Biology subjects

Brone, B.

Publications and source records attributed to Brone, B..

6 recordsLinked to original sources

bioRxiv2025

Identification of low copy synaptic glycine receptors in the mouse brain using single molecule localisation microscopy

Glycine receptors (GlyRs) are heteropentameric chloride channels that mediate fast inhibitory neurotransmission in the brainstem and spinal cord, where they regulate motor and sensory processes. GlyRs are clustered in the post-synaptic membrane by strong interactions of the {beta} subunit with the scaffold protein gephyrin. Even though GlyR{beta} mRNA is highly expressed throughout the brain, the existence of synaptic GlyRs remains controversial as there is little conclusive evidence using conventional fluorescence microscopy and electrophysiological recordings. Here we exploit the high sensitivity and spatial resolution of single molecule localisation microscopy (SMLM) to investigate the presence of GlyRs at inhibitory synapses in the brain, focusing on several areas of the telencephalon. Making use of a knock-in mouse model expressing endogenous mEos4b-tagged GlyR{beta}, we identified few GlyRs in sub-regions of the hippocampus. Dual-colour SMLM revealed that these sparse receptors are integrated within the post-synaptic gephyrin domain, pointing to a possible role in maintaining the structural integrity of inhibitory synapses. In contrast, we found functionally relevant numbers of synaptic GlyRs at inhibitory synapses in the ventral striatum. Our results highlight the strength of SMLM to detect few and sparsely distributed synaptic molecules in complex samples and to analyse their organisation with high spatial precision.

neuroscience↗

bioRxiv2023

Stretch-injury promotes activation of microglia with enhanced phagocytic and synaptic stripping activities

Microglial cells must act as the first line of defense of the central nervous system, but they can be exposed to various mechanical signals that may trigger their activation. While the impact of chemical signaling on brain cells has been studied in detail, our current understanding of the mechanical signaling in microglia is still limited. To address this challenge, we exposed microglial cells to a single mechanical stretch and compared their behavior to chemical activation by lipopolysaccharide treatment. Here we show that stretching microglial cells results in their activation, demonstrating a strong mechanosensitivity. Stretched microglial cells exhibited higher Iba1 protein levels, a denser actin cytoskeleton and migrated more persistently. In contrary to LPS-treated cells, stretched microglia maintain a robust secretory profile of chemokines and cytokines, except for TNF-, highlighting the relevance of this model. Interestingly, a single stretch injury results in more compacted chromatin and DNA damage, suggesting possible long-term genomic instabilities in stretched microglia. Using neuronal networks in compartmentalized microfluidic chambers, we found that stretched microglial cells exhibit enhanced phagocytic and synaptic stripping activities. Altogether, our results propose that the immune potential of microglial cells can be unlocked by stretching events to maintain brain tissue homeostasis after mechanical injury.

biophysics↗

bioRxiv2022

Lack of the glycine alpha 2 receptor impairs reward-motivated behavior and striatal signal integration.

Distinct developmental pathologies, including autism spectrum disorder and schizophrenia, exhibit impaired reward-motivated behavior. Key to proper reward-motivated behavior is dopamine-mediated modulation of striatal activity. The glycine alpha 2 receptor (GlyR2) is the single functionally expressed glycine receptor in adult striatum, and is therefore ideally positioned to modulate striatal behavior and cellular activity. Here, we report excessive appetitive conditioning in male GlyR2 knockout mice. We next show that depletion of GlyR2 enhances dopamine-induced increases in the activity of putative dopamine D1-expressing striatal projection neurons, while not affecting dopamine neuron activity. Moreover, we found that excessive locomotor responses to amphetamine in GlyR2 KO mice correlate with immediate early gene c-fos expression in the dorsal striatum. 3-D modeling revealed an increase in the number of activated cell ensembles in the striatum in response to D-amphetamine in GlyR2 KO mice. Taken together, we show that depletion of GlyR2 impairs reward-motivated behavior and altered striatal signal integration. This sheds important light onto the cellular mechanisms that underlie reward function, and pave the way towards novel therapeutics for the treatment of e.g. schizophrenia and addiction. Significance statementThe glycine receptor alpha 2 has long been studied for its role in development, with expression assumed to decline throughout adulthood in favor of the glycine receptor alpha 1 and 3. Yet, we showed that in the dorsal striatum, the glycine alpha 2 receptor is the only functionally expressed glycine receptor at adult age (Molchanova et al., 2017). In the present work, we show for the first time that the glycine alpha 2 receptor crucially affects striatal cell activity, which lies at the basis of reward-motivated behaviors, and which is impaired in many psychiatric pathologies. Indeed, a link between the mutations in the glycine alpha 2 receptor and autism as well as schizophrenia has been described, but a functional role for the glycine alpha 2 receptor in adult brain structures that are involved in psychiatric pathologies, was never shown before.

neuroscience↗

bioRxiv2021

Glycine receptor α3K governs mobility and conductance of L/K splice variant heteropentamers

Glycine receptors (GlyRs) are ligand-gated pentameric chloride channels in the central nervous system. GlyR-3 is a possible target for chronic pain treatment and temporal lobe epilepsy. Alternative splicing into K or L variants determines the subcellular fate and function of GlyR-3, yet it remains to be shown whether its different splice variants can functionally co-assemble, and what the properties of such heteropentamers would be. Here, we subjected GlyR-3 to a combined fluorescence microscopy and electrophysiology analysis. We employ masked Pearsons and dual-color spatiotemporal correlation analysis to prove that GlyR-3 splice variants heteropentamerize, adopting the mobility of the K variant. Fluorescence-based single-subunit counting experiments revealed a variable and concentration ratio dependent hetero-stoichiometry. Via single-channel on-cell patch clamp we show heteropentameric conductances resemble those of the 3K splice variant. Our data are compatible with a model where 3 heteropentamerization fine-tunes mobility and activity of GlyR 3 channels, which is important to understand and tackle 3 related diseases. SummaryThe glycine receptor 3 is key to the central nervous systems physiology and involved in chronic pain and epilepsy. In this paper, Lemmens et al. reveal and functionally characterize 3 splice variant heteropentamerization via advanced single-molecule fluorescence image analysis. DeclarationsO_ST_ABSFundingC_ST_ABSWe acknowledge the UHasselt Advanced Optical Microscopy Centre (AOMC). Prof. Em. Marcel Ameloot, the Research Foundation Flanders (FWO, project G0H3716N) and the province of Limburg (Belgium) (tUL Impuls II) are acknowledged for funding the microscopy hardware. V. Lemmens is grateful for a doctoral scholarship from the UHasselt (17DOC11BOF) and KU Leuven (C14/16/053) Special Research Funds (BOF). Conflicts of interest / competing interestsNo conflicts of interest apply. Ethics approvalNot applicable Availability of data and materialAll data and material are available upon request. Code availabilityFluctuation imaging and co-localization analyses were performed in the software package PAM [71]. The software is available as source code, requiring MATLAB to run, or as pre-compiled standalone distributions for Windows or MacOS at http://www.cup.uni-muenchen.de/pc/lamb/software/pam.html or hosted in Git repositories under http://www.gitlab.com/PAM-PIE/PAM and http://www.gitlab.com/PAM-PIE/PAMcompiled. A detailed user manual is available at http://pam.readthedocs.io. Author contributionsConceptualization Meier J.C., Brone B. and Hendrix J.; Investigation and formal analysis Lemmens V. and Thevelein B.; Software development Hendrix J.; Writing the original draft Lemmens V., Thevelein B and Hendrix, J.; Review and editing by all authors.

biochemistry↗

bioRxiv2020

ATP-Citrate lyase fuels axonal transport across species

Microtubule (MT)-based transport is an evolutionary conserved processed finely tuned by posttranslational modifications. Among them, α-tubulin acetylation, which is catalyzed by the α-tubulin N-acetyltransferase 1, Atat1, promotes the recruitment and processivity of molecular motors along MT tracks. However, the mechanisms that controls Atat1 activity remains poorly understood. Here, we show that a pool of vesicular ATP-citrate lyase Acly acts as a rate limiting enzyme to modulate Atat1 activity by controlling availability of Acetyl-Coenzyme-A (Acetyl-CoA). In addition, we showed that Acly expression is reduced upon loss of Elongator activity, further connecting Elongator to Atat1 in the pathway regulating α-tubulin acetylation and MT-dependent transport in projection neurons, across species. Remarkably, comparable defects occur in fibroblasts from Familial Dysautonomia (FD) patients bearing an autosomal recessive mutation in the gene coding for the Elongator subunit ELP1. Our data may thus shine new light on the pathophysiological mechanisms underlying FD.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2019

ATAT1-enriched vesicles promote microtubule acetylation via axonal transport

Microtubules are polymerized dimers of - and {beta}-tubulin that underlie a broad range of cellular activities. Acetylation of -tubulin by the acetyl-transferase ATAT1 modulates microtubule dynamics and functions in neurons. However, it remains unclear how and why this enzyme acetylates microtubules over long distances in axons. Here, we show that loss of ATAT1 impairs axonal transport in neurons and cell free motility assays confirm a requirement of tubulin acetylation for proper bidirectional vesicular transport. Moreover, we demonstrate that the main cellular pool of ATAT1 is transported at the cytosolic side of neuronal vesicles that are moving along axons. Altogether, our data suggest that axonal transport of ATAT1-enriched vesicles is the predominant driver of -tubulin acetylation in axons.

neuroscience↗