Maple Syrup Decreases TDP-43 Proteotoxicity in a Caenorhabditis

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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

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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

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Abstract:

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

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

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

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

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

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

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neuroprotective activity. Maple syrup decreased several age-dependent phenotypes

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

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FOXO transcription factor DAF-16 to be effective.

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

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INTRODUCTION

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

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

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

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

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

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

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

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and sporadic ALS

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still poorly understood.

3,4

2

and mutations in the

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

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

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

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

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

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immunological and proteotoxic stress pathways

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

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

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

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integrity in several neuronal proteotoxicity models 13.

8-11

. Using our models, we have also 11,12

. Among

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

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

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

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


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

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only present in the syrup

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phenols

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

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

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combination of phenols is involved in this neuroprotection.

15-17

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and which includes sucrose, antioxidants and many different

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

. We

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MATERIALS AND METHODS

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Strains and maintenance

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

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previously described in Vaccaro et al. 2011

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

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

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

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NGM supplementation

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

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

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

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

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

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Paralysis assay

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

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

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

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

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

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Neurodegeneration


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

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

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

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

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

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Fluorescence intensity measurements

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

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

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

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

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tailed t-tests were used for statistical analysis.

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Analysis of maple syrup by GC-MS

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

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

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

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

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

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

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

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

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


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

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

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acid was added to each sample.

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

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

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

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

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

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

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

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the same manner as described here.

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

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

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

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

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

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

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

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

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

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RESULTS AND DISCUSSION

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

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

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including rats, mice or worms among others

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

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

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

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

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

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

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syrup14.

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20-22

. However, the effectiveness of these

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

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

. The

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

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

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

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by the expression of mutant TDP-43.

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

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

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

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and activation of the endoplasmic reticulum (ER) stress response

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

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


10,26

. We observed that

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

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

Maple Syrup Decreases TDP-43 Proteotoxicity in a Caenorhabditis

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