Leucine-rich Repeat Kinase 2 Protein Research Articles

Parkinson’s Disease: Biomarkers for Diagnosis and Disease Progression

Parkinson’s disease (PD) is a progressive neurological disease that causes both motor and nonmotor symptoms. While our understanding of putative mechanisms has advanced significantly, it remains challenging to verify biomarkers with sufficient evidence for regular clinical use. Clinical symptoms are the primary basis for diagnosing the disease, which can be mild in the early stages and overlap with other neurological disorders. As a result, clinical testing and medical records are mostly relied upon for diagnosis, posing substantial challenges during both the initial diagnosis and the continuous disease monitoring. Recent biochemical, neuroimaging, and genetic biomarkers have helped us understand the pathophysiology of Parkinson’s disease. This comprehensive study focuses on these biomarkers, which were chosen based on their relevance, methodological excellence, and contribution to the field. Biochemical biomarkers, including α-synuclein and glial fibrillary acidic protein (GFAP), can predict disease severity and progression. The dopaminergic system is widely used as a neuroimaging biomarker to diagnose PD. Numerous genes and genome wide association study (GWAS) sites have been related to the development of PD. Recent research on the SNCA gene and leucine-rich repeat protein kinase 2 (LRRK2) has shown promising results. By evaluating current studies, this review intends to uncover gaps in biomarker validation and use, while also highlighting promising improvements. It emphasizes the need for dependable and reproducible indicators in improving PD diagnosis and prognosis. These biomarkers may open up new avenues for early diagnosis, disease progression tracking, and the development of personalized treatment programs.

The R1441C-Lrrk2 mutation induces myeloid immune cell exhaustion in an age- and sex-dependent manner in mice.

Age is the greatest risk factor for many neurodegenerative diseases, yet immune system aging, a contributor to neurodegeneration, is understudied. Genetic variation in the LRRK2 gene affects risk for both familial and sporadic Parkinson's disease (PD). The leucine-rich repeat kinase 2 (LRRK2) protein is implicated in peripheral immune cell signaling, but the effects of an aging immune system on LRRK2 function remain unclear. We analyzed peritoneal macrophages from R1441C-Lrrk2 knock-in mice and observed a biphasic, age-dependent effect of the R1441C-Lrrk2 mutation on peritoneal macrophage function. We report increases in antigen presentation, anti-inflammatory cytokine production, lysosomal activity, and pathogen uptake in peritoneal macrophages from young (2- to 3-month-old) female R1441C-Lrrk2 mice. Conversely, macrophages from aged (18- to 21-month-old) female R1441C-Lrrk2 mice exhibited decreased antigen presentation after inflammatory insult, decreased lysosomal function, and pathogen uptake, with a concomitant increase in DNA fragmentation in the presence of pathogens. This immune cell exhaustion phenotype was not observed in male R1441C-Lrrk2 mice and was driven by increased LRRK2 protein kinase activity. This phenotype was also observed in human peripheral myeloid cells, with monocyte-derived macrophages from patients with PD who had either the R1441C- or Y1699C-LRRK2 mutation exhibiting decreased pathogen uptake and increased PDL1 expression, consistent with immune cell exhaustion. Our findings that LRRK2 mutations conferred an immunological advantage at a young age but could predispose the carrier to age-acquired immune cell exhaustion have implications for the therapeutic development of LRRK2 inhibitors.

CK and LRRK2 Involvement in Neurodegenerative Diseases.

Neurodegenerative diseases (NDDs) are currently the most widespread neuronal pathologies in the world. Among these, the most widespread are Alzheimer's disease (AD), dementia, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD)-all characterized by a progressive loss of neurons in specific regions of the brain leading to varied clinical symptoms. At the basis of neurodegenerative diseases, an emerging role is played by genetic mutations in the leucine-rich repeat kinase 2 (LRRK2) gene that cause increased LRRK2 activity with consequent alteration of neuronal autophagy pathways. LRRK2 kinase activity requires GTPase activity which functions independently of kinase activity and is required for neurotoxicity and to potentiate neuronal death. Important in the neurodegeneration process is the upregulation of casein kinase (CK), which causes the alteration of the AMPK pathway by enhancing the phosphorylation of α-synuclein and huntingtin proteins, known to be involved in PD and HD, and increasing the accumulation of the amyloid-β protein (Aβ) for AD. Recent research has identified CK of the kinases upstream of LRRK2 as a regulator of the stability of the LRRK2 protein. Based on this evidence, this review aims to understand the direct involvement of individual kinases in NDDs and how their crosstalk may impact the pathogenesis and early onset of neurodegenerative diseases.

Clinical characteristics and pathophysiological properties of newly discovered LRRK2 variants associated with Parkinson's disease

Leucine-rich repeat kinase 2 (LRRK2) is the most common gene responsible for familial Parkinson's disease (PD). The gene product of LRRK2 contains multiple protein domains, including armadillo repeat, ankyrin repeat, leucine-rich repeat (LRR), Ras-of-complex (ROC), C-terminal of ROC (COR), kinase, and WD40 domains. In this study, we performed genetic screening of LRRK2 in our PD cohort, detecting sixteen LRRK2 rare variants. Among them, we selected seven variants that are likely to be familial and characterized them in terms of LRRK2 protein function, along with clinical information and one pathological analysis. The seven variants were S1120P and N1221K in the LRR domain; I1339M, S1403R, and V1447M in the ROC domain; and I1658F and D1873H in the COR domain. The kinase activity of the LRRK2 variants N1221K, S1403R, V1447M, and I1658F toward Rab10, a well-known phosphorylation substrate, was higher than that of wild-type LRRK2. LRRK2 D1873H showed enhanced self-association activity, whereas LRRK2 S1403R and D1873H showed reduced microtubule-binding activity. Pathological analysis of a patient with the LRRK2 V1447M variant was also performed, which revealed Lewy pathology in the brainstem. No functional alterations in terms of kinase activity, self-association activity, and microtubule-binding activity were detected in LRRK2 S1120P and I1339M variants. However, the patient with PD carrying LRRK2 S1120P variant also had a heterozygous Glucosylceramidase beta 1 (GBA1) L444P variant. In conclusion, we characterized seven LRRK2 variants potentially associated with PD. Five of the seven variants in different LRRK2 domains exhibited altered properties in kinase activity, self-association, and microtubule-binding activity, suggesting that each domain variant may contribute to disease progression in different ways.

Microglial Sp1 induced LRRK2 upregulation in response to manganese exposure, and 17β-estradiol afforded protection against this manganese toxicity

Chronic exposure to elevated levels of manganese (Mn) causes a neurological disorder referred to as manganism, presenting symptoms similar to those of Parkinson’s disease (PD), yet the mechanisms by which Mn induces its neurotoxicity are not completely understood. 17β-estradiol (E2) affords neuroprotection against Mn toxicity in various neural cell types including microglia. Our previous studies have shown that leucine-rich repeat kinase 2 (LRRK2) mediates Mn-induced inflammatory toxicity in microglia. The LRRK2 promoter sequences contain three putative binding sites of the transcription factor (TF), specificity protein 1 (Sp1), which increases LRRK2 promoter activity. In the present study, we tested if the Sp1-LRRK2 pathway plays a role in both Mn toxicity and the protection afforded by E2 against Mn toxicity in BV2 microglial cells. The results showed that Mn induced cytotoxicity, oxidative stress, and tumor necrosis factor-α production, which were attenuated by an LRRK2 inhibitor, GSK2578215A. The overexpression of Sp1 increased LRRK2 promoter activity, mRNA and protein levels, while inhibition of Sp1 with its pharmacological inhibitor, mithramycin A, attenuated the Mn-induced increases in LRRK2 expression. Furthermore, E2 attenuated the Mn-induced Sp1 expression by decreasing the expression of Sp1 via the promotion of the ubiquitin-dependent degradation pathway, which was accompanied by increased protein levels of RING finger protein 4, the E3-ligase of Sp1, Sp1 ubiquitination, and SUMOylation. Taken together, our novel findings suggest that Sp1 serves as a critical TF in Mn-induced LRRK2 expression as well as in the protection afforded by E2 against Mn toxicity through reduction of LRRK2 expression in microglia.

RedOx regulation of LRRK2 kinase activity by active site cysteines

Mutations of the human leucine-rich repeat kinase 2 (LRRK2) have been associated with both, idiopathic and familial Parkinson’s disease (PD). Most of these pathogenic mutations are located in the kinase domain (KD) or GTPase domain of LRRK2. In this study we describe a mechanism in which protein kinase activity can be modulated by reversible oxidation or reduction, involving a unique pair of adjacent cysteines, the “CC” motif. Among all human protein kinases, only LRRK2 contains this “CC” motif (C2024 and C2025) in the Activation Segment (AS) of the kinase domain. In an approach combining site-directed mutagenesis, biochemical analyses, cell-based assays, and Gaussian accelerated Molecular Dynamics (GaMD) simulations we could attribute a role for each of those cysteines. We employed reducing and oxidizing agents with potential clinical relevance to investigate effects on kinase activity and microtubule docking. We find that each cysteine gives a distinct contribution: the first cysteine, C2024, is essential for LRRK2 protein kinase activity, while the adjacent cysteine, C2025, contributes significantly to redox sensitivity. Implementing thiolates (R-S-) in GaMD simulations allowed us to analyse how each of the cysteines in the “CC” motif interacts with its surrounding residues depending on its oxidation state. From our studies we conclude that oxidizing agents can downregulate kinase activity of hyperactive LRRK2 PD mutations and may provide promising tools for therapeutic strategies.

LRRK2 regulates ferroptosis through the system Xc-GSH-GPX4 pathway in the neuroinflammatory mechanism of Parkinson's disease.

Parkinson's disease (PD) is the most prevalent neurodegenerative disorder. Neuroinflammation mediated by activated microglia and apoptosis of dopaminergic (DA) neurons in the midbrain are its primary pathological manifestations. Leucine-rich repeat protein kinase 2 (LRRK2) kinase has been observed to increase expression during neuroinflammation,however, the effect of LRRK2 on microglia activation remains poorly understood. In this study, we have established lipopolysaccharide (LPS) treated BV2 cells and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) models for both in vivo and in vitro investigation. Our data in vivo reveal that LRRK2 can promote microglia activation by regulating ferroptosis and activatingnuclear factor-κB. Inhibition of LRRK2 expression effectively suppressed the LPS-induced pro-inflammatory cytokines and facilitated the secretion of neuroprotective factors. Importantly, by co-overexpressing LRRK2 and glutathione peroxidase 4 (GPX4), we identified the system Xc-GSH-GPX4 pathway as a crucial component in LRRK2-mediated microglial ferroptosis and inflammatory responses. Using a microglial culture supernatant (MCS) transfer model, we found that inhibiting LRRK2 or downregulating ferroptosis in BV2 cells prevented SH-SY5Y cell apoptosis. Additionally, we observed abundant expression of LRRK2 and P-P65 in the midbrain, which was elevated in the MPTP-induced PD model, along with microglia activation. LRRK2 and P-P65 expression inhibition with PF-06447475 attenuated microglia activation in the nigrostriatal dense part of MPTP-treated mice. Based on our findings, it is evident that LRRK2 plays a critical role in promoting the neuroinflammatory response during the pathogenesis of PD by regulating the system Xc-GSH-GPX4 pathway. Taken together, our data highlights the potential research and therapeutic value of targeting LRRK2 to regulate neuroinflammatory response in PD through ferroptosis.

Identification of novel LRRK2 inhibitors by structure-based virtual screening and alchemical free energy calculation.

The Leucine-rich repeat kinase 2 (LRRK2) target has been identified as a promising drug target for Parkinson's disease (PD) treatment. This study focuses on optimizing the activity of LRRK2 inhibitors using alchemical relative binding free energy (RBFE) calculations. Initially, we assessed various free energy calculation methods across different LRRK2 kinase inhibitor scaffolds. The results indicate that alchemical free energy calculations are promising for prospective predictions on LRRK2 inhibitors, especially for the aminopyrimidine scaffold with an RMSE of 1.15 kcal mol-1 and Rp of 0.83. Following this, we optimized a potent LRRK2 kinase inhibitor identified from previous virtual screenings, featuring a novel scaffold. Guided by RBFE predictions using alchemical methods, this optimization led to the discovery of compound LY2023-001. This compound, with a [1,2,4]triazolo[5,6-b]indole scaffold, exhibited enhanced inhibitory activity against G2019S LRRK2 (IC50 = 12.9 nM). Molecular dynamics (MD) simulations revealed that LY2023-001 formed stable hydrogen bonds with Glu1948, and Ala1950 in the G2019S LRRK2 protein. Additionally, its phenyl substituents engage in strong electrostatic interactions with Lys1906 and van der Waals interactions with Leu1885, Phe1890, Val1893, Ile1933, Met1947, Leu1949, Leu2001, Ala2016, and Asp2017. Our findings underscore the potential of computational methods in the successful optimization of small molecules, offering important insights for the development of novel LRRK2 inhibitors.

Differential LRRK2 Signalling and Gene Expression in WT-LRRK2 and G2019S-LRRK2 Mouse Microglia Treated with Zymosan and MLi2.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause autosomal dominant Parkinson's 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 suggests 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. Here, 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. We 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 that 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. Overall, 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.

Targeting Rab-RILPL interactions as a strategy to downregulate pathogenic LRRK2 in Parkinson's disease.

Familial Parkinson's disease (PD) is frequently linked to multiple disease-causing mutations within Leucine-Rich Repeat Protein Kinase 2 (LRRK2), leading to aberrant kinase activity. Multiple pathogenic effects of enhanced LRRK2 activity have been identified, including loss of cilia and centrosomal cohesion defects. When phosphorylated by LRRK2, Rab8a and Rab10 bind to phospho-specific RILPL effector proteins. RILPL-mediated accumulation of pRabs proximal to the mother centriole is critical for initiating deficits in ciliogenesis and centrosome cohesion mediated by LRRK2. We hypothesized that Rab-derived phospho-mimics may serve to block phosphorylated Rab proteins from docking with RILPL in the context of hyperactive LRRK2 mutants. This would serve as an alternative strategy to downregulate pathogenic signaling mediated by LRRK2, rather than targeting LRRK2 kinase activity itself. To test this theory, we designed a series of constrained peptides mimicking phosphorylated Switch II derived from Rab8. These RILPL interacting peptides, termed RIP, were further shown to permeate cells. Further, several peptides were found to bind RILPL2 and restore ciliogenesis and centrosomal cohesion defects in cells expressing PD-associated mutant LRRK2. This research demonstrates the utility of constrained peptides as downstream inhibitors to target pathogenic LRRK2 activity and may provide an alternative approach to target specific pathways activated by LRRK2.

Genome-Wide Association Study and Genomic Prediction of Fusarium Wilt Resistance in Common Bean Core Collection.

The common bean (Phaseolus vulgaris L.) is a globally cultivated leguminous crop. Fusarium wilt (FW), caused by Fusarium oxysporum f. sp. phaseoli (Fop), is a significant disease leading to substantial yield loss in common beans. Disease-resistant cultivars are recommended to counteract this. The objective of this investigation was to identify single nucleotide polymorphism (SNP) markers associated with FW resistance and to pinpoint potential resistant common bean accessions within a core collection, utilizing a panel of 157 accessions through the Genome-wide association study (GWAS) approach with TASSEL 5 and GAPIT 3. Phenotypes for Fop race 1 and race 4 were matched with genotypic data from 4740 SNPs of BARCBean6K_3 Infinium Bea Chips. After ranking the 157-accession panel and revealing 21 Fusarium wilt-resistant accessions, the GWAS pinpointed 16 SNPs on chromosomes Pv04, Pv05, Pv07, Pv8, and Pv09 linked to Fop race 1 resistance, 23 SNPs on chromosomes Pv03, Pv04, Pv05, Pv07, Pv09, Pv10, and Pv11 associated with Fop race 4 resistance, and 7 SNPs on chromosomes Pv04 and Pv09 correlated with both Fop race 1 and race 4 resistances. Furthermore, within a 30 kb flanking region of these associated SNPs, a total of 17 candidate genes were identified. Some of these genes were annotated as classical disease resistance protein/enzymes, including NB-ARC domain proteins, Leucine-rich repeat protein kinase family proteins, zinc finger family proteins, P-loopcontaining nucleoside triphosphate hydrolase superfamily, etc. Genomic prediction (GP) accuracy for Fop race resistances ranged from 0.26 to 0.55. This study advanced common bean genetic enhancement through marker-assisted selection (MAS) and genomic selection (GS) strategies, paving the way for improved Fop resistance.

The Parkinson’s disease-associated mutation LRRK2 G2385R alters mitochondrial biogenesis via the PGC-1α-TFAM pathway

Mutations in the Leucine-rich repeat protein kinase 2 (LRRK2) gene are the most frequent cause of familial Parkinson’s disease (PD). Although LRRK2 has been extensively studied, the pathogenic mechanism of the LRRK2 G2385R mutation, which is most common in Asian populations, especially in the Chinese Han population, remains unclear. In this study, we demonstrated that the LRRK2 G2385R mutation in HEK293T cells led to a reduction in cellular PGC-1α protein expression and inhibition of mitochondrial biogenesis through the PGC-1α-TFAM pathway. This resulted in a decrease in mitochondrial genome expression, which in turn impaired the normal electron transfer process of the oxidative phosphorylation respiratory chain, leading to mitochondrial dysfunction and onset of apoptosis. The mitochondrial dysfunction and apoptosis caused by the LRRK2 G2385R mutation were significantly alleviated by antioxidant Idebenone, which provides a theoretical basis for the subsequent development of precise treatment specifically for PD patients with LRRK2 G2385R mutation. Further validation of our findings in neurons and animal models are necessary.

Leucine-rich repeat kinase 2 limits dopamine D1 receptor signaling in 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 requires 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 Parkinson’s-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. Constitutive deletion of the Lrrk2 gene specifically from D1R-expressing neurons potentiated D1R signaling at the cellular and synaptic level and enhanced alcohol-related behaviors and drinking. Mice with cell-specific deletion of Lrrk2 were more prone to heavy alcohol drinking, and consumption was insensitive to punishment. These findings identify a potential novel role for LRRK2 function in the striatum in promoting resilience against heavy and persistent alcohol drinking.

Inhibition of the Exocyst Complex Attenuates the LRRK2 Pathological Effects.

Pathological mutations in leucine-rich repeat kinase 2 (LRRK2) gene are the major genetic cause of Parkinson's disease (PD). Multiple lines of evidence link LRRK2 to the control of vesicle dynamics through phosphorylation of a subset of RAB proteins. However, the molecular mechanisms underlying these processes are not fully elucidated. We have previously demonstrated that LRRK2 increases the exocyst complex assembly by Sec8 interaction, one of the eight members of the exocyst complex, and that Sec8 over-expression mitigates the LRRK2 pathological effect in PC12 cells. Here, we extend this analysis using LRRK2 drosophila models and show that the LRRK2-dependent exocyst complex assembly increase is downstream of RAB phosphorylation. Moreover, exocyst complex inhibition rescues mutant LRRK2 pathogenic phenotype in cellular and drosophila models. Finally, prolonged exocyst inhibition leads to a significant reduction in the LRRK2 protein level, overall supporting the role of the exocyst complex in the LRRK2 pathway. Taken together, our study suggests that modulation of the exocyst complex may represent a novel therapeutic target for PD.

Capturing Differences in the Regulation of LRRK2 Dynamics and Conformational States by Small Molecule Kinase Inhibitors.

Mutations in the human leucine rich repeat protein kinase-2 (LRRK2) create risk factors for Parkinson's disease, and pathological functions of LRRK2 are often correlated with aberrant kinase activity. Past research has focused on developing selective LRRK2 kinase inhibitors. In this study, we combined enhanced sampling simulations with HDX-MS to characterize the inhibitor-induced dynamic changes and the allosteric communications within the C-terminal domains of LRRK2, LRRK2RCKW. We find that the binding of MLi-2 (a type I kinase inhibitor) stabilizes a closed kinase conformation and reduces the global dynamics of LRRK2RCKW, leading to a more compact LRRK2RCKW structure. In contrast, the binding of Rebastinib (a type II kinase inhibitor) stabilizes an open kinase conformation, which promotes a more extended LRRK2RCKW structure. By probing the distinct effects of the type I and type II inhibitors, key interdomain interactions are found to regulate the communication between the kinase domain and the GTPase domain. The intermediate states revealed in our simulations facilitate the efforts toward in silico design of allosteric modulators that control LRRK2 conformations and potentially mediate the oligomeric states of LRRK2 and its interactions with other proteins.

Secretion of VGF relies on the interplay between LRRK2 and post-Golgi v-SNAREs

The neuropeptide VGF was recently proposed as a neurodegeneration biomarker. The Parkinson's disease-related protein leucine-rich repeat kinase 2 (LRRK2) regulates endolysosomal dynamics, a process that involves SNARE-mediated membrane fusion and could regulate secretion. Here we investigate potential biochemical and functional links between LRRK2 and v-SNAREs. We find that LRRK2 directly interacts withthe v-SNAREs VAMP4 and VAMP7. Secretomics reveals VGF secretory defects in VAMP4 and VAMP7knockout (KO) neuronal cells. In contrast, VAMP2 KO "regulated secretion-null" and ATG5 KO "autophagy-null" cells release more VGF. VGF is partially associated with extracellular vesicles and LAMP1+ endolysosomes. LRRK2 expression increases VGF perinuclear localization and impairs its secretion. Retention using selective hooks (RUSH) assays show that a pool of VGF traffics through VAMP4+ and VAMP7+ compartments, and LRRK2 expression delays its transport to the cell periphery. Overexpression of LRRK2 or VAMP7-longin domain impairs VGF peripheral localization in primary cultured neurons. Altogether, our results suggest that LRRK2 might regulate VGF secretion via interaction with VAMP4 and VAMP7.

The function of Golgi apparatus in LRRK2-associated Parkinson's disease.

Parkinson's disease (PD) is a chronic neurodegenerative disease associated with the intracellular organelles. Leucine-rich repeat kinase 2 (LRRK2) is a large multi-structural domain protein, and mutation in LRRK2 is associated with PD. LRRK2 regulates intracellular vesicle transport and function of organelles, including Golgi and lysosome. LRRK2 phosphorylates a group of Rab GTPases, including Rab29, Rab8, and Rab10. Rab29 acts in a common pathway with LRRK2. Rab29 has been shown to recruit LRRK2 to the Golgi complex (GC) to stimulate LRRK2 activity and alter the Golgi apparatus (GA). Interaction between LRRK2 and Vacuolar protein sorting protein 52 (VPS52), a subunit of the Golgi-associated retrograde protein (GARP) complex, mediates the function of intracellular soma trans-Golgi network (TGN) transport. VPS52 also interacts with Rab29. Knockdown of VPS52 leads to the loss of LRRK2/Rab29 transported to the TGN. Rab29, LRRK2, and VPS52 work together to regulate functions of the GA, which is associated with PD. We highlight recent advances in the roles of LRRK2, Rabs, VPS52, and other molecules, such as Cyclin-dependent kinase 5 (CDK5) and protein kinase C (PKC) in the GA, and discuss their possible association with the pathological mechanisms of PD.

Hybrid enhanced sampling approaches to gain structural insights into leucine-rich repeat kinase 2 (LRRK2) Parkinson's disease mutation.

Parkinson's disease (PD) is a neurodegenerative disease affecting millions worldwide. A commonly mutated gene in PD is leucine-rich repeat kinase 2 (LRRK2), which contains multiple domains, including but not limited to kinase, ATPase, and GTPase. LRRK2 has been found to increase kinase activity and colocalize with microtubules. LRRK2 protein dynamics are affected by its conformation, regulating microtubule interactions and potentially enhancing intracellular trafficking. Despite recent advances in the structural insights of LRRK2, detailed studies on the effects of single-point mutations on the dynamics and conformations of LRRK2 are limited. Consequently, an all-atomistic molecular dynamics simulation is required to understand the allosteric and dynamical effects of single-point disease mutations in LRRK2, but it would be computationally expensive. We thereby employ enhanced molecular dynamics simulations to study single-point mutations in LRRK2, particularly the G2019S, R1441C, R1441G, R1441H, and Y1699C mutations. Our two-phase approach incorporates implementing Gaussian-accelerated molecular dynamics (GaMD) for enhanced sampling, followed by the weighted ensemble (WE) method. GaMD is an enhanced MD approach where a boost potential is added to the total potential energy of the system to accelerate the conformational search, while the WE method readjusts the weight of the simulation trajectories by frequent resampling to obtain the kinetic properties of interest accurately. Investigating the dynamics of such single-point mutations in LRRK2 could explain why specific mutations result in increased kinase activation and dynamicity of LRRK2.

Gaussian-accelerated molecular dynamics to probe mutations in Parkinson's LRRK2 gene.

Parkinson's disease is the second most common neurodegenerative disease followed by Alzheimer's disease. Leucine-rich repeat kinase 2 (LRRK2) is one of many mutated genes associated with Parkinson's disease. LRRK2 has been shown to function in intracellular trafficking and wrap around microtubules. While an open LRRK2 conformation is considered to be inactive, a closed conformation favors oligomerization around microtubules, thus affecting the motility of microtubule-based motor proteins. Despite recent advances exhibiting the effects of single-point mutations on LRRK2, structural dynamics from a computational perspective are still limited. Mutations in LRRK2 can affect protein dynamics and conformation, but can also induce allosteric effects. Gaussian-accelerated molecular dynamics (GaMD) is therefore employed to understand the effect of mutants on the dynamics of the LRRK2 protein. Unlike traditional molecular dynamics, GaMD is an enhanced sampling technique where the boost potentials of the system are raised in order to lower energy barriers. This allows for the investigation of complex biological processes while sampling the potential energy landscape of the entire system. Analyzing the dynamics of particular mutations in LRRK2 could explain why specific mutations result in kinase activation and oligomerization.

Generation of a human iPSC line (CIBi014-A) from a patient with Parkinson’s disease carrying a novel heterozygotic PARK8 (LRRK2) mutation

Leucine-rich repeat kinase 2 (LRRK2), also known as Dardarin (derived from the Basque word “dardara,” meaning tremor), is a protein kinase encoded by the PARK8 gene. This protein is mainly distributed in the cytoplasm; however, it is also present in the mitochondrial outer membrane. Mutations in the LRRK2 protein cause Parkinson's disease (PD). We generated a human induced pluripotent stem cell (iPSC) line (CIBi014-A) expressing pluripotency markers. This cell line exhibited a normal karyotype and could be differentiated into three germ layers in vitro. This line will be valuable for investigating the mechanism underlying PD and identifying potential therapeutic targets.