The CaMKII/MLC1 Axis Confers Ca2+-Dependence to Volume-Regulated Anion Channels (VRAC) in Astrocytes.

Astrocytes, the main glial cells of the central nervous system, play a key role in brain volume control due to their intimate contacts with cerebral blood vessels and the expression of a distinctive equipment of proteins involved in solute/water transport. Among these is MLC1, a protein highly expressed in perivascular astrocytes and whose mutations cause megalencephalic leukoencephalopathy with subcortical cysts (MLC), an incurable leukodystrophy characterized by macrocephaly, chronic brain edema, cysts, myelin vacuolation, and astrocyte swelling. Although, in astrocytes, MLC1 mutations are known to affect the swelling-activated chloride currents (ICl,swell) mediated by the volume-regulated anion channel (VRAC), and the regulatory volume decrease, MLC1's proper function is still unknown. By combining molecular, biochemical, proteomic, electrophysiological, and imaging techniques, we here show that MLC1 is a Ca2+/Calmodulin-dependent protein kinase II (CaMKII) target protein, whose phosphorylation, occurring in response to intracellular Ca2+ release, potentiates VRAC-mediated ICl,swell. Overall, these findings reveal that MLC1 is a Ca2+-regulated protein, linking volume regulation to Ca2+ signaling in astrocytes. This knowledge provides new insight into the MLC1 protein function and into the mechanisms controlling ion/water exchanges in the brain, which may help identify possible molecular targets for the treatment of MLC and other pathological conditions caused by astrocyte swelling and brain edema.

Megalencephalic Leukoencephalopathy with Subcortical Cysts Disease-Linked MLC1 Protein Favors Gap-Junction Intercellular Communication by Regulating Connexin 43 Trafficking in Astrocytes.

Astrocytes, the most numerous cells of the central nervous system, exert critical functions for brain homeostasis. To this purpose, astrocytes generate a highly interconnected intercellular network allowing rapid exchange of ions and metabolites through gap junctions, adjoined channels composed of hexamers of connexin (Cx) proteins, mainly Cx43. Functional alterations of Cxs and gap junctions have been observed in several neuroinflammatory/neurodegenerative diseases. In the rare leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC), astrocytes show defective control of ion/fluid exchanges causing brain edema, fluid cysts, and astrocyte/myelin vacuolation. MLC is caused by mutations in MLC1, an astrocyte-specific protein of elusive function, and in GlialCAM, a MLC1 chaperon. Both proteins are highly expressed at perivascular astrocyte end-feet and astrocyte-astrocyte contacts where they interact with zonula occludens-1 (ZO-1) and Cx43 junctional proteins. To investigate the possible role of Cx43 in MLC pathogenesis, we studied Cx43 properties in astrocytoma cells overexpressing wild type (WT) MLC1 or MLC1 carrying pathological mutations. Using biochemical and electrophysiological techniques, we found that WT, but not mutated, MLC1 expression favors intercellular communication by inhibiting extracellular-signal-regulated kinase 1/2 (ERK1/2)-mediated Cx43 phosphorylation and increasing Cx43 gap-junction stability. These data indicate MLC1 regulation of Cx43 in astrocytes and Cx43 involvement in MLC pathogenesis, suggesting potential target pathways for therapeutic interventions.

Megalencephalic Leukoencephalopathy with Subcortical Cysts Protein-1 (MLC1) Counteracts Astrocyte Activation in Response to Inflammatory Signals.

Megalencephalic leukoencephalopathy with subcortical cysts protein-1 (MLC1) is a membrane protein expressed by perivascular astrocytes. MLC1 mutations cause MLC, an incurable leukodystrophy characterized by macrocephaly, brain edema, cysts, myelin vacuolation, and astrocytosis, leading to cognitive/motor impairment and epilepsy. Although its function is unknown, MLC1 favors regulatory volume decrease after astrocyte osmotic swelling and down-regulates intracellular signaling pathways controlling astrocyte activation and proliferation. By combining analysis of human brain tissues with in vitro experiments, here we investigated MLC1 role in astrocyte activation during neuroinflammation, a pathological condition exacerbating patient symptoms. MLC1 upregulation was observed in brain tissues from multiple sclerosis, Alzheimer's, and Creutzfeld-Jacob disease, all pathologies characterized by strong astrocytosis and release of inflammatory cytokines, particularly IL-1ß. Using astrocytoma lines overexpressing wild-type (WT) or mutated MLC1 and astrocytes from control and Mlc1 knock-out (KO) mice, we found that IL-1ß stimulated WT-MLC1 plasma membrane expression in astrocytoma cells and control primary astrocytes. In astrocytoma, WT-MLC1 inhibited the activation of IL-1ß-induced inflammatory signals (pERK, pNF-kB) that, conversely, were constitutively activated in mutant expressing cells or abnormally upregulated in KO astrocytes. WT-MLC1+ cells also expressed reduced levels of the astrogliosis marker pSTAT3. We then monitored MLC1 expression timing in a demyelinating/remyelinating murine cerebellar organotypic culture model where, after the demyelination and release of inflammatory cytokines, recovery processes occur, revealing MLC1 upregulation in these latter phases. Altogether, these findings suggest that by modulating specific pathways, MLC1 contributes to restore astrocyte homeostasis after inflammation, providing the opportunity to identify drug target molecules to slow down disease progression.

Megalencephalic leukoencephalopathy with subcortical cysts protein-1 regulates epidermal growth factor receptor signaling in astrocytes.

Mutations in the MLC1 gene, which encodes a protein expressed in brain astrocytes, are the leading cause of MLC, a rare leukodystrophy characterized by macrocephaly, brain edema, subcortical cysts, myelin and astrocyte vacuolation. Although recent studies indicate that MLC1 protein is implicated in the regulation of cell volume changes, the exact role of MLC1 in brain physiology and in the pathogenesis of MLC disease remains to be clarified. In preliminary experiments, we observed that MLC1 was poorly expressed in highly proliferating astrocytoma cells when compared with primary astrocytes, and that modulation of MLC1 expression influenced astrocyte growth. Because volume changes are key events in cell proliferation and during brain development MLC1 expression is inversely correlated to astrocyte progenitor proliferation levels, we investigated the possible role for MLC1 in the control of astrocyte proliferation. We found that overexpression of wild type but not mutant MLC1 in human astrocytoma cells hampered cell growth by favoring epidermal growth factor receptor (EGFR) degradation and by inhibiting EGF-induced Ca(+) entry, ERK1/2 and PLCγ1 activation, and calcium-activated KCa3.1 potassium channel function, all molecular pathways involved in astrocyte proliferation stimulation. Interestingly, MLC1 did not influence AKT, an EGFR-stimulated kinase involved in cell survival. Moreover, EGFR expression was higher in macrophages derived from MLC patients than from healthy individuals. Since reactive astrocytes proliferate and re-express EGFR in response to different pathological stimuli, the present findings provide new information on MLC pathogenesis and unravel an important role for MLC1 in other brain pathological conditions where astrocyte activation occurs.

Native metastable prefibrillar oligomers are the most neurotoxic species among amyloid aggregates.

Many proteins belonging to the amyloid family share the tendency to misfold and aggregate following common steps, and display similar neurotoxicity. In the aggregation pathway different kinds of species are formed, including several types of oligomers and eventually mature fibers. It is now suggested that the pathogenic aggregates are not the mature fibrils, but the intermediate, soluble oligomers. Many kinds of aggregates have been described to exist in a metastable state and in equilibrium with monomers. Up to now it is not clear whether a specific structure is at the basis of the neurotoxicity. Here we characterized, starting from the early aggregation stages, the oligomer populations formed by an amyloid protein, salmon calcitonin (sCT), chosen due to its very slow aggregation rate. To prepare different oligomer populations and characterize them by means of photoinduced cross-linking SDS-PAGE, Energy Filtered-Transmission Electron Microscopy (EF-TEM) and Circular Dichroism (CD) spectroscopy, we used Size Exclusion Chromatography (SEC), a technique that does not influence the aggregation process leaving the protein in the native state. Taking advantage of sCT low aggregation rate, we characterized the neurotoxic potential of the SEC-separated, non-crosslinked fractions in cultured primary hippocampal neurons, analyzing intracellular Ca(2+) influx and apoptotic trend. We provide evidence that native, globular, metastable, prefibrillar oligomers (dimers, trimers and tetramers) were the toxic species and that low concentrations of these aggregates in the population was sufficient to render the sample neurotoxic. Monomers and other kind of aggregates, such as annular or linear protofibers and mature fibers, were totally biologically inactive.

Phosphorylation and nitration of tyrosine residues affect functional properties of Synaptophysin and Dynamin I, two proteins involved in exo-endocytosis of synaptic vesicles.

Phosphorylation and nitration of protein tyrosine residues are thought to play a role in signaling pathways at the nerve terminal and to affect functional properties of proteins involved in the synaptic vesicle (SV) exo-endocytotic cycle. We previously demonstrated that the tyrosine residues in the C-terminal domain of the SV protein Synaptophysin (SYP) are targets of peroxynitrite (PN). Here, we have characterized the association between SYP and c-src tyrosine kinase demonstrating that phosphorylation of Tyr(273) in the C-terminal domain of SYP is crucial in mediating SYP binding to and activation of c-src. SYP forms a complex with Dynamin I (DynI), a GTPase required for SV endocytosis, which may be regulated by tyrosine phosphorylation of SYP. We here report that, in rat brain synaptosomes treated with PN, the formation of SYP/DynI complex was impaired. Noteworthy, we found that DynI was also modified by PN. DynI tyrosine phosphorylation was down-regulated in a dose-dependent manner, while DynI tyrosine nitration increased. Using mass spectrometry analysis, we identified Tyr(354) as one nitration site in DynI. In addition, we tested DynI self-assembly and GTPase activity, which are enhanced by c-src-dependent tyrosine phosphorylation of DynI, and found that both were inhibited by PN. Our results suggest that the site-specific tyrosine residue modifications may modulate the association properties of SV proteins and serve as a regulator of DynI function via control of self-assembly, thus influencing the physiology of the exo-endocytotic cycle.

MLC1 trafficking and membrane expression in astrocytes: role of caveolin-1 and phosphorylation.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare congenital leukodystrophy caused by mutations in the MLC1 gene that encodes a membrane protein of unknown function. In the brain MLC1 protein is mainly expressed in astrocyte end-feet, localizes in lipid rafts and associates with the dystrophin glycoprotein complex (DGC). Using pull-down and co-fractionation assays in cultured human and rat astrocytes, we show here that MLC1 intracellular domains pull-down the DGC proteins syntrophin, dystrobrevin, Kir4.1 and caveolin-1, the structural protein of caveolae, thereby supporting a role for DGC and caveolar structures in MLC1 function. By immunostaining and subcellular fractionation of cultured rat or human astrocytes treated with agents modulating caveolin-mediated trafficking, we demonstrate that MLC1 is also expressed in intracellular vesicles and endoplasmic reticulum and undergoes caveolae/raft-mediated endocytosis. Inhibition of endocytosis, cholesterol lowering and protein kinases A- and C-mediated MLC1 phosphorylation favour the expression of membrane-associated MLC1. Because pathological mutations prevent MLC1 membrane expression, the identification of substances regulating MLC1 intracellular trafficking is potentially relevant for the therapy of MLC.

Peroxynitrite induces tyrosine residue modifications in synaptophysin C-terminal domain, affecting its interaction with src.

Peroxynitrite is a potent oxidant that contributes to tissue damage in neurodegenerative disorders. We have previously reported that treatment of rat brain synaptosomes with peroxynitrite induced post-translational modifications in pre- and post-synaptic proteins and stimulated soluble N-ethylmaleimide sensitive fusion proteins attachment receptor complex formation and endogenous glutamate release. In this study we show that, following peroxynitrite treatment, the synaptic vesicle protein synaptophysin (SYP) can be both phosphorylated and nitrated in a dose-dependent manner. We found that tyrosine-phosphorylated, but not tyrosine-nitrated, SYP bound to the src tyrosine kinase and enhanced its catalytic activity. These effects were mediated by direct and specific binding of the SYP cytoplasmic C-terminal tail with the src homology 2 domain. Using mass spectrometry analysis, we mapped the SYP C-terminal tail tyrosine residues modified by peroxynitrite and found one nitration site at Tyr250 and two phosphorylation sites at Tyr263 and Tyr273. We suggest that peroxynitrite-mediated modifications of SYP may be relevant in modulating src signalling of synaptic terminal in pathophysiological conditions.

Biochemical characterization of MLC1 protein in astrocytes and its association with the dystrophin-glycoprotein complex.

MLC1 gene mutations have been associated with megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare neurologic disorder in children. The MLC1 gene encodes a membrane protein (MLC1) with unknown function which is mainly expressed in astrocytes. Using a newly developed anti-human MLC1 polyclonal antibody, we have investigated the biochemical properties and localization of MLC1 in cultured astrocytes and brain tissue and searched for evidence of a relationship between MLC1 and proteins of the dystrophin-glycoprotein complex (DGC). Cultured astrocytes express two MLC1 components showing different solubilisation properties and subcellular distribution. Most importantly, we show that the membrane-associated component of MLC1 (60-64 kDa) localizes in astrocytic lipid rafts together with dystroglycan, syntrophin and caveolin-1, and co-fractionates with the DGC in whole rat brain tissue. In the human brain, MLC1 protein is expressed in astrocyte processes and ependymal cells, where it colocalizes with dystroglycan and syntrophin. These data indicate that the DGC may be involved in the organization and function of the MLC1 protein in astrocyte membranes.

Synaptic remodeling induced by axotomy of superior cervical ganglion neurons: Involvement of metalloproteinase-2.

We previously demonstrated the involvement of the dystrophin-dystroglycan (Dys-DG) complex in the stabilization of intraganglionic synapses in rodent superior cervical ganglion (SCG) by investigating changes in the organization of their post-synaptic apparatus induced either by ganglionic neuron axotomy or by the lack of Dys in genetically dystrophic mdx mice, or by the combination of the two. A role of the matrix metalloproteinases (MMPs) MMP-2 and MMP-9 in the degradation of DG and, hence, in disrupting the connection between the extracellular matrix (ECM) and the cortical cytoskeleton, has recently been proposed. We hypothesized that the degradation by MMPs of ECM proteins and DG in ganglionic neurons may be involved in injury-induced synaptic detachment observed in rodent SCG. In this review, we report changes in MMP-2 and in the proteins involved in one of the enzymatic cascades of activation induced by axotomy of rat SCG neurons. This will be preceded by a description of our previous observations that led to investigate the role of MMP-2 in this experimental model.

Immunodetection of partially glycosylated isoforms of alpha-dystroglycan by a new monoclonal antibody against its beta-dystroglycan-binding epitope.

The alpha/beta dystroglycan (DG) complex links the extracellular matrix to the actin cytoskeleton. The extensive glycosylation of alpha-DG is believed to be crucial for the interaction with its extracellular matrix-binding partners. We characterized a monoclonal antibody, directed against the beta-DG-binding epitope ( approximately positions 550-565), which recognizes preferentially hypoglycosylated alpha-DG. In Western blot, the antibody was able to detect a number of partially glycosylated alpha-DG isoforms from rat brain and chicken skeletal muscle tissue samples. In addition, we demonstrated its inhibitory effect on the interaction between alpha- and beta-DG in vitro and preliminary immunostaining experiments suggest that such hypoglycosylated alpha-DG isoforms could play a role within cells.