The viral theory of schizophrenia revisited: Abnormal placental gene expression and structural changes with lack of evidence for H1N1 viral presence in placentae of infected mice or brains of exposed offspring

Abstract

Researchers have long noted an excess of patients with schizophrenia were born during the months of January and March. This winter birth effect has been hypothesized to result either from various causes such as vitamin D deficiency (McGrath, 1999, McGrath et al., 2010), or from maternal infection during pregnancy. Infection with a number of viruses during pregnancy including influenza, and rubella are known to increase the risk of schizophrenia in the offspring (Brown, 2006). Animal models using influenza virus or Poly I:C, a viral mimic, have been able to replicate many of the brain morphological, genetic, and behavioral deficits of schizophrenia (Meyer et al., 2006, Meyer et al., 2008a, Meyer et al., 2009, Bitanihirwe et al., 2010, Meyer and Feldon, 2010, Short et al., 2010). Using a murine model of prenatal viral infection, our laboratory has shown that viral infection on embryonic days 9, 16, and 18 leads to abnormal expression of brain genes and brain structural abnormalities in the exposed offspring (Fatemi et al., 2005, Fatemi et al., 2008a, Fatemi et al., 2008b, Fatemi et al., 2009a, Fatemi et al., 2009b). The purpose of the current study was to examine gene expression and morphological changes in the placenta, hippocampus, and prefrontal cortex as a result of viral infection on embryonic day 7 of pregnancy. Pregnant mice were either infected with influenza virus [A/WSN/33 strain (H1N1)] or sham-infected with vehicle solution. At E16, placentas were harvested and prepared for either microarray analysis or for light microscopy. We observed significant, upregulation of 77 genes and significant downregulation of 93 genes in placentas. In brains of exposed offspring following E7 infection, there were changes in gene expression in prefrontal cortex (6 upregulated and 24 downregulated at P0; 5 upregulated and 14 downregulated at P56) and hippocampus (4 upregulated and 6 downregulated at P0; 6 upregulated and 13 downregulated at P56). QRT-PCR verified the direction and magnitude of change for a number of genes associated with hypoxia, inflammation, schizophrenia, and autism. Placentas from infected mice showed a number of morphological abnormalities including presence of thrombi and increased presence of immune cells. Additionally, we searched for presence of H1N1 viral-specific genes for M1/M2, NA, and NS1 in placentas of infected mice and brains of exposed offspring and found none. Our results demonstrate that prenatal viral infection disrupts structure and gene expression of the placenta, hippocampus, and prefrontal cortex potentially explaining deleterious effects in the exposed offspring without evidence for presence of viral RNAs in the target tissues.

This article is part of a Special Issue entitled ‘Schizophrenia’.

Introduction

Increased rates of schizophrenia have been associated with higher latitude and colder climate (Kinney et al., 2009). Different hypotheses have been proposed for this increase including vitamin D deficiency (McGrath, 1999, McGrath et al., 2010) and prenatal viral infection (Lewis, 2001, Fatemi, 2005, Brown, 2006). First proposed by McGrath (1999), prenatal vitamin D deficiency as a risk factor for schizophrenia could potentially explain several epidemiological findings including season of birth, latitude, differences in rates of schizophrenia between urban vs. rural populations, and the increased risk in 2nd generation migrants to northern countries (McGrath, 1999, Kinney et al., 2009, McGrath et al., 2010). Animal models of developmental vitamin D (DVD) deficiency have shown changes in brain development (Eyles et al., 2009), synaptic plasticity (Grecksch et al., 2009), and behavior (Grecksch et al., 2009, Fernandes de Abreu et al., 2010, Kesby et al., 2010) consistent with schizophrenia.

Over the past thirty years, numerous reports have established that viral infections during pregnancy increase the risk for the development of schizophrenia in the offspring (Lewis, 2001, Fatemi, 2005, Brown, 2006). An excess of schizophrenic patients are born during late winter and spring indicating the potential of influenza infections for these cases. Indeed, studies suggest that 5–15% excess schizophrenic births in the Northern Hemisphere occur during the months of January and March (Hare et al., 1972; Machon et al., 1983, Susser et al., 1999, Boyd et al., 1986, Pallast et al., 1994). Currently, there is debate whether direct infection of the offspring leads to changes ultimately resulting in schizophrenia or whether production of maternal cytokines in response to infection is responsible (Aronsson et al., 2001, Aronsson et al., 2002, Shi et al., 2009).

The current prevailing opinion is in favor of maternal cytokines as the causative agent of pathology in the developing offspring. A series of studies in different animal models for schizophrenia (Carpenter and Koenig, 2008) shows that maternal infection with human influenza or poly I:C can cause abnormalities in behavioral indices such as PPI and latent inhibition (LI) (Shi et al., 2003, Smith et al., 2007, Zuckerman and Weiner, 2003), both of which are similarly disrupted in subjects with schizophrenia. Recently, it has been demonstrated that injection of pregnant mice with IL-6, but not IFNγ, at E12.5 resulted in deficits in PPI and LI in the adult offspring but not juveniles (Smith et al., 2007), similar to what is observed in schizophrenia. Smith et al. (2007) also found that poly I:C-induced behavioral deficits in PPI, LI, and open field tests could be reversed if the poly I:C was co-administered with anti-IL-6 antibodies (Smith et al., 2007). Using IL-6 knockout mice, injection of poly I:C had no effect on PPI, open field test, and social interaction test (Smith et al., 2007). Inhibition of IL-6-mediated JAK2/STAT3 signaling using luteolin and its structural analog diosmin, has been shown to reduce behavioral deficits in social interaction in the offspring of mice born to dams injected with IL-6 on E12.5 of pregnancy (Parker-Athill et al., 2009). Interestingly, overexpression of anti-inflammatory cytokine IL-10 modulates poly I:C-induced behaviors such as PPI and LI (Meyer et al., 2008b). Taken together, these results suggest that pro-inflammatory cytokines, in particular IL-6, are key to mediating the effects of poly I:C infection on behavioral deficits in rodents that mimic similar deficits in schizophrenia and autism (Smith et al., 2007).

There is also evidence that the virus itself crosses the placenta to directly infect the fetus. Using a mouse animal model Aronsson et al., 2001, Aronsson et al., 2002 have shown that viral RNA for influenza A/WSN/33 strain (H1N1) persisted in the brains of offspring of virally infected dams. Recently a human postmortem study (Gu et al., 2007) demonstrated that the avian influenza virus (H5N1) can be transmitted across the placenta from mother to fetus. Taken together, these data show that both strains, at the expected doses contracted, can infect and be transmitted transplacentally to the fetus. These studies further provide clear evidence that one does not need viremia to induce placental passage of the virus.

An important aspect neglected in this debate is that the site of pathology may be the placenta. Whether the virus, traveling from the lungs, crosses the placenta; or whether in response to infection, maternal cytokines are produced which cross the placenta remains unknown. In the current study we examined the placenta as a site of pathology following prenatal viral infection on E7, in addition to prefrontal cortex and hippocampus in the exposed offspring. We hypothesized that viral infection would lead to gene expression and anatomical abnormalities in the exposed mice.