Heat shock alters the expression of schizophrenia and autism candidate genes in an induced pluripotent stem cell model of the human telencephalon

Abstract

Schizophrenia (SZ) and autism spectrum disorders (ASD) are highly heritable neuropsychiatric disorders, although environmental factors, such as maternal immune activation (MIA), play a role as well. Cytokines mediate the effects of MIA on neurogenesis and behavior in animal models. However, MIA stimulators can also induce a febrile reaction, which could have independent effects on neurogenesis through heat shock (HS)-regulated cellular stress pathways. However, this has not been well-studied. To help understand the role of fever in MIA, we used a recently described model of human brain development in which induced pluripotent stem cells (iPSCs) differentiate into 3-dimensional neuronal aggregates that resemble a first trimester telencephalon. RNA-seq was carried out on aggregates that were heat shocked at 39°C for 24 hours, along with their control partners maintained at 37°C. 186 genes showed significant differences in expression following HS (p<0.05), including known HS-inducible genes, as expected, as well as those coding for NGFR and a number of SZ and ASD candidates, including SMARCA2, DPP10, ARNT2, AHI1 and ZNF804A. The degree to which the expression of these genes decrease or increase during HS is similar to that found in copy loss and copy gain copy number variants (CNVs), although the effects of HS are likely to be transient. The dramatic effect on the expression of some SZ and ASD genes places HS, and perhaps other cellular stressors, into a common conceptual framework with disease-causing genetic variants. The findings also suggest that some candidate genes that are assumed to have a relatively limited impact on SZ and ASD pathogenesis based on a small number of positive genetic findings, such as SMARCA2 and ARNT2, may in fact have a much more substantial role in these disorders - as targets of common environmental stressors.

Figure 1. Neuronal aggregates, day 50.

Top panels: SOX2+ structures containing radial glial cells with surrounding field of neurons (MAP+, TUJ1+ cells). Bottom left panel: Neurons contain layers of predominantly GABAergic and glutamatergic neurons (GABA+ and VGLUT2+, respectively). Bottom right panel: The neurons express pre and post synaptic proteins (synaptophysin/SYN, gephyrin/GEPH, respectively).

Figure 2. Heat map showing relative expression of 186 genes that exhibited significant change in gene expression following heat shock at a nominally significant level (p<0.05: 105 increased in expression; 81 decreased).

Figure 3. qPCR validation for C3 and HS3.

Between 4–8 aggregates were analyzed individually in triplicate using the 2^−ΔΔCt relative expression method with β2-microglobulin (β2M) as a control gene. Each sample was normalized against a common control RNA. Mean values +/− standard deviation are shown and a student's t-test was performed. A single asterisk indicates a p<0.05; two asterisks indicate a p<0.01. The actual p-values are ZNF804A (0.0002), SMARCA2 (0.0001), HSP90AB1 (0.03), NGFR (0.04), HIST1H2BD (0.02), KAT2A (0.05), ARNT2 (0.002), AHI1 (0.006).

Figure 4. Ingenuity Pathway Analysis showing top diseases and biological functions of differentially expressed genes.

All genes that were significant at a p<0.05 level were subjected to pathway analysis (Ingenuity Pathway Analysis; IPA). The top diseases and biological functions are shown.