Biology subjects

Costa, M. R.

Publications and source records attributed to Costa, M. R..

8 recordsLinked to original sources

bioRxiv2026

The Alzheimer's disease-associated rs28834970 variant in PTK2B impairs electrical activity and regulates Tau phosphorylation and APP processing in human neuronal cultures

Background: Genome-wide association studies have identified the PTK2B gene, which encodes Proline-rich tyrosine kinase 2 (PYK2), as a genetic risk factor for Alzheimer's disease (AD). However, its role in AD aetiology remains to be understood, as a complex contribution to amyloid {beta} (A{beta}) toxicity, tauopathy and neuronal dysfunction is suggested by contradictory evidence. Determining how the causal variant among AD-associated PTK2B variants impairs PYK2 function to trigger AD hallmarks could bring clarification. Methods: We combined in silico prediction tools with luciferase reporter assays in neuroblastoma cells to prioritize potential AD-associated PTK2B functional variants. We generated isogenic KOLF2.1J induced pluripotent stem cells (iPSCs) carrying the most relevant functional variant using CRISPR-Cas9 and differentiated them into human neuronal cultures (hNCs). We assessed PYK2 expression via immunoblotting, performed transcriptomic profiling using RNA-sequencing, analyzed neuronal network activity via multielectrode array (MEA) recordings, quantified synaptic density through immunocytochemistry in microfluidic devices, and assessed Tau phosphorylation and amyloid-precursor protein (APP) metabolism. Results: The rs28834970 regulatory variant was identified as the most functionally relevant in our model, with the C risk allele promoting transcription factor C/EBP{beta} binding within a cis-regulatory element. In hNCs, the homozygous rs28834970 CC genotype reduced total PYK2 levels by more than 80%, while increasing the proportion of phosphorylated (activated) PYK2. Transcriptomic analysis revealed 724 downregulated and 113 upregulated gene sets, with altered expression of genes involved in extracellular matrix organization, cytoplasmic translation and neurotransmitter secretion. MEA recordings showed reduced network recruitment and altered burst organization. Immunocytochemistry in microfluidic devices consistently revealed that rs28834970 impacts dendritic development and postsynaptic maturation. The risk genotype significantly elevated Tau phosphorylation at pathological sites (Thr181, Thr217, Thr231, Ser202-Thr205, Ser396-Ser404), and enhanced APP processing, resulting in decreased levels of APP and CTF but increased levels of sAPP, sAPP{beta} and A{beta}. Conclusion: The rs28834970 C risk allele mechanistically links AD susceptibility to downregulation of PTK2B/PYK2 expression and to the increase in Tau pathology and A{beta} levels while compromising synaptic structure and function.

genetics↗

bioRxiv2026

Cell-Type Specific Molecular and Functional Consequences of TDP-43 Loss-of-Function in Human Induced Neurons

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disorder characterized by the cytoplasmic aggregation and nuclear depletion of the TDP-43 protein. The latter impairs TDP-43 function as an RNA-binding protein and compromises the repression of cryptic splicing events, affecting both glutamatergic upper motor neurons and cholinergic lower motor neurons. This study systematically investigated the molecular and functional consequences of TDP-43 knockdown in human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons (iGNs) and cholinergic motor neurons (iMNs) using antisense oligonucleotides. The results demonstrated that TDP-43 loss elicits widespread, cell-type-specific changes in gene expression and alternative splicing. Notably, a shared subset of ALS-associated targets, including STMN2 and UNC13A, were consistently downregulated and mis-spliced across both neuronal subtypes. Functionally, Microelectrode Array (MEA) electrophysiology recordings revealed that TDP-43 knockdown induces a hyperexcitable phenotype in both neuronal populations, though they exhibited distinct network patterns: iGNs displayed synchronized bursting and significant shifts in overall electrophysiological profiles, while iMNs showed asynchronous firing. Furthermore, the inclusion of astrocytes in co-culture models expanded the repertoire of detectable cryptic splicing, including an event in HDGFL2 previously identified in patient cerebrospinal fluid. Despite these profound molecular and functional deficits, TDP-43 depletion did not impact neuronal viability or increase susceptibility to glutamate-induced excitotoxicity. These findings validate hiPSC-derived iGNs and iMNs as relevant models for ALS and highlight the critical necessity of considering cell-type specificity when elucidating pathogenesis and developing targeted therapies.

neuroscience↗

bioRxiv2025

Rational design of potent small molecule SMARCA2/A4 (BRM/BRG1) degraders acting via the recruitment of FBXO22

Target-anchored monovalent degraders are more drug-like than their bivalent counterparts, Proteolysis Targeting Chimeras (PROTACs), while offering greater target specificity control than the E3 ligase-anchored monovalent degraders, also known as molecular glues. However, their discovery has typically been serendipitous, and the rules governing their identification remain unclear. This study focused on the intentional discovery of SMARCA2/A4 monovalent degraders using a library based on SMARCA2/A4 bromodomain-binding ligands. Compound G-6599 emerged as a lead candidate, showing exceptional degradation potency and specificity for SMARCA2/A4. Mechanistic studies revealed that G-6599 operates through the ubiquitin-proteasome pathway and the E3 ligase FBXO22. G-6599 was shown to promote ternary complex formation between SMARCA2 and FBXO22 involving covalent conjugation to a cysteine residue on the latter. Unlike other recently identified FBXO22-dependent degraders, it does not require biotransformation. The selective degradation ability of G-6599, along with its unique mechanism, highlights the therapeutic potential of target-anchored monovalent degraders.

cancer biology↗

bioRxiv2022

BIN1 regulates electrical activity and network synchronization in hiPSC-derived neurons

BackgroundBridging Integrator 1 (BIN1) is the second most important Alzheimers disease (AD) risk gene, but its physiological roles in neurons and its contribution to brain pathology remain largely elusive. In this work, we show that BIN1 plays a critical role in the regulation of calcium homeostasis, electrical activity, and gene expression of glutamatergic neurons. MethodsWe generated 3D cerebral organoids and 2D enriched neuronal cell cultures from isogenic BIN1 wild-type (WT), heterozygous (HET) and homozygous knockout (KO) human-induced pluripotent stem cells (hiPSCs). Using single-cell RNA-sequencing, biochemical assays, immunocytochemistry and multi-electrode array(MEA) electrophysiology, we characterized the molecular and functional consequences of reduced BIN1 expression in different neural cell types. ResultsWe show that BIN1 is mainly expressed by oligodendrocytes and glutamatergic neurons of cerebral organoids, like in the human brain. Both BIN1 HET and KO cerebral organoids show specific transcriptional alterations, mainly associated with ion transport and synapses in glutamatergic neurons. We then demonstrate that BIN1 cell-autonomously regulates gene expression in glutamatergic neurons by using a novel protocol to generate pure culture of human-derived induced neurons (hiNs). Using this system, we also show that BIN1 plays a key role in the regulation of neuronal calcium transients and electrical activity via its interaction with the L-type voltage-gated calcium channel Cav1.2. BIN1 KO hiNs show reduced activity-dependent internalization and higher Cav1.2 expression compared to WT hiNs. Pharmacological treatment with clinically relevant doses of nifedipine, a calcium channel blocker, partly rescues neuronal electrical and gene expression alterations in BIN1 KO glutamatergic neurons. Further, we show that transcriptional alterations in BIN1 KO hiNs affecting biological processes related to calcium homeostasis are also present in glutamatergic neurons of the human brain at late stages of AD pathology. ConclusionsTogether, our findings suggest that BIN1-dependent alterations in neuronal properties could contribute to AD pathophysiology and that treatment with low doses of clinically approved calcium blockers should be considered as an option to dampen disease onset and progression.

neuroscience↗

bioRxiv2021

The Alzheimer's disease genetic risk factor BIN1 induces isoform-dependent neurotoxicity through early endosome defects

The Bridging Integrator 1 (BIN1) gene is a major susceptibility gene for Alzheimers disease (AD). Deciphering its pathophysiological role is challenging due to its numerous isoforms. Here we observed in Drosophila that human BIN1 isoform1 (BIN1iso1) overexpression, contrary to BIN1iso8 and BIN1iso9, induced an accumulation of endosomal vesicles and neurodegeneration. Systematic search for endosome regulators able to prevent BIN1iso1-induced neurodegeneration indicated that a defect at the early endosome level is responsible for the neurodegeneration. In human induced neurons (hiNs) and cerebral organoids, BIN1 knock-out resulted in the narrowing of early endosomes. This phenotype was rescued by BIN1iso1 but not BIN1iso9 expression. Finally, BIN1iso1 overexpression also led to an increase in the size of early endosomes and neurodegeneration in hiNs. Altogether, our data demonstrate that the AD susceptibility gene BIN1, and especially BIN1iso1, contributes to early-endosome size deregulation, which is an early pathophysiological hallmark of AD pathology.

neuroscience↗

bioRxiv2020

Evidence of progenitor cell lineage rerouting in the adult mouse hippocampus

Cell lineage in the adult hippocampus comprises multipotent and neuron-committed progenitors. In the present work, we fate-mapped neuronal progenitors using Dcx-CreERT2 and CAG-CAT-EGFP double-transgenic mice (cDCX/EGFP). We show that three days after tamoxifen-mediated recombination in cDCX/EGFP adult mice, GFP+ cells in the dentate gyrus co-expresses DCX and about 6% of these cells are proliferative neuronal progenitors. After 30 days, 20% of GFP+ generated from these progenitors differentiate into GFAP+ astrocytes. Administration of the chemoconvulsants kainic acid (KA) or pilocarpine (PL) led to a significant increase in the number of GFP+ cells in both ipsi and contralateral dentate gyrus. However, while PL favored the differentiation of neurons in both ipsi- and contralateral sides, KA stimulated neurogenesis only in the contralateral side. In the ipsilateral side, KA injection led to an unexpected increase of astrogliogenesis in the Dcx-lineage. These different effects of KA and PL in the Dcx-lineage are associated with distinct alterations of the parvalbuminergic plexus and inflammatory responses in the hippocampi. Finally, we also observed a small number of GFP+/GFAP+ cells displaying radial-glia morphology ipsilaterally 3 days after KA administration, indicating that Dcx-progenitors could regress to a multipotent stage. Altogether, our data suggest that cell lineage in the adult hippocampus is not unidirectional and can be modulated by environmental signals.

neuroscience↗

bioRxiv2018

Evidence of Muller glia conversion into retina ganglion cells using Neurogenin2

Macular Degeneration, Glaucoma, and Retinitis Pigmentosa are all leading causes of irreversible visual impairment in the elderly, affecting hundreds of millions of patients. Muller glia cells (MGC), the main type of glia found in the vertebrate retina, can resume proliferation in the adult injured retina and contribute to tissue repair. Also, MGC can be genetically reprogrammed through the expression of the transcription factor (TF) Achaete-scute homolog 1 (ASCL1) into induced neurons (iNs), displaying key hallmarks of photoreceptors, bipolar and amacrine cells, which may contribute to regenerate the damaged retina. Here, we show that the TF neurogenin 2 (NEUROG2) is also sufficient to lineage-reprogram MGC into iNs. The efficiency of MGC lineage conversion by NEUROG2 is similar to that observed after expression of ASCL1. However, reprogramming efficiency is affected by previous exposure to EGF and FGF2 during the expansion of MGC population. Transduction of either Neurog2 or Ascl1 led to the upregulation of key retina neuronal genes in MGC-derived iNs, but only NEUROG2 induced a consistent increase in the expression of putative retinal ganglion cell (RGC) genes. In vivo electroporation of Neurog2 in the neonatal retina also induced a shift in the generation of retinal cell subtypes, favoring the differentiation RGCs at the expense of MGCs. Altogether, our data indicate that Neurog2 induces lineage conversion of MGCs into RGC-like iNs.

neuroscience↗

bioRxiv2018

Multiple-gene targeting and mismatch tolerance can confound analysis of genome-wide pooled CRISPR screens

Genome-wide loss-of-function screens using the CRISPR/Cas9 system allow the efficient discovery of cancer cell vulnerabilities. While several studies have focused on correcting for DNA cleavage toxicity biases associated with copy number alterations, the effects of sgRNAs co-targeting multiple genomic loci in CRISPR screens have not been discussed yet. In this work, we analyze CRISPR essentiality screen data from 391 cancer cell lines to characterize biases induced by multi-target sgRNAs. We investigate two types of multi-targets: on-targets predicted through perfect sequence complementarity, and off-targets predicted through sequence complementarity with up to two nucleotide mismatches. We found that the number of on-targets and off-targets both increase sgRNA activity in a cell line-specific manner, and that existing additive models of gene knockout effects fail at capturing genetic interactions that may occur between co-targeted genes. We use synthetic lethality between paralog genes to show that genetic interactions can introduce biases in essentiality scores estimated from multi-target sgRNAs. We further show that single-mismatch tolerant sgRNAs can confound the analysis of gene essentiality and lead to incorrect co-essentiality functional networks.

bioinformatics↗