G. Stanley McKnight

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.

Cell-type-specific isolation of ribosome-associated mRNA from complex tissues

Gene profiling techniques allow the assay of transcripts from organs, tissues, and cells with an unprecedented level of coverage. However, most of these approaches are still limited by the fact that organs and tissues are composed of multiple cell types that are each unique in their patterns of gene expression. To identify the transcriptome from a single cell type in a complex tissue, investigators have relied upon physical methods to separate cell types or in situ hybridization and immunohistochemistry. Here, we describe a strategy to rapidly and efficiently isolate ribosome-associated mRNA transcripts from any cell type in vivo. We have created a mouse line, called RiboTag, which carries an Rpl22 allele with a floxed wild-type C-terminal exon followed by an identical C-terminal exon that has three copies of the hemagglutinin (HA) epitope inserted before the stop codon. When the RiboTag mouse is crossed to a cell-type-specific Cre recombinase-expressing mouse, Cre recombinase activates the expression of epitope-tagged ribosomal protein RPL22ha, which is incorporated into actively translating polyribosomes. Immunoprecipitation of polysomes with a monoclonal antibody against HA yields ribosome-associated mRNA transcripts from specific cell types. We demonstrate the application of this technique in brain using neuron-specific Cre recombinase-expressing mice and in testis using a Sertoli cell Cre recombinase-expressing mouse.

A genetic test of the effects of mutations in PKA on mossy fiber LTP and its relation to spatial and contextual learning.

Using a genetic approach, we assessed the effects of mutations in protein kinase A (PKA) on long-term potentiation (LTP) in the mossy fiber pathway and its relationship to spatial and contextual learning. Ablation by gene targeting of the C beta 1 or the RI beta isoform of PKA produces a selective defect in mossy fiber LTP, providing genetic evidence for the role of these isoforms in the mossy fiber pathway. Despite the elimination of mossy fiber LTP, the behavioral responses to novelty, spatial learning, and conditioning to context are unaffected. Thus, contrary to current theories about hippocampal function, mossy fiber LTP does not appear to be required for spatial or contextual learning. In the absence of mossy fiber LTP, adequate spatial and contextual information might reach the CA1 region via other pathways from the entorhinal cortex.

Compensatory Regulation of RIα Protein Levels in Protein Kinase A Mutant Mice

The cAMP-dependent protein kinase holoenzyme is assembled from regulatory (R) and catalytic (C) subunits that are expressed in tissue-specific patterns. Despite the dispersion of the R and C subunit genes to different chromosomal loci, mechanisms exist that coordinately regulate the intracellular levels of R and C protein such that cAMP-dependent regulation is preserved. We have created null mutations in the RIβ and RIIβ regulatory subunit genes in mice, and find that both result in an increase in the level of RIα protein in tissues that normally express the β isoforms. Examination of RIα mRNA levels and the rates of RIα protein synthesis in wild type and RIIβ mutant mice reveals that the mechanism of this biochemical compensation by RIα does not involve transcriptional or translational control. These in vivo findings are consistent with observations made in cell culture, where we demonstrate that the overexpression of Cα in NIH 3T3 cells results in increased RIα protein without increases in the rate of RIα synthesis or the level of RIα mRNA. Pulse-chase experiments reveal a 4-5-fold increase in the half-life of RIα protein as it becomes incorporated into the holoenzyme. Compensation by RIα stabilization may represent an important biological mechanism that safeguards cells from unregulated catalytic subunit activity.

Distinct patterns of cAMP-dependent protein kinase gene expression in mouse brain

In situ hybridization was used to localize cAMP-dependent protein kinase (PKA) mRNAs in the adult mouse CNS. The PKA holoenzyme contains two catalytic (C) subunits and a regulatory (R) subunit dimer. Our studies demonstrate expression of two isoforms of C (C alpha and C beta) and four isoforms of R (RI alpha, RI beta, RII alpha, and RII beta) in the CNS. mRNAs for C alpha, RI alpha, and RI beta preferentially localize in the neocortex, caudate-putamen, hypothalamus, thalamus, and hippocampus. Hybridization with C beta and RII beta probes is clearly distinguished from the C alpha-like pattern by a reduced level of hybridization in the thalamus and by a relative increase in expression in the dentate gyrus compared with cell layers CA1-3 in the hippocampus. RII alpha transcripts are very specifically localized in the medial habenula. The differential expression of PKA subunit genes suggests that functional differences in cAMP responses within neural tissues may be mediated by the biochemical properties of specific PKA isoforms.

PKA isoforms, neural pathways, and behaviour: making the connection

In mammals, the cAMP-dependent protein kinase (PKA) family of enzymes is assembled from the products of four regulatory and two catalytic subunit genes, all of which are expressed in neurons. Specific isoforms of PKA display differences in biochemical properties and subcellular localization, but it has been difficult to ascribe specific physiological functions to any given isoform. The recent development of gene knockout and transgenic mouse models has allowed for a more integrated examination of the in vivo roles of specific PKA isoforms in gene expression, synaptic plasticity, and behaviour.

Cyclic AMP second messenger systems

Cells carefully regulate the generation and destruction of cAMP using diverse families of adenylate cyclases and phosphodiesterases. Genes for several cyclases have now been cloned, giving structural information about the enzymes and providing access to the remaining members of this family. A much larger family of phosphodiesterases has been uncovered and the regulatory properties of both the cyclases and phosphodiesterases provide diverse mechanisms to modulate intracellular cAMP. Most of the actions of cAMP are mediated through phosphorylation of substrates of the cAMP-dependent protein kinases. Recent progress has helped define the pathway between cAMP and the activation of gene transcription.

The induction of ovalbumin and conalbumin mRNA by estrogen and progesterone in chick oviduct explant cultures.

Abstract Estrogen pretreated chick oviduct tissue can be restimulated in vitro by physiological concentrations of estrogen and progesterone. The rates of synthesis of the major egg white proteins, ovalbumin and conalbumin, as well as the cellular levels of their respective mRNAs, increase after characteristic lag periods; this confirms previously reported results in vivo and demonstrates that both the lag phenomena and the mRNA induction are a function of the direct interaction of steroids with oviduct cells. The antagonistic action of progesterone on an estrogen-mediated induction of conalbumin mRNA also occurs in vitro, and the kinetics of this response are examined. Progesterone terminates the estradiol-induced accumulation of conalbumin mRNA within 30 min after addition to the medium; progesterone alone or in combination with estrogen, however, is capable of inducing conalbumin mRNA after a 4 hr lag period. The temporary nature of this antagonism and the fact that it does not occur with ovalbumin induction indicate the complexity of the oviducts response to steroids. The role of protein synthesis in the induction of both ovalbumin and conalbumin was examined by including protein synthesis inhibitors in the culture medium. Puromycin, cycloheximide, emetine, pactamycin and high salt all block the induction of both ovalbumin and conalbumin mRNA when added together with either estrogen or progesterone. The effect of puromycin is reversible. After the drug is removed from the medium, the mRNA accumulation begins with the same characteristic lag period seen when no inhibitors are added. When given 2 hr after estrogen, puromycin stops the accumulation of conalbumin mRNA within 30 min, whereas cycloheximide and emetine allow the mRNA to accumulate for another 2 hr before causing complete inhibition. There is no effect of protein synthesis inhibitors on the number of estrogen receptors localized in the nucleus. The data suggest a direct link between protein synthesis and the steroid-induced accumulation of specific mRNAs in this system.

Sympathetic Stimulation of Adult Cardiomyocytes Requires Association of AKAP5 With a Subpopulation of L-Type Calcium Channels

Rationale: Sympathetic stimulation of the heart increases the force of contraction and rate of ventricular relaxation by triggering protein kinase (PK)A-dependent phosphorylation of proteins that regulate intracellular calcium. We hypothesized that scaffolding of cAMP signaling complexes by AKAP5 is required for efficient sympathetic stimulation of calcium transients. Objective: We examined the function of AKAP5 in the &bgr;-adrenergic signaling cascade. Methods and Results: We used calcium imaging and electrophysiology to examine the sympathetic response of cardiomyocytes isolated from wild type and AKAP5 mutant animals. The &bgr;-adrenergic regulation of calcium transients and the phosphorylation of substrates involved in calcium handling were disrupted in AKAP5 knockout cardiomyocytes. The scaffolding protein, AKAP5 (also called AKAP150/79), targets adenylyl cyclase, PKA, and calcineurin to a caveolin 3–associated complex in ventricular myocytes that also binds a unique subpopulation of Cav1.2 L-type calcium channels. Only the caveolin 3–associated Cav1.2 channels are phosphorylated by PKA in response to sympathetic stimulation in wild-type heart. However, in the AKAP5 knockout heart, the organization of this signaling complex is disrupted, adenylyl cyclase 5/6 no longer associates with caveolin 3 in the T-tubules, and noncaveolin 3–associated calcium channels become phosphorylated after &bgr;-adrenergic stimulation, although this does not lead to an enhanced calcium transient. The signaling domain created by AKAP5 is also essential for the PKA-dependent phosphorylation of ryanodine receptors and phospholamban. Conclusions: These findings identify an AKAP5-organized signaling module that is associated with caveolin 3 and is essential for sympathetic stimulation of the calcium transient in adult heart cells.

The Essential Role of RIα in the Maintenance of Regulated PKA Activity

Abstract: Cloning of the individual regulatory (R) and catalytic (C) subunits of the cAMP‐dependent protein kinase (PKA) and expression of these subunits in cell culture have provided mechanistic answers about the rules for PKA holoenzyme assembly. One of the central findings of these studies is the essential role of the RIα regulatory subunit in maintaining the catalytic subunit under cAMP control. The role of RIα as the key compensatory regulatory subunit in this enzyme family was confirmed by gene knockouts of the three other regulatory subunits in mice. In each case, RIα has demonstrated the capacity for significant compensatory regulation of PKA activity in tissues where the other regulatory subunits are expressed, including brain, brown and white adipose tissue, skeletal muscle, and sperm. The essential requirement of the RIα regulatory subunit in maintaining cAMP control of PKA activity was further corroborated by the knockout of RIα in mice, which results in early embryonic lethality due to failed cardiac morphogenesis. Closer examination of RIα knockout embryos at even earlier stages of development revealed profound deficits in the morphogenesis of the mesodermal embryonic germ layer, which gives rise to essential structures including the embryonic heart tube. Failure of the mesodermal germ layer in RIα knockout embryos can be rescued by crossing RIα knockout mice to Cα knockout mice, supporting the conclusion that inappropriately regulated PKA catalytic subunit activity is responsible for the phenotype. Isolation of primary embryonic fibroblasts from RIα knockout embryos reveals profound alterations in the actin‐based cytoskeleton, which may account for the failure in mesoderm morphogenesis at gastrulation.