The authors would like to thank Dr. Hui Zheng (Baylor College of Medicine) for the APP cDNA. This work was funded by NIH grants R21AG031948 (D.B., J.M.B.), F30AG030878 (E.S.S.), R01DK073394 (AFO), the John Sealy Memorial Endowment Fund for Biomedical Research (AFO), and the Jean C. and William D. Willis Neuroscience Research Endowment (E.S.S.). J.M.B. is a scholar in the Translational Research Scholar Program and a member of the University Of Texas Medical Branch Claude E. Pepper Older Americans Independence Center (supported by NIH Grants UL1RR029876 and P30-AG-024832, respectively).

1. Expression of Recombinant APP Intracellular Domain (AICD)
<ol>
 <li>Transform E. coli strain BL21 with human AICD (residues 649-695 of APP, neuronal isoform numbering) cloned into vector pGEX-4T-1 (GE Healthcare). This vector will express AICD as the C-terminal moiety of a fusion protein of glutathione-S-transferase (GST). This vector also encodes a thrombin cleavage sequence to facilitate removal of the GST moiety. Details of cloning AICD into pGEX-4T-1 can be found in our previous publication4.</li>
 <li>From a single colony, inoculate 10 ml of LB broth with ampicillin (100 &mu;g/ml), and incubate overnight at 37 &deg;C with vigorous shaking.</li>
 <li>The following morning, inoculate 400 ml LB/ampicillin in a one-liter flask with 5 ml of the overnight culture.</li>
 <li>Incubate at 37 &deg;C with vigorous shaking until the optical density at 600 nm has reached 0.4 to 0.6. This will normally take 2 to 2.5 hr.</li>
</ol>
At this point it may be desirable to take out a small sample (~10 &mu;l) to follow the purification by SDS-PAGE (see Figure 1A,B). Each subsequent purification step should also have a small aliquot removed for analysis by SDS-PAGE.
<ol start="5">
 <li>Induce expression of GST-AICD by adding 0.4 mM of isopropyl-beta-D-thiogalactopyranoside (IPTG). Incubate at 37 &deg;C with vigorous shaking for 5 hr.</li>
 <li>Centrifuge the cells at 6,000 x g for 15 min at 4 &deg;C. The cell pellet can be stored at -80 &deg;C for several months at this point of the procedure.</li>
</ol>
2. Purification of GST-AICD
<ol>
 <li>Prepare 50 ml lysis buffer as follows: 1 mM dithiothreitol, 1mM ethylenediaminetetraacetic acid (EDTA), 0.1% Triton-X100, 1 mM phenylmethylsulfonyl fluoride (PMSF), Complete protease inhibitor cocktail (Roche Applied Science), and phosphate buffered saline. Chill on ice.</li>
 <li>Thaw bacterial pellet (if previously frozen) on ice.</li>
</ol>
At this point, it is critical that all steps are performed at 4 &deg;C to limit proteolysis. All tubes, buffers, and other reagents should be pre-chilled. If using a French press or emulsifier for lysis, these should be pre-chilled as well.
<ol start="3">
 <li>Thoroughly resuspend the bacterial pellet in 20 ml of lysis buffer. A combination of vortexing and trituration with 10 ml pipette on ice will ensure complete resuspension of the pellet.</li>
 <li>Pass the resuspended bacterial cells through an emulsifier (e.g., Avestin EmulsiFlex-C3) or French pressure cell, pre-chilled to 4 &deg;C at 30,000 psi. Pass the lysate through the emulsifier a second time. Collect the lysate into a pre-chilled 50 ml tube.</li>
</ol>
The main advantage of these two instruments is that they minimize heating of the sample. The commonly used technique of sonication generates significant heat at the tip of the sonication probe and can result in aggregation of recombinant proteins.
<ol start="5">
 <li>Centrifuge the lysate at 15,000 x g for 30 min. Separate and keep the supernatant into a pre-chilled 50 ml tube.</li>
 <li>Add 500 &mu;l of a 50% slurry of glutathione-agarose (Sigma-Aldrich). Rotate for 30 min at 4 &deg;C. While the sample is rotating, make fresh elution buffer: 50 mM Tris 7.8, 0.1% Triton-X100, 0.5 mM dithiothreitol, 10 mM reduced glutathione.</li>
</ol>
The only step of the protocol which is performed at room temperature is the elution step. Thus, the elution buffer is kept at room temperature. Elution buffer should be prepared fresh.
<ol start="7">
 <li>Pour the lysate and slurry into a disposable 5" polystyrene chromatography column with coarse (90-130 &mu;m) filters (Evergreen Scientific). Wash the column 5 times with 3 ml of phosphate buffered saline.</li>
 <li>Cap the bottom of the column and add 500 &mu;l of room temperature elution buffer. Cap the top of the column and rotate for 5 min at room temperature. Collect the eluate into a chilled 1.5 ml microfuge tube. Repeat the elution two more times (1.5 ml total eluate).</li>
 <li>Dialyze the eluate in a 10,000 molecular weight cutoff dialysis membrane (e.g., Thermo Scientific Slide-A-Lyzer dialysis cassettes) in 4 liters of phosphate buffered saline overnight at 4 &deg;C. Dialyze against an additional 4 liters of phosphate buffered saline at 4 &deg;C for 1 hr the following day.</li>
 <li>Remove the protein from the dialysis cassette and perform a protein quantification assay to determine yield of GST-AICD. A typical purification from 400 ml of culture will result in yields of &gt;20 mg of purified protein at concentrations of 10-20 mg/ml. Aliquot the protein into 200 &mu;l aliquots and freeze at -80 &deg;C.</li>
</ol>
3. Thrombin Cleavage of GST-AICD and Purification of AICD
<ol>
 <li>Thaw 200 &mu;l of purified GST-AICD and add 20 &mu;l of 1 U/&mu;l thrombin. Incubate overnight at 37 &deg;C.</li>
</ol>
The quality and purity of thrombin varies considerably among manufacturers, and can be a significant source of contamination. We use thrombin from GE Healthcare (see table of reagents). After overnight incubation, &gt;95% of the fusion protein should be cleaved (Figure 1B).
<ol start="2">
 <li>After overnight cleavage with thrombin, it is likely that a fraction of the liberated AICD underwent aggregation and the solution may appear cloudy. Clear the aggregated material by centrifugation at 20,000 x g for 30 min at 4 &deg;C. Remove the supernatant into a pre-chilled microfuge tube.</li>
 <li>Add 50 &mu;l of 50% glutathione-agarose slurry and 50 &mu;l 50% p-aminobenzamidine-agarose slurry to remove GST and thrombin, respectively. Incubate with rotation for 5 min at room temperature.</li>
 <li>Centrifuge briefly to sediment the agarose beads, and remove the supernatant into a fresh tube. Extract the GST four more times with glutathione-agarose. A single extraction with p-aminobenzamidine-agarose is sufficient to remove the 20 U of thrombin.</li>
 <li>Perform a protein quantitation assay on 5 &mu;l of the purified AICD. Typical yields from a 400 ml culture are 50-100 &mu;g at a concentration of 0.2 to 0.5 mg/ml. Higher yields are possible by cleaving larger volumes of GST-AICD.</li>
</ol>
Purified AICD should be prepared fresh for biochemical/biophysical characterization. Unused material should be discarded. It is also possible to concentrate the AICD by lyophilizing the sample and resuspending in a smaller volume of buffer6. If detecting AICD requires blotting (such as after a filter trap or dot blot assay), antigen retrieval on the membrane should be performed as described by Pimplikar and Suryanarayana12.
4. AICD Aggregation for Atomic Force Microscopy (AFM)
<ol>
 <li>Dilute freshly prepared AICD in phosphate buffered saline to a concentration of 0.1 mg/ml in a final volume of 15 &mu;l. At this point, various compounds and/or proteins may be added to determine if they inhibit AICD aggregation. For example, we have shown that equimolar concentrations of ubiquilin-1 prevent aggregation of AICD in this assay4.</li>
</ol>
It is also possible to aggregate larger reaction volumes for biochemical experiments such as filter trap assays and light scattering4. We have had success performing filter trap assays on reaction mixtures as large as 400 &mu;l with AICD diluted to concentrations as low as 1 &mu;M.
<ol start="2">
 <li>Induce thermal aggregation by incubating the samples at 43 &deg;C with shaking at 800 rpm in an Eppendorf Thermomixer for 48 hr. This temperature was chosen based upon chaperone assays which examine the thermal aggregation of the model chaperone substrate citrate synthase4,13.</li>
</ol>
Thermal aggregation is a commonly used method to induce the structural transition of a protein from a native to a non-native structure (which is usually beta sheet rich) which results in the formation of intermolecular aggregates. Thermal aggregation is also used to examine the function of molecular chaperones, which shield hydrophobic segments from forming inappropriate intermolecular interactions (and thus retard or prevent thermal aggregation).
<ol start="3">
 <li>Dilute the sample 20-fold in deionized water and spot 2 &mu;l onto freshly cleaved mica. Dry the sample in a dessicator overnight. It will be necessary to empirically determine the optimal dilution for imaging, and thus several dilutions ranging from 10- to 100-fold would be prudent.</li>
 <li>Visualize the aggregated AICD using the AFM operating in tapping mode14. The cantilevers used are gold-coated micro sharp nitride levers (MSNL, Bruker) with a nominal tip radius of 2 nm. Typical tapping amplitudes during imaging are 10-20 nm at the resonance frequency of the cantilevers (30-50 kHz).</li>
</ol>
We found that image quality and contrast of different protein aggregates depended on the applied force and the duration of the experiment. To minimize sample perturbation by the tip, we maintained the applied force relatively low by keeping a set-point ratio above 95% and limiting the scanning of each sample to 5-10 min. Image processing was performed with WSxM software (Nanotec). Standard image processing consisted of plane subtraction and flattening. Our laboratories use a "home-built" AFM15 interfaced to a commercial scanning probe microscope control system (Nanotec).
5. Representative Results
The expression and relative enrichment of GST-AICD is shown in Figure 1A. After lysis, the majority of the fusion protein is present in the soluble fraction, and therefore extraction from inclusion bodies is not required. The material eluted from the glutathione agarose column is &gt;95% pure. In the preparation shown in Figure 1, the concentration of protein eluted from the column was 14.5 mg/ml, and the yield was 22 mg (from a starting culture of 400 ml). Cleavage of 200 &mu;l of this preparation overnight with 20 U of thrombin resulted in almost 100% cleavage of the fusion protein (Figure 1B). The amount of thrombin used is low enough as to not be detectable by Coomassie blue staining (see "Cleaved" lane, Figure 1B). Removal of the thrombin and GST resulted in some loss of material, however it was &gt;90% pure with a yield in this prep of 70 &mu;g of AICD at a concentration of 0.35 mg/ml. Figure 1C shows the flow chart for aggregation of AICD and subsequent analysis. AICD aggregates imaged by AFM are typically spheroid/amorphous, and range in size from 50 to 100 nm (Figure 1D,F). Aggregated material is not detectable when an equimolar amount of the chaperone ubiquilin-1 is added to the aggregation reaction (Figure 1E)4.
<img alt="Figure 1" src="/files/ftp_upload/4204/4204fig1.jpg" /> 
Figure 1. Purification and aggregation of AICD. A) Coomassie blue staining of samples collected at each step of the purification process. For the uninduced and induced samples, 20 &mu;l of total bacteria were run on the gel. For the supernatant fraction, 5 &mu;l of lysate (from a total volume of 20 ml) were run on the gel. For the pellet fraction, the pellet was resuspended in 20 ml of lysis buffer, and 5 &mu;l of this material were sonicated in a water bath sonicator for 30 min at 4 &deg;C prior to loading onto the gel. For the flowthrough, 5 &mu;l were run on the gel. The amounts of eluate loaded onto the gel are indicated. B) Coomassie blue stained gel of uncleaved and cleaved GST-AICD (2 &mu;l) and 1 and 5 &mu;l purified AICD. C) Flowchart of AICD purification, aggregation, and analysis. D,E) Representative AFM image of thermally aggregated AICD in the absence (D) and presence (E) of the molecular chaperone ubiquilin-1. F) Histogram of the size distribution of AICD aggregates.

<ol>
	<li>De Strooper, B., Vassar, R., Golde, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secretases:+enzymes+with+therapeutic+potential+in+Alzheimer+disease.">The secretases: enzymes with therapeutic potential in Alzheimer disease.</a> Nature reviews. Neurology. 6, 99-107 (2010).</li><li>Chang, K. A., Suh, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+roles+of+amyloid+intracellular+domain+of+amyloid+precursor+protein.">Possible roles of amyloid intracellular domain of amyloid precursor protein.</a> BMB reports. 43, 656-663 (2010).</li><li>McLoughlin, D. M., Miller, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+FE65+proteins+and+Alzheimer's+disease.">The FE65 proteins and Alzheimer's disease.</a> J. Neurosci. Res. 86, 744-754 (2008).</li><li>Stieren, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquilin-1+is+a+molecular+chaperone+for+the+amyloid+precursor+protein.">Ubiquilin-1 is a molecular chaperone for the amyloid precursor protein.</a> J. Biol. Chem. 286, 35689-35698 (2011).</li><li>Ramelot, T. A., Nicholson, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation-induced+structural+changes+in+the+amyloid+precursor+protein+cytoplasmic+tail+detected+by+NMR.">Phosphorylation-induced structural changes in the amyloid precursor protein cytoplasmic tail detected by NMR.</a> J. Mol. Biol. 307, 871-884 (2001).</li><li>Ramelot, T. A., Gentile, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+structure+of+the+amyloid+precursor+protein+cytoplasmic+tail+indicates+preordering+of+structure+for+binding+to+cytosolic+factors.">Transient structure of the amyloid precursor protein cytoplasmic tail indicates preordering of structure for binding to cytosolic factors.</a> Biochem. 39, 2714-2725 (2000).</li><li>Radzimanowski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+intracellular+domain+of+the+amyloid+precursor+protein+in+complex+with+Fe65-PTB2.">Structure of the intracellular domain of the amyloid precursor protein in complex with Fe65-PTB2.</a> EMBO Rep. 9, 1134-1140 (2008).</li><li>Ohkawara, T., Nagase, H., Koh, C. S., Nakayama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+precursor+protein+intracellular+domain+alters+gene+expression+and+induces+neuron-specific+apoptosis.">The amyloid precursor protein intracellular domain alters gene expression and induces neuron-specific apoptosis.</a> Gene. 475, 1-9 (2011).</li><li>von Rotz, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+APP+intracellular+domain+forms+nuclear+multiprotein+complexes+and+regulates+the+transcription+of+its+own+precursor.">The APP intracellular domain forms nuclear multiprotein complexes and regulates the transcription of its own precursor.</a> J. Cell Sci. 117, 4435-4448 (2004).</li><li>Hamid, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+precursor+protein+intracellular+domain+modulates+cellular+calcium+homeostasis+and+ATP+content.">Amyloid precursor protein intracellular domain modulates cellular calcium homeostasis and ATP content.</a> J. Neurochem. 102, 1264-1275 (2007).</li><li>Ghosal, K., Stathopoulos, A., Pimplikar, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=APP+intracellular+domain+impairs+adult+neurogenesis+in+transgenic+mice+by+inducing+neuroinflammation.">APP intracellular domain impairs adult neurogenesis in transgenic mice by inducing neuroinflammation.</a> PLoS ONE. 5, e11866 (2010).</li><li>Pimplikar, S. W., Suryanarayana, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+APP+intracellular+domain+in+brain+tissue.">Detection of APP intracellular domain in brain tissue.</a> Met. Molecul. Biol. 670, 85-91 (2011).</li><li>Buchner, J., Grallert, H., Jakob, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+chaperone+function+using+citrate+synthase+as+nonnative+substrate+protein.">Analysis of chaperone function using citrate synthase as nonnative substrate protein.</a> Met. Enzymol. 290, 323-338 (1998).</li><li>Hansma, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+atomic+force+microscopy+of+DNA.">Recent advances in atomic force microscopy of DNA.</a> Scanning. 15, 296-299 (1993).</li><li>Valbuena, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quasi-simultaneous+imaging/pulling+analysis+of+single+polyprotein+molecules+by+atomic+force+microscopy.">Quasi-simultaneous imaging/pulling analysis of single polyprotein molecules by atomic force microscopy.</a> Rev. Sci. Instrum. 78, 113707 (2007).</li><li>Radzimanowski, J., Beyreuther, K., Sinning, I., Wild, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overproduction,+purification,+crystallization+and+preliminary+X-ray+analysis+of+human+Fe65-PTB2+in+complex+with+the+amyloid+precursor+protein+intracellular+domain.">Overproduction, purification, crystallization and preliminary X-ray analysis of human Fe65-PTB2 in complex with the amyloid precursor protein intracellular domain.</a> Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 64, 409-412 (2008).</li><li>Chen, T. Y., Liu, P. H., Ruan, C. T., Chiu, L., Kung, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intracellular+domain+of+amyloid+precursor+protein+interacts+with+flotillin-1,+a+lipid+raft+protein.">The intracellular domain of amyloid precursor protein interacts with flotillin-1, a lipid raft protein.</a> Biochem. Biophys. Res. Commun. 342, 266-272 (2006).</li><li>Kim, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Notch1+signaling+by+the+APP+intracellular+domain+facilitates+degradation+of+the+Notch1+intracellular+domain+and+RBP-Jk.">Regulation of Notch1 signaling by the APP intracellular domain facilitates degradation of the Notch1 intracellular domain and RBP-Jk.</a> J. Cell Sci. 124, 1831-1843 (2011).</li><li>Lazarov, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport,+amyloid+precursor+protein,+kinesin-1,+and+the+processing+apparatus:+revisited.">Axonal transport, amyloid precursor protein, kinesin-1, and the processing apparatus: revisited.</a> J. Neurosci. 25, 2386-2395 (2005).</li><li>Kakuda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equimolar+production+of+amyloid+beta-protein+and+amyloid+precursor+protein+intracellular+domain+from+beta-carboxyl-terminal+fragment+by+gamma-secretase.">Equimolar production of amyloid beta-protein and amyloid precursor protein intracellular domain from beta-carboxyl-terminal fragment by gamma-secretase.</a> J Biol. Chem. 281, 14776-14786 (2006).</li><li>Gosal, W. S., Myers, S. L., Radford, S. E., Thomson, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+under+the+atomic+force+microscope.">Amyloid under the atomic force microscope.</a> Protein Pept. Lett. 13, 261-270 (2006).</li></ol>

No conflicts of interest declared.

In this protocol we have outlined a procedure for obtaining highly pure AICD for structural, biophysical, and biochemical analyses. This procedure does not require sophisticated chromatography equipment and is therefore accessible to most laboratories. Other groups have purified AICD5-7,16, including GST-AICD17-19, for biochemical/structural analyses. Disadvantages to previous protocols include poor solubility of AICD16, less than ideal purity17, and the requirement for size ex...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>pGEX-4T-1</td>
			<td>GE Healthcare</td>
			<td>28-9545-49</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>GE Healthcare</td>
			<td>27-0846-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Fisher Scientific</td>
			<td>BP1760</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bradford protein assay reagent</td>
			<td>Bio-Rad</td>
			<td>500-0002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Coomassie blue</td>
			<td>Bio-Rad</td>
			<td>161-0786</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IPTG ( isopropyl-beta-D thiogalactopyranoside)</td>
			<td>Sigma-Aldrich</td>
			<td>I6758</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glutathione-agarose</td>
			<td>Sigma-Aldrich</td>
			<td>G4510</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>p-aminobenzamidine-agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A7155</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Complete protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Slide-A-Lyzer dialysis cassettes</td>
			<td>Thermo Scientific</td>
			<td>66380</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chromatography columns</td>
			<td>Evergreen Scientific</td>
			<td>208-3367-050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Emulsifier</td>
			<td>Avestin, Inc</td>
			<td>EmulsiFlex-C3</td>
			<td>Highly recommended</td>
		</tr>
		<tr>
			<td>Eppendorf Thermomixer</td>
			<td>Eppendorf</td>
			<td>022670107</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mica Disks</td>
			<td>Ted Pella</td>
			<td>50-12</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>AFM cantilevers</td>
			<td>Bruker</td>
			<td>MSNL-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>WSxM software</td>
			<td>Nanotec</td>
			<td>N/A</td>
			<td>Free download</td>
		</tr>
	</tbody>
</table>

purification and aggregation of the amyloid precursor protein intracellular domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

Watch this Scientific Journal Video about Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain at JoVE.com

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

purification-aggregation-amyloid-precursor-protein-intracellular

Research

JoVE Journal

Medicine

11.7K Views.  University of Texas Medical Branch. A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

Video: Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Performing and Processing FNA of Anterior Fat Pad for Amyloid

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis. Since the clinical presentation of this disease is so variable and non-specific, the associated risks of these biopsies are too great for the diagnostic yield. Other sites that have a lower biopsy risk, such as skin or gingival, are also relatively invasive and expensive. In addition, these biopsies may not always have sufficient amyloid deposits to establish a diagnosis. Fat pad aspiration has demonstrated good clinical correlation with low cost and minimal morbidity. However, there are no standardized protocols for performing this procedure or processing the aspirated specimen, which leads to variable and nonreproducible results. The most frequently utilized modality for detecting amyloid in tissue is an apple-green birefringence on Congo red stained sections using a polarizing microscope. This technique requires cell block preparation of aspirated material. Unfortunately, patients presenting in early stage of amyloidosis have minimal amounts of amyloid which greatly reduces the sensitivity of Congo red stained cell block sections of fat pad aspirates. Therefore, ultrastructural evaluation of fat pad aspirates by electron microscopy should be utilized, given its increased sensitivity for amyloid detection. This article demonstrates a simple and reproducible procedure for performing anterior fat pad aspiration for the detection of amyloid utilizing both Congo red staining of cell block sections and electron microscopy for ultrastructural identification.

Fat pad aspiration is a preferred, minimally invasive, and low cost approach as compared to other methods to detect amyloid for diagnosis of systemic amyloidosis. This video article demonstrates a procedural outline for performing fat pad aspiration with appropriate processing of the specimen for the optimal diagnostic outcome.

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&amp;#946; and Tau Neuropathology

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or in buffer (washed platelets), by the use of one or a combination of agonists. The use of washed platelets separated from their plasma environment and in the absence of anticoagulants allows for studying intrinsic platelet properties. Among the large panel of agonists, arachidonic acid (AA), adenosine di-phosphate (ADP), thrombin and collagen are the most frequently used. The aggregation response is quantified by measuring the relative optical density (OD) over time of platelet suspension under continuous stirring. Platelets in homogeneous suspension limit the passage of light after the addition of an agonist, platelet shape change occurs producing a small transitory increase in OD. Following this initial activation step, platelet clots form gradually, allowing the passage of light through the suspension as a result of decreased OD. The aggregation process is ultimately expressed as a percentage, compared to the OD of platelet-poor plasma or buffer. Rigorous calibration is thus essential at the beginning of each experiment. As a general rule: calibration to 0% is set by measuring the OD of a non-stimulated platelet suspension while measuring the OD of the suspension medium containing no platelets represents a value of 100%. Platelet aggregation is generally visualized as a real-time aggregation curve. Turbidimetry is one of the most commonly used laboratory techniques for the investigation of platelet function and is considered as the historical gold standard and used for the development of new pharmaceutical agents aimed at inhibiting platelet aggregation. Here, we describe detailed protocols for 1) preparation of human washed platelets and 2) turbidimetric analysis of collagen-induced aggregation of human washed platelets pretreated with the food dye Brilliant Blue FCF that was recently identified as an inhibitor of Pannexin1 (Panx1) channels.

We describe a straightforward method for the isolation of washed platelets from human blood followed by agonist-induced platelet aggregation measurements by turbidimetry. As an example we apply this method for studying the aggregation response of human platelets to collagen after a pre-incubation with the Pannexin1 channel inhibitor Brilliant Blue FCF.

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or ...

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca2+ reservoir for contraction, which reuptakes intracellular Ca2+ during relaxation. The SR Ca2+ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca2+ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca2+ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa2+ reserve and diastolic Ca2+ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca2+ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca2+ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) to diastolic calcium removal was evaluated.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca2+ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Isolation and Purification of Murine Cardiac Pericytes

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier integrity. However, their tissue-specific functions in the heart are not well understood. Moreover, there is currently no protocol utilizing readily accessible materials to isolate and purify pericytes of cardiac origin. Our protocol focuses on using the widely used mammalian model, the mouse, as our source of cells. Using the enzymatic digestion and mechanical dissociation of heart tissue, we obtained a crude cell mixture that was further purified by fluorescence activating cell sorting (FACS) by a plethora of markers. Because there is no single unequivocal marker for pericytes, we gated for cells that were CD31-CD34-CD45-CD140b+NG2+CD146+. Following purification, these primary cells were cultured and passaged multiple times without any changes in morphology and marker expression. With the ability to regularly obtain primary murine cardiac pericytes using our protocol, we hope to further understand the role of pericytes in cardiovascular physiology and their therapeutic potential.

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

Ultracentrifuge-Based Isolation of Extracellular Vesicles from Human ADSCs

Purification and Characterization of Extracellular Vesicles from Human Adipose-Derived Mesenchymal Stem Cells

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research suggests that their regenerative function may be attributed to cell-cell contact and cell paracrine effects. The paracrine effect is an important way for cells to interact and transfer information over short distances, in which extracellular vesicles (EVs) play a functional role as carriers. There is significant potential for ADSC EVs in regenerative medicine. Multiple studies have reported on the effectiveness of these methods. Various methods for extracting and isolating EVs are currently described based on principles such as centrifugation, precipitation, molecular size, affinity, and microfluidics. Ultracentrifugation is regarded as the gold standard for isolating EVs. Nevertheless, a meticulous protocol to highlight precautions during ultracentrifugation is still absent. This study presents the methodology and crucial steps involved in ADSC culture, supernatant collection, and EV ultracentrifugation. However, even though ultracentrifugation is cost-effective and requires no further treatment, there are still some inevitable drawbacks, such as a low recovery rate and EV aggregation.

Author Spotlight: Standardizing and Improving the Extraction and Purification of Extracellular Vesicles from Human ADSCs

This protocol describes all procedures, from culturing human adipose-derived mesenchymal stem cells (ADSCs) and collecting supernatant to extracting extracellular vesicles (EVs) using ultracentrifugation.

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research ...