Establishing an Alzheimer's Disease Model in Zebrafish Brain Through Amyloid Peptide Injection

Abstract

Bhattarai, P., et al. Modeling Amyloid-β42 Toxicity and Neurodegeneration in Adult Zebrafish Brain. J. Vis. Exp. (2017).

This video demonstrates the procedure of injecting monomeric amyloid peptides into the forebrain of adult zebrafish to generate amyloid toxicity, mimicking the neuronal damage seen in Alzheimer's disease

Protocol

1. Preparation of Aβ42 Peptide

1. Synthesize peptides (see Table 1) using the standard 9-fluorenyl methoxycarbonyl (Fmoc) chemistry with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronoiumhexafluorphosphate (HBTU) as the coupling reagent on an automated solid-phase peptide synthesizer. The scale of the synthesis was 100 µmol.
2. Load the automated peptide synthesizer with 500 mg of the Fmoc-protected resin as the solid phase (loading capacity of 0.2 mmol/g). Load the dissolved Fmoc-protected amino acids at a concentration of 0.5 M in the volume required and as calculated for the respective synthesizer.
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Results

Table 1: Aβ42 and TR-Aβ42 peptide sequences.

Peptide	Peptide Sequence	MW (g/mol)
Aβ42	DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA	4514.1
TR-Aβ42	GWTLNSAGYLLGKINLKALAALAKKILDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLM.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fmoc-protected amino acids	IRIS Biotech GmbH (Marktredwitz, Germany)		Fmoc-based amino acids for solid phase peptide synthesis (SPPS)
N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU)	IRIS Biotech GmbH (Marktredwitz, Germany)	RL-1030	Activator
Oxyma	IRIS Biotech GmbH (Marktredwitz, Germany)	RL-1180	Racemization supressor
N,N-Diisopropylethylamine	IRIS Biotech GmbH (Marktredwitz, Germany)	SOL-003	Base
Dimethylformamide	IRIS Biotech GmbH (Marktredwitz, Germany)	SOL-004	Solvent
N-Methylmorpholine	Thermo Fisher (Kandel) GmbH, Germany	A12158	Base
1-Hydroxybenzotriazole hydrate (HOBT)	Sigma-Aldrich Co. LLC. (St. Louis, MO, USA)	157260 ALDRICH	Activator
Piperidine	MERCK KGaA (Darmstadt, Germany)	822299	Fmoc deprotection reagent
Dichlormethane (DCM)	MERCK KGaA (Darmstadt, Germany)	106050	Solvent
Formic acid (FA)	MERCK KGaA (Darmstadt, Germany)	100264	Buffer component for HPLC
Trifluoroacetic acid (TFA)	MERCK KGaA (Darmstadt, Germany)	808260	Clevage Mixture reagent
Triisopropylsilane(TIS)	MERCK KGaA (Darmstadt, Germany)	233781 ALDRICH	Clevage Mixture reagent
Acetonitrile (for UPLC/LCMS)	Sigma-Aldrich Laborchemikalien GmbH	34967-1L	Solvent
Acetonitrile (for HPLC)	VWR International Ltd, England	83639.32	Solvent
Diethylether	VWR International Ltd, England	23811.326	Solvent for peptide precipitation
Dithiotritol (DTT)	VWR International Ltd, England	0281-25G	Clevage Mixture reagent
TentaGel S RAM Fmoc rink amide resin	Rapp Polymere GmbH (Tuebingen, Germany)	S30023	Solid phase for SPPS
Peptide synthesis 5 ml syringes with included filters	Intavis AG (Cologne, Germany)	34.274	Reaction tube for SPPS and for clevage from the Solid Phase
Polytetrafluoroethylene (PTFE) filter	Sartorius Stedtim (Aubagne, France)	11806-50-N	Filteration of precipitated peptides
Polyvinylidenefluoride (PVDF) syringe filter	Carl Roth GmbH + Co. KG Karlsruhe	KC78.1	Pre-filteration for HPLC
Peptide Synthesizer	Intavis, Cologne, Germany	ResPep SL	Automated solid-phase peptide synthesizer
Water Alliance HPLC	Waters, Milford Massachusetts, USA	Waters 2998, Waters e2695	Semi-preparative reverse-phase high pressure liquid chromatography (HPLC)
PolymerX, bead size 10μm, 250x10 mm	Phenomenex Ltd. Germany	00G-4328-N0	Porous polystyrene divinylbenzene HPLC column
Milli-Q Advantage A10, with a Milli-Q filter	EMD Millipore Corporation, Billerica, MA, USA	LCPAK0001	Water purification system
Filtration Unit	Sartorius Stedtim (Aubagne, France)	16307	Filtration unit for peptide precipitation
UPLC Aquity with UV Detector	Waters, Milford Massachusetts, USA	M09UPA 664M	Analytical reverse phase ultra HPLC for LC-MS
ACQUITY UPLC BEH C18, bead size 1.7 μm, 50x2.1 mm	Waters, Milford Massachusetts, USA	186002350	Analytical C18 column
ACQUITY TQ Detector	Waters, Milford Massachusetts, USA	QBB908	Electrospray ionization mass spectrometry (ESI-MS)
CHRIST ALPHA 2-4 LD plus + vacuubrand RZ6	Martin Christ Gefriertrocknungsanlagen GmbH, Germany	16706, 101542	Lyophilizer with vaccum pump
Paradigm plate reader	Beckman Coulter
MESAB (ethyl-m-aminobenzoate methanesulphonate)	Sigma-Aldrich	A5040
Petri dishes	Sarstedt	821.472
Phosphate-buffered saline	Life Technologies, GIBCO	10010-056
Needle	Becton-Dickinson	305178
Dissecting microscope	Olympus, Leica, Zeiss	Varies with the manufacturer
Dumont Tweezers	World Precision Instruments	501985
Gillies Dissecting Forceps	World Precision Instruments	501265
Glass injection capillaries	World Precision Instruments	TWF10
PicoNozzle	World Precision Instruments	5430-12
Pneumatic PicoPump	World Precision Instruments	SYS-PV820
Ring illuminator; Ring Light Guide	Parkland Scientific	ILL-RLG