Apoe4 and Alzheimer's Disease Pathogenesis-Mitochondrial Deregulation and Targeted Therapeutic Strategies.

APOE ε4 allele (ApoE4) is the primary genetic risk factor for sporadic Alzheimer's disease (AD), expressed in 40-65% of all AD patients. ApoE4 has been associated to many pathological processes possibly linked to cognitive impairment, such as amyloid-ß (Aß) and tau pathologies. However, the exact mechanism underlying ApoE4 impact on AD progression is unclear, while no effective therapies are available for this highly debilitating neurodegenerative disorder. This review describes the current knowledge of ApoE4 interaction with mitochondria, causing mitochondrial dysfunction and neurotoxicity, associated with increased mitochondrial Ca2+ and reactive oxygen species (ROS) levels, and it effects on mitochondrial dynamics, namely fusion and fission, and mitophagy. Moreover, ApoE4 translocates to the nucleus, regulating the expression of genes involved in aging, Aß production, inflammation and apoptosis, potentially linked to AD pathogenesis. Thus, novel therapeutical targets can be envisaged to counteract the effects induced by ApoE4 in AD brain.

Nuclear localization and phosphorylation modulate pathological effects of alpha-synuclein.

Alpha-synuclein (aSyn) is a central player in Parkinson's disease (PD) but the precise molecular mechanisms underlying its pathogenicity remain unclear. It has recently been suggested that nuclear aSyn may modulate gene expression, possibly via interactions with DNA. However, the biological behavior of aSyn in the nucleus and the factors affecting its transcriptional role are not known. Here, we investigated the mechanisms underlying aSyn-mediated transcription deregulation by assessing its effects in the nucleus and the impact of phosphorylation in these dynamics. We found that aSyn induced severe transcriptional deregulation, including the downregulation of important cell cycle-related genes. Importantly, transcriptional deregulation was concomitant with reduced binding of aSyn to DNA. By forcing the nuclear presence of aSyn in the nucleus (aSyn-NLS), we found the accumulation of high molecular weight aSyn species altered gene expression and reduced toxicity when compared with the wild-type or exclusively cytosolic protein. Interestingly, nuclear localization of aSyn, and the effect on gene expression and cytotoxicity, was also modulated by phosphorylation on serine 129. Thus, we hypothesize that the role of aSyn on gene expression and, ultimately, toxicity, may be modulated by the phosphorylation status and nuclear presence of different aSyn species. Our findings shed new light onto the subcellular dynamics of aSyn and unveil an intricate interplay between subcellular location, phosphorylation and toxicity, opening novel avenues for the design of future strategies for therapeutic intervention in PD and other synucleinopathies.

Revisiting cell and gene therapies in Huntington's disease.

Neurodegenerative movement disorders, such as Huntington's disease (HD), share a progressive and relentless course with increasing motor disability, linked with neuropsychiatric impairment. These diseases exhibit diverse pathophysiological processes and are a topic of intense experimental and clinical research due to the lack of therapeutic options. Restorative therapies are promising approaches with the potential to restore brain circuits. However, there were less compelling results in the few clinical trials. In this review, we discuss cell replacement therapies applied to animal models and HD patients. We thoroughly describe the initial trials using fetal neural tissue transplantation and recent approaches based on alternative cell sources tested in several animal models. Stem cells were shown to generate the desired neuron phenotype and/or provide growth factors to the degenerating host cells. Besides, genetic approaches such as RNA interference and the CRISPR/Cas9 system have been studied in animal models and human-derived cells. New genetic manipulations have revealed the capability to control or counteract the effect of human gene mutations as described by the use of antisense oligonucleotides in a clinical trial. In HD, innovative strategies are at forefront of human testing and thus other brain genetic diseases may follow similar therapeutic strategies.

Calcium Signaling in Aging and Neurodegenerative Diseases 2019.

The European Calcium Society (ECS) workshop, which is held every 2 years, is a dedicated meeting of scientists interested in the elucidation of the action of calcium binding, calcium signaling and the study of proteins and organelles, such as mitochondria and endoplasmic reticulum, thereby involved, either in health and disease conditions. The 8th edition of the ECS workshop was organized by a group of researchers from the University of Coimbra, Portugal, in close collaboration with ECS board members. Thanks to the central role of "Calcium Signaling in Aging and Neurodegenerative Disorders", the ECS 2019 workshop was attended by 62 experts who presented their results in a plenary lecture and five regular symposia, two oral communication sessions and two poster sessions, followed by a hands-on session on calcium imaging. All the scientific and social events were fully participated by the scientific community that allowed a close and fruitful interaction and discussion between junior researchers and senior experts in the field. In this report, the contributions in individual sessions are summarized.

Sodium butyrate rescues dopaminergic cells from alpha-synuclein-induced transcriptional deregulation and DNA damage.

Alpha-synuclein (aSyn) is considered a major culprit in Parkinson's disease (PD) pathophysiology. However, the precise molecular function of the protein remains elusive. Recent evidence suggests that aSyn may play a role on transcription regulation, possibly by modulating the acetylation status of histones. Our study aimed at evaluating the impact of wild-type (WT) and mutant A30P aSyn on gene expression, in a dopaminergic neuronal cell model, and decipher potential mechanisms underlying aSyn-mediated transcriptional deregulation. We performed gene expression analysis using RNA-sequencing in Lund Human Mesencephalic (LUHMES) cells expressing endogenous (control) or increased levels of WT or A30P aSyn. Compared to control cells, cells expressing both aSyn variants exhibited robust changes in the expression of several genes, including downregulation of major genes involved in DNA repair. WT aSyn, unlike A30P aSyn, promoted DNA damage and increased levels of phosphorylated p53. In dopaminergic neuronal cells, increased aSyn expression led to reduced levels of acetylated histone 3. Importantly, treatment with sodium butyrate, a histone deacetylase inhibitor (HDACi), rescued WT aSyn-induced DNA damage, possibly via upregulation of genes involved in DNA repair. Overall, our findings provide novel and compelling insight into the mechanisms associated with aSyn neurotoxicity in dopaminergic cells, which could be ameliorated with an HDACi. Future studies will be crucial to further validate these findings and to define novel possible targets for intervention in PD.

Oxidative stress involving changes in Nrf2 and ER stress in early stages of Alzheimer's disease.

Oxidative stress and endoplasmic reticulum (ER) stress have been associated with Alzheimer's disease (AD) progression. In this study we analyzed whether oxidative stress involving changes in Nrf2 and ER stress may constitute early events in AD pathogenesis by using human peripheral blood cells and an AD transgenic mouse model at different disease stages. Increased oxidative stress and increased phosphorylated Nrf2 (p(Ser40)Nrf2) were observed in human peripheral blood mononuclear cells (PBMCs) isolated from individuals with mild cognitive impairment (MCI). Moreover, we observed impaired ER Ca2+ homeostasis and increased ER stress markers in PBMCs from MCI individuals and mild AD patients. Evidence of early oxidative stress defense mechanisms in AD was substantiated by increased p(Ser40)Nrf2 in 3month-old 3xTg-AD male mice PBMCs, and also with increased nuclear Nrf2 levels in brain cortex. However, SOD1 protein levels were decreased in human MCI PBMCs and in 3xTg-AD mice brain cortex; the latter further correlated with reduced SOD1 mRNA levels. Increased ER stress was also detected in the brain cortex of young female and old male 3xTg-AD mice. We demonstrate oxidative stress and early Nrf2 activation in AD human and mouse models, which fails to regulate some of its targets, leading to repressed expression of antioxidant defenses (e.g., SOD-1), and extending to ER stress. Results suggest markers of prodromal AD linked to oxidative stress associated with Nrf2 activation and ER stress that may be followed in human peripheral blood mononuclear cells.

Overexpression of BDNF and Full-Length TrkB Receptor Ameliorate Striatal Neural Survival in Huntington's Disease.

BACKGROUND: Several cellular mechanisms have been proposed to explain the pathogenesis of Huntington's disease (HD), including the lack of striatal brain-derived neurotrophic factor (BDNF). Thus, by preferentially binding to tropomyosin receptor kinase B (TrkB) receptor, BDNF is an important neurotrophin implicated in striatal neuronal survival. OBJECTIVE: To study the influence of BDNF and TrkB receptors in intracellular signaling pathways and caspase-3 activation in HD striatal cells. METHODS: HD mutant knockin and wild-type striatal cells were transduced with preproBDNF or full-length TrkB receptors to analyze BDNF processing, AKT and extracellular signal-regulated kinase (ERK) activation and the activity of caspase-3 in the absence or presence of staurosporine (STS). RESULTS: HD mutant cells transduced with preproBDNF-mCherry (mCh) expressed similar levels of pro- and mature BDNF compared to WT cells, but HD cells released lower levels of pro- and mature BDNF. Despite this, BDNF-mCh overexpression rescued decreased AKT phosphorylation and reduced the caspase-3 activation observed in HD cells. Activated ERK was also enhanced in HD BDNF-mCh/TrkB-eGFP receptor co-cultures. Of relevance, overexpression of TrkB-eGFP in HD cells decreased caspase-3 activation, and stimulation of TrkB-eGFP-transduced mutant cells with recombinant human BDNF reduced both basal and STS-induced caspase-3 activation. CONCLUSION: The results highlight the importance of BDNF-induced TrkB receptor signaling in rescuing HD-mediated apoptotic features in striatal cells.

Restoration of c-Src/Fyn Proteins Rescues Mitochondrial Dysfunction in Huntington's Disease.

Aims: Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder with no effective therapies. Mutant huntingtin protein (mHTT), the main HD proteinaceous hallmark, has been linked to reactive oxygen species (ROS) formation and mitochondrial dysfunction, among other pathological mechanisms. Importantly, Src-related kinases, c-Src and Fyn, are activated by ROS and regulate mitochondrial activity. However, c-Src/Fyn involvement in HD is largely unexplored. Thus, in this study, we aimed at exploring changes in Src/Fyn proteins in HD models and their role in defining altered mitochondrial function and dynamics and redox regulation. Results: We show, for the first time, that c-Src/Fyn phosphorylation/activation and proteins levels are decreased in several human and mouse HD models mainly due to autophagy degradation, concomitantly with mHtt-expressing cells showing enhanced TFEB-mediated autophagy induction and autophagy flux. c-Src/Fyn co-localization with mitochondria is also reduced. Importantly, the expression of constitutive active c-Src/Fyn to restore active Src kinase family (SKF) levels improves mitochondrial morphology and function, namely through improved mitochondrial transmembrane potential, mitochondrial basal respiration, and ATP production, but it did not affect mitophagy. In addition, constitutive active c-Src/Fyn expression diminishes the levels of reactive species in cells expressing mHTT. Innovation: This work supports a relevant role for c-Src/Fyn proteins in controlling mitochondrial function and redox regulation in HD, revealing a potential HD therapeutic target. Conclusion: c-Src/Fyn restoration in HD improves mitochondrial morphology and function, precluding the rise in oxidant species and cell death. Antioxid. Redox Signal. 38, 95-114.

Mitochondrial and Redox-Based Therapeutic Strategies in Huntington's Disease.

Significance: The molecular processes that determine Huntington's disease (HD) pathogenesis are not yet fully understood, and until now no effective neuroprotective therapeutic strategies have been developed. Mitochondria are one of most important organelles required for neuronal homeostasis, by providing metabolic pathways relevant for energy production, regulating calcium homeostasis, or controlling free radical generation and cell death. Because augmented reactive oxygen species (ROS) accompanied by mitochondrial dysfunction are relevant early HD mechanisms, targeting these cellular mechanisms may constitute relevant therapeutic approaches. Recent Advances: Previous findings point toward a close relationship between mitochondrial dysfunction and redox changes in HD. Mutant huntingtin (mHTT) can directly interact with mitochondrial proteins, as translocase of the inner membrane 23 (TIM23), disrupting mitochondrial proteostasis and favoring ROS production and HD progression. Furthermore, abnormal brain and muscle redox signaling contributes to altered proteostasis and motor impairment in HD, which can be improved with the mitochondria-targeted antioxidant mitoquinone or resveratrol, an SIRT1 activator that ameliorates mitochondrial biogenesis and function. Critical Issues: Various antioxidants and metabolic enhancers have been studied in HD; however, the real outcome of these molecules is still debatable. New compounds have proven to ameliorate mitochondrial and redox-based signaling pathways in early stages of HD, potentially precluding selective neurodegeneration. Future Directions: Unraveling the molecular etiology of deregulated mitochondrial function and dynamics, and oxidative stress opens new prospects for HD therapeutics. In this review, we explore the role of redox unbalance and mitochondrial dysfunction in HD progression, and further describe advances on clinical trials in HD based on mitochondrial and redox-based therapeutic strategies.

Macroautophagy and Mitophagy in Neurodegenerative Disorders: Focus on Therapeutic Interventions.

Macroautophagy, a quality control mechanism, is an evolutionarily conserved pathway of lysosomal degradation of protein aggregates, pathogens, and damaged organelles. As part of its vital homeostatic role, macroautophagy deregulation is associated with various human disorders, including neurodegenerative diseases. There are several lines of evidence that associate protein misfolding and mitochondrial dysfunction in the etiology of Alzheimer's, Parkinson's, and Huntington's diseases. Macroautophagy has been implicated in the degradation of different protein aggregates such as Aß, tau, alpha-synuclein (α-syn), and mutant huntingtin (mHtt) and in the clearance of dysfunctional mitochondria. Taking these into consideration, targeting autophagy might represent an effective therapeutic strategy to eliminate protein aggregates and to improve mitochondrial function in these disorders. The present review describes our current understanding on the role of macroautophagy in neurodegenerative disorders and focuses on possible strategies for its therapeutic modulation.

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.

MPV17 Mutations Are Associated With a Quiescent Energetic Metabolic Profile.

Mutations in the MPV17 gene are associated with hepatocerebral form of mitochondrial depletion syndrome. The mechanisms through which MPV17 mutations cause respiratory chain dysfunction and mtDNA depletion is still unclear. The MPV17 gene encodes an inner membrane mitochondrial protein that was recently described to function as a non-selective channel. Although its exact function is unknown, it is thought to be important in the maintenance of mitochondrial membrane potential (ΔΨm). To obtain more information about the role of MPV17 in human disease, we investigated the effect of MPV17 knockdown and of selected known MPV17 mutations associated with MPV17 disease in vitro. We used different approaches in order to evaluate the cellular consequences of MPV17 deficiency. We found that lower levels of MPV17 were associated with impaired mitochondrial respiration and with a quiescent energetic metabolic profile. All the mutations studied destabilized the protein, resulting in reduced protein levels. We also demonstrated that different mutations caused different cellular abnormalities, including increased ROS production, decreased oxygen consumption, loss of ΔΨm, and mislocalization of MPV17 protein. Our study provides novel insight into the molecular effects of MPV17 mutations and opens novel possibilities for testing therapeutic strategies for a devastating group of disorders.

Mitochondrial and Redox Modifications in Huntington Disease Induced Pluripotent Stem Cells Rescued by CRISPR/Cas9 CAGs Targeting.

Mitochondrial deregulation has gained increasing support as a pathological mechanism in Huntington's disease (HD), a genetic-based neurodegenerative disorder caused by CAG expansion in the HTT gene. In this study, we thoroughly investigated mitochondrial-based mechanisms in HD patient-derived iPSC (HD-iPSC) and differentiated neural stem cells (NSC) versus control cells, as well as in cells subjected to CRISPR/Cas9-CAG repeat deletion. We analyzed mitochondrial morphology, function and biogenesis, linked to exosomal release of mitochondrial components, glycolytic flux, ATP generation and cellular redox status. Mitochondria in HD cells exhibited round shape and fragmented morphology. Functionally, HD-iPSC and HD-NSC displayed lower mitochondrial respiration, exosomal release of cytochrome c, decreased ATP/ADP, reduced PGC-1α and complex III subunit expression and activity, and were highly dependent on glycolysis, supported by pyruvate dehydrogenase (PDH) inactivation. HD-iPSC and HD-NSC mitochondria showed ATP synthase reversal and increased calcium retention. Enhanced mitochondrial reactive oxygen species (ROS) were also observed in HD-iPSC and HD-NSC, along with decreased UCP2 mRNA levels. CRISPR/Cas9-CAG repeat deletion in HD-iPSC and derived HD-NSC ameliorated mitochondrial phenotypes. Data attests for intricate metabolic and mitochondrial dysfunction linked to transcriptional deregulation as early events in HD pathogenesis, which are alleviated following CAG deletion.

Cellular and molecular mechanisms involved in the neurotoxicity of opioid and psychostimulant drugs.

Substance abuse and addiction are the most costly of all the neuropsychiatric disorders. In the last decades, much progress has been achieved in understanding the effects of the drugs of abuse in the brain. However, efficient treatments that prevent relapse have not been developed. Drug addiction is now considered a brain disease, because the abuse of drugs affects several brain functions. Neurological impairments observed in drug addicts may reflect drug-induced neuronal dysfunction and neurotoxicity. The drugs of abuse directly or indirectly affect neurotransmitter systems, particularly dopaminergic and glutamatergic neurons. This review explores the literature reporting cellular and molecular alterations reflecting the cytotoxicity induced by amphetamines, cocaine and opiates in neuronal systems. The neurotoxic effects of drugs of abuse are often associated with oxidative stress, mitochondrial dysfunction, apoptosis and inhibition of neurogenesis, among other mechanisms. Understanding the mechanisms that underlie brain dysfunction observed in drug-addicted individuals may contribute to improve the treatment of drug addiction, which may have social and economic consequences.

Amyloid ß Peptide Compromises Neural Stem Cell Fate by Irreversibly Disturbing Mitochondrial Oxidative State and Blocking Mitochondrial Biogenesis and Dynamics.

Alzheimer's disease (AD) is the most common neurodegenerative disease and is characterized by the accumulation of amyloid ß peptide (Aß). Although most AD mouse models present a decline in neurogenesis, they express mutated genes which regulate neurogenesis per se and are not present in most AD patients, thus masking the real impact of Aß on adult neurogenesis. Mitochondrion, a well-known target of Aß in neurons, is a main regulator of neural stem cell (NSC) fate. Here, we aimed to investigate the impact of Aß on NSC mitochondria and cell fate decisions, namely whether and how Aß affects neurogenesis. NSC fate and mitochondrial parameters, including biogenesis, dynamics, and oxidative stress, were evaluated. Our results showed that Aß impaired NSC viability and proliferation and indirectly blocked neurogenic differentiation, by disrupting mitochondrial signaling of self-renewing NSCs. Importantly, Aß decreased ATP levels, generated oxidative stress, and affected the radical scavenger system through SOD2 and SIRT3. Aß also reduced mtDNA and mitochondrial biogenesis proteins, such as Tfam, PGC-1α, and NRF1, and inhibited activation of PGC-1α-positive regulator CREB. Moreover, Aß triggered mitochondrial fragmentation in self-renewing NSCs and reduced mitochondrial fusion proteins, such as Mfn2 and ERRα. Notably, Aß compromised NSC commitment and survival by irreversibly impairing mitochondria and thwarting any neurogenic rescue through mitochondrial biogenesis, dynamics, or radical scavenger system. Altogether, this study brings new perspective to rethink the molecular targets relevant for endogenous NSC-based strategies in AD.

Chronic stress triggers divergent dendritic alterations in immature neurons of the adult hippocampus, depending on their ultimate terminal fields.

Chronic stress, a suggested precipitant of brain pathologies, such as depression and Alzheimer's disease, is known to impact on brain plasticity by causing neuronal remodeling as well as neurogenesis suppression in the adult hippocampus. Although many studies show that stressful conditions reduce the number of newborn neurons in the adult dentate gyrus (DG), little is known about whether and how stress impacts on dendritic development and structural maturation of these newborn neurons. We, herein, demonstrate that chronic stress impacts differentially on doublecortin (DCX)-positive immature neurons in distinct phases of maturation. Specifically, the density of the DCX-positive immature neurons whose dendritic tree reaches the inner molecular layer (IML) of DG is reduced in stressed animals, whereas their dendritic complexity is increased. On the contrary, no change on the density of DCX-positive neurons whose dendritic tree extends to the medial/outer molecular layer (M/OML) of the DG is found under stress conditions, whereas the dendritic complexity of these cells is diminished. In addition, DCX+ cells displayed a more complex and longer arbor in the dendritic compartments located in the granular cell layer of the DG under stress conditions; on the contrary, their dendritic segments localized into the M/OML were shorter and less complex. These findings suggest that the neuroplastic effects of chronic stress on dendritic maturation and complexity of DCX+ immature neurons vary based on the different maturation stage of DCX-positive cells and the different DG sublayer, highlighting the complex and dynamic stress-driven neuroplasticity of immature neurons in the adult hippocampus.

Mechanisms of neurodegeneration in Huntington's disease.

Huntington's disease (HD) is caused by an expansion of cytosine-adenine-guanine (CAG) repeats in the huntingtin gene, which leads to neuronal loss in the striatum and cortex and to the appearance of neuronal intranuclear inclusions of mutant huntingtin. Huntingtin plays a role in protein trafficking, vesicle transport, postsynaptic signaling, transcriptional regulation, and apoptosis. Thus, a loss of function of the normal protein and a toxic gain of function of the mutant huntingtin contribute to the disruption of multiple intracellular pathways. Furthermore, excitotoxicity, dopamine toxicity, metabolic impairment, mitochondrial dysfunction, oxidative stress, apoptosis, and autophagy have been implicated in the progressive degeneration observed in HD. Nevertheless, despite the efforts of a multidisciplinary scientific community, there is no cure for this devastating neurodegenerative disorder. This review presents an overview of the mechanisms that may contribute for HD pathogenesis. Ultimately, a better understanding of these mechanisms will lead to the development of more effective therapeutic targets.

Coriolus versicolor biomass increases dendritic arborization of newly-generated neurons in mouse hippocampal dentate gyrus.

Brain cognitive reserve refers to the ability of the brain to manage different challenges that arise throughout life, making it resilient to neuropathology. Hippocampal adult neurogenesis has been considered to be a relevant contributor for brain cognitive reserve and brain plasticity. Coriolus versicolor (CV), a common healthful mushroom, has been receiving increasing attention by its antitumoral, anti-inflammatory, antioxidant, antibacterial, and immunomodulatory properties, including in the hippocampus. Herein, we evaluated whether CV biomass oral administration for 2.5 months enhances hippocampal neurogenic reserve under normal/physiological conditions, by quantifying hippocampal dentate gyrus (DG) granular cell layer (GCL) and subgranular zone (SGZ) volumes, proliferation, number and dendritic complexity features of hippocampal newly-generated neurons. We also analyzed ß-catenin levels in DG newly-generated immature neurons, because it plays a major role in neurogenesis. Although no differences were observed in the volume of GCL and SGZ layers, in proliferation and in the number of newly-generated neurons of controls and CV-administered mice, we found that CV administration promotes a significant increase in dendritic length and branching and total dendritic volume of immature neurons, suggesting a positive effect of oral CV administration in the hippocampal neurogenic reserve. We also observed that ß-catenin levels are increased both in the nucleus and cytoplasm of DG immature neurons, suggesting that Wnt/ß-catenin signalling may play an important role in the CV positive effect on the differentiation of these cells. These data unveil a so far unexplored neurogenic potential of CV supplementation, which emerges as a possible preventive strategy for different neurological conditions.

Dominant-Negative Effects of Adult-Onset Huntingtin Mutations Alter the Division of Human Embryonic Stem Cells-Derived Neural Cells.

Mutations of the huntingtin protein (HTT) gene underlie both adult-onset and juvenile forms of Huntington's disease (HD). HTT modulates mitotic spindle orientation and cell fate in mouse cortical progenitors from the ventricular zone. Using human embryonic stem cells (hESC) characterized as carrying mutations associated with adult-onset disease during pre-implantation genetic diagnosis, we investigated the influence of human HTT and of an adult-onset HD mutation on mitotic spindle orientation in human neural stem cells (NSCs) derived from hESCs. The RNAi-mediated silencing of both HTT alleles in neural stem cells derived from hESCs disrupted spindle orientation and led to the mislocalization of dynein, the p150Glued subunit of dynactin and the large nuclear mitotic apparatus (NuMA) protein. We also investigated the effect of the adult-onset HD mutation on the role of HTT during spindle orientation in NSCs derived from HD-hESCs. By combining SNP-targeting allele-specific silencing and gain-of-function approaches, we showed that a 46-glutamine expansion in human HTT was sufficient for a dominant-negative effect on spindle orientation and changes in the distribution within the spindle pole and the cell cortex of dynein, p150Glued and NuMA in neural cells. Thus, neural derivatives of disease-specific human pluripotent stem cells constitute a relevant biological resource for exploring the impact of adult-onset HD mutations of the HTT gene on the division of neural progenitors, with potential applications in HD drug discovery targeting HTT-dynein-p150Glued complex interactions.

Limited Effect of Chronic Valproic Acid Treatment in a Mouse Model of Machado-Joseph Disease.

Machado-Joseph disease (MJD) is an inherited neurodegenerative disease, caused by a CAG repeat expansion within the coding region of ATXN3 gene, and which currently lacks effective treatment. In this work we tested the therapeutic efficacy of chronic treatment with valproic acid (VPA) (200mg/kg), a compound with known neuroprotection activity, and previously shown to be effective in cell, fly and nematode models of MJD. We show that chronic VPA treatment in the CMVMJD135 mouse model had limited effects in the motor deficits of these mice, seen mostly at late stages in the motor swimming, beam walk, rotarod and spontaneous locomotor activity tests, and did not modify the ATXN3 inclusion load and astrogliosis in affected brain regions. However, VPA chronic treatment was able to increase GRP78 protein levels at 30 weeks of age, one of its known neuroprotective effects, confirming target engagement. In spite of limited results, the use of another dosage of VPA or of VPA in a combined therapy with molecules targeting other pathways, cannot be excluded as potential strategies for MJD therapeutics.