Name: modeling amyloid-β42 toxicity and neurodegeneration in adult zebrafish brain
Uploaded: 2017-10-25T12:30:00.000+00:00
Duration: 10 min 1 s
Description: Watch this Scientific Journal Video about Modeling Amyloid-Î²42 Toxicity and Neurodegeneration in Adult Zebrafish Brain at JoVE.com

This work was supported by DZNE and the Helmholtz Association (VH-NG-1021), CRTD, TU Dresden (FZ-111, 043_261518), and DFG (KI1524/6) (C.K.); and by the Leibniz Association (SAW-2011-IPF-2) and BMBF (BioLithoMorphie 03Z2E512) (Y.Z.). We would also like to thank Ulrike Hofmann for peptide synthesis, and to Nandini Asokan, Prayag Murawala, and Elly Tanaka for help during filming the procedure.

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 A&#946;42 Peptide
<ol>
	<li>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 &#181;mol.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#181;mol.</li>
	<li>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 &#181;m and wash with 20 mL ice-cold diethyl ether.</li>
	<li>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.</li>
	<li>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 &#181;m.
	<ol>
		<li>Pre-heat the column and maintain at 50 &#176;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.</li>
		<li>Monitor the chromatogram at 220 nm, collect the appropriate peaks, and analyze using the LC-MS.</li>
	</ol>
	</li>
	<li>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 &#181;m). Confirm the peptide product by mass spectrometry. 
	NOTE: The UPLC is coupled to an electrospray ionization mass spectrometry (ESI-MS) and Tandem Quadrupole Detector.</li>
	<li>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 &#176;C. Store at -80 &#176;C, indefinitely.</li>
	<li>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.</li>
</ol>
2. Preparation of the Injection Mixture
<ol>
	<li>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.</li>
	<li>Dissolve the peptide to a final concentration of 20 &#181;M 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.</li>
</ol>
3. Anesthesia
<ol>
	<li>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).</li>
	<li>Remove the desired number of fish from their tanks into a transport container with 5 L of system water.</li>
	<li>Half-fill a plastic Petri dish (90 mm) with 40 mL anesthetics. Use this dish for injections.</li>
	<li>Incubate the fish in the anesthetics until the opercular movement has ceased.</li>
</ol>
4. Cerebroventricular Microinjection
<ol>
	<li>Preparation of injection apparatus
	<ol>
		<li>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.</li>
		<li>Bring the pressure setting on the pressure source to 25 psi.</li>
		<li>Set the microinjector parameters to the following: hold pressure 20 psi; eject pressure 10 psi; period value 2.5; gating value 100 ms.</li>
		<li>Load the glass capillary with the injection solution. Insert the glass capillary into the microinjection holder. Adjust the injection angle to 45&#176;. 
		NOTE: More detailed protocols can be found as described in references27,46.</li>
	</ol>
	</li>
	<li>Place one fish into a new Petri dish filled with anaesthetization solution (as in step 3.3).</li>
	<li>Hold the fish with the forceps and orient for injection.</li>
	<li>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 references27,45,46 for additional details.</li>
	<li>Insert the glass capillary into the slit.
	<ol>
		<li>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.</li>
		<li>Orient the tip of the glass capillary towards the telencephalon at a 45&#176; angle. Inject 1 &#181;L of the solution. The liquid disperses immediately after injection.</li>
	</ol>
	</li>
</ol>
5. Recovery
<ol>
	<li>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).</li>
</ol>
6. Tissue Preparation and Sectioning
<ol>
	<li>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.</li>
	<li>Sacrifice the fish using the appropriate method depending on the ethical regulations (e.g., treat the fish with 0.1 M MESAB).</li>
	<li>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.</li>
	<li>Fix the heads using 2% paraformaldehyde (PFA) overnight at 4 &#176;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.</li>
	<li>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 &#176;C.</li>
	<li>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 &#176;C or continue to the next step (cryosectioning).</li>
	<li>Section the heads into 12 &#181;m thick cryosections using a cryostat as described28,42. Transfer the cryosections directly onto glass slides and store at -20 &#176;C for long-term usage.</li>
</ol>
7. Immunohistochemical Staining and Microscopy
NOTE: Perform all incubation steps in a humidified chamber. And, all the washing steps are for 10 min each.
<ol>
	<li>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).</li>
	<li>Incubate with the primary antibody (anti-A&#946;42 antibody; 1:500 dilution in PBSTx) at 4 &#176;C overnight.&#160; Following day, wash once in PBS and twice in PBSTx.</li>
	<li>Incubate the sections with the secondary antibody (fluorescence-coupled detection, 1:500 dilution) along with DAPI (1 &#956;g/mL) in PBSTx for 2 h at room temperature.</li>
	<li>Wash three times in PBSTx. After the final washing, mount the slides with a coverslip&#160; using 100 &#956;L 70% glycerol.</li>
	<li>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.</li>
</ol>

The authors have nothing to disclose

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 and analyses of various peptides that can cause toxic aggregations50,51. Therefore, our system offers a versatile method for manual injection of toxic proteins into zebrafish brain and analyzing the cellular effects of such toxicity.
Amyloid peptides aggregate quickly, and therefore, the stocks must be kept in the water-DMF-acetonitrile solution, and should be mixed with PBS only 0.5 h before the injection. If there are large aggregates in the solution, the injection solution should be prepared only 10 min before the injection. Injection efficiency is an important parameter for yielding consistent results. Please refer to our previous JoVE paper46 for performing a good and consistent injection. If no aggregation is seen after injection, the injection method must be optimized.
Our method is limited in terms of the size of the molecules to be injected. We previously showed that the plasmids or short oligomers can be taken up efficiently by the ventricular cells45,46, however, for efficient delivery into deep brain tissues, large molecules (e.g., antibodies, large proteins) cannot be used. Additionally, lipophilic molecules may not easily penetrate the deep tissues because such molecules will be sequestered by the first few layers of ventricular cells.
The generation of mouse models of neurodegeneration is time-consuming and maintenance of these models is quite expensive. Our injection method is a rapid model for toxic amyloid deposition, and neurodegeneration. Additionally, the zebrafish has a superior regenerative ability compared to mammalian models, and investigations focusing on how vertebrate brains could mount a regeneration response after neurodegeneration will surely benefit from rapid assay systems in zebrafish.
The quality of the synthesized peptide and its purity are important for the success of the experiment. To ensure this quality, synthesized peptides must be thoroughly characterized using liquid chromatography and mass spectroscopy. Additionally, circular dichroism and aggregation studies as described39 are suggested.
Injection into the fish brain is a critical step to ensure a consistent aggregation and pathological outcome. A longitudinal study that analyzes the aggregation and clearance dynamics of the amyloid peptides can be performed, if desired. We use an amyloid peptide coupled to a carrier peptide to ensure equal distribution and penetration into the brain tissue. Uncoupled amyloid peptides also give similar results but the timeline of distribution and aggregation is different39. The experimenter can choose the desired version of amyloid peptides depending on the aim.
There is great potential for use of our method in combination with other manipulation studies. CVMI of amyloid peptides can be combined with drug treatments or injection of other compounds to test the synergistic effects of various molecules in a disease condition. Ultimately, our rapid and novel model can also be used for small-scale drug-screening approaches in an easy laboratory setting.
Our manual microinjection method can be efficiently used to inject amyloid peptides into the adult zebrafish brain to mimic amyloid deposition. Amyloid depositions in the brain elicit cytotoxic effect and results in AD pathology, hence displaying an acute neurodegenerative condition. The future outlook for this method would be to utilize it in an acute degenerative model to study the neuropathology in zebrafish brain and the regenerative response thereof. Such understanding will provide an important platform to design new therapeutic strategies.

AD is a chronic progressive disease characterized by the loss of neurons and synapses in the cerebral cortex1,2,3,4,5. 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-&#946; (A&#946;) peptides that form &#946;-pleated structures in the brain parenchyma5. The accumulation of A&#946;42 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 loss7,8,9,10.
The adult brain of teleost zebrafish serves as an excellent model to study the regulation of stem cell plasticity11,12,13,14,15,16,17,18,19,20 and various diseases in the central nervous system (CNS), including AD21,22,23,24. Owing to a vast array of available experimental methods19,20,25,26,27,28,29,30,31, these studies are informative and feasible. Zebrafish can replenish the CNS13,15,32,33,34,35,36,37,38, in part by using molecular programs activated after neuronal loss19,39,40,41,42,43,44. 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 A&#946;42 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, A&#946;42 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) method27,39,45,46 to mimic amyloid deposition (Figure 1). CVMI provides a novel way of delivering the peptides, which aggregate upon injection as &#946;-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.

<table><tbody><tr><td>Fmoc-protected amino acids</td><td>IRIS Biotech GmbH (Marktredwitz, Germany)</td><td></td><td>Fmoc-based amino acids for solid phase peptide synthesis (SPPS)</td></tr><tr><td>N,N,N&amp;prime;,N&amp;prime;-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU)</td><td>IRIS Biotech GmbH (Marktredwitz, Germany)</td><td>RL-1030</td><td>Activator</td></tr><tr><td>Oxyma</td><td>IRIS Biotech GmbH (Marktredwitz, Germany)</td><td>RL-1180</td><td>Racemization supressor</td></tr><tr><td>N,N-Diisopropylethylamine</td><td>IRIS Biotech GmbH (Marktredwitz, Germany)</td><td>SOL-003</td><td>Base</td></tr><tr><td>Dimethylformamide</td><td>IRIS Biotech GmbH (Marktredwitz, Germany)</td><td>SOL-004</td><td>Solvent</td></tr><tr><td>N-Methylmorpholine</td><td>Thermo Fisher (Kandel) GmbH, Germany</td><td>A12158</td><td>Base</td></tr><tr><td>1-Hydroxybenzotriazole hydrate (HOBT)</td><td>Sigma-Aldrich Co. LLC. (St. Louis, MO, USA)</td><td>157260 ALDRICH</td><td>Activator</td></tr><tr><td>Piperidine</td><td>MERCK KGaA (Darmstadt, Germany)</td><td>822299</td><td>Fmoc deprotection reagent</td></tr><tr><td>Dichlormethane (DCM)</td><td>MERCK KGaA (Darmstadt, Germany)</td><td>106050</td><td>Solvent</td></tr><tr><td>Formic acid (FA)</td><td>MERCK KGaA (Darmstadt, Germany)</td><td>100264</td><td>Buffer component for HPLC</td></tr><tr><td>Trifluoroacetic acid (TFA)</td><td>MERCK KGaA (Darmstadt, Germany)</td><td>808260</td><td>Clevage Mixture reagent</td></tr><tr><td>Triisopropylsilane(TIS)</td><td>MERCK KGaA (Darmstadt, Germany)</td><td>233781 ALDRICH</td><td>Clevage Mixture reagent</td></tr><tr><td>Acetonitrile (for UPLC/LCMS)</td><td>Sigma-Aldrich Laborchemikalien GmbH</td><td>34967-1L</td><td>Solvent</td></tr><tr><td>Acetonitrile (for HPLC)</td><td>VWR International Ltd, England</td><td>83639.320</td><td>Solvent</td></tr><tr><td>Diethylether</td><td>VWR International Ltd, England</td><td>23811.326</td><td>Solvent for peptide precipitation</td></tr><tr><td>Dithiotritol (DTT)</td><td>VWR International Ltd, England</td><td>0281-25G</td><td>Clevage Mixture reagent</td></tr><tr><td>TentaGel S RAM Fmoc rink amide resin</td><td>Rapp Polymere GmbH (Tuebingen, Germany)</td><td>S30023</td><td>Solid phase for SPPS</td></tr><tr><td>Peptide synthesis 5 ml syringes with included filters</td><td>Intavis AG (Cologne, Germany)</td><td>34.274</td><td>Reaction tube for SPPS and for clevage from the Solid Phase</td></tr><tr><td>Polytetrafluoroethylene (PTFE) filter</td><td>Sartorius Stedtim (Aubagne, France)</td><td>11806-50-N</td><td>Filteration of precipitated peptides</td></tr><tr><td>Polyvinylidenefluoride (PVDF) syringe filter</td><td>Carl Roth GmbH + Co. KG Karlsruhe</td><td>KC78.1</td><td>Pre-filteration for HPLC</td></tr><tr><td>Peptide Synthesizer</td><td>Intavis, Cologne, Germany</td><td>ResPep SL</td><td>Automated solid-phase peptide synthesizer</td></tr><tr><td>Water Alliance HPLC</td><td>Waters, Milford Massachusetts, USA</td><td>Waters 2998, Waters e2695</td><td>Semi-preparative reverse-phase high pressure liquid chromatography (HPLC)</td></tr><tr><td>PolymerX, bead size 10&amp;mu;m, 250x10 mm</td><td>Phenomenex Ltd. Germany</td><td>00G-4328-N0</td><td>Porous polystyrene divinylbenzene HPLC column</td></tr><tr><td>Milli-Q Advantage A10, with a Milli-Q filter</td><td>EMD Millipore Corporation, Billerica, MA, USA</td><td>LCPAK0001</td><td>Water purification system</td></tr><tr><td>Filtration Unit</td><td>Sartorius Stedtim (Aubagne, France)</td><td>16307</td><td>Filtration unit for peptide precipitation</td></tr><tr><td>UPLC Aquity with UV Detector</td><td>Waters, Milford Massachusetts, USA</td><td>M09UPA 664M</td><td>Analytical reverse phase ultra HPLC for LC-MS</td></tr><tr><td>ACQUITY UPLC BEH C18, bead size 1.7 &amp;mu;m, 50x2.1 mm</td><td>Waters, Milford Massachusetts, USA</td><td>186002350</td><td>Analytical C18 column</td></tr><tr><td>ACQUITY TQ Detector</td><td>Waters, Milford Massachusetts, USA</td><td>QBB908</td><td>Electrospray ionization mass spectrometry (ESI-MS)</td></tr><tr><td>CHRIST ALPHA 2-4 LD plus + vacuubrand RZ6</td><td>Martin Christ Gefriertrocknungsanlagen GmbH, Germany</td><td>16706, 101542</td><td>Lyophilizer with vaccum pump</td></tr><tr><td>Paradigm plate reader</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>MESAB (ethyl-m-aminobenzoate methanesulphonate)</td><td>Sigma-Aldrich</td><td>A5040</td><td></td></tr><tr><td>Petri dishes</td><td>Sarstedt</td><td>821.472</td><td></td></tr><tr><td>Phosphate-buffered saline</td><td>Life Technologies, GIBCO</td><td>10010-056</td><td></td></tr><tr><td>Needle</td><td>Becton-Dickinson</td><td>305178</td><td></td></tr><tr><td>Dissecting microscope</td><td>Olympus, Leica, Zeiss</td><td>Varies with the manufacturer</td><td></td></tr><tr><td>Dumont Tweezers</td><td>World Precision Instruments</td><td>501985</td><td></td></tr><tr><td>Gillies Dissecting Forceps</td><td>World Precision Instruments</td><td>501265</td><td></td></tr><tr><td>Glass injection capillaries</td><td>World Precision Instruments</td><td>TWF10</td><td></td></tr><tr><td>PicoNozzle</td><td>World Precision Instruments</td><td>5430-12</td><td></td></tr><tr><td>Pneumatic PicoPump</td><td>World Precision Instruments</td><td>SYS-PV820</td><td></td></tr><tr><td>Ring illuminator; Ring Light Guide</td><td>Parkland Scientific</td><td>ILL-RLG</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM1950</td><td></td></tr></tbody></table>

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.

HPLC was used to purify the synthesized peptide and mass spectrometry has been used to characterize the purified amyloid &#946; peptides. The HPLC column was heated to 50 &#176;C to improve the separation of the A&#946; 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., the correct mass to charge ratio of the required amyloid &#946; peptide) was further processed for experiments (Figure 2).
The functionality of the peptides in forming aggregates can be assessed using various spectroscopic methods39,48,49, and also by incubating the peptides in PBS at room temperature for more than 1 h, which will yield aggregates. In this case, precipitates and large aggregates should be seen in solution.
After cerebroventricular microinjection, the peptides aggregate in the brain and form amyloid depositions (Figure 3). These aggregates are mostly seen as intracellular depositions, but also around the blood vessels. Such aggregations are indications of a successful accumulation of amyloid peptides in the tissue.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56014/56014fig1.jpg" /> 
Figure 1: Schematic representation of the peptide synthesis, purification, injection, and analyses. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56014/56014fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56014/56014fig2.jpg" /> 
Figure 2: Characterization of native amyloid-&#946;42 peptide. Mass spectrometry characterization of native amyloid-&#946;42 peptide (A) and the CPP-tagged amyloid-&#946;42 peptide (TR-A&#946;42; B). (C) The chromatograph of the purified, native, and TR-A&#946;42 peptide on the Ultra-high Pressure Liquid Chromatography (UPLC). X-axes denote the mass-to-charge ratio (m/z) (A, B) and the elution time in minutes (C). <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56014/56014fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56014/56014fig3.jpg" /> 
Figure 3: Immunohistochemical staining of amyloid deposition in an adult zebrafish brain section 1 day after injection. A&#946;42 is in green, and nuclei are in blue. Detection of green clusters indicates efficient A&#946;42 aggregation. Scale bars = 100 &#181;m. This figure has been modified from reference39.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Peptide</td>
			<td colspan="4">Peptide Sequence</td>
			<td>MW (g/mol)</td>
		</tr>
		<tr>
			<td>A&#946;42</td>
			<td colspan="4">DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA</td>
			<td>4514.1</td>
		</tr>
		<tr>
			<td>TR-A&#946;42</td>
			<td colspan="4">GWTLNSAGYLLGKINLKALAALAKKILDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA</td>
			<td>5919.8</td>
		</tr>
	</tbody>
</table>
Table 1: A&#946;42 and TR-A&#946;42 peptide sequences.

Watch this Scientific Journal Video about Modeling Amyloid-Î²42 Toxicity and Neurodegeneration in Adult Zebrafish Brain at JoVE.com

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

modeling-amyloid-42-toxicity-neurodegeneration-adult-zebrafish

Nanyang Technological University

Research

JoVE Journal

Neuroscience

11.1K Views.  German Centre for Neurodegenerative Diseases (DZNE) Dresden within Helmholtz Association. 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.

Video: Modeling Amyloid-β42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled Molecules

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological problems after stroke and is shown to impair neurogenic processes. Interestingly, the adult zebrafish has recently emerged as a relevant and useful model to mimic hyperglycemia/diabetes and to investigate constitutive and regenerative neurogenesis. This work provides methods to develop zebrafish models of hyperglycemia to explore the impact of hyperglycemia on brain cell proliferation under homeostatic and brain repair conditions. Acute hyperglycemia is established using the intraperitoneal injection of D-glucose (2.5 g/kg bodyweight) into adult zebrafish. Chronic hyperglycemia is induced by immersing adult zebrafish in D-glucose (111 mM) containing water for 14 days. Blood-glucose-level measurements are described for these different approaches. Methods to investigate the impact of hyperglycemia on constitutive and regenerative neurogenesis, by describing the mechanical injury of the telencephalon, dissecting the brain, paraffin embedding and sectioning with a microtome, and performing immunohistochemistry procedures, are demonstrated. Finally, the method of using zebrafish as a relevant model for studying the biodistribution of radiolabeled molecules (here,[18F]-FDG) using PET/CT is also described.

This work describes methods to establish acute and chronic hyperglycemia models in zebrafish. The aim is to investigate the impact of hyperglycemia on physiological processes, such as constitutive and injury-induced neurogenesis. The work also highlights the use of zebrafish to follow radiolabeled molecules (here, [18F]-FDG) using PET/CT.

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological ...

Developmental Biology

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae are a preeminent model for the neurobehavioral study of environmental pollutants. Here, a detailed experimental protocol is provided for the evaluation of the neurotoxicity of environmental pollutants using zebrafish larvae, including the collection of the embryos, the exposure process, neurobehavioral indicators, the test process, and data analysis. Also, the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the assay. The protocol has been used in the development of psychopathic drugs, research on environmental neurotoxic pollutants, and can be optimized to make corresponding studies or be helpful for mechanistic studies. The protocol demonstrates a clear operation process for studying neurobehavioral effects on zebrafish larvae and can reveal the effects of various neurotoxic substances or pollutants.

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

Environment

The Three-Chamber Choice Behavioral Task using Zebrafish as a Model System

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or cognitive impairment. We have modified and developed a three-chamber choice cognitive task similar to that used with rodents for use with hyperglycemic zebrafish. The testing chamber consists of a centrally located starting chamber and two choice compartments on either side, with a shoal of conspecifics used as the reward. We provide data showing that once acquired, zebrafish remember the task at least 8 weeks later. Our data indicate that zebrafish respond robustly to this reward, and we have identified cognitive deficits in hyperglycemic fish after 4 weeks of treatment. This behavioral assay may also be applicable to other studies related to cognition and memory.

We present a behavioral chamber designed to assess cognitive performance. We provide data showing that once acquired, zebrafish remember the task 8 weeks later. We also show that hyperglycemic zebrafish have altered cognitive performance, indicating that this paradigm is applicable to studies assessing cognition and memory.

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or ...

Behavior

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.

Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Biology

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research and develop alternative in vivo neuroinflammation models to understand the role of neuroinflammation in neurodegeneration. In this study, a larval zebrafish model of neuroinflammation mediated by ventricular microinjection of lipopolysaccharide (LPS) to induce an immune response and neurotoxicity was developed and validated. The transgenic zebrafish lines elavl3:mCherry, ETvmat2:GFP, and mpo:EGFP were used for real-time quantification of brain neuron viability by fluorescence live imaging integrated with fluorescence intensity analysis. The locomotor behavior of zebrafish larvae was recorded automatically using a video-tracking recorder. The content of nitric oxide (NO), and the mRNA expression levels of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1&#946; (IL-1&#946;), and human tumor necrosis factor &#945; (TNF-&#945;) were investigated to assess the LPS-induced immune response in the larval zebrafish head. At 24 h after the brain ventricular injection of LPS, loss of neurons and locomotion deficiency were observed in zebrafish larvae. In addition, LPS-induced neuroinflammation increased NO release and the mRNA expression of IL-6, IL-1&#946;, and TNF-&#945; in the head of 6 days post fertilization (dpf) zebrafish larvae, and resulted in the recruitment of neutrophils in the zebrafish brain. In this study, injection of zebrafish with LPS at a concentration of 2.5-5 mg/mL at 5 dpf was determined as the optimum condition for this pharmacological neuroinflammation assay. This protocol presents a new, quick, and efficient methodology for brain ventricle microinjection of LPS to induce LPS-mediated neuroinflammation and neurotoxicity in a zebrafish larva, which is useful for studying neuroinflammation and could also be used as a high-throughput in vivo drug screening assay.

This protocol demonstrates the microinjection of lipopolysaccharide into the brain ventricular region in a zebrafish larval model to study the resulting neuroinflammatory response and neurotoxicity.

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research ...

Behavioral Test Battery to Assess Neurotoxic Effects in Adult Zebrafish

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

Author Spotlight: Assessment of Environmental and Drug-Induced Behavioral Changes in Adult Zebrafish 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. ...

Modeling Drug-Induced Hemorrhage in Zebrafish Larva

Source: Crilly, S., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/59716">Using zebrafish larvae to study the pathological consequences of hemorrhagic stroke.</a> J. Vis. Exp. (2019)
In this video, fertilized zebrafish embryos are dechorionated under a stereo microscope and then exposed to a cholesterol synthesis inhibitor, leading to the development of blood vessel ruptures and brain hemorrhages in the larvae. Hemorrhagic larvae, identifiable by distinct red spots, are subsequently separated under a microscope for further analysis.

Modeling Amyloid-β42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

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Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled Molecules

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

The Three-Chamber Choice Behavioral Task using Zebrafish as a Model System

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

In Vivo Modeling of the Morbid Human Genome using Danio rerio

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

Modelowanie krwotoku wywołanego lekami u larw danio pręgowanego