Cytoplasm Of Astrocytes Research Articles

Spinal cord injury induces endoplasmic reticulum stress with different cell‐type dependent response

The mechanisms of injury-induced apoptosis of neurons within the spinal cord are poorly understood. In this study, we show that spinal cord injury (SCI) induces endoplasmic reticulum stress revealed by the activation of an unbalanced unfolded protein response (UPR). Using a weight-drop contusion model of SCI, the UPR activation was characterized by a quick transient phosphorylation of alpha subunit of eukaryotic initiation factor 2 soon restored by the up-regulation of its regulator Gadd34; an effective cleavage/activation of the ATF6alpha transcription factor leading to up-regulation of the canonical UPR target genes Chop, Xbp1 and Grp78; the presence of the processing of Xbp1 mRNA indicative of inositol requiring kinase 1 activation, and a gradual accumulation of C/EBP homologous transcription factor protein (CHOP) with concomitant caspase-12 activation. Interestingly, the subcellular distribution of CHOP was found in the nucleus of neurons and oligodendrocytes but in the cytoplasm of astrocytes. Considering the pro-apoptotic action attributed to this transcription factor, this phenomenon might account for the different susceptibility of cell types to dye after SCI.

Neuronal mdr-1 gene expression after experimental focal hypoxia: A new obstacle for neuroprotection?

Neuronal damage after stroke-associated brain hypoxia is a leading cause of long-term disability and death. The refractoriness to therapeutic strategies for neuroprotection after 3 h post brain ischemia is poorly understood. P-glycoprotein (P-gp), the multidrug resistance gene (MDR-1) product is normally expressed at blood–brain-barrier. P-gp neuronal expression has been demonstrated in refractory epilepsy and after brain ischemia. In this report we investigated the hypoxia-induced neuronal P-gp expression after local injection of CoCl 2 (1–200 mM) in the fronto-parietal cortex of male adult rats (Bregma − 1.30 mm) by stereotaxic surgery. P-gp immunostaining of brain slides was analyzed using specific monoclonal antibodies and double immunolabeling was done with specific astrocytic and neuronal markers. Five days after injection of 1 mM CoCl 2, P-gp expression surrounding the lesion site was observed in neurons, astrocytic end-foot on capillary blood vessels and endothelial cells on blood vessels. Higher CoCl 2 doses (200 mM) resulted in additional P-gp immunostaining of the entire astrocytic and neuronal cytoplasm. Electron microscopy (EM) studies showed alterations in neurons as early as 6 h after the CoCl 2 injection. P-gp expression in hypoxic neurons and astrocytic end-foot could potentially impair of drugs access to the brain parenchyma thus suggesting the presence of two P-gp-based pumping systems (one in astrocytes and other in the hypoxic neurons) that are able to behave as a previously unnoticed obstacle for pharmacological strategies of neuroprotection.

Evidence for BAG3 modulation of HIV‐1 gene transcription

A family of co-chaperone proteins that share the Bcl-2-associated athanogene (BAG) domain are involved in a number of cellular processes, including proliferation and apoptosis. Among these proteins, BAG3 has received increased attention due to its high levels in several disease models and ability to associate with Hsp70 and a number of other molecular partners. BAG3 expression is stimulated during cell response to stressful conditions, such as exposure to high temperature, heavy metals, and certain drugs. Here, we demonstrate that BAG3 expression is elevated upon HIV-1 infection of human lymphocytes and fetal microglial cells. Furthermore, BAG3 protein was detectable in the cytoplasm of reactive astrocytes in HIV-1-associated encephalopathy biopsies, suggesting that induction of BAG3 is part of the host cell response to viral infection. To assess the impact of BAG3 upregulation on HIV-1 gene expression, we performed transcription assays and demonstrated that BAG3 can suppress transcription of the HIV-1 long terminal repeat (LTR) in microglial cells. This activity was mapped to the kappaB motif of the HIV-1 LTR. Results from in vitro and in vivo binding assays revealed that BAG3 suppresses interaction of the p65 subunit of NF-kappaB with the kappaB DNA motif of the LTR. Results from binding and transcriptional assay identified the C-terminus of BAG3 as a potential domain involved in the observed inhibitory effect of BAG3 on p65 activity. These observations reveal a previously unrecognized cell response, that is, an increase in BAG3, elicited by HIV-1 infection, and may provide a new avenue for the suppression of HIV-1 gene expression.

Receptors for leptin and estrogen in the subcommissural organ of rabbits are differentially modulated by fasting

In rabbits, the fasting-dependent reduction of LH secretion is likely mediated by leptin and estrogens via receptors in the brain. For the first time, using immunohistochemistry, the presence and regulation of receptors for leptin (Ob-R) and estradiol-17β subtype α (ERα) were studied in the subcommissural organ (SCO) of rabbits, which were fed either ad libitum (control) or fasted for 48 h (treated) to verify whether this brain structure is a potential site of integration for metabolism and reproduction. In control rabbits, the cytoplasm of glial cells lining the SCO evidenced strong Ob-R immunoreactivity, whereas both ependymal and hypendymal cells of this glandular-like structure were negative. The Ob-R positive glial cells were identified as fibrous astrocytes using the phosphotungstic acid–hematoxylin histochemical (PTAH) and glial fibrillary acidic protein (GFAP) immunohistochemical techniques. ERα immunoreactive nuclei were detectable exclusively in the specialized cells forming the SCO, whereas surrounding astrocytes and neurons were negative. Compared to controls, in fasted rabbits, the staining of Ob-R immunoreaction was reduced in the cytoplasm of positive astrocytes, but greatly enhanced in plasma membranes, whereas the number of ERα immunoreactive SCO cells was increased (13.2 ± 2.7 vs. 5.2 ± 2.0, P < 0.01). Ependymal cells lining the third ventricle were negative for both Ob-R and ERα. Our results indicate, although indirectly, that the SCO, together with the astrocytes in close contact with this structure, is a likely target for nutritional and gonadal signals carried by leptin and estrogens, suggesting that these specialized glial cells may regulate reproduction and metabolism through mechanisms still unknown.

WEST NILE VIRUS INFECTION OF PRIMARY MOUSE NEURONAL AND NEUROGLIAL CELLS: THE ROLE OF ASTROCYTES IN CHRONIC INFECTION

Primary cultures of embryonic murine neurons and newborn mouse astrocytes were inoculated with West Nile virus (WNV) strain NY385-99 to compare the pathogenesis of WNV infection in these types of CNS cells. Two different outcomes were observed. WNV infection in the neurons was rapidly progressive and destructive; within 5 days, all of the neurons were destroyed through apoptosis. WNV infection in the astrocytes evolved more slowly and did not seem to be highly lethal to the cells. The infected astrocytes continued to produce infectious virus (10(4.6)-10(6.5) PFU/mL) for 114 days, in a permissive, persistent infection. During this period, WNV antigen could be shown in the cytoplasm of the infected astrocytes by immunocytochemical assay, transmission electron microscopy of ultrathin sections, and in the cell culture medium by complement fixation test. Our results with this in vitro experimental murine cell model indicate that astrocytes can develop chronic or persistent infection with WNV, suggesting that these cells may play a role in the maintenance of WNV in the CNS.

Kinetic Parameters and Lactate Dehydrogenase Isozyme Activities Support Possible Lactate Utilization by Neurons

Lactate is potentially a major energy source in brain, particularly following hypoxia/ischemia; however, the regulation of brain lactate metabolism is not well understood. Lactate dehydrogenase (LDH) isozymes in cytosol from primary cultures of neurons and astrocytes, and freshly isolated synaptic terminals (synaptosomes) from adult rat brain were separated by electrophoresis, visualized with an activity-based stain, and quantified. The activity and kinetics of LDH were determined in the same preparations. In synaptosomes, the forward reaction (pyruvate + NADH + H(+ )--> lactate + NAD(+)), which had a V (max) of 1,163 micromol/min/mg protein was 62% of the rate in astrocyte cytoplasm. In contrast, the reverse reaction (lactate + NAD(+ )--> pyruvate + NADH + H(+)), which had a V (max) of 268 micromol/min/mg protein was 237% of the rate in astrocytes. Although the relative distribution was different, all five isozymes of LDH were present in synaptosomes and primary cultures of cortical neurons and astrocytes from rat brain. LDH1 was 14.1% of the isozyme in synaptic terminals, but only 2.6% and 2.4% in neurons and astrocytes, respectively. LDH5 was considerably lower in synaptic terminals than in neurons and astrocytes, representing 20.4%, 37.3% and 34.8% of the isozyme in these preparations, respectively. The distribution of LDH isozymes in primary cultures of cortical neurons does not directly reflect the kinetics of LDH and the capacity for lactate oxidation. However, the kinetics of LDH in brain are consistent with the possible release of lactate by astrocytes and oxidative use of lactate for energy in synaptic terminals.

Linker Histone H1 Binds to Disease Associated Amyloid-like Fibrils

Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most prevalent neurodegenerative diseases of the central nervous system. These two diseases share a common feature in that a normally soluble peptide (amyloid-β) or protein (α-synuclein) aggregates into an ordered fibrillar structure. As well as structural similarities observed between fibrillar aggregates related to these diseases, common pathological processes of increased oxidative injury, excitotoxicity and altered cell cycle are also evident. It was the aim of this study to identify novel interacting proteins to the amyloid-like motif and therefore identify common potential pathways between neurodegenerative diseases that share biophysical properties common to classical amyloid fibrils. Optimal ageing of recombinant proteins to form amyloid-like fibrils was determined by electron microscopy, Congo red birefringement and photo-induced cross-linking. Using pull-down assays the strongest detected interacting protein to the amyloid-like motifs of amyloid-β, α-synuclein and lysozyme was identified as histone H1. The interaction with the amyloid-like motif was confirmed by techniques including surface plasmon resonance and immunohistochemistry. Histone H1 is known to be an integral part of chromatin within the nucleus, with a primary role of binding DNA that enters and exits from the nucleosome, and facilitating the shift in equilibrium of chromatin towards a more condensed form. However, phosphorylated histone H1 is predominantly present in the cytoplasm and as yet the functional significance of this translocation is unknown. This study also found that histone H1 is localised within the cytoplasm of neurons and astrocytes from areas affected by disease as well as amyloid plaques, supporting the hypothesis that histone H1 favoured binding to an ordered fibrillar motif. We conclude that the binding of histone H1 to a general amyloid-like motif indicates that histone H1 may play an important common role in diseases associated with amyloid-like fibrils.

Characterization of STAT3-expressing cells in the postnatal rat brain

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor abundantly expressed in the postnatal brain that is involved in the differentiation of cultured astrocytes. Thus far, the cellular identity and anatomical distribution of STAT3-expressing cells in the postnatal brain is poorly known. This study identifies the cell type(s), anatomical location, and temporal distribution of STAT3-expressing cells by using immunohistochemistry and confocal microscopy on postnatal day 3 (P3), 10 (P10), and 21 (P21) rat brain sections. Furthermore, the phosphorylation of STAT3 on tyrosine and serine residues was analyzed at these different stages by immunoprecipitation followed by Western blot. STAT3 immunoreactivity was observed in the cytoplasm and nucleus of many maturating astrocytes positive for nestin (at P3) or positive for GFAP (at P10) distributed throughout the white and grey matter. Moreover, robust nuclear immunoreactivity was observed in brainstem motoneurons. Phosphorylation on tyrosine and serine was observed at P3 and increased at P10, which suggests an augmented activation of STAT3 at the mid-postnatal period. At P21, STAT3 immunoreactivity dramatically decreased to remain visible only in the cytoplasm of white matter astrocytes and hypothalamic and brainstem neuronal groups. Furthermore, while the phosphorylation of tyrosine residues tended to decrease, that of serine residues further increased. In summary, our study reveals a complex regulation of STAT3 phosphorylation in the postnatal brain and provides in vivo evidence of the specific expression of STAT3 in maturating astrocytes and brainstem motoneurons.

Alexander disease

Alexander disease has always been considered a classic leukodystrophy (the term literally meaning abnormal white matter nutrition, but usually used more broadly to indicate any hereditary or metabolic white matter abnormality). Although rare (the true incidence is not yet known), the disease is always included in the differential diagnosis when encountering an infant with macrocephaly, delayed development, and an abnormal neuroimaging study primarily affecting the frontal lobes. Confirmation of the diagnosis previously required brain biopsy or autopsy, and demonstration of protein aggregates known as Rosenthal fibers within the cytoplasm of astrocytes. MRI criteria for diagnosis, as refined by van der Knaap et al.,1 noted additional features besides frontal white matter defects (a periventricular rim with high signal on T1-weighted images and low signal on T2-weighted images, abnormalities of the basal ganglia and thalami, brainstem abnormalities, and contrast enhancement of particular gray and white matter structures) and are now highly reliable benchmarks for clinical practice. The realization that mutations in the astrocyte intermediate filament GFAP are responsible for nearly all cases transformed the clinical approach for the most common, infantile, form. …

February 28 Highlights

The MRI phenotypic variation of Alexander disease continues to broaden. van der Knaap et al. describe seven patients with a DNA-confirmed diagnosis, who display medulla atrophy, a spinal cord lesion, or ventricular garlands on MRI, whereas cerebral white matter abnormalities were mild or virtually absent. see page 494 The editorial by Barkovich and Messing notes that Alexander disease has usually been characterized by macrocephaly, delayed development, and MRI evidence of leukodystrophy affecting the frontal lobes. Diagnosis previously required brain biopsy or autopsy and demonstration of protein aggregates known as Rosenthal fibers with the cytoplasm of astrocytes. Now molecular diagnosis has expanded the clinical phenotype of juvenile and adult forms of Alexander disease to include children without obvious leukodystrophy but possessing a newly recognized and peculiar anomaly in the walls of the lateral ventricles termed garlands. From a genetic standpoint Alexander disease is a …

Co‐localization of β‐peptide and phosphorylated tau in astrocytes in a patient with corticobasal degeneration

The co-localization of amyloid beta (Abeta) and phosphorylated tau in astrocytes in a patient with corticobasal degeneration is described. At autopsy, the present case exhibited neuropathological findings compatible with those of corticobasal degeneration, including atrophy of the frontal and temporal lobes, neuronal loss and gliosis in the cortical and subcortical regions, and presence of cortical ballooned neurons and astrocytic plaques. Moreover, many senile plaques were found in the cerebral cortex. There were also clusters of Abeta-positive granules associated with astrocytic cytoplasm and processes in the subiculum and entorhinal cortex. In the entorhinal cortex, the Abeta-positive granules were occasionally co-localized with phosphorylated tau-positive fibrillary structures in the astrocytic cytoplasm. To our knowledge, this is the first demonstration of co-localization of Abeta and phosphorylated tau in astrocytes. This phenomenon implies that phagocytosis of Abeta coincides with production of phosphorylated tau in the same reactive astrocytes.

Interferon-α2b may impair myelinization of rat optic nerve

This story investigated the effects of interferon-alpha-2b (IFN-alpha2b) on the optic nerves of 17 adult male Wistar albino rats. Animals were divided into 3 groups: 6 rats (group 1) received 7.5 units (5 mIU/m2) IFN-alpha2b-a normal treatment dose, and 6 (groups 2) received 30.0 units (20 mIU/m2)-a high dose; 5 rats (control group) received 0.5 mL saline. Test substances were delivered by intraperitoneal injection 3 times a week for 3 weeks with animals under inhalation anesthesia. After the rats were sacrificed, their optic nerves were dissected, sectioned, and examined under an electron microscope. The mean thicknesses of the basal membranes of blood vessels were 86.354 nm in the control group, 104.297 nm in group 1, and 140.181 nm in group 2. Basal membrane changes in IFN groups were dose dependent. Mitochondrial swelling, degeneration, increased diameter of vacuoles, and vacuolization in the cytoplasm of oligodendrocytes and astrocytes were also observed. IFN-alpha2b has histopathologic effects on blood vessels and cells of the optic nerve.

The mitochondrial complex I inhibitor rotenone triggers a cerebral tauopathy

Reduced activity of the mitochondrial respiratory chain--particularly complex I--may be implicated in the etiology of both Parkinson's disease and progressive supranuclear palsy, although these neurodegenerative diseases differ substantially as to their distinctive pattern of neuronal cell loss and the predominance of cerebral alpha-synuclein or tau protein pathology. To determine experimentally whether chronic generalized complex I inhibition has an effect on the distribution of alpha-synuclein or tau, we infused rats systemically with the plant-derived isoflavonoid rotenone. Rotenone-treated rats with a pronounced metabolic impairment had reduced locomotor activity, dystonic limb posture and postural instability. They lost neurons in the substantia nigra and in the striatum. Spherical deposits of alpha-synuclein were observed in a few cells, but cells with abnormal cytoplasmic accumulations of tau immunoreactivity were significantly more numerous in the striatum of severely lesioned rats. Abnormally high levels of tau immunoreactivity were found in the cytoplasm of neurons, oligodendrocytes and astrocytes. Ultrastructurally, tau-immunoreactive material consisted of straight 15-nm filaments decorated by antibodies against phosphorylated tau. Many tau+ cell bodies also stained positive for thioflavin S, nitrotyrosine and ubiquitin. Some cells with abnormal tau immunoreactivity contained activated caspase 3. Our data suggest that chronic respiratory chain dysfunction might trigger a form of neurodegeneration in which accumulation of hyperphosphorylated tau protein predominates over deposits of alpha-synuclein.

1420 POSTER Stereotactic body radiation therapy for lung metastases: impact on overall survival

The localization of angiotensin II-like immunoreactivity (A II-LI) has been studied at an ultrastructural level in the thoracic and sacral spinal cord vegetative regions of male and female guinea pigs. Most of the AII-LI structures are axon terminals and fibers localized in both vegetative networks which connect bilaterally and cranio-caudally and/or caudo-cranially the preganglionic vegetative nuclei in the thoracic and sacral spinal cord intermediate zone. The A II-LI product is localized predominantly in large granular and small vesicles of axon terminals and varicosites which form axo-somatic, axo-dendritic and axo-axonal synapses. Occasionally A II-LI is observed over cysterns of the endoplasmatic reticulum and throughout the cytoplasm of astrocytes which are neighbouring on blood vessels and are in contact with neuronal processi. Immunoreactive terminals are observed in direct contacts with endothelial cells too. These observations confirm recently described light-microscopic localization of Ang II-LI in the studied spinal cord areas, as well as its suggested role as transmitter and/or modulator of the neuronal function and regulatory role over the cardiovascular function and blood circulation.

Glyoxal inactivates glutamate transporter‐1 in cultured rat astrocytes

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive motor paralysis and selective motor neuron death. There is increasing evidence that motor neuron death in ALS is mediated by glutamate toxicity resulting from reduced activity of astrocytic glutamate transporter-1 (GLT-1). Recent morphological studies have shown that Nepsilon-(carboxymethyl)lysine (CML) accumulates in reactive astrocytes of ALS spinal cords. CML is a product of post-translational protein modification by glyoxal, a reactive aldehydic intermediate. In considering these documents, it is important to determine whether GLT-1 protein modification by glyoxal might cause reduced GLT-1 activity. To address this issue, we investigated the effects of glyoxal on GLT-1 properties in cultured rat astrocytes. High performance liquid chromatography showed reduced glutamate uptake activity in the glyoxal-exposed cells. Immunocytochemical analysis displayed CML accumulation in the cytoplasm of astrocytes by glyoxal exposure. Immunoblots of immunoprecipitated GLT-1 disclosed GLT-1 CML adduct formation in the glyoxal-exposed cells. Our results indicate that glyoxal modifies GLT-1 to form CML and simultaneously deprives its glutamate uptake activity. Thus, these toxic effects of glyoxal on astrocytes might be implicated in motor neuron death in ALS.

Expression of the interferon-α/β-inducible MxA protein in brain lesions of subacute sclerosing panencephalitis

The type-I interferon (IFN) inducible human MxA protein exhibits antiviral activity against a variety of RNA viruses including the measles virus (MV). In this study, we investigated the association between the expression of MV antigens and MxA in subacute sclerosing panencephalitis (SSPE) brains. We analyzed the MxA expression in and around lesions in brains of three SSPE patients and compared it with normal brains. Double staining with antibodies against MxA and the MV nucleocapsid revealed that MxA was highly expressed in a belt surrounding MV-antigen-positive lesions in SSPE brains. In normal appearing regions distant from a lesion in SSPE brains and in normal brains, MxA was not detected. Furthermore, MxA was often less or not expressed in the center of lesions expressing high amounts of MV antigens. Such a pattern of MxA expression in SSPE brains clearly indicates that newly infected cells release type I IFN and will become demarcated by a protecting barrier of MxA expressing cells. Double staining with antibodies against MxA and glial fibrillary acidic protein (GFAP) showed that the MxA protein was expressed mainly in the cytoplasm of astrocytes. MxA expression did not correlate with the presence of cellular infiltrates of inflammatory cells, although some lymphoid cells were also positive for MxA. Since MxA inhibits the replication of MV, these findings suggest that the IFN-induced MxA protein plays an important role in slowing down the viral spread in SSPE brains and by doing so may contribute to the persistence of the MV-infection.

A behavioral and ultrastructural dissection of the interference of aluminum with aging

The persistence of neuroscientists in exploring aluminium's (Al) possible contribution to the pathogenesis of Alzheimer's disease (AD) has resulted in a wealth of researches detailing the biological toxicity of this metal. However, to date, there have been few accounts of the interference of Al with aging and its relevance to the pathogenesis of AD. We investigated the behavioral and the ultrastructural signatures of Al in the hippocampus on young and aging rats which were exposed for three months to aluminium gluconate. The aging animals displayed decreased scores of activity and emotionality, and the Al-exposed aging males had altered emotional reactivity behaviors. The electron-microscopic analysis indicated that Al promoted in the aging hippocampus a variety of cellular and ultrastructural degenerative signs, such as granulo-vacuolar degenerations, deposition of lipofuscin and amyloid in the cytoplasm of neurons and astrocytes, and in extracellular compartments, Hirano bodies, demyelination and the atrophy of the mitochondria. Moreover, the quantitation of myelin sheath width and the diameter of mitochondria measured on randomly selected samples confirmed that myelin and mitochondria are primary targets of Al's toxicity. Demyelination and mitochondrial atrophy seemed more advanced in the hippocampus of Al-exposed aging males, supporting the effect of sex suggested by the behavioral results. These findings and other collateral results also reported here are discussed in the context of a possible involvement of Al in AD, mediated by aging and catalyzed by hepatic morphopathology.

Trans-endocytosis via spinules in adult rat hippocampus.

Locations of a distinctive mode of trans-endocytosis involving dendrites, axons, and glia were quantified through serial section electron microscopy. Short vesicular or long vermiform evaginations emerged from dendrites and axons and were engulfed by presynaptic or neighboring axons, astrocytes, and, surprisingly, a growth cone to form double-membrane structures called spinules. In total, 254 spinules were evaluated in 326 microm(3) of stratum radiatum in area CA1 of mature rat hippocampus. Spinules emerged from spine heads (62%), necks (24%), axons (13%), dendritic shafts (1%), or nonsynaptic protrusions (<1%) and invaginated into axons (approximately 90%), astrocytic processes (approximately 8%), or a growth cone (approximately 1%). Coated pits occurred on the engulfing membrane at the tips of most spinules (69%), and double-membrane structures occurred freely in axonal and astrocytic cytoplasm, suggesting trans-endocytosis. Spinule locations differed among mushroom and thin spines. For mushroom spines, most (84%) of the spinules were engulfed by presynaptic axons, 16% by neighboring axons, and none by astrocytic processes. At thin spines, only 17% of the spinules were engulfed by presynaptic axons, whereas 67% were engulfed by neighboring axons and 14% by astrocytic processes. Spinules engulfed by astrocytic processes support the growing evidence that perisynaptic glia interact directly with synapses at least on thin spines. Spinules with neighboring axons may provide a mechanism for synaptic competition in the mature brain. Trans-endocytosis of spinules by presynaptic axons suggest retrograde signaling or coordinated remodeling of presynaptic and postsynaptic membranes to remove transient perforations and assemble the postsynaptic density of large synapses on mushroom spines.

Traumatic injury-induced midkine expression in the adult rat spinal cord during the early stage.

Spinal cord injury is a debilitating condition. Midkine (MK) is involved in the generation of the central nervous system during development; however, the role of MK in the mature spinal cord has not been clarified. We examined the expression of MK, which has neurotrophic activity, before and after traumatic injury to the adult rat spinal cord. Following laminectomy, the rat spinal cord was injured at the T-9 level by applying extradural static weight-compression, in which a cylindrical compressor was used to induce complete and irreversible transverse spinal cord injury with paralysis of the lower extremities. The expression of MK was examined up to 14 days after the injury by immunohistochemical and Western blot analyses. Intense MK immunoreactivity was observed in the gray matter around the injury site but not in the necrotic lesion 1-7 days postinjury, although it was slightly positive 14 days after the injury. MK immunoreactivity was not detected in the normal spinal cord. The expression of MK was an early event, and its expression was compared to the increased production of glial fibrillary acidic protein (GFAP), a marker of reactive astrocytes, that was elevated at 2 days postinjury and continued over a 14 day period following the injury. Double immunostaining with anti-MK and anti-GFAP showed the existence of MK in the astrocytic cytoplasm. These findings suggest that MK was produced in astrocytes approximating the damaged region and may represent a reparative neurotrophic factor during the early phase of traumatic injury of the spinal cord.

Evidence for involvement of transforming growth factor beta1 signaling pathway in activation of JC virus in human immunodeficiency virus 1-associated progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy is a fatal demyelinating disease of the central nervous system frequently seen in patients with impaired immune systems, particularly acquired immunodeficiency syndrome. JC virus (JCV), a human neurotropic polyomavirus, is the etiologic infectious agent of this disease. The significantly higher incidence of progressive multifocal leukoencephalopathy in patients with acquired immunodeficiency syndrome than in patients with other immunosuppressive conditions suggests that molecular interactions between human immunodeficiency virus 1 and JCV, via the Tat protein, are responsible for the activation of the JCV enhancer/promoter and the development of progressive multifocal leukoencephalopathy. An indirect mechanism through activation of cytokines, such as transforming growth factor beta1 and Smads 3 and 4, may also be responsible for the enhancement of JCV gene expression. Immunohistochemical analysis in progressive multifocal leukoencephalopathy samples and chloramphenicol acetyl transferase assays on cell cultures were performed to corroborate this hypothesis. The JCV capsid protein VP-1 was found in the nuclei of oligodendrocytes and in the nuclei and cytoplasm of bizarre astrocytes. Human immunodeficiency virus proteins, including p24 and Tat, were detected in the cytoplasm of astrocytes. Tat, but not p24, was detected in oligodendrocytes, suggesting that extracellular Tat accumulates in the nuclei of oligodendrocytes, where JCV gene transcription takes place. High levels of transforming growth factor beta1 and Smads 3 and 4 were detected in JCV-infected oligodendrocytes. Results from in vitro studies confirm activation of the JCV early and late promoters by Smads 3 and 4. These observations support our model, suggesting that the induction of transforming growth factor beta1 by human immunodeficiency virus 1 Tat can stimulate its downstream factors, including Smads 3 and 4, which in turn augment transcription of the JCV promoter in glial cells.