Estrogen protects neuronal cells from amyloid beta-induced apoptosis via regulation of mitochondrial proteins and function
© Nilsen et al; licensee BioMed Central Ltd. 2006
Received: 02 August 2006
Accepted: 03 November 2006
Published: 03 November 2006
Neurodegeneration in Alzheimer's disease is associated with increased apoptosis and parallels increased levels of amyloid beta, which can induce neuronal apoptosis. Estrogen exposure prior to neurotoxic insult of hippocampal neurons promotes neuronal defence and survival against neurodegenerative insults including amyloid beta. Although all underlying molecular mechanisms of amyloid beta neurotoxicity remain undetermined, mitochondrial dysfunction, including altered calcium homeostasis and Bcl-2 expression, are involved in neurodegenerative vulnerability.
In this study, we investigated the mechanism of 17β-estradiol-induced prevention of amyloid beta-induced apoptosis of rat hippocampal neuronal cultures. Estradiol treatment prior to amyloid beta exposure significantly reduced the number of apoptotic neurons and the associated rise in resting intracellular calcium levels. Amyloid beta exposure provoked down regulation of a key antiapoptotic protein, Bcl-2, and resulted in mitochondrial translocation of Bax, a protein known to promote cell death, and subsequent release of cytochrome c. E2 pretreatment inhibited the amyloid beta-induced decrease in Bcl-2 expression, translocation of Bax to the mitochondria and subsequent release of cytochrome c. Further implicating the mitochondria as a target of estradiol action, in vivo estradiol treatment enhanced the respiratory function of whole brain mitochondria. In addition, estradiol pretreatment protected isolated mitochondria against calcium-induced loss of respiratory function.
Therefore, we propose that estradiol pretreatment protects against amyloid beta neurotoxicity by limiting mitochondrial dysfunction via activation of antiapoptotic mechanisms.
A growing body of evidence supports the critical role of amyloid beta peptide (Aβ)2 in Alzheimer's disease (AD) pathogenesis. Early onset AD is associated with overproduction of the 42-amino acid form of Aβ (Aβ1–42) and Aβ1–42 is toxic to neurons in vitro and in vivo [1–3]. To develop therapeutic strategies for reducing neuronal loss in AD, much effort has been extended to determine the molecular interactions underlying Aβ-induced neurotoxicity. Several lines of evidence suggest that calcium plays a key role in age-related changes in the brain that lead to AD and dementia [4–7]. Free intracellular calcium is a key activator of many signal transduction pathways of neurons, and alterations in intracellular calcium homeostasis are pivotal regulators of brain aging, memory and cell death [4–6, 8–11]. According to a "calcium hypothesis" of AD, disturbances in calcium homeostasis are the proximal cause of neurodegeneration in AD, in which calcium dysfunction augments tau hyperphosphorylation, Aβ formation and neurotoxicity [4, 5, 9, 12].
Calcium (Ca2+) transients in neurons are largely determined by mitochondria due to their large Ca2+ capacity . Mitochondrial Ca2+ uptake is driven by the mitochondrial membrane potential (ΔΨm) and occurs at the threshold of cytosolic Ca2+, followed by slow release, leading to a net accumulation of mitochondrial calcium ([Ca2+]m) and an alteration of physiological [Ca2+]i transients [14, 15]. Mitochondrial dysfunction often leads to dysregulation of Ca2+ homeostasis and consequent adverse downstream effects, including further damage to mitochondria setting up the mitochondrial spiral that is associated with multiple central nervous system disorders . During aging, especially during the development and progression of neurodegenerative diseases, including AD, damaged mitochondria are unable to maintain the energy demands of the cell [17, 18]. This can lead to an increased production of free radicals, which induces the interruption of oxidative phosphorylation, resulting in decreased levels of ATP that are necessary for normal energy homeostasis . Apoptosis of degenerating neurons occurs in association with an accumulation of abnormal mitochondria in perikaryal regions and oxidative damage to the nucleus . The same pattern of mitochondria lesions is observed in human AD brain biopsy samples . Thus, therapeutic interventions that promote mitochondrial function could be a strategy to prevent neuronal dysfunction and death.
A prevention mode of estrogen exposure can prevent neurotoxicity both in vitro and in vivo [20–25]. Many of the proximate effects of estrogen treatment that have been associated with estrogen-induced neuroprotection converge upon the mitochondria [7, 26]. We have previously shown that estrogen-induced neuroprotection is mediated through regulation of calcium homeostasis [22, 26]. Further, we have shown that 17β-estradiol restores calcium homeostasis in neurons from middle-aged and old rats . That the estrogen-induced regulation of calcium homeostasis is dependent upon maintenance of mitochondrial respiration and ΔΨm  indicates a direct role of mitochondria in the neuroprotective effects of estrogen.
In the present study, we investigated the mechanisms whereby 17β-estradiol (E2) exposure can prevent Aβ-induced neuronal apoptosis and mitochondrial function. Results of these analyses indicate that E2 protects rat hippocampal neurons against Aβ toxicity and that such protection is associated with maintenance of calcium homeostasis, a decrease of cytochrome c release, a decrease of Bax translocation to the mitochondria and enhanced mitochondrial respiratory function. These data together with previously published findings indicate that E2 exposure prior to Aβ insult leads to a complex array of responses that coalesce in an organized cellular strategy to prevent loss of mitochondrial calcium homeostasis while simultaneously promoting Bcl-2 family protein strategies that prevent opening of the membrane permeability transition pore.
17β-estradiol prevents amyloid beta-induced neuronal death
17β-estradiol prevents amyloid beta1–42-induced dysregulation of calcium homeostasis
17β-estradiol prevents amyloid beta1–42-induced depletion of mitochondrial Bcl-2
17β-estradiol prevents amyloid beta1–42-induced translocation of Bax
Neurotoxicity resulting from apoptosis often results from the induction of Bax translocation from the cytosol to the mitochondria where it can mediate the release of apoptotic factors such as cytochrome c [36–39]. To determine if the neuroprotective effect of E2 is associated with regulation of Bax translocation or cytochrome c release, primary hippocampal neurons were pretreated with E2 (10 ng/mL) or vehicle control for 48 hr prior to Aβ1–42 (1.5 μM) exposure for 24 hr and assessed for Bax localization by Western blot and immunocytochemical analyses.
17β-estradiol prevents amyloid beta1–42-induced release of cytochrome c
17β-estradiol prevents neurotoxic-induced decline in mitochondria respiration
To determine if E2-treatment serves a protective function against increased calcium loads, mitochondrial respiratory function was assessed following a 2 min calcium exposure in the presence of glutamate and malate. Ovariectomized adult rats were administered E2 (30 μg/kg in sesame oil) or vehicle control (sesame oil) 24 hr prior to mitochondrial isolation from whole brain tissue. Brain mitochondria from E2-treated rats displayed significantly greater oxygen consumption following Ca2+ challenge in State 3 respiration and a significantly higher RCR than mitochondria from control rats (Fig. 8C; p < 0.05; n = 7). Further there was a smaller decrease in the RCR values following Ca2+ challenge in the brain mitochondria from E2-treated rats than control (Fig. 8C; p < 0.05; n = 7).
Neurodegenerative diseases, like Alzheimer's disease, are associated with disruption of calcium homeostasis and mitochondrial dysfunction leading to apoptotic events and neuronal cell death. Previously we demonstrated that 17β-estradiol protects against age-related calcium dysregulation . In this study we show that estrogen pretreatment prevents amyloid beta-induced calcium dysregulation, mitochondrial failure and resultant apoptosis in hippocampal neuronal cultures. These results are consistent with previous data that demonstrate that estrogen is neuroprotective against Aβ-induced neurotoxicity [21, 27, 40], but further the field by providing evidence for the underlying mechanism of E2-induced neuroprotection through regulation of mitochondrial signals that initiate and activate apoptotic processes.
During aging, especially during the development and progression of neurodegenerative diseases, including Alzheimer's disease (AD), damaged mitochondria are unable to maintain the energy demands of the cell. Interrupted energy metabolism is observed in many instances of neurodegeneration [16, 18, 19, 41, 42], including cerebral ischemia and Alzheimer's disease , two neurological conditions that account for the majority of all neurodegenerative conditions. Reductions in cerebral metabolic rate often occur before the development of clinical disabilities. Impairment of oxidative energy metabolism leads to increased expression of amyloid precursor protein (APP)  and to cytoskeletal disorganization, including the appearance of epitopes associated with paired helical filaments/tangles [43, 44]. This can lead to an increased production of free radicals, which induces the interruption of oxidative phosphorylation, resulting in decreased levels of ATP that are necessary for normal energy homeostasis. Subsequently, cerebral metabolism may be unable to meet the energy demands required to restore dissipated membrane potentials, as ATP in the injured brain is significantly reduced. Furthermore, a limited energy supply is evident from the reductions in mitochondrial oxygen consumption, mitochondrial membrane potential, and mitochondrial enzymatic activity. In this study we demonstrated that E2 prevented the neurotoxic-induced decline in mitochondrial respiratory function. This is consistent with the previous reports that E2 is protective against cell death induced by energy depletion  and blocks the decrease in mitochondrial membrane potential induced by Aβ in PC12 cells . Thus E2 may be able to prevent interruption of neuronal energy metabolism associated with neurodegeneration and may reduce APP expression and paired helical filament formation.
Underlying the neurodegeneration-associated mitochondrial dysfunction appears to be dysregulation of calcium homeostasis. Several lines of evidence suggest that calcium plays a key role in age-related changes in the brain that lead to AD and dementia. Free intracellular calcium is one of the most important messengers for many signal transduction pathways of neurons, and alterations in intracellular calcium homeostasis are critically involved in brain aging, memory and cell death. Landfield's group showed that altered intracellular calcium is directly correlated with impaired neuronal plasticity such that elevated intracellular calcium and frequency facilitation were negatively correlated in individual old neurons within hippocampal slices . This finding led these investigators to postulate that intracellular calcium is likely elevated in old hippocampal neurons and frequency facilitation would thus be impaired in old hippocampal neurons during the theta frequencies associated with cognitive processing. Consistent with this postulate are recent in vivo data from studies in old rats. Foy and colleagues found that E2 suppressed the calcium-dependent induction of long-term depression in CA1 hippocampal neurons of old rats . A later analysis by Foster and co-workers showed that E2 decreased the Ca2+-activated afterhyperpolarization which is larger in old rats compared to young rat CA1 neurons and is enhanced by a higher density of L-type calcium channels in the old rat neuron . More recently we have shown that estrogen can reverse the age-associated calcium dysregulation in primary neuronal cultures obtained from the hippocampus of aged rats .
According to a "calcium hypothesis" of AD, arising from numerous preclinical in vitro studies, disturbances in calcium homeostasis are the proximal cause of neurodegeneration in AD . There is a large body of evidence from preclinical experimental models and from human subjects that alterations in calcium signalling occur during initial phases of AD, even before the development of overt symptoms or any obvious extracellular amyloid-beta pathology . In the current studies, we showed that E2 pretreatment prevented the Aβ-induced rise in resting calcium concentration in primary hippocampal neurons, an effect similar to the E2-mediated reduction in calcium rise induced by excitotoxic glutamate [22, 26]. More recently we have shown that estrogen can reverse the age-associated calcium dysregulation in primary neuronal cultures obtained from the hippocampus of aged rats . A mitochondrial site of action for estrogen-mediated neuroprotection is supported by the functional mitochondria dependence of the attenuation of the glutamate-induced Ca2+ rise .
One mechanism by which estrogen may regulate mitochondrial calcium homeostasis and maintain energy metabolism is via the antiapoptotic protein Bcl-2. The magnitude of Ca2+ accumulation by mitochondria can be altered by Bcl-2 [29, 30], which is localized to the mitochondrial membrane and its expression has been shown to significantly enhance mitochondrial Ca2+ sequestration [29, 30, 50]. In the current studies we demonstrated that E2 increases, and prevents the Aβ-induced decrease in, the mitochondrial expression of Bcl-2, consistent with previous reports of E2-induced increases in Bcl-2 in reproductive tissues and brain [22, 32–35, 51]. In addition to increasing the magnitude of Ca2+ sequestered by mitochondria, Bcl-2 enhances the tolerability of mitochondria for increased levels of [Ca2+]i that otherwise result in dissipation of ΔΨm and cell death . Consistent with the increase in mitochondrial Ca2+ load tolerability demonstrated here, Mattson et al. showed that a supraphysiological concentration of E2 (10 μM) can preserve ΔΨm in PC12 cells expressing mutant presenilin that were exposed to Aβ . We propose that by increasing [Ca2+]m uptake capacity, and the Bcl-2-induced resistance to Ca2+-induced respiratory inhibition, E2 prevents Bax translocation and cytochrome c release, limiting the loss of viability initiated by neurotoxic insults.
In summary, we have shown that E2 provides neuroprotection in rat hippocampal neurons subjected to Aβ toxicity and that such protection is associated with maintenance of calcium homeostasis, a decrease of cytochrome c release, a decrease of Bax translocation to the mitochondria and enhanced mitochondrial respiratory function. Taken together these data indicate that mechanisms of estrogen-inducible neuroprotection against degenerative insults are a function of estrogen activation of cellular mechanisms whose ultimate outcome is promotion of mitochondrial viability. Thus mitochondria are ideal therapeutic targets of estrogen and estrogen-like surrogates in brain. Further elucidation of the mitochondrial sites of estrogen action will allow for development of selective therapeutic agents for use in estrogen therapy for prevention of neurodegenerative diseases.
Culture materials were from Gibco BRL (Rockville, MD). Chemicals were from MP Biomed (Irvine, CA) unless otherwise noted. Steroids were dissolved in ethanol and diluted in culture medium with final ethanol concentration <0.001%. Fura2-AM, Calcein AM and ethidium homodimer-1 were from Molecular Probes (Eugene, OR). Amyloid beta1–42 was from American Peptide Company (Sunnyvale, CA).
Hippocampal neurons from embryonic day 18 (E18) rat fetuses were cultured as previously described and generated cultures 98% neuronal in phenotype . Briefly, embryonic rat hippocampi were dissociated by passage through fire-polish constricted Pasteur pipettes. Neurons plated on poly-d-lysine coated coverslips (22 mm round), chamberslides (Falcon; 4 well) or polyethylenimine coated 6-well plates were grown in Neurobasal medium supplemented with 5 U/ml penicillin, 5 mg/ml streptomycin, and B27 supplement at 37°C in humidified 5% CO2 atmosphere for 10–12 days prior to experimentation.
Amyliod beta-induced injury
Neurons were pretreated with 17β-estradiol (10 ng/mL) or vehicle control for 48 hr prior to exposure to fibrillar amyloid beta (1.5 μM) for the indicated times. Fibrillar Aβ1–42 was prepared by solubilizing Aβ1–42 in HCL (10 mM) at a concentration of 1 mM. Aβ1–42 was diluted in PBS to 100 μM and incubated for 72 hr at 22°C . At the beginning of each experiment Aβ1–42 was further diluted to 1.5 μM in complete Neurobasal medium.
Cell viability was measured by calcein/ethidium homodimer staining. Neurons were incubated in phosphate buffered saline (PBS) containing calcein AM (1 μM) and ethidium homodimer-1 (2 μM) for 30 min at room temperature. Fluorescent intensity was measured using a dual-wavelength fluorescent plate reader (GeniosPro; Molecular Devices) at 485/530 nm Ex/Em and 530/645 nm Ex/Em for calcein and ethidium, respectively. Data represents percent live or dead cells normalized to control fluorescent values. Data is presented as mean ± S.E.M. from 4 independent experiments with 8 wells per condition per experiment. Images were captured using the Marianas imaging system with Slidebook software (Intelligent Imaging Innovations, Inc., Santa Monica, CA) based on a Zeiss 200 M inverted microscope.
Neurons grown on chamber slides were treated as for Neuronal Survival above and fixed in 4% paraformaldehyde for 15 at room temperature and processed for TUNEL staining by the In Situ Cell Death Detection Kit, Fluorescein kit (Roche Applied Science; Indianapolis, IN) according to the manufacturer's instructions. Neurons were counterstained with DAPI to label nuclei. Three random fields per well were captured using the Marianas imaging system with Slidebook software (Intelligent Imaging Innovations, Inc., Santa Monica, CA) based on a Zeiss 200 M inverted microscope. Images were captured from 3 wells per condition per experiment (total of 9 fields per condition). Number of TUNEL positive neurons and number of nuclei (DAPI) were determined using Slidebook software and percent TUNEL positive neurons was calculated from the ratio of number of TUNEL positive neurons to number of total nuclei. Data represents the mean ± SEM from 4 independent experiments.
Measurement of cytoplasmic Ca2+using Fura2-AM
Hippocampal neurons grown on glass coverslips were pretreated with E2 (10 ng/mL) or vehicle control for 48 hr prior Aβ1–42 exposure for 24 hr. Neurons were loaded in the dark with Fura2-AM (2 μM) in Hank's Buffered Saline (HBS) (45 min.; 37°C). Cytosolic calcium concentrations were determined by comparing the 340/380 ratio to a standard curve as previously described . At least 10 neurons per coverslip were assessed with at least 3 coverslips per condition per experiment. Data represents the mean ± SEM from 4 independent experiments. Equal dye loading was determined as previously described .
Western blot analysis
Cytosolic and mitochondrial fractions were obtained using the Cytosol/Mitochondrial Fractionation Kit (Calbiochem; Sand Diego, CA) according to the manufacturer's instructions. Protein concentration was determined by the BCA assay. 25 μg of total protein were loaded per well and electorphoresed in a 2% SDS-PAGE gel. Protein was electrotransfered to PVDF membranes and probed with primary antibodies to Bcl-2 (1:250; BD Transduction Laboratories (610539); San Jose, CA), Bax (1:1000; Cell Signaling Technology (2772); Beverly, MA) and cytochrome c (1:1000; BD Transduction Laboratories (5564333); San Jose, CA) and respective horseradish peroxidase (HRP)- conjugated secondary antibodies (1:10,000; 1 hr room temperature). Porin (Mitosciences, Eugen, OR) and Actin (Santa Cruz, Santa Cruz CA) were used as loading controls for mitochondrial and cytosolic fraction, respectively (Data not shown). Bands were visualized with TMB peroxidase kit (Vector Laboratories) and quantitated by scanning and densitometry with Un-Scan-It software (Silk Scientific; Orem, UT). Data are presented as means ± S.E.M. from 4 independent experiments.
Treated neurons grown on chamberslides were fixed in 4% paaraformaldehyde for 15 at room temperature 24 hr following Aβ exposure. For mitochondrial labeling, neurons were incubated in Mitotracker Red CMXRos for 10 min at 37°C for 10 min prior to fixation. Neurons were washed in PBS and permeabilized in PBS + 0.01% triton X-100 for 5 min prior to incubation in primary antibody (Bax: 1:250; Cell Signaling Technology (2772); Beverly, MA; cytochrome c: 1:250; Pharmingen (556432); San Jose, CA) for 2 hr at room temperature. Antibody-antigen complexes were visualized by incubating in FITC (1:250; Vector Laboratories; Burlingame, CA)- or CY3 (1:1000; Amersham; Piscataway, NJ)-conjugated secondary antibodies for 45 min at room temperature, coverslipping with DAPI containing mounting medium (Vector Laboratories; Burlingame, CA) and capturing images using the Marianas imaging system with Slidebook software (Intelligent Imaging Innovations, Inc., Santa Monica, CA).
Adult (4–6 months old) female ovariectomized Sprague-Dawley rats were injected subcutaneously with 17b-estradiol (30 μg/kg) in sesame oil 2 weeks following surgery. 24 hr later, rats were sacrificed and whole brain tissue was homogenized in mitochondrial isolation buffer (0.32 M sucrose, 1 mM EDTA and 10 mM Tris; pH 7.4). Homogenates were centrifuged at 1,330 × g for 5 min at 4°C. Pellets were re-homogenized and centrifuged. The two postnuclear supernantants were combined and centrifuged at 21,200 × g for 10 min at 4°C. The resulting crude mitochondrial pellets were resuspended in 15% Percoll and layered over a 23%/40% discontinuous Percoll gradient and centrifuged at 27, 000 × g for 10 min at 4°C. The fraction accumulating at the 23%/40% interface was collected and diluted 1:4 with isolation buffer and centrifuged at 16,000 × g for 10 min at 4°C.
The pellet was transferred to a 1.5 ml tube centrifuged at 7,300 × g for 10 min at 4°C. The pellet will be resuspended in 40 μL isolation buffer. Protein concentration will be determined by the BCA assay. Mitochondrial integrity was assessed assessing cytochrome c oxidase activity (Cytochrome c oxidase activity kit, Sigma) in intact and lysed mitochondrial samples. Cytochrome c oxidase in inaccessible in intact mitochondria and high activity in these samples relative to total activity (lysed samples) indicates poor mitochondrial integrity.
Measurement of respiration in mitochondria isolated from rat brains
Aliquots of mitochondria (1 mg/mL) were used in measurements of respiratory activity using a Clark-type oxygen electrode (Hansatech Oxygraph) as previously described. Oxygen electrode buffer (130 mM KCl, 2 mM KH2PO4, 3 mM HEPES, 2 mM MgCl2, 1 mM EGTA) was incubated for 1 min in a magnetically stirred chamber at 28°C. The respiratory substrates, glutamate (5 mM) and malate (2.5 mM), were added followed by the isolated mitochondria (100 μg). Basal respiration was first measured in the absence of ADP for 1 min, followed by Ca2+ (100 μM) or equivalent volume of buffer for an additional 2 min. Subsequently, State 3 respiration was measured in the presence of ADP, by addition of 20 mL ADP (0.027 M in 0.067 M NaPO4) to determine the maximal rate of coupled ATP synthesis. Then State 4 respiration was induced by addition of the adenine nucleotide translocator inhibitor atractyloside (50 μM). The respiratory control ratio was calculated using the ratio of State 3 to State 4 respiratory rates.
Statistically significant differences were determined by one-way ANOVA followed by Student-Neuman Keuls post hoc analysis.
- The abbreviations used are:
Aβ, amyloid beta
Amyloid beta 1–42
mitochondrial membrane potential
mitochondrial calcium concentration
cytosolic calcium concentration
amyloid precursor protein.
This work was supported by grants from the National Institutes of Mental Health (1RO1 MH67159-01A1, R.D.B and J.N.), the Kenneth T. and Eileen L. Norris Foundation (R.D.B.) and the L.K. Whittier Foundation (R.D.B.).
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