Assessing Au ophagy in Mic oglia:
A Two-S ep Model o De e mine
Au ophagosome Fo ma ion,
Deg ada ion, and Ne Tu no e
Ainhoa Plaza-Zabala
1,2
*, Vi ginia Sie a-To e
2,3
and Amanda Sie a
2,3,4
*
1
Depa men o Pha macology, Achuca o Basque Cen e o Neu oscience, Leioa, Spain,
2
Glial Cell Biology Lab, Achuca o
Basque Cen e o Neu oscience, Uni e si y o he Basque Coun y UPV/EHU, Leioa, Spain,
3
Depa men o Neu oscience,
Achuca o Basque Cen e o Neu oscience, Leioa, Spain,
4
Ike basque Founda ion, Bilbao, Spain
Au ophagy is a complex p ocess ha encompasses he enclosu e o cy oplasmic deb is
o dys unc ional o ganelles in memb anous esicles, he au ophagosomes, o hei
elimina ion in he lysosomes. Au ophagy is inc easingly ecognized as a c i ical p ocess
in mac ophages, including mic oglia, as i finely egula es inna e immune unc ions such as
inflamma ion. A gold-s anda d me hod o assess i s induc ion is he analysis o he
au ophagic flux using as a su oga e he exp ession o he mic o ubule-associa ed ligh
chain p o ein 3 conjuga ed o phospha idyle hanolamine (LC3-II) by Wes e n blo , in he
p esence o lysosomal inhibi o s. The e o e, he cu en defini ion o au ophagy flux
ac ually pu s he ocus on he deg ada ion s age o au ophagy. In con as , he mos
impo an au ophagy con olling genes ha ha e been iden ified in he las ew yea s in ac
a ge ea ly s ages o au ophagosome o ma ion. F om a biological s andpoin is he e o e
concei able ha au ophagosome o ma ion and deg ada ion a e independen ly egula ed
and we a gue ha bo h s ages need o be sys ema ically analyzed. He e, we p opose a
simple wo-s ep model o unde s and changes in au ophagosome o ma ion and
deg ada ion using da a om con en ional LC3-II Wes e n blo , and es i using wo
models o au ophagy modula ion in cul u ed mic oglia: apamycin and he ULK1/2
inhibi o , MRT68921. Ou wo-s ep model will help o un a el he e ec o gene ic,
pha macological, and en i onmen al manipula ions on bo h o ma ion and deg ada ion
o au ophagosomes, con ibu ing o dissec ou he ole o au ophagy in physiology and
pa hology in mic oglia as well as o he cell ypes.
Keywo ds: au ophagy, au ophagosome, o ma ion, deg ada ion, LC3, mic oglia
F on ie s in Immunology | www. on ie sin.o g Janua y 2021 | Volume 11 | A icle 6206021
Edi ed by:
Jo ge Co eale,
Fundacio
´n Pa a la Lucha Con a las
En e medades Neu olo
´gicas de la
In ancia (FLENI), A gen ina
Re iewed by:
Daniela Bakula,
Uni e si y o Copenhagen, Denma k
Xiaohong Zhuang,
The Chinese Uni e si y o Hong Kong,
China
Eisuke I aku a,
Chiba Uni e si y, Japan
*Co espondence:
Amanda Sie a
[email p o ec ed]
Ainhoa Plaza-Zabala
[email p o ec ed]
Special y sec ion:
This a icle was submi ed o
Mul iple Scle osis
and Neu oimmunology,
a sec ion o he jou nal
F on ie s in Immunology
Recei ed: 23 Oc obe 2020
Accep ed: 11 Decembe 2020
Published: 29 Janua y 2021
Ci a ion:
Plaza-Zabala A, Sie a-To e V and
Sie a A (2021) Assessing Au ophagy in
Mic oglia: A Two-S ep Model o
De e mine Au ophagosome Fo ma ion,
Deg ada ion, and Ne Tu no e .
F on . Immunol. 11:620602.
doi: 10.3389/ immu.2020.620602
METHODS
published: 29 Janua y 2021
doi: 10.3389/ immu.2020.620602
INTRODUCTION
Au ophagy is a complex phenomenon dedica ed o elimina e
in acellula deb is, om p o ein agg ega es o dys unc ional
o ganelles, and is hus essen ial o main ain cell fi ness (1,2).
In he b ain, ini ial s udies ocused on i s majo ole in neu onal
su i al (3,4), bu mo e ecen e idence sugges s ha au ophagy
also con ols heal h and unc ion o o he b ain cell ypes,
including mic oglia, he b ain mac ophages (1,5). Au ophagy
con ols se e al p ocesses in mic oglia, including me abolic
fi ness (6), inflamma ion, phagocy osis o amyloid be a in
oden models o Alzheime ’sdisease(7), deg ada ion o
ex acellula be a-amyloid fib ils (8) and synuclein (9), myelin
phagocy osis in acu e expe imen al encephalomyeli is (10), as
well as synap ic p uning and social beha io in mice (11).
O e all, au ophagy is eme ging as a majo con olle o
immune cell unc ion, egula ing inna e and adap i e immune
esponses (12).
Assessing au ophagy is complica ed and cu en guidelines
ecommend using se e al complemen a y me hods (13).
None heless, he gold s anda d emains he analysis o he
au ophagic flux using LC3 (mic o ubule-associa ed ligh chain
p o ein 3). Du ing au ophagy, cy osolic LC3 (LC3-I) is conjuga ed
o phospha idyle hanolamine and ec ui ed o he nascen
phagopho e memb anes (LC3-II). The phagopho e hen
encloses cy osolic ma e ial o o ganelles o ming a double-
memb ane au ophagosome, which is hen edi ec ed owa ds he
lysosome o i s enzyma ic deg ada ion. The au ophagic flux is
calcula ed as he di e en ial amoun o LC3-II in he p esence/
absence o lysosomal inhibi o s, such as bafilomycin o
chlo oquine, among o he s. As lysosomal deg ada ion is
inhibi ed au ophagosomes accumula e and, he e o e, he change
in LC3-II exp ession in o ms abou he au ophagosomes ha
would ha e been deg aded, e go, i is a measu e o
au ophagosome deg ada ion. Howe e , LC3-II Wes e n blo aw
da a con ains in o ma ion abou bo h au ophagosome o ma ion
and deg ada ion (Figu e 1A).
Impo an ly, o ma ion and deg ada ion a e egula ed by
conce ed bu independen mechanisms: mos au ophagic-
egula o y genes a e in ol ed in he ea ly s ages o au ophagy,
as is he case o he ATG amily encoding p o eins ha a e
mainly in ol ed in au ophagosome o ma ion and ma u a ion
(1,14). In con as , au ophagosome deg ada ion la gely depends
on lysosomal p o eins and enzymes (Figu e 1B). The e o e, bo h
ea ly and la e s ages o au ophagy should be sys ema ically
analyzed o unde s and he au ophagosome u no e in any
gi en condi ion.
METHODS
Cell Cul u e
The mu ine mic oglial BV2 cell line and p ima y mic oglia we e
used o es au ophagy modula ing compounds. BV2 mic oglia
we e g own and main ained in Dulbecco’sModified Eagle
Medium (DMEM) (Gibco) supplemen ed wi h Fe al Bo ine
Se um 10% (FBS, Gibco) and a mix u e o an ibio ics/
an imyco ic (1%) including, penicillin, s ep omycin, and
ampho e icin (all om Gibco). Fo expe imen s, 1x10
6
cells
adhe ed o uncoa ed plas ic pla es we e used. P ima y
mic oglia cul u es we e pe o med as p e iously desc ibed (15,
16). Pos na al day 0–1 (P0-P1) ms-EGFP mice pup b ains we e
ex ac ed, and he meninges we e peeled o . The ol ac o y bulb
and ce ebellum we e disca ded, and he es o he b ain was hen
mechanically homogenized by ca e ul pipe ing and
enzyma ically diges ed wi h papain (20 U/ml, Sigma), and
deoxy ibonuclease (DNAse; 150 U/µl, In i ogen) o 15 min
a 37°C. The esul ing cell suspension was hen fil e ed h ough a
40 mm nylon cell s aine (Fishe ) and ans e ed o a 50 ml
Falcon ube quenched by 5 ml o 20% FBS (Gibco) in HBSS.
A e wa ds, he cell suspension was cen i uged a 200 g o
5 min, he pelle was esuspended in 1 ml DMEM (Gibco)
supplemen ed wi h 10% FBS and 1% An ibio ic/An imyco ic
(Gibco), and seeded in T75 Poly L-lysine-coa ed (15 ml/ml,
Sigma) cul u e flasks a a densi y o wo b ains pe flask.
Medium was changed he day a e and hen e e y 3–4 days,
always en iched wi h G anulocy e Mac ophage Colony
S imula ing Fac o (5 ng/ml GM-CSF, Sigma). A e confluence
(a 37°C, 5% CO
2
o app oxima ely 14 d), mic oglia cells we e
ha es ed by shaking a 100–150 pm, 37°C, 4 h. Isola ed cells
we e coun ed and pla ed a a densi y o 2x10
6
cells/well on poly-
l-lysine-coa ed plas ic pla es. BV2 and p ima y mic oglia we e
allowed o se le o a leas 24 h be o e expe imen s.
D ug T ea men s
BV2 mic oglia we e ea ed wi h apamycin 100 nM (Fishe
Scien ific) o 6 h in he p esence and absence o bafilomycin 100
nM (SelleckChem) o au ophagy induc ion. P ima y mic oglia
we e ea ed wi h he au ophagy inhibi o MRT68921 1, 10, o 30
µM (Sigma) o 3 o 6 h wi h o wi hou bafilomycin 100
nM (SelleckChem).
P o ein Ex ac ion and Wes e n Blo
Mic oglia we e di ec ly lysed in plas ic pla es wi h RIPA bu e
con aining p o ease inhibi o cock ail (100x) (The moFishe ).
The cell suspension was hen sonica ed o 5s and cen i uged
(10,000 g, 10 min) o ob ain solubilized p o ein in he
supe na an . Sample p o ein con en was quan ified in
iplica es by BCA (Bicinchoninic Acid) assay ki
(The moFishe ) a 590 nm using a mic opla e eade (Syne gy
HT, BioTek). b-me cap oe hanol dena u ed p o eins (15–20 ug)
we e loaded on o 14% T is-glycine polyac ylamide gels
(The moFishe ) and un a 120V o 90min. P o ein samples
we e hen blo ed o ni ocellulose memb anes (0.45 µm po e
size) (The moFishe ) a 200 mA o 90 min o using he T ans-
Blo Tu bo Mini Ni ocelluloseT ans e Pack(Bio-Rad).
Abb e ia ions: APh, Au ophagosome; ATG, Au ophagy- ela ed p o ein; BAF,
Bafilomycin A1; BCA, Bicinchoninic Acid; DegR, Deg ada ion a io; DMEM,
Dulbecco’sModified Eagle Medium; ECL, Enhanced Chemoluminescence;
EXP
-
, basal condi ion; EXP
+
, expe imen al condi ion; FBS, Fe al Bo ine Se um;
Fo mR, Fo ma ion a io; GM-CSF, G anulocy e-Mac ophage Colony S imula ing
Fac o ; LC3-II, mic o ubule-associa ed ligh chain p o ein 3 conjuga ed o
phospha idyle hanolamine; mTORC1, Mechanis ic Ta ge o Rapamycin
Complex 1; Ne R, Ne a io; PD, Pa kinson’s disease; ss, s eady-s a e; TBS-T,
T is Bu e ed Saline con aining 0.1% Tween 20; TFEB, ansc ip ion ac o -EB;
ULK1/2, unc-51-like kinases 1/2.
Plaza-Zabala e al. Two-S ep Analysis o Au ophagy
F on ie s in Immunology | www. on ie sin.o g Janua y 2021 | Volume 11 | A icle 6206022
T ans e e ficiency was e ified by Ponceau S (Sigma) s aining.
Fo immunoblo ing, memb anes we e insed in T is Bu e ed
Saline con aining 0.1% Tween 20 (Sigma) (TBS-T) and hen
blocked o 1 h in TBS-T con aining 5% powde milk.
Memb anes we e a e wa ds incuba ed wi h abbi p ima y
an ibody o LC3 (1:3,000, NB100-2220, No us Biologicals),
and mouse p ima y an ibody o b-ac in (1:5,000, Sigma), in
TBS-T con aining 4% Bo ine Se um Albumin (BSA) o e nigh
(4°C, shake ). Nex day, memb anes we e insed and incuba ed
wi h Ho se adish Pe oxidase (HRP) conjuga ed an i- abbi
(1:5,000) and an i-mouse (1:5,000) seconda y an ibodies (Cell
Signaling) o he apamycinblo o wi h hefluo escen
S a B igh Blue 700 an i-mouse (1:5,000) and S a B igh Blue
700 an i- abbi (1:5,000) seconda y an ibodies (Bio-Rad) o he
MRT68921 blo s in TBS-T con aining 5% powde milk. A e
insing memb anes, p o ein was isualized by Enhanced
ChemiLuminescence (ECL) using Supe signal Wes Fem o
Maximum Sensi i i y Subs a e (The moFishe ) o he
apamycin blo o by immunofluo escence o he MRT68921
blo s, in a ChemiDoc imaging sys em (BioRad). Band in ensi y
was quan ified using he Gel Analyze me hod o Fiji so wa e.
S a is ics
S a is ical analysis was pe o med wi h SigmaPlo . No mali y
and homoscedas ici y we e assessed p io o analysis. Raw LC3
da a was ini ially analyzed by wo-way ANOVA, bu since an
in e ac ion be ween ea men ( apamycin, MRT68921) and
bafilomycin was ound, he global e ec o he ea men was
subsequen ly analyzed by one-way ANOVA. In addi ion, flux,
and o ma ion and deg ada ion a es we e analyzed by one- ail
S uden - es (Figu es 5A, B) o by one-way ANOVA ollowed
by a Holm-Sidak pos hoc es (Figu e 5C). Fo ma ion and
deg ada ion a es we e compa ed o one using a one- ail
S uden - es . Da a is shown as mean ± SEM. Only es s wi h
p <0.05 a e conside ed significan ly di e en ; es s wi h p <0.1 a e
epo ed o ha e a endency.
MODELING AUTOPHAGOSOME
FORMATION AND DEGRADATION
He e we p opose a simple wo-s ep model o analyze au ophagy,
in which he ne numbe o au ophagosomes (i.e., he
au ophagosome pool) a any gi en ime is ea ed as a black
box o which he e is an inpu ( o ma ion) and an ou pu
(deg ada ion) (Figu e 2A). The o ma ion phase encompasses
phagopho e o ma ion, ca go seques a ion, and au ophagosome
closu e, and he deg ada ion phase summa izes he lysosomal
usion and he enzyma ic deg ada ion o he au ophagosome
con en s. None heless, he p ecise defini ion o o ma ion/
deg ada ion in each expe imen al se up depends on he
physiological p ocess blocked by he pa icula lysosomal
inhibi o used: usion inhibi o s, such as inblas ine, which
blocks anspo o au ophagosomes by mic o ubules; p o ease
A
B
FIGURE 1 | Es ima ion o au ophagy flux a ia ions using LC3 u no e assay. (A) To al p o ein homogena es ob ained om mic oglia unde con ol (EXP
-
) and
expe imen al condi ions (EXP
+
) a e analyzed by Wes e n Blo o e alua e LC3 le els in he p esence and absence o lysosomal inhibi o s. When au ophagy is
ac i a ed, LC3-I (soluble o m) is lipida ed o he phopha idyle hanolamine o he nascen phagopho e o ming LC3-II (memb ane-bound o m). LC3-II accumula es
along he ex ension o he au ophagic acuoles as i closes and is used as an es ima e o he numbe o au ophagosomes. Upon usion wi h lysosomes, LC3-II le els
dec ease due o he deg ada ion o he inne au ophagosomal memb ane simul aneously wi h he luminal ca go. In he p esence o lysosomal inhibi o s,no
deg ada ion occu s and LC3-II le els a e main ained. The sub ac ion o LC3-II quan i ies in he p esence and absence o lysosomal inhibi o s p o ides an es ima e o
he au ophagosomes ha ha e been deg aded du ing he expe imen al pe iod o ime. (B) Ea ly s ages o au ophagy, which lead o he de no o o ma ion o
au ophagosomes, a e mainly egula ed by ATG p o eins. The LC3 amily o p o eins (ATG8) pa icipa e in he o ma ion o au ophagosomes and p og essi ely
disappea a e lysosomal usion and ca go deg ada ion in au olysosomes. La e s ages o au ophagy depend on he unc ionali y o lysosomal p o eins and enzymes.
Plaza-Zabala e al. Two-S ep Analysis o Au ophagy
F on ie s in Immunology | www. on ie sin.o g Janua y 2021 | Volume 11 | A icle 6206023
inhibi o s, such as E64d and leupep in; o p o on pump
inhibi o s, such as bafilomycin. This concep ual ame can be
easily modeled by a simple equa ion in which he size o he
au ophagosome (APh) pool in a gi en ime poin depends on
he numbe o au ophagosomes in he s eady-s a e (ss) plus he
numbe o au ophagosomes o med minus he au ophagosomes
deg aded in a ce ain pe iod o ime:
APh = APHss + APh Fo ma ion −APh Deg ada ion
The a io be ween deg ada ion and o ma ion is he ne
au ophagic u no e , which is a measu e o he ela i e eloci y
o au ophagosome o ma ion e sus deg ada ion. A gi en
A
B
DE
C
FIGURE 2 | A wo-s ep model o au ophagy o analyze o ma ion and deg ada ion o au ophagosomes. (A) The model ep esen s he au ophagosomes as a box
wi h an inpu (au ophagosome o ma ion, pu ple do s) and an ou pu (au ophagosome deg ada ion, g een do s) ha de e mines he au ophagosome ne u no e .
A1–A3 ep esen di e en possible scena ios wi h no changes (A1), an inc ease (A2) and a dec ease (A3) in he au ophagosome ne u no e . (B) G aph ep esen ing
he amoun o LC3-II (au, a bi a y uni s) in wo expe imen al condi ions ep esen ing (EXP
-
and EXP
+
) in he p esence o absence o he lysosomal inhibi o
bafilomycin (BAF
-
and BAF
+
), and he o mulas used o calcula e o ma ion, deg ada ion, and ne u no e . The do ed ed a ows ma k he LC3-II aw da a alues
used o calcula e he o ma ion and deg ada ion a es and a ios. (C) Simula ed aw LC3-II da a (au) (le ) used o calcula e he o ma ion and deg ada ion a es and
a ios ( igh ) used in he g aphs shown in (B, D,E).(D,E) G aphs ep esen ing he a e o change o o ma ion, deg ada ion and ne u no e be ween he wo
expe imen al condi ions.
Plaza-Zabala e al. Two-S ep Analysis o Au ophagy
F on ie s in Immunology | www. on ie sin.o g Janua y 2021 | Volume 11 | A icle 6206024
s imulus could ac p opo ionally bo h on he o ma ion and he
deg ada ion, main aining he size o he APh pool and esul ing
in a cons an ne u no e a io (Figu e 2A1). Howe e , unde
some condi ions, he egula ion o he o ma ion and
deg ada ion o au ophagosomes may be dissocia ed: an
inc eased deg ada ion would dec ease he size o he APh pool
and inc ease he ne u no e (Figu e 2A2); and an inc eased
o ma ion would inc ease he size o he APh pool and dec ease
he ne u no e (Figu e 2A3). Thus, we p opose ha o
unde s and he complexi y o he biology unde nea h he
au ophagosome u no e we need o analyze sepa a ely
o ma ion, deg ada ion, and he ne au ophagic u no e .
This analysis can be pe o med using he da a a ailable in
con en ional LC3 assays by Wes e n blo . In his ype o analysis,
cells o issue om wo expe imen al condi ions (EXP
-
: con ol
and EXP
+
: expe imen al s imulus) a e incuba ed in he p esence
o absence o lysosomal inhibi o s such as bafilomycin (BAF
-
and
BAF
+
) o a ce ain pe iod o ime. P o ein om hese ou
condi ions is ex ac ed and he exp ession o LC3-II is analyzed
by Wes e n blo and, ideally, no malized o e e ence p o eins
such as ac in (13). In addi ion, he complemen a y no maliza ion
o LC3-II o LC3-I may acili a e he unde s anding o he ull
pic u e o he au ophagy esponse (13).
In he basal condi ion (EXP
-
), he amoun o LC3-II in he
absence o lysosomal inhibi o s (BAF
-
) ep esen s he APh pool
in he s eady s a e, analogous o aking a snapsho o he
au ophagic p ocess (Figu e 2B). The di e ence be ween he
amoun o LC3-II in cells incuba ed wi h and wi hou
lysosomal inhibi o s (BAF
+
–BAF
-
) in he basal condi ion
ep esen s he au ophagosomes ha ha e disappea ed (i.e., he
deg ada ion phase), which is wha is con en ionally called
au ophagic flux. To calcula e he au ophagosomes ha ha e
o med, ou model s ems om he assump ion ha in he
basal condi ion au ophagy is a an equilib ium because
o ma ion and deg ada ion occu a he same speed:
Basal condi ion :
APh Fo ma ion = APh Deg ada ion
APhequilib ium = APhss
Thus, in he basal condi ion (EXP
-
) he au ophagosomes ha
ha e o med a e iden ical o he au ophagosomes ha ha e
deg aded, and hus a e also ep esen ed by he amoun o LC3-
II wi h and wi hou lysosomal inhibi o s (BAF
+
–BAF
-
)(Figu es
2B, C).
In he expe imen al condi ion (EXP
+
), he amoun o LC3-II
in he absence o lysosomal inhibi o s (BAF
-
in EXP
+
) ep esen s
he size o he APh pool unde he s imulus. Again, deg ada ion
can be calcula ed as he di e ence in LC3-II wi h and wi hou
lysosomal inhibi o s [(BAF
+
in EXP
+
)–(BAF
-
in EXP
+
)].
Fo ma ion can be calcula ed as he di e ence be ween he
amoun o LC3-II in he p esence o lysosomal inhibi o s
minus he size o he ini ial APh pool in s eady-s a e
condi ions [(BAF
+
in EXP
+
) - (BAF
-
in EXP
-
)] (Figu es 2B,
C). This p ocedu e allows us o calcula e he o ma ion and
deg ada ion o au ophagosomes in con ol and expe imen al
condi ions (Figu e 2D). To hen compa e whe he he
s imulus ac s p opo ionally in bo h o ma ion and
deg ada ion, we can calcula e he a io be ween expe imen al
and basal condi ions (EXP
+
/EXP
-
) o bo h o ma ion and
deg ada ion (Figu e 2E). Finally, o compa e he ela i e
magni ude o deg ada ion compa ed o o ma ion, we can
calcula e he a io be ween bo h as he ne u no e a io
(Figu e 2E), which has a alue o one in basal condi ions,
because au ophagosome o ma ion and deg ada ion occu a
he same a e ( ed do ed line in Figu e 2E).
DISSECTING OUT AUTOPHAGOSOME
FORMATION AND DEGRADATION
This model allows us o disc imina e and quan i y di e en
po en ial biological scena ios ha may a ec au ophagosome
o ma ion, deg ada ion, o bo h. Fo ins ance, a ypical
au ophagic s imulus would be expec ed o p opo ionally
inc ease au ophagosome o ma ion and deg ada ion,
main aining a balanced au ophagy (Figu e 3A1). Examples o
his scena io a e ea men wi h he well-known au ophagy
ac i a o apamycin, an inhibi o o he mTORC1 complex
(Mechanis ic Ta ge O Rapamycin Complex 1) (17); o
ac i a ion o he ansc ip ion ac o -EB (TFEB), which
coo dina ely egula es he biogenesis o au ophagosomes and
lysosomes (18), main aining he equilib ium be ween o ma ion
and deg ada ion.
To exempli y his scena io, we simula ed aw LC3-II Wes e n
blo da a om a canonical au ophagy s imulus (Figu e 3A2), and
om he e we calcula ed he classic au ophagy flux, showing he
expec ed inc ease (Figu e 3A3). We hen applied ou model o
he aw da a and obse ed ha he canonical au ophagic
s imulus inc eased bo h o ma ion and deg ada ion (Figu e
3A4). Impo an ly, bo h o ma ion and deg ada ion a ios we e
simila and, as a esul , he ne au ophagy a io was cons an
(Figu e 3A5), implying a main enance o he ne au ophagic
u no e bu a a highe a e/ eloci y, ha could be possibly
main ained in he long e m.
In con as , he e a e o he biological scena ios ha a e no so
easily disc imina ed using he con en ional calcula ion o he
au ophagy flux (Figu es 3B, C). Examples o hese scena ios a e
si ua ions in which au ophagosome o ma ion is inc eased
(Figu e 3B) o dec eased (Figu e 3C), wi hou concomi an ly
a ec ing deg ada ion. Fo ins ance, o e exp ession o ATG
p o eins o accumula ion o in acellula deb is would lead o
inc eased au ophagosome o ma ion. Bu i lysosomal e ficiency
(i.e., deg ada ion) is no p opo ionally inc eased,
au ophagosomes will s all in he lysosomes wi hou deg ading
he au ophagic ca go, leading o a dec eased ne u no e a io
and inc eased au ophagosome pool. This e ec has been o
example obse ed in cells ha o e exp ess A g5 bu whose
lysosomal unc ion is comp omised (19). In his case,
calcula ion o he au ophagy flux would no e eal any changes
(Figu es 3B1, C1), al hough he aw LC3-II da a is e iden ly
di e en (Figu es 3B2, C2). Ou model would help o quan i y
Plaza-Zabala e al. Two-S ep Analysis o Au ophagy
F on ie s in Immunology | www. on ie sin.o g Janua y 2021 | Volume 11 | A icle 6206025
he specific e ec on o ma ion (Figu es 3B3, C3), and he
al e a ion o he ne au ophagy a io (Figu es 3B4, C4),
e ealing an unbalanced au ophagy (Figu es 3B5, C5), and a
po en ially ca as ophic si ua ion o he cell ha could no
possibly be main ained o e ime.
O he biological scena ios ha canno be disc imina ed using
con en ional analysis o he au ophagy flux a e shown in Figu es
4,5. Some s imuli may selec i ely inc ease au ophagosome
deg ada ion wi hou a ec ing hei o ma ion coo dina ely
(Figu e 4A), o e en educing i (Figu e 4B). Fo example,
enhanced lysosomal biogenesis o lysosomal enzymes e ficiency
migh lead o inc eased au ophagosome deg ada ion, esul ing in
an inc eased ne u no e a io and educed au ophagosome
pool size. This imbalance has been epo ed in mice gene ically
deficien o he ca hepsin inhibi o cys a in B, which exhibi
enhanced lysosomal p o eolysis (20). Whe eas in his case he
calcula ion o he au ophagy flux would sugges an enhanced
au ophagy, ou model would e eal he imbalance be ween
o ma ion and deg ada ion, sugges ing ha in ac cellula
deb is would no be emo ed any as e om he cy oplasm.
Ano he scena io in which ou model may p o e use ul is one
whe e au ophagosome deg ada ion is educed bu o ma ion is
A
BC
FIGURE 3 | Theo e ical examples o a ia ions in he o ma ion o au ophagosomes ha lead o balanced o unbalanced au ophagy. (A) Example o a balanced flux
wi h p opo ional inc ease in au ophagosome o ma ion and deg ada ion. The model o balanced flux wi h equal o ma ion (pu ple do s) and deg ada ion (g een do s)
(A1), he aw LC3-II/ac in Wes e n blo da a (A2), he con en ional au ophagy flux (A3), he o ma ion and deg ada ion a es (A4), and he o ma ion, deg ada ion, and
ne a ios (A5) a e shown. The ed do ed line ep esen s he h eshold o one o de e mine a significan change (o e 1, basal condi ions) in he o ma ion,
deg ada ion and ne u no e a ios. (B,C) Show examples wi h simila con en ional flux (B1, C1), which a e in ac de i ed om dissimila aw LC3-II/ac in Wes e n
blo da a (B2, C2). In (B) Ou model would e eal inc eased au ophagosome o ma ion a e and no changes in deg ada ion a e (B3), leading o an inc eased
o ma ion a io and educed ne a io (B4), and an unbalanced au ophagy (B5). In con as , in (C) ou model would e eal dec eased au ophagosome o ma ion a e
and no changes in deg ada ion a e (C3), leading o an inc eased o ma ion a io and educed ne a io (C4), and an unbalanced au ophagy (C5).
Plaza-Zabala e al. Two-S ep Analysis o Au ophagy
F on ie s in Immunology | www. on ie sin.o g Janua y 2021 | Volume 11 | A icle 6206026
main ained (Figu e 5A) o e en inc eased (Figu e 5B). An
exampleo hisscena ioisapa hologicalcondi ionwhe e
dys unc ional o ganelles accumula e and he cell ies o
enclose hem in au ophagosomes bu lysosomal unc ionali y is
comp omised, o ins ance because lysosomes a e de ec i e o
engaged in o he deg ada ion pa hways such as phagocy osis o
endocy osis. This e ec could be obse ed in Pa kinson’s disease
(PD) dopamine gic neu ons, which con ain LC3-posi i e Lewy
bodies, and ha e s alled au ophagosomes, and lysosomal
deple ion (21). This complex e ec canno be ully unde s ood
by simply analyzing he educ ion in he au ophagy flux bu
would be ins ead clea ly desc ibed by ou wo-s ep model.
TESTING THE MODEL IN VITRO
We ha e di ec ly es ed ou model wi h expe imen al da a using
wo well-cha ac e ized au ophagy modula o s: he au ophagy
induce apamycin, which inhibi s mTORC1 (22); and he
au ophagy inhibi o MRT68921, which blocks ULK1/2 (unc-
51-like kinases 1/2) (22,23). Bo h mTORC1 and ULK1/2 a e
ea ly checkpoin s o canonical au ophagy: mTORC1 ansduces
signals om ene gy and damage senso s and is inhibi ed unde
s ess ul si ua ions, eleasing ULK1/2 (unc-51-like kinase 1/2) by
a se ies o phospho yla ion and dephospho yla ion e en s o
ini ia e he au ophagy cascade (1,14). As a cell model we used
cul u es o mic oglia (BV2 cells o p ima y cul u es) and
analyzed he amoun o LC3-II by Wes e n blo as a
measu emen o he size o he au ophagosome pool.
In BV2 mic oglia apamycin (6 h, 100 nM) showed he
expec ed esponse and a endency o inc eased LC3-II flux
(Figu e 6A). In addi ion, ou model unco e ed a pa allel
inc ease in o ma ion and deg ada ion o au ophagosomes,
esul ing in a cons an size o he APh pool and no changes in
he ne au ophagosome u no e . Thus, apamycin allowed he
main enance o he equilib ium be ween o ma ion and
deg ada ion (Figu e 6A), indica ing a sus ained au ophagy ha
he cell can main ain o e ime.
On he o he hand, MRT68921 (3 h, 30 mM) esul ed in he
expec ed dec ease in he LC3-II flux in p ima y mic oglia (Figu e
6B). Howe e , analysis wi h ou model e ealed ha only
deg ada ion was educed whe eas au ophagosome o ma ion
emained cons an (Figu e 6B). This da a is in appa en
con adic ion wi h he desc ibed ole o MRT in blocking he
au ophagy p e-ini ia ion complex (22,23). To add ess his
disc epancy, we used a second pa adigm o MRT68921 wi h a
longe ea men and lowe dosage (6 h, 1–10 mM; Figu e 6C), and
obse ed ha he ups eam e ec o inhibi ion o au ophagosome
o ma ion wi h MRT 10µM ansla ed in o a simila dec ease in
deg ada ion (Figu e 6C). The e o e, ou model p o es use ul o
disc imina e he e ec o expe imen al manipula ions on he
o ma ion and/o deg ada ion o au ophagosomes.
AB
FIGURE 4 | Theo e ical examples o inc eased au ophagosome deg ada ion ha lead o unbalanced au ophagy. (A, B) show examples wi h simila con en ional flux
(A1, B1), de i ed om appa en ly simila aw LC3-II/ac in Wes e n blo da a (A2, B2). In (A) ou model would e eal an inc eased au ophagosome deg ada ion a e
and no changes in he o ma ion a e (A3), leading o an inc eased deg ada ion a io and ne a io (A4), and an unbalanced au ophagy (A5). In con as , in (B) ou
model would e eal dec eased au ophagosome o ma ion a e bu inc eased deg ada ion (B3), leading o dec eased o ma ion a io, inc eased deg ada ion a io, and
a s ong inc ease in he ne a io (C4), ul ima ely esul ing in a highly unbalanced au ophagy (B5).
Plaza-Zabala e al. Two-S ep Analysis o Au ophagy
F on ie s in Immunology | www. on ie sin.o g Janua y 2021 | Volume 11 | A icle 6206027
FUTURE DIRECTIONS
Au ophagyisacomplexmul i-s epphenomenonandi s assessmen
is a complica ed ask ha equi es using complimen a y me hods,
as mos cu en guidelines ecommend (13,24). Visualiza ion o
double-memb ane au ophagosomes by ansmission elec on
mic oscopy, li e imaging o LC3 acidifica ion using a iome ic
analysis o fluo opho es, o analysis o subs a e deg ada ion should
co obo a e he da a ob ained by analysis o LC3-II exp ession as a
p oxy o au ophagosome o ma ion and deg ada ion. I is also
impo an o no e ha au ophagy is a ime-dependen p ocess and,
as such, i s dynamics should be assessed o e ime (25). In addi ion,
LC3-II immunoblo ing assays ha e se e al limi a ions, such as he
e e ence p o ein used o no malize LC3-II alues, he iming and
concen a ion o he lysosomal inhibi o used, o he in insic
nonlinea de ec ion o p o eins by enhanced chemoluminescence
(ECL) (26). The mos widely used me hod o asses au ophagy is,
none heless, he analysis o he LC3-II flux in he p esence o
lysosomal inhibi o s. Howe e , he complexi ies associa ed o
in e p e ing LC3-II flux ha e been ho oughly poin ed ou be o e,
in he ques o an op imal “au ophagomome e ”(26). One o he
key poin s is ha au ophagosomes o ma ion and deg ada ion a e
spa ially and empo ally dissocia ed (27) and ha he e o e hey
need o be assessed independen ly.
To add ess his issue, we he e p opose a simple concep ual
ame o help in e p e ing LC3-II flux expe imen s. Ou wo-s ep
model concei es he s eady-s a e le els o LC3-II as an indi ec
measu e o he pool o au ophagosomes p esen when he snap-
sho is aken. Assuming ha in he basal condi ion he cells o
issue o in e es a e in some so o equilib ium, he amoun o
au ophagosomes o med and deg aded should be oughly he
same. Thus, he au ophagosome pool can be ea ed as a black
box o which he inpu ( o ma ion) and ou pu (deg ada ion) a e
iden ical, and can be es ima ed as he di e ence be ween LC3-II
le els in he p esence and absence o lysosomal inhibi o s. In he
expe imen al condi ion, deg ada ion can be simila ly calcula ed
as he di e ence be ween LC3-II le els in he p esence and
absence o lysosomal inhibi o s (i.e., he con en ional LC3-II
flux). In addi ion, we p opose ha he o ma ion o
au ophagosomes in he expe imen al condi ion can be
es ima ed by sub ac ing he s eady-s a e au ophagosome pool
o he au ophagosomes ha ha e accumula ed in he p esence o
lysosomal inhibi o s. This model allows us o dissec ou he
e ec s o he expe imen al condi ions o au ophagosome
o ma ion and deg ada ion. In addi ion, i also allows us o
unde s and he ne changes in he size o he au ophagosomal
pool ha a e he esul o main aining (o no ) he ne u no e
a io a equilib ium.
We ha e es ed he wo-s ep model using pha macological
au ophagy modula o s such as he au ophagy induce apamycin
and he au ophagy inhibi o MRT68921 in mic oglia. As
expec ed, apamycin enhanced au ophagy flux inc easing bo h
AB
FIGURE 5 | Theo e ical examples o dec eased au ophagosome deg ada ion ha lead o unbalanced au ophagy. (A, B) show examples wi h simila con en ional
flux (A1, B1), de i ed om appa en ly simila aw LC3-II/ac in Wes e n blo da a (A2, B2). In (A) ou model would e eal a dec eased au ophagosome deg ada ion
a e and no changes in he o ma ion a e (A3), leading o a educed deg ada ion a io and ne a io (A4), and an unbalanced au ophagy (A5). In con as ,in(B) ou
model would e eal inc eased au ophagosome o ma ion a e bu dec eased deg ada ion (B3), leading o inc eased o ma ion a io, educed deg ada ion a io, and a
s ong educ ion in he ne a io (C4), ul ima ely esul ing in a highly unbalanced au ophagy (B5).
Plaza-Zabala e al. Two-S ep Analysis o Au ophagy
F on ie s in Immunology | www. on ie sin.o g Janua y 2021 | Volume 11 | A icle 6206028
au ophagosome o ma ion and deg ada ion a he concen a ion
(100 nM) and ime poin (6 h) es ed. Howe e , he au ophagy
inhibi o MRT68921 exhibi ed concen a ion and ime-
dependen di e en ial e ec s. A a medium concen a ion (10
mM) and long ime-poin (6 h), MRT68921 dec eased bo h
au ophagosome o ma ion and deg ada ion, in line wi h he
inhibi o y e ec s desc ibed o e ULK1/2 kinase ac i i y, while
no e ec was obse ed a a lowe concen a ion (1 mM).
Ne e heless, a high concen a ion (30 µM) and sho ime-
poin (3h), MRT68921 selec i ely dec eased au ophagosome
deg ada ion while main aining hei o ma ion. This was an
unexpec ed esul since MRT68921 inhibi s ULK1/2 kinase, a
p o ein mainly known o i s ole in au ophagy ini ia ion (28).
Howe e , ULK1/2 kinase also egula es he ec ui men o o he
au ophagy- ela ed p o eins o he p oduc i e o ma ion o
au ophagosomes (23,29). Thus, inhibi ion o ULK1/2 kinase
ac i i y a high concen a ions and sho ime-poin s could
p e e en ially a ec au ophagosome deg ada ion ac i i y,
A
B
C
FIGURE 6 | Valida ion o he wo-s ep model wi h au ophagy modula ing compounds. (A) Au ophagy induc ion assessed a e ea men wi h apamycin (100 nM,
6 h) in he p esence and absence o Bafilomycin (100 nM) in he BV2 mic oglia cell line. A ep esen a i e blo , he aw da a ob ained, and he calcula ions o flux,
au ophagosome o ma ion and deg ada ion, and ne u no e a ios a e shown. Da a is p esen ed as % o e con ol (LC3-II/ac in). (B, C) Au ophagy inhibi ion
assessed a e ea men wi h MRT68921 (30 mM, 3 h in (B); 1 and 10 mM, 6 h in (C) in he p esence and absence o Bafilomycin (100 nM) in mouse p ima y
mic oglia. A ep esen a i e blo , he aw da a ob ained, and he calcula ions o flux, au ophagosome o ma ion and deg ada ion, and ne u no e a ios a e shown.
Da a is p esen ed as % o e con ol (LC3-II/ac in). Da a ep esen mean ± SEM o 3 independen expe imen s.
#
ep esen s p < 0.1, * ep esen s p < 0.05 and
** ep esen s p < 0.01 by one ailed S uden - es (A, B), o Holm-Sidak a e a significan e ec o he ea men was ound wi h 1-way ANOVA (C).
Plaza-Zabala e al. Two-S ep Analysis o Au ophagy
F on ie s in Immunology | www. on ie sin.o g Janua y 2021 | Volume 11 | A icle 6206029
