Molecular and Behavioral Effects of a Null Mutation in All PKA Cβ Isoforms

doi:10.1006/mcne.2002.1119

Molecular and Cellular Neuroscience

Volume 20, Issue 3, July 2002, Pages 515-524

https://doi.org/10.1006/mcne.2002.1119 Get rights and content

Abstract

The cAMP-dependent protein kinase (PKA) Cβ gene encodes three isoforms, two of which (Cβ2 and Cβ3) are transcribed from neural-specific promoters. Here we report the effects of knocking out all PKA Cβ subunit isoforms in mice. Total PKA activity was unaffected in the hippocampus and amygdala, while basal PKA activity was reduced by 26% in the brains of Cβall^−/− mice despite a compensatory increase in Cα protein. Cued fear conditioning was disrupted in Cβall^−/− mice when tested on a mixed C57BL/6/129 background but was indistinguishable from wild type mice when bred onto a 98% C57BL/6 background. This suggests an amygdala-specific deficit in the Cβall null mice that is sensitive to strain-specific genetic modifiers. Behavioral testing including locomotor activity, contextual fear conditioning, and conditioned taste aversion was normal in Cβall null mice on the 50% C57BL/6J background. We conclude that Cβ protein is not essential for neuronal development or function but may play a more subtle role in memory that is modulated by strain-specific genetic modifiers.

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PKA CβKO mice, lacking all three isoforms (C1, C2, C3) of Prkacb were resistant to DIO and the associated metabolic dysregulation including glucose tolerance and insulin insensitivity (Enns et al., 2009; London et al., 2019). Transcription of Prkarcb C2 and C3 isoforms occurs exclusively under neural promoters (Desseyn, Burton, & Mcknight, 2000; Guthrie, Skalhegg, & Mcknight, 1997), and accordingly, CβKO mice had reductions in brain PKA enzymatic activity (Howe, Wiley, & Mcknight, 2002) that increased sympathetic outflow presumably by decreased inhibition of NE exocytosis through decreased PKA activation. While neither insulin nor leptin act directly through GPCRs, both modulate cAMP in hypothalamic neurons to control energy intake (Zhao, 2005) and mediate the effects of glucagon (Zhao et al., 2000).
The cAMP-dependent protein kinase (PKA) system represents a primary cell-signaling pathway throughout systems and across species. PKA facilitates the actions of hormones, neurotransmitters and other signaling molecules that bind G-protein coupled receptors (GPCR) to modulate cAMP levels. Through its control of synaptic events, exocytosis, transcriptional regulation, and more, PKA signaling regulates cellular metabolism and emotional and stress responses making it integral in the maintenance and dysregulation of energy homeostasis. Neural PKA signaling is regulated by afferent and peripheral efferent signals that link specific neural cell populations to the regulation of metabolic processes in adipose tissue, liver, pancreas, adrenal, skeletal muscle, and gut. Mouse models have provided invaluable information on the roles for PKA subunits in brain and key metabolic organs. While limited, human studies infer differential regulation of the PKA system in obese compared to lean individuals. Variants identified in PKA subunit genes cause Cushing syndrome that is characterized by metabolic dysregulation associated with endogenous glucocorticoid excess. Under healthy physiologic conditions, the PKA system is exquisitely regulated by stimuli that activate GPCRs to alter intracellular cAMP concentrations, and by PKA cellular localization and holoenzyme stability. Adenylate cyclase activity generates cAMP while phosphodiesterase-mediated cAMP degradation to AMP decreases cAMP levels downstream of GPCRs. Chronic perturbations in PKA signaling appear to be capable of resetting PKA regulation at several levels; in addition, sex differences in PKA signaling regulation, while not well understood, impact the physiologic consequences of metabolic dysregulation and obesity. This review explores the roles for PKA signaling in the pathogenesis of metabolic diseases including obesity, type 2 diabetes mellitus and associated co-morbidities through neural-peripheral crosstalk and cAMP/PKA signaling pathway targets that hold therapeutic potential.
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The cAMP-dependent protein kinase, more commonly referred to as protein kinase A (PKA), is one of the most-studied enzymes in biology. PKA is ubiquitously expressed in mammalian cells, can be activated in response to a plethora of biological stimuli, and phosphorylates more than 250 known substrates. Indeed, PKA is of central importance to a wide range of organismal processes, including energy homeostasis, memory formation and immunity. It serves as the primary effector of the second-messenger molecule 3′,5′-cyclic adenosine monophosphate (cAMP), which is believed to have mostly inhibitory effects on the adaptive immune response. In particular, elevated levels of intracellular cAMP inhibit the activation of conventional T cells by limiting signal transduction through the T-cell receptor and altering gene expression, primarily in a PKA-dependent manner. Regulatory T cells have been shown to increase the cAMP levels in adjacent T cells by direct and indirect means, but the role of cAMP within regulatory T cells themselves remains incompletely understood. Paradoxically, cAMP has been implicated in promoting T-cell activation as well, adding another functional dimension beyond its established immunosuppressive effects. Furthermore, PKA can phosphorylate the NF-κB subunit p65, a transcription factor that is essential for T-cell activation, independently of cAMP. This phosphorylation of p65 drastically enhances NF-κB-dependent transcription and thus is likely to facilitate immune activation. How these immunosuppressive and immune-activating properties of PKA balance in vivo remains to be elucidated. This review provides a brief overview of PKA regulation, its ability to affect NF-κB activation, and its diverse functions in T-cell biology.
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In some brain areas, such as in the PVH, Prkaca+/− mice appeared to have more PKA activity as compared to the WT mice, probably indicating a compensatory mechanism. Prior studies report that compensatory increases in Cβ levels occurred in brain of Cα knockout mice [67]. Also, it has previously been shown that, although accounting for just 5–10% of total PKA activity in mouse cells, Cβ compensates not only for Cα deficiency in certain situations [29] but it may also modify significantly overall PKA activity in the setting of strain-specific and other modifiers [26,68].
Cyclic adenosine mono-phosphate-dependent protein kinase (PKA) is critically involved in the regulation of behavioral responses. Previous studies showed that PKA’s main regulatory subunit, R1α, is involved in anxiety-like behaviors. The purpose of this study was to determine how the catalytic subunit, Cα, might affect R1α’s function and determine its effects on anxiety-related behaviors.
The marble bury (MB) and elevated plus maze (EPM) tests were used to assess anxiety-like behavior and the hotplate test to assess nociception in wild type (WT) mouse, a Prkar1a heterozygote (Prkar1a^+/−) mouse with haploinsufficiency for the regulatory subunit (R1α), a Prkaca heterozygote (Prkaca^+/−) mouse with haploinsufficiency for the catalytic subunit (Cα), and a double heterozygote mouse (Prkar1a^+/−/Prkaca^+/−) with haploinsufficiency for both R1α and Cα. We then examined specific brain nuclei involved in anxiety.
Results of MB test showed a genotype effect, with increased anxiety-like behavior in Prkar1a^+/− and Prkar1a^+/−/Prkaca^+/− compared to WT mice. In the EPM, Prkar1a^+/− spent significantly less time in the open arms, while Prkaca^+/− and Prkar1a^+/−/Prkaca^+/− mice displayed less exploratory behavior compared to WT mice. The loss of one Prkar1a allele was associated with a significant increase in PKA activity in the basolateral (BLA) and central (CeA) amygdala and ventromedial hypothalamus (VMH) in both Prkar1a^+/− and Prkar1a^+/−/Prkaca^+/− mice.
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Two tissue-specific Cα variants, Cα1 and CαS, are encoded by alternative use of 2 exons located 5′ to exon 2 in the Cα gene: Cα1 is ubiquitously expressed and CαS is seen only in sperm (Vetter et al., 2011). There are numerous splice variants of the Cβ gene but only the Cβ1 variant is ubiquitously expressed, while the Cβ2 variant is mostly associated with lymphoid, and the Cβ3 and Cβ4 variants with neural tissues; Cγ’s presence is limited (Howe et al., 2002; Zhang and Daaka, 2011). Thus, PKA activity in most tissues (other than brain, kidney and spleen where there is Cβ activity) appears to be Cα-dependent (Skålhegg et al., 2002).
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The R subunit isoforms exhibit major differences in the tissue distribution, biochemical and physical properties encoded by four genes (RI-Alpha, RI-Beta, RII-Alpha and RII-Beta) (Tasken et al., 1993). Several research groups have reported CNS effects in knock-out models for RI-Beta (Brandon et al., 1995, Huang et al., 1995), RII-Alpha (Rao et al., 2004) and RII-Beta (Brandon et al., 1998), as well as catalytic unit knockouts (Huang et al., 1995; Qi et al., 1996; Howe et al., 2002). RI-Alpha (R1a−/−), RII-Alpha(RIIa−/−), & R1-Beta (R1B−/−), – subunit deficient mice were shown to have reduced cAMP – stimulated (and PKI-inhibited) PKA activity, while only the RI-Alpha (R1a−/−) mice showed significantly increased baseline (non cAMP-stimulated, but PKI inhibited) PKA activity.
The role of cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signaling in the molecular pathways involved in fear and memory is well established. Prior studies in our lab reported that transgenic mice with an inactivating mutation in Prkar1a gene (codes for the 1-alpha regulatory subunit (R1α) of PKA) exhibited behavioral abnormalities including anxiety and depression. In the present study, we examined the role of altered PKA signaling on anxiety-like behaviors in Prkar1a^+/− mice compared to wild-type (WT) littermates. The elevated plus maze (EPM) and marble bury (MB) tests were used to assess anxiety-like behavior. The hotplate test was performed to evaluate analgesia. We further examined the impact of the Prkar1a inactivating mutation on PKA activity in specific nuclei of the brain associated with anxiety-like behavior. Results for the MB test showed a genotype effect, with increased anxiety-like behavior in Prkar1a^+/− mice, compared to WT littermates (p < 0.05). MANOVA analysis showed a significant genotype difference in anxiety-like behavior in the EPM between WT and Prkar1a^+/− mice on combined dependent variables (open arm time and open to total time ratio; p < 0.05). Results of hotplate testing showed no genotype effect however; the expected sex difference was noted. Analysis of PKA activity showed the loss of one Prkar1a allele led to an increase in basal and cAMP-stimulated kinase activity in both the basolateral and central amygdala. These results suggest that the alteration in PKA signaling in Prkar1a^+/− mice is not a ubiquitous effect; and supports the importance of cAMP/PKA pathway in neurobiological processes involved in anxiety and fear sensitization.

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Regular ArticleMolecular and Behavioral Effects of a Null Mutation in All PKA Cβ Isoforms

Abstract

Cell

J. Biol. Chem.

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J. Biol. Chem.

Behav. Brain Res.

Behav. Brain Res.

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J. Biol. Chem.

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Neuron

Cell

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Trends Neurol.

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The unique catalytic subunit of sperm cAMP-dependent protein kinase is the product of an alternative Calpha mRNA expressed specifically in spermatogenic cells

Mol. Biol. Cell

Synaptic plasticity in the lateral amygdala: A cellular hypothesis of fear conditioning

Learn. Mem.

Regular Article
Molecular and Behavioral Effects of a Null Mutation in All PKA Cβ Isoforms