LRRK2G2019S acts as a dominant interfering mutant in the context of iron overload

A key pathological feature of Parkinson’s Disease (PD) is the loss of neuromelanin, an iron chelator within the dopaminergic neurons, which results in iron toxicity thought to result in selective neuronal vulnerability. This implicated iron handling pathways as an early target of research in PD. Given the critical role of PD-related activating mutations in LRRK2 (leucine-rich repeat protein kinase 2) within membrane trafficking pathways we examined the impact of mutant LRRK2G2019S on iron homeostasis within a model macrophage cell line known to have high iron capacity. Proteomics analysis revealed a dysregulation of iron-related proteins in steady state with highly elevated levels of ferritin light chain and a reduction of ferritin heavy chain. LRRK2 mutant cells showed efficient ferritinophagy upon iron chelation, but upon iron overload there was a near complete block in the degradation of the ferritinophagy adaptor NCOA4. Surprisingly, NCOA4 levels were not rescued upon inhibition of the LRRK2 kinase activity in iron overload conditions, nor was the phosphorylation of substrate Rab GTPases. We therefore generated a CRISPR mutation to delete the kinase domain of LRRK2 and express only the Rab-binding armadillo repeat domain. Although the kinase domain was deleted, the truncation mutant of LRRK2 showed strong Rab8 phosphorylation in conditions of iron overload, similar to LRRK2G2019S cells, with the phosphorylated Rab8 accumulating at the plasma membrane. These data indicate that the G2019S mutation acts as a kinase independent, dominant-interfering mutant specifically in conditions of iron overload. Together, our data implicate LRRK2 as a key regulator of iron homeostasis and point to the need for an increased focus on the mechanisms of iron dysregulation in PD.