Novel Role of Copper Transporter CTR1 and Therapeutic Potential of Copper Chelators in Retinal Ischemia-Reperfusion Injury

Abstract

Purpose: Retinal ischemia contributes to vision loss in ischemic and diabetic retinopathies through oxidative stress, neurovascular injury, and inflammation. Copper (Cu), whereas an essential micronutrient, can be toxic in excess and is regulated by Cu transporters such as CTR1. However, the role of CTR1 in ischemic retinopathy remains unclear.

Methods and results: Retinal ischemia-reperfusion (IR) injury was induced by elevating intraocular pressure (IOP) to 110 millimeters of mercury (mm Hg) for 40 minutes in the right eyes of Ctr1 heterozygous (Ctr1+/-) and wild-type (WT) mice. In WT mice, IR triggered rapid CTR1 upregulation and increased retinal Cu levels (measured by inductively coupled plasma mass spectrometry [ICP-MS]). IR injury caused retinal ganglion cell (RGC) loss, inner retinal thinning, vascular degeneration, and apoptosis, all of which were significantly attenuated in Ctr1+/- mice. Ctr1+/- mice also exhibited reduced microglial (Iba1⁺) and glial cells (GFAP⁺) activation and preserved visual function, as assessed by electroretinography. Mechanistically, IR-induced reactive oxygen species (\({{\rm{O}}_{2}}^{-}\)) production (DHE staining), upregulation of NADPH oxidase components (NOX2 and p47phox), and NF-κB activation were markedly suppressed in Ctr1+/- mice. Treatment with the Cu chelator tetrathiomolybdate (TTM) similarly reduced retinal thinning, neurovascular damage, apoptosis, gliosis, and oxidative stress after IR injury.

Conclusions: CTR1 plays a central role in mediating Cu-dependent oxidative stress, neurovascular degeneration, and inflammation following retinal IR injury. Targeting the CTR1-Cu axis may represent a novel therapeutic strategy for ischemic retinopathy.

Figure 1.

CTR1 protein expression and Cu levels are increased following retinal IR injury in mice. (A) Western blotting analysis of CTR1 and β-actin (loading control) protein expression in the retinal tissues of WT mice subjected to sham surgery (0 hours) or at 3, 6, and 24 hours after IR injury. Bar graphs show fold changes relative to the sham surgery group, normalized to β-actin (n = 5–8 per group). (B). Representative immunofluorescence images of retinal tissues stained with CTR1 Ab or IgG (negative control) along with DAPI nuclear staining at 3 hours post-IR injury in WT mice. Scale bar = 50 µm. (C) Cu, zinc, and iron contents in the retinal tissues were measured using inductively coupled plasma mass spectrometry (ICP-MS) at sham surgery, and 3, 6, and 24 hours post-IR injury (n = 6–7 per group). *P < 0.05. NS, not significant.

Figure 2.

Ctr1^+/− mice exhibit protection against neurovascular degeneration following IR injury. (A) Representative images of retinal flat-mounts labeled with the neuronal marker (NeuN) and quantification from WT and CTR1^+/− mice 7 days post-IR injury. Bar graph represents numbers of NeuN-positive cells expressed by the percentage of WT sham (n = 3–4 per group). (B) Representative images of retinal vascular digests in WT and CTR1^+/− mice at 14 days post-IR injury. The red arrows indicate degenerate capillaries. The bar graph represents the number of acellular capillaries per mm² in the retina (n = 6 per group). (C) Representative H&E-stained retinal sections from WT and CTR1^+/− mice 7 days post-IR injury. The bar graph represents the percentage of WT sham (n = 8–9 per group). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS/OS, inner segment/outer segment layer; RPE, retinal pigment epithelium layer. (D) OCT images in the retina of WT retina or CTR1^+/− retina at 7 days post-IR injury. The yellow arrow indicates retinal detachment in IR-injured WT retina. *P < 0.05, **P < 0.01, and ***P < 0.001, NS, not significant. Scale bar = 100 µm.

Figure 3.

Ctr1^+/− mice exhibit reduced retinal apoptosis and inflammation following IR injury. (A) TUNEL staining with DAPI nuclear counterstaining in retinal sections from WT and CTR1^+/− mice 3 days post-IR injury. The bar graph represents numbers of apoptotic cells per retinal section (n = 4–5 per group). (B) Iba1 staining shows microglia activation, with DAPI nuclear counterstaining, in retinal sections from WT and CTR1^+/− mice at 3 days post-IR injury (n = 4–5 per group). (C) GFAP staining shows Müller cell activation, with DAPI nuclear counterstaining, in retinal sections from WT and CTR1^+/− mice at 5 days post-IR injury (n = 5–6 per group). *P < 0.05, **P < 0.01, and ***P < 0.001, NS, not significant. Scale bar = 50 µm. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer.

Figure 4.

Ctr1^+/− mice exhibit preserved visual function following retinal IR injury. (A) Representative dark-adapted electroretinogram (ERG) intensity showing retinal function by a- and b-waveforms elicited by 1 log cd·s/m² flashes in WT or Ctr1^+/− mice at 7 days post-IR injury. (B) The bar graph represents quantification of a- and b-waveforms amplitudes (n = 4–5 each group). *P < 0.05, and **P < 0.01, NS, not significant.

Figure 5.

Ctr1^+/− mice exhibit reduced oxidative stress and NADPH oxidase subunits upregulation following retinal IR injury. (A) DHE imaging of O₂⁻ formation in the retina sections from WT and Ctr1^+/− mice at 6 hours after IR injury. The Bar graph represents quantification of fluorescence intensity expressed by the average values for each slide (n = 6 per group). *P < 0.05, ns, not significant. Scale bar = 100 µm. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. (B) The qPCR analysis for Nox2, p22phox, and p47phox mRNA expression in retina tissues at 6 hours after IR injury (n = 3–5 per group). (C) Western blot analysis for phospho-NFkB (p-p65) or total p65 protein expression at 3 hours post-IR injury. The bar graph represents the relative values of each group expressed as a percentage of its respective sham (n = 5 per group). *P < 0.05, **P < 0.01, and ***P < 0.001, NS, not significant.

Figure 6.

Cu chelator TTM treatment protects against neurovascular degeneration following retinal IR injury. (A) Representative images of retinal flat-mounts labeled with the neuronal marker (NeuN) and quantification from vehicle- or TTM-treated mice 7 days post-IR injury. The bar graph represents numbers of NeuN-positive cells expressed by the percentage of vehicle-treated sham (n = 4 per group). (B) Representative images of retinal vascular digests in vehicle- or TTM-treated mice at 14 days post-IR injury. The red arrows indicate degenerate capillaries. The bar graph represents the number of acellular capillaries per mm² in the retina (n = 4–6 per group). (C) Representative H&E-stained retinal sections from vehicle- or TTM-treated mice at 7 days post-IR injury. The bar graph represents the percentage of vehicle-treated sham (n = 7–9 per group). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS/OS, inner segment/outer segment layer; RPE, retinal pigment epithelium layer. (D) OCT images in the retina of vehicle- or TTM-treated mice at 7 days post-IR injury. The yellow arrow indicates retinal detachment in IR-injured vehicle-treated mice. *P < 0.05, **P < 0.01, and ***P < 0.001, NS, not significant. Scale bar = 100 µm.

Figure 7.

Cu chelator TTM treatment reduces retinal apoptosis and inflammation following IR injury. (A) TUNEL staining with DAPI nuclear counterstaining in retinal sections from vehicle- or TTM-treated mice at 3 days post-IR injury. The bar graph represents numbers of apoptotic cells per retinal section (n = 4–5 per group). (B) Iba1 staining shows microglia activation, with DAPI nuclear counterstaining, in retinal sections from vehicle- or TTM-treated mice at 3 days post-IR injury (n = 4 per group). (C) GFAP staining shows Müller cell activation, with DAPI nuclear counterstaining, in retinal sections from vehicle- or TTM-treated mice at 5 days post-IR injury (n = 4–5 per group). (A–C) *P < 0.05, **P < 0.01, and ***P < 0.001, NS, not significant. Scale bar = 50 µm. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer.

Figure 8.

Cu chelator TTM treatment attenuates oxidative stress following retinal IR injury. (A) DHE imaging of ${{\mathrm{O}}_2}^{-}$ formation in the retina sections from vehicle- or TTM-treated mice at 6 hours after IR injury. The bar graph represents quantification of fluorescence intensity expressed by the average values for each slide (n = 4–6 per group). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. **P < 0.01, and ***P < 0.001, NS, not significant. Scale bar = 100 µm. (B) Proposed model illustrating the role of CTR1-Cu axis in IR-induced inflammation, cell death and neurovascular degeneration leading to visual impairment. Retinal IR injury triggers a rapid increase in CTR1 expression and Cu levels in the retina, which upregulates the expression of NOX components, leading to elevated ROS production. This promotes the activation of p-NFkB, inflammation, and apoptosis, driving neurovascular degeneration, and visual impairment. These IR-induced retinal damages were significantly mitigated in Ctr1^+/− mice or through treatment with the Cu chelator, TTM.