Inhibition of lipopolysaccharide-induced microglial activation by preexposure to neurotrophin-3

Microglia activated by neural injuries produce proinflammatory mediators, but activated microglia also appear in developing neural tissue to phagocytose cell debris resulting from programmed cell death without inducing tissue damage. Thus, factors associated with the developing CNS may modulate microglial activities. Previously we reported that pretreatment with neurotrophin-3 (NT-3), a factor known to regulate neural development, inhibits the production of proinflammatory mediators, nitric oxide (NO), tumor necrosis factor-alpha, and interleukin-1beta, in BV2 activated by inflammagen lipopolysaccharide (LPS). In this study, the inhibition of proinflammatory mediators by NT-3 pretreatment (preNT-3) in primary microglia with LPS stimulation was corroborated. Moreover, pretreatment of LPS-activated microglia with NT-3 induced a trend of reduction in phagocytotic ability. By using LPS-activated BV2 cells, we further found that reduced expression of inducible NO synthetase by preNT-3 was mediated by MAP kinase and PI3 kinase signaling pathways. Moreover, pretreatment of BV2 cells with NT-3 led to reduced levels of the p65 subunit of nucleus factor-kappaB (NFkappaB) and its DNA binding activity. Accordingly, our results indicate that preexposure of microglia to NT-3 leads to a reduced production of proinflammatory mediators in activated microglia by the induction of MAP kinase and PI3 kinase signaling, which in turn may reduce NFkappaB DNA binding activity. This suggests that an NT-enriched microenvironment may be favorable for preventing the inflammatory reaction of microglia.     

Tripchlorolide protects neuronal cells from microglia‐mediated β‐amyloid neurotoxicity through inhibiting NF‐κB and JNK signaling

Recent research has focused on soluble oligomeric assemblies of β‐amyloid peptides (Aβ) as the proximate cause of neuroinflammation, synaptic loss, and the eventual dementia associated with Alzheimer's disease (AD). In this study, tripchlorolide (T₄), an extract of Tripterygium wilfordii Hook. F (TWHF), was studied as a novel agent to suppress neuroinflammatory process in microglial cells and to protect neuronal cells against microglia‐mediated oligomeric Aβ toxicity. T₄ significantly attenuated oligomeric Aβ(1‐42)‐induced release of inflammatory productions such as tumor necrosis factor‐α, interleukin‐1β, nitric oxide (NO), and prostaglandin E₂. It also downregulated the protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) in microglial cells. Further molecular mechanism study demonstrated that T₄ inhibited the nuclear translocation of nuclear factor‐κB (NF‐κB) without affecting I‐κBα phosphorylation. It repressed Aβ‐induced JNK phosphorylation but not ERK or p38 MAPK. The inhibition of NF‐κB and JNK by T₄ is correlated with the suppression of inflammatory mediators in Aβ‐stimulated microglial cells. These results suggest that T₄ protects neuronal cells by blocking inflammatory responses of microglial cells to oligomeric Aβ(1‐42) and that T₄ acts on the signaling of NF‐κB and JNK, which are involved in the modulation of inflammatory response. Therefore, T₄ may be an effective agent in modulating neuroinflammatory process in AD. © 2009 Wiley‐Liss, Inc.     

β-Secretase activity in rat astrocytes: translational block of BACE1 and modulation of BACE2 expression

BACE1 and BACE2 are two closely related membrane-bound aspartic proteases. BACE1 is widely recognized as the neuronal β-secretase that cleaves the amyloid-β precursor protein, thus allowing the production of amyloid-β, i.e. the peptide that has been proposed to trigger the neurodegenerative process in Alzheimer's disease. BACE2 has ubiquitous expression and its physiological and pathological role is still unclear. In light of a possible role of glial cells in the accumulation of amyloid-β in brain, we have investigated the expression of these two enzymes in primary cultures of astrocytes. We show that astrocytes possess β-secretase activity and produce amyloid-β because of the activity of BACE2, but not BACE1, the expression of which is blocked at the translational level. Finally, our data demonstrate that changes in the astrocytic phenotype during neuroinflammation can produce both a negative as well as a positive modulation of β-secretase activity, also depending on the differential responsivity of the brain regions.     

Low-dose 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine causes inflammatory activation of astrocytes in nuclear factor-κB reporter mice prior to loss of dopaminergic neurons

Neuroinflammation is implicated in the progression of numerous disease states of the CNS, but early inflammatory signaling events in glial cells that may predispose neurons to injury are not easily characterized in vivo. To address this question, we exposed transgenic mice expressing a nuclear factor-κB (NF-κB)-driven enhanced green fluorescent protein (EGFP) reporter construct to low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and examined inflammatory activation of astrocytes in relation to neurobehavioral and neuropathological outcomes. The highest dose of MPTP (60 mg/kg total dose) caused a decrease in locomotor activity and a reduction in stride length. No significant loss of dopaminergic neurons in the substantia nigra was apparent at any dose. In contrast, expression of tyrosine hydroxylase in striatal fibers was reduced at 60 mg/kg MPTP, as were levels of dopamine and DOPAC. Colocalized expression of EGFP and inducible nitric oxide synthase (NOS2) occurred in astrocytes at 30 and 60 mg/kg MPTP and was associated with increased protein nitration in nigral dopaminergic neurons. Inhibition of NF-κB in primary astrocytes by expression of mutant IκBα suppressed expression of NOS2 and protected cocultured neurons from astrocyte-mediated apoptosis. These data indicate that inflammatory activation of astrocytes and enhanced nitrosative stress occurs at low doses of MPTP prior to loss of dopaminergic neurons. NF-κB-mediated expression of NOS2 appears to be a sensitive indicator of neuroinflammation that correlates with MPTP-induced neurochemical and neurobehavioral deficits prior to loss of dopaminergic neurons in the subtantia nigra.     

In vivo and in vitro effects of aluminum on the activity of mouse brain acetylcholinesterase

Cholinesterases are a large family of enzymatic proteins widely distributed throughout both neuronal and non-neuronal tissues. In Alzheimer's disease (AD), analytical as well as epidemiological studies suggest an implication of an abnormal focal accumulation of aluminum in the brain. In this devastating disease, aluminum may interfere with various biochemical processes including acetylcholine metabolism, and can thus act as a possible etiopathogenic cofactor. Acetylcholinesterase (AChE) exists in several molecular forms that differ in solubility and mode of membrane attachment rather than in catalytic activity. Mice were treated orally with aluminum chloride or aluminum lactate (Al(lac)(3)), and AChE activity in their brain homogenates was then assayed. Results showed that this in vivo treatment augmented the activity of the enzyme. An activating effect was also observed in vitro, when the aluminum compounds were added directly to mouse brain homogenates. However, the activating effect observed in vivo was much more marked than that observed in vitro. In addition, the activation produced by Al(lac)(3) was higher than that obtained after aluminum chloride treatment. Kinetics measurements of AChE activity in the absence and presence of treatment with aluminum both in vivo and in vitro are reported. The influence of the metal speciation on enzymatic activity is discussed in relation to a possible implication of aluminum in some neurodegenerative diseases.     

Enhanced long-term potentiation in the hippocampus of rats expressing mutant presenillin-1 is age related

Electrophysiological recordings were made from Fischer rats engineered to express the human presenilin 1 gene carrying the M146V mutation. Extracellular recordings of field excitatory post-synaptic potential (EPSPs) were made to investigate EPSP properties, paired pulse responses, posttetanic potentiation, and long-term potentiation in the stratum radiatum and dentate gyrus of hippocampal slices maintained in vitro. Transgenic rats aged approximately 6 months showed no differences from their wild-type littermates in any of these properties. However, at 18 months, long-term potentiation in the CA1 was facilitated in the transgenic rats with a different pattern of synaptic enhancement. No changes were observed in paired pulse facilitation (PPF) or post-tetanic potentiation (PPT) and no changes were seen in the dentate gyrus. Field potential amplitudes were significantly greater and PPF was enhanced in the CA1 of all older rats. Intracellular recordings from CA1 pyramidal cells of the older group of rats revealed no differences in the passive or active membrane properties of cells in the two groups, but intracellularly recorded EPSPs were significantly longer.     

Baicalein attenuates astroglial activation in the 1‐methyl‐4‐phenyl‐1,2,3,4‐tetrahydropyridine‐induced Parkinson's disease model by downregulating the activations of nuclear factor‐κB,ERK, and JNK

In Parkinson's disease (PD), neuroinflammation plays a critical role in the neurodegenerative process. Furthermore, activated microglia and astrocytes, responsible for activated immune response in the central nervous system, are found in regions associated with dopaminergic neuronal death. The flavonoid baicalein is known to have antibacterial, antiviral, and antiinflammatory activities. In the present study, the neuroprotective effects of baicalein were examined in a murine 1‐methyl‐4‐phenyl‐1,2,3,4‐tetrahydropyridine (MPTP) model of PD. Low doses of baicalein improved motor ability and prevented dopaminergic neuron loss caused by MPTP. In addition, microglial and astrocyte activations were reduced in PD mice pretreated with baicalein. Further study of primary astrocytes revealed that baicalein suppressed the 1‐methyl‐4‐phenylpyridine‐induced nuclear translocation of nuclear factor‐κB and reduced the activations of JNK and ERK, suggesting that the neuroprotective effects of baicalein in our PD model were due to attenuated astrocyte activation. The findings of this study indicate that baicalein could be useful for the treatment of PD and other neuroinflammation‐related neurodegenerative diseases. © 2013 Wiley Periodicals, Inc.     

Astrocytes contribute to neuronal impairment in beta A toxicity increasing apoptosis in rat hippocampal neurons

Astrocytosis is a common feature of amyloid plaques, the hallmark of Alzheimer's disease (AD), along with activated microglia, neurofibrillary tangles, and beta-amyloid (beta A) deposition. However, the relationship between astrocytosis and neurodegeneration remains unclear. To assess whether beta A-stimulated astrocytes can damage neurons and contribute to beta A neurotoxicity, we studied the effects of beta A treatment in astrocytic/neuronal co-cultures, obtained from rat embryonic brain tissue. We found that in neuronal cultures conditioned by beta A-treated astrocytes, but not directly in contact with beta A, the number of apoptotic cells increased, doubling the values of controls. In astrocytes, beta A did not cause astrocytic cell death, nor did produce changes in nitric oxide or prostaglandin E(2) levels. In contrast, S-100 beta expression was remarkably increased. Our data show for the first time that beta A--astrocytic interaction produces a detrimental effect on neurons, which may contribute to neurodegeneration in AD.     

Anti-amyloid beta activity of metallothionein-III is different from its neuronal growth inhibitory activity: structure-activity studies

Recent studies have shown that metallothionein-III (MT-III), but not MT-I or -II, antagonizes both the neurotrophic and neurotoxic effects of amyloid β peptides (Aβs). Further, its anti-Aβ-toxicity effect was attributed to the fact that it inhibits the formation of fibrillar Aβ. MT-III alone also affects neuron survival in culture—promoting at low but inhibiting at high concentrations. To characterize these biological activities of MT-III in relation to its neuronal growth inhibitory activity discovered by Uchida et al. [Neuron 7 (1991) 337–347], we here studied effects of the P7S/P9A double mutant, and the N- and C-terminal domains of MT-III on primary cultures of rat embryonic cortical neurons in the presence and absence of Aβ. Results show that (i) only the wild-type MT-III inhibited the formation of SDS-resistant Aβ aggregates and protected cortical neurons from the toxic effect of Aβ, and (ii) both the wild type and the N-terminal domain of MT-III promote neuron survival at low concentrations but inhibited it at high concentrations. On the basis of these findings, we conclude that the anti-Aβ activity of MT-III is different from its neuronal growth inhibitory activity and suggest that the increased trophic activity of AD brain extracts could be attributed to its low MT-III content.     

Alzheimer's disease: usefulness of the Family Strain Questionnaire and the Screen for Caregiver Burden in the study of caregiving-related problems

OBJECTIVES: The economic and psychosocial impacts of Alzheimer's disease (AD) on caregivers are so well documented that they have stimulated socioeconomic regulations that are international in scope. In Italy caregivers have the right to receive economic and psychosocial aid. However, to distribute such aid the needs of caregivers, must be properly assessed. Here we have attempted to integrate two measures, the Family Strain Questionnaire (FSQ) and the Screen for Caregiver Burden (SCB), in order to evaluate caregiver needs that are both general and specific to AD. MATERIAL AND METHODS: The SCB and FSQ were administered to 91 primary caregivers of home-based patients with AD. Caregivers also were asked to rate the activities of daily living of their care recipients, the latter of which were administered the Mini-Mental State Examination. RESULTS: The SCB and FSQ provide different, but complementary assessments of the needs of AD caregivers. The SCB identifies situations that are potentially stressful to AD caregivers and the FSQ identifies the needs of specific caregivers (e.g. men vs women, spouses vs children, unemployed vs employed). CONCLUSION: Together these measures may help government agencies to assess caregiver needs beyond those assessed by each individual measure.     

Signaling pathways of bisphenol A-induced apoptosis in hippocampal neuronal cells: role of calcium-induced reactive oxygen species, mitogen-activated protein kinases, and nuclear factor-kappaB

In the present study, we investigated the neurotoxicity of bisphenol A [BPA; 2,2-bis-(4 hydroxyphenyl) propane] and the underlying mechanisms of action in mouse hippocampal HT-22 cells. BPA, known to be a xenoestrogen, is used in the production of water bottles, cans, and teeth suture materials. BPA-treated HT-22 cells showed lower cell viability than did controls at concentrations of BPA over 100 microM. BPA induced apoptotic cell death as indicated by staining with Hoechst 33258, costaining with Annexin V/propidium iodide, and activation of caspase 3. BPA regulated the generation of reactive oxygen species (ROS) by increasing intracellular calcium. BPA activated phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), and nuclear translocation of nuclear factor (NF)-kappaB. Pretreatment with specific inhibitors for calcium, ROS, ERK, and JNK decreased BPA-induced cell death; however, inhibitor for NF-kappaB increased BPA-induced cell death. The results suggest that calcium, ROS, ERK, and JNK are involved in BPA-induced apoptotic cell death in HT-22 cells. In contrast, an NF-kappaB cascade was activated for survival signaling after BPA treatment.     

Neuroprotective signaling and the aging brain: take away my food and let me run

It is remarkable that neurons are able to survive and function for a century or more in many persons that age successfully. A better understanding of the molecular signaling mechanisms that permit such cell survival and synaptic plasticity may therefore lead to the development of new preventative and therapeutic strategies for age-related neurodegenerative disorders. We all know that overeating and lack of exercise are risk factors for many different age-related diseases including cardiovascular disease, diabetes and cancers. Our recent studies have shown that dietary restriction (reduced calorie intake) can increase the resistance of neurons in the brain to dysfunction and death in experimental models of Alzheimer's disease, Parkinson's disease, Huntington's disease and stroke. The mechanism underlying the beneficial effects of dietary restriction involves stimulation of the expression of 'stress proteins' and neurotrophic factors. The neurotrophic factors induced by dietary restriction may protect neurons by inducing the production of proteins that suppress oxyradical production, stabilize cellular calcium homeostasis and inhibit apoptotic biochemical cascades. Interestingly, dietary restriction also increases numbers of newly-generated neural cells in the adult brain suggesting that this dietary manipulation can increase the brain's capacity for plasticity and self-repair. Work in other laboratories suggests that physical and intellectual activity can similarly increase neurotrophic factor production and neurogenesis. Collectively, the available data suggest the that dietary restriction, and physical and mental activity, may reduce both the incidence and severity of neurodegenerative disorders in humans. A better understanding of the cellular and molecular mechanisms underlying these effects of diet and behavior on the brain is also leading to novel therapeutic agents that mimick the beneficial effects of dietary restriction and exercise.     

Neural progenitor number is regulated by nuclear factor-kappaB p65 and p50 subunit-dependent proliferation rather than cell survival

The number of cells generated by a proliferating stem or precursor cell can be influenced both by proliferation and by the degree of cell death/survival of the progeny generated. In this study, the extent to which cell survival controls progenitor number was examined by comparing the growth characteristics of neurosphere cultures derived from mice lacking genes for the death-inducing Bcl-2 homologue Hara Kiri (Hrk), apoptosis-associated protein 1 (Apaf1), or the prosurvival nuclear factor-kappaB (NFkappaB) subunits p65, p50, or c-rel. We found no evidence that Hrk or Apaf1, and by inference the mitochondrial cell death pathway, are involved in regulating the number of neurosphere-derived progeny. However, we identified the p65p50 NFkappaB dimer as being required for the normal growth and expansion of neurosphere cultures. Genetic loss of both p65 and p50 NFkappaB subunits resulted in a reduced number of progeny but an increased proportion of neurons. No effect on cell survival was observed. This suggests that the number and fate of neural progenitor cells are more strongly regulated by cell cycle control than survival.     

Ryanodine receptor-mediated [Ca(2+)](i) release in glomus cells is independent of natural stimuli and does not participate in the chemosensory responses of the rat carotid body

Advanced glycation endproducts (AGEs), protein-bound oxidation products of sugars, have been shown to be involved in the pathophysiological processes of Alzheimer’s disease (AD). AGEs induce the expression of various pro-inflammatory cytokines and the inducible nitric oxide synthase (iNOS) leading to a state of oxidative stress. AGE modification and resulting crosslinking of protein deposits such as amyloid plaques may contribute to the oxidative stress occurring in AD. The aim of this study was to immunohistochemically compare the localization of AGEs and β-amyloid (Aβ) with iNOS in the temporal cortex (Area 22) of normal and AD brains. In aged normal individuals as well as early stage AD brains (i.e. no pathological findings in isocortical areas), a few astrocytes showed co-localization of AGE and iNOS in the upper neuronal layers, compared with no astrocytes detected in young controls. In late AD brains, there was a much denser accumulation of astrocytes co-localized with AGE and iNOS in the deeper and particularly upper neuronal layers. Also, numerous neurons with diffuse AGE but not iNOS reactivity and some AGE and iNOS-positive microglia were demonstrated, compared with only a few AGE-reactive neurons and no microglia in controls. Finally, astrocytes co-localized with AGE and iNOS as well as AGE and were found surrounding mature but not diffuse amyloid plaques in the AD brain. Our results show that AGE-positive astrocytes and microglia in the AD brain express iNOS and support the evidence of an AGE-induced oxidative stress occurring in the vicinity of the characteristic lesions of AD. Hence activation of microglia and astrocytes by AGEs with subsequent oxidative stress and cytokine release may be an important progression factor in AD.     

Association of platelet-derived soluble glycoprotein VI in plasma with Alzheimer's disease

Accumulating evidence from epidemiological, clinical and experimental studies suggests that vascular risk factors and angiopathic mechanisms are involved in the pathogenesis of Alzheimer’s disease (AD). Platelets could be the missing link between AD and the vasculature.

Soluble glycoprotein VI (sGPVI) and β-thromboglobulin (β-TG) plasma and cerebrospinal fluid (CSF) levels as markers of platelet activity were measured in 30 AD patients and 20 age-matched healthy elderly controls by ELISA. The severity of dementia was assessed by mini-mental state examination (MMSE).

We found in AD patients significantly decreased sGPVI plasma levels (0.55 ± 0.18 ng/ml) as compared to healthy controls (0.75 ± 0.43 ng/ml; p = 0.033). In AD patients, sGPVI levels were positively correlated with β-TG plasma levels (r = 0.244, p = 0.05) and with cognitive status as measured by MMSE score (r = 0.271; p = 0.048). In unconcentrated CSF samples, levels of β-TG and sGPVI were below the detection limit of the assays in AD patients and healthy controls.

Our results suggest an association of sGPVI with the pathogenesis of AD. These findings encourage future research into whether sGPVI plasma levels may reflect or even mediate neuroprotective mechanisms in AD.     

Cysteine proteases are the major beta-secretase in the regulated secretory pathway that provides most of the beta-amyloid in Alzheimer's disease: role of BACE 1 in the constitutive secretory pathway

This article focuses on beta-amyloid (Abeta) peptide production and secretion in the regulated secretory pathway and how this process relates to accumulation of toxic Abeta in Alzheimer's disease. New findings are presented demonstrating that most of the Abeta is produced and secreted, in an activity-dependent manner, through the regulated secretory pathway in neurons. Only a minor portion of cellular Abeta is secreted via the basal, constitutive secretory pathway. Therefore, regulated secretory vesicles contain the primary beta-secretases that are responsible for producing the majority of secreted Abeta. Investigation of beta-secretase activity in regulated secretory vesicles of neuronal chromaffin cells demonstrated that cysteine proteases account for the majority of the beta-secretase activity. BACE 1 is present in regulated secretory vesicles but provides only a small percentage of the beta-secretase activity. Moreover, the cysteine protease activities prefer to cleave the wild-type beta-secretase site, which is relevant to the majority of AD cases. In contrast, BACE 1 prefers to cleave the Swedish mutant beta-secretase site that is expressed in a minor percentage of the AD population. These new findings lead to a unifying hypothesis in which cysteine proteases are the major beta-secretases for the production of Abeta in the major regulated secretory pathway and BACE 1 is the beta-secretase responsible for Abeta production in the minor constitutive secretory pathway. These results indicate that inhibition of multiple proteases may be needed to decrease Abeta production as a therapeutic strategy for Alzheimer's disease.     

Age-related changes in plasma dehydroepiandrosterone levels in adults with Down's syndrome and the risk of dementia

People with Down's syndrome (DS) are at high risk of developing early onset dementia. Recent studies suggest a link between age-related decreases in dehydroepiandrosterone (DHEA) concentrations and dementia in the general population. The present study investigates the relationship between DHEA serum levels and age and the risk of dementia in adults with DS. The DHEA plasma concentrations of 67 adults with DS and 65 age-matched controls were determined. Participants with DS were assessed for the presence of dementia using the CAMDEX informant interview. The DHEA plasma concentrations decreased with age in subjects with DS as well as in controls. Age significantly predicted DHEA levels in both groups (B = -0.06, t = -4.536, P < 0.001 in the DS group and B = -0.04, t = -2.928, P < 0.005 in control participants). The mean ± SD DHEA level was 3.47 ± 1.41 μmol/l in controls and 2.79 ± 1.24 μmol/l in participants with DS. This difference was significant (t = -2.981, P < 0.01). Within the DS population, ancova revealed a significant relationship between DHEA concentrations and dementia (F(1,65) = 4.348, P < 0.05). We found that DHEA levels declined significantly with age in people with DS and controls and were lower, in comparison to age-matched controls, in people with DS across all ages studied. Those with DS and evidence of dementia have lower DHEA concentrations than those with DS (controlling for age) but without dementia.     

一氧化氮在神经退行性疾病中的作用:非类固醇性抗炎药的治疗前景(英文)

一氧化氮(nitric oxide, NO)是一类胞内信使。研究表明,神经退行性病人脑组织中催化合成NO的酶的表达水平显著提高,提示NO与神经退行性疾病密切相关。此外,在这些组织中还检测到硝化的蛋白,提示NO在这些组织中具有生物活性。在神经免疫应答中,神经元和胶质细胞(包括小胶质细胞和星形胶质细胞)内都发生了NO水平的改变。很多神经退行性疾病都伴随有神经炎症,抑制神经炎症的信号通路能延迟这些疾病的发展。因此,NO及其释放通路已逐渐成为神经退行性疾病研究领域的热点,对它们的理解能帮助我们找到合适的方案来预防、减缓或者治愈这些疾病。