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

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.

Figure 1. shRNA-mediated Knockdown of Endogenous Mecp2 Reduced Dendritic Length in Pyramidal Hippocampal Neurons, without Affecting Axonal Morphology

A. Representative examples of 4 div hippocampal neurons transfected with a shRNA control sequence (left) or shRNA interfering sequence to knockdown endogenous Mecp2 (right). Neurons were co-transfected with eGFP (green) to perform quantitative morphological analyses (scale bar = 10μm). B. Population data on axonal length and branch points. C. Population data on dendritic length and branch points. In this and all subsequent figures, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001, from unpaired Student's t test or one-way ANOVA (see text for details). Additionally, in this and all subsequent figures, arrowheads indicate the position of the axons.

Figure 2. The Overexpression of Wildtype MECP2 Increased Dendritic Branching and Axonal Growth, while the Overexpression of RTT-Associated MECP2 Mutations Reduced Dendritic and Axonal Development

A. Representative examples of neurons transfected with either a control empty vector, a plasmid to overexpress wildtype human MECP2, or plasmids to overexpress to different missense MECP2 mutations (R106W or T158M) commonly found in Rett syndrome patients. Neurons were co-transfected with eGFP to perform quantitative morphological analyses (scale bar = 10μm). B. Population data on axonal length and branch points. C. Population data on dendritic length and branch points.

Figure 3. Mecp2 Knockdown or Wildtype MECP2 Overexpression Increased Intracellular BDNF Levels, while RTT-associated MECP2 Mutants Did Not Affect BDNF Levels

A. Representative examples of BDNF immunostaining (red) in hippocampal neurons transfected with different expression plasmids (green). Neurons were transfected with expression plasmids to overexpress BDNF-GFP, wildtype MECP2 or MECP2 mutations tagged with GFP. Neurons were also transfected with an shRNA sequence to knockdown endogenous Mecp2 and eGFP (scale bar = 10μm). Images fields always included two or more transfected neurons (i.e. GFP positive) and untransfected neurons (i.e. GFP negative) to perform quantitative comparisons of BDNF immunofluorescence staining. B. Population data on BDNF immunofluorescence intensity normalized to untransfected neurons within the same fields of view.

Figure 4. The BDNF Scavenger TrkB-Fc Prevented the Increase in Dendritic Branching Induced by Wildtype MECP2 Overexpression, without Affecting Axonal Growth

A. Representative examples of hippocampal neurons co-transfected with a control GFP plasmid and a plasmid to overexpress wildtype MECP2 (scale bar = 10μm). For quantitative morphological analysis, the neurons were co-transfected with eGFP (green) or stained with anti-α-tubulin antibodies (blue). B. Population data on axonal length and branch points. C. Population data on dendritic length and branch points.

Figure 5. Bdnf Overexpression Rescued Dendritic Atrophy Caused by Mecp2 Knockdown, Increasing Dendritic Length as much as in BDNF-Transfected Neurons

A. Representative examples of neurons transfected with an shRNA control plasmid, an shRNA plasmid to knockdown endogenous Mecp2, and a plasmid to overexpress Bdnf (scale bar = 10μm). For quantitative morphological analyses, neurons were co-transfected with eGFP (green) or stained with anti-α-tubulin antibodies (blue). B. Population data on axonal length and branch points. C. Population data on dendritic length and branch points.

Figure 6. Bdnf Overexpression Partially Rescued Dendritic Atrophy in Neurons Expressing RTT-Associated MECP2 Mutations, Increasing Dendrite Length Only to Control Levels

A. Representative examples of neurons transfected with a control GFP plasmid, plasmids to overexpress to different missense MECP2 mutations (R106W or T158M) commonly found in Rett syndrome patients, and a plasmid to overexpress Bdnf (scale bar = 10μm). For quantitative morphological analyses, neurons were co-transfected with eGFP (green) or stained with anti-α-tubulin antibodies (blue). B. Population data on axonal length and branch points. C. Population data on dendritic length and branch points.