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A 24-Residue Peptide (p5), Derived from p35, the Cdk5 Neuronal Activator, Specifically Inhibits Cdk5-p25 Hyperactivity and Tau Hyperphosphorylation*

Open AccessPublished:August 18, 2010DOI:https://doi.org/10.1074/jbc.M110.134643
      The activity of Cdk5-p35 is tightly regulated in the developing and mature nervous system. Stress-induced cleavage of the activator p35 to p25 and a p10 N-terminal domain induces deregulated Cdk5 hyperactivity and perikaryal aggregations of hyperphosphorylated Tau and neurofilaments, pathogenic hallmarks in neurodegenerative diseases, such as Alzheimer disease and amyotrophic lateral sclerosis, respectively. Previously, we identified a 125-residue truncated fragment of p35 called CIP that effectively and specifically inhibited Cdk5-p25 activity and Tau hyperphosphorylation induced by Aβ peptides in vitro, in HEK293 cells, and in neuronal cells. Although these results offer a possible therapeutic approach to those neurodegenerative diseases assumed to derive from Cdk5-p25 hyperactivity and/or Aβ induced pathology, CIP is too large for successful therapeutic regimens. To identify a smaller, more effective peptide, in this study we prepared a 24-residue peptide, p5, spanning CIP residues Lys245–Ala277. p5 more effectively inhibited Cdk5-p25 activity than did CIP in vitro. In neuron cells, p5 inhibited deregulated Cdk5-p25 activity but had no effect on the activity of endogenous Cdk5-p35 or on any related endogenous cyclin-dependent kinases in HEK293 cells. Specificity of p5 inhibition in cortical neurons may depend on the p10 domain in p35, which is absent in p25. Furthermore, we have demonstrated that p5 reduced Aβ(1–42)-induced Tau hyperphosphorylation and apoptosis in cortical neurons. These results suggest that p5 peptide may be a unique and useful candidate for therapeutic studies of certain neurodegenerative diseases.

      Introduction

      The activity of Cdk5, a multifunctional serine/threonine kinase, is critical for neuronal development and synaptic activity; it sustains neurite outgrowth, neuronal migration, cortical lamination, and survival (
      • Chae T.
      • Kwon Y.T.
      • Bronson R.
      • Dikkes P.
      • Li E.
      • Tsai L.H.
      ,
      • Cheung Z.H.
      • Fu A.K.
      • Ip N.Y.
      ,
      • Dhavan R.
      • Tsai L.H.
      ,
      • Gilmore E.C.
      • Ohshima T.
      • Goffinet A.M.
      • Kulkarni A.B.
      • Herrup K.
      ,
      • Lai K.O.
      • Ip N.Y.
      ,
      • Nikolic M.
      • Dudek H.
      • Kwon Y.T.
      • Ramos Y.F.
      • Tsai L.H.
      ,
      • Ohshima T.
      • Gilmore E.C.
      • Longenecker G.
      • Jacobowitz D.M.
      • Brady R.O.
      • Herrup K.
      • Kulkarni A.B.
      ,
      • Ohshima T.
      • Ward J.M.
      • Huh C.G.
      • Longenecker G.
      • Veeranna Pant H.C.
      • Brady R.O.
      • Martin L.J.
      • Kulkarni A.B.
      ,
      • Zheng Y.L.
      • Li B.S.
      • Kanungo J.
      • Kesavapany S.
      • Amin N.
      • Grant P.
      • Pant H.C.
      ). Its activity depends on the binding of its neuron-specific, cyclin-related activators, p35 and p39 (
      • Hisanaga S.
      • Saito T.
      ,
      • Tang D.
      • Wang J.H.
      ). Cdk5 has also been implicated as a key player in learning and memory (
      • Angelo M.
      • Plattner F.
      • Giese K.P.
      ,
      • Fischer A.
      • Sananbenesi F.
      • Pang P.T.
      • Lu B.
      • Tsai L.H.
      ,
      • Fischer A.
      • Sananbenesi F.
      • Schrick C.
      • Spiess J.
      • Radulovic J.
      ,
      • Fischer A.
      • Sananbenesi F.
      • Spiess J.
      • Radulovic J.
      ).
      Normally, Cdk5 activity is tightly regulated, but under conditions of neuronal stress, it is deregulated, leading to hyperactivity, neuronal pathology, and cell death. Accordingly, Cdk5 has been implicated in certain neurodegenerative disorders, such as AD.
      The abbreviations used are: AD
      Alzheimer disease
      EV
      empty vector
      DIC
      days in culture
      β-amyloid.
      A model of the role of Cdk5 in neurodegeneration suggests that a stress-induced influx of calcium ions into neurons activates calpain, a Ca2+-activated protease, which cleaves p35 into p25 and a p10 fragment. p25, in turn, forms a more stable Cdk5-p25 hyperactive complex, which hyperphosphorylates Tau and induces cell death (
      • Ahlijanian M.K.
      • Barrezueta N.X.
      • Williams R.D.
      • Jakowski A.
      • Kowsz K.P.
      • McCarthy S.
      • Coskran T.
      • Carlo A.
      • Seymour P.A.
      • Burkhardt J.E.
      • Nelson R.B.
      • McNeish J.D.
      ,
      • Cruz J.C.
      • Tseng H.C.
      • Goldman J.A.
      • Shih H.
      • Tsai L.H.
      ,
      • Kusakawa G.
      • Saito T.
      • Onuki R.
      • Ishiguro K.
      • Kishimoto T.
      • Hisanaga S.
      ,
      • Lee M.S.
      • Kwon Y.T.
      • Li M.
      • Peng J.
      • Friedlander R.M.
      • Tsai L.H.
      ,
      • Nguyen M.D.
      • Mushynski W.E.
      • Julien J.P.
      ,
      • Noble W.
      • Olm V.
      • Takata K.
      • Casey E.
      • Mary O.
      • Meyerson J.
      • Gaynor K.
      • LaFrancois J.
      • Wang L.
      • Kondo T.
      • Davies P.
      • Burns M.
      • Veeranna Nixon R.
      • Dickson D.
      • Matsuoka Y.
      • Ahlijanian M.
      • Lau L.F.
      • Duff K.
      ). Indeed, increased levels of p25 and Cdk5 activity have been reported in AD brains. The finding that p25 may be toxic comes from studies of cortical neurons treated with β-amyloid (Aβ), a key marker of AD pathology, where p35 is converted to p25 accompanied by activated Cdk5, Tau hyperphosphorylation, and apoptosis (
      • Town T.
      • Zolton J.
      • Shaffner R.
      • Schnell B.
      • Crescentini R.
      • Wu Y.
      • Zeng J.
      • DelleDonne A.
      • Obregon D.
      • Tan J.
      • Mullan M.
      ,
      • Zheng Y.L.
      • Kesavapany S.
      • Gravell M.
      • Hamilton R.S.
      • Schubert M.
      • Amin N.
      • Albers W.
      • Grant P.
      • Pant H.C.
      ).
      Expression of the Cdk5-p25 complex seems to be primarily responsible for the Tau pathology and suggests that a therapeutic approach directed specifically at this target might prove successful (
      • Helal C.J.
      • Kang Z.
      • Lucas J.C.
      • Gant T.
      • Ahlijanian M.K.
      • Schachter J.B.
      • Richter K.E.
      • Cook J.M.
      • Menniti F.S.
      • Kelly K.
      • Mente S.
      • Pandit J.
      • Hosea N.
      ,
      • Lau L.F.
      • Seymour P.A.
      • Sanner M.A.
      • Schachter J.B.
      ,
      • Shiradkar M.R.
      • Padhalingappa M.B.
      • Bhetalabhotala S.
      • Akula K.C.
      • Tupe D.A.
      • Pinninti R.R.
      • Thummanagoti S.
      ). For most of these studies, however, the focus has been on aminothiazol compounds resembling roscovitine, a kinase inhibitor that competes with the ATP binding site in Cdk5 and other kinases. These drugs do not act specifically on Cdk5-p25 but also inhibit Cdk5-p35 and other Cdks essential for normal development and function. This could be responsible for serious secondary side effects and thereby compromise any therapeutic value.
      Our approach to this problem, however, is based on earlier studies, where we identified a peptide of 125 amino acid residues of p35 (CIP) that inhibited Cdk5-p25 activity and rescued cortical neurons from Aβ-induced apoptosis without affecting Cdk5-p35 activity (
      • Zheng Y.L.
      • Li B.S.
      • Amin N.D.
      • Albers W.
      • Pant H.C.
      ). This approach might prove to be a more effective way to suppress deregulated Cdk5-p25 hyperactivity inducing neurodegenerative pathology. For a therapy to be effective, however, it must be small enough to pass the blood-brain barrier; a large peptide of 125 residues may be problematic, to say the least. For that reason, we set out to produce a smaller peptide of CIP with equivalent specificity. Based on an analysis of Cdk5-p25 structure and molecular dynamics, several smaller peptides were produced and tested. We identified a 24-residue peptide, called p5, that more effectively inhibited Cdk5-p25 activity in cortical neurons than CIP without affecting endogenous Cdk5-p35 or other Cdks. The small size and specificity of p5 inhibition make it an excellent candidate for therapeutic trials in animal models of AD.

      DISCUSSION

      Neurodegenerative disorders leading to dementia, such as Alzheimer disease, are chronic, long term processes resulting from an accumulation of various lesions and insults. Among the latter are oxidative stress (
      • Smith D.S.
      • Tsai L.H.
      ), inflammation, hormonal deficits, abnormal cholesterol metabolism (
      • Luque F.A.
      • Jaffe S.L.
      ,
      • Martins I.J.
      • Berger T.
      • Sharman M.J.
      • Verdile G.
      • Fuller S.J.
      • Martins R.N.
      ), and exocitotoxic stress (
      • Dong X.X.
      • Wang Y.
      • Qin Z.H.
      ). Such defects result in synaptic function deficits, neuronal death, and dementia. The hallmark pathologies of AD brains are the β-amyloid-rich senile plaques and the neurofibrillary tangles containing hyperphosphorylated Tau among other constituents (neurofilaments, kinases, etc). These pathologies have generally been regarded as the primary cause of cell death and dementia, and the factors responsible for their formation have been extensively studied. β-Amyloid is toxic in a variety of neuronal preparations (
      • Yankner B.A.
      • Duffy L.K.
      • Kirschner D.A.
      ) and is known to induce hyperphosphorylated Tau accumulations and cytoskeletal defects in cortical neurons in vitro (
      • Zheng Y.L.
      • Kesavapany S.
      • Gravell M.
      • Hamilton R.S.
      • Schubert M.
      • Amin N.
      • Albers W.
      • Grant P.
      • Pant H.C.
      ,
      • Zheng Y.L.
      • Li B.S.
      • Amin N.D.
      • Albers W.
      • Pant H.C.
      ) and in β-amyloid-expressing transgenic mice (
      • Smith W.W.
      • Gorospe M.
      • Kusiak J.W.
      ). Accumulation of hyperphosphorylated Tau in neurofibrillary tangles is correlated with the activation of kinases, such as GSK3 (
      • Liu F.
      • Liang Z.
      • Shi J.
      • Yin D.
      • El-Akkad E.
      • Grundke-Iqbal I.
      • Iqbal K.
      • Gong C.X.
      ) and Cdk5 (
      • Ahlijanian M.K.
      • Barrezueta N.X.
      • Williams R.D.
      • Jakowski A.
      • Kowsz K.P.
      • McCarthy S.
      • Coskran T.
      • Carlo A.
      • Seymour P.A.
      • Burkhardt J.E.
      • Nelson R.B.
      • McNeish J.D.
      ,
      • Noble W.
      • Olm V.
      • Takata K.
      • Casey E.
      • Mary O.
      • Meyerson J.
      • Gaynor K.
      • LaFrancois J.
      • Wang L.
      • Kondo T.
      • Davies P.
      • Burns M.
      • Veeranna Nixon R.
      • Dickson D.
      • Matsuoka Y.
      • Ahlijanian M.
      • Lau L.F.
      • Duff K.
      ,
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      ,
      • Tseng H.C.
      • Zhou Y.
      • Shen Y.
      • Tsai L.H.
      ), as well as the down-regulation of phosphatases, such as PP2A (
      • Liu M.
      • Choi S.
      • Cuny G.D.
      • Ding K.
      • Dobson B.C.
      • Glicksman M.A.
      • Auerbach K.
      • Stein R.L.
      ). Our laboratory, along with others (
      • Cruz J.C.
      • Kim D.
      • Moy L.Y.
      • Dobbin M.M.
      • Sun X.
      • Bronson R.T.
      • Tsai L.H.
      ,
      • Maccioni R.B.
      • Otth C.
      • Concha I.I.
      • Muñoz J.P.
      ,
      • Rockenstein E.
      • Torrance M.
      • Mante M.
      • Adame A.
      • Paulino A.
      • Rose J.B.
      • Crews L.
      • Moessler H.
      • Masliah E.
      ), has focused on Cdk5 and its deregulation as a principal “player” responsible for Tau pathology, neurofibrillary tangle accumulation, and cell death in AD. Several lines of evidence are consistent with this hypothesis. Cdk5 is stabilized and hyperactivated when complexed with p25, a truncated fragment of the normal p35 regulator (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      ). Neuronal stress converts p35 to its truncated form p25 by activating calpain, a calcium-dependent protease (
      • Lee M.S.
      • Kwon Y.T.
      • Li M.
      • Peng J.
      • Friedlander R.M.
      • Tsai L.H.
      ,
      • Nath R.
      • Davis M.
      • Probert A.W.
      • Kupina N.C.
      • Ren X.
      • Schielke G.P.
      • Wang K.K.
      ), resulting in long term accumulation of p25, a major factor in the formation of hyperactivated Cdk5. Consistent with this hypothesis, the ratio of p25 to p35 has been reported to be significantly higher in AD brains than in control brains, particularly in the frontal cortex (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      ,
      • Tseng H.C.
      • Zhou Y.
      • Shen Y.
      • Tsai L.H.
      ), although other laboratories report either a decrease in p25 or no evidence of an increased p25-p35 ratio in AD brains (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de La Monte S.
      • Dikkes P.
      • Tsai L.H.
      ,
      • Taniguchi S.
      • Fujita Y.
      • Hayashi S.
      • Kakita A.
      • Takahashi H.
      • Murayama S.
      • Saido T.C.
      • Hisanaga S.
      • Iwatsubo T.
      • Hasegawa M.
      ). Nevertheless, overexpression of p25 in cultured neurons leads to cytoskeletal disruption, hyperphosphorylated Tau, and apoptotic cell death (
      • Ahlijanian M.K.
      • Barrezueta N.X.
      • Williams R.D.
      • Jakowski A.
      • Kowsz K.P.
      • McCarthy S.
      • Coskran T.
      • Carlo A.
      • Seymour P.A.
      • Burkhardt J.E.
      • Nelson R.B.
      • McNeish J.D.
      ). Cdk5 activity is deregulated in hippocampal neurons by Aβ fibrils, resulting in the activation of Cdk5 and hyperphosphorylation of Tau (
      • Alvarez A.
      • Muñoz J.P.
      • Maccioni R.B.
      ,
      • Alvarez A.
      • Toro R.
      • Cáceres A.
      • Maccioni R.B.
      ). Probably because of its multifunctional role in the nervous system, aberrant Cdk5-p25 activity looms as a major factor in neurodegenerative disorders. This suggests that inhibitors of Cdk5 activity may be effective candidates for therapeutic development (
      • Lau L.F.
      • Seymour P.A.
      • Sanner M.A.
      • Schachter J.B.
      ,
      • Glicksman M.A.
      • Cuny G.D.
      • Liu M.
      • Dobson B.
      • Auerbach K.
      • Stein R.L.
      • Kosik K.S.
      ,
      • Tsai L.H.
      • Lee M.S.
      • Cruz J.
      ).
      Although several potent chemical inhibitors of Cdk5 have been identified and studied (
      • Shiradkar M.R.
      • Padhalingappa M.B.
      • Bhetalabhotala S.
      • Akula K.C.
      • Tupe D.A.
      • Pinninti R.R.
      • Thummanagoti S.
      ,
      • Knockaert M.
      • Wieking K.
      • Schmitt S.
      • Leost M.
      • Grant K.M.
      • Mottram J.C.
      • Kunick C.
      • Meijer L.
      ,
      • Helal C.J.
      • Kang Z.
      • Lucas J.C.
      • Bohall B.R.
      ), most compete with ATP at the catalytic binding site. Accordingly, these compounds are relatively nonspecific because other cyclin-dependent kinases (as well as other kinases) are equally dependent on ATP binding. Acknowledging this problem, a group, in search of non-competitive ATP inhibitors, are screening an extensive library of compounds that affect the kinetics of Cdk5-p25 interactions with Tau to distinguish competitive from non-competitive inhibitors of Cdk5 activity (
      • Liu M.
      • Choi S.
      • Cuny G.D.
      • Ding K.
      • Dobson B.C.
      • Glicksman M.A.
      • Auerbach K.
      • Stein R.L.
      ,
      • Glicksman M.A.
      • Cuny G.D.
      • Liu M.
      • Dobson B.
      • Auerbach K.
      • Stein R.L.
      • Kosik K.S.
      ). The ultimate challenge for such compounds is to selectively inhibit Cdk5-p25 in biological systems without affecting the activity of Cdk5-p35, the active complex essential for neural development and function.
      As an approach to this problem, in our previous studies, we have demonstrated that CIP, a 125-amino acid truncated peptide derived from p35, specifically inhibited Cdk5-p25 activity and significantly decreased hyperphosphorylation of Tau and apoptosis induced by Aβ treatment in both HEK293 cells and cortical neurons (
      • Zheng Y.L.
      • Kesavapany S.
      • Gravell M.
      • Hamilton R.S.
      • Schubert M.
      • Amin N.
      • Albers W.
      • Grant P.
      • Pant H.C.
      ,
      • Zheng Y.L.
      • Li B.S.
      • Amin N.D.
      • Albers W.
      • Pant H.C.
      ). An effective therapeutic drug, however, should be much smaller to increase absorption efficiency (particularly across the blood-brain barrier) after injection or oral administration. Here we tested several truncated peptides derived from CIP and identified p5 (only 24 residues) as a more effective inhibitor of Cdk5-p25 activity than CIP in vitro. Furthermore, we confirmed that p5 rescued cortical neurons from Aβ toxicity, Tau pathology, and cell death. At low doses, it specifically inhibited Cdk5-p25 activity without affecting endogenous Cdk5-p35 activity in cortical neurons. At higher doses, the activities of closely related Cdk kinases, such as Cdc2, Cdk2, Cdk4, and Cdk6, in proliferating HEK293 cells were unaffected by p5.
      Although both complexes are inhibited equally by p5 in vitro, it is noteworthy that the specificity of inhibition is displayed within cells; in cortical neurons, hyperactive Cdk5-p25 is more effectively inhibited by p5 than the normal endogenous Cdk5-p35 complex. Although the factors responsible for p5 specificity in cortical neurons are not understood, it is critical to the therapeutic potential of p5. We propose that the p10 myristoylated N-terminal domain of p35, absent in p25, determines the specificity of p5 inhibition in cortical neurons.
      The p10 interaction in vivo with other cellular proteins, such as tubulin (microtubules) and calmodulin (
      • Hou Z.
      • Li Q.
      • He L.
      • Lim H.Y.
      • Fu X.
      • Cheung N.S.
      • Qi D.X.
      • Qi R.Z.
      ,
      • He L.
      • Hou Z.
      • Qi R.Z.
      ), may protect the activity of the Cdk5-p35 complex (and not the Cdk5-p25 complex) from p5 inhibition. In our experiments (Fig. 9), we showed that after preincubation of the Cdk5-p35 complex with tubulin, under conditions favoring polymerization, the addition of 0.45 μm p5 had no effect on activity, whereas Cdk5-p25 was inhibited under identical conditions. Without preincubation, however, p5 inhibited both complexes equally; p35 interaction with soluble tubulin did not prevent p5 inhibition. These data suggest that in cortical neurons, the relatively high affinity of p35 to microtubules sequesters the Cdk5-p35 complex from p5, interfering with p5 binding to Cdk5. The significant finding is that in cortical neurons, p5 inhibits the hyperactive Cdk5-p25 complex responsible for neuronal pathology without affecting the activity of the endogenous, functionally dependent Cdk5-p35.
      We have identified and characterized p5 as a small, more readily diffusible peptide that specifically inhibits Cdk5-p25 activity at low doses, does not affect endogenous Cdk5-p35 essential for neuronal development and function, does not inhibit related Cdks in proliferating cells (and hence would have significantly reduced side effects), and rescues neurons from Aβ toxicity, Tau hyperphosphorylation, and cell death. Accordingly, we believe that p5 is an excellent therapeutic candidate to rescue neurons from the debilitating onslaught of Aβ toxicity and hyperactivated Cdk5-p25 that characterizes some neurodegenerative disorders.

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