Valentina Benfenati

An aquaporin-4/transient receptor potential vanilloid 4 (AQP4/TRPV4) complex is essential for cell-volume control in astrocytes

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 brains volume homeostasis by acting as an osmosensor that couples osmotic stress to downstream signaling cascades.

Expression and functional characterization of transient receptor potential vanilloid-related channel 4 (TRPV4) in rat cortical astrocytes.

Cell-cell communication in astroglial syncytia is mediated by intracellular Ca(2+) ([Ca(2+)](i)) responses elicited by extracellular signaling molecules as well as by diverse physical and chemical stimuli. Despite the evidence that astrocytic swelling promotes [Ca(2+)](i) elevation through Ca(2+) influx, the molecular identity of the channel protein underlying this response is still elusive. Here we report that primary cultured cortical astrocytes express the transient receptor potential vanilloid-related channel 4 (TRPV 4), a Ca(2+)-permeable cation channel gated by a variety of stimuli, including cell swelling. Immunoblot and confocal microscopy analyses confirmed the presence of the channel protein and its localization in the plasma membrane. TRPV4 was functional because the selective TRPV4 agonist 4-alpha-phorbol 12,13-didecanoate (4alphaPDD) activated an outwardly rectifying cation current with biophysical and pharmacological properties that overlapped those of recombinant human TRPV4 expressed in COS cells. Moreover, 4alphaPDD and hypotonic challenge promoted [Ca(2+)](i) elevation mediated by influx of extracellular Ca(2+). This effect was abolished by low micromolar concentration of the TRPV4 inhibitor Ruthenium Red. Immunofluorescence and immunogold electron microscopy of rat brain revealed that TRPV4 was enriched in astrocytic processes of the superficial layers of the neocortex and in astrocyte end feet facing pia and blood vessels. Collectively, these data indicate that cultured cortical astroglia express functional TRPV4 channels. They also demonstrate that TRPV4 is particularly abundant in astrocytic membranes at the interface between brain and extracerebral liquid spaces. Consistent with its roles in other tissues, these results support the view that TRPV4 might participate in astroglial osmosensation and thus play a key role in brain volume homeostasis.

A transparent organic transistor structure for bidirectional stimulation and recording of primary neurons

Real-time stimulation and recording of neural cell bioelectrical activity could provide an unprecedented insight in understanding the functions of the nervous system, and it is crucial for developing advanced in vitro drug screening approaches. Among organic materials, suitable candidates for cell interfacing can be found that combine long-term biocompatibility and mechanical flexibility. Here, we report on transparent organic cell stimulating and sensing transistors (O-CSTs), which provide bidirectional stimulation and recording of primary neurons. We demonstrate that the device enables depolarization and hyperpolarization of the primary neuron membrane potential. The transparency of the device also allows the optical imaging of the modulation of the neuron bioelectrical activity. The maximal amplitude-to-noise ratio of the extracellular recording achieved by the O-CST device exceeds that of a microelectrode array system on the same neuronal preparation by a factor of 16. Our organic cell stimulating and sensing device paves the way to a new generation of devices for stimulation, manipulation and recording of cell bioelectrical activity in vitro and in vivo.

The Increased Activity of TRPV4 Channel in the Astrocytes of the Adult Rat Hippocampus after Cerebral Hypoxia/Ischemia

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.

Water transport between CNS compartments: functional and molecular interactions between aquaporins and ion channels.

The physiological ability of the mammalian CNS to integrate peripheral stimuli and to convey information to the body is tightly regulated by its capacity to preserve the ion composition and volume of the perineuronal milieu. It is well known that astroglial syncytium plays a crucial role in such process by controlling the homeostasis of ions and water through the selective transmembrane movement of inorganic and organic molecules and the equilibration of osmotic gradients. Astrocytes, in fact, by contacting neurons and cells lining the fluid-filled compartments, are in a strategic position to fulfill this role. They are endowed with ion and water channel proteins that are localized in specific plasma membrane domains facing diverse liquid spaces. Recent data in rodents have demonstrated that the precise dynamics of the astroglia-mediated homeostatic regulation of the CNS is dependent on the interactions between water channels and ion channels, and their anchoring with proteins that allow the formation of macromolecular complexes in specific cellular domains. Interplay can occur with or without direct molecular interactions suggesting the existence of different regulatory mechanisms. The importance of molecular and functional interactions is pinpointed by the numerous observations that as consequence of pathological insults leading to the derangement of ion and volume homeostasis the cell surface expression and/or polarized localization of these proteins is perturbed. Here, we critically discuss the experimental evidence concerning: (1) molecular and functional interplay of aquaporin 4, the major aquaporin protein in astroglial cells, with potassium and gap-junctional channels that are involved in extracellular potassium buffering. (2) the interactions of aquaporin 4 with chloride and calcium channels regulating cell volume homeostasis. The relevance of the crosstalk between water channels and ion channels in the pathogenesis of astroglia-related acute and chronic diseases of the CNS is also briefly discussed.

Low-threshold blue lasing from silk fibroin thin films

Silk is a natural biocompatible material that can be integrated in a variety of photonic systems and optoelectronic devices. The silk replication of patterned substrates with features down to tens of nanometers is exploited to realize highly transparent, mechanically stable, and free-standing structures with optical wavelength size. We demonstrate organic lasing from a blue-emitting stilbene-doped silk film spin-coated onto a one-dimensional distributed feedback grating (DFB). The lasing threshold is lower than that of organic DFB lasers based on the same active dye. These findings pave the way to the development of an optically active biocompatible technological platform based on silk.

Carbenoxolone inhibits volume-regulated anion conductance in cultured rat cortical astroglia.

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.

Functional down-regulation of volume-regulated anion channels in AQP4 knockdown cultured rat cortical astrocytes

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.

A silk platform that enables electrophysiology and targeted drug delivery in brain astroglial cells

Astroglial cell survival and ion channel activity are relevant molecular targets for the mechanistic study of neural cell interactions with biomaterials and/or electronic interfaces. Astrogliosis is the most typical reaction to in vivo brain implants and needs to be avoided by developing biomaterials that preserve astroglial cell physiological function. This cellular phenomenon is characterized by a proliferative state and altered expression of astroglial potassium (K(+)) channels. Silk is a natural polymer with potential for new biomedical applications due to its ability to support in vitro growth and differentiation of many cell types. We report on silk interactions with cultured neocortical astroglial cells. Astrocytes survival is similar when plated on silk-coated glass and on poly-D-lysine (PDL), a well known polyionic substrate used to promote astroglial cell adhesion to glass surfaces. Comparative analyses of whole-cell patch-clamp experiments reveal that silk- and PDL-coated cells display depolarized resting membrane potentials (-40 mV), very high input resistance, and low specific conductance, with values similar to those of undifferentiated glial cells. Analysis of K(+) channel conductance reveals that silk-astrocytes express large outwardly delayed rectifying K(+) current (K(DR)). The magnitude of K(DR) in PDL- and silk-coated astrocytes is similar, indicating that silk does not alter the resting K(+) current. We also demonstrate that guanosine- (GUO) embedded silk enables the direct modulation of astroglial K(+) conductance in vitro. Astrocytes plated on GUO-embedded silk are more hyperpolarized and express inward rectifying K(+) conductance (K(ir)). The K(+) inward current increases and this is paralleled by upregulation and membrane polarization of K(ir)4.1 protein signal. Collectively these results indicate that silk is a suitable biomaterial platform for the in vitro studies of astroglial ion channel responses and related physiology.

Photostimulation of Whole-Cell Conductance in Primary Rat Neocortical Astrocytes Mediated by Organic Semiconducting Thin Films

Astroglial ion channels are fundamental molecular targets in the study of brain physiology and pathophysiology. Novel tools and devices intended for stimulation and control of astrocytes ion channel activity are therefore highly desirable. The study of the interactions between astrocytes and biomaterials is also essential to control and minimize reactive astrogliosis, in view of the development of implantable functional devices. Here, the growth of rat primary neocortical astrocytes on the top of a light sensitive, organic polymer film is reported; by means of patch-clamp analyses, the effect of the visible light stimulation on membrane conductance is then determined. Photoexcitation of the active material causes a significant depolarization of the astroglial resting membrane potential: the effect is associated to an increase in whole-cell conductance at negative potentials. The magnitude of the evoked inward current density is proportional to the illumination intensity. Biophysical and pharmacological characterization suggests that the ion channel mediating the photo-transduction mechanism is a chloride channel, the ClC-2 channel. These results open interesting perspectives for the selective manipulation of astrocyte bioelectrical activity by non-invasive, label-free, organic-based, photostimulation approaches.