Defining the ligand-dependent proximatome of the sigma 1 receptor

Sigma 1 Receptor (S1R) is a therapeutic target for a wide spectrum of pathological conditions ranging from neurodegenerative diseases to cancer and COVID-19. S1R is ubiquitously expressed throughout the visceral organs, nervous, immune and cardiovascular systems. It is proposed to function as a ligand-dependent molecular chaperone that modulates multiple intracellular signaling pathways. The purpose of this study was to define the S1R proximatome under native conditions and upon binding to well-characterized ligands. This was accomplished by fusing the biotin ligase, Apex2, to the C terminus of S1R. Cells stably expressing S1R-Apex or a GFP-Apex control were used to map proximal proteins. Biotinylated proteins were labeled under native conditions and in a ligand dependent manner, then purified and identified using quantitative mass spectrometry. Under native conditions, S1R biotinylates over 200 novel proteins, many of which localize within the endomembrane system (endoplasmic reticulum, Golgi, secretory vesicles) and function within the secretory pathway. Under conditions of cellular exposure to either S1R agonist or antagonist, results show enrichment of proteins integral to secretion, extracellular matrix formation, and cholesterol biosynthesis. Notably, Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) displays increased binding to S1R under conditions of treatment with Haloperidol, a well-known S1R antagonist; whereas Low density lipoprotein receptor (LDLR) binds more efficiently to S1R upon treatment with (+)-Pentazocine ((+)-PTZ), a classical S1R agonist. Furthermore, we demonstrate that the ligand bound state of S1R correlates with specific changes to the cellular secretome. Our results are consistent with the postulated role of S1R as an intracellular chaperone and further suggest important and novel functionalities related to secretion and cholesterol metabolism

PPIP5K2 and PCSK1 are Candidate Genetic Contributors to Familial Keratoconus

Keratoconus (KC) is the most common corneal ectatic disorder affecting >300,000 people in the US. KC normally has its onset in adolescence, progressively worsening through the third to fourth decades of life. KC patients report significant impaired vision-related quality of life. Genetic factors play an important role in KC pathogenesis. To identify novel genes in familial KC patients, we performed whole exome and genome sequencing in a four-generation family. We identified potential variants in the PPIP5K2 and PCSK1 genes. Using in vitro cellular model and in vivo gene-trap mouse model, we found critical evidence to support the role of PPIP5K2 in normal corneal function and KC pathogenesis. The gene-trap mouse showed irregular corneal surfaces and pathological corneal thinning resembling KC. For the first time, we have integrated corneal tomography and pachymetry mapping into characterization of mouse corneal phenotypes which could be widely implemented in basic and translational research for KC diagnosis and therapy in the future

Silencing p75NTR prevents proNGF-induced endothelial cell death and development of acellular capillaries in rat retina

Accumulation of the nerve growth factor precursor (proNGF) and its receptor p75NTR have been associated with several neurodegenerative diseases in both brain and retina. However, whether proNGF contributes to microvascular degeneration remain unexplored. This study seeks to investigate the mechanism by which proNGF/p75NTR induce endothelial cell (EC) death and development of acellular capillaries, a surrogate marker of retinal ischemia. Stable overexpression of the cleavage-resistant proNGF and molecular silencing of p75NTR were utilized in human retinal EC and rat retinas in vivo. Stable overexpression of proNGF decreased NGF levels and induced retinal vascular cell death evident by 1.9-fold increase in acellular capillaries and activation of JNK and cleaved-PARP that were mitigated by p75NTRshRNA. In vitro, overexpression of proNGF did not alter TNF-α level, reduced NGF, however induced EC apoptosis evident by activation of JNK and p38 MAPK, cleaved-PARP. Silencing p75NTR using siRNA restored expression of NGF and TrkA activation and prevented EC apoptosis. Treatment of EC with human-mutant proNGF induced apoptosis that coincided with marked protein interaction and nuclear translocation of p75NTR and the neurotrophin receptor interacting factor. These effects were abolished by a selective p75NTR antagonist. Therefore, targeting p75NTR represents a potential therapeutic strategy for diseases associated with aberrant expression of proNGF

Sigma 1 receptor regulates ERK activation and promotes survival of optic nerve head astrocytes

<div><p>The sigma 1 receptor (S1R) is a unique transmembrane protein that has been shown to regulate neuronal differentiation and cellular survival. It is expressed within several cell types throughout the nervous system and visceral organs, including neurons and glia within the eye. S1R ligands are therapeutic targets for diseases ranging from neurodegenerative conditions to neoplastic disorders. However, effects of S1R activation and inhibition within glia cells are not well characterized. Within the eye, the astrocytes at the optic nerve head are crucial to the health and survival of the neurons that send visual information to the brain. In this study, we used the S1R-specific agonist, (+)-pentazocine, to evaluate S1R activation within optic nerve head-derived astrocytes (ONHAs). Treatment of ONHAs with (+)-pentazocine attenuated the level and duration of stress-induced ERK phosphorylation following oxidative stress exposure and promoted survival of ONHAs. These effects were specific to S1R activation because they were not observed in ONHAs that were depleted of S1R using siRNA-mediated knockdown. Collectively, our results suggest that S1R activation suppresses ERK1/2 phosphorylation and protects ONHAs from oxidative stress-induced death.</p></div

Effect of PTZ on ROS generation when ONHAs exposed to H₂O₂.

<p>(A) Representative images of ONHAs treated with 100μM H₂O₂ for 24 hours in the presence or absence of PTZ (10μM, 1 hour pretreatment followed by co-treatment). ROS generation was visualized using CellROX Green reagent. Scale bar: 100μm. (B) Quantitative analysis of intracellular ROS. For each group, three coverslips were quantified, and eight images were taken from each coverslip. Mean signal intensity was quantified by ImageJ. Intracellular ROS generation increased when ONHAs were exposed to H₂O₂. The ROS generation was inhibited by PTZ. Data were analyzed using one-way ANOVA followed by Tukey-Kramer post hoc test for multiple comparisons. Significantly different from control ****P<0.0001. Significantly different between groups ####P<0.0001. Experiments were repeated 3 times.</p

S1R knockdown in HeLa cells and ONHAs.

<p>HeLa cells were transfected with human scrambled siRNA or with human S1R siRNA. Western blot analysis of S1R levels at 3 days (A) and 6 days (B) following transfection is shown. Quantitation of western blot results shows S1R levels normalized to GAPDH as the internal control (C, D). Results are presented as fold change of S1R levels derived from the S1R siRNA-transfected cells compared to S1R levels derived from scrambled siRNA-transfected cells. Data were analyzed using t-test. Significantly different from control *p<0.05, **p<0.01. Experiments were repeated 3 times. MTT assay was performed to assess viability at 3 days (E) and 6 days (F) after scrambled or S1R siRNA transfection. Viability was significantly decreased in S1R siRNA-transfected HeLa cells compared to scrambled siRNA-transfected cells. Transfection with scrambled siRNA did not cause significant HeLa cell death compared to non-transfection control (G, H). Data were analyzed using t-test. Significantly different from control ****p<0.0001. Experiments were performed in quadruplicate and repeated 3 times. ONHAs were transfected with rat scrambled siRNA or with rat S1R siRNA. Western blot analysis of S1R levels at 3 days (I) and 6 days (J) following transfection is shown. Quantitation of western blot results shows S1R levels normalized to GAPDH as the internal control (K, L). Results are presented as fold change of S1R levels derived from the S1R siRNA-transfected cells compared to S1R levels derived from scrambled siRNA-transfected cells. Data were analyzed using t-test. Significantly different from control: **p<0.01, ***p<0.001. Experiments were repeated 3 times. MTT assay was performed to assess viability at 3 days (M) and 6 days (N) after scrambled or S1R siRNA transfection. Viability was not significantly changed in S1R siRNA-transfected ONHAs compared to scrambled siRNA-transfected cells. Transfection with scrambled siRNA did not cause significant ONHA death compared to non-transfection control (O, P). Experiments were performed in quadruplicate and repeated 3 times.</p

Knockdown of S1R within ONHAs blocks (+)-pentazocine-mediated suppression of ROS generation.

<p>(A) Five days following transfection with S1R siRNA, ONHAs were treated with 100μM H₂O₂ for 24 hours in the presence or absence of PTZ (10μM, 1 hour pretreatment followed by co-treatment). MTT assay was performed to assess viability.100μM H₂O₂ induced 50% cell death, and PTZ treatment did not significantly increase cell viability. (B-C) Effect of S1R knockdown on ROS generation in ONHAs. Data were analyzed using one-way ANOVA followed by Tukey-Kramer post hoc test for multiple comparisons. Significantly different from control: ***p<0.001, ****p<0.0001. (B) Representative images of ONHAs six days following transfection with either scrambled siRNA or S1R siRNA. ROS generation was visualized using CellROX Green reagent. Scale bar: 100μm. (C) Quantitative analysis of intracellular ROS. For each group, three coverslips were quantified, and eight images were taken from each coverslip. Mean signal intensity was quantified by ImageJ. Transfection with scrambled siRNA did not significantly increase ROS generation compared with non-transfected control cells. S1R siRNA-transfected ONHAs showed increased intracelluar ROS compared with scrambled siRNA-transfected ONHAs. Data were analyzed using one-way ANOVA followed by Tukey-Kramer post hoc test for multiple comparisons. Significantly different from control: **p<0.01. Significantly different between groups: #p<0.05. (D) Representative images of S1R siRNA-transfected ONHAs treated with 100μM H₂O₂ with or without PTZ for 24 hours. Scale bar: 100μm. (E) Quantitative analysis of intracellular ROS. For each group, three coverslips were quantified, and eight images were taken from each coverslip. Mean signal intensity was quantified by ImageJ. ROS generation increased when cells were incubated with H₂O₂. The ROS generation was not inhibited by PTZ treatment. Data were analyzed using one-way ANOVA followed by Tukey-Kramer post hoc test for multiple comparisons. Significantly different from control *p<0.05. Experiments were repeated 3 times.</p

Characterization of cultured primary rat optic nerve head astrocytes (ONHA).

<p>(A) ONHAs were fixed and probed with antibodies against GFAP (red) and NCAM (green), S1R (red), OSP (red), and Iba-1 (red). The cells were counterstained with DAPI to label DNA (blue) as a marker for nuclei. Scale bar: 50μm. (B) Quantitative analysis shows that more than 95% of the cells in culture express GFAP. (C) The cell lysates from ONHAs (lane 3) were positive for GFAP, a marker for astrocytes and S1R, but negative for Iba-1, a marker for microglial cells, and OSP, a marker for oligodendrocytes. The protein extract from rat brain (lane 1) and from rat optic nerve tissue (lane 2) were used as positive controls.</p

The effect of (+)-pentazocine and H₂O₂ on ONHAs viability.

<p>(A) ONHAs were treated with (+)-pentazocine (PTZ) at varying concentrations (1,3,10 or 50μM) for 24 hours. MTT assay was performed to assess viability. Treatment with PTZ did not cause a significant change in percentage viability compared to the untreated control cells. At the highest concentration of PTZ (50μM), there was a trend toward decrease in viability that was not significant. (B) ONHAs were exposed to various H₂O₂ concentrations (50,100,150, 200, 250, 500μM) for 24 hours. H₂O₂ induced ONHA death in a dose dependent manner. (C) ONHAs were treated with 100μM H₂O₂ for 24 hours in the presence or absence of PTZ (10μM, 1 hour pretreatment followed by co-treatment). Exposure to H₂O₂ significantly decreased viability compared to non-exposed cells. Compared to H₂O₂-exposure with no PTZ treatment, the H₂O₂-exposed, PTZ-treated ONHAs showed significantly increased viability. Data were analyzed using one-way ANOVA followed by Tukey-Kramer post hoc test for multiple comparisons. Significantly different from control *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Significantly different between groups ###P<0.001. Experiments were performed in quadruplicate and repeated 3 times.</p

Knockdown of S1R within ONHAs blocks the (+)-pentazocine-mediated suppression of ERK1/2 phosphorylation.

<p>Phophorylation of ERK was detected after 3 days (A) and 6 days (C) following transfection of scrambled or S1R siRNA in ONHA. At 3 days (A) and 6 days (C) following S1R siRNA transfection, pERK was increased compared to scrambled siRNA control. (B, D) Quantitative analysis of pERK levels represented as pERK normalized to total ERK. Results are presented as fold change of the pERK/total ERK ratio derived from S1R siRNA transfected cells versus scrambled siRNA transfected control cells. Data were analyzed using t-test. Significantly different from control: *p<0.05. Experiments were repeated 3 times. (E) Effect of PTZ on H₂O₂–exposed S1R siRNA transfected ONHA. 5 days after S1R siRNA transfection, ONHA were incubated with 100μM H₂O₂ at 37°C for 15 minutes, 30 minutes, 1 hour, 3 hours and 24 hours in the presence or absence of PTZ (10μM, 1 hour pretreatment followed by cotreatment). Western blot analysis is shown. (F) Quantitative analysis of pERK levels represented as pERK normalized to total ERK. Results are presented as fold change of the pERK/total ERK ratio derived from H₂O₂ exposed, S1R siRNA transfected cells versus control non-H₂O₂ exposed, S1R siRNA transfected cells. Data were analyzed using two-way ANOVA followed by Tukey-Kramer post hoc test for multiple comparisons.</p