Distribution of Smad mRNA and proteins in the rat brain.

Smad proteins are known to transduce the action of TGF-ß superfamily proteins including TGF-ßs, activins, and bone morphogenetic proteins (BMPs). In this study, we examined the expression of Smad1, -2, -3, -4, -5, and -8 mRNA in the rat brain by means of RT-PCR and in situ hybridization (ISH). In addition, we examined the nuclear accumulation of Smad1, -2, -3, -5, and -8 proteins after intracerebroventricular injection of TGF-ß1, activin A, or BMP6 with immunohistochemistry to investigate whether TGF-ß, activin, and/or BMP activate Smads in the rat brain. RT-PCR analysis revealed that Smad1, -2, -3, -4, -5, and -8 mRNA was expressed in the brain and that the Smad3 and Smad8 mRNA differed in the expression level between brain regions. For example, there were high levels of expression of Smad3 mRNA in the cerebral cortex, caudate putamen/globus pallidus, and cerebellum, but low levels in the thalamus and midbrain. Expression of Smad8 mRNA was higher in the midbrain, cerebellum, and pons/medulla oblongata in comparison to the olfactory bulb, cerebral cortex, caudate putamen/globus pallidus, hippocampus/dentate gyrus, and thalamus. ISH signals for Smad1 mRNA were widely detected in the brain except for a small number of regions including the olfactory tubercle, posterior region of hypothalamus, and cerebellar nuclei. ISH signals for Smad2 mRNA were abundantly observed in several brain regions including the olfactory bulb, piriform cortex, basal ganglia, cingulate cortex, epithalamus, including the pineal gland and medial habenular nuclei, hypothalamus, inferior colliculi of the midbrain, and some nuclei in the pons, cerebellar cortex, and choroid plexus. ISH signals for Smad3 mRNA were also abundantly observed in several brain regions. Especially strong signals for Smad3 mRNA were observed in the olfactory tubercle, piriform cortex, basal ganglia, dentate gyrus, and cingulate cortex. ISH signals for Smad5 and Smad8 mRNA were restricted to a small number of brain regions, the signal intensity of which was weak. ISH signals for Smad4 mRNA were detected in all regions examined. Intracerebroventricular injection of activin A induced nuclear accumulation of Smad2 and Smad3 immunoreactivity in neurons. In contrast, intracerebroventricular injection of TGF-ß1 or BMP6 did not induce nuclear accumulation of the immunoreactivity for any Smad in neurons. These results suggest that activin-Smad signaling plays an important role in brain homeostasis.

Proteomic analysis of the hippocampus in naïve and ischemic-preconditioned rat.

The hippocampus exhibits regional differences in vulnerability to ischemia, wherein pyramidal cells in the CA1 region are vulnerable to ischemia while pyramidal cells in the CA3 region and granule cells in the dentate gyrus (DG) region are relatively ischemia resistant. However, pyramidal cells in the CA1 region reportedly exhibit ischemic tolerance following exposure to a brief non-lethal period of ischemia known as ischemic preconditioning. In this study, we used proteomic analysis to examine the difference in protein expression between naïve rat CA1 and CA3/DG regions, as well as the altered protein expression in the CA1 region after 3min of ischemic preconditioning. Proteomic analysis identified ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1), glutathione S-transferase µ5 (GSTµ5), glutamine synthetase (GS), and dynamin-1 as proteins with differential expression levels in naïve CA1 and CA3/DG regions. The difference in expression levels of GSTµ5 and GS between these two regions was further confirmed by western blot. Our analysis also identified aconitase2, α-tubulin, protein-l-isoaspartate O-methiltransferase (PIMT), and voltage-dependent anion channel 1 (VDCA1) as proteins with down-regulated expression levels in the CA1 region following 3min ischemic preconditioning. The decrease in the expression of aconitase2 was also confirmed by western blot and immunohistochemical staining. The present results suggest that GSTµ5 and GS may be associated with ischemia-resistance in the CA3/DG region and that aconitase2 may play a part in the ischemic tolerance in the CA1 region.