Calpain-Mediated Alterations in Astrocytes Before and During Amyloid Chaos in Alzheimer's Disease.

One of the changes found in the brain in Alzheimer's disease (AD) is increased calpain, derived from calcium dysregulation, oxidative stress, and/or neuroinflammation, which are all assumed to be basic pillars in neurodegenerative diseases. The role of calpain in synaptic plasticity, neuronal death, and AD has been discussed in some reviews. However, astrocytic calpain changes sometimes appear to be secondary and consequent to neuronal damage in AD. Herein, we explore the possibility of calpain-mediated astroglial reactivity in AD, both preceding and during the amyloid phase. We discuss the types of brain calpains but focus the review on calpains 1 and 2 and some important targets in astrocytes. We address the signaling involved in controlling calpain expression, mainly involving p38/mitogen-activated protein kinase and calcineurin, as well as how calpain regulates the expression of proteins involved in astroglial reactivity through calcineurin and cyclin-dependent kinase 5. Throughout the text, we have tried to provide evidence of the connection between the alterations caused by calpain and the metabolic changes associated with AD. In addition, we discuss the possibility that calpain mediates amyloid-ß clearance in astrocytes, as opposed to amyloid-ß accumulation in neurons.

Lack of Cdk5 activity is involved on Dopamine Transporter expression and function: Evidences from an animal model of Attention-Deficit Hyperactivity Disorder.

Attention deficit/Hyperactivity disorder (ADHD) is one of the most diagnosed psychiatric disorders nowadays. The core symptoms of the condition include hyperactivity, impulsiveness and inattention. The main pharmacological treatment consists of psychostimulant drugs affecting Dopamine Transporter (DAT) function. We have previously shown that genetically modified mice lacking p35 protein (p35KO), which have reduced Cdk5 activity, present key hallmarks resembling those described in animal models useful for studying ADHD. The p35KO mouse displays spontaneous hyperactivity and shows a calming effect of methylphenidate or amphetamine treatment. Interestingly, dopaminergic neurotransmission is altered in these mice as they have an increased Dopamine (DA) content together with a low DA turnover. This led us to hypothesize that the lack of Cdk5 activity affects DAT expression and/or function in this animal model. In this study, we performed biochemical assays, cell-based approaches, quantitative fluorescence analysis and functional studies that allowed us to demonstrate that p35KO mice exhibit decreased DA uptake and reduced cell surface DAT expression levels in the striatum (STR). These findings are supported by in vitro observations in which the inhibition of Cdk5 activity in N2a cells induced a significant increase in constitutive DAT endocytosis with a concomitant increase in DAT localization to recycling endosomes. Taken together, these data provide evidences regarding the role of Cdk5/p35 in DAT expression and function, thus contributing to the knowledge of DA neurotransmission physiology and also providing therapeutic options for the treatment of DA pathologies such as ADHD.

L5-6 Spinal Nerve Ligation-induced Neuropathy Changes the Location and Function of Ca2+ Channels and Cdk5 and Affects the Compound Action Potential in Adjacent Intact L4 Afferent Fibers.

Voltage-gated Ca2+ (CaV) channels regulate multiple cell processes, including neurotransmitter release, and have been associated with several pathological conditions, such as neuropathic pain. Cdk5, a neuron-specific kinase, may phosphorylate CaV channels, altering their functional expression. During peripheral nerve injury, upregulation of CaV channels and Cdk5 in the dorsal root ganglia (DRG) and the spinal cord, has been correlated with allodynia. We recently reported an increase in the amplitude of the C component of the compound action potential (cAP) of afferent fibers in animals with allodynia induced by L5-6 spinal nerve ligation (SNL), recorded in the corresponding dorsal roots. This was related to an increase in T-type (CaV3.2) channels generated by Cdk5-mediated phosphorylation. Here, we show that CaV channel functional expression is also altered in the L4 adjacent intact afferent fibers in rats with allodynia induced by L5-6 SNL. Western blot analysis showed that both Cdk5 and CaV3.2 total levels are not increased in the DRG L3-4, but their subcellular distribution changes by concentrating on the neuronal soma. Likewise, the Cdk5 inhibitor olomoucine affected the rapid and the slow C components of the cAP recorded in the dorsal roots. Patch-clamp recordings revealed an increase in T- and N-type currents recorded in the soma of acute isolated L3-4 sensory neurons after L5-6 SNL, which was prevented by olomoucine. These findings suggest changes in CaV channels location and function in L3-4 afferent fibers associated with Cdk5-mediated phosphorylation after L5-6 SNL, which may contribute to nerve injury-induced allodynia.

CDK5 Targeting as a Therapy for Recovering Neurovascular Unit Integrity in Alzheimer's Disease.

The neurovascular unit (NVU) is responsible for synchronizing the energetic demand, vasodynamic changes, and neurochemical and electrical function of the brain through a closed and interdependent interaction of cell components conforming to brain tissue. In this review, we will focus on cyclin-dependent kinase 5 (CDK5) as a molecular pivot, which plays a crucial role in the healthy function of neurons, astrocytes, and the endothelium and is implicated in the cross-talk of cellular adhesion signaling, ion transmission, and cytoskeletal remodeling, thus allowing the individual and interconnected homeostasis of cerebral parenchyma. Then, we discuss how CDK5 overactivation affects the integrity of the NVU in Alzheimer's disease (AD) and cognitive impairment; we emphasize how CDK5 is involved in the excitotoxicity spreading of glutamate and Ca2+ imbalance under acute and chronic injury. Additionally, we present pharmacological and gene therapy strategies for producing partial depletion of CDK5 activity on neurons, astrocytes, or endothelium to recover neuroplasticity and neurotransmission, suggesting that the NVU should be the targeted tissue unit in protective strategies. Finally, we conclude that CDK5 could be effective due to its intervention on astrocytes by its end feet on the endothelium and neurons, acting as an intermediary cell between systemic and central communication in the brain. This review provides integrated guidance regarding the pathogenesis of and potential repair strategies for AD.

PPARγ S273 Phosphorylation Modifies the Dynamics of Coregulator Proteins Recruitment.

The nuclear receptor PPARγ is essential to maintain whole-body glucose homeostasis and insulin sensitivity, acting as a master regulator of adipogenesis, lipid, and glucose metabolism. Its activation through natural or synthetic ligands induces the recruitment of coactivators, leading to transcription of target genes such as cytokines and hormones. More recently, post translational modifications, such as PPARγ phosphorylation at Ser273 by CDK5 in adipose tissue, have been linked to insulin resistance trough the dysregulation of expression of a specific subset of genes. Here, we investigate how this phosphorylation may disturb the interaction between PPARγ and some coregulator proteins as a new mechanism that may leads to insulin resistance. Through cellular and in vitro assays, we show that PPARγ phosphorylation inhibition increased the activation of the receptor, therefore the increased recruitment of PGC1-α and TIF2 coactivators, whilst decreases the interaction with SMRT and NCoR corepressors. Moreover, our results show a shift in the coregulators interaction domains preferences, suggesting additional interaction interfaces formed between the phosphorylated PPARγ and some coregulator proteins. Also, we observed that the CDK5 presence disturb the PPARγ-coregulator's synergy, decreasing interaction with PGC1-α, TIF2, and NCoR, but increasing coupling of SMRT. Finally, we conclude that the insulin resistance provoked by PPARγ phosphorylation is linked to a differential coregulators recruitment, which may promote dysregulation in gene expression.

Cdk5 phosphorylates CaV1.3 channels and regulates GABAA-mediated miniature inhibitory post-synaptic currents in striato-nigral terminals.

Neurotransmission is one of the most important processes in neuronal communication and depends largely on Ca2+ entering synaptic terminals through voltage-gated Ca2+ (CaV) channels. Although the contribution of L-type CaV channels in neurotransmission has not been unambiguously established, increasing evidence suggests a role for these proteins in noradrenaline, dopamine, and GABA release. Here we report the regulation of L-type channels by Cdk5, and its possible effect on GABA release in the substantia nigra pars reticulata (SNpr). Using patch-clamp electrophysiology, we show that Cdk5 inhibition by Olomoucine significantly increases current density through CaV1.3 (L-type) channels heterologously expressed in HEK293 cells. Likewise, in vitro phosphorylation showed that Cdk5 phosphorylates residue S1947 in the C-terminal region of the pore-forming subunit of CaV1.3 channels. Consistent with this, the mutation of serine into alanine (S1947A) prevented the regulation of Cdk5 on CaV1.3 channel activity. Our data also revealed that the inhibition of Cdk5 increased the frequency of high K+-evoked miniature inhibitory postsynaptic currents in rat SNpr neurons, acting on L-type channels. These results unveil a novel regulatory mechanism of GABA release in the SNpr that involves a direct action of Cdk5 on L-type channels.

Cdk5-Dependent Phosphorylation of CaV3.2 T-Type Channels: Possible Role in Nerve Ligation-Induced Neuropathic Allodynia and the Compound Action Potential in Primary Afferent C Fibers.

Voltage-gated T-type Ca2+ (CaV3) channels regulate diverse physiological events, including neuronal excitability, and have been linked to several pathological conditions such as absence epilepsy, cardiovascular diseases, and neuropathic pain. It is also acknowledged that calcium/calmodulin-dependent protein kinase II and protein kinases A and C regulate the activity of T-type channels. Interestingly, peripheral nerve injury induces tactile allodynia and upregulates CaV3.2 channels and cyclin-dependent kinase 5 (Cdk5) in dorsal root ganglia (DRG) and spinal dorsal horn. Here, we report that recombinant CaV3.2 channels expressed in HEK293 cells are regulatory targets of Cdk5. Site-directed mutagenesis showed that the relevant sites for this regulation are residues S561 and S1987. We also found that Cdk5 may regulate CaV3.2 channel functional expression in rats with mechanical allodynia induced by spinal nerve ligation (SNL). Consequently, the Cdk5 inhibitor olomoucine affected the compound action potential recorded in the spinal nerves, as well as the paw withdrawal threshold. Likewise, Cdk5 expression was upregulated after SNL in the DRG. These findings unveil a novel mechanism for how phosphorylation may regulate CaV3.2 channels and suggest that increased channel activity by Cdk5-mediated phosphorylation after SNL contributes nerve injury-induced tactile allodynia.SIGNIFICANCE STATEMENT Neuropathic pain is a current public health challenge. It can develop as a result of injury or nerve illness. It is acknowledged that the expression of various ion channels can be altered in neuropathic pain, including T-type Ca2+ channels that are expressed in sensory neurons, where they play a role in the regulation of cellular excitability. The present work shows that the exacerbated expression of Cdk5 in a preclinical model of neuropathic pain increases the functional expression of CaV3.2 channels. This finding is relevant for the understanding of the molecular pathophysiology of the disease. Additionally, this work may have a substantial translational impact, since it describes a novel molecular pathway that could represent an interesting therapeutic alternative for neuropathic pain.

Rapid Estrogen and Progesterone Signaling to Dendritic Spine Formation via Cortactin/Wave1-Arp2/3 Complex.

BACKGROUND: Synaptic plasticity is the neuronal capacity to modify the function and structure of dendritic spines (DS) in response to neuromodulators. Sex steroids, particularly 17ß-estradiol (E2) and progesterone (P4), are key regulators in the control of DS formation through multiprotein complexes including WAVE1 protein, and are thus fundamental for the development of learning and memory. OBJECTIVES: The aim of this work was to evaluate the molecular switch Cdk5 kinase/protein phosphatase 2A (PP2A) in the control of WAVE1 protein (phosphorylation/dephosphorylation) and the regulation of WAVE1 and cortactin to the Arp2/3 complex, in response to rapid treatments with E2 and P4 in cortical neuronal cells. RESULTS: Rapid treatment with E2 and P4 modified neuronal morphology and significantly increased the number of DS. This effect was reduced by the use of a Cdk5 inhibitor (Roscovitine). In contrast, inhibition of PP2A with PP2A dominant negative construct significantly increased DS formation, evidencing the participation of kinase/phosphatase in the regulation of WAVE1 in DS formation induced by E2 and P4. Cortactin regulates DS formation via Src and PAK1 kinase induced by E2 and P4. Both cortactin and WAVE1 signal to Arp2/3 complex to synergistically promote actin nucleation. CONCLUSION: These results suggest that E2 and P4 dynamically regulate neuron morphology through nongenomic signaling via cortactin/WAVE1-Arp2/3 complex. The control of these proteins is tightly orchestrated by phosphorylation, where kinases and phosphatases are essential for actin nucleation and, finally, DS formation. This work provides a deeper understanding of the biological actions of sex steroids in the regulation of DS turnover and neuronal plasticity processes.

Hypothermia treatment ameliorated cyclin-dependent kinase 5-mediated inflammation in ischemic stroke and improved outcomes in ischemic stroke patients.

OBJECTIVES: The inflammatory response is a key mechanism of neuronal damage and loss during acute ischemic stroke. Hypothermia has shown promise as a treatment for ischemic stroke. In this study, we investigated the molecular signaling pathways in ischemic stroke after hypothermia treatment. METHODS: Cyclin-dependent kinase 5 (CDK5) was overexpressed or silenced in cultured cells. Nuclear transcription factor-κB (NF-κB) activity was assessed by measurement of the luciferase reporter gene. An ischemic stroke model was established in Sprague-Dawley (SD) rats using the suture-occluded method. Animals were assigned to three groups: sham operation control, ischemic stroke, and ischemic stroke + hypothermia treatment groups. Interleukin 1ß (IL-1ß) levels in the culture supernatant and blood samples were assessed by ELISA. Protein expression was measured by Western blotting. RESULTS: In HEK293 cells and primary cortical neuronal cultures exposed to hypothermia, CDK5 overexpression was associated with increased IL-1ß, caspase 1, and NF-κB levels. In both a murine model of stroke and in patients, increased IL-1ß levels were observed after stroke, and hypothermia treatment was associated with lower IL-1ß levels. Furthermore, hypothermia-treated patients showed significant improvement in neurophysiological functional outcome. CONCLUSIONS: Overall, hypothermia offers clinical benefit, most likely through its effects on the inflammatory response.

Exploring the mechanism of PPARγ phosphorylation mediated by CDK5.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor with a key role in metabolic processes and is target of CDK5 kinase phosphorylation at S245 (S273 in PPARγ isoform 2), thereby inducing insulin resistance. A remarkable effort has been addressed to find PPARγ ligands that inhibit S245 phosphorylation, but the poor understanding in this field challenges the design of such ligands. Here, through computational and biophysical methods, we explored an experimentally validated model of PPARγ-CDK5 complex, and we presented K261, K263 or K265, which are conserved in mammals, as important anchor residues for this interaction. In addition, we observed, from structural data analysis, that PPARγ ligands that inhibit S245 phosphorylation are not in direct contact with these residues; but induce structural modifications in PPARγ:CDK5/p25 interface. In summary, our PPARγ and CDK5/p25 interaction analyses open new possibilities for the rational design of novel inhibitors that impair S245 phosphorylation.

CDK5: A Unique CDK and Its Multiple Roles in the Nervous System.

The cyclin-dependent kinase 5 (CDK5) is known as an exceptional component of the CDK family, due to its characteristic regulatory pathways and its atypical roles in comparison to the classical cyclins. Despite its functional uniqueness, CDK5 shares a great part of its structural similarity with other members of the cyclin-dependent kinase family. After its discovery 26 years ago, a progressive set of cellular functions has been associated with this protein kinase, ranging from neuronal migration, axonal guidance, and synaptic plasticity in diverse stages of brain development, including specific and complex cognitive functions. More than 30 substrates for CDK5 have been found in different cellular pathways. Together with its essential physiological roles, a major discovery was the finding twenty years ago that CDK5 participates in neurodegenerative diseases responsible for tau hyperphosphorylations, and, as a consequence, it becomes a neurotoxic factor. This review focuses on the wide roles of CDK5 in the central nervous system, its implications in neurodegeneration, and provides an integrative insight of its involvement in pain modulation, Alzheimer's disease, and other contexts.

Hypothermia treatment ameliorated cyclin-dependent kinase 5-mediated inflammation in ischemic stroke and improved outcomes in ischemic stroke patients

OBJECTIVES: The inflammatory response is a key mechanism of neuronal damage and loss during acute ischemic stroke. Hypothermia has shown promise as a treatment for ischemic stroke. In this study, we investigated the molecular signaling pathways in ischemic stroke after hypothermia treatment. METHODS: Cyclin-dependent kinase 5 (CDK5) was overexpressed or silenced in cultured cells. Nuclear transcription factor-κB (NF-κB) activity was assessed by measurement of the luciferase reporter gene. An ischemic stroke model was established in Sprague-Dawley (SD) rats using the suture-occluded method. Animals were assigned to three groups: sham operation control, ischemic stroke, and ischemic stroke + hypothermia treatment groups. Interleukin 1β (IL-1β) levels in the culture supernatant and blood samples were assessed by ELISA. Protein expression was measured by Western blotting. RESULTS: In HEK293 cells and primary cortical neuronal cultures exposed to hypothermia, CDK5 overexpression was associated with increased IL-1β, caspase 1, and NF-κB levels. In both a murine model of stroke and in patients, increased IL-1β levels were observed after stroke, and hypothermia treatment was associated with lower IL-1β levels. Furthermore, hypothermia-treated patients showed significant improvement in neurophysiological functional outcome. CONCLUSIONS: Overall, hypothermia offers clinical benefit, most likely through its effects on the inflammatory response.

[Neurogenesis and gliogenesis modulation in cerebral ischemia by CDK5 RNAi-based therapy].

INTRODUCTION: Cerebral ischemia is the third cause of death risk in Colombia and the first cause of physical disability worldwide. Different studies on the silencing of the cyclin-dependent kinase 5 (CDK5) have shown that reducing its activity is beneficial in ischemic contexts. However, its effect on neural cell production after cerebral ischemia has not been well studied yet. OBJECTIVE: To evaluate CDK5 silencing on the production of neurons and astrocytes after a focal cerebral ischemia in rats. MATERIALS AND METHODS: We used 40 eight-week-old male Wistar rats. Both sham and ischemia groups were transduced at CA1 hippocampal region with an adeno-associated viral vector using a noninterfering (shSCRmiR) and an interfering sequence for CDK5 (shCDK5miR). We injected 50 mg/kg of bromodeoxyuridine intraperitoneally from hour 24 to day 7 post-ischemia. We assessed the neurological abilities during the next 15 days and we measured the immunoreactivity of bromodeoxyuridine (BrdU), doublecortin (DCX), NeuN, and glial fibrillary acid protein (GFAP) from day 15 to day 30 post-ischemia. RESULTS: Our findings showed that CDK5miR-treated ischemic animals improved their neurological score and presented increased BrdU+ cells 15 days after ischemia, which correlated with higher DCX and lower GFAP fluorescence intensities, and, although mature neurons populations did not change, GFAP immunoreactivity was still significantly reduced at 30 days post-ischemia in comparison with untreated ischemic groups. CONCLUSION: CDK5miR therapy generated the neurological recovery of ischemic rats associated with the induction of immature neurons proliferation and the reduction of GFAP reactivity at short and longterm post-ischemia.

Neurogenesis and gliogenesis modulation in cerebral ischemia by CDK5 RNAi-based therapy / Modulación de la neurogénesis y la gliogénesis en la isquemia cerebral mediante una terapia basada en la interferencia de CDK5

Abstract Introduction: Cerebral ischemia is the third cause of death risk in Colombia and the first cause of physical disability worldwide. Different studies on the silencing of the cyclin-dependent kinase 5 (CDK5) have shown that reducing its activity is beneficial in ischemic contexts. However, its effect on neural cell production after cerebral ischemia has not been well studied yet. Objective: To evaluate CDK5 silencing on the production of neurons and astrocytes after a focal cerebral ischemia in rats. Materials and methods: We used 40 eight-week-old male Wistar rats. Both sham and ischemia groups were transduced at CA1 hippocampal region with an adeno-associated viral vector using a noninterfering (shSCRmiR) and an interfering sequence for CDK5 (shCDK5miR). We injected 50 mg/kg of bromodeoxyuridine intraperitoneally from hour 24 to day 7 post-ischemia. We assessed the neurological abilities during the next 15 days and we measured the immunoreactivity of bromodeoxyuridine (BrdU), doublecortin (DCX), NeuN, and glial fibrillary acid protein (GFAP) from day 15 to day 30 post-ischemia. Results: Our findings showed that CDK5miR-treated ischemic animals improved their neurological score and presented increased BrdU+ cells 15 days after ischemia, which correlated with higher DCX and lower GFAP fluorescence intensities, and, although mature neurons populations did not change, GFAP immunoreactivity was still significantly reduced at 30 days post-ischemia in comparison with untreated ischemic groups. Conclusion: CDK5miR therapy generated the neurological recovery of ischemic rats associated with the induction of immature neurons proliferation and the reduction of GFAP reactivity at short and longterm post-ischemia.

Resumen Introducción. La isquemia cerebral es la tercera causa de riesgo de muerte en Colombia y la primera causa de discapacidad física en el mundo. En diversos estudios en los que se silenció la cinasa 5 dependiente de la ciclina (CDK5) se ha demostrado que la reducción de su actividad es beneficiosa frente a la isquemia. Sin embargo, su efecto sobre la neurogénesis después de la isquemia no se ha dilucidado suficientemente. Objetivo. Evaluar el silenciamiento de la CDK5 en la neurogénesis y la gliogénesis después de la isquemia cerebral focal en ratas. Materiales y métodos. Se usaron 40 machos de rata Wistar de ocho semanas de edad. Los grupos de control y los isquémicos sometidos a transducción en la región del hipocampo CA1, se inyectaron intraperitonealmente por estereotaxia con 50 mg/kg de bromodesoxiuridina (BrdU) a partir de las 24 horas y hasta el día 7 después de la isquemia, con un vector viral asociado a adenovirus usando una secuencia no interferente (SCRmiR) y una interferente (CDK5miR). Se evaluó la capacidad neurológica durante los quince días siguientes y se detectó la capacidad de inmunorreacción para la BrdU, la proteína doblecortina (DCX), los núcleos neuronales (NeuN), y la proteína fibrilar acídica de la glía (Glial Fibrillary Acidic Protein, GFAP) a los 15 y 30 días de la isquemia. Resultados. Los animales isquémicos tratados con CDK5miR mejoraron su puntuación neurológica y presentaron un incremento de la BrdU+ a los 15 días de la isquemia, lo cual se correlacionó con una mayor intensidad de la DCX+ y una menor de la GFAP+. No hubo modificación de los NeuN+, pero sí una reducción significativa de la GFAP+ a los 30 días de la isquemia en los animales tratados comparados con los animales isquémicos no tratados. Conclusión. La terapia con CDK5miR generó la recuperación neurológica de ratas isquémicas asociada con la inducción de la neurogénesis y el control de la capacidad de reacción de la proteína GFAP a corto y largo plazo después de la isquemia.

Recovery of Neurovascular Unit Integrity by CDK5-KD Astrocyte Transplantation in a Global Cerebral Ischemia Model.

Astrocytes play metabolic and structural support roles and contribute to the integrity of the blood-brain barrier (BBB), linking communication between neurons and the endothelium. Cyclin-dependent kinase 5 (CDK5) likely exerts a dual effect on the endothelium and astrocytes due to its involvement in migration and angiogenesis; the overactivation of CDK5 is associated with dysfunction in glutamate recapture and hypoxia. Recently, we proposed that CDK5-targeted astrocytes facilitate the recovery of neurological and motor function in transplanted ischemic rats. In the current study, we treated cerebral ischemic rats and endothelial cells exposed to glutamate toxicity with CDK5 knock-down (CDK5-KD) astrocytes to determine the role of CDK5 in neurovascular integrity. We found that the effects of CDK5-KD were sustained for 4 months, preventing neuronal and astrocyte loss, facilitating the recovery of the BBB via the production of BDNF by endogenous astrocytes (GFP-) surrounding vessels in the motor cortex and the corpus callosum of global ischemic rats, and improving neurological performance. These findings were supported by the in vitro findings of increased transendothelial resistance, p120-ctn+ adhesion and reduced intercellular gaps induced by a CDK5 inhibitor (roscovitine) in bEnd.3 cells in a glutamate-toxicity model. Additionally, CDK5-KD astrocytes in co-culture protected the endothelial cell viability, increased BDNF release from astrocytes, increased BDNF immunoreactivity in neighboring astrocytes and endothelial cells and enhanced cell adhesion in a glutamate-toxicity model. Altogether, these findings suggest that a CDK5 reduction in astrocytes protects the endothelium, which promotes BDNF release, endothelial adhesion, and the recovery of neurovascular unit integrity and brain function in ischemic rats.

Cyclin-dependent kinase 5 modulates the P2X2a receptor channel gating through phosphorylation of C-terminal threonine 372.

The purinergic P2X2 receptor (P2X2R) is an adenosine triphosphate-gated ion channel widely expressed in the nervous system. Here, we identified a putative cyclin-dependent kinase 5 (Cdk5) phosphorylation site in the full-size variant P2X2aR (TPKH), which is absent in the splice variant P2X2bR. We therefore investigated the effects of Cdk5 and its neuronal activator, p35, on P2X2aR function. We found an interaction between P2X2aR and Cdk5/p35 by co-immunofluorescence and co-immunoprecipitation in HEK293 cells. We also found that threonine phosphorylation was significantly increased in HEK293 cells co-expressing P2X2aR and p35 as compared to cells expressing only P2X2aR. Moreover, P2X2aR-derived peptides encompassing the Cdk5 consensus motif were phosphorylated by Cdk5/p35. Whole-cell patch-clamp recordings indicated a delay in development of use-dependent desensitization (UDD) of P2X2aR but not of P2X2bR in HEK293 cells co-expressing P2X2aR and p35. In Xenopus oocytes, P2X2aRs showed a slower UDD than in HEK293 cells and Cdk5 activation prevented this effect. A similar effect was found in P2X2a/3R heteromeric currents in HEK293 cells. The P2X2aR-T372A mutant was resistant to UDD. In endogenous cells, we observed similar distribution between P2X2R and Cdk5/p35 by co-localization using immunofluorescence in primary culture of nociceptive neurons. Moreover, co-immunoprecipitation experiments showed an interaction between Cdk5 and P2X2R in mouse trigeminal ganglia. Finally, endogenous P2X2aR-mediated currents in PC12 cells and P2X2/3R mediated increases of intracellular Ca in trigeminal neurons were Cdk5 dependent, since inhibition with roscovitine accelerated the desensitization kinetics of these responses. These results indicate that the P2X2aR is a novel target for Cdk5-mediated phosphorylation, which might play important physiological roles including pain signaling.

Targeting CDK5 post-stroke provides long-term neuroprotection and rescues synaptic plasticity.

Post-stroke cognitive impairment is a major cause of long-term neurological disability. The prevalence of post-stroke cognitive deficits varies between 20% and 80% depending on brain region, country, and diagnostic criteria. The biochemical mechanisms underlying post-stroke cognitive impairment are not known in detail. Cyclin-dependent kinase 5 is involved in neurodegeneration, and its dysregulation contributes to cognitive disorders and dementia. Here, we administered cyclin-dependent kinase 5-targeting gene therapy to the right hippocampus of ischemic rats after transient right middle cerebral artery occlusion. Cyclin-dependent kinase 5 RNA interference prevented the impairment of reversal learning four months after ischemia as well as neuronal loss, tauopathy, and microglial hyperreactivity. Additionally, cyclin-dependent kinase 5 silencing increased the expression of brain-derived neurotrophic factor in the hippocampus. Furthermore, deficits in hippocampal long-term potentiation produced by excitotoxic stimulation were rescued by pharmacological blockade of cyclin-dependent kinase 5. This recovery was blocked by inhibition of the TRKB receptor. In summary, these findings demonstrate the beneficial impact of cyclin-dependent kinase 5 reduction in preventing long-term post-ischemic neurodegeneration and cognitive impairment as well as the role of brain-derived neurotrophic factor/TRKB in the maintenance of normal synaptic plasticity.

Neuroprotection Induced by Transplanted CDK5 Knockdown Astrocytes in Global Cerebral Ischemic Rats.

Cerebral ischemia is a cerebrovascular episode that generates a high incidence of death and physical and mental disabilities worldwide. Excitotoxicity, release of free radicals, and exacerbated immune response cause serious complications in motor and cognitive areas during both short and long time frames post-ischemia. CDK5 is a kinase that is widely involved in the functions of neurons and astrocytes, and its over-activation is implicated in neurodegenerative processes. In this study, we evaluated the brain parenchymal response to the transplantation of CDK5-knockdown astrocytes into the somatosensory cortex after ischemia in rats. Male Wistar rats were subjected to the two-vessel occlusion (2VO) model of global cerebral ischemia and immediately transplanted with shCDK5miR- or shSCRmiR-transduced astrocytes or with untransduced astrocytes (Control). Our findings showed that animals transplanted with shCDK5miR astrocytes recovered motor and neurological performance better than with those transplanted with WT or shSCRmiR astrocytes. Cell transplantation produced an overall prevention of neuronal loss, and CDK5-knockdown astrocytes significantly increased the immunoreactivity (IR) of endogenous GFAP in branches surrounding blood vessels, accompanied by the upregulation of PECAM-1 IR in the walls of vessels in the motor and somatosensory regions and by an increase in Ki67 IR in the subventricular zone (SVZ), partially associated with the production of BDNF. Together, our data suggest that transplantation of shCDK5miR astrocytes protects the neurovascular unit in ischemic rats, allowing the motor and neurological function recovery.