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.

1. Construction of Transgenic Worms
<ol>
	<li>Transformation.
	<ol>
		<li>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 &deg;C O/N.</li>
		<li>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 broken open at a later stage.</li>
		<li>Prepare injection mix. Make the injection mixture containing the DNA of interest (20 ng/&mu;l per construct) plus empty plasmid DNA to a final concentration of 150 ng/&mu;l. Centrifuge the injection mixture to remove any contaminant. This step is crucial because contaminants reduce transformation efficiency. Transfer the top 5 &mu;l (debris and other contaminants collect in the bottom) to a fresh tube to be used in injection.</li>
		<li>Load injection mix by capillarity. The bottom of the pipette is immersed in the injection mix. Typically 0.5 &mu;l are sufficient to inject 100 worms.</li>
		<li>Load injection pipette. Insert the pipette onto the holder of the micromanipulator and break it open by rubbing its tip against the edge of a cover slip mounted on a glass slide or alternatively against debris on the agarose pad. This is a crucial step as too large tips damage the worm and too small tips are easily clogged. The quality of the broken tip can be judged by the shape and most importantly by the flow rate. The flow rate can be assessed by the size of the bubbles flowing from the open tip immersed in a drop of halocarbon 700 oil in response to an injection pressure.</li>
		<li>Transfer worms to injection pad. Place a drop of 700 halocarbon oil on an injection pad and transfer several (1 or 2 for beginners) worms to the oil. Use a worm pick to push the worms down onto the pad until they adhere to the agarose. Worms should be oriented in rows with ventral sides facing the same direction. Avoid touching worm's head. If after several attempts the worms fail to adhere to the pad, replace with a fresh pad or increase agarose thickness and/or concentration.</li>
		<li>Insert the pipette into the worm. By moving the stage, position the worm under the pipette. Position the pipette in the central core of the gonad because its cytoplasm is shared by many germ cell nuclei. This increases the likelihood to deliver injected DNA to many progeny. The pipette should lie almost parallel to the worm (~15&deg;-25&deg; angle). Push vertically the tip of the pipette down the worm's body until the skin is depressed. Then gently tap on the manipulator to induce tip's insertion. For best results inject both gonads.</li>
		<li>Inject the DNA solution. Apply pressure to the pipette until the gonad swells. Stop the flow and pull the worm off the pipette. Test the needle for flow and then move to the next worm and repeat injection steps.</li>
		<li>Recover the worms: Add a drop (~10 &mu;l) of recovery buffer on the worms and incubate until the worms begin swimming. Add an equal volume of M9, wait until worms resume swimming and repeat several times until the solution is mostly M9. Individually transfer worms to seeded plates.</li>
	</ol>
	</li>
	<li>Integration.
	<ol>
		<li>Place 40 healthy, well-fed, L4 worms into a fresh plate</li>
		<li>Irradiate with &gamma;-ray with 4,000 rads for 40 min. Transfer irradiated worms (P0) in fresh, OP50 seeded plates (4 worms/plate). Worms can alternatively be irradiated with a dose of 300 J/m2 UVs.</li>
		<li>Transfer 10-20 F1 transformants from each plate to individual plates, label the plates with the corresponding P0 origin.</li>
		<li>Single out 2-4 F2 transformants for each F1 to separate plates. Check the F3 progeny for 100% transmission of the transformation marker. Typically, 1-3% of progeny from an irradiated worm will have an integration event.</li>
		<li>Make stocks of three or more independent transformant lines.</li>
	</ol>
	</li>
</ol>
2. Behavioral Assays
<ol>
	<li>Age-synchronization.
	<ol>
		<li>Grow worms in standard 10 cm NGM plates + OP50 E. coli until a large population of gravid adults is reached (3-5 days).</li>
		<li>Collect the worms in 50 ml Falcon tubes by suspending them in 1 ml M9 buffer.</li>
		<li>Add 5 ml M9 buffer and centrifuge at 450 x g for 3 min. Discard supernatant. Repeat 3-4x.</li>
		<li>At the end of the last centrifugation, remove the supernatant and add 10 volumes of basic hypochlorite solution (0.5 M NaOH, 1% hypochlorite freshly mixed) to the pelleted worms. Incubate at RT for ~10 min. The lysis reaction can be monitored by placing a drop of the lysis reaction on a coverslip and examining the worms under a microscope. When roughly 80% of worms are broken the reaction should be stopped.</li>
		<li>Stop the lysis reaction by adding the same volume of sterile egg buffer.</li>
		<li>Collect the eggs (and carcasses) by centrifugation at 450 x g for 5 min.</li>
		<li>Wash the eggs with sterile egg buffer 2-3x.</li>
		<li>Incubate the eggs O/N in M9 buffer and seed them on standard NGM plates.</li>
	</ol>
	</li>
	<li>Chemotaxis assay.
	<ol>
		<li>Prepare several pieces of agar roughly 0.5x0.5x0.5 cm. We deposit the agar in a 10-cm plate and we cut the agar chunks from there.</li>
		<li>Soak the agar chunk in a solution containing the desired attractant (at saturating, near-saturating concentrations) for 2 hr. Typical attractants are lysine (0.5 M), biotin (0.2 M).</li>
		<li>Deposit an agar chunk in a 10 cm test plate in which the location of a test spot and a control spot have been marked (Figure 1A). Allow equilibration and formation of a gradient O/N (Figure 1B). Prepare 5 plates for a single experiment.</li>
		<li>Prior the experiment add 10 &mu;l of 20 mM NaN3 (anesthetic) to each spot.</li>
		<li>Place 20 age-synchronized worms in the center of the plate. Place the plate in the incubator at 20 &deg;C.</li>
		<li>After 1 hr, count the animals on the test/control spots and calculate the chemotaxis index, (C.I.) as follow: <img alt="Equation 1" fo:content-width=".863in" fo:src="/files/ftp_upload/50435/50435eq1highres.jpg" src="/files/ftp_upload/50435/50435eq1.jpg" /> where N, Ntest and NCnt., indicate the total number of animals, the number of animals in the test spot and the number of animals in the control spot.</li>
	</ol>
	</li>
</ol>
3. Primary Embryonic Cell Culture
<ol>
	<li>Lyse worms as described in age-synchronization.</li>
	<li>Stop the lysis reaction by adding the same volume of sterile egg buffer and centrifuge at 450 x g for 5 min. Gently discard supernatant being careful to not lose pelleted eggs. Repeat 2-3x or until supernatant is clear.</li>
	<li>Resuspend pelleted eggs (and carcasses) in 2 ml sterile egg buffer and add 2 ml of sterile 60% sucrose in egg buffer. Mix this solution until eggs are completely resuspended (as they tend to form clumps under centrifugation) by hand or by vortexing.</li>
	<li>Centrifuge at 450 x g for 15 min.</li>
	<li>Carefully transfer supernatant (containing the eggs) to a sterile tube. Discard pellet which contains carcasses and other by-products of lysis.</li>
	<li>Remove residual sucrose by resuspending the eggs in egg buffer and centrifuging at 450 x g for 5 min. Gently collect and discard the supernatant. Repeat 3x.</li>
	<li>Under a laminar hood resuspend pelleted eggs in sterile egg buffer containing 1 U/ml chitinase at RT to digest the eggshells. After 30 min start to monitor the reaction (under an inverted cell culture microscope). Each batch of chitinase has a slightly different activity. Typically digestion is completed in 1 hr.</li>
	<li>When roughly 70-80% eggshells are digested by chitinase add CM-15 (L-15 cell culture medium containing 10% fetal bovine serum, 50 U/ml penicillin, and 50 &mu;g/ml streptomycin). Dissociate cells using a syringe with a 27 gauge. Filter the cell suspension with a 5.0 &mu;m filter to remove intact embryos, clumps of cells and larvae.</li>
	<li>Pellet the dissociated cell suspension by centrifugation at 450 x g for 15 min. Remove the supernatant and resuspend the pellet in CM-15 cell culture medium.</li>
	<li>Plate dissociated cells on glass cover slips previously coated with peanut lectin (0.1 mg/ml) dissolved in water. Note: cells must adhere to the substrate in order to differentiate.</li>
	<li>Cells can be maintained at RT (16-20 &deg;C) in air for more than 2 weeks.</li>
</ol>

<ol>
	<li>Hardy, J., Allsop, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+deposition+as+the+central+event+in+the+aetiology+of+Alzheimer's+disease.">Amyloid deposition as the central event in the aetiology of Alzheimer's disease.</a> Trends in Pharmacological Sciences. 12 (10), 383-388 (1991).</li><li>Kotzbauer, P. T., Trojanowsk, J. Q., Lee, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lewy+body+pathology+in+Alzheimer's+disease.">Lewy body pathology in Alzheimer's disease.</a> Journal of Molecular Neuroscience. 17 (2), 225-232 (2001).</li><li>Greenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inclusion+body+myositis:+review+of+recent+literature.">Inclusion body myositis: review of recent literature.</a> Current Neurology and Neuroscience Reports. 9 (1), 83-89 (2009).</li><li>Revesz, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sporadic+and+familial+cerebral+amyloid+angiopathies.">Sporadic and familial cerebral amyloid angiopathies.</a> Brain Pathology. 12 (3), 343-357 (2002).</li><li>Hartmann, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+sites+of+intracellular+production+for+Alzheimer's+disease+A+beta40/42+amyloid+peptides.">Distinct sites of intracellular production for Alzheimer's disease A beta40/42 amyloid peptides.</a> Nature Medicine. 3 (9), 1016-1020 (1997).</li><li>Ohnishi, S., Takano, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+fibrils+from+the+viewpoint+of+protein+folding.">Amyloid fibrils from the viewpoint of protein folding.</a> Cellular and Molecular Life sciences: CMLS. 61 (5), 511-524 (2004).</li><li>DL, R. i. d. d. l. e., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+II.">C. elegans II.</a> Cold Spring Harbor Monograph. 1, 1222 (1997).</li><li>Cotella, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxic+role+of+k++channel+oxidation+in+Mammalian+brain.">Toxic role of k+ channel oxidation in Mammalian brain.</a> The Journal of Neuroscience: the official journal of the Society for Neuroscience. 32 (12), 4133-4144 (2012).</li><li>Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+human+beta-amyloid+peptide+in+transgenic+Caenorhabditis+elegans.">Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans.</a> Proceedings of the National Academy of Sciences of the United States of America. 92 (20), 9368-9372 (1995).</li><li>Cai, S. Q., Sesti, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation+of+a+potassium+channel+causes+progressive+sensory+function+loss+during+aging.">Oxidation of a potassium channel causes progressive sensory function loss during aging.</a> Nature Neuroscience. 12 (5), 611-617 (2009).</li><li>Yu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guanylyl+cyclase+expression+in+specific+sensory+neurons:+a+new+family+of+chemosensory+receptors.">Guanylyl cyclase expression in specific sensory neurons: a new family of chemosensory receptors.</a> Proc. Natl. Acad. Sci. U.S.A. 94, 3384-3387 (1997).</li><li>Bargmann, C. I., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensory+neurons+with+overlapping+functions+direct+chemotaxis+to+multiple+chemicals+in+C.+elegans.">Chemosensory neurons with overlapping functions direct chemotaxis to multiple chemicals in C. elegans.</a> Neuron. 7 (5), 729-742 (1991).</li><li>Caserta, T. 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The authors declare that they have no competing financial interests.

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...

Amyloid beta (A&beta;) is a peptide of 36-43 amino acids that is formed after sequential cleavage of the amyloid precursor protein (APP) by &beta; and &gamma; secretases 1. The &gamma; secretase processes the C-terminal end of the A&beta; peptide and is responsible for its variable lengths 5. The most common forms of A&beta; are A&beta;40 and A&beta;42, the latter being more commonly associated to pathologic conditions such as AD 5. At high concentrations A&beta; form &beta;-sheets that aggregate to form amyloid fibrils 6. Fibrils deposits are the main component of senile plaques surrounding neurons. Both plaques and diffusible, non-plaque A&beta; oligomers, are thought to constitute the underlying pathogenic forms of A&beta;.
Laboratory study of neuronal amylopathies is complicated by the fact that these conditions progress with time. Therefore, it is important to develop genetically tractable animal models-complementary to mice-with short life span. These models can be used to elucidate specific aspects of amylopathies-typically cellular and molecular-and by virtue of their simplicity, help to capture the essence of the problem. The worm Caenorhabditis elegans falls is this category. It has a short life span, ~20 days and in addition basic cellular processes including regulation of gene expression, protein trafficking, neuronal connectivity, synaptogenesis, cell signaling, and death are similar to mammalian 7. Unique features of the worm include powerful genetics and lack of a vessel system, which enables to study neuronal damage independently of vascular damage. On the other hand, the lack of a brain limits the use of C. elegans to studying many aspects of neurodegeneration. In addition, the reproduction and identification of anatomical distributions of lesions cannot be performed in this organism. Other limitations include the difficulty to assess both differences in gene expression profiles and impairment of complex behavior and memory function. Here we describe methods to generate C. elegans models of amylopathies.

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

a caenorhabditis elegans model system for amylopathy study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; 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 A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 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 A&beta;42 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.

With our protocols we study the effects of human A&beta;42 oligomer on neuronal function 8. A fragment encoding human A&beta;42 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 <...

Watch this Scientific Journal Video about A Caenorhabditis elegans Model System for Amylopathy Study at JoVE.com

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 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.

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

a-caenorhabditis-elegans-model-system-for-amylopathy-study

Research

JoVE Journal

Neuroscience

10.4K Views.  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.