NRF2 Activation Restores Disease Related Metabolic Deficiencies in Olfactory Neurosphere-Derived Cells from Patients with Sporadic Parkinson's Disease

Extent: 14p.Background: Without appropriate cellular models the etiology of idiopathic Parkinson’s disease remains unknown. We recently reported a novel patient-derived cellular model generated from biopsies of the olfactory mucosa (termed olfactory neurosphere-derived (hONS) cells) which express functional and genetic differences in a disease-specific manner. Transcriptomic analysis of Patient and Control hONS cells identified the NRF2 transcription factor signalling pathway as the most differentially expressed in Parkinson’s disease. Results: We tested the robustness of our initial findings by including additional cell lines and confirmed that hONS cells from Patients had 20% reductions in reduced glutathione levels and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium, inner salt] metabolism compared to cultures from healthy Control donors. We also confirmed that Patient hONS cells are in a state of oxidative stress due to higher production of H2O2 than Control cultures. siRNA-mediated ablation of NRF2 in Control donor cells decreased both total glutathione content and MTS metabolism to levels detected in cells from Parkinson’s Disease patients. Conversely, and more importantly, we showed that activation of the NRF2 pathway in Parkinson’s disease hONS cultures restored glutathione levels and MTS metabolism to Control levels. Paradoxically, transcriptomic analysis after NRF2 pathway activation revealed an increased number of differentially expressed mRNAs within the NRF2 pathway in L-SUL treated Patient-derived hONS cells compared to L-SUL treated Controls, even though their metabolism was restored to normal. We also identified differential expression of the PI3K/AKT signalling pathway, but only post-treatment. Conclusions: Our results confirmed NRF2 as a potential therapeutic target for Parkinson’s disease and provided the first demonstration that NRF2 function was inducible in Patient-derived cells from donors with uniquely varied genetic backgrounds. However, our results also demonstrated that the response of PD patient-derived cells was not co-ordinated in the same way as in Control cells. This may be an important factor when developing new therapeutics.Anthony L. Cook, Alejandra M. Vitale, Sugandha Ravishankar, Nicholas Matigian, Greg T. Sutherland, Jiangou Shan, Ratneswary Sutharsan, Chris Perry, Peter A. Silburn, George D. Mellick, Murray L. Whitelaw, Christine A. Wells, Alan Mackay-Sim and Stephen A. Woo

A Systems Biology Approach Reveals the Role of a Novel Methyltransferase in Response to Chemical Stress and Lipid Homeostasis

Using small molecule probes to understand gene function is an attractive approach that allows functional characterization of genes that are dispensable in standard laboratory conditions and provides insight into the mode of action of these compounds. Using chemogenomic assays we previously identified yeast Crg1, an uncharacterized SAM-dependent methyltransferase, as a novel interactor of the protein phosphatase inhibitor cantharidin. In this study we used a combinatorial approach that exploits contemporary high-throughput techniques available in Saccharomyces cerevisiae combined with rigorous biological follow-up to characterize the interaction of Crg1 with cantharidin. Biochemical analysis of this enzyme followed by a systematic analysis of the interactome and lipidome of CRG1 mutants revealed that Crg1, a stress-responsive SAM-dependent methyltransferase, methylates cantharidin in vitro. Chemogenomic assays uncovered that lipid-related processes are essential for cantharidin resistance in cells sensitized by deletion of the CRG1 gene. Lipidome-wide analysis of mutants further showed that cantharidin induces alterations in glycerophospholipid and sphingolipid abundance in a Crg1-dependent manner. We propose that Crg1 is a small molecule methyltransferase important for maintaining lipid homeostasis in response to drug perturbation. This approach demonstrates the value of combining chemical genomics with other systems-based methods for characterizing proteins and elucidating previously unknown mechanisms of action of small molecule inhibitors

Kumulacja wybranych metali ciężkich w kości udowej drobnych ssaków lądowych

The accumulation of lead, cadmium, iron, nickel, copper and zinc in the femora of yellow-necked mouse (Apodemus flavicollis) and bank vole (Clethrionomys glareolus) living near the site of coal power station Novaky (Slovakia) was investigated. The content of heavy metals in the bones was assessed by atomic absorption spectrophotometry method. Altogether 20 femora of adult individuals were analysed. Higher concentrations of Cd, Ni, Cu and Zn were detected in the bones of bank vole. Significant differences were observed for the concentrations of Cd, Ni and Zn (p < 0.05). On the contrary, higher concentrations of Pb and Fe were found in the femora of yellow necked mouse. However, the differences were not significant. Our results indicate that Clethrionomys glareolus may be considered as more bone loaded zoomonitor in comparison with Apodemus flavicollis.Zbadano kumulację ołowiu, kadmu, żelaza, niklu, miedzi i cynku w kości udowej myszy leśnej (Apodemus flavicollis) i nomicy rudej (Clethrionomys glareolus) zasiedlających tereny w pobliżu elektrowni Novaky na Słowacji. Zawartość metali ciężkich w kościach zmierzono metodą spektrofotometrii atomowej. Przebadano 20 kości udowych podchodzących od dorosłych osobników. Większe zawartości Cd, Ni, Cu i Zn stwierdzono w kościach nornicy rudej niż w kościach myszy leśnej. Istotne statystycznie różnice między badanymi ssakami dotyczyły zawartości Cd, Ni i Zn (p < 0.05). Z drugiej strony kości myszy leśnej zawierały więcej Pb i Fe niż kości nomicy rudej. Różnice te nie były jednak istotne statystycznie. Uzyskane wyniki wskazują, że kości nornicy rudej kumulują więcej metali ciężkich niż kości myszy leśnej, co może mieć znaczenie dla przyszłych badań monitoringowych