Microglial Apoptosis Research Articles

Abstract PR06: CSF1 signaling is a potential therapeutic target for glioma

Abstract Current therapies for high-grade gliomas only modestly extend survival. Given that intrinsic characteristics of glioma cancer stem cells (gCSCs) make them hard to effectively target with current therapies, cells in the tumor microenvironment such as microglia/macrophages are being investigated as therapy targets. We previously identified a microglial/macrophage growth factor, Macrophage colony stimulating factor (Csf1), as a candidate glioma oncogene in a mouse somatic mutagenesis screen. CSF1 overexpression, CSF1 receptor (CSF1R) overexpression, and phosphorylated CSF1R have also been detected in human gliomas. We are using genetic and pharmacological strategies to investigate the role of CSF1 signaling in gliomagenesis in immunocompetent autochthonous mouse models to examine the therapeutic utility of targeting the CSF1R signaling axis. To determine the relevant CSF1R ligand(s) in human gliomas, we performed semi-quantitative RT-PCR on patient-derived gCSCs to examine expression of splice variants encoding membrane bound or secreted CSF1 as well as the additional CSF1R ligand IL34. We found that gCSCs primarily express the mRNA splice variant encoding secreted CSF1, but a splice variant encoding a membrane bound form was also detected. Most gCSCs lines did not express detectable levels of IL34 mRNA. We therefore developed a tissue-specific secreted CSF1 overexpressing transgenic model to study the effect of modulating CSF1 levels in the central nervous system. Since CSF1 is a growth factor for microglia, we previously studied the effects of increased CSF1 levels on microglial phenotypes and found that CSF1 overexpression increased microglial numbers and promoted microglial proliferation in vivo, but did not inherently polarize microglia toward a M2-like phenotype. Moreover, CSF1 signaling is required for survival of adult microglia in vivo, since inhibition of CSF1 signaling by the small molecule inhibitor PLX3397 induced microglial apoptosis. We now show that CSF1 overexpression accelerates gliomagenesis in a genetic model of spontaneous gliomagenesis driven by activated Ras. Both high- and low-grade gliomas form in mice with activated Ras, but CSF1 overexpression causes a dramatic increase in the percentage of tumors that are high-grade. Additionally, gliomas do not form in mice expressing activated Ras that are also CSF1-deficient. Therefore our data in this in vivo model indicate that CSF1 signaling plays a fundamental role in glioma development. Additional studies are underway to determine the impact of CSF1 on microglial and glioma phenotypes, and to explore how to best target CSF1 signaling for glioma therapy. Citation Format: Ishani De, Megan D. Steffen, Emily Sokn, Clayton Patros, Suzanne Litscher, Paul A. Clark, John S. Kuo, Fausto J. Rodriguez, Lara S. Collier. CSF1 signaling is a potential therapeutic target for glioma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr PR06.

Protection by beta-Hydroxybutyric acid against insulin glycation, lipid peroxidation and microglial cell apoptosis.

BackgroundDiabetes mellitus is characterized jointly by hyperglycemia and hyperinsulinemia that make insulin more prone to be glycated and evolve insulin advanced glycation end products (Insulin- AGE). Here, we report the effect of beta-hydroxy butyrate (BHB) (the predominant ketone body) on the formation of insulin-AGE, insulin glycation derived liposomal lipid peroxidation and insulin-AGE toxicity in microglial cells.MethodsThe inhibitory effect of BHB was monitored as a result of insulin incubation in the presence of glucose or fructose using AGE-dependent fluorescence, Tyr fluorescence as well as anilinonaphthalenesulfonate (ANS) andthioflavin T (ThT) binding, and circular dichroism (CD) investigations. To study lipid peroxidation induced by insulin glycation, thiobarbituric acid (TBA) assay and thiobarbituric acid reactive substance (TBARS) monitoring were used. The effect of insulin–AGE on microglial viability was investigated by 3-(4, 5 dimethylthiazol-2-yl)—2, 5-diphenyltetrazoliumbromide (MTT) cell assay and Annexin V/propidium iodide (PI) staining.ResultsHere we are reporting the inhibitory effect of BHB on insulin glycation and generation of insulin-AGE as a possible explanation for insulin resistance. Moreover, the protective effect of BHB on consequential glycation derived liposomal lipid peroxidation as a causative event in microglial apoptosis is reported.ConclusionThe reduced insulin fibril formation, structural inertia to glycation involved conformational changes, anti-lipid peroxidation effect, and increasing microglia viability indicated the protective effect of BHB that disclose insight on the possible preventive effect of BHB on Alzheimer’s disease.

Isoflurane preconditioning provides neuroprotection against stroke by regulating the expression of the TLR4 signalling pathway to alleviate microglial activation.

Excessive microglial activation often contributes to inflammation-mediated neurotoxicity in the ischemic penumbra during the acute stage of ischemic stroke. Toll-like receptor 4 (TLR4) has been reported to induce microglial activation via the NF-κB pathway. Isoflurane preconditioning (IP) can provide neuroprotection and inhibit microglial activation. In this study, we investigated the roles of the TLR4 signalling pathway in IP to exert neuroprotection following ischemic stroke in vivo and in vitro. The results showed that 2% IP alleviated neurological deficits, reduced the infarct volume, attenuated apoptosis and weakened microglial activation in the ischemic penumbra. Furthermore, IP down-regulated the expression of HSP 60, TLR4 and MyD88 and up-regulated inhibitor of IκB-α expression compared with I/R group in vivo. In vitro, 2% IP and a specific inhibitor of TLR4, CLI-095, down-regulated the expression of TLR4, MyD88, IL-1β, TNF-α and Bax, and up-regulated IκB-α and Bcl-2 expression compared with OGD group. Moreover, IP and CLI-095 attenuated microglial activation-induced neuronal apoptosis, and overexpression of the TLR4 gene reversed the neuroprotective effects of IP. In conclusion, IP provided neuroprotection by regulating TLR4 expression directly, alleviating microglial activation and neuroinflammation. Thus, inhibiting the activation of microglial activation via TLR4 may be a new avenue for stroke treatment.

Activated microglia/macrophage whey acidic protein (AMWAP) inhibits NFκB signaling and induces a neuroprotective phenotype in microglia

BackgroundMicroglia reactivity is a hallmark of neurodegenerative diseases. We have previously identified activated microglia/macrophage whey acidic protein (AMWAP) as a counter-regulator of pro-inflammatory response. Here, we studied its mechanisms of action with a focus on toll-like receptor (TLR) and nuclear factor κB (NFκB) signaling.MethodsRecombinant AMWAP was produced in Escherichia coli and HEK293 EBNA cells and purified by affinity chromatography. AMWAP uptake was identified by fluorescent labeling, and pro-inflammatory microglia markers were measured by qRT-PCR after stimulation with TLR ligands. NFκB pathway proteins were assessed by immunocytochemistry, Western blot, and immunoprecipitation. A 20S proteasome activity assay was used to investigate the anti-peptidase activity of AMWAP. Microglial neurotoxicity was estimated by nitrite measurement and quantification of caspase 3/7 levels in 661W photoreceptors cultured in the presence of microglia-conditioned medium. Microglial proliferation was investigated using flow cytometry, and their phagocytosis was monitored by the uptake of 661W photoreceptor debris.ResultsAMWAP was secreted from lipopolysaccharide (LPS)-activated microglia and recombinant AMWAP reduced gene transcription of IL6, iNOS, CCL2, CASP11, and TNFα in BV-2 microglia treated with LPS as TLR4 ligand. This effect was replicated with murine embryonic stem cell-derived microglia (ESdM) and primary brain microglia. AMWAP also diminished pro-inflammatory markers in microglia activated with the TLR2 ligand zymosan but had no effects on IL6, iNOS, and CCL2 transcription in cells treated with CpG oligodeoxynucleotides as TLR9 ligand. Microglial uptake of AMWAP effectively inhibited TLR4-dependent NFκB activation by preventing IRAK-1 and IκBα proteolysis. No inhibition of IκBα phosphorylation or ubiquitination and no influence on overall 20S proteasome activity were observed. Functionally, both microglial nitric oxide (NO) secretion and 661W photoreceptor apoptosis were significantly reduced after AMWAP treatment. AMWAP promoted the filopodia formation of microglia and increased the phagocytic uptake of apoptotic 661W photoreceptor cells.ConclusionsAMWAP is secreted from reactive microglia and acts in a paracrine fashion to counter-balance TLR2/TLR4-induced reactivity through NFκB inhibition. AMWAP also induces a neuroprotective microglial phenotype with reduced neurotoxicity and increased phagocytosis. We therefore hypothesize that anti-inflammatory whey acidic proteins could have a therapeutic potential in neurodegenerative diseases of the brain and the retina.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0296-6) contains supplementary material, which is available to authorized users.

A disintegrin and metalloprotease 17 promotes microglial cell survival via epidermal growth factor receptor signalling following spinal cord injury.

Tumour necrosis factor-α (TNF-α) converting enzyme (TACE), also termed a disintegrin and metallopro-tease 17 (ADAM17), is involved in multiple cell signalling pathways. Through the secretion of epidermal growth factor receptor (EGFR) ligands, ADAM17 can activate the EGFR and is involved in various downstream signalling pathways. The present study aimed to investigate whether ADAM17-induced EGFR transactivation is involved in microglial cell survival following spinal cord injury (SCI). Reverse transcription quantitative polymerase chain reaction and western blot analysis revealed that ADAM17 was overexpressed in a mouse model following SCI. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay demonstrated that the viability of human microglia and oligodendrocytes were significantly reduced in a time- and dose-dependent manner following treatment with the ADAM17 antagonist, TNF protease inhibitor 2. Hoechst 33258 staining and flow cytometric analysis revealed that inhibiting ADAM17 increased the rate of cellular apoptosis in neuronal and glial cell cultures, which was accompanied by increased cleavage of caspase-3. Western blot analysis demonstrated that inhibiting ADAM17 resulted in a reduction in the phosphorylation of the EGFR signalling pathway components and thereby impaired functional recovery, inhibited cell viability and prompted microglial apoptosis following SCI. Pre-treatment with the EGFR inhibitor, AG1478, rescued the ADAM17-mediated proliferation of microglial cells. These data demonstrated that ADAM17 contributed to microglial cell survival, predominantly by EGFR signalling, following SCI.

Monitoring of Short-Term Erythropoietin Therapy in Rats with Acute Spinal Cord Injury Using Manganese-Enhanced Magnetic Resonance Imaging.

To evaluate the short-term outcome of erythropoietin (EPO) therapy in rats with spinal cord injury (SCI) using manganese-enhanced magnetic resonance imaging (MEMRI). Rats were divided in an EPO and a control group. Laminectomy at Th11 was performed, followed by SCI. MnCl2 was applied into the cisterna magna and functional recovery was examined after injury using BBB-scoring. Then, rats were euthanized and the spinal cord was extracted for MEMRI. Finally, histological analysis was performed and correlated with MEMRI. EPO-treated animals showed significantly better functional recovery (P = .008, r = .62) and higher mean signal-to-noise ratio (SNR) in MEMRI compared to controls for slices 10-13 (P = .017, R(2) = .31) at the level of the lesion epicenter. Functional recovery correlated significantly with higher SNR values, determined using the mean SNR between slices 10 and 13 (P = .047, R(2) = .36). In this region, histology revealed a significantly decreased number of microglia cells and apoptosis in EPO-treated animals. MEMRI successfully depicts the therapeutic effect of EPO in early SCI that leads to a significant recovery in rats, a significantly reduced immune response and significantly reduced number of apoptotic cells at the height of the lesion epicenter.

Microglial numbers attain adult levels after undergoing a rapid decrease in cell number in the third postnatal week.

During postnatal development, microglia, CNS resident innate immune cells, are essential for synaptic pruning, neuronal apoptosis and remodeling. During this period microglia undergo morphological and phenotypic transformations; however, little is known about how microglial number and density is regulated during postnatal CNS development. We found that after an initial increase during the first 14 postnatal days, microglial numbers in mouse brain began declining in the third postnatal week and were reduced by 50% by 6weeks of age; these "adult" levels were maintained until at least 9months of age. Microglial CD11b levels increased, whereas CD45 and ER-MP58 declined between P10 and adulthood, consistent with a maturing microglial phenotype. Our data indicate that both increased microglial apoptosis and a decreased proliferative capacity contribute to the developmental reduction in microglial numbers. We found no correlation between developmental reductions in microglial numbers and brain mRNA levels of Cd200, Cx3Cl1, M-Csf or Il-34. We tested the ability of M-Csf-overexpression, a key growth factor promoting microglial proliferation and survival, to prevent microglial loss in the third postnatal week. Mice overexpressing M-Csf in astrocytes had higher numbers of microglia at all ages tested. However, the developmental decline in microglial numbers still occurred, suggesting that chronically elevated M-CSF is unable to overcome the developmental decrease in microglial numbers. Whereas the identity of the factor(s) regulating microglial number and density during development remains to be determined, it is likely that microglia respond to a "maturation" signal since the reduction in microglial numbers coincides with CNS maturation.

Puerarin protects against β-amyloid-induced microglia apoptosis via a PI3K-dependent signaling pathway.

Puerarin extracted from Radix puerariae is well-known for its anti-oxidative and neuroprotective activities. In this study, we investigated the protective effect of puerarin on amyloid-β protein (Aβ)-induced cytotoxicity and its potential mechanisms in BV-2 and primary microglial cells. We found that pretreatment with puerarin afforded protection against Aβ-induced cytotoxicity through inhibiting apoptosis in BV-2 and primary microglial cells. This result was also confirmed by the activated caspase-3 assay. Phospho-Akt and Bcl-2 expression increased after pretreatment with puerarin in BV-2 and primary microglial cells exposed to Aβ, whereas Bax expression and cytochrome c release decreased. In addition, puerarin treatment prevented the loss of mitochondrial membrane potential and reactive oxygen species production. Interestingly, these effects of puerarin against Aβ insult were abolished by LY294002, an inhibitor of PI3K phosphorylation. Taken together, these findings suggest that puerarin prevents Aβ-induced microglial apoptosis via the activation of PI3K/Akt signaling pathway, and might be a potential preventive or therapeutic agent for Alzheimer's disease.

7. The role of microglia in stress-induced depression

Despite impressive progress in understanding the molecular, cellular and circuit-level correlates of major depression, the biological mechanisms that causally underlie this disorder are still unclear, possibly due to excessive focus on the dysfunctioning of neurons, as compared with other types of brain cells. Therefore, we examined the role of dynamic alterations in microglia activation status in the development of chronic unpredictable stress (CUS)-induced depressive-like condition in rodents. We found that following an initial period (2–3 days) of stress-induced microglial proliferation and activation, some microglia underwent apoptosis, leading to reductions in their numbers within the hippocampus following 5 weeks of CUS exposure. At that time, microglia displayed reduced expression of activation markers as well as dystrophic morphology. Blockade of the initial stress-induced microglial activation by minocycline, imipramine or by transgenic interleukin-1 receptor antagonist over-expression rescued the subsequent microglial apoptosis and decline, as well as the CUS-induced depression and suppressed neurogenesis. Treatment of CUS-exposed mice with LPS (endotoxin), M-CSF or GM-CSF, which all stimulated hippocampal microglial proliferation, reversed the depressive-like behavior and dramatically increased hippocampal neurogenesis, whereas treatment with imipramine or minocycline had minimal anti-depressive effects in these mice. These findings provide direct causal evidence that disturbances in microglial functioning has an etiological role in chronic stress-induced depression, suggesting that microglia stimulators could serve as fast-acting anti-depressants in some forms of depressive and stress-related conditions.

CSF1 overexpression has pleiotropic effects on microglia in vivo.

Macrophage colony stimulating factor (CSF1) is a cytokine that is upregulated in several diseases of the central nervous system (CNS). To examine the effects of CSF1 overexpression on microglia, transgenic mice that overexpress CSF1 in the glial fibrillary acidic protein (GFAP) compartment were generated. CSF1 overexpressing mice have increased microglial proliferation and increased microglial numbers compared with controls. Treatment with PLX3397, a small molecule inhibitor of the CSF1 receptor CSF1R and related kinases, decreases microglial numbers by promoting microglial apoptosis in both CSF1 overexpressing and control mice. Microglia in CSF1 overexpressing mice exhibit gene expression profiles indicating that they are not basally M1 or M2 polarized, but they do have defects in inducing expression of certain genes in response to the inflammatory stimulus lipopolysaccharide. These results indicate that the CSF1 overexpression observed in CNS pathologies likely has pleiotropic influences on microglia. Furthermore, small molecule inhibition of CSF1R has the potential to reverse CSF1-driven microglial accumulation that is frequently observed in CNS pathologies, but can also promote apoptosis of normal microglia.

CSF1 over‐expression has pleiotropic effects on microglia in vivo (844.13)

Increased microglial numbers and activated microglial phenotypes are associated with many central nervous system (CNS) disorders including gliomas, neurodegeneration and brain injury. Thus, microglia are being tested as potential drug targets. Macrophage Colony Stimulating Factor (CSF1), an important cytokine for microglia and macrophages, is upregulated in these CNS disorders, but the effects of CSF1 on microglial phenotypes have not been well‐characterized in vivo. To study the effects of CSF1 over‐expression on microglia in the unperturbed CNS in vivo, we generated a transgenic mouse model in which CSF1 is inducibly over‐expressed in the GFAP compartment. We found that CSF1 over‐expression results in increased microglial proliferation, increased microglial numbers and an altered microglial phenotype compared to control mice. Microglia in CSF1 over‐expressing mice do not exhibit gene expression profiles typical of either M1 or M2 polarization, however they are defective in their response to the inflammatory stimulus LPS. Treatment with a small molecule inhibitor of the CSF1 receptor (CSF1R) and related kinases, decreased microglial numbers through apoptosis in both CSF1 over‐expressing and control mice. These results indicate that CSF1 signaling has multiple effects on microglia in vivo, and pharmacological inhibition of this signaling pathway may have efficacy in reversing the pathological levels of microglial accumulation in various CNS diseases by promoting microglial apoptosis. Moreover, our CSF1 over‐expressing genetic model should have utility for studying the impact of CSF1 over‐expression in mouse models of human disease.Grant Funding Source: Goldhirsh Foundation, the University of Wisconsin Graduate School and the NIH

Plexin-A1 is required for Toll-like receptor-mediated microglial activation in the development of lipopolysaccharide-induced encephalopathy

Recent investigations have suggested that semaphorins, which are known repulsive axon guidance molecules, may play a crucial role in maintaining brain homeostasis by regulating microglial activity. Sema3A, secreted in higher amounts from injured neurons, is considered to suppress excessive inflammatory responses by inducing microglial apoptosis through its binding to Plexin-A1 receptors on activated microglia. To clarify the in vivo role of Plexin-A1-mediated signaling in lipopolysaccharide (LPS)-induced injury in mouse brain, we examined the neuroinflammatory changes initiated by LPS administration to the cerebral ventricles of wild-type (WT) and Plexin-A1-deficient (−/−) mice. WT mice administered LPS exhibited a significantly higher expression of COX-2, iNOS, IL-1β and TNF-α in the hippocampus, and a significantly greater ventricular enlargement and intracerebral infiltration of leukocytes, as compared with the saline-treated group. By contrast, Plexin-A1−/− mice administered LPS did not exhibit a significantly increased expression of COX-2, iNOS, IL-1β or TNF-α in the hippocampus as compared with the saline-treated group. Plexin-A1−/− mice administered LPS did not show significant increases in ventricle size or infiltration of leukocytes into the brain, as compared with the saline-treated group. In WT, but not in the Plexin-A1−/− primary microglia treated with LPS, Sema3A induced significantly more nitric oxide production than in the immunoglobulin G control. These results revealed the crucial role of the Sema3A-Plexin-A1 interaction in the Toll-like receptor 4-mediated signaling of the LPS-induced activation of microglia. Thus, results of the present study revealed the essential role of Plexin-A1 in the development of LPS-induced neuroinflammation in mice, suggesting the possible application of microglial control of the semaphorin-plexin signaling system to the treatment of LPS-induced encephalopathy and other psychiatric diseases associated with neuroinflammation.

Basal CD38/cyclic ADP‐ribose‐dependent signaling mediates ATP release and survival of microglia by modulating connexin 43 hemichannels

It is necessary to investigate the mechanisms underlying ATP release from neural cells, because extracellular ATP plays multiple important biological roles in the brain. CD38 is an ectoenzyme that consumes NAD(+) to produce cyclic ADP-ribose (cADPR), a potent agonist of ryanodine receptors. Our previous study showed that CD38 reductions led to microglial apoptosis. In this study, we used both murine microglial BV2 cells and primary microglial cultures as cellular models to test our hypothesis that basal CD38/cyclic ADP-ribose (CD38/cADPR)-dependent signaling plays a key role in ATP release, which mediates basal survival of microglia. We found that inhibition of CD38/cADPR-dependent signaling by CD38 silencing or 8-Bromo-cADPR, a ryanodine receptor antagonist, produced significant ATP release from BV2 microglia. Cx43 small interfering RNA and Cx43 hemichannel blocker 18-α-glycyrrhetinic acid completely prevented the CD38 silencing or 8-Bromo-cADPR-induced ATP release. Prevention of the ATP release could also be due to P2X7 receptor antagonists. Our study has further suggested a key role of ATP release in the microglial apoptosis induced by decreased CD38/cADPR-dependent signaling. In addition, by using primary microglial cultures, we found that 8-Bromo-cADPR also induced significant ATP release, which could be attenuated by 18-α-glycyrrhetinic acid. 8-Bromo-cADPR was also found to induce death of primary microglial cultures. In conclusion, our results have suggested novel roles of basal activation of CD38/cADPR-dependent signaling in mediating microglial functions and survival: It mediates ATP release from microglia by modulating Cx43 hemichannels, which can significantly affect microglial survival.

4-Hydroxy TEMPO Attenuates Dichlorvos Induced Microglial Activation and Apoptosis

Microglial cells have been implicated in various neurodegenerative diseases. Previous studies from our lab have shown that dichlorvos (an organophosphate) could induce Parkinson's like features in rats. Recently, we have shown that dichlorvos can induce microglial activation, and if not checked in time could ultimately induce neuronal apoptosis. However, this activation does not always pose a threat to the neurons. Activated microglia also secrete various neuronal growth factors, suggesting that they have beneficial roles in CNS repair. Therefore, it is essential to control their detrimental functions selectively. Here, we tried to find out how microglial cells behave when exposed to dichlorvos in either the presence or absence of potent nitric oxide scavenger and superoxide dismutase mimetic, 4-hydroxy TEMPO (4-HT). Wistar rat pups (1 day) were used to isolate and culture primary microglial cells. We found 4-HT pretreatment successfully attenuated the dichlorvos mediated microglial activation. Moreover, 4-HT pretreatment decreased the up-regulated levels of p53 and its downstream effector, p21. The expression of various cell cycle regulators such as Chk2, CDC25a, and cyclin A remained close to their basal levels when 4-HT pretreatment was given. DNA fragmentation analysis showed significant reduction in the DNA damage of 4-HT pretreated microglia as compared to dichlorvos treated cells. In addition to this, we found 4-HT pretreatment prevented the microglial cells from undergoing apoptotic cell death even after 48 h of dichlorvos exposure. Taken together, our results showed 4-HT pretreatment could successfully ameliorate the dichlorvos induced microglial cell damage.

Dynamic microglial alterations underlie stress-induced depressive-like behavior and suppressed neurogenesis

The limited success in understanding the pathophysiology of major depression may result from excessive focus on the dysfunctioning of neurons, as compared with other types of brain cells. Therefore, we examined the role of dynamic alterations in microglia activation status in the development of chronic unpredictable stress (CUS)-induced depressive-like condition in rodents. We report that following an initial period (2-3 days) of stress-induced microglial proliferation and activation, some microglia underwent apoptosis, leading to reductions in their numbers within the hippocampus, but not in other brain regions, following 5 weeks of CUS exposure. At that time, microglia displayed reduced expression of activation markers as well as dystrophic morphology. Blockade of the initial stress-induced microglial activation by minocycline or by transgenic interleukin-1 receptor antagonist overexpression rescued the subsequent microglial apoptosis and decline, as well as the CUS-induced depressive-like behavior and suppressed neurogenesis. Similarly, the antidepressant drug imipramine blocked the initial stress-induced microglial activation as well as the CUS-induced microglial decline and depressive-like behavior. Treatment of CUS-exposed mice with either endotoxin, macrophage colony-stimulating factor or granulocyte-macrophage colony-stimulating factor, all of which stimulated hippocampal microglial proliferation, partially or completely reversed the depressive-like behavior and dramatically increased hippocampal neurogenesis, whereas treatment with imipramine or minocycline had minimal or no anti-depressive effects, respectively, in these mice. These findings provide direct causal evidence that disturbances in microglial functioning has an etiological role in chronic stress-induced depression, suggesting that microglia stimulators could serve as fast-acting anti-depressants in some forms of depressive and stress-related conditions.

ADAM17 is a survival factor for microglial cells in vitro and in vivo after spinal cord injury in mice

A disintegrin and metalloprotease 17 (ADAM17) is a sheddase with important substrates including tumor necrosis factor-α (TNF-α) and its receptors, the p75 neurotrophin receptor (p75NTR), and members of the epidermal growth factor family. The rationale of this study was to inhibit ADAM17-induced shedding of soluble TNF-α in order to reduce detrimental inflammation after spinal cord injury (SCI). However, using the specific ADAM17 blocker BMS-561392 in neuronal and glial cell cultures, we show that proper functioning of ADAM17 is vital for oligodendrocyte and microglia survival in a p44 MAPK-dependent manner. In contrast, genetic ablation of ADAM17 specifically increases microglial death. Surprisingly, although blocking ADAM17 in vivo does not substantially change the ratio between membrane-bound and soluble TNF-α, it increases expression of the pro-apoptotic marker Bax and microglial apoptosis while impairing functional recovery after SCI. These data suggest that ADAM17 is a key survival factor for microglial cells after SCI.

Inhibition of mitochondrial fission attenuates Aβ-induced microglia apoptosis

Mitochondrial division inhibitor 1 (mdivi-1), a selective inhibitor of mitochondrial fission protein dynamin-related protein 1 (Drp1), has been reported to display neuroprotective properties in different animal models. In the present study, we investigated the protective effect of mdivi-1 on β-amyloid protein (Aβ)-induced cytotoxicity and its potential mechanisms in BV-2 and primary microglial cells. We found that mitochondrial fission was increased in Aβ treatment and inhibition of mitochondrial fission by mdivi-1 significantly reduced Aβ-induced expression of CD11b (a marker of microglial activation), viability loss and apoptotic rate increase in BV-2 and primary microglial cells. Moreover, we also found that mdivi-1 treatment markedly reversed mitochondrial membrane potential loss, cytochrome c (CytC) release and caspase-3 activation. Altogether, our data suggested that mdivi-1 exerts neuroprotective effects against Aβ-induced microglial apoptosis, and the underlying mechanism may be through inhibiting mitochondrial membrane potential loss, CytC release and suppression of the mitochondrial apoptosis pathway.

Autologous bone marrow mononuclear cells therapy attenuates activated microglial/macrophage response and improves spatial learning after traumatic brain injury

Autologous bone marrow-derived mononuclear cells (AMNCs) have shown therapeutic promise for central nervous system insults such as stroke and traumatic brain injury (TBI). We hypothesized that intravenous injection of AMNC provides neuroprotection, which leads to cognitive improvement after TBI. A controlled cortical impact (CCI) rodent TBI model was used to examine blood-brain barrier (BBB) permeability, neuronal and glial apoptosis, as well as cognitive behavior. Two groups of rats underwent CCI with AMNC treatment (CCI-autologous) or without AMNC treatment (CCI-alone), consisting of 2 million AMNC per kilogram body weight harvested from the tibia and intravenously injected 72 hours after injury. CCI-alone animals underwent sham harvests and received vehicle injections. Ninety-six hours after injury, AMNC significantly reduced the BBB permeability in injured animals, and there was an increase in apoptosis of proinflammatory activated microglia in the ipsilateral hippocampus. At 4 weeks after injury, we observed significant improvement in probe testing of CCI-Autologous group in comparison to CCI-Alone in the Morris Water Maze paradigm. Our data demonstrate that the intravenous injection of AMNC after TBI leads to neuroprotection by preserving early BBB integrity, increasing activated microglial apoptosis and improving cognitive function.

Reciprocal modulation of C/EBP‐α and C/EBP‐β by IL‐13 in activated microglia prevents neuronal death

In response to aggravation by activated microglia, IL-13 can significantly enhance ER stress induction, apoptosis, and death via reciprocal signaling through CCAAT/enhancer-binding protein alpha (C/EBP-α) and C/EBP-beta (C/EBP-β). This reciprocal signaling promotes neuronal survival. Since the induction of cyclooxygenase-2 (COX-2) and peroxisome proliferator-activated receptor gamma/heme oxygenase 1 (PPAR-γ/HO-1) by IL-13 plays a crucial role in the promotion of and protection from activated microglia, respectively; here, we investigated the role of IL-13 in regulating C/EBPs in activated microglia and determined its correlation with neuronal function. The results revealed that IL-13 significantly enhanced C/EBP-α/COX-2 expression and PGE2 production in LPS-treated microglial cells. Paradoxically, IL-13 abolished C/EBP-β/PPAR-γ/HO-1 expression. IL-13 also enhanced ER stress-evoked calpain activation by promoting the association of C/EBP-β and PPAR-γ. SiRNA-C/EBP-α effectively reversed the combined LPS-activated caspase-12 activation and IL-13-induced apoptosis. In contrast, siRNA-C/EBP-β partially increased microglial cell apoptosis. By NeuN immunochemistry and CD11b staining, there was improvement in the loss of CA3 neuronal cells after intrahippocampal injection of IL-13. This suggests that IL-13-enhanced PLA2 activity regulates COX-2/PGE2 expression through C/EBP-α activation. In parallel, ER stress-related calpain downregulates the PPAR-γ/HO-1 pathway via C/EBP-β and leads to aggravated death of activated microglia via IL-13, thereby preventing cerebral inflammation and neuronal injury.

Chemokine Fractalkine Attenuates Overactivation and Apoptosis of BV-2 Microglial Cells Induced by Extracellular ATP

Microglia, the resident macrophages of the central nervous system (CNS), are activated by a myriad of signaling molecules including ATP, an excitatory neurotransmitter and neuron-glial signal with both neuroprotective and neurotoxic effects. The "microglial dysfunction hypothesis" of neurodegeneration posits that overactivated microglia have a reduced neuroprotective capacity and instead promote neurotoxicity. The chemokine fractalkine (FKN), one of only two chemokines constitutively expressed in the CNS, is neuroprotective in several in vivo and in vitro models of CNS pathology. It is possible, but not yet demonstrated, that high ATP concentrations induce microglial overactivation and apoptosis while FKN reduces ATP-mediated microglial overactivation and cytotoxicity. In the current study, we examined the effects of FKN on ATP-induced microglial apoptosis and the underlying mechanisms in the BV-2 microglial cell line. Exposure to ATP induced a dose-dependent reduction in BV-2 cell viability. Prolonged exposure to a high ATP concentration (3 mM for 2 h) transformed ramified (quiescent) BV-2 cells to the amoebic state, induced apoptosis, and reduced Akt phosphorylation. Pretreatment with FKN significantly inhibited ATP-induced microglial apoptosis and transformed amoebic microglia to ramified quiescent cells. These protective effects were blocked by chemical inhibition of PI3 K, strongly implicating the PI3 K/Akt signaling pathway in FKN-mediated protection of BV-2 cells from cytotoxic ATP concentrations. Prevention of ATP-induced microglial overactivation and apoptosis may enhance the neuroprotective capacity of these cells against both acute insults and chronic CNS diseases.