Cdk5 Activity Research Articles

Morphine and HIV-1 Tat interact to cause region-specific hyperphosphorylation of tau in transgenic mice

Opiate abuse is prevalent among HIV-infected individuals and may exacerbate HIV-associated age-related neurocognitive disorders. However, the extent to which HIV and opiates converge to accelerate pathological traits indicative of brain aging remains unknown. The pathological phospho-isotypes of tau (pSer396, pSer404, pThr205, pSer202, and pThr181) and the tau kinases GSK3β and CDK5/p35 were explored in the striatum, hippocampus, and prefrontal cortex of inducible male and female HIV-1 Tat-transgenic mice, with some receiving escalating doses of morphine for 2 weeks. In the striatum of male mice, pSer396 was increased by co-exposure to morphine and Tat as compared to all other groups. Striatal pSer404 and pThr205 were increased by Tat alone, while pSer202 and pThr181 were unchanged. A comparison between Tat-transgenic female and male mice revealed disparate outcomes for pThr205. No other sex-related changes to tau phosphorylation were observed. In the hippocampus, Tat increased pSer396, while other phosphorylation sites were unchanged and pSer202 was not detected. In the prefrontal cortex, morphine increased pSer396 levels, which were unaffected by Tat, while other phosphorylation sites were unaffected. Assessment of tau kinases revealed no changes to striatal GSK3β (phosphorylated or total) or the total CDK5 levels. Striatal levels of phosphorylated CDK5 and p35, the activator of CDK5, were increased by Tat and with morphine co-exposure, respectively. P35 levels positively correlated with those of pSer396 with Tat and morphine co-exposure. The results reveal region-specific hyperphosphorylation of tau induced by exposure to morphine, Tat, and unique morphine and Tat interactions.

P39-associated Cdk5 activity regulates dendritic morphogenesis

Dendrites, branched structures extending from neuronal cell soma, are specialized for processing information from other neurons. The morphogenesis of dendritic structures is spatiotemporally regulated by well-orchestrated signaling cascades. Dysregulation of these processes impacts the wiring of neuronal circuit and efficacy of neurotransmission, which contribute to the pathogeneses of neurological disorders. While Cdk5 (cyclin-dependent kinase 5) plays a critical role in neuronal dendritic development, its underlying molecular control is not fully understood. In this study, we show that p39, one of the two neuronal Cdk5 activators, is a key regulator of dendritic morphogenesis. Pyramidal neurons deficient in p39 exhibit aberrant dendritic morphology characterized by shorter length and reduced arborization, which is comparable to dendrites in Cdk5-deficient neurons. RNA sequencing analysis shows that the adaptor protein, WDFY1 (WD repeat and FYVE domain-containing 1), acts downstream of Cdk5/p39 to regulate dendritic morphogenesis. While WDFY1 is elevated in p39-deficient neurons, suppressing its expression rescues the impaired dendritic arborization. Further phosphoproteomic analysis suggests that Cdk5/p39 mediates dendritic morphogenesis by modulating various downstream signaling pathways, including PI3K/Akt-, cAMP-, or small GTPase-mediated signaling transduction pathways, thereby regulating cytoskeletal organization, protein synthesis, and protein trafficking.

Phosphorylation of STAT3 by axonal Cdk5 promotes axonal regeneration by modulating mitochondrial activity

Cyclin-dependent kinase 5 (Cdk5) is involved in neural organization and synaptic functions in developing and adult brains, yet its role in axonal regeneration is not known well. Here, we characterize Cdk5 function for axonal regeneration after peripheral nerve injury. Levels of Cdk5 and p25 were elevated in sciatic nerve axons after injury. Cdk5 activity was concomitantly induced from injured nerve and increased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) on the serine 727 residue. Pharmacological and genetic blockades of Cdk5 activity phosphorylating STAT3 resulted in the inhibition of axonal regeneration as evidenced by reduction of retrograde labeling of dorsal root ganglion (DRG) sensory neurons and spinal motor neurons and also of neurite outgrowth of preconditioned DRG neurons in culture. Cdk5 and STAT3 were found in mitochondrial membranes of the injured sciatic nerve. Cdk5-GFP, which was translocated into the mitochondria by the mitochondrial target sequence (MTS), induced STAT3 phosphorylation in transfected DRG neurons and was sufficient to induce neurite outgrowth. In the mitochondria, Cdk5 activity was positively correlated with increased mitochondrial membrane potential as measured by fluorescence intensity of JC-1 aggregates. Our data suggest that Cdk5 may play a role in modulating mitochondrial activity through STAT3 phosphorylation, thereby promoting axonal regeneration.

CDK5 Inhibition Abrogates TNBC Stem-Cell Property and Enhances Anti-PD-1 Therapy.

Triple‐negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, in which the higher frequency of cancer stem cells (CSCs) correlates with the poor clinical outcome. An aberrant activation of CDK5 is found to associate with TNBC progression closely. CDK5 mediates PPARγ phosphorylation at its Ser 273, which induces CD44 isoform switching from CD44s to CD44v, resulting in an increase of stemness of TNBC cells. Blocking CDK5/pho‐PPARγ significantly reduces CD44v+ BCSCs population in tumor tissues, thus abrogating metastatic progression in TNBC mouse model. Strikingly, diminishing stemness transformation reverses immunosuppressive microenvironment and enhances anti‐PD‐1 therapeutic efficacy on TNBC. Mechanistically, CDK5 switches the E3 ubiquitin ligase activity of PPARγ and directly protects ESRP1 from a ubiquitin‐dependent proteolysis. This finding firstly indicates that CDK5 blockade can be a potent strategy to diminish stemness transformation and increase the response to PD‐1 blockade in TNBC therapy.

RPS23RG1 modulates tau phosphorylation and axon outgrowth through regulating p35 proteasomal degradation.

Tauopathies are a group of neurodegenerative diseases characterized by hyperphosphorylation of the microtubule-binding protein, tau, and typically feature axon impairment and synaptic dysfunction. Cyclin-dependent kinase5 (Cdk5) is a major tau kinase and its activity requires p35 or p25 regulatory subunits. P35 is subjected to rapid proteasomal degradation in its membrane-bound form and is cleaved by calpain under stress to a stable p25 form, leading to aberrant Cdk5 activation and tau hyperphosphorylation. The type Ib transmembrane protein RPS23RG1 has been implicated in Alzheimer's disease (AD). However, physiological and pathological roles for RPS23RG1 in AD and other tauopathies are largely unclear. Herein, we observed retarded axon outgrowth, elevated p35 and p25 protein levels, and increased tau phosphorylation at major Cdk5 phosphorylation sites in Rps23rg1 knockout (KO) mice. Both downregulation of p35 and the Cdk5 inhibitor roscovitine attenuated tau hyperphosphorylation and axon outgrowth impairment in Rps23rg1 KO neurons. Interestingly, interactions between the RPS23RG1 carboxyl-terminus and p35 amino-terminus promoted p35 membrane distribution and proteasomal degradation. Moreover, P301L tau transgenic (Tg) mice showed increased tau hyperphosphorylation with reduced RPS23RG1 levels and impaired axon outgrowth. Overexpression of RPS23RG1 markedly attenuated tau hyperphosphorylation and axon outgrowth defects in P301L tau Tg neurons. Our results demonstrate the involvement of RPS23RG1 in tauopathy disorders, and implicate a role for RPS23RG1 in inhibiting tau hyperphosphorylation through homeostatic p35 degradation and suppression of Cdk5 activation. Reduced RPS23RG1 levels in tauopathy trigger aberrant Cdk5-p35 activation, consequent tau hyperphosphorylation, and axon outgrowth impairment, suggesting that RPS23RG1 may be a potential therapeutic target in tauopathy disorders.

Small-molecule modulation of the p75 neurotrophin receptor inhibits a wide range of tau molecular pathologies and their sequelae in P301S tauopathy mice

In tauopathies, phosphorylation, acetylation, cleavage and other modifications of tau drive intracellular generation of diverse forms of toxic tau aggregates and associated seeding activity, which have been implicated in subsequent synaptic failure and neurodegeneration. Suppression of this wide range of pathogenic species, seeding and toxicity mechanisms, while preserving the physiological roles of tau, presents a key therapeutic goal. Identification and targeting of signaling networks that influence a broad spectrum of tau pathogenic mechanisms might prevent or reverse synaptic degeneration and modify disease outcomes. The p75 neurotrophin receptor (p75NTR) modulates such networks, including activation of multiple tau kinases, calpain and rhoA-cofilin activity. The orally bioavailable small-molecule p75NTR modulator, LM11A-31, was administered to tauP301S mice for 3 months starting at 6 months of age, when tau pathology was well established. LM11A-31 was found to reduce: excess activation of hippocampal cdk5 and JNK kinases and calpain; excess cofilin phosphorylation, tau phosphorylation, acetylation and cleavage; accumulation of multiple forms of insoluble tau aggregates and filaments; and, microglial activation. Hippocampal extracts from treated mice had substantially reduced tau seeding activity. LM11A-31 treatment also led to a reversal of pyramidal neuron dendritic spine loss, decreased loss of dendritic complexity and improvement in performance of hippocampal behaviors. These studies identify a therapeutically tractable upstream signaling module regulating a wide spectrum of basic mechanisms underlying tauopathies.

Flavonoids from Stem and Leaf of Scutellaria Baicalensis Georgi Inhibit the Phosphorylation on Multi-sites of Tau Protein Induced by Okadaic Acid and the Regulative Mechanism of Protein Kinases in Rats.

The present study aims to investigate the effect of flavonoids from stem and leaf of Scutellaria Baicalensis Georgi (SSF) on multi-sites phosphorylation of tau protein in the cerebral cortex and hippocampus of rats induced by okadaic acid (OA) and the regulative mechanism of the protein kinases. The model of AD-like memory impairment and neuronal injuries was established in male SD rats who were microinjected with OA (200 ng/kg) to establish a memory impairment model and screened for successful model rats by Morris water maze on day 21 after surgery. The successful model rats were continuously administered with intragastric infusion (ig) SSF 25, 50 and 100 mg/kg or Ginkgo biloba leaves flavonoids (GLF) 200 mg/kg for 36 d. The relative protein expressed levels of phosphorylated tau protein at sites of Ser199, Ser202, Ser214, Ser404 and Thr231, protein kinases (CDK5, PKA, pTyr216-GSK3β and pSer9-GSK3β) were detected by Western blotting. The relative protein expressed levels of p-tau(Ser199), p-tau(Ser202), p-tau(Ser214), p-- tau(Ser404), p-tau(Thr231) and pTyr216-GSK3β were significantly increased in both cerebral cortex and hippocampus regions of the model rats subjected to intracerebroventricular injection of OA (P<0.01), while the protein expressed levels of CDK5, PKA and pSer9-GSK3β (P<0.01) were reduced. SSF can dramatically reverse these increments in phosphorylated tau protein levels (P<0.01) and differently regulate the protein expressed levels of CDK5, PKA and GSK3β (P<0.01) in rats' cerebral cortex and hippocampus induced by OA. GLF also exhibit a similar effect to SSF. The results demonstrated that SSF could inhibit the hyperphosphorylation of tau in rats' cerebral cortex and hippocampus induced by microinjection of OA, which may be related to the activities of protein kinase CDK5, PKA and GSK3β.

Amelioration of Tau pathology and memory deficits by targeting 5-HT7 receptor.

Tauopathies comprise a heterogeneous family of neurodegenerative diseases characterized by pathological accumulation of hyperphosphorylated Tau protein. Pathological changes in serotonergic signaling have been associated with tauopathy etiology, but the underlying mechanisms remain poorly understood. Here, we studied the role of the serotonin receptor 7 (5-HT7R), in a mouse model of tauopathy induced by overexpressing the human Tau[R406W] mutant associated with inherited forms of frontotemporal dementia. We showed that the constitutive 5-HT7R activity is required for Tau hyperphosphorylation and formation of highly bundled Tau structures (HBTS) through G-protein-independent, CDK5-dependent mechanism. We also showed that 5-HT7R physically interacts with CDK5. At the systemic level, 5-HT7R-mediated CDK5 activation induces HBTS leading to neuronal death, reduced long-term potentiation (LTP), and impaired memory in mice. Specific blockade of constitutive 5-HT7R activity in neurons that overexpressed Tau[R406W] prevents Tau hyperphosphorylation, aggregation, and neurotoxicity. Moreover, 5-HT7R knockdown in the prefrontal cortex fully abrogates Tau[R406W]-induced LTP deficits and memory impairments. Thus, 5-HT7R/CDK5 signaling emerged as a new, promising target for tauopathy treatments.

Identification of Quinazolinone Analogs Targeting CDK5 Kinase Activity and Glioblastoma Cell Proliferation.

CDK5/p25 kinase plays a major role in neuronal functions, and is hyperactivated in several human cancers including glioblastoma and neurodegenerative pathologies such as Alzheimer's and Parkinson's. CDK5 therefore constitutes an attractive pharmacological target. Since the successful discovery and development of Roscovitine, several ATP-competitive inhibitors of CDK5 and peptide inhibitors of CDK5/p25 interface have been developed. However, these compounds suffer limitations associated with their mechanism of action and nature, thereby calling for alternative targeting strategies. To date, few allosteric inhibitors have been developed for successful targeting of protein kinases. Indeed, although this latter class of inhibitors are believed to be more selective than compounds targeting the active site, they have proven extremely difficult to identify in high throughput screens. By implementing a fluorescent biosensor that discriminates against ATP-pocket binding compounds to screen for allosteric inhibitors that target conformational activation of CDK5, we have identified a novel family of quinazolinones. Characterization of these hits and several of their derivatives revealed their inhibitory potential toward CDK5 kinase activity in vitro and to inhibit glioblastoma cell proliferation. The quinazolinone derivatives described in this study are the first small molecules reported to target CDK5 at a site other than the ATP pocket, thereby constituting attractive leads for glioblastoma therapeutics and providing therapeutic perspectives for neurodegenerative diseases. These compounds offer alternatives to conventional ATP-competitive inhibitors or peptides targeting CDK5/p25 interface with the potential of bypassing their limitations.

Cdk5-Mediated Phosphorylation of Sirt1 Contributes to Podocyte Mitochondrial Dysfunction in Diabetic Nephropathy.

Aims: Mitochondrial dysfunction contributes to podocyte injury, which is the leading cause of proteinuria in diabetic nephropathy (DN). In this study, we explored the role of cyclin-dependent kinase 5 (Cdk5) in mitochondrial dysfunction of podocytes under diabetic conditions. Results: Our results showed that the expression and activity of Cdk5 were significantly upregulated in vivo and in vitro under diabetic conditions, accompanied by the downregulation of synaptopodin and nephrin, as well as structural and functional mitochondrial dysfunction. Inhibition of Cdk5 with roscovitine or dominant-negative Cdk5 led to the attenuation of podocyte injury by upregulating synaptopodin and nephrin. The inhibition of Cdk5 also ameliorated mitochondrial dysfunction by decreasing reactive oxygen species levels and cytochrome c release, while increasing adenosine triphosphate production. Sirt1, an NAD+-dependent deacetylase, was decreased in podocytes with high glucose (HG) treatment; however, its phosphorylation level at S47 was significantly upregulated. We demonstrated that HG levels cause overactive Cdk5 to phosphorylate Sirt1 at S47. Suppression of Cdk5 reduced Sirt1 phosphorylation levels and mutation of S47 to nonphosphorable alanine (S47A), significantly attenuated podocyte injury and mitochondrial dysfunction in diabetic condition in vivo and in vitro. Innovation and Conclusion: Our study has demonstrated the role of Cdk5 in regulating mitochondrial function through Sirt1 phosphorylation and thus can potentially be a new therapeutic target for DN treatment. IRB number: 20190040. Antioxid. Redox Signal. 34, 171-190.

The role of cyclin-dependent kinase 5 in neuropathic pain.

The chronification of pain can be attributed to changes in membrane receptors and channels underlying neuronal plasticity and signal transduction largely within nociceptive neurons that initiate and maintain pathological pain states. These proteins are subject to dynamic modification by posttranslational modifications, creating a code that controls protein function in time and space. Phosphorylation is an important posttranslational modification that affects ∼30% of proteins in vivo. Increased phosphorylation of various nociceptive ion channels and of their modulators underlies sensitization of different pain states. Cyclin-dependent kinases are proline-directed serine/threonine kinases that impact various biological and cellular systems. Cyclin-dependent kinase 5 (Cdk5), one member of this kinase family, and its activators p35 and p39 are expressed in spinal nerves, dorsal root ganglia, and the dorsal horn of the spinal cord. In neuropathic pain conditions, expression and/or activity of Cdk5 is increased, implicating Cdk5 in nociception. Experimental evidence suggests that Cdk5 is regulated through its own phosphorylation, through increasing p35's interaction with Cdk5, and through cleavage of p35 into p25. This narrative review discusses the molecular mechanisms of Cdk5-mediated regulation of target proteins involved in neuropathic pain. We focus on Cdk5 substrates that have been linked to nociceptive pathways, including channels (eg, transient receptor potential cation channel and voltage-gated calcium channel), proteins involved in neurotransmitter release (eg, synaptophysin and collapsin response mediator protein 2), and receptors (eg, glutamate, purinergic, and opioid). By altering the phosphoregulatory "set point" of proteins involved in pain signaling, Cdk5 thus appears to be an attractive target for treating neuropathic pain conditions.

Effects of Single-Dose and Long-Term Ketamine Administration on Tau Phosphorylation-Related Enzymes GSK-3β, CDK5, PP2A, and PP2B in the Mouse Hippocampus.

Ketamine is a recreational drug that causes emotional and cognitive impairments, but its specific mechanisms of action are still unclear. Recent evidence suggests that Tau protein phosphorylation and targeted delivery to the postsynaptic area are closely related to its neurotoxicity, and our recent studies have shown that long-term ketamine administration causes excessive Tau protein phosphorylation. However, the regulatory mechanism of Tau protein phosphorylation induced by ketamine has not been clarified. In the present study, we administered a single ketamine injection and long-term (6months) ketamine injections in C57BL/6 mice, to investigate the effects of different doses of ketamine on the expression levels of Tau protein and its phosphorylation, the expression levels and activities of the related protein phosphokinases GSK-3β and CDK5, and the expression levels and activities of the related protein phosphatases PP2A and PP2B in the mouse hippocampus. Our results showed that both single-dose and long-term ketamine administration induced excessive phosphorylation of the Tau protein at ser202/thr205 and ser396. A single ketamine administration caused an increase in the activity of GSK-3β (at high doses) and a decrease in the activity of PP2A. On the other hand, long-term ketamine administration resulted in an increase in the activities of GSK-3β (at high doses) and CDK5, and a decrease in the activity of PP2A. Our results indicate that GSK-3β, CDK5, and PP2A may be involved in ketamine-induced Tau protein phosphorylation.

CDKAL1 (rs10946398) and TCF7L2 (rs7903146) gene polymorphisms and their association with risk of type-2 diabetes mellitus in population of Uttarakhand, India

PurposeType 2 diabetes mellitus (T2DM), a non- insulin dependent form of diabetes is predominantly the outcome of multivariate factors ranging from genetic, hereditary to environmental factors. CDKAL1 ((rs10946398)), gene play a permissive role in β-cell function by inhibiting activity of CDK5, the decrease of insulin gene expression. TCF7L2 ((rs7903146)) gene involved in the Wnt signalling pathway, has its direct effects on glucose-induced, which may risk prevalence of T2DM. The present study aims to understand the association of CDKAL1 and TCF7L2 Gene polymorphism in T2DM patients among the population of Uttarakhand, India. Materials and methodsThe present study included 400 T2DM subjects and 400 healthy controls. DNA extraction done from blood serum collected in plain vials and CDKAL1, TCF7L2 gene polymorphism was done by the PCR-RFLP method. ResultsSignificant differences were observed in BMI (kg/m2), FPG (mg/dl), PPG (mg/dl), HbA1c, FPI, systolic BP (mmHg), diastolic BP (mmHg) among the subjects and controls (all p < .0001). Genotypes of CDKAL1 and TCF7L2 genes among T2DM and healthy controls was found to be statistically significantly (p < .0001).Among the T2DM subjects, CDKAL1 genotypes GG, GC and CC were 32.5%, 60% and 7.5% while in healthy control, CDKAL1 genotypes GG, GC and CC were 58%, 39% and 3% respectively. Among the T2DM subjects, TCF7L2 genotype CC, CT and TT were 35%, 63% and TT 2.5% while in healthy control CC, CT, TT were 53%, 46% and 1% respectively. Higher heterozygous GC genotype and mutant CC genotype. Higher C allele frequency (0.37%) noticed in patients as compare to control C allele (0.22%). Odd ratio with 95% CI were calculated for CDKAL1, GC and CC had 2.79 (2.08–3.75) and 4.13 (2.08–8.20) risk taking GG as reference and TCF7L2, CT and TT had 2.09 (1.57–2.78), 3.04 (1.01–9.09) risk taking CC in T2DM subjects. ConclusionStudy concluded that CDKAL1 heterozygous GC, mutant CC were higher in among T2DM and TCF7L2 heterozygous CT, mutant TT were higher among T2DM subjects suggested connected with T2DM. Higher odds and relative risk was found with CDKAL1, TCF7L2 heterozygous GC, CT and mutant CC, TT genotypes respectively.

CDK4 and CDK5 Inhibition Have Comparable Mild Hypothermia Effects in Preventing Drp1-Dependent Mitochondrial Fission and Neuron Death Induced by MPP.

Mild hypothermia has promising effects in the treatment of acute brain insults and also affects cell cycle progression. Mitochondrial dynamics, fusion and fission, are changed along with the cell cycle and disrupted in neurodegenerative diseases, including Parkinson's disease (PD). However, the effects of hypothermia on aberrant mitochondrial dynamics in PD remain unknown. We hypothesized that mild hypothermia protects neurons by regulating cell cycle-dependent protein expression and mitochondrial dynamics in a 1-methyl-4-phenylpyridinium (MPP+)-induced cell model of PD. We found that the hypothermia treatment at 32°C prevented MPP+-induced neuron death; however, 32°C treatment itself also reduced cell viability. This reduction was associated with cell cycle arrest and downregulation of cyclin-dependent kinase 4 (CDK4) in proliferating human SK-N-SH neuroblastoma cells but upregulation in well-differentiated primary rat cortical neurons. In both types of neurons, hypothermia upregulated p27 (an endogenous inhibitor of CDKs) and p35 (CDK5 activator) protein expression. Treatment with hypothermia, or a selective CDK4 inhibitor, or roscovitine (CDK5 inhibitor) prevented MPP+-induced mitochondrial fission, upregulation of mitochondrial fission protein dynamin-related protein 1 (Drp1), and neuron death. In addition, overexpression of dominant negative mutant Drp1K38A improved MPP+-induced mitochondrial fission while overexpression of wild-type Drp1 blunted the prevention of mitochondrial fission by hypothermia as well as CDK4 inhibitor and roscovitine. These results elucidate that hypothermia may inhibit CDK4 and CDK5 activation by upregulating p27 and p35 expression to prevent Drp1-dependent mitochondrial fission and neuron loss after MPP+ treatment. CDK4 and CDK5 inhibition imitates the neuroprotective functions of hypothermia as a potential therapy for PD.

Metformin Ameliorates Synaptic Defects in a Mouse Model of AD by Inhibiting Cdk5 Activity.

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase that is activated by the neuron-specific activators p35/p39 and plays important roles in neuronal development, synaptic plasticity, and cognitive behavior. However, the proteolytic cleavage of p35 to p25 leads to prolonged and aberrant Cdk5 activation and results in synaptic depression, highly mimicking the early pathology of Alzheimer’s disease (AD). Therefore, Cdk5 inhibition is a potential promising strategy for AD drug development. Here in the present study, we showed that metformin, the most widely used drug for type 2 diabetes, suppressed Cdk5 hyper-activation and Cdk5-dependent tau hyper-phosphorylation in the APP/PS1 mouse hippocampus. We also identified the underlying molecular and cellular mechanism that metformin prevented Cdk5 hyper-activation by inhibiting the calpain-dependent cleavage of p35 into p25. Moreover, chronic metformin treatment rescued the core phenotypes in APP/PS1 mice as evidenced by restored spine density, surface GluA1 trafficking, Long-term potentiation (LTP) expression, and spatial memory. Altogether our study discovered an unidentified role of metformin in suppressing Cdk5 hyper-activation and thus preventing AD pathogenesis and suggested that metformin is a potential promising AD therapeutic drug.

Fluorescent Biosensor of CDK5 Kinase Activity in Glioblastoma Cell Extracts and Living Cells

CDK5 plays a major role in neuronal functions, and is hyperactivated in neurodegenerative pathologies as well as in glioblastoma and neuroblastoma. Although this kinase constitutes an established biomarker and pharmacological target, there are few means of probing its activity in cell extracts or in living cells. To this aim a fluorescent peptide reporter of CDK5 kinase activity, derived from a library of CDK5-specific substrates, is engineered and its ability to respond to recombinant CDK5/p25 is established and CDK5 activity in glioblastoma cell extracts is reported on through sensitive changes in fluorescence intensity. A cell-penetrating variant of this biosensor which can be implemented to image CDK5 activation dynamics in space and in time is further implemented. This original biosensor constitutes a potent tool for quantifying differences in CDK5 activity following treatment with selective inhibitors and for monitoring CDK5 activation, following inhibition or stimulation, in a physiologically relevant environment. As such it offers attractive opportunities to develop a diagnostic assay for neuronal pathologies associated with hyperactivated CDK5, as well as a companion assay to evaluate response to new therapies targeting this kinase.

Cdk5 Phosphorylation of STAT3 in Dorsal Root Ganglion Neurons Is Involved in Promoting Axonal Regeneration After Peripheral Nerve Injury.

PurposeThe goal of this study is to investigate the role of cyclin-dependent kinase 5 (Cdk5) in axonal regeneration in dorsal root ganglion (DRG) neurons after peripheral nerve injury.MethodsCrush injury was given on the sciatic nerve in rats. The DRG tissues were prepared 1, 3, and 7 days after injury and used for western blotting and immunofluorescence staining experiments. Primary DRG neurons were prepared and treated with Cdk5 inhibitor roscovitine or used for transfections with plasmid constructs. After immunofluorescence staining, neurite length of DRG neurons was analyzed and compared among experimental groups. In addition, roscovitine was injected into the DRG in vivo, and the sciatic nerve after injury was prepared and used for immunofluorescence staining to analyze axonal regeneration in nerve sections.ResultsLevels of Cdk5 and p25 were increased in DRG neurons after sciatic nerve injury (SNI). Levels of S727-p-STAT3, but not Y705-p-STAT3, were increased in the DRG. Immunofluorescence staining revealed that Cdk5 and STAT3 proteins were mostly colocalized in DRG neurons and Y705-p-STAT3 signals were localized within the nucleus area of DRG neurons. A blockade of Cdk5 activity by roscovitine or by transfection with dominant negative Cdk5 (dn-Cdk5) and nonphosphorylatable forms of STAT3 (S727A or Y705F) resulted in significant reductions of the neurite outgrowth of cultured DRG neurons. In vivo administration of roscovitine into the DRG markedly attenuated distal elongation of regenerating axons in the sciatic nerve after injury.ConclusionsOur study demonstrated that Cdk5 activity induced from DRG neurons after SNI increased phosphorylation of STAT3. The activation of Cdk5-STAT3 pathway may be involved in promoting axonal regeneration in the peripheral nerve after injury.

Dendrobium nobile Lindl alkaloid and metformin ameliorate cognitive dysfunction in senescence-accelerated mice via suppression of endoplasmic reticulum stress

The senescence-accelerated mouse prone 8 (SAMP8) mice have many pathological features of Alzheimer’s disease (AD) with aging. We previously reported that Dendrobium nobile Lindl alkaloid (DNLA) effectively improved cognitive deficits in multiple Alzheimer’s disease (AD) models. This study further used SAMP8 mice to study the anti-aging effects of DNLA, focusing on endoplasmic reticulum (ER) stress. DNLA and metformin were orally administered to SAMP8 mice starting at 4-month of age for 6 months. Behavioral tests were performed in 10-month-old SAMP8 mice and age-matched SAMR1 control mice. At the end of experiment, neuron damage was evaluated by histology and transmission electron microscopy. ER stress-related proteins were analyzed with Western-blot. DNLA improved learning and memory impairments, reduced the loss of neurons and Nissl bodies in the hippocampus and cortex. DNLA ameliorated ER dilation and swelling in the hippocampal neurons. DNLA down-regulated the protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling pathway, decreased calpain 1, GSK-3β and Cdk5 activities and the Tau hyper-phosphorylation. The effects of DNLA were comparable to metformin. In summary, DNLA was effective in improving cognitive deficits in aged SAMP8 mice, possibly via suppression of ER stress-related PERK signaling pathway, sequential inhibition of calpain 1, GSK-3β and Cdk5 activities, and eventually reducing the hyper-phosphorylation of Tau.

Retrospective assessment of cyclin-dependent kinase 5 mRNA and protein expression and its association with patient survival in breast cancer.

Cyclin‐dependent kinase 5 (Cdk5) is an atypical member of the cyclin‐dependent kinase family and functions as a serine/threonine kinase that can be activated by non‐cyclin binding activators p35 or p39. Cdk5 expression and activity has been linked with the development and progression of cancer; however, its expression in breast cancer has not been fully described. Protein expression of Cdk5 was determined in a large cohort of early‐stage invasive breast cancer tumours (n = 1110) with long‐term follow‐up data using immunohistochemistry. Expression of CDK5 mRNA was assessed in the METABRIC cohort (n = 1980). Low nuclear and cytoplasmic expression of Cdk5 expression was significantly associated with shorter breast cancer‐specific survival (P = .004 and P = .001, respectively). Importantly, low nuclear and cytoplasmic expression of Cdk5 remained associated with survival in multivariate analysis, including potentially confounding factors (hazard ratio (HR) = 0.612, 95% confidence interval (CI) = 0.418‐0.896, P = .011 and HR = 0.507, 95% CI = 0.318‐0.809, P = .004, respectively). In addition, low nuclear and cytoplasmic expression of Cdk5 was significantly associated with clinicopathological criteria associated with adverse patient prognosis. Low CDK5 mRNA expression was associated with shorter patient survival (P = .005) in the METABRIC cohort; no associations between copy gain or loss and survival were observed. These data suggest that low Cdk5 expression is associated with poor clinical outcome of breast cancer patients and may be of clinical relevance.

Physiological and Pathological Roles of Cdk5: Potential Directions for Therapeutic Targeting in Neurodegenerative Disease

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine (ser)/threonine (Thr) kinase that has been demonstrated to be one of the most functionally diverse kinases within neurons. Cdk5 is regulated via binding with its neuron-specific regulatory subunits, p35 or p39. Cdk5-p35 activity is critical for a variety of developmental and cellular processes in the brain, including neuron migration, memory formation, microtubule regulation, and cell cycle suppression. Aberrant activation of Cdk5 via the truncated p35 byproduct, p25, is implicated in the pathogenesis of several neurodegenerative diseases. The present review highlights the importance of Cdk5 activity and function in the brain and demonstrates how deregulation of Cdk5 can contribute to the development of neurodegenerative conditions such as Alzheimer's and Parkinson's disease. Additionally, we cover past drug discovery attempts at inhibiting Cdk5-p25 activity and discuss which types of targeting strategies may prove to be the most successful moving forward.