Abstract
Posttraumatic stress disorder (PTSD) after the pandemic has emerged as a major neuropsychiatric component of post-acute COVID-19 syndrome, yet the current pharmacotherapy for PTSD is limited. The use of adrenergic drugs to treat PTSD has been suggested; however, it is hindered by conflicting clinical results and a lack of mechanistic understanding of drug actions. Our studies, using both genetically modified mice and human induced pluripotent stem cell-derived neurons, reveal a novel α2A adrenergic receptor (α2AAR)-spinophilin-cofilin axis in the hippocampus that is critical for regulation of contextual fear memory reconsolidation. In addition, we have found that two α2 ligands, clonidine and guanfacine, exhibit differential abilities in activating this signaling axis to disrupt fear memory reconsolidation. Stimulation of α2AAR with clonidine, but not guanfacine, promotes the interaction of the actin binding protein cofilin with the receptor and with the dendritic spine scaffolding protein spinophilin to induce cofilin activation at the synapse. Spinophilin-dependent regulation of cofilin is required for clonidine-induced disruption of contextual fear memory reconsolidation. Our results inform the interpretation of differential clinical observations of these two drugs on PTSD and suggest that clonidine could provide immediate treatment for PTSD symptoms related to the current pandemic. Furthermore, our study indicates that modulation of dendritic spine morphology may represent an effective strategy for the development of new pharmacotherapies for PTSD.
Introduction
Posttraumatic stress disorder (PTSD) affects approximately 7–8% of the general population [1] and 11–20% of veterans [2] in the United States, when considering lifetime risk. The number of PTSD cases has further surged as a result of the COVID-19 pandemic; the prevalence of PTSD reaches over 30% both in patients who survived severe viral infection [3] and in frontline healthcare workers dealing with the pandemic [4]. Therefore, proper treatment of PTSD could have profound long-term impacts on human health and healthcare systems. However, currently there are only two FDA-approved medications for PTSD, and only 20–30% of patients achieve complete remission with treatment [5], indicating an urgent need to develop more effective pharmacotherapies for this mental illness.
The brain noradrenergic system plays an essential role in regulating emotional memory [6]. Noradrenergic dysfunction acts as a key contributing factor in the pathophysiology of PTSD [7], and the use of adrenergic ligands, including α2 adrenergic receptor (AR) agonists, has been proposed as an attractive treatment for PTSD symptoms [8, 9]. Preclinical studies have demonstrated that pharmacological manipulations of α2AR activities have profound impact on fear memory formation and retention [10,11,12], although the underlying molecular mechanism remains elusive. Importantly, multiple case reports and double-blinded clinical trials have demonstrated that clonidine, an α2AR agonist, improves multiple symptoms associated with PTSD including nightmare, agitation, and poor sleep quality [13,14,15,16,17,18,19]. However, α2-adrenergic modulation of PTSD-related processes has been challenged by clinical trials of another α2AR agonist, guanfacine; in placebo-controlled trials, guanfacine fails to show significant efficacy for PTSD, arguing against the effectiveness of targeting α2AR in PTSD treatment [20, 21]. The conflicting clinical effects and the lack of mechanistic understanding of drug actions have largely hindered the use of α2AR agonists to treat PTSD. In this study, we attempt to resolve these conflicting clinical observations on α2AR agonists by examining the mechanisms of action of these two drugs on fear memory using animal models and neurons derived from human induced pluripotent stem cells (hiPSCs).
Mounting evidence supports that previously stored memories can be reactivated by retrieval, entering a labile or destabilized state, and restabilized in an updated form [22,23,24]. This process is referred to as memory reconsolidation. Disruption of reconsolidation using pharmacological agents has been suggested as a promising therapeutic strategy for the treatment of PTSD [25]. Currently, a complete understanding of molecular mechanisms underlying memory reconsolidation is still lacking. Here we show, for the first time, that reconsolidation of fear memory requires dynamic changes in the activity and synaptic localization of cofilin.
Cofilin is an actin-severing protein and controls dendritic spine morphology and synaptic plasticity through regulating actin dynamics [26, 27]. Morphological changes of dendritic spines have been indicated as an essential cellular event that underlies learning and memory [28, 29] and disruption of these events is associated with many neuropsychiatric disorders including PTSD [30,31,32]. Activation of cofilin by dephosphorylation at Ser3 causes dendritic spine remodeling in hippocampal neurons, leading to transformation of mature mushroom-shaped spines into immature long thin spines [33]. Consistently, cofilin-deficient neurons show an increase in the number of mature neurons with large spine heads [34]. Changes in cofilin activity are required for both long-term potentiation (LTP) [35, 36] and long-term depression (LTD) [37, 38], the two key cellular/synaptic mechanisms for learning and memory. While cofilin inactivation leads to actin assembly and spine enlargement in LTP [35, 36], cofilin activation drives actin disassembly and spine shrinkage in LTD [37, 38]. Given the pivotal role of cofilin in these processes, precise regulation of its synaptic activity is essential to support proper actin reorganization in learning and memory. To date, our knowledge regarding the spatial and temporal control of cofilin activity during a physiological process, and how this can be manipulated by neurotransmitters and hormones as a means to regulate learning and memory, remains largely scarce. In the present study, we provide the first example that the spatial and temporal dynamics of cofilin at the synapse can be regulated by an FDA-approved adrenergic drug to modulate fear memory.
We identified cofilin as a novel downstream signaling effector that mediates α2AAR-elicited regulation of fear memory reconsolidation. The two α2AAR agonists, clonidine and guanfacine, show distinct ability in activating cofilin. Clonidine, but not guanfacine, promotes the interaction of cofilin with the receptor and with a synaptic scaffolding protein, spinophilin, which is essential in enhancing cofilin activity at the synapse. This ligand-selective activation of the α2AAR-spinophilin-cofilin signaling axis leads to distinct regulation of contextual fear memory reconsolidation by clonidine and guanfacine and informs interpretation of differential clinical observations of these drugs on PTSD. Our study further suggests that pharmacological manipulation of spine morphology modulators such as cofilin represents an effective strategy for modification of fear memory reconsolidation, and thus has far-reaching implications for the development of active pharmacotherapies for PTSD.
Materials and methods
Animals
All experiments conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committees (IACUC) of University of Alabama at Birmingham and Augusta University. Mice were maintained on a 12-h light/dark cycle with food and water continuously available and used at 3–5 months of age. α2AAR deficient (Adra2a−/−) were originally obtained from The Jackson Laboratory (stock number 004367) and bred and maintained on site at the C57BL/6 background. The generation of spinophilin deficient (Ppp1r9b−/−) mice has been described previously [39], and the line has been backcrossed more than 10 generations to the C57BL/6 background. Both males and females were used, and we did not observe a significant difference between the sexes.
Reagents and antibodies
All peptides were synthesized and purified by GenScript, USA, Inc. Peptides containing a 16 aa sequence of the cofilin Ser3 site (MASGVAVSDGVIKVFN, referred to as S3 peptides) or phosphor-Ser3 site [MAS(p)GVAVSDGVIKVFN, referred to as pS3 peptides] were used. These peptides were fused to a TAT-like polyarginine membrane permeability sequence (GRRRRRRRRRRR) to facilitate its entrance into cells and to a biotin molecule to allow detection. The TAT-like peptide (GRRRRRRRRRRR) was used as a control. Antibodies to cofilin (cat #5175S, dilution 1:1000), phosphorylated cofilin (p-cofilin) (cat# 3311S, dilution 1:500) and Myc-Tag (9B11) (cat# #2276S, dilution 1:1000) were purchased from Cell Signaling Technology. HA.11 antibody (cat#901515) was from Biolegend. Clonidine (cat# C7897), guanfacine (cat#G1043), BRL44408 (cat# B4559) were from Sigma-Aldrich, and JP1302 (cat# 26-661-0) and imiloxan (cat# 09-861-0) were purchased from Thermo Fisher Scientific.
