Control of interneuron dendritic growth through NRG1/erbB4-mediated kalirin-7 disinhibition.

Neuregulin 1 (NRG1) is a secreted trophic factor that activates the postsynaptic erbB4 receptor tyrosine kinase. Both NRG1 and erbB4 have been repeatedly associated with schizophrenia, but their downstream targets are not well characterized. ErbB4 is highly abundant in interneurons, and NRG1-mediated erbB4 activation has been shown to modulate interneuron function, but the role for NRG1-erbB4 signaling in regulating interneuron dendritic growth is not well understood. Here we show that NRG1/erbB4 promote the growth of dendrites in mature interneurons through kalirin, a major dendritic Rac1-GEF. Recent studies have shown associations of the KALRN gene with schizophrenia. Our data point to an essential role of phosphorylation in kalirin-7's C terminus as the critical site for these effects. As reduced interneuron dendrite length occurs in schizophrenia, understanding how NRG1-erbB4 signaling modulates interneuron dendritic morphogenesis might shed light on disease-related alterations in cortical circuits

Evolution of genes and repeats in the Nimrod superfamily

The recently identified Nimrod superfamily is characterized by the presence of a special type of EGF repeat, the NIM repeat, located right after a typical CCXGY/W amino acid motif. On the basis of structural features, nimrod genes can be divided into three types. The proteins encoded by Draper-type genes have an EMI domain at the N-terminal part and only one copy of the NIM motif, followed by a variable number of EGF-like repeats. The products of Nimrod B-type and Nimrod C-type genes (including the eater gene) have different kinds of N-terminal domains, and lack EGF-like repeats but contain a variable number of NIM repeats. Draper and Nimrod C-type (but not Nimrod B-type) proteins carry a transmembrane domain. Several members of the superfamily were claimed to function as receptors in phagocytosis and/or binding of bacteria, which indicates an important role in the cellular immunity and the elimination of apoptotic cells. In this paper, the evolution of the Nimrod superfamily is studied with various methods on the level of genes and repeats. A hypothesis is presented in which the NIM repeat, along with the EMI domain, emerged by structural reorganizations at the end of an EGF-like repeat chain, suggesting a mechanism for the formation of novel types of repeats. The analyses revealed diverse evolutionary patterns in the sequences containing multiple NIM repeats. Although in the Nimrod B and Nimrod C proteins show characteristics of independent evolution, many internal NIM repeats in Eater sequences seem to have undergone concerted evolution. An analysis of the nimrod genes has been performed using phylogenetic and other methods and an evolutionary scenario of the origin and diversification of the Nimrod superfamily is proposed. Our study presents an intriguing example how the evolution of multigene families may contribute to the complexity of the innate immune response

High-Throughput Single-Cell Manipulation in Brain Tissue

The complexity of neurons and neuronal circuits in brain tissue requires the genetic manipulation, labeling, and tracking of single cells. However, current methods for manipulating cells in brain tissue are limited to either bulk techniques, lacking single-cell accuracy, or manual methods that provide single-cell accuracy but at significantly lower throughputs and repeatability. Here, we demonstrate high-throughput, efficient, reliable, and combinatorial delivery of multiple genetic vectors and reagents into targeted cells within the same tissue sample with single-cell accuracy. Our system automatically loads nanoliter-scale volumes of reagents into a micropipette from multiwell plates, targets and transfects single cells in brain tissues using a robust electroporation technique, and finally preps the micropipette by automated cleaning for repeating the transfection cycle. We demonstrate multi-colored labeling of adjacent cells, both in organotypic and acute slices, and transfection of plasmids encoding different protein isoforms into neurons within the same brain tissue for analysis of their effects on linear dendritic spine density. Our platform could also be used to rapidly deliver, both ex vivo and in vivo, a variety of genetic vectors, including optogenetic and cell-type specific agents, as well as fast-acting reagents such as labeling dyes, calcium sensors, and voltage sensors to manipulate and track neuronal circuit activity at single-cell resolution

PAK1 Protein Expression in the Auditory Cortex of Schizophrenia Subjects

Deficits in auditory processing are among the best documented endophenotypes in schizophrenia, possibly due to loss of excitatory synaptic connections. Dendritic spines, the principal post-synaptic target of excitatory projections, are reduced in schizophrenia. p21-activated kinase 1 (PAK1) regulates both the actin cytoskeleton and dendritic spine density, and is a downstream effector of both kalirin and CDC42, both of which have altered expression in schizophrenia. This study sought to determine if there is decreased auditory cortex PAK1 protein expression in schizophrenia through the use of quantitative western blots of 25 schizophrenia subjects and matched controls. There was no significant change in PAK1 level detected in the schizophrenia subjects in our cohort. PAK1 protein levels within subject pairs correlated positively with prior measures of total kalirin protein in the same pairs. PAK1 level also correlated with levels of a marker of dendritic spines, spinophilin. These latter two findings suggest that the lack of change in PAK1 level in schizophrenia is not due to limited sensitivity of our assay to detect meaningful differences in PAK1 protein expression. Future studies are needed to evaluate whether alterations in PAK1 phosphorylation states, or alterations in protein expression of other members of the PAK family, are present in schizophrenia

Rac1 and Rac3 GTPases Regulate the Development of Hilar Mossy Cells by Affecting the Migration of Their Precursors to the Hilus

We have previously shown that double deletion of the genes for Rac1 and Rac3 GTPases during neuronal development affects late developmental events that perturb the circuitry of the hippocampus, with ensuing epileptic phenotype. These effects include a defect in mossy cells, the major class of excitatory neurons of the hilus. Here, we have addressed the mechanisms that affect the loss of hilar mossy cells in the dorsal hippocampus of mice depleted of the two Rac GTPases. Quantification showed that the loss of mossy cells was evident already at postnatal day 8, soon after these cells become identifiable by a specific marker in the dorsal hilus. Comparative analysis of the hilar region from control and double mutant mice revealed that synaptogenesis was affected in the double mutants, with strongly reduced presynaptic input from dentate granule cells. We found that apoptosis was equally low in the hippocampus of both control and double knockout mice. Labelling with bromodeoxyuridine at embryonic day 12.5 showed no evident difference in the proliferation of neuronal precursors in the hippocampal primordium, while differences in the number of bromodeoxyuridine-labelled cells in the developing hilus revealed a defect in the migration of immature, developing mossy cells in the brain of double knockout mice. Overall, our data show that Rac1 and Rac3 GTPases participate in the normal development of hilar mossy cells, and indicate that they are involved in the regulation of the migration of the mossy cell precursor by preventing their arrival to the dorsal hilus

The Actin Binding Domain of βI-Spectrin Regulates the Morphological and Functional Dynamics of Dendritic Spines

Actin microfilaments regulate the size, shape and mobility of dendritic spines and are in turn regulated by actin binding proteins and small GTPases. The βI isoform of spectrin, a protein that links the actin cytoskeleton to membrane proteins, is present in spines. To understand its function, we expressed its actin-binding domain (ABD) in CA1 pyramidal neurons in hippocampal slice cultures. The ABD of βI-spectrin bundled actin in principal dendrites and was concentrated in dendritic spines, where it significantly increased the size of the spine head. These effects were not observed after expression of homologous ABDs of utrophin, dystrophin, and α-actinin. Treatment of slice cultures with latrunculin-B significantly decreased spine head size and decreased actin-GFP fluorescence in cells expressing the ABD of α-actinin, but not the ABD of βI-spectrin, suggesting that its presence inhibits actin depolymerization. We also observed an increase in the area of GFP-tagged PSD-95 in the spine head and an increase in the amplitude of mEPSCs at spines expressing the ABD of βI-spectrin. The effects of the βI-spectrin ABD on spine size and mEPSC amplitude were mimicked by expressing wild-type Rac3, a small GTPase that co-immunoprecipitates specifically with βI-spectrin in extracts of cultured cortical neurons. Spine size was normal in cells co-expressing a dominant negative Rac3 construct with the βI-spectrin ABD. We suggest that βI-spectrin is a synaptic protein that can modulate both the morphological and functional dynamics of dendritic spines, perhaps via interaction with actin and Rac3

Age-Dependent Targeting of Protein Phosphatase 1 to Ca2+/Calmodulin-Dependent Protein Kinase II by Spinophilin in Mouse Striatum

Mechanisms underlying age-dependent changes of dendritic spines on striatal medium spiny neurons are poorly understood. Spinophilin is an F-actin- and protein phosphatase 1 (PP1)-binding protein that targets PP1 to multiple downstream effectors to modulate dendritic spine morphology and function. We found that calcium/calmodulin-dependent protein kinase II (CaMKII) directly and indirectly associates with N- and C-terminal domains of spinophilin, but F-actin can displace CaMKII from the N-terminal domain. Spinophilin co-localizes PP1 with CaMKII on the F-actin cytoskeleton in heterologous cells, and spinophilin co-localizes with synaptic CaMKII in neuronal cultures. Thr286 autophosphorylation enhances the binding of CaMKII to spinophilin in vitro and in vivo. Although there is no change in total levels of Thr286 autophosphorylation, maturation from postnatal day 21 into adulthood robustly enhances the levels of CaMKII that co-immunoprecipitate with spinophilin from mouse striatal extracts. Moreover, N- and C-terminal domain fragments of spinophilin bind more CaMKII from adult vs. postnatal day 21 striatal lysates. Total levels of other proteins that interact with C-terminal domains of spinophilin decrease during maturation, perhaps reducing competition for CaMKII binding to the C-terminal domain. In contrast, total levels of α-internexin and binding of α-internexin to the spinophilin N-terminal domain increases with maturation, perhaps bridging an indirect interaction with CaMKII. Moreover, there is an increase in the levels of myosin Va, α-internexin, spinophilin, and PP1 in striatal CaMKII immune complexes isolated from adult and aged mice compared to those from postnatal day 21. These changes in spinophilin/CaMKII interactomes may contribute to changes in striatal dendritic spine density, morphology, and function during normal postnatal maturation and aging

Ephrin-A5 and EphA5 Interaction Induces Synaptogenesis during Early Hippocampal Development

Synaptogenesis is a fundamental step in neuronal development. For spiny glutamatergic synapses in hippocampus and cortex, synaptogenesis involves adhesion of pre and postsynaptic membranes, delivery and anchorage of pre and postsynaptic structures including scaffolds such as PSD-95 and NMDA and AMPA receptors, which are glutamate-gated ion channels, as well as the morphological maturation of spines. Although electrical activity-dependent mechanisms are established regulators of these processes, the mechanisms that function during early development, prior to the onset of electrical activity, are unclear. The Eph receptors and ephrins provide cell contact-dependent pathways that regulate axonal and dendritic development. Members of the ephrin-A family are glycosyl-phosphatidylinositol-anchored to the cell surface and activate EphA receptors, which are receptor tyrosine kinases.Here we show that ephrin-A5 interaction with the EphA5 receptor following neuron-neuron contact during early development of hippocampus induces a complex program of synaptogenic events, including expression of functional synaptic NMDA receptor-PSD-95 complexes plus morphological spine maturation and the emergence of electrical activity. The program depends upon voltage-sensitive calcium channel Ca2+ fluxes that activate PKA, CaMKII and PI3 kinase, leading to CREB phosphorylation and a synaptogenic program of gene expression. AMPA receptor subunits, their scaffolds and electrical activity are not induced. Strikingly, in contrast to wild type, stimulation of hippocampal slices from P6 EphA5 receptor functional knockout mice yielded no NMDA receptor currents.These studies suggest that ephrin-A5 and EphA5 signals play a necessary, activity-independent role in the initiation of the early phases of synaptogenesis. The coordinated expression of the NMDAR and PSD-95 induced by eprhin-A5 interaction with EphA5 receptors may be the developmental switch that induces expression of AMPAR and their interacting proteins and the transition to activity-dependent synaptic regulation

RhoGTPase Regulators Orchestrate Distinct Stages of Synaptic Development

Small RhoGTPases regulate changes in post-synaptic spine morphology and density that support learning and memory. They are also major targets of synaptic disorders, including Autism. Here we sought to determine whether upstream RhoGTPase regulators, including GEFs, GAPs, and GDIs, sculpt specific stages of synaptic development. The majority of examined molecules uniquely regulate either early spine precursor formation or later matura- tion. Specifically, an activator of actin polymerization, the Rac1 GEF β-PIX, drives spine pre- cursor formation, whereas both FRABIN, a Cdc42 GEF, and OLIGOPHRENIN-1, a RhoA GAP, regulate spine precursor elongation. However, in later development, a novel Rac1 GAP, ARHGAP23, and RhoGDIs inactivate actomyosin dynamics to stabilize mature synap- ses. Our observations demonstrate that specific combinations of RhoGTPase regulatory pro- teins temporally balance RhoGTPase activity during post-synaptic spine development

The protocadherin 17 gene affects cognition, personality, amygdala structure and function, synapse development and risk of major mood disorders

Major mood disorders, which primarily include bipolar disorder and major depressive disorder, are the leading cause of disability worldwide and pose a major challenge in identifying robust risk genes. Here, we present data from independent large-scale clinical data sets (including 29 557 cases and 32 056 controls) revealing brain expressed protocadherin 17 (PCDH17) as a susceptibility gene for major mood disorders. Single-nucleotide polymorphisms (SNPs) spanning the PCDH17 region are significantly associated with major mood disorders; subjects carrying the risk allele showed impaired cognitive abilities, increased vulnerable personality features, decreased amygdala volume and altered amygdala function as compared with non-carriers. The risk allele predicted higher transcriptional levels of PCDH17 mRNA in postmortem brain samples, which is consistent with increased gene expression in patients with bipolar disorder compared with healthy subjects. Further, overexpression of PCDH17 in primary cortical neurons revealed significantly decreased spine density and abnormal dendritic morphology compared with control groups, which again is consistent with the clinical observations of reduced numbers of dendritic spines in the brains of patients with major mood disorders. Given that synaptic spines are dynamic structures which regulate neuronal plasticity and have crucial roles in myriad brain functions, this study reveals a potential underlying biological mechanism of a novel risk gene for major mood disorders involved in synaptic function and related intermediate phenotypes