Cellular and Molecular Mechanisms of R/S-Roscovitine and CDKs Related Inhibition under Both Focal and Global Cerebral Ischemia: A Focus on Neurovascular Unit and Immune Cells.

Ischemic stroke is the second leading cause of death worldwide. Following ischemic stroke, Neurovascular Unit (NVU) inflammation and peripheral leucocytes infiltration are major contributors to the extension of brain lesions. For a long time restricted to neurons, the 10 past years have shown the emergence of an increasing number of studies focusing on the role of Cyclin-Dependent Kinases (CDKs) on the other cells of NVU, as well as on the leucocytes. The most widely used CDKs inhibitor, (R)-roscovitine, and its (S) isomer both decreased brain lesions in models of global and focal cerebral ischemia. We previously showed that (S)-roscovitine acted, at least, by modulating NVU response to ischemia. Interestingly, roscovitine was shown to decrease leucocytes-mediated inflammation in several inflammatory models. Specific inhibition of roscovitine majors target CDK 1, 2, 5, 7, and 9 showed that these CDKs played key roles in inflammatory processes of NVU cells and leucocytes after brain lesions, including ischemic stroke. The data summarized here support the investigation of roscovitine as a potential therapeutic agent for the treatment of ischemic stroke, and provide an overview of CDK 1, 2, 5, 7, and 9 functions in brain cells and leucocytes during cerebral ischemia.

Principal component analysis, a useful tool to study cyclin-dependent kinase-inhibitor's effect on cerebral ischaemia.

Stroke is a leading cause of acute death related in part to brain oedema, blood-brain barrier disruption and glial inflammation. A cyclin-dependant kinase inhibitor, (S)-roscovitine, was administered 90 min after onset on a model of rat focal cerebral ischaemia. Brain swelling and Evans Blue tissue extravasation were quantified after Evans Blue injection. Combined tissue Evans Blue fluorescence and immunofluorescence of endothelial cells (RECA1), microglia (isolectin-IB4) and astrocytes (glial fibrillary acidic protein) were analysed. Using a Student's t-test or Mann-Whitney test, (S)-roscovitine improved recovery by more than 50% compared to vehicle (Mann-Whitney, P < 0.001), decreased significantly brain swelling by 50% (t-test, P = 0.0128) mostly in the rostral part of the brain. Main analysis was therefore performed on rostral cut for immunofluorescence to maximize biological observations (cut B). Evans Blue fluorescence decreased in (S)-roscovitine group compared to vehicle (60%, t-test, P = 0.049) and was further supported by spectrophotometer analysis (Mann-Whitney, P = 0.0002) and Evans Blue macroscopic photonic analysis (t-test, P = 0.07). An increase of RECA-1 intensity was observed in the ischaemic hemisphere compared to non-ischaemic hemisphere. Further study showed, in the ischaemic hemisphere that (S)-roscovitine treated group compared to vehicle, showed a decrease of: (i) endothelial RECA-1 intensity of about 20% globally, mainly located in the cortex (-28.5%, t-test, P = 0.03); (ii) Microglia's number by 55% (t-test, P = 0.006) and modulated reactive astrocytes through a trend toward less astrocytes number (15%, t-test, P = 0.05) and astrogliosis (21%, t-test, P = 0.076). To decipher the complex relationship of these components, we analysed the six biological quantitative variables of our study by principal component analysis from immunofluorescence studies of the same animals. Principal component analysis differentiated treated from non-treated animals on dimension 1 with negative values in the treated animals, and positive values in the non-treated animals. Interestingly, stroke recovery presented a negative correlation with this dimension, while all other biological variables showed a positive correlation. Dimensions 1 and 2 allowed the identification of two groups of co-varying variables: endothelial cells, microglia number and Evans Blue with positive values on both dimensions, and astrocyte number, astrogliosis and brain swelling with negative values on dimension 2. This partition suggests different mechanisms. Correlation matrix analysis was concordant with principal component analysis results. Because of its pleiotropic complex action on different elements of the NeuroVascular Unit response, (S)-roscovitine may represent an effective treatment against oedema in stroke.

Sustained (S)-roscovitine delivery promotes neuroprotection associated with functional recovery and decrease in brain edema in a randomized blind focal cerebral ischemia study.

Stroke is a devastating disorder that significantly contributes to death, disability and healthcare costs. In ischemic stroke, the only current acute therapy is recanalization, but the narrow therapeutic window less than 6 h limits its application. The current challenge is to prevent late cell death, with concomitant therapy targeting the ischemic cascade to widen the therapeutic window. Among potential neuroprotective drugs, cyclin-dependent kinase inhibitors such as (S)-roscovitine are of particular relevance. We previously showed that (S)-roscovitine crossed the blood-brain barrier and was neuroprotective in a dose-dependent manner in two models of middle cerebral artery occlusion (MCAo). According to the Stroke Therapy Academic Industry Roundtable guidelines, the pharmacokinetics of (S)-roscovitine and the optimal mode of delivery and therapeutic dose in rats were investigated. Combination of intravenous (IV) and continuous sub-cutaneous (SC) infusion led to early and sustained delivery of (S)-roscovitine. Furthermore, in a randomized blind study on a transient MCAo rat model, we showed that this mode of delivery reduced both infarct and edema volume and was beneficial to neurological outcome. Within the framework of preclinical studies for stroke therapy development, we here provide data to improve translation of pre-clinical studies into successful clinical human trials.

EPA/DHA and Vitamin A Supplementation Improves Spatial Memory and Alleviates the Age-related Decrease in Hippocampal RXRγ and Kinase Expression in Rats.

Studies suggest that eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and vitamin A are critical to delay aged-related cognitive decline. These nutrients regulate gene expression in the brain by binding to nuclear receptors such as the retinoid X receptors (RXRs) and the retinoic acid receptors (RARs). Moreover, EPA/DHA and retinoids activate notably kinase signaling pathways such as AKT or MAPK, which includes ERK1/2. This suggests that these nutrients may modulate brain function in a similar way. Therefore, we investigated in middle-aged rats the behavioral and molecular effects of supplementations with EPA/DHA and vitamin A alone or combined. 18-month-old rats exhibited reference and working memory deficits in the Morris water maze, associated with a decrease in serum vitamin A and hippocampal EPA/DHA contents. RARα, RXRß, and RXRγ mRNA expression and CAMKII, AKT, ERK1/2 expression were decreased in the hippocampus of middle-aged rats. A combined EPA/DHA and vitamin A supplementation had a beneficial additive effect on reference memory but not in working memory in middle-aged rats, associated with an alleviation of the age-related decrease in RXRγ, CAMKII, AKT, and ERK1 expression in the hippocampus. This study provides a new combined nutritional strategy to delay brain aging.

Erythrocyte DHA level as a biomarker of DHA status in specific brain regions of n-3 long-chain PUFA-supplemented aged rats.

n-3 Long-chain PUFA (n-3 LC-PUFA), particularly EPA and DHA, play a key role in the maintenance of brain functions such as learning and memory that are impaired during ageing. Ageing is also associated with changes in the DHA content of brain membranes that could contribute to memory impairment. Limited studies have investigated the effects of ageing and n-3 LC-PUFA supplementation on both blood and brain fatty acid compositions. Therefore, we assessed the relationship between fatty acid contents in plasma and erythrocyte membranes and those in the hippocampus, striatum and cerebral cortex during ageing, and after a 5-month period of EPA/DHA supplementation in rats. In the blood, ageing was associated with an increase in plasma DHA content, whereas the DHA content remained stable in erythrocyte membranes. In the brain, ageing was associated with a decrease in DHA content, which was both region-specific and phospholipid class-specific. In EPA/DHA-supplemented aged rats, DHA contents were increased both in the blood and brain compared with the control rats. The present results demonstrated that n-3 LC-PUFA level in the plasma was not an accurate biomarker of brain DHA status during ageing. Moreover, we highlighted a positive relationship between the DHA levels in erythrocyte phosphatidylethanolamine (PE) and those in the hippocampus and prefrontal cortex in EPA/DHA-supplemented aged rats. Within the framework of preventive dietary supplementation to delay brain ageing, these results suggest the possibility of using erythrocyte PE DHA content as a reliable biomarker of DHA status in specific brain regions.