WDR23 mediates NRF2 proteostasis and cytoprotective capacity in the hippocampus.

Pathogenic brain aging and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease are characterized by chronic neuroinflammation and the accumulation of dysfunctional or misfolded proteins that lead to progressive neuronal cell death. Here we demonstrate that a murine model with global loss of the CUL4-DDB1 substrate receptor WDR23 (Wdr23KO) results in changes in multiple age-related hippocampal-dependent behaviors. The behavioral differences observed in Wdr23KO animals accompany the stabilization of the NRF2/NFE2L2 protein, an increase in RNA transcripts regulated by this cytoprotective transcription factor, and an increase in the steady state level of antioxidant defense proteins. Taken together, these findings reveal a role for WDR23-proteostasis in mediating cytoprotective capacity in the hippocampus and reveal the potential for targeting WDR23-NRF2 signaling interactions for development of therapies for neurodegenerative disorders.

Loss of WDR23 proteostasis impacts mitochondrial homeostasis in the mouse brain.

Mitochondrial adaptation is important for stress resistance throughout life. Here we show that WDR23 loss results in an enrichment for genes regulated by nuclear respiratory factor 1 (NRF1), which coordinates mitochondrial biogenesis and respiratory functions, and an increased steady state level of several nuclear coded mitochondrial resident proteins in the brain. Wdr23KO also increases the endogenous levels of insulin degrading enzyme (IDE) and the relaxin-3 peptide (RLN3), both of which have established roles in mediating mitochondrial metabolic and oxidative stress responses. Taken together, these studies reveal an important role for WDR23 as a component of the mitochondrial homeostat in the murine brain.

WDR23 mediates NRF2 proteostasis and cytoprotective capacity in the hippocampus.

Pathogenic brain aging and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease are characterized by chronic neuroinflammation and the accumulation of dysfunctional or misfolded proteins that lead to progressive neuronal cell death. Here we demonstrate that a murine model with global loss of the CUL4-DDB1 substrate receptor WDR23 ( Wdr23KO ) results in changes in multiple age-related hippocampal-dependent behaviors. The behavioral differences observed in Wdr23KO animals accompany the stabilization of the NRF2/NFE2L2 protein, an increase in RNA transcripts regulated by this cytoprotective transcription factor, and an increase in the steady state level of antioxidant defense proteins. Taken together, these findings reveal a role for WDR23-proteostasis in mediating cytoprotective capacity in the hippocampus and reveal the potential for targeting WDR23-NRF2 signaling interactions for development of therapies for neurodegenerative disorders. HIGHLIGHTS: WDR23 regulates NRF2/NFE2L2 stability in the mouse hippocampus Loss of Wdr23 significantly increases the expression of NFE2L2/NRF2 target genes Global loss of WDR23 influences age-related behaviors differentially in males and females.

Near-Infrared Monoamine Oxidase Inhibitor Biodistribution in a Glioma Mouse Model.

PURPOSE: The biodistribution imaging kinetics of near-infrared monoamine oxidase inhibitor (NMI) are reported here. METHODS: NMI was administered intravenously or orally to mice and detected by NIR fluorescence optical imaging within minutes and the longitudinal signal distribution was measured for up to 1 week after a single dose. RESULTS: NMI rapidly reached 3.7-fold higher ventral and 3.2-fold higher brain region fluorescent signal intensity compared to oral route at 24 h. Similar patterns of NMI biodistribution were found in mice with or without intracranial implanted GL26 brain tumors. NMI was highly associated with tumors in contrast to adjacent non-tumor brain, confirming diagnostic utility. NMI 5 mg/kg imaging signal in brain at 48 h was optimal (tumor/non-tumor ratio > 3.5) with minimum off-target distribution. Intravenous NMI imaging signal peaked between 24 h and 48 h for lung, liver, kidney, blood, brain, and most other tissues. Clearance (signal weaker, but still present) from most tissues occurred by day 7. Intravenous low dose (0.5 mg/kg) minimally labeled tumor and other tissues, 5 mg/kg showed optimal imaging signal in glioma at a dose we previously reported as efficacious, and 50 mg/kg was tolerable but saturated the tissue signals beyond tumor specificity. Gel electrophoresis showed two major bands present in brain tumor and tissue protein lysates. CONCLUSIONS: Intravenous 5 mg/kg was optimal dose to target brain tumor and identified off-target organs of concern: lungs, liver, and kidneys. These results demonstrate the biodistribution and optimal dose range of NMI for treatment and diagnostic monitoring of glioma.

Allopregnanolone preclinical acute pharmacokinetic and pharmacodynamic studies to predict tolerability and efficacy for Alzheimer's disease.

To develop allopregnanolone as a therapeutic for Alzheimer's disease, we investigated multiple formulations and routes of administration in translationally relevant animal models of both sexes. Subcutaneous, topical (transdermal and intranasal), intramuscular, and intravenous allopregnanolone were bolus-administered. Pharmacokinetic analyses of intravenous allopregnanolone in rabbit and mouse indicated that peak plasma and brain levels (3-fold brain/plasma ratios) at 5min were sufficient to activate neuroregenerative responses at sub-sedative doses. Slow-release subcutaneous suspension of allopregnanolone displayed 5-fold brain/plasma ratio at Cmax at 30min. At therapeutic doses by either subcutaneous or intravenous routes, allopregnanolone mouse plasma levels ranged between 34-51ng/ml by 30min, comparable to published endogenous human level in the third trimester of pregnancy. Exposure to subcutaneous, topical, intramuscular, and intravenous allopregnanolone, at safe and tolerable doses, increased hippocampal markers of neurogenesis including BrdU and PCNA in young 3xTgAD and aged wildtype mice. Intravenous allopregnanolone transiently and robustly phosphorylated CREB within 5min and increased levels of neuronal differentiation transcription factor NeuroD within 4h. Neurogenic efficacy was achieved with allopregnanolone brain exposure of 300-500hr*ng/g. Formulations were tested to determine the no observable adverse effect level (NOAEL) and maximally tolerated doses (MTD) in male and female rats by sedation behavior time course. Sex differences were apparent, males exhibited ≥40% more sedation time compared to females. Allopregnanolone formulated in sulfobutyl-ether-beta-cyclodextrin at optimized complexation ratio maximized allopregnanolone delivery and neurogenic efficacy. To establish the NOAEL and MTD for Allo-induced sedation using a once-per-week intravenous regenerative treatment regimen: In female rats the NOAEL was 0.5mg/kg and MTD 2mg/kg. The predicted MTD in human female is 0.37mg/kg. In male rats the NOAEL and MTD were less than those determined for female. Outcomes of these PK/PD studies predict a safe and efficacious dose range for initial clinical trials of allopregnanolone for Alzheimer's disease. These findings have translational relevance to multiple neurodegenerative conditions.

Frontiers in therapeutic development of allopregnanolone for Alzheimer's disease and other neurological disorders.

Allopregnanolone (Allo), a neurosteroid, has emerged as a promising promoter of endogenous regeneration in brain. In a mouse model of Alzheimer's disease, Allo induced neurogenesis, oligodendrogenesis, white matter generation and cholesterol homeostasis while simultaneously reducing ß-amyloid and neuroinflammatory burden. Allo activates signaling pathways and gene expression required for regeneration of neural stem cells and their differentiation into neurons. In parallel, Allo activates systems to sustain cholesterol homeostasis and reduce ß-amyloid generation. To advance Allo into studies for chronic human neurological conditions, we examined translational and clinical parameters: dose, regimen, route, formulation, outcome measures, and safety regulations. A treatment regimen of once per week at sub-sedative doses of Allo was optimal for regeneration and reduction in Alzheimer's pathology. This regimen had a high safety profile following chronic exposure in aged normal and Alzheimer's mice. Formulation of Allo for multiple routes of administration has been developed for both preclinical and clinical testing. Preclinical evidence for therapeutic efficacy of Allo spans multiple neurological diseases including Alzheimer's, Parkinson's, multiple sclerosis, Niemann-Pick, diabetic neuropathy, status epilepticus, and traumatic brain injury. To successfully translate Allo as a therapeutic for multiple neurological disorders, it will be necessary to tailor dose and regimen to the targeted therapeutic mechanisms and disease etiology. Treatment paradigms conducted in accelerated disease models in young animals have a low probability of successful translation to chronic diseases in adult and aged humans. Gender, genetic risks, stage and burden of disease are critical determinants of efficacy. This review focuses on recent advances in development of Allo for Alzheimer's disease (AD) that have the potential to accelerate therapeutic translation for multiple unmet neurological needs.

Allopregnanolone as regenerative therapeutic for Alzheimer's disease: translational development and clinical promise.

Herein, we review a translational development plan to advance allopregnanolone to the clinic as a regenerative therapeutic for neurodegenerative diseases, in particular Alzheimer's. Allopregnanolone, an endogenous neurosteroid that declines with age and neurodegenerative disease, was exogenously administered and assessed for safety and efficacy to promote neuro-regeneration, cognitive function and reduction of Alzheimer's pathology. Allopregnanolone-induced neurogenesis correlated with restoration of learning and memory function in a mouse model of Alzheimer's disease and was comparably efficacious in aged normal mice. Critical to success was a dosing and treatment regimen that was consistent with the temporal requirements of systems biology of regeneration in brain. A treatment regimen that adhered to regenerative requirements of brain was also efficacious in reducing Alzheimer's pathology. With an optimized dosing and treatment regimen, chronic allopregnanolone administration promoted neurogenesis, oligodendrogenesis, reduced neuroinflammation and beta-amyloid burden while increasing markers of white matter generation and cholesterol homeostasis. Allopregnanolone meets three of the four drug-like physicochemical properties described by Lipinski's rule that predict the success rate of drugs in development for clinical trials. Pharmacokinetic and pharmacodynamic outcomes, securing GMP material, development of clinically translatable formulations and acquiring regulatory approval are discussed. Investigation of allopregnanolone as a regenerative therapeutic has provided key insights into mechanistic targets for neurogenesis and disease modification, dosing requirements, optimal treatment regimen, route of administration and the appropriate formulation necessary to advance to proof of concept clinical studies to determine efficacy of allopregnanolone as a regenerative and disease modifying therapeutic for Alzheimer's disease.

A dominant negative ERß splice variant determines the effectiveness of early or late estrogen therapy after ovariectomy in rats.

The molecular mechanisms for the discrepancy in outcome of initiating estrogen therapy (ET) around peri-menopause or several years after menopause in women are unknown. We hypothesize that the level of expression of a dominant negative estrogen receptor (ER) ß variant, ERß2, may be a key factor determining the effectiveness of ET in post-menopausal women. We tested this hypothesis in ovariectomized nine month-old (an age when irregular estrous cycles occur) female Sprague Dawley rats. Estradiol treatment was initiated either 6 days (Early ET, analogous to 4 months post-menopause in humans), or 180 days (Late ET, analogous to 11 years post-menopause in humans) after ovariectomy. Although ERß2 expression increased in all OVX rats, neurogenic and neuroprotective responses to estradiol differed in Early and Late ET. Early ET reduced ERß2 expression in both hippocampus and white blood cells, increased the hippocampal cell proliferation as assessed by Ki-67 expression, and improved mobility in the forced swim test. Late ET resulted in either no or modest effects on these parameters. There was a close correlation between the degree of ERß2 expression and the preservation of neural effects by ET after OVX in rats, supporting the hypothesis that persistent elevated levels of ERß2 are a molecular basis for the diminished effectiveness of ET in late post-menopausal women. The correlation between the expression of ERß2 in circulating white blood cells and brain cells suggests that ERß2 expression in peripheral blood cells may be an easily accessible marker to predict the effective window for ET in the brain.

Allopregnanolone restores hippocampal-dependent learning and memory and neural progenitor survival in aging 3xTgAD and nonTg mice.

We previously demonstrated that allopregnanolone (APα) increased proliferation of neural progenitor cells and reversed neurogenic and cognitive deficits prior to Alzheimer's disease (AD) pathology (Wang, J.M., Johnston, P.B., Ball, B.G., Brinton, R.D., 2005. The neurosteroid allopregnanolone promotes proliferation of rodent and human neural progenitor cells and regulates cell-cycle gene and protein expression. J. Neurosci. 25, 4706-4718; Wang, J.M., Singh, C., Liu, L., Irwin, R.W., Chen, S., Chung, E.J., Thompson, R.F., Brinton, R.D., 2010. Allopregnanolone reverses neurogenic and cognitive deficits in mouse model of Alzheimer's disease. Proc. Natl. Acad. Sci. U. S. A. 107, 6498-6503). Herein, we determined efficacy of APα to restore neural progenitor cell survival and associative learning and memory subsequent to AD pathology in male 3xTgAD mice and their nontransgenic (nonTg) counterparts. APα significantly increased survival of bromodeoxyuridine positive (BrdU+) cells and hippocampal-dependent associative learning and memory in 3xTgAD mice in the presence of intraneuronal amyloid beta (Aß) whereas APα was ineffective subsequent to development of extraneuronal Aß plaques. Restoration of hippocampal-dependent associative learning was maximal by the first day and sustained throughout behavioral training. Learning and memory function in APα-treated 3xTgAD mice was 100% greater than vehicle-treated and comparable to maximal normal nonTg performance. In aged 15-month-old nonTg mice, APα significantly increased survival of bromodeoxyuridine-positive cells and hippocampal-dependent associative learning and memory. Results provide preclinical evidence that APα promoted survival of newly generated cells and restored cognitive performance in the preplaque phase of AD pathology and in late-stage normal aging.

Allopregnanolone promotes regeneration and reduces ß-amyloid burden in a preclinical model of Alzheimer's disease.

Previously, we demonstrated that allopregnanolone (APα) promoted proliferation of rodent and human neural progenitor cells in vitro. Further, we demonstrated that APα promoted neurogenesis in the hippocampal subgranular zone (SGZ) and reversed learning and memory deficits in the male triple transgenic mouse model of Alzheimer's (3xTgAD). In the current study, we determined the efficacy of APα to promote the survival of newly generated neural cells while simultaneously reducing Alzheimer's disease (AD) pathology in the 3xTgAD male mouse model. Comparative analyses between three different APα treatment regimens indicated that APα administered 1/week for 6 months was maximally efficacious for simultaneous promotion of neurogenesis and survival of newly generated cells and reduction of AD pathology. We further investigated the efficacy of APα to impact Aß burden. Treatment was initiated either prior to or post intraneuronal Aß accumulation. Results indicated that APα administered 1/week for 6 months significantly increased survival of newly generated neurons and simultaneously reduced Aß pathology with greatest efficacy in the pre-pathology treatment group. APα significantly reduced Aß generation in hippocampus, cortex, and amygdala, which was paralleled by decreased expression of Aß-binding-alcohol-dehydrogenase. In addition, APα significantly reduced microglia activation as indicated by reduced expression of OX42 while increasing CNPase, an oligodendrocyte myelin marker. Mechanistic analyses indicated that pre-pathology treatment with APα increased expression of liver-X-receptor, pregnane-X-receptor, and 3-hydroxy-3-methyl-glutaryl-CoA-reductase (HMG-CoA-R), three proteins that regulate cholesterol homeostasis and clearance from brain. Together these findings provide preclinical evidence for the optimal treatment regimen of APα to achieve efficacy as a disease modifying therapeutic to promote regeneration while simultaneously decreasing the pathology associated with Alzheimer's disease.

Neuroregenerative mechanisms of allopregnanolone in Alzheimer's disease.

The proliferative pool and regenerative potential of neural stem cells diminishes with age, a phenomenon that may be exacerbated in prodromal and mild Alzheimer's disease (AD) brains. In parallel, the neuroactive progesterone metabolite, allopregnanolone (APα), along with a host of other factors, is decreased in the AD brain. Results of preclinical analyses demonstrate that APα is a potent inducer of neural progenitor proliferation of both rodent and human derived neural progenitor cells in vitro. In vivo, APα significantly increased neurogenesis within the subgranular zone of the dentate gyrus and subventricular zone of the 3xTgAD mouse model. Functionally, APα reversed the learning and memory deficits of 3xTgAD mice prior to and following the onset of AD pathology and was comparably efficacious in aged normal mice. In addition to inducing regenerative responses in mouse models of AD, APα significantly reduced beta-amyloid burden, beta-amyloid binding alcohol dehydrogenase load, and microglial activation. In parallel, APα increased markers of white matter generation and cholesterol homeostasis. Analyses to determine the optimal treatment regimen in the 3xTgAD mouse brain indicated that a treatment regimen of APα once per week was optimal for both inducing neurogenesis and reducing AD pathology. Pharmacokinetic analyses indicated that APα is rapidly increased in both plasma and brain following a single dose. APα is most efficacious when administered once per week which will contribute to its margin of safety. Further, analyses in both animals and humans have provided parameters for safe APα dosage exposure in humans. From a translational perspective, APα is a small molecular weight, blood brain barrier penetrant molecule with substantial preclinical efficacy data as a potential Alzheimer's therapeutic with existing safety data in animals and humans. To our knowledge, APα is the only small molecule that both promotes neural progenitor regeneration in brain and simultaneously reduces AD pathology burden.

Medroxyprogesterone acetate antagonizes estrogen up-regulation of brain mitochondrial function.

The impact of clinical progestins used in contraception and hormone therapies on the metabolic capacity of the brain has long-term implications for neurological health in pre- and postmenopausal women. Previous analyses indicated that progesterone and 17ß-estradiol (E2) sustain and enhance brain mitochondrial energy-transducing capacity. Herein we determined the impact of the clinical progestin, medroxyprogesterone acetate (MPA), on glycolysis, oxidative stress, and mitochondrial function in brain. Ovariectomized female rats were treated with MPA, E2, E2+MPA, or vehicle with ovary-intact rats serving as a positive control. MPA alone and MPA plus E2 resulted in diminished mitochondrial protein levels for pyruvate dehydrogenase, cytochrome oxidase, ATP synthase, manganese-superoxide dismutase, and peroxiredoxin V. MPA alone did not rescue the ovariectomy-induced decrease in mitochondrial bioenergetic function, whereas the coadministration of E2 and MPA exhibited moderate efficacy. However, the coadministration of MPA was detrimental to antioxidant defense, including manganese-superoxide dismutase activity/expression and peroxiredoxin V expression. Accumulated lipid peroxides were cleared by E2 treatment alone but not in combination with MPA. Furthermore, MPA abolished E2-induced enhancement of mitochondrial respiration in primary cultures of the hippocampal neurons and glia. Collectively these findings indicate that the effects of MPA differ significantly from the bioenergetic profile induced by progesterone and that, overall, MPA induced a decline in glycolytic and oxidative phosphorylation protein and activity. These preclinical findings on the basis of acute exposure to MPA raise concerns regarding neurological health after chronic use of MPA in contraceptive and hormone therapy.

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

Our previous analyses showed that allopregnanolone (APalpha) significantly increased proliferation of rodent and human neural progenitor cells in vitro. In this study, we investigated the efficacy of APalpha 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 Alzheimer's (3xTgAD) and the correlation between APalpha-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. APalpha 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. APalpha reversed the cognitive deficits to restore learning and memory performance to the level of normal non-Tg mice. In 3xTgAD mice, APalpha-induced survival of neural progenitors was significantly correlated with APalpha-induced memory performance. These findings suggest that early neurogenic deficits, which were evident before immunodetectable Abeta, may contribute to the cognitive phenotype of AD, and that APalpha could serve as a regenerative therapeutic to prevent or delay neurogenic and cognitive deficits associated with mild cognitive impairment and Alzheimer's disease.

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 Alzheimer's disease (AD). To address whether mitochondrial dysfunction precedes the development of AD pathology, we conducted mitochondrial functional analyses in female triple transgenic Alzheimer's 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 (Abeta) level in the 3xTg-AD mice was significantly increased at 9 months and temporally correlated with increased level of Abeta 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 and estrogen regulate oxidative metabolism in brain mitochondria.

The ovarian hormones progesterone and estrogen have well-established neurotrophic and neuroprotective effects supporting both reproductive function and cognitive health. More recently, it has been recognized that these steroids also regulate metabolic functions sustaining the energetic demands of this neuronal activation. Underlying this metabolic control is an interpretation of signals from diverse environmental sources integrated by receptor-mediated responses converging upon mitochondrial function. In this study, to determine the effects of progesterone (P4) and 17beta-estradiol (E2) on metabolic control via mitochondrial function, ovariectomized rats were treated with P4, E2, or E2 plus P4, and whole-brain mitochondria were isolated for functional assessment. Brain mitochondria from hormone-treated rats displayed enhanced functional efficiency and increased metabolic rates. The hormone-treated mitochondria exhibited increased respiratory function coupled to increased expression and activity of the electron transport chain complex IV (cytochrome c oxidase). This increased respiratory activity was coupled with a decreased rate of reactive oxygen leak and reduced lipid peroxidation representing a systematic enhancement of brain mitochondrial efficiency. As such, ovarian hormone replacement induces mitochondrial alterations in the central nervous system supporting efficient and balanced bioenergetics reducing oxidative stress and attenuating endogenous oxidative damage.

Regenerative potential of allopregnanolone.

The neuroendocrine status of the brain has been linked to the quality of the aging process, to the risk of Alzheimer's disease and to progression of neurodegenerative pathology. Data from multiple levels of analysis ranging from in vitro cellular models to in vivo animal models to clinical investigations indicate that the decline of neurosteroids play a key role in successful aging and prevention of neurodegenerative disease Alzheimer's. Among the neurosteroids in decline during aging is allopregnanolone (APalpha, a metabolite of progesterone, which is reduced in the serum, plasma and brain of aged vs. young subjects. Further, Alzheimer's disease (AD) victims exhibit an even greater reduction in plasma and brain levels of APalpha relative to age-matched neurologically normal controls. Our earlier work has shown that APalpha is a neurogenic agent for rodent hippocampal neural progenitors and for human neural progenitor cells derived from the cerebral cortex. Our ongoing research seeks to determine the neurogenic potential of APalpha in the triple transgenic mouse model of Alzheimer's disease (3 x TgAD) as AD related pathology progresses from imperceptible to mild to severe. Initial analyses suggest that APalpha may maintain the regenerative ability of the brain, modify progression of AD related pathology and reverse learning and memory deficits in 3 x TgAD mice. This review summarizes current APalpha research in different animal models, neural progenitor regeneration within a degenerative milieu and the challenge for developing neuroregenerative therapeutics.

Estradiol in vivo regulation of brain mitochondrial proteome.

We used a combined proteomic and functional biochemical approach to determine the overall impact of 17beta-estradiol (E2) on mitochondrial protein expression and function. To elucidate mitochondrial pathways activated by E2 in brain, two-dimensional (2D) gel electrophoresis was conducted to screen the mitoproteome. Ovariectomized adult female rats were treated with a single injection of E2. After 24 h of E2 exposure, mitochondria were purified from brain and 2D analysis and liquid chromatography-tandem mass spectrometry protein identification were conducted. Results of proteomic analyses indicated that of the 499 protein spots detected by image analysis, a total of 66 protein spots had a twofold or greater change in expression. Of these, 28 proteins were increased in expression after E2 treatment whereas 38 proteins were decreased in expression relative to control. E2 regulated key metabolic enzymes including pyruvate dehydrogenase, aconitase, and ATP-synthase. To confirm that E2-inducible changes in protein expression translated into functional consequences, we determined the impact of E2 on the enzymatic activity of the mitochondrial electron transport chain. In vivo, E2 treatment enhanced brain mitochondrial efficiency as evidenced by increased respiratory control ratio, elevated cytochrome-c oxidase activity and expression while simultaneously reducing free radical generation in brain. Results of these analyses provide insights into E2 mechanisms of regulating brain mitochondria, which have the potential for sustaining neurological health and prevention of neurodegenerative diseases associated with mitochondrial dysfunction such as Alzheimer's disease.

Regeneration in a degenerating brain: potential of allopregnanolone as a neuroregenerative agent.

Confronting the efficacy of a regenerative therapeutic is the degenerative environment that is characterized by neuronal loss, physical plague and glial scar barriers and inflammation. But perhaps more fundamental from a regenerative perspective, are changes in the biochemical milieu of steroid and peptide growth factors, cytokines and neurotransmitter systems. Data from multiple levels of analysis indicate that gonadal steroid hormones and their metabolites can promote neural health whereas their decline or absence are associated with decline in neural health and increased risk of neurodegenerative disease including Alzheimer's. Among the steroids in decline, is allopregnanolone (APbeta, a neurosteroid metabolite of progesterone, which was found to be reduced in the serum [1,2] and plasma [3] and brain of aged vs. young subjects [4]. Further, Alzheimer disease (AD) victims showed an even further reduction in plasma and brain levels of APalpha relative to age-matched neurologically normal controls [1,4,5]. Our earlier work has shown that APalpha is a neurogenic agent for rodent hippocampal neural progenitors and for human neural progenitor cells derived from the cerebral cortex[6]. Our ongoing research seeks to determine the neurogenic potential of APalpha in the triple transgenic mouse model of Alzheimer's disease (3xTgAD) as AD related pathology progresses from imperceptible to mild to severe. Initial analyses suggest that neurogenic potential changes with age in nontransgenic mice and that the neurogenic profile differs between non-transgenic and 3xTgAD mice. Comparative analyses indicate that APalpha modifies neurogenesis in both nontransgenic and 3xTgAD mice. Preliminary data suggest that APalpha may modify Alzheimer's pathology progression. Together the data indicate that APalpha may maintain the regenerative ability of the brain and modify progression of AD related pathology. Challenges for efficacy of regenerative agents within a degenerative milieu are discussed.

Estrogen protects neuronal cells from amyloid beta-induced apoptosis via regulation of mitochondrial proteins and function.

BACKGROUND: 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. RESULTS: In this study, we investigated the mechanism of 17beta-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. CONCLUSION: Therefore, we propose that estradiol pretreatment protects against amyloid beta neurotoxicity by limiting mitochondrial dysfunction via activation of antiapoptotic mechanisms.

Activation of estrogen receptor alpha increases and estrogen receptor beta decreases apolipoprotein E expression in hippocampus in vitro and in vivo.

Previous evidence indicates that, in carriers of apolipoprotein E4 (ApoE4), estrogen therapy increased the risk of late-onset Alzheimer's disease (AD), whereas in individuals carrying ApoE2/3, estrogen therapy reduced the risk of AD [Cauley JA, Zmuda JM, Yaffe K, Kuller LH, Ferrell RE, Wisniewski SR, Cummings SR (1999) J Bone Miner Res 14:1175-1181; Yaffe K, Haan M, Byers A, Tangen C, Kuller L (2000) Neurology 54:1949-1954]. Estrogen mechanisms of action are mediated by two estrogen receptors (ERs), ERalpha and ERbeta. In this study, we determined the relationship between ER subtype and estrogen regulation of ApoE expression in HT-22 cells ectopically transfected with ERalpha or ERbeta, in primary cultured rat hippocampal neurons in vitro and in rat hippocampus in vivo by both molecular biological and pharmacological analyses. Results of these analyses demonstrated that activation of ERalpha either by 17beta-estradiol or a specific-agonist, propylpyrazole triol, up-regulated ApoE mRNA and protein expression. In contrast, the ERbeta-selective agonist, diarylpropionitrile, down-regulated ApoE mRNA and protein expression. These results demonstrate that, in vitro and in vivo, ApoE expression can be differentially regulated depending on activation of ER subtypes. These data suggest that use of ER-selective ligands could provide therapeutic benefit to reduce the risk of AD by increasing ApoE expression in ApoE2/3 allele carriers and decreasing ApoE expression in ApoE4 allele carriers.