LRRC8A is essential for swelling-activated chloride current and for regulatory volume decrease in astrocytes

Consolidated evidence indicates that astroglial cells are critical in the homeostatic regulation of cellular volume by means of ion channels and aquaporin-4. Volume-regulated anion channel (VRAC) is the chloride channel that is activated upon cell swelling and critically contributes to cell volume regulation in astrocytes. The molecular identity of VRAC has been recently defined, revealing that it belongs to the leucine-rich repeat-containing 8 (LRRC8) protein family. However, there is a lack of evidence demonstrating that LRRC8A underpins VRAC currents in astrocyte. Nonetheless, direct evidence of the role of LRRC8A in astrocytic regulatory volume decrease remains to be proved. Here, we aim to bridge this gap in knowledge by combining RNA interference specific for LRRC8A with patch-clamp analyses and a water-permeability assay. We demonstrated that LRRC8A molecular expression is essential for swelling-activated chloride current via VRAC in primary-cultured cortical astrocytes. The knockdown of LRRC8A with a specific short interference RNA abolished the recovery of the cell volume after swelling induced by hypotonic challenge. In addition, immunoblotting, immunofluorescence, confocal imaging, and immunogold electron microscopy demonstrated that LRRC8A is expressed in the plasma membrane of primary cortical astrocytes and in situ in astrocytes at the perivascular interface with endothelial cells. Collectively, our results suggest that LRRC8A is an essential subunit of VRAC and a key factor for astroglial volume homeostasis.-Formaggio, F., Saracino, E., Mola, M. G., Rao, S. B., Amiry-Moghaddam, M., Muccini, M., Zamboni, R., Nicchia, G. P., Caprini, M., Benfenati, V. LRRC8A is essential for swelling-activated chloride current and for regulatory volume decrease in astrocytes

Widespread distribution of lymphatic vessels in human dura mater remote from sinus veins

Background and purpose: Previous experimental studies have shown that meningeal lymphatic vessels are located primarily along the walls of the dural sinus veins. Whether they are more widespread throughout human dura mater has presently not been characterized. The present study explored in humans whether meningeal lymphatic vessels may be identified remote from the sinus veins and whether they differ in the various location of dura mater.Methods: We included 15 patients who underwent neurosurgery, in whom dura mater was removed as part of the planned procedure. Tissue was prepared for immunohistochemistry using the lymphatic endothelial cell markers lymphatic vessel endothelial hyaluronan receptor 1 protein (LYVE-1), podoplanin and vascular endothelial growth factor receptor 3 (VEGFR3).Results: Lymphatic endothelial cell positive cells were found in dura mater at the posterior fossa (n = 8), temporal skull base (n = 5), frontal convexity (n = 1), and cranio-cervical junction (n = 1). They were most commonly seen remote from blood vessels, but also occurred along blood vessels, and seemed to be most abundant at the skull base.Conclusion: The present observations show that human lymphatic vessels are widespread in dura mater, not solely lining the dural sinuses

Mechanisms underlying glial polarization in retina and brain

Glial cells, astrocytes and Müller glia, are involved in homeostatic functions in the CNS. Emanating from these cells are fine processes that interact with vasculature to form ‘glio-vascular’ interface. At this interface, the fine processes enwrap the vasculature and are called endfeet. At this site, K+ channel Kir4.1 and water channel aquaporin-4 (AQP4) are highly expressed in a polarized manner and are involved in buffering K+ and water, respectively. In several pathologies, this polarized distribution is lost suggesting an underlying mechanism that influence the polarity. In this thesis, Shreyas has explored the molecular mechanisms that are responsible for Kir4.1 and AQP4 polarity by use of novel transgenic mouse model. Evidence is provided that β1-syntrophin is involved in tethering Kir4.1 to the endfeet and that both α1- and β1-syntrophin are involved in polarized distribution of AQP4 in retina. Interestingly, β1-syntrophin plays no role in polarized distribution of AQP4 in the brain. These novel insights help us understand the basic physiology and provide future directions in search of causes and potential treatment strategies for diseases affecting the retina and brain