P62/SQSTM1 is a novel leucine-rich repeat kinase 2 (LRRK2) substrate that enhances neuronal toxicity

Autosomal-dominant, missense mutations in the leucine-rich repeat protein kinase 2 (LRRK2) gene are the most common genetic predisposition to develop Parkinson’s disease (PD). LRRK2 kinase activity is increased in several pathogenic mutations (N1437H, R1441C/G/H, Y1699C, G2019S), implicating hyperphosphorylation of a substrate in the pathogenesis of the disease. Identification of the downstream targets of LRRK2 is a crucial endeavor in the field to understand LRRK2 pathway dysfunction in the disease. We have identified the signaling adapter protein p62/SQSTM1 as a novel endogenous interacting partner and a substrate of LRRK2. Using mass spectrometry and phospho-specific antibodies, we found that LRRK2 phosphorylates p62 on Thr138 in vitro and in cells. We found that the pathogenic LRRK2 PD-associated mutations (N1437H, R1441C/G/H, Y1699C, G2019S) increase phosphorylation of p62 similar to previously reported substrate Rab proteins. Notably, we found that the pathogenic I2020T mutation and the risk factor mutation G2385R displayed decreased phosphorylation of p62. p62 phosphorylation by LRRK2 is blocked by treatment with selective LRRK2 inhibitors in cells. We also found that the amino-terminus of LRRK2 is crucial for optimal phosphorylation of Rab7L1 and p62 in cells. LRRK2 phosphorylation of Thr138 is dependent on a p62 functional ubiquitin-binding domain at its carboxy-terminus. Co-expression of p62 with LRRK2 G2019S increases the neurotoxicity of this mutation in a manner dependent on Thr138. p62 is an additional novel substrate of LRRK2 that regulates its toxic biology, reveals novel signaling nodes and can be used as a pharmacodynamic marker for LRRK2 kinase activity.</p

Detection of alpha-synuclein conformational variants from gastro-intestinal biopsy tissue as a potential biomarker for Parkinson's disease

Aims Gastrointestinal (GI) a-synuclein (aSyn) detection as a potential biomarker of Parkinson’s disease (PD) is challenged by conflicting results of recent studies. To increase sensitivity and specificity, we applied three techniques to detect different conformations of aSyn in GI biopsies obtained from a longitudinal, clinically wellcharacterized cohort of PD patients and healthy controls (HC). Methods With immunohistochemistry (IHC), we used antibodies reactive for total, phosphorylated and oligomeric aSyn; with aSyn proximity ligation assay (AS-PLA), we targeted oligomeric aSyn species specifically; and with paraffin-embedded tissue blot (AS-PET-blot) we aimed to detect fibrillary, synaptic aSyn. Results A total of 163 tissue blocks were collected from 51 PD patients (113 blocks) and 21 HC (50 blocks). In 31 PD patients, biopsies were taken before the PD diagnosis (Prodromal); while in 20 PD patients biopsies were obtained after diagnosis (Manifest). The majority of tissues blocks were from large intestine (62%), followed by small intestine (21%), stomach (10%) and oesophagus (7%). With IHC, four staining patterns were detected (neuritic, ganglionic, epithelial and cellular), while two distinct staining patterns were detected both with AS-PLA (cellular and diffuse signal) and with AS-PET-blot (aSyn-localized and pericrypt signal). The level of agreement between different techniques was low and no single technique or staining pattern reliably distinguished PD patients (Prodromal or Manifest) from HC. Conclusions Our study suggests that detection of aSyn conformational variants currently considered pathological is not adequate for the diagnosis or prediction of PD. Future studies utilizing novel ultrasensitive amyloid aggregation assays may increase sensitivity and specificity

Selective vulnerability in α-synucleinopathies

Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy are neurodegenerative disorders resulting in progressive motor/cognitive deficits among other symptoms. They are characterised by stereotypical brain cell loss accompanied by the formation of proteinaceous aggregations of the protein α-synuclein (α-syn), being, therefore, termed α-synucleinopathies. Although the presence of α-syn inclusions is a common hallmark of these disorders, the exact nature of the deposited protein is specific to each disease. Different neuroanatomical regions and cellular populations manifest a differential vulnerability to the appearance of protein deposits, cell dysfunction, and cell death, leading to phenotypic diversity. The present review describes the multiple factors that contribute to the selective vulnerability in α-synucleinopathies. We explore the intrinsic cellular properties in the affected regions, including the physiological and pathophysiological roles of endogenous α-syn, the metabolic and genetic build-up of the cells and their connectivity. These factors converge with the variability of the α-syn conformational strains and their spreading capacity to dictate the phenotypic diversity and regional vulnerability of each disease. Finally, we describe the exogenous and environmental factors that potentially contribute by igniting and modulating the differential pathology in α-synucleinopathies. In conclusion, we think that it is the confluence of this disruption of the cellular metabolic state and α-syn structural equilibrium through the anatomical connectivity which appears to initiate cascades of pathological processes triggered by genetic, environmental, or stochastic events that result in the "death by a thousand cuts" profile of α-synucleinopathies

mTOR independent regulation of macroautophagy by Leucine Rich Repeat Kinase 2 via Beclin-1

Leucine rich repeat kinase 2 is a complex enzyme with both kinase and GTPase activities, closely linked to the pathogenesis of several human disorders including Parkinson’s disease, Crohn’s disease, leprosy and cancer. LRRK2 has been implicated in numerous cellular processes; however its physiological function remains unclear. Recent reports suggest that LRRK2 can act to regulate the cellular catabolic process of macroautophagy, although the precise mechanism whereby this occurs has not been identi ed. To investigate the signalling events through which LRRK2 acts to in uence macroautophagy, the mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) and Beclin-1/phosphatidylinositol 3-kinase (PI3K) pathways were evaluated in astrocytic cell models in the presence and absence of LRRK2 kinase inhibitors. Chemical inhibition of LRRK2 kinase activity resulted in the stimulation of macroautophagy in a non-canonical fashion, independent of mTOR and ULK1, but dependent upon the activation of Beclin 1-containing class III PI3-kinase

Increasing microtubule acetylation rescues axonal transport and locomotor deficits caused by LRRK2 Roc-COR domain mutations

​Leucine-rich repeat kinase 2 (​LRRK2) mutations are the most common genetic cause of Parkinson’s disease. ​LRRK2 is a multifunctional protein affecting many cellular processes and has been described to bind microtubules. Defective microtubule-based axonal transport is hypothesized to contribute to Parkinson’s disease, but whether ​LRRK2 mutations affect this process to mediate pathogenesis is not known. Here we find that ​LRRK2 containing pathogenic Roc-COR domain mutations (R1441C, Y1699C) preferentially associates with deacetylated microtubules, and inhibits axonal transport in primary neurons and in Drosophila, causing locomotor deficits in vivo. In vitro, increasing microtubule acetylation using deacetylase inhibitors or the tubulin acetylase ​αTAT1 prevents association of mutant ​LRRK2 with microtubules, and the deacetylase inhibitor ​trichostatin A (​TSA) restores axonal transport. In vivo knockdown of the deacetylases ​HDAC6 and ​Sirt2, or administration of ​TSA rescues both axonal transport and locomotor behavior. Thus, this study reveals a pathogenic mechanism and a potential intervention for Parkinson’s disease

The LRRK2 signalling system

The LRRK2 gene is a major contributor to genetic risk for Parkinson's disease and understanding the biology of the leucine-rich repeat kinase 2 (LRRK2, the protein product of this gene) is an important goal in Parkinson's research. LRRK2 is a multi-domain, multi-activity enzyme and has been implicated in a wide range of signalling events within the cell. Because of the complexities of the signal transduction pathways in which LRRK2 is involved, it has been challenging to generate a clear idea as to how mutations and disease associated variants in this gene are altered in disease. Understanding the events in which LRRK2 is involved at a systems level is therefore critical to fully understand the biology and pathobiology of this protein and is the subject of this review

LRRK2 at the interface of autophagosomes, endosomes and lysosomes

Over the past 20 years, substantial progress has been made in identifying the underlying genetics of Parkinson’s disease (PD). Of the known genes, LRRK2 is a major genetic contributor to PD. However, the exact function of LRRK2 remains to be elucidated. In this review, we discuss how familial forms of PD have led us to hypothesize that alterations in endomembrane trafficking play a role in the pathobiology of PD. We will discuss the major observations that have been made to elucidate the role of LRRK2 in particular, including LRRK2 animal models and high-throughput proteomics approaches. Taken together, these studies strongly support a role of LRRK2 in vesicular dynamics. We also propose that targeting these pathways may not only be beneficial for developing therapeutics for LRRK2-driven PD, but also for other familial and sporadic cases

Cell state-dependent allelic effects and contextual Mendelian randomization analysis for human brain phenotypes

Gene expression quantitative trait loci are widely used to infer relationships between genes and central nervous system (CNS) phenotypes; however, the effect of brain disease on these inferences is unclear. Using 2,348,438 single-nuclei profiles from 391 disease-case and control brains, we report 13,939 genes whose expression correlated with genetic variation, of which 16.7-40.8% (depending on cell type) showed disease-dependent allelic effects. Across 501 colocalizations for 30 CNS traits, 23.6% had a disease dependency, even after adjusting for disease status. To estimate the unconfounded effect of genes on outcomes, we repeated the analysis using nondiseased brains (n = 183) and reported an additional 91 colocalizations not present in the larger mixed disease and control dataset, demonstrating enhanced interpretation of disease-associated variants. Principled implementation of single-cell Mendelian randomization in control-only brains identified 140 putatively causal gene-trait associations, of which 11 were replicated in the UK Biobank, prioritizing candidate peripheral biomarkers predictive of CNS outcomes

LRRK2 protein levels are determined by kinase function and are crucial for kidney and lung homeostasis in mice

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinson's disease (PD), but the underlying pathophysiological mechanisms and the normal function of this large multidomain protein remain speculative. To address the role of this protein in vivo, we generated three different LRRK2 mutant mouse lines. Mice completely lacking the LRRK2 protein (knock-out, KO) showed an early-onset (age 6 weeks) marked increase in number and size of secondary lysosomes in kidney proximal tubule cells and lamellar bodies in lung type II cells. Mice expressing a LRRK2 kinase-dead (KD) mutant from the endogenous locus displayed similar early-onset pathophysiological changes in kidney but not lung. KD mutants had dramatically reduced full-length LRRK2 protein levels in the kidney and this genetic effect was mimicked pharmacologically in wild-type mice treated with a LRRK2-selective kinase inhibitor. Knock-in (KI) mice expressing the G2019S PD-associated mutation that increases LRRK2 kinase activity showed none of the LRRK2 protein level and histopathological changes observed in KD and KO mice. The autophagy marker LC3 remained unchanged but kidney mTOR and TCS2 protein levels decreased in KD and increased in KO and KI mice. Unexpectedly, KO and KI mice suffered from diastolic hypertension opposed to normal blood pressure in KD mice. Our findings demonstrate a role for LRRK2 in kidney and lung physiology and further show that LRRK2 kinase function affects LRRK2 protein steady-state levels thereby altering putative scaffold/GTPase activity. These novel aspects of peripheral LRRK2 biology critically impact ongoing attempts to develop LRRK2 selective kinase inhibitors as therapeutics for PD