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Unbiased and Targeted Mass Spectrometry Provides Insight into Huntington’s Disease Pathogenesis
Todd Greco, Joel Federspiel, Jaime Hutton, Jeff Cantle, Jeff Carroll, & Ileana Cristea
May 27th, 2020
Nat Rev Dis Primers (2015).
• Monogenic neurogenerative disorder Huntingtin (Htt) gene
• Htt gene Increased CAG repeat Expanded polyQ
• Massive cell loss in striatum and cortex
• Liver also selective target in HD
Normal Huntington’s Disease
Huntington’s Disease: A Polyglutamine Expansion Disorder
Expanded PolyQ
(~350 kDa)
Questions
Biology of huntingtin (Htt) Consequence of Htt lowering therapies?
Identification of proximal disease-modifiers using discovery-based and targeted MS
Pathophysiology of polyQ expansion (mHTT) Gain/loss of function?
Approaches
Altered protein interaction dynamics in the brain
Proteome dysfunction in the liver
Tissue-selective pathology Proteome signatures of HD?
Defining Protein Markers of HD in Liver using Targeted MS• Goal: Define liver proteome signatures for expanded polyQ Htt or loss of Htt • Protein candidate selection
• Unbiased liver proteome analysis (collected by Carroll lab & Evotec) • Genetic variants linked to age of disease onset (GeM-HD Consortium)• Diverse roles, including metabolism (34), cell adhesion (14), RNA processing/transport (16)
• Approach: Design targeted relative quantification 1D-LC assays using Skyline• Experimental spectra supplemented with Prosit predicted spectra (Gessulat et al., 2019)
Knock-in HD Mouse Models
Hun
tingt
on’s
D
isea
se Htt-20QHtt-111QHtt LKO
6
Age(Months)
WT
PolyQ LKOvs.
Peptide inclusion list
- SSWPASTQEGLR
- ASEAGATAPK- IQLEEYSNTR
- GTAEPITVTIVPAYR
- GYPDFNLIVNPYEPIR- QQIEDQQGLPK
Htt
155 Target Proteins
PRM-MS/MSTargeted
MS Assays
Spectral Libraries
(DDA +Prosit)
Refinement
Striatal protein marker showing
cell loss
(N = 3)
Kovalenko et al., (2018). J Huntingtons Dis. 7(1).
Proteome dysfunction in the liver
Acetyl-CoA carboxylase 2
Metabolic Proteins
Dysregulated proteins in metabolism in PolyQ and KO
WT LKO PolyQ71.5
Retention Time
0
100
200
300
400
500
600
700
Inte
nsity
(10^
3)
71.5-0.1 ppm
71.5Retention Time
0
100
200
300
400
500
600
700
Inte
nsity
(10^
3)
71.60 ppm
71.4 71.6 71.8Retention Time
0
100
200
300
400
500
600
700
Inte
nsity
(10^
3)
71.6+0.2 ppm
y9 - 1147.6081+y8 - 1050.5553+y7 - 937.4713+y6 - 806.4308+y5 - 693.3467+y4 - 546.2783+
WT LKO PolyQ
R.LPLMIFANWR.G (2+)max dotp = 0.93
Acetyl-coenzyme A synthetase
WT LKO PolyQ49 50Retention Time
0
0.5
1
1.5
Inte
nsity
(10^
6)
49.8+2.1 ppm
50Retention Time
0
0.5
1
1.5
Inte
nsity
(10^
6)
49.9+1.8 ppm
49.0 49.5 50.0 50.5Retention Time
0
0.5
1
1.5
Inte
nsity
(10^
6) 49.8+2.3 ppm
K.VAFYWEGNEPGETTK.I (2+)max dotp = 0.94
Dysregulated protein involved in DNA repair in Liver KO
Wheeler et al. (2003). Human Mol. Genetics. 12(3):273-81
Msh2
Norm
alize
d Pe
ak A
rea
WTLKO
HET0.6
0.8
1.0
1.2
1.4 p=0.049
MSH2
WT LKO PolyQ
MSH2-/-MSH2+/+
• Loss of MSH2 in mouse brain is protective
39.0 39.2Retention Time
010203040506070
Inte
nsity
(10^
3)
39.1+2.9 ppm
39.0 39.2Retention Time
010203040506070
Inte
nsity
(10^
3) 39.1+3.3 ppm
39.0 39.2Retention Time
010203040506070
Inte
nsity
(10^
3)
39.1+3.8 ppm
WT LKO PolyQ
K.DIYQDLNR.L (2+)max dotp = 0.97
Dysregulation of proteins in cell adhesion and actin cytoskeleton in Liver KO
Efnb1
Norm
alize
d Pe
ak A
rea
WTLKO
HET0.7
0.8
0.9
1.0
1.1
1.2
1.3
Ephrin B1p=0.039
WT LKO PolyQ
Integrin α1p=0.098
WT LKO PolyQ
p=0.082Claudin3
WT LKO PolyQ
Farp2
Norm
alize
d Pe
ak A
rea
WTLKO
HET0.6
0.8
1.0
1.2
1.4
Farp2 (GEF)p=0.024
WT LKO PolyQ 59.5Retention Time
0
20
40
60
80
100
Inte
nsity
(10^
3)
59.7-1.4 ppm
59.5 60.0Retention Time
0
20
40
60
80
100
59.7+1.3 ppm
59.5 60.0Retention Time
0
20
40
60
80
100
K.LLDSTVELFDIEPK.C (2+)max dotp = 0.98
48.8 49.0 49.2Retention Time
050
100150200250300350
Inte
nsity
(10^
3)
49.0-0.3 ppm
48.8 49.0 49.2Retention Time
050
100150200250300350
49.0+2.7 ppm
48.8 49.0 49.2Retention Time
050
100150200250300350
49.0-0.9 ppm
WT LKO PolyQ
K.GGSGTAGTEPSDIIIPLR.T (2+) max dotp = 0.96
HAP40, a known Htt PPI, is reduced in PolyQ and LKO mice
Guo et al. (2019). Nature. 555(7694):117-120
F8a1N
orm
aliz
ed P
eak
Are
a
WTLK
OHET
0.0
0.5
1.0
1.5
2.0 p=0.01 HAP40
WT LKO PolyQ
WT LKO PolyQ
R.DYTGALALFTR.M (2+)max dotp = 0.97
58.0Retention Time
0
10
20
30
40
50
Inte
nsity
(10^
3)
58.1+2.2 ppm
58.0Retention Time
0
10
20
30
40
50
58.1-0.1 ppm
58.0Retention Time
0
10
20
30
40
50
58.1+3.2 ppm
Immunoaffinity Purification MS to prioritize PolyQ-dependent interactions in the brain
-20
-10
0
10
20
-20 0 20 40PC1 (59.5%)
PC2
(14.
3%)
140Q-10m140Q-2m20Q-10m20Q-2m
-40
PolyQ shift in PPI profileKnown Htt PPIs (IP Abundance)
20Q-2m140Q-2m
20Q-10m140Q-10m
0
1.0×108
2.0×108
3.0×108
*HAP40
0
5.0×106
1.0×107
1.5×107
2.0×107
*Contactin 1 (Cntn1)
Knock-in FLAG HD Mouse strains
Dounce tissue lysis
FLAG-Htt IgG
Label-free IP
Trypsin digestion
Label-free Quantitative nLC-MS/MSPeptide
separation
SAINT PPI scoring
PolyQ dependent PPI
Brain dissection
Choi, H et al (2011). Nature Methods, 8:70-3
Hun
tingt
on’s
D
isea
se Htt-20Q
Htt-140Q
2 10
Age(Months)
Altered protein interaction dynamics in the brain
IP A
bund
ance
,M
S In
tens
ity
Cluster (# of proteins)
Age and PolyQ-dependent Htt Interactions Have Distinct Functional Classes
(PolyQ)
Late Disease Early Disease
Clusters 1, 2, & 3 (Synapse Trans)Cluster 5 (Actin Network)
Cluster 6 & 7 (Synapse Trans)
(PolyQ)
20Q-2m140Q-2m20Q-10m140Q-10m
1.0 5.0 10.0 15.0
# of Proteins / Pathway # of Proteins / Pathway
Pre-symptomatic (Early)
Distinct PolyQ-dependent Htt Interactions in Pre-symptomatic & HD Mice
Huntington’s Disease (Late)• Increased: SNARE complex members: syntaxin1b (Stx1b), SNAP25, NEM-sensitive factor (Nsf)
• Decreased:Mitogen-activated protein kinase 1 (Mapk1)
• 184 differential interactions
Contribution of proteome abundance?
(10) (112) (0) (139)
HD
Pre
45
62
77
Pre-symptomatic (Early)
PolyQ-dependent Interactions are not Driven by Proteome Abundance
Huntington’s Disease (Late)
What mechanisms drive change in
interaction levels?
• Only 7 of the differential interactions are also regulated at the proteome level• Similarly low overlap at the transcriptome level
Langfelder et al. (2016). Nat. Neurosci. 19(4).
Federpsiel et al. (2019). Mol. Cell. Proteomics.18:S92-S113
Parallel Isotope-labeled IP-MS Integrates PolyQ-dependent interaction stability
Dounce tissue lysis
FLAG-Htt IgG
Label-free IP
Trypsin digestion
Label-free Quantitative nLC-MS/MSPeptide
separation
SAINT PPI scoring
PolyQ dependent
PPI
Brain dissection
FLAG-Htt Isotope-labeled
Quantitative nLC-MS/MSIsotope labeled IP PolyQ PPI
Stability
Htt*Wild-type,
age matched, 13C-Lys labeled
Mix 1:1
Greco et al. (2013). Mol Syst Biol. 9:672Greco et al. (2015). Methods in Mol Biol.
Hun
tingt
on’s
D
isea
se Htt-20Q
Htt-140Q
2 10
Age(Months)
Mouse strains
Label-free
+
Metabolic labeling Relative Stability
Interaction Relative Stabilities are PolyQ and Age DependentInteraction Stabilty Profile
for HTT-Q20 2mo
I-DIRT Ratio
SAIN
T Sc
ore
0.4 0.6 0.8 1.00.0
0.2
0.4
0.6
0.8
1.020Q-2m (72)
Interaction Stabilty Profilefor HTT-Q140 2mo
I-DIRT Ratio
SAIN
T Sc
ore
0.4 0.6 0.8 1.00.0
0.2
0.4
0.6
0.8
1.0140Q-2m (106) Interaction Stabilty Profile
for HTT-Q140 10mo
I-DIRT Ratio
SAIN
T Sc
ore
0.4 0.6 0.8 1.00.0
0.2
0.4
0.6
0.8
1.0140Q-10m (36)
Interaction Stabilty Profilefor HTT-Q20 10mo
I-DIRT Ratio
SAIN
T Sc
ore
0.4 0.6 0.8 1.00.0
0.2
0.4
0.6
0.8
1.020Q-10m (24)
Pres
ympt
omat
icPo
lyQ
HD
man
ifest
Pol
yQ
• Age-dependent decrease in stability
• PolyQ-dependentincrease in stability
How to classify PPIs stabilities versus interaction levels?
Hun
tingt
on’s
D
isea
se Htt-20Q
Htt-140Q
Age(Months)
2 10
• Differential proteins > Late disease
• Differential protein + increased stability> Early disease
PolyQ-dependent Htt Interaction Dynamics
Pre-symptomatic HD
Pre-symptomatic
Manifest HD
• Suggests functional divergence within SNARE complex at stability level
HD is a whole-body disease
• Reinforce role of Htt in DNA repair • Cell adhesion proteins in normal Htt function?• Highlight proteome-interactome relationship (HAP40)
Biology of normal huntingtin (Htt) Consequence of Htt lowering therapies?
Pathophysiology of polyQ expansion (mHTT) Gain/loss of function?
Tissue-selective pathology Proteome signatures of HD?
• Potential for metabolic protein dysregulation, e.g. in fatty acid synthesis• Distinct regulation of Htt PPIs in early and late state disease
• Differential effects of PolyQ on SNARE protein interaction levels and stability
• Continued application of targeted MS assays across tissues (proteome, metabolome, and lipidome)
PolyQ
Dr. Joel Federspiel Dr. Jaime HuttonDr. Joshua JusticeXinlei ShengBokai SongLaura Murray-NergerCora BetsingerKatelyn CookTimothy HowardMichelle KennedyDawei LiuWilliam HofstadterMatthew TylPranav RekapalliCaroline TaberElene TsopurashviliJulia EdgarBrett PhelanEmily Cheng
AcknowledgmentsDr. Ileana Cristea
Funding
Dr. Scott Zeitlin
Dr. Jeff CarrollDr. Jeff Cantle
Collaborators