Rab10 phosphorylation at Thr73 (pRab10) is a well-established readout of the kinase LRRK2, a protein whose mutations are associated with Parkinson’s disease. Here, we present a protocol for high-content quantification of endogenous pRab10-positive vesicles in primary astrocyte-enriched cultures using the Operetta CLS system. We describe steps for cell culture, immunofluorescence, image acquisition, and quantitative analysis using Harmony software. This protocol is applicable to other LRRK2-expressing cells and offers a scalable platform for quantitative studies of LRRK2 physiological and pathological activity.

Abstract 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.

Parkinson’s 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). A body of evidence indicates that synapse demise precedes by years neuronal death. Still, early synaptic dysfunctions in PD are poorly deciphered. Here we combined literature metanalysis, proteomics and phosphoproteomics with biochemical, imaging and electrophysiological measurements in neurons, brains and synaptosomes from knockout and knockin mouse models, as well as human iPSC-derived neurons associated with the PD-kinase LRRK2. We show that phosphorylation of LRRK2 at Ser935, which controls LRRK2 subcellular localization, rapidly increases upon brain-derived neurotrophic factor (BDNF) stimulation of differentiated SH-SY5Y cells and primary mouse neurons. Affinity-purification coupled with mass spectrometry (AP-MS/MS) analysis revealed that LRRK2 interactome is significantly reshaped upon BDNF stimulation, with an interconnected network of actin cytoskeleton-associated proteins increasing their binding to LRRK2. Accordingly, LRRK2 knockout neurons exhibit decreased TrkB signaling and fail to induce BDNF-dependent spinogenesis. In vivo , one-month old Lrrk2 knockout mice display defects in spine maturation, a phenotype that disappears with age. In human iPSC-derived cortical neurons, BDNF increases the frequency of miniature excitatory post-synaptic currents (mEPSC) in wild-type but not in the presence of LRRK2 knockout, functionally supporting a distinctive role of LRRK2 in BDNF-synaptic signaling. Finally, Lrrk2 G2019S PD mutant synaptosomes display differentially phosphorylated proteins enriched in categories related to postsynaptic structural organization. Taken together, our study discloses a critical function of LRRK2 in BDNF-dependent synaptic processes and identifies the postsynaptic actin cytoskeleton as a convergent site of LRRK2 pathophysiological activity.

14-3-3 constitute a highly conserved family of proteins that participate in the regulation of essential cellular processes by establishing extensive protein-protein interactions. Consequently, perturbation of the 14-3-3s interactome can be implicated in the pathogenesis of several diseases. Phosphorylation has emerged as a key mechanism by which 14-3-3s interactome can be regulated, and aberrantly phosphorylated 14-3-3s have been observed in patients with neurological disorders, among which Parkinson’s disease. Here, we specifically investigate phosphorylation of 14-3-3γ at Ser59 and assess its impact on α-Synuclein aggregation. Consistent with observations performed by others with different 14-3-3 isoforms, we observed that phosphorylation reduces the protective abilities of 14-3-3γ both in vitro and in cellular models. Importantly, this effect is also evident in cerebrospinal fluid (CSF), suggesting that phosphorylation of 14-3-3γ may directly contribute to α-Synuclein aggregation in vivo and may be relevant to disease-associated mechanisms. Using bioinformatics approaches, we further examined how Ser59 phosphorylation reshapes the global 14-3-3γ interactome, revealing novel interaction networks and pathways potentially implicated in neurodegenerative disease pathogenesis.

Abstract Parkinson’s disease (PD) is defined pathologically by loss of dopamine-producing neurons in the substantia nigra pars compacta (SNc). Yet synaptic dysfunction emerges much earlier, making it essential to define the mechanisms that drive early nigrostriatal deregulation. In the SNc, molecularly distinct dopamine neuron subtypes show differential susceptibility to PD. Here, we used intersectional genetic mouse models to determine how the PD-linked kinase LRRK2 affects vulnerable dopamine subtypes. Immunofluorescence and proximity-labeling proteomics revealed enriched LRRK2 expression in vulnerable dopamine neuron subclusters. High-resolution imaging showed that pathogenic LRRK2 disrupts presynaptic release-site organization in vulnerable dopamine axons, leading to reduced spontaneous and evoked striatal dopamine release in vivo. Proteomic analyses further showed that mutant LRRK2 increases phosphorylation of RAB3 proteins, impairing their interaction with the active-zone effectors RIM1 and RIM2. Together, these findings highlight a subtype-specific, cell-autonomous mechanism by which pathogenic LRRK2 impairs PD-vulnerable nigrostriatal synapses and provide a framework for therapeutic strategies targeting early synaptic deficits in PD.

ABSTRACT Mutations in the protein DJ-1 are linked to familial forms of Parkinson’s 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.

Summary Coding mutations in the Leucine-rich repeat kinase 2 ( LRRK2 ) gene represent the most common cause of familial Parkinson’s 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.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of late-onset familial and idiopathic Parkinson's disease (PD), known to date. Importantly, recent data from postmortem tissue as well as biomarker studies suggest that independent of mutations, increased kinase activity of LRRK2 plays an essential role in idiopathic PD pathogenesis. Despite extensive research on LRRK2, its activation mechanism(s) and how the various mutations result in increased kinase activity and neuronal death are still not completely understood. Accumulating evidence points to LRRK2 phosphoregulation, both autophosphorylation and phosphorylation by other kinases, as one potential molecular trigger of its activation. LRRK2 activation and localization are regulated by phosphatases such as protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A); however, the exact mechanism of this phosphoregulation is not known. Our data reveal that in vitro PP2A dephosphorylates sites within the RocCOR-GTPase domain of LRRK2 and, as a result, destabilizes LRRK2 dimers, with consequent reduction of its kinase activity. Strikingly, our data further highlight that LRRK2 in turn phosphorylates the catalytic subunit of the PP2A holoenzyme PPP2CA at its critical residue T304. Furthermore, LRRK2-mediated phosphorylation of PP2CA T304 alters the methylation of the C-terminus, which is crucial for both holoenzyme formation and catalytic activity. Importantly, expression of WT-PPP2CA protects from LRRK2-G2019S-induced neuronal cell death, while the PPP2CA-T304A mutant fails to do so, suggesting that impaired PP2A holoenzyme formation might be detrimental for LRRK2-PD.

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of late-onset familial and idiopathic Parkinson’s disease (PD), known to date. Importantly, recent data from post-mortem tissue as well as biomarker studies suggest that independent of mutations, increased kinase activity of LRRK2 plays an essential role in idiopathic PD pathogenesis. Despite extensive research on LRRK2, its activation mechanism(s) and how the various mutations result in increased kinase activity and neuronal death is still not completely understood. Accumulating evidence points to LRRK2 phospho-regulation, both auto-phosphorylation and phosphorylation by other kinases, as one potential molecular trigger of its activation. LRRK2 activation and localization is regulated by phosphatases such as Protein phosphatase 1 (PP1) and Protein phosphatase 2A (PP2A), however the exact mechanism of this phospho-regulation is not known. Our data reveal that PP2A dephosphorylates sites within the RocCOR-GTPase domain of LRRK2 and as a result de-stabilizes LRRK2 dimers, with consequent reduction of its kinase activity. Strikingly, our data further highlight that LRRK2 in turn phosphorylates the catalytic subunit of the PP2A holoenzyme PPP2CA at its critical residue T304, crucial for both holoenzyme formation and catalytic activity. Importantly, expression of WT-PPP2CA protects from LRRK2-G2019S induced neuronal cell death, while PPP2CA-T304 mutants fail to do so, suggesting that impaired PP2A holoenzyme formation might be detrimental for LRRK2-PD. Significance statement Increased kinase activity of LRRK2 is a hallmark of Parkinson’s disease. Accumulating evidence points to LRRK2 phospho-regulation as one potential molecular trigger of its activation. However, the exact regulation of phosphorylation was not well understood. Our data reveal a feedback loop between LRRK2 and PP2A (a phosphatase) activity, which explains the neuroprotective properties of PP2A’s enzymatic activity in LRRK2-induced parkinsonism. Interestingly, PP2A activity is downregulated in alpha-synuclein PD models, having the same net-effect on PP2A activity as with hyper-active mutant LRRK2, and the methylation status of the C-terminal of PPP2CA plays an essential role in this regulation and progression of the disease. Understanding the regulation of PP2A, by post-translational modifications such as phosphorylation and methylation, can thus give important insight into the onset and progression of PD in general. ### Competing Interest Statement The authors have declared no competing interest. Michael J. Fox Foundation, 6709.3.

The end-stage pathology of Parkinson's 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 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 RIM1/2 effector proteins and impacting their synaptic functions. Overall, this research highlights the cell-autonomous dysfunctions caused by mutant LRRK2 in the neurons that are primarily affected by the disease. It also provides a framework for therapeutic strategies for early nigrostriatal synaptic deficits in PD.

The enzyme glucocerebrosidase (GCase) catalyses the hydrolysis of glucosylceramide to glucose and ceramide within lysosomes. Homozygous or compound heterozygous mutations in the GCase-encoding GBA1 gene cause the lysosomal storage disorder Gaucher disease, while heterozygous and homozygous mutations are the most frequent genetic risk factor for Parkinson's disease. These mutations commonly affect GCase stability, trafficking or activity. Here, we report the development and characterization of nanobodies (Nbs) targeting and acting as molecular chaperones for GCase. We identify several Nb families that bind with nanomolar affinity to GCase. Based on biochemical characterization, we group the Nbs in two classes: Nbs that improve the activity of the enzyme and Nbs that increase GCase stability in vitro. A selection of the most promising Nbs is shown to improve GCase function in cell models and positively impact the activity of the N370S mutant GCase. These results lay the foundation for the development of new therapeutic routes.

Neuroinflammation is increasingly recognized as a central pathological mechanism in Parkinson's disease (PD), a progressive neurodegenerative disorder marked by dopaminergic neuron loss and diverse motor and non-motor symptoms. The NLRP3 inflammasome and its adaptor protein ASC are critical to initiating and sustaining inflammatory responses in the central nervous system. Although acute inflammasome activation supports host defence responses, chronic activation has been linked to the pathogenesis of PD. Increasing evidence indicates that mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2), particularly the PD-associated G2019S mutation, enhance inflammatory signalling in microglia and peripheral immune cells. However, how LRRK2 intersects with the NLRP3 pathway remains unclear. Here, we investigate the role of LRRK2-G2019S in the priming and activation of the inflammasome in mouse primary microglia and human monocyte-derived microglia-like cells (hMDMi). Under unstimulated conditions, LRRK2-G2019S microglia displayed elevated NLRP3 expression and spontaneous formation of ASC specks within the nucleus, a subcellular localization not previously reported in microglia. Nuclear ASC specks also emerged in Wild Type microglia and hMDMi after lipopolysaccharide priming, but progressed to cytosolic ASC specks and IL-1β release only after canonical activation of NLRP3. These findings suggest that nuclear ASC specks mark a primed state of inflammasome activation in microglia. The LRRK2-G2019S mutation enhances this phenotype, potentially predisposing microglia to exaggerated inflammatory responses. This work identifies a novel cellular feature associated with PD-linked LRRK2 and uncovers a previously unrecognized layer of inflammasome regulation in microglia, offering new avenues to understand and target neuroinflammation in PD.

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 Parkinson’s disease and present a major challenge in clinical settings. The underlying molecular mechanisms responsible for these side effects remain poorly understood. Parkinson’s disease-associated leucine-rich repeat kinase 2 (LRRK2) has an essential 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 Parkinson’s disease pathophysiology.

Parkinson's disease (PD) is a multisystem disorder presenting motor and non-motor symptoms. Motor dysfunction is the most debilitating, caused by the degeneration of dopamine-producing neurons. Increasing evidence indicates that synapse demise occurs years before neuronal death. Yet, the early synaptic dysfunctions in PD remain poorly understood. Leucine-Rich Repeat Kinase 2 (LRRK2), a serine/threonine kinase and GTPase relevant for both familial and sporadic forms of PD, has been increasingly associated with synaptic processes. These include the phosphorylation of key synaptic proteins and interactions with cytoskeletal components. Brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) are fundamental for synapse maturation, maintenance, and plasticity. Recent findings indicate that neurotrophic signaling is impaired in PD. In this review, we critically discuss the significance of identifying and clarifying the early molecular events leading to synaptic dysfunction in PD. We examine how mutant LRRK2 affects these processes and the relationship between LRRK2 and BDNF signaling from both mechanistic and therapeutic perspectives.

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. ### Competing Interest Statement The authors have declared no competing interest. * AD : Alzheimer Disease ALP : autophagic lysosomal pathway α-syn : alpha-synuclein AMPK : AMP-activated protein kinase CLEAR : Coordinated Lysosomal Expression and Regulation DA : dopamine FCS : fluorescence correlation spectroscopy GCase : Glucocerebrosidase KD : knockdown KO : knockout LRRK2 : leucine-rich repeat kinase 2 mbt : mushroom bodies tiny mTORC1 : mechanistic target of rapamycin complex 1 OE : overexpressing PAK4 : p21 activated kinase 4 PAK5 : p21 activated kinase 5 PAK6 : p21 activated kinase 6 PD : Parkinson Disease WT : wild type

Microglia contribute to the outcomes of various pathological conditions including Parkinson's disease (PD). Microglia are heterogenous, with a variety of states recently identified in aging and neurodegenerative disease models. Here, we delved into the diversity of microglia in a preclinical PD model featuring the G2019S mutation in LRRK2, a known pathological mutation associated with PD. Specifically, we investigated the ‘dark microglia’ (DM) and the ‘disease-associated microglia’ (DAM) which present a selective enrichment of CLEC7A expression. In the dorsal striatum - a region affected by PD pathology - extensive ultrastructural features of cellular stress as well as reduced direct cellular contacts, were observed for microglia from old LRRK2 G2019S mice versus controls. In addition, DM were more prevalent while CLEC7A-positive microglia had extensive phagocytic ultrastructural characteristics in the LRRK2 G2019S mice. Furthermore, our findings revealed a higher proportion of DM in LRRK2 G2019S mice, and an increased number of CLEC7A-positive cells with age, exacerbated by the pathological mutation. These CLEC7A-positive cells exhibited a selective enrichment of ameboid morphology and tended to cluster in the affected animals. In summary, we provide novel insights into the occurrence and features of recently defined microglial states, CLEC7A-positive cells and DM, in the context of LRRK2 G2019S PD pathology.

The enzyme glucocerebrosidase (GCase) catalyses the hydrolysis of glucosylceramide to glucose and ceramide within lysosomes. Homozygous or compound heterozygous mutations in the GCase-sencoding GBA1 gene cause the lysosomal storage disorder Gaucher disease, while heterozygous mutations are the most frequent genetic risk factor for Parkinson’s disease. These mutations commonly affect GCase stability, trafficking or activity. Here, we report the development and characterization of nanobodies (Nbs) targeting and acting as chaperones for GCase. We identified several Nb families that bind with nanomolar affinity to GCase. Based on biochemical characterization, we grouped the Nbs in two classes: Nbs that improve the activity of the enzyme and Nbs that increase GCase stability in vitro . A selection of the most promising Nbs was shown to improve GCase function in cell models and positively impact the activity of the N370S mutant GCase. These results lay the foundation for the development of new therapeutic routes.### Competing Interest StatementT. D. M, W.V., N. P., E. G. and C. S. are inventors on filed patent covering findings described in this manuscript (application number: EP 24159588.3). All other authors declare no competing interests.

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 Parkinson's 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.