Subpial Astrocytes Research Articles

Changes in Cx43 and AQP4 Proteins, and the Capture of 3 kDa Dextran in Subpial Astrocytes of the Rat Medial Prefrontal Cortex after Both Sham Surgery and Sciatic Nerve Injury.

A subpopulation of astrocytes on the brain's surface, known as subpial astrocytes, constitutes the "glia limitans superficialis" (GLS), which is an interface between the brain parenchyma and the cerebrospinal fluid (CSF) in the subpial space. Changes in connexin-43 (Cx43) and aquaporin-4 (AQP4) proteins in subpial astrocytes were examined in the medial prefrontal cortex at postoperative day 1, 3, 7, 14, and 21 after sham operation and sciatic nerve compression (SNC). In addition, we tested the altered uptake of TRITC-conjugated 3 kDa dextran by reactive subpial astrocytes. Cellular immunofluorescence (IF) detection and image analysis were used to examine changes in Cx43 and AQP4 protein levels, as well as TRITC-conjugated 3 kDa dextran, in subpial astrocytes. The intensity of Cx43-IF was significantly increased, but AQP4-IF decreased in subpial astrocytes of sham- and SNC-operated rats during all survival periods compared to naïve controls. Similarly, the uptake of 3 kDa dextran in the GLS was reduced following both sham and SNC operations. The results suggest that both sciatic nerve injury and peripheral tissue injury alone can induce changes in subpial astrocytes related to the spread of their reactivity across the cortical surface mediated by increased amounts of gap junctions. At the same time, water transport and solute uptake were impaired in subpial astrocytes.

Silencing GFAP isoforms in astrocytoma cells disturbs laminin‐dependent motility and cell adhesion

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed in astrocytes and neural stem cells. The GFAP gene is alternatively spliced, and expression of GFAP is highly regulated during development, on brain damage, and in neurodegenerative diseases. GFAPα is the canonical splice variant and is expressed in all GFAP-positive cells. In the human brain, the alternatively spliced transcript GFAPδ marks specialized astrocyte populations, such as subpial astrocytes and the neurogenic astrocytes in the human subventricular zone. We here show that shifting the GFAP isoform ratio in favor of GFAPδ in astrocytoma cells, by selectively silencing the canonical isoform GFAPα with short hairpin RNAs, induced a change in integrins, a decrease in plectin, and an increase in expression of the extracellular matrix component laminin. Together, this did not affect cell proliferation but resulted in a significantly decreased motility of astrocytoma cells. In contrast, a down-regulation of all GFAP isoforms led to less cell spreading, increased integrin expression, and a >100-fold difference in the adhesion of astrocytoma cells to laminin. In summary, isoform-specific silencing of GFAP revealed distinct roles of a specialized GFAP network in regulating the interaction of astrocytoma cells with the extracellular matrix through laminin.-Moeton, M., Kanski, R., Stassen, O. M. J. A., Sluijs, J. A., Geerts, D., van Tijn, P., Wiche, G., van Strien, M. E., Hol, E. M. Silencing GFAP isoforms in astrocytoma cells disturbs laminin dependent motility and cell adhesion.

Protective role of brain water channel AQP4 in murine cerebral malaria

Tragically common among children in sub-Saharan Africa, cerebral malaria is characterized by rapid progression to coma and death. In this study, we used a model of cerebral malaria appearing in C57BL/6 WT mice after infection with the rodent malaria parasite Plasmodium berghei ANKA. Expression and cellular localization of the brain water channel aquaporin-4 (AQP4) was investigated during the neurological syndrome. Semiquantitative real-time PCR comparing uninfected and infected mice showed a reduction of brain AQP4 transcript in cerebral malaria, and immunoblots revealed reduction of brain AQP4 protein. Reduction of brain AQP4 protein was confirmed in cerebral malaria by quantitative immunogold EM; however, polarized distribution of AQP4 at the perivascular and subpial astrocyte membranes was not altered. To further examine the role of AQP4 in cerebral malaria, WT mice and littermates genetically deficient in AQP4 were infected with P. berghei. Upon development of cerebral malaria, WT and AQP4-null mice exhibited similar increases in width of perivascular astroglial end-feet in brain. Nevertheless, the AQP4-null mice exhibited more severe signs of cerebral malaria with greater brain edema, although disruption of the blood-brain barrier was similar in both groups. In longitudinal studies, cerebral malaria appeared nearly 1 d earlier in the AQP4-null mice, and reduced survival was noted when chloroquine rescue was attempted. We conclude that the water channel AQP4 confers partial protection against cerebral malaria.

GFAP isoforms in adult mouse brain with a focus on neurogenic astrocytes and reactive astrogliosis in mouse models of Alzheimer disease.

Glial fibrillary acidic protein (GFAP) is the main astrocytic intermediate filament (IF). GFAP splice isoforms show differential expression patterns in the human brain. GFAPδ is preferentially expressed by neurogenic astrocytes in the subventricular zone (SVZ), whereas GFAP+1 is found in a subset of astrocytes throughout the brain. In addition, the expression of these isoforms in human brain material of epilepsy, Alzheimer and glioma patients has been reported. Here, for the first time, we present a comprehensive study of GFAP isoform expression in both wild-type and Alzheimer Disease (AD) mouse models. In cortex, cerebellum, and striatum of wild-type mice, transcripts for Gfap-α, Gfap-β, Gfap-γ, Gfap-δ, Gfap-κ, and a newly identified isoform Gfap-ζ, were detected. Their relative expression levels were similar in all regions studied. GFAPα showed a widespread expression whilst GFAPδ distribution was prominent in the SVZ, rostral migratory stream (RMS), neurogenic astrocytes of the subgranular zone (SGZ), and subpial astrocytes. In contrast to the human SVZ, we could not establish an unambiguous GFAPδ localization in proliferating cells of the mouse SVZ. In APPswePS1dE9 and 3xTgAD mice, plaque-associated reactive astrocytes had increased transcript levels of all detectable GFAP isoforms and low levels of a new GFAP isoform, Gfap-ΔEx7. Reactive astrocytes in AD mice showed enhanced GFAPα and GFAPδ immunolabeling, less frequently increased vimentin and nestin, but no GFAPκ or GFAP+1 staining. In conclusion, GFAPδ protein is present in SVZ, RMS, and neurogenic astrocytes of the SGZ, but also outside neurogenic niches. Furthermore, differential GFAP isoform expression is not linked with aging or reactive gliosis. This evidence points to the conclusion that differential regulation of GFAP isoforms is not involved in the reorganization of the IF network in reactive gliosis or in neurogenesis in the mouse brain.

Clinical aspects and pathology of Alexander disease, and morphological and functional alteration of astrocytes induced by GFAP mutation

Alexander disease (AxD) is pathologically characterized by the presence of Rosenthal fibers (RF), which are made up of GFAP, αB-crystallin and heat shock protein 27, in the cytoplasm of perivascular and subpial astrocyte endfeet. Since GFAP mutation has been confirmed in reported cases of AxD, clinical or experimental research is being conducted on the relationship between GFAP mutation and the onset pathology as well as the clinical form. We conducted a nationwide survey and a clinical study, and classified AxD into three types: cerebral AxD (type 1), which primarily has an infantile onset with presence of seizures, psychomotor developmental retardation, macrocephaly, and abnormalities in the superior frontal cerebral white matter observed in a brain MRI; bulbospinal AxD (type 2), which primarily has an adult onset with presence of muscle weakness, hyperreflexia, bulbar or pseudobulbar symptoms, signal abnormalities, and atrophy observed in an MRI of the medulla oblongata and upper cervical spinal cord; and an intermediate form (type 3) which has the characteristics of both. A research on GFAP mutations and aggregate formation concluded that GFAP mutations decreased the solubility of GFAP. According to our cell model experiment, the formation of mutant GFAP aggravates depending on the site of the GFAP mutation. Furthermore, there is a possibility that polymorphism in the GFAP promoter gene regulates the degree to which GFAP is expressed; it may have an effect on clinical heterogeneity. Recent research using cell and animal models suggests that the pathology of AxD involves not only mere functional abnormalities in intermediate filaments but also functional abnormalities in astrocytes as well as in neurons. Clarification of the glia-neuron interactions will prove the disease to be very interesting.

Connexin hemichannel-mediated CO2-dependent release of ATP in the medulla oblongata contributes to central respiratory chemosensitivity.

Arterial PCO2, a major determinant of breathing, is detected by chemosensors located in the brainstem. These are important for maintaining physiological levels of PCO2 in the blood and brain, yet the mechanisms by which the brain senses CO(2) remain controversial. As ATP release at the ventral surface of the brainstem has been causally linked to the adaptive changes in ventilation in response to hypercapnia, we have studied the mechanisms of CO(2)-dependent ATP release in slices containing the ventral surface of the medulla oblongata. We found that CO(2)-dependent ATP release occurs in the absence of extracellular acidification and correlates directly with the level of PCO2. ATP release is independent of extracellular Ca(2+) and may occur via the opening of a gap junction hemichannel. As agents that act on connexin channels block this release, but compounds selective for pannexin-1 have no effect, we conclude that a connexin hemichannel is involved in CO(2)-dependent ATP release. We have used molecular, genetic and immunocytochemical techniques to demonstrate that in the medulla oblongata connexin 26 (Cx26) is preferentially expressed near the ventral surface. The leptomeninges, subpial astrocytes and astrocytes ensheathing penetrating blood vessels at the ventral surface of the medulla can be loaded with dye in a CO(2)-dependent manner, suggesting that gating of a hemichannel is involved in ATP release. This distribution of CO(2)-dependent dye loading closely mirrors that of Cx26 expression and colocalizes to glial fibrillary acidic protein (GFAP)-positive cells. In vivo, blockers with selectivity for Cx26 reduce hypercapnia-evoked ATP release and the consequent adaptive enhancement of breathing. We therefore propose that Cx26-mediated release of ATP in response to changes in PCO2 is an important mechanism contributing to central respiratory chemosensitivity.

Phosphorylation in the C-terminal domain of Aquaporin-4 is required for Golgi transition in primary cultured astrocytes

Aquaporin-4 (AQP4) is expressed in the perivascular and subpial astrocytes end-feet in mammalian brain, and plays a critical component of an integrated water and potassium homeostasis. Here we examine whether AQP4 is phosphorylated in primary cultured mouse astrocytes. Astrocytes were metabolically labeled with [32P]phosphoric acid, then AQP4 was immunoprecipitated with anti-AQP4 antibody. We observed that AQP4 was constitutively phosphorylated, which is reduced by treatment with protein kinase CK2 inhibitors. To elucidate the phosphorylation of AQP4 by CK2, myc-tagged wild-type or mutant AQP4 was transiently transfected in primary cultured astrocytes. Substitution of Ala residues for four putative CK2 phosphorylation sites in the C terminus abolished the phosphorylation of AQP4. Immunofluorescent microscopy revealed that the quadruple mutant was localized in the Golgi apparatus. These observations indicate that the C-terminal domain of AQP4 is constitutively phosphorylated at least in part by protein kinase CK2 and it is required for Golgi transition.

Subpial astrocytosis and focal leptomeningeal angiotropic astrocytosis leading to vascular compression: observations made in a case of mitochondrial encephalopathy

We performed standard histological, immunohistochemical, and ultrastructural examinations of formalin Wxed samples of the brain (frontal cortex, brain stem, basal ganglia, and spinal medulla) of a male patient, aged 12, who succumbed to a mitochondrial disorder, characterized as MERRF (8344A > G). Neuropathology revealed regressive neuronal changes including necrotic foci [9]. Necroses were localized subependymally in the brain stem and in the ventral part of the spinal medulla, where it communicated with the leptomeningeal space. There was prominent generalized astrocytosis in all samples examined. GFAP and S100 immunostaining showed remarkably dense networks of astrocyte processes, which often had a beaded appearance. In the cerebral cortex, astrocytosis was restricted to the Wrst two cortical layers. The subarachnoid space was frequently thickened by a loose, variably cellular population of spindle-shaped cells positive for vimentin and S100 protein, but negative for GFAP. In several subarachnoid regions, the cells were condensed to relatively dense bands which were not only vimentin and S100 protein positive, but also strongly stained for GFAP. They were frequently in contact with the adventitia of otherwise normal appearing subarachnoid arteries (Fig. 1a). In the brain stem region (lateral side of the tegmentum) small leptomeningeal arteries were completely encircled by a thick, dense concentric cuV-like layer of Wbrous astrocytes (Fig. 1b–d) which were either isolated or only in partial contact with the subpial astrocytic mass (Fig. 1e, f). Cells of the astrocytic cuVs stained strongly for GFAP (Fig. 1b) and S100 protein; however, only a few expressed vimentin. The perivascular concentric astrocytic bands displayed discrete laminin positivity at the periphery. The entangled vessels were compressed, leaving only slit-like lumens or were completely occluded (Fig. 1c, d). Electron microscopy, carried out in paraYn sections from several leptomeningeal lesions, showed aggregates of intermediate Wlaments (t10 nm thick) identical to those found in the subpial astrocytes of the brain. Our observation of remarkable numbers of astrocytes in the leptomeningeal space is exceptional. Comparable observations are very rare [3–5, 7, 13]. Their presence might be related to the activation of proposed residential meningeo-glial network [6] which may lead to parallel activation of brain astrocytes. It is interesting that some of the tumors arising in the meningeal region express GFAP [1, 11, 12]. It is also worth mentioning that the astrocytic immunophenotype (GFAP, S100) has been seen in extracerebral mesenchymal cells, e.g., Wbroblasts and chondrocytes [2]. What is unique is the presence of perivascular cuV-like astrocytic bands leading to vascular compression. Linkage to subpial astrocytic hypertrophy suggests that they originated from this astrocyte subpopulation [8]. The presence of laminin at the periphery of the cuVs suggests a highly order arrangement comparable with the subpial gliobasal membrane. The spatial relationship between the vascular compressive lesions and the brain stem necrosis suggests H. H4lkova · M. Elleder (&) 1st Faculty of Medicine, Institute of Inherited Metabolic Disorders, Division B, Bldg.D, Ke Karlovu 2, 128 08 Prague 2, Czech Republic e-mail: melleder@cesnet.cz

Aquaporin-4, homeostasis, and neurologic disease

Aquaporins constitute a family of water channels that regulate the transport of water in essentially all organs, including the kidney, gastrointestinal tract, secretory glands, inner ear, nervous system, and muscle.1–3 Because of his seminal discovery of aquaporins as the long-sought water channels,1 Peter Agre of Johns Hopkins University was awarded the Nobel Prize in Chemistry in 2003. Aquaporin-4 (AQP4) is the main aquaporin in the brain and is expressed in highest density in the perivascular and subpial astrocyte end-feet. Aquaporin-4 is a critical component of an integrated mechanism of regulation of cell volume and potassium (K+) homeostasis. Aquaporin-4 has been implicated in several neurologic conditions, including cerebral edema and seizures, and is the target of autoimmune attack in neuromyelitis optica (NMO). Recent studies on expression of and regulation of AQP4 provide testable hypotheses on the mechanisms of neural damage and make AQP4 a potential therapeutic target in these and other conditions. Aquaporins are water channels that consist of four identical subunits, each with six membrane-spanning α-helices.2,3 Aquaporin-4 is the most abundant aquaporin in the brain and is concentrated at glial–fluid interfaces, particularly in the perivascular and subpial end feet of the astrocytes (figure 1). Aquaporin-4 is also present on ependymal cells, and in low levels, on brain endothelial cells.3 The polarized distribution of AQP4 in the perivascular and subpial domains of the astrocytic plasma membrane results from its interaction with cytoskeleton and extracellular matrix proteins as part of the dystroglycan complex.4 At the astrocyte end foot process, AQP4 co-localizes with the …

The astrocytic response towards invasive meningiomas

meningiomas disrupt the pial-glial basement membrane. This membrane is closely attached to the subpial glial endfeet forming the glia limitans. The fate of subpial astrocytes during the course of brain invasion by meningiomas is not known. In the present study we immunolabelled sections of 35 brain-invasive meningiomas (14 meningothelial meningiomas WHO grade I, 11 atypical WHO grade II and 10 anaplastic WHO grade III) using anti-collagen IV to label basement membrane material, and antibodies against astrocytic markers CD44, SPARC (secreted protein, acidic and rich in cysteine) and glial fibrillary acidic protein (GFAP). Astrocytes were present at the tumour-brain interface of 14/14 WHO grade I meningiomas, 9/11 WHO grade II tumours and 9/10 WHO grade III tumours. The presence of astrocytes was associated with an intact basement membrane in 11/14 WHO grade I meningiomas (P < 0.01), 6/9 WHO grade II tumours, and 6/9 WHO grade III tumours. However, tumour embedded in deep brain parenchyma lacked both an astrocytic reaction and basement membrane material. In conclusion, astrocytes eventually disappear from the tumour-brain interface during the course of meningioma infiltration. This observation might indicate that the survival of subpial astrocytes is dependent on an intact pial-glial basement membrane.

Extravasation of plasma proteins can confound interpretation of proteomic studies of brain: A lesson from apo A-I in mesial temporal lobe epilepsy

Apolipoprotein A-I (apo A-I), a major component of high density lipoproteins, has been shown to be involved in lipid metabolism, cholesterol homeostasis and degeneration/regeneration of brain tissues and was proposed as a useful marker for the extent and severity of CNS injury. We searched for aberrant protein expression in hippocampus from patients with mesial temporal lobe epilepsy (MTLE) by an analytical method based on two-dimensional gel electrophoresis coupled with mass spectrometry (MALDI-TOF/TOF) and unambiguously identified 2 spots as apolipoprotein A-I forms in brain of MTLE patients with 7.5-fold increased levels (controls: 0.046 ± 0.046; MTLE patients: 0.343 ± 0.154, mean ± SD, P = 0.003). Western blot analysis confirmed increased apo A-I levels in MTLE. Immunohistochemistry detected staining for apo A-I extracellularly in perivasal brain parenchyma, neuropil and areas with increased glial fibrillary acidic protein (GFAP) expression as well as some pyramidal neurons and subpial astrocytes. These findings indicate that the increase of apo A-I in MTLE was possibly not reflecting a pathogenetic role but was rather due to extravasates, bleedings or increase of microvascular endothelial cells known to synthesize apo A-I. Care has to be taken when protein expressional findings are to be interpreted in the presence of plasma proteins, including apo A-I, thus clearly representing a confounding factor.

Induction and temporal changes of osteopontin mRNA and protein in the brain following systemic lipopolysaccharide injection

We analyzed expression of osteopontin (OPN), a cytokine regulating tissue repair and inflammation, in astrocytes and microglia in response to systemic lipopolysaccharide (LPS) administration (250 μg/100 g). OPN mRNA expression appeared in subpial astrocytes as early as 6 h, and then spread over the brain parenchyma. The signal for OPN mRNA reached a peak at 24 h post-injection, and returned to basal levels after 48 h. Changes in OPN immunoreactivity in the LPS-injected rat mirrored OPN mRNA induction patterns. These results provide the first evidence of OPN induction in astrocytes and microglia following peripheral immune challenge, and suggest that OPN may play a key role in the pathogenesis of neuroinflammation.

Temporal progressive antigen expression in radial glia after contusive spinal cord injury in adult rats.

In the development of the CNS, radial glial cells are among the first cells derived from neuroepithelial cells. Recent studies have reported that radial glia possess properties of neural stem cells. We analyzed the antigen expression and distribution of radial glia after spinal cord injury (SCI). Sprague-Dawley rats had a laminectomy at Th11-12, and spinal cord contusion was created by compression with 30 g of force for 10 min. In the injury group, rats were examined at 24 h and 1, 4, and 12 weeks after injury. Frozen sections of 20-microm thickness were prepared from regions 5 and 10 mm rostral and caudal to the injury epicenter. Immunohistochemical staining was performed using antibodies to 3CB2 (a specific marker for radial glia), nestin, and glial fibrillary acidic protein (GFAP). At 1 week after injury, radial glia that bound anti-3CB2 MAb had spread throughout the white matter from below the pial surface. From 4 weeks after injury, 3CB2 expression was also observed in the gray matter around the central canal, and was especially strong around the ependymal cells and around blood vessels. In double-immunohistochemical assays for 3CB2 and GFAP or 3CB2 and nestin, coexpression was observed in subpial structures that extended into the white matter as arborizing processes and around blood vessels in the gray matter. The present study demonstrated the emergence of radial glia after SCI in adult mammals. Radial glia derived from subpial astrocytes most likely play an important role in neural repair and regeneration after SCI.

Embryonic intermediate filament, nestin, expression following traumatic spinal cord injury in adult rats

Precursor cells in the ependyma of the lateral ventricles of adult mammalian brain have been reported in brain, and also in the spinal cord. The present study used antibody to the intermediate filament protein (nestin) as an immunohistochemical marker for neural stem cells and precursor cells in a rat model of spinal cord trauma. Male Sprague–Dawley rats ( n=25) had a laminectomy at Thll–Thl2, and spinal cord contusion was created by compression with 30 g of force for 10 min. The rats were killed at 24 h, 1 week and 4 weeks after injury, and four levels of the spinal cord were examined: 5 mm and 10 mm, both rostral and caudal region to the injury center. Time- and region-dependent alterations of nestin immunoreactivity were analyzed. Revealed at 24 h post-injury, 5 mm rostral and caudal to the lesions, nestin expression was observed in ependymal cells and around the hemorrhagic and necrotic lesion located in dorsal spinal cord, peaking at 1 week after injury. Moreover, nestin expression was also observed in the white matter of ventral spinal cord, extending into arborizing processes centripetally from the pial surface toward the central canal. At 4 weeks after injury, nestin expression in ependyma decreased 10 mm from the injury site. But nestin expression in white matter increased dramatically with a 100-fold increase in nestin originating from the pial surface, and extension now to all the white matter. The latter was accompanied by glial fibrillary acidic protein positivity into very long arborizing processes, morphologically compatible with radial glia. The findings suggest two possible sources of precursor cells in adult mammalian spinal cord; ependyma of the central canal and subpial astrocytes. Subpial astrocytes may be associated with neural repair and regeneration after spinal cord injury.

HSP70 constitutive expression in rat central nervous system from postnatal development to maturity.

We studied the level of the basal (constitutive) HSP70 expression (inducible and constitutive forms) in the central nervous system (CNS) of male and female rats from the postnatal period to maturity. HSP70 levels were analyzed by immunoblotting in five different areas (cortex, hippocampus, hypothalamus, cerebellum, and spinal cord). The highest levels of HSP70 were found in juvenile rats and decreased progressively until reaching baseline levels between 2 and 4 months. A slight and nonsignificant increase in aged (2-year-old) rats compared with adult subjects was observed in some cerebral areas (cerebral cortex, hippocampus, and cerebellum). In the first weeks of postnatal development, HSP70 immunoreactivity was distributed throughout CNS sections and no specific immunopositive cells could be clearly determined. In adult animals, strong immunostaining was observed in some large neurons (Purkinje neurons and mesencephalic and spinal cord motor neurons), some perivascular and subpial astrocytes, and ependymocytes. Immunoelectron microscopy revealed that HSP70 in these cells is located in the perinuclear area and in mitochondria, rough endoplasmic reticulum, and microtubules. In neurons, strong immunolabeling was also observed in synaptic membranes. The postnatal time course of HSP70 levels and the location and size of HSP70-immunopositive cells suggest that HSP70 constitutively expressed in the rat CNS may be mainly determined by the degree of development and metabolic activity of the neural cells.

Ultrastructural Features of Pleomorphic Xanthoastrocytoma: A Comparative Study with Glioblastoma Multiforme

Eighteen tumors (15 cases) were ultrastructurally examined and compared to 11 examples of glioblastoma multiforme (GBM) [to determine the cellular nature of pleomorphic xanthoastrocytoma (PXA).] PXA, as well as GBM, were principally composed of pleomorphic astrocytes containing numerous intermediate filaments. Lipid droplets, lysosomes, and basal laminas were more numerous and fully developed in PXA, but were not specific to the tumor. Aggregates of secondary lysosomes, light microscopically evident as eosinophilic granular bodies, and ribosome-lamella complexes were exclusively seen in PXA. Also noted in PXA were centrioles, occasional Rosenthal fibers, hemidesmosomes in small number, rudimentary cell junctions, and interstitial calcifications. In addition to astrocytic constituents, 20% of PXA contained cells with neuronal features, as evidenced by the presence of dense-core granules, microtubules, and clear vesicles. In contrast, GBMs consisted solely of astrocytic cells. In the present study, the salient ultrastructural findings of PXA were largely degenerative in nature, including numerous lipid droplets and secondary lysosomes, as well as basal laminas, ribosome-lamella complexes, and occasional neuronal differentiation. The presence of basal lamina does not necessarily imply a histogenetic derivation from subpial astrocytes, in that it is also a common feature of GBM. Given the occurrence of biphenotypic, glioneuronal differentiation in some cases, PXA may be derived from a neuroepithelial stem cell. The authors conclude that PXA is fundamentally an astrocytic tumor, albeit one with a significant tendency to undergo neuronal differentiation.

Neuronal expression of the fukutin gene.

Fukuyama-type congenital muscular dystrophy (FCMD), a relatively common autosomal recessive disorder in Japan, is characterized by severe congenital muscular dystrophy in combination with cortical dysgenesis (polymicrogyria). The gene responsible for FCMD encodes a novel protein, fukutin, which is likely to be an extracellular protein. Pathological study of brain tissue from FCMD fetuses revealed frequent breaks in the glia limitans and basement membrane complex. Disruption of the basal lamina in FCMD muscle was also seen. Thus, structural alteration of the basal lamina appears to play a key role in the pathophysiology of FCMD. To investigate the role of fukutin in brain anomalies, we examined fukutin mRNA expression in the human brain. Northern blot and RT-PCR analysis revealed that the fukutin gene is expressed at similar levels in fetal and adult brain, whereas its expression is much reduced in FCMD brains. Tissue in situ hybridization analysis revealed fukutin mRNA expression in the migrating neurons, including Cajar-Retzius cells and adult cortical neurons, as well as in hippocampal pyramidal cells and cerebellar Purkinje cells. However, we observed no expression in the glia limitans, the subpial astrocytes (which contribute to basement membrane formation) or other glial cells. In the FCMD brain, neurons in regions with no dysplasia showed fair expression, whereas transcripts were nearly undetectable in the overmigrated dysplastic region. These observations suggest that fukutin function may influence neuronal migration itself rather than formation of the basement membrane. Furthermore, differences in mRNA levels among neurons in early developmental stages may partially differentiate normal and abnormal regions.

Connexin-43 in rat spinal cord: localization in astrocytes and identification of heterotypic astro-oligodendrocytic gap junctions

Connexin-43 in relation to gap junctions between astrocytes and between other cell types in rat spinal cord was investigated immunohistochemically. In gray matter, connexin-43 was distributed thoughout all laminae, but was more concentrated in the substantia gelatinosa and around the central canal. Ultrastructurally, immunostaining was present in the cytoplasm of, and at gap junctions between, fine astrocytic processes, most of which ensheathed neuronal elements. In white matter, connexin-43 was localized to somata of fibrous astrocytes, their glial fibrillary acidic protein-positive processes running parallel to myelinated axons, and at gap junctions between these processes. Labelling was also evident in thick radially-directed astrocytic processes displaying pockets of staining near immunopositive gap junctions. Near the cord surface, staining was present in cell bodies of subpial astrocytes and at gap junctions between their tangential processes which formed most of the glia limitans. Radially-directed processes of subpial astrocytes formed symmetrically- and asymmetrically-labelled gap junctions with each other and extended fine branches into surrounding white matter where they made contact and often formed gap junctions with oligodendrocytic processes at the outer surface of myelinated fibres. Immunopositive astrocyte processes also made heterologous gap junctions with unstained oligodendrocyte cell bodies. Ependymal cells lining the central canal exhibited apical cytoplasmic labelling, as well as symmetrically-labelled gap junctions at their apices. Ependymal cells also formed asymmetrically-labelled gap junctions at which the junctional membranes of unlabelled cells, presumed to be tanycytes, were unstained.The results indicate the expression of connexins in addition to connexin-43 at asymmetrically-labelled gap junctions between some astrocytic processes, between astrocytes and oligodendrocytes and between some ependymal cells. The presence of gap junctions between astrocyte and oligodendrocyte processes at the outer surface of myelin suggests incorporation of the latter into the extensive gap junctionally-coupled astrocytic syncytium.

Abundant expression of HIV Nef and Rev proteins in brain astrocytes in vivo is associated with dementia

To relate the expression of HIV regulatory proteins and HIV-specific mRNA in the brain cells of infected individuals with clinical neurological disease. Formalin-fixed postmortem brain tissue from 14 HIV-infected adult patients, with previous repeated neurological and neuroradiological examinations, was studied by immunohistochemical and molecular biological methods. Samples from non-infected brains served as controls. Immunohistochemistry with monoclonal antibodies (MAb) was combined with in situ RNA hybridization. Target cells were identified with MAb to glial fibrillary acidic protein (GFAP; astrocytes), CD68 (activated macrophages) and Ricinus communis agglutinin (RCA-1; microglia, endothelial cells). For HIV, a panel of MAb against HIV Nef, Tat, Rev and Env proteins or probes specific for all classes of mRNA (nef), for singly or non-spliced mRNA (env) and for non-spliced mRNA (gag/pol) were used. Nef protein was detected in subcortical or subpial astrocytes in seven out of 14 samples, and in multinucleated giant cells in two cases. Gag/pol or env mRNA-expressing astrocytes were detected in four cases. In four out of five cases studied, HIV Rev, but not Tat, was also expressed in astrocytes. Six out of the seven patients with Nef-positive astrocytes had suffered from moderate to severe dementia. The patient with most rapidly progressing severe dementia showed extensive HIV mRNA expression together with Nef and Rev expression in astrocytes. In adult human brain, astrocytes are infected by HIV and preferentially express HIV Nef and Rev proteins but are also sometimes productively infected. Astrocyte infection is associated with moderate to severe dementia which agrees with recent knowledge on the housekeeping activities of astrocytes and their eventual role in learning and memory.

MRI of pleomorphic xanthoastrocytoma: case report

MRI showed a cortically-based partially cystic and markedly enhancing mass in the uncus of the right temporal lobe in a patient with long standing refractory partial complex seizures. Histopathological examination revealed a pleomorphic xanthoastrocytoma, a rare, usually benign tumour thought to originate from subpial astrocytes.