Vertebrate-specific glutaredoxin is essential for brain development Lars Bräutigama,b, Lena Dorothee Schüttec, José Rodrigo Godoyc, Timour Prozorovskid, Manuela Gellertc, Giselbert Hauptmannb, Arne Holmgrena, Christopher Horst Lilligc,1, and Carsten Berndta,d,1 aDivision of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles Väg 2, 17177 Stockholm, Sweden; bDepartment of Biosciences and Nutrition, Karolinska Institutet, Novum, 14183 Huddinge, Sweden; cDepartment for Clinical Cytobiology and Cytopathology, Philipps-University, Robert-Koch-Strasse 6, 35037 Marburg, Germany; and dDepartment of Neurology, Medical Faculty, Heinrich-Heine University, Merowinger Platz 1A, 40225 Düsseldorf, Germany Edited by Pasko Rakic, Yale University, New Haven, CT, and approved October 13, 2011 (received for review June 22, 2011) Cellular functions and survival are dependent on a tightly controlled Knockdown of the mitochondrial Grx2 isoform sensitized HeLa redox potential. Currently, an increasing amount of data supports cells toward oxidative stress-induced apoptosis, whereas its over- the concept of local changes in the redox environment and specific expression increased resistance against various apoptotic stimuli redox signaling events controlling cell function. Specific protein thiol (14, 15). Protection against mitochondrial oxidative stress is con- groups are the major targets of redox signaling and regulation. nected to Grx2-mediated redox modifications of specific cysteine Thioredoxins and glutaredoxins catalyze reversible thiol-disulfide residues of mitochondrial complex I (16). exchange reactions and are primary regulators of the protein thiol Although the importance of cellular redox control during redox state. Here, we demonstrate that embryonic brain develop- embryonic development is well-established, no specific thiol re- ment depends on the enzymatic activity of glutaredoxin 2. Zebrafish dox regulation events have been identified so far. Here, we have with silenced expression of glutaredoxin 2 lost virtually all types of discovered an essential role of the vertebrate-specific oxidore- neurons by apoptotic cell death and the ability to develop an axonal ductase Grx2 for brain development. Using the powerful model scaffold. As demonstrated in zebrafish and in a human cellular organism zebrafish (zf), Danio rerio, as well as a human cellular model for neuronal differentiation, glutaredoxin 2 controls axonal model, we discovered that regulation of axonal outgrowth, survi- outgrowth via thiol redox regulation of collapsin response mediator val of neuronal cells, and subsequent formation of a functioning protein 2, a central component of the semaphorin pathway. This neuronal network depends on the enzymatic activity of cytosolic study provides an example of a specific thiol redox regulation essen- Grx2. tial for vertebrate embryonic development. Results The genome of zebrafish is fully sequenced and its latest annota- embryonic development ∣ axonogenesis tion (zv9) contains open reading frames for two dithiol Grxs. Sequence analysis revealed that the protein encoded by the gene he development of any multicellular organism depends on the 436677 (zgc: 92698) is the zebrafish homologue to hGrx2 (17). It Tcomplex interaction of fundamental cellular processes like lacks a mitochondrial target sequence and is most closely related proliferation, differentiation, migration, and apoptosis. Intensive to hGrx2c (Fig. S1A). In contrast to hGrx2, zfGrx2 possesses the research during the last decade has established an essential role common Grx active site motif C-P-Y-C. The two conserved extra of the cellular redox state in all these processes and thereby in structural cysteines are present, which connected zfGrx2 to a embryonic development (1, 2). It has been shown that the redox vertebrate-specific group (Fig. S1B). We determined with the state regulates differentiation of neural progenitors, naturally Grx-specific hydroxyethyl disulfide (HED) assay an enzymatic occurring death of postmitotic neurons during development, and k ¼ 4 23 Æ 1 08 −1 activity of cat . s , which is comparable to mamma- neuronal function via redox regulated transcription factors (3, 4). lian Grx2 (18). Currently, we experience a paradigm shift from the importance ZfGrx2 was expressed in the four cell stage and ubiquitously of a global cellular oxidant/antioxidant balance toward specific distributed in zebrafish embryos throughout development signaling events mediated by reversible redox modifications of (Fig. 1A, Fig. S2). Silencing of the zfGrx2 expression by injection protein thiol groups (5, 6). The oxidoreductases that control the of a specific antisense morpholino (Fig. 1 A and B) induced apop- redox state of these thiol groups are thioredoxins (Trxs) and glu- tosis in the CNS from midsomitogenesis until approximately taredoxins (Grxs). They are members of the thioredoxin family 30 hours post fertilization (hpf) (Fig. 1 C–F). Apoptotic cells were of proteins which are characterized by a common fold of three confined to the eye, the anterior brain, the hindbrain, and the beta sheets and four alpha helices including a cis-proline and a spinal cord in all embryos lacking zfGrx2 (n ¼ 125), whereas C-X-X-C (where C is cysteine and X any other amino acid) active uninjected control embryos showed only few scattered apoptotic site motif (7). Grxs reduce protein disulfides using both cysteinyl cells (n ¼ 76). residues of their active site, whereas the reduction of disulfides Knockdown of zfGrx2 was accompanied by extensive loss of between protein thiols and glutathione depends only on the early differentiated neurons at 24 hpf in 97% (n ¼ 34) of ana- N-terminal cysteinyl residue (6). The genome of most vertebrates lyzed embryos (Fig. 2A). The number and distribution of newborn encodes four Grxs, the dithiol Grxs 1 and 2 and the monothiol neurons did not substantially differ between morpholino injected Grxs 3 and 5, with the latter being a subfamily characterized by a C-X-X-S active site motif (6). Author contributions: L.B., C.H.L., and C.B. designed research; L.B., L.D.S., J.R.G., T.P., and Human Grx2 contains an atypical active site motif (C-S-Y-C, M.G. performed research; G.H. contributed new reagents/analytic tools; L.B., C.H.L., and consensus sequence: C-P-Y-C) and two extra cysteines forming a C.B. analyzed data; and L.B., A.H., C.H.L., and C.B. wrote the paper. – structural disulfide bond (8 10). Alternative splicing and tran- The authors declare no conflict of interest. scription initiation of the human gene GLRX2 gives rise to three This article is a PNAS Direct Submission. different isoforms, the mitochondrial Grx2a and the cytosolic 1To whom correspondence may be addressed. E-mail: [email protected] or horst@ Grx2b and Grx2c (11). It was proposed that under conditions lillig.de. ½2 2 2þ of oxidative stress Grx2a is activated by the loss of its Fe S This article contains supporting information online at www.pnas.org/lookup/suppl/ cluster, which might therefore serve as a redox sensor (12, 13). doi:10.1073/pnas.1110085108/-/DCSupplemental. 20532–20537 ∣ PNAS ∣ December 20, 2011 ∣ vol. 108 ∣ no. 51 www.pnas.org/cgi/doi/10.1073/pnas.1110085108 Downloaded by guest on September 30, 2021 reacted to the stimulus but swam away slowly in an uncoordi- nated, circlewise fashion (Movie S1). The onset of neuronal degradation coincides with the time window of initial axon tract formation. Therefore we visualized the axon scaffold and revealed a severe reduction of tracts in nearly all zfGrx2 knockdown embryos at 24 hpf (94%, n ¼ 49) compared to uninjected controls (n ¼ 21). Both anterior brain commissures as well as the major longitudinal axon tracts were strongly reduced in knockdown embryos and the trigeminal gang- lion was almost undetectable (Fig. 2C). Already the maturation of axon tracts was disturbed as essentially no axons could be detected at 18, 20, and 22 hpf, the time frame when the scaffold developed in wild-type embryos (Fig. 2C). Length and number of branching points of single axons were significantly reduced in embryos lacking zfGrx2 (32.86 Æ 5.50 μm, 0.18 Æ 0.09) com- pared to wild-type embryos (133.76 Æ 7.73 μm, 2.04 Æ 0.29) (mean Æ SEM, n ¼ 25, Fig. 3 A and B). To confirm the effect of cytosolic Grx2 on the outgrowth of neurites in a cellular system, we chose the human cell line SH-SY5Y as a model for neuronal differentiation. Cells were transfected with constructs allowing transient expression of the cytosolic isoform of human Grx2, hGrx2c. Morphology of control cells and hGrx2c overexpressing cells was compared with and Fig. 1. Knockdown of zfGrx2 induced apoptosis in central nervous system. þ Injection of a morpholino blocking zfGrx2 translation reduced the levels without induction of differentiation by RA (Fig. 3C). hGrx2c of the corresponding protein as visualized by whole mount immunohisto- SH-SY5Y cells displayed significantly longer neurites with chemistry (A and B). Loss of zfGrx2 induced apoptosis in the central nervous 42.2 Æ 2.7 μm(37.4 Æ 1.4 μm without RA) compared to those of system as demonstrated by in vivo caspase 3 measurements (C and D) and wild-type cells with 31.1 Æ 1.2 μm(24.0 Æ 0.7 μm without RA). TUNEL staining (E and F). Scale bar, 50 μm. The number of branching points per neurite increased signifi- cantly from 0.46 Æ 0.059 (0.52 Æ 0.08 without RA) in control – A and uninjected embryos at 16 22 hpf (Fig. 2 ), indicating that cells to 1.12 Æ 0.11 (0.95 Æ 0.19 without RA) in hGrx2cþ cells the majority of neurons were lost between 22 and 24 hpf. In fact, (Fig. 3D). neurons were identified as the predominant cell type undergoing We verified that the above described phenotypes in zebrafish apoptosis at 24 hpf following knockdown of Grx2 (Fig. S3). embryos were caused specifically by the loss of zfGrx2 by injection To investigate if specific neuronal subpopulations were af- of zfGrx2 capped mRNA together with the morpholino (Fig.