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By Hua Wang, Zhiyou Peng, Yiwen Li et al.

Excerpt from the article published in Retinal Ganglion Cell Degeneration, 15 March 2022, PREPRINT (Version 1) available at Research Square Doi: https://doi.org/10.21203/rs.3.rs-1439890/v1

Editor’s Highlights

• The sigma 2 receptor (σ2R) plays a signicant role in the retinal ganglion cell (RGC) degeneration pathway and that inhibiting σ2R/TMEM97 function is neuroprotective.
• The results presented herein also provide persuasive experimental support a new therapeutic strategy to protect RGCs and other neurons in neurodegenerative diseases by inhibiting σ2R/TMEM97.

Abstract

The sigma 2 receptor (σ₂R) was recently identified as an ER (endoplasmic reticulum) membrane protein known as transmembrane protein 97 (TMEM97). Studies have shown that compounds that bind to σ₂R/TMEM97 are neuroprotective, suggesting that σ₂R/TMEM97 is involved in pathways leading to neurodegeneration. We explore the role of σ₂R/TMEM97 in neurodegeneration by characterizing ischemia-induced retinal ganglion cell (RGC) degeneration in TMEM97^−/− mice and demonstrate that in the absence of σ₂R/TMEM97, RGCs are resistant to degeneration. In addition, we show that DKR-1677, a selective σ₂R/TMEM97 ligand, significantly protects RGCs from ischemia-induced degeneration in wild-type mice. These results provide conclusive evidence that σ₂R/TMEM97 plays a significant role in RGC death to facilitate neurodegeneration following ischemic injury. Blocking the function of σ₂R/TMEM97 thus is neuroprotective. This work is a breakthrough toward elucidating the biology and function of σ₂R/TMEM97 in RGCs and likely in other σ₂R/TMEM97-expressing neurons. Moreover, these findings support future studies to develop new neuroprotective approaches to treat RGC degenerative diseases by inhibiting σ₂R/TMEM97.

Introduction

Sigma receptors were originally identified by radioactive ligand-binding assays as a subclass of opioid receptors 1, but it was subsequently shown that they are a distinct group of receptors with two members: the sigma-1 receptor (σ1R) and the sigma-2 receptor (σ2R) 2–5. σ1R was cloned in 1996, and the protein sequence showed no homology to any other mammalian protein 6. The molecular nature of σ2R remained elusive until in 2017 when a membrane protein was puried using a resin coupled with a high-affinity σ2R- binding ligand 7. This protein was transmembrane protein 97 (TMEM97), and because this protein has ligand-binding properties matching those of the enigmatic σ2R, it was finally possible to identify σ2R as TMEM97 7. The structure of σ2R/TMEM97 and its complexes with several ligands have recently been reported 8.

The biological function of σ2R/TMEM97 is not well understood, but its wide distribution in the body, including the brain, the liver, pancreas, testis, and other organs 9–11 suggests that it may have distinct functions in different cells. σ2R/TMEM97 is known to regulate intracellular Ca2+ levels 12, and to play a role in cholesterol tracking and uptake 13–15. The high expression of σ2R/TMEM97 in proliferating cancer cells makes it a biomarker and target for cancer diagnosis and therapy 16–22. σ2R/TMEM97 is also involved in various neurological disorders 23. Recent studies in animal models of Alzheimer’s disease and traumatic brain injury demonstrate that small molecules selectively binding to σ2R/TMEM97 are neuroprotective 24,25, suggesting that σ2R/TMEM97 is involved in pathways relevant to neurodegeneration.

To explore the role of σ2R/TMEM97 in neurodegenerative processes, we characterized ischemia-induced retinal ganglion cell (RGC) degeneration in mice with or without σ2R/TMEM97 (i.e., TMEM97+/+or TMEM97−/− mice). Herein we report that in TMEM97−/− mice, the absence of σ2R/TMEM97 renders RGCs resistant to ischemia-induced cell death. In addition, intravitreal injection of the selective, high-affinity σ2R/TMEM97 ligand DKR-1677 in wildtype (TMEM97+/+) mice protects RGCs from ischemia damage. These findings demonstrate that σ2R/TMEM97 is involved in RGC degeneration, and its presence facilitates the degeneration process. The present work is a breakthrough in understanding the biological role of σ2R/TMEM97 in neurodegenerative processes in RGCs and likely in other TMEM97 expressing neurons. Importantly, these results support novel therapeutic approaches to prevent or slow the progression of RGC degenerative diseases by inhibiting the function of σ2R/TMEM97.

Results

Retinal structure and TMEM97 expression in TMEM97−/− mice TMEM97−/− mice are viable and fertile with no visible gross abnormalities. The retinal structure of a TMEM97−/− mouse is indistinguishable from the retina of a wild-type animal (Fig. 1), indicating that σ2R/TMEM97 is not critical for the development and maintenance of the retinal structure.

The distribution of σ2R/TMEM97 in the retina was studied by immunocytochemical analysis in wild-type mice. σ2R/TMEM97 immunoactivity is primarily present in RGCs (Fig. 2a, 2b), but it is also found in photoreceptor inner segments (IS), the RPE (retinal pigment epithelium), cells in the inner nuclear layer (INL), and sparsely in the outer nuclear layer (ONL), (Fig. 2a, 2b).

TMEM97 −/− mice were generated by replacing the TMEM97 gene with a Velocigene cassette ZEN-Ub1 that has a lacZ reporter gene under the control of the TMEM97 promoter 26. Accordingly, the expression of lacZ (encoding β-galactosidase) reects the expression of TMEM97, which can be characterized by X- gal staining. As shown in Fig. 2c, blue X-gal staining is present in the RGCs, the IS, cells in INL, the RPE, and sparsely in the ONL (Fig. 2c). The blue staining in the RPE cells is mostly masked by the heavy melanin pigment in the 20 μm thick section (Fig. 2c). The outer plexiform (OPL) and inner plexiform (IPO) are also stained (Fig. 2c).

The σ2R/TMEM97 immunostaining pattern in the wildtype mice (Fig. 2a) overlaps the X-gal staining pattern in TMEM97−/− mice, except in the IPL and OPL where σ2R/TMEM97 immunostaining is absent, but the blue X-gal staining is present. This discrepancy is likely caused by differences in the subcellular localization of σ2R/TMEM97 and β-galactosidase, because σ2R/TMEM97 is an ER membrane protein whereas β-galactosidase is not.

The absence of TMEM97 expression in the retinas of TMEM97−/−mice was conrmed by reverse transcription PCR (RT-PCR). Robust TMEM97 mRNA expression was found in the wildtype retinas, but no TMEM97 mRNA was detected in the retinas of TMEM97−/− mice (Fig. 2d).

Ischemia-induced RGC degeneration in the retinas of TMEM97−/− mice Retinal ischemia was created by elevating the intraocular pressure (IOP) to 120 mm Hg for 45 min in the left eyes of wildtype and TMEM97−/− mice. The right eyes in each group were untouched and served as controls. Eyes were collected 7 days after ischemia, and RGCs were identified by immunostaining of the RGC marker RBPMS (RNA binding protein with multiple splicing) 27. There was a significant loss of RGCs in the left eyes of wild-type mice as compared with RGCs in the untouched right eye (Fig. 3a, 3b). In contrast, many RGCs are present in the left eyes of TMEM97−/− mice after retinal ischemia (Fig. 3d). Figure 3c shows the retina of the untouched fellow eye from the same animal as shown in Fig. 3d. The RGC survival rate was calculated as the ratio of RBPMS-positive cells in the left eye vs those in the right eye in an animal, and the RGC survival rate in the TMEM97−/− mice (0.59 ± 0.02, n = 9) is significantly higher than that in the wildtype TMEM97+/+ mice animals (0.18 ± 0.01, n = 8, P < 0.001) (Fig. 3e). These results demonstrate that without σ2R/TMEM97, RGCs are resistant to ischemia-induced cell death.

Pattern ERG measurement The pattern electroretinogram (PERG) has been used as a sensitive measurement to assess the electrophysiological function of RGCs 28. To monitor changes in RGC electrophysiological activities, we recorded a baseline PERG in wildtype and TMEM97−/− mice. The left eyes in each of these groups of animals were then subjected to retinal ischemia, and the PERG was measured again seven days after ischemia.

Retinal ischemia in wild-type mice resulted in a marked decrease in mean PERG amplitude (~ 60%, P < 0.001, n = 5) (Fig. 4a, WT, Post-IS) as compared to the baseline (Fig. 4a, WT, Pre-IS). Conversely, there was no significant alteration of mean PERG amplitude in TMEM97−/− mice following ischemia (Fig. 4a, TM−/−, Pre- IS and Post-IS). The change in PERG amplitude in each animal after ischemia (∆Amp, Post-IS minus Pre-IS) was calculated, and the results show that the average ∆Amp is significantly different from zero in the wild-type mice (P < 0.01, n = 5) (Fig. 4b). In contrast, the average ∆Amp in TMEM97−/− mice is not significantly different from zero (Fig. 4b).

Retinal ischemia also induced an increase in mean PERG latency in wildtype mice(~ 20 ms, P < 0.01) (Fig. 4c, WT, Post-IS) as compared with the baseline (Fig. 4c WT, Per-IS), but no such increase was observed in TMEM97−/− mice (Fig. 4c, TM−/−, Per-IS and Post-IS). Calculations of the PERG latency difference in each animal (∆Lat, Post-IS minus Per-IS) show that the average ∆Lat in wildtype mice are significantly different from zero (P < 0.01, n = 5) (Fig. 4d), whereas the average ∆Lat in TMEM97−/− mice is not significantly different from zero (Fig. 4d).

Protection of RGCs by σ 2 R/TMEM97 ligand DKR-1677 DKR-1677 is a selective and high-affinity σ2R/TMEM97 ligand (Fig. 5f) that has been shown to be neuroprotective in two models of traumatic brain injury 25. We investigated the effect of DKR-1677 on RGCs in wild-type mice following ischemia. Animals were divided into a DKR-1677 group and a vehicle control group. The left eyes of animals in each group were intravitreally injected with either DKR-1677 (20 μg/μL in DMSO, 2 μL/eye) or vehicle (DMSO 2 μL/eye), and the right eyes were not injected. Five days after injection, retinal ischemia was performed on the left (injected) eyes in each group, whereas the right eyes were untouched. RGC survival was assessed 7 days after ischemia.

There was a significant loss of RGCs in the left eyes in the vehicle group after ischemia, as compared with the untouched right eyes (Fig. 5a, 5b). In DKR-1677 injected eyes, however, many surviving RGCs were present after ischemia (Fig. 5d). Figure 5c shows the retina of the untouched fellow eye from the same animal as shown in Fig. 5d. Quantitative analysis showed that the RGC survival rate in the DKR- 1677 group (0.59 ± 0.04, n = 5) was significantly higher than that in the vehicle group (0.22 ± 0.10, n = 3, P < 0.01) (Fig. 5e). These results demonstrate that the σ2R/TMEM97 ligand DKR-1677 protects RGCs against ischemia-induced cell death.

Discussion

The most significant finding of this work is the loss-of-function phenotype of TMEM97−/− in RGCs, wherein ablation of the TMEM97 gene renders cells resistant to ischemic damage. RGCs in TMEM97−/− mice had significantly higher survival rates after ischemia than cells in wild-type mice with fully functional σ2R/TMEM97. Moreover, RGC electrophysiological activities in TMEM97−/− mice were much better maintained after ischemia than cells in wild-type mice. These findings unequivocally demonstrate that σ2R/TMEM97 is involved in the degenerative process in RGCs, and its presence facilitates ischemia-induced degeneration.

We also demonstrate that the σ2R/TMEM97 ligand DKR-1677 protects RGCs in wild-type mice from ischemia-induced cell death. DKR-1677 binds to σ2R/TMEM97 with high affinity (Ki=5.1 nM), and it is highly selective for σ2R/TMEM97 (45-fold over σ1R) (Fig. 5f) 25. The neuroprotective effect of DKR-1677 on RGCs of wild-type mice is consistent with the loss-of-function phenotype in TMEM97−/− mice and indicates that DKR-1677 protects RGCs by blocking the action of σ2R/TMEM97.

Our present work demonstrates that σ2R/TMEM97 facilitates RGC degeneration, whereas studies have shown showed that σ1R promotes RGC survival 29. For example, genetic ablation of σ1R in mice results in age-related inner retinal dysfunction and loss of RGCs 30, and σ1R agonists were shown to protect RGC 29,31. It seems that σ2R/TMEM97 and σ1R have opposite roles in RGC degeneration. The question is whether σ1R and σ2R/TMEM97 are involved in the same mechanism to regulate neurodegeneration, and/or whether they interact with each other to regulate the degeneration process. Opposite roles of σ1R and σ2R/TMEM97 have been reported in human uveal melanoma cells in which σ1R is found to be pro-proliferative and σ2R/TMEM97 is anti-proliferative 32. σ1R and σ2R/TMEM97 also play opposite roles in mediating the effects of cocaine exposure in mice 33.

Compounds binding to σ2R/TMEM97 have been reported to be neuroprotective in the brain in several studies. For example, the σ2R/TMEM97-binding compound SAS-0132 was found to significantly improve cognitive performance in a mouse model of Alzheimer’s disease 24, and CT1812, which also binds selectively to σ2R/TMEM97, is in clinical trials for Alzheimer’s disease therapy 34. DKR-1677, the σ2R/TMEM97 ligand used in this work, was found to be neuroprotective in two models of traumatic brain injury 25. The results presented herein show that the mechanism of action of DKR-1677 for neuroprotection in RGCs is to block the function of σ2R/TMEM97. It therefore seems likely that the neuroprotective effects of small molecules that bind to σ2R/TMEM97 in brain neurons is also a result of σ2R/TMEM97 inhibition.

Although the biological functions of σ2R/TMEM97 are not well understood, σ2R/TMEM97 is known to be involved in regulating cellular Ca2+ levels and controlling cellular cholesterol levels 12,13. Treating cells with σ2R/TMEM97 ligands induces a biphasic increase in cytosolic Ca2+ levels 12, suggesting that σ2R/TMEM97 signaling could be mediated by intracellular Ca2+ levels. σ2R/TMEM97 is also known to regulate cellular cholesterol by controlling cholesterol tracking from lysosomes to the ER and cholesterol uptake13–15. Furthermore, the high levels of σ2R/TMEM97 expression in proliferating cancers have been exploited as a target for cancer therapy and as a biomarker for diagnosis 18,20,22. σ2R/TMEM97 is believed to be associated with a number of neurological disorders, including anxiety, depression, and addiction 23, although the biological mechanisms are not well understood. In animal models, σ2R/TMEM97 binding compounds were found to mitigate alcohol withdrawal symptoms 35,36 and effects of cocaine 37. σ2R/TMEM97 ligands also induce long-lasting relief of neuropathic pain 38. Two recent reports described a potential role σ2R/TMEM97 in oxidative damage to RPE cells, but the results are contradictory 39,40. Further studies are needed to clarify how σ2R/TMEM97 is associated with oxidative damage in RPE cells.

We used ischemia-induced RGC degeneration in the present work as a model to study the role of σ2R/TMEM97 in neurodegeneration, but the ndings that inhibiting σ2R/TMEM97 protects RGCs could have specic clinical relevance. RGCs are retinal output neurons that process and convey visual information from the retina to the visual cortex 41. Clinical conditions that affect RGC functions, including glaucoma, ischemic optic neuropathies, hereditary optic neuropathies, and demyelinating disease, can lead to blindness 42–44. For example, glaucoma, a leading cause of irreversible vision loss, is a heterogeneous group of optic neuropathies characterized by RGC degeneration. 45–48. A promising therapeutic strategy for glaucoma is to protect RGCs thus to stop or slow down disease progression 43,49. The present ndings provide experimental data that support new approaches to protect RGCs by inhibiting σ2R/TMEM97. Because compounds that bind to σ2R/TMEM97 readily pass the blood-retinal barrier, synthetic σ2R/TMEM97 ligands that improve RGC survival might be used in the development of novel therapies for glaucoma and other retinal diseases.

In summary, the present work is a breakthrough in elucidating the mechanism and function of σ2R/TMEM97 in RGCs and in other σ2R/TMEM97 expressing neurons. Our data show that σ2R/TMEM97 plays a signicant role in the RGC degeneration pathway and that inhibiting σ2R/TMEM97 function is neuroprotective. The results presented herein also provide persuasive experimental support a new therapeutic strategy to protect RGCs and other neurons in neurodegenerative diseases by inhibiting σ2R/TMEM97.