Increased ER–mitochondria tethering promotes axon regeneration

Significance

Small organelles such as endoplasmic reticulum (ER) and mitochondria play key roles in cellular functions and survival. Furthermore, physical tethering between ER–mitochondria allows communication between these 2 organelles, and proteins involved in the contact sites have been identified in various cells. However, physiological significances of ER–mitochondria contacts in axons are unknown. Here, we discover that Grp75 mRNA is locally translated in injured axonal tips. Up-regulation of Grp75 promotes ER–mitochondria tethering and enhances axon regeneration in vitro. Increased levels ofGrp75 in dorsal root ganglion neurons facilitates axon regeneration and functional recovery of animals with nerve injury. These results imply that overexpression of Grp75 may provide a therapeutic strategy to treat and enhance axonal regeneration following nerve injury.

Abstract

Translocation of the endoplasmic reticulum (ER) and mitochondria to the site of axon injury has been shown to facilitate axonal regeneration; however, the existence and physiological importance of ER–mitochondria tethering in the injured axons are unknown. Here, we show that a protein linking ER to mitochondria, the glucose regulated protein 75 (Grp75), is locally translated at axon injury site following axotomy, and that overexpression of Grp75 in primary neurons increases ER–mitochondria tethering to promote regrowth of injured axons. We find that increased ER–mitochondria tethering elevates mitochondrial Ca²⁺ and enhances ATP generation, thereby promoting regrowth of injured axons. Furthermore, intrathecal delivery of lentiviral vector encoding Grp75 to an animal with sciatic nerve crush injury enhances axonal regeneration and functional recovery. Together, our findings suggest that increased ER–mitochondria tethering at axonal injury sites may provide a therapeutic strategy for axon regeneration.

The endoplasmic reticulum (ER) and mitochondria have their own distinct functions in the cell, but the ER and mitochondria form contacts at the mitochondrial-associated membranes (MAMs), which allows these organelles to communicate and perform independent functions (1–3). MAMs participate in regulation of cellular signaling and metabolism, such as Ca²⁺ homeostasis, lipid exchange, mitochondrial energy generation, and apoptosis (1, 2, 4–6). Furthermore, ER–mitochondria contacts mediate mitochondrial fission as well as neural stem cell development (3, 7). While a number of ER–mitochondria tethering proteins have been identified and the role of ER–mitochondria contacts have been actively studied in various systems, the presence and functional significance of ER–mitochondria contacts in axons have not been examined.

Axons of the mammalian central nervous system lack the ability to regenerate following injury, while axons of the mammalian peripheral nervous system show limited regeneration capacity (8). When axonal damage occurs, various cellular responses are initiated to repair damaged axons. The level of Ca²⁺ has been shown to increase upon axonal injury, which then activates the retrograde transport of signaling molecules to induce gene expression in the neuronal cell body (8, 9). Furthermore, local protein synthesis of regeneration-associated genes in injured axons provides materials for resealing the ruptured membrane and for the formation of new growth cone (10, 11). The cytoskeleton and membranes also reorganize upon axon injury (12). In addition, relocation of organelles in injured axons occurs. Translocations of the ER and mitochondria to the injured axon tip have been reported (13–15); however, it is not known whether accumulated ER and mitochondria form physical contacts or affect axon regeneration.

Glucose regulated protein 75 (Grp75) is a protein found at the interface of MAMs that links the ER to mitochondria by simultaneously interacting with the inositol 1,4,5-trisphosphate receptor (IP₃R) in the ER and voltage-dependent anion channel 1 (VDAC1) in the outer membrane of mitochondria (16). Thus, Grp75 regulates Ca²⁺ shuttling from the ER to mitochondria (2, 16). A proteomics study identified Grp75 as one of the proteins synthesized in injured axons (11). Taken together, we hypothesized that axonal injury induces local translation of Grp75, which then increases ER–mitochondria contacts. We report that axonal injury indeed increases ER–mitochondria tethering in axons, and this promotes axon regrowth and functional regeneration. We further demonstrate that local translation of Grp75 upon axonal injury is a mechanism contributing to increased ER–mitochondria tethering and axonal regeneration.

Results and Discussion

Grp75 Is Locally Translated.

We found that upon axonal injury, the ER and mitochondria translocate into injured axon sites (Fig. 1 A–C), consistent with previous reports (13–15). Furthermore, the ER and mitochondria showed a high degree of colocalization (Fig. 1D), raising the possibility that the ER and mitochondria form physical contacts in injured axons. As a first step to understanding whether the ER forms physical contacts with mitochondria in the axon, we monitored the level of Grp75 before and after axonal injury. Grp75 is a protein found at the interface of MAMs and links the ER to mitochondria by simultaneously interacting with IP₃R in the ER and VDAC1 in the outer membrane of mitochondria (16). We found that the Grp75 mRNA transcript is present both in the cell body and axon of primary hippocampal neurons, and there was no glial cell contamination in our axonal preparations by examining the presence of GFAP for astrocytes and myelin basic protein for oligodendrocytes (SI Appendix, Fig. S1A). Furthermore, Grp75 protein in axon increased significantly after axotomy, while Grp75 in the cell body remained unchanged (SI Appendix, Fig. S1 C and D).

Fig. 1.

Grp75 is locally translated in injured axons and interacts with IP₃R1 and VDAC1. (A–C) ER and mitochondria were accumulated in the injured axon tip. Sec61β tagged with EGFP was used to label the ER, and mCherry with mitochondria targeting sequence was used to mark mitochondria. Fluorescent intensity was measured from the axon tip to a 20-µm distal segment before and after axonal injury. Arrowhead indicates the injured tip. (Scale bar, 20 μm.) Five axons for the ER and 6 axons for mitochondria were used. Fluorescent intensity was compared between paired samples and tested for statistical significance by paired t test: *P < 0.05 (B) and *P < 0.01 (C). (D) Mander’s correlation coefficient showed that colocalization between ER and mitochondria increased after axotomy. Statistical significance was measured by paired t test, *P < 0.05. (E) Pseudocolor images of the green fluorescent Dendra-2 in the axon tip after axotomy. Images were taken at 10-min intervals, but shown here with 30-min intervals. Red arrowhead indicates incision site of axon. (Scale bars, 10 μm.) (F) Change in fluorescence intensity was analyzed in the injured axon tip. Primary hippocampal neurons at DIV 4 were used. n = 8 axons in each condition, *P < 0.01. (G) Schematic drawing that showed the BiFC assay. (H) Cytoplasmic Grp75 links the ER and mitochondria in axons. Axons expressing ETS-IP₃R1[LBD]-VN and VC-∆MTS-Grp75 (IP₃R1[LBD]/Grp75) or VDAC1-VN and VC-∆MTS-Grp75 (VDAC1/Grp75) showed BiFC fluorescence. (Scale bar, 10 μm.)

Studies have shown that local translation of mRNA transcripts occurs in injured axons upon axonal injury (8, 10, 17), and inhibition of local protein synthesis prevents axonal regeneration (8, 17). This prompted us to examine whether Grp75 is locally translated upon axon injury. To this end, we generated a vector that contains the photo-switchable Dendra-2 protein fused with the 5′UTR and 3′UTR of Grp75 mRNA together with 2 copies of the palmitoylation sequence (5′UTR of Grp75-Dendra2-3′UTR of Grp75) (18). The existing green fluorescent Dendra-2 protein at the tip of the injured axon was irreversibly photo-converted to red fluorescent Dendra-2 protein using UV illumination, and newly synthesized Dendra-2 protein (green) was quantified by analyzing time-lapse images taken every 10 min for 90 min after axotomy. As seen in Fig. 1 E and F, newly synthesized green fluorescent Dendra-2 was detected at the injured axonal tip and its level increased gradually over time. However, addition of anisomycin, a translation inhibitor, blocked the synthesis of green fluorescent Dendra-2, suggesting that axotomy triggers local protein synthesis of Grp75 at the injured axon tip. As control, Dendra-2 containing UTRs of GAPDH was also monitored, but axotomy did not trigger any change in its levels following axonal insult. It is important to note that only the injured tip displayed a significant increase in green fluorescence and not the entire length of the axon (SI Appendix, Fig. S1 E and F), suggesting that the increased in Dendra-2 at the tip is not likely due to transport from the cell body. Together, these results indicate that Grp75 mRNA is locally translated at the axonal tip after axotomy.

Grp75 Links IP₃R1 and VDAC1.

The local increase in Grp75 protein further suggests increased ER–mitochondria tethering in axons. We thus first devised a strategy to visualize Grp75 interaction with IP₃R1 or VDAC1 using biomolecular fluorescence complementation (BiFC) (19) (Fig. 1G). Grp75 contains a mitochondrial targeting signal (MTS), and has been shown to act both as a linker in the cytoplasm to couple the ER to mitochondria (4, 16), and as a chaperon protein inside of mitochondria (20). Indeed, when we expressed Grp75 tagged with Myc (Grp75-Myc), it distributed both in the cytoplasm and mitochondria of NIH/3T3 cells (SI Appendix, Fig. S2). However, deletion of the MTS (Myc-∆MTS-Grp75) led to localization of Myc-∆MTS-Grp75 in the cytoplasm, while replacing the MTS with the mitochondrial protein Cox8a [MTS^(Cox8a)-Myc-∆MTS-Grp75] resulted in exclusive localization of MTS^(Cox8a)-Myc-∆MTS-Grp75 in the mitochondria (SI Appendix, Fig. S2). We took advantage of the cytoplasmic distribution of ∆MTS-Grp75 to monitor ER–mitochondria tethering, since Grp75 distribution in the cytoplasm is required for its function as a linker of ER–mitochondria. For BiFC, we generated truncated fluorescent protein mVenus: VN (mVenus amino acids #1 to 172) and VC (mVenus amino acids #155 to 238) (Fig. 1 G and H and SI Appendix, Fig. S3A). Each of the two truncated proteins showed no fluorescence unless they are in close contact, thus allowing the individual mVenus fragments to form its native configuration.

To monitor VDAC1 and Grp75 interaction, we fused the VN fragment to the C terminus of VDAC1 (VDAC1-VN) and the VN fragments to the N terminus of ∆MTS-Grp75 (VC-∆MTS-Grp75). To monitor IP₃R1 and Grp75 interaction, we also tagged the ligand binding domain of IP₃R1 (LBD, amino acids #224 to 605) with VN in the C terminus and added an ER targeting sequence (ETS) to the N terminus (ETS-IP₃R1[LBD]-VN) (16). Coupled expression of VDAC1-VN and VC-∆MTS-Grp75 or VC-∆MTS-Grp75 and ETS-IP₃R1[LBD]-VN clearly displayed fluorescent BiFC signals in axons (Fig. 1H) and the cell body (SI Appendix, Fig. S3C). As a control, we also tested whether fluorescent signals can be detected from neurons containing 1) individual expression of VC-∆MTS-Grp75, VDAC1-VN, or ETS-IP₃R1[LBD]-VN; 2) coexpression of VC-∆MTS-Grp75 and VDAC1 deleting a part of the C terminus (VDAC1-∆265-283-VN); and 3) VC-∆MTS-Grp75 and the suppressor domain of IP₃R1(amino acids #1 to 223) (ETS-IP₃R1[SD]-VN) (SI Appendix, Fig. S3 B and C). None of these constructs displayed BiFC signals. Together, these results suggest that Grp75 indeed interacts with both IP₃R1 and VDAC1 in axons and the cell body of primary hippocampal neurons. Furthermore, BiFC signals coming from interaction between Grp75 and IP₃R1 or between Grp75 and VDAC1 increased in injured axons (Fig. 2 A and B), indicating that ER–mitochondria tethering is enhanced after axonal insults.

Fig. 2.

Overexpression of Grp75 facilitates regrowth of injured axon. (A and B) BiFC images showed that interaction between Grp75 and IP₃R1 or between Grp75 and VDAC1 in injured axons was increased 2 h after axon injury. BiFC signals were measured from the injured axon tip to the 20-µm distal segment. n = 7 axons, *P < 0.05. Arrowheads indicate injured axonal tip. (Scale bars, 10 µm.) (C and D) Three days after axotomy, axon length was measured in each condition. For axotomy, vacuum aspiration was applied to the exit border of microgrooves, which severs any axons grown to the axonal compartment in the microfluidic device. (Scale bar, 200 μm.) Total axon numbers used for calculation were 294 for control, 680 for Grp75, 943 for ∆MTS-Grp75, 613 for MTS^(Cox8a)-∆MTS-Grp75. n = 4 independent experiments, *P < 0.0001; n.s., not significant.

Overexpression of Grp75 Promotes Axon Regeneration.

What is the physiological significance of this increased ER–mitochondria tethering? We hypothesized that up-regulation of Grp75 and increased ER–mitochondria contacts may promote regrowth of injured axons. To test this hypothesis, we first transfected primary hippocampal neurons with Grp75, Grp75 without MTS (∆MTS-Grp75), so that it can act as a linker in the cytoplasm, and Grp75 with MTS replaced with Cox8a’s MTS so that it is localized in mitochondria [MTS^(Cox8a)-Myc-∆MTS-Grp75] (SI Appendix, Fig. S4A). Of transfection efficiency, 65 to 80% was achieved (SI Appendix, Fig. S4B). These neurons were cultured in a microfluidic device where neurons are plated on one side of the device (21) (SI Appendix, Fig. S5A). Only axons can grow through the microgrooves and reach to the other compartment, thus allowing differentiation of axons from cell bodies. Axon length of neurons expressing different types of Grp75 was measured at day in vitro (DIV) 4, and all showed similar length when stained with Tau-1 antibody (SI Appendix, Fig. S5B). In contrast, reduction of Grp75 by Grp75 siRNA transfection to neurons hindered normal axon development (SI Appendix, Fig. S5 C and D). Furthermore, we found that while reduction of Grp75 by Grp75 siRNA has a mild effect on normal axon development, it significantly inhibited axon regrowth after axonal injury (SI Appendix, Fig. S5E).

Next, to assess if overexpression of Grp75 promotes regrowth of axons following axonal injury, vacuum aspiration was applied to the exit borders of microgrooves, which severs the axons grown to the axonal compartment in the microfluidic device (SI Appendix, Fig. S5A). We performed axotomy at DIV 4 and continued to culture the injured neurons for 3 additional days (Fig. 2 C and D). A striking increase in axonal regrowth was detected in neurons with ∆MTS-Grp75 overexpression, with axon length almost 2 times longer than that of control. The length of axons cultured for 7 d without damage is similar to that of injured axons overexpressing ∆MTS-Grp75 (SI Appendix, Fig. S5F). Axons containing Grp75 with its own MTS also regenerated, but the length only increased about one-third. It is likely that the difference between Grp75 with or without MTS on axonal regeneration capability is due to the amount of Grp75 located in cytoplasm. Indeed, when we overexpressed Grp75 construct with exclusive localization in the mitochondria [MTS^(Cox8a)-Myc-∆MTS-Grp75], it failed to enhance axon regrowth following axotomy (Fig. 2C).

Next, we tested whether overexpressing ∆MTS-Grp75 is effective in promoting growth of injured axons at a later time point in neuron culture. To this end, we transfected neurons with ∆MTS-Grp75 at DIV 10, performed axotomy at DIV 12, and then monitored axon growth after another 6 d in culture. The results clearly showed that ∆MTS-Grp75 overexpression still effectively induced regrowth of injured axons even at a more mature stage (SI Appendix, Fig. S5G). Furthermore, we tested whether ∆MTS-Grp75 overexpression postinjury can still enhance axon regeneration. We performed axotomy at DIV 4, and then waited for 3 d before ∆MTS-Grp75 transfection. These neurons were cultured for another 3 d before measuring the axon length measurement. We discovered that delayed Grp75 expression still enhanced growth of previously injured axons (SI Appendix, Fig. S5H), suggesting that ∆MTS-Grp75 overexpression can promote axon regeneration regardless of timing of the treatment before or after axon damage. Together, these results indicate that an increase in cytoplasmic Grp75, and hence an increased ER–mitochondria tethering, promote axonal regrowth following injury.

In addition to enhancing the regrowth of axons of injured hippocampal neurons, we tested whether Grp75 overexpression modulates the regrowth of dorsal root ganglion (DRG) axons following injury. In SI Appendix, Fig. S6, we confirmed: (i) the presence of Grp75 mRNA transcript in axons; (ii) obstruction of axon development by Grp75 siRNA transfection; and (iii) promotion of axon regrowth after axotomy by overexpression of Grp75 without MTS. Together, these results verify that Grp75 located in cytoplasm promotes regrowth of injured axon in DRG primary neurons as well.

Grp75 Overexpression Increases ATP Production in Injured Axons.

Next, we investigated how enhanced ER–mitochondria contacts trigger regrowth of the injured axon tip. As ER–mitochondria tethering had been shown to play critical roles in modulating Ca²⁺ homeostasis and ATP production (2), we tested whether altered Ca²⁺ level or ATP production underlies enhanced axonal regrowth. First, we monitored cytoplasmic and mitochondrial Ca²⁺ level in the tip of axons. To this end, we transfected the genetically encoded Ca²⁺ indicators for cytoplasm and mitochondria: GEM-GECO and mito-case12, respectively (22, 23) (Fig. 3 A and B). When insult was applied to the axonal tip, both control axons and axons overexpressing ∆MTS-Grp75 showed increased Ca²⁺ level in cytoplasm, which is consistent with previous reports showing the increase in Ca²⁺ concentration after axotomy (9, 12). However, mitochondrial Ca²⁺ level was not changed in control axons, while axons overexpressing ∆MTS-Grp75 significantly increased mitochondrial Ca²⁺ level after axonal injury. Also, axons overexpressing MTS^Cox8a-∆MTS-Grp75 showed increased cytosolic Ca²⁺ level without altering mitochondrial Ca²⁺ level (SI Appendix, Fig. S7 A and B). These results suggest that increased ER–mitochondria tethering due to Grp75 overexpression does not alter cytoplasmic Ca²⁺ level, but facilitates Ca²⁺ influx to mitochondria upon axon injury. Furthermore, we assessed the ATP level in the injured axonal tip when ∆MTS-Grp75 was overexpressed. To measure the ATP level in axons, we used genetically encoded FRET-based ATP indicators: ATeam1.03 for cytoplasm and mito-ATeam1.03 for mitochondria (24) (Fig. 3 C and D). Upon axonal injury, local ATP level in the cytoplasm and mitochondria was increased in the axonal tip having ∆MTS-Grp75. Our results suggest that increasing Grp75 enhances ER and mitochondria tethering in the injured axonal tips, leading to increase Ca²⁺ transfer from the ER to mitochondria, which then activates the TCA cycle and the enzymes in electron transport chain to increase ATP generation and promote axon regeneration.

Fig. 3.

Grp75 overexpression elevates mitochondrial Ca²⁺ level and ATP generation in injured axons. (A) Ca²⁺ levels in the injured axon tip were measured using genetically encoded Ca²⁺ indicator, GEM-GECO; n = 6 and *P < 0.05 (between 0 and 120 min). (Scale bar, 10 μm.) (B) Analysis of Ca²⁺ influx to mitochondria using mito-Case12 showed that mitochondrial Ca²⁺ was increased in injured axons containing ∆MTS-Grp75. Fluorescent intensity was measured from the injured axon tip to a 20-µm distal segment; n = 7 and *P < 0.05. (Scale bar, 10 μm.) (C and D) FRET signals indicating increased ATP were found in cytoplasm (C) and mitochondria (D) of injured axons having ∆MTS-Grp75. Arrowheads indicate the injured axon tip. Quantification of ATP in the injured axon tip overexpressing ∆MTS-Grp75 by using genetically encoded FRET-based ATP indicators: ATeam1.03 for cytoplasm (C) and mito-ATeam1.03 for mitochondria (D). n = 5 (C), n = 7 (D), *P < 0.05. (Scale bar, 10 μm.)

To verify that Ca²⁺ release from the ER can regulate the regrowth of injured axons, we blocked IP₃R1 by adding a selective and membrane-permeable inhibitor, xestospongin C (Xes C) (25), or stimulated IP₃R1 by treating neurons with IP₃R1 agonist, adenophostin A (Ad A) (26) (SI Appendix, Fig. S7 C and D). Injured axons treated with Xes C showed decreased axon regrowth after axotomy compared with that of control. In contrast, axons treated with Ad A after axotomy profoundly enhanced axonal regrowth. We also assessed levels of mitochondrial Ca²⁺ and ATP in injured axons that were treated with these drugs (SI Appendix, Fig. S7 E and F). As anticipated, we observed Ca²⁺ and ATP levels in mitochondria were reduced with Xes C treatment, while Ad A elevated mitochondrial Ca²⁺ and ATP levels. Together, these results further indicate that Ca²⁺ transfer from the ER to mitochondria plays a key role in axonal regeneration. We also confirmed whether Grp75 expression and the effect of Ad A share the same pathways on regrowth of injured axons (SI Appendix, Fig. S7 C and D). Indeed, Grp75 reduction by Grp75 siRNA diminished the effect of Ad A on the capability of injured axon regrowth. Taken together, these results indicate that Ca²⁺ transfer from the ER to mitochondria plays a key role in axonal regeneration.

Grp75 Overexpression Restores Sciatic Nerve Injury in Mice.

Finally, we tested whether overexpression of Grp75 enhances axonal regeneration following injury in vivo by monitoring both axon regrowth and functional recovery of animals following sciatic nerve injury (Fig. 4 and SI Appendix, Fig. S8). Sciatic nerve crush injury was induced in mice with or without lentiviral delivery of Grp75. Two weeks before sciatic nerve crush, we performed intrathecal delivery of lentivirus containing Myc-∆MTS-Grp75 (Fig. 4A). Three days after the crush, we then assessed the regrowth of injured axons by staining with GAP43, a marker for axon regeneration, on cryostat sectioned sciatic nerve (Fig. 4 B and C). Compared with control, a significantly increased number of GAP43-positive axons was detected distal to the crush site in mice injected with Myc-∆MTS-Grp75. This result confirms that Grp75 overexpression promotes axon regeneration in animals following injured axons. To further verify axon regeneration, we performed retrograde labeling of DRG neurons with a tracing dye, DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) that was injected 10-mm distal from the injury site (27). DRG sections of mice overexpressing Grp75 showed increased the number of neurons that labeled retrogradely with DiI compared with control (Fig. 4 D and E). This result indicates that Grp75 overexpression restores damaged axons through axon regeneration.

Fig. 4.

Grp75 overexpression restores sciatic nerve injury in mice by facilitating axon regeneration. (A) Cryostat section of injured sciatic nerve containing a control lentiviral vector with Myc tag or a vector expressing Myc-∆MTS-Grp75. Arrowheads indicate the crush site. (Scale bar, 1 mm.) (B) Regrowth of injured axons was stained by GAP43 antibody, a marker for axon regeneration. Sciatic nerve section was prepared 3 d after sciatic nerve crush. Arrowheads indicate the crush site. (Scale bar, 1 mm.) (C) Quantification of the number of GAP43⁺ axons moved further distal to the crush site. Nine animals for control and 5 animals having ∆MTS-Grp75, *P < 0.01. (D) Axon regeneration was examined by retrograde labeling of DRG neurons with tracing dye, DiI that was injected into the sciatic nerve. (Scale bar, 200 μm.) (E) Quantification of DiI⁺ neurons in DRG sections. Three mice were used for each condition; *P < 0.0001 and **P < 0.01; n.s., not significant. (F) Recovery of sensory function was assessed by performing Von Frey test, *P < 0.001, n = 6. (G) Functional recovery from cold allodynia was tested by performing cold plate test, *P < 0.001, n = 13. (H) Motor recovery was analyzed by using a treadmill; *P < 0.01, n = 6. (I and J) Morphological recovery was measured by toe-spreading geometry of the injured hind paw (I), and sciatic functional index was determined every 3 d postinjury; *P < 0.05, n = 5.

We next evaluated functional recovery of the animal. First, we investigated sensory function by performing a Von Frey test and cold-plate test that can measure the pain response (Fig. 4 F and G). Control mice with sciatic nerve crush injury exhibited increased sensitivity to manual Von Frey hair application and cold plate. In contrast, mice injected with lentiviral vector containing Myc-∆MTS-Grp75 showed gradual recovery from mechanical and cold allodynia. To assess motor recovery after sciatic nerve injury, we then tested mice on the treadmill (Fig. 4H). Mice with Grp75 overexpression showed increased duration on the treadmill at day 9 after the nerve injury. We also evaluated a morphological parameter using sciatic functional index, which measures toe-spreading geometry of the injured hind paw compared with the normal contralateral paw (28) (Fig. 4 I and J). Mice with Grp75 overexpression showed a significantly faster rate of recovery, with almost fully restored sciatic functional index 15 d after injury.

When axons are injured, the ER and mitochondria translocate to the injured site to reseal the membrane and to provide ATP for axon regeneration (13, 15); however, whether physical tethering of ER–mitochondria occurs in injured axons and whether ER–mitochondria contacts contribute to axon regeneration are not known. In this study, we discovered that Grp75 is locally translated in injured axon, and overexpression of Grp75 promotes axon regeneration by increasing ER and mitochondria contact in an injured axon tip. The increased ER–mitochondria contacts enhance Ca²⁺ transfer from the ER to mitochondria, thereby elevating ATP generation required for axon regeneration. Grp75 is known as a mitochondrial protein containing a mitochondrial targeting sequence, but also found at the interface of MAMs. It is interesting to note that our results reveal that cytoplasmic and mitochondrial Grp75 play different roles in axon regeneration. We found that when Grp75 is solely located in mitochondria [MTS^(Cox8a)-Myc-∆MTS-Grp75], axon length is similar to that of control, suggesting that Grp75 in mitochondria contributes little to axonal regeneration. On the other hand, expression of Grp75 without a mitochondrial targeting sequence (∆MTS-Grp75) significantly increased axon length following injury (Fig. 2 C and D), indicating that cytoplasmic Grp75 (∆MTS-Grp75), rather than mitochondrial Grp75, plays a key role in axon regeneration rather than mitochondrial Grp75.

The role of Grp75 in regulating growth and survival is not well understood. There are several reports indicating that beneficial effects of Grp75 overexpression under different stresses, such as metabolic stress, glucose deprivation, cytotoxins, or oxidative damage (29–31). However, a recent study by Honrath et al. (4) showed that Grp75 is critical in ER–mitochondria coupling and oxidative stress-mediated cell death. Moreover, Grp75 overexpression in HT22 neuronal cells increases sensitivity to glutamate-induced oxidative cell death. Our findings are consistent with the beneficial effects of Grp75 by showing enhanced axon regeneration when ∆MTS-Grp75 is expressed in injured axons. The discrepancy between different studies may come from the distribution of Grp75 in cells. It is interesting to note that Grp75 in the cytoplasm and mitochondria plays opposite roles in induction of apoptosis (32, 33). Interaction between Grp75 and p53 in the cytoplasm inhibits translocation of p53 to the nucleus or mitochondria, which prevents induction of apoptosis, while mitochondrial Grp75 promotes translocation of p53 to the mitochondria and triggers apoptosis under oxidative-stress conditions. Our findings also support that Grp 75 in different cellular compartments may have different roles. It will thus be interesting to further dissect the functions of cytoplasmic 75 or mitochondrial Grp75 in regulating different cellular processes in the future.

In summary, we demonstrate that cytoplasmic Grp75 links IP₃R1 in the ER and VDAC1 in mitochondria. Furthermore, Grp75 is locally translated at the injured axonal tips, and overexpression of Grp75 increases ER–mitochondria contacts to provide the energy need to regrow injured axons. Finally, we found that an increased level of Grp75 in DRG neurons promotes axon regeneration and functional recovery of animals with nerve injury. Together, our results raise the exciting possibility that overexpression of cytoplasmic Grp75 may be a therapeutic strategy to treat and enhance axonal regeneration following nerve injury.

Materials and Methods

Animals were used in accordance with protocols approved by the Animal Care and Use Committees of the Ulsan National Institute of Science and Technology. The C57BL/6 mouse strain was purchased from Hyochang Science. Animals were housed in a 12-h light- and dark-cycle cage room. For behavioral studies, 12-wk-old male mice were used. Details of experimental procedures are provided in SI Appendix.