Aberrant alterations of PrPSc, GFAP and Iba1 in the brain samples of 139A- and ME7-infected mice collected at mid-early, mid-late and terminal stages
The clinical, neuropathological and pathogenic features of scrapie agents 139A- and ME7-infected C57 mice were thoroughly described previously. The mean incubation times of the mice infected with agents 139A and ME7 were 183.9 ± 23.1 (154-226) and 184.2 ± 11.8 (165-193) days, respectively [24]. To test the changes of main hallmarks of prion disease in the brain samples of scrapie infected mice collected at mid-early, mid-late and terminal stages, which were employed into further proteomic assays, the levels of PrPSc, GFAP and Iba1 were evaluated by the individual specific Western blots. PK-resistant PrP signals (PrPres or PrPSc) were detected in all brain samples of scrapie infected mice, showing three bands ranging from 20-30 kDa (Fig 1A). Quantitative assay of the signal intensities showed that the PrPSc in the samples of mid-early stage were weak and became much stronger in those of mid-late and late stage (Fig 1A). As expected, the signals of GFAP (Fig 1B) and Iba1 (Fig 1C) in the brain samples of the scrapie infected mice displayed time-dependent increased, while maintained almost unchanged in the brain tissues of normal mice (data not shown).
Global acetylated proteins profiles of the brain samples of 139A- and ME7-infected mice collected at the mid-early, mid-late and terminal stages
The homogenates of cortex regions of three individual mice infected with scrapie agents 139A and ME7 collected at mid-early, mid-late and final stages were pooled as the samples of 139A-80 dpi, 139A-120 dpi, 139A-180 dpi, as well as ME7-80 dpi, ME7-120 dpi, ME7-180 dpi, respectively. Meanwhile, the homogenates of cortex regions of three age-matched individual normal mice were pooled as the normal control. The quality of extracted proteins, quantitative accuracy of proteins, trypsin hydrolysis efficiency, mass spectral mass deviation, mass spectrometry acquisition intensity and data volume fulfilled the requirements of the quality control for proteomic assays. Totally, 271,383 MS/MS spectra were achieved and 63,365 of them were matched spectra. 2,250 proteins, elicited from 7,831 unique peptides, were identified with 95% confidence interval by the Peptide Prophet Algorithm. A total of 1,485 acetylated peptides were identified.
The DEAPs in the brains between scrapie infected and normal mice were calculated. Out of 1,485 acetylated peptides, the DEAPs (changing >1.5 fold) in the brains of 139A-80 dpi, 139A-120 dpi and 139A-180 dpi were 118 (7.9%), 42 (2.8%) and 51 (3.4%), while those in the brains of ME7-80 dpi, ME7-120 dpi and ME7-180 dpi were 390 (26.3%), 227 (15.3%) and 75 (5.1%), respectively (Fig 2A). The DEAPs (changing >2.0 fold) in the brains of 139A-80 dpi, 139A-120 dpi and 139A-180 dpi were 8 (0.54%), 5 (0.34%) and 6 (0.4%), while those in the brains of ME7-80 dpi, ME7-120 dpi and ME7-180 dpi were 12 (0.81%), 36 (2.4%) and 91 (6.1%), respectively (Fig 2B). Obviously, much more decreased DEAPs in the brain samples of the early stage, while more increased DEAPs in the mid-late and late stages of 139A- and ME7-infected mice were identified both in the groups of >1.5 fold and >2.0 fold (Fig 2). Additionally, more DEAPs were found in the brains of ME7-infected mice at every time-point than those of 139A-infected mice. For exploring the potential changes in proteomic profiles among the different brain samples maximally, we used the peptides that were >1.5-fold increased or decreased as DEAPs in this study.
The profiles of DEAPs in mitochondria of the brain samples of 139A- and ME7-infected mice collected at the mid-early, mid-late and terminal stages
Out of 1,485 identified acetylated peptides, 328 peptides were marked as mitochondrial OS, which accounting for 22.1% of total acetylated peptides. 135 acetylated peptides (41.1%) showed differentially expressed (>1.5-fold increased and/or <0.666-fold decreased) in one or more tested samples of scrapie infected mice. The numbers of DEAPs in the samples each time-point of those two scrapie infected mice were illustrated in Fig 3A. Similar as the global profile of DEAPs, the numbers of DEAPs in the brain samples of mid-early stages of scrapie infected mice were much more than those of mid-late and terminal stages. More DEAPs were observed in the samples of ME7 than 139A infected mice. In the samples of 80 dpi, 99 (98%) out of 101 DEAPs in ME7 and 30 (96.8%) out of 31 DEAPs in 139A were down-regulated ones. In the samples of 120 dpi, the decreased DEAPs reduced to 15.9% (7/44) in ME7 and 54.5% (6/11) in 139A. In the samples of late stage (180 dpi), all 4 DEAPs in 139A were increased ones, while 3 (20%) out of 15 DEAPs in ME7 showed increased.
Further, the consistency and dissimilarity of the mitochondrial associated DEAPs each time-point between two scrapie infected mice were analyzed. The acetylated peptides showing differentially expressed in both those two scrapie experimental mice were considered as overlapped ones. The acetylated peptides showing differentially expressed in one infected model, but not differentially expressed while expressed at the same tendency (>1.5 and <1.0 for the increased ones and >1.0 and <0.666 for the decreased ones) in another model were considered as consistent ones. The acetylated peptides showing differentially expressed in one infected model, whereas expressed at the opposite tendency were considered as the dissimilar ones. As shown in Fig 3B, in the samples of mid-early stage, 29 acetylated peptides were overlapped, 73 were consistent and none was dissimilar between 139A and ME7 infected mice. In the samples of mid-late stage, 6 were overlapped, 37 were consistent and 4 were dissimilar. In the samples of late stage, none was overlapped, but 17 were consistent and only one was dissimilar. It indicates that predominantly majority of the identified acetylated mitochondrial associated peptides were consistently expressed in the brains of those two kinds of scrapie infected mice at three time-points during infection, although the samples of ME7-infected mice contains much more DEAPs than 139A-infected ones.
Bioinformatic assay revealed that 328 identified acetylated mitochondrial associated peptides belonged to 74 different proteins, among them 44 (59.5%) proteins showed differentially expressed at least at one tested time-point. The names of those 44 proteins, as well as the numbers of the identified peptides, the numbers of DEAPs, the acetylated positions of the individual differentially expressed proteins and the DEAPs in the tested samples were summarized in Table 1. 9 (20.5%) out of 44 proteins contained 10 or more than 10 identified peptides, including aconitate hydratase (with 6 DEAPs), aspartate aminotransferase (with 14 DEAPs), ATP synthase subunit alpha (with 8 DEAPs), ATP synthase subunit beta (with 5 DEAPs), dihydrolipoyl dehydrogenase (with 6 DEAPs), glutamate dehydrogenase 1 (with DEAPs), isocitrate dehydrogenase [NADP] (with 2 DEAPs), malate dehydrogenase (with 8 DEAPs), pyruvate dehydrogenase E1 component subunit alpha (with 8 DEAPs). In addition, 24 (54.5%) differentially changed proteins contained 2 to 9 identified peptides and 11 (25%) proteins contained only one identified peptide.
Only three up-regulated DEAPs were identified in the samples of mid-early stage, two in ME7- and one in 139A-infected mice. All three up-regulated DEAPs belonged to ATP synthase subunit alpha, and the acetylated sites were at aa. 194 (for both ME7 and 139A) and aa. 541 (for ME7), respectively. Further analysis revealed that the level of the acetylated peptide at aa.541 in the sample of 139A-80 dpi was 1.4675-fold increased. Those two acetylated peptides turned to the normal ranges in the samples of 120 and 180 dpi of both ME7- and 139A-infected mice. 7 down-regulated DEAPs in the samples of 80 dpi changed to significantly up-regulated in 120 dpi. They were acyl-coenzyme A thioesterase 2 acetylated at aa.83, acyl-coenzyme A thioesterase 9 acetylated at aa.102, dihydrolipoyl dehydrogenase acetylated at aa.122, dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex acetylated at aa.268, isocitrate dehydrogenase [NAD] subunit alpha acetylated at aa.200 and aa.214, peptidyl-prolyl cis-trans isomerase F acetylated at aa.85, pyruvate dehydrogenase E1 component subunit alpha acetylated at aa.244 and 313, succinate--CoA ligase [ADP-forming] subunit beta acetylated at aa.88. In parallel, 79 down-regulated DEAPs in the samples of 80 dpi turned to normal ranges in that of 120 and 180 dpi, majority showing increasing trends in 120 dpi and decreasing slightly in 180 dpi. Only 12 down-regulated DEAPs in the samples of 80 dpi maintained still significantly down-regulated in the subsequent samples. In the samples of 120 dpi, 26 DEAPs showed up-regulated only in this time-point, while 7 up-regulated ones were down-regulated significantly in 80 dpi and 6 up-regulated ones maintained in the increased status in 180 dpi. In addition, all 11 down-regulated and 7 up-regulated DEAPs in the samples of 180 dpi were also identified as DEAPs in those of 80 and/or 120 dpi. Those data indicate that during scrapie infection, the acetylation of the mitochondrial related proteins in the whole brain tissues is markedly down-regulated in the mid-early stage, become active in the mid-late stage and turn to relatively silent in the terminal stage.
Involvement of the significant pathways in the brain samples of 139A- and ME7-infected mice collected at the mid-early, mid-late and terminal stages
To analyze the possible involved biological pathway of the DEAPs, Kyoto Encycloppedia of Genes and Genomes pathway analysis was conducted using the software of KOBAS2.0. As shown in Fig 4A, 55, 25, 18 and 39, 13, 10 biological pathways were significantly changed (P<0.05) in the samples of 80, 120 and 180 dpi of ME7- and 139A-infected mice, respectively. The top 10 involved pathways based on their P values and the numbers of the differentially expressed acetylated proteins each tested samples were summarized in Table 2. Six significantly changed biological pathways were identified in all tested samples, including Carbon metabolism, Metabolic pathways, Biosynthesis of amino acids, Glycolysis/Gluconeogenesis, Pyruvate metabolism and Citrate cycle (TCA cycle). Subsequently, the numbers of the involved up- and down-regulated DEAPs of those six pathways at each tested sample were counted (Fig 4B-G). All six pathway showed the similar distributing patterns of DEAPs, with absolutely predominantly down-regulated DEAPs in the mid-early stage and up-regulated DEAPs in the mid-late and late stage. Meanwhile, the numbers of the involved DEAPs in those pathways reduced along with the incubation, particularly in the brain samples of ME7-infected mice. Two kinds of scrapie infected mice models also displayed other differences beside of the numbers of the involved DEAPs. In the samples of 139A-infected mice, all involved DEAPs in the six pathways were down-regulated in 80 dpi and up-regulated in 180 dpi. In the samples of 120 dpi, the DEAPs in three pathways were all up-regulated, while the other three ones contained only one down-regulated DEAP. Similar pattern was also observed in the samples of ME7-infected mice, overwhelming majority of down-regulated DEAPs in that of 80 dpi and majority of up-regulated DEAPs in 120 dpi. In the samples of 180 dpi, the numbers of DEAPs were much less and the ratios of up- and down-regulated DEAPs in the most (5/6) involved pathways were almost same.
Changes of the involved DEAPs in the pathway of citrate cycle (TCA cycle) in the brain samples of 139A- and ME7-infected mice collected at the mid-early, mid-late and terminal stages
Citrate cycle or TCA cycle is an essential metabolism pathway in the aerobic body, which locates in cytoplasm of prokaryote and mitochondria of eukaryote. The acetylation processes of dozens of enzymes in TCA cycles showed significantly changed in the brain tissues during prion infections, covering 13 and 8 down-regulated DEAPs in 80 dpi, 8 and 2 up-regulated ones in 120 dpi, 2 and 2 up-regulated ones in 180 dpi of ME7- and 139A-infected mice, respectively (Fig 4C). Furthermore, the status of each DEAP at those three time-points were analyzed (Table 3). 7 out of 8 down-regulated DEAPs in 80 dpi of 139A-infected mice were also significantly decreased in that of ME7-infected mice. The rest of the involved DEAPs observed only in one type of scrapie infected mouse (5 in ME7 and 1 in 139A) also showed down-regulated tendencies in another model despite of insignificance. Most of those down-regulated DEAPs turned to raise in 120 dpi, among them 4 DEAPs were even significantly up-regulated. All down-regulated DEAPs in 80 dpi dropped down to the normal ranges in 180 dpi. All eight DEAPs in TCA cycle identified in 120 dpi were up-regulated ones, among them, two DEAPs were commonly detected in both 139A-and ME7-infected mice. 3 out of 8 up-regulated DEAPs in 120 dpi still maintained as increased ones, while other 5 ones turned to normal ranges in 180 dpi. In the samples of 180 dpi, only 4 DEAPs were identified in TCA cycle, all of them being up-regulated. Those data strongly indicate that the acetylating processes of the enzymes of TCA cycle in the brain tissues were negatively regulated in the early stage and positively regulated in the mid-late and terminal stage of prion infection.
The potential affected steps in TCA cycle due to the changes of DEAPs in the brain tissues at different time-points during prion infection were evaluated. Dozens of steps in TAC cycle were affected via negatively regulated acetylation for the relevant enzymes in the samples of 80 dpi (left panels in Fig 5A and B). Many steps were also affected in 120 dpi via up-regulated acetylation for the enzymes, particularly in ME7-infected mice (middle panels in Fig 5A and B). In the samples of 180 dpi, the affected steps focused on the processes from succinate to fumarate, fumarate to malate and malate to oxaloacetate, via positively regulated acetylation for succinate dehydrogenase, fumarate hydratase and malate dehydrogenase (right panels in Fig 5A and B). It indicates that the activity of TCA cycle in the context of whole brain is strongly influenced by deacetylation in the early stage and by acetylation in the mid-late and late stage during prion infection.