SIRT2 Deficiency Exacerbates Hepatic Steatosis via a Putative Role of the ER Stress Pathway.

Nonalcoholic fatty liver disease (NAFLD), a condition strongly associated with obesity and insulin resistance, is characterized by hepatic lipid accumulation and activation of the endoplasmic reticulum (ER) stress response. The sirtuin 2 (SIRT2) protein deacetylase is emerging as a new player in metabolic homeostasis, but its role in the development of hepatic steatosis and its link with ER stress activation remains unknown. SIRT2-knockout (SIRT2-KO) and wild-type mice were fed either a control or a high-fat diet (HFD) for 4 weeks. Genetic manipulation of SIRT2 levels was performed in human hepatic cells. Although apparently normal under a control diet, SIRT2-KO mice showed accelerated body weight gain and adiposity on a HFD, accompanied by severe insulin resistance. Importantly, SIRT2-KO mice exhibited worsened hepatic steatosis independently from diet, consistent with upregulated gene expression of lipogenic enzymes and increased expression of ER stress markers. Exposure of hepatic cells to palmitate induced lipid accumulation, increased ER stress, and decreased SIRT2 expression. Moreover, SIRT2-silenced cells showed enhanced lipid accumulation and ER stress activation under basal conditions, whereas SIRT2 overexpression abrogated palmitate-induced lipid deposition and ER stress activation. Our findings reveal a role for SIRT2 in the regulation of hepatic lipid homeostasis, potentially through the ER stress response, suggesting that SIRT2 activation might constitute a therapeutic strategy against obesity and its metabolic complications.

Identification of the calpain-generated toxic fragment of ataxin-3 protein provides new avenues for therapy of Machado-Joseph disease| Spinocerebellar ataxia type 3.

AIMS: Machado-Joseph disease (MJD) is the most frequent dominantly inherited cerebellar ataxia worldwide. Expansion of a CAG trinucleotide in the MJD1 gene translates into a polyglutamine tract within ataxin-3, which upon proteolysis may lead to MJD. The aim of this work was to understand the in vivo contribution of calpain proteases to the pathogenesis of MJD. Therefore, we investigated (a) the calpain cleavage sites in ataxin-3 protein, (b) the most toxic ataxin-3 fragment generated by calpain cleavage and (c) whether targeting calpain cleavage sites of mutant ataxin-3 could be a therapeutic strategy for MJD. METHODS: We generated truncated and calpain-resistant constructs at the predicted cleavage sites of ataxin-3 using inverse PCR mutagenesis. Lentiviral vectors encoding these constructs were transduced in the adult mouse brain prior to western blot and immunohistochemical analysis 5 and 8 weeks later. RESULTS: We identified the putative calpain cleavage sites for both wild-type and mutant ataxin-3 proteins. The mutation of these sites eliminated the formation of the toxic fragments, namely, the 26-kDa fragment, the major contributor for striatal degeneration. Nonetheless, reducing the formation of both the 26- and 34-kDa fragments was required to preclude the intranuclear localisation of ataxin-3. A neuroprotective effect was observed upon mutagenesis of calpain cleavage sites within mutant ataxin-3 protein. CONCLUSIONS: These findings suggest that the calpain system should be considered a target for MJD therapy. The identified calpain cleavage sites will contribute to the design of targeted drugs and genome editing systems for those specific locations.

Trehalose alleviates the phenotype of Machado-Joseph disease mouse models.

BACKGROUND: Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3, is the most common of the dominantly inherited ataxias worldwide and is characterized by mutant ataxin-3 aggregation and neuronal degeneration. There is no treatment available to block or delay disease progression. In this work we investigated whether trehalose, a natural occurring disaccharide widely used in food and cosmetic industry, would rescue biochemical, behavioral and neuropathological features of an in vitro and of a severe MJD transgenic mouse model. METHODS: Two MJD animal models, a lentiviral based and a transgenic model, were orally treated with 2% trehalose solution for a period of 4 and 30 weeks, respectively. Motor behavior (rotarod, grip strength and footprint patterns) was evaluated at different time points and neuropathological features were evaluated upon in-life phase termination. RESULTS: Trehalose-treated MJD mice equilibrated for a longer time in the rotarod apparatus and exhibited an improvement of ataxic gait in footprint analysis. Trehalose-mediated improvements in motor behaviour were associated with a reduction of the MJD-associated neuropathology, as MJD transgenic mice treated with trehalose presented preservation of cerebellar layers thickness and a decrease in the size of ataxin-3 aggregates in Purkinje cells. In agreement, an improvement of neuropathological features was also observed in the full length lentiviral-based mouse model of MJD submitted to 2% trehalose treatment. CONCLUSIONS: The present study suggests trehalose as a safety pharmacological strategy to counteract MJD-associated behavioural and neuropathological impairments.

Molecular Mechanisms and Cellular Pathways Implicated in Machado-Joseph Disease Pathogenesis.

Machado-Joseph disease (MJD) is a dominantly inherited disorder originally described in people of Portuguese descent, and associated with the expansion of a CAG tract in the coding region of the causative gene MJD1/ATX3. The CAG repeats range from 10 to 51 in the normal population and from 55 to 87 in SCA3/MJD patients. MJD1 encodes ataxin-3, a protein whose physiological function has been linked to ubiquitin-mediated proteolysis. Despite the identification of the causative mutation, the pathogenic process leading to the neurodegeneration observed in the disease is not yet completely understood. In the past years, several studies identified different molecular mechanisms and cellular pathways as being impaired or deregulated in MJD. Autophagy, proteolysis or post-translational modifications, among other processes, were implicated in MJD pathogenesis. From these studies it was possible to identify new targets for therapeutic intervention, which in some cases proved successful in models of disease.

Dipeptidyl peptidase IV (DPP-IV) inhibition prevents fibrosis in adipose tissue of obese mice.

BACKGROUND: During the development of obesity the expansion of white adipose tissue (WAT) leads to a dysregulation and an excessive remodeling of extracellular matrix (ECM), leading to fibrosis formation. These ECM changes have high impact on WAT physiology and may change obesity progression. Blocking WAT fibrosis may have beneficial effects on the efficacy of diet regimen or therapeutical approaches in obesity. Since dipeptidyl peptidase IV (DPP-IV) inhibitors prevent fibrosis in tissues, such as heart, liver and kidney, the objective of this study was to assess whether vildagliptin, a DPP-IV inhibitor, prevents fibrosis in WAT in a mouse model of obesity, and to investigate the mechanisms underlying this effect. METHODS: We evaluated the inhibitory effect of vildagliptin on fibrosis markers on WAT of high-fat diet (HFD)-induced obese mice and on 3T3-L1 cell line of mouse adipocytes treated with a fibrosis inducer, transforming growth factor beta 1 (TGFß1). RESULTS: Vildagliptin prevents the increase of fibrosis markers in WAT of HFD-fed mice and reduces blood glucose, serum triglycerides, total cholesterol and leptin levels. In the in vitro study, the inhibition of DPP-IV with vildagliptin, neuropeptide Y (NPY) treatment and NPY Y1 receptor activation prevents ECM deposition and fibrosis markers increase induced by TGFß1 treatment. CONCLUSIONS: Vildagliptin prevents fibrosis formation in adipose tissue in obese mice, at least partially through NPY and NPY Y1 receptor activation. GENERAL SIGNIFICANCE: This study highlights the importance of vildagliptin in the treatment of fibrosis that occur in obesity.

Unravelling Endogenous MicroRNA System Dysfunction as a New Pathophysiological Mechanism in Machado-Joseph Disease.

Machado-Joseph disease (MJD) is a genetic neurodegenerative disease caused by an expanded polyglutamine tract within the protein ataxin-3 (ATXN3). Despite current efforts, MJD's mechanism of pathogenesis remains unclear and no disease-modifying treatment is available. Therefore, in this study, we investigated (1) the role of the 3' UTR of ATXN3, a putative microRNA (miRNA) target, (2) whether miRNA biogenesis and machinery are dysfunctional in MJD, and (3) which specific miRNAs target ATXN3-3' UTR and whether they can alleviate MJD neuropathology in vivo. Our results demonstrate that endogenous miRNAs, by targeting sequences in the 3' UTR, robustly reduce ATXN3 expression and aggregation in vitro and neurodegeneration and neuroinflammation in vivo. Importantly, we found an abnormal MJD-associated downregulation of genes involved in miRNA biogenesis and silencing activity. Finally, we identified three miRNAs-mir-9, mir-181a, and mir-494-that interact with the ATXN3-3' UTR and whose expression is dysregulated in human MJD neurons and in other MJD cell and animal models. Furthermore, overexpression of these miRNAs in mice resulted in reduction of mutATXN3 levels, aggregate counts, and neuronal dysfunction. Altogether, these findings indicate that endogenous miRNAs and the 3' UTR of ATXN3 play a crucial role in MJD pathogenesis and provide a promising opportunity for MJD treatment.

Caloric restriction blocks neuropathology and motor deficits in Machado-Joseph disease mouse models through SIRT1 pathway.

Machado-Joseph disease (MJD) is a neurodegenerative disorder characterized by an abnormal expansion of the CAG triplet in the ATXN3 gene, translating into a polyglutamine tract within the ataxin-3 protein. The available treatments only ameliorate symptomatology and do not block disease progression. In this study we find that caloric restriction dramatically rescues the motor incoordination, imbalance and the associated neuropathology in transgenic MJD mice. We further show that caloric restriction rescues SIRT1 levels in transgenic MJD mice, whereas silencing SIRT1 is sufficient to prevent the beneficial effects on MJD pathology. In addition, the re-establishment of SIRT1 levels in MJD mouse model, through the gene delivery approach, significantly ameliorates neuropathology, reducing neuroinflammation and activating autophagy. Furthermore, the pharmacological activation of SIRT1 with resveratrol significantly reduces motor incoordination of MJD mice. The pharmacological SIRT1 activation could provide important benefits to treat MJD patients.

Neuropeptide Y mitigates neuropathology and motor deficits in mouse models of Machado-Joseph disease.

Machado-Joseph disease (MJD) is a fatal, dominantly inherited neurodegenerative disorder associated with an expanded polyglutamine tract within the ataxin-3 protein, and characterized by progressive impairment of motor coordination, associated with neurodegeneration of specific brain regions, including cerebellum and striatum. The currently available therapies do not allow modification of disease progression. Neuropeptide Y (NPY) has been shown to exert potent neuroprotective effects by multiple pathways associated with the MJD mechanisms of disease. Thus, we evaluated NPY levels in MJD and investigated whether raising NPY by gene transfer would alleviate neuropathological and behavioural deficits in cerebellar and striatal mouse models of the disease. For that, a cerebellar transgenic and a striatal lentiviral-based models of MJD were used. NPY overexpression in the affected brain regions in these two mouse models was obtained by stereotaxic injection of adeno-associated viral vectors encoding NPY. Up to 8 weeks after viral injection, balance and motor coordination behaviour and neuropathology were analysed. We observed that NPY levels were decreased in two MJD patients' cerebella and in striata and cerebella of disease mouse models. Furthermore, overexpression of NPY alleviated the motor coordination impairments and attenuated the related neuropathological parameters, preserving cerebellar volume and granular layer thickness, reducing striatal lesion and decreasing mutant ataxin-3 aggregation. Additionally, NPY mediated increase of brain-derived neurotrophic factor levels and decreased neuroinflammation markers. Our data suggest that NPY is a potential therapeutic strategy for MJD.