Boecker, C. A. | Search bioRxiv

bioRxiv2022

Regulatory imbalance between LRRK2 kinase, PPM1H phosphatase, and ARF6 GTPase disrupts the axonal transport of autophagosomes

Gain-of-function mutations in the LRRK2 gene cause Parkinsons disease (PD), increasing phosphorylation of RAB GTPases through hyperactive kinase activity. We found that LRRK2-hyperphosphorylated RABs disrupt the axonal transport of autophagosomes by perturbing the coordinated regulation of cytoplasmic dynein and kinesin motors. In iPSC-derived human neurons, knock-in of the strongly-hyperactive LRRK2-p.R1441H mutation caused striking impairments in autophagosome transport, inducing frequent directional reversals and pauses. Knock-out of the opposing Protein Phosphatase 1H (PPM1H) phenocopied the effect of hyperactive LRRK2. Overexpression of ADP-ribosylation factor 6 (ARF6), a GTPase that acts as a switch for selective activation of dynein or kinesin, attenuated transport defects in both p.R1441H knock-in and PPM1H knock-out neurons. Together, these findings support a model where a regulatory imbalance between LRRK2-hyperphosphorylated RABs and ARF6 induces an unproductive "tug-of-war" between dynein and kinesin, disrupting processive autophagosome transport. This disruption may contribute to PD pathogenesis by impairing the essential homeostatic functions of axonal autophagy.

neuroscience↗

bioRxiv2022

Distinct adaptations revealed by unbiased proteomic analysis of autophagy cargos in the brain in PINK1 and LRRK2 models of Parkinson's disease

Autophagy is essential for neuronal homeostasis, while defects in autophagy are implicated in Parkinson disease (PD), a prevalent and progressive neurodegenerative disorder. We used unbiased proteomics to compare cargos degraded by basal autophagy in the brain from two mouse models of PD, PINK1-/- and LRRK2G2019S mice. We find evidence for the upregulation of adaptive pathways to support homeostasis in both PD models. In PINK1-/- mice, we observed increased expression of the selective receptor BNIP3 along with evidence of engagement of other alternative pathways for mitophagy. Despite these changes, we find the rate of autophagic flux in PINK1-/- neurons is decreased. In LRRK2G2019S mice, hyperactive kinase activity known to impair autophagosomal and lysosomal function results in increased secretion of extracellular vesicles and autophagy cargo. In support of this observation, we find reduced levels of PIKFYVE, a negative regulator of extracellular vesicle secretion, in both brain and cortical neurons from LRRK2G2019S mice. Thus, distinct adaptive pathways are activated to compensate for perturbations induced by either loss of PINK1 or hyperactivation of LRRK2. Our findings highlight the engagement of compensatory pathways to maintain homeostasis in the brain, and provide insights into the vulnerabilities these compensatory changes may introduce that may further contribute to PD progression.

molecular biology↗