Inducible and conditional activation of ERK5 MAP kinase rescues mice from cadmium-induced olfactory memory deficits.

Cadmium (Cd) is a heavy metal that is one of the most toxic environmental pollutants throughout the world. We previously reported that Cd exposure impairs olfactory memory in mice. However, the underlying mechanisms for its neurotoxicity for olfactory function are not well understood. Since adult Subventricular zone (SVZ) and Olfactory Bulb (OB) neurogenesis contributes to olfaction, olfactory memory defects caused by Cd may be due to inhibition of neurogenesis. In this study, using bromodeoxyuridine (BrdU) labeling and immunohistochemistry, we found that 0.6 mg/L Cd exposure through drinking water impaired adult SVZ/OB neurogenesis in C57BL/6 mice. To determine if the inhibition of olfactory memory by Cd can be reversed by stimulating adult neurogenesis, we utilized the transgenic caMEK5 mouse strain to conditional stimulate of adult neurogenesis by activating the endogenous ERK5 MAP kinase signaling pathway. This was accomplished by conditionally induced expression of active MEK5 (caMEK5) in adult neural stem/progenitor cells. The caMEK5 mice were exposed to 0.6 mg/L Cd for 38 weeks, and tamoxifen was administered to induce caMEK5 expression and stimulate adult SVZ/OB neurogenesis during Cd exposure. Short-term olfactory memory test and sand-digging based, odor-cued olfactory learning and memory test were conducted after Cd and tamoxifen treatments to examine their effects on olfaction. Here we report that Cd exposure impaired short-term olfactory memory and odor-cued associative learning and memory in mice. Furthermore, the Cd-impaired olfactory memory deficits were rescued by the tamoxifen-induction of caMEK5 expression. This suggests that Cd exposure impairs olfactory function by affecting adult SVZ/OB neurogenesis in mice.

Phosphodiesterase7 Inhibition Activates Adult Neurogenesis in Hippocampus and Subventricular Zone In Vitro and In Vivo.

The phosphodiesterase 7 (PDE7) enzyme is one of the enzymes responsible for controlling intracellular levels of cyclic adenosine 3',5'-monophosphate in the immune and central nervous system. We have previously shown that inhibitors of this enzyme are potent neuroprotective and anti-inflammatory agents. In addition, we also demonstrated that PDE7 inhibition induces endogenous neuroregenerative processes toward a dopaminergic phenotype. Here, we show that PDE7 inhibition controls stem cell expansion in the subgranular zone of the dentate gyrus of the hippocampus (SGZ) and the subventricular zone (SVZ) in the adult rat brain. Neurospheres cultures obtained from SGZ and SVZ of adult rats treated with PDE7 inhibitors presented an increased proliferation and neuronal differentiation compared to control cultures. PDE7 inhibitors treatment of neurospheres cultures also resulted in an increase of the levels of phosphorylated cAMP response element binding protein, suggesting that their effects were indeed mediated through the activation of the cAMP/PKA signaling pathway. In addition, adult rats orally treated with S14, a specific inhibitor of PDE7, presented elevated numbers of proliferating progenitor cells, and migrating precursors in the SGZ and the SVZ. Moreover, long-term treatment with this PDE7 inhibitor shows a significant increase in newly generated neurons in the olfactory bulb and the hippocampus. Also a better performance in memory tests was observed in S14 treated rats, suggesting a functional relevance for the S14-induced increase in SGZ neurogenesis. Taken together, our results indicate for the first time that inhibition of PDE7 directly regulates proliferation, migration and differentiation of neural stem cells, improving spatial learning and memory tasks. Stem Cells 2017;35:458-472.

Expression of Idh1R132H in the Murine Subventricular Zone Stem Cell Niche Recapitulates Features of Early Gliomagenesis.

Isocitrate dehydrogenase 1 mutations drive human gliomagenesis, probably through neomorphic enzyme activity that produces D-2-hydroxyglutarate. To model this disease, we conditionally expressed Idh1R132H in the subventricular zone (SVZ) of the adult mouse brain. The mice developed hydrocephalus and grossly dilated lateral ventricles, with accumulation of 2-hydroxyglutarate and reduced α-ketoglutarate. Stem and transit amplifying/progenitor cell populations were expanded, and proliferation increased. Cells expressing SVZ markers infiltrated surrounding brain regions. SVZ cells also gave rise to proliferative subventricular nodules. DNA methylation was globally increased, while hydroxymethylation was decreased. Mutant SVZ cells overexpressed Wnt, cell-cycle and stem cell genes, and shared an expression signature with human gliomas. Idh1R132H mutation in the major adult neurogenic stem cell niche causes a phenotype resembling gliomagenesis.

3ß-hydroxysteroid dehydrogenase in the brain ependymocytes.

We studied activity of 3ß-hydroxysteroid dehydrogenase in rat brain ependymocytes. Enzyme activity was found in the cytoplasm of cells lining the villi in the vascular plexuses in the lateral ventricles and cells lining the ventricles. These data suggest that ependymocyte can synthesize neurosteroids.

Querkopf is a key marker of self-renewal and multipotency of adult neural stem cells.

Adult neural stem cells (NSCs) reside in the subventricular zone (SVZ) and produce neurons throughout life. Although their regenerative potential has kindled much interest, few factors regulating NSCs in vivo are known. Among these is the histone acetyltransferase querkopf (QKF, also known as MYST4, MORF, KAT6B), which is strongly expressed in a small subset of cells in the neurogenic subventricular zone. However, the relationship between Qkf gene expression and the hierarchical levels within the neurogenic lineage is currently unknown. We show here that the 10% of SVZ cells with the highest Qkf expression possess the defining NSC characteristics of multipotency and self-renewal and express markers previously shown to enrich for NSCs. A fraction of cells expressing Qkf at medium to high levels is enriched for multipotent progenitor cells with limited self-renewal, followed by a population containing migrating neuroblasts. Cells low in Qkf promoter activity are predominantly ependymal cells. In addition, we show that mice deficient for Bmi1, a central regulator of NSC self-renewal, show an age-dependent decrease in the strongest Qkf-expressing cell population in the SVZ. Our results show a strong relationship between Qkf promoter activity and stem cell characteristics, and a progressive decrease in Qkf gene activity as lineage commitment and differentiation proceed in vivo.

[3beta-Hydroxysteroid dehydrogenase in ependimocytes of brain lateral ventricle lining and vascular plexus villi in rats of different age].

Using the histochemical method, the activity of 3beta-hydroxysteroid dehydrogenase (HSDH) was studied in the brain of laboratory male albino rats of different age groups: 5-6 days (n = 6), 45-50 days (n = 12), and 6 months (n = 15). The quantitative assessment of reaction intensity was performed with the cytospectrophotometer. The results obtained indicate that the ependimocytes lining the brain lateral ventricles and covering the villi of their vascular plexuses are characterized by the presence of HSDH activity typical to that of steroid-producing cells. In this regard ependimocytes may be attributed to the cells that can produce neurosteroids. It was established that HSDH activity in ependimocytes was minimal in the early postnatal period and considerably increased by the prepuberty period, remaining at this level in adult animals.

Cyclin-dependent kinase 5 is required for control of neuroblast migration in the postnatal subventricular zone.

At the lateral wall of the lateral ventricles in the adult rodent brain, neuroblasts form an extensive network of elongated cell aggregates called chains in the subventricular zone and migrate toward the olfactory bulb. The molecular mechanisms regulating this migration of neuroblasts are essentially unknown. Here, we report a novel role for cyclin-dependent kinase 5 (Cdk5), a neuronal protein kinase, in this process. Using in vitro and in vivo conditional knock-out experiments, we found that Cdk5 deletion impaired the chain formation, speed, directionality, and leading process extension of the neuroblasts in a cell-autonomous manner. These findings suggest that Cdk5 plays an important role in neuroblast migration in the postnatal subventricular zone.

NADPH oxidase- and mitochondrion-derived superoxide at rostral ventrolateral medulla in endotoxin-induced cardiovascular depression.

We evaluated the contribution of superoxide anion (O2*-) generated by NADPH oxidase or mitochondria in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons for arterial pressure maintenance are located, on cardiovascular depression induced by inducible nitric oxide synthase-derived NO after Escherichia coli lipopolysaccharide (LPS) treatment. In Sprague-Dawley rats maintained under propofol anesthesia, microinjection of LPS bilaterally into the RVLM induced progressive hypotension, bradycardia, and reduction in sympathetic vasomotor outflow over our 240-min observation period. This was accompanied by an increase in O2*- production (60-240 min) in the RVLM, alongside phosphorylation of p47(phox) or p67(phox), upregulation of gp91(phox) or p47(phox) protein, and increase in Rac-1 or NADPH oxidase activity (60-120 min), and a depression of mitochondrial respiratory enzyme activity (120-240 min). Whereas inhibition of NADPH oxidase or knockdown of the gp91(phox) or p47(phox) gene blunted the early phase (60-150 min), coenzyme Q10 or mitochondrial K(ATP) channel inhibitor antagonized the delayed phase (120-240 min) of LPS-induced increase in O2*- production in RVLM and cardiovascular depression. We conclude that, whereas NADPH oxidase-derived O2*- in RVLM participates predominantly in the early phase, O2*- generated by depression in mitochondrial respiratory enzyme activity or opening of mitoK(ATP) channels mediates the delayed phase of LPS-induced cardiovascular depression.

Activities of 3beta-HSD and aromatase in slices of developing and adult zebra finch brain.

Sex steroids influence the development and function of the songbird brain. Developmentally, the neural circuitry underlying song undergoes masculine differentiation under the influence of estradiol. In adults, estradiol stimulates song behavior and the seasonal growth of song control circuits. There is good reason to believe that these neuroactive estrogens are synthesized in the brain. At all ages, estrogens could act at the lateral ventricle, during migration, or where song nuclei exist or will form. We investigated the activity of two critical steroidogenic enzymes, 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) and aromatase, using a slice culture system. Sagittal brain slices were collected from juvenile (posthatch day 20) and adult zebra finches containing either the lateral ventricle, where neurons are born, or the telencephalic song nuclei HVC and RA. The slices were incubated with (3)H-dehydroepiandrosterone or (3)H-androstenedione. Activity was determined by isolating certain products of 3beta-HSD (5alpha-androstanedione, 5beta-androstanedione, estrone, and estradiol) and aromatase (estrone and estradiol). Activities of both 3beta-HSD and aromatase were detected in all slices and were confirmed using specific enzyme inhibitors. We found no significant difference in activity between adult males and females in either region for either enzyme. Juvenile female slices containing the lateral ventricle, however, showed greater levels of 3beta-HSD activity than did similar slices from age-matched males. Determination of the activity of these critical steroidogenic enzymes in slice culture has implications for the role of neurosteroids in brain development.

Amino acids and transaminases activity in ventricular CSF and in brain of normal and Alzheimer patients.

The present study was conducted to determine the concentration of amino acids in the cerebrospinal spinal fluid (CSF) and the activities of two tramsaminases: glutamic oxaloacetate transaminase (GOT) and glutamic pyruvate transaminase (GPT) in human Alzheimer disease (AD) and normal brain. L-glutamic acid, L-glutamine and L-alanine are the most abundant amino acids in the CSF (50-55% of total amino acids). L-glutamine occurs at much higher levels in Alzheimer CSF compared to the normal CSF (229+/-91.8 nmol/ml in AD versus 107+/-47.2 nmol/ml in normal; P=0.0041). In contrast, L-aspartate occurs at significantly lower concentrations in Alzheimer CSF than normal CSF (46.1+/-25.7 nmol/ml in Alzheimer versus 95.2+/-52.6 nmol/ml in normal; P=0.020). In Alzheimer brain (frontal, parietal and occipital cortices) GOT is present at significantly higher activities than in normal brain cortices (about 1.5 times higher; P<0.01). No significant differences for GPT activity occurred between normal and AD brain. Since CSF receives amino acids from brain tissues, and since GOT catalyzes the conversion of L-aspartate to L-glutamate, the higher concentrations of L-glutamine (which is derived from L-glutamate), and the lower concentrations of L-aspartate found in Alzheimer CSF could be considered as a consequence of the higher activity of GOT that occurs in Alzheimer brain.

Gene expression of EXT1 and EXT2 during mouse brain development.

Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPGs) play significant roles in various biological processes. There is a wealth of circumstantial and experimental evidence suggesting the roles of HS in mammalian neural development. HS synthesis is governed by a series of enzymes. Among them, two enzymes, EXT1 and EXT2, catalyze polymerization of glucuronic acid and N-acetylglucosamine, the crucial step of HS synthesis. To obtain insight into the roles of HS in neural development, we examined the spatiotemporal expression patterns of EXT1 and EXT2 during mice brain development. RT-PCR analyses showed that expression of EXT1 and EXT2 peaks during early postnatal period in the cerebrum and around birth in the cerebellum. In situ hybridization revealed that in the embryonic brain, EXT1 and EXT2 were localized primarily in the neuroepithelial cells surrounding the lateral ventricles, the mesencephalic vesicle, and the fourth ventricle. In the early postnatal stage, intense expression of EXT1 and EXT2 was observed in the cerebral cortex and the hippocampus formation. In the postnatal cerebellum, expression of EXT1 and EXT2 was mainly observed in external and internal granular layers. Our results demonstrate that EXT1 and EXT2 are highly expressed in the developing brain, and that their expression is developmentally regulated, suggesting that HS is involved in various neurodevelopmental processes.

Choroid plexus epithelial cells in adult rats show structural alteration but not apoptosis following an exposure to hypobaric hypoxia.

The choroid plexus in adult rats was examined for any structural alteration or apoptotic cell death following a high altitude exposure which leads to the development of hypobaric hypoxia due to reduced oxygen tension in the atmospheric air. Caspase-3 (a protease which mediates apoptosis) immunoreactivity was absent in the choroid plexus epithelial cells in the control rats and following altitude exposure; Bcl-2 (anti-apoptotic protein) and Bax (pro-apoptotic protein) immunoreactivity were upregulated at 3 h-2 days following the altitude exposure when compared to the controls but not in longer surviving rats. At the ultrastructural level, glycogen particles and vacuoles were observed in some epithelial cells at 7 days following the altitude exposure. It is suggested that transient exposure to high altitude may not cause much damage to the choroid plexus epithelial cells except for some structural alteration which may be due to altered metabolism of the cells in response to hypobaric hypoxia.

Localization of 5-oxo-L-prolinase mRNA in the murine choroid plexus by in situ hybridization.

The gamma-glutamyl cycle is thought to play a significant role in the transport of amino acids between the blood and the cerebrospinal fluid. In the present study we investigated the expression of 5-oxo-L-prolinase, an enzyme of this pathway, in mouse brain and kidney by in situ hybridization using digoxygenin-labeled RNA probes. Expression of 5-OPase mRNA was found in epithelial cells of the proximal tubules in kidney as well as in the epithelium and vascular endothelial cells of the choroid plexus but not in periventricular capillaries.