1. Sex Hormones in Autism - Androgens and Estrogens Differentially and Reciprocally Regulate RORA, a Novel Candidate Gene for Autism 이진세, 임민혁, 육근영, 나미라, 이지수

2. Contents Abstract Results Method Discussion Introduction

3. Abstract  Autism, a pervasive neurodevelopmental disorder manifested by deficits in social behavior and interpersonal communication is inexplicably biased towards males by a ratio of,4:1, with no clear understanding.  Here, we show that male and female hormones differentially regulate the expression of a novel autism candidate gene, retinoic acid-related orphan receptor-alpha (RORA) in a neuronal cell line, SH-SY5Y.  We further show that aromatase protein is RORA has the potential to be under both negative and positive feedback regulation by male and female hormones, through one of its transcriptional aromatase, and further suggest a mechanism for introducing sex bias in autism.

4. Introduction  Autism refers to a spectrum of neurodevelopmental disorders with a prevalence of,1:110 that are characterized by deficits in social understanding and interactions, aberrant language development and/or usage, and repetitive, stereotyped behaviors, often with restricted interests[1,2].  The disorder is inexplicably biased towards males by a ratio of at least 4:1, prompting theories that fetal or perinatal exposure to elevated levels of male hormones may increase susceptibility towards autism[3].  Despite some evidence linking elevated fetal testosterone levels in amniotic fluid to autistic symptomatology[4,5] and increasing rightward asymmetry of the corpus callosum[6], and our own studies which have identified deregulated genes involved in androgen biosynthesis as well as higher testosterone levels in lymphoblastoid cells from autistic individuals [8], no sex hormone-sensitive candidate genes for autism have been reported. At present, there is still no clear understanding of the molecular mechanisms through which the sex hormones may play a role in autism susceptibility.

5. Introduction  We have recently identified a novel autism candidate gene, retinoic acid-related (RAR) orphan receptor-alpha (RORA)[9], which is a hormone-dependent transcription factor. In brief, our combined studies have demonstrated: a) reduced expression of RORA in lymphoblastoid cell lines (LCL) derived from autistic individuals[10,11]; b) increased methylation and reduced protein expression of RORA in the LCL[9]; and c) decreased expression of RORA protein in autistic brain[9].  Together, these results link molecular changes in RORA in peripheral cells to molecular pathology in the brain of autistic individuals. These findings are particularly relevant to ASD as RORA is involved in several key processes negatively impacted in autism, including Purkinje cell differentiation[12], cerebellar development[13,14], protection of neurons against oxidative stress[15], suppression of inflammation[ 16], and regulation of circadian rhythm[17].  Behavioral studies on the RORA-deficient staggerer (RORA+/sg) mouse, primarily used as a model to study ataxia and dystonia[13], further show that RORA is also associated with restricted behaviors reminiscent of ASD, such as perseverative tendencies[ 18], limited maze patrolling[19], anomalous object exploration[20] as well as deficits in spatial learning[21].

6. Introduction  Here, we show that the level of RORA expression can be regulated by both male and female hormones through their respective receptors, and that one of its transcriptional targets is CYP19A1 (aromatase), an enzyme responsible for the conversion of testosterone to estrogen. We further show that the amount of aromatase protein in brain tissues from autistic and nonautistic donors correlates with the amount of RORA protein, with a statistically significant reduction of both RORA and aromatase in autistic tissues. We thus propose that in ASD, down-regulation of RORA is exacerbated by a negative feedback mechanism involving reduction of aromatase that can result in build-up of its substrate testosterone which, in turn, can further suppress RORA expression.

7. Result Male and female hormones oppositely regulate RORA expression in human neuroblastoma cells. RORA: a hormone-dependent transcription factor that is involved in a variety of functions impacted in autism Estrogen: neuroprotective effects on the brain and RORA has been reported to be neuroprotective against oxidative stress and inflammation

8. Result - ① concentration

9. Result - ② time

10. Result - ③ chromatin immunoprecipitation (ChIP)-PCR analyses RORA is a potential transcriptional target of both AR and ER.`

11. Result - ④ chromatin immunoprecipitation (ChIP)-PCR analyses

12. Result - ⑤ chromatin immunoprecipitation (ChIP)-PCR analyses Aromatase expression is enhanced by RORA overexpression. Aromatase is a potential transcriptional target of RORA.

13. Result - ⑥ confocal immunofluorescence analyses of tissue arrays RORA and aromatase proteins are decreased in postmortem brain tissues from autistic subjects.

14. Result - ⑦ confocal immunofluorescence analyses of tissue arrays RORA and aromatase proteins are decreased in postmortem brain tissues from autistic subjects.

15. Result - ⑧ confocal immunofluorescence analyses of tissue arrays Aromatase protein is decreased and correlated with RORA protein Level in postmortem brain tissues of autistic individuals expression of aromatase protein in the neurons was also strongly correlated with RORA expression

16. Discussion A model for reciprocal hormonal effects on RORA. the observed reduction in RORA in autistic brain may lead to increased testosterone levels through down regulation of aromatase. Through AR, testosterone negatively modulates RORA, whereas estrogen upregulates RORA through ER.

17. Materials and Methods cell culture SH-SY5Y? The human neuroblastoma cells How to culture? 1:1 Modified Eagle’s Medium + Ham’s F12 media (15% fetal bovine serum, 1% penicillin/streptomycin) at 37uC with 5% CO2 split 1:2 every 3–4 days when the cells reached,80% confluency. How to harvest? the cells were treated with trypsin-EDTA for 2–3 min to release them from the surface of the culture flask, followed by the addition of media to inactivate the trypsin. The detached cells were then pelleted by spinning at 2,000 rpm for 5 min at 4uC and gently washed twice with cold PBS.

18. Materials and Methods Hormone treatment ConcentrationTime 0.1 nM, 1 nM, and 10 nM of DHT or estradiol for 2 hrs. 10 nM DHT or 1 nM estradiol → harvested at 1 hr, 2 hrs, 4 hrs, 8 hrs, and 24 hrs after treatment. An equal volume of absolute ethanol ⇒ used control for both experiments. SH-SY5Y cells were carefully washed twice in situ with phenol red-free 1:1 DMEM/F12 media supplemented with 15% charcoal dextran-treated serum and 1% P/S, and then cultured in the phenol red-free medium for 24 hours to deplete the medium of exogenous hormones.

19. Materials and Methods Plasmid Transfection The DNA sequence coding for the DNA binding and transcription activation sites of human RORA was cloned into the pSG5. To enhance RORA expression, the plasmid was transfected into the cells using Lipofectamine LTX and PLUS reagents. Briefly, SH-SY5Y cells were plated in a 6-well culture plate containing complete growth media without antibiotics and incubated until the cell density was 50–60% confluent. The plasmid was diluted in 500ml of Opti-MEM I Reduced Serum Medium and 1.25 ml of PLUS reagent was added to the diluted plasmid solution.

20. Materials and Methods Plasmid Transfection Lipofectamine LTX (25 ml) was then added to the plasmid solution and incubated for 25 min at room temperature to form plasmid-Lipofectamine LTX- PLUS complexes. The growth medium was aspirated without disturbing the cells, and 500ml of the plasmid- Lipofectamine LTX-PLUS complexes were added. The cells were incubated with the complexes For the negative control: the empty plasmid was transfected into the cells. Mock treatment without plasmid was also conducted to serve as an additional negative control

21. Materials and Methods Quantitative RT-PCR analyses Quantitative RT-PCR analyses were performed as described. 1. total RNA was isolated from the cells using the TRIzol isolation method 2. cDNA was synthesized using iScript cDNA Synthesis. (at 25uC for 5 min, 42uC for 30 min, ending with 85uC for 5 min) 3. After reverse transcription, the cDNA reaction was diluted to a volume of 50 ml with nuclease-free water and used as a template for qPCR analyses.

22. Materials and Methods Identification of potential binding sites for AR, ER, and RORA Putative binding sites for the transcription factors were identified using PROMO 3.0, a web-based program that employs the TRANSFAC database version 8.3 to construct specific binding site weight matrices for prediction of transcription factor binding sites. (JASPAR & ChampionChIP databases & UCSC Genome Browser) - selected for ChIP-qPCR analyses

23. Materials and Methods ChIP-qPCR analyses the SH-SY5Y neuroblastoma cells were cultured as previously described and treated with DHT or estradiol for 2 hrs. The cells were then treated with 37% formaldehyde for exactly 10 min to crosslink the proteins to the DNA. Nuclear extraction was performed using the Active Motif Nuclear Extract Kit Nuclear pellets were sonicated on wet ice to shear the chromatin to 200–1,000 bp. Sonicated chromatin was then divided into aliquots for 3–4 immunoprecipitation (IP) reactions.

24. Materials and Methods ChIP-qPCR analyses Each IP reaction was conducted using 1 mg of mouse monoclonal anti-AR, mouse monoclonal anti-Era, or rabbit polyclonal anti-RORA antibodies. Negative control: Sonicated chromatin was also immunoprecipitated with normal mouse IgG or normal rabbit IgG antibodies Positive control: Anti-RNA polymerase II was also included.

25. Materials and Methods ChIP-qPCR analyses IP-DNA was then reverse- crosslinked to separate it from the proteins and purified using Millipore DNA purification columns. Real-Time qPCR analyses were conducted to measure the enrichment of each putative transcription factor binding site in the IP-DNA. Fold-enrichment of each binding site was determined by normalizing the Ct value of the transcription factor binding site in immunoprecipitated DNA with the Ct value of that transcription factor binding site in IgG-IP DNA.

26. Materials and Methods Confocal immunofluorescence microscopy

27. Materials and Methods Statistical analyses ANOVA analyses were performed to determine significance of the differences in expression levels between the hormonally- treated and untreated samples as well as the fluorescence intensities between the autistic and the control groups.