Bdnf overexpression in hippocampal neurons prevents dendritic atrophy caused by Rett-associated MECP2 mutations

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Abstract

The expression of the methylated DNA-binding protein MeCP2 increases during neuronal development, which suggests that this epigenetic factor is crucial for neuronal terminal differentiation. We evaluated dendritic and axonal development in embryonic day-18 hippocampal neurons in culture by measuring total length and counting branch point numbers at 4 days in vitro, well before synapse formation. Pyramidal neurons transfected with a plasmid encoding a small hairpin RNA (shRNA) to knockdown endogenous Mecp2 had shorter dendrites than control untransfected neurons, without detectable changes in axonal morphology. On the other hand, overexpression of wildtype (wt) human MECP2 increased dendritic branching, in addition to axonal branching and length. Consistent with reduced neuronal growth and complexity in Rett syndrome (RTT) brains, overexpression of human MECP2 carrying missense mutations common in RTT individuals (R106W or T158M) reduced dendritic and axonal length. One of the targets of MeCP2 transcriptional control is the Bdnf gene. Indeed, endogenous Mecp2 knockdown increased the intracellular levels of BDNF protein compared to untransfected neurons, suggesting that MeCP2 represses Bdnf transcription. Surprisingly, overexpression of wt MECP2 also increased BDNF levels, while overexpression of RTT-associated MECP2 mutants failed to affect BDNF levels. The extracellular BDNF scavenger TrkB-Fc prevented dendritic overgrowth in wt MECP2-overexpressing neurons, while overexpression of the Bdnf gene reverted the dendritic atrophy caused by Mecp2-knockdown. However, this effect was only partial, since Bdnf increased dendritic length only to control levels in mutant MECP2-overexpressing neurons, but not as much as in Bdnf-transfected cells. 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.

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

References (101)

  • CusackS.M. et al.

    Suppression of MeCP2beta expression inhibits neurite extension in PC12 cells

    Exp. Cell. Res.

    (2004)
  • GreschO. et al.

    New non-viral method for gene transfer into primary cells

    Methods

    (2004)
  • HagbergB. et al.

    Rett syndrome: criteria for inclusion and exclusion

    Brain Dev.

    (1985)
  • JugloffD.G. et al.

    Increased dendritic complexity and axonal length in cultured mouse cortical neurons overexpressing methyl-CpG-binding protein MeCP2

    Neurobiol. Dis.

    (2005)
  • KangH. et al.

    Neurotrophins and time: different roles for TrkB signaling in hippocampal long-term potentiation

    Neuron

    (1997)
  • KaufmannW.E. et al.

    Cyclooxygenase-2 expression during rat neocortical development and in Rett syndrome

    Brain Dev.

    (1997)
  • KaufmannW.E. et al.

    MeCP2 expression and function during brain development: implications for Rett syndrome's pathogenesis and clinical evolution

    Brain Dev.

    (2005)
  • KishiN. et al.

    MECP2 is progressively expressed in post-migratory neurons and is involved in neuronal maturation rather than cell fate decisions

    Mol. Cell. Neurosci.

    (2004)
  • KloseR.J. et al.

    DNA binding selectivity of MeCP2 due to a requirement for A/T sequences adjacent to methyl-CpG

    Mol. Cell

    (2005)
  • KudoS. et al.

    Functional analyses of MeCP2 mutations associated with Rett syndrome using transient expression systems

    Brain Dev.

    (2001)
  • LaSalleJ.M.

    Paradoxical role of methyl-CpG-binding protein 2 in Rett syndrome

    Curr. Top. Dev. Biol.

    (2004)
  • MatarazzoV. et al.

    The transcriptional repressor Mecp2 regulates terminal neuronal differentiation

    Mol. Cell. Neurosci.

    (2004)
  • McAllisterA.K. et al.

    Opposing roles for endogenous BDNF and NT-3 in regulating cortical dendritic growth

    Neuron

    (1997)
  • MullaneyB.C. et al.

    Developmental expression of methyl-CpG binding protein 2 is dynamically regulated in the rodent brain

    Neuroscience

    (2004)
  • NanX. et al.

    MeCP2 is a transcriptional repressor with abundant binding sites in genomic chromatin

    Cell

    (1997)
  • PhilippartM.

    Rett and Angelman's syndromes: models of arrested development

    Pediatr. Neurol.

    (2001)
  • ShahbazianM.D. et al.

    Rett syndrome and MeCP2: linking epigenetics and neuronal function

    Am. J. Hum. Genet.

    (2002)
  • SunY.E. et al.

    The ups and downs of BDNF in Rett syndrome

    Neuron

    (2006)
  • TaoX. et al.

    Ca2+ influx regulates BDNF transcription by a CREB family transcription factor-dependent mechanism

    Neuron

    (1998)
  • WanM. et al.

    Rett syndrome and beyond: recurrent spontaneous and familial MECP2 mutations at CpG hotspots

    Am. J. Hum. Genet.

    (1999)
  • ZivN.E. et al.

    Evidence for a role of dendritic filopodia in synaptogenesis and spine formation

    Neuron

    (1996)
  • AbuhatziraL. et al.

    MeCP2 deficiency in the brain decreases BDNF levels by REST/CoREST-mediated repression and increases TRKB production

    Epigenetics

    (2007)
  • AmaralM.D. et al.

    TRPC3 channels are necessary for brain-derived neurotrophic factor to activate a nonselective cationic current and to induce dendritic spine formation

    J. Neurosci.

    (2007)
  • AmirR.E. et al.

    Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2

    Nat. Genet.

    (1999)
  • Andres-BarquinP.J. et al.

    Id genes in nervous system development

    Histol. Histopathol.

    (2000)
  • ArmstrongD.D.

    Neuropathology of Rett syndrome

    Ment. Retard. Dev. Disabil. Res. Rev.

    (2002)
  • ArmstrongD. et al.

    Selective dendritic alterations in the cortex of Rett syndrome

    J. Neuropathol. Exp. Neurol.

    (1995)
  • BallestarE. et al.

    The impact of MECP2 mutations in the expression patterns of Rett syndrome patients

    Hum. Genet.

    (2005)
  • BankerG. et al.

    Culturing Nerve Cells

    (1998)
  • BienvenuT. et al.

    Molecular genetics of Rett syndrome: when DNA methylation goes unrecognized

    Nat. Rev., Genet.

    (2006)
  • BienvenuT. et al.

    MECP2 mutations account for most cases of typical forms of Rett syndrome

    Hum. Mol. Genet.

    (2000)
  • CaballeroI.M. et al.

    MeCP2 in neurons: closing in on the causes of Rett syndrome

    Hum. Mol. Genet.

    (2005)
  • ChahrourM. et al.

    MeCP2, a key contributor to neurological disease, activates and represses transcription

    Science

    (2008)
  • ChenG. et al.

    Relative contribution of endogenous neurotrophins in hippocampal long-term potentiation

    J. Neurosci.

    (1999)
  • ChenW.G. et al.

    Derepression of BDNF transcription involves calcium-dependent phosphorylation of MeCP2

    Science

    (2003)
  • ClineH. et al.

    The regulation of dendritic arbor development and plasticity by glutamatergic synaptic input: a review of the synaptotrophic hypothesis

    J. Physiol.

    (2008)
  • CohenS. et al.

    Medicine. Activating a repressor

    Science

    (2008)
  • CraigA.M. et al.

    Neuronal polarity

    Annu. Rev. Neurosci.

    (1994)
  • DaniV.S. et al.

    Reduced cortical activity due to a shift in the balance between excitation and inhibition in a mouse model of Rett syndrome

    Proc. Natl. Acad. Sci. U. S. A.

    (2005)
  • DelgadoI.J. et al.

    Expression profiling of clonal lymphocyte cell cultures from Rett syndrome patients

    BMC Med. Genet.

    (2006)
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