This work is supported by grants to W.C. from National Institutes of Health Grant AI074809 and The University of Texas M. D. Anderson Cancer Center Institutional Research Grant Program.

1. Prepare MES Buffer
<ol>
	<li>Use MES buffer (0.1 M 2-(N-morpholino)ethanesulfonic acid and 0.9 M NaCl) to prepare both protein-only amyloid as well as stabilized soluble protein oligomers. Weigh 1.95 g of MES and 5.27 g of NaCl. Add H2O and adjust pH to 4.7. Make up the final volume to 100 ml. Store at 4&#186; C for up to 6 months.</li>
</ol>
2. Prepare Protein-only Amyloid Directly from Native Proteins
<ol>
	<li>Weigh protein of choice (any native protein or peptide) and reconstitute to 10 mg/ml in MES buffer.</li>
	<li>Incubate at 65 &#186;C in a waterbath for 4 hr. By the end of the period, white precipitate is visible in solution.</li>
	<li>Neutralize the sample with 10% (v/v) of Tris-HCl (1 M, pH 10.5).</li>
	<li>Dialyze the protein in a dialysis cassette overnight to exchange to the desired buffer for further analysis.</li>
	<li>A control sample of native protein can be prepared by following steps 2.1, 2.3, and 2.4 without heat precipitation (step 2.2).</li>
</ol>
3. Prepare Hybrid Amyloid from Stabilized Soluble Oligomers of Native Proteins
<ol>
	<li>Prepare stabilized soluble protein oligomers
	<ol>
		<li>Weigh protein of choice and reconstitute to 10 mg/ml in MES buffer. Leave at room temperature for 15 min.</li>
		<li>Weigh 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and prepare a fresh stock of 20 mg/ml in MES buffer.</li>
		<li>Mix protein and EDC solutions at a ratio of 2:5 (v/v), i.e. 2 mg of protein for every 10 mg of EDC.</li>
		<li>Incubate at room temperature for 2 hr.</li>
		<li>Neutralize the sample with 10% (v/v) of Tris-HCl (1 M, pH 10.5).</li>
		<li>Dialyze the protein in a dialysis cassette overnight to exchange to the desired buffer for further analysis.</li>
		<li>Store the stabilized soluble protein oligomer at 4 &#186;C up to 4 weeks.</li>
	</ol>
	</li>
	<li>Prepare hybrid amyloids from stabilized soluble protein oligomer.
	<ol>
		<li>To prepare DNA-containing amyloid, mix DNA and stabilized soluble protein oligomer at 1:1 ratio (w/w). Leave at 4 &#186;C for at least 2 hr. During this period, insoluble precipitate is visible in solution when the initial protein concentration is 0.5 mg/ml or higher.</li>
		<li>To prepare RNA-containing amyloid, mix RNA and stabilized soluble protein oligomer at 1:1 ratio (w/w). Leave at 4 &#186;C for at least 2 hr. During this period, insoluble precipitate is visible in solution when the initial protein concentration is 0.5 mg/ml or higher.</li>
		<li>To prepare heparin-containing amyloid, mix heparin and stabilized soluble protein oligomer at 1:1 ratio (w/w). Leave at 4 &#186;C for at least 2 hrs. During this period, insoluble precipitate is visible in solution when the initial protein concentration is 0.5 mg/ml or higher.</li>
	</ol>
	</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+misfolding,+functional+amyloid,+and+human+disease.">Protein misfolding, functional amyloid, and human disease.</a> Annu. Rev. Biochem. 75, 333-366 (2006).</li><li>Goldschmidt, L., Teng, P. K., Riek, R., Eisenberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+the+amylome,+proteins+capable+of+forming+amyloid-like+fibrils.">Identifying the amylome, proteins capable of forming amyloid-like fibrils.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 3487-3492 (2010).</li><li>Fowler, D. M., Koulov, A. V., Balch, W. E., Kelly, J. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+classification+of+toxic+amyloid+oligomers.">Structural classification of toxic amyloid oligomers.</a> J. Biol. Chem. 283, 29639-29643 (1074).</li><li>Di Domizio, 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=Binding+with+nucleic+acids+or+glycosaminoglycans+converts+soluble+protein+oligomers+to+amyloid.">Binding with nucleic acids or glycosaminoglycans converts soluble protein oligomers to amyloid.</a> J. Biol. Chem. 287, 736-747 (2012).</li><li>Zhang, X., Li, J. -. P., Lijuan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparan+sulfate+proteoglycans+in+amyloidosis.">Heparan sulfate proteoglycans in amyloidosis.</a> Prog. Mol. Biol. Transl. Sci. 93, 309-334 (2010).</li><li>Jim&#233;nez, 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=Protein-DNA+Interaction+at+the+Origin+of+Neurological+Diseases:+A+Hypothesis.">Protein-DNA Interaction at the Origin of Neurological Diseases: A Hypothesis.</a> J. Alzheimer's Dis. 22, 375-391 (2010).</li><li>Bhattacharya, M., Jain, N., Mukhopadhyay, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+Mechanism+of+Aggregation+and+Fibril+Formation+from+Bovine+Serum+Albumin.">Insights into the Mechanism of Aggregation and Fibril Formation from Bovine Serum Albumin.</a> J. Phys. Chem. 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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=Partially+Unfolded+States+of+b2-Microglobulin+and+Amyloid+Formation+in+vitro.">Partially Unfolded States of b2-Microglobulin and Amyloid Formation in vitro.</a> Biochemistry. 39, 8735-8746 (2000).</li><li>Liu, K., Cho, H. S., Lashuel, H. A., Kelly, J. W., Wemmer, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+glimpse+of+a+possible+amyloidogenic+intermediate+of+transthyretin.">A glimpse of a possible amyloidogenic intermediate of transthyretin.</a> Nat. Struct. Mol. Biol. 7, 754-757 (2000).</li><li>Pfefferkorn, C. M., McGlinchey, R. P., Lee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+pH+on+aggregation+kinetics+of+the+repeat+domain+of+a+functional+amyloid.">Effects of pH on aggregation kinetics of the repeat domain of a functional amyloid.</a> Pmel17. Proc. Natl. Acad. Sci. U.S.A. 107, 21447-21452 (2010).</li><li>Di Domizio, 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=Nucleic+acid-containing+amyloid+fibrils+potently+induce+type+I+interferon+and+stimulate+systemic+autoimmunity.">Nucleic acid-containing amyloid fibrils potently induce type I interferon and stimulate systemic autoimmunity.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 14550-14555 (2012).</li><li>Halle, A., Hornung, V., Petzold, G. C., Stewart, C. R., Monks, B. G., Reinheckel, T., Fitzgerald, K. A., Latz, E., Moore, K. J., Golenbock, D. 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We have nothing to disclose.

The method described here offers a rapid and flexible means to prepare amyloid fibrils in vitro from virtually any protein or peptide of choice. Several different types of amyloid can be reliably prepared - protein-only fibrils or hybrid aggregates containing DNA, RNA, &#160;or glycosaminoglycans. The procedures involved are simple, straight forward, and do not require advanced technical training. The amyloid prepared by this method is quite stable and can be safely stored at 4 &#186;C or frozen at -80 &#186;C f...

Proteins are the most abundant biological macromolecules present in all types of cells. They occur in a great variety of sizes, structures, and post-translational modifications, and fulfill an enormous range of important biological functions when in their native forms. More than two dozens of aberrant polypeptides have been implicated in numerous human pathological conditions, such as Alzheimer&#39;s disease, Parkinson disease, &#160;and Type 2 Diabetes1-4. The terminal misfolded proteins accumulate as amyloid fibrils&#160;the insoluble stable aggregates that occur extracellularly or intracellularly.
Despite the implication of specific proteins in certain diseases, increasing evidence supports the notion that all polypeptides have intrinsic properties that enable amyloid transformation. A genome-wide sequence survey identified the &#8220;amylome&#8221;, by which amyloid-prone fragments constitute roughly 15% of all coding peptide segments from E. coli to humans5. Accordingly, an increasing number of functional amyloids, which participate in various important cellular processes, have been discovered in recent years. For example, bacteria assemble amyloids to form biofilm and spore structures that are critical for their survival and pathogenesis6-9. Peptide hormones form amyloid deposits during storage within mammalian secretory granules before being released, further implying that the protein amyloid form can serve beneficial biological functions6,10,11. Moreover, proteins can also assemble into amyloid fibrils to relay critical cellular signals12,13.
For many years, the most studied amyloids are prepared from individual peptides that are associated with human diseases, such as amyloid beta or prion peptide. These peptides usually lack well defined structure and spontaneously form amyloid in solution over time, a process mediated by a partially misfolded intermediate that serves as the precursor of insoluble amyloid. The precursor of amyloid is also called soluble protein oligomer, which is rare and unstable when generated from natural peptides14,15. However, as described earlier, almost any protein in principle can adopt amyloid conformation under the appropriate conditions. We recently established a method to prepare stabilized soluble oligomers of native proteins, which readily form amyloid in the presence of various cofactors16. The resulting amyloid fibrils reflect the complex nature of the terminal protein misfolding aggregates17,18. Here we use the term protein-only amyloid to refer the protein fibrils without cofactors and hybrid amyloid for the fibrils containing nonproteinaceous components.
Previously, various methods have been developed to generate amyloid in vitro by applying conditions known to favor protein misfolding, such as high temperature, hydrophobic environment, and pH variations19-23. However, they associate with various problems, such as low efficiency, reversible folding, large batch variations or slow kinetics. The approach described here is reproducible, easy to scale up, and allows one to precisely control the timing and condition of amyloid conversion.

<table><tbody><tr><td>2-(N-morpholino)ethanesulfonic acid (MES)</td><td>Sigma-Aldrich</td><td>M8250</td><td></td></tr><tr><td>NaCl</td><td>Fisher</td><td>BP358-10</td><td></td></tr><tr><td>1-ethyl-3-[3-dimethyl-aminopropyl] carbodiimide hydrochloride (EDC)</td><td>Thermo/Pierce</td><td>22980</td><td></td></tr><tr><td>DNA from salmon sperm</td><td>Sigma</td><td>D1626</td><td></td></tr><tr><td>RNA from torula yeast</td><td>Sigma</td><td>R6625</td><td></td></tr><tr><td>Heparin from porcine intestinal mucosa</td><td>Sigma</td><td>H3149</td><td></td></tr><tr><td>Tris base</td><td>Fisher</td><td>BP152-1</td><td></td></tr><tr><td>Equipment:</td><td></td><td></td><td></td></tr><tr><td>Water bath</td><td>Fisher Science</td><td>Isotemp 210</td><td></td></tr><tr><td>Slide-A-Lyzer dialysis cassette</td><td>Thermo/Pierce</td><td>66380</td><td></td></tr></tbody></table>

rapid generation of amyloid from native proteins in vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

The ability to convert native proteins directly to amyloid relies on the conformational change incurred at high temperature in the MES buffer. Precipitation in the solution is a good indication of protein aggregation and possibly amyloid formation. On the other hand, to allow soluble protein oligomer to stabilize, EDC is used to crosslink the proteins to solidify the oligomeric conformation induced in MES buffer. As a result, the stabilized protein oligomers are multimeric proteins (Figure 1), distinct f...

Watch this Scientific Journal Video about Rapid Generation of Amyloid from Native Proteins In vitro at JoVE.com

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

rapid-generation-of-amyloid-from-native-proteins-in-vitro

Research

JoVE Journal

Biology

6.1K Views.  The University of Texas MD Anderson Cancer Center. Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Video: Rapid Generation of Amyloid from Native Proteins In vitro

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (&gt;95%) A&#946;42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the A&#946;40 peptide with identical success and without the need to alter the method.

Herein we report a tailored HPLC purification protocol that yields high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides, capable of oligomer formation. Amyloid beta is a highly aggregation prone, hydrophobic peptide implicated in Alzheimer&#39;s disease. The amyloidogenic nature of the peptide makes its purification a challenge.

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

Biochemistry

A11-positive &#946;-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer is widely accepted as the leading neurotoxin in the progress of Alzheimer&#39;s disease (AD) and is considered to be the crucial event in the pathogenesis of AD. Therefore, A&#946; oligomer inhibitors might prevent neurodegeneration and have the potential to be developed as disease-modifying treatments of AD. However, different formation protocols of A&#946; oligomer might lead to oligomers with different characteristics. Moreover, there are not many methods to effectively screen A&#946;1-42 oligomer inhibitors. An A11 antibody can react with a subset of toxic A&#946;1-42 oligomer with anti-parallel &#946;-sheet structures. In this protocol, we describe how to prepare an A11-positive A&#946;1-42 oligomer-rich sample from a synthetic A&#946;1-42 peptide in vitro and to evaluate relative amounts of A11-positive A&#946;1-42 oligomer in samples by a dot blotting analysis using A11 and A&#946;1-42-specific 6E10 antibodies. Using this protocol, inhibitors of A11-positive A&#946;1-42 oligomer can also be screened from semi-quantitative experimental results.

This protocol describes how to prepare A&#946; oligomers from a synthetic peptide in vitro and to evaluate relative amounts of A&#946; oligomer by a dot blotting analysis.

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer ...

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.

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 ...

Medicine

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

Neuroscience

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 ...

Characterization of pH-Dependent Reversible Self-Assembly of Amyloid Beta 1-40-Coated Gold Colloids

The characterization of A&#946;1-40 coated over the nano-gold colloidal particle surfaces was conducted using surface plasmon resonance (SPR) spectroscopy and transmission electron microscopy (TEM). The observed pH-dependent shift of the SPR band of A&#946;1-40-coated 20 nm gold particles was correlated with alternation of aggregation and disaggregation observed in the TEM images. A pH of ~4&#160;induced an unfolded conformation, and a pH of ~10&#160;induced a folded conformation of A&#946;1-40 on the gold surface. This reversible aggregation process was observed by Raman imaging as the pH was gradually changed from pH 4 to pH 10. We observed a pH-dependent morphology change in A&#946;1-40 on the gold surface, where clear aggregates were observed at pH 4. However, we observed very subtle differences in the surface-enhanced Raman spectroscopy (SERS) spectrum between pH 4 and pH 10 conditions, with the most striking difference being spectral density in the region of 250 cm-1 and 1750 cm-1. Specifically, the mode analysis of the reversible aggregation indicated that the aggregates were formed by the unfolded conformation of A&#946;1-40 which involved the benzene ring section of Tyrosine and Phenylalanine. Conversely, the disassembly of the aggregates was associated with conformational changes in protein folding of A&#946;1-40 involving Histidine, Glutamine, Methionine, and Aspartic acid.

Here, we present a systematic method describing the quasi-reversible self-assembly of the amyloid beta 1-40 (A&#946;1-40)-coated 20 nm gold aggregates. The nano-size-dependent quasi-reversible networking between peptides was correlated with specific amino acids or sections of the A&#946;1-40 monomer.

The characterization of A&#946;1-40 coated over the nano-gold colloidal particle surfaces was conducted using surface plasmon resonance (SPR) spectroscopy ...

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson&#39;s disease synucleinopathy and nigrostriatal degeneration. The standardization of &#945;-syn PFF generation and in vivo application is critical in order to ensure consistent, robust &#945;-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric &#945;-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of &#945;-syn PFFs into rats or mice. Starting with monomeric &#945;-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed &#945;-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated &#945;-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.&#160;

The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model ...

Detecting Amyloid-&#946; Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies on accumulation of amyloid-&#946; (A&#946;) in the brain. A&#946; is produced from the proteolytic cleavage of the beta-amyloid precursor protein (APP) by &#946;-secretase and &#947;-secretase. In pathological circumstances, the increased &#946;-cleavage of APP leads to overproduction of A&#946;, which aggregates into A&#946; plaques. Since A&#946; plaques are a characteristic of AD pathology, detecting the amount of A&#946; is very important in AD research. In this protocol, we introduce the immunofluorescent staining method to visualize A&#946; deposition. The mouse model used in our experiments is 5&#215;FAD, which carries five mutations found in human familial AD. The neuropathological and behavioral deficits of 5xFAD mice are well-documented, which makes it a good animal model to study A&#946; pathology. We will introduce the procedure including transcardial perfusion, cryosectioning, immunofluorescent staining and quantification to detect A&#946; accumulation in 5&#215;FAD mice. With this protocol, researchers can investigate A&#946; pathology in an AD mouse model.

In the neuropathology of Alzheimer's disease, one of the most crucial characteristics is the deposition of amyloid-&#946;. In this protocol, we describe the method of immunofluorescent staining in 5&#215;FAD transgenic mouse to detect amyloid-&#946; accumulation in plaques. The process of perfusion, cryosectioning, staining and quantification will be described in detail.

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies ...

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.

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 ...

Rapid Generation of Amyloid from Native Proteins In vitro

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A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

A11-positive β-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

Intracerebroventricular Injection of Amyloid-β Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Modeling Amyloid-β42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Characterization of pH-Dependent Reversible Self-Assembly of Amyloid Beta 1-40-Coated Gold Colloids

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Detecting Amyloid-β Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

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