Identification of molluscan nicotinic acetylcholine receptor (nAChR) subunits involved in formation of cation- and anion-selective nAChRs

Acetylcholine (ACh) is a neurotransmitter commonly found in all animal species. It was shown to mediate fast excitatory and inhibitory neurotransmission in the molluscan CNS. Since early intracellular recordings, it was shown that the receptors mediating these currents belong to the family of neuronal nicotinic acetylcholine receptors and that they can be distinguished on the basis of their pharmacology. We previously identified 12 Lymnaea cDNAs that were predicted to encode ion channel subunits of the family of the neuronal nicotinic acetylcholine receptors. These Lymnaea nAChRs can be subdivided in groups according to the residues supposedly contributing to the selectivity of ion conductance. Functional analysis in Xenopus oocytes revealed that two types of subunits with predicted distinct ion selectivities form homopentameric nicotinic ACh receptor (nAChR) subtypes conducting either cations or anions. Phylogenetic analysis of the nAChR gene sequences suggests that molluscan anionic nAChRs probably evolved from cationic ancestors through amino acid substitutions in the ion channel pore, a mechanism different from acetylcholine-gated channels in other invertebrates

A Gene Network Perspective on Axonal Regeneration

The regenerative capacity of injured neurons in the central nervous system is limited due to the absence of a robust neuron-intrinsic injury-induced gene response that supports axon regeneration. In peripheral neurons axotomy induces a large cohort of regeneration-associated genes (RAGs). The forced expression of some of these RAGs in injured neurons has some beneficial effect on axon regeneration, but the reported effects are rather small. Transcription factors (TFs) provide a promising class of RAGs. TFs are hubs in the regeneration-associated gene network, and potentially control the coordinate expression of many RAGs simultaneously. Here we discuss the use of combined experimental and computational methods to identify novel regeneration-associated TFs with a key role in initiating and maintaining the RAG-response in injured neurons. We propose that a relatively small number of hub TFs with multiple functional connections in the RAG network might provide attractive new targets for gene-based and/or pharmacological approaches to promote axon regeneration in the central nervous system

A Structural and Mutagenic Blueprint for Molecular Recognition of Strychnine and d-Tubocurarine by Different Cys-Loop Receptors

Cys-loop receptors (CLR) are pentameric ligand-gated ion channels that mediate fast excitatory or inhibitory transmission in the nervous system. Strychnine and d-tubocurarine (d-TC) are neurotoxins that have been highly instrumental in decades of research on glycine receptors (GlyR) and nicotinic acetylcholine receptors (nAChR), respectively. In this study we addressed the question how the molecular recognition of strychnine and d-TC occurs with high affinity and yet low specificity towards diverse CLR family members. X-ray crystal structures of the complexes with AChBP, a well-described structural homolog of the extracellular domain of the nAChRs, revealed that strychnine and d-TC adopt multiple occupancies and different ligand orientations, stabilizing the homopentameric protein in an asymmetric state. This introduces a new level of structural diversity in CLRs. Unlike protein and peptide neurotoxins, strychnine and d-TC form a limited number of contacts in the binding pocket of AChBP, offering an explanation for their low selectivity. Based on the ligand interactions observed in strychnine- and d-TC-AChBP complexes we performed alanine-scanning mutagenesis in the binding pocket of the human α1 GlyR and α7 nAChR and showed the functional relevance of these residues in conferring high potency of strychnine and d-TC, respectively. Our results demonstrate that a limited number of ligand interactions in the binding pocket together with an energetic stabilization of the extracellular domain are key to the poor selective recognition of strychnine and d-TC by CLRs as diverse as the GlyR, nAChR, and 5-HT3R

Novel Candidate Genes Associated with Hippocampal Oscillations

The hippocampus is critical for a wide range of emotional and cognitive behaviors. Here, we performed the first genome-wide search for genes influencing hippocampal oscillations. We measured local field potentials (LFPs) using 64-channel multi-electrode arrays in acute hippocampal slices of 29 BXD recombinant inbred mouse strains. Spontaneous activity and carbachol-induced fast network oscillations were analyzed with spectral and cross-correlation methods and the resulting traits were used for mapping quantitative trait loci (QTLs), i.e., regions on the genome that may influence hippocampal function. Using genome-wide hippocampal gene expression data, we narrowed the QTLs to eight candidate genes, including Plcb1, a phospholipase that is known to influence hippocampal oscillations. We also identified two genes coding for calcium channels, Cacna1b and Cacna1e, which mediate presynaptic transmitter release and have not been shown to regulate hippocampal network activity previously. Furthermore, we showed that the amplitude of the hippocampal oscillations is genetically correlated with hippocampal volume and several measures of novel environment exploration

The Lymnaea Cardioexcitatory Peptide (LyCEP) Receptor: A G-Protein–Coupled Receptor for a Novel Member of the RFamide Neuropeptide Family

A novel G-protein–coupled receptor (GRL106) resembling neuropeptide Y and tachykinin receptors was cloned from the molluscLymnaea stagnalis. Application of a peptide extract from the Lymnaea brain to Xenopus oocytes expressing GRL106 activated a calcium-dependent chloride channel. Using this response as a bioassay, we purified the ligand for GRL106,Lymnaea cardioexcitatory peptide (LyCEP), an RFamide-type decapeptide (TPHWRPQGRF-NH2) displaying significant similarity to the Achatina cardioexcitatory peptide (ACEP-1) as well as to the recently identified family of mammalian prolactin-releasing peptides. In the Lymnaeabrain, the cells that produce egg-laying hormone are the predominant site of GRL106 gene expression and appear to be innervated by LyCEP-containing fibers. Indeed, LyCEP application transiently hyperpolarizes isolated egg-laying hormone cells. In theLymnaea pericardium, LyCEP-containing fibers end blindly at the pericardial lumen, and the heart is stimulated by LyCEPin vitro. These data confirm that LyCEP is an RFamide ligand for GRL10

Single-cell isoform RNA sequencing characterizes isoforms in thousands of cerebellar cells

Full-length RNA sequencing (RNA-Seq) has been applied to bulk tissue, cell lines and sorted cells to characterize transcriptomes1–11, but applying this technology to single cells has proven to be difficult, with less than ten single-cell transcriptomes having been analyzed thus far12,13. Although single splicing events have been described for ≤200 single cells with statistical confidence14,15, full-length mRNA analyses for hundreds of cells have not been reported. Single-cell short-read 3′ sequencing enables the identification of cellular subtypes16–21, but full-length mRNA isoforms for these cell types cannot be profiled. We developed a method that starts with bulk tissue and identifies single-cell types and their full-length RNA isoforms without fluorescence-activated cell sorting. Using single-cell isoform RNA-Seq (ScISOr-Seq), we identified RNA isoforms in neurons, astrocytes, microglia, and cell subtypes such as Purkinje and Granule cells, and cell-type-specific combination patterns of distant splice sites6–9,22,23. We used ScISOr-Seq to improve genome annotation in mouse Gencode version 10 by determining the cell-type-specific expression of 18,173 known and 16,872 novel isoforms

Functional characterisation of human synaptic genes expressed in the Drosophila brain

Drosophila melanogaster is an established and versatile model organism. Here we describe and make available a collection of transgenic Drosophila strains expressing human synaptic genes. The collection can be used to study and characterise human synaptic genes and their interactions and as controls for mutant studies. It was generated in a way that allows the easy addition of new strains, as well as their combination. In order to highlight the potential value of the collection for the characterisation of human synaptic genes we also use two assays, investigating any gain-of-function motor and/or cognitive phenotypes in the strains in this collection. Using these assays we show that among the strains made there are both types of gain-of-function phenotypes investigated. As an example, we focus on the three strains expressing human tyrosine protein kinase Fyn, the small GTPase Rap1a and human Arc, respectively. Of the three, the first shows a cognitive gain-of-function phenotype while the second a motor gain-of-function phenotype. By contrast, Arc, which has no Drosophila ortholog, shows no gain-of-function phenotype

Aralar Sequesters GABA into Hyperactive Mitochondria, Causing Social Behavior Deficits

Social impairment is frequently associated with mitochondrial dysfunction and altered neurotransmission. Although mitochondrial function is crucial for brain homeostasis, it remains unknown whether mitochondrial disruption contributes to social behavioral deficits. Here, we show that Drosophila mutants in the homolog of the human CYFIP1, a gene linked to autism and schizophrenia, exhibit mitochondrial hyperactivity and altered group behavior. We identify the regulation of GABA availability by mitochondrial activity as a biologically relevant mechanism and demonstrate its contribution to social behavior. Specifically, increased mitochondrial activity causes gamma aminobutyric acid (GABA) sequestration in the mitochondria, reducing GABAergic signaling and resulting in social deficits. Pharmacological and genetic manipulation of mitochondrial activity or GABA signaling corrects the observed abnormalities. We identify Aralar as the mitochondrial transporter that sequesters GABA upon increased mitochondrial activity. This study increases our understanding of how mitochondria modulate neuronal homeostasis and social behavior under physiopathological conditions

Genetics of the Hippocampal Transcriptome in Mouse: A Systematic Survey and Online Neurogenomics Resource

Differences in gene expression in the CNS influence behavior and disease susceptibility. To systematically explore the role of normal variation in expression on hippocampal structure and function, we generated an online microarray database for a diverse panel of strains of mice, including most common inbred strains and numerous recombinant inbred lines (www.genenetwork.org). Using this resource, coexpression networks for families of genes can be generated rapidly to test causal models related to function. The data set is optimized for quantitative trait locus (QTL) mapping and was used to identify over 5500 QTLs that modulate mRNA levels. We describe a wide variety of analyses and novel synthetic approaches that take advantage of this resource, and demonstrate how both the data and associated tools can be applied to the study of gene regulation in the hippocampus and relations to structure and function

AMPAR Auxiliary Protein SHISA6 Facilitates Purkinje Cell Synaptic Excitability and Procedural Memory Formation

The majority of excitatory postsynaptic currents in the brain are gated through AMPA-type glutamate receptors, the kinetics and trafficking of which can be modulated by auxiliary proteins. It remains to be elucidated whether and how auxiliary proteins can modulate synaptic function to contribute to procedural memory formation. In this study, we report that the AMPA-type glutamate receptor (AMPAR) auxiliary protein SHISA6 (CKAMP52) is expressed in cerebellar Purkinje cells, where it co-localizes with GluA2-containing AMPARs. The absence of SHISA6 in Purkinje cells results in severe impairments in the adaptation of the vestibulo-ocular reflex and eyeblink conditioning. The physiological abnormalities include decreased presence of AMPARs in synaptosomes, impaired excitatory transmission, increased deactivation of AMPA receptors, and reduced induction of long-term potentiation at Purkinje cell synapses. Our data indicate that Purkinje cells require SHISA6-dependent modification of AMPAR function in order to facilitate cerebellar, procedural memory formation.Peter et al. show that the SHISA6 protein modulates the synaptic function of Purkinje cells in mice. In the absence of SHISA6, memory formation during classical eyeblink conditioning and eye movement adaptations is severely impaired as a result of a major synaptic excitability phenotype in Purkinje cells