Roberta Diaz Brinton

Mitochondrial bioenergetic deficit precedes Alzheimer's pathology in female mouse model of Alzheimer's disease

Mitochondrial dysfunction has been proposed to play a pivotal role in neurodegenerative diseases, including Alzheimers disease (AD). To address whether mitochondrial dysfunction precedes the development of AD pathology, we conducted mitochondrial functional analyses in female triple transgenic Alzheimers mice (3xTg-AD) and age-matched nontransgenic (nonTg). Mitochondrial dysfunction in the 3xTg-AD brain was evidenced by decreased mitochondrial respiration and decreased pyruvate dehydrogenase (PDH) protein level and activity as early as 3 months of age. 3xTg-AD mice also exhibited increased oxidative stress as manifested by increased hydrogen peroxide production and lipid peroxidation. Mitochondrial amyloid beta (Aβ) level in the 3xTg-AD mice was significantly increased at 9 months and temporally correlated with increased level of Aβ binding to alcohol dehydrogenase (ABAD). Embryonic neurons derived from 3xTg-AD mouse hippocampus exhibited significantly decreased mitochondrial respiration and increased glycolysis. Results of these analyses indicate that compromised mitochondrial function is evident in embryonic hippocampal neurons, continues unabated in females throughout the reproductive period, and is exacerbated during reproductive senescence. In nontransgenic control mice, oxidative stress was coincident with reproductive senescence and accompanied by a significant decline in mitochondrial function. Reproductive senescence in the 3xTg-AD mouse brain markedly exacerbated mitochondrial dysfunction. Collectively, the data indicate significant mitochondrial dysfunction occurs early in AD pathogenesis in a female AD mouse model. Mitochondrial dysfunction provides a plausible mechanistic rationale for the hypometabolism in brain that precedes AD diagnosis and suggests therapeutic targets for prevention of AD.

Progesterone Receptors: Form and Function in Brain

Emerging data indicate that progesterone has multiple non-reproductive functions in the central nervous system to regulate cognition, mood, inflammation, mitochondrial function, neurogenesis and regeneration, myelination and recovery from traumatic brain injury. Progesterone-regulated neural responses are mediated by an array of progesterone receptors (PR) that include the classic nuclear PRA and PRB receptors and splice variants of each, the seven transmembrane domain 7TMPRbeta and the membrane-associated 25-Dx PR (PGRMC1). These PRs induce classic regulation of gene expression while also transducing signaling cascades that originate at the cell membrane and ultimately activate transcription factors. Remarkably, PRs are broadly expressed throughout the brain and can be detected in every neural cell type. The distribution of PRs beyond hypothalamic borders, suggests a much broader role of progesterone in regulating neural function. Despite the large body of evidence regarding progesterone regulation of reproductive behaviors and estrogen-inducible responses as well as effects of progesterone metabolite neurosteroids, much remains to be discovered regarding the functional outcomes resulting from activation of the complex array of PRs in brain by gonadally and/or glial derived progesterone. Moreover, the impact of clinically used progestogens and developing selective PR modulators for targeted outcomes in brain is a critical avenue of investigation as the non-reproductive functions of PRs have far-reaching implications for hormone therapy to maintain neurological health and function throughout menopausal aging.

Divergent impact of progesterone and medroxyprogesterone acetate (Provera) on nuclear mitogen-activated protein kinase signaling.

The impact of progestins on estrogen-inducible mechanisms of neuroprotection was investigated. Previously, we showed that estrogen and progesterone are neuroprotective against excitotoxicity, whereas the synthetic progestin medroxyprogesterone acetate (MPA; Provera) is not. Here, we demonstrate that 17β-estradiol (E2) and progesterone (P4) treatment of hippocampal neurons attenuated the excitotoxic glutamate-induced rise in intracellular calcium concentration. Although MPA had no effect alone, MPA completely antagonized E2-induced attenuation of intracellular calcium concentration. Activation of extracellular receptor kinase (ERK) is required for estrogen-induced neuroprotection and calcium regulation. Paradoxically, E2, P4, and MPA all elicited similar rapid and transient activation of ERK, presenting a contradiction between the dependence on ERK for gonadal hormone-induced neuroprotection and the lack of neuroprotection induced by MPA. Subcellular analysis of ERK demonstrated that the phospho-ERK signal is transduced to the nucleus only by E2 and P4, not by MPA. These results indicate that the profile of nuclear translocation of ERK is consistent with the neuroprotective profile. Further, the E2-induced nuclear translocation of ERK was blocked by coadministration of MPA. Results of this study reveal that nuclear ERK induction by ovarian steroids is predictive of the neuroprotective effects of estrogen and progestin treatments, revealing a hitherto unrecognized divergence of progestin signaling through the src/MAPK pathway. These results have much broader implications encompassing the impact of progestins on estrogen-mediated effects in multiple tissues. The recent results from the Womens Health Initiative trial, which used MPA as the progestinal agent, indicate that differences between progestin formulations are crucial to health outcomes in women.

Estrogen, menopause, and the aging brain : How basic neuroscience can inform hormone therapy in women

As neuroscientists, we can be so taken by the complexity and unique capabilities of the brain that we occasionally forget that it is part of a larger integrated biological system reliant on signaling and communication throughout the organism. The interactions between key organs that release hormones

Mechanism of estrogen-mediated neuroprotection: Regulation of mitochondrial calcium and Bcl-2 expression

Estrogens are neuroprotective against glutamate excitotoxicity caused by an excessive rise in intracellular calcium ([Ca2+]i). In this study, we demonstrate that 17β-estradiol (E2) treatment of hippocampal neurons attenuated the excitotoxic glutamate-induced rise in bulk-free [Ca2+]i despite potentiating the influx of Ca2+ induced by glutamate. E2-induced attenuation of bulk-free [Ca2+]i depends on mitochondrial sequestration of Ca2+, which is blocked in the presence of the combination of rotenone and oligomycin or in the presence of antimycin, which collapse the mitochondrial membrane potential, thereby preventing mitochondrial Ca2+ transport. Release of mitochondrial Ca2+ by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) after excitotoxic glutamate treatment resulted in a greater [Ca2+]i in E2-treated cells, indicating an E2-induced increase in the mitochondrial calcium ([Ca2+]m) load. The increased [Ca2+]m load was accompanied by increased expression of Bcl-2, which can promote mitochondrial Ca2+ load tolerance. These findings provide a mechanism of E2-induced neuronal survival by attenuation of excitotoxic glutamate [Ca2+]i rise via increased mitochondrial sequestration of cytosolic Ca2+ coupled with an increase in Bcl-2 expression to sustain mitochondrial Ca2+ load tolerance and function.

17β-estradiol induced Ca2+ influx via L-type calcium channels activates the Src/ERK/cyclic-AMP response element binding protein signal pathway and BCL-2 expression in rat hippocampal neurons: A potential initiation mechanism for estrogen-induced neuroprotection

Our group and others have demonstrated that 17beta-estradiol (E2) induces neurotrophic and neuroprotective responses in hippocampal and cortical neurons which are dependent upon the Src/extracellular signal-regulated kinase (ERK) signaling pathways. The purpose of this study was to determine the upstream mechanism(s) that initiates the signaling cascade leading to E2-inducible neuroprotection. We tested the hypothesis that E2 activates rapid Ca(2+) influx in hippocampal neurons, which would lead to activation of the Src/ERK signaling cascade and up-regulation of Bcl-2 protein expression. Using fura-2 ratiometric Ca(2+) imaging, we demonstrated that E2 induced a rapid rise of intracellular Ca(2+) concentration ([Ca(2+)](i)) within minutes of exposure which was blocked by an L-type Ca(2+) channel antagonist. Inhibition of L-type Ca(2+) channels resulted in a loss of E2 activation of the Src/ERK cascade, activation of cyclic-AMP response element binding protein (CREB) and subsequent increase in Bcl-2. Real-time intracellular Ca(2+) imaging combined with pERK immunofluorescence, demonstrated that E2 induced [Ca(2+)](i) was coincident with ERK activation in the same neuron. Small interfering RNA knockdown of CREB resulted in a loss of E2 activation of CREB and subsequent E2-induced increase of Bcl-2 expression. We further demonstrated the presence of specific membrane E2 binding sites in hippocampal neurons. Together, these data indicate that E2-induced Ca(2+) influx via the L-type Ca(2+) channel is required for E2 activation of the Src/ERK/CREB/Bcl-2 signaling. Implications of these data for understanding estrogen action in brain and use of estrogen therapy for prevention of neurodegenerative disease are discussed.

Estrogen receptor subtypes alpha and beta contribute to neuroprotection and increased Bcl-2 expression in primary hippocampal neurons

Estrogen receptor (ER) mediated neuroprotection has been demonstrated in both in vitro and in vivo model systems. However, the relative contribution by either ER subtype, ERalpha or ERbeta, to estrogen-induced neuroprotection remains unresolved. To address this question, we investigated the impact of selective ER agonists for either ERalpha, PPT, or ERbeta, DPN, to prevent neurodegeneration in cultured hippocampal neurons exposed to excitotoxic glutamate. Using three indicators of neuronal viability and survival, we demonstrated that both the ERalpha selective agonist PPT and the ERbeta selective agonist DPN protected hippocampal neurons against glutamate-induced cell death in a dose-dependent manner, with the maximal response occurring at 100 pM. Further analyses showed that both PPT and DPN enhanced Bcl-2 expression in hippocampal neurons, with an efficacy comparable to their neuroprotective capacity. Collectively, the present data indicate that activation of either ERalpha or ERbeta can promote neuroprotection in hippocampal neurons, suggesting that both receptor subtypes could be involved in estrogen neuroprotection. As ERbeta is highly expressed in the brain and has little or no expression in the breast or uterus, discovery and design of ERbeta selective molecules could provide a strategy for activating the beneficial effects of estrogen in the brain without activating untoward effects of estrogen in reproductive organs.

The Neurosteroid Allopregnanolone Promotes Proliferation of Rodent and Human Neural Progenitor Cells and Regulates Cell-Cycle Gene and Protein Expression

Our previous research demonstrated that the neuroactive progesterone metabolite allopregnanolone (3α-hydroxy-5α-pregnan-20-one) rapidly induced hippocampal neuron neurite regression (Brinton, 1994). We hypothesized that allopregnanolone-induced neurite regression was a prelude to mitogenesis initiated by a rise in intracellular calcium. Supporting this hypothesis, the current data demonstrate that allopregnanolone, in a dose-dependent manner, induces a significant increase in proliferation of neuroprogenitor cells (NPCs) derived from the rat hippocampus and human neural stem cells (hNSCs) derived from the cerebral cortex. Proliferation was determined by incorporation of bromodeoxyuridine and [3H]thymidine, fluorescence-activated cell sorter analysis of murine leukemia virus-green fluorescent protein-labeled mitotic NPCs, and total cell number counting. Allopregnanolone-induced proliferation was isomer and steroid specific, in that the stereoisomer 3β-hydroxy-5β-pregnan-20-one and related steroids did not increase [3H]thymidine uptake. Immunofluorescent analyses for the NPC markers nestin and Tuj1 indicated that newly formed cells were of neuronal lineage. Furthermore, microarray analysis of cell-cycle genes and real-time reverse transcription-PCR and Western blot validation revealed that allopregnanolone increased the expression of genes that promote mitosis and inhibited the expression of genes that repress cell proliferation. Allopregnanolone-induced proliferation was antagonized by the voltage-gated L-type calcium channel (VGLCC) blocker nifedipine, consistent with the finding that allopregnanolone induces a rapid increase in intracellular calcium in hippocampal neurons via a GABA type A receptor-activated VGLCC (Son et al., 2002). These data demonstrate that allopregnanolone significantly increased rat NPC and hNSC proliferation with concomitant regulation in mitotic cell-cycle genes via a VGLCC mechanism. The therapeutic potential of allopregnanolone as a neurogenic molecule is discussed.

17 β-Estradiol Enhances the Outgrowth and Survival of Neocortical Neurons in Culture

Results of this investigation demonstrate that exposure to 17 β-estradiol differentially and significantly regulates cortical nerve cell outgrowth depending on the cortical region. Parietal and occipital neurons treated with 1 nM 17 β-estradiol showed a greater magnitude of neuronal outgrowth whereas outgrowth of temporal cortex neurons was decreased in the presence of 1 nM 17 β-estradiol. Frontal cortex neurons showed a consistent enhancement of neuronal outgrowth that did not reach statistical significance. The dose response profile for 17 β-estradiol regulation of the macromorphological features exhibited a bimodal dose response relationship whereas the dose response profile for 17 β-estradiol regulation of the micromorphological features exhibited a dose response more characteristic of an inverted V-shaped function. An antagonist to the NMDA receptor antagonist, AP5, abolished the growth promoting effect of 17 β-estradiol whereas the nuclear estrogen receptor antagonist ICI 182,780 did not. Lastly, neocortical neurons exposed to 17 β-estradiol exhibited greater viability and survival than control neurons over a two week period. These data indicate that 17 β-estradiol can enhance the growth and viability of select populations of neocortical neurons and that the growth promoting effects of 17 β-estradiol can be blocked by an antagonist to the NMDA glutamate receptor and not by an antagonist to the estrogen nuclear receptor.

Allopregnanolone reverses neurogenic and cognitive deficits in mouse model of Alzheimer's disease

Our previous analyses showed that allopregnanolone (APα) significantly increased proliferation of rodent and human neural progenitor cells in vitro. In this study, we investigated the efficacy of APα to promote neurogenesis in the hippocampal subgranular zone (SGZ), to reverse learning and memory deficits in 3-month-old male triple transgenic mouse model of Alzheimers (3xTgAD) and the correlation between APα-induced neural progenitor cell survival and memory function in 3xTgAD mice. Neural progenitor cell proliferation was determined by unbiased stereological analysis of BrdU incorporation and survival determined by FACS for BrdU+ cells. Learning and memory function was assessed using the hippocampal-dependent trace eye-blink conditioning paradigm. At 3 months, basal level of BrdU+ cells in the SGZ of 3xTgAD mice was significantly lower relative to non-Tg mice, despite the lack of evident AD pathology. APα significantly increased, in a dose-dependent manner, BrdU+ cells in SGZ in 3xTgAD mice and restored SGZ proliferation to normal magnitude. As with the deficit in proliferation, 3xTgAD mice exhibited deficits in learning and memory. APα reversed the cognitive deficits to restore learning and memory performance to the level of normal non-Tg mice. In 3xTgAD mice, APα-induced survival of neural progenitors was significantly correlated with APα-induced memory performance. These findings suggest that early neurogenic deficits, which were evident before immunodetectable Aβ, may contribute to the cognitive phenotype of AD, and that APα could serve as a regenerative therapeutic to prevent or delay neurogenic and cognitive deficits associated with mild cognitive impairment and Alzheimers disease.