Quantitative and functional interrogation of parent-of-origin allelic expression biases in the brain.

The maternal and paternal genomes play different roles in mammalian brains as a result of genomic imprinting, an epigenetic regulation leading to differential expression of the parental alleles of some genes. Here we investigate genomic imprinting in the cerebellum using a newly developed Bayesian statistical model that provides unprecedented transcript-level resolution. We uncover 160 imprinted transcripts, including 41 novel and independently validated imprinted genes. Strikingly, many genes exhibit parentally biased--rather than monoallelic--expression, with different magnitudes according to age, organ, and brain region. Developmental changes in parental bias and overall gene expression are strongly correlated, suggesting combined roles in regulating gene dosage. Finally, brain-specific deletion of the paternal, but not maternal, allele of the paternally-biased Bcl-x, (Bcl2l1) results in loss of specific neuron types, supporting the functional significance of parental biases. These findings reveal the remarkable complexity of genomic imprinting, with important implications for understanding the normal and diseased brain.

The major histocompatibility complex and autism spectrum disorder.

Autism spectrum disorder (ASD) is a complex disorder that appears to be caused by interactions between genetic changes and environmental insults during early development. A wide range of factors have been linked to the onset of ASD, but recently both genetic associations and environmental factors point to a central role for immune-related genes and immune responses to environmental stimuli. Specifically, many of the proteins encoded by the major histocompatibility complex (MHC) play a vital role in the formation, refinement, maintenance, and plasticity of the brain. Manipulations of levels of MHC molecules have illustrated how disrupted MHC signaling can significantly alter brain connectivity and function. Thus, an emerging hypothesis in our field is that disruptions in MHC expression in the developing brain caused by mutations and/or immune dysregulation may contribute to the altered brain connectivity and function characteristic of ASD. This review provides an overview of the structure and function of the three classes of MHC molecules in the immune system, healthy brain, and their possible involvement in ASD.

MHCI negatively regulates synapse density during the establishment of cortical connections.

Major histocompatibility complex class I (MHCI) molecules modulate activity-dependent refinement and plasticity. We found that MHCI also negatively regulates the density and function of cortical synapses during their initial establishment both in vitro and in vivo. MHCI molecules are expressed on cortical neurons before and during synaptogenesis. In vitro, decreasing surface MHCI (sMHCI) on neurons increased glutamatergic and GABAergic synapse density, whereas overexpression decreased it. In vivo, synapse density was higher throughout development in ß2m(-/-) mice. MHCI also negatively regulated the strength of excitatory, but not inhibitory, synapses and controlled the balance of excitation and inhibition onto cortical neurons. sMHCI levels were modulated by activity and were necessary for activity to negatively regulate glutamatergic synapse density. Finally, acute changes in sMHCI and activity altered synapse density exclusively during early postnatal development. These results identify a previously unknown function for immune proteins in the negative regulation of the initial establishment and function of cortical connections.

MHC class I molecules are present both pre- and postsynaptically in the visual cortex during postnatal development and in adulthood.

Immune molecules have been discovered recently to play critical roles in the development, function, and plasticity of the cerebral cortex. MHC class I (MHCI) molecules are expressed in the central nervous system and regulate activity-dependent refinement of visual projections during late postnatal development. They have also been implicated in neurodevelopmental diseases such as schizophrenia and autism. Despite the excitement generated by these unique roles for immune proteins in the brain, little is known about how these molecules regulate cortical connections. The first step toward elucidating the mechanism is to identify the spatial and temporal distribution of MHCI proteins throughout development. Using a pan-specific antibody that recognizes many MHCI variants for biochemistry and immunohistochemistry, we found that MHCI proteins are expressed in the rat visual cortex at all ages examined-during the peak of synaptogenesis, the critical period of synaptic refinement, and adulthood. Their abundance in the cortex peaked during early postnatal development, declining during periods of plasticity and adulthood. In contrast to current assumptions, pre- and postembedding immunogold electron microscopy (EM) revealed that MHCI proteins were present both pre- and postsynaptically at all ages examined. They were often found in the postsynaptic density and were closely associated with synaptic vesicles in the presynaptic terminal. These results suggest a previously undescribed model in which MHCI molecules function on both sides of the synapse to regulate connectivity in the mammalian visual cortex before, during, and after the establishment of connections.

Seeing the light: insulin receptors and the CNS.

Although insulin clearly affects brain function, the role of insulin receptor (IR) signaling in the establishment and function of circuits in vivo remains largely unknown. In this issue of Neuron, Chiu et al. show a role for IRs in regulating synapse density and dendritic plasticity required for visual responses in Xenopus.

Actinfilin is a Cul3 substrate adaptor, linking GluR6 kainate receptor subunits to the ubiquitin-proteasome pathway.

Kainate receptors have been implicated in excitotoxic neuronal death induced by diseases such as epilepsy and stroke. Actinfilin, a synaptic member of the BTB-Kelch protein family, is known to bind to the actin cytoskeleton. However, little is understood about its function at the synapse. Here, we report that actinfilin is able to bind to GluR6, a kainate-type glutamate receptor subunit, and target GluR6 for degradation. Like many members of its protein family, actinfilin acts as a substrate adaptor, binding Cullin 3 (Cul3) and linking GluR6 to the E3 ubiquitin-ligase complex. We map this interaction to the Kelch repeat domain of actinfilin and the GluR6 C terminus. Co-immunoprecipitation and immunofluorescence studies show that GluR6 is ubiquitinated, and that GluR6 levels are decreased by actinfilin overexpression but increased when actinfilin levels are reduced by specific RNA interference. Furthermore, actinfilin-Cul3 interactions appear to be important for regulating surface GluR6 expression. Synaptic GluR6 levels are elevated in mice with lowered neuronal Cul3 expression and when dominant-negative forms of Cul3 are transfected into hippocampal neurons. Together our data demonstrate that actinfilin acts as a scaffold, linking GluR6 to the Cul3 ubiquitin ligase to provide a novel mechanism for kainate receptor degradation.

Insulin-like growth factor-1 modulation of CaV1.3 calcium channels depends on Ca2+ release from IP3-sensitive stores and calcium/calmodulin kinase II phosphorylation of the alpha1 subunit EF hand.

In neurons, L-type calcium channels (CaV1.2 and CaV1.3) regulate an extensive range of functions. However, the roles of CaV1.3-containing L channels, which are physiologically and pharmacologically distinct from the better understood CaV1.2 channels, are only beginning to be determined. We find that CaV1.3 channels are modulated by the insulin-like growth factor-1/receptor tyrosine kinase (IGF-1/RTK) through a signaling pathway that involves phospholipase C, calcium release from IP3-sensitive internal stores, and calcium/calmodulin kinase II. In addition, we find that the IGF-1-induced modulation requires phosphorylation of a specific serine residue, S1486, in the EF hand motif of the CaV1.3 subunit. This modulation alters CaV1.3 activity, causing a left shift in the current-voltage relationship and strongly potentiating peak currents at hyperpolarized membrane potentials. We also find that CaV1.3 channels and their RTK-dependent potentiation contribute to the regulation of the survival-promoting transcription factor cAMP response element-binding protein (CREB): in both cortical and hippocampal neurons, depolarization and IGF-1 rapidly increase phospho-CREB levels in a manner that requires CaV1.3 activity and the S1486 phosphorylation site to achieve a full effect. Although the full effects of CaV1.3 channels remain to be determined, their preferential localization to dendritic shafts and spine heads coupled with their ability to activate at relatively hyperpolarized and even subthreshold potentials suggests that CaV1.3 activity may subserve different cellular functions from CaV1.2 and, in particular, may be important in transducing signals initiated by excitatory neurotransmission.

Cell surface expression of GluR5 kainate receptors is regulated by an endoplasmic reticulum retention signal.

Kainate receptors (KARs) are mediators of excitatory neurotransmission in the mammalian central nervous system, and their efficient targeting and trafficking is critical for normal synaptic function. A key step in the delivery of KARs to the neuronal plasma membrane is the exit of newly assembled receptors from the endoplasmic reticulum (ER). Here we report the identification of a novel ER retention signal in the alternatively spliced C-terminal domain of the GluR5-2b subunit, which controls receptor trafficking in both heterologous cells and neurons. The ER retention motif consists of a critical arginine (Arg-896) and surrounding amino acids, disruption of which promotes ER exit and surface expression of the receptors, as well as altering their physiological properties. The Arg-896-mediated ER retention of GluR5 is regulated by a mutation that mimics phosphorylation of Thr-898, but not by PDZ interactions. Furthermore, two positively charged residues (Arg-900 and Lys-901) in the C terminus were also found to regulate ER export of the receptors. Taken together, our results identify novel trafficking signals in the C-terminal domain of GluR5-2b and demonstrate that alternative splicing is an important mechanism regulating KAR function.

N-cadherin regulates ingrowth and laminar targeting of thalamocortical axons.

Thalamocortical axons are precisely targeted to cortical layer IV, but the identity of specific molecules that govern the establishment of laminar specificity in the thalamocortical projection has been elusive. In this study, we test the role of N-cadherin, a homophilic cell adhesion molecule, in laminar targeting of thalamocortical axons using cocultured thalamic and cortical slice explants exposed to N-cadherin function-blocking antibodies or inhibitory peptides. In untreated cocultures, labeled thalamocortical axons normally grow to and stop in layer IV, forming terminal-like arbors. In the N-cadherin-blocked cocultures, thalamic axons reach layer IV by growing through deep layers at the same rate as those in the untreated cocultures, but instead of terminating in layer IV, they continue growing uninterruptedly through layer IV and extend into supragranular layers to reach the outermost cortical edge, where some form terminal-like arbors in this aberrant laminar position. In cocultures in which the cortical slice is taken at an earlier maturational stage, one that corresponds to a time when thalamic axons are normally growing through deep layers before the emergence of layer IV from the cortical plate, thalamic axon ingrowth through deep layers is significantly attenuated by N-cadherin blocking reagents. These data indicate that N-cadherin has multifaceted roles in establishing the thalamocortical projection, governing aspects of both thalamic axon ingrowth and laminar targeting by acting as a layer IV stop signal, which progressively change in parallel with the maturational state of the cortex.