Biology subjects

Tombesi, G.

Publications and source records attributed to Tombesi, G..

5 recordsLinked to original sources

bioRxiv2026

Loss of catalytic activity and impaired proteostasis in guanosine nucleotide-depleted LRRK2

Coding mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene represent the most common cause of familial Parkinsons disease (PD), and are frequently observed in idiopathic PD. In addition, variation around the LRRK2 locus has been shown to alter PD risk by genome-wide association studies. Disease-causing mutations cluster within the catalytic core of LRRK2 - composed of GTPase (ROC) and serine-threonine kinase domains - and lead to an increase in kinase activity, resulting in hyperphosphorylation of a subset of RAB GTPases and consequent cellular toxicity. However, the interplay between LRRK2 GTPase and kinase domains, and with the surrounding scaffold regions has remained underexplored, with implications for the prediction of on- and off-target effects associated with kinase inhibition. To address this gap, here we dissected the contributions of kinase, GTPase and scaffold domains to LRRK2 function in murine macrophages and tissues expressing endogenous levels of GTP/GDP-binding deficient Lrrk2 T1348N. Guanosine nucleotide-free Lrrk2 is devoid of both GTPase and kinase activities but maintains the scaffold shell, leading to significant reshaping of Lrrk2 interactome and engagement in novel interactions. This altered functional state leads to impaired autophagy and accumulation of enlarged lysosomes and autophagic cargo in macrophages and kidneys. Since pharmacological inhibition of LRRK2 is under clinical evaluation, our results reveal retained scaffold functions upon loss of catalytic activity that warrant careful consideration.

cell biology↗

bioRxiv2025

Leucine-rich repeat kinase 2 impairs the release sites of Parkinson's disease vulnerable dopamine axons

The end-stage pathology of Parkinsons disease (PD) involves the loss of dopamine-producing neurons in the substantia nigra pars compacta (SNc). However, synaptic deregulation of these neurons begins much earlier. Understanding the mechanisms behind synaptic deficits is crucial for early therapeutic intervention, yet these remain largely unknown. In the SNc, different dopamine neuron subtypes show varying susceptibility patterns to PD, complicating our understanding. This study uses intersectional genetic mouse models to uncover synaptic perturbations in vulnerable dopamine neurons, focusing on the LRRK2 kinase, a protein closely linked to PD. Through a combination of immunofluorescence and advanced proximity labeling methods, we found higher LRRK2 expression in the most vulnerable dopamine neuron subclusters. High-resolution imaging revealed that pathogenic LRRK2 disrupts release sites in vulnerable dopamine axons, leading to decreased in vivo spontaneous and evoked striatal dopamine release in mice with LRRK2 mutations. Proteomic and biochemical analyses indicate that mutant LRRK2 increases the phosphorylation of RAB3 proteins, reducing their interactions with RIM1and RIM2 effector proteins and impacting their synaptic functions. Overall, this research highlights the cell-autonomous dysfunctions caused by mutant LRRK2 in neurons, which are primarily affected by the disease. It also provides a framework for future therapeutic strategies for early nigrostriatal synaptic deficits in PD.

neuroscience↗

bioRxiv2024

LRRK2 mediates haloperidol-induced changes in indirect pathway striatal projection neurons

Haloperidol is used to manage psychotic symptoms in several neurological disorders through mechanisms that involve antagonism of dopamine D2 receptors that are highly expressed in the striatum. Significant side effects of haloperidol, known as extrapyramidal symptoms, lead to motor deficits similar to those seen in Parkinsons disease and present a major challenge in clinical settings. The underlying molecular mechanisms responsible for these side effects remain poorly understood. Parkinsons disease-associated LRRK2 kinase has an important role in striatal physiology and a known link to dopamine D2 receptor signaling. Here, we systematically explore convergent signaling of haloperidol and LRRK2 through pharmacological or genetic inhibition of LRRK2 kinase, as well as knock-in mouse models expressing pathogenic mutant LRRK2 with increased kinase activity. Behavioral assays show that LRRK2 kinase inhibition ameliorates haloperidol-induced motor changes in mice. A combination of electrophysiological and anatomical approaches reveals that LRRK2 kinase inhibition interferes with haloperidol-induced changes, specifically in striatal neurons of the indirect pathway. Proteomic studies and targeted intracellular pathway analyses demonstrate that haloperidol induces a similar pattern of intracellular signaling as increased LRRK2 kinase activity. Our study suggests that LRRK2 kinase plays a key role in striatal dopamine D2 receptor signaling underlying the undesirable motor side effects of haloperidol. This work opens up new therapeutic avenues for dopamine-related disorders, such as psychosis, also furthering our understanding of Parkinsons disease pathophysiology. SummaryChen et al. demonstrate that haloperidol mediated changes in the striatal indirect pathway neurons and circuits are linked to Parkinsons disease associated LRRK2. Inhibiting LRRK2 kinase activity ameliorates the motoric side effects of haloperidol, suggesting a potential approach to alleviating the unwanted side effects of antipsychotics.

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↗

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↗