Drosophila phosphatidylinositol-4 kinase fwd promotes mitochondrial fission and can suppress Pink1/parkin phenotypes

Balanced mitochondrial fission and fusion play an important role in shaping and distributing mitochondria, as well as contributing to mitochondrial homeostasis and adaptation to stress. In particular, mitochondrial fission is required to facilitate degradation of damaged or dysfunctional units via mitophagy. Two Parkinson’s disease factors, PINK1 and Parkin, are considered key mediators of damage-induced mitophagy, and promoting mitochondrial fission is sufficient to suppress the pathological phenotypes in Drosophila Pink1/parkin mutants. We sought additional factors that impinge on mitochondrial dynamics and which may also suppress Pink1/parkin phenotypes. We found that the Drosophila phosphatidylinositol 4-kinase IIIβ homologue, Four wheel drive (Fwd), promotes mitochondrial fission downstream of the pro-fission factor Drp1. Previously described only as male sterile, we identified several new phenotypes in fwd mutants, including locomotor deficits and shortened lifespan, which are accompanied by mitochondrial dysfunction. Finally, we found that fwd overexpression can suppress locomotor deficits and mitochondrial disruption in Pink1/parkin mutants, consistent with its function in promoting mitochondrial fission. Together these results shed light on the complex mechanisms of mitochondrial fission and further underscore the potential of modulating mitochondrial fission/fusion dynamics in the context of neurodegeneration.


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Mitochondria are dynamic organelles that are transported to the extremities of the cell and frequently 53 undergo fusion and fission events, which influences their size, branching and degradation. Many of 54 the core components of the mitochondrial fission and fusion machineries have been well 55 characterised, these include the pro-fusion factors Mfn1/2 and Opa1, and pro-fission factors Drp1 56 and Mff (1). Maintaining an appropriate balance of fission and fusion, as well as transport dynamics, 57 is crucial for cellular health and survival as mutations in many of the core components cause severe 58 neurological conditions in humans and model organisms (2). 59 The mitochondrial fission/fusion cycle has been linked to the selective removal of damaged 60 mitochondria through the process of autophagy (termed mitophagy), in which defective mitochondria 61 are engulfed into autophagosomes and degraded by lysosomes (3, 4). Two genes that have been 62 firmly linked to the mitophagy process are PINK1 and PRKN (5-7). Mutations in these genes cause To identify genes involved in mitochondrial quality control and homeostasis, we previously 73 performed an RNAi screen in Drosophila S2 cells to identify kinases and phosphatases that 74 phenocopy or suppress hyperfused mitochondria caused by loss of Pink1 (24). We identified the 75 phosphatidylinositol 4-kinase IIIβ homologue, four wheel drive (fwd), whose knockdown 76 phenocopied Pink1 RNAi, resulting in excess mitochondrial fusion. Drosophila mutant for fwd have 77 been reported to be viable but male sterile due to incomplete cytokinesis during spermatogenesis 78 (25-28); however, no other organismal phenotypes or mitochondrial involvement have been described to date. Thus, we sought to better understand the role of Fwd in mitochondrial 80 homeostasis. 81 In this study, we have characterised fwd mutants for organismal phenotypes associated with 82 of branches) were increased upon loss of fwd ( Fig. 2D-F). These results are consistent with the 136 previous cell-based study indicating loss of fwd causes mitochondrial hyperfusion. 137 We next assessed mitochondrial function, analysing maximal respiratory capacity in intact 138 mitochondria and overall ATP levels in whole animals. Respiration measured by the oxygen 139 consumption rate in energised mitochondria, stimulated via either complex I or complex II substrates, 140 was significantly reduced in fwd mutants (Fig. 3A). However, the overall level of ATP was not 141 significantly affected (Fig. 3B). These results indicate that mitochondrial respiration is affected by 142 loss of fwd but compensatory mechanisms could still maintain normal steady-state ATP levels in the 143 organism. 144 145 fwd mutant phenotypes are suppressed by loss of fusion factors 146 The results above substantiate that loss of fwd causes excess mitochondrial fusion in vivo. We next 147 addressed whether the mitochondrial hyperfusion may contribute to the locomotor deficit. To do this 148 we combined ubiquitous expression of fwd RNAi with genetic manipulations that reduce fusion 149 (partial loss of pro-fusion factors Marf or Opa1) or promote fission (overexpression of pro-fission 150 factor Drp1), and assessed climbing behaviour. Heterozygous loss of either Marf (the fly homologue 151 of MFN1/2) or Opa1, which did not affect climbing alone, was sufficient to significantly suppress the 152 climbing deficit caused by fwd RNAi (Fig. 4A, B). However, contrary to what we expected, 153 overexpression of Drp1 was not able to ameliorate the climbing defect (Fig. 4C). 154 To better understand these results, we analysed the mitochondrial morphology in neuronal 155 cell bodies of these genotypes. As with the fwd mutant, fwd RNAi caused a significant elongation of 156 mitochondria and increased branching ( Fig. 4D-F). Consistent with the effects on climbing, 157 heterozygous loss of Marf or Opa1 reverted the increase in mitochondrial length, whereas Drp1 158 overexpression did not (Fig. 4D, E). Interestingly, the increased branching caused by loss of fwd 159 was suppressed by heterozygous loss of Marf or Drp1 overexpression, but not by heterozygous loss 160 of Opa1 (Fig. 4D, F) overexpression was sufficient to significantly suppress the climbing deficit in both mutants (Fig. 5A, 175 B). In addition, the thoracic indentations caused by degeneration of the underlying flight muscle were 176 also significantly improved (Fig. 5C). Disruption of mitochondrial integrity in the flight muscles was 177 also visibly improved when fwd was overexpressed in muscles (Fig. 5D). These results are 178 consistent with Fwd overexpression promoting mitochondrial fission and partially reverting the 179 hyperfusion caused by Pink1/parkin loss. 180

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We were intrigued by the earlier observation that heterozygous loss of Marf or Opa1 could 182 revert the aberrant mitochondrial morphology and climbing defect of fwd RNAi, but the 183 overexpression of Drp1 did not (Fig. 4). These results suggested that the activity of Drp1 might 184 require Fwd, which we sought to test further. As a paradigm for Drp1 activity, overexpression of Drp1 185 is sufficient to substantially suppress the climbing deficit and mitochondrial disruption in Pink1 and 186 parkin mutants (Fig. 6A-D), as previously reported (18). Remarkably, coincident knockdown of fwd 187 completely prevented the ability of Drp1 to rescue the Pink1/parkin mutant phenotypes ( Fig. 6A-D). 188 These results further indicates that Drp1 requires the activity of Fwd. 189

Discussion
We previously identified knockdown of fwd to induce mitochondrial hyperfusion in cultured cells, 192 similar to loss of Pink1 (24). Here we have validated that genetic loss or knockdown of fwd also 193 causes excess mitochondrial fusion in neuronal cells in vivo, leading to increased mitochondrial 194 length and branching (Fig. 2). As mitochondrial fission/fusion dynamics have been shown to be 195 important for proper mitochondrial homeostasis (2), it is not surprising that this also has an impact 196 on respiration at the organismal level (Fig. 3). Furthermore, it follows that this in turn has an impact 197 on organismal fitness and vitality (Fig. 1). While fwd mutants have previously been shown to be male 198 sterile, we describe for the first time new phenotypes associated with loss of fwd: profound locomotor 199 deficits and shortened lifespan. Interestingly, there is a stronger requirement for fwd in the nervous 200 system compared to the musculature. 201 The robust locomotor phenotype allowed us to test the genetic relationship between fwd and 202 core components of the mitochondrial fission/fusion machinery. Given the excess mitochondrial 203 fusion in fwd mutants, suppression of the organismal phenotypes by reduction of fusion factors Marf 204 and Opa1 was expected. However, it was surprising that overexpression of the fission factor Drp1 205 was unable to ameliorate organismal phenotypes or even the mitochondrial morphology (Fig. 4). 206 These results suggested that Drp1 requires Fwd to function. Consistent with this, Drp1 207 overexpression was no longer able to rescue Pink1/parkin mutant phenotypes in the absence of fwd 208 (Fig. 6). These genetic experiments strongly hint at a functional link between Drp1 and Fwd but do 209 not illuminate the molecular mechanism underpinning it. Fwd, is the Drosophila homologue of 210 phosphatidylinositol 4-kinase IIIβ [PI(4)KB], which mediates the phosphorylation of 211 phosphatidylinositol to generate phosphatidylinositol 4-phosphate [PI(4)P] (31). PI(4)P is one of the 212 most abundant phosphoinositides, which is usually concentrated in the trans-Golgi network (32); 213 thus, an obvious mechanism by which PI(4)P may influence mitochondrial dynamics is not 214 immediately apparent. However, while this manuscript was in preparation, Nagashima and 215 colleagues reported that Golgi-derived PI(4)P-containing vesicles were required for the final stages 216 of mitochondrial fission (33). In that study, the authors found that loss of PI(4)KIIIβ led to a 217 hyperfused and branched mitochondrial network, consistent with what we observed here (Fig. 2). 218 Moreover, they described that while Drp1 was still recruited, it was unable to fully execute the scission event, although the reason is unclear, leading to extended mitochondrial constriction sites. 220 Our genetic evidence that the action of Drp1 requires Fwd is consistent with these findings, and 221 provide an in vivo validation of Nagashima and colleagues' results. Further, it is interesting to note 222 that while the study by Nagashima et al. suggests a universal role for PI(4)P in mitochondrial fission, 223 our in vivo analysis revealed that while fwd affected mitochondrial morphology in the nervous 224 system, it appeared to have no major impact in the musculature. These tissue-specific requirements 225 were borne out in the strong locomotor deficits caused by neuronal loss of fwd but much less so by 226 knockdown in muscles. Clearly, further work is required to better understand the complexities of 227 regulated fission/fusion events in different cell contexts in vivo. 228 A key role of mitochondrial fission/fusion dynamics is in contributing to a quality control 229 mechanism of mitochondrial sorting to eliminate dysfunctional units via mitophagy (3, 4). A 230 substantial body of evidence from cellular models indicates that mammalian PINK1/Parkin act to 231 promote damage-induced mitophagy (5-7), and some in vivo evidence from Drosophila also 232 supports this (34, 35). However, the precise nature of PINK1/Parkin-mediated mitochondrial 233 turnover in vivo is debated with contradictory results emerging (36-40). Nevertheless, interventions 234 to combat the decline in mitochondrial homeostasis remain a key challenge to combatting 235 PINK1/PRKN related pathologies. One mechanism that seems to provide substantial benefit in 236 physiological contexts is through augmenting mitochondrial fission, which presumably facilitates the 237 flux of damaged mitochondrial components towards turnover (17-20). Here, we provide further 238 evidence that augmenting a pro-fission pathway is beneficial against Pink1/parkin dysfunction. As 239 phosphoinositides can be interconverted by the action of multiple enzymes that may be druggable, 240 these findings suggest another potential route towards a therapeutic intervention. were taken at a resolution of 2048x2048 pixels and they were prepared using Fiji software 295 (RRID:SCR_002285). 296

Analysis of mitochondrial morphology 298
Motoneuron cell bodies from larvae ventral nerve cord expressing CCAP-GAL4 were used to 299 analyse mitochondrial branches marked by mitoGFP. All images were processed using Fiji software 300 (RRID:SCR_002285). Z-stacks of individual neurons were cropped to a size of 232 × 232 pixels. The 301 mitoGFP signal was enhanced and smoothed using two filters: unsharp mask (radius =10.0 pixels, 302 Mask strength 0.9) and median filtering (radius =3). Then binary masks were created using "Otsu 303 method" in auto and Dark background, and 'skeletonized' from the Process and Binary menu served 304 to generate the branches. These skeletonized images were analysed using Analyse Skeleton 305 (2D/3D). Finally, Median branch length per cell was calculated using Branch Length column from 306 "Branch information" window, and the proportion of individual vs interconnected branches per cell 307 was calculated by taking the "Number of branches" column from the "Results" window. 308 309

Transmission electron-microscopy 310
Thoraces were prepared from 5-day-old adult flies and treated as previously described (8). Ultra-311 thin sections were examined using a FEI Tecnai G2 Spirit 120KV transmission electron-microscope. 312

ATP levels 326
The ATP assay was performed as described previously (24). Briefly, five male flies for each 327 genotype were homogenized in 100 µL 6 M guanidine-Tris/EDTA extraction buffer and subjected to 328 rapid freezing in liquid nitrogen. Homogenates were diluted 1/100 with the extraction buffer and 329 mixed with the luminescent solution (CellTiter-Glo Luminescent Cell Viability Assay, Promega).