Cell-Free and In Vivo Characterization of the Inhibitory Activity of Lavado Cocoa Flavanols on the Amyloid Protein Ataxin-3: Toward New Approaches against Spinocerebellar Ataxia Type 3.

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder characterized by ataxia and other neurological manifestations, with a poor prognosis and a lack of effective therapies. The amyloid aggregation of the ataxin-3 protein is a hallmark of SCA3 and one of the main biochemical events prompting its onset, making it a prominent target for the development of preventive and therapeutic interventions. Here, we tested the efficacy of an aqueous Lavado cocoa extract and its polyphenolic components against ataxin-3 aggregation and neurotoxicity. The combination of biochemical assays and atomic force microscopy morphological analysis provided clear evidence of cocoa flavanols' ability to hinder ATX3 amyloid aggregation through direct physical interaction, as assessed by NMR spectroscopy. The chemical identity of the flavanols was investigated by ultraperformance liquid chromatography-high-resolution mass spectrometry. The use of the preclinical model Caenorhabditis elegans allowed us to demonstrate cocoa flavanols' ability to ameliorate ataxic phenotypes in vivo. To the best of our knowledge, Lavado cocoa is the first natural source whose extract is able to directly interfere with ATX3 aggregation, leading to the formation of off-pathway species.

Sodium valproate increases activity of the sirtuin pathway resulting in beneficial effects for spinocerebellar ataxia-3 in vivo.

Machado-Joseph disease (MJD, also known as spinocerebellar ataxia type 3) is a fatal neurodegenerative disease that impairs control and coordination of movement. Here we tested whether treatment with the histone deacetylase inhibitor sodium valproate (valproate) prevented a movement phenotype that develops in larvae of a transgenic zebrafish model of the disease. We found that treatment with valproate improved the swimming of the MJD zebrafish, affected levels of acetylated histones 3 and 4, but also increased expression of polyglutamine expanded human ataxin-3. Proteomic analysis of protein lysates generated from the treated and untreated MJD zebrafish also predicted that valproate treatment had activated the sirtuin longevity signaling pathway and this was confirmed by findings of increased SIRT1 protein levels and sirtuin activity in valproate treated MJD zebrafish and HEK293 cells expressing ataxin-3 84Q, respectively. Treatment with resveratrol (another compound known to activate the sirtuin pathway), also improved swimming in the MJD zebrafish. Co-treatment with valproate alongside EX527, a SIRT1 activity inhibitor, prevented induction of autophagy by valproate and the beneficial effects of valproate on the movement in the MJD zebrafish, supporting that they were both dependent on sirtuin activity. These findings provide the first evidence of sodium valproate inducing activation of the sirtuin pathway. Further, they indicate that drugs that target the sirtuin pathway, including sodium valproate and resveratrol, warrant further investigation for the treatment of MJD and related neurodegenerative diseases.

Small Molecule Rescue of ATXN3 Toxicity in C. elegans via TFEB/HLH-30.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is a polyglutamine expansion disease arising from a trinucleotide CAG repeat expansion in exon 10 of the gene ATXN3. There are no effective pharmacological treatments for MJD, thus the identification of new pathogenic mechanisms, and the development of novel therapeutics is urgently needed. In this study, we performed a comprehensive, blind drug screen of 3942 compounds (many FDA approved) and identified small molecules that rescued the motor-deficient phenotype in transgenic ATXN3 Caenorhabditis elegans strain. Out of this screen, five lead compounds restoring motility, protecting against neurodegeneration, and increasing the lifespan in ATXN3-CAG89 mutant worms were identified. These compounds were alfacalcidol, chenodiol, cyclophosphamide, fenbufen, and sulfaphenazole. We then investigated how these molecules might exert their neuroprotective properties. We found that three of these compounds, chenodiol, fenbufen, and sulfaphenazole, act as modulators for TFEB/HLH-30, a key transcriptional regulator of the autophagy process, and require this gene for their neuroprotective activities. These genetic-chemical approaches, using genetic C. elegans models for MJD and the screening, are promising tools to understand the mechanisms and pathways causing neurodegeneration, leading to MJD. Positively acting compounds may be promising candidates for investigation in mammalian models of MJD and preclinical applications in the treatment of this disease.

Protective role of vitamin B6 against mitochondria damage in Drosophila models of SCA3.

Polyglutamine (polyQ)-mediated mitochondria damage is one of the prime causes of polyQ toxicity, which leads to the loss of neurons and the injury of non-neuronal cells. With the discovery of the crucial role of the gut-brain axis and gut microbes in neurological diseases, the relationship between visceral damage and neurological disorders has also received extensive attention. This study successfully simulated the polyQ mitochondrial damage model by expressing 78 or 84 polyglutamine-containing Ataxin3 proteins in Drosophila intestinal enterocytes. In vivo, polyQ expression can reduce mitochondrial membrane potential, mitochondrial DNA damage, abnormal mitochondrial morphology, and loose mitochondrial cristae. Expression profiles evaluated by RNA-seq showed that mitochondrial structural genes and functional genes (oxidative phosphorylation and tricarboxylic acid cycle-related) were significantly down-regulated. More importantly, Bioinformatic analyses demonstrated that pathological polyQ expression induced vitamin B6 metabolic pathways abnormality. Active vitamin B6 participates in hundreds of enzymatic reactions and is very important for maintaining mitochondria's activities. In the SCA3 Drosophila model, Vitamin B6 supplementation significantly suppressed ECs mitochondria damage in guts and inhibited cellular polyQ aggregates in fat bodies, indicating a promising therapeutic strategy for the treatment of polyQ. Taken together, our results reveal a crucial role for the Vitamin B6-mediated mitochondrial protection in polyQ-induced cellular toxicity, which provides strong evidence for this process as a drug target in polyQ diseases treatment.

Protocol for the Characterization of the Cytosine-Adenine-Guanine Tract and Flanking Polymorphisms in Machado-Joseph Disease: Impact on Diagnosis and Development of Gene-Based Therapies.

Polyglutamine spinocerebellar ataxias (SCAs) constitute a group of autosomal dominantly inherited neurodegenerative disorders with considerable phenotypic overlap. Definitive diagnoses rely on the detection of a mutation in each associated locus, comprising the abnormal expansion of the trinucleotide cytosine-adenine-guanine (CAG) in coding exons. Assessment of single nucleotide polymorphisms associated with the CAG expansion in the context of SCAs is also relevant for improving molecular diagnosis and for generating novel therapeutic strategies. The current study is focused on Machado-Joseph disease/SCA type 3, with the aim of developing a protocol for the accurate determination of the CAG length in exon 10 of the human ATXN3 gene and to characterize flanking polymorphisms. A single pair of primers was designed and validated, and two complementary PCR-based methods were established. In method I, PCR amplicons were cloned and sequenced, allowing the assessment of three single nucleotide polymorphisms in the vicinity of the CAG repeat (C987GG/G987GG, TAA1118/TAC1118, and C1178/A1178), which can constitute potential targets for personalized gene-based therapies. Method II combines PCR, capillary electrophoresis, and a size correction formula, enabling a time and cost-effective determination of the number of CAGs. The established protocol paves the way to overcome technical difficulties related to the molecular characterization of the CAG motif and intragenic polymorphisms in the context of Machado-Joseph disease/SCA type 3 and may prove useful when applied to other polyglutamine SCAs.

Allele-selective lowering of mutant HTT protein by HTT-LC3 linker compounds.

Accumulation of mutant proteins is a major cause of many diseases (collectively called proteopathies), and lowering the level of these proteins can be useful for treatment of these diseases. We hypothesized that compounds that interact with both the autophagosome protein microtubule-associated protein 1A/1B light chain 3 (LC3)1 and the disease-causing protein may target the latter for autophagic clearance. Mutant huntingtin protein (mHTT) contains an expanded polyglutamine (polyQ) tract and causes Huntington's disease, an incurable neurodegenerative disorder2. Here, using small-molecule-microarray-based screening, we identified four compounds that interact with both LC3 and mHTT, but not with the wild-type HTT protein. Some of these compounds targeted mHTT to autophagosomes, reduced mHTT levels in an allele-selective manner, and rescued disease-relevant phenotypes in cells and in vivo in fly and mouse models of Huntington's disease. We further show that these compounds interact with the expanded polyQ stretch and could lower the level of mutant ataxin-3 (ATXN3), another disease-causing protein with an expanded polyQ tract3. This study presents candidate compounds for lowering mHTT and potentially other disease-causing proteins with polyQ expansions, demonstrating the concept of lowering levels of disease-causing proteins using autophagosome-tethering compounds.

Vulnerability of frontal brain neurons for the toxicity of expanded ataxin-3.

Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of CAG repeats in the ATXN3 gene leading to an elongated polyglutamine tract in the ataxin-3 protein. Previously, we demonstrated that symptoms of SCA3 are reversible in the first conditional mouse model for SCA3 directing ataxin-3 predominantly to the hindbrain. Here, we report on the effects of transgenic ataxin-3 expression in forebrain regions. Employing the Tet-off CamKII-promoter mouse line and our previously published SCA3 responder line, we generated double transgenic mice (CamKII/MJD77), which develop a neurological phenotype characterized by impairment in rotarod performance, and deficits in learning new motor tasks as well as hyperactivity. Ataxin-3 and ubiquitin-positive inclusions are detected in brains of double transgenic CamKII/MJD77 mice. After turning off the expression of pathologically expanded ataxin-3, these inclusions disappear. However, the observed phenotype could not be reversed, very likely due to pronounced apoptotic cell death in the frontal brain. Our data demonstrate that cerebellar expression is not required to induce a neurological phenotype using expanded ATXN3 as well as the pronounced sensibility of forebrain neurons for toxic ataxin-3.

Unbiased screen identifies aripiprazole as a modulator of abundance of the polyglutamine disease protein, ataxin-3.

No disease-modifying treatment exists for the fatal neurodegenerative polyglutamine disease known both as Machado-Joseph disease and spinocerebellar ataxia type 3. As a potential route to therapy, we identified small molecules that reduce levels of the mutant disease protein, ATXN3. Screens of a small molecule collection, including 1250 Food and Drug Administration-approved drugs, in a novel cell-based assay, followed by secondary screens in brain slice cultures from transgenic mice expressing the human disease gene, identified the atypical antipsychotic aripiprazole as one of the hits. Aripiprazole increased longevity in a Drosophila model of Machado-Joseph disease and effectively reduced aggregated ATXN3 species in flies and in brains of transgenic mice treated for 10 days. The aripiprazole-mediated decrease in ATXN3 abundance may reflect a complex response culminating in the modulation of specific components of cellular protein homeostasis. Aripiprazole represents a potentially promising therapeutic drug for Machado-Joseph disease and possibly other neurological proteinopathies.

Wake-promoting effects of ONO-4127Na, a prostaglandin DP1 receptor antagonist, in hypocretin/orexin deficient narcoleptic mice.

Prostaglandin (PG)D2 is an endogenous sleep substance, and a series of animal studies reported that PGD2 or PGD2 receptor (DP1) agonists promote sleep, while DP1 antagonists promote wakefulness. This suggests the possibility of use of PG DP1 antagonists as wake-promoting compounds. We therefore evaluated the wake-promoting effects of ONO-4127Na, a DP1 antagonist, in a mouse model of narcolepsy (i.e., orexin/ataxin-3 transgenic mice) and compared those to effects of modafinil. ONO-4127Na perfused in the basal forebrain (BF) area potently promoted wakefulness in both wild type and narcoleptic mice, and the wake-promoting effects of ONO-4127Na at 2.93 × 10(-4) M roughly corresponded to those of modafinil at 100 mg/kg (p.o.). The wake promoting effects of ONO-4127Na was observed both during light and dark periods, and much larger effects were seen during the light period when mice slept most of the time. ONO-4127Na, when perfused in the hypothalamic area, had no effects on sleep. We further demonstrated that wake-promoting effects of ONO-4127Na were abolished in DP1 KO mice, confirming that the wake-promoting effect of ONO-4127Na is mediated by blockade of the PG DP1 receptors located in the BF area. ONO-4127Na reduced DREM, an EEG/EMG assessment of behavioral cataplexy in narcoleptic mice, suggesting that ONO-4127Na is likely to have anticataplectic effects. DP1 antagonists may be a new class of compounds for the treatment of narcolepsy-cataplexy, and further studies are warranted.

Dystonia in Machado-Joseph disease: Clinical profile, therapy and anatomical basis.

INTRODUCTION: Dystonia is frequent in Machado-Joseph disease, but several important aspects are not yet defined, such as the detailed clinical profile, response to treatment and anatomical substrate. METHODS: We screened 75 consecutive patients and identified those with dystonia. The Burke-Marsden-Fahn Dystonia Rating Scale was employed to quantify dystonia severity. Patients with dystonia received levodopa 600 mg/day for 2 months and were videotaped before and after treatment. A blinded evaluator rated dystonia in the videos. Patients with disabling dystonia who failed to respond to levodopa treatment received botulinum toxin. Finally, volumetric T1 and diffusion tensor imaging sequences were obtained in the dystonic group using a 3T-MRI scanner to identify areas of gray and white matter that were selectively damaged. RESULTS: There were 21 patients with dystonia (28%): 9 classified as generalized and 12 as focal/segmental. Patients with dystonia had earlier onset and larger (CAG) expansions (28.9 ± 11.7 vs 40.6 ± 11.4; p < 0.001 and 75 vs 70; p < 0.001, respectively). Although group analyses failed to show benefit on levodopa (p = 0.07), some patients had objective improvement. In addition, ten patients received botulinum toxin resulting in a significant change in dystonia scores after 4 weeks (p = 0.03). Patients with dystonia had atrophy at pre- and paracentral cortices; whereas, non-dystonic patients had occipital atrophy. Basal ganglia volume was reduced in both groups, but atrophy at the thalami, cerebellar white matter and ventral diencephali was disproportionately higher in the dystonic group. CONCLUSION: Dystonia in Machado-Joseph disease is frequent and often disabling, but may respond to levodopa. It is associated predominantly with structural abnormalities around the motor cortices and in the thalami.

T1-11 and JMF1907 ameliorate polyglutamine-expanded ataxin-3-induced neurodegeneration, transcriptional dysregulation and ataxic symptom in the SCA3 transgenic mouse.

More studies are required to develop therapeutic agents for treating spinocerebellar ataxia type 3 (SCA3), which is caused by mutant polyglutamine-expanded ataxin-3 and is the most prevalent subtype of spinocerebellar ataxias. T1-11 [N6-(4-Hydroxybenzyl) adenosine], isolated from a Chinese medicinal herb Gastordia elata, is an adenosine A2A receptor agonist. SCA3 and Huntington's disease (HD) belong to a family of polyglutamine neurodegenerative diseases. T1-11 exerted a therapeutic effect on HD transgenic mouse by decreasing protein level of polyglutamine-expanded huntingtin in the striatum. In the present study, we test the possibility that T1-11 or JMF1907 [N6-(3-Indolylethyl) adenosine], a synthetic analog of T1-11, alleviates pontine neuronal death, cerebellar transcriptional downregulation and ataxic symptom in the SCA3 transgenic mouse expressing HA-tagged polyglutamine-expanded ataxin-3-Q79 (ataxin-3-Q79HA). Daily oral administration of T1-11 or JMF1907 prevented neuronal death of pontine nuclei in the SCA3 mouse with a dose-dependent manner. Oral application of T1-11 or JMF1907 reversed mutant ataxin-3-Q79-induced cerebellar transcriptional repression in the SCA3 transgenic mouse. T1-11 or JMF1907 ameliorated the symptom of motor incoordination displayed by SCA3 mouse. Oral administration of T1-11 or JMF1907 significantly decreased protein level of ataxin-3-Q79HA in the pontine nuclei or cerebellum of SCA3 mouse. T1-11 or JMF1907 significantly augmented the chymotrypsin-like activity of proteasome in the pontine nuclei or cerebellum of SCA3 mouse. Our results suggests that T1-11 and JMF1907 alleviate pontine neuronal death, cerebellar transcriptional downregulation and ataxic symptom of SCA3 transgenic mouse by augmenting the proteasome activity and reducing the protein level of polyglutamine-expanded ataxin-3-Q79 in the pontine nuclei and cerebellum.

Genetic deletion of melanin-concentrating hormone neurons impairs hippocampal short-term synaptic plasticity and hippocampal-dependent forms of short-term memory.

The cognitive role of melanin-concentrating hormone (MCH) neurons, a neuronal population located in the mammalian postero-lateral hypothalamus sending projections to all cortical areas, remains poorly understood. Mainly activated during paradoxical sleep (PS), MCH neurons have been implicated in sleep regulation. The genetic deletion of the only known MCH receptor in rodent leads to an impairment of hippocampal dependent forms of memory and to an alteration of hippocampal long-term synaptic plasticity. By using MCH/ataxin3 mice, a genetic model characterized by a selective deletion of MCH neurons in the adult, we investigated the role of MCH neurons in hippocampal synaptic plasticity and hippocampal-dependent forms of memory. MCH/ataxin3 mice exhibited a deficit in the early part of both long-term potentiation and depression in the CA1 area of the hippocampus. Post-tetanic potentiation (PTP) was diminished while synaptic depression induced by repetitive stimulation was enhanced suggesting an alteration of pre-synaptic forms of short-term plasticity in these mice. Behaviorally, MCH/ataxin3 mice spent more time and showed a higher level of hesitation as compared to their controls in performing a short-term memory T-maze task, displayed retardation in acquiring a reference memory task in a Morris water maze, and showed a habituation deficit in an open field task. Deletion of MCH neurons could thus alter spatial short-term memory by impairing short-term plasticity in the hippocampus. Altogether, these findings could provide a cellular mechanism by which PS may facilitate memory encoding. Via MCH neuron activation, PS could prepare the day's learning by increasing and modulating short-term synaptic plasticity in the hippocampus.