Modelling of BCS1L-related human mitochondrial disease in Drosophila melanogaster

Abstract

Mutations in BCS1L are the most frequent cause of human mitochondrial disease linked to complex III deficiency. Different forms of BCS1L-related diseases and more than 20 pathogenic alleles have been reported to date. Clinical symptoms are highly heterogenous, and multisystem involvement is often present, with liver and brain being the most frequently affected organs. BCS1L encodes a mitochondrial AAA + -family member with essential roles in the latest steps in the biogenesis of mitochondrial respiratory chain complex III. Since Bcs1 has been investigated mostly in yeast and mammals, its function in invertebrates remains largely unknown. Here, we describe the phenotypical, biochemical and metabolic consequences of Bcs1 genetic manipulation in Drosophila melanogaster. Our data demonstrate the fundamental role of Bcs1 in complex III biogenesis in invertebrates and provide novel, reliable models for BCS1L-related human mitochondrial diseases. These models recapitulate several features of the human disorders, collectively pointing to a crucial role of Bcs1 and, in turn, of complex III, in development, organismal fitness and physiology of several tissues.

Measurement of mitochondrial respiratory chain enzymatic activities in Drosophila melanogaster samples

“… (B) Complex II activity recordings of wild-type mitochondria in absence (Mito) or presence of the inhibitor (Mito + Malonate). (C) Complex III activity recording of wild-type mitochondria in absence (Mito) or presence of the inhibitor (Mito + Antimycin A) plus recording of mitochondria from complex III assembly factor Bcs1 gene knockdown D. melanogaster individuals (Mito CIII KD), ( Brischigliaro et al., 2021 ). (D) Complex IV activity recording of wild-type mitochondria in absence (Mito) or presence of the inhibitor (Mito + KCN) plus recording of mitochondria from complex IV assembly factor Coa8 gene knockdown D. melanogaster individuals (Mito CIV KD), (Brischigliaro et al., 2019).…”

“… (C) Complex III activity recording of wild-type mitochondria in absence (Mito) or presence of the inhibitor (Mito + Antimycin A) plus recording of mitochondria from complex III assembly factor Bcs1 gene knockdown D. melanogaster individuals (Mito CIII KD), ( Brischigliaro et al., 2021 ). …”

“…The MRC activities measured in this way are often normalized to the activity of the citrate synthase, a mitochondrial matrix enzyme of the Krebs cycle that is considered as a good marker of mitochondrial volume. This detailed protocol builds on previously described methods for the isolation of mitochondria from D. melanogaster tissues ( Brischigliaro et al., 2019 ; Brischigliaro et al., 2021 ) and on the procedures for measuring MRC enzymatic activities in human samples ( Bugiani et al., 2004 ; Kirby et al., 2007 ; Spinazzi et al., 2012 ; Trounce et al., 1996 ). However, extensive optimization was carried out by the authors in order to provide a detailed and step-by-step procedure easy to follow also for newcomers.…”

“…Here, we provide a verified and reliable protocol for mitochondria isolation from various D. melanogaster samples and subsequent measurement of the activity of MRC complexes I–V plus citrate synthase (CS) through UV-VIS spectrophotometry. For complete details on the use and execution of this protocol, please refer to Brischigliaro et al. (2021) .…”

“…For complete details on the use and execution of this protocol, please refer to Brischigliaro et al. (2021) .…”