A. Marie Phillips

Identification of a Drosophila gene encoding a calmodulin-binding protein with homology to the trp phototransduction gene

We have isolated a number of Drosophila cDNAs on the basis of their encoding calmodulin-binding proteins. A full-length cDNA clone corresponding to one of these genes has been cloned and sequenced. Conservation of amino acid sequence and tissue-specific expression are observed between this gene and the transient receptor potential (trp) gene. We propose the name transient receptor potential-like (trpl) to describe this newly isolated gene. The trpl protein contains two possible calmodulin-binding sites, six transmembrane regions, and a sequence homologous to an ankyrin-like repeat. Structurally, the trpl and trp proteins resemble cation channel proteins, particularly the brain isoform of the voltage-sensitive Ca2+ channel. The identification of a protein similar to the trp gene product, yet also able to bind Ca2+/calmodulin, allows for a reinterpretation of the phenotype of the trp mutations and suggests that both genes may encode light-sensitive ion channels.

Glucose transporter 1 deficiency in the idiopathic generalized epilepsies

We examined whether glucose transporter 1 (GLUT1) deficiency causes common idiopathic generalized epilepsies (IGEs).

HCN channelopathies: pathophysiology in genetic epilepsy and therapeutic implications

Hyperpolarization‐activated cyclic nucleotide‐gated channels (HCN) can act as pacemakers in the brain making them strong candidates for driving aberrant hypersynchronous network activity seen in epilepsy. Transcriptional changes in HCN channels occur in several animal models of epilepsy. However, only recently have genetic studies demonstrated sequence variation in HCN1 and HCN2 genes associated with human epilepsy. These include a triple proline deletion in HCN2 that increases channel function and occurs more often in patients with febrile seizure syndromes. Other HCNx gene variants have been described in idiopathic generalized epilepsy although the functional consequence of these remains unclear. In this review we explore potential cellular and network mechanisms involving HCN channels in the genetic epilepsies. We suggest how new genetic sequencing technology, medium‐throughput functional assays and the ability to develop syndrome‐specific animal models will provide a more comprehensive understanding of how Ih contributes to pathogenic mechanisms underlying human genetic epilepsy. We also discuss what is known about the pharmacological manipulation of HCN channels in the context of epilepsy and how this may help future efforts in developing HCN‐channel‐based therapy.

Molecular and genetic characterization of the interactions between the Drosophila stoned-B protein and DAP-160 (intersectin)

The stoned locus of Drosophila produces a dicistronic transcript and encodes two proteins, stoned-A (STNA) and stoned-B (STNB). Both proteins are located at synaptic terminals. The STNB protein contains a domain that has homology with the mu-subunit of the AP (adaptor protein) complex, as well as a number of NPF (Asp-Pro-Phe) motifs known to bind EH (Eps15 homology) domains. Mutations at the stoned locus interact synergistically with mutations at the shibire (dynamin) locus and alter synaptic vesicle endocytosis. The STNB protein has also been shown to interact with synaptic vesicles via synaptogamin-I. We initiated an investigation of the possible interaction of DAP-160 (dynamin-associated protein of 160 kDa), a Drosophila member of the intersectin family, with the STNB protein. We show here that both of the viable stoned alleles interacted with a genetic construct that reduces DAP-160 levels to 25% of normal. One of these stoned alleles contains a substitution resulting in a stop codon in the open reading frame encoding STNB. This allele also shows markedly reduced levels of both DAP-160 and dynamin. As anticipated, the NPF motifs in STNB are found to be high-affinity binding motifs for the EH domains of DAP-160. One of the SH3 (Src homology 3) domains of DAP-160 also interacts with STNB. Finally, we show that immunoprecipitation of STNB from fly head extracts co-precipitates with DAP-160, and we conclude that the interaction of the STNB protein with both synaptotagmin I and DAP-160 may regulate synaptic vesicle recycling by recruiting dynamin to a pre-fission complex.

A Neural Gene from Drosophila melanogaster with Homology to Vertebrate and Invertebrate Glutamate Decarboxylases

Abstract: Cross‐species hybridization has been used to isolate a second Drosophila gene, with homology to a feline glutamate decarboxylase (Gad) cDNA. The gene differs in sequence, chromosomal location, and spatial expression from the previously reported Drosophila Gad gene, but both encode proteins of 58 kDa. The derived amino acid sequence reveals a typical pyridoxal phosphate binding site and sequence homology consistent with a glutamate decarboxylase function. The protein includes an amino‐terminal polyasparagine sequence, and a /β‐pleated sheet region, with regularly spaced glutamine and arginine residues, not found in other decarboxylases. Expression in the adult is limited to the neuropil of the first optic ganglion and to regions of the thoracic musculature that may correspond to the location of motor neuron axons. This is consistent with a glial localization for the transcript. There is no overlap with the reported expression of Drosophila Gad. Although the molecular evidence suggests that this gene encodes a pyridoxal phosphate‐dependent decarboxylase, glutamate decarboxylase activity associated with this gene could not be demonstrated, and the in vivo substrate is unknown. It is possible that the protein encoded by this gene is novel, not only in sequence and spatial expression, but also in substrate specificity.

Effective Translation of the Second Cistron in Two Drosophila Dicistronic Transcripts Is Determined by the Absence of In-frame AUG Codons in the First Cistron

The novel dicistronic transcript encoded by the Drosophila melanogaster stoned gene was recognized as being unusual in that the protein encoded by the first open reading frame, stoned-A (STNA), contains no internal methionine residues in a protein of 93 kDa. The dicistronic nature of the stoned locus and the lack of methionine residues in STNA is conserved across dipteran species. A second methionine-free cistron, encoding Snapin, was identified in Drosophila and also found to be dicistronic, the second open reading frame (ORF) encoding a methyltransferase. We have replaced the methyltransferase cistron with green fluorescent protein (GFP) and used this dicistronic construct to show that the GFP cistron is translated in Drosophila S2 cells. The insertion of in-frame AUG codons into the snapin ORF attenuates the translation of GFP, and the level of attenuation correlates with the number of inserted AUGs. Increasing the efficiency of translation-initiation of the Snapin cistron also attenuates the translation of GFP. This indicates that failure to initiate translation at the first AUG allows ribosomes to scan through the Snapin ORF and to initiate translation of the second cistron, unless new AUG codons are inserted. These data are used to interpret the expression of the stoned locus and in particular, to explain the altered stoned protein levels in the stoned-temperature-sensitive mutant allele, which replaces a lysine with a methionine codon early in the first, stonedA, cistron.

Identification of a gene family from Drosophila melanogaster encoding proteins with homology to invertebrate sarcoplasmic calcium-binding proteins (SCPS)

Using antibodies raised against the Drosophila Ca(2+)-binding protein DCABP-23, we have isolated two distinct cDNA clones that encode Ca(2+)-binding proteins of the invertebrate sarcoplasmic calcium-binding protein (SCP) family. Southern blot analysis of whole genomic DNA has shown that one of the clones, Dcabp-A.1, is present in more than one copy in the genome of the fly, and is located in the beta-heterochromatic region at cytological division 80 on chromosome III. The expression pattern of this transcript shows that it is present in the tubular but not the fibrillar muscles of the adult thorax. This expression pattern is consistent with this being a true SCP. In contrast, the expression pattern of the transcript corresponding to the second cDNA clone is exclusive to neural tissue. This transcript derives from a single copy gene, and is located at cytological position 89 D on chromosome III. Comparative analysis of the amino acid sequences from the proteins encoded by the two cDNAs with that of the original DCABP-23 protein indicates that the purified DCABP-23 contained mainly the DCABP-A.1 protein. The identification of members of the SCP family of proteins in Drosophila, will allow for a future genetic investigation of the function of these ubiquitous proteins.

Excitotoxic-mediated transcriptional decreases in HCN2 channel function increase network excitability in CA1

Changes in the conductance of the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel that mediates Ih are proposed to contribute to increased network excitability. Synchronous neuronal burst activity is a good reflection of network excitability and can be generated in isolated hippocampal slice cultures by removing Mg2+ from the extracellular fluid. We demonstrate that Ih contributes to this activity by increasing both the frequency and duration of bursting events. Changes in HCN channel function are also implicated in altered seizure susceptibility. Short-term application of kainic acid (KA) is known to initiate long lasting changes in neuronal networks that result in seizures, and in slice cultures was found to alter HCN mRNA levels in an isoform and hippocampal sub-region specific manner. These changes correlate with the ability of each sub-region to develop synchronous burst activity following KA that we have previously reported. Specifically, a loss of synchronous activity in the CA3 correlated with an increase in HCN2 mRNA levels that normalized concomitantly with the restoration of CA3 burst activity 7 days post insult. In contrast, in CA1 an increase in synchronous burst duration correlated with a reduction in HCN2 mRNA levels and both changes were still evident for 7 days post insult. Lamotrigine, known to increase Ih, reversed the impact of KA on burst duration in CA1 at both time-points linking a transcriptional reduction in HCN2 function to increased burst duration.

Loss of synaptic Zn2+ transporter function increases risk of febrile seizures.

Febrile seizures (FS) are the most common seizure syndrome and are potentially a prelude to more severe epilepsy. Although zinc (Zn2+) metabolism has previously been implicated in FS, whether or not variation in proteins essential for Zn2+ homeostasis contributes to susceptibility is unknown. Synaptic Zn2+ is co-released with glutamate and modulates neuronal excitability. SLC30A3 encodes the zinc transporter 3 (ZNT3), which is primarily responsible for moving Zn2+ into synaptic vesicles. Here we sequenced SLC30A3 and discovered a rare variant (c.892C > T; p.R298C) enriched in FS populations but absent in population-matched controls. Functional analysis revealed a significant loss-of-function of the mutated protein resulting from a trafficking deficit. Furthermore, mice null for ZnT3 were more sensitive than wild-type to hyperthermia-induced seizures that model FS. Together our data suggest that reduced synaptic Zn2+ increases the risk of FS and more broadly support the idea that impaired synaptic Zn2+ homeostasis can contribute to neuronal hyperexcitability.

Sodium channel β1 subunit localizes to axon initial segments of excitatory and inhibitory neurons and shows regional heterogeneity in mouse brain

The β1 subunit of voltage‐gated sodium channels, Navβ1, plays multiple roles in neurons spanning electrophysiological modulation of sodium channel α subunits to cell adhesion and neurite outgrowth. This study used immunohistochemistry to investigate Navβ1 subneuronal and regional expression. Navβ1 was enriched at axon initial segments (AIS) and nodes of Ranvier. Navβ1 expression at the AIS was detected throughout the brain, predominantly in the hippocampus, cortex, and cerebellum. Despite expression of Navβ1 in both excitatory and inhibitory AIS, it displayed a marked and fine‐grained heterogeneity of expression. Such heterogeneity could have important implications for the tuning of single neuronal and regional excitability, especially in view of the fact that Navβ1 coexpressed with Nav1.1, Nav1.2, and Nav1.6 subunits. The disruption of Navβ1 AIS expression by a human epilepsy‐causing C121W genetic mutation in Navβ1 was also investigated using a mouse model. AIS expression of Navβ1 was reduced by approximately 50% in mice heterozygous for the C121W mutation and was abolished in homozygotes, suggesting that loss of Navα subunit modulation by Navβ1 contributes to the mechanism of epileptogenesis in these animals as well as in patients. J. Comp. Neurol. 523:814–830, 2015.