Mitochondrial Dynamics Impacts Stem Cell Identity and Fate Decisions by Regulating a Nuclear Transcriptional Program

Mitochondrial Dynamics Impacts Stem Cell Identity and Fate Decisions by Regulating a Nuclear Transcriptional Program

Article Mitochondrial Dynamics Impacts Stem Cell Identity and Fate Decisions by Regulating a Nuclear Transcriptional Program Graphical Abstract Auth...

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Article

Mitochondrial Dynamics Impacts Stem Cell Identity and Fate Decisions by Regulating a Nuclear Transcriptional Program Graphical Abstract

Authors Mireille Khacho, Alysen Clark, Devon S. Svoboda, ..., Mary-Ellen Harper, David S. Park, Ruth S. Slack

Correspondence [email protected]

In Brief

Highlights d

Mitochondrial dynamics regulates the fate and identity of stem cells

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Mitochondrial dynamics regulates stem cell fate by modifying ROS signaling

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ROS activate developmental gene expression via an NRF2dependent retrograde pathway

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Aberrant mitochondrial dynamics impairs stem cell selfrenewal and maintenance

Khacho et al., 2016, Cell Stem Cell 19, 232–247 August 4, 2016 ª 2016 Elsevier Inc. http://dx.doi.org/10.1016/j.stem.2016.04.015

Khacho et al. report that mitochondrial dynamics regulates neural stem cell fate during development and in the adult mouse brain. Using acute loss-offunction approaches to uncouple mitochondrial bioenergetics from the fission/fusion machinery, they find that the latter independently regulates ROS levels upstream of NRF2. Thus, defects in mechanisms that maintain the pool of mitochondria also impair neural stem cell function, which has important implications for aging and neurodegenerative diseases.

Cell Stem Cell

Article Mitochondrial Dynamics Impacts Stem Cell Identity and Fate Decisions by Regulating a Nuclear Transcriptional Program Mireille Khacho,1 Alysen Clark,1 Devon S. Svoboda,1 Joelle Azzi,1 Jason G. MacLaurin,1 Cynthia Meghaizel,1 Hiromi Sesaki,2 Diane C. Lagace,1 Marc Germain,1,4 Mary-Ellen Harper,3 David S. Park,1 and Ruth S. Slack1,* 1Department

of Cellular and Molecular Medicine, University of Ottawa Brain and Mind Research Institute, Ottawa, ON K1H 8M5, Canada of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA 3Department of Biochemistry, Microbiology and Immunology, Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, ON K1H 8M5, Canada 4Present address: De ´ partement de Biologie Me´dicale, Universite´ du Que´bec Trois-Rivie`res, Trois-Rivie`res, Que´bec, QC G9A 5H7, Canada *Correspondence: [email protected] http://dx.doi.org/10.1016/j.stem.2016.04.015 2Department

SUMMARY

Regulated mechanisms of stem cell maintenance are key to preventing stem cell depletion and aging. While mitochondrial morphology plays a fundamental role in tissue development and homeostasis, its role in stem cells remains unknown. Here, we uncover that mitochondrial dynamics regulates stem cell identity, self-renewal, and fate decisions by orchestrating a transcriptional program. Manipulation of mitochondrial structure, through OPA1 or MFN1/2 deletion, impaired neural stem cell (NSC) self-renewal, with consequent age-dependent depletion, neurogenesis defects, and cognitive impairments. Gene expression profiling revealed ectopic expression of the Notch self-renewal inhibitor Botch and premature induction of transcription factors that promote differentiation. Changes in mitochondrial dynamics regulate stem cell fate decisions by driving a physiological reactive oxygen species (ROS)-mediated process, which triggers a dual program to suppress self-renewal and promote differentiation via NRF2-mediated retrograde signaling. These findings reveal mitochondrial dynamics as an upstream regulator of essential mechanisms governing stem cell selfrenewal and fate decisions through transcriptional programming.

INTRODUCTION The capacity of stem cells to self-renew is integral for the lifelong maintenance of a stem cell pool and essential for the homeostatic preservation and regenerative potential of tissues. As such, the preservation of stem cells relies on the intricate regulation of cell fate decisions, whereby an imbalance toward cell lineage commitment at the expense of self-renewal can have detrimental effects. In recent years, the decline of stem cells has been intimately linked to aging and observed in some degen232 Cell Stem Cell 19, 232–247, August 4, 2016 ª 2016 Elsevier Inc.

erative disorders (Oh et al., 2014; Signer and Morrison, 2013), but the mechanisms underlying this decline remain unclear. Mitochondria are essential organelles with an established role in organismal longevity (Bratic and Larsson, 2013; Hwang et al., 2012) given their central contribution to energy metabolism, cell death, and signaling pathways (Green and Kroemer, 2004). In particular, balanced mitochondrial dynamics and morphology, through regulated cycles of fission and fusion (Chen and Chan, 2004), have emerged as being crucial to the maintained function of these organelles and vital for tissue homeostasis. Given the importance of mitochondrial dynamics during aging and degenerative diseases (Khacho and Slack, 2015), there is surprisingly little knowledge in relation to a developmental function and whether mitochondrial structure plays a role in stem cells. For example, during aging and in many neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease, aberrant mitochondrial fission leads to progressive loss of mitochondrial function and oftentimes contributes to the pathological development of diseases (Burte´ et al., 2015; Itoh et al., 2013; Scheckhuber et al., 2007). While the neurological defects associated with impaired dynamics have been attributed to loss of mitochondrial function in post-mitotic cells (Burte´ et al., 2015), nothing is known as to how this may impact the neural stem cell pool. RESULTS Disruption of Mitochondrial Dynamics Impairs Stem Cell Self-Renewal Presently, little is known regarding the impact of mitochondrial dynamics on stem cell function. To gain a clear depiction of mitochondrial morphology within neural stem and progenitor cells during neurogenesis, we began with embryonic cortical development as a model system. At mid-neurogenesis (embryonic day 15.5 [E15.5]), distinct cell populations within the developing cortex have unique mitochondrial morphologies (Figures 1A and 1B). First, mitochondria of Sox2-expressing uncommitted cells in the ventricular zone (VZ), which contain neural stem cells (NSCs), exhibited an elongated morphology (Figures 1A and 1B). Notably, this morphology differs from the documented description of mitochondria, as fragmented and underdeveloped, in

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embryonic and hematopoietic stem cells (Simsek et al., 2010; St John et al., 2005; Xu et al., 2013; Zhang et al., 2011). Progression of cells to a committed fate was paralleled by transition of mitochondria to a fragmented state in Tbr2 committed neural progenitors in the subventricular zone (SVZ) (Figures 1A and 1B). Mitochondria then regain an elongated phenotype in Tuj1 postmitotic neurons (Figures 1A and 1B), consistent with reported transitions in mitochondrial morphology as cells undergo differentiation (Kasahara et al., 2013). The novel and striking distinction in mitochondrial morphology between Sox2 uncommitted and Tbr2 committed cells suggested a role for mitochondrial dynamics in NSCs and that changes in mitochondrial structure may regulate cell fate decisions. To investigate the role of mitochondrial dynamics in stem cells, mitochondrial structure was disrupted in uncommitted cells of the early telencephalon (E9.5–E10.5), prior to the onset of neurogenesis. This was achieved by in vivo conditional genetic deletion of the classical fusion/fission machinery, including the outer mitochondrial membrane fusion proteins MFN1/2 (Chen et al., 2003) or the dynamin-GTPase fission protein DRP1 (Smirnova et al., 2001). In these models, loss of MFN1/2 caused severe mitochondrial fragmentation in all cell populations of the developing cortex, whereas DRP1 loss promoted excessive mitochondrial elongation (Figures 1C, 1D, S1A, and S1B) (Chen and Chan, 2004). Of note, the shortened mitochondrial length in Sox2+ cells in MFN1/2 double knockouts (DKOs), was now comparable to that of Tbr2+ progenitor cells in wildtype brains (Figure S1A). Initial assessment of the Sox2+ population that contains NSCs showed no detectable effect of disrupted mitochondrial dynamics on cell number or viability (Figures S1C and S1D). Upon examination of the replicating population of uncommitted cells at E15.5 (mid-neurogenesis), there was a noticeable change in the division angle of Sox2 anaphase cells along the VZ. MFN1/2 deficiency, which results in mitochondrial fragmentation, significantly increased the number of dividing cells with a horizontal cleavage plane relative to the ventricular surface, at the expense of anaphase cells with vertical cleavage planes (Figure 1E). This bias of Sox2 anaphase cells toward horizontally oriented cleavage is consistent with a

decrease in NSC self-renewal (Siller and Doe, 2009). Interestingly, the opposite was observed following mitochondrial elongation by DRP1 loss (Figure 1E). Importantly, increased mitochondrial fragmentation by MFN1/2 loss affects the cleavage angle of Sox2+ cells as early as E12.5 (Figures 1F and 1G), confirming that the observed shift was not due to secondary effects of long-term disruption of mitochondrial structure. Since the division angle of Sox2+ cells along the VZ suggests a change in self-renewal versus committed fate decisions, we questioned whether mitochondrial structure might impact the self-renewal capacity and fate of NSCs. Stem cells possess the dual capacity to either self-renew, in order to maintain their population, or commit and differentiate into a specified cell type. To directly test whether mitochondrial dynamics plays a role in the regulation of stem cell self-renewal and fate decisions, several parameters were investigated using in vitro, in vivo, and in utero experiments. We began by assessing the consequence of disrupted mitochondrial dynamics on NSC self-renewal. This was achieved by in vitro measurement of self-renewal capacity, using neurosphere assays, following acute manipulation of MFN1/2 or DRP1 in Sox2+ uncommitted cells obtained from E11.5 embryonic cortices. Disruption of mitochondrial dynamics demonstrated that loss of MFN1/2 in uncommitted cells results in a drastic inability to form neurospheres in culture (Figure 1H), while DRP1 loss showed a modest increase in neurosphere number (Figure 1I). Furthermore, acute RNAi-mediated loss of the mitochondrial inner membrane fusion protein OPA1 (Meeusen et al., 2006) resulted in decreased primary and secondary neurospheres, which provide an estimation of stem cell self-renewal capacity (Figure 1J). In addition, analysis of the Notch-mediated self-renewal pathway revealed that disruption of mitochondrial dynamics both in vivo and in our acute culture model resulted in a concurrent alteration in the levels of cytoplasmic Notch1 and its target gene, Hes5 (Figures 1K and S1E). These data suggest that mitochondrial dynamics plays an important role in the self-renewal capacity of NSCs. Next, we hypothesized that the decrease in self-renewal of NSCs by increased mitochondrial fragmentation was due to altered cell fate decisions and that enhanced

Figure 1. Disruption of Mitochondrial Dynamics Impairs Neural Stem Cell Self-Renewal (A) Representative confocal images of mitochondrial morphology (Tom20) in Sox2 (uncommitted cells containing NSCs), Tbr2 (committed progenitors), and Tuj1 (post-mitotic neurons) cells in coronal sections of wild-type (WT) E15.5 cortex. Insets are zoomed views. (B) Quantified mitochondrial length (n = 3). (C) Representative confocal images of mitochondrial morphology in Sox2+ cells in VZ of E15.5 coronal sections (MFN1/2-DKO, MFN1/2-Foxg1Cre; DRP1-KO, DRP1-Emx1Cre). Insets are zoomed views. (D) Mitochondrial length quantification from (C) (n = 3). (E) Division angle measurements of Sox2+ anaphase cells in E15.5 coronal sections. Horizontal (0 –30 ), intermediate (30 –60 ), and vertical (60 –90 ) cleavage angles (yellow line) relative to the apical surface of the lateral vertical (white line). Chromatin is visualized by DAPI (n = 3). (F) Representative confocal images of mitochondrial morphology in Sox2+ cells in VZ of E12.5 coronal sections. (G) Division angle measurements of Sox2+ anaphase cells in E12.5 coronal sections (n = 3). (H and I) Primary neurospheres derived from cells of the dorsal cortex of E15.5 MFN1/2-NestinCreErt2 treated with vehicle or 200nM Tamoxifen (H), or DRP1-floxed infected with GFP or Cre-GFP lentiviruses (I) (n = 5). (J) Primary and secondary neurospheres from WT cells of the dorsal cortex infected with lentivirus encoding shCtr or shOPA1 (n = 6, primary assay and n = 3, secondary assay). (K) Immunoblots from whole-cell lysates of E12.5 MFN1/2-Foxg1Cre-WT and MFN1/2- Foxg1Cre-DKO dorsal cortical tissue, shCtr or shOPA1-infected cultured neurospheres, or E15.5 DRP1-Emx1Cre-WT or DRP1-Emx1Cre-KO dorsal cortical tissue. (L) Representative confocal images following 48 hr of in utero co-electroporations of shCtr or shOPA1 with GFP at E13.5 in the dorsal forebrain. Scale bar, 50 mm. (M) Quantification of GFP+ cell populations colabeled with Sox2 or DCX. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate; LV, lateral ventricle. For (A)–(M), results are presented as mean ± SD (*p < 0.05, **p < 0.01, and ***p < 0.001, Student’s t test). Scale bar, 10 mm. See also Figure S1.

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commitment decisions may occur at the expense of NSC selfrenewal. Consistent with this hypothesis, the MFN1/2 DKO cortex exhibited an enhancement in the percentage of cells transitioning to a committed progenitor state (Sox2+/Tbr+ and Tbr2+ cells) (Figure S1F). Next, to confirm that mitochondrial fragmentation induces a change in cell fate at the cellular level and that the observed phenotype was not a consequence of secondary defects due to abnormal brain development, we used in utero electroporations. Acute short hairpin RNA (shRNA)-mediated knockdown of the fusion protein OPA1 in Sox2+ uncommitted cells, in the context of a healthy wild-type developing cortex, caused a significant cell-autonomous increase in the number of GFP+ electroporated cells that had committed to a neuronal fate (GFP+/DCX+ cells) at the expense of the Sox2 population (GFP+/Sox2+ cells) after only 48 hr (Figures 1L and 1M). Together, these data implicate mitochondrial structure in stem cell self-renewal regulation and suggest that mitochondrial dynamics act upstream to direct cell fate decisions. Impairment of NSC Self-Renewal by Loss of Mitochondrial Dynamics Is Not due to Mitochondrial Dysfunction To uncover the underlying origin for the influence of aberrant mitochondrial dynamics on NSC self-renewal versus cell commitment decisions, we first investigated whether changes in NSC fate arose from impairments in mitochondrial function. Long-term dysregulation of mitochondrial dynamics has been shown to lead to mitochondrial dysfunction, due to impaired complementation of mtDNA (Chen et al., 2010). However, acute manipulation of mitochondrial dynamics, as done in our in vitro and in utero studies, caused rapid and robust alterations in NSC cell fate. Thus, we asked whether acute manipulations of mitochondrial structure, consistent with short-term term loss of mitochondrial dynamics, could impair mitochondrial function. The use of an acute model system would permit for a more direct evaluation of mitochondrial function at the onset of NSC alterations by dysregulated mitochondrial dynamics. Acute disruption of mitochondrial dynamics in E12.5 Sox2+ uncommitted cells by loss of MFN1/2, OPA1, or DRP1, which leads to changes in neurosphere number as early as 3–4 days following knockout or knockdown of the gene of interest, did not affect total cellular ATP levels (Figure 2A). In addition, no detectable impairment in

the ATP-generating function of mitochondria was observed in these cells. First, inhibition of mitochondrial ATP production using the ATP synthase inhibitor oligomycin showed no detectable difference in oligomycin-sensitive ATP levels (Figures 2B–2D). Second, MFN1/2-, OPA1-, or DRP1-deficient cells did not show any detectable dysfunction in ATP generation when forced to rely solely on mitochondrial metabolism (Figures 2E–2G). Furthermore, mitochondrial membrane potential was not significantly different between wild-type and MFN1/2-, OPA1-, or DPR1-deficient cells (Figures 2H–2K). This is in contrast to what is observed in cells treated with antimycin A (a complex III inhibitor) or carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), which impair mitochondrial function (Figures 2L and 2M) and decrease membrane potential (Figure 2N). In addition, treatment of OPA1-depleted cells with antimycin A or FCCP to induce mitochondrial dysfunction now resulted in detectable changes in ATP levels and mitochondrial ATP production (Figures 2O and 2P), thus validating the sensitivity of our assay to detect mitochondrial dysfunction if present. Together, these data demonstrate that promoting mitochondrial fragmentation in NSCs, which results in loss of NSC self-renewal capacity and an apparent push toward neuronal commitment, was not due to a cellular energy deficit or a general consequence of mitochondrial dysfunction. Loss of MFN1/2 Causes Adult NSC Depletion and Cognitive Defects The capacity of stem cells to continuously self-renew is integral for the lifelong maintenance of a stem cell pool. The data presented thus far provide evidence that aberrant mitochondrial dynamics impairs the self-renewal capacity of NSCs. As such, an ultimate hallmark of impaired self-renewal of the NSC pool would be the eventual depletion of this population. Given that disruption in mitochondrial morphology is a common theme in aging and neurodegenerative diseases in adults, we used adult neurogenesis within the hippocampal dentate gyrus (DG) as a model system to confirm an essential role for mitochondrial structure in the self-renewal and maintenance of the NSC population. To test whether induction of aberrant mitochondrial fragmentation in adult NSCs would have a major impact on stem cell maintenance and neurogenesis within the context of normal brain development, an inducible knockout of MFN1/2

Figure 2. Acute Manipulation of Mitochondrial Dynamics Does Not Impair Mitochondrial Function in NSCs and Progenitors (A) Total ATP levels after acute (4 days) deletion of MFN1/2, OPA1, or DRP1. MFN1/2-NestinCreErt2 cells were treated with 100 nM tamoxifen (n = 5), wild-type cells were infected with lentiviruses for shCtr or shOPA1 (n = 5), and DRP1-flox cells were infected with lentiviruses encoding GFP or CreGFP (n = 4). (B–D) ATP in cells subjected to the same conditions as in (A) and treated with oligomycin for 1 hr (n = 4 for MFN1/2 and DRP1; n = 5 for OPA1). (E–G) ATP in cells subjected to the same conditions as in (A) and harvested in control (with glucose) or galactose (without glucose) media followed by a 1-hr oligomycin treatment (n = 4 for MFN1/2 and DRP1; n = 5 for OPA1). (H–K) Measurements of mitochondrial membrane potential in cells subjected to the same condition as in (A) using tetramethylrhodamine ethyl ester (TMRE). Graph in (H) shows quantification relative to control (black bars) (n = 3). (I–K) Representative histograms from flow cytometry analysis of TMRE. (L) Total ATP levels following treatment with Antimycin A or FCCP in wild-type cells (n = 4). (M) ATP in wild-type cells subjected to same conditions as in (L) and harvested in control (with glucose) or galactose (without glucose) media followed by a 30-min oligomycin treatment (n = 4). (N) Measurements of mitochondrial membrane potential in cells subjected to same condition as in (L) showing quantification and representative histograms from flow cytometry analysis of TMRE. (O) Total ATP levels following treatment with Antimycin A or FCCP in cells infected with shCtr or shOPA1 lentivirus (n = 4). (P) ATP in lentiviral shCtr or shOPA1-infected cells subjected to same conditions as in (O) and harvested in control (with glucose) or galactose (without glucose) media followed by a 30-min oligomycin treatment (n = 4). Data presented as mean ± SEM (ns, not significant; *p < 0.05, **p < 0.01, and ***p < 0.001, Student’s t test).

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Figure 3. Deletion of MFN1/2 Causes Stem Cell Depletion, Impaired Neurogenesis, and Cognitive Defects

previously associated with impairments in adult hippocampal neurogenesis (CanH I G MWM/Reversal-training cino et al., 2013). Deletion of MFN1/2 MWM/Reversal-probe MWM/Reversal-probe using the inducible NestinCreERT2 model MFN1/2 WT showed impaired outcomes in the MFN1/2 WT MFN1/2 DKO recall of fear conditioning tests (FigMFN1/2 DKO 50 50 ure 3C) and defects in spatial and 40 40 reversal learning in the Morris water 30 maze (Figures 3D–3I and S2). These 30 data highlight the sensitivity of the stem 20 20 cell population to alterations in mitochon10 10 drial morphology and provide the first 0 0 evidence that disruption of mitochon1 2 3 drial dynamics, as observed during aging Reversal training (days) and neurodegenerative diseases, impairs the long-term maintenance and function was generated with a NestinCreERT2 model. Inducible knockout of stem cells from direct, as well as potential indirect, defects of MFN1/2 specifically in NSCs within the adult neurogenic resulting from impaired mitochondrial dynamics. niche showed a significant reduction in Sox2+ uncommitted cells in the SGZ of the adult DG as early as 4 weeks following Mitochondria Mediate an ATP-Independent Function in disruption of mitochondrial dynamics (Figures 3A and 3B). NSCs and Undergo a Metabolic Switch upon Furthermore, the impact of MFN1/2 loss on adult neurogenesis Commitment to a Progenitor Fate was evident by a decrease in DCX+ newborn neurons (Figures Mitochondria in various stem cells are generally described as 3A and 3B). Next, behavioral studies were employed to assess underdeveloped and fragmented, with little or no bioenergetic the functional consequence of mitochondrial alterations on adult activity (Norddahl et al., 2011). However, little is known regarding neurogenesis. Given that adult neurogenesis contributes to the metabolic characteristics of NSCs and the functional signifhippocampus-dependent learning and memory (Aimone et al., icance of mitochondria in this system. In order to gain insight 2014), we predicted that dysregulated mitochondrial dynamics into the role of mitochondrial structure in NSCs, we first investiwould reveal significant deficits in stringent cognitive testing gated the metabolism of these cells. Metabolic analysis was 1

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performed directly from in vivo E11.5 cortical tissue, which is highly enriched in NSC-containing Sox2+ uncommitted cells with elongated mitochondria (Figures 4A, S3A, and S3B). These cells showed mostly (80%–90%) non-mitochondrial ATP generation (ie. glycolytic) as inhibition of mitochondrial oxidative phosphorylation (OXPHOS) using oligomycin caused minor alterations in cellular ATP levels (Figure 4B). This was further corroborated by high expression of the key glycolytic enzymes hexokinase-II (HXKII) and pyruvate dehydrogenase kinase-I (PDK1) (Figure 4D). Thus, Sox2 uncommitted NSCs isolated directly from embryonic brains exhibit a glycolytic metabolism, as observed in other stem cells, though they contain highly elongated mitochondria (Shyh-Chang et al., 2013; Simsek et al., 2010; Takubo et al., 2013). Despite a glycolytic metabolism, uncommitted cells contain functional respiratory complexes with the capability of driving efficient OXPHOS when forced to rely on mitochondrial metabolism (Figures 4C and 4D). This suggests that NSCs have active mechanisms to limit mitochondrial respiration, potentially by high expression of UCP2 and IF1 (Figure 4D), proteins previously implicated in the suppression of OXPHOS in stem cells (Sa´nchez-Arago´ et al., 2013; Zhang et al., 2011). In contrast, neurosphere cultures, containing mostly committed progenitors (Figure S3B), as well as post-mitotic cortical neurons, undergo a progressive shift to mitochondrialdependent energy production with a concomitant decrease in expression of the glycolytic enzymes HXKII and PDKI, as well as the OXPHOS inhibitors UCP2 and IF1 (Figures 4B and 4D), consistent with a metabolic switch to mitochondrial OXPHOS during progenitor commitment (Chung et al., 2007; Mandal et al., 2011; Yu et al., 2013). The progressive changes in mitochondrial-dependent OXPHOS was further assessed by mitochondrial oxygen consumption studies. Seahorse analysis of Sox2+ uncommitted cells isolated directly from embryonic brains at E11.5 and progenitor-enriched neurosphere cultures revealed a modification in the respiratory profile, whereby a significant increase in basal oxygen consumption rate (OCR) and ATP-linked OCR occurs as cells undergo a cell fate transition (Figures 4E, 4F, S3C). This was also confirmed using cells derived from embryonic cortical tissue at E12.5 and subjected to fluorescence-activated cell sorting (FACS) based on expression of the stem cell surface marker CD15 (Figure S3D) (Capela and Temple, 2002). Comparison of respiration characteristics from tissue-derived CD15+ NSCs and CD15 committed cells demonstrated a similar shift in mitochondrial function with an increase in basal OCR and ATP-linked OCR in committed progenitors (Figures 4G, 4H, and S3E). The respiratory profiles observed here are consistent with previous studies showing low O2 consumption in stem cells and an increase in mitochondrial respiration in differentiating cells (Panopoulos et al., 2012; Varum et al., 2011; Zhang et al., 2011). Therefore, comparisons between uncommitted NSCs and committed progenitor cells showed a unique metabolic profile for each cell population and an ATP-independent function of mitochondria in NSCs. Whereas Sox2+ uncommitted cells are mainly glycolytic, contain elongated mitochondria, and exhibit a unique respiratory profile, the transition of NSCs to a committed fate is paralleled by drastic shortening of mitochondrial length and induction of a metabolic switch, as observed by concomitant alterations in the metabolic and respiratory profiles. These observations, together with the 238 Cell Stem Cell 19, 232–247, August 4, 2016

changes in self-renewal and cell fate decisions (Figure 1), are consistent with an ATP-independent role for mitochondrial dynamics and function in the regulation of stem cell identity. Aberrant Mitochondrial Fragmentation Alters the Metabolic Identity of NSCs Data obtained from the metabolic studies led us to postulate that manipulation of mitochondrial dynamics in NSCs, to recapitulate the fragmented mitochondrial morphology found in committed cells, could generate a metabolic and respiratory profile comparable to such cells. This would be consistent with findings presented in this study that mitochondrial fragmentation promotes NSC fate commitment at the expense of NSC self-renewal. Interestingly, inducing mitochondrial fragmentation in Sox2 uncommitted cells altered their metabolic signature to that which resembled the metabolic profile of wild-type committed neural progenitors with short mitochondria (Figure 4I), as evidenced by decreased levels of IF1, HXKII, and UCP2. This was confirmed by respiratory analysis of mitochondria. Acute loss of MFN1/2 altered the respiratory profile of mitochondria with increased basal and ATP-linked OCR (Figures 4J, 4K, and S3F), which mimicked that observed in committed progenitors (Figures 4E–4H). In essence, the fragmented mitochondria generated by genetic manipulations in our model systems resemble the fragmented mitochondria observed in Tbr2+ progenitors, both in length and in respiratory signature. These observations, together with the changes in self-renewal and cell fate decisions, confirmed a role for mitochondrial dynamics in the upstream regulation of stem cell identity. Mitochondrial Structure Regulates ROS Levels in Uncommitted Cells Because mitochondria have recently gained more recognition as signaling centers, we sought to investigate the mechanism by which mitochondrial structure can dictate the identity and fate of stem cells. Wild-type neural progenitor cells exhibit fragmented mitochondria (Tbr2+ population in Figure 1A) and have a unique metabolic signature, which notably includes reduced levels of complex I and the antioxidant SOD1 (cultured NS in Figure 4D). This was intriguing since it mirrored the profile of NSCs with fragmented mitochondria due to loss of MFN1/2 or OPA1 (Figures 4I). Given the function of SOD1 in reducing cellular ROS levels and the established role of complex I as a major site of ROS production (Hirst et al., 2008), we postulated that NSCs and committed progenitors might exhibit a physiological change in ROS levels. Indeed, cells obtained from the cortex of E10.5 Sox2-GFP expressing embryos (Ellis et al., 2004) and sorted by FACS based on Sox2-GFP expression revealed an increase in mitochondrial superoxide (mtROS) (Figures 5A–5C) and cytoplasmic ROS (Figures 5D and 5E) levels in Sox2GFP( ) progenitor cells compared to Sox2-GFP(+) NSCs. This was further validated using FACS sorted CD15-labeled cells derived from E12.5 cortical tissue, which showed a moderate increase in mtROS levels in CD15 committed cells when compared to CD15+ stem cells (Figures S4A and S4B). Next, we hypothesized that changes in mitochondrial structure, from NSCs to progenitors, may mediate these physiological changes in ROS levels as a signaling mechanism to regulate NSC selfrenewal and cell fate decisions. Consistent with this hypothesis,

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enhanced mitochondrial fragmentation by acute loss of MFN1/2 or OPA1 resulted in a moderate increase in mtROS levels and cytoplasmic ROS without any signs of oxidative damage (Figures 5F, 5G, S4C, and S4D), implying a modification in ROS signaling rather than an oxidative-stress-induced response. Importantly, the general anti-oxidant N-acetylcysteine (NAC) and the more specific superoxide scavengers MitoTEMPO and EUK134 were able to rescue the neurosphere defects incurred by acute OPA1 loss (Figure 5H). These data suggest that mitochondrial dynamics is an upstream regulator of physiological ROS to control stem cell self-renewal versus commitment decisions. Consistent with this conclusion was the observation that loss of DRP1, which causes mitochondrial elongation and promotes NSC self-renewal (Figures 1C, 1D, and 1I), resulted in decreased levels of mtROS and increased oxygen consumption driven by proton leak (Figures 4L, 4M, 5F, and S4C). To further confirm that the complex-I-mediated physiological increase in ROS, by mitochondrial fragmentation, regulates the fate of NSCs, both pharmacological and genetic methods were employed. First, treatment of wild-type uncommitted cells with a chronic low dosage of the complex I inhibitor rotenone, to mimic the decreased levels of complex I and ROS generation observed in our studies (Figure S4E), resulted in a significant decrease in neurosphere formation in culture that was rescued by treatment with NAC (Figure 5I). Second, using an in vivo conditional genetic deletion or acute loss of apoptosis-inducing factor (AIF), a classical model of complex I deficiency and ROS generation, to generate a moderate increase in mitochondrial superoxide and cytoplasmic ROS levels without any signs of oxidative damage or cell death (Figures 5J and S4F–S4H) (Apostolova et al., 2006), was able to recapitulate all the phenotypes observed by aberrant mitochondrial fragmentation. Specifically, measurements of the division angle of Sox2 anaphase cells in E15.5 AIF knockout (KO) cortices revealed a significant increase in the number of dividing cells with a horizontal cleavage plane relative to the ventricular surface, at the expense of anaphase cells with vertical cleavage planes (Figure 5L). This shift in the NSC mode of division by AIF loss was observed as early as E12.5 (Figure S4I). Consistent with an increase in differentiative divisions of AIF-deficient Sox2 anaphase cells, there was an increase in the percentage of cells transitioning to committed progenitor fate (Sox2/Tbr2 and Tbr2 cells) and to newborn neurons (DCX+

cells) (Figure S4J). Furthermore, acute loss of AIF using in vitro RNAi-mediated knockdown, which did not affect total ATP levels or mitochondrial-mediated ATP production (Figures S4K and S4L), resulted in decreased primary and secondary neurospheres (Figure 5K). Consistent with a loss of NSC self-renewal, a decreased level of Notch1 and its target gene, Hes5, was also observed (Figure 5M and S4M). Importantly, re-expression of mitochondrial-targeted AIF (mito-AIF) was sufficient to rescue the neurosphere defect in acute cre-mediated AIF loss (Figure 5N). Metabolic analysis of AIF KO tissue revealed deceased levels of HKII, IF1, UCP2, complex I, and SOD1 (Figure 5O) and an altered respiratory profile (Figures 5P, 5Q, and S4N), which mimicked that observed in wild-type Tbr2+ committed progenitors, as well as that of cells with manipulated mitochondrial dynamics. These data demonstrate that physiological modifications in ROS are sufficient to modify NSC fate decisions. Furthermore, the findings presented here validate that changes in mitochondrial dynamics act as a physiological mechanism in the regulation of NSC self-renewal and fate, through modification of ROS levels. Mitochondrial Dynamics Regulates the Nuclear Developmental Program of NSCs While ROS signaling has been associated with stem cell maintenance and cell fate decisions, the mechanism by which this occurs is poorly understood (Ha¨ma¨la¨inen et al., 2015; Ito et al., 2004; Maryanovich and Gross, 2013; Owusu-Ansah and Banerjee, 2009). To understand how mitochondrial dynamics and ROS levels can modify self-renewal and fate decisions, the nuclear developmental program of NSCs was investigated. RNA-sequencing (RNA-seq) differential gene expression analysis of E12.5 Sox2+ uncommitted cells from AIF WT and AIF KO cortical tissue demonstrated a significant upregulation of genes involved in neuronal differentiation (Isl1, Olig2, Lhx5, Nkx2.1, Sim1, and Six3), redox response (Slc7a11/xCT and Aldh1l2), and the Notch pathway (Botch/CHAC1) (Figure 6A). Gene Ontology (GO) annotation revealed enrichment in neurogenic transcription factors and genes that regulate cell fate and neuronal commitment (Figure 6B). Upregulation of these target genes was validated by qRT-PCR in AIF KO cortical tissue (Figure 6C) and in acute AIF loss in culture (Figure 6D). Importantly, the changes in expression of the identified target genes

Figure 4. Disruption of Mitochondrial Structure Mimics a Physiological Shift in the Metabolic Profile of Neural Stem Cells (A) Representative confocal images of coronal sections showing mitochondrial morphology (Tom20) in Sox2+ uncommitted cells from WT E11.5 cortex. Higher magnification in right panel. Scale bar, 100 mm. (B) ATP levels relative to initial values (black) following 1-hr oligomycin treatment in WT E11.5 dissociated cortical tissue, long-term (6 days) cultured neurospheres, and cortical neuronal cultures (n = 3–4). (C) ATP levels relative to initial values (black) following 1-hr oligomycin treatment from dissociated E12.5 cortical tissue harvested in control (with glucose) or galactose media (without glucose) (n = 3). (D) Immunoblot from whole-cell lysates derived from similar conditions as in (B). (E and F) Oxygen consumption rate (OCR) in cells derived from either E11.5 cortical tissue or neurosphere cultures using a Seahorse XF24 Extracellular Flux Analyzer. Mean and SEM (n = 4 replicates from three independent experiments). (G and H) OCR (as in E and F) in cells derived from E12.5 cortical tissue and labeled with anti-CD15 Alexa Fluor 647 antibody followed by FACS sorting (n = 4 replicates from three independent experiments). (I) Immunoblot from whole-cell lysates derived from WT cultured cells infected with shCtr or shOPA1 lentiviruses or from cortical tissue of E12.5 MFN1/2Fog1Cre-WT and MFN1/2-Foxg1Cre-DKO. CI, complex I; CIII, complex III; CV, complex V. (J–M) Respiratory analysis using Seahorse XF24 Extracellular Flux Analyzer of cells derived from E12.5 MFN1/2-flox (J and K) or DRP1-flox (L and M) cortices and infected with lentivirus encoding GFP or CreGFP to induce acute loss of MFN1/2 or DRP1 (n = 4 replicates from four independent experiments). Data are presented as (B and C) mean ± SD or (E, H, and J–M) mean ± SEM (*p < 0.05, **p < 0.01, and ***p < 0.001, Student’s t test). See also Figure S3.

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Figure 5. Mitochondrial Dynamics Regulates Physiological ROS Levels (A–C) Flow cytometry analysis of mitochondrial superoxide levels by mitosox staining in Sox2-GFP+ uncommitted cells and Sox2-GFP committed progenitors. (A) Representative scatterplots. (B) Representative histogram. (C) Quantification of the mean fluorescence intensity (MFI) from the flow cytometry analysis (n = 4 independent samples). (D and E) Flow cytometry analysis of cytoplasmic ROS levels by DHE staining in Sox2-GFP+ uncommitted cells and Sox2-GFP committed progenitors. (D) Representative histogram. (E) Quantification of the mean fluorescence intensity (MFI) (n = 4 independent samples). (legend continued on next page)

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were similarly observed in acute OPA1 and MFN1/2 loss in culture (Figures 6D and 6E), confirming that mitochondrial dynamics is implicated in the upstream regulation of developmental gene expression. Highly relevant was the striking upregulation of Botch (Figure 6C), a newly identified inhibitor of Notch and NSC self-renewal (Chi et al., 2012). shRNA-mediated knockdown of Botch (Figure S5A) was sufficient to restore neurosphere formation (Figure 6F) and Notch1 signaling (Figure 6G) in shOPA1 cells. Furthermore, the upregulation of Botch by mitochondrial fragmentation is ROS mediated, since treatment of shOPA1 cells with NAC, which rescues the self-renewal of stem cells (Figure 5H), was capable of reducing ectopic expression of Botch (Figure 6H). Also, treatment with a chronic low dosage of rotenone, to mimic the decreased levels of complex I and ROS generation (Murphy, 2009) observed in our study, was sufficient to mediate a ROS-dependent increase in Botch RNA levels (Figure 6I) that can be restored to normal levels upon treatment with NAC or MitoTEMPO (Figures 6J and 6K). Together, these data demonstrate that mitochondrial dynamics can dictate neural stem cell fate through alterations of nuclear gene expression, such as Botch, that is triggered by complex-I-mediated ROS signaling. These data also indicate that changes in mitochondrial structure during development regulate physiological ROS levels to inhibit self-renewal and promote differentiation. Thus, these findings uncover a previously unappreciated role for mitochondrial dynamics in the physiological upstream coordination of stem cell fate decisions. Mitochondrial Dynamics Regulates a Nuclear Transcriptional Program via NRF2-Mediated Retrograde Signaling Next, we examined the mechanism that could mediate such mitochondrial retrograde signaling to alter the transcriptional profile of NSCs. Screening for over-represented transcription factors using the upregulated genes identified by RNA-seq revealed several candidates (Figure 7A). Highly relevant was the presence of NRF2, a master regulator of the antioxidant response, whose activity was apparent in our system as observed by the upregulation of its direct target gene Slc7a11 (Sasaki et al., 2002) in AIF KO tissue and shOPA1, shAIF, and MFN1/2 DKO cultures (Figures 5C–5E). The role of NRF2 in

relaying the ROS-dependent regulation of NSC fate, by changes in mitochondrial dynamics, was tested by manipulating its expression or activity. Loss of NRF2 expression (Figures 7B and 7C) or activity (overexpression of a dominant-negative form of NRF2 [dnNRF2]) (Figure 7D) was sufficient to prevent Botch upregulation by OPA1 or AIF deficiency. Importantly, loss of NRF2 expression or activity in shOPA1 cells restored self-renewal capacity, as measured by neurosphere formation (Figures 7E and 7F), and rescued Notch signaling (Figure 7G). As aberrant NSC self-renewal has never been associated with NRF2 deficiency, these data led us to question whether NRF2 plays a biological role during normal NSC cell fate decisions. Assessment of cortical neurogenesis in NRF2 KO brains at E15.5 showed abnormal cortical development (Figure 7H) and decreased ability of progenitor cells to commit to a neuronal fate in the absence of NRF2 (Figures 7I–7K). As whole-embryo knockouts of NRF2 could cause non-specific effects, in utero electroporation was also used to induce a more cell-autonomous manipulation of NRF2. In utero electroporation of dnNRF2, in the context of a wild-type embryonic cortex, resulted in a significant decrease in the number of electroporated Sox2+ cells that had committed to a neuronal fate (mCherry+/DCX+ cells) (Figures 7L and 7M). Furthermore, analysis of molecular targets identified by RNA-seq revealed that loss of NRF2 expression decreased basal levels of Botch1 RNA (Figure 7N). In addition to Botch, NRF2 was also required for upregulation of neurogenic transcription factors mediated by disruption of mitochondrial dynamics (Figure 7O). Thus, these results identify the stem cell selfrenewal inhibitor Botch (Chi et al., 2012), as well as pro-neuronal transcription factors such as Isl1, Nkx2.1, Lhx5, and Sim1, as targets of NRF2. In addition, these data demonstrate that mitochondrial structure regulates a physiological NRF2-mediated developmental retrograde pathway, through ROS signaling, to activate a dual program that inhibits self-renewal decisions while activating a neurogenic differentiation program. DISCUSSION The endowed capacity of stem cells to undergo fate decisions, self-renew, or commit to a specified cell fate is essential for tissue homeostasis and stem cell maintenance throughout life.

(F) Mitosox fluorescence measurements in cells derived from E12.5 MFN1/2-flox or DRP1-flox cortices and infected with LV-GFP or LV-CreGFP or wild-type cortices and infected with shCtr or shOPA1 lentiviruses (n = 3). (G) DHE fluorescence measurements in cells derived from wild-type cells infected with shCtr or shOPA1 lentiviruses (n = 3). (H) Primary neurosphere assay from E12.5 wild-type cells derived from the dorsal cortex and infected with shCtr or shOPA1 lentiviruses with or without NAC, EUK134, or MitoTEMPO treatment (n = 4). (I) Primary neurosphere assay from E12.5 wild-type cells derived from the dorsal cortex and subjected to chronic treatment with 10 nM rotenone with or without NAC treatment (n = 3). (J) Mitosox and DHE fluorescence measurements in cells derived from wild-type cells infected with shCtr or shAIF lentiviruses (n = 3). (K) Division angle measurements of Sox2+ anaphase cells in E15.5 AIF WT and AIF KO coronal sections. Horizontal (0 –30 ), intermediate (30 –60 ), and vertical (60 –90 ) cleavage angles (yellow line) relative to the apical surface of the lateral vertical (white line). Chromatin is visualized by DAPI (n = 3). (L) Primary and secondary neurospheres from WT cells of the dorsal cortex infected with lentivirus encoding shCtr or shAIF (n = 6, primary assay and n = 3, secondary assay). (M) Immunoblot from whole-cell lysates of E12.5 AIF-WT and AIF-KO dorsal cortical tissue. (N) Primary neurosphere assay of AIF-floxed cells from the dorsal cortex infected with lentivirus encoding GFP or Cre-GFP or co-infected with Cre-GFP and mitochondrial-targeted AIF (mito-AIF) (n = 4). (O) Immunoblot from whole-cell lysates derived from E12.5 AIF-WT and AIF-KO cortical tissue. CI, complex I; CIII, complex III; CV, complex V. (P and Q) Respiratory analysis using Seahorse XF24 Extracellular Flux Analyzer of cells derived from E12.5 WT cortex and infected with lentivirus encoding shCtr or shAIF to induce acute loss of AIF (n = 4 replicates from four independent experiments). Data are presented as (C and E–N) mean ± SD or (P and Q) as mean ± SEM (*p < 0.05, **p < 0.01, and ***p < 0.001, Student’s t test). See also Figure S4.

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Figure 6. Mitochondrial Dynamics Regulates a ROS-Dependent Nuclear Transcriptional Program (A) Pie chart of enriched biological processes from RNA-seq analysis of upregulated genes in uncommitted cells from E12.5 AIF-WT and AIF-KO cortical tissue. Values show number of genes significantly upregulated by adjusted p values. (B) Gene Ontology (GO) enrichment analysis of genes from (A). (C–E) Validation of RNA-seq results by qRT-PCR using (C) E12.5 AIF-WT and AIF-KO cortical tissue; (D) cultured neurospheres derived from wild-type E12.5 dorsal cortex and infected with shCtr, shAIF, or shOPA1 lentiviruses; and (E) cultured neurospheres derived from E12.5 MFN1/2-flox dorsal cortex and infected with GFP or CreGFP lentiviruses (n = 3). (F) Primary neurosphere assay of E12.5 WT cells derived from the dorsal cortex and infected with shCtr or shOPA1 lentiviruses alone or co-infected with Botch shRNA (shBotch) (n = 3). (G) Immunoblots using cell lysates generated from same conditions as (E). (H–K) qRT-PCR analysis of Slc7a11 and Botch RNA levels in neurospheres following (H) infection with shCtr or shOPA1 lentiviruses in the presence or absence of 1 mM NAC (n = 4), (I) in untreated and Rotenone treated neurospheres (n = 4), or (J and K) following additional treatment with 1 mM NAC or MitoTEMPO (n = 3). Data are presented as (C, D, E, and H–K) mean ± SEM and (F) mean ± SD (*p < 0.05, **p < 0.01, and ***p < 0.001, Student’s t test). All qRT-PCR data were normalized to GAPDH. See also Figure S5.

Here, we report that the dynamic properties of mitochondria play an upstream regulatory role in the fate decisions of stem cells. Mitochondrial dynamics and morphology, established through regulated fission and fusion, have a major impact on stem cell

identity, self-renewal capacity, and fate decisions by regulating physiological complex-I-mediated ROS levels to mediate NSCs commitment. We show that mitochondrial dynamics directs stem cell fate through modification of ROS signaling to Cell Stem Cell 19, 232–247, August 4, 2016 243

Figure 7. Mitochondrial Dynamics Regulates the Developmental Program of Neural Stem Cells by NRF2-Dependent Retrograde Signaling (A) Bar plot showing a sample of overrepresented transcription factors within the upregulated genes identified by RNA-seq analysis. (B and C) qRT-PCR analysis in neurospheres generated from E12.5 dorsal cortex of NRF2-WT or NRF2-KO infected with (B) lentivirus encoding shCtr or shOPA1 or (C) shAIF (n = 4). (D) qRT-PCR analysis in neurospheres generated from E12.5 wild-type dorsal cortex and co-infected with lentiviruses encoding mCherry or a dominant-negative form of NRF2 (dnNRF2) with lentiviruses encoding control shCtr or shOPA1 (n = 3). (legend continued on next page)

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activate an NRF2-dependent retrograde developmental pathway, which triggers a dual program that suppresses self-renewal while promoting commitment and differentiation (Figures 7P and 7Q). These results provide insight into the fundamental mechanisms of stem cell self-renewal and fate decisions during development and in the context of long-term maintenance of stem cells in aging and diseases. During development and in the context of tissue regeneration, there are molecular mechanisms in place, at the transcriptional and translational levels, to regulate stem cell function. We now uncover that the state of an organelle, namely mitochondria, has the capacity to coordinate self-renewal versus differentiation of stem cells. Mitochondria are dynamic organelles that undergo morphological changes through fission and fusion. Although major changes in mitochondrial structure have been generally attributed as a cellular response to stress, we now present evidence that mitochondrial dynamics can act as a functional regulatory factor, beyond ATP generation, in the context of physiological and developmental processes. In fact, mitochondrial dynamics serves as a regulatory point for the coordination of a nuclear developmental program in NSCs, by dictating the cellular redox state. ROS have been historically viewed as toxic byproducts of cellular redox reactions, but they have recently gained a more positive perspective as physiological signaling molecules (Sena and Chandel, 2012). Work in the stem cell field has proposed a role for ROS in the differentiation of stem cells, but the mechanisms are not fully understood. Furthermore, it is currently unknown as to how ROS levels are modified in stem cells. Here, we identify mitochondrial dynamics as the mechanism by which physiological levels of ROS can be fine-tuned, and we establish a fundamental role for ROS as physiological signaling molecules that modify the nuclear transcriptional profile of stem cells through an NRF2-mediated mitochondrial to nuclear retrograde pathway. In essence, we present a model whereby changes in mitochondrial structure direct the fate of stem cells. In this model, elongated mitochondria in NSCs maintain low ROS levels and promote self-renewal, while a transition of mitochondria to a more fragmented state results in a modest increase in ROS levels, thereby inducing the expression of genes that inhibit self-renewal (Botch) and promote commitment and differentiation (Figures 7P and 7Q). The ability of stem cells to continuously self-renewal is key to preventing stem cell depletion and aging. Although it is clear that aging results in stem cell depletion, the underlying reasons for

this decline remain unclear. Given our findings, we propose that disruption of mitochondrial dynamics, as observed during aging and in degenerative diseases, is an important factor in the eventual depletion of the stem cell pool. Though mitochondrial fragmentation is generally perceived as a sign of mitochondrial dysfunction and oxidative stress (Detmer and Chan, 2007), data presented in this study modify this view and present changes in mitochondrial morphology as a means to induce intracellular signaling. Within the biological setting of development or regeneration, mitochondrial fragmentation is required for the transient passage of cells to committed progenitors. However, dysregulation of mitochondrial dynamics and a imbalance toward a chronically fragmented state, such as that observed during aging and many neurodegenerative diseases (Archer, 2013; Khacho and Slack, 2015), impair the self-renewal capacity of stem cells and lead to depletion of the stem cell pool. With this in mind, the classical view of regulated mitochondrial dynamics as an essential requirement for maintained energy production and survival of post-mitotic cells can now be revised to include a basic function in stem cells. This modified view has far-reaching implications in the basic understanding of aging and many degenerative diseases that were once thought to only affect the existing differentiated population of cells or tissues. EXPERIMENTAL PROCEDURES Detailed procedures can be found in Supplemental Experimental Procedures. Animals All experiments were approved by the University of Ottawa’s Animal Care Ethics Committee and adhered to the guidelines of the Canadian Council on Animal Care. Littermate controls were used for all experiments. Details for the generation of all animal models in this study can be found in Supplemental Experimental Procedures. Mitochondrial Length Mitochondrial length was assessed by staining with Tom20 (translocase of outer mitochondria). Mitochondrial length was measured by tracing the mitochondria using ImageJ software. Mitochondrial length was either binned into different categories (<0.5 mm, 0.5–1 mm, 1–2 mm, or >2 mm) or taken as an average. ATP Assay ATP concentrations were measured with the CellTiter-Glo Luminescent Assay (Promega) using a LUMIstar Galaxy luminometer (BMG Labtechnologies) according to manufacturer’s protocol. Data were collected from multiple replicate wells for each experiment. Viability of cells under all conditions was ensured by propidium iodide (PI) staining. Cell counts were determined by

(E) Primary neurosphere assay from E12.5 NRF2-WT or NRF2-KO cells derived from the dorsal cortex and infected with shCtr or shOPA1 lentiviruses (n = 3). (F) Primary neurosphere assay performed on E12.5 WT cells derived from the dorsal cortex and infected the same as in (D) (n = 4). (G) Immunoblot using cells lysates generated from same conditions as (D). (H) Representative confocal images of E15.5 coronal sections of NRF2-het and NRF2-KO cortex stained with DAPI. (I–K) Representative confocal images and quantification of bromodeoxyuridine (BrdU)+ cells following a 24 hr pulse in E15.5 NRF2-het and NRF2-KO. (L and M) Representative confocal images and quantification following 48 hr of in utero electroporations of constructs encoding mCherry or dnNRF2-mCherry at E13.5 in the dorsal forebrain. (N) qRT-PCR analysis in neurospheres generated from E12.5 dorsal cortex of NRF2-WT or NRF2-KO (n = 4). (O) qRT-PCR analysis of RNA levels of additional targets from same conditions as in (D) (n = 3–4 independent experiments). (P) Schematic diagram summarizing the mitochondrial, metabolic, and redox changes that occur as stem cells undergo cell fate decisions. (Q) Schematic diagram illustrating the fundamental role of mitochondrial morphology in the regulation of a ROS and NRF2-dependent transcriptional program that dictates self-renewal versus differentiation of NSCs. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate; vent., ventricle. Results are presented as (B–D, N, and O) mean ± SEM and (E, F, K, and M) as mean ± SD (*p < 0.05, **p < 0.01, and ***p < 0.001, Student’s t test).

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trypan blue. For experiments using galactose, DMEM media without glucose was used and supplemented with 5 mM galactose.

(CPSR), and the Parkinson’s Research Consortium (PRC). Equipment was supported by the University of Ottawa Brain and Mind Research Institute.

Oxygen Consumption The Seahorse XF24 Extracellular Flux Analyzer (Seahorse Biosciences) was used to measure oxygen consumption in cells. Cells derived from cortical tissue or from neurosphere cultures were seeded onto Cell Takcoated (22.4 mg/ml) 24-well Seahorse plates at a density of 2.0 3 105 cells/well in 100 ml stem cell media (SCM) media supplemented with additional 0.5 mM sodium pyruvate. Plates were immediately spun at 200 3 g for 1 min and allowed to stop without brakes and then placed at 37 C for 25 min. 500 ml SCM was slowly added to each well, followed by an additional incubation of 20 min at 37 C prior to loading into the XF Analyzer. Following measurements of resting respiration, cells were treated sequentially with oligomycin (1 mg/ml) to measure the nonphosphorylating OCR, FCCP (2 mM) to get the maximal OCR, and Antimycin A (1 mM) to measure the extramitochondrial OCR. Each measurement was taken over a 2-min interval followed by 2 min of mixing and 2 min of incubation. Three measurements were taken for the resting OCR: after oligomycin treatment, after FCCP, and after Antimycin A treatment.

Received: September 24, 2015 Revised: March 1, 2016 Accepted: April 28, 2016 Published: May 26, 2016

qRT-PCR Total RNA was extracted from cortical tissue or cultured neurospheres using the PureLink RNA Mini Kit (Ambion) following the manufacturer’s protocol. One-step qRT-PCR gene expression analysis was performed using the Rotor-gene SYBR green RT-PCR kit (QIAGEN, 204174). For all experiments, primer sequences are provided in Table S1. All reactions were run in triplicate or quadruplicate and averaged. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control for mRNA.

Bratic, A., and Larsson, N.G. (2013). The role of mitochondria in aging. J. Clin. Invest. 123, 951–957.

Statistical Analysis Statistical comparisons in this study were performed using a two-tailed t test. Differences were considered significant with a p value of p < 0.05 (*), **p < 0.01, or ***p < 0.001. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, five figures and one table and can be found with this article online at http:// dx.doi.org/10.1016/j.stem.2016.04.015. AUTHOR CONTRIBUTIONS M.K. and R.S.S. conceptualized the study. M.K. designed and performed all experiments, analyzed and interpreted the data, and wrote the paper. A.C. assisted and performed experiments and maintained mice colonies. D.S.S. assisted with in vivo experiments. J.A. provided technical assistance. J.G.M. provided technical assistance and generated tools for the study. C.M. assisted in maintaining MFN1/2-DKO mice and performed some assays. H.S. generated DRP1-floxed mice. D.C.L. supervised behavioral experiments and interpreted the behavioral data. M.G. provided input and interpreted the data. M.-E.H. provided the Seahorse Analyzer, reagents, and advice and interpreted the metabolic analysis. D.S.P. provided reagents, assisted in NRF2 experiments, and interpreted the data. R.S.S. provided input to experimental design, interpreted the data, and directed the research. All authors of this study reviewed the manuscript. ACKNOWLEDGMENTS We thank Dr. Lisa Julian for critical review of the manuscript, as well as Linda Jui, Delphie Dugal-Tessier, Bensun Fong, Sarah Hewitt, Paul Marcogliese, and Steve Callaghan for technical assistance. We also thank Dr. Vera Tang and the Flow Cytometry and Virometry (FCV) Core Facility at the University of Ottawa for expertise in flow cytometry and FACS. This research was funded by grants from the Canadian Institutes of Health Research (CIHR), the Heart and Stroke Foundation of Canada (HSFC), and the Brain Canada/Krembil Foundation (to R.S.S.), as well as NIH grant GM089853 (to H.S.). M.K. was supported by fellowships from the HSFC, the Canadian Partnership for Stroke Recovery

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