in-vitro models of LRRK2

LRRK2 protein is relatively rare in cells compared with other kinases, and overexpression of the human wild-type protein in cell lines or primary neuronal cultures is well tolerated, with relatively minimal increases in markers of cell
death or other morphologic indicators. In contrast, overexpression of wild-type mixed-lineage kinase 2 protein, the closest kinase to LRRK2 apart from LRRK1, based
on sequence similarity of the kinase domain, results in immediate apoptosis. However, overexpression of LRRK2 protein with the G2019S mutation results in a significant increase in markers of cell death, including neurite shortening
and changes in membrane permeability. The effects appear to track with kinase activity, because mutations that ablate kinase activity also nullify the upregulation
of markers of toxicity associated with LRRK2-G2019S expression.A phenotype derived from WT-LRRK2 protein expression is more subtle, but expression of WT-LRRK2 protein in neurons may leave cells more susceptible to other toxicities, including peroxide exposure. Perhaps a more relevant phenotype for target validation would derive from the knockdown of LRRK2 protein or ablation of LRRK2 kinase activity in concert with exposure to agents commonly used in models of PD, such as overexpression of human a-syn or chemical toxins selective for dopaminergic neurons. However, numerous technical challenges include the lack of commercially available antibodies
with sufficient specificity to detect human and (more so) mouse LRRK2 protein, coupled with incomplete knowledge of the half-life of the endogenous protein in primary cells that constitute in vitro models. The criteria for relevant in vitro models of PD are still hotly contested because the defining end-point requirements such as cell death, protein aggregation, or other biochemical changes in signaling or metabolism are not agreed on. In vitro data that demonstrate kinase-dependent changes in LRRK2 overexpression paradigms are provocative but not necessarily
relevant, unless the endogenous and physiologically important targets for kinase activity are verified in these systems. The fact that a phenotype is observed for PD-associated mutations that link with kinase overactivity does not imply
that the protein is correctly functioning in these cells. Ultimately, in vivo model systems that demonstrate LRRK2-dependent phenotype more universally agreed as important for PD (neurodegeneration and inclusion formation in dopaminergic
neurons within the SNpc in intact animals) may be required before in vitro model systems can be validated as useful tools for validating therapeutic compounds before use in humans.

LRRK2 target validation

The implication of LRRK2 as a potential target for neuroprotection strategies in PD clearly originated from human genetic studies. The LRRK2 protein had not been previously identified as a critical mediator of cell-death pathways or mechanisms important in neuron survival before the identification of mutations causative of PD. Further, most individuals with PD do not harbor known LRRK2 mutations, and
missense mutations are relatively rare in accounting for a small percentage of PD in most populations. Model systems that demonstrate an LRRK2-dependent phenotype are necessary to validate LRRK2 as an appropriate potential therapeutic target and to help address whether LRRK2 would serve as a general target for therapy in PD, because most individuals living with PD presumably do not harbor coding mutations in LRRK2. Thus, the question of whether LRRK2 represents a valid target in PD cases without LRRK2 mutations may not be fully answered until safe and effective modifiers of LRRK2 action are available in the clinic.