Rat neuron-specific enolase (NSE) promoter plasmid containing hPGC-1α was constructed by inserting 3.1 kb cDNA fragment with entire coding region of hPGC-1α (NM_013261.2, OriGene Technologies, Inc. Rockville, MD) in Not I site of the plasmid vector. A cassette of ~8 kb SalI fragment containing NSE promoter and hPGC-1α was gel purified and microinjected into one-cell mouse egg (C57BL6 × SJL)as described previously [31, 32]. TgPGC-1α founders were identified by PCR-based genotyping.
Male TgSOD1-G93A mutant transgenic mice (C57BL6 × SJL) were purchased from the Jackson Laboratory and bred with female PGC-1α transgenic mice in our transgenic mouse facility to generate TgSOD1-G93A/TgPGC-1α double transgenic mice and their TgSOD1-G93A, TgPGC-1α or wild-type littermates. Mice were housed on a 12-hour-light, 12-hour-dark cycle and allowed ad libitum access to food. Mice were weighed weekly starting from 8 weeks of age. The survival study endpoint was defined as meeting any one of the following conditions: no spontaneous breathing or movement for 60 seconds with no response to pain; the animal is unable to roll over the normal position within 10 seconds following a push over; or complete hind limb paralysis. The Institutional Animal Care Committee of Mount Sinai School of Medicine reviewed and approved all experimental protocols used in this study.
The brains of 5 months old Wild type and Tg2576 mice were dissected, cut into two hemispheres and fixed in 4% Paraformadehyde. After extensive wash with PBS, equilibrated with 30% sucrose and embedded in Tissue Freezing Medium (Triangle Biomedical Sciences). The frozen brains were transversely sectioned (14 uM). The tissue sections were incubated with PGC-1α antibody (Santa Cruz Technology, H-300, 1:500 dilution) overnight at 4°C. The sections were then washed and incubated with FITC-conjugated secondary antibody (1:250 dilution) for 1 hour. Following several washes with PBS, images were acquired under fluorescence microscope.
Motor Function Assessment
TgSOD1-G93A/TgPGC-1α mice and TgSOD1-G93A mice were tested on an accelerating rotarod (7650 Ugo Basile Biological Research Apparatus, Comerio, Italy) as previously described. In brief, mice are placed onto a grooved cylinder (facing away from the experimenter) rotating at a predetermined speed that incrementally increases to a maximal rotation at 300s; the time maintained on the rod by each mouse (latency) is then recorded (300 s max). A diminishing latency indicates declining performance and at values of 0 s is suggestive of severe muscular weakness and impaired coordination. Mice were tested beginning at 80 days of age weekly until they could no longer perform the test. Before testing, mice underwent a 1 week training period wherein they were introduced to the apparatus and handled by the experimenter daily. Testing was conducted during the last 4 h of the day portion of the light cycle in an environment with minimal stimuli such as noise, movement, or changes in light or temperature.
Glucose Tolerance Test
Mice were fasted overnight in clean cages with free access to water in new clean bottles. The next morning each mouse was weighed, and a baseline fasted blood glucose measurement was taken by applying tail blood to a Contour Blood Glucose Monitoring System (Bayer). Each mouse was injected intraperitonealy with a filter-sterilized solution of 20% (w/v) D-glucose, with the size of the bolus determined by animal weight (2 mg glucose/g body weight). Blood glucose measurements were taken as described above for each animal at 15, 30, 60 and 120 minutes. The data were plotted as blood glucose concentration (mg/dL) over time (minutes).
A Bead-based multiplex luminex assay was performed using MILLIPLEX MAP 8-Plex Multi-Pathway Signaling Kit, Phosphoprotein (Millipore, Billerica, MA) following the manufacturer's protocols. Briefly, the spinal cords were homogenized in ice-cold Milliplex lysis buffer with protease inhibitors and then centrifuged at 12,000 rpm for 10 minutes at 4°C. Protein concentration was measured using the Bradford method. 25 μg protein of each sample was used for analysis.
Preparation of Mouse Spinal Cord Sections
Age and gender-matched wild type, TgSOD1-G93A, TgPGC-1α and TgSOD1-G93A/TgPGC-1α double transgenic mice were euthanized by ketamine and their spinal cords were dissected out. The lumbar region was separated and rapidly frozen under 2-methylbutane on dry ice. The samples were stored at -80°C until sectioned. For sectioning, samples were embedded in OCT compound and transverse sections were cut at -20°C using a Leica CM3050 cryostat. The sections were collected on positively-charged glass slides (Superfrost Plus, Fisher Scientific) and stored at -80°C till use.
Ten serial sections (25 μm thick) were cut 350 μm apart through the lumbar (L3-L5) spinal cord of each animal (n = 5). The sections were mounted onto Superfrost Plus slides and Nissl staining was performed using cresyl violet as previously described . Large (>25 μm), Nissl stained neurons were counted within the ventral horns under a light microscope at a magnification of 200.
Histochemical Reaction, Imaging and Analysis
8 μm thick sections of the lumbar spinal cord were air dried for 30 min and used for activity staining of mitochondrial complexes [1, 18]. All the staining reactions were carried out at room temperature in the dark. To quantify complex I activity, the sections were incubated in 0.1 M Tris-HCl (pH 7.4), 0.14 mM NADH, 1 mg/ml nitroblue tetrazolium, 2 μg/ml antimycin, 84 mM malonate and 2 mM potassium cyanide. For complex II histochemistry, the enzyme was activated [34, 35] by a 10 min incubation in 0.05 M potassium phosphate buffer (pH 7.4), followed by the addition of the reaction mix consisting of 4.5 mM EDTA, 2 mg/ml nitroblue tetrazolium, 50 mM succinate, 0.2 mM phenazonium methosulfate, 2 μg/ml antimycin, 60 μM rotenone and 2 mM potassium cyanide in the same buffer. The complex IV reaction required 75 mg/ml sucrose, 1 mg/ml diaminobenzidine HCl, 24 U/ml catalase, 1 mg/ml cytochrome c, 2 μg/ml antimycin, 60 μM rotenone and 84 mM malonate in 0.05 M potassium phosphate buffer (pH 7.4). The negative controls contained 60 μM rotenone, 84 mM malonate or 2 mM potassium cyanide to specifically inhibit complexes I, II or IV respectively. After incubations of 20 min for the complex I and IV reactions or 10 min for the complex II reaction, the sections were washed twice in phosphate buffered saline, once in distilled water and then mounted in glycerin jelly.
Stained sections were visualized under an Olympus MVX10 Macroview stereomicroscope controlled by MicroSuite Five Biological Suite software and photographed using the attached Hamamatsu C8484 monochrome camera. Images were taken with particular care to use uniform gray scales and below the level of saturation. Optical intensities of the ventral horn area on these sections were quantified using ImageJ software (NIH). Optical intensities were converted to optical densities (OD) by the formula: OD = log10 (Ibk/Im), where Ibk is background intensity and Im is measured intensities from different regions of the sections.