S100 Beta Gene Research Articles

Age-dependent expression of S100beta in the brain of mice.

S100beta is a soluble calcium binding protein released by glial cells. It has been reported as a neurotrophic factor that promotes neurite maturation and outgrowth during development. This protein also plays a role in axonal stability and in long term potentiation in the adult brain. The ability of S100beta to modulate neuronal morphology raises the important question whether there is an age-related difference in the expression of S100beta in the cerebral and cerebellar cortices of AKR strain mice and is this change is region specific. Our RT-PCR and Western blotting experiments show that the expression of S100beta gene in the cerebral and cerebellar cortices starts from 0 day, peaks at about 45 days. However, in 70-week old mice its expression is significantly up-regulated as compared to that of 20-week old mice. S100beta follows the same age-related pattern in both cerebral and cerebellar cortices. These results suggest that S100beta is important for brain development and establishment of proper brain functions. Up-regulation of S100beta in old age may have some role in development of age-related pathological systems in the brain.

Evidence for the association of the S100β gene with low cognitive performance and dementia in the elderly

Variations in the S100beta gene may be instrumental in producing a continuum from mild cognitive decline to overt dementia. After screening 25 single nucleotide polymorphisms (SNPs) in S100beta, we observed association of the rs2300403 intron 2 SNP with poorer cognitive function in three independent populations. Moreover, we detected a significant association of this SNP with increased risk of developing dementia or Alzheimer's disease (AD) in six independent populations, especially in women and in the oldest. Furthermore, we characterised a new primate-specific exon within intron 2 (the corresponding mRNA isoform was called S100beta2). S100beta2 expression was increased in AD brain compared with controls, and the rs2300403 SNP was associated with elevated levels of S100beta2 mRNA in AD brains, especially in women. Therefore, this genetic variant in S100beta increases the risk of low cognitive performance and dementia, possibly by favouring a splicing event increasing S100beta2 isoform expression in the brain.

GFAP and S100beta expression in the cortex and hippocampus in response to mild cortical contusion.

We studied the acute response of glial fibrillary acidic protein (GFAP) and S100beta gene expression in the cerebral cortex and hippocampus to mild unilateral cortical contusion. Our goal was to evaluate and compare the expression patterns of each gene in the early stages of the astrocytic response to brain injury. RNA was extracted from the cerebral cortex and hippocampus of male rats at 0, 3, 12, 24, or 96 h after lesion or sham-operation, then quantified using an RNase protection assay. Contusion produced a robust elevation in GFAP mRNA by 12 h in both brain regions on the ipsilateral side to the contusion. In the cortex, but not the hippocampus, this elevation was sustained at 96 h. S100beta mRNA levels were elevated bilaterally in lesioned animals at 24 h in both brain regions. However, these data are difficult to interpret because sham mRNA levels decreased with time, making it unclear whether contusion stimulates S100beta gene expression or whether it mitigates the inhibitory effect of sham. We further analyzed the effect of contusion on GFAP and S100beta immunoreactive astrocyte density at 96 h postlesion or postsham by double-label immunocytochemistry. All detectable astrocytes under all conditions were S100beta immunoreactive in both brain regions. Furthermore, all S100beta immunoreactive astrocytes in the lesioned ipsilateral cortex were also GFAP immunoreactive, whereas only about 11% of S100beta positive cells were also GFAP labeled in the contralateral lesioned or the ipsilateral sham cortex. In the hippocampus, all S100beta immunoreactive cells were also GFAP immunoreactive under all conditions. These data correlate with the gene expression data at 96 h, and suggest that, at least in the cortex, resident S100beta-expressing astrocytes produce GFAP at levels that are undetectable by immunocytochemistry until they are activated in response to injury.

Transcriptional regulation of the human S100 beta gene.

S100 beta is a calcium-binding protein produced and secreted by glial cells in the central and peripheral nervous systems. S100 beta promotes neuronal differentiation and survival but may be detrimental to cells if overexpressed. The selective overproduction of S100 beta has been implicated in the progression of the neuropathological changes in Alzheimer's disease. In addition, at high concentrations, S100 beta stimulates toxic intracellular pathways in cultured cells. To begin to define the regulation of S100 beta expression, we characterized the human S100 beta promoter and mapped its upstream regulatory elements by using a luciferase reporter system. The functional S100 beta promoter was localized to a region -168/ +697 containing 168 bp upstream of the transcription initiation site of the gene. This minimal promoter was active in a variety of cell types, including those of glial, neuronal, and non-neural origin. The human S100 beta promoter activity is regulated by both positive and negative regulatory elements located upstream in the 5' flanking DNA regions. The regions -788/ -391 and -1012/ -788 contain strong positive, cell type-specific regulatory elements. Negative regulatory elements were mapped to the more distal -4437/ -1012 and -1012/ -788 regions of the gene. The -4437/ -1012 negative element suppressed promoter activity in all cell types examined, except C6 glioma cells. These data demonstrate that the expression of the human S100 beta gene is under complex transcriptional regulation that allows for precise control of the S100 beta level in the nervous system.

Accumulation of S100 beta mRNA and protein in cerebellum during infancy in Down syndrome and control subjects.

S100 protein is a 20 kDA calcium-binding protein that accumulates during CNS maturation in mammals. The human gene coding for the beta subunit of S100 protein (S100 beta) is located on chromosome 21, in a subtelomeric position in 21q22.3. In order to investigate the effect of trisomy 21 on S100 beta gene expression, we performed Southern, Northern and Western blot analysis on DNA, RNA and protein, respectively, extracted from the cerebellum of control and Down syndrome (DS) subjects aged 1-18 months. Southern blot analysis revealed a novel EcoRI polymorphism in the S100 beta gene in two of 15 DNA samples examined, and a 1.5 gene dosage for S100 beta in DS. Northern and Western blot analysis showed an approximately 10-fold increase in S100 beta mRNA and protein levels between 1 and 18 months. No differences in the rates of accumulation of S100 beta mRNA and protein were observed between DS and normal subjects. These results demonstrate an increase in S100 beta mRNA and protein levels during infancy indicative of postnatal astrocytic maturation and show that there is no gross deregulation in the expression of the S100 beta gene in DS as a consequence of trisomy 21.

Regulation of the rat S100β gene expression: The role of the 2 kb 5′‐upstream sequence in glial specific expression

We have studied the role of the 2047 bp 5'-upstream region and 232 bp first exon sequence of the rat S100 beta gene in glial specific expression. S100 beta luciferase expression vectors carrying serial deletions of the S100 beta 5' upstream sequence were constructed and transiently transfected into the peripheral nervous system (PNS) glial-type cell line RT4-D6, the PNS neuronal-type cell line RT4-E5, and the central nervous system (CNS) glial-type cell line C6. The hepatoma cell line HTC was also transfected as a nonneural control. From this functional analysis, we found a glial-specific positive regulatory sequence(s) mapped between -583 and -106 relative to the transcriptional start site. This region confers a significantly higher level of luciferase expression in the glial-type cell lines RT4-D6 and C6 than in the neuronal cell line RT4-E5 and the hepatoma cell line HTC. Also, a non-cell type specific positive regulatory element was identified in the first exon sequence between +78 and +232. Though non-cell type-specific, it was found to have a predominant effect in glial cells. From these observations, we have concluded that the 2047 bp 5'-upstream region and 232 bp of the first exon sequence confers the high levels of S100 beta expression in glial cells through these two positive elements.

Female specific hyperactivity in S100 beta transgenic mice does not habituate in open-field.

S100 beta, a calcium-binding brain specific protein, may affect brain development and long-term potentiation. Its gene maps to a region of chromosome 21 duplicated in Down's Syndrome (DS), and its levels are elevated in DS. To test the hypothesis that elevated S100 beta levels cause brain dysfunction in a mammalian system, transgenic mice carrying multiple copies of the human S100 beta gene have been generated and their locomotory patterns are analyzed in open field situations. Female-specific hyperactivity was observed in 2-month-old and in 12-month-old transgenic mice, which rules out the previous speculation that postmenopausal hormonal changes constitute a necessary factor in this behavioral abnormality. Analysis of temporal patterns of activity showed a profound abnormality in transgenic females: the initially elevated activity quickly habituated in males and in normal females, however, its level remained high in the transgenic females throughout the 9-min recording session. These observations are compatible with the suggestion that hippocampal function is abnormal in the females of S100 beta transgenic mice.

Organization, sequence, and expression of the murine S100 beta gene. Transcriptional regulation by cell type-specific cis-acting regulatory elements.

The organization, sequence, and transcriptional regulation of expression of the murine S100 beta gene are reported. The gene is approximately 9 kilobase pairs in length and is composed of three exons and two introns. The deduced murine S100 beta protein sequence differs from the human S100 beta protein by only 1 amino acid. The murine S100 beta gene contains a TATA box (AATAA) and a reverse CCAAT box (ATTGG) located at 30 nucleotides and 92 nucleotides upstream of the cap site, respectively. A 149-base pair DNA fragment (-157/-9) spanning the TATA box and the reverse CCAAT box functions as a promoter. The murine S100 beta promoter drives a 4-fold higher level of transcription in glial (C6) than in non-glial (3T3) cells, suggesting the existence of a potential cell type-specific regulatory element within the promoter region. The 5'-flanking region suppresses transcription from the homologous S100 beta as well as the heterologous SV40 promoters in an orientation-independent fashion. However, the 5'-flanking region exhibits cell type specificity when suppressing the S100 beta promoter-dependent transcription, indicating its involvement in the cell type-specific expression of S100 beta gene. In order to map cell type-specific regulatory elements, transcription analyses of various deletions of the 5'-region were carried out in C6 and 3T3 cells. Two cell type-specific negative regulatory elements, one active in non-glial cells and another active in glial cells, were mapped to the regions -1552/-1234 and -1234/-551, respectively. A strong negative regulatory element and a relatively weak negative element were located in the regions -551/-157 and -1669/-1552, respectively. The murine S100 beta gene is under complex transcriptional regulation involving tonic negative control exerted by combination of multiple cis-acting regulatory elements including cell type-specific elements.

Cell-specific expression of high levels of human S100 beta in transgenic mouse brain is dependent on gene dosage

The beta-subunit of S100 protein (S100 beta) is highly conserved in the mammalian brain. The gene coding for human S100 beta has been mapped to chromosome 21. In order to study the consequences of overexpression of the S100 beta gene, transgenic mice were generated by microinjection of a 17.3 kilobase human genomic fragment containing the three exons and the transcription control elements of the human S100 beta gene. Mice from four transgenic lines carried approximately 10-100 transgene copies. Northern blotting demonstrated a tissue-specific and gene dose-dependent expression of human S100 beta mRNA in mouse brain. Increased expression of S100 beta mRNA was correlated with an increased production of S100 beta protein. Examination of brain sections by in situ hybridization and immunocytochemistry indicated that S100 beta was localized globally to astrocytes, as well as to discrete neurons in the mesencephalic and motor trigeminal, facial, and lemniscus nuclei in both normal and transgenic mice. In peripheral tissues, human S100 beta was expressed at 10-50-fold lower levels than in brain. The strict gene dosage dependence and cell specificity of transgene expression suggest the presence of a locus control region (LCR) in the human S100 beta gene. The mice tolerated 10-100-fold higher than normal levels of S100 beta gene expression in brain without any gross physical or behavioral abnormalities. The high-level expression and cell specificity of the S100 beta promoter/LCR suggest that it may provide a valuable tool to direct the expression of other transgenic products to specific cell types in the CNS.

Cloning and expression of the human S100 beta gene.

S100 protein is a low molecular weight, EF-hand, Ca2(+)-binding protein widely distributed and conserved in the central nervous system of vertebrates. The gene coding for the beta subunit of human S100 protein (S100 beta) has been recently mapped to chromosome 21. In order to study the expression of this gene in normal and abnormal brain development, we have isolated and characterized overlapping genomic clones spanning the region coding for human S100 beta and its flanking sequences. The intron-exon organization of the human S100 beta gene is similar to that of the genes coding for several other members of the S100 protein subfamily of EF-hand proteins. The human S100 beta gene is composed of 3 exons, the first of which specifies the 5'-untranslated region, while the second and third each encode a single EF-hand, Ca2(+)-binding domain. The promoter region contains several potential regulatory transcription elements including the cAMP-responsive elements CRE and AP-2. A novel sequence motif, the S100 protein element, situated in close proximity to the TATA box of the genes of several members of the S100 protein subfamily, has been identified. In addition, multiple repeats with similar nucleotide sequence and location to the recently reported beta globin direct repeat elements have been also found in the human S100 beta promoter. A full length (17.3 kilobases) copy of the human S100 beta gene was constructed and transfected into rat glioma C6 cells. Stable transfectants were shown to express correctly initiated transcripts of the human S100 beta gene, indicating that the cloned sequences contain functional regulatory transcription elements.