listed. For example, when "comprising" is used, a reference to "a" feature is also intended to be a reference to "at least one" of that feature. Terms such as "consisting", "consist", etc., may be considered subsets of terms such as "comprising", in appropriate context. Thus, in embodiments disclosed herein using terms such as "comprising", it will be understood that these embodiments provide teachings of corresponding embodiments using terms such as "consisting". Additionally, terms such as "about," "approximately," and the like, whenever used, typically refer to a reasonable variation, such as ±5% variation of the disclosed value, or a 4% variation of the disclosed value, or a 3% variation of the disclosed value, or a 2% variation of the disclosed value, or a 1% variation of the disclosed value.

[0037] Furthermore, in the description herein, certain values ​​may be disclosed in ranges. The values ​​at the end of the range are intended to indicate the preferred range. Whenever a range is described, it is intended that the range encompasses and teaches all possible subranges in addition to the individual values ​​within the range. That is, the end points of the range should not be interpreted as invariant limits. For example, a description of a range of 1% to 5% is intended to specifically disclose subranges such as 1% to 2%, 1% to 3%, 1% to 4%, 2% to 3%, etc., in addition to values ​​within the range such as 1%, 2%, 3%, 4%, and 5%, respectively. It is also to be understood that the individual values ​​within the range include integers, fractions, and decimals. Furthermore, whenever a range is described, it is also intended that the range encompasses and teaches values ​​from the end points of the numerical values ​​set forth to a further decimal point or to two significant digits (where appropriate). For example, a description of a range of 1% to 5% is intended to specifically disclose the range of 1.00% to 5.00%, as well as the range of 1.0% to 5.0%, and all intermediate values ​​throughout that range (e.g., 1.01%, 1.02%...4.98%, 4.99%, 5.00% and 1.1%, 1.2%...4.8%, 4.9%, 5.0%, etc.). The above specific disclosure is applicable to ranges of any depth / breadth.

[0038] Furthermore, when describing some embodiments, the present disclosure may disclose a method and / or process as a particular sequence of steps. However, it will be understood that unless specifically required, the method or process should not be limited to the particular sequence of steps disclosed. Other sequences of steps may be possible. The particular order of steps disclosed herein should not be construed as unduly limiting. Unless specifically required, the method and / or process disclosed herein should not be limited to steps performed in the order described. The sequence of steps may be varied and still be within the scope of the present disclosure.

[0039] Furthermore, while the present disclosure provides embodiments having one or more of the features / characteristics discussed herein, it will be understood that one or more of these features / characteristics may be denied in other alternative embodiments, and the present disclosure provides support for such denials and these related alternative embodiments. [Brief description of the drawings]

[0040] [Figure 1] ALS iPSC-derived MNs display a pathological phenotype. (A) Schematic of MN differentiation protocol. (B) Immunostaining of wild-type (BJ-iPS, 18a, and GM23720), familial ALS (29d, 47a, and 19f), and sporadic ALS (sALS1, sALS2, sALS3) iPSC-derived cultures at day 28 showing induction of ISL1+SMI32+MNs. Cell nuclei were counterstained with DAPI. Scale bar, 50 μm. (C) Quantification of ISL1+MNs from days 25 to 35 demonstrating that wild-type MNs (BJ-iPS, 18a, and GM23720) remain viable, whereas familial and sporadic ALS MNs show a significant decrease in viability over time. (D) Quantification of ER stress transcripts CHOP and spliced ​​XBP1 (sXBP1) by qPCR in MN cultures at day 28. Fold changes are normalized to the expression levels of each mRNA in BJ-iPS. (E) Immunostaining of isogenic ALS (BJ-SOD1L144F and BJ-TDP43G298S) iPSC-derived cultures at day 28 showing the formation of ISL1+SMI32+MN. Cell nuclei were counterstained with DAPI. Scale bar, 50 μm. (F) Quantification of ISL1+MN from BJ-SOD1L144F and BJ-TDP43G298S at days 25-35 revealed an accelerated death phenotype similar to other ALS strains. (G) MN cultures derived from BJ-SOD1L144F and BJ-TDP43G298S show upregulation of CHOP and sXBP1 compared to the isogenic control line, BJ-iPS. In (D) and (G), gene expression was normalized to ACTINB and HPRT. ***p<0.001, ns not significant; two-tailed t-test. [Diagram 2]Sporadic and familial ALS MNs exhibit low oxidative and high glycolytic metabolic profiles. (A) Metabolic flux plots of sorted neurons from healthy controls and ALS patients, measuring oxygen consumption rate (OCR) as a function of time. Bioenergetic parameters were measured using MitoStress assay by adding complex V inhibitor oligomycin, mitochondrial uncoupler FCCP, and complex I and III inhibitors rotenone and antimycin A (AA). (B) Basal respiration, ATP production, and spare respiration were calculated for sorted neurons from each cell line, demonstrating reduced mitochondrial respiration in ALS MNs. (C) Metabolic flux plots of sorted neurons from healthy controls and ALS patients, measuring extracellular acidification rate (ECAR) as a function of time. Bioenergetic parameters were measured using glycolytic stress assay by adding glucose, complex V inhibitor oligomycin, and hexokinase inhibitor 2-DG. (D) Basal acidification, glycolysis, and glycolytic capacity were calculated for sorted neurons from each cell line, demonstrating increased glycolysis in ALS MN. (E) OCR measurements were performed and calculated for healthy, ALS, and diseased isogenic iPSC-derived cortical neurons at day 28 using the MitoStress assay. (F) Metabolic flux analysis was performed using the MitoStress assay. Basal respiration, ATP production, and spare respiration were calculated for BJ-iPS, BJ-SOD1L144F, and BJ-TDP43G298S cortical neurons, showing no significant changes in basal respiration and ATP production. Similarly, no significant changes in basal respiration and ATP production are observed for healthy and ALS patient iPSC-derived cortical neurons. (G) OCR measurements were performed and calculated for healthy, ALS, and diseased isogenic iPSC-derived cardiomyocytes at day 28 using the MitoStress assay. (H) Metabolic flux analysis was performed using the MitoStress assay.Basal respiration, ATP production, and spare respiration were calculated for BJ-iPS, BJ-SOD1L144F, and BJ-TDP43G298S cardiomyocytes, and no significant changes were observed in basal respiration and ATP production. Similarly, no significant changes were observed in basal respiration and ATP production in cardiomyocytes derived from iPSCs from healthy subjects and ALS patients. ***p<0.001, ns not significant; two-tailed t-test. [Diagram 3]Hyperacetylation of mitochondrial proteins in familial and sporadic ALS MN. (A) Western blot analysis of iPSC-derived MN at day 28, probed with SIRT3, total MnSOD, MnSOD specifically acetylated at lysine 68 (MnSOD K68ac) on whole cell lysates, and with acetyl-lysine protein on purified mitochondrial extracts. (B) Densitometric analysis of Western blot bands shows no significant change in SIRT3 protein levels in ALS MN versus healthy MN. (C) Densitometric analysis of MnSOD(K68ac) normalized to total MnSOD revealed that MnSOD(K68ac) was upregulated approximately 5-fold in all ALS iPSC-derived MN, indicating reduced activity of SIRT3 in ALS MN. (D) Immunohistochemistry of lumbar sections from control and sporadic ALS patients (SALS) revealed increased MnSOD(K68ac) signal in lumbar motor neurons (arrows) from SALS patients. Lipofuscin is seen in large, healthy motor neurons, which is a function of normal cellular aging and is unrelated to disease (*). Scale bars, 2.5 mm (left panel) and 100 μm (right panel). (E) Quantification of MnSOD(K68ac) signal in control (n=380) and SALS (n=216) lumbar motor neurons demonstrates increased MnSOD(K68ac) signal in SALS patients. (F) Densitometric analysis of acetyl-lysine signal normalized to TOMM20 revealed approximately 3-fold increased mitochondrial protein acetylation in all ALS iPSC-derived MNs. (G) Measurement of complex I activity in MNs from healthy controls and ALS patients revealed a significant reduction of 30–80% in ALS MNs. *p<0.05, **p<0.01, ***p<0.001, ns not significant; two-tailed t-test. [Figure 4]Loss of SIRT3 in MNs results in an ALS-like phenotype. (A) Western blot analysis of day 28 MNs derived from BJ-iPS and two isogenic SIRT3+ / - (#6 and #17) clones confirmed a decrease in SIRT3 protein as well as an increase in MnSOD (K68ac) and increased mitochondrial protein acetylation. (B) Densitometric analysis of Western blot bands reveals a 50% decrease in SIRT3 protein levels and an increase in MnSOD (K68ac) in both SIRT3+ / -#6 and #17 versus healthy MNs. (C) qPCR measurement of CHOP and sXBP1 shows that both ER stress transcripts are significantly upregulated in SIRT3+ / -#6 and #17 versus isogenic BJ-iPS controls. (D) Measurement of OCR using MitoStress assay of day 28 MNs differentiated from BJ-iPS, SIRT3+ / -#6 and #17. (E) Measurement of basal respiration, ATP production, and spare respiration of day 28 MNs differentiated from BJ-iPS, SIRT3+ / -#6, and #17. (F) Measurement of ECAR using glycolytic stress assay of day 28 MNs differentiated from BJ-iPS, SIRT3+ / -#6, and #17. (G) Measurement of basal acidification, glycolysis, and glycolytic capacity of day 28 MNs differentiated from BJ-iPS, SIRT3+ / -#6, and #17. (H) Quantification of ISL1+MNs derived from both BJ-SIRT3+ / - clones from day 25 to day 35 revealed a progressive death phenotype compared to their healthy controls (BJ-iPS). (I) Representative images of ISL1+SMI32+MNs derived from BJ-iPS, BJ-SIRT3+ / -#6, and #17 iPSCs showing the size of soma from day 28 and day 31 MNs (white dotted box). Scale bar, 50 μm. (J) Quantification of mean soma size and number of primary neurites in both BJ-SIRT3+ / - clones shows a deterioration in neuronal health from days 28 to 31. **p<0.01, ***p<0.001, ns not significant; two-tailed t-test. [Diagram 5]NAM supplementation reverses mitochondrial respiratory defects and improves neuronal morphology in ALS MNs. (A) Mitochondrial NAD+ levels were measured in healthy and ALS MNs after NAM supplementation, revealing increased mitochondrial NAD+ utilization in ALS MNs. (B) WT and ALS iPSC-derived MNs were treated with either HO or NAM from day 28 to day 35. The number of ISL1+ MNs was quantified and normalized to the number of ISL1+ MNs in each cell line at day 28. NAM supplementation prevents MN death in ALS MNs. (C-E) Measurements of basal respiration, ATP production, and spare respiration in healthy and ALS MNs treated with water or 0.5 mM NAM as control, respectively. (F) Representative images of BJ-SOD1L144F and BJ-TDP43G298S ISL1+SMI32+ MNs showing that NAM supplementation promotes healthier neuronal morphology. The size of MN cell bodies is outlined by white dotted lines. Scale bar, 50 μm. (G) Neuronal cell body size and primary neurites were measured, showing an overall improvement in neuronal morphology in ALS MNs supplemented with NAM. ***p<0.001, ns not significant; two-tailed t-test. [Figure 6]Small molecule activators of SIRT3, but not riluzole or edaravone, improve neuronal morphology in ALS MNs. (A) Representative Western blots and CETSA melt curves in intact cells for SIRT3 targeting with C12 (20 μM). (B) Western blot analysis at day 31 revealed decreased mitochondrial Ac-K signal in ALS MNs upon treatment with C12. (C) WT and ALS iPSC-derived MNs were treated with either DMSO / H2O or C12 / riluzole / edaravone from day 28 to day 35. The number of ISL1+ MNs was quantified and normalized to the number of ISL1+ MNs in each cell line at day 28. Treatment with C12, riluzole, and edaravone prevents MN death in ALS MNs. (D-F) Quantification by qPCR of ER stress transcripts CHOP and spliced ​​XBP1 (sXBP1) in control or C12 / riluzole / edaravone-treated day 31 MN cultures. Fold changes are normalized to the expression levels of each mRNA in DMSO- or water-treated BJ-iPS MNs. Gene expression was normalized to ACTINB and HPRT. ***p<0.001, ns not significant; two-tailed t-test. [Figure 7]Small molecule activators of SIRT3 reverse respiratory abnormalities in ALS MNs, whereas neither riluzole nor edaravone improves. (A-C) Measurements of basal respiration, ATP production, and spare respiration in healthy and ALS MNs treated with DMSO and C12, respectively. Of note, C12 treatment rescued ATP production to healthy levels. (D-F) Measurements of basal respiration, ATP production, and spare respiration in healthy and ALS MNs treated with DMSO and riluzole, respectively. Of note, riluzole treatment fails to reverse mitochondrial bioenergetic abnormalities in ALS MNs. (G-I) Measurements of basal respiration, ATP production, and spare respiration in healthy and ALS MNs treated with water and edaravone, respectively. Of note, edaravone treatment fails to reverse mitochondrial bioenergetic abnormalities in ALS MNs. (J) Representative images of BJ-iPS, BJ-SOD1L144F, and BJ-TDP43G298S ISL1+SMI32+MNs showing the size of cell bodies (white dotted lines) from MNs at day 28 and day 31, demonstrating that only C12 treatment promotes healthier neuronal morphology. Scale bar, 50 μm. ***p<0.001, ns not significant; two-tailed t-test. [Figure 8] Knockdown of GCN5L1 rescues ATP production and ameliorates metabolic defects in ALS MN. Measurements of basal respiration, ATP production, and spare respiration in healthy and ALS MN treated with non-targeting and GCN5L1 siRNA, respectively, show that knockdown of GCN5L1 rescued ATP production to healthy levels. [Figure 9] Knockdown of GCN5L1 reduced the hyperglycolytic phenotype in ALS MN. Measurements of basal acidification, basal glycolysis, and glycolytic capacity in healthy and ALS MN treated with non-targeting and GCN5L1 siRNA, respectively. In summary, knockdown of GCN5L1 reduced the hyperglycolytic phenotype in ALS MN. [Figure 10]Knockdown of GCN5L1 promotes MN survival in ALS MN. WT and ALS iPSC-derived MN were treated with either non-targeting or GCN5L1 siRNA from day 28 to day 35. The number of ISL1+ MN was quantified and normalized to the number of ISL1+ MN in each cell line at day 28. Knockdown of GCN5L1 prevents MN death in ALS MN. [Figure 11] Knockdown of GCN5L1 promotes healthier neuronal morphology. Representative images of BJ-SOD1L144F and BJ-TDP43G298S ISL1+SMI32+ MNs showing that knockdown of GCN5L1 promotes healthier neuronal morphology at day 31. Scale bar, 50 μm. [Figure 12] Confirmation of GCN5L1 knockdown by siRNA. MNs transfected with siRNA against human GCN5L1 showed a 60% reduction in GCN5L1, which was sufficient to promote healthier morphology and correct the metabolic phenotype associated with ALS MNs. [Figure 13]Generation of isogenic lines using CRISPR / Cas9 technology (related to Figures 1 and 5). Schematic summarizing the strategy for generating isogenic knock-ins for SOD1L144F and TDP43G298S mutations and isogenic knock-outs for SIRT3 in the BJ-iPS background. Guide RNA sequences are underlined and the sequence of the PAM is highlighted in grey. (A) Single-stranded oligonucleotides carrying the mutations serve as repair templates to promote a G to C transition in exon 5 conferring a leucine (L) to phenylalanine (F) mutation. (B) Single-stranded oligonucleotides carrying the mutations serve as repair templates to promote a G to A transition in exon 5 conferring a glycine (G) to serine (S) mutation. (C) DNA sequencing confirms a 6-bp and 9-bp deletion in exon 1 of BJ-SIRT3+ / -#6 and #17, respectively. (D) Both isogenic SIRT3 haploinsufficient clones differentiated well into ISL1+SMI32+MN with efficiency comparable to BJ-iPS. Cell nuclei were counterstained with DAPI. Scale bar, 50 μm. ns not significant. [Figure 14]Metabolic flux was measured in NPCs at day 10, showing no significant changes between ALS and healthy cells (related to Figure 2). (A) OCR measurements using MitoStress assay were performed and calculated for wild-type and ALS NPCs at day 10. (B) Basal respiration, ATP production, and spare respiration were calculated for NPCs from each cell line, showing no significant difference in ATP production, but a significant decrease in spare respiration in ALS NPCs. (C) OCR measurements using MitoStress assay were performed and calculated for wild-type and isogenic ALS NPCs at day 10. (D) Basal respiration, ATP production, and spare respiration were calculated for NPCs from each cell line, showing no significant difference in ATP production, but a significant decrease in spare respiration in isogenic ALS NPCs. (E) ECAR measurements using glycolysis stress assay were performed and calculated for wild-type and ALS NPCs at day 10. (F) Basal acidification, glycolysis, and glycolytic capacity were calculated for NPCs from each cell line, showing no significant differences in glycolysis. (G) ECAR measurements using a glycolytic stress assay were performed and calculated for wild-type and isogenic ALS NPCs at day 10. (H) Basal acidification, glycolysis, and glycolytic capacity were calculated for NPCs from each cell line, showing no significant differences in glycolysis. ***p<0.001, ns not significant. [Figure 15] Metabolic flux measurements in other metabolically active cell types show that the changes between ALS and healthy cells are not significant (related to FIG. 3). (A) Immunostaining of cultures from iPSCs at different time points of differentiation of cortical neurons. Cell nuclei were counterstained with DAPI. Scale bar, 50 μm. (B) OCR measurements using the MitoStress assay were performed and calculated for cortical neurons derived from healthy, ALS, and diseased isogenic iPSCs at day 28. (C) OCR measurements using the MitoStress assay were performed and calculated for cardiomyocytes derived from healthy, ALS, and diseased isogenic iPSCs at day 28. ***p<0.001, ns not significant. [Figure 16]Activation of SIRT3 or inhibition of GCN5L1 ameliorates the pathological phenotype of ALS MN (related to FIG. 5). (A) Western blot analysis at day 28 confirming knockdown of SIRT3 and elevated MnSOD(K68ac) levels in si-SIRT3 conditions. (B) Densitometric analysis of western blot bands shows that MnSOD(K68ac) levels are significantly elevated in si-SIRT3 conditions. (C) Measurement of basal neuronal respiration, ATP production, and spare respiration in si-NT (dark grey) and si-SIRT3 (light grey) conditions. (D) Measurement of intracellular NAD+:NADH ratio in healthy and ALS MN reveals reduced NAD+ utilization in ALS MN. (E) Measurement of neuronal soma size and primary neurites shows overall improvement of neuronal morphology in NAM-supplemented ALS MN. (F) Western blot analysis at day 31 confirming that overexpression of SIRT3 in ALS MN did not reduce MnSOD(K68ac) levels, further validating that SIRT3 activity is affected in ALS MN. (GI) Measurement of neuronal basal respiration, ATP production, and spare respiration in control (black) and SIRT3 overexpression (grey) conditions. (J) Quantification of GCN5L1 transcripts by qPCR in cultures of GCN5L1 knockdown MN at day 31. Fold changes are normalized to non-targeted MN cultures. (K) Measurement of neuronal soma size and primary neurites show an overall improvement in neuronal morphology in GCN5L1 knockdown ALS MN. *p<0.05, **p<0.01, ***p<0.001, ns not significant. [Figure 17]C12 activates SIRT3 and alleviates ALS pathological phenotypes (related to Figures 6 and 7). (A) Western blot analysis at day 31 reveals that as C12 concentrations increase, MnSOD(K68ac) levels decrease in a dose-dependent manner, but SIRT3 protein expression does not change significantly. (B) Densitometric analysis of Western blot bands reveals that in C12-treated MNs, SIRT3 levels do not change significantly, but MnSOD(K68ac) levels decrease significantly in a dose-dependent manner. (C) ALS MNs treated with C12 show improved motor neuron survival at 5 μM. (D) Representative images of ISL1+SMI32+ALS MNs show that C12 treatment improves motor neuron survival at 5 μM. (E-F) Quantification by qPCR of ER stress transcripts CHOP and spliced ​​XBP1 (sXBP1) in DMSO or C12-treated day 31 MN cultures. Fold changes are normalized to the expression levels of each mRNA in BJ-iPS MNs treated with DMSO or water. (G-I) Glycolysis and glycolytic capacity were measured in healthy and ALS MNs treated with DMSO (light grey) or 5 μM C12 (dark grey) as control, respectively. *p<0.05, **p<0.01, ***p<0.001, ns not significant. [Figure 18]C12 treatment improves neuronal morphology in ALS MNs, but neither riluzole nor edaravone (related to FIG. 7). (A-B) Measurements of neuronal cell body size and primary neurites show an overall improvement in neuronal morphology in ALS MNs treated with C12. (C) Representative images of healthy and ALS iPSC-derived ISL1+SMI32+MNs showing the size of MN cell bodies (white dotted lines) at 28 days and 31 days after treatment with C12. Scale bar, 50 μm. (D) Representative images of healthy and ALS iPSC-derived ISL1+SMI32+MNs showing the size of MN cell bodies (white dotted lines) at 28 days and 31 days after treatment with riluzole or edaravone. Scale bar, 50 μm. (E-H) Treatment of ALS MNs with riluzole or edaravone did not improve neuronal morphology. Measurements of neuronal soma size and number of primary neurites of treated neurons showed no significant changes compared to the respective controls. ***p<0.001, ns not significant. [Figure 19] C12 targets / activates SIRT3 and has no effect in SIRT3-deficient clones. (A-C) Treatment of SIRT3+ / - clones #6 and #17 with C12 did not improve MN survival, MN morphology, or mitochondrial respiration. [Figure 20]SIRT3 activation rescued mitochondrial respiratory defects specific to ALS MNs. (Related to Figure 7). (A) OCR measurements using MitoStress assay were performed and calculated for day 28 healthy (BJ-iPS) and SMA type I (1-38G) MNs. (B) Basal respiration, ATP production, and spare respiration were calculated, revealing reduced mitochondrial bioenergetics in SMA type I. (C) OCR measurements using MitoStress assay in BJ-iPS and SMA type I MNs treated with DMSO (light grey) or 5 μM C12 (dark grey). (D) Basal respiration, ATP production, and spare respiration were measured in healthy MNs (BJ-iPS) and SMA type I (1-38G) MNs treated with DMSO (light grey) or 5 μM C12 (dark grey) as a control, respectively, showing no improvement in mitochondrial bioenergetics in SMA MNs, suggesting that SIRT3 activation is specific to rescue ALS MNs. ***p<0.001, ns not significant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Exemplary, non-limiting embodiments of methods for promoting motor neuron survival and / or function, and related methods, uses, agents, and compositions, are disclosed below.

[0042] In various embodiments, a method of promoting survival and / or function of a motor neuron is provided, comprising modulating / controlling acetylation of a mitochondrial product in the motor neuron. In some embodiments, the method is a method of promoting survival of a motor neuron. The method embodiments can increase cell / motor neuron survival and / or reduce cell / motor neuron loss. In some embodiments, the method is a method of promoting / improving function, optionally metabolic function, of a motor neuron. The method embodiments can restore proper function, optionally metabolic function and / or mitochondrial function, in the motor neuron, at least to some extent. For example, the method embodiments can restore proper function, optionally metabolic function and / or mitochondrial function, in the motor neuron, such that the function is similar or more similar to that in a healthy / normal motor neuron. In some embodiments, modulating / controlling acetylation of a mitochondrial product comprises reducing / inhibiting acetylation of a mitochondrial product. In some embodiments, the mitochondrial product comprises a mitochondrial protein. Without being bound by theory, it is believed that increased acetylation or hyperacetylation of mitochondrial products, such as mitochondrial proteins, in motor neurons leads to metabolic or mitochondrial respiratory disorders in motor neurons, resulting in death. In certain motor neuron diseases, such as amyotrophic lateral sclerosis, hyperacetylation of mitochondrial products, such as mitochondrial proteins, may be observed.

[0043] Thus, in various embodiments, a method for promoting survival and / or function of motor neurons in motor neuron diseases is provided, comprising reducing / inhibiting acetylation of mitochondrial proteins in motor neurons. Examples of motor neuron diseases include amyotrophic lateral sclerosis, primary lateral sclerosis, progressive bulbar palsy, pseudobulbar palsy, progressive muscular atrophy, spinal muscular atrophy, Kennedy's disease, and post-polio syndrome. In one embodiment, the motor neuron disease includes amyotrophic lateral sclerosis (ALS) or an ALS-like disease. The ALS-like disease may be a disease in which or characterized by increased / excessive acetylation or hyperacetylation of mitochondrial proteins in motor neurons causes metabolic abnormalities and death of motor neurons. ALS-like disease can be identified, for example, by measuring the level / relative level of mitochondrial protein acetylation in diseased motor neurons and comparing the level of acetylation with control motor neurons, for example, non-diseased or healthy motor neurons, and if the diseased motor neurons show an increase / increase in the level / relative level of acetylation compared to control motor neurons, the disease can be classified or considered as an ALS-like disease.Thus, in some embodiments, the method can further comprise measuring / determining the acetylation of mitochondrial proteins in motor neurons.If the level / relative level of acetylation of motor neurons is measured / determined to be increased / increased compared to control motor neurons, then the acetylation of mitochondrial proteins in motor neurons can be reduced or inhibited to promote the survival and / or function of motor neurons.

[0044] In various embodiments, reducing acetylation of mitochondrial proteins in motor neurons comprises reducing the amount / fraction / percentage of acetylated mitochondrial proteins in motor neurons and / or reducing the degree of acetylation (e.g., number of acetylated sites) in mitochondrial proteins in motor neurons. In some embodiments, the acetylation comprises lysine acetylation. In various embodiments, the reduction is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%. It may be at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100%. In some embodiments, the reduction is at least about 25%. In various examples, the degree of reduction in mitochondrial protein acetylation achieved by agents such as nicotinamide (Nam), 7-hydroxy-3-(4'-methoxyphenyl)coumarin (C12), and GCN5L1 siRNA is at least about 25%.

[0045] In various embodiments, the acetylation of mitochondrial proteins in motor neurons can be reduced by contacting the motor neurons with an agent. The agent can reduce the amount / fraction / percentage of acetylated mitochondrial proteins in motor neurons and / or reduce the degree of acetylation in mitochondrial proteins in motor neurons. An "agent" can be anything (physical, chemical, biological, etc.) that a motor neuron can be exposed to, for example, to reduce acetylation of mitochondrial proteins in motor neurons. Non-limiting examples of an "agent" include chemicals, compounds, composition molecules, small molecules, nucleic acid sequences, nucleic acid analogs, proteins, peptides, aptamers, antibodies, or fragments thereof. The term "agent" does not in any way exclude the use of more than one such agent. Thus, the term "agent" also contemplates mixtures, fusions, combinations, compositions, and conjugates, such as mixtures, fusions, combinations, compositions, and conjugates of any chemicals, compounds, compositions, molecules, small molecules, nucleic acid sequences, nucleic acid analogs, proteins, peptides, aptamers, antibodies, or fragments thereof.

[0046] The nucleic acid sequence may be RNA and / or DNA and may be single-stranded or double-stranded. In one example, the nucleic acid sequence comprises an oligonucleotide. The nucleic acid sequence may be composed of natural bases, chemically modified bases, artificial bases, nucleotide analogs, and combinations thereof. The nucleic acid sequence may also comprise modification(s) to its ribose or sugar moiety and / or modification(s) to its phosphate linkage or phosphodiester bond. Examples of ribose or sugar modifications include modifications to the 2' position of the ribose ring, such as 2'-amino, 2'-fluoro, 2'-O-methyl, and 2'-O-methoxy-ethyl. Non-limiting examples of modified phosphate linkages or phosphodiester bonds include phosphorothioate linkages, boranophosphate linkages, methylphosphonates, phosphorothioate analogs, substitution with triazole linkages, and the like. In one example, the nucleic acid sequence comprises phosphoramidite nucleotides.

[0047] Non-limiting examples of nucleic acid analogs include peptide nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), locked nucleic acid (LNA), morpholino nucleotides, threose nucleic acid (TNA), glycol nucleic acid (GNA), arabinose nucleic acid (ANA), 2'-deoxy-2'-fluoro-β-D-arabinonucleic acid (2'F-ANA), 2'-fluoroarabinose nucleic acid (FANA), 2'-deoxy-2'-fluororibonucleic acid (2'-F-ANA), 2'-fluoroarabinose nucleic acid (FANA), 2'-deoxy-2'-fluororibonucleic acid (2'-F-ANA), 2'-fluoro-β-D-arabinon ... RNA or FRNA), cyclohexene nucleic acid (CeNA), anhydrohexitol nucleic acid (HNA), unlocked nucleic acid (UNA), ethylene-bridged nucleic acid (ENA), (4'→6') linked oligo 2',3'-dideoxy-β-D-glucopyranose nucleic acid (homo-DNA or hDNA), xylonucleic acid (XyNA), deoxyxylonucleic acid (dXyNA), aminoallyl uridine (aa-UTP), N3'→P5'-phosphoramidite (NP), tricycloDNA (tcDNA), phosphorodiamidite morpholino (PMO), and derivatives thereof.

[0048] Non-limiting examples of proteins include mutant proteins, genetically engineered proteins, peptides, synthetic peptides, recombinant proteins, chimeric proteins, antibodies, humanized proteins, modified proteins, and fragments thereof (e.g., antigen-binding fragments). As used herein, the term "antibody" refers to an immunoglobulin or fragment thereof and encompasses any polypeptide that contains an antigen-binding fragment or an antigen-binding domain. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific (such as bispecific), humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies. The term "antibody" can include antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function. Antibodies are not necessarily obtained from a particular source or produced by any particular method.

[0049] In various embodiments, the motor neurons may be naturally occurring or non-naturally occurring / genetically engineered (e.g., iPSC-derived motor neurons). Typically, motor neurons include upper motor neurons and lower motor neurons.

[0050] Thus, in various embodiments, a method of promoting survival and / or function of ALS or ALS-like motor neurons is provided, the method comprising contacting the motor neurons with an agent capable of reducing mitochondrial protein acetylation.

[0051] In various embodiments, the ALS or ALS-like motor neurons have or are characterized by increased / hyperacetylation or hyperacetylation of mitochondrial proteins. In various examples, the acetylation level of mitochondrial proteins or the expression / signal of acetylated mitochondrial proteins (e.g., acetyl lysine protein or acetyl lysine 68 on manganese superoxide dismutase (MnSOD K68ac)) in ALS or ALS-like motor neurons is greater than about 1-fold, e.g., at least about 1.05-fold, at least about 1.1-fold, at least about 1.15-fold, at least about 1.2-fold, at least about 1.25-fold, at least about 1.3-fold, at least about 1.35-fold, at least about 1.4-fold, at least about 1.45-fold, at least about 1.5-fold, at least about 1.55-fold, at least about 1.6-fold, at least about 1.65-fold, at least about The increase is 1.7 times, at least about 1.75 times, at least about 1.8 times, at least about 1.85 times, at least about 1.9 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times, at least about 5 times, at least about 5.5 times, at least about 6 times, at least about 6.5 times, at least about 7 times, at least about 7.5 times, at least about 8 times, at least about 8.5 times, at least about 9 times, at least about 9.5 times, or at least about 10 times.In various embodiments, the ALS or ALS-like motor neurons comprise one or more of the following characteristics: mutations in one or more genes selected from SOD1 (e.g., G93A, A4V, L144F, etc.), TDP43 (e.g., G298S), C9ORF72 (e.g., expanded GGGGCC repeats), and others such as FUS, HNRNPA1, VAPB, etc., mitochondrial dysfunction, metabolic respiratory abnormalities, altered morphology compared to control / healthy motor neurons, abnormal mitochondrial morphology compared to mitochondria of control / healthy motor neurons, accelerated death phenotype compared to control / healthy motor neurons, decreased basal survival compared to control / healthy motor neurons, increased endoplasmic reticulum (ER) stress compared to control / healthy motor neurons, upregulated / increased expression of the CHOP gene compared to control / healthy motor neurons, increased mitochondrial function ... increased expression of spliced ​​XBP1 (sXBP1) compared to control / healthy motor neurons, decreased basal respiration compared to control / healthy motor neurons, decreased ATP-related oxygen consumption rate (OCR) compared to control / healthy motor neurons, decreased ATP production compared to control / healthy motor neurons, increased spare respiratory capacity compared to control / healthy motor neurons, increased extracellular acidification rate (ECAR) compared to control / healthy motor neurons, increased glycolysis compared to control / healthy motor neurons, increased glycolytic capacity compared to control / healthy motor neurons, reduced / impaired oxidative phosphorylation compared to control / healthy motor neurons, decreased expression and / or activity of deacetylases (e.g. sirtuins such as SIRT3) compared to control / healthy motor neurons, decreased activity of complex I compared to control / healthy motor neurons, decreased mitochondrial NAD compared to control / healthy motor neurons. +levels (e.g., at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% reduced), elevated / increased mitophagy compared to control / healthy motor neurons, decreased neuronal cell body size compared to control / healthy motor neurons, decreased primary neurites compared to control / healthy motor neurons, and increased excitability relative to control / healthy motor neurons. In some embodiments, the change (e.g., increase or decrease) in one or more of the above characteristics (e.g., gene expression, basal survival, ATP production, spare respiratory capacity, etc.) in ALS or ALS-like motor neurons compared to control / healthy motor neurons is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100%. In some embodiments, the change is at least about 0.1 fold, at least about 0.2 fold, at least about 0.3 fold, at least about 0.4 fold, at least about 0.5 fold, at least about 0.6 fold, at least about 0.7 fold, at least about 0.8 fold, or at least about 0.9 fold.In some embodiments, the change is more than about 1-fold, e.g., at least about 1.05-fold, at least about 1.1-fold, at least about 1.15-fold, at least about 1.2-fold, at least about 1.25-fold, at least about 1.3-fold, at least about 1.35-fold, at least about 1.4-fold, at least about 1.45-fold, at least about 1.5-fold, at least about 1.55-fold, at least about 1.6-fold, at least about 1.65-fold, at least about 1.7-fold, at least about 1.75-fold, at least about 1.8-fold, at least about 1.85-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, or at least about 10-fold. In one embodiment, ALS or ALS-like motor neurons do not show differential expression, substantially increased expression, or substantially decreased expression of SIRT3 compared to control / healthy motor neurons. In one embodiment, the expression of SIRT3 in ALS or ALS-like motor neurons is substantially similar or equivalent to the expression of SIRT3 in control / healthy motor neurons.

[0052] In various embodiments, the ALS can be sporadic ALS and / or familial ALS.

[0053] In various embodiments, the mitochondrial protein comprises a protein or subunit thereof that can be localized to the mitochondria of the motor neuron. In some embodiments, the mitochondrial protein comprises a protein or subunit thereof that is synthesized in the mitochondria (or encoded in the mitochondria) from mitochondrial DNA. In some embodiments, the mitochondrial protein comprises a protein or subunit thereof that is synthesized outside the mitochondria and imported into the mitochondria. The term "protein" includes any chain of amino acids, such as proteins, peptides, polypeptides, and complex proteins such as glycoproteins, lipoproteins, phosphoproteins, metalloproteins, cytochromes, etc. In some embodiments, the mitochondrial protein comprises an enzyme. In some embodiments, the mitochondrial protein comprises a metabolic enzyme. In some embodiments, the mitochondrial protein comprises an antioxidant enzyme. In some embodiments, the mitochondrial protein is involved in one or more of the following pathways: cell death, oxidative phosphorylation, production of energy, fatty acid metabolism, sugar metabolism, and amino acid metabolism. In some embodiments, the mitochondrial protein interacts with or is a target (e.g., a direct or indirect downstream target) of a deacetylase, optionally a sirtuin, and optionally SIRT3. In some embodiments, the mitochondrial protein interacts with or is a target (e.g., a direct or indirect downstream target) of an acetyltransferase, optionally GCN5L1 (GCN5 (general control of amino acid synthesis 5)-like 1). In some embodiments, the mitochondrial protein comprises manganese superoxide dismutase (MnSOD). In some embodiments, the acetylation of the mitochondrial protein comprises acetylation of lysine. In some embodiments, the acetylation of the mitochondrial protein comprises acetylation of lysine 68 in manganese superoxide dismutase (MnSOD K68ac). In various examples, microarray / gene expression analysis performed on healthy and sporadic ALS iPSC-derived motor neurons revealed over-representation of one or more of the above pathways.In silico association predictions may indicate that mitochondrial abnormalities contribute to the pathogenesis of sporadic ALS.

[0054] In various embodiments, reducing / inhibiting mitochondrial protein acetylation includes increasing the expression and / or activity of deacetylase. In some embodiments, reducing / inhibiting mitochondrial protein acetylation does not include increasing the expression of deacetylase or only increasing the expression of deacetylase, for example, without increasing the expression of deacetylase or its associated activity. In various embodiments, reducing / inhibiting mitochondrial product acetylation includes reducing / inhibiting the expression and / or activity of acetyltransferase. Thus, in various embodiments, the agent is selected from the group consisting of an activator or agonist of deacetylase, an inhibitor or antagonist of acetyltransferase, and combinations thereof.

[0055] In some embodiments, the deacetylase comprises a mitochondrially enriched deacetylase. In some embodiments, the deacetylase comprises a major mitochondrial deacetylase. In some embodiments, inducing or increasing the activity of a deacetylase in ALS or ALS-like motor neurons rescues a pathological phenotype and / or metabolic abnormality. In some embodiments, the deacetylase comprises a NAD +In some embodiments, the deacetylase comprises a sirtuin family of proteins. The sirtuin family can include SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7, which can be assigned to five subclasses (I, II, III, IV, and U). In some embodiments, the deacetylase comprises a mitochondrial sirtuin. In some embodiments, the mitochondrial sirtuin is selected from the group consisting of SIRT3, SIRT4, SIRT5, and combinations thereof. In one embodiment, the deacetylase comprises SIRT3. In various examples, SIRT3 levels in ALS mice are compared to SIRT3 levels in healthy mice, and SIRT3 levels in human healthy spinal cords are compared to SIRT3 levels in ALS postmortem spinal cords. In examples, there is an increase in SIRT3 mRNA and protein in ALS spinal cords in humans. Because SIRT3 is an enzyme, its activity may provide more information than just expression levels. In various instances, similar SIRT3 protein levels were found in ALS iPSC-derived motor neurons despite a very large reduction in SIRT3 activity, and in various instances, loss of SIRT3 function results in the ALS phenotype.

[0056] Thus, in various embodiments, the deacetylase activator comprises a SIRT3 activator. The SIRT3 activator can promote deacetylation of targets downstream of SIRT3. In some embodiments, the SIRT3 activator can promote deacetylation of mitochondrial proteins. In some embodiments, the SIRT3 activator can promote deacetylation of manganese superoxide dismutase (MnSOD). In some embodiments, the SIRT3 activator can promote deacetylation of lysine 68 in manganese superoxide dismutase (MnSOD K68ac). In various embodiments, the SIRT3 activator can reduce the level of acetylation of mitochondrial proteins, MnSOD, and / or MnSOD K68ac. In various embodiments, the reduction is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100%.

[0057] In some embodiments, the SIRT3 activator comprises a cofactor and / or a cosubstrate of SIRT3. In some embodiments, the SIRT3 activator comprises a NAD + and / or NAD + In various embodiments, NAD is a precursor, or an analog, derivative, or combination thereof. + The precursor is selected from the group consisting of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (Nam), nicotinamide mononucleotide (NMN) nicotinamide riboside (NR), nicotinic acid, and derivatives, analogs, and combinations thereof. In some embodiments, NAD +The precursor comprises vitamin B3 or niacin, or analogs, derivatives, and combinations thereof. In some aspects, vitamin B3 or niacin is selected from the group consisting of NA, Nam, NR, and combinations thereof. In some embodiments, the method comprises contacting the motor neuron with a food, such as a food for medical purposes, that can reduce mitochondrial protein acetylation. Examples of food include health foods, food additives, food supplements, dietary supplements, nutritional supplements, nutrients, vitamins, and / or nutraceuticals. In various examples, increasing the NAD+ level of astrocytes in ALS reduces astrocyte-mediated toxicity in a co-culture model of astrocytes and motor neurons.

[0058] In various embodiments, the SIRT3 activator, NAD + , or NAD + Precursor is NAD in motor neuron mitochondria + The levels can be increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100%. In various embodiments, the SIRT3 activator, NAD + , or NAD + Precursor is NAD in motor neuron mitochondria +To increase the level to greater than about 1 fold, e.g., at least about 1.05 fold, at least about 1.1 fold, at least about 1.15 fold, at least about 1.2 fold, at least about 1.25 fold, at least about 1.3 fold, at least about 1.35 fold, at least about 1.4 fold, at least about 1.45 fold, at least about 1.5 fold, at least about 1.55 fold, at least about 1.6 fold, at least about 1.65 fold, at least about 1.7 fold, at least about 1.75 fold, at least about 1.8 fold, at least about 1.9 fold, at least about 2.0 fold, at least about 2.1 fold, at least about 2.2 fold, at least about 2.3 fold, at least about 2.4 fold, at least about 2.5 fold, at least about 2.6 fold, at least about 2.7 fold, at least about 2.8 fold, at least about 2.9 fold, at least about 2.8 fold, at least about 2.9 fold, at least about 2.1 fold, at least about 2.2 fold, at least about 2.3 fold, at least about 2.4 fold, at least about 2.5 fold, at least about 2.6 fold, at least about 2.7 fold, at least about 2.8 fold, at least about 2.9 ... The increase can be about 1.85-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, or at least about 10-fold.

[0059] In various embodiments, the SIRT3 activator, NAD + , or NAD + The concentration of the precursor is about 0.01 mM to about 10 mM, about 0.05 mM to about 5 mM, about 0.07 mM to about 3 mM, about 0.08 mM to about 2 mM, about 0.1 mM to about 1 mM, about 0.2 mM to about 0.8 mM, about 0.3 mM to about 0.7 mM, or about 0.4 mM to about 0.6 mM. + , or NAD + The concentration of the precursor is about 10 mM or less, about 9 mM or less, about 8 mM or less, about 7 mM or less, about 6 mM or less, about 5 mM or less, about 4 mM or less, about 3 mM or less, about 2 mM or less, about 1 mM or less, about 0.9 mM or less, about 0.8 mM or less, about 0.7 mM or less, about 0.6 mM or less, or about 0.5 mM or less. + , or NAD +The concentration of the precursor is at least about 0.01 mM, at least about 0.05 mM, at least about 0.1 mM, at least about 0.15 mM, at least about 0.2 mM, at least about 0.25 mM, at least about 0.3 mM, at least about 0.35 mM, at least about 0.4 mM, at least about 0.45 mM, or at least about 0.5 mM. In one embodiment, the SIRT3 activator or NAD + The concentration of the precursor is about 0.5 mM. SIRT3 activator, NAD + , or NAD + The concentration / dose / amount of the precursor concentration may be further varied to achieve the desired response for a particular motor neuron, patient, composition, and mode of administration with no or minimal toxicity to the cells or the patient. In various embodiments, the SIRT3 activator, NAD + , or NAD + The concentration of the precursor is below that which would cause toxicity, e.g., cellular or hepatic toxicity. In various embodiments, the SIRT3 activator, NAD + , or NAD + The concentration of the precursor causes substantially no toxicity or only minimal toxicity, e.g., cellular or hepatic toxicity. In various embodiments, the concentration of the SIRT3 activator, NAD required to reduce mitochondrial protein acetylation levels by a desired amount. + , or NAD + If the concentration / dose / amount of precursor may cause toxicity, e.g., cellular or hepatic toxicity, a second / further agent, e.g., a different SIRT3 activator and / or a GCN5L1 inhibitor, may be used in combination with a SIRT3 activator or NAD + The precursor may be used at a lower concentration / dosage / amount. In various embodiments, the second / further agent inhibits NAD in the mitochondria of the motor neuron. +The second / further agent does not substantially affect the level. In various embodiments, the second / further agent does not include tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (Nam), nicotinamide mononucleotide (NMN) nicotinamide riboside (NR), nicotinic acid, or derivatives or analogs thereof. In some embodiments, the second / further agent includes a GCN5L1 inhibitor. In some embodiments, the agent or SIRT3 activator does not include / consist of vitamin B3 or niacin or food, for example, as the only / only agent for activating SIRT3 or reducing mitochondrial protein acetylation.

[0060] In various embodiments, the SIRT3 activator can bind to SIRT3, e.g., the active site of SIRT3. In various embodiments, the SIRT3 activator has a binding affinity, e.g., a high binding affinity, for SIRT3. In various embodiments, the SIRT3 activator can change the conformation of the active site on SIRT3. In various embodiments, the SIRT3 activator is specific for SIRT3. For example, the SIRT3 activator embodiment cannot activate and / or bind to another sirtuin, such as SIRT1, SIRT2, SIRT4, SIRT5, SIRT6, and / or SIRT7. In some embodiments, the SIRT3 activator is a low molecular weight. In some embodiments, the SIRT3 activator is about 1500 Daltons (Da) or less, about 1400 Da or less, about 1300 Da or less, about 1200 Da or less, about 1100 Da or less, about 1000 Da or less, about 900 Da or less, about 800 Da or less, about 700 Da or less, about 600 Da or less, about 500 Da or less, about 400 Da or less, about 300 Da or less, about 200 Da or less, about 100 Da, or about 50 Da or less. In some embodiments, the SIRT3 activator comprises a small molecule. The small molecule may comprise an organic compound. The small molecule may be a natural compound (e.g., a compound derived from a plant) and / or a chemically synthesized / man-made compound. The small molecule may be a flavonoid, optionally a flavanonol. In some embodiments, the small molecule comprises a small polyphenol. In some embodiments, the small molecule is selected from the group consisting of 7-hydroxy-3-(4'-methoxyphenyl)coumarin (C12), honokiol, dihydromyricetin (DHM), and derivatives, analogs, and combinations thereof. In some embodiments, the small molecule has the chemical structure:

[0061] [ka] or derivatives, analogs, or combinations thereof.

[0062] As used herein, the term "derivative," "analog," or "functional analog" of a parent molecule refers to a molecule that is structurally related to the parent molecule. For example, a derivative, analog, or functional analog of a parent molecule may share common structural features, basic structure, and / or underlying chemical basis with the parent molecule. A derivative may be generated or obtained from a parent molecule, but is not limited to being generated or obtained from a parent molecule. In some embodiments, a derivative is, at least theoretically, derivable from a parent molecule through modification of the parent molecule. The term "derivative" also includes conjugates, salts, metabolites, and prodrugs of a parent molecule (e.g., modified derivatives that can be converted to the original compound under physiological conditions). However, in some embodiments, an analog or functional analog does not necessarily have to be generated or obtained using the parent molecule as a starting material. In various embodiments, a derivative, analog, or functional analog of a parent molecule shares or retains at least to some extent the functions, chemical properties, biological properties, chemical activities, and / or biological activities associated with the parent molecule. On a case-by-case basis, one skilled in the art will be able to identify common structural features, basic structure, and / or underlying chemical basis of molecules that must be maintained in derivatives, analogs, or functional analogs in order to retain function, chemical properties, biological properties, chemical activity, and / or biological activity. One skilled in the art will also be able to identify assays that can demonstrate retention of function, chemical properties, biological properties, chemical activity, and / or biological activity. For example, assays such as cellular thermal shift assay (CETSA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, Western blot analysis, metabolic flux analysis MitoStress assay, motor neuron survival assay, enzyme-linked immunosorbent assay (ELISA), electrophoretic mobility shift assay (EMSA), surface plasmon resonance (SPR), biolayer interferometry (BLI), etc., can be performed to determine function, chemical properties, biological properties, chemical activity, and / or biological activity of molecules.

[0063] In various embodiments, the concentration of the SIRT3 activator or small molecule or derivative or analog is about 0.1 μM to about 100 μM, about 0.5 μM to about 50 μM, about 1 μM to about 30 μM, about 5 μM to about 15 μM, about 1 μM to about 10 μM, about 2 μM to about 8 μM, or about 3 μM to about 7 μM. In various embodiments, the concentration of the SIRT3 activator or small molecule or derivative or analog is about 100 μM or less, about 50 μM or less, about 30 μM or less, about 15 μM or less, about 10 μM or less, about 9 μM or less, about 8 μM or less, about 7 μM or less, about 6 μM or less, or about 5 μM or less. In various embodiments, the concentration of the SIRT3 activator or small molecule or derivative or analog is at least about 0.1 μM, at least about 0.5 μM, at least about 1 μM, at least about 1.5 μM, at least about 2 μM, at least about 2.5 μM, at least about 3 μM, at least about 3.5 μM, at least about 4 μM, at least about 4.5 μM, at least about 5 μM, at least about 5.5 μM, at least about 6 μM, at least about 6.5 μM, at least about 7 μM, at least about 7.5 μM, at least about 8 μM, at least about 8.5 μM, at least about 9 μM, at least about 9.5 μM, or at least about 10 μM. In one embodiment, the concentration of the SIRT3 activator or small molecule or derivative or analog is about 5 μM. In one embodiment, the concentration of the SIRT3 activator or small molecule or derivative or analog is less than about 10 μM. The concentration / dosage / amount of SIRT3 activator or small molecule or derivative or analog may further be varied to achieve the desired response for a particular motor neuron, patient, composition, SIRT3 activator, small molecule, and mode of administration while causing no or minimal toxicity to the cell or patient. In various embodiments, the concentration of SIRT3 activator or small molecule or derivative or analog is below that which causes toxicity to the cell or patient.

[0064] In some embodiments, the deacetylase activator increases / is capable of increasing the activity of the deacetylase, hi some embodiments, the deacetylase activator does not / is unable to increase the expression or expression level of the deacetylase.

[0065] In some embodiments, the acetyltransferase comprises a mitochondrially enriched acetyltransferase. In some embodiments, the acetyltransferase counteracts the effects of deacetylases, such as SIRT3, on acetylation and / or respiration. In some embodiments, knockdown or depletion of acetyltransferases in ALS or ALS-like motor neurons rescues pathological phenotypes and / or metabolic disorders. In some embodiments, the acetyltransferase comprises a protein with sequence homology and / or loose sequence alignment with the nuclear acetyltransferase general control of amino acid synthesis 5 (GCN5). In some embodiments, the acetyltransferase comprises general control of amino acid synthesis 5-like 1 (GCN5L1), also known as lysosome-related organelle complex 1 subunit 1 of biogenesis (BLOC1S1).

[0066] Thus, in various embodiments, the acetyltransferase inhibitor comprises a GCN5L1 inhibitor. In some embodiments, the GCN5L1 inhibitor can reduce / inhibit acetylation of mitochondrial proteins. In some embodiments, the GCN5L1 inhibitor can reduce / inhibit acetylation of manganese superoxide dismutase (MnSOD). In some embodiments, the GCN5L1 inhibitor can reduce / inhibit acetylation of lysine 68 in manganese superoxide dismutase (MnSOD K68ac). In various embodiments, the GCN5L1 inhibitor can reduce the level of acetylation of mitochondrial proteins, MnSOD, and / or MnSOD K68ac. In various embodiments, the reduction is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100%.

[0067] In some embodiments, the acetyltransferase inhibitor comprises a chemical, i.e., a chemical inhibitor. The chemical compound may comprise an organic or inorganic compound. In some embodiments, the chemical inhibitor comprises a small molecule. In some embodiments, the acetyltransferase inhibitor comprises a nucleic acid sequence. In some embodiments, the acetyltransferase inhibitor comprises about 200 nucleotides / base or less, about 190 nucleotides / base or less, about 180 nucleotides / base or less, about 170 nucleotides / base or less, about 160 nucleotides / base or less, about 150 nucleotides / base or less, about 140 nucleotides / base or less, about 130 nucleotides / base or less, about 120 nucleotides / base or less, about 110 nucleotides / base or less, about 100 nucleotides / base or less, about 90 nucleotides / base or less, about 80 nucleotides / base or less, about 70 nucleotides / base or less, about 60 nucleotides / base or less, about 50 nucleotides / base or less, about 40 nucleotides / base or less, about 30 nucleotides / base or less, about 20 nucleotides / base or less, or about 10 nucleotides / base or less. In some embodiments, the acetyltransferase comprises about 10 to about 200 nucleotides / base, about 10 to about 150 nucleotides / base, about 10 to about 100 nucleotides / base, about 10 to about 50 nucleotides / base, about 10 to about 40 nucleotides / base, about 10 to about 30 nucleotides / base, about 10 to about 20 nucleotides / base, about 15 to about 40 nucleotides / base, about 15 to about 30 nucleotides / base, or about 15 to about 20 nucleotides / base. In various embodiments, the acetyltransferase inhibitor comprises an oligonucleotide. In various embodiments, the acetyltransferase inhibitor comprises an RNA. In various embodiments, the acetyltransferase inhibitor comprises an inhibitory RNA. In various embodiments, the acetyltransferase inhibitor is selected from the group consisting of an antisense oligonucleotide (ASO), a gapmer, a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), a guide RNA (gRNA), a single guide RNA (sgRNA), and combinations thereof.In some embodiments, the acetyltransferase inhibitor comprises a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) complex, such as a CRISPR-Cas9 complex, or a portion thereof.Antisense oligonucleotides (ASOs) can knock down genes, such as GCN5L1 or BLOC1S1, through exon skipping and subsequent mRNA degradation or translation inhibition.SiRNAs can induce gene knockdown, such as GCN5L1 or BLOC1S1, via the endoribonuclease DICER.

[0068] In various embodiments, the oligonucleotide is a) a sequence complementary to the coding sequence (CDS) of the BLOC1S1 gene or SEQ ID NO:2, optionally the coding sequence (CDS) of the BLOC1S1 gene or SEQ ID NO:3 and / or SEQ ID NO:4, or a portion thereof, optionally a linear sequence, or b) a sequence complementary to the sequence of a) by at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or a sequence that differs from the sequence in a by about 1 nucleotide / base, about 2 nucleotides / base, about 3 nucleotides / base, about 4 nucleotides / base, about 5 nucleotides / base, about 6 nucleotides / base, about 7 nucleotides / base, about 8 nucleotides / base, about 9 nucleotides / base, or about 10 nucleotides / base. In various embodiments, the oligonucleotide comprises a) a sequence complementary to the coding sequence (CDS) of the BLOC1S1 gene, SEQ ID NO:3 and / or SEQ ID NO:4, or a portion thereof, or b) a sequence that shares at least about 75% sequence identity with the sequence in a).In various embodiments, the oligonucleotide has a sequence similar to SEQ ID NO:1 (or AGAGGAGGCGAGGCUAU), or b) a sequence similar to SEQ ID NO:1 (or AGAGGAGGCGAGGCUAU), or c) a sequence similar to SEQ ID NO:1 (or AGAGGAGGCGAGGCUAU), or d) a sequence similar to SEQ ID NO:1 (or AGAGGAGGCGAGGCUAU), or d) a sequence similar to SEQ ID NO:1 (or AGAGGAGGCGAGGCUAU), or e) a sequence similar to SEQ ID NO:1 (or AGAGGAGGCGAGGCUAU), or f ... %, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or a sequence that differs from the sequence of a) by about 1 nucleotide / base, about 2 nucleotides / base, about 3 nucleotides / base, about 4 nucleotides / base, about 5 nucleotides / base, about 6 nucleotides / base, about 7 nucleotides / base, about 8 nucleotides / base, about 9 nucleotides / base, or about 10 nucleotides / base. In various embodiments, the oligonucleotide comprises a sequence that shares at least about 75% sequence identity with SEQ ID NO: 1, or a sequence that differs from SEQ ID NO: 1 by about 1 nucleotide, about 2 nucleotides, about 3 nucleotides, about 4 nucleotides, or about 5 nucleotides. In various embodiments, the acetyltransferase inhibitor reduces / inhibits the expression of GCN5L1.

[0069] In various embodiments, an oligonucleotide comprising a sequence complementary to a portion of the BLOC1S1 gene, a portion of the coding sequence (CDS) of the BLOC1S1 gene, or a portion of any of SEQ ID NOs: 2-4 comprises a sequence complementary to a stretch of sequence / nucleic acid residues in the BLOC1S1 gene, the coding sequence (CDS) of the BLOC1S1 gene, or SEQ ID NOs: 2-4 such that hybridization of the oligonucleotide to at least a portion of the gene or sequence is achievable. In various embodiments, the stretch of sequence / nucleic acid residues comprises at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, or at least about 30 consecutive nucleic acid residues in the BLOC1S1 gene, the coding sequence (CDS) of the BLOC1S1 gene, or any of SEQ ID NOs: 2-4. In some embodiments, the oligonucleotide may comprise a sequence complementary to more than one stretch, e.g., about 2, about 3, of sequence / nucleic acid residues in the BLOC1S1 gene, the coding sequence (CDS) of the BLOC1S1 gene, or SEQ ID NOs: 2-4.

[0070] In various embodiments, the acetyltransferase inhibitor can reduce / reduce the expression of the acetyltransferase (e.g., gene or protein expression) by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100%. In one embodiment, the acetyltransferase inhibitor can reduce / reduce the expression of the acetyltransferase by about 50%-70%.

[0071] In various embodiments, the agent is capable of inhibiting the expression and / or activity of the following characteristics in ALS or ALS-like motor neurons: mitochondrial dysfunction, metabolic respiratory abnormalities, morphological changes, abnormal mitochondrial morphology, survival / basal survival, neuronal cell body size, amount of primary neurites, endoplasmic reticulum (ER) stress, basal respiration, ATP-related oxygen consumption rate (OCR), ATP production, spare respiratory capacity, oxidative phosphorylation, expression and / or activity of deacetylases (e.g., sirtuins such as SIRT3), activity of complex I, mitochondrial NAD +The agent may improve one or more of the following characteristics, for example, to be similar or more similar to healthy / normal motor neurons: expression of the CHOP gene, amount / level of spliced ​​XBP1 (sXBP1), extracellular acidification rate (ECAR), and mitophagy. In some embodiments, the agent may change / modulate (e.g., increase / promote or decrease / reduce) one or more of the above characteristics to be similar or more similar to healthy / normal motor neurons by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100%. In some embodiments, the change is at least about 0.1 fold, at least about 0.2 fold, at least about 0.3 fold, at least about 0.4 fold, at least about 0.5 fold, at least about 0.6 fold, at least about 0.7 fold, at least about 0.8 fold, or at least about 0.9 fold. In some embodiments, the change is more than about 1 fold, e.g., at least about 1.05 fold, at least about 1.1 fold, at least about 1.15 fold, at least about 1.2 fold, at least about 1.25 fold, at least about 1.3 fold, at least about 1.35 fold, at least about 1.4 fold, at least about 1.45 fold, at least about 1.5 fold, at least about 1.55 fold, at least about 1.6 fold, at least about 1.65 fold, at least about 1.7 fold, at least about 1.75 fold, at least about 1.8-fold, at least about 1.85-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, or at least about 10-fold.

[0072] In various embodiments, the methods include in vitro or ex vivo methods.

[0073] In various embodiments, an agent or composition is provided that can regulate / control the acetylation of mitochondrial products in motor neurons. In various embodiments, the agent or the composition can reduce / inhibit the acetylation of mitochondrial products, such as mitochondrial proteins, in motor neurons. The agent or the composition can be used in treatment. The agent or the composition can be a pharmaceutical composition. The agent or the composition can include one or more of a suitable carrier, additive, adjuvant, diluent, and excipient. In various embodiments, an agent or composition for use in treatment is provided. In various embodiments, an agent or composition for use in treatment is provided. In various embodiments, an agent or composition for use in treatment of ALS or ALS-like disease is provided. In some embodiments, a therapeutically effective amount of an agent or composition for use in treatment of ALS or ALS-like disease is provided, for example, an amount that can reduce the acetylation of mitochondrial proteins in motor neurons by a desired amount. Thus, in various embodiments, an agent or composition for use in treatment of ALS or ALS-like disease is provided that can reduce the acetylation of mitochondrial proteins.

[0074] In various embodiments, the agent or composition is selected from the group consisting of deacetylase activators or agonists, acetyltransferase inhibitors or antagonists, and combinations thereof. In various embodiments, the deacetylase activator comprises a SIRT3 activator. In various embodiments, the SIRT3 activator is selected from the group consisting of a small molecule, NAD or a precursor thereof, and combinations thereof. In various embodiments, the small molecule is selected from the group consisting of 7-hydroxy-3-(4'-methoxyphenyl)coumarin (C12), honokiol, dihydromyricetin (DHM), and derivatives, analogs, and combinations thereof. In various embodiments, the NAD precursor is selected from the group consisting of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (Nam), nicotinamide mononucleotide (NMN) nicotinamide riboside (NR), nicotinic acid, and derivatives, analogs, and combinations thereof. In various embodiments, the acetyltransferase inhibitor comprises a GCN5L1 inhibitor. In various embodiments, the GCN5L1 inhibitor comprises an oligonucleotide and / or a chemical entity. In various embodiments, the oligonucleotide is selected from the group consisting of an antisense oligonucleotide (ASO), a gapmer, a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), a guide RNA (gRNA), a single guide RNA (sgRNA), and combinations thereof. In various embodiments, the oligonucleotide comprises a) a sequence complementary to the CDS of the BLOC1S1 gene or a portion thereof, or SEQ ID NO:3 or a portion thereof, or SEQ ID NO:4 or a portion thereof, or a sequence that shares at least about 75% sequence identity with the sequence of a). In various embodiments, the oligonucleotide comprises a sequence that shares at least about 75% sequence identity with SEQ ID NO:1, or a sequence that differs from SEQ ID NO:1 by about 1 nucleotide, about 2 nucleotides, about 3 nucleotides, about 4 nucleotides, or about 5 nucleotides.

[0075] In some embodiments, the agent or composition can increase the level of expression (e.g., gene and / or protein expression) of SIRT3 (or any complex containing SIRT3). In some embodiments, the agent or composition can increase the level of RNA encoding SIRT3. In some embodiments, the agent or composition can increase the transcription of nucleic acid encoding SIRT3. In some embodiments, the agent or composition can promote proper post-transcriptional processing (e.g., splicing, translation, post-translational processing) of RNA encoding SIRT3. In some embodiments, the agent or composition can increase the level of SIRT3 protein (or any protein complex containing SIRT3). In some embodiments, the agent or composition can reduce degradation of SIRT3 (or any protein complex containing SIRT3). In some embodiments, the agent or composition can promote the interaction between SIRT3 (or any protein complex containing SIRT3) and an interaction partner of SIRT3 (or an interaction partner of any protein complex containing SIRT3). In some embodiments, the agent or composition can increase / increase the level of function of SIRT3 (or any complex containing SIRT3). In some embodiments, the agent or composition can activate SIRT3 (or any complex containing SIRT3). In some embodiments, the agent or composition can increase the level of deacetylase activity by SIRT3 (and / or any complex containing SIRT3). In some embodiments, the agent or composition can reduce / inhibit mitochondrial protein hyperacetylation resulting from decreased SIRT3 activity. In some embodiments, the agent or composition can promote deacetylation of targets downstream of SIRT3.

[0076] In some embodiments, the agent or composition can reduce the level of expression (e.g., gene and / or protein expression) of GCN5L1 (or any complex containing GCN5L1). In some embodiments, the agent or composition can reduce the level of RNA encoding GCN5L1. In some embodiments, the agent or composition can reduce / inhibit the transcription of nucleic acid encoding GCN5L1. In some embodiments, the agent or composition can reduce / inhibit post-transcriptional processing (e.g., splicing, translation, post-translational processing) of RNA encoding GCN5L1. In some embodiments, the agent or composition can reduce / inhibit the level of GCN5L1 protein (or any protein complex containing GCN5L1). In some embodiments, the agent or composition can increase / promote the degradation of GCN5L1 (or any protein complex containing GCN5L1). In some embodiments, the agent or composition can reduce / inhibit / disrupt the interaction between GCN5L1 (or any protein complex containing GCN5L1) and an interaction partner of GCN5L1 (or an interaction partner of any protein complex containing GCN5L1). In some embodiments, the agent or composition can reduce the level of function of GCN5L1 (or any complex containing GCN5L1). In some embodiments, the agent or composition can inhibit the acetyltransferase activity of GCN5L1 (or any complex containing GCN5L1). In some embodiments, the agent or composition can reduce the level of acetyltransferase activity by GCN5L1 (or any complex containing GCN5L1). In some embodiments, the agent or composition can reduce / inhibit the hyperacetylation of mitochondrial proteins caused by increased GCN5L1 activity. In some embodiments, the agent or composition can reduce / inhibit the acetylation of targets downstream of GCN5L1.

[0077] In some embodiments, the agent or composition can reduce mitochondrial protein acetylation in motor neurons, hi some embodiments, the agent or composition can reduce acetylation of manganese superoxide dismutase (MnSOD) in motor neurons, such as acetylation of lysine 68 in manganese superoxide dismutase (MnSOD K68ac).

[0078] Agents or compositions can be assessed for the properties listed in the preceding paragraph using a suitable assay, which may be, for example, an in vitro assay, optionally a cell-based assay or a cell-free assay.

[0079] Gene expression can be analyzed by means well known to those skilled in the art. The level of RNA encoding a given gene can be determined, for example, by techniques such as RT-qPCR. Protein expression can also be determined by means well known to those skilled in the art. The level of a given protein / its isoforms can be determined, for example, by antibody-based methods including Western blot, immunohistochemistry / immunohistochemistry / cytochemistry, flow cytometry, ELISA, etc.

[0080] An increase in gene or protein expression levels for a given gene is greater than 1-fold the level of expression observed in the uninduced state (i.e., in the absence of an inducer of SIRT3 expression / activity), e.g., ≥1.01-fold, ≥1.05-fold, ≥1.1-fold, ≥1.15-fold, ≥1.2-fold, ≥1.25-fold, ≥1.3-fold, ≥1.35-fold, ≥1.4-fold, ≥1.45-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥1.10-fold, ≥1.12-fold, ≥1.14-fold, ≥1.16-fold, ≥1.18-fold, ≥1.19-fold, ≥1.20-fold, ≥1.21-fold, ≥1.22-fold, ≥1.23-fold, ≥1.24-fold, ≥1.25-fold, ≥1.25-fold, ≥1.26-fold, ≥1.27-fold, ≥1.28-fold, ≥1.29-fold, ≥1.30-fold, ≥1.31-fold, ≥1.32-fold, ≥1.33-fold, ≥1.34-fold, ≥1.35-fold, ≥1.35-fold, ≥1.35-fold, ≥1.36-fold, ≥1.37-fold, ≥1.38-fold, ≥1.39-fold, ≥1.40-fold, ≥1.41-fold, ≥1.42-fold, ≥1.43-fold, ≥1.44-fold, ≥1.45-fold, ≥1.45-fold, ≥1.4 The increase may be one of: 5x, ≧1.55x, ≧1.6x, ≧1.65x, ≧1.7x, ≧1.75x, ≧1.8x, ≧1.85x, ≧1.9x, ≧2x, ≧3x, ≧4x, ≧5x, ≧6x, ≧7x, ≧8x, ≧9x, ≧10x, ≧20x, ≧30x, ≧40x, ≧50x, ≧60x, ≧70x, ≧80x, ≧90x, or ≧100x. In some embodiments, an agent or composition capable of increasing gene or protein expression increases gene expression by more than 5% of expression observed in the uninduced state, e.g., by one of: >10%, >15%, >20%, >25%, >30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%, >100%, >200%, >300%, >400%, >500%, >600%, >700%, >800%, >900%, or >1000%.

[0081] The reduction in gene or protein expression of a given gene may be less than 1-fold the level of expression observed in an uninhibited state (i.e., in the absence of a GCN5L1 inhibitor), for example, a reduction to one of the following: ≦0.99-fold, ≦0.95-fold, ≦0.9-fold, ≦0.85-fold, ≦0.8-fold, ≦0.75-fold, ≦0.7-fold, ≦0.65-fold, ≦0.6-fold, ≦0.55-fold, ≦0.5-fold, ≦0.45-fold, ≦0.4-fold, ≦0.35-fold, ≦0.3-fold, ≦0.25-fold, ≦0.2-fold, ≦0.15-fold, ≦0.1-fold, ≦0.0.05-fold, or ≦0.01-fold. In some embodiments, an agent capable of reducing / inhibiting expression of a gene or protein inhibits more than 5% of expression observed in the uninhibited state, e.g., ≧10%, ≧15%, ≧20%, ≧25%, ≧30%, ≧35%, ≧40%, ≧45%, ≧50%, ≧55%, ≧60%, ≧65%, ≧70%, ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%, or 100%.

[0082] Agents or compositions that can increase the gene expression of SIRT3 (e.g., increase the level of RNA encoding SIRT3, increase the transcription of the nucleic acid encoding SIRT3, and / or decrease the degradation of RNA encoding SIRT3) or reduce / inhibit the gene expression of GCN5L1 (e.g., decrease the level of RNA encoding GCN5L1, decrease / inhibit the transcription of the nucleic acid encoding GCN5L1, and / or increase / promote the degradation of RNA encoding GCN5L1) can be identified using an assay that includes detecting the level of RNA encoding SIRT3 or GCN5L1, for example, by RT-qPCR. Such an assay can include treating cells / tissues, such as motor neurons, with an agent or composition, and then comparing the level of RNA encoding SIRT3 or GCN5L1 in such cells / tissues with the level of RNA encoding SIRT3 or GCN5L1 in cells / tissues under appropriate control conditions (e.g., untreated / vehicle-treated cells / tissues).

[0083] Agents or compositions that can increase the protein expression of SIRT3 (e.g., increase the level of SIRT3 protein or any protein complex containing SIRT3, or decrease the degradation of SIRT3 protein or any protein complex containing SIRT3) or reduce / inhibit the protein expression of GCN5L1 (e.g., decrease the level of GCN5L1 protein or any protein complex containing GCN5L1, or increase / promote the degradation of GCN5L1 protein or any protein complex containing GCN5L1) can be identified using an assay that includes detecting the level of SIRT3 protein, GCN5L1 protein, or any of these complexes, for example, using antibody / reporter-based methods (Western blot, ELISA, immunohisto / cytochemistry, etc.). Such assays can include treating cells / tissues, such as motor neurons, with an agent or composition, and then comparing the level of the protein / protein complex in such cells / tissues with the level in cells / tissues under appropriate control conditions (e.g., untreated / vehicle-treated cells / tissues).

[0084] Agents or compositions that can promote the interaction between SIRT3 (or any complex containing SIRT3) and SIRT3's interaction partner (or any complex containing SIRT3's interaction partner), or reduce / inhibit / disrupt the interaction between GCN5L1 (or any complex containing GCN5L1) and GCN5L1's interaction partner (or any complex containing GCN5L1's interaction partner), can be identified using an assay that includes detecting the level of interaction, for example, using an antibody / reporter-based method. The level of interaction between SIRT3 (or any complex containing SIRT3) and SIRT3's interaction partner (or any complex containing SIRT3's interaction partner), or between GCN5L1 (or any complex containing GCN5L1) and GCN5L1's interaction partner (or any complex containing GCN5L1's interaction partner), can be analyzed, for example, using resonance energy transfer technology (e.g., FRET, BRET), or a method for analyzing the correlation of interaction. The assay may include treating cells / tissues, such as motor neurons, with an agent or composition, and then comparing the level of interaction between SIRT3 (or any complex containing SIRT3) and an interaction partner of SIRT3 (or an interaction partner of any complex containing SIRT3) or between GCN5L1 (or any complex containing GCN5L1) and an interaction partner of GCN5L1 (or an interaction partner of a complex containing GCN5L1) in such cells / tissues with the level of interaction observed in cells / tissues in appropriate control conditions (e.g., untreated / vehicle-treated cells / tissues). The level of interaction may be analyzed using techniques such as, for example, ELISA, surface plasmon resonance, or biolayer interferometry. The assay may include comparing the level of interaction in the presence of the agent or composition with the level of interaction in appropriate control conditions (e.g., in the absence of the agent or composition).

[0085] The promotion / increase in the interaction between SIRT3 (or any complex comprising SIRT3) and an interaction partner of SIRT3 (or an interaction partner of any complex comprising SIRT3) is more than 1-fold, e.g., ≧1.01-fold, of the level of interaction observed in the uninduced state (i.e., in the absence of an agent or composition that promotes / increases the interaction between SIRT3 (or any complex comprising SIRT3) and an interaction partner of SIRT3 (or an interaction partner of a complex comprising SIRT3)). , ≧1.05-fold, ≧1.1-fold, ≧1.15-fold, ≧1.2-fold, ≧1.25-fold, ≧1.3-fold, ≧1.35-fold, ≧1.4-fold, ≧1.45-fold, ≧1.5-fold, ≧1.55-fold, ≧1.6-fold, ≧1.65-fold, ≧1.7-fold, ≧1.75-fold, ≧1.8-fold, ≧1.85-fold, ≧1.9-fold, ≧2-fold, ≧3-fold, ≧4-fold, ≧5-fold, ≧6-fold, ≧7-fold, ≧8-fold, ≧9-fold, ≧10-fold, ≧20-fold, ≧30-fold, ≧40-fold, ≧50-fold, ≧60-fold, ≧70-fold, ≧80-fold, ≧90-fold, or ≧100-fold. In some embodiments, an agent capable of promoting / increasing an interaction between (SIRT3 or any complex comprising SIRT3) and an interaction partner of SIRT3 (or an interaction partner of a complex comprising SIRT3) increases the interaction by more than 5%, e.g., ≥ 10%, ≥ 15%, ≥ 20%, ≥ 25%, ≥ 30%, ≥ 35%, ≥ 40%, ≥ 50%, ≥ 60%, ≥ 70%, ≥ 80%, ≥ 90%, ≥ 100%, ≥ 110%, ≥ 120%, ≥ 130%, ≥ 140%, ≥ 150%, ≥ 160%, ≥ 170%, ≥ 180%, ≥ 190%, ≥ 210%, ≥ 220%, ≥ 230%, ≥ 240%, ≥ 250%, ≥ 260%, ≥ 270%, ≥ 280%, ≥ 300%, ≥ 350%, ≥ 360%, ≥ 370%, ≥ 380%, ≥ 390%, ≥ 400%, ≥ 410%, ≥ 420%, ≥ 430%, ≥ 440%, ≥ 450%, ≥ 460%, ≥ 470%, ≥ 480%, ≥ 490%, ≥ 500%, ≥ 510%, ≥ 520%, ≥ 530%, ≥ 540%, ≥ 550%, ≥ 560%, ≥ 570%, ≥ 580%, ≥ 590%, ≥ 600%, ≥ 610%, ≥ 620%, ≥ 630%, ≥ 640%, ≥ 650%, ≥ 660%, ≥ 670%, ≥ 680%, An increase of only one of the following: ≧45%, ≧50%, ≧55%, ≧60%, ≧65%, ≧70%, ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%, ≧100%, ≧200%, ≧300%, ≧400%, ≧500%, ≧600%, ≧700%, ≧800%, or ≧900%, ≧1000%.

[0086] The reduction / inhibition / disruption of the interaction between GCN5L1 (or any complex containing GCN5L1) and an interaction partner of GCN5L1 (or an interaction partner of any complex containing GCN5L1) is equivalent to the uninhibited state (i.e., an agent that reduces / inhibits / disrupts the interaction between GCN5L1 (or any complex containing GCN5L1) and an interaction partner of GCN5L1 (or an interaction partner of any complex containing GCN5L1)). or in the absence of the composition), to less than 1-fold, e.g., to one of: <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold. In some embodiments, an agent or composition capable of reducing / inhibiting / disrupting the interaction between GCN5L1 (or any complex comprising GCN5L1) and an interaction partner of GCN5L1 (or an interaction partner of a complex comprising GCN5L1) inhibits more than 5% of the interaction observed in the uninhibited state, for example, ≧10%, ≧15%, ≧20%, ≧25%, ≧30%, ≧35%, ≧40%, ≧45%, ≧50%, ≧55%, ≧60%, ≧65%, ≧70%, ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%, or 100%.

[0087] Agents or compositions that can modulate the function of SIRT3 or GCN5L1 (or any of these complexes) can be identified using assays for the relevant function. Such assays can include treating cells / tissues expressing SIRT3 or GCN5L1 (or any of these complexes), such as motor neurons, with the agent or composition, and then comparing the level of the relevant function to that observed in appropriate control conditions (e.g., untreated / vehicle-treated cells / tissues). Agents or compositions can also be identified using assays that include detecting the level of a correlate of the function of SIRT3 or GCN5L1 (or any of these complexes) (e.g., gene and / or protein expression and / or activity of one or more proteins whose expression is directly / indirectly up-regulated or down-regulated as a result of the function of SIRT3 / GCN5L1 (or any of these complexes). Such an assay may involve treating cells / tissues expressing SIRT3 or GCN5L1 (or any of these complexes) function, such as motor neurons, with an agent, and then comparing the level of a functional correlate of SIRT3 or GCN5L1 (or any of these complexes) in such cells / tissues with the level of the relevant functional correlate in an appropriate control condition (e.g., untreated / vehicle-treated cells / tissues). The function of SIRT3 (and / or any complex containing SIRT3) may be, for example, deacetylase activity. The function of SIRT3 (and / or any complex containing SIRT3) may be, for example, a product of deacetylase activity. The function of GCN5L1 (and / or any complex containing GCN5L1) may be, for example, an acetyltransferase activity. The function of GCN5L1 (and / or any complex containing GCN5L1) may be, for example, a product of acetyltransferase activity.

[0088] Agents or compositions that can increase deacetylase activity by SIRT3 (or any complex containing SIRT3) or reduce / inhibit acetyltransferase activity by GCN5L1 (or any complex containing GCN5L1) can be identified using an assay that includes detecting the level of deacetylase or acetyltransferase activity, for example, using an antibody / reporter-based method. The assay can include treating cells / tissues, such as motor neurons, expressing SIRT3, GCN5L1 (or any complex), with the agent or composition, and then comparing the level of acetylation (e.g., in mitochondrial proteins or in representative mitochondrial proteins) to the level of acetylation observed in appropriate control conditions (e.g., untreated / vehicle-treated cells / tissues). The assay can also include comparing the level of relevant activity exhibited by SIRT3, GCN5L1 (or any complex containing GCN5L1) in the presence of the agent or composition to the level observed in the absence of the agent or composition. Examples of assays that can be performed are described in the Examples.

[0089] Enhancement / increase in function (e.g., deacetylase activity) of SIRT3 (or any complex containing SIRT3) can be more than 1-fold the level of activity observed in the uninduced state, e.g., ≥1.01-fold, ≥1.05-fold, ≥1.1-fold, ≥1.15-fold, ≥1.2-fold, ≥1.25-fold, ≥1.3-fold, ≥1.35-fold, ≥1.4-fold, ≥1.45-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥1.10-fold, ≥1.12-fold, ≥1.14-fold, ≥1.16-fold, ≥1.18-fold, ≥1.19-fold, ≥1.20-fold, ≥1.21-fold, ≥1.22-fold, ≥1.23-fold, ≥1.24-fold, ≥1.25-fold, ≥1.25-fold, ≥1.35-fold, ≥1.36-fold, ≥1.37-fold, ≥1.38-fold, ≥1.39-fold, ≥1.40-fold, ≥1.41-fold, ≥1.42-fold, ≥1.43-fold, ≥1.44-fold, ≥1.45-fold, ≥1.45-fold, ≥1.46-fold, ≥1.47-fold, ≥1.48-fold, ≥1.49-fold, ≥1.50-fold, ≥1.51-fold, ≥1.52-fold, ≥1.53-fold, ≥1.54-fold, ≥1.55-fold, ≥1.55-fold, ≥1 The enhancement / increase may be one of the following: .55-fold, ≥1.6-fold, ≥1.65-fold, ≥1.7-fold, ≥1.75-fold, ≥1.8-fold, ≥1.85-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, ≥10-fold, ≥20-fold, ≥30-fold, ≥40-fold, ≥50-fold, ≥60-fold, ≥70-fold, ≥80-fold, ≥90-fold, or ≥100-fold. In some embodiments, an agent or composition capable of enhancing / increasing the function (e.g., deacetylase activity) of SIRT3 (or any complex comprising SIRT3) may enhance / increase activity by more than 5%, e.g., ≥10%, ≥15%, ≥20%, ≥25%, ≥30%, ≥35%, ≥40%, ≥45%, ≥50%, ≥55% of the relevant activity observed in the uninduced state. , ≥60%, ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, ≥100%, ≥200%, ≥300%, ≥400%, ≥500%, ≥600%, ≥700%, ≥800%, ≥900%, or ≥1000% enhanced / increased.

[0090] The reduction / inhibition of the function (e.g., acetyltransferase activity) of GCN5L1 (or any complex containing GCN5L1) may be a reduction / inhibition to one of the following levels of activity less than 1-fold the level observed in the uninhibited state: ≦0.99-fold, ≦0.95-fold, ≦0.9-fold, ≦0.85-fold, ≦0.8-fold, ≦0.75-fold, ≦0.7-fold, ≦0.65-fold, ≦0.6-fold, ≦0.55-fold, ≦0.5-fold, ≦0.45-fold, ≦0.4-fold, ≦0.35-fold, ≦0.3-fold, ≦0.25-fold, ≦0.2-fold, ≦0.15-fold, ≦0.1-fold, ≦0.0.05-fold, or ≦0.01-fold. In some embodiments, an agent or composition capable of reducing / inhibiting the function (e.g., acetyltransferase activity) of GCN5L1 (and / or any complex containing GCN5L1) inhibits more than 5% of the relevant activity observed in the uninhibited state, e.g., ≧10%, ≧15%, ≧20%, ≧25%, ≧30%, ≧35%, ≧40%, ≧45%, ≧50%, ≧55%, ≧60%, ≧65%, ≧70%, ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%, or 100%.

[0091] In some embodiments, the agent is and includes a SIRT3 binding molecule, a SIRT3 complex binding molecule, a molecule capable of increasing the level of SIRT3, a molecule capable of increasing the level of the SIRT3 complex, a GCN5L1 binding molecule, a GCN5L1 complex binding molecule, a molecule capable of decreasing the level of GCN5L1, or a molecule capable of decreasing the level of the GCN5L1 complex. As used herein, a "SIRT3 binding molecule" refers to a molecule capable of binding to a SIRT3-containing complex. A "SIRT3 complex binding molecule" refers to a molecule capable of binding to a GCN5L1-containing complex. A "GCN5L1 binding molecule" refers to a molecule capable of binding to a GCN5L1-containing complex. A SIRT3 binding molecule or a GCN5L1 binding molecule can be identified using any suitable assay for detecting the binding of a molecule to a related factor (i.e., SIRT3, GCN5L1, or any complex containing SIRT3 and / or GCN5L1). Such an assay may include detecting the formation of a complex between a related factor and a molecule.

[0092] SIRT3 binding molecule, GCN5L1 binding molecule (or binding molecule of any complex containing SIRT3 or GCN5L1) can be analyzed by suitable assay to identify agonist of SIRT3 or antagonist of GCN5L1.For example, molecule that specifically binds to SIRT3 (and / or any complex containing SIRT3) can be evaluated for its ability to activate deacetylase or activate deacetylase activity.For example, molecule that specifically binds to GCN5L1 (and / or any complex containing GCN5L1) can be evaluated for its ability to inhibit acetyltransferase or inhibit acetyltransferase activity.

[0093] In some embodiments, a SIRT3 binding molecule (or a binding molecule of any complex that includes SIRT3) can promote the ability of its target (i.e., SIRT3 or any complex that includes SIRT3) to interact with an interaction partner. In some embodiments, a SIRT3 binding molecule (or a binding molecule of any complex that includes SIRT3) can change the conformation of the active site on SIRT3. In some embodiments, a GCN5L1 binding molecule (or a binding molecule of any complex that includes GCN5L1) can inhibit the ability of its target (i.e., GCN5L1 or any complex that includes GCN5L1) to interact with an interaction partner. In some embodiments, a GCN5L1 binding molecule (or a binding molecule of any complex that includes GCN5L1) behaves as a competitive inhibitor of the interaction between its target and its interaction partner. A binding molecule can occupy or otherwise reduce access to a region of a target that is required for binding to its interaction partner. The ability of a SIRT3-binding molecule or a GCN5L1-binding molecule (or a binding molecule of any complex containing SIRT3 or GCN5L1) to promote or inhibit the interaction between its target and its interaction partner can be evaluated, for example, by analyzing the interaction in the presence of one or both of the interaction partners or after incubating one or both of the interaction partners with the relevant binding molecule. Examples of assays suitable for determining whether a given binding agent can promote or inhibit the interaction between its target and its interaction partner include ELISA and competitive ELISA.

[0094] In various embodiments, the SIRT3 binding molecule, GCN5L1 binding molecule (or binding molecule of any complex containing SIRT3 or GCN5L1) comprises a chemical, i.e., a chemical inhibitor. In some embodiments, the chemical comprises a small molecule. The chemical compound or small molecule may be an organic or inorganic compound. In some embodiments, the chemical compound or small molecule comprises an organic compound. The chemical compound or small molecule may be identified by screening a chemical compound library or small molecule library. The chemical compound library or small molecule library may be a plurality of chemical compounds or small molecules collected from any of a number of sources, including chemically synthesized compounds or molecules and natural products, or produced by combinatorial chemistry techniques.

[0095] Small molecules can have low molecular weights of about 1500 Da or less, about 1400 Da or less, about 1300 Da or less, about 1200 Da or less, about 1100 Da or less, about 1000 Da or less, about 900 Da or less, about 800 Da or less, about 700 Da or less, about 600 Da or less, about 500 Da or less, about 400 Da or less, about 300 Da or less, about 200 Da or less, about 100 Da or less, or about 50 Da or less. In some embodiments, small molecules have a molecular weight of about 50 Da to about 1500 Da, about 100 Da to about 1000 Da, or about 300 Da to about 700 Da.

[0096] In various embodiments, the SIRT3 binding molecule, GCN5L1 binding molecule (or binding molecule of any complex containing SIRT3 or GCN5L1) comprises an aptamer. Nucleic acid aptamers are reviewed, for example, in Zhou and Rossi Nat Rev Drug Discov.2017 16(3):181-202, and can be identified and / or generated by sequential evolution of molecules (SELEX) or by developing SOMAmers (modified aptamers with slow off-rates) (Gold L et al.(2010)PLoS ONE 5(12):e15004). Aptamers and SELEX are described in Tuerk and Gold, Science(1990)249(4968):505-10 and WO 91 / 19813. Nucleic acid aptamers can comprise DNA and / or RNA and can be single-stranded or double-stranded. Nucleic acid aptamers may include chemically modified nucleic acids, for example, where the sugar and / or phosphate and / or base are chemically modified. Such modifications may improve the stability of the aptamer or make it less susceptible to degradation, and may include modifications at the 2' position of the ribose. Nucleic acid aptamers may be chemically synthesized, for example, on a solid support. Solid-phase synthesis may use phosphoramidite chemistry. Briefly, a solid-supported nucleotide is detritylated and then coupled with a suitably activated nucleoside phosphoramidite to form a phosphite triester bond. Capping may then be performed, followed by oxidation of the phosphite triester using an oxidizing agent, typically iodine. This cycle can then be repeated to assemble the aptamer (see, e.g., Sinha, ND; Biernat, J.; McManus, J.; Koster, H. Nucleic Acids Res. 1984, 12, 4539; and Beaucage, SL; Lyer, RP (1992). Tetrahedron 48(12):2223).Peptide aptamers and methods for their generation and identification are reviewed in Reverdatto et al., Curr Top Med Chem. (2015) 15(12):1082-101, which is incorporated by reference in its entirety.

[0097] Molecules capable of increasing the level of SIRT3 (or a complex comprising SIRT3) include molecules capable of increasing the expression of the gene and / or protein of SIRT3 (or a complex comprising SIRT3). In some embodiments, molecules capable of increasing the level of SIRT3 (or a complex comprising SIRT3) induce or increase the expression of a polypeptide encoded by the SIRT3 gene. Increasing the expression of a gene or protein of a given target gene (e.g., SIRT3 gene) can include, for example, increasing / promoting the transcription of the gene, promoting proper post-transcriptional processing (e.g., splicing) of the RNA transcribed from the gene, increasing the stability of the RNA transcribed from the gene, reducing / preventing the degradation of the RNA transcribed from the gene, increasing / promoting the translation of the RNA transcribed from the gene into a protein, promoting proper post-translational processing of the polypeptide encoded by the gene, increasing the stability of the polypeptide encoded by the gene, or reducing / preventing the degradation of the polypeptide encoded by the gene.

[0098] Molecules capable of reducing the level of GCN5L1 (or a complex containing GCN5L1) include molecules capable of reducing the expression of the gene and / or protein of GCN5L1 (or a complex containing GCN5L1). In some embodiments, molecules capable of reducing the level of GCN5L1 (or a complex containing GCN5L1) reduce or prevent the expression of a polypeptide encoded by the BLOC1S1 gene. Inhibiting the expression of a gene or protein of a given target gene (e.g., the BLOC1S1 gene) can include, for example, inhibiting the transcription of the gene, inhibiting post-transcriptional processing (e.g., splicing) of the RNA transcribed from the gene, reducing the stability of the RNA transcribed from the gene, promoting the degradation of the RNA transcribed from the gene, inhibiting the translation of the RNA transcribed from the gene into a protein, inhibiting post-translational processing of the polypeptide encoded by the gene, reducing the stability of the polypeptide encoded by the gene, or promoting the degradation of the polypeptide encoded by the gene.

[0099] Inhibition or induction / activation / promotion / upregulation of gene or protein expression can be achieved, for example, by altering / disrupting the nucleotide sequence of the gene or altering / disrupting the nucleotide sequence required for expression of the gene (e.g., a regulatory sequence governing expression of the gene). In some embodiments, inhibition or induction / activation / promotion / upregulation of gene or protein expression can include altering the nucleotide sequence, for example, by substitution, deletion, or insertion of one or more nucleotides. For example, in certain aspects and embodiments, the present disclosure contemplates inhibiting expression of a gene or protein by deleting all or a portion of the nucleotide sequence of the relevant gene. For example, in certain aspects and embodiments, the present disclosure contemplates inducing / activating / promoting / upregulating expression of a gene or protein by introducing an activating mutation, introducing a regulatory sequence that enhances expression of the gene, and / or amplifying all or a portion of the nucleotide sequence of the relevant gene.

[0100] Altering / disrupting a nucleotide sequence to inhibit / prevent gene or protein expression from a gene is sometimes referred to as "knocking out" the gene. A "knock-in" of a gene can alter (e.g., increase) gene or protein expression, for example, by introducing one or more copies of the target gene or by operably inserting a regulatory sequence that enhances expression of the target gene.

[0101] The nucleotide sequence can be disrupted, for example, by homologous recombination or by modification of the target nucleic acid with site-specific nucleases (SSNs).

[0102] Modification by homologous recombination can involve the exchange of nucleic acid sequences through crossover events guided by homologous sequences, as reviewed, for example, in Mortensen Curr Protoc Neurosci. (2007) Chapter 4:Unit 4.29 and Vasquez et al., PNAS (2001) 98(15):8403-8410, both of which are incorporated herein by reference in their entireties.

[0103] Gene editing using SSNs is reviewed, for example, in Eid and Mahfouz, Exp Mol Med. (2016) 48(10):e265, which is incorporated herein by reference in its entirety. Enzymes capable of creating double-strand breaks (DSBs) can be engineered to introduce site-specific DSBs into a target nucleic acid sequence of interest. DSBs can be repaired by error-prone non-homologous end joining (NHEJ), in which both ends of the break are rejoined, often with the insertion or deletion of nucleotides. Alternatively, DSBs can be repaired by high-homology directed repair (HDR), in which a DNA template with ends homologous to the break site is provided and introduced into the DSB site. SSNs that can be engineered to generate target nucleic acid sequence-specific DSBs include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats / CRISPR-associated-9 (CRISPR / Cas9) system.

[0104] The ZFN system is reviewed, for example, in Umov et al., Nat Rev Genet. (2010) 11(9):636-46, which is incorporated herein by reference in its entirety. ZFNs contain a programmable zinc finger DNA binding domain and a DNA cleavage domain (e.g., a FokI endonuclease domain). The DNA binding domain can be identified by screening zinc finger arrays capable of binding to a target nucleic acid sequence. The TALEN system is reviewed, for example, in Mahfouz et al., Plant Biotechnol J. (2014) 12(8):1006-14, which is incorporated herein by reference in its entirety. TALENs contain a programmable DNA binding TALE domain and a DNA cleavage domain (e.g., a FokI endonuclease domain). TALEs contain a repeat domain consisting of 33-39 amino acid repeats that are identical except for two residues at positions 12 and 13 of each repeat, which are the repeat variable dipeptides (RVDs). Each RVD can determine the binding of the repeat to a nucleotide in a target DNA sequence according to the following relationship: "HD" binds to C, "NI" binds to A, "NG" binds to T, and "NN" or "NK" binds to G (Moscou and Bogdanove, Science (2009) 326 (5959): 1501). CRISPR / Cas9 and related systems, e.g., CRISPR / Cpf1, CRISPR / C2c1, CRISPR / C2c2, and CRISPR / C2c3, are reviewed, e.g., in Nakade et al., Bioengineered (2017) 8 (3): 265-273, which is incorporated herein by reference in its entirety. These systems include an endonuclease (e.g., Cas9, Cpf1, etc.) and a single guide RNA (sgRNA) molecule. The sgRNA can be engineered to target the endonuclease activity to a nucleic acid sequence of interest.

[0105] Disruption of nucleotide sequences that utilize SSN can be accomplished in animals, for example, by administering to the animal a nucleic acid encoding a component of the relevant SSN system. For example, one or more vectors containing a nucleic acid encoding a component of the SSN system may be administered to the animal to target the relevant gene.

[0106] Inhibition of gene or protein expression can also be achieved, for example, by treatment with an agent capable of decreasing gene or protein expression. For example, inhibition of gene or protein expression can be achieved using an inhibitory nucleic acid, such as an antisense nucleic acid. An antisense nucleic acid can bind to a target nucleic acid by complementary base pairing. When the target nucleic acid is an RNA (e.g., an RNA transcribed from an associated gene), an antisense nucleic acid can bind to the target RNA to promote degradation of the RNA and / or inhibit translation. An inhibitory nucleic acid can be an antisense oligonucleotide (ASO).

[0107] The use of inhibitory nucleic acids for gene silencing is reviewed, for example, in O'Keefe, Mater Methods (2013) 3:197, which is incorporated herein by reference in its entirety. The inhibitory nucleic acid can inhibit gene or protein expression by RNA interference (RNAi). RNAi involves inhibiting gene expression and translation by targeting and neutralizing mRNA molecules. In some embodiments, the inhibitory nucleic acid is a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a microRNA (miRNA).

[0108] As used herein, the terms "small interfering RNA" or "siRNA," "short hairpin RNA" or "shRNA," "microRNA" or "miRNA" encompass both naturally occurring and synthetically produced sequences.

[0109] miRNA design is discussed in John et al, PLoS Biology, 11(2), 1862-1879, 2004. Suitable siRNA, shRNA, and miRNA sequences for targeting a given gene can be designed using, for example, siRNA Wizard (invivoGEN) or BLOCK-IT RNAi Designer (Invitrogen).

[0110] By way of background, siRNAs can be obtained by processing of long double-stranded RNAs, and when found in nature, they are typically of exogenous origin. Microinterfering RNAs (miRNAs) include endogenously encoded small non-coding RNAs. Both siRNAs and miRNAs can inhibit the translation of mRNAs with partially complementary target sequences without RNA cleavage, and degrade mRNAs with fully complementary sequences. siRNAs are typically double-stranded. To optimize the effectiveness of RNA-mediated inhibition of target gene function, the length of the siRNA molecule may be optimized for accurate recognition of the siRNA by the RISC complex, which mediates siRNA recognition of the mRNA target. The DNA sequence encoding the miRNA includes the sequence of the miRNA and its adjacent reverse complement. When this DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse complement can base-pair to form a partially double-stranded RNA segment.

[0111] ShRNAs are typically more stable than synthetic siRNAs. shRNAs may contain short inverted repeats separated by small loop sequences. One inverted repeat may be complementary to a gene target. In cells, shRNAs are typically processed by DICER into siRNAs, which degrade the mRNA of the target gene and suppress its expression. shRNAs may be generated intracellularly, for example, by transcription from a vector. In various embodiments, the use of the agent or composition in the manufacture of a medicament for treating motor neuron disease is provided. In various embodiments, the use of the agent or composition in the manufacture of a medicament for treating ALS or ALS-like disease is provided.

[0112] In various embodiments, a method of treating ALS or an ALS-like disease in a subject is provided, comprising administering to the subject an agent or composition, optionally in a therapeutically effective amount. In various embodiments, the method further comprises determining the suitability of the subject for the treatment. In various embodiments, determining the suitability of the subject for the treatment comprises determining / measuring the level of mitochondrial protein acetylation in the subject's motor neurons (or motor neurons derived from the subject) and comparing it to the level of mitochondrial protein acetylation in healthy / control motor neurons, where an elevated level of mitochondrial protein acetylation in the subject's motor neurons compared to the level in healthy / control motor neurons indicates that the subject is suitable for the treatment. In various embodiments, the method comprises determining / measuring the level of mitochondrial protein acetylation in the subject's motor neurons and comparing it to the level of mitochondrial protein acetylation after administration of the agent or composition. In various embodiments, a decrease in the level of mitochondrial protein acetylation in the subject's motor neurons after administration of the agent or composition may indicate that the treatment is effective and / or may indicate an improvement in the disease in the subject. In various embodiments, an increase in the level of mitochondrial protein acetylation in the motor neurons of the subject after administration of the agent or composition may indicate that the treatment is ineffective and / or that the disease in the subject is worsening. Thus, in various embodiments, the method may be a method of determining the effectiveness of the treatment or a method of prognosis. It will be understood that the method may also be useful for determining / monitoring the progression of disease in a subject by comparing the mitochondrial protein acetylation levels in samples from the subject at different time points. Here, a decrease in the mitochondrial protein acetylation level at a later time point compared to an earlier time point indicates that the disease is improving, while an increase in the mitochondrial protein acetylation level at a later time point compared to an earlier time point indicates that the disease is worsening.

[0113] In various embodiments, a method for identifying / diagnosing ALS or an ALS-like disease in a subject is provided, comprising determining / measuring the level of mitochondrial protein acetylation in the subject's motor neurons and comparing it with the level of acetylation in healthy / control motor neurons, indicating that the subject has or is susceptible to ALS or an ALS-like disease if the level of mitochondrial protein acetylation in the subject's motor neurons is elevated compared to the level in healthy / control motor neurons.

[0114] In various embodiments, the subject comprises a mammalian subject. In various embodiments, the subject comprises a human subject. In various embodiments, the motor neuron comprises a mammalian motor neuron. In various embodiments, the motor neuron comprises a human motor neuron.

[0115] In various embodiments, (a) a method of identifying a molecule capable of binding to a mitochondrial protein deacetylase (e.g., SIRT3) and / or a mitochondrial protein acetyltransferase (e.g., GCN5L1), comprising contacting the deacetylase and / or acetyltransferase with a candidate molecule and determining whether the candidate molecule binds to the deacetylase and / or acetyltransferase; (b) a method for identifying a modulator of a mitochondrial protein deacetylase (e.g., SIRT3) and / or a mitochondrial protein acetyltransferase (e.g., GCN5L1), comprising contacting a cell (e.g., a motor neuron) with a candidate molecule and detecting an increase or decrease in expression, amount, or activity of the deacetylase and / or acetyltransferase in or on the cell; (c) a method for identifying a molecule suitable for the treatment, prevention, or amelioration of ALS or an ALS-like disease, comprising determining whether the candidate molecule is an agonist or antagonist of a mitochondrial protein deacetylase (e.g., SIRT3) and / or a mitochondrial protein acetyltransferase (e.g., GCN5L1), preferably by exposing the candidate molecule to a deacetylase and / or acetyltransferase or a cell expressing the deacetylase and / or acetyltransferase (e.g., a motor neuron) to determine whether the candidate molecule is an agonist or antagonist thereof; or (d) A method for identifying an agonist or antagonist of a mitochondrial protein deacetylase (e.g., SIRT3) and / or a mitochondrial protein acetyltransferase (e.g., GCN5L1), comprising administering a candidate molecule to an animal and determining whether the animal exhibits increased or decreased expression, amount, or activity of the deacetylase and / or acetyltransferase; Optionally, methods are provided which include isolating or synthesizing said molecule, said modulator, said agonist, or said antagonist.

[0116] In some embodiments, the molecules, regulators, agonists, or antagonists thus identified include deacetylase activators, e.g., SIRT3 activators, and / or acetyltransferase inhibitors, e.g., GCN5L1 inhibitors. In some embodiments, the molecules, regulators, agonists, or antagonists thus identified have a similar affinity to SEQ ID NO:1 by at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about The oligonucleotides share 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, or a sequence that differs from SEQ ID NO: 1 by about 1 nucleotide / base, about 2 nucleotides / base, about 3 nucleotides / base, about 4 nucleotides / base, about 5 nucleotides / base, about 6 nucleotides / base, about 7 nucleotides / base, about 8 nucleotides / base, about 9 nucleotides / base, or about 10 nucleotides / base. In some embodiments, the molecules, modulators, agonists, or antagonists thus identified include oligonucleotides that share at least about 75% sequence identity with SEQ ID NO: 1, or a sequence that differs from SEQ ID NO: 1 by about 1 nucleotide, about 2 nucleotides, about 3 nucleotides, about 4 nucleotides, or about 5 nucleotides. In some embodiments, oligonucleotides for use in therapy are provided.

[0117] In various embodiments, a method for screening / identifying an agent or composition for treating ALS or ALS-like disease is provided, comprising contacting a cell, e.g., an ALS or ALS-like motor neuron, with a candidate agent or composition, and determining whether mitochondrial protein acetylation is reduced in the cell, e.g., motor neuron, after contacting, and concluding that the candidate agent or composition is an agent or composition for treating ALS or ALS-like disease if mitochondrial protein acetylation is reduced in the cell, e.g., motor neuron, after contacting, and concluding that the candidate agent or composition is not an agent or composition for treating ALS or ALS-like disease if mitochondrial protein acetylation is not reduced in the cell, e.g., motor neuron, after contacting. Protein acetylation can be measured by methods well known to those skilled in the art. For example, protein acetylation can be measured by detecting lysine acetylation through the use of an antibody specific for acetyl-lysine residues. Measurement / determination can be qualitative, quantitative, or semi-quantitative. Suitable methods for detecting lysine acetylation status include Western blot analysis, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA) mass spectrometry, and combinations of these methods. In one embodiment, determining whether mitochondrial protein acetylation is reduced in the motor neuron after contacting includes detecting acetylation at lysine-68.

[0118] In various embodiments, the screening / identification method involves determining whether the level of expression (e.g., gene and / or protein expression) of SIRT3 (or any complex comprising SIRT3) is increased; whether the level of RNA encoding SIRT3 is increased; whether transcription of nucleic acid encoding SIRT3 is increased; whether proper post-transcriptional processing (e.g., splicing, translation, post-translational processing) of RNA encoding SIRT3 is promoted; whether the level of SIRT3 protein (or any protein complex comprising SIRT3) is increased; whether degradation of SIRT3 (or any protein complex comprising SIRT3) is reduced; whether interaction between SIRT3 (or any protein complex comprising SIRT3) and an interacting partner of SIRT3 (or an interacting partner of any protein complex comprising SIRT3) is promoted; determining whether the level of function of SIRT3 (or any protein complex containing SIRT3) is increased / enhanced, whether SIRT3 (or any protein complex containing SIRT3) is activated, whether the level of deacetylase activity by SIRT3 (and / or any protein complex containing SIRT3) is increased, whether hyperacetylation of mitochondrial proteins due to decreased SIRT3 activity is reduced / inhibited, and / or whether deacetylation of downstream targets of SIRT3 is promoted, and / or determining whether the candidate agent binds to SIRT3 (or a complex thereof) after the contacting, and if one or more of the above are achieved after the contacting, concluding that the candidate agent is an agent for treating ALS or an ALS-like disease, and if one or more of the above are not achieved after the contacting, concluding that the candidate agent is not an agent for treating ALS or an ALS-like disease.

[0119] In various embodiments, the screening / identification method involves determining whether the level of expression (e.g., gene and / or protein expression) of GCN5L1 (or any complex comprising GCN5L1) is reduced, whether the level of RNA encoding GCN5L1 is reduced, whether transcription of nucleic acid encoding GCN5L1 is reduced / inhibited, whether post-transcriptional processing (e.g., splicing, translation, post-translational processing) of RNA encoding GCN5L1 is reduced / inhibited, whether the level of GCN5L1 protein (or any protein complex comprising GCN5L1) is reduced / inhibited, whether degradation of GCN5L1 (or any protein complex comprising GCN5L1) is increased / promoted, whether the interaction between GCN5L1 (or any protein complex comprising GCN5L1) and an interacting partner of GCN5L1 (or an interacting partner of any protein complex comprising GCN5L1) is reduced / inhibited / disrupted, whether the level of GCN5L1 protein (or any protein complex comprising GCN5L1) is reduced / inhibited ... determining whether the level of function of GCN5L1 (or any protein complex comprising GCN5L1) is reduced, whether acetyltransferase activity of GCN5L1 (or any protein complex comprising GCN5L1) is inhibited, whether the level of acetyltransferase activity by GCN5L1 (or any protein complex comprising GCN5L1) is reduced, whether hyperacetylation of mitochondrial proteins due to increased GCN5L1 activity is reduced / inhibited, and / or whether acetylation of downstream targets of GCN5L1 is reduced / inhibited; and / or determining whether the candidate agent binds to GCN5L1 (or a complex thereof) after contacting, and if one or more of the above is achieved after contacting, concluding that the candidate agent is an agent for treating ALS or an ALS-like disease, and if one or more of the above is not achieved after contacting, concluding that the candidate agent is not an agent for treating ALS or an ALS-like disease.

[0120] In various embodiments, the screening / identification method comprises determining whether a candidate agent induces or inhibits the expression of any of the following characteristics in a cell, e.g., an ALS or ALS-like motor neuron, after contact: mitochondrial dysfunction, metabolic respiratory abnormalities, morphological changes, abnormal mitochondrial morphology, survival / basal survival, neuronal cell body size, amount of primary neurites, endoplasmic reticulum (ER) stress, basal respiration, ATP-related oxygen consumption rate (OCR), ATP production, spare respiratory capacity, oxidative phosphorylation, expression and / or activity of deacetylases (e.g., sirtuins such as SIRT3), activity of complex I, mitochondrial NAD + The method may include, or may further include, determining whether the candidate agent can improve one or more of the following characteristics, for example, to be similar or more similar to healthy / normal motor neurons: level, expression of CHOP gene, amount / level of spliced ​​XBP1 (sXBP1), extracellular acidification rate (ECAR), and mitophagy, and concluding that the candidate agent is an agent for treating ALS or an ALS-like disease if the candidate agent can improve one or more of the above characteristics, and concluding that the candidate agent is not an agent for treating ALS or an ALS-like disease if the candidate agent cannot improve one or more of the above characteristics.

[0121] The determination of the above characteristics can be carried out by methods known to those of skill in the art or described herein, and further, the determination can be qualitative, quantitative, or semi-quantitative. For example, in some embodiments, determining whether a characteristic has been changed / modulated (e.g., reduced / increased / promoted / improved, etc.) may refer to whether the characteristic has been altered / modulated to a certain extent, e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100%, or at least about 0.1 fold, at least about 0.2 fold, at least about 0.3 fold, at least about 0.4 fold, at least about 0.5 fold, at least about 0.6 fold, at least about 0.7 fold, at least about 0.8 fold, at least about 0.9 fold, or more than about 1 fold, e.g., at least about At least about 1.05 times, at least about 1.1 times, at least about 1.15 times, at least about 1.2 times, at least about 1.25 times, at least about 1.3 times, at least about 1.35 times, at least about 1.4 times, at least about 1.45 times, at least about 1.5 times, at least about 1.55 times, at least about 1.6 times, at least about 1.65 times, at least about 1.7 times, at least about 1.75 times, at least about 1.8 times, at least about 1.85 times, at least about 1. 9 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 4.5 fold, at least about 5 fold, at least about 5.5 fold, at least about 6 fold, at least about 6.5 fold, at least about 7 fold, at least about 7.5 fold, at least about 8 fold, at least about 8.5 fold, at least about 9 fold, at least about 9.5 fold, or at least about 10 fold changed / modulated.

[0122] In various embodiments, the amino acid sequence of SEQ ID NO:1 is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121%, at least about 122%, at least about 123%, at least about 124%, at least about 125%, at least about 126%, at least about 127%, at least about 128%, at least about 129%, at least about 130%, at least about 131%, at least about 132%, at least about 133%, at least about 134%, at least about 1 Provided are oligonucleotides that share at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or sequences that differ from SEQ ID NO: 1 by about 1 nucleotide / base, about 2 nucleotides / base, about 3 nucleotides / base, about 4 nucleotides / base, about 5 nucleotides / base, about 6 nucleotides / base, about 7 nucleotides / base, about 8 nucleotides / base, about 9 nucleotides / base, or about 10 nucleotides / base. In some embodiments, provided are oligonucleotides that share at least about 75% sequence identity with SEQ ID NO: 1, or sequences that differ from SEQ ID NO: 1 by about 1 nucleotide, about 2 nucleotides, about 3 nucleotides, about 4 nucleotides, or about 5 nucleotides. In some embodiments, oligonucleotides for use in therapy are provided.

[0123] In various embodiments, a method is provided for generating a heterozygous L144F mutation in the SOD1 gene in a healthy human iPSC line, thereby generating a pair of isogenic iPSCs. In various embodiments, a method is provided for generating a heterozygous G298S mutation in the TDP43 gene in a healthy human iPSC line, thereby generating a pair of isogenic iPSCs. In various embodiments, a method is provided for generating a SIRT3 haploinsufficient human / mouse iPSC model. In various embodiments, a motor neuron disease model, such as an ALS or ALS-like disease model, is provided, in which expression of SIRT3 is depleted and / or SIRT3 is haploinsufficient. In various embodiments, a method is provided for enriching motor neurons for metabolic flux measurements. In various embodiments, a method is provided for in vitro characterization of iPSC-derived neurons, in which low oxidative and high glycolytic metabolic profiles are indicative of ALS motor neurons. In various embodiments, a method is provided for activating SIRT3 in human motor neurons, thereby promoting healthy mitochondrial respiration and reducing the hyperglycolytic phenotype of ALS motor neurons. In various embodiments, methods are provided for depleting expression of GCN5L1 in human motor neurons, thereby promoting healthy mitochondrial respiration and reducing the hyperglycolytic phenotype of ALS motor neurons.

[0124] In various embodiments, methods, products, or uses are provided herein. EXAMPLES

[0125] Exemplary embodiments of the present disclosure will be better understood and readily apparent to those skilled in the art from the following discussion and, where applicable, in conjunction with the drawings. It should be understood that several modifications can be made without departing from the scope of the present invention. The exemplary embodiments are not necessarily mutually exclusive, as some may be combined with one or more embodiments to form new exemplary embodiments.

[0126] result MNs generated from ALS patient iPSCs and isogenic ALS knock-in iPSCs show reduced mitochondrial respiration and ATP production Using established protocols, we generated three healthy iPSC lines: BJ-iPS, 18a, and GM23720, three sporadic ALS lines: sALS1, sALS2, and sALS3, and a familial ALS iPSC with the following mutations: 29d (SOD1 L144F ), 49a(TDP43 G298S ), and 19f (C9ORF72 expanded GGGGCC repeat) (Figure 1A). Using this chemically defined protocol, ISL1 was isolated from all of these iPSC lines at day 28. + SMI32 + MN was effectively induced (Fig. 1B). To confirm the previously reported ALS phenotype in these cell lines, we used ISL1 + Basal survival of MNs was measured from days 25 to 35 and an accelerated death phenotype was found to be associated with ALS MNs (Figure 1C). As increased ER stress is also a molecular signature of ALS MNs, we also measured the mRNA levels of key genes in the ER stress pathway, finding significant upregulation of CHOP and spliced ​​XBP1 (sXBP1) (Figure 1D).

[0127] To confirm that the ALS-specific phenotypic and molecular profile is not due to inherent variability between cell lines, we used CRISPR / Cas9 technology to identify SOD1 L144F and TDP43 G298S An isogenic control was generated in which the mutation was introduced into a healthy BJ-iPSC line (Figure 1E; Figures 13A, 13B). The mutation was then confirmed by DNA sequencing (Figures 13A, 13B). Similar to what was observed in the patient iPSC line, the isogenic SOD1 L144F (BJ-SOD1 L144F ) and TDP43 G298S (BJ-TDP43 G298S) knock-in lines also showed reduced MN survival and increased expression of the ER stress genes CHOP and sXBP1 (Figures 1F, 1G).

[0128] Although ALS neurons have been shown to have abnormal mitochondria, it remains to be established whether these morphological abnormalities have any impact on mitochondrial function in ALS MNs. As oxidative phosphorylation is essential for maintaining neuronal metabolism and survival, we investigated whether mitochondrial respiration might be impaired in ALS MNs. To enrich for MNs in iPSC-derived cultures, we performed magnetic sorting using a cocktail of PSA-NCAM and CD171 antibodies. Using this sorting strategy, we were able to selectively enrich for ISL1 without the use of AraC, which is sometimes used to deplete neural progenitor cells (NPCs) in culture but can also induce neuronal cell death due to oxidative stress. + MNs were enriched by approximately 60% (Fig. 14A, 14B). To investigate whether ALS MNs exhibit metabolic respiratory abnormalities, the oxygen consumption rate (OCR) of these sorted neurons was measured as a function of time using an extracellular flux analyzer. Both familial and sporadic ALS strains were found to have significantly reduced basal respiration, reduced ATP-related OCR and spare respiratory capacity compared to healthy MNs (Fig. 2A, 2B). Similarly, BJ-SOD1 L144F and BJ-TDP43 G298S Isogenic iPSC-derived MNs showed similar decreases in basal respiration (p<0.0001), ATP production (p<0.0001), and spare respiratory capacity (p<0.01) as 29d and 47a MNs, which also harbor heterozygous L144F and G298S mutations in SOD1 and TDP43, respectively (Figures 2A, 2B).

[0129] Since ATP production in diseased MNs is reduced by at least 25% in various ALS strains, it made sense that these neurons would have to rely on other energy sources to meet their metabolic demands. To investigate whether these neurons would switch to glycolysis to meet their energy demands, we measured the extracellular acidification rate (ECAR) and found that ALS MNs exhibited increased glycolysis and glycolytic capacity compared to healthy MNs (Figures 2C, 2D), demonstrating a shift in cellular metabolism. Similarly, BJ-SOD1 L144F and BJ-TDP43 G298S MNs recapitulated this shift in cellular metabolism from oxidative phosphorylation to glycolysis compared to BJ-iPS MNs, revealing that this is not due to cell line variability but is an ALS-specific metabolic feature (Fig. 2C, 2D). To assess the specificity of these metabolic changes in MNs, we measured the OCR of day 10 NPCs and found that ALS NPCs did not show significant changes in basal respiration and ATP-related OCR. However, the reduced spare respiratory capacity was reduced by approximately half in ALS NPCs (Fig. 14C, 14D). Moreover, the glycolytic profile of day 10 NPCs does not show significant changes between healthy and ALS cells (Fig. 14E, 14F).

[0130] Other metabolically active cell types derived from ALS iPSCs do not show an obvious decrease in mitochondrial respiration. We next asked whether the ALS-causing mutation affects all actively respiring cell types or specifically MNs. To this end, we transformed all iPSCs into BRN2 cells using a 28-day protocol. + SATB2 + These neurons were then replated in 96-well plates for metabolic flux analysis. OCR measurements revealed that BJ-SOD1 cells were more potent than isogenic BJ-iPS cortical neurons. L144F and BJ-TDP43 G298SCortical neurons revealed no significant differences in basal respiration and ATP production (Figures 2E, 2F). Similarly, cortical neurons derived from familial and sporadic ALS iPSCs showed no significant differences in basal respiration and ATP production compared to the three healthy controls (Figures 2E, 2F). However, it should be noted that the spare respiratory capacity was reduced by more than half in all ALS cortical neurons, suggesting that these cells may be more sensitive to metabolic stress.

[0131] To investigate non-neuronal cell types, we used cTnT from all iPSCs. + Cardiomyocytes were also induced (Figure 15C) and metabolic flux analysis was performed. L144F and BJ-TDP43 G298S Although cardiomyocytes from ALS MNs were not significantly different from isogenic healthy controls in terms of basal respiration and ATP production, they also had reduced total respiratory capacity (Fig. 2G, H). Similarly, similar results were obtained for cardiomyocytes from familial and sporadic ALS patients (Fig. 2G, H). Collectively, these results reveal that reduced mitochondrial respiration and a concomitant increase in glycolysis are metabolic hallmarks of ALS MNs.

[0132] Metabolic abnormalities in ALS MN associated with hyperacetylation of mitochondrial proteins Having established that mitochondrial respiratory abnormalities are common and unique to both familial and sporadic ALS, understanding the mechanisms underlying these abnormalities was sought. Mitochondrial respiration is known to be fine-tuned by mitochondrial protein acetylation, a process controlled by SIRT3, a mitochondrial deacetylase, and GCN5L1, a proposed acetyltransferase that catalyzes the reverse reaction. Since depletion of SIRT3 has been shown to cause hyperacetylation of mitochondrial proteins in muscle, heart, and liver tissues, we investigated whether ALS-specific metabolic abnormalities could be observed as a result of SIRT3 loss. However, Western blot analysis did not reveal any significant changes in SIRT3 levels between healthy and ALS MNs or MNs derived from a set of isogenic iPSC lines (Figures 3A, 3B). It was speculated that SIRT3 activity may be affected even if expression levels were not altered. Therefore, to determine SIRT3 activity, we measured the relative acetylation of lysine 68 in MnSOD (MnSOD K68ac), one of the best-characterized SIRT3 targets. Indeed, Western blot analysis revealed that all cultures of ALS MNs, including sporadic ALS MNs and isogenic MNs, had much more MnSOD K68ac compared to healthy controls, suggesting reduced SIRT3 activity (Figure 3A, 3C). Human postmortem lumbar spinal cord sections were also analyzed by immunohistochemistry, confirming that lumbar αMNs from sporadic ALS patients had higher MnSOD K68ac signals compared to non-ALS controls (Figure 3D, 3E). As SIRT3 has multiple targets in mitochondria, it is expected that loss of SIRT3 activity would affect global mitochondrial acetylation. To confirm this, mitochondrial extracts were collected from all iPSC-derived MNs and immunoblotted for acetylated proteins using a specific antibody against acetyl-lysine.Western blot analysis revealed significantly higher intensities of acetylated proteins in all ALS lines compared to healthy controls, as did the MnSOD K68ac assay (Figure 3A, 3F). Interactome studies of SIRT3 revealed several complex I subunits among its downstream targets. Elaborating further, we found that reduced complex I activity was observed in all ALS MNs (Figure 3G), explaining the decreased basal mitochondrial respiration observed in these ALS MNs (Figure 2).

[0133] Loss of SIRT3 function leads to ALS-like phenotypes To assess the function of SIRT3 in mitochondrial bioenergetics control, we created a genetic model of SIRT3 depletion and performed RNA interference to examine the effects of SIRT3 depletion on MN survival and function. Using a CRISPR / Cas9 approach, we generated multiple isogenic SIRT3 haploinsufficiency (SIRT3 + / - ) iPSC lines were generated (Figure 13C). Of a total of 66 clones screened, 31 were heterozygous knockouts (47%), but none were completely knockouts, suggesting that SIRT3-deficient iPSCs are not viable. + / - Clones #6 and #17 were randomly selected and both clones were shown to differentiate into MNs with approximately the same efficiency as the isogenic control BJ-iPS (Figure 13D). + / - Both #6 and #17 were confirmed by Western blot to show a 50% reduction in SIRT3 protein. BJ-SIRT3 + / -MnSOD K68ac levels were also significantly higher in #6 and #17, suggesting that SIRT3 is important in controlling the acetylation status of mitochondrial proteins (Figures 4A, 4B). Furthermore, purified mitochondrial extracts also showed that clones #6 and #17 had more acetyl-lysine residues (Figure 4A), consistent with the hyperacetylated mitochondrial profile seen in ALS iPSC-derived MNs (Figure 3A). A series of in vitro ALS phenotypes have already been demonstrated, including accelerated MN death, increased ER stress signaling, reduced cell body, and a low oxidative / high glycolytic metabolic profile (Figure 2). Therefore, we sought to determine whether loss of SIRT3 could induce ALS phenotypes in vitro. Both SIRT3 + / - Measurement of ER stress transcripts in clonal day 28 MNs revealed that CHOP and sXBP1 mRNAs were significantly upregulated (Fig. 4C), similar to what was seen in all ALS MNs examined (Fig. 1D). Metabolic flux measurements revealed that both SIRT3 and SIRT4 were upregulated. + / - We confirmed that clonally derived MNs exhibited decreased mitochondrial respiration (Figures 4D, 4E) and increased glycolysis (Figures 4F, 4G). Phenotypically, both SIRT3 + / - Clone-derived motor neurons showed decreased viability (Figure 4H), and significantly reduced neuronal cell body size and primary neurites at 31 days (Figures 4I, 4J). + / - Given that MN displays an ALS-like phenotype, this suggests that partial loss of SIRT3 activity contributes to ALS pathogenesis.

[0134] Next, SIRT3 + / -To confirm previous findings in MNs, we performed small interfering RNA (siRNA)-mediated knockdown to transiently deplete SIRT3 levels in healthy BJ-iPS MNs at day 25. At day 28, 70% knockdown was confirmed at both the mRNA and protein levels, along with an increase in MnSOD K68ac, indicative of reduced SIRT3 activity (Figures 16A, 16B). Analysis of these neurons at day 28 also revealed reduced OCR parameters (Figure 16C). Collectively, these results suggest that SIRT3 is important in maintaining mitochondrial bioenergetics in MNs and that depletion of SIRT3 causes an ALS-like phenotype.

[0135] Activation of SIRT3 or inhibition of GCN5L1 ameliorates metabolic abnormalities in ALS MN SIRT3 is a NAD + Since SIRT3 is a mitochondrial NAD-dependent deacetylase, one possibility for decreased SIRT3 activity is the reduction of mitochondrial NAD in ALS MN. + To investigate this, we investigated the NAD levels in isolated mitochondria extracted from iPSC-derived MNs. + When measured, NAD levels were significantly lower in mitochondria from sporadic, familial, and isogenic ALS MN compared to healthy controls. + The levels were found to be reduced by 60-80% (Figure 5A). + The use of 0.5 mM nicotinamide (NAM), a precursor of mitochondrial NAD, reduced the mitochondrial NAD levels in ALS MN. +It was demonstrated that NAM significantly increased mitochondrial NAD+ levels (Fig. 5A) and promoted ALS MN survival (Fig. 5B). Supplementation with NAM elevated mitochondrial NAD+ levels in ALS MNs (Fig. 5A) and significantly improved basal mitochondrial respiration, ATP production, and spare respiratory capacity (Fig. 5C-E). It is worth pointing out that supplementation with NAM did not increase mitochondrial NAD+ levels in healthy MNs, nor did it significantly affect mitochondrial respiration, indicating that ALS MNs are a target for rescue. Furthermore, the addition of exogenous NAM to cell culture medium could promote healthier neuronal morphology similar to that of wild-type MNs (Fig. 5F, 5G).

[0136] We next asked whether overexpressing SIRT3 could improve the metabolic abnormalities of ALS MNs. We infected all iPSC-derived MNs on day 28 with either an inducible SIRT3-expressing lentivirus or a control GFP virus and collected them on day 31 for analysis. Using immunoblotting, we confirmed that SIRT3 was robustly overexpressed in both healthy and ALS iPSC-derived MNs, but the MnSOD K68ac signal was not reduced in ALS MNs overexpressing SIRT3 (Figure 16D). Of note, overexpressing SIRT3 significantly improved basal respiration, ATP production, and spare respiration in healthy MNs but had no significant effect in ALS MNs (Figure 16E-G), supporting previous results that SIRT3 activity, not expression, is rate-limiting in ALS MNs. Thus, NAM or other NAD + It is not surprising that simply overexpressing SIRT3 without added precursors does not significantly affect mitochondrial respiration in ALS MN.

[0137] Since GCN5L1 has been suggested to promote acetylation in mitochondria, we next investigated whether silencing GCN5L1 could improve the metabolic abnormalities and other associated phenotypes in ALS MNs. To this end, iPSC-derived MNs were transfected with siRNA targeting GCN5L1, achieving 60% knockdown (Fig. 16J). Metabolic flux analysis using the MitoStress kit revealed that basal respiration, ATP production, and spare respiration were significantly improved in GCN5L1 knockdown ALS MNs (Fig. 8). Furthermore, ECAR measurements demonstrated a decrease in basal acidification and glycolysis rates, suggesting that knockdown of GCN5L1 corrects the metabolic abnormalities characteristic of ALS MNs (Fig. 9). Knocking down GCN5L1 also reduced the ISL1 + MN survival and healthier neuronal morphology were promoted (FIG. 10; FIG. 16K).

[0138] Small molecule activators of SIRT3, but not riluzole or edaravone, improve mitochondrial bioenergetics in ALS MN. Currently, there are no known specific GCN5L1 inhibitors, but a few SIRT3 activators have been identified, including a specific SIRT3 agonist previously identified as 7-hydroxy-3-(4'-methoxyphenyl)coumarin or C12, which alters the conformation of the SIRT3 active site with high affinity and promotes deacetylation of downstream targets. To assess the specificity of C12 binding to SIRT3, we used a cellular thermal shift assay (CETSA) and confirmed that C12 promotes the thermostability of SIRT3 but not another abundantly expressed sirtuin, SIRT1 (Figure 6A). We then investigated the effect of C12 on the thermostability of SIRT3 in one of the ALS strains (BJ-SOD1 L144FA dose-response assay of C12 was performed by treating MNs derived from ALS MNs (Fig. 6B) with increasing doses of C12. Western blot analysis showed that SIRT3 and total MnSOD protein levels were unchanged, but MnSOD K68ac signal was dose-dependently decreased (Fig. 17A, 17B). It should also be noted that treatment with 10 μM C12 was cytotoxic (Fig. 17C, 17D), and subsequent experiments were performed with 5 μM C12. Treatment of a panel of ALS MNs with 5 μM C12 further demonstrated a significant decrease in mitochondrial acetyl-lysine signal in all ALS cultures (Fig. 6B), confirming that C12 promotes mitochondrial deacetylation.

[0139] Next, the effect of C12 was compared relative to FDA-approved ALS drugs, namely, riluzole and edaravone. Riluzole functions by blocking sodium channels, and edaravone acts as an ROS scavenger. However, it is currently unknown whether riluzole and edaravone regulate mitochondrial respiration. To this end, iPSC-derived MNs were treated with riluzole, edaravone, or DMSO, and water control on days 28-31. A repertoire of iPSC-derived MNs was treated with C12, riluzole, edaravone, or DMSO, and water control on days 28-31. First, all three compounds were found to promote ALS MN survival (Figure 6C). Similarly, all three compounds significantly reduced the expression of ER stress transcripts CHOP and sXBP1 in all ALS MNs (Figure 6D-F).

[0140] Metabolic flux analysis at day 31 revealed that C12 promoted mitochondrial respiration (Fig. 7A-C), decreased glycolysis (Fig. 17E-G), and increased mitochondrial complex I activity (Fig. 17H) in ALS MNs. Metabolic flux analysis revealed that treatment with riluzole and edaravone did not show any significant changes in the metabolic profile of ALS MNs (Fig. 7D-I), indicating that neither riluzole nor edaravone treatment improved mitochondrial bioenergetics. C12 treatment also improved neuronal morphology, resulting in an enlarged neuronal cell body size and an increased number of primary neurites (Fig. 7J, Fig. 18A-C), similar to healthy MNs. However, treatment with either riluzole or edaravone did not improve neuronal morphology (Fig. 7J, Fig. 18D-H). Taken together, these data suggest that activation of SIRT3 is effective in promoting motor neuron survival and maintaining mitochondrial bioenergetics in ALS MNs.

[0141] Of note, BJ-SIRT3 + / - Treatment of #6 and #17 with C12 did not promote survival or improve neuronal morphology (Figures 19A, 19B), and C12 treatment did not improve mitochondrial respiration in both SIRT3-deficient clones (Figure 19C), providing further evidence that C12 acts through promoting SIRT3 activity rather than through nonspecific off-target effects.

[0142] Finally, we wondered whether SIRT3 activation has a general neuroprotective effect or is specific to the rescue of ALS MNs. To investigate this, we decided to analyze the effect of C12 treatment on MNs derived from patients with spinal muscular atrophy (SMA). SMA is an autosomal recessive motor neuron disorder caused by mutations in both copies of the SMN1 gene, resulting in a dramatic reduction in full-length functional SMN protein. iPSCs from severe type I SMA patients (1-38G) were differentiated into MNs and MitoStress assays were performed on day 28. SMA MNs showed significantly reduced basal respiration and ATP production compared to healthy BJ MNs (Figures 20A, 20B). Subsequent treatment of these SMA MNs with C12 did not improve the metabolic abnormalities (Figures 20C, 20D), suggesting that SIRT3 activation is specific to the rescue of ALS MNs.

[0143] Consideration Although ALS is a heterogeneous motor neuron disease, all patients have similar clinical findings, suggesting the possibility of converging pathogenesis pathways independent of the various genetic mutations known to cause ALS. In this disclosure, we have identified metabolic signatures of both sporadic and familial ALS MN, characterized by low oxidation and high glycolysis. Increased production and release of lactate, the end product of glycolysis, leads to the upregulation of SOD1. G93A This explains the central nervous system acidosis seen in mice. Reduced cellular respiration was also observed in postmortem spinal cords of sporadic ALS patients. High glycolytic metabolism is likely a compensatory mechanism in ALS MN to overcome the lack of ATP generated through oxidative phosphorylation, because restoration of ATP production by NAM supplementation and SIRT3 activation also corrects the high glycolytic metabolic profile.

[0144] Although the functional relevance of other mitochondrial sirtuins in ALS pathogenesis remains to be elucidated, this disclosure demonstrates the importance of SIRT3 in controlling mitochondrial function in MN. SIRT3 deficiency has been observed in many neurodegenerative diseases as well as metabolic disorders. Loss of SIRT3 results in the downregulation of SOD1 G93A Mitochondrial fragmentation occurs in spinal motor neurons of mice, leading to neuronal death. One of the key findings was that reduced mitochondrial respiration and hyperacetylation of mitochondrial proteins are molecular hallmarks of ALS MN. iPSCs from familial and sporadic patients and SOD1 L144F and TDP43 G298S MNs derived from isogenic iPSC lines carrying the mutations show a consistent increase in acetylated mitochondrial proteins, including MnSOD K68ac, a well-characterized target of SIRT3. Postmortem analysis of ALS spinal cords further supported this finding. This molecular abnormality was accompanied by a resulting mitochondrial respiratory defect that was reversed by activation of SIRT3.

[0145] To confirm the finding that loss of SIRT3 activity is responsible for mitochondrial metabolic defects and a range of ALS-like phenotypes, in addition to using patient-derived iPSCs, isogenic cell lines were generated. - / - It should be noted that mice with complete Sirt3 knockout in iPSCs developed normally and were not embryonic lethal, but neuronal survival and function appeared to be affected, which may be due to species-specific differences. Nevertheless, these results confirmed that partial loss of SIRT3 was sufficient to cause the ALS phenotype in iPSC-derived MNs.

[0146] GCN5L1 has been shown to promote acetylation in mitochondria and shares a subset of mitochondrial targets with SIRT3. Although the mechanism by which GCN5L1 initiates acetylation remains largely unknown, knockdown of GCN5L1 has been shown to reduce mitochondrial protein acetylation and improve mitochondrial OCR. Similarly, knockdown of GCN5L1 in ALS MNs improves mitochondrial respiration and reduces glycolytic capacity, demonstrating that GCN5L1 plays a role in controlling mitochondrial respiration. Improvements in MN survival and neuronal morphology were also observed in ALS MNs treated with siRNA targeting GCN5L1, suggesting that GCN5L1 is a potential target for restoring metabolic abnormalities and slowing disease pathology in ALS MNs.

[0147] Apart from reversing metabolic abnormalities in ALS MNs, activation of SIRT3 and / or inhibition of GCN5L1 also rescued other in vitro ALS phenotypes, suggesting that early ALS neuronal defects are reversible and treatable and that the SIRT3-GCN5L1 axis is a critical upstream pathway controlling MN function and integrity. In support of this data, Sirt3 - / - Mouse cortical neurons have also been shown to be particularly vulnerable to excitatory, oxidative, and metabolic stress. These data support the notion that NAD may be a risk factor for ALS patients. + It has been shown that NAD levels are lower than in healthy subjects. + It has been confirmed that supplementation with NAM, a precursor of mammalian NAD, can reverse the ALS phenotype. Furthermore, knockout of intracellular nicotinamide phosphoribosyltransferase (iNAMPT) in the projection neurons of adult mice leads to ALS-like MN degeneration phenotypes. iNAMPT is a mammalian NAD + It functions as the rate-limiting enzyme in the biosynthetic salvage pathway. Depletion of iNAMPT in motor neurons leads to hyperacetylation of mitochondrial proteins, which is associated with increased NAD +It should be noted that these findings are most likely due to reduced SIRT3 activity caused by low levels of NAD. + We demonstrate that SIRT3 is important in regulating mitochondrial function and metabolic processes, possibly through activation of SIRT3.

[0148] Of interest in this field, two drugs approved by the FDA for the treatment of ALS, riluzole and edaravone, were also found to be ineffective in restoring the mitochondrial metabolic abnormalities characteristic of ALS MN. This could explain the drugs' marginal efficacy - riluzole was found to be effective in patients with bulbar ALS, but not in subjects with limb ALS, which account for the majority of ALS patients. Riluzole extended life expectancy by an average of three months, whereas edaravone was only effective in a small proportion of ALS patients.

[0149] In conclusion, our findings using patient-derived and isogenic iPSCs reveal that decreased mitochondrial respiration and increased glycolysis are metabolic hallmarks of ALS MN. We also establish that mitochondrial SIRT3 activation is a target for reversing disease phenotypes in both sporadic and familial ALS. Finally, these data confirm that NAM supplementation and small molecule SIRT3 agonists reverse some ALS phenotypes in vitro and have therapeutic potential for development into effective treatments.

[0150] method

[0151] [Table 1-1]

[0152] [Table 1-2]

[0153] [Table 1-3]

[0154] [Table 1-4]

[0155] [Table 1-5]

[0156] Culturing hiPSCs in iPS-Brew hPSC Medium All hiPSCs were cultured feeder-free on Matrigel-coated dishes using iPS-Brew XF according to the manufacturer's instructions. Routine passaging with ReLeSR (Stem Cell Technologies) was performed once every 6-7 days. The iPSC lines and their variants used in this study are listed in Table S1 below.

[0157] [Table 2]

[0158] Directed differentiation into motor neurons Pluripotent stem cells were differentiated into spinal motor neuron fates according to established protocols previously described (4). Briefly, human iPSCs were first neuronized by activating the Wnt pathway with CHIR99021 treatment (4.25 μM) while simultaneously blocking bone morphogenetic protein (BMP) signaling with LDN-193189 treatment (0.5 μM). On day 3, cultures were caudalized and ventralized with retinoic acid (1 μM) and purmorphamine (1 μM), respectively. Neurotrophic factors BDNF (10 ng / mL) and GDNF (10 ng / mL) were added to the neuronal cultures on day 17 to promote neuronal maturation into motor neurons. N2B27 medium (50% DMEM / F12, 50% Neuro medium, 1% L-Glutamax, 1% MEM non-essential amino acids supplemented with 1% N2 supplement and 2% B27 supplement) was used throughout motor neuron differentiation.

[0159] Directed differentiation into cortical neurons Pluripotent stem cells were differentiated towards a cortical neuronal fate according to an established protocol previously described (5) with slight modifications. First, human iPSCs were neuronized by SB431542 treatment (8 μM) to inhibit the TGF-β pathway while simultaneously blocking bone morphogenetic protein (BMP) signaling with LDN-193189 treatment (0.5 μM). On day 14, DAPT (2.5 μM) was added to further differentiate the cortical neural progenitors for another 7 days. Neurotrophic factors BDNF (10 ng / mL) and GDNF (10 ng / mL) were added to the neuronal cultures on day 21 to promote neuronal maturation into cortical neurons. Vitamin A-free N2B27 medium (50% DMEM / F12, 50% Neuro medium, 1% L-Glutamax, 1% MEM non-essential amino acids supplemented with 1% N2 supplement and 2% B27 supplement without vitamin A) was used throughout the differentiation of cortical neurons.

[0160] Directed differentiation into cardiomyocytes Pluripotent stem cells were differentiated towards a cardiomyocyte fate according to established protocols previously described (6). Briefly, human iPSCs were first induced towards mesoderm by treatment with CHIR99021 (12 μM) in insulin-free RPMI / B27 for 1 day to activate the Wnt pathway. To direct these mesodermal progenitor cells towards a cardiac fate, IWP2 (5 μM) was added to the cultures on day 3 for 2 days in insulin-free RPMI / B27. On day 7, cardiac mesoderm cells were cultured in insulin-containing RPMI / B27 to generate functional contracting cardiomyocytes.

[0161] Cas9-mediated knockout / knockin in BJ-iPS lines Guide RNAs (gRNAs) were designed using the guide design tool (crispr.mit.edu) from the Feng Zhang lab before the service was shut down. gRNAs were cloned into the Cas9-containing plasmid PX458 or PX459 and transfected into 293T cells using Lipofectamine 2000 transfection reagent for surveyor nuclease assays. Validated gRNAs and ssODNs (Table S2) were transfected into BJ-iPS hiPSCs using Lipofectamine Stem transfection reagent. Two days after transfection, cultures were sorted for GFP+ cells or selected with 1 μM puromycin. Single cells were then plated out and expanded before screening. gDNA of colonies was harvested and subjected to PCR amplification followed by Sanger sequencing (Applied Biosystems 3730xl).

[0162] [Table 3]

[0163] Motor neuron survival assay Motor neuron cultures were treated with AraC on day 23 and plated at 75,000 cells / well in 96-well plates on day 24. Cultures were then fixed with 4% PFA on days 25, 28, 31, and 35, respectively, to quantify motor neuron survival. Fixed cultures were stained with the motor neuron marker ISL1, and cell nuclei were counterstained with DAPI before being photographed under a high-content microscope Phenix (Perkin Elmer). The percentage of ISL1+ cells was normalized to the total number of nuclei. To calculate a normalized survival index, the percentage of ISL1+ / DAPI on day 25 was arbitrarily set as 1.0, and motor neuron survival on subsequent days was then normalized to the day 25 culture. Biological triplicates were performed, each containing a minimum of five technical replicates.

[0164] Neuron sorting with magnetic microbeads After dissociation with Accutase, the cells were blocked with a solution containing phosphate-buffered saline, 0.5% bovine serum albumin (BSA), and 2 mM EDTA. The cells were then incubated with CD171-APC antibody (Miltenyi Biotec) and PSA-NCAM-APC antibody (Miltenyi Biotec) for 10 min at 4°C. After washing, the cells were incubated with anti-APC microbeads for 15 min at 4°C. The cells were then washed twice and filtered before being loaded onto a separation column (LS column) attached to a magnetic stand (all from Miltenyi Biotec). After three rounds of washing, the column was removed from the magnetic stand and eluted into culture medium for replating the labeled cells.

[0165] Metabolic flux analysis using the Seahorse XFe96 analyzer Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using an XFe96 Seahorse Biosciences Extracellular Flux Analyzer (Agilent Technologies). Purified motor neurons or neural progenitor cells were plated at 125,000 neurons / well on Seahorse 96-well plates precoated with Matrigel 24 hours prior to the assay. 45 minutes prior to the assay, the culture medium was replaced with 175 μL of fresh Seahorse DMEM basal medium. The inlet of the Seahorse analyzer was filled with 1 μM oligomycin, 1 μM FCCP, or 0.5 μM each of rotenone and antimycin A for OCR. For ECAR, 10 mM glucose, 1 μM oligomycin, and 50 mM 2-DG were used according to the manufacturer's instructions. OCR and ECAR levels were recorded, normalized, and quantified according to the manufacturer's instructions. Biological triplicates were performed, each containing a minimum of five technical replicates.

[0166] Small molecule treatment in MN cultures Riluzole was reconstituted in DMSO and used at a final concentration of 5 μM based on previous studies (7), whereas edaravone was dissolved in water and used at a final concentration of 100 μM (8). NAM and C12 were reconstituted in water and DMSO, respectively, and diluted in culture medium to the desired concentrations of 0.5 mM (9) and 5 μM, respectively. Day 27 motor neurons were plated at 75,000 cells / well in 96-well plates. Treatment with each small molecule compound began on day 28 for a total of 3 days. Biological triplicates were performed, each containing a minimum of five technical replicates.

[0167] RNA interference in MN cultures Day 27 motor neuron cultures were dissociated with Accutase and seeded at 2 million cells / well in 6-well plates. On day 28, non-targeting siRNA or siRNA were individually complexed with Lipofectamine RNAiMAX according to the manufacturer's instructions and added to the motor neuron cultures. 10 pmol siRNA and 8 μL Lipofectamine RNAiMAX were used per well. Cells were harvested for RNA and protein analysis and fixed for immunostaining or metabolic flux analysis 3 days after siRNA transfection.

[0168] RNA extraction and RT-qPCR Cells were harvested in Trizol reagent for RNA extraction according to the manufacturer's instructions. Purified RNA was converted to cDNA using a High-Capacity cDNA Reverse Transcription Kit, and quantitative PCR (qPCR) was performed using PowerUp™ SYBR™ Green Master Mix (all from Applied Biosystems) on a QuantStudio 5 Real-Time PCR System. Gene expression was normalized to the expression of HPRT and ACTB unless otherwise stated. Primers used are listed in Table S3.

[0169] [Table 4]

[0170] SDS-PAGE and Western Blot Protein lysates were resolved on 12% SDS-PAGE gels or 4–20% precast gels in Tris-glycine-SDS buffer. Proteins were then transferred to nitrocellulose membranes and blocked with 5% milk in TBST buffer. Primary antibodies were diluted in 5% milk and incubated with the membranes overnight at 4 °C. The primary antibodies used are listed in Table S4. Membranes were washed three times with TBST buffer. Corresponding horseradish peroxidase secondary antibodies (Life Technologies) were then diluted 1:5000 in 5% milk and incubated for 90 min at room temperature. Blots were washed three times and then exposed to ECL for imaging.

[0171] [Table 5]

[0172] Cellular thermal shift assay (CETSA) CETSA was performed as previously described (3) and experiments were carried out with HEK293T cells. Briefly, 30 million cells were exposed to C12 or DMSO at a final concentration of 20 μM for 1 h in low-attachment plates. After incubation, cells were harvested, washed, pelleted, and resuspended in 1 mL of PBS. Equal volumes of cell suspension were dispensed into Eppendorf tubes. Cell suspensions were then heated (48-68 °C) and lysed using two cycles of freeze-thaw. The soluble fraction was isolated and analyzed by Western blot analysis as described above.

[0173] Isolation of mitochondria Mitochondria were isolated from motor neuron cultures using MACS technology. First, motor neuron cultures were Accutase-treated and washed twice with PBS before being resuspended in ice-cold lysis buffer. Cells were then homogenized with 15 strokes of a Dounce homogenizer (Pestle B). The homogenate was diluted with 1x isolation buffer based on the manufacturer's instructions. Anti-TOM22 was added to magnetically label mitochondria, followed by incubation at 4°C for 1 h. Cells were then washed twice and filtered before being loaded onto a separation column (LS column) attached to a magnetic stand (all from Miltenyi Biotec). After three rounds of washing, the column was removed from the magnetic stand and the labeled mitochondria were eluted into storage buffer for downstream applications.

[0174] Measurement of complex I activity Cell lysates were prepared using a Complex I Enzyme Activity Microplate Assay Kit (Abcam) according to the manufacturer's instructions. Complex I activity was recorded, normalized, and quantified based on the manufacturer's instructions. Biological triplicates were performed, each containing a minimum of three technical replicates.

[0175] NAD+ / NADH quantification Purified motor neurons were plated at 50,000 neurons / well in 96-well plates pre-coated with Matrigel 24 hours prior to the assay. Cultures were then prepared for measuring NAD+ / NADH using the NAD / NADH-Glo™ Assay (Promega) according to the manufacturer's instructions. Levels of NAD+ / NADH were recorded, normalized, and quantified according to the manufacturer's instructions. Biological triplicates were performed, each containing a minimum of three technical replicates.

[0176] Mitochondrial NAD + Quantification of Mitochondria were isolated (10 million cells) from motor neuron cultures using MACS technology (as described above). Mitochondrial NAD+ was prepared and quantified using the NAD / NADH-Glo™ Assay (Promega) according to the manufacturer's instructions. Briefly, isolated mitochondria were first diluted with a 2:1:1 ratio of PBS, bicarbonate-based buffer, and 1% DTAB. 0.4 N HCl was added to the mitochondrial suspension, followed by heating at 60°C for 15 minutes. After heating, 0.5 M Trizma base was added to the suspension, and mitochondrial NAD+ was determined according to the manufacturer's instructions (Promega). + Record your levels and NAD + Normalized and quantified against a standard curve. Biological triplicates were performed, each containing a minimum of three technical replicates.

[0177] Immunostaining of cultured cells Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 15 min, and blocked with a buffer containing 5% FBS and 1% BSA for 1 h at room temperature. Primary antibodies (Table S4) were diluted in blocking buffer and incubated overnight at 4 °C. Cells were washed three times with PBS. Respective secondary antibodies were diluted 1:1500 in blocking buffer and incubated for 90 min at room temperature in the dark. DAPI was used at 0.1 μg / mL to visualize cell nuclei.

[0178] Immunohistochemical study of tissue from ALS patients Lumbar spinal cord tissue sections were cut from blocks of paraffin-embedded ALS (n=4) and control tissue (n=4) obtained from the UCSD CNS biorepository. Tissue sections, 6 μm thick, were deparaffinized in histology-grade CitriSolv (2×15 min each) followed by a graded alcohol series (100, 90, 70, and 50% ethanol (vol / vol), 3 min each) and then washed in water (2×3 min). Endogenous peroxidase activity was then quenched with 0.6% hydrogen peroxide (vol / vol) in methanol for 15 min. After a 20 min permeabilization step with 1×PBS, 0.2% TritonX100, antigen retrieval was performed in a pressure cooker at 120°C for 20 min in a high pH solution (1% Tris-based). Sections were blocked with 2% fetal bovine serum (vol / vol) and incubated with MnSOD(K68ac) antibody (1:100) overnight at 4°C.

[0179] The next day, after equilibration to room temperature, the sections were washed three times with 1x PBS and then incubated with 150 μL of secondary antibody for 60 min at room temperature. Signals were detected via a color reaction using NovaRed for 1–3 min per section until the desired staining was achieved. Counterstaining was performed with hematoxylin for 10 s. Sections were dehydrated and then coverslips were added.

[0180] Image acquisition and image analysis Images were acquired using a high content microscope Phenix (Perkin Elmer) with a 20x air objective. Image analysis, including cell number and intensity measurements, was performed using a Columbus (Perkin Elmer).

[0181] For primary neurite analysis, neurites were determined from the neuronal cell body size based on SMI-32 staining. For neuronal cell body size analysis, cell nuclei were identified by DAPI and the cytoplasmic area around the nucleus was determined based on SMI-32 staining. The area of ​​neuronal cell body size was measured by image analysis software (ImageJ, NIH) based on the cytoplasmic area around the nucleus excluding neurites.

[0182] For patient tissues, all slides were scanned with a Hamamatsu Nanozoomer 2.0HT Slide Scanner at the UCSD Microscopy Core. Scanned slides were evaluated at 1x and 20x magnification using the viewing software NDP.view2. All neurons were evaluated in both anterior horn sections from a total of four non-consecutive tissue sections per patient to ensure no overlapping neurons. K68Ac expression patterns and intensity were determined for all neurons using Fiji. Color deconvolution was performed using "HDAB" as the defined vector. Neurons were measured and quantified using "Colour_2", which represents the VectorRed signal without background from the counterstain (Colour_1 is hematoxylin). Regions of interest were determined for each neuron and intensity was quantified using "Mean gray value". To convert intensity to optical density (OD), the formula used was: OD=log(maximum intensity / mean intensity) for 8-bit images. From the resulting OD, the average darkness of the image due to the DAB signal was quantified (thus representing the staining of MnSOD-K68ac).

[0183] statistical analysis At least three biological replicates were performed for each experiment. Measurements were obtained from different samples for each analysis. Statistical analysis comparing two groups was performed using a two-tailed unpaired Student's t-test. P values ​​less than 0.05 were considered significant. All results are presented as mean ± standard deviation unless otherwise stated.

[0184] Declaration of Human Ethics All tissues were collected with consent under IRB (Institutional Review Board) oversight in compliance with HIPAA (Health Insurance Portability and Accountability Act of 1996). Patients were given the option to donate their CNS tissue postmortem to the ALS biorepository and provided consent prior to death. All consent forms and other legal documentation are handled by clinical research support staff and kept anonymous to the basic research team. To maintain patient autonomy and anonymity, each patient is assigned separate clinical, CNS, and fibroblast identification numbers.

[0185] References 1. Boulting GL, Kiskinis E, Croft GF, Amoroso MW, Oakley DH, Wainger BJ, et al.A functionally characterized test set of human induced pluripotent stem cells.Nat Biotechnol.2011;29(3):279-86. 2. Kiskinis E, Sandoe J, Williams LA, Boulting GL, Moccia R, Wainger BJ, et al. Pathways disrupted in human ALS motor neurons identified through genetic correction of mutant SOD1.Cell Stem Cell.2014;14(6):781-95. 3. Jafari R, Almqvist H, Axelsson H, Ignatushchenko M, Lundback T, Nordlund P, et al.The cellular thermal shift assay for evaluating drug target interactions in cells.Nat Protoc.2014;9(9):2100-22. 4. Hor JH,Soh ES,Tan LY,Lim VJW,Santosa MM,Winanto,et al.Cell cycle inhibitors protect motor neurons in an organoid model of Spinal Muscular Atrophy.Cell Death Dis.2018;9(11):1100. 5. Muratore CR,Srikanth P,Callahan DG,Young-Pearse TL.Comparison and optimization of hiPSC forebrain cortical differentiation protocols.PLoS One.2014;9(8):e105807. 6. Lian X,Zhang J,Azarin SM,Zhu K,Hazeltine LB,Bao X,et al.Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt / beta-catenin signaling under fully defined conditions.Nat Protoc.2013;8(1):162-75. 7. Sperling S,Aung T,Martin S,Rohde V,Ninkovic M.Riluzole:a potential therapeutic intervention in human brain tumor stem-like cells.Oncotarget.2017;8(57):96697-709. 8. Lee BJ,Egi Y,van Leyen K,Lo EH,Arai K.Edaravone,a free radical scavenger,protects components of the neurovascular unit against oxidative stress in vitro.Brain Res.2010;1307:22-7. 9. Schondorf DC, Ivanyuk D, Baden P, Sanchez-Martinez A, De Cicco S, Yu C, et al. The NAD+ Precursor Nicotinamide Riboside Rescues Mitochondrial Defects and Neuronal Loss in iPSC and Fly Models of Parkinson’s Disease. Cell Rep. 2018;23(10):2976-88. 10. Ng SY, Soh BS, Rodriguez-Muela N, et al. Genome-wide RNA-Seq of Human Motor Neurons Implicates Selective ER Stress Activation in Spinal Muscular Atrophy. Cell Stem Cell. 2015;17(5):569-584. doi:10.1016 / j.stem.2015.08.003. 11. Rodriguez-Muela N, Litterman NK, Norabuena EM, et al. Single-Cell Analysis of SMN Reveals Its Broader Role in Neuromuscular Disease. Cell Rep. 2017;18(6):1484-1498. doi:10.1016 / j.celrep.2017.01.035. 12. Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676-682. Published 2012 Jun 28. doi:10.1038 / nmeth.2019.

[0186] Use Motor neurons are highly energetic cells. They rely primarily on oxidative phosphorylation to meet high metabolic demands, and any deviation from this norm can lead to neurological disorders. By using induced pluripotent stem cells (iPSCs) from healthy controls, familial ALS patients, and sporadic ALS patients (including isogenic pairs) differentiated into spinal MNs, cortical neurons, and cardiomyocytes, we demonstrated that alterations in energy metabolism precede motor neuron loss and identified a distinct putative mechanism linking metabolic changes to motor neuron death in the motor neuron disease ALS. We demonstrated that mitochondrial respiratory abnormalities are a common pathway involved in both sporadic and familial ALS.

[0187] We developed a method to enrich the above-mentioned MNs for metabolic flux measurements. In various examples, we identified reduced mitochondrial respiration / low oxidation and increased glycolysis / high glycolysis, which were not observed in NPCs, as metabolic indicators of ALS MNs by excluding neural progenitor cells (NPCs) and other non-neuronal cells that do not respond to differentiation in metabolic assays. In various examples, metabolic flux analysis shows motor neuron-specific mitochondrial respiration defects in ALS. In various examples, all forms of familial and sporadic ALS MNs tested showed similar metabolic profiles / mitochondrial respiration abnormalities, which were attributed to hyperacetylation of mitochondrial proteins.

[0188] In mitochondria, SIRT3 functions as a mitochondrial deacetylase to maintain mitochondrial function and integrity, while GCN5L1 is a mitochondrially abundant acetyltransferase that catalyzes reactions that antagonize SIRT3 or counteract the acetylation and respiratory effects of SIRT3. In various instances, mitochondrial hyperacetylation has been shown to be controlled by the deacetylase SIRT3 and GCN5L1, which promotes mitochondrial protein acetylation. In various instances, SIRT3 haploinsufficient motor neurons recapitulate ALS-like phenotypes. In various instances, activation of SIRT3, for example with nicotinamide or small molecule activators or inhibitors of GCN5L1, can reverse the abnormal metabolic profile in all ALS motor neurons and correct a range of ALS-associated phenotypes / morphologies. In some instances, activation of SIRT3 promotes ALS MN survival and neurite outgrowth by restoring healthy mitochondrial respiration, but does not rescue SMA. Correspondingly, in various instances, knockdown / depletion of GCN5L1 in ALS motor neurons also rescues ALS-associated phenotypes / morphologies and promotes motor neuron survival and neurite outgrowth by promoting healthy levels of mitochondrial respiration.

[0189] The present inventors have demonstrated that hyperacetylation of mitochondrial proteins is indicative of both sporadic and familial ALS, and have identified the SIRT3-GCN5L1 axis as a common pathogenesis node for both familial and sporadic ALS. Advantageously, the present disclosure reveals that targeting the SIRT3-GCN5L1 axis to regulate mitochondrial protein acetylation (e.g., by increasing SIRT3 activity and / or inhibiting GCN5L1) represents a promising therapeutic strategy for ALS.

[0190] Those skilled in the art will appreciate that other changes and / or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the present disclosure as broadly described. For example, in the description herein, features of different illustrative embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, subsumed, etc. among the different illustrative embodiments. The present embodiments are therefore considered in all respects to be illustrative and not restrictive.

Claims

1. 1. A GCN5L1 (GCN5 (general control of amino acid synthesis 5)-like 1) inhibitor for use in the treatment of amyotrophic lateral sclerosis (ALS) or ALS-like motor neuron survival and / or function, comprising an oligonucleotide: the oligonucleotide is selected from the group consisting of an antisense oligonucleotide (ASO), a gapmer, a small interfering RNA (siRNA), a small hairpin RNA (shRNA), and combinations thereof; The inhibitor, wherein said oligonucleotide comprises a sequence complementary to the coding sequence of the BLOC1S1 gene or a part thereof, or SEQ ID NO:3 or a part thereof, or SEQ ID NO:4 or a part thereof.

2. The GCN5L1 inhibitor of claim 1, wherein the oligonucleotide comprises the sequence of SEQ ID NO:

1.

3. 3. Use of the GCN5L1 inhibitor of claim 1 or 2 in the manufacture of a medicament for treating ALS or an ALS-like disease, wherein the GCN5L1 inhibitor comprises an oligonucleotide.

4. Use of an oligonucleotide comprising the sequence of SEQ ID NO:1 in the manufacture of a medicament for treating ALS or an ALS-like disease.

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