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Alexander disease (AxD) is a devastating leukodystrophy caused by gain-of-function mutations in GFAP, and the only available treatments are supportive. Recent advances in antisense oligonucleotide (ASO) therapy have demonstrated that transcript targeting can be a successful strategy for human neurodegenerative diseases amenable to this approach. We have previously used mouse models of AxD to show that Gfap-targeted ASO suppresses protein accumulation and reverses pathology; however, the mice have a mild phenotype with no apparent leukodystrophy or overt clinical features and are therefore limited for assessing functional outcomes. In this report, we introduce a rat model of AxD that exhibits hallmark pathology with GFAP aggregation in the form of Rosenthal fibers, widespread astrogliosis, and white matter deficits. These animals develop normally during the first postnatal weeks but fail to thrive after weaning and develop severe motor deficits as they mature, with about 14% dying of unknown cause between 6 and 12 weeks of age. In this model, a single treatment with Gfap-targeted ASO provides long-lasting suppression, reverses GFAP pathology, and, depending on age of treatment, prevents or mitigates white matter deficits and motor impairment. In this report, we characterize an improved animal model of AxD with myelin pathology and motor impairment, recapitulating prominent features of the human disease, and use this model to show that ASO therapy has the potential to not only prevent but also reverse many aspects of disease.

Purpose: The clinical diagnosis and classification of Alexander disease (AxD) relies in part on qualitative neuroimaging biomarkers; however, these biomarkers fail to distinguish and discriminate different subtypes of AxD, especially in the presence of overlap in clinical symptoms. To address this gap in knowledge, we applied neurite orientation dispersion and density imaging (NODDI) to an innovative CRISPR‐Cas9 rat genetic model of AxD to gain quantitative insights into the neural substrates and brain microstructural changes seen in AxD and to potentially identify novel quantitative NODDI biomarkers of AxD. Methods: Multi‐shell DWI of age‐ and sex‐matched AxD and wild‐type Sprague Dawley rats (n = 6 per sex per genotype) was performed and DTI and NODDI measures calculated. A 3 × 2 × 2 analysis of variance model was used to determine the effect of genotype, biological sex, and laterality on quantitative measures of DTI and NODDI across regions of interest implicated in AxD. Results: There is a significant effect of genotype in the amygdala, hippocampus, neocortex, and thalamus in measures of both DTI and NODDI brain microstructure. A genotype by biological sex interaction was identified in DTI and NODDI measures in the corpus callosum, hippocampus, and neocortex. Conclusion: We present the first application of NODDI to the study of AxD using a rat genetic model of AxD. Our analysis identifies alterations in NODDI and DTI measures to large white matter tracts and subcortical gray nuclei. We further identified genotype by sex interactions, suggesting a possible role for biological sex in the neuropathogenesis of AxD. [ABSTRACT FROM AUTHOR]

Anastasis is a recently described process in which cells recover after late-stage apoptosis activation. The functional consequences of anastasis for cells and tissues are not clearly understood. Using Drosophila, rat and human cells and tissues, including analyses of both males and females, we present evidence that glia undergoing anastasis in the primary astrogliopathy Alexander disease subsequently express hallmarks of senescence. These senescent glia promote non-cell autonomous death of neurons by secreting interleukin family cytokines. Our findings demonstrate that anastasis can be dysfunctional in neurologic disease by inducing a toxic senescent population of astroglia. [ABSTRACT FROM AUTHOR]

Abstract Background Increased expression of glial fibrillary acidic protein (GFAP) within macroglia is commonly seen as a hallmark of glial activation after damage within the central nervous system, including the retina. The increased expression of GFAP in glia is also considered part of the pathologically inhibitory environment for regeneration of axons from damaged neurons. Recent studies have raised the possibility that reactive gliosis and increased GFAP cannot automatically be assumed to be negative events for the surrounding neurons and that the context of the reactive gliosis is critical to whether neurons benefit or suffer. We utilized transgenic mice expressing a range of Gfap to titrate the amount of GFAP in retinal explants to investigate the relationship between GFAP concentration and the regenerative potential of retinal ganglion cells. Findings Explants from Gfap-/- and Gfap+/- mice did not have increased neurite outgrowth compared with Gfap+/+ or Gfap over-expressing mice as would be expected if GFAP was detrimental to axon regeneration. In fact, Gfap over-expressing explants had the most neurite outgrowth when treated with a neurite stimulatory media. Transmission electron microscopy revealed that neurites formed bundles, which were surrounded by larger cellular processes that were GFAP positive indicating a close association between growing axons and glial cells in this regeneration paradigm. Conclusions We postulate that glial cells with increased Gfap expression support the elongation of new neurites from retinal ganglion cells possibly by providing a scaffold for outgrowth.

The role that glia play in neurological disease is poorly understood but increasingly acknowledged to be critical in a diverse group of disorders. Here we use a simple genetic model of Alexander disease, a progressive and severehumandegenerative nervous system disease caused by a primary astroglial abnormality, to perform an in vivo screen of 1987 compounds, including many FDA-approved drugs and natural products. We identify four compounds capable of dose-dependent inhibition of nervous system toxicity. Focusing on one of these hits, glycopyrrolate, we confirm the role for muscarinic cholinergic signaling in pathogenesis using additional pharmacologic reagents and genetic approaches. We further demonstrate that muscarinic cholinergic signaling works through downstream Gq to control oxidative stress and death of neurons and glia. Importantly, we document increased muscarinic cholinergic receptor expression in Alexander disease model mice and in postmortem brain tissue from Alexander disease patients, and that blocking muscarinic receptors in Alexander disease model mice reduces oxidative stress, emphasizing the translational significance of our findings. We have therefore identified glial muscarinic signaling as a potential therapeutic target in Alexander disease, and possibly in other gliopathic disorders as well. [ABSTRACT FROM AUTHOR]

Glial fibrillary acidic protein (GFAP) is the major intermediate filament of mature astrocytes in the mammalian CNS. Dominant gain of function mutations inGFAPlead to the fatal neurodegenerative disorder, Alexander disease (AxD), which is characterized by cytoplasmic protein aggregates known as Rosenthal fibers along with variable degrees of leukodystrophy and intellectual disability. The mechanisms by which mutantGFAPleads to these pleiotropic effects are unknown. In addition to astrocytes,GFAPis also expressed in other cell types, particularly neural stem cells that form the reservoir supporting adult neurogenesis in the hippocampal dentate gyrus and subventricular zone of the lateral ventricles. Here, we show that mouse models of AxD exhibit significant pathology in GFAP-positive radial glia-like cells in the dentate gyrus, and suffer from deficits in adult neurogenesis. In addition, they display impairments in contextual learning and spatial memory. This is the first demonstration of cognitive phenotypes in a model of primary astrocyte disease. [ABSTRACT FROM AUTHOR]

Alexander disease is a fatal neurodegenerative disease caused by dominant mutations in glial fibrillary acidic protein (GFAP). The disease is characterized by protein inclusions called Rosenthal fibers within astrocyte cell bodies and processes, and an antioxidant response mediated by the transcription factor Nrf2. We sought to test whether further elevation of Nrf2 would be beneficial in a mouse model of Alexander disease. Forcing overexpression of Nrf2 in astrocytes of R236H GFAP mutant mice decreased GFAP protein in all brain regions examined (olfactory bulb, hippocampus, cerebral cortex, brainstem, cerebellum, and spinal cord) and decreased Rosenthal fibers in olfactory bulb, hippocampus, corpus callosum, and brainstem. Nrf2 overexpression also restored body weights of R236H mice to near wild-type levels. Nrf2 regulates several genes involved in homeostasis of the antioxidant molecule glutathione, and the neuroprotective effects of Nrf2 in other neurological disorders may reflect restoration of glutathione to normal levels. However, glutathione levels in R236H mice were not decreased. Nrf2 overexpression did not change glutathione levels or ratio of reduced to oxidized glutathione (indicative of oxidative stress) in olfactory bulb, where Nrf2 dramatically reduced GFAP. Depletion of glutathione through knock-out of the GCLM (glutamate-cysteine ligase modifier subunit) also did not affect GFAP levels or body weight of R236H mice. These data suggest that the beneficial effects of Nrf2 are not mediated through glutathione. [ABSTRACT FROM AUTHOR]

Glial fibrillary acidic protein (GFAP) is the major intermediate filament protein of astrocytes, and its expression changes dramatically during development and following injury. To facilitate study of the regulation of GFAP expression, we have generated dual transgenic mice expressing both firefly luciferase under the control of a 2.2 kb human GFAP promoter and Renilla luciferase under the control of a 0.5 kb human Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) promoter for normalization of the GFAP signal. The GFAP-fLuc was highly expressed in brain compared to other tissues, and was limited to astrocytes, whereas the GAPDH-RLuc was more widely expressed. Normalization of the GFAP signal to the GAPDH signal reduced the inter-individual variability compared to using the GFAP signal alone. The GFAP/GAPDH ratio correctly reflected the up-regulation of GFAP that occurs following retinal degeneration in FVB/N mice because of the rd mutation. Following kainic acid-induced seizures, changes in the GFAP/GAPDH ratio precede those in total GFAP protein. In knock-in mice expressing the R236H Alexander disease mutant, GFAP promoter activity is only transiently elevated and may not entirely account for the accumulation of GFAP protein that takes place. [ABSTRACT FROM AUTHOR]

Mutations in the gene for the astrocyte specific intermediate filament, glial fibrillary acidic protein (GFAP), cause the rare leukodystrophy Alexander disease (AxD). To study the pathology of this primary astrocyte defect, we have generated knock-in mice with missense mutations homologous to those found in humans. In this report, we show that mice with GFAP-R76H and -R236H mutations develop Rosenthal fibers, the hallmark protein aggregates observed in astrocytes in AxD, in the hippocampus, corpus callosum, olfactory bulbs, subpial, and periventricular regions. Astrocytes in these areas appear reactive and total GFAP expression is elevated. Although general white matter architecture and myelination appear normal, when crossed with an antioxidant response element reporter line, the mutant mice show a distinct pattern of reporter-gene induction that is especially prominent in the corpus callosum, and histochemical staining reveals accumulation of iron in the same region. The mutant mice have a normal lifespan and show no overt behavioral defects, but are more susceptible to kainate-induced seizures. Although these mice demonstrate increased GFAP expression by themselves, further elevation of GFAP via crosses to GFAP transgenic animals leads to a shift in GFAP solubility, an increased stress response, and ultimately death. The mice do not display the full spectrum of pathology observed in human infantile AxD, but may more closely resemble the adult form of the disease. These studies provide formal proof linking GFAP mutations with Rosenthal fibers and oxidative stress, and correlate gliosis and GFAP protein levels to the severity of the disease. [ABSTRACT FROM AUTHOR]

Alexander disease is a fatal neurodegenerative disease caused by dominant mutations in glial fibrillary acidic protein (GFAP). The disease is characterized by protein inclusions called Rosenthal fibers within astrocyte cell bodies and processes, and an antioxidant response mediated by the transcription factor Nrf2. We sought to test whether further elevation of Nrf2 would be beneficial in a mouse model of Alexander disease. Forcing overexpression of Nrf2 in astrocytes of R236H GFAP mutant mice decreased GFAP protein in all brain regions examined (olfactory bulb, hippocampus, cerebral cortex, brainstem, cerebellum, and spinal cord) and decreased Rosenthal fibers in olfactory bulb, hippocampus, corpus callosum, and brainstem. Nrf2 overexpression also restored body weights of R236H mice to near wild-type levels. Nrf2 regulates several genes involved in homeostasis of the antioxidant molecule glutathione, and the neuroprotective effects of Nrf2 in other neurological disorders may reflect restoration of glutathione to normal levels. However, glutathione levels in R236H mice were not decreased. Nrf2 overexpression did not change glutathione levels or ratio of reduced to oxidized glutathione (indicative of oxidative stress) in olfactory bulb, where Nrf2 dramatically reduced GFAP. Depletion of glutathione through knock-out of the GCLM (glutamate-cysteine ligase modifier subunit) also did not affect GFAP levels or body weight of R236H mice. These data suggest that the beneficial effects of Nrf2 are not mediated through glutathione.