The Cdk5-Mcl-1 Axis Promotes Mitochondrial Dysfunction and Neurodegeneration in a Model of Alzheimer’S Disease Kumar Nikhil and Kavita Shah*

RESEARCH ARTICLE The Cdk5-Mcl-1 axis promotes mitochondrial dysfunction and neurodegeneration in a model of Alzheimer’s disease Kumar Nikhil and Kavita Shah*

ABSTRACT cyclin D and cyclin E, which activate other family members. Instead, Cdk5 deregulation is highly neurotoxic in Alzheimer’s disease (AD). it possesses its own specific protein partners, p35 (also known as We identified Mcl-1 as a direct Cdk5 substrate using an innovative Cdk5r1), p39 (also known as Cdk5r2) and cyclin I (CCNI), which are chemical screen in mouse brain lysates. Our data demonstrate that highly expressed in the central nervous system, particularly Mcl-1 levels determine the threshold for cellular damage in response to in postmitotic neurons (Tsai et al., 1994; Tang et al., 1995; neurotoxic insults. Mcl-1 is a disease-specific target of Cdk5, which Brinkkoetter et al., 2010). Cyclin I has not been shown to activate associates with Cdk5 under basal conditions, but is not regulated by it. any other Cdks, suggesting that it might be a unique Cdk5 activator. Neurotoxic insults hyperactivate Cdk5 causing Mcl-1 phosphorylation Likewise, Cdk5 also interacts with its own set of negative at T92. This phosphorylation event triggers Mcl-1 ubiquitylation, which regulators. It is not inhibited by p27Kip1 (also known as Cdkn1b), directly correlates with mitochondrial dysfunction. Consequently, p21Cip1 (also known as Cdkn1a) or p57Kip2 (also known as ectopic expression of phosphorylation-dead T92A-Mcl-1 fully Cdkn1c), which inhibit other Cdk family members. Instead, Cdk5 prevents mitochondrial damage and subsequent cell death triggered activity is inhibited by binding to glutathione-S-transferase P by neurotoxic treatments in neuronal cells and primary cortical (GSTP1), cyclin D1 (CCND1) and cyclin E (CCNE) (Sun et al., neurons. Notably, enhancing Mcl-1 levels offers comparable 2011; Modi et al., 2012; Odajima et al., 2011). Additionally, Cdk5 neuroprotection to that observed upon Cdk5 depletion, suggesting is not phosphorylated at T14, and phosphorylation at Y15 by c-Abl, that Mcl-1 degradation by direct phosphorylation is a key mechanism Eph receptor A4 (EphA4) or Fyn was shown to activate it, instead of by which Cdk5 promotes neurotoxicity in AD. The clinical significance inhibition observed for its family members (Zukerberg et al., 2000; of the Mcl-1-Cdk5 axis was investigated in human AD clinical Sasaki, et al., 2002; Fu et al., 2007). Notably, one study reported that specimens, revealing an inverse correlation between Mcl-1 levels phosphorylation at Y15 has no impact on Cdk5 activity (Kobayashi and disease severity. These results emphasize the potential of Mcl-1 et al., 2014). upregulation as an attractive therapeutic strategy for delaying or Although all Cdk family members are crucial orchestrators of the preventing neurodegeneration in AD. cell cycle, Cdk5 does not participate in the normal cell cycle. However, in postmitotic neurons, deregulation of Cdk5 activity KEY WORDS: Cdk5, Mcl-1, Alzheimer’s disease, Neurodegeneration, aberrantly activates various components of the cell cycle, resulting Chemical genetic, Neuroprotection in neuronal death (Chang et al., 2012). Cdk5 is highly expressed and active in postmitotic neurons, where other Cdks are minimally INTRODUCTION expressed. During embryogenesis, Cdk5 is essential for brain Alzheimer’s disease (AD) is a progressive neurodegenerative development and regulates various neuronal pathways, including disease, which currently affects an estimated 5.3 million neuronal migration, neurite outgrowth, axon guidance and synapse Americans. This number is expected to increase to 16 million by formation (Ohshima et al., 1996). Postbirth, Cdk5 activity is crucial 2050, with an estimated increase of ∼1 million new cases per year. for higher cognitive functions such as synaptic plasticity, learning Currently, the only available drugs for AD are N-methyl D-aspartate and memory formation, long term behavioral changes and drug receptor antagonists and acetylcholinesterase inhibitors, which target addiction (Heller et al., 2016; Hernandez et al., 2016). Reduced symptoms, but do not prevent neurodegeneration (Sun et al., 2012). activity and hyperactivity of Cdk5 are equally neurotoxic, and give In this study, we focused on cyclin-dependent kinase 5 (Cdk5), which rise to various neurodevelopmental and neurological disorders. For is deregulated in AD and contributes to all three hallmarks: formation example, reduction or loss of Cdk5 activity is associated with of β-amyloid (Aβ) plaques and neurofibrillary tangles (NFT), and mental retardation, schizophrenia, epilepsy and attention-deficit/ extensive neurodegeneration (Shukla et al., 2012). hyperactivity disorder (ADHD) (Venturin et al., 2004; Engmann Cdk5 is an atypical cyclin-dependent kinase (Cdk) family et al., 2011; Patel et al., 2004; Drerup et al., 2010). By contrast, member, which has evolved to be functionally divergent from its hyperactivation of Cdk5 occurs in many neurodegenerative family (Dhavan and Tsai, 2001; Shah and Lahiri, 2014, 2017; Shah diseases, including AD, Parkinson’s disease (PD), ischemic stroke and Rossie, 2017). Cdk5 requires a binding partner for activation; and amyotrophic lateral sclerosis (ALS), and is highly destructive however, it is not activated by classical cyclins such as cyclin A, (Hisanaga and Endo, 2010; Meyer et al., 2014). In AD, a variety of neurotoxic signals such as excitotoxicity, Aβ, 2+ Department of Chemistry and Purdue University Center for Cancer Research, ischemia and oxidative stress increase intracellular Ca levels in Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA. neurons, causing the activation of calpain, which cleaves p35 into p25 and p10. p25 has a 5- to 10-fold longer half-life than p35 and *Author for correspondence ([email protected]) lacks the membrane anchoring signal. This results in the constitutive K.S., 0000-0002-0451-1305 activation and mislocalization of the Cdk5-p25 complex, which results in the phosphorylation of a variety of nonphysiological

Received 28 April 2017; Accepted 24 July 2017 targets, culminating in the formation of neurotoxic NFT and Journal of Cell Science

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β-amyloid, and neuronal death (Sun et al., 2009; Chang et al., 2010). contributor to AD pathogenesis (Hosie et al., 2012). HT22 cells Cdk5 regulates neurotoxic β-amyloid formation both at were treated with glutamate for 1.5, 3 and 6 h, and Cdk5 immune transcriptional and post-translational levels. Cdk5 increases complexes were isolated and Mcl-1 levels analyzed. Surprisingly, β-secretase transcription via Stat3, leading to increased β-amyloid Mcl-1 and Cdk5 were associated in control cells, and the level of formation (Wen et al., 2008). Cdk5 increases β-secretase activity by association remained almost the same until 1.5 h of glutamate directly phosphorylating it at T252 (Song et al., 2015). We have treatment. However, as time progressed, Mcl-1 association with shown that phosphorylation of FOXO3a (also known as FOXO3) at Cdk5 decreased and only ∼20% of Mcl-1 remained associated after S43, S173, S294 and S325 by deregulated Cdk5 also contributes to 6 h (Fig. 1B). This result suggested that either glutamate treatment neurotoxic Aβ formation (Shi et al., 2016). A crucial role of Cdk5 in results in the dissociation of Cdk5 and Mcl-1 or that the levels of exacerbating AD pathogenesis was recently shown in a mouse Mcl-1 decline with time. We have previously observed that Cdk5 model, in which specific inhibition of aberrant Cdk5 activity using a levels remain constant during glutamate treatment in HT22 cells. peptide inhibitor attenuated NFT formation and restored synaptic Therefore, Mcl-1 levels were analyzed in glutamate-treated HT22 and cognitive functions (Shukla et al., 2017). cells, revealing a time-dependent decrease in Mcl-1 levels (Fig. 1B). In this study, we uncovered a novel mechanism by which We conducted the reciprocal experiment, in which Mcl-1 was deregulated Cdk5 harnesses induced myeloid cell leukemia isolated from control and glutamate-treated cells (for 1.5, 3 and 6 h), sequence 1 (Mcl-1) and promotes neuronal death. Using an which also confirmed that Cdk5 and Mcl-1 associate under basal innovative chemical genetic screen, we identified Mcl-1 as a direct conditions. Similarly, there was a time-dependent decrease in Cdk5 substrate of Cdk5 kinase in mouse brain lysates. Mcl-1 is a and Mcl-1 association due to the decrease in Mcl-1 levels (Fig. 1C). prosurvivor member of the B-cell lymphoma 2 (Bcl-2) family, We also isolated the Mcl-1 immune complex from control and which is essential for neuronal development (Arbour et al., 2008). glutamate-treated cells, which revealed decreased levels of Mcl-1 as Germline knockout of Mcl-1 causes peri-implantation lethality on time progressed (Fig. 1D). This result suggests that the observed embryonic day 3.5 (Rinkenberger et al., 2000). During cortical decrease in Cdk5 levels in Mcl-1 immune complexes is not due to neurogenesis, Mcl-1 is highly expressed in neural precursors and decreased binding of Cdk5, but instead results from overall reduced newly committed neurons in the cortical plate. Not surprisingly, Mcl-1 levels. loss of Mcl-1 in neuronal progenitors results in extensive apoptosis. To examine whether the interaction between Cdk5 and Mcl-1 was Mcl-1 is also highly expressed in postmitotic neurons and is associated direct, we conducted an in vitro pull-down assay using recombinant with neuronal protection following prolonged seizures (Mori et al., Cdk5 and Mcl-1. Initially, Mcl-1 immune complexes were isolated. 2004). Loss of Mcl-1 sensitizes neurons to DNA damage-induced They were then incubated with recombinant Cdk5, which pulled death (Arbour et al., 2008). In this study, we investigated the down Cdk5 (Fig. 1E). Similarly, we isolated a Cdk5 immune mechanism of Mcl-1 regulation upon neurotoxic conditions and its complex and incubated it with recombinant Mcl-1, which pulled consequences in a mouse hippocampal cell line (HT22 cells), mouse down Mcl-1 (Fig. 1F). These in vitro binding assays confirmed that primary neurons and AD human clinical specimens. Cdk5 and Mcl-1 bind each other directly. This finding prompted us to investigate whether Mcl-1 binds to RESULTS the Cdk5 activator p35 or p25 in HT22 cells. p35/p25 immune Mcl-1 is a direct substrate of Cdk5 complexes were isolated from untreated and glutamate-treated HT22 The chemical-genetic approach utilizes an engineered kinase that cells, and their potential binding to Mcl-1 analyzed. Glutamate possesses a ‘hole’ in the active site, which in the presence of a treatment triggered the formation of p25 as expected (Fig. 1G). radioactive orthogonal ATP analog [e.g. N6-(phenethyl) ATP], Contrary to Cdk5 immune complexes, which showed Mcl-1 binding specifically transfers the radioactive tag (32P) to its substrates. These in both untreated and treated cells, Mcl-1 was only present in ATP analogs are not accepted by wild-type kinases, permitting glutamate-treated cells, suggesting that Mcl-1 presumably associates unbiased identification of direct substrates of the engineered kinase in with p35 and/or p25 via Cdk5 (Fig. 1G). Therefore, we next a global environment (Shah et al., 1997; Shah and Shokat, 2003; investigated Cdk5 levels in p35/p25 immune complexes, which Shah and Vincent, 2005; Kim and Shah, 2007; Johnson et al., 2011, revealed that Cdk5 associates with p35/p25 only in glutamate-treated 2012; Wang et al., 2017a,b). Using these design criteria, we cells (Fig. 1H). Taken together, these findings indicate that glutamate previously generated an analog-sensitive mutant of Cdk5 (named treatment triggers Cdk5 binding to p35/p25, which brings Mcl-1 to Cdk5-as1) that efficiently accepted N-6-phenethyl-ATP (PE-ATP) as p35/p25 immune complexes in glutamate-treated cells (Fig. 1G). the orthogonal ATP analog. Using Cdk5-as1 and [32P] PE-ATP, we Because Mcl-1 associates with Cdk5 under basal conditions, we previously identified several direct Cdk5 substrates including GM130 next investigated whether p35 or p25 was present in this complex in (also known as Golga2), peroxiredoxin 1, peroxiredoxin 2, lamin A, the absence of any neurotoxic signal. Cdk5 immune complexes lamin B, Cdc25a, Cdc25b, Cdc25c and Foxo3 (Sun et al., 2008a,b; were isolated from control and glutamate-treated cells, and p35/p25 Chang et al., 2011, 2012; Shi et al., 2016). In this study, we focused levels analyzed. As shown in Fig. 1I, a negligible amount of p35 on Cdk5-mediated regulation of Mcl-1 signaling. As proteomic associates with Cdk5 in control cells. However, glutamate-treatment screens can often lead to false positives, we tested whether Cdk5 increased the formation of p25, which associated with Cdk5 in a directly phosphorylates Mcl-1 using an in vitro kinase assay. We time-dependent manner (Fig. 1I). More importantly, the absence of found that Cdk5 directly phosphorylates Mcl-1, confirming the result any p35 or p25 in the Cdk5 immune complex in control cells obtained from the chemical genetic screen (Fig. 1A). indicated that Mcl-1 associates with monomeric inactive Cdk5 in the absence of any neurotoxic signal. Mcl-1 is a disease-specific target of Cdk5 This finding also indicated that glutamate treatment should To uncover the significance of Mcl-1 phosphorylation by Cdk5, we increase the binding of p25 to Cdk5 in the Cdk5-Mcl-1 complex. determined whether they associate with each other in HT22 cells Therefore, we isolated Mcl-1 immune complexes from control under normal and/or neurotoxic conditions. We chose glutamate and glutamate-treated HT22 cells, and investigated the levels of p35 as the neurotoxic signal. Glutamate excitotoxicity is a major and p25. As expected, no p35 or p25 was present in the Mcl-1 Journal of Cell Science

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Fig. 1. Mcl-1 is a disease-specific target of Cdk5. (A) Mcl-1 is a direct substrate of Cdk5. Cdk5-p25 complex was subjected to a kinase assay with either [32P]ATP alone (lane 1), or with 6x-His-Mcl-1 and [32P]ATP (lane 3) for 15 min. Lane 2 shows Mcl-1 incubated with [32P]ATP. (B) Cdk5 and Mcl-1 association under normal and neurotoxic conditions in HT22 cells. Cdk5 was immunoprecipitated from either control or glutamate-treated HT22 cells (for 0–6 h), and the association of Cdk5 and Mcl-1 was analyzed. Total Mcl-1 and actin levels were probed in whole-cell lysates. (C) Cdk5 and Mcl-1 association under normal and neurotoxic conditions in HT22 cells. Mcl-1 was immunoprecipitated from either control or glutamate-treated HT22 cells (for 0–6 h), and Mcl-1 and Cdk5 association was analyzed. Total Mcl-1, Cdk5 and actin levels were probed in whole-cell lysates. (D) Mcl-1 levels decrease in Mcl-1 immune complexes. Mcl-1 was immunoprecipitated from control and glutamate-treated HT22 cells (for 0–6 h), and its levels analyzed. Actin was used as a control. (E) Cdk5 binds Mcl-1 directly. Mcl-1 and Cdk5 association was analyzed using recombinant Cdk5 and Mcl-1 in an in vitro pull-down assay. Mcl-1 on beads was incubated with 6×-His-Cdk5 and binding analyzed. The lower panel shows the input (6×-His-Cdk5 and 6×-His-Mcl-1). (F) Cdk5 and Mcl-1 bind directly. Mcl-1 and Cdk5 association was analyzed using recombinant Cdk5 and Mcl-1 in an in vitro pull-down assay. The lower panel shows the input (6×-His-Cdk5 and 6×-His-Mcl-1). (G) Glutamate stimulates the association of p35/p25 with Mcl-1. p35/p25 were immunoprecipitated from either control or glutamate-treated HT22 cells (for 0–6 h), and the association of p35/ p25 with Mcl-1 analyzed. The lower panel shows the relative levels of p35 and p25 in whole-cell lysate from untreated and glutamate-treated cells. Actin was used as a loading control. (H) Cdk5 associates with p35/p25 upon glutamate stimulation. p35/p25 were immunoprecipitated from either control or glutamate-treated HT22 cells (for 0–6 h), and the association of p35/p25 with Cdk5 analyzed. (I) Cdk5 associates with p25 upon glutamate stimulation. (J) Glutamate stimulates association of p25 and Mcl-1. Mcl-1 was immunoprecipitated from either control or glutamate-treated HT22 cells (for 0–6 h), and the association of Mcl-1 and p35/p25 was analyzed. Results in A-J are from at least three independent experiments. (K) Cdk5 activity increases upon glutamate treatment in HT22 cells. The Cdk5 kinase assay was performed as described in the Materials and Methods. *P<0.05, compared with untreated HT22 cells. Journal of Cell Science

3025 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 3023-3039 doi:10.1242/jcs.205666 immune complex in control cells; however, glutamate treatment in residues. This preference suggested T92 as a potential Cdk5 site on HT22 cells resulted in increased presence of p25 in the Mcl-1 Mcl-1. Therefore, we generated the corresponding phospho-dead immune complex (Fig. 1J). Since p25 binding increases Cdk5 mutant (T92A-Mcl-1) and subjected it to an in vitro kinase assay activity, we also measured Cdk5 activity in control and glutamate- using Cdk5-p25. Wild-type Mcl-1 was efficiently phosphorylated, treated cells using an in vitro kinase assay. As shown in Fig. 1K, but T92A-Mcl-1 showed no phosphorylation, confirming that T92 Cdk5 activity increases ∼2-fold after 45 min of glutamate treatment. is the only Cdk5 phosphorylation site on Mcl-1 (Fig. 3A). Taken together, these findings suggest that monomeric inactive Cdk5 binds to Mcl-1 under basal conditions, but does not Cdk5 deregulation triggers Mcl-1 nuclear translocation phosphorylate it. Glutamate treatment activates Cdk5 leading to independent of its phosphorylation at T92 Mcl-1 phosphorylation, suggesting that Mcl-1 is a disease-specific We next investigated whether phosphorylation of Mcl-1 at T92 is target of Cdk5. responsible for nuclear translocation of Mcl-1 upon glutamate treatment. Therefore, we generated stable HT22 cells expressing Cdk5 regulates the subcellular localization of Mcl-1 either Flag-tagged wild-type Mcl-1 or Flag-tagged T92A-Mcl-1, and in HT22 cells analyzed their subcellular localization with or without glutamate Association of Cdk5 and Mcl-1 upon glutamate treatment prompted exposure for 1.5, 3, 6 and 12 h. As expected, wild-type Mcl-1- us to investigate their subcellular localization in HT22 cells. Both expressing HT22 cells revealed nuclear translocation of Mcl-1 upon Cdk5 and Mcl-1 were predominantly cytosolic in HT22 cells glutamate treatment, which increased to ∼50% after 12 h of treatment (Fig. 2A). Glutamate treatment resulted in increased nuclear (Fig. 3B,C). Inhibition of Cdk5 activity using roscovitine, or translocation of both Cdk5 and Mcl-1 in a time-dependent manner depletion of Cdk5 using corresponding shRNA, largely prevented (Fig. 2A,B). After 6–12 h of glutamate treatment, ∼35% cells nuclear translocation of Mcl-1 (Fig. 3B–E). Finally, these findings displayed nuclear Mcl-1, whereas >80% revealed nuclear Cdk5. were corroborated by analyzing the nuclear and cytosolic fractions of Notably, when Cdk5 was inhibited using a small molecule inhibitor wild-type Mcl-1-HT22 cells, which were either control, glutamate- roscovitine, both Mcl-1 and Cdk5 remained cytoplasmic, suggesting treated, glutamate and roscovitine-treated, or glutamate and Cdk5 that Cdk5 not only shows a similar subcellular localization to that of shRNA-treated for varying periods. As shown in Fig. 3F, glutamate Mcl-1, but is also responsible for nuclear translocation of Mcl-1 upon treatment increased the nuclear population of Mcl-1, which was fully glutamate stimulation in HT22 cells (Fig. 2A,B). prevented by Cdk5 inhibition or depletion. These results thus confirm To further validate these results, we performed cytosolic and that deregulated Cdk5 triggers nuclear translocation of wild-type nuclear fractionation of control and HT22 cells treated with Mcl-1 in neuronal cells. glutamate for different time periods. Similar to the results Interestingly, when T92A-Mcl-1-expressing HT22 cells were obtained using immunofluorescence, subcellular fractionation also exposed to glutamate, mutant Mcl-1 still migrated to the nucleus, showed a time-dependent increase in the translocation of Mcl-1 to suggesting that phosphorylation of T92 is not responsible for its the nucleus, which increased with increased glutamate exposure nuclear translocation (Fig. 4A,B). These findings were confirmed in time (Fig. 2C). Cdk5 inhibition using roscovitine fully abrogated cytosolic and nuclear fractions of glutamate-exposed T92A-Mcl-1 this translocation, confirming that Cdk5 activity is responsible for HT22 cells, which revealed both cytosolic and nuclear residence of the migration of Mcl-1 to the nucleus (Fig. 2D). T92A-Mcl-1 in glutamate-exposed cells (Fig. 4C). Taken together, these results demonstrate that although Depletion of Cdk5 inhibits nuclear translocation of Mcl-1 deregulated Cdk5 is responsible for the nuclear translocation of Roscovitine is a highly potent inhibitor of Cdk5; however, it is not Mcl-1, it is not caused by T92 phosphorylation. Our data show that monospecific and inhibits Cdk1 and Cdk2 with almost equal Cdk5 deregulation also causes dispersion of the nuclear envelope, potency (Meijer et al., 1997). Therefore, we knocked down Cdk5 as is evident by the presence of nuclear lamina in the cytosolic and analyzed Mcl-1 subcellular localization upon glutamate fractions in Fig. 2C, Fig. 3F and Fig. 4C (Chang et al., 2011). treatment. Ablation of Cdk5 fully prevented the nuclear Therefore, we predict that disruption of the nuclear lamina translocation of Mcl-1 upon glutamate treatment (analyzed up to mislocalizes Mcl-1 to the nucleus. When Cdk5 is inhibited or 12 h), thereby supporting the results obtained using roscovitine depleted, the nuclear lamina remains intact (Fig. 2D,G and Fig. 3F), (Fig. 2E,F). Finally, we also generated nuclear and cytosolic and maintains the cytoplasmic residence of Mcl-1 despite glutamate fractions of control, glutamate-treated and Cdk5 shRNA-treated treatment. cells, which too revealed complete inhibition of Mcl-1 nuclear translocation in Cdk5-depleted cells (Fig. 2G). Collectively, these Cdk5-mediated phosphorylation of Mcl-1 triggers its results demonstrate that Cdk5 controls the migration of Mcl-1 to the degradation by ubiquitylation nucleus upon deregulation. Importantly, cellular fractionation An increase in Mcl-1 levels is often linked with cell survival (Mori experiments showed increased levels of lamin A in the cytosolic et al., 2004). Therefore, we investigated the consequences of Mcl-1 fraction of glutamate-treated cells (Fig. 2C), but significantly lower phosphorylation in HT22 cells under neurotoxic conditions. Mcl-1 levels in Cdk5-inhibited (Fig. 2D) or Cdk5-depleted (Fig. 2G) cells. levels rapidly decreased upon glutamate treatment in a time- This finding is consistent with our previous study showing rapid dependent manner (Fig. 4D). As shown in Fig. 4E, we observed a dispersion of the nuclear envelope in glutamate-treated HT22 cells >70% decrease in Mcl-1 levels in HT22 cells exposed to glutamate and primary neurons, triggered by Cdk5-mediated phosphorylation for 24 h in three independent experiments. Importantly, Cdk5 of lamin A and lamin B1 (Chang et al., 2011). inhibition or Cdk5 depletion largely prevented this decrease in Mcl- 1 levels under neurotoxic conditions, suggesting that Cdk5 is Cdk5 phosphorylates Mcl-1 at T92 responsible for the observed reduction in Mcl-1 levels (Fig. 4F). To uncover the mechanism by which Cdk5 regulates Mcl-1, we Fig. 4G depicts the impact of Cdk5 inhibition on Mcl-1 levels in examined Cdk5-mediated phosphorylation sites on Mcl-1. Cdk5 glutamate-treated cells from three independent experiments prefers to phosphorylate SP/TP sites which are flanked by basic (Fig. 4G). Journal of Cell Science

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Fig. 2. Cdk5 promotes nuclear localization of Mcl-1 upon glutamate stimulation. (A) Glutamate stimulates nuclear translocation of Cdk5 and Mcl-1. HT22 cells were treated with glutamate for 0–12 h with or without roscovitine, and then immunostained as described in the Materials and Methods. Representative images are shown. (B) Percentage of cells showing nuclear translocation of Mcl-1. At least 100 cells from 10 random frames were counted. Data are mean±s.e.m. from three independent experiments. *P<0.05 vs nucleus fraction control; #P<0.05 vs cytoplasmic fraction control, analyzed by two-way analysis of variance. (C,D) Subcellular fractionation of Mcl-1 in glutamate-treated HT22 cells in the absence or presence of roscovitine, as described in the Materials and Methods, with α-tubulin as the cytoplasmic marker and lamin A as the nuclear marker. N, nuclear fraction; C, cytoplasmic fraction. (E) Cdk5 knockdown inhibits nuclear translocation of Mcl-1. HT22 cells were treated with Cdk5 shRNA for 30 h, followed by glutamate treatment for 0–12 h. (F) The percentage of cells showing nuclear translocation of Mcl-1. Data are mean±s.e.m. from three independent experiments. (G) Cdk5 shRNA-infected HT22 cells were treated with glutamate (0–12 h)

and fractionated as described in the Materials and Methods, with α-tubulin as the cytoplasmic marker and lamin A as the nuclear marker. Scale bars: 20 μm. Journal of Cell Science

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Fig. 3. Cdk5 directly phosphorylates Mcl-1 at T92. (A) The Cdk5-p25 complex (Cdk5/p25) phosphorylates Mcl-1 at T92. Recombinant 6×-His-tagged wild type and Mcl-1 mutant were subjected to a kinase assay with Cdk5-p25. (B) Glutamate stimulates nuclear translocation of Cdk5 and Mcl-1 in Mcl-1-HT22 cells. Mcl-1-HT22 cells were treated with glutamate for 0–12 h with or without roscovitine, and then immunostained with anti-Flag and Cdk5 antibody. Representative images are shown. (C) The percentage of cells showing nuclear translocation of Mcl-1. Data are mean±s.e.m. from three independent experiments. *P<0.05 vs nucleus fraction control; #P<0.05 vs cytoplasmic fraction control, analyzed by two-way analysis of variance. (D) Cdk5 knockdown inhibits nuclear translocation of Mcl-1. Cdk5 shRNA-infected Mcl-1-HT22 cells were treated with glutamate (0–12 h). Representative images are shown. (E) The percentage of cells showing nuclear translocation of Mcl-1. Data are mean±s.e.m. from three independent experiments. (F) Subcellular fractionation of Mcl-1 in glutamate-treated Mcl-1-HT22 cells in the absence or presence of roscovitine or Cdk5 shRNA, with α-tubulin as the cytoplasmic marker and lamin A as the nuclear marker. Scale bars: 20 μm. Journal of Cell Science

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Fig. 4. Mcl-1 translocation to the nucleus is phosphorylation independent. (A) Mcl-1 phospho-dead mutant translocates to the nucleus upon glutamate stimulation. T92A-Mcl-1-HT22 cells were treated with glutamate for 0–12 h, and then immunostained with anti-Flag antibody. Representative images are shown. Scale bar: 20 μm. (B) The percentage of cells showing nuclear translocation of Mcl-1. Data are mean±s.e.m. from three independent experiments. *P<0.05 vs nucleus fraction control; #P<0.05 vs cytoplasmic fraction control, analyzed by two-way analysis of variance. (C) Subcellular fractionation of T92A-Mcl-1 in glutamate-treated T92A-Mcl-1-HT22 cells, with α-tubulin as the cytoplasmic marker and lamin A as the nuclear marker. T92A-Mcl-1 was visualized using anti-Flag antibody. (D) Mcl-1 levels decrease upon glutamate treatment. HT22 cells were treated with glutamate for 0–24 h and the total levels of Mcl-1 analyzed. (E) Average relative ratios of Mcl-1 band intensities to alpha-tubulin band intensities upon glutamate treatment, as obtained from three independent experiments. *P<0.05, compared with untreated HT22 cells. (F) Cdk5 inhibition or ablation prevents the decrease in Mcl-1 levels in glutamate-treated HT22 cells. HT22 cells were treated with glutamate for 12 h, in the presence and absence of roscovitine or Cdk5 shRNA and then the total levels of Mcl-1 were analyzed. (G) Mcl-1 protein levels in HT22 cells in response to glutamate treatment with or without roscovitine and Cdk5 shRNA. Data are mean±s.e.m. from three independent experiments. *P<0.05, compared with untreated HT22 cells; #P<0.05, compared with glutamate-treated cells. (H) Wild-type Flag-Mcl-1 levels decrease upon glutamate treatment. Flag-Mcl-1-HT22 cells were treated similarly to in D and total levels of Flag-Mcl-1 analyzed using Flag antibody. (I) Average relative ratios of Flag (Mcl-1) band intensities to α-tubulin band intensities upon glutamate treatment, as obtained from three independent experiments. *P<0.05, compared with untreated Mcl-1-HT22 cells. (J) Cdk5 inhibition or ablation prevents the decrease in wild-type Flag-Mcl-1 levels in glutamate-treated HT22 cells. Flag-Mcl-1-HT22 cells were treated similarly to in F and Mcl-1 levels analyzed using Flag antibody. (K) Wild-type Flag-Mcl-1 protein levels in HT22 cells in response to glutamate treatment with or without roscovitine and Cdk5 shRNA. Data are mean±s.e.m. of three independent experiments. *P<0.05, compared with untreated HT22 cells. #P<0.05, compared with glutamate-treated cells. (L) T92A-Mcl-1 levels do not vary significantly with glutamate treatment. Flag-tagged T92A-Mcl-1-HT22 cells were treated similarly to in D and total levels of Mcl-1 analyzed using Flag antibody. (M) Average relative ratios of Flag (Mcl-1) band intensities to α-tubulin band intensities upon glutamate treatment, as obtained from three independent experiments. (N) T92A-Mcl-1 levels do not vary significantly in the absence or presence of Cdk5 inhibitor or Cdk5 shRNA in glutamate-treated cells. T92A-Mcl-1-HT22 cells were treated similarly to in F and Mcl-1 levels analyzed. (O) Flag-T92A-Mcl-1 protein levels in HT22 cells in response to glutamate treatment with or without roscovitine and Cdk5 shRNA. Data are mean±s.e.m. from three independent experiments. Journal of Cell Science

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To investigate a potential role of T92 phosphorylation in this treated neuronal cells, confirming a central role of deregulated Cdk5 event, both wild-type Flag-tagged Mcl-1 and Flag-tagged T92A- in promoting neurotoxic cascades. Mcl-1 HT22 cells were treated with glutamate for increasing time The correlation between Mcl-1 degradation and increased cell periods. As expected, Flag-Mcl-1 was degraded in wild-type Mcl-1 death prompted us to overexpress Mcl-1 in HT22 cells as a potential HT22 cells (Fig. 4H). Fig. 4I shows the average impact of glutamate therapeutic approach to rescue cells exposed to neurotoxic conditions. treatment on Mcl-1 levels from three independent experiments. Overexpression of Mcl-1 was indeed highly neuroprotective and Importantly, Mcl-1 degradation was prevented if Cdk5 activity was >80% of the cells survived, compared to 50% of regular HT22 cells. inhibited or depleted (Fig. 4J,K). By contrast, T92A-Mcl-1 HT22 Phosphorylation-dead T92A-Mcl-1 expression rescued >90% of the cells showed no change in Mcl-1 levels upon glutamate exposure cells from neurotoxicity, which underscores the significance of Mcl-1 (Fig. 4L,M), and were unaffected by Cdk5 inhibition or depletion upregulation as an approach for AD therapy (Fig. 6B). (Fig. 4N,O). Taken together, these results confirm that Cdk5- mediated phosphorylation of Mcl-1 is responsible for the decrease Phosphorylation-resistant Mcl-1 protects cells from in Mcl-1 levels. mitochondrial dysfunction Mcl-1 has a short half-life of <1 hour (Adams and Cooper, 2007). Mcl-1 serves as a critical regulatory point in mitochondrial-induced Therefore, we investigated whether glutamate exposure resulted in apoptosis by regulating the permeability of cytochrome c (Thomas the degradation of Mcl-1. HT22 cells were treated with et al., 2010). Release of cytochrome c triggers the formation of an cycloheximide to inhibit protein synthesis, and their degradation apoptosome complex, thereby initiating a caspase cascade leading to kinetics analyzed with and without glutamate treatment. Exposure neuronal apoptotic death. Therefore, we next investigated to glutamate resulted in reduced Mcl-1 levels, suggesting that the whether protection of Mcl-1 from Cdk5-mediated degradation Cdk5-mediated decrease in Mcl-1 levels upon glutamate stimulation rescues mitochondrial dysfunction triggered by glutamate is caused by increased degradation (Fig. 5A,B). To investigate the stimulation in neuronal cells. Potential mitochondrial impact of Mcl-1 phosphorylation on its stability, we also analyzed depolarization was analyzed using JC-1 dye in HT22, Mcl-1- its protein degradation profile in Flag-Mcl-1-HT22 and Flag-T92A- HT22 and T92A-Mcl-1-HT22 cells exposed to glutamate for Mcl-1-HT22 cells upon glutamate treatment. These cells were 12 h and 24 h. JC-1 is a cationic dye, used as an indicator of exposed to cycloheximide, then treated with glutamate for varying mitochondrial potential. When mitochondria are hyperpolarized, periods, and the levels of ectopically expressed wild-type and JC-1 accumulates at high concentration in the matrix, leading to mutant Mcl-1 were examined using Flag antibody. Although wild- its aggregation, resulting in red to orange emission. When type Mcl-1 was rapidly degraded upon glutamate treatment mitochondria are depolarized, JC-1 is present predominantly as a (Fig. 5C,D), T92A-Mcl-1 was significantly resistant to glutamate monomer that yields green fluorescence. Importantly, the ratio of toxicity (Fig. 5E,F). Taken together, these results indicate that red to green fluorescence depends only on the membrane Cdk5-mediated phosphorylation of Mcl-1 triggers its degradation. potential and is independent of mitochondrial size, shape, and We next examined whether Mcl-1 degradation is mediated by density. Use of dual-color fluorescence detection thus allows ubiquitylation. We expressed 6×-His-ubiquitin in HT22 cells, researchers to perform ratiometric semiquantitative assessment which were exposed to glutamate for 12 h. Mcl-1 was then of mitochondrial polarization states (Perry et al., 2011). immunoprecipitated and potential ubiquitylation analyzed. As As shown in Fig. 6C, glutamate stimulation for 12 h and 24 h shown in Fig. 5G, glutamate treatment resulted in robust initiated robust mitochondrial depolarization in HT22 cells at 12 h, ubiquitylation of Mcl-1, which was significantly inhibited upon which significantly increased at 24 h. Although wild-type Mcl-1- Cdk5 inhibition or depletion. Similar results were obtained in Flag- expressing HT22 cells were relatively more resistant to mitochondrial Mcl-1-HT22 cells upon glutamate treatment, confirming that Cdk5 dysfunction at 12 h and 24 h, T92A-Mcl-1-HT22 exhibited higher promotes Mcl-1 degradation by ubiquitylation in glutamate- resilience to glutamate-induced neurotoxicity (Fig. 6D,E exposed HT22 cells (Fig. 5H). By contrast, T92A-Mcl-1 cells respectively). Fig. 6F displays the ratio of JC-1 aggregates versus showed little ubiquitylation of mutant Mcl-1 (analyzed using Flag monomers in HT22, Mcl-1-HT22 and T92A-Mcl-1-HT22 cells. antibody) upon glutamate exposure, thereby suggesting that Cdk5- mediated phosphorylation of Mcl-1 at T92 contributes to Mcl-1 Deregulated Cdk5 triggers nuclear translocation of Mcl-1 degradation (Fig. 5I). in primary neurons upon excitotoxicity To examine a potential role of Cdk5 in regulating Mcl-1 levels and Mcl-1 levels set the threshold for neuronal vulnerability its consequences under more pathologically relevant conditions, we As Mcl-1 is an anti-apoptotic protein, we hypothesized that Cdk5- exposed mature primary neurons to 100 µM glutamate to induce mediated degradation of Mcl-1 may be one of the mechanisms by excitotoxicity (Hosie et al., 2012). We initially analyzed whether which Cdk5 promotes neurotoxicity. We observed ∼50% loss of glutamate stimulation affects the subcellular localization of Mcl-1 cell viability after 24 h of glutamate treatment in HT22 cells. Mcl-1 and if this event was regulated by Cdk5. Similar to the results knockdown in these cells resulted in a modest decrease in cell obtained in HT22 cells, glutamate stimulation of primary cortical viability (∼60%) (Fig. 6A). This result was not surprising, as Mcl-1 neurons exhibited a time-dependent increase in nuclear had already been depleted by >70% following glutamate treatment translocation of Mcl-1, which was essentially prevented when (Fig. 4D,E); therefore, its knockdown did not reveal any significant Cdk5 was inhibited using roscovitine or knocked down using Cdk5 impact on cell viability. By contrast, in normal HT22 cells, Mcl-1 shRNA (Fig. 7A–D). These results confirm that Cdk5 deregulation knockdown resulted in ∼30% loss of cell viability, suggesting that upon excitotoxicity triggers nuclear translocation of Mcl-1. In Mcl-1 is important for cell survival under normal and neurotoxic parallel, we also examined Cdk5 activity in primary cortical conditions in neuronal cells. Cdk5 inhibition using roscovitine was neurons, which confirmed Cdk5 activation upon exposure to partially neuroprotective, which is presumably due to its inhibition glutamate (Fig. 7E). To further confirm that Cdk5 deregulation of Cdk1 and Cdk2, both of which are required for the normal cell was caused by p25, we transduced TAT-p25, which too revealed cycle. Cdk5 depletion was highly neuroprotective in glutamate- similar levels of Cdk5 activation in primary neurons (Fig. 7F). Journal of Cell Science

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Fig. 5. Cdk5-mediated phosphorylation of Mcl-1 at T92 triggers Mcl-1 degradation by ubiquitylation. (A) Glutamate stimulation promotes Mcl-1 degradation. HT22 cells were treated with 10 µg/ml of the protein synthesis inhibitor cycloheximide for 30 min, followed by control or 5 mM glutamate treatment. Cells were harvested at various time points (0, 45 and 90 min) and Mcl-1 protein levels determined. CHX, cycloheximide. (B) Graphical representation of Mcl-1 degradation rate. The results of densitometric scanning are shown with Mcl-1 signal normalized to α-tubulin signal. Data are mean±s.e.m. from three independent experiments. (C) Flag-Mcl-1-HT22 cells were treated as described above and total levels of Mcl-1 analyzed using Flag antibody. (D) Graphical representation of Flag (Mcl-1) degradation rate. The results of densitometric scanning are shown with Flag (Mcl-1) signal normalized to α-tubulin signal. Data are mean±s.e.m. from three independent experiments. (E) T92A-Mcl-1 is resistant to glutamate-induced degradation. T92A-Mcl-1-HT22 cells were treated as described above and T92A-Mcl-1 levels analyzed using Flag antibody. (F) Graphical representation of Mcl-1 degradation rate. The results of densitometric scanning are shown with Flag (Mcl-1) signal normalized to α-tubulin signal. Data are mean±s.e.m. from three independent experiments. (G) Mcl-1 degradation is mediated by ubiquitylation. 6×-His-ubiquitin was expressed in HT22 cells, followed by glutamate treatment for 12 h in the presence and absence of roscovitine or Cdk5 shRNA. Mcl-1 was immunoprecipitated and potential ubiquitylation analyzed using 6×-His antibody. Representative data are shown from at least three independent experiments. (H) Wild-type Mcl-1 is ubiquitylated in response to glutamate treatment. Flag-Mcl-1-HT22 cells were treated as described above and ubiquitylated proteins were isolated and analyzed. Representative data are shown from at least three independent experiments. (I) T92A-Mcl-1 is resistant to ubiquitylation upon glutamate treatment. T92A-Mcl-1-HT22 cells were treated similarly and ubiquitylated proteins were isolated and analyzed. Representative data are shown from at least three independent experiments.

The fusion of the TAT sequence with proteins enables protein transduction efficiently activates Cdk5 in cells (Sun et al., 2008a). transduction into cells when directly added to the culture (Becker- TAT-fusion red fluorescent protein (TAT-RFP) was used as a

Hapak et al., 2001). We have previously shown that TAT-p25 control. Journal of Cell Science

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Fig. 6. Mcl-1 degradation promotes mitochondrial dysfunction and neurotoxicity. (A) Mcl-1 ablation is neurotoxic in HT22 cells. Scrambled-shRNA infected, Cdk5-shRNA- infected, Mcl-1-shRNA-infected or roscovitine-exposed HT22 cells were treated with glutamate for 24 h. Cell viability was examined using an MTT assay. *P<0.05, compared with untreated HT22 cells. (B) Ectopic expression of T92A-Mcl-1 rescues cells from glutamate toxicity. HT22, Mcl-1-HT22 and T92A-Mcl-1-HT22 cells were treated with glutamate in the absence or presence of roscovitine and Cdk5 shRNA. Cell viability was examined using an MTT assay. *P<0.05, compared with respective control cells. (C) Glutamate stimulation induces mitochondrial depolarization in HT22 cells. Mitochondrial depolarization was analyzed using JC-1 dye. (D) Glutamate stimulation induces mitochondrial depolarization in Mcl-1-HT22 cells. (E) T92A-Mcl-1-HT22 cells are more resistant to mitochondrial depolarization than control HT22 cells. (F) Ratio of JC-1 aggregates versus monomers in HT22, Mcl-1-HT22 and T92A- Mcl-1-HT22 cells. Data are mean±s.e.m. from three independent experiments. *P<0.05, compared with untreated cells. Scale bars: 20 μm. Journal of Cell Science

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Fig. 7. See next page for legend. Journal of Cell Science

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Fig. 7. Mcl-1 signaling in primary neurons. (A) Cdk5 promotes nuclear finding is highly significant, as glutamate treatment already reduces translocation of Mcl-1 in primary neurons. Primary cortical neurons were Mcl-1 levels to ∼20% of its original level (Fig. 7G,H), and additional treated with glutamate (100 µM) for 12 h in the presence or absence of reduction in Mcl-1 levels via knockdown further sensitizes these roscovitine (10 µM), and then immunostained. (B) The percentage of cells showing nuclear translocation of Mcl-1. Data are mean±s.e.m. from three neurons, suggesting that Mcl-1 degradation is an important independent experiments. *P<0.05 vs nucleus fraction control; #P<0.05 vs contributor to Cdk5-induced neurotoxicity. Cdk5 depletion fully cytoplasmic fraction control, analyzed by two-way analysis of variance. (C) The prevented the loss of neuronal viability induced by excitotoxicity, percentage of cells showing nuclear translocation of Cdk5. Data are mean± thus highlighting a central role of Cdk5 in this process. s.e.m. from three independent experiments. *P<0.05 vs nucleus fraction Finally, we analyzed the role of Cdk5 in inducing synaptic toxicity # control; P<0.05 vs cytoplasmic fraction control analyzed by two-way analysis under excitotoxic conditions by investigating the distribution of of variance. (D) Cdk5 depletion inhibits nuclear translocation of Mcl-1. Primary cortical neurons were treated with glutamate (100 µM) for 12 h in the presence PSD95, a postsynaptic protein (also known as DLG4), in primary or absence of Cdk5 shRNA, and then immunostained. (E) Cdk5 kinase activity cortical neurons. Untreated cortical neurons immunostained with increases upon glutamate treatment in primary cortical neurons. Primary PSD95 (green) and MAP2 (a neuronal protein, red) antibodies cortical neurons were treated with glutamate for 0.5 h and 1 h, and Cdk5 kinase displayed long neurites, which shrank severely upon glutamate activity was analyzed as described in the Materials and Methods. Data are treatment with a concomitant decrease in PSD95 levels (Fig. 7L,M). mean±s.e.m. from three independent experiments. *P<0.05, compared with Inhibition or ablation of Cdk5 prevented PSD95 downregulation, untreated primary neurons. (F) TAT-p25 transduction increases Cdk5 activity highlighting a role of deregulated Cdk5 in synaptic toxicity. in primary cortical neurons. 200 nM TAT-p25 was transduced in primary neurons and Cdk5 kinase activity measured as described in the Materials and Methods. Data are mean±s.e.m. from three independent experiments. Mcl-1 levels show inverse correlation with disease severity *P<0.05, compared with untreated primary neurons. (G) Mcl-1 is degraded by in AD pathogenesis glutamate treatment. Primary cortical neurons were treated with glutamate for To investigate the clinical significance of our findings, we examined 0–24 h and the total levels of Mcl-1 analyzed. (H) Average relative ratios of Mcl- Mcl-1 levels in human AD and age-matched control brain tissues. α 1 band intensities to -tubulin band intensities upon glutamate treatment, as We obtained the tissues from prefrontal cortex area 9 (Brodmann’s obtained from three independent experiments. *P<0.05, compared with untreated primary neurons. (I) Cdk5 depletion or inhibition prevents the area 9), a prefrontal association region located in the superior frontal decrease in Mcl-1 levels in glutamate-treated cells. Mcl-1 levels were analyzed gyrus of the brain, which is severely affected in AD (Braak and following glutamate (12 h), roscovitine and Cdk5 shRNA treatments. (J) Mcl-1 Braak, 1991). These neurons are extremely vulnerable in AD, and protein levels in primary cortical neurons in response to glutamate treatment their loss strongly associates with the severity of the disease, with with or without roscovitine and Cdk5 shRNA. Data are mean±s.e.m. from three >90% loss occurring in the end stages of AD (Bussierè et al., 2003). independent experiments. *P<0.05, compared with untreated primary Mcl-1 levels were analyzed in tissues obtained from patients with neurons; #P<0.05, compared with glutamate-treated cells. (K) Inhibition and mild (n=1), moderate (n=2) and severe (n=2) AD, along with age- ablation of Cdk5 inhibits neurotoxicity while ablation of Mcl-1 promotes n neurotoxicity in primary cortical neurons. Cdk5-shRNA- or Mcl-1-shRNA- matched controls ( =3). Although a limited number of specimens infected primary neurons (30 h) were treated with glutamate (24 h). Cell were analyzed in this study, among these clinical tissues, Mcl-1 viability was tested by an MTT assay. *P<0.05, compared with untreated levels exhibited a strong correlation with disease severity and neurons. (L) Cdk5 inhibition or ablation prevents synaptic neurotoxicity in decreased substantially as the disease progressed, with minimal primary neurons. Primary cortical neurons pre-treated with roscovitine or Cdk5 Mcl-1 levels observed in the patients with severe AD (Fig. 8A). shRNA were treated with glutamate for 12 h, and then immunostained for Fig. 8B shows the mean Mcl-1 levels in age-matched controls and PSD95 and MAP2. Representative images are shown. (M) Quantification of relative neurite length in cortical neurons exposed to various treatments. AD patients from three independent experiments (Fig. 8B). These *P<0.05, compared with untreated primary neurons; #P<0.05, compared with results supported our finding that Mcl-1 degradation is one of the glutamate-treated neurons. ImageJ 1.46r with add-in NeuriteQuant was used key mechanisms in the progression of AD pathology. for morphological analysis. *P<0.05, compared with untreated neurons; # P<0.05, compared with glutamate-treated cells. Scale bars: 20 μm. DISCUSSION Three spliced variants of Mcl-1 are known to exist in human tissues: full length or the long isoform (Mcl-1L), with an antiapoptotic Cdk5 is responsible for Mcl-1 degradation under excitotoxic function, the short isoform (Mcl-1S), with a proapoptotic function, conditions in primary neurons and Mcl-1ES, also with a proapoptotic function (Kozopas et al., We next analyzed whether excitotoxicity affects the levels of Mcl-1 1993; Bae et al., 2000; Kim et al., 2009). Previous studies have and whether this process is Cdk5 dependent. Mature primary established that only the long isoform of Mcl-1 is expressed in the cortical neurons were treated with glutamate for varying periods and brain and is associated with neuronal survival (Mori et al., 2004). Mcl-1 levels analyzed. As shown in Fig. 7G, excitotoxicity Although a crucial oncogenic role of Mcl-1 in cancer has been well triggered a rapid loss in Mcl-1 levels (>50% in 6 h), similar to established, only a handful of studies have analyzed its contribution that observed in HT22 cells (Fig. 4D). Fig. 7H displays the in neurological disorders. A recent study revealed that β-amyloid alterations in Mcl-1 levels in response to excitotoxicity from three injection into the lateral ventricles of mice results in downregulation independent experiments. of Mcl-1 due to a decrease in the levels of myocardin-related A potential role of Cdk5 in degrading Mcl-1 upon excitotoxicity transcription factor-A (MRTF-A, also known as Mkl1), a was examined using roscovitine and Cdk5 shRNA, both of which transcriptional activator of Mcl-1 (Zhang et al., 2016). By prevented Mcl-1 degradation under neurotoxic conditions (Fig. 7I,J), contrast, another recent study reported that injection of β-amyloid confirming that deregulated Cdk5 is responsible for degrading Mcl-1 in mice upregulates Mcl-1 mRNA leading to neuroprotection (Tao in primary neurons. To investigate the significance of Mcl-1 levels in et al., 2017). Similarly, preconditioning of neurons with a sublethal Cdk5-induced neurotoxicity, we examined cell viability in mature dose of 5-aminoimidazole-4-carboxamide riboside (AICAR), primary neurons treated with 100 µM glutamate. Excitotoxicity activates AMP-activated protein kinase (AMPK), which in turn resulted in ∼60% loss of neuronal viability over 24 h (Fig. 7K). More upregulates the transcription of Mcl-1, providing neuroprotection importantly, Mcl-1 knockdown further sensitized the neurons to (Anilkumar et al., 2013). Taken together, these findings highlight excitotoxic conditions resulting in >70% loss of cell viability. This a neuroprotective role of Mcl-1; however, it remains unclear Journal of Cell Science

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Fig. 8. Mcl-1 levels show inverse correlation with disease severity in AD clinical specimens. (A) Mcl-1 levels in human AD and age-matched control brain tissues. Mcl-1 levels were analyzed in tissues obtained from patients with mild (n=1), moderate (n=2) and severe (n=2) AD, along with age-matched controls (n=3). (B) Average Mcl-1 levels in age-matched controls and AD patients from three independent experiments. (C) Schematic of our model showing the link between Cdk5-mediated phosphorylation of Mcl-1 and neuronal death. Monomeric Cdk5 associates with Mcl-1. Glutamate stimulation triggers the formation of p25 formation, which binds the Cdk5-Mcl-1 complex, activating Cdk5. Deregulated Cdk5 phosphorylates Mcl-1 at T92, triggering its degradation by ubiquitylation. Mcl-1 degradation causes mitochondrial dysfunction and subsequent neuronal death. whether Mcl-1 is upregulated or downregulated in response to In this study, we have unraveled the mechanism by which Cdk5 neurotoxic insults in AD models. Similarly, it is not known whether deregulation directly leads to the degradation of Mcl-1 resulting in Mcl-1 levels are regulated post-translationally in AD models. Mcl-1 neurotoxicity in a model of AD (Fig. 8C). Our data demonstrate that levels have also not been analyzed in AD patient specimens and Mcl-1 levels determine the threshold for cellular damage in response their correlation with disease progression remains unknown. to neurotoxic insults. Interestingly, under basal conditions, inactive Journal of Cell Science

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Cdk5 remains associated with Mcl-1, although it does not affect its Isolation of primary cortical neurons levels. We also investigated potential binding of p35 and p25 to The primary cortical neurons were isolated from E17 CD1 mice embryos as Mcl-1 independent of Cdk5 in an in vitro binding assay using purified reported previously (Chang et al., 2012). All experiments were performed recombinant Mcl-1, p35 and p25. Recombinant p25 and p35 on after 12 days in culture. Briefly, cortices were isolated and treated with beads were independently incubated with 6×-His-Mcl-1. p35 and p25 trypsin (0.2 mg/ml) at 37°C for 30 min, followed by trituration in minimum essential media (MEM) supplemented with DNase (130 U/ml) and 10% immune complexes showed no binding with Mcl-1, confirming that FBS. They were then passed through a cell strainer (40 µM mesh). After p35 or p25 do not bind Mcl-1 directly (data not shown). However, centrifugation at 200 g for 4 min, the isolated cortical neurons were seeded glutamate stimulation triggers the association of p25 with the Cdk5- on poly-L-lysine-coated coverslips in a 24-well plate at a density of 5×104 Mcl-1 complex, leading to Mcl-1 phosphorylation at T92 and rapid cells, or in a six-well plate at a density of 1×106 cells, in MEM supplemented degradation via ubiquitylation. Degradation of Mcl-1 directly with 5% FBS, 5% horse serum, 0.5 mM glutamine, 2.6 g/l glucose, 2.2 g/l correlates with mitochondrial dysfunction and subsequent neuronal NaHCO3 and 1 mM pyruvate. The medium was changed after 12 h and half death (Fig. 8C). the amount of medium was replaced every 5 days. Importantly, a recent study has shown that serum and KCl Expression plasmids and constructs deprivation triggers apoptosis in cerebral granule neurons via Flag-tagged human Mcl-1 was cloned into TAT-HA and VIP3 vectors at phosphorylation of Mcl-1 at S140 and T144 by JNK1. BamHI and EcoRI sites. Flag-tagged Mcl-1 mutant was generated using Phosphorylation of Mcl-1 at T144 primes it for phosphorylation by overlapping PCR. GSK3β which phosphorylates it further at S159, triggering its degradation (Magiera et al., 2013). Consequently, the phospho-dead Expression and purification of Cdk5, p25 and Mcl-1 S140A, T144A double mutant of Mcl-1 is degradation-resistant and 6×-His-Cdk5 and 6×-His-p25 were generated and purified as reported rescues cells from apoptosis. previously (Chang et al., 2012). 6×-His tagged wild-type and mutant Mcl-1 were expressed in E. coli and purified according to our published procedures In contrast to the results from Magiera et al. (2013), our results (Sun et al., 2008a,b). indicated a crucial role of Cdk5 in mediating neurotoxicity in a Mcl-1- dependent manner. We observed that ectopic expression of phospho- Transfection and retroviral infection resistant T92A-Mcl-1 is fully capable of rescuing neurons from cell For generating stable cell lines, Flag-tagged wild-type and mutant Mcl-1 death by preventing mitochondrial dysfunction upon neurotoxic plasmid (Addgene plasmid #25371, deposited by Roger Davis) (Morel insults, which was comparable to the neuroprotection observed upon et al., 2009) were transiently transfected using calcium phosphate into Cdk5 depletion. This finding suggests that Mcl-1 degradation is one of Phoenix cells. The retroviruses were harvested and used to infect HT22 cells the key mechanisms by which Cdk5 promotes neurodegeneration. as reported previously (Shah and Shokat, 2002). Furthermore, the clinical significance of our finding was confirmed in specimens from AD patients, in which a strong inverse correlation was In vitro kinase assays in vitro observed between Mcl-1 levels and disease severity compared to age- For labeling, 2 µg of 6×-His-tagged wild-type or mutant Mcl-1 were incubated with Cdk5-p25 and 0.5 mCi of [γ-32P]ATP in a kinase buffer. matched controls. These findings strongly emphasize the potential of Reactions were terminated by adding SDS sample buffer, after which the Mcl-1 upregulation as an attractive therapeutic strategy for the proteins were separated by SDS-PAGE, transferred to PVDF membrane and treatment of AD and perhaps other neurological disorders. This is in exposed for autoradiography. For determining Cdk5 activity in control and sharp contrast with cancer, for which degradation of Mcl-1 is highly glutamate-treated cells, Cdk5 immune complexes were isolated, and subjected desirable for eradicating diseased cells. Therefore, Mcl-1 upregulation to an in vitro kinase assay using [32P]ATP and 5 µg Cdk5 substrate peptide in the brain needs to be highly tissue-specific, and could delay, and (KHHKSPKHR) in a final volume of 30 µl kinase buffer (50 mM Tris-HCl, possibly prevent, neurodegeneration. pH 8.00, 20 mM MgCl2). After 30 min, the reactions were terminated by In conclusion, Cdk5 deregulation is known to trigger many neurotoxic spotting 25 µl of the reaction mixture on p81 phosphocellulose disks cascades in AD pathogenesis, rendering Cdk5 a highly attractive (Whatman) and immersing in 100 ml 10% acetic acid for 25 min, followed – therapeutic target for the treatment of AD. This study identified a new by three washes in 0.5% phosphoric acid (3 5 min each) and finally rinsing with acetone. Radioactivity was measured in a liquid scintillation counter mechanism by which Cdk5 deregulation leads to rapid degradation of a (Packard Instrument Company, CA). pro-survival protein Mcl-1, a process that is extremely neurotoxic. Together, these results strongly emphasize the potential of Mcl-1 Mcl-1 shRNA upregulation or Cdk5 inhibition as attractive therapeutic strategies for Cdk5 shRNA (mouse) was generated in our previous study (Chang et al., delaying and perhaps preventing neurodegeneration in AD patients. 2011). Mcl-1 shRNA was cloned into pLKO.1 vector (Addgene plasmid #10878, deposited by David Root) (Moffat et al., 2006). Mcl-1 shRNA MATERIALS AND METHODS (mouse) primer sequences were as follows: forward primer: 5′-CCGGGG- Glutamate, 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide GACTGGCTAGTTAAACAAACTCGAGTTTGTTTAACTAGCCAGTC- (MTT) and poly-l-lysine were obtained from Sigma-Aldrich. Roscovitine CCTTTTTG-3′; reverse primer: 5′-AATTCAAAAAGGGACTGGCTAG- was purchased from LC Laboratories. Antibodies against Cdk5 (C-8), Cdk5 TTAAACAAACTCGAGTTTGTTTAACTAGCCAGTCCC-3′. Scrambled (DC-17), p35 (C-19), actin (C-2), α-tubulin (TU-02), lamin A (H-102), shRNA, Cdk5 and Mcl-1 shRNA lentiviruses were generated and used for MAP2 (H300), PSD95 (7E3) and enolase (N-14) were purchased from infecting HT22 cells for 30 h. Mcl-1 and Cdk5 shRNA validation are shown Santa Cruz Biotechnology (Chang et al., 2011, 2012). Validated antibodies in Fig. S1A,B. against Mcl-1 were purchased from One World Lab. All antibodies were used at 1:1000 dilution and are listed in Table S1. Validation of the Mcl-1 Western blotting and Cdk5 antibodies is shown in Fig. S1. Cells were lysed in modified RIPA buffer, supplemented with protease inhibitors. Equal amounts of cell extracts were used for western blotting. Cell culture HT22 cells were a kind gift from David Schubert (Salk Institute, La Jolla, MTT assay CA). HEK293T cells were purchased from ATCC. HT22 cells, HEK293T Cells were seeded in 96-well plates at 5×103 cells per 100 µl per well and cultured and Phoenix cells were cultured as previously reported (Sun et al., 2011). All for 24 h. The MTT assay was conducted as reported previously (Sun et al., cells were tested for potential contamination. 2008a). At the end of treatment, MTT (0.5 mg/ml) was added and incubated for Journal of Cell Science

3036 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 3023-3039 doi:10.1242/jcs.205666 an additional 30 min. The media were aspirated and the cells on wells dissolved Ubiquitylation assay in DMSO (100 µl). The absorbance value at 590 nm was measured using a HT22 cells were transfected with 6×-His-ubiquitin followed by glutamate microplate reader (Tecan Spectrafluor Plus, Männedorf). Experiments were treatment for 12 h in the presence and absence of roscovitine or Cdk5 repeated three times in triplicate wells to ensure reproducibility. shRNA. After 6 h of glutamate treatment, MG132 (Sigma-Aldrich) was added to a final concentration of 10 μM for an additional 6 h to stabilize Immunoprecipitation of Cdk5 and Mcl-1 from HT22 cells ubiquitylated proteins. After 12 h of glutamate treatment, cells were lysed HT22 cells were treated with 5 mM glutamate for 1.5, 3 or 6 h. Cells were and Mcl-1 was immunoprecipitated using anti-Mcl-1 antibody. The proteins then harvested and lysed in RIPA buffer. After centrifugation, whole-cell were then separated by SDS-PAGE and potential ubiquitylation was lysate was mixed with protein A Sepharose beads (Sigma-Aldrich) and analyzed using anti-6×-His antibody. Each experiment was performed at antibody for either Cdk5 or Mcl-1. After incubation overnight, least three times independently. immunocomplexes were washed and then subjected to western blotting using either Cdk5 or Mcl-1 antibodies. Mitochondria polarization measurement Mitochondrial depolarization was measured as reported previously (Sun Cdk5 and Mcl-1 in vitro binding assay et al., 2008b). Briefly, HT22, Mcl-1-HT22 and T92A-Mcl-1-HT22 cells To investigate whether Mcl-1 directly binds Cdk5 in an in vitro assay, were treated with glutamate for 12 h or 24 h. Subsequently, 0.8 µg/µl JC-1 recombinant 6×-His-Mcl-1 was incubated with either Mcl-1 antibody or IgG was added from a 40 µg/µl stock solution in DMSO and incubated for control in the presence of protein A Sepharose beads for 4 h at 4°C. Both 10 min. JC-1 was removed and PBS was added for imaging. Representative immune complexes were washed and incubated with 6×-His-Cdk5 for an images were taken from each sample. The percentage of cells with additional 4 h. The beads were washed, loaded on SDS-PAGE gels, transferred depolarized mitochondria was counted as the average of 100 cells from at to PVDF membrane and probed using Cdk5 antibody. For reciprocal least five random fields. immunoprecipitation, recombinant 6×-His-Cdk5 was incubated with either Cdk5 antibody or IgG control. Both immune complexes were washed and Statistical analysis incubated with 6×-His-Mcl-1. Subsequently, the beads were washed, loaded on Data are expressed as mean±s.e.m. and were statistically evaluated with one- SDS-PAGE, transferred onto PVDF membrane and probed using Mcl-1 way ANOVA followed by the Bonferroni post hoc test using GraphPad antibody. Prism 5.04 software (GraphPad Software). P<0.05 was considered statistically significant. Subcellular fractionation Subcellular fractionation of HT22 cells was conducted as described previously Acknowledgements (Chang et al., 2011). Briefly, cells were lysed in subcellular fractionation buffer We thank Dr David Schubert for HT22 cells and New York Brain Bank for providing (250 mM sucrose, 20 mM HEPES, pH 7.4, 10 mM KCl, 1.5 mM MgCl2, clinical specimens. 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 1 mM DTT) and then passed through a 25 G needle (10×). The cell lysate was further incubated on ice for Competing interests 20 min. The nuclear pellet was obtained after centrifugation at 900 g for 5 min. The authors declare no competing or financial interests. The supernatant was centrifuged at 6000 g for 10 min. The cytosolic fraction was obtained after the removal of supernatant. Both nuclear and cytosolic Author contributions fractions were mixed with SDS loading dye. Proteins were separated using Conceptualization: K.S.; Methodology: K.N.; Validation: K.N.; Formal analysis: K.N., SDS-PAGE and transferred to PVDF membrane. Tubulin was used as the K.S.; Investigation: K.N., K.S.; Data curation: K.N.; Writing - original draft: K.S.; Writing - review & editing: K.N., K.S.; Visualization: K.S.; Supervision: K.S.; Project cytoplasmic marker and lamin A as the nuclear marker. administration: K.S.; Funding acquisition: K.S. Immunofluorescence Funding HT22 cells were grown on poly-L-lysine-coated coverslips for 24 h before This work was supported by the National Institute on Aging (R21-AG 47447 to K.S. subjecting to various treatments. Roscovitine (10 μM) was added 0.5 h before and P50 AG008702 to the New York Brain Bank, New York). Deposited in PMC for glutamate treatment and Cdk5 shRNA lentivirus was added 30 h prior to release after 12 months. glutamate treatment. Cells were fixed with 4% formaldehyde in PBS for 20 min at room temperature, and then washed three times with PBS. The cells Supplementary information were blocked in 1% FBS, 2% BSA and 0.1% triton X-100 in PBS for 1 h at Supplementary information available online at 25°C. Cells were labeled with antibodies (Cdk5, Mcl-1, MAP2, PSD95 or http://jcs.biologists.org/lookup/doi/10.1242/jcs.205666.supplemental Flag) for 2 h in PBS, followed by incubation with fluorescein isothiocyanate- conjugated goat anti-mouse-IgG secondary antibody (1:1000 dilution, References cat. no. 115-095-003, Jackson Immunoresearch), fluorescein isothiocyanate- Adams, K. W. and Cooper, G. M. (2007). Rapid turnover of mcl-1 couples conjugated donkey anti-rabbit-IgG secondary antibody (1:1000 dilution, cat. translation to cell survival and apoptosis. J. Biol. Chem. 282, 6192-6200. no. 711-095-152, Jackson Immunoresearch), Texas Red-conjugated goat Anilkumar, U., Weisová, P., Düssmann, H., Concannon, C. G., König, H.-G. and anti-rabbit-IgG secondary antibody (1:500 dilution, T6391, Invitrogen) or Prehn, J. H. M. (2013). AMP-activated protein kinase (AMPK)-induced Texas Red-conjugated goat anti-mouse-IgG secondary antibody (1:500 preconditioning in primary cortical neurons involves activation of MCL-1. dilution, cat. no. T6390, Invitrogen). Cells were visualized using an Eclipse J. Neurochem. 124, 721-734. E600 microscope (Nikon Instruments). To quantify cytoplasmic and nuclear Arbour, N., Vanderluit, J. L., Le Grand, J. N., Jahani-Asl, A., Ruzhynsky, V. A., Cheung, E. C. C., Kelly, M. A., MacKenzie, A. E., Park, D. S., Opferman, J. T. localization, the average fluorescence intensity in the nucleus was compared et al. (2008). Mcl-1 is a key regulator of apoptosis during CNS development and with the average fluorescence intensity of an area of equal size in the cytosol. after DNA damage. J. Neurosci. 28, 6068-6078. This value was plotted with respect to different treatment times. The ratio Bae, J., Leo, C. P., Hsu, S. Y. and Hsueh, A. J. W. (2000). MCL-1S, a splicing between the two was used to define whether the localization of the protein in variant of the antiapoptotic BCL-2 family member MCL-1, encodes a proapoptotic the cell is cytoplasmic or nuclear. If the ratio was more than 1, the protein was protein possessing only the BH3 domain. J. Biol. Chem. 275, 25255-25261. considered to be positive for nuclear localization. Becker-Hapak, M., McAllister, S. S. and Dowdy, S. F. (2001). TAT-mediated protein transduction into mammalian cells. Methods 24, 247-256. Protein stability assay Braak, H. and Braak, E. (1991). Neuropathological stageing of Alzheimer-related – μ changes. Acta Neuropathol. 82, 239-259. HT22 cells were grown to 60 70% confluency and treated with 10 g/ml Brinkkoetter, P. T., Pippin, J. W. and Shankland, S. J. (2010). Cyclin I-Cdk5 protein synthesis inhibitor cycloheximide (30 min pretreatment) with or governs survival in post-mitotic cells. Cell Cycle 9, 1729-1731. without glutamate treatment. Cells were harvested at various time points and Bussiere,̀ T., Giannakopoulos, P., Bouras, C., Perl, D. P., Morrison, J. H. and Hof, protein levels analyzed. P. R. (2003). Progressive degeneration of nonphosphorylated neurofilament protein- Journal of Cell Science

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