Supplementary results
Deletion of ASIC1a gene remodeled mouse brain mitochondrial proteins
If mtASIC1a is involved in mitochondrial functions, it should functionally and/or physically interact with other mitochondrial proteins within a network. Therefore, deletion of theASIC1a gene could disturb this protein network and alter the status of mtASIC1a-related mitochondrial proteins within it. To test this hypothesis, we performed 2-D gelelectrophoresis and MS analysis to examine alterations in protein expressions in the ASIC1a-/- brains. In 2-D gel analysis, we found 85 different protein spots between WT and ASIC1a null brain samples (Fig. S6a).Among them, 28 significantly (T > 4, equals to p < 0.01) and markedly changed (> 2 folds) proteins were analyzed by MS (Fig. S6a, lower). In particular, 11 were up-regulated, and the rest were down-regulated after deletion of ASIC1a gene (Fig. S6a, lower). These proteins can be classified into four categories according to their localizations: 3 synaptic proteins, 5 cytoskeletal proteins, 11 cytosolic proteins, 2 acidic organelle proteins (Atp6v1a and Atp6v1b2) and 7 mitochondrial proteins (although Pick1 is not typicallya mitochondrial protein, Fig. S6a, lower & 6b). Notably, up-regulated in the ASIC1a-/- brain areglycerol-3-phosphate dehydrogenase 2 (Gpd2) and peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (PIN1), which both are involved in ROS generation1-2. The up-regulation of these proteins may contribute to the elevated ROS level in ASIC1a-/- brains. Furthermore, we found that aldehyde dehydrogenase 7 family member a1 (Aldh7a1), which displays a neuroprotective effect against ROS-induced damage2-3, was up-regulated and 3-hydroxyisobutyryl-CoA hydrolase (Hibch) was down-regulated in ASIC1a-/- brains. Although it remains unclear the role of Hibch in ROS-induced death, the study in fruit fly showed that mutation of Hibch could delay the injury-induced synapse and axon degeneration 4. Changes of these two proteins may be the result of adaptation to the relatively higher level of basal ROSas an enhanced defense system against further ROS-induced damage in ASIC1a-/- brains. It remains to be determined about the link between ROS and the following three proteins, solute carrier family 25 member 25 (Slc25a25), mitochondria transcription termination factordomain containing 2 (Mterfd2), and protein interacting with protein kinase C 1 (Pick1). We should point out that Pick 1 is not exclusively in mitochondria. It is also found in synapses5-6. Because most altered proteins are related to ROS generation and signaling pathways, the remodel of proteins in the ASIC1a null brain may reflect the redox imbalance that occurredin vivo. However, since we have compared the total brain proteins between WTand ASIC1a-/-mice, the subcellular location(s) for the observed alterations was not necessarily mitochondria, but could be other organelles. More detailed examinations are required to further clarify the changes of mitochondrial proteins in ASIC1a null brains.
Supplementary discussion
Moreover, we observed increased mitochondrial volume and ROS levels in the brain of ASIC1a-/- mice. We found that the expression levels of several mitochondrion-associated proteins were altered inASIC1a-deficient mouse brains (Fig. S6). Given the presence of ASIC1a in mitochondria, it is reasonable to speculate that mtASIC1a may contribute to a large array of pathophysiological mitochondrial function(s) including regulation of ROS homeostasis. Indeed,changes were observed in ASIC1a null mouse brains for several proteinsinvolved in ROS generation (Fig. S6b). However, the mechanism(s) underlying the elevated ROS level remains undefined. Considering the up-regulation of Gpd2 and PIN 1 inASIC1a-/- brains, the elevated ROS level could result from an increased ROS generation rather than a decreased ROS scavenging (Fig. S6b). On the one hand, not only severe oxidative stress causes damage to DNA, lipid, carbohydrates and proteins, often leading to loss of their function(s)7,but also oxidative stress induces adaptation through up-regulation of defense systems and neutralization of ROS7. In ASIC1a-/- brains we found a significant up-regulation of an antioxidant factor, Aldh7a1 3and a down regulation of Hibch, which is reported to contribute to neuronal degeneration 4 (Fig. S6b). These could help explain the apparent paradox that although ROS levels were increased in the ASIC1a-/- neurons, the cells were actually more resistant to oxidation-induced death than the WT neurons (Fig.1). It is important to point out that we have only selected the most dramatically changed (> 2 folds) proteins from our 2-D gels for MS analysis. Therefore, the data represent a small fraction of all the altered mitochondrial proteins in the mouse brain resulting from deletion of the ASIC1a gene. Additional more detailed proteomic assays are warranted in order to reveal more complete protein changes in ASIC1a-/- brains and functional assays should be implemented to clarify the meanings of these changes. Furthermore, the oxidative stress caused damages accumulate with aging and may eventually overwhelm the protective effects due to impaired MPT. Such an effect may contribute to age-related diseases such as AD, PD and HD 7-9. Thus mtASIC1a may be of particular relevance to the treatment and prevention of neurodegeneration.
Supplementary references
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Supplementary legends
Figure S1. (a) Fluorescent measurement of Ca2+ uptake using isolated mitochondria from ASIC1a+/+ (upper), in the presence of 100 µM AMI (middle) and 1 M PcTX1 (bottom). (b) Representative images for Calcein-CoCl2 bleaching assays (upper) and the summary data (lower) for WT neurons infected with control virus (Vector) or lentivirus for shRNA of ASIC1a (shRNA). The neurons were either untreated (CRTL) or treated with ionomycin (5 µM).
Figure S2. MS/MS fragmentation of peptide fragments. (a) MS/MS fragmentation of peptide fragment TAVAPIER. Monoisotopic mass of neutral peptide Mr(calc): 855.4814; Matches (Bold Red):14/99 fragment ions using 36 most intense peaks. (b) MS/MS fragmentation of peptide fragment EQGVLSFWR. Monoisotopic mass of neutral peptide Mr(calc): 1120.5665; Matches (Bold Red): 26/128 fragment ions using 46 most intense peaks. immon., immonium ion; Seq, sequence.
Figure S3. ASIC1a is present in mitochondrial fractions. (a) Presence of ASIC1a and organelle markers in mitochondrial preparations by sucrose-based method. (b) Identification of ASIC1a in mitochondrial fractions from HEK 293 cells expressing EGFP-tagged ASIC1a. A protein band of ~ 95 kDa (indicated by the arrow in b1) was subjected to MS analysis (b2) and identified to be ASIC1a (b2, specific peptide fragments of ASIC1a, SFKPKPFNMR and GHPAGMTYAANILPHHPAR. See Figs. S5-1 & S5-2 for details).
Figure S4. Analysis the co-localization of EGFP-ASIC1a and Mito-DsRed in transfected cultured mouse cortical neurons. (a) Same imagesas that shown in Fig. 4b, with white lines indicating the area of analysis. (b1 & b2) Line profiles of the indicated areas. Co-localization of EGFP-ASIC1a (Green) and Mito-DsRed (Red) is intracellular (c1), but not in plasma membrane (c2). (c1 & c2) Cytofluorograms reflect the distribution ofredandgreen pixelsfrom their respective channels of the image. EGFP-ASIC1a and Mito-DsRed show a high degree of co-localization (c1). The cross-correlation function (CCF) reflects the overall trend of co-localization for the double labeled image (c2).
Figures S5-1 & 5-2. MS/MS fragmentation of peptide fragments. (a) MS/MS fragmentation of peptide fragment SFKPKPFNMR. Monoisotopic mass of neutral peptide Mr(calc): 1250.6594; Matches (Bold Red):25/140 fragment ions using 28 most intense peaks. (b) MS/MS fragmentation of peptide fragment GHPAGMTYAANILPHHPAR. Monoisotopic mass of neutral peptide Mr(calc): 2010.0006; Matches (Bold Red):14/310 fragment ions using 11 most intense peaks. immon., immonium ion; Seq, sequence.
Figure S6∣Remodeling of mitochondrial proteins in ASIC1a-/- mouse brains. (a) Representativeimages of 2-D gels (upper) and the distributions of altered proteins in the whole brain of ASIC1anull mice (lower). Others include 11 cytosolic proteins and2 acidic organelle proteins. (b) Table of significantly and markedly (>2 folds) changedmitochondrial proteins (n = 3 from each genotype, T > 4 equals to p < 0.01, by unpaired t test; *, atypical mitochondrial protein, N.A., not available).
Table S1. A list of proteins identified by MSanalysis of the ~ 30 kDa band. The top score is ANT2 (91), followed by ANT1 (64) & ANT3 (64).
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