Activation of cyclin-dependent kinase 5 broadens action potentials in human sensory neurons.

Chronic pain is one of the most devastating and unpleasant conditions, associated with many pathological states. Tissue or nerve injuries induce extensive neurobiological plasticity in nociceptive neurons, which leads to chronic pain. Recent studies suggest that cyclin-dependent kinase 5 (CDK5) in primary afferents is a key neuronal kinase that modulates nociception through phosphorylation under pathological conditions. However, the impact of the CDK5 on nociceptor activity especially in human sensory neurons is not known. To determine the CDK5-mediated regulation of human dorsal root ganglia (hDRG) neuronal properties, we have performed the whole-cell patch clamp recordings in neurons dissociated from hDRG. CDK5 activation induced by overexpression of p35 depolarized the resting membrane potential (RMP) and reduced the rheobase currents as compared to the control neurons. CDK5 activation changed the shape of the action potential (AP) by increasing AP -rise time, -fall time, and -half width. The application of a prostaglandin E2 (PG) and bradykinin (BK) cocktail in control hDRG neurons induced the depolarization of RMP and the reduction of rheobase currents along with increased AP rise time. However, PG and BK applications failed to induce any significant changes in the p35-overexpressing group. We conclude that, in dissociated hDRGs neurons, CDK5 activation through the overexpression of p35 broadens the AP and that CDK5 may play important roles in the modulation of AP properties in human primary afferents under the condition in which CDK5 is upregulated, contributing to chronic pain.

ACTIVATION OF CYCLIN-DEPENDENT KINASE 5 BROADENS ACTION POTENTIALS IN HUMAN SENSORY NEURONS.

Chronic pain is one of the most devastating and unpleasant conditions, associated with many pathological conditions. Tissue or nerve injuries induce comprehensive neurobiological plasticity in nociceptive neurons, which leads to chronic pain. Recent studies suggest that cyclin-dependent kinase 5 (CDK5) in primary afferents is a key neuronal kinase that modulates nociception through phosphorylation-dependent manner under pathological conditions. However, the impact of the CDK5 on nociceptor activity especially in human sensory neurons are not known. To determine the CDK5-mediated regulation of human dorsal root ganglia (hDRG) neuronal properties, we have performed the whole-cell patch clamp recordings in neurons dissociated from hDRG. CDK5 activation induced by overexpression of p35 depolarized the resting membrane potential and reduced the rheobase currents as compared to the uninfected neurons. CDK5 activation evidently changed the shape of the action potential (AP) by increasing AP rise time, AP fall time, and AP half width. The application of a prostaglandin E2 (PG) and bradykinin (BK) cocktail in uninfected hDRG neurons induced the depolarization of RMP and the reduction of rheobase currents along with increased AP rise time. However, PG and BK applications failed to induce any further significant changes in addition to the aforementioned changes of the membrane properties and AP parameters in the p35-overexpressing group. We conclude that CDK5 activation through the overexpression of p35 in dissociated hDRG neurons broadens AP in hDRG neurons and that CDK5 may play important roles in the modulation of AP properties in human primary afferents under pathological conditions, contributing to chronic pain.

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.

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.

Therapeutic effects of TP5, a Cdk5/p25 inhibitor, in in vitro and in vivo models of Parkinson's disease.

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.

TP5, a Peptide Inhibitor of Aberrant and Hyperactive CDK5/p25: A Novel Therapeutic Approach against Glioblastoma.

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.

Quantum capacitance-limited MoS2 biosensors enable remote label-free enzyme measurements.

We have demonstrated atomically thin, quantum capacitance-limited, field-effect transistors (FETs) that enable the detection of pH changes with 75-fold higher sensitivity (≈4.4 V per pH) over the Nernst value of 59 mV per pH at room temperature when used as a biosensor. The transistors, which are fabricated from monolayer films of MoS2, use a room temperature ionic liquid (RTIL) in place of a conventional oxide gate dielectric and exhibit very low intrinsic noise resulting in a pH resolution of 92 × 10-6 at 10 Hz. This high device performance, which is a function of the structure of our device, is achieved by remotely connecting the gate to a pH sensing element allowing the FETs to be reused. Because pH measurements are fundamentally important in biotechnology, the increased resolution demonstrated here will benefit numerous applications ranging from pharmaceutical manufacturing to clinical diagnostics. As an example, we experimentally quantified the function of the kinase Cdk5, an enzyme implicated in Alzheimer's disease, at concentrations that are 5-fold lower than physiological values, and with sufficient time-resolution to allow the estimation of both steady-state and kinetic parameters in a single experiment. The high sensitivity, increased resolution, and fast turnaround time of the measurements will allow the development of early diagnostic tools and novel therapeutics to detect and treat neurological conditions years before currently possible.

Overexpression of the Cdk5 inhibitory peptide in motor neurons rescue of amyotrophic lateral sclerosis phenotype in a mouse model.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects motor nerve cells in the brain and the spinal cord. Etiological mechanisms underlying the disease remain poorly understood; recent studies suggest that deregulation of p25/Cyclin-dependent kinase 5 (Cdk5) activity leads to the hyperphosphorylation of Tau and neurofilament (NF) proteins in ALS transgenic mouse model (SOD1G37R). A Cdk5 involvement in motor neuron degeneration is supported by analysis of three SOD1G37R mouse lines exhibiting perikaryal inclusions of NF proteins and hyperphosphorylation of Tau. Here, we tested the hypothesis that inhibition of Cdk5/p25 hyperactivation in vivo is a neuroprotective factor during ALS pathogenesis by crossing the new transgenic mouse line that overexpresses Cdk5 inhibitory peptide (CIP) in motor neurons with the SOD1G37R, ALS mouse model (TriTg mouse line). The overexpression of CIP in the motor neurons significantly improves motor deficits, extends survival and delays pathology in brain and spinal cord of TriTg mice. In addition, overexpression of CIP in motor neurons significantly delays neuroinflammatory responses in TriTg mouse. Taken together, these data suggest that CIP may serve as a novel therapeutic agent for the treatment of neurodegenerative diseases.

Phosphorylation of the Transient Receptor Potential Ankyrin 1 by Cyclin-dependent Kinase 5 affects Chemo-nociception.

Cyclin-dependent kinase 5 (Cdk5) is a key neuronal kinase that is upregulated during inflammation, and can subsequently modulate sensitivity to nociceptive stimuli. We conducted an in silico screen for Cdk5 phosphorylation sites within proteins whose expression was enriched in nociceptors and identified the chemo-responsive ion channel Transient Receptor Potential Ankyrin 1 (TRPA1) as a possible Cdk5 substrate. Immunoprecipitated full length TRPA1 was shown to be phosphorylated by Cdk5 and this interaction was blocked by TFP5, an inhibitor that prevents activation of Cdk5. In vitro peptide-based kinase assay revealed that four of six TRPA1 Cdk5 consensus sites acted as substrates for Cdk5, and modeling of the ankyrin repeats disclosed that phosphorylation would occur at characteristic pockets within the (T/S)PLH motifs. Calcium imaging of trigeminal ganglion neurons from genetically engineered mice overexpressing or lacking the Cdk5 activator p35 displayed increased or decreased responsiveness, respectively, to stimulation with the TRPA1 agonist allylisothiocyanate (AITC). AITC-induced chemo-nociceptive behavior was also heightened in vivo in mice overexpressing p35 while being reduced in p35 knockout mice. Our findings demonstrate that TRPA1 is a substrate of Cdk5 and that Cdk5 activity is also able to modulate TRPA1 agonist-induced calcium influx and chemo-nociceptive behavioral responses.

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

Abstract: 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.

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 Alzheimer's 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-ß 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 peptide's 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.

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.

Knockdown of Expression of Cdk5 or p35 (a Cdk5 Activator) Results in Podocyte Apoptosis.

Podocytes are terminally differentiated glomerular epithelial cells. Podocyte loss has been found in many renal diseases. Cdk5 is a cyclin-dependent protein kinase which is predominantly regulated by p35. To study the role of Cdk5/p35 in podocyte survival, we first applied western blotting (WB) analysis to confirm the time-course expression of Cdk5 and p35 during kidney development and in cultured immortalized mouse podocytes. We also demonstrated that p35 plays an important role in promoting podocyte differentiation by overexpression of p35 in podocytes. To deregulate the expression of Cdk5 or p35 in mouse podocytes, we used RNAi and analyzed cell function and apoptosis assaying for podocyte specific marker Wilms Tumor 1 (WT1) and cleaved caspase 3, respectively. We also counted viable cells using cell counting kit-8. We found that depletion of Cdk5 causes decreased expression of WT1 and apoptosis. It is noteworthy, however, that downregulation of p35 reduced Cdk5 activity, but had no effect on cleaved caspase 3 expression. It did, however, reduce expression of WT1, a transcription factor, and produced podocyte dysmorphism. On the other hand increased apoptosis could be detected in p35-deregulated podocytes using the TUNEL analysis and immunofluorescent staining with cleaved caspase3 antibody. Viability of podocytes was decreased in both Cdk5 and p35 knockdown cells. Knocking down Cdk5 or p35 gene by RNAi does not affect the cycline I expression, another Cdk5 activator in podocyes. We conclude that Cdk5 and p35 play a crucial role in maintaining podocyte differentiation and survival, and suggest these proteins as targets for therapeutic intervention in podocyte-damaged kidney diseases.

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.

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

Parkinson's disease (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. Recent evidence indicates that cyclin-dependent kinase 5 (Cdk5) is inappropriately activated in several neurodegenerative conditions, including PD. To date, strategies to specifically inhibit Cdk5 hyperactivity have not been successful without affecting normal Cdk5 activity. Previously we reported that TFP5 peptide has neuroprotective effects in animal models of Alzheimer's disease. Here we show that TFP5/TP5 selective inhibition of Cdk5/p25 hyperactivation in vivo and in vitro 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 selective inhibition of Cdk5/p25 -hyperactivation by TFP5/TP5 peptide, which identifies the kinase as a potential therapeutic target to reduce neurodegeneration in Parkinson's disease.

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 Alzheimer's 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 ß 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-1ß, 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.

Activation of cyclin-dependent kinase 5 mediates orofacial mechanical hyperalgesia.

BACKGROUND: Cyclin-dependent kinase 5 (Cdk5) is a unique member of the serine/threonine kinase family. This kinase plays an important role in neuronal development, and deregulation of its activity leads to neurodegenerative disorders. Cdk5 also serves an important function in the regulation of nociceptive signaling. Our previous studies revealed that the expression of Cdk5 and its activator, p35, is upregulated in nociceptive neurons during peripheral inflammation. The aim of the present study was to characterize the involvement of Cdk5 in orofacial pain. Since mechanical hyperalgesia is the distinctive sign of many orofacial pain conditions, we adapted an existing orofacial stimulation test to assess the behavioral responses to mechanical stimulation in the trigeminal region of the transgenic mice with either reduced or increased Cdk5 activity. RESULTS: Mice overexpressing or lacking p35, an activator of Cdk5, showed altered phenotype in response to noxious mechanical stimulation in the trigeminal area. Mice with increased Cdk5 activity displayed aversive behavior to mechanical stimulation as indicated by a significant decrease in reward licking events and licking time. The number of reward licking/facial contact events was significantly decreased in these mice as the mechanical intensity increased. By contrast, mice deficient in Cdk5 activity displayed mechanical hypoalgesia. CONCLUSIONS: Collectively, our findings demonstrate for the first time the important role of Cdk5 in orofacial mechanical nociception. Modulation of Cdk5 activity in primary sensory neurons makes it an attractive potential target for the development of novel analgesics that could be used to treat multiple orofacial pain conditions.

Cdk5 inhibitory peptide (CIP) inhibits Cdk5/p25 activity induced by high glucose in pancreatic beta cells and recovers insulin secretion from p25 damage.

Cdk5/p25 hyperactivity has been demonstrated to lead to neuron apoptosis and degenerations. Chronic exposure to high glucose (HG) results in hyperactivity of Cdk5 and reduced insulin secretion. Here, we set out to determine whether abnormal upregulation of Cdk5/p25 activity may be induced in a pancreatic beta cell line, Min6 cells. We first confirmed that p25 were induced in overexpressed p35 cells treated with HG and increased time course dependence. Next, we showed that no p25 was detected under short time HG stimulation (4-12 hrs), however was detectable in the long exposure in HG cells (24 hrs and 48 hrs). Cdk5 activity in the above cells was much higher than low glucose treated cells and resulted in more than 50% inhibition of insulin secretion. We confirmed these results by overexpression of p25 in Min6 cells. As in cortical neurons, CIP, a small peptide, inhibited Cdk5/p25 activity and restored insulin secretion. The same results were detected in co-infection of dominant negative Cdk5 (DNCdk5) with p25. CIP also reduced beta cells apoptosis induced by Cdk5/p25. These studies indicate that Cdk5/p25 hyperactivation deregulates insulin secretion and induces cell death in pancreatic beta cells and suggests that CIP may serve as a therapeutic agent for type 2 diabetes.

Impaired neurogenesis and neurite outgrowth in an HIV-gp120 transgenic model is reversed by exercise via BDNF production and Cdk5 regulation.

Human immunodeficiency virus (HIV) infection-associated neurocognitive disorders is accompanied with brain atrophy. In these patients, impairment of adult neurogenesis and neurite outgrowth in the hippocampus may contribute to cognitive dysfunction. Although running exercises can enhance neurogenesis and normalize neurite outgrowth, the underlying molecular mechanisms are not well understood. The HIV envelope protein, gp120, has been shown to impair neurogenesis. Using a gp120 transgenic mouse model, we demonstrate that exercise stimulated neural progenitor cell (NPC) proliferation in the hippocampal dentate gyrus and increased the survival rate and generation of newborn cells. However, sustained exercise activity was necessary as the effects were reversed by detraining. Exercise also normalized dendritic outgrowth of neurons. Furthermore, it increased the expression of hippocampal brain-derived neurotrophic factor (BDNF) and normalized hyperactivation of cyclin-dependent kinase 5 (Cdk5). Hyperactivated Cdk5 or gp120 treatment led to aberrant neurite outgrowth and BDNF treatment normalized the neurite outgrowth in NPC cultures. These results suggest that sustained exercise has trophic activity on the neuronal lineage which is mediated by Cdk5 modulation of the BDNF pathway.