MLC1: A New Calcium-regulated Protein Conferring Calcium Dependence to Volume-regulated Anion Channels (VRAC) in Astrocytes.

MLC1 is a membrane protein highly expressed by brain perivascular astrocytes. Mutations in the MLC1 gene account for megalencephalic leukoencephalopathy with subcortical cysts (MLC), an incurable leukodystrophy characterized by macrocephaly, brain edema and cysts, myelin vacuolation and astrocyte swelling, causing cognitive and motor dysfunctions. It has been demonstrated that MLC1 mutations affect the swelling-activated Cl - currents (I Cl,swell ) mediated by volume-regulated anion channel (VRAC) and the consequent regulatory volume decrease (RVD) and lead to abnormal activation of intracellular signaling pathways linked to inflammation/osmotic stress. Despite this knowledge, the MLC1 physiological role and MLC molecular pathogenesis are still elusive. Following the observations that Ca 2+ regulates all the MLC1-modulated processes and that intracellular Ca 2+ homeostasis is altered in MLC1-defective cells, we applied a multidisciplinary approach including biochemistry, molecular biology, video imaging, electrophysiology and proteomic techniques on cultured astrocytes to uncover new Ca 2+ -dependent signaling pathways controlling MLC1 function. Here, we revealed that MLC1 binds the Ca 2+ effector proteins calmodulin (CaM) and Ca 2+ /CaM-dependent protein kinase II (CaMKII) and, as result, changes its assembly, localization and functional properties in response to Ca 2+ changes. Noteworthy, CaM binding to the COOH terminal promotes MLC1 trafficking to the plasma membrane, while CaMKII phosphorylation of the NH 2 -terminal potentiates MLC1 activation of I Cl,swell . Overall, these results revealed that MLC1 is a Ca 2+ -regulated protein linking VRAC function and, possibly, volume regulation to Ca 2+ signaling in astrocytes. These findings open new avenues of investigations aimed at clarifying the abnormal molecular pathways underlying MLC and other diseases characterized by astrocyte swelling and brain edema.

Therapeutic Applications of Resveratrol in Hepatic Encephalopathy through Its Regulation of the Microbiota, Brain Edema, and Inflammation

Hepatic encephalopathy is a common complication in patients with liver cirrhosis and portosystemic shunting. Patients with hepatic encephalopathy present a variety of clinical features, including neuropsychiatric manifestations, cognitive dysfunction, impaired gut barrier function, hyperammonemia, and chronic neuroinflammation. These pathogeneses have been linked to various factors, including ammonia-induced oxidative stress, neuronal cell death, alterations in the gut microbiome, astrocyte swelling, and blood-brain barrier disruptions. Many researchers have focused on identifying novel therapeutics and prebiotics in the hope of improving the treatment of these conditions. Resveratrol is a natural polyphenic compound and is known to exert several pharmacological effects, including antioxidant, anti-inflammatory, and neuroprotective activities. Recent studies suggest that resveratrol contributes to improving the neuropathogenic effects of liver failure. Here, we review the current evidence describing resveratrol’s effects in neuropathogenesis and its impact on the gut-liver axis relating to hepatic encephalopathy. We highlight the hypothesis that resveratrol exerts diverse effects in hepatic encephalopathy and suggest that these effects are likely mediated by changes to the gut microbiota, brain edema, and neuroinflammation.

Malignant astrocyte swelling and impaired glutamate clearance drive the expansion of injurious spreading depolarization foci

Spreading depolarizations (SDs) indicate injury progression and predict worse clinical outcome in acute brain injury. We demonstrate in rodents that acute brain swelling upon cerebral ischemia impairs astroglial glutamate clearance and increases the tissue area invaded by SD. The cytotoxic extracellular glutamate accumulation (>15 µM) predisposes an extensive bulk of tissue (4–5 mm2) for a yet undescribed simultaneous depolarization (SiD). We confirm in rat brain slices exposed to osmotic stress that SiD is the pathological expansion of prior punctual SD foci (0.5–1 mm2), is associated with astrocyte swelling, and triggers oncotic neuron death. The blockade of astrocytic aquaporin-4 channels and Na+/K+/Cl− co-transporters, or volume-regulated anion channels mitigated slice edema, extracellular glutamate accumulation (<10 µM) and SiD occurrence. Reversal of slice swelling by hyperosmotic mannitol counteracted glutamate accumulation and prevented SiD. In contrast, inhibition of glial metabolism or inhibition of astrocyte glutamate transporters reproduced the SiD phenotype. Finally, we show in the rodent water intoxication model of cytotoxic edema that astrocyte swelling and altered astrocyte calcium waves are central in the evolution of SiD. We discuss our results in the light of evidence for SiD in the human cortex. Our results emphasize the need of preventive osmotherapy in acute brain injury.

Mechanism of Aquaporin-4 Up-Regulation After Traumatic Brain Injury and Preventative Action of Astragalus Polysaccharides in Mice

Here, we aimed to clarify the anti-inflammatory function of Astragalus Polysaccharides (APS), a chemical compound derived from Astragalus membranaceus, and the action of AQP4 on brain injury. We hypothesized that APS could improve the traumatic brain injury (TBI) outcome via inhibiting expression of AQP4 in astrocytes. The present study elucidated that AQP4 was up-regulated and was effectively blocked by APS in mice with severe controlled cortical impact (CCI). Pre-treatment with APS effectively inhibited the up-regulation of AQP4 and diminished the neurological deficits in mice. Additionally, primary astrocytes treated with mechanically-injured astrocyte supernatant, to mimic TBI in vitro, showed a significant up-regulation in swelling. We confirmed various signal molecules (NF-ĸB, MAPKs, and ERK) to have a role in astrocyte swelling, after activation in trauma, and to be involved in the up-regulation of AQP4. These signal molecules also significantly decreased with APS treatment. In conclusion, our study suggests that APS attenuated neurological deficits and brain edema by decreasing AQP4 up-regulation in astrocytes following TBI in mice, via reducing NF-ĸB, MAPKs, and the ERK signal molecules.

Ammonia induced microglia activation was associated with limited effects on connexin 43 and aquaporin 4 expression in an astrocyte-microglia co-culture model

Background Hepatic encephalopathy (HE) is a neurological complication resulting from acute or chronic liver disease. Hyperammonemia leading to astrocyte swelling and cerebral edema in combination with neuroinflammation including microglia activation, mainly contribute to the pathogenesis of HE. However, little is known about microglia and their inflammatory response, as well as their influence on astrocytic channels and astrocyte swelling under hyperammonemia. Objective To investigate the effects of ammonia on the microglial activation and morphology in different set-ups of an in vitro astrocyte-microglia co-culture model. Further, potential effects on glial viability, connexin 43 (Cx43) and aquaporin 4 (AQP4) expression were tested. Methods Primary rat glial co-cultures of astrocytes containing 5% (M5, representing "physiological" conditions) or 30% (M30, representing "pathological" conditions) of microglia were incubated with 3 mM, 5 mM, 10 mM and 20 mM ammonium chloride (NH4Cl) for 6 h and 24 h in order to mimic the conditions of HE. An MTT assay was performed to measure the viability, proliferation and cytotoxicity of cells. The microglial phenotypes were analyzed by immunocytochemistry. The expression of Cx43 and AQP4 were quantified by immunoblot analysis. Results A significant reduction of glial viability was observed in M30 co-cultures after incubation with 20 mM NH4Cl for 6 h, whereas in M5 co-cultures the viability remained unchanged. Microglial activation was detected by immunocytochemistry after incubation with 3 mM, 5 mM and 10 mM NH4Cl for 6 h and 24 h in M5 as well as in M30 co-cultures. The Cx43 expression was slightly increased in M30 co-cultures after 6 h incubation with 5 mM NH4Cl. Also, the AQP4 expression was slightly increased only in M5 co-cultures treated with 10 mM NH4Cl for 6 h. Under the other conditions, Cx43 and AQP4 expression was not affected by NH4Cl. Conclusions The novel aspect of our study was the significant microglial activation and decrease of viability after NH4Cl incubation in different set-ups of an in vitro astrocyte-microglia co-culture model, contributing to better understanding of pathophysiological mechanisms of HE. Hyperammonemia led to limited effects on Cx43 and AQP4 expression, the relevance of these minimal changes should be viewed with caution.

Malignant astrocyte swelling and impaired glutamate clearance drive the expansion of injurious spreading depolarization foci

Spreading depolarizations (SD) indicate infarct maturation and predict worse clinical outcome in ischemic stroke. We demonstrate here in rodents that brain edema formation upon ischemic stroke impairs astroglial glutamate clearance and increases the tissue area invaded by SD. The cytotoxic glutamate accumulation predisposes an extensive bulk of tissue for a yet undescribed simultaneous depolarization (SiD). We confirm in rat brain slices under hypo-osmotic stress that SiD is the pathological expansion of prior SD foci, is associated with astrocyte swelling and triggers oncotic neuron death. The blockade of astrocytic aquaporin-4 channels and Na+/K+/Cl- co-transporters, or volume-regulated anion channels mitigated slice edema, glutamate accumulation and SiD occurrence. Reversal of slice edema by hyperosmotic treatment counteracted glutamate accumulation and prevented SiD. In contrast, paralysis of astrocyte metabolism or inhibition of astrocyte glutamate uptake reproduced the SiD phenotype. We discuss our results in the light of evidence for SiD in the human cortex. Our results emphasize the need of preventive osmotherapy in ischemic stroke.

Ammonia Induced Microglial Activation Modulates Connexin 43 and Aquaporin 4 Expression in Astrocyte-microglia Co-culture Model

Background Hepatic encephalopathy (HE) is a neurological complication resulting from acute or chronic liver disease. Hyperammonemia leading to astrocyte swelling and cerebral edema in combination with neuroinflammation including microglia activation, mainly contribute to the pathogenesis of HE. However, little is known about microglia and their inflammatory response, as well as their influence on astrocytic channels and astrocyte swelling under hyperammonemia.Objective To investigate the effects of ammonia on the microglial activation, glial viability, connexin 43 (Cx43) and aquaporin 4 (AQP4) expression in astrocytes in in vitro astrocyte-microglia co-culture model.Methods Primary rat glial cocultures of astrocytes containing 5% (M5, representing "physiological" conditions) or 30% (M30, representing "pathological" conditions) of microglia were incubated with 3 mM, 5 mM, 10 mM and 20 mM ammonium chloride (NH4Cl) for 6 h and 24 h in order to mimic the conditions of HE. An MTT assay was performed to measure the viability, proliferation and cytotoxicity of cells. The microglial phenotypes were analysed by immunocytochemistry. The expression of Cx43 and AQP4 were quantified by immunoblot analysis.Results A significant reduction of glial viability was observed in M30 co-cultures after incubation with 20 mM NH4Cl for 6 h, whereas in M5 co-cultures the viability remained unchanged. Microglial activation was detected by immunocytochemistry after incubation with 3 mM, 5 mM and 10 mM NH4Cl for 6 h and 24 h in M5 as well as in M30 co-cultures. The Cx43 expression was increased significantly in M30 co-cultures after 6 h incubation with 5 mM NH4Cl. The AQP4 expression was increased significantly in M5 co-cultures treated with 10 mM NH4Cl for 6 h.Conclusions Our findings showed a significant microglial activation, decrease of viability and increase in Cx43 and AQP4 expression after NH4Cl incubation in astrocyte-microglia co-culture model. Based on previous in vitro studies suggesting that microglia activation influences astrocytic networks, it can be assumed that the microglial activation under hyperammonemia can modulate the Cx43 and AQP4 expression in astrocytes in a dynamic way and this can contribute to astrocytic dysfunction in HE.