Insulin‐like growth factor‐1 abrogates microglial oxidative stress and TNF‐α responses to spreading depression

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.

     

Diet‐induced obesity accelerates the onset of terminal phenotypes in α‐synuclein transgenic mice

Parkinson's disease (PD) and diabetes belong to the most common neurodegenerative and metabolic syndromes, respectively. Epidemiological links between these two frequent disorders are controversial. The neuropathological hallmarks of PD are protein aggregates composed of amyloid‐like fibrillar and serine‐129 phosphorylated (pS129) α‐synuclein (AS). To study if diet‐induced obesity could be an environmental risk factor for PD‐related α‐synucleinopathy, transgenic (TG) mice, expressing the human mutant A30P AS in brain neurons, were subjected after weaning to a lifelong high fat diet (HFD). The TG mice became obese and glucose‐intolerant, as did the wild‐type controls. Upon aging, HFD significantly accelerated the onset of the lethal locomotor phenotype. Coinciding with the premature movement phenotype and death, HFD accelerated the age of onset of brainstem α‐synucleinopathy as detected by immunostaining with antibodies against pathology‐associated pS129. Amyloid‐like neuropathology was confirmed by thioflavin S staining. Accelerated onset of neurodegeneration was indicated by Gallyas silver‐positive neuronal dystrophy as well as astrogliosis. Phosphorylation of the activation sites of the pro‐survival signaling intermediate Akt was reduced in younger TG mice after HFD. Thus, diet‐induced obesity may be an environmental risk factor for the development of α‐synucleinopathies. The molecular and cellular mechanisms remain to be further elucidated.

     

Testing the potency of anti‐TNF‐α and anti‐IL‐1β drugs using spheroid cultures of human osteoarthritic chondrocytes and donor‐matched chondrogenically differentiated mesenchymal stem cells

Inflammation plays a major role in progression of rheumatoid arthritis, a disease treated with antagonists of tumor necrosis factor‐alpha (TNF‐α) and interleukin 1β (IL‐1β). New in vitro testing systems are needed to evaluate efficacies of new anti‐inflammatory biological drugs, ideally in a patient‐specific manner. To address this need, we studied microspheroids containing 10,000 human osteoarthritic primary chondrocytes (OACs) or chondrogenically differentiated mesenchymal stem cells (MSCs), obtained from three donors. Hypothesizing that this system can recapitulate clinically observed effects of anti‐inflammatory drugs, spheroids were exposed to TNF‐α, IL‐1β, or to supernatant containing secretome from activated macrophages (MCM). The anti‐inflammatory efficacies of anti‐TNF‐α biologicals adalimumab, infliximab, and etanercept, and the anti‐IL‐1β agent anakinra were assessed in short‐term microspheroid and long‐term macrospheroid cultures (100,000 OACs). While gene and protein expressions were evaluated in microspheroids, diameters, amounts of DNA, glycosaminoglycans, and hydroxiproline were measured in macrospheroids. The tested drugs significantly decreased the inflammation induced by TNF‐α or IL‐1β. The differences in potency of anti‐TNF‐α biologicals at 24 h and 3 weeks after their addition to inflamed spheroids were comparable, showing high predictability of short‐term cultures. Moreover, the data obtained with microspheroids grown from OACs and chondrogenically differentiated MSCs were comparable, suggesting that MSCs could be used for this type of in vitro testing. We propose that in vitro gene expression measured after the first 24 h in cultures of chondrogenically differentiated MSCs can be used to determine the functionality of anti‐TNF‐α drugs in personalized and preclinical studies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1045–1058, 2018     

Rapid mitochondrial dysfunction mediates TNF‐alpha‐induced neurotoxicity

Tumor necrosis factor alpha (TNF‐α) is known to exacerbate ischemic brain injury; however, the mechanism is unknown. Previous studies have evaluated the effects of TNF‐α on neurons with long exposures to high doses of TNF‐α, which is not pathophysiologically relevant. We characterized the rapid effects of TNF‐α on basal respiration, ATP production, and maximal respiration using pathophysiologically relevant, post‐stroke concentrations of TNF‐α. We observed a reduction in mitochondrial function as early as 1.5 h after exposure to low doses of TNF‐α, followed by a decrease in cell viability in HT‐22 cells and primary neurons. Subsequently, we used the HT‐22 cell line to determine the mechanism by which TNF‐α causes a rapid and profound reduction in mitochondrial function. Pre‐treating with TNF‐R1 antibody, but not TNF‐R2 antibody, ameliorated the neurotoxic effects of TNF‐α, indicating that TNF‐α exerts its neurotoxic effects through TNF‐R1. We observed an increase in caspase 8 activity and a decrease in mitochondrial membrane potential after exposure to TNF‐α which resulted in a release of cytochrome c from the mitochondria into the cytosol. These novel findings indicate for the first time that an acute exposure to pathophysiologically relevant concentrations of TNF‐α has neurotoxic effects mediated by a rapid impairment of mitochondrial function.

     

Identification of new Presenilin‐1 phosphosites: implication for γ‐secretase activity and Aβ production

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.

     

Inhibition of interleukin‐1β production by extracellular acidification through the TDAG8/cAMP pathway in mouse microglia

Interleukin‐1β (IL‐1β) is released from activated microglia and involved in the neurodegeneration of acute and chronic brain disorders, such as stroke and Alzheimer's disease, in which extracellular acidification has been shown to occur. Here, we examined the extracellular acidic pH regulation of IL‐1β production, especially focusing on TDAG8, a major proton‐sensing G‐protein‐coupled receptor, in mouse microglia. Extracellular acidification inhibited lipopolysaccharide ‐induced IL‐1β production, which was associated with the inhibition of IL‐1β cytoplasmic precursor and mRNA expression. The IL‐1β mRNA and protein responses were significantly, though not completely, attenuated in microglia derived from TDAG8‐deficient mice compared with those from wild‐type mice. The acidic pH also stimulated cellular cAMP accumulation, which was completely inhibited by TDAG8 deficiency. Forskolin and a cAMP derivative, which specifically stimulates protein kinase A (PKA), mimicked the proton actions, and PKA inhibitors reversed the acidic pH‐induced IL‐1β mRNA expression. The acidic pH‐induced inhibitory IL‐1β responses were accompanied by the inhibition of extracellular signal‐related kinase and c‐Jun N‐terminal kinase activities. The inhibitory enzyme activities in response to acidic pH were reversed by the PKA inhibitor and TDAG8 deficiency. We conclude that extracellular acidic pH inhibits lipopolysaccharide‐induced IL‐1β production, at least partly, through the TDAG8/cAMP/PKA pathway, by inhibiting extracellular signal‐related kinase and c‐Jun N‐terminal kinase activities, in mouse microglia.

     

IL‐1β and TNF‐α induce neurotoxicity through glutamate production: a potential role for neuronal glutaminase

Glutaminase 1 is the main enzyme responsible for glutamate production in mammalian cells. The roles of macrophage and microglia glutaminases in brain injury, infection, and inflammation are well documented. However, little is known about the regulation of neuronal glutaminase, despite neurons being a predominant cell type of glutaminase expression. Using primary rat and human neuronal cultures, we confirmed that interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α), two pro‐inflammatory cytokines that are typically elevated in neurodegenerative disease states, induced neuronal death and apoptosis in vitro. Furthermore, both intracellular and extracellular glutamate levels were significantly elevated following IL‐1β and/or TNF‐α treatment. Pre‐treatment with N‐Methyl‐d ‐aspartate (NMDA) receptor antagonist MK‐801 blocked cytokine‐induced glutamate production and alleviated the neurotoxicity, indicating that IL‐1β and/or TNF‐α induce neurotoxicity through glutamate. To determine the potential source of excess glutamate production in the culture during inflammation, we investigated the neuronal glutaminase and found that treatment with IL‐1β or TNF‐α significantly upregulated the kidney‐type glutaminase (KGA), a glutaminase 1 isoform, in primary human neurons. The up‐regulation of neuronal glutaminase was also demonstrated in situ in a murine model of HIV‐1 encephalitis. In addition, IL‐1β or TNF‐α treatment increased the levels of KGA in cytosol and TNF‐α specifically increased KGA levels in the extracellular fluid, away from its main residence in mitochondria. Together, these findings support neuronal glutaminase as a potential component of neurotoxicity during inflammation and that modulation of glutaminase may provide therapeutic avenues for neurodegenerative diseases.     

2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin promotes astrocyte activation and the secretion of tumor necrosis factor‐α via PKC/SSeCKS‐dependent mechanisms

2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is a ubiquitous environmental pollutant that could induce significant toxic effects in the human nervous system. However, the underlying molecular mechanism has not been entirely elucidated. Reactive astrogliosis has implicated in various neurological diseases via the production of a variety of pro‐inflammatory mediators. Herein, we investigated the potential role of TCDD in facilitating astrocyte activation and the underlying molecular mechanisms. We showed that TCDD induced rapid astrocyte activation following TCDD exposure, which was accompanied by significantly elevated expression of Src‐Suppressed‐C Kinase Substrate (SSeCKS), a protein involved in protein kinase C (PKC)‐mediated Nuclear Factor kappa B signaling, suggesting a possible involvement of PKC‐induced SSeCKS activation in TCDD‐triggered reactive astroglia. In keeping with the finding, we found that the level of phosphorylated Nuclear Factor kappa B p65 was remarkably increased after TCDD treatment. Furthermore, interference of SSeCKS attenuated TCDD‐induced inducible nitric oxide synthase, glial fibrillary acidic protein, phospho‐p65 expression, and tumor necrosis factor‐α secretion in astrocytes. In addition, pre‐treatment with PKC inhibitor also attenuated TCDD‐induced astrocyte activation, as well as SSeCKS expression. Interestingly, we found that TCDD treatment could lead to SSeCKS perinuclear localization, which could be abolished after treatment with PKC inhibitor. Finally, we showed that inhibition of PKC activity or SSeCKS expression would impair TCDD‐triggered tumor necrosis factor‐α secretion. Our results suggested that TCDD exposure could lead to astrocyte activation through PKC/SSeCKS‐dependent mechanisms, highlighting that astrocytes might be important target of TCDD‐induced neurotoxicity.

     

Allosteric modulation of sigma‐1 receptors by SKF83959 inhibits microglia‐mediated inflammation

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.

     

Divergent effects of the H50Q and G51D SNCA mutations on the aggregation of α‐synuclein

The discoveries of mutations in SNCA were seminal findings that resulted in the knowledge that α‐synuclein (αS) is the major component of Parkinson's disease‐associated Lewy bodies. Since the pathologic roles of these protein inclusions and SNCA mutations are not completely established, we characterized the aggregation properties of the recently identified SNCA mutations, H50Q and G51D, to provide novel insights. The properties of recombinant H50Q, G51D, and wild‐type αS to polymerize and aggregate into amyloid were studied using (trans,trans)‐1‐bromo‐2,5‐bis‐(4‐hydroxy)styrylbenzene fluorometry, sedimentation analyses, electron microscopy, and atomic force microscopy. These studies showed that the H50Q mutation increases the rate of αS aggregation, whereas the G51D mutation has the opposite effect. However, H50Q and G51D αS could still be similarly induced to form intracellular aggregates from the exposure to exogenous amyloidogenic seeds under conditions that promote their cellular entry. Both mutant αS proteins, but especially G51D, promoted cellular toxicity under cellular stress conditions. These findings reveal that the novel pathogenic SNCA mutations, H50Q and G51D, have divergent effects on aggregation properties relative to the wild‐type protein, with G51D αS demonstrating reduced aggregation despite presenting with earlier disease onset, suggesting that these mutants promote different mechanisms of αS pathogenesis.

     

Microglia in juvenile neuronal ceroid lipofuscinosis are primed toward a pro‐inflammatory phenotype

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 CLN3^Δex7/8 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 CLN3^Δex7/8 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 CLN3^Δex7/8 microglia exist in primed state and hyper‐respond to several inflammatory stimuli compared to WT cells. CLN3^Δex7/8 microglia displayed constitutive caspase‐1 activity that when blocked led to increased glutamate release that coincided with hemichannel opening. Conditioned medium from activated CLN3^Δex7/8 or WT microglia induced significant cell death in CLN3^Δex7/8 but not WT neurons, demonstrating that intrinsically diseased CLN3^Δex7/8 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.

     

Leptin inhibits amyloid β‐protein fibrillogenesis by decreasing GM1 gangliosides on the neuronal cell surface through PI3K/Akt/mTOR pathway

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.

     

DJ‐1 deficiency impairs autophagy and reduces alpha‐synuclein phagocytosis by microglia

Parkinson's disease (PD) is a progressive neurodegenerative disorder, of which 1% of the hereditary cases are linked to mutations in DJ‐1, an oxidative stress sensor. The pathological hallmark of PD is intercellular inclusions termed Lewy Bodies, composed mainly of α‐Synuclein (α‐Syn) protein. Recent findings have shown that α‐Syn can be transmitted from cell to cell, suggesting an important role of microglia, as the main scavenger cells of the brain, in clearing α‐Syn. We previously reported that the knock down (KD) of DJ‐1 in microglia increased cells’ neurotoxicity to dopaminergic neurons. Here, we discovered that α‐Syn significantly induced elevated secretion of the proinflammatory cytokines IL‐6 and IL‐1β and a significant dose‐dependent elevation in the production of nitric oxide in DJ‐1 KD microglia, compared to control microglia. We further investigated the ability of DJ‐1 KD microglia to uptake and degrade soluble α‐Syn, and discovered that DJ‐1 KD reduces cell‐surface lipid raft expression in microglia and impairs their ability to uptake soluble α‐Syn. Autophagy is an important mechanism for degradation of intracellular proteins and organelles. We discovered that DJ‐1 KD microglia exhibit an impaired autophagy‐dependent degradation of p62 and LC3 proteins, and that manipulation of autophagy had less effect on α‐Syn uptake and clearance in DJ‐1 KD microglia, compared to control microglia. Further studies of the link between DJ‐1, α‐Syn uptake and autophagy may provide useful insights into the role of microglia in the etiology of the PD.