All the strains of transgenic mice used in this study have been previously described. The PrP.SOD1-G37R SOD1 line 110 mice were generated by a vector that utilized the mouse prion protein promoter (MoPrP.Xho) containing a hSOD1
cDNA with the G37R mutation (20
). These mice were maintained in a hybrid background of C57BL/6J and C3H/HeJ. All other strains of mice used in this study were generated with genomic fragments of the hSOD1
gene. The L126Z SOD1 line 45 used a modified genomic fragment in which exons 3–5 of the human gene were fused (19
) and a stop codon was inserted at codon 127. These mice were maintained in a hybrid background of C57BL/6J and C3H/HeJ. Mice expressing WT hSOD1 that we used included a line developed by Gurney et al
), maintained in both hybrid backgrounds of C57BL/6J × SJL and in a congenic background of C57BL/6J (these strains are designated SJLWT and CgWT, respectively), and WT line 76 mice (designated L76WT) (16
) maintained in a congenic background of C57BL/6J mice. The congenic Tg(SOD1)Gur2/J mice were graciously provided by Dr Gregory Cox (Jackson Laboratories, Bar Harbor, ME, USA). All studies involving mice were approved by the Institutional Animal Care and Use Committee at the University of Florida.
For the identification of genotype, DNA from mouse tail biopsy was extracted by digesting tissue (0.6 cm length) in 600 μl of TNES buffer (50 mm Tris base, pH 7.5; 400 mm NaCl; 100 mm EDTA, pH 8.0; 0.5% SDS) with 18 μl of Proteinase K at 20 mg/ml. After being kept overnight in a 55°C water bath, 167 μl of supersaturated 6 m NaCl was added to each sample, mixed and then centrifuged at 16 100 g for 5 min. A total of 650 μl of supernatant was removed and placed into a new microfuge tube, then centrifuged again before 600 μl of supernatant was moved to a new tube and mixed with an equal volume of cold 100% ethanol. DNA was then pelleted by centrifuging at 14 000 r.p.m. for 5 min and washed with 1 ml of cold 70% ethanol before centrifuging again for 2 min. The final DNA pellet was resuspended in 200 μl of TE buffer (10 mm Tris base, pH 7.5; 1 mm EDTA, pH 8.0).
The DNA extracted from mouse tail was analyzed by PCR using the following primers. For the L126Z and WT hSOD1 lines of mice, we used two primers: Hu-S primer (5′-TCA AGC GAT TCT CCT GCC T); H/M-AS primer (5′-CAC ATT GCC CAR GTC TCC A; R = A/G). For the G37R line 110 hSOD1 mice, we used three primers: PrP-S primer (5′-GGG ACT ATG TGG ACT GAT GTC GG); PrP-AS primer (5′-CCA AGC CTA GAC CAC GAG AAT GC); HuSOD1-S (5′-GTC GAC AAG CAT GGC CAC GAA GGC CGT GTG C). PCR reactions were performed using Taq DNA polymerase and PCR reagents from New England Biolabs (Ipswich, MA, USA) following the manufacturer's protocol. The settings for the thermocycler were as follows: 94°C for 5 min; 35 cycles of 94°C for 30 s; 60°C for 1 min; 72°C for 5 min; 72°C for 10 min. The PCR products were the following: the L126Z hSOD1 transgene produced a fragment of ~500 bp, the WT hSOD1 transgene produced a fragment of ~1200 bp and G37R hSOD1 transgene produced a fragment of ~500 bp. Endogenous mouse prn-p genes produce fragments of ~750 bp and served as a positive control.
SOD1 cDNA expression plasmids
All of the non-tagged WT SOD1
and ALS-associated mutant hSOD1
cDNAs were expressed from plasmids based on the mammalian pEF-BOS expression vector, and have been previously described (19
). GFP was expressed in pcDNA3.1 as described previously (12
SOD1 fusion protein cDNA variants were created from an expression vector (pPD30.38) that contains WT hSOD1 fused to eYFP (yellow fluorescent protein), which was kindly provided by Dr Rick Morimoto (Northwestern University). This SOD1::eYFP construct contains a 27 bp linker (translated sequence—LQLKLQASA) between SOD1 and YFP that was modified to include an SalI restriction site (translated sequence—LQSTLQASA). This SOD1::YFP DNA fusion construct was then cloned into our mammalian pEF-BOS expression vector. From this initial SOD1::YFP expression plasmid, we generated vectors for A4V::YFP and G37R::YFP. Additionally, we also created a control plasmid in pEF-BOS for eYFP (termed YFP). Similar to SOD1::YFP fusion proteins, we created SOD1 fusion proteins with a red fluorescent protein tag [RFP, Turbo RFP cDNA obtained from the pTRIPZ empty vector available at Open Biosystems (Huntsville, AL, USA)] by replacing the YFP tag with the RFP tag. In this way, we created WT::RFP, A4V::RFP and G37R::RFP constructs along with a vector to express RFP alone. All cell culture studies of SOD1 aggregation used HEK293FT cells (Invitrogen, Carlsbad, CA, USA).
Fresh spinal cords were harvested from 4-month-old mice, with one-half of the cord immediately used for RNA extraction and the other half frozen for analyses of protein levels. Extraction of spinal cord mRNA was performed using TRIzol (Invitrogen) as described by the manufacturer. Five micrograms of total RNA was electrophoresed in formaldehyde–agarose gels and then transferred onto a nylon membrane by capillary action using 10x SSC buffer (1.5 m NaCl, 150 mm sodium citrate, in distilled water treated with diethyl pyrocarbonate). The nylon membrane was then cross-linked, and pre-hybridized for 1 h at 65°C with hybridization buffer (1% BSA, 1 mm EDTA at pH 8.0, 0.5 m NaHPO4 at pH 7.2, 7% SDS) followed by hybridization at 65°C with the 32P-labeled cDNA probes (at ~1.4 × 106 c.p.m./ml) for 12 h. As probes, we generated a fragment of hSOD1 cDNA and a fragment that recognizes endogenous mouse PrP mRNA, the latter serving as a loading control. Probes were labeled using the DNA-Ready to Go-label beads 32P-dCTP from GE Healthcare (Pittsburgh, PA, USA) and purified with Illustra ProbeQuant G-50 Micro Columns from GE Healthcare as described by the manufacturer. After hybridization, membranes were washed three times for 30 min with wash buffer 1 (0.1% BSA, 1 mm EDTA at pH 8.0, 40 mm NaHPO4 at pH 7.2, 5% SDS) at 65°C. Two more washes were then performed with wash buffer 2 (1 mm EDTA at pH 8.0, 40 mm NaHPO4 at pH 7.2, 1% SDS). Membranes were then exposed to film and developed after 24 h or imaged by phosphorimaging on a Molecular Dynamics Instrument (Bio-Rad Laboratories, Hercules, CA, USA) . Northern blot analyses were performed on a total of three spinal cords from each of WT mouse strains. Band intensities from the northern blots quantified by phosphorimaging were calculated using Quantity One 4.6.5 software (Bio-Rad Laboratories). In the quantification of SOD1 mRNA levels, we normalized the values of endogenous prion protein mRNA, which served as a loading control. The statistical significance of differences in mRNA levels was determined by unpaired Student's t-tests.
Determination of total SOD1 protein levels in the spinal cord
Spinal cords were dissolved by sonication in five volumes (by weight) of 1x TEN buffer (10 mm
Tris, pH 7.5; 1 mm
EDTA, pH 8.0; 100 mm
NaCl) with protease inhibitor cocktail at 1:100. A brief 800g
centrifugation for 10 min was performed to discard cell debris. Protein concentrations in the supernatant fraction, which represents total extracted protein, were measured by BCA assay (Pierce Biotechnology, Rockford, IL, USA). Five microgram of protein was electrophoresed in 18% SDS–PAGE gels, transferred to membranes and immunoblotted with an antibody (1:2500, overnight) that recognizes mouse and hSOD1 (34
) or an antibody (1:2500 overnight) that recognizes only hSOD1 (33
) as indicated in the figure legends. Blots were also probed (1:5000, overnight) with an antibody that recognizes β-tubulin-III for loading control. A secondary goat anti-rabbit IgG (1:5000, 1 h) was used to detect primary antibodies. Band intensities from the immunoblots were calculated using the Fujifilm imaging system (FUJIFILM Life Science, Valhalla, NY, USA). In the quantification of SOD1 protein levels, we normalized the values to the tubulin-loading control.
Detergent extraction of SOD1 aggregates
This assay generates two protein fractions termed S1 (detergent-soluble) and P2 (detergent-insoluble), the latter containing aggregated forms of mutant SOD1, as described previously (3
). Detergent extraction of cultured cells was performed as described in the foregoing reports; however, for spinal cord samples, slight modifications of the protocol were followed to obtain higher levels of extracted protein. A total of 300 μl of spinal cord crude supernatant was extracted by adding an equal volume of buffer to produce a final concentration of 10 mm
Tris, pH 7.5; 1 mm
EDTA, pH 8.0; 100 mm
NaCl; 0.5% NP-40, 1:100 v/v protease inhibitor cocktail. The mixture was sonicated three times for 10 s each before centrifugation at >100 000g
for 5 min in a Beckman AirFuge (Brea, CA, USA) to produce supernatant 1 (S1) and pellet (P1) fractions. The P1 fractions were then washed with the same extraction buffer (sonicated twice, 15 s each) and centrifuged at >100 000g
for 5 min. The supernatant was discarded and the remaining pellet represented the detergent-insoluble (P2) fraction, which was resuspended in a buffer containing SDS and deoxycholate (10 mm
Tris, pH 7.5; 1 mm
EDTA, pH 8.0; 100 mm
NaCl; 0.5% NP-40, 0.25% SDS, 0.5% deoxycholate, 1:100 v/v protease inhibitor cocktail). The total volumes corresponding to S1 and P2 fractions were of 600 and 100 μl, respectively. Protein concentrations of S1 and P2 fractions were determined by BCA assay, as described by the manufacturer (Pierce Biotechnology). For SDS–PAGE and immunoblot analyses, 5 μg of protein from S1 and 20 μg of protein from P2 were used. In some of the SDS–PAGE analyses, reducing agents were omitted from the loading and running buffers in order to separate and visualize reduced and oxidized forms of SOD1 following procedures described previously (6
Analyses of S1 and P2 fractions by FTMS
FTMS analyses of S1 and P2 of spinal cords of two different G37R/SJLWT samples were performed as described previously (12
). Here, each spinal cord sample was extracted in detergent to obtain S1 and P2 fractions from three combined spinal cords (1.2 ml of S1 and 600 μl of P2). The entire P2 fraction was analyzed by HPLC followed by FTMS to identify WT and mutant hSOD1 mass signatures.
Transfection of HEK293FT for immunocytochemistry was performed on glass coverslips that were previously coated with 0.5 mg/ml poly-l-lysine in 1x phosphate-buffered saline (PBS) solution. Lipofectamine 2000 was used to transfect 2 μg of expression vector per well following the manufacturer's protocol (Invitrogen). Transfected cells were then fixed with 4% paraformaldehyde in 1x PBS solution for 15 min. Additional staining was performed to visualize the nuclei, using DAPI solution (4′,6-diamidino-2-phenylindole, dihydrochloride, stock 14.3 mm from Invitrogen) at 1:2000 for a minimum of 10 min. Fluorescence was visualized by epifluorescence on an Olympus BX60 microscope with a color camera and by confocal microscopy on an Olympus IX81-DSU spinning disk confocal microscope indicated in the figure legends.
Quantitation of immunoblots
Quantification of the SOD1 protein in detergent-insoluble and detergent-soluble fractions was performed by measuring the band intensity of SOD1 in each lane using a Fuji Imaging system (FUJIFILM Life Science, Stamford, CT USA). For immunoblots of S1 fractions, we load 5 μg of total protein, and for the P2 fractions, we load 20 μg of total protein. Protein concentrations of S1 and P2 fractions were determined by BCA assay, as described by the manufacturer (Pierce Biotechnology). The untransfected cells served as a control. Aggregation index was calculated as the ratio of the band intensity in the detergent-insoluble fraction (P2) to that of the detergent-soluble fraction (S1). Note that the amount of protein loaded on the gel for P2 fractions is 4-fold more than the S1 fraction. The mean and standard error of the mean (SEM) were calculated for the aggregation index of each sample in each experiment. In each cell culture experiment, we included a positive control, which consisted of transfection of expression vectors for A4V SOD1. To normalize variation in data between experiments, relative values of aggregation index are normalized to P2/S1 value of A4V at 24 h transfection (set to 1) (3
All statistical analyses were performed using GraphPad PRISM 5.01 Software (La Jolla, CA, USA). The specific statistical method used in analyzing the data is noted in the figure legends.