Studies On The Expression Of RAE-1 Molecules In The Central Nervous System

Studies in recent years have revealed that MHC-I molecules and their receptors are expressed in the developing central nervous system(CNS) as well as specific regions of the adult brain, where they inhibit the growth and regeneration of neural fibers and suppress synaptic plasticity. In previous studies, we identified a family of retinoic acid-inducible, MHC-I-like proteins named RAE-1. The mouse RAE-1 family is consisted of five RAE-1 members: RAE-1a, RAE-1b, RAE-1d, RAE-1g and RAE-1e. Like MHC-I molecules, RAE-1 proteins contain a-1 and a-2 domains but lack the a-3 and trans-membrane domains, and so they are anchored on the cell surface by a GPI-tail. Since the identification of RAE-1, a large number of studies by other laboratories found that in the immune system, RAE-1 activates NK cells by binding to the activating NK cell receptor NKG2 D, while MHC-1 inhibits NK cells through MHC-1 receptors such as Pir B. Hence, it seems that RAE-1 and MHC-1 act to antagonize each other’s function.In the nervous system, preliminary studies by Northern blot showed that Rae-1 m RNA was highly expressed in the developing embryonic forebrain(E11-E18), but their expression was sharply down-regulated before birth and was barely detectable in the adult brain. Given that most neurons and their associated dendrites and axons are generated in the mid- and late-stages of embryonal development, and retinoic acid plays important roles in neural development, the results above suggest that RAE-1 molecules may be involved in the developmental regulation of neural structures and function by retinoic acid. The structural similarity, sequence homology, and functional antagonism in the immune system between RAE-1 and MHC-I suggest that they may also work together in the developing nervous system to construct the structure and maintain the dynamic function of the CNS. However, until today, no information about the expression, distribution and function of RAE-1 in the CNS is available. Therefore, here we used RT-PCR, Western blot and immunohistochemical staining to examine the expression of Rae-1 m RNA and RAE-1 protein in the CNS, as well as the intracellular localization of RAE-1 protein in neurons.First, we used RT-PCR to analyze expression levels of Rae-1 m RNA in Neuro-2a cells and primary cultures of embryonic neural stem cells, as well as fetal and adult brains. The results showed that Rae-1 m RNA was undetectable in undifferentiated Neuro-2a and primary neural stem cells, but was highly expressed in differentiated Neuro-2a and neural stem cells, as well as E11-E18 mouse brains; the expression became barely detectable in late-stage(E18) embryonic and adult brains. These results suggest that Rae-1 is expressed in cells that are differentiating along neuronal direction and the expression is sharply down-regulated in mature neurons. However, the RAE-1 receptor NKG2 D is undetectable in any of these cells and tissues, suggesting that there may be a different RAE-1 receptor in neurons.Next, we used Western blot and immunostaining to examine RAE-1 protein levels in these cells. Consistent with the expression pattern of Rae-1 m RNA, Western blot result showed that RAE-1 protein was undetectable in undifferentiated Neuro-2a, but were abundant in differentiated Neuro-2a as well as E11-E18 embryonic mouse brains, and weak expression was detected in the adult mouse brain. Because RAE-1 is a highly glycosylated cell surface protein, there were several bands on the Western blot which were bigger than the theoretical size of RAE-1; however, the size of the detected protein bands was reduced after treatment by the N-glycosylation inhibitor tunicamycin, providing support to the authenticity of the detected protein bands. To examine the intracellular localization of RAE-1 protein, we used immunofluorescent staining of differentiated Neuro-2a cells. The results showed that RAE-1 signal was distributed throughout the entire neuronal cell body, with the most abundant RAE-1 signal detected on the tips of growing neurites(likely the growth cones). After treatment by PI-PLC to remove GPI-anchored proteins from the cell surface, RAE-1 signal became much weaker, demonstrating that the detected signal was likely RAE-1 protein. At the same time, immunohistochemical staining of brain tissue sections showed that RAE-1 protein was detectable in the SVZ and hippocampus of developing mouse brains, as well as the SVZ, hippocampus and Purkinje cell layer of the adult mouse brain.Taking together, these results reveal that Rae-1 m RNA and RAE-1 protein have similar expression patterns, suggesting that Rae-1 m RNA is translated into RAE-1 protein in time. RAE-1 molecules are highly expressed in differentiating and maturating neurons, and are abundantly concentrated in the growing tip of extending neural fibers, but are sharply down-regulated in mature neurons, indicating that RAE-1 likely plays important roles in promoting neuronal differentiation, axon guidance or synaptic formation. Low level of RAE-1 in the adult brain suggests that RAE-1 may play a role in maintaining and regulating neural plasticity. However, the detailed biological roles of RAE-1 remain to be investigated in future studies.