A Conserved Role for LRRK2 and Roco Proteins in the Regulation of Mitochondrial Activity

Abstract

Parkinson’s Disease (PD) is the second most common neurodegenerative disease world-wide. Mutations in the multidomain protein Leucine Rich Repeat Kinase 2 (LRRK2) are the most frequent cause of hereditary PD. Furthermore, recent data suggest that independent of mutations, increased kinase activity of LRRK2 plays an essential role in PD pathogenesis. Isolated mitochondria of tissue samples from PD patients carrying LRRK2 mutations display a significant impairment of mitochondrial function. However, due to the complexity of the mitochondrial signaling network, the role of LRRK2 in mitochondrial metabolism is still not well understood. Previously we have shown that D. discoideum Roco4 is a suitable model to study the activation mechanism of LRRK2 in vivo. To get more insight in the LRRK2 pathways regulating mitochondrial activity we used this Roco4 model system in combination with murine RAW macrophages. Here we show that both Dictyostelium roco4 knockout and cells expressing PD-mutants show behavioral and developmental phenotypes that are characteristic for mitochondrial impairment. Mitochondrial activity measured by Seahorse technology revealed that the basal respiration of D. discoideum roco4- cells is significantly increased compared to the WT strain, while the basal and maximal respiration values of cells overexpressing Roco4 are reduced compared to the WT strain. Consistently, LRRK2 KO RAW 264.7 cells exhibit higher maximal mitochondrial respiration activity compared to the LRRK2 parental RAW264.7 cells. Measurement on isolated mitochondria from LRRK2 KO and parental RAW 264.7 cells revealed no difference in activity compared to the parental cells. Furthermore, neither D. discoideum roco4- nor LRRK2 KO RAW 264.7 showed a difference in either the number or the morphology of mitochondria compared to their respective parental strains. This suggests that the observed effects on the mitochondrial respiratory in cells are indirect and that LRRK2/Roco proteins most likely require other cytosolic cofactors to elicit mitochondrial effects.

Dictyostelium discoideum: A Model System for Neurological Disorders

“…The developmental defect was rescued by ectopic overexpression of the wild type Roco4 protein, or its kinase domain only, or a chimeric Roco4/Lrrk2 protein in which the Roco4 kinase domain was replaced with the human LRRK2 kinase domain. However, the mutant Roco4 G1179S protein only partially rescued the developmental defect in the null background [43]. Although the Roco4 G1179S strains had no defect in the number of fruiting bodies, their development was still significantly delayed, and their morphology was aberrant with smaller stalks.…”

“…Roco3/QkgA plays a role in cell proliferation with null mutants growing quicker in shaking culture and strains overexpressing QkgA growing slower [41]. Null mutants of Roco4 and Roco11 display aberrant fruiting body morphology revealing that both proteins are essential to normal multicellular development [43].…”

“…Specific human LRKK2 pathogenic variants are rare and often confined to a few families, however, the most studied mutation, a substitution at residue 2019 (G2019S) in the kinase domain, accounts for ~4% of familial and ~1% of sporadic cases worldwide [49]. This G residue is conserved in D. discoideum Roco4 at residue 1179 and the equivalent mutant form has been created (G1179S) and expressed in the Roco 4 null background [43]. As in human LRRK2, mutation of this residue in Roco 4 results in increased kinase activity in vitro [42].…”

“…Its ablation results in normal aggregation but delayed tip formation and subsequent multicellular development with culmination occurring after 70 h compared to 12 h in the wild type (WT). The Roco4 null mutant also displayed a reduction in the number of fruiting bodies that formed and had a severe defect in the fruiting body morphology with very few stalk cells [43]. The reduction in stalk cells was determined to be due to a lack of cellulose rather than a defect in the stalk differentiation pathway [38].…”

Dictyostelium discoideum as a Model for Investigating Neurodegenerative Diseases

“…Furthermore, PD-related mutations in Roco4 revealed correlating decreases in GTPase activity and increasing kinase activity except for L1180T (LRRK2 I2020T), which shows reduced kinase activity like its LRRK2 counterpart (Jaleel et al, 2007;Ohta et al, 2010;Kortholt et al, 2012). Functionally, deletion of Roco4 or LRRK2 or expression of mutant forms of LRRK2 in Dictyostelium and in human macrophages indirectly leads to mitochondrial dysfunction (Rosenbusch et al, 2021). Therefore, Roco4 is an attractive candidate for investigating the role of LRRK2 in normal physiology and in PD.…”