Introduction
The large number of neurodegenerative diseases that are associated with the accumulation of insoluble protein aggregates suggests an important role for dysfunction of proteostasis during aging [
1]. The two predominant autophagic processes are cell mediated autophagy and macro-autophagy [
2,
3]. Increasing evidence suggests that macroautophagy is the predominant process regulating the elimination of the protein aggregates that accumulate in age-related neurodegenerative diseases [
4,
5].
Autophagy proceeds through a process of phagophore initiation, assembly, fusion with the lysosome and degradation [
6,
7]. Initiation proceeds through pathways mediated by Ulk proteins and beclin/VPS34. Membrane elongation involves a series of Atg proteins (Atg 5, 7, 10 and 12), which prime phospholipids to interact with Microtubule-associated protein 1A/1B-light chain 3 (LC3), form the autophagic membrane, identify ubiquitinated species and engulf the target [
7]. The resulting autophagosome then fuses with the lysosome, leading to degradation of the autolysosomal material, including LC3. The appearance of LC3 labeled vesicles is now routinely used to identify autophagy [
3].
Increasing evidence suggests that defects in autophagy contribute to the pathophysiology of Parkinson’s disease. Many of the genes associated with familial Parkinson’s disease are required for autophagy. This includes β-glucocerebroside and ATP13A2 [
8]. Parkin and PINK1 are two proteins linked to autosomal recessive Parkinsonism that appear to regulate mitophagy [
9‐
11]. These proteins interact to recruit LC3 to mitochondria in peripheral cells, although the applicability of this pathway to neurons remains unclear. α-Synuclein is the principle protein that accumulates in sporadic Parkinson’s disease. α-Synuclein has been shown to interact with the cell mediated autophagy pathway through a process that is inhibited by mutant A53T α-synuclein [
12].
Mutations in LRRK2 are common in familial Parkinson’s disease. LRRK2 exhibits pleiotropic functions, perhaps best shown by recent network studies [
13]. Recent evidence raises the possibility that the toxic actions of LRRK2 are mediated by α-synuclein [
14]. Studies using cell culture first indicated that mutations in LRRK2 interfere with autophagy, including cell mediated autophagy [
15‐
17]. Knockout studies proved that endogenous LRRK2 is required for proper autophagic function [
18‐
20]. The knockout studies were notable for demonstrating strong deficits in autophagic function in the kidney, but autophagic deficits were observed in the dopaminergic neurons or elsewhere in the brain [
18‐
20]. The limited neuronal effects of LRRK2 knockout might reflect compensation by LRRK1, which is a close homologue of LRRK2 present in all mammals.
C. elegans provides a potentially important system to examine the actions of LRRK2 because they have only one LRRK2, termed lrk-1. They also lack endogenous α-synuclein, which enables study of LRRK2 function with or without α-synuclein. We have now used C. elegans to investigate how LRRK2 and α-synuclein affect macroautophagy, and whether the two proteins interact to modify macroautophagy over the nematode lifespan. We created lines of C. elegans that express mCherry fused lgg-1, the nematode homolog of LC3, in dopaminergic neurons, and followed the expression of the lgg-1 reporter throughout the lifespan. We now report that autophagy begins to decline after egg-laying in adults is accomplished. Expressing human mutant LRRK2 enhances the age-related decline. Introducing α-synuclein into the system promotes autophagy at a young age but interacts in a synergistic manner with both WT and mutant LRRK2 to decrease autophagy and promote dopaminergic death in an age-dependent manner. Thus, the interaction between α-synuclein and LRRK2 interferes with cellular function predominantly in aging tissues.
Discussion
The current study presents a new tool for studying autophagy in C. elegans, and then uses this tool to evaluate the interactions between LRRK2, α-synuclein, autophagy and aging in dopaminergic neurons. The generation of LC3::mCherry provides a valuable reporter for monitoring autophagic flux. The LC3::mCherry reporter has been used extensively in mammalian systems, and is widely accepted as an accurate reporter of autophagic flux [
28]. In extending the reporter to the nematode, we used lgg-1, which is the nematode homolog of LC3, to insure that it would interact appropriately with the nematode autophagic system. The lgg-1 construct was designed using the dopamine transporter promoter, which drives selective expression in dopaminergic neurons. Restricting expression to the eight DA neurons simplifies the complexity of the visual field, and allows analysis of autophagy in the specific neuronal type that is most affected by the pathophysiology of PD.
A large number of studies indicate that levels of LC3 are inversely proportional to autophagic flux [
3,
7]. The current study used an lgg-1 (nematode LC3 homolog) construct driven by the
dat-
1 promoter. We quantified lgg-1 levels by fluorescence intensity (Figs.
1,
2,
3,
4 and
5), immunoblotting (Fig.
1,
3 and
5) and finally by counting puncta. Quantification of the strength of
dat-1 promoter activity over the lifespan showed a modest effect of the aging process. Fluorescence from the
dat-1::GFP promoter decreased with aging, which was opposite to the increase in fluorescence observed with the
dat-1::lgg-1::mCherry reporter. This indicates that increases in the lgg-1 reporter with aging did not reflect age-related increases in activity of the
dat-1 promoter. The increase in activity of the lgg-1::mCherry reporter with bafilomycin and ATG-5 deletion were also consistent with prior studies in mammalian cells, in which deficits in autophagy increase activity of the LC3::GFP reporter, which suggests that lgg-1::mCherry levels correlate with autophagic flux. The number of lgg-1::mCherry granules also reflects changes in autophagy, increasing with bafilomycin treatment as autophagic flux becomes stalled (Fig.
1a) and reflecting genotype status in nematodes expressing LRRK2 and/or α-synuclein (Fig.
4d). An additional concern was lgg-1 cleavage. One prior study show that lgg-1 can be cleaved at the C-terminus
in vitro, however neither our study nor a prior study observed evidence of significant cleavage
in vivo [
21,
22]. Work from Alberti et al. shows that lgg-1 and lgg-2 exhibit functional overlap with respect to autophagy and complement the autophagic activity of the companion protein [
29]. Finally, we also generated a
dat-
1::lmp-1::GFP lysosomal reporter. The readout from this reporter provides a strong comparison with the
dat-
1::lgg-1::mCherry reporter, and was striking because it exhibited no changes in response to expression of LRRK2 constructs; these results support the hypothesis that the changes in lgg-1::mCherry reporter reflect autophagic flux rather than transcription from the
dat-1 promoter or other factors. Thus, multiple independent lines of evidence support the hypothesis that the lgg-1::mCherry reporter reliably reflects autophagic flux.
We characterized autophagy over the lifespan, and observed progressive age-related inhibition of autophagy once the nematodes had finished their reproductive period. WT LRRK2 increased autophagic flux in young nematodes, while mutant LRRK2 (G2019S and R1441C) inhibited autophagy. We observed that the
dat-1::lgg-1::mCherry reporter was responsive to concomitant expression of LRRK2 constructs, while introducing α-synuclein into C. elegans dopamine neurons increased autophagy in young adult nematodes, and the effect of α-synuclein was dominant over concomitant expression of LRRK2 (WT or mutant). During aging, both mutant LRRK2 and α-synuclein inhibited autophagy and increased dopaminergic degeneration. Although these proteins are beneficial at young ages, competitive actions of LRRK2 and α-synuclein on similar uptake systems might impede removal of α-synuclein aggregates, producing a synergistic inhibition of autophagy, a corresponding accumulation of insoluble, oligomeric α-synuclein and synergistic increases degeneration of DA neurons. In addition, although WT LRRK2 improves autophagy throughout the lifespan when expressed in absence of α-synuclein, co-expressing α-synuclein with WT LRRK2 lead to an age dependent inhibition of autophagy, and a synergistic increase in degeneration of DA neurons. These data suggest that LRRK2 and α-synuclein affect autophagy through interacting pathways that lead to synergistic effects, and supports other studies suggesting they might act through similar pathways [
14].
LRRK2 and α-synuclein are both known to modulate vesicular function [
30‐
36]. Knockout of LRRK2 in the mouse reduces autophagic flux in the mouse kidney [
18]. In addition, a LRRK2 regulatory network that we recently developed indicates that several autophagy linked genes are part of the LRRK2 network, including VPS-34 and HDAC-6 [
37]. Interpretation of WT LRRK2 over-expression studies in mammalian cells is less clear because the effects are frequently modest. However, WT LRRK2 expression in C. elegans produces a striking reduction in lgg-1::mCherry levels, suggesting increased autophagic flux. The stronger effect of LRRK2 in nematodes might reflect the simpler biology of these organisms. Nematodes (and
drosophila) have only one LRRK, lrk-1. LRRK2 might possess a stronger ability to impact on the autophagic system than lrk-1, which would mean that expressing LRRK2 in the nematode induces a strong gain of function.
The actions of α-synuclein and LRRK2 that we observed fit well with the prior studies suggesting that α-synuclein and LRRK2 promote vesicular function, and also fits well with our clinical understanding of the pathophysiology of PD. The increase in autophagy that we observed induced by expressing α-synuclein in young adult nematodes is consistent with other studies showing that α-synuclein promotes vesicular dynamics. Loss of α-synuclein inhibits formation of synaptic vesicles, and reduces dopaminergic function [
30‐
32]; conversely, expressing α-synuclein can compensate for the deleterious of CSPα deletion at the synapse [
38]. Analysis of α-synuclein actions might be particularly striking in C. elegans because it lacks an endogenous homolog of α-synuclein, thus the transgene is introducing a novel function to the nematode.
LRRK2 also exhibits a biology that is appears linked to vesicular dynamics. LRRK2 is associated with vesicles and endosomal uptake [
33,
36]. WT LRRK2 showed a strong ability to increase autophagy throughout the lifespan, which might also reflect activity directed towards vesicular functions, although many pathways regulate autophagy. In contrast, mutant LRRK2 (G2019S and R1441C) inhibits autophagy, which is similar to reports by other groups as well [
15,
17,
25,
39‐
41]. The ability of both G2019S and R1441C LRRK2 to decrease autophagy below that of nematodes lacking even endogenous nematode lrk-1 points to an activity extending beyond a simple loss of function, and suggests active inhibition of autophagy. Competition for similar autolysosomal uptake sites would account for the increase in α-synuclein levels upon co-expression with WT or mutant LRRK2, which provides further support that the two proteins act on mutually interacting pathways. With increasing age, this competition appears to also increase levels of oligomeric α-synuclein, leading to enhanced degeneration of dopaminergic neurons. Thus, LRRK2 and α-synuclein appear to act through intersecting pathways, which would be beneficial at young ages, but deleterious at old ages.
The deleterious mix of LRRK2 and α-synuclein becomes increasingly apparent with aging. When autophagy is examined in C. elegans lines expressing LRRK2 without α-synuclein, lines expressing G2019S LRRK2 exhibit a 75 % increase in lgg-1::mCherry fluorescence by day 12 (indicating low autophagic flux), while lines expressing WT LRRK2 maintains consistently minimal fluorescence (indicating rapid autophagic flux). In contrast, in the presence of α-synuclein, lines expressing G2019S LRRK2 exhibit a 4-fold increase in lgg-1::mCherry fluorescence by day 12, while lines expressing WT LRRK2 show almost a doubling of fluorescence and indicating strongly reduced autophagic flux. G2019S LRRK2 and other genetic factors implicated in PD, might impact on a subtle aspect of the autophagic pathway, rather than interfering with autophagy generally. For instance, β-glucocerebrosidase mutations impact on a pathway that appears to selectively affect the ability of neuron to degrade α-synuclein, while also increasing the tendency of α-synuclein to oligomerize [
42]. The ability of aggregated α-synuclein to inhibit autophagy suggests a synergistic mechanism in which LRRK2 inhibits degradation of α-synuclein, which leads to the accumulation of oligomeric/aggregated α-synuclein, which adds to age-related autophagic inhibition associated with LRRK2 expression.
The age-related interaction between α-synuclein and LRRK2 also translates to enhanced neurodegeneration. Our prior study showed discordant effects of WT and G2019S LRRK2 on age-linked degeneration of dopaminergic neurons, with WT LRRK2 being protective and G2019S LRRK2 being detrimental [
25]. Introducing α-synuclein into the system produces a response that reflects the human condition much better. In the presence of α-synuclein, both WT and G2019S LRRK2 enhance age-linked degeneration of DA neurons. These results show that α-synuclein interacts with both WT and G2019S LRRK2 to cause a synergistic inhibition of autophagy, and an age-linked degeneration of DA neurons. The age-linked degeneration associated with G2019S LRRK2 expression parallels work in mouse, where induced expression of G2019S LRRK2 elicited age-linked degeneration of DA neurons [
43]. However, to the best of our knowledge, our report is the first report showing age-linked degeneration of DA neurons associated with expression of WT LRRK2.
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Competing interests
BW has equity in Aquinnah Pharmaceuticals, Inc. The other authors have no competing interests.
Authors’ contributions
SS performed experiments, analyzed data and edited the manuscript. PEA contributed to the experiments and edited the manuscript. VG and LL contributed to the experiments. OS contributed to the experimental design. BW conceived of and designed the project, analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.