Leucine Rich Repeat Kinase 2 (LRRK2) is one of the most important genetic contributors to Parkinson's disease. LRRK2 has been implicated in a number of cellular processes, including macroautophagy. To test whether LRRK2 has a role in regulating autophagy, a specific inhibitor of the kinase activity of LRRK2 was applied to human neuroglioma cells and downstream readouts of autophagy examined. The resulting data demonstrate that inhibition of LRRK2 kinase activity stimulates macroautophagy in the absence of any alteration in the translational targets of mTORC1, suggesting that LRRK2 regulates autophagic vesicle formation independent of canonical mTORC1 signaling. This study represents the first pharmacological dissection of the role LRRK2 plays in the autophagy/lysosomal pathway, emphasizing the importance of this pathway as a marker for LRRK2 physiological function. Moreover it highlights the need to dissect autophagy and lysosomal activities in the context of LRRK2 related pathologies with the final aim of understanding their aetiology and identifying specific targets for disease modifying therapies in patients.

Mammalian glycogen synthase kinase-3 (GSK3) is generated from two genes, GSK3alpha and GSK3beta, while a splice variant of GSK3beta (GSK3beta2), containing a 13 amino acid insert, is enriched in neurons. GSK3alpha and GSK3beta deletions generate distinct phenotypes. Here, we show that phosphorylation of CRMP2, CRMP4, beta-catenin, c-Myc, c-Jun and some residues on tau associated with Alzheimer's disease, is altered in cortical tissue lacking both isoforms of GSK3. This confirms that they are physiological targets for GSK3. However, deletion of each GSK3 isoform produces distinct substrate phosphorylation, indicating that each has a different spectrum of substrates (e.g. phosphorylation of Thr509, Thr514 and Ser518 of CRMP is not detectable in cortex lacking GSK3beta, yet normal in cortex lacking GSK3alpha). Furthermore, the neuron-enriched GSK3beta2 variant phosphorylates phospho-glycogen synthase 2 peptide, CRMP2 (Thr509/514), CRMP4 (Thr509), Inhibitor-2 (Thr72) and tau (Ser396), at a lower rate than GSK3beta1. In contrast phosphorylation of c-Myc and c-Jun is equivalent for each GSK3beta isoform, providing evidence that differential substrate phosphorylation is achieved through alterations in expression and splicing of the GSK3 gene. Finally, each GSK3beta splice variant is phosphorylated to a similar extent at the regulatory sites, Ser9 and Tyr216, and exhibit identical sensitivities to the ATP competitive inhibitor CT99021, suggesting upstream regulation and ATP binding properties of GSK3beta1 and GSK3beta2 are similar

Myoclonus-dystonia (M-D) is a rare movement disorder characterized by a combination of non-epileptic myoclonic jerks and dystonia. SGCE mutations represent a major cause for familial M-D being responsible for 30%-50% of cases. After excluding SGCE mutations, we identified through a combination of linkage analysis and whole-exome sequencing KCTD17 c.434 G>A p.(Arg145His) as the only segregating variant in a dominant British pedigree with seven subjects affected by M-D. A subsequent screening in a cohort of M-D cases without mutations in SGCE revealed the same KCTD17 variant in a German family. The clinical presentation of the KCTD17-mutated cases was distinct from the phenotype usually observed in M-D due to SGCE mutations. All cases initially presented with mild myoclonus affecting the upper limbs. Dystonia showed a progressive course, with increasing severity of symptoms and spreading from the cranio-cervical region to other sites. KCTD17 is abundantly expressed in all brain regions with the highest expression in the putamen. Weighted gene co-expression network analysis, based on mRNA expression profile of brain samples from neuropathologically healthy individuals, showed that KCTD17 is part of a putamen gene network, which is significantly enriched for dystonia genes. Functional annotation of the network showed an over-representation of genes involved in post-synaptic dopaminergic transmission. Functional studies in mutation bearing fibroblasts demonstrated abnormalities in endoplasmic reticulum-dependent calcium signaling. In conclusion, we demonstrate that the KCTD17 c.434 G>A p.(Arg145His) mutation causes autosomal dominant M-D. Further functional studies are warranted to further characterize the nature of KCTD17 contribution to the molecular pathogenesis of M-D.

The specific autophagic elimination of mitochondria (mitophagy) plays the role of quality control for this organelle. Deregulation of mitophagy leads to an increased number of damaged mitochondria and triggers cell death. The deterioration of mitophagy has been hypothesized to underlie the pathogenesis of several neurodegenerative diseases, most notably Parkinson disease. Although some of the biochemical and molecular mechanisms of mitochondrial quality control are described in detail, physiological or pathological triggers of mitophagy are still not fully characterized. Here we show that the induction of mitophagy by the mitochondrial uncoupler FCCP is independent of the effect of mitochondrial membrane potential but dependent on acidification of the cytosol by FCCP. The ionophore nigericin also reduces cytosolic pH and induces PINK1/PARKIN-dependent and -independent mitophagy. The increase of intracellular pH with monensin suppresses the effects of FCCP and nigericin on mitochondrial degradation. Thus, a change in intracellular pH is a regulator of mitochondrial quality control.Continuous ATP production is essential for cell survival. Mitochondria are the major ATP producers in the cell; these are distributed throughout different tissues in varying amounts depending on energy demand. The number of mitochondria within cells is regulated by the balance between mitochondrial biogenesis (1) and the removal of damaged mitochondria. Furthermore, mitochondria also regulate cell death, which is triggered by release of intramitochondrial proteins (2). Considering this, the removal of dysfunctional mitochondria is an important process for ensuring cell survival.Damaged mitochondria, as well as other impaired organelles and proteins in cells are degraded by specific autophagy. Mitochondrial autophagy (mitophagy) 2 plays a role in the quality control of this organelle, whereas dysfunction in mitophagy can lead to an increase in the number of damaged mitochondria that can trigger the activation of cell death pathways (3, 4). Over the last several years, a number of studies have provided substantial evidence showing the PINK1-Parkin pathway (familial forms of Parkinson disease caused by mutations in the genes encoding the PTEN (phosphatase and tensin homolog)-induced putative kinase-1 (PINK1) and Parkin E3 ubiquitin ligase) promotes mitophagy. PINK1 accumulates at the outer mitochondrial membrane of depolarized mitochondria, which recruits cytoplasmic Parkin to the damaged organelle. This results in the ubiquitination of mitofusins (Mfns) by Parkin, the engulfment of the dysfunctional mitochondria by autophagosomes, and subsequent mitophagy (5-8). In recent years, abnormal autophagy and mitophagy has been shown for toxininjured dopaminergic neurons as well as in all the major genetic models of Parkinson disease (PD), including ␣-synuclein, leucine-rich repeat kinase 2 (LRRK2), parkin, PTEN-induced kinase 1 (PINK1), and DJ-1 (9, 10). Historically, mitophagy research focuses on the biochemical and molecular mechanisms of PD; ...

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 identified. To investigate the signalling events through which LRRK2 acts to influence 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.