Tumor necrosis factor alpha induces a metalloprotease-disintegrin, ADAM8 (CD 156): implications for neuron-glia interactions during neurodegeneration

Abstract

ADAM proteases, defined by extracellular disintegrin and metalloprotease domains, are involved in protein processing and cell-cell interactions. Using wobbler (WR) mutant mice, we investigated the role of ADAMs in neurodegeneration and reactive glia activation in the CNS. We found that ADAM8 (CD 156), a suspected leukocyte adhesion molecule, is expressed in the CNS and highly induced in affected CNS areas of WR mice, in brainstem and spinal cord. ADAM8 mRNA and protein are found at low levels throughout the normal mouse CNS, in neurons and oligodendrocytes. In the WR CNS regions in which neurodegeneration occurs, ADAM8 is induced in neurons, reactive astrocytes, and activated microglia. Similarly, the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) is upregulated and shows the same cellular distribution. In primary astrocytes from wild-type and WR mice, in primary cerebellar neurons, and in mouse motoneuron-like NSC19 cells, ADAM8 expression was induced up to 15-fold by mouse TNF-alpha, in a dose-dependent manner. In both cell types, ADAM8 was also induced by human TNF-alpha, indicating that TNF receptor type I (p55) is involved. Induction of ADAM8 mRNA was suppressed by treatment with an interferon-regulating factor 1 (IRF-1) antisense oligonucleotide. We conclude that IRF-1-mediated induction of ADAM8 by TNF-alpha is a signaling pathway relevant for neurodegenerative disorders with glia activation, proposing a role for ADAM8 in cell adhesion during neurodegeneration.

Fig. 1.

ADAM8 mRNA in the CNS of normal mice as shown byin situ hybridization (coronal and transversal sections; dorsal is up). A, Cerebral cortex, anterior part with hippocampus (lateral is left).B, Brainstem with part of cerebellum (lateral isright). C, Cortical layer (detail ofA). D, Granular layer of the cerebellum with Purkinje cells (detail of B). E, Gyrus dentatus (detail of A). F, Negative control (sense probe), detail of cerebellum, as in D.G, H, Cervical spinal cord, ventral part; comparison between wild type (G) and wobbler (H). Dashed lines indicate borders between gray (top) and white (bottom) substance. Scale bars: A,B, 500 μm; (in C) C,E, 100 μm; (in D) D,F, 100 μm; (in G) G,H, 50 μm.

Fig. 2.

ADAM8 mRNA levels in the CNS and testis of WT and WR mice. A, RT-PCR analysis of ADAM8 mRNA in different brain regions of 40-d-old WR mice and their normal WT littermates. After reverse transcription, PCR was performed in 25 cycles as a duplex PCR with ADAM8 and L7 primers, the latter serving as an amplification standard. Chain lengths of marker bands are given on theleft. −RT, Negative control, no reverse transcriptase. B, Quantitation of ADAM8 mRNA in WT and WR mice, given as ratio WR/WT, derived by radioactive hybridization of amplificates normalized to the L7 reference signal. C, Immunodetection of ADAM8 in Western blots of WT and WR brainstem, spinal cord, and lung extracts. Membrane filters were incubated with polyclonal anti-ADAM8, and the bands were visualized by chemiluminescence. As loading control, a monoclonal β-actin-specific antibody was used to stain a parallel blot. The molecular mass marker positions are indicated on the left. Thepro-A8 on the right marks the position of the pro-ADAM8 at ∼100 kDa, and the filled triangle indicates the position of the processed form of ADAM8. The band indicated by an asteriskcorresponds to unspecific binding of the ADAM8 antibody.

Fig. 3.

Localization of ADAM8 in glia cells in the spinal cord of 40-d-old WT (A–C, G–I) and WR (D–F, J–L) mice, as demonstrated by immunofluorescence. A–C, WT, anti-ADAM8 (green, A), anti-CD45 (red, B), or both (yellow, C); insetsin A–C, staining of cells in the white matter with anti-ADAM8 (in A), oligodendrocyte-specific antigen CNPase (in B) and merged (in C).D–F, WR, ADAM8 (green,D), CD45 (red, E), or both (yellow, F). Note that all microglia cells express ADAM8. G–I, Spinal cord of WT mouse, stained with anti-ADAM8 (G), GFAP (H), or with both (I). J–L, WR, stained for ADAM8 (J), GFAP (K), or both (L). Note that, in J–L, astrocytes appear more compacted than usual because of very short exposure times. GFAP staining in astrocytic processes is not visible. Scale bar (in A): A–L, 50 μm.

Fig. 4.

Cytokine mRNA expression in 40-d-old wild-type and wobbler mice. The indicated cytokines were detected in an RT-PCR analysis with 25 cycles of PCR. Control, no reverse transcriptase (−RT), except for IL-1β and IFN-γ for which mRNA from mouse spleen was used as positive control to verify PCR conditions.

Fig. 5.

Double immunolabeling of TNF-α and glia-specific markers CD45 (A, B) or GFAP (C, D) in the ventral spinal cord of 40-d-old WT (A, C) or WR (B, D) mice. Arrowheads inD indicate a degenerating motoneuron surrounded by astrocytes in the ventral horn of the spinal cord. Scale bar (inA): A–D, 20 μm.

Fig. 6.

ADAM8 transcriptional activation by mouse TNF-α in granular cells and NSC19 cells. A, Granular cells (GC) and NSC19 cells were incubated with indicated units of mouse TNF-α for 24 hr; the relative amounts of ADAM8, IRF-1, and L7 (reference) mRNA levels were determined by RT-PCR and visualized by ethidium bromide staining. B, ADAM8 immunofluorescence of NSC19 cells treated with indicated units of TNF-α. Note the staining toward the cell membrane. Even in the presence of 3000 U of TNF-α, NSC19 cells did not show signs of cell damage. Scale bar, 50 μm.

Fig. 7.

Upregulation of ADAM8 mRNA by exogenous TNF-α. Quantitation of ADAM8 mRNA levels in primary WT and WR astrocytes (A), granular cells (B), and NSC19 cells (C), as determined by RT-PCR.A, Induction of ADAM8 mRNA by TNF-α in primary astrocyte cultures from WT and WR mice. In all cases, values were normalized to those of untreated WT cells, 1.0 by definition.B, ADAM8 induction by TNF-α in granular cells.C, Dose response obtained with recombinant mouse (m) and human (h) TNF-α in NSC19 cells. D, ADAM8 mRNA induction by TNF-α in NSC19 cells after preincubation with cycloheximide (CHX); untreated NSC cells, 1.0 by definition. E, Induction of ADAM8 mRNA by TNF-α in the presence of either IRF-1 antisense (aIRF1) or sense (sIRF1) oligonucleotide. Values were normalized to untreated NSC19 cells with sIRF1. Data were given as mean values of at least three independent experiments. When the same experiment was performed on L929 cells, there was no stimulation of ADAM8 mRNA by TNF-α at the concentrations used above (data not shown).