Objective
Proteasomal dysfunction in the hippocampus CA1 region after transient global ischemia in gerbil was reported previously. Recently the accumulation of ubiquitin-B (UBB)+1 protein was observed in the neurons of patients with tauopathy that proteasome activity also impaired. UBB+1 is an aberrant protein that was generated by a transcriptional dinucleotide deletion. UBB+1 has a novel COOH-terminus and lacks the functional carboxyl-terminal residue. UBB+1 lose the ability of ubiquitination and blocks the proteasome function. We investigate the contribution of UBB+1 in delayed neuronal death after transient global ischemia.
Methods and materials
Male Mongolian Gerbils weighing 60 to 80 g were anesthetized with 1.5% isoflurane in a mixture of 30% O2 and 70% N2O via a face mask with spontaneous breathing. The bilateral common carotid arteries were transiently occluded using aneurysm clips for 5 minutes. Then the clips were removed to restore the cerebral blood flow. Gerbils were sacrificed under deep anesthesia at 1, 2, 3, 4 days after ischemia (n=6 at each time points) and brains were removed. The hippocampus CA1 region, CA3 and dentate gyrus were separately dissected under microscope and RT-PCR was performed to analyze UBB+1 mRNA in each region of hippocampus. For immunohistochemistry, the brains were perfusion-fixed with 4% paraformaldehyde in PBS and frozen in dry ice. Coronal sections were prepared using a cryostat. Immunostaining of UBB+1 protein by the avidin-biotin-peroxidase complex (ABC) method and double immunofluorescence staining with UBB+1 and TUNEL were performed.
Results
UBB+1 mRNA was detected in every region of hippocampus at every time point after ischemia and also in the non-ischemic control animals. No UBB+1 protein immunoreactivity was observed in every region of hippocampus at 1 day after ischemia and non-ischemic control. In CA1 region, UBB+1 immunoreactivity appeared in the cytoplasm of pyramidal cells at 2 days after ischemia. The cytoplasmic immunoreactivity increased from 2 days to 4 days after ischemia. At 4 days after ischemia, double immunofluorescence staining revealed that UBB+1 immunoreactive CA1 pyramidal neurons co-localized with TUNEL-positive cells. In contrast, UBB+1 protein was transiently detected at 2 days after ischemia in CA3 and dentate gyrus, however, UBB+1 immunoreactive cells disappeared at 3 and 4 days after ischemia. No TUNEL-positive cells were observed in CA3 and dentate gyrus.
Discussion
In human brain, UBB+1 mRNA is generated in both neurodegenerative disease and nondemented control, while, the accumulation of UBB+1 protein is only observed in tauopathy that caused by impaired protein degradation via the ubiquitin-proteasome system. In this study, UBB+1 mRNA was present not only in the dying cells of CA1 but also in the surviving cells of CA3 and Dentate gyrus. In contrast to UBB+1 mRNA, UBB+1 protein only accumulated in CA1 region. We concluded that mutant ubiquitin may be a key mediator of the ubiquitin-proteasome dysfunction-indused neuronal signaling pathway and may have an important role in modifying delayed neuronal death.