Jun Ming Wang

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.

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.

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.

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.

Therapeutic Potential of Neurogenesis for Prevention and Recovery from Alzheimer's Disease: Allopregnanolone as a Proof of Concept Neurogenic Agent

A major challenge not yet addressed by current therapeutic interventions for Alzheimers disease (AD) is the regeneration of lost neurons and neural circuitry to restore cognitive function. Therapies that lead to cessation of the degenerative process still leave the brain riddled with deteriorated neural circuits and reduced neuron number. The discovery of neurogenesis in the adult brain and the regenerative potential of neural stem cells holds the promise for restoration of neural populations and regeneration of neural circuits necessary for cerebral function. While the regenerative potential of neural stem cells is great, so too is the challenge of delivering neural stem cells to the brain. Basic science analyses and human trials indicate that constituents of microenvironments within the brain determine the neurogenic potential, phenotypic differentiation of neural stem cells and magnitude of the neural stem cell pool. Multiple analyses have documented that dentate neurogenesis is regulated by multiple growth factors which are abundant during development and which dramatically decline with age. While the cause(s) of age-associated decline in neurogenesis remains to be fully determined, loss in growth factors, FGF-2, IGF-1 and VEGF, in the microenvironment of the subgranular zone (SGZ) are prime contributors to the reduced neurogenic potential. The decline in dentate neurogenesis can be observed as early as middle age. In the aged and AD brain, both the pool of neural stem cells and their proliferative potential are markedly diminished. In parallel, the level of potential regenerative factors is diminished in the brains of Alzheimers patients compared to age-matched controls. Our efforts have been directed towards discovery and development of small, blood brain barrier penetrant molecules to promote endogenous proliferation of neural stem cells within the brain. These endeavors have led to the discovery that the neurosteroid alloprognanolone (APalpha) is a potent and highly efficacious proliferative agent in vitro and in vivo of both rodent and human neural stem cells. Results of our in vitro studies coupled with our more recent analyses in the triple transgenic mouse model of AD suggest that APalpha is a promising strategy for promoting neurogenesis in the aged brain and potentially for restoration of neuronal populations in brains recovering from neurodegenerative disease or injury. A brief overview of issues impacting the therapeutic potential of neurogenesis and the factors used to promote neurogenesis in the aging and degenerating brain is presented. Also included is a review of our current research into the neurogenic potential of the small molecule, blood brain barrier penetrating, neurosteroid allopregnanolone (APalpha).

Allopregnanolone Promotes Regeneration and Reduces β-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 Alzheimers (3xTgAD). In the current study, we determined the efficacy of APα to promote the survival of newly generated neural cells while simultaneously reducing Alzheimers 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 Aβ burden. Treatment was initiated either prior to or post intraneuronal Aβ accumulation. Results indicated that APα administered 1/week for 6 months significantly increased survival of newly generated neurons and simultaneously reduced Aβ pathology with greatest efficacy in the pre-pathology treatment group. APα significantly reduced Aβ generation in hippocampus, cortex, and amygdala, which was paralleled by decreased expression of Aβ-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 Alzheimers disease.

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 Alzheimers 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 Alzheimers 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 (Aβ) whereas APα was ineffective subsequent to development of extraneuronal Aβ 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.

Activation of estrogen receptor α increases and estrogen receptor β 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 Alzheimers 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), ERα and ERβ. In this study, we determined the relationship between ER subtype and estrogen regulation of ApoE expression in HT-22 cells ectopically transfected with ERα or ERβ, 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 ERα either by 17β-estradiol or a specific-agonist, propylpyrazole triol, up-regulated ApoE mRNA and protein expression. In contrast, the ERβ-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.

Clinically Relevant Progestins Regulate Neurogenic and Neuroprotective Responses in Vitro and in Vivo

Previously, we demonstrated that progesterone (P(4)) promoted adult rat neural progenitor cell (rNPC) proliferation with concomitant regulation of cell-cycle gene expression via the P(4) receptor membrane component/ERK pathway. Here, we report the efficacy of seven clinically relevant progestins alone or in combination with 17β-estradiol (E(2)) on adult rNPC proliferation and hippocampal cell viability in vitro and in vivo. In vitro analyses indicated that P(4), norgestimate, Nestorone, norethynodrel, norethindrone, and levonorgestrel (LNG) significantly increased in rNPC proliferation, whereas norethindrone acetate was without effect, and medroxyprogesterone acetate (MPA) inhibited rNPC proliferation. Proliferative progestins in vitro were also neuroprotective. Acute in vivo exposure to P(4) and Nestorone significantly increased proliferating cell nuclear antigen and cell division cycle 2 expression and total number of hippocampal 5-bromo-2-deoxyuridine (BrdU)-positive cells, whereas LNG and MPA were without effect. Mechanistically, neurogenic progestins required activation of MAPK to promote proliferation. P(4), Nestorone, and LNG significantly increased ATP synthase subunit α (complex V, subunit α) expression, whereas MPA was without effect. In combination with E(2), P(4), Nestorone, LNG, and MPA significantly increased BrdU incorporation. However, BrdU incorporation induced by E(2) plus LNG or MPA was paralleled by a significant increase in apoptosis. A rise in Bax/Bcl-2 ratio paralleled apoptosis induced by LNG and MPA. With the exception of P(4), clinical progestins antagonized E(2)-induced rise in complex V, subunit α. These preclinical translational findings indicate that the neurogenic response to clinical progestins varies dramatically. Progestin impact on the regenerative capacity of the brain has clinical implications for contraceptive and hormone therapy formulations prescribed for pre- and postmenopausal women.

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 (APα), 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 APα relative to age-matched neurologically normal controls [1,4,5]. Our earlier work has shown that APα 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 APα 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 APα modifies neurogenesis in both non-transgenic and 3xTgAD mice. Preliminary data suggest that APα may modify Alzheimer’s pathology progression. Together the data indicate that APα 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.