Objective: To determine the pathogenic mechanisms underlying RAB39B-mediated Parkinson’s disease (PD).

Background: PD is a neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra and presence of α-synuclein (αSN) aggregates. We previously demonstrated loss of function mutations in Ras Analog in Brain 39B (RAB39B) cause early onset PD, with dysregulation of αSN observed in vitro and in vivo [1]. However, the normal cellular role(s) of RAB39B, and how loss of function causes neurological disease, are currently unknown.

Methods: We utilised CRISPR/CAS9 genome editing to generate isogenic pluripotent stem cell (PSC) lines with deletion/knockout of RAB39B, in addition to PSC from RAB39B deletion patient fibroblasts. We also utilised CRISPR/CAS9 to generate Rab39b knockout (KO) C57BL/6 mice and investigated the temporal and spatial distribution of Rab39b mRNA and protein in the mouse brain by real-time PCR, in situ hybridisation and western blot analysis of micro-dissected brain.

Results: We successfully differentiated cortical and dopaminergic neurons from the isogenic and patient stem cell lines. Western blot analysis demonstrated a significant increase in steady-state levels of αSN in the mature RAB39B knockout neurons compared to controls. By two weeks of age steady-state levels of Rab39b were high in the hippocampus, cerebral cortex and substantia nigra. Rab39b-KO mice displayed sustained hind-limb clasping and behavioural analysis at three time points (11, 18 and 22 months) identified motor deficits. On the balance beam, knockout mice performed significantly worse than littermate controls, slipping more than five times as often at 22 months (Wildtype 2.3±1.0, n=6; Knockout 12.8±3.3, n=6, p=0.006). Similarly, there was a significant deficit recorded by accelerating rotarod at 22 months (Wildtype 169.2±17.6, n=6; Knockout 83.2±21.4, n=6, p=0.010).

Conclusions: We have generated unique models that recapitulate aspects of the human disease; these will be useful tools to determine the neuropathological mechanisms underlying RAB39B-mediated PD.

References: Mutations in RAB39B cause X-linked early onset Parkinson disease with α-synuclein pathology. Wilson GR, Sim JC, McLean C, Giannandrea M, Galea CA, Riseley JR, Stephenson SE, Fitzpatrick E, Haas SA, Pope K, Hogan KJ, Gregg RG, Bromhead CJ, Wargowski DS, Lawrence CH, James PA, Churchyard A, Gao Y, Phelan DG, Gillies G, Salce N, Stanford L, Marsh AP, Mignogna ML, Hayflick SJ, Leventer RJ, Delatycki MB, Mellick GD, Kalscheuer VM, D’Adamo P, Bahlo M, Amor DJ, Lockhart PJ. Am J Hum Genet 2014, 95(6):729-35.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/cell-and-animal-models-lacking-rab39b-show-biochemical-and-behavioural-phenotypes-that-model-aspects-of-parkinsons-disease/