Interferon-Alpha Reduces Human Hippocampal Neurogenesis and Increases Apoptosis via Activation of Distinct STAT1-Dependent Mechanisms

Abstract

Background: In humans, interferon-α treatment for chronic viral hepatitis is a well-recognized clinical model for inflammation-induced depression, but the molecular mechanisms underlying these effects are not clear. Following peripheral administration in rodents, interferon-α induces signal transducer and activator of transcription-1 (STAT1) within the hippocampus and disrupts hippocampal neurogenesis.

Methods: We used the human hippocampal progenitor cell line HPC0A07/03C to evaluate the effects of 2 concentrations of interferon-α, similar to those observed in human serum during its therapeutic use (500 pg/mL and 5000 pg/mL), on neurogenesis and apoptosis.

Results: Both concentrations of interferon-α decreased hippocampal neurogenesis, with the high concentration also increasing apoptosis. Moreover, interferon-α increased the expression of interferon-stimulated gene 15 (ISG15), ubiquitin-specific peptidase 18 (USP18), and interleukin-6 (IL-6) via activation of STAT1. Like interferon-α, co-treatment with a combination of ISG15, USP18, and IL-6 was able to reduce neurogenesis and enhance apoptosis via further downstream activation of STAT1. Further experiments showed that ISG15 and USP18 mediated the interferon-α-induced reduction in neurogenesis (potentially through upregulation of the ISGylation-related proteins UBA7, UBE2L6, and HERC5), while IL-6 mediated the interferon-α-induced increase in apoptosis (potentially through downregulation of aquaporin 4). Using transcriptomic analyses, we showed that interferon-α regulated pathways involved in oxidative stress and immune response (e.g., Nuclear Factor (erythroid-derived 2)-like 2 [Nrf2] and interferon regulatory factor [IRF] signaling pathway), neuronal formation (e.g., CAMP response element-binding protein [CREB] signaling), and cell death regulation (e.g., tumor protein(p)53 signaling).

Conclusions: We identify novel molecular mechanisms mediating the effects of interferon-α on the human hippocampus potentially involved in inflammation-induced neuropsychiatric symptoms.

Keywords: apoptosis; depression; inflammation; interferon-alpha; neurogenesis.

Figure 1.

Interferon (IFN)-α reduces differentiation, increases apoptosis, and upregulates signal transducer and activator of transcription-1 (STAT1), interferon-stimulated gene 15 (ISG15), and ubiquitin-specific peptidase 18 (USP18) gene expression. IFN-α (500 and 5000 pg/mL) decreased doublecortin (DCX)+ (a) and microtubule-associated protein 2 (MAP2)+ cells (b). IFN-α 5000 pg/mL increased both caspase 3 (CC3)+ cells (c) and CC3+/MAP2+ cells (d). IFN-α (500 and 5000 pg/mL) increased STAT1 (e), ISG15 (f), and USP18 (g) gene expression. Three independent experiments were conducted on independent cultures (n=3). Data are shown as mean±SEM. 1-way ANOVA, Newman–Keuls’ posthoc test ^*P<.05, ^**P<.01, ^***P<.001, ^****P<.0001, compared with vehicle treatment or as indicated.

Figure 2.

Cotreatment with interferon-stimulated gene 15 (ISG15), ubiquitin-specific peptidase 18 (USP18), and interleukin (IL)-6 mimics the effect of interferon (IFN)-α on neurogenesis and apoptosis. ISG15 and all proteins reduced doublecortin (DCX)+ cells (a). ISG15, USP18, and all proteins reduced microtubule-associated protein 2 (MAP2)+ cells (b). USP18 also decreased caspase 3 (CC3)+ cells, whereas IL-6 and all proteins significantly increased both CC3+ and CC3/MAP2+ cells (c-d). Three independent experiments were conducted on independent cultures (n=3). Data are shown as mean±SEM. 1-way ANOVA, Newman–Keuls’ posthoc test ^*P<.05, ^**P<.01, ^***P<.001, compared with vehicle treatment or as indicated.

Figure 3.

Interleukin (IL)-6A prevents the effects of interferon (IFN)-α on apoptosis. Treatment with IL-6A prevented the increase in caspase 3 (CC3)+ and CC3/microtubule-associated protein 2 (MAP2)+ cells detected upon IFN-α 5000 pg/mL (c-d). Three independent experiments were conducted on independent cultures (n=3). Data are shown as mean±SEM. 1-way ANOVA, Newman–Keuls’ posthoc test ^*P<.05, ^**P<.01, ^****P<.0001, compared with vehicle treatment or as indicated.

Figure 4.

Treatment with a signal transducer and activator of transcription-1 (STAT1) inhibitor reverts the effects of interferon (IFN)-α on neurogenesis and apoptosis by preventing the upregulation of interferon-stimulated gene 15 (ISG15), ubiquitin-specific peptidase 18 (USP18), and interleukin (IL)-6. Fludarabine prevented the decrease in doublecortin (DCX)+ and microtubule-associated protein 2 (MAP2)+ cells caused by IFN-α (500 and 5000 pg/mL) (a-b), as well as the decrease in caspase 3 (CC3)+ and CC3+/MAP2+ cells detected upon IFN-α 5000 pg/mL (c-d). Fludarabine prevented the increase in ISG15 (e) and USP18 (f) genes and IL-6 protein expression (g) observed upon IFN-α (500 and 5000 pg/mL). Three independent experiments were conducted on independent cultures (n=3). Data are shown as mean±SEM. 1-way ANOVA, Newman–Keuls’ posthoc test ^**P<.01, ^***P<.001, ^****P<.0001, compared with vehicle treatment or as indicated.

Figure 5.

Treatment with a signal transducer and activator of transcription-1 (STAT1) inhibitor prevents the effect of interferon-stimulated gene 15 (ISG15) on neurogenesis and of interleukin (IL)-6 on apoptosis. Fludarabine prevented the decrease in microtubule-associated protein 2 (MAP2)+ cells caused by ISG15 and all proteins (b), as well as the increase in caspase 3 (CC3)+ and CC3+/MAP2+ cells detected upon treatment with IL-6 (c) and all proteins (d). Three independent experiments were conducted on independent cultures (n=3). Data are shown as mean±SEM. One-way ANOVA, Newman–Keuls’ posthoc test ^*P<.05, ^**P<.01, ^****P<.0001, compared with vehicle treatment or as indicated.

Figure 6.

Treatment with interferon (IFN)-α, interferon-stimulated gene 15 (ISG15), and all proteins increase UBA7, UBE2L6, and HERC5 gene expression via activation of signal transducer and activator of transcription-1 (STAT1) protein. IFN-α (500 and 5000 pg/mL) increased UBA7 (a), UBE2L6 (b), and HERC5 (c) gene expression, whereas cotreatment with fludarabine prevented such upregulation (a-c). ISG15 and all proteins induced UBA7 (d), UBE2L6 (e), and HERC5 (f). Cotreatment with either ISG15 or all proteins and fludarabine prevented such increase (d-f). Three independent experiments were conducted on independent cultures (n=3). Data are shown as mean±SEM. One-way ANOVA, Newman–Keuls’ posthoc test ^**P<.01, ^***P<.001, ^****P<.0001, compared with vehicle treatment or as indicated.

Figure 7.

Treatment with interferon (IFN)-α, interleukin (IL)-6, and all proteins decrease AQP4 gene expression via activation of signal transducer and activator of transcription-1 (STAT1) protein. IFN-α 5000 pg/mL downregulated AQP4 gene expression, whereas cotreatment with IFN-α 5000 pg/mL and fludarabine prevented such downregulation (a). IL-6A blocked the reduction in AQP4 seen upon IFN-α 5000 pg/mL (b). Treatment with IL-6 and all proteins decreased AQP4 gene expression, whereas cotreatment of IL-6 or all proteins with fludarabine prevented the reduction in AQP4 gene expression (c). Three independent experiments were conducted on independent cultures (n=3). Data are shown as mean±SEM. 1-way ANOVA, Newman–Keuls’ posthoc test ^*P<.05, ^***P<.001, compared with vehicle treatment or as indicated.