Nazario Rubio

Mouse Astrocytes Store and Deliver Brain‐derived Neurotrophic Factor Using the Non‐catalytic gp95trkB Receptor

A study of the brain‐derived neurotrophic factor (BDNF)‐binding capacity of pure astrocytes demonstrated that these cells bind and endocytose [125l]BDNF rapidly using the gp95trkb truncated receptor. A linear Scatchard plot indicated the presence of only one type of receptor that bound the ligand, with a low Kd of 1.24 × 10−8 M. There were an average of 36 468 copies of this receptor on untreated astrocytes. Interestingly, the neurotrophin was not degraded intracellularly, as demonstrated by HPLC experiments. Furthermore, the stored molecule was released by a mechanism regulated by the extracellular BDNF concentration as a bioactive neurotrophic molecule that supports neuron survival, in a time‐ and temperature‐dependent manner. The data demonstrate that astrocytes exert an active role in the bioavailability of this neurotrophin, which is further enhanced in an inflammatory‐like situation induced experimentally in culture using interferon‐SgM.

In vitro myelination by oligodendrocyte precursor cells transfected with the neurotrophin-3 gene

Oligodendrocyte precursor cells require exogenous neurotrophin‐3 (NT‐3) for differentiation into oligodendrocytes. We transfected precursor cells with the gene for NT‐3 and looked for changes in their development into myelin‐forming cells. The expression of NT‐3 in transfected cells was demonstrated by reverse transcription followed by PCR as well as by Northern blots. Direct synthesis of the neurotrophin product and its release to the culture supernatants were also shown by specific ELISA. Transfection converts precursor cells into actively dividing cells that can incorporate 3H‐thymidine into DNA. In the absence of growth factors, a parallel increase in the survival of the transfected cultures was also demonstrated by the MTT test. The final demonstration of biological changes in transfected versus untreated cells was a 10‐fold increase in myelin basic protein production observed in Western blots and the direct observation by phase‐contrast and electron microscopy of myelin membranes in cocultures with hippocampal neurons. We discuss the future use of this transfected cells in regeneration and functional recovery in experimental models of multiple sclerosis.

Demonstration of the presence of a specific interferon-γ receptor on murine astrocyte cell surface

Abstract Interferon gamma (IFN-γ) is a pleiotropic lymphokine produced by T-lymphocytes which acts as a soluble mediator in immunological reactions. In addition to several immune target cells, such as monocytes and macrophages, it acts on the principal glial population, the astrocytes, inducing Ia antigen expression. We have developed a binding assay for 125I-labeled recombinant murine IFN-γ, and show that, using this assay, IFN-γ interacts with a single specific receptor on the murine astrocyte cell membrane. The binding is specific and saturable and it takes place with a K d = 1.64 × 10−9 M, with 11,100 receptor molecules per astrocytic cell. The binding shows, as for macrophages, species specificity. Using an immune assay including rabbit antibodies to IFN-γ and 125I-labeled protein A, we have demonstrated an internalization of the ligand. This is an energy-dependent process, as around 50% of the bound IFN-γ is endocytosed after 4 h at 37°C when cultures are maintained in complete culture medium.

High-neurovirulence GDVII virus induces apoptosis in murine astrocytes through tumor necrosis factor (TNF)-receptor and TNF-related apoptosis-inducing ligand

Abstract We carried out a study to determine if the high-neurovirulence GDVII strain of Theiler’s murine encephalomyelitis virus (TMEV) and the demyelinating, low-neurovirulence BeAn strain induced apoptosis in murine astrocytes. Astrocytes, the major glial cell population of the central nervous system, were semipermissive for GDVII virus replication. Programmed cell death, demonstrated by apoptosis-specific caspase-3 protease activity, was maximal 8 h after GDVII infection at an m.o.i. of 1. Purified TMEV capsid proteins VP1, VP2, and VP3 did not induce apoptosis but antibodies to VP1 and VP2 inhibited it. Antibody inhibition of caspase-3 activity as well as flow cytometry experiments implicated TNF-related apoptosis-inducing ligand (TRAIL) and TNF-α-receptor (TNF-R) in apoptosis signaling. Converselly, TNF-α and the TRAIL-receptor were not upregulated. Furthermore, the number of functional TNF-α receptors, but not their affinity, was increased in apoptotic GDVII virus-infected astrocytes, as confirmed in binding experiments with 125I-labeled recombinant murine TNF-α. In vivo studies showed that most of the cells loaded with the virus when injected in the brains of SJL mice were neurons but very few showed TUNEL costaining. Conversely, many of the apoptotic cells found were also positive for GFAP staining.

Theiler's murine encephalomyelitis virus-binding activity on neural and non-neural cell lines and tissues

Three categories of cell lines are described with respect to their activity in binding Theilers murine encephalomyelitis virus (TMEV). High, medium and low densities of viral receptors can be detected on cell lines from different species and origins by using an immunological binding assay. Nevertheless, TMEV acts as a fastidious virus that only infects a few cell types productively. No correlation between virion binding and degree of permissiveness to infection could be detected. The presence of viral receptors in both susceptible and resistant strains of mice seemed to have a widespread tissue distribution, the thymus being an exception. When primary cerebral cultures, enriched in neurons, astrocytes or oligodendrocytes, were checked in the immunological assay, a higher density of viral receptors was detected in the neuronal population. The number of virus-binding sites in the BHK-21 cell line is reported here to be 5 x 10(3) per cell; approximately 15 x 10(3) and 2.5 x 10(3) are the estimates of binding sites per cultured neuron and macroglial cell, respectively.

Lack of cross-reaction between myelin basic proteins and putative demyelinating virus envelope proteins

No cross-reaction could be detected between purified myelin basic proteins (MBP) from mouse, rat or human origins and envelope proteins of viruses suspected of inducing demyelinating processes. In the experimental model using Theilers murine encephalomyelitis virus, competition radioimmunoassay failed to detect any cross-reaction between MBP and VP1, VP2 and VP3 envelope antigens. In the human situation, antibodies against SV5 and measles viruses, both etiologically linked with multiple sclerosis, also failed to recognize MBP. These results rule out molecular mimicry as a cause of demyelination.

Interferon‐γ induces the expression of immediate early genes c‐fos and c‐jun in astrocytes

The expression of the proto‐oncogenes, c‐fos and c‐jun, in cultured mouse astrocytes and its induction by the potent astrocyte activator interferon‐γ (IFN‐γ), were examined by Northern blot and flow cytometry. Both proto‐oncogenes were induced in a dose‐dependent manner, peaking around 100 U/ml of IFN‐γ. The kinetics of expression is very transient for c‐fos, reaching a maximum at 30 min and decreasing rapidly thereafter. The c‐jun remained high throughout the stages analysed. Cycloheximide superinduced c‐fos and c‐jun induction by IFN‐γ, thus indicating that both act as immediate early genes. The products of c‐fos and c‐jun, proteins FOS and JUN, that act in conjunction forming the regulatory factor AP‐1, were detected 1 hr after stimulation in virtually all cells, using flow cytometry. The induction in astrocytes of both proto‐oncogenes could be the first stage of immunological activation of these central nervous system cells by immune interferon.

Interferon-γ induces proliferation but not apoptosis in murine astrocytes through the differential expression of the myc proto-oncogene family

Interferon-gamma (IFN-gamma) is a cytokine mainly secreted by activated T-lymphocytes. Treatment of mouse astrocytes in vitro with IFN-gamma augmented the basal expression of the primary response proto-oncogenes c-myc and L-myc as detected by Northern blotting. Such inductions were maximal at doses of 10 U/ml and after 60 min of treatment. Astrocytes fully differentiated in vitro do not express N-myc mRNA nor are induced to express it by exposure to IFN-gamma. As demonstrated by flow cytometry, the common protein product of the myc family was present in the nucleus of the cells. The specificity of IFN-gamma induction was demonstrated when antibodies against IFN-gamma completely suppressed the overinduction of these mRNAs. No apoptotic death can be detected in astrocytes treated with IFN-gamma at doses that induce c-myc expression. Conversely, treatment with a dose of 10 U/ml induced cells proliferation in astrocytic cells as measured by (3)H-thymidine incorporation.

Rapid purification of the main allergen of lolium perenne by high-performance liquid chromatography

The main allergen from rye grass (Lolium perenne) pollen was purified by size-exclusion high-performance liquid chromatography. The purified allergen had a molecular weight of 32,000 daltons and was significantly more active in solid phase radioimmunoassay than the whole extract. The highly purified antigen can be obtained very rapidly and with a recovery of 30%.

An in vitro experimental model of neuroinflammation: the induction of interleukin-6 in murine astrocytes infected with Theiler’s murine encephalomyelitis virus, and its inhibition by oestrogenic receptor modulators

This paper describes an experimental model of neuroinflammation based on the production of interleukin‐6 (IL‐6) by neural glial cells infected with Theiler’s murine encephalomyelitis virus (TMEV). Production of IL‐6 mRNA in mock‐infected and TMEV‐infected SJL/J murine astrocytes was examined using the Affymetrix murine genome U74v2 DNA microarray. The IL‐6 mRNA from infected cells showed an eightfold increase in hybridization to a sequence encoding IL‐6 located on chromosome number 5. Quantitative real‐time reverse transcription PCR (qPCR) was used to study the regulation of IL‐6 expression. The presence of IL‐6 in the supernatants of TMEV‐infected astrocyte cultures was quantified by ELISA and found to be weaker than in cultures of infected macrophages. The IL‐6 was induced by whole TMEV virions, but not by Ad.βGal adenovirus, purified TMEV capsid proteins, or UV‐inactivated virus. Two recombinant inflammatory cytokines, IL‐1α and tumour necrosis factor‐α were also found to be potent inducers of IL‐6. The secreted IL‐6 was biologically active because it fully supported B9 hybridoma proliferation in a [3H]thymidine incorporation bioassay. The cerebrospinal fluid of infected mice contained IL‐6 during the acute encephalitis phase, peaking at days 2–4 post‐infection. Finally, this in vitro neuroinflammation model was fully inhibited, as demonstrated by ELISA and qPCR, by five selective oestrogen receptor modulators.