Niranjana D. Amin

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.

Calpain mediates calcium-induced activation of the Erk1,2 MAPK pathway and cytoskeletal phosphorylation in neurons relevance to Alzheimer's disease

Aberrant phosphorylation of the neuronal cytoskeleton is an early pathological event in Alzheimers disease (AD), but the underlying mechanisms are unclear. Here, we demonstrate in the brains of AD patients that neurofilament hyperphosphorylation in neocortical pyramidal neurons is accompanied by activation of both Erk1,2 and calpain. Using immunochemistry, Western blot analysis, and kinase activity measurements, we show in primary hippocampal and cerebellar granule (CG) neurons that calcium influx activates calpain and Erk1,2 and increases neurofilament phosphorylation on carboxy terminal polypeptide sites known to be modulated by Erk1,2 and to be altered in AD. Blocking Erk1,2 activity either with antisense oligonucleotides to Erk1,2 mRNA sequences or by specifically inhibiting its upstream activating kinase MEK1,2 markedly reduced neurofilament phosphorylation. Calpeptin, a cell-permeable calpain inhibitor, blocked both Erk1,2 activation and neurofilament hyperphosphorylation at concentrations that inhibit calpain-mediated cleavage of brain spectrin. By contrast, inhibiting Erk1,2 with U-0126, a specific inhibitor of Mek1,2, had no appreciable effect on ionomycin-induced calpain activation. These findings demonstrate that, under conditions of calcium injury in neurons, calpains are upstream activators of Erk1,2 signaling and are likely to mediate in part the hyperphosphorylation of neurofilaments and tau seen at early stages of AD as well as the neuron survival-related functions of the MAP kinase pathway.

Neuronal Cyclin-Dependent Kinase 5 Activity Is Critical for Survival

Cyclin-dependent kinase 5 (Cdk5) null mice exhibit a unique phenotype characterized by perinatal mortality, disrupted cerebral cortical layering attributable to abnormal neuronal migration, lack of cerebellar foliation, and chromatolytic changes of neurons in the brainstem and the spinal cord. Because Cdk5 is expressed in both neurons and astrocytes, it has been unclear whether this phenotype is primarily attributable to defects in neurons or in astrocytes. Herein we report reconstitution of Cdk5 expression in neurons in Cdk5 null mice and its effect on the null phenotype. Unlike the Cdk5 null mice, the reconstituted Cdk5 null mice that express the Cdk5 transgene under the p35 promoter (TgKO mice) were viable and fertile. Because Cdk5 expression is mainly limited to neurons in these mice and rescues the defects in the nervous system of the Cdk5 null phenotype, it clearly demonstrates that Cdk5 activity is necessary for normal development and survival of p35-expressing neurons.

Phosphorylation of human high molecular weight neurofilament protein (hNF-H) by neuronal cyclin-dependent kinase 5 (cdk5).

Neurofilaments (NFs), the neuron-specific intermediate (i.e. approximately 10-nm diameter) filaments are major cytoskeletal components of most neurons. In a mature mammalian neuron, NFs are co-assembled from three subunits, NF-L (low), NF-M (medium), and NF-H (high), with molecular masses of 68, 95, and 115 kDa, respectively. Neurofilament proteins (NF-Ps), particularly, NF-H, are most extensively phosphorylated in large myelinated axons under normal conditions. This phosphorylation occurs on the serine residues of the lysine (Lys)-serine (Ser)-proline (Pro) (KSP) multiple amino acid repeats of the carboxy-terminal tail domain. Phosphorylation of KSP motifs affects physical, biochemical, and immunological properties of NF-H. For example, phosphorylation is thought to play a pivotal role in the maintenance of the neuronal cytoskeletal structure which influences the conduction velocity of the nerve fiber. The key components responsible for phosphorylation are not known. In this study, an identified cyclin-dependent kinase 5 (cdk5), isolated from nervous tissue, has been shown to phosphorylate the human NF-H (hNF-H) and affects its electrophoretic mobility. On the basis of the following observations, it is suggested that neuronal cdk5 (cdk5) phosphorylates KSPXK motifs in the human high molecular weight neurofilament (hNF-H) and affects its electrophoretic mobility. (1) A 14-mer synthetic peptide (KSPEKAKSPVKEEA) derived from hNF-H; (2) a bacterially expressed protein containing 14 KSPXK multiple repeats of hNF-H in C-terminal tail domain; and (3) a dephosphorylated hNF-H in neurofilament preparation are phosphorylated by cdk5. The decrease in molecular mass of hNF-H caused by dephosphorylated was completely recovered upon cdk5 phosphorylation. It is proposed that neuronal cdk5 regulates phosphorylation of the KSPXK motif in hNF-H and other cytoskeletal proteins with similar motifs in the nervous system.

Cyclin-dependent kinase 5 (cdk5) activation requires interaction with three domains of p35†

Cyclin‐dependent kinase 5 (cdk5), in contrast to other members of the cyclin‐dependent kinase family, is not activated by cyclins but instead is activated by complexing with neuron‐specific activator molecules (p35, p39, and p67). The most effective activator of cdk5 both in vitro and in vivo is p35. We have taken a kinetic approach to study the interaction between p35, its various truncated forms, and cdk5 to understand better the mechanism of its activation. The cdk5 complexes formed with the truncated forms p25 and p21 produced similar maximum active kinase, whereas the cdk5 complexed with full‐length p35 and a further truncated form spanning amino acid residues from 138 to 291, with approximate molecular weight of 16 kDa (p16), produced slightly less (80%) activation than p25. P16 was the smallest fragment of p35 that produced activation equal to or greater than that of full‐length p35. By examination of further truncations of p16, we found that a small number of residues, 11 and 4 at the N‐ and C‐termini, respectively, of p16, are essential for cdk5 activation. Further truncation, removing both essential N‐ and C‐terminal domains, produces a peptide with markedly higher affinity for cdk5 compared with the peptides that retain either of these domains. Using these inactive truncated peptides as inhibitors, we examined the kinetics of activation. From these studies we conclude that activation involves at least three cdk5‐interacting domains, one located at each end of p16 and at least one located in a central domain. The cdk5 activation process is slow: The second‐order rate constant for p16 is about 1.2 μM−1 hr−1. On the basis of kinetic data, we suggest that cdk5 exists in two conformations. The inactive kinase conformation predominates in the absence of the activator. Activation occurs in two stages: a rapid and reversible interaction of cdk5 with its activator, which involves only one or two binding domains, followed by a slow stabilization of the active conformation as interaction with all three domains is achieved. Published 2002 Wiley‐Liss, Inc.

A truncated peptide from p35, a Cdk5 activator, prevents Alzheimer's disease phenotypes in model mice

Alzheimers disease (AD), one of the leading neurodegenerative disorders of older adults, which causes major socioeconomic burdens globally, lacks effective therapeutics without significant side effects. Besides the hallmark pathology of amyloid plaques and neurofibrillary tangles (NFTs), it has been reported that cyclin‐dependent kinase 5 (Cdk5), a critical neuronal kinase, is hyperactivated in AD brains and is, in part, responsible for the above pathology. Here we show that a modified truncated 24‐aa peptide (TFP5), derived from the Cdk5 activator p35, penetrates the blood‐brain barrier after intraperitoneal injections, inhibits abnormal Cdk5 hyperactivity, and significantly rescues AD pathology (up to 70–80%) in 5XFAD AD model mice. The mutant mice, injected with TFP5 exhibit behavioral rescue, whereas no rescue was observed in mutant mice injected with either saline or scrambled peptide. However, TFP5 does not inhibit cell cycle Cdks or normal Cdk5/p35 activity, and thereby has no toxic side effects (even at 200 mg/kg), a common problem in most current therapeutics for AD. In addition, treated mice displayed decreased inflammation, amyloid plaques, NFTs, cell death, and an extended life by 2 mo. These results suggest TFP5 as a potential therapeutic, toxicity‐free candidate for AD.—Shukla, V., Zheng, Y.‐L., Mishra, S. K., Amin, N. D., Steiner, J., Grant, P., Kesavapany, S., Pant, H. C. A truncated peptide from p35, a Cdk5 activator, prevents Alzheimers disease phenotypes in model mice. FASEB J. 27, 174–186 (2013). www.fasebj.org

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.

Bacterial Adenylyl Cyclases

Publisher Summary This chapter describes the enzyme (adenylyl cyclase) that effect the synthesis of Adenosine 3’,5’-cyclic monophosphate (cAMP) in various bacterial species. The content will rather reflect current major interests. The adenylyl cyclase from Escherichia coli has been a major subject of research interest ever since it was identified as the probable point for physiological regulation of cAMP levels in that organism and therefore a prime candidate for a protein mediator of the catabolite repression response mechanism. cAMP functions as a cytoplasmic element mediating some reactions crucial for efficient cellular function. An activity that has been found only in eukaryotic cells is the CAMP-dependent protein kinase . This enzyme is a well-known target of the action of cAMP as a second messenger, in which action this ligand transmits a signal generated by an extracellular hormone. The manner in which cAMP acts on the cAMP-dependent protein kinase involves a release of the catalytic moiety of the enzyme from a complex in which its activity is inhibited as a result of binding to a regulatory subunit.

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.