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Research Articles: Cellular/Molecular

Ubiquitin C-terminal hydrolase L1 (UCH-L1) promotes hippocampus-dependentmemory via its deubiquitinating effect on TrkB

Yun-Yun Guo1, Lu Yi1, Yuan Zheng2, Xiao-Rong Chen1, Jun-Lu Dong1, Rong-Rong Yuan1, Shu-Hong Huang1, Hui

Yu1, Yue Wang1, Zhe-Yu Chen1 and Bo Su1

1Department of Neurobiology, Shandong Provincial Key Laboratory of Mental Disorders, School of Medicine,Shandong University, Jinan, Shandong 250012, P.R. China.2Department of Pediatrics, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong, 250012,P.R. China.

DOI: 10.1523/JNEUROSCI.3148-16.2017

Received: 8 October 2016

Revised: 21 April 2017

Accepted: 4 May 2017

Published: 12 May 2017

Author contributions: Y.G., Y.L., Y.Z., X.-R.C., J.-L.D., R.-r.Y., and B.S. performed research; Y.G., S.-H.H., H.Y., Y.W., Z.-Y.C., and B.S. analyzed data; Y.G. and B.S. wrote the paper; Z.-Y.C. and B.S. designedresearch.

This work was supported by National 973 Basic Research Program of China (No. 2012CB911004), NationalNatural Science Foundation of China (No. 331571449, 31401219), China Postdoctoral Science Foundation (No.2016M600534) and Shandong Provinical Natural Science Foundation, China (No. ZR2014CQ023). The authorsdeclare no competing financial interests.

Correspondence should be addressed to To whom correspondence should be addressed: Bo Su,Department of Neurobiology, School of Medicine, Shandong University, No.44 Wenhua Xi Road, Jinan,Shandong 250012, P. R. China. Tel: 86-531-88382329, Fax: 86-531-88382329, E-mail: bxs103@sdu.edu.cn

Cite as: J. Neurosci ; 10.1523/JNEUROSCI.3148-16.2017

Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formattedversion of this article is published.

In vivo

in vivo

in vitro

in vivo

°

Experiment 1. UCH-L1 promotes CFC memory by regulating TrkB

Experiment 2: UCH-L1 interacts with TrkB

In vitro

Experiment 3: Mapping the TrkB/UCH-L1 interaction domain

Experiment 4: UCH-L1 regulates TrkB ubiquitination as a deubiquitinating enzyme

in

vitro

Experiment 5: Study of the ubiquitination site in TrkB regulated by UCH-L1

Experiment 6: The effects of Tat-UCH-L175-85 on ubiquitination of TrkB, TrkB

degradation and intracellular trafficking

Experiment 7: The effects of Tat-UCH-L175-85 on TrkB and its downstream

signaling pathways.

Experiment 8: The effects of Tat-UCH-L175-85 on CFC learning and memory

Statistical Analysis

in vivo

F

F

F

F

F

F

F

in vitro in vivo

F

in vivo

in vitro

F

F

α

α

α

F

F

in vitro

in vivo.

in vivo

in vivo

in vivo

In vitro

in

vitro

in vitro

α. ΗΕΚ293 α

α

AFigure1.

shRNA TrkB UCH-L1 DAPI Merge

B

TrkB

Con shTrkB Con shTrkB

DG Other hip Cortex StriatumDG Other hip Cortex Striatum

Con UCH-L1

UCH-L1 Con shTrkB Con shTrkB Con UCH-L1 Con UCH-L1 Con UCH-L1

C

D E

P4D1

TrkB

TrkB

UCH-L1

OE UCH-L1

sh TrkBOE UCH-L1 & sh TrkB

145kD

25kD55kD

IP:TrkB

Lysate

F H

G

OE UCH-L1 sh TrkB OE UCH-L1& shTrkB

OE UCH-L1 sh TrkB OE UCH-L1& shTrkB

I

T T T0

20

40

60

80OE UCH-L1ANA-12OE UCH-L1 & ANA-12

0

20

400

60

80

OE UCH-L1 ANA12 OE UCH-L1& ANA12

*

J

pY99

TrkB

UCH-L1

IP:T

rkB

Lysa

te

TrkB

OE UCH-L1

ANA-12

OE UCH-L1 & ANA-12

K

OE UCH-L1 ANA12 OE UCH-L1& ANA12

L

**

**pY99

/Trk

B

0

0.4

0.8

1.2

1.4

**

15 days

Conditioning STM

1 Hour

Contro S r shTrkB shTrkB & TrkB

wt UCH-L1

C90S UCH-L1

M N

0

0.2

0.4

0.6

0.8

1

1.2

#****

kB

0

0.5

1

1.5

2

2.5

*

**

145kD

145kD

145kD

25kD42kD

Con S r shTrkBshTrkB & TrkB

wt UCH-L1

C90S UCH-L1

0

20

40

60

80

100

&

TrkB

Con

100

#

OE wt UCH-L1

shTrkB

OE C90S UCH-L1

shTrkB & OE TrkB

shTrkB & OE UCH-L1

0

20

40

60

80**##

##

##100

shTrkBwt UCH-L1

25kD55kD

145kD

55kD

25kD42kD

145kD

42kD

##

UCH-L1

145kD

145kD

##

Flag-TrkB

Myc-UCH-L1

FL T1

+ + +

145kD

35kD

145kD

35kD

Myc

Flag

Flag

Myc

IP:Flag

Lysate

A B

Flag-TrkB FL

Myc-UCH-L1 -

+

+

+

Flag

Flag

Myc

Myc

IP: Myc

Lysate

145kD

35kD

145kD

35kD

145kD

145kD

55kD

IgG TrkB

25kD

25kD

TrkB

TrkB

α-tubulin

UCH-L1

UCH-L1

Lysate

IP

C

D E

HA-UCH-L1 +

+

+

Flag

HA

Flag

HA

IP:Flag

Lysate25kD

145kD

25kD

145kD

F

Figure2.

-

-

35kD

145kD

35kD

145kD

-Flag-TrkB KD

Flag-TrkB FL

Myc-UCH-L1

+

+

+++

-

Myc

Flag

Flag

Myc

IP:Flag

Lysate

0

0.2

0.4

0.6

0.8

1

1.2

1.4

The C

O-I

P o

f T

rkB

with U

CH

-L1

TrkB WT TrkB KD

Flag-TrkB FL

IP:

-

-

Myc-UCH-L1

Flag-TrkB FLFlag-JM1

Flag-JM0Flag-ΔJM1

+ + + + +

+- - - -- - - + -

- - -+ -- - -+ -

Myc-UCH-L1

Flag-IL2RFlag-IL2R JM1

+ + + +

- - - +- - + -- + --

C

DFlag-TrkB FL

TrkB FL

TrkB T1

JM1

JM0

ΔJM1

EC TM

JM1

JM TK CT

CT1

AEC TM

Flag-IL 2R

Flag-IL 2R JM1

JM1

B

Figure 3.

145kD 35kD

145kD

35kD

Myc

Flag

Flag

Myc

IP:Flag

Lysate

Myc

Flag

Flag

Myc

IP:Flag

Lysate

145kD 35kD

145kD 35kD

40kD

40kD

UCH-L1 FL 223aa

UCH-L1 1-150aa

UCH-L1 60-223aa

α-helix β-sheet

A

WT

1-150

60-223

+

-

-

-

+

+

-

-

+

+

-

-

+

+

-

-

Myc

Flag

Flag

Myc

IP:Myc

Lysate

145kD

35kD

145kD

35kD

B

C

+ + + + ++ +

1 2 3 4 5 6

Myc

Flag

Flag

Myc

IP:Myc

Lysate

D

Flag-TrkB FL

Myc-UCH-L1

145kD

40kD

145kD

40kD

GFP

Flag

GFP

Flag

25kDIP:Flag

Lysate

GFP-75-85

GFP

- +

+

-+

+

145kD

25kD

145kD

Δ60-75

Δ75-85

Δ85-100

Δ110-125

Δ125-147

Myc-UCH-L11

2

3

4

5

6

WT

60-75

75-85

85-100

110-125

125-147

C90S

D30A

-

+

-

-

-

+

-

+

-

-

+

+

-

+

-

+

Myc

Flag

Flag

Myc

IP:Flag

Lysate

35kD

145kD

35kD145kD 0

0.2

0.4

0.6

0.8

1

1.2

1.4

Th

e C

O-I

P o

f U

CH

-L1

with T

rkB WT

E F G

Wt C90S D30A

*

Figure 4.

-

Myc-U

CH

-L1

Flag-TrkB FL

Myc-U

CH

-L1

Flag-TrkB FLFlag-TrkB FL

0 15 BDNF(min)P4D1 FK1

Ub

TrkB

TrkB

145kD

145kD

145kD

55kD

A

Ub

P4D1 FK1BkrT: PI

0 15

92kD

92kD

92kD

55kD

70kD :PI

nina

B C

0 15 0 15BDNF(min)

Ub

TrkB

TrkB

145kD

145kD

25kD

145kD

55kD

D

BDNF(min) 0 15 0 15

145kD

145kD

25kD

145kD

55kD

TrkB

TrkB

IP:T

rkBUb

E

C90S

++

+

D30A

WT

++

+

++

+++

145kD

145kD

35kD

145kD

+

145kD

25kD

145kD

145kD

+++

++

FIP

:Trk

B

0 15

0 15 0 151 2 3

145kD

145kD

145kD

55kD

Ub

TrkB

TrkB

0 15 BDNF(min)P4D1 K48

0 15 K63

0 15

G

Con BDNF

*

0

25kD

Figure 6

A

K454R

--

+-

++

+-

++

+-

++

+-

++

+-

++

+-

++

+-

++

C

K460R

K464R

K506R

K524R

K533R

FL

145kD

145kD

35kD

145kD HA

Flag

Flag Lysate

IP:Flag

0

0.2

0.4

0.6

0.8

1

1.2

FL K460R K464RK454R K506R K524R K533R

**

D

--

+-

++

+-

++

+-

++

HA-UbMyc-UCHL-1

Myc-UCHL-1HA-Ub

HA

Flag

FlagMyc

Lysate

IP:Flag

145kD

145kD

35kD

145kD

0

0.2

0.4

0.6

0.8

1

1.2

FL KJMR R460K

**

Ubi

quiti

natio

n le

vel o

f Trk

B

**

***** * **

##

B

Flag-TrkB FL WT KJMR R460K

TrkBNO UCH-L1UCH-L1

NO UCH-L1UCH-L1

Ubi

quiti

natio

n le

vel o

f Trk

B

Myc

##

Myc-UCHL1-+

++

++

Flag-TrkB FLTat Con Tat UCH-L175-85

145kD

35kD

145kD

35kD

Myc

Flag

Flag

Myc

A-

-+

--

++

--

++

++

++

+

B

HA

Flag

Myc

Flag

145kD

145kD

35kD

145kD

70kD

Tat UCH-L175-85

HA-UbFlag-TrkB FL

Myc-UCH-L1

C

145kD

145kD

145kD

70kD

55kD

BDNFTat Con Tat UCH-L175-85

P4D1

TrkB

TrkB

BkrT:PI

Tat Con Tat UCH-L175-85

TrkB

Lysa

teIP

:An

D

****

level BkrT ecafrS

)ezla

mroN(

00.2

0.40.60.81.01.2

Tat Con Tat UCH-L175-85

E

F 7

IP:F

lag

Lysa

te IP:F

lag

Lysa

te Lysa

te

ConBDNF

- + - +

BDNF - + - +

##

-

-+

--

++

--

++

++

++

+Tat UCH-L175-85

HA-UbMyc-UCH-L1

HA

Myc

IP:

sLy

sate

100kD100kD

35kD

100kDF 55kD

145kDwt TrkB K460R TrkB

TrkB

G

H

level BkrT ecafrS

)ezla

mroN(

0.20.40.60.81.01.2

0

**

*

wt TrkB K460R TrkB

1.4

- + - +

ConBDNF

42kD 100kD

145kD

55kD

B

DN

F +

A

B

+

-

+

+

-

+

+

-

-

+

+

+

IP:Avidin

Lysate

TrkB

α-tubulin

C

D

Internalized Recycled Merge

BD

NF

B

DN

F +

Ta

t U

CH

L1

75

-85

% R

ecycle

d

H

G

A

mo

un

t a

fte

r R

ew

arm

ing

145kD

55kD

J

BDNF

Strip

of S

urf

ace T

rkB

-FL

Internal+Surface Surface Merge

BD

NF

Ta

t U

CH

L1

75

-85

Ve

hic

le

Figure 8.

Tat Con

**

0

20

40

60

80

100

Tat Con Tat UCH-L175-85

*

80

**

Tat Con Tat UCH-L175-850

20

40

60

100

**

Tat Con Tat UCH-L175-85

1.S

urface

2. S

trip

3. In

tern

aliz

atio

n4. re

warm

ing

IP:Avidin

Lysate

TrkB

α-tubulin

145kD

55kD

% o

f In

tern

aliz

atio

n

% o

f In

tern

aliz

atio

n

1 2 3 1 2 3

Tat UCH-L175-85

1.S

urface

2. S

trip

3. In

tern

aliz

atio

n4. re

warm

ing

Tat Con

Tat UCH-L175-85

80

0

20

40

60

100

0

20

40

60

80

100

Tat Con Tat UCH-L175-85

E

Fof S

urf

ace T

rkB

-FL

% o

f In

tern

aliz

atio

n

40

0

10

20

30

50

**

wt TrkB

wt TrkB K460R TrkB

K460R TrkB

80

0

20

40

60

100

I

% R

ecycle

d

of S

urf

ace T

rkB

-FL

% o

f In

tern

aliz

atio

n

pTrkB

TrkB

pAkt

AktpErk1/2

Erk1/2

GAPDH

BDNF(min) 0 15 0 15

Tat Con Tat UCH-L175-85

6060145kD

62kD

42kD

42kD37kD

145kD

62kD

fold

indu

ctio

n(p

TrkB

/Trk

B)

**

0

0.2

0.4

0.6

0.8

1.0

1.2

**

D

**

*

***

C

A BFigure 9

Tat ConTat UCH-L175-85

Tat ConTat UCH-L175-855 5

0 15 60BDNF(min)

5

0 15 60BDNF(min)

5

fold

indu

ctio

n(p

Akt/A

kt)

00.5

1.0

1.52.02.53.0

3.5

0 15 60BDNF(min)

5

fold

indu

ctio

n(p

ERK1

/2/E

RK1

/2)

0.20.4

0.6

0.8

1.0

1.2

0

Tat ConTat UCH-L175-85

pTrkB

TrkB

BDNF(min) 0 15 0 15 6060wt TrkB K460R TrkB

-tubulin

E

00.5

11.5

22.5

33.5

4

0 15 60

Fold

indu

ctio

n(pT

rkB/

TrkB

)

TrkB WTTrkB K460R

F

**

**

*

BDNF(min)

B C

F

Per

cent

Tim

e Fr

eezi

ng (%

)

80

60

40

20

0

**Tat Con Tat UCH-L175-85

A

D

Figure 10.

Ub

TrkB-FL

TrkB-FL

IP:T

rkB

Lysa

te

70kD

145kD

145kD

145kD

55kD

25kD

25kD

UCH-L1

UCH-L1

Tat ConTat UCH-L175-85

CFC - - + +- -+ ++ +- -

E

Perc

ent T

ime

Free

zing

(%) Tat Con

Tat UCH-L175-85

80

60

40

20

0

TrkB

Ubi

quiti

natio

n

T

NO CFC CFC

Tat ConTat UCH-L175-85

0

2

4

6

8

10

#

**

0

0.2

0.4

0.6

0.8

1

1.2##

**

NO CFC CFC

Tat ConTat UCH-L175-85

NO CFC CFC

*

0

0.5

1.0

1.5

2.0

2.5

Inte

ract

ion

betw

een

UC

H-L

1&Tr

kB)