Ser46 Phosphorylation and Prolyl-Isomerase Pin1- Mediated Isomerization of P53 Are Key Events in P53- Dependent Apoptosis Induced by Mutant Huntingtin

Ser46 phosphorylation and prolyl-isomerase Pin1- mediated isomerization of p53 are key events in p53- dependent apoptosis induced by mutant huntingtin

Alice Grisona,1,2, Fiamma Mantovania,b,1, Anna Comela,b, Elena Agostonic, Stefano Gustincichc,d,e, Francesca Persichettic,f, and Giannino Del Sala,b,3

aLaboratorio Nazionale Consorzio Interuniversitario per le Biotecnologie, 34149 Trieste, Italy; bDepartment of Life Sciences, University of Trieste, 34100 Trieste, Italy; cSector of Neurobiology, International School for Advanced Studies, 34136 Trieste, Italy; dInternational School for Advanced Studies Unit, Italian Institute of Technology, 34136 Trieste, Italy; eGiovanni Armenise–Harvard Foundation Laboratory, International School for Advanced Studies, 34136 Trieste, Italy; and fDepartment of Environmental and Life Sciences, University of Eastern Piedmont, 15121 Alessandria, Italy

Edited* by Carol Prives, Columbia University, New York, NY, and approved September 13, 2011 (received for review April 18, 2011)

Huntington disease (HD) is a neurodegenerative disorder caused by and PKCδ. The subsequent transduction of stress-dependent a CAG repeat expansion in the gene coding for huntingtin protein. phosphorylation into speciﬁc conformational changes of p53 that Several mechanisms have been proposed by which mutant hunting- fully unleash its apoptotic activity is performed by the prolyl tin (mHtt) may trigger striatal neurodegeneration, including mito- isomerase Pin1. This enzyme catalyzes cis/trans isomerization of chondrial dysfunction, oxidative stress, and apoptosis. Furthermore, proline bonds preceded by phosphorylated serine or threonine mHtt induces DNA damage and activates a stress response. In this residues (pSer/Thr-Pro), thereby altering structure and functions context, p53 plays a crucial role in mediating mHtt toxic effects. Here of its substrates (10, 11). Upon genotoxic insults, Pin1 binds mul- we have dissected the pathway of p53 activation by mHtt in human tiple sites on p53, promoting its accumulation in stressed cells, the activation of its transcriptional functions, and the induction of its neuronal cells and in HD mice, with the aim of highlighting critical – nodes that may be pharmacologically manipulated for therapeutic apoptotic activity (12 14). intervention. We demonstrate that expression of mHtt causes in- Phosphorylation-dependent prolyl isomerization triggered by creased phosphorylation of p53 on Ser46, leading to its interaction Pin1 represents an essential mechanism in modulating several signaling pathways involved in DNA damage and apopotosis. In with phosphorylation-dependent prolyl isomerase Pin1 and conse- the CNS, Pin1 is highly expressed and regulates several substrates, quent dissociation from the apoptosis inhibitor iASPP, thereby including the hyperphosphorylated form of tau in Alzheimer’s inducing the expression of apoptotic target genes. Inhibition of disease (15), whereas in Parkinson disease (PD) Pin1 facilitates Ser46 phosphorylation by targeting homeodomain-interacting pro- α δ formation of -synuclein inclusions (16). tein kinase 2 (HIPK2), PKC , or ataxia telangiectasia mutated kinase, Based on these considerations, we reasoned that Pin1 could be as well as inhibition of the prolyl isomerase Pin1, prevents mHtt- critical for mediating p53-dependent mHtt toxicity. Therefore, dependent apoptosis of neuronal cells. These results provide a ratio-

by investigating its role in this context, we may highlight crucial CELL BIOLOGY nale for the use of small-molecule inhibitors of stress-responsive upstream events involved in HD pathogenesis and unveil at- protein kinases and Pin1 as a potential therapeutic strategy for HD tractive targets for development of novel therapeutic options to treatment. counteract HD.

untington disease is a dominantly inherited neurodegenera- Results Htive disorder due to an expanded CAG repeat sequence in the Expression of Mutant Huntingtin Promotes the Interaction of p53 HD gene that elongates a segment of glutamine residues in the with Pin1. Analysis of postmortem brains of HD patients revealed protein huntingtin (Htt) (1). The most striking pathological man- high levels of p53 relative to healthy controls (Fig. 1A), in agree- ifestation of HD is a gradual loss of neurons, predominantly in the ment with previous reports (5). To study the stress pathways re- striatum, causing motor abnormalities and cognitive decline (2). sponsible for p53 activation in HD neurons, we then analyzed p53 Toxic properties of mutant huntingtin (mHtt) are believed to cause phosphorylation. Interestingly, in HD brains, p53 was phosphory- HD. Among them, mitochondrial dysfunction and generation of lated on Ser46 (Fig.1A), a modification that has been associated reactive oxygen species (ROS) lead to DNA lesions (3, 4). In- with activation of its apoptotic function upon stress (14, 17). Nu- terestingly, expression of full-length mHtt protein and N-terminal clear accumulation of mHtt N-terminal fragments is observed in fragments containing the polyQ expansion elicit a DNA damage HD brains (18) and animal models (18). Expression of these response, with activation of the ATM/ATR pathways (3, 4) and truncated forms recapitulates many molecular and neurological their downstream effectors, including the tumor suppressor p53 (3, HD phenotypes (19). The N-terminal fragment (residues 1–171) of 5, 6). p53 mediates mitochondrial dysfunction and cytotoxicity in either wild-type or mutant Htt (bearing 21 and 150 polyQ, re- HD cells and in transgenic animal models, whereas its inhibition spectively) were thus expressed in SH-SY5Y human neuroblas- prevents these phenotypes (5). p53 governs a wide array of pathways involved in genomic stability, antioxidant activities, and energy metabolism in addition to Author contributions: F.M. and G.D.S. designed research; A.G., F.M., A.C., and E.A. per- promoting either cytostatic or cytotoxic responses to intrinsic and formed research; E.A., S.G., and F.P. contributed new reagents/analytic tools; F.M. and exogenous sources of cellular stress (7). Given the complexity of G.D.S. analyzed data; and F.M., F.P., and G.D.S. wrote the paper. p53 functions within the cell, a better understanding of how sig- The authors declare no conflict of interest. naling networks converge on this hub to modulate mHtt-dependent toxicity is required to shed light on the reduced ability of *This Direct Submission article had a prearranged editor. the brain neurons to survive. Regulation of p53 activities relies on Freely available online through the PNAS open access option. a complex network of posttranslational modifications and protein 1A.G and F.M. contributed equally to this work. interactions (8, 9), which ultimately determine the outcome of the 2Present address: Sector of Neurobiology, International School for Advanced Studies, p53 response. This process entails site-specific phosphorylation by 34136 Trieste, Italy. several DNA damage-activated protein kinases, including, among 3To whom correspondence should be addressed. E-mail: [email protected]. others, ataxia telangiectasia mutated (ATM), ATM and Rad3-re- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lated (ATR), homeodomain-interacting protein kinase 2 (HIPK2), 1073/pnas.1106198108/-/DCSupplemental.

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Fig. 1. Expression of mutant huntingtin induces the interaction of p53 with Pin1. (A) Phosphorylation of p53 on Ser46 in postmortem brains was compared

between HD patients (HD1 and HD2) and healthy controls (C) by Western blot. (Middle and Bottom) Levels of total p53 and actin as loading control. (B) SH- SY5Y cells were transfected with constructs expressing the N-terminal 1–171 Htt fragment with either 21Q or 150Q. p53 was immunoprecipitated from equal amounts of total cell lysates and analyzed by Western blot with antibodies speciﬁc for phosphorylated Ser46 and total p53. The levels of actin and Htt proteins in input lysates are shown. (C) SH-SY5Y were transfected with indicated constructs. Cell lysates normalized for p53 protein levels were subjected to coimmunoprecipitation to analyze interaction of endogenous p53 and Pin1. (D) H1299 cells were transfected with indicated constructs, and the interaction of p53 with recombinant Pin1 protein was analyzed by GST pull-down of cell lysates normalized for p53 levels.

toma cells to verify whether p53 Ser46 phosphorylation was a We then analyzed activation of the p53 response in the brains of consequence of mHtt expression. Interestingly, mutant but not Hdh CAG knock-in mice in which the glutamine tract of mouse Htt wild-type Htt induced the phosphorylation of endogenous p53 on is extended to 111 residues (HdhQ111) (20). These mice show striatal Ser46, in addition to the previously reported phosphorylation of neurodegeneration, reactive gliosis, and gait abnormalities at older Ser15 (6) (Fig. 1B). Because Ser46 phosphorylation generates age (after 24 mo) (21). However, we observed stabilization of p53 in a target site for the prolyl isomerase Pin1 (12, 14), we asked whether brain extracts and the consequent transcriptional induction of the Pin1 might play a role in mediating activation of p53 upon mHtt p53 target gene p21WAF1 in the striatum of 12-mo-old HdhQ111 mice expression in neuronal cells. Strikingly, expression of the mHtt- compared with their WT littermates, HdhQ7 (Fig. 2D). This finding 150Q fragment was sufficient to promote the interaction of en- suggests that activation of the p53 pathway by mHtt-associated dogenous p53 and Pin1 proteins in SH-SY5Y cells (Fig. 1C). stress is an early event in HD pathogenesis and could precede neu- Moreover, mHtt expression stimulated direct interaction of p53 rological symptoms. HdhQ111 mice were then crossed with Pin1KO with Pin1 as demonstrated by GST-Pin1 pull-down assays (Fig. 1D), mice (22) to verify whether Pin1 is required for p53 activation. The and this effect was proportional to the amount of mHtt. Of note, interaction between p53 and Pin1 was clearly detectable in protein Pin1 neither interacted with Htt (1–171) protein fragments (Fig. S1 extracts obtained from HdhQ111/Pin1WT mouse brains (Fig. S3A), A and B) nor affected mHtt protein levels (Fig. S1 C and D). confirming what was observed in human cells. Importantly, in contrast to HdhQ111/Pin1WT mice, p53 transcriptional activity was Pin1 Mediates Activation of the p53 Pathway by mHtt. It has been not induced in HdhQ111/Pin1KO mice (Fig. 2D), and the expression previously shown that expression of mHtt in SH-SY5Y cells trig- of the apoptotic p53 target PUMA in HdhQ111 mouse brains was gers a p53-dependent response involving the activation of apo- indeed dependent on Pin1 (Fig. 2E). We therefore analyzed ptotic genes, including Bax and PUMA (5). As shown in Fig. 2A, striatal neurodegeneration in 24-mo-old mice (Fig. S3 B and D). Pin1 potentiated the induction of p53 transcriptional activity by In agreement with published data (21), moderate neuronal loss mHtt. Moreover, induction of endogenous PUMA in response to was observed in HdhQ111 mice compared with WT Htt littermates mHtt expression in these cells also required Pin1 (Fig. 2B). on a Pin1 WT background (Fig. S3D). Pin1 KO mice also showed In agreement with previous reports (5), expression of mHtt in a similar reduction of striatal neurons number compared with WT SH-SY5Y cells provoked apoptosis, which was reduced by 50% littermates, which could possibly be ascribed to the reported age- upon silencing p53 expression (Fig. 2C). The same effect was ob- dependent neurodegeneration of mice lacking Pin1 (23). Impor- served upon silencing the expression of Pin1. Importantly, mHtt- tantly, in Pin1 KO mice the numbers of striatal neurons did not induced apoptosis could be reestablished in Pin1-depleted cells by further decrease on mHtt expression, suggesting that lack of p53 overexpression of a siRNA-resistant Pin1 construct; this was not activation (Fig. 2 D and E) might indeed prevent Htt-dependent effective in cells depleted of p53 (Fig. 2C), suggesting that the effect neurodegeneration in mice devoid of Pin1. of Pin1 relies on p53. It is noteworthy that expression of a catalyt- These results indicate that Pin1 plays a critical role for p53 ically inactive Pin1 mutant was unable to rescue knockdown of activation in response to mHtt expression in striatal neurons of endogenous Pin1, proving that the prolyl isomerase activity is es- a mouse model of HD pathogenesis. sential for transducing mHtt-dependent stress into p53 activation. Similar results were also observed in another neuroblastoma cell Phosphorylation of p53 on Ser46 by HIPK2 and PKCδ Is an Upstream line, SK-N-SH (Fig. S2). These data indicate that the Pin1/p53 Event in the mHtt-Pin1-p53 Pathway. Our observations indicated pathway plays a major role in neuronal apoptosis induced by mHtt. that phosphorylation of p53 on Ser46 is triggered by mHtt

17980 | www.pnas.org/cgi/doi/10.1073/pnas.1106198108 Grison et al. Downloaded by guest on September 24, 2021 AB (Fig. 3B). Both p53 WT and p53 3M-S46WT were then able to induce the proapoptotic p53 target PUMA upon transfection of mHtt, whereas p53 S46A was almost inactive (Fig. 3B). Of note, similar experiments performed in mouse neuroblastoma cells demonstrated that phosphorylation of Ser58, the mouse homolog of p53 Ser46, is essential for mHtt-induced apoptosis (Fig. S4B). Therefore, we concluded that phosphorylation of p53 on Ser46 is a crucial event in the pathway leading to neuronal death induced by mHtt in human and mouse cells. Our data also indicate that modification at this site is sufficient for Pin1 to enhance p53’s C apoptotic function in cells expressing mHtt. Among the protein kinases that catalyze phosphorylation of p53 on Ser46, HIPK2 plays a key role in unleashing p53’s apoptotic activity upon DNA damage (24). HIPK2 is induced by cytotoxic stimuli through the ATM/ATR pathway (25), which becomes activated upon mHtt-dependent stress (3). Interestingly, expression of mHtt was sufficient to up-regulate HIPK2 protein levels in SH-SY5Y cells, and this effect required ATM kinase activity because it was prevented by treatment with the ATM-specific D inhibitor KU55933 (Fig. 3C). We thus inhibited HIPK2 expression by RNAi, which dampened mHtt-dependent phosphorylation of p53 on Ser46 with concomitant decrease of apoptosis (Fig. 3D). This result indicates a major role for HIPK2 in the activation of p53 by mHtt. Interestingly, depletion of HIPK2 did not fully prevent Ser46 phosphorylation triggered by mHtt (Fig. 3D), implying the involvement of other kinases in inducing apoptosis downstream of mHtt. We postulated that PKCδ might concur to this effect, because its activation has been reported to regulate p53 during neuronal death (26). E Knockdown of PKCδ by RNAi strongly decreased phospho-Ser46 (Fig. 3D), consistent with the notion that this enzyme phosphorylates Ser46 upon DNA damage (27). Moreover, mHtt-dependent apoptosis was also reduced. Importantly, concomitant knockdown of both HIPK2 and PKCδ synergized to inhibit both Ser46 phosphorylation and mHtt-induced apoptosis (Fig. 3D). The reduction of apoptosis exceeded that obtained by silencing p53, suggesting Fig. 2. Pin1 activity is required for induction of p53-dependent apoptosis by δ mHtt. (A) The cooperative effect of mHtt and Pin1 on p53 transcriptional that HIPK2 and PKC might also regulate p53-independent apo- CELL BIOLOGY activity was evaluated by transfecting SH-SY5Y cells with pG13-Luc reporter ptotic pathways downstream to mHtt. and vectors expressing mHtt(1–171)150Q and increasing amounts (+, ++) of Pin1-HA. The graph shows means and SD of three independent experiments. Pin1 Unlocks p53 from iASPP in Response to mHtt Expression. We (B) SH-SY5Y cells were transfected with indicated constructs and siRNA oli- have previously shown that Pin1-mediated isomerization of phos- gonucleotides, and the expression of the p53 target PUMA was evaluated by phorylated Ser46-Pro47 site unleashes p53’s apoptotic potential Western blot after 48 h. (C) SH-SY5Y cells were transfected with the indicated upon DNA damage, by leading to its dissociation from the apo- combinations of constructs expressing mHtt(1–171)150Q-GFP, Pin1 siRNA ptosis inhibitor iASPP (14). Because mHtt triggers both DNA oligonucleotides, and siRNA-resistant wild-type Pin1-HA (WT) or the catalyt- damage (3) and Ser46 phosphorylation, we investigated whether it ically inactive mutant S67E (CI). Apoptosis of mHtt GFP-expressing cells was can induce p53 dissociation from iASPP. Coimmunoprecipitation evaluated by TUNEL assay after 48 h. The histograms show mean and SD of experiments highlighted that the interaction between p53 and WAF1 three independent experiments. (D) Expression of p21 mRNA was ana- iASPP was progressively lost upon expression of increasing lyzed by qRT-PCR from the striatum of 12-mo-old mice of the indicated gen- amounts of mHtt in H1299 cells (Fig. 4A). This effect correlated otypes (at least three mice for each genotype), normalizing for expression of B β with and required Ser46 phosphorylation (Fig. 4 ): indeed, WT -actin. A t test was performed using homoschedastic variance through Htt caused neither p53 Ser46 phosphorylation (Fig. 4B) nor iASPP groups and one tail parameter. p53 was immunoprecipitated from equal detachment (Fig. 4C), and S46A mutation impaired the dissocia- amounts of brain lysates of the same mice and analyzed by Western blot. C Actin protein levels in input lysates are shown. (E) The expression of the p53 tion of p53 from iASPP on mHtt expression (Fig. S4 ). Impor- tantly, when Pin1 expression was silenced, p53 remained bound to target PUMA in total brain lysates of 12-mo-old mice of the indicated geno- D types (two mice for each genotype, 1 and 2) was analyzed by Western blot. iASPP regardless of mHtt expression (Fig. 4 ). These data indicate that blocking Pin1 prevents p53 from inducing proapoptotic effectors due to the sustained inhibition by iASPP even in presence (Fig. 1 A and B). To define whether this or other phosphorylations of mHtt-dependent stress. are responsible for activating p53 apoptotic response upon expression of mHtt, we used p53 phosphorylation mutants with single Interfering with p53 Activation by Pin1 Prevents Apoptosis and multiple substitutions of Ser/Thr with Ala residues within Pin1 Downstream of mHtt. Given that p53 activation mediates the cy- binding sites (14) (Fig. 3A). Expression of these proteins in p53- totoxic effects of mHtt, we hypothesized that pharmacologic inhibition of catalytic activity of either Pin1 or of the kinases that null H1299 cells demonstrated that Ser46 is required for mHtt- phosphorylate p53 on the Pin1 target site Ser46 could prove ef- induced apoptosis, because a Ser46-Ala p53 mutant was unable to B fective in preventing mHtt-induced apoptosis. In fact, treatment cause apoptosis in response to mHtt expression (Fig. 3 ). In with the specific Pin1 inhibitor PiB (28) reduced mHtt-dependent contrast, a p53 mutant (p53 3M-S46wt) that lacked the remaining apoptosis (Fig. 5A) with efficiency similar to knockdown of Pin1 or three major Pin1 binding sites (i.e., Ser33, Thr81, and Ser315) p53 (Fig. 2C). Intriguingly, inhibition of Pin1 also appeared to could efficiently induce apoptosis downstream to mHtt expres- reduce Ser46 phosphorylation. Treatment of SH-SY5Y cells with sion. This protein was phosphorylated on Ser46 in cells expressing rottlerin, a compound widely used to inhibit PKCδ activity, also mHtt (Fig. S4A), and its apoptotic activity was potentiated by Pin1 reduced mHtt-dependent cellular toxicity and dampened Ser46

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Fig. 3. p53 Ser46 phosphorylation is promoted by mHtt through HIPK2 and PKCδ and is required for apoptosis induction by Pin1. (A) p53 scheme indicating Pin1 consensus sites (phospho–Ser/Thr-Pro). DBD, DNA binding domain; NLS, nuclear localization signal; TA, transactivation domain. p53 mutants have Ser/ Thr-to-Ala substitutions in Pin1 consensus sites at residue 46 (p53 S46A) or at the three other major Pin1-binding sites at residues 33, 81, and 315 (p53 3M- S46wt). (B) p53-null H1299 cells were transfected with the indicated constructs (see also A). Apoptosis of mHtt GFP-expressing cells was evaluated by TUNEL assay after 48 h. The histograms show mean and SD of three independent experiments. (C) SH-SY5Y cells were transfected with mHtt(1–171)150Q-GFP and treated with the ATM inhibitor KU-55933 10 μM for 24 h. HIPK2 protein levels were evaluated by Western blot. (D) SH-SY5Y cells were transfected with the indicated combinations of mHtt(1–171)150Q-GFP expression construct and siRNA oligonucleotides for HIPK2 and PKCδ. Apoptosis of mHtt GFP-expressing cells was evaluated by TUNEL assay after 48 h. The histograms show mean and SD of three independent experiments.

phosphorylation triggered by mHtt (Fig. 5B) similar to PKCδ neurodegeneration, the identity of druggable upstream mediators knockdown (Fig. 3D). Because specific inhibitors of HIPK2 are of polyQ-dependent toxicity still remains elusive. In this work we not available, we attempted to pharmacologically interfere with have focused on an emerging model of HD pathogenesis, where this pathway by inhibiting the upstream kinases. Treatment of SH- mHtt evokes a canonical DNA damage response in neuronal cells, SY5Y cells with caffeine, a well-known inhibitor of ATM/ATR with induction of ATM/ATR kinases and consequent activation of activities, strongly reduced mHtt-dependent cellular toxicity and p53 (3). We have observed that an important mark of p53 acti- Ser46 phosphorylation (Fig. 5C). We then focused on ATM, vation, i.e., Ser46 phosphorylation, is induced downstream of which, besides inducing HIPK2, directly phosphorylates p53 on mHtt expression and is also evident in the brains of HD patients. Ser46, in addition to Ser15 (29). Strikingly, the ATM-specific in- Ser46 phosphorylation is specifically triggered by severe or per- hibitor KU55933 was effective in preventing mHtt-induced apo- sistent stress and represents a major event in shifting the p53 re- ptosis by reducing phosphorylation of p53 on both Ser46 and sponse from cell-cycle arrest to apoptosis (17, 30, 31). Here we Ser15 (Fig. 5D). have shown that this modification is a prerequisite for execution of Together, our experimental evidences support a model (Fig. apoptosis downstream of mHtt. We have previously demonstrated 5E) where stress generated by mHtt triggers activation of ATM, that recognition of phosphorylated Ser46 by the prolyl isomerase HIPK2, and PKCδ kinases, which lead to phosphorylation of p53 Pin1 leads to dissociation of p53 from the apoptosis inhibitor on Ser46. This process preludes Pin1-dependent prolyl isomeri- iASPP (14). Our results indicate that in cells expressing mHtt, zation and consequent dissociation of p53 from iASPP as a pre- isomerization by Pin1 is an essential step for unleashing the ap- requisite for taking the apoptotic route. optotic potential of p53 from iASPP, thus allowing induction of apoptotic effectors. Discussion HD neuropathology is characterized by massive loss of medium Despite the huge amount of data accumulated so far, a cure for spiny neurons in the striatum. The influence of p53 in HD patho- HD is not yet available. Though attention has been especially genesis in vivo has been clearly shown by using mHtt-transgenic fly devoted to the roles of downstream effectors, such as caspases, in and mouse models (5). Here we demonstrate that genetic ablation

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Fig. 4. mHtt induces dissociation of p53 from iASPP in a Ser46- and Pin1- dependent fashion. (A) H1299 cells were transfected with constructs expressing p53 and increasing amounts (+, ++) of mHtt(1–171)150Q; the interaction of p53 with endogenous iASPP protein was then analyzed by coimmunoprecipitation. (B) H1299 cells were transfected with constructs expressing p53 and Htt(1–171) fragments bearing either 21Q or 150Q. Total cell lysates were analyzed by Western blot with antibodies specific for Ser46- phosphorylated and for total p53. (C) The interaction between p53 and iASPP proteins after transfection of increasing amounts of wt Htt(1–171)21Q in H1299 cells was determined as in A.(D) The effect of RNAi-mediated knockdown of Pin1 on the dissociation of p53 from iASPP induced by Fig. 5. Inhibition of p53 phosphorylation on Ser46 or of Pin1-mediated isom- overexpressed mHtt was determined as in A. erization reduces mHtt-dependent apoptosis. (A–D) SH-SY5Y cells were transfected with a construct expressing mHtt(1–171)150Q-GFP and treated with the Pin1 inhibitor PiB, 5 μM(A), the PKCδ inhibitor rottlerin, 5 μM(B), the Q111 of Pin1 in Hdh KI mice prevents precocious activation of p53, ATM/ATR inhibitor caffeine, 3 mM (C), the ATM-specific inhibitor KU-55933, suggesting that Pin1 is required for induction of the p53 response, 10 μM(D), or with same amount of solvent as a control (–). Apoptosis of mHtt at least in early stages of HD. In fact, we observed that p53 tran- GFP-expressing cells was evaluated by TUNEL assay after 24 h. Graphs show CELL BIOLOGY scriptional activity is induced in striatal neurons in vivo more than means and SD of three independent experiments. To detect p53 phosphory- 1 y before cell death, implying that these cells possess the ability to lation, p53 was immunoprecipitated from equal amounts of total cell lysates deal with chronic stress for long periods of time. Analysis of older and analyzed by Western blot. The protein levels of Pin1, actin, and mHtt in animals also suggested that interference with p53 activation by input lysates are shown. (E) Model for regulation of p53 by Pin1 upon cellular stress generated by mutant huntingtin. In cells expressing mHtt, the activities of targeting Pin1 might reduce Htt-induced neurodegeneration. δ fi ATM, HIPK2, and PKC lead to phosphorylation of p53 on Ser46. Subsequent Our ndings also imply that p53-independent pathways may prolyl isomerization of the phospho–Ser46-Pro47 site by Pin1 then unlocks p53 concur to HD-related toxicity. The p53 family member p73 has from the apoptosis inhibitor iASPP, leading to induction of apoptotic genes. been found relevant for mHtt-induced neuronal death (32), and Pharmacologic interference with this pathway can be accomplished by use of the ability of Pin1 to potentiate the apoptotic activity of both p53 small-molecule inhibitors that target ATM, PKCδ, or Pin1, thereby preventing and p73 (33) might be critical in this respect. p53 cytotoxic activity. By describing how mHtt stimulates the activation of p53 by triggering its phosphorylation-induced, Pin1-dependent isomeri- E zation, we suggest that this polyQ-expanded protein causes neu- emerged from our data (Fig. 5 ) details potential pharmacological rotoxicity by acting as an upstream inducer rather than through targets. First, we demonstrated that small-molecule inhibitors of direct interaction with p53 (5, 34); this implies that p53 activation Pin1 can protect neuronal cells from mHtt-induced apoptosis might represent a general pathogenic mechanism for polyglut- in vitro and may therefore be effective as a therapeutic strategy for amine diseases sharing the occurrence of DNA lesions (4). In the treatment of HD. Although it is arguable that development of CNS, p53 mediates neuronal death in response to excitotoxicity clinically useful inhibitors of Pin1 awaits further improvement, our and oxidative stress (35), and its activity has been implicated in results may also indicate stress-induced p53 kinases as druggable other neurodegenerative diseases, such as PD (36). Intriguingly, therapeutic targets. The dissociation of p53 from iASPP and the p53 has been shown to increase mHtt expression (37). Therefore, induction of apoptotic effectors can indeed be prevented by in- by activating p53, Pin1 might enforce a noxious loop triggered by terfering with phospho-Ser46 isomerization by Pin1 (14). Here we Htt mutation, enhancing its toxic effects. In this respect, common have succeeded in reducing mHtt-dependent apoptosis of neuro- genetic polymorphisms (SNPs) affecting stress-induced p53 acti- nal cells by inhibiting the activity of PKCδ and ATM kinases, which vation (38) might impinge on HD pathogenesis (39). However, lead to Ser46 phosphorylation either directly or indirectly. Protein SNPs in the Pin1 promoter region have also been described as kinases are a growing drug target class for diseases of peripheral affecting protein expression (40, 41), and it would be thus in- tissues, and several candidate therapeutics targeting CNS kinases teresting to investigate whether Pin1 may act as a modifier of are now in various stages of preclinical and clinical development. HD pathogenesis. For instance, specific inhibitors of PKCδ have shown preclinical Identification of upstream pathogenic events in HD is crucial for in vivo efficacy in treatment of PD (42). designing therapeutic interventions, and the vast knowledge Because clinical trials of molecules that may restore function- available on the p53 pathway provides an advantageous standpoint ality to a single cellular pathway have failed, special attention has to highlight regulatory nodes suitable for manipulation. The model been devoted to the identification of drugs that may interfere with

Grison et al. PNAS | November 1, 2011 | vol. 108 | no. 44 | 17983 Downloaded by guest on September 24, 2021 different pathways at the same time. To this purpose, caffeine Antibodies were anti-Pin1 polyclonal (12) and monoclonal (G-8 Santa Cruz); seems particularly interesting for its neuroprotective properties as anti-p53 Pab240, FL-393, and DO-1 (Santa Cruz); anti-iASPP pAbiASPPN1 and a blocker of adenosine A2A receptors (ADORA2A) (43). Re- mAbiASPP49.3 (14); anti-Huntingtin MAB5490 (Millipore); anti–phospho- cently, a genetic variant of ADORA2A has been identiﬁed as Ser15-p53 (Cell Signaling Technology); anti–phospho–Ser46-p53 (BD Phar- a modiﬁer of age at onset in HD (44). The combined actions as mingen); anti-PUMA ab-9643 (Abcam); anti-actin and anti-tubulin (Sigma), inhibitor of p53-mediated apoptotic pathway as well as neuro- and anti-HSP90 F-8 (Santa Cruz). Anti-HIPK2 monoclonal antibody was a gift protective molecule acting at adenosine receptors may increase of L. Schmitz (University of Giessen, Giessen, Germany). caffeine’s chances as a pharmacological intervention for HD. Mice Strains. HdhQ111 knock-in mice expressing the complete endogenous Htt Q7 Methods gene with 111 polyQ (20) and their wild-type littermates (Hdh )inC57BL background were provided by M. MacDonald (Massachusetts General Hospital, Cell Lines and HD Tissues. SH-SY5Y and SK-N-SH human neuroblastoma cells Boston). Pin1 KO mice in C57BL background (22) were provided by A. Means ’ ’ were cultured in 1:1 Eagle s minimal essential medium (MEM)/F12 Ham s (Duke University, Durham, NC). HdhQ7/Q111:Pin1WT/KO mice were intercrossed to medium with 15% FBS, 0.5% GlutaMAX (Gibco), 1% nonessential amino give double-homozygous and heterozygous littermates. Genotyping for both acids, and antibiotics. Neuro-2a mouse neuroblastoma cells were cultured in loci was performed by PCR on tail DNA as described (20, 22). MEM with 10% FBS, 1% GlutaMAX, 1% nonessential amino acids, and antibiotics. H1299 human lung carcinoma cells were cultured in Roswell Park RT-PCR. Total RNA was extracted with QIAzol, and cDNA was transcribed using Memorial Institute medium with 10% FBS and antibiotics. PPIase-Parvulin QuantiTect Reverse Transcription Kit (Qiagen). RT-PCR was performed with Quanti- Inhibitor was from Calbiochem; Rottlerin and caffeine were from Sigma. Fast SYBR Green PCR Kit (Qiagen). Primer sequences are reported in Table S2. Plasmids and siRNA oligonucleotides (Table S1) are described in SI Methods. HD and control postmortem brain tissues were collected by the Harvard Apoptosis Assays. TUNEL assays were performed with TMR Red in Situ Cell Brain Tissue Resource Center, McLean Hospital (Belmont, MA). HD brains Death Detection Kit (Roche) following manufacturer’s instructions. were assigned Vonsattel grade-3 pathology. Postmortem intervals were from 12 to 32 h for controls and from 8 to 30 h for HD brains. ACKNOWLEDGMENTS. We thank colleagues at Laboratorio Nazionale Consorzio Interuniversitario per le Biotecnologie for advice, G. Pastore and In Vitro Binding and Western Blot. Analysis of p53-Pin1 interaction by GST pull- M. Maurutto for technical support, S. Soddu, X. Lu, and T. Hofmann for down and coimmunoprecipitation was done as described (12). p53-iASPP reagents, and G. Leanza for mouse immunohistochemistry. This work was coimmunoprecipitation was performed as described (14). More details are supported by Telethon Grant GGP07185 (to G.D.S. and F.P.) and Associazione provided in SI Methods. Italiana per la Ricerca sul Cancro (G.D.S.).

1. The Huntington’s Disease Collaborative Research Group (1993) A novel gene con- 24. Di Stefano V, Rinaldo C, Sacchi A, Soddu S, D’Orazi G (2004) Homeodomain-inter- taining a trinucleotide repeat that is expanded and unstable on Huntington’s disease acting protein kinase-2 activity and p53 phosphorylation are critical events for cis- chromosomes. Cell 72:971–983. platin-mediated apoptosis. Exp Cell Res 293:311–320. 2. Vonsattel JP, DiFiglia M (1998) Huntington disease. J Neuropathol Exp Neurol 57: 25. Winter M, et al. (2008) Control of HIPK2 stability by ubiquitin ligase Siah-1 and 369–384. checkpoint kinases ATM and ATR. Nat Cell Biol 10:812–824. 3. Illuzzi J, Yerkes S, Parekh-Olmedo H, Kmiec EB (2009) DNA breakage and induction of 26. Lee SJ, Kim DC, Choi BH, Ha H, Kim KT (2006) Regulation of p53 by activated protein DNA damage response proteins precede the appearance of visible mutant huntingtin kinase C-delta during nitric oxide-induced dopaminergic cell death. J Biol Chem 281: aggregates. J Neurosci Res 87:733–747. 2215–2224. 4. Bertoni A, et al. (2011) Early and late events induced by polyQ-expanded proteins: 27. Yoshida K, Liu H, Miki Y (2006) Protein kinase C delta regulates Ser46 phosphorylation Identification of a common pathogenic property of polyQ-expanded proteins. J Biol of p53 tumor suppressor in the apoptotic response to DNA damage. J Biol Chem 281: Chem 286:4727–4741. 5734–5740. 5. Bae BI, et al. (2005) p53 mediates cellular dysfunction and behavioral abnormalities in 28. Uchida T, et al. (2003) Pin1 and Par14 peptidyl prolyl isomerase inhibitors block cell Huntington’s disease. Neuron 47:29–41. proliferation. Chem Biol 10:15–24. 6. Illuzzi JL, Vickers CA, Kmiec EB (2011) Modifications of p53 and the DNA damage 29. Kodama M, et al. (2010) Requirement of ATM for rapid p53 phosphorylation at Ser46 response in cells expressing mutant form of the protein huntingtin. J Mol Neurosci 45: without Ser/Thr-Gln sequences. Mol Cell Biol 30:1620–1633. 256–268. 30. Oda K, et al. (2000) p53AIP1, a potential mediator of p53-dependent apoptosis, and 7. Vousden KH, Prives C (2009) Blinded by the light: The growing complexity of p53. Cell its regulation by Ser-46-phosphorylated p53. Cell 102:849–862. 137:413–431. 31. Smeenk L, et al. (2011) Role of p53 serine 46 in p53 target gene regulation. PLoS ONE 8. Kruse JP, Gu W (2009) Modes of p53 regulation. Cell 137:609–622. 6:e17574. 9. Collavin L, Lunardi A, Del Sal G (2010) p53-family proteins and their regulators: Hubs 32. Hoshino M, et al. (2006) Transcriptional repression induces a slowly progressive and spokes in tumor suppression. Cell Death Differ 17:901–911. atypical neuronal death associated with changes of YAP isoforms and p73. J Cell Biol 10. Yeh ES, Means AR (2007) PIN1, the cell cycle and cancer. Nat Rev Cancer 7:381–388. 172:589–604. 11. Lu KP, Zhou XZ (2007) The prolyl isomerase PIN1: A pivotal new twist in phosphory- 33. Mantovani F, et al. (2004) Pin1 links the activities of c-Abl and p300 in regulating p73 lation signalling and disease. Nat Rev Mol Cell Biol 8:904–916. function. Mol Cell 14:625–636. 12. Zacchi P, et al. (2002) The prolyl isomerase Pin1 reveals a mechanism to control p53 34. Steffan JS, et al. (2000) The Huntington’s disease protein interacts with p53 and CREB- functions after genotoxic insults. Nature 419:853–857. binding protein and represses transcription. Proc Natl Acad Sci USA 97:6763–6768. 13. Zheng H, et al. (2002) The prolyl isomerase Pin1 is a regulator of p53 in genotoxic 35. Mattson MP, Duan W, Pedersen WA, Culmsee C (2001) Neurodegenerative disorders response. Nature 419:849–853. and ischemic brain diseases. Apoptosis 6:69–81. 14. Mantovani F, et al. (2007) The prolyl isomerase Pin1 orchestrates p53 acetylation and 36. Alves da Costa C, Checler F (2011) Apoptosis in Parkinson’s disease: Is p53 the missing dissociation from the apoptosis inhibitor iASPP. Nat Struct Mol Biol 14:912–920. link between genetic and sporadic Parkinsonism? Cell Signal 23:963–968. 15. Lu PJ, Wulf G, Zhou XZ, Davies P, Lu KP (1999) The prolyl isomerase Pin1 restores the 37. Feng Z, et al. (2006) p53 tumor suppressor protein regulates the levels of huntingtin function of Alzheimer-associated phosphorylated tau protein. Nature 399:784–788. gene expression. Oncogene 25:1–7. 16. Ryo A, et al. (2006) Prolyl-isomerase Pin1 accumulates in Lewy bodies of Parkinson dis- 38. Grochola LF, Zeron-Medina J, Mériaux S, Bond GL (2010) Single-nucleotide poly- ease and facilitates formation of alpha-synuclein inclusions. JBiolChem281:4117–4125. morphisms in the p53 signaling pathway. Cold Spring Harb Perspect Biol 2:a001032. 17. Mayo LD, et al. (2005) Phosphorylation of human p53 at serine 46 determines pro- 39. Chattopadhyay B, Baksi K, Mukhopadhyay S, Bhattacharyya NP (2005) Modulation of moter selection and whether apoptosis is attenuated or amplified. J Biol Chem 280: age at onset of Huntington disease patients by variations in TP53 and human caspase 25953–25959. activated DNase (hCAD) genes. Neurosci Lett 374:81–86. 18. DiFiglia M, et al. (1997) Aggregation of huntingtin in neuronal intranuclear inclusions 40. Segat L, et al. (2007) PIN1 promoter polymorphisms are associated with Alzheimer’s and dystrophic neurites in brain. Science 277:1990–1993. disease. Neurobiol Aging 28:69–74. 19. Schilling G, et al. (1999) Intranuclear inclusions and neuritic aggregates in transgenic mice 41. Lu J, et al. (2009) A novel functional variant (−842G>C) in the PIN1 promoter con- expressing a mutant N-terminal fragment of huntingtin. Hum Mol Genet 8:397–407. tributes to decreased risk of squamous cell carcinoma of the head and neck by di- 20. White JK, et al. (1997) Huntingtin is required for neurogenesis and is not impaired by minishing the promoter activity. Carcinogenesis 30:1717–1721. the Huntington’s disease CAG expansion. Nat Genet 17:404–410. 42. Chico LK, Van Eldik LJ, Watterson DM (2009) Targeting protein kinases in central 21. Wheeler VC, et al. (2002) Early phenotypes that presage late-onset neurodegenera- nervous system disorders. Nat Rev Drug Discov 8:892–909. tive disease allow testing of modifiers in Hdh CAG knock-in mice. Hum Mol Genet 11: 43. Prediger RD (2010) Effects of caffeine in Parkinson’s disease: From neuroprotection to 633–640. the management of motor and non-motor symptoms. J Alzheimers Dis 20(Suppl1): 22. Atchison FW, Capel B, Means AR (2003) Pin1 regulates the timing of mammalian S205–S220. primordial germ cell proliferation. Development 130:3579–3586. 44. Dhaenens CM, et al.; Huntington French Speaking Network (2009) A genetic variation 23. Liou YC, et al. (2003) Role of the prolyl isomerase Pin1 in protecting against age- in the ADORA2A gene modifies age at onset in Huntington’s disease. Neurobiol Dis dependent neurodegeneration. Nature 424:556–561. 35:474–476.

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