Mitochondrial Dysfunction in LRRK2 PD Fibroblasts

Previously described (Smith et al., 2016 (link), Korecka & Thomas et al., 2019) deidentified healthy and LRRK2 G2019S patient fibroblasts were obtained from the Coriell and the NIA Aging Cell biorepositories or the Mayo clinic. Conducting research with patient fibroblasts was not considered to be human subject research as it does not involve intervention or interaction with the individual and the samples do not contain identifiable private information. The average age of the control and the mutant fibroblasts was matched to 66 and 67 years, respectively. Mitochondrial super oxide (MitoSOX^™ Red, ThermoFisher Scientific, M36008) levels, induced by 24-hour treatment with the mitochondrial depolarization agent valinomycin (10μM, SigmaAldrich, V0627), were measured in human fibroblasts obtained from healthy individuals (N=4) and PD patients carrying a LRRK2 G2019S mutation (N=5), in the presence of sepiapterin or SPRi3 (pretreatment for 24-hours before addition of valinomycin), using the live cell imaging system IncuCyte.

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Cronin S.J., Yu W., Hale A., Licht-Mayer S., Crabtree M.J., Korecka J.A., Tretiakov E.O., Sealey-Cardona M., Somlyay M., Onji M., An M., Fox J.D., Turnes B.L., Gomez-Diaz C., da Luz Scheffer D., Cikes D., Nagy V., Weidinger A., Wolf A., Reither H., Chabloz A., Kavirayani A., Rao S., Andrews N., Latremoliere A., Costigan M., Douglas G., Freitas F.C., Pifl C., Walz R., Konrat R., Mahad D.J., Koslov A.V., Latini A., Isacson O., Harkany T., Hallett P.J., Bagby S., Woolf C.J., Channon K.M., Je H.S, & Penninger J.M. (2023). Crucial neuroprotective roles of the metabolite BH4 in dopaminergic neurons. bioRxiv.

Publication Preprint 2023

MitoSOX™ Red valinomycin IncuCyte

Aging cell Cell Fibroblasts Human Lrrk2 Mitochondrial Mutation Oxide Patients Sepiapterin Valinomycin

Corresponding Organization : University of British Columbia

Other organizations : McLean Hospital, Harvard University, Medical University of Vienna, Max Perutz Labs, Vienna Biocenter, Boston Children's Hospital, Universidade Federal de Santa Catarina, Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases, Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Southern Medical University, Nanfang Hospital, Johns Hopkins University, Johns Hopkins Medicine, Governador Celso Ramos Hospital, Polydoro Ernani de São Thiago University Hospital, University of Edinburgh, Karolinska Institutet, University of Bath

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Variable analysis

independent variables

Treatment with the mitochondrial depolarization agent valinomycin (10μM)
Pretreatment with sepiapterin or SPRi3 for 24 hours before addition of valinomycin

dependent variables

Mitochondrial super oxide (MitoSOX™ Red) levels

control variables

Healthy individual fibroblasts (N=4)
LRRK2 G2019S mutant fibroblasts (N=5)
Matched average age of 66 years for control and 67 years for mutant fibroblasts

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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