Mutations in GFAP, encoding glial fibrillary acidic protein, are associated with Alexander disease.

Alexander disease is a rare disorder of the central nervous system of unknown etiology. Infants with Alexander disease develop a leukoencephalopathy with macrocephaly, seizures and psychomotor retardation, leading to death usually within the first decade; patients with juvenile or adult forms typically experience ataxia, bulbar signs and spasticity, and a more slowly progressive course. The pathological hallmark of all forms of Alexander disease is the presence of Rosenthal fibers, cytoplasmic inclusions in astrocytes that contain the intermediate filament protein GFAP in association with small heat-shock proteins. We previously found that overexpression of human GFAP in astrocytes of transgenic mice is fatal and accompanied by the presence of inclusion bodies indistinguishable from human Rosenthal fibers. These results suggested that a primary alteration in GFAP may be responsible for Alexander disease. Sequence analysis of DNA samples from patients representing different Alexander disease phenotypes revealed that most cases are associated with non-conservative mutations in the coding region of GFAP. Alexander disease therefore represents the first example of a primary genetic disorder of astrocytes, one of the major cell types in the vertebrate CNS.

Border-associated macrophages mediate the neuroinflammatory response in an alpha-synuclein model of Parkinson disease.

Dopaminergic cell loss due to the accumulation of α-syn is a core feature of the pathogenesis of Parkinson disease. Neuroinflammation specifically induced by α-synuclein has been shown to exacerbate neurodegeneration, yet the role of central nervous system (CNS) resident macrophages in this process remains unclear. We found that a specific subset of CNS resident macrophages, border-associated macrophages (BAMs), play an essential role in mediating α-synuclein related neuroinflammation due to their unique role as the antigen presenting cells necessary to initiate a CD4 T cell response whereas the loss of MHCII antigen presentation on microglia had no effect on neuroinflammation. Furthermore, α-synuclein expression led to an expansion in border-associated macrophage numbers and a unique damage-associated activation state. Through a combinatorial approach of single-cell RNA sequencing and depletion experiments, we found that border-associated macrophages played an essential role in immune cell recruitment, infiltration, and antigen presentation. Furthermore, border-associated macrophages were identified in post-mortem PD brain in close proximity to T cells. These results point to a role for border-associated macrophages in mediating the pathogenesis of Parkinson disease through their role in the orchestration of the α-synuclein-mediated neuroinflammatory response.

PDGF stimulates the massive expansion of glial progenitors in the neonatal forebrain.

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.

Isolation and characterization of glial filaments from human brain.

Intermediate (8--9 nm) filaments of human central nervous system astrocytes were isolated from the gliosed white matter of cases of adrenoleukodystrophy (ALD). This hereditary lipidosis is characterized pathologically by demyelination, loss of axons, and replacement of the white matter of the caudal cerebrum by a glial scar. Glial filaments were composed largely of a single protein component with a mol wt of about 49,000 daltons. Smaller components (44,000--39,000 daltons) were detected in some samples, and appear to represent degradation products of the filament protein. Human neurofilaments were isolated from the normal frontal white matter of ALD cases by the standard myelin-free axon technique. Isolated glial and neurofilament proteins comigrated during acrylamide gel electrophoresis in SDS. Polypeptides resulting from cyanogen bromide cleavage of the two filament proteins were the same. Both proteins reacted with rabbit antisera raised against isolated bovine neurofilament protein and human glial fibrillary acidic protein.

Sense and antisense modification of glial alpha B-crystallin production results in alterations of stress fiber formation and thermoresistance.

The phenotypic effects of selectively altering the levels of alpha B-crystallin in cultured glial cells were analyzed using sense and antisense approaches. Rat C6 glioma cells and human U-373MG glioma cells were transfected with a rat alpha B-crystallin sense cDNA or an antisense cDNA regulated by a Rous sarcoma virus promoter to alter cellular levels of alpha B-crystallin. The antisense strategy resulted in decreased alpha B-crystallin levels, as revealed by Western blot and immunocytochemical analyses. The reduced alpha B-crystallin expression was accompanied by alterations in cellular phenotype: (a) a reduction of cell size and/or a slender cell morphology; (b) a disorganized microfilament network; and (c) a reduction of cell adhesiveness. Like HSP27, the presence of additional alpha B-crystallin protein confers a thermoresistant phenotype to stable transfectants. Thus, alpha B-crystallin in glioma cells plays a role in their thermal resistance and may contribute to the stability of cytoskeletal organization.

Axonal transport of newly synthesized glycoproteins in a single identified neuron of Aplysia californica.

Increasing amounts of glycoprotein synthesized from L-[(3)H]fucose injected into the cell body of R2, an identified Aplysia neuron, were found in the right pleuro-abdominal connective. Autoradiography revealed that the glycoproteins were localized in the axon of R2. Glycoproteins appearing in the axon presumably were synthesized in the cell body, since no significant incorporation was observed when [(3)H]fucose was injected directly into the axon. [(3)H]glycoproteins were detected in the connective after a delay of 1 h after intrasomatic injection. Thereafter, transport from the cell body was rapid, and by 10 h after injection, 45% of the total neuronal [(3)H]glycoprotein had appeared in the axon. By analysing the radioactivity in cell body and connective 4, 10, and 15 h after injection, we found that [(3)H]glycoproteins were transported selectively compared to nonmacromolecular material. Sequential sectioning of the connective revealed that [(3)H]glycoproteins were transported in discrete waves. The population of membrane-associated [(3)H]glycoproteins in the axon differed from that in the cell body. Two of the five somatic components appeared to be transported preferentially. In addition a new component appeared in the axon 10 h after injection.

Axonal transport of [3H]serotonin in an identified neuron of Aplysia californica.

A population of characteristic ellipsoidal dense-core vesicles was identified in axons of the giant cerebral neuron of the mollusc Aplysia. We injected [3H]serotonin into the cell body of this identified serotonergic neuron in the isolated central nervous system in order to study the subcellular components associated with the neurotransmitter. Subcellular fractionation by differential centrifugation indicated that injected serotonin was rapidly taken up into particulate form. [3H]Serotonin appeared in the axons within 2 h after injection, and export continued at a constant rate of 6% of the total in the neuron/h thereafter. The dependence of the total amounts of [3H]serotonin which appeared in the axons in 6 h (export from the cell body) on the amounts injected was consistent with the idea that export is a saturable process, possibly depending on the capacity of somatic vesicles or of some unidentified carrier for serotonin. [3H]Serotonin moved into both major branches of the axon, where it was translocated rapidly. The transmitter, which was shown by autoradiography to be restricted to the axons of the injected cell, was distributed along axons in accumulations of radioactivity; in contrast, its precursor, [5-3H]hydroxytryptophan, moved slowly along axons in a smooth, declining curve, its kinetics consistent with diffusion. Quantitative electron microscope autoradiography revealed that the dense-core vesicles and the cytosol of axons fixed with glutaraldehyde were labeled with [3H]serotonin.

Synthesis of glycoproteins in a single identified neuron of Aplysia californica.

Incorporation of L-[(3)H]fucose into glycoproteins was studied in R2, the giant neuron in the abdominal ganglion of Aplysia. [(3)H]fucose injected directly into the cell body of R2 was readily incorporated into glycoproteins which, as shown by autoradiography, were confined almost entirely to the injected neuron. Within 4 h after injection, 67% of the radioactivity in R2 had been incorporated into glycoproteins; at least 95% of these could be sedimented by centrifugation at 105,000 g, suggesting that they are associated with membranes. Extraction of the particulate fraction with sodium dodecyl sulfate (SDS), followed by gel filtration on Sephadex G-200 and polyacrylamide gel electrophoresis in SDS revealed the presence of only five major radioactive glycoprotein components which ranged in apparent molecular weight from 100,000 to 200,000 daltons. Similar results were obtained after intrasomatic injection of [(3)H]N-acetylgalactosamine. Mild acid hydrolysis of particulate fractions released all of the radioactivity in the form of fucose. When ganglia were incubated in the presence of [(3)H]fucose, radioactivity was preferentially incorporated into glial cells and connective tissue. In contrast to the relatively simple electrophoretic patterns obtained from cells injected with [(3)H]fucose, gel profiles of particulate fractions labeled with [(14)C]valine were much more complex.

Lewy bodies of Parkinson's disease contain neurofilament antigens.

The Lewy body, a histological hallmark of Parkinson's disease, is a filamentous inclusion residing most prominently in pigmented neurons of the brainstem. Immunocytochemical reactions of Lewy bodies were examined with antisera to several filamentous proteins of the nervous system and positive reactions were found with those against neurofilaments. An abnormal organization of the neuronal cytoskeleton may be a pathological feature of Parkinson's disease.

Patterns and dynamics of SVZ cell migration in the postnatal forebrain: monitoring living progenitors in slice preparations.

Glial progenitors colonize the CNS widely in the perinatal period, but the pathways and mechanisms of migration are not well understood. We investigated the migration of progenitors from the neonatal rat forebrain subventricular zone (SVZ) by labeling them in vivo with a retrovirus encoding green fluorescent protein and visualizing movements by time lapse microscopy in slices. Cells within the dorsolateral SVZ moved in an undirected fashion but migrated radially and tangentially after emigration into white matter, cortex, and striatum. Cells in the striatal SVZ migrated parallel to the ventricular surface. During migration, elongation of the leading process and nuclear translocation were independent or linked. Orthogonal turning involved either cessation of cell body movement and formation of a new leading process or continuous cell body movement and bending of the leading process.

Generation of cerebellar interneurons from dividing progenitors in white matter.

The traditional view of the external granular layer of the cerebellar cortex giving rise to interneurons has been challenged by recent studies with quail-chick chimeras. To clarify the time and site of origins of interneurons, a retrovirus carrying the beta-galactosidase gene was injected into the deep cerebellar tissue or external granular layer of postnatal day 4/5 rats to label dividing progenitors. After deep cerebellar tissue injection, unipolar cells were found initially in white matter at 2 days postinjection and subsequently in the internal granule and molecular layers 4-6 days postinjection. Morphologically defined basket, stellate, and Golgi neurons were clearly identified by 20 days postinjection. In contrast, retroviral labeling of cells in the external granular layer produced only granule neurons in the internal granule layer. Thus, dividing progenitors in the cerebellar white matter migrate through the white matter into the cortex before differentiating into a variety of cortical interneurons.

A clonal analysis of glial lineages in neonatal forebrain development in vitro.

Retrovirus-mediated gene transfer combined with triple immunostaining for astro- and oligodendroglial markers (antibodies to glial fibrillary acidic protein, GD3 ganglioside, and galactocerebroside, and the O4 antibody) was used to study clonal aspects of glial lineage in primary cultures of the neonatal rat striatum. We found two major clonal populations: astrocyte clones containing GFAP+, but GD3-, O4-, and GC- cells, and oligodendrocyte clones containing cells expressing various combinations of GD3, O4, and GC, with rare GFAP+ cells. These results indicate that astrocytes and oligodendrocytes belong to separate lineages in forebrain postnatal development.

Endogenous progenitors remyelinate demyelinated axons in the adult CNS.

Remyelination occurs in demyelinated CNS regions in diseases such as multiple sclerosis. Identification of the cell type(s) responsible for this remyelination, however, has been elusive. Here, we examine one potential source of remyelinating oligodendrocytes-immature, cycling cells endogenous to adult white matter-and demonstrate that this population responds to demyelination by differentiating into myelinating oligodendrocytes. Dividing cells in subcortical white matter of adult rats were labeled by stereotactic injection of a replication-deficient lacZ-encoding retrovirus (BAG). Following a focal demyelination induced with lysolecithin, many of the BAG-labeled cells differentiated into myelinating oligodendrocytes engaging in repair of the lesion. Identification of endogenous cells capable of remyelination provides a target for the study of CNS repair processes in demyelinating diseases.

Both oligodendrocytes and astrocytes develop from progenitors in the subventricular zone of postnatal rat forebrain.

The developmental fates of subventricular zone (SVZ) cells of the postnatal rat forebrain were determined by retroviral-mediated gene transfer and immunolabeling for glial antigens. A beta-galactosidase-containing retrovirus injected stereotactically into the SVZ infected small, immature cells. By 28 days post-injection labeled cells had appeared in both gray and white matter of the ipsilateral hemisphere. White matter contained labeled oligodendrocytes, but few astrocytes, while neocortex and striatum contained both glial types, often appearing in tightly knit clusters. An analysis after simultaneously injecting alkaline phosphatase- and beta-galactosidase-containing retroviruses showed that cells in each cortical cluster were related. Most clusters contained a single cell type, but approximately 15% contained both astrocytes and oligodendrocytes. These observations strongly suggest that a single SVZ cell can differentiate into both glial types.

Aldolase C/zebrin II expression in the neonatal rat forebrain reveals cellular heterogeneity within the subventricular zone and early astrocyte differentiation.

During late gestational and early postnatal development, proliferating cells in the subventricular zones of the lateral ventricles (SVZ) migrate into the gray and white matter of the forebrain and differentiate into astrocytes and oligodendrocytes. Because the cellular composition and structure of the neonatal SVZ is poorly understood, we performed a differential display PCR screen to identify genes preferentially expressed therein. One highly expressed gene encoded aldolase C. We used a specific monoclonal antibody, aldolase C/zebrin II (ALDC/ZII), in combination with markers of glial lineage and proliferation, to characterize the cells that express this gene. In the neonatal SVZ, ALDC/ZII-positive cells, which are generally polygonal and display several processes, have a nonuniform spatial distribution. They do not express vimentin, GFAP, or NG2. A subset of ALDC/ZII-positive cells incorporates bromodeoxyuridine, but progenitors identified by beta-galactosidase expression after infection with recombinant BAG virus do not show ALDC/ZII immunoreactivity. Outside of the SVZ, beta-galactosidase-positive/ALDC/ZII-positive cells have an astrocytic phenotype, suggesting that immunoreactivity was acquired after exit from the SVZ. These studies demonstrate that the neonatal SVZ is composed of different populations of cells that can be characterized by their antigenic phenotype, their proliferative capacity, and their spatial distributions. Nonrandom distributions of different cell types within the SVZ may permit the formation of microenvironments that stimulate the production of cells with specific potentials at appropriate points in development. Analysis of ALDC/ZII expression by astrocyte lineage cells in the neonatal cerebral cortex and white matter may reveal insights into the phenotype and behavior of undifferentiated astrocyte progenitors.

Atypical pleomorphic neoplasms of the pineal gland: Case report and review of the literature.

BACKGROUND: Pineal region tumors are rare and diverse. Among them exist reports of pleomorphic xanthroastrocytoma (PXA) and pleomorphic granular cell astrocytoma (PGCA) of the pineal gland. These related tumors are remarkably similar sharing pleomorphic histologic features with only minor immunohistochemical and ultrastructural differences. CASE DESCRIPTION: We present a case of a 42-year old right-handed woman presented with a longstanding history of migraine headaches which had worsened over the two months leading up to her hospitalization. MRI revealed a 1.7 × 1.3 × 1.6 cm intensely enhancing lesion originating in the pineal gland. The tumor closely resembled PGCA but did not strictly fit the diagnostic requirements of either PGCA or PXA. CONCLUSION: The present case highlights the exotic nature of pineal region tumors with pleomorphic cell histology. Given the diverse range of tumors encountered in the pineal region, pathological confirmation is mandatory. Favorable clinical outcomes demonstrate that surgical resection alone can yield excellent long-term results for tumors falling within the spectrum of pleomorphic lesions of the pineal gland.

The association of actin with Hirano bodies.

Ultrastructural studies of the rod-shaped eosinophilic inclusions of the hippocampus (Hirano bodies) have demonstrated thin, filamentous components. The size of the filaments suggests that actin polymers (microfilaments) might form part of the Hirano body. To investigate this possibility, sections of human hippocampus and neocortex were stained, using a peroxidase-antiperoxidase (PAP) technique, with an antiserum against actin. Dense reaction product was seen over rod-shaped bodies, the location and morphology of which were typical of Hirano bodies. Immunocytochemical reactions on tissue previously stained with hematoxylin and eosin allowed a direct comparison between Hirano bodies, identified by their shape and eosinophilia, and the PAP reaction product. These results suggest an abnormal organization of a major cytoskeletal protein in hippocampal neurons, especially those of aged brains.

Alexander disease: new insights from genetics.

Prior to finding that GFAP mutations underlie many cases of Alexander disease, it was unclear whether the disease originated in astrocytes or if the formation of Rosenthal fibers was a response to an external insult. It was also unclear whether the etiology of the disease was environmental or genetic. For many cases of Alexander disease, these questions have now been answered. An immediate clinical benefit of this discovery is the possibility of diagnosing most cases of Alexander disease through analysis of patient DNA samples, rather than resorting to brain biopsy. In addition, fetal testing is now an option for parents who have had an Alexander disease child with an identified mutation and who wish to have additional children. For the future, these mutations should provide a unique window for illuminating the mechanism of the disease.

Altered response of fibroblasts from aged and Alzheimer donors to drugs that elevate cytosolic free calcium.

Previous studies demonstrate that resting intracellular calcium in cultured skin fibroblasts declines due to in vivo aging and is further depressed by Alzheimer's disease. These data suggest that altered calcium homeostasis may underlie the deficits in cell function (e.g., cell spreading) that also occur in these cells. Depressed cytosolic free calcium in fibroblasts from aged and Alzheimer donors can be elevated by various drug treatments. 3,4-Diaminopyridine, serum, N-formyl-methionyl-leucyl-phenylalanine and bradykinin increased cytosolic free calcium transiently although the rate of the increase was slower and the magnitude of the rise was less in cells from aged and Alzheimer donors when compared to young donors. Four minutes after N-formyl-methionyl-leucyl-phenylalanine or bradykinin treatment cytosolic free calcium returned to resting levels in all six cell lines. Six minutes after either serum or 3,4-diaminopyridine treatments, however, cytosolic free calcium in cells from aged and Alzheimer donors remained elevated at concentrations similar to the resting calcium level in young cells. Bradykinin and serum were effective in the absence of extracellular calcium but 3,4-diaminopyridine and N-formyl-methionyl-leucyl-phenylalanine were not. These demonstrate that dynamic, as well as resting calcium homeostasis, is altered in cultured skin fibroblasts from aged and Alzheimer donors.