mTOR independent alteration in ULK1 Ser758 phosphorylation following chronic LRRK2 kinase inhibition

Unc-51 Like Kinase 1 (ULK1) is a critical regulator of the biogenesis of autophagosomes, the central component of the catabolic macroautophagy pathway. Regulation of ULK1 activity is dependent upon several phosphorylation events acting to repress or activate the enzymatic function of this protein. Phosphorylation of Ser758 ULK1 has been linked to repression of autophagosome biogenesis and was thought to be exclusively dependent upon mTOR complex 1 kinase activity. In this study, a novel regulation of Ser758 ULK1 phosphorylation is reported following prolonged inhibition of the Parkinson's disease linked protein Leucine Rich Repeat Kinase 2 (LRRK2). Here, modulation of Ser758 ULK1 phosphorylation following LRRK2 inhibition is decoupled from the repression of autophagosome biogenesis and independent of mTOR complex 1 activity

Regulation of the autophagy protein LC3 by phosphorylation

PKA puts the brakes on autophagy by inhibiting LC3 recruitment to autophagosomes

The LRRK2-macroautophagy axis and its relevance to Parkinson's Disease

A wide variety of different functions and an impressive array of interactors have been associated with leucine-rich repeat kinase 2 (LRRK2) over the years. Here, I discuss the hypothesis that LRRK2 may be capable of interacting with different proteins at different times and places, therefore, controlling a plethora of diverse functions based on the different complexes formed. Among these, I will then focus on macroautophagy in the general context of the endolysosomal system. First, the relevance of autophagy in Parkinson's disease will be evaluated giving a brief overview of all the relevant Parkinson's disease genes; then, the association of LRRK2 with macroautophagy and the endolysosomal pathway will be analyzed based on the supporting literature

Mutations in the LRRK2 Roc-COR tandem domain link Parkinson's disease to Wnt signalling pathways

Mutations in PARK8, encoding LRRK2, are the most common known cause of Parkinson's disease. The LRRK2 Roc-COR tandem domain exhibits GTPase activity controlling LRRK2 kinase activity via an intramolecular process. We report the interaction of LRRK2 with the dishevelled family of phosphoproteins (DVL1-3), key regulators of Wnt (Wingless/Int) signalling pathways important for axon guidance, synapse formation and neuronal maintenance. Interestingly, DVLs can interact with and mediate the activation of small GTPases with structural similarity to the LRRK2 Roc domain. The LRRK2 Roc-COR domain and the DVL1 DEP domain were necessary and sufficient for LRRK2–DVL1 interaction. Co-expression of DVL1 increased LRRK2 steady-state protein levels, an effect that was dependent on the DEP domain. Strikingly, LRRK2–DVL1-3 interactions were disrupted by the familial PARK8 mutation Y1699C, whereas pathogenic mutations at residues R1441 and R1728 strengthened LRRK2–DVL1 interactions. Co-expression of DVL1 with LRRK2 in mammalian cells resulted in the redistribution of LRRK2 to typical cytoplasmic DVL1 aggregates in HEK293 and SH-SY5Y cells and co-localization in neurites and growth cones of differentiated dopaminergic SH-SY5Y cells. This is the first report of the modulation of a key LRRK2-accessory protein interaction by PARK8 Roc-COR domain mutations segregating with Parkinson's disease. Since the DVL1 DEP domain is known to be involved in the regulation of small GTPases, we propose that: (i) DVLs may influence LRRK2 GTPase activity, and (ii) Roc-COR domain mutations modulating LRRK2–DVL interactions indirectly influence kinase activity. Our findings also link LRRK2 to Wnt signalling pathways, suggesting novel pathogenic mechanisms and new targets for genetic analysis in Parkinson's disease

Identification of novel genetic determinants in the high prevalence early-onset inflammatory bowel disease population in Scotland

Grant no. 072789/Z/03/ZBackground & aims: The inflammatory bowel diseases (IBD), Crohn‟s disease (CD) and ulcerative colitis (UC), are common causes of chronic gastrointestinal morbidity, affecting up to 1 in 250 of the general population in Northern Europe. Up to 25% of IBD is diagnosed during childhood or adolescence. The aims for this thesis were to study the epidemiology, natural history and novel genetic determinants of childhood onset IBD in Scotland. Methods: The existing repository of childhood onset and adult onset IBD patients, established at the Western General Hospital in Edinburgh, was used and expanded. Thus, anatomical location and behaviour of disease were assessed in 416 childhood onset (276 CD, 99 UC, 41 IBDU diagnosed before 17th birthday) and 1297 adult patients (596 CD, 701 UC) using the Montreal classification. Additional phenotypic (at diagnosis and at regular follow-up intervals) and epidemiological data were gathered. In this cohort, genotyping of germline variants in putative susceptibility genes (NOD1/CARD4, IL23R, ATG16L1, IRGM, FLG) was performed to enable single variant and haplotype-tagging association studies. Genotypic data of population-matched healthy controls were obtained locally (n=342) and from the Wellcome Trust Case Control Consortium (n=2937). Results: Compared with adults, childhood-onset CD was characterized by a more extensive, “panenteric” phenotype (ileocolonic plus upper GI; p<0.0001 OR23.3; 95% CI (13.4–40.6) with less isolated ileal (p<0.0001 OR 0.06 (0.03–0.1) or colonic disease (p<0.0001, OR 0.3 (0.2–0.5)). In 39%, the anatomic extent increased within 2 years. UC was also more extensive in children at diagnosis vs adults (p0.05 after Bonferroni correction). We found that the allelic frequency of rs11209026*A located within the IL23R gene, differed significantly between IBD / CD cases and controls (p=0.01 OR 0.51(0.3-0.9) and p=0.04 OR 0.5 (0.3-0.98)). Using a gene-wide haplotype-tagging strategy, we demonstrated that the multiple association signals of the IL23R locus are independent of rs11209026 in childhood onset IBD and CD. In Scottish children, the effect of germline variation of ATG16L1 and IRGM on CD susceptibility was relatively small (OR< 1.4), and appeared less than in adult disease. Genotype–phenotype analysis demonstrated an association of pure ileal disease with the ATG16L1 rs2241880G-allele (p=0.02 OR 1.3 (1.03–1.7)). Using binary logistic regression analysis, we confirmed the effect of rs2241880 genotype (GG) on ileal disease versus colonic disease (p=0.03 OR 2.4 (1.05–5.6)). Null alleles of the epithelial barrier protein FLG have no important effect on IBD susceptibility (p>0.4), but contribute to the high prevalence of atopy, notably co-existent eczema and food allergy (p=0.0003 OR 3.3 (1.7–6.6) and p=0.0001 OR 4.5 (2.0–10.0), respectively). Conclusion: Childhood onset IBD is characterised by extensive intestinal involvement and progression of disease after diagnosis. Genetic association studies in childhood and adult IBD have provided evidence for a large number of new genomic loci. These loci encode genes involved in a number of homeostatic mechanisms: innate pattern recognition receptors, the differentiation of Th17-lymphocytes, autophagy, maintenance of epithelial barrier integrity and the orchestration of the secondary immune response

A Direct Interaction between Leucine-rich Repeat Kinase 2 and Specific β-Tubulin Isoforms Regulates Tubulin Acetylation

Mutations in LRRK2, encoding the multifunctional protein leucine-rich repeat kinase 2 (LRRK2), are a common cause of Parkinson disease. LRRK2 has been suggested to influence the cytoskeleton as LRRK2 mutants reduce neurite outgrowth and cause an accumulation of hyperphosphorylated Tau. This might cause alterations in the dynamic instability of microtubules suggested to contribute to the pathogenesis of Parkinson disease. Here, we describe a direct interaction between LRRK2 and β-tubulin. This interaction is conferred by the LRRK2 Roc domain and is disrupted by the familial R1441G mutation and artificial Roc domain mutations that mimic autophosphorylation. LRRK2 selectively interacts with three β-tubulin isoforms: TUBB, TUBB4, and TUBB6, one of which (TUBB4) is mutated in the movement disorder dystonia type 4 (DYT4). Binding specificity is determined by lysine 362 and alanine 364 of β-tubulin. Molecular modeling was used to map the interaction surface to the luminal face of microtubule protofibrils in close proximity to the lysine 40 acetylation site in α-tubulin. This location is predicted to be poorly accessible within mature stabilized microtubules, but exposed in dynamic microtubule populations. Consistent with this finding, endogenous LRRK2 displays a preferential localization to dynamic microtubules within growth cones, rather than adjacent axonal microtubule bundles. This interaction is functionally relevant to microtubule dynamics, as mouse embryonic fibroblasts derived from LRRK2 knock-out mice display increased microtubule acetylation. Taken together, our data shed light on the nature of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 might contribute to the pathogenesis of Parkinson disease

Leucine-rich repeat kinase 2 interacts with p21-activated kinase 6 to control neurite complexity in mammalian brain

Leucine-rich repeat kinase 2 (LRRK2) is a causative gene for Parkinson's disease, but the physiological function and the mechanism(s) by which the cellular activity of LRRK2 is regulated are poorly understood. Here, we identified p21-activated kinase 6 (PAK6) as a novel interactor of the GTPase/ROC domain of LRRK2. p21-activated kinases are serine-threonine kinases that serve as targets for the small GTP binding proteins Cdc42 and Rac1 and have been implicated in different morphogenetic processes through remodeling of the actin cytoskeleton such as synapse formation and neuritogenesis. Using an in vivo neuromorphology assay, we show that PAK6 is a positive regulator of neurite outgrowth and that LRRK2 is required for this function. Analyses of post-mortem brain tissue from idiopathic and LRRK2 G2019S carriers reveal an increase in PAK6 activation state, whereas knock-out LRRK2 mice display reduced PAK6 activation and phosphorylation of PAK6 substrates. Taken together, these results support a critical role of LRRK2 GTPase domain in cytoskeletal dynamics in vivo through the novel interactor PAK6, and provide a valuable platform to unravel the mechanism underlying LRRK2-mediated pathophysiology. We propose p21-activated kinase 6 (PAK6) as a novel interactor of leucine-rich repeat kinase 2 (LRRK2), a kinase involved in Parkinson's disease (PD). In health, PAK6 regulates neurite complexity in the brain and LRRK2 is required for its function, (a) whereas PAK6 is aberrantly activated in LRRK2-linked PD brain (b) suggesting that LRRK2 toxicity is mediated by PAK6

Neuronal autophagy and neurodegenerative diseases

Autophagy is a dynamic cellular pathway involved in the turnover of proteins, protein complexes, and organelles through lysosomal degradation. The integrity of postmitotic neurons is heavily dependent on high basal autophagy compared to non-neuronal cells as misfolded proteins and damaged organelles cannot be diluted through cell division. Moreover, neurons contain the specialized structures for intercellular communication, such as axons, dendrites and synapses, which require the reciprocal transport of proteins, organelles and autophagosomes over significant distances from the soma. Defects in autophagy affect the intercellular communication and subsequently, contributing to neurodegeneration. The presence of abnormal autophagic activity is frequently observed in selective neuronal populations afflicted in common neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis. These observations have provoked controversy regarding whether the increase in autophagosomes observed in the degenerating neurons play a protective role or instead contribute to pathogenic neuronal cell death. It is still unknown what factors may determine whether active autophagy is beneficial or pathogenic during neurodegeneration. In this review, we consider both the normal and pathophysiological roles of neuronal autophagy and its potential therapeutic implications for common neurodegenerative diseases