Quantitative diffusion imaging and genotype-by-sex interactions in a rat model of Alexander 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.

Plasma concentrations of glial fibrillary acidic protein, neurofilament light, and tau in Alexander disease.

INTRODUCTION: Alexander disease (AxD) is a rare leukodystrophy caused by dominant gain-of-function mutations in the gene encoding the astrocyte intermediate filament, glial fibrillary acidic protein (GFAP). However, there is an urgent need for biomarkers to assist in monitoring not only the progression of disease but also the response to treatment. GFAP is the obvious candidate for such a biomarker, as it is measurable in body fluids that are readily accessible for biopsy, namely cerebrospinal fluid and blood. However, in the case of ASOs, the treatment that is furthest in development, GFAP is the target of therapy and presumably would go down independent of disease status. Hence, there is a critical need for biomarkers that are not directly affected by the treatment strategy. METHODS: We explored the potential utility of biomarkers currently being studied in other neurodegenerative diseases and injuries, specifically neurofilament light protein (NfL), phosphorylated forms of tau, and amyloid-ß peptides (Aß42/40). RESULTS AND CONCLUSIONS: Here, we report that GFAP is elevated in plasma of all age groups afflicted by AxD, including those with adult onset. NfL and p-tau are also elevated, but to a much lesser extent than GFAP. In contrast, the levels of Aß40 and Aß42 are not altered in AxD.

Anastasis Drives Senescence and Non-Cell Autonomous Neurodegeneration in the Astrogliopathy Alexander Disease.

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.SIGNIFICANCE STATEMENT Under some conditions cells otherwise destined to die can be rescued just before death in a process called anastasis, or "rising from the dead." The fate and function of cells undergoing a near death experience is not well understood. Here, we find that in models and patient cells from Alexander disease, an important brain disorder in which glial cells promote neuronal dysfunction and death, anastasis of astrocytic glia leads to secretion of toxic signaling molecules and neurodegeneration. These studies demonstrate a previously unexpected deleterious consequence of rescuing cells on the brink of death and suggest therapeutic strategies for Alexander disease and related disorders of glia.

Pexidartinib treatment in Alexander disease model mice reduces macrophage numbers and increases glial fibrillary acidic protein levels, yet has minimal impact on other disease phenotypes.

BACKGROUND: Alexander disease (AxD) is a rare neurodegenerative disorder that is caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP), an intermediate filament that is primarily expressed by astrocytes. In AxD, mutant GFAP in combination with increased GFAP expression result in astrocyte dysfunction and the accumulation of Rosenthal fibers. A neuroinflammatory environment consisting primarily of macrophage lineage cells has been observed in AxD patients and mouse models. METHODS: To examine if macrophage lineage cells could serve as a therapeutic target in AxD, GFAP knock-in mutant AxD model mice were treated with a colony-stimulating factor 1 receptor (CSF1R) inhibitor, pexidartinib. The effects of pexidartinib treatment on disease phenotypes were assessed. RESULTS: In AxD model mice, pexidartinib administration depleted macrophages in the CNS and caused elevation of GFAP transcript and protein levels with minimal impacts on other phenotypes including body weight, stress response activation, chemokine/cytokine expression, and T cell infiltration. CONCLUSIONS: Together, these results highlight the complicated role that macrophages can play in neurological diseases and do not support the use of pexidartinib as a therapy for AxD.

Type II Alexander disease caused by splicing errors and aberrant overexpression of an uncharacterized GFAP isoform.

Alexander disease results from gain-of-function mutations in the gene encoding glial fibrillary acidic protein (GFAP). At least eight GFAP isoforms have been described, however, the predominant alpha isoform accounts for ∼90% of GFAP protein. We describe exonic variants identified in three unrelated families with Type II Alexander disease that alter the splicing of GFAP pre-messenger RNA (mRNA) and result in the upregulation of a previously uncharacterized GFAP lambda isoform (NM_001363846.1). Affected members of Family 1 and Family 2 shared the same missense variant, NM_001363846.1:c.1289G>A;p.(Arg430His) while in Family 3 we identified a synonymous variant in the adjacent nucleotide, NM_001363846.1:c.1290C>A;p.(Arg430Arg). Using RNA and protein analysis of brain autopsy samples, and a mini-gene splicing reporter assay, we demonstrate both variants result in the upregulation of the lambda isoform. Our approach demonstrates the importance of characterizing the effect of GFAP variants on mRNA splicing to inform future pathophysiologic and therapeutic study for Alexander disease.

AP-1 and the injury response of the GFAP gene.

Increased GFAP gene expression is a common feature of CNS injury, resulting in its use as a reporter to investigate mechanisms producing gliosis. AP-1 transcription factors are among those proposed to participate in mediating the reactive response. Prior studies found a consensus AP-1 binding site in the GFAP promoter to be essential for activity of reporter constructs transfected into cultured cells, but to have little to no effect on basal transgene expression in mice. Since cultured astrocytes display some properties of reactive astrocytes, these findings suggested that AP-1 transcription factors are critical for the upregulation of GFAP in injury, but not for its resting level of expression. We have examined this possibility by comparing the injury response in mice of lacZ transgenes driven by human GFAP promoters that contain the wild-type AP-1 binding site to those in which the site is mutated. An intact AP-1 site was found critical for a GFAP promoter response to the three different injury models used: physical trauma produced by cryoinjury, seizures produced by kainic acid, and chronic gliosis produced in an Alexander disease model. An unexpected additional finding was that the responses of the lacZ transgenes driven by the wild-type promoters were substantially less than that of the endogenous mouse GFAP gene. This suggests that the GFAP gene has previously unrecognized injury-responsive elements that reside further upstream of the transcription start site than the 2.2 kb present in the GFAP promoter segments used here.

Antisense suppression of glial fibrillary acidic protein as a treatment for Alexander disease.

OBJECTIVE: Alexander disease is a fatal leukodystrophy caused by autosomal dominant gain-of-function mutations in the gene for glial fibrillary acidic protein (GFAP), an intermediate filament protein primarily expressed in astrocytes of the central nervous system. A key feature of pathogenesis is overexpression and accumulation of GFAP, with formation of characteristic cytoplasmic aggregates known as Rosenthal fibers. Here we investigate whether suppressing GFAP with antisense oligonucleotides could provide a therapeutic strategy for treating Alexander disease. METHODS: In this study, we use GFAP mutant mouse models of Alexander disease to test the efficacy of antisense suppression and evaluate the effects on molecular and cellular phenotypes and non-cell-autonomous toxicity. Antisense oligonucleotides were designed to target the murine Gfap transcript, and screened using primary mouse cortical cultures. Lead oligonucleotides were then tested for their ability to reduce GFAP transcripts and protein, first in wild-type mice with normal levels of GFAP, and then in adult mutant mice with established pathology and elevated levels of GFAP. RESULTS: Nearly complete and long-lasting elimination of GFAP occurred in brain and spinal cord following single bolus intracerebroventricular injections, with a striking reversal of Rosenthal fibers and downstream markers of microglial and other stress-related responses. GFAP protein was also cleared from cerebrospinal fluid, demonstrating its potential utility as a biomarker in future clinical applications. Finally, treatment led to improved body condition and rescue of hippocampal neurogenesis. INTERPRETATION: These results demonstrate the efficacy of antisense suppression for an astrocyte target, and provide a compelling therapeutic approach for Alexander disease. Ann Neurol 2018;83:27-39.

Glial fibrillary acidic protein exhibits altered turnover kinetics in a mouse model of Alexander disease.

Mutations in the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP) lead to the rare and fatal disorder, Alexander disease (AxD). A prominent feature of the disease is aberrant accumulation of GFAP. It has been proposed that this accumulation occurs because of an increase in gene transcription coupled with impaired proteasomal degradation, yet this hypothesis remains untested. We therefore sought to directly investigate GFAP turnover in a mouse model of AxD that is heterozygous for a disease-causing point mutation (GfapR236H/+) (and thus expresses both wild-type and mutant protein). Stable isotope labeling by amino acids in cell culture, using primary cortical astrocytes, indicated that the in vitro half-lives of total GFAP in astrocytes from wild-type and mutant mice were similar at ∼3-4 days. Surprisingly, results obtained with stable isotope labeling of mammals revealed that, in vivo, the half-life of GFAP in mutant mice (15.4 ± 0.5 days) was much shorter than that in wild-type mice (27.5 ± 1.6 days). These unexpected in vivo data are most consistent with a model in which synthesis and degradation are both increased. Our work reveals that an AxD-causing mutation alters GFAP turnover kinetics in vivo and provides an essential foundation for future studies aimed at preventing or reducing the accumulation of GFAP. In particular, these data suggest that elimination of GFAP might be possible and occurs more quickly than previously surmised.

An In Vivo Pharmacological Screen Identifies Cholinergic Signaling as a Therapeutic Target in Glial-Based Nervous System Disease.

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 severe human degenerative 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 Gαq 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. SIGNIFICANCE STATEMENT: Despite the urgent need for better treatments for neurological diseases, drug development for these devastating disorders has been challenging. The effectiveness of traditional large-scale in vitro screens may be limited by the lack of the appropriate molecular, cellular, and structural environment. Using a simple Drosophila model of Alexander disease, we performed a moderate throughput chemical screen of FDA-approved drugs and natural compounds, and found that reducing muscarinic cholinergic signaling ameliorated clinical symptoms and oxidative stress in Alexander disease model flies and mice. Our work demonstrates that small animal models are valuable screening tools for therapeutic compound identification in complex human diseases and that existing drugs can be a valuable resource for drug discovery given their known pharmacological and safety profiles.

Astrocytes: a central element in neurological diseases.

The neurone-centred view of the past disregarded or downplayed the role of astroglia as a primary component in the pathogenesis of neurological diseases. As this concept is changing, so is also the perceived role of astrocytes in the healthy and diseased brain and spinal cord. We have started to unravel the different signalling mechanisms that trigger specific molecular, morphological and functional changes in reactive astrocytes that are critical for repairing tissue and maintaining function in CNS pathologies, such as neurotrauma, stroke, or neurodegenerative diseases. An increasing body of evidence shows that the effects of astrogliosis on the neural tissue and its functions are not uniform or stereotypic, but vary in a context-specific manner from astrogliosis being an adaptive beneficial response under some circumstances to a maladaptive and deleterious process in another context. There is a growing support for the concept of astrocytopathies in which the disruption of normal astrocyte functions, astrodegeneration or dysfunctional/maladaptive astrogliosis are the primary cause or the main factor in neurological dysfunction and disease. This review describes the multiple roles of astrocytes in the healthy CNS, discusses the diversity of astroglial responses in neurological disorders and argues that targeting astrocytes may represent an effective therapeutic strategy for Alexander disease, neurotrauma, stroke, epilepsy and Alzheimer's disease as well as other neurodegenerative diseases.

Astrocytic TDP-43 pathology in Alexander disease.

Alexander disease (AxD) is a rare neurodegenerative disorder characterized pathologically by the presence of eosinophilic inclusions known as Rosenthal fibers (RFs) within astrocytes, and is caused by dominant mutations in the coding region of the gene encoding glial fibrillary acidic protein (GFAP). GFAP is the major astrocytic intermediate filament, and in AxD patient brain tissue GFAP is a major component of RFs. TAR DNA binding protein of 43 kDa (TDP-43) is the major pathological protein in almost all cases of the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and ∼50% of frontotemporal lobar degeneration (FTLD), designated as FTLD-TDP. In ALS and FTLD-TDP, TDP-43 becomes insoluble, ubiquitinated, and pathologically phosphorylated and accumulates in cytoplasmic inclusions in both neurons and glia of affected brain and spinal cord regions. Previously, TDP-43 was detected in RFs of human pilocytic astrocytomas; however, involvement of TDP-43 in AxD has not been determined. Here we show that TDP-43 is present in RFs in AxD patient brains, and that insoluble phosphorylated full-length and high molecular weight TDP-43 accumulates in white matter of such brains. Phosphorylated TDP-43 also accumulates in the detergent-insoluble fraction from affected brain regions of Gfap(R236H/+) knock-in mice, which harbor a GFAP mutation homologous to one that causes AxD in humans, and TDP-43 colocalizes with astrocytic RF pathology in Gfap(R236H/+) mice and transgenic mice overexpressing human wild-type GFAP. These findings suggest common pathogenic mechanisms in ALS, FTLD, and AxD, and this is the first report of TDP-43 involvement in a neurological disorder primarily affecting astrocytes.

Disease specific therapies in leukodystrophies and leukoencephalopathies.

Leukodystrophies are a heterogeneous, often progressive group of disorders manifesting a wide range of symptoms and complications. Most of these disorders have historically had no etiologic or disease specific therapeutic approaches. Recently, a greater understanding of the pathologic mechanisms associated with leukodystrophies has allowed clinicians and researchers to prioritize treatment strategies and advance research in therapies for specific disorders, some of which are on the verge of pilot or Phase I/II clinical trials. This shifts the care of leukodystrophy patients from the management of the complex array of symptoms and sequelae alone to targeted therapeutics. The unmet needs of leukodystrophy patients still remain an overwhelming burden. While the overwhelming consensus is that these disorders collectively are symptomatically treatable, leukodystrophy patients are in need of advanced therapies and if possible, a cure.

Deficits in adult neurogenesis, contextual fear conditioning, and spatial learning in a Gfap mutant mouse model of Alexander disease.

Glial fibrillary acidic protein (GFAP) is the major intermediate filament of mature astrocytes in the mammalian CNS. Dominant gain of function mutations in GFAP lead 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 mutant GFAP leads to these pleiotropic effects are unknown. In addition to astrocytes, GFAP is 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.

Large-scale gene expression changes in APP/PSEN1 and GFAP mutation models exhibit high congruence with Alzheimer's disease.

Alzheimer's disease (AD) is a complex neurodegenerative disorder with both genetic and non-genetic causes. Animal research models are available for a multitude of diseases and conditions affecting the central nervous system (CNS), and large-scale CNS gene expression data exist for many of these. Although there are several models specifically for AD, each recapitulates different aspects of the human disease. In this study we evaluate over 500 animal models to identify those with CNS gene expression patterns matching human AD datasets. Approaches included a hypergeometric based scoring system that rewards congruent gene expression patterns but penalizes discordant gene expression patterns. The top two models identified were APP/PS1 transgenic mice expressing mutant APP and PSEN1, and mice carrying a GFAP mutation that is causative of Alexander disease, a primary disorder of astrocytes in the CNS. The APP/PS1 and GFAP models both matched over 500 genes moving in the same direction as in human AD, and both had elevated GFAP expression and were highly congruent with one another. Also scoring highly were the 5XFAD model (with five mutations in APP and PSEN1) and mice carrying CK-p25, APP, and MAPT mutations. Animals with the APOE3 and 4 mutations combined with traumatic brain injury ranked highly. Bulbectomized rats scored high, suggesting anosmia could be causative of AD-like gene expression. Other matching models included the SOD1G93A strain and knockouts for SNORD116 (Prader-Willi mutation), GRID2, INSM1, XBP1, and CSTB. Many top models demonstrated increased expression of GFAP, and results were similar across multiple human AD datasets. Heatmap and Uniform Manifold Approximation Plot results were consistent with hypergeometric ranking. Finally, some gene manipulation models, including for TYROBP and ATG7, were identified with reversed AD patterns, suggesting possible neuroprotective effects. This study provides insight for the pathobiology of AD and the potential utility of available animal models.

Beneficial effects of Nrf2 overexpression in a mouse model of Alexander disease.

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.

Protein changes in immunodepleted cerebrospinal fluid from a transgenic mouse model of Alexander disease detected using mass spectrometry.

Cerebrospinal fluid (CSF) is a low protein content biological fluid with a dynamic range spanning at least 9 orders of magnitude in protein content and is in direct contact with the brain. A modified IgY-14 immunodepletion treatment was performed to enhance analysis of the low volumes of CSF that are obtainable from mice. As a model system in which to test this approach, we utilized transgenic mice that overexpress the intermediate filament glial fibrillary acidic protein (GFAP). These mice are models for Alexander disease (AxD), a severe leukodystrophy in humans. From the CSF of control and transgenic mice we report the identification of 289 proteins, with relative quantification of 103 proteins. Biological and technical triplicates were performed to address animal variability as well as reproducibility in mass spectrometric analysis. Relative quantitation was performed using distributive normalized spectral abundance factor (dNSAF) spectral counting analysis. A panel of biomarker proteins with significant changes in the CSF of GFAP transgenic mice has been identified with validation from enzyme-linked immunosorbent assay (ELISA) and microarray data, demonstrating the utility of our methodology and providing interesting targets for future investigations on the molecular and pathological aspects of AxD.

Transgenic analysis of GFAP promoter elements.

Transcriptional regulation of the glial fibrillary acidic protein gene (GFAP) is of interest because of its astrocyte specificity and its upregulation in response to CNS injuries. We have used a transgenic approach instead of cell transfection to identify promoter elements of the human GFAP gene, since previous observations show that GFAP transcription is regulated differently in transfected cultured cells from in the mouse. We previously showed that block mutation of enhancer regions spanning from bp -1488 to -1434 (the C1.1 segment) and -1443 to -1399 (C1.2) resulted in altered patterns of expression and loss of astrocyte specificity, respectively. This analysis has now been extended upstream to bp -1612 to -1489 (the B region), which previously has been shown especially important for expression. Block mutation of each of four contiguous sequences, which together span the B region, each decreased the level of transgene activity by at least 50%, indicating that multiple sites contribute to the transcriptional activity in a cooperative manner. Several of the block mutations also altered the brain region pattern of expression, astrocyte specificity and/or the developmental time course. Transgenes were then analyzed in which mutations were limited to specific transcription factor binding sites in each of the 4 B block segments as well as in C1.1 and C1.2. Whereas mutation of the conserved consensus AP-1 site unexpectedly had little effect on transgene expression; NFI, SP1, STAT3, and NF-κB were identified as having important roles in regulating the strength of GFAP promoter activity and/or its astrocyte specificity.

P0S63del impedes the arrival of wild-type P0 glycoprotein to myelin in CMT1B mice.

More than 120 mutations in the Myelin Protein Zero gene (MPZ, P0) cause various forms of hereditary neuropathy. Two human mutations encoding either P0S63C or P0S63del have been shown to cause demyelination in mice through different gain of function pathomechanisms. P0S63del, for example, is retained in the endoplasmic reticulum (ER) and elicits a pathogenetic unfolded protein response (UPR). As P0 likely forms oligomers, another gain of abnormal function could include a dominant-negative interaction between P0S63del and normal P0 (P0wt). To test this idea, we generated a transgenic mouse that expressed a form of P0wt with a myc epitope tag at the C terminus (P0ct-myc). We show that P0ct-myc is trafficked and functions like P0wt, thus providing a new tool to study P0 in vivo. In mice that express both P0ct-myc and P0S63del, P0S63del specifically delays the transit of P0ct-myc through the ER and reduces the level of P0wt in the myelin sheath by half-a level previously shown to cause demyelination in mice and humans. Surprisingly, P0ct-myc does not co-immunoprecipitate with P0S63del, suggesting an indirect interaction. Thus, P0S63del causes not only a UPR-related toxic mechanism, but also a dominant-negative effect on P0wt that probably contributes to demyelinating neuropathy.

Early inactivation of p53 tumor suppressor gene cooperating with NF1 loss induces malignant astrocytoma.

Malignant astrocytoma, the most prevalent primary brain tumor, is resistant to all known therapies and frequently harbors mutations that inactivate p53 and activate Ras signaling. We have generated mouse strains that lack p53 and harbor a conditional allele of the NF1 tumor suppressor that negatively regulates Ras signaling. The mice develop malignant astrocytomas with complete penetrance. The majority of tumors display characteristics of glioblastoma multiforme with concomitant alteration of signaling pathways previously described in the human counterparts of this neoplasm. We find that the sequence of tumor suppressor inactivation influences tumorigenicity and that earliest evidence of tumor formation localizes to regions of the brain that contain a multipotent stem cell population capable of in vivo differentiation into neurons and glia.