Prenatal viral infection lead to altered gene expression of genes 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 western blotting revealed significant reductions in Mbp isoform expression at P14 and P56, significant reductions in Mag at P14 and P35, and significant upregulation of DM20 at P35 and P56. No Mbp bands were detected for both control and infected P0 mice. This result is consistent with what has been previously determined by other research groups (Mathisen et al., 1993
; Freude et al., 2008
). Morphometric analysis revealed reduced cerebellar and brain volumes at P14, reduced hippocampal volume at P35, reduced white matter thickness of the IC-r at P0, and increased white matter thickness in the CC at P14 and the MCP-r at P56. summarizes the changes observed for myelination genes and brain structure at the four postnatal time points.
Comparison of myelin gene mRNA and protein changes with brain volume and fractional anisotropic changes
While the cerebellum has been underutilized in the study of psychiatric disorders, we chose to investigate the effects of prenatal viral infection in this region because a number of studies have demonstrated changes in cerebellar structure and function in subjects with schizophrenia. Both increased (DeLisi et al., 1997
; Nopoulos et al., 1999
; Uematsu and Kaiya, 1989
) and decreased (Goldman et al., 2008
) cerebellar volumes have been previously observed in subjects with schizophrenia as measured by MRI. Additionally, disruption of the cortico-thalamic-cerebellar-cortical circuit (CTCCC) has been identified in subjects with schizophrenia (Andreasen et al., 1996
). Functional MRI scans have revealed reduced activation of the cerebellum during cognitive tests (Meyer-Lindberg et al., 2001
; Kumari et al., 2002
) and PET scans have revealed reduced blood flow during tests to evaluate “Theory of Mind” (Andreasen et al., 2008
). Taken together, these deficits point to impaired cerebellar function, which may contribute to cognitive and behavioral disturbances in subjects with schizophrenia.
Several reports using MRI and diffusion tensor imaging (DTI) techniques have shown reduced white matter diffusion anisotropy (diffusion changes in water in white matter) in subjects with schizophrenia (Lim and Helpern, 2002
; Ardekani et al., 2003
; Kubcki et al., 2003; Carpenter et al., 2008
). Reductions in white matter anisotropy reflect disrupted white matter connections (Ardekani et al., 2003
), which supports the disconnection model of schizophrenia (Bullmore et al., 1997
). Marked declines in FA have been observed over the duration of illness (Carpenter et al., 2008
; Friedman et al., 2008
). Other research groups have identified little or no changes in fractional anisotropy during first episode schizophrenia (Peters et al., 2008) and there are also negative findings showing no white matter abnormalities in schizophrenia (Steel et al., 2001
; Foong et al., 2002
; Peters et al., 2008). It is conceivable that downregulation of genes affecting production of myelin-related proteins, as well as other components of axons, may lay the foundation for white matter abnormalities which develop later in life in subjects who become schizophrenic (Hakak et al., 2001
; Tkachev et al., 2003
). Consistent with this hypothesis, we observed reduction in gene expression of Mbp, Mobp, Mog, and Plp1via microarray and qRT-PCR at P56, which corresponds to adulthood in mice. Moreover, we also observed decreased Mbp 18.5 kDa and 17.2 kDa protein at this time point.
Developmentally, reduced FA suggests delayed maturation as FA has been shown to increase with brain maturation from juvenile to adult as myelination decreases the space between neurons resulting in increased anisotropy (Neil et al., 1998
; Nomura et al., 1994
). Reduced FA in the right internal capsule observed at P0 may suggest delayed maturation while the increased FA in the corpus callosum at P14 and the right middle cerebellar peduncle at P56 might suggest accelerated maturation. The increased FA of the corpus callosum at P14 may be due to the upregulation of myelination genes: Mal, Mbp, Mog, Mag, and Mobp at the same time point (). However, qRT-PCR did not validate these findings and protein data revealed decreases in Mbp and Mag at P14 (, , and ). The increased FA of the CC at P14 and the MCP at P56 may be due to other factors. As mentioned in the introduction, hypermyelination and accelerated brain growth are common during the first two years of life for children with autism (Ben Bashat et al., 2007
; Courchesne and Pierce, 2005
, Redcay and Courchesne, 2005
). It is conceivable that our animal model may mimic some of the structural/genetic markers of autism (Fatemi et al., 2002a
; Patterson, 2007
). Further studies are also needed to clarify the role, if any, that myelination genes play in changes in fractional anisotropy observed in subjects with schizophrenia or in animal models of schizophrenia.
Mbp comprises about 30% of the myelin membrane and is believed to be involved in myelin compaction (Chambers and Perrone-Bizzozero, 2004
). Chambers and Perrone-Bizzozero (2004)
found reduced Mbp immunoreactivity in hippocampus of female subjects with schizophrenia. Animal models for schizophrenia have also shown alterations in Mbp expression following prenatal viral infection at E9 (Fatemi et al., 2005
) and injection of PolyI:C on E9.5 (Makinodan et al., 2008
). Moreover, transgenic mice that lack Mbp (known as shiverer) show delayed action potentials (Lehman and Harrison, 2002
). The observed significant reductions of Mbp protein isoforms at P14 and P56 are consistent with these findings.
Plp1 is a major component of mammalian CNS myelin with possible functions in myelin stability and maintenance (Hudson, 2004
). PLP1 has shown to be downregulated in the temporal cortex of subjects with schizophrenia (Aston et al., 2004
). Genetic association studies have been inconsistent with a weak association between a single nucleotide polymorphism of PLP1 and susceptibility to schizophrenia in Han Chinese males (Qin et al., 2005
), while a recent Japanese population-based association study showed no association with schizophrenia (Aleksic et al., 2008
). While our microarray and qRT-PCR results showed a significant downregulation of Plp1 mRNA at P56, we did not observe any significant changes in Plp1 protein expression although the DM20 splice variant was increased at P35 and P56.
Mag is involved with mediating glial-axon interactions during myelination (Marcus et al., 2002
). Microarray analysis has revealed reduced MAG expression in the temporal cortex (Aston et al., 2004
), anterior cingulate cortex (Dracheva et al., 2006
; McCullersmith et al., 2007
), hippocampus (Dracheva et al., 2006
), and prefrontal cortex (Hakak et al., 2001
; Tkachev et al., 2003
) of subjects with schizophrenia. In contrast Mitkus et al., (2008)
found no significant difference in MAG mRNA expression in dorsolateral prefrontal cortex between subjects with schizophrenia and controls (Mitkus et al., 2008
). Two association studies have shown an association between MAG and schizophrenia in Han Chinese (Wan et al., 2005
; Yang et al., 2005
). A recent study by Voineskos et al., (2008)
shows no association between MAG variants and schizophrenia based on single marker and haplotype analysis. While we observed an increase in MAG mRNA in P14 cerebella, qRT-PCR did not validate this finding. However, qRT-PCR did reveal significant reductions in Mag mRNA at P0 and P56 (). We did observe significant reductions in Mag protein levels between the offspring of control and infected dams (, ).
Three other myelination genes that were altered in cerebellum were: 1) Mog, which is specific to the CNS and is located on the surface of oligodendrocytes and myelin sheaths (Brunner et al., 1989
). MOG mRNA has been shown to be decreased in prefrontal cortex of subjects with schizophrenia (Tkachev et al., 2003
). We also observed a reduction in Mog mRNA at P56 as measured by microarray and qRT-PCR (); 2) Mobp, which is believed to have a role in myelin compaction or stabilization (Montague et al., 2006
). MOBP has been identified as a candidate gene for schizophrenia (Lewis et al., 2003
). We observed reduced Mobp via microarray at P56, which was verified by qRT-PCR; and 3) Mal is thought to be involved in the organization, transport, and maintenance of glycosphingolipid-enriched membranes (Kamsteeg et al., 2007
). MAL has been identified it as a candidate gene for schizophrenia (DeLisi et al., 2002
; Straub et al., 2002
; Lewis et al., 2003
). We observed increased Mal mRNA in cerebella of infected offspring ().
Middle second trimester is a period when prenatal infection has been linked to the development of schizophrenia later in life (Suvasari et al., 1999
; Limonson et al., 2003
; Brown et al., 2006
). Compared with other time points (E9, E18), infection at E16 (middle second trimester in mice) resulted in changes in expression for a greater number of genes including those related to myelination (). Moreover, there were more changes in FA than at E18 (). Unfortunately, we do not have data on white matter changes following infection at E9 for a comparison although we have previously shown reduced neocortical 1-VI layers, and intermediate zone, and hippocampus at P0 (Fatemi et al., 1999
). Why infection at mid-pregnancy leads to greater changes in myelination gene expression and white matter abnormalities remains to be determined by future experiments.
Summary changes following prenatal viral infection in Cerebellum
Some limitations of our study include: 1) there was a small sample size with n=3 or N=4 per group; 2) we were unable to measure additional candidate genes at each time point due to the extensive list of genes involved in the infection process; and 3) lack of behavioral, motor and cognitive data following infection at E16. Future studies will focus on obtaining behavioral data at adulthood to investigate the functional consequences of infection at E16.
Our results demonstrate that infection at E16 results in the following: 1) altered expression of a number of schizophrenia and autism candidate genes; 2) altered expression of genes related to myelination, 5 of which are verified by qRT-PCR at P56; 3) altered protein expression for Mag, Mbp, and DM20 suggesting changes in myelin composition; 4) atrophy in cerebellar volume at P14; 5) changes in fractional anisotropy in multiple regions suggesting changes in white matter maturation; and 6) more changes in altered expression of total genes and genes related to myelination and white matter than infection at E9 or E18. These results suggest that infection at mid-second trimester, a time point during which infection has been linked to development of schizophrenia in humans, causes more severe effects on brain development that either mid-first (E9) or late second (E18) trimesters.