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

AQP4-independent TRPV4 modulation of plasma membrane water permeability

: Despite of the major role of aquaporin (AQP) water channels in controlling transmembrane water fluxes, alternative ways for modulating water permeation have been proposed. In the Central Nervous System (CNS), Aquaporin-4 (AQP4) is reported to be functionally coupled with the calcium-channel Transient-Receptor Potential Vanilloid member-4 (TRPV4), which is controversially involved in cell volume regulation mechanisms and water transport dynamics. The present work aims to investigate the selective role of TRPV4 in regulating plasma membrane water permeability in an AQP4-independent way. Fluorescence-quenching water transport experiments in Aqp4-/- astrocytes revealed that cell swelling rate is significantly increased upon TRPV4 activation and in the absence of AQP4. The biophysical properties of TRPV4-dependent water transport were therefore assessed using the HEK-293 cell model. Calcein quenching experiments showed that chemical and thermal activation of TRPV4 overexpressed in HEK-293 cells leads to faster swelling kinetics. Stopped-flow light scattering water transport assay was used to measure the osmotic permeability coefficient (Pf, cm/s) and activation energy (Ea, kcal/mol) conferred by TRPV4. Results provided evidence that although the Pf measured upon TRPV4 activation is lower than the one obtained in AQP4-overexpressing cells (Pf of AQP4 = 0.01667 ± 0.0007; Pf of TRPV4 = 0.002261 ± 0.0004; Pf of TRPV4 + 4αPDD = 0.007985 ± 0.0006; Pf of WT = 0.002249 ± 0.0002), along with activation energy values (Ea of AQP4 = 0.86 ± 0.0006; Ea of TRPV4 + 4αPDD = 2.73 ± 1.9; Ea of WT = 8.532 ± 0.4), these parameters were compatible with a facilitated pathway for water movement rather than simple diffusion. The possibility to tune plasma membrane water permeability more finely through TRPV4 might represent a protective mechanism in cells constantly facing severe osmotic challenges to avoid the potential deleterious effects of the rapid cell swelling occurring via AQP channels

Cell Volume Regulation Mechanisms in Differentiated Astrocytes

The ability of astrocytes to control extracellular volume homeostasis is critical for brain function and pathology. Uncovering the mechanisms of cell volume regulation by astrocytes will be important for identifying novel therapeutic targets for neurological conditions, such as those characterized by imbalances to hydro saline challenges (as in edema) or by altered cell volume regulation (as in glioma). One major challenge in studying the astroglial membrane channels involved in volume homeostasis in cell culture model systems is that the expression patterns of these membrane channels do not resemble those observed in vivo. In our previous study, we demonstrated that rat primary astrocytes grown on nanostructured interfaces based on hydrotalcite-like compounds (HTlc) in vitro are differentiated and display molecular and functional properties of in vivo astrocytes, such as the functional expression of inwardly rectifying K+ channel (Kir 4.1) and Aquaporin-4 (AQP4) at the astrocytic microdomain. Here, we take advantage of the properties of differentiated primary astrocytes in vitro to provide an insight into the mechanism underpinning astrocytic cell volume regulation and its correlation with the expression and function of AQP4, Transient Receptor Potential Vanilloid 4 (TRPV4), and Volume Regulated Anion Channel (VRAC)

Alpha-synuclein/synapsin III pathological interplay boosts the motor response to methylphenidate

: Loss of dopaminergic nigrostriatal neurons and fibrillary α-synuclein (α-syn) aggregation in Lewy bodies (LB) characterize Parkinson's disease (PD). We recently found that Synapsin III (Syn III), a phosphoprotein regulating dopamine (DA) release with α-syn, is another key component of LB fibrils in the brain of PD patients and acts as a crucial mediator of α-syn aggregation and toxicity. Methylphenidate (MPH), a monoamine reuptake inhibitor (MRI) efficiently counteracting freezing of gait in advanced PD patients, can bind α-syn and controls α-syn-mediated DA overflow and presynaptic compartmentalization. Interestingly, MPH results also efficient for the treatment of attention deficits and hyperactivity disorder (ADHD), a neurodevelopmental psychiatric syndrome associated with Syn III and α-syn polymorphisms and constituting a risk factor for the development of LB disorders. Here, we studied α-syn/Syn III co-deposition and longitudinal changes of α-syn, Syn III and DA transporter (DAT) striatal levels in nigrostriatal neurons of a PD model, the human C-terminally truncated (1-120) α-syn transgenic (SYN120 tg) mouse, in comparison with C57BL/6J wild type (wt) and C57BL/6JOlaHsd α-syn null littermates. Then, we analyzed the locomotor response of these animals to an acute administration of MPH (d-threo) and other MRIs: cocaine, that we previously found to stimulate Syn III-reliant DA release in the absence of α-syn, or the selective DAT blocker GBR-12935, along aging. Finally, we assessed whether these drugs modulate α-syn/Syn III interaction by fluorescence resonance energy transfer (FRET) and performed in silico studies engendering a heuristic model of the α-syn conformations stabilized upon MPH binding. We found that only MPH was able to over-stimulate a Syn III-dependent/DAT-independent locomotor activity in the aged SYN120 tg mice showing α-syn/Syn III co-aggregates. MPH enhanced full length (fl) α-syn/Syn III and even more (1-120) α-syn/Syn III interaction in cells exhibiting α-syn/Syn III inclusions. Moreover, in silico studies confirmed that MPH may reduce α-syn fibrillation by stabilizing a protein conformation with increased lipid binding predisposition. Our observations indicate that the motor-stimulating effect of MPH can be positively fostered in the presence of α-syn/Syn III co-aggregation. This evidence holds significant implications for PD and ADHD therapeutic management

Sulfonates-PMMA nanoparticles conjugates: A versatile system for multimodal application

a b s t r a c t We report herein the viability of a novel nanoparticles (NPs) conjugated system, namely the attachment, based on ionic and hydrophobic interactions, of different sulfonated organic salts to positively charged poly(methylmethacrylate) (PMMA)-based core-shell nanoparticles (EA0) having an high density of ammonium groups on their shells. In this context three different applications of the sulfonates@EA0 systems have been described. In detail, their ability as cytotoxic drugs and pro-drugs carriers was evaluated in vitro on NCI-H460 cell line and in vivo against human ovarian carcinoma IGROV-1 cells. Besides, 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS) was chosen for NPs loading, and its internalization as bioimaging probe was evaluated on Hep G2 cells. Overall, the available data support the interest for these PMMA NPs@sulfonates systems as a promising formulation for theranostic applications. In vivo biological data strongly support the potential value of these core-shell NPs as delivery system for negatively charged drugs or biologically active molecules. Additionally, we have demonstrated the ability of these PMMA core-shell nanoparticles to act as efficient carriers of fluorophores. In principle, thanks to the high PMMA NPs external charge density, sequential and very easy post-loading of different sulfonates is achievable, thus allowing the preparation of nanocarriers either with bi-modal drug delivery behaviour or as theranostic systems

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

Biomimetic graphene for enhanced interaction with the external membrane of astrocytes

Graphene and graphene substrates display huge potential as material interfaces for devices and biomedical tools targeting the modulation or recovery of brain functionality. However, to be considered reliable neural interfaces, graphene-derived substrates should properly interact with astrocytes, favoring their growth and avoiding adverse gliotic reactions. Indeed, astrocytes are the most abundant cells in the human brain and they have a crucial physiological role to maintain its homeostasis and modulate synaptic transmission. In this work, we describe a new strategy based on the chemical modification of graphene oxide (GO) with a synthetic phospholipid (PL) to improve interaction of GO with brain astroglial cells. The PL moieties were grafted on GO sheets through polymeric brushes obtained by atom-transfer radical-polymerization (ATRP) between acryloyl-modified PL and GO nanosheets modified with a bromide initiator. The adhesion of primary rat cortical astrocytes on GO–PL substrates increased by about three times with respect to that on glass substrates coated with standard adhesion agents (i.e. poly-D-lysine, PDL) as well as with respect to that on non-functionalized GO. Moreover, we show that astrocytes seeded on GO–PL did not display significant gliotic reactivity, indicating that the material interface did not cause a detrimental inflammatory reaction when interacting with astroglial cells. Our results indicate that the reported biomimetic approach could be applied to neural prosthesis to improve cell colonization and avoid glial scar formation in brain implants. Additionally, improved adhesion could be extremely relevant in devices targeting neural cell sensing/modulation of physiological activity