Kimberly A. Burton

Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy

Voltage-gated sodium channels (NaV) are critical for initiation of action potentials. Heterozygous loss-of-function mutations in NaV1.1 channels cause severe myoclonic epilepsy in infancy (SMEI). Homozygous null Scn1a−/− mice developed ataxia and died on postnatal day (P) 15 but could be sustained to P17.5 with manual feeding. Heterozygous Scn1a+/− mice had spontaneous seizures and sporadic deaths beginning after P21, with a notable dependence on genetic background. Loss of NaV1.1 did not change voltage-dependent activation or inactivation of sodium channels in hippocampal neurons. The sodium current density was, however, substantially reduced in inhibitory interneurons of Scn1a+/− and Scn1a−/− mice but not in their excitatory pyramidal neurons. An immunocytochemical survey also showed a specific upregulation of NaV1.3 channels in a subset of hippocampal interneurons. Our results indicate that reduced sodium currents in GABAergic inhibitory interneurons in Scn1a+/− heterozygotes may cause the hyperexcitability that leads to epilepsy in patients with SMEI.

Deletion of Type IIα Regulatory Subunit Delocalizes Protein Kinase A in Mouse Sperm without Affecting Motility or Fertilization

Cyclic AMP stimulates sperm motility in a variety of mammalian species, but the molecular details of the intracellular signaling pathway responsible for this effect are unclear. The type IIα isoform of protein kinase A (PKA) is induced late in spermatogenesis and is thought to localize PKA to the flagellar apparatus where it binds cAMP and stimulates motility. A targeted disruption of the type IIα regulatory subunit (RIIα) gene allowed us to examine the role of PKA localization in sperm motility and fertility. In wild type sperm, PKA is found primarily in the detergent-resistant particulate fraction and localizes to the mitochondrial-containing midpiece and the principal piece. In mutant sperm, there is a compensatory increase in RIα protein and a dramatic relocalization of PKA such that the majority of the holoenzyme now appears in the soluble fraction and colocalizes with the cytoplasmic droplet. Unexpectedly the RIIα mutant mice are fertile and have no significant changes in sperm motility. Our results demonstrate that the highly localized pattern of PKA seen in mature sperm is not essential for motility or fertilization.

Simultaneous visualization of two cellular mRNA species in individual neurons by use of a new double in Situ hybridization method

We present a new method for the simultaneous detection of two mRNA species within individual neurons. The technique involves the use of radio-labeled and digoxigenin-labeled cRNA probes, the application of which confers a high specificity and sensitivity to the in situ hybridization analysis. We demonstrate the use of this method by illustrating the coexpression of preprogonadotropin-releasing hormone (GnRH) mRNA and preprogalanin mRNA in neurons in the rat forebrain and report a distinct sexual dimorphism in galanin gene expression by GnRH neurons. Coupling this technology with semi-quantitative analysis of the mRNA species hybridized with the isotopically labeled mRNA would permit studies of gene regulation in individual cells among the heterogeneous populations of the brain.

Comparison of 2 Commercially Available Next-Generation Sequencing Platforms in Oncology.

Comparison of 2 Commercially Available NextGeneration Sequencing Platforms in Oncology The growing use of next generation-sequencing to identify cancer-associated alterations as well as the increasing number of targeted drugs holds promise for better matching patients with cancer with effective therapies. The FoundationOne (F1; Foundation Medicine) test sequences clinical tumor samples to characterize the exons of 315 cancer-associated genes and introns from 28 genes involved in rearrangements. The Guardant360 (G360; Guardant Health) test uses cell-free circulating DNA from blood to sequence 70 genes. Both the F1 and G360 tests have high specificities (>99%) and somewhat lower sensitivities.1,2 However, little is known about how different next-generation sequencing tests compare when used in the same patients with cancer. We compared reports from F1 and G360 testing in 9 patients from a community oncology practice to determine the level of concordance between the platforms.

Loss of the Synaptic Vesicle Protein SV2B results in reduced neurotransmission and altered synaptic vesicle protein expression in the retina.

The Synaptic Vesicle Protein 2 (SV2) family of transporter-like proteins is expressed exclusively in vesicles that undergo calcium-regulated exocytosis. Of the three isoforms expressed in mammals, SV2B is the most divergent. Here we report studies of SV2B location and function in the retina. Immunolabeling studies revealed that SV2B is detected in rod photoreceptor synaptic terminals where it is the primary isoform. In mice lacking SV2B, synaptic transmission at the synapse between photoreceptors and bipolar neurons was decreased, as evidenced by a significant reduction in the amplitude of the b-wave in electroretinogram recordings. Quantitative immunoblot analyses of whole eyes revealed that loss of SV2B was associated with reduced levels of synaptic vesicle proteins including synaptotagmin, VAMP, synaptophysin and the vesicular glutamate transporter V-GLUT1. Immunolabeling studies revealed that SV2B is detected in rod photoreceptor synaptic terminals where it is the primary isoform. Thus, SV2B contributes to the modulation of synaptic vesicle exocytosis and plays a significant role in regulating synaptic protein content.

Deficient Gene Expression in Protein Kinase Inhibitor α Null Mutant Mice

ABSTRACT Protein kinase inhibitor (PKI) is a potent endogenous inhibitor of the cyclic AMP (cAMP)-dependent protein kinase (PKA). It functions by binding the free catalytic (C) subunit with a high affinity and is also known to export nuclear C subunit to the cytoplasm. The significance of these actions with respect to PKIs physiological role is not well understood. To address this, we have generated by homologous recombination mutant mice that are deficient in PKIα, one of the three isoforms of PKI. The mice completely lack PKI activity in skeletal muscle and, surprisingly, show decreased basal and isoproterenol-induced gene expression in muscle. Further examination revealed reduced levels of the phosphorylated (active) form of the transcription factor CREB (cAMP response element binding protein) in the knockouts. This phenomenon stems, at least in part, from lower basal PKA activity levels in the mutants, arising from a compensatory increase in the level of the RIα subunit of PKA. The deficit in gene induction, however, is not easily explained by current models of PKI function and suggests that PKI may play an as yet undescribed role in PKA signaling.

The Role of Protein Kinase A Anchoring via the RIIα Regulatory Subunit in the Murine Immune System

Intracellular cAMP may inhibit T cell activation and proliferation via activation of the cAMP-dependent protein kinase, PKA. PKA signaling is maintained through interactions of the regulatory subunit with A-kinase anchoring proteins (AKAPs). We demonstrated that T cells contain AKAPs and now ask whether PKA anchoring to AKAPs via the RIIα regulatory subunit is necessary for cAMP-mediated inhibition of T cell activation. We studied the immune systems of mice lacking the RIIα regulatory subunit of PKA (−/−) and the ability of cells isolated from these mice to respond to cAMP. Dissection of spleen and thymus from wild-type (WT) and −/− mice, single cell suspensions generated from these organs, and flow cytometry analysis illustrate that the gross morphology, cell numbers, and cell populations in the spleen and thymus of the −/− mice are similar to WT controls. In vitro, splenocytes from −/− mice respond to anti-CD3/anti-CD28 and PMA/ionomycin stimulation and produce IL-2 similar to WT. Cytokine analysis revealed no significant difference in Th1 or Th2 differentiation. Finally, equivalent frequencies of CD8+ IFN-γ producing effector cells were stimulated upon infection of WT or −/− mice with Listeria monocytogenes. These data represent the first study of the role of RIIα in the immune system in vivo and provide evidence that T cell development, homeostasis, and the generation of a cell-mediated immune response are not altered in the RIIα −/− mice, suggesting either that RIIα is not required for normal immune function or that other proteins are able to compensate for RIIα function.

GROWTH HORMONE (GH) RECEPTOR GENE EXPRESSION IN THE HYPOTHALAMUS IS INCREASED DURING PUBERTY

The development of a pulsatile pattern of GH secretion occurs at puberty. Although the mechanisms responsible for the generation of GH pulses are unknown, a direct feedback effect of GH on the hypothalamus (via GH receptors) appears to play an important role. We tested the hypothesis that GH receptor gene expression changes over puberty by comparing GH receptor mRNA levels in the periventricular nucleus (PeN) of prepubertal (25 days) and adult (70 days) male rats. We performed in situ hybridization on coronal brain slices using a 35S-UTP-labeled cRNA probe and measured autoradiographic silver grains above GH receptor mRNA-containing cells. We observed that cellular levels of GH receptor mRNA (reported as grains/cell±SE) were significantly higher in adult compared to prepubertal animals (116±6 vs 92±4, respectively; p<0.05). The factors responsible for this pubertal increase in GH receptor message are unclear; however, mean plasma levels of GH increase during puberty and GH has been shown to increase GH receptor mRNA levels in other tissues. Therefore, we tested the hypothesis that GH regulates GH receptor gene expression in the PeN by comparing cellular levels of GH receptor mRNA among groups of control and GH-deficient rats (both dwarfs and GHRH antibody-treated). GH deficiency did not significantly alter the GH receptor mRNA levels when compared with control levels.Conclusion: An increase in GH receptor gene expression may be involved in the pubertal development of pulsatile GH secretion. This increase in GH receptor mRNA most likely occurs independent of increased mean plasma GH levels.

Participant Attitudes Toward an Intensive Trial of Multiple Biopsies, Multidimensional Molecular Analysis, and Reporting of Results in Metastatic Triple-Negative Breast Cancer

Purpose Multidimensional molecular analysis of tumor tissue intensively over space and time can provide insight into how cancers evolve and escape treatment. Attitudes of participants in such trials have not been assessed. We explored patient views regarding an intensive study incorporating multiple biopsies, multidimensional molecular testing, and drug response predictions that are reported to the oncologist and patient. Patients and Methods A structured, self-administered survey was conducted among the first 15 patients enrolled in ITOMIC-001 (Intensive Trial of Omics in Cancer). Patients with metastatic triple-negative breast cancer were accrued at two sites in Washington state. Surveys containing 17 items were administered at enrollment and after the return of results. Surveys explored perceptions regarding risks, personal benefits, benefits to others, uncertainties associated with interpreting complex molecular results, concerns regarding multiple biopsies, and potential loss of confidentiality. At follow-up, three additional unique items explored patient coping. Results All participants expressed a strong desire for their experiences to benefit others, and all perceived a higher likelihood of deriving benefit than described during detailed consent discussions. Loss of confidentiality ranked lowest among patient concerns. Despite acknowledging uncertainties and risks inherent in complex molecular testing for clinical reporting, participants wanted access to findings in evaluating treatment choices, even if the best available evidence was weak. Follow-up surveys demonstrated relatively little change in attitudes, although concern about study biopsies generally declined. Study participation helped several patients cope better with their disease. Conclusion In advanced breast cancer, these findings demonstrate the feasibility of engaging motivated patients in trials that navigate the uncertainties associated with intensive spatial and longitudinal multidimensional molecular testing for the purpose of advancing precision medicine.

Cis-Compound Mutations are Prevalent in Triple Negative Breast Cancer and Can Drive Tumor Progression

About 16% of breast cancers fall into a clinically aggressive category designated triple negative (TNBC) due to a lack of ERBB2, estrogen receptor and progesterone receptor expression1-3. The mutational spectrum of TNBC has been characterized as part of The Cancer Genome Atlas (TCGA)4; however, snapshots of primary tumors cannot reveal the mechanisms by which TNBCs progress and spread. To address this limitation we initiated the Intensive Trial of OMics in Cancer (ITOMIC)-001, in which patients with metastatic TNBC undergo multiple biopsies over space and time5. Whole exome sequencing (WES) of 67 samples from 11 patients identified 426 genes containing multiple distinct single nucleotide variants (SNVs) within the same sample, instances we term Multiple SNVs affecting the Same Gene and Sample (MSSGS). We find that >90% of MSSGS result from cis-compound mutations (in which both SNVs affect the same allele), that MSSGS comprised of SNVs affecting adjacent nucleotides arise from single mutational events, and that most other MSSGS result from the sequential acquisition of SNVs. Some MSSGS drive cancer progression, as exemplified by a TNBC driven by FGFR2(S252W;Y375C). MSSGS are more prevalent in TNBC than other breast cancer subtypes and occur at higher-than-expected frequencies across TNBC samples within TCGA. MSSGS may denote genes that play as yet unrecognized roles in cancer progression.