Background
Alexander disease (AxD) is a neurodegenerative disorder that primarily affects the white matter of the central nervous system (CNS) [1–5]. It was first reported in 1949 by W. Stewart Alexander in a 15-month-old boy with megalencephaly, hydrocephalus and psychomotor retardation. The brain pathology of the boy showed “progressive fibrinoid degeneration of fibrillary astrocytes,” [6] which was later identified as Rosenthal fibers that were initially described by Werner Rosenthal in ependymoma in 1898 [7]. Rosenthal fibers are homogeneous eosinophilic inclusions stained by hematoxylin and eosin, and consist mainly of glial fibrillary acidic protein (GFAP), αB-crystallin, heat shock protein (HSP) 27 and cyclin D2 [2, 3, 5]. Messing and colleagues reported that AxD was elicited by mutations in the gene encoding GFAP, a type III intermediate filament predominantly found in astrocytes. They suggested that the mutations act in a gain-of-function fashion based on their finding that the phenotypes of Gfap null mice did not parallel those of AxD [8]. Since then, many different GFAP mutations have been reported in AxD patients [9].
AxD has been classified into three clinical subtypes depending on age at onset (AAO). Infantile AxD (birth to 2 years), the most frequent subtype, is characterized by progressive megalencephaly and/or hydrocephalus, developmental delay, psychomotor retardation, epileptic seizures. Juvenile AxD (2–14 years) features spastic paraplegia, progressive bulbar signs and ataxia with spared cognitive function. Adult AxD (late adolescence and beyond), the least frequent subtype and often misdiagnosed with multiple sclerosis, shows variable manifestations including progressive ataxia, tetraparesis, bulbar and pseudobulbar signs [3, 10]. A revised classification system was proposed based on statistical analysis of clinical, radiologic, and genetic features of 215 cases of AxD. In the revised system, patients with type I AxD show early AAO, macrocephaly, developmental delay and typical brain magnetic resonance imaging (MRI) features. In contrast, patients with type II AxD exhibit various AAO, bulbar symptoms, ocular movement abnormality and atypical MRI findings [11].
Although AxD can be diagnosed based on comprehensive evaluation of patient history, physical examination, brain MRI, GFAP sequencing and cerebral biopsy, GFAP sequencing and cerebral biopsy remain to be the best diagnostic approaches [3, 10]. Detection of Rosenthal fibers through cerebral biopsy is considered to be one of the best diagnostic approaches. However, most putative AxD patients with GFAP mutations did not undergo cerebral biopsy [12–14] as it is an invasive procedure. In addition, Rosenthal fibers are not a pathognomonic feature of AxD because they are also occasionally found in astrocytic tumors, ependymoma, hamartomas, craniopharyngioma, pineal cysts, glial scars and multiple sclerosis [3, 15]. Hence, DNA sequencing is the only definitive diagnostic approach for AxD under most circumstances. However, identification of GFAP mutations in putative AxD patients does not guarantee that these mutations are associated with AxD because it is feasible that these mutations are just variants of unknown significance. Therefore, it is imperative to determine whether the GFAP mutations found in tentative AxD patients are disease-causing. To this end, two methods have been employed. First, an in vitro assembly assay was performed with recombinant mutant GFAPs purified from E. coli and the formation of aggregates was then assessed. Second, an expression plasmid encoding the mutant GFAP was transfected into various mammalian cell lines, which were then observed for GFAP aggregates [13, 16–20]. However, these methods might not be suitable for testing the causality of the GFAP mutations, because both methods do not reflect the in vivo environment around astrocytes and the second method adopts a strong exogenous promoter to express mutant GFAP.
Zebrafish (Danio rerio) are tropical freshwater fish and a vertebrate model organism that is used to study vertebrate development because of transparent embryos, and rapid and external development. Especially, zebrafish have been extensively used to research nervous system development and to establish vertebrate models of neurodegenerative diseases [21, 22]. Zebrafish have astrocytes [23], and zebrafish Gfap shares 67% identity and 77% similarity with human GFAP, along with well-conserved hot spot amino acids mutated in AxD (Fig. 1a) [24]. In addition, regulatory elements that drive the specific expression of zebrafish gfap in astrocytes were identified [25].
Fig. 1 Clinical features and GFAP sequences of the proband. a Comparison between human and zebrafish GFAP, and location of amino acid residues whose mutations are discussed in this study. Human GFAP: NCBI accession number NP_002046; zebrafish Gfap: NP_571448. D: aspartate; R: arginine. b Pedigree of individuals with p.Asp128Asn GFAP shown as solid symbols. Symbols and nomenclature follow established guidelines [44]. A small circle within a square or a circle indicates an individual who tested negative for a GFAP mutation. P, proband. c-e Brain MR images of the proband. c Sagittal T2-weighted MR image shows marked atrophy of the medullar oblongata (arrow). d Sagittal T1-weighted MR image reveals prominent atrophy in the upper cervical cord (arrow) and cerebellar hemisphere (arrowhead). e Fluid-attenuated inversion recovery (FLAIR) image shows high signal intensity lesions in the bilateral cerebellar dentate nuclei (arrow). f and g DNA sequence analysis of the GFAP. Arrows indicate c.382G. f Electropherogram of the proband reveals a heterozygous G-to-A substitution at position 382 of the GFAP, which is predicted to substitute asparagine for aspartic acid (p.Asp128Asn). g Representative electropherogram of GFAP sequences in 200 control subjects
We saw a patient who presented with slowly progressive gait disturbance and a missense mutation in the GFAP, and made a tentative diagnosis of AxD based on clinical and radiological findings. To determine whether the mutation is disease-causing, we set out to develop a zebrafish model that would be useful for molecular diagnosis of AxD.