Genetic dissection reveals that Akt is the critical kinase downstream ...

Genetic dissection reveals that Akt is the criticalkinase downstream of LRRK2 to phosphorylateand inhibit FOXO1, and promotes neuron survival

Chia-Lung Chuang1,2,{, Yu-Ning Lu1,2,{, Hung-Cheng Wang1,2 and Hui-Yun Chang1,2,∗

1Institute of Systems Neuroscience, Department of Medical Science, National Tsing Hua University, 101, Section 2,

Kuang-Fu Road, Hsinchu 30013, Taiwan and 2Brain Research Center, National Tsing Hua University, 101, Section 2,

Kuang-Fu Road, Hsinchu 30013, Taiwan

Received February 6, 2014; Revised May 7, 2014; Accepted June 5, 2014

Leucine-rich repeatkinase2(LRRK2) isacomplexkinaseandmutations inLRRK2areperhapsthemostcommongenetic cause of Parkinson’s disease (PD). However, the identification of the normal physiological function ofLRRK2 remains elusive. Here, we show that LRRK2 protects neurons against apoptosis induced by theDrosophila genes grim, hid and reaper. Genetic dissection reveals that Akt is the critical downstream kinaseof 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 impairthe ability of LRRK2 to activate Akt, and fail to prevent apoptotic death. Ectopic expression of a constitutiveactive form of Akt hence is sufficient to rescue this functional deficit. These data establish that LRRK2 can pro-tect neurons from apoptotic insult through a survival pathway in which LRRK2 signals to activate Akt, and theninhibits FOXO1. These results might indicate that a LRRK-Akt therapeutic pathway to promote neuron survivaland 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 toeliminate unnecessary or damaged neurons. How do the devel-oping neurons decide whether to die or to survive? Viktor Ham-burger and Rita Levi-Montalcini described that the survival ofdeveloping neurons is vitally dependent on the availability ofneurotrophic supports from their innervated targets (1). Neuro-trophic factors trigger the activation of critical protein kinasecascades that promote survival and inhibit apoptosis (2). Whilemature neurons no longer require trophic factors to sustaintheir survival, activation of pathways that promote survivaland inhibit apoptosis might still be indispensable. Thus, dis-turbances in survival signaling and/or activation of apoptoticpathways in response to stress and toxic proteins might be anunderlying disease mechanism of a number of neurodegenera-tive diseases such as Alzheimer’s disease and Parkinson’sdisease (PD) (3).

PD is the second most common neurodegenerative diseasecharacterized by progressive loss of dopaminergic neuronsin the substantia nigra and the deposition of Lewy bodies (4).Although familial forms of PD are rare, some monogenicforms of PD resemble a sporadic disease in terms of their clin-ical and pathological manifestations (5), the associated genesmay offer functional and pathogenic insights into the diseasemechanism. Several genes associated with PD have beenshown to directly or indirectly inhibit apoptosis. For example,PINK1 can interact with Bcl-xL to prevent pro-apoptotic cleav-age of Bcl-xL, thereby promoting cell survival, and DJ-1 inter-acts with Daxx to suppress cell death (6,7). In addition, the E3ligase Parkin appears to protect neurons by negatively regulatingthe pro-apoptotic protein Bax, and the anti-apoptotic effect ofa-synuclein is associated with inhibition of caspase-3 cleavage(8,9). Furthermore, both PINK1 and Parkin are crucial for mito-chondrial quality control by targeting dysfunctional mitochon-drial proteins to the lysosome (10). All of these facts raise thepossibility that these genes mutated in PD might normally

†These authors contributed equally to this work.

∗To whom correspondence should be addressed at: Institute of Systems Neuroscience, Department of Medical Science, National Tsing Hua University, 101,Section 2, Kuang-Fu Road, Hsinchu 30013, Taiwan. Tel: +886 35742778; Email: [email protected]

# The Author 2014. Published by Oxford University Press. All rights reserved.For Permissions, please email: [email protected]

Human Molecular Genetics, 2014, Vol. 23, No. 21 5649–5658doi:10.1093/hmg/ddu281Advance Access published on June 10, 2014

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protect neurons by promoting survival and inhibiting apoptosisin mature neurons.

Genetic mutations in the PARK8 gene-encoding LRRK2 arethe most common cause of PD (11,12). LRRK2 appears to be asignal transduction protein with kinase domains in the centralcatalytic core and several motifs, including a Ras of complex(ROC) domain, a C-terminal of ROC (COR) domain, aleucine-rich repeat (LRR) and a WD40 domain (13,14). Thedownstream effectors of LRRK2, and the molecular and cellularmechanisms 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 variousregions of the human brain including the cortex, hippocampus,midbrain, brainstem and dopaminergic neurons in the substantianigra (15). Compelling evidence suggests that the most commonPD-linked LRRK2 mutations are located in the central catalyticcore. These include the autosomal-dominant R1441C andG2019S mutations, and their aberrant kinase and GTPase activ-ities appear central to inducing neurotoxicity and neurodegen-eration (16). Previous studies using cell lines and modelorganisms have implicated pathological LRRK2 in the restric-tion of neurite outgrowth, mitochondria dysfunction, increasedprotein translation and impairments in endocytosis and autop-hagy (13). Although LRRK2-associated PD pathogenesis hasbeen demonstrated in a number of models, identification of thephysiological role of LRRK2 to corroborate its kinase cascaderemains unidentified. Moreover, the important question of howPD-linked LRRK2 mutations affect signaling function and the

relevance of these effects to neurodegeneration remainsunanswered (13).

Here, we used Drosophila compound eyes to explore theeffects of LRRK2 signaling on apoptosis in vivo. Our geneticand biochemical studies demonstrated a role for human LRRK2in neuroprotection. Expression of wild-type LRRK2 promotedneuronal survival against apoptosis through activation of thedownstream effector, Akt by phosphorylation of Ser473. Phos-phorylated Akt in turn inhibited FOXO 1 signaling. In contrast,this pro-survival role was inhibited in two PD-linked LRRK2mutants, R1441C and G2019S. These results define, for thefirst time, the function and molecular mechanism underlyinghow LRRK2 expression promotes neuronal survival that maybe compromised in LRRK2-linked monogenic PD.

RESULTS

LRRK2 has anti-apoptotic function

Previous studies reported that LRRK2 knockout mice displayedapoptotic cell death in the kidney, and that a null mutant of the flyLRRK2 homolog, lrrk, exhibits dopaminergic degeneration(17,18). These studies suggested that LRRK2 might exert ananti-apoptotic function. To address this question, we studiedthe Drosophila compound eye, a model commonly used forstudying apoptosis (Fig. 1A). By expressing three pro-apoptoticgenes, reaper, hid and grim using the eye-specific GMR-GAL4driver, we found that the normal compound eye size wasgreatly reduced (Fig. 1B–D). The Drosophila compound eye

Figure 1. Expression of human LRRK2 inhibits cell death in Drosophila. Scanning electron microscope (SEM) micrographs of Drosophila compound eyes. (A) Rep-resentative 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-typehuman LRRK2 transgene expression in the eye using the GMR-Gal4 driver (GMR::LRRK2) produced an eye morphology similar to the wild-type control inA. (F–H) Co-expression of LRRK2 with any one of the three apoptotic genes partially rescued eye morphology, resulting in relatively larger eyes with moreevident ommatidia. In all panels, anterior is to the left and posterior is to the right. Scale bar, 100 mm.

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has highly regular ommatidia. Expression of these pro-apoptoticgenes severely disrupted the regular pattern and boundaries ofeach unit eye (Fig. 1B–D). Western blotting analysis ofprotein lysates from eyes expressing the wild-type humanLRRK2 transgene yielded a �280 kDa band (SupplementaryMaterial, Fig. S1). There were no observable defects in these eyes(Fig. 1E). Remarkably, when human LRRK2 was co-expressingwith reaper, hid and grim, signs of cell death were markedly sup-pressed, resembling a normal eye. The suppression of apoptosiswas particularly distinctive in the GMR::grim strain, whichwe used for all subsequent studies (Fig. 1F–H). Interestingly,co-expression of fly lrrk also suppressed cell death induced bygrim (Supplementary Material, Fig. S2). To confirm the anti-apoptotic function of lrrk, we examined lrrk null mutant flies(lrrk2/2). The overall organization and structure of lrrk2/2

flies appeared normal except reduced body size, including com-pound eyes. Ommatidial counts revealed 766+ 20 ommatidia inwild-type eyes, 736+ 26 ommatidia in lrrk2/+ heterozygouseyes, and only 602+ 32 ommatidia, �20% reduction as com-pared with wild-type, in lrrk2/2 eyes. Interestingly, thereduced compound eye size of lrrk2/2 flies could be rescuedby expressing human LRRK2 (752+ 25 ommatidia) (Supple-mentary Material, Fig. S3A and B). Taken together, these datasuggest that the anti-apoptotic function of human LRRK2 isevolutionary conserved.

The anti-apoptotic function of LRRK2 requires Akt

To investigate whether the observed anti-apoptotic function ofhuman LRRK2 might be mediated through the activation of

other kinases, we tested 100 RNAi lines in the Drosophilakinome. Expression of grim alone showed an apoptotic eyephenotype (Fig. 2A). We found that knockdown of fly Akt(AktRNAi) markedly reversed the ability of LRRK2 to suppressthe grim-induced eye apoptosis phenotype (Fig. 2B and C). Akthas been reported as a LRRK2 substrate in vitro (19) indicatingthat the screen was capable of identifying a relevant target. Tofurther validate the downstream role of Akt in the pro-survivalfunction of LRRK2, wild-type Akt was overexpressed in thestarter background (co-expression of grim and LRRK2).Indeed, increasing Akt level enhanced the anti-apoptotic effectof human LRRK2 (Fig. 2D), suggesting that these two kinasesexert pro-survival effects in the apoptotic eye model. It is import-ant to note that while a number of kinases, including phosphoi-nositide 3-kinase (PI3K), extracellular signal-regulated kinase(ERK; fly rolled or rl), mitogen-activated protein kinasekinase 7 (MKK7; fly hemipterious or hep), and MAPKK (flyDownstream of raf1, Dsor1), that have been proposed to likelyinteract with LRRK2, knockdown of these kinases did not alterthe starter line phenotype (Fig. 2E–H). Our findings indicatethat the genetic interaction between LRRK2 and Akt in anti-apoptosis function is specific, thereby implying that LRRK2signaling mediates cell survival through Akt pathways.

LRRK2 selectively increases phosphorylated Akt to inhibitFOXO but not ERK

The aforementioned genetic results suggest that Akt, a well char-acterized intracellular signaling pathway that promotes cell sur-vival (20), is the downstream target of LRRK2. Therefore, we

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 besuppressed 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 thestarter line background, knockdown of Akt through the expression of an RNAi construct inhibited the anti-apoptotic effect of LRRK2 (C). Expression of wild-typeAkt enhanced the anti-apoptotic effect of LRRK2 resulting in a normal sized compound eye and mostly regular ommatidia (D). (E–H) Expression of the RNAi con-structs to knockdown PI3K (E), rl (Drosophila ERK) (F), hep (Drosophila MKK7) (G) and dsor (Drosophila MKK) (H) in the starter line displaced an eye phenotypecomparable to the starter line suggesting that unlike Akt-RNAi in (C) these genes do not function downstream of anti-apoptotic LRRK2. Scale bar, 100 mm.

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assessed whether human LRRK2 expression is sufficient to acti-vate the fly Akt signaling pathway. Western blot analysis showedthat LRRK2 expression in fly eyes led to a robust increase inp-Akt Ser473 as compared with wild-type, GMR::GFP orUAS-LRRK2 controls (Fig. 3A and B). Drosophila genome con-tains a single Akt gene which encodes two splice forms with twodistinct length of 513 and 611 amino acids, and both isoforms canbe recognized by p-Akt Ser 473 antibody as previously sug-gested (21,22). To confirm this, we treated protein extractswith phosphatase; while both bands could still be detected byAkt antibody, none of them could be detected by phospho-AktSer473 antibody after dephosphorylation, thereby suggestingthat both bands represent phosphorylated Akt Ser473 (Supple-mentary Material, Fig. S4). Under ectopically expressedconditions, LRRK2 enhanced the phosphorylation of the corre-sponding Ser residues of both isoforms (Fig. 3A and B); and ithad a stronger impact on the high molecular weight isoform(a 9-fold increase) compared with control. Consistent with thegenetic result that LRRK2/lrrk targets on Akt, we observedthat lacking lrrk in lrrk2/2 flies led to a decrease in p-AktSer473 as compared with wild-type and heterozygous lrrk2/+

flies (Fig. 3C). In contrast, LRRK2 may specifically act on thisSer residue of Akt because its expression did not affect thelevels of p-Akt Thr308 and the total Akt that were comparableacross genotypes (Fig. 3A and B). It is also noteworthy thatwhile PI3K is a canonical upstream kinase regulating Akt activ-ity, PI3K levels did not significantly change upon expressinghuman LRRK2 as compared with control (Fig. 3A and B), sug-gesting that LRRK2-regulated Akt activation is unlikely to bemediated through PI3K. Together, these data strengthen thenotion that LRRK2 activates downstream Akt via specific phos-phorylation of a conserved Ser473 residue.

The ERK (MAPK) signaling pathway is also regulated byPI3K and could promote cell survival (23). We further exploredwhether human LRRK2 might also affect this kinase activitythrough phosphorylation. We found LRRK2 expression didnot significantly affect the level of total ERK protein or p-ERKas compared with control (Fig. 3A and B). It is noteworthy thatthe effect of human LRRK2 on fly Akt is not an eye-specificevent, because expression of LRRK2 in fly dopaminergicneurons also yielded similar results (Supplementary Material,Fig. S5).

Figure 3. Expression of human LRRK2 specifically promotes Akt phosphorylation at Ser473 leading to phosphorylation of downstream targets FOXO1 and GSK-3b.(A) Protein lysates from the heads of wild-type (CS), controls (GMR::GFP, UAS-LRRK2) and LRRK2 expressing (GMR::LRRK2) flies were analyzed by westernblotting using antibodies against Akt, Akt-pSer308, Akt-pSer473, PI3K, ERK and ERK-pThr202/Tyr204.a-tubulin was used as a loading control. (B) Quantificationof 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 ¼ 4). Both bands to represent pAkt 473. (C) The relative levels of Akt-pSer473 and total Akt were analyzed by western blotting using proteinlysates from the fly heads of wild-type (w1118), lrrk null (lrrk2/2), lrrk heterozygous (lrrk2/+) and human LRRK2 expression (GMR::LRRK2) flies. a-tubulinserved as a loading control. (D) Immunoblotting of Akt downstream targets detected FOXO1, FOXO1-pSer256, GSK-3b and GSK-3b-pSer9 protein levels in theindicated protein lysates.







Activation of Akt signaling could promote cell survival,growth and metabolism through the regulation of a variety ofdownstream molecules (24). Given the anti-apoptotic role ofAkt, we speculated that it might negatively regulate the Fork-head family member FOXO 1 through phosphorylation, whichwould block FOXO 1-induced pro-apoptotic changes in geneexpression. To test this, we compared the level of FOXO 1 andphosphorylated FOXO 1 Ser256 protein levels in the humanLRRK2 overexpressing fly. As shown in Figure 3D, the levelof phosphorylated FOXO 1 was substantially increased byLRRK2 expression compared with control. Notably, FOXO 1protein level was reduced in this condition. Moreover, Akt caninactivate glycogene synthase kinase 3b (GSK-3b) throughdirect phosphorylation which might in turn inhibit the facilita-tion of apoptosis (25). GSK-3b activation has been implicatedin a number of neurodegenerative diseases that may contributeto tauopathy (26,27). We tested whether LRRK2 expressionmight negatively regulate GSK-3b by phosphorylation Similarto the effect of LRRK2 on FOXO1, LRRK2 expression produceda robust increase in GSK-3b Ser9 phosphorylation.

To identify whether FOXO1 is the downstream effector ofLRRK2 that regulates neuronal survival (Fig. 4A), we examinedif expression of wild-type FOXO1 or non-phosphorylatedFOXO S259A might attenuates the anti-apoptotic function ofLRRK2. As expected, co-expression of FOXO1 or FOXO1S259A, diminished the robust reversed effect of LRRK2 onapoptosis, and resulted in a relative reduced eye compared

with the starter line (Fig. 4A and B–C). However, co-expressionof GSK-3bS9A produced no change of eye phenotype (Fig. 4D).Therefore, our biochemical and genetic data provide further mo-lecular basis for how LRRK2-Akt survival signal is mediatedthrough FOXO1, a negative downstream regulator of LRRK2-Akt but not through GSK-3b.

Loss of LRRK2 anti-apoptotic function in PD-linked LRRK2mutants coincides with failure to activate Akt

The pathogenic mechanism of monogenic PD-linked mutationsin LRRK2 is still not well understood (28). The anti-apoptoticfunction of wild-type human LRKK2, through activation ofAkt signaling, led us to ask whether PD-linked LRRK2 muta-tions might compromise this signaling pathway. To answerthis, we utilized two common PD-associated LRRK2 mutants,G2019S and R1441C. Using an antibody specifically recognizesphosphorylated Akt at Ser473, we found that unlike wild-typeLRRK2 which could enhance Akt phosphorylation at Ser473,both LRRK2R1441C and LRRK2G2019S failed to phosphoryl-ate Akt (Fig. 5A). These data implicate a loss of normal anti-apoptotic function in these LRRK2 mutations.

Next, to further corroborate our finding that LRRK2R1441Cand LRRK2G2019S have indeed partially lost their anti-apoptotic effect, we expressed each individual mutation inthe GMR::grim fly. Unlike wild-type LRRK2, which showedrobust suppression of the grim-induced eye phenotype, expres-sing these PD-linked LRRK2 mutants could not significantlysuppress grim-induced apoptosis, thereby resulting in the reducedeye size phenotype (Fig. 5B–E). Together, these genetic andbiochemical findings suggest that wild-type LRRK2 exerts apro-survival role through the Akt signaling pathway, which maybe compromised in the PD-linked mutations, LRRK2G2019Sand LRRK2R1441C.

A constitutive active form of Akt restores the impairedanti-apoptotic function in LRRK2G2019S andLRRK2R1441C mutants

Because the PD-linked LRRK2G2019S and LRRK2R1441Cmutants were largely lost their ability to activate Akt survivalpathways and failed to alleviate grim-induced apoptosis(Fig. 5), we asked whether this functional loss of anti-apoptosisfunction might be rescued by co-expression of a constitutiveactive form of Akt with these LRRK2 mutants. To this end, weco-expressed three transgenes: grim to induce the apoptotic eyephenotype, LRRK2G2019S or LRRK2R1441C, and a constitu-tive active form of Akt (Drosophila Akt.T342D.S505D that cor-responds to mammalian Akt.T308D.S473D). We observed thatthe compromised pro-survival function of LRRK2R1441C orLRRK2G2019S on grim-induced apoptosis (Fig. 5) could bemarkedly rescued by co-expression with a constitutive activeAkt (Fig. 6). Interestingly, co-expression of constitutive activeAkt with LRRK2R1441C or LRRK2G2019S produced a strongerrescue effect than expression of constitutive active Akt alone(Fig. 6). Taken together, these data suggest that human LRRK2exerts its anti-apoptotic function through activation of the Akt sig-naling pathway, and that activation of this pathway alone is suffi-cient to promote neuron survival.

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) wasdramatically impeded by the expression of dFOXO1 S259S or wild-typedFOXO1, which results in a severely reduced eye phenotype (B and C). GFPwas 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 mm.



DISCUSSION

Here, we present the functional characterization of humanLRRK2 in a Drosophila genetic model system. Remarkably,we found that LRRK2 can rescue neuronal apoptosis inducedby ectopic expression of death genes, grim, hid and reaper inthe fly visual system. Furthermore, our genetic screen and bio-chemical analyses provide novel insight into the role ofLRRK2 in promoting neuronal survival, at least in part by acti-vating its downstream Akt effector. We showed that wild-typeLRRK2, but not its PD-linked mutants, phosphorylates the Aktat Ser473 thereby inhibiting the pro-apoptotic transcriptionfactor FOXO1 in neurons. Our data reveal for the first time invivo, an anti-apoptotic role of LRRK2. This survival promotingfunction of LRRK2 was compromised in autosomal-dominantmutants, R1441C and G2019S, but could be reversed by aconstitutive active form of Akt, that further confers that wild-type LRRK2 plays a pro-survival role. Taken together, ourdata provide novel mechanistic insights into how PD-linked

LRRK2 mutations might be detrimental for neurons via theloss-of-function mechanism.

LRRK2 has kinase and GTPase domains; and its kinase activ-ity is thought to be similar to the signaling molecule MAPKKK(13). Our data demonstrate that while LRRK2 plays a role inphosphorylating Akt, a proto-oncogene that promotes neuronsurvival this LRRK2-Akt pathway is distinct from the canonicalsignaling cascades regulated by MAPKKK. Indeed, Akt hasbeen shown to suppress apoptotic cell death in a variety of cellu-lar contexts (29–31). Our work has revealed that LRRK2 canactivate the Akt signaling pathway. These data identify an anti-apoptotic function for LRRK2 and provide a mechanism bywhich it exerts its protective role. Moreover, this conclusion isin agreement with reports showing that elevated LRRK2 pro-motes cell survival in papillary renal and thyroid cancers andin stress-induced cell death. Furthermore, LRRK2 inhibitionwas found to elicit apoptosis in human mid-brain derived neur-onal progenitor cells and in rodent kidney cells (17,32–34).Our study extends these observations by establishing the

Figure 5. PD-linked LRRK2 mutants G2019S and R1441C fail to increase Akt phosphorylation and promote cell survival. (A) Western blotting analyses for pAktSer473 in head lysates from transgenic flies expressing wild-type LRRK2 (GMR::LRRK2, upper section), R1441C (GMR::LRRK2R1441C, middle section) andG2019S (GMR::LRRK2G2019S, lower section) mutants. Immunoreactivity detected with anti-a-tubulin served as a loading control. (B–E) SEM micrographs ofDrosophila 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 mm.

Figure 6. Constitutive active form of Akt can induce cell survival in pathogenic LRRK2 mutants. SEM micrographs of Drosophila compound eyes show how theapoptotic 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 constitutiveactive 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 mm.



pro-survival role of LRRK2 is mediated via Akt activation.Interestingly, LRRK2 is functionally analogous to anothermonogenic PD-linked gene DJ1 (Park 7), which is an oncogenethat exerts a pro-survival role via activating the Ras/MAPKsignaling pathway. Therefore, despite different genetic traits,LRRK2 and DJ1 may mediate parallel survival signaling path-ways to prevent DA neurons from degeneration (35).

The anti-apoptotic role of the Akt signal pathway has beenwell established; phosphorylation at Thr308 in the activationloop and Ser473 in the hydrophobic motif are required for theanti-apoptotic activity of Akt (36). Interestingly, we found thatLRRK2-mediated Akt phosphorylation is specific to Ser473,but not to Thr308, suggesting that LRRK2 mediates its anti-apoptotic function by regulating Akt activation via Ser473phosphorylation in the hydrophobic motif only. Similar to ourobservation of LRRK2, several kinases, such as integrin-linkedkinase and the Rictor-mTOR complex, phosphorylate Akt atSer473 to promote cell survival (37,38). To further characterizehow activated Akt mediates cell survival, we identified thedownstream targets of Akt which were inactivated upon ectopicLRKK2 expression. Indeed, our observation that LRRK2 inhib-ited the pro-apoptotic transcription factors including FOXO1 viaAkt substantiates the role of this signaling pathway in neuronalprotection (24). However, these findings are contradictory to areport that suggested a role for LRRK2 in modifying thepro-apoptotic signal of FOXO 1 (39). However, the previousstudy was distinct in the fact that of FOXO was overexpressed,whereas in our study grim was overexpressed. This also suggeststhe possibility that differences in the molecular context acrossstudies might contribute to important discrepancies betweenresults on LRRK2 function. Interestingly, as FOXO1 is oneof the major inhibited downstream targets of LRRK2-Akt,co-overexpression of Drosophila FOXO1 with PD-linked LRRK2mutants would be expected to compromise the survival effectof LRRK2. While some studies support an anti-apoptotic rolefor LRRK2, additional studies are needed to determine whetherthis anti-apoptotic function of LRRK2-Akt signaling pathwayis also conserved in mammal systems. These functional datasuggest that an increase in Akt phosphorylation by LRRK2may account for the underlying signaling mechanism ofLRRK2 that is recruited to actively suppress apoptotic deathduring aging in response to stress. Furthermore, identifyingwhether the LRRK2-Akt signaling pathway is triggered by spe-cific upstream activators such as stress and understand how it isregulated in cells are questions that warrant further studies.

Currently, the underlying pathogenesis of PD-linked LRRK2mutations is not well understood (40). Many studies pro-pose various toxic gain-of-function mechanisms for PD-linkedLRRK2 mutations, including aberrant 4E-BP phosphorylation,increased eukaryotic initiation factor (eIF) 4E mediated proteintranslation (41), promotion of aberrant interactions with micro-RNAs, dysregulated protein translation (42), damaged proteinsorting (43), impaired neurite outgrowth (44), increased tauphosphorylation (45) and transduction of death signals via phys-ical interactions with Fas-associated protein death domains (46).In this study, we found that wild-type LRRK2 has a protectiverole in neurons. To assess whether this pro-survival function ofLRRK2-Akt signaling (Fig. 7A) might be compromised in thecontext of PD-related mutations, we tested two well describedLRRK2 mutants, G2019S and R1441C. Our findings indicated

that these mutant forms of LRRK2 fail to phosphorylate AktSer473, blunting the pro-survival function that is observed inwild-type LRRK2. Indeed, unlike normal LRRK2 which canrescue grim-induced apoptosis in the fly eye, G2019S andR1441C mutants are ineffective in exerting this anti-apoptoticfunction (Fig. 7B). Moreover, a constitutive active form ofAkt, and to a lesser extend of wild-type Akt, can rescue theloss of anti-apoptotic function of R1441C and G2019S mutants.These data lead to a plausible scenario in which PD-linkedLRKK2 mutations may have compromised anti-apoptotic func-tion due to a defect in their effective ability to activate Akt, whichmight cause cells to be more susceptible to neurodegeneration.The functional characterization of LRRK2 and its mutantsraises the possibility that the pathological effect of G2019Sand R1441C mutants is caused by a toxic gain-of-functionmechanism 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 reporteddiminished levels of total Akt and pAkt Ser473 in postmortemPD brains (47) and that a defective Akt signaling is associatedwith the loss of dopaminergic neurons (48). Additionally, Aktsignaling has been demonstrated to prevent injury-inducedneuronal death and to promote axon and dendrite regeneration(49). Moreover, the constitutive active form of Akt has beenfound to induce profound trophic effects on dopaminergicneurons against neurotoxin-induced apoptotic death in murinemodels of PD (50). These data imply a therapeutic potentialfor wild-type LRRK-Akt to reverse or slow PD-associatedneurodegeneration.

Figure 7. A survival-signaling pathway regulated by wild-type LRRK2, but notLRRK2 R1441C and G2019S mutants. (A) LRRK2 (blue) activates the down-stream effector Akt (purple) to mediate its anti-apoptotic effect via inhibitionand phosphorylation of FOXO 1 (red) and thereby promotes cell survival(green). (B) LRRK2 G2019S and R1441C fail to activate the downstream effect-or Akt and thereby show loss of anti-apoptotic function.



In conclusion, our work provides novel mechanistic insightinto the physiological role of LRRK2. LRRK2 recruitsthe Akt signaling pathway in the inhibiting molecules thatpromote cell death. To our knowledge, these results pro-vide the first in vivo evidence to support the pro-survival func-tion of LRRK2 via activation of anti-apoptotic signalingmechanisms. This study is expected to shed light on the devel-opment of novel therapeutic strategies for PD prevention andtreatment.

MATERIALS AND METHODS

Fly strains and epigenetic analysis

All flies were cultured on corn meal-yeast-agar medium at 258C,with an automated light/dark cycle. Characterization ofUAS-LRRK2.1 (II) and UAS-LRRK2.2 (III) is described in theMolecular cloning and transgene expression sections. UAS-LRRK2G2019 and UAS-LRRK2R1441C were used as previouslydescribed (41,51). Drosophila lrrk null mutant, lrrk2/2, hasbeen reported previously (18). GMR-GAL4 was obtained fromLarry Zipursky (University of California, Los Angeles).Several crosses of these fly strains were used in this study suchas 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. Flystrains for epigenetic experiments including, GMR-rpr, GMR-hid, GMR-grim, UAS-Akt, UAS-AktRNAi, UAS-rlRNAi, UAS-hemipterousRNAi, UAS-downstream of raf1(Dsor)RNAi andUAS-PI3KRNAi and 100 kinase RNAi lines were either obtainedfrom the Bloomington Drosophila stock center (Department ofBiology, Indiana University, Bloomington, IN) and the ViennaDrosophila RNAi center, or the Taiwan Drosophila stockcenter. The experimenters were blindly to the fly genotypes.Ten to 20 individual flies were imaged per group and most, ifnot all, the genetic interaction experiments were performed atleast four times.

Molecular cloning and transgene expression

Human wild-type LRRK2 cDNA (Origene Technologies, Rock-ville, MD) was subcloned into a pUAST vector and was con-firmed by DNA sequencing. We obtained multiple UAS-LRRK2transformation lines on the second and third chromosomesusing germ line transformation. UAS-LRRK2.1 (II) and UAS-LRRK2.2 (III) which showed comparable protein expressionlevels were selected for this study.

Scanning electron microscopy (SEM) andimmunohistochemistry

To analyze the Drosophila compound eyes, newly enclosedadult flies were used for SEM analysis. SEM was performed aspreviously described (52). Whole-mount eyes were performedas previously described (53). F-actin enriched retina waslabeled with Rhodomine-conjugated phalloidin (Sigma). Thefly eyes were observed using a Zeiss LSM-510 or 710 confocalmicroscope for fluorescent imaging.

Western blot analysis

To detect phosphorylated and total protein levels, tissue homo-genates 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% bro-mophenol blue). SDS-polyacrylamide gel electrophoresis using6–8% gels and immunoblotting were performed to analyzesamples of freshly prepared protein extracts as previouslydescribed (54). The following primary antibodies (1:1000 dilu-tions) 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 Tech-nology, Danvers, MA); anti-phospho-Akt (Thr308) (ThermoScientific); and anti-PI3K anti-GSK-3b, anti-phospho-GSK-3b(Ser9) (Gene Tex, Irvine, CA). Western blots were developedwith the appropriate peroxidase-conjugated secondary anti-bodies (Jackson ImmunoResearch Laboratories, West Grove,PA) and Enhanced Chemiluminescence reagents (Thermo Sci-entific), and visualized by ImageQuant 350 (GE Healthcare).

Statistical analysis

Data are shown as mean+SEM, and represent at least three bio-logical replicates with 20 flies per experiment. Statistical com-parisons between groups were performed using one-wayANOVA with Bonferroni’’s multiple comparison tests withGraphPad Prism Software.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online.

ACKNOWLEDGEMENTS

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

Conflict of Interest statement. None declared.

FUNDING

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

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Genetic dissection reveals that Akt is the critical kinase downstream ...

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