Frank H. Yu

Autistic-like behaviour in Scn1a +/− mice and rescue by enhanced GABA-mediated neurotransmission

Haploinsufficiency of the SCN1A gene encoding voltage-gated sodium channel NaV1.1 causes Dravet’s syndrome, a childhood neuropsychiatric disorder including recurrent intractable seizures, cognitive deficit and autism-spectrum behaviours. The neural mechanisms responsible for cognitive deficit and autism-spectrum behaviours in Dravet’s syndrome are poorly understood. Here we report that mice with Scn1a haploinsufficiency exhibit hyperactivity, stereotyped behaviours, social interaction deficits and impaired context-dependent spatial memory. Olfactory sensitivity is retained, but novel food odours and social odours are aversive to Scn1a+/− mice. GABAergic neurotransmission is specifically impaired by this mutation, and selective deletion of NaV1.1 channels in forebrain interneurons is sufficient to cause these behavioural and cognitive impairments. Remarkably, treatment with low-dose clonazepam, a positive allosteric modulator of GABAA receptors, completely rescued the abnormal social behaviours and deficits in fear memory in the mouse model of Dravet’s syndrome, demonstrating that they are caused by impaired GABAergic neurotransmission and not by neuronal damage from recurrent seizures. These results demonstrate a critical role for NaV1.1 channels in neuropsychiatric functions and provide a potential therapeutic strategy for cognitive deficit and autism-spectrum behaviours in Dravet’s syndrome.

Too many roads not taken

Most protein research focuses on those known before the human genome was mapped. Work on the slew discovered since, urge Aled M. Edwards and his colleagues.

Specific deletion of NaV1.1 sodium channels in inhibitory interneurons causes seizures and premature death in a mouse model of Dravet syndrome

Heterozygous loss-of-function mutations in the brain sodium channel NaV1.1 cause Dravet syndrome (DS), a pharmacoresistant infantile-onset epilepsy syndrome with comorbidities of cognitive impairment and premature death. Previous studies using a mouse model of DS revealed reduced sodium currents and impaired excitability in GABAergic interneurons in the hippocampus, leading to the hypothesis that impaired excitability of GABAergic inhibitory neurons is the cause of epilepsy and premature death in DS. However, other classes of GABAergic interneurons are less impaired, so the direct cause of hyperexcitability, epilepsy, and premature death has remained unresolved. We generated a floxed Scn1a mouse line and used the Cre-Lox method driven by an enhancer from the Dlx1,2 locus for conditional deletion of Scn1a in forebrain GABAergic neurons. Immunocytochemical studies demonstrated selective loss of NaV1.1 channels in GABAergic interneurons in cerebral cortex and hippocampus. Mice with this deletion died prematurely following generalized tonic-clonic seizures, and they were equally susceptible to thermal induction of seizures as mice with global deletion of Scn1a. Evidently, loss of NaV1.1 channels in forebrain GABAergic neurons is both necessary and sufficient to cause epilepsy and premature death in DS.

Na(V)1.1 channels are critical for intercellular communication in the suprachiasmatic nucleus and for normal circadian rhythms.

NaV1.1 is the primary voltage-gated Na+ channel in several classes of GABAergic interneurons, and its reduced activity leads to reduced excitability and decreased GABAergic tone. Here, we show that NaV1.1 channels are expressed in the suprachiasmatic nucleus (SCN) of the hypothalamus. Mice carrying a heterozygous loss of function mutation in the Scn1a gene (Scn1a+/−), which encodes the pore-forming α-subunit of the NaV1.1 channel, have longer circadian period than WT mice and lack light-induced phase shifts. In contrast, Scn1a+/− mice have exaggerated light-induced negative-masking behavior and normal electroretinogram, suggesting an intact retina light response. Scn1a+/− mice show normal light induction of c-Fos and mPer1 mRNA in ventral SCN but impaired gene expression responses in dorsal SCN. Electrical stimulation of the optic chiasm elicits reduced calcium transients and impaired ventro-dorsal communication in SCN neurons from Scn1a+/− mice, and this communication is barely detectable in the homozygous gene KO (Scn1a−/−). Enhancement of GABAergic transmission with tiagabine plus clonazepam partially rescues the effects of deletion of NaV1.1 on circadian period and phase shifting. Our report demonstrates that a specific voltage-gated Na+ channel and its associated impairment of SCN interneuronal communication lead to major deficits in the function of the master circadian pacemaker. Heterozygous loss of NaV1.1 channels is the underlying cause for severe myoclonic epilepsy of infancy; the circadian deficits that we report may contribute to sleep disorders in severe myoclonic epilepsy of infancy patients.

Di-(2 ethylhexyl) phthalate and flutamide alter gene expression in the testis of immature male rats

We previously demonstrated that the androgenic and anti-androgenic effects of endocrine disruptors (EDs) alter reproductive function and exert distinct effects on developing male reproductive organs. To further investigate these effects, we used an immature rat model to examine the effects of di-(2 ethylhexyl) phthalate (DEHP) and flutamide (Flu) on the male reproductive system. Immature male SD rats were treated daily with DEHP and Flu on postnatal days (PNDs) 21 to 35, in a dose-dependent manner. As results, the weights of the testes, prostate, and seminal vesicle and anogenital distances (AGD) decreased significantly in response to high doses of DEHP or Flu. Testosterone (T) levels significantly decreased in all DEHP- treated groups, whereas luteinizing hormone (LH) plasma levels were not altered by any of the two treatments at PND 36. However, treatment with DEHP or Flu induced histopathological changes in the testes, wherein degeneration and disorders of Leydig cells, germ cells and dilatation of tubular lumen were observed in a dose-dependent manner. Conversely, hyperplasia and denseness of Leydig, Sertoli and germ cells were observed in rats given with high doses of Flu. The results by cDNA microarray analysis indicated that 1,272 genes were up-regulated by more than two-fold, and 1,969 genes were down-regulated in response to DEHP, Flu or both EDs. These genes were selected based on their markedly increased or decreased expression levels. These genes have been also classified on the basis of gene ontology (e.g., steroid hormone biosynthetic process, regulation of transcription, signal transduction, metabolic process, biosynthetic process...). Significant decreases in gene expression were observed in steroidogenic genes (i.e., Star, Cyp11a1 and Hsd3b). In addition, the expression of a common set of target genes, including CaBP1, Vav2, Plcd1, Lhx1 and Isoc1, was altered following exposure to EDs, suggesting that they may be marker genes to screen for the anti-androgenic or androgenic effects of EDs. Overall, our results demonstrated that exposure to DEHP, Flu or both EDs resulted in a alteration of gene expression in the testes of immature male rats. Furthermore, the toxicological effects of these EDs on the male reproductive system resulted from their anti-androgenic effects. Taken together, these results provide a new insight into the molecular mechanisms underlying the detrimental impacts of EDs, in regards to anti-androgenic effects in humans and wildlife.

Mechanisms by which the inhibition of specific intracellular signaling pathways increase osteoblast proliferation on apatite surfaces

Osteoblasts proliferate slowly on the surface of calcium phosphate apatite which is widely used as a substrate biomaterial in bone regeneration. Owing to poor adhesion signaling in the cells grown on the calcium phosphate surface, inadequate growth factor signaling is generated to trigger cell cycle progression. The present study investigated an intracellular signal transduction pathway involved in the slow cell proliferation in osteoblasts grown on the calcium phosphate surface. Small GTPase RhoA and phosphatase and tensin homolog (PTEN) were more activated in cells grown on the surface of calcium phosphate apatite than on tissue culture plate. Specific inhibition of RhoA and PTEN induced the cells on calcium phosphate apatite surface to proliferate at a similar rate as cells on tissue culture plate surface. Specific inhibition of ROCK, which is a downstream effector of RhoA and an upstream activator of PTEN also increased proliferation of these osteoblasts. Present results indicate that physical property of calcium phosphate crystals that impede cell proliferation may be surmounted by the inhibition of the RhoA/ROCK/PTEN pathway to rescue delayed proliferation of osteoblasts on the calcium phosphate apatite surface. In addition, specific inhibition of ROCK promoted cell migration and osteoblast differentiation. Inhibition of the RhoA/ROCK/PTEN intracellular signaling pathway is expected to enhance cell activity to promote and accelerate bone regeneration on the calcium phosphate apatite surface.

Estrogen receptor α is involved in the induction of Calbindin-D9k and progesterone receptor by parabens in GH3 cells: A biomarker gene for screening xenoestrogens

The effects of paraben, a xenoestrogen with known endocrine disrupting bioactivity were evaluated. We used the induction of an estrogenic biomarker gene - Calbindin-D(9k) (CaBP-9k) to investigate the xenoestrogenic activity of a panel of parabens (methyl-, ethyl-, propyl-, isopropyl-, butyl-, and isobutylparabens) in GH3 rat pituitary cancer cell line. Following 24-h treatment, a significant increase in CaBP-9k expression of transcript and protein was dependent on the concentration-treated as well as the linear length of the alkyl chain from methyl- to isobutylparabens. Interestingly, co-treatment with fulvestrant, a pure antiestrogen largely reversed the paraben-dependent induction of CaBP-9k mRNA and protein in GH3 cell line. To better understand the mechanism of CaBP-9k induction by these endocrine disrupting compounds, we measured the levels of estrogen receptor (ERα) and progesterone receptor (PR) expression following parabens exposure. Also, we monitored the transiently transfected with plasmids containing of estrogen response element (ERE) sequence into GH3. In the GH3 cells, a large increase in PR mRNA and protein was observed in a concentration-dependent manner after parabens treatment that was effectively blocked in the presence of antagonist of 17β-estradiol (fulvestrant). And, luciferase activity was expressed from the putative ERE and expression was stimulated by parabens. To confirm that ERα signaling is involved in parabens induction of CaBP-9k and PR mRNA and protein, we treated GH3 cells with an antiestrogen, fulvestrant, which blocked the paraben-induced upregulation of CaBP-9k and PR. Taken together, these results indicate that CaBP-9k and PR is induced by parabens via the ER pathway in GH3 cell line.

Effects of 17β-estradiol and xenoestrogens on mouse embryonic stem cells

Xenoestrogens such as 4-tert-octylphenol (OP) and 4-nonylphenol (NP) can adversely affect the reproductive and immune systems from their estrogenic effects in target cells. In this study, we investigated the effects of xenoestrogens on the expression of undifferentiation markers in mouse embryonic stem (ES) cells and of cardiomyocyte differentiation markers in mouse embryoid body (EB) cells induced to differentiate into cardiomyocytes from ES cells. The expressions of undifferentiation markers (Oct4, Sox2, Zfp206, and Rex-1) and cardiomyocyte differentiation markers (alpha-MHC, beta-MHC, ANF, and MLC-2V) were determined by semi- and quantitative real-time PCR. Treatment with E(2) or OP and NP induced an increase in Oct4 expression at the transcriptional level in a dose- and time-dependent manner. However, no difference was observed in the expression of Sox2, Zfp206 or Rex-1 genes in ES cells, suggesting that E(2) may be an Oct4 enhancer in ES cells. Induction of Oct4 expression by E(2) and xenoestrogens (OP and NP) did not change the methylation pattern of the Oct4-promoter and was not affected by treatment with a demethylating agent, 5-azacytidine. Taken together, these results suggest that E(2) and xenoestrogens may impact on the undifferentiation process of ES and EB cells, and retain ES cells in an undifferentiated state.

Parathyroid hormone-related protein and glucocorticoid receptor beta are regulated by cortisol in the kidney of male mice

AIMSnParathyroid hormone-related protein (PTHrP) is a peptide growth factor produced in a wide range of tissues from brain and parathyroid, to kidney and uterus. The purpose of this study was to determine whether the adrenal cortical hormones, hydrocortisone (cortisol), modulate PTHrP expression and glucocorticoid receptor (GR)β in mice kidney.nnnMAIN METHODSnChanges in PTHrP gene expression were determined by real-time PCR and its protein level was examined by Western blot analysis. In addition, expression of renal PTHrP protein was localized by immunohistochemistry. Effects of RU486 on the expression levels of GRα/β or PTHrP gene in the kidneys were analyzed by Western blot analysis.nnnKEY FINDINGSnWe found that renal expression levels of PTHrP mRNA were higher in males than in females up to 9weeks of age. Using immunohistochemistry, we observed higher levels of PTHrP expression within the cortex than in the medulla in both male and female mice, and this expression was localized in the epithelial cells of the renal proximal tubules. Treatment of 4-week-old mice with aldosterone and cortisol for three days showed larger increases in both PTHrP mRNA and protein levels in males compared with females. The expression of GRβ in male, but not female, kidneys was significantly upregulated after treatment with cortisol, but not after treatment with aldosterone. Inhibition of glucocorticoid signaling by pre-treatment with a GR antagonist prior to cortisol administration largely abolished this cortisol-dependent increase in PTHrP and GRβ expressions.nnnSIGNIFICANCEnThese results suggest that PTHrP expression and GRβ in the kidneys of male mice may be regulated by cortisol.

Cytotoxic effects of one-step self-etching adhesives on an odontoblast cell line.

The aim of this study was to evaluate the cytotoxic effects of one-step self-etching adhesives. Cells from an immortalized mouse odontoblast cell line (MDPC-23) were cultured with six different dental adhesive systems (diluted to concentrations of 0.5% for 4 h): Adper Easy Bond (EB), Xeno V (XV), iBond (IB), AdheSE One (AO), Clearfil SE primer (CS), and Adper Single Bond 2 (SB). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and flow cytometric apoptosis assays were used to evaluate cell viability and the rate of apoptosis. The odontoblasts were also examined under a scanning electron microscope. While all of the cultures with adhesives showed reduced viability, the viabilities in the IB and SB groups were not significantly different from the control group. Although increased apoptosis rates were observed in all of the cultures with adhesives, the rate in the SB group was not significantly different from the rate in the control. The control group showed the lowest apoptosis rate followed by the SB, AO, IB, EB, XV, and CS groups. When examined under a scanning electron microscope, control odontoblasts and the SB group exhibited relatively large cytoplasmic extensions. In contrast, in the EB and CS groups, fewer fibroblasts remained adhered to the plate surface. Cytoplasmic membrane shrinkage and cell-free areas with residual membrane fragments from dead cells were observed. In conclusion, all cultures with one-step self-etching adhesives showed increased apoptotic activity. SB, an etch-and-rinse adhesive, was comparable to the control group, and CS and EB showed the lowest odontoblast viabilities according to the MTT assay.