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Background: Overactivated microglia that cluster at neuritic plaques constantly release neurotoxins, which actively contribute to progressive neurodegeneration in Alzheimer's disease (AD). Therefore, attenuating microglial clustering can reduce focal neuroinflammation at neuritic plaques. Previously, we identified CCL5 and CCL2 as prominent chemokines that mediate the chemotaxis of microglia toward beta-amyloid (Abeta)aggregates. Although transforming growth factor-beta1 (TGF-beta1) has been shown to down-regulate the expression of chemokines in activated microglia, whether TGF-beta1 can reduce the chemotaxis of microglia toward neuritic plaques in AD remains unclear.Methods: In the present study, we investigated the effects of TGF-beta1 on Abeta-induced chemotactic migration of BV-2 microglia using time-lapse recording, transwell assay, real-time PCR, ELISA, and western blotting.Results: The cell tracing results suggest that the morphological characteristics and migratory patterns of BV-2 microglia resemble those of microglia in slice cultures. Using this model system, we discovered that TGF-beta1 reduces Abeta-induced BV-2 microglial clustering in a dose-dependent manner. Chemotactic migration of these microglial cells toward Abeta aggregates was significantly attenuated by TGF-beta1. However, these microglia remained actively moving without any reduction in migration speed. Pharmacological blockade of TGF-beta1 receptor I (ALK5) by SB431542 treatment reduced the inhibitory effects of TGF-beta1 on Abeta-induced BV-2 microglial clustering, while preventing TGF-beta1-mediated cellular events, including SMAD2 phosphorylation and CCL5 down-regulation.Conclusions: Our results suggest that TGF-beta1 reduces Abeta-induced microglial chemotaxis via the SMAD2 pathway. The down-regulation of CCL5 by TGF-beta1 at least partially contributes to the clustering of microglia at Abeta aggregates. The attenuating effects of SB431542 upon TGF-beta1-suppressed microglial clustering may be mediated by restoration of CCL5 to normal levels. TGF-beta1 may ameliorate microglia-mediated neuroinflammation in AD by preventing activated microglial clustering at neuritic plaques. [ABSTRACT FROM AUTHOR]

High-fat diet (HFD)-induced fatty liver disease is a deteriorating risk factor for Alzheimer's disease (AD). Mitigating fatty liver disease has been shown to attenuate AD-like pathology in animal models. However, it remains unclear whether enhancing Aβ clearance through immunotherapy would in turn attenuate HFD-induced fatty liver or whether its efficacy would be compromised by long-term exposure to HFD. Here, the therapeutic potentials of an anti-Aβ antibody, NP106, was investigated in APP/PS1 mice by HFD feeding for 44 weeks. The data demonstrate that NP106 treatment effectively reduced Aβ burden and pro-inflammatory cytokines in HFD-fed APP/PS1 mice and ameliorated HFD-aggravated cognitive impairments during the final 18 weeks of the study. The rejuvenating characteristics of microglia were evident in APP/PS1 mice with NP106 treatment, namely enhanced microglial Aβ phagocytosis and attenuated microglial lipid accumulation, which may explain the benefits of NP106. Surprisingly, NP106 also reduced HFD-induced hyperglycemia, fatty liver, liver fibrosis, and hepatic lipids, concomitant with modifications in the expressions of genes involved in hepatic lipogenesis and fatty acid oxidation. The data further reveal that brain Aβ burden and behavioral deficits were positively correlated with the severity of fatty liver disease and fasting serum glucose levels. In conclusion, our study shows for the first time that anti-Aβ immunotherapy using NP106, which alleviates AD-like disorders in APP/PS1 mice, ameliorates fatty liver disease. Minimizing AD-related pathology and symptoms may reduce the vicious interplay between central AD and peripheral fatty liver disease, thereby highlighting the importance of developing AD therapies from a systemic disease perspective., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Brainstem encephalitis, a manifestation of severe enterovirus 71 (EV71) infection, is an acute excessive inflammatory response. The mechanisms underlying its development remain poorly understood. Usually neurotropic viruses trigger acute host immune response by engaging cell surface or intracellular receptors. Here, we show that EV71 engagement with intracellular receptor TLR9 elicits IL-12p40-iNOS signaling causing encephalitis in mice. We identified IL-12p40 to be the only prominent cytokine-induced at the early infection stage in the brainstem of mice subjected to a lethal dose of EV71. The upregulated IL-12p40 proteins were expressed in glial cells but not neuronal cells. To better understand the role of IL-12p40 in severe EV71 infection, we treated the EV71-infected mice with an antibody against IL-12p40 and found the mortality rate, brainstem inflammation, and gliosis to be markedly reduced, suggesting that the acute IL-12p40 response plays a critical role in the pathogenesis of brainstem encephalitis. Mechanistically, intracellular TLR9 was found essential to the activation of the IL-12p40 response. Blocking TLR9 signaling with CpG-ODN antagonist ameliorated IL-12p40 response, brainstem inflammation, and limb paralysis in mice with EV71-induced encephalitis. We further found the glial IL-12p40 response might damage neurons by inducing excess production of neurotoxic NO by iNOS. Overall, EV71 engagement with intracellular TLR9 was found to elicit a neurotoxic glial response via IL12p40-iNOS signaling contributing to the neurological manifestation of EV71 infection. This pathway could potentially be targeted for the treatment of brainstem encephalitis., (© 2022. The Author(s).)

Diabetes is a risk factor for Alzheimer's disease (AD), a chronic neurodegenerative disease. We and others have shown prediabetes, including hyperglycemia and obesity induced by high fat and high sucrose diets, is associated with exacerbated amyloid beta (Aβ) accumulation and cognitive impairment in AD transgenic mice. However, whether hyperglycemia reduce glial clearance of oligomeric amyloid-β (oAβ), the most neurotoxic Aβ aggregate, remains unclear. Mixed glial cultures simulating the coexistence of astrocytes and microglia in the neural microenvironment were established to investigate glial clearance of oAβ under normoglycemia and chronic hyperglycemia. Ramified microglia and low IL-1β release were observed in mixed glia cultures. In contrast, amoeboid-like microglia and higher IL-1β release were observed in primary microglia cultures. APPswe/PS1dE9 transgenic mice are a commonly used AD mouse model. Microglia close to senile plaques in APPswe/PS1dE9 transgenic mice exposed to normoglycemia or chronic hyperglycemia exhibited an amoeboid-like morphology; other microglia were ramified. Therefore, mixed glia cultures reproduce the in vivo ramified microglial morphology. To investigate the impact of sustained high-glucose conditions on glial oAβ clearance, mixed glia were cultured in media containing 5.5 mM glucose (normal glucose, NG) or 25 mM glucose (high glucose, HG) for 16 days. Compared to NG, HG reduced the steady-state level of oAβ puncta internalized by microglia and astrocytes and decreased oAβ degradation kinetics. Furthermore, the lysosomal acidification and lysosomal hydrolysis activity of microglia and astrocytes were lower in HG with and without oAβ treatment than NG. Moreover, HG reduced mitochondrial membrane potential and ATP levels in mixed glia, which can lead to reduced lysosomal function. Overall, continuous high glucose reduces microglial and astrocytic ATP production and lysosome activity which may lead to decreased glial oAβ degradation. Our study reveals diabetes-induced hyperglycemia hinders glial oAβ clearance and contributes to oAβ accumulation in AD pathogenesis., Competing Interests: The authors have declared that no competing interests exist.

Alzheimer's disease (AD) is a progressive neurodegenerative disease with a multifactorial etiology. A multitarget treatment that modulates multifaceted biological functions might be more effective than a single-target approach. Here, the therapeutic efficacy of combination treatment using anti-Aβ antibody NP106 and curcumin analog TML-6 versus monotherapy was investigated in an APP/PS1 mouse model of AD. Our data demonstrate that both combination treatment and monotherapy attenuated brain Aβ and improved the nesting behavioral deficit to varying degrees. Importantly, the combination treatment group had the lowest Aβ levels, and insoluble forms of Aβ were reduced most effectively. The nesting performance of APP/PS1 mice receiving combination treatment was better than that of other APP/PS1 groups. Further findings indicate that enhanced microglial Aβ phagocytosis and lower levels of proinflammatory cytokines were concurrent with the aforementioned effects of NP106 in combination with TML-6. Intriguingly, combination treatment also normalized the gut microbiota of APP/PS1 mice to levels resembling the wild-type control. Taken together, combination treatment outperformed NP106 or TML-6 monotherapy in ameliorating Aβ pathology and the nesting behavioral deficit in APP/PS1 mice. The superior effect might result from a more potent modulation of microglial function, cerebral inflammation, and the gut microbiota. This innovative treatment paradigm confers a new avenue to develop more efficacious AD treatments.

Observational epidemiological studies have associated vitamin D deficiency with Alzheimer's disease (AD). However, whether vitamin D deficiency would result in some impacts on the vitamin D binding receptor (VDR) remains to be characterized in AD. Vitamin D helps maintain adult brain health genomically through binding with and activating a VDR/retinoid X receptor (RXR) transcriptional complex. Thus, we investigated the role of VDR in AD using postmortem human brains, APP/PS1 mice, and cell cultures. Intriguingly, although vitamin D was decreased in AD patients and mice, hippocampal VDR levels were inversely increased. The abnormally increased levels of VDR were found to be colocalized with Aβ plaques, gliosis and autophagosomes, implicating a non-genomic activation of VDR in AD pathogenesis. Mechanistic investigation revealed that Aβ upregulated VDR without its canonical ligand vitamin D and switched its heterodimer binding-partner from RXR to p53. The VDR/p53 complex localized mostly in the cytosol, increased neuronal autophagy and apoptosis. Chemically inhibiting p53 switched VDR back to RXR, reversing amyloidosis and cognitive impairment in AD mice. These results suggest a non-genomic rewiring of VDR to p53 is key for the progression of AD, and thus VDR/p53 pathway might be targeted to treat people with AD., (© 2021 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Increasing evidence suggests that elderly people with dementia are vulnerable to the development of severe coronavirus disease 2019 (COVID-19). In Alzheimer's disease (AD), the major form of dementia, β-amyloid (Aβ) levels in the blood are increased; however, the impact of elevated Aβ levels on the progression of COVID-19 remains largely unknown. Here, our findings demonstrate that Aβ 1-42 , but not Aβ 1-40 , bound to various viral proteins with a preferentially high affinity for the spike protein S1 subunit (S1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the viral receptor, angiotensin-converting enzyme 2 (ACE2). These bindings were mainly through the C-terminal residues of Aβ 1-42 . Furthermore, Aβ 1-42 strengthened the binding of the S1 of SARS-CoV-2 to ACE2 and increased the viral entry and production of IL-6 in a SARS-CoV-2 pseudovirus infection model. Intriguingly, data from a surrogate mouse model with intravenous inoculation of Aβ 1-42 show that the clearance of Aβ 1-42 in the blood was dampened in the presence of the extracellular domain of the spike protein trimers of SARS-CoV-2, whose effects can be prevented by a novel anti-Aβ antibody. In conclusion, these findings suggest that the binding of Aβ 1-42 to the S1 of SARS-CoV-2 and ACE2 may have a negative impact on the course and severity of SARS-CoV-2 infection. Further investigations are warranted to elucidate the underlying mechanisms and examine whether reducing the level of Aβ 1-42 in the blood is beneficial to the fight against COVID-19 and AD.

Prenatal exposure to buprenorphine renders offspring vulnerable to cerebral impairments. In this study, our data demonstrate, for the first time, that prenatal exposure to buprenorphine escalates astrocyte activation concurrent with indications of endoplasmic reticulum (ER) stress in the hippocampi of neonates, and this can be prevented by the coadministration of dextromethorphan with buprenorphine. Furthermore, dextromethorphan can inhibit the accumulation of GPR37 in the hippocampus of newborns caused by buprenorphine and is accompanied by the proapoptotic ER stress response that involves the procaspase-3/CHOP pathway. Primary astrocyte cultures derived from the neonates of the buprenorphine group also displayed aberrant ER calcium mobilization and elevated basal levels of cyclooxygenase-2 (COX-2) at 14 days in vitro while showing sensitivity to lipopolysaccharide-activated expression of COX-2. Similarly, these long-lasting defects in the hippocampus and astrocytes were abolished by dextromethorphan. Our findings suggest that prenatal exposure to buprenorphine might instigate long-lasting effects on hippocampal and astrocytic functions. The beneficial effects of prenatal coadministration of dextromethorphan might be, at least in part, attributed to its properties in attenuating astrocyte activation and hippocampal ER stress in neonates., Competing Interests: The authors declare no competing financial interests., (Copyright © 2021 Chun-Hua Lin et al.)

High-fat and high-sugar diets contribute to the prevalence of type 2 diabetes and Alzheimer's disease (AD). Although the impact of high-fat diets on AD pathogenesis has been established, the effect of high-sucrose diets (HSDs) on AD pathogenesis remains unclear. This study sought to determine the impact of HSDs on AD-related pathologies. Male APPswe/PS1dE9 (APP/PS1) transgenic and wild-type mice were provided with HSD and their cognitive and hypothalamus-related noncognitive parameters, including feeding behaviors and glycemic regulation, were compared. HSD-fed APP/PS1 mice showed increased neuroinflammation, as well as increased cortical and serum levels of amyloid-β. HSD-fed APP/PS1 mice showed aggravated obesity, hyperinsulinemia, insulin resistance, and leptin resistance, but there was no induction of hyperphagia or hyperleptinemia. Leptin-induced phosphorylation of signal transducer and activator of transcription 3 in the dorsomedial and ventromedial hypothalamus was reduced in HSD-fed APP/PS1 mice, which might be associated with attenuated food-anticipatory activity, glycemic dysregulation, and AD-related noncognitive symptoms. Our study demonstrates that HSD aggravates metabolic stresses, increases AD-related pathologies, and attenuates hypothalamic leptin signaling in APP/PS1 mice., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The original version of this article unfortunately contained a mistake. The authors observed inadvertent error in Fig. 7d, in which the image of the GFAP/DAPI in the WT saline treated mice was rotated left 90-degree by mistake. The corrected representative image is given below.

Astrocytes play pivotal roles in regulating glutamate homeostasis at tripartite synapses. Inhibition of soluble epoxide hydrolase (sEHi) provides neuroprotection by blocking the degradation of 14,15-epoxyeicosatrienoic acid (14,15-EET), a lipid mediator whose synthesis can be activated downstream from group 1 metabotropic glutamate receptor (mGluR) signaling in astrocytes. However, it is unclear how sEHi regulates glutamate excitotoxicity. Here, we used three primary rat cortical culture systems, neuron-enriched (NE), astrocyte-enriched glia-neuron mix (GN), and purified astrocytes, to delineate the underlying mechanism by which sEHi and 14,15-EET attenuate excitotoxicity. We found that sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) and 14,15-EET both attenuated N-methyl-D-aspartate (NMDA)-induced neurite damage and cell death in GN, not NE, cortical cultures. The anti-excitotoxic effects of 14,15-EET and AUDA were both blocked by the group 1 mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), as were their protective effects against NMDA-disrupted perineuronal astrocyte processes expressing glutamate transporter-1 (GLT-1) and subsequent glutamate uptake. Knockdown of sEH expression also attenuated NMDA neurotoxicity in mGluR5- and GLT-1-dependent manners. The 14,15-EET/AUDA-preserved astroglial integrity was confirmed in glutamate-stimulated primary astrocytes along with the reduction of the c-Jun N-terminal kinase 1 phosphorylation, in which the 14,15-EET effect is mGluR5-dependent. In vivo studies validated that sEHi and genetic deletion of sEH (Ephx2-KO) ameliorated excitotoxic kainic acid-induced seizure, memory impairment, and neuronal loss while preserving GLT-1-expressing perineuronal astrocytes in hippocampal CA3 subregions. These results suggest that 14,15-EET mediates mGluR5-dependent anti-excitotoxicity by protecting astrocytes to maintain glutamate homeostasis, which may account for the beneficial effect of sEH inhibition in excitotoxic brain injury and diseases.

Background: Astrocyte activation is a common pathological feature in many brain diseases with neuroinflammation, and revealing the underlying mechanisms might shed light on the regulatory processes of the diseases. Recently, soluble epoxide hydrolase (sEH) has been proposed to affect neuroinflammation in brain injuries. However, the roles of astrocytic sEH in brains with neurodegeneration remain unclear., Methods: The expression of astrocytic sEH in the brains of APPswe/PSEN1dE9 (APP/PS1) mice developing Alzheimer's disease (AD)-like pathology was evaluated by confocal imaging. LPS-activated primary astrocytes with mRNA silencing or overexpression of sEH were used to investigate its regulatory roles in astrocyte activation and the induction of pro-inflammatory markers. Primary astrocytes isolated from a sEH knockout (sEH -/- ) background were also applied., Results: The immunoreactivity of sEH was increased in activated astrocytes in parallel with the progression of AD in APP/PS1 mice. Our data from primary astrocyte cultures further demonstrate that the overexpression of sEH ameliorated, while the silencing of sEH mRNA enhanced, the lipopolysaccharides (LPS)-induced expression of pro-inflammatory markers, such as inducible nitric oxide, cyclooxygenase 2 (COX-2), and pro-inflammatory cytokines. These findings suggest that sEH negatively regulates astrocyte immune responses. Enhanced immune responses found in LPS-activated sEH -/- astrocytes also support the notion that the expression of sEH could suppress the immune responses during astrocyte activation. Similarly, sEH -/- mice that received intraperitoneal injection of LPS showed exacerbated astrocyte activation in the brain, as observed by the elevated expression of glial fibrillary acidic protein (GFAP) and pro-inflammatory markers. Moreover, our data show that the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) was upregulated in activated astrocytes from sEH mouse brains, and the pharmacological blockade of STAT3 activity alleviated the pro-inflammatory effects of sEH deletion in LPS-activated primary astrocytes., Conclusions: Our results provide evidence, for the first time, showing that sEH negatively regulates astrocytic immune responses and GFAP expression, while the underlying mechanism at least partly involves the downregulation of STAT3 phosphorylation. The discovery of a novel function for sEH in the negative control of astrocytic immune responses involving STAT3 activation confers further insights into the regulatory machinery of astrocyte activation during the development of neurodegeneration.

Alzheimer's disease (AD), a progressive neurodegenerative disease is highly associated with metabolic syndromes. We previously demonstrated that glycemic dysregulation and obesity are augmented in high fat diet (HFD)-treated APPswe/PS1dE9 (APP/PS1) transgenic mice. In the current study, the underlying mechanism mediating exacerbated metabolic stresses in HFD APP/PS1 transgenic mice was further examined. APP/PS1 mice developed insulin resistance and, consequently, impaired glucose homeostasis after 10 weeks on HFD. [ 18 F]-2-fluoro-2-deoxy-d-glucose ([ 18 F]-FDG) positron emission tomography showed that interscapular brown adipose tissue is vulnerable to HFD and AD-related pathology. Chronic HFD induced hyperphagia, with limited effects on basal metabolic rates in APP/PS1 transgenic mice. Excessive food intake may be caused by impairment of leptin signaling in the hypothalamus because leptin failed to suppress the food intake of HFD APP/PS1 transgenic mice. Leptin-induced pSTAT3 signaling in the arcuate nucleus was attenuated. Dysregulated energy homeostasis including hyperphagia and exacerbated obesity was elicited prior to the presence of the amyloid pathology in the hypothalamus of HFD APP/PS1 transgenic mice; nevertheless, cortical neuroinflammation and the level of serum Aβ and IL-6 were significantly elevated. Our study demonstrates the pivotal role of AD-related pathology in augmenting HFD-induced insulin and leptin resistance and impairing hypothalamic regulation of energy homeostasis.

In this study, we examine whether an anti-inflammatory thiourea derivative, compound #326, actions on ion channels. The effects of compound #326 on Ca 2+ -activated K + channels were evaluated by patch-clamp recordings obtained in cell-attached, inside-out or whole-cell configuration. In pituitary GH 3 cells, compound #326 increased the amplitude of Ca 2+ -activated K + currents (I K(Ca) ) with an EC 50 value of 11.6 μM, which was reversed by verruculogen, but not tolbutamide or TRAM-34. Under inside-out configuration, a bath application of compound #326 raised the probability of large-conductance Ca 2+ -activated K + (BK Ca ) channels. The activation curve of BK Ca channels was shifted to less depolarised potential with no modification of the gating charge of the curve; consequently, the difference of free energy was reduced in the presence of this compound. Compound #326-stimulated activity of BK Ca channels is explained by a shortening of mean closed time, despite its inability to alter single-channel conductance. Neither delayed-rectifier nor erg-mediated K + currents was modified. Compound #326 decreased the peak amplitude of voltage-gated Na + current with no clear change in the overall current-voltage relationship of this current. In HEK293T cells expressing α-hSlo, compound #326 enhanced BK Ca channels effectively. Intriguingly, the inhibitory actions of compound #326 on interleukin 1β in lipopolysaccharide-activated microglia were significantly reversed by verruculogen, whereas BK Ca channel inhibitors suppressed the expressions of inducible nitric oxide synthase. The BK Ca channels could be an important target for compound #326 if similar in vivo results occur, and the multi-functionality of BK Ca channels in modulating microglial immunity merit further investigation., (© 2017 Wiley Periodicals, Inc.)

Ethnopharmacological Relevance: Metabolic syndrome and vascular dysfunction was suggested to be the risk factors for Alzheimer's disease (AD). Xuefu Zhuyu decoction (XZD) is a traditional Chinese medicine used to treat metabolic syndrome and cardiac-cerebral vascular disease. The effects of XZD on ameliorating metabolic syndrome, amyloid-related pathologies and cognitive impairment in an animal model of AD with metabolic stress was investigated., Materials and Method: The animal model of AD with metabolic stress was created by administrating high-fat diet and a low-dose injection of streptozotocin prior to the appearance of senile plaques in APP/PS1 transgenic mice. The diabesity-associated metabolic changes and AD-related pathological alterations were examined., Results: We found that XZD reduced body weight, insulin and leptin level, HOMA-IR, hepatic triglyceride, serum Aβ42 in the metabolic stressed AD animal. XZD also ameliorated oral glucose tolerant, Aβ deposition, astrocyte and microglia activation in the vicinity of plaques, and nesting behavior in the metabolic stressed AD animal., Conclusion: The results of this study suggest that XZD is able to reduce the peripheral metabolic stress-mediated vascular hypoperfusion, neuroinflammation and AD-related pathology in APP/PS1 mice., (Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.)

Background: The accumulation of soluble oligomeric amyloid-β peptide (oAβ) proceeding the formation of senile plaques contributes to synaptic and memory deficits in Alzheimer's disease. Our previous studies have indentified scavenger receptor A (SR-A), especially SR-A type I (SR-AI), as prominent scavenger receptors on mediating oAβ clearance by microglia while glycan moiety and scavenger receptor cysteine-rich (SRCR) domain may play the critical role. Macrophage receptor with collagenous structure (MARCO), another member of class A superfamily with a highly conserved SRCR domain, may also play the similar role on oAβ internalization. However, the role of N-glycosylation and SRCR domain of SR-AI and MARCO on oAβ internalization remains unclear., Result: We found that oAβ internalization was diminished in the cells expressing SR-AI harboring mutations of dual N-glycosylation sites (i.e. N120Q-N143Q and N143Q-N184Q) while they were normally surface targeted. Normal oAβ internalization was observed in 10 SR-AI-SRCR and 4 MARCO-SRCR surface targeted mutants. Alternatively, the SRCR mutants at β-sheet and α-helix and on disulfide bone formation obstructed receptor's N-glycosylation and surface targeting., Conclusion: Our study reveals that N-glycan moiety is more critical than SRCR domain for SR-A-mediated oAβ internalization.

Growth-associated protein 43 (GAP43), a protein kinase C (PKC)-activated phosphoprotein, is often implicated in axonal plasticity and regeneration. In this study, we found that GAP43 can be induced by the endotoxin lipopolysaccharide (LPS) in rat brain astrocytes both in vivo and in vitro. The LPS-induced astrocytic GAP43 expression was mediated by Toll-like receptor 4 and nuclear factor-κB (NF-κB)- and interleukin-6/signal transducer and activator of transcription 3 (STAT3)-dependent transcriptional activation. The overexpression of the PKC phosphorylation-mimicking GAP43(S41D) (constitutive active GAP43) in astrocytes mimicked LPS-induced process arborization and elongation, while application of a NF-κB inhibitory peptide TAT-NBD or GAP43(S41A) (dominant-negative GAP43) or knockdown of GAP43 all inhibited astrogliosis responses. Moreover, GAP43 knockdown aggravated astrogliosis-induced microglial activation and expression of proinflammatory cytokines. We also show that astrogliosis-conditioned medium from GAP43 knock-down astrocytes inhibited GAP43 phosphorylation and axonal growth, and increased neuronal damage in cultured rat cortical neurons. These proneurotoxic effects of astrocytic GAP43 knockdown were accompanied by attenuated glutamate uptake and expression of the glutamate transporter excitatory amino acid transporter 2 (EAAT2) in LPS-treated astrocytes. The regulation of EAAT2 expression involves actin polymerization-dependent activation of the transcriptional coactivator megakaryoblastic leukemia 1 (MKL1), which targets the serum response elements in the promoter of rat Slc1a2 gene encoding EAAT2. In sum, the present study suggests that astrocytic GAP43 mediates glial plasticity during astrogliosis, and provides beneficial effects for neuronal plasticity and survival and attenuation of microglial activation., Significance Statement: Astrogliosis is a complex state in which injury-stimulated astrocytes exert both protective and harmful effects on neuronal survival and plasticity. In this study, we demonstrated for the first time that growth-associated protein 43 (GAP43), a well known growth cone protein that promotes axonal regeneration, can be induced in rat brain astrocytes by the proinflammatory endotoxin lipopolysaccharide via both nuclear factor-κB and signal transducer and activator of transcription 3-mediated transcriptional activation. Importantly, LPS-induced GAP43 mediates plastic changes of astrocytes while attenuating astrogliosis-induced microglial activation and neurotoxicity. Hence, astrocytic GAP43 upregulation may serve to indicate beneficial astrogliosis after CNS injury., (Copyright © 2016 the authors 0270-6474/16/362028-17$15.00/0.)

Although metabolic syndrome was suggested to be a risk factor for Alzheimer's disease (AD), the role of metabolic stress in the initiation of AD pathology remains unclear. In this study, metabolic stress was induced by a high-fat diet and low-dose injection of streptozotocin (HFSTZ) before the appearance of senile plaques in APP/PS1 transgenic mice. We found that, HFSTZ treatment exacerbated amyloid beta burden and astrocyte activation in the vicinity of plaques. Moreover, we observed an upregulation of astrocytic S100B expression in the brain parenchyma of HFSTZ-treated APP/PS1 mice concurrent with increased interleukin-6 expression in cerebral microvascular cells. To determine the impact of HFSTZ treatment on brain function, we performed [(18)F]fludeoxyglucose-positron emission tomography and analyzed nesting behavior. HFSTZ treatment impaired nest construction and cerebral glucose metabolism in several brain regions of APP/PS1 mice during the early stage of AD. These results suggest that HFSTZ-induced peripheral metabolic stress may contribute to vascular inflammation and astrocyte reactivity in the parenchyma and may impair activity of daily living skill and cerebral glucose metabolism in APP/PS1 mice., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Diabesity-associated metabolic stresses modulate the development of Alzheimer's disease (AD). For further insights into the underlying mechanisms, we examine whether the genetic background of APPswe/PS1dE9 at the prodromal stage of AD affects peripheral metabolism in the context of diabesity. We characterized APPswe/PS1dE9 transgenic mice treated with a combination of high-fat diet with streptozotocin (HFSTZ) in the early stage of AD. HFSTZ-treated APPswe/PS1dE9 transgenic mice exhibited worse metabolic stresses related to diabesity, while serum β-amyloid levels were elevated and hepatic steatosis became apparent. Importantly, two-way analysis of variance shows a significant interaction between HFSTZ and genetic background of AD, indicating that APPswe/PS1dE9 transgenic mice are more vulnerable to HFSTZ treatment. In addition, body weight gain, high hepatic triglyceride, and hyperglycemia were positively associated with serum β-amyloid, as validated by Pearson's correlation analysis. Our data suggests that the interplay between genetic background of AD and HFSTZ-induced metabolic stresses contributes to the development of obesity and hepatic steatosis. Alleviating metabolic stresses including dysglycemia, obesity, and hepatic steatosis could be critical to prevent peripheral β-amyloid accumulation at the early stage of AD.

The aryl hydrocarbon receptor (AhR) regulates peripheral immunity; but its role in microglia-mediated neuroinflammation in the brain remains unknown. Here, we demonstrate that AhR mediates both anti-inflammatory and proinflammatory effects in lipopolysaccharide (LPS)-activated microglia. Activation of AhR by its ligands, formylindolo[3,2-b]carbazole (FICZ) or 3-methylcholanthrene (3MC), attenuated LPS-induced microglial immune responses. AhR also showed proinflammatory effects, as evidenced by the findings that genetic silence of AhR ameliorated the LPS-induced microglial immune responses and LPS-activated microglia-mediated neurotoxicity. Similarly, LPS-induced expressions of tumor necrosis factor α (TNFα) and inducible nitric oxide synthase (iNOS) were reduced in the cerebral cortex of AhR-deficient mice. Intriguingly, LPS upregulated and activated AhR in the absence of AhR ligands via the MEK1/2 signaling pathway, which effects were associated with a transient inhibition of cytochrome P450 1A1 (CYP1A1). Although AhR ligands synergistically enhance LPS-induced AhR activation, leading to suppression of LPS-induced microglial immune responses, they cannot do so on their own in microglia. Chromatin immunoprecipitation results further revealed that LPS-FICZ co-treatment, but not LPS alone, not only resulted in co-recruitment of both AhR and NFκB onto the κB site of TNFα gene promoter but also reduced LPS-induced AhR binding to the DRE site of iNOS gene promoter. Together, we provide evidence showing that microglial AhR, which can be activated by LPS, exerts bi-directional effects on the regulation of LPS-induced neuroinflammation, depending on the availability of external AhR ligands. These findings confer further insights into the potential link between environmental factors and the inflammatory brain disorders., (© 2015 Wiley Periodicals, Inc.)

Intrauterine infection (chorioamnionitis) aggravates neonatal hypoxic-ischemic (HI) brain injury, but the mechanisms linking systemic inflammation to the CNS damage remain uncertain. Here we report evidence for brain influx of T-helper 17 (TH17)-like lymphocytes to coordinate neuroinflammatory responses in lipopolysaccharide (LPS)-sensitized HI injury in neonates. We found that both infants with histological chorioamnionitis and rat pups challenged by LPS/HI have elevated expression of the interleukin-23 (IL-23) receptor, a marker of early TH17 lymphocytes, in the peripheral blood mononuclear cells. Post-LPS/HI administration of FTY720 (fingolimod), a sphingosine-1-phosphate receptor agonist that blocks lymphocyte trafficking, mitigated the influx of leukocytes through the choroid plexus and acute induction of nuclear factor-κB signaling in the brain. Subsequently, the FTY720 treatment led to attenuated blood-brain barrier damage, fewer cluster of differentiation 4-positive, IL-17A-positive T-cells in the brain, less proinflammatory cytokine, and better preservation of growth and white matter functions. The FTY720 treatment also provided dose-dependent reduction of brain atrophy, rescuing >90% of LPS/HI-induced brain tissue loss. Interestingly, FTY720 neither opposed pure-HI brain injury nor directly inhibited microglia in both in vivo and in vitro models, highlighting its unique mechanism against inflammation-sensitized HI injury. Together, these results suggest that the dual hit of systemic inflammation and neonatal HI injury triggers early onset of the TH17/IL-17-mediated immunity, which causes severe brain destruction but responds remarkably to the therapeutic blockade of lymphocyte trafficking., (Copyright © 2014 the authors 0270-6474/14/3416467-15$15.00/0.)

Background: Insufficient clearance of soluble oligomeric amyloid-β peptide (oAβ) in the central nervous system leads to the synaptic and memory deficits in Alzheimer's disease (AD). Previously we have identified scavenger receptor class A (SR-A) of microglia mediates oligomeric amyloid-β peptide (oAβ) internalization by siRNA approach. SR-A is a member of cysteine-rich domain (SRCR) superfamily which contains proteins actively modulating the innate immunity and host defense, however the functions of the SRCR domain remain unclear. Whether the SRCR domain of SR-AI modulates the receptor surface targeting and ligand internalization was investigated by expressing truncated SR-A variants in COS-7 cells. Surface targeting of SR-A variants was examined by live immunostaining and surface biotinylation assays. Transfected COS-7 cells were incubated with fluorescent oAβ and acetylated LDL (AcLDL) to assess their ligand-internalization capabilities., Result: Genetic ablation of SR-A attenuated the internalization of oAβ and AcLDL by microglia. Half of oAβ-containing endocytic vesicles was SR-A positive in both microglia and macrophages. Clathrin and dynamin in SR-AI-mediated oAβ internalization were involved. The SRCR domain of SR-AI is encoded by exons 10 and 11. SR-A variants with truncated exon 11 were intracellularly retained, whereas SR-A variants with further truncations into exon 10 were surface-targeted. The fusion of exon 11 to the surface-targeted SR-A variant lacking the SRCR domain resulted in the intracellular retention and the co-immunoprecipitation of Bip chaperon of the endoplasmic reticulum. Surface-targeted variants were N-glycosylated, whereas intracellularly-retained variants retained in high-mannose states. In addition to the collagenous domain, the SRCR domain is a functional binding domain for oAβ and AcLDL. Our data suggest that inefficient folding of SR-AI variants with truncated SRCR domain was recognized by the endoplasmic reticulum associated degradation which leads to the immature N- glycosylation and intracellular retention., Conclusion: The novel functions of the SRCR domain on regulating the efficacy of receptor trafficking and ligand binding may lead to possible approaches on modulating the innate immunity in Alzheimer's disease and atherosclerosis.

Curcumin has been proposed for treatment of various neuroinflammatory and neurodegenerative conditions, including post-traumatic inflammation during acute spinal cord injury (SCI). In this study, we examined whether curcumin anti-inflammation involves regulation of astrocyte reactivation, with special focus on the injury-induced RANTES (regulated on expression normal T-cell expressed and secreted) from astrocytes in acute SCI. Male Sprague-Dawley (SD) rats were subjected to impact injury of the spinal cord followed by treatment with curcumin (40 mg/kg i.p.). RANTES and inducible nitric oxide synthase expression as well as RANTES-positive astrocytes were all induced by injury accompanied by the elevation of lipid peroxidation, and attenuated by the curcumin treatment. In primary cultured rat astrocytes challenged with lipopolysaccharide (LPS) to mimic astrocyte reactivation following SCI, LPS induces robust increase of RANTES expression and the effect was also reduced by 1 μM curcumin treatment. Furthermore, cortical neurons cultured with astrocyte conditioned medium (ACM) conditioned with both LPS and curcumin (LPS-curcumin/ACM), which characteristically exhibited decreased RANTES expression when compared with ACM from astrocytes treated with LPS alone (LPS/ACM), showed higher level of cell viability and lower level of cell death as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity assay and lactate dehydrogenase release assay, respectively. Knockdown of RANTES expression by siRNA (siRANTES) shows reduced RANTES expression and release from LPS-reactivated astrocytes, and ACM obtained from this condition (LPS-siRANTES/ACM) becomes less cytotoxic as compared with the LPS-ACM. Therefore, curcumin reduction of robust RANTES production in reactivated astrocytes both in vitro and in vivo may contribute to its neuroprotection and potential application in SCI.

The accumulation of soluble oligomeric amyloid-β peptide (oAβ) proceeds the formation of senile plaques and contributes to synaptic and memory deficits in Alzheimer's disease (AD). The mechanism of mediating microglial oAβ clearance remains unclear and thought to occur via scavenger receptors (SRs) in microglia. SRs respond to their ligands in a subtype-specific manner. Therefore, we sought to identify the specific subtypes of SRs that mediate oAβ internalization and proteases that degrade oAβ species in naïve primary microglia. The component of oAβ species were characterized by western blot analysis, analytical ultracentrifugation analysis, and atomic force microscopy. The oAβ species remained soluble in the medium and microglial lysates during incubation at 37 °C. SR-A, but not CD36, mediated oAβ internalization in microglia as suggested by the use of subtype-specific neutralizing antibodies and small interfering RNAs (siRNAs). Immunoprecipitation analysis showed that oAβ interacted with SR-A on the plasma membrane. After internalization, over 40% of oAβ vesicles were trafficked toward lysosomes and degraded by cysteine proteases, including cathepsin B. The inhibitors of proteasome, neprilysin, matrix metalloproteinases, and insulin degrading enzyme failed to protect internalized oAβ from degradation. Our study suggests that SR-A and lysosomal cathepsin B are critical in microglial oAβ clearance, providing insight into how microglia are involved in the clearance of oAβ and their roles in the early stages of AD., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Neuroimmune pharmacology is a newly emerging field that intersects with neuroscience, immunology, and pharmacology and that is seeking avenues for translational research and better understanding of disease mechanisms. It focuses on the immunity of the central nervous system (CNS) which is greatly influenced by endogenous effectors, such as cytokines and neurotransmitters, and by exogenous substances, including therapeutic compounds, infectious pathogens, and drugs of abuse. In this article, we attempt to raise awareness of the pivotal discovery of how those mediators affect the immunity of the CNS in both physiological conditions and processes of certain mental illnesses, including psychiatric disorders, neurodegenerative diseases, and cerebral dysfunctions due to drugs of abuse. The abnormality in cytokine networks, neurotransmitter homeostasis, and other immune responses may be involved in the neuropathology associated with those mental illnesses, and the therapeutic effects of the potential treatments can be attributed, at least partially, to their immunomodulatory activities. However, the resulting inflammatory cytokines from certain treatments frequently cause psychiatric complications. In addition, the poor neuropathological outcomes frequently found among drug abusers with HIV-1 infection appear to be related to the neurotoxic and immunomodulatory effects of the drugs used. Importantly, glial cells, especially microglia and astrocytes, are key players in the immunomodulatory activities in the CNS, and the functioning CNS is largely dependent upon the reciprocal interactions between neurons and glial cells. Therefore, glia-neuron interactions have become a critical issue for further understanding the disease mechanism. From this review, readers will gain insights into the new field of neuroimmune pharmacology, with a focus on the impacts of CNS immunity on the mental illnesses.

Increasing evidence indicates that microglial activation plays an important role in the pathogenesis of Alzheimer's disease (AD). In AD, activated microglia may facilitate the clearance of beta-amyloid (Abeta), a neurotoxic component in AD pathogenesis. However, microglial activation comes at the cost of triggering neuro-inflammation, which contributes to cerebral dysfunction. Thus, pharmacological approaches that can achieve a favorable combination of a reduced microglia-mediated neuro-inflammation, and an enhanced Abeta clearance may be beneficial for preventing the progression of the disease. Here, we show that some newly synthesized compounds may exert such a combination of functions. Using mouse primary microglia and RAW264.7 cells, we found that some thiourea derivatives significantly enhanced microglial Abeta phagocytosis and suppressed microglial immune responses, as evidenced by the reduced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2). Of note, some commercially available inhibitors for iNOS and/or COX-2, such as ibuprofen, dextromethorphan, and N(G)-methyl-l-arginine (l-NMA), show negligible effects on microglial Abeta phagocytosis. Among the thiourea derivatives, our data show that a lead compound, designated as compound #326, (1-Naphthalen-1-yl-3-[5-(3-thioureido-phenoxy)-pentyl]-thiourea) appears to be the most potent in promoting Abeta phagocytosis and in inhibiting the LPS-induced expression of iNOS and COX-2 (when used at concentrations in the low muM range). The potency of compound #326 may have beneficial effects on modulating microglial activation in AD. The structure-activity relationship indicates that the thiourea group, alkyl linker, and the hydrophobic aryl group largely influence the dual functions of the compounds. These findings may indicate a structural basis for the improved design of future drug therapies for AD., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

The number of microglia surrounding senile plaques is correlated with the size of plaques in Alzheimer's disease (AD). It is unclear whether more microglia are passively recruited toward larger senile plaques or, conversely, microglia recruited to senile plaques directly contribute to the growth of plaques. In this study, BV-2 microglia were used to delineate the role of microglia in the growth of plaques using time-lapse recording. Aggregated beta amyloid peptide (Abeta)-induced BV-2 microglia to form clusters. The recruitment of BV-2 microglia bearing membrane-adhered Abeta enlarged preexisting Abeta aggregates. The receptors involved in the microglial uptake of Abeta, including integrin, formyl peptide like receptor 1, and scavenger receptors, also mediated the microglial clustering. Neutralization antibodies against chemokines significantly attenuated Abeta-induced microglial clustering and the enlargement of Abeta aggregates. Our results reveal a novel role of microglia in directly increasing the size of Abeta aggregates and suggest the targeting of Abeta-mediated microglial chemotactic migration in developing therapeutic interventions for AD.

Alzheimer's disease (AD) is the leading cause of dementia in the elderly. Although the etiology of AD remains unclear, microglia-mediated neuroinflammation is believed to play an important role in its pathogenesis. Microglial activation occurs in AD and is characterized by apparent phagocytic activity and by increased production and secretion of several cytokines, chemokines, reactive oxygen and nitrogen species, prostaglandin (PG)E2, and neurotrophic factors. Microglial activation can be neuroprotective through the release of neurotrophic factors and by phagocytosing Abeta, a critical neurotoxic component in AD brain. Concurrently, microglial activation causes elevated inflammatory responses that lead to paracrine damage to neurons. Therefore, a well-controlled microglial activation that diminishes microglial-mediated oxidative damage while promoting neuronal protection may be the key for AD therapy. Peroxisome proliferator-activated receptor gamma (PPARgamma) has recently gained increasing attention in AD due to its function as a molecular target for non-steroidal anti-inflammatory drugs (NSAIDs). In this review, we will discuss the role of PPARgamma in microglial innate immunity in AD and how pharmacological manipulation of microglial activation using PPARgamma ligands might facilitate the treatment of AD.

Oxidative stress, protein misfolding, protein complex formation, and detergent insolubility are biochemical features of Alzheimer's disease (AD). We tested the cause-and-effect relationships among these using MC65 human neuroblastoma cells that exhibit toxicity upon conditional expression of carboxy-terminal fragments (CTFs) of the human amyloid precursor protein (APP). Treatments with three different antioxidants (alpha-tocopherol, N-acetyl cysteine, and alpha-lipoic acid) or three different compounds (glycerol, trimethylamine-N-oxide, and 4-phenylbutyric acid) that have been described to have a "chemical chaperone" function in promoting protein folding all had a protective effect on MC65 cells and decreased markers of oxidative damage and accumulation of high molecular weight amyloid (A) beta-immunoreactive (IR) species. However, chaperones partially reduced detergent insolubility of the remaining Abeta-IR species, while antioxidants did not. These results suggest that protein misfolding associated with overexpression of APP CTFs promotes oxidative stress and cytotoxicity and contributes to formation of detergent-insoluble species that appear unrelated to cytotoxicity.

Background: The pathogenesis of idiopathic Parkinson's disease (PD) remains elusive, although evidence has suggested that neuroinflammation characterized by activation of resident microglia in the brain may contribute significantly to neurodegeneration in PD. It has been demonstrated that aggregated alpha-synuclein potently activates microglia and causes neurotoxicity. However, the mechanisms by which aggregated alpha-synuclein activates microglia are not understood fully., Methods: We investigated the role of prostaglandin E2 receptor subtype 2 (EP2) in alpha-synuclein aggregation-induced microglial activation using ex vivo, in vivo and in vitro experimental systems., Results: Results demonstrated that ablation of EP2(EP2-/-) significantly enhanced microglia-mediated ex vivo clearance of alpha-synuclein aggregates (from mesocortex of Lewy body disease patients) while significantly attenuating neurotoxicity and extent of alpha-synuclein aggregation in mice treated with a parkinsonian toxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Furthermore, we report that reduced neurotoxicity by EP2-/- microglia could be attributed to suppressed translocation of a critical cytoplasmic subunit (p47-phox) of NADPH oxidase (PHOX) to the membranous compartment after exposure to aggregated alpha-synuclein., Conclusion: Thus, it appears that microglial EP2 plays a critical role in alpha-synuclein-mediated neurotoxicity.

Alzheimer's disease (AD) is the leading cause of dementia. Although the etiology of AD remains controversial, the amyloid hypothesis suggests that beta-amyloid (Abeta) peptides may contribute to brain dysfunction, and microglial activation has become increasingly regarded as a potential contributor to disease pathogenesis. Microglial activation is characterized by morphological changes and by production of various effectors, and activated neuroinflammation concurrent with increased oxidative stress may contribute to damage to neurons. However, recently there has been a recognition that microglia may also play a neuroprotective role through their release of neurotrophic factors and through phagocytosis of Abeta. Thus, there is growing consensus that a favorable combination of diminished microglia-mediated neuroinflammation and enhanced Abeta clearance may be critical in AD therapy. In this review, we will discuss the role of microglial activation in AD and how pharmacologic manipulation of microglia might bear upon the treatment of AD.

Reactive oxygen species (ROS) are known to be involved in the pathogenesis of traumatic brain injury (TBI). Previous studies have shown that the susceptibility of mice to TBI-induced formation of cortical lesion is determined by the expression levels of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD, respectively). However, the underlying biochemical mechanisms are not understood. In this study, we measured the efficiency of mitochondrial respiration in mouse brains with altered expression of these two enzymes. While controlled cortical impact injury (CCII) with a deformation depth of 2 mm caused a drastic decrease in NAD-linked bioenergetic capacity in brain mitochondria of wild-type mice, the functional decrease was not observed in brains of littermate transgenic mice overexpressing CuZnSOD or MnSOD. In addition, a 1 mm CCII greatly compromised brain mitochondrial function in mice deficient in CuZnSOD or MnSOD, but not wild-type mice. Inclusion of the calcium-chelating agent, EGTA, in the assay solution could completely prevent dysfunction of oxidative phosphorylation in all mitochondrial samples, suggesting that the observed impairment of mitochondrial function was a result of calcium overloading. In conclusion, our results imply that mitochondrial dysfunction induced by superoxide anion radical contributes to lesion formation in mouse brain following physical trauma.

Prostaglandin (PG) E(2) acts via four functionally antagonistic E-prostanoid (EP) receptors that are expressed on multiple cell types in the nervous system; these are designated EP1-4. We showed previously that EP2 null mice are protected from CD14-dependent neuronal damage in vivo following intracerebroventricular (ICV) injection of lipopolysaccharide (LPS). Clear interpretation of this neuroprotective outcome is limited because EP2 is expressed on glia and neurons. We tested the hypothesis that microglial EP2 is required for paracrine neurotoxicity following activation of innate immunity, using primary murine microglia and neuron co-cultures. We demonstrated that microglial EP2 was necessary for lipopolysaccharide (LPS)-activated microglia-mediated neurotoxicity, as well as induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2). Genetic deletion of microglial iNOS, pharmacological suppression of COX-2 activity, or addition of exogenous superoxide dismutase (SOD) and catalase in the presence of EP2 also abolished neurotoxicity. This loss of paracrine neurotoxicity by EP2(-/-) microglia occurred in the absence of reduced cytokine levels. We conclude that microglial EP2 is critical to innate immunity-mediated paracrine damage to neurons involving COX-2 and iNOS. EP2 should be considered as a therapeutic target for suppression of microglial innate immunity-mediated damage in neurodegenerative diseases., ((c) 2005 Wiley-Liss, Inc.)

Epidemiologic and animal model data support a role for the prostaglandin pathway in AD pathogenesis. However, unexpected toxicity from protracted use of some nonsteroidal anti-inflammatory drugs (NSAIDs) compels investigation of therapeutic targets in this pathway other than COX inhibitors. Previously, we have shown that mice lacking one specific receptor for PGE2, EP2 (EP2-/-), are protected from the indirect neurotoxic effects of cerebral innate immune response mediated by CD14-dependent activation. Here we review data showing that EP2-/- microglia have a highly desirable combination of features: ablated indirect neurotoxicity following exposure to Abeta(1-42) coupled with enhanced phagocytosis of Abeta peptides, both synthetic and those deposited in human brain. These data point to microglial EP2 as a more focused target within the PG pathway for therapy in AD.

Experimental therapies for Alzheimer's disease (AD) are focused on enhanced clearance of neurotoxic Abeta peptides from brain. Microglia can be neuroprotective by phagocytosing Abeta; however, this comes at the cost of activated innate immunity that causes paracrine damage to neurons. Here, we show that ablation of E prostanoid receptor subtype 2 (EP2) significantly increased microglial-mediated clearance of Abeta peptides from AD brain sections and enhanced microglial Abeta phagocytosis in cell culture. The enhanced phagocytosis was PKC-dependent and was associated with elevated microglial secretion of the chemoattractant chemokines, macrophage inflammatory protein-1alpha and macrophage chemoattractant protein-1. This suggested that microglial activation is negatively regulated by EP2 signaling through suppression of prophagocytic cytokine secretion. However, despite this enhancement of Abeta phagocytosis, lack of EP2 completely suppressed Abeta-activated microglia-mediated paracrine neurotoxicity. These data demonstrate that blockade of microglial EP2 is a highly desirable mechanism for AD therapy that can maximize neuroprotective actions while minimizing bystander damage to neurons.

The mechanisms underlying Parkinson's disease (PD) and Lewy body (LB) formation, a pathological hallmark of PD, are incompletely understood; however, mitochondrial dysfunction is likely to be at least partially responsible. To study the processes that might be related to nigral neurodegeneration and LB formation, we employed nonbiased quantitative proteomics with isotope-coded affinity tag (ICAT) to compare the mitochondrial protein profiles in the substantia nigra (SN) between controls and mice treated chronically with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a potent mitochondrial toxicant, and an adjuvant, probenecid (prob), for 5 weeks, which produced selective nigrostriatal neurodegeneration with formation of LB-like cytoplasmic inclusions in the remaining nigral neurons. This method identified a total of more than 300 proteins; of these proteins, more than 100 displayed significant changes in relative abundance in the MPTP/prob-treated mice compared to the controls. We validated one of these proteins, DJ-1, whose mutation has been implicated in familial PD, with Western blot analysis, followed by immunohistochemical studies of its distribution in the SN in relation to cytoplasmic inclusions in mice, as well as in classical LBs in PD patients. The results demonstrated that DJ-1 was not only colocalized with alpha-synuclein in dopaminergic neurons but also to cytoplasmic inclusions in mice treated with MPTP/prob. In addition, DJ-1 was present in the halo but not in the core of classical LBs in patients with PD. Our findings suggested that DJ-1 might play an important role in mitochondrial dysfunction, as well as LB formation in PD.

The cause-and-effect relationship between innate immune activation and neurodegeneration has been difficult to prove in complex animal models and patients. Here we review findings from a model of direct innate immune activation via CD14 stimulation using intracerebroventricular injection of lipopolysaccharide. These data show that CD14-dependent innate immune activation in cerebrum leads to the closely linked outcomes of neuronal membrane oxidative damage and dendritic degeneration. Both forms of neuronal damage could be blocked by ibuprofen and alpha-tocopherol, but not naproxen or gamma-tocopherol, at pharmacologically relevant concentrations. This model provides a convenient method to determine effective agents and their appropriate dose ranges for protecting neurons from CD14-activated innate immunity-mediated damage, and can guide drug development for diseases, such as Alzheimer disease, that are thought to derive in part from CD14-activated innate immune response.

Reactive oxygen species are believed to participate in the pathogenesis of traumatic brain injury (TBI). To evaluate the role of cellular glutathione peroxidase (Gpx1), a selenium-containing enzyme functioning in reduction of hydrogen peroxide and alkyl hyperoxides, in protecting animals against TBI, a line of Gpx1 transgenic mice was generated. Overexpression of Gpx1 was found in many organs including the brain of the transgenic mice. This line of transgenic mice and knockout mice deficient in Gpx1 were used in a model of controlled cortical impact injury and the efficiency of oxidative phosphorylation in brain mitochondria was determined. Although a 2-mm depth of mechanical impact caused a drastic decrease in NAD-linked electron transfer activities and energy-coupling capacities in brain mitochondria of nontransgenic mice, the decrease in mitochondrial function was completely prevented by overexpression of Gpx1 in Gpx1 transgenic mice. In addition, a 1-mm deformation depth hardly affected brain mitochondrial function in wild-type (Gpx1+/+) mice, yet resulted in a significant decrease in mitochondrial bioenergetic capacity in brains of homozygous Gpx1 knockout (Gpx1-/-) mice. Further experiments showed that inclusion of calcium chelator egtazic acid in measurement of mitochondrial respiration could completely restore the efficiency of mitochondrial respiration in injured brains of nontransgenic mice and Gpx1-/- mice, suggesting that the observed mitochondrial dysfunction is a direct result of increase in mitochondrion-associated calcium, which is secondary to the increased oxidative stress. These studies not only establish the role of Gpx1 in preventing mitochondrial dysfunction in mouse brain after TBI, but also suggest the species of reactive oxygen responsible for this event.

Ischemic injury to brain is associated with both disruption of the blood-brain barrier and increased oxidative stress. Given the neurotoxicity associated with exposure to oxidized low-density lipoprotein (oxLDL) in vitro, we tested the hypothesis that oxLDL may be present in parenchymal cells of cerebrum after infarction and that oxLDL may influence the pathophysiology of cerebral infarction. Our results showed that the subacute phase of cerebral infarction in patients was characterized by the appearance of oxLDL epitopes in astrocytes, but not neurons or microglia, in the perinecrotic zone. We further demonstrated that minimally oxLDL was most effectively internalized by primary cultures of rat astrocytes, and that exposure to minimal oxLDL stimulated astrocyte interleukin-6 secretion but did not alter nitric oxide production. These results demonstrate for the first time that oxLDL is present in brain parenchyma of patients with ischemic infarction and suggest a potential mechanism by which oxLDL may activate innate immunity and thereby indirectly influence neuronal survival.

Niemann-Pick type C disease (NPC) is characterized by neurodegeneration secondary to impaired cholesterol trafficking and excessive glycosphingolipid storage. Abnormal cholesterol and ganglioside metabolism may influence the generation and aggregation of amyloidogenic fragments (ie, C99 and Abeta) from amyloid-beta precursor protein (APP), crucial factors causing neurodegeneration in Alzheimer's disease. To reveal whether abnormal accumulation and aggregation of APP fragments also occurs in NPC, we studied their expression in cultured cortical neurons treated with U18666A, a compound widely used to induce NPC defects, and also in brain tissues from NPC patients. U18666A treatment resulted in increased intraneuronal levels of C99 and insoluble Abeta42, which were distributed among early and late endosomes, in compartments distinct from where endogenous cholesterol accumulates. Analyses of NPC brains revealed that C99 or other APP C-terminal fragments (APP-CTF), but not Abeta42, accumulated in Purkinje cells, mainly in early endosomes. In contrast, in hippocampal pyramidal neurons, the major accumulated species was Abeta42, in late endosomes. Similar to what has been shown in Alzheimer's disease, cathepsin D, a lysosomal hydrolase, was redistributed to early endosomes in NPC Purkinje cells, where it co-localized with C99/APP-CTF. Our results suggest that endosomal abnormalities related to abnormal lipid trafficking in NPC may contribute to abnormal APP processing and Abeta42/C99/APP-CTF deposition.

Maneb, a widely used fungicide, has been associated with Parkinsonism in humans. In experimental models, maneb and its major active element, manganese ethylene-bis-dithiocarbamate (Mn-EBDC) cause selective nigrostriatal neurodegeneration in mice and in rats, respectively. To investigate the mechanisms underlying this neurodegeneration, we studied the effects of Mn-EBDC on proteasomal function, which is decreased in patients with Parkinson's disease (PD), in a dopaminergic neuronal cell line (MES 23.5 or MES). The results demonstrated that exposure of MES cells to 6 microM Mn-EBDC for 7 days produced not only significant neurotoxicity but also inhibition of proteasomal chymotrypsin-like and postglutamyl peptidase activities. Proteasomal dysfunction was accompanied by formation of cytoplasmic inclusions that were positive for alpha-synuclein immunostaining and significantly increased sodium dodecyl sulfate-insoluble alpha-synuclein aggregation seen by Western blot analysis. In addition, there was a significant increase in oxidative stress, evidenced by elevated total protein carbonyl content, in cells treated with Mn-EBDC. Manipulation of intracellular reduced glutathione levels with N-acetyl-L-cysteine or L-buthionine sulfoximine pretreatment to modulate Mn-EBDC-mediated oxidative stress altered Mn-EBDC-mediated neurotoxicity, proteasomal dysfunction, and alpha-synuclein aggregation in these cells. These data suggest that neurotoxicity-induced by Mn-EBDC is at least partially attributable to Mn-EBDC-mediated proteasomal inhibition, and that the proteasome may be an important target by which environmental exposure modifies the risk for developing PD in vulnerable populations.

Increasing evidence suggests that intraneuronal amyloid-beta (Abeta) accumulation may be an early event in Alzheimer's disease (AD) pathogenesis. However direct in vivo evidence regarding initial Abeta seeding is missing. Using an APP transgenic mouse model, our sensitive immunocytochemical procedures revealed a novel intraneuronal Abeta deposition in the somas of hippocampal CA1/subiculum neurons far in advance of the occurrence of extracellular Abetaplaques. These deposits increased exponentially with age and were elevated approximately 4-fold (p < 0.001) by high fat/high cholesterol diet. Abeta40 and Abeta42 were the major constituents of these deposits and were co-localized with lysosomal markers. Our results are consistent with the notion that the earliest Abeta deposition occurs intraneuronally, prior to extracellular amyloid plaque formation., (Copyright 2003 Lippincott Williams & Wilkins)

The axonal microtubule stabilizing protein tau is hyperphosphorylated in several neurodegenerative conditions, including Alzheimer's disease, yet the genes that regulate tau phosphorylation are largely unknown. Disabled-1 (Dab1) is a cytoplasmic adapter protein that interacts with apolipoprotein E (ApoE) receptors and controls neuronal positioning during embryonic brain development. We have investigated the role of Dab1 in tau phosphorylation. We found that wild-type Dab1, but not a mutant lacking tyrosine phosphorylation sites, protects mice from the hyperphosphorylation of tau. However, the absence of Dab1 is not sufficient to cause tau hyperphosphorylation, because hyperphosphorylation is manifested only when Dab1 is mutated in specific mouse strain backgrounds. Tau hyperphosphorylation correlates with early death in susceptible mouse strains, and it occurs in the neurons of the hippocampus and dentate gyrus. By quantitative trait locus (QTL) analysis of Dab1-deficient mice on a hybrid strain background, we uncovered one significant and three suggestive chromosomal loci that modulate tau phosphorylation. Two of these QTL regions contain genes that are defective in early onset Alzheimer's disease. Our findings suggest that Dab1 gene disruption sensitizes mice to tau hyperphosphorylation contingent on specific haplotypes that are linked to Alzheimer's disease loci. Dab1 mutant mice provide an animal model for studying the relationships between ApoE receptors, tau hyperphosphorylation, and Alzheimer's disease.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by the progressive and global loss of cognitive functions. Pathological features include a loss of neurons in vulnerable brain regions and the extracellular deposition of abnormal protein aggregates known as amyloid plaques. Amyloid-beta protein (A beta is the major component of amyloid plaques and is derived from a larger transmembrane glycoprotein, termed amyloid beta protein precursor (APP), by proteolysis. The AD research has focused on A beta production and metabolism, its extracellular deposition, and its cellular toxicity. Recent evidence, however, suggests that A beta as well as the C-terminal fragments (CTF) of APP can accumulate intraneuronally. The neuronal loss and synaptic transmission deficit in AD may therefore depend on intraneuronal accumulation of A beta/CTF rather than on extracellular plaque formation. Accordingly, we propose that one of the primary targets of therapeutic intervention should be intracellular A beta/CTF and its toxic cellular effect. We have established a cell-culture model in which the neurons degenerate on induction of endogenous expression of A beta/CTF of APP. These cultures have been used to test whether tricyclic pyrone (TP) compounds may prevent A beta/CTF-mediated neuronal death. The results to date have been encouraging. Lead compounds will now be selected for their abilities to ameliorate A beta/CTF-mediated pathology in transgenic mice. Our hope is that these compounds may eventually prove beneficial for the prevention and treatment of AD.

Epidemiological data show correlations between hypercholesterolemia and Alzheimer's disease (AD). We test the hypothesis that hypercholesterolemia modulates Abeta deposition in mice overexpressing the human APP695 Swedish mutation (K670N and M671L) (TgAPPsw). Feeding mice a high fat/high cholesterol (HFHC) diet for 7-10 months increased total cholesterol levels by 4-fold. The extent of Abeta immunostained plaque-like deposits were significantly higher for mice fed the HFHC diet as compared with mice fed rodent chow. Extent of deposits correlated inversely with plasma levels of HDL and directly to apolipoprotein E. Overall, plasma lipoproteins may be an important factor in induction of AD-like plaques in mice. The lowering of plasma lipids may be therapeutic for AD patients.

Objective: The purpose of the study was to evaluate the effect of a low carotenoid diet (83 micrograms Beta-carotene) on malondialdehyde-thiobarbituric acid (MDA-TBA) concentrations of nine pre-menopausal women., Methods: Subjects lived on the metabolic research unit of the Western Human Nutrition Research Center (WHNRC), where diet, exercise and other activities were controlled. Five subjects (Group C, control group) consumed a low carotenoid diet and received an additional 0.5 mg/day of Beta-carotene while four subjects (Group P, placebo group) received only the low carotenoid diet during days 1 to 60 (period 1). All subjects received 0.5 mg/day of Beta-carotene during days 60 to 100 (period 2), plus three capsules/day mixed carotenoid supplement (Neo-Life Company) during study days 100 to 120. Changes in MDA-TBA concentrations were analyzed during the study periods and between the groups., Results: At the start of the study (day 1), no significant difference in the MDA-TBA concentration was observed between the control (Group C) and the placebo (Group P) subjects. During period 1 (days 2 to 60), when Group P subjects consumed the low carotenoid diet without supplementation, the MDA-TBA values for Group P rose markedly and were significantly (p < 0.05) higher than the MDA-TBA values for Group C subjects who were receiving carotenoid supplementation. During period 2 (days 60 to 100) when both groups received carotenoid supplementation, the MDA-TBA values of Group P subjects were significantly (p < 0.05) reduced to the point where they were similar to the MDA-TBA values for Group C subjects., Conclusions: These findings provide evidence to support the beneficial effects of carotenoids in preventing lipid peroxidation in the cells. Further studies are needed to identify the exact mechanism by which carotenoids prevent lipid peroxidation and the amount needed for normal activity.