Vertebrate-specific 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 potential. Currently, an increasing amount of data supports cells toward -induced , 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 (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 and catalyze reversible thiol- 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 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 1 08 −1 activity of cat . . s , which is comparable to mamma- neuronal function via redox regulated 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 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 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- 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 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 and 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). 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 5.50 μm, 0.18 0.09) com- pared to wild-type embryos (133.76 7.73 μm, 2.04 0.29) (mean 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 2.7 μm(37.4 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 1.2 μm(24.0 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 0.059 (0.52 0.08 without RA) in control – A and uninjected embryos at 16 22 hpf (Fig. 2 ), indicating that cells to 1.12 0.11 (0.95 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. 4, fected by reduced levels of zfGrx2, we examined secondary motor Fig. S3B). Whereas injection of the morpholino alone resulted in neurons, dopaminergic neurons, and glutamatergic excitatory the knockdown phenotype in all embryos (n ¼ 15), simultaneous interneurons. Independent from neuronal cell type, the number injection with zfGrx2 mRNA inhibited significantly induction of of those specific neurons was dramatically diminished (Fig. 2B), apoptosis (in 86% of embryos, n ¼ 15) as well as reduction of which resulted in severe behavioral changes of embryos. In con- axon tracts (in 68% of embryos, n ¼ 22). Moreover, oxidoreduc- trast to the stereotypical fast escape response which wild-type em- tase activity of zfGrx2 was required to rescue the knockdown bryos display upon mechanical stimuli, embryos lacking zfGrx2 phenotype. Simultaneous injection of mRNAs coding for mutants BIOCHEMISTRY

Fig. 2. Knockdown of zfGrx2 suppressed formation of a neuronal network. Morpholino-induced knockdown of zfGrx2 led to loss of early HuC/D positive neurons between 22 and 24 hpf (A). As demonstrated by different techniques, all tested neuronal subgroups were affected at 24 hpf: glutamatergic excitatory interneurons, dopaminergic neurons, and secondary motor neurons (B). Embryos lacking zfGrx2 were not able to develop an axonal scaffold (C). Ac, anterior commissure; dlt, dorsal longitudinal tract; nMLF, nucleus of the medial longitudinal fascicle; poc, posterior commissure; tg, trigeminal ganglion; vlt, ventral longitudinal tract. Scale bar, 50 μm.

Bräutigam et al. PNAS ∣ December 20, 2011 ∣ vol. 108 ∣ no. 51 ∣ 20533 Downloaded by guest on September 30, 2021 Coimmunoprecipitation experiments identified collapsin re- sponse mediator protein 2 (CRMP2) as potential interaction partner of hGrx2 in HeLa cells [Schütte and Lillig (20)]. We detected colocalization of zfGrx2 and zfCRMP2 in the of developing neurons (Fig. S5). Knockdown of zfCRMP2 by injection of a specific antisense morpholino as well as inhibition of zfCRMP2 by lacosamide mimicked the zfGrx2 knockdown phenotype. Axonal tracts and anterior brain commissures were reduced in 64% of 55 analyzed embryos by zfCRMP2 knockdown (Fig. 5A) and in 97% of 46 analyzed embryos by treatment with 2 μM lacosamide (Fig. S6A). Simultaneous injection of zfCRMP2 mRNA together with the morpholino blocking zfGrx2 expression decreased the amount of embryos with reduced axon scaffold from 81% in controls (n ¼ 16) to 37% (n ¼ 19) (Fig. 5B). This rescue indicated a functional interaction of zfGrx2 and zfCRMP2 during axonal outgrowth. To investigate if Grx2 changed the thiol redox state of CRMP2, we developed a two-dimensional (2D) redox blotting technique which is based on different pI values de- pendent on the thiol redox state via addition of two carboxyl groups to oxidized protein thiols and found indeed that zfCRMP2 thiols in zfGrx2 knockdown embryos were more oxidized com- pared to those in control embryos (Fig. 5C). Accordingly, rescue of the zfGrx2 knockdown phenotype by coinjection of zfCRMP2 mRNA was explained by increased amounts of reduced forms Fig. 3. Outgrowth of neurites depends on the presence of Grx2. (A) Length B and number of branching points of axons in embryos with silenced zfGrx2 of zfCRMP2 (Fig. S6 ). The redox state of CRMP2 was shifted expression were dramatically reduced 24 hpf, as demonstrated by immuno- to reduced forms in SH-SY5Y cells overexpressing hGrx2c histochemistry using antiacetylated tubulin antibodies (asterisks mark cell (Fig. 5D). To investigate, if CRMP2 is a direct substrate of Grx2, bodies). (B) Quantitative data obtained from A. White bars, wild-type; black we incubated recombinantly expressed and purified proteins bars, zfGrx2 knockdown (mean SEM, n ¼ 25, two-tailed Student’s t test, in different redox buffers based on the glutathione/glutathione −7 *** p < 3 × 10 ). (C) Three-dimensional reconstruction of the cytoskeleton disulfide (GSH/GSSG) couple (−180 and −300 mV) and re- (red, DNA; blue, tubulin; green, actin; single confocal layers as Insets; blue, þ∕− vealed a direct reduction of CRMP2 by the GSH/zfGrx2 redox DNA; yellow, actin) of SH-SY5Y cells grown for 8 d RA. (D) Quantification system (Fig. 5E). The redox state affected the secondary structure of axon lengths (−RA, n ¼ 247; +RA, n ¼ 156) and branching points (−RA, n ¼ 106; +RA, n ¼ 213) of three independent experiments corresponding of zfCRMP2. Using CD spectroscopy, we detected a time-depen- to C; white bars, control cells; black bars, hGrx2cþ cells (mean SEM, two- dent increase in ellipticity between 210 and 230 nm when reduced tailed Student’s t test, ***p < 0.008). zfCRMP2 was incubated with H2O2 for 1, 15, or 30 min (Fig. 5F). These spectroscopic changes induced by oxidation indicated a lacking the N- (C37S) or the C-terminal active site cysteine resi- conformational change of the protein that was reversed by the due (C40S) rescued formation of the axon scaffold only in 8% GSH/zfGrx2 redox system, or zfGrx2 alone (Fig. 5F). Observed (n ¼ 45) and 17% (n ¼ 26) of the embryos, respectively (Fig. 4). changes in both, thiol redox state and conformation were not Because the activity of zfGrx2 was essential, we dependent on the formation of a mixed disulfide between oxi- wondered if a general increase in oxidative stress was a cause of dized zfCRMP2 and zfGrx2 (expected molecular mass 75 kDa) the observed phenotype. However, determination of carbony- (Fig. 5G). lated proteins (Fig. S4A) and rescue experiments with zfGpx1 mRNA (Fig. S4B)—glutathione peroxidase (Gpx1) is a general Discussion antioxidant protein reducing hydrogen peroxide and is used to This study provides clear evidence that proper development of the embryonic nervous system is dependent on the oxidoreductase rescue oxidative stress-induced phenotypes (19)—demonstrated activity of cytosolic Grx2. We found that Grx2 regulates axonal out- that loss of zfGrx2 did not induce oxidative stress. Moreover, growth, survival of neurons, and development of a functional brain comparison of protein thiols in the whole zebrafish proteome by via the thiol redox state of CRMP2 representing a thiol redox regu- redox differential gel electrophoresis (redox-DIGE) (Fig. S4C) lated pathway essential for vertebrate embryonic development. excluded an effect of zfGrx2 knockdown on the overall redox So far, only monothiol Grxs were described to be essential for state of the proteome. vertebrate development (21, 22). In mice, transcription of Grx1 as The lack of a general effect on the redox state as well as well as mitochondrial and cytosolic Grx2 have been demonstrated requirement of both active site cysteine residues to rescue the in early embryogenesis (23). Our analysis showed that also zfGrx2 zfGrx2 knockdown phenotype suggested that redox regulation of is already present in the fertilized zebrafish egg and expressed a specific protein thiol group caused the disturbed formation of ubiquitously throughout the first 24 h of development, the critical the neuronal network. period when all major organs, including the brain, are formed (24). Zebrafish embryos lacking zfGrx2 lost virtually all types of neurons by apoptosis and the ability to establish an axonal scaf- fold, which severely impaired larval movements. We were not able to detect an increase of general oxidative stress in embryos lacking zfGrx2, but we could confirm that development of axon tracts depends on the oxidoreductase activity of Grx2 indicating Fig. 4. Formation of the axonal network depends on oxidoreductase activity that knockdown of zfGrx2 disrupts specific signaling pathways. of zfGrx2. Impaired formation of an axonal network (antiacetylated tubulin antibodies) could be rescued by injection of 20 pg∕embryo capped zfGrx2 Axon growth and guidance depends strongly on dynamic mRNA simultaneously to morpholino injection. Injection of mRNA coding modifications of the cytoskeleton (25). It was described before for active site mutants of zfGrx2 (zfGrx2C37S, zfGrx2C40S) did not rescue. that activation of actin and tubulin, the essential components of Scale bar, 50 μm. the cytoskeleton, are regulated by the GSH/Grx system (26, 27).

20534 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1110085108 Bräutigam et al. Downloaded by guest on September 30, 2021 Fig. 5. Grx2 acts via CRMP2. Knockdown of zfCRMP2 by morpholino injection impaired formation of axon scaffold (A). Impaired formation of axon scaffold by knockdown of zfGrx2 was rescued by simultaneous injection of 20 pg∕embryo capped zfCRMP2 mRNA (B). Densitometric analyses of the resulting pattern of CRMP2-specific spots after separation by 2D redox blots demonstrated Grx2-dependent changes in the thiol redox state of CRMP2 (C–E). At 24 hpf zfCRMP2 was more oxidized in embryos lacking zfGrx2 compared to wild-type (C), whereas in SH-SY5Y cells overexpressing hGrx2c the redox state of CRMP2 was more reduced compared to control cells (D). The redox state of recombinant zfCRMP2 was more reduced after incubation with recombinant zfGrx2 for 1 h under anaerobic conditions in a GSH/GSSG buffer adjusted to −300 mV compared to a buffer with a potential of −180 mV (E). Thiol redox modifications changed secondary structure of zfCRMP2 (F). (F, I ) Ellipticity of reduced recombinant zfCRMP2 (solid line) and after incubation with H2O2 for 1 min (dashed/dotted line), 15 min (dotted line), and 30 min (dashed line). (F, ii) Spectra of oxidized zfCRMP2 before (dashed line) and after incubation with GSH/zfGrx2 (solid line). (F, iii) Spectra of oxidized zfCRMP2 before (dashed line) and after incubation with reduced zfGrx2 at a ratio of 1∶1.2 (solid line). (G) A Coomassie blue-stained PAGE comparing quarternary structure of oxidized zfCRMP (60 kDa) before and after incubation with reduced zfGrx2 (16 kDa) at a ratio of 1∶1.2. Furthermore, a Grx-like domain of TXNRD1_v3, an isoform a tightly controlled balance of oxidized and reduced CRMP2 of 1, induces formation of cytoplasmic fila- might be essential to regulate both neuronal survival and axonal ments and filopodia in HEK293 cells (28). Interestingly, this outgrowth (Fig. 6). domain shares high homology with hGrx2 (29) and zfGrx2. CRMP2 is involved in several neurological disorders and it has One of the most important signaling pathways for the establish- been proposed that manipulation of CRMP2 activity, e.g., by the ment of an axonal scaffold is the semaphorin pathway (30). When epilepsy drug lacosamide (46), may offer unique opportunities for axon steering is required, the guidance cue semaphorin 3A therapy against some of these disorders (35, 47). (Sema3A) binds to the receptor pair neuropilin-1/plexin3A. Sub- In the brains of adult mice, rats, and humans Grx2 is mainly sequently, plexin3A conveys the signal via molecule interacting expressed in neurons (48–50) and it is suggested that mitochon- with CasL (MICAL) to CRMP2, which induces growth cone col- drial Grx2 may protect neurons against cell death in Parkinson’s lapse (31). Thereby CRMP2 controls axonal branching, guidance, disease by maintaining function of mitochondrial complex I and number of neurites (32–34). CRMP2 is essential during brain (48, 51). The interaction demonstrated here between Grx2 and development (35) and highly conserved across vertebrate species CRMP2 indicates that also cytosolic Grx2 might be important for (36). It is expressed in the major neural clusters of developing protection against neurological disorders. Alzheimer’s disease zebrafish embryos, especially between 16 and 24 hpf when the for- patients displayed a larger pool of oxidized CRMP2 (52) suggest- mation of the neuronal network takes place (37). The activity of ing a relation between a functional Grx2-dependent regulation of CRMP2 is subject to complex regulation, e.g., phosphorylation and CRMP2 and the ability to regenerate damaged adult neuronal alternative splicing (38). Here, we demonstrated that activity of networks. CRMP2 also depends on redox regulation by Grx2 both in zebra- In synopsis, our report highlights the significance of glutare- fish and human cells. Our results show that one or more disulfides

doxin-based thiol redox regulation during vertebrate embryonic BIOCHEMISTRY formed by the eight (mammalian CRMP2), respectively nine development and its potential importance in physiology and (zfCRMP2) cysteine residues are reduced by Grx2. Oxidation of pathophysiology of the brain. CRMP2 through MICAL was suggested before (39). During pre- paration of this manuscript it has been shown that CRMP2, which Materials and Methods was oxidized via hydrogen peroxide formation by MICAL upon Zebrafish Maintenance and Cell Culturing. Zebrafish maintenance and staging Sema3A stimulation, is a substrate for Trx (40). It was proposed followed standard protocols (24). SH-SY5Y cells were propagated in MEM that oxidized CRMP2 forms a complex with Trx which facilitates medium including 10% fetal calf serum, 2 mM penicillin/streptavidin, and 2 mM glutamine. For transient expression of tag-free human Grx2c, phosphorylation and growth cone collapse. Because of the differ- 6 ent pKa values of the active site cysteine residues and the resulting 5.5 × 10 SH-SY5Y cells were resuspended in 550 μL transfection buffer (21 mM Hepes, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4,6mMD-glucose, reaction mechanism and kinetics of Trx and other oxidoreductases, μ a stable mixed disulfide complex between Trx and CRMP2 is highly pH 7.15), mixed with 20 g of plasmid pEGFP-N1-Grx2c (11) and electro- unlikely (41). Between Grx2 and CRMP2 there is no formation of a stable complex. Therefore, we favor an alternative model in which redox regulation of CRMP2 induces conformational changes supported by the in vitro data using CD spectrocopy. These changes could, for instance, be essential for the exposure of phos- phorylation sites. A delicate balance of Sema3A signaling is also essential for survival of immature neurons (42, 43). Disruption of the sema- phorin signaling pathway decreases the rate of cell death in dorsal Fig. 6. Model of the role of Grx2 during brain development. A redox circuit consisting of Grx2 and MICAL regulates the thiol redox state of CRMP2. In root ganglions (DRG), whereas overexpression of Sema3A in- response to semaphorin signaling MICAL oxidizes CRMP2, whereas Grx2 re- duces apoptosis in embryonic DRG (44, 45). This overexpression duces CRMP2. The balance between reduced and oxidized CRMP2 regulates might lead, as described above, to overactivation of MICAL and axonal outgrowth. Disturbance of thiol redox regulation of CRMP2 can lead to subsequently to an increased pool of oxidized CRMP2, which we loss of the axonal scaffold and to neuronal apoptosis. Therefore, oxidoreduc- also have observed in zfGrx2 knockdown embryos. Therefore, tase activity of Grx2 is essential for the formation of a neuronal network.

Bräutigam et al. PNAS ∣ December 20, 2011 ∣ vol. 108 ∣ no. 51 ∣ 20535 Downloaded by guest on September 30, 2021 porated by a single pulse of 250 V and 1,500 μF. Differentiation of SH-SY5Y phosphine (TCEP). After removal of the excess reductant using an NAP-5 col- cells was induced by 10 μM RA. umn (GE healthcare), spectra were recorded using a Jasco spectropolari- ometer. Subsequently, reduced protein was incubated with 0.5 mM H2O2. Cloning and Recombinant Protein Expression. The ESTs IMAPp9981714884Q1 Next, in an enzymatic approach oxidized zfCRMP2 was incubated with (zfGrx2) and IRAKp961K11293Q (zfCRMP2) were purchased through Ima- 0.5 mM GSH and 0.1 μM recombinant zfGrx2 before ellipticity of rereduced Genes and the respective ORFs amplified by PCR (for all primer sequences zfCRMP2 was recorded. In a single turnover approach 10 μM oxidized see Table S1). Mutations were generated by rolling circle PCR. zfGpx1 was zfCRMP2 was incubated with 12 μM reduced zfGrx2 (incubated for 30 min amplified from whole adult zebrafish cDNA. PCR products were subcloned with 10 mM DTT and 10 mM TCEP followed by gel filtration using an into pet15b (Novagene) to express the respective His-tagged proteins in NAP-5 column). Ellipticity of reduced zfGrx2 was used as reference. Escherichia coli BL21 codon plus (DE3)-RIL cells (Stratagene) as previously described (13). Protein concentrations were determined by absorption coef- Preparation of Whole Zebrafish Embryo Extracts and Cell Extracts. Whole zeb- ficients (zfGrx2, 3;480 M∕cm at 280 nm; zfCRMP2, 64;200 M∕cm at 280 nm ). rafish embryo extracts were prepared following a previously published pro- The enzymatic activity of glutaredoxins was measured by the HED assay (53). tocol (54) with some modifications. Embryos were dechorionated and incubated in preblocking buffer (100 mM N-ethyl maleimide in PBS) for Treatment of Zebrafish Embryos. The morpholino oligomer knocking-down 30 min. Yolk sack was removed by pipetting up and down in deyolking buffer zfGrx2 (5′-GTTGAAGATACTAGGAAAGCAAACG-3′) targets the direct up- (55 mM NaCl, 1.8 mM KCl, 1.25 mM NaHCO3) followed by centrifugation for stream sequence of the ATG codon, the morpholino knocking-down 30 s at 300 g. SH-SY5Y cells were harvested by trypsination and washed zfCRMP2 (5′-TCACTCTGGAAACACAGATAAACAC-3′) targets the splice accep- with PBS. Pellets of zebrafish and SH-SY5Y cells were taken up in preblocking tor of the second and thereby inhibits correct splicing of both isoforms buffer and the fourfold volume of lysis buffer (150 mM NaCl, 5 mM EDTA, zfCRMP2a and b. Both morpholinos (GeneTools) were dissolved to a concen- 100 mM N-ethylmaleimide, 8 M urea in 100 mM Na-phosphate buffer tration of 3 mM in dH2O and diluted 1∕40 (Grx2) or 1∕10 (CRMP2) in injection pH 7.2), and 2% (wt∕vol) CHAPS was added followed by a 20 min anaerobic buffer (9 μM spermine, 0.21 mM spermidine, and 0.3% phenol red in PBS). incubation at room temperature and snap freezing. Single cell eggs were injected with 1.5 nL using a Femtojet microinjector (Eppendorf). Capped mRNA was generated with the mMessage/Machine SDS-PAGE and Western Blotting. SDS-PAGE and Western blotting were per- Kit (Ambion). The ORFs of zfGrx2, zfGrx2C37S, zfGrx2C40S, zfCRMP2, and formed using Novex MiniCells (Invitrogen), precasted Precise gels (Thermo zfGpx1 were cloned into pCS2 and used as template. Lacosamide (VIMPAT) Scientific, Invitrogen), and PVDF membranes (Macherey and Nagel). Horse was purchased through a local pharmacy. radish peroxidase labeled immunocomplexes were stained with SuperSignal West Pico/Femto (9∶1) luminescence kit (Thermo Scientific). Carbonylated In Situ Hybridization and Immunohistochemistry. In situ hybridization including proteins were visualized by formation and detection of derivatives with generation of riboprobes as well as zebrafish whole mount immunohisto- 2,4-dinitrophenylhydrazin after SDS-PAGE and Western blotting transfer chemistry was performed according to standard protocols. To stain sections, of 6.4 μg zebrafish extract using antidinitrophenol antibodies (BP424, Acris embryos were fixed in 4% paraformaldehyde and either embedded in par- Antibodies). affin, or in tissue tek (Sakura Finetek) and frozen in liquid nitrogen. Sections (5–10 μm) were cut with a microtom (Micron) or a CM1900 UV cryostat (Leica). Two-Dimensional Redox Blot and Redox-DIGE. For 2D redox blots and redox- Paraffin sections were deparaffinized and antigens were demasked by DIGE, pellets of N-ethyl maleimide treated proteins [2D redox blots, 200 μgof heating in 10 mM citrate buffer pH 6.0 for 10 min and nonspecific antibody zebrafish extract; 10 μg of SH-SY5Y extract; or recombinant proteins, 4.8 μM binding sites were blocked with 5% normal goat serum (Invitrogen). For im- zfCRMP2 was incubated for 1 h under anaerobic conditions with 0.048 μM munohistochemical analysis of SH-SY5Y cultures, cells were seeded on glass zfGrx2 in GSH/GSSG buffers adjusted to −180 (6.06 mM GSH/3.94 mM GSSG) plates coated with 0.5 μg∕μL fibronectin and propagated as described above. and −300 mV (9.999 mM GSH/0.001 mM GSSG); redox-DIGE, 10 μg zebrafish After washing with PBS and fixation with 4% paraformaldehyde, cells were extract] were resuspended in 100 mM Na-phosphate buffer pH 7.2 containing permeabilized for 1 h in PBS containing 10 mM Hepes, 3% BSA, 0.3% Triton 150 mM NaCl, 5 mM EDTA, and 8 M urea. Previously oxidized thiols were X-100. Primary antibodies were dissolved in this buffer and incubated over- reduced with 10 or 0.2 mM TCEP. For 2D blots, the newly generated thiols night at 4 °C, secondary antibodies for 1 h at room temperature. Fixed cells were labeled by incubation with 20 mM 5-maleimido isophtalic acid. After and embryo sections were mounted using Mowiol 4–88 (Carl Roth). Rabbit precipitation with 12% trichloroacetic acid, proteins were washed with anti-zfGrx2 was produced as described before (50), other primary antibodies aceton, rehydrated in 8 M urea, 1% Nonidet P-40 (NP-40), 20 mM DTT, obtained from different suppliers and sources: actin (sc-47778, Santa Cruz) , 0.5% ampholytes, separated by two dimensions (first pH 3–10 for fish ex- tubulin (T9026, Sigma), CRMP2 (C2993, Sigma; sc-25895, Santa Cruz), acety- – lated tubulin (T6793, Sigma), zn5 (ZIRC), HuC/D (A21271, Invitrogen). Nuclei tracts, pH 4 7 for SH-SY5Y extracts, second molecular weight) according to ’ were counterstained with Hoechst 33342 (Sigma), mitochondria with prohi- supplier s manual (Invitrogen), and transferred to PVDF membrane as de- bitin (Calbiochem), cytosol/nuclei with DNaseI labeled with Alexa-488 (Invi- scribed above. For redox-DIGE, the newly generated thiols in the different trogen), and actin fibers with phalloidin labeled with Alexa Fluor-546 samples were labeled by incubation with 0.4 mM Cy3, respectively, Cy5 mini- (Invitrogen). As secondary antibodies horseradish peroxidase conjugated mal CyDye (GE healthcare). After inactivation of free dye by 65 mM DTT, un- μ IgGs (BioRad, Sigma), alkaline phosphatase conjugated antibodies (Sigma), labeled extract (40 g) was added, the two samples were pooled in 8 M urea, Alexa Fluor-488 and -633 labeled antibodies, and Cy2, and Cy5 (Invitrogen), 20 mM DTT, 1% NP-40, 0.5% ampholytes, and separated as described before and Cy3 (Millipore) labeled antibodies were used. at the same gel. The 2D gel was scanned using a Typhoon Trio scanner (GE healthcare). Yellow color is the result of an overlay of Cy3 and Cy5. Detection of Apoptosis. Active caspase 3 was detected in vivo using the Caspase-3 DEVD-R110 Fluorometric HTS Assay Kit (Biotium). Embryos were Computational Analysis and Statistics. Primary sequence data was analyzed dechorionized and transferred into 200 μL PBS containing 7 μL substrate using BLAST and Align2seq. Deconvolution, three-dimensional reconstruc- solution, incubated 15 min and washed several times with PBS containing tion, and maximum intensity projection of confocal microscopy pictures were 0.1% Tween-20. Prior to analysis by fluorescence microscopy, embryos were computed using the software package Huygens (Scientific Volume Imaging). anesthetized with tricaine and mounted in 3% methylcellulose. In addition, Neurite lengths were measured using Adobe Photoshop CS5, densitometric apoptotic cells were detected using the in situ cell death detection Kit (Roche) measurements were performed using ImageJ. For statistical analyses the two- in conjunction with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phos- tailed Student’s t test was used. phate staining. ACKNOWLEDGMENTS. We thank Sabrina Oesteritz, Gisela Lesch (Philipps- Microscopy. Stained cells or zebrafish were analyzed under a Leica MZ16 University) and Iris Söll, Lena Ringdén (Karolinska Institutet) for technical microscope equipped with a Leica DFC300FX camera, a Zeiss Axioplan micro- and administrative assistance, Vadim N. Gladyshev, Leonard I. Zon (Harvard scope equipped with a Zeiss Axiocam digital camera, a Leica Diaplan micro- Medical School), and Orhan Aktas (Heinrich-Heine University) for valuable scope equipped with a MicroPublisher camera (Qimaging), a Leica TCS SP2 discussions as well as Shin-ichi Higashijima (Okazaki Institute for Integrative Bioscience) for providing the zebrafish alx-GFP transgenic line. This work was instrument using a 40x oil plan apochromat lens (Leica), or an Olympus supported by the Deutsche Forschungsgemeinschaft BE 3259/2 (to C.B.), BX51 microscope equipped with a U-CMAD3 digital camera. SFB593-N01 (to C.H.L.), the Karolinska Institutet (G.H., C.B.), Karolinska Insti- tutet fellowship for new PhD students Dnr. 2379/07-225 (to L.B.), the Swedish CD Spectroscopy. Twenty-five micromolars recombinant zfCRMP2 were com- Cancer Society Grant 961 (to A.H.), the Swedish Research Council (A.H.), and pletely reduced by incubation with 1 mM DTT and 1 mM Tris(2 carboxyethyl) the P. E. Kempkes foundation (C.B.).

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