Neuroprotection and lifespan extension in Ppt1-/- mice by ... · Normal PPT1-deficient Failure of...

Supplementary information

Title

Neuroprotection and lifespan extension in Ppt1-/-mice by NtBuHA: therapeutic implications for INCL

Authors

Chinmoy Sarkar, Goutam Chandra, Shyiong Peng, Zhongjian Zhang, Aiyi Liu and Anil B. Mukherjee

Nature Neuroscience: doi:10.1038/nn.3526

Plasma membrane

Endolysosome

PPT1-deficientNormal

Failure ofdepalmitoylation

Lysosomalstorage of ceroid

Degradationproducts

Protein

Palmitate

Supplementary data Fig. 1. In normal cells, the membrane-anchored palmitoylated proteins are depalmitoylated by lysosomal PPT1 and degraded by lysosomal hydrolases clearing the lysosomes. However in PPT1-deficient (INCL) cells failure of depalmitoylation makes these proteins refractory to lysosomal hydrolases. Consequently, accumulation of the palmitoylated proteins leads to lysosomal storage of ceroid causing INCL pathogenesis.

Supplementary Fig. 1 Schematic depiction of the mechanism of ceroid accumulation in INCL.


Supplementary data Fig. 2 INCL fibroblasts were incubated with increasing concentrations of (a) N-t-butyl hydroxylamine, (b) N-benzylhydroxylamine, (c) N-Methyl hydroxylamine, (d) N-cyclohexyl hydroxylamine, (e) N-benzyloxycarbonyl hydroxylamine, (f) N,N-dimethyl hydroxylamine, (g) N,N-diethyl hydroxylamine, (h) N-benzoyl-N-phenyl hydroxylamine, (i) N,O-bis(trimethylsilyl) hydroxylamine, (j) N,O-Di-BOC hydroxylamine for 48 hours. Viability of the treated cells was estimated by MTT assay (see method section). Note that most of the derivatives are nontoxic up to 1 mM concentration. * P<0.05.

a

c

e

g

i j

h

f

d

b

NtBuHA (mM)

0

50

100

150

Control 0.1 0.5 1 2.5

Via

bili

ty (

%co

ntr

ol)

N-benzylHA (mM)

020406080

100120140

Control 0.1 0.5 1 2.5

Via

bili

ty (

%co

ntr

ol)

N-methylHA (mM)

0

20

40

60

80

100

120

Control 0.1 0.5 1 2.5

Via

bili

ty (

% c

on

tro

l)

N-cyclohexylHA (mM)

020406080

100120140

Control 0.1 0.5 1 2.5

Via

bili

ty (

% c

on

tro

l)

N-benzyloxycarbonylHA (mM)

020406080

100120140

Control 0.1 0.5 1 2.5

Via

bili

ty (

% c

on

tro

l)

N,N-dimethyl HAControl 0.1 0.5 1 2.5

0

50

100

150

200

Via

bili

ty (

% c

on

tro

l)

N,N-diethyl HA (mM)

020406080

100120140

Control 0.1 0.5 1 2.5

Via

bili

ty (

% c

on

tro

l)

N-benzoyl-N-phenyl HA (mM)

020406080

100120

Control 0.1 0.5 1 2.5

Via

bili

ty (

% c

on

tro

l)

N,O-bis(trimethylsilyl) HA (mM)

0

50

100

150

Control 0.1 0.5 1 2.5

Via

bili

ty (

% c

on

tro

l)

N,O-Di-BOC-HA (mM)

0

50

100

150

200

Control 0.1 0.5 1 2.5

Via

bili

ty (

% c

on

tro

l)

* *

*

Supplementary Fig. 2. Effect of hydroxylamine derivatives on viability of INCL fibroblasts.


Supplementary Fig. 3 Viability of INCL lymphoblasts treated with NtBuHA: Time Course and plating efficiency of NtBuHA treated INCL fibroblasts.

Supplementary data Fig 3. (a). Lymphoblasts isolated from INCL patients were cultured for varying length of time in presence of 500 µM NtBuHA. Viability of those cells at the end of incubation period was estimated by MTT assay and expressed as percent of viability of untreated control. The data is presented as the mean of three independent experiments ±SD. (b). Skin fibroblasts from an INCL patient were treated with varying doses (0 to 2.5 mM) of NtBuHA for 48 h. At the end of the treatment period the cells were trypsinized and re-plated at a density of 100 cells per plate. The colonies formed from 100 cells in more than 10 replicates for each treatment were stained with Crystal violet, air dried and photographed.

0

20

40

60

80

100

120

140

0 12 24 36 48 60

Time (h)

Via

bilit

y (

% c

ontr

ol)

Control 0.1 mM NtBuHA 0.5 mM NtBuHA

1 mM NtBuHA 2.5 mM NtBuHA

a

b


a. b.

1 2 3 4 5 6

Time course

0

5000

10000

15000

20000

25000

30000

35000

40000

0 10 50 100 250 500

NtBuHA (uM)

Den

sity

(IN

Tpe

rm

m2 )

Dose response

1 2 3 4 5 6 7

0

10000

20000

30000

40000

50000

60000

0 15 30 60 120

Time (m)

Den

sity

(IN

T p

er m

m2 )

Supplementary Fig. 4 NtBuHA-mediated cleavage of thioester linkage in [14C] PalmitoylCoA.

Supplementary data Fig. 4 (a) Dose-response: The release of free [14C]palmitic acid from [14C] palmitoyl CoA by varying doses of NtBuHA at room temperature for 1 h. Lanes: (1) [14C]palmitic acid standard; (2) [14C]-palmitoyl CoA standard; (3) 10 µM NtBuHA; (4) 50 µM NtBuHA; (5) 100 µM NtBuHA; (6) 250 µM NtBuHA and (7) 500 µM NtBuHA. (b) Time-course of [14C]-palmitic acid release from [14C] palmitoyl CoA by 500 µM of NtBuHA. Lanes: (1) [14C]palmitic acid standard; (2) 0 min; (3) 15 min; (4) 30 min; (5) 60 min; (6) 120 min. Arrows indicate free [14C]Palmitate released. The densitometricquantitation of free [14C]palmitate bands are shown graphically below the autoradiographsof the TLCs (n=3). Note that the release of [14C]palmitate from [14C]-palmitoyl CoA by NtBuHA is dose- and time-dependent. The results were analyzed using student t test and P<0.01 was considered significant (in panel a) and P<0.05 in panel b at 60 and 120 minutes.


Supplementary Fig. 5 Densitometric quantitations of lipid thioester bands in untreated-and NtBuHA-treated INCL lymphoblasts.

Supplementary data Fig. 5 (a). [35S]Cysteine-labeled lipid thioester bands resolved by TLC as shown in Figure 1f. Eight bands were readily identifiable (arrows) and marked as bands 1 to 8 (from the top). The density of each of these bands from untreated and NtBuHA-treated INCL patients’ cells were quantified using QuantityOne software (Biorad). The densitometric data are presented as the mean intensity/mm2 (INT/mm2) from three independent experiments ±SD. (b). [35S]Cysteine-labeled lipid thioester bands in untreated and NtBuHA-treated lymphoblasts from 9 INCL patients are resolved by TLC as shown in Figure 1g (right panel). Each of the 8 readily identifiable bands as indicated in Fig.1f was marked as bands 1 to 8 (from the top). The results of densitometric quantitation are presented as the mean of 3 independent experiments ±SD.

0 10000

20000

30000

40000

50000

60000

band 1

band 2

band 3

band 4

band 5

band 6

band 7

band 8

Normal

INCL

INCL- NtBuHA

P<0.05

P<0.01

P<0.05

P<0.05

P<0.01

P<0.01

Density (INT per mm2)

0 5000 10000 15000 20000 25000 30000 35000 40000 45000

band8

band7

band6

band5

band4

band3

band2

band1

Density (INT per mm2)

12h NtBuHA

12h Control

24h NtBuHA

24h Control

48h NtBuHA

48h Control

a b


a.

b.

Unt

reat

ed c

ontr

olN

tBuH

A-t

reat

edMagnified inset

c.

0

50

100

150

200

250

Untreated control NtBuHA treated

Num

ber

of G

RO

Ds

per

cell P<0.0001

Supplementary Fig. 6 Depletion of GRODs in INCL fibroblasts after treatment with NtBuHA.

Supplementary data Fig. 6 INCL fibroblast cells were treated without (a) or with (b) 250 µM of NtBuHA for 3 weeks. Cells were then fixed in 2.5% glutaraldehyde and processed for electron microscopy. The number of GRODs/cell from untreated- (n=7) and NtBuHA-treated (n=10) INCL fibroblasts are graphically represented in panel c. Note a clear reduction in GROD level in INCL cells treated with NtBuHA. Scale bar: 1µm.


Supplementary Fig. 7 Western blot analyses of ER- and oxidative stress markers in WT and Ppt1-/- mouse brains.

Grp94

0

1

2

WT

Rel

ativ

e de

nsity

Grp78

0

2

4

Rel

ativ

e de

nsity

ATF6

0

2

4

SOD2

0

1

2

Catalase

0

2

4

WT

WT

Untre

ated

-Ppt1-/-

WT

WT

Rel

ativ

e de

nsity

Rel

ativ

e de

nsity

Rel

ativ

e de

nsity

P<0.01 P<0.01P<0.01 P<0.01

P<0.01 p<0.01 P<0.01 P<0.01P<0.01 P<0.01

NuBuH

A-trea

ted-Ppt1-/-

Untre

ated

-Ppt1-/-

NuBuH

A-trea

ted-Ppt1-/-

Untre

ated

-Ppt1-/-

NuBuH

A-trea

ted-Ppt1-/-

Untre

ated

-Ppt1-/-

NuBuH

A-trea

ted-Ppt1-/-

Untre

ated

-Ppt1-/-

NuBuH

A-trea

ted-Ppt1-/-

1 2 3 1 2 3 1 2 3

β‐actin

Catalase

SOD 2

ATF 6

Grp 78

Grp 94

WTUntreated Ppt1/-

NtBuHA-treated Ppt1-/-

Supplementary data Fig. 7 Cortical tissue homogenates prepared from 6-month old WT, untreated-Ppt1-/-

and NtBuHA-treated Ppt1-/- mice (n=6 in each group) were analyzed for ER-stress markers: ATF6, Grp94 and Grp78 and oxidative stress-markers: SOD2 and catalase, respectively. The results of densitometricquantitation of the protein bands (mean +SDs) are represented in the bar graphs.


Supplementary Fig. 8 Protection of superficial and deep cortical neurons of Ppt1-/-

mice by NtBuHA.

10 µm100 µm

Cux1 Ctip2 DAPI Merge Cux1/DAPI Ctip2/DAPI

WT

NtB

uHA

-tre

ated

-Ppt1-/

-

100 µm

100 µm

10 µm

10 µm

II&III

IV

V

VI

VI

VI

V

V

III&III

IV

II&III

IV

a b

c d

I

I

0

5

10

15

20

25

Num

ber

of C

ux1-

posi

tive

Cel

ls p

er 0

.01m

m2

0

5

10

Num

ber

of C

tip2-

posi

tive

Cel

ls p

er 0

.01m

m2P<0.05

P<0.05

P<0.05 P<0.05

Unt

reat

ed-Ppt1-/

-

WT Untreated Ppt1-/-




Supplementary data Fig. 8 Superficial (II-IV) and deep (IV-VI) cortical layers of WT (a; upper panel),untreated Ppt1-/- (a; middle panel), and NtBuHA-treated Ppt1-/- (a; lower panel) brain sections were immunostained with Cux1 and Ctip2 respectively. For each group, representative images of Cux1 and Ctip2 –positive cells in layers II & III and layer V respectively are shown (b). Quantification of Cux1- (c) and Ctip2- (d) positive cells per 0.01 mm2 in these layers revealed significant protection of both these cells in NtBuHA-treated Ppt1-/- mice over their untreated littermates (n=4).


Supplementary Fig. 9 Cortical thickness in WT, untreated- and NtBuHA-treated Ppt1-/- mice; evaluation of motor coordination by rotarod rest in 3-month old WT and Ppt1-/- littermates and rotarod endurance test of untreated and NtBuHA-treated Ppt1-/- mice.

b

c

V2ML

a

Corpus callosum

60 µm

WTUntreated Ppt1-/-


0100200300400500600700800

Cor

tical

thic

knes

s (µ

m)

P<0.0001P<0.0001

WTUntreated Ppt1-/-


Supplementary data Fig. 9 The sections of the brain from 6-month old WT (n= 4), untreated Ppt1-/-

(n=4) and NtBuHA-treated Ppt1-/- (n=4) mice were stained with cresyl violet and photomicrographs were analyzed for cortical thickness (a). Graphic representation of the brain region (V2ML, secondary visual cortex mediolateral) where the cortical thickness was measured (Arrow); (b) cresyl violet stained cortical sections from WT, untreated- and NtBuHA-treated Ppt1-/- mice; (c) graphic representation of the cortical thickness among WT mice and their untreated- and NtBuHA-treated littermates. The results are presented as the mean + SD; (d) Three-month old WT (n=6) and Ppt1-/- mice (n=6) were tested on rotarod at 4, 8 and 12 rpm for 60 seconds. No significant difference in motor coordination was observed at this age between WT and Ppt1-/- mice; (e) Six-months old WT (n= 6), untreated Ppt1-/- (n=8) and NtBuHA-treated Ppt1-/- (n=7) mice were allowed to stay on rotarod at 12 rpm for up to 300 seconds. Note a significant, albeit modest, improvement in performance of NtBuHA-treated Ppt1-/- mice in the rotarod endurance test.

0

50

100

150

200

250

300

350

Tim

e on

rot

arod

(s)

P<0.001

P<0.001



0

10

20

30

40

50

60

70

RPM

Tim

e on

rot

arod

(s)

WT

Ppt1-/-

4 8 12

ed


kDa250

130 100

7055

35

25

15

kDa250

130 100

7055

35

25

15

kDa100

70

55

35

Supplementary Fig. 10 Full length pictures of the cropped blots presented in the mainfigures: 4e and 5e .

kDa250

130 100

7055

3525

1510

kDa250

130 100

7055

3525

1510

kDa250

130 100

7055

3525

1510

1 2 3 1 2 3 1 2 3

WTUntreated PPT1-/-

NtBuHA-treated PPT1-/-

CleavedCaspase-9

ß-Actin

Cleaved PARP-1

a b

1 2 3 1 2 3 1 2 3

WTUntreated PPT1-/-

NtBuHA-treated PPT1-/-

NeuN

GFAP

ß-Actin

Supplementary data Fig. 10: panel a, full length picture of the cropped gel in main Figure 4e; panel b, full length picture of the cropped gel in main Figure 5e.


Supplemenary Table 1: Hydroxylamine and its Derivatives

Chemical Formula Description

NH2OH · HCl Hydroxylamine hydrochloride

1. C6H15NO3 N-(tert-Butyl)hydroxylamine acetate

2. C7H9NO · HCl N-Benzylhydroxylamine hydrochloride

3. CH5NO · HCl N-Methylhydroxylamine hydrochloride

4. C6H13NO · HCl N-Cyclohexylhydroxylamine hydrochloride

5. C2H7NO · HCl N,N-Dimethylhydroxylamine hydrochloride

6. C4H11NO N,N-Diethylhydroxylamine

7. C6H19NOSi2 N,O-Bis(trimethylsilyl) hydroxylamine

8. C8H9NO3 N-Benzyloxycarbonyl hydroxylamine

9. C14H15NO N,N-Dibenzylhydroxylamine

10. C13H11NO2 N-Benzoyl-N-phenyl hydroxylamine

11. C23H32ClNO N-tert-Butyl-O-[1-[4-(chloromethyl) phenyl]ethyl]- N-(2-methyl-1-phenylpropyl) hydroxylamine

12. C10H19NO5 N,O-Di-Boc-hydroxylamine


Supplementary Table 2: PPT1-deficient cell lines from INCL patients

Cell line PPT1-mutations

Lymphoblasts*

1. C11568 MissenseMissense

A364T (R122W)G541A (V181M)

2. C12275 MissenseNonsense

G353A (G118D)C451T (R151X)

3. C11796 NonsenseNonsense

C451T (R151X)C451T (R151X)

4. C10949 NonsenseNonsense

C490T (R161X)C490T (R161X)

5. C1045LT NonsenseNonsense

C451T (R151X)C451T (R151X)

6. C7142L NonsenseMissense

C451T (R151X)G541A (V181M)

7. C10320LT NonsenseMissense

C451T (R151X)G749T (G250V)

8. C11560 MissenseNonsense

A223C (T75P)C451T (R151X)

9. C12488 MissenseMissense

A223C (T75P)A223C (T75P)

10.

C9982 (N1) Normal lymphoblasts (NL)

Fibroblasts

INCL Fibroblasts MissenseMissense

A364T (R122W)A364T (R122W)

* All lymphoblasts and their genotypes were generously provided by Late Dr. K.E. Wisniewski.

11.

GM00131(N2) Normal lymphoblasts (NL)

12.

GM00536 (N3) Normal lymphoblasts (NL)

13.

C9955 (N4) Normal lymphoblasts (NL)


Supplemental Video: Rotarod Test

Rotarod test (60 seconds) in WT, untreated Ppt1-/- and NtBuHA-treated Ppt1-/- mice. Mouse#1 & 2 (left), NtBuHA-treated; Mouse #3 & 4 (middle), untreated and Mouse#5 (right), WT.


Neuroprotection and lifespan extension in Ppt1-/- mice by ... · Normal PPT1-deficient Failure of...

Documents

Transcript of Neuroprotection and lifespan extension in Ppt1-/- mice by ... · Normal PPT1-deficient Failure of...