Astrocytes provide metabolic support for neurons and modulate their functions by releasing a plethora of neuroactive molecules diffusing to neighboring cells. Here we report that astrocytes also play ...a role in cortical neurons' vulnerability to Herpes simplex virus type‐1 (HSV‐1) infection through the release of extracellular ATP. We found that the interaction of HSV‐1 with heparan sulfate proteoglycans expressed on the plasma membrane of astrocytes triggered phospholipase C‐mediated IP3‐dependent intracellular Ca2+ transients causing extracellular release of ATP. ATP binds membrane purinergic P2 receptors (P2Rs) of both neurons and astrocytes causing an increase in intracellular Ca2+ concentration that activates the Glycogen Synthase Kinase (GSK)‐3β, whose action is necessary for HSV‐1 entry/replication in these cells. Indeed, in co‐cultures of neurons and astrocytes HSV‐1‐infected neurons were only found in proximity of infected astrocytes releasing ATP, whereas in the presence of fluorocitrate, an inhibitor of astrocyte metabolism, switching‐off the HSV‐1‐induced ATP release, very few neurons were infected. The addition of exogenous ATP, mimicking that released by astrocytes after HSV‐1 challenge, restored the ability of HSV‐1 to infect neurons co‐cultured with metabolically‐inhibited astrocytes. The ATP‐activated, P2R‐mediated, and GSK‐3‐dependent molecular pathway underlying HSV‐1 infection is likely shared by neurons and astrocytes, given that the blockade of either P2Rs or GSK‐3 activation inhibited infection of both cell types. These results add a new layer of information to our understanding of the critical role played by astrocytes in regulating neuronal functions and their response to noxious stimuli including microbial agents via Ca2+‐dependent release of neuroactive molecules.
Main Points
Ca2+‐dependent release of ATP from astrocytes affects neurons' vulnerability to HSV‐1 infection
P2R‐dependent GSK‐3 activation is involved in HSV‐1 entry/replication in both astrocytes and neurons
Extensive research provides evidence that neuroinflammation underlies numerous brain disorders. However, the molecular mechanisms by which inflammatory mediators determine synaptic and cognitive ...dysfunction occurring in neurodegenerative diseases (e.g., Alzheimer’s disease) are far from being fully understood. Here we investigated the role of interleukin 1β (IL-1β), and the molecular cascade downstream the activation of its receptor, to the synaptic dysfunction occurring in the mouse model of multiple Herpes simplex virus type-1 (HSV-1) reactivations within the brain. These mice are characterized by neuroinflammation and memory deficits associated with a progressive accumulation of neurodegenerative hallmarks (e.g., amyloid-β protein and tau hyperphosphorylation). Here we show that mice undergone two HSV-1 reactivations in the brain exhibited increased levels of IL-1β along with significant alterations of: (1) cognitive performances; (2) hippocampal long-term potentiation; (3) expression synaptic-related genes and pre- and post-synaptic proteins; (4) dendritic spine density and morphology. These effects correlated with activation of the epigenetic repressor MeCP2 that, in association with HDAC4, affected the expression of synaptic plasticity-related genes. Specifically, in response to HSV-1 infection, HDAC4 accumulated in the nucleus and promoted MeCP2 SUMOylation that is a post-translational modification critically affecting the repressive activity of MeCP2. The blockade of IL-1 receptors by the specific antagonist Anakinra prevented the MeCP2 increase and the consequent downregulation of gene expression along with rescuing structural and functional indices of neurodegeneration. Collectively, our findings provide novel mechanistic evidence on the role played by HSV-1-activated IL-1β signaling pathways in synaptic deficits leading to cognitive impairment.
Several studies including ours reported the detrimental effects of extracellular tau oligomers (ex-oTau) on glutamatergic synaptic transmission and plasticity. Astrocytes greatly internalize ex-oTau ...whose intracellular accumulation alters neuro/gliotransmitter handling thereby negatively affecting synaptic function. Both amyloid precursor protein (APP) and heparan sulfate proteoglycans (HSPGs) are required for oTau internalization in astrocytes but the molecular mechanisms underlying this phenomenon have not been clearly identified yet. Here we found that a specific antibody anti-glypican 4 (GPC4), a receptor belonging to the HSPG family, significantly reduced oTau uploading from astrocytes and prevented oTau-induced alterations of Ca2+-dependent gliotransmitter release. As such, anti-GPC4 spared neurons co-cultured with astrocytes from the astrocyte-mediated synaptotoxic action of ex-oTau, thus preserving synaptic vesicular release, synaptic protein expression and hippocampal LTP at CA3-CA1 synapses. Of note, the expression of GPC4 depended on APP and, in particular, on its C-terminal domain, AICD, that we found to bind Gpc4 promoter. Accordingly, GPC4 expression was significantly reduced in mice in which either APP was knocked-out or it contained the non-phosphorylatable amino acid alanine replacing threonine 688, thus becoming unable to produce AICD. Collectively, our data indicate that GPC4 expression is APP/AICD-dependent, it mediates oTau accumulation in astrocytes and the resulting synaptotoxic effects.
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•Extracellular tau oligomers need GPC4 to enter astrocytes.•GPC4-mediated oligomeric tau-upload affects synaptic function.•The expression of GPC4 depends on Amyloid Precursor Protein Intracellular Domain.
Aims
Several studies reported that astrocytes support neuronal communication by the release of gliotransmitters, including ATP and glutamate. Astrocytes also play a fundamental role in buffering ...extracellular glutamate in the synaptic cleft, thus limiting the risk of excitotoxicity in neurons. We previously demonstrated that extracellular tau oligomers (ex‐oTau), by specifically targeting astrocytes, affect glutamate‐dependent synaptic transmission via a reduction in gliotransmitter release. The aim of this work was to determine if ex‐oTau also impair the ability of astrocytes to uptake extracellular glutamate, thus further contributing to ex‐oTau‐dependent neuronal dysfunction.
Methods
Primary cultures of astrocytes and organotypic brain slices were exposed to ex‐oTau (200 nM) for 1 h. Extracellular glutamate buffering by astrocytes was studied by: Na+ imaging; electrophysiological recordings; high‐performance liquid chromatography; Western blot and immunofluorescence. Experimental paradigms avoiding ex‐oTau internalisation (i.e. heparin pre‐treatment and amyloid precursor protein knockout astrocytes) were used to dissect intracellular vs extracellular effects of oTau.
Results
Ex‐oTau uploading in astrocytes significantly affected glutamate‐transporter‐1 expression and function, thus impinging on glutamate buffering activity. Ex‐oTau also reduced Na‐K‐ATPase activity because of pump mislocalisation on the plasma membrane, with no significant changes in expression. This effect was independent of oTau internalisation and it caused Na+ overload and membrane depolarisation in ex‐oTau‐targeted astrocytes.
Conclusions
Ex‐oTau exerted a complex action on astrocytes, at both intracellular and extracellular levels. The net effect was dysregulated glutamate signalling in terms of both release and uptake that relied on reduced expression of glutamate‐transporter‐1, altered function and localisation of NKA1A1, and NKA1A2. Consequently, Na+ gradients and all Na+‐dependent transports were affected.
Extracellularly, tau oligomers cause NKAs mislocalisation on the plasma membrane inducing intracellular Na+ overload in astrocytes leading to reduced Na+ gradient across the membrane.
Intracellularly, tau oligomers downregulate GLT‐1 expression.
The net effects of extracellular tau oligomers are a reduction of Na+‐driven uploading of extracellular glutamate from astrocytes.
Abstract Epidemiological and experimental findings suggest that chronic infection with Herpes simplex virus type 1 (HSV-1) may be a risk factor for Alzheimer's disease (AD), but the molecular ...mechanisms underlying this association have not been fully identified. We investigated the effects of HSV-1 on excitability and intracellular calcium signaling in rat cortical neurons and the impact of these effects on amyloid precursor protein (APP) processing and the production of amyloid-β peptide (Aβ). Membrane depolarization triggering firing rate increases was observed shortly after neurons were challenged with HSV-1 and was still evident 12 hours postinfection. These effects depended on persistent sodium current activation and potassium current inhibition. The virally induced hyperexcitability triggered intracellular Ca2+ signals that significantly increased intraneuronal Ca2+ levels. It also enhanced activity- and Ca2+ -dependent APP phosphorylation and intracellular accumulation of Aβ42. These findings indicate that HSV-1 causes functional changes in cortical neurons that promote APP processing and Aβ production, and they are compatible with the co-factorial role for HSV-1 in the pathogenesis of AD suggested by previous findings.
Alzheimer's disease (AD) is a well-studied neurodegenerative disorder; nevertheless, significant therapeutic agents for the pharmacological treatment of this neuropathology are unavailable to date. ...The toxicity of amyloid beta -peptide (A beta ) has been implicated as a critical cause in the development of AD, and A beta -amyloid-induced toxicity is typically associated with apoptosis. Here, we investigated the effect of 17 beta -estradiol (E2) on A beta -induced toxicity in cerebellar granule cells (CGCs). Our data showed a significant induction of apoptosis in neurons treated with A beta , and the addition of E2 reduced this effect. In addition, E2 reduced the A beta -induced up-regulation of Bax and down-regulation of Bcl-xL, and inhibited the subsequent mitochondrial release of cytochrome c and activation of caspase-3. Moreover, E2 inhibited A beta -induced c-Jun N-terminal protein kinase (JNK) activation. Taken together, these findings indicate that E2 protects against A beta -induced apoptosis in neuronal cells by preventing mitochondrial dysfunction and interfering with the JNK signalling cascade.
Throughout life, new neurons are continuously generated in the hippocampus, which is therefore a major site of structural plasticity in the adult brain. We recently demonstrated that extremely ...low-frequency electromagnetic fields (ELFEFs) promote the neuronal differentiation of neural stem cells
in vitro by up-regulating Ca
v1-channel activity. The aim of the present study was to determine whether 50-Hz/1 mT ELFEF stimulation also affects adult hippocampal neurogenesis
in vivo, and if so, to identify the molecular mechanisms underlying this action and its functional impact on synaptic plasticity. ELFEF exposure (1 to 7
h/day for 7
days) significantly enhanced neurogenesis in the dentate gyrus (DG) of adult mice, as documented by increased numbers of cells double-labeled for 5-bromo-deoxyuridine (BrdU) and doublecortin. Quantitative RT-PCR analysis of hippocampal extracts revealed significant ELFEF exposure-induced increases in the transcription of pro-neuronal genes (
Mash1, NeuroD2, Hes1) and genes encoding Ca
v1.2 channel α
1C subunits. Increased expression of NeuroD1, NeuroD2 and Ca
v1 channels was also documented by Western blot analysis. Immunofluorescence experiments showed that, 30
days after ELFEF stimulation, roughly half of the newly generated immature neurons had survived and become mature dentate granule cells (as shown by their immunoreactivity for both BrdU and NeuN) and were integrated into the granule cell layer of the DG. Electrophysiological experiments demonstrated that the new mature neurons influenced hippocampal synaptic plasticity, as reflected by increased long-term potentiation. Our findings show that ELFEF exposure can be an effective tool for increasing
in vivo neurogenesis, and they could lead to the development of novel therapeutic approaches in regenerative medicine.
► Extremely low-frequency electromagnetic fields (ELFEFs) enhance
in vivo neurogenesis. ► ELFEF stimulation increases the expression of pro-neuronal genes and Ca
v1.2 channels. ► Surviving newly ELFEF-generated neurons integrate in the hippocampal circuitry. ► New mature neurons influence synaptic plasticity increasing long-term potentiation. ► ELFEF stimulation may lead to novel therapeutic approach in regenerative medicine.
Exposure to extremely low-frequency electromagnetic fields (ELFEF) influences the expression of key target genes controlling adult neurogenesis and modulates hippocampus-dependent memory. Here, we ...assayed whether ELFEF stimulation affects olfactory memory by modulating neurogenesis in the subventricular zone (SVZ) of the lateral ventricle, and investigated the underlying molecular mechanisms. We found that 30 days after the completion of an ELFEF stimulation protocol (1 mT; 50 Hz; 3.5 h/day for 12 days), mice showed enhanced olfactory memory and increased SVZ neurogenesis. These effects were associated with upregulated expression of mRNAs encoding for key regulators of adult neurogenesis and were mainly dependent on the activation of the Wnt pathway. Indeed, ELFEF stimulation increased Wnt3 mRNA expression and nuclear localization of its downstream target β-catenin. Conversely, inhibition of Wnt3 by Dkk-1 prevented ELFEF-induced upregulation of neurogenic genes and abolished ELFEF's effects on olfactory memory. Collectively, our findings suggest that ELFEF stimulation increases olfactory memory via enhanced Wnt/β-catenin signaling in the SVZ and point to ELFEF as a promising tool for enhancing SVZ neurogenesis and olfactory function.
Astrocytes provide metabolic support for neurons and modulate their functions by releasing a plethora of neuroactive molecules diffusing to neighboring cells. Here we report that astrocytes also play ...a role in cortical neurons' vulnerability to Herpes simplex virus type-1 (HSV-1) infection through the release of extracellular ATP. We found that the interaction of HSV-1 with heparan sulfate proteoglycans expressed on the plasma membrane of astrocytes triggered phospholipase C-mediated IP
-dependent intracellular Ca
transients causing extracellular release of ATP. ATP binds membrane purinergic P2 receptors (P2Rs) of both neurons and astrocytes causing an increase in intracellular Ca
concentration that activates the Glycogen Synthase Kinase (GSK)-3β, whose action is necessary for HSV-1 entry/replication in these cells. Indeed, in co-cultures of neurons and astrocytes HSV-1-infected neurons were only found in proximity of infected astrocytes releasing ATP, whereas in the presence of fluorocitrate, an inhibitor of astrocyte metabolism, switching-off the HSV-1-induced ATP release, very few neurons were infected. The addition of exogenous ATP, mimicking that released by astrocytes after HSV-1 challenge, restored the ability of HSV-1 to infect neurons co-cultured with metabolically-inhibited astrocytes. The ATP-activated, P2R-mediated, and GSK-3-dependent molecular pathway underlying HSV-1 infection is likely shared by neurons and astrocytes, given that the blockade of either P2Rs or GSK-3 activation inhibited infection of both cell types. These results add a new layer of information to our understanding of the critical role played by astrocytes in regulating neuronal functions and their response to noxious stimuli including microbial agents via Ca
-dependent release of neuroactive molecules.
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