Juvenile neuronal ceroid lipofuscinosis (JNCL) is a lysosomal storage disease caused by an autosomal recessive mutation in CLN3. Regions of microglial activation precede and predict areas of neuronal loss in JNCL; however, the functional role of activated microglia remains to be defined. The inflammasome is a key molecular pathway for activating pro‐IL‐1β in microglia, and IL‐1β is elevated in the brains of JNCL patients and can induce neuronal cell death. Here, we utilized primary microglia isolated from CLN3Δex7/8 mutant and wild‐type (WT) mice to examine the impact of CLN3 mutation on microglial activation and inflammasome function. Treatment with neuronal lysates and ceramide, a lipid intermediate elevated in the JNCL brain, led to inflammasome activation and IL‐1β release in CLN3Δex7/8 microglia but not WT cells, as well as increased expression of additional pro‐inflammatory mediators. Similar effects were observed following either TNF‐α or IL‐1β treatment, suggesting that CLN3Δex7/8 microglia exist in primed state and hyper‐respond to several inflammatory stimuli compared to WT cells. CLN3Δex7/8 microglia displayed constitutive caspase‐1 activity that when blocked led to increased glutamate release that coincided with hemichannel opening. Conditioned medium from activated CLN3Δex7/8 or WT microglia induced significant cell death in CLN3Δex7/8 but not WT neurons, demonstrating that intrinsically diseased CLN3Δex7/8 neurons are less equipped to withstand cytotoxic insults generated by activated microglia. Collectively, aberrant microglial activation may contribute to the pathological chain of events leading to neurodegeneration during later stages of JNCL.
DJ‐1 is an oxidative stress sensor that localizes to the mitochondria when the cell is exposed to oxidative stress. DJ‐1 mutations that result in gene deficiency are linked to increased risk of Parkinson's disease (PD). Activation of microglial stress conditions that are linked to PD may result in neuronal death. We postulated that DJ‐1 deficiency may increase microglial neurotoxicity. We found that down‐regulation of DJ‐1 in microglia using an shRNA approach increased cell sensitivity to dopamine as measured by secreted pro‐inflammatory cytokines such as IL‐1β and IL‐6. Furthermore, we discovered that DJ‐1‐deficient microglia had increased monoamine oxidase activity that resulted in elevation of intracellular reactive oxygen species and nitric oxide leading to increased dopaminergic neurotoxicity. Rasagaline, a monoamine oxidase inhibitor approved for treatment of PD, reduced the microglial pro‐inflammatory phenotype and significantly reduced neurotoxicity. Moreover, we discovered that DJ‐1‐deficient microglia have reduced expression of triggering receptor expressed on myeloid cells 2 (TREM2), previously suggested as a risk factor for pro‐inflammation in neurodegenerative diseases. Further studies of DJ‐1‐mediated cellular pathways in microglia may contribute useful insights into the development of PD providing future avenues for therapeutic intervention.
Alzheimer's disease (AD) is a neurodegenerative disorder that represents the most common type of dementia among elderly people. Amyloid beta (Aβ) peptides in extracellular Aβ plaques, produced from the amyloid precursor protein (APP) via sequential processing by β‐ and γ‐secretases, impair hippocampal synaptic plasticity, and cause cognitive dysfunction in AD patients. Here, we report that Aβ peptides also impair another form of synaptic plasticity; cerebellar long‐term depression (LTD). In the cerebellum of commonly used AD mouse model, APPswe/PS1dE9 mice, Aβ plaques were detected from 8 months and profound accumulation of Aβ plaques was observed at 18 months of age. Biochemical analysis revealed relatively high levels of APP protein and Aβ in the cerebellum of APPswe/PS1dE9 mice. At pre‐Aβ accumulation stage, LTD induction, and motor coordination are disturbed. These results indicate that soluble Aβ oligomers disturb LTD induction and cerebellar function in AD mouse model.
Tuftsin (Thr‐Lys‐Pro‐Arg) is a natural immunomodulating peptide found to stimulate phagocytosis in macrophages/microglia. Tuftsin binds to the receptor neuropilin‐1 (Nrp1) on the surface of cells. Nrp1 is a single‐pass transmembrane protein, but its intracellular C‐terminal domain is too small to signal independently. Instead, it associates with a variety of coreceptors. Despite its long history, the pathway through which tuftsin signals has not been described. To investigate this question, we employed various inhibitors to Nrp1's coreceptors to determine which route is responsible for tuftsin signaling. We use the inhibitor EG00229, which prevents tuftsin binding to Nrp1 on the surface of microglia and reverses the anti‐inflammatory M2 shift induced by tuftsin. Furthermore, we demonstrate that blockade of transforming growth factor beta (TGFβ) signaling via TβR1 disrupts the M2 shift similar to EG00229. We report that tuftsin promotes Smad3 phosphorylation and reduces Akt phosphorylation. Taken together, our data show that tuftsin signals through Nrp1 and the canonical TGFβ signaling pathway.
Recent studies have shown that sigma‐1 receptor orthodox agonists can inhibit neuroinflammation. SKF83959 (3‐methyl‐6‐chloro‐7,8‐hydroxy‐1‐[3‐methylphenyl]‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepine), an atypical dopamine receptor‐1 agonist, has been recently identified as a potent allosteric modulator of sigma‐1 receptor. Here, we investigated the anti‐inflammatory effects of SKF83959 in lipopolysaccharide (LPS)‐stimulated BV2 microglia. Our results indicated that SKF83959 significantly suppressed the expression/release of the pro‐inflammatory mediators, such as tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), inducible nitric oxide synthase (iNOS), and inhibited the generation of reactive oxygen species. All of these responses were blocked by selective sigma‐1 receptor antagonists (BD1047 or BD1063) and by ketoconazole (an inhibitor of enzyme cytochrome c17 to inhibit the synthesis of endogenous dehydroepiandrosterone, DHEA). Additionally, we found that SKF83959 promoted the binding activity of DHEA with sigma‐1 receptors, and enhanced the inhibitory effects of DHEA on LPS‐induced microglia activation in a synergic manner. Furthermore, in a microglia‐conditioned media system, SKF83959 inhibited the cytotoxicity of conditioned medium generated by LPS‐activated microglia toward HT‐22 neuroblastoma cells. Taken together, our study provides the first evidence that allosteric modulation of sigma‐1 receptors by SKF83959 inhibits microglia‐mediated inflammation.
In the present study, the effects of the two classical anti‐epileptic drugs, carbamazepine and valproic acid, and the non‐classical anti‐seizure drug vinpocetine were investigated on the expression of the pro‐inflammatory cytokines IL‐1β and TNF‐α in the hippocampus of rats by PCR or western blot after the administration of one or seven doses. Next, the effects of the anti‐seizure drugs were investigated on the rise in cytokine expression induced by lipopolysaccharides (LPS) inoculation in vivo. To validate our methods, the changes induced by the pro‐convulsive agents 4‐aminopyridine, pentylenetetrazole and pilocarpine were also tested. Finally, the effect of the anti‐seizure drugs on seizures and on the concomitant rise in pro‐inflammatory cytokine expression induced by 4‐aminopyridine was explored. Results show that vinpocetine and carbamazepine reduced the expression of IL‐1β and TNF‐α from basal conditions, and the increase in both pro‐inflammatory cytokines induced by LPS. In contrast, valproic acid failed to reduce both the expression of the cytokines from basal conditions and the rise in IL‐1β and TNF‐α expression induced by LPS. Tonic‐clonic seizures induced either by 4‐aminopyridine, pentylenetetrazole or pilocarpine increased the expression of IL‐1β and TNF‐α markedly. 4‐aminopyridine‐induced changes were reduced by all the tested anti‐seizure drugs, although valproic acid was less effective. We conclude that the anti‐seizure drugs, vinpocetine and carbamazepine, whose mechanisms of action involve a decrease in ion channels permeability, also reduce cerebral inflammation.
Beta amyloid (Aβ) oligomers are thought to contribute to the pathogenesis of Alzheimer's disease. However, clinical trials using Aβ immunization were unsuccessful due to strong brain inflammation, the mechanisms of which are poorly understood. In this study we tested whether monoclonal antibodies to oligomeric Aβ would prevent the neurotoxicity of Aβ oligomers in primary neuronal‐glial cultures. However, surprisingly, the antibodies dramatically increased the neurotoxicity of Aβ. Antibodies bound to monomeric Aβ fragments were non‐toxic to cultured neurons, while antibodies to other oligomeric proteins: hamster polyomavirus major capsid protein, human metapneumovirus nucleocapsid protein, and measles virus nucleocapsid protein, strongly potentiated the neurotoxicity of their antigens. The neurotoxicity of antibody‐oligomeric antigen complexes was abolished by removal of the Fc region from the antibodies or by removal of microglia from cultures, and was accompanied by inflammatory activation and proliferation of the microglia in culture. In conclusion, we find that immune complexes formed by Aβ oligomers or other oligomeric/multimeric antigens and their specific antibodies can cause death and loss of neurons in primary neuronal‐glial cultures via Fc‐dependent microglial activation. The results suggest that therapies resulting in antibodies to oligomeric Aβ or oligomeric brain virus proteins should be used with caution or with suppression of microglial activation.
Microglia are the resident macrophages of the central nervous system that survey the microenvironment for signals of injury or infection. The response to such signals induces an inflammatory response involving macrophages derived from both resident microglia and recruited circulating monocytes. Although implicated as contributors to autoimmune‐mediated injury, microglia/ macrophages have recently been shown to be critical for the important central nervous system regenerative process of remyelination. This functional dichotomy may reflect their ability to be polarized along a continuum of activation states including the well‐characterized cytotoxic M1 and regenerative M2 phenotypes. Here, we review the roles of microglia, monocytes and the macrophages which they give rise to in creating lesion environments favourable to remyelination, highlighting the specific roles of M1 and M2 phenotypes and how the pro‐regenerative role of the innate immune system is altered by ageing.
Metabotropic glutamate receptor 5 (mGluR5) regulates excitatory post‐synaptic signaling in the central nervous system (CNS) and is implicated in various CNS disorders. Protein kinase A (PKA) signaling is known to play a critical role in neuropsychiatric disorders such as Parkinson's disease, schizophrenia, and addiction. Dopamine signaling is known to modulate the properties of mGluR5 in a cAMP‐ and PKA‐dependent manner, suggesting that mGluR5 may be a direct target for PKA. Our study identifies mGluR5 at Ser870 as a direct substrate for PKA phosphorylation and demonstrates that this phosphorylation plays a critical role in the PKA‐mediated modulation of mGluR5 functions such as extracellular signal‐regulated kinase phosphorylation and intracellular Ca2+ oscillations. The identification of the molecular mechanism by which PKA signaling modulates mGluR5‐mediated cellular responses contributes to the understanding of the interaction between dopaminergic and glutamatergic neuronal signaling.
Inheritance of the apolipoprotein E4 (apoE4) genotype has been identified as the major genetic risk factor for late‐onset Alzheimer's disease (AD). Studies have shown that the binding between apoE and amyloid‐β (Aβ) peptides occurs at residues 244–272 of apoE and residues 12–28 of Aβ. ApoE4 has been implicated in promoting Aβ deposition and impairing clearance of Aβ. We hypothesized that blocking the apoE/Aβ interaction would serve as an effective new approach to AD therapy. We have previously shown that treatment with Aβ12‐28P can reduce amyloid plaques in APP/PS1 transgenic (Tg) mice and vascular amyloid in TgSwDI mice with congophilic amyloid angiopathy. In the present study, we investigated whether the Aβ12‐28P elicits a therapeutic effect on tau‐related pathology in addition to amyloid pathology using old triple transgenic AD mice (3xTg, with PS1M146V, APPSwe and tauP30IL transgenes) with established pathology from the ages of 21 to 26 months. We show that treatment with Aβ12‐28P substantially reduces tau pathology both immunohistochemically and biochemically, as well as reducing the amyloid burden and suppressing the activation of astrocytes and microglia. These affects correlate with a behavioral amelioration in the treated Tg mice.
Interleukin‐1β (IL‐1β) is essential for eliciting protective immunity during the acute phase of Staphylococcus aureus (S. aureus) infection in the central nervous system (CNS). We previously demonstrated that microglial IL‐1β production in response to live S. aureus is mediated through the Nod‐like receptor protein 3 (NLRP3) inflammasome, including the adapter protein ASC (apoptosis‐associated speck‐like protein containing a caspase‐1 recruitment domain), and pro‐caspase 1. Here, we utilized NLRP3, ASC, and caspase 1/11 knockout (KO) mice to demonstrate the functional significance of inflammasome activity during CNS S. aureus infection. ASC and caspase 1/11 KO animals were exquisitely sensitive, with approximately 50% of mice succumbing to infection within 24 h. Unexpectedly, the survival of NLRP3 KO mice was similar to wild‐type animals, suggesting the involvement of an alternative upstream sensor, which was later identified as absent in melanoma 2 (AIM2) based on the similar disease patterns between AIM2 and ASC KO mice. Besides IL‐1β, other key inflammatory mediators, including IL‐6, CXCL1, CXCL10, and CCL2 were significantly reduced in the CNS of AIM2 and ASC KO mice, implicating autocrine/paracrine actions of IL‐1β, as these mediators do not require inflammasome processing for secretion. These studies demonstrate a novel role for the AIM2 inflammasome as a critical molecular platform for regulating IL‐1β release and survival during acute CNS S. aureus infection.
Leptin is a centrally acting hormone that controls metabolic pathways. Recent epidemiological studies suggest that plasma leptin is protective against Alzheimer's disease. However, the mechanism that underlies this effect remains uncertain. To investigate whether leptin inhibits the assembly of amyloid β‐protein (Aβ) on the cell surface of neurons, we treated primary neurons with leptin. Leptin treatment decreased the GM1 ganglioside (GM1) levels in the detergent‐resistant membrane microdomains (DRMs) of neurons. The increase in GM1 expression induced by leptin was inhibited after pre‐treatment with inhibitors of phosphatidylinositol 3‐kinase (LY294002), Akt (triciribine) and the mammalian target of rapamycin (i.e. rapamycin), but not by an inhibitor of extracellular signal‐regulated kinase (PD98059). In addition, pre‐treatment with these reagents blocked the induction of GM1 in DRMs by leptin. Furthermore, Aβ assembly on the cell surface of neurons was inhibited greatly after treatment with leptin. This reduction was markedly inhibited after pre‐treatment with LY294002, triciribine, and rapamycin. These results suggest that leptin significantly inhibits Aβ assembly by decreasing GM1 expression in DRMs of the neuronal surface through the phosphatidylinositol 3‐kinase/Akt/mammalian target of rapamycin pathway.
Inflammation within the CNS is a major component of many neurodegenerative diseases. A characteristic feature is the generation of microglia‐derived factors that play an essential role in the immune response. IL‐1β is a pro‐inflammatory cytokine released by activated microglia, able to exacerbate injury at elevated levels. In the presence of caspase‐1, pro‐IL‐1β is cleaved to the mature cytokine following NOD‐like receptor pyrin domain containing 3 (NLRP3) inflammasome activation. Growing evidence suggests that ceramide plays a critical role in NLRP3 inflammasome assembly, however, the relationship between ceramide and inflammasome activation in microglia remains unknown. Here, we investigated potential mechanistic links between ceramide as a modulator of NLRP3 inflammasome assembly and the resulting secretion of IL‐1β using small bioactive enzyme stimulators and inhibitors of ceramide signaling in wild‐type and apoptosis‐associated speck‐like protein containing a CARD knockout (ASC?/?) primary microglia. To induce the expression of inflammasome components, microglia were primed prior to experiments. Treatment with sodium palmitate (PA) induced de novo ceramide synthesis via modulation of its synthesizing protein serine palmitoyl transferase resulting in increased IL‐1β secretion in microglia. Exposure of microglia to the serine palmitoyl transferase‐inhibitor l ‐cycloserine significantly prevented PA‐induced IL‐1β secretion. Application of the ceramide analogue C2 and the sphingosine‐1‐phosphate‐receptor agonist Fingolimod (FTY720) up‐regulated levels of IL‐1β and cleaved caspase‐1 in wild‐type microglia, whereas ASC?/? microglia were unaffected. HPA‐12 inhibition of ceramide transport did not affect inflammasome activation. Taken together, our findings reveal a critical role for ceramide as a positive modulator of NLRP3 inflammasome assembly and the resulting release of IL‐1β.
We systematically investigated the purification process of post‐synaptic density (PSD) and post‐synaptic membrane rafts (PSRs) from the rat forebrain synaptic plasma membranes by examining the components and the structures of the materials obtained after the treatment of synaptic plasma membranes with TX‐100, n‐octyl β‐d ‐glucoside (OG) or 3‐([3‐cholamidopropyl]dimethylammonio)‐2‐hydroxy‐1‐propanesulfonate (CHAPSO). These three detergents exhibited distinct separation profiles for the synaptic subdomains. Type I and type II PSD proteins displayed mutually exclusive distribution. After TX‐100 treatment, type I PSD was recovered in two fractions: a pellet and an insoluble fraction 8, which contained partially broken PSD‐PSR complexes. Conventional PSD was suggested to be a mixture of these two PSD pools and did not contain type II PSD. An association of type I PSD with PSRs was identified in the TX‐100 treatment, and those with type II PSD in the OG and CHAPSO treatments. An association of GABA receptors with gephyrin was easily dissociated. OG at a high concentration solubilized the type I PSD proteins. CHAPSO treatment resulted in a variety of distinct fractions, which contained certain novel structures. Two different pools of GluA, either PSD or possibly raft‐associated, were identified in the OG and CHAPSO treatments. These results are useful in advancing our understanding of the structural organization of synapses at the molecular level.
An important pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid‐beta (Aβ) peptides in the brain parenchyma, leading to neuronal death and impaired learning and memory. The protease γ‐secretase is responsible for the intramembrane proteolysis of the amyloid‐β precursor protein (APP), which leads to the production of the toxic Aβ peptides. Thus, an attractive therapeutic strategy to treat AD is the modulation of the γ‐secretase activity, to reduce Aβ42 production. Because phosphorylation of proteins is a post‐translational modification known to modulate the activity of many different enzymes, we used electrospray (LC‐MS/MS) mass spectrometry to identify new phosphosites on highly purified human γ‐secretase. We identified 11 new single or double phosphosites in two well‐defined domains of Presenilin‐1 (PS1), the catalytic subunit of the γ‐secretase complex. Next, mutagenesis and biochemical approaches were used to investigate the role of each phosphosite in the maturation and activity of γ‐secretase. Together, our results suggest that the newly identified phosphorylation sites in PS1 do not modulate γ‐secretase activity and the production of the Alzheimer's Aβ peptides. Individual PS1 phosphosites shall probably not be considered therapeutic targets for reducing cerebral Aβ plaque formation in AD.
Spreading depression (SD), the most likely cause of migraine aura and perhaps migraine, occurs with increased oxidative stress (OS). SD increases reactive oxygen species (ROS), and ROS, in turn, can signal to increase neuronal excitability, which includes increased SD susceptibility. SD also elevates tumor necrosis factor‐α (TNF‐α), which increases neuronal excitability. Accordingly, we probed for the cellular origin of OS from SD and its relationship to TNF‐α, which might promote SD, using rat hippocampal slice cultures. We observed significantly increased OS from SD in astrocytes and microglia but not in neurons or oligodendrocytes. Since insulin‐like growth factor‐1 (IGF‐1) mitigates OS from SD, we determined the cell types responsible for this effect. We found that IGF‐1 significantly decreased microglial but not astrocytic OS from SD. We also show that IGF‐1 abrogated the SD‐induced TNF‐α increase. Furthermore, TNF‐α application increased microglial but not astrocytic OS, an effect abrogated by IGF‐1. Next, we showed that SD increased SD susceptibility, and does so via TNF‐α. This work suggests that microglia promote SD via increased and interrelated ROS and TNF‐α signaling. Thus, IGF‐1 mitigation of microglial ROS and TNF‐α responses may be targets for novel therapeutics development to prevent SD, and perhaps migraine.
Accumulating evidence indicates that activated microglia contribute to the neuropathology involved in many neurodegenerative diseases and after traumatic injury to the CNS. The cytokine transforming growth factor‐beta 1 (TGF‐β1), a potent deactivator of microglia, should have the potential to reduce microglial‐mediated neurodegeneration. It is therefore perplexing that high levels of TGF‐β1 are found in conditions where microglia are chronically activated. We hypothesized that TGF‐β1 signaling is suppressed in activated microglia. We therefore activated primary rat microglia with lipopolysaccharide (LPS) and determined the expression of proteins important to TGF‐β1 signaling. We found that LPS treatment decreased the expression of the TGF‐β receptors, TβR1 and TβR2, and reduced protein levels of Smad2, a key mediator of TGF‐β signaling. LPS treatment also antagonized the ability of TGF‐β to suppress expression of pro‐inflammatory cytokines and to induce microglial cell death. LPS treatment similarly inhibited the ability of the TGF‐β related cytokine, Activin‐A, to down‐regulate expression of pro‐inflammatory cytokines and to induce microglial cell death. Together, these data suggest that microglial activators may oppose the actions of TGF‐β1, ensuring continued microglial activation and survival that eventually may contribute to the neurodegeneration prevalent in chronic neuroinflammatory conditions.
Histone deacetylase (HDAC) inhibitors prevent neural cell death in in vivo models of cerebral ischaemia, brain injury and neurodegenerative disease. One mechanism by which HDAC inhibitors may do this is by suppressing the excessive inflammatory response of chronically activated microglia. However, the molecular mechanisms underlying this anti‐inflammatory effect and the specific HDAC responsible are not fully understood. Recent data from in vivo rodent studies have shown that inhibition of class I HDACs suppresses neuroinflammation and is neuroprotective. In our study, we have identified that selective HDAC inhibition with inhibitors apicidin, MS‐275 or MI‐192, or specific knockdown of HDAC1 or 2 using siRNA, suppresses the expression of cytokines interleukin‐6 (IL‐6) and tumour necrosis factor‐alpha (TNF‐α) in BV‐2 murine microglia activated with lipopolysaccharide (LPS). Furthermore, we found that in the absence of HDAC1, HDAC2 is up‐regulated and these increased levels are compensatory, suggesting that these two HDACs have redundancy in regulating the inflammatory response of microglia. Investigating the possible underlying anti‐inflammatory mechanisms suggests an increase in protein expression is not important. Taken together, this study supports the idea that inhibitors selective towards HDAC1 or HDAC2, may be therapeutically useful for targeting neuroinflammation in brain injuries and neurodegenerative disease.
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