Fig. 3

A Response- and homeostasis-associated protein levels of astrocyctes derived from MBP29-hα-syn and non-transgenic littermates. Upper left panel: Cortical and striatal levels of GFAP were increased in MBP29-hα-syn mice by ~ 4-(p < 0.001) and ~ 8-fold (p < 0.001), respectively. Similarly, there was an increased cortical and striatal level for VIM in MBP29-hα-syn mice by ~ 2-(p = 0.007) and ~ 2.5-fold (p < 0.001), respectively. Quantification of WBs (n = 5/ each group/ region) is shown as mean ± SD. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as control. kDa = kilo Dalton. *p < 0.05, **p < 0.01, ***p < 0.005. Upper right panel: AQP-4 levels are highly increased by ~ 3-fold in the cortex of MBP29-hα-syn mice (p < 0.001). Note the much lower, but not significant increase of AQP-4 levels in the striatum of MBP29-hα-syn mice (p = 0.054). A proportional and similar upregulation is present for growth associated protein-43 (GAP-43) levels showing a ~ 2-fold (p < 0.001) and ~ 1.5-fold (p = 0.074) increase in the cortex and the striatum of MBP29-hα-syn mice. A similar pattern is observed for GS by a ~ 2.5-fold increase (p < 0.001) in the cortex of MBP29-hα-syn mice, however without changing striatal levels. Quantification of WBs (n = 5 animals/group/region) is shown as mean ± SD. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as control. kDa = kilo Dalton. *p < 0.05, **p < 0–01, ***p < 0.005. Lower left panel: Protein levels of glutamate reuptake transporters glutamate transporter-1 (GLT-1) and glutamate-aspartate transporter 1 (GLAST) are not altered in the cortex of MBP29-hα-syn mice. In contrast, both glutamate reuptake transporters were reduced by ~ 2-fold (GLT-1, p = 0.001; GLAST, p = 0.005). Quantification of WBs (n = 5 animals/group/region) is shown as mean ± SD. Due to overlapping WB signals of GLT-1, GLAST, and GAPDH, whole protein Ponceau-S staining served as control. *p < 0.05, **p < 0.01, ***p < 0.005. B Isolation of astrocytes via MACS and cellular phenotyping. Flow cytometry analysis of magnetic activated cell sorting (MACS) of striatal and cortical astrocytes using the astrocyte cell surface antigen-2 (ACSA2) without (standard procedure; ACSA-2⁺) or with a preceding, negative sorting step for the oligodendrocyte marker 4 (negative pre-sorting, O4⁻, ACSA2⁺). Without removing O4⁺-cells, the purity of ACSA2⁺-cells amounts to 61.5% (left panel); a substantial O4⁺ cell population (35.1%) was still present. By adding the O4 presorting step, a highly improved purity of ACSA2⁺-cells (86.3%) was achieved; much less O4⁺-cells (5.8%) were detected (middle panel). Y-axis: logarithmic fluorescence of allophycocyanin (APC)-signal. X-axis: logarithmic fluorescence of phycoerythrin (PE)-signal. Each cell population analyzed is marked by a black rectangle, the proportion of ACSA2⁺ and O4⁺-cells is given in % of the entire cell population. Enrichment of the 50 most highly expressed genes in the isolated cell population using clusterProfiler. Enrichment analysis revealed the most significant enriched genes associated with astrocytes (right panel). Neural stem cells show a similar gene expression pattern