Dynamics of astrocytes Ca2+ signaling: a low-cost fluorescence customized system for 2D cultures.

In an effort to help reduce the costs of fluorescence microscopy and expand the use of this valuable technique, we developed a low-cost platform capable of visualising and analysing the spatio-temporal dynamics of intracellular Ca2+ signalling in astrocytes. The created platform, consisting of a specially adapted fluorescence microscope and a data analysis procedure performed with Imagej Fiji software and custom scripts, allowed us to detect relative changes of intracellular Ca2+ ions in astrocytes. To demonstrate the usefulness of the workflow, we applied the methodology to several in vitro astrocyte preparations, specifically immortalised human astrocyte cells and wild-type mouse cells. To demonstrate the reliability of the procedure, analyses were conducted by stimulating astrocyte activity with the agonist dihydroxyphenylglycine (DHPG), alone or in the presence of the antagonist 2-methyl-6-phenylethyl-pyridine (MPEP).

Group I and group II metabotropic glutamate receptors are upregulated in the synapses of infant rats prenatally exposed to valproic acid.

RATIONALE: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impaired social interaction and restricted/stereotyped behavior. Prenatal exposure to valproic acid (VPA) is associated with an increased risk of developing ASD in humans and autistic-like behaviors in rodents. Increasing evidence indicates that dysfunctions of glutamate receptors at synapses are associated with ASD. In the VPA rat model, an involvement of glutamate receptors in autism-like phenotypes has been suggested; however, few studies were carried out on metabotropic glutamate (mGlu) receptors. OBJECTIVES: We examined the protein expression levels of group I (mGlu1 and mGlu5) and group II (mGlu2/3) mGlu receptors in rats prenatally exposed to VPA and evaluated the effect of mGlu receptor modulation on an early autism-like phenotype in these animals. METHODS: We used western blotting analysis on synaptosomes obtained from forebrain of control and VPA rats at different ages (postnatal day P13, 35, 90) and carried out ultrasonic vocalization (USV) emission test in infant control and VPA rats. RESULTS: The expression levels of all these receptors were significantly increased in infant VPA rats. No changes were detected in adolescent and adult rats. An acute treatment with the preferential mGlu2/3 antagonist, LY341495, attenuated the impairment in the USV emission in VPA rats. No effect was observed after a treatment with the mGlu5 selective antagonist, MTEP. CONCLUSIONS: Our findings demonstrate that the expression of group I and group II mGlu receptors is upregulated at synapses of infant VPA rats and suggest that mGlu2/3 receptor modulation may have a therapeutic potential in ASD.

Blunted type-5 metabotropic glutamate receptor-mediated polyphosphoinositide hydrolysis in two mouse models of monogenic autism.

The involvement of the mGlu5 receptors in the pathophysiology of several forms of monogenic autism has been supported by numerous studies following the seminal observation that mGlu5 receptor-dependent long-term depression was enhanced in the hippocampus of mice modeling the fragile-X syndrome (FXS). Surprisingly, there are no studies examining the canonical signal transduction pathway activated by mGlu5 receptors (i.e. polyphosphoinositide - PI - hydrolysis) in mouse models of autism. We have developed a method for in vivo assessment of PI hydrolysis based on systemic injection of lithium chloride followed by treatment with the selective mGlu5 receptor PAM, VU0360172, and measurement of endogenous inositolmonophosphate (InsP) in brain tissue. Here, we report that mGlu5 receptor-mediated PI hydrolysis was blunted in the cerebral cortex, hippocampus, and corpus striatum of Ube3am-/p+ mice modeling Angelman syndrome (AS), and in the cerebral cortex and hippocampus of Fmr1 knockout mice modeling FXS. In vivo mGlu5 receptor-mediated stimulation of Akt on threonine 308 was also blunted in the hippocampus of FXS mice. These changes were associated with a significant increase in cortical and striatal Homer1 levels and striatal mGlu5 receptor and Gαq levels in AS mice, and with a reduction in cortical mGlu5 receptor and hippocampal Gαq levels, and an increase in cortical phospholipase-Cß and hippocampal Homer1 levels in FXS mice. This is the first evidence that the canonical transduction pathway activated by mGlu5 receptors is down-regulated in brain regions of mice modeling monogenic autism.

Aberrant mitochondrial bioenergetics in the cerebral cortex of the Fmr1 knockout mouse model of fragile X syndrome.

Impaired energy metabolism may play a role in the pathogenesis of neurodevelopmental disorders including fragile X syndrome (FXS). We checked brain energy status and some aspects of cell bioenergetics, namely the activity of key glycolytic enzymes, glycerol-3-phosphate shuttle and mitochondrial respiratory chain (MRC) complexes, in the cerebral cortex of the Fmr1 knockout (KO) mouse model of FXS. We found that, despite a hyperactivation of MRC complexes, adenosine triphosphate (ATP) production via mitochondrial oxidative phosphorylation (OXPHOS) is compromised, resulting in brain energy impairment in juvenile and late-adult Fmr1 KO mice. Thus, an altered mitochondrial energy metabolism may contribute to neurological impairment in FXS.

EEG Abnormalities as a Neurophysiological Biomarker of Severity in Autism Spectrum Disorder: A Pilot Cohort Study.

To date, the phenotypic significance of EEG abnormalities in patients with ASD is unclear. In a population affected by ASD we aimed to evaluate: the phenotypic characteristics; the prevalence of EEG abnormalities; the potential correlations between EEG abnormalities and behavioral and cognitive variables. Sixty-nine patients with ASD underwent cognitive or developmental testing, language assessment, and adaptive behavior skills evaluation as well as sleep/wake EEG recording. EEG abnormalities were found in 39.13% of patients. EEG abnormalities correlated with autism severity, hyperactivity, anger outbursts, aggression, negative or destructive behavior, motor stereotypies, intellectual disability, language impairment and self-harm. Our findings confirmed that EEG abnormalities are present in the ASD population and correlate with several associated phenotypic features.

Activation of Serotonin 5-HT7 Receptors Modulates Hippocampal Synaptic Plasticity by Stimulation of Adenylate Cyclases and Rescues Learning and Behavior in a Mouse Model of Fragile X Syndrome.

We have previously demonstrated that activation of serotonin 5-HT7 receptors (5-HT7R) reverses metabotropic glutamate receptor-mediated long term depression (mGluR-LTD) in the hippocampus of wild-type (WT) and Fmr1 Knockout (KO) mice, a model of Fragile X Syndrome (FXS) in which mGluR-LTD is abnormally enhanced. Here, we have investigated intracellular mechanisms underlying the effect of 5-HT7R activation using patch clamp on hippocampal slices. Furthermore, we have tested whether in vivo administration of LP-211, a selective 5-HT7R agonist, can rescue learning and behavior in Fmr1 KO mice. In the presence of an adenylate cyclase blocker, mGluR-LTD was slightly enhanced in WT and therefore the difference between mGluR-LTD in WT and Fmr1 KO slices was no longer present. Conversely, activation of adenylate cyclase by either forskolin or Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) completely reversed mGluR-LTD in WT and Fmr1 KO. 5-HT7R activation reversed mGluR-LTD in WT and corrected exaggerated mGluR-LTD in Fmr1 KO; this effect was abolished by blockade of either adenylate cyclase or protein kinase A (PKA). Exposure of hippocampal slices to LP-211 caused an increased phosphorylation of extracellular signal regulated kinase (ERK), an intracellular effector involved in mGluR-LTD, in WT mice. Conversely, this effect was barely detectable in Fmr1 KO mice, suggesting that 5-HT7R-mediated reversal of mGluR-LTD does not require ERK stimulation. Finally, an acute in vivo administration of LP-211 improved novel object recognition (NOR) performance in WT and Fmr1 KO mice and reduced stereotyped behavior in Fmr1 KO mice. Our results indicate that mGluR-LTD in WT and Fmr1 KO slices is bidirectionally modulated in conditions of either reduced or enhanced cAMP formation. Activation of 5-HT7 receptors reverses mGluR-LTD by activation of the cAMP/PKA intracellular pathway. Importantly, a systemic administration of a 5-HT7R agonist to Fmr1 KO mice corrected learning deficits and repetitive behavior. We suggest that selective 5-HT7R agonists might become novel pharmacological tools for FXS therapy.

Altered surface mGluR5 dynamics provoke synaptic NMDAR dysfunction and cognitive defects in Fmr1 knockout mice.

Metabotropic glutamate receptor subtype 5 (mGluR5) is crucially implicated in the pathophysiology of Fragile X Syndrome (FXS); however, its dysfunction at the sub-cellular level, and related synaptic and cognitive phenotypes are unexplored. Here, we probed the consequences of mGluR5/Homer scaffold disruption for mGluR5 cell-surface mobility, synaptic N-methyl-D-aspartate receptor (NMDAR) function, and behavioral phenotypes in the second-generation Fmr1 knockout (KO) mouse. Using single-molecule tracking, we found that mGluR5 was significantly more mobile at synapses in hippocampal Fmr1 KO neurons, causing an increased synaptic surface co-clustering of mGluR5 and NMDAR. This correlated with a reduced amplitude of synaptic NMDAR currents, a lack of their mGluR5-activated long-term depression, and NMDAR/hippocampus dependent cognitive deficits. These synaptic and behavioral phenomena were reversed by knocking down Homer1a in Fmr1 KO mice. Our study provides a mechanistic link between changes of mGluR5 dynamics and pathological phenotypes of FXS, unveiling novel targets for mGluR5-based therapeutics.

The antineoplastic drug flavopiridol reverses memory impairment induced by Amyloid-ß1-42 oligomers in mice.

The ectopic re-activation of cell cycle in neurons is an early event in the pathogenesis of Alzheimer's disease (AD), which could lead to synaptic failure and ensuing cognitive deficits before frank neuronal death. Cytostatic drugs that act as cyclin-dependent kinase (CDK) inhibitors have been poorly investigated in animal models of AD. In the present study, we examined the effects of flavopiridol, an inhibitor of CDKs currently used as antineoplastic drug, against cell cycle reactivation and memory loss induced by intracerebroventricular injection of Aß1-42 oligomers in CD1 mice. Cycling neurons, scored as NeuN-positive cells expressing cyclin A, were found both in the frontal cortex and in the hippocampus of Aß-injected mice, paralleling memory deficits. Starting from three days after Aß injection, flavopiridol (0.5, 1 and 3mg/kg) was intraperitoneally injected daily, for eleven days. Here we show that a treatment with flavopiridol (0.5 and 1mg/kg) was able to rescue the loss of memory induced by Aß1-42, and to prevent the occurrence of ectopic cell-cycle events in the mouse frontal cortex and hippocampus. This is the first evidence that a cytostatic drug can prevent cognitive deficits in a non-transgenic animal model of AD.

The FMRP/GRK4 mRNA interaction uncovers a new mode of binding of the Fragile X mental retardation protein in cerebellum.

Fragile X syndrome (FXS), the most common form of inherited intellectual disability, is caused by the silencing of the FMR1 gene encoding an RNA-binding protein (FMRP) mainly involved in translational control. We characterized the interaction between FMRP and the mRNA of GRK4, a member of the guanine nucleotide-binding protein (G protein)-coupled receptor kinase super-family, both in vitro and in vivo. While the mRNA level of GRK4 is unchanged in the absence or in the presence of FMRP in different regions of the brain, GRK4 protein level is increased in Fmr1-null cerebellum, suggesting that FMRP negatively modulates the expression of GRK4 at the translational level in this brain region. The C-terminal region of FMRP interacts with a domain of GRK4 mRNA, that we called G4RIF, that is folded in four stem loops. The SL1 stem loop of G4RIF is protected by FMRP and is part of the S1/S2 sub-domain that directs translation repression of a reporter mRNA by FMRP. These data confirm the role of the G4RIF/FMRP complex in translational regulation. Considering the role of GRK4 in GABAB receptors desensitization, our results suggest that an increased GRK4 levels in FXS might contribute to cerebellum-dependent phenotypes through a deregulated desensitization of GABAB receptors.

Synapses as therapeutic targets for autism spectrum disorders: an international symposium held in pavia on july 4th, 2014.

New progresses into the molecular and cellular mechanisms of autism spectrum disorders (ASDs) have been discussed in 1 day international symposium held in Pavia (Italy) on July 4th, 2014 entitled "synapses as therapeutic targets for autism spectrum disorders" (satellite of the FENS Forum for Neuroscience, Milan, 2014). In particular, world experts in the field have highlighted how animal models of ASDs have greatly advanced our understanding of the molecular pathways involved in synaptic dysfunction leading sometimes to "synaptic clinical trials" in children.

5-HT7 receptors as modulators of neuronal excitability, synaptic transmission and plasticity: physiological role and possible implications in autism spectrum disorders.

Serotonin type 7 receptors (5-HT7) are expressed in several brain areas, regulate brain development, synaptic transmission and plasticity, and therefore are involved in various brain functions such as learning and memory. A number of studies suggest that 5-HT7 receptors could be potential pharmacotherapeutic target for cognitive disorders. Several abnormalities of serotonergic system have been described in patients with autism spectrum disorder (ASD), including abnormal activity of 5-HT transporter, altered blood and brain 5-HT levels, reduced 5-HT synthesis and altered expression of 5-HT receptors in the brain. A specific role for 5-HT7 receptors in ASD has not yet been demonstrated but some evidence implicates their possible involvement. We have recently shown that 5-HT7 receptor activation rescues hippocampal synaptic plasticity in a mouse model of Fragile X Syndrome, a monogenic cause of autism. Several other studies have shown that 5-HT7 receptors modulate behavioral flexibility, exploratory behavior, mood disorders and epilepsy, which include core and co-morbid symptoms of ASD. These findings further suggest an involvement of 5-HT7 receptors in ASD. Here, we review the physiological roles of 5-HT7 receptors and their implications in Fragile X Syndrome and other ASD.

Viability of olfactory ensheathing cells after hypoxia and serum deprivation: Implication for therapeutic transplantation.

Olfactory ensheathing cells (OECs) represent glial cells supporting neuronal turnover in the olfactory system. In vitro, OECs promote axonal growth as a source of neurotrophic growth factors; in vivo, they produce myelin, promoting remyelination of damaged axons. Consequently, OEC transplantation appears to be a promising treatment for spinal cord injury, although the functional recovery is limited. This might be ascribed to the microenvironment at the lesion site, lacking growth factors (GFs), nutrients, and oxygen. To mimic this condition, we used an in vitro approach by growing primary neonatal mouse OECs under hypoxic conditions and/or serum deprivation. In addition, we compared OECs survival/proliferation with that of primary cultures of Schwann cells (SCs) and astrocytes under the same experimental conditions. Cultures were analyzed by immunocytochemistry, and cell viability was evaluated by MTT assay. Different GFs, such as NGF, bFGF, and GDNF, and their combination were used to rescue cells from serum and/or oxygen deprivation. We show that the cell types were differently sensitive to the tested stress conditions and that OECs were the most sensitive among them. Moreover, OEC viability was rescued by bFGF under serum-deprived or hypoxic condition but not under conditions of drastic serum deprivation and hypoxia. bFGF was effective also for the other cell types, whereas the effect of the other GFs was negligible. This model suggests that administration of bFGF might be considered useful to sustain cell survival/proliferation after transplantation of OECs either alone or in combination with other glial cell types.

Changes in mGlu5 receptor-dependent synaptic plasticity and coupling to homer proteins in the hippocampus of Ube3A hemizygous mice modeling angelman syndrome.

Angelman syndrome (AS) is caused by the loss of Ube3A, an ubiquitin ligase that commits specific proteins to proteasomal degradation. How this defect causes autism and other pathological phenotypes associated with AS is unknown. Long-term depression (LTD) of excitatory synaptic transmission mediated by type 5 metabotropic glutamate (mGlu5) receptors was enhanced in hippocampal slices of Ube3A(m-/p+) mice, which model AS. No changes were found in NMDA-dependent LTD induced by low-frequency stimulation. mGlu5 receptor-dependent LTD in AS mice was sensitive to the protein synthesis inhibitor anisomycin, and relied on the same signaling pathways as in wild-type mice, e.g., the mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycine pathway, and protein tyrosine phosphatase. Neither the stimulation of MAPK and PI3K nor the increase in Arc (activity-regulated cytoskeleton-associated protein) levels in response to mGlu5 receptor activation were abnormal in hippocampal slices from AS mice compared with wild-type mice. mGlu5 receptor expression and mGlu1/5 receptor-mediated polyphosphoinositide hydrolysis were also unchanged in the hippocampus of AS mice. In contrast, AS mice showed a reduced expression of the short Homer protein isoform Homer 1a, and an increased coupling of mGlu5 receptors to Homer 1b/c proteins in the hippocampus. These findings support the link between Homer proteins and monogenic autism, and lay the groundwork for the use of mGlu5 receptor antagonists in AS.

Dysregulation of group-I metabotropic glutamate (mGlu) receptor mediated signalling in disorders associated with Intellectual Disability and Autism.

Activation of group-I metabotropic glutamate receptors, mGlu1 and mGlu5, triggers a variety of signalling pathways in neurons and glial cells, which are differently implicated in synaptic plasticity. The earliest and much of key studies discovered abnormal mGlu5 receptor function in Fragile X syndrome (FXS) mouse models which then motivated more recent work that finds mGlu5 receptor dysfunction in related disorders such as intellectual disability (ID), obsessive-compulsive disorder (OCD) and autism. Therefore, mGlu1/5 receptor dysfunction may represent a common aetiology of these complex diseases. Furthermore, many studies have focused on dysregulation of mGlu5 signalling to synaptic protein synthesis. However, emerging evidence finds abnormal mGlu5 receptor interactions with its scaffolding proteins in FXS which results in mGlu5 receptor dysfunction and phenotypes independent of signalling to protein synthesis. Finally, both an increased and reduced mGlu5 functioning seem to be associated with ID and autism spectrum disorders, with important consequences for potential treatment of these developmental disorders.

Endothelin-1 is over-expressed in amyotrophic lateral sclerosis and induces motor neuron cell death.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive loss of motor neurons (MNs) and astrogliosis. Recent evidence suggests that factors secreted by activated astrocytes might contribute to degeneration of MNs. We focused on endothelin-1 (ET-1), a peptide which is strongly up-regulated in reactive astrocytes under different pathological conditions. We show that ET-1 is abundantly expressed by reactive astrocytes in the spinal cord of the SOD1-G93A mouse model and sporadic ALS patients. To test if ET-1 might play a role in degeneration of MNs, we investigated its effect on MN survival in an in vitro model of mixed rat spinal cord cultures (MSCs) enriched of astrocytes exhibiting a reactive phenotype. ET-1 exerted a toxic effect on MNs in a time- and concentration-dependent manner, with an exposure to 100-200nM ET-1 for 48h resulting in 40-50% MN cell death. Importantly, ET-1 did not induce MN degeneration when administered on cultures treated with AraC (5µM) or grown in a serum-free medium that did not favor astrocyte proliferation and reactivity. We found that both ETA and ETB receptors are enriched in astrocytes in MSCs. The ET-1 toxic effect was mimicked by ET-3 (100nM) and sarafotoxin S6c (10nM), two selective agonists of endothelin-B receptors, and was not additive with that of ET-3 suggesting the involvement of ETB receptors. Surprisingly, however, the ET-1 effect persisted in the presence of the ETB receptor antagonist BQ-788 (200nM-2µM) and was slightly reversed by the ETA receptor antagonist BQ-123 (2µM), suggesting an atypical pharmacological profile of the astrocytic receptors responsible for ET-1 toxicity. The ET-1 effect was not undone by the ionotropic glutamate receptor AMPA antagonist GYKI 52466 (20µM), indicating that it is not caused by an increased glutamate release. Conversely, a 48-hour ET-1 treatment increased MN cell death induced by acute exposure to AMPA (50µM), which is indicative of two distinct pathways leading to neuronal death. Altogether these results indicate that ET-1 exerts a toxic effect on cultured MNs through mechanisms mediated by reactive astrocytes and suggest that ET-1 may contribute to MN degeneration in ALS. Thus, a treatment aimed at lowering ET-1 levels or antagonizing its effect might be envisaged as a potential therapeutic strategy to slow down MN degeneration in this devastating disease.

Differential patterns of NOTCH1-4 receptor expression are markers of glioma cell differentiation.

Background Notch signaling is deregulated in human gliomas and may play a role in their malignancy. However, the role of each Notch receptor in glioma cell differentiation and progression is not clear. We examined the expression pattern of Notch receptors and compared it with differentiation markers in glioma cell lines, primary human cultures, and biopsies of different grades. Furthermore, the effects of a γ-secretase inhibitor (GSI) on cell survival were assessed. Methods Notch receptors and markers of cellular differentiation were analyzed by reverse transcriptase PCR, Western blotting, immunohistochemistry, and immunocytochemistry. GSI sensitivity was assessed in both cell lines and primary cultures grown as monolayers or tumorspheres, by MTT assay. Results In cell lines, Notch1 and Notch2/4 levels paralleled those of glial fibrillary acidic protein (GFAP) and vimentin, respectively. In human gliomas and primary cultures, Notch1 was moderate/strong in low-grade tumors but weak in glioblastoma multiforme (GBM). Conversely, Notch4 increased from astrocytoma grade II to GBM. Primary GBM cultures grown in serum (monolayer) showed moderate/high levels of CD133, nestin, vimentin, and Notch4 and very low levels of GFAP and Notch1, which were reduced in tumorspheres. This effect was drastic for Notch4. GSI reduced cell survival with stronger effect in serum, whilst human primary cultures showed different sensitivity. Conclusion Data from cell lines and human gliomas suggest a correlation between expression of Notch receptors and cell differentiation. Namely, Notch1 and Notch4 are markers of differentiated and less differentiated glioma cells, respectively. We propose Notch receptors as markers of glioma grading and possible prognostic factors.