Brain Proteomic Research Of Senescence-Accelerated Mouse During Aging Process And The Functional Investigation Of Differential Prtein

The senescence-accelerated prone mouse strain 8 (SAMP8) is an accelerated aging model, which is usually used to investigate aging and age-related disorders. SAM/resistantl (SAMR1) mouse shows normal aging process and is used as a control to SAMP 8. In the present study, in order to find the new age-related protein, we performed the proteomic analysis of hippocampus and cortex in SAMP8 and SAMR1 mice at 5,10, and 15 months of age. Comparison was carried out not only between SAMP8 and SAMR1 mice at the same age, but also between the 5-,10-and 15-month old mice in both strains.In addition, we detected expression level and enzymatic activity of the important differential protein-----ubiquitin carboxy-terminal hydrolase (UCH) in SAMP8 mice and natural aged rat. Furthermore, we explored the effects UCH on the accumulation of phosphorylated tau in primary cultured cortical neurons.Part I.Brain Proteomic Research of SAMP8 during Aging Process1.2-DE protein patterns of hippocampus and cortex from SAM miceReproducible high-resolution 2-DE maps were constructed for hippocampal and cortical proteins of SAM mice respectively. For each gel, more than 1,700 spots were visualized by silver-stained.2. Protein spots changed in SAMP8 compared with SAMR1 miceCompared with age-matched SAMR1 mice,13 spots were found to be differentially expressed in SAMP8 mice. Among them,12 spots were shown to exhibit similar change pattern in the hippocampus and cortex of all the three age grades of SAMP8, in which 1 protein (spot no.1481) were up-regulated,2 proteins (spot no.173a and 293) disappeared,4 proteins (spot no.173b,173c, 1144a and 1404) down-regulated, and 5 proteins (spot no.173d,316,915,1144b, and 1475) presented only in SAMP8. In addition,1 spot (spot no.2379) was found down-regulated only in the hippocampus of 15-month old SAMP8.3. Protein spots changed with age in both strains of SAM miceThe proteomic study of SAMP8 mice indicated that 4 protein spots were differentially expressed with age in the hippocampus, in which 2 proteins (spot no.1333 and 2440) were up-regulated, and 2 proteins (spot no.2524 and 2379) were down-regulated,In the cortex,7 protein spots were found differentially expressed with age. Besides spot no.1333,2440 and 2524, another 4 differential spots were found.1 protein (spot no.2309) was up-regulated, and 3 proteins (spot no.1427,1507 and 1862) were down-regulated.Above 7 differential spots were also found differentially expressed with age in SAMR1 mice, and exhibited similar change pattern. No other fresh differential spots were observed with age only in SAMR1 mice.4. Identification of differential proteinsAmong the differential spots in SAMP8,9 spots were identified by Mascot searches in the NCBI nr database:they were mitochondrial inner membrane protein (also termed mitofilin, spot no.173a-d), UCH-L3 (spot no.1404), adenylate kinase 4 (AK4, spot no.1481), heme binding protein 1 (spot no. 2379), and an unnamed protein product with G1 accession number of gi|12847201 (spot no.1144a and 1144b). Among these proteins, except mitofilin and UCH-L3, the others were reported to be related to aging for the first time by us.And for the spots which were differentially expressed with age,7 spots were successfully identified as N-myc downstream regulated gene 2 (NDRG2, spot no.1333), enolase 2 (spot no.1507), myosin, light polypeptide 3 (spot no.2309), Cu/Zn superoxide dismutase (Cu/Zn SOD, spot no.2524), heme binding protein 1 (spot no.2379), and two unnamed protein product with G1 accession number of gi|74214304 (spot no.1427) and gi|74178239 (spot no.1862). Among these proteins, except Cu/Zn SOD, the others were reported to be related to aging for the first time by us. PartⅡExpression and Functional Investigation of the Important Differential Protein----Ubiquitin Carboxy-terminal Hydrolase (UCH)Section A. Expression and Functional Studies of UCH in SAMP8 Mice and Aged RatsTo verify the proteomic results, we further detected the mRNA, protein level, enzymatic activity of UCH-L3 and its important isozyme UCH-L1, and the downstream product mono-ubiqutin (mono-Ub) and the phosphorylated tau in 5-and 15-month old SAM mice. In addition, we compared the protein level and enzymatic activity of UCH between the natural aged rats (26 month old) and the young rats (3 month old).1. Expression and functional studies of UCH-L1The mRNA level of UCH-L1 in the cortex showed no difference between 15-month old SAMP8 and age-matched SAMR1 mice. And the protein level and enzymatic activity were decreased only in the cortex of 15-month old SAMP8. In the hippocampus and cortex of 5 months and the hippocampus of 15 months, the protein level and enzymatic activity in SAMP8 mice were not changed. In addition, although the protein level of UCH-L1 were not changed, the enzymatic activity were significantly decreased in the hippocampus and cortex of aged rats compared with young rats.2. Expression and functional studies of UCH-L3The mRNA level of UCH-L3 in the cortex showed no difference between 15-month old SAMP8 and age-matched SAMR1 mice. And the protein level and enzymatic activity were both significantly decreased in the hippocampus and cortex of 5,15-month old SAMP8 compared with age-matched SAMR1. In addition, the protein level and enzymatic activity of UCH-L3 in the cortex of aged rats were significantly decreased in comparison with young rats. And the protein level was not change in the hippocampus of aged rats; however, its enzymatic activity was significantly decreased.3. The levels of Mono-Ub and hyperphosphorylated tau in the hippocampus and cortex of 5-, 15-month old SAM miceThe protein level of mono-Ub was significantly decreased in the cortex of 15-month old SAMP8 compared with SAMR1 mice. However, in the hippocampus and cortex of 5 months and the hippocampus of 15 months, mono-Ub level in SAMP8 mice was not changed. In addition, mono-Ub level was decreased with age in both SAMP8 and SAMR1 mice.The phosphorylated tau at Ser 199/202 and Ser396 sites were significantly increased in the hippocampus and cortex of 5,15-month old SAMP8 in comparison with age-matched SAMR1.Section B. Effects of ubiquitin carboxy-terminal hydrolase (UCH) activity on the phosphorylated tau and the underlying mechanisms1. The dose-, and time-dependent effects of UCH-L1 inhibitor (LDN-57444) on its activity in primary cortical neuron cultures2.5,5,7.5 and 10μM UCH-L1 inhibitor decreased its activity in primary cultured cortical neurons in a dose-dependent manner. Then we chose the dose of 7.5μM, and treated the cell for 30 min,1 h,2 h,6 h,12 h,24 h and 48 h. It was shown that UCH-L1 activity was decreased in a time-dependent manner in the period of 30min-12 h, and the enzymatic activity was not further decreased after 12 h.2. The dose-, and time-dependent effect of UCH-L3 inhibitor (4,5,6,7-Tetrachloroindan-1,3-dione) on its activity in primary cortical neuron cultures2.5,5,10 and 20μM UCH-L3 inhibitor decreased its activity in primary cultured cortical neurons in a dose-dependent manner. Then we chose the dose of 10μM, and treated the cell for 30 min,1 h,2 h,6 h,12 h,24 h and 48 h. It was shown that UCH-L3 activity was decreased in a time-dependent manner in the period of 30min～12 h, and the enzymatic activity was not further decreased after 12 h.The results of the dose-dependent relationship showed that 7.5μM UCH-L1 inhibitor and 10μM UCH-L3 inhibitor decreased the same degree of respective enzymatic activity, about 60%.3. Effects of UCH-L1 inhibitor, UCH-L3 inhibitor respectively or simultaneously on the phosphorylated tau in primary cultured neurons.After the primary cultured cortical neurons were treated with 7.5μM UCH-L1 inhibitor and 10μM UCH-L3 inhibitor respectively or simultaneously for 2 h,6 h,12 h,24 h and 48 h, the phosphorylated tau at Ser 199/202 and Ser396 sites were detected by western blot. When compared with control group, the level of pSer199/202 tau and pSer396 tau were significantly increased to the top level after respective or simultaneous treatment with UCH-L3 inhibitor and UCH-L1 inhibitor for 2 h, and decreased to the control level at 6 h and 12 h, then increased again at 24 h and 48 h but still lower than the level at 2 h. The effect of UCH-L3 inhibitor was stronger than UCH-L1 inhibitor under the same inhibitory degree.4. The involvement of glycogen synthase kinase-3β(GSK-3β) and protein phosphatase 2A (PP2A) in UCH inhibitor-induced accumulation of phosphorylated tauThe phosphorylation of tau was dual regulated by protein kinases and phosphatases. Therefore we also detected the effects of UCH inhibitors on the protein levels of pY216/279 GSK-3P (the active form of GSK-3β), total GSK-3βand PP2A.The pY216/279 GSK-3βresults showed that the level of pY216/279 GSK-3βwas significantly increased after respective or simultaneous treatment of UCH-L3 inhibitor and UCH-L1 inhibitor for 2 h, but was not changed after 2 h. The total GSK-3βresults showed that the treatment of above 3 groups did not change total GSK-3βlevel at the time points of 2 h,6 h and 12 h, but its level was significantly decreased at 24 h and 48 h. And the level of PP2A C subunit was evidently decreased after the treatment of above 3 groups for 2 h,6 h,12 h,24 h and 48 h, which did not show time-dependent relationship.These results suggested that GSK-3βactivation and PP2A inhibition were involved in the UCH inhibitor-induced accumulation of hyperphosphorylated tau in the primary cultured neurons.5. Effects of UCH inhibitors on okadaic acid (OA)-induced hyperphosphorylated tau and the underlying mechanisms5.1 Effects of OA on tau hyperphosphorylationThe dose-dependent study showed that OA induced tau hyperphosphorylation at multiple AD sites with a dose-dependent manner. And the time-dependent study showed that tau hyperphosphorylation at multiple sites were significantly increased after treatment with 25 nM OA for 3 h, and continually increased to the top level at 6 h, then gradually decreased. But the hyperphosphorylated level at 12 h and 24 h were still higher than control group, and decreased to the control level at 48 h. It was indicated that OA induced a transient accumulation of hyperphosphorylated tau, and these aggregates could be removed within 48 h, which pointed to an efficient unimpaired proteolytic system in the primary cultured neurons.5.2 Effects of UCH inhibitosr on OA-induced hyperphosphorylated tauPrimary cortical neurons were treated with 25 nM OA in the presence of 10μM UCH-L3 inhibitor or 7.5μM UCH-L1 inhibitor, for 24 h and 48 h. It was showed that in the presence of UCH-L3 inhibitor for 24 h, the level of pSer199/202 tau was significantly increased in comparison with OA treatment alone. But the level of pSer 199/202 tau in the neurons treated with OA and UCH-L1 inhibitor for 24 h had no difference with that of the neurons treated with OA alone. When the neurons were treated with OA and UCH-L3 inhibitor, or treated with OA and UCH-L1 inhibitor for 48 h, the level of pSer 199/202 tau was significantly increased compared with OA treatment alone.The results of pSer396 tau showed that in the presence of UCH-L3 inhibitor for 24 h, the level of pSer396 tau was significantly increased in comparison with OA treatment alone. But the level of pSer396 tau in the neurons treated with OA and UCH-L1 inhibitor for 24 h had no difference with that of the neurons treated with OA alone. For the time point of 48 h, the level of pSer396 tau in the neurons treated with OA and UCH-L3 inhibitor, or treated with OA and UCH-L1 inhibitor had increasing trend in comparison with that of neurons treated with OA alone, but there was no statistical difference.These results suggested that the accumulation of hyperphosphorylated tau induced by OA could not be removed when UCH activity was simultaneously inhibited. And this effect of UCH-L1 inhibitor was tenuous, but the effect of UCH-L3 inhibitor was significantly stronger than UCH-L1 inhibitor.5.3 The involvement of GSK-3βin the accumulation of phosphorylated tau induced by UCH inhibitor and OAThe level of pY216/279 GSK-3P was significantly increased after treatment with 25 nM OA alone for 3 h, and continually increased to the top level at 6 h, then gradually decreased. But its level at 12 h were still higher than control group, and then decreased to the control level at 24 h and 48 h. And the expression level of total GSK-3βwas not changed during the whole period of OA treatment.In addition, in the presence of UCH-L3 inhibitor or UCH-L1 inhibitor for 24 h, the levels of pY216/279 GSK-3P and total GSK-3P were not changed in comparison with OA treatment alone for 24 h. For the time point of 48 h, the level of pY216/279 GSK-3βwas increased and the level of total GSK-3βwas decreased in the presence of UCH-L3 inhibitor, and the level of pY216/279 GSK-3βwas not changed and the level of total GSK-3P was decreased in the presence of UCH-L1 inhibitor in comparison with OA treatment alone. It was suggested that GSK-3βactivation was involved in the stabilization of OA-induced phosphorylated tau by UCH inhibitor.5.4 The involvement of PP2A in the accumulation of phosphorylated tau induced by UCH inhibitor and OAThe expression level of PP2A C subunit was not changed during the whole period of OA treatment alone. In the presence of UCH-L3 inhibitor for 24 h and 48 h, the expression level of PP2A C subunit had no difference with OA treatment alone. However, the expression level of PP2A C subunit was significantly decreased in the presence of UCH-L1 inhibitor for 24 h and 48 h. These results indicated that PP2A inhibition was involved in the stabilization of OA-induced phosphorylated tau by UCH-L1 inhibitor, but not involved in this effects of UCH-L3 inhibitor.