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Neuroscience

A Caenorhabditis elegans Model System for Amylopathy Study

Published: May 17th, 2013

DOI:

10.3791/50435

1Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human Aβ42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (Aβ) in tissues with time. Aβ is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study Aβ-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human Aβ42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing Aβ42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

Amyloid beta (Aβ) is a peptide of 36-43 amino acids that is formed after sequential cleavage of the amyloid precursor protein (APP) by β and γ secretases 1. The γ secretase processes the C-terminal end of the Aβ peptide and is responsible for its variable lengths 5. The most common forms of Aβ are Aβ40 and Aβ42, the latter being more commonly associated to pathologic conditions such as AD 5. At high concentrations Aβ form β-sheets that aggregate to form amyloid fibrils 6. Fibrils deposits are the main component of senile plaques surrounding ne....

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1. Construction of Transgenic Worms

  1. Transformation.
    1. Prepare injection pads. Place a drop of hot, 2% agarose dissolved in water, onto a glass coverslip. Quickly place a second coverslip on the drop and lightly tap it. After the agarose is solidified, slide coverslips apart, and bake the coverslip-pad in a vacuum oven at 80 °C O/N.
    2. Pull pipettes. We use a Sutter P-97 puller to pull 1/0.5 mm O.D./I.D. borosilicate capillaries with filament. Pipettes are forged with closed tip which is .......

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With our protocols we study the effects of human Aβ42 oligomer on neuronal function 8. A fragment encoding human Aβ42 and the artificial signal peptide coding sequence of Fire vector pPD50.52 was amplified from construct PCL12 9 using primers that introduced a Sma 1 restriction endonuclease site at the ends. The fragment was then inserted into a construct containing a 2,481-bp flp-6 promoter sequence in the pPD95.75 Fire vector between the unique Sma 1 site <.......

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Here we describe a combined approach, to study cellular and molecular aspects of amylopathies using C. elegans. The advantages of this approach include: 1) low cost. C.elegans is maintained in normal Petri dish seeded with bacteria, at room temperature. 2) Powerful genetics. Transgenic animals can be obtained in few months and a wide array of promoter sequences is available to drive expression of the desired gene in specific neurons. 3) Simple, well-characterized, nervous system. C. elegans pos.......

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We thank Dr. Shuang Liu for critical reading of the manuscript. The PCL12 construct was a gift form Dr. Christopher D. Link. This work was supported by two National Science Foundation grants (0842708 and 1026958) and an AHA grant (09GRNT2250529) to FS.

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Name Company Catalog Number Comments
Name of Reagent Company Catalog Number Comments
      1. NGM
Sodium Chloride Sigma-Aldrich S5886 3 g
Bacteriological agar AMRESCO J637 17 g
Bacto-peptone AMRESCO J636 2.5 g
Distilled Water     Bring to 975 ml
      Sterilized by autoclaving, then add the following items and mix well
Magnesium sulfate Sigma-Aldrich M2643 1 ml of 1 M stock
Calcium Chloride Sigma-Aldrich C5670 1 ml of 1 M stock
Cholesterol Sigma-Aldrich C3045 1 ml of 5 mg/ml stock( in ethanol)
Potassium phosphate buffer     25 ml of 1M stock
      2. Potassium phosphate buffer
Potassium phosphate monobasic Sigma-Aldrich P5655 108.3 g
Potassium phosphate dibasic Sigma-Aldrich P3786 35.6 g
Distilled Water     Bring to 1 L
      Sterilized by autoclaving
      3. M9 buffer
Potassium phosphate monobasic Sigma-Aldrich P5655 3 g
Sodium phosphate dibasic Sigma-Aldrich S5136 6 g
Sodium Chloride Sigma-Aldrich S5886 5 g
Magnesium sulfate Sigma-Aldrich M2643 1 ml of 1 M stock
Distilled Water     Bring to 1 L
      Sterilized by autoclaving
      4. Egg buffer (pH 7.3, 340 mOsm)
Sodium Chloride Sigma-Aldrich S5886 118 mM
Potassium Chloride Sigma-Aldrich P5405 48 mM
Calcium Chloride Sigma-Aldrich C5670 2 mM
Magnesium Chloride Sigma-Aldrich M4880 2 mM
HEPES Fisher Scientific BP310 25 mM
Distilled Water     Bring to 1 L
      Sterilized by autoclaving
      5. CM-15
L-15 culture medium Gibco 11415 450 ml
Fetal Bovine Serum Gibco 10437-028 50 ml
Penicillin Gibco 15140 50 units/ml
Streptomycin Gibco 15140 50 g/ml
      Adjust to 340 mOsm with sucrose then sterile filter into autoclaved bottles and store at 4 °C
      6. Other Reagents
Halocarbon 700 oil Halocarbon Products 9002-83-9  
5 μm Acrodisc Syringe Filter PALL Co. 4199  
Chitinase Sigma-Aldrich C6137-5UN  
Lectin (peanut) Sigma-Aldrich L0881-10MG  
Sodium hydroxide Fisher Scientific S320  
Lysine Sigma-Aldrich L5501  
Biotin Sigma-Aldrich B4639  
Sodium hypochlorite solution Sigma-Aldrich 425044  
Sodium azide Sigma-Aldrich 71289  
Sucrose Sigma-Aldrich S0389  

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