β-Amyloid-Induced Cholinergic Denervation Correlates with Enhanced Nitric Oxide Synthase Activity in Rat Cerebral Cortex: Reversal by NMDA Receptor Blockade

Ample experimental evidence indicates that acute β-amyloid infusion into the nucleus basalis of rats elicits abrupt degeneration of the magnocellular cholinergic neurons projecting to the cerebral cortex. In fact, involvement of a permanent Ca²⁺ overload, partially via N-methyl-

Full-size image

-aspartate (NMDA) receptors, was proposed as a pivotal mechanism in β-amyloid-induced neurodegeneration. A definite measure of NMDA receptor-mediated processes and subsequent Ca²⁺ entry is the induction of Ca²⁺/calmodulin-activated neuronal nitric oxide synthase (nNOS) in nerve cells. In the present account we therefore assessed activation of nNOS in correlation with cholinergic decline after β-amyloid_(1–42) or β-amyloid_(25–35) infusion into the rat nucleus basalis. The results demonstrate the β-amyloid conformation-dependent enhancement of cortical nitric oxide synthase (NOS) activity. Furthermore, chronic application of the polyamine site NMDA receptor blocker ifenprodil effectively attenuated β-amyloid neurotoxicity. We propose that nNOS activation reflects the degree of β-amyloid-induced excitotoxic injury in a proportional manner. Moreover, Ca²⁺-mediated processes via NMDA receptors, or direct binding of β-amyloid to this receptor may be a critical step in the neurotoxic mechanisms in vivo.     

Activation of nuclear factor-κB by β-amyloid peptides and interferon-γ in murine microglia

An increasing body of evidence suggests that amyloid-β (Aβ) peptides and microglia are crucially involved in the pathogenesis of Alzheimer's disease. In an effort to further elucidate the biological effects of Aβ towards microglia, we investigated the ability of Aβ peptides to activate nuclear factor (NF)-κB in the N9 murine microglial cell line. Co-stimulation of microglia with suboptimal concentrations of Aβ(25–35) and 100 U/ml IFNγ resulted in the detection of a specific NF-κB DNA-binding activity in nuclear extracts, as determined in gel mobility shift assays. This response required at least 120 min to be evident and supershift experiments revealed that the NF-κB complex contains both RelA and p50. Accordingly, immunoblot experiments showed that amongst NF-κB/Rel proteins, RelA and p50 are mobilized to the nucleus following microglial cell stimulation with Aβ(25–35) plus IFNγ. Higher concentrations of Aβ(25–35) were effective by themselves in inducing NF-κB activation, both in the N9 microglial cell line and in rat primary microglia, as well as in human monocytes. For purposes of comparison, microglia were also stimulated with bacterial LPS, a known NF-κB inducer. As expected, LPS strongly induced the formation of two NF-κB DNA-binding activities, one of which was identified as RelA/p50. The LPS response was also more rapid, as it was already evident by 40 min and remained sustained for up to 3 h. Collectively, these findings indicate that NF-κB activation might constitute one of the mechanisms underlying the inducible expression of κB-dependent genes in microglia stimulated by Aβ peptides and IFNγ, or by LPS.     

Amyloid-β peptide activates cultured astrocytes: morphological alterations, cytokine induction and nitric oxide release

A common feature of many neurodegenerative disorders is an abundance of activated glial cells (astrocytes and microglia). In Alzheimer's disease (AD), activated astrocytes are in close apposition to and surrounding the amyloid plaques. The mechanisms by which the astrocytes become activated in AD and the consequences of reactive astrocytosis to disease progression are not known. We examined the possibility that the amyloid-β (Aβ) peptide, a major constituent of the amyloid plaque, could act as a stimulus leading to activation. We found that treatment of rat cortical astrocyte cultures with aggregated Aβ 1–42 peptide induces activation, as assessed by reactive morphological changes and upregulation of selective glial mRNA and proteins, such as the inflammatory cytokine interleukin-1β. Aβ also stimulates inducible nitric oxide synthase (iNOS) mRNA levels and nitric oxide (NO) release. Aβ 1–42, a major form of amyloid associated with neurotoxicity, activated astrocytes in a time- and dose-dependent manner, whereas a scrambled Aβ 1–42 sequence or Aβ 17–42 had little or no effect. We also determined that the Aβ activity can be found in a supernatant fraction containing soluble Aβ oligomers. Our data suggest that Aβ plays a role in the reactive astrocytosis of AD and that the inflammatory response induced upon glial activation is a critical component of the neurodegenerative process.     

Transforming growth factor β's 1, 2 and 3 inhibit proliferation of ramified microglia on an astrocyte monolayer

The transforming growth factor β's (TGFβ) are a multipotent family of cytokines with strong immunosuppressive and neurotrophic effects. In the current study, we examined the effect of the TGFβ's 1, 2 and 3 on the proliferation of ramified microglia cultured on top of a confluent astrocyte monolayer. All three TGFβ isoforms inhibited proliferation. PCR analysis also showed the presence of mRNA for the TGFβ receptors type I and II and for all 3 TGFβ isoforms in microglia, astrocytes and in co-cultures. Moreover, removal of this endogenous TGFβ activity with antibodies against TGFβ1 and TGFβ3 strongly stimulated microglial proliferation. These inhibitory effects on the proliferation of ramified microglia suggest that TGFβ's may play an important role in the regulation of the microglial population under normal conditions and after injury or disease in the central nervous system.     

Activation of microglial cells by PrP and β-amyloid fragments raises intracellular calcium through L-type voltage sensitive calcium channels

The prion protein (PrP) and the amyloid β (Aβ) precursor protein (APP) are two normal proteins constitutively synthesised in human brain. An altered form of PrP accumulates in Creutzfeldt–Jakob disease, while Aβ is involved in the pathogenesis of Alzheimer's disease. Synthetic fragments of both proteins, PrP106–126 and β25–35 (β25–35), have been demonstrated to induce neurodegeneration and microglia activation. This study was undertaken to compare PrP106–126 and β25–35 capability of activating human resting microglial cells. Our results show that both peptides are able to induce microglial activation and to elicit an increase in [Ca²⁺]_i levels in cells loaded with calcium-green 1. Inhibitors of L-type voltage-sensitive calcium channels (verapamil, nifedipine and diltiazem) prevented the increase in [Ca²⁺]_i concentration as observed after treatment with PrP106–126 and β25–35, thus indicating a transmembrane calcium influx through these channels. In addition, verapamil abolished the proliferative effect of both PrP106–126 and β25–35.     

Promotion of transition metal-induced reactive oxygen species formation by β-amyloid

β-amyloid protein appears to be involved in the neural degeneration associated with Alzheimer's disease. However, its mechanism of action is poorly understood. The ability of the neurotoxic peptide fragment (25–35) derived from β-amyloid, to promote the generation of reactive oxygen species (ROS) by a postmitochondrial fraction (P2) derived from rat cerebrocortex, has been examined. The peptide fragment, when incubated together with P2, did not cause excess ROS formation. However, 10 μM FeSO₄ or 10 μM CuSO₄ were able to enhance ROS production in the P2 fraction and this was increased further in the concurrent presence of the 25–35 fragment. The corresponding inverse sequence non-neurotoxic peptide (35–25) had no parallel ability to augment iron-stimulated ROS production suggesting a degree of specificity for the observed effect. There was no formation of excess ROS when the 25–35 peptide and 0.5 mM Al₂(SO₄)₃ were incubated with the P2 fraction. However in the presence of both aluminum and iron salts together with the 25–35 peptide, ROS production was augmented to a level significantly higher than that in the absence of aluminum. Polyglutamate, a peptide reported to mitigate aluminum toxicity had no effect on iron-related ROS generation but completely prevented its further potentiation by aluminum. The results indicate that β-amyloid is able to potentiate the free-radical promoting capacity of metal ions such as iron, copper and aluminum. Such potentiation may be a relevant mechanism underlying β-amyloid-induced degeneration of nerve cells.     

Amnesia induced in mice by centrally administered β-amyloid peptides involves cholinergic dysfunction

Substantial evidences suggest that the increased cerebral deposition, and neurotoxic action of the β-amyloid peptide, the major constituent of senile plaques, may represent the underlying cause of the cognitive deficits observed in Alzheimer's disease. Herein, we attempted to verify this hypothesis by inducing a potential Alzheimer's-type amnesia after direct intracerebroventricular administration of aggregatedβ_25–35-amyloid peptide in mice. In this aim, mnesic capacities were evaluated after 6–13 days, using spontaneous alternation in the Y-maze, step-down type passive avoidance and place learning in a water-maze. Pretraining administration of aggregatedβ_25–35 peptide induced dose-dependent decreases in both alternation behaviour and passive avoidance, at doses of 3 and 9 nmol/mouse. A reduced but still significant impairment was observed when the peptide was not aggregated, or ‘aged’, by preincubation for 4 days at 37°C. Theβ_1–28 peptide, at 3 nmol/mouse, also induced a marked decrease in step-down latency. Posttraining, but not preretention, administration of_25–35 peptide also significantly impaired learning. The beneficial effects of cholinergic agents onβ_25–35-induced amnesia was examined using the cholinesterase inhibitor tacrine (THA, 1.3 and 4.3 μmol/kg i.p.) and the nicotinic receptor agonist (−)-nicotine (NIC, 0.06 and 0.2 μmol/kg i.p.). Both drugs induced a dose-dependent abrogation of theβ_25–35-induced decreases in alternation behaviour and passive avoidance. Furthermore, THA, at 1.3 μmol/kg, and NIC, at 0.2 μmol/kg, also reversed theβ_25–31-induced impairment of place learning and retention in the water-maze. Histological examination of Cresyl violet-stained brain sections indicated a moderate but significant cell loss within the frontoparietal cortex and the hippocampal formation of mice treated with agedβ_25–35 peptide (9 nmol). Examination of Congo red-stained sections in the same animals demonstrated the presence of numerous amyloid deposits throughout these brain areas. These results confirm that the deposition of β-amyloid peptide in the brain is in some way related to impairment of learning and cholinergic degeneration and suggest that the [25–35] fragment of the β-amyloid protein, sufficient to induce neuronal death in cultures, also induces an Alzheimer's-type amnesia in mice.     

Cultured Rat Microglia Express C1q and Receptor for C1q: Implications for Amyloid Effects on Microglia

Senile plaques, the pathological hallmark of Alzheimer's disease (AD), are associated with complement components, including C1q. Reactive microglia appear to be involved in the later stages of plaque development. Since tissue macrophages are known to synthesize C1q, cultured rat microglia were examined for C1q immunoreactivity. Anti-C1q staining was detected, particularly in process-bearing microglia, indicating constitutive expression of C1q. Thus, microglia could provide a source of C1q for plaques even before becoming reactive. Since it has been previously shown that C1q binds β1-42, the major constituent of senile plaques, and since β1-42 is toxic to microglia in vitro , we asked if preincubation of β1-42 with C1q alters either metabolic indices of amyloid-induced degeneration in microglial cultures or the formation of amyloid deposits on these cells. While electron microscopic analysis of negatively stained amyloid fibrils confirmed that preincubation with C1q induced the association of C1q with the fibrils, no effect of the binding of C1q to β1-42 on β1-42 toxicity in microglia was observed. Interestingly, immunoreactivity for the C1q receptor that is known to modulate phagocytosis was found and was up-regulated in non-process-bearing microglia by interferon-γ. While these data exclude a role for the C1q receptor in β1-42 toxicity in microglia, the observed expression and up-regulation of C1q receptor on microglia by interferon-γ would be consistent with a role for C1q in complement-mediated inflammatory responses in AD and as a potential activator of microglial function in plaques.     

cAMP potentiates beta-amyloid-induced nitric oxide release from microglia

The beta-amyloid peptide (Abeta) has been known to activate microglia and to induce release of nitric oxide (NO). In this study, we examined the effect of cAMP on Abeta-induced microglial activation using cultured rat brain microglia. Dibutyryl-cAMP (dbcAMP) and 3-isobutyl-1-methylxanthine (IBMX) significantly potentiated Abeta(25-35)- or Abeta(1-42)-induced NO release in a dose-dependent manner. The increase in NO release was due to the increased expression of inducible nitric oxide synthase (iNOS). However, forskolin, an adenylate cyclase activator, weakly increased NO release at 10-50 microM but caused a decrease at 100 microM. These results suggest that increase in intracellular cAMP could potentiate microglial activation induced by Abeta.     

Microglial development is altered in immature spinal cord by exposure to radiation

Previous studies in this laboratory have documented that the microglial environment of the immature spinal cord is altered by exposure to ionizing radiation. As a result, the lumbosacral spinal cord is markedly depleted of both oligodendrocytes and astrocytes, while leaving axons and the overall cytoarchitecture intact. The status of the microglia in the irradiated region is unknown and is of interest given the interactions between microglia and astrocytes recently elucidated by others. This study uses both in vivo and in vitro approaches to examine the microglial population in normal and irradiated immature spinal cord. The lectin, Griffonia (Bandeiraea) simplicifolia, was selected since it marks microglia both in paraffin embedded sections and in cell cultures. Light microscopic examination of spinal cord sections revealed a reduced microglial population in the irradiated region when compared to littermate controls, and a change in morphology of the remaining microglia to that described by others as ‘‘activated’’. Cultures prepared from lumbosacral spinal cords harvested from 3-day-old rats within 2–4 hr following irradiation were compared with cultures derived from their non-irradiated littermates after 8 days in vitro. Cultures from the irradiated spinal cords revealed trends similar to those observed in vivo, i.e. a reduced microglial population and altered morphology. Although all glial cell types were reduced in cultures from irradiated spinal cords, the few microglia present were usually positioned atop astrocytes. The consistency of reduction in all glial populations in this model shows the microglia to be a novel microenvironment for further studies of roles of microglia within the spinal cord.     

β-Amyloid stimulates glial cells in vitro to produce growth factors that accumulate in senile plaques in Alzheimer's disease

The effects of a synthetic homolog of β-amyloid (β1–42) on the secretion of interleukin-1 (IL-1) and basic fibroblast growth factor (bFGF) from cultures of microglia and astrocytes, cells that surround β-amyloid-containing plaques in Alzheimer's disease, were examined. Our results show that β-amyloid not only enhances glial cell secretion of these factors, it stimulates the proliferation and morphological transformation of microglia. Since IL-1 and bFGF are known to elevate the synthesis of the β-amyloid precursor protein and other plaque components, it is suggested that in this way, cascades may arise that contribute to the process of plaque development.     

Autoradiographical imaging of PPARγ agonist effects on PBR/TSPO binding in TASTPM mice

Chronic inflammation is known to occur in the brains of Alzheimer's Disease (AD) patients, including the presence of activated microglia close to amyloid plaques. We utilised real time autoradiography and immunohistochemistry to investigate microglial activation and the potential anti-inflammatory effects of PPARγ agonists in the Thy-1 APP695swe/Thy-1 PS-1.M146V (TASTPM) overexpressing transgenic mouse model of AD. An age dependent increase in specific [³H](R)-PK11195 binding to peripheral benzodiazepine receptors (PBR)/translocator protein (18 kDa) (TSPO) was observed in the cortex of TASTPM mice compared to wild type mice, indicative of microglial activation. This was consistent with immunohistochemical data showing age-dependent increases in CD68 immunoreactivity co-localised with amyloid β (Aβ) deposits. In 10 month old TASTPM mice, pioglitazone (20 mg/kg) and ciglitazone (50 mg/kg) significantly reduced [³H](R)-PK11195 and [³H]DPA-713 binding in cortex and hippocampus, indicative of reduced microglial activation. In AD brain, significant [³H](R)-PK11195 and [³H]DPA-713 binding was observed across all stages of the disease. These results support the use of PBR/TSPO autoradiography in TASTPM mice as a functional readout of microglial activation to assess anti-inflammatory drugs prior to evaluation in AD patients.     

Co-localization of β-amyloid peptides, apolipoprotein E and glial markers in senile plaques in the prefrontal cortex of old rhesus monkeys

Based on the homology of human and monkey amyloid precursor proteins and the derived β-amyloid peptides (Aβ) the investigation of brains from old monkeys might be useful for the understanding of β-amyloidosis in the aetiology of Alzheimer's disease. In the present study, the prefrontal cortex, which is known to be highly susceptible to the deposition of Aβ, was screened for the occurrence of senile plaques in perfused tissue of aged rhesus monkeys (Macaca mulatta). Aβ deposits were immunocytochemically detected in five of six macaques aged about 28 years. Differently N-terminal truncated Aβ species in the senile plaques were simultaneously detected by a carbocyanine double fluorescence method applying the bright red fluorescent Cy3 and the novel green fluorescent Cy2. In a few cases, immunoreactivity for the shortened fragment containing the amino acids 17–42 (Aβ_17–42; p3 fragment with a molecular weight of 3 kDa) was demonstrated in deposits apparently devoid of Aβ_8–17. Senile plaques were further characterized by carbocyanine double labelling of Aβ and astrocytes, microglia and apolipoprotein E.© 1997 Elsevier Science B.V. All rights reserved.     

Human microglia mediate anti-Cryptococcus neoformans activity in the presence of specific antibody

The interaction of the opportunistic fungus Cryptococcus neoformans with human microglia was studied in vitro in the presence and absence of capsule binding antibody. In the absence of capsule binding antibody there was little or no phagocytosis. Addition of the murine monoclonal antibody (mAb) 2H1 (IgG1, kappa) to the capsular glucuronoxylomannan (GXM) produced a dose-dependent enhancement of C. neoformans phagocytosis by microglia. Phagocytosis resulted in marked inhibition of fungal proliferation. Microglial antifungal activity was studied by colony forming unit assay, l-[³H]leucine incorporation assay, and phase contrast microscopy. At microglia: C. neoformans ratios of 10:1 to 80:1 fungal growth was reduced by 61–95%. Inhibitors of nitric oxide synthase and reactive oxygen intermediates did not prevent antifungal activity mediated by human microglia. Transmission electron microscopic studies revealed that although some internalized yeast cells were killed, the majority were intact consistent with fungistasis. Human microglia cells are potent effector cells against C. neoformans in vitro in the presence of specific antibody. Enhancement of microglial activity in vivo by opsonins may be a useful therapeutic strategy.     

Generation and characterization of mouse microglial cell lines

A murine cell line (MMGT1) has been established after transfection of primary microglial cell cultures with a v-myc-containing plasmid. This cell line was comparable with primary microglial cells with respect to morphology, presence of acetylated low density lipoprotein receptor, non-specific estrase, CD63, major histocompatibility complex antigens and CD11, and binding for Ricinus communis agglutinin. Primary microglia as well as MMGT1 cells were negative for glial fibrillary acidic protein. Different MMGT1 strains were obtained after subcloning, two of which resembled histocytes (F4/80 and BM-8). These cell strains, MMGT12 and 16, were able to opsonize latex beads, and could be induced by endotoxins (LPS) to secrete TNF-α, IL-1, IL-6, TGF-β, and EGF. The other subclones had intermediate (MCA519, ER-MP20) or mixed macrophage characteristics and did not react to endotoxin by an increase in TNF-α, IL-1, and TGF-β. Our newly established murine microglia lines may prove to be useful models to study inflammation and repair in the brain.     

β-amyloid peptide decreases membrane fluidity

The β-amyloid peptide-25–35 (βA25–35) decreases the fluidity of mouse brain membranes in a concentration-depending fashion. First effects were already seen at a βA25–35 concentration of 100 nmol/1. β-Amyloid peptide (1–40) was similarly active. βA25–35 also decreases the fluidity of human lymphocyte membranes and of membranes from the cortex, hippocampus, striatum, and cerebellum of the rat, although the effects in the rat cerebellum are only weak. Scrambled βA25–35 when investigated under similar conditions showed no effects on membrane fluidity. It is suggest that the effect on cellular calcium-signalling but also the neurotoxic properties of β-amyloid might be the result of its concentration depending effects on membrane properties.     

Bexarotene Blocks Calcium-Permeable Ion Channels Formed by Neurotoxic Alzheimer’s β-Amyloid Peptides

相似文献   

Noradrenaline- and time-dependent changes in neocortical α2- and β1-adrenoceptor binding in dorsal noradrenergic bundle-lesioned rats

A 6-hydroxydopamine-induced lesion of the dorsal noradrenergic bundle (DNB) in rats markedly decreased neocortical noradrenaline concentration (NNC) by 72–100% as measured 1, 3 and 13 months after the lesioning procedure. The concomitant assessment of neocortical α₂- and β₁-adrenoceptor binding (NAAB and NBAB, respectively) usually indicated significant increases of 25–74% for these two variables. There were, however, cases of unchanged NAAB and NBAB which presumably reflected an incomplete DNB lesion and a consequent time-related, partial recovery of NNC. The results emphasize the potential for long-term sequelae of the DNB lesion, and the existence of a critical NNC threshold (10–30% of control NNC values) which modulates postsynaptic α₂ and β₁-adrenoceptor density.