Biology subjects

Bubacco, L.

Publications and source records attributed to Bubacco, L..

7 recordsLinked to original sources

bioRxiv2026

Evolutionary divergence of LRRK2 interaction domains contributes to human- and mouse-specific protein interaction networks

Leucine-rich repeat kinase 2 (LRRK2) is a complex multidomain protein whose catalytic and protein-protein interaction domains regulate a wide range of cellular processes. To investigate whether evolutionary divergence of these domains contributes to species-specific differences in LRRK2 biology, we combined phylogenetic, sequence, interactome and structural analyses of human and mouse LRRK2. Phylogenetic analysis revealed that the catalytic core predates the acquisition of the N-terminal and C-terminal protein-protein interaction domains during LRRK2 evolution. Accordingly, despite the high overall sequence similarity between human and mouse LRRK2, sequence divergence was not uniformly distributed across the protein but was concentrated within protein-protein interaction domains, whereas the catalytic ROC-COR- kinase core displayed markedly higher conservation. Consistent with this pattern, comparison of curated human and mouse interactomes revealed substantial differences in protein interaction networks and associated biological pathways. Structural modelling of a subset of interactors further showed that predicted interaction interfaces are enriched for residues that differ between the two species, providing a structural rationale for altered interaction specificity. Together, these findings support the view that evolutionary divergence of LRRK2 protein-protein interaction domains contributes to species-specific interactome organization. These results provide an evolutionary framework for interpreting differences between human and mouse LRRK2 and highlight the importance of considering species-specific interaction networks when translating findings from experimental models.

bioinformatics↗

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

Nuclear ASC speck formation in microglia is associated with inflammasome priming and is exacerbated in LRRK2-G2019S Parkinson disease

Neuroinflammation is increasingly recognized as a central pathological mechanism in Parkinsons disease (PD), a progressive neurodegenerative disorder characterized by the selective loss of dopaminergic neurons and variety of motor and non-motor symptoms. The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome and its adaptor protein ASC play a critical role in initiating and maintaining inflammatory responses in the central nervous system. Although its acute activation is beneficial for host defense and homeostasis, chronic activation of the inflammasome has been associated with the pathogenesis of PD. Another key contributor to neuroinflammation is the leucine-rich repeat kinase 2 (LRRK2), particularly the G2019S mutation associated with PD, which has been shown to exacerbate inflammatory signaling in microglia and peripheral immune cells. However, the interaction between LRRK2 and the NLRP3 inflammasome pathway remains poorly understood. In this study, we investigated the role of LRRK2-G2019S in the priming and activation dynamics of the NLRP3 inflammasome using mouse primary microglia and human monocyte-derived microglia-like cells (hMDMi). We observed that LRRK2-G2019S microglia exhibit increased expression of NLRP3 under basal conditions and spontaneous formation of ASC specks within the nucleus, an unexpected subcellular location not previously reported in microglia. Interestingly, nuclear ASC specks also formed in wild-type microglia and hMDMi after lipopolysaccharide (LPS) priming but only progressed to cytosolic ASC specks and interleukin-1{beta} release after subsequent exposure to canonical NLRP3 activators. These findings suggest that nuclear ASC specks may represent a primed state of inflammasome activation and propose a novel cellular phenotype associated with LRRK2-G2019S. Altogether, our results reveal a new layer of inflammasome regulation in microglia and implicate LRRK2-G2019S in the promotion of a pro-inflammatory state, which may predispose to chronic neuroinflammation in PD. These findings advance our understanding of glial immune regulation and highlight potential therapeutic targets in PD.

physiology↗

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

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↗

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↗

bioRxiv2021

DOPAL initiates αSynuclein-mediated impaired proteostasis in neuronal projections leading to enhanced vulnerability in Parkinson's disease.

Dopamine dyshomeostasis has been acknowledged to be among the determinants of nigrostriatal neuron degeneration in Parkinsons disease (PD). Several studies in experimental models and postmortem PD patients underlined increasing levels of the aldehydic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is highly reactive towards proteins. DOPAL has been shown to covalently modify the presynaptic protein Synuclein (Syn), whose misfolding and aggregation represent a major trait of PD pathology, triggering Syn oligomerization in dopaminergic neurons. Here, we demonstrated that DOPAL elicits Syn neuronal accumulation and hampers Syn clearance at synapses and the soma. By combining cellular and in vivo models, we provided evidence that DOPAL-induced Syn buildup lessens neuronal resilience, compromises synaptic integrity, and overwhelms protein quality control pathways, specifically at neuronal projections. The resulting progressive decline of neuronal homeostasis leads to dopaminergic neuron loss and motor impairment, corroborating the Syn-DOPAL interplay as an early event in PD neurodegeneration.

neuroscience↗