Ya-Li Zheng

A Cdk5 inhibitory peptide reduces tau hyperphosphorylation and apoptosis in neurons

The extracellular aggregation of amyloid β (Aβ) peptides and the intracellular hyperphosphorylation of tau at specific epitopes are pathological hallmarks of neurodegenerative diseases such as Alzheimers disease (AD). Cdk5 phosphorylates tau at AD‐specific phospho‐epitopes when it associates with p25. p25 is a truncated activator, which is produced from the physiological Cdk5 activator p35 upon exposure to Aβ peptides. We show that neuronal infections with Cdk5 inhibitory peptide (CIP) selectively inhibit p25/Cdk5 activity and suppress the aberrant tau phosphorylation in cortical neurons. Furthermore, Aβ1−42‐induced apoptosis of these cortical neurons was also reduced by coinfection with CIP. Of particular importance is our finding that CIP did not inhibit endogenous or transfected p35/Cdk5 activity, nor did it inhibit the other cyclin‐dependent kinases such as Cdc2, Cdk2, Cdk4 and Cdk6. These results, therefore, provide a strategy to address, and possibly ameliorate, the pathology of neurodegenerative diseases that may be a consequence of aberrant p25 activation of Cdk5, without affecting ‘normal’ Cdk5 activity.

Cyclin-Dependent Kinase 5 Phosphorylation of Human Septin SEPT5 (hCDCrel-1) Modulates Exocytosis

Cyclin-dependent kinase 5 (Cdk5) is predominantly expressed in the nervous system, where it is involved in neuronal migration, synaptic transmission, and survival. The role of Cdk5 in synaptic transmission is mediated by regulating the cellular functions of presynaptic proteins such as synapsin, Munc18, and dynamin 1. Its multifunctional role at the synapse is complex and probably involves other novel substrates. To explore this possibility, we used a yeast two-hybrid screen of a human cDNA library with p35 as bait and isolated human septin 5 (SEPT5), known also as hCDCrel-1, as an interacting clone. Here we report that p35 associates with SEPT5 in GST (glutathione S-transferase)-pull-down and coimmunoprecipitation assays. We confirmed that Cdk5/p35 phosphorylates SEPT5 in vitro and in vivo and identified S327 of SEPT5 as a major phosphorylation site. A serine (S)-to-alanine (A) 327 mutant of SEPT5 bound syntaxin more efficiently than SEPT5 wild type. Additionally, coimmunoprecipitation from synaptic vesicle fractions and Cdk5 wild-type and knock-out lysates showed that phosphorylation of septin 5 by Cdk5/p35 decreases its binding to syntaxin-1. Moreover, mutant nonphosphorylated SEPT5 potentiated regulated exocytosis more than the wild type when each was expressed in PC12 cells. These data suggest that Cdk5 phosphorylation of human septin SEPT5 at S327 plays a role in modulating exocytotic secretion.

p35/Cyclin-Dependent Kinase 5 Phosphorylation of Ras Guanine Nucleotide Releasing Factor 2 (RasGRF2) Mediates Rac-Dependent Extracellular Signal-Regulated Kinase 1/2 Activity, Altering RasGRF2 and Microtubule-Associated Protein 1b Distribution in Neurons

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed kinase the activity of which is dependent on association with its neuron-specific activators, p35 and p39. Cdk5 activity is critical for the proper formation of cortical structures and lamination during development. In the adult nervous system, Cdk5 function is implicated in cellular adhesion, dopamine signaling, neurotransmitter release, and synaptic activity. In addition, Cdk5 is also involved in “cross-talk” with other signal transduction pathways. To further examine its involvement in cross-talk with other pathways, we identified proteins that interacted with p35 using the yeast two-hybrid system. We report here that p35 associates with Ras guanine nucleotide releasing factor 2 (RasGRF2) in coimmunoprecipitation and colocalization studies using transfected cell lines as well as primary cortical neurons. Additionally, Cdk5 phosphorylates RasGRF2 both in vitro and in vivo, leading to a decrease in Rac–guanidine exchange factor activity and a subsequent reduction in extracellular signal-regulated kinase 1/2 activity. We show that p35/Cdk5 phosphorylates RasGRF2 on serine737, which leads to an accumulation of RasGRF2 in the neuronal cell bodies coinciding with an accumulation of microtubule-associated protein 1b. The membrane association of p35 and subsequent localization of Cdk5 activity toward RasGRF2 and Rac provide insights into important cellular signaling processes that occur at the membrane, resulting in downstream effects on signal transduction cascades.

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

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.

Cyclin-dependent kinase 5 increases perikaryal neurofilament phosphorylation and inhibits neurofilament axonal transport in response to oxidative stress

Cyclin‐dependent kinase 5 (cdk5) phosphorylates the high molecular weight neurofilament (NF) protein. Overexpression of cdk5 inhibits NF axonal transport and induces perikaryal accumulation of disordered phospho‐NF cables. Experimental and clinical motor neuron disease is characterized by oxidative stress, increased cdk5 activity, and accumulation of phospho‐NFs within perikarya or proximal axons. Because oxidative stress increases cdk5 activity in experimental motor neuron disease, we examined whether oxidative stress induced cdk5‐mediated NF phosphorylation. Treatment of cultured neuronal cells with hydrogen peroxide inhibited axonal transport of green fluorescent protein‐tagged NF subunits and induced perikaryal accumulation of NF phosphoepitopes normally confined to axons. These effects were prevented by treatment with the cdk5 inhibitor roscovitine or transfection with a construct expressing the endogenous cdk5 inhibitor peptide. These findings indicate that oxidative stress can compromise NF dynamics via hyperactivation of cdk5 and suggest that antioxidants may alleviate multiple aspects of neuropathology in motor neuron disease.

Phosphorylation of the head domain of neurofilament protein (NF-M): A factor regulating topographic phosphorylation of NF-M tail domain KSP sites in neurons

In neurons the phosphorylation of neurofilament (NF) proteins NF-M and NF-H is topographically regulated. Although kinases and NF subunits are synthesized in cell bodies, extensive phosphorylation of the KSP repeats in tail domains of NF-M and NF-H occurs primarily in axons. The nature of this regulation, however, is not understood. As obligate heteropolymers, NF assembly requires interactions between the core NF-L with NF-M or NF-H subunits, a process inhibited by NF head domain phosphorylation. Phosphorylation of head domains at protein kinase A (PKA)-specific sites seems to occur transiently in cell bodies after NF subunit synthesis. We have proposed that transient phosphorylation of head domains prevents NF assembly in the soma and inhibits tail domain phosphorylation; i.e. assembly and KSP phosphorylation in axons depends on prior dephosphorylation of head domain sites. Deregulation of this process leads to pathological accumulations of phosphorylated NFs in the soma as seen in some neurodegenerative disorders. To test this hypothesis, we studied the effect of PKA phosphorylation of the NF-M head domain on phosphorylation of tail domain KSP sites. In rat cortical neurons we showed that head domain phosphorylation of endogenous NF-M by forskolin-activated PKA inhibits NF-M tail domain phosphorylation. To demonstrate the site specificity of PKA phosphorylation and its effect on tail domain phosphorylation, we transfected NIH3T3 cells with NF-M mutated at PKA-specific head domain serine residues. Epidermal growth factor stimulation of cells with mutant NF-M in the presence of forskolin exhibited no inhibition of NF-tail domain phosphorylation compared with the wild type NF-M-transfected cells. This is consistent with our hypothesis that transient phosphorylation of NF-M head domains inhibits tail domain phosphorylation and suggests this as one of several mechanisms underlying topographic regulation.

Cyclin-Dependent Kinase 5 Modulates the Transcriptional Activity of the Mineralocorticoid Receptor and Regulates Expression of Brain-Derived Neurotrophic Factor

Glucocorticoids, major end effectors of the stress response, play an essential role in the homeostasis of the central nervous system (CNS) and contribute to memory consolidation and emotional control through their intracellular receptors, the glucocorticoid and mineralocorticoid receptors. Cyclin-dependent kinase 5 (CDK5), on the other hand, plays important roles in the morphogenesis and functions of the central nervous system, and its aberrant activation has been associated with development of neurodegenerative disorders. We previously reported that CDK5 phosphorylated the glucocorticoid receptor and modulated its transcriptional activity. Here we found that CDK5 also regulated mineralocorticoid receptor-induced transcriptional activity by phosphorylating multiple serine and threonine residues located in its N-terminal domain through physical interaction. Aldosterone and dexamethasone, respectively, increased and suppressed mRNA/protein expression of brain-derived neurotrophic factor (BDNF) in rat cortical neuronal cells, whereas the endogenous glucocorticoid corticosterone showed a biphasic effect. CDK5 enhanced the effect of aldosterone and dexamethasone on BDNF expression. Because this neurotrophic factor plays critical roles in neuronal viability, synaptic plasticity, consolidation of memory, and emotional changes, we suggest that aberrant activation of CDK5 might influence these functions through corticosteroid receptors/BDNF.

Targeting Cdk5 Activity in Neuronal Degeneration and Regeneration

The major priming event in neurodegeneration is loss of neurons. Loss of neurons by apoptotic mechanisms is a theme for studies focused on determining therapeutic strategies. Neurons following an insult, activate a number of signal transduction pathways, of which, kinases are the leading members. Cyclin-dependent kinase 5 (Cdk5) is one of the kinases that have been linked to neurodegeneration. Cdk5 along with its principal activator p35 is involved in multiple cellular functions ranging from neuronal differentiation and migration to synaptic transmission. However, during neurotoxic stress, intracellular rise in Ca2+ activates calpain, which cleaves p35 to generate p25. The long half-life of Cdk5/p25 results in a hyperactive, aberrant Cdk5 that hyperphosphorylates Tau, neurofilament and other cytoskeletal proteins. These hyperphosphorylated cytoskeletal proteins set the groundwork to forming neurofibrillary tangles and aggregates of phosphorylated proteins, hallmarks of neurodegenerative diseases like Alzheimer’s disease, Parkinson’s disease and Amyotropic Lateral Sclerosis. Attempts to selectively target Cdk5/p25 activity without affecting Cdk5/p35 have been largely unsuccessful. A polypeptide inhibitor, CIP (Cdk5 inhibitory peptide), developed in our laboratory, successfully inhibits Cdk5/p25 activity in vitro, in cultured primary neurons, and is currently undergoing validation tests in mouse models of neurodegeneration. Here, we discuss the therapeutic potential of CIP in regenerating neurons that are exposed to neurodegenerative stimuli.

Pin1-dependent Prolyl Isomerization Modulates the Stress-induced Phosphorylation of High Molecular Weight Neurofilament Protein

Aberrant phosphorylation of neuronal cytoskeletal proteins is a key pathological event in neurodegenerative disorders such as Alzheimer disease (AD) and amyotrophic lateral sclerosis, but the underlying mechanisms are still unclear. Previous studies have shown that Pin1, a peptidylprolyl cis/trans-isomerase, may be actively involved in the regulation of Tau hyperphosphorylation in AD. Here, we show that Pin1 modulates oxidative stress-induced NF-H phosphorylation. In an in vitro kinase assay, the addition of Pin1 substantially increased phosphorylation of NF-H KSP repeats by proline-directed kinases, Erk1/2, Cdk5/p35, and JNK3 in a concentration-dependent manner. In vivo, dominant-negative (DN) Pin1 and Pin1 small interfering RNA inhibited epidermal growth factor-induced NF-H phosphorylation. Because oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, we studied the role of Pin1 in stressed cortical neurons and HEK293 cells. Both hydrogen peroxide (H2O2) and heat stresses induce phosphorylation of NF-H in transfected HEK293 cells and primary cortical cultures. Knockdown of Pin1 by transfected Pin1 short interference RNA and DN-Pin1 rescues the effect of stress-induced NF-H phosphorylation. The H2O2 and heat shock induced perikaryal phospho-NF-H accumulations, and neuronal apoptosis was rescued by inhibition of Pin1 in cortical neurons. JNK3, a brain-specific JNK isoform, is activated under oxidative and heat stresses, and inhibition of Pin1 by Pin1 short interference RNA and DN-Pin1 inhibits this pathway. These results implicate Pin1 as a possible modulator of stress-induced NF-H phosphorylation as seen in neurodegenerative disorders like AD and amyotrophic lateral sclerosis. Thus, Pin1 may be a potential therapeutic target for these diseases.

Cdk5-Mediated Phosphorylation of δ-Catenin Regulates Its Localization and GluR2-Mediated Synaptic Activity

Cyclin-dependent kinase 5 (Cdk5)-mediated phosphorylation plays an important role in proper synaptic function and transmission. Loss of Cdk5 activity results in abnormal development of the nervous system accompanied by massive disruptions in cortical migration and lamination, therefore impacting synaptic activity. The Cdk5 activator p35 associates with δ-catenin, the synaptic adherens junction protein that serves as part of the anchorage complex of AMPA receptor at the postsynaptic membrane. However, the implications of Cdk5-mediated phosphorylation of δ-catenin have not been fully elucidated. Here we show that Cdk5-mediated phosphorylation of δ-catenin regulates its subcellular localization accompanied by changes in dendritic morphogenesis and synaptic activity. We identified two Cdk5 phosphorylation sites in mouse δ-catenin, serines 300 and 357, and report that loss of Cdk5 phosphorylation of δ-catenin increased its localization to the membrane. Furthermore, mutations of the serines 300 and 357 to alanines to mimic nonphosphorylated δ-catenin resulted in increased dendritic protrusions accompanied by increased AMPA receptor subunit GluR2 localization at the membrane. Consistent with these observations, loss of Cdk5 phosphorylation of δ-catenin increased the AMPA/NMDA ratio. This study reveals how Cdk5 phosphorylation of the synaptic mediator protein δ-catenin can alter its localization at the synapse to impact neuronal synaptic activity.