Biology subjects

Nobre, R. J.

Publications and source records attributed to Nobre, R. J..

10 recordsLinked to original sources

bioRxiv2026

Disease-associated variants alter a novel physiological role of hnRNP H2 in neuronal development

Heterogeneous nuclear ribonucleoprotein H2 (hnRNP H2) is a member of the hnRNP family of RNA-binding proteins that regulate neuronal function through multiple aspects of RNA metabolism. Mutations in the nuclear localisation signal (NLS) and quasi-RNA recognition motifs (qRRMs) of hnRNP H2 have recently been linked to a severe developmental syndrome, termed HNRNPH2-related neurodevelopmental disorder. Despite its emerging importance, the physiological roles of hnRNP H2 in neuronal development and synaptic maturation, and how disease-associated variants alter these processes to drive pathology, remain poorly understood. Here, we show that hnRNP H2 is expressed in hippocampal neurons and localises to the synaptic compartment. Knockdown of hnRNP H2 revealed essential roles of the protein in the regulation of neuronal morphology, dendritic spine maturation, synaptic transmission, and cortical neuronal migration in vivo. Disease-associated hnRNP H2 variants altered protein localisation and activity-dependent synaptic recruitment, resulting in defects in neuronal morphology, in vivo cortical neuronal migration and laminar orientation, dendritic spine maturation, and synaptic function. Together, these findings establish hnRNP H2 as a critical regulator of neurodevelopment and identify a molecular basis for HNRNPH2-related neurodevelopmental disorder.

neuroscience↗

bioRxiv2026

Mesenchymal Stromal Cell Therapeutic Potential to RestoreNeurovascular Integrity in Machado-Joseph Disease

BackgroundMachado-Joseph disease (MJD), or Spinocerebellar Ataxia type 3 (SCA3), is a neurodegenerative disorder caused by an expansion of the CAG repeat in the MJD1/ATXN3 gene, which encodes for a polyglutamine-expansion in Ataxin-3. Similar to what occurs in other neurodegenerative disorders, the cerebrovascular system, particularly the blood-brain barrier (BBB), is impaired in MJD. BBB is an important cellular barrier that controls homeostasis of the central nervous system, and its disruption compromises neuronal function. To date, there is no disease-modifying therapy for MJD. Our group previously demonstrated that MSC administration can ameliorate the phenotype of MJD transgenic mice. However, the effect of MSC on the neurovascular unit and BBB integrity remains unexplored in the context of MJD. In the present work, we aimed to evaluate the therapeutic potential of MSC in mitigating vascular abnormalities and BBB disruption in MJD. MethodsEight-month-old MJD transgenic mice were treated with two consecutive bone marrow-MSC intravenous injections (1 week apart). Behavioral tests to assess motor coordination were conducted before and after treatment. Vascular structure, function, and BBB disruption were evaluated by immunohistochemistry and western blot. ResultsMSC administration partially restored vascular impairment in MJD transgenic mice. Treated mice exhibited improved gait and motor performance footprint and beam walking tests) and concurrently attenuated vascular dysfunction. Specifically, MSC-treated mice exhibited a decrease in collagen IV surface area and stabilized cerebellar blood supply. Additionally, MSC treatment diminished BBB permeability to the Evans blue (EB) dye in MJD mice and restored the levels of important adherent and tight junction-associated proteins in the cellular membrane, including vascular endothelial cadherin (VE-cadherin), claudin-5, and occludin, in a sex-specific manner. Conclusionsour results highlight significant cerebrovascular dysregulations in MJD, which are sex-dependent and progress with the disease stage. Moreover, our findings suggest that MSC administration partially reverted vascular impairments, providing valuable insights into the mechanisms underlying the therapeutic potential of MSC in MJD. Graphical AbstractMSC treatment partially reverts neurovascular dysregulation in MJD. MJD mice exhibit increased vessel coverage, increased BBB permeability, and altered TJ and AJ expression and subcellular localization. MSC administration modulates vessel coverage, reduces BBB leakage, and partially normalizes TJ/AJ subcellular localization. List of abbreviations: AJ - Adherent junction; BBB - Blood-Brain barrier; MJD- Machado-Joseph disease; MSC - mesenchymal stromal cell; TJ - thigh junction. Created with BioRender.com O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/703043v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@8fa0a2org.highwire.dtl.DTLVardef@17b13f9org.highwire.dtl.DTLVardef@15c5ce3org.highwire.dtl.DTLVardef@d67539_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

rAAV-Delivered Bicistronic Artificial microRNAs for Allele-Specific Silencing Improve Motor and Molecular Outcomes in Spinocerebellar Ataxia Type 3

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is an autosomal dominant neurodegenerative disorder caused by the expansion of CAG trinucleotide repeats in the ATXN3 gene. This mutation induces a toxic gain-of-function of the ATXN3 protein, leading to neurodegeneration, particularly in the cerebellum and brainstem. Despite extensive research, no disease-modifying treatments are available for SCA3 patients. In this study, we developed and tested a novel therapeutic strategy using recombinant adeno-associated virus (rAAV) to deliver bicistronic artificial microRNAs designed to selectively silence the mutant ATXN3 allele. Through in vitro screening, we identified a lead construct (miATXN3-10x2) that effectively and specifically silenced the mutant allele by targeting of a single nucleotide polymorphism (SNP) associated with the repeat expansion. This construct was packaged into rAAV9 and delivered via intra-cerebellar administration into two mouse models of SCA3, resulting in robust suppression of mutant ATXN3 in the cerebellum. To assess long-term efficacy, we performed intra-cisterna magna (ICM) injections of rAAV9-miATXN3-10x2 in a severe SCA3 transgenic mouse model. Widespread distribution of viral vectors and miATXN3 copies was observed in disease-relevant brain regions. Treated animals exhibited significant and sustained improvements in motor function at 5, 8, and 11 weeks post-injection. Histological analyses showed a reduction in mutant ATXN3 aggregates and a trend toward preventing shrinkage of cerebellar molecular layer. These findings were supported by dose-dependent reductions in mutant ATXN3 mRNA levels and decreased expression of neuroinflammatory markers in the cerebellum. Additionally, a significant increase of the neuronal marker NeuN was also observed in treated animals. Finally, transcriptomic profiling of the cerebellum demonstrated that treated transgenic animals exhibited an improved transcriptomic signature, shifting toward a wild-type profile. In conclusion, our findings highlight the therapeutic potential of a single administration of rAAVs encoding bicistronic artificial microRNAs for allele-specific gene silencing in SCA3. This study provides compelling preclinical evidence supporting the translation of this approach into clinical applications for SCA3 patients.

molecular biology↗

bioRxiv2025

AAV-mediated allele-specific silencing alleviates neuropathology in a novel non-human primate model of Spinocerebellar ataxia type 3

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an autosomal dominant neurodegenerative disorder caused by an abnormal expansion of the cytosine-adenine-guanine (CAG) repeats in the ATXN3 gene. This mutation results in the production of an Ataxin-3 protein with an extended polyglutamine sequence, contributing to the diseases neuropathology. Currently, no treatment is available that can slow or halt the progression of SCA3. Gene-targeted therapies have gained significant attention for their potential to address the root cause of SCA3. Preliminary studies in transgenic mice using adeno-associated viral vector serotype 9 (AAV9) encoding artificial microRNAs targeting the mutant ATXN3 allele (AAV9-miR-ATXN3) have shown promising results. However, to advance this therapeutic approach toward clinical application, further studies in an animal model that more closely resembles human biology are essential. In this exploratory study, we assessed the biodistribution and target engagement of AAV9-miR-ATXN3 delivered via intracisterna magna (ICM) injection in non-human primates (NHPs). Using a lentiviral vector (LV) to introduce a mutant Ataxin-3 cDNA with 72 glutamines (LV-mutATXN3-Q72) into the NHP cerebellum, we successfully overexpressed SCA3 in the NHP brain. SCA3 NHP exhibited Ataxin-3 aggregation in the cerebellum, recruitment of inflammatory cells and reduced cerebellar volume. ICM administration of AAV9-miR-ATXN3 effectively directed transgene expression to key brain regions impacted by SCA3 pathology and enabled specific, dose-dependent silencing of mutant Ataxin-3. Furthermore, the therapeutic dose prevented the cerebellar morphological and biochemical alterations induced by the overexpression of mutant ATXN3. These proof-of-concept experiments are crucial, not only for advancing AAV9-miR-ATXN3 toward clinical use but also for establishing a valuable platform for validating future therapeutic interventions.

molecular biology↗

bioRxiv2025

Innate immunocompetent iNSpheroids: A hiPSC-derived 3D model to study the central nervous system captures an early CNS response to rAAV

Gene therapies using adeno-associated viruses (AAVs) for central nervous system (CNS) disorders face challenges due to host immune responses not represented in classical preclinical models. Here, we present a human-induced pluripotent stem cell (hiPSC)-derived innate immunocompetent 3D CNS model that recapitulates neuroinflammatory hallmarks, serving as a platform for preclinical gene therapy development. Utilizing various scales of stirred-tank bioreactor systems, we generated (neurospheroids) iNSpheroids composed of neurons, astrocytes, and oligodendrocytes, alongside microglial cells (iMGL) to mimic the neuro-immune axis. These systems enabled large-scale production of iNSpheroids and subsequent miniaturization for co-culture experiments and screening of inflammatory stimuli, while maintaining a highly controlled environment. The iMGL-iNSpheroids demonstrated active neuron-microglia crosstalk and exhibited distinct inflammatory responses to a series of neuroinflammatory factors. iMGL-iNSpheroids mounted a mild and transient response to rAAV9, mediated by the activation of inflammatory pathways (e.g., TNF-via NF-{kappa}B activation) in glial cell populations. This model offers a valuable tool to dissect neuroinflammatory mechanisms, accelerating gene therapy development. TeaserImmune-competent 3D human CNS model recapitulates glial responses to rAAVs, enabling reliable preclinical gene therapy screening.

bioengineering↗

bioRxiv2025

Astrocytic mGluR5 signaling tunes emotional and cognitive processing in the adult brain

The hippocampus is a brain region involved in both emotion regulation and higher cognitive functions. Astrocytes have emerged as active modulators of synaptic activity, capable of sensing, integrating, and responding to neuronal signals. At glutamatergic synapses, astrocytes detect glutamate through the activation of the metabotropic glutamate receptor 5 (mGluR5). However, most existing research has focused on the role of mGluR5 in developing rodents or in pathological contexts, likely because of the reported lower astrocytic mGluR5 expression levels in adulthood compared to postnatal stages. Importantly, prior studies and our preliminary data have demonstrated mGluR5-mediated signaling in astrocytes of adult mice, supporting a role for this receptor. Therefore, the main objectives of this study were (1) to determine whether these lower levels of mGluR5 are sufficient to activate astrocytes in the adult brain and (2) to investigate whether this activation is involved in regulating circuit function and behavior. To address these objectives, we evaluated adult mice employing a combination of calcium imaging in astrocytes, and loss- and gain-of-function manipulations to assess synaptic plasticity and behavior in adult mice. First, we found that astrocytes of adult mice display fully functional mGluR5-dependent calcium activity. To examine the role of this activity, we induced the deletion of mGluR5 in astrocytes across the entire brain of adult mice. These mice developed anxious- and depression-like behaviors, along with reduced sociability and recognition memory, but showed increased behavioral flexibility. These results highlighted the hippocampus as a key region for mGluR5-mediated astrocytic influence on behavior, leading us to specifically target hippocampal astrocytes. A viral-driven ablation in this area demonstrated that astrocytic mGluR5 plays a role in both basal transmission and the regulation of synaptic plasticity. Behaviorally, the deletion of astrocytic mGluR5 in the hippocampus recapitulated anxious-like behaviors, social deficits, and impaired long-term recognition memory. Surprisingly, it improved place recognition memory but reduced behavioral flexibility. Lastly, overexpressing this receptor to enhance mGluR5 signaling specifically in hippocampal astrocytes impaired place recognition memory but improved behavioral flexibility, revealing a role for astrocytic mGluR5 in regulating these behaviors. Overall, our results confirmed the biological relevance of astrocytic mGluR5 during adulthood, specifically in modulating hippocampal function.

neuroscience↗

bioRxiv2025

Gene Editing for ATXN3 Inactivation in Machado-Joseph disease: CRISPR-Cas9 as a Therapeutic Alternative to TALEN-Induced Toxicity

Machado-Joseph disease (MJD) is an autosomal dominantly-inherited neurodegenerative disorder, caused by an over-repetition of the polyglutamine-codifying region in the ATXN3 gene. Strategies based on the suppression of the deleterious gene products have demonstrated promising results in pre-clinical studies. Nonetheless, these strategies do not target the root cause of the disease. In order to prevent the downstream toxic pathways, our goal was to develop gene editing-based strategies to permanently inactivate the human ATXN3 gene. TALENs and CRISPR-Cas9 systems were designed to target exon 2 of this gene and functional characterization was performed in a human cell line. After the demonstration of TALENs and CRISPR-Cas9 efficiency on gene disruption, a sequence of each system was selected for further in vivo experiments. Although both TALENs and CRISPR-Cas9 systems led to a drastic reduction of ATXN3 aggregates in the striatum of a lentiviral-based mouse model of MJD/SCA3, only CRISPR-Cas9 system allowed the improvement of key neuropathological markers of the disease. Importantly, the administration of the engineered system in YAC-MJD84.2/84.2 mice mediated a delay in disease progression, when compared with non-treated littermates. These data provide the first in vivo evidence of the efficacy of a CRISPR-Cas9-based approach to permanently inactivate the ATXN3 gene in the brain of two mouse models of the disease, supporting its potential as a new therapeutic avenue in the context of MJD/SCA3.

neuroscience↗

bioRxiv2024

Small non-coding RNA content in plasma-derived extracellular vesicles distinguish ataxic SCA3 mutation carriers from pre-ataxic and control subjects

Spinocerebellar ataxia type 3 (SCA3), a neurodegenerative disorder caused by a CAG expansion in the ATXN3 gene, is the most common spinocerebellar ataxia subtype worldwide. Currently, there is no therapy to stop or prevent disease progression. Promising therapeutic strategies are emerging, but their translation into clinical practice requires sensitive and reliable biomarkers. Blood circulating extracellular vesicles constitute a promising source of biomarkers with potential to track alterations of the central nervous system due to their ability to cross the blood brain barrier. Here, we perform sequencing analysis of small RNAs from plasma-derived extracellular vesicles from SCA3 mutation carriers (10 pre-ataxic and 10 ataxic) and 12 control subjects to identify potential RNA biomarker candidates for this disease. Data showed that plasma-derived extracellular vesicles from ataxic SCA3 mutation carriers are enriched in mitochondrial, nuclear, and nucleolar RNA biotypes compared to pre-ataxic and control subjects. Moreover, ataxic mutation carriers could be discriminated from control and pre-ataxic subjects based on the miRNAs or piRNAs content, but not tRNA. Furthermore, we identified a subset of differentially expressed miRNAs and piRNAs that clearly differentiate ataxic mutation carriers from pre-ataxic and control subjects. These findings open new avenues for further investigation on the role of these RNAs in the pathogenesis of SCA3 and their potential as biomarkers for this disease.

neuroscience↗

bioRxiv2023

A systematic screening assay identifies efficient small guide RNAs for CRISPR activation

CRISPR-mediated gene activation (CRISPRa) encompasses a growing field of biotechnological approaches with exciting implications for gene therapy. However, there is a lack of experimental validation tools for selecting efficient sgRNAs for downstream applications. Here, we present a screening assay capable of identifying efficient single- and double sgRNAs through fluorescence quantification in vitro. In addition, we provide a tailored Golden Gate cloning workflow for streamlined incorporation of selected sgRNA candidates into lentiviral (LVs) or adeno-associated vectors (AAVs). The overall workflow was validated using therapeutically relevant genes for neurodegenerative diseases, such as Tfeb, Adam17, and Sirt1. The most efficient sgRNAs also demonstrated activation of endogenous gene expression at mRNA and protein levels. Further proof- of-principle assays using Tfeb indicated that gene activation was accompanied by increased levels of Lc3b. This data demonstrates the potential of the screening assay to identify functionally efficient sgRNA candidates across multiple genes along with streamlined cloning of viral vectors and may assist in accelerating future developments of CRISPRa-focused applications.

molecular biology↗

bioRxiv2023

Isolation of Biologically Active Extracellular Vesicles-Associated AAVs for Gene Delivery to the Brain by Size Exclusion Chromatography

Extracellular vesicles-associated adeno-associated viral vectors (EV-AAVs) emerged as a new opportunity for non-invasive gene therapy targeting the central nervous system (CNS). However, in previous reports, only AAV serotypes with known ability to cross the blood-brain barrier (BBB) have been used for EV-AAV production and testing through non-invasive strategies. In this work, we aimed at optimizing a size exclusion chromatography (SEC) protocol for the production and isolation of natural and biologically active brain-targeting EV-AAVs, that could be applied to any AAV serotype and further used for non-invasive gene delivery to the CNS. We performed a comparison between SEC and differential ultracentrifugation (UC) isolation protocols in terms of yield, contaminants, and transgene expression efficiency. We found that SEC allows a higher recovery of EV-AAVs, free of cell contaminating proteins and with less solo AAVs than UC. Remarkably, SEC-purified EV-AAVs also showed to be more potent at transgene expression than solo AAVs in neuronal cell lines. EV-AAVs exhibited the ability to cross the BBB in neonatal mice upon intravenous administration. In conclusion, SEC-purified brain-targeting EV-AAVs show to be a promising gene delivery vector for therapy of brain disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/542901v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@7016b8org.highwire.dtl.DTLVardef@e3a5f3org.highwire.dtl.DTLVardef@1a43286org.highwire.dtl.DTLVardef@f110c5_HPS_FORMAT_FIGEXP M_FIG C_FIG During the production of AAV vectors, a small percentage of AAVs is secreted in association with extracellular vesicles, named "EV-AAVs". EV-AAVs can be efficiently isolated by size exclusion chromatography (SEC). When intravenously injected in mice, brain targeting EV-AAVs can cross the blood brain barrier (BBB) and transduce neuronal cells.

molecular biology↗