Interferon gamma induces retrograde dendritic retraction and inhibits synapse formation

Abstract

The expression of interferon gamma (IFNgamma) increases after neural injury, and it is sustained in chronic inflammatory conditions such as multiple sclerosis and infection with human immunodeficiency virus. To understand how exposure to this proinflammatory cytokine might affect neural function, we examined its effects on cultures of neurons derived from the central and peripheral nervous systems. IFNgamma inhibits initial dendritic outgrowth in cultures of embryonic rat sympathetic and hippocampal neurons, and this inhibitory effect on process growth is associated with a decrease in the rate of synapse formation. In addition, in older cultures of sympathetic neurons, IFNgamma also selectively induces retraction of existing dendrites, ultimately leading to an 88% decrease in the size of the arbor. Dendritic retraction induced by IFNgamma represents a specific cellular response because it occurs without affecting axonal outgrowth or cell survival, and it is not observed with tumor necrosis factor alpha or other inflammatory cytokines. IFNgamma-induced dendritic retraction is associated with the phosphorylation and nuclear translocation of signal transducer and activator of transcription 1 (STAT1), and expression of a dominant-negative STAT1 construct attenuates the inhibitory effect of IFNgamma. Moreover, retrograde dendritic retraction is observed when distal axons are selectively exposed to IFNgamma. These data imply that IFNgamma-mediated STAT1 activation induces both dendritic atrophy and synaptic loss and that this occurs both at the sites of IFNgamma release and at remote loci. Regressive actions of IFNgamma on dendrites may contribute to the neuropathology of inflammatory diseases.

Fig. 1.

Effect of BMP-7 and IFNγ on the morphology of cultured sympathetic neurons. Shown are phase-contrast (A, C, E, G) and fluorescence (B, D, F, H) micrographs of neurons immunostained with a monoclonal antibody (mAb) to MAP-2 after 5 d of treatment. Neurons grown in control medium lacked dendrites (A, B). Exposure to BMP-7 (5 ng/ml) caused the neurons to form multiple dendrites (C, D). IFNγ (10 ng/ml) alone had no effect on dendritic outgrowth (E, F), but it inhibited the growth of these processes in cultures that were exposed to BMP-7 (G, H). Scale bar, 30 μm.

Fig. 2.

IFNγ inhibits BMP-7-induced dendritic outgrowth. Sympathetic neurons were treated with BMP-7 (5 ng/ml), IFNγ (10 ng/ml), or BMP-7 and IFNγ. On days 8 and 15, cellular morphology (n ≥ 60 cells/condition) was visualized by immunostaining with mAb to MAP-2. A, Number of dendrites per cell. B, Total dendritic length. C, Number of MAP-2-positive cells per culture. *p < 0.05 versus BMP-7.

Fig. 3.

Dose-dependent inhibition of dendritic outgrowth by IFNγ. The effects of varying concentrations of IFNγ were assessed in cultures treated with either a maximally effective dose of BMP-7 (50 ng/ml; open circles) or one close to the ED₅₀ (5 ng/ml; open triangles). After 5 d of cotreatment with BMP-7 and IFNγ cultures were immunostained with mAb to MAP-2.

Fig. 4.

IFNγ inhibits dendritic growth and synaptogenesis in hippocampal neurons. Hippocampal neurons were grown in the absence or presence of IFNγ (30 ng/ml) for 5 d.A, Fluorescence micrograph of neuron grown in control medium. Dendritic morphology (n > 25 cells/condition) and presynaptic specializations were analyzed by double-labeled immunostaining with antibodies to MAP-2 (red) and SV-2 (green). Cells were examined with a Bio-Rad confocal microscope, using 1 μm optical sections. SV-2-positive puncta that are associated with dendrites have been shown previously to represent sites of synaptic contact (Fletcher et al., 1991). Scale bar, 10 μm. B, Total dendritic length. C, Synapses per neuron. *p < 0.05 versus control.

Fig. 5.

IFNγ induces dendritic retraction. Sympathetic neurons were treated initially with 5 ng/ml BMP-7 (A, B, D) or 20 ng/ml (C) for 5 d. Subsequently, they were divided into two groups. One was treated continuously with BMP-7, and the other was treated with both IFNγ (10 ng/ml) and BMP7 for an additional 6 d. Dendritic morphology was assessed by immunostaining with mAb to MAP-2 (n ≥ 60 cells/condition). A, C, Number of dendrites per cell.B, Total dendritic length. D, Number of MAP-2-positive cells per culture. *p < 0.05 versus BMP-7 on day 5.

Fig. 6.

Lack of effect of IFNγ on axonal growth.A, Explants of superior cervical ganglia were maintained for 2 d in the presence or absence of IFNγ (10 ng/ml). Subsequently, the ganglia were dissociated, and cells were plated onto coverslips coated with laminin (2 μg/ml). Cells then were exposed continuously to control medium or IFNγ for 12 hr. Axons were immunostained with mAb that recognizes phosphorylated forms of H and M neurofilament subunits. Cellular morphology was analyzed using Image software (n ≥ 60 cells/condition).B, Cultures were treated with BMP-7 (5 ng/ml) for 8 d; during the last 3 d of treatment some were also exposed to IFNγ (10 ng/ml). Expression of cytoskeletal proteins was detected by Western blotting with mAb to tau and a polyclonal antibody to actin. Equal amounts of protein were loaded into each lane.

Fig. 7.

TNFα does not potentiate the effect of IFNγ on dendritic retraction. Sympathetic neurons were exposed to BMP-7 (5 ng/ml) for 9 d. Beginning on day 5, some of them were treated additionally with IFNγ (10 ng/ml), TNFα (10 ng/ml), or the combination of both of the agents. On day 9, dendritic morphology was assessed by immunostaining with mAb to MAP-2. *p < 0.05 versus BMP-7 on day 5.

Fig. 8.

IFNγ induces phosphorylation and nuclear translocation of STAT1. Sympathetic neurons were treated with BMP-7 (5 ng/ml) for 5 d. Subsequently, IFNγ (10 ng/ml) was added for 1 hr to the medium of some cultures (C, D), whereas others were treated with BMP-7 alone (A, B). The cellular location of STAT1 was detected by immunostaining with an antibody that reacts with a phosphorylated form of STAT1 (Tyr 701); the cells were examined with a Bio-Rad confocal microscope, using 1 μm optical sections. Shown are phase-contrast (A, C) and fluorescence (B, D) micrographs.

Fig. 9.

Dominant-negative STAT1 overcomes the inhibitory effect of IFNγ on dendrites. Sympathetic neurons were cotransfected with plasmids containing the genes for EGFP and (A) wild-type (STAT1 WT) or mutant STAT1 (STAT1 F) or (B) wild-type or mutant STAT3 or mutant STAT1 by Lipofectamine reagent. After 48 hr the cells were treated with BMP-7 (10 ng/ml), IFNγ (20 ng/ml), or BMP-7 and IFNγ. On day 6, cellular morphology was visualized by immunostaining with mAb to MAP-2. Transfected cells were assessed by the expression of EGFP. *p < 0.05 versus BMP-7 and IFNγ in the culture transfected with the control vector.

Fig. 10.

Dendritic retraction in response to retrograde signaling by IFNγ. A, Schematic representation of the three-compartment culture of sympathetic neurons. Dissociated neurons were plated in the Center compartment, and axons extended into the Left and Rightcompartments. B, Negative control cultures were maintained in control medium (C₂) in the absence of BMP-7 or IFNγ; positive control and experimental cultures were exposed to BMP-7 (10 ng/ml) added to C₂ in the Center compartment for 7 d. Three days after the addition of BMP-7, IFNγ (10 or 50 ng/ml) was added to the C₂ in the Right compartment of experimental cultures (indicated by the arrow in C). Then 4 d later, dendritic growth was quantified in cultures immunostained with mAb to MAP-2 (n ≥ 60 cells/condition). Addition of IFNγ to the distal axons and axon terminals caused a dose-dependent reduction in total dendritic length (C). ∗p < 0.05 versus BMP-7 on day 7.