The Endocannabinoid System in Glial Cells and Their Profitable Interactions to Treat Epilepsy: Evidence from Animal Models

Epilepsy is one of the most common neurological conditions. Yearly, five million people are diagnosed with epileptic-related disorders. The neuroprotective and therapeutic effect of (endo)cannabinoid compounds has been extensively investigated in several models of epilepsy. Therefore, the study of specific cell-type-dependent mechanisms underlying cannabinoid effects is crucial to understanding epileptic disorders. It is estimated that about 100 billion neurons and a roughly equal number of glial cells co-exist in the human brain. The glial population is in charge of neuronal viability, and therefore, their participation in brain pathophysiology is crucial. Furthermore, glial malfunctioning occurs in a wide range of neurological disorders. However, little is known about the impact of the endocannabinoid system (ECS) regulation over glial cells, even less in pathological conditions such as epilepsy. In this review, we aim to compile the existing knowledge on the role of the ECS in different cell types, with a particular emphasis on glial cells and their impact on epilepsy. Thus, we propose that glial cells could be a novel target for cannabinoid agents for treating the etiology of epilepsy and managing seizure-like disorders.

Transient Hypoperfusion to Ischemic/Anoxic Spreading Depolarization is Related to Autoregulatory Failure in the Rat Cerebral Cortex

Background In ischemic stroke, cerebral autoregulation and neurovascular coupling may become impaired. The cerebral blood flow (CBF) response to spreading depolarization (SD) is governed by neurovascular coupling. SDs recur in the ischemic penumbra and reduce neuronal viability by the insufficiency of the CBF response. Autoregulatory failure and SD may coexist in acute brain injury. Here, we set out to explore the interplay between the impairment of cerebrovascular autoregulation, SD occurrence, and the evolution of the SD-coupled CBF response. Methods Incomplete global forebrain ischemia was created by bilateral common carotid artery occlusion in isoflurane-anesthetized rats, which induced ischemic SD (iSD). A subsequent SD was initiated 20–40 min later by transient anoxia SD (aSD), achieved by the withdrawal of oxygen from the anesthetic gas mixture for 4–5 min. SD occurrence was confirmed by the recording of direct current potential together with extracellular K+ concentration by intracortical microelectrodes. Changes in local CBF were acquired with laser Doppler flowmetry. Mean arterial blood pressure (MABP) was continuously measured via a catheter inserted into the left femoral artery. CBF and MABP were used to calculate an index of cerebrovascular autoregulation (rCBFx). In a representative imaging experiment, variation in transmembrane potential was visualized with a voltage-sensitive dye in the exposed parietal cortex, and CBF maps were generated with laser speckle contrast analysis. Results Ischemia induction and anoxia onset gave rise to iSD and aSD, respectively, albeit aSD occurred at a longer latency, and was superimposed on a gradual elevation of K+ concentration. iSD and aSD were accompanied by a transient drop of CBF (down to 11.9 ± 2.9 and 7.4 ± 3.6%, iSD and aSD), but distinctive features set the hypoperfusion transients apart. During iSD, rCBFx indicated intact autoregulation (rCBFx < 0.3). In contrast, aSD was superimposed on autoregulatory failure (rCBFx > 0.3) because CBF followed the decreasing MABP. CBF dropped 15–20 s after iSD, but the onset of hypoperfusion preceded aSD by almost 3 min. Taken together, the CBF response to iSD displayed typical features of spreading ischemia, whereas the transient CBF reduction with aSD appeared to be a passive decrease of CBF following the anoxia-related hypotension, leading to aSD. Conclusions We propose that the dysfunction of cerebrovascular autoregulation that occurs simultaneously with hypotension transients poses a substantial risk of SD occurrence and is not a consequence of SD. Under such circumstances, the evolving SD is not accompanied by any recognizable CBF response, which indicates a severely damaged neurovascular coupling.

Senescence in Primary Rat Astrocytes Induces Loss of the Mitochondrial Membrane Potential and Alters Mitochondrial Dynamics in Cortical Neurons

The decline in brain function during aging is one of the most critical health problems nowadays. Although senescent astrocytes have been found in old-age brains and neurodegenerative diseases, their impact on the function of other cerebral cell types is unknown. The aim of this study was to evaluate the effect of senescent astrocytes on the mitochondrial function of a neuron. In order to evaluate neuronal susceptibility to a long and constant senescence-associated secretory phenotype (SASP) exposure, we developed a model by using cellular cocultures in transwell plates. Rat primary cortical astrocytes were seeded in transwell inserts and induced to premature senescence with hydrogen peroxide [stress-induced premature senescence (SIPS)]. Independently, primary rat cortical neurons were seeded at the bottom of transwells. After neuronal 6 days in vitro (DIV), the inserts with SIPS-astrocytes were placed in the chamber and cocultured with neurons for 6 more days. The neuronal viability, the redox state [reduced glutathione/oxidized glutathione (GSH/GSSG)], the mitochondrial morphology, and the proteins and membrane potential were determined. Our results showed that the neuronal mitochondria functionality was altered after being cocultured with senescent astrocytes. In vivo, we found that old animals had diminished mitochondrial oxidative phosphorylation (OXPHOS) proteins, redox state, and senescence markers as compared to young rats, suggesting effects of the senescent astrocytes similar to the ones we observed in vitro. Overall, these results indicate that the microenvironment generated by senescent astrocytes can affect neuronal mitochondria and physiology.

Azoxystrobin Impairs Neuronal Migration and Induces ROS Dependent Apoptosis in Cortical Neurons

Fungicides often cause genotoxic stress and neurodevelopmental disorders such as autism (ASD). Fungicide-azoxystrobin (AZOX) showed acute and chronic toxicity to various organisms, and remained a concern for ill effects in developing neurons. We evaluated the neurotoxicity of AZOX in developing mouse brains, and observed prenatal exposure to AZOX reduced neuronal viability, neurite outgrowth, and cortical migration process in developing brains. The 50% inhibitory concentration (IC50) of AZOX for acute (24 h) and chronic (7 days) exposures were 30 and 10 μM, respectively. Loss in viability was due to the accumulation of reactive oxygen species (ROS), and inhibited neurite outgrowth was due to the deactivation of mTORC1 kinase activity. Pretreatment with ROS scavenger- N-acetylcysteine (NAC) reserved the viability loss and forced activation of mTORC1 kinase revived the neurite outgrowth in AZOX treated neurons. Intra-amniotic injection of AZOX coupled with in utero electroporation of GFP-labelled plasmid in E15.5 mouse was performed and 20 mg/kg AZOX inhibited radial neuronal migration. Moreover, the accumulation of mitochondria was significantly reduced in AZOX treated primary neurons, indicative of mitochondrial deactivation and induction of apoptosis, which was quantified by Bcl2/Bax ratio and caspase 3 cleavage assay. This study elucidated the neurotoxicity of AZOX and explained the possible cure from it.

Loss of SORCS2 is Associated with Neuronal DNA Double-Strand Breaks

SORCS2 is one of five proteins that constitute the Vps10p-domain receptor family. Members of this family play important roles in cellular processes linked to neuronal survival, differentiation and function. Genetic and functional studies implicate SORCS2 in cognitive function, as well as in neurodegenerative and psychiatric disorders. DNA damage and DNA repair deficits are linked to ageing and neurodegeneration, and transient neuronal DNA double-strand breaks (DSBs) also occur as a result of neuronal activity. Here, we report a novel role for SORCS2 in DSB formation. We show that SorCS2 loss is associated with elevated DSB levels in the mouse dentate gyrus and that knocking out SORCS2 in a human neuronal cell line increased Topoisomerase IIβ-dependent DSB formation and reduced neuronal viability. Neuronal stimulation had no impact on levels of DNA breaks in vitro, suggesting that the observed differences may not be the result of aberrant neuronal activity in these cells. Our findings are consistent with studies linking the VPS10 receptors and DNA damage to neurodegenerative conditions.

Adaptogenic potential of Centella lujica supplement in sleep deprived mice

Background: Stress, whether internal or external, has been shown to play a role in the accumulation of oxidative free radicals which leads to cellular modification of normal organ or body function. Centella lujica (C. lujica) is a commonly cultivated herb with therapeutic benefits in various studies. This study aims to evaluate its beneficial effect on the brain and liver of mice exposed to sleep deprivation-induced stress.Methods: Albino mice were treated with distilled water (control), C. lujica (50 and 100 mg/kg) or astaxanthin (50 mg/kg) for seven days. All groups except control were then subjected to three-days of sleep deprivation using the grid suspended over water model beginning from day 4 of treatment. Behavioural assessments followed by biochemical assays and histological analysis were carried out thereafter.Results: Sleep deprivation caused an increase in blood glucose and triglycerides levels but reduced high density lipoproteins. Brain pro-oxidant levels were increased with a concomitant decrease in antioxidants, recognition memory was diminished while depressive-like symptoms were enhanced and neuronal viability of hippocampal CA1 as well as prefrontal cortex cells was reduced in sleep-deprived mice. However, supplementation with C. lujica reversed these effects as significantly as astaxanthin.Conclusions: C. lujica possesses antioxidant property that makes it an effective adaptogen against stress induced responses in mice.

Bisphenol a Induces Autophagy Defects and AIF-Dependent Apoptosis via HO-1 and AMPK to Degenerate N2a Neurons

Bisphenol A (BPA) is an environmental contaminant widely suspected to be a neurological toxicant. Epidemiological studies have demonstrated close links between BPA exposure, pathogenetic brain degeneration, and altered neurobehaviors, considering BPA a risk factor for cognitive dysfunction. However, the mechanisms of BPA resulting in neurodegeneration remain unclear. Herein, cultured N2a neurons were subjected to BPA treatment, and neurotoxicity was assessed using neuronal viability and differentiation assays. Signaling cascades related to cellular self-degradation were also evaluated. BPA decreased cell viability and axon outgrowth (e.g., by down-regulating MAP2 and GAP43), thus confirming its role as a neurotoxicant. BPA induced neurotoxicity by down-regulating Bcl-2 and initiating apoptosis and autophagy flux inhibition (featured by nuclear translocation of apoptosis-inducing factor (AIF), light chain 3B (LC3B) aggregation, and p62 accumulation). Both heme oxygenase (HO)-1 and AMP-activated protein kinase (AMPK) up-regulated/activated by BPA mediated the molecular signalings involved in apoptosis and autophagy. HO-1 inhibition or AIF silencing effectively reduced BPA-induced neuronal death. Although BPA elicited intracellular oxygen free radical production, ROS scavenger NAC exerted no effect against BPA insults. These results suggest that BPA induces N2a neurotoxicity characterized by AIF-dependent apoptosis and p62-related autophagy defects via HO-1 up-regulation and AMPK activation, thereby resulting in neuronal degeneration.

Targeting Oxidative Stress with Antioxidant Duotherapy After Experimental Traumatic Brain Injury

We assessed the effect of antioxidant therapy using the Food and Drug Administration-approved respiratory drug N-acetylcysteine (NAC) or sulforaphane (SFN) as monotherapies or duotherapy in vitro in neuron-BV2 microglial co-cultures and validated the results in a lateral fluid-percussion model of TBI in rats. As in vitro measures, we assessed neuronal viability by microtubule-associated-protein 2 immunostaining, neuroinflammation by monitoring tumor necrosis factor (TNF) levels, and neurotoxicity by measuring nitrite levels. In vitro, duotherapy with NAC and SFN reduced nitrite levels to 40% (p < 0.001) and neuroinflammation to –29% (p < 0.001) compared with untreated culture. The treatment also improved neuronal viability up to 72% of that in a positive control (p < 0.001). The effect of NAC was negligible, however, compared with SFN. In vivo, antioxidant duotherapy slightly improved performance in the beam walking test. Interestingly, duotherapy treatment decreased the plasma interleukin-6 and TNF levels in sham-operated controls (p < 0.05). After TBI, no treatment effect on HMGB1 or plasma cytokine levels was detected. Also, no treatment effects on the composite neuroscore or cortical lesion area were detected. The robust favorable effect of duotherapy on neuroprotection, neuroinflammation, and oxidative stress in neuron-BV2 microglial co-cultures translated to modest favorable in vivo effects in a severe TBI model.

Implication of exosomes derived from cholesterol-accumulated astrocytes in Alzheimer's disease pathology

Amyloid β (Aβ) peptides generated from the amyloid precursor protein (APP) play a critical role in the development of Alzheimer's disease (AD) pathology. Aβ-containing neuronal exosomes, which represent a novel form of intercellular communication, have been shown to influence function/vulnerability of neurons in AD. Unlike neurons, the significance of exosomes derived from astrocytes remains unclear. In this study, we evaluated the significance of exosomes derived from U18666A-induced cholesterol-accumulated astrocytes in the development of AD pathology. Our results show that cholesterol accumulation decreases exosome secretion, whereas lowering cholesterol level increases exosome secretion from cultured astrocytes. Interestingly, exosomes secreted from U18666A-treated astrocytes contain higher levels of APP, APP-CTFs, soluble APP, APP secretases and Aβ1-40 than exosomes secreted from control astrocytes. Furthermore, we show that exosomes derived from U18666A-treated astrocytes can lead to neurodegeneration, which is attenuated by decreasing Aβ production or by neutralizing exosomal Aβ peptide with an Aβ antibody. These results, taken together, suggest that exosomes derived from cholesterol-accumulated astrocytes can play an important role in trafficking APP/Aβ peptides and influencing neuronal viability in the affected regions of the AD brain.

Tilianin Ameliorates Cognitive Dysfunction and Neuronal Damage in Rats with Vascular Dementia via p-CaMKII/ERK/CREB and ox-CaMKII-Dependent MAPK/NF-κB Pathways

Vascular dementia (VaD) is a common cause of cognitive decline and dementia of vascular origin, but the precise pathological mechanisms are unknown, and so effective clinical treatments have not been established. Tilianin, the principal active compound of total flavonoid extract from Dracocephalum moldavica L., is a candidate therapy for cardio-cerebrovascular diseases in China. However, its potential in the treatment of VaD is unclear. The present study is aimed at investigating the protective effects of tilianin on VaD and exploring the underlying mechanism of the action. A model of VaD was established by permanent 2-vessel occlusion (2VO) in rats. Human neurons (hNCs) differentiated from human-induced pluripotent stem cells were used to establish an oxygen-glucose deprivation (OGD) model. The therapeutic effects and potential mechanisms of tilianin were identified using behavioral tests, histochemistry, and multiple molecular biology techniques such as Western blot analysis and gene silencing. The results demonstrated that tilianin modified spatial cognitive impairment, neurodegeneration, oxidation, and apoptosis in rats with VaD and protected hNCs against OGD by increasing cell viability and decreasing apoptosis rates. A study of the mechanism indicated that tilianin restored p-CaMKII/ERK1/2/CREB signaling in the hippocampus, maintaining hippocampus-independent memory. In addition, tilianin inhibited an ox-CaMKII/p38 MAPK/JNK/NF-κB associated inflammatory response caused by cerebral oxidative stress imbalance in rats with VaD. Furthermore, specific CaMKIIα siRNA action revealed that tilianin-exerted neuroprotection involved increase of neuronal viability, inhibition of apoptosis, and suppression of inflammation, which was dependent on CaMKIIα. In conclusion, the results suggested the neuroprotective effect of tilianin in VaD and the potential mechanism associated with dysfunction in the regulation of p-CaMKII-mediated long-term memory and oxidation and inflammation involved with ox-CaMKII, which may lay the foundation for clinical trials of tilianin for the treatment of VaD in the future.