GPCR endocytosis rewires neuronal gene expression and cellular architecture

GPCR endocytosis rewires neuronal gene expression and cellular architecture | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results GPCR endocytosis rewires neuronal gene expression and cellular architecture Katherine L. Hall , Matthew J. Klauer , View ORCID Profile Nikoleta G. Tsvetanova doi: https://doi.org/10.1101/2025.08.26.672159 Katherine L. Hall 1 Department of Pharmacology and Cancer Biology, Duke University , Durham, NC 27710 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Matthew J. Klauer 1 Department of Pharmacology and Cancer Biology, Duke University , Durham, NC 27710 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nikoleta G. Tsvetanova 1 Department of Pharmacology and Cancer Biology, Duke University , Durham, NC 27710 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nikoleta G. Tsvetanova For correspondence: nikoleta.tsvetanova{at}duke.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT In the brain, G protein-coupled receptors (GPCRs) regulate neuronal excitability, synaptic transmission, and behavior by engaging transcriptional and translational programs that produce enduring changes in cellular function and architecture. However, the molecular mechanisms that couple GPCR activation to these adaptations remain poorly understood. Here, we demonstrate that the beta-adrenergic receptor (β2AR), a mediator of noradrenaline in the central nervous system, remodels neuronal morphology through compartmentalized signaling pathways that orchestrate distinct layers of gene regulation. Following stimulation, β2ARs remain active on endosomes, and their intracellular signaling promotes dendritic growth and synapse formation. These structural effects are driven by two coordinated regulatory axes: PKA/CREB-dependent transcription of morphogenesis-related genes and PKA/mTOR-dependent translation of components of the protein synthesis machinery. Altogether, this work defines novel spatial and biochemical principles by which GPCR signaling drives structural reorganization and functional adaptations in neurons. INTRODUCTION G protein-coupled receptors (GPCRs) make up the largest and most diverse family of membrane receptors in humans. The vast majority of non-sensory GPCRs are expressed in the brain where they regulate neuronal excitability and synaptic plasticity to support key functions, including learning, memory, reward processing, and motor control 1 . Not surprising, disruptions in GPCR signaling have been linked to a wide range of neurological and psychiatric disorders, making these receptors major targets for neuropharmacological therapies 1 – 3 . Despite their widespread relevance, how GPCRs drive long-term changes in neuronal function remains poorly defined. These adaptations depend on precise regulation of gene expression, including transcriptional and translational reprogramming that reshapes neuronal identity and connectivity. Such regulatory events are particularly important for sustained forms of plasticity, such as long-term potentiation 4 , 5 . Yet, the nature of these gene expression programs, and how these are coordinated downstream of GPCRs, have not been systematically characterized. At the cellular level, GPCRs transduce their effects by activation of heterotrimeric G proteins and regulation of the abundance of second messengers. The cyclic AMP (cAMP)-protein kinase A (PKA) cascade is a major signaling pathway engaged by neuronal GPCRs. Upon ligand binding, receptors coupled to the G protein, Gαs, stimulate adenylyl cyclase, leading to increase in intracellular cAMP. Elevated levels of cAMP in turn activate PKA and other effectors, which influence a broad range of cellular targets and neuronal processes, including development and excitability 6 . Achieving this functional diversity requires mechanisms that can direct signals with both spatial and temporal precision. Recent studies suggest that the subcellular location of GPCR activation contributes to this specificity. In particular, GPCR signaling is not restricted to the plasma membrane, but can continue from intracellular compartments, prolonging and/or shaping activation of select downstream pathways 7 – 10 . However, most of these findings are based on heterologous systems or non-neuronal models, and it is not clear whether these mechanisms are conserved in neurons. Given the size and morphological complexity of neurons, we reasoned that intracellular GPCR signaling may be particularly important for relaying extracellular signals across extended cellular domains and into specialized compartments to regulate gene expression. We address this in the context of the beta2 adrenergic receptor (β2AR), a well-suited model for investigating how GPCR/cAMP signaling influences neuronal function. The β2AR is one of the most extensively studied GPCRs and is expressed across multiple tissues, including in the brain. In the central nervous system, it mediates key noradrenergic effects on synaptic plasticity by engaging transcription- and translation-dependent mechanisms that regulate attention, arousal, learning, and memory 11 – 14 . In addition, prior work in HEK293 cells has shown that the β2AR remains active on endosomes after internalization 15 , 16 , making it a useful system for assessing whether such spatial organization is conserved in neurons. Here, we integrate imaging-based analyses of dendritic and synaptic architecture with global transcriptional and translational profiling to show that β2AR activation induces extensive structural remodeling through compartment-specific regulation of gene expression. RESULTS β2AR signaling promotes dendritogenesis and synapse formation Because cortical excitatory neurons are a primary site of adrenergic receptor function 14 , 17 – 19 , we employed a previously validated human iPSC-derived neuronal system (iNeurons) that expresses the β2AR ( Fig. 1a ). iNeurons are scalable and homogeneous, allowing robust transcriptomic and translatomic profiling, and provide a unique cellular platform for dissecting GPCR signaling and downstream gene regulation in a human neuronal context. Download figure Open in new tab Figure 1. β2AR activation promotes structural remodeling of neurons. (A) Left : Schematic of the workflow for generation of human iPSC-derived cortical neurons. Right : Fixed cell fluorescence microscopy staining of iNeuron cultures (DIV21) with antibodies for the cortical marker, NGN2 (green), and neuronal marker, MAP2 (red). (B) Representative images of neurons treated with vehicle (no drug, ‘ND’) or 1 μM Isoproterenol (Iso) for 2 h, fixed and stained with antibody for MAP2 three days later (left) ; quantification of numbers of crossings by Sholl analysis (right) . Data are mean of n = 21-25 cells from 3 independent experiments. (C) Left: Representative images of neurons stained for vGLUT1 as presynaptic marker (green) and PSD95 as postsynaptic marker (red). Right: Quantification of synaptic pairs based on colocalization analysis of vGLUT1/PSD95 puncta per 35 μ m . Neurons were treated with vehicle (‘ND’) or 1 μM Isoproterenol (‘Iso’) for 24 h. Data are mean of n = 12 cells from 3 independent experiments. Error bars represent standard error of the mean. **** = p < 0.0001 by unpaired Student’s t test. Scale bars, 50 μm in (B) and 10 μm in (C) . β2AR activity promotes excitability and synaptic potentiation, processes that are generally coupled to structural remodeling of the neuron. We therefore first asked whether β2AR signaling has effects on dendritic growth and synapse formation by stimulating iNeurons with the synthetic beta-adrenergic receptor agonist, isoproterenol. To assess dendritic arbor complexity, we performed Sholl analysis which revealed a significant ligand-dependent increase in the number of dendritic branches ( Fig. 1b ). To measure synapse formation, we quantified colocalization of the pre- and postsynaptic markers, VGLUT1 and PSD95, respectively. Treatment with agonist caused an increase in the number of synaptic pairs ( Fig. 1c ). Therefore, activation of the β2AR enhances both dendritic complexity and synapse formation, indicating a prominent role of the cascade in promoting neuronal structural remodeling. Next, we sought to define the spatial organization of the β2AR signaling pathway and the molecular programs that underlie its role in neuronal morphogenesis. Intracellular β2AR signaling is required for neuronal structural remodeling It was previously reported in HEK293 cells that the β2AR initiates G protein-dependent signaling from the plasma membrane and from endosomes 15 , 16 . To assess the activity of β2ARs in neuronal compartments, we used two validated single-chain antibody (nanobody) sensors, Nb80 and Nb37, that recognize the active state of the receptor and Gαs protein, respectively 15 . Each biosensor tagged with green fluorescent protein (GFP) was co-expressed with the β2AR in iNeurons. Under unstimulated conditions, the receptor was confined to the plasma membrane, while the nanobody was diffusely distributed in the cytoplasm by live confocal microscopy ( Fig. 2a-b ). Following stimulation with isoproterenol, both biosensors co-localized with the internalized receptor on endosomal vesicles ( Fig. 2a-b ). These support that β2AR and Gαs are active on endosomes in neurons. To provide evidence that intracellular activation contributes to the resulting cellular response, we measured receptor-induced cAMP production. To parse out the endosomal response, we restricted β2AR signaling to the plasma membrane by acute endocytic inhibition with the compound Dyngo-4a, an established method to block dynamin-dependent trafficking 15 , 16 . Blockade of receptor endocytosis significantly blunted the cAMP response ( Fig. 2c ). Hence, endosomal β2ARs play an active role in shaping downstream second messenger production. Download figure Open in new tab Figure 2. Endosomal signaling by β2AR is required for dendrite growth and synapse formation. (A-B) Detection of active β2AR / Gαs on endosomal vesicles. Left : Representative confocal images of neurons expressing Flag-tagged receptor (red) and GFP-tagged conformational biosensors for detection of active β2AR (Nb80, green) (A) and Gαs (Nb37, green) (B) . 1 μM Isoproterenol (Iso) was added at t=0. Right : Line scan analyses of colocalization between the β2AR and the biosensor. Yellow line: line scan analysis of gray value in each channel. Arrows indicate colocalization. (C) Endocytosis is required for cAMP accumulation. Neurons were pretreated with vehicle (DMSO) or 30 μM Dyngo-4a for 30 min, then stimulated with 1 μM Iso for 5 min, lysed and cAMP levels were measured using an ELISA assay. Data are mean of n = 3 independent experiments. (D-E) Analyses of dendritogenesis and synapse formation. Representative images are shown for each condition. Neurons were fixed and stained for MAP2 (D) or vGLUT1/PSD95 (E) . Neurons were pretreated with vehicle (DMSO) or 30 μM Dyngo-4a for 30 min, then stimulated with 1 μM Iso or 100 μM cAMP analog (8Br-cAMP). Quantification of number of crossings by Sholl analysis (D) and synaptic pairs (E) is shown. Data are mean of n = 18-25 cells in (D) , n = 9-12 (E) from 2-3 independent experiments. Error bars represent standard error of the mean. **** = p < 0.0001, *** = p < 0.001, ** = p < 0.01, * = p < 0.05 by two-way ANOVA with Sidak correction in (C) , one-way ANOVA with Dunnett correction in (D-E) . ND = no drug; Veh = vehicle. Scale bars, 10 μm in (A), (B), (E) and 50 μm in (D) . Next, we asked whether the intracellular β2AR population is necessary to induce dendrite growth and synapse formation. Remarkably, pre-treatment of cells with Dyngo-4a completely abolished the effects of isoproterenol on neuron morphology ( Fig. 2d-e ). Notably, the endocytic inhibitor had no impact on dendritic growth and synapse formation following direct stimulation of neurons with a cell-permeable cAMP analog ( Fig. 2d-e ), supporting that loss of structural plasticity reflects a selective requirement for endosomal β2ARs. Collectively, these results demonstrate an active role for intracellular receptors in mediating neuronal signaling and structural reorganization. β2AR activation induces extensive neuronal transcriptional and translational reprogramming Changes in neuronal morphology depend on de novo protein synthesis to provide the molecular machinery needed for structural adaptation. Notably, these are regulated by transcription-dependent and -independent mechanisms, as neuronal proteins necessary for synapse remodeling are translated from newly transcribed as well as pre-existing mRNAs 20 . Therefore, to determine the molecular underpinnings of β2AR-dependent structural remodeling, we set out to delineate the global neuronal transcriptional and translational programs regulated by the receptor. To identify the β2AR-dependent transcriptional responses, we conducted RNA sequencing (RNA-seq) analysis of iNeurons treated with isoproterenol for 2 hours. Through differential expression analysis, we identified ∼400 neuronal transcriptional targets, the majority of which were upregulated by agonist ( Fig. 3a , Dataset S1) . A fraction of these genes were classified as β2AR transcriptional targets in other cell models 16 , 21 and likely represent a conserved gene group regulated by this receptor (39/402 genes, p < 2.0 x 10 -16 by Fisher’s exact test) (Dataset S1) . These included well-known immediate early genes (IEGs) that are expressed ubiquitously and expected to be induced by cAMP, such as FOS , CGA and DUSP1 (Supplementary Fig. 1a) . The remaining genes represented neuronal targets and included at least 40 genes expressed predominantly in the brain (e.g., NPAS4, NGN2, SYT4, KCNA2 ). Accordingly, GO term analysis pointed to regulation of several pathways selectively associated with neuronal function, such as “neuron differentiation”, “neuropeptide signaling” and “anatomical structure development” ( Fig. 3b ). The latter category was of particular mechanistic salience to the effects of β2AR on neuronal morphology, as it encompassed factors with established roles in neurite growth and axon development ( Fig. 3a in red, Dataset S1) . Notably, one of these transcriptional targets was brain-derived neurotrophic factor ( BDNF ), which encodes a signaling molecule tied to axon growth and pathfinding, dendritic arbor growth and synaptic connectivity 22 . Specifically, BDNF expression was robustly upregulated following isoproterenol treatment. We used RT-qPCR to validate the transcriptional induction of BDNF and UNCX , another gene with known roles in neuronal development and connectivity 23 (Supplementary Fig. 1a) . Download figure Open in new tab Figure 3. Transcriptional and translational responses to GPCR activation. (A) Volcano plot of RNA-seq analysis showing neuronal gene fold induction following activation with 1 μM Isoproterenol for 2 h (log 2 Isoproterenol/no drug). Blue dots represent β2AR-dependent transcriptional targets. Red dots represent transcriptional targets that make up the Gene Ontology category “Anatomical structure development” in (B) . Data are mean of n = 3 independent experiments. (B) Gene ontology categories enriched among the transcriptional targets. (C) Scatter plot of changes in mRNA (‘Transcriptional change’) and RPF abundance (‘Translational change’) following activation with 1 μM Isoproterenol for 2 h from integrated RNA-seq and Ribo-seq analyses. Values are log 2 (Isoproterenol/no drug). Dark blue dots represent forwarded targets. Light blue dots represent buffered targets. Red dots represent exclusively translationally regulated targets. The parentheses indicate number of target genes. Data are mean of n = 3 (RNA-seq) or mean of n = 4 (Ribo-seq) independent experiments. (D) Gene ontology categories enriched among the exclusive translationally regulated β2AR-dependent targets. Next, we assessed how activation of the receptor cascade impacts the neuronal translational landscape. To this end, we carried out ribosome profiling (Ribo-seq) under matched stimulation conditions (Supplementary Fig. 1b) . The sequenced ribosome footprints (RPFs) exhibited the expected size distribution, three-nucleotide periodicity and enrichment primarily in coding sequences supporting that this analysis is measuring ribosome-occupied status of actively translating neuronal mRNAs (Supplementary Fig. 1c-d) . To evaluate the extent of overlap between transcriptional and translational responses to β2AR, we first examined the correlation between RNA-seq and Ribo-seq replicates. Overall, there was a strong correlation between RNA and ribosome footprint abundance (Supplementary Fig. 1e) . However, replicates within each assay displayed higher similarity, supporting both common and distinct layers of transcriptional and translational regulation (Supplementary Fig. 1e , compare scatter plots on top vs bottom). Considering genes with uniquely mapped reads across the two experimental platforms, we compared the agonist-dependent changes in RPF and RNA counts for each transcript to determine its mode of regulation. We identified 129 “forwarded” targets, for which changes in ribosome occupancy correlated directly with changes in RNA expression levels ( Fig. 3c in dark blue , Dataset S2) . For these, β2AR-dependent translation can be attributed primarily to changes in mRNA abundance, and by extension, transcriptional signaling. In agreement, all forwarded targets were also classified as transcriptional hits, including mRNAs encoding regulators of neuronal structural remodeling. In contrast, 143 genes displayed discordant agonist-induced changes in RPF and mRNA levels, indicating regulation at the level of translation. Of these, 89 genes showed mRNA abundance changes without corresponding significant shifts in ribosome occupancy. These genes are regulated at multiple levels, with translational control “buffering” changes in mRNA abundance (“buffered” targets, Fig. 3c in light blue , Dataset S2 ). This set of targets was enriched primarily for genes encoding transcription factors (Supplementary Fig. 1f). The remaining 54 genes changed solely at the level of ribosome occupancy without changes in mRNA abundance and were classified as “exclusive” translationally regulated targets of the β2AR ( Fig. 3c in red , Dataset S2) . GO analysis of the isoproterenol-dependent exclusive translational hits revealed that these orchestrated distinct cellular functions compared to the transcriptional targets. Notably, factors involved in the process of “translation” were over-represented and comprised one third of the “exclusive” genes ( Fig. 3d ). We selected and validated one gene from each regulatory category by RT-qPCR and Western blot analyses that measured agonist-induced changes in mRNA and protein abundance, respectively (Supplementary Fig. 1g-h) . These genomic analyses indicated that β2AR signaling elicits widespread changes in neuronal gene expression by engaging transcriptional and translational regulatory mechanisms. Specifically, activation of the cascade promoted the transcription of genes involved in neuronal morphology and growth, while independently enhancing the selective translation of mRNAs encoding components of the protein synthesis machinery. GPCR endocytosis is required for coordinated gene expression control Given that β2AR endocytosis was required for isoproterenol-driven neuronal remodeling, we hypothesized that the agonist-induced gene expression changes may be critically dependent on intracellular receptor signaling. To interrogate the contribution of endosomal β2ARs to differential gene expression, we conducted parallel transcriptional and translational analyses in unstimulated and isoproterenol-treated iNeurons, in which receptor trafficking was blocked acutely with Dyngo-4a. Application of inhibitor alone did not interfere with steady-state neuronal gene expression (Supplementary Fig. 2a-b) . By contrast, Dyngo-4a markedly suppressed the agonist-driven responses at both the transcriptional and translational levels ( p < 2.0 × 10 - 16 for β2AR transcriptional targets; p < 7.0 × 10 - 7 for exclusive β2AR translational targets, Wilcoxon test). To further understand the scope and nature of the regulation, we classified genes within each dataset as targets of endosomal or plasma membrane signaling based on inhibitor effects. This revealed that endocytosis was broadly required for β2AR-mediated transcriptional responses, as all targets underwent significant loss of regulation in the presence of Dyngo-4a (FDR < 0.1 by multiple t- test analysis, Fig. 4a ). RT-qPCR analysis of select genes, including IEGs and factors enriched in the neuronal GO categories “anatomical structure development” and “neuron differentiation”, confirmed that their expression was regulated by β2AR in an endocytosis-dependent manner (Supplementary Fig. 2c) . We similarly stratified the exclusive translational hits according to the impact of Dyngo-4a on their translation efficiency. As with the transcriptional responses, endosomal β2ARs emerged as the primary drivers of translation and were required for the regulation of ∼85% of target RNAs, ( Fig. 4b , “endo”). These included genes that fell into the significant Ribo-seq GO categories identified in cells with intact endocytosis, including “translation” (Supplementary Fig. 2d) . However, while none of the transcriptional targets exhibited intact regulation upon endocytic blockade, we found that the regulation of ∼15% of translational targets did not require internalization. Hence, these RNAs represented targets of plasma membrane β2ARs ( Fig. 4b , “PM”). Download figure Open in new tab Figure 4. β2AR-induced gene reprogramming requires endocytosis. (A-B) Heatmaps of transcriptional (A) and exclusive translational (B) targets from RNA-seq/Ribo-seq analyses. Neurons were pretreated with vehicle (DMSO) or 30 μM Dyngo-4a for 30 min, then stimulated with 1 μM Isoproterenol (Iso) for 2 h. Colors represent log 2 fold changes between Iso-treated and the corresponding untreated samples in terms of mRNA abundance (A) or changes in translation efficiency (ΔTE, the ratio between Iso-dependent changes in ribosomal occupancy and transcript abundance). Color-coding in (B) indicates whether a gene is classified as endosomal (‘Endo’, green) or plasma membrane target (‘PM’, yellow) based on multiple unpaired t test analysis with FDR < 0.1 cut-off. K-means clustering analysis was performed with pheatmap in R. Together, these findings indicated that both transcriptional and translational responses to β2AR activation are predominantly endosome-dependent, although the translational regulation of a subset of mRNAs persisted even in the absence of intracellular receptors. Endosomal β2ARs remodel neuronal morphology through PKA/CREB and PKA/mTOR signaling axes To uncover which molecular pathways are engaged by endosomal receptor activity to drive gene regulation, we analyzed motif enrichment patterns across target gene sets. Transcription factor analysis revealed CREB as the major driver of β2AR-induced transcriptional signaling (Supplementary Fig. 3a) . This aligned with the established understanding that GPCRs signaling via cAMP activate PKA to promote CREB-dependent transcription 16 . Consistent with this model, isoproterenol stimulation increased CREB Ser133 phosphorylation, an effect impaired by the PKA inhibitor H89 (Supplementary Fig. 3b) . Therefore, we asked whether endosomal β2ARs are required for localized activation of PKA. We monitored kinase activity using single-fluorophore biosensors targeted to the cytosol and nucleus 24 , 25 and found that isoproterenol robustly activated PKA in both compartments, and this effect was inhibited by endocytic blockade (Supplementary Fig. 3c-d) . Moreover, treatment with H89 prevented isoproterenol-induced dendritic growth and synapse formation ( Fig. 5a-b , red bars), indicating that the structural adaptations elicited by β2ARs depend on PKA as the principal downstream effector. Download figure Open in new tab Figure 5. Compartmentalized β2AR activation modulates neuronal architecture via distinct PKA-linked pathways. (A-B) PKA and mTOR are required for β2AR-induced neuronal morphogenesis. Representative images of dendritogenesis and synapse formation analyses are shown. Neurons were fixed and stained for MAP2 (A) or vGLUT1/PSD95 (B) . Neurons were pretreated with vehicle (DMSO), 10 μM H89 or 100 nM Torin for 30 min, then stimulated with 1 μM Iso. Quantification of number of crossings by Sholl analysis (A) and synaptic pairs (B) is shown. Data are mean of n = 13-17 cells in (A) and n = 10 cells in (B) from 2-3 independent experiments. (C-D) Endosomal β2AR signaling and PKA are required for mTOR activation. Neurons pretreated with vehicle (DMSO), 10 μM H89, 30 μM Dyngo-4a or 100 nM Torin for 30 min, then stimulated with 1 μM Iso for 30 min. Representative Western blots of 4E-BP1 phosphorylation (Thr37/46) are shown. (E) Model: Endosomal GPCR signaling elicits morphogenesis through transcriptional and translational changes that together boost the cell’s protein synthesis capacity to provide key structural effectors. Error bars represent standard error of the mean. **** = p < 0.0001 by one-way ANOVA with Dunnett correction. ND = no drug; Veh = vehicle. Scale bars, 50 μm in (A) and 10 μm in (B) . In parallel with the transcriptional programs, we next examined the translationally regulated gene set that exhibited dependence on β2AR endocytosis. These showed striking enrichment of mRNAs related to ribosomal structure and translational processes (15/53 targets; p < 1.0x10 - 43 , Fisher’s exact test, Supplementary Fig. 2d ). Moreover, translation of these genes was significantly reduced by endocytic blockade ( p = 1.0x10 - 3 , Wilcoxon test). Notably, this gene set was characterized by the presence of a 5′ terminal oligopyrimidine (5′TOP) motif, a signature conferring sensitivity to mTOR-dependent translational control 26 . Prompted by this, we examined further the effects of β2AR stimulation on TOP mRNAs. Of ∼100 annotated mammalian TOP mRNAs 26 , 89 genes were detected in our Ribo-seq datasets. Although only a subset passed our threshold for classification as ‘exclusive’ translational hit, TOP mRNAs as a group showed robust induction of translation efficiency in response to isoproterenol compared to all other genes ( p = 8.4 x 10 -22 by Wilcoxon test, Supplementary Fig. 3e) . These findings raised the possibility that intracellular β2AR signaling regulates neuronal mRNA translation through the mTOR pathway. Indeed, isoproterenol increased phosphorylation of the mTOR substrate 4EBP-1, an effect blocked by inhibitors of mTOR, PKA, and dynamin ( Fig. 5c-d ). These supported that mTOR activation lies downstream of PKA and requires receptor endocytosis. Lastly, because mTOR is a well-established driver of structural plasticity downstream of receptor tyrosine kinases 27 , 28 , we hypothesized that endosomal β2ARs engage a similar mechanism to control morphogenesis. Indeed, application of the mTOR inhibitor, Torin, blocked β2AR-induced dendritic arborization and synapse formation ( Fig. 5a-b , blue bars). Collectively, these results indicate that intracellular β2ARs signaling coordinates gene expression through PKA/CREB and PKA/mTOR to induce dendritic and synaptic remodeling. DISCUSSION While the classical view held that GPCRs signal primarily from the plasma membrane, growing evidence has highlighted that they can also be activated from intracellular compartments 7 – 10 . Despite the prevalence and critical roles of these pathways in the brain, endosomal GPCR signaling, particularly through Gαs/cAMP, is only beginning to be elucidated in neurons. Here, we discover that activation of β2ARs promotes dendrite growth and synapse formation through spatially restricted cAMP signaling, which is critical for initiating downstream transcriptional and translational regulatory programs. Our data therefore support that compartmentalized GPCR signaling selectively engages distinct downstream effectors to rewire the molecular landscape and cellular architecture of neurons. Activation of endosomal β2ARs triggers two key discrete molecular outcomes: transcriptional upregulation of genes associated with neuronal development and morphogenesis, and selective enhancement of translation efficiency for mRNAs encoding components of the protein synthesis machinery ( Fig. 3 , 4) . This is consistent with a coordinated two-tiered mechanism for promoting structural plasticity driven by the internal receptor: a rapid translational response that amplifies the protein synthesis capacity of the cell coupled with a transcriptional program that supplies key structural regulators ( Fig. 5e ). Although in principle multiple cascades can be activated by endosomal β2ARs 16 , 21 , 29 – 31 , our data support PKA as the primary downstream effector that decodes the signal to modulate neuronal gene expression and the subsequent effects on neuronal morphogenesis. Specifically, we find that these processes are mediated by PKA-dependent activation of transcription through CREB and translation through mTOR ( Fig. 5 , Supplementary Fig. 3) . Notably, CREB and mTOR have each been associated with the regulation of neuronal structural plasticity 27 , 32 , 33 . However, the precise neuronal mRNA targets of each that mediate these effects had not been clearly defined, a gap addressed by our study. Further, prevailing models have placed neuronal mTOR regulation under the control of extracellular signal-regulated kinase (Erk) and phosphoinositide 3-kinase (PI3K) cascades downstream of receptor tyrosine kinases 34 – 36 . In the context of cAMP signaling, Gobert et al. similarly reported that forskolin-induced mTOR activation and translational control in organotypic neuronal cultures required Erk and PI3K 37 . On the other hand, studies in non-neuronal models have found that cAMP signaling can regulate mTOR via PKA-dependent mechanisms 21 , 38 – 40 , in agreement with the pathway defined here. Therefore, the discrepancy between our findings and previous reports on neuronal mTOR regulation may reflect the broader lack of mechanistic studies on GPCR and cAMP signaling in neurons. Interestingly, we find that neuronal translation, unlike transcription, exhibits a more nuanced dependence on intracellular β2ARs ( Fig. 4 ). Specifically, some genes retain isoproterenol-induced translational regulation in the absence of endocytosis, suggesting that these targets may be governed by distinct spatial and temporal requirements for GPCR/cAMP signaling. This distinction may stem from the established capacity of neurons to locally translate certain mRNAs at synaptic sites in response to stimulation 41 , 42 . Hence, ribosomes and/or RNA-binding proteins involved in the translation of these transcripts could be poised to respond to compartmentalized cues that do not propagate to the nucleus to drive transcription. Examination of the subcellular localization and translation of target mRNAs that do and do not require endosomal GPCR signaling would be instrumental in evaluating this as a tentative mechanism. Given the extreme morphological complexity of neurons, endosomal receptors offer an elegant solution to a fundamental problem: how neurons transmit information from the process into the soma. According to our data, PKA activity remains limited across neuronal compartments when β2AR endocytosis is blocked (Supplementary Fig. 3) . Based on prior work, we anticipate that this compartmentalization of cAMP/PKA signaling arises not just from cell geometry, but also from local signalosome scaffolding by A-kinase anchoring proteins and cAMP breakdown by phosphodiesterase (PDE) enzymes 43 , 44 . In HEK293 cells, we have shown that cell surface-delimited β2AR signaling is ‘buffered’ by higher PDE activity at the plasma membrane relative to endosomes 16 , 24 , 45 . While it remains to be determined if, and how, these mechanisms operate in neurons, intracellular GPCRs may be especially effective at bridging long distances and evading PDE hydrolysis by enabling spatially discrete signaling waves and sustaining the response. We note that the idea of signaling vesicles as platforms that mediate long-range intracellular communication is not without precedent: it has been shown that retrograde endosomes are critical for neurotrophic signal transmission from axons to the cell body downstream of receptor tyrosine kinases 46 , 47 . While the regulatory mechanisms and downstream effects may differ, our findings support that intracellular GPCR activation serves a similar function. More broadly, the requirement for endosomal β2AR signaling in promoting structural plasticity raises the intriguing possibility that neurotransmitter concentration and kinetics may serve as a biological gate for this spatial mode of signaling. Unlike brief, low-concentration neurotransmitter exposure that may activate surface receptors transiently, higher or sustained levels of norepinephrine, as might occur during heightened arousal or attention, is expected to promote efficient β2AR internalization. This in turn would facilitate the selective activation of the endosome-restricted PKA/CREB and mTOR pathways triggering extensive gene adaptation. In this model, the intensity and duration of neuromodulator release would determine whether β2AR signaling remains confined to rapid, membrane-delimited events or transitions to a gene expression-driven, plasticity-permissive state. Future studies dissecting the precise signaling thresholds and temporal dynamics that govern this mechanism will provide additional critical insights and means to test this model. Altogether, the present work illuminates new spatial and biochemical mechanisms, by which GPCR signaling modulates neuronal function. Moving forward, it will be important to establish how broadly these mechanisms apply across neuronal types and circuits, and how they ultimately affect network activity and behavior. AUTHOR CONTRIBUTIONS K.L.H., M.J.K. and N.G.T. designed research; K.L.H. and M.J.K. performed and analyzed the experiments; K.L.H. and N.G.T. wrote the paper; K.L.H., M.J.K. and N.G.T. edited the paper. MATERIALS AND METHODS Chemicals and antibodies (−)-Isoproterenol hydrochloride (Sigma-Aldrich, Cat #I6504) was purchased from, dissolved in 100 mM ascorbic acid to 10 mM stock, and used at indicated concentrations. Dyngo-4a (Abcam, Cat #120689) was resuspended in DMSO to 30 mM stock and used at 30-45 μM final concentration. Torin-1 (MedChemExpress, Cat #HY-13003) was resuspended to 100 μM in DMSO and used at a final concentration of 100 nM. H89 (Cayman Chemical, Cat #10010556) was dissolved in DMSO to 10 mM and used at 10 μM final concentration. 8-bromo-cAMP (Abcam, #ab141448) was dissolved in water to 100mM and used at 100 μM final concentration. Pitstop 2 (Abcam, #ab120687) was dissolved in DMSO to 30 mM and used at 30 μM final concentration. Rabbit anti-4E-BP1 monoclonal antibody (Cell Signaling Technologies, Cat #9644), rabbit anti-phospho-4E-BP1 monoclonal antibody (Thr37/Thr46) (Cell Signaling Technologies, Cat #2855), rabbit anti-FTH1 monoclonal antibody (Cell Signaling Technologies, Cat #4393), rabbit anti-FOS monoclonal antibody (Cell Signaling Technologies, Cat #2250T), rabbit anti-NPAS4 monoclonal antibody (Activity Signaling, Cat #AS-AB18A-100), mouse anti-beta-Actin monoclonal antibody (Santa Cruz Biotechnology Cat #sc-69879 were used at 1:1,000 for Western blots. Chicken anti-MAP2 monoclonal antibody (Phosphosolutions, Cat #1099-MAP2), rabbit anti-NGN2 polyclonal antibody (Invitrogen Cat #PA5-78556), guinea pig anti-VGLUT1 polyclonal antibody (Millipore, Cat #AB5905), rabbit anti-PSD95 polyclonal antibody (Abcam, Cat #AB18258) were used at 1:1,000 for immunofluorescent staining. The following secondary antibodies were used at 1:10,000 for Western and immunofluorescence: donkey anti-mouse IRDye 680RD (Licor, Cat #926-68072), donkey anti-rabbit IRDye 800CW (Licor, Cat #925-32213), goat anti-guinea pig Alexa Fluor 488 (Thermo Fisher Scientific, Cat #A11073), goat anti-chicken Alexa Fluor 568 (Thermo Fisher Scientific, Cat #A11041), donkey anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific, Cat #A21206), donkey anti-rabbit Alexa Fluor 647 (Thermo Fisher Scientific, Cat #A31573). Lentivirus production Lentivirus was used to express ExRai-AKAR2, ExRai-AKAR2-NLS, Nb37-eGFP, and Nb80-eGFP. HEK293T were transfected with lentiviral constructs and packaging vectors (psPAX2 and VSVG) using Lipofectamine 2000 (Thermo Fisher Scientific, Cat #11668019) and OptiMEM (Thermo Fisher Scientific, Cat #31985062). Media was changed 16 h post transfection and the supernatant was harvested 72 h post transfection. The viral supernatant was concentrated using LentiX concentrator (Takara, Cat #631231), resuspended in PBS, and snap-frozen or used immediately. Construct cloning The lentiviral GFP-tagged Nb37 and Nb80 constructs were described previously 24 . To generate pFUGW-ExRai-AKAR2 plasmid, AKAR2 sequence was PCR amplified from the original ExRai-AKAR2 and ExRai-AKAR2-NLS constructs 24 , 25 and inserted into a digested pFUGW-GFP backbone by In-Fusion cloning (Takara Bio, Cat #638947) following recommended protocols. Cell culture The human iPSC line expressing Flag-tagged β2AR was previously generated and characterized in Ref 48 . Briefly, hiPSCs expressing Ngn2 under a doxycycline-inducible promoter integrated into the AAVS1 safe harbor locus 49 were transduced with Flag-β2AR under ubiquitin promoter and low-expressing clones were isolated by fluorescent cell sorting. iPSCs were grown in mTeSR media (StemCell Technologies, Cat #100-1130) with 10 μM Rock inhibitor (MedChem Express, Cat #HY-10583-10mg) on plates with Matrigel Basement Membrane Matrix (Corning, Cat #356231) diluted in Knockout DMEM (Thermo Fisher Scientific, Cat #10829018) to 100 μg/mL. Media changes were performed daily. For differentiation into iNeurons, iPSCs were grown for 3 days in Knockout DMEM/F12 (Thermo Fisher Scientific, Cat #12660012) supplemented with 1X N2 (ThermFisher Cat #17502048), 10 ng/mL Recombinant Human NT-3 (PeproTech Cat #450-03), 2 μg/mL Doxycycline (Sigma, Cat #D3447), 10 ng/mL Recombinant Human/Murine/Rat BDNF (PeproTech, Cat #450-02), 1 μg/mL Mouse Laminin (Thermo Fisher Scientific, Cat #23017015) and 1X MEM Non-Essential Amino Acids (NEAA) (Thermo Fisher Scientific, Cat #11140050). Then, neurons were replated and grown on BioCoat PDL-coated plates (Corning, Cat #354413) in 0.5X Neurobasal-A media (Thermo Fisher Scientific, Cat #10888022) and 0.5X DMEM/F-12 media (Thermo Fisher Scientific, Cat #11330032) supplemented with 0.5X N2 supplement, 2 μg/mL Doxycycline, 10 ng/mL recombinant NT3, 10 ng/mL recombinant BDNF, 0.5X B-27 supplement (Thermo Fisher Scientific, Cat #12587010), 0.5X Glutamax (Thermo Fisher Scientific, Cat #35050-061) and 1X NEAA. On day 7, half of the culture medium was replaced with an equal amount of fresh culture medium without doxycycline. On day 14, half of the medium was again removed, and fresh culture medium without doxycycline was added at twice that volume. RNA-seq and Ribo-seq sample prep iNeurons were plated and differentiated at ∼1.2 million cells per well in BioCoat PDL-coated 6-well plates (Corning Cat, #354413) and 6 wells were pooled per biological replicate per condition. At 21 days of differentiation, cells were treated with either 45 μM of Dyngo-4a or vehicle (DMSO) for 20 min to ensure uniform and robust inhibition of dynamin, and then stimulated with 1uM Isoproterenol for 2 h. Cells were then washed with ice-cold PBS and lysed in freshly prepared RNase-free Lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 1% Triton X-100, 100 µg/mL cycloheximide, 25 U/mL TURBO DNase (Thermo Fisher Scientific, Cat #AM2238). Cells were triturated 10 times by passing through a 25G needle and clarified by centrifuging at 20,000x g for 10 minutes at 4°C. Each lysate was split for subsequent RNA-seq and Ribo-seq library preparation. For RNA-seq, total RNA was isolated using Zymo RNA Clean & Concentrator-25 kit (Genesee, Cat #11-353) and 1 µg total RNA was used as input for library generation using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Cat #E7490) and NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs, Cat #E7775). For Ribo-seq libraries, an aliquot of lysate containing around 10 µg was used as input. Ribo-seq samples were generated from these lysates following previously published protocols 21 . RNA-seq and Ribo-seq analysis Raw data files were trimmed by removing adaptors, sequences aligning to ncRNA, tRNA, and rRNA were removed using Bowtie (version 2.3.5), and the remaining reads were aligned to the Genome Reference Consortium Human Build 38 (GRCh38) using STARaligner (version 2.7.5). RPF reads were extracted using length reads of 26-34 nucleotides using STARaligner FeatureCounts (Subread version 1.6.3). R was used for building an annotation hub database and DESeq2 (version 3.16) with Wald test was implemented for differential expression analysis of raw reads 50 . Transcriptional targets were defined as RNAs with significant changes from the RNA-seq dataset using an adjusted p -value cutoff of 0.01 and an absolute gene log₂-fold change (Isoproterenol-treated/untreated) > 0.5. ΔTE values were computed by incorporating unstimulated RNA samples as an additional covariate in the DESeq input, as detailed in Ref 51 , and z-scores were calculated based on ΔTE distribution for each gene. Significant RPF changes for the Riboseq analysis were determined using adjusted p -value of 0.05 as cut-off. Genes were categorized as “Forwarded” if they showed significant RNAseq and RPF changes. “Exclusive” genes were defined as those with significant RPF changes, ΔTE values at least 1.5 SD from the mean in the same direction, and no significant RNA change (RNA-seq p -adj > 0.01). “Buffered” genes were classified as those with significant RNA changes, non-significant RPF changes, and ΔTE ≥ 1.5 SD below the mean in the same direction as the RNA change. Transcriptional and translational hits were further categorized as driven by endosomal or plasma membrane signaling based on their dependence on endocytosis. For this, multiple unpaired t- test analyses were carried out comparing the RNAseq log₂-fold change values (Transcriptional targets) or ΔTE values (Exclusive translational targets) between vehicle- and Dyngo-4a-treated conditions. Cut-offs were established based on FDR using the two-stage step-up method of Benjamini, Krieger and Yekutieli. Targets with significant Dyngo-4a effects (FDR < 0.1) were designated as ‘endosomal’ hits; the remaining genes were classified as ‘plasma membrane’ hits. For quality control metrics, Ribotish 52 and Ribotoolkit 53 R packages were used for feature count analysis and periodicity. The package bigPint 54 was used to calculate Pearson correlation coefficients between drug-treated conditions and library prep replicates. GOrilla GO 55 was used for gene ontology analysis and SRplot 56 was used to generate GO dot plot graphs. Spinning disk confocal imaging and analysis The Andor Dragonfly Spinning Disk Confocal microscope equipped with 405 nm, 488 nm, 561 nm, 637 nm diode lasers, CO 2 - and temperature-controlled chamber was used for live- and fixed-cell imaging experiments and Fusion software was used for data collection. For fixed cell microscopy-based analyses, neurons were differentiated for 21 days on PDL- and laminin-coated glass coverslips. Following differentiation, cells were washed with PBS and then fixed at room temperature using 4% paraformaldehyde diluted in a 4% sucrose/PBS solution for 20 min. Coverslips were then washed twice in PBS, followed by 15 min in 50 mM NH 4 Cl solution and a 5-min incubation in 0.1% Triton-X 100/PBS solution. Primary antibodies were diluted in a 1% BSA/0.1% Triton-X/PBS solution and incubated with the coverslips for 2 h at room temperature or overnight at 4°C. Coverslips were washed 3 times in a 1% BSA/0.1% Triton-X/PBS solution and incubated for 30 min with secondary antibody. Coverslips were mounted with Prolong Gold antifade reagent with DAPI (Invitrogen, Cat #P36935) and stored at 4°C. For dendritogenesis experiments, neurons were plated at ∼20k cells/well on coverslips placed in 24-well plates and grown for 3 days. Immature neurons were used for this analysis for ease of tracing and fully capturing dendritic complexity before axons get too long due to the rapid differentiation of iNeurons. On DIV3, cells were treated with inhibitors for 30 min, then stimulated with 1 μM Isoproterenol for 2 h. Following agonist treatment, the media was aspirated and replaced with the preconditioned drug-free media. Neurons were then fixed using the protocol above at DIV6 staining with anti-MAP2 primary antibody for dendrite visualization. Coverslips were imaged at 20x to allow for full visualization of neuron lengths. For best image analysis, regions of the slides where neurons were seeded at low confluency were captured. For dendrite quantification, the paint tool in ImageJ (Version 1.54p) was used to isolate cells from background fluorescence. Color thresholding was used to create a mask for the MAP2 fluorescence channel which highlights the fluorescence making it easier to be converted to binary for Sholl Analysis with the SNT plugin in ImageJ 57 , 58 . The center of the neuron was marked as the middle of the cell body and the shell radius was kept the same for all neurons. The intersection output from the Sholl profile was captured from the cell body to the furthest dendrite tip and averaged to normalize varying neurite lengths across conditions. The average number of intersections of each neuron was then plotted. For synapse pair formation experiments, neurons were plated at ∼30k cell/well on laminin- and PDL-coated glass coverslips placed in 24-well plates and differentiated for 21 days. On DIV21, neurons were pretreated with inhibitor for 30 min, then stimulated with 1 μM Isoproterenol for 24 h. Following that, cells were fixed using anti-vGLUT1 and anti-PSD95 antibodies for pre- and post-synaptic protein visualization, respectively. MAP2 staining was also used for whole-neuron visualization. Slides were then imaged at 100x and analyzed with ImageJ. The fluorescent channels for vGLUT1 and PSD95 were deconvoluted for puncta visualization and merged, and color thresholding of the merged channel fluorescence was isolated and converted into a mask. The anti-MAP2 channel was used for ensuring 35 micron length was used for each neuron to normalize across conditions with the Line Tool in ImageJ. Number of puncta from the mask was recorded and plotted. For nanobody imaging, neurons were differentiated at ∼500k on matrigel-coated glass-bottom imaging dishes in clear N2 media with a combination of Neurobasal-A without phenol red (Thermo Fisher Scientific, Cat #12349015) and DMEM/F12 without phenol Red (Thermo Fisher Scientific, Cat #21041025). Neurons were transduced with lentiviral constructs (eGFP-Nb37 or eGFP-Nb80) for three days. Alexa-647 conjugated M1 mouse anti-flag antibody was added directly to the medium for 5 min to label cell-surface Flag-tagged β2AR, and neurons were stimulated as described and imaged before stimulation and after 15 min of ligand. For imaging of the ExRai-AKAR2 sensors, iPSCs were infected with lentiviral constructs and differentiated on matrigel-coated glass bottom imaging dishes for live-cell analysis. Pitstop 2 was used instead of Dyngo-4a to inhibit endocytosis, because Dyngo-4a exhibits fluorescence in the green channel, which interferes with imaging-based readouts. Using the 37°C chamber attached to the Andor Dragonfly Spinning Disk, neurons were pre-treated for 20 min with 30 μM Pitstop or vehicle (DMSO). Next, 3 baseline reads were taken every 30 sec, neurons were stimulated with 10 nM Isoproterenol and imaged for every 30 sec for 10 min total. Fluorescence was quantified by defining ROIs for individual cells, measuring signals from the 480 nm channel in each frame, and determining ΔF/F values. Based on these, area under the curve values were calculated. ELISA cAMP Neurons were plated in 12-well BioCoat PDL-coated plates (Corning, Cat #354413) for 21 days at ∼250k cells/well, then pretreated with vehicle (DMSO) or 30 µM Dyngo-4a for 30 min. Neurons were stimulated for 1 µM Isoproterenol for 5 min and the Cayman ELISA cAMP assay (VWR, Cat #75817–364) was used to quantify cAMP, following manufacturer protocols. All values were normalized to the total protein concentration of the respective sample. RT-qPCR Neurons were differentiated for 21 days in 12-well BioCoat PDL-coated plates (Corning, Cat #354413) at ∼250k cells/well, pretreated with 45 µM Dyngo-4a or vehicle (DMSO) for 30 min, then stimulated with 1 µM Isoproterenol for 2 h. RNA was extracted using QIAshredder (Qiagen, Cat #79656) and the Qiagen RNAeasy RNA-isolation kit (Qiagen Cat #74106) and reverse transcription was performed using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, Cat #18064014) following manufacturer protocols. For quantitative PCR, Power SYBR Green PCR MasterMix (AppliedBiosystems, Cat #4364344) was used with the following primers: GAPDH Forward: GTCTCCTCTGACTTCAACAGCG; GAPDH Reverse: ACCACCCTGTTGCTGTAGCCAA; FOS Forward: GCCTCTCTTACTACCACTCACC; FOS Reverse: AGATGGCAGTGACCGTGGGAAT; FTH1 Forward: TGAAGCTGCAGAACCAACGAGG; FTH1 Reverse: GCACACTCCATTGCATTCAGCC; NPAS4 Forward: TGGCTCTACTGGACATCTCCGA; NPAS4 Reverse: CGGTAGTGTTGAGCAGAAGCGT; BDNF Forward: CATCCGAGGACAAGGTGGCTTG; BDNF Reverse: GCCGAACTTTCTGGTCCTCATC; UNCX Forward: AGAAGGCGTTCAACGAGAGCCA; UNCX Reverse: CGTGTTCTCCTTCTTCTTCCAC; CGA Forward: TCCATTCCGCTCCTGATGTGA; CGA Reverse: CGTCTTGGACCTTAGTGGAG; DUSP1 Forward: CAACCACAAGGCAGACATCAGC; DUSP1 Reverse: GTAAGCAAGGCAGATGGTGGCT . All gene levels were normalized to the housekeeping gene GAPDH . The CFX-384 Touch Real-Time PCR System equipped with CFXMaestro software (BioRad) was used for analysis. Western Blot Analysis Neurons growing on BioCoat PDL-coated 6-well plates (Corning, Cat #354413) were treated on DIV20-22 as specified, washed with PBS and then lysed with ice-cold RIPA buffer (Sigma, Cat #R0278) containing phosphatase inhibitors (Sigma, Cat #P0044), protease-inhibitor cocktail (Sigma, Cat #P8340), and 1 mM PMSF (Sigma, Cat #329-98-6) on a shaker for 15 min at 4°C before. Cells were scraped off the wells and transferred into pre-chilled microcentrifuge tubes. After cold centrifugation, the supernatant was collected and protein concentration was measured using the Peirce Protein Assay kit (Thermo Fisher Scientific, Cat #23227). Equal amounts of total protein were then boiled in 4X Laemmli buffer with 10% 2-mercaptoethanol for 5 min at 90°C. Protein was loaded into Mini-PROTEAN TGX Stain-Free 4–15% gels (BioRad, Cat #4561094). Gels were transferred to PVDF membrane (Sigma, Cat #IPFL00010). Membranes were blocked with Intercept Blocking buffer/5% BSA (Licor, Cat #927-60001) for 1 h. Primary antibodies were added to the Intercept Blocking buffer and incubated overnight with shaking at 4°C. After washing in Tris Buffer Saline (TBS) solution with 1% Tween (Sigma, Cat #P9416-50ML), secondary antibodies were diluted in the Blocking buffer and incubated for 1 h at RT with shaking. The membrane was washed with TBS and imaged using the Odyssey imager system equipped with ImageStudio software (LICOR). ACKNOWLEDGEMENTS We thank members of the Tsvetanova lab for their thoughtful feedback on the study. All microscopy imaging was performed using resources from the Duke Light Microscopy Core Facility (LMCF) with training under Drs. Lisa Cameron and Yasheng Gao. Dr. Jin Zhang (UCSD, CA) generously provided the ExRai-AKAR2 constructs used to image PKA activity. This work was supported by the NIH (R01NS127847 to N.G.T.) and American Heart Association (25PRE1377010 to K.L.H.). 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