Misfolding of Lysosomal α-Galactosidase a in a Fly Model and Its Alleviation by the Pharmacological Chaperone Migalastat

Abstract

Fabry disease, an X-linked recessive lysosomal disease, results from mutations in the GLA gene encoding lysosomal α-galactosidase A (α-Gal A). Due to these mutations, there is accumulation of globotriaosylceramide (GL-3) in plasma and in a wide range of cells throughout the body. Like other lysosomal enzymes, α-Gal A is synthesized on endoplasmic reticulum (ER) bound polyribosomes, and upon entry into the ER it undergoes glycosylation and folding. It was previously suggested that α-Gal A variants are recognized as misfolded in the ER and undergo ER-associated degradation (ERAD). In the present study, we used Drosophila melanogaster to model misfolding of α-Gal A mutants. We did so by creating transgenic flies expressing mutant α-Gal A variants and assessing development of ER stress, activation of the ER stress response and their relief with a known α-Gal A chaperone, migalastat. Our results showed that the A156V and the A285D α-Gal A mutants underwent ER retention, which led to activation of unfolded protein response (UPR) and ERAD. UPR could be alleviated by migalastat. When expressed in the fly’s dopaminergic cells, misfolding of α-Gal A and UPR activation led to death of these cells and to a shorter life span, which could be improved, in a mutation-dependent manner, by migalastat.

The Uncovered Function of the Drosophila GBA1a-Encoded Protein

“…The results did not reveal any lysosomal function for GBA1a -encoded protein; however, we tested whether its mutant form leads to activation of UPR and of ERAD, like the fly GBA1b m ortholog [ 5 ] and the human GBA1 -encoded GCase [ 22 , 23 , 24 ]. With no available anti GBA1a -encoded protein antibodies and with difficulties to grow flies in the presence of a proteasome inhibitor, we tested ERAD of GBA1a -encoded protein in transfected HEK293T cells expressing the normal or the mutant variants of GBA1a , treated with the proteasome inhibitor MG132 ( Figure 3 A,B).…”

AGAL misprocessing-induced ER stress and the unfolded protein response: lysosomal storage-independent mechanism of Fabry disease pathogenesis?

“…For many missense AGAL variants associated with the classic FD, enzyme deficiency results from misfolding and endoplasmic reticulum associated protein degradation (ERAD) [28]. In amenable variants, pharmacological chaperones allow correct folding, stabilization and trafficking of mutated AGAL proteins through the secretory pathway to the lysosomes to catabolize accumulated substrates [4, 29, 30].…”

Exploiting the Potential of Drosophila Models in Lysosomal Storage Disorders: Pathological Mechanisms and Drug Discovery

“…Patients often present vascular remodeling with an increased thickness of the carotid intima-media, due to the huge proliferation of smooth muscle cells [ 131 , 132 ]. Recently, a new Drosophila model for Fabry disease was generated by integrating wild type and mutant α-Gal A gene under the control of UAS/GAL4 system in the fly genome [ 133 ]. Adult flies expressing mutated α-Gal A display significant locomotor dysfunction and shortened lifespan, compared to flies expressing the wild type form.…”

“…Moreover, selective expression of mutated variants in dopaminergic cells, lead to cell death. Treatment of flies from first day of eclosion for 22 days with migalastat, a molecular chaperone, rescues lifespan, locomotor defects and dopaminergic cells deaths, but fails in ameliorating UPR parameters [ 133 ].…”