Metabolic Enzyme Alterations and Astrocyte Dysfunction in a Murine Model of Alexander Disease With Severe Reactive Gliosis

Abstract

Alexander disease (AxD) is a rare and fatal neurodegenerative disorder caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). In this report, a mouse model of AxD (GFAP^Tg;Gfap^+/R236H) was analyzed that contains a heterozygous R236H point mutation in murine Gfap as well as a transgene with a GFAP promoter to overexpress human GFAP. Using label-free quantitative proteomic comparisons of brain tissue from GFAP^Tg;Gfap^+/R236H versus wild-type mice confirmed upregulation of the glutathione metabolism pathway and indicated proteins were elevated in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which had not been reported previously in AxD. Relative protein-level differences were confirmed by a targeted proteomics assay, including proteins related to astrocytes and oligodendrocytes. Of particular interest was the decreased level of the oligodendrocyte protein, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (Ugt8), since Ugt8-deficient mice exhibit a phenotype similar to GFAP^Tg;Gfap^+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAP^Tg;Gfap^+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAP^Tg;Gfap^+/R236H mice was also selected for further investigation due to its uncharacterized association to AxD, critical function in astrocyte proliferation, and functional ability to inhibit the anti-inflammatory PPAR signaling pathway in models of amyotrophic lateral sclerosis (ALS). Within Gfap⁺ astrocytes, Fabp7 was markedly increased in the hippocampus, a brain region subjected to extensive pathology and chronic reactive gliosis in GFAP^Tg;Gfap^+/R236H mice. Last, to determine whether the findings in GFAP^Tg;Gfap^+/R236H mice are present in the human condition, AxD patient and control samples were analyzed by Western blot, which indicated that Type I AxD patients have a significant fourfold upregulation of FABP7. However, immunohistochemistry analysis showed that UGT8 accumulates in AxD patient subpial brain regions where abundant amounts of Rosenthal fibers are located, which was not observed in the GFAP^Tg;Gfap^+/R236H mice.

Keywords: Alexander disease; Fabp7; Ugt8; astrocytes; reactive gliosis.

Graphical abstract

Fig. 1

Elevation of GFAP in the GFAP^Tg;Gfap^+/R236Hmice quantified by μDIA mass spectrometry.A and B, representative Gfap MS2 chromatograms are shown for the peptide sequence LADVYQAELR, which is present in both mouse Gfap (residues 109–118) and human GFAP (residues 112–121). The arrow points to the peptide apex intensity in each chromatogram. A, shows the peptide is more abundant in GFAP^Tg;Gfap^+/R236H mice compared with (B), which shows a wild-type animal. C, average normalized intensity of GFAP across 37 peptides in the GFAP^Tg;Gfap^+/R236H and wild-type mice. Error bars are standard deviation (stdev).

Fig. 2

RF and reactive gliosis proteins are among the most changed in the quantified proteome of GFAP^Tg;Gfap^+/R236Hmice versus wild type. Heat map of proteins previously documented in AxD to have altered abundance, as well as three reactive gliosis proteins (Fabp7, Aldh1l1, Ndrg2) that have not previously been linked to AxD phenotype. Data shown are the individual mouse Log2 normalized protein fold-change values in every sample. Proteins without detectable amounts in a particular sample were assigned a Log₂ normalized fold-change value of −1 (dark blue in the heat map).

Fig. 3

PPAR pathway proteins are elevated in GFAP^Tg;Gfap^+/R236Hmice. Data shown are untargeted μDIA proteomic quantification results of 4938 proteins (67 proteins that are listed in supplemental Table S2 but detected only in the GFAP^Tg;Gfap^+/R236H or wild-type mice are not included in this plot). PPAR pathway proteins are highlighted in red with Swiss-Prot gene abbreviations. The vertical lines denote fold-change values of −1.3 and +1.3 (without Log transformation), and the horizontal line represents a p-value = 0.05 (without Log transformation).

Fig. 4

Ugt8 is decreased in GFAP^Tg;Gfap^+/R236Hmice.A, Western blot of Ugt8 and Gapdh (loading control). Data from the molecular weight ladder is not shown. Each (rep) is a biological replicate from each genotype. B, average Western blot fold-change of Ugt8 normalized to Gapdh in the GFAP^Tg;Gfap^+/R236H mice relative to wild type was −13.7 fold. Error bars are standard error of the mean (SEM).

Fig. 5

Brain region alterations in Fabp7 and Ugt8 in wild type compared with GFAP^Tg;Gfap^+/R236Hmice.A and B, Fabp7 immunoreactivity is increased in GFAP^Tg;Gfap^+/R236H hippocampal regions relative to the same regions in wild-type littermates. The fifth cerebellar lobe (5 CB) is shown for reference. C, double immunolabeling with Gfap (Ci, red) and Fabp7 (Cii, green) indicates significant colocalization in the merged image (Ciii, merge, DAPI- a nuclear stain is shown in blue). Wild-type mice (Ai–Aiii) and GFAP^Tg;Gfap^+/R236H mice (Bi–Ciii). D and E, Ugt8 immunoreactivity is decreased across the hippocampus (Ei), corpus callosum (Eii), and cortex (Eiii) in GFAP^Tg;Gfap^+/R236H mice relative to wild-type littermates (Di–Diii) (Scale bar = 100 μm). CA2, CA2 hippocampal region; CC, corpus callosum; DG, dentate gyrus; LI, LII/III and LVI, cortical layers 1, 2/3, and 6; ML, molecular layer; OR, oriens layers.

Fig. 6

UGT8, FABP7, and GFAP Western blotting in AxD patient and human controls.A–F, in all Western blots the samples loaded in each lane from left-to-right were: lane 1 is a 15-year-old male control, lane 2 is a 50-year-old female control, lane 3 is a 1-year-old male control, lane 4 is a 6-year-old male R239C AxD patient, lane 5 is a 14-year-old male R79C AxD patient, lane 6 is a 50-year-old female S247P AxD patient, and lane 7 is a 1-year-old male R239H AxD patient. Additional demographics for these samples are provided in supplemental Table S4. A, Western blot of FABP7, ACTIN (loading control), and GFAP. B, average Western blot fold-change for FABP7 normalized to actin with adult-onset patient sample removed (error bars SEM). C, relationship between FABP7 and GFAP for each control and AxD tissue sample. D, Western blot of UGT8, actin (loading control), and GFAP. E, average Western blot fold-change for UGT8 normalized to actin with adult-onset patient sample removed (error bars SEM). F, relationship between UGT8 and GFAP for each control and AxD tissue sample.

Fig. 7

AxD patient and human control immunohistochemistry analysis of FABP7 and UGT8.A and B, the demographics of the samples analyzed are as follows: AxD patient #1 is a 3-year-old female with an R239H GFAP mutation, and AxD patient #2 is an 8-year-old male with an R416W GFAP mutation, and the control is a 4-year-old female with no neuropathology. A, anti-FABP7 staining. Ai, normal subcortical white matter from a control with no neuropathology. The antibody labels cells with several fine, branching processes, indicative of astrocytes. The arrow points to one astrocyte. The normal appearing white matter in the AxD patients is similar (data not shown). Aii, in this image of AxD white matter from AxD patient #1, the normal structures have been replaced by astrocytes and RFs, one of the largest is pointed out (arrow). The scale bars are 100 μm. B, anti-UGT8 staining. Bi, normal subcortical white matter from a control with no neuropathology. The antibody labels the cytoplasm of small cells with round nuclei, indicative of oligodendrocytes (arrows point to two oligodendrocytes). Just above the bottom oligodendrocyte is a blood vessel, which is negative. Bii, in this image of subpial area of the cerebral cortex of patient #2, where RFs accumulate, astrocytes contain punctate, positive signal in the cell body (black arrows). Round to oval profiles represent RFs (red arrow), many of which stain peripherally, a common feature of RF immunostaining. The astrocyte at the top right is multinucleated, a common feature of AxD astrocytes and is shown at higher magnification in the inset. Biii, in this section of AxD subcortical white matter from AxD patient #1, showing an astrocyte (black arrow), positive staining of astrocyte end feet around a blood vessel (short arrow), RFs (one shown with red arrow), and an oligodendrocyte (green arrow). In the normal appearing white matter in the AxD sections, the antibody stained only oligodendrocytes, as in controls (data not shown). Scale bars are 50 μm.