Pridopidine rescues BDNF/TrkB trafficking dynamics and synapse homeostasis in a Huntington disease brain-on-a-chip model.

Huntington disease (HD) is a neurodegenerative disorder caused by polyglutamine-encoding CAG repeat expansion in the huntingtin (HTT) gene. HTT is involved in the axonal transport of vesicles containing brain-derived neurotrophic factor (BDNF). In HD, diminished BDNF transport leads to reduced BDNF delivery to the striatum, contributing to striatal and cortical neuronal death. Pridopidine is a selective and potent sigma-1 receptor (S1R) agonist currently in clinical development for HD. The S1R is located at the endoplasmic reticulum (ER)-mitochondria interface, where it regulates key cellular pathways commonly impaired in neurodegenerative diseases. We used a microfluidic device that reconstitutes the corticostriatal network, allowing the investigation of presynaptic dynamics, synaptic morphology and transmission, and postsynaptic signaling. Culturing primary neurons from the HD mouse model HdhCAG140/+ provides a "disease-on-a-chip" platform ideal for investigating pathogenic mechanisms and drug activity. Pridopidine rescued the trafficking of BDNF and TrkB resulting in an increased neurotrophin signaling at the synapse. This increased the capacity of HD neurons to release glutamate and restored homeostasis at the corticostriatal synapse. These data suggest that pridopidine enhances the availability of corticostriatal BDNF via S1R activation, leading to neuroprotective effects.

Pridopidine reduces mutant huntingtin-induced endoplasmic reticulum stress by modulation of the Sigma-1 receptor.

The endoplasmic reticulum (ER)-localized Sigma-1 receptor (S1R) is neuroprotective in models of neurodegenerative diseases, among them Huntington disease (HD). Recent clinical trials in HD patients and preclinical studies in cellular and mouse HD models suggest a therapeutic potential for the high-affinity S1R agonist pridopidine. However, the molecular mechanisms of the cytoprotective effect are unclear. We have previously reported strong induction of ER stress by toxic mutant huntingtin (mHtt) oligomers, which is reduced upon sequestration of these mHtt oligomers into large aggregates. Here, we show that pridopidine significantly ameliorates mHtt-induced ER stress in cellular HD models, starting at low nanomolar concentrations. Pridopidine reduced the levels of markers of the three branches of the unfolded protein response (UPR), showing the strongest effects on the PKR-like endoplasmic reticulum kinase (PERK) branch. The effect is S1R-dependent, as it is abolished in cells expressing mHtt in which the S1R was deleted using CRISPR/Cas9 technology. mHtt increased the level of the detergent-insoluble fraction of S1R, suggesting a compensatory cellular mechanism that responds to increased ER stress. Pridopidine further enhanced the levels of insoluble S1R, suggesting the stabilization of activated S1R oligomers. These S1R oligomeric species appeared in ER-localized patches, and not in the mitochondria-associated membranes nor the ER-derived quality control compartment. The colocalization of S1R with the chaperone BiP was significantly reduced by mHtt, and pridopidine restored this colocalization to normal, unstressed levels. Pridopidine increased toxic oligomeric mHtt recruitment into less toxic large sodium dodecyl sulfate-insoluble aggregates, suggesting that this in turn reduces ER stress and cytotoxicity.

Sigma-1 and dopamine D2/D3 receptor occupancy of pridopidine in healthy volunteers and patients with Huntington disease: a [18F] fluspidine and [18F] fallypride PET study.

PURPOSE: Pridopidine is an investigational drug for Huntington disease (HD). Pridopidine was originally thought to act as a dopamine stabilizer. However, pridopidine shows highest affinity to the sigma-1 receptor (S1R) and enhances neuroprotection via the S1R in preclinical studies. Using [18F] fluspidine and [18F] fallypride PET, the purpose of this study was to assess in vivo target engagement/receptor occupancy of pridopidine to the S1R and dopamine D2/D3 receptor (D2/D3R) at clinical relevant doses in healthy volunteers (HVs) and as proof-of-concept in a small number of patients with HD. METHODS: Using [18F] fluspidine PET (300 MBq, 0-90 min), 11 male HVs (pridopidine 0.5 to 90 mg; six dose groups) and three male patients with HD (pridopidine 90 mg) were investigated twice, without and 2 h after single dose of pridopidine. Using [18F] fallypride PET (200 MBq, 0-210 min), four male HVs were studied without and 2 h following pridopidine administration (90 mg). Receptor occupancy was analyzed by the Lassen plot. RESULTS: S1R occupancy as function of pridopidine dose (or plasma concentration) in HVs could be described by a three-parameter Hill equation with a Hill coefficient larger than one. A high degree of S1R occupancy (87% to 91%) was found throughout the brain at pridopidine doses ranging from 22.5 to 90 mg. S1R occupancy was 43% at 1 mg pridopidine. In contrast, at 90 mg pridopidine, the D2/D3R occupancy was only minimal (~ 3%). CONCLUSIONS: Our PET findings indicate that at clinically relevant single dose of 90 mg, pridopidine acts as a selective S1R ligand showing near to complete S1R occupancy with negligible occupancy of the D2/D3R. The dose S1R occupancy relationship suggests cooperative binding of pridopidine to the S1R. Our findings provide significant clarification about pridopidine's mechanism of action and support further use of the 45-mg twice-daily dose to achieve full and selective targeting of the S1R in future clinical trials of neurodegenerative disorders. Clinical Trials.gov Identifier: NCT03019289 January 12, 2017; EUDRA-CT-Nr. 2016-001757-41.

Sigma-1 Receptor (S1R) Interaction with Cholesterol: Mechanisms of S1R Activation and Its Role in Neurodegenerative Diseases.

The sigma-1 receptor (S1R) is a 223 amino acid-long transmembrane endoplasmic reticulum (ER) protein. The S1R modulates the activity of multiple effector proteins, but its signaling functions are poorly understood. S1R is associated with cholesterol, and in our recent studies we demonstrated that S1R association with cholesterol induces the formation of S1R clusters. We propose that these S1R-cholesterol interactions enable the formation of cholesterol-enriched microdomains in the ER membrane. We hypothesize that a number of secreted and signaling proteins are recruited and retained in these microdomains. This hypothesis is consistent with the results of an unbiased screen for S1R-interacting partners, which we performed using the engineered ascorbate peroxidase 2 (APEX2) technology. We further propose that S1R agonists enable the disassembly of these cholesterol-enriched microdomains and the release of accumulated proteins such as ion channels, signaling receptors, and trophic factors from the ER. This hypothesis may explain the pleotropic signaling functions of the S1R, consistent with previously observed effects of S1R agonists in various experimental systems.

Pridopidine protects neurons from mutant-huntingtin toxicity via the sigma-1 receptor.

Huntington's disease (HD) is a neurodegenerative disease caused by a CAG repeat expansion in the Huntingtin gene (HTT), translated into a Huntingtin protein with a polyglutamine expansion. There is preferential loss of medium spiny neurons within the striatum and cortical pyramidal neurons. Pridopidine is a small molecule showing therapeutic potential in HD preclinical and clinical studies. Pridopidine has nanomolar affinity to the sigma-1 receptor (sigma-1R), which is located predominantly at the endoplasmic reticulum (ER) and mitochondrial associated ER membrane, and activates neuroprotective pathways. Here we evaluate the neuroprotective effects of pridopidine against mutant Huntingtin toxicity in mouse and human derived in vitro cell models. We also investigate the involvement of the sigma-1 receptor in the mechanism of pridopidine. Pridopidine protects mutant Huntingtin transfected mouse primary striatal and cortical neurons, with an EC50 in the mid nanomolar range, as well as HD patient-derived induced pluripotent stem cells (iPSCs). This protection by pridopidine is blocked by NE-100, a purported sigma-1 receptor antagonist, and not blocked by ANA-12, a reported TrkB receptor antagonist. 3PPP, a documented sigma-1 receptor agonist, shows similar neuroprotective effects. Genetic knock out of the sigma-1 receptor dramatically decreases protection from pridopidine and 3PPP, but not protection via brain derived neurotrophic factor (BDNF). The neuroprotection afforded by pridopidine in our HD cell models is robust and sigma-1 receptor dependent. These studies support the further development of pridopidine, and other sigma-1 receptor agonists as neuroprotective agents for HD and perhaps for other disorders.

Pridopidine stabilizes mushroom spines in mouse models of Alzheimer's disease by acting on the sigma-1 receptor.

There is evidence that cognitive decline in Alzheimer's disease (AD) results from deficiencies in synaptic communication (e.g., loss of mushroom-shaped 'memory spines') and neurodegenerative processes. This might be treated with sigma-1 receptor (S1R) agonists, which are broadly neuroprotective and modulate synaptic plasticity. For example, we previously found that the mixed muscarinic/S1R agonist AF710B prevents mushroom spine loss in hippocampal cultures from APP knock-in (APP-KI) and presenilin-1-M146 V knock-in (PS1-KI) mice. We also found that the "dopaminergic stabilizer" pridopidine (structurally similar to the S1R agonist R(+)-3-PPP), is a high-affinity S1R agonist and is synaptoprotective in a mouse model of Huntington disease. Here we tested whether pridopidine and R(+)-3-PPP are synaptoprotective in models of AD and whether this requires S1R. We also examined the effects of pridopidine on long-term potentiation (LTP), endoplasmic reticulum calcium and neuronal store-operated calcium entry (nSOC) in spines, all of which are dysregulated in AD, contributing to synaptic pathology. We report here that pridopidine and 3-PPP protect mushroom spines from Aß42 oligomer toxicity in primary WT hippocampal cultures from mice. Pridopidine also reversed LTP defects in hippocampal slices resulting from application of Aß42 oligomers. Pridopidine and 3-PPP rescued mushroom spines in hippocampal cultures from APP-KI and PS1-KI mice. S1R knockdown from lenti-viral shRNA expression destabilized WT mushroom spines and prevented the synaptoprotective effects of pridopidine in PS1-KI cultures. Knockout of PS1/2 destabilized mushroom spines and pridopidine was unable to prevent this. Pridopidine lowered endoplasmic reticulum calcium levels in WT, PS1-KO, PS1-KI and PS2 KO neurons, but not in PS1/2 KO neurons. S1R was required for pridopidine to enhance spine nSOC in PS1-KI neurons. Pridopidine was unable to rescue PS1-KI mushroom spines during pharmacological or genetic inhibition of nSOC. Oral pridopidine treatment rescued mushroom spines in vivo in aged PS1-KI-GFP mice. Pridopidine stabilizes mushroom spines in mouse models of AD and this requires S1R, endoplasmic reticulum calcium leakage through PS1/2 and nSOC. Thus, pridopidine may be useful to explore for the treatment of AD.

Pridopidine, a clinic-ready compound, reduces 3,4-dihydroxyphenylalanine-induced dyskinesia in Parkinsonian macaques.

BACKGROUND: Pridopidine, in development for Huntington's disease, may modulate aberrant l-dopa-induced effects including l-dopa-induced dyskinesia (LID). OBJECTIVE: This study investigated whether pridopidine could reduce LID in the MPTP macaque model of Parkinson's disease and characterized the observed behavioral effects in terms of receptor occupancy. METHODS: The pharmacokinetic profile and effects of pridopidine (15-30 mg/kg) on parkinsonism, dyskinesia, and quality of on-time, in combination with l-dopa, were assessed in MPTP macaques with LID. Pridopidine receptor occupancy was estimated using known in vitro binding affinities to σ1 and dopamine D2 receptors, in vivo PET imaging, and pharmacokinetic profiling across different species. RESULTS: Pridopidine produced a dose-dependent reduction in dyskinesia (up to 71%, 30 mg/kg) and decreased the duration of on-time with disabling dyskinesia evoked by l-dopa by 37% (20 mg/kg) and 60% (30 mg/kg). Pridopidine did not compromise the anti-parkinsonian benefit of l-dopa. Plasma exposures following the ineffective dose (15 mg/kg) were associated with full σ1 occupancy (>80%), suggesting that σ1 engagement alone is unlikely to account for the antidyskinetic benefits of pridopidine. Exposures following effective doses (20-30 mg/kg), while providing full σ1 occupancy, provide only modest dopamine D2 occupancy (<40%). However, effective pridopidine doses clearly engage a range of receptors (including adrenergic-α2C , dopamine-D3 , and serotoninergic-5-HT1A sites) to a higher degree than D2 and might contribute to the antidyskinetic actions. CONCLUSIONS: In MPTP macaques, pridopidine produced a significant decrease in LID without compromising the antiparkinsonian benefit of l-dopa. Although the actions of pridopidine were associated with full σ1 occupancy, effective exposures are more likely associated with occupancy of additional, non-sigma receptors. This complex pharmacology may underlie the effectiveness of pridopidine against LID. © 2018 International Parkinson and Movement Disorder Society.

Pridopidine activates neuroprotective pathways impaired in Huntington Disease.

Pridopidine has demonstrated improvement in Huntington Disease (HD) motor symptoms as measured by secondary endpoints in clinical trials. Originally described as a dopamine stabilizer, this mechanism is insufficient to explain the clinical and preclinical effects of pridopidine. This study therefore explored pridopidine's potential mechanisms of action. The effect of pridopidine versus sham treatment on genome-wide expression profiling in the rat striatum was analysed and compared to the pathological expression profile in Q175 knock-in (Q175 KI) vs Q25 WT mouse models. A broad, unbiased pathway analysis was conducted, followed by testing the enrichment of relevant pathways. Pridopidine upregulated the BDNF pathway (P = 1.73E-10), and its effect on BDNF secretion was sigma 1 receptor (S1R) dependent. Many of the same genes were independently found to be downregulated in Q175 KI mice compared to WT (5.2e-7 < P < 0.04). In addition, pridopidine treatment upregulated the glucocorticoid receptor (GR) response, D1R-associated genes and the AKT/PI3K pathway (P = 1E-10, P = 0.001, P = 0.004, respectively). Pridopidine upregulates expression of BDNF, D1R, GR and AKT/PI3K pathways, known to promote neuronal plasticity and survival, as well as reported to demonstrate therapeutic benefit in HD animal models. Activation of S1R, necessary for its effect on the BDNF pathway, represents a core component of the mode of action of pridopidine. Since the newly identified pathways are downregulated in neurodegenerative diseases, including HD, these findings suggest that pridopidine may exert neuroprotective effects beyond its role in alleviating some symptoms of HD.

The sigma-1 receptor mediates the beneficial effects of pridopidine in a mouse model of Huntington disease.

The tri-nucleotide repeat expansion underlying Huntington disease (HD) results in corticostriatal synaptic dysfunction and subsequent neurodegeneration of striatal medium spiny neurons (MSNs). HD is a devastating autosomal dominant disease with no disease-modifying treatments. Pridopidine, a postulated "dopamine stabilizer", has been shown to improve motor symptoms in clinical trials of HD. However, the target(s) and mechanism of action of pridopidine remain to be fully elucidated. As binding studies identified sigma-1 receptor (S1R) as a high-affinity receptor for pridopidine, we evaluated the relevance of S1R as a therapeutic target of pridopidine in HD. S1R is an endoplasmic reticulum - (ER) resident transmembrane protein and is regulated by ER calcium homeostasis, which is perturbed in HD. Consistent with ER calcium dysregulation, we observed striatal upregulation of S1R in aged YAC128 transgenic HD mice and HD patients. We previously demonstrated that dendritic MSN spines are lost in aged corticostriatal co-cultures from YAC128 mice. We report here that pridopidine and the chemically similar S1R agonist 3-PPP prevent MSN spine loss in aging YAC128 co-cultures. Spine protection was blocked by neuronal deletion of S1R. Pridopidine treatment suppressed supranormal ER Ca2+ release, restored ER calcium levels and reduced excessive store-operated calcium (SOC) entry in spines, which may account for its synaptoprotective effects. Normalization of ER Ca2+ levels by pridopidine was prevented by S1R deletion. To evaluate long-term effects of pridopidine, we analyzed expression profiles of calcium signaling genes. Pridopidine elevated striatal expression of calbindin and homer1a, whereas their striatal expression was reduced in aged Q175KI and YAC128 HD mouse models compared to WT. Pridopidine and 3-PPP are proposed to prevent calcium dysregulation and synaptic loss in a YAC128 corticostriatal co-culture model of HD. The actions of pridopidine were mediated by S1R and led to normalization of ER Ca2+ release, ER Ca2+ levels and spine SOC entry in YAC128 MSNs. This is a new potential mechanism of action for pridopidine, highlighting S1R as a potential target for HD therapy. Upregulation of striatal proteins that regulate calcium, including calbindin and homer1a, upon chronic therapy with pridopidine, may further contribute to long-term beneficial effects of pridopidine in HD.

Drug resistance confounding prion therapeutics.

There is not a single pharmaceutical that halts or even slows any neurodegenerative disease. Mounting evidence shows that prions cause many neurodegenerative diseases, and arguably, scrapie and Creutzfeldt-Jakob disease prions represent the best therapeutic targets. We report here that the previously identified 2-aminothiazoles IND24 and IND81 doubled the survival times of scrapie-infected, wild-type mice. However, mice infected with Rocky Mountain Laboratory (RML) prions, a scrapie-derived strain, and treated with IND24 eventually exhibited neurological dysfunction and died. We serially passaged their brain homogenates in mice and cultured cells. We found that the prion strain isolated from IND24-treated mice, designated RML[IND24], emerged during a single passage in treated mice. Although RML prions infect both the N2a and CAD5 cell lines, RML[IND24] prions could only infect CAD5 cells. When passaged in CAD5 cells, the prions remained resistant to high concentrations of IND24. However, one passage of RML[IND24] prions in untreated mice restored susceptibility to IND24 in CAD5 cells. Although IND24 treatment extended the lives of mice propagating different prion strains, including RML, another scrapie-derived prion strain ME7, and chronic wasting disease, it was ineffective in slowing propagation of Creutzfeldt-Jakob disease prions in transgenic mice. Our studies demonstrate that prion strains can acquire resistance upon exposure to IND24 that is lost upon passage in mice in the absence of IND24. These data suggest that monotherapy can select for resistance, thus intermittent therapy with mixtures of antiprion compounds may be required to slow or stop neurodegeneration.

Biaryl amides and hydrazones as therapeutics for prion disease in transgenic mice.

The only small-molecule compound demonstrated to substantially extend survival in prion-infected mice is a biaryl hydrazone termed "Compd B" (4-pyridinecarboxaldehyde,2-[4-(5-oxazolyl)phenyl]hydrazone). However, the hydrazone moiety of Compd B results in toxic metabolites, making it a poor candidate for further drug development. We developed a pharmacophore model based on diverse antiprion compounds identified by high-throughput screening; based on this model, we generated biaryl amide analogs of Compd B. Medicinal chemistry optimization led to multiple compounds with increased potency, increased brain concentrations, and greater metabolic stability, indicating that they could be promising candidates for antiprion therapy. Replacing the pyridyl ring of Compd B with a phenyl group containing an electron-donating substituent increased potency, while adding an aryl group to the oxazole moiety increased metabolic stability. To test the efficacy of Compd B, we applied bioluminescence imaging (BLI), which was previously shown to detect prion disease onset in live mice earlier than clinical signs. In our studies, Compd B showed good efficacy in two lines of transgenic mice infected with the mouse-adapted Rocky Mountain Laboratory (RML) strain of prions, but not in transgenic mice infected with human prions. The BLI system successfully predicted the efficacies in all cases long before extension in survival could be observed. Our studies suggest that this BLI system has good potential to be applied in future antiprion drug efficacy studies.

Nucleoporin POM121 signals TFEB-mediated autophagy via activation of SIGMAR1/sigma-1 receptor chaperone by pridopidine.

Macroautophagy/autophagy is an essential process for cellular survival and is implicated in many diseases. A critical step in autophagy is the transport of the transcription factor TFEB from the cytosol into the nucleus, through the nuclear pore (NP) by KPNB1/importinß1. In the C9orf72 subtype of amyotrophic lateral sclerosis-frontotemporal lobar degeneration (ALS-FTD), the hexanucleotide (G4C2)RNA expansion (HRE) disrupts the nucleocytoplasmic transport of TFEB, compromising autophagy. Here we show that a molecular chaperone, the SIGMAR1/Sigma-1 receptor (sigma non-opioid intracellular receptor 1), facilitates TFEB transport into the nucleus by chaperoning the NP protein (i.e., nucleoporin) POM121 which recruits KPNB1. In NSC34 cells, HRE reduces TFEB transport by interfering with the association between SIGMAR1 and POM121, resulting in reduced nuclear levels of TFEB, KPNB1, and the autophagy marker LC3-II. Overexpression of SIGMAR1 or POM121, or treatment with the highly selective and potent SIGMAR1 agonist pridopidine, currently in phase 2/3 clinical trials for ALS and Huntington disease, rescues all of these deficits. Our results implicate nucleoporin POM121 not merely as a structural nucleoporin, but also as a chaperone-operated signaling molecule enabling TFEB-mediated autophagy. Our data suggest the use of SIGMAR1 agonists, such as pridopidine, for therapeutic development of diseases in which autophagy is impaired.Abbreviations: ALS-FTD, amyotrophic lateral sclerosis-frontotemporal dementiaC9ALS-FTD, C9orf72 subtype of amyotrophic lateral sclerosis-frontotemporal dementiaCS, citrate synthaseER, endoplasmic reticulumGSS, glutathione synthetaseHRE, hexanucleotide repeat expansionHSPA5/BiP, heat shock protein 5LAMP1, lysosomal-associated membrane protein 1MAM, mitochondria-associated endoplasmic reticulum membraneMAP1LC3/LC3, microtubule-associated protein 1 light chain 3NP, nuclear poreNSC34, mouse motor neuron-like hybrid cell lineNUPs, nucleoporinsPOM121, nuclear pore membrane protein 121SIGMAR1/Sigma-1R, sigma non-opioid intracellular receptor 1TFEB, transcription factor EBTMEM97/Sigma-2R, transmembrane protein 97.

Pridopidine Does Not Significantly Prolong the QTc Interval at the Clinically Relevant Therapeutic Dose.

INTRODUCTION: Pridopidine is a highly selective sigma-1 receptor (S1R) agonist in development for the treatment of Huntington's disease (HD) and amyotrophic lateral sclerosis (ALS). Pridopidine's activation of S1R enhances cellular processes that are crucial for neuronal function and survival but are impaired in neurodegenerative diseases. Human brain positron emission tomography (PET) imaging studies show that at the therapeutic dose of 45 mg twice daily (bid), pridopidine selectively and robustly occupies the S1R. We conducted concentration-QTc (C-QTc) analyses to assess pridopidine's effect on the QT interval and investigated its cardiac safety profile. METHODS: C-QTc analysis was conducted using data from PRIDE-HD, a phase 2, placebo-controlled trial evaluating four pridopidine doses (45, 67.5, 90, 112.5 mg bid) or placebo over 52 weeks in HD patients. Triplicate electrocardiograms (ECGs) with simultaneous plasma drug concentrations were determined in 402 patients with HD. The effect of pridopidine on the Fridericia-corrected QT interval (QTcF) was evaluated. Cardiac-related adverse events (AEs) were analyzed from PRIDE-HD alone and from pooled safety data of three double-blind, placebo-controlled trials with pridopidine in HD (HART, MermaiHD, and PRIDE-HD). RESULTS: A concentration-dependent effect of pridopidine on the change from baseline in the Fridericia-corrected QT interval (ΔQTcF) was observed, with a slope of 0.012 ms (ms) per ng/mL (90% confidence interval (CI), 0.0109-0.0127). At the therapeutic dose of 45 mg bid, the predicted placebo-corrected ΔQTcF (ΔΔQTcF) was 6.6 ms (upper bound 90% CI, 8.0 ms), which is below the level of concern and not clinically relevant. Analysis of pooled safety data from three HD trials demonstrates that at 45 mg bid, pridopidine cardiac-related AE frequencies are similar to those with placebo. No patients reached a QTcF of 500 ms and no patients experienced torsade de pointes (TdP) at any pridopidine dose. CONCLUSIONS: At the 45 mg bid therapeutic dose, pridopidine demonstrates a favorable cardiac safety profile, with an effect on the QTc interval that is below the level of concern and not clinically relevant. TRIAL REGISTRATION: PRIDE-HD (TV7820-CNS-20002) trial registration: ClinicalTrials.gov identifier, NCT02006472, EudraCT 2013-001888-23; HART (ACR16C009) trial registration: ClinicalTrials.gov identifier, NCT00724048; MermaiHD (ACR16C008) trial registration: ClinicalTrials.gov identifier, NCT00665223, EudraCT No. 2007-004988-22.

A mouse model for eukaryotic translation initiation factor 2B-leucodystrophy reveals abnormal development of brain white matter.

Eukaryotic translation initiation factor 2B is a major housekeeping complex that governs the rate of global protein synthesis under normal and stress conditions. Mutations in any of its five subunits lead to leucoencephalopathy with vanishing white matter, an inherited chronic-progressive fatal brain disease with unknown aetiology, which is among the most prevalent childhood white matter disorders. We generated the first animal model for the disease by introducing a point mutation into the mouse Eif2b5 gene locus, leading to R132H replacement corresponding to the clinically significant human R136H mutation in the catalytic subunit. In contrast to human patients, mice homozygous for the mutant Eif2b5 allele (Eif2b5(R132H/R132H) mice) enable multiple analyses under a defined genetic background during the pre-symptomatic stages and during recovery from a defined brain insult. Time-course magnetic resonance imaging revealed for the first time the delayed development of the brain white matter due to the mutation. Electron microscopy demonstrated a higher proportion of small-calibre nerve fibres. Immunohistochemistry detected an abnormal abundance of oligodendrocytes and astrocytes in the brain of younger animals, as well as an abnormal level of major myelin proteins. Most importantly, mutant mice failed to recover from cuprizone-induced demyelination, reflecting an increased sensitivity to brain insults. The anomalous development of white matter in Eif2b5(R132H/R132H) mice underscores the importance of tight translational control to normal myelin formation and maintenance.

Neuroprotection of retinal ganglion cells by the sigma-1 receptor agonist pridopidine in models of experimental glaucoma.

Optic neuropathies such as glaucoma are characterized by retinal ganglion cell (RGC) degeneration and death. The sigma-1 receptor (S1R) is an attractive target for treating optic neuropathies as it is highly expressed in RGCs, and its absence causes retinal degeneration. Activation of the S1R exerts neuroprotective effects in models of retinal degeneration. Pridopidine is a highly selective and potent S1R agonist in clinical development. We show that pridopidine exerts neuroprotection of retinal ganglion cells in two different rat models of glaucoma. Pridopidine strongly binds melanin, which is highly expressed in the retina. This feature of pridopidine has implications to its ocular distribution, bioavailability, and effective dose. Mitochondria dysfunction is a key contributor to retinal ganglion cell degeneration. Pridopidine rescues mitochondrial function via activation of the S1R, providing support for the potential mechanism driving its neuroprotective effect in retinal ganglion cells.

The Sigma-1 Receptor Mediates Pridopidine Rescue of Mitochondrial Function in Huntington Disease Models.

Pridopidine is a selective Sigma-1 receptor (S1R) agonist in clinical development for Huntington disease (HD) and amyotrophic lateral sclerosis. S1R is a chaperone protein localized in mitochondria-associated endoplasmic reticulum (ER) membranes, a signaling platform that regulates Ca2+ signaling, reactive oxygen species (ROS) and mitochondrial fission. Here, we investigate the protective effects of pridopidine on various mitochondrial functions in human and mouse HD models. Pridopidine effects on mitochondrial dynamics were assessed in primary neurons from YAC128 HD mice expressing the mutant human HTT gene. We observe that pridopidine prevents the disruption of mitochondria-ER contact sites and improves the co-localization of inositol 1,4,5-trisphosphate receptor (IP3R) and its chaperone S1R with mitochondria in YAC128 neurons, leading to increased mitochondrial activity, elongation, and motility. Increased mitochondrial respiration is also observed in YAC128 neurons and in pridopidine-treated HD human neural stem cells (hNSCs). ROS levels were assessed after oxidative insult or S1R knockdown in pridopidine-treated YAC128 neurons, HD hNSCs, and human HD lymphoblasts. All HD models show increased ROS levels and deficient antioxidant response, which are efficiently rescued with pridopidine. Importantly, pridopidine treatment before H2O2-induced mitochondrial dysfunction and S1R presence are required for HD cytoprotection. YAC128 mice treated at early/pre-symptomatic age with pridopidine show significant improvement in motor coordination, indicating a delay in symptom onset. Additionally, in vivo pridopidine treatment reduces mitochondrial ROS levels by normalizing mitochondrial complex activity. In conclusion, S1R-mediated enhancement of mitochondrial function contributes to the neuroprotective effects of pridopidine, providing insight into its mechanism of action and therapeutic potential.

Effects of Pridopidine on Functional Capacity in Early-Stage Participants from the PRIDE-HD Study.

BACKGROUND: No pharmacological treatment has been demonstrated to provide a functional benefit for persons with Huntington's disease (HD). Pridopidine is a sigma-1-receptor agonist shown to have beneficial effects in preclinical models of HD. OBJECTIVE: To further explore the effect of pridopidine on Total Functional Capacity (TFC) in the recent double-blind, placebo-controlled PRIDE-HD study. METHODS: We performed post-hoc analyses to evaluate the effect of pridopidine on TFC at 26 and 52 weeks. Participants were stratified according to baseline TFC score and analyzed using repeated measures (MMRM) and multiple imputation assuming missing not-at-random (MNAR) and worst-case scenarios. RESULTS: The pridopidine 45 mg bid dosage demonstrated a beneficial effect on TFC for the entire population at week 52 of 0.87 (nominal p = 0.0032). The effect was more pronounced for early HD participants (HD1/HD2, TFC = 7-13), with a change from placebo of 1.16 (nominal p = 0.0003). This effect remained nominally significant using multiple imputation with missing not at random assumption as a sensitivity analysis. Responder analyses showed pridopidine 45 mg bid reduced the probability of TFC decline in early HD patients at Week 52 (nominal p = 0.02). CONCLUSION: Pridopidine 45 mg bid results in a nominally significant reduction in TFC decline at 52 weeks compared to placebo, particularly in patients with early-stage HD.

Additional Safety and Exploratory Efficacy Data at 48 and 60 Months from Open-HART, an Open-Label Extension Study of Pridopidine in Huntington Disease.

BACKGROUND: Open-HART was an open-label extension of HART, a randomized, double-blind, placebo-controlled study of pridopidine in Huntington disease (HD). Previously, we reported safety and exploratory efficacy data after 36 months of treatment with pridopidine 45 mg twice daily. In the interim, emerging data suggests pridopidine may have neuroprotective effects mediated by sigma-1 receptor agonism. OBJECTIVE: To report additional safety and exploratory efficacy data for continued open-label use of 45 mg BID pridopidine at 48 and 60 months. METHODS: Patients in Open-HART were followed up to or greater than 60 months. Adverse events, concomitant medications, vital signs, laboratory values, and ECG data were monitored. Rates of decline in total functional capacity (TFC) and total motor score (TMS) over 60 months were evaluated in an exploratory analysis and compared between Open-HART and placebo recipients from the 2CARE trial. To account for missing data, sensitivity analyses were performed. RESULTS: Of the original Open-HART baseline cohort (N = 118), 40 remained in the study at 48 months and 33 at 60 months. Pridopidine remained safe and well tolerated over the 60-month interval. TFC and TMS at 48 and 60 months remained stable, showing less decline at these timepoints compared to historical placebo controls from the 2CARE trial. TFC differences at 48 and 60 months observed remained nominally significant after sensitivity analysis. CONCLUSION: The 45 mg BID pridopidine dosage remained safe and tolerable over 60 months. Exploratory analyses show TFC and TMS stability at 48 and 60 months, in contrast to placebo historical controls from the 2CARE trial. Results are consistent with data reported from the recent Phase 2 PRIDE-HD trial showing less functional decline in the pridopidine 45 mg BID treated group at 52 weeks.

Pridopidine Induces Functional Neurorestoration Via the Sigma-1 Receptor in a Mouse Model of Parkinson's Disease.

Pridopidine is a small molecule in clinical development for the treatment of Huntington's disease. It was recently found to have high binding affinity to the sigma-1 receptor, a chaperone protein involved in cellular defense mechanisms and neuroplasticity. Here, we have evaluated the neuroprotective and neurorestorative effects of pridopidine in a unilateral 6-hydroxydopamine (6-OHDA) lesion model of parkinsonism in mice. By 5 weeks of daily administration, a low dose of pridopidine (0.3 mg/kg) had significantly improved deficits in forelimb use (cylinder test, stepping test) and abolished the ipsilateral rotational bias typical of hemiparkinsonian animals. A higher dose of pridopidine (1 mg/kg) significantly improved only the rotational bias, with a trend towards improvement in forelimb use. The behavioral recovery induced by pridopidine 0.3 mg/kg was accompanied by a significant protection of nigral dopamine cell bodies, an increased dopaminergic fiber density in the striatum, and striatal upregulation of GDNF, BDNF, and phosphorylated ERK1/2. The beneficial effects of pridopidine 0.3 mg/kg were absent in 6-OHDA-lesioned mice lacking the sigma-1 receptor. Pharmacokinetic data confirmed that the effective dose of pridopidine reached brain concentrations sufficient to bind S1R. Our results are the first to show that pridopidine promotes functional neurorestoration in the damaged nigrostriatal system acting via the sigma-1 receptor.

Targeting the Sigma-1 Receptor via Pridopidine Ameliorates Central Features of ALS Pathology in a SOD1G93A Model.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease affecting both the upper and lower motor neurons (MNs), with no effective treatment currently available. Early pathological events in ALS include perturbations in axonal transport (AT), formation of toxic protein aggregates and Neuromuscular Junction (NMJ) disruption, which all lead to axonal degeneration and motor neuron death. Pridopidine is a small molecule that has been clinically developed for Huntington disease. Here we tested the efficacy of pridopidine for ALS using in vitro and in vivo models. Pridopidine beneficially modulates AT deficits and diminishes NMJ disruption, as well as motor neuron death in SOD1G93A MNs and in neuromuscular co-cultures. Furthermore, we demonstrate that pridopidine activates the ERK pathway and mediates its beneficial effects through the sigma-1 receptor (S1R). Strikingly, in vivo evaluation of pridopidine in SOD1G93A mice reveals a profound reduction in mutant SOD1 aggregation in the spinal cord, and attenuation of NMJ disruption, as well as subsequent muscle wasting. Taken together, we demonstrate for the first time that pridopidine improves several cellular and histological hallmark pathologies of ALS through the S1R.