Common genetic risk for Parkinson's disease and dysfunction of the endo-lysosomal system.

Parkinson's disease is a progressive neurological disorder, characterized by prominent movement dysfunction. The past two decades have seen a rapid expansion of our understanding of the genetic basis of Parkinson's, initially through the identification of monogenic forms and, more recently, through genome-wide association studies identifying common risk variants. Intriguingly, a number of cellular pathways have emerged from these analysis as playing central roles in the aetiopathogenesis of Parkinson's. In this review, the impact of data deriving from genome-wide analyses for Parkinson's upon our functional understanding of the disease will be examined, with a particular focus on examples of endo-lysosomal and mitochondrial dysfunction. The challenges of moving from a genetic to a functional understanding of common risk variants for Parkinson's will be discussed, with a final consideration of the current state of the genetic architecture of the disorder. This article is part of a discussion meeting issue 'Understanding the endo-lysosomal network in neurodegeneration'.

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

Introduction: 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 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. Methods: 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. Results: 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 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. Discussion: 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.

Artificial intelligence for neurodegenerative experimental models.

INTRODUCTION: Experimental models are essential tools in neurodegenerative disease research. However, the translation of insights and drugs discovered in model systems has proven immensely challenging, marred by high failure rates in human clinical trials. METHODS: Here we review the application of artificial intelligence (AI) and machine learning (ML) in experimental medicine for dementia research. RESULTS: Considering the specific challenges of reproducibility and translation between other species or model systems and human biology in preclinical dementia research, we highlight best practices and resources that can be leveraged to quantify and evaluate translatability. We then evaluate how AI and ML approaches could be applied to enhance both cross-model reproducibility and translation to human biology, while sustaining biological interpretability. DISCUSSION: AI and ML approaches in experimental medicine remain in their infancy. However, they have great potential to strengthen preclinical research and translation if based upon adequate, robust, and reproducible experimental data. HIGHLIGHTS: There are increasing applications of AI in experimental medicine. We identified issues in reproducibility, cross-species translation, and data curation in the field. Our review highlights data resources and AI approaches as solutions. Multi-omics analysis with AI offers exciting future possibilities in drug discovery.

Leucine-rich repeat kinase 2 at a glance.

Leucine-rich repeat kinase 2 (LRRK2) is a multidomain scaffolding protein with dual guanosine triphosphatase (GTPase) and kinase enzymatic activities, providing this protein with the capacity to regulate a multitude of signalling pathways and act as a key mediator of diverse cellular processes. Much of the interest in LRRK2 derives from mutations in the LRRK2 gene being the most common genetic cause of Parkinson's disease, and from the association of the LRRK2 locus with a number of other human diseases, including inflammatory bowel disease. Therefore, the LRRK2 research field has focused on the link between LRRK2 and pathology, with the aim of uncovering the underlying mechanisms and, ultimately, finding novel therapies and treatments to combat them. From the biochemical and cellular functions of LRRK2, to its relevance to distinct disease mechanisms, this Cell Science at a Glance article and the accompanying poster deliver a snapshot of our current understanding of LRRK2 function, dysfunction and links to disease.

Microglia produce the amyloidogenic ABri peptide in familial British dementia.

Mutations in ITM2B cause familial British, Danish, Chinese and Korean dementias. In familial British dementia (FBD) a mutation in the stop codon of the ITM2B gene (also known as BRI2 ) causes a C-terminal cleavage fragment of the ITM2B/BRI2 protein to be extended by 11 amino acids. This fragment, termed amyloid-Bri (ABri), is highly insoluble and forms extracellular plaques in the brain. ABri plaques are accompanied by tau pathology, neuronal cell death and progressive dementia, with striking parallels to the aetiology and pathogenesis of Alzheimer's disease. The molecular mechanisms underpinning FBD are ill-defined. Using patient-derived induced pluripotent stem cells, we show that expression of ITM2B/BRI2 is 34-fold higher in microglia than neurons, and 15-fold higher in microglia compared with astrocytes. This cell-specific enrichment is supported by expression data from both mouse and human brain tissue. ITM2B/BRI2 protein levels are higher in iPSC-microglia compared with neurons and astrocytes. Consequently, the ABri peptide was detected in patient iPSC-derived microglial lysates and conditioned media but was undetectable in patient-derived neurons and control microglia. Pathological examination of post-mortem tissue support ABri expression in microglia that are in proximity to pre-amyloid deposits. Finally, gene co-expression analysis supports a role for ITM2B/BRI2 in disease-associated microglial responses. These data demonstrate that microglia are the major contributors to the production of amyloid forming peptides in FBD, potentially acting as instigators of neurodegeneration. Additionally, these data also suggest ITM2B/BRI2 may be part of a microglial response to disease, motivating further investigations of its role in microglial activation. This has implications for our understanding of the role of microglia and the innate immune response in the pathogenesis of FBD and other neurodegenerative dementias including Alzheimer's disease.

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

Whilst the majority of Parkinson's Disease (PD) cases are sporadic, much of our understanding of the pathophysiological basis of the disease can be traced back to the study of rare, monogenic forms of PD. 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 pathogenesis. The NSL interactome has been built, using 3 online tools: PINOT, HIPPIE and MIST, to mine curated, literature-derived protein-protein interaction (PPI) data. We built (i) the 'mitochondrial' NSL interactome exploring its relevance to PD genetics and (ii) the PD-oriented NSL 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. In addition, we find nuclear processes to be amongst those most significantly enriched within the PD-associated NSL interactome. These findings strengthen the role of the NSL complex in sporadic and familial PD, mediated by both its mitochondrial and nuclear functions.

Tissue specific LRRK2 interactomes reveal a distinct striatal functional unit.

Mutations in LRRK2 are the most common genetic cause of Parkinson's 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 evaluated taking into consideration the differential expression patterns and the co-expression behaviours of the LRRK2 interactors in 15 different healthy tissue types. 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. The general LRRK2 interactome paired with the expression profiles of its members constitutes a powerful tool to generate tissue-specific LRRK2 interactomes. We exemplified the generation of the tissue-specific LRRK2 interactomes and explored the functions highlighted by the "core LRRK2 interactors" in the striatum in comparison with the cerebellum. Finally, we illustrated how the LRRK2 general interactome reported in this manuscript paired with the expression profiles can be used to trace the relationship between LRRK2 and specific interactors of interest, here focusing on the LRRK2 interactors belonging to the Rab protein family.

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.

Lysosomal positioning regulates Rab10 phosphorylation at LRRK2+ lysosomes.

Genetic variation at the leucine-rich repeat kinase 2 (LRRK2) locus contributes to an enhanced risk of familial and sporadic Parkinson's disease. Previous data have demonstrated that recruitment to various membranes of the endolysosomal system results in LRRK2 activation. However, the mechanism(s) underlying LRRK2 activation at endolysosomal membranes and the cellular consequences of these events are still poorly understood. Here, we directed LRRK2 to lysosomes and early endosomes, triggering both LRRK2 autophosphorylation and phosphorylation of the direct LRRK2 substrates Rab10 and Rab12. However, when directed to the lysosomal membrane, pRab10 was restricted to perinuclear lysosomes, whereas pRab12 was visualized on both peripheral and perinuclear LRRK2+ lysosomes, suggesting that lysosomal positioning provides additional regulation of LRRK2-dependent Rab phosphorylation. Anterograde transport of lysosomes to the cell periphery by increasing the expression of ARL8B and SKIP or by knockdown of JIP4 blocked the recruitment and phosphorylation of Rab10 by LRRK2. The absence of pRab10 from the lysosomal membrane prevented the formation of a lysosomal tubulation and sorting process we previously named LYTL. Conversely, overexpression of RILP resulted in lysosomal clustering within the perinuclear area and increased LRRK2-dependent Rab10 recruitment and phosphorylation. The regulation of Rab10 phosphorylation in the perinuclear area depends on counteracting phosphatases, as the knockdown of phosphatase PPM1H significantly increased pRab10 signal and lysosomal tubulation in the perinuclear region. Our findings suggest that LRRK2 can be activated at multiple cellular membranes, including lysosomes, and that lysosomal positioning further provides the regulation of some Rab substrates likely via differential phosphatase activity or effector protein presence in nearby cellular compartments.

Regulation of mitophagy by the NSL complex underlies genetic risk for Parkinson's disease at 16q11.2 and MAPT H1 loci.

Parkinson's disease is a common incurable neurodegenerative disease. The identification of genetic variants via genome-wide association studies has considerably advanced our understanding of the Parkinson's disease 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 Parkinson's disease, but its relevance to idiopathic Parkinson's disease is unclear. We used a mitophagy screening assay to evaluate the functional significance of risk genes identified through genome-wide association studies. We identified two new regulators of PINK1-dependent mitophagy initiation, KAT8 and KANSL1, previously shown to modulate lysine acetylation. These findings suggest PINK1-mitophagy is a contributing factor to idiopathic Parkinson's disease. KANSL1 is located on chromosome 17q21 where the risk associated gene has long been considered to be MAPT. While our data do not exclude a possible association between the MAPT gene and Parkinson's disease, they provide 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.

Cytosolic sequestration of spatacsin by Protein Kinase A and 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.

The emerging role of LRRK2 in tauopathies.

Parkinson's disease (PD) is conventionally described as an α-synuclein aggregation disorder, defined by Lewy bodies and neurites, and mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common autosomal dominant cause of PD. However, LRRK2 mutations may be associated with diverse pathologies in patients with Parkinson's syndrome including tau pathology resembling progressive supranuclear palsy (PSP). The recent discovery that variation at the LRRK2 locus is associated with the progression of PSP highlights the potential importance of LRRK2 in tauopathies. Here, we review the emerging evidence and discuss the potential impact of LRRK2 dysfunction on tau aggregation, lysosomal function, and endocytosis and exocytosis.

Seventy-Two-Hour LRRK2 Kinase Activity Inhibition Increases Lysosomal GBA Expression in H4, a Human Neuroglioma Cell Line.

Mutations in LRRK2 and GBA1 are key contributors to genetic risk of developing Parkinson's disease (PD). To investigate how LRRK2 kinase activity interacts with GBA and contributes to lysosomal dysfunctions associated with the pathology of PD. The activity of the lysosomal enzyme ß-Glucocerebrosidase (GCase) was assessed in a human neuroglioma cell model treated with two selective inhibitors of LRKK2 kinase activity (LRRK2-in-1 and MLi-2) and a GCase irreversible inhibitor, condutirol-beta-epoxide (CBE), under 24 and 72 h experimental conditions. We observed levels of GCase activity comparable to controls in response to 24 and 72 h treatments with LRRK2-in-1 and MLi-2. However, GBA protein levels increased upon 72 h treatment with LRRK2-in-1. Moreover, LC3-II protein levels were increased after both 24 and 72 h treatments with LRRK2-in-1, suggesting an activation of the autophagic pathway. These results highlight a possible regulation of lysosomal function through the LRRK2 kinase domain and suggest an interplay between LRRK2 kinase activity and GBA. Although further investigations are needed, the enhancement of GCase activity might restore the defective protein metabolism seen in PD.

Leucine-rich repeat kinase 2 (LRRK2): an update on the potential therapeutic target for Parkinson's disease.

AREAS COVERED: In this review, we will provide an update on the current status of drugs and other technologies that have emerged in recent years and provide an overview of their efficacy in ameliorating LRRK2 kinase activity and overall safety in animal models and humans. EXPERT OPINION: The growth of both target discovery and innovative drug design has sparked a lot of excitement for the future of how we treat Parkinson's disease. Given the immense focus on LRRK2 as a therapeutic target, it is expected within the next decade to determine its therapeutic properties, or lack thereof, for PD.

A step forward for LRRK2 inhibitors in Parkinson's disease.

A phase 1 clinical trial for kinase inhibitors targeting LRRK2 provides the foundation for testing the efficacy of LRRK2 kinase inhibitors in Parkinson's disease.

Directing LRRK2 to membranes of the endolysosomal pathway triggers RAB phosphorylation and JIP4 recruitment.

Coding mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene, which are associated with dominantly inherited Parkinson's disease (PD), lead to an increased activity of the encoded LRRK2 protein kinase. As such, kinase inhibitors are being considered as therapeutic agents for PD. It is therefore of interest to understand the mechanism(s) by which LRRK2 is activated during cellular signaling. Lysosomal membrane damage represents one way of activating LRRK2 and leads to phosphorylation of downstream RAB substrates and recruitment of the motor adaptor protein JIP4. However, it is unclear whether the activation of LRRK2 would be seen at other membranes of the endolysosomal system, where LRRK2 has also shown to be localized, or whether these signaling events can be induced without membrane damage. Here, we use a rapamycin-dependent oligomerization system to direct LRRK2 to various endomembranes including the Golgi apparatus, lysosomes, the plasma membrane, recycling, early, and late endosomes. Irrespective of membrane location, the recruitment of LRRK2 to membranes results in local accumulation of phosphorylated RAB10, RAB12, and JIP4. We also show that endogenous RAB29, previously nominated as an activator of LRRK2 based on overexpression, is not required for activation of LRRK2 at the Golgi nor lysosome. We therefore conclude that LRRK2 signaling to RAB10, RAB12, and JIP4 can be activated once LRRK2 is accumulated at any cellular organelle along the endolysosomal pathway.

The Roc domain of LRRK2 as a hub for protein-protein interactions: a focus on PAK6 and its impact on RAB phosphorylation.

Leucine-rich repeat kinase 2 (LRRK2) has taken center stage in Parkinson's disease (PD) research as mutations cause familial PD and more common variants increase lifetime risk for disease. One unique feature in LRRK2 is the coexistence of GTPase/Roc (Ras of complex) and kinase catalytic functions, bridged by a COR (C-terminal Of Roc) platform for dimerization. Multiple PD mutations are located within the Roc/GTPase domain and concomitantly lead to defective GTPase activity and augmented kinase activity in cells, supporting a crosstalk between GTPase and kinase domains. In addition, biochemical and structural data highlight the importance of Roc as a molecular switch modulating LRRK2 monomer-to-dimer equilibrium and building the interface for interaction with binding partners. Here we review the effects of PD Roc mutations on LRRK2 function and discuss the importance of Roc as a hub for multiple molecular interactions relevant for the regulation of cytoskeletal dynamics and intracellular trafficking pathways. Among the well-characterized Roc interactors, we focused on the cytoskeletal-related kinase p21-activated kinase 6 (PAK6). We report the affinity between LRRK2-Roc and PAK6 measured by microscale thermophoresis (MST). We further show that PAK6 can modulate LRRK2-mediated phosphorylation of RAB substrates in the presence of LRRK2 wild-type (WT) or the PD G2019S kinase mutant but not when the PD Roc mutation R1441G is expressed. These findings support a mechanism whereby mutations in Roc might affect LRRK2 activity through impaired protein-protein interaction in the cell.

Dissecting the Phenotype and Genotype of PLA2G6-Related Parkinsonism.

BACKGROUND: Complex parkinsonism is the commonest phenotype in late-onset PLA2G6-associated neurodegeneration. OBJECTIVES: The aim of this study was to deeply characterize phenogenotypically PLA2G6-related parkinsonism in the largest cohort ever reported. METHODS: We report 14 new cases of PLA2G6-related parkinsonism and perform a systematic literature review. RESULTS: PLA2G6-related parkinsonism shows a fairly distinct phenotype based on 86 cases from 68 pedigrees. Young onset (median age, 23.0 years) with parkinsonism/dystonia, gait/balance, and/or psychiatric/cognitive symptoms were common presenting features. Dystonia occurred in 69.4%, pyramidal signs in 77.2%, myoclonus in 65.2%, and cerebellar signs in 44.6% of cases. Early bladder overactivity was present in 71.9% of cases. Cognitive impairment affected 76.1% of cases and psychiatric features 87.1%, the latter being an isolated presenting feature in 20.1%. Parkinsonism was levodopa responsive but complicated by early, often severe dyskinesias. Five patients benefited from deep brain stimulation. Brain magnetic resonance imaging findings included cerebral (49.3%) and/or cerebellar (43.2%) atrophy, but mineralization was evident in only 28.1%. Presynaptic dopaminergic terminal imaging was abnormal in all where performed. Fifty-four PLA2G6 mutations have hitherto been associated with parkinsonism, including four new variants reported in this article. These are mainly nontruncating, which may explain the phenotypic heterogeneity of childhood- and late-onset PLA2G6-associated neurodegeneration. In five deceased patients, median disease duration was 13.0 years. Brain pathology in three cases showed mixed Lewy and tau pathology. CONCLUSIONS: Biallelic PLA2G6 mutations cause early-onset parkinsonism associated with dystonia, pyramidal and cerebellar signs, myoclonus, and cognitive impairment. Early psychiatric manifestations and bladder overactivity are common. Cerebro/cerebellar atrophy are frequent magnetic resonance imaging features, whereas brain iron deposition is not. Early, severe dyskinesias are a tell-tale sign. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Vesicular dysfunction and pathways to neurodegeneration.

Cellular control of vesicle biology and trafficking is critical for cell viability, with disruption of these pathways within the cells of the central nervous system resulting in neurodegeneration and disease. The past two decades have provided important insights into both the genetic and biological links between vesicle trafficking and neurodegeneration. In this essay, the pathways that have emerged as being critical for neuronal survival in the human brain will be discussed - illustrating the diversity of proteins and cellular events with three molecular case studies drawn from different neurological diseases.

SORL1 mutation in a Greek family with Parkinson's disease and dementia.

Whole exome sequencing and linkage analysis were performed in a three generational pedigree of Greek origin with a broad phenotypic spectrum spanning from Parkinson's disease and Parkinson's disease dementia to dementia of mixed type (Alzheimer disease and vascular dementia). We identified a novel heterozygous c.G1135T (p.G379W) variant in SORL1 which segregated with the disease in the family. Mutation screening in sporadic Greek PD cases identified one additional individual with the mutation, sharing the same 12.8Mb haplotype. Our findings provide support for SORL1 mutations resulting in a broad range of additional phenotypes and warrants further studies in neurodegenerative diseases beyond AD.