Combined knockout ofLrrk2andRab29does not result in behavioral abnormalitiesin vivo

Abstract

Coding mutations in the LRRK2 gene, encoding for a large protein kinase, have been shown to cause familial Parkinson's disease (PD). The immediate biological consequence of LRRK2 mutations is to increase kinase activity, leading to the suggestion that inhibition of this enzyme might be useful therapeutically to slow disease progression. Genome-wide association studies have identified the chromosomal loci around LRRK2 and one of its proposed substrates, RAB29, as contributors towards the lifetime risk of sporadic PD. Considering the evidence for interactions between LRRK2 and RAB29 on the genetic and protein levels, here we generated a double knockout mouse model and determined whether there are any consequences on brain function with aging. From a battery of motor and non-motor behavioral tests, we noted only that 18-24 month Rab29 -/and double (Lrrk2 -/-/Rab29 -/-) knockout mice had diminished locomotor behavior in open field compared to wildtype mice. However, no genotype differences were seen in number of substantia nigra pars compacta (SNc) dopamine neurons or in tyrosine hydroxylase levels in the SNc and striatum, which might reflect a PD-like pathology. These results suggest that depletion of both Lrrk2 and Rab29 is tolerated, at least in mice, and support that this pathway might be able to be safely targeted for therapeutics in humans. Significance statement:Genetic variation in LRRK2 that result in elevated kinase activity can cause Parkinson's disease (PD), suggesting LRRK2 inhibition as a therapeutic strategy. RAB29, a substrate of LRRK2, has also been associated with increased PD risk. Evidence exists for an interactive relationship between LRRK2 and RAB29. Mouse models lacking either LRRK2 or RAB29 do not show brain pathologies. We hypothesized that the loss of both targets would result in additive effects across in vivo and post-mortem assessments in aging mice. We found that loss of both LRRK2 and RAB29 did not result in significant behavioral deficits or dopamine neuron loss. This evidence suggests that chronic inhibition of this pathway should be tolerated clinically.

Endogenous Rab29 does not impact basal or stimulated LRRK2 pathway activity

“…As a readout for LRRK2 pathway activity, we measured phosphorylation of Rab10 at Thr73 [ 7 ], employing a well-characterized phospho-specific antibody [ 51 ]. Consistent with previous reports [ 44 ], the Rab29 knock-out mice were viable and displayed no overt phenotypes. We analyzed wildtype, heterozygous and Rab29 knock-out MEFs ( Figure 2A ) as well as wildtype and Rab29 knock-out lung ( Figure 2B ), spleen ( Figure 2C ), kidney ( Figure 2D ) and various brain sections ( Supplementary Figure S1 ) derived from littermate 6-month-old mice treated ± MLi-2 LRRK2 inhibitor (30 mg/kg, 2 h).…”

“…Fox Foundation (see Material and methods). These mice have also been deployed in other studies mentioned above [ 43 , 44 , 46 ]. As a readout for LRRK2 pathway activity, we measured phosphorylation of Rab10 at Thr73 [ 7 ], employing a well-characterized phospho-specific antibody [ 51 ].…”

“…Furthermore, analysis of genetic models reveals that Rab29 and LRRK2 operate co-ordinately to control axon elongation in Caenorhabditis elegans , and lysosomal trafficking and kidney pathology in mice [ 43 ]. A recent study reports that combined knock-out of LRRK2 and Rab29 does not result in a PD-relevant neuronal pathology or behavioral abnormalities [ 44 ]. Rab29 has been implicated in maintaining Golgi morphology and in mediating the retrograde trafficking of the mannose-6-phosphate receptor (M6PR), which recognizes and delivers lysosomal enzymes from the trans Golgi to late endosomes and lysosomes [ 45 , 46 ].…”

Glucocerebrosidase 1 and leucine‐rich repeat kinase 2 in Parkinson disease and interplay between the two genes

“…Over‐expression of RAB29 supresses lysosomal abnormalities in G2019S mutant neurons. However, knockout of both LRRK2 and RAB29 in mice does not result in behavioural changes (Mazza et al, 2020), and neither knockout nor over‐expression of RAB29 affects basal phosphorylation of RAB10 and RAB12 by LRRK2 kinase in vivo (Kalogeropulou et al, 2020). This suggests that RAB29 is not a mediator of basal or pathogenic LRRK2 phosphorylation activity.…”

Endogenous Rab38 regulates LRRK2’s membrane recruitment and substrate Rab phosphorylation in melanocytes

“…17 Critically, almost all studies thus far demonstrate that knockdown/knockout of endogenous Rab29 in multiple cell lines does not decrease LRRK2 kinase function and does not augment Parkinsonian phenotypes in animals, calling into question the physiologic role of Rab29 in LRRK2 activation. 18,19 An important exception is a study of Rab29 in the mouse macrophage cell line Raw264.7, in which Rab29 knockdown decreases LRRK2 activation following lysosomotropic stress, suggesting that Rab regulation of LRRK2 may be cell-type and context dependent. 20,21 Rab32 and Rab38 are highly homologous to Rab29 (56% and 52% identity to Rab29, respectively).…”