The PKG Inhibitor CN238 Affords Functional Protection of Photoreceptors and Ganglion Cells against Retinal Degeneration.

Hereditary retinal degeneration (RD) is often associated with excessive cGMP signalling in photoreceptors. Previous research has shown that inhibition of cGMP-dependent protein kinase G (PKG) can reduce photoreceptor loss in two different RD animal models. In this study, we identified a PKG inhibitor, the cGMP analogue CN238, which preserved photoreceptor viability and functionality in rd1 and rd10 mutant mice. Surprisingly, in explanted retinae, CN238 also protected retinal ganglion cells from axotomy-induced retrograde degeneration and preserved their functionality. Furthermore, kinase activity-dependent protein phosphorylation of the PKG target Kv1.6 was reduced in CN238-treated rd10 retinal explants. Ca2+-imaging on rd10 acute retinal explants revealed delayed retinal ganglion cell repolarization with CN238 treatment, suggesting a PKG-dependent modulation of Kv1-channels. Together, these results highlight the strong neuroprotective capacity of PKG inhibitors for both photoreceptors and retinal ganglion cells, illustrating their broad potential for the treatment of retinal diseases and possibly neurodegenerative diseases in general.

The photoreceptor protective cGMP-analog Rp-8-Br-PET-cGMPS interacts with cGMP-interactors PKGI, PDE1, PDE6, and PKAI in the degenerating mouse retina.

The inherited eye disease retinitis pigmentosa (RP) causes the loss of photoreceptors by a still unknown cell death mechanism. During this degeneration, cyclic guanosine-3',5'-monophosphate (cGMP) levels become elevated, leading to over-activation of the cGMP-binding protein cGMP-dependent protein kinase (PKG). cGMP analogs selectively modified to have inhibitory actions on PKG have aided in impeding photoreceptor death, and one such cGMP analog is Rp-8-Br-PET-cGMPS. However, cGMP analogs have previously been shown to interact with numerous targets, so to better understand the therapeutic action of Rp-8-Br-PET-cGMPS, it is necessary to elucidate its target-selectivity and hence what potential cellular mechanism(s) it may affect within the photoreceptors. Here, we, therefore, applied affinity chromatography together with mass spectrometry to isolate and identify Rp-8-Br-PET-cGMPS interactors from retinas derived from three different murine RP models (i.e., rd1, rd2, and rd10 mice). Our findings revealed that Rp-8-Br-PET-cGMPS bound seven known cGMP-binding proteins, including PKG1ß, PDE1ß, PDE1c, PDE6α, and PKA1α. Furthermore, an additional 28 proteins were found to be associated with Rp-8-Br-PET-cGMPS. This latter group included MAPK1/3, which is known to connect with cGMP/PKG in other systems. However, in organotypic retinal cultures, Rp-8-Br-PET-cGMPS had no effect on photoreceptor MAPK1/3 expression or activity. To summarize, Rp-8-Br-PET-cGMPS is more target specific compared to regular cGMP.

Pathomechanisms of Inherited Retinal Degeneration and Perspectives for Neuroprotection.

The precise processes causing photoreceptor cell death in retinal degeneration (RD) are still largely unknown but are likely to follow a variety of degenerative mechanisms. While different genetic insults can trigger distinct molecular pathways, eventually these may converge into a limited number of common cell death mechanisms. These mechanisms often involve deregulation of cyclic guanosine monophosphate (cGMP)-signaling and proteostasis, which both may increase photoreceptor energy expenditure. Comprehensive information on these mechanisms may allow for targeted interventions to delay or prevent photoreceptor loss. Here, we review the current knowledge on photoreceptor degenerative mechanisms, focusing on processes triggered by aberrant cGMP-signaling, proteostasis, and energy metabolism. Afterward, we discuss how these pathways could potentially be used to treat photoreceptor degeneration, highlighting data from a number of recent studies on inhibitory cGMP analogs, proteostasis blockers, and interventions aimed at fortifying energetic status. Finally, we provide perspectives on how such experimental approaches could be translated into future clinical applications.

In vitro Model Systems for Studies Into Retinal Neuroprotection.

Therapy development for neurodegenerative diseases of the retina constitutes a major unmet medical need, and this may be particularly relevant for inherited diseases of the retina, which are largely untreatable to this day. Therapy development necessitates appropriate models to improve the understanding of the underlying degenerative mechanisms, as well as for the testing and evaluation of novel treatment approaches. This review provides an overview of various in vitro model systems used to study retinal neuroprotection. The in vitro methods and technologies discussed range from primary retinal cell cultures and cell lines, to retinal organoids and organotypic retinal explants, to the cultivation of whole eyeballs. The advantages and disadvantages of these methods are compared and evaluated, also in view of the 3R principles (i.e., the refinement, reduction, and replacement of live animal testing), to identify suitable in vitro alternatives for in vivo experimentation. The article further expands on the use of in vitro models to test and evaluate neuroprotective treatments and to aid the development of retinal drug delivery systems. Among the pharmacological agents tested and characterized in vitro are such that interfere with aberrant cyclic guanosine monophosphate (cGMP) -signaling or such that inhibit the activities of poly (ADP-ribose) polymerase (PARP), histone deacetylases (HDAC), calpain-type proteases, as well as unfolded protein response-related stress. We then introduce nanoparticle-based drug delivery systems and discuss how different in vitro systems may be used to assess their efficacy in the treatment of retinal diseases. The summary provides a brief comparison of available in vitro models and relates their advantages and limitations to the various experimental requirements, for instance, for studies into disease mechanisms, novel treatments, or retinal toxicity. In many cases, combinations of different in vitro models may be required to obtain a comprehensive view of the efficacy of a given retinal neuroprotection approach.

Kinase activity profiling identifies putative downstream targets of cGMP/PKG signaling in inherited retinal neurodegeneration.

Inherited retinal diseases (IRDs) are a group of neurodegenerative disorders that lead to photoreceptor cell death and eventually blindness. IRDs are characterised by a high genetic heterogeneity, making it imperative to design mutation-independent therapies. Mutations in a number of IRD disease genes have been associated with a rise of cyclic 3',5'-guanosine monophosphate (cGMP) levels in photoreceptors. Accordingly, the cGMP-dependent protein kinase (PKG) has emerged as a new potential target for the mutation-independent treatment of IRDs. However, the substrates of PKG and the downstream degenerative pathways triggered by its activity have yet to be determined. Here, we performed kinome activity profiling of different murine organotypic retinal explant cultures (diseased rd1 and wild-type controls) using multiplex peptide microarrays to identify proteins whose phosphorylation was significantly altered by PKG activity. In addition, we tested the downstream effect of a known PKG inhibitor CN03 in these organotypic retina cultures. Among the PKG substrates were potassium channels belonging to the Kv1 family (KCNA3, KCNA6), cyclic AMP-responsive element-binding protein 1 (CREB1), DNA topoisomerase 2-α (TOP2A), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (F263), and the glutamate ionotropic receptor kainate 2 (GRIK2). The retinal expression of these PKG targets was further confirmed by immunofluorescence and could be assigned to various neuronal cell types, including photoreceptors, horizontal cells, and ganglion cells. Taken together, this study confirmed the key role of PKG in photoreceptor cell death and identified new downstream targets of cGMP/PKG signalling that will improve the understanding of the degenerative mechanisms underlying IRDs.

The role of cGMP-signalling and calcium-signalling in photoreceptor cell death: perspectives for therapy development.

The second messengers, cGMP and Ca2+, have both been implicated in retinal degeneration; however, it is still unclear which of the two is most relevant for photoreceptor cell death. This problem is exacerbated by the close connections and crosstalk between cGMP-signalling and calcium (Ca2+)-signalling in photoreceptors. In this review, we summarize key aspects of cGMP-signalling and Ca2+-signalling relevant for hereditary photoreceptor degeneration. The topics covered include cGMP-signalling targets, the role of Ca2+ permeable channels, relation to energy metabolism, calpain-type proteases, and how the related metabolic processes may trigger and execute photoreceptor cell death. A focus is then put on cGMP-dependent mechanisms and how exceedingly high photoreceptor cGMP levels set in motion cascades of Ca2+-dependent and independent processes that eventually bring about photoreceptor cell death. Finally, an outlook is given into mutation-independent therapeutic approaches that exploit specific features of cGMP-signalling. Such approaches might be combined with suitable drug delivery systems for translation into clinical applications.

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium.

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2-9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day. The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

Guanylyl Cyclase A/cGMP Signaling Slows Hidden, Age- and Acoustic Trauma-Induced Hearing Loss.

In the inner ear, cyclic guanosine monophosphate (cGMP) signaling has been described as facilitating otoprotection, which was previously observed through elevated cGMP levels achieved by phosphodiesterase 5 inhibition. However, to date, the upstream guanylyl cyclase (GC) subtype eliciting cGMP production is unknown. Here, we show that mice with a genetic disruption of the gene encoding the cGMP generator GC-A, the receptor for atrial and B-type natriuretic peptides, display a greater vulnerability of hair cells to hidden hearing loss and noise- and age-dependent hearing loss. This vulnerability was associated with GC-A expression in spiral ganglia and outer hair cells (OHCs) but not in inner hair cells (IHCs). GC-A knockout mice exhibited elevated hearing thresholds, most pronounced for the detection of high-frequency tones. Deficits in OHC input-output functions in high-frequency regions were already present in young GC-A-deficient mice, with no signs of an accelerated progression of age-related hearing loss or higher vulnerability to acoustic trauma. OHCs in these frequency regions in young GC-A knockout mice exhibited diminished levels of KCNQ4 expression, which is the dominant K+ channel in OHCs, and decreased activation of poly (ADP-ribose) polymerase-1, an enzyme involved in DNA repair. Further, GC-A knockout mice had IHC synapse impairments and reduced amplitudes of auditory brainstem responses that progressed with age and with acoustic trauma, in contrast to OHCs, when compared to GC-A wild-type littermates. We conclude that GC-A/cGMP-dependent signaling pathways have otoprotective functions and GC-A gene disruption differentially contributes to hair-cell damage in a healthy, aged, or injured system. Thus, augmentation of natriuretic peptide GC-A signaling likely has potential to overcome hidden and noise-induced hearing loss, as well as presbycusis.

The cGMP Pathway and Inherited Photoreceptor Degeneration: Targets, Compounds, and Biomarkers.

Photoreceptor physiology and pathophysiology is intricately linked to guanosine-3',5'-cyclic monophosphate (cGMP)-signaling. Here, we discuss the importance of cGMP-signaling for the pathogenesis of hereditary retinal degeneration. Excessive accumulation of cGMP in photoreceptors is a common denominator in cell death caused by a variety of different gene mutations. The cGMP-dependent cell death pathway may be targeted for the treatment of inherited photoreceptor degeneration, using specifically designed and formulated inhibitory cGMP analogues. Moreover, cGMP-signaling and its down-stream targets may be exploited for the development of novel biomarkers that could facilitate monitoring of disease progression and reveal the response to treatment in future clinical trials. We then briefly present the importance of appropriate formulations for delivery to the retina, both for drug and biomarker applications. Finally, the review touches on important aspects of future clinical translation, highlighting the need for interdisciplinary cooperation of researchers from a diverse range of fields.