Histone H2AX Promotes Neuronal Health by Controlling Mitochondrial Homeostasis

Histone H2AX promotes neuronal health by controlling mitochondrial homeostasis

Urbain Weyemia, Bindu D. Paula, Deeya Bhattacharyaa, Adarsha P. Mallaa, Myriem Boufraqechb, Maged M. Harraza, William M. Bonnerc, and Solomon H. Snydera,d,e,1

aThe Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205; bEndocrine Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; cDevelopmental Therapeutics Branch, Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; dDepartment of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and eDepartment of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205

Contributed by Solomon H. Snyder, February 27, 2019 (sent for review December 3, 2018; reviewed by Olga A. Martin and Robert Schwarcz) Phosphorylation of histone H2AX is a major contributor to efficient may reflect well their common roles in DNA repair. We recently DNA repair. We recently reported neurobehavioral deficits in mice provided evidence that H2AX facilitates redox homeostasis by lacking H2AX. Here we establish that this neural failure stems from controlling an NRF2-regulated antioxidant response pathway (18). impairment of mitochondrial function and repression of the mito- Mitochondrial defects are a major source of impaired redox ho- chondrial biogenesis gene PGC-1α. H2AX loss leads to reduced levels meostasis and are often associated with age-related neurological of the major subunits of the mitochondrial respiratory complexes in diseases (19, 20). A major risk factor for several of these diseases is mouse embryonic fibroblasts and in the striatum, a brain region the concomitant dysfunction of mitochondrial activity leading to particularly vulnerable to mitochondrial damage. These defects are increased ROS and decreased DNA repair (21). Here we show that substantiated by disruption of the mitochondrial shape in H2AX mu- H2AX loss leads to substantially diminished mitochondrial activity tant cells. Ectopic expression of PGC-1α restores mitochondrial oxida- associated with disturbed mitochondrial shape. These defects re- tive phosphorylation complexes and mitigates cell death. H2AX flect repression of the mitochondrial biogenesis gene peroxisome knockout mice display increased neuronal death in the brain when proliferator-activated receptor gamma coactivator 1-aplha (PGC- challenged with 3-nitropronionic acid, which targets mitochondria. 1α) and the oxidative phosphorylation subunits. Ectopic expression NEUROSCIENCE This study establishes a role for H2AX in mitochondrial homeostasis of PGC-1α partially rescued cells from mitochondrial defects and associated with neuroprotection. restored toxicity associated with mitochondrial damage. We also report that H2AX mutant mice were uniquely vulnerable to mito- mitochondrial homeostasis | neuroprotection | oxidative stress | chondrial damage in the brain. These findings identify histone histone H2AX | DNA repair H2AX as a key regulator of mitochondrial homeostasis which promotes neuroprotection and clarify links between genomic in- enome integrity is maintained by a number of tightly regu- stability and redox homeostasis in neurodegeneration and aging. Glated pathways that lead to efficient DNA repair in response to endogenous or exogenous genotoxic agents (1). Compromis- Results and Discussion ing DNA repair or increasing mutation loads can lead to a broad Histone H2AX Deficiency Leads to Repression of Mitochondrial Biogenesis range of human diseases, including those related to immune Genes and Impairment of Oxidative Phosphorylation. Deficits of DNA deficiency, cancer, inflammation, aging, and developmental dis- repair genes are associated with diverse forms of neurodegeneration – orders affecting the nervous system (2 5). During early devel- (1). Neurologic disorders arising from deficient DNA repair involve opment, especially when progenitor cells expand and differentiate oxidative stress as well as the inability to repair oxidative DNA into mature neurons, efficient DNA repair machinery is crucial (1). Brain is uniquely vulnerable to genomic instability (1, 6, 7). Defects Significance in DNA repair genes often lead to age-associated neurological disorders, but underlying mechanisms are still obscure (1, 7–9). Several neurological disorders originating from deficient DNA re- Histone H2AX elicits proper DNA repair through its phosphory- pair often involve oxidative stress (1, 6, 10). DNA repair proteins lation by ataxia-telangiectasia mutated (ATM) kinase. While ATM whose deficiencies are associated with oxidative stress in the brain senses reactive oxygen species, the role of H2AX in the mainte- (3, 11) include ataxia-telangiectasia mutated (ATM) kinase, meiotic nance of redox homeostasis remains unknown. Here we establish recombination 11 (MRE11), Nijmegen breakage syndrome 1 that H2AX deletion leads to impairment of mitochondrial func- (NBS1), aprataxin (APTX), and tyrosyl-DNA phosphodiesterase 1 tion and repression of the mitochondrial biogenesis gene PGC-1α. (TDP1) (1, 3). Murine ATM models facilitate clarification of links Restoring PGC-1α abrogates mitochondrial deficits and mitigates between genomic instability and impaired redox homeostasis. cell death. This study unveils a role for H2AX in mitochondrial When ATM is absent or mutated, the cell suffers from faulty DNA homeostasis associated with neuroprotection. repair and genomic instability and harbors elevated levels of reactive oxygen species (ROS) originating from the impairment of Author contributions: U.W. and S.H.S. designed research; U.W., B.D.P., D.B., A.P.M., M.B., – and M.M.H. performed research; W.M.B. contributed new reagents/analytic tools; U.W. ROS-sensing functions of ATM (12 14). These deficiencies lead to and S.H.S. analyzed data; and U.W. and S.H.S. wrote the paper. Ataxia telangiectasia (AT). We have shown that diminution of ROS Reviewers: O.A.M., Peter MacCallum Cancer Centre; and R.S., University of Maryland reduces phenotypic damage associated with AT (15). ATM elicits School of Medicine. DNA repair by phosphorylating the histone variant H2AX fol- Conflict of interest statement: U.W. and O.A.M. are coauthors on a 2016 article; they did lowing double-stranded DNA breaks (16). This process is sub- not collaborate directly on the paper. The authors declare no competing financial stantiated by the well-established role of H2AX in DNA damage interests. repair (5). Similarities between ATM and H2AX are evident in the Published under the PNAS license. phenotypes of their knockout mouse models. In both instances, 1To whom correspondence should be addressed. Email: [email protected]. males are sterile, and there is increased genomic instability evi- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. denced by abnormalities in chromosome structure, immunodefi- 1073/pnas.1820245116/-/DCSupplemental. ciency, and enhanced radiosensitivity (12, 17). These similarities

www.pnas.org/cgi/doi/10.1073/pnas.1820245116 PNAS Latest Articles | 1of6 Downloaded by guest on September 29, 2021 A MEFs B PGC1⍺ (MEFs) C BRAIN: STRIATUM 0.15 H2AX WT H2AX KO H2AX WT H2AX KO 15 kDa ** H2AX 0.10 130 kDa 130 kDa PGC1⍺ PGC1⍺ 0.05 15 kDa H2AX 38 kDa GAPDH Relative protein amount 0.00 38 kDa T O GAPDH W K X X A A 2 2 D PGC1⍺ (Striatum) H H 0.010 H2AX WT 0.6 E *** H2AX KO *** 0.008

0.4 0.006

0.2 0.004

mRNA levelsmRNA *** Relative protein amount 0.002 * 0.0 ** T O W K X X A A 2 2 0.000 H H M B 2 AM RMT B C1 MEFs F TF T F POL ARG H2AX WT H2AX KO PP 1.5 G * 55 kDa H2AX WT CV-ATP5A H2AX KO 37 kDa CIV-MTCO1 * 48 kDa 1.0 CIII-UQCRC2 *** 25 kDa CII-SDHB * 15 kDa CI-NDUFB8 0.5 * 15 kDa H2AX Relative protein amount 38 kDa 0.0 GAPDH I V III xI xI x e plex V plex I pl ple m m o om o om C H BRAIN: STRIATUM C Comple C C H2AX WT H2AX KO 2.0 I H2AX WT ** H2AX KO 55 kDa CV-ATP5A 1.5 37 kDa CIV-MTCO1 * * 1.0 48 kDa CIII-UQCRC2 ** *** 25 kDa CII-SDHB 0.5

15 kDa CI-NDUFB8 amount Relative protein 0.0 15 kDa H2AX V I I x II x III e le 38 kDa plex V p pl GAPDH mplex m o om Complex Com Co C C

Fig. 1. Histone H2AX depletion elicits pronounced diminution of mitochondrial biogenesis genes and reduced expression of OXPHOS complexes. (A–D) H2AX deletion reduces PGC-1α, a major mitochondrial biogenesis gene. Protein lysates from wild-type and H2AX knockout MEFs, as well as lysates from the striatum, were processed for immunoblot detection of H2AX and PGC-1α. Actin was used as a loading control. (A) Representative image of PGC-1α expression in MEFs and (B) quantification. (C) Representative image of PGC-1α expression in the striatum and (D) quantification. Data are means ± SEM (n = 3). Statistical significance was determined by a two-tailed, unpaired Student’s t test. (E) Analysis of POLRMT, TFB2M, TFAM, and PPARGC1B transcript levels in wild-type and H2AX knockout MEFs by RT-PCR. Expression values are relative fold changes for gene transcripts normalized to GAPDH. Error bars represent SEM (n = 3). (F and G) Western blot analysis of OXPHOS subunits in wild-type and H2AX knockout MEFs. We performed the immunoblot detection using total OXPHOS Human WB Antibody Mixture, enabling concomitant analysis of main proteins of each complex in the electron transport chain, including complex I subunit NDUFB8, complex II subunit 30 kDa, complex III subunit Core 2, complex IV subunit I, and ATP synthase subunit alpha; (F) representative image; (G) quantification. Data are means ± SEM (n = 3). (H and I) Western blot analysis of the OXPHOS subunits in the striatum of 4-mo-old wild-type and H2AX knockout mice; (H) representative image; (I) quantification. Data are means ± SEM (n = 3; *P < 0.05; **P < 0.001; ***P < 0.0001).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1820245116 Weyemi et al. Downloaded by guest on September 29, 2021 lesions (6, 8). These features are exemplified in AT, a form of pro- component of chromatin and an important player in DNA repair gressive cerebellar degeneration associated with oxidative stress (12, pathways, we speculated that loss of H2AX can alter chromatin 14). We have shown that diminution of ROS reduces phenotypic and DNA damage responses, thereby impacting mitochondrial damage associated with AT (15). ATM kinase elicits DNA repair by biogenesis and eliciting oxidative stress. Deletion of H2AX led to phosphorylating the histone variant H2AX following double- pronounced diminution of PGC-1α protein in mouse embryonic stranded DNA breaks, which is consistent with the well-established fibroblasts (MEFs) as well as the brain (Fig. 1 A–D). Similar role of H2AX in DNA damage repair (5). We observed neuro- decreases occurred for the PGC-1α targets, TFAM, TFB2M, and behavioral defects and impaired redox disposition in mice with ge- POLRMT, as well as for PPARGC1B, another homolog of PGC- netic deletion of H2AX (18). In the present study, we report a role 1α (Fig. 1E). Western blot analysis revealed substantial decreases for H2AX in promoting mitochondrial homeostasis and mediating of the key subunits of the five OXPHOS complexes in both neuronal health. H2AX-deficient MEFs and the brains of mutant mice (Fig. 1 F– Most mitochondrial proteins are encoded in the nucleus (22). I). The reduction in the OXPHOS complexes expression seems Examples include basal transcription factors such as TFAM, more pronounced in the striatum, since only a partial depletion TFB2M, TFB1M, and POLRMT, as well as the mitochondrial of the OXPHOS complexes I and II was observed in the cortex biogenesis gene PGC-1α (22, 23). PGC-1α is a transcription (SI Appendix, Fig. S1A). Similar pattern of OXPHOS expression coactivator that regulates the expression of TFAM, TFB2M, was detected in peripheral tissue such as the liver (SI Appendix, TFB1M, and POLRMT, as well as the oxidative phosphorylation Fig. S1B), suggesting an increased vulnerability of the striatum to complex subunits (OXPHOS) through interaction with the H2AX loss-induced mitochondrial damage. Taken together, transcription factor nuclear respiratory factor 1 (NRF1) (24, 25). H2AX deletion impairs expression of PGC-1α, the master reg- It is well established that mitochondrial biogenesis and respira- ulator of mitochondrial biogenesis, leading to alteration of the tion are stimulated by PGC-1α through induction of NRF1 and principal components of mitochondrial function. NRF2 gene expression (26). Changes in chromatin configuration H2AX knockout cells displayed disorganized mitochondrial or alteration of chromatin-based DNA repair pathways are fac- cristae and slightly enlarged mitochondria (Fig. 2 A and B). tors in regulating transcription factors (27, 28), but regulatory Cristae are functionally dynamic compartments, serving as sites mechanisms are often unclear. As histone H2AX is a key for OXPHOS complexes. A substantial amount of the main NEUROSCIENCE A MEF STRIATUM B a1 c1 H2AX WT

500 nm 500 nm b1 d1 C Oligomycin Ant A / Rot 250 FCCP H2AX WT 200 H2AX KO 150

100 H2AX KO

OCR (pmol/min)/ Cell number Cell (pmol/min)/ OCR 0 7 8 8 0 0 .37 .7 .3 .9 .6 .0 2 4 7 9 2 5 500 nm 500 nm 3 6 9 3 1 16 Time (min)

Fig. 2. H2AX deletion leads to disorganized cristae, enlarged mitochondria, and impairment of oxidative phosphorylation. (A) Illustrative images of mitochondrial shape of wild-type and H2AX knockout cells in MEFs (a1, b1) and in the striatum (c1, d1) using TEM. Arrows in a1 and b1 indicate mitochondria cristae. Images were taken at 33,000×.(B) Diameter of 30 individual mitochondria in both wild-type and H2AX knockout striatum were quantified. H2AX KO cells display 20% increase in the size of their mitochondrial diameter; **P < 0.001. (C) Significantly higher baseline mitochondrial respiration (0 min to 34 min), ATP production (34.77 min to 70 min), and maximal respiration and sparse respiratory capacity (70 min to 120 min) are observed in H2AX wild-type MEFs compared with H2AX knockout cells. These measurements were assessed using Seahorse MitoStress Kit (n = 5).

Weyemi et al. PNAS Latest Articles | 3of6 Downloaded by guest on September 29, 2021 A WTKO KO+PGC1 130 kDa PGC1⍺

55 kDa CV-ATP5A 37 kDa CIV-MTCO1 48 kDa CIII-UQCRC2

25 kDa CII-SDHB

15 kDa CI-NDUFB8

38 kDa GAPDH B C

D E

Fig. 3. Ectopic expression of PGC-1α restores OXPHOS subunits and protects from the cytotoxicity induced by mitochondrial damage. (A) Immunoblot analysis of OXPHOS subunits in H2AX wild-type cells (H2AX WT), H2AX knockout cells (H2AX KO), and H2AX knockout cells in which PGC-1α expression was ectopically and transiently restored for 48 h. Actin was used as a loading control. Both wild-type and knockout cells were transfected with control vector. Most of the subunits were partially restored, except for complex IV and complex V. (Left) Representative image. (Right) Quantification. (B) Cells deficient in H2AX are vulnerable to 3-nitropropionic acid (3-NP), an inhibitor of OXPHOS complex II. This cytotoxicity was reversed by ectopic expression of PGC-1α in H2AX knockout cells. Viable cells were quantified using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were treated with increasing doses of 0,0.1,1,and2mM3-NP for 48 h before MTT was added. Data are means ± SD; n = 3. Statistical significance was determined by a two-tailed, unpaired Student’s t test. (C–E) Mitochondrial complex inhibitors induce lower survival in H2AX mutant cells, and H2AX ectopic expression mitigates these cytotoxic effects. (C and D) Parental cells (H2AX WT), H2AX knockout cells (H2AX KO), and H2AX knockout cells in which H2AX expression was restored (REV) were treated with increasing concentrations of mitochondrial complex I inhibitors 1-methyl-4-phenylpyridinium (MPP+) and rotenone. Cell survival was estimated 48 h posttreatment using MTT assay. Cells were treated with increasing doses of 0, 10, 50, and 100 μMMPP+, or with 0, 0.1, 1, and 10 nM rotenone for 48 h before MTT was added. (E) Cell vulnerability to 3-nitropropionic acid (3-NP) was analyzed using MTT assay. Cells were treated with increasing doses of 0, 0.1, 1, and 2 mM 3-NP for 48 h before MTT was added. Data are means ± SD; n = 3. Statistical significance was determined by a two-tailed, unpaired Student’s t test; *P < 0.05; **P < 0.001; ns, nonsignificant.

subunits of the complex III and ATP synthase, and the cyto- Ectopic Expression of PGC-1α Restores OXPHOS Complexes and chrome c, are stored in the cristae (29). Shapes of cristae and Reverses Mitochondria Damage-Induced Cytotoxicity. Overexpressing OXPHOS function are closely linked with direct impact on cel- PGC-1α in the H2AX knockout cells partially restored OXPHOS lular metabolism (29). The loss of cristae in H2AX mutants fits subunit levels (Fig. 3A) and diminished cytotoxicity elicited by the with the diminished levels of OXPHOS subunits in mutant cells. mitochondrial complex II inhibitor 3-nitropropionic acid (3-NP) We monitored OXPHOS function, employing the Seahorse (Fig. 3B). Enhanced mitochondrial functions, especially those in- technique. We observed diminished baseline oxygen consump- volving the respiratory oxidative phosphorylation complexes, are tion rate (OCR) in H2AX mutant cells, as well as reduced ATP associated with PGC-1α.PGC-1α is a master integrator of production and impaired spare respiratory capacity (Fig. 2C). cellular signals that govern mitochondrial biogenesis, oxidative

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1820245116 Weyemi et al. Downloaded by guest on September 29, 2021 A B CORONAL SECTION among others. How changes in chromatin dynamic affect the H2AX WT H2AX KO metabolism of mitochondria via regulation of PGC-1α and its transcriptional targets is unclear.

H2AX Deficiency Increases Vulnerability to 3-Nitropropionic Acid That Targets Mitochondria in the Brain: Role in Neuroprotection. We Control 3-NP TH challenged wild-type and H2AX mutant mice with 3-NP as il- DAPI lustrated in Fig. 4A. In H2AX mutants, acute 3-NP treatment resulted in a 50% loss of striatal dopaminergic nerve terminals as measured by immunostaining and immunoblot analyses of tyro- H2AX WT Control 3-NP Control 3-NP sine hydroxylase (TH) expression (Fig. 4 B–D). These observa- tions were substantiated by signs of systemic mitochondrial impairment in H2AX mutant mice and in mice treated with 3-NP as revealed by measurement of body temperature (Fig. 4E). The Sections and Tissues collection damages seem limited to DA neurons, since other populations

H2AX KO remain relatively unaffected, as indicated by unchanged NeuN SI Appendix C BRAIN: STRIATUM immunoreactivity ( , Fig. S2). Several models of H2AX WT H2AX KO ns neurodegeneration employ drugs that target mitochondrial D 150 CTRL 3-NP CTRL 3-NP ns function (31, 32). For instance, PGC-1α−deficient mice are * uniquely vulnerable to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyr- 52 kDa TH 100 idine−induced degeneration of dopamine neurons in the sub- 15kDa H2AX stantia nigra and striatum (25). Overexpressing PGC-1α protects 50 neuronal cells from oxidative stress and cell death (25). Our 38 kDa GAPDH

Relative protein amount findings that H2AX knockout mice exhibit increased vulnerability 0 * L P P to 3-NP in the striatum suggest enhanced neurodegeneration in 40 R -N N C) 3 TRL 3- E ° ns CT C * T O mice lacking H2AX following damage to mitochondria, further 38 O K W WT K substantiating evidence that H2AX promotes neuronal health 36 via control of mitochondrial homeostasis. NEUROSCIENCE 34 In summary, the present study demonstrates that H2AX, a 32 histone variant and classic DNA repair protein, promotes mi-

Rectal ( Temperature 30 tochondrial biogenesis and mediates responses to neurotoxic L P -NP R -N 3 CT drugs targeting mitochondria. In addition, we show that cells O O 3 WT CTRL WT K K lacking H2AX have impaired oxygen consumption, as well as disturbances in mitochondrial shape. Histone variant H2AX has Fig. 4. H2AX deficiency leads to increased neuronal damage following mitochondrial damage. (A) Wild-type and H2AX knockout mice were in- been studied primarily as a mediator of DNA repair (5). The jected with either 3-nitropropionic acid or PBS following instructions de- present study extends the role of this protein to the arena of scribed in Materials and Methods. After 24 h, mice were killed, and brain mitochondrial function and neuronal health. Our findings reveal tissues were either fixed in NBF or collected for biochemical analyses. (B) how alterations in the nuclear genome influence mitochondrial Coronal sections of the brain were taken through the striatum and analyzed activity and cell metabolism. Other reports describe concomitant for TH-positive cells by immunohistochemistry. Purple, TH; blue, DAPI. (Scale defects in DNA repair and mitochondrial activity in neurode- bars, 100 μm.) (C and D) Western blot analysis of TH in the striatum. (C) generative diseases such as AT (7, 33). Several features of aging ± = Representative image; (D) quantification. Data are means SD; n 4 mice and neurodegeneration in AT are suppressed by complementation per group. Statistical significance was determined by a two-tailed, unpaired with antiaging compounds or selective activators of mitochondrial Student’s t test. (E) H2AX loss and 3-nitropionic acid treatment cause reduction of body temperature. We measured the rectal temperature in wild-type and biogenesis (7). One of the hallmarks of neurodegeneration is the H2AX knockout mice. H2AX mutant mice display a significant reduction in elevated risk of impaired mitochondrial function combined with the body temperature, and this reduction progresses with 3-nitropropionic acid cell’s inability to properly handle DNA lesions. These phenomena treatment. These changes are reflective of lower mitochondrial activity. Data occur in diverse diseases as well as during aging (21, 34). Our are means ± SD; n = 4 mice per group. Statistical significance was determined finding that deficiency of the DNA repair gene H2AX leads to by a two-tailed, unpaired Student’s t test; *P < 0.05; ns, nonsignificant. impaired mitochondrial homeostasis and increased neuronal damage reflects a key role for histone H2AX and chromatin-based DNA repair in neurodegenerative diseases and aging. Accord- phosphorylation, adaptive thermoregulation, and fatty acid bio- ingly, global genomic instability resulting from mutations or de- synthesis/degradation (22, 30). ficiencies in DNA repair genes may impact mitochondrial To further assess the link between the loss of H2AX and mi- function leading to neurodegeneration. tochondrial damage, we explored effects of additional inhibitors of mitochondrial complexes on the viability of cells deficient for Materials and Methods H2AX. H2AX knockout cells were vulnerable to inhibitors of Cell Culture. MEF from both wild-type and H2AX mutant mice were obtained mitochondrial OXPHOS complexes I and II with toxicity re- from the Developmental Therapeutics Branch, Laboratory of Molecular versed by overexpressing H2AX (Fig. 3 C–E). These observa- Pharmacology, National Cancer Institute, National Institutes of Health. Cells tions show that the deficits observed in H2AX mutants are not were grown at 37 °C with 5% CO2 in DMEM (Invitrogen), supplemented with the result of other unrelated differences between wild-type and 10% FBS (Atlanta Biologicals). All media were supplemented with 2 mM mutant cells. Taken together, these findings establish that the glutamine, penicillin, and streptomycin (Invitrogen). mitochondrial defects induced by H2AX deletion lead to in- − creased cytotoxicity which can be reversed by restoring markers Animals. Animals were housed on a 12-h light dark schedule and received food and water ad libitum. William Bonner and Andre Nussenzweig (Na- of mitochondrial biogenesis. Histone H2AX is a key component tional Cancer Institute/National Institutes of Health) kindly provided the of a cluster of proteins involved in genome stability and chro- H2AX heterozygote mice. All animals were treated in accordance with matin remodeling following DNA double-strand breaks. Exam- the recommendations of the National Institutes of Health and approved by ples include proteins such as ATM, NBS1, RAD50, and 53BP1, the Johns Hopkins University Committee on Animal Care.

Weyemi et al. PNAS Latest Articles | 5of6 Downloaded by guest on September 29, 2021 Four to five-month-old animals were used to analyze effects of 3- increased energy demand. The third injection is a mix of rotenone, a com- Nitropropionic acid (3-NP). Injections were based on protocols previously plex I inhibitor, and antimycin A, a complex III inhibitor. This combination reported for mice (32). In brief, 3-NP (Sigma) was dissolved at 10 mg/mL in shuts down mitochondrial respiration and enables the calculation of sterile 0.1 ml PBS and adjusted to pH 7.4 with sodium hydroxide. For TH nonmitochondrial respiration driven by processes outside the mitochondria. detection in the striatum, mice received i.p. injections of 60 mg/kg of 3-NP Data were normalized to total cell survival. The assay results were analyzed twice, with 2 h between injections, and tissues were collected 24 h using Wave program 2.3.0 (Seahorse Bioscience), and data were exported in postinjection. GraphPad Prism 7.

Real-Time PCR. Total RNA was extracted from cells using RNeasy Mini Kit Brain Tissue Processing and Immunostaining. Mice were anesthetized by i.p. ’ (Qiagen) according to the manufacturer s instructions. Quality of RNA injection of sodium pentobarbital (80 mg/kg), then the heart was exposed, preparation, based on the 28S/18S ribosomal RNAs ratio, was assessed using and transcardiac perfusion was performed using PBS for 5 min followed by the RNA 6000 Nano Lab-On-chip (Agilent Technologies). Reverse transcrip- ice-cold 10% neutral buffered formalin (NBF; vol/vol) for 30 min (using 3 mL/min tion and real-time PCR (RT-PCR) were performed as previously described (35). rate of perfusion) and maintained in NBF for 24 h. Tissues were transferred to Oligonucleotides were predesigned and validated, and were considered to 30% sucrose and maintained at 4 °C for 24 h. Brains were sectioned on a be proprietary information by Thermo Fisher Scientific. However, the assays freezing stage sliding microtome into a series of 35-μm sections. Sections were IDs are available and are referenced as follow: PPARGC1B (Mm00504730_m1), TFAM (Mm00447485_m1), TFB2M (Mm01620397_s1), and POLRMT permeabilized with 0.5% Triton X-100 in Tris buffered saline, then blocked with (Mm00553272_m1) 5% normal goat serum. Mouse anti-TH was used as a primary antibody at the dilution of 1:1,000. Anti-mouse IgG (H+L) Alexa Fluor 647 conjugate was used Mitochondrial Stress Assay Using Seahorse. Mitochondrial function was de- as a secondary antibody. ZEISS LSM 800 confocal microscopy was used to image termined by measuring OCR of each cell line using XF Cell Mito Stress Test Kit immunostained sections. (Agilent Technologies). Wild-type and H2AX knockout MEFs were seeded in an XF96 cell culture microplate. Media were prepared by adding 1 mmol/L of Statistical Analysis. Statistical analyses were performed using GraphPad Prism pyruvate, 2 mmol/L of glutamine, and 10 mmol/L of glucose and stored as per 7 software (GraphPad Software) and Microsoft Excel 2010. Parametric data the manufacturer’s instructions. Seahorse assay was run in XF96 Extracellular were analyzed using a two-tailed t test. A value of P < 0.05 was considered Flux Analyzer (Agilent Technologies). Following three baseline OCR mea- statistically significant. Data are presented as mean ± SD or as mean ± SEM. surements, cells were exposed sequentially to oligomycin (0.5 μmol/L), car- Generation of cells expressing H2AX-WT [H2AX-rescued cells or revertant bonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP; 1 μmol/L), and (REV)], Western blots, cell viability assay, Transmission Electron Microscopy rotenone/antimycin A (0.5 μmol/L). Oligomycin inhibits ATP synthase (com- (TEM), Mitochondrial extraction, and immunoblot can be found in SI Ap- plex V), and the decrease in OCR following injection of oligomycin correlates pendix, SI Materials and Methods. with the mitochondrial respiration associated with cellular ATP production. FCCP is an uncoupling agent that collapses the proton gradient and disrupts ACKNOWLEDGMENTS. We thank Roxanne Barrow and Lauren Albacarys of the mitochondrial membrane potential, allowing cells to achieve maximal The Solomon H. Snyder Department of Neuroscience (Johns Hopkins Univer- OCR. As a result, electron flow through the electron transfer chain is uninhib- sity) for help with experiments. We thank Barbara Smith (TEM Specialist at ited, and oxygen is maximally consumed by complex IV. The FCCP-stimulated Johns Hopkins School of Medicine) for her help in preparation and imaging OCR can then be used to calculate spare respiratory capacity, defined as of TEM samples. This work was supported by the National Institutes of the difference between maximal respiration and basal respiration. Spare Health and United States Public Health Service (USPHS) Grants MH18501 respiratory capacity is a measure of the ability of the cell to respond to and DA000266.

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