Mecp2 deficiency leads to delayed maturation and altered gene expression in hippocampal neurons
Introduction
Rett Syndrome (RTT) is a neurodevelopmental disorder that affects one of every 15,000 female births. RTT patients develop normally until 6 to 18 months of age, but then regress rapidly experiencing a wide range of neurological defects, such as seizures, ataxia and stereotypical hand movements. Individuals affected by RTT often survive into adulthood, and while some symptoms stabilize, others may worsen (Hagberg et al., 1983, Hagberg and Witt-Engerstrom, 1986, Kriaucionis and Bird, 2003). In most cases, RTT can be linked to loss-of-function mutations in the X-linked MECP2 gene (Amir et al., 1999), which encodes a methylated-CpG binding protein that recruits additional factors such as histone deacetylase to repress transcription (Bird, 2002). Several lines of Mecp2 mutant mice (KO) have been generated and these mice develop similar symptoms to those seen in RTT patients and have been widely used to study the etiology of human RTT (Chen et al., 2001, Guy et al., 2001, Shahbazian et al., 2002, Pelka et al., 2006). Nevertheless, the neurodevelopmental pathways and specific genes targeted by the disruption of this epigenetic regulatory control have not been determined.
Recent experimental evidence indicates that MeCP2 may play a vital role in neuronal maturation (Bienvenu and Chelly, 2006). A critical step in the process of neuronal maturation is synaptogenesis, which coincides with the increased expression of MeCP2 in developing neurons (Akbarian et al., 2001, Zoghbi, 2003) (Shahbazian and Zoghbi, 2002), suggesting that epigenetic modulation of gene regulation during this period might be critical for brain development. In fact, postmortem analysis has demonstrated reduced numbers of axonal and dendritic processes, decreased dendritic spine density and lowered levels of the dendritic cytoskeletal protein MAP2 in RTT brains (Kaufmann and Moser, 2000, Armstrong, 2002). Consistent with human pathology, pyramidal neurons in the cortex of adult Mecp2 null mutant (KO) mice were found to have smaller soma and less complex dendrites, though the morphology and density of dendritic spines were not determined in this study (Kishi and Macklis, 2004). Exogenous Mecp2 expression could also lead to increased neurite complexity in cultured neurons (Jugloff et al., 2005), further suggesting a role of MeCP2 in dendritic development. However, in another study, analyses of Golgi-stained cortical and subcortical neurons of Mecp2 truncation mutant mice (Mecp2y/308) did not reveal significant abnormalities in either dendritic arbor or spine density (Moretti et al., 2006). The discrepancy between these results could be due to differences in either the model systems analyzed or the methods used. Abnormalities in dendritic spines have been found in several developmental disorders [reviewed by Fiala et al., 2002]. Therefore it is critical to clarify whether Mecp2 mutations affect spine development by monitoring the maturation of single neurons in a well defined cell population in order to understand the function of MeCP2 in neural development and the etiology of RTT.
Unlike most other brain regions, neurogenesis in the adult dentate gyrus (DG) persists throughout life. In adult mice, newborn DG neurons develop properties similar to mature granule neurons after approximately 4–8 weeks of differentiation. The properties of newborn neurons in the adult DG recapitulate embryonic hippocampal development (Song et al., 2005), providing a unique model system for studying the generation and maturation of neurons in postnatal brains (Gage, 2002). The hippocampus also provides a logical framework to study the pathogenesis of MeCP2 deficiency because the morphological maturation, functional properties and molecular mechanisms of the hippocampus have been extensively characterized due to their potentially critical roles in learning and memory (Ziv and Garner, 2004, Nicoll and Schmitz, 2005), and because Mecp2 KO mice have been shown to have impaired long-term potentiation and depression, impaired excitatory neurotransmission and altered expression of neurotransmitter receptors in hippocampal neurons (Asaka et al., 2006, Moretti et al., 2006, Nelson et al., 2006).
Mecp2 has been found to be expressed in neural stem cells (NSCs) (Jung et al., 2003, Namihira et al., 2004). While MeCP2 was shown to be involved in embryonic neurogenesis in Xenopus, studies have indicated that this is not the case in mice (Stancheva et al., 2003, Kishi and Macklis, 2004). Recent evidence has revealed that adult NSCs are different from embryonic NSCs in both the cellular environment they encounter and in their intrinsic genetic and epigenetic properties (Zhao et al., 2003, Cheng et al., 2005). Moreover, deletion of Mecp2-related Methyl-CpG binding protein 1 (Mbd1) specifically affects postnatal, but not embryonic, neurogenesis (Zhao et al., 2003), suggesting that postnatal neurogenesis may be particularly vulnerable to altered epigenetic regulation. Therefore, analyzing postnatal neurogenesis in the absence of Mecp2 will provide critical information for understanding the function of this protein.
In this study, we have determined that Mecp2 is not critical for the early stages of neurogenesis. In contrast, we show that immature neurons in the DG of KO mice exhibit deficits in their ability to transition into later mature stages of development. This deficit results in adult Mecp2 KO mice retaining characteristic features of immature brains, suggesting a stalled maturation. At a single neuronal level in the postnatal hippocampus, Mecp2-deficient neurons exhibited a reduced number of dendritic spines. By analyzing gene expression profiles of a homogeneous population of DG neurons isolated from KO brains, we have found that the expression levels of several genes encoding proteins that are likely to be involved in synaptogenesis were altered. Together, these data suggest that Mecp2 is critical for the maturation of young neurons, possibly through regulating synaptogenic factors.
Section snippets
Animals
All animal procedures were performed according to protocols approved by the University of New Mexico Animal Care and Use Committee. The Mecp2 KO mice (Mecp2tm1.1Jae) used in this study were created by deleting exons 3 containing the MBD domain of Mecp2 (Chen et al., 2001). These mice have been bred over 40 generations onto ICR background. They start to show neurological symptoms between 5 and 7 weeks of age and die before 10 weeks of age. For histological analyses, mice were euthanized by
Early postnatal neurogenesis appears normal in Mecp2-deficient mice
We have previously found that mice deficient for Mbd1, an Mecp2-related protein, exhibited reduced adult hippocampal neurogenesis both in vivo and in vitro (Zhao et al., 2003). Because RTT manifests at 6–18 months of age in patients, well after primary neurogenesis, we asked whether a lack of functional Mecp2 causes deficits in postnatal neurogenesis that might be linked to neurological symptoms comparable to those seen in RTT patients. We therefore compared neural stem/progenitor cells (NSCs)
Discussion
With the identification of MECP2 as the gene responsible for RTT, it becomes critically important to understand the role of MECP2 in postnatal neural development. In this study, we provide strong evidence that while the lack of Mecp2 does not affect the production of NSCs, it does significantly impair subsequent steps in the maturation of neurons. First, we determined that the expression of specific markers defining the transition from immature to mature neurons was delayed in Mecp2-deficient
Acknowledgments
Images in this paper were generated in the University of New Mexico Cancer Center Fluorescence Microscopy Facility, supported as detailed on the webpage (http://hsc.unm.edu/crtc/microscopy/facility.html). We thank R. Lee and G. Phillips for technical assistance. We thank Dr L.A. Cunningham for advice in stereotaxic grafting. We thank L.C. Tafoya and Drs. M.C. Wilson, C.F. Valenzuela, P. Jin, X. Li and H. van Praag for critical reading of the manuscript and helpful comments. We thank M.L. Gage
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