Modeling Amyloid-β42 Toxicity and Neurodegeneration in Adult Zebrafish Brain
Summary
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.
Abstract
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.
Introduction
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-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 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 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) method27,39,45,46 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.
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 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, 250×10 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, 50×2.1 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 |
References
- LaFerla, F. M., Green, K. N. Animal models of Alzheimer disease. Cold Spring Harb Perspect Med. 2 (11), (2012).
- Selkoe, D. J. Alzheimer’s disease: genes, proteins, and therapy. Physiol Rev. 81 (2), 741-766 (2001).
- Serpell, L. C. Alzheimer’s amyloid fibrils: structure and assembly. Biochim Biophys Acta. 1502 (1), 16-30 (2000).
- Beyreuther, K., Masters, C. L. Alzheimer’s disease. The ins and outs of amyloid-beta. Nature. 389 (6652), 677-678 (1997).
- Glenner, G. G., Wong, C. W. Alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun. 120 (3), 885-890 (1984).
- Blennow, K., de Leon, M. J., Zetterberg, H. Alzheimer’s disease. Lancet. 368 (9533), 387-403 (2006).
- Hardy, J. The amyloid hypothesis for Alzheimer’s disease: a critical reappraisal. J Neurochem. 110 (4), 1129-1134 (2009).
- McGowan, E., et al. Abeta42 is essential for parenchymal and vascular amyloid deposition in mice. Neuron. 47 (2), 191-199 (2005).
- Hardy, J., Selkoe, D. J. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science. 297 (5580), 353-356 (2002).
- Tincer, G., Mashkaryan, V., Bhattarai, P., Kizil, C. Neural stem/progenitor cells in Alzheimer’s disease. Yale J Biol Med. 89 (1), 23-35 (2016).
- Diotel, N., et al. Effects of estradiol in adult neurogenesis and brain repair in zebrafish. Horm Behav. 63 (2), 193-207 (2013).
- Grandel, H., Brand, M. Comparative aspects of adult neural stem cell activity in vertebrates. Dev Genes Evol. 223 (1-2), 131-147 (2013).
- Kizil, C., Kaslin, J., Kroehne, V., Brand, M. Adult neurogenesis and brain regeneration in zebrafish. Dev Neurobiol. 72 (3), 429-461 (2012).
- Diotel, N., et al. Cxcr4 and Cxcl12 expression in radial glial cells of the brain of adult zebrafish. J Comp Neurol. 518 (24), 4855-4876 (2010).
- Zupanc, G. K. Adult neurogenesis and neuronal regeneration in the brain of teleost fish. J Physiol Paris. 102 (4-6), 357-373 (2008).
- Adolf, B., et al. Conserved and acquired features of adult neurogenesis in the zebrafish telencephalon. Dev Biol. 295 (1), 278-293 (2006).
- Grandel, H., Kaslin, J., Ganz, J., Wenzel, I., Brand, M. Neural stem cells and neurogenesis in the adult zebrafish brain: origin, proliferation dynamics, migration and cell fate. Dev Biol. 295 (1), 263-277 (2006).
- Kaslin, J., Ganz, J., Brand, M. Proliferation, neurogenesis and regeneration in the non-mammalian vertebrate brain. Philos Trans R Soc Lond B Biol Sci. 363 (1489), 101-122 (2008).
- Alunni, A., Bally-Cuif, L. A comparative view of regenerative neurogenesis in vertebrates. Development. 143 (5), 741-753 (2016).
- Than-Trong, E., Bally-Cuif, L. Radial glia and neural progenitors in the adult zebrafish central nervous system. Glia. 63 (8), 1406-1428 (2015).
- Santana, S., Rico, E. P., Burgos, J. S. Can zebrafish be used as animal model to study Alzheimer’s disease?. Am J Neurodegener Dis. 1 (1), 32-48 (2012).
- Newman, M., Verdile, G., Martins, R. N., Lardelli, M. Zebrafish as a tool in Alzheimer’s disease research. Biochim Biophys Acta. 1812 (3), 346-352 (2010).
- Paquet, D., et al. A zebrafish model of tauopathy allows in vivo imaging of neuronal cell death and drug evaluation. J Clin Invest. 119 (5), 1382-1395 (2009).
- Xi, Y., Noble, S., Ekker, M. Modeling neurodegeneration in zebrafish. Curr Neurol Neurosci Rep. 11 (3), 274-282 (2011).
- Barbosa, J. S., et al. Live imaging of adult neural stem cell behavior in the intact and injured zebrafish brain. Science. 348 (6236), 789-793 (2015).
- Dray, N., et al. Large-scale live imaging of adult neural stem cells in their endogenous niche. Development. 142 (20), 3592-3600 (2015).
- Kizil, C., Brand, M. Cerebroventricular microinjection (CVMI) into adult zebrafish brain is an efficient misexpression method for forebrain ventricular cells. PLoS One. 6 (11), e27395 (2011).
- Chapouton, P., Godinho, L. Neurogenesis. Methods Cell Biol. 100, 73-126 (2010).
- Chen, C. H., Durand, E., Wang, J., Zon, L. I., Poss, K. D. zebraflash transgenic lines for in vivo bioluminescence imaging of stem cells and regeneration in adult zebrafish. Development. 140 (24), 4988-4997 (2013).
- McKenna, A., et al. Whole-organism lineage tracing by combinatorial and cumulative genome editing. Science. 353 (6298), (2016).
- Mokalled, M. H., et al. Injury-induced ctgfa directs glial bridging and spinal cord regeneration in zebrafish. Science. 354 (6312), 630-634 (2016).
- Kishimoto, N., Shimizu, K., Sawamoto, K. Neuronal regeneration in a zebrafish model of adult brain injury. Dis Model Mech. 5 (2), 200-209 (2012).
- Fleisch, V. C., Fraser, B., Allison, W. T. Investigating regeneration and functional integration of CNS neurons: lessons from zebrafish genetics and other fish species. Biochim Biophys Acta. 1812 (3), 364-380 (2010).
- Chapouton, P., Jagasia, R., Bally-Cuif, L. Adult neurogenesis in non-mammalian vertebrates. Bioessays. 29 (8), 745-757 (2007).
- Becker, T., et al. Readiness of zebrafish brain neurons to regenerate a spinal axon correlates with differential expression of specific cell recognition molecules. J Neurosci. 18 (15), 5789-5803 (1998).
- Rothenaigner, I., et al. Clonal analysis by distinct viral vectors identifies bona fide neural stem cells in the adult zebrafish telencephalon and characterizes their division properties and fate. Development. 138 (8), 1459-1469 (2011).
- Marz, M., Schmidt, R., Rastegar, S., Strahle, U. Regenerative response following stab injury in the adult zebrafish telencephalon. Dev Dyn. 240 (9), 2221-2231 (2012).
- Kroehne, V., Freudenreich, D., Hans, S., Kaslin, J., Brand, M. Regeneration of the adult zebrafish brain from neurogenic radial glia-type progenitors. Development. 138 (22), 4831-4841 (2011).
- Bhattarai, P., et al. IL4/STAT6 signaling activates neural stem cell proliferation and neurogenesis upon Amyloid-β42 aggregation in adult zebrafish brain. Cell Reports. 17 (4), 941-948 (2016).
- Cosacak, M. I., Papadimitriou, C., Kizil, C. Regeneration, Plasticity, and Induced Molecular Programs in Adult Zebrafish Brain. Biomed Res Int. , (2015).
- Kizil, C., et al. The chemokine receptor cxcr5 regulates the regenerative neurogenesis response in the adult zebrafish brain. Neural Dev. 7, 27 (2012).
- Kizil, C., et al. Regenerative neurogenesis from neural progenitor cells requires injury-induced expression of Gata3. Dev Cell. 23 (6), 1230-1237 (2012).
- Kyritsis, N., et al. Acute inflammation initiates the regenerative response in the adult zebrafish brain. Science. 338 (6112), 1353-1356 (2012).
- Katz, S., et al. . Cell Rep. 17 (5), 1383-1398 (2016).
- Kizil, C., et al. Efficient cargo delivery using a short cell-penetrating peptide in vertebrate brains. PLoS One. 10 (4), e0124073 (2015).
- Kizil, C., Iltzsche, A., Kaslin, J., Brand, M. Micromanipulation of gene expression in the adult zebrafish brain using cerebroventricular microinjection of morpholino oligonucleotides. J Vis Exp. (75), e50415 (2013).
- Sewald, N., Jakubke, H. . Peptides: Chemistry and Biology. , (2009).
- Beyer, I., et al. Solid-Phase Synthesis and Characterization of N-Terminally Elongated Abeta-3-x -Peptides. 化学. 22 (25), 8685-8693 (2016).
- Zheng, Y., et al. Kinesin-1 inhibits the aggregation of amyloid-beta peptide as detected by fluorescence cross-correlation spectroscopy. FEBS Lett. 590 (7), 1028-1037 (2016).
- Balducci, C., Forloni, G. In Vivo Application of Beta Amyloid Oligomers: a Simple Tool to Evaluate Mechanisms of Action and New Therapeutic Approaches. Curr Pharm Des. 20 (15), 2491-2505 (2013).
- Schiffer, N. W., et al. Identification of anti-prion compounds as efficient inhibitors of polyglutamine protein aggregation in a zebrafish model. J Biol Chem. 282 (12), 9195-9203 (2007).
- Wieduwild, R., Tsurkan, M., Chwalek, K., Murawala, P., Nowak, M., Freudenberg, U., Neinhuis, C., Werner, C., Zhang, Y. Minimal peptide motif for non-covalent peptide-heparin hydrogels. Journal of the American Chemical Society. 135 (8), 2919-2922 (2013).
