Expression in the human brain of retinoic acid induced 1, a protein associated with neurobehavioural disorders
- 1.9k Downloads
Retinoic acid induced 1 (RAI1) is a protein of uncertain mechanism of action which nevertheless has been the focus of attention because it is a major contributing factor in several human developmental disorders including Smith–Magenis and Potocki–Lupski syndromes. Further, RAI1 may be linked to adult neural disorders with developmental origins such as schizophrenia and autism. The protein has been extensively examined in the rodent but very little is known about its distribution in the human central nervous system. This study demonstrated the presence of RAI1 transcript in multiple regions of the human brain. The cellular expression of RAI1 protein in the human brain was found to be similar to that described in the mouse, with high levels in neurons, but not glia, of the dentate gyrus and cornus ammonis of the hippocampus. In the cerebellum, a second region of high expression, RAI1 was present in Purkinje cells, but not granule cells. RAI1 was also found in neurons of the occipital cortex. The expression of this retinoic acid-induced protein matched well in the hippocampus with expression of the retinoic acid receptors. The subcellular distribution of human neuronal RAI1 indicated its presence in both cytoplasm and nucleus. Overall, human RAI1 protein was found to be a highly expressed neuronal protein whose distribution matches well with its role in cognitive and motor skills.
KeywordsTranscription Cytoplasmic Smith–Magenis Potocki–Lupski Retinoic acid RAR Cerebellum Hippocampus Cerebral cortex
The retinoic acid induced 1 (RAI1) gene is highly conserved through mammalian evolution (Girirajan et al. 2005), while the corresponding protein is known to be expressed at high levels in the heart and neuronal structures (Toulouse et al. 2003). Its precise action is unclear, but from its similarity to the transcriptional regulator TCF20 (also called stromelysin-1 platelet-derived growth factor-responsive element binding protein, SPBP), it may be part of a complex that regulates transcription and indeed it can act as a transactivator (Seranski et al. 2001; Bi et al. 2004; Carmona-Mora et al. 2010). A lot of interest surrounds RAI1 because of its involvement in neurobehavioral disorders in an intriguing dosage-sensitive manner (Carmona-Mora and Walz 2010). Haploinsufficiency of the RAI1 gene is associated with Smith–Magenis syndrome, a rare disorder that includes craniofacial, behavioural and neurological signs including intellectual difficulties and sleep disturbance, as well as obesity (Slager et al. 2003; Girirajan et al. 2005). Many features of Smith–Magenis syndrome, originally described as resulting from an interstitial deletion in chromosome 17p11.2 (Smith et al. 1986), are caused by the loss of RAI1 which is located in this chromosomal region. In contrast, duplication of this same chromosomal region results in another rare disorder, Potocki–Lupski syndrome, whose features also include neurobehavioral difficulties including features of autism, hypotonia and cardiovascular anomalies. Again, the change in expression in RAI1 is proposed to contribute to the disorder (Cao et al. 2013). RAI1 is also associated with neurodevelopmental disorders that are pervasive into adulthood including schizophrenia (Toulouse et al. 2003) and autism (Carmona-Mora and Walz 2010) as well as adult diseases such as Parkinson’s disease (Do et al. 2011) and cerebellar ataxia (Hayes et al. 2000). The potential action of RAI1 as a regulator of transcription is thus a key to normal function of the adult brain.
RAI1 was first described as a retinoic acid-regulated gene (Imai et al. 1995) from whence derived its name. Retinoic acid is the bioactive metabolite of vitamin A, acting through the ligand-gated nuclear receptor RAR, and which influences motor function, memory and behaviour (Shearer et al. 2012). The initial discovery of RAI1 came from study of the P19 mouse embryonic carcinoma cell line, showing that the expression of GT1, a splice variant of RAI1, was significantly up-regulated after treatment with retinoic acid to induce neuronal differentiation (Imai et al. 1995). The RAI1 promoter contains multiple retinoic acid response elements (RAREs) in its promoter (Laperriere et al. 2007) although there has been no description since that time of regulation of RAI1 by retinoic acid.
Despite the growing knowledge of the role of RAI1 in human neurological and psychiatric diseases, and its high expression in brain (Toulouse et al. 2003), the distribution of this protein in the human brain has yet to be described. The present study aimed to investigate the expression of RAI1 in human hippocampus, cortex and cerebellum, areas likely involved in cognitive and motor functions of RAI1 neural expression (Elsea and Girirajan 2008). In these regions, RAI1 was expressed in neurons, but not GFAP-positive glia. The subcellular distribution of endogenous RAI1 implied both nuclear and cytoplasmic localization, differing from what has been described when RAI1 is overexpressed in cell lines.
The present study was approved by the Ethics Committee of Universidade Metropolitana de Santos, SP, Brazil, and by the Brazilian Health Research Committee on April 4th 2011, designated CONEP 16168, documents registered as 25000.169694/2010-18. Caudal human hippocampi, cerebellar and cerebral cortices were obtained from six male individuals aged 55 years or less who did not present any neurological or psychiatric disease and were collected during necropsy procedures. Brains from individuals whose death was related to head trauma, extensive infection or toxic, anoxic or metabolic injuries were excluded from this study. For immunohistochemistry, samples from the hippocampus, cerebral cortex and cerebellum, measuring typically 0.5 cm3, were fixed in 10 % phosphate-buffered formalin within 24 h of death and processed into paraffin wax blocks within the following 24 h. Further samples from the same areas were collected in RNAlater RNA Stabilization Reagent (Qiagen, Venlo, The Netherlands) and stored at 4 °C for qPCR.
Fluorescence immunohistochemistry was performed as previously described in (Fragoso et al. 2012), adapted from an earlier study (Makitie et al. 2010). Wax-embedded tissue samples were sectioned at 7 μm, mounted onto A380-bond slides (Electron Microscopy Sciences, Hatfield PA 19440, USA) and dried overnight at 37 °C. Mounted sections were dewaxed in xylene and rehydrated through decreasing ethanol concentrations (100, 95, 80 and 70 %). Sections were then boiled for 10 min in sodium citrate buffer, pH 6.0, allowed to cool on the bench for 20 min and then washed in phosphate-buffered saline (PBS) pH 7.4, containing 0.1 % Tween 20 (Sigma) and 1 % pooled human serum (BioSera). Sections were then blocked for 1 h at room temperature in PBS containing 0.3 % Tween 20, 5 % normal goat serum, 5 % bovine serum albumin and 5 % pooled human serum. The sections were next incubated overnight at 4 °C in primary antibody diluted in blocking solution. The following primary antibodies were used: mouse anti-Calbindin (1:500; Sigma, C9848), mouse anti-GFAP (1:500; Sigma, G3893), chicken anti-MAP2 (1:1000; Abcam, ab5392), mouse anti-RAI1 (1:500; Santa Cruz, D-11, sc-365065), rabbit anti-RAI1 (1:500; Abcam, ab58658). After incubation, the slides were washed in blocking solution, then incubated with a fluorescent secondary antibody (anti-rabbit 1:300, Invitrogen or anti chicken 1:400, Jackson Immunoresearch) diluted in blocking solution for 1 h at room temperature. Slides were washed in blocking solution and incubated for 1 min with 10 % Sudan Black (Acros Organics) in 70 % isopropanol to reduce autofluorescence (Schnell et al. 1999; Neumann and Gabel 2002). The slides were then thoroughly washed in distilled water and mounted with mounting medium containing 1,4-diazabicyclo[2.2.2]octane (DABCO, Sigma) and bisbenzimide (Sigma). The sections were digitally imaged on a Zeiss Axio Imager M2 or a Zeiss LSM710 confocal microscope on an inverted Axio Observer Z1 stand.
Quantitative polymerase chain reaction (qPCR)
qPCR was carried out as previously described (Fragoso et al. 2012). Total RNA was extracted from tissue samples using an Isolate RNA kit (Bioline) and cDNA synthesized using a High Capacity RNA-to-cDNA kit (Applied Biosystems). qPCR was carried out using SensiMix SYBR qPCR master mix (Bioline) and primers designed to amplify human RAI1: forward: CCC AGG AGC ACT GGG TGC ATG A, reverse: GCA GCT GGA ACA CAT CAT GTC CAC G. The reference gene used was GAPDH, forward: TCT TTT GCG TCG CCA GCC GA, reverse: AGT TAA AAG CAG CCC TGG TGA CCA. Standard curves and blank controls were run for both genes. Samples (n = 3) were run on a LightCycler 480 thermal cycler (Roche) and analysed using the efficiency-corrected E-Method in Roche LightCycler 480 v1.5 software. Regional differences in RAI1 expression levels were analysed by ANOVA, followed by post hoc t tests.
Human hippocampal protein was extracted in 0.01 M phosphate buffer containing a protease inhibitor cocktail (Sigma) using mechanical homogenization and 3 freeze–thaw cycles. Homogenates were centrifuged for 10 min at 12,000 rpm at 4 °C. Total protein levels in each sample were quantified by the BCA assay (Pierce) and 50 μg total protein per lane was loaded onto a NuPAGE Novex Tris–acetate 3–8 % gradient mini-gel (Life Technologies). After separation, proteins were transferred onto a Hybond ECL nitrocellulose membrane (GE Healthcare) using a Mini Trans-Blot Cell (Bio-Rad) for 4 h and loading was checked with Ponceau S (Sigma). Membranes were probed with the primary antibody that was to be used for anti-RAI1 immunohistochemistry, rabbit anti-RAI1 (1:1000; Abcam). Labelled proteins were detected using a horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Sigma; 1:5000) and enhanced chemiluminescence (ECL; Millipore), incubated for 5 min followed by exposure to X-ray film (Thermo Fisher Scientific).
SH-SY5Y human neuroblastoma cells were grown in Dulbecco’s Modified Eagle (DMEM)/F-12 medium (Gibco), containing 10 % fetal calf serum and penicillin–streptomycin. Cells were harvested by trypsinization, pelleted by centrifugation and the medium discarded. Subcellular fractionation was carried out immediately using a protein and RNA isolation system kit (PARIS kit, Ambion), following the manufacturer’s protocol. Briefly, the cell pellet was resuspended in ice-cold PARIS cell fractionation buffer and incubated on ice for 10 min to lyse the cells. Nuclei were pelleted by centrifugation at 4 °C (500g, 5 min), and then the cytoplasmic fraction was removed into another tube and kept on ice. To reduce contamination by cytoplasmic proteins, the nuclear pellet was washed by gentle resuspension in cell fractionation buffer then centrifuged again. The supernatant was discarded and the nuclei were lysed in PARIS cell disruption buffer. A second cell pellet obtained at the same time was homogenized in ice-cold cell disruption buffer without fractionation to give a whole cell lysate. Anti-RAI1 western blotting was performed as described above. Western blotting against the nuclear protein histone H3 (rabbit anti-histone H3, 1:10.000; Abcam) was performed to confirm that the cytoplasmic fraction was free of nuclear contamination.
Interest in RAI1 has rapidly increased in the past few years because of its association with disease. Deletion of the chromosomal region 17p11.2 results in Smith–Magenis syndrome and haploinsufficiency of RAI1 makes a major contribution to the resulting craniofacial abnormalities, behavioural problems and mental retardation. The reciprocal duplication of this region leads to Potocki–Lupski syndrome, which similarly results in mental retardation, but also hyperactivity and some autism-like features. This study shows, for the first time, the distribution of RAI1 protein in the adult human brain. RAI1 was found to be present in the majority of neurons in both hippocampus and cortex, as expected from earlier studies showing the human brain to be the tissue with highest RAI1 expression and mRNA to be present in all brain regions examined except for the corpus callosum (Toulouse et al. 2003), similar to the distribution in the mouse (Imai et al. 1995). As found in the mouse (Bi et al. 2007), we found that RAI1 is absent from glial cells in the hippocampus and is strongly expressed in cerebellar Purkinje cells but not granule cells. The subcellular distribution of RAI1 was suggestive of expression of RAI1 in both the nucleus and cytoplasm.
The RAI1 protein has multiple putative nuclear localization sequences (NLS) (Slager et al. 2003; Carmona-Mora and Walz 2010) and when expressed in a cell line is transported into the nucleus (Carmona-Mora et al. 2010), while mutant forms of RAI1 lacking the NLS localize to the cytoplasm (Bi et al. 2005; Carmona-Mora et al. 2010; Carmona-Mora et al. 2012). Several studies points to RAI1 functioning as a transactivator (Seranski et al. 2001; Bi et al. 2004; Carmona-Mora et al. 2010) and it induces expression of the central circadian circadian locomotor output cycles kaput (CLOCK) gene while haploinsufficiency of RAI1 disrupts many circadian genes (Williams et al. 2012). RAI1’s regulation of BDNF in the hypothalamus may contribute to the hyperphagia and obesity seen in Smith–Magenis syndrome (Burns et al. 2010) and haploinsufficiency of RAI1 results in abnormal expression of a number of genes associated with obesity, including proopiomelanocortin (POMC). Major gene changes in the hypothalamus in this condition also include alterations in growth hormone together with a number of homeobox-containing transcription factors (Burns et al. 2010).
These results point to RAI1’s presence in the nucleus to regulate transcription. When transfected into cell lines it localizes to the nucleus and is associated with chromatin (Bi et al. 2005, 2010; Carmona-Mora et al. 2012) while a study of lymphoblastoid cells from normal or Smith–Magenis patients indicated expression in the nucleus (Carmona-Mora et al. 2012). Although the smallest predicted splicing variant of RAI1 lacks the NLS and localizes to the cytoplasm (Burns et al. 2010), this is not the large form of RAI1 seen by western blotting in this study. However, it is possible that the protein conformation of this smaller form is such that it is only recognized by antibody in fixed cells and not once denatured for gel electrophoresis and western blotting. It is of interest though that the original identification of the endogenous RAI1 gene, in the mouse P19 embryonic carcinoma cell line (Imai et al. 1995), also described the protein to be present in the cytoplasm of neurons of the adult mouse brain and thus a cytoplasmic role was proposed. It is possible that this is also the smaller form of RAI1. This present study suggests that forms of endogenous RAI1 in the human brain can be expressed in both the nucleus and cytoplasm in neurons suggesting a role for this cytoplasmic expression, even if just to inhibit RAI1’s nuclear action to regulate transcription. The relative intensity of expression of RAI1 in the nucleus and cytoplasm differed even between neurons of the same type implying a variance between these cells that may reflect, for instance, functional activity.
The inducibility by retinoic acid that gives RAI1 its name was reflected in the parallels in this study between the neuronal expression of RAI1 and overlap with retinoic acid receptor expression. Retinoic acid can regulate RAI1 (Imai et al. 1995) and there is a retinoic acid response element (RARE) just upstream of exon 1 (Toulouse et al. 2003); an analysis of the RAI1 promoter found three DR2-type RAREs which are bound by retinoic acid receptors as determined by chromatin immunoprecipitation assay (Laperriere et al. 2007). In the contrary direction, there may be some element of retinoid signalling downstream of RAI1 action. Cell line analysis of genes altered by haploinsufficiency of RAI1 found retinoid x receptor beta (RXRB) as one of the ten main genes up-regulated and also proposed an interrelatedness in phenotype with several diseases that included DiGeorge syndrome and fragile X syndrome (Girirajan et al. 2009). Both of these developmental disorders have been associated with abnormal retinoic acid signalling (Guris et al. 2006; Soden and Chen 2010). The interrelationship between retinoic acid, its receptors (RARs) and RAI1 are not yet understood. Given that RAI1 promotes transcription (Carmona-Mora et al. 2012) and may act as transcriptional co-activator like its homologue TCF20 [also called SPBP (Darvekar et al. 2012)], perhaps RAI1 and the RARs themselves interact. The action of nuclear receptors, such as the androgen and oestrogen receptors, as well as the RARs, are tightly controlled by co-activators and co-repressors (Rochette-Egly and Germain 2009). It is of note that TCF20 acts as a co-activator for the androgen receptor (Elvenes et al. 2011) but is a repressor of phosphorylated estrogen receptor (Gburcik et al. 2005).
The findings of this report provide the first demonstration of high expression of the essential protein RAI1 in neurons of several regions of the human brain. Its presence in both nucleus and cytoplasm may indicate shuttling, and so control, of this protein whose function includes regulation of transcription. Alternatively, different splice variants may be localized to the two subcellular compartments. The coexpression of RAI1 with the retinoic acid receptors supports a functional relationship between these two transcription regulatory proteins.
Funding was provided by the Wellcome Trust and Tenovus Scotland. Prof Fragoso is the recipient of a Post Doctoral Science without Borders grant from the Brazilian National Council for Scientific and Technological Development (CNPq, 237450/2012-7). We also thank Aberdeen Proteomics for assistance with the western blots as well as the Microscopy and Histology Core Facility at the University of Aberdeen for confocal microscopy.
- Bi W, Yan J, Shi X, Yuva-Paylor LA, Antalffy BA, Goldman A, Yoo JW, Noebels JL, Armstrong DL, Paylor R, Lupski JR (2007) Rai1 deficiency in mice causes learning impairment and motor dysfunction, whereas Rai1 heterozygous mice display minimal behavioral phenotypes. Hum Mol Genet 16(15):1802–1813. doi: 10.1093/hmg/ddm128 CrossRefPubMedGoogle Scholar
- Burns B, Schmidt K, Williams SR, Kim S, Girirajan S, Elsea SH (2010) Rai1 haploinsufficiency causes reduced Bdnf expression resulting in hyperphagia, obesity and altered fat distribution in mice and humans with no evidence of metabolic syndrome. Hum Mol Genet 19(20):4026–4042. doi: 10.1093/hmg/ddq317 CrossRefPubMedGoogle Scholar
- Carmona-Mora P, Encina CA, Canales CP, Cao L, Molina J, Kairath P, Young JI, Walz K (2010) Functional and cellular characterization of human retinoic acid induced 1 (RAI1) mutations associated with Smith–Magenis Syndrome. BMC Mol Biol 11:63. doi: 10.1186/1471-2199-11-63 CrossRefPubMedCentralPubMedGoogle Scholar
- Do CB, Tung JY, Dorfman E, Kiefer AK, Drabant EM, Francke U, Mountain JL, Goldman SM, Tanner CM, Langston JW, Wojcicki A, Eriksson N (2011) Web-based genome-wide association study identifies two novel loci and a substantial genetic component for Parkinson’s disease. PLoS Genet 7(6):1002141. doi: 10.1371/journal.pgen.1002141 CrossRefGoogle Scholar
- Williams SR, Zies D, Mullegama SV, Grotewiel MS, Elsea SH (2012) Smith–Magenis syndrome results in disruption of CLOCK gene transcription and reveals an integral role for RAI1 in the maintenance of circadian rhythmicity. Am J Hum Genet 90(6):941–949. doi: 10.1016/j.ajhg.2012.04.013 CrossRefPubMedCentralPubMedGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.