Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria

Mitochondrial stress response is essential for cell survival, and damaged mitochondria are a hallmark of neurodegenerative diseases. Thus, it is fundamental to understand how mitochondria relay information within the cell. Here, by investigating mitochondrial-endosomal contact sites we made the surprising observation that the small GTPase Rab5 translocates from early endosomes to mitochondria upon oxidative stress. This process is reversible and accompanied by an increase in Rab5-positive endosomes in contact with mitochondria. Interestingly, activation of Rab5 on mitochondria depends on the Rab5-GEF ALS2/Alsin, encoded by a gene mutated in amyotrophic lateral sclerosis (ALS). Alsin-deficient human-induced pluripotent stem cell-derived spinal motor neurons are defective in relocating Rab5 to mitochondria and display increased susceptibility to oxidative stress. These findings define a novel pathway whereby Alsin catalyzes the assembly of the Rab5 endocytic machinery on mitochondria. Defects in stress-sensing by endosomes could be crucial for mitochondrial quality control during the onset of ALS.

What could be the signal that drives Rab5 recruitment? Several possible scenarios such as 244 morphological changes to mitochondria and/or release of mitochondrial-derived factor(s) may be 245 accounted for. Morphological changes such as matrix condensation or swelling of mitochondria are 246 often associated with MOMP, cytochrome c release, and subsequent activation of caspases (Gottlieb, 247 Armour et al., 2003). However, this is not a prerequisite. For example, the protonophore carbonyl 248 cyanide m-chlorophenyl hydrazone (CCCP) causes mitochondrial swelling and rounding without 249 immediate cytochrome c release nor cell death (Gao, Pu et al., 2001, Lim, Minamikawa et al., 2001. 250 On the other hand, hydrogen peroxide (H2O2) was reported to induce mitochondrial rounding 251 associated with cytochrome c release and caspase activation (Takeyama, Miki et al., 2002). To address 252 this question, we tested CCCP and H2O2 on the effect of Rab5 localization. In DMSO control cells, 253 mitochondria were mostly tubular ( Figure 5A,B, top panels). The exposure of cells to either CCCP or 254 H2O2 for 2 h resulted in mitochondrial rounding, similar to the effect with laser-treatment ( Figure  255 5A,B, bottom panels). Interestingly, Rab5 enrichment on mitochondria was only observed in H2O2-256 treated cells and not in CCCP-treated cells ( Figure 5A,B, arrowheads). The enrichment of Rab5 to 257 mitochondria in H2O2 condition was ~4-fold higher compared to control cells, as revealed by co-258 localization analysis ( Figure 5C,D). 259 To corroborate these morphological observations with an independent method, we also tested 260 the effect of H2O2 on Rab5 association with endosomes and mitochondria by subcellular fractionation.
We isolated cytosolic and mitochondrial fractions via differential centrifugation and assessed the purity 262 by Western blot analysis using TOM20 as mitochondrial marker and EEA1 as endosomal marker. 263 Consistent with the observation that Rab5 is translocated to stressed mitochondria, we found that cells 264 challenged with H2O2 showed a strong increase in the amount of Rab5 present in the mitochondrial 265 fraction ( Figure 5E). 266 We then asked whether the observed difference between CCCP and H2O2 could be related to In the course of H2O2 treatment by live-cell imaging, we found that mitochondria appeared to 283 respond with different kinetics within an individual cell (Video 6). At the 60-min time point, distinct regions of the mitochondrial network were more prone to rounding and membrane permeabilization 285 than others, as revealed by the differential loss of MitoTracker-Red signal when compared to control at 286 0 min ( Figure 6A, inset image). Interestingly, these regions correlated exclusively with Rab5 ring-like 287 recruitment ( Figure 6A, inset image, arrowheads). This suggests that Rab5 may be involved in either 288 facilitating or preventing the apoptotic process. To address this, we over-expressed either GFP or GFP-289 Rab5 in HeLa cells and measured the amount of cytosolic cytochrome c at different time points after 290 H2O2 addition. We found a significant delay in cytochrome c release from mitochondria in GFP-Rab5 291 transfected cells compared to control cells ( Figure 6B,C). These results suggest that Rab5 plays a 292 protective role in mitochondrial-induced apoptosis by down-regulating the release of pro-apoptotic 293 factors into cytosol. 294 Given the key role of Rab5 in the biogenesis of the endosomal system (Zeigerer, Gilleron et al.,295 2012), the dramatic translocation of Rab5 to mitochondria upon oxidative stress by H2O2 led us to ask 296 whether endocytic trafficking is affected. To address this, we stimulated HeLa cells with Alexa-647 297 Tfn continuously for 5 and 10 min. In the absence of H2O2, transferrin was present in endosomes 298 throughout the cells and at 10 min accumulated in perinuclear recycling endosomes (Maxfield &  299 McGraw, 2004) ( Figure 6D, top). In contrast, cells treated with 250 µM H2O2 showed a severe block in 300 Tfn accumulation at both 5 min and 10 min ( Figure 6D,E). This suggests that endosomal trafficking is 301 inhibited during mitochondrial stress, consistent with the reduction of Rab5 on endosomes and its re-302 location to mitochondria. 303 304

Rab5 enrichment on the OMM is accompanied by specific effector recruitment 305
Since Rab5 translocates from early endosomes to mitochondria, with consequent reduction in 306 endocytic uptake, we next asked whether endosomal Rab5 effectors are also recruited onto 307 mitochondria. We were able to systematically assess the localization of various endosomal effectors 308 such as Rabenosyn-5/ZFYVE20, EEA1 and APPL1/2 in BAC GFP-Rab5 HeLa cells labeled with 309 MitoTracker-Red CMXRos via immunostaining by pair-wise combinations. We aimed at detecting the 310 endogenous rather than the tagged proteins as the latter often cause perturbations and do nor 311 recapitulate the native protein function (Kalaidzidis JCB 2016). The specific antibodies were first 312 tested in untreated control cells, which all showed significant levels of co-localization with GFP-Rab5 313 The weak recruitment of Rabex-5 led us to hypothesize that another GEF might be principally 346 involved. We turned our attention to Alsin as a potential candidate GEF for Rab5 on mitochondria 347 based on several lines of evidence. Alsin is the gene product of ALS2, which is mutated in multiple iPSC-MNs was less tubular and consist of more numerous and smaller rounded mitochondria than 393 those in HeLa cells ( Figure 8B, Ctrl). We next wanted to verify whether iPSC-MNs would exhibit the 394 same mitochondrial response to oxidative stress as observed in HeLa cells. Interestingly, we found that 395 iPSC-MNs to be more susceptible to detachment and apoptosis than HeLa cells when challenged with 396 250 µM H2O2 for 2 h under the same conditions (data not shown). As a result, we optimized the H2O2 397 concentration to 100 µM for 1 hour such that no immediate cell detachment was observed during the 398 treatment. Using a lower concentration and shorter incubation time, we then examined the morphology 399 of mitochondria, translocation of endogenous Rab5, association of Rab5 with endosomes and 400 mitochondria, and release of cytochrome c into cytosol. Interestingly, we did not observe significant 401 alterations to mitochondria morphology in both WT and Alsin -/-iPSC-MNs. On the other hand, WT 402 iPSC-MNs challenged with H2O2 showed robust enrichment of Rab5 on OMM, as observed in HeLa 403 cell, but not in Alsin -/-iPSC-MNs ( Figure 8B, right panel). 404 To further corroborate these results, we also performed subcellular fractionation in iPSC-MNs. 405 In untreated control cells, endogenous Rab5 was present solely in the cytosolic fraction and 406 undetectable in the purified mitochondrial fraction ( Figure 8C). However, upon challenge with H2O2, 407 Rab5 was found to co-fractionate with the mitochondrial fraction in WT iPSC-MNs but only weakly in 408 Alsin -/-iPSC-MNs ( Figure 8C). The loss of Rab5 in mutant cells correlated with greater susceptibility 409 to H2O2-induced apoptosis, as assessed by the rapid release of cytochrome c to the cytosol within an 410 hour and further accumulation at later time points, in comparison to WT cells ( Figure 8D). 411 Collectively, our findings demonstrate that Alsin is a key regulator for recruiting Rab5 to 412 mitochondria, which together, confer cytoprotective function against oxidative stress. 413

Discussion: 415
We discovered a novel cytoprotective mechanism during oxidative stress that entails the 416 translocation of Rab5 from early endosomes to mitochondria. Interestingly, the activation of Rab5 417 requires ALS2/Alsin, that has also been implicated in early onset ALS. Our results provide an 418 unexpected mechanistic link between the endosomal system and mitochondria that could be of primary 419 importance for the understanding of the mechanism that cause ALS and other neurodegenerative 420 Our findings suggest that the endocytic system is a primary responder to mitochondria under stress. 426 We found that laser-or exogenous ROS (e.g. H2O2)-induced damage causes MOMP, mitochondrial 427 swelling, and release of cytochrome c, leading to caspase activation and apoptosis. In these conditions, 428 mitochondria appear to elicit a "cue" to the endosomal system, which results in the recruitment of 429 Alsin and subsequently Rab5 onto OMM to inhibit cytochrome c release and thus, promote cell 430 survival ( Figure 9). In the course of this study, Hammerling et al. (Hammerling, Najor et al., 2017) 431 have reported a mitochondrial clearance mechanism by which Rab5-positive early endosomes 432 sequester mitochondria via the ESCRT machinery when cells are treated with FCCP (carbonyl 433 cyanide-p-trifluoromethoxypenylhydrazone), a derivative compound of CCCP. Our mechanism 434 appears to be distinct from this and previously reported autophagic mechanisms. First, we did not 435 observe engulfment of mitochondria into Rab5-positive early endosomes but recruitment of Alsin, 436 Rab5 and Rabenosyn-5 on mitochondria as well as early endosome-mitochondria interactions in 437 response to stress. Second, we did not observe such Rab5 recruitment on mitochondria in CCCP-438 treated cells. One explanation could be attributed to different cell types and lower concentration of 439 CCCP employed in our experiments. Third, the recruitment of Rab5 to damaged mitochondria occurs 440 very rapidly, i.e. within min, well preceding any autophagic components that we analyzed in this study. 441 We found that autophagy is restricted to only a subset of small mitochondrial fragments that are LC3 + 442 whereas the majority are devoid of established autophagic markers such as Parkin, LC3 and Lamp1. 443 We cannot rule out that mitochondrial clearance mechanism may still be activated at a later time (see 444 below). We attempted to track the fate of damaged mitochondria in a localized region after laser 445 treatment, but the continuous photoirradation required to achieve high spatio-temporal resolution also 446 We postulate that the pathological condition of mitochondrial defects in Alsin KO cells is related to a 527 deficiency in Rab5 recruitment to mitochondria, thereby leading to low-levels of protection from ROS accompanying aging. In ALS patients, motor neurons accumulate likely more damaged mitochondria 529 as they age, which eventually become an overload for cells. 530 The cause for ALS is still not fully understood, but oxidative stress is considered to be a major (1:500) (Abcam, #ab19857), rabbit anti-PAX6 (1:300) (Covance, #PRB-278P), and rabbit anti-SOX2 612 (1:500) (Abcam, #ab97959). 613

Transferrin uptake 615
Cells were seeded in a 384-well plate and incubated with either complete medium or in the presence of 616 250 μM H2O2 for 2 hours at 37ºC. Cells were then pulsed with Alexa-647 Tfn (10 µg/ml) for 5 min or 617 10 min, followed by 3x PBS wash, fixed with 3.7% PFA for 15 min, and then stained with DAPI 618 (1:1000) and CellMask Blue (1:2000) (ThermoFisher). Image acquisition was performed via the 619 automated confocal imaging system, CV7000S Yogokawa. Images analysis were performed using 620 MotionTracking software. cbg.de/get/) and described previously (Gilleron, Querbes et al., 2013). The y-axis is expressed as the 700 ratio of co-localized objects (eg. A to B) to total objects found in A. Final images were assembled 701      Laser / H2O2