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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.

Background The 2019 novel coronavirus disease (COVID-19) pandemic has highlighted the importance of health research and fostered clinical research as never before. A huge number of clinical trials for potential COVID-19 interventions have been launched worldwide. Therefore, the effort of monitoring and characterizing the ongoing research portfolio of COVID-19 clinical trials has become crucial in order to fill evidence gaps that can arise, define research priorities and methodological issues, and eventually, formulate valuable recommendations for investigators and sponsors. The main purpose of the present work was to analyze the landscape of COVID-19 clinical research in Italy, by mapping and describing the characteristics of planned clinical trials investigating the role of drugs and convalescent plasma for treatment or prevention of COVID-19 disease. Methods During an 11-month period between May 2020 and April 2021, we performed a survey of the Italian COVID-19 clinical trials on therapeutic and prophylactic drugs and convalescent plasma. Clinical trials registered in the Italian Medicines Agency (AIFA) and ClinicalTrials.gov websites were regularly monitored. In the present paper, we report an analysis of study design characteristics and other trial features at 6 April 2021. Results Ninety-four clinical trials planned to be carried out in Italy were identified. Almost all of them (91%) had a therapeutic purpose; as for the study design, the majority of them adopted a parallel group (74%) and randomized (76%) design. Few of them were blinded (33%). Eight multiarm studies were identified, and two of them were multinational platform trials. Many therapeutic strategies were investigated, mostly following a drug repositioning therapeutic approach. Conclusions Our study describes the characteristics of COVID-19 clinical trials planned to be carried out in Italy over about 1 year of pandemic emergency. High level quality clinical trials were identified, although some weaknesses in study design and replications of experimental interventions were observed, particularly in the early phase of the pandemic. Our findings provide a critical view of the clinical research strategies adopted for COVID-19 in Italy during the early phase of the pandemic. Further actions could include monitoring and follow-up of trial results and publications and focus on non-pharmacological research areas.

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.

Our previous studies have demonstrated that specific peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists play a fundamental role in oligodendrocyte progenitor (OP) differentiation, protecting them against oxidative and inflammatory damage. The antihypertensive drug Telmisartan (TLM) was shown to act as a PPAR-γ modulator. This study investigates the TLM effect on OP differentiation and validates its capability to restore damage in a pharmacological model of Niemann-Pick type C (NPC) disease through a PPAR-γ-mediated mechanism. For the first time in purified OPs, we demonstrate that TLM-induced PPAR-γ activation downregulates the type 1 angiotensin II receptor (AT1), the level of which naturally decreases during differentiation. Like other PPAR-γ agonists, we show that TLM promotes peroxisomal proliferation and promotes OP differentiation. Furthermore, TLM can offset the OP maturation arrest induced by a lysosomal cholesterol transport inhibitor (U18666A), which reproduces an NPC1-like phenotype. In the NPC1 model, TLM also reduces cholesterol accumulation within peroxisomal and lysosomal compartments and the contacts between lysosomes and peroxisomes, revealing that TLM can regulate intracellular cholesterol transport, crucial for myelin formation. Altogether, these data indicate a new potential use of TLM in hypomyelination pathologies such as NPC1, underlining the possible repositioning of the drug already used in other pathologies.

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-&gamma, response element (PPAR-&gamma, /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-&gamma, 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-&alpha, ) insult, with pio showing faster kinetics of action and compound 1 a higher activity than DMF. In conclusion, NRF2 and PPAR-&gamma, 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.

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.

The ERCC8/CSA gene encodes a WD-40 repeat protein (CSA) that is part of a E3-ubiquitin ligase/COP9 signalosome complex. When mutated, CSA causes the Cockayne Syndrome group A (CS-A), a rare recessive progeroid disorder characterized by sun sensitivity and neurodevelopmental abnormalities. CS-A cells features include ROS hyperproduction, accumulation of oxidative genome damage, mitochondrial dysfunction and increased apoptosis that may contribute to the neurodegenerative process. In this study, we show that CSA localizes to mitochondria and specifically interacts with the mitochondrial fission protein dynamin-related protein (DRP1) that is hyperactivated when CSA is defective. Increased fission is not counterbalanced by increased mitophagy in CS-A cells thus leading to accumulation of fragmented mitochondria. However, when mitochondria are challenged with the mitochondrial toxin carbonyl cyanide m-chloro phenyl hydrazine, CS-A fibroblasts undergo mitophagy as efficiently as normal fibroblasts, suggesting that this process remains targetable to get rid of damaged mitochondria. Indeed, when basal mitophagy was potentiated by overexpressing Parkin in CSA deficient cells, a significant rescue of the dysfunctional mitochondrial phenotype was observed. Importantly, Parkin overexpression not only reactivates basal mitophagy, but plays also an anti-apoptotic role by significantly reducing the translocation of Bax at mitochondria in CS-A cells. These findings provide new mechanistic insights into the role of CSA in mitochondrial maintenance and might open new perspectives for therapeutic approaches.

Huntington disease (HD) is a neurodegenerative disease caused by expansion of CAG repeats in the huntingtin (Htt) gene. The expression of hMTH1, the human hydrolase that degrades oxidized purine nucleoside triphosphates, grants protection in a chemical HD mouse model in which HD-like features are induced by the mitochondrial toxin 3-nitropropionic acid (3-NP). To further examine the relationship between oxidized dNTPs and HD-like neurodegeneration, we studied the effects of hMTH1 expression in a genetic cellular model for HD, such as striatal cells expressing mutant htt (HdhQ111). hMTH1 expression protected these cells from 3-NP and H2O2-induced killing, by counteracting the mutant htt-dependent increased vulnerability and accumulation of nuclear and mitochondrial DNA 8-hydroxyguanine levels. hMTH1 expression reverted the decreased mitochondrial membrane potential characteristic of HdhQ111 cells and delayed the increase in mitochondrial reactive oxygen species associated with 3-NP treatment. Further indications of hMTH1-mediated mitochondrial protection are the partial reversion of 3-NP-induced alterations in mitochondrial morphology and the modulation of DRP1 and MFN1 proteins, which control fusion/fission rates of mitochondria. Finally, in line with the in vitro findings, upon 3-NP in vivo treatment, 8-hydroxyguanine levels in mitochondrial DNA from heart, muscle and brain are significantly lower in transgenic hMTH1-expressing mice than in wild-type animals., Highlights ► hMTH1 provides defence against cell death in mutant htt-expressing striatal cells ► hMTH1 improves mitochondrial functionality in mutant htt-expressing striatal cells ► hMTH1 protects in vivo mitochondria from endogenous and 3-NP induced oxidation

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.

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.

We have previously shown that natural (15-deoxy-Δ12,14-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 Ca2+ waves; the latter closely correlated with the presence of mitochondria and were inhibited by the mitochondrial respiratory chain Complex I inhibitor rotenone. Because Ca2+ 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.

J. Neurochem. (2010) 115, 450–459. Abstract 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-β (TGF-β) 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 Ca2+ 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-β 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-β treatment of mixed glial cultures were clearly distinct. LPS induced a set of classical pro-inflammatory genes, whereas TGF-β increased the expression of genes associated with atypical microglial phenotype, namely arginase-1 and TGF-β 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.

On the basis of a Janssen's patent, we approached a new synthesis of some 1,3,5-triazin-4,6-diones as potential non peptidic prokineticin receptor antagonists, containing the following substitutions: (N(1) and N(5) link a 4-methoxybenzyl and a 4-ethylbenzyl, respectively; C(2) can link an amino-ethyl-guanidine (reference compound 1) or an ethylendiamine (2) or an amino-ethyl-amino-2-imidazoline (3). New compounds were assessed for PKR1 and PKR2 affinity. Antagonist properties were evaluated as inhibition of 1 nM Bv8-induced intracellular Ca2+ mobilization.

An autosomal dominant protein aggregate myopathy, characterized by high plasma creatine kinase and calsequestrin-1 (CASQ1) accumulation in skeletal muscle, has been recently associated with a missense mutation in CASQ1 gene. The mutation replaces an evolutionarily-conserved aspartic acid with glycine at position 244 (p.D244G) of CASQ1, the main sarcoplasmic reticulum (SR) Ca2+ binding and storage protein localized at the terminal cisternae of skeletal muscle cells. Here, immunocytochemical analysis of myotubes, differentiated from muscle-derived primary myoblasts, shows that sarcoplasmic vacuolar aggregations positive for CASQ1 are significantly larger in CASQ1-mutated cells than control cells. A strong co-immuno staining of both RyR1 and CASQ1 was also noted in the vacuoles of myotubes and muscle biopsies derived from patients. Electrophysiological recordings and sarcoplasmic Ca2+ measurements provide evidence for less Ca2+ release from the SR of mutated myotubes when compared to that of controls. These findings further clarify the pathogenic nature of the p.D244G variant and point out defects in sarcoplasmic Ca2+ homeostasis as a mechanism underlying this human disease, which could be distinctly classified as "CASQ1-couplonopathy"., peer-reviewed

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.

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.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) 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 (

The ERCC8/CSA gene encodes a WD-40 repeat protein that is part of a E3-ubiquitin ligase/COP9 signalosome complex. When mutated it gives rise to a rare recessive progeroid disorder, Cockayne Syndrome group A (CSA), characterized by sun sensitivity and neurodevelopmental abnormalities. CS-A cells are characterized by redox unbalance and mitochondrial dysfunction that may contribute to the neurodegenerative process. In this study we show that CSA localizes to mitochondria and its deficiency leads to accumulation of fragmented mitochondria. The analysis of mitochondrial dynamics showed that in CS-A cells the dynamin-related protein (DRP1) is hyperactivated concomitantly to p53 activation and the mitophagic flux is increased. Efficient rescue of the dysfunctional mitochondrial phenotype and inhibition of apoptotic BCL2-associated X protein (Bax) at mitochondria of CS-A cells was achieved by improving the cell cleaning system via overexpression of Parkin. These findings provide new promising tools for limiting cell loss in CS diseased brain regions.

International audience; 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.

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 A2Areceptors (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 Ca2+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.

Peroxisome proliferator-activated receptor-γ (PPAR-γ) is one of the most studied nuclear receptor since its identification as a target to treat metabolic and neurological diseases. In addition to exerting anti-inflammatory and neuroprotective effects, PPAR-γ agonists, such as the insulin-sensitizing drug pioglitazone, promote the differentiation of oligodendrocytes (OLs), the myelin-forming cells of the central nervous system (CNS). In addition, PPAR-γ agonists increase OL mitochondrial respiratory chain activity and OL’s ability to respond to environmental signals with oscillatory Ca2+ waves. Both OL maturation and oscillatory Ca2+ waves are prevented by the mitochondrial inhibitor rotenone and restored by PPAR-γ agonists, suggesting that PPAR-γ promotes myelination through mechanisms involving mitochondria.

Presenilin-1 (PS1) and presenilin-2 (PS2) are transmembrane proteins widely expressed in the central nervous system, which function as the catalytic subunits of γ-secretase, the enzyme that releases amyloid-β protein (Aβ) from ectodomain cleaved amyloid precursor protein (APP) by intramembrane proteolysis. Mutations in PS1, PS2, and Aβ protein precursor are involved in the etiology of familial Alzheimer’s disease (FAD), while the cause of the sporadic form of AD (SAD) is still not known. However, since similar neuropathological changes have been observed in both FAD and SAD, a common pathway in the etiology of the disease has been suggested. Given that age-related deranged Ca2+ regulation has been hypothesized to play a role in SAD pathogenesis via PS gene regulation and γ-secretase activity, we studied the in vitro regulation of PS1 and PS2 in the human neuron-like SK-N-BE cell line treated with the specific endoplasmic reticulum (ER) calcium ATPase inhibitor Thapsigargin (THG), to introduce intracellular Ca2+ perturbations and mimic the altered Ca2+ homeostasis observed in AD. Our results showed a consistent and significant down-regulation of PS2, while PS1 appeared to be unmodulated. These events were accompanied by oxidative stress and a number of morphological alterations suggestive of the induction of apoptotic machinery. The administration of the antioxidant N-acetylcysteine (NAC) did not revert the THG-induced effects reported, while treatment with the Ca2+-independent ER stressor Brefeldin A did not modulate basal PS1 and PS2 expression. Collectively, these results suggest that Ca2+ fluctuation rather than ER stress and/or oxidative imbalance seems to play an essential role in PS2 regulation and confirm that, despite their strong homology, PS1 and PS2 could play different roles in AD.

International audience; Neuropeptide S (NPS) is a recently identified bioactive peptide that activates an orphan G-protein coupled receptor, called the NPS receptor (NPSR). In rats, NPS and NPSR constitute a novel neuropeptide system expressed both in the central nervous system and in peripheral tissues, controlling visceromotor, neuroendocrine, nociceptive and behavioural responses. To improve the knowledge of the role of the NPS-NPSR system in the gastrointestinal (GI) tract, we investigated: 1- the supraspinal effect of NPS on motor functions of the upper (gastric emptying and gastrointestinal transit) and lower (distal colonic transit and faecal output) Cl tract under basal conditions, 2- during pathological states (restraint stress and corticotropin releasing factor (CRF)-induced defecation) in the rat, and 3- the receptor type involved in treatment with NPS using NPS, tachykinin NK3 and opioid receptor antagonists (([D-Cys(tBu)(5)]NPS), SR142801 and naloxone, respectively). Intracerebroventricular injection of NPS failed to modify basal gastric emptying, gastrointestinal transit and distal colon propulsion, but significantly and dose-dependently reduced faecal pellet excretion and weight stimulated by restraint stress and CRF. The inhibitory effect of NPS on stress-induced defecation was unmodified by pre-treatment with either the tachykinin or opioid receptor antagonists, but was counteracted by a NPSR antagonist. The present study demonstrates, for the first time, that the supraspinal NPS system, which does not participate in the physiological control of Cl motility, plays an inhibitory role on defecation stimulated by restraint stress and CRF. The combination of the ability of NPS to inhibit faecal output together with its known anxiolytic effect may be promising, especially in pathological conditions such as irritable bowel syndrome, where stress and the hyperactivity of the CRF system contribute to the co-morbidity of anxiety with colonic motor symptoms such as diarrhoea.

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-β (TGF-β) 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-β 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-β treatment of mixed glial cultures were clearly distinct. LPS induced a set of classical pro-inflammatory genes, whereas TGF-β increased the expression of genes associated with atypical microglial phenotype, namely arginase-1 and TGF-β 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.

Purpose Curcumin, a phenolic compound extracted from the rhizome of Curcuma longa, was found to attenuate NMDA-induced excitotoxicity in primary retinal cultures. This study was conducted to further characterize curcumin neuroprotective ability and analyze its effects on NMDA receptor (NMDAr). Methods NMDAr modifications were analyzed in primary retinal cell cultures using immunocytochemistry, whole-cell patch-clamp recording and western blot analysis. Cell death was evaluated with the TUNEL assay in primary retinal and hippocampal cultures. Optical fluorometric recordings with Fura 2-AM were used to monitor [Ca(2+)](i). Results Curcumin dose- and time-dependently protected both retinal and hippocampal neurons against NMDA-induced cell death, confirming its anti-excitotoxic property. In primary retinal cultures, in line with the observed reduction of NMDA-induced [Ca(2+)](i) rise, whole-cell patch-clamp experiments showed that a higher percentage of retinal neurons responded to NMDA with low amplitude current after curcumin treatment. In parallel, curcumin induced an increase in NMDAr subunit type 2A (NR2A) level, with kinetics closely correlated to time-course of neuroprotection and decrease in [Ca(2+)](i). The relation between neuroprotection and NR2A level increase was also in line with the observation that curcumin neuroprotection required protein synthesis. Electrophysiology confirmed an increased activity of NR2A-containing NMDAr at the plasma membrane level. Conclusions These results confirm the neuroprotective activity of curcumin against NMDA toxicity, possibly related to an increased level of NR2A, and encourage further studies for a possible therapeutic use of curcumin based on neuromodulation of NMDArs.

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., Highlights • We studied MLC1 intracellular expression and trafficking in astrocytes. • MLC1 traffics along early/late endosomes and is subjected to Rab11-mediated recycling. • MLC1 interacts with the V-ATPase and modulates early endosome organelle acidity. • MLC1 accelerates the recycling of transferrin and TRPV4 calcium channel. • Pathological mutations impair MLC1 regulation of organelle acidity and protein recycling.

The branched-chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids involved in several important brain functions. Although commonly used as nutritional supplements, excessive intake of BCAAs might favour the establishment of neurotoxic conditions as indicated by the severe neurological symptoms characterising inherited disorders of BCAA catabolism such as maple syrup urine disease (MSUD). Recent evidence indicates that BCAAs induce excitotoxicity through mechanisms that require the presence of astrocytes. In the present study, we evaluated the effects of BCAAs on microglia, the main immune cells of the brain. As an experimental model we used primary microglial cells harvested from mixed glial cultures that had been kept in normal or high BCAA medium (H-BCAA). We show that H-BCAA microglial cells exhibit a peculiar phenotype characterized by a partial skewing toward the M2 state, with enhanced IL-10 expression and phagocytic activity but also increased free radical generation and decreased neuroprotective functions. We suggest that such an intermediate M1/M2 phenotype might result in a less efficient microglial response, which would promote the establishment of a low grade chronic inflammation and increase the likelihood of neurodegeneration. Although based on in vitro evidence, our study adds on to an increasing literature indicating that the increasing use of dietary integrators might deserve consideration for the possible drawbacks. In addition to excitotoxicity, the altered immune profile of microglia might represent a further mechanism by which BCAAs might turn into toxicants and facilitate neurodegeneration.