JCV agnoprotein-induced reduction in CXCL5/LIX secretion by oligodendrocytes is associated with activation of apoptotic signaling in neurons

Abstract

An indispensable role for oligodendrocytes in the protection of axon function and promotion of neuronal survival is strongly supported by the finding of progressive neuron/axon degeneration in human neurological diseases that affect oligodendrocytes. Imaging and pathological studies of the CNS have shown the presence of neuroaxonal injury in progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the CNS, resulting from destruction of oligodendrocytes upon productive replication of the pathogenic neurotropic polyomavirus JC. Here, we examined the extracellular factors involved in communication between oligodendrocytes and neurons. Culturing cortical neurons with conditioned medium (CM) from rat CG4 oligodendrocytic cells that express the JCV agnoprotein showed that CXCL5/LIX, which is a chemokine closely related to the human CXCL5/ENA78 and CXCL6/GCP-2 chemokines, is essential for neuronal cell survival. We found that in CM from agnoprotein-producing CG-4 cells level of CXC5/LIX is decreased compared to control cells. We also demonstrated that a reduced expression of CXCL5/LIX by CG4 GFP-Agno cells triggered a cascade of signaling events in cortical neurons. Analysis of mitogen-activated protein kinases (MAPK) and glycogen synthase kinase (GSK3) pathways showed that they are involved in mechanisms of neuronal apoptosis in response to the depletion of CXCL5/LIX signaling. These data suggest that agnoprotein-induced dysregulation of chemokine production by oligodendrocytes may contribute to neuronal/axonal injury in the pathogenesis of PML lesions.

Figure 1. Structural alterations in rat cortical neurons exposed to CM from CG4-Ol constitutively expressing JCV agnoprotein

A and B. Phase-contrast images of CG4 cells expressing GFP and GFP-Agno induced to differentiate into oligodendrocyte lineage. Scale bar, 20 µm. C. Quantification of levels of mRNAs for PLP and DM-20 by QPCR. Relative levels of mRNAs from CG4 cells, un-induced and induced to differentiate into oligodendrocytic lineage for 2 or 4 days, were expressed as the ratio to the number of the target gene copies relative to the number of reference gene glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) copies and was referred as arbitrary units. D. Immunoblot analysis demonstrating the presence of GFP-agnoprotein using an antibody against agnoprotein. The position of the GFP-Agnoprotein band (35 kDa) is indicated by an arrow. Grb2 serves as a loading control (low panel). E and F. Rat cortical neurons isolated from rat embryos (E17) were incubated with CM from CG4-Ol cells expressing GFP or GFP-Agno. After 16 hours of incubation, neurons were fixed and immunolabeled with antibody to class III β-tubulin (green fluorescence). Nuclei were counterstained with DAPI (blue). Scale bar, 20 µm.

Figure 2. Comparison of CM from agnoprotein-expressing CG4-Ol to CG4-Ol control for levels of the CXCL5/LIX chemokine

CM collected from agnopositive and agnonegative CG4 cells at the 4th day of differentiation into oligodendrocytic lineage was analyzed by cytokine antibody array (A) and quantified (C). Positions of the cytokines on the membrane is depicted on cytokine array map provided by the Manufacturer (B). CXCL5/LIX levels in the supernatants of CG4 cells, CG4 GFP and CG4 GFP-Agno cells at the 4^th day of differentiation as measured by ELISA ND - below detectable level. (D). Experiments were repeated three times.

Figure 3. Neuronal survival after treatment with conditioned medium from agnoprotein-expressing CG4 cells

A. Analysis of the effect of CXCL5/LIX on the survival of rat cortical neurons in the MTT assay. Rat cortical neurons were treated with CM and cell viability was evaluated after 16 hours. The order of samples is as follows: 1. neuronal culture medium; 2. CG4-Ol CM; 3. CG4-Ol GFP CM; 4. CG4-Ol GFP-Agno CM; 5. CG4-Ol GFP CM + neutralizing anti-LIX antibodies (3 µg/ml); 6. 1h pretreatment with rLIX (100 ng/ml) + CG4-Ol GFP-Agno CM; 7. CG4-Ol GFP CM + IgG1 (3µg/ml); 8. CG4-Ol GFP-Agno CM + BSA (100ng/ml). The relative cell viability (percent) for each sample was determined as the ratio of average absorbance for treated to that for untreated cells (sample 1). B. Effect of CXCL5/ LIX on the apoptosis of rat cortical neurons in the nexin assay. Flow cytometry with Annexin V-PE and Nexin 7-ADD. Rat cortical neurons were treated with CM and cell viability was evaluated after 16 hours and presented as percentage of double-positive cells. Sample order is the same as in panel A.

Figure 4. Effect of reduced level of CXCL5/LIX in CM on pro-survival signal transduction pathways in neurons

A. Western blot analysis of total lysates prepared from rat cortical neurons treated with: 1. neuronal CM; 2. CG4-Ol CM; 3. CG4-Ol GFP CM; 4. CG4-Ol GFP-Agno CM; 5. CG4-Ol GFP CM + neutralizing anti-LIX antibodies (3 µg/ml); 6. 1h pretreatment with rLIX (100 ng/ml) + CG4-Ol GFP-Agno CM; 7. CG4-Ol GFP CM + IgG1 (3 µg/ml); 8. CG4-Ol GFP-Agno CM + BSA (100 ng/ml). B. Effect of SB 202190 on p38 MAPK, activation of caspase 3 and apoptotic signaling in neurons treated with CM from agnoprotein-expressing cells.

Figure 5. Effect of agnoprotein on GSK3β activity

A. Treatment of neurons with CM from CG4 cells with silenced agnoprotein and LiCl, an inhibitor of GSK3. The level of expression of total GSK3β is also shown. B. Western blot for agnoprotein expression in the cells used in Panel A is shown together with α-Tubulin as a loading control.