Abnormal expression of myelination genes and alterations in white matter fractional anisotropy following prenatal viral influenza infection at E16 in mice

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Abstract

Prenatal viral infection has been associated with the development of schizophrenia and autism. Our laboratory has previously shown that viral infection causes deleterious effects on brain structure and function in mouse offspring following late first trimester (E9) and late second trimester (E18) administration of influenza virus. We hypothesized that middle second trimester infection (E16) in mice may lead to a different pattern of brain gene expression and structural defects in the developing offspring. C57BL6 mice were infected on E16 with a sublethal dose of human influenza virus or sham-infected using vehicle solution. Male offspring of the infected mice were collected at P0, P14, P35, and P56, their brains removed and cerebella dissected and flash frozen. Microarray, DTI and MRI scanning, as well as qRT-PCR and SDS-PAGE and western blotting analyses were performed to detect differences in gene expression and brain atrophy. Expression of several genes associated with myelination, including Mbp, Mag, and Plp1 were found to be altered, as were protein levels of Mbp, Mag, and DM20. Brain imaging revealed significant atrophy in cerebellum at P14, reduced fractional anisotropy in white matter of the right internal capsule at P0, and increased fractional anisotropy in white matter in corpus callosum at P14 and right middle cerebellar peduncle at P56. We propose that maternal infection in mouse impacts myelination genes.

Introduction

There is robust epidemiologic evidence indicating that environmental contributions, including prenatal infections, may lead to genesis of schizophrenia (Suvisaari et al., 1999, Limosin et al., 2003 Fatemi, 2005, Fatemi, 2008, Brown, 2006) and autism (Spear, 2000, Spear, 2004, Arndt et al., 2005, Libbey et al., 2005). Several groups, including our laboratory, have shown evidence for viral infections and/or immune challenges as being responsible for production of abnormal brain structure and function in rodents whose mothers were exposed to viral insults throughout pregnancy (Fatemi et al., 1999, Fatemi et al., 2002a, Fatemi et al., 2005, Fatemi et al., 2008a, Fatemi et al., 2008b, Fatemi et al., 2008c, Meyer et al., 2006, Meyer et al., 2007, Meyer et al., in press).

Previous reports have implicated various gene families in the etiopathology of schizophrenia, including genes involved in myelination (Hakak et al., 2001, Tkachev et al., 2003, Le-Niculescu et al., 2007). Hakak et al. (2001) using mostly elderly schizophrenic and matched control dorsolateral prefrontal cortex (DLPFC) homogenates, showed downregulation of 5 genes [myelin-associated glycoprotein (MAG), myosin and lymphocyte protein (MAL), transferrin, neuregulin receptor ERBB3, and gelsolin] whose expression is enriched in myelin-forming oligodendrocytes, which have been implicated in the formation and maintenance of myelin sheaths. Later, Tkachev et al. (2003) using Brodmann Area 9 (BA9) homogenates from Stanley Neuropathology Consortium brain collection showed significant downregulation in several myelin and oligodendrocyte related genes, such as proteolipid protein 1 (PLP1; Pongrac et al., 2002), MAG, oligodendrocyte specific protein CLDN11, myelin oligodendrocyte glycoprotein (MOG), and myelin basic protein (MBP) (Tkachev et al., 2003). Applying a functional genomics approach integrating data from postmortem studies, human genetic linkage studies, and an animal model, Le-Niculescu et al. (2007) identified six genes (MAL, MBP, phospholipid protein 1 (PLP1), glial fibrillary acidic protein (GFAP), myelin-associated oligodendrocyte basic protein, and cyclic nucleotide phosphodiesterase) as schizophrenia candidate genes.

Hypermyelination has previously been observed in autism (Ben Bashat et al., 2007), Sturge–Weber syndrome (Moritani et al., 2008), and a case report of merosin deficiency in which the subject displayed mental retardation and epilepsy (Deodato et al., 2002). More than 70% of children with autism experience accelerated brain growth during their first 2 years of life (Courchesne and Pierce, 2005, Redcay and Courchesne, 2005). Significantly, Ben Bashat et al. (2007) observed accelerated maturation of white matter in a sample of children with autism between the ages of 1.8 and 3.3 years, the critical ages for accelerated brain growth.

Our group has demonstrated that influenza infection at E9 and E18 of pregnancy in mice leads to abnormal corticogenesis, brain atrophy, pyramidal cell atrophy, and alterations in levels of several neuroregulatory proteins, such as Reelin, and Gfap, in the exposed mouse progeny (Fatemi et al., 1999, Fatemi et al., 2002a, Fatemi et al., 2002b, Fatemi et al., 2008a, Shi et al., 2003). We have demonstrated altered expression of myelination genes using this model (Fatemi et al., 2005, Fatemi et al., 2008a). Balb/c mice infected with influenza at E9 displayed reduced expression of Mbp and Plp1 mRNA at birth (postnatal day 0 (P0)) (Fatemi et al., 2005). Following infection at E18, myelin transcription factor 1-like (Mytl1) was upregulated in cerebellum and hippocampus at P0 (Fatemi et al., 2008a, Fatemi et al., 2008b, Fatemi et al., 2008c).

We hypothesized that middle second trimester infection (E16) in mice may lead to a different pattern of brain gene expression and structural defects in the developing offspring. Here, we present genetic, imaging, and protein data showing that a similar sublethal dose of human influenza virus (H1N1) in C57BL6 mice at E16, also leads to altered expression of many brain genes, including myelination genes, which may underlie brain atrophy and white matter changes in the exposed mouse offspring.

Section snippets

Viral infection

C57BL6 mice were infected on E16 with a sublethal dose of influenza A/NWS/33 (H1N1) virus or sham-infected using vehicle solution. After infection, drinking water was supplemented with 0.006% oxytetracycline (Pfizer, New York, NY) to control possible bacterial infections. Pregnant mice were allowed to deliver pups and the day of delivery was considered P0.

Brain collection and dissection

Male offspring of the infected and sham-infected mice were collected at P0, 14, 35, and 56 and deeply anesthetized and sacrificed with

Results

Gene expression data showed a significant (p < 0.05) at least 1.5 fold up- or downregulation of genes in cerebella (27 upregulated and 73 downregulated at P0; 205 upregulated and 16 downregulated at P14; and 450 upregulated and 205 downregulated at P56) of mouse offspring (Supporting Table 1, online). Several genes, which have been previously implicated in the etiopathology of autism and schizophrenia, were shown to be affected significantly (p < 0.05) by DNA microarray including: autism

Discussion

Prenatal viral infection leads to altered gene expression in cerebellum at P0, P14, and P56, including numerous schizophrenia candidate genes, such as Rgs4, Auts2, Mbp, Mag, Mog, Mobp, Plp1, and Ncam1. qRT-PCR verified the direction and magnitude of change for Auts2, Mbp, Mobp, Mog, Plp1, and Rgs4 at P56. Further investigation of myelination genes via SDS-PAGE and western blotting revealed significant reductions in Mbp isoform expression at P14 and P56, significant reductions in Mag at P14 and

Role of funding source

Funding for this study was provided by the National Institute of Child Health and Human Development grant 5R01-HD046589-04 (SHF). The funding source had no further role in the study design; in the collection, analysis and interpretation of the data; in the writing of the report; and in the decision to submit the paper for publication.

Contributors

All authors have contributed to and approved the final manuscript.

Conflict of interest

None.

Acknowledgements

Grant support by the National Institute of Child Health and Human Development (#5R01-HD046589-04) to SHF is gratefully acknowledged. Assistance with microarray data interpretation by Dr. Chuanning Tang and Dr. Tongbin Li of the University of Minnesota, Department of Neuroscience is gratefully acknowledged.

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