Topical administration of GLP-1 eyedrops improves retinal ganglion cell function by facilitating presynaptic GABA release in early experimental diabetes.

ABSTRACT: Diabetic retinopathy is a prominent cause of blindness in adults, with early retinal ganglion cell (RGC) loss contributing to visual dysfunction or blindness. In the brain, defects in y-aminobutyric acid (GABA) synaptic transmission are associated with pathophysiological and neurodegenerative disorders, whereas glucagon-like peptide-1 (GLP-1) has demonstrated neuroprotective effects. However, it is not yet clear whether diabetes causes alterations in inhibitory input to RGCs and whether and how GLP-1 protects against neurodegeneration in the diabetic retina through regulating inhibitory synaptic transmission to RGCs. In the present study, we used the patch-clamp technique to record GABA subtype A receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) in RGCs from streptozotocin-induced diabetes model rats. We found that early diabetes (4 weeks of hyperglycemia) decreased the frequency of GABAergic mIPSCs in RGCs without altering their amplitude, suggesting a reduction in the spontaneous release of GABA to RGCs. Topical administration of GLP-1 eyedrops over a period of 2 weeks effectively countered the hyperglycemia-induced downregulation of GABAergic mIPSC frequency, subsequently enhancing the survival of RGCs. Concurrently, the protective effects of GLP-1 on RGCs in diabetic rats were eliminated by topical administration of exendin-9-39, a specific GLP-1 receptor antagonist, or SR95531, a specific antagonist of the GABA subtype A receptor. Furthermore, extracellular perfusion of GLP-1 was found to elevate the frequencies of GABAergic mIPSCs in both ON- and OFF-type RGCs. This elevation was shown to be mediated by activation of the phosphatidylinositol-phospholipase C/inositol 1,4,5-trisphosphate receptor/Ca2+/protein kinase C signaling pathway downstream of GLP-1 receptor activation. Moreover, multielectrode array recordings revealed that GLP-1 functionally augmented the photoresponses of ON-type RGCs. Optomotor response tests demonstrated that diabetic rats exhibited reductions in visual acuity and contrast sensitivity that were significantly ameliorated by topical administration of GLP-1. These results suggest that GLP-1 facilitates the release of GABA onto RGCs through the activation of GLP-1 receptor, leading to the de-excitation of RGC circuits and the inhibition of excitotoxic processes associated with diabetic retinopathy. Collectively, our findings indicate that the GABA system has potential as a therapeutic target for mitigating early-stage diabetic retinopathy. Furthermore, the topical administration of GLP-1 eyedrops represents a non-invasive and effective treatment approach for managing early-stage diabetic retinopathy.

Exendin-4 promotes retinal ganglion cell survival and function by inhibiting calcium channels in experimental diabetes.

Progressive damage of retinal ganglion cells (RGCs) is observed in early diabetic retinopathy. Intracellular Ca2+ overload mediated by Ca2+ influx through voltage-gated Ca2+ channels (VGCCs) is involved in neurodegeneration, whereas glucagon-like peptide-1 (GLP-1) provides neuroprotection. However, whether GLP-1 plays a neuroprotective role in diabetic retinas by modulating VGCCs remains unknown. We found that eye drops of exendin-4, a long-acting GLP-1 receptor (GLP-1R) agonist, prevented the increase of L-type Ca2+ current (ILCa) densities of RGCs induced by 4-week hyperglycemia and promoted RGC survival by suppressing L-type VGCC (L-VGCC) activity in streptozotocin-induced diabetic rats. Moreover, exendin-4-induced suppression of ILCa in RGCs may be mediated by a GLP-1R/Gs/cAMP-PKA/ryanodine/Ca2+/calmodulin/calcineurin/PP1 signaling pathway. Furthermore, exendin-4 functionally improved the light-evoked spiking ability of diabetic RGCs. These results suggest that GLP-1R activation enhances cAMP to PP1 signaling and that PP1 inactivates L-VGCCs by dephosphorylating them, thereby reducing Ca2+ influx, which could protect RGCs against excitotoxic Ca2+ overload.

Short-term acute bright light exposure induces a prolonged anxiogenic effect in mice via a retinal ipRGC-CeA circuit.

Light modulates mood through various retina-brain pathways. We showed that mice treated with short-term acute bright light exposure displayed anxiety-related phenotypes in a prolonged manner even after the termination of the exposure. Such a postexposure anxiogenic effect depended upon melanopsin-based intrinsically photosensitive retinal ganglion cell (ipRGC) activities rather than rod/cone photoreceptor inputs. Chemogenetic manipulation of specific central nuclei demonstrated that the ipRGC-central amygdala (CeA) visual circuit played a key role in this effect. The corticosterone system was likely to be involved in this effect, as evidenced by enhanced expression of the glucocorticoid receptor (GR) protein in the CeA and the bed nucleus of the stria terminalis and by the absence of this effect in animals treated with the GR antagonist. Together, our findings reveal a non-image forming visual circuit specifically designed for "the delayed" extinction of anxiety against potential threats, thus conferring a survival advantage.

The role of ipRGCs in ocular growth and myopia development.

The increasing global prevalence of myopia calls for elaboration of the pathogenesis of this disease. Here, we show that selective ablation and activation of intrinsically photosensitive retinal ganglion cells (ipRGCs) in developing mice induced myopic and hyperopic refractive shifts by modulating the corneal radius of curvature (CRC) and axial length (AL) in an opposite way. Melanopsin- and rod/cone-driven signals of ipRGCs were found to influence refractive development by affecting the AL and CRC, respectively. The role of ipRGCs in myopia progression is evidenced by attenuated form-deprivation myopia magnitudes in ipRGC-ablated and melanopsin-deficient animals and by enhanced melanopsin expression/photoresponses in form-deprived eyes. Cell subtype-specific ablation showed that M1 subtype cells, and probably M2/M3 subtype cells, are involved in ocular development. Thus, ipRGCs contribute substantially to mouse eye growth and myopia development, which may inspire novel strategies for myopia intervention.

Signaling Mechanism for Modulation by GLP-1 and Exendin-4 of GABA Receptors on Rat Retinal Ganglion Cells.

Glucagon-like peptide-1 (GLP-1) is expressed in retinal neurons, but its role in the retina is largely unknown. Here, we demonstrated that GLP-1 or the GLP-1 receptor (GLP-1R; a G protein-coupled receptor) agonist exendin-4 suppressed γ-aminobutyric acid receptor (GABAR)-mediated currents through GLP-1Rs in isolated rat retinal ganglion cells (GCs). Pre-incubation with the stimulatory G protein (Gs) inhibitor NF 449 abolished the exendin-4 effect. The exendin-4-induced suppression was mimicked by perfusion with 8-Br-cAMP (a cAMP analog), but was eliminated by the protein kinase A (PKA) inhibitor Rp-cAMP/KT-5720. The exendin-4 effect was accompanied by an increase in [Ca2+]i of GCs through the IP3-sensitive pathway and was blocked in Ca2+-free solution. Furthermore, when the activity of calmodulin (CaM) and CaM-dependent protein kinase II (CaMKII) was inhibited, the exendin-4 effect was eliminated. Consistent with this, exendin-4 suppressed GABAR-mediated light-evoked inhibitory postsynaptic currents in GCs in rat retinal slices. These results suggest that exendin-4-induced suppression may be mediated by a distinct Gs/cAMP-PKA/IP3/Ca2+/CaM/CaMKII signaling pathway, following the activation of GLP-1Rs.

Orexin-A Intensifies Mouse Pupillary Light Response by Modulating Intrinsically Photosensitive Retinal Ganglion Cells.

We show for the first time that the neuropeptide orexin modulates pupillary light response, a non-image-forming visual function, in mice of either sex. Intravitreal injection of the orexin receptor (OXR) antagonist TCS1102 and orexin-A reduced and enhanced pupillary constriction in response to light, respectively. Orexin-A activated OX1Rs on M2-type intrinsically photosensitive retinal ganglion cells (M2 cells), and caused membrane depolarization of these cells by modulating inward rectifier potassium channels and nonselective cation channels, thus resulting in an increase in intrinsic excitability. The increased intrinsic excitability could account for the orexin-A-evoked increase in spontaneous discharges and light-induced spiking rates of M2 cells, leading to an intensification of pupillary constriction. Orexin-A did not alter the light response of M1 cells, which could be because of no or weak expression of OX1Rs on them, as revealed by RNAscope in situ hybridization. In sum, orexin-A is likely to decrease the pupil size of mice by influencing M2 cells, thereby improving visual performance in awake mice via enhancing the focal depth of the eye's refractive system.SIGNIFICANCE STATEMENT This study reveals the role of the neuropeptide orexin in mouse pupillary light response, a non-image-forming visual function. Intravitreal orexin-A administration intensifies light-induced pupillary constriction via increasing the excitability of M2 intrinsically photosensitive retinal ganglion cells by activating the orexin receptor subtype OX1R. Modulation of inward rectifier potassium channels and nonselective cation channels were both involved in the ionic mechanisms underlying such intensification. Orexin could improve visual performance in awake mice by reducing the pupil size and thereby enhancing the focal depth of the eye's refractive system.

Egr-1 and RNA POL II facilitate glioma cell GDNF transcription induced by histone hyperacetylation in promoter II.

The specific mechanisms for epigenetic regulation of gene transcription remain to be elucidated. We previously demonstrated that hyperacetylation of histone H3K9 in promoter II of glioma cells promotes high transcription of the glial cell line-derived neurotrophic factor (GDNF) gene. This hyperacetylation significantly enhanced Egr-1 binding and increased the recruitment of RNA polymerase II (RNA POL II) to that region (P < 0.05). Egr-1 expression was abnormally increased in C6 glioma cells. Further overexpression of Egr-1 significantly increased Egr-1 binding to GDNF promoter II, while increasing RNA POL II recruitment, thus increasing GDNF transcription (P < 0.01). When the acetylation of H3K9 in the Egr-1 binding site was significantly reduced by the histone acetyltransferase (HAT) inhibitor curcumin, binding of Egr-1 to GDNF promoter II, RNA POL II recruitment, and GDNF mRNA expression were significantly downregulated (P < 0.01). Moreover, curcumin attenuated the effects of Egr-1 overexpression on Egr-1 binding, RNA POL II recruitment, and GDNF transcription (P < 0.01). Egr-1 and RNA POL II co-existed in the nucleus of C6 glioma cells, with overlapping regions, but they were not bound to each other. In conclusion, highly expressed Egr-1 may be involved in the recruitment of RNA POL II in GDNF promoter II in a non-binding manner, and thereby involved in regulating GDNF transcription in high-grade glioma cells. This regulation is dependent on histone hyperacetylation in GDNF promoter II.