DnaJ-1 and karyopherin α3 suppress degeneration in a newDrosophilamodel of Spinocerebellar Ataxia Type 6

Abstract

Spinocerebellar ataxia type 6 (SCA6) belongs to the family of CAG/polyglutamine (polyQ)-dependent neurodegenerative disorders. SCA6 is caused by abnormal expansion in a CAG trinucleotide repeat within exon 47 of CACNA1A, a bicistronic gene that encodes α1A, a P/Q-type calcium channel subunit and a C-terminal protein, termed α1ACT. Expansion of the CAG/polyQ region of CACNA1A occurs within α1ACT and leads to ataxia. There are few animal models of SCA6. Here, we describe the generation and characterization of the first Drosophila melanogaster models of SCA6, which express the entire human α1ACT protein with a normal or expanded polyQ. The polyQ-expanded version of α1ACT recapitulates the progressively degenerative nature of SCA6 when expressed in various fly tissues and the presence of densely staining aggregates. Additional studies identify the co-chaperone DnaJ-1 as a potential therapeutic target for SCA6. Expression of DnaJ-1 potently suppresses α1ACT-dependent degeneration and lethality, concomitant with decreased aggregation and reduced nuclear localization of the pathogenic protein. Mutating the nuclear importer karyopherin α3 also leads to reduced toxicity from pathogenic α1ACT. Little is known about the steps leading to degeneration in SCA6 and the means to protect neurons in this disease are lacking. Invertebrate animal models of SCA6 can expand our understanding of molecular sequelae related to degeneration in this disorder and lead to the rapid identification of cellular components that can be targeted to treat it.

Inhibition of alpha-synuclein aggregation by multifunctional dopamine agonists assessed by a novel in vitro assay and an in vivo Drosophila synucleinopathy model

“…As eye cells degenerate, GFP fluorescence is lost and can be quantified when imaged through a fluorescent microscope (Figs 7 and 8). The GFP reporter faithfully tracks internal eye degeneration and the technique can be used to identify modifiers of various neurotoxic proteins4849.…”

“…Dissected heads were imaged using an Olympus BX53 microscope equipped with a DP72 digital camera, and fluorescence from fly eyes was quantified as previously described4849. All flies in each experimental setup were examined and imaged using the same conditions.…”

USP5 Is Dispensable for Monoubiquitin Maintenance in Drosophila

“…SDS-PAGE, Western Blotting, and Quantification-Larvae, pupae, or flies, as indicated in figures and legends, were homogenized in boiling SDS lysis buffer (50 mM Tris, pH 6.8, 2% SDS, 10% glycerol, and 100 mM DTT), sonicated, boiled for 10 min, centrifuged for 10 min at 13,000 ϫ g at room temperature, loaded onto SDS-PAGE gels, electrophoresed at 160 -170 V, and transferred onto PVDF membranes (Bio-Rad) for Western blotting, as previously described (19,(22)(23)(24)(25). 10 larvae, 5 pupae, or 5 adults were collected per group in 15 l of lysis buffer per larva, 20 l of lysis buffer per pupa, or 30 l of buffer per adult.…”

A novel iron (II) preferring dopamine agonist chelator D-607 significantly suppresses α-syn- and MPTP-induced toxicities in vivo

“…At specific time points, fly heads were dissected for fluorescence imaging with an Olympus BX53 microscope equipped with a DP72 digital camera; fluorescence from each eye was quantified using the publicly available ImageJ software. Average retinal fluorescence for each treatment condition were calculated as previously described (Burr et al, 2014; Tsou et al, 2015a; Yedlapudi et al, 2016). …”