Loss of Nardilysin, a Mitochondrial Co-chaperone for α-Ketoglutarate Dehydrogenase, Promotes mTORC1 Activation and Neurodegeneration - PubMed (original) (raw)

. 2017 Jan 4;93(1):115-131.

doi: 10.1016/j.neuron.2016.11.038. Epub 2016 Dec 22.

Hector Sandoval 2, Sonal Nagarkar-Jaiswal 1, Manish Jaiswal 1, Shinya Yamamoto 3, Nele A Haelterman 4, Nagireddy Putluri 5, Vasanta Putluri 5, Arun Sreekumar 5, Tulay Tos 6, Ayse Aksoy 7, Taraka Donti 2, Brett H Graham 8, Mikiko Ohno 9, Eiichiro Nishi 9, Jill Hunter 10, Donna M Muzny 11, Jason Carmichael 12, Joseph Shen 12, Valerie A Arboleda 13, Stanley F Nelson 13, Michael F Wangler 3, Ender Karaca 2, James R Lupski 14, Hugo J Bellen 15

Affiliations

Loss of Nardilysin, a Mitochondrial Co-chaperone for α-Ketoglutarate Dehydrogenase, Promotes mTORC1 Activation and Neurodegeneration

Wan Hee Yoon et al. Neuron. 2017.

Abstract

We previously identified mutations in Nardilysin (dNrd1) in a forward genetic screen designed to isolate genes whose loss causes neurodegeneration in Drosophila photoreceptor neurons. Here we show that NRD1 is localized to mitochondria, where it recruits mitochondrial chaperones and assists in the folding of α-ketoglutarate dehydrogenase (OGDH), a rate-limiting enzyme in the Krebs cycle. Loss of Nrd1 or Ogdh leads to an increase in α-ketoglutarate, a substrate for OGDH, which in turn leads to mTORC1 activation and a subsequent reduction in autophagy. Inhibition of mTOR activity by rapamycin or partially restoring autophagy delays neurodegeneration in dNrd1 mutant flies. In summary, this study reveals a novel role for NRD1 as a mitochondrial co-chaperone for OGDH and provides a mechanistic link between mitochondrial metabolic dysfunction, mTORC1 signaling, and impaired autophagy in neurodegeneration.

Keywords: DNAJA3; NRD1; OGDHL; TCA cycle; alpha-ketoglutarate; autophagy; metabolism; mitochondrial chaperones; rapamycin.

Copyright © 2017 Elsevier Inc. All rights reserved.

PubMed Disclaimer

Figures

Figure 1

Figure 1. dNrd1 Mutants Exhibit Progressive Neurodegenerative Features in PRs

(A) ERGs of 1 day-old and 35 day-old mutant clones in PRs of wild-type (wt), four dNrd1 alleles (dNrd1A, B, C, and D), and dNrd1A mutants carrying an 80kb P[acman] genomic rescue transgene (gR-WT). An ERG trace consists of an on-transient (red dotted circles), an amplitude (red arrow), and an off-transient (blue dotted circles). Quantification of the on-transients (B) and off-transients (C) of ERG traces in (A). Error bars indicate s.e.m. P values were calculated using Student’s t-test. **P <0.01, ***P<0.001. (D) Light micrographs of cross sections of retina of 1-day- and 35-day-old flies in wt, dNrd1A and dNrd1A; gR-WT. (E) Schematic of protein domains of fly and vertebrate NRD1. Arrows indicate molecular lesions and alleles of dNrd1. Blue boxes indicate mitochondria targeting sequences (MTSs) predicted by MitoProt and numbers beside the blue boxes indicate the probability that each NRD1 localizes to the mitochondria. Abbreviations: Dm = Drosophila melanogaster, Hs = Homo sapiens, Bt = Bos taurus, Mm = Mus musculs, Rn = Rattus norvegicus, Gg = Gallus gallus. See also Figure S1.

Figure 2

Figure 2. NRD1 Localizes to the Mitochondria

(A) Confocal micrographs of S2 cells co-expressing mito-GFP (green) and full-length V5-tagged dNrd1 (DNRD1-wt), or V5-tagged dNrd1 cDNA lacking N-terminal MTS (DNRD1-ΔMTS) (red). Scale bars, 10μm. (B) Confocal micrographs of dNrd1C mutant larvae muscle expressing dNrd1_-V5 (DNRD1-wt), or dNrd1-Δ_MTS-V5 (DNRD1-ΔMTS) (red), using a muscle driver (C57-Gal4). ATP5A (green) labels mitochondria. Scale bars, 10μm. (C) Confocal micrographs of motor neuron axons co-expressing mito-GFP (green) and _dNrd1_-V5 (red) (D42-GAL4>UAS-dNrd1-V5, UAS-mito-GFP). Scale bar, 5μm. (D) Confocal micrographs of S2 cells co-expressing mito-GFP (green) and V5-tagged full-length human NRD1 (NRD1-WT), or NRD1 cDNA lacking N-terminal MTS (_NRD1-Δ_MTS) (red). Scale bars, 10μm. (E) Confocal micrographs of dNrd1A mutant larvae muscle expressing human NRD1 (red) (da-Gal4>UAS-human NRD1-V5). Gdh (green) labels mitochondria. Scale bars, 5μm. See also Figure S2.

Figure 3

Figure 3. Loss of dNrd1 does not Interfere with ATP Levels

(A) Relative amount of mitochondrial DNA copy number measured in dNrd1 mutant larvae and controls. (B) Western blots for mitochondrial proteins – PDH-E1α, NDUFS3, ATP5A, Porin, Cyt C and Actin in dNrd1 mutants and controls. (C) Mitochondrial membrane potential is measured by TMRE dye in larva muscle (left). Quantification of membrane potential in dNrd1A mutant and control (right). (D) Relative levels of NADH in dNrd1 mutants and control. (E) Relative levels of ATP in dNrd1 mutants and controls. (F) Aconitase activity in both native and reactivation conditions in dNrd1 mutant larvae and controls. (G) The ETC complex activity (I-IV) in dNrd1 mutant larvae and controls. Errors bars in (A), (C), (D), (E), (F), and (G) indicate s.e.m. P values were calculated using Student’s t-test. *P <0.05, **P <0.01, ***P <0.001. N.S. indicates not statistically significant.

Figure 4

Figure 4. NRD1 is Required for the Activity of OGDH

(A) Quantification of ratio of metabolites in the TCA cycle of dNrd1A mutants to those in control larvae. (B) Quantification of ratio of metabolites in the TCA cycle of Nrd1KO mutant MEF (mNrd1−/−) to those in wild-type MEF (mNrd1+/+). (C-F) The activity of enzymes in the TCA cycle is measured from purified mitochondria (C, D, and F) or total larva extract (E) in dNrd1 mutants and controls. (G-H) Western blots for protein level of DOGDH-GFP (G) or protein level of Mdh2-myc expressed from genomic rescue transgene (H) and ATP5A in total (T), cytosolic (C) and mitochondrial (M) fraction in dNrd1A mutant and control larvae. (I) Western blots for protein level of OGDH, CS and Actin in Nrd1 mutant MEF (mNrd1−/−) and wild-type MEF (mNrd1+/+). (J) The activity of OGDH in Nrd1 mutant MEF (mNrd1−/−) and wild-type MEF (mNrd1+/+). (K) Quantification of the ratio of metabolites in the TCA cycle of dOgdh RNAi larvae (Act-Gal4/UAS-dOgdh RNAi) compared to those in control larvae (Act-Gal4/+). (A-F J, and K) Error bars indicate s.e.m. P values were calculated using Student’s t-test. *P <0.05, **P <0.01, ***P <0.001. (L) Schematic representation of the TCA cycle. Metabolites that increase in both dNrd1 mutant and mNrd1−/− MEF are shown in red color. Note that in dNrd1 mutant flies (Figure 4A), dOgdh knockdown flies (Figure 4K), and in mNrd1 knockout MEFs (Figure 4B), there is a loss of some other metabolites (green) produced in the TCA cycle. Abbreviations: Citrate synthase (CS); Aconitase (ACO); Isocitrate dehydrogenase 3 (IDH3); Oxoglutarate dehydrogenase (OGDH); Glutamate dehydrogenase (GDH); Succinate-CoA ligase (SUCLG); Succinate dehydrogenase complex (SDHc); Fumarate hydratase (FH); Malate dehydrogenase 2 (MDH2); Pyruvate dehydrogenase complex (PDHc). See also Figure S3 and S4.

Figure 5

Figure 5. NRD1 Interacts with Mitochondrial Chaperones and OGDH

(A) The table shows 20 mitochondrial proteins that are identified by IP/MS using V5 antibody from DNRD1-expressing larvae (da-Gal4>UAS-dNrd1-V5). These mitochondrial proteins are selected from proteins whose spectral counts are at least 9-fold higher in DNRD1-expressing larvae compared to control (da-Gal4). The proteins in the TCA cycle are marked with green color, amino acid catabolism with red color and mitochondrial chaperone proteins with blue color. (B) Co-immunoprecipitation using anti-V5 antibody from S2 cell lysates overexpressing C-terminally V5-tagged DNRD1 and C-terminally Flag-tagged DOGDH. Western bots were performed using either anti-Flag or anti-V5 antibody. (C) Co-immunoprecipitation using anti-Myc antibody from S2 cell lysates overexpressing C-terminally V5-tagged DNRD1 and C-terminally Myc-tagged mitochondrial chaperone proteins. Western bots were performed using either anti-V5 or anti-Myc antibody. (D) Import of C-terminal Flag-tagged DOGDH into isolated mitochondria from dNrd1A and control larvae. An arrow indicates DOGDH-Flag proteins detected by anti-Flag antibody. S indicates 10% of starting protein for the assay. The reactions were stopped at different time points (2, 5, 10, 20, and 30 minutes) after mixing mitochondria and DOGDH-Flag protein. The isolated mitochondria were subjected to western blots with anti-Flag antibody. (E) Co-immunoprecipitation using anti-V5 antibody from S2 cell lysates overexpressing C-terminally V5-tagged DNRD1 and C-terminally Flag-tagged DOGDH, together with one of C-terminally Myc-tagged mitochondrial chaperone proteins. Western bots were performed using anti-Flag, anti-V5 or anti-Myc antibody. (F) Mitochondria isolated from wild-type or dNrd1 mutants expressing DOGDH-Flag (+/Y; UAS-dOgdh-Flag /+;da-Gal4/+ or dNrd1A/Y; UAS-dOgdh-Flag /+;da-Gal4/+) were incubated at the indicated temperature and aggregated proteins were precipitated by ultracentrifugation. Supernatants (S) and pellets (P) were analyzed by western blot with an antibody against Flag or ATP5A. (G) Mitochondrial chaperone assay of DNRD1 for DOGDH-Renilla luciferase (DOGDH-Rluc). In the presence of extra DNRD1, DOGDH-Rluc is ~2.2 fold more active (left) and upon heat-shock the loss of OGDH-Rluc activity is milder (right). Error bars indicate s.e.m. P values were calculated using Student’s t-test. **P <0.01, ***P <0.001. N.S. indicates not statistically significant. (H) Schematic representation of mitochondrial chaperones that play a role in import and protein folding, and metabolic pathways centered in the TCA cycle. See also Figure S5.

Figure 6

Figure 6. Identification of Patients with Neurodegenerative Phenotypes with Homozygous Deleterious Variants in NRD1 and OGDHL

(A) Familial segregation of the NRD1 variant (NM_002525.2:exon 17:c.1906_1907delAT: p.M636VfsX2). The NRD1 variant is homozygous in the proband and heterozygous or wild type in the parents and unaffected siblings. A sister (Red circle) was born with early onset developmental delay and failure to thrive. She died of unknown cause at 16 months of age. No additional clinical information and DNA was available. (B) Schematic representation of protein domains of human and fly NRD1 and position of stop codons of human NRD1 variant and fly alleles. (C) A T1 sagittal MRI image of the patient at age 8 years showing mild cerebellar volume loss (green asterisk). A T2 midaxial image shows enlarged extracerebral spaces (green arrows) compared to those in a control MRI. (D) Quantification of the on- and off-transients of ERG traces in 1 day-old and 35 day-old dNrd1A clones in PRs and 35 day-old dNrd1A clones expressing human NRD1 cDNA. Error bars indicate s.e.m. P values were calculated using Student’s t-test. **P <0.01. N.S. indicates not statistically significant. (E) Familial segregation of the _OGDHL_ variant (Chr10:50946295_G>A; OGDHL: NM_018245:exon18:c.C2333T:p.S778L). The OGDHL variant is homozygous in the proband and heterozygous in the parents and an unaffected sibling. (F) Schematic of protein domains of OGDHL and amino acid sequences around S778L. Dotted box (red) indicates a serine residue (red asterisk) that is mutated in the patient 2. Arrow (red) indicates a catalytic histidine. (G) A T1 sagittal MRI image from the patient 2 done at age 6 years showing a severely hypoplastic corpus callosum (green arrow) as well as an abnormal cerebellum (green asterisk). A control MRI image T1 sagittal sequence from a 5 year old child who was evaluated by neurology for migraine headaches showing a normal cerebellum and corpus callosum. (H) A schematic of the generation of dOgdh-T2A-Gal4 by Recombinase-Mediated Cassette Exchange (RMCE), and the translation of a Gal4 protein by a ribosomal skipping mechanism. The location of the Mi{MIC} insertion MI06026 is indicated by the red triangle. The T2A-Gal4 cassette consists of a splice acceptor (SA, light gray) followed by a linker (L, dark gray), a ribosomal skipping T2A peptide sequence (red), a Gal4 coding sequence (pale purple), a polyadenylation signal (pA, turquoise), and a splice donor (SD, light gray). Two inverted attB sites (blue) are positioned at the 5’- and 3’- end of the cassette (I) (Left table) Complementation test results of dOgdh-T2A-Gal4 alleles. +, complement; -, failure to complement. dOgdh-T2A-Gal4 complements the original MiMIC insertion (MI06026) that is not mutagenic (as it is inserted in the reverse orientation to the gene). dOgdh-T2A-Gal4 fails to complement a deficiency (Df(3L)ED4674) that lacks the dOgdh locus. These data indicate that dOgdh-T2A-Gal4 is a loss-of-function mutant. (Right table) dOgdh lethality can be rescued by expression of wild-type DOGDH or its human homologue but not by the mutant forms. Gal4 expression from dOgdh-T2A-Gal4 allowed dOgdh, dOgdhS793L, OGDH, or OGDHS791L to be expressed in dOgdh mutant background. See also Figure S6.

Figure 7

Figure 7. Loss of NRD1 or OGDH leads to an increase in TORC1 activity and decreases autophagy

(A-B) Western blots for the levels of phospho-S6K, S6K, phospho-4E-BP, p62, and Actin in dNrd1 mutants (dNrd1B) and its control (A), and in dOgdh RNAi (Act-Gal4/UAS-dOgdh RNAi) and control animals (UAS-dOgdh RNAi /+) (B). (C) Western blots for the levels of phospho-S6K, S6K, phospho-4E-BP, 4E-BP, LC3B and Actin in Nrd1ko MEFs (mNrd1−/−) and wild-type MEFs (mNrd1+/+) with or without rapamycin (100 nM). (D) Quantification of the on- and off-transients of ERGs in 35-day-old dNrd1B mutant clones in PRs with or without treatment of 3μM rapamycin and control PRs (dNrd1B; gRes-WT) (E) Quantification of the on- and off-transients of ERGs in 28-day-old PRs where dOgdh is knocked-down by RNAi (Rh1-Gal4/UAS-dOgdh RNAi) with or without treatment of 3μM rapamycin and control PRs (Rh-1-Gal4/UAS-empty vector). (F) Quantification of the on- and off-transients of ERGs in 35-day-old dNrd1B mutant clones in PRs with or without expressing Atg1 (ey-Gal4>UAS-Atg1) and control PRs (dNrd1B; gRes-WT) (D-F) Error bars indicate s.e.m. P values were calculated using Student’s t-test. *P <0.05, **P <0.01, ***P <0.001. N.S. indicates not statistically significant. (G) Model of neurodegeneration in NRD1 or OGDH mutants: Loss of NRD1 or OGDH leads to an elevation of α-KG and glutamine, which induces aberrant activation of mTORC1. Hyperactive mTORC1 signaling causes a decrease in autophagy, which leads to neurodegeneration. Restoring mTORC1 activity by treatment of rapamycin rescues neuronal defects in NRD1 and OGDH mutants. See also Figure S8.

References

    1. Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR. A method and server for predicting damaging missense mutations. Nat. Methods. 2010;7:248–249. -PMC -PubMed
    1. Ali YO, Allen HM, Yu L, Li-Kroeger D, Bakhshizadehmahmoudi D, Hatcher A, McCabe C, Xu J, Bjorklund N, Taglialatela G, et al. NMNAT2:HSP90 Complex Mediates Proteostasis in Proteinopathies. PLoS Biol. 2016;14:e1002472. -PMC -PubMed
    1. Barcelo H, Stewart MJ. Altering Drosophila S6 kinase activity is consistent with a role for S6 kinase in growth. Genesis. 2002;34:83–85. -PubMed
    1. Bellen HJ, Yamamoto S. BenchMarks Morgan ’ s Legacy : Fruit Flies and the Functional Annotation of Conserved Genes. Cell. 2015;163:12–14. -PMC -PubMed
    1. Bender T, Lewrenz I, Franken S, Baitzel C, Voos W. Mitochondrial enzymes are protected from stress-induced aggregation by mitochondrial chaperones and the Pim1/LON protease. Mol Biol Cell. 2011;22:541–554. -PMC -PubMed

MeSH terms

Substances

Grants and funding

LinkOut - more resources