Role of Wnt signalling pathway in mechanotransduction pathway in SV-40 immortalised human chondrocyte cell lines

The mechanotransduction pathway in chondrocytes is facilitated by a αSßI mechanoreceptor at the cell surface and involves tyrosine phosphorylation of paxillin, focal adhesion kinase and ß catenin. The availability of ß-catenin at the cell membrane, in the cytoplasm and in the nucleus plays a key part in the process of mesenchymal condensation during chondrogenesis, which is regulated by Wnt signalling and interaction with other signalling pathways. GSK3ß (glycogen synthase kinase 3ß), is a key mediator in the Wnt pathway and is involved in regulating ß catenin cytoplasmic and nuclear distribution. Wnt binds to its receptor Frizzled (Fz) and subsequent canonical signalling leads to inhibition of GSK3ß, and cytoplasmic accumulation of ß catenin. This, in turn, promotes ß catenin binding to LEF/TCF transcription factors and induction of target gene expression. In the absence of a Wnt signal, GSK3p, as a part of an axin and APC (adenomatous polyposis coli) complex, phosphorylates ß catenin and induces its degradation via the ubiquitin/ proteosome pathway.This thesis has set out to investigate whether Wnt pathway components are expressed in human chondrocyte cell lines and to explore whether the Wnt pathway plays any role in mechanotransduction pathway in chondrocytes.Using RT-PCR, cloning, immunofluorescence and confocal microscopy it was for the first time demonstrated that the Wnt signalling components,Wnt-l, Fz-2, Fzrp and ß catenin were expressed in human chondrocyte cell lines. Using confocal microscopy, 7 fibronectin and CD44 were identified in association with chondrocyte Wnt-Fz complexes, suggesting that they may be coreceptors necessary for transducing Wnt signals intracellularly. A kinase assay demonstrated that GSK3ß activity is increased following 40 minute ofmechanical stimulation and that a phosphoinositol-3 OHkinase (PI3K) inhibitor decreased the activity ofthis kinase. A Wnt agonist, Lithium, on the other hand, increased the GSK3ß activity following 20 and 60 minute of mechanical stimulation. Western blotting suggested that in this study the formation of GSK3ß/ß catenin complexes were induced in the presence of Lithium and mechanical stimulation delayed this process. However, this evidence of the complex formation ofGSK3ß with ß catenin in Western blots was not supported by preliminary analysis of densitometric data and further investigation is required to confirm these findings.The results indicate that Wnt signalling components are expressed in chondrocyte cell lines and may be involved in mechanical signalling in these cells. However, the induction ofGSK3ß activity following mechanical stimulation was mediated by a PI3K dependent pathway rather than a Wnt pathway. This, in turn, may influence the stabilisation of GSK3ß/ßcatenin complex following recruitment of activated protein kinase B (PKB)by PI3K and phosphorylation of GSK3ß. These, in turn, control the cytoplasmic/nuclear distribution of ß-catenin which affects the regulation of downstream target genes such as CD44, fibronectin and some metalloproteinases. CD44 and fibronectin are essential components of cartilage, which are involved in matrix assembly and the maintenance of cartilage integrity

Vitamin B12 modulates Parkinson’s disease LRRK2 kinase activity through allosteric regulation and confers neuroprotection

Missense mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) cause the majority of familial and some sporadic forms of Parkinson’s disease (PD). The hyperactivity of LRRK2 kinase induced by the pathogenic mutations underlies neurotoxicity, promoting the development of LRRK2 kinase inhibitors as therapeutics. Many potent and specific small molecule LRRK2 inhibitors have been reported with promise. However, nearly all inhibitors are ATP competitive – some with unwanted side effects and unclear clinical outcome - alternative types of LRRK2 inhibitors are lacking. Herein we find 5’-deoxyadenosylcobalamin (AdoCbl), a physiological form of the essential micronutrient vitamin B12 as a mixed-type allosteric inhibitor of LRRK2 kinase activity. Multiple assays show that AdoCbl directly binds LRRK2, leading to the alterations of protein conformation and ATP binding in LRRK2. STD-NMR analysis of a LRRK2 homologous kinase reveals the contact sites in AdoCbl that interface with the kinase domain. Furthermore, we provide evidence that AdoCbl modulates LRRK2 activity through disruption of LRRK2 dimerization. Treatment with AdoCbl inhibits LRRK2 kinase activity in cultured cells and brain tissue, and importantly prevents neurotoxicity in primary rodent cultures as well as in transgenic C. elegans and D. melanogaster expressing LRRK2 disease variants. Finally, AdoCbl alleviates deficits in dopamine release sustainability caused by LRRK2 disease variants in mouse models. Our study uncovers vitamin B12 as a novel class of LRRK2 kinase modulator with a distinct mechanism, which can be harnessed to develop new LRRK2-based PD therapeutics in the futur

Abnormal visual gain control in a Parkinson's disease model

Our understanding of Parkinson's disease (PD) has been revolutionized by the discovery of disease-causing genetic mutations. The most common of these is the G2019S mutation in the LRRK2 kinase gene, which leads to increased kinase activity. However, the link between increased kinase activity and PD is unclear. Previously, we showed that dopaminergic expression of the human LRRK2-G2019S transgene in flies led to an activity-dependent loss of vision in older animals and we hypothesized that this may have been preceded by a failure to regulate neuronal activity correctly in younger animals. To test this hypothesis, we used a sensitive measure of visual function based on frequency-tagged steady-state visually evoked potentials. Spectral analysis allowed us to identify signals from multiple levels of the fly visual system and wild-type visual response curves were qualitatively similar to those from human cortex. Dopaminergic expression of hLRRK2-G2019S increased contrast sensitivity throughout the retinal network. To test whether this was due to increased kinase activity, we fed Drosophila with kinase inhibitors targeted at LRRK2. Contrast sensitivity in both day 1 and day 14 flies was normalized by a novel LRRK2 kinase inhibitor 'BMPPB-32'. Biochemical and cellular assays suggested that BMPPB-32 would be a more specific kinase inhibitor than LRRK2-IN-1. We confirmed this in vivo, finding that dLRRK- null flies show large off-target effects with LRRK2-IN-1 but not BMPPB-32. Our data link the increased Kinase activity of the G2019S-LRRK2 mutation to neuronal dysfunction and demonstrate the power of the Drosophila visual system in assaying the neurological effects of genetic diseases and therapies