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, 220 (2), 1195-203

Expression in the Human Brain of Retinoic Acid Induced 1, a Protein Associated With Neurobehavioural Disorders

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Expression in the Human Brain of Retinoic Acid Induced 1, a Protein Associated With Neurobehavioural Disorders

Yara Dadalti Fragoso et al. Brain Struct Funct.

Abstract

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.

Figures

Fig. 1
Fig. 1
Expression of RAI1 mRNA transcript in regions of the human brain and immunohistochemistry for RAI1 in the dentate gyrus. qPCR analysis of RAI1 in the human cerebellum, hippocampus and occipital lobe of the cerebral cortex indicated its presence in all these regions (a). Cerebellar and occipital lobe expression is shown relative to hippocampal expression. Expression was significantly greater in the cerebellum compared to the hippocampus (P < 0.05; ANOVA followed by post hoc t test) and the occipital lobe (P < 0.01). Mouse (b) and rabbit (c) anti-RAI1 antibodies were compared and found to show similar strong expression in the human dentate gyrus and, when combined for double labelling, were found to be identical in distribution in both nucleus and cytoplasm in cells of the dentate gyrus, although expression varied between individual neurons (d). Confocal analysis of RAI1 expression also indicated expression of RAI1 in nuclei and cytoplasm, and expression in the same cells as RARγ (e). Scale bars 50 μm in b, c; 25 μm in d, e
Fig. 2
Fig. 2
RAI1 expression in nucleus and cytoplasm of neurons in the hippocampal CA1 subfield. Double labelling of rabbit anti-RAI1 with MAP2 antibodies demonstrates expression of RAI1 in neurons of CA1 (a) while labelling with GFAP shows its absence in glia in this region (b). Of note was the differing subcellular distribution of RAI1—using the rabbit anti-RAI1 antibody, expression was confined to the nucleus in some cells (c), whereas RAI1 was present in both the nucleus and cytoplasm of others (d). Scale bars 25 μm
Fig. 3
Fig. 3
Expression of RAI1 in the cytoplasm of human SH-SY5Y cells. The expression of RAI1 in the cytoplasm was examined in a cell line in which RAI1 was apparently present in both the cytoplasm and nucleus (a and higher magnification view of three cells in a ı). The rabbit anti-RAI1 detected a protein of approximately 280 kDa by western blotting (b). When SH-SY5Y cells were lysed and separated into nuclear and cytoplasmic fractions, the 280 kDa band was detected by western blotting in the whole cell (WCL) and cytosolic (CYTO) fraction but very little was present in the nuclear (NUC) fraction (c). The cytosolic fraction was uncontaminated by the nuclear fraction as indicated by the absence of the 17 kDa histone H3 protein (c). Scale bar in (a) 25 μm
Fig. 4
Fig. 4
Expression of RAI1 in the human cerebellum and cerebral cortex. RAI1 was strongly expressed in cerebellar Purkinje cells (PC) and was observed in both the nucleus and the cytoplasm of the larger dendrites (a, b), but was absent from cerebellar granule neurons (GC in b). RAI1 was present in cells of layers II to IV of the occipital cortex (c). Scale bars 25 μm
Fig. 5
Fig. 5
Colocalization of RAI1 with the retinoic acid receptors (RARs). In the hippocampal CA1 subfield, RAI1 was expressed in cells that also express RARα, which is predominantly in the nucleus (a), RARβ, present in both the nucleus and cytoplasm (b) and RARγ, which is mainly nuclear in expression (c). Scale bars 25 μm

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