Jennifer L. Larimore

Dendritic spine pathologies in hippocampal pyramidal neurons from Rett syndrome brain and after expression of Rett-associated MECP2 mutations.

Rett syndrome (RTT) is an X chromosome-linked neurodevelopmental disorder associated with the characteristic neuropathology of dendritic spines common in diseases presenting with mental retardation (MR). Here, we present the first quantitative analyses of dendritic spine density in postmortem brain tissue from female RTT individuals, which revealed that hippocampal CA1 pyramidal neurons have lower spine density than age-matched non-MR female control individuals. The majority of RTT individuals carry mutations in MECP2, the gene coding for a methylated DNA-binding transcriptional regulator. While altered synaptic transmission and plasticity has been demonstrated in Mecp2-deficient mouse models of RTT, observations regarding dendritic spine density and morphology have produced varied results. We investigated the consequences of MeCP2 dysfunction on dendritic spine structure by overexpressing ( approximately twofold) MeCP2-GFP constructs encoding either the wildtype (WT) protein, or missense mutations commonly found in RTT individuals. Pyramidal neurons within hippocampal slice cultures transfected with either WT or mutant MECP2 (either R106W or T158M) showed a significant reduction in total spine density after 48 h of expression. Interestingly, spine density in neurons expressing WT MECP2 for 96 h was comparable to that in control neurons, while neurons expressing mutant MECP2 continued to have lower spine density than controls after 96 h of expression. Knockdown of endogenous Mecp2 with a specific small hairpin interference RNA (shRNA) also reduced dendritic spine density, but only after 96 h of expression. On the other hand, the consequences of manipulating MeCP2 levels for dendritic complexity in CA3 pyramidal neurons were only minor. Together, these results demonstrate reduced dendritic spine density in hippocampal pyramidal neurons from RTT patients, a distinct dendritic phenotype also found in neurons expressing RTT-associated MECP2 mutations or after shRNA-mediated endogenous Mecp2 knockdown, suggesting that this phenotype represent a cell-autonomous consequence of MeCP2 dysfunction.

Bdnf Overexpression in Hippocampal Neurons Prevents Dendritic Atrophy Caused by Rett-Associated MECP2 Mutations

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.

Quantitative Proteomic and Genetic Analyses of the Schizophrenia Susceptibility Factor Dysbindin Identify Novel Roles of the Biogenesis of Lysosome-Related Organelles Complex 1

The Biogenesis of Lysosome-Related Organelles Complex 1 (BLOC-1) is a protein complex containing the schizophrenia susceptibility factor dysbindin, which is encoded by the gene DTNBP1. However, mechanisms engaged by dysbindin defining schizophrenia susceptibility pathways have not been quantitatively elucidated. Here, we discovered prevalent and novel cellular roles of the BLOC-1 complex in neuronal cells by performing large-scale Stable Isotopic Labeling of Cells in Culture (SILAC) quantitative proteomics combined with genetic analyses in dysbindin-null mice (Mus musculus) and the genome of schizophrenia patients. We identified 24 proteins that associate with the BLOC-1 complex, many of which were altered in content/distribution in cells or tissues deficient in BLOC-1. New findings include BLOC-1 interactions with the COG complex, a Golgi apparatus tether, and antioxidant enzymes peroxiredoxins 1–2. Importantly, loci encoding eight of the 24 proteins are affected by genomic copy number variation in schizophrenia patients. Thus, our quantitative proteomic studies expand the functional repertoire of the BLOC-1 complex and provide insight into putative molecular pathways of schizophrenia susceptibility.

Modulation of dendritic spine development and plasticity by BDNF and vesicular trafficking: fundamental roles in neurodevelopmental disorders associated with mental retardation and autism

The process of axonal and dendritic development establishes the synaptic circuitry of the central nervous system (CNS) and is the result of interactions between intrinsic molecular factors and the external environment. One growth factor that has a compelling function in neuronal development is the neurotrophin brain-derived neurotrophic factor (BDNF). BDNF participates in axonal and dendritic differentiation during embryonic stages of neuronal development, as well as in the formation and maturation of dendritic spines during postnatal development. Recent studies have also implicated vesicular trafficking of BDNF via secretory vesicles, and both secretory and endosomal trafficking of vesicles containing synaptic proteins, such as neurotransmitter and neurotrophin receptors, in the regulation of axonal and dendritic differentiation, and in dendritic spine morphogenesis. Several genes that are either mutated or deregulated in neurodevelopmental disorders associated with mental retardation have now been identified, and several mouse models of these disorders have been generated and characterized. Interestingly, abnormalities in dendritic and synaptic structure are consistently observed in human neurodevelopmental disorders associated with mental retardation, and in mouse models of these disorders as well. Abnormalities in dendritic and synaptic differentiation are thought to underlie altered synaptic function and network connectivity, thus contributing to the clinical outcome. Here, we review the roles of BDNF and vesicular trafficking in axonal and dendritic differentiation in the context of dendritic and axonal morphological impairments commonly observed in neurodevelopmental disorders associated with mental retardation.

The actions of BDNF on dendritic spine density and morphology in organotypic slice cultures depend on the presence of serum in culture media.

We have previously shown that brain-derived neurotrophin factor (BDNF) increases dendritic spine density and the proportion of stubby spines in apical dendrites of CA1 pyramidal neurons of hippocampal slice cultures maintained in serum-free media. We show here that serum withdrawal causes an increase in the proportion of thin spines and a decrease in the fraction of stubby spines, without changing the overall density of dendritic spines. When slices are maintained in serum-containing media, BDNF also increased spine density but had the opposite effect on spine morphology: it increased the proportion of mushroom and thin spines and decreased the proportion of stubby spines. Intriguingly, slices maintained in serum media showed a lower p75NTR-to-TrkB expression level than serum-free slices, even after BDNF exposure. The differential actions of BDNF on spine morphology depending on the presence of serum in culture media, together with the difference in neurotrophin receptor expression are reminiscent of opposing functional signaling by p75NTR and Trk receptors, and reveal a complex modulation of dendritic morphology by BDNF signaling.

The schizophrenia susceptibility factor dysbindin and its associated complex sort cargoes from cell bodies to the synapse

A novel vesicle transport mechanism is described that requires dysbindin-associated complexes for cargo targeting from neuronal cell bodies to neurites and nerve terminals. The results suggest that mistargeting of specific vesicular cargoes may underlie, in part, the molecular pathogenesis of schizophrenia.

Activity-dependent BDNF release and TRPC signaling is impaired in hippocampal neurons of Mecp2 mutant mice

Dysfunction of the neurotrophin brain-derived neurotrophic factor (BDNF) is implicated in Rett syndrome (RTT), but the state of its releasable pool and downstream signaling in mice lacking methyl-CpG-binding protein-2 (Mecp2) is unknown. Here, we show that membrane currents and dendritic Ca2+ signals evoked by recombinant BDNF or an activator of diacylglycerol (DAG)-sensitive transient receptor potential canonical (TRPC) channels are impaired in CA3 pyramidal neurons of symptomatic Mecp2 mutant mice. TRPC3 and TRPC6 mRNA and protein levels are lower in Mecp2 mutant hippocampus, and chromatin immunoprecipitation (ChIP) identified Trpc3 as a target of MeCP2 transcriptional regulation. BDNF mRNA and protein levels are also lower in Mecp2 mutant hippocampus and dentate gyrus granule cells, which is reflected in impaired activity-dependent release of endogenous BDNF estimated from TRPC currents and dendritic Ca2+ signals in CA3 pyramidal neurons. These results identify the gene encoding TRPC3 channels as a MeCP2 target and suggest a potential therapeutic strategy to boost impaired BDNF signaling in RTT.

Cell Biology of the BLOC-1 Complex Subunit Dysbindin, a Schizophrenia Susceptibility Gene

There is growing interest in the biology of dysbindin and its genetic locus (DTNBP1) due to genetic variants associated with an increased risk of schizophrenia. Reduced levels of dysbindin mRNA and protein in the hippocampal formation of schizophrenia patients further support involvement of this locus in disease risk. Here, we discuss phylogenetically conserved dysbindin molecular interactions that define its contribution to the assembly of the biogenesis of lysosome-related organelles complex-1 (BLOC-1). We explore fundamental cellular processes where dysbindin and the dysbindin-containing BLOC-1 complex are implicated. We propose that cellular, tissue, and system neurological phenotypes from dysbindin deficiencies in model genetic organisms, and likely individuals affected with schizophrenia, emerge from abnormalities in few core cellular mechanisms controlled by BLOC-1-dysbindin-containing complex rather than from defects in dysbindin itself.

MeCP2 Regulates the Synaptic Expression of a Dysbindin-BLOC-1 Network Component in Mouse Brain and Human Induced Pluripotent Stem Cell-Derived Neurons

Clinical, epidemiological, and genetic evidence suggest overlapping pathogenic mechanisms between autism spectrum disorder (ASD) and schizophrenia. We tested this hypothesis by asking if mutations in the ASD gene MECP2 which cause Rett syndrome affect the expression of genes encoding the schizophrenia risk factor dysbindin, a subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), and associated interacting proteins. We measured mRNA and protein levels of key components of a dysbindin interaction network by, quantitative real time PCR and quantitative immunohistochemistry in hippocampal samples of wild-type and Mecp2 mutant mice. In addition, we confirmed results by performing immunohistochemistry of normal human hippocampus and quantitative qRT-PCR of human inducible pluripotent stem cells (iPSCs)-derived human neurons from Rett syndrome patients. We defined the distribution of the BLOC-1 subunit pallidin in human and mouse hippocampus and contrasted this distribution with that of symptomatic Mecp2 mutant mice. Neurons from mutant mice and Rett syndrome patients displayed selectively reduced levels of pallidin transcript. Pallidin immunoreactivity decreased in the hippocampus of symptomatic Mecp2 mutant mice, a feature most prominent at asymmetric synapses as determined by immunoelectron microcopy. Pallidin immunoreactivity decreased concomitantly with reduced BDNF content in the hippocampus of Mecp2 mice. Similarly, BDNF content was reduced in the hippocampus of BLOC-1 deficient mice suggesting that genetic defects in BLOC-1 are upstream of the BDNF phenotype in Mecp2 deficient mice. Our results demonstrate that the ASD-related gene Mecp2 regulates the expression of components belonging to the dysbindin interactome and these molecular differences may contribute to synaptic phenotypes that characterize Mecp2 deficiencies and ASD.

The neuronal Arf GAP centaurin α1 modulates dendritic differentiation

Centaurin α1 is an Arf GTPase-activating protein (GAP) that is highly expressed in the nervous system. In the current study, we show that endogenous centaurin α1 protein is localized in the synaptosome fraction, with peak expression in early postnatal development. In cultured dissociated hippocampal neurons, centaurin α1 localizes to dendrites, dendritic spines and the postsynaptic region. siRNA-mediated knockdown of centaurin α1 levels or overexpression of a GAP-inactive mutant of centaurin α1 leads to inhibition of dendritic branching, dendritic filopodia and spine-like protrusions in dissociated hippocampal neurons. Overexpression of wild-type centaurin α1 in cultured hippocampal neurons in early development enhances dendritic branching, and increases dendritic filopodia and lamellipodia. Both filopodia and lamellipodia have been implicated in dendritic branching and spine formation. Following synaptogenesis in cultured neurons, wild-type centaurin α1 expression increases dendritic filopodia and spine-like protrusions. Expression of a GAP-inactive mutant diminishes spine density in CA1 pyramidal neurons within cultured organotypic hippocampal slice cultures. These data support the conclusion that centaurin α1 functions through GAP-dependent Arf regulation of dendritic branching and spines that underlie normal dendritic differentiation and development.