MeCP2 deficiency disrupts axonal guidance, fasciculation, and targeting by altering Semaphorin 3F function

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Abstract

Rett syndrome (RTT) is an autism spectrum disorder that results from mutations in the transcriptional regulator methyl-CpG binding protein 2 (MECP2). In the present work, we demonstrate that MeCP2 deficiency disrupts the establishment of neural connections before synaptogenesis. Using both in vitro and in vivo approaches, we identify dynamic alterations in the expression of class 3 semaphorins that are accompanied by defects in axonal fasciculation, guidance, and targeting with MeCP2 deficiency. Olfactory axons from Mecp2 mutant mice display aberrant repulsion when co-cultured with mutant olfactory bulb explants. This defect is restored when mutant olfactory axons are co-cultured with wild type olfactory bulbs. Thus, a non-cell autonomous mechanism involving Semaphorin 3F function may underlie abnormalities in the establishment of connectivity with Mecp2 mutation. These findings have broad implications for the role of MECP2 in neurodevelopment and RTT, given the critical role of the semaphorins in the formation of neural circuits.

Introduction

Methyl-CpG binding protein 2 (MeCP2) is a multifunctional protein that induces transcriptional repression by recruitment of co-repressors and chromatin remodeling (reviewed in Chahrour and Zoghbi, 2007, Francke, 2006, Klose and Bird, 2006). Mutations in the Mecp2 gene have been linked to human diseases such as Rett syndrome (RTT, MIM 312750), a neurodevelopmental disorder associated with mental retardation, autistic behavior, and loss of previously acquired milestones, including purposeful hand use and expressive language (Amir et al., 1999). Alterations in MECP2 expression have also been detected in other autism spectrum disorders (ASD) as well as in nonsyndromic mental retardation (Chahrour and Zoghbi, 2007). The observed correlation between MECP2 expression/function and neurological disorders clearly indicates a requirement for MECP2 in the normal development of the nervous system. However, the mechanisms whereby MECP2 mutation disrupts neurodevelopment remain unclear.

To elucidate the role of MeCP2 in neurodevelopment, mouse models that either lack MeCP2 or express a mutant form of MeCP2 have been generated. These animals share many of the features of the human disorder (Chen et al., 2001, Guy et al., 2001, Pelka et al., 2006, Shahbazian et al., 2002). Studies in such animal models indicate that MeCP2 functions in maturation/terminal differentiation of CNS neurons (Kishi and Macklis, 2004, Matarazzo et al., 2004, Smrt et al., 2007), axonal and dendritic growth and morphology (Belichenko et al., 2009a, Cusack et al., 2004, Jugloff et al., 2005, Larimore et al., 2009, Zhou et al., 2006), synaptic formation and function (Asaka et al., 2006, Chao et al., 2007, Dani et al., 2005, Fukuda et al., 2005, Moretti et al., 2006, Nelson et al., 2006) and neurotransmission (Chao et al., 2007, Dani et al., 2005, Medrihan et al., 2008, Nelson et al., 2006). In addition, the absence of MeCP2 in mouse models is accompanied by deficits in learning, memory (Moretti et al., 2006, Pelka et al., 2006) and social behavior (Moretti et al., 2005). The current view proposes that MeCP2 dysfunction affects synaptic plasticity via failure of dendritic function. However, the molecular basis for these defects remains unclear, and the effect that MeCP2 deficiency may have on axonal function has not been explored.

Our laboratory has used the olfactory system as a developmental model. We demonstrated that MeCP2 expression correlates with the maturation of olfactory sensory neurons (OSNs) and precedes synaptogenesis (Cohen et al., 2003). We also found that both MeCP2 deficiency and dysfunction impair OSN terminal differentiation at the time of synaptogenesis (Matarazzo et al., 2004, Palmer et al., 2008). These acute and transient defects in synaptogenesis are followed by a separate and persistent phase of chronic compromise in synaptic architecture. Since the olfactory system undergoes continuous neurogenesis, sampling this epithelium from patients allowed us to perform in vivo analyses not possible to do from brain. Our studies of olfactory nasal biopsies from RTT patients have revealed similarities in the neurodevelopmental defects found in RTT patients and mouse models of RTT, validating the olfactory system as a model to study RTT and MeCP2 dysfunction (Ronnett et al., 2003). Using this system, we identified functional groups of proteins that are dysregulated in Mecp2-null mice; in particular, a group of proteins involved in cytoskeletal arrangements and axonal guidance (Matarazzo and Ronnett, 2004).

Therefore, we hypothesized that MeCP2 deficiency alters axonal guidance events during development to ultimately compromise the establishment of neural connections and synaptic function. Using the olfactory system for both in vitro and in vivo approaches, we identify a novel function for MeCP2 in axonal guidance, fasciculation, and targeting, and a role for class 3 semaphorins in these defects. These findings have broad implications for the role of MeCP2 in neurodevelopment and RTT, given the critical role of semaphorins in the formation and maintenance of neural circuits (Yazdani and Terman, 2006).

Section snippets

Laminar targeting is altered in the main olfactory bulb (MOB) from Mecp2-null mice

OSN axons enter the MOB forming the olfactory nerve layer (ONL) and terminating in regions of neuropil called glomeruli, where they form synapses with mitral and tufted cells. The MOB has a distinct lamellar structure, each layer containing a different cell type (Mori et al., 1999). During the first postnatal weeks, axons from OSNs target the glomerular layer but can also be found overshooting the external plexiform layer (EPL). By P10–20, this excessive axonal growth is normally refined and

Discussion

In the present study we demonstrate that 1) MeCP2 deficiency disrupts the establishment of neural connections well before synaptogenesis; 2) MeCP2 deficient mice have defects in axonal guidance, fasciculation, and targeting; 3) guidance defects are accompanied by temporal/regional alterations in the expression of class 3 semaphorins and receptors; and 4) non-cell autonomous dysfunction of Sema3F signaling is responsible for some of the defects in connectivity observed with Mecp2 mutation. These

Mice

We used a MeCP2-null mouse model generated by the Cre LoxP recombination system to delete exon 3 of Mecp2 (Chen et al., 2001). Mice were generously provided by Dr. R. Jaenisch and maintained on a Balb/c background. M72-IRES-tauLacZ transgenic mice were obtained from Dr Peter Mombaerts et al. (1996), and were crossed with the Mecp2-null mice to generate double mutants. All the experiments were performed using hemizygous Mecp2 males (Mecp2-null) and WT littermate controls. Animal procedures were

Acknowledgments

We thank A. Puschel for the Sema3 vectors, D. D. Ginty for providing the anti-Npn2 antibody, P. Mombaerts for providing M72-IRES-tauLacZ mice and members of the Ronnett Lab for critical reading of the manuscript.

This work was supported by grants from the National Institute of Neurological Disorders and Stroke (NINDS), the National Institute on Deafness and Other Communication Disorders (NIDCD) and the National Institute of Child Health and Human Development (NICHD).

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