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.

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.

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.

Anti-Tribbles Pseudokinase 2 (TRIB2)-Immunization Modulates Hypocretin/Orexin Neuronal Functions.

Study Objectives: Recent findings showed that 16%-26% of narcolepsy patients were positive for anti-tribbles pseudokinase 2 (TRIB2) antibody, and the intracerebroventricular administration of immunoglobulin-G purified from anti-TRIB2 positive narcolepsy patients caused hypocretin/orexin neuron loss. We investigated the pathophysiological role of TRIB2 antibody using TRIB2-immunized rats and hypocretin/ataxin-3 transgenic (ataxin-3) mice. Methods: Plasma, cerebrospinal fluid (CSF), and hypothalamic tissues from TRIB2-immunized rats were collected. Anti-TRIB2 titers, hypocretin contents, mRNA expressions, the cell count of hypocretin neurons, and immunoreactivity of anti-TRIB2 antibodies on hypocretin neurons were investigated. The plasma from ataxin-3 mice was also used to determine the anti-TRIB2 antibody titer changes following the loss of hypocretin neurons. Results: TRIB2 antibody titers increased in the plasma and CSF of TRIB2-immunized rats. The hypothalamic tissue immunostained with the sera from TRIB2-immunized rats revealed positive signals in the cytoplasm of hypcretin neurons. While no changes were found regarding hypothalamic hypocretin contents or cell counts, but there were significant decreases of the hypocretin mRNA level and release into the CSF. The plasma from over 26-week-old ataxin-3 mice, at the advanced stage of hypocretin cell destruction, showed positive reactions against TRIB2 antigen, and positive plasma also reacted with murine hypothalamic hypocretin neurons. Conclusions: Our results suggest that the general activation of the immune system modulates the functions of hypocretin neurons. The absence of a change in hypocretin cell populations suggested that factors other than anti-TRIB2 antibody play a part in the loss of hypocretin neurons in narcolepsy. The increased anti-TRIB2 antibody after the destruction of hypocretin neurons suggest that anti-TRIB2 antibody in narcolepsy patients is the consequence rather than the inciting cause of hypocretin cell destruction.

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.

Hypocretin/orexin loss changes the hypothalamic immune response.

Hypocretin, also known as orexin, maintains the vigilance state and regulates various physiological processes, such as arousal, sleep, food intake, energy expenditure, and reward. Previously, we found that when wild-type mice and hypocretin/ataxin-3 littermates (which are depleted of hypothalamic hypocretin-expressing neurons postnatally) were administered lipopolysaccharide (LPS), the two genotypes exhibited significant differences in their sleep/wake cycle, including differences in the degree of increase in sleep periods and in recovery from sickness behaviour. In the present study, we examined changes in the hypothalamic vigilance system and in the hypothalamic expression of inflammatory factors in response to LPS in hypocretin/ataxin-3 mice. Peripheral immune challenge with LPS affected the hypothalamic immune response and vigilance states. This response was altered by the loss of hypocretin. Hypocretin expression was inhibited after LPS injection in both hypocretin/ataxin-3 mice and their wild-type littermates, but expression was completely abolished only in hypocretin/ataxin-3 mice. Increases in the number of histidine decarboxylase (HDC)-positive cells and in Hdc mRNA expression were found in hypocretin/ataxin-3 mice, and this increase was suppressed by LPS. Hypocretin loss did not impact the change in expression of hypothalamic inflammatory factors in response to LPS, except for interferon gamma and colony stimulating factor 3. The number of c-Fos-positive/HDC-positive cells in hypocretin/ataxin-3 mice administered LPS injections was elevated, even during the rest period, in all areas, suggesting that there is an increase in the activity of histaminergic neurons in hypocretin/ataxin-3 mice following LPS injection. Taken together, our results suggest a novel role for hypocretin in the hypothalamic response to peripheral immune challenge. Our findings contribute to the understanding of the pathophysiology of narcolepsy.

Combined therapy with m-TOR-dependent and -independent autophagy inducers causes neurotoxicity in a mouse model of Machado-Joseph disease.

A major pathological hallmark in several neurodegenerative disorders, like polyglutamine disorders (polyQ), including Machado-Joseph disease (MJD), is the formation of protein aggregates. MJD is caused by a CAG repeat expansion in the ATXN3 gene, resulting in an abnormal protein, which is prone to misfolding and forms cytoplasmic and nuclear aggregates within neurons, ultimately inducing neurodegeneration. Treatment of proteinopathies with drugs that up-regulate autophagy has shown promising results in models of polyQ diseases. Temsirolimus (CCI-779) inhibits the mammalian target of rapamycin (m-TOR), while lithium chloride (LiCl) acts by inhibiting inositol monophosphatase, both being able to induce autophagy. We have previously shown that chronic treatment with LiCl (10.4 mg/kg) had limited effects in a transgenic MJD mouse model. Also, others have shown that CCI-779 had mild positive effects in a different mouse model of the disease. It has been suggested that the combination of mTOR-dependent and -independent autophagy inducers could be a more effective therapeutic approach. To further explore this avenue toward therapy, we treated CMVMJD135 transgenic mice with a conjugation of CCI-779 and LiCl, both at concentrations known to induce autophagy and not to be toxic. Surprisingly, this combined treatment proved to be deleterious to both wild-type (wt) and transgenic animals, failing to rescue their neurological symptoms and actually exerting neurotoxic effects. These results highlight the possible dangers of manipulating autophagy in the nervous system and suggest that a better understanding of the potential disruption in the autophagy pathway in MJD is required before successful long-term autophagy modulating therapies can be developed.

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.