Bdnf overexpression in hippocampal neurons prevents dendritic atrophy caused by Rett-associated MECP2 mutations
Introduction
Neurodevelopmental disorders caused by genetic disruptions or chemical/physical insults during brain development affect neuronal terminal differentiation, i.e. axon and dendrite growth and branching, as well as synaptogenesis, thus impairing synaptic transmission and plasticity leading to altered network connectivity (Chechlacz and Gleeson, 2003, Johnston et al., 2001). Reduced dendritic length and branching have been demonstrated in many disorders associated with mental retardation (Kaufmann and Moser, 2000). One disorder associated with impaired brain development is Rett syndrome (RTT), a disease that affects nearly 1:15,000 females worldwide (Hagberg et al., 1985). Early development appears normal in individuals with RTT until approximately 6–18 months of age, when physical, motor and social–cognitive behavior enter a period of regression (Hagberg, 2002, Percy and Lane, 2005). Indeed, the neuropathology of RTT reveals several areas of abnormal brain development. Head circumference is decelerated, and postmortem observations of RTT brains demonstrate a reduction in brain weight (Armstrong et al., 1999, Jellinger et al., 1988, Schultz et al., 1993). Microscopically, brain autopsy material from RTT patients revealed impaired dendritic growth and complexity of pyramidal cells in the frontal and motor cortices, as well as in the subiculum (Armstrong et al., 1995), and reduced levels of MAP-2, a dendritic protein involved in microtubule stabilization (Kaufmann et al., 2000, Kaufmann et al., 1995, Kaufmann et al., 1997). These observations support the notion that RTT is a disorder caused by impaired dendritic, axonal and eventually synapse formation and maturation, leaving afflicted individuals in a state of arrested brain development (Armstrong, 2002, Johnston et al., 2001, Johnston et al., 2003, Kaufmann et al., 2005, LaSalle, 2004, Naidu, 1997, Philippart, 2001).
The majority of RTT cases are associated with mutations in a gene located on the X chromosome that encodes methyl-CpG-binding protein 2 (MeCP2) (Amir et al., 1999, Bienvenu et al., 2000, Van den Veyver and Zoghbi, 2001, Wan et al., 1999). MeCP2 is a nuclear protein that binds DNA specifically to A/T rich sites in close proximity to methylated CpG (cytidine-phospodiester-guanosine) islands. After binding to these methylated CpG sites, MeCP2 recruits the mSin3a co-repressor, which contains histone deacetylase complexes, thereby altering the structure of genomic DNA and regulating the transcription of specific target genes (Jones et al., 1998, Klose et al., 2005, Nan and Bird, 2001, Nan et al., 1997, Nan et al., 1998). DNA methylation is a common mechanism of silencing genes during cellular differentiation, and MeCP2 may play a critical role in neuronal terminal differentiation by reading this epigenetic code. Furthermore, MeCP2 expression in cortical neurons increases during brain development (Akbarian et al., 2001, Cohen et al., 2003, Jung et al., 2003; LaSalle et al., 2001, Shahbazian et al., 2002, Zoghbi, 2003). The developmental increase in its expression strongly suggests that MeCP2 plays a critical role in neuronal terminal differentiation, i.e. the development of axons, dendrites and dendritic spines leading to proper synapse formation and maturation (Cassel et al., 2004, Kaufmann et al., 2005, Kishi and Macklis, 2004, Matarazzo et al., 2004, Matarazzo and Ronnett, 2004, Mullaney et al., 2004). Missense mutations in MECP2 identified in RTT patients cluster in the methyl-binding domain (MBD) and transcriptional repressor domain (TRD) of the protein, suggesting that they represent loss-of-function mutations. Indeed, many of these mutations on the MBD of MeCP2 (like R106W and T158M used in the present studies; see below), reduce the affinity of MeCP2 for methylated DNA, thus impairing its ability to localize to heterochromatin and repress transcription in gene reporter assays (Kudo et al., 2001, Kudo et al., 2003).
To extend our knowledge of its role in neuronal terminal differentiation in the context of RTT, we manipulated MeCP2 expression levels in primary cultures of embryonic day-18 hippocampal neurons, a well-established experimental model for studies of neuronal differentiation (Banker and Goslin, 1998, Craig and Banker, 1994). At the time of plating, dissociated neurons were transfected with expression cDNA plasmids encoding either: (1) a small hairpin RNA (shRNA) interfering sequence to knockdown endogenous Mecp2 expression; (2) wildtype (wt) human MECP2; (3) one of two of the most common missense mutations in MECP2 found in RTT patients, R106W or T158M (www.mecp2.org.uk). Quantitative analyses of dendritic and axonal morphology (total length and number of branch points) were performed after 4 days in vitro, when a single defined axon and several dendrites are well defined (Dotti et al., 1988), and before synapse formation (Ziv and Smith, 1996). Considering that Bdnf (the gene coding for the neurotrophin brain-derived neurotrophic factor, BDNF) is a prominent gene shown to be regulated by MeCP2 (Abuhatzira et al., 2007, Chahrour et al., 2008, Chang et al., 2006, Chen et al., 2003, Klein et al., 2007, Martinowich et al., 2003, Ogier et al., 2007, Wang et al., 2006), we estimated intracellular BDNF protein levels by quantitative immunocytochemistry. Lastly, we tested whether Bdnf overexpression or extracellular BDNF scavenging with TrkB-Fc could revert the morphological effects of manipulations of MeCP2 levels. Our results demonstrate that MeCP2 plays varied roles in dendritic and axonal development during neuronal terminal differentiation, and that some of these effects are mediated by autocrine actions of BDNF.
Section snippets
Cell culture of dissociated hippocampal neurons
All animal procedures followed national and international ethical guidelines, and were reviewed and approved by the IACUC at UAB on an annual basis. Hippocampal neurons were cultured from embryonic day-18 (E18) Sprague–Dawley rat embryos (Charles River, Wilmington, MA) as previously described (Moore et al., 2007). Briefly, both hippocampi were dissected and neurons were re-suspended in Neurobasal medium containing B-27 supplement with penicillin–streptomycin and l-glutamine (InVitrogen;
Results
We studied the role of MeCP2 on neuronal terminal differentiation using hippocampal neurons from 18 day-old rat embryos maintained in dissociated primary cell culture. Pyramidal neurons in this culture preparation undergo a well-characterized morphological differentiation of axons and dendrites (Banker and Goslin, 1998, Craig and Banker, 1994). Quantitative analyses of dendritic and axonal morphology in pyramidal neurons transfected with eGFP or stained with anti-α-tubulin antibodies were
Discussion
Here, we presented several new observations regarding the role of MeCP2 in the terminal differentiation of hippocampal neurons, specifically the growth and branching of their dendrites and axons. First, endogenous Mecp2 knockdown reduced dendritic length without affecting axonal morphology. Second, overexpression of wildtype human MECP2 increased dendritic branching, as well as axonal length and branching. Third, overexpression of missense MECP2 mutations common in Rett syndrome reduced
Acknowledgments
Supported by NIH grants NS40593 and NS057780, IRSF and the Civitan International Foundation. We also thank the assistance of the UAB Intellectual and Developmental Disabilities Research Center (P30-HD38985) and the UAB Neuroscience Cores (P30-NS47466, P30-NS57098). We thank Drs. Masami Kojima (Research Institute for Cell Engineering, NIAIST, Osaka, Japan) for the generous gift of BDNF-xFP encoding plasmids, and Carolyn Schanen (Nemours Biomedical Research, Alfred I. duPont Hospital for
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JLL and CAC contributed equally to this work.