LRRK2 KO models: clues for a physiological role of LRRK2 in autophagy and lysosomal function
Important evidence for a physiological role of LRRK2 in regulating autophagy came from knockout animals. Specifically, there is an accumulation of lipofuscin granules, aggregated α-synuclein and increased levels of the autophagosomal marker LC3-II in LRRK2 knockout kidneys [
52]. These effects are age-dependent, in that there are bi-phasic alterations in autophagy, with an initial increase of p62 and LC3-II at 7 months and a decrease at 20 months. No changes in LC3-II were observed in an independent study of kidneys of 14 month-old LRRK2 KO mice [
53].
However, no apparent signs of neurodegeneration have been observed in LRRK2 KO rodents. The 6-fold higher expression levels of LRRK2 in kidney compared to brain and the absence of its homologue LRRK1 may explain this severe kidney phenotype [
52,
54]. Knockout of dLrrk, the single
Drosophila homologue of LRRK1/2, has been shown to cause alterations in lysosomal positioning [
55]. Along the same lines, knockout of the single
C elegans homologue, Lrk-1 m causes defects in synaptic vesicle protein positioning in neurons [
56].
Several studies in cells have indicated a role for LRRK2 in the regulation of autophagy. Under conditions that stimulate autophagy but prevent fusion to lysosomes, knockdown of LRRK2 led to a decreased accumulation of autophagosomes [
57]. LRRK2 kinase inhibition has also been shown to increase levels of the lipdated autophagosome marker LC3-II and the adaptor protein p62 [
58,
59]. Recent findings have shown that this kinase-dependent regulation of LC3 lipidation is mediated through Beclin-1 signaling but independent of mTOR/ULK1 signaling, suggesting non-canonincal regulation of autophagy [
60].
There is a potential discrepancy between LC3-II levels, which generally increase with LRRK2 knockout or kinase inhibition [
58,
59], and accumulation of autophagosomes, which decrease under similar conditions [
57]. Iit is important to note that at steady state these two measures can be difficult to interpret in terms of overall flux through the autophagy pathway. For example, both induction of autophagy and inhibition of autophagosome clearance results in the accumulation of lipidated LC3-II. In H-4 cells, a combined treatment with a LRRK2 kinase inhibitor and bafilomycin, to block lysosomal acidification, results in an additive increase in LC3-II [
58]. This suggests that LRRK2 inhibition does not block flux through the overall autophagy pathway but rather increases formation of autophagosomes. By extension, these considerations suggest that LRRK2 normally functions to block autophagosome formation.
However, even these data are complicated by the observation that, in microglial cells, knockdown of LRRK2 can decrease LC3-II formation after lysosomal inhibition [
57], in contrast to increases in mice [
52] and H4 cells [
58,
59]. It is possible therefore that there are cell-type specific signaling events that can modulate the direction of effect of LRRK2 on autophagy markers, indicating that autophagy regulation may be a downstream consequence of LRRK2 deficiency rather than a primary event.
In addition, higher levels of lysosomal markers and the lysosomal protease cathepsin D are seen in LRRK2 knockout mouse kidneys compared to their wild type counterparts irrespective of age [
54]. Similar phenotypic changes, including lipofuscin accumulation and increase in lysosomal markers have been observed in LRRK2 KO rats [
61,
62]. Therefore, while influencing autophagosome formation, LRRK2 may also play a role in lysosomal maturation and/or trafficking. How these two events are related is not immediately clear and, given then age-dependence of some changes [
52,
54], it remains possible that alterations in one part of the autophagy-lysosome system are compensated for by alterations in other degradative processes.
Pathogenic mutations in LRRK2 KO affect vesicular events in vitro and in vivo
The above data show that the normal function of LRRK2 appears to be related to vesicular trafficking. Several observations in different systems further suggest that LRRK2 mutations across multiple domains of the protein also alter vesicular dynamics.
Fibroblasts derived from PD patients carrying mutations across several enzymatic domains of LRRK2 (G2019S, Y1669C, R1441C) show a diminished autophagic response to starvation, measured by LC-3 conversion, compared to control fibroblasts [
63]. Cells overexpressing R1441C LRRK2 show an increase in MVBs and autophagic vacuoles [
51]. Overexpression of G2019S in cells also results in an increase in autophagic vacuoles and decreased neuronal process length. Knockdown of the conserved autophagy genes LC3 and Atg7 as well as inhibition of ERK signaling reversed this effect [
64]. Overexpression of wild type LRRK2 in cells has also been reported to result in an increase of autophagosomes [
65].
iPSC derived dopaminergic neurons from G2019S mutation carriers show an increase of autophagic vacuoles and an accumulation of aggregated α-synuclein [
66,
67]. In these cells, there were no changes in
SNCA transcription, suggesting an impaired degradation of α-synuclein [
67]. G2019S LRRK2 iPSC showed a decrease in neurite length compared to control iPSC and induction of autophagy further exacerbated this phenotype [
66]. An independent study of G2019S iPSC derived dopaminergic neurons and isogenic controls also showed neurite shortening in an ERK-dependent way [
67]. Notably, G2019S LRRK2-mediated effects on autophagy in cells have also been reported to be mediated through ERK signaling [
68]. Finally, in vivo, mice carrying the G2019S mutation show an accumulation of autophagic vacuoles in the cerebral cortex, as do R1441C LRRK2 transgenic mice [
69].
The collective data available therefore suggests that mutant forms of LRRK2 decrease LC3 lipidation and result in the accumulation of autophagic vacuoules. The observations with LC3 are consistent with the data from knockout and inhibition models that LRRK2 normal function is to block autophagosome formation and that dominant mutations enhance this activity. However, the subsequent accumulation of autophagic vesicles suggests that there are additional effects of mutations in LRRK2 on the overall function of the autophagy-lysosomal pathway. One possible explanation for this apparent discrepancy comes from the observec concurrent increase in autophagic vacuoles and accumulation of α-synuclein in cells with G2019S LRRK2 [
66,
67]. Because α-synuclein is degraded by the lysosome [
70], the available data could suggest that G2019S mutant of LRRK2 simultaneously block autophagosome formation and lysosomal function, which contrasts perhaps with the accumulation of lysosomal enzymes in LRRK2 knockout animals [
52,
54].
Candidate mechanisms for LRRK2 effects on vesicular trafficking
There are several potential mechanisms by which LRRK2 may affect vesicular trafficking. Indirect mechanism, such as those where LRRK2 has direct effects on metabolic or cellular signaling pathways that then indirectly affect autophagy, may explain some of the observed correlated changes noted above. However, here we will focus on regulation of vesicular trafficking events that are potentially mediated by direct protein-protein interactions. The rationale for this limitation on discussion of mechanisms is that as LRRK2 has multiple protein interaction domains, these are likely important effectors of its function in cells.
Unbiased proteomics approaches have provided important insights into the functional roles of LRRK2. Rab5 was first found to interact with LRRK2 using a yeast-two-hybrid screening approach [
71]. Conversely, LRRK2 was identified as an interaction partner in a yeast-two-hybrid screen for Rab32 [
72]. High-throughput protein-protein interaction arrays have shown that LRRK2 physically interacts with Rab7L1 (also known as Rab29) [
73]. In the latter case, we have found that Rab7L1/Rab29 is important for recruiting LRRK2 to the TGN, along with the clathrin-uncoating protein cyclin-G associated kinase (GAK) and the co-chaperone BAG5. This protein complex may be conserved as similar proteins are important for the recruitment of Lrk-1 to the golgi apparatus in
C elegans [
74], Importantly, Rab7L1 and GAK are nominated to be risk factors for sporadic PD [
14]. Clearance of Golgi-derived vesicles by the LRRK2 complex including Rab7L1 is enhanced by mutations across all enzymatic domains of LRRK2 whereas hypothesis testing LRRK2 mutations, including those that are kinase dead or cannot bind GDP/GTP, were ineffective in TGN vesicle clearance [
73]. This suggests that enzymatic activities of LRRK2 are required to promote TGN clustering and clearance and that pathogenic mutations result in a gain-of-function that enhance this phenotype [
73].
In addition, LRRK2 was shown to interact with a number of other Rab GTPases, including Rab32 and Rab38 [
72]. Recently, phosphoproteomic screens were performed in an effort to identify
bona fide LRRK2 kinase substrates [
75]. Two screens were performed using cells from mice engineered to have either the kinase hyperactive G2019S or kinase inhibitor resistant A2016T LRRK2, in combination with treatment of distinct LRRK2 kinase inhibitors. Overlap of these screens resulted in the identification of a single LRRK2 kinase substrate, Rab10. Further analysis in HEK293FT cells indicated that Rab10 as well as Rab8 and Rab12 are direct physiological LRRK2 substrates [
75].
Although publication of independent confirmation of these findings is still awaited, they suggest that one of the key functions of LRRK2, kinase activity, is important in control of Rabs and, hence vesicular trafficking events. Furthermore, in cells (but not in vitro), mutations in several different regions of LRRK2 consistently result in increased Rab phosphorylation, supporting the contention that different LRRK2 domains work together to produce functional output [
75]. Along the same lines, all pathogenic mutations in LRRK2 increase Rab7L1-dependent retention at the TGN [
73]. However, the precise mechanism(s) by which LRRK2 domains interact in cells remain to be determined.
Collectively, these data place LRRK2 at the scene of the crime for vesicle sorting. A recent computational analysis of the LRRK2 interactome further supports a potential role for LRRK2 in vesicular dynamics such as endocytosis and autophagy [
76]. However, the range of Rabs identified suggests multiple roles for LRRK2 at different intracellular membranes. It is also of interest that LRRK2 has a different set of Rabs that appear to be direct substrates from those that were nominated as stronger binding partners, perhaps suggesting that depending on the Rab, LRRK2 may have different modes of action. Further confirmation of the binding and phosphorylation events are needed before we can be certain of the precise role that LRRK2 plays in Rab biology and vice-versa. Nonetheless, because Rab proteins are important in vesicular dynamics, these results suggest that the mechanism by which LRRK2 affects intracellular membranes is mediated via Rab interactions.
There are several pieces of evidence to suggest that, in different tissues and systems, the physiological interaction with Rabs is important for mediating the effects of mutations in LRRK2 on membrane trafficking. As well as causing changes in autophagy, pathogenic LRRK2 mutations have also been shown to lead to alterations in synaptic vesicle trafficking in neurons. Rab5 has a particularly strong role in synaptic vesicle endocytosis. Overexpression of WT LRRK2 impaired synaptic vesicle endocytosis and this effect was further enhanced by overexpression of G2019S LRRK2, whereas-expression of Rab5 rescued this phenotype [
77].
Further supporting the idea that LRRK2 and Rabs co-operate to modulate vesicular trafficking, Rab7L1 KO mice have the same lysosomal pathology in the kidneys as LRRK2 KO mice and the combined deficiency of both proteins also results in a similar phenotype suggesting a genetic interaction with consistent direction between these two proteins [
78]. Whether this is true for other Rabs that are direct substrates of LRRK2 is not known, and future studies are required to further substantiate the relationship between LRRK2, Rabs and regulation of the autophagy-lysosome system.
Studies in
C. elegans neurons suggest that suggests that the LRRK2 nematode ortholog acts downstream of Rab7L1 ortholog in endo-lysosomal trafficking. Furthermore, cellular work showed that LRRK2 interacts with AP-3 as a downstream effector, essential for trafficking of lysosomal membrane proteins from the Golgi to the lysosomes [
78]. The
Drosophila homolog of LRRK2 (dLrrk) colocalizes with endosomes and lysosomes and interacts late endosomal protein Rab7. dLrrk loss-of-function mutants have abnormalities in the endosome and dLrrk can negatively regulate Rab7-dependent perinuclear localization of lysosome [
55]. In contrast, a mutation in dLrrk corresponding to the G2019S mutation in LRRK2 promotes Rab7-dependent perinuclear positioning of lysosomes [
55]. Accumulation of autophagosomes and presence of enlarged lysosomes and endosomes were also observed in
dLrrk loss-of-function mutants [
79]. This phenotype was rescued by overexpression of Rab9, which promotes recycling of endosomes to the TGN via the retromer, again possibly due to a direct interaction [
79]. As noted above, dLrrk is paralog of LRRK1/LRRK2 [
80] and therefore may interact with a slightly different or broader set of Rabs than LRRK2. Nonetheless, these collective data strongly suggest that the effects of LRRK2 across several species depend on Rab GTPases in different tissues and cells, not just in neurons.
Fibroblasts of PD patients carrying the G2019S mutation showed decreased Rab7 activity. Overexpression of G2019S as well as R1441C LRRK2 cause a decrease of Rab7 activity in cells [
81]. Moreover, expression of mutant LRRK2 caused a delay in early to late endosomal trafficking, as evidenced by a decreased Rab5 to Rab7 transitioning [
81]. A dramatic delay of trafficking out of late endosomes was observed in cells overexpressing G2019S and R1441C LRRK2. These late endosomes showed a marked increase in Rab7-positive tubules [
81].
However, in addition to Rab proteins, LRRK2 may also mechanistically alter membrane dynamics via other important interacting proteins. LRRK2 has been shown to interact and colocalize with Sec16, a key protein involved in ER-Golgi transport [
82]. The R1441C LRRK2 mutation impaired this interaction and mouse primary fibroblasts from R1441C transgenic mice showed impaired ER to Golgi trafficking [
82].
LRRK2 and its
Drosophila homologue dLRRK were shown to phosphorylate the synaptic vesicle endocytosis protein endophilin-A in vitro [
83,
84]. In
Drosophila, increased endophilinA phosphorylation by G2019S dLrrk resulted impaired synaptic endocytosis [
83]. Moreover, dLRRK-dependent phosphorylation of endophilinA was recently shown to stimulate autophagy in at Drosophila synapses, highlighting cross-talk between endosomal and autophagosomal signaling networks [
85].
Collectively, these data show that LRRK2 can interact with multiple vesicle-associated proteins. One of the most important remaining questions for LRRK2 biology is how binding to Rabs or other proteins influences the observed alterations in autophagy and lysosomal markers seen in cells and animal models, or whether other mechanisms are at play. A particular complexity of vesicular trafficking is that events are often inter-related as, for example, multiple Rabs co-operate to influence overall protein and vesicle sorting [
35]. Thus, overall flux through a pathway may depend on interactions between multiple partners some of which may anatogonize each other. Further complicating interpretation, presumably most tissues and cells have compensatory mechanisms that will at least partially recover function in vesicle sorting. It will therefore be important to examine multiple steps of vesicular sorting to see which are consistently and directly affected by LRRK2 deficiency and mutations to determine which events are direct and which are consequential.