Biology subjects

Mamais, A.

Publications and source records attributed to Mamais, A..

9 recordsLinked to original sources

bioRxiv2026

Vps35 p.D620N causes Lrrk2 kinase hyperactivity, chronic microglial activation and inflammation

Pathogenic variants in leucine-rich repeat kinase 2 (LRRK2), RAB32, and vacuolar protein sorting 35 (VPS35) cause dominantly inherited, late-onset Parkinson's disease (PD). Mutations in all three genes constitutively activate LRRK2 kinase activity, enhancing innate immune defense against pathogens; however, chronic activation is linked to dopaminergic neuronal degeneration. LRRK2 and RAB32 are highly expressed in all myeloid cells including brain microglia, whereas VPS35, a core component of the retromer, is ubiquitously expressed. Nevertheless, the VPS35 p.D620N substitution induces the greatest constitutive increase in endogenous LRRK2 kinase activity, and its consequences for immune function remain unclear. We therefore examined the transcriptomic and functional effects of wild type (WT) and Vps35 p.D620N knock-in mice (VKI) from microglia, isolated from brains of naive animals at six-month of age, and initially analyzed by single-cell RNA sequencing (scRNAseq). Despite the absence of overt behavioral differences, differential gene expression revealed genotype-dependent changes in antimicrobial humoral immunity, lysosomal stress sensing, and phagocytosis. S100 family genes and lipocalin 2 were markedly upregulated, a finding subsequently validated by gene expression and immunohistochemistry. Downregulation was also observed in pathways governing microglial regulation of synaptic transmission, neuronal development and homeostatic immune signaling. Peripherally-administered lipopolysaccharide (LPS) stimulated microglial activation and phagocytic markers in WT brain, and further increased morphological activation and synaptic engulfment in VKI animals. Compared to WT, Vps35 p.D620N drives a chronic pro-inflammatory microglial state characterized by elevated innate immune signaling, lysosomal stress and enhanced phagocytic activity. Hence, heightened microglial sensitivity, further exacerbated by a peripheral immune challenge, offers a parsimonious, if multicellular, explanation for the multifactorial etiology of PD, and may help to account for its penetrance and variable expressivity. Together, these findings provide mechanistic insight into how retromer dysfunction and LRRK2 kinase hyperactivity intersect with microglial biology to influence PD pathogenesis, potentially promoting synaptic remodeling and neurodegenerative vulnerability.

neuroscience↗

bioRxiv2025

Parkinson's disease LRRK2 mutations dysregulate iron homeostasis and promote oxidative stress and ferroptosis in human neurons and astrocytes

BackgroundIron accumulation is a hallmark of sporadic and familial Parkinsons disease (PD) and correlates with clinical motor symptom severity. The biochemical mechanisms driving iron dyshomeostasis in PD brain and whether these are early or late events in the neurodegenerative process remain unknown. Nigral iron levels in LRRK2-PD patients have been reported to be even higher than in idiopathic PD, and greater in non-manifesting LRRK2 carriers than controls, suggesting that iron accumulation precedes clinical onset in LRRK2-associated PD. However, the cells affected and mechanisms governing iron dyshomeostasis in PD remain unclear. MethodsHere, we investigated multiple independent measures of iron homeostasis in iPSCs, iPSC-derived cortical and dopaminergic neurons, and astrocytes from control and PD patient-derived iPSCs and an isogenic iPSC panel of three independent pathogenic PD mutations. High-content and super-resolution microscopy of iron-specific probes and ICP-MS were used to determine iron content and distribution across different cell types and LRRK2 genotypes. The upstream effectors and downstream consequences of iron dyshomeostasis on ferroptosis signaling were also examined. ResultsWe found that heterozygous LRRK2 mutations dysregulate cellular iron levels across iPSCs, neurons and astrocytes, in a kinase-dependent manner. Lysosomal ferrous iron was specifically and consistently elevated across iPSCs, iPSC-derived cortical and dopaminergic neurons, and astrocytes carrying LRRK2 mutations and rescued by treatment with the selective LRRK2 inhibitor, MLi-2. Importantly, we show that lipid peroxidation and ROS levels are elevated in isogenic LRRK2 mutant neurons, while iron chelation or MLi-2 reduced LRRK2-dependent ROS damage. LRRK2 regulates the function of over a dozen Rab GTPase proteins through direct phosphorylation, and our prior work revealed a unique correlation with Rab8a trafficking and iron. Here, we report that CRISPR/Cas9 knockout of Rab8a recapitulates LRRK2-driven effects on intracellular iron in different cell types and that exogenous Rab8a normalizes lysosomal iron levels in R1441C LRRK2 iPSCs. ConclusionsTogether, our findings demonstrate that LRRK2 mutations disrupt iron homeostasis across multiple cell types, including dopaminergic neurons, and establish a discrete biochemical pathway linking LRRK2 and vulnerability to ferroptosis signaling.

neuroscience↗

bioRxiv2025

Peripheral inflammation mediates midbrain Lrrk2 kinase activity via Rab32 expression

Mutations that increase leucine-rich repeat kinase 2 (LRRK2) activity confer significant risk for Parkinsons disease (PD), yet incomplete disease penetrance suggest additional factors are required to manifest disease. We recently identified RAB32 Ser71Arg as a Mendelian gene for PD. Here, we establish Rab32 as a key mediator linking peripheral inflammation to Lrrk2 activation. We show that Rab32 and Rab38 expression are modestly, but inversely, correlated with their homolog Rab29. In vivo, peripheral lipopolysaccharide (LPS)-induced inflammation selectively induced Rab32 expression in midbrain Iba1+ microglia but not dopaminergic neurons, where it localized to Lamp1+ lysosomal compartments and correlated with Lrrk2 kinase activity. LPS similarly induces Rab32 expression in human induced pluripotent stem cell-derived microglia, demonstrating a unified biological response to inflammation across species. Promoter analysis identified Tfe3, a master regulator of lysosomal biogenesis and autophagy, as a key driver of Rab32 expression induced Lrrk2 kinase activation. During inflammation, Tfe3 translocated to the nucleus of midbrain Iba1+ microglia to induce Rab32 expression and Lrrk2 kinase activity. Knockdown of Tfe3, but not Tfeb, mitigates these effects, establishing Rab32 as a physiological rheostat of Lrrk2 activity. This mechanistic pathway enables peripheral inflammation to modulate LRRK2 activity and highlights Rab32/Tfe3 as a therapeutic targeting for neuroprotection in PD.

cell biology↗

bioRxiv2023

Differential LRRK2 signalling and gene expression in WT-LRRK2 and G2019S-LRRK2 mouse microglia treated with zymosan and MLi2.

IntroductionMutations in the Leucine Rich Repeat Kinase 2 (LRRK2) gene cause autosomal dominant Parkinsons disease (PD) with the most common causative mutation being the LRRK2 p.G2019S within the kinase domain. LRRK2 protein is highly expressed in the human brain and also in the periphery, and high expression of dominant PD genes in immune cells suggest involvement of microglia and macrophages in inflammation related to PD. LRRK2 is known to respond to extracellular signalling including TLR4 resulting in alterations in gene expression, with the response to TLR2 signalling through zymosan being less known. MethodsHere, we investigated the effects of zymosan, a TLR2 agonist and the potent and specific LRRK2 kinase inhibitor MLi-2 on gene expression in microglia from LRRK2-WT and LRRK2 p.G2019S knock-in mice by RNA-Sequencing analysis. ResultsWe observed both overlapping and distinct zymosan and MLi-2 mediated gene expression profiles in microglia. At least two candidate Genome-Wide Association (GWAS) hits for PD, CathepsinB (Ctsb) and Glycoprotein-nmb (Gpnmb), were notably downregulated by zymosan treatment. Genes involved in inflammatory response and nervous system development were up and downregulated respectively with zymosan treatment while MLi-2 treatment particularly exhibited upregulated genes for ion transmembrane transport regulation. Furthermore, we observed the top twenty most significantly differentially expressed genes in LRRK2 p.G2019S microglia show enriched biological processes in iron transport and response to oxidative stress. DiscussionOverall, these results suggest that microglial LRRK2 may contribute to PD pathogenesis through altered inflammatory pathways. Our findings should encourage future investigations of these putative avenues in the context of PD pathogenesis.

neuroscience↗

bioRxiv2023

The LRRK2 kinase substrates Rab8a and Rab10 contribute complementary but distinct disease-relevant phenotypes in human neurons

Mutations in the LRRK2 gene cause familial Parkinsons disease presenting with pleomorphic neuropathology that can involve -synuclein or tau accumulation. LRRK2 mutations are thought to converge toward a pathogenic increase in LRRK2 kinase activity. A subset of small Rab GTPases have been identified as LRRK2 substrates, with LRRK2-dependent phosphorylation resulting in Rab inactivation. We used CRISPR/Cas9 genome editing to generate a novel series of isogenic iPSC lines deficient in the two most well validated LRRK2 substrates, Rab8a and Rab10, from two independent, deeply phenotyped healthy control lines. Thorough characterization of NGN2-induced neurons revealed divergent effects of Rab8a and Rab10 deficiency on lysosomal pH, LAMP1 association with Golgi, -synuclein insolubility and tau phosphorylation, while parallel effects on lysosomal numbers and Golgi clustering were observed. Our data demonstrate largely antagonistic effects of genetic Rab8a or Rab10 inactivation which provide discrete insight into the pathologic features of their biochemical inactivation by pathogenic LRRK2 mutation. HighlightsO_LIRab8a and Rab10 deficiency induce lysosomal and Golgi defects C_LIO_LIRab8a and Rab10 deficiency induce opposing effects on lysosomal pH C_LIO_LIRab8a KO and Rab10 KO neurons show divergent effects on synuclein and tau proteostasis C_LIO_LIInactivation of different Rab GTPases can induce distinct disease-relevant phenotypes C_LI

neuroscience↗

bioRxiv2022

Leucine-Rich Repeat Kinase 2 limits dopamine D1 receptor signaling in the striatum and biases against heavy persistent alcohol drinking

The transition from hedonic alcohol drinking to problematic drinking is a hallmark of alcohol use disorder that occurs only in a subset of drinkers. This transition is known to require long-lasting changes in the synaptic drive and the activity of striatal neurons expressing dopamine D1 receptor (D1R). The molecular mechanisms that generate vulnerability in some individuals to undergo the transition are less understood. Here, we report that the Parkinsons-related protein leucine-rich repeat kinase 2 (LRRK2) modulates striatal D1R function to affect the behavioral response to alcohol and the likelihood that mice transition to heavy, persistent alcohol drinking. Deletion of the Lrrk2 gene specifically from D1R-expressing neurons potentiates D1R signaling at the cellular and synaptic level, enhancing alcohol-related behaviors and drinking. Mice with cell-specific deletion of Lrrk2 are more prone to heavy alcohol drinking and consumption is insensitive to punishment. These findings identify a novel role for LRRK2 function in the striatum in promoting resilience against heavy and persistent alcohol drinking.

neuroscience↗

bioRxiv2020

Pathogenic mutations in Leucine-rich repeat kinase 2 sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinsons disease (PD) while polymorphic LRRK2 variants are associated with sporadic PD. PD-linked mutations increase LRRK2 kinase activity and induce neurotoxicity in vitro and in vivo. The small GTPase Rab8a is a LRRK2 kinase substrate and is involved in receptor-mediated recycling and endocytic trafficking of transferrin, but the effect of PD-linked LRRK2 mutations on the function of Rab8a are poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a into lysosomes in cells while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive accumulation of endocytosed transferrin into Rab8a-positive lysosomes leading to a dysregulation of iron transport. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in post-mortem PD tissue. Here, we show that iPSC-derived microglia from patients carrying the most common LRRK2 mutation, G2019S, mistraffic transferrin to lysosomes proximal to the nucleus in proinflammatory conditions. Furthermore, G2019S knock-in mice show significant increase in iron deposition in microglia following intrastriatal LPS injection compared to wild type mice, accompanied by striatal accumulation of ferritin. Our data support a role of LRRK2 in modulating iron uptake and storage in response to proinflammatory stimuli in microglia.

cell biology↗

bioRxiv2020

Sequential screening nominates the Parkinson's disease associated kinase LRRK2 as a regulator of Clathrin-mediated endocytosis

Mutations in leucine-rich repeat kinase 2 (LRRK2) are an established cause of inherited Parkinsons disease (PD). LRRK2 is expressed in both neurons and glia in the central nervous system, but its physiological function(s) in each of these cell types is uncertain. Through sequential screens, we report a functional interaction between LRRK2 and Clathrin adaptor protein complex 2 (AP2). Analysis of LRRK2 KO tissue revealed a significant dysregulation of AP2 complex components, suggesting LRRK2 may act upstream of AP2. In line with this hypothesis, expression of LRRK2 was found to modify recruitment and phosphorylation of AP2. Furthermore, expression of LRRK2 containing the R1441C pathogenic mutation resulted in impaired clathrin-mediated endocytosis (CME). A decrease in activity-dependent synaptic vesicle endocytosis was also observed in neurons harboring an endogenous R1441C LRRK2 mutation. Alongside LRRK2, several PD-associated genes intersect with membrane-trafficking pathways. To investigate the genetic association between Clathrin-trafficking and PD, we used polygenetic risk profiling from IPDGC genome wide association studies (GWAS) datasets. Clathrin-dependent endocytosis genes were found to be associated with PD across multiple cohorts, suggesting common variants at these loci represent a cumulative risk factor for disease. Taken together, these findings suggest CME is a LRRK2-mediated, PD relevant pathway.

molecular biology↗

bioRxiv2020

LRRK2 mediates tubulation and vesicle sorting from membrane damaged lysosomes

Mutations in the leucine rich repeat kinase 2 (LRRK2) gene are a cause of familial and sporadic Parkinsons disease (PD). Nonetheless, the biological functions of LRRK2 remain incompletely understood. Here, we observed that LRRK2 is recruited to lysosomes that have a ruptured membrane. Using unbiased proteomics, we observed that LRRK2 is able to recruit the motor adaptor protein JIP4 to permeabilized lysosomes in a kinase-dependent manner through the phosphorylation of RAB35 and RAB10. Super-resolution live cell imaging microscopy and FIB-SEM revealed that once at the lysosomal membrane, JIP4 promotes the formation of LAMP1-negative lysosomal tubules that release membranous content from ruptured lysosomes. Released vesicular structures are able to interact with other lysosomes. Thus, we described a new process that uses lysosomal tubulation to release vesicular structures from permeabilized lysosomes. LRRK2 orchestrates this process that we name LYTL (LYsosomal Tubulation/sorting driven by LRRK2) that, given the central role of the lysosome in PD, is likely to be disease relevant.

cell biology↗