Conceived and designed the experiments: YW. Performed the experiments: YW EM. Analyzed the data: YW. Contributed reagents/materials/analysis tools: YF KD KY MK TN KN. Wrote the paper: YW.
The authors have declared that no competing interests exist.
Multiple cellular functions are compromised in amyotrophic lateral sclerosis (ALS). In familial ALS (FALS) with Cu/Zn superoxide dismutase (SOD1) mutations, the mechanisms by which the mutation in SOD1 leads to such a wide range of abnormalities remains elusive.
To investigate underlying cellular conditions caused by the SOD1 mutation, we explored mutant SOD1-interacting proteins in the spinal cord of symptomatic transgenic mice expressing a mutant SOD1, SOD1Leu126
These results indicate that disease-causing mutant SOD1 likely leads to inadequate protein-protein interactions. This could be an early and crucial process in the pathogenesis of FALS.
Amyotrophic lateral sclerosis (ALS), a progressive and fatal disorder of the central nervous system (CNS), selectively affects both upper and lower motor neurons in the cerebral cortex, brain stem and spinal cord. Approximately 10 percent of ALS cases are of the hereditary type (familial ALS; FALS), and about 20% of FALS cases are associated with Cu/Zn superoxide dismutase (SOD1) mutations
To investigate the nature of the mutant SOD1, we have studied a SOD1 mutation characterized by a 2-bp deletion at codon 126 (SOD1Leu126delTT)
Two lines of transgenic mice were used in this experiment. The DF (
Simulation models of the FLAG-tagged SOD1s were constituted using Discovery Studio 1.7 software (Accelrys, USA) from Protein Data Bank (PDB) data; 1MFM for DF, 2V0A for WF as templates. The red parts indicate the FLAG epitopes. The DF mutant SOD1 possibly exposes a hydrophobic structure, which is normally buried by the dimer interface.
DF | WF | NTG | ||||||||||
Protein | Abbreviation | Accession no. | Probability score | No. of peptide | Protein | Accession no. | Probability score | No. of peptide | Protein | Accession no. | Probability score | No. of peptide |
heat shock cognate 70 | Hsc70, HspA8 | 13242237 | 170.22 | 20 | heat shock cognate 70 | 13242237 | 30.17 | 3 | ||||
heat shock protein 70-2 | Hsp70-2, HspA2 | 123621 | 80.25 | 10 | heat shock protein 70-2 | 123621 | 30.16 | 3 | ||||
heat shock 70 protein 1-like | Hapa1L | 56757584 | 40.25 | 4 | ||||||||
heat shock protein 9A | HspA9A | 6754256 | 30.16 | 3 | ||||||||
ubiquitin carboxy-terminal hydrolase L1 | UCHL1 | 61098212 | 20.24 | 3 | ||||||||
Na/K ATPase α 2 subunit | ATP1A2 | 30409956 | 120.19 | 12 | ||||||||
Na/K ATPase α 3 subunit | ATP1A3 | 27552786 | 100.20 | 10 | ||||||||
Na/K ATPase α 1 subunit | ATP1A1 | 21450277 | 40.18 | 4 | ||||||||
ATP synthase β subunit | ATP5B | 31980648 | 80.25 | 9 | ||||||||
ATP synthase α subunit | ATP5A | 6680748 | 50.20 | 5 | ||||||||
ATP synthase γ subunit | ATP5C | 11602916 | 30.17 | 3 | ||||||||
α (non-neuron) enolase |
ENO1 | 12963491 | 70.25 | 7 | pyruvate kinase | 2506796 | 50.22 | 5 | ||||
lactate dehydrogenase | LDH | 6678674 | 60.22 | 6 | ||||||||
brain glycogen phosphorylase |
PYGB | 24418919 | 30.25 | 3 | ||||||||
phosphofructokinase 1 | PFKA, PFKM | 13638207 | 30.20 | 3 | ||||||||
malate dehydrogenase 1 | MDH1 | 31982178 | 30.17 | 3 | ||||||||
aconitase 2 | ACO2 | 18079339 | 30.16 | 3 | ||||||||
glyceraldehyde-3-phosphate dehydrogenase | GAPDH | 6679937 | 20.33 | 3 | ||||||||
α tubulin | TUBA | 34740335 | 140.22 | 14 | keratin | 6678643 | 30.21 | 3 | keratin | 627899 | 50.21 | 6 |
β tubulin | TUBB | 5174735 | 120.25 | 13 | β actin | 4501885 | 30.21 | 3 | ||||
β actin | ACTB | 49868 | 60.20 | 6 | ||||||||
cofilin 1 | CFL1 | 6680924 | 50.26 | 5 | ||||||||
microtubule-associated protein 2 | MAP2 | 126741 | 50.23 | 5 | ||||||||
myelin basic protein |
MBP | 6754658 | 30.16 | 3 | ||||||||
syntaxin binding protein 1 | STXBP1 | 21594764 | 40.19 | 5 | ||||||||
clathrin heavy chain | CLTC | 51491845 | 40.20 | 4 | ||||||||
eukaryotic translation elongation factor 1 | eEF1A | 51873060 | 40.17 | 4 | ||||||||
2′,3′-cyclic-nucleotide 3′-phosphodiesterase I |
CNP | 2160434 | 80.19 | 8 | mouse Cu/Zn superoxide dismutase | 45597447 | 20.22 | 3 | ||||
dihydropyrimidinase-like 2 | DPYSL2 | 40254595 | 50.20 | 5 | ||||||||
latexin | LXN | 31980632 | 40.25 | 5 | ||||||||
solute carrier family 25, member 4 | SLC25A4 | 82917335 | 40.22 | 4 | ||||||||
solute carrier family 25, member 5 | SLC25A5 | 22094075 | 30.22 | 3 | ||||||||
solute carrier family 25, member 12 | SLC25A12 | 27369581 | 30.21 | 4 | ||||||||
glutamate oxaloacetate transaminase 2 | GOT2 | 6754036 | 40.21 | 4 |
Proteins expressed in non-neural cells.
To confirm if region and age specificities existed in the DF mice, a proteomic profile of spinal cords aged 145 days was compared with similar profiles of spinal cords aged 35 days (presymptomatic) and the cerebellum aged 145 days (
DF (35) spinal cord | DF (145) spinal cord |
DF (145) cerebellum | ||||||
Protein | Accession no. | Probability score | No. of peptide | Protein | Protein | Accession no. | Probability score | No. of peptide |
heat shock cognate 70 | 13242237 | 200.28 | 22 | heat shock cognate 70 | heat shock cognate 70 | 13242237 | 160.25 | 17 |
heat shock protein 70-2 | 123621 | 80.27 | 9 | heat shock protein 70-2 | heat shock protein 70-2 | 123621 | 70.26 | 7 |
heat shock protein 70 |
56757667 | 40.25 | 4 | heat shock 70 protein 1-like | heat shock protein 70 |
56757667 | 40.24 | 4 |
heat shock protein 9A |
heat shock 70 protein 1-like | 56757584 | 30.26 | 3 | ||||
ubiquitin carboxy-terminal hydrolase L1 |
||||||||
Na/K ATPase α 3 subunit | 27552786 | 90.26 | 10 | Na/K ATPase α 2 subunit | Na/K ATPase α 3 subunit | 27552786 | 100.23 | 11 |
Na/K ATPase α 2 subunit | 30409956 | 80.26 | 9 | Na/K ATPase α 3 subunit | Na/K ATPase α 2 subunit | 30409956 | 90.23 | 10 |
Na/K ATPase α 1 subunit | 21450277 | 60.26 | 7 | Na/K ATPase α 1 subunit | Na/K ATPase α 1 subunit | 21450277 | 80.23 | 9 |
Na/K ATPase α 4 subunit |
18203577 | 20.21 | 3 | ATP synthase β subunit |
Na/K ATPase α 4 subunit |
18203577 | 20.22 | 3 |
ATP synthase α subunit | 6680748 | 40.30 | 4 | ATP synthase α subunit | ATP synthase α subunit | 6680748 | 70.25 | 7 |
ATP synthase γ subunit |
potassium-transporting ATPase α subunit | 20137339 | 30.19 | 3 | ||||
glyceraldehyde-3-phosphate dehydrogenase | 6679937 | 60.34 | 7 | α enolase (non-neuron) |
glyceraldehyde-3-phosphate dehydrogenase | 6679937 | 40.36 | 5 |
lactate dehydrogenase |
pyruvate kinase | 551295 | 40.23 | 4 | ||||
brain glycogen phosphorylase |
aconitase 2 | 18079339 | 40.27 | 4 | ||||
phosphofructokinase 1 |
||||||||
malate dehydrogenase 1 |
||||||||
aconitase 2 | ||||||||
glyceraldehyde-3-phosphate dehydrogenase | ||||||||
β tubulin | 12963615 | 80.34 | 9 | α tubulin | β tubulin | 5174735 | 90.37 | 11 |
α tubulin | 34740335 | 70.28 | 7 | β tubulin | α tubulin | 34740335 | 70.29 | 8 |
keratin |
112983636 | 30.20 | 4 | β actin | β actin | 49868 | 30.22 | 3 |
β actin | 4501885 | 40.20 | 4 | cofilin 1 |
keratin |
54607171 | 20.21 | 3 |
myelin basic protein | 6754658 | 30.19 | 3 | microtubule-associated protein 2 |
||||
myelin basic protein | ||||||||
clathrin heavy chain | 51491845 | 30.25 | 3 | syntaxin binding protein 1 | syntaxin binding protein 1 | 21594764 | 40.21 | 4 |
clathrin heavy chain | clathrin heavy chain | 51491845 | 30.26 | 3 | ||||
eukaryotic translation elongation factor 1 | 51873060 | 30.19 | 4 | eukaryotic translation elongation factor 1 | eukaryotic translation initiation factor 4B |
55976513 | 50.24 | 6 |
eukaryotic translation initiation factor 4B |
55976513 | 30.22 | 3 | |||||
2′,3′-cyclic-nucleotide 3′-phosphodiesterase I | 2160434 | 90.27 | 10 | 2′,3′-cyclic-nucleotide 3′-phosphodiesterase I | dihydropyrimidinase-like 2 | 40254595 | 40.30 | 4 |
dihydropyrimidinase-like 2 | 40254595 | 40.30 | 4 | dihydropyrimidinase-like 2 | adenylate cyclase-associated protein 2 | 13385554 | 40.28 | 4 |
latexin |
protein phosphatase 1B | 33859600 | 40.30 | 4 | ||||
solute carrier family 25, member 4 |
||||||||
solute carrier family 25, member 5 |
||||||||
solute carrier family 25, member 12 |
||||||||
glutamate oxaloacetate transaminase 2 |
* Data are same as
The DF mutant SOD1 represents a relatively small proportion of the total SOD1 content of the spinal cord compared to both the WF and wild-type mouse SOD1s (
The FLAG-tagged SOD1 and the ligand proteins were immunoprecipitated using the FLAG affinity resin and detected by the anti FLAG antibody (A) and anti-SOD1 polyclonal antibody (uSOD1) that recognizes both human and mouse SOD1 (B). The DF mutant SOD1 shows a distinct single band (A and B). In contrast, the WF shows high molecular weight bands (A) and a low molecular weight band which is possibly the mouse intrinsic SOD1 (B), indicating that the WF forms the heterodimer with the mouse SOD1. Crude spinal cord extracts (5% input) were analyzed using antibodies for human specific SOD1 (hSOD1) (C) and for uSOD1 (D). Compared to the WF, the amount of the DF SOD1 protein is considerably lower (C). The WF itself is lower still compared to the mouse intrinsic SOD1 (D). The immunoprecipitation products with the FLAG were analyzed by antibodies for ATP1A (E) and Hsp/Hsc70 (G). In this analysis, only the DF preparations show the presence of ATP1A (E). In the Hsp/Hsc70 analysis in (G), there are non-specific precipitations also in WF and NTG. In contrast, the immunoprecipitated samples for ATP1A (F), Hsp/Hsc70 (H) and ATP5B (I) were analyzed using antibodies for hSOD1 (F) or uSOD1 (H and I). Correspondingly, the human form of SOD1 is only seen to be co-immunoprecipitated in DF preparation (F, H and I). Lanes from the left are: DF, WF, NTG, and a purified human SOD1 (A, B, C, D, F, H, and I); and DF, WF, NTG, and a crude soluble fraction of NTG mice (E and G).
Immunohistochemical analysis was performed using the aforementioned antibodies for ATP1A and ATP5B, as well as for a number of respective subunits: Na/K ATPase subunit β (ATP1B) and ATP synthase α subunit (ATP5A). Immunoreactivity for ATP1A was similar in both the DF (
Fixed-frozen sections of the spinal cord (10 µm thickness) derived from symptomatic DF mice at 145 days of life (A, C, E, and G) and age-matched NTG mice (B, D, F, and H) were stained with antibodies for ATP1A (A and B), ATP1B (C and D), ATP5A (E and F), ATP5B (G and H). The tissue was counterstained with hematoxylin. Bar: 200 µm.
As ATP5A and ATP5B are the major components of the mitochondrial ATP synthase, spotty expression is seen in the cell body in NTG mice (
We first hypothesized that there could be a specific ligand protein(s) for the mutant SOD1 only in the spinal cord and motor cortex. However, the results instead revealed that the mutant SOD1-interacting proteins were rather ubiquitously expressed. As Rakhit et al. suggested
Previous reports indicate that Na/K ATPase enzymatic activity decreases significantly in the motor neurons of SOD1G93A mice and chronically from the presymptomatic stage until the end stage
It is evident that a fraction of SOD1 is localized in the mitochondria
A wide range of proteins were shown to interact with the monomer-misfolded mutant SOD1, which is not necessarily an exceptional form in FALS-associated homodimeric mutant SOD1s. The mutant SOD1-interacting proteins might be compromised in their innate functions in a normal cellular compartment by bonding with the mutant SOD1. Among them, the impairment of Na/K ATPase and/or ATP synthase alone could cause devastating cellular consequences.
Two lines of transgenic mice were used in this experiment. The DF (
All animal experiments were carried out in accordance with Guidelines for Animal Experimentation at the Faculty of Medicine, Tottori University, and all efforts were made to minimize animal numbers and suffering during the experiments. Mice were sacrificed by intraperitoneal injection of pentobarbital. The spinal cords were rapidly removed (n = 5 in case of DF mice age 35, otherwise n = 4) and immediately stored in liquid nitrogen until use.
Spinal cords as well as cerebellum were suspended 1∶10 (w/v) in a lysis buffer (0.15 M NaCl, 10 mM Tris-HCl and 1% Triton X-100, pH 7.4) containing a cocktail of protease inhibitors (Roche Diagnostics, Switzerland) and homogenized
CNS tissue extracts were prepared in the same way for proteomic analysis. Immunoprecipitation was performed using the anti-FLAG M2 affinity gel or otherwise with a combination (10 µg antibody/10 µl resin) of protein A/G plus-agarose and an anti-Na/K-ATPase α, rabbit polyclonal antibody (H-300, Santa Cruz biotechnology, USA) that recognizes all Na/K-ATPase α isoforms; anti-Hsp72/73 mouse monoclonal antibody (W27, Calbiochem, USA) that recognizes both Hsp70 and Hsc70; or anti-ATP synthase beta mouse monoclonal antibody (ab5432, abcam, USA). Immobilized antibody/bound antigen was collected by centrifugation (5,000
Immunoblot was performed using a rabbit anti-Cu/Zn superoxide dismutase polyclonal antibody (SOD-101, Stressgen, USA) for both human and mouse SOD1, rabbit anti superoxide dusmutase 1 (SOD1) human specific polyclonal antibody (AB5480, Chemicon, USA), monoclonal anti-FLAG M2 antibody (Sigma), or otherwise using the aforementioned antibodies for ATP1A and Hsp/Hsc70. Aliquots of 10 µg were electrophoresed and transferred onto PVDF membranes (Hybond-P; Amersham Biosciences, UK). For chemiluminescense detection, we used a conventional ECL system (Amersham Biosciences), as well as ECL advance and rabbit IgG TrueBlot (eBioscince, USA).
Symptomatic DF mice at ∼145 days of life (within a week of symptom onset) as well as age-matched NTG mice were sacrificed and perfused with 4% paraformaldehyde. Fixed-frozen sections of the spinal cord (10 µm thickness) were stained immunohistochemically by the streptavidin-biotin method using the aforementioned antibodies for ATP1A and ATP5B, as well as anti-Na/K ATPase β1 monoclonal antibody (464.9, Santa Cruz biotechnology, USA) for ATP1B, and ATP5A monoclonal antibody (15H4, Santa Cruz biotechnology, USA). The tissue was counterstained with hematoxylin.