Regulation of neuronal excitability by release of proteins from glial cells.
ABSTRACT: Effects of glial cells on electrical isolation and shaping of synaptic transmission between neurons have been extensively studied. Here we present evidence that the release of proteins from astrocytes as well as microglia may regulate voltage-activated Na(+) currents in neurons, thereby increasing excitability and speed of transmission in neurons kept at distance from each other by specialized glial cells. As a first example, we show that basic fibroblast growth factor and neurotrophin-3, which are released from astrocytes by exposure to thyroid hormone, influence each other to enhance Na(+) current density in cultured hippocampal neurons. As a second example, we show that the presence of microglia in hippocampal cultures can upregulate Na(+) current density. The effect can be boosted by lipopolysaccharides, bacterial membrane-derived stimulators of microglial activation. Comparable effects are induced by the exposure of neuron-enriched hippocampal cultures to tumour necrosis factor-?, which is released from stimulated microglia. Taken together, our findings suggest that release of proteins from various types of glial cells can alter neuronal excitability over a time course of several days. This explains changes in neuronal excitability occurring in states of thyroid hormone imbalance and possibly also in seizures triggered by infectious diseases.
Project description:We explored the hypothesis that injured neurons release lipocalin-2 as a help me signal.In vivo lipocalin-2 responses were assessed in rat focal cerebral ischemia and human stroke brain samples using a combination of ELISA and immunostaining. In vitro, microglia and astrocytes were exposed to lipocalin-2, and various markers and assays of glial activation were quantified. Functional relevance of neuron-to-glia lipocalin-2 signaling was examined by transferring conditioned media from lipocalin-2-activated microglia and astrocytes onto neurons to see whether activated glia could protect neurons against oxygen-glucose deprivation and promote neuroplasticity.In human stroke samples and rat cerebral ischemia, neuronal expression of lipocalin-2 was significantly increased. In primary cell cultures, exposing microglia and astrocytes to lipocalin-2 resulted in glial activation. In microglia, lipocalin-2 converted resting ramified shapes into a long-rod morphology with reduced branching, increased interleukin-10 release, and enhanced phagocytosis. In astrocytes, lipocalin-2 upregulated glial fibrillary acid protein, brain-derived neurotropic factor, and thrombospondin-1. Conditioned media from lipocalin-2-treated astrocytes upregulated synaptotagmin, and conditioned media from lipocalin-2-treated microglia upregulated synaptophysin and post-synaptic density 95 (PSD95) and protected neurons against oxygen-glucose deprivation.These findings provide proof of concept that lipocalin-2 is released by injured neurons as a help me distress signal that activates microglia and astrocytes into potentially prorecovery phenotypes.
Project description:Inborn errors of metabolism are often associated with neurodevelopmental disorders and brain injury. A deficiency of aminopeptidase P1, a proline-specific endopeptidase encoded by the Xpnpep1 gene, causes neurological complications in both humans and mice. In addition, aminopeptidase P1-deficient mice exhibit hippocampal neurodegeneration and impaired hippocampus-dependent learning and memory. However, the molecular and cellular changes associated with hippocampal pathology in aminopeptidase P1 deficiency are unclear. We show here that a deficiency of aminopeptidase P1 modifies the glial population and neuronal excitability in the hippocampus. Microarray and real-time quantitative reverse transcription-polymerase chain reaction analyses identified 14 differentially expressed genes (Casp1, Ccnd1, Myoc, Opalin, Aldh1a2, Aspa, Spp1, Gstm6, Serpinb1a, Pdlim1, Dsp, Tnfaip6, Slc6a20a, Slc22a2) in the Xpnpep1<sup>-/-</sup> hippocampus. In the hippocampus, aminopeptidase P1-expression signals were mainly detected in neurons. However, deficiency of aminopeptidase P1 resulted in fewer hippocampal astrocytes and increased density of microglia in the hippocampal CA3 area. In addition, Xpnpep1<sup>-/-</sup> CA3b pyramidal neurons were more excitable than wild-type neurons. These results indicate that insufficient astrocytic neuroprotection and enhanced neuronal excitability may underlie neurodegeneration and hippocampal dysfunction in aminopeptidase P1 deficiency.
Project description:The neuronal ceroid lipofuscinoses (NCLs) are the most common cause of childhood dementia and are invariably fatal. Early localized glial activation occurs in these disorders, and accurately predicts where neuronal loss is most pronounced. Recent evidence suggests that glial dysfunction may contribute to neuron loss, and we have now explored this possibility in infantile NCL (INCL, CLN1 disease). We grew primary cultures of astrocytes, microglia, and neurons derived from Ppt1 deficient mice (Ppt1-/-) and assessed their properties compared to wildtype (WT) cultures, before co-culturing them in different combinations (astrocytes with microglia, astrocytes or microglia with neurons, all three cell types together). These studies revealed that both Ppt1-/- astrocytes and microglia exhibit a more activated phenotype under basal unstimulated conditions, as well as alterations to their protein expression profile following pharmacological stimulation. Ppt1- /- astrocytes also displayed abnormal calcium signalling and an elevated cytoplasmic Ca2+ level, and a profound defect in their survival. Ppt1-/- neurons displayed decreased neurite outgrowth, altered complexity, a reduction in cell body size, and impaired neuron survival with prolonged time in culture. In co-cultures, the presence of both astrocytes and microglia from Ppt1-/- mice further impaired the morphology of both wild type and Ppt1-/- neurons. This negative influence was more pronounced for Ppt1-/- microglia, which appeared to trigger increased Ppt1-/- neuronal death. In contrast, wild type glial cells, especially astrocytes, ameliorated some of the morphological defects observed in Ppt1-/- neurons. These findings suggest that both Ppt1-/- microglia and astrocytes are dysfunctional and may contribute to the neurodegeneration observed in CLN1 disease. However, the dysfunctional phenotypes of Ppt1-/- glia are different from those present in CLN3 disease, suggesting that the pathogenic role of glia may differ between NCLs.
Project description:<h4>Background</h4>Exposure to increased manganese (Mn) causes inflammation and neuronal injury in the cortex and basal ganglia, resulting in neurological symptoms resembling Parkinson's disease. The mechanisms underlying neuronal death from exposure to Mn are not well understood but involve inflammatory activation of microglia and astrocytes. Expression of neurotoxic inflammatory genes in glia is highly regulated through the NF-?B pathway, but factors modulating neurotoxic glial-glial and glial-neuronal signaling by Mn are not well understood.<h4>Methods</h4>We examined the role of NF-?B in Mn-induced neurotoxicity by exposing purified microglia, astrocytes (from wild-type and astrocyte-specific IKK knockout mice), and mixed glial cultures to varying Mn concentrations and then treating neurons with the conditioned media (GCM) of each cell type. We hypothesized that mixed glial cultures exposed to Mn (0-100 ?M) would enhance glial activation and neuronal death compared to microglia, wild-type astrocytes, or IKK-knockout astrocytes alone or in mixed cultures.<h4>Results</h4>Mixed glial cultures treated with 0-100 ?M Mn for 24 h showed the most pronounced effect of increased expression of inflammatory genes including inducible nitric oxide synthase (Nos2), Tnf, Ccl5, Il6, Ccr2, Il1b, and the astrocyte-specific genes, C3 and Ccl2. Gene deletion of IKK2 in astrocytes dramatically reduced cytokine release in Mn-treated mixed glial cultures. Measurement of neuronal viability and apoptosis following exposure to Mn-GCM demonstrated that mixed glial cultures induced greater neuronal death than either cell type alone. Loss of IKK in astrocytes also decreased neuronal death compared to microglia alone, wild-type astrocytes, or mixed glia.<h4>Conclusions</h4>This suggests that astrocytes are a critical mediator of Mn neurotoxicity through enhanced expression of inflammatory cytokines and chemokines, including those most associated with a reactive phenotype such as CCL2 but not C3.
Project description:Repetitive correlated activation of pre- and postsynaptic neurons induced long-term potentiation (LTP) of synaptic transmission among hippocampal neurons grown on a layer of astrocytes (mixed cultures) but not among neurons cultured in glial conditioned medium. Supplement of D-serine, an agonist for the glycine-binding site of N-methyl-D-aspartate (NMDA) receptors, enhanced NMDA receptor activation and enabled LTP induction in glial conditioned medium cultures. The induction of LTP in both mixed cultures and hippocampal slices was suppressed by NMDA receptor antagonists, glycine-binding-site blockers of NMDA receptors, or an enzyme that degrades endogenous D-serine. By providing extracellular D-serine that facilitates activation of NMDA receptors, astrocytes thus play a key role in long-term synaptic plasticity.
Project description:Activity-dependent signaling between neurons and astrocytes contributes to experience-dependent plasticity and development of the nervous system. However, mechanisms responsible for neuron-glial interactions and the releasable factors that underlie these processes are not well understood. The pro-inflammatory cytokine, leukemia-inhibitory factor (LIF), is transiently expressed postnatally by glial cells in the hippocampus and rapidly up-regulated by enhanced neural activity following seizures. To test the hypothesis that spontaneous neural activity regulates glial development in hippocampus via LIF signaling, we blocked spontaneous activity with the sodium channel blocker tetrodotoxin (TTX) in mixed hippocampal cell cultures in combination with blockers of LIF and purinergic signaling. TTX decreased the number of GFAP-expressing astrocytes in hippocampal cell culture. Furthermore, blocking purinergic signaling by P2Y receptors contributed to reduced numbers of astrocytes. Blocking activity or purinergic signaling in the presence of function-blocking antibodies to LIF did not further decrease the number of astrocytes. Moreover, hippocampal cell cultures prepared from LIF -/- mice had reduced numbers of astrocytes and activity-dependent neuron-glial signaling promoting differentiation of astrocytes was absent. The results show that endogenous LIF is required for normal development of hippocampal astrocytes, and this process is regulated by spontaneous neural impulse activity through the release of ATP.
Project description:Fine control of neuronal activity is crucial to rapidly adjust to subtle changes of the environment. This fine tuning was thought to be purely neuronal until the discovery that astrocytes are active players of synaptic transmission. In the adult hippocampus, microglia are the other major glial cell type. Microglia are highly dynamic and closely associated with neurons and astrocytes. They react rapidly to modifications of their environment and are able to release molecules known to control neuronal function and synaptic transmission. Therefore, microglia display functional features of synaptic partners, but their involvement in the regulation of synaptic transmission has not yet been addressed. We have used a combination of pharmacological approaches with electrophysiological analysis on acute hippocampal slices and ATP assays in purified cell cultures to show that activation of microglia induces a rapid increase of spontaneous excitatory postsynaptic currents. We found that this modulation is mediated by binding of ATP to P2Y1R located on astrocytes and is independent of TNF? or NOS2. Our data indicate that, on activation, microglia cells rapidly release small amounts of ATP, and astrocytes, in turn, amplified this release. Finally, P2Y1 stimulation of astrocytes increased excitatory postsynaptic current frequency through a metabotropic glutamate receptor 5-dependent mechanism. These results indicate that microglia are genuine regulators of neurotransmission and place microglia as upstream partners of astrocytes. Because pathological activation of microglia and alteration of neurotransmission are two early symptoms of most brain diseases, our work also provides a basis for understanding synaptic dysfunction in neuronal diseases.
Project description:<h4>Aims</h4>Ethyl pyruvate (EP) mediates protective effects after neuronal injury. Besides a direct conservation of damaged neurons, the modulation of indigenous glial cells has been suggested as one important mechanism for EP-related neuroprotection. However, the specific contribution of glial cells is still unknown.<h4>Methods</h4>Organotypic hippocampal slice cultures (OHSC) were excitotoxically lesioned by 50 ?mol/L N-methyl-D-aspartate (NMDA, for 4 hours) or left untreated. In an additional OHSC subset, microglia was depleted using the bisphosphonate clodronate (100 ?g/mL) before lesion. After removal of NMDA, EP containing culture medium (0.84 ?mol/L, 8.4 ?mol/L, 42 ?mol/L, 84 ?mol/L, 168 ?mol/L) was added and incubated for 72 hours. OHSC were stained with propidium iodide to visualize degenerating neurons and isolectin IB<sub>4</sub> -FITC to identify microglia. Effects of EP at concentrations of 0.84, 8.4, and 84 ?mol/L (0-48 hours) were analyzed in the astrocytic scratch wound assay.<h4>Results</h4>EP significantly reduced neurodegeneration following induced excitotoxicity except for 168 ?mol/L. For 84 ?mol/L, a reduction in the microglia cells was observed. Microglia depletion did not affect neuronal survival after EP treatment. EP decelerated astrocytic wound closure at 48 hours after injury.<h4>Conclusion</h4>EP-mediated neuroprotection seems to be mediated by astrocytes and/or neurons.
Project description:Stromal interaction molecules (STIM) 1 and 2 are sensors of the calcium concentration in the endoplasmic reticulum. Depletion of endoplasmic reticulum calcium stores activates STIM proteins which, in turn, bind and open calcium channels in the plasma membrane formed by the proteins ORAI1, ORAI2, and ORAI3. The resulting store-operated calcium entry (SOCE), mostly controlled by the principal components STIM1 and ORAI1, has been particularly characterized in immune cells. In the nervous system, all STIM and ORAI homologs are expressed. This review summarizes current knowledge on distribution and function of STIM and ORAI proteins in central neurons and glial cells, i.e. astrocytes and microglia. STIM2 is required for SOCE in hippocampal synapses and cortical neurons, whereas STIM1 controls calcium store replenishment in cerebellar Purkinje neurons. In microglia, STIM1, STIM2, and ORAI1 regulate migration and phagocytosis. The isoforms ORAI2 and ORAI3 are candidates for SOCE channels in neurons and astrocytes, respectively. Due to the role of SOCE in neuronal and glial calcium homeostasis, dysfunction of STIM and ORAI proteins may have consequences for the development of neurodegenerative disorders, such as Alzheimer's disease.
Project description:Astrocytic GFAP expression increases during normal aging in many brain regions and in primary astrocyte cultures derived from aging rodent brains. As shown below, we unexpectedly found that the age-related increase of GFAP expression was suppressed in mixed glia (astrocytes+microglia). However, the age-related increase of GFAP was observed when E18 neurons were co-cultured with mixed glia. Thus, the presence of microglia can suppress the age-related increase of GFAP, in primary cultures of astrocytes. To more broadly characterize how aging and co-culture with neurons alters glial gene expression, we profiled gene expression in mixed glia from young (3 mo) and old (24 mo) male rat cerebral cortex by Affymetrix microarray (Rat230 2.0). The majority of age changes were independent of the presence of neurons. Overall, the expression of twofold more genes increased with age than decreased with age. The minority of age changes that were either suppressed or revealed by the presence of neurons may be useful to analyze glial-neuron interaction during aging. Some in vitro changes are shared with those of aging rat hippocampus in studies from the Landfield group (Rowe et al., 2007; Kadish et al., 2009).