Résumé
The G2019S substitution in the kinase domain of LRRK2 (LRRK2(G2019s)) is the most prevalent mutation associated with Parkinson's disease (PD). Neurotoxic effects of LARK2(G2019S) are thought to result from an increase in its kinase activity as compared to wild type LRRK2. However, it is unclear whether the kinase domain of LRRK2(G2019s) is sufficient to trigger degeneration or if the full length protein is required. To address this question, we generated constructs corresponding to the C-terminal domain of LRRK2 (Delta LRRK2). A kinase activity that was increased by G2019 -> S substitution could be detected in Delta LRRK2. However biochemical experiments suggested it did not bind or phosphorylate the substrate RAB10, in contrast to full length LRRK2. The overexpression of Delta LRRK2(G2019s) in the rat striatum using lentiviral vectors (LVs) offered a straightforward and simple way to investigate its effects in neurons in vivo. Results from a RT-qPCR array analysis indicated that Delta LRRK2(G2019s) led to significant mRNA expression changes consistent with a kinase-dependent mechanism. We next asked whether Delta LRRK2 could be sufficient to trigger neurodegeneration in the substantia nigra pars compacta (SNc) in adult rats. Six months after infection of the substantia nigra pars compacta (SNc) with LV-Delta LRRK2(WT) or LV-Delta LRRK2(G2019s), the number of DA neurons was unchanged. To examine whether higher levels of Delta LRRK2(G2019s) could trigger degeneration we cloned Delta LRRK2 in AAV2/9 construct. As expected, AAV2/9 injected in the SNc led to neuronal expression of Delta LRRK2(WT) and Delta LRRK2(G2019s) at much higher levels than those obtained with LVs. Six months after injection, unbiased stereology showed that AAV-Delta LRRK2(G2019s) produced a significant 30% loss of neurons positive for tyrosine hydroxylase- and for the vesicular dopamine transporter whereas AAV-Delta LRRK2(WT) did not. These findings show that overexpression of the C-terminal part of LRRK2 containing the mutant kinase domain is sufficient to trigger degeneration of DA neurons, through cell-autonomous mechanisms, possibly independent of RAB10.