Biology subjects

Bossi, E.

Publications and source records attributed to Bossi, E..

7 recordsLinked to original sources

bioRxiv2025

LRRK2 G2019S disrupts GABAergic signaling and shifts excitatory/inhibitory balance in the striatum

The excitatory/inhibitory (E/I) balance within neural circuits is essential for proper brain function, and its disruption is a hallmark of several neurodegenerative diseases. In Parkinsons disease (PD), widespread alterations in the basal ganglia circuitry lead to an E/I imbalance in the striatum, contributing to excitotoxicity. Leucine-rich repeat kinase 2 (LRRK2) has recently emerged as a key contributor to both familial and sporadic forms of PD, with the pathogenic Gly2019Ser (G2019S) mutation representing one of the most frequently observed variants. This mutation is known to exacerbate excitotoxicity by impairing glutamate reuptake mechanisms, particularly through dysregulation of EAAT2 activity and its membrane localization. In contrast, the role of LRRK2 in GABAergic transmission remains poorly understood. Here, we reveal a clear modulation of inhibitory signaling by LRRK2 through a comprehensive approach combining mouse striatal slices and Xenopus laevis oocytes. Our results demonstrate, for the first time, that LRRK2 G2019S induces a significant reduction in GABA-evoked current amplitudes. Moreover, we identified an altered distribution of receptor isoforms in pathological tissue, affecting both tonic and phasic GABA currents. Specifically, synaptic GABAA receptors containing the {gamma}2 subunit were functionally modulated by LRRK2 G2019S. The reduced availability of gephyrin in the presence of the G2019S variant may impair the gephyrin-GABAA receptor complex, leading to decreased receptor surface expression and further shifting the glutamate/GABA current ratio toward excitatory dominance. This is supported by the increased activity of AMPA and NMDA receptors observed in the pathological striatum. Overall, our findings highlight a previously underappreciated role of LRRK2 G2019S in impairing GABAergic transmission and disrupting the E/I balance. These insights point to novel circuit-level mechanisms underlying LRRK2-linked PD and suggest new avenues for the development of disease-modifying therapies targeting inhibitory dysfunction. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/684189v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1b685b3org.highwire.dtl.DTLVardef@3ea39org.highwire.dtl.DTLVardef@1064a58org.highwire.dtl.DTLVardef@1fb0304_HPS_FORMAT_FIGEXP M_FIG C_FIG The LRRK2 G2019S mutation contributes to excitatory/inhibitory imbalance by reducing GABA-evoked currents. Specifically, it is associated with diminished phasic GABAergic transmission and enhanced tonic inhibition, suggesting an altered subcellular distribution of GABAA receptor subtypes.

neuroscience↗

bioRxiv2025

Delineating plant responses to the 3',5'- and 2',3'-cAMP isomers

Similar to animals, both the 3,5- and the 2,3-cAMP isomers are present in plants. The former is the enzymatic product of adenylate cyclases (ACs), the latter is an RNA degradation product. While there is increasing evidence that both isomers can elicit or modulate a broad range of physiological responses, the question of isomer specificity of responses has remained largely unresolved. To delineate isomer-specific responses in Arabidopsis thaliana at the systems level, we have combined a comparative proteomics and electrophysiological approaches. Both isomers cause distinct systemic effects on the proteome, with the 2,3 isomer notably affecting systems-level functions like transcriptional regulation. None of the isomers affects net ion fluxes in the root under control conditions, but both were able to attenuate the magnitude of oxidative stress-induced K+ net loss and Ca2+ uptake by 2-fold. Isomer-specific responses of single molecular targets were assessed in the cyclic nucleotide-gated channels 2 and 18 (CNGC2 and CNGC18). Both channels are gated by the 3,5-cAMP isomer only, suggesting that the gating is isomer-specific and this implies that gating in vivo depends on catalytically active ACs. HighlightsDistinct Arabidopsis responses to 3,5- and 2,3-cAMP uncover isomer-specific molecular targets and physiological effects in cyclic nucleotide signaling.

plant biology↗

bioRxiv2024

Unveiling the crucial role of betaine: Modulation of GABA homeostasis via SLC6A1 transporter (GAT1)

Betaine is an endogenous osmolyte that exhibits therapeutic potential by mitigating various neurological disorders. However, the underlying cellular and molecular mechanisms responsible for its neuroprotective effects remain puzzling. In this study, we describe a possible mechanism behind the positive impact of betaine in preserving neurons from excitotoxicity. Using electrophysiology, mass spectroscopy, radiolabelled cellular assay, and molecular dynamics simulation we demonstrate that betaine at mM concentration acts as a slow substrate of GAT1 (slc6a1), the predominant GABA transporter in the central nervous system. Intriguingly, when betaine is present at low concentration (0.01-3 mM) with GABA (at concentration <K0.5), it blocks the GABA reuptake. This GAT1 modulation occurs through the temporal inhibition of the transporter, i.e., the prolonged occupancy by betaine impedes the rapid transition of the transporter to the inward conformation. The temporal inhibition results in a crucial regulatory mechanism contributing to the maintenance of GABA homeostasis, preserving neurons from excitotoxicity.

neuroscience↗

bioRxiv2024

Bioisosteric analogs of MDMA with improved pharmacological profile

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is re-emerging in clinical settings as a candidate for the treatment of specific psychiatric disorders (e.g. post-traumatic stress disorder) in combination with psychotherapy. MDMA is a psychoactive drug, typically regarded as an empathogen or entactogen, which leads to transporter-mediated monoamine release. Despite its therapeutic potential, MDMA can induce dose-, individual-, and context-dependent untoward effects outside safe settings. In this study, we investigated whether three new methylenedioxy bioisosteres of MDMA improve its off-target profile. In vitro methods included radiotracer assays, transporter electrophysiology, bioluminescence resonance energy transfer and fluorescence-based assays, pooled human liver microsome/S9 fraction incubation with isozyme mapping, and liquid chromatography coupled to high-resolution mass spectrometry. In silico methods included molecular docking. Compared with MDMA, all three MDMA bioisosteres (ODMA, TDMA, and SeDMA) showed similar pharmacological activity at human serotonin and dopamine transporters (hSERT and hDAT, respectively) but decreased activity at 5-HT2A/2B/2C receptors. Regarding their hepatic metabolism, they differed from MDMA, with N-demethylation being the only metabolic route shared, and without forming phase II metabolites. Additional screening for their interaction with human organic cation transporters (hOCTs) and plasma membrane transporter (hPMAT) revealed a weaker interaction of the MDMA analogs with hOCT1, hOCT2, and hPMAT. Our findings suggest that these new MDMA analogs might constitute appealing therapeutic alternatives to MDMA, sparing the primary pharmacological activity at hSERT and hDAT, but displaying a reduced activity at 5-HT2A/2B/2C receptors and reduced hepatic metabolism. Whether these MDMA bioisosteres may pose lower risk alternatives to the clinically re-emerging MDMA warrants further studies.

pharmacology and toxicology↗

bioRxiv2022

Functional characterization of Atlantic salmon (Salmo salar L.) PepT2 transporters

The high-affinity/low-capacity system Slc15a2 (PepT2) is responsible for the reuptake of di/tripeptides from the renal proximal tubule, but it also operates in many other tissues/organs. Information regarding PepT2 in teleost fish is limited and to date functional data are available from the zebrafish (Danio rerio) only. Here, we report the identification of two slc15a2 genes in the Atlantic salmon (Salmo salar) genome, namely slc15a2a and slc15a2b. The two encoded PepT2 proteins share 87% identity and resemble both structurally and functionally to the canonical vertebrate PepT2 system. The mRNA tissue distribution analyses reveal a widespread distribution of slc15a2a transcripts, being more abundant in the brain and gills, while slc15a2b transcripts are mainly expressed in kidney and distal part of gastrointestinal tract. The function of the two transporters was investigated by heterologous expression in Xenopus laevis oocytes and two- electrode voltage-clamp recordings of transport and presteady-state currents. Both PepT2a and PepT2b in the presence of Gly-Gln elicit pH-dependent and Na+ independent inward currents. The biophysical and kinetic analysis of the recorded currents defined the transport properties, confirming that the two Atlantic salmon PepT2 proteins behave as high-affinity/low-capacity transporters. The recent structures and the previous kinetic schemes of rat and human PepT2 qualitatively account for the characteristics of the two Atlantic salmon proteins. This study is the first to report on the functional expression of two PepT2-type transporters that operate in the same vertebrate organism as a result of (a) gene duplication process(es). Key points summaryO_LITwo slc15a2-type genes, slc15a2a and slc15a2b coding for PepT2-type peptide transporters were found in the Atlantic salmon. C_LIO_LIslc15a2a transcripts, widely distributed in the fish tissues, are abundant in brain and gills, while slc15a2b transcripts are mainly expressed in kidney and distal gastrointestinal tract. C_LIO_LIAmino acids involved in vertebrate Slc15 transport function are conserved in PepT2a and PepT2b proteins. C_LIO_LIDetailed kinetic analysis indicates that both PepT2a and PepT2b operate as high-affinity transporters. C_LIO_LIThe kinetic schemes and structures proposed for the mammalian models of PepT2 are suitable to explain the function of the two Atlantic salmon transporters. C_LI

physiology↗

bioRxiv2021

Bile acids gate dopamine transporter mediated currents

Bile acids (BAs) are molecules derived from cholesterol that are involved in dietary fat absorption. New evidence supports an additional role for BAs as regulators of brain function. Interestingly, sterols such as cholesterol interact with monoamine transporters (MAT), including the dopamine (DA) transporter (DAT) which plays a key role in DA neurotransmission and reward circuitries in the brain. The present study explores interactions of the BA, obeticholic acid (OCA), with DAT and mechanistically defines the regulation of DAT activity via both electrophysiology and molecular modeling. We express murine DAT (mDAT) in Xenopus laevis oocytes and confirm that DA induces an inward current that reaches a steady-state at a negative membrane voltage. Next, we show that OCA triggers an inward current through DAT that is Na+ dependent and not regulated by intracellular calcium. OCA also inhibits the DAT-mediated Li+ leak current, a feature that parallels DA action and indicates direct binding to the transporter. Interestingly, OCA does not alter DA affinity nor the ability of DA to promote a DAT-mediated inward current, suggesting that the interaction of OCA with the transporter is non-competitive, in regard to DA. The current induced by OCA is transient in nature, returning to baseline in the continued presence of the BA. To understand the molecular mechanism of how OCA affects DAT electrical activity, we performed docking simulations. These simulations revealed two potential binding sites that provide important insights into the potential functional relevance of the OCA-DAT interaction. First, in the absence of DA, OCA binds DAT through interactions with D421, a residue normally involved in coordinating the binding of the Na+ ion to the Na2 binding site (Borre et al., 2014;Cheng and Bahar, 2015). Furthermore, we uncover a separate binding site for OCA on DAT, of equal potential functional impact, that is facilitated through the residues DAT R445 and D436. This binding may stabilize the inward-facing open (IFo) state by preventing the re-formation of the IF gating salt bridges, R60-D436 and R445-E428, that are required for DA transport. This study suggests that BAs may represent novel pharmacological tools to regulate DAT function, and possibly, associated behaviors.

neuroscience↗

bioRxiv2021

Trafficking of the glutamate transporter is impaired in LRRK2-related Parkinson's disease

The Excitatory Amino Acid Transporter 2 (EAAT2) accounts for 80 % of brain glutamate clearance and is mainly expressed in astrocytic perisynaptic processes. EAAT2 function is finely regulated by endocytic events, recycling to the plasma membrane and degradation. Noteworthy, deficits in EAAT2 have been associated with neuronal excitotoxicity and neurodegeneration. In this study, we show that EAAT2 trafficking is impaired by the leucine-rich repeat kinase 2 (LRRK2) pathogenic variant G2019S, a common cause of late-onset familial Parkinsons disease (PD). In LRRK2 G2019S human brains and experimental animal models, EAAT2 protein levels are significantly decreased, which is associated with elevated gliosis. The decreased expression of the transporter correlates with its reduced functionality in mouse LRRK2 G2019S purified astrocytic terminals and in Xenopus laevis oocytes expressing human LRRK2 G2019S. In LRRK2 G2019S knockin mouse brain, the correct surface localization of the endogenous transporter is impaired, resulting in its interaction with a plethora of endo-vesicular proteins. Mechanistically, we report that pathogenic LRRK2 kinase activity delays the recycling of the transporter to the plasma membrane via Rabs inactivation, causing its intracellular relocalization and degradation. Taken together, our results demonstrate that pathogenic LRRK2 interferes with the physiology of EAAT2, pointing to extracellular glutamate overload as a possible contributor to neurodegeneration in PD.

neuroscience↗