From the disruption of RNA metabolism to the targeting of RNA-binding proteins: The case of polyglutamine spinocerebellar ataxias.

Polyglutamine spinocerebellar ataxias (PolyQ SCAs) represent a group of monogenetic diseases in which the expanded polyglutamine repeats give rise to a mutated protein. The abnormally expanded polyglutamine protein produces aggregates and toxic species, causing neuronal dysfunction and neuronal death. The main symptoms of these disorders include progressive ataxia, motor dysfunction, oculomotor impairment, and swallowing problems. Nowadays, the current treatments are restricted to symptomatic alleviation, and no existing therapeutic strategies can reduce or stop the disease progression. Even though the origin of these disorders has been associated with polyglutamine-induced toxicity, RNA toxicity has recently gained relevance in polyQ SCAs molecular pathogenesis. Therefore, the research's focus on RNA metabolism has been increasing, especially on RNA-binding proteins (RBPs). The present review summarizes RNA metabolism, exposing the different processes and the main RBPs involved. We also explore the mechanisms by which RBPs are dysregulated in PolyQ SCAs. Finally, possible therapies targeting the RNA metabolism are presented as strategies to reverse neuropathological anomalies and mitigate physical symptoms.

PolyQ Database-an integrated database on polyglutamine diseases.

Polyglutamine (polyQ) diseases are neurodegenerative disorders caused by abnormally expanded Cytosine, Adenine, Guanine (CAG) triplet repeat sequences in the coding region of otherwise unrelated genes. Until now, nine different polyQ diseases have been described: Huntington's disease, dentatorubral-pallidoluysian atrophy, spinal and bulbar muscular atrophy and six types of spinocerebellar ataxias-1, 2, 3, 6, 7 and 17. The pathogenic expansion translates into an aberrant tract of glutamines in the encoded proteins, compromising several cellular functions and biological processes. There is currently no cure available for the progressive neurodegenerative disorders caused by the ensuing cytotoxic alterations. Although each disease is considered rare, polyQ diseases constitute the largest group of monogenic neurodegenerative disorders. Information about these disorders is scattered among several books, articles and general databases, hindering exploration by students and researchers, but also by patients and their families. Therefore, we aimed to develop a free online database to fill this gap, by centralizing relevant available information. The PolyQ Database is a platform that focuses on all nine polyQ diseases and offers information about topics that are pertinent for scientists, clinicians and the general public, including epidemiology, the characteristics of the causative genes and the codified proteins, the pathophysiology of the diseases and the main clinical manifestations. The database is available at https://polyq.pt/, and it is the first of its kind, focusing exclusively on this group of rare diseases. The database was conceived to be continuously updated and allow incorporation and dissemination of the latest information on polyQ diseases.

Reprogramming iPSCs to study age-related diseases: Models, therapeutics, and clinical trials.

The unprecedented rise in life expectancy observed in the last decades is leading to a global increase in the ageing population, and age-associated diseases became an increasing societal, economic, and medical burden. This has boosted major efforts in the scientific and medical research communities to develop and improve therapies to delay ageing and age-associated functional decline and diseases, and to expand health span. The establishment of induced pluripotent stem cells (iPSCs) by reprogramming human somatic cells has revolutionised the modelling and understanding of human diseases. iPSCs have a major advantage relative to other human pluripotent stem cells as their obtention does not require the destruction of embryos like embryonic stem cells do, and do not have a limited proliferation or differentiation potential as adult stem cells. Besides, iPSCs can be generated from somatic cells from healthy individuals or patients, which makes iPSC technology a promising approach to model and decipher the mechanisms underlying the ageing process and age-associated diseases, study drug effects, and develop new therapeutic approaches. This review discusses the advances made in the last decade using iPSC technology to study the most common age-associated diseases, including age-related macular degeneration (AMD), neurodegenerative and cardiovascular diseases, brain stroke, cancer, diabetes, and osteoarthritis.

Measuring healthy ageing: current and future tools.

Human ageing is a complex, multifactorial process characterised by physiological damage, increased risk of age-related diseases and inevitable functional deterioration. As the population of the world grows older, placing significant strain on social and healthcare resources, there is a growing need to identify reliable and easy-to-employ markers of healthy ageing for early detection of ageing trajectories and disease risk. Such markers would allow for the targeted implementation of strategies or treatments that can lessen suffering, disability, and dependence in old age. In this review, we summarise the healthy ageing scores reported in the literature, with a focus on the past 5 years, and compare and contrast the variables employed. The use of approaches to determine biological age, molecular biomarkers, ageing trajectories, and multi-omics ageing scores are reviewed. We conclude that the ideal healthy ageing score is multisystemic and able to encompass all of the potential alterations associated with ageing. It should also be longitudinal and able to accurately predict ageing complications at an early stage in order to maximize the chances of successful early intervention.

Autophagy in Spinocerebellar Ataxia Type 3: From Pathogenesis to Therapeutics.

Machado-Joseph disease (MJD) or spinocerebellar ataxia 3 (SCA3) is a rare, inherited, monogenic, neurodegenerative disease, and the most common SCA worldwide. MJD/SCA3 causative mutation is an abnormal expansion of the triplet CAG at exon 10 within the ATXN3 gene. The gene encodes for ataxin-3, which is a deubiquitinating protein that is also involved in transcriptional regulation. In normal conditions, the ataxin-3 protein polyglutamine stretch has between 13 and 49 glutamines. However, in MJD/SCA3 patients, the size of the stretch increases from 55 to 87, contributing to abnormal protein conformation, insolubility, and aggregation. The formation of aggregates, which is a hallmark of MJD/SCA3, compromises different cell pathways, leading to an impairment of cell clearance mechanisms, such as autophagy. MJD/SCA3 patients display several signals and symptoms in which the most prominent is ataxia. Neuropathologically, the regions most affected are the cerebellum and the pons. Currently, there are no disease-modifying therapies, and patients rely only on supportive and symptomatic treatments. Due to these facts, there is a huge research effort to develop therapeutic strategies for this incurable disease. This review aims to bring together current state-of-the-art strategies regarding the autophagy pathway in MJD/SCA3, focusing on evidence for its impairment in the disease context and, importantly, its targeting for the development of pharmacological and gene-based therapies.

How can we modulate aging through nutrition and physical exercise? An epigenetic approach.

The World Health Organization predicts that by 2050, 2.1 billion people worldwide will be over 60 years old, a drastic increase from only 1 billion in 2019. Considering these numbers, strategies to ensure an extended "healthspan" or healthy longevity are urgently needed. The present study approaches the promotion of healthspan from an epigenetic perspective. Epigenetic phenomena are modifiable in response to an individual's environmental exposures, and therefore link an individual's environment to their gene expression pattern. Epigenetic studies demonstrate that aging is associated with decondensation of the chromatin, leading to an altered heterochromatin structure, which promotes the accumulation of errors. In this review, we describe how aging impacts epigenetics and how nutrition and physical exercise can positively impact the aging process, from an epigenetic point of view. Canonical histones are replaced by histone variants, concomitant with an increase in histone post-translational modifications. A slight increase in DNA methylation at promoters has been observed, which represses transcription of previously active genes, in parallel with global genome hypomethylation. Aging is also associated with deregulation of gene expression - usually provided by non-coding RNAs - leading to both the repression of previously transcribed genes and to the transcription of previously repressed genes. Age-associated epigenetic events are less common in individuals with a healthy lifestyle, including balanced nutrition, caloric restriction and physical exercise. Healthy aging is associated with more tightly condensed chromatin, fewer PTMs and greater regulation by ncRNAs.

Lipoprotein Metabolism, Protein Aggregation, and Alzheimer's Disease: A Literature Review.

Alzheimer's disease (AD) is the most common form of dementia. The physiopathology of AD is well described by the presence of two neuropathological features: amyloid plaques and tau neurofibrillary tangles. In the last decade, neuroinflammation and cellular stress have gained importance as key factors in the development and pathology of AD. Chronic cellular stress occurs in degenerating neurons. Stress Granules (SGs) are nonmembranous organelles formed as a response to stress, with a protective role; however, SGs have been noted to turn into pathological and neurotoxic features when stress is chronic, and they are related to an increased tau aggregation. On the other hand, correct lipid metabolism is essential to good function of the brain; apolipoproteins are highly associated with risk of AD, and impaired cholesterol efflux and lipid transport are associated with an increased risk of AD. In this review, we provide an insight into the relationship between cellular stress, SGs, protein aggregation, and lipid metabolism in AD.

The stress granule protein G3BP1 alleviates spinocerebellar ataxia-associated deficits.

Polyglutamine diseases are a group of neurodegenerative disorders caused by an abnormal expansion of CAG repeat tracts in the codifying regions of nine, otherwise unrelated, genes. While the protein products of these genes are suggested to play diverse cellular roles, the pathogenic mutant proteins bearing an expanded polyglutamine sequence share a tendency to self-assemble, aggregate and engage in abnormal molecular interactions. Understanding the shared paths that link polyglutamine protein expansion to the nervous system dysfunction and the degeneration that takes place in these disorders is instrumental to the identification of targets for therapeutic intervention. Among polyglutamine diseases, spinocerebellar ataxias (SCAs) share many common aspects, including the fact that they involve dysfunction of the cerebellum, resulting in ataxia. Our work aimed at exploring a putative new therapeutic target for the two forms of SCA with higher worldwide prevalence, SCA type 2 (SCA2) and type 3 (SCA3), which are caused by expanded forms of ataxin-2 (ATXN2) and ataxin-3 (ATXN3), respectively. The pathophysiology of polyglutamine diseases has been described to involve an inability to properly respond to cell stress. We evaluated the ability of GTPase-activating protein-binding protein 1 (G3BP1), an RNA-binding protein involved in RNA metabolism regulation and stress responses, to counteract SCA2 and SCA3 pathology, using both in vitro and in vivo disease models. Our results indicate that G3BP1 overexpression in cell models leads to a reduction of ATXN2 and ATXN3 aggregation, associated with a decrease in protein expression. This protective effect of G3BP1 against polyglutamine protein aggregation was reinforced by the fact that silencing G3bp1 in the mouse brain increases human expanded ATXN2 and ATXN3 aggregation. Moreover, a decrease of G3BP1 levels was detected in cells derived from patients with SCA2 and SCA3, suggesting that G3BP1 function is compromised in the context of these diseases. In lentiviral mouse models of SCA2 and SCA3, G3BP1 overexpression not only decreased protein aggregation but also contributed to the preservation of neuronal cells. Finally, in an SCA3 transgenic mouse model with a severe ataxic phenotype, G3BP1 lentiviral delivery to the cerebellum led to amelioration of several motor behavioural deficits. Overall, our results indicate that a decrease in G3BP1 levels may be a contributing factor to SCA2 and SCA3 pathophysiology, and that administration of this protein through viral vector-mediated delivery may constitute a putative approach to therapy for these diseases, and possibly other polyglutamine disorders.

Ataxin-2 in the hypothalamus at the crossroads between metabolism and clock genes.

ATXN2 gene, encoding for ataxin-2, is located in a trait locus for obesity. Atxn2 knockout (KO) mice are obese and insulin resistant; however, the cause for this phenotype is still unknown. Moreover, several findings suggest ataxin-2 as a metabolic regulator, but the role of this protein in the hypothalamus was never studied before. The aim of this work was to understand if ataxin-2 modulation in the hypothalamus could play a role in metabolic regulation. Ataxin-2 was overexpressed/re-established in the hypothalamus of C57Bl6/Atxn2 KO mice fed either a chow or a high-fat diet (HFD). This delivery was achieved through stereotaxic injection of lentiviral vectors encoding for ataxin-2. We show, for the first time, that HFD decreases ataxin-2 levels in mouse hypothalamus and liver. Specific hypothalamic ataxin-2 overexpression prevents HFD-induced obesity and insulin resistance. Ataxin-2 re-establishment in Atxn2 KO mice improved metabolic dysfunction without changing body weight. Furthermore, we observed altered clock gene expression in Atxn2 KO that might be causative of metabolic dysfunction. Interestingly, ataxin-2 hypothalamic re-establishment rescued these circadian alterations. Thus, ataxin-2 in the hypothalamus is a determinant for weight, insulin sensitivity and clock gene expression. Ataxin-2's potential role in the circadian clock, through the regulation of clock genes, might be a relevant mechanism to regulate metabolism. Overall, this work shows hypothalamic ataxin-2 as a new player in metabolism regulation, which might contribute to the development of new strategies for metabolic disorders.

Mutant Ataxin-2 Expression in Aged Animals Aggravates Neuropathological Features Associated with Spinocerebellar Ataxia Type 2.

Spinocerebellar ataxia type 2 (SCA2) is a rare autosomal, dominantly inherited disease, in which the affected individuals have a disease onset around their third life decade. The molecular mechanisms underlying SCA2 are not yet completely understood, for which we hypothesize that aging plays a role in SCA2 molecular pathogenesis. In this study, we performed a striatal injection of mutant ataxin-2 mediated by lentiviral vectors, in young and aged animals. Twelve weeks post-injection, we analyzed the striatum for SCA2 neuropathological features and specific aging hallmarks. Our results show that aged animals had a higher number of mutant ataxin-2 aggregates and more neuronal marker loss, compared to young animals. Apoptosis markers, cleaved caspase-3, and cresyl violet staining also indicated increased neuronal death in the aged animal group. Additionally, mRNA levels of microtubule-associated protein 1 light-chain 3B (LC3) and sequestosome-1 (SQSTM1/p62) were altered in the aged animal group, suggesting autophagic pathway dysfunction. This work provides evidence that aged animals injected with expanded ataxin-2 had aggravated SCA2 disease phenotype, suggesting that aging plays an important role in SCA2 disease onset and disease progression.

The autophagy-enhancing drug carbamazepine improves neuropathology and motor impairment in mouse models of Machado-Joseph disease.

AIMS: Machado-Joseph disease (MJD), or spinocerebellar ataxia type 3 (SCA3), is the most common autosomal dominantly-inherited ataxia worldwide and is characterised by the accumulation of mutant ataxin-3 (mutATXN3) in different brain regions, leading to neurodegeneration. Currently, there are no available treatments able to block disease progression. In this study, we investigated whether carbamazepine (CBZ) would activate autophagy and mitigate MJD pathology. METHODS: The autophagy-enhancing activity of CBZ and its effects on clearance of mutATXN3 were evaluated using in vitro and in vivo models of MJD. To investigate the optimal treatment regimen, a daily or intermittent CBZ administration was applied to MJD transgenic mice expressing a truncated human ATXN3 with 69 glutamine repeats. Motor behaviour tests and immunohistology was performed to access the alleviation of MJD-associated motor deficits and neuropathology. A retrospective study was conducted to evaluate the CBZ effect in MJD patients. RESULTS: We found that CBZ promoted the activation of autophagy and the degradation of mutATXN3 in MJD models upon short or intermittent, but not daily prolonged, treatment regimens. CBZ up-regulated autophagy through activation of AMPK, which was dependent on the myo-inositol levels. In addition, intermittent CBZ treatment improved motor performance, as well as prevented neuropathology in MJD transgenic mice. However, in patients, no evident differences in SARA scale were found, which was not unexpected given the small number of patients included in the study. CONCLUSIONS: Our data support the autophagy-enhancing activity of CBZ in the brain and suggest this pharmacological approach as a promising therapy for MJD and other polyglutamine disorders.

ULK overexpression mitigates motor deficits and neuropathology in mouse models of Machado-Joseph disease.

Machado-Joseph disease (MJD) is a fatal neurodegenerative disorder clinically characterized by prominent ataxia. It is caused by an expansion of a CAG trinucleotide in ATXN3, translating into an expanded polyglutamine (polyQ) tract in the ATXN3 protein, that becomes prone to misfolding and aggregation. The pathogenesis of the disease has been associated with the dysfunction of several cellular mechanisms, including autophagy and transcription regulation. In this study, we investigated the transcriptional modifications of the autophagy pathway in models of MJD and assessed whether modulating the levels of the affected autophagy-associated transcripts (AATs) would alleviate MJD-associated pathology. Our results show that autophagy is impaired at the transcriptional level in MJD, affecting multiple AATs, including Unc-51 like autophagy activating kinase 1 and 2 (ULK1 and ULK2), two homologs involved in autophagy induction. Reinstating ULK1/2 levels by adeno-associated virus (AAV)-mediated gene transfer significantly improved motor performance while preventing neuropathology in two in vivo models of MJD. Moreover, in vitro studies showed that the observed positive effects may be mainly attributed to ULK1 activity. This study provides strong evidence of the beneficial effect of overexpression of ULK homologs, suggesting these as promising instruments for the treatment of MJD and other neurodegenerative disorders.

Autophagy in Spinocerebellar ataxia type 2, a dysregulated pathway, and a target for therapy.

Spinocerebellar ataxia type 2 (SCA2) is an incurable and genetic neurodegenerative disorder. The disease is characterized by progressive degeneration of several brain regions, resulting in severe motor and non-motor clinical manifestations. The mutation causing SCA2 disease is an abnormal expansion of CAG trinucleotide repeats in the ATXN2 gene, leading to a toxic expanded polyglutamine segment in the translated ataxin-2 protein. While the genetic cause is well established, the exact mechanisms behind neuronal death induced by mutant ataxin-2 are not yet completely understood. Thus, the goal of this study is to investigate the role of autophagy in SCA2 pathogenesis and investigate its suitability as a target for therapeutic intervention. For that, we developed and characterized a new striatal lentiviral mouse model that resembled several neuropathological hallmarks observed in SCA2 disease, including formation of aggregates, neuronal marker loss, cell death and neuroinflammation. In this new model, we analyzed autophagic markers, which were also analyzed in a SCA2 cellular model and in human post-mortem brain samples. Our results showed altered levels of SQSTM1 and LC3B in cells and tissues expressing mutant ataxin-2. Moreover, an abnormal accumulation of these markers was detected in SCA2 patients' striatum and cerebellum. Importantly, the molecular activation of autophagy, using the compound cordycepin, mitigated the phenotypic alterations observed in disease models. Overall, our study suggests an important role for autophagy in the context of SCA2 pathology, proposing that targeting this pathway could be a potential target to treat SCA2 patients.

Stress granules, RNA-binding proteins and polyglutamine diseases: too much aggregation?

Stress granules (SGs) are membraneless cell compartments formed in response to different stress stimuli, wherein translation factors, mRNAs, RNA-binding proteins (RBPs) and other proteins coalesce together. SGs assembly is crucial for cell survival, since SGs are implicated in the regulation of translation, mRNA storage and stabilization and cell signalling, during stress. One defining feature of SGs is their dynamism, as they are quickly assembled upon stress and then rapidly dispersed after the stress source is no longer present. Recently, SGs dynamics, their components and their functions have begun to be studied in the context of human diseases. Interestingly, the regulated protein self-assembly that mediates SG formation contrasts with the pathological protein aggregation that is a feature of several neurodegenerative diseases. In particular, aberrant protein coalescence is a key feature of polyglutamine (PolyQ) diseases, a group of nine disorders that are caused by an abnormal expansion of PolyQ tract-bearing proteins, which increases the propensity of those proteins to aggregate. Available data concerning the abnormal properties of the mutant PolyQ disease-causing proteins and their involvement in stress response dysregulation strongly suggests an important role for SGs in the pathogenesis of PolyQ disorders. This review aims at discussing the evidence supporting the existence of a link between SGs functionality and PolyQ disorders, by focusing on the biology of SGs and on the way it can be altered in a PolyQ disease context.

Current Status of Gene Therapy Research in Polyglutamine Spinocerebellar Ataxias.

Polyglutamine spinocerebellar ataxias (PolyQ SCAs) are a group of 6 rare autosomal dominant diseases, which arise from an abnormal CAG repeat expansion in the coding region of their causative gene. These neurodegenerative ataxic disorders are characterized by progressive cerebellar degeneration, which translates into progressive ataxia, the main clinical feature, often accompanied by oculomotor deficits and dysarthria. Currently, PolyQ SCAs treatment is limited only to symptomatic mitigation, and no therapy is available to stop or delay the disease progression, which culminates with death. Over the last years, many promising gene therapy approaches were investigated in preclinical studies and could lead to a future treatment to stop or delay the disease development. Here, we summed up the most promising of these therapies, categorizing them in gene augmentation therapy, gene silencing strategies, and gene edition approaches. While several of the reviewed strategies are promising, there is still a gap from the preclinical results obtained and their translation to clinical studies. However, there is an increase in the number of approved gene therapies, as well as a constant development in their safety and efficacy profiles. Thus, it is expected that in a near future some of the promising strategies reviewed here could be tested in a clinical setting and if successful provide hope for SCAs patients.

Lauroylated Histidine-Enriched S413-PV Peptide as an Efficient Gene Silencing Mediator in Cancer Cells.

PURPOSE: This study aimed to endow the cell-penetrating peptide (CPP) S413-PV with adequate features towards a safe and effective application in cancer gene therapy. METHODS: Peptide/siRNA complexes were prepared with two new derivatives of the CPP S413-PV, which combine a lauroyl group attached to the N- or C-terminus with a histidine-enrichment in the N-terminus of the S413-PV peptide, being named C12-H5-S413-PV and H5-S413-PV-C12, respectively. Physicochemical characterization of siRNA complexes was performed and their cytotoxicity and efficiency to mediate siRNA delivery and gene silencing in cancer cells were assessed in the absence and presence of serum. RESULTS: Peptide/siRNA complexes prepared with the C12-H5-S413-PV derivative showed a nanoscale (ca. 100 nm) particle size, as revealed by TEM, and efficiently mediated gene silencing (37%) in human U87 glioblastoma cells in the presence of 30% serum. In addition, the new C12-H5-S413-PV-based siRNA delivery system efficiently downregulated stearoyl-CoA desaturase-1, a key-enzyme of lipid metabolism overexpressed in cancer, which resulted in a significant decrease in the viability of U87 cells. Importantly, these complexes were able to spare healthy human astrocytes. CONCLUSIONS: These encouraging results pave the way for a potential application of the C12-H5-S413-PV peptide as a promising tool in cancer gene therapy.

Mapping gene expression in social anxiety reveals the main brain structures involved in this disorder.

Social Anxiety Disorder (SAD) is characterized by emotional and attentional biases as well as distorted negative self-beliefs. According this, we proposed to identify the brain structures and hub genes involved in SAD. An analysis in Pubmed and TRANSFAC was conducted and 72 genes were identified. Using Microarray data, from Allen Human Brain Atlas, it was possible to identify three modules of co-expressed genes from our gene set (R package WGCNA). Higher mean gene expression was found in cortico-medial group, basomedial nucleus, ATZ in amygdala and in head and tail of the caudate nucleus, nucleus accumbens and putamen in striatum. Our enrichment analysis identified the followed hub genes: DRD2, HTR1A, JUN, SP1 and HDAC4. We suggest that SAD is explained by delayed extinction of circuitry for conditioned fear. Caused by reduced activation of the dopaminergic and serotonergic systems,due diminished expectation of reward during social interactions.