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

Abstract

Gain-of-function mutations in the LRRK2 gene cause Parkinson's 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.

Axonal transport of autophagosomes is regulated by dynein activators JIP3/JIP4 and ARF/RAB GTPases

“…Interestingly, overexpression of GTP-locked ARF6 is sufficient to ameliorate the AV motility phenotype observed in hyperactive LRRK2 mutant conditions (Dou et al, 2022). Under increased LRRK2 activity, JIP3/4 seem to exhibit enhanced interaction with kinesin and phosphorylated RABs (Dou et al, 2022; Boecker et al, 2021; Kluss et al, 2022; Bonet-Ponce et al, 2020).…”

“…Interestingly, overexpression of GTP-locked ARF6 is sufficient to ameliorate the AV motility phenotype observed in hyperactive LRRK2 mutant conditions (Dou et al, 2022). Under increased LRRK2 activity, JIP3/4 seem to exhibit enhanced interaction with kinesin and phosphorylated RABs (Dou et al, 2022; Boecker et al, 2021; Kluss et al, 2022; Bonet-Ponce et al, 2020). In these conditions, GTP-locked ARF6 expression presumably scaffolds the formation of more JIP3/4-dynein-dynactin complexes to compete with the RAB-JIP3/4-kinesin-1 complexes (Dou et al, 2022).…”

“…Under increased LRRK2 activity, JIP3/4 seem to exhibit enhanced interaction with kinesin and phosphorylated RABs (Dou et al, 2022; Boecker et al, 2021; Kluss et al, 2022; Bonet-Ponce et al, 2020). In these conditions, GTP-locked ARF6 expression presumably scaffolds the formation of more JIP3/4-dynein-dynactin complexes to compete with the RAB-JIP3/4-kinesin-1 complexes (Dou et al, 2022). ARF6 and phospho-RAB10 (or RAB35) may compete to bind the same limited pool of JIP3/4, or excess JIP3/4 may be available for recruitment into multiple motor complexes concurrently (Bonet-Ponce et al, 2020; Miyamoto et al, 2014; Kobayashi and Fukuda, 2012).…”

“…The opposing motor kinesin-1 also localizes to axonal AVs where it may compete with dynein (Wong and Holzbaur, 2014; Maday et al, 2012). Kinesin inactivation on AVs is essential for autophagic transport and flux, and its dysregulation can be observed in the context of neurodegenerative disease (Fu et al, 2014; Boecker et al, 2021; Dou et al, 2022).…”

Proteomics analysis of autophagy cargos reveals distinct adaptations in PINK1 and LRRK2 models of Parkinson disease

“…Hyperactive LRRK2 leads to increased phosphorylation of RAB proteins, direct LRRK2 kinase targets [22,23] and key regulators of vesicle trafficking pathways [24]. Elevated LRRK2 activity in neurons is sufficient to disrupt both the transport and the maturation of autophagic vesicles in the axon, leading to a block in autophagic degradation [25,26]. Rare familial mutations in the machinery required for the selective autophagic degradation of damaged mitochondria (mitophagy), including mutations in PINK1, cause more acute forms of PD with earlier age of onset and higher penetrance than the LRRK2 mutations.…”