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.
   NOTE: The calculations are made to enable the coupling of each Fmoc-protected amino acid twice with 5 times excess of each building block to the resin.
3. Dissolve the required reagents for peptide synthesis in dimethylformamide (DMF). For example, prepare the activator, HBTU, at a concentration of 0.48 M, 45% v/v N-Methylmorpholine (NMM) (the base), and 5% v/v Acetic Anhydride (the capping mixture) to cap the non-reacted amino groups.
4. Cleave all the peptides from the resin by continuously mixing the solid support using an agitator in a freshly prepared cleavage mixture consisting of Trifluoroacetic acid (TFA): triisopropylsilane (TIS): water: dithiothreitol (DTT) at 90 (v/v): 5 (v/v): 2.5 (v/v): 2.5 (m/v) for 4 h. Use 10 mL for the synthesis scale of 100 µmol.
5. Precipitate the cleaved product by adding the cleavage mixture to 100 mL ice-cold diethyl ether. Pass through a filtration unit containing a Polytetrafluoroethylene (PTFE) filter with a pore size of 0.45 µm and wash with 20 mL ice-cold diethyl ether.
6. Collect the filtered peptide from the filter paper and dissolve 100 mg of precipitated peptide in 5 mL distilled deionized water: acetonitrile at 1:1.
7. Purify via reverse-phase high-pressure liquid chromatography (HPLC) on a semi-preparative HPLC equipped with a porous polystyrene divinylbenzene column with a bead size of 10 µm.
   1. Pre-heat the column and maintain it at 50 °C using a column heating device. Collect all the major fractions using an automated fraction collector by applying a gradient from 5% to 100% solvent B over 25 min at 4 mL/min.
      NOTE: Solvent A is 0.1% TFA in water, and solvent B is 0.1% TFA in acetonitrile.
   2. Monitor the chromatogram at 220 nm, collect the appropriate peaks, and analyze using the liquid chromatography-mass spectrometry (LC-MS).
8. Confirm the purity by analytical reverse-phase ultra-high-pressure liquid Chromatography (UPLC) at 220 nm, monitoring with a ultraviolet (UV) Detector while passing the sample through an analytical C18 column (bead size 1.7 µm). Confirm the peptide product by mass spectrometry.
   NOTE: The UPLC is coupled to an electrospray ionization mass spectrometry (ESI-MS) and Tandem Quadrupole Detector.
9. Lyophilize the correct fractions of the desired peptide in a round-bottom flask by applying a vacuum of 0.052 mbar to a fluffy powder. Couple the vacuum pump to a freezing unit maintained at -78 °C. Store at -80 °C indefinitely.
10. Use the lyophilized peptide to prepare a stock solution of 1 mM in a mixture of acetonitrile, DMF, and analytical-grade water at 1:1:1 for the experiments. This solution can be stored for at least 6 months, as the peptides do not aggregate in it.

2. Preparation of the Injection Mixture

1. Prepare phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4) for diluting the lyophilized peptides.
2. Dissolve the peptide to a final concentration of 20 µM in PBS. Prepare this solution fresh. Mix well and store on ice until the injection. Do not exceed 30 min to prevent aggregation in the solution.

3. Anesthesia

1. Prepare the stock solution of the anesthetics, 0.1% ethyl-m-aminobenzoate methanesulphonate (MESAB), in regular fish water from the circulating system. Prepare the anaesthetization solution to a final concentration of 0.0025% (v/v).
2. Remove the desired number of fish from their tanks into a transport container with 5 L of system water.
3. Half-fill a plastic Petri dish (90 mm) with 40 mL anesthetics. Use this dish for injections.
4. Incubate the fish in the anesthetics until the opercular movement has ceased.

4. Cerebroventricular Microinjection

1. Preparation of injection apparatus
   1. Prepare the glass injection capillaries using a needle puller with the following parameters: heating cycle, 537; pulling cycle, 250; velocity, 1.5 s; time, 80 ms.
   2. Bring the pressure setting on the pressure source to 25 psi.
   3. Set the microinjector parameters to 20 psi for hold and 10 psi for eject, 2.5 for period, and 100 ms for gating.
   4. Load the glass capillary with the injection solution. Insert the glass capillary into the microinjection holder. Adjust the injection angle to 45°.
2. Place one fish into a new Petri dish filled with anaesthetization solution.
3. Hold the fish with the forceps and orient for injection.
4. Generate a slit using the tip of a 30 G needle in the skull over the optic tectum where the two lateral plates meet. Do not insert the tip into the brain tissue, this would cause bleeding and damage.
5. Insert the glass capillary into the slit.
   1. Use only the tip of the needle and do not penetrate more than 1 mm through the skull. Keep holding the fish and insert the tip of the glass capillary through the incision site.
   2. Orient the tip of the glass capillary towards the telencephalon at a 45° angle. Inject 1 µL of the solution. The liquid disperses immediately after injection.

5. Recovery

1. Place the fish back in a transport container until it recovers. Connect the container to the regularly circulating fish water to ensure optimum water quality.
   NOTE: The recovery should normally take 1 minute. If it takes longer, the fish must be kept in the anesthetics for a shorter time (the experimenter needs to optimize this).

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