TP5: A Novel Therapeutic Approach Targeting Aberrant and Hyperactive CDK5/p25 for the Treatment of Colorectal Carcinoma.

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.

Human Endogenous Retrovirus K Envelope in Spinal Fluid of Amyotrophic Lateral Sclerosis Is Toxic.

OBJECTIVE: Human endogenous retroviruses have been implicated in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Expression of human endogenous retrovirus K (HERV-K) subtype HML-2 envelope (Env) in human neuronal cultures and in transgenic mice results in neurotoxicity and neurodegeneration, and mice expressing HML-2 Env display behavioral and neuromuscular characteristics resembling ALS. This study aims to characterize the neurotoxic properties of HML-2 Env. METHODS: Env neurotoxicity was detected in ALS cerebrospinal fluid and confirmed using recombinant Env protein in a cell-based assay and a mouse model. The mechanism of neurotoxicity was assessed with immunoprecipitation followed by mass spectrometry and Western blot, and by screening a panel of inhibitors. RESULTS: We observed that recombinant HML-2 Env protein caused neurotoxicity resulting in neuronal cell death, retraction of neurites, and decreased neuronal electrical activity. Injection of the Env protein into the brains of mice also resulted in neuronal cell death. HML-2 Env protein was also found in the cerebrospinal fluid of patients with sporadic ALS. The neurotoxic properties of the Env and the cerebrospinal fluid could be rescued with the anti-Env antibody. The Env was found to bind to CD98HC complexed to ß1 integrin on the neuronal cell surface. Using a panel of compounds to screen for their ability to block Env-induced neurotoxicity, we found that several compounds were protective and are linked to the ß1 integrin pathway. INTERPRETATION: HERV-K Env is released extracellularly in ALS and causes neurotoxicity via a novel mechanism. Present results pave the way for new treatment strategies in sporadic ALS. ANN NEUROL 2022;92:545-561.

Protocols for Characterization of Cdk5 Kinase Activity.

Cyclin-dependent kinases (Cdks) are generally known to be involved in controlling the cell cycle, but Cdk5 is a unique member of this protein family for being most active in post-mitotic neurons. Cdk5 is developmentally important in regulating neuronal migration, neurite outgrowth, and axon guidance. Cdk5 is enriched in synaptic membranes and is known to modulate synaptic activity. Postnatally, Cdk5 can also affect neuronal processes such as dopaminergic signaling and pain sensitivity. Dysregulated Cdk5, in contrast, has been linked to neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). Despite primarily being implicated in neuronal development and activity, Cdk5 has lately been linked to non-neuronal functions including cancer cell growth, immune responses, and diabetes. Since Cdk5 activity is tightly regulated, a method for measuring its kinase activity is needed to fully understand the precise role of Cdk5 in developmental and disease processes. This article includes methods for detecting Cdk5 kinase activity in cultured cells or tissues, identifying new substrates, and screening for new kinase inhibitors. Furthermore, since Cdk5 shares homology and substrate specificity with Cdk1 and Cdk2, the Cdk5 kinase assay can be used, with modification, to measure the activity of other Cdks as well. © 2021 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: Measuring Cdk5 activity from protein lysates Support Protocol 1: Immunoprecipitation of Cdk5 using Dynabeads Alternate Protocol: Non-radioactive protocols to measure Cdk5 kinase activity Support Protocol 2: Western blot analysis for the detection of Cdk5, p35, and p39 Support Protocol 3: Immunodetection analysis for Cdk5, p35, and p39 Support Protocol 4: Genetically engineered mice (+ and - controls) Basic Protocol 2: Identifying new Cdk5 substrates and kinase inhibitors.

Phosphorylation, Dephosphorylation, and Multiprotein Assemblies Regulate Dynamic Behavior of Neuronal Cytoskeleton: A Mini-Review.

Cellular localization, assembly and abnormal aggregation of neurofilaments depend on phosphorylation. Pathological processes associated with neurodegeneration exhibit aberrant accumulation of microtubule associated aggregated forms of hyperphosphorylated neuronal protein tau in cell bodies. These processes are critical for the disease progression in patients suffering from Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis. In healthy cells, tau is localized in axons. Topographic regulation suggests that whereas the sites of synthesis of kinases and neurofilaments are the cell bodies, and sites of their functional assemblies are axons, phosphorylation/dephosphorylation are the key processes that arrange the molecules at their precise locations. Phosphorylation sites in the dynamic developmental and degenerative processes differ. Not all these processes are well understood. New advancements identify epigenetic factors involved in AD which account for the influence of age-related environment/genome interactions leading to the disease. Progress in proteomics highlights previously found major proteins and adds more to the list of those involved in AD. New key elements of specificity provide determinants of molecular recognition important for the assembly of macromolecular complexes. In this review, we discuss aberrant spatial distribution of neuronal polypeptides observed in neuropathies: aggregation, association with proteins of the neuronal cytoskeleton, and phosphorylation dependent dynamics. Particularly, we emphasize recent advancements in understanding the function and determinants of specific association of molecules involved in Alzheimer's disease with respect to the topographic regulation of phosphorylation in neuronal cytoskeleton and implications for the design of new therapies. Further, we address the role of various filament systems in maintenance of the shape, rigidity and dynamics of the cytoskeleton.

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.

In a series of studies, we have identified TFP5, a truncated fragment of p35, the Cdk5 kinase regulatory protein, which 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.

Profiling of p5, a 24 Amino Acid Inhibitory Peptide Derived from the CDK5 Activator, p35 CDKR1 Against 70 Protein Kinases.

Cyclin-dependent kinase 5 (CDK5) is a multifunctional serine/threonine kinase that regulates a large number of neuronal processes essential for nervous system development and function with its activator p35 CDK5R1. Upon neuronal insults, p35 is proteolyzed and cleaved to p25 producing deregulation and hyperactivation of CDK5 (CDK5/p25), implicated in tau hyperphosphorylation, a pathology in some neurodegenerative diseases. A truncated, 24 amino acid peptide, p5, derived from p35 inhibits the deregulated CDK5 phosphotransferase activity and ameliorates Alzheimer's disease (AD) phenotypes in AD model mice. In the present study, we have screened a diverse panel of 70 human protein kinases for their sensitivities to p5, and a subset of these to p35. At least 16 of the tested protein kinases exhibited IC50 values that were 250 µM or less, with CAMK4, ZAP70, SGK1, and PIM1 showing greater sensitivity to inhibition by p5 than CDK5/p35 and CDK5/p25. In contrast, the p5 peptide modestly activated LKB1 and GSK3ß. A sub set of kinases screened against p35 showed that activity of CAMK4 in the absence of calcium and calmodulin was also markedly inhibited by p35. The Cyclin Y-dependent kinases PFTK1 (CDK14) and PCTK1 (CDK16) were activated by p35 at least 10-fold in the absence of Cyclin Y and by approximately 50% in its presence. These findings provide additional insights into the mechanisms of action for p5 and p35 in the regulation of protein phosphorylation in the nervous system.

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

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 GSK3ß. 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 GSK3ß.

Ecological attributes of Hepatozoon lacertilis Gupta et al., 2011 susceptibility in Indian lizards, Hemidactylus flaviviridis (Gekkonidae) and Calotes versicolor (Agamidae).

Ecological attributes of haematozoan parasites are poorly understood. In this study, we report haematozoan prevalence in two species of Indian lizards, Hemidactylus flaviviridis (Family: Gekkonidae) and Calotes versicolor (Family: Agamidae) under three macro-environmental variables: host location, weight and seasonal variations. Hemidactylus flaviviridis (n= 199) and Calotes versicolor (n= 34) were sampled from Bareilly, Chandausi and Mirzapur, Uttar Pradesh, India belonging to different weight groups [Group I (0-5 gm), Group II (5-10 gm) and Group III (10-15 gm)] and during various seasons [Summer (May-July), Rainy (August-October), Winter (November-January), Spring (February-April)] of the year. A haemogregarine, Hepatozoon Miller, 1908 was discovered from both host species. Test for identity of the parasites was conducted by feeding infected Culex quinquefasciatus (Diptera: Culicidae) on infection-free H. flaviviridis and C. versicolor and blood examinations on 22(nd) day (H. flaviviridis) and 25(th) day (C. versicolor) post feeding (pf) revealed similar haematozoan parasites and were identified as Hepatozoon lacertilis Gupta et al., 2011. Infectivity from different locations indicated a prevalence of 5.26% (Bareilly) and 16.36% (Mirzapur) in H. flaviviridis whereas infectivity was comparatively higher (19.23%) in C. versicolor. In different weight groups, Group III indicated highest infectivity in both lizards being 21.42% (C. versicolor) and 17.85% (H. flaviviridis). Parasites showed highest prevalence during spring season (H. flaviviridis : 9.52%; C. versicolor : 25%). Values of significance were determined by chi-square test to compare the prevalence within different variables (host location, weight and season). The study has importance for its contribution to the knowledge on the diversity of reptilian hosts infected by haemogregarines. It is the first record of Hepatozoon infectivity in both lizard species with respect to the three macro-environmental variables.

Biomarkers in Alzheimer's disease: a review.

Alzheimer's disease is the most common form of dementia affecting millions of individuals worldwide. It is currently diagnosed only via clinical assessments and confirmed by postmortem brain pathology. The development of validated biomarkers for Alzheimer's disease is essential to improve diagnosis and accelerate the development of new therapies. Biochemical and neuroimaging markers could facilitate diagnosis, predict AD progression from a pre-AD state of mild cognitive impairment (MCI), and be used to monitor efficacies of disease-modifying therapies. Cerebrospinal fluid (CSF) levels of Aß40, Aß42, total tau, and phosphorylated tau have diagnostic values in AD. Measurements of the above CSF markers in combination are useful in predicting the risk of progression from MCI to AD. New potential biomarkers are emerging, and CSF or plasma marker profiles may eventually become part of the clinician's toolkit for accurate AD diagnosis and management. These biomarkers along with clinical assessment, neuropsychological testing, and neuroimaging could achieve a much higher diagnostic accuracy for AD and related disorders in the future.

Exploring the possible mechanisms of action behind the antinociceptive activity of Bacopa monniera.

AIM: Earlier studies have demonstrated that Bacopa monniera (BM), a plant described in Ayurveda for many CNS actions was found to exhibit antidepressant (methanolic extract at 20mg/kg and 40mg/kg p.o.) as well as antinociceptive activity (aqueous extract (AE) at 80 mg/kg, 120 mg/kg and 160 mg/kg p.o.). The present study sought to explore the possible mechanisms of antinociceptive effects of aqueous extract of Bacopa monniera (AEBM) at 80 mg/kg, 120 mg/kg and 160 mg/kg given orally. MATERIALS AND METHODS: AEBM was given singly as well as with selective α2 receptor blocker Yohimbine, selective ß1 receptor blocker Atenolol, serotonin receptor antagonist Cyproheptadine and a non-selective opioid receptor antagonist naloxone in experimental groups of mice and rats under strict protocols and conditions. RESULTS: We observed that the antinociceptive effects of AEBM in the acetic acid writhing test was prevented by prior treatment with the selective Yohimbine (1 mg/kg, i.p; 14.50 ± 2.26 and 37.17 ± 2.14 writhes in the AEBM-treated and yohimbine pre-treated AEBM groups, respectively) and selective ß1 Atenolol receptor blocker (1 mg/kg, i.p; 14.50 ± 2.26 and 31.00 ± 5.44 writhes in the AEBM-treated and yohimbine pre-treated AEBM groups, respectively). In the formalin test, the reduction in licking time with AEBM was found to be reversed by prior treatment with serotonin receptor antagonist Cyproheptadine (1 mg/kg, i.p; 47.33 ± 2.25s and 113.50 ± 3.83s (during phase I i.e. 0-5 min) and 26.67 ± 3.83s and 88.17 ± 7.27s (during phase II i.e. 20-30 min) in the AEBM-treated and Cyproheptadine pre-treated AEBM groups, respectively). The % increase in tail flick latency with AEBM was prevented by prior treatment with the non-selective opioid receptor antagonist naloxone (2mg/kg, i.p; 282.35 and 107.35 in the AEBM-treated and naloxone-treated groups, respectively). CONCLUSIONS: Our results indicate, that the endogenous adrenergic, serotonergic and opioidergic systems are involved in the analgesic mechanism of action of the aqueous extract of Bacopa monniera.