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

Abstract

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 SCA13 R420H 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.

Infant and adult SCA13 mutations differentially affect Purkinje cell excitability, maturation, and viability in vivo

“…As discussed below, the differential effects of aR3H and iR4H on Purkinje cell excitability strongly suggest that significant overexpression was not a confounding factor in our experiments. This likely accounts for the differences between our results and those of Namikawa et al, although we cannot rule out other possibilities, such as expression in different subsets of Purkinje cells due to the use of different drivers (Namikawa et al, 2019).…”

“…Cell death during this early period of cerebellar development is incompatible with the delayed onset of SCA13 in human patients carrying the aR3H mutation (Subramony et al, 2013;Waters et al, 2005;Waters et al, 2006). One possibility is that the abnormally early death of aR3H-expressing cells in that study resulted from overexpression of the mutant protein (Namikawa et al, 2019). Expression was driven by multiple copies of a Purkinje cell-specific enhancer sequence.…”

“…Strikingly, the onset of death in aR3H-expressing cells depended on expression level, as controlled by the number of copies of the enhancer element. Increasing expression of aR3H significantly accelerated the onset of Purkinje cell death (Namikawa et al, 2019). In contrast, we used the native promoter of the aldoca gene, which is specifically expressed in zebrafish Purkinje cells starting early in cerebellar development.…”

Functionally distinct Purkinje cell types show temporal precision in encoding locomotion

“…The lack of topography in conjunction with the fact that all of the Purkinje cells in zebrafish are ZebrinII positive (33) could imply that the mammalian Purkinje cell compartmentalization developed later in the evolution. We cannot rule out the possibility that the Purkinje cell diversity can also reflect the age of the neurons, although in zebrafish the vast majority of the Purkinje cells are generated early during development, within the first week (2-7 dpf), and their number remains relatively stable afterward (30). By that time, the Purkinje cells shape their dendritic morphologies and establish their connections (27).…”

Erinacine C Activates Transcription from a Consensus ETS DNA Binding Site in Astrocytic Cells in Addition to NGF Induction

“…Luciferase reporter constructs were generated with the help of the pBluescriptII-SK (Agilent Technologies, Inc., Santa Clara, CA, USA) containing recognition sites for the Tol2 transposase [ 20 ]—pBTolmini. 4 × tandem repeats of consensus sites of transcriptions factors were cloned as annealed oligonucleotides with DNA overhang for Kpn I (5′) and Sbf I (3′), respectively, and cloned into Kpn I/ Pst I-sites of a pBTolmini-E1b-Firefly luciferase SV40polyA vector.…”