Regulation of Nuclear TDP-43 by NR2A-Containing NMDA Receptors and PTEN

1556 Research Article Regulation of nuclear TDP-43 by NR2A-containing NMDA receptors and PTEN

Mei Zheng1,2, Mingxia Liao2, Tianyuan Cui2, Honglin Tian3, Dong-Sheng Fan1,* and Qi Wan2,* 1Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Beijing, 100191, China 2Department of Physiology and Cell Biology, University of Nevada School of Medicine, 1664 North Virginia Street, MS0352, Reno, NV 89557, USA 3Department of Pharmacology, University of Nevada School of Medicine, 1664 North Virginia Street, MS0352, Reno, NV 89557, USA *Author for correspondence ([email protected]; [email protected])

Accepted 27 October 2011 Journal of Cell Science 125, 1556–1567 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.095729

Summary The dysfunction of TAR DNA-binding protein-43 (TDP-43) is implicated in neurodegenerative diseases. However, the function of TDP- 43 is not fully elucidated. Here we show that the protein level of endogenous TDP-43 in the nucleus is increased in mouse cortical neurons in the early stages, but return to basal level in the later stages after glutamate accumulation-induced injury. The elevation of TDP-43 results from a downregulation of phosphatase and tensin homolog (PTEN). We further demonstrate that activation of NR2A- containing NMDA receptors (NR2ARs) leads to PTEN downregulation and subsequent reduction of PTEN import from the cytoplasm to the nucleus after glutamate accumulation. The decrease of PTEN in the nucleus contributes to its reduced association with TDP-43, and thereby mediates the elevation of nuclear TDP-43. We provide evidence that the elevation of nuclear TDP-43, mediated by NR2AR activation and PTEN downregulation, confers protection against cortical neuronal death in the late stages after glutamate accumulation. Thus, this study reveals a NR2AR–PTEN–TDP-43 signaling pathway by which nuclear TDP-43 promotes neuronal survival. These results suggest that upregulation of nuclear TDP-43 represents a self-protection mechanism to delay neurodegeneration in the early stages after glutamate accumulation and that prolonging the upregulation process of nuclear TDP-43 might have therapeutic significance.

Key words: TAR DNA-binding protein-43, NR2A-containing NMDA receptor, PTEN, Glutamate neurotoxicity, Neuroprotection, Neurodegeneration

Introduction NMDA receptors are the major subtypes of ionotropic glutamate

Journal of Cell Science The TAR DNA-binding protein-43 (TDP-43) is a highly receptors to mediate glutamate neurotoxicity-induced neuronal conserved, heterogeneous ribonucleoprotein. TDP-43 has both death or neurodegeneration (Annis and Vaughn, 1998; Arundine nuclear export and import signals, but its distribution is primarily and Tymianski, 2004; Brunet et al., 2009; Hanson et al., 2010; nuclear (Ayala et al., 2008; Winton et al., 2008). It has been shown Hardingham et al., 2002; Lee et al., 1999; Sarraf-Yazdi et al., that TDP-43 regulates gene transcription, exon splicing and exon 1998; Sen et al., 2005). However, how NMDA receptors induce inclusion (Sreedharan et al., 2008). However, the function of TDP- neuronal death or neurodegeneration remains unclear. The NMDA 43 and it underlying mechanisms are not fully understood. receptors containing NR2A and NR2B subunits (NR2ARs and Evidence from in vivo models of murine TDP-43 deletion NR2BRs) are the major subtypes of NMDA receptors expressed in indicates that TDP-43 is required for embryogenesis and is the central nervous system (CNS) (Dingledine et al., 1999). Studies essential for viability (Kraemer et al., 2010; Sephton et al., 2010; demonstrate that NR2ARs and NR2BRs play opposing role in Wu et al., 2010). Recently, the major proteins of the pathological regulating neuronal survival or death (Chen et al., 2008; DeRidder inclusions in amyotrophic lateral sclerosis (ALS) have been et al., 2006; Hardingham et al., 2002; Liu et al., 2007; Ning et al., identified as TDP-43 and its C-terminal fragments of ,20–25 kDa 2004; Vanhoutte and Bading, 2003). This might explain why use (Arai et al., 2006; Igaz et al., 2008; Mackenzie et al., 2007; of NMDA receptor antagonists as neuroprotective agents has been Neumann et al., 2006). TDP-43 has also been identified as a disappointing in clinical trials (Gredal et al., 1997; Traynor et al., component in the inclusions of frontotemporal lobar degeneration 2006). Thus, investigating the specific effects differentially (FTLD) and other neurodegenerative diseases (Arai et al., 2009; mediated by NR2AR- and NR2BR-dependent intracellular Hasegawa et al., 2007; Neumann et al., 2006). signaling would provide molecular evidence for the development Glutamate accumulation-mediated neurotoxicity is known to of selective neuroprotection therapies. play a crucial role in traumatic and ischemic brain injuries, as well Studies by others and us have revealed that suppression of as in neurodegenerative diseases including ALS (Culcasi et al., PTEN (phosphatase and tensin homolog) protects against 1994; Fiszman et al., 2010; Grosskreutz et al., 2010; Lafon-Cazal neuronal death (Chang et al., 2007; Ning et al., 2004). et al., 1993; Perry et al., 1987; Plaitakis and Caroscio, 1987). The Although it functions in the cytoplasm, PTEN can enter the elevation of glutamate concentration causes neurotoxicity through nucleus to regulate transcription, alternative splicing and mRNA overactivation of ionotropic glutamate receptors (Arundine and stability (Planchon et al., 2008). Under normal conditions, PTEN Tymianski, 2004; Hanson et al., 2010; Hardingham et al., 2002; shuttles between the cytoplasm and nucleus (Gil et al., 2006; Lee et al., 1999; Rothstein et al., 1990; Sarraf-Yazdi et al., 1998). Planchon et al., 2008). In the nucleus, PTEN has been shown to Regulation of nuclear TDP-43 1557

cause downregulation of extracellular signal-regulated kinase was confined to the nucleus (Fig. 1D). We did not observe (ERK), leading to a decrease in cyclin D1 levels and G0–G1 significant expression of TDP-43 in the cytoplasm in either arrest (Planchon et al., 2008). By interacting with CENP-C, control or THA-treated neurons (Fig. 1D). Taken together, these PTEN enhances centromere stability and overall genomic results indicate that TDP-43 expression is increased only in the stability (Planchon et al., 2008). Similar to its role in the nucleus in the early stages after glutamate accumulation. cytoplasm, nuclear PTEN also induces apoptosis (Planchon et al., 2008). Downregulation of PTEN contributes to the upregulation of To understand the cellular and molecular mechanisms that TDP-43 after glutamate accumulation mediate the role of TDP-43 in both physiological and Given that the phosphatase PTEN is involved in regulating neurodegenerative conditions, we investigated the effect of neuronal survival and exerts its effects in both cytoplasm and NMDA receptors and PTEN on TDP-43 expression in an in nucleus, we tested the effect of PTEN on TDP-43. We first vitro glutamate accumulation-induced neurodegeneration model measured the level of PTEN protein in cortical neurons at 3 days using cultured mouse cortical neurons. We show that the protein after THA treatment. We showed that, in contrast to the increase level of endogenous TDP-43 in the nucleus is increased in the of TDP-43, PTEN was decreased at 3 days after THA treatment early stages after glutamate accumulation. The increase of TDP- (Fig. 2A). To determine whether PTEN downregulation 43 is mediated through a reduced association of PTEN with TDP- contributed to TDP-43 upregulation, we tested the effect of a 43 in the nucleus, which results from the activation of NR2ARs selective PTEN inhibitor bisperoxovanadium [bpV(pic)] on the and subsequent downregulation of nuclear PTEN. We also protein expression of TDP-43 in the cortical neurons (Schmid provide evidence that the upregulation of nuclear TDP-43 by et al., 2004). We found that PTEN inhibition by bpV(pic) not NR2AR activation and PTEN downregulation in the early stages only enhanced TDP-43 expression in control cortical neurons after glutamate accumulation is neuroprotective. Thus, the (Fig. 2B), but also increased TDP-43 expression at 6 days after NR2AR–PTEN–TDP-43 signaling pathway might represent THA treatment (Fig. 2C). However, bpV(pic) treatment had no a general mechanism against neuronal death in the early significant effect on TDP-43 expression at 3 days after THA stages of glutamate neurotoxicity-induced neuronal injury and treatment (Fig. 2D). To provide further evidence for the neurodegeneration. regulation of TDP-43 by PTEN, the cultured neurons were transfected with PTEN siRNAs (siRNApten) or PTEN cDNAS Results (Fig. 2E–H). Our data showed that suppression or overexpression The protein level of nuclear TDP-43 is increased in cortical of PTEN increased and inhibited TDP-43 expression, neurons in the early stages after endogenous glutamate respectively (Fig. 2E–H). These results suggest that TDP-43 is accumulation negatively regulated by endogenous PTEN, and that the To investigate the functional consequences of TDP-43 in the upregulation of TDP-43 expression is mediated by PTEN CNS, we established an in vitro glutamate-induced neurotoxicity downregulation in the early stages after glutamate accumulation. model in cultured mouse cortical neurons. DL-threo-beta- To determine how TDP-43 was regulated by PTEN, two hydroxyaspartate (THA), an inhibitor of glutamate transporters, different dominant-negative forms of phosphatase-inactivated

Journal of Cell Science was used to induce neuronal injury. By promoting extracellular PTEN mutants, C124A and G129E, were used to inhibit glutamate accumulation, THA treatment causes glutamate endogenous lipid and protein phosphatase activities of PTEN neurotoxicity and has been used to induce neuronal injury in (Myers et al., 1998; Tamura et al., 1998; Weng et al., 2001). The vitro (Corse et al., 1999; Kidd and Isaac, 2000; Matyja et al., C124A mutation causes a loss of both lipid and protein 2006; Nagan´ska et al., 2010; Tolosa et al., 2008; Van Westerlaak phosphatase activities, and the mutant G129E results only in et al., 2001). To characterize this model, we first performed a loss of lipid phosphatase activity with retention of protein lactate dehydrogenase (LDH) release assay to measure THA- phosphatase activity. Our results demonstrated that the level of induced neuronal damage in the cortical cultures. We showed TDP-43 was increased in neurons transfected with both C124A that the rate of neuronal death was not significantly altered during and G129E (Fig. 3A,B), suggesting that the downregulation of the first 3 days after treatment with 100 mM THA, but increased lipid phosphatase activity of PTEN contributes to upregulation of at 6 days after treatment (Fig. 1A). We then performed western TDP-43. blot assays to measure the protein level of TDP-43 in the specific We also performed cycloheximide chase experiments to injury paradigm. We found that the level of TDP-43 was determine the effect of PTEN on TDP-43 stability. The remarkably increased in cortical neurons during the first 3 neuronal cultures were first treated with vehicle or THA days after THA treatment (Fig. 1B). However, the increased (100 mM) for 3 days and then the cultures were treated with expression of TDP-43 returned to basal levels at 6 days after cycloheximide (20 mg/ml) for 1, 3, 6 and 12 hours (Turgeon et al., THA insult (Fig. 1B). Our data also showed that there were no C- 2001). We found that the protein level of TDP-43 was terminal fragments of TDP-43 in THA-treated groups or in the significantly decreased in control neurons at 6 hours after respective controls (Fig. 1C). cycloheximide treatment (Fig. 3C). By contrast, in THA-treated Because TDP-43 is mainly expressed in the nucleus, we next neurons, the protein level of TDP-43 remained unchanged tested whether the distribution of TDP-43 was altered in following treatment with cycloheximide for 12 hours (Fig. 3D). the nucleus of cortical neurons after glutamate accumulation. Moreover, our data showed that bpV(pic) treatment stabilized the Immunocytochemical staining was performed to determine TDP- level of TDP-43 after 12 hours of treatment with cycloheximide 43 expression with the use of propidium iodide (PI) to label the (Fig. 3E). These results indicate that downregulation of PTEN nucleus in cultured cortical neurons (Fig. 1D). Although we leads to the enhancement of TDP-43 protein stability, which confirmed that TDP-43 expression was increased at 3 days after might contribute to THA-induced increase of TDP-43 expression THA treatment (Fig. 1D), we found that the increased TDP-43 in the early stages after glutamate accumulation. 1558 Journal of Cell Science 125 (6) Journal of Cell Science

Fig. 1. Protein expression of nuclear TDP-43 is increased in mouse cortical neurons in the early stages after THA treatment. (A) Time course of THA- induced neuronal damage. Summarized data indicate that LDH release is remarkably increased at 6 days after 100 mM THA treatment (mean 6 s.e.m.; n56 animals for each group; *P,0.05 vs control). (B) Representative immunoblots (top panel) and summarized data (bottom panel) show that the protein level of TDP-43 is increased at 3 days after 100 mM THA treatment (n58 animals for each group; *P,0.05 vs 3d/control; data are normalized to 1d/control). (C) A full blot image of the sample TDP-43 blot in B. (D) Sample images (left) and summarized data (right) show that the protein expression of TDP-43 in the nucleus of cultured cortical motor neurons is increased at 3 days after 100 mM THA treatment (for each group, n530 cells from three independent experiments; *P,0.05 vs 3d/control; data are normalized to 3d/control).

NR2AR activation leads to PTEN downregulation and showed that the NR2AR antagonist NVP-AAM077 (0.4 mM) but subsequent TDP-43 upregulation after glutamate not NR2BR antagonist Ro25-6981 (0.5 mM) attenuated THA- accumulation induced PTEN downregulation after glutamate accumulation To further reveal the upstream signaling to regulate TDP-43, we (Fig. 4A). Although NVP-AAM077 has small degree of cross- tested the effects of NMDA receptors on the protein expression inhibition on NR2BRs (Liu et al., 2007), this cross-effect can be of both PTEN and TDP-43 after glutamate accumulation. We ignored because our data indicated that NR2BR inhibition had Regulation of nuclear TDP-43 1559 Journal of Cell Science

Fig. 2. The increase of TDP-43 is mediated by PTEN downregulation soon after THA treatment. (A) Representative immunoblots (left) and summarized data (right) show that the protein expression of PTEN is decreased at 3 days after 100 mM THA treatment (n57 for each group; *P,0.05 vs 3d/control; data are normalized to 3d/control). (B) Sample immunoblots (left) and summarized data (right) show that treatment with the PTEN inhibitor bpV(pic) (100 nM) for 24 hours increases the protein expression of TDP-43 in normal cortical neurons (n58 for each group; *P,0.05 vs control). (C) Representative immunoblots (left) and summarized data (right) show that treatment with bpV(pic) (100 nM) at 5 days after 100 mM THA insult increases the protein expression of TDP-43 in cortical neurons at 6 days after THA insult (n56 for each group; *P,0.05 vs 6d/THA; data are normalized to 6d/control). (D) Sample immunoblots (left) and summarized data (right) show that treatment withbpV(pic) (100 nM) at 2 days after 100 mM THA insult has no effect on the protein expression of TDP-43 at 3 days after THA insult (n56 for each group; *P,0.05 vs 3d/control; #P.0.05 vs 3d/THA). (E) Treatment with PTEN siRNA (siRNApten) but not the non- targeting control siRNA (NsiRNA) in cultured cortical neurons suppresses PTEN expression (n55 for each group; *P,0.05 vs NsiRNA). (F) Treatment with siRNApten but not NsiRNA increases TDP-43 expression (n55 for each group; *P,0.05 vs NsiRNA). (G) Transfection of PTEN cDNA increases PTEN expression in cultured cortical neurons (n54 for each group; *P,0.05 vs GFP). (H) PTEN overexpression reduces TDP-43 expression in cultured cortical neurons (n54 for each group. *P,0.05 vs. GFP). All bar graphs show means 6 s.e.m.

no effect on TDP-43 expression (Fig. 4A). Thus, PTEN glutamate accumulation. To verify this possibility, the effects of downregulation is in part mediated by NR2AR activation after NVP-AAM077 (0.4 mM) and Ro25-6981 (0.5 mM) on TDP-43 glutamate accumulation. expression were tested in cortical neurons treated with THA As our data indicated that TDP-43 was a downstream effector (100 mM). We showed that the inhibition of NR2ARs but not of PTEN (Fig. 2), we reasoned that NR2AR activation would NR2BRs blocked the upregulation of TDP-43 expression at 3 lead to TDP-43 upregulation through PTEN downregulation after days after THA treatment (Fig. 4B). We also showed that 1560 Journal of Cell Science 125 (6)

Fig. 3. The lipid phosphatase activity of PTEN mediates the regulation of TDP-43 by PTEN. (A) Transfection of C124A induces increased expression of TDP-43 in cultured cortical neurons (n54 for each group; *P,0.05 vs GFP). (B) Transfection of G129E increases TDP-43 expression in cultured cortical neurons (n54 for each group; *P,0.05 vs GFP). (C) The level of TDP-43 is decreased in control neurons at 6 hours after cycloheximide treatment in cultured cortical neurons (n56 for each group; *P,0.05 vs 0 h). (D) The protein level of TDP-43 is not altered following Journal of Cell Science treatment for 12 hours with cycloheximide in THA-treated neurons. (E) The protein level of TDP-43 remains unchanged following treatment with cycloheximide for 12 hours in bpV(pic)- treated neurons. All bar graphs show means 6 s.e.m.

inhibiting NR2AR activity prevented THA-induced upregulation from cytoplasm into the nucleus leads to the decrease of PTEN in of TDP-43, and this effect was blocked by treatment of PTEN the nucleus. inhibitor bpV(pic) (Fig. 4C). These data suggest that NR2AR To understand how reduced nuclear PTEN led to the activation leads to PTEN downregulation, which in turn results upregulation of TDP-43 in the nucleus, we performed a co- in TDP-43 upregulation in the early stages after glutamate immunoprecipitation assay to test whether PTEN could accumulation. physically associate with TDP-43 to regulate TDP-43 expression. Indeed, our data indicated that TDP-43 was co- immunoprecipitated with PTEN by an anti-PTEN antibody in the Decreased association of PTEN with TDP-43 in the nucleus homogenates of cultured cortical neurons (Fig. 5B). Conversely, mediates TDP-43 upregulation after glutamate immunoprecipitation with an anti-TDP-43 antibody co- accumulation precipitated PTEN (Fig. 5C). These data indicate that PTEN Because the downregulation of PTEN might occur in the nucleus forms a protein complex with TDP-43 in the nucleus to to regulate TDP-43 expression, we performed subcellular negatively regulate TDP-43 expression. To further determine fractionation assays to measure the PTEN levels in both the whether this was also true in cortical neurons subjected to cytoplasm and nucleus. Our data showed that the protein glutamate insult, we measured the association levels of PTEN expression of PTEN was decreased not only in the cytosolic with TDP-43 in cortical neurons in both control and THA-treated fraction but also in the nuclear fraction at 3 days after THA conditions. Our data showed that both the level of co-precipitated treatment (Fig. 5A), suggesting that a reduced import of PTEN TDP-43 by anti-PTEN antibody and the level of co-precipitated Regulation of nuclear TDP-43 1561

Fig. 4. NR2AR activation mediates PTEN downregulation and TDP-43 upregulation in early stages after THA treatment. (A) Sample immunoblots (left) and summarized data (right) show that the NR2AR antagonist NVP-AAM077 (0.4 mM) but not the NR2BR antagonist Ro25-6981 (0.5 mM) reduces THA-induced PTEN downregulation at 3 days after 100 mM THA treatment (n57 for each group; *P,0.05 vs control; **P.0.05 vs 3d/THA; #P,0.05 vs. 3d/THA; data are normalized to control). (B) Representative immunoblots (left) and summarized data (right) show that NVP-AAM077 (0.4 mM) but not Ro25-6981 (0.5 mM) prevented upregulation of TDP-43 expression at 3 days after 100 mM THA treatment (n56 for each group; *P,0.05 vs control; #P,0.05 vs 3d/THA; data are normalized to control). (C) Sample immunoblots (left) and summarized data Journal of Cell Science (right) show that bpV(pic) (100 nM) prevented NR2AR-inhibition-induced blockade of TDP-43 upregulation in cortical neurons at 3 days after 100 mM THA treatment (n56 for each group; *P,0.05 vs control; **P,0.05 vs 3d/THA; #P,0.05 vs NVP-AAM077+3d/THA; data are normalized to control). All bar graphs show means 6 s.e.m.

PTEN by anti-TDP-43 antibody were significantly lower at 3 with THA or PTEN inhibitor (Wan et al., 1997a). TDP-43 was days after THA treatment compared with those in control cortical immunoprecipitated by antibody against TDP-43, and then anti- neurons (Fig. 5D,E), and that the NR2AR antagonist NVP- phosphoserine/phosphotyrosine antibodies were used to detect AAM077 blocked the reduction of co-precipitated TDP-43 or the phosphorylation level of precipitated TDP-43 proteins. We PTEN induced by THA treatment (Fig. 5D,E). Together, these found that both serine and tyrosine phosphorylation of TDP-43 results suggest that NR2AR activation/PTEN downregulation were not altered at 3 days after THA treatment and that PTEN may contribute to the reduced association of PTEN with TDP-43 inhibitor bpV(pic) had no significant effects on serine and in the nucleus, which leads to the upregulation of TDP-43 after tyrosine phosphorylation of TDP-43 (Fig. 5G,H). These data glutamate accumulation. indicate that regulation of TDP-43 by PTEN is not mediated To determine whether PTEN regulated TDP-43 through direct through a dephosphorylation process. binding, we performed an in vitro binding assay (Ning et al., 2004). Our data showed that PTEN was not directly associated Upregulation of nuclear TDP-43 confers protection against with TDP-43 (Fig. 5F). To determine whether PTEN-dependent glutamate accumulation-induced neuronal death dephosphorylation was involved in the regulation of TDP-43 Because NR2AR activation and PTEN downregulation are expression after glutamate insult, we measured the levels of believed to promote neuronal survival (Chang et al., 2007; serine and tyrosine phosphorylation of TDP-43 in neurons treated Chen et al., 2008; Liu et al., 2007; Ning et al., 2004), it is possible 1562 Journal of Cell Science 125 (6) Journal of Cell Science

Fig. 5. The association of PTEN with TDP-43 is reduced at the early stage after glutamate accumulation. (A) Sample immunoblots (left) and summarized data (right) from subcellular fractionation assays show that the protein expression of PTEN is decreased in both cytosolic and nuclear fractions at 3 days after 100 mM THA treatment (n56 for each group; *P,0.05 vs cytoplasm or nucleus control; data were normalized to cytoplasm control). Tubulin, a marker of cytoplasmic fraction; p85, a marker of nucleus fraction. (B) Representative immunoblots from co-immunoprecipitation assays show that TDP-43 is co- immunoprecipitated by an antibody against PTEN. No Ab, no antibody added to the assay. (C) Sample immunoblots from co-immunoprecipitation assays show that PTEN is co-immunoprecipitated by by an antibody against TDP-43. (D) Representative immunoblots (left) and summarized data (right) from co- immunoprecipitation assays show that the level of co-precipitated TDP-43 by anti-PTEN antibody is significantly reduced at 3 days after 100 mM THA treatment compared with that in control cortical neurons, and that the NR2AR antagonist NVP-AAM077 (0.4 mM) blocks the reduction of coprecipitated TDP-43 induced by THA treatment (n56 for each group; *P,0.05 vs control; #P,0.05 vs 3d/THA; data are normalized to control). (E) Representative immunoblots (left) and summarized data (right) from co-immunoprecipitation assays show that the level of coprecipitated PTEN by anti-TDP-43 antibody is significantly decreased at 3 days after 100 mM THA treatment compared with that in control cortical neurons, and that the NR2AR antagonist NVP-AAM077 (0.4 mM) blocks the reduction of coprecipitated PTEN induced by THA treatment (n56 for each group; *P,0.05 vs control; #P,0.05 vs 3d/THA; data are normalized to control). (F) In vitro 35 binding assay showing the direct binding of [ S]PTEN to GST–NR1-1aCT but not GST–TDP-43. (G) Representative immunoblot (left) and summarized data (right) show that THA (treatment for 3 days) and bpV(pic) have no significant effect on the serine phosphorylation of TDP-43 (n53 for each group). (H) Representative immunoblot (left) and summarized data (right) show that THA (treatment for 3 days) and bpV(pic) have no significant effect on the tyrosine phosphorylation of TDP-43 (n53 for each group). All bar graphs show means 6 s.e.m. Regulation of nuclear TDP-43 1563

that the effect delivered by nuclear TDP-43 upregulation in pro-survival signal in nature. To determine whether NR2AR and the early stages after glutamate accumulation represents a PTEN were upstream of TDP-43 to regulate neuronal survival, we neuroprotective response. To test this possibility, we used first measured the effect of the NR2AR antagonist NVP-AAM077 siRNA to knock down TDP-43 (siRNAtdp43) in the nucleus of on neuronal death in cortical neurons subjected to glutamate insult. cultured cortical neurons (Fig. 6A). We showed that the cell death We found that NR2AR inhibition by NVP-AAM077 (0.4 mM) in cortical neurons transfected with siRNAtdp43 was enhanced at 3 remarkably increased cell death in cortical neurons at 3 days after days after THA treatment compared with the control neurons THA treatment (Fig. 7A). However, the transient overexpression transfected with non-targeting control siRNA (NsiRNA) (Fig. 6B). of TDP-43 (Fig. 7B) or inhibition of PTEN by bpV(pic) attenuated These data indicate that the endogenous TDP-43 in the nucleus is a the NR2AR inhibition-induced increase of neuronal death after Journal of Cell Science

Fig. 6. Suppressing nuclear TDP-43 increases the death of cortical neurons at early stages after THA treatment. (A) Sample images (left) and summarized data (right) indicate that TDP-43 siRNA (siRNAtdp43) but not the non-targeting control siRNA (NsiRNA) inhibits TDP-43 expression in the nucleus of normal cortical neurons (means 6 s.e.m.; for each group, n535 cells from three independent experiments; *P,0.05 vs NsiRNAtdp43; data are normalized to NsiRNA). (B) Representative images (left) and summarized data (right) show that knockdown of TDP-43 with siRNAtdp43 promotes cortical neuronal death at 3 days after 100 mM THA treatment compared with the control neurons transfected with NsiRNA (means 6 s.e.m.; for each group, n550 cells from three independent experiments; *P,0.05 vs GFP; data are normalized to GFP). The neurons were transfected with siRNAs at 1 day after THA treatment. 1564 Journal of Cell Science 125 (6) Journal of Cell Science

Fig. 7. Upregulation of nuclear TDP-43 protects against cortical neuronal death. (A) Representative images (left) and summarized data (right) show that overexpression of TDP-43 or treatment with bpV(pic) reduces NR2AR inhibition-mediated increase of neuronal death at 3 days after 100 mM THA treatment (means 6 s.e.m.; for each group, n550 cells from three independent experiments; *P,0.05 vs GFP; **P,0.05 vs 3d/THA+GFP+NVP-AAM077; data are normalized to GFP). The neurons were transfected with cDNA encoding TDP-43 at 1 day after THA treatment. (B) Sample images (left) and summarized data (right) indicate that transient transfection of TDP-43 cDNA increases TDP-43 expression in nucleus in normal cortical neurons (means 6 s.e.m.; for each group, n530 cells from three independent experiments; *P,0.05 vs GFP; data are normalized to GFP).

glutamate insult (Fig. 7A). Thus, the upregulation of TDP-43 by neurodegeneration (Fiesel et al., 2010; Iguchi et al., 2009). NR2AR activation and PTEN downregulation plays a However, there is no direct evidence showing the neuroprotective neuroprotective role in the early stages after glutamate effect of endogenous TDP-43 in neuronal injury and neurotoxicity-induced injury. neurodegeneration. Our study provides the first evidence that nuclear TDP-43 expression is increased at the early stages in an in Discussion vitro glutamate accumulation-induced neurodegeneration model By knockdown or deletion of TDP-43 in vitro and in vivo, recent (Corse et al., 1999; Kidd and Isaac 2000; Matyja et al., 2006; studies suggest that TDP-43 is a pro-survival protein in the Nagan´ska et al., 2010; Tolosa et al., 2008; Van Westerlaak et al., nucleus and that the loss of function of nuclear TDP-43 promotes 2001). We demonstrate that glutamate accumulation results in an Regulation of nuclear TDP-43 1565

upregulation of nuclear TDP-43, and that the TDP-43 PTEN with TDP-43 in the nucleus, which contributes to the upregulation confers a neuroprotective effect. These results increased nuclear expression of TDP-43 after glutamate reveal a self-protection mechanism mediated by TDP-43 accumulation. It is unclear how the decreased association of upregulation at the early stage of neurodegeneration. It would PTEN and TDP-43 affects TDP-43 levels. However, based on our be interesting in the future to test whether TDP-43 also protects evidence that: (1) TDP-43 expression is negatively regulated from other forms of neuronal death or cytotoxicity in different by PTEN (Fig. 2); (2) PTEN does not directly interact with cell types. TDP-43 (Fig. 5F); (3) PTEN has no effect on serine/tyrosine Aberrant regulation of TDP-43 has been found to play complex phosphorylation of TDP-43 (Fig. 5G,H); and (4) PTEN roles in both the nucleus and cytoplasm in the pathogenesis of downregulation enhances the stability of TDP-43, we reason ALS (Barmada et al., 2010; Winton et al., 2008). In ALS patients, that the decrease in the PTEN–TDP-43 association might TDP-43 expression is remarkably reduced in the nucleus in the indirectly lead to a relief of TDP-43 inhibition by PTEN in a affected neurons with cytoplasmic inclusions (Cairns et al., 2007; phosphorylation-independent manner, which enhances the Hasegawa et al., 2007; Neumann et al., 2006). In transgenic mice stability of TDP-43 and thus increases TDP-43 expression. with TDP-43 overexpression, TDP-43 accumulates in the nucleus Future studies are required to further address how this process and aggregates in the cytoplasm (Wils et al., 2010). These occurs. findings indicate that a translocation of TDP-43 from the nucleus In summary, the present study provides the first evidence that to cytoplasm might contribute to the accumulation of TDP-43 in upregulation of TDP-43, mediated by NR2AR activation and the cytoplasmic inclusions. In the in vitro neurodegeneration PTEN downregulation, confers neuroprotection in the early model, we demonstrate that in response to glutamate stages after glutamate accumulation. Given that NMDA accumulation, endogenous TDP-43 is only increased in the receptors, PTEN and TDP-43 are involved in various CNS nucleus and does not translocate to cytoplasm. Thus, although disorders, the NR2AR–PTEN–TDP-43 signaling might TDP-43 behaves as a pro-survival signaling protein in the represent novel therapeutic targets for the development of a nucleus, the upregulation of endogenous TDP-43 in the nucleus neuroprotective strategy. would not lead to its translocation to the cytoplasmic compartment. Materials and Methods To understand the cellular and molecular mechanism by which Culture of mouse cortical motor neurons and THA treatment The cortical neuronal cultures were prepared from C57BL/6 mice at gestational TDP-43 plays a neuroprotective role during the neurodegeneration day 17 using a modified protocol (Shan et al., 2009). After removing meninges, process, we investigated the effect of NMDA receptors on TDP-43 cortices were placed in ice-cold plating medium (Neurobasal medium, 2% B-27 expression in an in vitro neurodegeneration model. We supplement, 0.5% FBS, 0.5 mM L-glutamax and 25 mM glutamic acid). Dissociated motor neurons were prepared from precentral gyrus that was demonstrate for the first time that the upregulation of TDP-43 carefully dissected from the cortices. The neurons were suspended in plating is mediated by the activation of NR2AR after glutamate medium and plated on Petri dishes coated with poly-D-lysine. After 3 days in accumulation. Recent evidence shows that NR2AR activation culture, half of the plating medium was removed and replaced with maintenance promotes neuronal survival in acute CNS injury such as cerebral medium (Neurobasal medium, 2% B-27 supplement, and 0.5 mM L-glutamine). Thereafter, maintenance medium was changed in the same manner every 3 days. ischemia and traumatic spinal injury (Alilain and Goshgarian, To induce chronic glutamate neurotoxicity, the cultures were exposed to THA

Journal of Cell Science 2008; Liu et al., 2007; Terasaki et al., 2010). However, the (100 mM) at 8 days after plating. The cultured neurons were collected for underlying mechanisms remain largely unknown. In the present experiments at 1, 3 and 6 days after THA treatment. study, we identify TDP-43 as a novel downstream signaling Western blotting, co-immunoprecipitation and in vitro binding assays protein of NR2ARs in a glutamate neurotoxicity-induced Western blotting, co-immunoprecipitation and in vitro binding assays were neurodegeneration model. Our results suggest that upregulation performed as reported previously (Ning et al., 2004). For western blotting, total of TDP-43 by NR2AR activation counteracts neurodegeneration in proteins were extracted with lysis buffer. Equal amounts of proteins were separated the early stages after glutamate insult. By contrast, NR2BR is by 8–10% SDS polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were electronically transferred to polyvinylidene difluoride membrane and found to have no effect on TDP-43 expression in our experimental incubated with a blocking buffer for 1 hour at room temperature. The model, although previous studies, including ours, indicate that membranes were incubated with primary antibodies overnight at 4˚C, and then NR2BR overactivation increases neuronal death by suppression of incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. The anti-TDP-43 antibody was purchased from Santa Cruz Biotech CREB-, ERK- and PINK1-dependent survival signaling pathways (Santa Cruz, CA) and the anti-PTEN antibody was purchased from Chemicon (Hardingham et al., 2002; Shan et al., 2009; Wang et al., 2004). (Temecula, CA). The protein bands were imaged using ECL (Amersham Because blocking NMDA receptors would suppress NR2AR- Pharmacia Biotech). The membranes were re-probed with anti-GAPDH antibody mediated neuroprotection while inhibiting NR2BR-mediated as a loading control. For co-immunoprecipitation experiments, the cell lysates (500 mg proteins in 1 ml) were pre-absorbed with 20 ml protein A/G agarose beads neuronal death, simply inhibiting NMDA receptors would not be at 4˚C for 30 minutes, spun at 10,000 r.p.m. for 10 seconds, and the supernatant a proper strategy to prevent neurodegeneration. This might explain was incubated with specific primary antibody at 4˚C overnight. After incubation why the use of glutamate antagonists as neuroprotective agents has with 20 ml protein A/G agarose beads for 1.5 hours at 4˚C, the immunocomplexes were collected by centrifugation and washed three times with ice-cold washing been disappointing in clinical trials (Gredal et al., 1997; Traynor buffer. The final products were boiled for 5 minutes and resolved with SDS- et al., 2006). Thus, investigating cellular and molecular PAGE, and immunoblotted with specific antibodies. Images were analyzed using mechanism mediating the differential role of NR2AR and ImageJ software (Version 1.34). For in vitro binding assay, GST–NR1-1aCT, GST– TDP-43 or GST (10 mg) alone was incubated with [35S]PTEN probe for 4 hours at NR2BR in neuronal survival or death is crucial for us to develop room temperature. The beads were washed six times with tissue homogenizing a selective neuroprotection strategy for the treatment of ischemic buffer and eluted with 10 mM glutathione. Eluates were separated by SDS-PAGE or traumatic CNS injury and neurodegenerative diseases. and visualized by autoradiography (Ning et al., 2004). Our results also show that PTEN downregulation mediates NR2AR-dependent elevation of nuclear TDP-43 following Immunocytochemical staining Immunocytochemical staining was performed as described previously (Liu et al., glutamate accumulation. Interestingly, we provide evidence that 2006; Wan et al., 1997b). Briefly, the transfected cells were fixed with 4% the downregulation of PTEN leads to a reduced association of paraformaldehyde and blocked in 5% normal goat serum. The specimen was 1566 Journal of Cell Science 125 (6)

incubated with primary antibody overnight at 4˚C and then incubated with References fluorochrome-conjugated secondary antibody for 1 hour at room temperature. The Alilain, W. J. and Goshgarian, H. G. (2008). Glutamate receptor plasticity and primary anti-TDP-43 antibody was purchased from Santa Cruz Biotech (Santa activity-regulated cytoskeletal associated protein regulation in the phrenic motor Cruz, CA). Secondary antibodies, Alexa Fluor 594 (red fluorescence) and Alexa nucleus may mediate spontaneous recovery of the hemidiaphragm following chronic Fluor 488 (green fluorescence), were purchased from Invitrogen (Carlsbad, CA). cervical spinal cord injury. Exp. Neurol. 212, 348-357. DAPI or propidium iodide (PI) was used to label the nuclei. Fluorescent-labeled Annis, C. M. and Vaughn, J. E. (1998). Differential vulnerability of autonomic and proteins were imaged using a 636 or 406 objective mounted on a Zeiss LSM 510 somatic motor neurons to N-methyl-D-aspartate-induced excitotoxicity. Neuroscience META confocal microscope (Oberkochen, Germany) as described previously (Liu 83, 239-249. et al., 2006; Wan et al., 1997b). Images were acquired using a Zeiss AxioCam Arai, T., Hasegawa, M., Akiyama, H., Ikeda, K., Nonaka, T., Mori, H., Mann, D., digital camera in the linear range with constant settings and were analyzed using Tsuchiya, K., Yoshida, M., Hashizume, Y. et al. (2006). TDP-43 is a component of ImageJ software. Each image was a z-series of 6–13 images, taken at 0.75-mm- ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and depth intervals. The resultant stack was ‘flattened’ into a single image using a amyotrophic lateral sclerosis. Biochem. Biophys. Res. Commun. 351, 602-611. maximum projection. For individual experiments, all images in all experiments Arai, T., Mackenzie, I. R., Hasegawa, M., Nonoka, T., Niizato, K., Tsuchiya, K., were analyzed using identical acquisition parameters. During data acquisition and Iritani, S., Onaya, M. and Akiyama, H. (2009). Phosphorylated TDP-43 in analysis, the investigator was blind to the treatment group. Alzheimer’s disease and dementia with Lewy bodies. Acta Neuropathol. 117,125- 136. Arundine, M. and Tymianski, M. (2004). Molecular mechanisms of glutamate- Transfection dependent neurodegeneration in ischemia and traumatic brain injury. Cell. Mol. Life The small interfering RNAs specific to mouse TDP-43 and PTEN (siRNAtdp43 Sci. 61, 657-668. and siRNApten) and non-targeting control siRNA (NsiRNA) were purchased Ayala, Y. M., Zago, P., D’Ambrogio, A., Xu, Y. F., Petrucelli, L., Buratti, E. and from Santa Cruz Biotech (Santa Cruz, CA). The cortical neurons were transfected Baralle, F. E. (2008). Structural determinants of the cellular localization and with using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the shuttling of TDP-43. J. Cell Sci. 121, 3778-3785. manufacturer’s protocol (Ning et al., 2004). cDNA encoding green fluorescent Barmada, S. J., Skibinski, G., Korb, E., Rao, E. J., Wu, J. Y. and Finkbeiner, S. protein (GFP) was used as a marker of successful neuronal transfection. The cells (2010). Cytoplasmic mislocalization of TDP-43 is toxic to neurons and enhanced by a mutation associated with familial amyotrophic lateral sclerosis. J. Neurosci. 30,639- were fixed for immunocytochemical labeling at 48 hours after transfection. 649. Brunet, N., Tarabal, O., Esquerda, J. E. and Caldero´, J. (2009). Excitotoxic Subcellular fractionation assay motoneuron degeneration induced by glutamate receptor agonists and mitochondrial The assay was performed as described (Ayala et al., 2008). Briefly, cultured toxins in organotypic cultures of chick embryo spinal cord. J. Comp. Neurol. 516, neurons were harvested and washed in PBS by repeated centrifugation. The 277-290. experiment was carried out at 4˚C. The pellet was resuspended in five volumes of Cairns, N. J., Neumann, M., Bigio, E. H., Holm, I. E., Troost, D., Hatanpaa, K. J., buffer N [15 mM Tris-HCl, pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, Foong, C., White, C. L., 3rd, Schneider, J. A., Kretzschmar, H. A. et al. (2007). 1 mM CaCl2, 1 mM DTT, 2 mM Na3VO4, 1 mM PMSF, 0.25 M sucrose, TDP-43 in familial and sporadic frontotemporal lobar degeneration with ubiquitin Complete protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN)]. inclusions. Am. J. Pathol. 171, 227-240. Cell lysis was obtained by adding an equal amount of buffer N plus 0.6% NP-40. Chang, N., El-Hayek, Y. H., Gomez, E. and Wan, Q. (2007). Phosphatase PTEN in After incubation for 5 minutes, nuclei were pelleted and gently resuspended in neuronal injury and brain disorders. Trends Neurosci. 30, 581-586. 1 ml of buffer N. The nuclei were again pelleted and lysed using an equal volume Chen, M., Lu, T. J., Chen, X. J., Zhou, Y., Chen, Q., Feng, X. Y., Xu, L., Duan, W. H. and Xiong, Z. Q. (2008). Differential roles of NMDA receptor subtypes in of solution 2 (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA, 0.1 mM DTT, 0.5 mM PMSF, 5% glycerol, 0.4 M NaCl). The nuclear ischemic neuronal cell death and ischemic tolerance. Stroke 39, 3042-3048. fraction was cleared by centrifugation following incubation for 30 minutes. Corse, A. M., Bilak, M. M., Bilak, S. R., Lehar, M., Rothstein, J. D. and Kuncl, Nuclear and cytoplasmic fractions were quantified and visualized by western R. W. (1999). Preclinical testing of neuroprotective neurotrophic factors in a model of chronic motor neuron degeneration. Neurobiol. Dis. 6, 335-346. blotting. Culcasi, M., Lafon-Cazal, M., Pietri, S. and Bockaert, J. (1994). 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Journal of Cell Science with intact plasma membranes and released from cells with damaged membranes. Meaney, D. F. (2006). Traumatic mechanical injury to the hippocampus in vitro LDH release was measured using a CytoTox 96 Cytotoxicity kit based on the causes regional caspase-3 and calpain activation that is influenced by NMDA receptor manufacturer’s instructions (Promega, Madison, WI) (Shan et al., 2009). The subunit composition. Neurobiol. Dis. 22, 165-176. levels of maximal neuronal death were measured by treating the cultures with 106 Dingledine, R., Borges, K., Bowie, D. and Traynelis, S. F. (1999). The glutamate lysis solution to yield complete lysis of the cells. Absorbance data were obtained receptor ion channels. Pharmacol. Rev. 51, 7-61. using a 96-well plate reader at 490 nm. Fiesel, F. C., Voigt, A., Weber, S. S., Van den Haute, C., Waldenmaier, A., Go¨rner,K., PI labeling was performed to detect neuronal death as described in our previous Walter, M., Anderson, M. L., Kern, J. V., Rasse, T. M. et al. (2010). Knockdown of study (Ning et al., 2004). Briefly, all the experimental cultures were plated with transactive response DNA-binding protein (TDP-43) downregulates histone deacetylase equal number of cells. The culture medium of cells co-transfected with GFP and 6. EMBO J. 29, 209-221. specific siRNAs or cDNAs was replaced by extracellular solution containing PI at Fiszman, M. L., Ricart, K. C., Latini, A., Rodrı´guez, G. and Sica, R. E. (2010). In a final concentration of 50 mg/ml. After incubation for 20 minutes in an ambient vitro neurotoxic properties and excitatory aminoacids concentration in the gas incubator at 37˚C, cultures were washed with extracellular solution and then cerebrospinal fluid of amyotrophic lateral sclerosis patients. Relationship with the fixed with 4% paraformaldehyde. Neuronal death (percentage) was determined by degree of certainty of disease diagnoses. Acta Neurol. Scand. 121, 120-126. calculating the number of PI-labeled neurons expressing GFP. The investigator for Gil, A., Andre´s-Pons, A., Ferna´ndez, E., Valiente, M., Torres, J., Cervera, J. and Pulido, R. (2006). Nuclear localization of PTEN by a Ran-dependent mechanism the cell count was blinded to the experimental treatment. enhances apoptosis: Involvement of an N-terminal nuclear localization domain and multiple nuclear exclusion motifs. Mol. Biol. Cell 17, 4002-4013. Statistics Gredal, O., Werdelin, L., Bak, S., Christensen, P. B., Boysen, G., Kristensen, M. O., All population data were expressed as means 6 s.e.m. The Student’s t-test or the Jespersen, J. H., Regeur, L., Hinge, H. H. and Jensen, T. S. (1997). A clinical trial ANOVA test was used when appropriate. Statistical significance was placed at of dextromethorphan in amyotrophic lateral sclerosis. Acta Neurol. Scand. 96, 8-13. P,0.05. Grosskreutz, J., Van Den Bosch, L. and Keller, B. U. (2010). Calcium dysregulation in amyotrophic lateral sclerosis. Cell Calcium 47, 165-174. Hanson, K. A., Kim, S. H., Wassarman, D. A. and Tibbetts, R. S. (2010). Ubiquilin Acknowledgements modifies TDP-43 toxicity in a Drosophila model of amyotrophic lateral sclerosis We thank James Shen and Virginia Lee for providing us with TDP- (ALS). J. Biol. Chem. 285, 11068-11072. 43 cDNA constructs. We also thank Y. P. Auberson for the gift of Hardingham, G. E., Fukunaga, Y. and Bading, H. (2002). Extrasynaptic NMDARs NVP-AAM077. oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways. Nat. Neurosci. 5, 405-414. Hasegawa, M., Arai, T., Akiyama, H., Nonaka, T., Mori, H., Hashimoto, T., Funding Yamazaki, M. and Oyanagi, K. (2007). TDP-43 is deposited in the Guam This study was supported by University of Nevada Start-up fund and parkinsonism-dementia complex brains. Brain 130, 1386-1394. grants from the National Center for Research Resources [grant Igaz, L. M., Kwong, L. K., Xu, Y., Truax, A. C., Uryu, K., Neumann, M., Clark, C. M., Elman, L. B., Miller, B. L., Grossman, M. et al. (2008). Enrichment of C- number RR024210] and the National Institute of General Medical terminal fragments in TAR DNA-binding protein-43 cytoplasmic inclusions in brain Sciences [grant number GM103554] from the National Institutes of but not in spinal cord of frontotemporal lobar degeneration and amyotrophic lateral Health. Deposited in PMC for release after 12 months. sclerosis. Am. J. Pathol. 173, 182-194. Regulation of nuclear TDP-43 1567

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