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

Michael R Heaven,Anthony W Herren,Daniel L Flint,Natasha L Pacheco,Jiangtao Li,Alice Tang,Fatima Khan,James E Goldman,Brett S Phinney,Michelle L Olsen

doi:10.1016/j.mcpro.2021.100180

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 (GFAPTg;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 GFAPTg;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 GFAPTg;Gfap+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAPTg;Gfap+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAPTg;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 GFAPTg;Gfap+/R236H mice. Last, to determine whether the findings in GFAPTg;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 GFAPTg;Gfap+/R236H mice.

Highlights

Models of Alexander disease (AxD) provide insight into functions of astrocytes related to neuropathology. peroxisome proliferator-activated receptor (PPAR) pathway found upregulated in AxD mice may be neuroprotective
The average coefficient of variation (CV) for all the peptides quantified in the GFAPTg;Gfap+/R236H and wild-type mice was 15.2% and 15.7%, respectively, indicating the measurements were highly reproducible across biological replicates (n = 4 per genotype)
The GFAPTg;Gfap+/R236H mouse model of AxD provides a means to assess the contribution of astrocytes to a neurological disorder, and in recent years there has been a growing body of evidence indicating that astrocytes play a critical role in the pathological processes occurring in other neurological conditions including autism [8], epilepsy [9], amyotrophic lateral sclerosis (ALS) [10, 70], major depressive disorder (MDD)

Summary

Graphical Abstract

In Brief The article contains the first whole brain proteomic survey from a mouse model of Alexander disease (AxD). To extend these previous reports to the protein level in whole brain, LC-MS/MS-based proteomics was performed on a severe mouse model of AxD (referred to hereafter as GFAPTg;Gfap+/R236H), which harbors a diseasecausing heterozygous point mutation at R236H in murine Gfap as well as elevated levels of GFAP due to a human GFAP transgene expressed in astrocytes [18] These mice develop motor abnormalities (front limb weakness) and die at approximately postnatal day 35 from convulsive seizures, recapitulating some of the clinical features of AxD [19]. GFAPTg;Gfap+/R236H mice display AxD-like neuropathological and molecular changes that include pronounced reactive gliosis, the presence of RFs [22], an inflammatory response characterized by microglial activation, and reduced levels of the astrocytic glutamate transporter [23]

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION