APOE genotype and biological sex regulate astroglial interactions with amyloid plaques in Alzheimer's disease mice.

The most significant genetic risk factor for developing late-onset Alzheimer's disease (AD) is the ε4 allele of apolipoprotein E (APOE4). APOE genotype and biological sex are key modulators of microglial and astroglial function, which exert multiple effects on AD pathogenesis. Here, we show astroglial interactions with amyloid plaques in the EFAD transgenic mouse model of AD. Using confocal microscopy, we observed significantly lower levels of astrocytic plaque coverage and plaque compaction (beneficial effects of glial barrier formation) with APOE4 genotype and female sex. Conversely, neurite damage and astrocyte activation in the plaque environment were significantly higher in APOE4 carriers and female mice. Astrocyte coverage of plaques was highest in APOE3 males and poorest in APOE4 females. Collectively, our findings provide new insights into the roles of astroglia and highlight the importance of addressing independent and interactive effects of APOE genotype and biological sex in understanding processes contributing to AD pathogenesis.

APOE genotype and sex affect microglial interactions with plaques in Alzheimer's disease mice.

Microglia affect Alzheimer's disease (AD) pathogenesis in opposing manners, by protecting against amyloid accumulation in early phases of the disease and promoting neuropathology in advanced stages. Recent research has identified specific microglial interactions with amyloid plaques that exert important protective functions including attenuation of early pathology. It is unknown how these protective microglial interactions with plaques are affected by apolipoprotein E (APOE) genotype and sex, two well-established AD risk factors that modulate microglial function. We investigated this question using quantitative confocal microscopy to compare microglial interactions with amyloid plaques in male and female EFAD mice across APOE3 and APOE4 genotypes at 6 months of age. We observed that microglial coverage of plaques is highest in male APOE3 mice with significant reductions in coverage observed with both APOE4 genotype and female sex. Plaque compaction, a beneficial consequence of microglial interactions with plaques, showed a similar pattern in which APOE4 genotype and female sex were associated with significantly lower values. Within the plaque environment, microglial expression of triggering receptor expressed on myeloid cells 2 (TREM2), a known regulator of microglial plaque coverage, was highest in male APOE3 mice and reduced by APOE4 genotype and female sex. These differences in plaque interactions were unrelated to the number of microglial processes in the plaque environment across groups. Interestingly, the pattern of amyloid burden across groups was opposite to that of microglial plaque coverage, with APOE4 genotype and female sex showing the highest amyloid levels. These findings suggest a possible mechanism by which microglia may contribute to the increased AD risk associated with APOE4 genotype and female sex.

Norepinephrine induces BDNF and activates the PI-3K and MAPK cascades in embryonic hippocampal neurons.

Both antidepressant treatment and physical exercise have been shown to increase circulating levels of norepinephine (NE) and hippocampal brain-derived neurotrophic factor (BDNF). Increases in BDNF have been shown to be associated with enhanced dendritic arborization and neuronal survival, which forms the theoretical basis of the Neurotrophin Hypothesis of antidepressant action. Using isolated embryonic hippocampal neurons and immunoblotting, we show that application of NE increases BDNF and phosphorylated Trk, and that these increases can be prevented by ERK and PI-3K inhibitors. In addition, NE-induced increases in phospho-ERK2 and PI-3K were each suppressed by a PI-3K and MAPK inhibitor, respectively. Furthermore, phosphorylation of cAMP-response element binding (CREB) protein was also increased by NE and brought down to baseline levels by MAPK and PI-3K inhibitors. And finally, because both the MAPK and PI-3K inhibitors suppress phosphorylation of both TrkB (upstream) and CREB (downstream), these results indicate that NE-induced BDNF expression follows a cyclic pathway, reminiscent of a positive feedback loop. The results of this study provide an in vitro model of the intracellular signaling mechanisms activated by NE, via ligand-G-protein-coupled receptor (GPCR)-to-BDNF-RTK transactivation, that is putatively thought to occur in vivo as a result of excitatory neural activity.

beta-Amyloid neurotoxicity: a discussion of in vitro findings.

Significant advances in Alzheimer's disease (AD) research require definitive, reproducible findings from all employed paradigms. Recently, the existing in vitro data addressing the possible contribution of beta-amyloid protein to AD neuropathology have been the subject of controversy. We summarize and interpret existing data and discuss relevant methodological issues. We suggest that in vitro data support the conclusion that beta-amyloid peptides decrease the viability of cultured neurons and that this effect can be enhanced by subsequent insults.

Beta-amyloid deposition and other measures of neuropathology predict cognitive status in Alzheimer's disease.

The relationship between progressive cognitive decline and underlying neuropathology associated with Alzheimer s disease (AD) is a key issue in defining the mechanisms responsible for functional loss. This has been a subject of much controversy, with separate studies comparing various clinical and neuropathological indices in AD. Further, it is difficult to compare studies with differences in histochemical staining protocols, brain regions examined, and data quantification criteria. There are many difficulties in designing a clinical-pathological correlative study involving AD patients. It is necessary to control for several key parameters. For example, a broad range of cognitively impaired subjects is needed, as well as short postmortem delays, brief intervals between cognitive testing and death, and the most sensitive detection and quantification techniques. In this study, we carefully controlled for each of these parameters to determine if there is a relationship between global cognitive dysfunction and multiple neuropathological indices. We selected 20 individuals representing a broad range of cognitive ability from normal to severely impaired based on the MMSE, Blessed IMC, and CDR. We counted plaque number, NFT number, dystrophic neurite number, and the relative extent of thioflavine positive plaques and neuritic involvement within plaques. We also quantified cortical area occupied by beta-amyloid immunoreactivity (A beta Load) and PHF-1 positive neuropil threads and tangles (PHF Load) using computer-based image analysis. Interestingly, we found that most pathologic measures correlated highly with the severity of dementia. However, the strongest predictor of premortem cognitive dysfunction on all three cognitive measures was the relative area of entorhinal cortex occupied by beta-amyloid deposition. In conclusion, our data show that in a carefully controlled correlative study, a variety of neuropathological variables are strongly correlated with cognitive impairment. Plaque related variables may be as strongly related to cognitive dysfunction as other established measures, including synapse loss, cell death and tau hyperphosphorylation, although no correlative study can demonstrate causality.

Alzheimer-associated presenilin-2 confers increased sensitivity to apoptosis in PC12 cells.

Presenilin-2 is a gene of unknown function recently identified based upon linkage with some forms of familial Alzheimer's disease. To investigate potential effects of PS-2 on cell viability, rat pheochromocytoma (PC12) cells were stably transfected with cDNA constructs encoding either full-length human PS-2 or, for comparison, mouse Bcl-X(L). Overexpression of PS-2 conferred increased sensitivity to the apoptotic stimuli staurosporine and hydrogen peroxide. In contrast, Bcl-X(L) overexpression significantly reduced cell death induced by these stimuli. These results suggest that one function of PS-2 may involve modulation of cell viability.

Cultured GABA-immunoreactive neurons are resistant to toxicity induced by beta-amyloid.

Neurodegeneration in Alzheimer's disease is characterized by a selective loss of particular cell populations. Several recent lines of evidence suggest that beta-amyloid protein directly contributes to the disease's progression and is likely responsible for the observed pattern of neuronal death. We have previously demonstrated that aggregated beta-amyloid peptides are neurotoxic to cultured neurons. We now report that a neuronal population exhibiting GABA-immunoreactivity is resistant to beta-amyloid-induced toxicity in vitro, a finding consistent with observations in the Alzheimer brain. Determination of the intrinsic neuronal characteristics responsible for resistance to beta-amyloid may prove beneficial in both understanding the mechanism(s) of beta-amyloid neurotoxicity and halting the disease's progressive neuronal degeneration.

Androgens modulate neuronal vulnerability to kainate lesion.

Testosterone has been shown to have multiple beneficial effects on neuronal viability in developing and adult animals. Most often, testosterone promotes neural health indirectly via enzymatic conversion to estradiol by aromatase. Unclear is whether androgens can directly modulate vulnerability to neuronal insults in adult animals. We investigated this issue by modulating androgen status in rats prior to challenge with the excitotoxin kainate. Adult male rats were maintained in the following conditions: i) gonadectomized (GDX) to deplete endogenous androgens, ii) GDX+replacement with dihydrotestosterone (DHT) the active and non-aromatizable testosterone metabolite, iii) sham-GDX. Animals were then lesioned with kainate and surviving hippocampal neurons quantified. In the CA2/3 and hilar regions of the hippocampus, a modest lesion was observed in sham-GDX animals corresponding to approximately 25% cell loss in comparison to non-lesioned rats. The depletion of endogenous androgens by GDX significantly augmented lesion severity, consistent with the hypothesis that androgens are involved in maintaining cell viability. Importantly, DHT hormone replacement in GDX rats significantly attenuated kainate-induced neuron loss in CA2/3, suggesting direct androgen neuroprotection. These results demonstrate that androgens act as endogenous modulators of neuron viability, a function that may be compromised in aging men as a consequence of normal, age-related androgen depletion.

Beta-amyloid-induced changes in cultured astrocytes parallel reactive astrocytosis associated with senile plaques in Alzheimer's disease.

One neuropathological characteristic of Alzheimer's disease is an abundance of reactive astrocytes, particularly in association with senile plaques. Neither the factor(s) responsible for initiating the reactive astrocytosis nor the effects of this event on disease progression are known. We investigated the possibility that beta-amyloid protein, the primary constituent of plaques, contributes to reactive astrocytosis by comparing results derived from both culture studies and immunohistochemical analyses of Alzheimer brain tissue. We report that beta-amyloid peptides, in an aggregation-dependent manner, rapidly induce a reactive phenotype in cultured rat astrocytes. Reactive morphological changes are accompanied by increased immunoreactivities for glial fibrillary acidic protein and basic fibroblast growth factor. Although toxic to other types of central nervous system cells, aggregated beta-amyloid peptides do not significantly decrease astrocyte viability. Rather, the processes of cultured astrocytes envelop aggregated deposits of beta-amyloid peptide. In Alzheimer brain, the processes of reactive astrocytes were also observed to engulf beta-amyloid deposits. Similar to the in vitro findings, the astrocytic response was associated only with beta-amyloid plaques exhibiting an aggregated structure. Further, the plaque-associated reactive astrocytes showed enhanced immunoreactivities for glial fibrillary acidic protein and basic fibroblast growth factor. These data suggest that beta-amyloid which has assembled into beta-sheet fibrils significantly contributes to the reactive astrocytosis characteristic of Alzheimer's disease. Thus, in addition to its hypothesized direct effects on neuronal viability, beta-amyloid may also influence disease progression indirectly via reactive astrocytosis.

beta-Amyloid induces neuritic dystrophy in vitro: similarities with Alzheimer pathology.

beta-Amyloid protein, the major component of neuritic plaques found in Alzheimer's disease, has been implicated as a potential contributor to the disease's progressive neuropathology. We report that within a two day exposure to aggregates of synthetic beta-amyloid peptide, the neurites of cultured rat hippocampal neurons adopt a dystrophic appearance. Observed morphological changes in the neurites include beading, fragmentation, terminal swelling and tortuous growth patterns. The degenerative changes are similar to those observed in neurites associated with neuritic plaques, suggesting that beta-amyloid may induce the neuritic abnormalities of Alzheimer neuropathology.

Estrogen regulates bcl-x expression in rat hippocampus.

In this study, we examined whether experimental alterations of circulating estrogen levels are associated with changes in the expression of bcl-x, an inhibitor of apoptosis. We report that bcl-x mRNA expression in rat hippocampus significantly decreases after reduction of estrogen levels resulting from ovariectomy. Exposure of ovariectomized rats to 17beta-estradiol for either 5 or 28 days restored bcl-x mRNA expression to levels at or above those observed in sham-ovariectomized control animals. These data demonstrate that physiological levels of estrogen regulate hippocampal expression of bcl-x, an important modulator of neuronal apoptosis. Estrogen-mediated regulation of bcl-x may be relevant to the maintenance of neuronal viability and may contribute to the mechanism of estrogen neuroprotection.

Testosterone attenuates beta-amyloid toxicity in cultured hippocampal neurons.

Accumulating evidence suggests that testosterone has neurotrophic and perhaps neuroprotective actions. Thus, age-related depletion of testosterone may increase the brain's vulnerability to Alzheimer's disease and related disorders. To begin investigating this issue, cultured neurons were exposed to the Alzheimer-related insult beta-amyloid in the presence of testosterone. beta-Amyloid neurotoxicity was significantly reduced by testosterone via a rapid, estrogen-independent mechanism. These data may provide additional insight into the treatment of age-related neurodegenerative disorders.

Calretinin-immunoreactive neurons are resistant to beta-amyloid toxicity in vitro.

The molecular pathways by which beta-amyloid protein (A beta) induces neurotoxicity in vitro are unknown. We report that cultured hippocampal neurons exhibiting immunoreactivity for the calcium binding protein calretinin are relatively resistant to degeneration resulting from exposure to either beta 25-35 or beta 1-42. These findings suggest that intrinsic characteristics of calretinin cells, possibly including enhanced calcium buffering capacity, underlie the resistance of these cells to A beta toxicity in vitro and perhaps similar insults in Alzheimer's disease.

beta-Amyloid peptides induce degeneration of cultured rat microglia.

Microglia are often associated with senile plaques, a primary pathological hallmark of Alzheimer's disease (AD) that consists largely of insoluble deposits of beta-amyloid (A beta) protein. Synthetic A beta peptides have been shown to induce neurite dystrophy and neuronal death in vitro when the peptides are assembled into aggregates. We now report that assembled A beta peptides induce morphological evidence of degeneration in process-bearing microglia in vitro, as well as metabolic dysfunction in microglial cultures, but a non-assembling scrambled sequence A beta peptide does not.

In vitro aging of beta-amyloid protein causes peptide aggregation and neurotoxicity.

beta-Amyloid peptide forms the senile plaques of Alzheimer's disease and has been previously demonstrated to have both trophic and toxic effects on neurons in vitro. We report here that synthetic beta-amyloid peptide shows both aggregation and neurotoxicity after a 2-4 day incubation period, but is neurite-promoting and not toxic in its initially solubilized state. SDS-PAGE characterization shows that newly solubilized beta-amyloid is predominantly monomeric whereas incubated peptide has several high molecular weight species.

Surface determinants of neuronal survival and growth on self-assembled monolayers in culture.

We have studied the modulation of hippocampal neuron morphological development in vitro using surfaces derivatized with aminosilane self-assembled monolayers (SAMs). The efficacies of model SAMs, alone, or in combination with adsorbed heparan sulfate glycosaminoglycan (HS), are related to the physical and chemical properties of the surfaces. These properties are determined using X-ray photoelectron spectroscopy (XPS), optical ellipsometry, and wettability measurements. The ability of surfaces to promote somal adhesion and the maintenance of discrete neurites appears to be sensitive to the density and accessibility of positively charged amine or amide groups, and has less of an apparent relationship to the surface density of uncharged amines. Aromatic ring-containing aminosilanes are ineffective in promoting neuron growth, while adsorbed HS augments the neurite-promoting capacity of one marginally adhesive SAM. These results are relevant to an improved understanding of the 'non-specific' contributions of the substrate in affecting neuronal development and the rational design of model surface coatings for neuronal culture.

Ultrastructural analysis of beta-amyloid-induced apoptosis in cultured hippocampal neurons.

Following treatment with the beta-amyloid (A beta) 25-35 analog, scanning and transmission electron microscopy were used to investigate the morphological changes in cultured hippocampal neurons during the course of degeneration. Ultrastructural analysis revealed focal cell surface blebbing and rapid condensation of nuclear chromatin. Changes in cytoplasmic morphology included prominent vacuole formation, dispersal of polyribosome rosettes and the disappearance of the golgi complex, smooth endoplasmic reticulum and microtubules with increased cytoplasmic electron density. Mitochondria and limited rough endoplasmic reticulum remained intact throughout the process of cell death. These results provide additional evidence suggesting A beta-induced cell death in vitro occurs via an apoptotic mechanism.

Androgens selectively protect against apoptosis in hippocampal neurones.

Androgens can protect neurones from injury, although androgen neuroprotection is not well characterised in terms of either specificity or mechanism. In the present study, we compared the ability of androgens to protect neurones against a panel of insults, empirically determined to induce cell death by apoptotic or non-apoptotic mechanisms. Three criteria defining but not inclusive of apoptosis are: protection by caspase inhibition, protection by protein synthesis inhibition and the presence of pyknotic nuclei. According to these criteria, beta-amyloid, staurosporine, and Apoptosis Activator II induced cell death involving apoptosis, whereas hydrogen peroxide (H(2)O(2)), iron, calcium ionophore and 3-nitropropionic acid induced cell death featuring non-apoptotic characteristics. Pretreatment of hippocampal neurones with testosterone or dihydrotestosterone attenuated cell death induced by beta-amyloid, staurosporine and Apoptosis Activator II, but none of the other insults. The anti-oxidant Trolox did not reduce cell death induced by beta-amyloid, staurosporine and Apoptosis Activator II, but did protect against H(2)O(2) and iron. Similarly, a supra-physiological concentration of oestrogen reduced cell death induced by H(2)O(2) and iron, an effect not observed with androgens. We also show that activation of oestrogen pathways was not necessary for androgen neuroprotection. These data suggest that androgens directly activate a neuroprotective mechanism specific to inhibition of cell death involving apoptosis. Determining the specificity of androgen neuroprotection may enable the development of androgen compounds for the treatment of neurodegenerative disorders.