CDK5 downregulation enhances synaptic plasticity.

CDK5 is a serine/threonine kinase that is involved in the normal function of the adult brain and plays a role in neurotransmission and synaptic plasticity. However, its over-regulation has been associated with Tau hyperphosphorylation and cognitive deficits. Our previous studies have demonstrated that CDK5 targeting using shRNA-miR provides neuroprotection and prevents cognitive deficits. Dendritic spine morphogenesis and forms of long-term synaptic plasticity-such as long-term potentiation (LTP)-have been proposed as essential processes of neuroplasticity. However, whether CDK5 participates in these processes remains controversial and depends on the experimental model. Using wild-type mice that received injections of CDK5 shRNA-miR in CA1 showed an increased LTP and recovered the PPF in deficient LTP of APPswe/PS1Δ9 transgenic mice. On mature hippocampal neurons CDK5, shRNA-miR for 12 days induced increased dendritic protrusion morphogenesis, which was dependent on Rac activity. In addition, silencing of CDK5 increased BDNF expression, temporarily increased phosphorylation of CaMKII, ERK, and CREB; and facilitated calcium signaling in neurites. Together, our data suggest that CDK5 downregulation induces synaptic plasticity in mature neurons involving Ca2+ signaling and BDNF/CREB activation.

p120-catenin is necessary for neuroprotection induced by CDK5 silencing in models of Alzheimer's disease.

Cyclin-dependent kinase 5 (CDK5) plays important roles in synaptic function. Its unregulated over-activation has been, however, associated with neurodegeneration in Alzheimer's disease. Our previous studies revealed that CDK5 silencing ameliorates tauopathy and spatial memory impairment in the 3xTgAD mouse model. However, how CDK5 targeting affects synaptic adhesion proteins, such as those involved in the cadherin/catenin system, during learning and memory processes is not completely understood. In this study, we detected reduced expression of p120 catenin (p120 ctn), N-cadherin, and ß-catenin in the brain of human Alzheimer's disease patients, in addition to a reduced PSD95 and GluN2B protein levels in a 3xTgAD mouse model. Such decrease in synaptic proteins was recovered by CDK5 silencing in mice leading to a better learning and memory performance. Additionally, CDK5 inhibition or knockout increased p120 ctn levels. Moreover, in a glutamate-induced excitotoxicity model, CDK5 silencing-induced neuroprotection depended on p120 ctn. Together, those findings suggest that p120 ctn plays an important role in the neuronal dysfunction of Alzheimer's disease models and contributes to CDK5 silencing-induced neuroprotection and improvement of memory function. p120ctn is part of the synaptic adhesion molecular complex N-cadh/p120ctn/B-ctn/PSD95, and it has a pivotal role in cell adhesion stabilization and dendritic spine modulation. Our data show that synaptic adhesion complex is affected in AD human brains and in AD models. This complex is recovered by the silencing of CDK5, preventing memory dysfunction in an AD mice model and contributing to the neuroprotection in a depend-mode of p120ctn.

CDK5 knockdown in astrocytes provide neuroprotection as a trophic source via Rac1.

Astrocytes perform metabolic and structural support functions in the brain and contribute to the integrity of the blood-brain barrier. Astrocytes influence neuronal survival and prevent gliotoxicity by capturing glutamate (Glu), reactive oxygen species, and nutrients. During these processes, astrocytic morphological changes are supported by actin cytoskeleton remodeling and require the involvement of Rho GTPases, such as Rac1. The protein cyclin-dependent kinase 5 (CDK5) may have a dual effect on astrocytes because it has been shown to be involved in migration, senescence, and the dysfunction of glutamate recapture; however, its role in astrocytes remains unclear. Treating a possible deregulation of CDK5 with RNAi is a strategy that has been proposed as a therapy for neurodegenerative diseases. Models of glutamate gliotoxicity in the C6 astroglioma cell line, primary cultures of astrocytes, and co-cultures with neurons were used to analyze the effects of CDK5 RNAi in astrocytes and the role of Rac1 in neuronal viability. In C6 cells and primary astrocytes, CDK5 RNAi prevented the cell death generated by glutamate-induced gliotoxicity, and this finding was corroborated by pharmacological inhibition with roscovitine. This effect was associated with the appearance of lamellipodia, protrusions, increased cell area, stellation, Rac1 activation, BDNF release, and astrocytic protection in neurons that were exposed to glutamate excitotoxicity. Interestingly, Rac1 inhibition in astrocytes blocked BDNF upregulation and the astrocyte-mediated neuroprotection. Actin cytoskeleton remodeling and stellation may be a functional phenotype for BDNF release that promotes neuroprotection. In summary, our findings suggest that CDK5- knockdown in astrocytes acts as a trophic source for neuronal protection in a Rac1-dependent manner.

p35 and Rac1 underlie the neuroprotection and cognitive improvement induced by CDK5 silencing.

CDK5 plays an important role in neurotransmission and synaptic plasticity in the normal function of the adult brain, and dysregulation can lead to Tau hyperphosphorylation and cognitive impairment. In a previous study, we demonstrated that RNAi knock down of CDK5 reduced the formation of neurofibrillary tangles (NFT) and prevented neuronal loss in triple transgenic Alzheimer's mice. Here, we report that CDK5 RNAi protected against glutamate-mediated excitotoxicity using primary hippocampal neurons transduced with adeno-associated virus 2.5 viral vector eGFP-tagged scrambled or CDK5 shRNA-miR during 12 days. Protection was dependent on a concomitant increase in p35 and was reversed using p35 RNAi, which affected the down-stream Rho GTPase activity. Furthermore, p35 over-expression and constitutively active Rac1 mimicked CDK5 silencing-induced neuroprotection. In addition, 3xTg-Alzheimer's disease mice (24 months old) were injected in the hippocampus with scrambled or CDK5 shRNA-miR, and spatial learning and memory were performed 3 weeks post-injection using 'Morris' water maze test. Our data showed that CDK5 knock down induced an increase in p35 protein levels and Rac activity in triple transgenic Alzheimer's mice, which correlated with the recovery of cognitive function; these findings confirm that increased p35 and active Rac are involved in neuroprotection. In summary, our data suggest that p35 acts as a mediator of Rho GTPase activity and contributes to the neuroprotection induced by CDK5 RNAi.

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.

Endothelial activation and injury by microparticles in patients with systemic lupus erythematosus and rheumatoid arthritis.

BACKGROUND: Endothelial activation and damage is commonly observed in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) and is related to development of atherosclerosis and cardiovascular diseases. Different components of the immune system seem to participate in the endothelial injury, such as generation of autoantibodies and formation of immune complexes (ICs). Microparticles (MPs) and their immune complexes (MPs-ICs) are increased in the circulation of patients with SLE and RA; therefore, we propose these extracellular vesicles could interact and modulate the function of endothelial cells. Hence, the effect of MPs and MPs-ICs from patients with SLE and RA in endothelial cells was evaluated. METHODS: Macrovascular and microvascular endothelial cells were exposed to MPs and MPs-ICs from healthy donors and patients with SLE and RA. Vesicles uptake/binding, expression of adhesion molecules, cytokine and chemokine production, monocyte adherence, and alterations of endothelial monolayer were evaluated by flow cytometry and fluorescence microscopy. RESULTS: Endothelial cells internalized MPs and MPs-ICs and increased CD54 and CD102 expression and CCL2, CCL5, and IL-6 production after the treatment with these extracellular vesicles, which led to an increase in the adherence of classic monocytes. These vesicles also induced low expression of VE-cadherin in membrane, depolymerization of actin filaments, and formation of intercellular spaces, which led to endothelial death and increased permeability after MPs and MPs-ICs exposure. CONCLUSIONS: MPs and MPs-ICs from patients with SLE and RA increase adhesion molecules expression, chemokine production, and structural alterations in macrovascular and microvascular endothelial cells. Therefore, high counts of these vesicles in patients would promote endothelial alterations and secondary tissue leukocyte infiltration.

Neuroprotective activity and acetylcholinesterase inhibition of five Amaryllidaceae species: a comparative study.

AIMS: Amaryllidaceae alkaloids exhibit a wide range of physiological effects, of which the acetylcholinesterase (AChE) inhibitory activity is the most relevant. However, scientific evidence related to their neuroprotective effectiveness against glutamate-induced toxicity has been lacking. Thus, the purpose of this study was to conduct a comparative study of the neuroprotective activity and the AChE inhibitory activity of species of Amaryllidaceae. MAIN METHODS: The neuroprotective activity against glutamate-induced toxicity was measured in rat cortical neurons and the Ellman method was employed for the quantification of acetylcholinesterase inhibitory activity of alkaloidal extracts of five species of Amaryllidaceae (Crinum jagus, Crinum bulbispermum, Hippeastrum barbatum, Hippeastrum puniceum and Zephyranthes carinata). The alkaloid Amaryllidaceae patterns based on GC/MS analyses were also investigated. KEY FINDINGS: The results showed that the alkaloidal extract from C. jagus presented a high neuroprotective activity in both pre- and post-treatments against a glutamate excitotoxic stimulus. Furthermore, the alkaloid extracts from C. jagus and Z. carinata revealed an inhibitory activity of AChE from the electric eel with IC50 values of 18.28±0.29 and 17.96±1.22µg/mL, respectively. In addition, 46 alkaloids were detected by GC/MS, and 20 of them were identified based on their mass spectra and retention index. The results suggest that the neuroprotective effects might be associated with lycorine and crinine-type alkaloids, whereas the acetylcholinesterase enzyme inhibitory activity could be related to galanthamine and lycorine-type alkaloids, although not based on synergistic processes. SIGNIFICANCE: In summary, Amaryllidaceae species are sources of alkaloids with potential use for Alzheimer's disease.

Protection after stroke: cellular effectors of neurovascular unit integrity.

Neurological disorders are prevalent worldwide. Cerebrovascular diseases (CVDs), which account for 55% of all neurological diseases, are the leading cause of permanent disability, cognitive and motor disorders and dementia. Stroke affects the function and structure of blood-brain barrier, the loss of cerebral blood flow regulation, oxidative stress, inflammation and the loss of neural connections. Currently, no gold standard treatments are available outside the acute therapeutic window to improve outcome in stroke patients. Some promising candidate targets have been identified for the improvement of long-term recovery after stroke, such as Rho GTPases, cell adhesion proteins, kinases, and phosphatases. Previous studies by our lab indicated that Rho GTPases (Rac and RhoA) are involved in both tissue damage and survival, as these proteins are essential for the morphology and movement of neurons, astrocytes and endothelial cells, thus playing a critical role in the balance between cell survival and death. Treatment with a pharmacological inhibitor of RhoA/ROCK blocks the activation of the neurodegeneration cascade. In addition, Rac and synaptic adhesion proteins (p120 catenin and N-catenin) play critical roles in protection against cerebral infarction and in recovery by supporting the neurovascular unit and cytoskeletal remodeling activity to maintain the integrity of the brain parenchyma. Interestingly, neuroprotective agents, such as atorvastatin, and CDK5 silencing after cerebral ischemia and in a glutamate-induced excitotoxicity model may act on the same cellular effectors to recover neurovascular unit integrity. Therefore, future efforts must focus on individually targeting the structural and functional roles of each effector of neurovascular unit and the interactions in neural and non-neural cells in the post-ischemic brain and address how to promote the recovery or prevent the loss of homeostasis in the short, medium and long term.

Atorvastatin requires geranylgeranyl transferase-I and Rac1 activation to exert neuronal protection and induce plasticity.

Statins are widely used cholesterol-lowering drugs that may reduce the incidence of stroke and the progression of Alzheimer's disease (AD). However, how statins exert these beneficial effects remains poorly understood. Thus, this study evaluated the roles of Rac1 geranylgeranylation and the relationship between Rac1 and αN-catenin in the protective activity of atorvastatin (ATV) in a cortical neuronal culture model of glutamate (GLU) excitotoxicity. We found that ATV-induced neuroprotection and plasticity were blocked by isoprenoids, such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), inhibition of farnesylation (FTI-277) and geranylgeranylation (GGTI-286), down-regulation of GGTase-Iß and Rac activity and promotion of active RhoA. Additionally, ATV rescued the distribution of dendritic αN-catenin and increased the number and length of dendritic branches; these effects were reversed by GGTI-286, GGTase-Iß shRNA, Rac1 shRNA and a dominant-negative version of Rac1 (T17N). In summary, our findings suggest that ATV requires GGTase-Iß, prenylation and active Rac1 to induce protection and plasticity. In this regard, αN-catenin is a marker for stable interactions between adhesion proteins and the actin cytoskeleton and is necessary for the neuroprotective action of ATV.