Research ReportEffects of postnatal dietary choline supplementation on motor regional brain volume and growth factor expression in a mouse model of Rett syndrome
Introduction
Neuronal developmental processes including neurogenesis, migration, maturation, and synapse refinement, occur predominantly in utero but also during postnatal periods (Rao and Jacobson, 2005). These processes are genetically regulated but also susceptible to environmental manipulation, such as nutritional status, which can alter the adult phenotype in several developmental disorders, including Rett syndrome (RTT; reviewed recently in Nag et al., in press-b). Using a mouse model of RTT, we have shown previously that postnatal dietary choline supplementation improves locomotor behavior in null male Mecp21lox mice (Nag and Berger-Sweeney, 2007) and increases total brain volume in heterozygous female but not null male Mecp21lox mice (Ward et al., 2008). Here, we extend those findings by examining how perinatal choline supplementation influences growth factors and brain volume in two motor regions of the brain, the cerebellum and striatum, in null male Mecp21lox mice and their wild-type littermates.
RTT is an autism spectrum disorder caused by mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2), a transcriptional repressor that binds methylated DNA (Amir et al., 1999). RTT girls, who are generally heterozygous with one normal and one mutated MECP2 gene, have variable MeCP2 expression (and varying phenotypes) due to random X-inactivation patterns. Whereas, males who are null for MeCP2 expression are more severely affected and often do not survive birth. The phenotype in RTT girls includes reduced head growth, regression of motor and cognitive skills (Hagberg et al., 1983, Hagberg and Witt-Engerstrom, 1986), and abnormalities in neuroanatomy and biochemistry (Armstrong et al., 1995, Armstrong, 2002, Jellinger, 2003, Wenk et al., 1991). These phenotypic features are also observed in Mecp2 mouse models of RTT and are particularly well-replicated in the null males as compared to the heterozygous females (Chen et al., 2001, Kishi and Macklis, 2004, Stearns et al., 2007). The most consistent neurochemical changes in RTT girls include decreased cholinergic markers in the basal forebrain, basal ganglia and midbrain tegmentum (Kitt and Wilcox, 1995, Wenk et al., 1991), suggesting that cholinergic deficits may underlie some of the behavioral abnormalities in RTT.
In rodents, choline supplementation during critical periods of brain development enhances transmission at cholinergic synapses (Blusztajn et al., 1998, Blusztajn and Wurtman, 1983), protects against neurodegeneration (Guo-Ross et al., 2002), improves performance on several behavioral tasks (Meck et al., 1988, Ricceri and Berger-Sweeney, 1998), and up-regulates growth factor expression (Sandstrom et al., 2002, Wong-Goodrich et al., 2008). In Mecp21lox null mice, dietary choline supplementation from postnatal day (PD) 1–21 improves motor deficits as measured by rotor-rod performance, while cognitive deficits are unaltered (Nag and Berger-Sweeney, 2007). These results suggest that postnatal choline supplementation acts on brain motor regions of Mecp21lox mice, therefore, in the current study we focus on two motor regions of the brain, the cerebellum and striatum. In addition, we focus these studies on Mecp21lox null male mice because their phenotype more closely resembles that of RTT girls than the heterozygous female Mecp21lox mice (Stearns et al., 2007), and we can detect behavioral improvements in the males in response to choline supplementation but not in the females, who have only very subtle motor and cognitive deficits in early adulthood (Nag and Berger-Sweeney, 2007). Furthermore, the Mecp21lox null male mice provide an interesting opportunity to examine how choline supplementation can improve behavioral responses in the absence of Mecp2 protein, in other words, in the absence of this transcriptional repressor that binds methylated DNA.
It is not clear how perinatal choline supplementation causes long-term alterations in brain and behavior. Choline is essential for the structural integrity of cell membranes, methyl metabolism, transmembrane signal transduction, and cholinergic neurotransmission (Blusztajn, 1998, Zeisel, 1981, Zeisel, 2006, Zeisel and Blusztajn, 1994). Given the essential functions of choline in the organism, one possible mechanism by which supplementation may improve behavior in a mouse model of RTT is by increasing neuronal integrity, which may be reflected globally in volumetric changes in the brain. Indeed, longitudinal magnetic resonance imaging (MRI) reveals increases in whole brain volume in heterozygous female Mecp21lox mice between PD 21 and 42 in response to postnatal choline supplementation, but not in null male Mecp21lox mice who lack the Mecp2 gene (Ward et al., in press). Using MRI, it is difficult to resolve specific regions within the brain. Therefore, in the current study, we measured specific brain regions associated with motor activity, namely the cerebellum and striatum, in histological sections using the computer program AMIRA™ to look for more subtle regional volume changes that may not be apparent by examining whole brain volume.
We hypothesized that choline supplementation improves motor behaviors in null male Mecp21lox mice by increasing brain volume and stimulating neuronal growth factors. Growth factors promote neurite outgrowth, neuronal differentiation, and maintenance of cell function and survival (Ebendal, 1992). Mecp2 null mice have reduced cortical dendritic arborizations (Kishi and Macklis, 2004) that may be partially restored with choline supplementation. Three extensively studied growth factors are insulin-like growth factor 1 (IGF-1), nerve growth factor (NGF), and brain derived growth factor (BDNF). The latter two are neurotrophins, a class of growth factors, and play a significant role in postnatal survival and neurite outgrowth. While all three growth factors have different expression profiles and act on different classes of neurons, they all promote neuronal integrity.
In the mouse brain, IGF-1 is associated with overall brain size. Over-expression of IGF-1 results in increased brain volume (Han, 1995), whereas IGF-1 null mice have reduced brain volume (Beck et al., 1995). IGF-1 mRNA is widely expressed in fetal brain tissue, with more discrete postnatal expression in olfactory bulb, hippocampus, and cerebellum (Bondy et al., 1992), being particularly important in cerebellar development (Torres-Aleman et al., 1994). RTT girls have unchanged levels of cerebrospinal and serum IGF-1 levels compared to controls (Riikonen, 2003, Vanhala et al., 2000), however no study has reported IGF-1 expression in the brains of RTT girls.
NGF and BDNF expression are evident in the hippocampus, cerebellum, cortex, striatum and basal forebrain (Large et al., 1986, Mobley et al., 1989, Senut et al., 1990, Shelton and Reichardt, 1986). NGF up-regulates the expression of choline acetyltransferase (ChAT), the synthetic enzyme for acetylcholine. NGF regulates expression of cholinergic cells of the striatum (Van Vulpen and Van Der Kooy, 1999), as well as the basal forebrain (Berse et al., 1999, Li et al., 1995, Tian et al., 1996). There are conflicting reports about NGF levels in RTT. It is either reduced (Lappalainen et al., 1996, Lipani et al., 2000, Riikonen, 2003, Riikonen and Vanhala, 1999) or unaltered compared to control subjects (Vanhala et al., 1998, Wenk and Hauss-Wegrzyniak, 1999), with the results being seemingly dependent on age, pathology, and sample collection site. We have found no reports of NGF levels in mouse models of RTT. BDNF levels are low in RTT (Abuhatzira et al., 2007), which has been confirmed in mouse models of the syndrome (Chang et al., 2006, Kondo et al., 2008). These reductions are not surprising given the evidence that Mecp2 regulates BDNF expression (Zhou et al., 2006). Because both NGF and BDNF are important for maintaining neuronal integrity, NGF being particularly critical for cholinergic neurons and increasing dendritic arborizations, it is possible that reduced growth factors contribute to the marked reduction of dendritic arborizations (Armstrong et al., 1995), and reduced number of ChAT-positive neurons in RTT (Wenk and Hauss-Wegrzyniak, 1999).
In rats, prenatal choline supplementation increases IGF-1 and BDNF expression in the hippocampus and NGF in both hippocampus and frontal cortex (Glenn et al., 2007, Sandstrom et al., 2002, Wong-Goodrich et al., 2008). We therefore hypothesized that perinatal choline supplementation would increase expression of these growth factors in the cerebellum and striatum of Mecp21lox mice, and that these increases could lead to increased neuronal survival and neurite outgrowth that would be reflected in increased brain regional volumes and associated improvements in motor behaviors.
In this study we measure brain volume using the computer program AMIRA™ and the expression of IGF-1, NGF, and BNDF via ELISA, in untreated and choline supplemented wild-type and Mecp21lox null male mice. We focus on the cerebellum and striatum as we found previously that choline supplementation improves motor behaviors. Understanding the interactions and underlying mechanisms between nutrition and the genetics of RTT may provide insights into the root causes of the disorder and provide opportunities for palliative treatments, even if they do not cure the disorder.
Section snippets
Mecp21lox mice have reduced volume in specific brain regions, which are not altered with choline supplementation
Compared to wild-type mice, whole brain volume at PD 43 was decreased in Mecp21lox mice (234.51 ± 6.56 mm3, and 199.91 ± 5.78 mm3 respectively; Fig. 1A). There was a significant genotype effect [F(1, 20) = 48.16, p ≪ 0.001], with post-hoc analysis showing reduced brain volume in Mecp21lox mice (p < 0.01; Fig. 1A) as compared to wild-type mice. Weights of dissected whole brains support these whole brain volume measurements, and validate the use of AMIRA™ in accurately measuring volume. Untreated
Discussion
We have found that whole brain, striatal and cerebellar volumes are reduced in adult Mecp21lox null mice as compared to wild-type controls. Postnatal choline supplementation does not significantly alter any of these volumes in the adult null mice. BDNF levels, but not IGF-1 or NGF levels, are reduced in adult Mecp21lox null mice as compared to wild-type controls. Postnatal choline supplementation increases BDNF levels in striatum, but not cerebellum, in the wild-type mice. However, BDNF levels
Animals and postnatal choline supplementation
All experiments were conducted on Mecp21lox null mice (Chen et al., 2001) and their C57/BL6 male wild-type littermates, with procedures approved by the Wellesley College Institutional Animal Care and Use Committee, conforming to the standards set forth in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mecp21lox female heterozygous founder mice (a gift from Dr. R. Jaenisch) were back-crossed with C57/BL6 males to establish and maintain a colony of Mecp21lox
Acknowledgments
We would like to thank U. Berger for genotyping, M-A Lopez-Gomez for technical assistance, L.R. Schaevitz for comments on the manuscript, and P. Carey and G. Quinan for animal care. This work was supported by funds from the National Science Foundation and a Wellesley College Faculty Award.
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