B.K. Binukumar

Peptide (TFP5/TP5), derived from Cdk5 activator P35, provides neuroprotection in the MPTP model of Parkinson's disease

TFP5/TP5 rescues dopaminergic neurodegeneration induced by MPTP in a mouse model of Parkinson’s disease (PD). The neuroprotective effect of TFP5/TP5 peptide is also associated with marked reduction in neuroinflammation and apoptosis. Selective inhibition of Cdk5/p25 by TFP5/TP5 peptide identifies the kinase as a potential target to reduce neurodegeneration in PD.

TFP5, a peptide derived from p35, a Cdk5 neuronal activator, rescues cortical neurons from glucose toxicity.

Multiple lines of evidence link the incidence of diabetes to the development of Alzheimers disease (AD). Patients with diabetes have a 50 to 75% increased risk of developing AD. Cyclin dependent kinase 5 (Cdk5) is a serine/threonine protein kinase, which forms active complexes with p35 or p39, found principally in neurons and in pancreatic β cells. Recent studies suggest that Cdk5 hyperactivity is a possible link between neuropathology seen in AD and diabetes. Previously, we identified P5, a truncated 24-aa peptide derived from the Cdk5 activator p35, later modified as TFP5, so as to penetrate the blood-brain barrier after intraperitoneal injections in AD model mice. This treatment inhibited abnormal Cdk5 hyperactivity and significantly rescued AD pathology in these mice. The present study explores the potential of TFP5 peptide to rescue high glucose (HG)-mediated toxicity in rat embryonic cortical neurons. HG exposure leads to Cdk5-p25 hyperactivity and oxidative stress marked by increased reactive oxygen species production, and decreased glutathione levels and superoxide dismutase activity. It also induces hyperphosphorylation of tau, neuroinflammation as evident from the increased expression of inflammatory cytokines like TNF-α, IL-1β, and IL-6, and apoptosis. Pretreatment of cortical neurons with TFP5 before HG exposure inhibited Cdk5-p25 hyperactivity and significantly attenuated oxidative stress by decreasing reactive oxygen species levels, while increasing superoxide dismutase activity and glutathione. Tau hyperphosphorylation, inflammation, and apoptosis induced by HG were also considerably reduced by pretreatment with TFP5. These results suggest that TFP5 peptide may be a novel candidate for type 2 diabetes therapy.

Topographic regulation of neuronal intermediate filaments by phosphorylation, role of peptidyl-prolyl isomerase 1: significance in neurodegeneration.

The neuronal cytoskeleton is tightly regulated by phosphorylation and dephosphorylation reactions mediated by numerous associated kinases, phosphatases and their regulators. Defects in the relative kinase and phosphatase activities and/or deregulation of compartment-specific phosphorylation result in neurodegenerative disorders. The largest family of cytoskeletal proteins in mammalian cells is the superfamily of intermediate filaments (IFs). The neurofilament (NF) proteins are the major IFs. Aggregated forms of hyperphosphorylated tau and phosphorylated NFs are found in pathological cell body accumulations in the central nervous system of patients suffering from Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis. The precise mechanisms for this compartment-specific phosphorylation of cytoskeletal proteins are not completely understood. In this review, we focus on the mechanisms of neurofilament phosphorylation in normal physiology and neurodegenerative diseases. We also address the recent breakthroughs in our understanding the role of different kinases and phosphatases involved in regulating the phosphorylation status of the NFs. In addition, special emphasis has been given to describe the role of phosphatases and Pin1 in phosphorylation of NFs.

TFP5, a Peptide Inhibitor of Aberrant and Hyperactive Cdk5/p25, Attenuates Pathological Phenotypes and Restores Synaptic Function in CK-p25Tg Mice

It has been reported that cyclin-dependent kinase 5 (cdk5), a critical neuronal kinase, is hyperactivated in Alzheimers disease (AD) and may be, in part, responsible for the hallmark pathology of amyloid plaques and neurofibrillary tangles (NFTs). It has been proposed by several laboratories that hyperactive cdk5 results from the overexpression of p25 (a truncated fragment of p35, the normal cdk5 regulator), which, when complexed to cdk5, induces hyperactivity, hyperphosphorylated tau/NFTs, amyloid-β plaques, and neuronal death. It has previously been shown that intraperitoneal (i.p.) injections of a modified truncated 24-aa peptide (TFP5), derived from the cdk5 activator p35, penetrated the blood-brain barrier and significantly rescued AD-like pathology in 5XFAD model mice. The principal pathology in the 5XFAD mutant, however, is extensive amyloid plaques; hence, as a proof of concept, we believe it is essential to demonstrate the peptides efficacy in a mouse model expressing high levels of p25, such as the inducible CK-p25Tg model mouse that overexpresses p25 in CamKII positive neurons. Using a modified TFP5 treatment, here we show that peptide i.p. injections in these mice decrease cdk5 hyperactivity, tau, neurofilament-M/H hyperphosphorylation, and restore synaptic function and behavior (i.e., spatial working memory, motor deficit using Rota-rod). It is noteworthy that TFP5 does not inhibit endogenous cdk5/p35 activity, nor other cdks in vivo suggesting it might have no toxic side effects, and may serve as an excellent therapeutic candidate for neurodegenerative disorders expressing abnormally high brain levels of p25 and hyperactive cdk5.

The interaction of Munc 18 (p67) with the p10 domain of p35 protects in vivo Cdk5/p35 activity from inhibition by TFP5, a peptide derived from p35

A truncated fragment of p35, the Cdk5 kinase regulatory protein (TFP5), inhibits specifically hyperactive Cdk5/p25 activity and rescues the Alzheimer’s disease and Parkinson’s disease phenotype in model mice. To account for the selective inhibition of Cdk5/p25 activity, the p10 N-terminal domain of p35, absent in p25, spares Cdk5/p35.

TFP5/TP5 peptide provides neuroprotection in the MPTP model of Parkinson's disease

Cyclin-dependent kinase 5 (Cdk5) is a member of the serine-threonine kinase family of cyclin-dependent kinases. Cdk5 is critical to normal mammalian nervous system development and plays important regulatory roles in multiple cellular functions. Recent evidence indicates that Cdk5 is inappropriately activated in several neurodegenerative conditions, including Parkinson′s disease (PD). PD is a chronic neurodegenerative disorder characterized by the loss of dopamine neurons in the substantia nigra, decreased striatal dopamine levels, and consequent extrapyramidal motor dysfunction. During neurotoxicity, p35 is cleaved to form p25. Binding of p25 with Cdk5 leads deregulation of Cdk5 resulting in number of neurodegenerative pathologies. To date, strategies to specifically inhibit Cdk5 hyperactivity have not been successful without affecting normal Cdk5 activity. Here we show that inhibition of p25/Cdk5 hyperactivation through TFP5/TP5, truncated 24-aa peptide derived from the Cdk5 activator p35 rescues nigrostriatal dopaminergic neurodegeneration induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP/MPP + ) in a mouse model of PD. TP5 peptide treatment also blocked dopamine depletion in the striatum and improved gait dysfunction after MPTP administration. The neuroprotective effect of TFP5/TP5 peptide is also associated with marked reduction in neuroinflammation and apoptosis. Here we show inhibition of Cdk5/p25-hyperactivation by TFP5/TP5 peptide, which identifies Cdk5/p25 as a potential therapeutic target to reduce neurodegeneration in PD.

Analysis of the Inhibitory Elements in the p5 Peptide Fragment of the CDK5 Activator, p35, CDKR1 Protein

Abstract Besides the hallmark pathology of amyloid plaques and neurofibrillary tangles, it is well documented that cyclin-dependent kinase 5 (CDK5), a critical neuronal protein kinase in nervous system development, function, and survival, when deregulated and hyperactivated induces Alzheimer’s disease (AD) and amyotrophic lateral sclerosis and Parkinson’s disease-like phenotypes in mice. In a recent study, we demonstrated that p5, a small, truncated fragment of 24 amino acid residues derived from the CDK5 activator protein 35 (NCK5A, p35), selectively inhibited deregulated CDK5 hyperactivity and ameliorated AD phenotypes in model mice. In this study, we identified the most inhibitory elements in the p5 peptide fragment. Each amino acid residue in p5 was systematically replaced with its homologous residues that may still be able to functionally substitute. The effects of these p5 peptide analogs were studied on the phosphotransferase activities of CDK5/p35, CDK5/p25, ERK1, and GSK3β. The mimetic p5 peptide (A/V substitution at the C-terminus of the peptide) in the sequence, KNAFYERALSIINLMTSKMVQINV (p5-MT) was the most effective inhibitor of CDK5 kinase activity of 79 tested mimetic peptides including the original p5 peptide, KEAFWDRCLSVINLMSSKMLQINA (p5-WT). Replacement of the residues in C-terminus end of the peptide affected CDK5 phosphotransferase activity most significantly. These peptides were strong inhibitors of CDK5, but not the related proline-directed kinases, ERK1 and GSK3β.

Candidate Bio-Markers of Alzheimer’s Disease

Alzheimer’s disease (AD) is a neurodegenerative disorder of the central nervous system characterized by a progressive loss of short-term memory accompanied by a gradual loss of cognitive functions (Ross et al., 2004). AD is among the most frequently encountered diseases in aging societies with an estimated 5million people in the United States and 17 million people worldwide suffering from the disease. It is expected that these numbers will quadruple by the year 2040, by which 1 out of 45 Americans will be affected, leading to a considerable public health burden (Fratiglioni et al., 1999). AD pathogenic mecha‐ nisms contributing to neuronal loss and brain dysfunction are still unclear. However, remarkable advances have taken place in understanding of both the genetics and molecular biological aspects of the intracellular processing of amyloid and tau and the changes leading to the pathologic formation of extracellular amyloid plaques and the intraneuronal aggregation of hyperphosphorylated tau into neurofibrillary tangles. This progress in our understanding of the molecular pathology has set the stage for clinical‐ ly meaningful advances in the development of biomarkers.

Peripheral and orofacial pain sensation is unaffected by the loss of p39

Cdk5 is a key neuronal kinase necessary for proper brain development, which has recently been implicated in modulating nociception. Conditional deletion of Cdk5 in pain-sensing neurons attenuates pain responses to heat in both the periphery and orofacial regions. Cdk5 activity is regulated by binding to the activators p35 and p39, both of which possess a cyclin box. Our previous examination of the nociceptive role of the well-characterized Cdk5 activator p35 using mice that either lack or overexpress this regulatory subunit demonstrated that Cdk5/p35 activity affects mechanical, chemical, and thermal nociception. In contrast, the nociceptive role of Cdk5’s other less-studied activator p39 is unknown. Here, we report that the knockout of p39 in mice did not affect orofacial and peripheral nociception. The lack of any algesic response to nociceptive stimuli in the p39 knockout mice contrasts with the hypoalgesic effects that result from the deletion of p35. Our data demonstrate different and nonoverlapping roles of Cdk5 activators in the regulation of orofacial as well as peripheral nociception with a crucial role for Cdk5/p35 in pain signaling.

Peptides Derived from Neuronal Cell Cycle like Kinase 5 Activator p35, inNeurodegeneration; Pathology and Therapy

Neurodegenerative disorders such as Alzheimer’s or Parkinson’s are diseases of aging. Mutations, lesions, neuronal insults, the genetic and stochastic “slings and arrows” of living, accumulate and downgrade organ function. Microarray analyses of aging brain identify patterns of genetic changes that correlate with neurodegenerative phenotypes. Genes involved in synaptic transmission, mitochondrial function and protein turnover are downregulated while significant numbers of genes regulating DNA repair, stress responses and inflammation are upregulated. This impact on the brain induces, a complex, multidimensional network of abnormal interactions leading to deposits of protein aggregates such as amyloid plaques, tau and neurofilament proteins tangles. For some, this pathology leads to neuron loss, behavioral defects, cognitive decline and death. Therapeutic approaches for AD, for example, target the molecular pathways leading to plaques and tangles; these include kinases and phosphatases. Among kinases, one stands out, Cdk5/p35, essential for neuronal migration, synapse formation, function and survival. Studies have shown that aging-induced neuronal stress deregulates and hyper activates Cdk5, a ubiquitous feature of neuronal disorders such as Alzheimer’s, Amyotropic lateral sclerosis (ALS) and Parkinson’s (PD). Among its many effects, hyperactive Cdk5 is implicated in the production of abnormal phosphorylated protein aggregates and is a therapeutic target. Roscovitine and related compounds inhibit Cdk5 activity but not specifically; cell cycle Cdks and other kinases are equally affected. In our laboratory two truncated peptides CIP (126a.a) and P5 (24a.a.), derived from p35, activator, of Cdk5 have been shown to specifically inhibit hyperactive Cdk5 in vitro, in cortical neurons and in AD, ALS and PD model mice. As a result the neurodegenerative phenotype was diminished; aggregates and inflammation were reduced, abnormal behavior was improved and increased animal’s longevity. We believe these peptides are excellent therapeutic candidates for those neurodegenerative disorders expressing hyperactive Cdk5 in the brain.