Biology subjects

Brito, V.

Publications and source records attributed to Brito, V..

7 recordsLinked to original sources

bioRxiv2026

Unscheduled DNA synthesis reveals a DNA repair hotspot and biomarker of somatic instability at the expanded-CAG repeat tract in the huntingtin gene

Somatic instability (SI) of expanded DNA repeats is a hallmark of repeat expansion disorder (REDs) and drives onset and progression in Huntingtons disease (HD) yet the absence of target engagement (TE) biomarkers for SI-modulating therapies represents a critical gap to clinical development. Here, we describe the development of the unscheduled repair synthesis assay (URSA)--combining 5-ethynyl-2-deoxyuridine (EdU) pulse-labeling with digital PCR or sequencing--and show that the CAG-expanded huntingtin (HTT) exon 1 allele is a highly active DNA repair hotspot in cells from people with HD (PwHD). Repair activity increases with repeat length, is allele-specific, and depends strongly on MSH3, a central driver of somatic expansion. URSA robustly quantifies MSH3 modulation in preclinical models within days compared to weeks or months required by conventional repeat-length measurements. Critically, substantial repair activity is detectable in peripheral blood mononuclear cells (PBMCs) from PwHD, where signal correlates with CAG length and improves predictive models of somatic expansion (SE) beyond age and CAG length alone. Unlike repeat-length changes, which require years to accumulate in blood, URSA signal is measurable within days. These findings establish DNA repair activity at the mutant HTT locus as a mechanistically grounded pharmacodynamic biomarker, enabling TE monitoring on a clinically actionable timescale, and with broad applicability to REDs and other diseases where modulation of the DNA damage response is therapeutically targeted.

neuroscience↗

bioRxiv2026

Targeted 3'-end RNA sequencing uncovers cryptic polyadenylation in Huntington's disease linked to somatic instability and CAG repeat purity

Huntingtons disease (HD) is a progressive neurodegenerative disorder caused by expanded CAG repeats in the first exon of the HTT gene, which encodes for huntingtin (HTT) protein. Full-penetrance is established at 40 repeats, but beyond, somatic repeat instability in the brain and CAG repeat purity modulate disease onset and severity. Previous studies have described that expanded repeats induce the incomplete splicing of HTT intron 1 to express the most pathogenic HTT isoform, known as HTT1a. Yet, the lack of a robust and sensitive method to evaluate HTT RNA-misprocessing has limited our understanding of HTT1a expression in HD pathophysiology. Here we describe a targeted RNA sequencing approach, known as 3-end targeted RNA sequencing or 3TRS, to simultaneously quantify multiple HTT transcripts generated by canonical and cryptic polyadenylation in several HD models. We show that activation of HTT cryptic polyadenylation is highly selective and requires long and uninterrupted CAG repeat expansions. In HD knock-in mice and human postmortem brain, cryptic HTT expression strongly correlates with brain-specific somatic repeat instability, supporting a model where ultralong and unstable CAG repeats drive toxicity by activating HTT RNA-misprocessing. Overall, 3TRS provides a robust framework to investigate HTT1a biogenesis and expression and to evaluate HTT-lowering therapeutic strategies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/697463v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@168d8e1org.highwire.dtl.DTLVardef@76ba3forg.highwire.dtl.DTLVardef@bdaf93org.highwire.dtl.DTLVardef@151e581_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Transcriptional and synaptic regulation of NMDA glutamate receptor-mediated hippocampal plasticity and memory

Synapse-to-nucleus signaling regulates activity-dependent synaptic plasticity underlying memory by linking N-methyl-D-aspartate (NMDA) glutamate receptors (GluN) to gene transcription mediated by the transcription factor cAMP-response element binding protein (CREB), but the underlying gene programs mediating potentiation at excitatory synapses are unknown. Here, we analyzed genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) datasets of mouse and human CREB and the synaptonuclear factor CREB-regulated transcription coactivator1 (CRTC1) to identify relevant target genes and biological pathways coupling neuronal activity to synaptic function/plasticity. Our analyses indicate that CRTC1 specifically couples neuronal activity with synaptic plasticity by binding to conserved promoters of CREB target genes comprising inducible transcription factors (including c fos, Crem, Npas4 and Nr4a1-3), and neuronal excitability and plasticity genes, including Ntrk2, Homer1, Dlg4 (PSD-95) and the NMDA receptor subunit Grin1 (GluN1). CRTC1/CREB target genes were highly enriched in gene ontology (GO) nuclear terms, including several members of the CREB family, and transcriptional modulators and repressors. Interestingly, GO enrichment and protein-protein interaction (PPI) network analyses revealed that genes mediating synapse-to-nucleus signaling (including most known synaptonuclear factors and direct interacting modulators) are collectively regulated by CREB/CRTC1, and that protein kinase C (PKC) is a key interactor of the CRTC1/14-3-3 complex at synapses. In agreement with these in silico analyses, we show that CRTC1 regulates synaptic activity-dependent phosphorylation and synaptic recruitment of GluN1 mediated by PKC in hippocampal neurons, and that PKC activation reverses NMDA receptor-mediated currents and long-term potentiation (LTP) deficits caused by CRTC1 silencing in the hippocampus. Consistent with genomics and functional data, morphological and behavioral analyses show crucial roles of CRTC1 on dendritic spine structure, plasticity, and hippocampal-dependent associative memory. Our results support a model in which neuronal activity and synaptic inputs are integrated in the nucleus through conserved CREB/CRTC1-regulated transcriptional programs sustaining global synapse-to-nucleus signaling pathways impacting on synaptic plasticity and memory.

neuroscience↗

bioRxiv2024

Characterization and Antiatherogenic Potential of P3R99 Monoclonal Antibody Against Sulfated Glycosaminoglycans: Physicochemical and Functional Insights

Atherosclerosis is initiated by the retention of ApoB-containing lipoproteins in the arterial wall, mediated by glycosaminoglycan chains of proteoglycans. At the Center for Molecular Immunology, we are developing the P3R99 monoclonal antibody (mAb) to target this process. This study characterizes new P3R99 mAb variants expressed in CHO-K1 and HEK-293 cell lines. We compared these variants with the parental mAb from NS0 cells using SDS-PAGE, size exclusion and cation exchange chromatography, dynamic light scattering, peptide mapping, far-UV circular dichroism, and PNGase F deglycosylation. All variants exhibited a molecular size of [~]150 kDa, [~]99% purity, and similar average particle sizes (12.5-13.7 nm). They displayed a high {beta}-sheet content (>40%) and basic amino acids on the surface, with minor differences in peptide maps compared to the parental mAb. Notable differences were found in the content of acidic and basic species and glycosylation profiles. NS0-derived P3R99 had lower G0F content (10.39%), higher G1F (38.29%) and G2F (30.44%) levels, with more terminal galactose (83.07%) and sialylation (15.33%). In contrast, CHO-K1 and HEK-293 variants showed similar glycosylation patterns. Despite these differences, the antigen and atherosclerotic lesion recognition properties of the mAb were unaffected in vitro. Biodistribution studies in Sprague Dawley rats (1 mg, IV, n=3) revealed preferential accumulation of the new P3R99 variants in aortas and reduced LDL arterial retention (1 mg, IP). Passive administration of the mAbs (2 mg every three days, three IV doses, n=6-7) in a Lipofundin 20%-induced atherosclerosis NZW rabbit model also demonstrated preferential accumulation in aortas and reduced atherosclerosis, with 60% of treated rabbits not developing lesions. These results suggest that the P3R99 mAb derived from CHO-K1 and HEK-293 cells retains its antiatherogenic properties despite structural differences from the NS0-derived mAb associated with the different expression systems.

immunology↗

bioRxiv2023

m6A RNA modification of mHtt intron 1 regulates the generation of Htt1a in Huntington's Disease

Huntingtons disease (HD) is a dominantly inherited neurodegenerative disorder caused by an expanded, somatically unstable CAG repeat in the first exon of the huntingtin gene (HTT). In the presence of an expanded CAG repeat, huntingtin mRNA undergoes an aberrant processing that generates HTT1a transcripts with exon 1 and intron 1 sequences, which encodes the aggregation-prone and pathogenic HTTexon 1 protein. The regulatory mechanisms that contribute to the production of HTT1a are not fully understood. In a previous transcriptome-wide m6A landscape study performed in Hdh+/Q111 knock-in mice, we have found that the proximal region of intron 1 to exon1-intron 1 splice site in Htt RNA is highly modified by m6A. Several pieces of evidence have demonstrated that m6A is involved in RNA processing and splicing. Therefore, in this study we set out to explore the impact of m6A RNA modifications in the generation of Htt1a. We show in the striatum of Hdh+/Q111 mice that m6A is enriched in intronic sequences 5 to the cryptic poly (A) sites (IpA1 and IpA2) at 680 and 1145 bp into intron 1 as well as in Htt1a polyadenylated mRNA. We also verified the presence of specific m6A-modified sites near the 5 exon1-intron1 splice donor site. Intronic HTT m6A methylation was recapitulated in human samples showing a significantly increased methylation ratio in HD putamen post-mortem samples and in HD fibroblast cell lines from pre-symptomatic and symptomatic patients. In order to test the hypothesis that the m6A modification is involved in mutant Htt RNA processing, we performed a pharmacological inhibition of METTL3 and a targeted demethylation of Htt intron 1 in HD cells using a dCas13-ALKBH5 system. We found that Htt1a transcript levels in HD cells are regulated by METTL3 and by methylation status in Htt intron 1. Site-specific manipulation with an RNA editing system resulted in decreased expression levels of Htt1a, which was accompanied by a reduction in DNA damage, a major hallmark in HD. Finally, we propose that m6A methylation in intron 1 is likely dependent on the expanded CAG repeats. These findings provide insight into the role of m6A in the generation of the aberrantly spliced mutant Htt transcripts with important implications for therapeutic strategies.

neuroscience↗

bioRxiv2023

Novel chP3R99 mAb reduces subendothelial retention of atherogenic lipoproteins in Insulin-Resistant rats: Acute treatment versus long-term protection as an idiotypic vaccine for atherosclerosis

BACKGROUNDAtherosclerosis is triggered by the retention of apolipoprotein B-containing lipoproteins by proteoglycans. In addition to LDL, remnant lipoproteins have emerged as pivotal contributors to this pathology, particularly in the context of insulin resistance and diabetes. We have previously reported anti-atherogenic properties of a monoclonal antibody (chP3R99) that recognizes sulfated glycosaminoglycans on arterial proteoglycans. METHODS AND RESULTSSolid-phase assays demonstrated that chP3R99 effectively blocked over 50% lipoprotein binding to chondroitin sulfate and vascular extracellular matrix in vitro. The pre-perfusion of chP3R99 (competitive effect) resulted in specific antibody-arterial accumulation and reduced fluorescent lipoprotein retention by [~]60% in insulin resistant JCR:LA-cp rats. This competitive reduction was dose-dependent (25 {micro}g/mL-250 {micro}g/mL), effectively decreasing deposition of cholesterol associated with lipoproteins. In a five-week vaccination study in insulin resistant rats with (200 {micro}g SC, once a week), chP3R99 reduced arterial lipoprotein retention, and was associated with the production of anti-chondroitin sulfate antibodies (Ab3) able to accumulate in the arteries (dot-blot). Neither the intravenous inoculation of chP3R99 (4.5 mg/kg), nor the immunization with this antibody displayed adverse effects on lipid or glucose metabolism, insulin resistance, liver function, blood cell indices, or inflammation pathways in JCR:LA-cp rats. CONCLUSIONSBoth acute (passive) and long-term administration (idiotypic cascade) of chP3R99 antibody reduced LDL and remnant lipoprotein interaction with proteoglycans in an insulin-resistant setting. These findings support the innovative approach of targeting pro-atherogenic lipoprotein retention by chP3R99 as a passive therapy or as an idiotypic vaccine for atherosclerosis. CLINICAL PERSPECTIVEO_ST_ABSWhat Is New?C_ST_ABSO_LIInnovative anti-atherosclerotic chP3R99 mAb interferes with proteoglycan binding of both LDL and remnant lipoproteins in vitro and in vivo. C_LIO_LIIn vivo kinetic studies that immunize with chP3R99 reveal a temporal induction of an anti-idiotypic antibody cascade in a model of insulin resistance (analogous to a vaccine). C_LIO_LIWe discovered that the idiotypic chP3R99 monoclonal antibody (Ab1) was able to induce protective anti-anti-idiotypic (Ab3) antibodies present in both sera as well as the aorta (target organ) in vivo. C_LI What Are The Clinical Implications?O_LIThe chP3R99 mAb has efficacy for reducing the arterial retention of both LDL and remnant-derived lipoproteins and may be relevant of those with Type-2 Diabetes and/or residual CVD risk. C_LIO_LIWe show efficacy of chP3R99 mAb under pro-inflammatory conditions and that it does not exacerbate other metabolic aberrations during insulin resistance. C_LIO_LIData support the targeting pro-atherogenic lipoprotein retention with chP3R99 as a passive therapy or as an idiotypic vaccine for atherosclerosis, complementary to lipid lowering approaches. C_LI

pathology↗

bioRxiv2020

Hippocampal Egr1-dependent neuronal ensemblesnegatively regulate motor learning

Motor skills learning is classically associated with brain regions including cerebral and cerebellar cortices and basal ganglia. Less is known about the role of the hippocampus in the acquisition and storage of motor skills. Here we show that mice receiving a long-term training in the accelerating rotarod display marked transcriptional changes in the striatum and hippocampus when compared with short-term trained mice. We identify Egr1 as a modulator of gene expression in the hippocampus during motor learning. Using mice in which neural ensembles are permanently labeled in an Egr1 activity-dependent fashion we identify ensembles of Egr1-expressing pyramidal neurons in CA1 activated in short- and long-term trained mice in the rotarod task. When Egr1 is downregulated or these neuronal ensembles are depleted, motor learning is improved whereas their chemogenetic stimulation impairs motor learning performance. Thus, Egr1 organizes specific CA1 neuronal ensembles during the accelerating rotarod task that limit motor learning.

neuroscience↗