After validating in vivo muscle deficits, α-glucosidase deficits in the brain and muscle, as well as elevated glycogen levels in the brain and muscle, our R&D team now evaluated several disease-relevant biomarkers in Pompe 6neo mice longitudinally. In a first step, liver, heart, diaphragm, tibialis anterior and quadriceps tissue were evaluated by PAS staining to detect polysaccharides, glycoproteins, and glycolipids. All analyzed tissues of 6neo mice presented with a more intense staining. Numerous dark pink-purple granules could be observed strongest in muscle tissues (Fig.1). These results suggest that tissue of 6neo mice is characterized by increased levels of polysaccharide, glycoprotein, and/or glycoprotein accumulations.
Figure 1: PAS staining of liver, heart, diaphragm, tibialis anterior and quadriceps sections of 6neo mice. Images on the top correspond to wild type mice, while images on the bottom correspond to 6neo mice. Analyzed mice were 24 weeks old. Note the intense staining in the heart, diaphragm, tibialis anterior and quadriceps tissue of 6neo mice, as well as the presence of numerous dark pink-purple granules, especially in muscle tissues.
As second step, we quantitatively evaluated LAMP2 as lysosomal marker, ubiquitin and LC3-B as marker for autophagy, Iba1 as marker for microgliosis, and CD45 as marker for hematopoietic cells, excluding mature erythrocytes and platelets. While all markers showed only weak signal in wild type mice of all age groups, they all showed highly increased expression levels in 6neo mice that further increased with age. LAMP2 (A), as well as ubiquitin (B) and the downstream of ubiquitin activated LC3-B (C) were already significantly increased at the age of 4 weeks. Whereas Iba1 (D) and CD45 (E) were significantly increased at the age of 8 weeks compared to age-matched wild type mice (Fig. 2).
Figure 2: Quantification of LAMP2, ubiquitin, LC3-B, Iba1 and CD45 immunoreactive area in the quadriceps of 4, 8, and 24 weeks old 6neo mice. Immunoreactive (IR) area in percent measured within the region of interest (RIO) on 5 quadriceps sections per mouse. Two-way ANOVA followed by Bonferroni’s multiple comparisons test. Mean ± SEM; n = 8 / group; *p<0.05; **p<0.01; ***p<0.001.
Together with our previous results showing first in vivo muscle deficits at the age of 8 weeks as well as glycogen substrate accumulation, these histological analyses provide further insight into pathological changes of 6neo mice as model of Pompe disease.
These results further promote 6neo mice as valuable tool to study Pompe disease and test new compounds for their efficacy to ameliorate the lysosomal storage disease’s devastating symptoms.
Further results about pathological changes in the brain and spinal cord of 6neo mice are already evaluated and will be presented soon. Stay tuned for more information about this mouse model for Pompe disease!
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