proteomic characterization of csf extracellular …
TRANSCRIPT
PROTEOMIC CHARACTERIZATION OF CSF EXTRACELLULAR VESICLES IN HIV PATIENTS Debjani Guha1, David R Lorenz1, Vikas Misra1, Sukrutha Che�mada1, Susan Morgello2, Dana Gabuzda1
1Department of Cancer Immunology and Virology, Dana-Farber Cancer Ins�tute, Boston, MA 2Departments of Neurology, Neuroscience and Pathology, Mount Sinai Medical Center, New York, NY
400 μl CSF (n=20)
Isolation of EVs by ExoQuick method
Depletion of abundant proteins Protein A/G beads + Protein L beadsProteome Purify-12 Immunodepletion
Resin
EV fraction EV-depleted CSF
Untargeted Proteomics by ABSciex 4800Plus MALDI-TOF/TOF
Data analysis, Gene ontology (GO)
CD9
FLOT-1
HSP70 1 2 3 4 5
CD81
70 kDa
47 kDa
25 kDa
25 kDa
b EV-1a
EV (n=20) EV-depleted CSF (n=20)
713 (26.1%) abundant proteins excluded
Number of proteins analyzed=2014
Number of proteins with > 2 unique peptides=1134 (56.2%)
Total number of proteins=1626
364 (22.4%) abundant proteins excluded
Number of proteins with > 2 unique peptides=702 (55.6%)
Total number of proteins=2727
Number of proteins analyzed=1262
c d
3442527295
140
17
26
MWkDa
260
EV-1
21%
15%
21%13%
11%
19%
Biological processes
24%
18%
27%
11%
11%
9%
Immune response
Stress responseMitochondriaInflammationMetabolic process
Vesicles
c
Stre
ss re
spon
se
Inflam
mation
Metabo
lic pr
oces
s
Mitoch
ondri
a0
2000
4000
6000
8000
10000 Biological processes
Tota
lpep
tide
coun
ts
Immun
e res
pons
e
Epithe
lial c
ells
0
2000
4000
6000
8000
10000 Cellular components
Tota
lpep
tide
coun
ts
Neuron
s
Endoth
elial
cells
Myeloi
d cell
s
Astroc
ytes
Choroi
d plex
us
Blood-b
rain b
arrier
Oligod
endro
cytes
22%
7%
22%
3%
10%18%
Cellular componentsNeuronsMyeloid cellsAstrocytesEndothelial cells
OligodendrocytesEpithelial cellsBlood-brain barrierChoroid plexus
20%
17%
10%18%2%
24%
3%6% 5%
#to
talp
eptid
e hi
ts
ALDH
1L1
GFAP
GLUL
NDRG
2PE
A15
S100
BSL
C1A3
SLIT
RK40
100
200
300
400 Astrocytes
CD14
CD16
3CD
H2CH
I3L1
CSF1
RM
ARCO M
IFM
MP2
MM
P9M
POM
RC1
MRC
20
100
200
300
400 Myeloid cells
CA2
CRYA
BEP
HX2
GLUL
GRIA
4M
BPM
OGPD
GFA
PEBP
1PL
P1RE
G3A
TNR
0
100
200
300
400 Oligodendrocytes
ALDH
1A2
ATP1
A1AT
P1A2
ATP1
B1AT
P2B1 EZR
GNAI
2GN
AI3
LAMA
5LA
MB1
LAM
P2NI
D1
0
100
200
300
400 Epithelial cells
ANGP
TL1
CAV1
CTNN
B1IC
AM1
ICAM
2IC
AM5
ITGB
1M
CAM
VCAM
1VW
F0
100
200
300
400 Endothelial cells
ACO1
ACO2
ADH1
AAD
H1B
ATP5
A1AT
P5B
CA2
CALM
1CA
LM2
CAT
CLIC
6DS
TNPP
M1J
SEPT
2TG
FBI
TIM
P1
0
100
200
300
400 Choroid plexus
AGRN
AQP1
AQP4
DAG1
FBLN
1FB
LN2
FBLN
5IC
AM1
ICAM
5LA
MA2
LAMA
4LA
MA5
LAM
C1LR
P1NI
D1NI
D2VC
AM1
VWF
0100200300400500600 Blood-brain barrier
ACP1
ANXA
1AN
XA2
ANXA
4AN
XA5
B2M
CAL
REI
F6H
LA-A
HLA
-BH
LA-D
RAIC
OSL
GM
BL2
MPO NC
LN
T5C
2PS
MA7
0
100
200
300
400 Immune response
CR
PEN
O1
GAS
6IL
6ST
ISG
15LT
A4H
MSN
PLTP
POST
NR
HO
ASI
RPA
THBS
1TR
OVE
20
100
200
300
400 Inflammation
ACO
T2AC
OT7
CU
TAD
NM
1LD
NPE
PG
LUD
1PC
MT1
PTR
FRA
NVD
AC1
VDAC
20
100
200
300
400 Mitochondrial function
PDCD
6IP
SDCB
PAR
F1AR
F3AR
F6CD
9CD
81RA
B11B
RAB1
4RA
B18
RAB1
ARA
B1C
RAB2
ARA
B2B
RAB3
ARA
B5A
RAB5
BRA
B5C
RAB6
ARA
B6C
RAB7
ARA
B8A
RAB8
BHS
P90A
A1HS
P90A
A2P
HSP9
0AB1
HSP9
0AB2
PHS
P90A
B3P
HSP9
0AB4
PHS
P90B
1HS
PA1B
HSPA
1LHS
PA2
HSPA
4HS
PA5
HSPA
8HS
PB1
HSPD
1
0
30
60
90
120
150
180
210
240
#tot
alpe
ptide
hits EV
EV-depleted CSF
#to
talp
eptid
e hi
ts#
tota
lpep
tide
hits
a
b
c
ENO2
GRIA
4L1
CAM
NCAM
1NC
AM2
NEFL
NFAS
CNP
TNNP
TX1
NPTX
2NR
XN1
NRXN
2NR
XN3
SNAP
91SY
N1
0
100
200
300
400 Neurons
a b
Fig. 2 Comparison of CSF EV and EV-depleted CSF proteins in HIV+ subjects (n=20). (a) Flowcharts summarizing numbers of total and abundant proteins detected in CSF EV fractions and EV-depleted CSF by untargeted proteomics. (b) Comparison of total peptide counts for proteins mapped to selected biological process and cellular component ontology terms in EV fractions and EV-depleted CSF. (c) Pie-charts illustrating the percentage of proteins mapped to different biological process and cellular component categories in EV fractions and EV-depleted CSF by GO analysis and expression annotation.
EV EV-depleted CSF
27%
20%14%
Fig. 3 Proteins associated with exosomes, cellular components, and biological processes are abundant in CSF EV fractions. (a) CSF EV fractions are enriched with exosomal proteins compared to EV-depleted CSF. Comparative abundance of proteins related to (b) neurons, astrocytes, myeloid cells, oligodendrocytes, endothelial cells, epithelial cells, blood-brain barrier (BBB), and choroid plexus (CP), and (c) biological processes including immune responses, inflammation, stress responses, and mitochondrial functions in EV fractions and EV-depleted CSF. Bar graphs show the number of total peptide counts for individual proteins among all EV fractions and EV-depleted CSF.
EV co
ncen
tratio
n(lo
g 10pa
rticle
s/ml)
a
c
9
10
11
12p=0.004
All HIV+ No HAND HAND0
200
400
600
800
1000
#an
alyz
edpr
otei
ns
No HANDHAND
p=0.007
p=0.11
EV fraction EV-depleted CSF
b
-10 -5 0 5 100
1
2
3
4
Log 2 Fold change
-Log
10(p
-val
ue)
LAMB1
LRP1LAMC1FBLN1 MFGE8LUM
ANXA2
SELENBP1PRDX6
FC=2
p=0.05
d eHAND
No HAND
43 507 579NPTN
NFASCCD14
VCAM1
Log2 Fold change
-Log
(p-v
alue
)
FC=-2
-10 -5 0 5 100
1
2
10
p=0.1
p=0.05CDH13 LAMB1
LAMB2LRP1 SELENBP1ANXA2
MFGE8
NFASCPRDX2
PRDX6CD14
CHI3L1MMP2
ANXA4
FC=2FC=-2ANI+MND
No HAND
55 493 462
f
Protein hits
Immune response
AARS, ACP1, AGRN, AHCY, ANP32A, ANPEP, ANXA1, ANXA2, ANXA3, ANXA4, ANXA5, ANXA6, ANXA11, ARF6, ATG7, ATP1B3, ATRN, BGN, BIN1, BPIFA1, BSG, CADM1, CALR, CAP1, CAT, CAV1, CD14, CD163, CD44, CD47, CD81, CD9, CDC42, CHI3L1, CHST15, CPE, CRP, CTSD, CTSF, CTSG, CTSL, CTSS, DBI, DCN, DDAH2, DEFA1, EFHD2, EGFR, EIF6, EPB41L2, EPHA4, EZR, FERMT2, FLNA, FUS, GAPDH, GAS6, GMDS, GNAI2, GNAS, GPI, GPX4, GSTP1, HARS, HLA -A, HLA -B, HLA- DRA, HMGB1, HSP90AA1, HSP90AA2P, HSP90AB1, HSP90AB2P, HSP90AB3P, HSP90AB4P, HSP90B1, HSPA1A, HSPA4, HSPA5, HSPB1, HSPD1, ICAM1, ICAM5, IFIT1, IFITM1, IFITM3, ,IL6ST, ILF2, ISG15, ITGB1, KARS, LAMP 2, LBP, LCP1, LTA4H, LY6H, LYZ, MAPK1, MAPK3, MARCKS, MASP1, MBL2, MBP, MFGE8, MIF, MMP9, MPO, MRC1, MVP, MX1, NACA, NAMPT, NCAM1, NCL, NDRG1, NQO1, NT5C2, NT5E, OAS3, PACSIN1, PARK7, PDE12, PNP, PPIA, PPIB, PPP2R1A, PRKCA, PROC, PSMA7, PSMB8, PSMB9, PSME1, PTPN11, PTX3, PZP, RAB7A, RAC1, RHOA, RNPEP, ROCK1, S100A11, S100A13, SAG, SEMA7A, SET, SH3KBP1, SIRPA, SLC3A2, SOD1, SOD2, STAB1, STAT1, THBS1,THY1, TPP2, TROVE2, TUBB, TXN, USP7, USP14, VCAM1, WARS, YARS
Inflammatory response
AKR1B1, AMBP, ANXA1, ANXA3, APEX1, ASS1, BPIFA1, CALR, CAP1, CAPNS1, CD14, CD163, CD44, CD47, CDC42, CFL1, CHI3L1, COPS5, CPNE1, CRP, CTSG, CTSS, DCN, DDX39B, DEFA1, EGFR, EIF5A, EIF6, ELN, ENO1, EPHX2, EZR, FBLN5, FCN2, FLII, FLNA, FLOT1, FTH1, GAS6, GCLC, GM2A, GNA13, GPI, GSTP1, HLA-DRA, HMGB1, HSP90AA1, HSPA1A, HSPB1, HSPD1, ICAM1, IL6ST, ISG15, ITGB1, LAMP2, LBP, LTA4H, LYZ, MAPK1, MAPK3, MARCH1, MBL2, MCAM, MFGE8, MIF, MMP2, MMP9, MPO, MRC1, MSN, NAMPT, NDRG1, NT5E, PDE12, PLEC, PLTP, POSTN, PRDX5, PRKCA, PROC, PTX3, RAB18, RAC1, RARS, REG3A , RHOA, S100A9, SCAMP4, SERPINA4, SIRPA, SLC3A2, SOD1, SOD3, STAB1, STAT1, THBS1, THY1, TROVE2, TXN, UBE2N, VCAM1, VIM, YARS, YWHAZ
Stress response
ACO1, AKR1C1, AMBP, ANXA1, ARRB1, ASS1, AXL, CALD1, CALR, CANX, CAP1, CAST, CAT, CCT2, CCT5 , CCT6A, CCT7, CD9, CDH13, CETN2, CGREF1, CLIC1, CLIC4, CTNNB1*, CRYAA, CRYAB, CUL3, DCN, DEFA1, DUSP3, DYNLRB1, EIF2S1, EIF3F, EIF5A, ENO1, ENOSF1, FLNA, FTH1, FTL, G6PD, GAPDH, GCLC, GLO1, GNAQ, GPD1, GPI, GPX1, GPX3, GPX4, GSR, GSS, GSTM2, GSTM3, GSTP1, GSTT1, HIVEP3, HSP90AA1, HSP90AA2P, HSP90AB1, HSP90AB2P, HSP90AB3P, HSP90AB4P, HSP90B1, HSPA1A, HSPA1B, HSPA1L, HSPA4, HSPA5, HSPA8, HSPA9, HSPB1, HSPD1, ITGB1, LAMB1, LDHB, MAPK1, MBL2, MMP9, MPO, MTPN, NAMPT, NAPRT, NCAM1, NCL, NDRG1, NQO1, OCLN, OLA1, PARK7, PDCD6IP, PDIA6, PFN1, PGLS, PGRMC1, PLIN3, PPIA, PPM1B, PRDX1, PRDX2, PRDX4, PRDX5, PRDX6, PTK7, PTPRZ1, RAB8A, RAC1, RAN, REG3A, RHOA, RNH1, S100A6, S100A9, S100A13, S100B, SAFB, SBDS, SCFD1, SELENBP1, SEPT11, SEPP1, SLC12A2, SLC16A1, SLC2A1, SLC9ASNCA, SNCB, SNCG, SOD1, SOD2, SOD3, SPTAN1, SSB, STIP1, STRAP, SVEP1, TCP1, TGM2, TIMP1, TLN1, TNC, TPI1, TXN, TXNRD1, UBXN1, UFM1, USP14, VCAM1, VGF, VIM, WARS
Metabolic process
ACAT2, ACBD7, ACOT7, ACP1, AHCYL2, AKR1B1, AKR1C2, ANXA1, ANXA3, AP1M1, APEH, ASAH1, ATP1A2, B4GALNT1, BDH2, BRD8, CBR1, CHAD, CLIC1, CPE, COX7A2, CPNE3, CRYL1, CRYM, DBI, DDX39A, DKK3, DLD, DNM2, EPHX1, ECHDC1, ELN, FABP4, FABP5, FABP7, FASN, GANAB, GGT5, GLO1, GM2A, GMDS, GMPPA, GPD1, GPI, GPLD1, GPX3, GPX4, GSS, GYG1, HADH, HEXB, IDH1, IDH2, IGFBP2, ISOC2, LANCL1, LMNA, LRP1, MAT2A, ME1, MGAT1, NAMPT, NCL, NIT2, NLGN2, NQO1, OLA1, OPLAH, PAFAH1B1, PAFAH1B2, PCYT2, PGD, PGM2, PITPNA, PLTP, PNPO, PPAP2B, PRDX2, PTPRN, QDPR, QSOX1, SAR1A, SHMT1, SLC1A2, SLC2A1, SLC2A5, SNCA, SOD1, SORT1, THBS1, TPP1, TXNRD1, UBE2NL, USO1, USP7, VIM, VPS13C, VPS35
Mitochondrial functions
ABAT, ACAT1, ACO2, ACOT2, ACOT7, ALDH2, ALDH6A1, ANXA5, ATP5A1, ATP5B, CAPN1, CCBL2, CD47, CLIC4, COX7A2, CRYAA, CS, CUTA, DBI, DDAH2, DES, DLD, DNM1L, DNPEP, EIF5A, ETFA, GLUD1, GOT2, GPX4, GSR, GYS1, HADH, HADHA, HK1, HSPA1A, HSPA1B, HSPA9, HSPB1, HSPD1, IDH2, KARS, MPST, MT-CO2, MTHFD1, NDUFA9, NPM1, PARK7, PCMT1, PDE12, PDIA3, PPA1, PRDX5, PRKAR2A, PRMT1, PTK7, PTRF, RALA, RAN, REEP5, RNH1, S100A1, SEMA3B, SH3GLB1, SLC25A4, SOD1, SOD2, TPP1, UBA1, USP7, VDAC1, VDAC2, VIM, VPS13C, VPS26A, YWHAQ
Vesicles
AHSA1, ANKFY1, ANXA2, ANXA3, ANXA7, AP1B1, AP1G1, AP1M1, AP1M2, AP2B1, AP2M1, ARF1, ARF3, ARF6, ARL3, ARRB1, ATRN, BIN1, CALR , CD81, CD9, CDC42, CLTC, COPA, COPB1, COPB2, COPG1, CORO1A, CPE, CPNE1, CRP, CSE1L, DNM1L, DPP7, EEA1, EHD2, FLOT1, GDI1, GDI2, GOLIM4, GPI, GPM6A, HLA- DRA, HSP90AA1, HSPA8, ITGB1, KIF5A, KIF5B, KTN1, L1CAM, LAMP2, LCAT, MAP4, MRC2, NAPA, NPTXR, NSF, NUDT3, PACSIN2, PDCD61P , PDGFR, PDGFRB, PFN1, PGRMC1, PITPNA, PLIN3, PPP3CC, PRKAR2A, PRKAR2B, RAB1A, RAB1B , RAB2A, RAB2B, RAB3A, RAB5A, RAB5B, RAB5C, RAB6A, RAB10, RAB11A, RAB14, RAB18, RAB21, RAB23, RALA, RALB, RAN, RHAB40A, RHAB18, RHOA, S100B, SAR1A, SCAMP4, SCFD1, SDCBP, SEC13, SEC23A, SEC24C, SEC24D, SEPT5, SEPT7, SEPT11, SERPINI1, SH3GL2, SH3GLB1, SH3KBP1, SNAP25, SNAP91, SNCA, SNCB, SNX1, SNX5, SORL1, SORT1, SPTBN2, STXBP1, SYN1, SYNGR1, SYNJ1, SYT1, TIMP1, TNC, USO1, VAMP2, VAMP5, VAT1, VPS26A, VWF, YKT6, YWHAB
Blood- brain barrier AGRN, AQP1, AQP4, CDH13, CDH15, CDH2, CTNNB1, DAG1, DES, ENO2, FBLN1, FBLN2, FBLN5, GFAP, GJA1, GJD4, GSTM3, ICAM1, ICAM5, INA, LAMA2, LAMA4, LAMA5, LAMB1, LAMB2, LAMC1, LAMC3, LCAT, LRP1, NID1, NID2, OCLN, PLTP, SLC2A1, SLC5A6, VCAM1, VWF
Choroid Plexus ACO1, ACO2, ADH1A, ADH1B, ADH5, AQP1, AQP4, ATP5A1, ATP5B, CA2, CALM1, CAT, CLIC6, COMT, DSTN, EPDR1, EZR, GPX1, GPX3, GSR, GSTM2, GSTM3, GSTM4, GSTP1, GSTT1, LAMA2, LAMA4,
LAMA5, LAMB1, LAMB2, LAMC1, LAMC3, MMP2, MMP9, MSN, NID1, NID2, PARK7, PPM1J, RAB7A, SELENOP, SEPT2, SOD1, SOD2, TGFBI, TIMP1 , TTR, TXN, VIM
ACO1ANXA1ANXA2ANXA4ANXA5ANXA6CRPCTSDDPYSL2ENO1EZRGAS6HLA−AHLA−DRAITGB1TIMP1TIMP2GPIGPX3GSTM2GSTM3GSTM4GSTP1HSP90AA1HSP90AA2PHSP90AB1HSP90AB3PHSPA1BHSPA1LHSPA4HSPD1NAMPTPARK7PRDX1PRDX2PRDX5PRDX6SNCASNCBSNCGSOD1SOD3STIP1TXNVIMCD14CD163CDH13CDH2CHI3L1MARCOMIFMMP2MMP9MRC1MRC2ALDH1L1BGNGFAPGLULPEA15S100BSLC1A3ENO2GLO1INANCAM1NCAM2NEFLNFASCNPTNNRCAMNRXN1NRXN2NRXN3SORL1AGRNAQP1AQP4DAG1FBLN1FBLN2FBLN5ICAM1ICAM5LAMA2LAMA4LAMA5LAMB1LAMB2LAMC1LBPNID1NID2VCAM1VWFATP1A1ATP1A2ATP1B1ATP5BCLIC6
Proteinintensity (log2)
0102030
Protein functionImmune/In�ammStress responseMyeloid cellsAstrocytesNeuronBBBCP
CSF particleconcentration (log10)
89101112
Plasma viral load (cp/ml, log10)
23456
NCINo HANDHAND
128
241
147
332
205
782
209
214
280
072
228
129
020
143
093
083
148
010
151
139
EV concentrations (particles/ml)Pool 1 Pool 2 Pool 3
HIV - 3.0 x 1011 2.7 x 1011 2.0 x 1011
No HAND 1.9 x10 11 1.2 x 1012 1.0 x 1011
ANI 1.1 x 1012 3.1 x 1011 4.5 x 1011
MND 1.1 x 1012 2.6 x 1011 2.8 x 1011
HAD 5.4 x 1011 7.5 x 1011 5.0 x 1011
Astrocytic markers
Stress response markers
25
70
25
PRDX2
FLOT-1
CD9
HIV-1
GLUL
GFAP
42
55
47
25
CRP
HSP70
PARK7
Inflammatory marker
Exosomal markers
22
No HAND-1
No HAND-2
ANIMND
HADMark
er
PRDX2 PARK7 HSP70
GLUL GFAP CD9 FLOT-1
CRP
Nor
mal
ized
inte
nsity
Nor
mal
ized
inte
nsity
a b
c
MW(kDa)
0
2000
4000
6000
8000
0
1000
2000
3000
4000
HIV-
N o HAND
ANI+MND
HAD0
1000
2000
3000
4000
HIV-
NoHAND
ANI+MND
HAD0
5000
10000
15000
20000
25000
0
5000
10000
15000
0
5000
10000
15000
20000
HIV-
No HAND
ANI+MND
HAD0
5000
10000
15000
HIV-
No HAND
ANI+MND
HAD0
2000
4000
6000
8000
10000
Mock 10 25 50 100 250 500 10000
30
60
90
120
%Ce
llvi
abili
ty
Mock 50 100 250 500
1
2
3
4
Intra
cellu
larR
OS(fo
ldch
ange
)
CD9
U87 WCL U87 EV
PARK7
GLUL
PRDX2
GFAP
42 kDa
50 kDa
25 kDa
GAPDH37 kDa 25 kDa
0
1
2
Norm
alize
din
tens
ity
48 kDa
0
1
2
3
4
Mock
100 µM 250 µM
50 µM
H2O2 dose (µM)
a
e
b
d
c
H2O2 dose (µM)
PRDX2
PRDX2GFA
PGLU
LGLU
LGFA
P
PARK7
PARK7
U87 WCL U87 EV
Mock
Mock50
µM50
µM25
0 µM
250 µM
100 µM
100 µM
Marker
Marker Mock
50100250
p=0.30p=0.09
p=0.07
p=0.25p=0.53 p=0.42
p=0.23 p=0.22
p=0.006
p=0.01
p=0.006p=0.004
p=0.02p=0.01
p=0.003p=0.002
p<0.0001p=0.001
p=0.003
Fig. 7 Effect of oxidative stress on astrocytic, stress response, and inflammation markers in U87 cells and EVs. (a) Cultured U87 cells were treated with 10, 25, 50, 100, 250, 500, and 1000 µM H2O2 for 4 hours. Cell viability was measured by MTT assay (n=3). Data represent mean + SEM. (b) U87 cells were treated with H2O2 (50, 100, 250, and 500 µM) for 4 hours and generation of intracellular ROS was measured spectrophotometrically (n=3). Significant differences in (a) and (b) were evaluated by t-test (p-value <0.05). (c) Fluorescence images of intracellular ROS following H2O2 treatment (50, 100, 250 µM) of U87 cells for 4 hours. Scale bar=100 µm (d) Immunoblotting for astrocytic (GFAP, GLUL) and stress response (PRDX2, PARK7) markers in U87 cells and EVs (n=3). GAPDH and CD9 were used as loading controls for U87 cells and U87 EVs, respectively. Results are representative of three independent experiments. (e) Densitometric quantification of normalized GFAP, GLUL, PARK7, PRDX2, and CRP bands from U87 cells and EVs. Significant differences between at least two of the four conditions in each set are indicated using one-way ANOVA (p-value <0.05).
Fig. 1 Characterization of CSF EVs from HIV+ subjects. (a) Transmission electron micrograph of whole-mounted CSF EVs from a representative HIV+ subject. EVs are indicated with black arrows. Scale bar=100 nm. (b) Histogram of CSF EV size distribution by nanoparticle tracking analysis (NTA) from a representative HIV+ subject. (c) Immunoblotting for exosome markers Hsp70, FLOT-1, CD81, and CD9 in CSF EV fractions from two representative HIV+ subjects. EV-depleted CSF from the corresponding samples were used as controls (Ctrl). (d) Silver staining of EV and EV-depleted CSF proteins (Ctrl) from two representative HIV+ subjects. Proteins were separated by SDS-PAGE prior to staining.
BACKGROUND: Extracellular vesicles (EVs) are nano -sized particles present in most body fluids including cerebrospinal fluid (CSF). Little is known about CSF EV proteins in HIV+ individuals. In this cross-sectional study, we characterized the CSF EV proteome in HIV+ subjects and its relationship to neuroinflammation, stress responses, and HIV-associated neurocognitive disorders (HAND). METHODS: CSF EVs isolated from 20 age-matched HIV+ subjects with (n=10) or without (n=10) cognitive impairment were characterized by electron microscopy, nanoparticle tracking analysis, immunoblotting, and untargeted LC/MS/MS mass spectrometry. Functional annotation was performed by gene ontology (GO) mapping and expression annotation using Biobase Transfac and PANTHER software. Cultured astrocytic U87 cells were treated with hydrogen peroxide for 4 hours to induce oxidative stress and EVs isolated by ultracentrifugation. Selected markers of astrocytes (GFAP, GLUL), inflammation (CRP), and stress responses (PRDX2, PARK7, HSP70) were evaluated in EVs released by U87 cells following induction of oxidative stress, and in CSF EVs from HIV+ patients by immunoblotting.
RESULTS: Mass spectrometry identified 2727 and 1626 proteins in EV fractions and EV-depleted CSF samples, respectively. CSF EV fractions were enriched with exosomal markers including Alix, syntenin, tetraspanins, and heatshock proteins, and a subset of neuronal (ENO2, NFL, NPTN, NRXNs), astrocyte (GFAP, PEA15, S100B, SLC1A3), oligodendrocyte (MAG, MBP, MOG), and choroid plexus (ACO2, CLIC6, COMT, EZR, TTR) markers in comparison to EV-depleted CSF. Proteins related to synapses, immune/inflammatory responses, stress responses, metabolic processes, mitochondrial functions, and blood- brain barrier were also identified in CSF EV fractions by GO mapping. HAND subjects had higher abundance of CSF EVs (p<0.005) and proteins mapping to GO terms for synapses, glial cells, inflammation, and stress responses compared to those without HAND. GFAP, GLUL, CRP, PRDX2, PARK7, and HSP70 were confirmed by immunoblotting of CSF EVs of HAND subjects and were also detected in EVs released by U87 cells under oxidative stress.
CONCLUSIONS: CSF EVs derived from neurons, glial cells, and choroid plexus carry synaptic, immune/inflammationrelated, and stress response proteins in HIV+ individuals with cognitive impairment, representing a valuable source for biomarker discovery.
GPX
3G
SRG
STM
2G
STM
3HS
P90A
A1HS
P90A
A2P
HSP9
0AB1
HSPA
4HS
PD1
NAM
PTPA
RK7
PRDX
1PR
DX5
PRDX
6SN
CA
SNC
BSO
D1SO
D3
0
100
200
300
400 Stress response
Abstract
Study design
Results
Conclusions • Our findings suggest that CSF EVs in HIV+ individuals are likely to originate from neurons, glial cells, choroid plexepithelial cells, and BBB, and may participate in diverse types of cell-to-cell communication in the CNS.
• Higher abundance of proteins related to synaptic function, immune/inflammation and stress responses, mitochondand BBB in CSF EVs of HAND compared to non-HAND subjects suggests that CSF EVs are likely to be involved in HIV-associated neurocognitive impairment and represent a valuablesource of candidate biomarkers for future studies. • Our untargeted approach identified a number of interesting CSF EV proteins that warrant further study in large prospective cohorts using sensitive targeted quantitative assays to evaluate their potential to serve as predictive disease-specific markers.
CSF samples from 20 HIV+ subjects were collected between 1998-2013 by the National NeuroAIDS Tissue Consortium (NNTC)
Inclusion criteria were use of combination ART, age >40 years, and CSF viral load <50 HIV RNA copies/ml
Untargeted LC- MS/MS proteomics of CSF EV
• Untargeted LC- MS/MS was performed by ABSciex 4800 Plus
MALDI-TOF/TOF mass spectrometry (n=20 subjects).
• Abundant CSF proteins were excluded from the analyses. • GO analysis was performed using geneXplain TRANSFAC, and
Panther softwares
Isolation and characterization of extracellular vesicles (EVs) • EVs were isolated from 400 μl CSF from HIV+ and control subjects using ExoQuick reagent • Twelve common abundant proteins and IgG were depleted with • Proteome Purify
-
-12 immunodepletion resin (R&D Systems) and A/G beads and L beads, respectively • EVs were evaluated by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) (ParticleMetrix Zetaview instrument), and immunoblotting for CD9, CD81, FLOT-1, and Hsp70
Results
support
This work was supported by National Institutes of Health grants to D.G. (R01 MH097659, R01 MH110259, R01 DA040391). Financial support for the NNTC was provided through the following cooperative agreements from the National Institutes of Health: U24MH100930; U24MH100931; U24MH100928; U24MH100929; U24MH100925.
Results
Abbreviations: HAND, HIV-associated neurocognitive disorders; IQR, interquartile range; VL, viral load; WBC, white blood cells; ART, antiretroviral therapy; RT, reverse transcriptase; Data represent median (IQR) unless otherwise indicated; *Not available for 3 subjects. ** HIV encephalitis was diagnosed at autopsy in 1 subject with MND.
HIV+ no HAND HAND (n=20) (n=10) (n=10) Age (years)* 52.2 (47– 59) 51.3 (46-61) 52.3 (50-58) Gender (Male, n, %) 20 (100) 10 (100) 10 (100) Race (n, %) Black 5 (25) 2 (20) 3 (30) White 15 (75) 8 (80) 7 (70) Smoking (n, %) 12 (75) 7 (70) 5 (83) Alcohol use (n, %) 3 (15) 1 (10) 2 (20) Cocaine use (n, %) 4 (20) 0 (0) 4 (40) Hepatitis C seropositivity (n, %) 9 (45) 5 (50) 4 (40) Cerebrovascular disease (n, %) 3 (15) 0 (0) 3 (30) Depression (n, %) 8 (44) 5 (50) 3 (37) Duration of HIV infection (years) 13.3 (10-21) 15.5 (13-21) 11.5 (6-19) HIV RNA Plasma VL 700 (48-44131) 38.4 (26-17774) 4064 (700-236292) Plasma (<50 copies/ml) 13 (65) 9 (90) 4 (40) CSF (<50 copies/ml) * 16 (94) 9 (100) 7 (87) CD4 count (cells/µl) 162 (116 – 373) 262.5 (146-411) 157 (75-182) <350 cells/µl 13 (65) 5 (50) 8 (80) Nadir CD4 count (cells/µl) 31.5 (12 – 64) 15 (12 - 46) 55 (23 - 71) < 200 cells/µl (n, %) 18 (90) 9 (90) 9 (90) CSF WBC (cells/µl) 1 (0-2) 2 (1-2) 0 (0-1) ART use (n, %) 18 (90) 10 (100) 8 (80) Protease inhibitors 16 (80) 8 (80) 8 (80) Nucleoside RT inhibitors 17 (85) 10 (100) 7 (70) Integrase inhibitors 4 (20) 2 (20) 2 (20) HIV encephalitis 1 (5) 0 (0) 1 (10)**
Table 1 Demographic and clinical characteristics of the study cohort
0
300
600
900
1200
#ab
unda
ntpr
otei
ns
EVEV-depleted CSF
HAND HANDNo HANDNo HAND
g
0
300
600
900
1200
#an
alyz
edpr
otei
ns
No HAND No HANDHAND HAND
•
•
ImmunoblottingGFAP, GLUL, CRP, PRDX2, PARK7, and HSP70 were confirmed by immunoblotting of CSF EVs of HAND subjects and were also detected in EVs released by U87
cells under oxdidative stress.
Table 2 CSF EV proteins from HIV+ subjects (n=20) mapped to biological processes and cellular components
Cellular Components
Neurons ADH5, AGRN, ANK2, ASAH1, ATP2B4, BASP1, CALB1, CAMK2A, CAMK2B, CBLN1, CD44, CDH2, CORO1C, CRYAB, DBN1, DCC, DCLK1, DNPEP, DPYSL2, DPYSL3, EEA1, EEF1A2, EGFR, ENO2, EPHX2, FLNA, FLNB, FMNL2, FSCN1, FSTL4, GAP43, GAS6, GLO1, GLUD1, GNAO1, GNAS, GNAZ, GPHN*, GPM6A, GRIA4, HK1, ICAM5, IGFBP7, INA, IQGAP1, KIF5A, L1CAM, LAMA2, LAMC1, LAMP2, LCAT, LINGO1, LRP1, LSAMP, MAP1A, MAP1B, MARCKS, MMP9, MPO, NCAM1, NCAM2, NDRG1, NEFH, NEFL, NEFM, NEGR1, NFASC, NLGN2, NLGN3, NPEPPS, NPTN, NPTX1, NPTX2*, NQO1, NRCAM, NRGN, NRP1, NRP2, NRXN1, NRXN2, NRXN3*, NTM, OLFM2, PACSIN1, PAFAH1B1, PARK7, PCDH1, PCLO, PCMT1, PEBP1, PFN1, PLTP, PLXNB2, PPP2R2A, PPP5C, PRDX5, PRDX6, PRKAR2B, PRKCA, PRSS3, PSMB8, PTPRF, PVALB, PYGB, RAB5A, RAB7A, RAC1, RELN, RHOG, ROBO1, RPL11, RTN4, S100A1, SEMA3B, SEMA3G, SEMA7A, SEPT6, SEPT9, SERPINE2, SERPINI1, SHANK1, SLC1A2, SLC1A3, SLC6A1, SLITRK4, SNAP25, SNAP91, SNCA, SNCB, SNCG, SOD1, SOD2, SORL1, SPON1, SPTBN1, STXBP1, SULT1A2, SYN1, SYN2, SYNGR1, SYNJ1, SYPL1, SYT1, TGM2, THBS4, TNC, TNR, TPP1, TWF2, TXN, UCHL1, USP47, VAT1, VGF, XRCC5, YWHAE, YWHAQ
Astrocytes ACO1, ADD3, ADH5, ADK, AKR1C1, AKR7A2, ALDH1A2, ALDH1L1*, ANPEP, ANXA2, ANXA4, ANXA7, AQP4, ASS1, BGN, CAMK2A, CAV1, CCS, CD44, CDH13, CRYAB, DBI, DDX1, ECM1, EEA1, EGFR, ENO1, ENO2, EPHX1, EPHX2, EZR, FLOT1, FMNL2, FSCN1, GFAP, GJA1, GLO1, GLUL, GNA11, GNA13, GNAS, GNB2L1, GNGI2, GRIA4, HK1, HSPA1A, HSPA9, HSPB1, HSPD1, ICAM1, IGFBP6, INA, ITGB1, LAMA4, LAMB1, LDHA, LRP1, MAPK1, MAPK3, MMP2, MMP9, MVP, NCAM1, NCAN, NDRG1, NDRG2, NPEPPS, NQO1, PARK7, PDGFRB, PEA15, PEBP1, PLTP, PPP2R2A, PRDX1, PRDX6, PRICKLE1, PRKCA, PRSS3, PTBP1, PYGB, REG3A, RTN4, S100A6, S100B, SEPT2, SEPT7, SERPINE2, SLC1A2, SLC1A3, SLC2A1, SLITRK4, SOD1, SOD2, SPOCK3, TIMP1, TIMP2, VCAM1, VIM, YWHAB
Myeloid cells ACO1, AHCY, AKR1C2, ANPEP, ANXA11, ANXA2, ARL8A, ARRB1, ASS1, ATP6V1B2, ATRN, AXL, B2M, BGN, BSG, CALD1, CAND1, CAP1, CAPG, CAT, CD14, CD163, CD44, CDC42, CHI3L1, CHORDC1, CPB2, CPE, CPVL, CRP, DCTN2, DEFA1, EGFR, EIF3A, ENO1, EZR, FABP4, FCN3, FKBP4, FLNB, FMNL2, FSCN1, FTH1, FTL, GJA1, GNA11, GNA13, GNAI2, GNAQ, GPI, GPLD1, GRHPR, HARS, HMGB1, HRSP12, HSP90AB1, HSPA8, HSPB1, HSPD1, ICAM1, IFI44L, IL6ST, ITGB1, L1CAM, LAMP2, LANCL1, LRP1, LTA4H, LYZ, MAP1A, MAPK1, MAPK3, MARCO, MFGE8, MIF, MMP2, MMP9, MPO, MRC1, MRC2, MSN, MTPN, MX2, NAMPT, NCAM1, NCL, NDRG1, NDRG2, NELL2, NME2, NQO1, NT5C2, NT5E, PBXIP1, PDGFRB, PEBP1, PGM1, PLIN3, PLP1, PLTP, POH1, PPP3R1, PRCP, PRELP, PRKCA, PROC, PTX3, PTPN11, RAB2A, RAB5C, RDX, RHOA, ROCK1, RUVBL1, S100A9, SERPINB1, SERPINI1, SLC2A5, SLC3A2, SND1, SNX6, SOD2, SOD3, SSB, ST13, STAB1, STAT1, TIMP1, TIMP2, TNC, TXN, TYMP, VCAM1, VCAN, VSIG4, XRCC5
Endothelial cells AHNAK, AKAP12, ANGPTL1, ANPEP, ANXA2, ANXA4, ANXA5, APRT, ARF1, ARF3, ARF6, ARRB1, ATP5A1, ATP5B, B2M, BGN, BSG, CADM1, CAMK2B, CAPG, CAPN2, CAPZA1, CAT, CAV1, CD109, CD14, CD44, CD47, CD81, CD9, CDC42, CDH13, CDH2, CFL1, COPS5, CPB2, CPE, CRABP1, CRP, CRYAB, CTNNB1, DCC, DCN, DDAH2, DKK3, EEF1A1, EEF1A2, EGFR, ENO1, FABP4, FABP5, FERMT2, FLNB, FLOT1, FMNL2, FSCN1, FTH1, FTL, GFAP, GFPT1, GJA1, GNA13, GNAO1, GNAS, GPX4, HLA-A, HLA-B, HLA-DRA, HMCN1, HMGB1, HNMT, HSP90B1, HSPA1A, HSPA5, HSPA8, HSPD1, ICAM1, ICAM2, ICAM5, IL6ST, ITGA1, ITGB1, LAMA2, LAMA4, LAMB1, LAMC1, LAMP2, LTA4H, LTBP2, MAPK1, MAPK3, MARCKS, MBL2, MCAM, MIF, MMP2, MMP9, MMRN2, MPO, MRC1, MRC2, MSN, MTPN, MVP, NDRG1, NPM1,NQO1, NRP1, NRP2, NRXN2, NT5E, OCLN, PDGFRB, PGD, PIK3R1, PLS3, PLXDC2, PRCP, PRDX1, PRDX2, PRDX5, PRKAR2B, PRKCA, PROC, PSMA2, PTK7, PTMA, PTPN11, PTX3, QSOX1, RAB14, RAB18, RAB1A, RAB2A, RAB5B, RAB6A, RAC1, RHOA, ROBO1, S100A1, S100A13, SDCBP, SEPP1, SEPT5, SEPT8, SERPINA4, SET, SLC12A2, SLC2A1, SLC2A5, SLC3A2, SNCA, SOD1, SOD3, SRPX, SSB, ST13, STAB1, STAT1, TGM2, THBS1, THY1, TIMP1, TIMP2, VASN, VCAM1, VCL, VIM, VWF, YWHA
Epithelial cells
ADD1, ADD3, ADH5, AHNAK, AKAP12, AKR1A1, AKR1B1, ALDH1A1, AMBP, ANPEP, ANXA1, ANXA2, ANXA4, AQP1, ASS1, ATP1A1, ATP1A2, ATP1A3, ATP1B1, ATP2B1, AXL, BCAM, BGN, BPIFA1, BSG, CA2, CADM1, CALR, CAPZA1, CAT, CAV1, CD109, CD14, CD44, CD9, CDC42, CETN2, CHI3L1, CLIC4, COMP, COPA, COPS5, CRABP1, CRP, CRYAA, CRYAB, CSE1L, CTNNB1, DBI, DCN, DCTN2, DDAH2, DDB1, DIP2B, DKK3, DNM2, EEF1G, EGFR, EHD2, EIF2S1, EIF3F, EIF6, ENO1, ENO2, EZR, FCN3, FERMT2, FHL1, FMNL2, FSCN1, FTH1, FUCA1, GALNT2, GCLC, GDA, GJA1, GM2A, GNA11, GNAI1, GNAI2, GNAI3, GNAQ, GNAS, GNAZ, GNB2L1, GPX3, GSTM2, GSTM3, HINT1, HLA-DRA, HMCN1, HMGB1, HPRT1, HRSP12, HSPA1A, HSPA5, HSPA8, HSPD1, ICAM1, IGFBP2, IGFBP6, IGFBP7, IL6ST, ISYNA1, ITGA1, ITGB1, KCNJ13, KIAA1199, KIF5B, L1CAM, LAMA2, LAMA5, LAMB1, LAMC1, LAMC3, LAMP2, LBP, LCP1, LTA4H, LTBP2, LTBP4, LUM, LZTFL1, MAPK1, MAPK3, MARCKS, MBP, MCAM, MFGE8, MGP, MIF, MMP2, MMP9, MRC2, MSN, MVP, NAP1L4, NCAM1, NDRG1, NEO1, NID1, NME2, NPEPPS, NQO1, NQO2, NRP1, NT5E, PARK7, PCDH1, PCMT1, PDGFRB, PEA15, PLCD1, PLP1, PLS3, POSTN, PPAP2B, PPIB, PPP1R1B, PRDX1, PRDX6, PRKAR2B, PRKCA, PRSS3, PSMA7, PSMB9, PSMD2, PTPRD, PTPRF, PTRF, PTX3, RAB10, RAB21, RAB3A, RAC1, RELN, RHOA, RLBP1, ROBO1, ROCK1, S100A9, SAG, SDCBP, SDPR, SELENBP1, SEPP1, SEPT5, SEPT7, SERPINA7, SLC22A6, SLC2A1, SLC2A5, SLC3A2, SLC4A1, SLC4A2, SLC9A3R1, SND1, SNX1, SOD2, SOD3, SPTAN1, STAT1, STOM, STX2, TGFBI, TGM2, THBS1, THBS2, THBS4, TIMP1, TIMP2, TUBB1, TXN, TYMP, VCAM1, VCL, VIM, XRCC5, XRCC6
Oligodendrocytes ANXA4, CA2, CRYAB, EPHX2, GLUL, GRIA4, HSPA1A, HSPD1, MBP, MAG*, MOG , PEBP1, PLP1, REG3A, , SLC1A3, TNR *
22%
Synaptic proteins are shown in italicsProteins curated manually;
2%
Biological processes
Protein hits
ExosomesProteins identified by one unique peptide hit are shown in blue;
Poster # 00408
Fig. 4 CSF EV protein abundance in rela-tion to cognitive status of HIV+ subjects (n=20). HAND subjects (n=10) had (a) higher EV concentration (solid red circles represent subjects with HAD) and (b) greater EV-associated protein abundance compared to HIV+ subjects without HAND (n=10). (c) The number of high abundance proteins detected in EV fractions and EV-depleted CSF was similar in subjects with versus without HAND (left panel), while HAND subjects had greater abun-dance of analyzed proteins compared to those without HAND (right panel). (d) Venn-diagram showing overlap of proteins identified in CSF EV fractions from subjects with and without HAND (e) Volcano plot showing differences in protein abundance for subjects with vs. without HAND among 507 proteins identified in both groups. Each dot represents a single protein; red dots correspond to proteins significantly increased by ≥ 2-fold (p<0.05). Selected proteins with high fold-changes, p-values <0.05, or biological relevance for HAND pathophysiology are labeled. (f) Venn-diagram showing overlap of proteins identified in CSF EV fractions from subjects with ANI or MND and without HAND. (g) Volcano plot showing differences in protein abundance for subjects with ANI or MND vs. without HAND among 493 proteins identified in both groups. Each dot represents a single protein; red dots correspond to proteins significantly increased by ≥ 2-fold (p<0.1). Selected proteins with high fold-changes, p-values <0.1, or biological relevance for HAND pathophysiology are labeled.
Fig. 5 Supervised heatmap of 101 CSF EV proteins identified in 20 HIV+ subjects with (n=10) and without (n=10) HAND. Proteins identified by >2 unique peptide counts in 6 or more subjects mapping to immune / inflammatory responses, stress response, myeloid cells, astrocytes, neurons, blood-brain barrier (BBB), and choroid plexus (CP) ontology terms are shown. Columns correspond to individual subjects (font color in black: no HAND; green: ANI; blue: MND; red: HAD; purple: NPI-O) and rows to individual proteins. Color scale (blue-yellow-red) illustrates relative log2 transformed peptide intensities. CSF EV concentrations (particles/ml) and plasma VL were log10 transformed. Triangles at the top illustrate increasing gradient of CSF particle concentrations in subjects with and without HAND. NCI: Neurocognitive impairment
Fig. 6 Abundance of astrocytic, stress responses, and inflammatory markers in CSF EVs of HIV- and HIV+ subjects with or without HAND. (a) EV concentrations measured in 300 µl pooled CSF samples by NTA in HIV-, HIV+ non-HAND, and HAND (ANI, MND, and HAD) sub-jects (n=3; 2-3 subjects per pool). (b) Detection of astrocytic markers GLUL and GFAP, stress response markers PRDX2, PARK7, and HSP70, inflammatory marker CRP, and exosomal markers CD9 and FLOT-1 in CSF EV fractions by immunoblotting. One representative blot is shown for individual markers. Results are representative of three independent experiments. (c) Den-sitometric quantification of HSP70, PARK7, PRDX2, CRP, GLUL, GFAP, CD9, and FLOT-1 in samples from HIV-, HIV+ non-HAND, ANI+MND, or HAD from immunoblotting (n=3, except PARK7, PRDX, and CD9 where n=2). Bands in each lane were normalized to corresponding EV concentrations. Bars denote mean, error bars denote standard error. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
We thank the Taplin Mass Spectrometry Facility at Harvard Medical School for proteomic analysis and Harvard Medical School Electron Microscopy Facility for TEM imaging.