Leucine-rich repeat kinase 2 (LRRK2) is a complex kinase and mutations in LRRK2 are perhaps the most common genetic cause of Parkinson's disease (PD). However, the identification of the normal physiological function of LRRK2 remains elusive. Here, we show that LRRK2 protects neurons against apoptosis induced by the Drosophila genes grim, hid and reaper. Genetic dissection reveals that Akt is the critical downstream kinase of LRRK2 that phosphorylates and inhibits FOXO1, and thereby promotes survival. Like human LRRK2, Drosophila lrrk also promotes neuron survival; lrrk loss-of-function mutant displays reduced cell numbers, which can be rescued by LRRK2 expression. Importantly, LRRK2 G2019S and LRRK2 R1441C mutants impair the ability of LRRK2 to activate Akt, and fail to prevent apoptotic death. Ectopic expression of a constitutive active form of Akt hence is sufficient to rescue this functional deficit. These data establish that LRRK2 can protect neurons from apoptotic insult through a survival pathway in which LRRK2 signals to activate Akt, and then inhibits FOXO1. These results might indicate that a LRRK-Akt therapeutic pathway to promote neuron survival and to prevent neurodegeneration in Parkinson's disease.

INTRODUCTION

Cells in the developing brain require trophic factors to survive. However, programmed cell death is also required in order to eliminate unnecessary or damaged neurons. How do the developing neurons decide whether to die or to survive? Viktor Hamburger and Rita Levi-Montalcini described that the survival of developing neurons is vitally dependent on the availability of neurotrophic supports from their innervated targets (1). Neurotrophic factors trigger the activation of critical protein kinase cascades that promote survival and inhibit apoptosis (2). While mature neurons no longer require trophic factors to sustain their survival, activation of pathways that promote survival and inhibit apoptosis might still be indispensable. Thus, disturbances in survival signaling and/or activation of apoptotic pathways in response to stress and toxic proteins might be an underlying disease mechanism of a number of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease (PD) (3).

PD is the second most common neurodegenerative disease characterized by progressive loss of dopaminergic neurons in the substantia nigra and the deposition of Lewy bodies (4). Although familial forms of PD are rare, some monogenic forms of PD resemble a sporadic disease in terms of their clinical and pathological manifestations (5), the associated genes may offer functional and pathogenic insights into the disease mechanism. Several genes associated with PD have been shown to directly or indirectly inhibit apoptosis. For example, PINK1 can interact with Bcl-xL to prevent pro-apoptotic cleavage of Bcl-xL, thereby promoting cell survival, and DJ-1 interacts with Daxx to suppress cell death (6,7). In addition, the E3 ligase Parkin appears to protect neurons by negatively regulating the pro-apoptotic protein Bax, and the anti-apoptotic effect of α-synuclein is associated with inhibition of caspase-3 cleavage (8,9). Furthermore, both PINK1 and Parkin are crucial for mitochondrial quality control by targeting dysfunctional mitochondrial proteins to the lysosome (10). All of these facts raise the possibility that these genes mutated in PD might normally protect neurons by promoting survival and inhibiting apoptosis in mature neurons.

Genetic mutations in the PARK8 gene-encoding LRRK2 are the most common cause of PD (11,12). LRRK2 appears to be a signal transduction protein with kinase domains in the central catalytic core and several motifs, including a Ras of complex (ROC) domain, a C-terminal of ROC (COR) domain, a leucine-rich repeat (LRR) and a WD40 domain (13,14). The downstream effectors of LRRK2, and the molecular and cellular mechanisms that govern its function have not been identified. However, LRRK2 most resembles the mixed-lineage kinases (MLK) which are functionally similar to MAPK kinase kinase (MAPKKK). LRRK2 expression is widespread in various regions of the human brain including the cortex, hippocampus, midbrain, brainstem and dopaminergic neurons in the substantia nigra (15). Compelling evidence suggests that the most common PD-linked LRRK2 mutations are located in the central catalytic core. These include the autosomal-dominant R1441C and G2019S mutations, and their aberrant kinase and GTPase activities appear central to inducing neurotoxicity and neurodegeneration (16). Previous studies using cell lines and model organisms have implicated pathological LRRK2 in the restriction of neurite outgrowth, mitochondria dysfunction, increased protein translation and impairments in endocytosis and autophagy (13). Although LRRK2-associated PD pathogenesis has been demonstrated in a number of models, identification of the physiological role of LRRK2 to corroborate its kinase cascade remains unidentified. Moreover, the important question of how PD-linked LRRK2 mutations affect signaling function and the relevance of these effects to neurodegeneration remains unanswered (13).

Here, we used Drosophila compound eyes to explore the effects of LRRK2 signaling on apoptosis in vivo. Our genetic and biochemical studies demonstrated a role for human LRRK2 in neuroprotection. Expression of wild-type LRRK2 promoted neuronal survival against apoptosis through activation of the downstream effector, Akt by phosphorylation of Ser473. Phosphorylated Akt in turn inhibited FOXO 1 signaling. In contrast, this pro-survival role was inhibited in two PD-linked LRRK2 mutants, R1441C and G2019S. These results define, for the first time, the function and molecular mechanism underlying how LRRK2 expression promotes neuronal survival that may be compromised in LRRK2-linked monogenic PD.

RESULTS

LRRK2 has anti-apoptotic function

Previous studies reported that LRRK2 knockout mice displayed apoptotic cell death in the kidney, and that a null mutant of the fly LRRK2 homolog, lrrk, exhibits dopaminergic degeneration (17,18). These studies suggested that LRRK2 might exert an anti-apoptotic function. To address this question, we studied the Drosophila compound eye, a model commonly used for studying apoptosis (Fig. 1A). By expressing three pro-apoptotic genes, reaper, hid and grim using the eye-specific GMR-GAL4 driver, we found that the normal compound eye size was greatly reduced (Fig. 1B–D). The Drosophila compound eye has highly regular ommatidia. Expression of these pro-apoptotic genes severely disrupted the regular pattern and boundaries of each unit eye (Fig. 1B–D). Western blotting analysis of protein lysates from eyes expressing the wild-type human LRRK2 transgene yielded a ∼280 kDa band (Supplementary Material, Fig. S1). There were no observable defects in these eyes (Fig. 1E). Remarkably, when human LRRK2 was co-expressing with reaper, hid and grim, signs of cell death were markedly suppressed, resembling a normal eye. The suppression of apoptosis was particularly distinctive in the GMR::grim strain, which we used for all subsequent studies (Fig. 1F–H). Interestingly, co-expression of fly lrrk also suppressed cell death induced by grim (Supplementary Material, Fig. S2). To confirm the anti-apoptotic function of lrrk, we examined lrrk null mutant flies (lrrk−/−). The overall organization and structure of lrrk−/− flies appeared normal except reduced body size, including compound eyes. Ommatidial counts revealed 766 ± 20 ommatidia in wild-type eyes, 736 ± 26 ommatidia in lrrk−/+ heterozygous eyes, and only 602 ± 32 ommatidia, ∼20% reduction as compared with wild-type, in lrrk−/− eyes. Interestingly, the reduced compound eye size of lrrk−/− flies could be rescued by expressing human LRRK2 (752 ± 25 ommatidia) (Supplementary Material, Fig. S3A and B). Taken together, these data suggest that the anti-apoptotic function of human LRRK2 is evolutionary conserved.

Figure 1.

Expression of human LRRK2 inhibits cell death in Drosophila. Scanning electron microscope (SEM) micrographs of Drosophila compound eyes. (A) Representative micrograph of the regular organization of ommatidia and bristles in the wild-type Canton S (CS) adult eye. Expression of the apoptosis-inducing genes (B) reaper, (C) hid and (D) grim with the eye-specific glass promoter resulted in a severe phenotype involving a reduced size and roughness of the eye. (E) Wild-type human LRRK2 transgene expression in the eye using the GMR-Gal4 driver (GMR::LRRK2) produced an eye morphology similar to the wild-type control in A. (FH) Co-expression of LRRK2 with any one of the three apoptotic genes partially rescued eye morphology, resulting in relatively larger eyes with more evident ommatidia. In all panels, anterior is to the left and posterior is to the right. Scale bar, 100 μm.

Figure 1.

Expression of human LRRK2 inhibits cell death in Drosophila. Scanning electron microscope (SEM) micrographs of Drosophila compound eyes. (A) Representative micrograph of the regular organization of ommatidia and bristles in the wild-type Canton S (CS) adult eye. Expression of the apoptosis-inducing genes (B) reaper, (C) hid and (D) grim with the eye-specific glass promoter resulted in a severe phenotype involving a reduced size and roughness of the eye. (E) Wild-type human LRRK2 transgene expression in the eye using the GMR-Gal4 driver (GMR::LRRK2) produced an eye morphology similar to the wild-type control in A. (FH) Co-expression of LRRK2 with any one of the three apoptotic genes partially rescued eye morphology, resulting in relatively larger eyes with more evident ommatidia. In all panels, anterior is to the left and posterior is to the right. Scale bar, 100 μm.

The anti-apoptotic function of LRRK2 requires Akt

To investigate whether the observed anti-apoptotic function of human LRRK2 might be mediated through the activation of other kinases, we tested 100 RNAi lines in the Drosophila kinome. Expression of grim alone showed an apoptotic eye phenotype (Fig. 2A). We found that knockdown of fly Akt (AktRNAi) markedly reversed the ability of LRRK2 to suppress the grim-induced eye apoptosis phenotype (Fig. 2B and C). Akt has been reported as a LRRK2 substrate in vitro (19) indicating that the screen was capable of identifying a relevant target. To further validate the downstream role of Akt in the pro-survival function of LRRK2, wild-type Akt was overexpressed in the starter background (co-expression of grim and LRRK2). Indeed, increasing Akt level enhanced the anti-apoptotic effect of human LRRK2 (Fig. 2D), suggesting that these two kinases exert pro-survival effects in the apoptotic eye model. It is important to note that while a number of kinases, including phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinase (ERK; fly rolled or rl), mitogen-activated protein kinase kinase 7 (MKK7; fly hemipterious or hep), and MAPKK (fly Downstream of raf1, Dsor1), that have been proposed to likely interact with LRRK2, knockdown of these kinases did not alter the starter line phenotype (Fig. 2E–H). Our findings indicate that the genetic interaction between LRRK2 and Akt in anti-apoptosis function is specific, thereby implying that LRRK2 signaling mediates cell survival through Akt pathways.

Figure 2.

LRRK2 genetically interacts with Akt to promote cell survival. (A and B) The apoptotic phenotype induced by expressing grim in the eye (A) could be suppressed by the co-expression of LRRK2 (B, starter line). GFP was included in (A) to maintain the same copy number of transgene as in (B). (C and D) In the starter line background, knockdown of Akt through the expression of an RNAi construct inhibited the anti-apoptotic effect of LRRK2 (C). Expression of wild-type Akt enhanced the anti-apoptotic effect of LRRK2 resulting in a normal sized compound eye and mostly regular ommatidia (D). (EH) Expression of the RNAi constructs to knockdown PI3K (E), rl (Drosophila ERK) (F), hep (Drosophila MKK7) (G) and dsor (Drosophila MKK) (H) in the starter line displaced an eye phenotype comparable to the starter line suggesting that unlike Akt-RNAi in (C) these genes do not function downstream of anti-apoptotic LRRK2. Scale bar, 100 μm.

Figure 2.

LRRK2 genetically interacts with Akt to promote cell survival. (A and B) The apoptotic phenotype induced by expressing grim in the eye (A) could be suppressed by the co-expression of LRRK2 (B, starter line). GFP was included in (A) to maintain the same copy number of transgene as in (B). (C and D) In the starter line background, knockdown of Akt through the expression of an RNAi construct inhibited the anti-apoptotic effect of LRRK2 (C). Expression of wild-type Akt enhanced the anti-apoptotic effect of LRRK2 resulting in a normal sized compound eye and mostly regular ommatidia (D). (EH) Expression of the RNAi constructs to knockdown PI3K (E), rl (Drosophila ERK) (F), hep (Drosophila MKK7) (G) and dsor (Drosophila MKK) (H) in the starter line displaced an eye phenotype comparable to the starter line suggesting that unlike Akt-RNAi in (C) these genes do not function downstream of anti-apoptotic LRRK2. Scale bar, 100 μm.

LRRK2 selectively increases phosphorylated Akt to inhibit FOXO but not ERK

The aforementioned genetic results suggest that Akt, a well characterized intracellular signaling pathway that promotes cell survival (20), is the downstream target of LRRK2. Therefore, we assessed whether human LRRK2 expression is sufficient to activate the fly Akt signaling pathway. Western blot analysis showed that LRRK2 expression in fly eyes led to a robust increase in p-Akt Ser473 as compared with wild-type, GMR::GFP or UAS-LRRK2 controls (Fig. 3A and B). Drosophila genome contains a single Akt gene which encodes two splice forms with two distinct length of 513 and 611 amino acids, and both isoforms can be recognized by p-Akt Ser 473 antibody as previously suggested (21,22). To confirm this, we treated protein extracts with phosphatase; while both bands could still be detected by Akt antibody, none of them could be detected by phospho-Akt Ser473 antibody after dephosphorylation, thereby suggesting that both bands represent phosphorylated Akt Ser473 (Supplementary Material, Fig. S4). Under ectopically expressed conditions, LRRK2 enhanced the phosphorylation of the corresponding Ser residues of both isoforms (Fig. 3A and B); and it had a stronger impact on the high molecular weight isoform (a 9-fold increase) compared with control. Consistent with the genetic result that LRRK2/lrrk targets on Akt, we observed that lacking lrrk in lrrk−/− flies led to a decrease in p-Akt Ser473 as compared with wild-type and heterozygous lrrk−/+ flies (Fig. 3C). In contrast, LRRK2 may specifically act on this Ser residue of Akt because its expression did not affect the levels of p-Akt Thr308 and the total Akt that were comparable across genotypes (Fig. 3A and B). It is also noteworthy that while PI3K is a canonical upstream kinase regulating Akt activity, PI3K levels did not significantly change upon expressing human LRRK2 as compared with control (Fig. 3A and B), suggesting that LRRK2-regulated Akt activation is unlikely to be mediated through PI3K. Together, these data strengthen the notion that LRRK2 activates downstream Akt via specific phosphorylation of a conserved Ser473 residue.

Figure 3.

Expression of human LRRK2 specifically promotes Akt phosphorylation at Ser473 leading to phosphorylation of downstream targets FOXO1 and GSK-3β. (A) Protein lysates from the heads of wild-type (CS), controls (GMR::GFP, UAS-LRRK2) and LRRK2 expressing (GMR::LRRK2) flies were analyzed by western blotting using antibodies against Akt, Akt-pSer308, Akt-pSer473, PI3K, ERK and ERK-pThr202/Tyr204. α-tubulin was used as a loading control. (B) Quantification of PI3K, Akt pAkt 308, pAkt 473, ERK and pERK protein levels according genotype. Fold change in protein levels is shown relative to wild-type protein levels (***P < 0.0001, n = 4). Both bands to represent pAkt 473. (C) The relative levels of Akt-pSer473 and total Akt were analyzed by western blotting using protein lysates from the fly heads of wild-type (w1118), lrrk null (lrrk−/−), lrrk heterozygous (lrrk−/+) and human LRRK2 expression (GMR::LRRK2) flies. α-tubulin served as a loading control. (D) Immunoblotting of Akt downstream targets detected FOXO1, FOXO1-pSer256, GSK-3β and GSK-3β-pSer9 protein levels in the indicated protein lysates.

Figure 3.

Expression of human LRRK2 specifically promotes Akt phosphorylation at Ser473 leading to phosphorylation of downstream targets FOXO1 and GSK-3β. (A) Protein lysates from the heads of wild-type (CS), controls (GMR::GFP, UAS-LRRK2) and LRRK2 expressing (GMR::LRRK2) flies were analyzed by western blotting using antibodies against Akt, Akt-pSer308, Akt-pSer473, PI3K, ERK and ERK-pThr202/Tyr204. α-tubulin was used as a loading control. (B) Quantification of PI3K, Akt pAkt 308, pAkt 473, ERK and pERK protein levels according genotype. Fold change in protein levels is shown relative to wild-type protein levels (***P < 0.0001, n = 4). Both bands to represent pAkt 473. (C) The relative levels of Akt-pSer473 and total Akt were analyzed by western blotting using protein lysates from the fly heads of wild-type (w1118), lrrk null (lrrk−/−), lrrk heterozygous (lrrk−/+) and human LRRK2 expression (GMR::LRRK2) flies. α-tubulin served as a loading control. (D) Immunoblotting of Akt downstream targets detected FOXO1, FOXO1-pSer256, GSK-3β and GSK-3β-pSer9 protein levels in the indicated protein lysates.

The ERK (MAPK) signaling pathway is also regulated by PI3K and could promote cell survival (23). We further explored whether human LRRK2 might also affect this kinase activity through phosphorylation. We found LRRK2 expression did not significantly affect the level of total ERK protein or p-ERK as compared with control (Fig. 3A and B). It is noteworthy that the effect of human LRRK2 on fly Akt is not an eye-specific event, because expression of LRRK2 in fly dopaminergic neurons also yielded similar results (Supplementary Material, Fig. S5).

Activation of Akt signaling could promote cell survival, growth and metabolism through the regulation of a variety of downstream molecules (24). Given the anti-apoptotic role of Akt, we speculated that it might negatively regulate the Forkhead family member FOXO 1 through phosphorylation, which would block FOXO 1-induced pro-apoptotic changes in gene expression. To test this, we compared the level of FOXO 1 and phosphorylated FOXO 1 Ser256 protein levels in the human LRRK2 overexpressing fly. As shown in Figure 3D, the level of phosphorylated FOXO 1 was substantially increased by LRRK2 expression compared with control. Notably, FOXO 1 protein level was reduced in this condition. Moreover, Akt can inactivate glycogene synthase kinase 3β (GSK-3β) through direct phosphorylation which might in turn inhibit the facilitation of apoptosis (25). GSK-3β activation has been implicated in a number of neurodegenerative diseases that may contribute to tauopathy (26,27). We tested whether LRRK2 expression might negatively regulate GSK-3β by phosphorylation Similar to the effect of LRRK2 on FOXO1, LRRK2 expression produced a robust increase in GSK-3β Ser9 phosphorylation.

To identify whether FOXO1 is the downstream effector of LRRK2 that regulates neuronal survival (Fig. 4A), we examined if expression of wild-type FOXO1 or non-phosphorylated FOXO S259A might attenuates the anti-apoptotic function of LRRK2. As expected, co-expression of FOXO1 or FOXO 1S259A, diminished the robust reversed effect of LRRK2 on apoptosis, and resulted in a relative reduced eye compared with the starter line (Fig. 4A and B–C). However, co-expression of GSK-3βS9A produced no change of eye phenotype (Fig. 4D). Therefore, our biochemical and genetic data provide further molecular basis for how LRRK2-Akt survival signal is mediated through FOXO1, a negative downstream regulator of LRRK2-Akt but not through GSK-3β.

Figure 4.

LRRK2 inhibits FOXO1 to promote cell survival. (A and B) The anti-apoptotic effect of LRRK2 in fly eyes co-expressing grim and LRRK2 (A) was dramatically impeded by the expression of dFOXO1 S259S or wild-type dFOXO1, which results in a severely reduced eye phenotype (B and C). GFP was included in (A) to maintain the same copy number of transgene as in (B) and (C). (D) Expression of active form of sggS9A did not interfere with the anti-apoptotic function of LRRK2 on grim-induced apoptosis. Scale bar, 100 μm.

Figure 4.

LRRK2 inhibits FOXO1 to promote cell survival. (A and B) The anti-apoptotic effect of LRRK2 in fly eyes co-expressing grim and LRRK2 (A) was dramatically impeded by the expression of dFOXO1 S259S or wild-type dFOXO1, which results in a severely reduced eye phenotype (B and C). GFP was included in (A) to maintain the same copy number of transgene as in (B) and (C). (D) Expression of active form of sggS9A did not interfere with the anti-apoptotic function of LRRK2 on grim-induced apoptosis. Scale bar, 100 μm.

Loss of LRRK2 anti-apoptotic function in PD-linked LRRK2 mutants coincides with failure to activate Akt

The pathogenic mechanism of monogenic PD-linked mutations in LRRK2 is still not well understood (28). The anti-apoptotic function of wild-type human LRKK2, through activation of Akt signaling, led us to ask whether PD-linked LRRK2 mutations might compromise this signaling pathway. To answer this, we utilized two common PD-associated LRRK2 mutants, G2019S and R1441C. Using an antibody specifically recognizes phosphorylated Akt at Ser473, we found that unlike wild-type LRRK2 which could enhance Akt phosphorylation at Ser473, both LRRK2R1441C and LRRK2G2019S failed to phosphorylate Akt (Fig. 5A). These data implicate a loss of normal anti-apoptotic function in these LRRK2 mutations.

Figure 5.

PD-linked LRRK2 mutants G2019S and R1441C fail to increase Akt phosphorylation and promote cell survival. (A) Western blotting analyses for pAkt Ser473 in head lysates from transgenic flies expressing wild-type LRRK2 (GMR::LRRK2, upper section), R1441C (GMR::LRRK2R1441C, middle section) and G2019S (GMR::LRRK2G2019S, lower section) mutants. Immunoreactivity detected with anti-α-tubulin served as a loading control. (BE) SEM micrographs of Drosophila compound eyes showed a grim-induced apoptotic phenotype (B). Co-expression of wild-type LRRK2 (C), but not LRRK2 mutants R1441C (D) or (E) G2019S, rescued the grim-induced apoptotic eye phenotype. Scale bar, 100 μm.

Figure 5.

PD-linked LRRK2 mutants G2019S and R1441C fail to increase Akt phosphorylation and promote cell survival. (A) Western blotting analyses for pAkt Ser473 in head lysates from transgenic flies expressing wild-type LRRK2 (GMR::LRRK2, upper section), R1441C (GMR::LRRK2R1441C, middle section) and G2019S (GMR::LRRK2G2019S, lower section) mutants. Immunoreactivity detected with anti-α-tubulin served as a loading control. (BE) SEM micrographs of Drosophila compound eyes showed a grim-induced apoptotic phenotype (B). Co-expression of wild-type LRRK2 (C), but not LRRK2 mutants R1441C (D) or (E) G2019S, rescued the grim-induced apoptotic eye phenotype. Scale bar, 100 μm.

Next, to further corroborate our finding that LRRK2R1441C and LRRK2G2019S have indeed partially lost their anti-apoptotic effect, we expressed each individual mutation in the GMR::grim fly. Unlike wild-type LRRK2, which showed robust suppression of the grim-induced eye phenotype, expressing these PD-linked LRRK2 mutants could not significantly suppress grim-induced apoptosis, thereby resulting in the reduced eye size phenotype (Fig. 5B–E). Together, these genetic and biochemical findings suggest that wild-type LRRK2 exerts a pro-survival role through the Akt signaling pathway, which may be compromised in the PD-linked mutations, LRRK2G2019S and LRRK2R1441C.

A constitutive active form of Akt restores the impaired anti-apoptotic function in LRRK2G2019S and LRRK2R1441C mutants

Because the PD-linked LRRK2G2019S and LRRK2R1441C mutants were largely lost their ability to activate Akt survival pathways and failed to alleviate grim-induced apoptosis (Fig. 5), we asked whether this functional loss of anti-apoptosis function might be rescued by co-expression of a constitutive active form of Akt with these LRRK2 mutants. To this end, we co-expressed three transgenes: grim to induce the apoptotic eye phenotype, LRRK2G2019S or LRRK2R1441C, and a constitutive active form of Akt (Drosophila Akt.T342D.S505D that corresponds to mammalian Akt.T308D.S473D). We observed that the compromised pro-survival function of LRRK2R1441C or LRRK2G2019S on grim-induced apoptosis (Fig. 5) could be markedly rescued by co-expression with a constitutive active Akt (Fig. 6). Interestingly, co-expression of constitutive active Akt with LRRK2R1441C or LRRK2G2019S produced a stronger rescue effect than expression of constitutive active Akt alone (Fig. 6). Taken together, these data suggest that human LRRK2 exerts its anti-apoptotic function through activation of the Akt signaling pathway, and that activation of this pathway alone is sufficient to promote neuron survival.

Figure 6.

Constitutive active form of Akt can induce cell survival in pathogenic LRRK2 mutants. SEM micrographs of Drosophila compound eyes show how the apoptotic eye phenotype induced by grim expression (A), could be partially rescued by the co-expression of a constitutive active form of Akt T342D.S505D (B). The PD-linked LRRK2 mutants R1441C and G2019S were unable to rescue the grim-induced apoptotic eye phenotype, but co-expression of the constitutive active Akt was able to compensate for the deficits in the two LRRK2 mutants R1441C and G2019S in promoting cell survival (C and D). Scale bar, 100 μm.

Figure 6.

Constitutive active form of Akt can induce cell survival in pathogenic LRRK2 mutants. SEM micrographs of Drosophila compound eyes show how the apoptotic eye phenotype induced by grim expression (A), could be partially rescued by the co-expression of a constitutive active form of Akt T342D.S505D (B). The PD-linked LRRK2 mutants R1441C and G2019S were unable to rescue the grim-induced apoptotic eye phenotype, but co-expression of the constitutive active Akt was able to compensate for the deficits in the two LRRK2 mutants R1441C and G2019S in promoting cell survival (C and D). Scale bar, 100 μm.

DISCUSSION

Here, we present the functional characterization of human LRRK2 in a Drosophila genetic model system. Remarkably, we found that LRRK2 can rescue neuronal apoptosis induced by ectopic expression of death genes, grim, hid and reaper in the fly visual system. Furthermore, our genetic screen and biochemical analyses provide novel insight into the role of LRRK2 in promoting neuronal survival, at least in part by activating its downstream Akt effector. We showed that wild-type LRRK2, but not its PD-linked mutants, phosphorylates the Akt at Ser473 thereby inhibiting the pro-apoptotic transcription factor FOXO1 in neurons. Our data reveal for the first time in vivo, an anti-apoptotic role of LRRK2. This survival promoting function of LRRK2 was compromised in autosomal-dominant mutants, R1441C and G2019S, but could be reversed by a constitutive active form of Akt, that further confers that wild-type LRRK2 plays a pro-survival role. Taken together, our data provide novel mechanistic insights into how PD-linked LRRK2 mutations might be detrimental for neurons via the loss-of-function mechanism.

LRRK2 has kinase and GTPase domains; and its kinase activity is thought to be similar to the signaling molecule MAPKKK (13). Our data demonstrate that while LRRK2 plays a role in phosphorylating Akt, a proto-oncogene that promotes neuron survival this LRRK2-Akt pathway is distinct from the canonical signaling cascades regulated by MAPKKK. Indeed, Akt has been shown to suppress apoptotic cell death in a variety of cellular contexts (2931). Our work has revealed that LRRK2 can activate the Akt signaling pathway. These data identify an anti-apoptotic function for LRRK2 and provide a mechanism by which it exerts its protective role. Moreover, this conclusion is in agreement with reports showing that elevated LRRK2 promotes cell survival in papillary renal and thyroid cancers and in stress-induced cell death. Furthermore, LRRK2 inhibition was found to elicit apoptosis in human mid-brain derived neuronal progenitor cells and in rodent kidney cells (17,3234). Our study extends these observations by establishing the pro-survival role of LRRK2 is mediated via Akt activation. Interestingly, LRRK2 is functionally analogous to another monogenic PD-linked gene DJ1 (Park 7), which is an oncogene that exerts a pro-survival role via activating the Ras/MAPK signaling pathway. Therefore, despite different genetic traits, LRRK2 and DJ1 may mediate parallel survival signaling pathways to prevent DA neurons from degeneration (35).

The anti-apoptotic role of the Akt signal pathway has been well established; phosphorylation at Thr308 in the activation loop and Ser473 in the hydrophobic motif are required for the anti-apoptotic activity of Akt (36). Interestingly, we found that LRRK2-mediated Akt phosphorylation is specific to Ser473, but not to Thr308, suggesting that LRRK2 mediates its anti-apoptotic function by regulating Akt activation via Ser473 phosphorylation in the hydrophobic motif only. Similar to our observation of LRRK2, several kinases, such as integrin-linked kinase and the Rictor-mTOR complex, phosphorylate Akt at Ser473 to promote cell survival (37,38). To further characterize how activated Akt mediates cell survival, we identified the downstream targets of Akt which were inactivated upon ectopic LRKK2 expression. Indeed, our observation that LRRK2 inhibited the pro-apoptotic transcription factors including FOXO1 via Akt substantiates the role of this signaling pathway in neuronal protection (24). However, these findings are contradictory to a report that suggested a role for LRRK2 in modifying the pro-apoptotic signal of FOXO 1 (39). However, the previous study was distinct in the fact that of FOXO was overexpressed, whereas in our study grim was overexpressed. This also suggests the possibility that differences in the molecular context across studies might contribute to important discrepancies between results on LRRK2 function. Interestingly, as FOXO1 is one of the major inhibited downstream targets of LRRK2-Akt, co-overexpression of Drosophila FOXO1 with PD-linked LRRK2 mutants would be expected to compromise the survival effect of LRRK2. While some studies support an anti-apoptotic role for LRRK2, additional studies are needed to determine whether this anti-apoptotic function of LRRK2-Akt signaling pathway is also conserved in mammal systems. These functional data suggest that an increase in Akt phosphorylation by LRRK2 may account for the underlying signaling mechanism of LRRK2 that is recruited to actively suppress apoptotic death during aging in response to stress. Furthermore, identifying whether the LRRK2-Akt signaling pathway is triggered by specific upstream activators such as stress and understand how it is regulated in cells are questions that warrant further studies.

Currently, the underlying pathogenesis of PD-linked LRRK2 mutations is not well understood (40). Many studies propose various toxic gain-of-function mechanisms for PD-linked LRRK2 mutations, including aberrant 4E-BP phosphorylation, increased eukaryotic initiation factor (eIF) 4E mediated protein translation (41), promotion of aberrant interactions with microRNAs, dysregulated protein translation (42), damaged protein sorting (43), impaired neurite outgrowth (44), increased tau phosphorylation (45) and transduction of death signals via physical interactions with Fas-associated protein death domains (46). In this study, we found that wild-type LRRK2 has a protective role in neurons. To assess whether this pro-survival function of LRRK2-Akt signaling (Fig. 7A) might be compromised in the context of PD-related mutations, we tested two well described LRRK2 mutants, G2019S and R1441C. Our findings indicated that these mutant forms of LRRK2 fail to phosphorylate Akt Ser473, blunting the pro-survival function that is observed in wild-type LRRK2. Indeed, unlike normal LRRK2 which can rescue grim-induced apoptosis in the fly eye, G2019S and R1441C mutants are ineffective in exerting this anti-apoptotic function (Fig. 7B). Moreover, a constitutive active form of Akt, and to a lesser extend of wild-type Akt, can rescue the loss of anti-apoptotic function of R1441C and G2019S mutants. These data lead to a plausible scenario in which PD-linked LRKK2 mutations may have compromised anti-apoptotic function due to a defect in their effective ability to activate Akt, which might cause cells to be more susceptible to neurodegeneration. The functional characterization of LRRK2 and its mutants raises the possibility that the pathological effect of G2019S and R1441C mutants is caused by a toxic gain-of-function mechanism and might also partially be caused by a loss-of-function mechanism that involves an inability to activate Akt. These data are supported by previous findings that reported diminished levels of total Akt and pAkt Ser473 in postmortem PD brains (47) and that a defective Akt signaling is associated with the loss of dopaminergic neurons (48). Additionally, Akt signaling has been demonstrated to prevent injury-induced neuronal death and to promote axon and dendrite regeneration (49). Moreover, the constitutive active form of Akt has been found to induce profound trophic effects on dopaminergic neurons against neurotoxin-induced apoptotic death in murine models of PD (50). These data imply a therapeutic potential for wild-type LRRK-Akt to reverse or slow PD-associated neurodegeneration.

Figure 7.

A survival-signaling pathway regulated by wild-type LRRK2, but not LRRK2 R1441C and G2019S mutants. (A) LRRK2 (blue) activates the downstream effector Akt (purple) to mediate its anti-apoptotic effect via inhibition and phosphorylation of FOXO 1 (red) and thereby promotes cell survival (green). (B) LRRK2 G2019S and R1441C fail to activate the downstream effector Akt and thereby show loss of anti-apoptotic function.

Figure 7.

A survival-signaling pathway regulated by wild-type LRRK2, but not LRRK2 R1441C and G2019S mutants. (A) LRRK2 (blue) activates the downstream effector Akt (purple) to mediate its anti-apoptotic effect via inhibition and phosphorylation of FOXO 1 (red) and thereby promotes cell survival (green). (B) LRRK2 G2019S and R1441C fail to activate the downstream effector Akt and thereby show loss of anti-apoptotic function.

In conclusion, our work provides novel mechanistic insight into the physiological role of LRRK2. LRRK2 recruits the Akt signaling pathway in the inhibiting molecules that promote cell death. To our knowledge, these results provide the first in vivo evidence to support the pro-survival function of LRRK2 via activation of anti-apoptotic signaling mechanisms. This study is expected to shed light on the development of novel therapeutic strategies for PD prevention and treatment.

MATERIALS AND METHODS

Fly strains and epigenetic analysis

All flies were cultured on corn meal-yeast-agar medium at 25°C, with an automated light/dark cycle. Characterization of UAS-LRRK2.1 (II) and UAS-LRRK2.2 (III) is described in the Molecular cloning and transgene expression sections. UAS-LRRK2G2019 and UAS-LRRK2R1441C were used as previously described (41,51). Drosophila lrrk null mutant, lrrk−/−, has been reported previously (18). GMR-GAL4 was obtained from Larry Zipursky (University of California, Los Angeles). Several crosses of these fly strains were used in this study such as the following: GMR-GAL4, UAS-LRRK2.1/CyO; GMR-GAL4;UAS-LRRK2.2; TH-GAL4, UAS-CD8GFP; GMR-GAL4; UAS-LRRK2R1441C. GMR-GAL4, UAS-LRRK2G2019S. Fly strains for epigenetic experiments including, GMR-rpr, GMR-hid, GMR-grim, UAS-Akt, UAS-AktRNAi, UAS-rlRNAi, UAS-hemipterousRNAi, UAS-downstream of raf1(Dsor)RNAi and UAS-PI3KRNAi and 100 kinase RNAi lines were either obtained from the Bloomington Drosophila stock center (Department of Biology, Indiana University, Bloomington, IN) and the Vienna Drosophila RNAi center, or the Taiwan Drosophila stock center. The experimenters were blindly to the fly genotypes. Ten to 20 individual flies were imaged per group and most, if not all, the genetic interaction experiments were performed at least four times.

Molecular cloning and transgene expression

Human wild-type LRRK2 cDNA (Origene Technologies, Rockville, MD) was subcloned into a pUAST vector and was confirmed by DNA sequencing. We obtained multiple UAS-LRRK2 transformation lines on the second and third chromosomes using germ line transformation. UAS-LRRK2.1 (II) and UAS-LRRK2.2 (III) which showed comparable protein expression levels were selected for this study.

Scanning electron microscopy (SEM) and immunohistochemistry

To analyze the Drosophila compound eyes, newly enclosed adult flies were used for SEM analysis. SEM was performed as previously described (52). Whole-mount eyes were performed as previously described (53). F-actin enriched retina was labeled with Rhodomine-conjugated phalloidin (Sigma). The fly eyes were observed using a Zeiss LSM-510 or 710 confocal microscope for fluorescent imaging.

Western blot analysis

To detect phosphorylated and total protein levels, tissue homogenates of dissected fly heads were obtained using a glassmicro-tissue grinder and sample buffer (10% glycerol, 5% beta-mercaptoethanol, 2% SDS, 62.5 mm Tris–HCL and 0.01% bromophenol blue). SDS-polyacrylamide gel electrophoresis using 6–8% gels and immunoblotting were performed to analyze samples of freshly prepared protein extracts as previously described (54). The following primary antibodies (1:1000 dilutions) were used for Western analysis: anti-Akt, anti-phospho-Akt (Ser473) anti-Erk, anti-phosph-Erk (Thr202/Tyr204), anti-FoxO1 and anti-phospho-FoxO1 (Ser256) (Cell Signaling Technology, Danvers, MA); anti-phospho-Akt (Thr308) (Thermo Scientific); and anti-PI3K anti-GSK-3β, anti-phospho-GSK-3β (Ser9) (Gene Tex, Irvine, CA). Western blots were developed with the appropriate peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) and Enhanced Chemiluminescence reagents (Thermo Scientific), and visualized by ImageQuant 350 (GE Healthcare).

Statistical analysis

Data are shown as mean ± SEM, and represent at least three biological replicates with 20 flies per experiment. Statistical comparisons between groups were performed using one-way ANOVA with Bonferroni’'s multiple comparison tests with GraphPad Prism Software.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online.

FUNDING

This work is supported by National Science Council Grant (B007002) and Mackay Memorial Hospital and National Tsing Hua University grant (102N2746E1).

ACKNOWLEDGEMENTS

We thank Y. Imai, C.T. Chien, D.S. Park and Horng-Dar Wang for providing the fly strains. We are grateful to Tzu-Kang Sang, Kathy J. Sang, Don Ready and Pien-Chien Huang for their critical comments and suggestions for this manuscript.

Conflict of Interest statement. None declared.

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Author notes

These authors contributed equally to this work.