DISORDERS, INCLUDING SYMPTOMS THEREOF USING PRIDOPIDINE

FIELD OF THE INVENSION

[001] The subject invention provides a method for treating a subject afflicted with a disease or disorder associated with mitochondrial disfunction, comprising administering to the subject a composition comprising pridopidine or pharmaceutically acceptable salt thereof.

BACKGROUND OF THE INVENTION

[002] Mitochondria are double-membrane organelles that are found in most eukaryotic cells and that execute many metabolic functions including ATP synthesis through oxidative phosphorylation (OXPHOS). Mitochondria are also involved in synthesis of biomolecules, maintenance of calcium homeostasis, production of reactive oxygen species (ROS), and apoptosis activation. Mitochondria are structurally complex and highly dynamic motile organelles. Mitochondria undergo constant morphological changes by the process of continuous cycles of fusion and fission that determines their morphology and most mitochondrial functions.

[003] Given their central role in cellular homeostasis, mitochondrial dysfunction has been linked to many age-related disorders including mitochondrial diseases, cancers, metabolic diseases and diabetes, inflammatory conditions, neurodegenerative disorders, neuropathy, and neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington disease. Pridopidine (4-[3-(methylsulfonyl)phenyl]-l-propyl-piperidine) (formerly known as ACR16, Huntexil®, TV-7820), is in clinical development for treatment of HD and ALS. Pridopidine was shown to exert neuroprotective properties in animal and cellular models of neurodegenerative diseases, including models of HD, PD, ALS and AD (Francardo, Veronica, Michal Geva, Francesco Bez, Quentin Denis, Lilach Steiner, Michael R. Hayden, and M. Angela Cenci. 2019.“Pridopidine Induces Functional Neurorestoration Via the Sigma- 1 Receptor in a Mouse Model of Parkinson’s Disease.” Neurotherapeutics 16 (2): 465-79; Ryskamp, Daniel A., Lili Wu, Jun Wu, Dabin Kim, Gerhard Rammes, Michal Geva, Michael Hayden, and Ilya Bezprozvanny. 2019“Pridopidine Stabilizes Mushroom Spines in Mouse Models of Alzheimer’s Disease by Acting on the Sigma-1 Receptor.” Neurobiology of Disease, 124, 489-504 ; Ryskamp, Daniel, Jun Wu, Michal Geva, Rebecca Kusko, Iris Grossman, Michael Hayden, and Ilya Bezprozvanny. 2017.“The Sigma-1 Receptor Mediates the Beneficial Effects of Pridopidine in a Mouse Model of Huntington Disease.” Neurobiology of Disease 97 (Pt A): 46-59; Ionescu, Ariel, Tal Gradus, Topaz Altman, Roy Maimon, Noi Saraf Avraham, Michal Geva, Michael Hayden, and Eran Perlson. 2019. “Targeting the Sigma-1 Receptor via Pridopidine Ameliorates Central Features of ALS Pathology in a SOD1G93A Model.” Cell Death & Disease 10 (3): 210; Garcia-Miralles, Marta, Michal Geva, Jing Ying Tan, Nur Amirah Binte Mohammad Yusof, Yoonjeong Cha, Rebecca Kusko, Liang Juin Tan, et al. 2017.“Early Pridopidine Treatment Improves Behavioral and Transcriptional Deficits in YAC128 Huntington Disease Mice.” JCI Insight). Mitochondrial dysfunction has been demonstrated in the pathology of each of these neurodegenerative diseases.

SUMMARY OF THE INVENTION

[004] In the first aspect the invention provides a method for treating a disease, disorder or any symptom thereof which is associated with mitochondrial disfunction, in a subject in need thereof comprising administering to the subject an effective dose of a composition comprising pridopidine or pharmaceutically acceptable salt thereof, thereby treating the subject.

[005] In a further aspect the invention provides a composition comprising pridopidine or pharmaceutically acceptable salt thereof for use in a method for treating a disease, disorder or any symptom thereof which is associated with mitochondrial disfunction.

[006] In some embodiments, said disease, disorder or any symptom thereof which is associated with mitochondrial disfunction is a disease, disorder or any symptom associated with mitochondrial myopathy.

[007] In other embodiments, said mitochondrial myopathy is selected from MELAS syndrome, MERRF syndrome, Leigh Disease, Chronic Progressive External Ophthalmoplegia (C/PEO), Diabetes mellitus and deafness (MIDD or DAD, Keams-Sayre syndrome (KSS), Alpers Syndrome, Mitochondrial DNA depletion syndrome (MDS), Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), Neuropathy, ataxia and retinitis pigmentosa (NARP), Pearson syndrome, Lebers Hereditary Optic Neuropathy (LHON), Dominant Optic Atrophy (DO A), Pigmentary retinopathy, Wolfram Syndrome, Friedrich’s Ataxia (FRDA), Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) and any combinations thereof.

[008] In further embodiments, said disease, disorder or any symptom thereof which is associated with mitochondrial disfunction is a disease, disorder or any symptom associated with a lysosomal storage disease.

[009] In other embodiments, said lysosomal storage disease is selected from Glycogenosis Type II (Pompe Disease), Multiple Sulphatase Deficiency (MSD), Mucopolysaccharidoses (MPS), Mucolipidoses (ML) Types I— III, G(Ml)-Gangliosidosis, Fabry Disease, Farber Disease, Gaucher Disease, Niemann-Pick Disease, Mucolipidoses (ML) Type IV, Cystinosis, Neuronal Ceroid-Lipofuscinoses, and any combinations thereof.

[0010] In some embodiments, said disease, disorder or any symptom thereof which is associated with mitochondrial disfunction is a disease, disorder or any symptom associated with a neurodegenerative disease.

[0011] In some embodiments, said neurodegenerative disease is selected from Parkinson's disease, Huntington disease, amyotrophic lateral sclerosis, Alzheimer's disease, Frontotemporal Dementia (FTD), Charcot-Marie-Tooth Disease (CMT) and any combinations thereof.

[0012] In some embodiments, said disease, disorder or any symptom thereof which is associated with mitochondrial disfunction is a bipolar disorder.

[0013] In some embodiments, said pridopidine is in its neutral/base form. In some embodiments, said pridopidine is in a pharmaceutically acceptable salt form. In some further embodiments, said pridopidine is pridopidine hydrochloride.

[0014] In some embodiments, said composition comprising pridopidine is administered orally.

[0015] In other embodiments, said composition comprising pridopidine is administered in the form of an inhalable powder, an injectable, a liquid, a gel, a solid, a capsule, eye drops or a tablet.

[0016] In some embodiments, the composition comprising pridopidine is administered periodically (i.e. said pridopidine is administered at regular pre-determined intervals of time, such as on a daily, hourly, weekly, monthly periods, each optionally also defining the dose to be administered and the number of administrations per time period). In further embodiments, the composition comprising pridopidine is administered once daily, twice daily or three times a day. In further embodiments, the composition comprising pridopidine is administered less often than once daily. In some embodiments, the composition comprising pridopidine is administered in one dose, two doses or three doses per day.

[0017] In some embodiments, pridopidine is administered in a daily dose of between 1 mg/day-400 mg/day. In some embodiments, pridopidine is administered in a daily dose of between 1 mg/day-300 mg/day. In other embodiments, pridopidine is administered in a daily dose of between lmg/day-90 mg/day. In other embodiments, pridopidine is administered in a daily dose of between 20 mg/day-90 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 45 mg/day-90 mg/day. In other embodiments, pridopidine is administered in a daily dose of between 20 mg/day-50 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 1 mg/day- 10 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 10 mg/day -20 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 20 mg/day-30 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 30 mg/day-40 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 40 mg/day-50 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 50 mg/day-60 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 60 mg/day-70 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 70 mg/day- 80 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 80 mg/day-90 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 90 mg/day-100 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 100 mg/day-150 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 150 mg/day-200 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 200 mg/day-250 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 250 mg/day-300 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 300 mg/day-350 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 350 mg/day-400 mg/day

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:

[0019] Figure 1A-1G show pridopidine rescues aberrant mitochondria morphology and restores mitochondrial-ER contacts in Y128 (YAC128, HD) neurons. Figure 1A: Visual representation of mitochondria network in WT and Y128 (HD) cortical/striatal neurons stained with Mitotracker dye. Figure IB: Quantification from 1A- Number of mitochondria are similar between WT and Y128 (HD) cortical/striatal neurons, before and after pridopidine treatment (n=4). Figures 1C and ID: Y128 HD mitochondria show impaired morphology with increased percentage of circular mitochondria (1C) and decreased percentage of elongated mitochondria (ID). Pridopidine treatment (1 mM) corrected Y128 morphology to normal levels. (n=4). Figure IE: WT and Y128 neurons were stained with mitochondria marker (IE middle column) and ER marker (IE right column) and co-localization of ER and mitochondria was analyzed (IE left column) (n=~15 projections/condition). Figure IF: Quantification of IE shows Mito-ER contacts are reduced in Y128 neurons compared to WT. Pridopidine (1 mM) significantly increased the Mito-ER contacts. Figure 1G: Aspect ratio, the ratio between mitochondrial axes is indicative of mitochondrial health, (see methods, paragraph 86 and results, paragraph 103) is reduced in HD neurons. Pridopidine treatment rescued aspect ratio in Y128 neurons (1G). *p<0.05, ***p<0.001 by Kruskal Wallis test followed by Dunn multiple comparison test [0020] Figures 2A-2B show pridopidine improves the impaired mitochondrial dynamics in Y128 HD neurons. Figure 2A: MitoDSRed transfected mitochondria were tracked in spinning disk confocal for 12 minutes and the velocity and directional trafficking were quantified using kymographs (n=7-9 projections/condition). WT - upper image shows mitochondria movement in both retrograde and anterograde directions. Y128 - middle image shows that in HD there is no mitochondria movement in either direction compared to WT. Y 128 treated with 1 mM pridopidine - lower image shows that pridopidine treatment restored mitochondria movement both retrogradely and anterogradely, similar to WT. Figure 2B: quantification of Figure 2A, Directional analysis of transport shows that the percent of stationary mitochondria increases in Y128 neurons, while both retrograde and anterograde transport is reduced. Pridopidine treatment reduced the percent of stationary mitochondria to WT levels and increased the percentage of both retrograde and anterograde transport to WT levels. Figure 2C: Total velocity is decreased in Y128 neurons. Pridopidine treatment enhances total mitochondrial velocity. *p<0.05, $p<0.05 (vs anterograde Y128 basal) by 2-way ANOVA followed by Tukey’s multiple comparison test.

[0021] Figures 3A-3H show that pridopidine rescues the impaired mitochondrial respiration in HD cellular models. Figures 3A-3D: Oxygen consumption and ATP production were evaluated in WT and Y128 cortical/striatal neurons treated with 1 and 5 mM pridopidine for 24h, with both doses showing rescue of basal and maximal respiration, and increased ATP production (n=3). Figures 3E-3H: Oxygen consumption and ATP production were reduced in HD neural stem cells (NSCs). Pridopidine treatment at 1 mM for 24h rescued both basal and maximal respiration, as well as ATP production. *p<0.05, **p<0.01 by Kruskal Wallis test followed by Dunn multiple comparison test.

[0022] Figures 4A-4C show pridopidine treatment protects Y128 neurons and HD lymphoblasts against induced mitochondrial dysfunction. Cortical (Figure 4A-left and Figure 4B) and striatal (Figure 4A-right and Figure 4C) WT and Y128 neurons treated with 0.1 or 1 mM pridopidine were evaluated for changes in mitochondrial membrane potential (MMP, DY _P1 ) after depolarization with oligomycin plus FCCP (Carbonyl cyanide- 4-(trifluoromethoxy)phenylhydrazone, an oxidative phosphorylation uncoupler) (n=7-10). Pridopidine (0.1 and lpM) increased MMP, which is reduced in Y128 neurons, back to levels comparable to WT in both cortical and striatal neurons. Figures 4D-4E: Oxidative stress was induced in Y 128 cortical/striatal co-cultures using H2O2, resulting in a significant decrease in MMP (Figure 4E) and reduced cell viability (Figure 4F). Pridopidine treatment (5 pM) rescued MMP (Figure 4D) and increased cell survival (Figure 4E), Figure 4F: Human lymphoblasts from HD patients or healthy controls were pre-treated with 1, 5 or 10 pM pridopidine and/or H2O2. All doses of pridopidine increased DY _hi , with the most significant effect achieved with 5 pM (n=4). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by Kruskal Wallis test followed by Dunn multiple comparison test.“Pri” refers to pridopidine.

[0023] Figures 5A-5E show that pridopidine reverts oxidative challenge-induced reactive oxygen species (ROS) production in Y128 neurons and HD NSCs and lymphoblasts. Figures 5A-5C: Figure 5A is a representative image of a cortical/striatal neuronal culture treated with MitoPY fluorescent probe for recording H2O2 levels. The mitochondrial complex III inhibitor Antimycin A (Ant A, 2 pM) was added, as indicated, to induce mitochondrial dysfunction (Left panel - before AntA treatment, middle panel- untreated neurons after AntA treatment, right panel - pridopidine-treated neurons after AntA treatment). Figure 5B: quantification of Figure 5A in cortical neurons. Figure 5C: quantification of Figure 5A in striatal neurons. Cortical and striatal neurons treated with 1 mM pridopidine show a reduction in mitochondrial H2O2 levels recorded by MitoPY fluorescent probe after AntA, as indicated (n=4, considering ~20 cells/condition for striatal neurons and ~10 cells/condition for cortical neurons). Scale bar = 30 mM. Figure 5D: Human NSCs were treated with the mitochondrial complex II inhibitor myxothiazol (Myxo, 3 pM) that also induces mitochondrial dysfunction and enhances mitochondrial H2O2 levels. Treatment with pridopidine (1 pM) for 24 h reduced mitochondrial H2O2 levels (n=4). Figure 5E: Control and HD lymphoblasts were challenged with H2O2 to induce mitochondrial dysfunction resulting in increased ROS levels (CellRox staining was used to determine ROS levels). Pre-treatment with 5 pM pridopidine for 24 hours reduced ROS levels significantly. (n=4). ^* p<0.05, ^** p<0.01, ^**** p<0.0001 by 2-way ANOVA followed by Tukey’s multiple comparison test. In Figure 5E, ^*** p<0.001, ^**** p<0.0001 by Kruskal Wallis test followed by Dunn multiple comparison test.

[0024] Figures 6A-6M show pridopidine treatment delays onset of motor deficits in Y128 mice, normalizes mitochondrial complexes activity and reduces H2O2 production in isolated Y128 striatal mitochondria. Figure 6A: Schematic representation of the in vivo experimental design: mice were treated with vehicle or pridopidine for 45 days rotarod behavioral test (RR) was measured before treatment on day 0 (1.5 months of age) and on day 44 (3 months of age); Figure 6B: At 1.5 months of age, before treatment there is no impairment in motor capacity in Y128 mice compared to WT. Figure 6C: At 3 months of age, significant impairment was observed in vehicle-treated Y128 mice. Pridopidine treatment significantly increased the latency to fall by Y128 mice compared to vehicle- treated controls in the rotarod motor test. Figures 6D-6H: Electron flow was evaluated in striatal mitochondria from vehicle- treated or pridopidine-treated wild-type and Y128 mice using Seahorse XF to measure oxygen consumption rate (OCR). Mitochondrial complexes inhibitors and substrates, 2 mM rotenone, 10 mM succinate, 4 pM antimycin A and 1 mM ascorbate/ lOOmM TMPD, were sequentially injected to calculate mitochondrial complex I, complex II, complex PI and complex IV activities, respectively. Complexes II, III and IV show increased OCR in Y128 mice, suggesting an early compensatory mechanism, and pridopidine rescues this effect. Figures 6I-6K: Antimycin A (2 pM) was used for inhibition of mitochondrial complex III. Mitochondrial levels of H2O2 are increased in mitochondria isolated from Y128 mice compared to WT mice. Pridopidine reduced Mitochondrial levels of H2O2 mice. XY lines show time-dependent changes in fluorescence after adding antimycin A. Figures 6L-6M: Calcium uptake is also improved in mitochondria from pridopidine-treated Y128 mice. Extracellular levels of calcium are reduced in response to pridopidine treatment (Figure 6L), which is evidence for increased mitochondrial calcium handling (Figure 6M) ^* p<0.05, ^** p<0.01 by non-parametric Kruskal -Wallis test.

[0025] Figures 7A-7B show pridopidine reduces mHtt-induced ER stress. H2a-GFP was transiently co-expressed with mutant Htt96Q-mCherry (HD exon 1) (Figure 7A) or Htt20Q-mCherry (WT) (Figure 7B) in STHdhQ7/7 cells. H2a-GFP aggregation is a marker of ER stress. Aggregation of mCherry and GFP were assessed and images acquired with a confocal microscope. Compared to untreated cells, Htt96Q enhances ER-stress and pridopidine treatment with 0.03 pM, 0.3 pM and 3 pM reduces ER stress in these cells (Figure 7A). Htt20Q (wt) does not induce ER stress (Figure 7B). For comparative purposes, 100% represents H2a-GFP relative intensity in untreated cells showing Htt96Q- mCherry aggregates, 0% is H2a-GFP relative intensity in untreated cells without Htt96Q- cherry aggregates. The graphs are averages of 3 experiments +-SE. *p<0.05 and **p<0.01 compared to untreated cells.

[0026] Figure 8 shows pridopidine reduces phosphorylated eIF2a (eIF2a-P) levels, a measure of ER stress, that are elevated in HD models. eIF2a-P levels were assayed in HEK293 cells transfected with mutant Htt (Htt96Q, solid line) or WT Htt (Htt20Q, dashed line) and treated with 0.3 and 3 mM pridopidine for 24 hours. The ratio of eIF2a -P to total eIF2a was quantified by immunoblot. Mutant Htt increased levels of phosphorylated eIF2a compared to WT, and both pridopidine concentrations reduced eIF2a-P levels. (*p<0.05)

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0027] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention.

[0028] In the first aspect the invention provides a method for treating a disease, disorder or any symptom thereof which is associated with mitochondrial disfunction, in a subject in need thereof comprising administering to the subject an effective dose of a composition comprising pridopidine or pharmaceutically acceptable salt thereof, thereby treating the subject.

[0029] In a further aspect the invention provides a composition comprising pridopidine or pharmaceutically acceptable salt thereof for use in a method for treating a disease, disorder or any symptom thereof which is associated with mitochondrial disfunction. [0030] When referring to a“ disease , disorder or any symptom thereof which is associated with mitochondrial disfunction’’ it should be understood to encompass any type of condition that risks the health of a subject wherein the impaired function of the mitochondria or any its parts plays a direct or indirect role.

[0031] In some embodiments, said disease, disorder or any symptom thereof which is associated with mitochondrial disfunction is a disease, disorder or any symptom associated with mitochondrial myopathy.

[0032] In other embodiments, said mitochondrial myopathy is selected from MELAS syndrome, MERRF syndrome, Leigh Disease, Alpers Syndrome, Chronic Progressive External Ophthalmoplegia (C/PEO), Diabetes mellitus and deafness (MIDD or DAD, Keams-Sayre syndrome (KSS), Mitochondrial DNA depletion syndrome (MDS), Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), Neuropathy, ataxia and retinitis pigmentosa (NARP), Pearson syndrome, Lebers Hereditary Optic Neuropathy (LHON), Dominant Optic Atrophy (DOA), Pigmentary retinopathy, Wolfram Syndrome, Friedrich’s Ataxia (FRDA), Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) and any combinations thereof.

[0033] When referring to “ Mitochondrial Myopathies'''’ it should be understood to encompass are any disease or disorder or any symptom caused by dysfunctional mitochondria. Mitochondrial diseases are sometimes (about 15% of the time) caused by mutations in the mitochondrial DNA that affect mitochondrial function. Other mitochondrial diseases are caused by mutations in genes of the nuclear DNA, whose gene products are imported into the mitochondria (mitochondrial proteins) as well as acquired mitochondrial conditions. Mitochondrial diseases take on unique characteristics both because of the way the diseases are often inherited and because mitochondria are so critical to cell function. The subclass of these diseases that have neuromuscular disease symptoms are often called a mitochondrial myopathy.

[0034] Mitochondrial myopathy diseases and disorders include, but are not limited to:

[0035] MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke). Progressive neurodegenerative disorder caused by mutations in mitochondrial DNA. Because the disease is poorly known and can be difficult to diagnose, it is not yet known how many individuals have developed MELAS throughout the world. The syndrome affects all ethnic groups and both males and females. Affected individuals usually begin showing symptoms between the ages of 4 and 40. The prognosis is poor; the disease often is fatal. There is no cure for MELAS syndrome; medical care is largely supportive. Symptoms: Because defective mitochondria exist in all the cells of patients with MELAS syndrome, many kinds of symptoms can develop, which are often debilitating. Strokes cause brain damage, leading to seizures, numbness, or partial paralysis. The encephalopathy (brain disease) causes tremors, muscle spasms, blindness, deafness, and may lead to dementia. Myopathy (muscle disease) causes difficulty walking, moving, eating, and speaking.

[0036] MERRF syndrome (or myoclonic epilepsy with ragged red fibers) Extremely rare disorder that begins in childhood and affects the nervous system and skeletal muscle as well as other body systems. The distinguishing feature in MERRF is myoclonus, consisting of sudden, brief, j erking spasms that can affect the arms and legs or the entire body. In addition, individuals with MERRF syndrome may have muscle weakness (myopathy), an impaired ability to coordinate movements (ataxia), seizures, and a slow deterioration of intellectual function (dementia). Short stature, degeneration of the optic nerve (optic atrophy), hearing loss, cardiomyopathy and abnormal sensation from nerve damage (peripheral neuropathy) are also common symptoms. Abnormal muscle cells are present and appear as ragged red fibers (RRF) when stained with the modified Gomori trichrome and viewed microscopically. MERRF is caused by mutations in mitochondrial DNA (mtDNA).

[0037] Leigh Disease prevalence at birth has been estimated at approximately 1 in 36 000. Typical onset of symptoms occurs before the age of 12 months but, in rare cases, the disease may manifest during adolescence or even early adulthood. Loss of motor milestones, hypotonia with poor head control, recurrent vomiting, and a movement disorder are common initial symptoms. Pyramidal and extrapyramidal signs, nystagmus, breathing disorders, ophthalmoplegia and peripheral neuropathy are often noted later. Epilepsy is relatively uncommon. There is no specific treatment for Leigh disease.

[0038] Chronic Progressive External Ophthalmoplegia (C/PEO) Characterized by slowly progressive paralysis of the extraocular muscles. Patients usually experience bilateral, symmetrical, progressive ptosis, followed by ophthalmoparesis months to years later. Ciliary and iris muscles are not involved. CPEO is the most frequent manifestation of mitochondrial myopathies. CPEO in association with mutations in mitochondrial DNA (mtDNA) may occur in the absence of any other clinical sign, but it is usually associated with skeletal muscle weakness. However, individuals with a similar clinical presentation may have various mitochondrial defects.

[0039] Diabetes mellitus and deafness (MIDD or DAD) MTDD represents 1% of people who have diabetes. Over 85% of people that carry the mutation in mitochondrial DNA at position 3243 present symptoms of diabetes. The average age at which people who have MIDD are typically diagnosed is 37 years old but has been seen to range anywhere between 11 years to 68 years old. Of these people with diabetes carrying the mitochondrial DNA mutation at position 3243, 75% experience sensorineural hearing loss. In these cases, hearing loss normally appears before the onset of diabetes and is marked by a decrease in perception of high tone frequencies. The associated hearing loss with diabetes is typically more common and more quickly declining in men than in women.

[0040] Kearns-Sayre syndrome (KSS) Onset: Before age 20. The prevalence of Keams- Sayre syndrome is approximately 1 to 3 per 100,000 individuals. Rare neuromuscular disorder. An important clinical symptomatic feature is the presence of a mono-or bilateral ptosis (partial closure of the eyelids). This disease is mostly characterized by three primary findings: progressive paralysis of certain eye muscles (chronic progressive external ophthalmoplegia [CPEO]); abnormal accumulation of colored (pigmented) material on the nerve-rich membrane lining the eyes (atypical retinitis pigmentosa), leading to chronic inflammation, progressive degeneration, and wearing-away of certain eye structures (pigmentary degeneration of the retina); and heart disease (cardiomyopathy) such as heart block. Other findings may include muscle weakness, short stature, hearing loss, and/or the loss of ability to coordinate voluntary movements (ataxia) due to problems affecting part of the brain (cerebellum). In some cases, KSS may be associated with other disorders and/or conditions.

[0041] Alpers Syndrome (Alpers-Huttenlocher Syndrome): Onset: Weeks to years after birth. Symptoms: psychomotor regression (dementia), seizures and liver disease. Severe, and continuous seizures lead to death within the first decade of life.

[0042] Mitochondrial DNA depletion syndrome (MDS) Onset: Infancy Symptoms: This disorder typically causes muscle weakness and/or liver failure, and more rarely, brain abnormalities.“Floppiness,” feeding difficulties and developmental delays are common symptoms; PEO and seizures are less common. [0043] Mitochondrial neuro-gastrointestinal encephalomyopathy (MNGIE) Onset: Usually before age 20. Symptoms: This disorder causes PEO, ptosis (droopy eyelids), limb weakness and gastrointestinal (digestive) problems, including chronic diarrhea and abdominal pain. Another common symptom is peripheral neuropathy (a malfunction of the nerves that can lead to sensory impairment and muscle weakness).

[0044] Neuropathy, ataxia and retinitis pigmentosa (NARP) Onset: Infancy to adulthood. Symptoms: NARP causes neuropathy (a malfunction of the nerves that can lead to sensory impairment and muscle weakness), ataxia and retinitis pigmentosa (degeneration of the retina in the eye, with resulting loss of vision). It also can cause developmental delay, seizures and dementia.

[0045] Pearson syndrome Onset: Infancy. Symptoms: This syndrome causes severe anemia and malfunction of the pancreas. Children who survive the disease usually go on to develop Keams-Sayre syndrome.

[0046] Lebers Hereditary Optic Neuropathy (LHON) Characterized by acute and painless central vision loss of both eyes in a sequential fashion over a period of days to months, LHON was the first maternally-inherited ophthalmologic disorder to be linked to a point mutation in mitochondrial DNA. LHON has a recognized disease prevalence estimated at 1 in 25,000 in England and other areas of Europe. Three mtDNA point mutations within mitochondrial respiratory chain complex I subunit genes (G11778A in ND4, G3460A in ND1, and T14484C in ND6) collectively cause 95% of LHON cases. Other pathogenic mtDNA mutations continue to be identified, particularly among non-Caucasian ethnic groups, such as the recently identified mtDNA T12338C mutation in ND5 that appears to be common in Han Chinese. [0047] Dominant Optic Atrophy (DOA) DO A is a genetic disease that primarily affects the retinal ganglion cells (RGC) and nerve fiber layer of the retina. The prevalence of DOA is estimated at 1 in 35,000 individuals in northern Europe. Visual acuity typically decreases over the first two decades of life to a mean of 20/80 to 20/120. Thinning of the neuroretinal rim appears to be a universal finding in DOA, with occasional findings including “saucerization” of the disc, a cup to disc ratio exceeding 0.5, and peripapillary atrophy. The early optic nerve appearance is often characterized by sectoral pallor of the optic nerve.

[0048] Pigmentary retinopathy and other ophthalmologic problems Pigmentary retinopathy is a non-specific finding that may be found in several mitochondrial diseases. The best described primary mtDNA disease in which pigmentary retinopathy may be seen is Neurogenic weakness, Ataxia, and Retinitis Pigmentosa (NARP), which results from a T8993C mtDNA mutation in the mitochondrial complex V subunit gene, ATPase 6.

[0049] Wolfram Syndrome an inherited condition that is typically associated with childhood-onset insulin-dependent diabetes mellitus and progressive optic atrophy. In addition, many people with Wolfram syndrome also develop diabetes insipidus and sensorineural hearing loss. An older name for the syndrome is DIDMOAD, which refers to diabetes insipidus, diabetes mellitus, optic atrophy, and deafness. Some people have mutations in the same gene that causes Wolfram syndrome but they do not get all the features of the syndrome, so they are said to have WFS1 -related disorders. The primary symptoms of Wolfram syndrome (diabetes mellitus, optic atrophy, diabetes insipidus and hearing loss) can emerge at different ages and change at different rates.

[0050] Friedrich’s Ataxia (FRDA) Genetic, progressive, neurodegenerative movement disorder, with atypical age of onset between 10 and 15 years. Initial symptoms may include unsteady posture, frequent falling, and progressive difficulty in walking due to impaired ability to coordinate voluntary movements (ataxia). Affected individuals often develop slurred speech (dysarthria), characteristic foot deformities, and an irregular curvature of the spine (scoliosis). FRDA is often associated with cardiomyopathy, a disease of cardiac muscle that may lead to heart failure or irregularities in heart rhythm (cardiac arrhythmias). About a third of the people with FRDA develop diabetes mellitus. The symptoms and clinical findings associated with FRDA result primarily from degenerative changes in the sensory nerve fibers at the point where they enter the spinal cord in structures known as dorsal root ganglia. This results in secondary degeneration of nerve fibers in the spinal cord which leads to a deficiency of sensory signals to the cerebellum, the part of the brain that helps to coordinate voluntary movements.

[0051] Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) Progressive metabolic disorder caused by thymidine phosphorylase (TP) enzyme deficiency. The lack of TP results in systemic accumulation of deoxyribonucleosides thymidine (dThd) and deoxyuridine (dUrd). In these patients, clinical features include mental regression, ophthalmoplegia, and fatal gastrointestinal complications. The accumulation of nucleosides also causes imbalances in mitochondrial DNA (mtDNA) deoxyribonucleoside triphosphates (dNTPs), which may play a direct or indirect role in the mtDNA depletion/deletion abnormalities, although the exact underlying mechanism remains unknown.

[0052] In further embodiments, said disease, disorder or any symptom thereof which is associated with mitochondrial disfunction is a disease, disorder or any symptom associated with a lysosomal storage disease.

[0053] In other embodiments, said lysosomal storage disease is selected from Glycogenosis Type II (Pompe Disease), Multiple Sulphatase Deficiency (MSD), Mucopolysaccharidoses (MPS), Mucolipidoses (ML) Types I— III, G(Ml)-Gangliosidosis, Fabry Disease, Farber Disease, Gaucher Disease, Niemann-Pick Disease, Mucolipidoses (ML) Type IV, Cystinosis, Neuronal Ceroid-Lipofuscinoses, and any combinations thereof.

[0054] When referring to“Lysosomal storage diseases (LSDs)” it should be understood to encompass any disease, disorder or symptom characterized by the progressive accumulation of undigested macromolecules in lysosomes. The massive accumulation of substances affects the function of lysosomes and impairs autophagic flux which may affect the cellular quality control of organelles such as mitochondria. LSDs exhibit signs of mitochondrial dysfunction, which include mitochondrial morphological changes, decreased mitochondrial membrane potential (DYih), diminished ATP production and increased generation of reactive oxygen species (ROS). Furthermore, reduced autophagic flux may lead to the persistence of dysfunctional mitochondria. Examples of lysosomal storage disease include, but are not limited to: Glycogenosis Type II (Pompe Disease), Multiple Sulphatase Deficiency (MSD), Mucopolysaccharidoses (MPS), Mucolipidoses (ML) Types I- III,G(Ml)-Gangliosidosis, Fabry Disease, Farber Disease, Gaucher Disease, Niemann-Pick Disease, Mucolipidoses (ML) Type IV, Cystinosis, Neuronal Ceroid-Lipofuscinoses

[0055] In some embodiments, said disease, disorder or any symptom thereof which is associated with mitochondrial disfunction is a disease, disorder or any symptom associated with a neurodegenerative disease.

[0056] Neurodegenerative diseases relate to any type of disabling disease, disorders or symptoms of the nervous system, characterized by the relative selective death of neuronal subtypes. Impaired mitochondrial function is key in the development of these diseases, such as impaired mitochondrial dynamics (shape, size, fission-fusion, distribution, movement etc.), abnormal mitochondrial membrane potential, oxygen consumption rate, ROS levels. [0057] In neurodegenerative diseases such as Parkinson's disease, Huntington disease, amyotrophic lateral sclerosis, Frontotemporal Dementia (FTD), Charcot-Marie-Tooth

Disease (CMT) and Alzheimer's disease mitochondrial function is impaired

[0058] In some embodiments, said neurodegenerative disease is selected from Parkinson's disease, Huntington disease, amyotrophic lateral sclerosis, Frontotemporal Dementia

(FTD), Charcot-Marie-Tooth Disease (CMT), Alzheimer's disease and any combinations thereof.

[0059] In some embodiments, said disease, disorder or any symptom thereof which is associated with mitochondrial disfunction is vanishing white matter (VWM) disease. Vanishing White Matter Disease (VWM) is one of more than 50 conditions that affect the white matter, or myelin, of the brain known collectively as Leukodystrophies. VWM, also known as Childhood Ataxia with Central Nervous System Hypomyelination (CACH), is an extremely rare neurological condition that destroys myelin, the brain’s white matter, or myelin. In doing so, it permanently affects transmission of brain signals to the rest of the body. Clinical conditions identified under VWM disease include but are not limited to: Childhood Ataxia with diffuse CNS Hypomyelination (CACH), Vanishing White Matter Leukodystrophy (VWM), Cree Leukoencephalopathy, Vanishing White Matter Leukodystrophy with Ovarian Failure, and any combinations thereof.

[0060] In further embodiments, the invention provides a method for treating a disease, disorder or any symptom thereof which is associated with mitochondrial disfunction, in a subject in need thereof comprising administering to the subject a composition comprising pridopidine or pharmaceutically acceptable salt thereof, wherein the mitochondrial disfunction is a bipolar disorder. [0061] Bipolar disorder a major mental disorder showing manic and depressive episodes, frequently accompanying psychotic symptoms. Mutations in mitochondrial DNA and mitochondrial dysfunction account for a subset of patients with the disorder.

[0062] In further embodiments, the invention provides a method for treating a disease, disorder or any symptom thereof which is associated with mitochondrial disfunction, in a subject in need thereof comprising administering to the subject a composition comprising pridopidine or pharmaceutically acceptable salt thereof, wherein the symptom of a disease or disorder associated with mitochondrial disfunction include any one or more of the following: poor growth, loss of muscle coordination, muscle weakness, neurological deficit, seizures, autism, autistic spectrum, autistic-like features, learning disabilities, heart disease, liver disease, kidney disease, gastrointestinal disorders, severe constipation, diabetes, increased risk of infection, thyroid dysfunction, adrenal dysfunction, autonomic dysfunction, confusion, disorientation, memory loss, poor growth, failure to thrive, poor coordination, sensory (vision, hearing) problems, reduced mental functions, disease of the organ, dementia, respiratory problems, hypoglycemia, apnea, lactic acidosis, seizures, swallowing difficulties, developmental delays, movement disorders (dystonia, muscle spasms, tremors, chorea), stroke, and brain atrophy.

[0063] In some embodiments, said pridopidine is in its neutral/base from. In other embodiments, said pridopidine is in a pharmaceutically acceptable salt form. In some embodiments, said pridopidine is pridopidine hydrochloride.

[0064] For the methods and use disclosed herein, the route of administration can be, e.g., oral. Routes of administration can also be classified by whether the effect is local (e.g., in topical administration) or systemic (e.g., in enteral or parenteral administration).“Local administration” as used herein shall mean administration of a compound or composition directly to where its action is desired, and specifically excludes systemic administration. “Topical administration” of a compound or composition as used herein shall mean application of the compound or composition to body surfaces such as the skin or mucous membranes such as eyes.“Ocular administration” as used herein shall mean application of a compound or composition to the eye of a subject or to the skin around the eye (periocular skin) or the mucosa around the eye, specifically the conjunctiva of a subject, i.e., local administration. The amount of pridopidine and the pharmaceutical compositions of the present invention may be administered by oral administration, topical administration, systemic administration, local administration, or ocular administration.

[0065] In some embodiments, said pridopidine is administered orally.

[0066] In further embodiments, said pridopidine is administered in the form of an inhalable powder, an injectable, a liquid, a gel, a solid, eye -drops, eye ointment, capsule or a tablet.

[0067] As used herein, “ pridopidine” means pridopidine base, a pharmaceutically acceptable salt thereof, derivative thereof, analogs thereof or combination of pridopidine and its analogs.

[0068] Example of pridopidine derivative is deuterium-enriched version of pridopidine and salts. Examples of deuterium-enriched pridopidine and salts and their methods of preparation may be found in U.S. Application Publication Nos. 2013-0197031, 2016- 0166559 and 2016-0095847, the entire content of each of which is hereby incorporated by reference.

[0069]“ Deuterium-enriched” means that the abundance of deuterium at any relevant site of the compound is more than the abundance of deuterium naturally occurring at that site in an amount of the compound. The naturally occurring distribution of deuterium is about 0.0156%. Thus, in a " deuterium-enriched’ compound, the abundance of deuterium at any of its relevant sites is more than 0.0156% and can range from more than 0.0156% to 100%. Deuterium-enriched compounds may be obtained by exchanging hydrogen with deuterium or synthesizing the compound with deuterium-enriched starting materials.

[0070] The invention also includes any salt of pridopidine, including any pharmaceutically acceptable salt, wherein pridopidine has a net charge (either positive or negative) and at least one counter ion (having a counter negative or positive charge) is added thereto to form said salt. The phrase "pharmaceutically acceptable salt(s)", as used herein, means those salts of compounds of the invention that are safe and effective for pharmaceutical use in mammals and that possess the desired biological activity. Pharmaceutically acceptable salts include salts of acidic or basic groups present in compounds of the invention. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p- toluenesulfonate and pamoate (i.e., l,T-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Certain compounds of the invention can form pharmaceutically acceptable salts with various amino acids. Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts. For a review on pharmaceutically acceptable salts see BERGE ET AL., 66 ./. PHARM. SCI. 1-19 (1977), which is incorporated herein by reference. In another embodiment the pridopidine salt of this invention is a hydrochloride salt. [0071] In some embodiments, the methods of this invention make use of a combination of pridopidine or pharmaceutically acceptable salt thereof with one or more of its analogs or pharmaceutically acceptable salt thereof.

[0072] In one embodiment, the analogs of pridopidine are represented by the following structures:

[0073] In other embodiments, the methods of this invention make use of a combination of pridopidine or pharmaceutically acceptable salt thereof and an analog of Compound (1) or pharmaceutically acceptable salt thereof.

[0074] In other embodiments, the methods of this invention make use of a combination of pridopidine or pharmaceutically acceptable salt thereof, an analog of Compound (1) and an analog of Compound (4) or pharmaceutically acceptable salt thereof.

[0075] The present invention thus also relates to pharmaceutical compositions comprising an agent of the subject invention in admixture with pharmaceutically acceptable auxiliaries, and optionally other therapeutic agents. The auxiliaries must be“ acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipients thereof.

[0076] Pharmaceutical compositions include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration or administration via an implant. The compositions may be prepared by any method well known in the art of pharmacy.

[0077] Pharmaceutical compositions include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration or administration via an implant. The compositions may be prepared by any method well known in the art of pharmacy.

[0078] Such methods include the step of bringing in association compounds used in the invention or combinations thereof with any auxiliary agent. The auxiliary agent(s), also named accessory ingredient(s), include those conventional in the art, such as carriers, fillers, binders, diluents, disintegrates, lubricants, colorants, flavoring agents, anti -oxidants, and wetting agents.

[0079] Pharmaceutical compositions suitable for oral administration may be presented as discrete dosage units such as pills, tablets, dragees or capsules, or as a powder or granules, or as a solution or suspension. The active ingredient may also be presented as a bolus or paste. The compositions can further be processed into a suppository or enema for rectal administration.

[0080] The invention further includes a pharmaceutical composition, as hereinbefore described, in combination with packaging material, including instructions for the use of the composition for a use as hereinbefore described.

[0081] For parenteral administration, suitable compositions include aqueous and non- aqueous sterile injection. The compositions may be presented in unit-dose or multi -dose containers, for example sealed vials and ampoules, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of sterile liquid carrier, for example water, prior to use. For transdermal administration, e.g. gels, patches or sprays can be contemplated. Compositions or formulations suitable for pulmonary administration e.g. by nasal inhalation include fine dusts or mists which may be generated by means of metered dose pressurized aerosols, nebulizers or insufflators.

[0082] The exact dose and regimen of administration of the composition will necessarily be dependent upon the therapeutic or nutritional effect to be achieved and may vary with the formula, the route of administration, and the age and condition of the individual subject to whom the composition is to be administered.

[0083] The term“ treatment’ as used herein refers to the administering of a therapeutic amount of the composition of the present invention which is effective to ameliorate undesired diseases, disorders, including symptoms associated with a diseases or disorders, to prevent the manifestation of such diseases, disorders, including symptoms associated with a diseases or disorders before they occur, to slow down the progression of the disease, slow down the deterioration of symptoms, to enhance the onset of remission period, slow down the irreversible damage caused in the progressive chronic stage of the disease, to delay the onset of said progressive stage, to lessen the severity or cure the disease, to improve survival rate or more rapid recovery, or to prevent the disease form occurring or a combination of two or more of the above. The "effective amount " for purposes disclosed herein is determined by such considerations as may be known in the art. The amount must be effective to achieve the desired therapeutic effect as described above, depending, inter alia, on the type and severity of the disease to be treated and the treatment regime. In some embodiment a composition comprising pridopidine or pharmaceutically acceptable salt thereof is between 1-400 mg, daily, twice daily, three times per day or less often than once a day. . As generally known, an effective amount depends on a variety of factors including the affinity of the ligand to the receptor, its distribution profile within the body, a variety of pharmacological parameters such as half-life in the body, on undesired side effects, if any, on factors such as age and gender, etc.

[0084] In some embodiments, pridopidine is administered in a daily dose of between 1 mg/day-400 mg/day. In some embodiments, pridopidine is administered in a daily dose of between lmg/day-300 mg/day. In other embodiments, pridopidine is administered in a daily dose of between lmg/day-90 mg/day In other embodiments, pridopidine is administered in a daily dose of between 20 mg/day-90 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 45 mg/day-90 mg/day. In other embodiments, pridopidine is administered in a daily dose of between 20 mg/day-50 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 1 mg/day-10 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 10 mg/day - 20 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 20 mg/day-30 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 30 mg/day -40 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 40 mg/day-50 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 50 mg/day-60 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 60 mg/day-70 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 70 mg/day-80 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 80 mg/day- 90 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 90 mg/day- 100 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 100 mg/day-150 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 150 mg/day -200 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 200 mg/day-250 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 250 mg/day-300 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 300 mg/day-350 mg/day. In further embodiments, pridopidine is administered in a daily dose of between 350 mg/day -400 mg/day

EXPERIMENTAL SECTION

Materials and Methods

[0085] Colonies of hemizygous YAC128 (Y128) [line HD53; mHTT high expresser] and WT mice, with FVB/N background, were housed under conditions of controlled temperature (22-23 °C) and under a 12-h light/12-h dark cycle. Food and water were available ad libitum. All mouse experiments were carried out in accordance with the guidelines of the Institutional Animal Care and Use of Committee and the European Community directive (2010/63/EU) and protocols approved by local committees for animal care. All efforts were made to minimize animal suffering and to reduce the number of animals used.

[0086] Primary neuronal cultures: Primary cortical, striatal and cortico-striatal co cultures were generated from the offspring of crosses between wild-type (WT) mice (used as controls) or hemizygous Y128 mice [line HD53] males and WT females from the same genetic background (FVB/N). Embryos from timed pregnant females were collected on day El 5.5-16.5 of gestation. For cortico-striatal co-cultures, cortex and striatum were micro- dissected, then diced and pooled for each genotype. The tissues were then dissociated and triturated. Cells were seeded on poly-D-lysine coated plates in enriched neurobasal medium. The cells were fed with 1/3 fresh medium every fifth day.

[0087] To obtain pure cortical and striatal neurons, tissue was micro-dissected and mechanically digested. Neurons were cultured in enriched Neurobasal medium and plated at a density of either 130 x 10 ³ cells/cm ² (high density) or 85 x 10 ³ (low density) on poly- D-lysine (0.1 mg/ml) coated plates. Cultures were maintained at 37°C in a 95/5% air/CCh incubator. At 3 days in vitro (DIV3), 5 mM 5-Fluoro-2'-deoxyuridine (5-FdU) was added to reduce dividing non-neuronal cells. Fresh medium was added at DIV7 and cells were used at DIV12.

[0088] Neuronal transfection: Striatal neurons were transfected with pDsRed2-Mito Vector at 8 DIV using the calcium phosphate precipitation method. The transfected neurons were then washed with neurobasal medium and transferred back to their original dishes containing conditioned culture medium until DIV12.

[0089] Lymphoblast cultures and transfection: Lymphoblasts from healthy controls (GM02174) and HD patients (NA04724) obtained from Coriell Institute were grown in supplemented RPMI medium. The lymphoblasts were passaged 1 :3 every 5-6 days.

[0090] Human neural stem cells culture: Neural stem cells (NSCs) were differentiated from heterozygous human induced pluripotent stem cells (iPSC), HD4-iPSC, with a normal (19 CAG repeats) and an expanded allele (72 CAG repeats), and control AMS4-iPSC. IPSC were maintain in Geltrex ^® (Thermo Fisher Sci., catalog no: A1413202) coated 6-well plates until they reached 90% confluence, at which point the neural induction protocol was applied. Neural differentiation was based on dual SMAD inhibition with SB431542 (Lefty/ Activin/transforming growth factor beta - TGFp inhibitor), dorsomorphin (bone morphogenetic protein - BMP inhibitor) and XAV-939 (b-catenin-transcription inhibitor and axin stabilizing agent). Neural induction occurred between day 0 and days 12-15. From day 0 to day 5, cells were maintained in iPSC medium without FGF2 and incubated with 5 mM dorsomorphin and 10 pM SB431542. Medium was changed every other day. From day 5 to day 12, the medium was gradually replaced by medium with increasing percentages of N2 medium. Between days 12 and 15, fields full of rosettes became morphologically visible. For differentiation, cells were replated in Geltrex ^® coated 12-well plates. Expression of the neural lineage marker proteins Nestin and SOX2 were confirmed by immunocytochemistry upon each differentiation process.

[0091] Pridopidine incubations: Pridopidine incubations were done for 24 h in all cellular models used, unless otherwise stated. Final concentrations are described in figures and figure legends. [0092] Mitochondrial network and ER co-localization: Cortico-striatal co-cultures were labelled with Mitotracker Deep Red FM dye for 30 min. The stained cells were washed prior to fixation with ice-cold methanol for 15 min at room temperature.

[0093] MitoDsRed-transfected striatal neurons were fixed with 4% paraformaldehyde, permeabilized and blocked before incubation with IP ₃ R3 antibody (1 : 1000, EMD Millipore, catalog no. AB9076). For nuclei detection, neurons were incubated with Hoechst 33342 and then mounted.

[0094] Confocal images were obtained as stacks, separated by 0.46 pm along the z axis, using a 63x lens on a Zeiss confocal microscope with LSM 710 software. FIJI software was used for image analysis. Z-stacks images were normalized for background, peak intensity regions denoting mitochondria-specific fluorescence were identified using Find Foci (Herbert, Alex D., Antony M. Carr, and Eva Hoffmann. 2014. “FindFoci: A Focus Detection Algorithm with Automated Parameter Training That Closely Matches Human Assignments, Reduces Human Inconsistencies and Increases Speed of Analysis.” Edited by Michael Lichten. PLoS ONE 9 (12)), and optimally resolved by filter application. Mitochondrial outlines were traced using Analyze Particles. Aspect ratio (the ratio between the major and minor axis of mitochondria) was used as an index of mitochondrial length. For IP ₃ R3 fluorescence, a threshold was set similarly to the one described above, and Integrated Density was calculated inside of mitochondrial regions of interest (ROIs) to obtain ER co-localization with mitochondria.

[0095] Mitochondrial movement analysis: MitoDsRed-transfected striatal neurons were washed and incubated in Na ⁺ medium, and mitochondrial movement studies were carried out at 37°C. Neuron projections were imaged every 5 seconds for a total of 145 frames, using a 63x objective on a spinning disc Zeiss inverted confocal. Mitochondrial movement analysis was done using the Kymograph Macro in Fiji. ROIs were designated using a segmented line following mitochondria trajectory across projections. Kymographs generated in a x-y dimension (distance us time) were used to obtain the slope to calculate mitochondrial velocity.

[0096] Seahorse oxygen respirometry: Oxygen consumption rate (OCR) in WT and hemizygous Y128 cortical/striatal co-cultures and NSCs was measured using Seahorse XFe-24/96 flux analyzers (Seahorse Bioscience). Cortical-striatal primary neurons were cultured in Seahorse XF96 V3 cell culture microplates at a density of 20,000 cells/well. NSCs were seeded 30,000 cells/well onto an XF24 cell culture microplate coated with Geltrex® and allowed to adhere for 24h at 37°C. Pridopidine (0.1, 1 and/or 5 mM) was added, when indicated in the graphs, 24 h before the experiment. The sensor cartridge plate was incubated with immersed sensors in a non-CCh incubator at 37°C for ~16 h. Three baseline measurements of OCR were sampled prior to sequential injection of mitochondrial complex V inhibitor oligomycin (1 mM), protonophore FCCP (Carbonyl cyanide-4- (trifluoromethoxy)phenylhydrazone, a oxidative phosphorylation uncoupler) (0.5 mM for neurons and 0.3 mM forNSCs) and antimycin A (0.5 mM for neurons and 1 mM for NSCs) plus rotenone (0.5 mM for neurons and 1 mM for NSCs) to completely inhibit mitochondrial respiration. Accordingly, mitochondrial basal respiration, maximal respiration and ATP production was automatically calculated and recorded by the Seahorse software. Data was normalized for protein levels.

[0097] Mitochondrial membrane potential: Mitochondrial membrane potential (MMP, Dy _ih ) was assessed in cortical and striatal neurons using the positively charged fluorescent probe tetramethylrhodamine methyl ester (TMRM ⁺ ) under quenched conditions and its accumulation in mitochondria was assessed after mitochondrial depolarization with oligomycin plus the mitochondrial respiration uncoupler FCCP. In cortical/striatal co cultures and lymphoblasts MMP was assessed using an equivalent probe, tetramethylrhodamine ethyl ester (TMRE ⁺ ) fluorescent probe, whose accumulation in mitochondria was directly evaluated by flow cytometry. Cortical and striatal neurons previously treated, when indicated, with pridopidine (0.1 and 1 mM; 24 h) were incubated with 150 nM TMRM (quenching conditions) in Na ⁺ medium for 30 min at 37°C. Under these conditions, retention of TMRM by mitochondria was studied to estimate changes in MMR/Dy. Basal fluorescence (503 nm excitation and 525 nm emission) was recorded for 4 min, followed by the addition of 2.5 mM FCCP + 2.5 pg/mL oligomycin to produce maximal mitochondrial depolarization and mitochondrial probe release. TMRM release was calculated based in the differences in fluorescence before and after addition of oligomycin/FCCP.

[0098] Primary neurons or lymphoblasts were cultured on 6-well plates. The cells were pretreated with/without pridopidine and hydrogen peroxide (H2O2) as per experimental condition followed by incubating with 25 nM TMRE methylester for 15 min at 37°C. After TMRE incubation, the cells were collected for FACS analysis.

[0099] Measurement of mitochondrial H2O2 levels: cortical and striatal neurons were pre-treated with pridopidine (0.1 and 1 pM) for 24 h and incubated with Mitochondria peroxy yellow 1 (MitoPYl) probe (8 pM) in Na ⁺ medium for 30 min at 37°C. MitoPY 1 was washed out and neurons were imaged in the same experimental medium every 1 min for 30 min using a 63x lens on a Zeiss inverted confocal spinning disc microscope with Zen Black 2012 software. Fluorescence was recorded by 503-nm excitation and enhanced emission at 528-nm (Dickinson, Bryan C, Vivian S Lin, and Christopher J Chang. 2013.“Preparation and Use of MitoPYl for Imaging Hydrogen Peroxide in Mitochondria of Live Cells.” Nature Protocols 8 (6)). After 10 min of basal reading, neurons were stimulated with antimycin A (2 mM). Specific MitoPY 1 fluorescence in mitochondria was confirmed by co incubating cells with MitoTracker Deep Red (300 nM). Fluorescence intensity at each time point was analyzed in FIJI using the time series analyzer plugin (v 3.0).

[00100] NSCs were plated 30,000/well in 96-well assay plates coated with Geltex® for 24 h at 37°C. Afterwards, NSC were incubated for another 24h with 1 mM pridopidine. Prior to acquisition, cells were washed and incubated for 20 minutes with 10 mM MitoPYl at 37°C and 5% CO2. Basal levels of MitoPYl fluorescence were measured for 10-15 minutes followed by exposure to myxothiazol (3 mM mitochondrial complex PI inhibitor) and measurement for an additional 30 minutes. The results were calculated as relative fluorescence units (RFU) per 30,000 cells. In isolated mitochondria, H2O2 levels were measured by resuspending 5 pg of isolated mitochondria in Amplex Red reagent with horseradish peroxidase (0.5 units/mL), and fluorescence measured at 570nm excitation and 585nm emission. After 10 min of basal reading, mitochondria were challenged with antimycin A (2 mM) and measured for an additional 10 minutes. Results were analyzed as time-dependent changes in fluorescence.

[00101] Reactive oxygen species (ROS) assay: Primary neurons and lymphoblasts attached to PDL-coated plates were treated with H2O2 (0-1 mM) for up to 6 hours. Cells were treated with 5 mM CellRox red reagent in complete medium followed by 30 min incubation. After washing, oxidative stress was measured by imaging on a Zeiss inverted microscope using a 40X objective using the same exposure settings for all samples. Eight random fields were sampled, and the fluorescence intensity was measured using ImageJ software. [00102] In vivo study design: 1.5 month-old WT and hemizygous Y128 mice (males and females in equal proportion) were divided into four groups. Mice received either pridopidine (30mg/kg at 100 pL/25g) or an equal volume of sterile water by oral gavage for 45 consecutive days, until the age of 4 months. Mice were housed 4 animals per cage enriched in corn-husk nesting material and paper rolls, each cage representing one individual experiment, for a total of 9 animals per group. Animals were weighed every week and the volume of treatment adjusted accordingly. Mice were behaviorally tested in rotarod immediately before treatment and on the day before the treatment ends. Tests were conducted blindly at a set time during the day. 24 h after the last gavage, mice were sacrificed, and mitochondria isolated from striatum.

[00103] Rotarod analysis: Motor learning and coordination and were assessed on a rotarod apparatus. In this test, mice must learn to run when placed on a constant rotating rod to prevent them from falling. Once the task is learned, the accelerating rotarod can be used to assess motor coordination and balance. Mice were allowed to acclimate to the behavior room for 2 h. Procedures were consistent for all subjects and tests made at minimum noise levels. Training consisted of four trails per day (120 s each), spaced by 1 hour, at a fixed speed of 14 rpm. The testing phase was carried out in the following day in an accelerated rotarod from 4 to 40 rpm over 5 minutes and consisted of 3 trials, spaced 2 hours apart. Rotarod scores are the average of 3 trials. Experiments were performed blinded for genotype and treatment. The motor coordination score was measured after training and the latency to fall was quantified in an accelerated rotarod from 5 to 40 rpm over 5 minutes.

[00104] Isolation of functional mitochondria: Striatum was dissected from mouse brains washed in mitochondria isolation buffer. Striatal mitochondria were isolated using discontinuous percoll density gradient centrifugation following homogenization. Protein content of isolated mitochondria was quantified by Bio-Rad assay.

[00105] Mitochondrial complexes activity: Complexes activity was evaluated by measuring oxygen consumption rate (OCR) using the Seahorse XF method (Agilent). 5 pg of isolated mitochondria diluted in mitochondrial assay solution were seeded in Poly(ethyleneimine)-coated XF24 seahorse plates in 450 pL, and the plate was allowed to equilibrate in a humidified C0 ₂ -free incubator at 37°C for 10-12 min. Sequential electron flow through the electron transport chain was evaluated by OCR measurement after sequential injection of rotenone (2 pM; complex I inhibitor), succinate (10 mM; complex P substrate), antimycin A (4 pM; complex PI inhibitor) and ascorbate/TMPD (10 mM/100 pM; electron donors to cytochrome C/complex IV).

[00106] Mitochondrial calcium handling: Calcium (Ca ²⁺ ) uptake by isolated mitochondria was measured using the Ca ²⁺ sensitive probe Calcium Green- 5N. Briefly, 5 pg mitochondria were incubated with 1 pM oligomycin and 150 nM Calcium Green-5N and fluorescence was measured in a spectrofluorometer microplate reader (excitation506 nm, emission 523 nm). After a baseline of 2 minutes, pulses of 10 pM CaC12 were added to mitochondria at 4 minute intervals. Mitochondrial Ca ²⁺ handling was calculated by the area under the curve after CaC12 pulses, which indicates the amount of extramitochondrial Ca ²⁺ taken up by mitochondria.

[00107] ER stress measurement: ER stress levels can be measured using H2a-GFP as protein indicator of early stages of ER stress. H2a-GFP is a misfolded secretory protein which in response to ER stress accumulates. The STHdhQ7/7 is a striatal derived cell line from a knock in transgenic mouse containing homozygous humanized Huntingtin gene (HTT) with 7 polyglutamine repeats (wild type). STHdhQ7/7 cells are transfected with either Htt96Q-mCherry (mutant, mimics the typical pathogenic expression of Htt in HD patients) or Htt20Q-mCherry (WT) constructs. When the polyQ-expanded Htt protein (96Q) exonl fused to fluorescent mCherry protein is expressed, the levels and aggregation of the protein in individual cells can be monitored using a fluorescent microscope.

[00108] Cell lysis and immunoblotting: Cells were lysed and phosphatase inhibitor cocktail 2 and 3 and lOmM b-glycerol phosphate were added to the lysis buffer to inhibit phosphatases for detection of phosphorylated proteins. After SDS-PAGE and transfer to nitrocellulose membrane, membranes were blocked and immunoblotted with primary antibody overnight at 4 °C, then washed and blotted with secondary antibody. After washing, enhanced chemiluminescence assay was performed and the membrane was exposed and quantified.

[00109] Statistical analysis: Results were expressed as mean ± SEM (standard error of the mean) of the number of independent experiments or animals indicated in figure legends. Comparisons between multiple groups were performed by non-parametric one-way analysis of variance (ANOVA) using Kruskal-Wallis test. Correction for multiple comparisons was done by two-way ANOVA followed by Tukey post-hoc test. Comparison between two groups was performed by non-parametric Mann- Whitney test or parametric Student's t-test. The F-test was performed to analyze the interaction term. Significance was accepted at p<0.05. All analyses were performed using Prism software (GraphPad Version 8.0). Mitochondrial parameters were evaluated in vitro using primary neurons isolated from Y128 HD mouse embryos, and human HD lymphoblasts and neural stem cells (NSCs). Striatal mitochondria isolated from Y128 mice treated with pridopidine or vehicle were used as an ex vivo model.

[00110] Results - [00111] Insights on mitochondrial function can be acquired by studying morphology and trafficking. Since events of fission and fusion are required for mitochondrial quality control, the mitochondrial aspect ratio (ratio between the major and minor axis of the ellipse equivalent to the mitochondrion) and mitochondrial trafficking, necessary for nourishing high energetic demands in synaptic terminals, are both measures of mitochondrial function. Cortico-striatal primary neurons harvested from the HD model YAC128 (Y128) and wild- type (WT) mice were stained with MitoTracker (Figure 1A) and mitochondria number (Figure IB) and morphology (Figure 1C and ID) assessed. HD neurons showed impairment in mitochondrial morphology: a significant increase in the number of circular mitochondria (Figure 1C) and a decrease in elongated mitochondria compared to age- matched wild-type neurons were observed (p<0.05) (Figure ID). Under these conditions mitochondrial fragmentation (fission) was favored despite no changes in mitochondrial mass (Figure IB), indicating decreased mitochondrial function. Pridopidine (1 mM) treatment rescued both number of circular and elongated mitochondria (p<0.05).

[00112] Poor co-localization of mitochondria with ER in Y128 HD neurons compared to WT was observed in striatal neurons transfected with mitoDsRed (mitochondria side) and stained with anti-IP3R (ER side) antibody for visualizing both organelles (Figure IE). Pridopidine (1 mM) highly increased mitochondria-ER colocalization in Y128 striatal neurons (Figure IE and IF, p<0.001). This result can account for increased ATP production and mitochondrial trafficking and velocity (observed in Figures 2A-2B). MitoDsRed-labelled mitochondria from Y128 striatal neurons also showed decreased aspect ratio (p<0.05) (Figure 1G), confirming previous results (Figure 1A-D). Treatment with pridopidine (lpM) reduced the number of fragmented mitochondria (p<0.05) (Figures IE, 1G) [00113] Mitochondrial anterograde transport was also greatly decreased in HD neurons, with approximately 90% of mitochondria in Y128 striatal neurons appearing stationary (p<0.05). Pridopidine reduces the percentage of stationary mitochondria, increasing both anterograde and retrograde transport (p<0.05). (Figures 2A, 2B - quantification). Mitochondrial transport velocity was also decreased, moving at half speed in Y 128 neurons compared to wild-type neurons (p<0.05). This decrease was ameliorated following pridopidine treatment (ImM) (p<0.05) (Figures 2A, 2C).

[00114] Y128 neurons showed reduced basal and maximal respiration as well as compromised ATP production (Figures 3A-3H). Reduced respiration can be a result of the impaired mitochondrial dynamics and morphology demonstrated in HD neurons (Figures 1A-1G and 2A-2B). Treatment with pridopidine at 1 and 5 mM doses rescued basal and maximal respiration (p<0.01) in HD Y128 corticostriatal neurons, as well as ATP production (p<0.05) (Figures 3B (basal), 3C (maximal) 3D (ATP production). Pridopidine 1 mM also increased basal (p<0.001) and maximal (p<0.05) mitochondrial respiration, as well as ATP production in human iPSC-derived neural stem cells (NSCs) from a heterozygous HD patient (HD-iPSC) (Figures 3E, 3F (basal), 3G (maximal), 3H (ATP production)).

[00115] Mitochondrial membrane potential (MMP, DY,, _i ) directly affects ATP production and is affected by mitochondrial Ca ²⁺ signaling. Y128 cortical and striatal neurons exhibited lower DY,, _i as a result of oligomycin and FCCP -induced mitochondrial dysfunction compared to wild-type cortical neurons (p<0.05), indicated by lower mitochondrial retention of TMRM (Figures 4A, 4B). Pridopidine (0.1 and 1 pM) increased DY , in both Y128 cortical and striatal neurons (p<0.05 for cortical neurons; p<0.05 for 0.1 pM pridopidine and p<0.01 for 1 pM pridopidine in striatal neurons) (Figures 4A-4C). [00116] Hydrogen peroxide (H2O2) was used as a more potent oxidative stimulus to evaluate DY _P1 In response to 0.1 mM H2O2 treatment, a marked decrease is observed in DY , in Y128 neurons (p<0.0001). Y128 coritcal/striatal neurons treated with pridopidine (5 mM), and then exposed to 0.1 mM H2O2 for 6 h, showed complete restoration of the DY _P1 loss induced by H2O2 (p<0.001)(Figure 4D), as well as restored cell viability (p<0.01)(Figure 4E). In lymphoblasts derived from HD patients, 0.1 mM H2O2 treatment caused a 50% decrease in DY _P1. Pridopidine doses 1, 5 and 10 pM pridopidine all increased DY _i , with the 5 pM dose showing the maximal protective effect after 0.1 mM H2O2 treatment (p<0.01) (Figure 4F).

[00117] Higher susceptibility of mitochondria from Y 128 neurons and HD lymphoblasts to H2O2 suggests that these cells exhibit increased oxidative stress. To test this, local H2O2 flux was measured with the fluorescent probe MitoPYl. The complex PI inhibitor Antimycin A (AntA), which induces reactive oxygen species (ROS) production, stimulated mitochondrial dysfunction in cortical (p<0.01) and striatal (p=0.0001) Y128 neurons to show a significant 2-fold increase in mitochondrial-driven H2O2 levels compared to WT neurons. In both cortical and striatal neurons 1 pM pridopidine reverted the increased levels of mito-ftCk prompted by AntA (pO.OOOl in cortical neurons, p<0.01 for 0.1 pM and pO.0001 for 1 pM in striatal neurons) (Figures 5A-5C).

[00118] In HD-NSCs treated with another mitochondrial complex III inhibitor myxothiazol (Myxo, 3 pM), cells showed a large increase in mito- H2O2 levels (p<0.0001). Pridopidine (1 pM) rescued the abnormal increase in H2O2 levels induced by inhibition of complex PI (p<0.01) (Figure 5D). Under basal conditions, HD lymphoblasts showed increased ROS production when compared to control lymphoblasts; when challenged with H2O2, pridopidine treatment decreased ROS levels in both basal and H ₂ 0 ₂ -challenged conditions (p<0.001) (Figure 5E). Thus, pridopidine reduced ROS levels in three HD cell models.

[00119] The neuroprotective effect of pridopidine was reproduced in vivo. WT and Y128 mice at 1.5 months of age (pre- symptomatic, males and females in equivalent proportion) were treated with either pridopidine 30 mg/kg or water for 45 consecutive days. Y128 mice exhibit motor deficits at 3 months of age in the rotarod performance test, therefore this test was applied before and after the treatment to test the efficacy of pridopidine at motor level. At 1.5 months (pre-treated) Y128 mice displayed the same motor coordination as wild-type mice (Figures 6A, 6B). At 3 months of age, vehicle-treated Y128 mice showed motor deficits compared to vehicle-treated wild-type mice, as observed by a reduced latency to fall during the accelerated rotarod test (p<0.05) (Figure 6C). Conversely, HD mice treated with pridopidine exhibited significant motor performance improvement in the accelerated rotarod (p<0.05) (Figure 6C) compared to vehicle-treated HD mice. After behavior analysis, functional mitochondria were isolated from striatum of all mice groups, and mitochondrial complexes activity was assessed by evaluating sequential electron flow through the electron transport chain by measuring OCR following sequential injections of rotenone, succinate, antimycin A and ascorbate/TMPD (h h N A ^f -tetramethyl p- phenylenediamine) that induced individual stimulation or inhibition of mitochondrial complexes I, II, III and IV, respectively, allowing the calculation of their activities (Figure 6D). Striatal mitochondria from vehicle-treated Y128 mice showed higher activity of complex I, II, III and IV, compared with vehicle-treated wild-type mice (p<0.01), suggesting an early compensatory mechanism (Figure 6D, 6F). Interestingly, increased complexes activities in Y128 HD mouse striatum was accompanied by increased mitochondrial H2O2 production before and after inhibition of complex III with AntA (p<0.05) (Figures 6D, 6G). These results suggest that abnormal complexes activities may underlie mitochondrial ROS production through increased electron leakage, leading to impaired ATP production. In vivo pridopidine treatment in Y128 mice normalized mitochondrial complexes activities, normalized H2O2 levels to the levels of WT vehicle- treated mice (p<0.05) (Figures 6I-6K) and increased mitochondrial Ca ²⁺ buffering capacity (Figures 6L, 6M).

[00120] Mitochondria and the endoplasmic reticulum (ER) are linked both functionally and physically. Physical contacts between mitochondria and ER occur at sites of mitochondria-associated membranes (MAM), highly specialized structures at which SIR is enriched. MAMs act as a conduit for the exchange of proteins, lipids, signaling molecules and, importantly, Ca ²⁺ . As a result, ER stress and mitochondrial dysfunction are closely linked (Morris, Gerwyn, Basant K. Puri, Ken Walder, Michael Berk, Brendon Stubbs, Michael Maes, and Andre F. Carvalho.“The Endoplasmic Reticulum Stress Response in Neuroprogressive Diseases: Emerging Pathophysiological Role and Translational Implications.” Molecular Neurobiology, (2018) 55:8765-8787).

[00121] Pridopidine decreases mHtt-induced ER stress: In STHdhQ7/7 cells transfected with the mutant Htt96Q-mCherry (expanded) construct, visible Htt96Q- mCherry aggregates can be observed (typically one large aggregate per cell) together with high levels of accumulated H2a-GFP, indicating of ER stress. STHdhQ7/7 cells expressing Htt20Q-mCherry (normal) or Htt96Q-mCherry without visible aggregates show low levels of H2a-GFP (no ER stress). Pridopidine significantly reduces H2a-GFP accumulation in cells positive for mHtt aggregates in a dose-dependent manner (Figure 7A), and does not alter H2a-GFP levels in cells without aggregates or in cells expressing Htt20Q-mCherry (Figure 7B). Thus, pridopidine decreases Htt-induced ER stress in a dose-dependent manner.

[00122] Phosphorylation of eIF2a is a hallmark of ER stress. In STHdhQ7/7 cells expressing Htt96Q-mCherry, eIF2a-phosphorylation (eIF2a-p) levels are 3.5-fold higher than in cells expressing Htt20Q-mCherry. Pridopidine treatment causes a significant reduction in eIF2a-P (measured by the ratio of eIF2a -P to total eIF2a), indicating a reduction in cellular ER stress (Figure 8).

[00123] In conclusion, the preclinical results show in in vitro and in vivo/ex vivo HD models that mitochondrial dysfunction is a hallmark of HD, precluding an effective antioxidant response against oxidative stimuli. This dysfunction may impact synaptic integrity and cellular survival. Pridopidine demonstrates rescue of different aspects of mitochondrial function in HD models, including the reduction of ROS levels in both HD human and mouse models and the increase of mitochondrial velocity as well as the percentage of elongated mitochondria, all indicative of mitochondrial dysfunction. Thus, pridopidine is efficacious for repairing mitochondrial dysfunction. Administration of pridopidine also delayed the appearance of first motor symptoms in Y128 mice, increased cell viability and rescued impaired oxidative phosphorylation. Additionally, in an in vitro HD model system pridopidine mitigates ER stress, which is closely linked to mitochondrial dysfunction, as SIR is located at MAM (mitochondria-associated membrane) sites at the ER membrane. These highly specialized sites have key roles in mitochondria function, including mitochondrial fission, Ca ²⁺ shuttling and oxidative stress.