Elsevier

Neuroscience

Volume 154, Issue 4, 17 July 2008, Pages 1255-1266
Neuroscience

Cellular neuroscience
Transmembrane protein 50b (C21orf4), a candidate for Down syndrome neurophenotypes, encodes an intracellular membrane protein expressed in the rodent brain

https://doi.org/10.1016/j.neuroscience.2008.01.089Get rights and content

Abstract

Transmembrane protein 50b, Tmem50b, previously referred to as C21orf4, encodes a predicted transmembrane protein and is one of few genes significantly over-expressed during cerebellar development in a Down syndrome mouse model, Ts1Cje. In order to assess potential mechanisms by which Tmem50b could contribute to Down syndrome–related phenotypes, we determined the expression patterns of Tmem50b mRNA, as well as Tmem50b protein distribution, expression and subcellular localization. In situ hybridization in mice at embryonic day 14.5 showed cortical plate and spinal cord mRNA expression. By postnatal day 7, strong mRNA expression was seen in the cerebellum, hippocampus and olfactory bulb, with diffuse cortical expression. Quantitative PCR of adult mouse tissue showed Tmem50b mRNA expression in the brain, heart and testis. A rabbit polyclonal antibody was generated against Tmem50b and rat and mouse tissue screening by Western blot, and immunohistochemistry showed that protein expression concurred with mRNA expression. Double immunofluorescence revealed that Tmem50b is highly expressed in rat and mouse glial fibrillary acidic protein–positive cells in vivo and in vitro, but less so in neuronal MAP2- or β-tubulin II-positive cells in vitro. Tmem50b is invariably expressed in cultured mouse neural precursor cells. In adult mouse cerebellum sections, Tmem50b immunoreactivity was found in Purkinje and Golgi cell somata and in Bergmann glial processes. Electron microscopy confirmed that Tmem50b was present on endoplasmic reticulum (ER) and Golgi apparatus membranes. Results indicate that Tmem50b is a developmentally-regulated intracellular ER and Golgi apparatus membrane protein that may prove important for correct brain development through functions associated with precursor cells and glia.

Section snippets

Animals

Animals used in the present study were Wistar rats, NMRI or C57BL/6 mice. Pregnant Wistar rats or Wistar pups were used for in vitro studies while male Wistar rats were used for protein extraction for Western blot. All other experiments were performed with C57BL/6 mice with the exception of embryonic in situ hybridization where NMRI mice were used to ensure maximum comparability with large-scale ongoing studies. Experiments were carried out in accordance with institutional guidelines that aim

Tmem50b mRNA expression

In situ hybridization at E14.5 (Fig. 1A–D) demonstrated high Tmem50b mRNA expression in the embryonic nervous system: the cortical plate, trigeminal ganglion, dorsal root ganglia and the spinal cord. Tmem50b is widely expressed in the postnatal brain at P7 (Fig. 1E–I) including the olfactory bulb, the cortex, the hippocampus and the cerebellum as well as many other regions. Expression is observed throughout layers II–VI of the postnatal cortex (Fig. 1F). In the olfactory bulb, expression is

Discussion

The expression patterns of many chromosome 21 (Hsa21) genes have been determined in a previous large-scale in situ hybridization screen which provided cursory evidence for expression of Tmem50b in the embryonic and adult mouse brain (Reymond et al 2002, Gitton et al 2002). During the current experiments, the Allen brain atlas (brain-map.org; Lein et al., 2007) confirmed the cursory results, suggesting that Tmem50b could be a candidate for DS brain phenotypes. In addition, we have previously

Conclusion

In conclusion, Tmem50b is an intracellular membrane protein expressed in key brain regions throughout development with a marked expression in glial cells that may be involved in cellular responses to cytokines.

Acknowledgments

R.X.M. is a CJ Martin Fellow and P.M.B. is a Senior Principal Research Fellow of the NHMRC, Australia. This project was supported by the Fondation Jérôme Lejeune. P.M.B. acknowledges the Joliot Curie Visiting Professorship and the hospitality of ESPCI. The authors wish to acknowledge the assistance of Sharon Mason, DanaKai Bradford, Luce Dauphinot, George Lutfalla, Jean Delabar, Florence Pouradier, Christophe Leterrier, Fabien Pasteau, Yann Herault, Jack-Christophe Cossec, Tania Vitalis and

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    Present address: Lawrence Berkeley National Laboratory, Genomics Division, Berkeley, CA 94720, USA.

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