Mitofusin-Dependent ER Stress Triggers Glial Dysfunction and Nervous System Degeneration in a Drosophila Model of Friedreich’s Ataxia

Abstract

Friedreich’s ataxia (FRDA) is the most important recessive ataxia in the Caucasian population. It is caused by a deficit of the mitochondrial protein frataxin. Despite its pivotal effect on biosynthesis of iron-sulfur clusters and mitochondrial energy production, little is known about the influence of frataxin depletion on homeostasis of the cellular mitochondrial network. We have carried out a forward genetic screen to analyze genetic interactions between genes controlling mitochondrial homeostasis and Drosophila frataxin. Our screen has identified silencing of Drosophila mitofusin (Marf) as a suppressor of FRDA phenotypes in glia. Drosophila Marf is known to play crucial roles in mitochondrial fusion, mitochondrial degradation and in the interface between mitochondria and endoplasmic reticulum (ER). Thus, we have analyzed the effects of frataxin knockdown on mitochondrial morphology, mitophagy and ER function in our fly FRDA model using different histological and molecular markers such as tetramethylrhodamine, ethyl ester (TMRE), mitochondria-targeted GFP (mitoGFP), p62, ATG8a, LAMP1, Xbp1 and BiP/GRP78. Furthermore, we have generated the first Drosophila transgenic line containing the mtRosella construct under the UAS control to study the progression of the mitophagy process in vivo. Our results indicated that frataxin-deficiency had a small impact on mitochondrial morphology but enhanced mitochondrial clearance and altered the ER stress response in Drosophila. Remarkably, we demonstrate that downregulation of Marf suppresses ER stress in frataxin-deficient cells and this is sufficient to improve locomotor dysfunction, brain degeneration and lipid dyshomeostasis in our FRDA model. In agreement, chemical reduction of ER stress by means of two different compounds was sufficient to ameliorate the effects of frataxin deficiency in three different fly FRDA models. Altogether, our results strongly suggest that the protection mediated by Marf knockdown in glia is mainly linked to its role in the mitochondrial-ER tethering and not to mitochondrial dynamics or mitochondrial degradation and that ER stress is a novel and pivotal player in the progression and etiology of FRDA. This work might define a new pathological mechanism in FRDA, linking mitochondrial dysfunction due to frataxin deficiency and mitofusin-mediated ER stress, which might be responsible for characteristic cellular features of the disease and also suggests ER stress as a therapeutic target.

Hedgehog Signaling Modulates Glial Proteostasis and Lifespan

“…The unfolded protein response (UPR) in the endoplasmic reticulum (ER) activates a signaling cascade that elevates pEIF2a levels (Halliday et al, 2017). We tested this using the pEIF2a antibody that has been used to quantify pEIF2a in Drosophila (Edenharter et al, 2018). Interestingly, we found that hh mutant adult brains exhibited a 2-fold increase in pEIF2a levels, which are strongly reduced to wild-type control levels in hh mutant adult flies expressing hsp68 in the glia (Figures 4M and 4N).…”

Disarrangement of Endoplasmic reticulum-mitochondria communication impairs Ca2+ homeostasis in FRDA

“…Mitochondrial dysfunction also affects its communication with other cellular compartments and the processes they regulate (González-Cabo & Palau, 2013). In relation to this, we and others have observed increased endoplasmic reticulum (ER) stress in different FRDA models (Bolinches-Amorós et al, 2014;Edenharter et al, 2018). The physical interaction between these compartments termed ER-mitochondria associated membranes (MAMs) (Pinton, 2018) requires specific protein networks.…”

“…Next, we decided to evaluate whether the observations obtained in our cellular model might be also relevant in a multicellular organism. We chose a Drosophila model in which frataxin silencing was targeted to glia cells because in this model, the interactions ER and mitochondria have been already shown to participate in the development and progression of the pathology (Edenharter et al, 2018). The loss of frataxin in Drosophila glia cells using the Repo-GAL4 line (Repo-G4) and a strong RNAi line (fhRNAi-1) induces 3 main defects, a locomotor dysfunction, a strong brain vacuolization and the accumulation of lipids within the fly brain (Navarro et al, 2010).…”

“…Importantly, we have also previously reported that frataxin-silenced cells show an impairment in Ca 2+ buffering, as a consequence of reduced mitochondrial Ca 2+ uptake capacity (Bolinches-Amorós et al, 2014). The involvement of MAMs in FRDA pathophysiology has been recently suggested in a Drosophila model of the disease in which downregulation of fly mitofusin (Mfn) was sufficient to counteract some frataxin-deficient phenotypes via reduction of ER stress (Edenharter et al, 2018). However, undoubtful and definitive evidences regarding the intimate link between frataxin and MAMs are still elusive.…”

Hedgehog Signalling Modulates Glial Proteostasis and Lifespan

“…The unfolded protein response (UPR) in the Endoplasmic reticulum (ER) activates a signalling cascade which elevates pEIF2a levels (Halliday et al, 2017). We tested this using the pEIF2a antibody, which has been used to quantify pEIF2a in Drosophila (Edenharter et al, 2018). Interestingly we found that hh mutant adult brains exhibited a two-fold increase in pEIF2a levels, which are strongly reduced to wild type control levels in hh mutant adult flies expressing hsp68 in the glia (Figure 4m-n).…”