This work was supported by the NIH grant R01AG061865 to R.J.V. and J.N.S. The authors thank Vassar and Savas research group members at Northwestern University for their thoughtful discussions. We also sincerely thank Dr(s). Ansgar Seimer and Ralf Langen at the University of South California for their crucial input. We thank Dr. Farida Korabova for sample preparation and negative staining electron microscopy imaging at Northwestern University Center for Advanced Microscopy.

This protocol involves the use of human or vertebrate brain tissues. All the research was performed in compliance with the approved institutional guidelines of the Northwestern University. The current workflow is standardized using APP-knock in (AppNL-G-F/NL-G-F) mouse brain cortical and hippocampal brain region extracts11. This protocol has been optimized for brain extracts from mice at 6-9 months of age, and it can effectively purify amyloids from both male and female animals.
NOTE: For a better understanding of the overall experimental procedure, see Figure 1 for a schematic of the workflow.
1. Tissue harvesting and amyloid purification
NOTE: Ideally, amyloid fibrils should be isolated from freshly dissected brain regions. However, this method also works well with snap- or flash-frozen brain tissues. Below is a brief outline of snap-freezing brain tissues for storage for use at a later time.
<ol>
	<li>Brain tissue harvesting and storage: Dissect mouse brains and quickly harvest the amyloid-laden regions (i.e., cortex and hippocampus), followed by snap freezing in liquid nitrogen or a dry ice alcohol bath. 
	NOTE: Snap freezing helps to preserve the structural attributes of the tissue&#39;s constituents. The mice are euthanized by isoflurane and cervical dislocation12,13. It is advised to avoid using CO2 since that can compromise brain proteome integrity.</li>
	<li>Post-dissection, cut and store fresh tissues in small blocks (e.g., 5 mm x 5 mm), as this facilitates more efficient homogenization before amyloid extraction. Transfer the tissue to the sterile cryogenic vial, tighten its cap, and rapidly submerge.</li>
	<li>Keep tissues frozen in ultracold conditions (i.e., -80 &#176;C or liquid nitrogen). If extraction is to be performed a few days later, store the tissues at -20 &#176;C and avoid freeze and thaw cycles. Thaw the frozen tissues on wet ice when ready for extraction and purification. 
	&#8203;NOTE: Direct contact of the tissues with liquid nitrogen can cause tissue and protein damage. Note that an alcohol bath can remove permanent markers from the tube labels.</li>
</ol>
2. Enrichment of SDS insoluble material
NOTE: Perform all the steps on ice and centrifuge at 4 &#176;C, unless stated otherwise. Details of all the buffers and solutions used in this protocol are provided in Supplementary File 1. Manufacturers and catalog numbers of chemicals and instruments are provided in the Table of Materials.
<ol>
	<li>Start with freshly dissected or snap-frozen brain tissue regions (thawed on ice) placed into a 2 mL tube containing 6-8 ceramic beads and freshly prepared ice-cold homogenization buffer (1 mL for 0.25-1 g of wet tissue mass).</li>
	<li>Transfer the tubes to the bead mill homogenizer and grind the tissue contents at 4000 rpm, with two cycles of 30 s each and an interval of 30 s in between. 
	NOTE: Alternatively, motorized stirrer or homogenizer systems can be used to grind and homogenize the tissues.</li>
	<li>Add 9 mL of ice-cold homogenization buffer to the 1 mL of brain whole tissue homogenate in 15 mL tubes, seal with laboratory wax film strips, and rotate end-to-end overnight at 4 &#176;C to ensure robust solubilization. 
	NOTE: To remove unwanted large cellular debris and lipids, on the next morning, spin the tubes at 800 x g at 4 &#176;C for 10 min and collect the supernatant in a fresh tube. Resuspend the remaining pellet in 2 mL of ice-cold homogenization buffer, mix well, spin again for 10 min at 4 &#176;C, and combine the two supernatants.</li>
	<li>Slowly add solid sucrose to the tissue extract suspension to a final concentration of 1.2 M, mix well, and centrifuge at 250,000 x g for 45 min at 4 &#176;C.</li>
	<li>Carefully remove and discard the supernatant using a pipette. Resuspend the pellet in 2 mL of homogenization buffer containing 1.9 M sucrose by triturating, followed by centrifugation at 125,000 x g for 45 min at 4 &#176;C. 
	NOTE: The buffer volume can be adjusted based on the amount of material recovered from the previous step. In general, 10 volumes of homogenization buffer (V/V) is ideal for this step.</li>
	<li>Collect the top white solid layer using a pipette, transfer it to a fresh tube, and solubilize in 1 mL of ice-cold wash buffer by pipetting up and down several times, without introducing air bubbles.</li>
	<li>Apart from the top layer, the pellet is also enriched with amyloid fibrils. For a higher yield, combine the two fractions and proceed. Carefully remove the middle aqueous layer using a pipette and discard.</li>
	<li>Centrifuge the combined fractions at 8000 x g for 20 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Add 1 mL of ice-cold digestion buffer to the washed pellet, resuspend, and incubate at room temperature (RT) for 3 h.</li>
	<li>Centrifuge at 8000 x g for 20 min at 4 &#176;C and remove the supernatant using a pipette.</li>
	<li>Resuspend and wash (same as Step 2.8.) the digested pellet two times in 1 mL of ice-cold Tris buffer.</li>
	<li>Resuspend the washed pellet in 1 mL of solubilization buffer containing 1% SDS and 1.3 M sucrose by pipetting up and down. Centrifuge quickly at 200,000 x g for 60 min at 4 &#176;C. 
	NOTE: Although the amyloid fibrils are highly resistant to detergents and denaturants, very long exposures to the detergent (1% SDS) may affect the integrity of the fibrils or remove the tightly bound proteins, which will compromise subsequent analyses. Therefore, immediately after resuspending the pellets, proceed to centrifugation.</li>
	<li>Save the pellet and increase the volume of the remaining supernatant by adding 50 mM Tris buffer (in ratio 1:0.3) to reduce the sucrose concentration from 1.3 to 1 M and centrifuge one more time at 200,000 x g for 45 min at 4 &#176;C.</li>
	<li>Dissolve the two pellets in 100 &#181;L of Tris buffer containing 0.5% SDS and pool for amyloid purification. Visible pellets are small and should appear opaque and off-white (see Figure 2A).</li>
</ol>
3. Amyloid purification
NOTE: Combine the two pellets, solubilize by pipetting until obtaining a uniform solution and proceed with the following steps of amyloid purification.
<ol>
	<li>For complete solubilization of the amyloid-rich material present in pellets, subject the tube to mechanical shearing produced by ultrasound waves in a bath sonication device running for 30 s ON and 30 s OFF at a medium-range frequency for 20 cycles.</li>
	<li>Immediately centrifuge the material at 20,000 x g for 30 min at 4 &#176;C and resuspend the pellet in 500 &#181;L of 0.5% SDS Tris buffer.</li>
	<li>Repeat Step 3.1. and Step 3.2. four times for a total of five washes. The number of washes can be increased to improve the purity. 
	NOTE: Sonication and washing steps are optimized at 0.5% SDS as higher concentrations may affect the structure, integrity, or protein composition of the fibrils. Increasing the SDS concentration at this step is not recommended.</li>
	<li>After the final centrifugation step, wash the pellet in 200 &#181;L of ultra-pure water and centrifuge at 20,000 x g for 30 min at 4 &#176;C to remove any remaining detergent. The washed pellet containing purified amyloid fibrils appears semi-transparent in color and is difficult to see sometimes.</li>
	<li>Dissolve the final pellet containing purified amyloid fibrils in 100 &#181;L of ultrapure water. Proceed to the next step immediately for downstream processing or save the fibril suspension at -20 &#176;C for further analysis. If frozen, thaw on ice before starting precipitation.</li>
</ol>
4. Methanol chloroform precipitation
NOTE: If the final goal is to perform protein analysis, it is recommended to desalt and remove additional non-proteinaceous impurities.
<ol>
	<li>Add 400 &#181;L of methanol to the purified 100 &#181;L of amyloid fibrils in a 1.5 mL tube and vortex well.</li>
	<li>Add 100 &#181;L of chloroform to the tube and vortex well.</li>
	<li>Add 300 &#181;L of ultrapure water and vortex thoroughly. This turns the mixture cloudy due to protein precipitation.</li>
	<li>Centrifuge at 12,000 x g for 2 min at RT.</li>
	<li>Carefully remove the aqueous layer (i.e., top) without disturbing the interface layer containing the protein flake.</li>
	<li>Add the same volume of methanol again and centrifuge at 12,000 x g for 2 min at RT.</li>
	<li>Discard the supernatant using a pipette and air-dry the pellet at RT. Avoid over-drying; amyloid fraction is difficult to redissolve if over-dried.</li>
</ol>
5. Trypsin digestion
<ol>
	<li>Dissolve the pellet in 50 &#181;L of guanidine hydrochloride (GuHCl) buffer. Sonicate in an ice-cold water bath and heat at 95 &#176;C for 5 min, if required.</li>
	<li>Vortex thoroughly at RT for 45 min to 1 h to dissolve it completely. The pellet will not be visible after this step, and the solution looks clear in appearance.</li>
	<li>Add the same volume of 0.2% surfactant solution. Solubilize at RT with vortexing for 60 min. This step enhances trypsin enzymatic activity.</li>
	<li>Add 1 &#181;L of tris-2-carboxyethylphosphine (TCEP) from the 500 mM stock solution. Incubate for 60 min.</li>
	<li>Add 2 &#181;L of 500 mM iodoacetamide (IAA) and incubate in the dark for 20 min.</li>
	<li>Quench the IAA with 5 &#181;L of TCEP solution for 15 min.</li>
	<li>Add the required volume of 50 mM ammonium bicarbonate solution to the tube to reduce the guanidine concentration to 1.5 M.</li>
	<li>Add 1% surfactant solution to the tube (1 &#181;L/50 &#181;g of protein).</li>
	<li>Add trypsin (1 &#181;g/100 &#181;g of protein) and leave the tube mixing at 37 &#176;C overnight.</li>
</ol>
6. Peptide cleanup
<ol>
	<li>Next morning, acidify the digested peptide solution by lowering the pH to 2.0 by adding formic acid.</li>
	<li>Activate the C18 (n-octadecyl) spin column in a 2 mL receiver tube by adding 200 &#181;L of 50% methanol solution and spin at 1500 x g for 2 min at RT. Repeat the activation step.</li>
	<li>Equilibrate the C18 column resin beds by adding 200 &#181;L of equilibration buffer and spinning for 2 min with the same conditions. Repeat this step.</li>
	<li>Load the acidified peptide solution on the C18 column and centrifuge at 1500 x g for 2 min at RT. Collect the flow-through and reload it one more time. Discard the second flow-through.</li>
	<li>Wash the peptides bound to the C18 resin with the wash buffer 2x, as done in Step 6.3. Use the same equilibration buffer for washing the column.</li>
	<li>Elute the peptides by adding 40 &#181;L of elution buffer followed by centrifugation at 1500 x g, for 2 min at RT. Repeat the elution step three times to increase the yield of the recovered peptides.</li>
	<li>Dry the peptides in a speed vacuum concentrator by evaporating the aqueous solution. Dry pellets can be saved at -20 &#176;C for a few weeks before the MS analysis.</li>
</ol>
7. Setting up mass spectrometer for peptide analysis
NOTE: For MS parameters, see Supplementary File 1 (adapted from a previous publication from the lab)14.
<ol>
	<li>Before loading the peptide samples for the MS analysis, dissolve the dry peptide pellets in 20 &#181;L of sample loading buffer and perform micro BCA to quantify the concentration of recovered peptides.</li>
	<li>Transfer the dissolved peptides in a glass vial and load 3 &#181;g (quantified from micro BCA) of peptides into the UPLC system autosampler.</li>
	<li>Load the samples onto a vented trap column (C18 HPLC column, 0.075 mm x 20 mm) with a flow rate of 250 nL/min.</li>
	<li>Arrange the trap column in line with an analytical column (0.075 &#181;m x 500 mm) and assemble an emitter tip to the electrospray ionization (ESI) source subjected to a spray voltage of 2000 V. 
	NOTE: In this study, ESI is performed, in which the mobile phase is subjected to high-voltage ionization into the gas phase. The spray created by this is directed to the vacuum chamber of the MS through a heated capillary. While under vacuum, the de-solvation of droplets and ejection of ions occurs in the presence of heat and voltage. Thereafter, the ions are accelerated toward the mass analyzer in the presence of a high voltage environment.</li>
	<li>Analyze the samples with 2 h acquisition runs. This study is performed using data-dependent acquisition with the most intense top 20 precursor ion selection paradigm. 
	NOTE: In data-dependent acquisition, a limited number of precursor peptides are detected in the MS1 scan and subjected to fragmentation for MS2 analysis. However, the dynamic exclusion is problematic here since highly abundant A&#946; peptides will be omitted for fragmentation and result in an underestimate of their quantity.</li>
	<li>For absolute quantification of A&#946; peptides, run the same samples one more time using the targeted MS/MS method. For this study, an exhaustive list of all m/z ratios for various possible tryptic A&#946; peptides for the APP knock-in mouse models is prepared (see Supplementary Table 1). Other groups should prepare a similar list in accordance with the available mouse model and the possible peptides generated from the protein of interest (e.g., A&#946; for AD). 
	NOTE: To address the exclusion of highly abundant peptides, use a targeted approach by providing a list of selected m/z values corresponding to A&#946; tryptic peptides. This approach addresses the problem of data-dependent exclusion of A&#946; peptides and can quantify the absolute quantities of different monomers (A&#946;38, 40, and 42 peptides) with high resolution.</li>
	<li>The mass spectrometer generates MS spectra of the peptide samples and saves raw data files on the target directory. Use this file to perform spectral matching using well-established statistical and bioinformatics tools. Multiple online and offline database search and analysis tools are available.</li>
</ol>
8. MS data analysis
<ol>
	<li>Extract the MS1 and MS2 files using the offline Rawconverter tool (http://www.fields.scripps.edu/rawconv/).</li>
	<li>Perform identification, quantification, and detailed analyses of the data on a web-based MS data search engine. In this study, Integrated Proteomics Pipeline - IP2 (Bruker: http://www.integratedproteomics.com/)&#160;was used. 
	NOTE: Several other online and offline MS data analysis tools are available and can also be used, such as MaxQuant (https://www.maxquant.org/).</li>
	<li>After uploading the MS1 and MS2 files, select an updated mouse proteome database. Here, an updated mouse database containing additional App knock-in specific mutations is selected for the identification of peptides using ProLuCId and SEQUEST algorithms11.</li>
	<li>In the IP2 analysis system, select the default parameters and modify them according to the experimental requirements. In this study, a peptide mass tolerance of 50 ppm for precursor and 600 ppm for fragments is used (refer to Supplementary File 1 for other parameters used in IP2). 
	NOTE: Researchers can modify the search parameters in accordance with the experimental objectives. For example, for identifying post-translational modifications, add differential modification values for various PTMs (e.g., ubiquitination, SUMOylation, phosphorylation).</li>
</ol>

<ol>
	<li>Willbold, D., Strodel, B., Schr&#246;der, G. F., Hoyer, W., Heise, 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-type+protein+aggregation+and+prion-like+properties+of+amyloids.">Amyloid-type protein aggregation and prion-like properties of amyloids.</a> Chemical Reviews. 121 (13), 8285-8307 (2021).</li><li>Rambaran, R. N., Serpell, L. C. <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:+abnormal+protein+assembly.">Amyloid fibrils: abnormal protein assembly.</a> Prion. 2 (3), 112-117 (2008).</li><li>Upadhyay, A., 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=Complex+inclusion+bodies+and+defective+proteome+hubs+in+neurodegenerative+disease:+New+clues,+new+challenges.">Complex inclusion bodies and defective proteome hubs in neurodegenerative disease: New clues, new challenges.</a> The Neuroscientist. , (2021).</li><li>Greiner, E. R., Kelly, J. W., Palhano, 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=Immunoprecipitation+of+amyloid+fibrils+by+the+use+of+an+antibody+that+recognizes+a+generic+epitope+common+to+amyloid+fibrils.">Immunoprecipitation of amyloid fibrils by the use of an antibody that recognizes a generic epitope common to amyloid fibrils.</a> PLOS ONE. 9 (8), 105433 (2014).</li><li>Kourelis, T. V., 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=A+proteomic+atlas+of+cardiac+amyloid+plaques.">A proteomic atlas of cardiac amyloid plaques.</a> JACC: CardioOncology. 2 (4), 632-643 (2020).</li><li>Mangione, P. P., 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=Increasing+the+accuracy+of+proteomic+typing+by+decellularisation+of+amyloid+tissue+biopsies.">Increasing the accuracy of proteomic typing by decellularisation of amyloid tissue biopsies.</a> Journal of Proteomics. 165, 113-118 (2017).</li><li>Rostagno, A., Neubert, T. A., Ghiso, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unveiling+brain+A&#946;+heterogeneity+through+targeted+proteomic+analysis.">Unveiling brain A&#946; heterogeneity through targeted proteomic analysis.</a> Methods in Molecular Biology. 1779, 23-43 (2018).</li><li>Roher, A. 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=Morphology+and+toxicity+of+A&#946;-(1-42)+dimer+derived+from+neuritic+and+vascular+amyloid+deposits+of+Alzheimer's+disease.">Morphology and toxicity of A&#946;-(1-42) dimer derived from neuritic and vascular amyloid deposits of Alzheimer's disease.</a> Journal of Biological Chemistry. 271 (34), 20631-20635 (1996).</li><li>Lu, J. -. X., 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=Molecular+structure+of+&#946;-amyloid+fibrils+in+Alzheimer's+disease+brain+tissue.">Molecular structure of &#946;-amyloid fibrils in Alzheimer's disease brain tissue.</a> Cell. 154 (6), 1257-1268 (2013).</li><li>Tycko, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-state+NMR+studies+of+amyloid+fibril+structure.">Solid-state NMR studies of amyloid fibril structure.</a> Annual Review of Physical Chemistry. 62, 279-299 (2011).</li><li>Saito, 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=Single+App+knock-in+mouse+models+of+Alzheimer's+disease.">Single App knock-in mouse models of Alzheimer's disease.</a> Nature Neuroscience. 17 (5), 661-663 (2014).</li><li>Meyerhoff, J., 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=Microdissection+of+mouse+brain+into+functionally+and+anatomically+different+regions.">Microdissection of mouse brain into functionally and anatomically different regions.</a> Journal of Visualized Experiments: JoVE. (168), e61941 (2021).</li><li>Spijker, S., Li, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+Rodent+Brain+Regions.">Dissection of Rodent Brain Regions.</a> Neuroproteomics. Neuromethods. 57, (2011).</li><li>Hark, T. J., 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=Pulse-chase+proteomics+of+the+App+knockin+mouse+models+of+Alzheimer's+disease+reveals+that+synaptic+dysfunction+originates+in+presynaptic+terminals.">Pulse-chase proteomics of the App knockin mouse models of Alzheimer's disease reveals that synaptic dysfunction originates in presynaptic terminals.</a> Cell Systems. 12 (2), 141-158 (2021).</li><li>Liu, 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=Highly+efficient+intercellular+spreading+of+protein+misfolding+mediated+by+viral+ligand-receptor+interactions.">Highly efficient intercellular spreading of protein misfolding mediated by viral ligand-receptor interactions.</a> Nature Communications. 12 (1), 5739 (2021).</li><li>Toyama, B. H., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+structure:+conformational+diversity+and+consequences.">Amyloid structure: conformational diversity and consequences.</a> Annual Review of Biochemistry. 80, 557-585 (2011).</li><li>Sundaria, N., 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=Neurodegeneration+&amp;+imperfect+ageing:+Technological+limitations+and+challenges.">Neurodegeneration &amp; imperfect ageing: Technological limitations and challenges.</a> Mechanisms of Ageing and Development. 200, 111574 (2021).</li><li>Cendrowska, U., 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=Unraveling+the+complexity+of+amyloid+polymorphism+using+gold+nanoparticles+and+cryo-EM.">Unraveling the complexity of amyloid polymorphism using gold nanoparticles and cryo-EM.</a> Proceedings of the National Academy of Sciences. 117 (12), 6866-6874 (2020).</li><li>Seuring, C., 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=Amyloid+fibril+polymorphism:+almost+identical+on+the+atomic+level,+mesoscopically+very+different.">Amyloid fibril polymorphism: almost identical on the atomic level, mesoscopically very different.</a> The Journal of Physical Chemistry B. 121 (8), 1783-1792 (2017).</li><li>Close, W., 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=Physical+basis+of+amyloid+fibril+polymorphism.">Physical basis of amyloid fibril polymorphism.</a> Nature Communications. 9 (1), 699 (2018).</li><li>Tycko, 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+polymorphism:+Structural+basis+and+neurobiological+relevance.">Amyloid polymorphism: Structural basis and neurobiological relevance.</a> Neuron. 86 (3), 632-645 (2015).</li><li>Konstantoulea, K., 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=Heterotypic+Amyloid+&#946;+interactions+facilitate+amyloid+assembly+and+modify+amyloid+structure.">Heterotypic Amyloid &#946; interactions facilitate amyloid assembly and modify amyloid structure.</a> The EMBO Journal. 41, 108591 (2022).</li><li>Hondius, D. C., 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=Proteomics+analysis+identifies+new+markers+associated+with+capillary+cerebral+amyloid+angiopathy+in+Alzheimer's+disease.">Proteomics analysis identifies new markers associated with capillary cerebral amyloid angiopathy in Alzheimer's disease.</a> Acta Neuropathologica Communications. 6 (1), 1-19 (2018).</li><li>Luo, J., W&#228;rml&#228;nder, S. K., Gr&#228;slund, A., Abrahams, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-interactions+between+the+Alzheimer+disease+amyloid-&#946;+peptide+and+other+amyloid+proteins:+a+further+aspect+of+the+amyloid+cascade+hypothesis.">Cross-interactions between the Alzheimer disease amyloid-&#946; peptide and other amyloid proteins: a further aspect of the amyloid cascade hypothesis.</a> Journal of Biological Chemistry. 291 (32), 16485-16493 (2016).</li><li>Hosp, F., 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=Spatiotemporal+proteomic+profiling+of+Huntington's+disease+inclusions+reveals+widespread+loss+of+protein+function.">Spatiotemporal proteomic profiling of Huntington's disease inclusions reveals widespread loss of protein function.</a> Cell Reports. 21 (8), 2291-2303 (2017).</li><li>Wallace, E. W. J., 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=Reversible,+specific,+active+aggregates+of+endogenous+proteins+assemble+upon+heat+stress.">Reversible, specific, active aggregates of endogenous proteins assemble upon heat stress.</a> Cell. 162 (6), 1286-1298 (2015).</li><li>Darling, A. L., Liu, Y., Oldfield, C. J., Uversky, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsically+disordered+proteome+of+human+membrane-less+organelles.">Intrinsically disordered proteome of human membrane-less organelles.</a> Proteomics. 18 (5-6), 1700193 (2018).</li><li>Kepchia, 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=Diverse+proteins+aggregate+in+mild+cognitive+impairment+and+Alzheimer's+disease+brain.">Diverse proteins aggregate in mild cognitive impairment and Alzheimer's disease brain.</a> Alzheimer's Research &amp; Therapy. 12 (1), 1-20 (2020).</li><li>Espay, A. J., 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=Revisiting+protein+aggregation+as+pathogenic+in+sporadic+Parkinson+and+Alzheimer+diseases.">Revisiting protein aggregation as pathogenic in sporadic Parkinson and Alzheimer diseases.</a> Neurology. 92 (7), 329-337 (2019).</li><li>F&#228;ndrich, M., Schmidt, M., Grigorieff, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+understanding+Alzheimer's+&#946;-amyloid+structures.">Recent progress in understanding Alzheimer's &#946;-amyloid structures.</a> Trends in Biochemical Sciences. 36 (6), 338-345 (2011).</li><li>Bonnin, E. A., Fornasiero, E. F., Lange, F., Turck, C. W., Rizzoli, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoSIMS+observations+of+mouse+retinal+cells+reveal+strict+metabolic+controls+on+nitrogen+turnover.">NanoSIMS observations of mouse retinal cells reveal strict metabolic controls on nitrogen turnover.</a> BMC Molecular and Cell Biology. 22 (1), 1-10 (2021).</li><li>Michno, W., 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=Following+spatial+A&#946;+aggregation+dynamics+in+evolving+Alzheimer's+disease+pathology+by+imaging+stable+isotope+labeling+kinetics.">Following spatial A&#946; aggregation dynamics in evolving Alzheimer's disease pathology by imaging stable isotope labeling kinetics.</a> Science Advances. 7 (25), (2021).</li><li>Toyama, B. H., 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=Identification+of+long-lived+proteins+reveals+exceptional+stability+of+essential+cellular+structures.">Identification of long-lived proteins reveals exceptional stability of essential cellular structures.</a> Cell. 154 (5), 971-982 (2013).</li><li>Bomba-Warczak, E., Edassery, S. L., Hark, T. J., Savas, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-lived+mitochondrial+cristae+proteins+in+mouse+heart+and+brain.">Long-lived mitochondrial cristae proteins in mouse heart and brain.</a> Journal of Cell Biology. 220 (9), 202005193 (2021).</li></ol>

The authors have nothing to disclose.

Developing a clear understanding of amyloid structure and composition is challenging for structural biologists and biochemists due to the biological complexities and experimental limitations in extracting purified fibrils from AD brain tissues16,17. Amyloid fibrils are polymorphic at the molecular level, showing a heterogeneous population of varying lengths and complexities18,19. To better understand thei...

Amyloid is an extremely stable supramolecular arrangement that is found in a diverse panel of proteins, some of which lead to pathological changes1. The accumulation of intra- or extracellular amyloid aggregates is observed in several neurodegenerative diseases2. Amyloid aggregates are heterogeneous and are enriched with a large number of proteins and lipids3. In recent years, interest in the amyloid proteome has generated substantial interest among basic and translational neuroscientists. Several methods have been developed to extract and purify amyloid aggregates from mouse and post-mortem human brain tissues. Laser-capture microdissection, immunoprecipitation, decellularization, and biochemical isolation of amyloid aggregates are widely used methods to extract and purify amyloid plaques, fibrils, and oligomers4,5,6,7. Many of these studies have focused on determining the protein composition of these tightly packed fibrillar deposits using semi-quantitative MS. However, the available results are inconsistent, and the surprisingly large number of co-purifying proteins previously reported are challenging to interpret.
The primary limitation of the existing literature describing the amyloid core proteome in AD and AD mouse model brains is that the purified material contains an unmanageable number of co-purifying proteins. The overall goal of this method is to overcome this limitation and develop a robust biochemical purification for isolating amyloid fibril cores. This strategy employs a previously described sucrose density gradient ultracentrifugation-based biochemical method for the isolation of SDS insoluble enriched amyloid fractions from post-mortem AD human and mouse brain tissues8,9. This method builds on the existing literature but goes further with ultrasonication and SDS washes to remove most of the loosely bound amyloid-associated proteins, leading to the isolation of highly purified amyloid fibrils (Figure 1). The fibrils purified by this protocol overcome several existing challenges frequently encountered in structural studies of amyloid fibrils isolated from brain extracts. Visualization of these fibrils with transmission electron microscopy (TEM) confirms the integrity and purity of the purified material (Figure 2). In this study, the isolated fibrils are solubilized and digested to peptides with trypsin, and label-free MS analysis can readily reveal the identity of the proteins forming the fibril core. Notably, some of these proteins have an inherent tendency to form supramolecular assemblies in non-membrane-bound organelles. In addition, many of the proteins identified in the analysis of amyloid-beta (A&#946;) fibrils are also associated with other neurodegenerative diseases, suggesting that these proteins may play a key role in multiple proteinopathies.
This SDS/ultrasonication method is unlikely to alter or disrupt the structure of the fibril cores. The purified material is also suitable for a wide range of top-down and bottom-up proteomic analysis approaches and additional MS-based structural analysis strategies, such as chemical crosslinking or hydrogen-deuterium exchange. The overall recovery using this method is relatively high and, thus, is suitable for detailed structural studies, which require micrograms to milligrams of the purified material. The purified material is also suitable for structural studies using cryoEM and atomic force microscopy. This protocol, in combination with the stable isotopic labeling of mammals, can facilitate solid-state nuclear magnetic resonance (NMR) studies of amyloid structure10.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acclaim PepMap 100 C18 HPLC column 0.075 mm x 20&#160;mm</td>
			<td>Thermo Scientific</td>
			<td>164535</td>
			<td rowspan="35">Alternative instruments, chemicals and antibodies from other manufacturers can be used</td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>9830</td>
		</tr>
		<tr>
			<td>anti-amyloid beta (1-16) 6E10 antibody</td>
			<td>Biolegend</td>
			<td>803001</td>
		</tr>
		<tr>
			<td>anti-amyloid beta (17-24) 4G8 antibody</td>
			<td>Biolegend</td>
			<td>800701</td>
		</tr>
		<tr>
			<td>anti-amyloid beta (N terminus 82E1) antibody</td>
			<td>IBL America</td>
			<td>10323</td>
		</tr>
		<tr>
			<td>anti-amyloid fibril LOC antibody</td>
			<td>&#160;EMD Millipore</td>
			<td>AB2287</td>
		</tr>
		<tr>
			<td>BCA kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23225</td>
		</tr>
		<tr>
			<td>Bioruptor Pico Plus</td>
			<td>Diagenode</td>
			<td>B01020001</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;C1016</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sigma-Aldrich</td>
			<td>C0130</td>
		</tr>
		<tr>
			<td>Complete&#160; Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>11697498001</td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Thermo Fisher Scientific</td>
			<td>EN0521</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>EDS</td>
		</tr>
		<tr>
			<td>Guanidine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>G4505</td>
		</tr>
		<tr>
			<td>HyperSep C18 Cartridges</td>
			<td>Thermo Fisher Scientific</td>
			<td>60108-302</td>
		</tr>
		<tr>
			<td>Integrated Proteomics Pipeline - IP2&#160;</td>
			<td>http://www.integratedproteomics.com/</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide (IAA)</td>
			<td>Sigma-Aldrich</td>
			<td>I1149</td>
		</tr>
		<tr>
			<td>K54 Tissue Homogenizing System Motor</td>
			<td>Cole Parmer</td>
			<td>Glas-Col 099C</td>
		</tr>
		<tr>
			<td>MaxQuant</td>
			<td>https://www.maxquant.org/</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro BCA kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23235</td>
		</tr>
		<tr>
			<td>Nanoviper 75 &#956;m x 50&#160;cm</td>
			<td>Thermo Scientific</td>
			<td>164942</td>
		</tr>
		<tr>
			<td>Optima L-90K Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>BR-8101P-E</td>
		</tr>
		<tr>
			<td>Orbitrap Fusion TribridMass Spectrometer</td>
			<td>Thermo Scientific</td>
			<td>IQLAAEGAAPFADBMBCX</td>
		</tr>
		<tr>
			<td>Pierce C18 Spin Columns</td>
			<td>Thermo Fisher Scientific</td>
			<td>89870</td>
		</tr>
		<tr>
			<td>Precellys 24 tissue homogenizer</td>
			<td>Bertin Instruments</td>
			<td>P000062-PEVO0-A</td>
		</tr>
		<tr>
			<td>ProteaseMAX(TM) Surfactant Trypsin Enhancer</td>
			<td>Promega</td>
			<td>V2072</td>
		</tr>
		<tr>
			<td>RawConverter</td>
			<td>http://www.fields.scripps.edu/rawconv/</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>VWR</td>
			<td>97064-646</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>74255</td>
		</tr>
		<tr>
			<td>Sorvall Legend Micro 21R Microcentrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>75002446</td>
		</tr>
		<tr>
			<td>Speed Vaccum Concentrator</td>
			<td>Labconco</td>
			<td>7315021</td>
		</tr>
		<tr>
			<td>Tris-2-carboxyethylphosphine (TCEP)</td>
			<td>Sigma-Aldrich</td>
			<td>C4706-2G</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Thermo Fisher Scientific</td>
			<td>15568025</td>
		</tr>
		<tr>
			<td>Trypsin Gold-Mass spec grade</td>
			<td>Promega</td>
			<td>V5280</td>
		</tr>
		<tr>
			<td>UltiMate 3000 RSLCnano System</td>
			<td>Thermo Scientific</td>
			<td>ULTIM3000RSLCNANO</td>
		</tr>
	</tbody>
</table>

biochemical purification and proteomic characterization of amyloid fibril cores from the brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

Here, a detailed method for the isolation and purification of amyloid fibrils using a modified sucrose density gradient ultracentrifugation purification method is summarized (see Figure 1). The innovation in this method is the inclusion of steps of ultrasonication-based washing using a water bath sonication system followed by SDS solubilization, which removes many loosely associated proteins from the amyloid fibrils that co-purify with the highly dense and clean fibrils. The ultrasonication ...

Watch this Scientific Journal Video about Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain at JoVE.com

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

biochemical-purification-proteomic-characterization-amyloid-fibril

Research

JoVE Journal

Neuroscience

2.9K Views.  Northwestern University Feinberg School of Medicine. This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

Video: Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3.
Amyloid &beta;-protein (A&beta;) is central to AD pathogenesis. Soluble, oligomeric A&beta; assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble A&beta; assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst A&beta; assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-&beta;17, a soluble, oligomeric, &beta;-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar &beta;2-microglobulin (&beta;2m) were found to bind fibrils of certain other amyloidogenic proteins besides &beta;2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric A&beta;4020. Unexpectedly, these aptamers bound fibrillar A&beta;40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric A&beta;4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of A&beta; and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.

Selection of Aptamers for Amyloid &#946;-Protein, the Causative Agent of Alzheimer's Disease

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid &beta;-protein, the causative agent of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

Purification of the Dendritic Filopodia-rich Fraction

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the dendritic filopodia establish contact with a target axon, they begin maturing into spines, leading to the formation of a synapse. Telencephalin (TLCN) is abundantly localized in dendritic filopodia and is gradually excluded from spines. Overexpression of TLCN in cultured hippocampal neurons induces dendritic filopodia formation. We showed that telencephalin strongly binds to an extracellular matrix molecule, vitronectin. Vitronectin-coated microbeads induced phagocytic cup formation on neuronal dendrites. In the phagocytic cup, TLCN, TLCN-binding proteins such as phosphorylated Ezrin/Radixin/Moesin (phospho-ERM), and F-actin are accumulated, which suggests that components of the phagocytic cup are similar to those of dendritic filopodia. Thus, we developed a method for purifying the phagocytic cup instead of dendritic filopodia. Magnetic polystyrene beads were coated with vitronectin, which is abundantly present in the culture medium of hippocampal neurons and which induces phagocytic cup formation on neuronal dendrites. After 24 h of incubation, the phagocytic cups were mildly solubilized with detergent and collected using a magnet separator. After washing the beads, the binding proteins were eluted and analyzed by silver staining and Western blotting. In the binding fraction, TLCN and actin were abundantly present. In addition, many proteins identified from the fraction were localized to the dendritic filopodia; thus, we named the binding fraction as the dendritic filopodia-rich fraction. This article describes details regarding the purification method for the dendritic filopodia-rich fraction.

In this protocol, we introduce a method for purifying the dendritic filopodia-rich fraction from the phagocytic cup-like protrusion structure on cultured hippocampal neurons by taking advantage of the specific and strong affinity between a dendritic filopodial adhesion molecule, TLCN, and an extracellular matrix molecule, vitronectin.

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the ...