Biology subjects

Civiero, L.

Publications and source records attributed to Civiero, L..

12 recordsLinked to original sources

bioRxiv2026

Parkinson disease-associated protein DJ-1 regulates the autophagic-lysosomal pathway through ROS-dependent modulation of the AMPK/mTORC1 axis.

Mutations in the protein DJ-1 are linked to familial forms of Parkinsons disease (PD). The protein has been well-documented to exert a role in energy metabolism and antioxidant defense, contributing to the maintenance of mitochondrial homeostasis. We and others have previously observed that DJ-1 can also influence autophagy, but the mechanisms are still incompletely defined. In this study, using complementary cellular and animal models, we characterize the impact of DJ-1 loss on the autophagic pathway. Our data demonstrate that DJ-1 deficiency impairs autophagosome-lysosome fusion and lysosomal degradation, resulting in the accumulation of dysfunctional autolysosomes and the subsequent buildup of autophagic substrates. Mechanistically, we show that elevated reactive oxygen species (ROS) in DJ-1-null models inhibit the energy-sensing AMP-activated protein kinase (AMPK), thereby activating the autophagy suppressor mechanistic target of rapamycin 1 (mTORC1). Collectively, these findings delineate a novel signaling axis linking oxidative stress to autophagic dysfunction, providing new insights into the cellular mechanisms underlying autophagic dysfunction in PD.

cell biology↗

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

SPROUTS_DB: an implemented database of contaminants for extracellular vesicle proteomics studies

BackgroundCurrent proteomics techniques allow rapid identification and quantification of proteins within any given biological source. In particular, nanoUHPLC/High-Resolution nanoESI-MS/MS enables the characterization of proteins in complex biological samples due to its high sensitivity, accuracy, and scalability. However, LC-MS/MS proteomics might still be susceptible to laboratory and sample-associated contaminants, which can significantly compromise the quality and reliability of data. Therefore, an accurate identification and annotation of such contaminants is crucial for the development of robust proteomics databases and spectral-libraries related search engines. This approach is of special interest in the field of secretome and extracellular vesicles (EVs), membrane-enclosed nanostructures that contain a variety of proteins crucial for cell-to-cell communication and translational applications. ResultsWhen working in ex vivo/in vitro settings, proteins from fetal bovine serum (FBS), commonly employed in standard cell culture media, may interfere with the proteome analysis. To address this issue, we conceived and designed SPROUTS_DB, Serum Protein Repository Of Unwanted Target(ed) Sequences DataBase, a dedicated resource to catalog serum-derived contaminants. Starting from media supplemented with EV-depleted FBS, we simulated cell growth conditions - in the absence of cells - followed by ultracentrifugation. LC-MS/MS analysis of these samples resulted in the identification of a novel set of 1,288 contaminant proteins, which has been deposited in the ProteomeXchange repository (identifier PXD044137). SPROUTS_DB contains primarily soluble proteins, mainly related to the Gene Ontology categories Extracellular Region and Extracellular Space, in line with the nature of the starting sample. In contrast, only a small fraction of the contaminants is classified as membrane-associated proteins, supporting the limited vesicle contamination in the complete medium, due to the use of EV-depleted FBS. Of note, we demonstrated that SPROUTS_DB outperforms existing contaminants databases, ensuring that only peptide spectra relevant to the examined sample are retained and identified as true positive data. ConclusionsConsidering that even proteins from phylogenetically distant organisms share extensive stretches of sequences, SPROUTS_DB is designed to discern contaminants from real sample proteins of interest, minimizing false positive identifications. To the best of our knowledge, SPROUTS_DB is the most updated database of contaminants useful for proteomics investigations of cellular secretomes and EV-containing samples.

cell biology↗

bioRxiv2024

Atypical chemokine receptor 3 regulates synaptic removal in disease astrocytes

Astrocytes participate in the clearance of obsolete or unwanted neuronal synapses. However, the molecular machinery involved in recognizing these synapses remains unclear, particularly under pathological conditions. Here, we investigated the phagocytic process of astrocytes through individual gene silencing with a druggable gene library. Our study demonstrates that astrocyte- mediated synapse engulfment is regulated by the Atypical chemokine receptor 3 (Ackr3). Mechanistically, we showed that Ackr3 recognizes phosphatidylethanolamine (PE)-bound C-X-C motif chemokine 12 (CXCL12) at synaptic terminals both in vitro and in vivo, thus serving as a novel marker of synaptic dysfunction. Notably, both the receptor and its ligand are upregulated in post- mortem human Alzheimers disease (AD) brains, and AD mouse models. Downregulation of the Ackr3 in AD mice significantly diminishes astrocyte-mediated synaptic elimination, and rescues pathological phenotypes, including synapse loss and cognitive impairment. Overall, this work unveils a novel, possibly targetable mechanism of astrocyte-mediated synaptic engulfment implicated in neurodegenerative disease.

neuroscience↗

bioRxiv2024

PAK6 promotes neuronal autophagy by regulating TFEB nuclear translocation.

Autophagy is a highly conserved homeostatic process essential for the bulk degradation of cytoplasmic components and aggregated proteins. Multiple evidence indicates that impairment of (macro)autophagy leads to neurodegeneration, such as Parkinson disease (PD). Our previous work showed that p21 activated kinase 6 (PAK6) interacts with the PD-associated leucine-rich repeat kinase (LRRK2) to promote neurite outgrowth in the mouse striatum; still the function of PAK6 in the brain is largely unknown. Here, we found that downregulation of neuronal but not glial mbt, the D. melanogaster homolog of PAK6, impairs autophagy-lysosomal function. PAK6 overexpression in cells and in C. elegans increases transcription factor EB (TFEB) nuclear translocation in a kinase activity-dependent manner. Mechanistically, PAK6 forms a complex with TFEB to regulate its nuclear localization in a manner dependent on phosphorylation of and binding to 14-3-3 proteins and phosphorylation of TFEB at S467. In line with its ability to promote neuronal autophagy, mbt downregulation exacerbates alpha-synuclein toxicity in Drosophila dopaminergic neurons. Moreover, PAK6 overexpression in the substantia nigra of mutant LRRK2 mice reduces the burden of phosphorylated alpha-synuclein in dopaminergic neurons. Altogether, our study uncovers a novel role of PAK6 as a positive regulator of autophagy via TFEB and suggests that modulating its activity may represent a way to selectively turn on autophagy in neurons, with implications for the treatment of neurodegenerative disorders.

neuroscience↗

bioRxiv2024

PAK6 rescues pathogenic LRRK2-mediated ciliogenesis and centrosomal cohesion defects in a mutation-specific manner

P21 activated kinase 6 (PAK6) is a serine-threonine kinase with physiological expression enriched in the brain and overexpressed in a number of human tumors. While the role of PAK6 in cancer cells has been extensively investigated, the physiological function of the kinase in the context of brain cells is poorly understood. Our previous work uncovered a link between PAK6 and the Parkinsons disease (PD)-associated kinase LRRK2, with PAK6 controlling LRRK2 activity and subcellular localization via phosphorylation of 14-3-3 proteins. Here, to gain more insights into PAK6 physiological function, we performed protein-protein interaction arrays and identified a subgroup of PAK6 binders related to ciliogenesis. We confirmed that endogenous PAK6 localizes at both the centrosome and the cilium, and positively regulates ciliogenesis not only in tumor cells but also in neurons and astrocytes. Strikingly, PAK6 rescues ciliogenesis and centrosomal cohesion defects associated with the G2019S but not the R1441C LRRK2 PD mutation. Since PAK6 binds LRRK2 via its GTPase/Roc-COR domain and the R1441C mutation is located in the Roc domain, we used microscale thermophoresis and AlphaFold2-based computational analysis to demonstrate that PD mutations in LRRK2 affecting the Roc-COR structure substantially decrease PAK6 affinity, providing a rationale for the differential protective effect of PAK6 toward the distinct forms of mutant LRRK2. Altogether, our study discloses a novel role of PAK6 in ciliogenesis and points to PAK6 as the first LRRK2 modifier with PD mutation-specificity.

neuroscience↗

bioRxiv2023

Investigation of microglial diversity in a mouse model of Parkinson's disease pathology

Microglia, the central nervous system resident immune cells, are now recognized to critically impact homeostasis maintenance and contribute to the outcomes of various pathological conditions including Parkinsons disease (PD). Microglia are heterogenous, with a variety of states recently identified in aging and neurodegenerative disease models, including the disease-associated microglia (DAM) which present a selective enrichment of CLEC7A encoding the CLEC7A or DECTIN1 protein, and the dark microglia (DM) displaying markers of cellular stress at the ultrastructural level. However, the roles of CLEC7A-positive microglia and DM in the pathology of PD have remained largely elusive. By applying immunofluorescence and scanning electron microscopy, we aimed to characterize 1) the CLEC7A -positive cell population, and 2) their possible relationships to DM in a mouse model harboring a G2019S pathogenic mutation of the LRRK2 gene, the most common mutation linked to PD. We examined 18-month-old mice, comparing between LRRK2 G2019S knock-in mice and wild-type controls. In the dorsal striatum, a region affected by PD pathology, extensive ultrastructural features of cellular stress (e.g., endoplasmic reticulum and Golgi apparatus dilation), as well as reduced direct cellular contacts, were observed for microglia from LRRK2 G2019S mice versus controls. CLEC7A-positive microglia exhibited extensive phagocytic ultrastructural characteristics in the LRRK2 G2019S mice. Additionally, the LRRK2 G2019S mice presented a higher proportion of DM. Lastly, immunofluorescence and biochemical analysis revealed higher number of CLEC7A-positive cells in Lrrk2 G2019S genotype versus controls both in tissues and in primary microglia cells. Of note, CLEC7A-positive cells present a selective enrichment of ameboid morphology and tend to cluster in the pathogenic animal. In summary, we provide novel insights into the involvement of recently-defined microglial states, CLEC7A-positive cells and DM, in the context of LRRK2 G2019S PD pathology.

neuroscience↗

bioRxiv2023

Prospective role of PAK6 and 14-3-3 gamma as biomarkers for Parkinson's disease

2.BackgroundParkinsons disease is a progressive neurodegenerative disorder mainly distinguished by sporadic aetiology, although a genetic component is also well established. Variants in the LRRK2 gene are associated with both familiar and sporadic disease. We have previously shown that PAK6 and 14-3-3{gamma} protein interact with and regulate the activity of LRRK2. ObjectivesThe aim of this study is to quantify PAK6 and 14-3-3{gamma} in plasma as a reliable biomarker strategy for the diagnosis of both sporadic and LRRK2-linked Parkinsons disease. MethodsAfter an initial quantification of PAK6 and 14-3-3{gamma} expression by means of Western blot in post-mortem human brains, we verified the presence of the two proteins in plasma by using quantitative ELISA tests. We analysed samples obtained from 39 healthy subjects, 40 patients with sporadic Parkinsons disease, 50 LRRK2-G2019S non-manifesting carriers and 31 patients with LRRK2-G2019S Parkinsons disease. ResultsThe amount of PAK6 and 14-3-3{gamma} is significantly different in patients with Parkinsons disease compared to healthy subjects. Moreover, the amount of PAK6 also varies with the presence of the G2019S mutation in the LRRK2 gene. Although the generalized linear models show a low association between the presence of PD and PAK6, the kinase can be added in a broader panel of biomarkers for the diagnosis of Parkinsons disease. ConclusionsChanges of PAK6 and 14-3-3{gamma} amount in plasma represent a shared readout for patients affected by sporadic and LRRK2-linked Parkinsons disease. Overall, they can contribute to the establishment of an extended panel of biomarkers for the diagnosis of Parkinsons disease.

neuroscience↗

bioRxiv2022

LRRK2 regulates dendritic spine dynamics through interaction with post-synaptic actin cytoskeleton

Parkinsons disease (PD) is a multisystemic disorder that manifests through motor and non-motor symptoms. Motor dysfunction is the most debilitating and it is caused by the degeneration of dopamine-producing neurons in the substantia nigra pars compacta (SNpc). Increasing evidence suggests that synapse dysfunction precedes neuronal loss by years. Still, early synaptic alterations in PD remain poorly understood. Here we integrate literature meta-analysis, proteomics and phosphoproteomics with biochemical, imaging and electrophysiological measurements in neurons and brains from knockout and knockin Lrrk2 mouse models, as well as human iPSC-derived neurons lacking LRRK2. We demonstrate that phosphorylation of LRRK2 at Ser935 and of RAB proteins is induced by brain-derived neurotrophic factor (BDNF) stimulation in differentiated SH-SY5Y cells and primary mouse neurons. Affinity-purification coupled with mass spectrometry (AP-MS/MS) revealed a significant remodelling of the LRRK2 interactome following BDNF treatment, with enhanced association of LRRK2 to a network of actin cytoskeleton-related proteins. Gene-ontology analyses of both literature-curated LRRK2 interactors and phospho-proteome from striatal tissues with elevated LRRK2 activity (G2019S knockin mice) highlight synapse-actin remodelling as major affected pathways. We further observed that loss of LRRK2 impairs BDNF signaling and alters postsynaptic density architecture. One month-old Lrrk2 knockout mice display structural alterations in dendritic protrusions, a phenotype that normalizes with age. In human iPSC-derived neurons, BDNF enhances the frequency of miniature excitatory post-synaptic currents (mEPSC) in wild-type cells, an effect that is abolished in the absence of LRRK2. Taken together, our study discloses a critical role of LRRK2 in BDNF-dependent synaptic modulation and identifies the synaptic actin cytoskeleton as a convergent site of LRRK2-associated pathophysiological processes in PD.

neuroscience↗

bioRxiv2021

The activities of LRRK2 and GCase are positively correlated in clinical biospecimens and experimental models of Parkinson's disease

LRRK2 is a kinase involved in different cellular functions, including autophagy, endolysosomal pathways and vesicle trafficking. Mutations in LRRK2 cause autosomal dominant forms of Parkinsons disease (PD). Heterozygous mutations in GBA1, the gene encoding the lysosomal enzyme glucocerebrosidase (GCase), are the most common genetic risk factors for PD. Moreover, GCase function is altered in idiopathic PD and in other genetic forms of the disease. Recent work suggests that LRRK2 kinase activity can regulate GCase function. However, both a positive and a negative correlation have been described. To gain insights into the impact of LRRK2 on GCase, we investigated GCase levels and activity in LRRK2 G2019S knockin mice, in clinical biospecimens from PD patients carrying this mutation and in patient-derived cellular models. In these models we found a positive correlation between the activities of LRRK2 and GCase, which was further confirmed in cell lines with genetic and pharmacological manipulation of LRRK2 kinase activity. Overall, our study indicates that LRRK2 kinase activity affects both the levels and the catalytic activity of GCase.

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↗

bioRxiv2020

PKA-mediated phosphorylation of SPG11/spatacsin regulates binding with a subset of 14-3-3 proteins

Mutations in SPG11, encoding spatacsin, constitute the major cause of autosomal recessive Hereditary Spastic Paraplegia (HSP) with thinning of the corpus callosum. Previous studies showed that spatacsin orchestrates cellular traffic events through the formation of a coat-like complex and its loss of function results in lysosomal and axonal transport impairments. However, the upstream mechanisms that regulate spatacsin trafficking are unknown. Here, using proteomics and CRISPR/Cas9-mediated tagging of endogenous spatacsin, we identified a subset of 14-3-3 proteins as physiological interactors of spatacsin. The interaction is modulated by Protein Kinase A (PKA)-dependent phosphorylation of spatacsin at Ser1955, which initiates spatacsin trafficking from the plasma membrane to the intracellular space. Our study provides novel insight in understanding spatacsin physio-pathological roles with mechanistic dissection of its associated pathways.

cell biology↗