Regulatory volume decrease (RVD) is a key mechanism for volume control that serves to prevent detrimental swelling in response to hypo-osmotic stress. The molecular basis of RVD is not understood. ...Here we show that a complex containing aquaporin-4 (AQP4) and transient receptor potential vanilloid 4 (TRPV4) is essential for RVD in astrocytes. Astrocytes from AQP4-KO mice and astrocytes treated with TRPV4 siRNA fail to respond to hypotonic stress by increased intracellular Ca²⁺ and RVD. Coimmunoprecipitation and immunohistochemistry analyses show that AQP4 and TRPV4 interact and colocalize. Functional analysis of an astrocyte-derived cell line expressing TRPV4 but not AQP4 shows that RVD and intracellular Ca²⁺ response can be reconstituted by transfection with AQP4 but not with aquaporin-1. Our data indicate that astrocytes contain a TRPV4/AQP4 complex that constitutes a key element in the brain's volume homeostasis by acting as an osmosensor that couples osmotic stress to downstream signaling cascades.
Through their ability to modulate synaptic transmission, glial cells are key regulators of neuronal circuit formation and activity. Kidins220/ARMS (kinase-D interacting substrate of 220 kDa/ankyrin ...repeat-rich membrane spanning) is one of the key effectors of the neurotrophin pathways in neurons where it is required for differentiation, survival, and plasticity. However, its role in glial cells remains largely unknown. Here, we show that ablation of Kidins220 in primary cultured astrocytes induced defects in calcium (Ca
) signaling that were linked to altered store-operated Ca
entry and strong overexpression of the transient receptor potential channel TRPV4. Moreover, Kidins220
astrocytes were more sensitive to genotoxic stress. We also show that Kidins220 expression in astrocytes is required for the establishment of proper connectivity of cocultured wild-type neurons. Altogether, our data reveal a previously unidentified role for astrocyte-expressed Kidins220 in the control of glial Ca
dynamics, survival/death pathways and astrocyte-neuron communication.
The polymodal transient receptor potential vanilloid 4 (TRPV4) channel, a member of the TRP channel family, is a calcium-permeable cationic channel that is gated by various stimuli such as cell ...swelling, low pH and high temperature. Therefore, TRPV4-mediated calcium entry may be involved in neuronal and glia pathophysiology associated with various disorders of the central nervous system, such as ischemia. The TRPV4 channel has been recently found in adult rat cortical and hippocampal astrocytes; however, its role in astrocyte pathophysiology is still not defined. In the present study, we examined the impact of cerebral hypoxia/ischemia (H/I) on the functional expression of astrocytic TRPV4 channels in the adult rat hippocampal CA1 region employing immunohistochemical analyses, the patch-clamp technique and microfluorimetric intracellular calcium imaging on astrocytes in slices as well as on those isolated from sham-operated or ischemic hippocampi. Hypoxia/ischemia was induced by a bilateral 15-minute occlusion of the common carotids combined with hypoxic conditions. Our immunohistochemical analyses revealed that 7 days after H/I, the expression of TRPV4 is markedly enhanced in hippocampal astrocytes of the CA1 region and that the increasing TRPV4 expression coincides with the development of astrogliosis. Additionally, adult hippocampal astrocytes in slices or cultured hippocampal astrocytes respond to the TRPV4 activator 4-alpha-phorbol-12,-13-didecanoate (4αPDD) by an increase in intracellular calcium and the activation of a cationic current, both of which are abolished by the removal of extracellular calcium or exposure to TRP antagonists, such as Ruthenium Red or RN1734. Following hypoxic/ischemic injury, the responses of astrocytes to 4αPDD are significantly augmented. Collectively, we show that TRPV4 channels are involved in ischemia-induced calcium entry in reactive astrocytes and thus, might participate in the pathogenic mechanisms of astroglial reactivity following ischemic insult.
Direct cell reprogramming enables direct conversion of fibroblasts into functional neurons and oligodendrocytes using a minimal set of cell-lineage-specific transcription factors. This approach is ...rapid and simple, generating the cell types of interest in one step. However, it remains unknown whether this technology can be applied to convert fibroblasts into astrocytes, the third neural lineage. Astrocytes play crucial roles in neuronal homeostasis, and their dysfunctions contribute to the origin and progression of multiple human diseases. Herein, we carried out a screening using several transcription factors involved in defining the astroglial cell fate and identified NFIA, NFIB, and SOX9 to be sufficient to convert with high efficiency embryonic and postnatal mouse fibroblasts into astrocytes (iAstrocytes). We proved both by gene-expression profiling and functional tests that iAstrocytes are comparable to native brain astrocytes. This protocol can be then employed to generate functional iAstrocytes for a wide range of experimental applications.
Accumulating evidence indicates that increased intracellular Na+ concentration (Na+i) in astroglial cells is associated with the development of brain edema under ischemic conditions, but the ...underlying mechanisms are still elusive. Here, we report that in primary cultured rat cortical astrocytes, elevations of Na+i reflecting those achieved during ischemia cause a marked decrease in hypotonicity‐evoked current mediated by volume‐regulated anion channel (VRAC). Pharmacological manipulations revealed that VRAC inhibition was not due to the reverse mode of the plasma membrane sodium/calcium exchanger. The negative modulation of VRAC was also observed in an astrocytic cell line lacking the predominant astrocyte water channel aquaporin 4, indicating that Na+i effect was not mediated by the regulation of aquaporin 4 activity. The inward rectifier Cl− current, which is also expressed by cultured astrocytes, was not affected by Na+i increase. VRAC depression by high Na+i was confirmed in adult astrocytes, suggesting that it was not developmentally regulated. Altogether, these results provide the first evidence that intracellular Na+ dynamics can modulate astrocytic membrane conductance that controls functional processes linked to cell volume regulation and add further support to the concept that limiting astrocyte intracellular Na+ accumulation might be a favorable strategy to counteract the development of brain edema.
We show that high elevation of intracellular sodium (Na+) concentration in cultured rat astrocytes decreases the activity of volume‐regulated anion channel (VRAC) measured by patch‐clamp technique. We speculate that in ischemic conditions intracellular Na+ elevation either through the augmented activity of the Na+‐dependent glutamate transporter (GLT‐1) or by Na+‐permeable channels could modulate the astrocytic volume and glutamate release (Glu−) regulated by VRAC. Whether VRAC inhibition is mediated through cytosolic mechanisms or by the alteration of mitochondrial activity remains to be determined.
We show that high elevation of intracellular sodium (Na+) concentration in cultured rat astrocytes decreases the activity of volume‐regulated anion channel (VRAC) measured by patch‐clamp technique. We speculate that in ischemic conditions intracellular Na+ elevation either through the augmented activity of the Na+‐dependent glutamate transporter (GLT‐1) or by Na+‐permeable channels could modulate the astrocytic volume and glutamate release (Glu−) regulated by VRAC. Whether VRAC inhibition is mediated through cytosolic mechanisms or by the alteration of mitochondrial activity remains to be determined.
In the brain, the astroglial syncytium is crucially involved in the regulation of water homeostasis. Accumulating evidence indicates that a dysregulation of the astrocytic processes controlling water ...homeostasis has a pathogenetic role in several brain injuries. Here, we have analysed by RNA interference technology the functional interactions occurring between the most abundant water channel in the brain, aquaporin-4 (AQP4), and the swelling-activated Cl⁻ current expressed by cultured rat cortical astrocytes. We show that in primary cultured rat cortical astrocytes transfected with control small interfering RNA (siRNA), hypotonic shock promotes an increase in cellular volume accompanied by augmented membrane conductance mediated by volume-regulated anion channels (VRAC). Conversely, astroglia in which AQP4 was knocked down (AQP4 KD) by transfection with AQP4 siRNA changed their morphology from polygonal to process-bearing, and displayed normal cell swelling but reduced VRAC activity. Pharmacological manipulations of actin cytoskeleton in rat astrocytes, and functional analysis in mouse astroglial cells, which retain their morphology upon knockdown of AQP4, suggest that stellation of AQP4 KD rat cortical astrocytes was not causally linked to reduction of VRAC current. Molecular analysis of possible candidates of swelling-activated Cl⁻ current provided evidence that in AQP4 KD astrocytes, there was a down-regulation of chloride channel-2 (CIC-2), which, however, was not involved in VRAC conductance. Inclusion of ATP in the intracellular saline restored VRAC activity upon hypotonicity. Collectively, these results support the view that in cultured astroglial cells, plasma membrane proteins involved in cell volume homeostasis are assembled in a functional platform.
In the brain, arachidonic acid (AA) plays a critical role in the modulation of a broad spectrum of biological responses, including those underlying neuroinflammation. By using microfluorometry, we ...investigated the action of extracellular AA in the modulation of the purinoceptor P2X7-mediated elevation of Ca(2+)(i) in cultured neocortical type-1 astrocytes and P2X7-, P2X2-transfected human embryonic kidney (HEK) 293 cells. We report that in cultured astrocytes, AA-induced Ca(2+)(i) elevation is coupled to depletion of intracellular Ca(2+) stores and to a sustained noncapacitative Ca(2+) entry. AA also induced a robust potentiation of the astrocytic P2X7-mediated Ca(2+)(i) rise evoked by the selective agonist 3'-O-(4-benzoyl)benzoyl-ATP (BzATP). Pharmacological studies demonstrate that the selective P2X7 antagonists oxidized ATP and Brilliant Blue G abrogated the AA-mediated potentiation of BzATP-evoked Ca(2+)(i) elevation. Fluorescent dye uptake experiments showed that the AA-induced increase in Ca(2+)(i) was not due to a switch of the P2X7 receptor from channel to the pore mode of gating. The synergistic effect of AA and BzATP was also observed in HEK293 cells stably expressing rat and human P2X7 but not in rat P2X2. Control HEK293 cells responded to AA exposure only with a transient Ca(2+)(i) elevation, whereas in those expressing the P2X7 receptor, AA elicited a potentiation of the BzATP-induced Ca(2+)(i) rise. Together, these findings indicate that AA mediates a complex regulation of Ca(2+)(i) dynamics also through P2X7-mediated Ca(2+) entry, suggesting that variations in AA production may be relevant to the control of both the temporal and spatial kinetics of Ca(2+)(i) signaling in astroglial cells.
Guanosine (Guo) is an endogenous neuroprotective molecule of the CNS, which has various acute and long‐term effects on both neurones and astroglial cells. Whether Guo also modulates the ...activity/expression of ion channels involved in homeostatic control of extracellular potassium by the astrocytic syncytium is still unknown. Here we provide electrophysiological evidence that chronic exposure (48 h) to Guo (500 μm) promotes the functional expression of an inward rectifier K+ (Kir) conductance in primary cultured rat cortical astrocytes. Molecular screening indicated that Guo promotes the up‐regulation of the Kir4.1 channel, the major component of the Kir current in astroglia in vivo. Furthermore, the properties of astrocytic Kir current overlapped those of the recombinant Kir4.1 channel expressed in a heterologous system, strongly suggesting that the Guo‐induced Kir conductance is mainly gated by Kir4.1. In contrast, the expression levels of two other Kir channel proteins were either unchanged (Kir2.1) or decreased (Kir5.1). Finally, we showed that inhibition of translational process, but not depression of transcription, prevents the Guo‐induced up‐regulation of Kir4.1, indicating that this nucleoside acts through de novo protein synthesis. Because accumulating data indicate that down‐regulation of astroglial Kir current contributes to the pathogenesis of neurodegenerative diseases associated with dysregulation of extracellular K+ homeostasis, these results support the notion that Guo might be a molecule of therapeutic interest for counteracting the detrimental effect of K+‐buffering impairment of the astroglial syncytium that occurs in pathological conditions.
Accumulating evidence indicate that the gap-junction inhibitor carbenoxolone (CBX) regulates neuronal synchronization, depresses epileptiform activity and has a neuroprotective action. These CBX ...effects do not depend solely on its ability to inhibit gap junction channels formed by connexins (Cx), but the underlying mechanisms remain to be elucidated. Here we addressed the questions whether CBX modulates volume-regulated anion channels (VRAC) involved in the regulatory volume decrease and regulates the associated release of excitatory amino acids in cultured rat cortical astrocytes. We found that CBX inhibits VRAC conductance with potency comparable to that able to depress the activity of the most abundant astroglial gap junction protein connexin43 (Cx43). However, the knock down of Cx43 with small interfering RNA (siRNA) oligonucleotides and the use of various pharmacological tools revealed that VRAC inhibition was not mediated by interaction of CBX with astroglial Cx proteins. Comparative experiments in HEK293 cells stably expressing another putative target of CBX, the purinergic ionotropic receptor P2X7, indicate that the presence of this receptor was not necessary for CBX-mediated depression of VRAC. Finally, we show that in COS-7 cells, which are not endowed with pannexin-1 protein, another astroglial plasma membrane interactor of CBX, VRAC current retained its sensitivity to CBX. Complementary analyses indicate that the VRAC-mediated release of excitatory amino acid aspartate was decreased by CBX. Collectively, these findings support the notion that CBX could affect astroglial ability to modulate neuronal activity by suppressing excitatory amino acid release through VRAC. They also provide a possible mechanistic clue for the neuroprotective effect of CBX in vivo.
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