Biology subjects

Bernal, J. A.

Publications and source records attributed to Bernal, J. A..

7 recordsLinked to original sources

bioRxiv2025

Long-term Nrf2-driven microglial repopulation mitigates microgliosis, neuronal loss and cognitive deficits in tauopathy

A prominent pathological feature of tauopathies, including Alzheimers disease, is a chronic microglial reactivity, which contributes to neuroinflammation and disease progression. Microglia, the innate immune cells of the brain, can be pharmacologically eliminated by inhibiting the colony stimulating factor 1 receptor (e.g. with PLX5622), which is essential for their survival and proliferation. Upon inhibitor withdrawal, microglia rapidly repopulate, replenishing the central nervous system niche with naive cells. In recent years, microglia repopulation strategies have gained great interest as a means to reprogram dysfunctional microglia, while avoiding the detrimental effects of prolonged immune depletion. Despite promising short-term results, the long-term efficacy and pharmacological modulation of repopulated microglial remain poorly understood. Here, we investigate whether repopulated microglia after PLX5622 treatment sustains their beneficial effects over time in an AAV-hTauP301L induced model. Additionally, we assessed whether activating the cytoprotective nuclear factor erythroid 2 p45-related factor 2 (Nrf2) during microglial repopulation enhanced and prolonged the therapeutic outcomes. While microglial repopulation alone failed to maintain its neuroprotection in the long-term, when combined with an Nrf2 inducer, it improved cognitive deficits, reverted hippocampal neuronal loss and restored microglial phenotypes and mitochondrial energetics homeostasis, in our tauopathy-induced model. These results highlight the importance of shaping the fate of self-renewed microglia and propose Nrf2-mediated microglia repopulation as a potential pharmacological strategy for the treatment of tauopathies. HIGHLIGHTSO_LINrf2 inducer ND523 boosts long-term repopulated microglia function in tauopathy. C_LIO_LIND523-mediated microglial repopulation mitigates tau-induced cognitive decline. C_LIO_LIND523-driven microglial repopulation reduces neuronal loss without altering p-tau. C_LIO_LIND523-microglial repopulation rescues microglial morphology and reduces reactivity. C_LIO_LIND523-mediated repopulation prevents mitochondrial energetics dysfunctions. C_LI

neuroscience↗

bioRxiv2024

A comprehensive genetic catalog of human double-strand break repair

The analysis of DNA sequence outcomes provides molecular insights into double-strand break (DSB) repair mechanisms. By employing parallel in-pool profiling of Cas9-induced indels within a genome-wide knockout library, we present a comprehensive catalog detailing how virtually every human gene influences the DSB repair process. This REPAIRome resource is validated through the identification of novel mechanisms, pathways and factors involved in DSB repair, including unexpected opposing roles for XLF and PAXX in DNA end processing, a molecular explanation for Cas9-induced multi-nucleotide insertions, the identification of HLTF as a DSB-repair factor, the involvement of the SAGA complex in microhomology-mediated end joining, and importantly, an indel mutational signature linked to VHL loss, renal carcinoma and hypoxia. Collectively, these results exemplify the potential of REPAIRome to drive future discoveries in DSB repair, CRISPR-Cas gene editing and the etiology of cancer mutational signatures.

molecular biology↗

bioRxiv2023

Extracellular cysteine disulfide bond break at Cys122 disrupts PIP2-dependent Kir2.1 channel function and leads to arrhythmias in Andersen-Tawil Syndrome

BackgroundAndersen-Tawil Syndrome Type 1 (ATS1) is a rare heritable disease caused by mutations in the strong inwardly rectifying K+ channel Kir2.1. The extracellular Cys122-to-Cys154 disulfide bond in the Kir2.1 channel structure is crucial for proper folding, but has not been associated with correct channel function at the membrane. We tested whether a human mutation at the Cys122-to-Cys154 disulfide bridge leads to Kir2.1 channel dysfunction and arrhythmias by reorganizing the overall Kir2.1 channel structure and destabilizing the open state of the channel. Methods and ResultsWe identified a Kir2.1 loss-of-function mutation in Cys122 (c.366 A>T; p.Cys122Tyr) in a family with ATS1. To study the consequences of this mutation on Kir2.1 function we generated a cardiac specific mouse model expressing the Kir2.1C122Y mutation. Kir2.1C122Y animals recapitulated the abnormal ECG features of ATS1, like QT prolongation, conduction defects, and increased arrhythmia susceptibility. Kir2.1C122Y mouse cardiomyocytes showed significantly reduced inward rectifier K+ (IK1) and inward Na+ (INa) current densities independently of normal trafficking ability and localization at the sarcolemma and the sarcoplasmic reticulum. Kir2.1C122Y formed heterotetramers with wildtype (WT) subunits. However, molecular dynamic modeling predicted that the Cys122-to-Cys154 disulfide-bond break induced by the C122Y mutation provoked a conformational change over the 2000 ns simulation, characterized by larger loss of the hydrogen bonds between Kir2.1 and phosphatidylinositol-4,5-bisphosphate (PIP2) than WT. Therefore, consistent with the inability of Kir2.1C122Y channels to bind directly to PIP2 in bioluminescence resonance energy transfer experiments, the PIP2 binding pocket was destabilized, resulting in a lower conductance state compared with WT. Accordingly, on inside-out patch-clamping the C122Y mutation significantly blunted Kir2.1 sensitivity to increasing PIP2 concentrations. ConclusionThe extracellular Cys122-to-Cys154 disulfide bond in the tridimensional Kir2.1 channel structure is essential to channel function. We demonstrated that ATS1 mutations that break disulfide bonds in the extracellular domain disrupt PIP2-dependent regulation, leading to channel dysfunction and life-threatening arrhythmias. CLINICAL PERSPECTIVEO_ST_ABSNOVELTY AND SIGNIFICANCEC_ST_ABSWhat is known? O_LIAndersen-Tawil Syndrome Type 1 (ATS1) is a rare arrhythmogenic disease caused by loss-of-function mutations in KCNJ2, the gene encoding the strong inward rectifier potassium channel Kir2.1 responsible for IK1. C_LIO_LIExtracellular Cys122 and Cys154 form an intramolecular disulfide bond that is essential for proper Kir2.1 channel folding but not considered vital for channel function. C_LIO_LIReplacement of Cys122 or Cys154 residues in the Kir2.1 channel with either alanine or serine abolished ionic current in Xenopus laevis oocytes. C_LI What new information does this article contribute? O_LIWe generated a mouse model that recapitulates the main cardiac electrical abnormalities of ATS1 patients carrying the C122Y mutation, including prolonged QT interval and life-threatening ventricular arrhythmias. C_LIO_LIWe demonstrate for the first time that a single residue mutation causing a break in the extracellular Cys122-to-Cys154 disulfide-bond leads to Kir2.1 channel dysfunction and arrhythmias in part by reorganizing the overall Kir2.1 channel structure, disrupting PIP2-dependent Kir2.1 channel function and destabilizing the open state of the channel. C_LIO_LIDefects in Kir2.1 energetic stability alter the functional expression of the voltage-gated cardiac sodium channel Nav1.5, one of the main Kir2.1 interactors in the macromolecular channelosome complex, contributing to the arrhythmias. C_LIO_LIThe data support the idea that susceptibility to arrhythmias and SCD in ATS1 are specific to the type and location of the mutation, so that clinical management should be different for each patient. C_LIO_LIAltogether, the results may lead to the identification of new molecular targets in the future design of drugs to treat a human disease that currently has no defined therapy. C_LI

molecular biology↗

bioRxiv2021

Dual Dysfunction of Kir2.1 Underlies Conduction and Excitation-Contraction Coupling Defects Promoting Arrhythmias in a Mouse Model of Andersen-Tawil Syndrome Type 1

Andersen-Tawil Syndrome (ATS) is associated with life threatening arrhythmias of unknown mechanism. We report on a mouse model carrying the trafficking-deficient mutant Kir2.1{Delta}314-315. The mouse recapitulates the electrophysiological phenotype of type 1 (ATS1), with slower conduction velocities in response to flecainide, QT prolongation exacerbated by isoproterenol, and increased vulnerability to calcium-mediated arrhythmias resembling catecholaminergic polymorphic ventricular tachycardia (CPVT). Kir2.1{Delta}314-315 expression significantly reduced inward rectifier K+ and Na+ inward currents, depolarized resting membrane potential and prolonged action potential duration. Immunolocalization in wildtype cardiomyocytes and skeletal muscle cells revealed a novel sarcoplasmic reticulum (SR) microdomain of functional Kir2.1 channels contributing to intracellular Ca2+ homeostasis. Kir2.1{Delta}314-315 cardiomyocytes showed defects in SR Kir2.1 localization and function, which contributed to abnormal spontaneous Ca2+ release events. This is the first in-vivo demonstration of a dual arrhythmogenic mechanism of ATS1 defects in Kir2.1 channel function at the sarcolemma and the SR, with overlap between ATS1 and CPVT.

cell biology↗

bioRxiv2020

Neuronal NOX4 knockdown alleviates pathological tau-related alterations in a humanized mouse model of tauopathy

Approximately 44 million people worldwide live with Alzheimers disease (AD) or a related form of dementia. Aggregates of the microtubule-associated protein tau are a common marker of these neurodegenerative diseases collectively termed as tauopathies. However, all therapeutic attempts based on tau have failed, suggesting that tau may only indicate a higher-level causal mechanism. For example, increasing levels of reactive oxygen species (ROS) may trigger protein aggregation or modulate protein degradation. Here we show that type 4 NADPH oxidase (NOX), the most abundant isoform of the only dedicated reactive oxygen producing enzyme family, is upregulated in dementia and AD patients and in a humanized mouse model of tauopathy. Both global knockout and neuronal knockdown of the Nox4 gene in mice, diminished the accumulation of pathological tau and positively modified established tauopathy by a mechanism that implicates modulation of the autophagy-lysosomal pathway (ALP). Moreover, neuronal-targeted NOX4 knockdown was sufficient to reduce neurotoxicity and prevented cognitive decline, suggesting a direct and causal role for neuronal NOX4. Thus, NOX4 is a previously unrecognized causal, mechanism-based target in tauopathies and blood-brain barrier permeable specific NOX4 inhibitors could have therapeutic potential even in established disease. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/338954v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1dac496org.highwire.dtl.DTLVardef@1a07dd5org.highwire.dtl.DTLVardef@1a5653aorg.highwire.dtl.DTLVardef@198f3e4_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2020

SARS-CoV-2 protein Nsp1 alters actomyosin cytoskeleton and phenocopies arrhythmogenic cardiomyopathy-related PKP2 mutant

Mutations in desmosomal Plakophilin-2 (PKP2) are the most prevalent drivers of arrhythmogenic cardiomyopathy (ACM) and a common cause of sudden cardiac death in young athletes. However, partner proteins that elucidate PKP2 cellular mechanism to understand cardiac dysfunction in ACM are mostly unknown. Here we identify the actin-based motor proteins Myh9 and Myh10 as key PKP2 interactors, and demonstrate that the expression of the ACM-related PKP2 mutant R735X alters actin fiber organization and cell mechanical stiffness. We also show that SARS-CoV-2 Nsp1 protein acts similarly to this known pathogenic R735X mutant, altering the actomyosin component distribution on cardiac cells. Our data reveal that the viral Nsp1 hijacks PKP2 into the cytoplasm and mimics the effect of delocalized R735X mutant. These results demonstrate that cytoplasmic PKP2, wildtype or mutant, induces the collapse of the actomyosin network, since shRNA-PKP2 knockdown maintains the cell structure, validating a critical role of PKP2 localization in the regulation of actomyosin architecture. The fact that Nsp1 and PKP2 mutant R735X share similar phenotypes also suggests that direct SARS-CoV-2 heart infection could induce a transient ACM-like disease in COVID-19 patients, which may contribute to right ventricle dysfunction, observed in patients with poor survival prognosis. HighlightsThe specific cardiac isoform Plakophilin-2a (PKP2) interacts with Myh9 and Myh10. PKP2 delocalization alters actomyosin cytoskeleton component organization. SARS-CoV-2 Nsp1 protein hijacks PKP2 from the desmosome into the soluble fraction where it is downregulated. Viral Nsp1 collapses the actomyosin cytoskeleton and phenocopies the arrhythmogenic cardiomyopathy-related mutant R735X.

molecular biology↗

bioRxiv2020

Conserved cysteines in titin sustain the mechanical function of cardiomyocytes

The protein titin determines cardiomyocyte contraction and truncating variants in the titin gene (TTN) are the most common cause of dilated cardiomyopathy (DCM). Different to truncations, missense variants in TTN are currently classified as variants of uncertain significance due to their high frequency in the population and the absence of functional annotation. Here, we report the regulatory role of conserved, mechanically active titin cysteines, which, contrary to current views, we uncover to be reversibly oxidized in basal conditions leading to isoform- and force-dependent modulation of titin stiffness and dynamics. Building on our functional studies, we demonstrate that missense mutations targeting a conserved titin cysteine alter myocyte contractile function and cause DCM in humans. Our findings have a direct impact on genetic counselling in clinical practice. One sentence summaryMutations targeting cysteines key to the mechanoredox control of titin cause human dilated cardiomyopathy

physiology↗