Valosin-containing protein is a key mediator between autophagic cell death and apoptosis in adult

14 Pages·2016·2.09 MB·English

by  Bo Kyoung Yeo

Yeoetal.MolecularBrain (2016) 9:31
DOI10.1186/s13041-016-0212-8
RESEARCH Open Access
Valosin-containing protein is a key
mediator between autophagic cell death
and apoptosis in adult hippocampal neural
stem cells following insulin withdrawal
Bo Kyoung Yeo1, Caroline Jeeyeon Hong1, Kyung Min Chung1, Hanwoong Woo1, Kyungchan Kim1,
Seonghee Jung1, Eun-Kyoung Kim1,2 and Seong-Woon Yu1,2*
Abstract
Background: Programmed cell death (PCD) plays essential roles in theregulationof survival and function ofneural
stemcells (NSCs). Abnormal regulation of this process is associated with developmental and degenerativeneuronal
disorders. However, the mechanismsunderlying thePCD ofNSCs remain largely unknown. Understanding the
mechanisms ofPCD inNSCs is crucial for exploring therapeutic strategies for the treatment ofneurodegenerative
diseases.
Result: We have previously reported that adult rat hippocampal neural stem (HCN) cellsundergo autophagic cell
death (ACD) following insulin withdrawal without apoptotic signsdespitetheir normal apoptotic capabilities. It is
unknown how interconnection between ACD and apoptosis is mediated inHCN cells. Valosin-containingprotein
(VCP)is knownto be essential for autophagosomematurationin mammalian cells. VCP is abundantly expressed in
HCNcells compared to hippocampal tissue and neurons. Pharmacological and genetic inhibitionof VCP atbasal
stateinthepresenceofinsulinmodestlyimpairedautophagicflux,consistentwithitsknownroleinautophagosome
maturation.Ofnote,VCPinactionininsulin-deprivedHCNcellssignificantlydecreasedACDanddown-regulated
autophagyinitiationsignalswithrobustinductionofapoptosis.Overallautophagylevelwasalsosubstantiallyreduced,
suggestingthenovelrolesofVCPatinitialstepofautophagy.
Conclusion:Takentogether,thesedatademonstratethatVCPmayplayanessentialroleintheinitiationofautophagy
andmediationofcrosstalkbetweenACDandapoptosisinHCNcellswhenautophagylevelishighuponinsulin
withdrawal.ThisisthefirstreportontheroleofVCPinregulationofNSCcelldeath.Elucidatingthemechanismby
whichVCPregulatesthecrosstalkofACDandapoptosiswillcontributetounderstandingthemolecularmechanism
ofPCDinNSCs.
Keywords:Autophagiccelldeath,Apoptosis,Adultneuralstemcells,Insulinwithdrawal,Valosin-containingprotein
*Correspondence:[email protected]
1DepartmentofBrainandCognitiveSciences,DaeguGyeongbukInstituteof
ScienceandTechnology(DGIST),Daegu711-873,RepublicofKorea
2NeurometabolomicsResearchCenter,DaeguGyeongbukInstituteof
ScienceandTechnology(DGIST),Daegu711-873,RepublicofKorea
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InternationalLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinkto
theCreativeCommonslicense,andindicateifchangesweremade.TheCreativeCommonsPublicDomainDedicationwaiver
(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Yeoetal.MolecularBrain (2016) 9:31 Page2of14
Background ACDtoapoptosis[14].Interestingly,activationofglycogen
Programmedcelldeath(PCD)isadeliberatecellularsui- synthasekinase-3β(GSK-3β),oneofthekeysignalingmol-
cide process. Dysfunction of PCD is implicated in vari- ecules in regulation of neuronal apoptosis, also promoted
ous human diseases, including developmental and ACD, not apoptosis, in insulin-deprived HCN cells [15].
neurodegenerative disorders, cancer and autoimmune These data suggest that there is the unique intrinsic cell
diseases [1]. PCD can be categorized by morphological death program that drives the cell death mode towards
criteria [2]. Type I cell death, known as apoptosis, is ACD rather than apoptosis in HCN cells following insulin
characterized by cell shrinkage, nuclear condensation, withdrawal. Currently,HCN cell death induced by insulin
membrane blebbing, mitochondria dysfunction, loss of withdrawal is regarded as the most genuine model of
selectivity in membrane permeabilization, and nuclear ACDinmammals[16].
DNA fragmentation. Lastly, cells are rapidly eliminated Valosin-containing protein(VCP)/p97isaubiquitously
by phagocytosis [3]. Type II PCD refers to autophagic expressed protein belonging to the AAA+ (ATPases As-
cell death (ACD). Autophagy is a catabolic process that sociated with diverse cellular Activities) protein family
disposes of various cytoplasmic components, including with two ATPase domains, D1 and D2 [17]. Following
protein aggregates and organelles [4]. The components binding of the substrates to the N and C terminal do-
are sequestered by autophagosomes, which fuse with mains, VCP hydrolyses ATP on its ATPase domains.
lysosomes for degradation. This process usually oc- Subsequently, VCP changes its complex formation with
curs in response to cellular stress to protect the cells. distinct interacting proteins or cofactors to exert its
However, prolonged autophagy can cause ACD [5]. multicellular functions [18–25]. Previous studies have
Type III cell death, called necrosis, is best defined by reported that VCP is involved in multiple cellular pro-
abnormal mitochondria morphology, swollen and rup- cesses, including cell cycle regulation, Golgi biogenesis,
tured cellular membrane, cell lysis and exposure of nuclear membrane formation, ubiquitin proteasome sys-
intracellular content to the extracellular space. There- tem (UPS), apoptosis and the autophagosome matur-
fore, inflammatory reactions are frequently related with ation [17, 26]. Cells with loss of VCP activity failed to
necrosis[6]. undergo autophagosome and lysosome fusion, thereby
Autophagy is the major degradation pathway for long- prevented autophagosome maturation, suggesting the
lived proteins and damaged organelles for clearance or positive regulation of autophagosome maturation by
recycling. Autophagosomes deliver cytoplasmic contents VCP in mammalian cells [27–29]. Mutations in human
to lysosome where autophagosomes become autolyso- VCP is associated with the multisystem disease called
somes for degradation. Autophagy process takes place in “inclusion body myopathy associated with Paget’s disease
eukaryotic cells from yeast to mammals for cellular re- of bone and frontotemporal dementia (IBMPFD)”, which
modeling during development as well as starvation, is featured with inclusion bodies in the brain or muscle
inflammatory reaction and cell death [7, 8]. Majority of [28, 30]. Furthermore, depletion or ATPase-inactive mu-
the studies on the physiological roles of autophagy sug- tants of VCP induced apoptosis in several different types
gest that autophagy is a protective process by helping to of cells [31]. These previous studies prompted us to
copewithcellularstressandmaintain cellular homeosta- examine the involvement of VCP in regulation of ACD
sis. However, there are reports on the causative role of inHCNcells following insulinwithdrawal.
autophagy in developmental cell death. Cell death of the In this study, we report the different actions of VCP
salivary glands during Drosophila development is medi- depending on the autophagy level. Inactivation of VCP
ated by autophagy genes (Atg) in a caspase-dependent at basal state in the presence of insulin led to mild im-
or independent manner [9, 10]. Also, excessive auto- pairment of autophagy, indicating involvement in autop-
phagy in response to stress or injury can cause ACD hagosome maturation, as previous reported by others.
[11, 12]. However,despitetheemergingroleofautophagy On the other hand, pharmacological and genetic inhib-
in regulation of PCD, the underlying mechanisms are ition of VCP in insulin-deprived HCN cells undergoing
poorlyunderstood. high level of autophagy decreased autophagy initiation
Previously we reported hippocampal neural stem signalingandreducedACD,concomitantwith robustin-
(HCN) cells undergo ACD upon insulin withdrawal [13]. duction of apoptosis. These results suggest a novel role
Cell death induced by insulin depletion did not show of VCP in mediation of autophagy, implicating VCP at
apoptotic signs. Instead autophagic markers were signifi- the early stage of autophagy as well as the late matur-
cantly increased, whereas anti-apoptotic/anti-autophagic ation step depending on the autophagy level. Our study,
proteins, Bcl-2 and Bcl-X , were decreased. Importantly, for the first time, reveals the critical role of VCP in
L
cell death rate was significantly decreased with knock- crosstalk between ACD and apoptosis, indicating VCP
down of Atg7 in insulin-deprived HCN cells. Of note, as a distinct regulator in survival and death of neural
high calpain activity switched the cell death mode from stemcells(NSCs).
Yeoetal.MolecularBrain (2016) 9:31 Page3of14
Results in the NSCs in the subgranular zone (SGZ) of the den-
VCPishighlyexpressedinHCNcellsanddegradedby tate gyrus, as revealed by co-localization with glia fibril-
autophagyfollowinginsulinwithdrawal lary acidic protein (GFAP), a NSC maker in SGZ [32].
Since VCP has not been studied in NSCs before, first, VCP was also expressed in the granule neurons of the
we examined the expression level of VCP in HCN cells. DG in adult hippocampus, when microtubule-associated
VCP was highly expressed in HCN stem cells in vitro, protein 2 (MAP2) was used as a neuronal marker [33]
compared to the total hippocampal tissue derived from (Fig. 1b). The time course analysis of VCP protein and
8-week-old rat and embryonic hippocampal primary mRNA expression levels was performed following insu-
neurons after 9 days in vitro (Fig. 1a). To further exam- lin withdrawal. From here, we denote the insulin-
ine whether VCP is abundantly expressed in HCN cells containing and insulin-depleted culture conditions as
than hippocampal neurons in vivo, we performed immu- I(+) and I(-), respectively. Consistent with our prior
nohistochemical analysis in 8-week-old rat brain tissue, studies [13–15], high level of autophagy was induced in
since HCN cells were derived from the adult rat hippo- I(-) (Fig. 1c). Autophagy induction was verified by an in-
campus of the same age [13]. VCP was highly expressed crease in the level of type II of microtubule-associated
D
A
HT HCN HN
VCP
-Actin
B
E
C
24h 48 h
Insulin : + - + - F
Insulin : + - -
VCP BafA1 : - - +
I VCP
LC3
II
I
LC3
II
-Actin
-Actin
Fig.1VCPishighlyexpressedinHCNcellsanddegradedthroughautophagyfollowinginsulinwithdrawal.aVCPwasabundantlyexpressedin
HCNcellscomparedwithhippocampaltissuepreparedfrom8-week-oldratandembryonicprimaryhippocampalneurons(9daysinvitro).bVCP
wasparticularlylocalizedinhippocampalneuralstemcellsinvivo,asindicatedbyarrowheads.GFAPandMAP2wereusedasNSCandneuronal
markers,respectively.Scalebar,20μm.cVCPproteinlevelwassignificantlydecreasedat48hfollowinginsulinwithdrawal.Quantitativeanalysis
ofVCPlevelswasnormalizedtoβ-actin.Quantitativedataarerepresentedatthemean±SD(n=3).Statisticalsignificancewasdeterminedwith
anOne-wayANOVAtestbyTukey’smultiplecomparisontest.*p<0.05.dVCPexpressionlevelsweredecreasedinexvivoorganotypichippocampal
sliceculturefollowinginsulinwithdrawalfor24h.ArrowindicatestheVCP-expressingHCNcellsinI(+)condition,whilearrowheadsshowthelossof
VCPexpressioninI(-)condition.Scalebar,20μm.eExpressionlevelsofVCPmRNAwerenotchangedbyinsulinwithdrawal.Quantitativedataare
representedatthemean±SD(n=3).n.s.notsignificant.fDegradationofVCPwaspreventedbyBafilomycinA1treatment(BafA1,30nMfor3h
beforeharvest)following48hinsulinwithdrawal
Yeoetal.MolecularBrain (2016) 9:31 Page4of14
protein1lightchain3(LC3-II),sincethisisthemostre- in I (-) HCN cells, which otherwise underwent genuine
liable biochemical marker of autophagy [34–36]. VCP ACD. Staurosporine (STS), a common apoptosis inducer,
protein level was decreased 48 h following insulin with- was used as a positive control of apoptosis in I(+) HCN
drawal (Fig. 1c). To further confirm the decrease of VCP cells. STS-induced cell death was efficiently prevented by
protein level following insulin withdrawal in ex vivo abroad-spectrumcaspaseinhibitor,Z-VAD-FMK(Fig.2b).
HCN culture, we measured VCP levels in organotypic Also, necrostatin-1, a widely used necroptosis inhibitor
hippocampal slice culture prepared from 8-week-old rat [41], did not change cell death rate in DBeQ-treated I(-)
withvimentin asaNSCmarker[37,38].VCPexpression HCN cells. This pharmacological inhibitor study suggests
level was highly enriched in vimentin-immunoreactive that VCP inhibition triggered a switch of cell death mode
stem cells in the SGZ in the presence of insulin, but its fromACDtoapoptosisinI(-)HCNcells.Furtheranalyses
level significantly decreased in insulin-deprived slices wereperformedtodemonstratetheinductionofapoptosis
(Fig. 1d). These results with organotypic slice cultures following VCP inhibition in I (-) HCN cells. Cleaved, ac-
were well accordant with the data obtained from HCN tive form of caspase 3 and chromatin condensation was
cell culture, demonstrating the decrease of VCP protein observed in DBeQ-treated I(-) HCN cells (Fig. 2c, d). A
level following insulin withdrawal in ex vivo and in vitro markedincreaseinAnnexinVpositivestainingindicating
conditions.Despite the decrease ofVCPprotein levels in thederangementofphosphatidylserinedistributionduring
HCN cells and hippocampal slices, the levels of VCP apoptosis was also observed in DBeQ-treated I(-) HCN
mRNA expression were not changed by insulin with- cells (Fig. 2e). Taken together, these data show that in-
drawal in HCN cells (Fig. 1e). These data suggest that hibition of VCP switches ACD to apoptosis in insulin-
VCP may be subject to post-translational regulation. To deprivedHCNcells.
testwhetherVCPitselfisa substrateforautophagicdeg- To further test whether genetic suppression of VCP
radation or proteasome in insulin-deprived HCN cells, expression also switches ACD to apoptosis, VCP-
autophagic flux inhibitor, Bafilomycin A1 (BafA1) was targeting small interfering RNA (siRNA) was transfected
treated following insulin withdrawal [39]. Interestingly, in HCN cells following the presented experimental
BafA1 (30 nM for 3 h) significantly prevented VCP deg- scheme (Fig. 3a–b). In support of the results obtained
radation suggesting that autophagy may be responsible with DBeQ treatment, knockdown of VCP did not alter
for degradation of VCP in insulin-deprived HCN cells cell death rate in I(+) condition, but significantly en-
(Fig. 1f). hanced cell death in I(-) HCN cells (Fig. 3c). Augmented
cell death in I(-) HCN cells by VCP knockdown was
PharmacologicalandgeneticinactivationofVCPswitched substantially diminished by Z-VAD-FMK (Fig. 3c) and
ACDtoapoptosisininsulin-deprivedHCNcells accompanied by caspase-3 activation (Fig. 3d). Collect-
VCP is known as a positive regulator in autophagosome ively, thesedata indicatethatgeneticknockdown ofVCP
maturation. To test whether VCP ATPase activity is re- expression induced a switch of PCD mode from ACD to
quired for ACD in HCN cells, HCN cells were treated apoptosis in I(-) HCN cells in a similar fashion as
with VCP ATPase activity inhibitor, N2, N4-dibenzylqui- pharmacologic inhibition of VCP. However, it should be
nazoline-2,4-diamine (DBeQ) [40], in insulin-depleted noted that VCP depletion or inactivation did not induce
media and cell death was measured. We tested a wide apoptosis at basal state in I(+) HCN cells, which is in
range of DBeQ concentrations both in I (+) and I (-) sharp contrast with the previous studies reported in
HCN cells and chose 0.5 μM concentration of DBeQ, yeast, Caenorhabditis. elegans, HeLa, and several other
based on marginal effect on cell death in I (+) but sig- mammaliancells[31,42–49].
nificant increaseofcelldeath in I(-)HCN cells (Fig. 2a). To exclude the possibility that DBeQ induces apop-
Hereafter, this concentration of DBeQ has been used tosis independent of inhibiting VCP, we treated VCP-
throughout this study. Since cell death in I(-) HCN cells suppressed I(-) HCN cells with DBeQ. Unlike the eleva-
was boosted by DBeQ, we wondered if this increase was tionofcelldeathinNTsiRNA-treated HCNcells,DBeQ
due to the continuance of the default ACD or switch to failed to increase cell death in I(-) HCN cells depleted of
different mode of PCD. To determine whether ACD was VCP (Fig. 3e). These data suggest that DBeQ-induced
affected by DBeQ in I(-) HCN cells, the different kinds apoptosis in I(-) condition was VCP-dependent. Also, to
of PCD inhibitors were treated (Fig. 2b). Consistent with test whether inhibition of VCP by DBeQ was due to
the absence of apoptotic indices, Z-VAD-FMK failed to perturbation of non-autophagy related functions of VCP
reduce cell death in I(-) HCN cells (data not shown), independently of autophagy, we stably knocked down
which was well documented in our prior studies [13–15]. Atg7 in HCN cells before DBeQ treatment. HCN cells
However, interestingly, Z-VAD-FMK significantly de- were transduced with the lentivirus expressing Atg7-
creasedcelldeathinDBeQ-treatedI(-)HCNcells(Fig.2b), targeting shRNA and subject to puromycin selection.
suggesting that apoptosis was induced by VCP inhibition Atg7 knockdown significantly decreased ACD in I(-)
Yeoetal.MolecularBrain (2016) 9:31 Page5of14
A C
D
B
100
24 h 48 h 12 h
n.s.
80
)
%
( * *** ***
h 60
at * ***
e
d 40
lle
C
20
E
0
Insulin : + - - - + - - - - + +
DBeQ: - - + + - - + + + - -
Z-VAD : - - - + - - - + - - +
STS : - - - - - - - - - + +
Necrostatin-1 : - - - - - - - - + - -
Fig.2PharmacologicalinhibitionofVCPswitchedACDtoapoptosisininsulin-deprivedHCNcells.aAVCPinhibitor,DBeQ(0.5μM)markedly
increasedcelldeathfollowinginsulinwithdrawalfor24h,butwithoutsignificanteffectonI(+)HCNcells.Quantitativedataweredetermined
usingOne-wayANOVAfollowedbyTukey’smultiplecomparisontest.*p<0.05,**p<0.01,***p<0.001.n.s.,notsignificant.bZ-VAD-FMK
significantlypreventedcelldeathinducedbyDBeQinI(-).Staurosporine(STS,1μMfor12h)wastreatedinI(+)HCNcellsasapositivecontrolfor
apoptosisinduction.However,additionofnecrostatin-1(10μM)upto48hdidnotdecreasecelldeathinDBeQ-treatedI(-)HCNcells.Quantitativedata
arerepresentedasthemean±SDbyOne-wayANOVAusingTukey’smultiplecomparisontest(n=3).**p<0.01,***p<0.001.n.s.,notsignificant.
cCaspase3wasactivatedinDBeQ-treatedI(-)HCNcells.dNuclearcondensationwasobservedbyHoechststaining,asindicatedbyarrow.Nuclear
condensationwasdetectedinDBeQ-treatedI(-)HCNcells,butpreventedbyZ-VAD-FMK.Scalebaris20μm.eApoptosisinductionwasconfirmedby
Annexin-VstainingandFACSanalysisfordetectionoftheexposureofphosphatidylserine
HCN cells, consistent with our previous studies [14, 15]. VCPinactivationreducedautophagyfluxininsulin-deprived
Of particular interest, DBeQ failed to increase cell death HCNcells
in I(-) HCN cells depleted of Atg7, nor did Z-VAD-FMK Our findings that loss of VCP activity switched ACD to
affect cell death (Fig. 3f). These data strongly demon- apoptosis suggest that VCP is required for the progress
strate that ACD is a prerequisite to DBeQ-induced of ACD in I(-) HCN cells. However, it is not known
switch to apoptosis in HCN cells and DBeQ-induced whether VCP functions at the autophagosome matur-
apoptosis proceeds following yet-to-be-discovered mech- ationstep,asinotherstudies[27–29],orplaysnewroles
anism for interaction between ACD and apoptosis, but in the autophagy of HCN cells upon insulin withdrawal.
isnotnon-specific cytotoxic event. To elucidate what roleVCP may play in the progression
Yeoetal.MolecularBrain (2016) 9:31 Page6of14
A
E
80
%) 60 n.s.
(
h *** ***
t
ae 40 ***
d
ell 20
C
B C
Insulin: + + 0
NT siRNA : + - 50 *** *** Insulin : + - - + - -
VCP siRNA : - + )% 40 NT siRNA : + + + - - -
( VCP siRNA : - - - + + +
h
VCP eat 30 DBeQ: - - + - - +
d 20
n.s.
ell 10
-Actin C
F
0 40
Insulin : + + - - -
***
NT siRNA : + - + - - %) 30 ***
VCP siRNA : - + - + + (
h
D Z-VAD : - - - - + at 20 n.s.
e
d
Insulin : + - - +
NT siRNA : + + - + ell 10
C
VCP siRNA : - - + -
STS : - - - + 0
Cleaved Insulin : + - - - - -
shScramble: + + - - - -
caspase-3
shAtg7: - - + + + +
DBeQ(µM) : - - - 0.25 0.5 0.25
-Actin
Z-VAD : - - - - - +
Fig.3GeneticinhibitionofVCPchangedACDtoapoptosisininsulin-deprivedHCNcells.aAnexperimentalschemefortheknockdownofVCP.
CDA,celldeathassay;WB,Westernblotting.bKnockdownofVCPwasconfirmedbyWesternblottingafter24htreatment.NT,non-targeting.
cVCPknockdownincreasedcelldeathandZ-VAD-FMKefficientlyblockedanincreaseofcelldeathinI(-)HCNcells.Quantitativedataare
determinedasthemean±SDbyOne-wayANOVAfollowedbyTukey’smultiplecomparisontest(n=3).***p<0.001.n.s.,notsignificant.
dCleavedcaspase3wasdetectedinI(-)HCNcellsfollowingVCPknockdownat48h.eCelldeathratewasnotaffectedbyDBeQinI(-)HCNcells
depletedofVCP.Quantitativedataaredeterminedasthemean±SDbyOne-wayANOVAfollowedbyTukey’smultiplecomparisontest(n=3).
***p<0.001.n.s.,notsignificant.fAtg7knockdowndecreasedcelldeathinI(-)HCNcells.However,celldeathratewasnotalteredbyDBeQor
Z-VAD-FMKinI(-)HCNcellswithstableknockdownofAtg7.Quantitativedataaredeterminedasthemean±SDbyOne-wayANOVAfollowedby
Tukey’smultiplecomparisontest(n=3).***p<0.001.n.s.,notsignificant
of autophagy in HCN cells, we depleted VCP function An increase in LC3-II level may indicate increased
by siRNA or using the same chemical inhibition ap- formation of autophagosomes with high autophagy flux.
proach of VCP activity and examined the autophagy flux Yet, impairment of autophagy flux due to blocking of
rate. VCP inactivation by DBeQ or siRNA knockdown in autophagosome-lysosome fusion and failure of autopha-
I(+) HCN cells led to a modest increase in LC3-II level gosome maturation can also lead to an increase in LC3-
(Fig. 4a–b). On the other hand, DBeQ treatment or VCP II. Therefore, mild increase following DBeQ-treatment
siRNA knockdown in I(-) HCN cells substantially de- or VCP depletion in I(+) HCN cells can be due to either
creased LC3-II levels (Fig. 4c–d). When autophagy flux increase or impairment in autophagy flux. If an increase
was blocked by addition of BafA1, there was a marked of LC3-II observed in VCP-depleted HCN cells in I(+)
increase in the amount of LC3-II and they reached the was due to high rate of autophagy flux, BafA1 treatment
same level between BafA1 control and BafA1/DBeQ or would lead to higher amount of LC3-II than BafA1 I(+)
BafA1/VCP siRNA in I(+) HCN cells (Fig. 4a–b). In the control cells. However, the amount of LC3-II was the
case of I(-) HCN cells, BafA1 also increased LC3-II same, suggesting that the overall autophagy flux rate
levels. However, interestingly, BafA1-induced accumula- remainedthesameregardlessofVCPinactivationduring
tion of LC3-II was significantly less in BafA1/DBeQ or the basal autophagy in I(+). Therefore, this modest in-
BafA1/VCP siRNA than BafA1 control in I(-) HCN cells crease in LC3-II by VCP depletion can be interpreted
(Fig. 4c–d). due to partial inhibition of autophagosome maturation
Yeoetal.MolecularBrain (2016) 9:31 Page7of14
A B
Insulin : + + + +
Insulin : + + + + NT siRNA : + - + -
DBeQ: - + - + VCP siRNA : - + - +
BafA1 : - - + + BafA1 : - - + +
I I
LC3 LC3
II II
-Actin -Actin
el el
levtin) 8 n.s. levtin) 8 n.s.
C3Ac 6 * C3Ac 6
L- L- *
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ativeC3-II/ 2 ativeC3-II/ 2
el(L 0 el(L 0
R R
C D
Insulin : - - - -
Insulin : - - - - NT siRNA : + - + -
DBeQ: - + - + VCP siRNA : - + - +
BafA1 : - - + + BafA1 : - - + +
I I
LC3 LC3
II II
-Actin -Actin
el * el
vn) vn) ***
leti 4 leti 5
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veL-II/- 2 * veL-II/- 23 *
atiC3 1 atiC3 1
el(L 0 el(L 0
R R
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el evn)
sionlev-Actin) 1.5 n.s. essionlo-Acti 12..50 n.s.
s rt
xpredto 1.0 expzed 1.0
NAealize 0.5 RNAmali 00..05
Rm 0.0 mor N A N A N A
LC3m(Nor I(+) I(-I)(-)/D BeQ LC3I((N+)/NT siI(R-)/NTIs(i-)R/V C P siR
Fig.4(Seelegendonnextpage.)
Yeoetal.MolecularBrain (2016) 9:31 Page8of14
(Seefigureonpreviouspage.)
Fig.4VCPinhibitionreducedautophagicfluxininsulin-deprivedHCNcells.a-bDBeQtreatment(a)orVCPknockdown(b)inducedamoderate
increaseinLC3-IIinI(+)HCNcells.BlockingofautophagyusingBafA1gaverisetoasignificantincreaseofLC3-IIwiththesamelevelsbetween
BafA1aloneandBafA1/DBeQorBafA1/VCPknockdowncellsinI(+).QuantitativeLC3-IIlevelsweredeterminedasthemean±SDbyOne-way
ANOVAfollowedbyTukey’smultiplecomparisontest(n=3).**p<0.01,***p<0.001.n.s.,notsignificant.c-dDBeQtreatment(c)orVCPknockdown
(d)ledtoadecreaseinLC3-IIinI(-)HCNcells.BlockingofautophagyusingBafA1gaverisetoasignificantincreaseofLC3-II,buttheamountof
accumulatedLC3-IIinBafA1/DBeQorBafA1/VCPknockdowncellswassignificantlylessthanBafA1aloneinI(-).QuantitativeLC3-IIlevelswere
determinedasthemean±SDbyOne-wayANOVAfollowedbyTukey’smultiplecomparisontest(n=3).**p<0.01,***p<0.001.n.s.,notsignificant.
emRNAexpressionlevelofLC3didnotshowdifferentchangesbyVCPinactivationinI(-).QuantitativeLC3mRNAexpressionlevelsarerepresented
asthemean±SD(n=3).QuantitativedatawasdeterminedbyStudent’st-test.*p<0.05.n.s.,notsignificant
with the yield of the same amount of LC3-II accumula-
A
tion following full inhibition of autophagy flux by BafA1. DAPI GFP-LC3 RFP-LC3 Merge
On the other hand, complete blocking of autophagy flux
by BafA1 disclosed considerably smaller amount of LC3-
I(+)
II in VCP depleted I(-) HCN cells than I(-) control, indi-
cating reduced rate of autophagy flux. Therefore, a de-
crease in LC3-II by DBeQ or VCP siRNA before BafA1
treatment imply the similar decrease of autophagy flux I(+)/
rate in I(-) HCN cells. Quantitative analysis of LC3 DBeQ
mRNA level did not show significant change by VCP
inactivation (Fig. 4e). Taken together, these data demon-
strate that VCP positively regulates autophagosome mat-
I(-)
uration at basal autophagy in I(+), as reported by others.
In contrast, VCP regulates overall autophagy flux rate
in insulin-deprived HCN cells when autophagy activity
ishigh. I(-)/
Modest changes in the level of LC3-II may not be DBeQ
detected due to saturation of Western blotting signals.
To further validate different roles of VCP for autophagy B
between I(+) and I(-) conditions, next, we adopted
monomeric RFP-GFP tandem fluorescent LC3 (mRFP-
GFP-LC3) and measured autophagy flux. Co-existence
of GFP and RFP signals in autophagosomes before mat-
uration and RFP signals in autolysosomes after matur-
ation are based on the acid-sensitive quenching of EGFP
protein. DBeQ treatment in I(+) increased the percent-
age of yellow puncta, but without significant increase in
the total puncta number, suggesting attenuation of
autophagosome maturation by DBeQ in I(+) HCN cells.
On the other hand, LC3 puncta formation was strikingly
increased in I(-) HCN cells and this increase was greatly
reduced by DBeQ. However, the percentages of yellow
puncta remained similar between I(-) and I(-)/DBeQ
Fig.5DifferentialregulationofautophagyfluxbyVCPinI(+)and
conditions, indicating that DBeQ inhibited autophagy I(-)HCNcells.amRFP-GFP-LC3wasusedforautopahgicfluxassay.
induction rather than maturation step in I(-) HCN cells DBeQtreatmentinI(+)ledtoanincreaseinthepercentageof
(Fig. 5). yellowpunctawithnomarkedincreaseinthenumberofthetotal
LC3puncta,suggestingimpairmentofautophagosomematuration.
DBeQtreatmentinI(-)greatlyreducedthenumberofthetotalLC3
VCPisrequiredforautophagyinitiationsignalingin
puncta,butthepercentageofyellowpunctaremainedsimilar
insulin-deprivedHCNcells betweenI(-)andI(-)/DBeQconditions.Scalebar,10μm.bQuantitative
Based on our results indicating the reduced autophagy redandyellowpunctanumbersweredeterminedasthemean±SD
levelbyVCPinactivationinI(-)HCNcells,wepostulated byOne-wayANOVAfollowedbyTukey’smultiplecomparisontest
(n=16–35).Blackandyellowasterisksindicatethecomparisonofthe
that VCP may regulate autophagy initiation signaling in
totalandyellowpuncta,respectively.*p<0.05,**p<0.01,***p<0.001
HCN cells following insulin withdrawal. Our previous
Yeoetal.MolecularBrain (2016) 9:31 Page9of14
study by Ha et al. demonstrated that glycogen synthase was rescued by VCP inaction surprisingly even in the
kinase 3β (GSK-3β) induced ACD in HCN cells following absence of insulin (Fig. 6a–b). In our previous report, we
insulinwithdrawal[15].Bothpharmacologicalandgenetic demonstrated that calpain 2 was degraded by UPS in I(-)
inactivationofGSK-3βsignificantlydecreasedACD,while HCNcells,butconditionsleadingtocalpainaccumulation
activation of GSK-3β increased autophagic flux and andhighcalpainactivityutterlyswitchedthedefaultACD
causedmorecelldeathwithoutinducingapoptosisfollow- toapoptosisinI(-)HCNcells[14].Inaccordancewiththe
ing insulin withdrawal. This report uncovered GSK-3β as induction of apoptosis in VCP-inactivated I(-) HCN cells,
one of the critical upstream signaling kinases for ACD in calpain2levelwasalsorestoredtomuchhigherlevelthan
I(-) HCN cells. GSK-3β activation, as revealed by a de- I(-) (Fig. 6a). Knockdown of VCP in I(+) induced mild
creaseintheinhibitoryphosphorylationofserineninefol- changes in the phosphorylation level of mTOR (data not
lowing insulin withdrawal, was significantly prevented by shown). Compared with dramatic recovery of mTOR
both pharmacological and genetic inactivation of VCP phosphorylationbyVCPknockdowninI(-),thesedatain-
(Fig. 6a–b). Mammalian target of rapamycin (mTOR) is dicatethatdeficitinautophagyinductioninI(-)HCNcells
known for a nutrient sensorand negativeregulator ofau- depletedofVCPislikeadirectresultofVCPinactivation
tophagy [50]. mTOR activation in relation to autophagy specificallyinI(-)condition.
suppression is reflected by various phosphorylation sites Double FYVE-domain-containing protein 1 (DFCP1) in-
including serine 2448 in response to insulin or nutrient teractswith phosphatidylinositol 3-phosphate (PtdIns(3)p),
stimulation [51, 52]. S2448 of mTOR was dephosphory- which is required for the early step of autophagosome
latedinI(-)HCNcells.However,itsphosphorylationlevel formation. Therefore,DFCP1isregardedasanautophagy
A B Insulin : + - -
Insulin: + - - NT siRNA : + + -
DBeQ: - - + VCP siRNA: - - +
p-mTOR(S2448) p-mTOR(S2448)
Calpain2 Calpain2
p-GSK3 (S9) p-
-Actin -Actin
C
DAPI DFCP1 Merge
I(+) ell
c
r
e
p 15 *** ***
1
P
C 10
F
I(-) D
f 5
o
r
e
b 0
mu I(+) I(-) BeQ
N D
DBIe(-Q)/ I(-)/
Fig.6VCPisrequiredforautophagyinitiationsignalinginHCNcellsfollowinginsulinwithdrawal.a-bNegativeregulatorsofautophagy,mTOR
andcalpain2weresuppressedinI(-),butreactivatedbyDBeQtreatment(a)orVCPknockdown(b).Onthecontrary,positiveinducersof
autophagysuchasGSK-3βwereactivatedfollowinginsulinwithdrawal,butdeactivatedbyDBeQtreatment(a)orVCPknockdown(b).cThehigh
numberofDFCP1punctaobservedinI(-)weregreatlyreducedbyDBeQtreatment.ThenumberofDFCP1punctawasquantifiedwithcut-off
diameterof0.5μm.Scalebar,5μm.QuantitativeDFCP1punctaweredeterminedasthemean±SDbyOne-wayANOVAfollowedbyTukey’s
multiplecomparisontest(n=10).***p<0.001
Yeoetal.MolecularBrain (2016) 9:31 Page10of14
Fig.7AschematicdiagramillustratingthenovelfunctionofVCPinACDandapoptosisinHCNcellsfollowinginsulinwithdrawal.HCNcells
undergoautophagiccelldeathfollowinginsulinwithdrawal.Inbasalautophagy,VCPpositivelyregulatesautophagosomematuration.However,
followinginsulinwithdrawal,VCPregulatesautophagyinitiationandthecrosstalkbetweenACDtoapoptosis
initiation marker[53].A transient transfectionofDFCP1- Our previous study revealed that calpain 2 inhibition
GFPconstructgavethehighnumberofDFCP1punctain and GSK-3β activation promoted ACD following insulin
I(-) HCN cells, compared with the negligible number in withdrawal HCN cells [14, 15]. In accordance with in-
I(+). However, DBeQ treatment strongly suppressed the hibition ofACDuponVCP inactivationinI(-)condition,
formation of DFCP1 puncta, again suggesting that VCP there was significant increase in calpain 2 and inactive
activity is required for autophagy initiation in I (-) HCN form of GSK-3β. GSK-3β and calpain 2 are anticipated
cells (Fig. 6c). This novel role of VCP in autophagy initi- as potential functional or physical interacting candidates
ation has not been reported before. Taken together, these ofVCPinHCNcells.
data suggest that VCP contributes to the maturation of We propose the following model for distinct roles of
autophagosomes to autolysosomes at basal autophagy VCP under different cellular stress conditions (Fig. 7). In
process under I(+) condition. However, when autophagy I(+) cells, VCP ensures the maturation of autophago-
flux is intensified upon insulin withdrawal, VCP is critic- somes and blocking of VCP activity impairs the late
ally involved in the initiation stage of autophagy, as well stage of autophagy flux at basal autophagy. When cells
exemplified in insulin-deprived HCN cells, which under- are encountered with high autophagy level following in-
goeshighautophagylevelandcelldeath. sulin withdrawal, depletion of VCP activity affects the
early stage of autophagy flux and diminishes the autoph-
Discussion agy initiating signals. Of note, our novel discovery also
Inthisreport,wedemonstratethatVCPisakeyregulator indicates another function of VCP in association with
of autophagy initiation signaling and a mediator of cross- crosstalk between autophagy and apoptosis, as the in-
talkbetweenACDandapoptosisininsulin-deprivedHCN action of VCP dramatically decreased autophagy initi-
cells. Theanalysisofthe phenotypes of VCP inhibitionor ation while the mode of cell death switched to apoptosis
knockdown revealed two important observations regard- in insulin-deprived HCN cells. Further studies should
ing autophagy process and cell death. First,VCP inactiva- clarify how VCPmovesfromalatestagetoanearlystage
tion did not alter autophagy induction under normal of autophagy under condition of insulin withdrawal. An-
conditionwheninsulinwasprovided.RatherVCPwasre- other important direction of future research will be to
quiredforautophagosomematuration.Ontheotherhand, understand how VCP affects the decision of cell death
VCP inhibition or depletion down-regulated autophagy modebetweenACDandapoptosis,andthemolecularde-
inductionsignalsinI(-)HCNcells.Second,ourresultsin- tailsregulatingthisVCPactivityduringACD.
dicatethatACDisthedominantmodeofPCDininsulin- Autophagy and apoptosis plays an essential role in
deprived HCN cells, but apoptosis can be an alternative development and survival of NSCs. Basal autophagy is
route to cell death when autophagy level is subdued by essential to protect cells against stress conditions by
VCP inhibition or depletion. However, unlike other stud- degrading damaged organelles and proteins. However,
ies, VCP inactivation itself, does not induce apoptosis in excessive autophagy can cause ACD. The abnormal
HCNcellsundernormalculturecondition.Ourdatasug- regulation of autophagy, such as too little autophagy or
gestthatregulationofswitchbetweenACDandapoptosis too much autophagy and ACD, plays an important role
by VCP constitutes a unique regulatory module of cell in aging and neurodegenerative diseases [54–58]. The
deathprogramwhenHCNcellsaredoomedtodiefollow- efforts of understanding the molecular mechanisms of
inginsulinwithdrawal. ACD have continued in recent years. Knowledge on the