Dopaminergic Neurodegeneration Induced by Parkinson’s Disease-Linked G2019S LRRK2 is Dependent on Kinase and GTPase Activity

Abstract

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant familial Parkinson's disease (PD) and represent the most common known cause of PD. LRRK2 can function as both a protein kinase and GTPase and PDlinked mutations are known to influence both of these enzymatic activities. While PD-linked LRRK2 mutations can commonly induce neuronal damage and toxicity in cellular models, the mechanisms underlying these pathogenic effects remain uncertain.Rodent models based upon familial LRRK2 mutations often lack the hallmark features of PD and robust neurodegenerative phenotypes in general. Here, we develop a robust pre-clinical model of PD in adult rats induced by the brain delivery of recombinant adenoviral vectors with neuronal-specific expression of full-length human LRRK2 harboring the most common G2019S mutation. In this model, G2019S LRRK2 induces the robust degeneration of substantia nigra dopaminergic neurons, a pathological hallmark of PD. Introduction of a stable kinase-inactive mutation or in-diet dosing with the selective kinase inhibitor, PF-360, attenuates neurodegeneration induced by G2019S LRRK2. Neuroprotection provided by pharmacological kinase inhibition is mediated by an unusual mechanism involving the selective and robust destabilization of human LRRK2 protein in the rat brain relative to endogenous LRRK2. Our study further demonstrates that dopaminergic neurodegeneration induced by G2019S LRRK2 critically requires normal GTPase activity. The introduction of hypothesis-testing mutations that increase GTP hydrolysis or impair GTP binding activity provide neuroprotection against G2019S LRRK2 via distinct mechanisms. Taken together, our data demonstrate that G2019S LRRK2 induces neurodegeneration in vivo via a mechanism that is dependent on kinase and GTPase activity. Our study provides a robust rodent model of LRRK2-linked PD and nominates kinase inhibition and modulation of GTPase activity as promising disease-modifying therapeutic targets.

LRRK2 and Protein Aggregation in Parkinson’s Disease: Insights From Animal Models

“…In this study, phosphorylated tau inclusions were detected in the substantia nigra across all LRRK2 variants, suggesting that the effect is not specific to the G2019S mutation or kinase activity (Nguyen et al, 2019). Concurrently, biochemical analysis determined that tau protein levels, phosphorylation state, and solubility were not altered in response to Ad5-LRRK2 vectors (Nguyen et al, 2019). Additionally, ubiquitin-positive inclusions in the striatum did not appear to be dependent on kinase activity and none of the LRRK2 vectors induced detectable α-synuclein pathology.…”

“…Additionally, ubiquitin-positive inclusions in the striatum did not appear to be dependent on kinase activity and none of the LRRK2 vectors induced detectable α-synuclein pathology. However, in this study, the G2019S LRRK2 vector alone induced APP-positive axonal inclusions and degenerative neuritic changes in the striatum (Nguyen et al, 2019). In a separate study, high-capacity adenoviral vectors (HC-AdV) expressing G2019S or G2019S/D1994N LRRK2 variants from a ubiquitous CAG promoter were delivered to the striatum of…”

“…Unfortunately, the G2019S/D1994N mutation was shown to alter LRRK2 protein stability, partly confounding the validity of this synthetic inactive mutation in determining kinase-dependency. However, an additional study performing intrastriatal injection of Ad5-LRRK2 vectors at a higher viral titer in rats utilized an alternative stable kinaseinactivating mutation, G2019S/K1906M (Nguyen et al, 2019). In this study, phosphorylated tau inclusions were detected in the substantia nigra across all LRRK2 variants, suggesting that the effect is not specific to the G2019S mutation or kinase activity (Nguyen et al, 2019).…”

“…However, an additional study performing intrastriatal injection of Ad5-LRRK2 vectors at a higher viral titer in rats utilized an alternative stable kinaseinactivating mutation, G2019S/K1906M (Nguyen et al, 2019). In this study, phosphorylated tau inclusions were detected in the substantia nigra across all LRRK2 variants, suggesting that the effect is not specific to the G2019S mutation or kinase activity (Nguyen et al, 2019). Concurrently, biochemical analysis determined that tau protein levels, phosphorylation state, and solubility were not altered in response to Ad5-LRRK2 vectors (Nguyen et al, 2019).…”

“…Familial PD caused by LRRK2 mutations (LRRK2-PD) generally develops as a late-onset autosomal dominant disorder and is thought to be driven by a toxic gain-of-function mechanism (Paisán-Ruıíz et al, 2004;Zimprich et al, 2004). A consensus view holds that elevated kinase activity underlies the pathogenic nature of LRRK2, as all causative mutations appear to enhance kinase activity, though GTPase activity seems to be required as well (Sheng et al, 2012;Steger et al, 2016;Nguyen et al, 2019). Despite LRRK2 mutations being one of the most common identifiable causes of PD, the vast majority of PD occurs via an unknown etiology.…”