RESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer , Placa Amiloide , Femenino , Masculino , Ratones , Animales , Placa Amiloide/genética , Astrocitos , Enfermedad de Alzheimer/genética , Apolipoproteína E4 , Apolipoproteínas E/genética , Genotipo , Ratones TransgénicosRESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Apolipoproteína E3/genética , Apolipoproteína E4/genética , Encéfalo/patología , Microglía/patología , Placa Amiloide/patología , Animales , Femenino , Genotipo , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones Transgénicos , Receptores Inmunológicos/metabolismo , Caracteres SexualesRESUMEN
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.
Asunto(s)
Andrógenos/farmacología , Apoptosis/efectos de los fármacos , Citoprotección/efectos de los fármacos , Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Animales , Animales Recién Nacidos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Evaluación Preclínica de Medicamentos , Embrión de Mamíferos , Hipocampo/fisiología , Neuronas/fisiología , Especificidad de Órganos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Estaurosporina/farmacologíaRESUMEN
Recent findings indicate that progesterone can attenuate the beneficial neural effects of oestrogen. In the present study, we investigated the hypothesis that progesterone can modulate oestrogen actions by regulating the expression and activity of oestrogen receptors, ERalpha and ERbeta. Our studies in cultured neurones demonstrate that progesterone decreases the expression of both ERalpha and ERbeta and, as a consequence, also reduces both ER-dependent transcriptional activity and neuroprotection. These results identify a potential mechanism by which progesterone antagonises neural oestrogen actions, a finding that may have important implications for hormone therapy in postmenopausal women.
Asunto(s)
Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Estrógenos/metabolismo , Regulación de la Expresión Génica , Neuronas/metabolismo , Fármacos Neuroprotectores/metabolismo , Progesterona/metabolismo , Animales , Células Cultivadas , Regulación hacia Abajo , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Femenino , Humanos , Neuronas/citología , RatasRESUMEN
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.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Neuronas/metabolismo , Norepinefrina/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Embrión de Mamíferos/citología , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Femenino , Hipocampo/citología , Neuronas/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Embarazo , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Receptor trkB/metabolismo , Transducción de SeñalRESUMEN
As a normal consequence of aging, men experience a significant decline in androgen levels. Although the neural consequences of age-related androgen depletion remain unclear, recent evidence suggests a link between low androgen levels and the development of Alzheimer's disease (AD). Here, we test the hypothesis that androgens act as endogenous modulators of beta-amyloid protein (Abeta) levels. To investigate this possibility, brain and plasma levels of Abeta were measured in male rats with varying hormonal conditions. Depletion of endogenous sex steroid hormones via gonadectomy (GDX) resulted in increased brain levels of Abeta in comparison to gonadally intact male rats. This GDX-induced increase in Abeta levels was reversed by DHT supplementation, demonstrating a functional role for androgens in modulating brain levels of Abeta. These findings suggest that age-related androgen depletion may result in accumulation of Abeta in the male brain and thereby act as a risk factor for the development of AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Andrógenos/fisiología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Péptidos beta-Amiloides/sangre , Andrógenos/administración & dosificación , Animales , Dihidrotestosterona/administración & dosificación , Implantes de Medicamentos , Estradiol/administración & dosificación , Terapia de Reemplazo de Hormonas , Masculino , Orquiectomía , Ratas , Ratas Sprague-Dawley , Receptores Androgénicos/metabolismo , Núcleos Septales/citología , Núcleos Septales/efectos de los fármacos , Núcleos Septales/metabolismoRESUMEN
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.
Asunto(s)
Andrógenos/farmacología , Dihidrotestosterona/farmacología , Agonistas de Aminoácidos Excitadores/toxicidad , Ácido Kaínico/toxicidad , Neuronas/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , Recuento de Células , Supervivencia Celular/efectos de los fármacos , Interacciones Farmacológicas , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Inmunohistoquímica/métodos , Masculino , Neuronas/metabolismo , Orquiectomía/métodos , Fosfopiruvato Hidratasa/metabolismo , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/efectos de los fármacos , Receptores Androgénicos/metabolismo , Convulsiones/inducido químicamente , Convulsiones/prevención & control , Tabique del Cerebro/citología , Tabique del Cerebro/efectos de los fármacos , Tabique del Cerebro/metabolismoRESUMEN
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.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Hormonas Esteroides Gonadales/farmacología , Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Testosterona/farmacología , Animales , Animales Recién Nacidos , Muerte Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Hipocampo/citología , Neuronas/citología , Ratas , Ratas Sprague-DawleyRESUMEN
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.
Asunto(s)
Apoptosis/genética , Estradiol/sangre , Regulación de la Expresión Génica/fisiología , Hipocampo/metabolismo , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Estradiol/farmacología , Femenino , Hipocampo/citología , Hipocampo/efectos de los fármacos , Degeneración Nerviosa/genética , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/fisiopatología , Fármacos Neuroprotectores/metabolismo , Fármacos Neuroprotectores/farmacología , Ovariectomía , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Proteína bcl-XRESUMEN
We investigated the possibility that estrogen and exercise interact in the hippocampus and regulate brain-derived neurotrophic factor (BDNF), a molecule increasingly recognized for its role in plasticity and neuron function. An important aspect of this study is to examine the effect of different time intervals between estrogen loss and estrogen replacement intervention. We demonstrate that in the intact female rat, physical activity increases hippocampal BDNF mRNA and protein levels. However, the exercise effect on BDNF up-regulation is reduced in the absence of estrogen, in a time-dependent manner. In addition, voluntary activity itself is stimulated by the presence of estrogen. In exercising animals, estrogen deprivation reduced voluntary activity levels, while estrogen replacement restored activity to normal levels. In sedentary animals, estrogen deprivation (ovariectomy) decreased baseline BDNF mRNA and protein, which were restored by estrogen replacement. Despite reduced activity levels in the ovariectomized condition, exercise increased BDNF mRNA levels in the hippocampus after short-term (3 weeks) estrogen deprivation. However, long-term estrogen-deprivation blunted the exercise effect. After 7 weeks of estrogen deprivation, exercise alone no longer affected either BDNF mRNA or protein levels. However, exercise in combination with long-term estrogen replacement increased BDNF protein above the effects of estrogen replacement alone. Interestingly, protein levels across all conditions correlated most closely with mRNA levels in the dentate gyrus, suggesting that expression of mRNA in this hippocampal region may be the major contributor to the hippocampal BDNF protein pool. The interaction of estrogen, physical activity and hippocampal BDNF is likely to be an important issue for maintenance of brain health, plasticity and general well-being, particularly in women.
Asunto(s)
Envejecimiento/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Estrógenos/deficiencia , Regulación de la Expresión Génica/fisiología , Hipocampo/metabolismo , Menopausia/metabolismo , Condicionamiento Físico Animal/fisiología , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Ensayo de Inmunoadsorción Enzimática , Terapia de Reemplazo de Estrógeno , Estrógenos/sangre , Estrógenos/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Recent findings indicate that estrogen is neuroprotective, a cellular effect that may contribute to its clinical benefits in delaying the development of Alzheimer's disease. In this report, we identify a novel neuronal action of estrogen that may contribute to its neuroprotective mechanism(s). Specifically, we report that estrogen significantly increases the expression of the antiapoptotic protein Bcl-xL in cultured hippocampal neurons. This effect presumably reflects classic estrogen transcriptional regulation, as we identified a putative estrogen response element in the bcl-x gene. Estrogen-induced enhancement of Bcl-xL is associated with a reduction in measures of beta-amyloid-induced apoptosis, including inhibition of both caspase-mediated proteolysis and neurotoxicity. A similar relationship between estrogen, Bcl-xL expression, and resistance to degeneration was also observed in human hippocampus. We report neuronal colocalization of estrogen receptor and Bcl-xL immunoreactivities that is most prominent in hippocampal subfield CA3, a region that shows relatively little immunoreactivity to paired helical filament-1, a marker of Alzheimer's disease neurodegeneration. These data suggest a novel mechanism of estrogen neuroprotection that may be relevant to estrogen's suggested ability to modulate neuronal viability across the life span, from neural sexual differentiation and development through age-related neurodegenerative conditions.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Apoptosis/efectos de los fármacos , Estradiol/farmacología , Neuronas/citología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Anciano , Anciano de 80 o más Años , Péptidos beta-Amiloides/metabolismo , Animales , Anticuerpos , Química Encefálica/efectos de los fármacos , Caspasas/metabolismo , Células Cultivadas , Expresión Génica/efectos de los fármacos , Hipocampo/química , Hipocampo/citología , Humanos , Inmunidad Innata , Masculino , Persona de Mediana Edad , Neuronas/efectos de los fármacos , Neuronas/enzimología , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Ratas , Ratas Sprague-Dawley , Receptores de Estrógenos/análisis , Receptores de Estrógenos/inmunología , Proteína bcl-XRESUMEN
Recent data from several groups suggest that the primary mechanism of beta-amyloid neurotoxicity may be mediated by reactive oxygen species. To evaluate this hypothesis, we first compared the efficacy of antioxidant agents in preventing toxicity caused by oxidative insults (iron, hydrogen peroxide, and tert-butyl hydroperoxide) and beta-amyloid peptides in cultured rat hippocampal neurons. Tested antioxidants (propyl gallate, Trolox, probucol, and promethazine) generally provided significant protection against oxidative insults but not beta-amyloid peptides. Next, we examined whether beta-amyloid causes oxidative stress, by comparing levels of lipid peroxidation after exposure to either iron or beta-amyloid. In a cell-free system, iron but not beta-amyloid generated lipid peroxidation. In culture, both insults caused rapid increases in lipid peroxidation, with iron inducing higher levels at later time points. Pretreatment with the antioxidant probucol significantly reduced lipid peroxidation caused by both insults but only attenuated iron toxicity, suggesting that lipid peroxidation does not contribute directly to cell death induced by beta-amyloid. Finally, we observed that increasing basal levels of oxidative stress by pretreating cultures with subtoxic doses of iron significantly increased neuronal vulnerability to beta-amyloid. The ability of beta-amyloid to induce oxidative stress and the demonstration that oxidative stress potentiates beta-amyloid toxicity support the clinical use of antioxidants for AD. However, these data do not support the theory that the primary mechanism of beta-amyloid toxicity involves oxidative pathways, indicating a continued need to identify additional cellular responses to beta-amyloid that underlie its neurodegenerative actions.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Antioxidantes/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neurotoxinas/farmacología , Estrés Oxidativo/fisiología , Animales , Muerte Celular/efectos de los fármacos , Hierro/farmacología , Peróxidos Lipídicos/metabolismo , Ratas/embriologíaRESUMEN
Thrombin activity is a factor in acute CNS trauma and may contribute to such chronic neurodegenerative diseases as Alzheimer's disease. Thrombin is a multifunctional serine protease that catalyses the final steps in blood coagulation. However, increasing evidence indicates that thrombin also elicits a variety of cellular and inflammatory responses, including responses from neural cells. Most recently, high concentrations of thrombin were shown to cause cell death in both astrocyte and hippocampal neuron cultures. The purpose of this study was to determine the mechanisms underlying thrombin-induced cell death. Our data show that thrombin appears to cause apoptosis as evidenced by cleavage of DNA into oligonucleosomal-sized fragments, fragmentation of nuclei, and prevention of death by inhibition of protein synthesis. Synthetic peptides that directly activate the thrombin receptor also induced apoptosis, indicating that thrombin-induced cell death occurred via activation of the thrombin receptor. The signal transduction cascade involves tyrosine and serine/threonine kinases and an intact actin cytoskeleton. Additional study revealed the involvement of the small GTP-binding protein RhoA. Thrombin induced RhoA activity in both astrocytes and hippocampal neurons, and inhibition of RhoA activity with exoenzyme C3 attenuated cell death, indicating that thrombin activation of RhoA was necessary for thrombin-induced cell death. Tyrosine kinase inhibitors blocked thrombin induction of RhoA, indicating that tyrosine kinase activity was required upstream of RhoA. These data suggest a sequential linkage of cellular events from which we propose a model for the second messenger cascade induced by thrombin in neural cells that can lead to apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Astrocitos/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Trombina/farmacología , Animales , Células Cultivadas/efectos de los fármacos , Humanos , Ratas , Ratas Sprague-DawleyRESUMEN
The amyloidogenic peptide beta-amyloid has previously been shown to bind to neurons in the form of fibrillar clusters on the cell surface, which induces neurodegeneration and activates a program of cell death characteristic of apoptosis. To further investigate the mechanism of Abeta neurotoxicity, we synthesized the all-D- and all-L-stereoisomers of the neurotoxic truncated form of Abeta (Abeta25-35) and the full-length peptide (Abeta1-42) and compared their physical and biological properties. We report that the purified peptides exhibit nearly identical structural and assembly characteristics as assessed by high performance liquid chromatography, electron microscopy, circular dichroism, and sedimentation analysis. In addition, both enantiomers induce similar levels of toxicity in cultured hippocampal neurons. These data suggest that the neurotoxic actions of Abeta result not from stereoisomer-specific ligand-receptor interactions but rather from Abeta cellular interactions in which fibril features of the amyloidogenic peptide are a critical feature. The promiscuous nature of these beta-sheet-containing fibrils suggests that the accumulation of amyloidogenic peptides in vivo as extracellular deposits represents a site of bioactive peptides with the ability to provide inappropriate signals to cells leading to cellular degeneration and disease.
Asunto(s)
Péptidos beta-Amiloides/química , Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Péptidos beta-Amiloides/síntesis química , Péptidos beta-Amiloides/toxicidad , Animales , Células Cultivadas , Cromatografía Líquida de Alta Presión , Isomerismo , RatasRESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/psicología , Péptidos beta-Amiloides/metabolismo , Encéfalo/patología , Manifestaciones Neuroconductuales , Placa Amiloide/patología , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Benzotiazoles , Recuento de Células , Corteza Entorrinal/patología , Femenino , Colorantes Fluorescentes , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Masculino , Ovillos Neurofibrilares/patología , Placa Amiloide/metabolismo , TiazolesRESUMEN
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.
Asunto(s)
Apoptosis , Proteínas de la Membrana/fisiología , Proteínas Proto-Oncogénicas c-bcl-2 , Enfermedad de Alzheimer , Animales , Supervivencia Celular , Expresión Génica , Humanos , Peróxido de Hidrógeno/farmacología , Proteínas de la Membrana/genética , Ratones , Células PC12 , Presenilina-2 , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Ratas , Estaurosporina/farmacología , Transfección , Proteína bcl-XRESUMEN
The cell death of cultured neurons triggered by beta-amyloid peptides has been theorized to model, at least in part, the neurodegeneration associated with Alzheimer's disease. To investigate potential strategies to interrupt beta-amyloid neurotoxicity in vitro, we examined the effects of potassium-induced membrane depolarization, a treatment previously demonstrated to reduce development-related apoptosis in cultured neurons. We report here that cultured rat hippocampal neurons pretreated for several hours with 30 mM KCl exhibit significantly reduced vulnerability to aggregated beta-amyloid peptides. The potassium-mediated neuroprotection was mimicked by activation of voltage-sensitive calcium channels using S(-)-Bay K8644 and was attenuated by R(+)-Bay K 8644, a blocker of voltage-dependent calcium channels, and KN-82, an inhibitor of calcium/calmodulin-dependent protein kinase II. The protein synthesis inhibitor cycloheximide also attenuated beta-amyloid neurotoxicity. Addition of cycloheximide following 30 mM KCl significantly increased protection offered by membrane depolarization, whereas cycloheximide addition during membrane depolarization blocked the protective effect. These data suggest that one cellular pathway that can inhibit neuronal death induced by beta-amyloid involves calcium influx through voltage-sensitive channels followed by stimulation of calcium/calmodulin-dependent protein kinase activity and synthesis of new protein(s).
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Hipocampo/patología , Neuronas/efectos de los fármacos , Neurotoxinas/toxicidad , Fragmentos de Péptidos/toxicidad , Potasio/farmacología , Enfermedad de Alzheimer/patología , Animales , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Muerte Celular/efectos de los fármacos , Células Cultivadas , Embrión de Mamíferos , Cinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas/patología , Neuronas/fisiología , Ratas , Factores de TiempoRESUMEN
Previous studies have reported that beta-amyloid peptides induce properties of reactivity in cultured astrocytes. We report here that aggregated A beta peptides increase expression of the enzyme glutamine synthetase in cultured astrocytes, as assessed by enzyme assay, Western blot analysis, and immunocytochemistry. The enhanced enzyme levels occur gradually over a period of 4 days after A beta exposure and maintain peak values for at least several days thereafter. These data suggest that A beta-related reactive astrocytosis in Alzheimer's disease brain may benefit local neurons by enhancing glial capacity to regulate levels of the excitotoxin glutamate.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Astrocitos/enzimología , Glutamato-Amoníaco Ligasa/biosíntesis , Animales , Astrocitos/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Células Cultivadas/enzimología , Corteza Cerebral/citología , Inducción Enzimática , Glutamato-Amoníaco Ligasa/metabolismo , RatasRESUMEN
beta-Amyloid protein has been implicated as a potential causative agent in the neuropathology associated with Alzheimer's disease. This possibility is supported by observations that beta-amyloid induces neuronal degeneration and astrocyte reactivity in vitro by as yet undefined mechanism(s). In this report, we present data demonstrating that the pathological effects of beta-amyloid on cultured cells are modulated by activation of the thrombin receptor. At concentrations between 50 and 500 nM, thrombin pretreatment significantly attenuates neurotoxicity mediated by fibrillar aggregates of beta 1-42 and beta 25-35 peptides. In cultured astrocytes, the stellate morphology induced by beta 1-42 and beta 25-35 aggregates can be prevented and reversed by thrombin exposures between 10 pM and 1 microM. In contrast, thrombin potentiates rather than attenuates the beta-amyloid-induced increased expression of basic fibroblast growth factor, suggesting that thrombin differentially modulates the effects of beta-amyloid on astrocytes. Thrombin's effects on both neurons and astrocytes are mimicked by thrombin receptor-activating peptide and inhibited by two potent thrombin inhibitors, hirudin and protease nexin-1. These data provide both new insight into the signaling pathways underlying the cellular effects of beta-amyloid and additional support for the role of thrombin as an important mediator of neuropathological events.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Astrocitos/citología , Neuronas/citología , Trombina/farmacología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/ultraestructura , Western Blotting , Muerte Celular/efectos de los fármacos , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/ultraestructura , Relación Dosis-Respuesta a Droga , Inmunohistoquímica , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Fármacos Neuroprotectores/farmacología , Neurotoxinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Trombina/fisiología , Transducción de Señal/fisiologíaRESUMEN
beta-Amyloid (A beta) is a 39-42 amino acid that is the primary component of plaques in Alzheimer's disease (AD). Previous studies from our laboratory and others have shown that A beta induces neurodegeneration via apoptosis in vitro, suggesting that A beta may also initiate an apoptotic pathway of cell death in AD. Apoptosis has been suggested to proceed by a gene-directed program in several systems. Accordingly, we have investigated whether A beta-mediated apoptosis is associated with the induction of genes that may regulate or play a role in cell death in vitro. Immediate early genes (IEGs) respond to cellular stimuli and participate in cellular signaling pathways. The protein products of some IEGs, e.g., c-jun, are capable of forming dimers and acting as transcriptional regulatory proteins, and have been implicated in apoptosis in both nonneuronal and neuronal cells. In this study, we report a selective and abnormally sustained induction of c-Jun in cultured hippocampal neurons treated with A beta. In addition, we describe the lack of induction of c-Jun in neurons that are relatively resistant to A beta-mediated toxicity, and a correspondence between immunoreactivity for c-Jun and changes in nuclear morphology that are indicative of apoptosis. These data demonstrate that c-Jun is induced in cultured neurons that undergo A beta-mediated apoptosis and suggest that c-Jun may participate in a cell death program in these neurons.