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By Michalina Hanzel, PhD
Alexander disease is a progressive and fatal neurological disease with phenotypes ranging from myelination abnormalities, gait ataxia and megalencephaly to predisposition to seizures. It is an autosomal dominant disease with mutations in the glial fibrillary acidic protein (GFAP) gene that result in astrocytic cytoplasmic inclusions, termed Rosenthal fibers, which contribute to the global neurological abnormalities. The role of GFAP in the disease is not known because murine and human astrocytes differ vastly in complexity and, therefore, the mutant mouse models generated lack the complete phenotypic presentation of the human disease.
A recent study1 has shed light on the role of GFAP in Alexander disease by using two independent patient-derived induced pluripotent stem cell (iPSC) lines. The lines were generated from dermal fibroblasts of Alexander disease patients and, following reprogramming into iPSCs, were de-differentiated into astrocytes using standard protocols. Patient GFAP mutations did not alter astrocyte differentiation efficiency and all lines expressed the astrocyte markers S100B and SOX9. Only the patient-derived cell lines, however, exhibited abnormalities associated with the disease, including the presence of Rosenthal fibers, as well as presence of heat shock protein alphaB-crystallin in cellular inclusions.
GFAP was detected in immersion fixed differentiated rat cortical stem cells using Mouse Anti-Human GFAP Monoclonal Antibody (Catalog # MAB2594) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm.
Detailed examination of the patient-derived astrocytes revealed that there are substantial changes in the localization of endoplasmic reticulum and lysosomes, especially around the perinuclear area. Immunostaining for the lysosomal marker LAMP2 revealed vesicles that varied in size and distribution in Alexander disease cells, compared to uniform vesicles in control cells. Endoplasmic reticulum protein ERp57 labelling revealed significant increase in non-reticular, somatic endoplasmic reticulum in the disease lines. These results, along with RNA-seq experiments, suggest disruption of endo- and exocytotic pathways, altered lipid biosynthesis and/or vesicular formation.
Propagation of calcium waves is a major way of astrocyte-to-astrocyte communication and requires proper endoplasmic reticulum and lysosome function. Stimulating Alexander disease astrocytes revealed that, while they are able to release intracellular calcium stores, they are defective at propagating calcium waves from one cell to another. Extracellular ATP is essential for calcium wave propagation and disruption of either production, release or detection of ATP by astrocytes can be responsible for disease phenotype. Careful experiments have revealed that the release of ATP is disrupted in the astrocytes from Alexander disease patients. Correcting the mutation in GFAP in patient cells using the CRISPR methodology restored normal astrocyte functions. Overall, results suggest that disrupted communication between astrocytes can account for the neurological phenotypes found in Alexander disease patients.
Studies on human derived cells reveal functions of proteins that are not necessarily present in model organisms. In this case, patient-derived astrocytes revealed fundamental biological roles for human GFAP in vesicle regulation, especially lysosome-mediated exocytosis of ATP. This is an interesting result because GFAP, an intermediate filament protein, has previously only been assigned structural functions. More studies on Alexander disease patient-derived cells are being reported2 and will hopefully expose detailed mechanistic roles for human GFAP and more fully explain the global neurological deficits in Alexander disease patients.
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Michalina Hanzel, PhD
Postdoctoral Associate at The Rockefeller University
Dr. Hanzel is currently studying synaptic function in the cerebellum to understand neurodevelopmental disorders and has a background in developmental neurobiology, molecular and cell biology.
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