A brain region-specific predictive gene map for autism derived by profiling a reference gene set.

Molecular underpinnings of complex psychiatric disorders such as autism spectrum disorders (ASD) remain largely unresolved. Increasingly, structural variations in discrete chromosomal loci are implicated in ASD, expanding the search space for its disease etiology. We exploited the high genetic heterogeneity of ASD to derive a predictive map of candidate genes by an integrated bioinformatics approach. Using a reference set of 84 Rare and Syndromic candidate ASD genes (AutRef84), we built a composite reference profile based on both functional and expression analyses. First, we created a functional profile of AutRef84 by performing Gene Ontology (GO) enrichment analysis which encompassed three main areas: 1) neurogenesis/projection, 2) cell adhesion, and 3) ion channel activity. Second, we constructed an expression profile of AutRef84 by conducting DAVID analysis which found enrichment in brain regions critical for sensory information processing (olfactory bulb, occipital lobe), executive function (prefrontal cortex), and hormone secretion (pituitary). Disease specificity of this dual AutRef84 profile was demonstrated by comparative analysis with control, diabetes, and non-specific gene sets. We then screened the human genome with the dual AutRef84 profile to derive a set of 460 potential ASD candidate genes. Importantly, the power of our predictive gene map was demonstrated by capturing 18 existing ASD-associated genes which were not part of the AutRef84 input dataset. The remaining 442 genes are entirely novel putative ASD risk genes. Together, we used a composite ASD reference profile to generate a predictive map of novel ASD candidate genes which should be prioritized for future research.

Animal model integration to AutDB, a genetic database for autism.

BACKGROUND: In the post-genomic era, multi-faceted research on complex disorders such as autism has generated diverse types of molecular information related to its pathogenesis. The rapid accumulation of putative candidate genes/loci for Autism Spectrum Disorders (ASD) and ASD-related animal models poses a major challenge for systematic analysis of their content. We previously created the Autism Database (AutDB) to provide a publicly available web portal for ongoing collection, manual annotation, and visualization of genes linked to ASD. Here, we describe the design, development, and integration of a new module within AutDB for ongoing collection and comprehensive cataloguing of ASD-related animal models. DESCRIPTION: As with the original AutDB, all data is extracted from published, peer-reviewed scientific literature. Animal models are annotated with a new standardized vocabulary of phenotypic terms developed by our researchers which is designed to reflect the diverse clinical manifestations of ASD. The new Animal Model module is seamlessly integrated to AutDB for dissemination of diverse information related to ASD. Animal model entries within the new module are linked to corresponding candidate genes in the original "Human Gene" module of the resource, thereby allowing for cross-modal navigation between gene models and human gene studies. Although the current release of the Animal Model module is restricted to mouse models, it was designed with an expandable framework which can easily incorporate additional species and non-genetic etiological models of autism in the future. CONCLUSIONS: Importantly, this modular ASD database provides a platform from which data mining, bioinformatics, and/or computational biology strategies may be adopted to develop predictive disease models that may offer further insights into the molecular underpinnings of this disorder. It also serves as a general model for disease-driven databases curating phenotypic characteristics of corresponding animal models.

Flotillin-1 promotes formation of glutamatergic synapses in hippocampal neurons.

Synapse malformation underlies numerous neurodevelopmental illnesses, including autism spectrum disorders. Here we identify the lipid raft protein flotillin-1 as a promoter of glutamatergic synapse formation. We cultured neurons from the hippocampus, a brain region important for learning and memory, and examined them at two weeks in vitro, a time period rich with synapse formation. Double-label immunocytochemistry of native flot-1 with glutamatergic and GABAergic synapse markers showed that flot-1 was preferentially colocalized with the glutamatergic presynaptic marker vesicular glutamate transporter 1 (VGLUT1), compared to the GABAergic presynaptic marker glutamic acid decarboxylase-65 (GAD-65). Triple-label immunocytochemistry of native flot-1, VGLUT1, and NR1, the obligatory subunit of NMDA receptors, indicates that Flot-1 was preferentially localized to synaptic rather than extrasynaptic NR1. Furthermore, electrophysiological results using whole-cell patch clamp showed that Flot-1 increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) but not miniature inhibitory postsynaptic currents (mIPSCs), whereas amplitude and decay kinetics of either type of synaptic current was not affected. Corresponding immunocytochemical data confirmed that the number of glutamatergic synapses increased with flot-1 overexpression. Overall, our anatomical and physiological results show that flot-1 enhances the formation of glutamatergic synapses but not GABAergic synapses, suggesting that the role of flot-1 in neurodevelopmental disorders should be explored.

Flotillin-1 mediates neurite branching induced by synaptic adhesion-like molecule 4 in hippocampal neurons.

Proper development of neurons in the hippocampus is essential for learning and memory. Our laboratory previously discovered a family of synaptic adhesion-like molecules (SALMs) which induce neurite outgrowth in this brain region (Wang et al., 2006). Here we establish flotillin-1 (flot-1) as a molecular mediator of neurite branching for SALM4. Knockdown of flot-1 alone in cultured hippocampal neurons using siRNA from 3-7days in vitro (DIV) impaired neurite branching, whereas overexpression of flot-1 during the same time period increased the number of processes and branching. We show that induction of neurite outgrowth by flot-1 depends on amino acids 134-151 as well as lipid raft microdomains, SoHo proteins to regulate the actin cytoskeleton, and the exocyst complex to deliver new membrane proteins to growing neurites. When each of SALMs 1-5 was overexpressed, siRNA knockdown of flot-1 prevented neurite branching by SALM4. Overall, our data reveal a flot-1 signaling pathway for hippocampal neurite branching that is regulated by SALM4.

NMDA receptors interact with flotillin-1 and -2, lipid raft-associated proteins.

N-methyl-D-aspartate receptors (NMDARs) mediate excitatory synaptic transmission in the brain. Here we demonstrate interactions between the NR2A and NR2B subunits of NMDARs with flotillin-1 (flot-1), a lipid raft-associated protein. When mapped, analogous regions in the far distal C-termini of NR2A and NR2B mediate binding to flot-1, and the prohibitin homology domain of flot-1 contains binding sites for NR2A and NR2B. Although NR2B can also directly bind to flot-2 via a separate region of its distal C-terminus, NMDARs were significantly more colocalized with flot-1 than flot-2 in cultured hippocampal neurons. Overall, this study defines a novel interaction between NMDARs and flotillins.

The role of the PDZ protein GIPC in regulating NMDA receptor trafficking.

The NMDA receptor is an important component of excitatory synapses in the CNS. In addition to its synaptic localization, the NMDA receptor is also present at extrasynaptic sites where it may have functions distinct from those at the synapse. Little is known about how the number, composition, and localization of extrasynaptic receptors are regulated. We identified a novel NMDA receptor-interacting protein, GIPC (GAIP-interacting protein, C terminus), that associates with surface as well as internalized NMDA receptors when expressed in heterologous cells. In neurons, GIPC colocalizes with a population of NMDA receptors on the cell surface, and changes in GIPC expression alter the number of surface receptors. GIPC is mainly excluded from the synapse, and changes in GIPC expression do not change the total number of synaptic receptors. Our results suggest that GIPC may be preferentially associated with extrasynaptic NMDA receptors and may play a role in the organization and trafficking of this population of receptors.

Development of gamma-aminobutyric acidergic synapses in cultured hippocampal neurons.

The formation and maturation of gamma-aminobutyric acid (GABA)-ergic synapses was studied in cultured hippocampal pyramidal neurons by both performing immunocytochemistry for GABAergic markers and recording miniature inhibitory postsynaptic currents (mIPSCs). Nascent GABAergic synapses appeared between 3 and 8 days in vitro (DIV), with GABAA receptor subunit clusters appearing first, followed by GAD-65 puncta, then functional synapses. The number of GABAergic synapses increased from 7 to 14 DIV, with a corresponding increase in frequency of mIPSCs. Moreover, these new GABAergic synapses formed on neuronal processes farther from the soma, contributing to decreased mIPSC amplitude and slowed mIPSC 19-90% rise time. The mIPSC decay quickened from 7 to 14 DIV, with a parallel change in the distribution of the alpha5 subunit from diffuse expression at 7 DIV to clustered expression at 14 DIV. These alpha5 clusters were mostly extrasynaptic. The alpha1 subunit was expressed as clusters in none of the neurons at 7 DIV, in 20% at 14 DIV, and in 80% at 21 DIV. Most of these alpha1 clusters were expressed at GABAergic synapses. In addition, puncta of GABA transporter 1 (GAT-1) were localized to GABAergic synapses at 14 DIV but were not expressed at 7 DIV. These studies demonstrate that mIPSCs appear after pre- and postsynaptic elements are in place. Furthermore, the process of maturation of GABAergic synapses involves increased synapse formation at distal processes, expression of new GABAA receptor subunits, and GAT-1 expression at synapses; these changes are reflected in altered frequency, kinetics, and drug sensitivity of mIPSCs.

Activity-dependent scaling of GABAergic synapse strength is regulated by brain-derived neurotrophic factor.

The homeostatic plasticity hypothesis suggests that neuronal activity scales synaptic strength. This study analyzed effects of activity deprivation on GABAergic synapses in cultured hippocampal neurons using patch clamp electrophysiology to record mIPSCs and immunocytochemistry to visualize presynaptic GAD-65 and the gamma2 subunit of the GABA(A) receptor. When neural activity was blocked for 48 h with tetrodotoxin (TTX, 1 microM), the amplitude of mIPSCs was reduced, corresponding with diminished sizes of GAD-65 puncta and gamma2 clusters. Treatment with the NMDA receptor antagonist APV (50 microM) or the AMPA receptor antagonist DNQX (20 microM) mimicked these effects, and co-application of brain-derived neurotrophic factor (BDNF, 100 ng/mL) overcame them. Moreover, when neurons were treated with BDNF alone for 48 h, these effects were reversed via the TrkB receptor. Overall, these results suggest that activity-dependent scaling of inhibitory synaptic strength can be modulated by BDNF/TrkB-mediated signaling.

Synaptic and extrasynaptic localization of brain-derived neurotrophic factor and the tyrosine kinase B receptor in cultured hippocampal neurons.

Brain-derived neurotrophic factor (BDNF) regulates synapses, but the distribution of BDNF and its receptor TrkB relative to the location of glutamatergic and gamma-aminobutyric acidergic (GABAergic) synapses is presently unknown. Immunocytochemistry was performed in primary hippocampal neuron cultures to determine whether BDNF and TrkB are preferentially localized to excitatory or inhibitory markers at 7, 14, and 21 days in vitro (DIV). Glutamatergic sites were localized with vesicular glutamate transporter type 1 (VGLUT1) as presynaptic marker and the NR1 subunit of the NMDA receptor and the GluR1 subunit of the AMPA receptor as receptor markers. GABAergic sites were labeled with the 65-kDa isoform of glutamic acid decarboxylase (GAD-65) as presynaptic marker and the gamma2 subunit of the GABAA receptor as receptor marker. During development, <30% of BDNF punctae and TrkB clusters were localized to glutamatergic and GABAergic markers. Because their rates of colocalization did not change from 7 to 21 DIV, this study details the distribution of BDNF and TrkB at 14 DIV. BDNF was preferentially colocalized with glutamatergic markers VGLUT1 and NR1 ( approximately 30% each). TrkB was also relatively highly colocalized with VGLUT1 and NR1 ( approximately 20% each) but was additionally highly colocalized with GABAergic markers GAD-65 ( approximately 20%) and gamma2 ( approximately 30%). NR1 clusters colocalized with BDNF puncta and TrkB clusters were mostly extrasynaptic, as were gamma2 clusters colocalized with TrkB clusters. These results show that, whereas most BDNF and TrkB protein is extrasynaptic, BDNF is preferentially associated with excitatory markers and that TrkB is associated equally with excitatory and inhibitory markers.