M98K-OPTN induces transferrin receptor degradation and RAB12-mediated autophagic death in retinal ganglion cells

Abstract

Mutations in the autophagy receptor OPTN/optineurin are associated with the pathogenesis of glaucoma and amyotrophic lateral sclerosis, but the underlying molecular basis is poorly understood. The OPTN variant, M98K has been described as a risk factor for normal tension glaucoma in some ethnic groups. Here, we examined the consequence of the M98K mutation in affecting cellular functions of OPTN. Overexpression of M98K-OPTN induced death of retinal ganglion cells (RGC-5 cell line), but not of other neuronal and non-neuronal cells. Enhanced levels of the autophagy marker, LC3-II, a post-translationally modified form of LC3, in M98K-OPTN-expressing cells and the inability of an LC3-binding-defective M98K variant of OPTN to induce cell death, suggested that autophagy contributes to cell death. Knockdown of Atg5 reduced M98K-induced death of RGC-5 cells, further supporting the involvement of autophagy. Overexpression of M98K-OPTN enhanced autophagosome formation and potentiated the delivery of transferrin receptor to autophagosomes for degradation resulting in reduced cellular transferrin receptor levels. Coexpression of transferrin receptor or supplementation of media with an iron donor reduced M98K-induced cell death. OPTN complexes with RAB12, a GTPase involved in vesicle trafficking, and M98K variant shows enhanced colocalization with RAB12. Knockdown of Rab12 increased transferrin receptor level and reduced M98K-induced cell death. RAB12 is present in autophagosomes and knockdown of Rab12 resulted in reduced formation of autolysosomes during starvation-induced autophagy, implicating a role for RAB12 in autophagy. These results also show that transferrin receptor degradation and autophagy play a crucial role in RGC-5 cell death induced by M98K variant of OPTN.

Keywords: RAB12; autophagy; glaucoma; optineurin; retinal ganglion cells; transferrin receptor.

Figure 1. Expression of M98K variant of OPTN induces cell death selectively in RGC-5 cells. (A) Schematic of OPTN showing various domains and position of different mutations. LIR, LC3-interacting region; UBD, ubiquitin-binding domain; ZF, zinc-finger domain. (B) RGC-5 cells were transfected with HA-tagged constructs of WT-OPTN or various mutants as indicated and stained with HA antibody to detect expressing cells. Panels show images captured using 63× objective of Axioplan2 microscope (Zeiss). DAPI and phase panels indicate nuclear and cell morphology. Arrows indicate apoptotic cells. (C) Quantitation of cell death induced by WT-OPTN and its mutants in RGC-5 cells upon 32 h of expression. Data represent mean ± s.d of percentage of expressing cells showing apoptotic morphology after subtraction of cell death seen in non-expressing cells, from at least three independent experiments using duplicate coverslips. n = 6, ***p < 0.001. (D) Western blot showing expression levels of various constructs of OPTN under conditions used for apoptotic assays. CDK2 was used as loading control. Ut, untransfected. (E) Left panel, quantitation of cell death induced by WT-OPTN and M98K in various cell lines. Data are represented as in (C). n = 6, ***p < 0.001. Right panel, levels of overexpressed M98K in whole cell lysates of indicated cell lines is shown in the western blot. (F) M98K expression causes cleavage of CASP3 and PARP1. Western blots showing cleavage of CASP3 and PARP1 upon overexpression of WT-OPTN, M98K-OPTN (in the presence and absence of chloroquine) and other mutants of M98K in RGC-5 cells. GAPDH was used as loading control. Positive control, RGC-5 cells treated with 10 ng/ml TNF and 20 μg/ml cycloheximide for 8 h; CQ, chloroquine. (G) Western blots showing cleavage of CASP3 and PARP1 upon overexpression of WT-OPTN and M98K in IMR-32 and HeLa cells.

Figure 2. M98K mutant shows enhanced interaction and colocalization with TFRC compared with WT-OPTN. (A) M98K forms more and larger vesicles. Upper panel, confocal images of RGC-5 cells showing vesicles formed by GFP-tagged construct of WT or M98K-OPTN after 24 h of expression. Scale bar: 10 μm. Lower panel, quantitation of vesicles formed by GFP-tagged WT or M98K-OPTN after 24 h of expression, based on size. n = 100 cells, ***p < 0.001. (B) Interaction of M98K with TFRC. RGC-5 cells were infected with adenoviruses expressing HA-tagged WT or M98K-OPTN. After 6 h of infection, cells were treated with 25 μM chloroquine for 12 h and cell lysates subjected to immunoprecipitation with HA antibody (HA) or control antibody (C) and western blotting with TFRC and HA antibodies. WCL, whole cell lysates. (C) Representative confocal images of RGC-5 cells showing colocalization of endogenous TFRC with GFP-tagged WT or M98K-OPTN after 24 h of expression. Scale bar: 10 μm. (D) The graph represents correlation coefficient of colocalization between GFP-WT, or GFP-M98K and endogenous TFRC. n = 30, ***p < 0.001. (E) Effect of M98K expression on transferrin uptake. RGC-5 cells expressing GFP-tagged WT or M98K were subjected to serum starvation for 2 h and then incubated with Alexa-546 conjugated transferrin (TF-Alexa-546) for 15 min. Cells were fixed and analyzed by confocal microscopy. Scale bar: 10 μm. Arrow heads denote cells with less transferrin uptake. (F) Quantitation of transferrin uptake by measuring the fluorescence intensity. Data represent the uptake of labeled transferrin in WT or M98K-OPTN-expressing cells (Exp), compared with nonexpressing cells (Non-Exp). n = 150 cells ***p < 0.001. (G) Representative confocal images showing colocalization of TF-Alexa-546 with TFRC in M98K-expressing cells after 24 h of expression. Scale bar: 10 μm.

Figure 3. M98K expression induces TFRC degradation through lysosomal pathway in RGC-5 cells. (A) Western blot shows TFRC levels in RGC-5 cells infected with control (con), WT, E50K or M98K adenoviruses after 24 and 30 h of expression. GAPDH was used as a loading control. The numbers below TFRC blot indicate relative TFRC levels after normalization. (B) Effect of M98K expression on TFRC levels in IMR-32 (left panel) and HeLa (right panel) cells. (C) M98K-mediated TFRC degradation is independent of proteasomal degradation. RGC-5 cells infected with control (con), WT or M98K adenoviruses for 12 h were treated with either DMSO or with 5 μM MG-132 for further 12 h. Cell lysates were then subjected to western blotting. CDK2 was used as a loading control. (D) Effect of lysosomal inhibitors on M98K-mediated TFRC degradation. After 6 h of infection with the indicated adenoviruses, RGC-5 cells were either left untreated or treated with 25 and 50 μM of chloroquine or 10 and 20 mM of ammonium chloride for 24 h. Whole cell lysates were subjected to western blotting. CQ, chloroquine.

Figure 4. M98K expression induces autophagy. (A) Effect of M98K expression on LC3-II levels. Western blot shows LC3-II levels in RGC-5 cell lysate after 24 h of adenovirus mediated expression of control (con), WT or M98K. GAPDH was used as a loading control. (B) Effect of M98K expression on autophagosome formation. Data represent the number of GFP-LC3B dots (autophagosomes) per cell in control (con), WT or M98K-expressing cells after 24 h of transfection. n = 45 cells ***p < 0.001. (C) RGC-5 cells were transfected with either control plasmid, HA-M98K or HA-M98K-D474N along with mCherry-GFP-LC3B construct for 24 h and were either kept untreated (upper panel) or in amino acid and serum-free media (EBSS) for 2 h (lower panel). Representative merged confocal images show autophagosomes (yellow dots) and autolysosomes (red dots only). (D) Quantitation of number of autophagosomes (green dots) and red dots per cell in control, HA-M98K or HA-M98K-D474N transfected in untreated (left panel) and EBSS treated (2 h) cells (right panel). n = 40 cells, ***p < 0.001; **p < 0.01; *p < 0.05. (E) Knockdown of Atg5 reduces M98K-mediated TFRC degradation. RGC-5 cells were transfected with either Con-Sh, Atg5-ShA or Atg5-ShB and after 24 h of knockdown, were either infected with WT or M98K adenoviruses for 24 h. Cell lysates were subjected to western blotting. GAPDH was used as a loading control. The numbers below TFRC and ATG5 blots indicate relative TFRC and ATG5 levels after normalization. (F) Knockdown of ATG5 reduced M98K-induced cell death. Data represent quantitation of cell death induced by M98K with or without Atg5 knockdown. n = 6, ***p < 0.001. (G) Effect of LC3 binding-defective mutation in M98K on cell death induced by M98K. Data show quantitation of cell death upon expression of M98K or M98K-F178A. n = 6 experiments ***p < 0.001. (H) Colocalization of GFP-LC3B with WT, M98K or their LC-3 binding deficient mutants. Panels show representative confocal images of RGC-5 cells expressing GFP-LC3B with indicated OPTN mutants. Scale bar: 10 μm. Magnified areas are shown as insets. (I) The graph shows correlation coefficient of colocalization of LC3B with indicated constructs of OPTN. n = 30 cells, ***p < 0.001. (J) Effect of M98K and LC3 binding-defective mutation in M98K, M98K-F178A, on endogenous TFRC levels after 32 h of transfection. Western blot showing TFRC levels upon expression of WT, M98K and M98K-F178A. GAPDH was used as a loading control.

Figure 5. M98K-OPTN enhances TFRC localization in autophagosomes. (A) TFRC colocalizes with GFP-LC3B positive structures in M98K-expressing cells. Panels show representative confocal images of RGC-5 cells expressing GFP-LC3B with HA-WT or HA-M98K OPTN, and costained for endogenous TFRC. Scale bar: 10 μm. Magnified areas are shown as insets. (B) The graph shows correlation coefficient of colocalization between GFP-LC3B and endogenous TFRC in WT or M98K-OPTN expressing cells. (C) Autophagy induction increases the localization of TFRC to GFP-LC3B positive structures. Panels show representative images of RGC-5 cells expressing GFP-LC3B with cellular TFRC grown in serum-containing media (UT) or in amino acid- and serum-free media (EBSS) for 3 h. Magnified areas are shown as insets. (D) Colocalization of cellular TFRC with endogenous LC3 in chloroquine (25 μM for 8 h) treated cells. (E) Colocalization of endogenous OPTN with GFP-LC3B-positive structures. Scale bar: 10 μm. (F) Induction of autophagy reduces TFRC levels. RGC-5 cells were either kept untreated or treated with EBSS for the indicated time. Western blots show levels of TFRC and endogenous OPTN. GAPDH is a loading control. The numbers below TFRC and OPTN blots indicate relative TFRC and OPTN levels after normalization. (G) Induction of autophagy enhances M98K-mediated cell death in a time-dependent manner. RGC-5 cells transfected with GFP-M98K were either kept untreated or were treated with EBSS for 2 h after indicated time of expression. Data represent cell death mediated by GFP-M98K in untreated and EBSS treated conditions. n = 3 experiments. ***p < 0.001. **p < 0.01. (H) Western blot shows TFRC levels in control and M98K-infected RGC-5 cells in untreated and EBSS treated conditions. The numbers below TFRC blot indicate relative TFRC levels after normalization.

Figure 6. Restoration of TFRC levels reduces M98K-induced cell death. (A) Effect of TFRC coexpression on M98K-induced cell death. Data represent mean ± s.d of percentage of expressing cells showing apoptotic morphology upon 32 h of expression. n = 6, ***p < 0.001. (B) Western blot shows M98K protein levels in the presence or absence of coexpressed TFRC. (C) Quantitation of cell death induced by M98K in the presence of chloroquine in RGC-5 cells. n = 6, **p < 0.001. CQ, chloroquine. (D) Quantitation of cell death induced by M98K in the presence of FAC, an iron supplement. n = 6 experiments, ***p < 0.001. FAC, ferric ammonium citrate; UT, untreated. (E) Quantitation of cell death induced by M98K with 4 h of EBSS treatment with or without FAC (30 μM, 24 h). ***p < 0.001.

Figure 7. TFRC degradation and cell death induced by M98K are dependent on its UBD. (A) Quantitation of cell death induced by WT, M98K and M98K-D474N (ubiquitin binding-defective mutant of M98K) in RGC-5 cells. n = 6, ***p < 0.001. (B) Representative confocal images showing colocalization of M98K and M98K-D474N mutants with TFRC. Scale bar: 10 μm. (C) Representative confocal images showing colocalization of M98K and M98K-D474N mutants with GFP-LC3B. Scale bar: 10 μm. (D) Western blots show levels of TFRC in RGC-5 cells after adenovirus-mediated expression of control (con), WT, M98K and M98K-D474N. GAPDH was used as a loading control.

Figure 8. Role of RAB12 in M98K-induced death of RGC-5 cells. (A) Western blot shows knockdown of HA-RAB12 by Rab12 shRNAs, ShA and ShB in RGC-5 cells. Tubulin was used as a loading control. Con-Sh, Control shRNA; TUBA, α-tubulin. (B) Knockdown of Rab12 reduced M98K-mediated cell death. Data represent quantitation of cell death induced by M98K with or without knockdown of Rab12. n = 6, ***p < 0.001. (C) Western blots show endogenous levels of TFRC and LC3-II after knockdown of Rab12 for 48 h by Rab12 ShB and ShA. GAPDH was used as a loading control. (D) Effect of Rab12 knockdown on TFRC degradation by M98K. RGC-5 cells were transfected with either Con-Sh or Rab12 ShB or ShA and after 24 h of knockdown, were infected with either WT or M98K adenoviruses for 24 h. Cell lysates were subjected to western blotting. GAPDH was used as a loading control. (E) Left panel, Representative confocal images showing colocalization of M98K and WT with HA-RAB12. Magnified areas are shown as insets. Scale bar: 10 μm. The graph on the right represents correlation coefficient of colocalization of WT or M98K with RAB12. n = 30 cells, ***p < 0.001. (F) Colocalization of endogenous OPTN and GFP-RAB12. Scale bar: 10 μm. (G) Interaction of OPTN with overexpressed RAB12. Cell lysates of RGC-5 cells expressing WT or M98K with HA-RAB12 were immunoprecipitated using HA (IP) or control antibody (C) and subjected to western blotting with HA and OPTN antibodies. WCL, whole cell lysates. (H) Colocalization of HA-RAB12 with GFP-LC3B positive structures. (I) Colocalization of GFP-RAB12 with endogenous LC3 in chloroquine (25 μM for 8 h) treated cells. Scale bar: 10 μm.

Figure 9. Knockdown of Rab12 decreases autolysosome formation. Representative confocal images showing number of autophagosomes (yellow dots) and autolysosomes (red dots only) upon Rab12 knockdown by control shRNA (Con-Sh) or Rab12 shRNA, shB in untreated conditions (A) and in amino acid and serum-starved conditions (B). RGC-5 cells were transfected with either Con-Sh or Rab12-ShB along with mCherry-GFP-LC3B construct for 32 h and kept untreated or in EBSS media for 3 h. Quantification of number of autophagosomes (yellow dots) and autolysosomes (red dots only) per cell in control or Rab12 knockdown cells in untreated (C) and in amino acid- and serum-starved conditions (D). n = 50 cells, ***p < 0.001.

Figure 10. Model depicting how M98K variant of OPTN alters cellular transferrin receptor dynamics leading to autophagic death in RGC-5 cells. Under steady-state conditions, membrane TFRC bound to transferrin-Fe complex is internalized by endocytosis and recycled back to the plasma membrane after intracellular release of Fe. A proportion of TFRC is degraded by lysosomes. OPTN interacts with LC3 and its role as an autophagy receptor has been proposed. In the presence of M98K-OPTN, cellular TFRC turnover dynamics is altered as this variant interacts more strongly with TFRC, and enhances its localization in autophagosomes. M98K-OPTN recruits RAB12, a GTPase involved in TFRC degradation, more efficiently. As a consequence, cellular TFRC is rapidly degraded in lysosomes, rather than being recycled to the plasma membrane through recycling endosomes. Reduced cellular TFRC level causes death of RGC-5 cells that can be prevented by expression of TFRC or inhibition of TFRC degradation or iron supplementation.