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