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Colorectal carcinoma (CRC) is a prevalent cancer worldwide with a high mortality rate. Evidence suggests that increased expression of Cyclin-dependent kinase 5 (CDK5) contributes to cancer progression, making it a promising target for treatment. This study examined the efficacy of selectively inhibiting CDK5 in colorectal carcinoma using TP5, a small peptide that selectively inhibits the aberrant and hyperactive CDK5/p25 complex while preserving physiological CDK5/p35 functions. We analyzed TP5’s impact on CDK5 activity, cell survival, apoptosis, the cell cycle, DNA damage, ATM phosphorylation, and reactive oxygen species (ROS) signaling in mitochondria, in CRC cell lines, both alone and in combination with chemotherapy. We also assessed TP5’s efficacy on a xenograft mouse model with HCT116 cells. Our results showed that TP5 decreased CDK5 activity, impaired cell viability and colony formation, induced apoptosis, increased DNA damage, and led to the G1 phase arrest of cell cycle progression. In combination with irinotecan, TP5 demonstrated a synergy by leading to the accumulation of DNA damage, increasing the γH2A.X foci number, and inhibiting G2/M arrest induced by Sn38 treatment. TP5 alone or in combination with irinotecan increased mitochondrial ROS levels and inhibited tumor growth, prolonging mouse survival in the CRC xenograft animal model. These results suggest that TP5, either alone or in combination with irinotecan, is a promising therapeutic option for colorectal carcinoma.

Parkinson’s Disease (PD) is a chronic progressive neurodegenerative disease. Current treatments for PD are symptomatic and only increase striatal dopamine levels. Proactive neuroprotective approaches that slow the progression of PD and maintain appropriate dopamine neuron populations are needed to treat the disease. One suggested mechanism contributing to the pathology of PD involves the binding of cyclin-dependent kinase 5 (Cdk5) to p25, creating a hyperactivated complex to induce cell death. The objective of this study is to investigate the neuroprotective and neurorestorative properties of Truncated Peptide 5 (TP5), a derivative of the p35 activator involved in Cdk5 regulation, via the inhibition of Cdk5/p25 complex function. SH-SY5Y cell line and the nematode Caenorhabditis elegans were exposed to paraquat (PQ), an oxidative stressor, to induce Parkinsonian phenotypes. TP5 was administered prior to PQ exposure to determine its neuroprotective effects and, in further experiments, after PQ exposure to examine its neurorestorative effects. In the SH-SY5Y cell line, TP5 was found to have neuroprotective effects using a cell viability assay and demonstrated neuroprotective and neurorestorative effects in C. elegans by examining dopaminergic neurons and dopamine-dependent behaviour. TP5 decreased elevated Cdk5 activation in worms that were exposed to PQ. TP5’s inhibition of Cdk5/p25 hyperactivity led to the protection of dopamine neurons in these PD models. This suggests that TP5 can act as a potential therapeutic drug towards PD.

We examined the efficacy of selective inhibition of cyclin-dependent kinase 5 (CDK5) in glioblastoma by TP5. We analyzed its impact in vitro on CDK5 expression and activity, cell survival, apoptosis and cell cycle. DNA damage was analyzed using the expression of γH2A.X and phosphorylated ATM. Its tolerance and efficacy were assessed on in vivo xenograft mouse models. We showed that TP5 decreased the activity but not the expression of CDK5 and p35. TP5 alone impaired cell viability and colony formation of glioblastoma cell lines and induced apoptosis. TP5 increased DNA damage by inhibiting the phosphorylation of ATM, leading to G1 arrest. Whereas CDK5 activity is increased by DNA-damaging agents such as temozolomide and irradiation, TP5 was synergistic with either temozolomide or irradiation due to an accumulation of DNA damage. Concomitant use of TP5 and either temozolomide or irradiation reduced the phosphorylation of ATM, increased DNA damage, and inhibited the G2/M arrest induced by temozolomide or irradiation. TP5 alone suppressed the tumor growth of orthotopic glioblastoma mouse model. The treatment was well tolerated. Finally, alone or in association with irradiation or temozolomide, TP5 prolonged mouse survival. TP5 alone or in association with temozolomide and radiotherapy is a promising therapeutic option for glioblastoma.

Neurofilaments (NFs) are thought to provide stability to the axon. We examined NF dynamics within axonal neurites of NB2a/d1 neuroblastoma by transient transfection with green fluorescent protein-tagged NF-heavy (GFP-H) under the control of a tetracycline-inducible promoter. Immunofluorescent and biochemical analyses demonstrated that GFP-H expressed early during neurite outgrowth associated with a population of centrally-situated, highly-phosphorylated crosslinked NFs along the length of axonal neurites (‘bundled NFs’). By contrast, GFP-H expressed after considerable neurite outgrowth displayed markedly reduced association with bundled NFs and was instead more evenly distributed throughout the axon. This differential localization was maintained for up to 2 weeks in culture. Once considerable neurite outgrowth had progressed, GFP that had previously associated with the NF bundle during early expression was irreversibly depleted by photobleaching. Cessation of expression allowed monitoring of NF turnover. GFP-H associated bundled NFs underwent slower decay than GFP-H associated with surrounding, less-phosphorylated NFs. Notably, GFP associated with bundled NFs underwent similar decay rates within the core and edges of this bundle. These results are consistent with previous demonstration of a resident NF population within axonal neurites, but suggest that this population is more dynamic than previously considered.

Abstract  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. [ABSTRACT FROM AUTHOR]

Abstract  Cyclin-dependent kinase 5 (cdk5) is a proline-directed serine/threonine kinase that is activated mostly by association with its activators, p35 and p39. Initially projected as a neuron-specific kinase, cdk5 is expressed ubiquitously and its kinase activity solely depends on the presence of its activators, which are also found in some non-neuronal tissues. As a multifunctional protein, cdk5 has been linked to axonogenesis, cell migration, exocytosis, neuronal differentiation and apoptosis. Cdk5 plays a critical role in functions other than normal physiology, especially in neurodegeneration. Its contribution to both normal physiological as well as pathological processes is mediated by its specific substrates. Cdk5-null mice are embryonically lethal, therefore making it difficult to study precisely what cdk5 does to the nervous system at early stages of development, be it neuron development or programmed cell death. Zebrafish model system bypasses the impediment, as it is amenable to reverse genetics studies. One of the functions that we have followed for the cdk5 ortholog in zebrafish in vivo is its effect on the Rohon-Beard (RB) neurons. RB neurons are the primary sensory spinal neurons that die during the first two days of zebrafish development eventually to be replaced by the dorsal root ganglia (DRG). Based on ours studies and others’, here we discuss possible mechanisms that may be involved in cdk5’s role in RB neuron development and survival. [ABSTRACT FROM AUTHOR]