Allopregnanolone reinstates tyrosine hydroxylase immunoreactive neurons and motor performance in an MPTP-lesioned mouse model of Parkinson's disease.

Restorative/protective therapies to restore dopamine neurons in the substantia nigra pars compacta (SNpc) are greatly needed to effectively change the debilitating course of Parkinson's disease. In this study, we tested the therapeutic potential of a neurogenic neurosteroid, allopregnanolone, in the restoration of the components of the nigrostriatal pathway in MPTP-lesioned mice by measuring striatal dopamine levels, total and tyrosine hydroxylase immunoreactive neuron numbers and BrdU-positive cells in the SNpc. An acute treatment (once/week for two weeks) with allopregnanolone restored the number of tyrosine hydroxylase-positive and total cell numbers in the SNpc of MPTP-lesioned mice, even though this did not increase striatal dopamine. It was also noted that MPTP treated mice to which allopregnanolone was administered had an increase in BrdU-positive cells in the SNpc. The effects of allopregnanolone in MPTP-lesioned mice were more apparent in mice that underwent behavioral tests. Interestingly, mice treated with allopregnanolone after MPTP lesion were able to perform at levels similar to that of non-lesioned control mice in a rotarod test. These data demonstrate that allopregnanolone promotes the restoration of tyrosine hydroxylase immunoreactive neurons and total cells in the nigrostriatal tract, improves the motor performance in MPTP-treated mice, and may serve as a therapeutic strategy for Parkinson'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.

High throughput analysis of neural progenitor cell proliferation in adult rodent hippocampus.

Extensive efforts have been made to determine the status on neural progenitor cell proliferation in specific pathological conditions and to evaluate the therapeutic efficacy of drugs for preventing neurogenic deficits in neurodegenerative diseases. However, the most commonly used stereological analysis using 5-bromo-2'-deoxyuridine (BrdU) immuno-positive sections is a time consuming and labor intensive process and is often a bottle neck in neurogenic drug development, particularly when large sample sizes are needed. In addition, BrdU is toxic to new born neurons and also labels DNA damage in old cells. In this study, we established a method that quantitatively measures the number of Ki-67, an endogenous cell proliferation marker, positive cells by flow cytometry which analyzes extracted cell nuclei from rodent hippocampi in suspension. Our results demonstrate that this approach can be applied to a large number of rodent samples, can be accomplished in a short period of time (1-3 days), and can be completed in a more accurately objective manner than by using 3-D cell counting with immunohistochemically processed sections.