Verification of AKT and CDK5 Gene and RNA Interference Combined with Irradiation to Mediate Fertility Changes in Plutella xylostella (Linnaeus).

Plutella xylostella (Linnaeus) mainly damages cruciferous crops and causes huge economic losses. Presently, chemical pesticides dominate its control, but prolonged use has led to the development of high resistance. In contrast, the sterile insect technique provides a preventive and control method to avoid the development of resistance. We discovered two genes related to the reproduction of Plutella xylostella and investigated the efficacy of combining irradiation with RNA interference for pest management. The results demonstrate that after injecting PxAKT and PxCDK5, there was a significant decrease of 28.06% and 25.64% in egg production, and a decrease of 19.09% and 15.35% in the hatching rate compared to the control. The ratio of eupyrene sperm bundles to apyrene sperm bundles also decreased. PxAKT and PxCDK5 were identified as pivotal genes influencing male reproductive processes. We established a dose-response relationship for irradiation (0-200 Gy and 200-400 Gy) and derived the irradiation dose equivalent to RNA interference targeting PxAKT and PxCDK5. Combining RNA interference with low-dose irradiation achieved a sub-sterile effect on Plutella xylostella, surpassing either irradiation or RNA interference alone. This study enhances our understanding of the genes associated with the reproduction of Plutella xylostella and proposes a novel approach for pest management by combining irradiation and RNA interference.

miR-4645-3p attenuates podocyte injury and mitochondrial dysfunction in diabetic kidney disease by targeting Cdk5.

Podocyte injury plays a critical role in the progression of diabetic kidney disease (DKD), but the underlying cellular and molecular mechanisms remain poorly understanding. MicroRNAs (miRNAs) can disrupt gene expression by inducing translation inhibition and mRNA degradation, and recent evidence has shown that miRNAs may play a key role in many kidney diseases. In this study, we identified miR-4645-3p by global transcriptome expression profiling as one of the major downregulated miRNAs in high glucose-cultured podocytes. Moreover, whether DKD patients or STZ-induced diabetic mice, expression of miR-4645-3p was also significantly decreased in kidney. In the podocytes cultured by normal glucose, inhibition of miR-4645-3p expression promoted mitochondrial damage and podocyte apoptosis. In the podocytes cultured by high glucose (30 mM glucose), overexpression of miR-4645-3p significantly attenuated mitochondrial dysfunction and podocyte apoptosis induced by high glucose. Furthermore, we found that miR-4645-3p exerted protective roles by targeting Cdk5 inhibition. In vitro, miR-4645-3p obviously antagonized podocyte injury by inhibiting overexpression of Cdk5. In vivo of diabetic mice, podocyte injury, proteinuria, and impaired renal function were all effectively ameliorated by treatment with exogenous miR-4645-3p. Collectively, these findings demonstrate that miR-4645-3p can attenuate podocyte injury and mitochondrial dysfunction in DKD by targeting Cdk5. Sustaining the expression of miR-4645-3p in podocytes may be a novel strategy to treat DKD.

Structural determinants for activation of the Tau kinase CDK5 by the serotonin receptor 5-HT7R.

BACKGROUND: Multiple neurodegenerative diseases are induced by the formation and deposition of protein aggregates. In particular, the microtubule-associated protein Tau leads to the development of so-called tauopathies characterized by the aggregation of hyperphosphorylated Tau within neurons. We recently showed that the constitutive activity of the serotonin receptor 7 (5-HT7R) is required for Tau hyperphosphorylation and aggregation through activation of the cyclin-dependent kinase 5 (CDK5). We also demonstrated physical interaction between 5-HT7R and CDK5 at the plasma membrane suggesting that the 5-HT7R/CDK5 complex is an integral part of the signaling network involved in Tau-mediated pathology. METHODS: Using biochemical, microscopic, molecular biological, computational and AI-based approaches, we investigated structural requirements for the formation of 5-HT7R/CDK5 complex. RESULTS: We demonstrated that 5-HT7R domains responsible for coupling to Gs proteins are not involved in receptor interaction with CDK5. We also created a structural model of the 5-HT7R/CDK5 complex and refined the interaction interface. The model predicted two conserved phenylalanine residues, F278 and F281, within the third intracellular loop of 5-HT7R to be potentially important for complex formation. While site-directed mutagenesis of these residues did not influence Gs protein-mediated receptor signaling, replacement of both phenylalanines by alanine residues significantly reduced 5-HT7R/CDK5 interaction and receptor-mediated CDK5 activation, leading to reduced Tau hyperphosphorylation and aggregation. Molecular dynamics simulations of 5-HT7R/CDK5 complex for wild-type and receptor mutants confirmed binding interface stability of the initial model. CONCLUSIONS: Our results provide a structural basis for the development of novel drugs targeting the 5-HT7R/CDK5 interaction interface for the selective treatment of Tau-related disorders, including frontotemporal dementia and Alzheimer's disease.

p35/CDK5 Regulates Bladder Cancer Proliferation and Migration and Promotes Higher Tumor Grade and Poor Survival Rate in Patients With Bladder Cancer.

BACKGROUND/AIM: Bladder cancer remains a significant global health concern, necessitating a deeper understanding of the molecular mechanisms underlying its progression. Cyclin-Dependent Kinase 5 (CDK5) has recently emerged as a potential player in bladder cancer pathogenesis. This study investigated the involvement of CDK5 in bladder cancer, emphasizing its potential as a therapeutic target. MATERIALS AND METHODS: The expression levels of CDK5 and p35 (CDK5 regulatory protein) and their roles in the tumor grade and malignancy of patient samples were evaluated using western blot analysis and immunohistochemistry. In addition, tumor cancer genome atlas (TCGA) was utilized to evaluate survival rate in patients with bladder cancer. We further confirmed the role of CDK5 with in vitro experiments using western blot analysis, immunocytochemistry, cell culture-based proliferation and migration assays. RESULTS: Higher CDK5 and p35 were associated with a higher tumor grade and poor survival rate in patients with bladder cancer. To confirm the role of CDK5 in vitro, we over-expressed CDK5 in bladder cancer cells. The results showed that the over-expression of CDK5 enhanced bladder cancer cell proliferation and migration. In addition, CDK5 inhibition by a pan-CDK inhibitor, Roscovitine (RV), significantly reduced proliferation of bladder cancer cells. Indeed, the migration and adhesion of bladder cancer cells were inhibited by RV treatment. CONCLUSION: CDK5 might play important roles in bladder cancer progression and be a potential diagnostic and therapeutic target in the near future.

Maintaining Drosha expression with Cdk5 inhibitors as a potential therapeutic strategy for early intervention after TBI.

Traumatic brain injury (TBI) is a major cause of death and disability in adults. The pathological process of TBI involves a multifactorial cascade in which kinases have been proven contribute to interactions between relevant factors and amplification of signaling cascades. Cyclin-dependent kinase 5 (Cdk5) is a promising kinase that has been implicated in various brain disorders, including TBI. However, the mechanism by which Cdk5 induces neuronal damage remains unclear. Here, we show for the first time that Drosha, a key enzyme in microRNA biogenesis, is a pivotal substrate of abnormally activated Cdk5. Cdk5-mediated phosphorylation decreases Drosha expression and exacerbates nerve injury in TBI. We proved that maintaining Drosha expression via the administration of repurposed Cdk5 inhibitors that were previously studied in clinical trials is a promising approach for the early treatment of TBI. Together, our work identifies Drosha as a novel target for neuroprotective strategies after TBI and suggests Cdk5-mediated regulation of Drosha expression as a potential therapeutic strategy for early TBI intervention.

TFP5, a Peptide Derived from Cdk5 Activator p35, Protects Pancreatic ß Cells from Glucose Toxicity.

We studied the effect of TFP5 on MIN6 cells (cultured mouse islet ß cells) treated with different concentrations of glucose (5 or 25 mM). The results were verified in C57BL/6J mice (control; n=12) and db/db mice with type 2 diabetes mellitus (n=12). To synthesize TFP5, peptide p5 (a derivative of p35 protein, activator of cyclin-dependent kinase 5, Cdk5) was conjugated with a FITC tag at the N-terminus and an 11-amino acid TAT protein transduction domain at the C-terminus. TFP5 was employed to inhibit Cdk5 activity and then to evaluate its efficiency in treating experimental type 2 diabetes mellitus. TFP5 effectively inhibited the pathological hyperactivity of Cdk5, enhanced insulin secretion, and protected pancreatic ß cells from apoptosis in vitro and in vivo. In addition, TFP5 inhibited inflammation in pancreatic islets by reducing the expression of inflammatory cytokines TGF-ß1, TNFα, and IL-1ß. These novel data indicates that TFP5 is a promising candidate for treatment of type 2 diabetes mellitus.

Mitigating aberrant Cdk5 activation alleviates mitochondrial defects and motor neuron disease symptoms in spinal muscular atrophy.

Spinal muscular atrophy (SMA), the top genetic cause of infant mortality, is characterized by motor neuron degeneration. Mechanisms underlying SMA pathogenesis remain largely unknown. Here, we report that the activity of cyclin-dependent kinase 5 (Cdk5) and the conversion of its activating subunit p35 to the more potent activator p25 are significantly up-regulated in mouse models and human induced pluripotent stem cell (iPSC) models of SMA. The increase of Cdk5 activity occurs before the onset of SMA phenotypes, suggesting that it may be an initiator of the disease. Importantly, aberrant Cdk5 activation causes mitochondrial defects and motor neuron degeneration, as the genetic knockout of p35 in an SMA mouse model rescues mitochondrial transport and fragmentation defects, and alleviates SMA phenotypes including motor neuron hyperexcitability, loss of excitatory synapses, neuromuscular junction denervation, and motor neuron degeneration. Inhibition of the Cdk5 signaling pathway reduces the degeneration of motor neurons derived from SMA mice and human SMA iPSCs. Altogether, our studies reveal a critical role for the aberrant activation of Cdk5 in SMA pathogenesis and suggest a potential target for therapeutic intervention.

CDK5 destabilizes PD-L1 via chaperon-mediated autophagy to control cancer immune surveillance in hepatocellular carcinoma.

BACKGROUND: In the past few years, immunotherapies of hepatocellular carcinoma (HCC) targeting programmed cell death protein 1 (PD-1) and its ligand programmed cell death ligand 1 (PD-L1), have achieved durable clinical benefits. However, only a fraction of HCC patients showed objective clinical response to PD-1/PD-L1 blockade alone. Despite the impact on post-translational modifications of PD-L1 being substantial, its significance in resistance to HCC immunotherapy remains poorly defined. METHODS: Cyclin-dependent kinase 5 (CDK5) expression was knocked down in HCC cells, CDK5 and PD-L1 protein levels were examined by Western blot. Coimmunoprecipitation was conducted to evaluate the interaction between proteins. Preclinical HCC mice model was constructed to evaluate the effect of CDK5 inhibitor alone or in combination with PD-1 antibody. Clinical HCC samples were used to elucidate the clinical relevance of CDK5, PD-L1, and PD-L1 T290 phosphorylation in HCC. RESULTS: We find that CDK5 deficiency upregulates PD-L1 protein expression in HCC cells and decipher a novel molecular mechanism under which PD-L1 is downregulated by CDK5, that is, CDK5 mediated PD-L1 phosphorylation at T290 promotes its binding with chaperon protein heat-shock cognate protein 70 (HSC70) and degradation through chaperon-mediated autophagy. Notably, treatment of CDK5 inhibitor, PNU112455A, effectively upregulates the tumorous PD-L1 level, promotes the response to anti-PD-1 immunotherapy,and prolongs the survival time of mice bearing HCC tumors. What is more, the T290 phosphorylation status of PD-L1 correlates with the prognosis of HCC. CONCLUSIONS: Targeting CDK5 can synergize with PD-1 blockade to suppress HCC growth, which may have clinical benefits. Our study reveals a unique regulation of the degradation of PD-L1 in HCC, and provides an attractive therapeutic target, a potential drug, and a new prognostic marker for the clinical treatment of HCC.

CDK5: an oncogene or an anti-oncogene: location location location.

Recent studies have uncovered various physiological functions of CDK5 in many nonneuronal tissues. Upregulation of CDK5 and/or its activator p35 in neurons promotes healthy neuronal functions, but their overexpression in nonneuronal tissues is causally linked to cancer of many origins. This review focuses on the molecular mechanisms by which CDK5 recruits diverse tissue-specific substrates to elicit distinct phenotypes in sixteen different human cancers. The emerging theme suggests that CDK5's role as an oncogene or anti-oncogene depends upon its subcellular localization. CDK5 mostly acts as an oncogene, but in gastric cancer, it is a tumor suppressor due to its unique nuclear localization. This indicates that CDK5's access to certain nuclear substrates converts it into an anti-oncogenic kinase. While acting as a bonafide oncogene, CDK5 also activates a few cancer-suppressive pathways in some cancers, presumably due to the mislocalization of nuclear substrates in the cytoplasm. Therefore, directing CDK5 to the nucleus or exporting tumor-suppressive nuclear substrates to the cytoplasm may be promising approaches to combat CDK5-induced oncogenicity, analogous to neurotoxicity triggered by nuclear CDK5. Furthermore, while p35 overexpression is oncogenic, hyperactivation of CDK5 by inducing p25 formation results in apoptosis, which could be exploited to selectively kill cancer cells by dialing up CDK5 activity, instead of inhibiting it. CDK5 thus acts as a molecular rheostat, with different activity levels eliciting distinct functional outcomes. Finally, as CDK5's role is defined by its substrates, targeting them individually or in conjunction with CDK5 should create potentially valuable new clinical opportunities.

P25/CDK5-mediated Tau Hyperphosphorylation in Both Ipsilateral and Contralateral Cerebra Contributes to Cognitive Deficits in Post-stroke Mice.

OBJECTIVE: Post-stroke cognitive impairment (PSCI) develops in approximately one-third of stroke survivors and is associated with ingravescence. Nonetheless, the biochemical mechanisms underlying PSCI remain unclear. The study aimed to establish an ischemic mouse model by means of transient unilateral middle cerebral artery occlusions (MCAOs) and to explore the biochemical mechanisms of p25/cyclin-dependent kinase 5 (CDK5)-mediated tau hyperphosphorylation on the PSCI behavior. METHODS: Cognitive behavior was investigated, followed by the detection of tau hyperphosphorylation, mobilization, activation of kinases and/or inhibition of phosphatases in the lateral and contralateral cerebrum of mice following ischemia in MACO mice. Finally, we treated HEK293/tau cells with oxygen-glucose deprivation (OGD) and a CDK5 inhibitor (Roscovitine) or a GSK3ß inhibitor (LiCl) to the roles of CDK5 and GSK3ß in mediating ischemia-reperfusion-induced tau phosphorylation. RESULTS: Ischemia induced cognitive impairments within 2 months, as well as causing tau hyperphosphorylation and its localization to neuronal somata in both ipsilateral and contralateral cerebra. Furthermore, p25 that promotes CDK5 hyperactivation had significantly higher expression in the mice with MCAO than in the shamoperation (control) group, while the expression levels of protein phosphatase 2 (PP2A) and the phosphorylation level at Tyr307 were comparable between the two groups. In addition, the CDK5 inhibitor rescued tau from hyperphosphorylation induced by OGD. CONCLUSION: These findings demonstrate that upregulation of CDK5 mediates tau hyperphosphorylation and localization in both ipsilateral and contralateral cerebra, contributing to the pathogenesis of PSCI.

Regulation of NLGN3 and the Synaptic Rho-GEF Signaling Pathway by CDK5.

Neuroligins (NLGNs) are postsynaptic cell adhesion molecules that are involved in synapse assembly and function. The NLGN gene family consists of 5 genes (NLGN1-3, 4X, and 4Y). NLGN3 forms heterodimers with other NLGNs and is expressed at both excitatory and inhibitory synapses, although the distinct role at different synapses is not fully understood. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase that targets various neuronal substrates to impact neuronal migration, neurite outgrowth, synaptic transmission, and plasticity. Both NLGNs and their presynaptic binding partners neurexins are highly associated with neurodevelopmental disorders. The NLGN3 gene is on the X chromosome and variants in NLGN3 have been linked to the pathophysiology in neurodevelopmental disorders. To better understand the endogenous modulation of NLGN3, we generated an HA-tagged knock-in mouse. We found that Cdk5 associates with NLGN3 in vivo and phosphorylates NLGN3 on serine 725 (S725) in the knock-in mouse of either sex. The phosphorylation affects the NLGN3 association with Kalirin-7, a postsynaptic guanine nucleotide exchange factors for Rho GTPase family proteins. We further observed that the phosphorylation modulates NLGN3 surface expression and NLGN3-mediated synaptic currents in cultured rat neurons. Thus, we characterized NLGN3 as a novel Cdk5 substrate and revealed the functional consequences of NLGN3 S725 phosphorylation in neurons. Our study provides a novel molecular mechanism underlying Cdk5-mediated regulation of postsynaptic cell adhesion molecules.SIGNIFICANCE STATEMENT NLGN3 is involved in synapse assembly and function at both excitatory and inhibitory synapses and has been associated with the pathophysiology of neurodevelopmental disorders. Cdk5 has brain-specific activity and is involved in neuronal transmission, synapse function, and plasticity. Here, we characterize NLGN3 as a Cdk5 substrate for the first time and show that Cdk5-mediated phosphorylation regulates NLGN3 function. We demonstrate that NLGN3 S725 is a Cdk5 phosphorylation site, and reveal that the site is important for NLGN3 association with Kalirin-7, NLGN3 surface expression, and NLGN3-mediated synaptic transmission.

Cdk5 phosphorylation-dependent C9orf72 degradation promotes neuronal death in Parkinson's disease models.

AIMS: Chromosome 9 open reading frame 72 (C9orf72) is one of the most dazzling molecules in neurodegenerative diseases, albeit that its role in Parkinson's disease (PD) remains unknown. This article aimed to explore the potential mechanism of C9orf72 involved in the pathogenesis of PD. METHODS: The expression and phosphorylation levels of C9orf72 were examined by Western blotting, RT-PCR, and immunoprecipitation using PD models. Multiple bioinformatics software was used to predict the potential phosphorylation sites of C9orf72 by Cdk5, followed by verification of whether Cdk5-inhibitor ROSCOVITINE could reverse the degradation of C9orf72 in PD. By constructing the sh-C9orf72-knockdown adenovirus and overexpressing the FLAG-C9orf72 plasmid, the effects of C9orf72 knockdown and overexpression, respectively, were determined. A short peptide termed Myr-C9orf72 was used to verify whether interfering with Cdk5 phosphorylation at the Ser9 site of the C9orf72 protein could alleviate autophagy disorder, neuronal death, and movement disorder in PD models. RESULTS: The expression level of the C9orf72 protein was significantly reduced, albeit the mRNA expression was not changed in the PD models. Moreover, the phosphorylation level was enhanced, and its reduction was mainly degraded by the ubiquitin-proteasome pathway. The key nervous system kinase Cdk5 directly phosphorylated the S9 site of the C9orf72 protein, which promoted the degradation of the C9orf72 protein. The knockdown of C9orf72 aggravated autophagy dysfunction and increased neuronal loss and motor dysfunction in substantia nigra neurons of PD mice. The overexpression of C9orf72 alleviated autophagy dysfunction in PD neurons. Specifically, interference with Cdk5 phosphorylation at the S9 site of C9orf72 alleviated autophagy dysfunction, neuronal death, and motor dysfunction mediated by C9orf72 protein degradation during PD. CONCLUSIONS: Cumulatively, our findings illustrate the importance of the role of C9orf72 in the regulation of neuronal death during PD progression via the Cdk5-dependent degradation.

Use of the dTAG system in vivo to degrade CDK2 and CDK5 in adult mice and explore potential safety liabilities.

The degradation tag (dTAG) system for target protein degradation can remove proteins from biological systems without the drawbacks of some genetic methods, such as slow kinetics, lack of reversibility, low specificity, and the inability to titrate dosage. These drawbacks can make it difficult to compare toxicity resulting from genetic and pharmacological interventions, especially in vivo. Because the dTAG system has not been studied extensively in vivo, we explored the use of this system to study the physiological sequalae resulting from CDK2 or CDK5 degradation in adult mice. Mice with homozygous knock-in of the dTAG sequence onto CDK2 and CDK5 were born at Mendelian ratios despite decreased CDK2 or CDK5 protein levels in comparison with wild-type mice. In bone marrow cells and duodenum organoids derived from these mice, treatment with the dTAG degrader dTAG-13 resulted in rapid and robust protein degradation but caused no appreciable change in viability or the transcriptome. Repeated delivery of dTAG-13 in vivo for toxicity studies proved challenging; we explored multiple formulations in an effort to maximize degradation while minimizing formulation-related toxicity. Degradation of CDK2 or CDK5 in all organs except the brain, where dTAG-13 likely did not cross the blood brain barrier, only caused microscopic changes in the testis of CDK2dTAG mice. These findings were corroborated with conditional CDK2 knockout in adult mice. Our results suggest that the dTAG system can provide robust protein degradation in vivo and that loss of CDK2 or CDK5 in adult mice causes no previously unknown phenotypes.

Favorable Immunotherapy Plus Tyrosine Kinase Inhibition Outcome of Renal Cell Carcinoma Patients with Low CDK5 Expression.

PURPOSE: Immunotherapy (IO) plus tyrosine kinase inhibitor (TKI) has become the first-line treatment for advanced renal cell carcinoma, despite the lack of prognostic biomarkers. Cyclin-dependent kinase 5 (CDK5) affects the tumor microenvironment, which may influence the efficacy of TKI+IO. MATERIALS AND METHODS: Two cohorts from our center (Zhongshan Metastatic Renal Cell Carcinoma [ZS-MRCC] cohort, Zhongshan High-risk Localized Renal Cell Carcinoma [ZS-HRRCC] cohort) and one cohort from a clinical trial (JAVELIN-101) were enrolled. The expression of CDK5 of each sample was determined by RNA sequencing. Immune infiltration and T cell function were evaluated by flow cytometry and immunohistochemistry. Response and progression-free survival (PFS) were set as primary endpoints. RESULTS: Patients of low CDK5 expression showed higher objective response rate (60.0% vs. 23.3%) and longer PFS in both cohorts (ZS-MRCC cohort, p=0.014; JAVELIN-101 cohort, p=0.040). CDK5 expression was enhanced in non-responders (p < 0.05). In the ZS-HRRCC cohort, CDK5 was associated with decreased tumor-infiltrating CD8+ T cells, which was proved by immunohistochemistry (p < 0.05) and flow cytometry (Spearman's ρ=-0.49, p < 0.001). In the high CDK5 subgroup, CD8+ T cells revealed a dysfunction phenotype with decreased granzyme B, and more regulatory T cells were identified. A predictive score was further constructed by random forest, involving CDK5 and T cell exhaustion features. The RFscore was also validated in both cohorts. By utilizing the model, more patients might be distinguished from the overall cohort. Additionally, only in the low RFscore did TKI+IO outperform TKI monotherapy. CONCLUSION: High-CDK5 expression was associated with immunosuppression and TKI+IO resistance. RFscore based on CDK5 may be utilized as a biomarker to determine the optimal treatment strategy.

Comprehensive analysis of CDK5 as a novel biomarker for progression in esophageal cancer.

BACKGROUND: Cyclin-dependent kinase 5 (CDK5) is a member of the cyclin-dependent kinase family, and unlike the rest of the members of the family, its kinase activity is independent of cyclins. Accumulating evidence has shown that CDK5 plays a significant role in the progress of tumorigenesis except in nervous system. In particular, the expression of CDK5 and its function in esophageal cancer (ESCA) remain unknown. METHODS: With TCGA and GEO databases, CDK5 was analyzed with the expression, predicted value, clinical relationship, functional enrichment, immune cell infiltration and immune molecules in ESCA. In addition, we explored the CDK5 expression with local datasets and the influence of CDK5 on proliferation, migration and invasion behaviors of the esophageal squamous cell carcinoma (ESCC) cells in vitro and in vivo experiments. RESULTS: CDK5 expression was upregulated in ESCA, and this regulation has been verified in cell lines of ESCC. Further analysis has found that the expression of CDK5 was correlated with race, weight, BMI, histological type and tumor central location in ESCA. KEGG analysis revealed that CDK5 was involved in the progress of cancers, innate immune system and PI3K-Akt signaling pathway. CDK5 was closely related to immune cells and immune molecules in ESCA. Functional experiments confirmed CDK5 was an oncogene in ESCC by in vivo and in vitro models. CONCLUSIONS: This study shows that CDK5 is a risk factor to promote tumor progression, and Roscovitine could be one of the effective tools in the therapy of ESCA.

MicroRNA-650 Regulates the Pathogenesis of Alzheimer's Disease Through Targeting Cyclin-Dependent Kinase 5.

Alzheimer's disease (AD) pathogenesis feature progressive neurodegeneration, amyloid-ß plaque formation, and neurofibrillary tangles. Ample evidence has indicated the involvement of epigenetic pathways in AD pathogenesis. Here, we show that the expression of microRNA 650 (miR-650) is altered in brains from AD patients. Furthermore, we found that the processing of primary miR-650 to mature miR-650 is misregulated. Bioinformatic analysis predicted that miR-650 targets the expression of three AD-associated components: Apolipoprotein E (APOE), Presenilin 1 (PSEN1), and Cyclin-Dependent Kinase 5 (CDK5), and we have experimentally confirmed that miR-650 is able to significantly reduce the expression of APOE, PSEN1, and CDK5 in vitro. Importantly, the overexpression of miR-650 was further shown to significantly alter the CDK5 level and ameliorate AD pathologies in APP-PSEN1 transgenic mice. Overall, our results indicate that miR-650 influences AD pathogenesis through regulation of CDK5.

Cyclin G1 induces maladaptive proximal tubule cell dedifferentiation and renal fibrosis through CDK5 activation.

Acute kidney injury (AKI) occurs in approximately 13% of hospitalized patients and predisposes patients to chronic kidney disease (CKD) through the AKI-to-CKD transition. Studies from our laboratory and others have demonstrated that maladaptive repair of proximal tubule cells (PTCs), including induction of dedifferentiation, G2/M cell cycle arrest, senescence, and profibrotic cytokine secretion, is a key process promoting AKI-to-CKD transition, kidney fibrosis, and CKD progression. The molecular mechanisms governing maladaptive repair and the relative contribution of dedifferentiation, G2/M arrest, and senescence to CKD remain to be resolved. We identified cyclin G1 (CG1) as a factor upregulated in chronically injured and maladaptively repaired PTCs. We demonstrated that global deletion of CG1 inhibits G2/M arrest and fibrosis. Pharmacological induction of G2/M arrest in CG1-knockout mice, however, did not fully reverse the antifibrotic phenotype. Knockout of CG1 did not alter dedifferentiation and proliferation in the adaptive repair response following AKI. Instead, CG1 specifically promoted the prolonged dedifferentiation of kidney tubule epithelial cells observed in CKD. Mechanistically, CG1 promotes dedifferentiation through activation of cyclin-dependent kinase 5 (CDK5). Deletion of CDK5 in kidney tubule cells did not prevent G2/M arrest but did inhibit dedifferentiation and fibrosis. Thus, CG1 and CDK5 represent a unique pathway that regulates maladaptive, but not adaptive, dedifferentiation, suggesting they could be therapeutic targets for CKD.

Cyclin-Dependent Kinase 5 Regulates cPLA2 Activity and Neuroinflammation in Parkinson's Disease.

Hyperactivation of cyclin-dependent kinase 5 (Cdk5) by p25, contributes to neuroinflammation causing neurodegeneration in Parkinson's disease (PD) and Alzheimer's disease. However, the mechanism by which Cdk5 induces neuroinflammation in the PD brain is largely unexplored. Here, we show that Cdk5 phosphorylates cytosolic phospholipase A2 (cPLA2) at Thr-268 and Ser-505 sites lead to its activation and generation of eicosanoid products. Mutational studies using site-directed mutagenesis and molecular simulations show that the architecture of the protein changes on each single-point mutation. Interestingly, double mutations also led to a severe decline in the activity of cPLA2 and to the disruption of its translocation to the plasma membrane. Further, the brain lysates of transgenic PD mouse models show hyperactivation of Cdk5, resulting in enhanced phosphorylation of Thr-268 and Ser-505 of cPLA2 and its heightened activity, confirming the findings observed in the cell culture model of PD. These phosphorylation sites of cPLA2 and Cdk5 could be explored as the future therapeutic targets against neuroinflammation in PD. Further, conjoint transcriptomic analysis of the publicly available human PD datasets strengthens the hypothesis that genes of the arachidonic acid, prostaglandin synthesis, and inflammatory pathways are significantly upregulated in the case of PD patients compared with that of healthy control subjects.

Cdk5 regulates IP3R1-mediated Ca2+ dynamics and Ca2+-mediated cell proliferation.

Loss of cyclin-dependent kinase 5 (Cdk5) in the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) increases ER-mitochondria tethering and ER Ca2+ transfer to the mitochondria, subsequently increasing mitochondrial Ca2+ concentration ([Ca2+]mt). This suggests a role for Cdk5 in regulating intracellular Ca2+ dynamics, but how Cdk5 is involved in this process remains to be explored. Using ex vivo primary mouse embryonic fibroblasts (MEFs) isolated from Cdk5-/- mouse embryos, we show here that loss of Cdk5 causes an increase in cytosolic Ca2+concentration ([Ca2+]cyt), which is not due to reduced internal Ca2+ store capacity or increased Ca2+ influx from the extracellular milieu. Instead, by stimulation with ATP that mediates release of Ca2+ from internal stores, we determined that the rise in [Ca2+]cyt in Cdk5-/- MEFs is due to increased inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release from internal stores. Cdk5 interacts with the IP3R1 Ca2+ channel and phosphorylates it at Ser421. Such phosphorylation controls IP3R1-mediated Ca2+ release as loss of Cdk5, and thus, loss of IP3R1 Ser421 phosphorylation triggers an increase in IP3R1-mediated Ca2+ release in Cdk5-/- MEFs, resulting in elevated [Ca2+]cyt. Elevated [Ca2+]cyt in these cells further induces the production of reactive oxygen species (ROS), which upregulates the levels of Nrf2 and its targets, Prx1 and Prx2. Cdk5-/- MEFs, which have elevated [Ca2+]cyt, proliferate at a faster rate compared to wt, and Cdk5-/- embryos have increased body weight and size compared to their wt littermates. Taken together, we show that altered IP3R1-mediated Ca2+ dynamics due to Cdk5 loss correspond to accelerated cell proliferation that correlates with increased body weight and size in Cdk5-/- embryos.

GSNOR deficiency attenuates MPTP-induced neurotoxicity and autophagy by facilitating CDK5 S-nitrosation in a mouse model of Parkinson's disease.

The S-nitrosoglutathione reductase (GSNOR) is a key denitrosating enzyme that regulates protein S-nitrosation, a process which has been found to be involved in the pathogenesis of Parkinson's disease (PD). However, the physiological function of GSNOR in PD remains unknown. In a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model, we found that GSNOR expression was significantly increased and accompanied by autophagy mediated by MPTP-induced cyclin dependent kinase 5 (CDK5), behavioral dyskinesias and dopaminergic neuron loss. Whereas, knockout of GSNOR, or treatment with the GSNOR inhibitor N6022, alleviated MPTP-induced PD-like pathology and neurotoxicity. Mechanistically, deficiency of GSNOR inhibited MPTP-induced CDK5 kinase activity and CDK5-mediated autophagy by increasing S-nitrosation of CDK5 at Cys83. Our study indicated that GSNOR is a key regulator of CDK5 S-nitrosation and is actively involved in CDK5-mediated autophagy induced by MPTP.