We as well as other laboratories around the world have shown 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 that hyperactive Cdk5 results from the overexpression of p25, (a truncated fragment of p35, the normal Cdk5 regulator), which, when complexes to Cdk5, induces hyperactivity, hyper phosphorylated tau / neurofilament tangles (NFTs), A-beta plaques and neuronal death. Our laboratory has recently shown that intraperitoneal (i.p.) injections of a modified truncated 24-amino acid 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, we show that peptide i.p. injections in these mice decrease Cdk5 activity, tau, neurofilament-M/H hyper phosphorylation, and restore synaptic function (LTP) and behavior (i.e., spatial working memory). It is noteworthy that TFP5 does not inhibitendogenous Cdk5/p35 activity, or 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. As a proof of concept, we have demonstrated that the peptide, injected into an AD model mouse overexpressing Cdk5/p25 (P25Tg mice), specifically targets the hyperactive kinase, reduces or eliminates AD pathology, and restores normal behavior. We suggest that the peptide may serve as a potential therapeutic candidate for those neurodegenerative disorders that overexpress p25, the hyperactive activator. Currently we have extended these neuroprotective effects of Peptide TFP5/TP5 in Parkinson's disease, MPTP animal models. A manuscript on this subject is published in Molecular Biology of Cell (2016). The editor of the journal made the following comments. This is a major advance that should be considered for clinical applications. In light of these studies presented in this manuscript, I am recommending this work be considered for MBOC Highlights and presentation to the public as an example of how basic research in cell biology advances our understanding of human life and health. This is an excellent extension of the neuroprotective role of the Cdk5 inhibitory peptide (TFP5/TP5) for symptoms of Parkinson's disease. This is a major advance that should be considered for clinical applications. I recommend publication as is, no revisions required. In a recent study, we also found that in addition of Cdk5 inhibiton, p5 also inhibited with higher efficacy 22 other kinases involved in neurodegenerative diseases (Binukumar et al., 2016). The profiling of kinase inhibitors is a critical and important step for drug development and their therapeutic application. The inhibitory profile of a compound across a broad collection of kinases is essential in understanding its biological and therapeutic implications. Obtaining any off-target activities, and in some cases identifying new targets, may lead to novel therapeutic applications of the inhibitor. Since there are multiple pathways producing neurodegenerative disorders reflecting the complex behavior of the diseases, undoubtedly, networks of cross talking kinases are involved. In order to characterize the mechanistic role of p5 peptide in these networks we have studied its effect in modulating the activity of seventy different kinases under identical in vitro assay conditions. This study reveals that P5 not only inhibits Cdk5 hyperactivation and deregulation but also inhibits many kinases some identified as relevant to neurodegeneration. This study may further support a novel role for P5 as a therapeutic candidate for multiple kinase pathways in neurodegenerative disease drug development. In a series of experiments, we have provided the evidence that the interaction of Munc 18 (p67), a synaptic vesicle associated protein, with the p10 domain of p35 protects in vivo Cdk5/p35 activity from inhibition by TFP5. It inhibits Cdk5/p35 and the hyperactive Cdk5/p25 activities in test tube experiments. In cortical neurons, however, and in vivo in Alzheimer's disease (AD) model mice, the peptide specifically inhibits the Cdk5/p25 complex and not the endogenous Cdk5/p35. To account for the selective inhibition of Cdk5/p25 activity, we propose that the p10 N-terminal domain of p35, absent in p25, spares Cdk5/p35 because p10 binds to macromolecules (e.g., tubulin and actin) as a membrane-bound multimeric complex that favors p35 binding to Cdk5 and catalysis. To test this hypothesis, we focused on Munc 18, a key synapse-associated neuronal protein, one of many proteins copurifying with Cdk5/p35 in membrane-bound multimeric complexes. Here we show that, in vitro, the addition of p67 protects Cdk5/p35 and has no effect on Cdk5/p25 activity in the presence of TFP5. In cortical neurons transfected with p67siRNA, we also show that TFP5 inhibits Cdk5/p35 activity, whereas in the presence of p67 the activity is protected. It does so without affecting any other kinases of the Cdk family of cyclin kinases. This difference may be of significant therapeutic value because the accumulation of the deregulated, hyperactive Cdk5/p25 complex in human brains has been implicated in pathology of AD and other neurodegenerative disorders. Mechanisms of specificity of TFP5 action will be studied in other kinases involved in neurodegenerative diseases. A key question is how do we explain the specificity of TFP5 action? In a test-tube kinase assay, P5, we found that the inhibitory peptide inhibits both Cdk5/p35 and Cdk5/p25 equally. We assume that p5 acts as an allosteric and/or competitive inhibitor, preventing binding of the regulator proteins (p35, p25) to the kinase. Interestingly, however, in vivo, in cortical neurons and model mice, it specifically inhibits the hyper- activated Cdk5/p25 but not the activity at lower concentration of the normal Cdk5/p35 complex, nor the activity of related cell cycle Cdks. It is this specificity at lower concentrations of the peptide action that distinguishes P5 as a valuable therapeutic candidate compared to those compounds, like roscovitine, that target the ATP binding site of the kinase. Our model, posits that the p10 (10kDa) of N- terminus of p35 that binds the Cdk5/p35 complex to the membrane selectively binds to cellular proteins in vivo including microtubules, Munc 18 (p67, n-sec1) provides a protective role. We have previously shown that microtubules (but not soluble tubulin) bind the Cdk5/p35 complex in a test-tube experiment and protect it from P5 inhibition. In contrast, Cdk5/p25, without the p10 domain, is inhibited by P5 in the presence of microtubules. Does TFP5 exhibit a similar pattern of specificity? We chose to test this using the synaptic protein Munc 18 (p67), a protein we isolated and cloned as part of a Cdk5 complex from rat brain during purification of active Cdk5. Although initially thought to be a regulator (because of its high binding specificity and activity), it turned out to be a Cdk5 substrate involved in synaptic activity.
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