Interferon standard therapy alters neuronal physiology in HCV patients

Background: Interferon-alpha (IFN) based therapy is the current standard therapy used for the treatment of chronic hepatitis C. Interestingly, IFN-mediated depressive side effects are much more frequent in HCV patients compared to HBV patients or individuals with malignant melanoma that receive this therapy. The physiological mechanisms underlying this hyper-responsiveness of HCV patients are still unknown. Therefore, we have analyzed the alterations prompted by IFN and poly(I:C) (which would mimic the presence of a HCV replicative intermediates) on crucial neuronal physiologic parameters related to the development of the depressive symptoms in the murine hippocampus (HP) and prefrontal cortex (PC), two brain areas closely related to depression development.

Methods: HP and PC neurons were isolated from 17-day-old embryos of C57 BL/6 mice, cultured in neurobasal medium and stimulated with murine IFN (mIFN), poly(I:C) and costimulated at 4 days in vitro. For the plasticity analysis, the neurons (2×104cell/well) were stimulated during 24h and incubated with anti-MAP2 (1:500) and anti-Tau1 (5µg/ml) O/N. For apoptosis analysis, neurons (3×104cell/well) were stimulated during 72h and incubated with anti-caspase3 (5µg/ml) O/N. Alexa-labelled secondary antibodies were used at 1:1000 for 1h. Nuclear staining was carried out using Hoechst (1:2000, 5min). Fluorescence micrographs were processed with Image J. The proliferation analysis was performed with the Quick Cell Proliferation Assay kit according to the manufacturer's instructions in neurons (2×104cell/well) treated for 72h.

Results: The proliferation assay revealed that neurogenesis is differenzially affected in both studied areas. Thus, while in PC neurogenesis only decreased significantly with the treatment of poly(I:C) at the highest concentration, in HP neurons it decreased with high concentrations of mIFN and poly(I:C), and with several of the costimulatory treatments. Concomitantly, apoptosis levels were increased in both studies tissues with all treatments. Plasticity features, assessed by the length of dendrites and axons, were altered in all cases, showing that treatments reduce the development of neurites in a similar way in both studied tissues. Concerning the number of dendrites/neuron, it was solely affected by the treatment with mIFN but not poly(I:C).

Conclusion: Exposition of murine HP and PC embryonary neurons to mIFN and poly(I:C) treatment affects not only the balance neurogenesis/apoptosis, but also plasticity features which is crucial for the right development and further maintenance of the neural network. This model may explain some physiopathological mechanisms underlying IFN-α therapy-associated depression.