CHIP Regulates Leucine-Rich Repeat Kinase-2 Ubiquitination, Degradation, and Toxicity

CHIP regulates leucine-rich repeat kinase-2 ubiquitination, degradation, and toxicity

Han Seok Koa,b, Rachel Baileyg, Wanli W. Smithe,f, Zhaohui Liue,f, Joo-Ho Shina,b, Yun-Il Leea,b, Yong-Jie Zhangg, Haibing Jiange,f, Christopher A. Rossc,e,f, Darren J. Moorea,b, Cam Pattersonh, Leonard Petrucellig, Ted M. Dawsona,b,c,1, and Valina L. Dawsona,b,c,d,1

aNeuroregeneration and Stem Cell Programs, Institute for Cell Engineering, bDepartment of Neurology, cSolomon H. Snyder Department of Neuroscience, dDepartment of Physiology, eDepartment of Psychiatry, and fDivision of Neurobiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205; gDepartment of Neuroscience, Mayo Clinic College of Medicine, Jacksonville, FL 32224; and hDivision of Cardiology, University of North Carolina, Chapel Hill, NC 27599

Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved December 15, 2008 (received for review October 9, 2008) Mutation in leucine-rich repeat kinase-2 (LRRK2) is the most com- are not known. Whether the levels of LRRK2 are linked to mon cause of late-onset Parkinson’s disease (PD). Although most toxicity also is unclear. cases of PD are sporadic, some are inherited, including those The carboxyl terminus of HSP70-interacting protein (CHIP) caused by LRRK2 mutations. Because these mutations may be plays a critical role in quality control of cellular proteins and associated with a toxic gain of function, controlling the expression stress recovery systems in most cell types (29, 30). CHIP contains of LRRK2 may decrease its cytotoxicity. Here we show that the multiple domains, including a tetratricopeptide repeat (TPR) carboxyl terminus of HSP70-interacting protein (CHIP) binds, ubiq- domain that allows it to interact with molecular chaperones, such uitinates, and promotes the ubiquitin proteasomal degradation of as HSP70 and HSP90, and a U-box domain that confers its E3 LRRK2. Overexpression of CHIP protects against and knockdown of ubiquitin ligase function (31, 32). Thus, CHIP functions as both CHIP exacerbates toxicity mediated by mutant LRRK2. Moreover, a co-chaperone and an E3 ubiquitin ligase and serves as a HSP90 forms a complex with LRRK2, and inhibition of HSP90 molecular link between cellular protein folding and degradation. chaperone activity by 17AAG leads to proteasomal degradation of CHIP mediates ubiquitin attachment to the chaperone substrate LRRK2, resulting in increased cell viability. Thus, increasing CHIP E3 and stimulates the degradation of chaperone substrates by the ligase activity and blocking HSP90 chaperone activity can prevent UPS (33, 34) CHIP has been linked to several neurodegenerative the deleterious effects of LRRK2. These ﬁndings point to potential diseases characterized by protein misfolding and aggregation. treatment options for LRRK2-associated PD. Because HSP90 interacts with LRRK2 and CHIP, we explored the potential for CHIP to regulate LRRK2 levels (33). We found LRRK2 ͉ Parkinson’s disease ͉ proteasome ͉ ubiquitin that CHIP interacts with and ubiqutinates LRRK2, leading to the latter’s proteasomal degradation through a HSP90 chaper- arkinson’s disease (PD) is a progressive neurodegenerative one–containing complex. In addition, we found that CHIP and Pdisorder pathologically characterized by loss of dopaminer- HSP90 levels are critical determinants of LRRK2 toxicity; thus, gic neurons from the substantia nigra and the presence of Lewy regulating the levels and activity of CHIP and HSP90 may be bodies (1–3). The etiology of PD is incompletely understood but potentially valid candidates for treating LRRK2-related PD. appears to involve both genetic and environmental factors. To Results date, 5 genes (␣-synuclein, parkin, DJ-1, PINK-1, and LRRK2) are associated with genetic forms of PD that closely resemble LRRK2 Interacts With CHIP. LRRK2 dimerizes and interacts with idiopathic PD (4–10). Mutation in LRRK2 is the most frequent HSP90 (18, 27, 28). Because CHIP is an ubiquitin ligase that genetic cause of PD (11). Patients with LRRK2 mutations exhibit interacts with HSP90, we explored the possibility that CHIP NEUROSCIENCE clinical and neurochemical phenotypes that are indistinguishable interacts with and ubiquitinates LRRK2 in SH-SY5Y neuroblas- from sporadic PD (9, 10). These patients suffer neuronal loss and toma cells (Fig. 1). To investigate a possible interaction between gliosis in the substantia nigra and development of Lewy bodies, CHIP and LRRK2, we conducted coimmunoprecipitation ex- and also exhibit pleomorphic neuropathology, including periments with Myc-tagged LRRK2 and HA-tagged CHIP, and ␣-synuclein and tau pathology (9, 12, 13). Thus, LRRK2 is found that LRRK2 pulled down CHIP (Fig. 1A) and Myc-tagged important for the pathogenesis of several major neurodegen- CHIP pulled down FLAG-tagged LRRK2 (Fig. 1B). In addition, erative disorders associated with parkinsonism. immunoprecipitation of FLAG-tagged wild-type (WT), LRRK2, a member of the ROCO protein family, contains a R1441C, or G2019S LRRK2 pulled down HA-tagged CHIP, guanosine triphosphatase (GTPase), a C-terminal of Ras do- demonstrating that CHIP also interacts with mutant LRRK2 main with a kinase effector domain (14), repeat sequences (Fig. 1C). We found no substantial differences between WT and beginning at the N terminus, and a leucine-rich repeat structure mutant LRRK2 in terms of binding to CHIP. We observed near its GTPase domain (15). LRRK2 is localized to membra- similar results in HeLa cells (data not shown). To explore nous structures, where it may be in involved in neuronal polarity (16–18). Mutations in LRRK2 are frequent in autosomal- Author contributions: H.S.K., L.P., T.M.D., and V.L.D. designed research; H.S.K., R.B., dominant PD as well as sporadic PD (19–23). PD-associated W.W.S., Z.L., J.-H.S., Y.-I.L., Y.-J.Z., and H.J. performed research; C.A.R., D.J.M., C.P., and L.P. LRRK2 mutants seem to enhance kinase activity, and mutant contributed new reagents/analytic tools; H.S.K., L.P., T.M.D., and V.L.D. analyzed data; and LRRK2-mediated neuronal toxicity requires GTP-binding and H.S.K., L.P., T.M.D., and V.L.D. wrote the paper. kinase activity (17, 24–26). The authors declare no conﬂict of interest. The ubiquitin proteosomal system (UPS) appears to regulate This article is a PNAS Direct Submission. LRRK2 level, with little influence from the autophagic and 1To whom correspondence may be addressed. E-mail: [email protected] or vdawson@ lysosomal degradation pathways (17). LRRK2 also dimerizes jhmi.edu. and interacts with HSP90 (18, 27, 28), which is somehow involved This article contains supporting information online at www.pnas.org/cgi/content/full/ in controlling LRRK2 levels. The identity of the E3 ligase and 0810123106/DCSupplemental. the mechanisms that regulate the stability of LRRK2 via HSP90 © 2009 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810123106 PNAS ͉ February 24, 2009 ͉ vol. 106 ͉ no. 8 ͉ 2897–2902 Downloaded by guest on September 30, 2021 _ C A HA-CHIP + + C 9S A _ Myc-CHIP _ WTWT ∆Ubox∆TPR Myc-LRRK2 + + kDa _ _ FLAG-LRRK2 + WT R1441 G201 FLAG-LRRK2 ++++ Input α-HA _ kDa 37 HA-CHIP + + + + α kDa Input -FLAG 250 α-HA 37 Input α-HA IP: 37 α-FLAG α 250 -Myc α-Myc α-HA 37 IP: 250 IP: 37 α-Myc α α-FLAG α-FLAG -Myc 250 Interaction TPR Ubox with LRRK2 WT-CHIP 1 303 + _ LRRK2 B Myc-CHIP + + D CHIP-∆Ubox1 189 + FLAG-LRRK2 _ + + WT KO kDa CHIP-∆TPR - kDa α 145 303 α Input -LRRK2 250 Input -FLAG 250 IP: α-LRRK2 250 α α-IgG WTWTF1F2F3F4F5F6F7F8 IP: -FLAG 250 α-LRRK2 B FLAG-LRRK2 +++ + + + + + + + α IP: 250 _ -Myc α-Myc 37 α HA-CHIP + + ++ + + + + + -CHIP α-CHIP 37 kDa Input α-FLAG 75 50 E DAPI LRRK2 CHIP Merged 37 α-FLAG 75 IP: 50 α-HA 37 α-HA 37 Interaction ANKLRR Roc COR KinaseWD40 with CHIP Fig. 1. LRRK2 interacts with CHIP. (A) Lysates from SH-SY5Y cells transfected LRRK2-WT 12527+ F1 (1-480) - with HA-tagged CHIP and Myc-tagged LRRK2 were subjected to IP with F2 (480-895) - F3 (895-1339) + anti-Myc, followed by anti-HA immunoblotting (Middle) or with anti-Myc F4 (981-1503) + antibody (Bottom) to show an equivalent amount of immunoprecipitated F5 (1534-1857) - F6 (981-1298) - LRRK2. (B). Lysates from SH-SY5Y cells transfected with Myc-tagged CHIP and F7 (1866-2139) - FLAG-tagged LRRK2 subjected to IP with anti-Myc, followed by anti-FLAG F8 (2125-2527) - immunoblotting (Middle) or anti-Myc antibody (Bottom). (C) Lysates from Fig. 2. LRRK2 interacts with the TPR domain of CHIP, and CHIP associates SH-SY5Y cells transfected with HA-tagged CHIP and FLAG-tagged WT, R1441C, with the ROC domain of LRRK2 in SH-SY5Y cells. (A) Lysates from SH-SY5Y cells or G2019S LRRK2 constructs subjected to IP with anti-FLAG, followed by transfected with FLAG-LRRK2 and Myc-tagged CHIP domain constructs were anti-HA immunoblotting (Middle) or anti-FLAG antibody (Bottom). (D) In vivo subjected to IP with anti-Myc, followed by anti-FLAG immunoblotting (Mid- interaction in mouse brain from lysates prepared from mouse brain subjected dle) or anti-Myc (Bottom). The deletion domains of CHIP used are shown at the to immunoprecipitation with anti-CHIP, anti-IgG with immunoblotting using bottom of the panel. (B) Lysates from SH-SY5Y cells transfected with HA- anti-CHIP or anti-LRRK2, respectively. (E) Primary cortical neurons were ﬁxed tagged CHIP and FLAG-tagged fragments of LRRK2 subjected to IP with and stained with primary antibodies against LRRK2 and CHIP, followed by anti-HA antibodies, followed by anti-FLAG immunoblotting (Middle)oran- detection with secondary antibodies conjugated to Cy2 (LRRK2; green) or Cy3 ti-HA (Bottom). A schematic representation of the different LRRK2 fragments (CHIP; red). Superimposing 2 colors (merged) resulted in a yellow signal, used is shown. indicating colocalization of the 2 proteins.

tation experiments in SH-SY5Y cells transfected with a series of whether LRRK2 interacts with CHIP, we conducted in vivo deletion mutants of FLAG-tagged LRRK2 constructs and HA- coimmunoprecipitation experiments with brains from WT mice tagged CHIP, and found that CHIP interacted with the ROC and LRRK2 knockout (KO) mice (Fig. 1D). Immunoprecipita- domain of LRRK2 (Fig. 2B). tion of endogenous CHIP pulled down LRRK2 from the WT mouse brain but not from the LRRK2 KO mouse brain (Fig. 1D). LRRK2 Is a Substrate for the CHIP E3 Ligase. To ascertain whether The failure to immunoprecipitate LRRK2 from the LRRK2 KO CHIP ubiquitinates LRRK2, we performed in vivo ubiquitina- mouse brain indicates that the coimmunoprecipitation of CHIP tion experiments. We cotransfected SH-SY5Y cells with FLAG- and LRRK2 from WT mouse brain was specific. tagged LRRK2, Myc-tagged WT CHIP, CHIP⌬U-box, Next, we used double-labeling immunofluorescence confocal CHIP⌬TPR, and HA-tagged ubiquitin. FLAG-tagged LRRK2 microscopic analysis to determine the cellular localizations of was immunoprecipitated with an anti-FLAG antibody from the endogenous CHIP and LRRK2 in primary cortical neurons. We total cell extract. LRRK2 was ubiquitinated by CHIP, as shown found significant colocalization between endogenous LRRK2 by the substantial anti-HA immunoreactivity in SH-SY5Y cells and CHIP in the soma and neurites of cortical neurons (Fig. 1E). (Fig. 3A). CHIP lacking the U-box still ubiquintinated LRRK2, These findings support a physiological interaction between whereas CHIP lacking the TPR domain failed to ubiquitinate CHIP and LRRK2. LRRK2 (Fig. 3A). Moreover, proteasome inhibition with the proteosomal inhibitor clasto-lactacystin ␤-lactone augmented LRRK2 Interacts With CHIP Through Its TPR Domain, and CHIP Associ- CHIP-mediated LRRK2 ubiquitination in HEK293 cells (Fig. ates With LRRK2 Through Its Ras of Complex Proteins (ROC) Domain. 3C). We also found that CHIP ubiquitinated mutant R1441C and CHIP contains 2 major structural motifs, a TPR motif and a G2019S LRRK2 in a manner similar to WT LRRK2 (Fig. 3B). U-box domain. The TPR domain is required for interaction with We noted substantially reduced ubiquitination of WT and HSC70 and HSP90, whereas the U-box domain has ubiquitin mutant LRRK2 in the presence of CHIP⌬TPR (Fig. 3B). ligase activity (Fig. 2A). To determine the domain of CHIP that To explore whether CHIP degrades LRRK2, we generated interacts with LRRK2, we transfected Myc-tagged WT CHIP, SH-SY5Y stable cell lines expressing FLAG-tagged WT, CHIP lacking the U-box domain (CHIP⌬Ubox), and CHIP G2019S, and R1441C LRRK2 and monitored the steady-state lacking the TPR domain (CHIP⌬TPR) into SH-SY5Y cells with levels of WT, G2019S, and R1441C LRRK2 in the presence of FLAG-tagged LRRK2. We found that LRRK2 interacted with WT CHIP, CHIP⌬TPR, and WT CHIP plus ␤-lactone (Fig. 3D). the TPR domain of CHIP, as demonstrated by a marked WT CHIP led to a significant and substantial reduction in the reduction in the coimmunoprecipitation of LRRK2 with the steady- state levels of WT, G2019S, and R1441C LRRK2, CHIP⌬TPR mutant compared with the interaction of LRRK2 whereas CHIP⌬TPR had no substantial effect, and ␤-lactone with WT CHIP (Fig. 2A). To determine the domain of LRRK2 prevented the degradation of WT, G2019S, and R1441C LRRK2 that interacts with the CHIP, we performed coimmunoprecipi- by CHIP (Fig. 3D). To investigate whether reducing the CHIP

2898 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810123106 Ko et al. Downloaded by guest on September 30, 2021 level leads to an increase in LRRK2 level, we measured the LRRK2 level in HeLa cells in the presence of CHIP siRNA compared with scrambled siRNA (Fig. 3E). Knocking down the expression of endogenous CHIP led to a substantial increase in the levels of FLAG-tagged LRRK2 in HeLa cells (Fig. 3E). To examine whether in vitro knockdown of endogenous CHIP leads to the accumulation of LRRK2, we measured LRRK2 levels in primary neurons after shRNA knockdown of CHIP compared with scrambled shRNA control. We found that shRNA- mediated knockdown of CHIP increased the steady-state level of endogenous LRRK2 protein compared with control to the same degree as proteasomal inhibition with MG132 (Fig. 3F). To- gether, proteasomal inhibition and shRNA knockdown of CHIP modestly increases the level of endogenous LRRK2 protein. To ascertain whether CHIP regulates the levels of LRRK2 in vivo, we compared LRRK2 levels in 1-year-old WT mice and CHIP KO mice. We found a significant (4-fold) increase in LRRK2 level in the CHIP KO mice compared with the WT mice in the soluble fraction (Fig. 3G). Western blot analysis and quantifi- cation of ␣-synuclein and DJ-1 levels failed to exhibit any significant change in soluble extracts relative to control brain extracts (Fig. S1). Taken together, these findings indicate that LRRK2 is a substrate for the CHIP E3 ligase and that CHIP regulates the level of LRRK2 through proteasomal degradation.

CHIP Rescues LRRK2 Toxicity. To determine whether CHIP can alter cell toxicity induced by LRRK2 (24, 25, 35), we investigated the effects of overexpression and knockdown of CHIP on LRRK2-induced cell death (Fig. 4). We cotransfected SH-SY5Y cells with GFP, CHIP, and G2019S or R1441C LRRK2 by Lipofectamine Plus (Invitrogen). Viable cells were defined as those having at least one smooth neuronal process that was twice the length of the cell body. Consistent with other observations (24, 25, 35), there was mild and decreased viability with WT Fig. 3. CHIP regulates the steady-state level of LRRK2 via UPS degrada- LRRK2. In contrast to WT LRRK2, the G2019S and R1441C tion. (A) Lysates from SH-SY5Y cells transfected with Myc-tagged WT CHIP, mutants caused significant cell toxicity compared with control Myc-tagged ⌬U-box or Myc-tagged ⌬TPR, HA-tagged ubiquitin, and FLAG- transfected cells (Fig. 4B and C). Importantly, CHIP overex- tagged LRRK2 subjected to immunoprecipitation with anti-FLAG, followed Ϸ by immunoblotting with anti-HA (Third panel) and anti-FLAG (Fourth pression reduced G2019S and R1441C toxicity by 50% in panel) for in vivo ubiquitination assays. Lysates also were probed with an SH-SY5Y cells (Fig. 4A, Top and B). In a similar manner, CHIP anti-Myc antibody (Second panel) and anti-HA (First panel) to demonstrate reduced G2019S and R1441C toxicity in primary cortical neu- ubiquitin and CHIP expression. Brackets indicate ubiquitinated LRRK2. (B) rons by Ϸ50% (Fig. 4A, Bottom and C). Knocking down CHIP Lysates from SH-SY5Y cells transfected with Myc-tagged WT CHIP and levels with siRNA significantly enhanced WT, G2019S, R1441C, Myc-tagged ⌬TPR, HA-tagged ubiquitin and FLAG-tagged WT and patho- and Y1699C LRRK2 toxicity compared with scrambled siRNA genic forms (R1441C and G2019S) of LRRK2 were subjected to immunopre- in SH-SY5Y cells (Fig. 4D). These results indicate that CHIP NEUROSCIENCE cipitation with anti-FLAG, followed by immunoblotting with anti-HA influences LRRK2 toxicity by regulating LRRK2 protein levels. (Third panel) and anti-FLAG (Fourth panel). Lysates also were probed with an anti-HA (First panel) and anti- Myc antibody (Second panel) to demonstrate ubiquitin and CHIP expression. Brackets indicate ubiquitinated HSP90 Regulates the Stability of LRRK2. Because CHIP and LRRK2 LRRK2. (C) HEK 293T cells were transfected with FLAG-tagged WT LRRK2 interact with HSP90 (18, 28), we performed immunoprecipita- and HA-tagged ubiquitin along with either Myc-CHIP or empty vector. tion experiments to further explore their interactions (Fig. 5). After 24 h, cells were treated with 10 ␮M ␤-lactone for 24 h. FLAG We transfected HeLa cells with FLAG-tagged WT LRRK2 and coimmunoprecipitation demonstrated that LRRK2-specific ubiquitination Myc-tagged CHIP, Myc-CHIP⌬U-box, Myc-CHIP⌬TPR, or was greatly increased in the presence of CHIP and/or ␤-lactone. (D) SH-SY5Y Myc-CHIP-K30A, followed by immunoprecipitation of Myc. We cells stably expressing FLAG-tagged LRRK2 and pathogenic forms (R1441C found that HSP90, HSP70, and LRRK2 coimmunoprecipitated and G2019S) of LRRK2 were transiently transfected with Myc-WT CHIP or with CHIP, but failed to coimmunoprecipitate with CHIP Myc-CHIP⌬TPR, and 24 h later treated with 5 ␮M ␤-lactone for 18 h. Total lysates were immunoblotted with anti-FLAG antibody to show the steady- lacking the TPR domain or CHIP K30A mutant within the TPR state LRRK2 level (Upper), with anti-Myc to show relative levels of CHIP domain (Fig. 5A). These findings suggest that the CHIP–LRRK2 (Middle), and with anti-actin to confirm equivalent loading (Bottom). (E) interaction depends on CHIP’s interaction with the HSPs. To HeLa cells were first transfected in triplicate with a nonsilencing control ascertain whether HSP90 could regulate the steady-state levels (scramble) or CHIP siRNA and then transfected with FLAG-tagged WT of LRRK2, we monitored LRRK2 levels in the presence of LRRK2 and Myc-CHIP or empty vector for 72 h, after which they were increasing concentration of the HSP90 inhibitor, 17AAG. harvested for Western blot analysis of CHIP and LRRK2. Cells exposed to the HSP90 and HSP70 levels increased after 17AAG treatment with CHIP siRNA exhibited a 20%–25% increase in LRRK2 level, whereas cells a concomitant reduction in WT LRRK2 levels (Fig. 5B). Next, exposed to the WT CHIP had a 20%–25% decrease in LRRK2 level on we monitored mutant LRRK2 levels in the presence of the quantitative densitometric analysis. (F) Primary cortical neurons were in- ␮ fected with lentiviruses delivering shRNAs for 2 days and then treated with HSP90 inhibitor, 17AAG (1 M). 17AAG significantly reduce 10 ␮M MG132 for 18 h. Proteins levels were monitored by Western blot the levels of WT and mutant G2019S and R1441C LRRK2 (by analysis, with actin as a loading control. (G) Mouse brains from WT and CHIP 78.5%, 67.8%, and 75.2%, respectively) (Fig. 5C). To investigate KO mice were analyzed by Western blot analysis (41). In panels D–G, values the mechanism by which HSP90 inhibition reduces LRRK2 represent optical density Ϯ SD, normalized to actin. *P Ͻ .05; **P Ͻ .005. expression, we used 17AAG to assess heat-shock factor 1 (HSF1)

Ko et al. PNAS ͉ February 24, 2009 ͉ vol. 106 ͉ no. 8 ͉ 2899 Downloaded by guest on September 30, 2021 Mock+CHIP Mock+G2019S G2019S+CHIP A B 120 SH-SY5Y 100 80 * # 60

SH-SY5Y SH-SY5Y 40 20

Relative viability (%) 0 Mock + _ _ _ _ _ CHIP _ + _ + _ +

Neuron G2019S _ _ + + _ _ R1441C _ _ _ _ + +

120 CD120 Neuron Scramble 100 100 siRNA CHIP # 80 * 80 * 60 60 * 40 * 40 * 20 20 Relative viability (%) 0 _ _ _ _ _ Relative viability (%) Mock + 0 CHIP _ + _ + _ + G2019S _ _ + + _ _ WT _ _ _ _ Mock R1441C + + G2019S Y1699C R1441C

Fig. 4. CHIP protects against and knockdown of CHIP exacerbates mutant LRRK2-induced toxicity. (A, Top) SH-SY5Y cells cotransfected with pcDNA3.1- GFP and vector pcDNA3.1, pcDNA3.1-FLAG-LRRK2-G2019S, or LRRK2-R1441C with or without Myc-CHIP at a 1:15 ratio. GFP-positive cells with 2-fold continuous extensions were counted as live cells. Representative photomicrographs for each experimental group are shown. The transfection efﬁciency of GFP was about 10%. (B) Quantitation of the data shown in panel A (Top), representing the cell viability of each experimental group normalized to that of cells cotransfected with empty vector and GFP. Data are mean Ϯ SE for 3 separate experiments performed in duplicate. *P Ͻ .05 vs. cells cotransfected with LRRK2-G2019S, vector, and GFP; #P Ͻ .05 vs. cells cotransfected with LRRK2-R1441C, vector, and GFP by ANOVA. (A, Bottom) Mouse primary cortical neurons cotransfected with pcDNA3.1-GFP and indicated constructs. GFP-positive viable neurons with 2-fold continuous neurites were counted as live neurons. Representative photomicrographs for each experimental group are shown. (C) Quantitation of the data shown in A (Bottom), representing the cell viability of each experimental group normalized to that of cells cotransfected with empty vector and GFP. Data are mean Ϯ SE for 3 separate Ͻ experiments performed in duplicate. *P .05 vs. cells cotransfected with Fig. 5. 17AAG decreases LRRK2 protein levels and associated toxicity in an Ͻ LRRK2-G2019S, vector, and GFP; #P .05 vs. cells cotransfected with LRRK2- HSP70/90-dependent manner. (A) HeLa cells transfected with FLAG-tagged R1441C, vector, and GFP by ANOVA. (D) SH-SY5Y cells transfected with non- WT LRRK2 and either Myc-tagged CHIP, Myc-tagged ⌬U-box, Myc-tagged silencing control or CHIP siRNA for 24 h. The cells were cotransfected with ⌬TPR, Myc-CHIP with the K30A mutation, or empty vector. Myc coimmuno- pcDNA3.1-GFP and pcDNA3.1, FLAG-tagged WT, G2019S, Y1699C, or R1441C precipitation shows that loss of the TPR domain or the K30A mutation abro- LRRK2. GFP-positive cells with 2-fold continuous extensions were counted as gates LRRK2–CHIP binding. (B) HeLa cells transfected with FLAG-tagged WT live cells. The quantitation of data represents the cell viability of each exper- LRRK2 were treated with the indicated concentrations of 17AAG or DMSO. imental group normalized to that of cells cotransfected with empty vector and Western blot analysis of cell lysates shows a 17AAG-mediated reduction of Ϯ GFP. Data are mean SE for 3 separate experiments performed in duplicate. LRRK2 protein levels, with a corresponding HSP70 induction in those cells. (C) Ͻ *P .05 vs. cells cotransfected with WT, LRRK2-G2019S, LRRK2-Y1699C, or HeLa cells transfected with WT, G2019S, or R1441C LRRK2 were treated with LRRK2-R1441C vector and GFP by ANOVA. 1 ␮M 17AAG or DMSO. LRRK2 WT, G2019S, and R1441C mutant LRRK2 protein levels are signiﬁcantly decreased (by 78.5%, 67.8%, and 75.2%, respectively) with 17AAG treatment (black bars) compared with vehicle treatment (gray and HSP90 expression in HeLa cells that overexpressed WT bars) after normalization to GAPDH and total LRRK2 levels (n ϭ 4). (D and E) LRRK2. We found that 17AAG reduced LRRK2 levels by 50% 17AAG-mediated degradation of LRRK2 requires a constitutive chaperone in cells transfected with nonsilencing siRNA, but siRNA sup- response. HeLa cells were transfected in duplicate with nonsilencing control, pression of HSP90 expression abrogated the 17AAG-mediated HSP90, and HSF1 siRNA and then transfected with FLAG-tagged WT LRRK2 and LRRK2 reductions (Fig. 5D). Suppression of HSF1 expression treated with 1 ␮M 17AAG or DMSO. Lysates were analyzed by Western blot. by siRNA had no effect on the 17AAG-mediated LRRK2 17AAG causes a strong decrease in LRRK2 in nonsilencing control. (D) Knock- E down of HSP90 increases LRRK2 levels in DMSO-treated cultures, but LRRK2 is reductions (Fig. 5 ). To determine whether 17AAG protects increased only marginally in 17AAG treated cells. (E) HSF1 siRNA increases against LRRK2 toxicity, we transfected SH-SY5Y cells with WT, LRRK2 levels compared with nonsilencing control. Treatment with 17AAG G2019S, and R1441C LRRK2 and monitored cell viability. We decreases LRRK2 levels comparably to the nonsilencing control treated with found that a 24-h treatment with 17AAG significantly protected 17AAG. (F) 17AAG protects against LRRK2 toxicity in SH-SY5Y cells treated against G2019S- and R1441C-induced cell death (Fig. 5F). with 10 nM 17AAG. Data are for 3 separate experiments done in duplicate and normalized to cells cotransfected with empty vector and GFP. Data are mean Ϯ Discussion SE. *P Ͻ .05. CHIP is implicated in various neurodegenerative diseases characterized by protein misfolding and aggregation (31, 36, 37). Our major finding in the current study is that CHIP is an ubiquitin E3 LRRK2, and the ROC domain of LRRK2 is required for the ligase for LRRK2 that regulates LRRK2 levels. LRRK2 inter- binding to CHIP. WT and mutant LRRK2 are polyubiquitinated acts, coimmunoprecipitates, and colocalizes with CHIP, and the by CHIP, and overexpression of CHIP decreases the steady-state 2 proteins coimmunoprecipitate from mouse brain. Familial level of WT and mutant LRRK2 in a proteasomal-dependent associated mutations in LRRK2 do not alter the interaction with manner. LRRK2 is an authentic CHIP substrate, as demon- CHIP. The TPR domain of CHIP is required for binding to strated by our findings that siRNA knockdown of CHIP leads to

2900 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810123106 Ko et al. Downloaded by guest on September 30, 2021 up-regulation of LRRK2 in SH-SY5Y cells, shRNA knockdown the chaperone system with aging may interfere with turnover of of CHIP leads to up-regulation of LRRK2 in primary neurons, LRRK2, leading to aggregation and toxicity and subsequent and knockout of CHIP in mice leads to the accumulation of Parkinson’s disease. Strategies that enhance HSP90- or CHIP- LRRK2 in aging CHIP KO brain tissue compared with age- mediated degradation could be possible therapeutic strategies to matched WT controls. Moreover, the up-regulation of LRRK2 treat LRRK2-linked PD. by CHIP knockdown is equivalent to the level of up-regulation induced by proteasomal inhibition. The toxicity of LRRK2 Materials and Methods toxicity depends on its level of expression, because overexpres- Plasmids, Antibodies, and Reagents. Details about the materials used in this sion of CHIP protects against mutant LRRK2 toxicity and study are provided in SI Materials and Methods. siRNA knockdown of CHIP enhances WT and mutant LRRK2 toxicity. HSP90 and HSP70 also interact with LRRK2 through Cell Culture and Transfection. Human neuroblastoma SH-SY5Y cells and HeLa cells were cultured as described previously (39, 40) and detailed in SI Materials CHIP’s TPR domain, and the binding of CHIP to LRRK2 is and Methods. mediated through these HSPs as a single point mutation in the TPR domain of CHIP that prevents its interaction with HSPs, Mouse Primary Cortical Neuronal Cultures and Electroporation Transfection. which abrogates CHIP’s interaction with LRRK2. Increasing the Mouse primary cortical neuronal cultures were derived from CD-1 mice (Jack- steady-state level of HSPs with the glendamycin derivative, son Laboratory) at E15–16. Cortices were dissociated, plated on laminin- and 17-AAG, reduces LRRK2 levels and toxicity. Together, these polyD-lysine–coated plates (BD Biosciences), and cultured in Neurobasal me- findings indicate that LRRK2 is a client of the CHIP–HSP dium with the addition of Glutamax, B-27 supplement, and penicillin/ chaperone system, and that regulation of LRRK2 levels through streptomycin (24). Under these culture conditions, 95% of cells were neurons. this system is potentially involved in LRRK2 toxicity. Transfection of LRRK2 constructs into mouse primary cortical neurons was carried out using Nucleofector (Amaxa Biosystems). HSP90 is a molecular chaperone crucial to the stability and function of many client proteins that promote cancer cell growth Immunoprecipitation and Western Blot Analysis. Coimmunoprecipitation and and survival (38). We and others have reported that HSP90 Western blot analysis experiments were conducted using standard tech- forms a complex with LRRK2 (18, 28), raising the possibility that niques. Details are provided in SI Materials and Methods. HSP90 plays an important role in the maintenance of LRRK2 protein quality by regulating the balance between the folding and In Vivo Ubiquitination Assay. SH-SY5Y cells were transiently transfected with degradation of LRRK2. The site of LRRK2–CHIP interaction 2 ␮g of Myc-tagged WT CHIP or Myc-tagged CHIP mutants, pcDNA3.1-FLAG- appears to be in the TPR domain, which is known to mediate tagged WT LRRK2, G2019S, R1441C and 2 ␮g of pMT123-HA-ubiquitin plas- interactions with chaperones, such as HSP90 and HSP70 (31). mids. 48 h later total cell lysates were harvested, the pellets were solubilized Thus, it appears that chaperone interaction is essential for in 2% SDS with sonication. The samples were split for use as input and for immunoprecipitation. Antibodies against FLAG were used for immunopre- LRRK2 ubiquitination and degradation by CHIP in vivo. In the cipitation followed by Western blot analysis with anti-HA, anti-Myc, and present study, we found that treatment with the HSP90 inhibitor anti-FLAG antibodies and detection with Super Signal West Pico and Femto 17AAG reduced the steady-state levels of WT LRRK2 and chemiluminescent substrates (Pierce Biotechnology). LRRK2 mutants. HSP90 knockdown suppressed the 17AAG- mediated reductions in LRRK2 levels (Fig. 5D), whereas sup- Measurement of Cell Viability. SH-SY5Y and neuron cell viability assay were pression of HSF1 expression by RNA interference prevented conducted as described previously (24) and as detailed in SI Materials and up-regulation of HSP70 (Fig. 5E), yet had no effect on 17AAG- Methods. mediated LRRK2 reductions. Together, these findings suggest that LRRK2 is processed through the constitutive HSP90 re- Statistical Analysis. Quantitative data are expressed as arithmetic mean Ϯ SE folding system, where it is prone to proteasomal degradation, based on at least 3 separate experiments performed in duplicate or quadru- plicate. The difference between 2 groups was analyzed using Student’s t-test rather than being dependent on de novo transcription of HSP or 1-way ANOVA. Signiﬁcance was deﬁned at P Ͻ .05. chaperones stimulated by HSF1. In summary, our findings provide evidence that LRRK2 is a ACKNOWLEDGMENTS. This work was supported by the Morris K. Udall Par- NEUROSCIENCE substrate for the CHIP E3 ligase and HSP90 chaperone system kinson’s Disease Research Center; National Institutes of Health/National Insti- and that CHIP and the HSP90 chaperone system are essential for tute of Neurological Disorders and Stroke Grants NS38377, NS54207, and the proper degradation of LRRK2. Modulating CHIP levels may NS04826, and National Institute of Aging Grant AG017216; the National Parkinson Foundation; and the American Parkinson Disease Association. be a critical determinant in LRRK2 toxicity. Because CHIP is T.M.D. is the Leonard and Madlyn Abramson Professor in Neurodegenerative part of the HSP90 chaperone system, perturbations in CHIP and Diseases.

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