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The brain of a 58-year-old woman was included as a civilian control in an ongoing autopsy study of military traumatic brain injury (TBI). The woman died due to a polysubstance drug overdose, with Coronavirus Disease 2019 (COVID-19) serving as a contributing factor. Immunohistochemical stains for β-amyloid (Aβ), routinely performed for the TBI study, revealed numerous, unusual neocortical Aβ deposits. We investigated the autopsied brains of 10 additional young patients (60 years old) who died of COVID-19, and found similar Aβ deposits in all, using two different Aβ antibodies across three different medical centers. The deposits failed to stain with Thioflavin-S. To investigate whether or not these deposits formed uniquely to COVID-19, we applied Aβ immunostains to the autopsied brains of COVID-19-negative adults who died with acute respiratory distress syndrome and infants with severe cardiac anomalies, and also biopsy samples from patients with subacute cerebral infarcts. Cortical Aβ deposits were also found in these cases, suggesting a link to hypoxia. The fate of these deposits and their effects on function are unknown, but it is possible that they contribute to the neurocognitive sequelae observed in some COVID-19 patients. Our findings may also have broader implications concerning hypoxia and its role in Aβ deposition in the brain.

Post-zygotically acquired genetic variants, or somatic variants, that arise during cortical development have emerged as important causes of focal epilepsies, particularly those due to malformations of cortical development. Pathogenic somatic variants have been identified in many genes within the PI3K-AKT-mTOR-signalling pathway in individuals with hemimegalencephaly and focal cortical dysplasia (type II), and more recently in SLC35A2 in individuals with focal cortical dysplasia (type I) or non-dysplastic epileptic cortex. Given the expanding role of somatic variants across different brain malformations, we sought to delineate the landscape of somatic variants in a large cohort of patients who underwent epilepsy surgery with hemimegalencephaly or focal cortical dysplasia. We evaluated samples from 123 children with hemimegalencephaly (n = 16), focal cortical dysplasia type I and related phenotypes (n = 48), focal cortical dysplasia type II (n = 44), or focal cortical dysplasia type III (n = 15). We performed high-depth exome sequencing in brain tissue-derived DNA from each case and identified somatic single nucleotide, indel and large copy number variants. In 75% of individuals with hemimegalencephaly and 29% with focal cortical dysplasia type II, we identified pathogenic variants in PI3K-AKT-mTOR pathway genes. Four of 48 cases with focal cortical dysplasia type I (8%) had a likely pathogenic variant in SLC35A2. While no other gene had multiple disease-causing somatic variants across the focal cortical dysplasia type I cohort, four individuals in this group had a single pathogenic or likely pathogenic somatic variant in CASK, KRAS, NF1 and NIPBL, genes previously associated with neurodevelopmental disorders. No rare pathogenic or likely pathogenic somatic variants in any neurological disease genes like those identified in the focal cortical dysplasia type I cohort were found in 63 neurologically normal controls (P = 0.017), suggesting a role for these novel variants. We also identified a somatic loss-of-function variant in the known epilepsy gene, PCDH19, present in a small number of alleles in the dysplastic tissue from a female patient with focal cortical dysplasia IIIa with hippocampal sclerosis. In contrast to focal cortical dysplasia type II, neither focal cortical dysplasia type I nor III had somatic variants in genes that converge on a unifying biological pathway, suggesting greater genetic heterogeneity compared to type II. Importantly, we demonstrate that focal cortical dysplasia types I, II and III are associated with somatic gene variants across a broad range of genes, many associated with epilepsy in clinical syndromes caused by germline variants, as well as including some not previously associated with radiographically evident cortical brain malformations.

Over 100 years ago, von Hösslein and Alzheimer described enlarged and multinucleated astrocytes in the brains of patients with Wilson disease. These odd astrocytes, now well known to neuropathologists, are present in a large variety of neurological disorders, and yet the mechanisms underlying their generation and their functional attributes are still not well understood. They undergo abnormal mitoses and fail to accomplish cytokinesis, resulting in multinucleation. Oxidative stress, hypoxia, and inflammation may be contributing pathologies to generate these astrocytes. The abnormal mitoses occur from changes in cell shape, the accumulation of cytoplasmic proteins, and the mislocalization of many of the important molecules whose coordination is necessary for proper mitotic spindle formation. Modern technologies will be able to characterize their abnormalities and solve century old questions of their form and function.

Pilocytic astrocytomas are the commonest childhood brain tumor. They are typically benign and usually are solitary neoplasms. To our knowledge, only one report of a pilocytic astrocytoma with leukemia has been previously issued. We herein describe the first case with documented histopathology of a 2-year-old boy who had a cerebellar pilocytic astrocytoma co-localized with an acute B-lymphoblastic leukemia. We speculate that chemotactic migration of leukemic cells to the pilocytic astrocytoma may be partly mediated through vascular endothelial growth factor (VEGF) and VEGF receptors.

BACKGROUND: In clinical practice, calcifications seen on CT studies within the large brain arteries are often referred as a surrogate marker for cholesterol-mediated atherosclerosis. However, limited data exists to support the association between calcification and atherosclerosis. In this study, we examined if intracranial arterial calcifications were associated with cholesterol-mediated intracranial large artery atherosclerosis (ILAA) within the arteries of the Circle of Willis in an autopsy based sample. METHODS: We carried out a cross-sectional analysis of histopathological characteristics of brain large arteries obtained from autopsy cases. Brain large arteries were examined for evidences of calcifications, which were rated as macroscopic (coalescent) and microscopic (scattered). In addition to calcification, we also obtained measurement of the arterial wall and the presence of ILAA and non-atherosclerotic arterial fibrosis. We built hierarchical models adjusted for demographic and vascular risk factors to assess the relationship between calcification and ILAA. RESULTS: In univariate analysis, the presence of any arterial calcifications was associated with cerebral infarcts (29% v. 14%, p

Alexander Disease (AxD) is a degenerative disorder caused by mutations in the GFAP gene, which encodes the major intermediate filament of astrocytes. As other cells in the CNS do not express GFAP, AxD is a primary astrocyte disease. Astrocytes acquire a large number of pathological features, including changes in morphology, the loss or diminution of a number of critical astrocyte functions and the activation of cell stress and inflammatory pathways. AxD is also characterized by white matter degeneration, a pathology that has led it to be included in the "leukodystrophies." Furthermore, variable degrees of neuronal loss take place. Thus, the astrocyte pathology triggers alterations in other cell types. Here, we will review the neuropathology of AxD and discuss how a disease of astrocytes can lead to severe pathologies in non-astrocytic cells. Our knowledge of the pathophysiology of AxD will also lead to a better understanding of how astrocytes interact with other CNS cells and how astrocytes in the gliosis that accompanies many neurological disorders can damage the function and survival of other cells.

Orbital schwannomas are typically slow-growing benign tumors that can cause gradual loss of vision, proptosis, and limitation of ocular motility. The authors present an atypical case of a rapidly growing orbital apex schwannoma in a patient with preexisting vision loss secondary to presumed sarcoidal optic neuritis. Contrary to the slowly progressive nature of a typical orbital schwannoma, the lesion was observed to enlarge from radiologically undiscernible to 3.5 cm over 4 years.

Alexander disease (AxD) is a neurodegenerative disorder characterized by astrocytic protein aggregates called Rosenthal fibers (RFs). We used mouse models of AxD to determine the protein composition of RFs to obtain information about disease mechanisms including the hypothesis that sequestration of proteins in RFs contributes to disease. A method was developed for RF enrichment, and analysis of the resulting fraction using isobaric tags for relative and absolute quantitation mass spectrometry identified 77 proteins not previously associated with RFs. Three of five proteins selected for follow-up were confirmed enriched in the RF fraction by immunobloting of both the AxD mouse models and human patients: receptor for activated protein C kinase 1 (RACK1), G1/S-specific cyclin D2, and ATP-dependent RNA helicase DDX3X. Immunohistochemistry validated cyclin D2 as a new RF component, but results for RACK1 and DDX3X were equivocal. None of these was decreased in the non-RF fractions compared to controls. A similar result was obtained for the previously known RF component, alphaB-crystallin, which had been a candidate for sequestration. Thus, no support was obtained for the sequestration hypothesis for AxD. Providing possible insight into disease progression, the association of several of the RF proteins with stress granules suggests a role for stress granules in the origin of RFs.

It is hypothesized that outward remodeling in systemic arteries is a compensatory mechanism for lumen area preservation in the face of increasing arterial stenosis. Large brain arteries have also been studied, but it remains unproven if all assumptions about arterial remodeling can be replicated in the cerebral circulation.The sample included 196 autopsied subjects with a mean age of 55 years; 63 % were men, and 74 % non-Hispanic whites. From each of 1396 dissected cadaveric large arteries of the circle of Willis, the areas of the lumen, intima, media, and adventitia were measured. Internal elastic lamina (IEL) area was defined as the area encircled by this layer. Stenosis was calculated by dividing the plaque area by the IEL area and multiplying by 100.Plotting stenosis against lumen area or stratified by arterial size showed no preservation of the lumen in the setting of growing stenosis. We could not find an association between greater IEL proportion and stenosis (B = 0.44, P = 0.86). Stratifying arteries by their size, we found that smaller arteries have greater lumen reduction at any degree of stenosis (B = -23.65, P ≤ 0.0001), and although larger arteries show a positive association between IEL proportion and stenosis, this was no longer significant after adjusting for covariates (B = 6.0, P = 0.13).We cannot confirm the hypothesis that large brain arteries undergo outward remodeling as an adaptive response to increasing degrees of stenosis. We found that the lumen decreases proportionally to the degree of stenosis.

Autism spectrum disorder (ASD) consists of a group of complex developmental disabilities characterized by impaired social interactions, deficits in communication and repetitive behavior. Multiple lines of evidence implicate mitochondrial dysfunction in ASD. In postmortem BA21 temporal cortex, a region that exhibits synaptic pathology in ASD, we found that compared to controls, ASD patients exhibited altered protein levels of mitochondria respiratory chain protein complexes, decreased Complex I and IV activities, decreased mitochondrial antioxidant enzyme SOD2, and greater oxidative DNA damage. Mitochondrial membrane mass was higher in ASD brain, as indicated by higher protein levels of mitochondrial membrane proteins Tom20, Tim23 and porin. No differences were observed in either mitochondrial DNA or levels of the mitochondrial gene transcription factor TFAM or cofactor PGC1α, indicating that a mechanism other than alterations in mitochondrial genome or mitochondrial biogenesis underlies these mitochondrial abnormalities. We further identified higher levels of the mitochondrial fission proteins (Fis1 and Drp1) and decreased levels of the fusion proteins (Mfn1, Mfn2 and Opa1) in ASD patients, indicating altered mitochondrial dynamics in ASD brain. Many of these changes were evident in cortical pyramidal neurons, and were observed in ASD children but were less pronounced or absent in adult patients. Together, these findings provide evidence that mitochondrial function and intracellular redox status are compromised in pyramidal neurons in ASD brain and that mitochondrial dysfunction occurs during early childhood when ASD symptoms appear.

Alexander Disease (AxD) is a primary disorder of astrocytes, caused by heterozygous mutations inGFAP, which encodes the major astrocyte intermediate filament protein, glial fibrillary acidic protein (GFAP). Astrocytes in AxD display hypertrophy, massive increases in GFAP, and the accumulation of Rosenthal fibers, cytoplasmic protein inclusions containing GFAP, and small heat shock proteins. To study the effects of GFAP mutations on astrocyte morphology and physiology, we have examined hippocampal astrocytes in three mouse models of AxD, a transgenic line (GFAPTg) in which the normal humanGFAPis expressed in several copies, a knock-in line (Gfap+/R236H) in which one of theGfapgenes bears an R236H mutation, and a mouse derived from the mating of these two lines (GFAPTg;Gfap+/R236H). We report changes in astrocyte phenotype in all lines, with the most severe in theGFAPTg;Gfap+/R236H, resulting in the conversion of protoplasmic astrocytes to cells that have lost their bushy-like morphology because of a reduction of distal fine processes, and become multinucleated and hypertrophic. Astrocytes activate the mTOR cascade, acquire CD44, and lose GLT-1. The altered astrocytes display a microheterogeneity in phenotypes, even neighboring cells. Astrocytes also show diminished glutamate transporter current, are significantly depolarized, and not coupled to adjacent astrocytes. Thus, the accumulation of GFAP in the AxD mouse astrocytes initiates a conversion of normal, protoplasmic astrocytes to astrocytes that display severely “reactive” characteristics, many of which may be detrimental to neighboring neurons and oligodendrocytes.

Astrocytes undergo important phenotypic changes in many neurological disorders, including strokes, trauma, inflammatory diseases, infectious diseases, and neurodegenerative diseases. We have been studying the astrocytes of Alexander disease (AxD), which is caused by heterozygous mutations in the GFAP gene, which is the gene that encodes the major astrocyte intermediate filament protein. AxD is a primary astrocyte disease because GFAP expression is specific to astrocytes in the central nervous system (CNS). The accumulation of extremely large amounts of GFAP causes many molecular changes in astrocytes, including proteasome inhibition, stress kinase activation, mechanistic target of rapamycin (mTOR) activation, loss of glutamate and potassium buffering capacity, loss of astrocyte coupling, and changes in cell morphology. Many of these changes appear to be common to astrocyte reactions in other neurological disorders. Using AxD to illuminate common mechanisms, we discuss the molecular pathology of AxD astrocytes and compare that to astrocyte pathology in other disorders.

We previously reported that retrovirally transduced platelet-derived growth factor-B (PDGFB) in glial progenitors of the rat cerebral white matter, subventricular zone, or brain stem induced malignant brain tumors closely resembling human glioblastoma (GBM). While human GBMs may progress over the period of several months to a few years, prospective, long-term in-vivo observation of histological changes of the tumor tissues is not feasible in these models, because the animals undergo rapid tumor progression and mortality within approximately 1 month. We thus performed successive, long-term in-vivo transplantation of the PDGFB-induced tumor cells into the rat cerebrum. Primary retroviral transduction of PDGFB in the glial progenitors of the rat basal ganglia induced malignant glioma resembling human GBM or anaplastic oligodendroglioma (AOL) consisting of relatively monomorphous tumor cells expressing markers for the oligodendrocyte lineage. In the course of long-term successive transplantation, tumor cells presented pleomorphism as well as focal GFAP expression. This suggests that secondary chromosomal aberration and dysregulation of gene expression following accelerated cell cycle by PDGFB stimulation would induce morphological and immunophenotypic changes in tumor cells. Furthermore, while the primary tumors contained only a minor fraction of proviral GFP-expressing or hemagglutinin-expressing cells, most tumor cells came to express these proviral genes in the course of serial transplantation suggesting a persistent role of PDGFB-expressing cells in maintenance and growth of the tumors. This model would be useful for investigation of the long-term effects of PDGFB stimulation in glioma tissues on anaplastic evolution.

Alexander disease (AxD) is a leukodystrophy caused by heterozygous mutations in the gene for glial fibrillary acidic protein, an intermediate filament protein expressed by astrocytes. The mutation causes prominent protein aggregates inside astrocytes; there is also loss of myelin and oligodendrocytes and neuronal degeneration. We show that immunohistochemical staining for glutamate transporter 1, the major brain glutamate transporter expressed primarily in astrocytes suggests decreased levels in the hippocampi of infantile AxD patients. A knock-in mouse model of AxD also shows significant reduction of glutamate transporter 1 in the hippocampus. To explore this phenomenon at the cellular level, wild-type and R239C mutant glial fibrillary acidic proteins (the most common mutation) were overexpressed in astrocytes in culture. Western blotting and whole-cell patch clamp recordings demonstrated that the R239C astrocytes exhibited markedly reduced glutamate transporter 1 protein levels; this resulted in attenuated or abolished glutamate-induced inward transporter current. Neurons cocultured with the R239C astrocytes exhibited increased death after glutamate challenge. These results indicate that aberrant astrocytes have decreased glutamate uptake, which may play an important role in the pathogenesis of neuronal and oligodendrocyte injury and death in AxD.

Platelet-derived growth factor (PDGF) plays a major role in regulating migration, proliferation, and differentiation of glial progenitors during normal brain development and in the abnormal proliferation and dispersion that drives the formation of malignant gliomas. To further explore the relationship between PDGF's effects on normal glial progenitors and its role in the formation of gliomas, we infected progenitor cells in the subventricular zone (SVZ) of the lateral ventricle of neonatal rat pups with a retrovirus that expresses PDGF and green fluorescent protein (GFP). At 3 days post-injection (dpi), a proliferation of PDGFRalpha+ progenitors was seen in the SVZ and white matter around the injection site and by 10 dpi the animals had large diffusely infiltrating tumors that resembled glioblastomas. The tumors contained a massive proliferation of both infected and uninfected PDGFRalpha+ progenitors, suggesting that PDGF was driving tumor formation via both autocrine and paracrine signaling. Rats co-injected with two retroviruses (one that expresses PDGF-IRES-DSRED and one that expresses only GFP) formed tumors that contained a mixture of DSRED+ cells (PDGF producers) and GFP+ cells (recruited progenitors). Time-lapse microscopy of slice cultures confirmed that both DSRED+ and GFP+ cells were highly migratory and proliferative. Furthermore, adding exogenous PDGF to slice cultures generated from nontumor-bearing brains (injected with control GFP retrovirus only) stimulated the migration and proliferation of GFP+ progenitors. These findings reveal the inherent growth factor responsiveness and tumorigenic potential of PDGFRalpha+ progenitors and highlight the importance of paracrine signaling in stimulating glioma growth and infiltration.

Glucose transporter type 1 (Glut-1) facilitates glucose flux across the blood-brain-barrier. In humans, Glut-1 deficiency causes acquired microcephaly, seizures and ataxia, which are recapitulated in our Glut-1 haploinsufficient mouse model. Postnatal brain weight deceleration and development of reactive astrogliosis were significant by P21 in Glut-1(+/-) mice. The brain weight differences remained constant after P21 whereas the reactive astrocytosis continued to increase and peaked at P90. Brain immunoblots showed increased phospho-mTOR and decreased phospho-GSK3-beta by P14. After fasting, the mature Glut-1(+/-) females showed a trend towards elevated phospho-GSK3-beta, a possible neuroprotective response. Lithium chloride treatment of human skin fibroblasts from control and Glut-1 DS patients produced a 45% increase in glucose uptake. Brain imaging of mature Glut-1(+/-) mice revealed a significantly decreased hippocampal volume. These subtle immunochemical changes reflect chronic nutrient deficiency during brain development and represent the experimental correlates to the human neurological phenotype associated with Glut-1 DS.

In the developing brain, cell migration is a crucial process for structural organization, and is therefore highly regulated to allow the correct formation of complex networks, wiring neurons, and glia. In the early postnatal brain, late developmental processes such as the production and migration of astrocyte and oligodendrocyte progenitors still occur. Although the brain is completely formed and structured few weeks after birth, it maintains a degree of plasticity throughout life, including axonal remodeling, synaptogenesis, but also neural cell birth, migration and integration. The subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus are the two main neurogenic niches in the adult brain. Neural stem cells reside in these structures and produce progenitors that migrate toward their ultimate location: the olfactory bulb and granular cell layer of the DG respectively. The aim of this review is to synthesize the increasing information concerning the organization, regulation and function of cell migration in a mature brain. In a normal brain, proteins involved in cell-cell or cell-matrix interactions together with secreted proteins acting as chemoattractant or chemorepellant play key roles in the regulation of neural progenitor cell migration. In addition, recent data suggest that gliomas arise from the transformation of neural stem cells or progenitor cells and that glioma cell infiltration recapitulates key aspects of glial progenitor migration. Thus, we will consider glioma migration in the context of progenitor migration. Finally, many observations show that brain lesions and neurological diseases trigger neural stem/progenitor cell activation and migration toward altered structures. The factors involved in such cell migration/recruitment are just beginning to be understood. Inflammation which has long been considered as thoroughly disastrous for brain repair is now known to produce some positive effects on stem/progenitor cell recruitment via the regulation of growth factor signaling and the secretion of a number of chemoattractant cytokines. This knowledge is crucial for the development of new therapeutic strategies. One of these strategies could consist in increasing the mobilization of endogenous progenitor cells that could replace lost cells and improve functional recovery.

Gliogenesis in the mammalian CNS continues after birth, with astrocytes being generated well into the first two postnatal weeks. In this study, we have isolated an A2B5+ astrocyte precursor (APC) from the postnatal rat forebrain, which is capable of differentiating into mature astrocytes in serum-free medium without further trophic support. Exposure to basic fibroblast growth factor (bFGF) selectively induces the APCs to proliferate, forming clusters of vimentin+ cells, which, within 2 weeks, differentiate into GFAP+ astrocytes. While bFGF functions as a potent mitogen, neither is it necessary to induce or maintain astrocyte differentiation, nor is it capable of maintaining the precursors in an immature, proliferative state. APCs exit the cell cycle and differentiate, even in the continued presence of fibroblast growth factor alone or in combination with other mitogenic factors such as platelet-derived growth factor. Under the culture conditions used, it was not possible to cause the astrocytes to re-enter cell cycle. After transplantation into the neonatal forebrain, APCs differentiated exclusively into astrocytes, regardless of brain region. Initially distributed widely within the forebrain, the precursors are most greatly concentrated within the subventricular zone (SVZ) and subcortical white matter, where they are maintained throughout postnatal development. APCs can be isolated from the SVZ and white matter of animals as late as 4 weeks after birth. © 2008 Wiley-Liss, Inc.

Forebrain subventricular zone (SVZ) progenitor cells give rise to glia and olfactory bulb interneurons during early postnatal life in rats. We investigated the potential of SVZ cells to alter their fate by transplanting them into a heterotypic neurogenic and gliogenic environment-the cerebellum. Transplanted cells were examined 1 to 7 weeks and 6 months post transplantation. Forebrain progenitors populated the cerebellum and differentiated into oligodendrocytes, cerebellar-specific Bergmann glia and velate astrocytes, and neurons. The transplanted cells that differentiated into neurons maintained an interneuronal fate: they were GABA-positive, expressed interneuronal markers, such as calretinin, and exhibited membrane properties that are characteristic of interneurons. However, the transplanted interneurons lost the expression of the olfactory bulb transcription factors Tbr2 and Dlx1, and acquired a cerebellar-like morphology. Forebrain SVZ progenitors thus have the potential to adapt to a new environment and integrate into diverse regions, and may be a useful tool in transplantation strategies.

Here we review how GFAP mutations cause Alexander disease. The current data suggest that a combination of events cause the disease. These include: (i) the accumulation of GFAP and the formation of characteristic aggregates, called Rosenthal fibers, (ii) the sequestration of the protein chaperones αB-crystallin and HSP27 into Rosenthal fibers, and (iii) the activation of both Jnk and the stress response. These then set in motion events that lead to Alexander disease. We discuss parallels with other intermediate filament diseases and assess potential therapies as part of this review as well as emerging trends in disease diagnosis and other aspects concerning GFAP.

During embryogenesis, dorsal radial glia generate pyramidal cell neurons but not interneurons, and are thought to degenerate or transform into astrocytes in the postnatal brain. Ventral radial glia, in contrast, retract their processes to form GFAP+ subventricular zone (SVZ) astrocytes that continue to generate interneurons into adulthood. We sought to fate map dorsal radial glia by codelivering an adenovirus expressing Cre recombinase and a lentivirus expressing dsRedExpress to the dorsal cortical surface of ROSA26R-YFP mice. Whereas the adenovirus is retrogradely transported to the cell body, the VSV-G (vesicular stomatitis virus G) pseudotyped lentivirus is not and allows us to control for viral diffusion from the site of infection. We found that dorsal radial glia give rise to gray and white matter astrocytes and oligodendrocytes. In addition, the dorsal radial glia fate map to astrocytes lining the dorsal aspect of the SVZ that persist at least 8 weeks postnatally. Finally, we found that dorsal radial glial-derived cells generate granule cell and periglomerular interneurons in the olfactory bulb and continue to form interneuronal precursors into adulthood.

Protein aggregates in astrocytes that contain glial fibrillary acidic protein (GFAP), small heat shock proteins, and ubiquitinated proteins are termed Rosenthal fibers and characterize Alexander disease, a leukodystrophy caused by heterozygous mutations in GFAP. The mechanisms responsible for the massive accumulation of GFAP in Alexander disease remain unclear. In this study, we show that overexpression of both wild type and R239C mutant human GFAP led to cytoplasmic inclusions. GFAP accumulation also led to a decrease of proteasome activity and an activation of the MLK2-JNK pathway. In turn, the expression of activated mixed lineage kinases (MLKs) induced JNK activation and increased GFAP accumulation, whereas blocking the JNK pathway decreased GFAP accumulation. Activated MLK also inhibited proteasome function. A direct inhibition of proteasome function pharmacologically further activated JNK. Our data suggest a synergistic interplay between the proteasome and the SAPK/JNK pathway in the context of GFAP accumulation. Feedback interactions among GFAP accumulation, SAPK/JNK activation, and proteasomal hypofunction cooperate to produce further protein accumulation and cellular stress responses.

Central neurocytoma (CN) is a rare periventricular tumor, whose derivation, lineage potential, and molecular regulation have been mostly unexplored. We noted that CN cells exhibited an antigenic profile typical of neuronal progenitor cellsin vivo, yetin vitrogenerated neurospheres, divided in response to bFGF (basic fibroblast growth factor), activated the neuroepithelial enhancer of the nestin gene, and gave rise to both neuron-like cells and astrocytes. When CN gene expression was compared with that of both normal adult VZ (ventricular zone) and E/nestin:GFP (green fluorescent protein)-sorted native neuronal progenitors, significant overlap was noted. Marker analysis suggested that the gene expression pattern of CN was that of a proneuronal population; glial markers were conspicuously absent, suggesting that the emergence of astroglia from CN occurred only with passage. The expression pattern of CN was distinguished from that of native progenitor cells by a cohort of differentially expressed genes potentially involved in both the oncogenesis and phenotypic restriction of neurocytoma. These included both IGF2 and several components of its signaling pathway, whose sharp overexpression implicated dysregulated autocrine IGF2 signaling in CN oncogenesis. Both receptors and effectors of canonical wnt signaling, as well as GDF8 (growth differentiation factor 8), PDGF-D, and neuregulin, were differentially overexpressed by CN, suggesting that CN is characterized by the concurrent overactivation of these pathways, which may serve to drive neurocytoma expansion while restricting tumor progenitor phenotype. This strategy of comparing the gene expression of tumor cells to that of the purified native progenitors from which they derive may provide a focused approach to identifying transcripts important to stem and progenitor cell oncogenesis.

De novo dominant mutations in the GFAP gene have recently been associated with nearly all cases of Alexander disease, a rare but devastating neurological disorder. These heterozygous mutations must occur very early in development and be present in nearly all cells in order to be detected by the sequencing methods used. To investigate whether the mutations may have arisen in the parental germ lines, we determined the parental chromosome bearing the mutations for 28 independent Alexander disease cases. These cases included 17 different missense mutations and one insertion mutation. To enable assignment of the chromosomal origin of the mutations, six new single nucleotide polymorphisms in the GFAP gene were identified, bringing the known total to 26. In 24 of the 28 cases analyzed, the paternal chromosome carried the GFAP mutation (P < 0.001), suggesting that they predominantly arose in the parental germ line, with most occurring during spermatogenesis. No effect of paternal age was observed. There has been considerable debate about the magnitude of the male to female germ line mutation rate; our ratio of 6:1 is consistent with indirect estimates based on the rate of evolution of the sex chromosome relative to the autosomic chromosomes.

Alexander disease is a progressive, usually fatal neurological disorder defined by the widespread and abundant presence in astrocytes of protein aggregates called Rosenthal fibers. The disease most often occurs in infants younger than 2 years and has been labeled a leukodystrophy because of an accompanying severe myelin deficit in the frontal lobes. Later onset forms have also been recognized based on the presence of abundant Rosenthal fibers. In these cases, clinical signs and pathology can be quite different from the infantile form, raising the question whether they share the same underlying cause. Recently, we and others have found pathogenic, de novo missense mutations in the glial fibrillary acidic protein gene in most infantile patients examined and in a few later onset patients. To obtain further information about the role of glial fibrillary acidic protein mutations in Alexander disease, we analyzed 41 new patients and another 3 previously described clinically, including 18 later onset patients. Our results show that dominant missense glial fibrillary acidic protein mutations account for nearly all forms of this disorder. They also significantly expand the catalog of responsible mutations, verify the value of magnetic resonance imaging diagnosis, indicate an unexpected male predominance for the juvenile form, and provide insights into phenotype-genotype relations.

We examined the role of IGF signaling in the remyelination process by disrupting the gene encoding the type 1 IGF receptor (IGF1R) specifically in the mouse brain by Cre-mediated recombination and then exposing these mutants and normal siblings to cuprizone. This neurotoxicant induces a demyelinating lesion in the corpus callosum that is reversible on termination of the insult. Acute demyelination and oligodendrocyte depletion were the same in mutants and controls, but the mutants did not remyelinate adequately. We observed that oligodendrocyte progenitors did not accumulate, proliferate, or survive within the mutant mice, compared with wild type, indicating that signaling through the IGF1R plays a critical role in remyelination via effects on oligodendrocyte progenitors.

Neural progenitors in the subventricular zone (SVZ) of the postnatal rat forebrain give rise to either olfactory interneurons or glia. To investigate the overall patterns of progenitor movement, we labeled neonatal rat SVZ cells by stereotactic injection of a GFP-encoding retrovirus into the SVZ at various coronal levels. We then studied the movements of labeled cells by time-lapse videomicroscopy in living brain slices cut in different orientations. We observed two migration patterns: (1) progenitors migrated radially into the overlying white matter and cortex, but only at the level of viral injection; these were previously shown to give rise to astrocytes and oligodendrocytes, (2) progenitors migrated in a bidirectional, rostrocaudal pattern along the entire extent of the SVZ; many of these cells eventually migrated into the olfactory bulb and developed into interneurons, but they did not turn to migrate radially out of the SVZ until they reached the olfactory bulb. Video imaging showed apparent boundaries to migration between the SVZ and adjacent structures. These observations indicate that there are at least two distinct migratory pathways within the SVZ used differentially by immature neurons and glia.

Chemokines are involved in recruitment and activation of hematopoietic cells in sites of infection and inflammation. The M3 gene of the gamma-herpesvirus gammaHV68 encodes an abundant secreted protein that binds CC chemokines with high affinity. We report here that this gene is essential for efficient induction of lethal meningitis by gammaHV68. An M3 mutant gammaHV68 (gammaHV68-M3.stop) was 100-fold less virulent than wild-type or marker rescue control (gammaHV68-M3.MR) viruses after intracerebral inoculation. After intracerebral inoculation, gammaHV68-M3.stop grew to lower titers than gammaHV68 or gammaHV68-M3.MR in the brain but spread to and grew normally in the spleen and lung. Expression of several CC chemokines was significantly induced in the CNS by gammaHV68 infection. Consistent with M3 acting by blockade of CC chemokine action, gammaHV68 induced a neutrophilic meningeal inflammatory infiltrate, while gammaHV68-M3.stop induced an infiltrate in which lymphocytes and macrophages predominated. In contrast to the important role of M3 in lethal meningitis, M3 was not required for establishment or reactivation from latent infection or induction of chronic arteritis. These data suggest a role for chemokines in the protection of the nervous system from viral infection and that the M3 protein acts in a tissue-specific fashion during acute but not chronic gammaHV68 infection to limit CC chemokine-induced inflammatory responses.

The heterogeneity and differentiation potential of mitotically active cells in the adult brain were studied by labeling adult rats with BrdU, and isolating an enriched population of cycling cells from neocortex and from subcortical white matter. The majority of this population isolated from either brain region labeled with O4, an early oligodendrocyte marker. In tissue culture, these O4+ progenitors acquired galactocerebroside, a glycolipid of mature oligodendrocytes, but not GFAP, an intermediate filament of astrocytes. A minority population expressed the intermediate filament protein, vimentin, but not O4. This population expressed GFAP after several days in culture. A third population of cycling cells, expressing the gangliosides labeled with the A2B5 antibody, represented a minority population in subcortical white matter, but one of the major cycling populations in cortex, with substantial overlap with O4. Small populations of cycling NG2+ cells also were observed. Thus, the cycling cells in the adult brain are heterogeneous, and the majority appear to belong to glial lineages. © 2001 John Wiley & Sons, Inc. J Neurobiol 48: 75–86, 2001

Since first being implicated in central nervous system disease 10 years ago, much has been learned concerning the regulation and function of the small heat shock protein alpha B-crystallin. Neuropathological, cellular and molecular studies all now point to a functional relationship between alpha B-crystallin and intermediate filaments. alpha B-crystallin accumulation marks reactive astrocytes in general in a wide variety of disorders and specifically intermediate filament-based glial inclusion bodies such as Rosenthal fibres found in astrocytes in Alexander's disease. In vitro, alpha B-crystallin expression suppresses intermediate filament aggregation and can prevent or reverse experimentally induced glial inclusion body formation. Conversely, dysregulation of glial fibrillary acidic protein expression in vivo results in Rosenthal fibre formation and upregulation of endogenous alpha B-crystallin expression. These data and those from studies recently carried out on other tissues strongly suggest that one function of this small heat shock protein is to modulate intermediate filament organization under conditions of physiological stress and neurodegenerative disease.

Gliogenesis in the mammalian central nervous system does not cease abruptly like neurogenesis. Instead, glia accumulate over a time period that extends into adulthood. To determine whether new glial cells in the adult cortex arise from resident progenitors and to determine the glial types to which these progenitors give rise to, cells in the perinatal subventricular zone (SVZ) were labeled with replication-deficient retroviral vectors, and clonal clusters of glia in the neocortex were examined from 1 week to 8 months of age. The average clonal cluster size increased during the first month of life. Interestingly, clusters containing oligodendrocyte lineage cells preferentially expanded with age, on average doubling every 3 months. Unexpectedly, the number of cells in astrocyte clusters decreased over time. In heterogeneous clusters, the numbers of oligodendroglia increased, whereas the number of astrocytes did not. Moreover, clonal clusters containing mature glia also contained less mature cells, indicating that clonally related progenitors do not differentiate synchronously in vivo. Thus, progenitors from the SVZ continue to cycle, resulting in an accumulation of oligodendroglia in the neocortex. These slowly cycling cells likely express the NG2 proteoglycan because a subset of the clonal clusters contained NG2+ cells and these NG2+ cells accumulated with time. J. Neurosci. Res. 57:435–446, 1999. © 1999 Wiley-Liss, Inc.

alpha 1-Antitrypsin (AAT) and alpha 2-macroglobulin (AMG) are elastase inhibitors that bind the enzyme and reduce measured levels of free elastase. It was recently demonstrated that some patients with intracranial aneurysms have significantly elevated plasma elastase (PE) levels. Although this elevation is unrelated to plasma AAT, it is unknown whether abnormal AAT phenotypes or reduced AMG levels play a role. Moreover, the pathological significance of this elevation is not understood.Plasma from 24 patients with aneurysms (ruptured, n = 15; unruptured, n = 9) and 10 age-matched patients who comprised a control group was analyzed for PE and AMG levels by enzyme-linked immunosorbent assay and for AAT phenotype by isoelectric focusing. Sections of superficial temporal temporal artery obtained from these patients at the time of surgery were examined for evidence of elastin degradation by using a van Gieson stain, with scoring on a nine-point quantitative scale.Patients with aneurysms showed significantly elevated PE levels (119 +/- 28 versus 17 +/- 7 micrograms/ml, P0.05), but AMG levels were not decreased. AAT phenotypic abnormalities were observed in 10% (2 of 20) of the patients with aneurysms, but this was not different from the expected population incidence (7%). Elastin degradation scores were significantly higher in patients with aneurysms than in patients control group (4.26 +/- 0.54 versus 1.21 +/- 0.43, P0.05). In addition, patients with higher elastase levels (80 micrograms/ml) demonstrated 55% higher degradation scores than did those with lower elastase levels (80 micrograms/ml).These data suggest that high PE levels may play a role in systemic arterial elastin degradation seen in patients with intracranial aneurysms. These data also support the contention that elevated elastase levels are not the result of decreased protease inhibitor levels. Although PE levels were significantly higher for the entire group of patients with aneurysms, this assay has relatively low sensitivity for predicting the presence of unruptured aneurysms. Additional study is necessary to determine whether serum elastase levels greater than 80 micrograms/ml, in the setting of other risk factors, are useful in identifying asymptomatic patients for additional screening.

Developmental studies have shown that both neurons and glia arise from the subventricular zone (SVZ) but there have been no clonal analyses to determine whether a single progenitor can produce both. Therefore, we used replication deficient retroviral vectors to analyze the clonal progeny of single rat SVZ cells that were maintained in culture media permissive or non-permissive for neuronal differentiation. When maintained in medium supplemented with 5% fetal bovine serum, all surviving progenitors generated glial cell clones. Within these glial clones we often observed both type 1 astrocytes and O-2A lineage cells. When SVZ cells were maintained in medium permissive for neurogenesis approximately 50% of the total clones contained at least one antigenically defined neuron. Of those clones that contained neurons, 60% contained neurons and glia. The other 50% of the total clones were either comprised of only astrocytes, astrocytes and oligodendrocytes, or were unidentifiable. Since the culture environment permitted multilineage clone formation, yet many homogeneous neuronal or astrocytic clones were obtained, some progenitors must become developmentally restricted while they are in the germinal zone. Therefore, we conclude that the perinatal SVZ is a mosaic of multipotential, bipotential, and lineage restricted precursors, and that the lack of postnatal neocortical neurogenesis is not due to the absence of potential neuroblasts.

Using replication-deficient retrovirus to transfer marker genes into immature cells, we have characterized spatial and temporal patterns of glial progenitor migration and differentiation in the early postnatal rat forebrain and cerebellum, and interneuron differentiation in the cerebellum. Progenitors do not migrate randomly, but follow discrete paths, largely confined to a coronal plane in forebrain and a sagittal plane in cerebellum. Radial glia provide one substrate for migration. In vitro studies suggest that radial glia contribute a permissive pathway along which progenitors in an immature, migratory state. Local environmental cues that progenitors encounter during migration may influence fate decisions substantially. Not all progenitors differentiate; some remain in an immature, proliferative state in which they do not complete differentiation, but can be induced to do so by pathological conditions.