Biology subjects

Carvalho, A. L.

Publications and source records attributed to Carvalho, A. L..

7 recordsLinked to original sources

bioRxiv2025

Allosteric Modulation of Pathological Ataxin-3 Aggregation: A Path to Spinocerebellar Ataxia Type-3 Therapies

Spinocerebellar ataxia type 3 (SCA3) is a rare inherited neurodegenerative disease caused by the expansion of a polyglutamine repeat in the protease ataxin-3 (Atx3). Despite extensive knowledge of the downstream pathophysiology, no disease-modifying therapies are currently available to halt disease progression. The accumulation of protein inclusions enriched in the polyQ-expanded Atx3 in neurons suggests that inhibiting its self-assembly may yield targeted therapeutic approaches. Here it is shown that a supramolecular tweezer, CLR01, binds to a lysine residue on a positively charged surface patch of the Atx3 catalytic Josephin domain. At this site, the binding of CLR01 decreases the conformational fluctuations of the distal flexible hairpin. This results in reduced exposure of the nearby aggregation-prone region, which overlaps with the substrate ubiquitin binding site and primes Atx3 self-assembly, ultimately delaying Atx3 amyloid fibril formation and reducing the secondary nucleation rate, a process linked to fibril proliferation and toxicity. These effects translate into the reversal of synapse loss in a SCA3 cultured cortical neuron model, an improved locomotor function in a C. elegans SCA3 model, and a delay in disease onset, accompanied by reduced severity of motor symptoms in a SCA3 mouse model. This study provides critical insights into Atx3 self-assembly, revealing a novel allosteric site for designing CLR01-inspired therapies targeting pathological aggregation pathways while sparing essential functional sites. These findings emphasize that targeting allosteric sites in amyloid-forming proteins may offer unique opportunities to develop safe therapeutic strategies for various protein misfolding disorders.

biophysics↗

bioRxiv2025

Permissive central tolerance plus defective peripheral checkpoints licence pathogenic memory B cells in CASPR2-antibody encephalitis

Autoimmunity affects 10% of the population. Within this umbrella, autoantibody-mediated diseases targeting one autoantigen provide a unique opportunity to comprehensively understand the developmental pathway of disease-causing B cells and autoantibodies. While such autoreactivities are believed to be generated during germinal centre reactions, the roles of earlier immune checkpoints in autoantigen-specific B cell tolerance are poorly understood. We address this concept in patients with CASPR2-autoantibody encephalitis and healthy controls. In both groups, comparable and high ([~]0.5%) frequencies of unmutated CASPR2-reactive naive B cells were identified. By contrast, CASPR2-reactive memory B cells were exclusive to patients, and their B cell receptors demonstrated affinity-enhancing somatic mutations with heterogenous binding kinetics. These effector molecules possessed epitope-dependent pathogenic effects in vitro neuronal cultures and in vivo. The unmutated common ancestors of these memory B cells showed a distinctive balance between strong CASPR2 reactivity and very limited binding across the remaining human proteome. Our results are the first to propose mechanisms underlying autoantigen-specific tolerance in humans. We identify permissive central tolerance, defective peripheral tolerance and heterogenous autoantibody binding properties as sequential pathogenic steps which licence CASPR2-directed pathology. By leveraging the basic immunobiology, we rationally direct tolerance-restoring approaches in CASPR2-antibody diseases. This paradigm is applicable across autoimmune conditions.

immunology↗

bioRxiv2024

Type I TARPs regulate Kv7.2 potassium channels andsusceptibility to seizures

The M-current is a low-threshold potassium current that modulates neuronal excitability and suppresses repetitive firing. However, the mechanisms regulating M-channel function remain unclear. We identified type I Transmembrane AMPA receptor Regulatory Proteins (TARPs) as M-channel Kv7.2 subunit interactors in cortical neurons, with their interaction increasing upon neuronal depolarization. Co-expression of TARPs with Kv7.2 increased channel surface expression and Kv7.2-mediated currents, while disrupting TARP-{gamma}2 expression in neurons perturbed dendritic Kv7.2 nano-clusters and decreased M-currents. Knock-in mice with an intellectual disability-associated TARP-{gamma}2 variant showed reduced hippocampal M-currents and increased seizure susceptibility, indicating that disrupting TARP-{gamma}2 regulation of Kv7.2-M-channels is epileptogenic. These findings show that TARP-{gamma}2, a synaptic protein crucial for excitatory transmission, also controls intrinsic excitability via M-channels. This discovery provides a link between synaptic transmission and neuronal excitability, with implications for disease, as the interplay between synaptic and intrinsic plasticity is pivotal to how the brain adapts to varying input signals. HighlightsO_LIType I TARPs bind to Kv7.2-M-channels and enhance Kv7.2-mediated currents. C_LIO_LITARP-{gamma}2 governs the neuronal nano-organization and function of Kv7.2 channels. C_LIO_LIIntellectual disability-associated TARP-{gamma}2 variant impairs M-currents and facilitates seizures. C_LIO_LIType I TARPs can serve as molecular integrators of synaptic and intrinsic excitability. C_LI

neuroscience↗

bioRxiv2024

Neuronal ARHGAP8 controls synapse structure and AMPA receptor-mediated synaptic transmission

The aberrant formation and function of neuronal synapses are recognized as major phenotypes in many cases of neurodevelopmental (NDDs) and -psychiatric disorders (NPDs). A growing body of research has identified an expanding number of susceptibility genes encoding proteins with synaptic function. Here, we present the first brain-focused characterization of a potential new susceptibility gene, ARHAGP8, which encodes a Rho GTPase activating protein (RhoGAP). Accumulating evidence suggests that ARHGAP8 plays a pivotal role in the pathogenesis of NPDs/NDDs. We provide the first evidence for ARHGAP8 as a novel player at excitatory synapses, with its synaptic localisation linked to the presence of the developmentally important NMDA receptor subunit GluN2B. By increasing ARHGAP8 levels in hippocampal neurons to mimic the copy number variant found in a subset of patients, we observed reductions in dendritic complexity and spine volume, accompanied by a significant decrease in synaptic AMPA receptor-mediated transmission. These results suggest that ARHGAP8 plays a role in shaping the morphology and function of excitatory synapses, and prompt further investigation of ARHGAP8 as a candidate gene in NDDs/NPDs.

neuroscience↗

bioRxiv2021

Aberrant hippocampal transmission and behavior in mice with a stargazin mutation linked to intellectual disability

Mutations linked to neurodevelopmental disorders, such as intellectual disability (ID), are frequently found in genes that encode for proteins of the excitatory synapse. Transmembrane AMPA receptor regulatory proteins (TARPs) are AMPA receptor auxiliary proteins that regulate crucial aspects of receptor function. Here, we investigate an ID-associated mutant form of the TARP family member stargazin. Molecular dynamics analyses showed that the stargazin V143L variant weakens the overall interface of the AMPAR:stargazin complex and hinders the stability of the complex. Knock-in mice for the V143L stargazin mutation manifest cognitive and social deficits and hippocampal synaptic transmission defects. In the hippocampus of stargazin V143L mice, CA1 neurons show impaired spine maturation in basal dendrites, and synaptic ultrastructural alterations. These data demonstrate a causal role for mutated stargazin in the pathogenesis of ID and highlight its role in the development and function of hippocampal synapses.

neuroscience↗

bioRxiv2021

Regulation of blood-brain barrier integrity and cognition by the microbiome-associated methylamines trimethylamine N-oxide and trimethylamine

BackgroundCommunication between the gut microbiota and the brain is primarily mediated via soluble microbe-derived metabolites, but the details of this pathway remain poorly defined. Methylamines produced by microbial metabolism of dietary choline and L-carnitine have received attention due to their proposed association with vascular disease, but their effects upon the cerebrovascular circulation have hitherto not been studied. ResultsHere we use an integrated in vitro/in vivo approach to show that physiologically relevant concentrations of the dietary methylamine trimethylamine N-oxide (TMAO) enhanced blood-brain barrier (BBB) integrity and protected it from inflammatory insult, acting through the tight junction regulator annexin A1. In contrast, the TMAO precursor trimethylamine (TMA) impaired BBB function and disrupted tight junction integrity. Moreover, we show that long-term exposure to TMAO protects murine cognitive function from inflammatory challenge, acting to limit astrocyte and microglial reactivity in a brain region-specific manner. ConclusionOur findings demonstrate the mechanisms through which microbiome-associated methylamines directly interact with the mammalian BBB, with consequences for cerebrovascular and cognitive function.

systems biology↗

bioRxiv2020

Constitutive ghrelin receptor activity modulates AMPA receptor traffic and supports memory formation

The ability of animals to store and retrieve food caches in the wild requires the integration of biological signals of hunger, satiety and memory. The role of ghrelin in regulating feeding and memory makes ghrelin receptors an important target to shape the required cellular and molecular responses. We investigated the effects of the high ligand-independent activity of the ghrelin receptor on the physiology of excitatory synapses. Blocking this type of activity produced a decrease in the synaptic content of AMPA receptors in hippocampal neurons and a reduction in GluA1 phosphorylation at Ser845. Impaired constitutive activity from the ghrelin receptor increased surface diffusion of AMPA receptors and impaired AMPA receptor synaptic delivery mediated by chemical long-term potentiation. These observations support a role for the constitutive activity of the ghrelin receptor in regulating AMPA receptor trafficking under basal conditions and synaptic plasticity. Accordingly, we found that blocking the ghrelin receptor constitutive activity impairs spatial and recognition memory. Impact statementThis work uncovers a role for the constitutive activity of the ghrelin receptor in memory, and in the regulation of the synaptic levels of AMPA receptors, their mobility and synaptic plasticity. Underscoring the importance of deciphering the physiological role of constitutive ghrelin receptor activity, ghrelin receptor inverse agonism is now being considered as a therapy to treat alcohol use disorder.

neuroscience↗