Dendritic spine pathologies in hippocampal pyramidal neurons from Rett syndrome brain and after expression of Rett-associated MECP2 mutations
Introduction
Deficits in dendritic architecture are common in disorders associated with mental retardation (MR), ranging from environmental (e.g. fetal alcohol syndrome) to autosomal (e.g. Down syndrome) and X chromosome-linked forms of MR (e.g. Fragile-X, Rett syndrome; reviewed by Fiala et al., 2002, Kaufmann and Moser, 2000). A series of groundbreaking studies published in the 1970s established the precedent of abnormalities in dendritic organization in cortical neurons from humans with MR (Huttenlocher, 1970, Huttenlocher, 1974, Marin-Padilla, 1972, 1976). These series of observations consistently described reductions in the density of dendritic spines, the postsynaptic compartment of excitatory synapses (reviewed by Peters et al., 1991). In addition, a prevalence of long and tortuous spines, thought to represent immature synapses, were also commonly observed in MR and combined, these dendritic spine anomalies were referred to as “spine dysgenesis” (Purpura, 1974). Since dendritic development is a process where the formation and maturation of spines are the result of interactions between intrinsic genetic factors and external environment, the study of spine development and maintenance in MR has significant clinical relevance.
Rett syndrome (RTT) is an X chromosome-linked mental retardation that results from sporadic mutations in the gene coding for the methylated DNA-binding transcription factor MeCP2 (Amir et al., 1999; reviewed by Percy, 2001). RTT affects approximately 1:10,000 females worldwide, and prominent symptoms include deceleration of body and head growth rate, hand stereotypies and regression in motor and speech capabilities, irregularities in motor activity and breathing patterns, in addition to cognitive impairments characteristic of an autism-spectrum disorder (reviewed by Percy and Lane, 2005). Although RTT occurs predominantly in females, mutations of MECP2 have also been identified in males, where the phenotypes range from severe encephalopathy, to classic RTT, to non-specific MR (Couvert et al., 2001, Kankirawatana et al., 2006, Masuyama et al., 2005, Moog et al., 2003). While mutations of MECP2 have been associated with RTT, duplications in the chromosomal region where MECP2 is located were shown to be related to neurological disorders associated with MR, suggesting that MECP2 is a critical dosage-sensitive gene (del Gaudio et al., 2006, Smyk et al., 2008).
Morphological studies in postmortem brain samples from RTT individuals described a characteristic neuropathology, which included decreased neuronal size and increased neuronal density in the cerebral cortex, hypothalamus and the hippocampal formation (Bauman et al., 1995a,b); decreased dendritic growth in pyramidal neurons of the subiculum and frontal and motor cortices (Armstrong et al., 1995); as well as the characteristic MR-associated spine dysgenesis, observed in pyramidal neurons of the motor cortex with regions of dendrites devoid of spines (Belichenko et al., 1994). The reduction in dendritic area together with the marked decrease in dendritic spine density strongly suggests that impaired synaptic transmission is a likely pathogenic consequence of MECP2 mutations causing RTT. Indeed, the increase in neuronal expression of MECP2/Mecp2 during early brain development suggests the importance of this protein in synapse formation and maintenance (Akbarian et al., 2001, Cassel et al., 2004, Jung et al., 2003, Kaufmann et al., 2005, Mullaney et al., 2004, Shahbazian et al., 2002b). While hippocampal and cortical synaptic dysfunction in Mecp2-based mouse models of RTT has been extensively studied, observations regarding neuronal, dendritic and synaptic pathologies have produced varied results (Asaka et al., 2006, Chao et al., 2007, Dani et al., 2005, Fukuda et al., 2005, Gemelli et al., 2006, Jugloff et al., 2005, Kishi and Macklis, 2004, Moretti et al., 2006, Nelson et al., 2006, Smrt et al., 2007, Zhou et al., 2006). Here, we present the first quantitative analyses of dendritic spine density in postmortem brain tissue from RTT individuals. To identify the consequences of cell-autonomous MeCP2 dysfunction on the morphology of dendrites and dendritic spines in hippocampal pyramidal neurons, we transfected organotypic slice cultures with either wildtype or RTT-associated MECP2 mutations.
Section snippets
Human postmortem brain
All procedures on human postmortem brain samples followed national and international ethics guidelines, and were reviewed and approved by the Institutional Review Board (IRB) at The University of Alabama at Birmingham (UAB). Brain sections containing the hippocampal formation were obtained from individuals diagnosed with RTT, and unaffected (non-MR) individuals served as controls. Postmortem human tissue was obtained from the NICHD Brain and Tissue Bank for Developmental Disorders at the
Hippocampal CA1 pyramidal neurons from RTT individuals have lower dendritic spine density than those from non-MR individuals
The only prior study on the features of dendritic spines in RTT individuals was performed on cortical samples and lacked quantitative statistical analyses (Belichenko et al., 1994). To perform quantitative analyses of spine density in pyramidal neurons from human hippocampus, postmortem tissue was obtained from female RTT individuals that were 1 to 42 years of age, and compared to age-matched unaffected non-MR female individuals (see Table 1 for all available information from the human brain
Discussion
Here, we present several novel observations regarding dendritic spine dysgenesis in RTT individuals and the role of the transcriptional regulator MeCP2 on dendritic complexity and dendritic spine density and morphology. First, we show that CA1 pyramidal neurons of the hippocampus in female individuals with RTT have lower dendritic spine density than age-matched unaffected (non-MR) female individuals, as observed qualitatively in pyramidal neurons of the motor cortex (Belichenko et al., 1994).
Acknowledgments
Supported by NIH grants NS40593 and NS057780, IRSF and the Civitan International Foundation (LP-M). We also thank the assistance of the UAB Intellectual and Developmental Disabilities Research Center (IDDRC; P30-HD38985) and the UAB Neuroscience Cores (P30-NS47466, P30-NS57098). Human tissue was obtained from the NICHD Brain and Tissue Bank for Developmental Disorders at the University of Maryland, Baltimore, MD, and the Harvard Brain Bank. We thank Dr. Carolyn Schanen (Nemours Biomedical
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