Effect Of NSSR1Gene Neuron-specific Knockout On The Development Of Mice Brain

NSSR1(neural salient serine/arginine rich protein-1) is one of the few SR proteins which expresses highly in nervous system. It regulates gene expression via regulating pre-mRNA alternative splicing. It has been reported that, the NSSR1transcripts are widely expressed in human and mouse tissues, while its proteins are expressed specifically in the brain, which shows a neuronal-specific expression character. NSSR1has also been reported to promote neural differentiation in cultured cells and in lower organisms, suggesting that it may play an important role in brain development. However, little has been known about the function of NSSR1in vivo in higher organisms up to now. The purpose of this thesis is to explore the in vivo role of NSSR1protein on neural development using NSSR1neuronal conditional knockout mice (cKO mice).We first investigated the distribution characteristic of NSSR1proteins in the brain of wild type (WT) mice using immunohistochemistry (IHC) analysis. The results showed that NSSR1proteins are highly expressed in the olfactory bulb, cerebral cortex, hippocampus, superior colliculus, inferior colliculus and cerebellum, however rarely or not expressed in other brain regions. Through investigating the co-localization of NSSR1proteins and NeuN (marker of maturated neurons) or GFAP (marker of astrocytes) by immunofluorescent analysis, we observed that the NSSR1protein is mainly expressed in neurons, especially in the nucleus of neurons, showing that the expression of NSSR1proteins is neural tissue and neuron cell-specific.It has been reported recently that systematic knockout of NSSR1leads to embryonic death of mice, implying the necessity of NSSR1cKO mice for exploring the in vivo role of NSSR1. Based on the neuron-specific expression character of NSSR1proteins, we constructed NSSR1neuronal conditional knockout mice using the Cre/loxP system. For the NSSR1knockout, frame-shift mutation was made on NSSR1gene by removing the exon3, and the translation of NSSR1terminated ahead of time, producing a truncated no-functional NSSR1protein. We first constructed the NSSR1Flox mice, which contain a loxP site at5’-and3’- ends of NSSR exon3, then by crossing the NSSR1Flox mice with the Nse-Cre mice, Cre+Flox+/+mice were obtained. The Cre+Flox+/+mice were proved to be neuron-specific NSSR1knockout mice by identifying the gene types and the expression level of NSSR1proteins in neurons of mice brain, which showed that exon3has been removed from the genome of neurons in the brain, and NSSR1protein expression decreases significantly in neurons.We further investigated the in vivo role of NSSR1protein in the development of mice brain using the NSSR1cKO mice by exploring the phenotype, brain structure, and behavior of the mice.We first compared the body weight, shape and weight of the brains between the cKO mice and the littermate wild type mice at different developmental stages, the results shows that at1d,7d,14d,21d,1month,2month after birth, no obvious difference existed either in the body weight or in the shape and weight of the brains between cKO and the littermate WT mice, which means that NSSRl gene cKO does not cause obvious change in the phenotype of the mice.We further analyzed the shape and structure of the cKO mice brain using crystal violet staining and immunohistochemistry staining of the neuronal marker NeuN and the astrocyte marker GFAP, the results showed that the CV staining of the neurons in cKO mice is obviously lighter, the depth of the cerebrum cortex is thicker, and the size of neuron cells in the cerebrum cortex became smaller, the organization between cells in the cerebrum cortex is looser, compared with those in the littermate WT mice. The results of the NeuN and GFAP immunohistochemistry staining showed that, in the cKO mice brain, the NeuN staining is lighter, the number of GFAP stained astrocytes, and the size and processes of astrocytes increased, compared with those in the littermate WT mice. The results showed that NSSR1cKO cause changes in the organization, number and cell character of neurons and astrocytes in the brain, which may be mediated by changing the proliferation, apoptosis, differentiation or maturation of the cells in the brain.To further investigate whether NSSR1conditional knockout cause change of cell proliferation, apoptosis, differentiation or maturation in the mice brain, we analyzed the mice sections with DCX and MAP2immunohistochemistry (immunofluorescent analysis), which stain neurons of different developing status, the results showed that, compared with those in the littermate WT mice, the number of DCX positive newborn neurons in the visual cortex of cKO mice increased, and the density and length of MAP2positive neuronal dendrites in cerebrum cortex decreased obviously. While in hippocampus, the DCX positive signal decreased, and the dendrites of DCX positive newborn neurons are shorter, while those of the matured MAP2positive neurons are longer in the cKO mice.As hippocampus is the brain region which plays important roles in learning and memory, and NSSR1conditional knockout leads to changes in the organization, number and cell character of neurons and astrocytes in hippocampus and the cerebrum cortex, we further want to know whether NSSR1conditional knockout has any effect on learning and memory function of mice using morris test and object recognition test, the results showed that the learning ability of cKO mice decreased obviously compared with that of the littermate WT mice.In conclusion, in this thesis, we successfully constructed NSSR1neuron-specific knockout mice, and investigated the in vivo role of NSSR1in the development of mice brain using this mice model by exploring the brain structure and behavior of the mice. The results showed that NSSR1conditional knockout changes the numbers and organization of neurons and astrocytes in cerebrum cortex and hippocampus, the differentiation and maturation of neurons, cell proliferation and apoptosis, as well as the learning and memory ability of the mice. These results imply that NSSR1is involved in the development of mice brain and furthermore, the learning and memory activity of the mice via regulating the cell proliferation, apoptosis, or neuron differentiation and maturation.