Biology subjects

Manzoni, C.

Publications and source records attributed to Manzoni, C..

15 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

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

Contrasting patterns of interleukin signalling in LRRK2 and idiopathic Parkinson's

Sporadic PD and LRRK2-associated Parkinsons Disease (PD) present with similar features although the familial form of disease is classically associated with less cognitive impairment, better REM sleep behaviour, and reduced incidence of hyposmia but more freezing gait and postural instability. Although being classified as the same disease from a clinical perspective there is an ongoing debate on whether these 2 conditions are linked to different causative molecular mechanisms. In this work we analysed the transcriptomics and proteomics profile of sporadic and LRRK2 PD patients to contrast and compare inflammatory markers in blood and CSF. Analyses showed molecular differences in the burden of inflammatory signals clearly differentiating the sporadic and the familial forms of disease. Finally, a cluster of closely connected proteins whose expression levels were changed in the familial forms of disease was highlighted. The levels of these inflammatory markers were associated with tau pathology suggesting this protein cluster might be a system model to evaluate the control of tau metabolism in the presence of LRRK2 mutations.

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 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

Transcriptomics analyses of the LRRK2 protein interactome reveal distinct molecular signatures for sporadic and LRRK2 Parkinson's Disease

Mutations in the LRRK2 gene are the most common genetic cause for familial Parkinsons Disease (LRRK2-PD) and an important risk factor for sporadic PD (sPD). Multiple clinical trials are ongoing to evaluate the benefits associated with the therapeutical reduction of LRRK2 kinase activity. In this study, we described the changes on transcriptomic profiles (whole blood mRNA levels) of LRRK2 protein interactors in the sPD and LRRK2-PD cases as compared to healthy controls with the aim of comparing the two PD conditions. We went on to model the protein-protein interaction (PPI) network around LRRK2, which was weighted to reflect the transcriptomic changes in the network based on the expression and co-expression levels. Our results showed that LRRK2 interactors present both similar but also different alterations in expression levels and co-expression behaviours in the sPD and LRRK2-PD cases. The similar changes result in decreased connectivity within a topological cluster of the LRRK2 PPI network associated with ribosomal functions and DNA/RNA metabolism in both the sPD and LRRK2-PD scenario; while the connectivity within the autophagy/mitophagy/neurotransmitter-transport related cluster was increased exclusively in the LRRK2-PD condition as compared to the healthy controls. These results suggest that, albeit being classified as the same disease based on clinical features, LRRK2-PD and sPD show some significant differences from a molecular perspective.

neuroscience↗

bioRxiv2023

Protein network analysis links the NSL complex to Parkinson's disease and mitochondrial biology

Whilst the majority of PD cases are sporadic, much of our understanding of the pathophysiological basis of disease can be traced back to the study of rare, monogenic forms of disease. In the past decade, the availability of Genome-Wide Association Studies (GWAS) has facilitated a shift in focus, toward identifying common risk variants conferring increased risk of developing PD across the population. A recent mitophagy screening assay of GWAS candidates has functionally implicated the non-specific lethal (NSL) complex in the regulation of PINK1-mitophagy. Here, a bioinformatics approach has been taken to investigate the proteome of the NSL complex, to unpick its relevance to PD progression. The mitochondrial NSL interactome has been built, mining 3 separate repositories: PINOT, HIPPIE and MIST, for curated, literature-derived protein-protein interaction (PPI) data. We built; i) the mitochondrial interactome, applying gene-set enrichment analysis (GSEA) to explore the relevance of the NSL mitochondrial interactome to PD and, ii) the PD-oriented interactome to uncover biological pathways underpinning the NSL /PD association. In this study, we find the mitochondrial NSL interactome to be significantly enriched for the protein products of PD associated genes, including the Mendelian PD genes LRRK2 and VPS35. Additionally, the PD associated interactome is enriched for mitochondrial processes; "mitochondrial cell death", "mitochondrial protein localisation", "membrane protein localisation" and "mitochondrial transport". Our data points to NSL complex members OGT and WDR5 as key drivers of this increased PD association. These findings strengthen a role for mitochondrial quality control in both familial and sporadic 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↗

bioRxiv2022

Tissue specific LRRK2 interactomes reveal a distinct functional unit within the striatum

Mutations in LRRK2 are the most common genetic cause of Parkinsons disease. Despite substantial research efforts, the physiological and pathological role of this multidomain protein remains poorly defined. In this study, we used a systematic approach to construct the general protein-protein interactome around LRRK2, which was then differentiated into 15 tissue-specific interactomes taking into consideration the differential expression patterns and the co-expression behaviours of the LRRK2 interactors in different healthy tissues. The LRRK2 interactors exhibited distinct expression features in the brain as compared to the peripheral tissues analysed. Moreover, a high degree of similarity was found for the LRRK2 interactors in putamen, caudate and nucleus accumbens, thus defining a potential LRRK2 functional cluster within the striatum. We also explored the functions highlighted by the "core LRRK2 interactors" within each tissue and illustrated how the LRRK2 interactomes can be used as a tool to trace the relationship between LRRK2 and specific interactors of interest, here exemplified with a study focused on the LRRK2 interactors belonging to the Rab protein family.

neuroscience↗

bioRxiv2021

Integrating protein networks and machine learning for disease stratification in the Hereditary Spastic Paraplegias

The Hereditary Spastic Paraplegias are a group of neurodegenerative diseases characterized by spasticity and weakness in the lower body. Despite the identification of causative mutations in over 70 genes, the molecular aetiology remains unclear. Due to the combination of genetic diversity and variable clinical presentation, the Hereditary Spastic Paraplegias are a strong candidate for protein-protein interaction network analysis as a tool to understand disease mechanism(s) and to aid functional stratification of phenotypes. In this study, experimentally validated human protein-protein interactions were used to create a protein-protein interaction network based on the causative Hereditary Spastic Paraplegia genes. Network evaluation as a combination of both topological analysis and functional annotation led to the identification of core proteins in putative shared biological processes such as intracellular transport and vesicle trafficking. The application of machine learning techniques suggested a functional dichotomy linked with distinct sets of clinical presentations, suggesting there is scope to further classify conditions currently described under the same umbrella term of Hereditary Spastic Paraplegias based on specific molecular mechanisms of disease.

systems biology↗

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↗

bioRxiv2020

Regulation of mitophagy by the NSL complex underlies genetic risk for Parkinson's disease at Chr16q11.2 and on the MAPT H1 allele.

Parkinsons disease (PD) is a common incurable neurodegenerative disease. The identification of genetic variants via genome-wide association studies (GWAS) has considerably advanced our understanding of the PD genetic risk. Understanding the functional significance of the risk loci is now a critical step towards translating these genetic advances into an enhanced biological understanding of the disease. Impaired mitophagy is a key causative pathway in familial PD, but its relevance to idiopathic PD is unclear. We used a mitophagy screening assay to evaluate the functional significance of risk genes identified through GWAS. We identified two new regulators of PINK1-mitophagy, KAT8 and KANSL1, previously shown to modulate lysine acetylation. We show that KAT8 and KANSL1 modulate PINK1 gene expression and subsequent PINK1-mitophagy. These findings suggest PINK1-mitophagy is a contributing factor to idiopathic PD. KANSL1 is located on chromosome 17q21 where the risk associated gene has long been considered to be MAPT. While our data does not exclude a possible association between the MAPT gene and PD, it provides strong evidence that KANSL1 plays a crucial role in the disease. Finally, these results enrich our understanding of physiological events regulating mitophagy and establish a novel pathway for drug targeting in neurodegeneration.

neuroscience↗

bioRxiv2019

An integrated genomic approach to dissect the genetic landscape regulating the cell-to-cell transfer of a-synuclein

Neuropathological and experimental evidence suggests that the cell-to-cell transfer of a-synuclein has an important role in the pathogenesis of Parkinsons disease (PD). However, the mechanism underlying this phenomenon is not fully understood. We undertook an siRNA, genome-wide high throughput screen to identify genes regulating the cell-to-cell transfer of a-synuclein. We transiently transfected HEK cells stably overexpressing a-synuclein with a construct encoding GFP-2a-aSynuclein-RFP. The cells expressing a-synuclein-RFP through transfection were double positive for GFP and RFP fluorescence, whereas the cells receiving it through transfer were positive only for RFP fluorescence. The amount of a-synuclein transfer was quantified by high content microscopy. A series of unbiased screens confirmed the involvement of 38 genes in the regulation of a-synuclein-RFP transfer. One of those hits was ITGA8, a candidate gene recently identified through a large PD genome wide association study (GWAS). Weighted gene co-expression network analysis (WGCNA) and weighted protein-protein network interaction analysis (WPPNIA) showed that the hits clustered in networks that included known PD Mendelian and GWAS risk genes more frequently than expected than random chance. Given the genetic overlap between a-synuclein transfer and PD, those findings provide supporting evidence for the importance of the cell-to-cell transfer of a-synuclein in the pathogenesis of PD, and expand our understanding of the mechanism of a-synuclein spread.

neuroscience↗

bioRxiv2019

PINOT: An Intuitive Resource for Integrating Protein-Protein Interactions

The past decade has seen the rise of omics data, for the understanding of biological systems in health and disease. This wealth of data includes protein-protein interaction (PPI) derived from both low and high-throughput assays, which is curated into multiple databases that capture the extent of available information from the peer-reviewed literature. Although these curation efforts are extremely useful, reliably downloading and integrating PPI data from the variety of available repositories is challenging and time consuming.\n\nWe here present a novel user-friendly web-resource called PINOT (Protein Interaction Network Online Tool; available at http://www.reading.ac.uk/bioinf/PINOT/PINOT_form.html) to optimise the collection and processing of PPI data from the IMEx consortium associated repositories (members and observers) and from WormBase for constructing, respectively, human and C. elegans PPI networks.\n\nUsers submit a query containing a list of proteins of interest for which PINOT will mine PPIs. PPI data is downloaded, merged, quality checked, and confidence scored based on the number of distinct methods and publications in which each interaction has been reported. Examples of PINOT applications are provided to highlight the performance, the ease of use and the potential applications of this tool.\n\nPINOT is a tool that allows users to survey the literature, extracting PPI data for a list of proteins of interest. The comparison with analogous tools showed that PINOT was able to extract similar numbers of PPIs while incorporating a set of innovative features. PINOT processes both small and large queries, it downloads PPIs live through PSICQUIC and it applies quality control filters on the downloaded PPI annotations (i.e. removing the need of manual inspection by the user). PINOT provides the user with information on detection methods and publication history for each of the downloaded interaction data entry and provides results in a table format that can be easily further customised and/or directly uploaded in a network visualization software.

bioinformatics↗

bioRxiv2019

Integration of eQTL and Parkinson’s disease GWAS data implicates 11 disease genes

Substantial genome-wide association study (GWAS) work in Parkinsons disease (PD) has led to an increasing number of loci shown reliably and robustly to be associated with the increased risk of the disease. Prioritising causative genes and pathways from these studies has proven problematic. Here, we present a comprehensive analysis of PD GWAS data with expression and methylation quantitative trait loci (eQTL/mQTL) using Colocalisation analysis (Coloc) and transcriptome-wide association analysis (TWAS) to uncover putative gene expression and splicing mechanisms driving PD GWAS signals. Candidate genes were further characterised by determining cell-type specificity, weighted gene co-expression (WGNCA) and protein-protein interaction (PPI) networks.\n\nGene-level analysis of expression revealed 5 genes (WDR6, CD38, GPNMB, RAB29, TMEM163) that replicated using both Coloc and TWAS analyses in both GTEx and Braineac expression datasets. A further 6 genes (ZRANB3, PCGF3, NEK1, NUPL2, GALC, CTSB) showed evidence of disease-associated splicing effects. Cell-type specificity analysis revealed that gene expression was overall more prevalent in glial cell-types compared to neurons. The WGNCA analysis showed that NUPL2 is a key gene in 3 modules implicated in catabolic processes related with protein ubiquitination (protein ubiquitination (p=7.47e-10) and ubiquitin-dependent protein catabolic process (p = 2.57e-17) in nucleus accumbens, caudate and putamen, while TMEM163 and ZRANB3 were both important in modules indicating regulation of signalling (p=1.33e-65] and cell communication (p=7.55e-35) in the frontal cortex and caudate respectively. PPI analysis and simulations using random networks demonstrated that the candidate genes interact significantly more with known Mendelian PD and parkinsonism proteins than would be expected by chance. The proteins core proteins this network were enriched for regulation of the ERBB receptor tyrosine protein kinase signalling pathways.\n\nTogether, these results point to a number of candidate genes and pathways that are driving the associations observed in PD GWAS studies.

neuroscience↗