Increased ER–mitochondria tethering promotesaxon regenerationSoyeon Leea,b, Wei Wanga,b,1, Jinyeon Hwangc, Uk Namgungc, and Kyung-Tai Mina,b,2

aDepartment of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology, 44919 Ulsan, South Korea; bNationalCreative Research Initiative Center for Proteostasis, Ulsan National Institute of Science and Technology, 44919 Ulsan, South Korea; and cDepartment ofOriental Medicine, Daejeon University, 34520 Daejeon, South Korea

Edited by Hee-Sup Shin, Institute for Basic Science, Daejeon, South Korea, and approved June 27, 2019 (received for review November 1, 2018)

Translocation of the endoplasmic reticulum (ER) and mitochondriato the site of axon injury has been shown to facilitate axonalregeneration; however, the existence and physiological importanceof ER–mitochondria tethering in the injured axons are unknown.Here, we show that a protein linking ER to mitochondria, the glu-cose regulated protein 75 (Grp75), is locally translated at axon injurysite following axotomy, and that overexpression of Grp75 in pri-mary neurons increases ER–mitochondria tethering to promoteregrowth of injured axons. We find that increased ER–mitochondriatethering elevates mitochondrial Ca2+ and enhances ATP genera-tion, thereby promoting regrowth of injured axons. Furthermore,intrathecal delivery of lentiviral vector encoding Grp75 to an animalwith sciatic nerve crush injury enhances axonal regeneration andfunctional recovery. Together, our findings suggest that increasedER–mitochondria tethering at axonal injury sites may provide a ther-apeutic strategy for axon regeneration.

axon regeneration | mitochondria | ER

The endoplasmic reticulum (ER) and mitochondria have theirown distinct functions in the cell, but the ER and mito-

chondria form contacts at the mitochondrial-associated mem-branes (MAMs), which allows these organelles to communicateand perform independent functions (1–3). MAMs participate inregulation of cellular signaling and metabolism, such as Ca2+

homeostasis, lipid exchange, mitochondrial energy generation,and apoptosis (1, 2, 4–6). Furthermore, ER–mitochondria con-tacts mediate mitochondrial fission as well as neural stem celldevelopment (3, 7). While a number of ER–mitochondria teth-ering proteins have been identified and the role of ER–mito-chondria contacts have been actively studied in various systems,the presence and functional significance of ER–mitochondriacontacts in axons have not been examined.Axons of the mammalian central nervous system lack the ability

to regenerate following injury, while axons of the mammalianperipheral nervous system show limited regeneration capacity (8).When axonal damage occurs, various cellular responses are initi-ated to repair damaged axons. The level of Ca2+ has been shownto increase upon axonal injury, which then activates the retrogradetransport of signaling molecules to induce gene expression in theneuronal cell body (8, 9). Furthermore, local protein synthesis ofregeneration-associated genes in injured axons provides materialsfor resealing the ruptured membrane and for the formation of newgrowth cone (10, 11). The cytoskeleton and membranes also reor-ganize upon axon injury (12). In addition, relocation of organellesin injured axons occurs. Translocations of the ER and mitochondriato the injured axon tip have been reported (13–15); however, it isnot known whether accumulated ER andmitochondria form physicalcontacts 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 si-multaneously interacting with the inositol 1,4,5-trisphosphatereceptor (IP3R) in the ER and voltage-dependent anion channel1 (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 proteinssynthesized in injured axons (11). Taken together, we hypothe-sized that axonal injury induces local translation of Grp75, whichthen increases ER–mitochondria contacts. We report that axonalinjury indeed increases ER–mitochondria tethering in axons, andthis promotes axon regrowth and functional regeneration. Wefurther demonstrate that local translation of Grp75 upon axonalinjury is a mechanism contributing to increased ER–mitochon-dria tethering and axonal regeneration.

Results and DiscussionGrp75 Is Locally Translated.We found that upon axonal injury, theER and mitochondria translocate into injured axon sites (Fig. 1A–C), consistent with previous reports (13–15). Furthermore, theER and mitochondria showed a high degree of colocalization(Fig. 1D), raising the possibility that the ER and mitochondriaform physical contacts in injured axons. As a first step to under-standing whether the ER forms physical contacts with mitochon-dria in the axon, we monitored the level of Grp75 before and afteraxonal injury. Grp75 is a protein found at the interface of MAMsand links the ER to mitochondria by simultaneously interactingwith IP3R in the ER and VDAC1 in the outer membrane of mi-tochondria (16). We found that the Grp75 mRNA transcript ispresent both in the cell body and axon of primary hippocampalneurons, and there was no glial cell contamination in our axonal

Significance

Small organelles such as endoplasmic reticulum (ER) and mito-chondria play key roles in cellular functions and survival. Fur-thermore, physical tethering between ER–mitochondria allowscommunication between these 2 organelles, and proteins in-volved in the contact sites have been identified in various cells.However, physiological significances of ER–mitochondria con-tacts in axons are unknown. Here, we discover that Grp75 mRNAis locally translated in injured axonal tips. Up-regulation ofGrp75 promotes ER–mitochondria tethering and enhances axonregeneration in vitro. Increased levels ofGrp75 in dorsal rootganglion neurons facilitates axon regeneration and functionalrecovery of animals with nerve injury. These results imply thatoverexpression of Grp75 may provide a therapeutic strategy totreat and enhance axonal regeneration following nerve injury.

Author contributions: S.L. and K.-T.M. designed research; S.L. performed research; W.W.,J.H., and U.N. contributed new reagents/analytic tools; S.L. and K.-T.M. analyzed data;and K.-T.M. wrote the paper with contribution from S.L.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1Present address: School of Mental Health, Wen Zhou Medical University, 325035Wen Zhou, Zhejiang Province, China.

2To whom correspondence may be addressed. Email: ktaimin@unist.ac.kr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1818830116/-/DCSupplemental.

Published online July 22, 2019.

16074–16079 | PNAS | August 6, 2019 | vol. 116 | no. 32 www.pnas.org/cgi/doi/10.1073/pnas.1818830116

Dow

nloa

ded

by g

uest

on

July

23,

202

1

preparations by examining the presence of GFAP for astrocytesand myelin basic protein for oligodendrocytes (SI Appendix, Fig.S1A). Furthermore, Grp75 protein in axon increased significantlyafter axotomy, while Grp75 in the cell body remained unchanged(SI Appendix, Fig. S1 C and D).Studies have shown that local translation of mRNA transcripts

occurs in injured axons upon axonal injury (8, 10, 17), and in-hibition of local protein synthesis prevents axonal regeneration(8, 17). This prompted us to examine whether Grp75 is locallytranslated upon axon injury. To this end, we generated a vectorthat contains the photo-switchable Dendra-2 protein fused withthe 5′UTR and 3′UTR of Grp75 mRNA together with 2 copiesof the palmitoylation sequence (5′UTR ofGrp75-Dendra2-3′UTRof Grp75) (18). The existing green fluorescent Dendra-2 proteinat the tip of the injured axon was irreversibly photo-converted tored fluorescent Dendra-2 protein using UV illumination, andnewly synthesized Dendra-2 protein (green) was quantified byanalyzing time-lapse images taken every 10 min for 90 min afteraxotomy. As seen in Fig. 1 E and F, newly synthesized greenfluorescent Dendra-2 was detected at the injured axonal tip andits level increased gradually over time. However, addition ofanisomycin, a translation inhibitor, blocked the synthesis of greenfluorescent Dendra-2, suggesting that axotomy triggers local pro-tein synthesis of Grp75 at the injured axon tip. As control,Dendra-2 containing UTRs of GAPDH was also monitored, butaxotomy did not trigger any change in its levels following axonalinsult. It is important to note that only the injured tip displayed asignificant increase in green fluorescence and not the entire lengthof the axon (SI Appendix, Fig. S1 E and F), suggesting that theincreased in Dendra-2 at the tip is not likely due to transport from

the cell body. Together, these results indicate thatGrp75mRNA islocally translated at the axonal tip after axotomy.

Grp75 Links IP3R1 and VDAC1. The local increase in Grp75 proteinfurther suggests increased ER–mitochondria tethering in axons.We thus first devised a strategy to visualize Grp75 interactionwith IP3R1 or VDAC1 using biomolecular fluorescence com-plementation (BiFC) (19) (Fig. 1G). Grp75 contains a mito-chondrial targeting signal (MTS), and has been shown to actboth as a linker in the cytoplasm to couple the ER to mito-chondria (4, 16), and as a chaperon protein inside of mito-chondria (20). Indeed, when we expressed Grp75 tagged withMyc (Grp75-Myc), it distributed both in the cytoplasm and mi-tochondria of NIH/3T3 cells (SI Appendix, Fig. S2). However,deletion of the MTS (Myc-ΔMTS-Grp75) led to localization ofMyc-ΔMTS-Grp75 in the cytoplasm, while replacing the MTSwith the mitochondrial protein Cox8a [MTS(Cox8a)-Myc-ΔMTS-Grp75] resulted in exclusive localization of MTS(Cox8a)-Myc-ΔMTS-Grp75 in the mitochondria (SI Appendix, Fig. S2). Wetook advantage of the cytoplasmic distribution of ΔMTS-Grp75to monitor ER–mitochondria tethering, since Grp75 distributionin the cytoplasm is required for its function as a linker of ER–

mitochondria. For BiFC, we generated truncated fluorescentprotein mVenus: VN (mVenus amino acids #1 to 172) and VC(mVenus amino acids #155 to 238) (Fig. 1 G and H and SIAppendix, Fig. S3A). Each of the two truncated proteins showedno fluorescence unless they are in close contact, thus allowing theindividual 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

B C D

E F

G

0 hr 2 hr

ER

Mito

Mer

ge

ER

Fluo

resc

ent i

nten

sity

(A.U

.)

1500

1000

500

00 hr 2 hr

*

A Mitochondria

Num

ber o

f mito

chon

dria 20

10

5

00 hr 2 hr

*15

Co-localization

Man

der’s

Coe

ffici

ent 1.0

0.6

0.4

0.00 hr 2 hr

*

0.8

0.2

After axotomy

Grp

75G

rp75

+Ani

soG

AP

DH High

Low

0 min 30 min 60 min 90 min

5’UTRGrp75-Dendra2-3’UTRGrp75

5’UTRGrp75-Dendra2-3’UTRGrp75+ Anisomycin5’UTRGAPDH-Dendra2-3’UTRGAPDH

Rel

ativ

e in

tens

ity4

3

2

1

00 10 20 30 40 50 60 70 80 90

Time (min)

*

IP R1

No interaction

No BiFC Signal

Mitochondria

ER

Grp75VN

VN

VC

VC

ER

Mitochondria

Interaction BiFC Signal

mVenus

mVenusVDAC1

Axon

BiFC Mito-mCherry Merge

IP3R

1(L

BD

)/G

rp75

VD

AC

1/G

rp75

H

Fig. 1. Grp75 is locally translated in injured axonsand interacts with IP3R1 and VDAC1. (A–C) ER andmitochondria were accumulated in the injured axontip. Sec61β tagged with EGFP was used to label theER, and mCherry with mitochondria targeting se-quence was used to mark mitochondria. Fluorescentintensity was measured from the axon tip to a 20-μmdistal segment before and after axonal injury. Ar-rowhead indicates the injured tip. (Scale bar, 20 μm.)Five axons for the ER and 6 axons for mitochondriawere used. Fluorescent intensity was compared be-tween paired samples and tested for statistical sig-nificance by paired t test: *P < 0.05 (B) and *P < 0.01(C). (D) Mander’s correlation coefficient showed thatcolocalization between ER and mitochondria in-creased after axotomy. Statistical significance wasmeasured by paired t test, *P < 0.05. (E) Pseudocolorimages of the green fluorescent Dendra-2 in theaxon tip after axotomy. Images were taken at 10-minintervals, but shown here with 30-min intervals. Redarrowhead indicates incision site of axon. (Scale bars,10 μm.) (F) Change in fluorescence intensity wasanalyzed in the injured axon tip. Primary hippo-campal neurons at DIV 4 were used. n = 8 axons ineach condition, *P < 0.01. (G) Schematic drawingthat showed the BiFC assay. (H) Cytoplasmic Grp75links the ER and mitochondria in axons. Axonsexpressing ETS-IP3R1[LBD]-VN and VC-ΔMTS-Grp75(IP3R1[LBD]/Grp75) or VDAC1-VN and VC-ΔMTS-Grp75 (VDAC1/Grp75) showed BiFC fluorescence.(Scale bar, 10 μm.)

Lee et al. PNAS | August 6, 2019 | vol. 116 | no. 32 | 16075

NEU

ROSC

IENCE

Dow

nloa

ded

by g

uest

on

July

23,

202

1

VN fragments to the N terminus of ΔMTS-Grp75 (VC-ΔMTS-Grp75). To monitor IP3R1 and Grp75 interaction, we also taggedthe ligand binding domain of IP3R1 (LBD, amino acids #224 to605) with VN in the C terminus and added an ER targetingsequence (ETS) to the N terminus (ETS-IP3R1[LBD]-VN) (16).Coupled expression of VDAC1-VN and VC-ΔMTS-Grp75 or VC-ΔMTS-Grp75 and ETS-IP3R1[LBD]-VN clearly displayed fluo-rescent BiFC signals in axons (Fig. 1H) and the cell body (SIAppendix, Fig. S3C). As a control, we also tested whether fluo-rescent signals can be detected from neurons containing 1)individual expression of VC-ΔMTS-Grp75, VDAC1-VN, or ETS-IP3R1[LBD]-VN; 2) coexpression of VC-ΔMTS-Grp75 andVDAC1 deleting a part of the C terminus (VDAC1-Δ265-283-VN);and 3) VC-ΔMTS-Grp75 and the suppressor domain of IP3R1(aminoacids #1 to 223) (ETS-IP3R1[SD]-VN) (SI Appendix, Fig. S3 Band C). None of these constructs displayed BiFC signals. To-gether, these results suggest that Grp75 indeed interacts withboth IP3R1 and VDAC1 in axons and the cell body of primaryhippocampal neurons. Furthermore, BiFC signals coming frominteraction between Grp75 and IP3R1 or between Grp75 andVDAC1 increased in injured axons (Fig. 2 A and B), indicatingthat ER–mitochondria tethering is enhanced after axonal insults.

Overexpression of Grp75 Promotes Axon Regeneration. What is thephysiological significance of this increased ER–mitochondriatethering? We hypothesized that up-regulation of Grp75 andincreased ER–mitochondria contacts may promote regrowth ofinjured axons. To test this hypothesis, we first transfected primaryhippocampal neurons with Grp75, Grp75 without MTS (ΔMTS-Grp75), so that it can act as a linker in the cytoplasm, and Grp75with MTS replaced with Cox8a’s MTS so that it is localized inmitochondria [MTS(Cox8a)-Myc-ΔMTS-Grp75] (SI Appendix, Fig.S4A). Of transfection efficiency, 65 to 80% was achieved (SI Ap-pendix, Fig. S4B). These neurons were cultured in a microfluidic

device where neurons are plated on one side of the device (21) (SIAppendix, Fig. S5A). Only axons can grow through the micro-grooves and reach to the other compartment, thus allowing dif-ferentiation of axons from cell bodies. Axon length of neuronsexpressing different types of Grp75 was measured at day in vitro(DIV) 4, and all showed similar length when stained with Tau-1antibody (SI Appendix, Fig. S5B). In contrast, reduction of Grp75by Grp75 siRNA transfection to neurons hindered normal axondevelopment (SI Appendix, Fig. S5 C and D). Furthermore, wefound that while reduction of Grp75 by Grp75 siRNA has a mildeffect on normal axon development, it significantly inhibited axonregrowth after axonal injury (SI Appendix, Fig. S5E).Next, to assess if overexpression of Grp75 promotes regrowth

of axons following axonal injury, vacuum aspiration was appliedto the exit borders of microgrooves, which severs the axons grownto the axonal compartment in the microfluidic device (SI Appen-dix, Fig. S5A). We performed axotomy at DIV 4 and continued toculture the injured neurons for 3 additional days (Fig. 2 C and D).A striking increase in axonal regrowth was detected in neuronswith ΔMTS-Grp75 overexpression, with axon length almost 2times longer than that of control. The length of axons cultured for7 d without damage is similar to that of injured axons over-expressing ΔMTS-Grp75 (SI Appendix, Fig. S5F). Axons contain-ing Grp75 with its own MTS also regenerated, but the length onlyincreased about one-third. It is likely that the difference betweenGrp75 with or without MTS on axonal regeneration capability isdue to the amount of Grp75 located in cytoplasm. Indeed, whenwe overexpressed Grp75 construct with exclusive localization inthe mitochondria [MTS(Cox8a)-Myc-ΔMTS-Grp75], it failed to en-hance axon regrowth following axotomy (Fig. 2C).Next, we tested whether overexpressing ΔMTS-Grp75 is ef-

fective in promoting growth of injured axons at a later time pointin neuron culture. To this end, we transfected neurons withΔMTS-Grp75 at DIV 10, performed axotomy at DIV 12, and

C DIV4 // +DIV3

Con

trol

Grp

75∆M

TS-

Grp

75

MTS

Cox

8a-

∆MTS

-G

rp75

0 hr 2 hr

Grp

75 /

IP3R

1B

iFC

mC

herr

y-S

ec61

β

A B

Fluo

resc

ent i

nten

sity

(A.U

.)

Grp75 / IP3R1 BiFC

*

0 hr 2 hr

3000

2000

1000

0

0 hr 2 hr

Grp

75/V

DA

C1

BiF

CM

ito-m

Che

rry

Fluo

resc

ent i

nten

sity

(A.U

.)

Grp75/VDAC1 BiFC

*

0 hr 2 hr

2000

1500

1000

0

500

DIV4 // +DIV3

800700

200

0

Axo

n Le

ngth

(μm

)

ControlGrp75

∆MTS-Grp75

MTSCox8a -∆MTS-Grp75

600500400300

100

n.s.

*

*

D

Overexpression

Fig. 2. Overexpression of Grp75 facilitates regrowthof injured axon. (A and B) BiFC images showed thatinteraction between Grp75 and IP3R1 or betweenGrp75 and VDAC1 in injured axons was increased 2 hafter axon injury. BiFC signals were measured fromthe injured axon tip to the 20-μm distal segment. n =7 axons, *P < 0.05. Arrowheads indicate injured ax-onal tip. (Scale bars, 10 μm.) (C and D) Three daysafter axotomy, axon length was measured in eachcondition. For axotomy, vacuum aspiration was ap-plied to the exit border of microgrooves, which sev-ers any axons grown to the axonal compartment inthe microfluidic device. (Scale bar, 200 μm.) Totalaxon numbers used for calculation were 294 forcontrol, 680 for Grp75, 943 for ΔMTS-Grp75, 613 forMTS(Cox8a)-ΔMTS-Grp75. n = 4 independent experi-ments, *P < 0.0001; n.s., not significant.

16076 | www.pnas.org/cgi/doi/10.1073/pnas.1818830116 Lee et al.

Dow

nloa

ded

by g

uest

on

July

23,

202

1

then monitored axon growth after another 6 d in culture. Theresults clearly showed that ΔMTS-Grp75 overexpression still ef-fectively induced regrowth of injured axons even at a more maturestage (SI Appendix, Fig. S5G). Furthermore, we tested whetherΔMTS-Grp75 overexpression postinjury can still enhance axonregeneration. We performed axotomy at DIV 4, and then waitedfor 3 d before ΔMTS-Grp75 transfection. These neurons werecultured for another 3 d before measuring the axon length mea-surement. We discovered that delayed Grp75 expression still en-hanced growth of previously injured axons (SI Appendix, Fig.S5H), suggesting that ΔMTS-Grp75 overexpression can promoteaxon regeneration regardless of timing of the treatment before orafter axon damage. Together, these results indicate that an in-crease in cytoplasmic Grp75, and hence an increased ER–mito-chondria tethering, promote axonal regrowth following injury.In addition to enhancing the regrowth of axons of injured

hippocampal neurons, we tested whether Grp75 overexpressionmodulates the regrowth of dorsal root ganglion (DRG) axonsfollowing injury. In SI Appendix, Fig. S6, we confirmed: (i) thepresence of Grp75 mRNA transcript in axons; (ii) obstruction ofaxon development by Grp75 siRNA transfection; and (iii) pro-motion of axon regrowth after axotomy by overexpression ofGrp75 without MTS. Together, these results verify that Grp75located in cytoplasm promotes regrowth of injured axon in DRGprimary neurons as well.

Grp75 Overexpression Increases ATP Production in Injured Axons.Next, we investigated how enhanced ER–mitochondria contactstrigger regrowth of the injured axon tip. As ER–mitochondriatethering had been shown to play critical roles in modulating Ca2+

homeostasis and ATP production (2), we tested whether alteredCa2+ level or ATP production underlies enhanced axonal regrowth.First, we monitored cytoplasmic and mitochondrial Ca2+ level inthe tip of axons. To this end, we transfected the genetically enco-ded Ca2+ indicators for cytoplasm and mitochondria: GEM-GECOand mito-case12, respectively (22, 23) (Fig. 3 A and B). When insultwas applied to the axonal tip, both control axons and axons over-expressing ΔMTS-Grp75 showed increased Ca2+ level in cyto-plasm, which is consistent with previous reports showing theincrease in Ca2+ concentration after axotomy (9, 12). However,mitochondrial Ca2+ level was not changed in control axons,while axons overexpressing ΔMTS-Grp75 significantly increasedmitochondrial Ca2+ level after axonal injury. Also, axons over-expressing MTSCox8a-ΔMTS-Grp75 showed increased cytosolic

Ca2+ level without altering mitochondrial Ca2+ level (SI Appendix,Fig. S7 A and B). These results suggest that increased ER–mito-chondria tethering due to Grp75 overexpression does not altercytoplasmic Ca2+ level, but facilitates Ca2+ influx to mitochon-dria upon axon injury. Furthermore, we assessed the ATP levelin the injured axonal tip when ΔMTS-Grp75 was overexpressed.To measure the ATP level in axons, we used genetically encodedFRET-based ATP indicators: ATeam1.03 for cytoplasm andmito-ATeam1.03 for mitochondria (24) (Fig. 3 C and D). Uponaxonal injury, local ATP level in the cytoplasm and mitochondriawas increased in the axonal tip having ΔMTS-Grp75. Our resultssuggest that increasing Grp75 enhances ER and mitochondriatethering in the injured axonal tips, leading to increase Ca2+

transfer from the ER to mitochondria, which then activates theTCA cycle and the enzymes in electron transport chain to in-crease ATP generation and promote axon regeneration.To verify that Ca2+ release from the ER can regulate the

regrowth of injured axons, we blocked IP3R1 by adding a selectiveand membrane-permeable inhibitor, xestospongin C (Xes C)(25), or stimulated IP3R1 by treating neurons with IP3R1 ago-nist, adenophostin A (Ad A) (26) (SI Appendix, Fig. S7 C and D).Injured axons treated with Xes C showed decreased axonregrowth after axotomy compared with that of control. In contrast,axons treated with Ad A after axotomy profoundly enhanced ax-onal regrowth. We also assessed levels of mitochondrial Ca2+ andATP in injured axons that were treated with these drugs (SI Ap-pendix, Fig. S7 E and F). As anticipated, we observed Ca2+ andATP levels in mitochondria were reduced with Xes C treatment,while Ad A elevated mitochondrial Ca2+ and ATP levels. To-gether, these results further indicate that Ca2+ transfer from theER to mitochondria plays a key role in axonal regeneration. Wealso confirmed whether Grp75 expression and the effect of Ad Ashare the same pathways on regrowth of injured axons (SI Ap-pendix, Fig. S7 C and D). Indeed, Grp75 reduction by Grp75siRNA diminished the effect of Ad A on the capability of injuredaxon regrowth. Taken together, these results indicate that Ca2+

transfer from the ER to mitochondria plays a key role in axonalregeneration.

Grp75 Overexpression Restores Sciatic Nerve Injury in Mice. Finally,we tested whether overexpression of Grp75 enhances axonalregeneration following injury in vivo by monitoring both axonregrowth and functional recovery of animals following sciaticnerve injury (Fig. 4 and SI Appendix, Fig. S8). Sciatic nerve crush

A B

C D

GEM-GECO

Con

trol

∆MTS

-G

rp75

High

Low

0 hr 2 hr

Rel

ativ

e in

tens

ity

Cytosolic Ca2+

*

Control∆MTS-Grp75

Time (min)0 20 40 60 80 10

012

00.0

0.5

1.0

1.5 Mito-case12

Con

trol

∆MTS

-G

rp75

0 hr 2 hr

AT1.03

Con

trol

∆MTS

-G

rp75

0 hr 2 hrMito-AT1.03

Con

trol

∆MTS

-G

rp75

0 hr 2 hr

Rel

ativ

e in

tens

ity

Mitochondrial Ca2+

*

Control∆MTS-Grp75

Time (min)

0 20 40 60 80 100

120

0.0

0.5

1.0

1.5

Rel

ativ

e FR

ET

inte

nsity

Mitochondrial ATP

Time (min)

0 20 40 60 80 100

120

0.0

0.5

1.0

1.5

*

Control∆MTS-Grp75

Rel

ativ

e FR

ET

inte

nsity

Cytosolic ATP

Control∆MTS-Grp75

Time (min)0 20 40 60 80 10

012

00.0

0.5

1.0

1.5

*

Fig. 3. Grp75 overexpression elevates mitochondrialCa2+ level and ATP generation in injured axons. (A)Ca2+ levels in the injured axon tip were measuredusing genetically encoded Ca2+ indicator, GEM-GECO; n = 6 and *P < 0.05 (between 0 and 120min). (Scale bar, 10 μm.) (B) Analysis of Ca2+ influx tomitochondria using mito-Case12 showed that mito-chondrial Ca2+ was increased in injured axons contain-ing ΔMTS-Grp75. Fluorescent intensity was measuredfrom the injured axon tip to a 20-μm distal segment;n = 7 and *P < 0.05. (Scale bar, 10 μm.) (C and D) FRETsignals indicating increased ATP were found in cyto-plasm (C) and mitochondria (D) of injured axons hav-ing ΔMTS-Grp75. Arrowheads indicate the injuredaxon tip. Quantification of ATP in the injured axon tipoverexpressing ΔMTS-Grp75 by using geneticallyencoded FRET-based ATP indicators: ATeam1.03 forcytoplasm (C) and mito-ATeam1.03 for mitochondria(D). n = 5 (C), n = 7 (D), *P < 0.05. (Scale bar, 10 μm.)

Lee et al. PNAS | August 6, 2019 | vol. 116 | no. 32 | 16077

NEU

ROSC

IENCE

Dow

nloa

ded

by g

uest

on

July

23,

202

1

injury was induced in mice with or without lentiviral delivery ofGrp75. Two weeks before sciatic nerve crush, we performed in-trathecal delivery of lentivirus containing Myc-ΔMTS-Grp75 (Fig.4A). Three days after the crush, we then assessed the regrowth ofinjured axons by staining with GAP43, a marker for axon re-generation, on cryostat sectioned sciatic nerve (Fig. 4 B and C).Compared with control, a significantly increased number ofGAP43-positive axons was detected distal to the crush site in miceinjected with Myc-ΔMTS-Grp75. This result confirms that Grp75overexpression promotes axon regeneration in animals followinginjured axons. To further verify axon regeneration, we performedretrograde labeling of DRG neurons with a tracing dye, DiI(1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlo-rate) that was injected 10-mm distal from the injury site (27).DRG sections of mice overexpressing Grp75 showed increasedthe number of neurons that labeled retrogradely with DiI com-pared with control (Fig. 4 D and E). This result indicates thatGrp75 overexpression restores damaged axons through axonregeneration.

We next evaluated functional recovery of the animal. First, weinvestigated sensory function by performing a Von Frey test andcold-plate test that can measure the pain response (Fig. 4 F andG). Control mice with sciatic nerve crush injury exhibited in-creased sensitivity to manual Von Frey hair application and coldplate. In contrast, mice injected with lentiviral vector containingMyc-ΔMTS-Grp75 showed gradual recovery from mechanicaland cold allodynia. To assess motor recovery after sciatic nerveinjury, we then tested mice on the treadmill (Fig. 4H). Mice withGrp75 overexpression showed increased duration on the tread-mill at day 9 after the nerve injury. We also evaluated a mor-phological parameter using sciatic functional index, whichmeasures toe-spreading geometry of the injured hind paw com-pared with the normal contralateral paw (28) (Fig. 4 I and J).Mice with Grp75 overexpression showed a significantly fasterrate of recovery, with almost fully restored sciatic functional index15 d after injury.When axons are injured, the ER and mitochondria translocate

to the injured site to reseal the membrane and to provide ATP foraxon regeneration (13, 15); however, whether physical tethering of

A B

C

D

E

GF H

I J

Myc

Con

trol-

LV-M

ycLV

-Myc

-∆M

TS-

Grp

75

Con

trol-

LV-M

ycLV

-Myc

-∆M

TS-

Grp

75

GAP43

100

80

60

40

20

00.0 1.0 2.0 3.0

Control∆MTS-Grp75

% o

f GA

P43

+ axo

ns

*

Distance from the crush site(mm)

Sham Control ∆MTS-Grp75

DiI

Retrograde tracing

No.

of D

iI+ se

nsor

y ne

uron

s 80

60

40

20

0

ShamControl

∆MTS-Grp75

n.s.

* **

Von Frey test

No.

of W

ithdr

awal

Post-injury (days)

ShamControl∆MTS-Grp75

*

14121086420

5 6 7 8 9

Cold plate test

No.

of L

ift

Post-injury (days)

ShamControl∆MTS-Grp75

*

302520

15

10

50

5 6 7 8 9

Treadmill test

No.

of F

allin

g

Post-injury (days)

ShamControl∆MTS-Grp75

*141210

86420

5 6 7 8 9

Sha

mC

ontro

l∆M

TS-

Grp

75

15(day)12963

Sci

atic

func

tiona

l ind

ex

Post-injury (days)

ShamControl∆MTS-Grp75

*

0

-50

-1003 6 15129

Fig. 4. Grp75 overexpression restores sciatic nerveinjury in mice by facilitating axon regeneration. (A)Cryostat section of injured sciatic nerve containing acontrol lentiviral vector with Myc tag or a vectorexpressing Myc-ΔMTS-Grp75. Arrowheads indicatethe crush site. (Scale bar, 1 mm.) (B) Regrowth ofinjured axons was stained by GAP43 antibody, amarker for axon regeneration. Sciatic nerve sectionwas prepared 3 d after sciatic nerve crush. Arrow-heads indicate the crush site. (Scale bar, 1 mm.) (C)Quantification of the number of GAP43+ axonsmoved further distal to the crush site. Nine animalsfor control and 5 animals having ΔMTS-Grp75, *P <0.01. (D) Axon regeneration was examined by ret-rograde 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 DRGsections. 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 per-forming Von Frey test, *P < 0.001, n = 6. (G) Func-tional recovery from cold allodynia was tested byperforming 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 wasmeasured by toe-spreading geometry of the injuredhind paw (I), and sciatic functional index was de-termined every 3 d postinjury; *P < 0.05, n = 5.

16078 | www.pnas.org/cgi/doi/10.1073/pnas.1818830116 Lee et al.

Dow

nloa

ded

by g

uest

on

July

23,

202

1

ER–mitochondria occurs in injured axons and whether ER–

mitochondria contacts contribute to axon regeneration are notknown. In this study, we discovered that Grp75 is locally translatedin injured axon, and overexpression of Grp75 promotes axon re-generation by increasing ER and mitochondria contact in an in-jured axon tip. The increased ER–mitochondria contacts enhanceCa2+ transfer from the ER to mitochondria, thereby elevatingATP generation required for axon regeneration. Grp75 is knownas a mitochondrial protein containing a mitochondrial targetingsequence, but also found at the interface of MAMs. It is in-teresting to note that our results reveal that cytoplasmic and mi-tochondrial Grp75 play different roles in axon regeneration. Wefound that when Grp75 is solely located in mitochondria[MTS(Cox8a)-Myc-ΔMTS-Grp75], axon length is similar to that ofcontrol, suggesting that Grp75 in mitochondria contributes little toaxonal regeneration. On the other hand, expression of Grp75without a mitochondrial targeting sequence (ΔMTS-Grp75) sig-nificantly increased axon length following injury (Fig. 2 C and D),indicating that cytoplasmic Grp75 (ΔMTS-Grp75), rather thanmitochondrial Grp75, plays a key role in axon regeneration ratherthan mitochondrial Grp75.The role of Grp75 in regulating growth and survival is not well

understood. There are several reports indicating that beneficialeffects of Grp75 overexpression under different stresses, such asmetabolic stress, glucose deprivation, cytotoxins, or oxidative dam-age (29–31). However, a recent study by Honrath et al. (4) showedthat Grp75 is critical in ER–mitochondria coupling and oxidativestress-mediated cell death. Moreover, Grp75 overexpression inHT22 neuronal cells increases sensitivity to glutamate-induced ox-idative cell death. Our findings are consistent with the beneficialeffects of Grp75 by showing enhanced axon regeneration whenΔMTS-Grp75 is expressed in injured axons. The discrepancy be-tween different studies may come from the distribution of Grp75 in

cells. It is interesting to note that Grp75 in the cytoplasm and mi-tochondria plays opposite roles in induction of apoptosis (32, 33).Interaction between Grp75 and p53 in the cytoplasm inhibitstranslocation of p53 to the nucleus or mitochondria, which preventsinduction of apoptosis, while mitochondrial Grp75 promotestranslocation of p53 to the mitochondria and triggers apoptosisunder oxidative-stress conditions. Our findings also support thatGrp 75 in different cellular compartments may have different roles.It will thus be interesting to further dissect the functions of cyto-plasmic 75 or mitochondrial Grp75 in regulating different cellularprocesses in the future.In summary, we demonstrate that cytoplasmic Grp75 links

IP3R1 in the ER and VDAC1 in mitochondria. Furthermore, Grp75is locally translated at the injured axonal tips, and overexpression ofGrp75 increases ER–mitochondria contacts to provide the energyneed to regrow injured axons. Finally, we found that an increasedlevel of Grp75 in DRG neurons promotes axon regeneration andfunctional recovery of animals with nerve injury. Together, our re-sults raise the exciting possibility that overexpression of cytoplasmicGrp75 may be a therapeutic strategy to treat and enhance axonalregeneration following nerve injury.

Materials and MethodsAnimals were used in accordancewith protocols approved by the Animal Careand Use Committees of the Ulsan National Institute of Science and Technology.The C57BL/6mouse strainwas purchased fromHyochang Science. Animals werehoused 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 inSI Appendix.

ACKNOWLEDGMENTS. This work was supported by Samsung Science andTechnology Foundation (SSTF-BA1301003) and a Leading Research Program,National Research Foundation of Korea grant funded by the Korea government(MEST) (2016R1A3B1905982).

1. O. M. de Brito, L. Scorrano, An intimate liaison: Spatial organization of the endo-plasmic reticulum-mitochondria relationship. EMBO J. 29, 2715–2723 (2010).

2. T. Hayashi, R. Rizzuto, G. Hajnoczky, T.-P. Su, MAM: More than just a housekeeper.Trends Cell Biol. 19, 81–88 (2009).

3. S. C. Lewis, L. F. Uchiyama, J. Nunnari, ER-mitochondria contacts couple mtDNA syn-thesis with mitochondrial division in human cells. Science 353, aaf5549 (2016).

4. B. Honrath et al., Glucose-regulated protein 75 determines ER-mitochondrial couplingand sensitivity to oxidative stress in neuronal cells. Cell Death Discov. 3, 17076 (2017).

5. S. Lahiri et al., A conserved endoplasmic reticulum membrane protein complex (EMC) fa-cilitates phospholipid transfer from the ER to mitochondria. PLoS Biol. 12, e1001969 (2014).

6. M. Krols, G. Bultynck, S. Janssens, ER-Mitochondria contact sites: A new regulator ofcellular calcium flux comes into play. J. Cell Biol. 214, 367–370 (2016).

7. S. Lee et al., Polo kinase phosphorylates miro to control ER-mitochondria contact sites andmitochondrial Ca(2+) homeostasis in neural stem cell development.Dev. Cell 37, 174–189 (2016).

8. F. M. Mar, A. Bonni, M. M. Sousa, Cell intrinsic control of axon regeneration. EMBORep. 15, 254–263 (2014).

9. Z. He, Y. Jin, Intrinsic control of axon regeneration. Neuron 90, 437–451 (2016).10. H. Jung, B. C. Yoon, C. E. Holt, Axonal mRNA localization and local protein synthesis in

nervous system assembly, maintenance and repair. Nat. Rev. Neurosci. 13, 308–324 (2012).11. D. Willis et al., Differential transport and local translation of cytoskeletal, injury-response,

and neurodegeneration protein mRNAs in axons. J. Neurosci. 25, 778–791 (2005).12. F. Bradke, J. W. Fawcett, M. E. Spira, Assembly of a new growth cone after axotomy:

The precursor to axon regeneration. Nat. Rev. Neurosci. 13, 183–193 (2012).13. S. M. Han, H. S. Baig, M. Hammarlund, Mitochondria localize to injured axons to

support regeneration. Neuron 92, 1308–1323 (2016).14. B. Zhou et al., Facilitation of axon regeneration by enhancing mitochondrial transport

and rescuing energy deficits. J. Cell Biol. 214, 103–119 (2016).15. K. Rao et al., Spastin, atlastin, and ER relocalization are involved in axon but not

dendrite regeneration. Mol. Biol. Cell 27, 3245–3256 (2016).16. G. Szabadkai et al., Chaperone-mediated coupling of endoplasmic reticulum and

mitochondrial Ca2+ channels. J. Cell Biol. 175, 901–911 (2006).17. I. Rishal, M. Fainzilber, Axon-soma communication in neuronal injury. Nat. Rev.

Neurosci. 15, 32–42 (2014).18. W. Wang, J. Z. Zhu, K. T. Chang, K. T. Min, DSCR1 interacts with FMRP and is required

for spine morphogenesis and local protein synthesis. EMBO J. 31, 3655–3666 (2012).19. C.-D. Hu, Y. Chinenov, T. K. Kerppola, Visualization of interactions among bZIP and

Rel family proteins in living cells using bimolecular fluorescence complementation.Mol. Cell 9, 789–798 (2002).

20. Z. Flachbartová, B. Kovacech, Mortalin—A multipotent chaperone regulating cellular

processes ranging from viral infection to neurodegeneration. Acta Virol. 57, 3–15 (2013).21. A. M. Taylor et al., A microfluidic culture platform for CNS axonal injury, regeneration

and transport. Nat. Methods 2, 599–605 (2005).22. Y. Zhao et al., An expanded palette of genetically encoded Ca2+ indicators. Science

333, 1888–1891 (2011).23. E. A. Souslova et al., Single fluorescent protein-based Ca2+ sensors with increased

dynamic range. BMC Biotechnol. 7, 37 (2007).24. H. Imamura et al., Visualization of ATP levels inside single living cells with fluores-

cence resonance energy transfer-based genetically encoded indicators. Proc. Natl.

Acad. Sci. U.S.A. 106, 15651–15656 (2009).25. P. De Smet et al., Xestospongin C is an equally potent inhibitor of the inositol 1,4,5-

trisphosphate receptor and the endoplasmic-reticulum Ca(2+) pumps. Cell Calcium 26,

9–13 (1999).26. H. Saleem, S. C. Tovey, A. M. Riley, B. V. Potter, C. W. Taylor, Stimulation of inositol

1,4,5-trisphosphate (IP3) receptor subtypes by adenophostin A and its analogues.

PLoS One 8, e58027 (2013).27. S. G. van Neerven et al., Retrograde tracing and toe spreading after experimental

autologous nerve transplantation and crush injury of the sciatic nerve: A descriptive

methodological study. J. Brachial Plex. Peripher. Nerve Inj. 7, e8–e14 (2012).28. A. S. Varejão, M. F. Meek, A. J. Ferreira, J. A. Patrício, A. M. Cabrita, Functional evalu-

ation of peripheral nerve regeneration in the rat: Walking track analysis. J. Neurosci.

Methods 108, 1–9 (2001).29. L. Xu, L. A. Voloboueva, Y. Ouyang, J. F. Emery, R. G. Giffard, Overexpression of mito-

chondrial Hsp70/Hsp75 in rat brain protects mitochondria, reduces oxidative stress, and

protects from focal ischemia. J. Cereb. Blood Flow Metab. 29, 365–374 (2009).30. L. Yang et al., Glucose-regulated protein 75 suppresses apoptosis induced by glucose

deprivation in PC12 cells through inhibition of Bax conformational change. Acta Bi-

ochim. Biophys. Sin. (Shanghai) 40, 339–348 (2008).31. J. Renaud, J. Bournival, X. Zottig, M. G. Martinoli, Resveratrol protects DAergic PC12

cells from high glucose-induced oxidative stress and apoptosis: Effect on p53 and

GRP75 localization. Neurotox. Res. 25, 110–123 (2014).32. R. Wadhwa et al., Inactivation of tumor suppressor p53 by mot-2, a hsp70 family

member. J. Biol. Chem. 273, 29586–29591 (1998).33. C. Londono, C. Osorio, V. Gama, O. Alzate, Mortalin, apoptosis, and neurodegeneration.

Biomolecules 2, 143–164 (2012).

Lee et al. PNAS | August 6, 2019 | vol. 116 | no. 32 | 16079

NEU

ROSC

IENCE

Dow

nloa

ded

by g

uest

on

July

23,

202

1