Maple Syrup Decreases TDP-43 Proteotoxicity in a Caenorhabditis

For a more comprehensive list of citations to this article, users are encouraged to ... maple syrup extract and its protective effects on normal human...
0 downloads 0 Views 1MB Size
Subscriber access provided by UOW Library

Article

Maple syrup decreases TDP-43 proteotoxicity in a C. elegans model of ALS Catherine Aaron, Gabrielle Beaudry, Alex Parker, and Martine Therrien J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05432 • Publication Date (Web): 13 Apr 2016 Downloaded from http://pubs.acs.org on April 14, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28

Journal of Agricultural and Food Chemistry

Maple syrup decreases TDP-43 proteotoxicity in a C. elegans model of ALS.

Catherine AaronŦŦ1, Gabrielle BeaudryŦŦ1, J. Alex Parker*1,2, Martine Therrien1,3 ŦŦ

These authors contributed equally to the work

Affiliations 1

Centre de recherche du Centre Hospitalier de l’Université de Montréal (CRCHUM),

900 St-Denis street, Montréal, Québec, Canada, H2X 0A9 2

Department of Neuroscience, University of Montréal,

2960 chemin de la tour, Montréal, Québec, Canada H3T 1J4 3

Department of Pathology and Cell Biology, University of Montreal,

2900 Edouard-Montpetit boul, Montréal, Québec, Canada H3T 1J4

*corresponding author JAP: [email protected], 514-890-8000 (28826),

ACS Paragon Plus Environment

1

Journal of Agricultural and Food Chemistry

Page 2 of 28

1

Abstract:

2

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease causing death of the

3

motor neurons. Proteotoxicity caused by TDP-43 protein is an important aspect of ALS

4

pathogenesis, with TDP-43 being the main constituent of the aggregates found in

5

patients. We have previously tested the effect of different sugars on the proteotoxicity

6

caused by the expression of mutant TDP-43 in C. elegans. Here we tested maple syrup, a

7

natural compound containing many active molecules including sugars and phenols, for

8

neuroprotective activity. Maple syrup decreased several age-dependent phenotypes

9

caused by the expression of TDP-43A315T in C. elegans motor neurons and requires the

10

FOXO transcription factor DAF-16 to be effective.

11 12

Key words: C. elegans, ALS, maple syrup, TDP-43

13

ACS Paragon Plus Environment

2

Page 3 of 28

14

Journal of Agricultural and Food Chemistry

INTRODUCTION

15 16

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting motor

17

neurons. Patients exhibit neurodegeneration leading to paralysis and ultimately to death

18

3-5 years after the onset of symptoms 1. So far, no cure is available for patients and the

19

only approved drug, riluzole, has modest effects 1. Like many other neurodegenerative

20

diseases, proteotoxicity is a major mechanism underlying neurodegeneration in ALS. In

21

most patients, TDP-43 is the main constituent of the aggregates

22

gene TARDBP, which encodes the TDP-43 protein, were found to be a cause of familial

23

and sporadic ALS

24

still poorly understood.

3,4

2

and mutations in the

. The proteotoxicity caused by the expression of mutant TDP-43 is

25

C. elegans is a free-living soil nematode that is widely used to study lifespan. Its

26

nervous system is well described and many groups have used this nematode to study

27

neurodegenerative diseases 5-7. Our group has previously described that mutant TDP-43 is

28

toxic to C. elegans motor neurons and induced neuronal loss and a cascade of cell death,

29

immunological and proteotoxic stress pathways

30

identified molecules that alleviate TDP-43 proteotoxicity in C. elegans

31

these, we have shown that supplementation of the worm’s diet with different types of

32

sugar, including glucose and sucrose, could improve motor function and neuronal

33

integrity in several neuronal proteotoxicity models 13.

8-11

. Using our models, we have also 11,12

. Among

34

To expand upon these findings, we aimed to identify a natural product with a high

35

sugar content of sugar that could have neuroprotective properties in C. elegans.

36

Therefore, we turned to maple syrup, a widely available compound that contains high

37

levels of sugar as well as phenols. Maple syrup is produced from the sap collected from

ACS Paragon Plus Environment

3

Journal of Agricultural and Food Chemistry

Page 4 of 28

38

maple tree and the boiling of the sap produces a cocktail of different compounds that are

39

only present in the syrup

40

phenols

41

their effect on C. elegans longevity, stress response and neuronal protection

42

show here that maple syrup is protective against TDP-43 proteotoxicity, and that a

43

combination of phenols is involved in this neuroprotection.

15-17

14

and which includes sucrose, antioxidants and many different

. The effect of phenols found in natural compounds have been studied for 18-20

. We

44 45

ACS Paragon Plus Environment

4

Page 5 of 28

Journal of Agricultural and Food Chemistry

46

MATERIALS AND METHODS

47

Strains and maintenance

48

Generation of the animal expressing unc-47p::TDP-43A315T ; unc-47p::GFP were

49

previously described in Vaccaro et al. 2011

50

were obtained from the Caenorhabditis elegans Genetics Center (CGC, Minnesota, US) .

51

All worms were maintained on normal NGM agar plates with OP-50 bacteria at 20 °C.

10

. daf-16(mu86) and zcIs4[hsp-4p::GFP]

52 53

NGM supplementation

54

Maple syrup (grade D, amber) was bought in local markets in Montreal in January 2014

55

and January 2015. Commercially available maple syrup was added directly to the NGM

56

plates at concentrations indicated (1%, 2% and 4%). Gallic acid, catechol, syringaldehyde

57

and 3,4 dihydroxybenzaldehyde (all from Sigma, Oakville, Canada) were dissolved in

58

DMSO and add to NGM at the indicated concentration (100 µM, 200 µM and 300 µM)

59 60

Paralysis assay

61

L4 animals were placed on normal or supplemented NGM plates and scored for 12 days.

62

Animals that failed to move upon being probed with a pick were counted as paralysed. At

63

least, 100 worms were counted for each condition over 3 trials. Animals were exposed to

64

compounds from hatching. Survival curves and statistics were produced using the Log-

65

rank (Mantel-Cox) test, standard-error is shown in figures.

66 67

Neurodegeneration

ACS Paragon Plus Environment

5

Journal of Agricultural and Food Chemistry

Page 6 of 28

68

To score gaps or breaks, worms at day 9 of adulthood were used for direct visualization

69

of GABAergic neurons. Worms were placed on a 2% agarose pad and immobilised with

70

5 mM levamisole. Visualization was done using Zeiss Axio Imager M2 microscope

71

microscope. At least 100 worms were counted over 3 trials. The mean and SEM were

72

calculated and two-tailed t-tests were used for statistical analysis.

73 74

Fluorescence intensity measurements

75

20 day-1 worms were visualized for the measurement of hsp-4p::GFP. Worms were put

76

on a 2% agarose pad and immobilised with 5 mM levamizole. Visualization was done

77

using Zeiss Axio Imager-M2 microscope and AxioCam HRM camera. Quantification of

78

the fluorescence was done using Image J. The mean and SEM were calculated and two-

79

tailed t-tests were used for statistical analysis.

80 81

Analysis of maple syrup by GC-MS

82

Extraction: Phenolic compounds excluding gallic acid were extracted as follows: A 1 ml

83

volume of commercially available maple syrup transferred to a 16 mm borosilicate test

84

tube. To this, 1 ml LC/MS grade water was added and enough NaCl to saturate the

85

solution. Following 60 sec of vortexing a volume of 2 ml ethyl acetate was added and

86

vortexed again for 60 sec. The samples were left at room temperature for 10 min to allow

87

for phase separation and then centrifuged at 4500 rpm for 5 minutes. The organic layer

88

was then dried under a stream of nitrogen. The samples were re-dissolved in 400 µL

89

ethyl acetate and transferred to eppendorf brand tubes. The samples were then dried

90

under a stream of nitrogen. Gallic acid was extracted under acidic conditions, where 1 ml

ACS Paragon Plus Environment

6

Page 7 of 28

Journal of Agricultural and Food Chemistry

91

of 1 N HCl was added to the maple syrup. Otherwise the extraction protocol remained

92

the same. A 1 µL volume of retention time locking standard, 840 ng/uL D27-myristic

93

acid was added to each sample.

94

Derivatization for GC/MS analysis: A two-step derivatization process was used. First

95

the samples were dissolved in 30 µL of 10 mg/ml methoxyamine HCl dissolved in

96

pyridine. The samples were allowed to incubate at room temperature for 30 min.The

97

samples were centrifuged at room temperature for 10 min at 15,000 rpm. The supernatant

98

was then transferred to GC/MS vials with glass inserts containing 70 µL MTBSTFA. The

99

samples were briefly vortexed and incubated at 70 oC for 1 hr. A volume of 1 µL was

100

injected for GC/MS analysis. A small amount of each authentic standard as prepared in

101

the same manner as described here.

102

GC/MS analysis: GC/MS data were acquired using an Agilent 5975C mass selective

103

detector coupled to a 7890A GC (Agilent technologies, Santa Clara, CA, USA). A DB-

104

5MS+DG capillary column (Agilent J&W, Santa Clara, CA, USA) was used to separate

105

extracted compounds. The GC temperature program started with a 1 min hold at 60 °C

106

followed by a 10 °C/min ramp to 300 °C. Bake-out was at 320 °C for 9 min. The injector

107

and interface to the MS were held at 285 °C. The helium carrier flow rate was held

108

constant at 1.5 ml/min (or a flow rate such that the D27-myristic acid standard has a

109

retention time of 18 min). The mass spectrometer was operated in full scan mode (50-

110

1000 da). Electron ionization was used at 70eV.

111 112 113

ACS Paragon Plus Environment

7

Journal of Agricultural and Food Chemistry

Page 8 of 28

114

RESULTS AND DISCUSSION

115

The neuroprotective effects of pure isolated active compounds such as resveratrol, as well

116

as other phenols or antioxidants have been reported in many in vitro and in vivo models,

117

including rats, mice or worms among others

118

compounds in combinations, or in concentrations that are found in natural products, is

119

less studied. Therefore, we aimed to identify a natural compound that could diminish

120

neuronal proteotoxicity, so we tested maple syrup for putative protective activity against

121

the cytotoxicity caused by the expression of mutant TDP-43 in C. elegans. Maple syrup is

122

produced from the sap collected from maple (Acer) tree species. It is thought that the

123

boiling of the sap produces a cocktail of different compounds that are only present in the

124

syrup14.

125

20-22

. However, the effectiveness of these

Maple syrup contains mainly sucrose (98%) but also includes antioxidants and 14-17

126

many different phenols, including Quebecol that is only found in maple syrup

. The

127

content of the syrup and concentrations of these active agents was shown to vary between

128

the different grade of maple syrup, therefore we used the darkest type, grade D23-25. We

129

used commercially available maple syrup and tested it against several phenotypes caused

130

by the expression of mutant TDP-43.

131

A transgenic C. elegans strain that expresses mutant TDP-43A315T only within the

132

worms GABAergic motor neurons was used as a genetic model of ALS. Our group has

133

previously shown that the expression of TDP-43A315T causes paralysis, neurodegeneration

134

and activation of the endoplasmic reticulum (ER) stress response

135

maple syrup has a dose-dependent effect on suppressing the paralysis caused by TDP-

136

43A315T, with 4% maple syrup being the most effective concentration (Figure 1A) (44%

ACS Paragon Plus Environment

10,26

. We observed that

8

Page 9 of 28

Journal of Agricultural and Food Chemistry

137

paralysis in untreated, 33% paralysis in 1% maple syrup (p value< 0.05), 24% in 2%

138

maple syrup (p value= 0.06) and 17% in 4% maple syrup (p value