A-kinase anchoring protein–mediated compartmentalization of pro-survival signaling in the retina

The retina plays a fundamental role in the process of vision, serving as the primary interface between external visual stimuli and the central nervous system. Because the retina is exposed to a variety of environmental stresses and deleterious insults, it is susceptible to a spectrum of pathological conditions that can detrimentally affect vision. This often leads to irreversible vision loss due to the injury of specific cell types. For instance, inherited retinal degeneration and age-related macular degeneration can lead to the death of photoreceptors, while conditions like glaucoma and optic nerve injury can result in the loss of ganglion cells. The precise pathological mechanisms driving retinal degeneration remain largely elusive, although research utilizing mouse models suggests that disruptions in intracellular signal transduction pathways may play a pivotal role. Signaling pathways within the retina orchestrate various aspects of retinal physiology, including phototransduction, synaptic transmission, and neuronal survival. Among these pathways, cyclic adenosine monophosphate (cAMP) signaling emerges as a central mechanism for coordinating pro-survival responses to a variety of insults.

cAMP is a ubiquitous second messenger that regulates a wide range of cellular processes in the retina. In response to extracellular stimuli, such as neurotransmitters and neurotrophic factors, adenylyl cyclases catalyze the conversion of adenosine triphosphate to cAMP, activating downstream effectors like protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC). Through the activation of these factors, cAMP signaling modulates ion channel activity, gene expression, and cellular metabolism within the retina (Murray et al., 2009). Elevated levels of cAMP signaling and neurotrophic factors like brain-derived neurotrophic factor have been demonstrated to prevent or delay cell death, supporting the survival and axon regeneration of retinal ganglion cells (RGCs) after injury (Wang et al., 2015). However, increasing cAMP alone does not significantly enhance RGC survival or the number of regenerating cells. On the other hand, intraocular injections of ciliary neurotrophic factor and a cAMP analog improve axonal regrowth but do not impact RGCs viability (Cui et al., 2003). Confirming this, Vigneswara et al. (2013) demonstrated that the combination treatment of pigment epithelium-derived factor and cAMP enhances RGCs axon regeneration. However, the mechanism by which cAMP exerts its protective effect on retinal neuronal survival and outgrowth is not well characterized.

It has been demonstrated the crucial role of cAMP signaling in the development and maturation of photoreceptors, as well as in the propagation of spontaneous activity waves during retina development. Elevating cAMP levels through A2 adenosine receptors located on developing amacrine and ganglion cells enhances the spatiotemporal properties of these waves. Furthermore, Coredorr et al. (2012) have investigated the expression and function of the soluble adenylyl cyclase (sAC, ADCY10) in RGCs. Their results demonstrate that activation of sAC, induced by electrical activity and bicarbonate, significantly enhances RGC survival and axonal growth. Conversely, the knockdown of sAC by siRNA markedly decreases RGC survival and axon growth in vitro, and survival in vivo. A recent study demonstrated that sAC in reactive astrocytes acts as a molecular switch for neuroprotective astrocyte reactivity to promote retinal ganglion cell survival (Cameron et al., 2024). In contrast, specific double knockdown of AC1/AC8 or their pharmacological inhibition does not impact the survival or physical characteristics of RGCs. Additionally, the regulation of downstream cAMP effectors also influences RGC survival. For example, it has been observed that H89, a selective and competitive inhibitor of PKA, may reduce RGC survival and neurite outgrowth. Interestingly, during treatment with forskolin, an AC activator, the same inhibitor does not decrease RGC survival. This evidence suggests that pro-survival signaling in retinal ganglion cells may rely on PKA-dependent pathways (Corredor et al., 2012). The PKA holoenzyme exists as a tetrameric protein and contains two catalytic (Cα or Cβ) and two regulatory (RIα, RIβ, RIIα, or RIIβ) subunits that bind cAMP. The biochemical and functional features of PKA holoenzymes are largely determined by the structure and the biochemical properties of the four functionally non-redundant regulatory subunits, RIα, RIβ, RIIα, and RIIβ. In the retina, each subunit was found to have a different spatial distribution. For example, the Cα subunit is localized throughout the cell body of all cell layers in the retina, and the Cβ subunit is highly expressed in photoreceptors, interneurons, and ganglion cells. On the other hand, RIIα and RIIβ are localized in photoreceptors and interneurons, while RIα and RIβ subunits are located in all retinal cells. However, how the spatial and cell-specific organization of PKA function is achieved? In the retina and many other tissues, this organization is ensured by large multimolecular scaffold proteins referred to as A-kinase anchoring proteins (AKAPs).

cAMP is localized within distinct signaling domains or compartments, enabling precise spatial-temporal regulation of individual phosphorylation events. AKAPs play a crucial role in orchestrating the formation of cAMP microdomains by organizing AC, phosphodiesterase (PDE), PKA, and a specific PKA substrate within the same intracellular locations (Figure 1). Disruption of any of these interactions leads to a loss of signaling efficiency and/or fidelity (Kapiloff et al., 2014). This spatially restricted control over cAMP levels allows AKAPs to finely tune the activation of downstream effectors, such as PKA, and regulate cellular responses to extracellular stimuli. Through dynamic interactions with PKA regulatory subunits and other binding partners, AKAPs modulate the activation state of PKA and influence the phosphorylation status of downstream targets in response to different intra- and extracellular cues. The interaction between AKAPs and PKA occurs through the binding of the dimerization-docking domain of the PKA-R subunit to an amphipathic α-helix on AKAPs. This spatially selective anchoring ensures that PKA is activated precisely where it is needed, contributing to the specificity and efficiency of signaling within the highly specialized cellular architecture of the retina.

Figure 1.

The examples of AKAP-organized pro-survival signaling in different neuronal compartments.

(A) By directly binding the PKA holoenzyme to the outer mitochondrial membrane, AKAP1 strategically coordinates various signal transduction cascades, including cAMP/PKA signaling, to regulate mitochondrial fusion and fission through interaction with Drp1. AKAP1 stimulates the phosphorylation of Drp1, resulting in subsequent mitochondrial elongation, increased ATP generation, and improved mitochondrial membrane potential. These combined changes contribute to neuroprotection. (B) AKAP6 located in the perinuclear region serves as a platform for the integration of signals induced by electrical activity and neurotrophins such as BDNF. Neuronal activity triggers calcium entry, activating calcium-sensitive ACs and leading to an increase in intracellular cAMP concentration, subsequently activating PKA. Within AKAP6 signalosomes, PKA has the capability to phosphorylate other proteins related to the cAMP degradation/synthesis machinery, such as PDE4. The phosphorylation events serve to regulate local cAMP signaling. BDNF acts through AKAP6-anchored MEK5, which triggers the activation of ERK5, leading to the subsequent phosphorylation of PDE4. This phosphorylation event serves to inhibit the hydrolysis of cAMP. This neurotrophin-dependent mechanism increases cAMP concentration around AKAP6 and increases the expression of pro-survival genes. The figure was prepared with freely accessible Server Medical Art using Corel Draw 8 graphic software. AC: Adenylyl cyclase; AKAP1: A-kinase anchoring protein 1; AKAP6: A-kinase anchoring protein 6; ATP: adenosine triphosphate; BDNF: brain-derived neurotrophic factor; cAMP: cyclic adenosine monophosphate; Drp1: dynamin-related protein 1; ERK5: extracellular signal-regulated kinase 5; MEK5: mitogen-activated protein kinase kinase 5; PDE4: phosphodiesterase 4D3; PKA: protein kinase A; RGCs: retinal ganglion cells.

AKAPs are integral to the intricate spatial and temporal organization of signaling pathways within the retina. AKAPs may primarily function as molecular scaffolds for PKA and other signaling enzymes and may interact with additional proteins involved in synaptic vesicle trafficking, cytoskeletal dynamics or calcium signaling. This functional diversity allows AKAPs to regulate a wide range of cellular processes within the retina, including neurotransmitter release, synaptic plasticity, and neuronal survival (Wild and Dell’Acqua, 2018). Among all members of the AKAP family, a key player is the muscle A-kinase anchoring protein (mAKAP; also known as AKAP6), localized at the perinuclear membrane via binding to the Klarsicht/ANC-1/Syne-1 homology domain, transmembrane protein nesprin-1α (Boczek et al., 2019). Besides binding PKA, mAKAP also anchors the cAMP target Epac1, AC (types II and V), and the cAMP-specific phosphodiesterase 4D3 (PDE4D) to discrete intracellular compartments. By binding together PKA and PDE4D, the AKAP6 signalosome can tightly modulate the specificity of cAMP signaling in the neuronal cells. Recent work has defined the neuroprotective role of AKAP6 in cultured primary RGCs. Wang and colleagues establish that AKAP6 expression is indispensable for RGC survival and neurite growth in vitro and in vivo (Wang et al., 2015). However, the genetic knockdown of AKAP6 does not regulate RGC survival during normal development or in the absence of injury. Notably, downregulation of AKAP6 in the adults promoted RGC death in the model of axonal degeneration of the optic nerve crush. In addition, AKAP6 expression was essential for the neuroprotective effects of brain-derived neurotrophic factor and cyclic AMP following injury. Our further investigation provides evidence for a neuronal perinuclear cAMP compartment organized by the scaffold protein AKAP6 that is required and sufficient for the promotion of neurite outgrowth in vitro and for the survival of RGCS in vivo following optic nerve injury (Boczek et al., 2019). We have demonstrated that the displacement of individual signaling enzymes from the signalosome, such as PDE4D3, using specific anchoring disruptor peptides leads to increased RGC survival in vivo after optic nerve injury. Besides regulating and shaping the dynamic of cAMP signaling, AKAP6 also anchors the mitogen-activated protein kinases MEK5 and ERK5, recognized to be crucial for neuronal survival. Additionally, other binding partners of AKAP6 have been identified to support the survival and axon growth of central nervous system neurons, including the calcium/calmodulin-dependent phosphatase calcineurin and the transcription factor MEF2. Moreover, the unique N-terminal domain of AKAP6 α isoform, absent in mAKAP6β, directly interacts with PDK1. This interaction between PDK1 and mAKAPα contributes to the activation of p90 ribosomal S6 kinase (RSK), which promotes neuronal survival in response to neurotrophic factors through both transcription-dependent and independent mechanisms. For instance, RSK phosphorylation of BAD, a pro-apoptotic protein family member, inhibits its activity and activates the transcription factor cAMP response element-binding protein (Passariello et al., 2015).

AKAP crucial for the proper development and maintenance of neuronal structures is A-kinase anchoring protein 1 (AKAP1), also known as D-AKAP1 (dual specificity AKAP1). Numerous AKAP1 splice variants are localized in a variety of tissues, including the brain, heart, liver, kidney, and skeletal muscle. AKAP1 is classified as a multifunctional mitochondrial scaffold protein that plays a pivotal role in the modulation of mitochondrial dynamics, bioenergetics, and calcium homeostasis, by recruiting multiple enzymes, including PKA to the outer mitochondrial membrane (Edwards et al., 2020). It is well known that progressive degeneration of the optic nerve and retinal ganglion cells are the main characteristics of glaucoma and can lead to permanent vision loss. To date, several risk factors have been implicated in the pathophysiology of glaucoma, for example, increased intraocular pressure, glial cell activation, and aging. However, the molecular mechanisms underlying RGC degeneration are still under investigation. Importantly, recent studies reveal that defects in mitochondria function may have a fundamental role in neuronal cell loss during glaucomatous neurodegeneration. It has been demonstrated that AKAP1 deficiency in RGCs induces dynamin-related protein 1 dephosphorylation-mediated mitochondrial fragmentation and cell death. Moreover, increased intraocular pressure causes a dramatic reduction in AKAP1 protein expression in the glaucomatous retina. Additionally, augmenting AKAP1 expression results in neuroprotection of RGCs from oxidative stress (Edwards et al., 2020).

Another AKAP essential for the retina is AKAP12, which serves as a crucial scaffolding molecule directing the localization of various signaling proteins to the plasma membrane. These proteins, including PKA, protein kinase C, calcineurin, Src-family kinases, cyclins, and calmodulin, play pivotal roles in regulating their respective signaling pathways. More recently, AKAP12 has emerged as a key player in maintaining vascular integrity and regulating endothelial cell function within the retina. It acts as a suppressor of angiogenesis and contributes to the formation of the human blood-retinal barrier. This function is achieved through mechanisms such as increasing angiopoietin-1 levels and decreasing vascular endothelial growth factor levels in astrocytes (Choi et al., 2007).

The role of AKAPs in retinal function, both in physiology and pathology, remains unresolved. However, emerging research continually provides new evidence for their role as nodal points for the survival of injured neurons. For instance, our results on cAMP/PKA signaling at AKAP6 suggest that the use of anchoring disruptors, designed to enhance local cAMP production or slow down its degradation, could be a potent tool for increasing the survival of retinal cells. Since specific AKAPs may influence local cAMP/PKA signaling differently, it is reasonable to investigate how these AKAP-organized discrete compartments orchestrate the sequence of events in retinal neuroprotection and neuroregeneration. Live-cell imaging of cAMP/PKA, facilitated by targeted and sensitive FRET sensors along with sensors for upstream stimuli such as Ca²⁺, is an excellent technique for such experiments. It not only enables imaging with spatial and temporal resolution but also allows tracking of changes in the submicromolar range. Understanding the mechanisms behind AKAP-mediated cAMP/PKA signaling compartmentalization should broaden the application of anchoring disruptors in gene therapies for retinal neuroprotection and/or neuroenhancement. In the context of human ocular diseases, particularly those associated with vision loss, such as glaucoma, it is imperative to advance in vivo cAMP/PKA imaging in animal models. This approach will help uncover early molecular changes in retinal signaling that lead to the subsequent loss of retinal ganglion cells. Additionally, such models provide an excellent platform for fast and efficient testing of novel pharmacological interventions.

This work was supported by National Science Center (Narodowe Centrum Nauki) grant No. UMO-2019/33/B/NZ4/00587 to TB.