Modeling Amyloid-β42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Podsumowanie

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-β42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer's disease model, followed by histological analyses and detection of aggregations.

Streszczenie

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-β42 (Aβ42) 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 Aβ42 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 Aβ42 peptides into zebrafish brain.

Wprowadzenie

AD is a chronic progressive disease characterized by the loss of neurons and synapses in the cerebral cortex^1,2,3,4,5. The classical neuropathological hallmarks of AD are the deposition of amyloid peptides and formation of the neurofibrillary tangles (NFTs)⁶. Senile plaques, also known as amyloid plaques, are composed of amyloid-β (Aβ) peptides that form β-pleated structures in the brain parenchyma⁵. The accumulation of Aβ42 in AD patients has an early and critical role in disease progression. AD triggers a cascade of events leading to synaptic dysfunction, impaired plasticity, and neuronal loss^7,8,9,10.

The adult brain of teleost zebrafish serves as an excellent model to study the regulation of stem cell plasticity^{11,12,13,14,15,16,17,18,19,20} and various diseases in the central nervous system (CNS), including AD^21,22,23,24. Owing to a vast array of available experimental methods^{19,20,25,26,27,28,29,30,31}, these studies are informative and feasible. Zebrafish can replenish the CNS^{13,15,32,33,34,35,36,37,38}, in part by using molecular programs activated after neuronal loss^{19,39,40,41,42,43,44}. Therefore, establishing a neurodegenerative disease model in zebrafish can help address novel questions regarding regenerative ability and stem cell biology in vertebrate brains.

Recently, we developed an amyloid toxicity model in adult zebrafish brain by injecting synthetic Aβ42 peptides (Table 1)³⁹. This injection caused neurodegeneration phenotypes reminiscent of human brain pathology (e.g., cell death, microglial activation, synaptic degeneration, and memory deficits), indicating that zebrafish can be used for eliciting neurodegeneration in zebrafish brain, Aβ42 peptides can be detected with immunohistochemical stainings, and molecular mechanisms of regeneration in adult zebrafish CNS can be identified³⁹. In this protocol, we demonstrate the injection of synthetic amyloid peptides into the zebrafish brain using a cerebroventricular injection (CVMI) method^27,39,45,46 to mimic amyloid deposition (Figure 1). CVMI provides a novel way of delivering the peptides, which aggregate upon injection as β-sheet structures and exert toxicity. The peptides are distributed evenly throughout the brain, targeting the ventricular area along the entire rostro-caudal axis⁴⁵. Additionally, this method allows for analyzing the morphological and molecular response of the NSPCs in adult zebrafish brain following amyloid inclusions. Such studies will provide us an insight for successful brain repair in mammals. Our method can be used to understand the necessary molecular mechanism of a successful regeneration response after AD-like symptoms to induce replenishment of lost neurons and functional recovery.

Protokół

This protocol is a standard procedure suggested by the EU guidelines (2010/63) and the European Society for Fish Models in Biology and Medicine (EuFishBioMed) in Karlsruhe Insitute of Technology (KIT). All methods described after here have been approved by the ethics commission (Landesdirektion Dresden; document number TVV-52/2015).

1. Preparation of Aβ42 Peptide

1. Synthesize peptides (see Table 1) using the standard 9-fluorenylmethoxycarbonyl (Fmoc) chemistry with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronoiumhexafluorphosphate (HBTU) as the coupling reagent on an automated solid-phase peptide synthesizer⁵². 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 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 resin⁴⁷.
3. Dissolve the required reagents for peptide synthesis in dimethlyformamide (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 of bead size 10 µm.
   1. Pre-heat the column and maintain 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 acetonitrile⁵².
   2. Monitor the chromatogram at 220 nm, collect the appropriate peaks, and analyze using the LC-MS.
8. Confirm the purity by analytical reverse phase ultra-high Pressure Liquid Chromatography (UPLC) at 220 nm by monitoring with a 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: 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 this solution.

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 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 the following: hold pressure 20 psi; eject pressure 10 psi; period value 2.5; gating value 100 ms.
   4. Load the glass capillary with the injection solution. Insert the glass capillary into the microinjection holder. Adjust the injection angle to 45°.
      NOTE: More detailed protocols can be found as described in references^27,46.
2. Place one fish into a new Petri dish filled with anaesthetization solution (as in step 3.3).
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. See references^27,45,46 for additional details.
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 to 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 min. If it takes longer, the fish must be kept in the anesthetics for a shorter time (this needs to be optimized by the experimenter).

6. Tissue Preparation and Sectioning

1. Wait for a desired period of time before sacrificing the fish.
   NOTE: This depends on the experimental question. Amyloid deposition can be seen as early as 1 day after injection.
2. Sacrifice the fish using the appropriate method depending on the ethical regulations (e.g., treat the fish with 0.1 M MESAB).
3. Cut open the skull above the optic tectum using pointed forcep on the dorsal side, and dissect the head just behind the pectoral fin using a scalpel.
4. Fix the heads using 2% paraformaldehyde (PFA) overnight at 4 °C. Use 3 mL of PFA per head in a plastic tube with a screw lid.
   CAUTION: Wear the appropriate personal protective equipment when handling PFA.
5. For cryoprotection and decalcification, wash the heads thrice in 0.1M Phosphate Buffer (pH 7.4), then transfer them into 20% sucrose/20% ethylenediaminetetraacetic (EDTA) solution and incubate overnight at 4 °C.
6. To embed the tissue into sectioning resin, freeze the heads in 7.5% gelatin/20% sucrose solution in plastic histology molds on dry ice (approximately 3-5 min to freeze a block). Store the samples at -80 °C or continue to the next step (cryosectioning).
7. Section the heads into 12 µm thick cryosections using a cryostat as described^28,42. Transfer the cryosections directly onto glass slides and store at -20 °C for long-term usage.

7. Immunohistochemical Staining and Microscopy

NOTE: Perform all incubation steps in a humidified chamber. And, all the washing steps are for 10 min each.

1. Dry the sections for 30 min at room temperature. After thawing, wash the sections twice in PBS and once in PBSTx (PBS with 0.03% Triton-X-100).
2. Incubate with the primary antibody (anti-Aβ42 antibody; 1:500 dilution in PBSTx) at 4 °C overnight.  Following day, wash once in PBS and twice in PBSTx.
3. Incubate the sections with the secondary antibody (fluorescence-coupled detection, 1:500 dilution) along with DAPI (1 μg/mL) in PBSTx for 2 h at room temperature.
4. Wash three times in PBSTx. After the final washing, mount the slides with a coverslip  using 100 μL 70% glycerol.
5. Acquire fluorescent images using a confocal microscope (for example with 20X PL APO N.A. 0.7 objective with 488 excitation range and a standard green filter; Figure 2)³⁹.

Wyniki

HPLC was used to purify the synthesized peptide and mass spectrometry has been used to characterize the purified amyloid β peptides. The HPLC column was heated to 50 °C to improve the separation of the Aβ peptides and all the fractions were collected. To identify the correctly synthesized peptide, mass spectroscopy analysis was performed for all fractions. The UPLC chromatogram shows the purity of the compound. The HPLC fraction that yielded one peak on the UPLC (i.e.

Dyskusje

The amyloid peptides can be modified to include sequence variations or various tags. For instance, a scrambled amyloid peptide can be generated, and the peptides can be labeled with fluorescent tags at the N-terminus of the peptide end or tagged with carrier peptides³⁹. Similarly, in this protocol, the carrier peptide is the cell-penetrating peptide TR because of its effectiveness to transport cargo deep into the brain tissue³⁹. Additionally, our method allows for injection...

Materiały

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	83639.320	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
Cryostat	Leica	CM1950