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.

Repurposing Dipyridamole in Niemann Pick Type C Disease: A Proof of Concept Study.

Niemann Pick type C disease (NPC) is a rare disorder characterized by lysosomal lipid accumulation that damages peripheral organs and the central nervous system. Currently, only miglustat is authorized for NPC treatment in Europe, and thus the identification of new therapies is necessary. The hypothesis addressed in this study is that increasing adenosine levels may represent a new therapeutic approach for NPC. In fact, a reduced level of adenosine has been shown in the brain of animal models of NPC; moreover, the compound T1-11, which is able to weakly stimulate A2A receptor and to increase adenosine levels by blocking the equilibrative nucleoside transporter ENT1, significantly ameliorated the pathological phenotype and extended the survival in a mouse model of the disease. To test our hypothesis, fibroblasts from NPC1 patients were treated with dipyridamole, a clinically-approved drug with inhibitory activity towards ENT1. Dipyridamole significantly reduced cholesterol accumulation in fibroblasts and rescued mitochondrial deficits; the mechanism elicited by dipyridamole relies on activation of the adenosine A2AR subtype subsequent to the increased levels of extracellular adenosine due to the inhibition of ENT1. In conclusion, our results provide the proof of concept that targeting adenosine tone could be beneficial in NPC.

Myelin Defects in Niemann-Pick Type C Disease: Mechanisms and Possible Therapeutic Perspectives.

Niemann-Pick type C (NPC) disease is a wide-spectrum clinical condition classified as a neurovisceral disorder affecting mainly the liver and the brain. It is caused by mutations in one of two genes, NPC1 and NPC2, coding for proteins located in the lysosomes. NPC proteins are deputed to transport cholesterol within lysosomes or between late endosome/lysosome systems and other cellular compartments, such as the endoplasmic reticulum and plasma membrane. The first trait of NPC is the accumulation of unesterified cholesterol and other lipids, like sphingosine and glycosphingolipids, in the late endosomal and lysosomal compartments, which causes the blockade of autophagic flux and the impairment of mitochondrial functions. In the brain, the main consequences of NPC are cerebellar neurodegeneration, neuroinflammation, and myelin defects. This review will focus on myelin defects and the pivotal importance of cholesterol for myelination and will offer an overview of the molecular targets and the pharmacological strategies so far proposed, or an object of clinical trials for NPC. Finally, it will summarize recent data on a new and promising pharmacological perspective involving A2A adenosine receptor stimulation in genetic and pharmacological NPC dysmyelination models.

Curcumin promotes oligodendrocyte differentiation and their protection against TNF-α through the activation of the nuclear receptor PPAR-γ.

Curcumin is a compound found in the rhizome of Curcuma longa (turmeric) with a large repertoire of pharmacological properties, including anti-inflammatory and neuroprotective activities. The current study aims to assess the effects of this natural compound on oligodendrocyte progenitor (OP) differentiation, particularly in inflammatory conditions. We found that curcumin can promote the differentiation of OPs and to counteract the maturation arrest of OPs induced by TNF-α by a mechanism involving PPAR-γ (peroxisome proliferator activated receptor), a ligand-activated transcription factor with neuroprotective and anti-inflammatory capabilities. Furthermore, curcumin induces the phosphorylation of the protein kinase ERK1/2 known to regulate the transition from OPs to immature oligodendrocytes (OLs), by a mechanism only partially dependent on PPAR-γ. Curcumin is also able to raise the levels of the co-factor PGC1-α and of the cytochrome c oxidase core protein COX1, even when OPs are exposed to TNF-α, through a PPAR-γ-mediated mechanism, in line with the known ability of PPAR-γ to promote mitochondrial integrity and functions, which are crucial for OL differentiation to occur. Altogether, this study provides evidence for a further mechanism of action of curcumin besides its well-known anti-inflammatory properties and supports the suggested therapeutic potential of this nutraceutical in demyelinating diseases.

NRF2 and PPAR-γ Pathways in Oligodendrocyte Progenitors: Focus on ROS Protection, Mitochondrial Biogenesis and Promotion of Cell Differentiation.

An adequate protection from oxidative and inflammatory reactions, together with the promotion of oligodendrocyte progenitor (OP) differentiation, is needed to recover from myelin damage in demyelinating diseases. Mitochondria are targets of inflammatory and oxidative insults and are essential in oligodendrocyte differentiation. It is known that nuclear factor-erythroid 2-related factor/antioxidant responsive element (NRF2/ARE) and peroxisome proliferator-activated receptor gamma/PPAR-γ response element (PPAR-γ/PPRE) pathways control inflammation and overcome mitochondrial impairment. In this study, we analyzed the effects of activators of these pathways on mitochondrial features, protection from inflammatory/mitochondrial insults and cell differentiation in OP cultures, to depict the specificities and similarities of their actions. We used dimethyl-fumarate (DMF) and pioglitazone (pio) as agents activating NRF2 and PPAR-γ, respectively, and two synthetic hybrids acting differently on the NRF2/ARE pathway. Only DMF and compound 1 caused early effects on the mitochondria. Both DMF and pio induced mitochondrial biogenesis but different antioxidant repertoires. Moreover, pio induced OP differentiation more efficiently than DMF. Finally, DMF, pio and compound 1 protected from tumor necrosis factor-alpha (TNF-α) insult, with pio showing faster kinetics of action and compound 1 a higher activity than DMF. In conclusion, NRF2 and PPAR-γ by inducing partially overlapping pathways accomplish complementary functions aimed at the preservation of mitochondrial function, the defense against oxidative stress and the promotion of OP differentiation.

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.

The activity of the Striatal-enriched protein tyrosine phosphatase in neuronal cells is modulated by adenosine A2A receptor.

We recently demonstrated that a tonic activation of adenosine A2A receptors (A2A Rs) is required for cocaine-induced synaptic depression and increase in the activity of STriatal-Enriched protein tyrosine Phosphatase (STEP). In this study, we elaborated on the relationship between A2A R and STEP using genetic, pharmacological, and cellular tools. We found that the activities of protein tyrosine phosphatases (PTPs), and in particular of STEP, are significantly increased in the striatum and hippocampus of a transgenic rat strain over-expressing the neuronal A2A R (NSEA2A ) with respect to wild-type (WT) rats. Moreover the selective A2A R agonist 4-[2-[[6-Amino-9-(N-ethyl-ß-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride up-regulates PTPs and STEP activities in WT but not in NSEA2A rats, while the selective A2A R antagonist 4-(-2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a} {1,3,5}triazin-5-yl-amino]ethyl)phenol restores the tyrosine phosphatase activities in NSEA2A , having no effects in WT rats. In addition, while cocaine induced the activation of PTP and STEP in WT rats, it failed to increase phosphatase activity in NSEA2A rats. A2A Rs modulate STEP activity also in the SH-SY5Y neuroblastoma cell line, where a calcium-dependent calcineurin/PP1 pathway was found to play a major role. In summary, the present study identified a novel interaction between A2A R and STEP that could have important clinical implications, since STEP has emerged as key regulator of signaling pathways involved in neurodegenerative and neuropsychiatric diseases and A2A Rs are considered a promising target for the development of therapeutic strategies for different diseases of the central nervous system. Read the Editorial Highlight for this article on page 270.

Adenosine A2A receptor stimulation restores cell functions and differentiation in Niemann-Pick type C-like oligodendrocytes.

Niemann Pick type C (NPC) disease is a rare neurovisceral disorder. Mutations in npc1 gene induce an intracellular accumulation of unesterified cholesterol in the endosomal/lysosomal system causing cell death. We recently showed that stimulation of adenosine A2A receptors (A2AR) restores cholesterol accumulation in late endosomes/lysosomes in human NPC fibroblasts and neural cell lines transiently transfected with NPC1 siRNA, suggesting that these receptors might be targeted to contrast the disease. Since NPC1 disease is characterized by dysmyelination and maturational arrest of oligodendrocyte progenitors (OPs), in this study, we investigated whether A2AR stimulation could promote oligodendrocyte differentiation and myelin formation, thus overcoming these important neurological abnormalities. We developed a NPC1 pharmacological model, in which primary cultures of OPs are exposed to a cholesterol transport inhibitor to induce a NPC1-like phenotype characterized by several typical features such as (i) cholesterol accumulation, (ii) altered mitochondrial morphology and membrane potential, (iii) defect of autophagy and (iv) maturation arrest. The A2AR agonist CGS21680 normalized all NPC1-like features. The ability of CGS21680 of rescuing OP from maturational arrest and promoting their differentiation to mature OL, suggests that A2AR stimulation might be exploited to correct dysmyelination in NPC1, further supporting their therapeutic potential in the disease.

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.

An altered redox balance and increased genetic instability characterize primary fibroblasts derived from xeroderma pigmentosum group A patients.

Xeroderma pigmentosum (XP)-A patients are characterized by increased solar skin carcinogenesis and present also neurodegeneration. XPA deficiency is associated with defective nucleotide excision repair (NER) and increased basal levels of oxidatively induced DNA damage. In this study we search for the origin of increased levels of oxidatively generated DNA lesions in XP-A cell genome and then address the question of whether increased oxidative stress might drive genetic instability. We show that XP-A human primary fibroblasts present increased levels and different types of intracellular reactive oxygen species (ROS) as compared to normal fibroblasts, with O2₋• and H2O2 being the major reactive species. Moreover, XP-A cells are characterized by decreased reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios as compared to normal fibroblasts. The significant increase of ROS levels and the alteration of the glutathione redox state following silencing of XPA confirmed the causal relationship between a functional XPA and the control of redox balance. Proton nuclear magnetic resonance (¹H NMR) analysis of the metabolic profile revealed a more glycolytic metabolism and higher ATP levels in XP-A than in normal primary fibroblasts. This perturbation of bioenergetics is associated with different morphology and response of mitochondria to targeted toxicants. In line with cancer susceptibility, XP-A primary fibroblasts showed increased spontaneous micronuclei (MN) frequency, a hallmark of cancer risk. The increased MN frequency was not affected by inhibition of ROS to normal levels by N-acetyl-L-cysteine.

The mitochondrial uncoupling protein-2 is a master regulator of both M1 and M2 microglial responses.

Microglial activation is a dynamic process, central to neuroinflammation, which can have beneficial or pathogenic effects to human health. Mitochondria are key players in neuroinflammatory and neurodegenerative processes, common to most brain diseases. To the best of our knowledge on the role of mitochondria in the modulation of neuroinflammation, we focused on the mitochondrial uncoupling protein-2 (UCP2), known to control mitochondrial functions and to be implicated in a variety of physiological and pathological processes. In primary microglial cultures, the M1 stimulus lipopolysaccharide induced an early and transitory decrease in UCP2 levels. The initial UCP2 down-regulation was paralleled by mitochondrial inner membrane potential (mMP) depolarization and increased mitochondrial reactive oxygen species production. The key role of UCP2 in controlling mMP and reactive oxygen species production was confirmed by both pharmacological inhibition and down-regulation by RNA interference. Additionally, UCP2-silenced microglia stimulated with lipopolysaccharide showed an enhanced inflammatory response, characterized by a greater production of nitric oxide and interleukin-6. UCP2 was differently regulated by M2 stimuli, as indicated by its persistent up-regulation by interleukin-4. In UCP2-silenced microglia, interleukin-4 failed to induce M2 genes (mannose receptor 1 and interleukin-10) and to reduce M1 genes (inducible nitric oxide synthase and tumour necrosis factor-α). Our findings indicate that UCP2 is central to the process of microglial activation, with opposite regulation of M1 and M2 responses, and point to UCP2 manipulation as a potential strategy for redirecting microglial response towards protective phenotypes in several brain diseases where neuroinflammation is recognized to contribute to neurodegeneration. We show that the mitochondrial uncoupling protein-2 (UCP2) is central to the process of microglial activation, with opposite regulation of M1 and M2 responses. In UCP2-silenced microglia, lipopolysaccharide (LPS) triggers an enhanced inflammatory response characterized by a greater expression of M1 genes, whereas interleukin-4 (IL-4) fails in inducing M2 genes and reducing M1 genes. We propose UCP2 manipulation as a potential strategy for redirecting microglial response towards protective phenotypes.

MLC1 protein: a likely link between leukodystrophies and brain channelopathies.

Megalencephalic leukoencephalopathy with subcortical cysts (MLCs) disease is a rare inherited, autosomal recessive form of childhood-onset spongiform leukodystrophy characterized by macrocephaly, deterioration of motor functions, epileptic seizures and mental decline. Brain edema, subcortical fluid cysts, myelin and astrocyte vacuolation are the histopathological hallmarks of MLC. Mutations in either the MLC1 gene (>75% of patients) or the GlialCAM gene (<20% of patients) are responsible for the disease. Recently, the GlialCAM adhesion protein was found essential for the membrane expression and function of the chloride channel ClC-2 indicating MLC disease caused by mutation in GlialCAM as the first channelopathy among leukodystrophies. On the contrary, the function of MLC1 protein, which binds GlialCAM, its functional relationship with ClC-2 and the molecular mechanisms underlying MLC1 mutation-induced functional defects are not fully understood yet. The human MLC1 gene encodes a 377-amino acid membrane protein with eight predicted transmembrane domains which shows very low homology with voltage-dependent potassium (K(+)) channel subunits. The high expression of MLC1 in brain astrocytes contacting blood vessels and meninges and brain alterations observed in MLC patients have led to hypothesize a role for MLC1 in the regulation of ion and water homeostasis. Recent studies have shown that MLC1 establishes structural and/or functional interactions with several ion/water channels and transporters and ion channel accessory proteins, and that these interactions are affected by MLC1 mutations causing MLC. Here, we review data on MLC1 functional properties obtained in in vitro and in vivo models and discuss evidence linking the effects of MLC1 mutations to brain channelopathies.

Nonenzymatic oxygenated metabolites of α-linolenic acid B1- and L1-phytoprostanes protect immature neurons from oxidant injury and promote differentiation of oligodendrocyte progenitors through PPAR-γ activation.

Phytoprostanes (PhytoP's) are formed in higher plants from α-linolenic acid via a nonenzymatic free radical-catalyzed pathway and act as endogenous mediators capable of protecting cells from damage under various conditions related to oxidative stress. Humans are exposed to PhytoP's, as they are present in relevant quantities in vegetable food and pollen. The uptake of PhytoP's through the olfactory epithelium of the nasal mucosa, upon pollen grain inhalation, is of interest as the intranasal pathway is regarded as a direct route of communication between the environment and the brain. On this basis, we sought to investigate the potential activities of PhytoP's on immature cells of the central nervous system, which are particularly susceptible to oxidative stress. In neuroblastoma SH-SY5Y cells, used as a model for undifferentiated neurons, B1-PhytoP's, but not F1-PhytoP's, increased cell metabolic activity and protected them from oxidant damage caused by H2O2. Moreover, B1-PhytoP's induced a moderate depolarization of the mitochondrial inner membrane potential. These effects were prevented by the PPAR-γ antagonist GW9662. When SH-SY5Y cells were induced to differentiate toward a more mature phenotype, they became resistant to B1-PhytoP activities. B1-PhytoP's also influenced immature cells of an oligodendroglial line, as they increased the metabolic activity of oligodendrocyte progenitors and strongly accelerated their differentiation to immature oligodendrocytes, through mechanisms at least partially dependent on PPAR-γ activity. However, B1-PhytoP's did not protect oligodendrocyte progenitors against oxidant injury. Taken together, these data suggest that B1-PhytoP's, through novel mechanisms involving PPAR-γ, can specifically affect immature brain cells, such as neuroblasts and oligodendrocyte progenitors, thereby conferring neuroprotection against oxidant injury and promoting myelination.

Megalencephalic leukoencephalopathy with subcortical cysts protein-1 modulates endosomal pH and protein trafficking in astrocytes: relevance to MLC disease pathogenesis.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy caused by mutations in the gene encoding MLC1, a membrane protein mainly expressed in astrocytes in the central nervous system. Although MLC1 function is unknown, evidence is emerging that it may regulate ion fluxes. Using biochemical and proteomic approaches to identify MLC1 interactors and elucidate MLC1 function we found that MLC1 interacts with the vacuolar ATPase (V-ATPase), the proton pump that regulates endosomal acidity. Because we previously showed that in intracellular organelles MLC1 directly binds Na, K-ATPase, which controls endosomal pH, we studied MLC1 endosomal localization and trafficking and MLC1 effects on endosomal acidity and function using human astrocytoma cells overexpressing wild-type (WT) MLC1 or MLC1 carrying pathological mutations. We found that WT MLC1 is abundantly expressed in early (EEA1(+), Rab5(+)) and recycling (Rab11(+)) endosomes and uses the latter compartment to traffic to the plasma membrane during hyposmotic stress. We also showed that WT MLC1 limits early endosomal acidification and influences protein trafficking in astrocytoma cells by stimulating protein recycling, as revealed by FITC-dextran measurement of endosomal pH and transferrin protein recycling assay, respectively. WT MLC1 also favors recycling to the plasma-membrane of the TRPV4 cation channel which cooperates with MLC1 to activate calcium influx in astrocytes during hyposmotic stress. Although MLC disease-causing mutations differentially affect MLC1 localization and trafficking, all the mutated proteins fail to influence endosomal pH and protein recycling. This study demonstrates that MLC1 modulates endosomal pH and protein trafficking suggesting that alteration of these processes contributes to MLC pathogenesis.

The stimulation of adenosine A2A receptors ameliorates the pathological phenotype of fibroblasts from Niemann-Pick type C patients.

Niemann-Pick type C1 (NPC1) disease is a rare neurovisceral disorder characterized by intracellular accumulation of unesterified cholesterol, sphingolipids, and other lipids in the lysosomal compartment. A deregulation of lysosomal calcium has been identified as one of the earliest steps of the degenerative process. Since adenosine A2A receptors (A2ARs) control lysosome trafficking and pH, which closely regulates lysosomal calcium, we hypothesized a role for these receptors in NPC1. The aim of this study was to evaluate the effects of the A2AR agonist CGS21680 on human control and NPC1 fibroblasts. We show that CGS21680 raises lysosomal calcium levels and rescues mitochondrial functionality (mitochondrial inner membrane potential and expression of the complex IV of the mitochondrial respiratory chain), which is compromised in NPC1 cells. These effects are prevented by the selective blockade of A2ARs by the antagonist ZM241385. The effects of A2AR activation on lysosomal calcium are not mediated by the cAMP/PKA pathway but they appear to involve the phosphorylation of ERK1/2. Finally, CGS21680 reduces cholesterol accumulation (Filipin III staining), which is the main criterion currently used for identification of a compound or pathway that would be beneficial for NPC disease, and such an effect is prevented by the Ca(2+) chelator BAPTA-AM. Our findings strongly support the hypothesis that A2AR agonists may represent a therapeutic option for NPC1 and provide insights on their mechanisms of action.

Megalencephalic leukoencephalopathy with subcortical cysts protein 1 functionally cooperates with the TRPV4 cation channel to activate the response of astrocytes to osmotic stress: dysregulation by pathological mutations.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare leukodystrophy characterized by macrocephaly, subcortical fluid cysts and myelin vacuolation, has been linked to mutations in the MLC1 gene. This gene encodes a membrane protein that is highly expressed in astrocytes. Based on MLC pathological features, it was proposed that astrocyte-mediated defects in ion and fluid homeostasis could account for the alterations observed in MLC-affected brains. However, the role of MLC1 and the effects of pathological mutations on astrocyte osmoregulatory functions have still to be demonstrated. Using human astrocytoma cells stably overexpressing wild-type MLC1 or three known MLC-associated pathological mutations, we investigated MLC1 involvement in astrocyte reaction to osmotic changes using biochemical, dynamic video imaging and immunofluorescence techniques. We have found that MLC1 overexpressed in astrocytoma cells is mainly localized in the plasma membrane, is part of the Na,K-ATPase-associated molecular complex that includes the potassium channel Kir4.1, syntrophin and aquaporin-4 and functionally interacts with the calcium permeable channel TRPV4 (transient receptor potential vanilloid-4 cation channel) which mediates swelling-induced cytosolic calcium increase and volume recovery in response to hyposmosis. Pathological MLC mutations cause changes in MLC1 expression and intracellular localization as well as in the astrocyte response to osmotic changes by altering MLC1 molecular interactions with the Na,K-ATPase molecular complex and abolishing the increase in calcium influx induced by hyposmosis and treatment with the TRPV4 agonist 4αPDD. These data demonstrate, for the first time, that MLC1 plays a role in astrocyte osmo-homeostasis and that defects in intracellular calcium dynamics may contribute to MLC pathogenesis.

Peroxisome proliferator-activated receptor γ agonists accelerate oligodendrocyte maturation and influence mitochondrial functions and oscillatory Ca(2+) waves.

We have previously shown that natural (15-deoxy-Δ-prostaglandin J2) and synthetic (pioglitazone) agonists of peroxisome proliferator-activated receptor γ (PPAR-γ) strengthen the intrinsic cellular mechanisms protecting oligodendrocyte (OL) progenitors (OPs) from oxidative insults and promote their differentiation. Here, we demonstrate that repeated administrations of PPAR-γ agonists to OP cultures accelerate their differentiation to OLs, as indicated by increased numbers of O4- and O1-positive cells that show increased myelin basic protein expression, elaborated cholesterol-enrichedmembranes and have increased peroxisomes. Moreover, PPAR-γ agonist-treated OLs show increased activity of the mitochondrial respiratory chain Complex IV and an increased ability to respond to environmental signals, such as adenosine diphosphate (ADP), with oscillatory Ca waves; the latter closely correlated with the presence of mitochondria and were inhibited by the mitochondrial respiratory chain Complex I inhibitor rotenone. Because Ca oscillations and mitochondrial respiratory chain activity play crucial roles in OL differentiation, these findings suggest that PPAR-γ agonists could protect OLs and promote myelination through several mechanisms, including those involving mitochondrial functions. Our studies support the therapeutic potential of PPAR-γ agonists in brain diseases in which mitochondrial alteration, oxidative stress, and demyelination occur and point to the need for a better understanding of the role of PPAR-γ and its agonists in OL biology.

TGF-ß and LPS modulate ADP-induced migration of microglial cells through P2Y1 and P2Y12 receptor expression.

Nucleotides act as early signals for microglial recruitment to sites of CNS injury. As microglial motility and activation can be influenced by several local factors at the site of the lesion, we investigated the effects of interferon-gamma, lipopolysaccharide (LPS) or transforming growth factor-ß (TGF-ß) addition to mixed glial cell cultures, on microglial migration in response to ADP, P2Y12 and P2Y1 mRNA expression as well as on the expression of an array of genes associated with the process of microglial activation. First, we demonstrated, by pharmacological inhibition and by using small interfering RNAs, that in addition to P2Y12, P2Y1 is involved in ADP-stimulated microglial migration. The ability of specific agonists to induce Ca(2+) mobilization further confirmed the expression of functional P2Y receptors in microglia. Then, we found that migratory capability and expression of both P2Y receptors were abrogated in microglial cells from LPS-stimulated mixed glial cultures, while TGF-ß increased ADP-induced migration and the expression of P2Y12 and P2Y1 receptors. Interferon-gamma did not influence receptor expression or microglial migration. Finally, the patterns of gene expression induced in microglia by LPS or TGF-ß treatment of mixed glial cultures were clearly distinct. LPS induced a set of classical pro-inflammatory genes, whereas TGF-ß increased the expression of genes associated with atypical microglial phenotype, namely arginase-1 and TGF-ß genes. These results imply that both P2Y1 and P2Y12 may guide microglia toward the lesion. They also suggest that the modulation of microglial purinergic receptors expression by local factors, through direct and/or astrocyte-mediated actions, may represent a novel mechanism affecting neuroinflammatory response.

Multiple actions of the human immunodeficiency virus type-1 Tat protein on microglial cell functions.

The human immunodeficiency virus type-1 (HIV-1) regulatory protein Tat is produced in the early phase of infection and is essential for virus replication. Together with other viral products, Tat has been implicated in the pathogenesis of HIV-1-associated dementia (HAD). As HIV-1 infection in the brain is very limited and macrophage/microglial cells are the only cellular type productively infected by the virus, it has been proposed that many of the viral neurotoxic effects are mediated by microglial products. We and others have shown that Tat affects the functional state of microglial cells, supporting the hypothesis that activated microglia play a role in the neuropathology associated with HIV-1 infection. This review describes the experimental evidence indicating that Tat stimulates microglia to synthesize potentially neurotoxic molecules, including proinflammatory cytokines and free radicals, and interferes with molecular mechanisms controlling cAMP levels, intracellular [Ca2+], and ion channel expression.

Inwardly rectifying K+ channels influence Ca2+ entry due to nucleotide receptor activation in microglia.

The expression in microglia of two K+ channel populations, inwardly- and delayed outwardly rectifying channels (Kir, Kdr), is under the control of a variety of signals among which inflammatory and immunomodulatory agents. This makes K+ channels good candidates for the control of cell activities and for their adaptation to the changes of the functional state of the cell. Here we investigated on the role played by Kir channels in the control of cytoplasmic Ca2+ movements. In particular, we focused on those linked to nucleotide receptors, which are known to regulate a variety of functions in microglia. By a Fura-2-based video-imaging approach we recorded Ca2+ transients induced by P2 activation. These were composed of an initial peak, mainly due to release from endoplasmic reticulum, and of a long lasting plateau linked to Ca2+ influx through cation non-selective and capacitative channels. In patch-clamp experiments, we observed that Ba2+ (1-100 microM) could inhibit Kir current, but was not effective on Kdr and ATP-induced K+ current. By using Ba2+ as a specific blocker of Kir channels, we found that their inhibition caused a decrease of the Ca2+ level, especially at the end of the 20s long agonist application period. The effect of Ba2+ was mimicked by high K(+)-induced depolarization. We conclude that Kir channels contribute to modulate the amplitude and time course of the ATP-induced Ca2+ transient through the control of membrane potential. We suggest that microglial cells adapt signal transduction mechanisms to the changes of their functional state also by varying the expression and modulating the activity of inwardly rectifying K+ channels.