Interferon-γ exposure of human iPSC-derived neurons alters major histocompatibility complex I and synapsin protein expression

Abstract

Human epidemiological data links maternal immune activation (MIA) during gestation with increased risk for psychiatric disorders with a putative neurodevelopmental origin, including schizophrenia and autism. Animal models of MIA provide evidence for this association and suggest that inflammatory cytokines represent one critical link between maternal infection and any potential impact on offspring brain and behavior development. However, to what extent specific cytokines are necessary and sufficient for these effects remains unclear. It is also unclear how specific cytokines may impact the development of specific cell types. Using a human cellular model, we recently demonstrated that acute exposure to interferon-γ (IFNγ) recapitulates molecular and cellular phenotypes associated with neurodevelopmental disorders. Here, we extend this work to test whether IFNγ can impact the development of immature glutamatergic neurons using an induced neuronal cellular system. We find that acute exposure to IFNγ activates a signal transducer and activator of transcription 1 (STAT1)-pathway in immature neurons, and results in significantly increased major histocompatibility complex I (MHCI) expression at the mRNA and protein level. Furthermore, acute IFNγ exposure decreased synapsin I/II protein in neurons but did not affect the expression of synaptic genes. Interestingly, complement component 4A (C4A) gene expression was significantly increased following acute IFNγ exposure. This study builds on our previous work by showing that IFNγ-mediated disruption of relevant synaptic proteins can occur at early stages of neuronal development, potentially contributing to neurodevelopmental disorder phenotypes.

Keywords: C4A; MHCI; iPSC; inflammation; interferon-γ; maternal immune activation; schizophrenia; synapsin.

Figure 1

SynGO synaptic gene ontology analyses of down-regulated DEG in D30 neurons acutely exposed to IFNγ. The sunburst plots represent synaptic annotated ontologies for CC (B) and BP (A) terms. The key color scale indicates -log10 FDR adjusted p-values. (A) Significantly enriched CC ontologies include synapse (red) presynaptic (magenta) clusters. (B) Annotated BP terms include synaptic (magenta), presynaptic (red) and signaling (blue) terms. (C,D) Plots of synaptic GO output showing the 3 CC (C) and 5 BC (D) significantly (FDR-adjusted) enriched terms for D30 IFNγ exposed neurons. The bar length indicates the number of genes, the order of each bar and numbers adjacent to each are the FDR adj. P-value. Analysis of data previously published inWarre-Cornish et al. (28).

Figure 2

Exposure of neurons to IFNγ results in increased MHCI. (A) Schematic of Opti-OX neural induction and IFNγ exposure at day 3 for 24 h. (B) Bar graphs of relative expression of selected IFNγ signaling-related genes, showing increased HLA-B and C4A expression in IFNγ-exposed neurons. The bars indicate the log(2^−ΔΔCt), which indicates the expression relative to housekeepers and normalized to the housekeepers of the control samples (See methods for details.) Expression in Day 4 NGN2-iNs exposed at day 3. N = 3. ^**** indicates P < 0.0001, ^*** indicates P = 0.000291 (unpaired t-test). (C) Western blot for p-STAT1, p-ERK1/2, ERK1/2, and GAPDH protein in Day 3 NGN2-iNs exposed to IFNγ (+) or vehicle (-) at day 3 for 15 min. Three biological replicates with different passage numbers are shown (D) Quantification of p-STAT1 and p-ERK1/2 blots shown in (C). The different data point colors represent biological replicates with different passage numbers. (E) ICC for MHCI. The top row shows control cells, the bottom row shows cells exposed to IFNγ at day 3 for 24hrs. The MHCI Fire LUT pseudo color shows higher intensity with warmer colors and lower intensity with cooler colors. The gray values corresponding to the colors are shown on the calibration bar on the right. (F–I) Scatter plots of MHCI intensity in control and IFNγ-exposed neurons. The horizontal bars represent the mean, the error bars represent the standard deviation. Each point in the intensity plots represents the mean intensity of one field of view i.e., image, of the respective object. The different data point colors represent biological replicates with different passage numbers. The IFNγ and control were compared using an unpaired T-test, where N = 3 and ****indicates P < 0.0001, ***indicates 0.001 <P>0.001, **indicates 0.001 <P>0.01.

Figure 3

Exposure of neurons to IFNγ results in decreased SYNI/II intensity in the cell bodies of some cells. (A) Characteristic localization of synapsin I/II in the cell body. Right image shows a detailed view of the highlighted region. The arrowhead indicates apparent synapsin vesicles within the cytoplasm. (B) IHC for synapsin I/II. The top row shows control cells, the bottom row shows cells exposed to IFNγ at day 3 for 24hrs. The SYN1 Fire LUT pseudo color shows higher intensity with warmer colors and lower intensity with cooler colors. Detailed view shown on right. The gray values corresponding to the colors are shown on the calibration bar on the right. (C–F) Scatter plots of synapsin I/II intensity in control and IFNγ-exposed neurons. The horizontal bars represent the mean, the error bars represent the standard deviation. Each point in the intensity plots represents the mean intensity of one field of view i.e., image, of the respective object. The different data point colors represent biological replicates with different passage numbers. The IFNγ and control were compared using an unpaired t-test, where N = 3 and **indicates d0.001 <P>0.01, *indicates 0.01 <P>0.05, and ns indicates P ≥ 0.05 (not significant).

Figure 4

Bar graphs of relative expression of selected synaptic genes and C4A, showing increased C4A expression in IFNγ-exposed neurons. The bars indicate the log(2^−ΔΔCt), which indicates the expression relative to housekeepers and normalized to the housekeepers of the control samples (See methods for details.) (A) Expression in Day 4 NGN2-iNs exposed at day 3. N = 3. ****indicates P < 0.0001, ***indicates P = 0.000291 (unpaired t-test). (B) Expression in day 27 conventionally differentiated 127_CTM iPSC line NPCs exposed at day 26. ****indicates P < 0.0001, **indicates P = 0.002947(unpaired t-test). The bar represents the mean, the error bars represent the standard deviation. Points of the same color represent the same biological replicate.