B6.SOD1-G93A transgenic mice are a highly translational model of amyotrophic lateral sclerosis (ALS) as they display common disease characteristics. Mice exhibit a slightly delayed progression of the ALS phenotype compared to the commonly used SOD1(*G93A)1Gur mice. B6.SOD1-G93A mice are bred on a congenic C57BL/6 background while SOD1(*G93A)1Gur mice are bred on a mixed C57BL/6xSJL background. The slower disease progression offers an extended window for therapeutic interventions, potentially leading to new treatment strategies. In a previous newsletter, we already presented data about strong muscle pathology in B6.SOD1-G93A mice by electromyography (EMG) and behavioral motor tests as well as first findings about neurodegeneration and autophagy in the spinal cord.
We now proceeded with the characterization of B6.SOD1-G93A mice by immunolabeling the lumbar spinal cord for choline acetyltransferase (ChAT), neuronal nuclear antigen (NeuN), ubiquitin and cathepsin D (Figure 1).
Figure 1: Immunofluorescent signal in lumbar spinal cord of wild type and B6.SOD1 mice. Images show examples of immunofluorescent labeling of ChAT (green), NeuN (light blue), ubiquitin (white) and cathepsin D (CTSD, red) on a lumbar cross-section of a wild type mouse (A) and a B6.SOD1-G93A mouse (B); nuclei were labeled with DAPI.
The number of ChAT- and NeuN-positive obejcts was significantly decreasing in B6.SOD1-G93A mice (Figure 2A), suggesting a strong motoneuron loss, while in parallel ubiquitin was increased in remaining ChAT-positive neurons (Figure 2B). The overall cathepsin D signal was not altered (Figure 2C) but significantly decreased in ChAT-positive neurons (Figure 2D), as already shown by Wootz et al., 2006 (doi: 10.1016/j.neuroscience.2006.07.048). This result might point at a disrupted autophagy pathway in motoneurons.
Figure 2: Quantification of mean immunofluorescent signal of ChAT, NeuN, ubiquitin and cathepsin D. Object density (A, C, D) and immunoreactive (IR) area (B) are shown. A, D: Unpaired t-test; B, C: Mann-Whitney test. Mean ± SEM (n = 8 per group, 3 sections per animal). **p<0.01, ***p<0.001.
Qualitative immunofluorescent labelling of the lumbar spinal cords with antibodies against HDAC6, human SOD1 (hSOD1) and ChAT suggests that the autophagy marker HDAC6 is expressed in SOD1-positive motoneurons in the ventral horn (Figure 3). Similar results were received from the cervical and thoracic spinal cord region (data not shown).
Figure 3: Immunofluorescent signal in lumbar spinal cord of a B6.SOD1-G93A mouse. Images show immunofluorescent labeling of HDAC6 (green), misfolded hSOD1 (red) and ChAT (light blue) on a lumbar cross-section; nuclei were labeled with DAPI (dark blue). Arrow points to a cell where HDAC6 and misfolded hSOD1 colocalize inside a ChAT-positive motoneuron.
The observed spinal cord pathology, including motoneuron loss and a disturbed autophagy signaling pathway, make the B6.SOD1-G93A mouse an indispensable model to evaluate underlying pathological ALS mechanisms and test the efficacy of new drugs against this dreadful disease.
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