Monday Article #55: The Role of Protein Kinase A in Anxiety Behaviors
Anxiety, experienced as excessive, uncontrollable worry about a variety of topics In the absence of respective stimuli or in a manner disproportionate to their potentially posed risk, is the key diagnostic criterion of generalized anxiety disorder (GAD).  Its etiological interrelatedness with dimensional measures of trait anxiety, such as pathological worry, fear of uncertainty, or neuroticism, and its high rate of treatment resistance have attracted the attention of psychiatric geneticists aiming at identifying biomarkers of disease risk and treatment response. 
Some people may experience clinically significant anxiety symptoms. This can manifest as anxiety in specific, non-threatening situations, panic symptoms, or generalized feelings of worry that occur most or all of the time. When anxiety symptoms cause distress or impairment, they are considered clinically significant and may result in a diagnosis of an anxiety disorder. 
Figure 1. An illustration of anxiety disorder.
Available data suggest that fear-related disorders are highly complex and polygenic, and despite substantial progress in genetics (and epigenetics), few responsible loci have been identified for these disorders. Recently, molecular genetic approaches, including genomic studies, have been applied to identify pathways that are associated with anxiety risk. There is now ample experimental and preclinical evidence showing that anxiety disorders are associated with abnormal neural processing of threat-related stimuli, which is mediated by the cyclic AMP (cAMP)-protein kinase A (PKA) pathway. In addition, stress in sensitive phases of development may influence structural integrity of specific brain regions and neural processing pathways involved in emotion regulation, consistent with a gene-environment-timing interaction in mood dysregulation. 
PKA is an evolutionarily conserved serine threonine kinase that regulates diverse signal transduction pathways, including cellular development, proliferation, differentiation, apoptosis, and tumorigenesis. PKA is considered the main target for cAMP in the cell, is widely distributed and serves as the principal effector mechanism for G-coupled receptors linked to adenylate cyclase . In the absence of cAMP, PKA is an inactive tetrameric holoenzyme consisting of two catalytic (C) subunits bound to a regulatory (R) subunit dimer, which is compartmentalized to distinct locations in the cell by A-kinase anchoring proteins (AKAPs) . AKAPs contribute to maintaining specificity of PKA signaling, but it is also believed that several isoforms and splice variants of especially the C subunits are important mediators of specificity.
Based on the elution profile on diethylaminoethyl cellulose exchange chromatography, two isoforms of the PKA heterotetrameric enzyme, PKA-I and -II, exist in most cells. The different PKA subtypes (types I and II) have different affinity for cAMP. This is due to the presence of either type I or II R subunits. There are four such subunits in humans (and mice): RIα and RIβ, and RIIα and RIIβ, coded by the PRKAR1A, PRKAR1β, PRKAR2A, and PRKAR2β genes, respectively .
R subunits form a homodimer that binds two C subunits (one each); there are four C subunits in humans (and mice): Cα, Cβ, Cγ, and protein kinase X gene (PRKX), coded by the PRKACA, PRKACB, PRKACG, and PRKX genes, respectively, in the PKA tetramer (R2C2). When the R and C subunits form a complex, the cAMP-catalytic activity is suppressed. As shown in knockout (KO) mouse studies, these four genes function in a tissue and cell type-specific manner to regulate accurately the activity of the C subunits .
Figure 2. The PKA enzyme is central in the regulation of the cAMP-signalling pathway.
The Role of PKA in Anxiety
PKA activity is affected by various neurotransmitters (i.e. acetylcholine, dopamine, norepinephrine, serotonin, and histamine) that are involved in alertness, anxiety, emotion, or mood indirectly through the stimulation of the G-protein-coupled receptor or adenyl cyclase or directly by cAMP . The CRE is present in many genes and functions as a promoter/enhancer element in many brain areas that respond to environmental stimuli . The observed differences in memory processes that are associated with mood, anxiety, or emotion are likely due to the effects of substances that affect adenyl cyclase activity. For example, dopaminergic D1 and B-noradrenergic receptors enhance, while serotonin receptors inhibit the activity of adenyl cyclase .
Signaling activity in neural circuits before or after stimuli may influence PKA activity and long-term potentiation (LTP), affecting fear learning and memory of the event . PKA has two peaks of activity in the process of long-term memory formation, with the first occurring a few minutes after the event, and the second occurring 2-3 h after the event (requires both transcription and protein synthesis). The PKA pathway is also an important component of short-term memory within the first hour after the event. The phosphorylated form of CREB also increases at these same time periods as PKA and contributes to the synthesis of new proteins that are essential for long-term memory formation .
Inhibitors of Protein Synthesis or PKA activity
Research with transgenic mouse models with inhibitors of protein synthesis or PKA activity demonstrate that inhibition of PKA activity blocks LTP in the hippocampus and interferes with memory consolidation for fear in the amygdala  Infusion of PKA inhibitors into BLA immediately following fear-conditioning training dose-dependently blocked consolidation of fear memory (24 h after training) but not short-term memory(4 h) . Infusion of inhibitor Rp-cAMPs into the CEA decreased CREB function and decreased neuropeptide Y (NPY) expression and provoked anxiety-like behavior and alcohol intake in nonpreferring rats .
Table 1: Preclinical research studies of anxiety or fear memory using stimulation, direct or indirect inhibition of PKA pathway, protein synthesis and various transgenic mice model
PKA-Related Targets Associated with Anxiety-Like Behaviors
Evidence from preclinical studies demonstrate that various targets in the PKA pathway are involved in the regulation of emotional behavior and alterations in this pathway are associated with anxiety and other comorbid behaviors and are therapeutic targets. For example, mice with targeted ablation of CREB during adulthood in forebrain neurons showed severe anxiety phenotype but unaltered hippocampal-dependent long-term memory in context-dependent fear conditioning. CREB protein during adulthood seems to be pivotal for the regulation of emotional behavior . Other PKA-related neural receptors involved in anxiety-like behavior include the glutamate receptor GLuR1  calmodulin signaling  corticotropin-releasing factor (CRF)-1 receptors within medial PFC , and cAMP-regulated phosphoprotein 32 kDa .
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