Age- and sex-related expression of norbin in the brain cortex of mice S.T. Mani, R.C. Kumar, M.K. Thakur * Biochemistry and Molecular Biology Laboratory, Centre of Advanced Study in Zoology, Banaras Hindu University, Varanasi 221005, India Received 22 March 2001; received in revised form 25 May 2001; accepted 29 May 2001 Abstract Norbin is a novel neuron speci®c protein that extends the neurites of neuronal cells. It is expressed in neural tissues like brain cortex, hippocampus, spinal cord and cerebellum. In this paper, we have studied the expression of norbin mRNA and protein in the brain cortex of male and female mice of different ages. Northern blot analysis showed that the level of norbin mRNA increased in both sexes during aging. However, Western blotting revealed that the protein increased in male but decreased in female with advancing age. These ®ndings suggest that norbin is involved in brain function which is dependent on age and sex. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aging; Norbin; Gene expression; Western blot; Sex; Mouse brain cortex The mammalian brain is remarkably complex and this complexity is evident from the observation that approxi- mately one third of the genome is devoted exclusively to the nervous system [12]. Sex differences in the brain have been widely recognized [15]. They are thought to arise during perinatal development through the action of testos- terone secreted by the developing testes and persist even in the absence of gonadal hormone during adult life. The basic plan of brain and body sex differences is the result of a cascade of events beginning with the role of sex-determin- ing genes in sexual differentiation and continuing with the actions of hormones in embryonic, neonatal, peripubertal and adult life [11]. Norbin (neurite-outgrowth-related brain protein) is a neuron speci®c protein which has been identi®ed by subtractive screening method [16] when long term potentiation (LTP) was induced in rat hippocampal slices following treatment with tetraethylammonium (TEA), a potassium channel blocker. LTP is a representative phenomenon of synaptic plasticity which is directly related to memory and is disrupted during aging [2]. Norbin consists of 729 amino acids and has a molecular weight of 79 kDa. Immunoblotting studies show a single 85 kDa band due to post-translational modi®cation of this protein. It is also expressed in other brain regions such as cortex, cere- bellum and spinal cord. As the brain cortex is involved in learning, memory, intellectual ability and other important functions [13] which change with age and there are sex differences in brain structure [11], we have examined the levels of norbin mRNA and protein in the brain cortex of male and female mice of different ages. Male and female mice of AKR strain representing young (6 weeks) adult (25 weeks) and old (65 weeks) were used. These mice have an average life span of 65 ^ 5 weeks. They were maintained in a colony at 25 ^ 28C with alternating 12 h dark and light schedule. They were fed with standard pellet diet and supplied drinking water ad libitum. The mice were sacri®ced by cervical dislocation and their brain cortex was dissected out. Total RNA from the brain cortex of male and female mice of different ages were isolated [3]. About 25 mg of total RNA was resolved on 1% agarose formaldehyde gel and transferred onto nitrocellulose membrane. The blot was air dried, baked at 808C for 2 h in a vacuum oven and then prehybridized in a solution containing 50% formamide, 5 £ Denhardt's solution, 5 £ SSPE, 50 mg/ml salmon sperm DNA and 0.1% SDS at 428C overnight. Random primer generated 32 P labelled norbin cDNA (5 £ 10 6 cpm) was added in the prehybridization solution and incubation was continued overnight. The blot was washed and exposed for autoradiography. For preparing total cellular extract, the brain cortex was homogenized in 2 vol of lysis buffer containing 175 mM NaCl, 50 mM EDTA, 50 mM Tris±Cl pH 7.5 and 2 mM PMSF. The homogenate was centrifuged at 25 000 £ g for 1 h and the supernatant was used for immunoblotting. The extract containing 25 mg of protein was analyzed by 10% SDS-PAGE [10] and transferred onto nitrocellulose Neuroscience Letters 308 (2001) 57±59 0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0304-3940(01)01981-4 www.elsevier.com/locate/neulet * Corresponding author. Fax: 191-542-368174. E-mail address: mkthakur@banaras.ernet.in (M.K. Thakur).