The C-terminal domain of LRRK2 with the G2019S mutation is sufficient to produce neurodegeneration of dopaminergic neurons in vivo

Abstract

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 LRRK2^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 (ΔLRRK2). A kinase activity that was increased by G2019➔S substitution could be detected in ΔLRRK2. However biochemical experiments suggested it did not bind or phosphorylate the substrate RAB10, in contrast to full length LRRK2. The overexpression of ΔLRRK2^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 ΔLRRK2^G2019S led to significant mRNA expression changes consistent with a kinase-dependent mechanism. We next asked whether ΔLRRK2 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-ΔLRRK2^WT or LV-ΔLRRK2^G2019S, the number of DA neurons was unchanged. To examine whether higher levels of ΔLRRK2^G2019S could trigger degeneration we cloned ΔLRRK2 in AAV2/9 construct. As expected, AAV2/9 injected in the SNc led to neuronal expression of ΔLRRK2^WT and ΔLRRK2^G2019S at much higher levels than those obtained with LVs. Six months after injection, unbiased stereology showed that AAV-ΔLRRK2^G2019S produced a significant ~30% loss of neurons positive for tyrosine hydroxylase- and for the vesicular dopamine transporter whereas AAV-ΔLRRK2^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.

Keywords: AAVs; Lentiviral vectors; Leucine-rich repeat kinase 2; Parkinson's disease.