Modeling Neurodegenerative Spinocerebellar Ataxia Type 13 in Zebrafish Using a Purkinje Neuron Specific Tunable Coexpression System.

Purkinje cells (PCs) are primarily affected in neurodegenerative spinocerebellar ataxias (SCAs). For generating animal models for SCAs, genetic regulatory elements specifically targeting PCs are required, thereby linking pathological molecular effects with impaired function and organismic behavior. Because cerebellar anatomy and function are evolutionary conserved, zebrafish represent an excellent model to study SCAs in vivo We have isolated a 258 bp cross-species PC-specific enhancer element that can be used in a bidirectional manner for bioimaging of transgene-expressing PCs in zebrafish (both sexes) with variable copy numbers for tuning expression strength. Emerging ectopic expression at high copy numbers can be further eliminated by repurposing microRNA-mediated posttranslational mRNA regulation.Subsequently, we generated a transgenic SCA type 13 (SCA13) model, using a zebrafish-variant mimicking a human pathological SCA13R420H mutation, resulting in cell-autonomous progressive PC degeneration linked to cerebellum-driven eye-movement deficits as observed in SCA patients. This underscores that investigating PC-specific cerebellar neuropathologies in zebrafish allows for interconnecting bioimaging of disease mechanisms with behavioral analysis suitable for therapeutic compound testing.SIGNIFICANCE STATEMENT SCA13 patients carrying a KCNC3R420H allele have been shown to display mid-onset progressive cerebellar atrophy, but genetic modeling of SCA13 by expressing this pathogenic mutant in different animal models has not resulted in neuronal degeneration so far; likely because the transgene was expressed in heterologous cell types. We developed a genetic system for tunable PC-specific coexpression of several transgenes to manipulate and simultaneously monitor cerebellar PCs. We modeled a SCA13 zebrafish accessible for bioimaging to investigate disease progression, revealing robust PC degeneration, resulting in impaired eye movement. Our transgenic zebrafish mimicking both neuropathological and behavioral changes manifested in SCA-affected patients will be suitable for investigating causes of cerebellar diseases in vivo from the molecular to the behavioral level.

Phage Display and Selections on Cells.

Antibody identification by phage display on protein or peptide targets is well established and many protocols are available. But there are many targets that cannot be expressed recombinantly or, like peptides, do not reflect correct folding of the protein. Most of these targets are cell surface receptors. Here, we describe a protocol for a panning strategy on cells to obtain specific binders to cell surface receptors. A depletion step is included to prevent enrichment of antibodies that bind to unwanted targets. Each step of the protocol is explained and variations of this protocol are given.

Sequence defined antibodies improve the detection of cadherin 2 (N-cadherin) during zebrafish development.

Cadherin-2 plays a fundamental role during zebrafish CNS and heart morphogenesis. Profiling zCdh2 expression via antibody staining is essential to achieving better understanding of its role during zebrafish development. Generation of recombinant human antibodies to zCdh2 by phage display was used to identify monoclonal antibodies with reduced unspecific binding patterns when compared to available commercial antibodies. Specificity was profiled using flow cytometry of wild type, zCdh2-defective mutant and zCdh2-GFP zebrafish cells. The epitopes recognized by the novel antibodies were mapped to peptides located in the first or second extracellular domains of zCdh2. These antibodies allowed improved observations of the spatial distribution of zCdh2 from imaging of whole mount zebrafish preparations. Since the generated antibodies are sequence defined, they can always be reconstituted from the information stored in the respective computer file, securing future reproducibility of respective experiments. The results further suggest that sequence defined antibodies with specificities thoroughly controlled by flow cytometry and genetic antigen-defective mutants or knockouts can substantially reduce the risk of misleading, false-positive results in whole mount imaging and other assays, and thus can improve the scientific value of such assays.