Intermittent hypoxia and stem cell implants preserve breathing capacity in a rodent model of amyotrophic lateral sclerosis.

RATIONALE: Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease causing paralysis and death from respiratory failure. Strategies to preserve and/or restore respiratory function are critical for successful treatment. Although breathing capacity is maintained until late in disease progression in rodent models of familial ALS (SOD1(G93A) rats and mice), reduced numbers of phrenic motor neurons and decreased phrenic nerve activity are observed. Decreased phrenic motor output suggests imminent respiratory failure. OBJECTIVES: To preserve or restore phrenic nerve activity in SOD1(G93A) rats at disease end stage. METHODS: SOD1(G93A) rats were injected with human neural progenitor cells (hNPCs) bracketing the phrenic motor nucleus before disease onset, or exposed to acute intermittent hypoxia (AIH) at disease end stage. MEASUREMENTS AND MAIN RESULTS: The capacity to generate phrenic motor output in anesthetized rats at disease end stage was: (1) transiently restored by a single presentation of AIH; and (2) preserved ipsilateral to hNPC transplants made before disease onset. hNPC transplants improved ipsilateral phrenic motor neuron survival. CONCLUSIONS: AIH-induced respiratory plasticity and stem cell therapy have complementary translational potential to treat breathing deficits in patients with ALS.

Dietary folate intakes and effects of folic acid supplementation on folate concentrations among Japanese pregnant women.

AIM: To quantify the consumed amount of dietary folate and to evaluate effects of folic acid and balanced diets on serum folate concentrations. MATERIAL & METHODS: Food records collected from 641 pregnant women were assessed to quantify the consumed amount of dietary folate for a 6-year period from 2003 to 2008. Changes in serum folate concentrations were evaluated among 80 pregnant women who either took folic acid tablets or consumed balanced diets in 2008. RESULTS: Though the dietary folate intake averaged 331 µg daily, 200 women in the 1st trimester consumed the least amount of dietary folate, 294 µg daily. It was observed that 42% of the 200 women either consumed at least 440 µg of dietary folate daily or took 400 µg folic acid supplements daily, but that 58% of them neither consumed dietary folate of 440 µg nor took folic acid supplements. Intakes of 400 µg folic acid supplements for 5 weeks resulted in a significant increase in serum folate concentrations but the consumption of balanced diets had no effect on increasing folate concentrations. CONCLUSIONS: The average intake of dietary folate did not fulfill the Recommended Dietary Allowance of 440 µg. Serum folate concentration significantly increased only among pregnant women who took folic acid supplements. Recommendations to consume balanced diets do not seem effective to decrease the incidence of neural tube defects.

Mitotic and neurogenic effects of dehydroepiandrosterone (DHEA) on human neural stem cell cultures derived from the fetal cortex.

Dehydroepiandrosterone (DHEA) is a neurosteroid with potential effects on neurogenesis and neuronal survival in humans. However, most studies on DHEA have been performed in rodents, and there is little direct evidence for biological effects on the human nervous system. Furthermore, the mechanism of its action is unknown. Here, we show that DHEA significantly increased the growth rates of human neural stem cells derived from the fetal cortex and grown with both epidermal growth factor (EGF) and leukemia inhibitory factor (LIF). However, it had no effect on cultures grown in either factor alone, suggesting a specific action on the EGF/LIF-responsive cell. Precursors of DHEA such as pregnenolone or six of its major metabolites, had no significant effect on proliferation rates. DHEA did not alter the small number (<3%) of newly formed neuroblasts or the large number (>95%) of nestin-positive precursors. However, the number of glial fibrillary acidic protein-positive cells, its mRNA, and protein were significantly increased by DHEA. We found both N-methyl-d-aspartate and sigma 1 antagonists, but not GABA antagonists, could completely eliminate the effects of DHEA on stem cell proliferation. Finally we asked whether the EGF/LIF/DHEA-responsive stem cells had an increased potential for neurogenesis and found a 29% increase in neuronal production when compared to cultures grown in EGF/LIF alone. Together these data suggest that DHEA is involved in the maintenance and division of human neural stem cells. Given the wide availability of this neurosteroid, this finding has important implications for future use.

Involvement of activin and inhibin in the regulation of food and water intake in the rat.

The expression of activin and inhibin has been demonstrated in the hypothalamus, but their physiological roles in the brain remain to be elucidated. In the present study, involvement of activin and inhibin in the regulation of food and water intake was examined. Male rats were deprived of food or water for 12 and 60 hr, and mRNA levels of activin/inhibin alpha, betaA and betaB subunits in the hypothalamus were estimated by RT-PCR. Gene expression of alpha subunit transiently decreased at 12 hr of food deprivation, while it did not change during water deprivation. Food and water deprivation for 60 hr increased mRNA levels of betaA and betaB subunits, respectively. These results indicated that gene expression of each subunit was independently regulated. Injection of activin A (0.5 and 4.0 microg) into the third ventricle decreased food intake. Water intake was suppressed by 4.0 microg, but not 0.5 microg, of activin A. Intracerebroventricular injection of inhibin A (0.5 and 4.0 microg) decreased water intake in a dose dependent manner without affecting food intake, suggesting that inhibin could act independently of activin. Taken together, it is suggested that activin and inhibin take part in the central regulation of nutrient and fluid balance, though further study is needed to determine precise molecular species involved.

Expression analyses of sex steroid-regulated genes in neonatal rat hypothalamus.

Estrogen plays an important role in sexual differentiation of the brain in rats during the perinatal period. To elucidate molecular mechanisms underlying sexual differentiation of the brain, in this study we investigated genes differentially expressed between sexes or induced to express by estrogen in neonatal rat hypothalamus using DNA microarray analysis in combination with real-time RT-PCR. It was found that the levels of expression of the genes encoding glutamic acid decarboxylase 65 and coronin 1b were higher in male than female hypothalamus on postnatal day (PN) 5 and those of collagen type 3 alpha1 and thioredoxin reductase 2 genes in female hypothalamus on PN5 were decreased and increased, respectively, by treatment with estradiol on PN2. Then the developmental changes in the expression of these 4 genes were examined from 1 day before the parturition to PN9, and they all showed sexual dimorphic patterns. In addition, dependence of the expression of these genes on either estradiol, testosterone or dihydrotestosterone during the neonatal period was confirmed. These results suggest that these four genes are involved in sexual differentiation of the rat brain, and that androgen per se as well as estrogen may take part in the processes.

Androgen induces p130 mRNA expression in the neonatal rat hypothalamus.

Our previous research using cDNA microarray analysis demonstrated that female rats displayed a higher p130 mRNA level than males in the hypothalamus at postnatal day (PN) 5. In the present study, it was shown that at PN3 males had a significantly elevated mRNA level over females, whereas at PN7 females displayed a higher expression level using a real-time reverse transcription-polymerase chain reaction. In situ hybridization analysis indicated relatively strong p130 mRNA signals in the ventromedial nucleus and the arcuate nucleus in the neonatal hypothalamus. Subcutaneous injection of 5alpha-dihydrotestosterone as well as testosterone propionate to PN2 neonatal rats significantly increased p130 gene expression at PN3, whereas estradiol benzoate did not have a significant effect. These results suggest that expression of the p130 gene in the neonatal rat hypothalamus is responsive to androgens and may be involved in sexual differentiation of the brain.

Sex-related differences in gene expression in neonatal rat hypothalamus assessed by cDNA microarray analysis.

Sexual differentiation of the rodent brain is recognized to involve transcriptional activation of multiple genes induced by gonadal steroids at developmental stages. To identify the genes differing in expression level between sexes, we analyzed gene expression in male and female rat hypothalami at postnatal day 5 by means of a cDNA microarray consisting of 2352 genes. By comparing the expression pattern between sexes, we identified 12 male-enriched genes and 20 female-enriched genes. Among them, the expression pattern of 1 male-enriched gene, jagged homolog 1, and those of 2 female-enriched genes, p27Kip1 and p130, were confirmed to be consistent with microarray data by RT-PCR. Investigation of these genes should help to elucidate the molecular and cellular mechanisms underlying sexual differentiation of the rodent central nervous system.

Granulin precursor gene: a sex steroid-inducible gene involved in sexual differentiation of the rat brain.

The mechanisms of sexual differentiation of the brain by sex steroids seem to be conserved throughout the mammalian species, although there may be some species differences. In rats, sex-dependent differentiation of the brain occurs in a sex steroid-dependent manner during the perinatal period known as the critical period. Androgen exposure during the perinatal period results in the development of structural and functional sexually dimorphic characteristics in the brain; the absence of testicular androgen leads the central nervous system to develop passively in a primarily female fashion, while the presence of androgen induces the masculinization of the brain. We attempted to characterize sex steroid-inducible genes that are involved in the sexually dimorphic function of the brain. Following the cDNA subtraction between hypothalami of 5-day-old intact and neonatally androgenized female rats, a granulin (grn) precursor gene was identified. The grn gene encodes a 6-kDa polypeptide known as a growth modulating factor of epithelial cells in vitro. Exogenous estrogen, as well as androgen, induced grn gene expression in the neonatal hypothalamus. In the brain of a 5-day-old male rat, grn mRNA was expressed in the ventromedial hypothalamic nucleus and the arcuate nucleus of the hypothalamus. Throughout the critical period for sexual differentiation of the brain, grn gene expression remained high in males, while in females it gradually decreased. Antisense oligodeoxynucleotide (ODN) complementary to grn mRNA was synthesized and infused into the third ventricle of male rats at 2 days of age. Two different control treatments were used; the first consisted of a control sequence ODN that had virtually no homology to known mRNAs, and the second consisted of vehicle alone. After maturation, the subject animals that were treated with antisense ODN of grn displayed significantly lower scores than the control males in various parameters assessing sexual behavior, i.e., mount, intromission, and ejaculation. The present results suggest that the grn gene, the expression of which is induced by sex steroids in the neonatal hypothalamus, plays a crucial role in the functional masculinization of the rat brain.