Molecular and Cellular Neuroscience 65 (2015) 135–142

Contents lists available at ScienceDirect

Molecular and Cellular Neuroscience journal homepage: www.elsevier.com/locate/ymcne

WNK1 is involved in Nogo66 inhibition of OPC differentiation Zhao-Huan Zhang b,1, Jiao-Jiao Li a,1, Qing-Jin Wang a, Wei-Qian Zhao a, Jiang Hong a, Shu-jie Lou c,⁎, Xiao-Hui Xu a,⁎ a b c

School of Life Sciences, Shanghai University, Shanghai 200444, China Department of Neurology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China Key Laboratory of Exercise and Health Sciences, Ministry of Education, Shanghai University of Sport, Shanghai 200438, China

a r t i c l e

i n f o

Article history: Received 28 May 2014 Revised 27 February 2015 Accepted 4 March 2015 Available online 6 March 2015 Keywords: LINGO-1 WNK1 Oligodendrocyte Differentiation Inhibition

a b s t r a c t LINGO-1 is a transmembrane receptor expressed primarily in the central nervous system (CNS) and plays an important role in myelination. Recent studies have indicated that it is also involved in oligodendrocyte precursor cell (OPC) survival and differentiation; however, the downstream signaling pathway underlying OPC development is unknown. In our previous study, we found that LINGO-1 is associated with WNK1 in mediating Nogoinduced neurite extension inhibition by RhoA activation. In an effort to identify the role of LINGO-1-WNK1 in OPCs, we first confirmed that WNK1 is also expressed in OPCs and co-localized with LINGO-1, which suppresses WNK1 expression by RNA interference-attenuated Nogo66-induced inhibition of OPC differentiation. Furthermore, we mapped the WNK1 kinase domain using several fragmented peptides to identify the key region of interaction with LINGO-1. We found that a sequence corresponding to the D6 peptide is necessary for the interaction. Finally, we found that using the TAT-D6 peptide to introduce D6 peptide into primary cultured OPC inhibits the association between LINGO-1 and WNK1 and significantly attenuates Nogo66-induced inhibition of OPC differentiation. Taken together, our results show that WNK1, via a specific region on WNK1 kinase domain, interacts with LINGO-1, thus mediating Nogo66-inhibited OPC differentiation. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Autoimmune-mediated demyelination of central nervous system (CNS) axons is a classic hallmark of multiple sclerosis (MS), which leads to axonal dysfunction and, eventually, transection (McFarland et al., 1992). Although remyelination of demyelinated axons by oligodendrocytes can occur during the early stages of the disease, this regenerative process is not sustained (Blakemore and Franklin, 2008; Jeffery and Blakemore, 1997). A likely contributing factor towards the loss of remyelination in later stages of the disease is the attenuation of the differentiation of oligodendrocyte precursor cells (OPCs), which actively migrate into demyelinated areas (Franklin, 2002). A possible explanation for the failure of OPC differentiation in MS is the presence of inhibitors within demyelinated lesions. A number of potential inhibitors have been proposed, including myelin debris, which accumulate in lesions as a result of demyelination (Kotter et al., 2006). Kotter et al. (2006) also suggested that myelin-mediated inhibition of OPC differentiation is mediated by Src-family (Fyn-1)–RhoA-ROCK-II signaling or protein kinase C (PKC) signaling (Baer et al., 2009); however, the complete signal transduction pathways downstream to the myelin debris remain unknown. Recently, the CNS transmembrane protein LINGO-1 (leucine rich repeat and Ig domain containing Nogo receptor interacting protein-1) ⁎ Corresponding authors. E-mail addresses: [email protected], [email protected] (S. Lou). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.mcn.2015.03.003 1044-7431/© 2015 Elsevier Inc. All rights reserved.

was shown to be a key negative regulator of oligodendrocyte differentiation, as well as myelination and remyelination (Lee et al., 2007; Mi et al., 2005, 2007). LINGO-1 is a component of the tripartite receptor complexes, which acts as a convergent mediator of the intracellular signaling in response to myelin-associated inhibitors which exist in myelin debris and lead to the inhibition of neurite extension (Mi et al., 2004). Our previous work has suggested that WNK1 (with no lysine (K) kinase 1)-mediated Nogo66 (one of the myelin inhibitors) induces LINGO-1 receptor activation and then activates Rho-A, thus inhibiting cultured neuron process extension (Zhang et al., 2009). In this work, we investigated whether WNK1 mediates Nogo66 inhibition on oligodendrocyte maturation through LINGO-1 as it does in neuron development. We also asked whether interrupting the interaction of endogenous LINGO-1 with WNK1 could disrupt the signal transduction leading to activating Rho-A and therefore promote oligodendrocyte maturation in the presence of myelin inhibitors. 2. Results 2.1. LINGO-1 and WNK1 are both expressed in oligodendrocytes and interact with each other We studied the expression of LINGO-1 and WNK1 in oligodendrocytes. Differentiation of oligodendrocyte precursor cells into myelinating oligodendrocytes can be defined in 7 stages (Boulanger and Messier,

136

Z.-H. Zhang et al. / Molecular and Cellular Neuroscience 65 (2015) 135–142

2014). Using the immunofluorescence method, we found that LINGO-1 and WNK1 were expressed in enriched cultures of oligodendrocytes at early (NG2 or A2B5 positive pre-oligodendrocytes) or late developmental stages (O4 positive immature oligodendrocytes, MBP positive nonmyelinating mature oligodendrocytes) (Fig. 1 A). In addition, the two proteins were co-immunoprecipitated from the cell lysates of cultured oligodendrocytes (Fig. 1 B), indicating mutual interaction. Next, we determined co-localization of LINGP-1 and WNK1 and found that both proteins did have some co-localizations in oligodendrocytes (Fig. 1C). Thus the LINGO-1–WNK1 signal transduction pathway may play a role in oligodendrocytes.

2.2. Nogo66 inhibits OPC differentiation It has been reported that OPCs also express the Nogo66 receptor (NgR) and LINGO-1 receptor (Guo et al., 2007; Huang et al., 2012), and that the NgR/LINGO-1 complex inhibits optic nerve oligodendrocyte differentiation (Kwon et al., 2014). We performed an in vitro assay to assess the effects of Nogo66 on OPC differentiation following the methods described. The neonatal OPCs were seeded on Nogo66 substrates produced by the incubation of GST-Nogo66 proteins on poly-Llysine covered culture dishes and the differentiation of oligodendrocyte

lineage cells in vitro was characterized by cell membrane area and immunological features. We found that cell membrane expansion was significantly reduced on GST-Nogo66-treated compared with the GSTtreated cells (Fig. S1A, B). We also found that MBP (mature oligodendrocyte marker) positive mature oligodendrocytes are significantly decreased when cultured on Nogo66 substrates while the NG2 (OPC marker) positive cells are not changed (Fig. 2A, B), which indicates that Nogo66 can significantly inhibit OPC differentiation.

2.3. TAT-LINGO-1-IC directly inhibits OPC differentiation The inhibition signal transduction of Nogo66 may be mediated by LINGO-1-IC, the intracellular domain of LINGO-1 (amino acids 580–620) (Zhang et al., 2009). The HIV-transactivator of transcription (TAT) contains a protein transduction domain that can be linked to other proteins to facilitate their delivery into cells. We expressed and purified TATLINGO-1-IC in vitro. OPCs were seeded on coverslips in differentiation medium with or without TAT-LINGO-1-IC (10 μM) for 48 h of incubation. As shown in Fig. 3A, the MBP positive mature oligodendrocytes are significantly decreased when treated with TAT-LINGO-1-IC, suggesting that TAT-LINGO-1-IC can significantly inhibit OPC differentiation as the Nogo66 does. These results indicate that excessive LINGO-IC can directly

A A2B5

Merge

WNK1

A2B5

Merge

LINGO-1

O4

Merge

WNK1

O4

Merge

LINGO-1

MBP

Merge

WNK1

MBP

Merge

LINGO-1

Merge

IP:

IB: WNK1

IB: LINGO-1

5% Input: WNK1

5% Input: LINGO-1

Anti-WNK1

C Con IgG

IP:

Con IgG

B

Anti-LINGO-1

LINGO-1

WNK1

Fig. 1. The developmental expression of LINGO-1 and WNK1 in cultured OPCs. (A) Primary cultured OPCs were differentiated for 1, 3, and 5 days and analyzed for LINGO-1 and WNK1 expression by immunocytochemical staining. The distribution of LINGO-1, WNK1 and A2B5, O4, or MBP is shown by immunofluorescence. Bar = 50 μm. (B) immunoprecipitation (IP) and immunoblot (IB) shows endogenous WNK1 interacts with LINGO-1 in cultured OPCs. (C) Primary cultured OPCs were differentiated for 5 days and then stained for endogenous LINGO-1 and WNK1 to reveal their co-localization. Bar = 50 μm.

Z.-H. Zhang et al. / Molecular and Cellular Neuroscience 65 (2015) 135–142

A

NG2

137

Merge

DAPI

n.s.

30 20 10

***

20 15 10 5 0

l

0

***

25

LI T-

on C TA

T-

C

tro

l tro on C

LI TTA

on C TA

T-

C

tro

l tro on C

TA

40

Percentage of MBP+ cells (%)

C

50

l

B

Percentage of NG2+ cells (%)

TAT-LIC

TAT-Control

Control

MBP

Fig. 2. Nogo66 inhibits OPC differentiation. (A) OPCs cultured in differentiation medium for 72 h; representative images of cells showing the NG2 or MBP positive cells in different groups. Bar = 50 μm. (B) Quantitative analysis of the relative percentages of NG2 or MBP positive cells in different treatment groups (n ≥ 1702 cells in each group from 3 independent experiments). n.s. = non-significant; ***P b 0.001 vs. control; one-way ANOVA with Student's t-test.

mediate OPC differentiation inhibition (Fig. 3B, C). Since we previously found that LINGO-1-IC directly binds to WNK1 when Nogo66 is stimulated (Zhang et al., 2009), we then investigated the role of WNK1 in OPC differentiation. 2.4. WNK1 mediates the inhibitory effect of myelin debris on oligodendrocyte maturation To identify a possible physiological role of WNK1, the RNAi approach was employed. OPCs were transfected with either shWNK1 or scrambled and cultured on Nogo66 substrates for 5 days. Due to the low transfection efficiency, we used the OPC membrane area since it rapidly increases during OPC development as a new statistic index. We have shown that cell membrane expansion is significantly decreased on GST-Nogo66-treated compared with the GST-treated cells (Fig. S1A, B). When shWNK1 was transfected, the cell membrane was significantly expanded in Nogo66-

treated cells (Fig. 4A, B) but not in GST-treated cells, suggesting that the knockdown of endogenous WNK1 specifically abrogates Nogo66 inhibition on OPC maturation. Furthermore, when a human full-length WNK1 (Zhang et al., 2009), which is resistant to the RNAi approach, was cotransfected with shWNK1, the effect of shWNK1 on OPC differentiation in the presence of Nogo66 was abolished, further indicating the specific role of shWNK1. Therefore, endogenous WNK1 mediates the inhibitory effect of Nogo66 on OPC maturation. We also found that the effects of TAT-LINGO-IC inhibit OPCs maturation is attenuated by WNK1 knockdown (Fig. S2A, B). 2.5. Interrupting the interaction of LINGO-1 with WNK1 by specific peptides promotes OPC maturation on Nogo66 substrates In order to map the WNK1 domains that mediate the interaction with LINGO-1, we generated seven constructs from WNK1 (123–

138

Z.-H. Zhang et al. / Molecular and Cellular Neuroscience 65 (2015) 135–142

A

MBP

DAPI

Merge

5 0

6 o6 og

6 o6 G

ST -N

og

G

***

10

ST -N

0

15

ST

10

20

G

20

25

G

30

C

Percentage of MBP+ cells (%)

n.s.

40

ST

B

Percentage of NG2+ cells (%)

GST-Nogo66

GST

NG2

Fig. 3. TAT-LINGO-IC directly inhibits OPC differentiation from immature to mature stage. (A) OPCs cultured in differentiation medium for 72 h with TAT-LINGO-IC peptide (TAT-LIC 10 μM), control peptide (10 μM), or not were immunostained with anti-NG2 and anti-MBP. (B, C) Quantification of the percentage of NG2 or MBP cells respectively (n ≥ 1796 cells in each group from 3 independent experiments). Bar = 50 μm. n.s. = non-significant; ***P b 0.001 vs. control, Student's t-test.

510aa) that had been identified to bind to LINGO-1-IC (Zhang et al., 2009). All of the constructs were fused with HA tags at the Nterminus: D1 (123–223aa), D2 (224–307aa), D3 (308–409aa), D4 (410–510aa), D5 (224–293aa), D6 (294–353aa), and D7 (354–409aa) (Fig. 5A). Our co-IP assays showed that LINGO-1 was associated with D2, D3, and D6 (Fig. 5B); thus, D6 is most likely the smallest peptide sequence in WNK1 protein that binds to LINGO-1. Then, we expressed and purified the TAT-D6 peptide by inserting the D6 encoding sequence into the PET28a-TAT vector. As expected (Fig. 6A), TAT-D6 prevented the association between WNK1 and LINGO-1. When added to cultured OPCs, TAT-D6 also caused a marked increase in OPC differentiation, compared with the vehicle control or TAT-Scr (a control peptide as methods described) (Fig. 6D, E) (Fig. S3A, B) on the Nogo66 substrate; this effect may be mediated by interrupting signal transduction since TAT-D6 can significantly inhibit Nogo66-induced RhoA activation (Fig. 6B, C). This study suggests that LINGO-1-mediated Nogo66 inhibition on OPC differentiation requires its interaction with WNK1. 3. Discussion Axons with functionally intact myelin sheaths are more resistant to injury or degeneration than denuded axons; thus, new approaches that focus on promotion of remyelination are beginning to be explored (Dubois-Dalcq et al., 2005). Although adult OPCs have the potential for in vivo remyelination by migrating to injured positions (Shields et al., 1999; Sim et al., 2002; Woodruff et al., 2004), the presence inhibitory molecules in myelin debris prevents OPC differentiation and subsequent remyelination (Baer et al., 2009; Kotter et al., 2006). OPCs are not like neurons, as adult OPCs have the same abilities and properties

as developmental OPCs; developmental and adult OPCs respond similarly to naked axons. In both cases, OPCs increase their rate of proliferation (Keirstead et al., 1998; Redwine and Armstrong, 1998), migrate into areas lacking myelination (Franklin and Blakemore, 1997), differentiate into mature myelinating oligodendrocytes (Deloulme et al., 2004; Kuhlmann et al., 2008), and myelinate axons (Caillava et al., 2011; Fancy et al., 2011). Developmental and adult OPCs also follow the same differentiation and maturation program (Fancy et al., 2011). Developmental OPCs are easily acquired from P1 SD rats. In this study, we used developmental OPCs for our experiments. OPC differentiation requires fast actin re-arrangement and membrane addition similar to axon growth in neurons; the signal pathway that affects axon growth may also affect OPCs differentiation. Since most myelin inhibitors bind to the LINGO-1/Nogo66R/p75(Troy) complex and induce axon growth inhibition in neurons, the role of the LINGO-1/Nogo66R/p75(Troy) complex in OPCs differentiation was investigated. Mi et al. (2007) showed that the OPC maturation and remyelination are significantly enhanced in vitro and in vivo by inhibiting LINGO-1 function using dominant-negative LINGO-1, LINGO-1 RNAi, or LINGO1-Fc but the specific protein that mediated the inhibition signal transduction was not described. Though our previous work has suggested that WNK1 and WNK3 are the downstream signaling molecules of LINGO-1 in neurons (Zhang et al., 2009, 2013), little is known about their function in oligodendrocytes. In the present study, we showed that WNK1 and LINGO-1 were highly expressed in the cultured oligodendrocytes and interact with each other. We also demonstrated that, as a downstream signaling molecule of LINGO-1, WNK1 is involved in Nogo66-induced OPC differentiation inhibition by activating RhoA.

Z.-H. Zhang et al. / Molecular and Cellular Neuroscience 65 (2015) 135–142

139

A eGFP

Merge

WNK1 ShRNA

GST

WNK1 ShRNA

GST-Nogo66

WNK1 ShRNA+Res

GST-Nogo66

B

ns 2.5

***

***

2.0 1.5 1.0

GST-Nogo66

WNK1 ShRNA

WNK1 ShRNA+Res

0.0

WNK1 ShRNA

0.5 Scramble ShRNA

oligendrocyte area index

DAPI

GST-Nogo66

Scramble ShRNA

MBP

GST

Fig. 4. WNK1 is important for Nogo66 mediated OPC mature inhibition. (A) OPCs were transfected with pSUPER encoding siRNAs against WNK1 (WNK1 shRNA) or scrambled sequence (scramble shRNA) either alone or together with WNK1 Rescuer (Res) and seed on GST or Nogo66 substrates. After 5 days, cultures were fixed and analyzed for membrane area. Scale bar = 50 μm. (B) Quantification for normalized membrane area. Data are shown as mean ± S.E.M. from three independent experiments. ***P b 0.001; one-way ANOVA with Student's t-test.

Though our previous work deduced that interaction of LINGO-1 with WNK1 may bring Rho-GDI–RhoA close to p75-bound guanine nucleotide exchange factors, such as Kalirin9, thereby facilitating RhoA activation (Zhang et al., 2009), the precise mechanism underlying the

regulation of RhoA activation by LINGO-1–WNK1 interaction in oligodendrocytes remained unknown. In this study, we found that suppressing WNK1 expression by RNA interference attenuated Nogo66-induced inhibition of OPC differentiation, further proving the important role of

Z.-H. Zhang et al. / Molecular and Cellular Neuroscience 65 (2015) 135–142

A

B

eGFP

LINGO-1

G O -1 -e LI G N FP G +D O -1 1 -e G LI FP N G +D O -1 2 -e LI G N F P+ G O D -1 3 -e G FP +D 4

140

LI N

1-352AA WNK1 KD 123-510AA

IP: anti-GFP IB: anti-HA D1

123-223 AA

HA

D3

308-409 AA

HA

410-510 AA HA

Input IB: anti-HA

224-307 AA

D2

G O -1 -e G FP LI +D N 5 G O -1 -e G LI FP N +D G O 6 -1 -e G FP +D 7

HA

D4

224-293 AA

D5 HA

D6 HA

LI N

HA

IP: anti-GFP IB: anti-HA

294-353 AA D7

354-409 AA

Input IB: anti-HA

Fig. 5. Domain 6 is mediated WNK1 interaction with LINGO-1. (A) Schematic structure of WNK1 (123–510aa) protein or domains. (B, C) Cell lysates from HEK293 cells co-transfected with HA-tagged WNK1 domains and LINGO-1-eGFP were subjected to IP with anti-GFP antibody, and then IB with anti-HA antibody, with HA as input control.

4. Experimental methods 4.1. Animals and reagents All of the animals in this study were obtained from Joint Ventures Sipper BK Experimental Animal (Shanghai, China). The animal experiments were undertaken in accordance with the National Institutes of

6+ og

o6

o6 og N

1.5 Active Rho-A (fold change)

TA

TA 6+

o6 og N

IB: anti-WNK1

C

T-

T-

D

6

Sc

r

B

6+

6+ o6 N

og

ST

1.0

***

0.5 0.0

o6 6

N

og N

E Nogo66+TAT-Scr

1.5 1.0 0.5

***

0.0

G ST +T N A og T -S o6 cr 6+ TA TD 6

Nogo66+TAT-D6

N

og

o6 6

GST

oligendrocyte area index

D

-D 6+ TA T

-S +T AT

Total Rho A

Total lysis IB: anti-actin

6

cr

Active Rho A IB: anti-LINGO-1

og o6

G

IP: anti-WNK1

TA

TA

T-

T-

D

6

Sc

A

LINGO-1 regulates oligodendrocyte differentiation by inhibiting ErbB2 translocation and activation in lipid rafts (Lee et al., 2014). The WNK1 involved in the ErbB2 translocation and activation should be investigated.

N

r

WNK1 on oligodendrocyte differentiation. Since other myelin inhibitors, such as MOG and OMgp also bind the LINGO-1/Nogo66R/p75 (Troy) complex, we proposed that their inhibition signals are also transduced by the LINGO-1/WNK1 pathway in OPCs. We found that the D6 peptide in WNK1 mediated the interaction of WNK1 with LINGO-1. We added excessive TAT-D6 peptides to cultured oligodendrocytes to prevent the association between endogenous WNK1 and LINGO-1. We also found that the TAT-D6 peptide can significantly attenuate the Nogo66-induced inhibition of OPC differentiation, which suggests a new treatment for MS disease as proven LINGO-1 inhibitors. Recently, there have reports that oligodendrocyte differentiation is positively regulated by the ErbB2 receptor tyrosine kinase and that

Fig. 6. WNK1 interaction with LINGO-1 is important for Nogo66 mediated OPC mature inhibition. (A) The association between WNK1 and LINGO-1 was blocked by TAT-D6 treatment in OPCs. Cell lysates from OPCs cultured on GST or Nogo66 substrates (with or without TAT-D6 20 μM treatment for 12 h) were subjected to IP with anti-WNK1 antibody and then IB with anti-WNK1 or LINGO-1 antibody. (B) TAT-D6 (20 μM for 12 h) treatment in OPCs cultured on Nogo66 substrates inhibits RhoA activation. The cell lysates were affinity-precipitated with GST-Rhotekin binding domain to detect GTP-bound Rho as mentioned under “Methods” and total Rho in cell lysate as inputs. Quantification shows in (C) where GTP Rho levels were normalized to total Rho protein levels in the cell lysate and presented as fold of control, data are shown as mean ± S.E.M. from three independent experiments. ***P b 0.001; Student's t-test. (D) Representative images show OPCs cultured in differentiation medium for 5 days with TAT-D6 (20 μM, added for every day) or not and quantification for the membrane area (E). Data are shown as mean ± S.E.M., n ≥ 216 cells for each group from three independent experiments. ***P b 0.001; One-way ANOVA with Student's t-test. Scale bar = 50 μm.

Z.-H. Zhang et al. / Molecular and Cellular Neuroscience 65 (2015) 135–142

Health Guide for the Care and Use of Laboratory Animals and with the approval of the Shanghai University Committee on Animal Care. The mouse antibodies against NG2 (MAB5384), A2B5 (MAB312), MBP (MAB381), and O4 (MAB345) were purchased from Millipore (Billerica, MA): rabbit-anti-LINGO-1 (07-678, Upstate), Goat-anti-LINGO-1 (sc48582, Santa Cruz), Mouse-anti-RhoA (sc-418, Santa Cruz), mouseanti-tubulin alpha (sc-58666, Santa Cruz), and rabbit-anti-WNK1 (WNK-11A, Alpha Diagnostic). The antibody for GAPDH was from Kangchen (Shanghai, China). 4.2. Primary cell culture and transfection Neuronal cells were isolated from postnatal day 1 (P1) Sprague– Dawley rats as previously described (Xu et al., 2006). Briefly, the forebrains were removed, cut into fragments in Hanks' buffered salt solution (HBSS), and incubated at 37 °C for 30 min with 0.125% trypsin. The dissociated cells were plated on poly-L-lysine (PLL)-coated tissue culture flasks and grown at 37 °C for 7–10 days in DMEM with 10% fetal calf serum (Invitrogen). The OPCs were collected by shaking the flask overnight at 280 rpm at 37 °C, resulting in 90% purity. Purified OPCs were transfected by electroporation using the Amaxa Nucleofector device with program O-003 as previously described (Xu et al., 2014). The OPCs were then cultured in neurobasal medium supplemented with 2% B27. To induce the differentiation of precursor cells, OPCs were plated in NB + B27 supplemented with 30 nM triiodothyronine for 2– 5 days and were identified using A2B5, NG2, O4, or MBP as markers. 4.3. Immunofluorescence staining

141

pSUPER vector to generate shRNAs as previously described (Zhang et al., 2009). The shWNK1 constructs were transfected into OPCs and cells were subjected to immunofluorescence staining at indicated times. 4.7. Nogo66 embedding The protein concentration of the preparation was determined (Biorad) and used immediately as a substrate in the OPC development assay. The culture slides were coated with 16.6 mg/mL poly-L-lysine at room temperature for 1 h and washed with 0.1 M NaHCO3. GSTNogo66 or GST was dried overnight onto the coated slides and used as a substrate (200 ng/cm2), then washed with ice-cold phosphatebuffered saline and seed with purify OPCs and cultured for 3–5 days in NB + B27 supplemented with 30 nM triiodothyronine. 4.8. Immunoprecipitation and immunoblotting Cell lysates were prepared as described previously (Zhang et al., 2013) and clarified by centrifugation at 11,200 g for 20 min at 4 °C. An equal amount (300–500 μl) of supernatants was incubated with 5 μl of the corresponding antibodies for 2 h at 4 °C. Protein G-agarose beads (Roche Applied Science) were then added for another 12 h rotation at 4 °C; immunoprecipitated samples were then washed 3 times with lysis buffer, boiled 3–5 min in sample-loading buffer, and then subjected to SDSPAGE, immunoblotted, and visualized with enhanced chemiluminescence (ECL, Pierce). The following antibodies were used: indicated first antibodies, and HRP-conjugated secondary antibody (1:10000, Santa Cruz). 4.9. Generation of TAT-D6 and TAT Scr peptides

Cells cultured on coverslips from P1 rat brains were washed in phosphate buffered saline and then fixed for 30 min with 4% paraformaldehyde at room temperature. The fixed samples were permeabilized with 0.1% Triton X-100 for 30 min, subsequently blocked with 1% bovine serum albumin in phosphate buffered saline, incubated at 4 °C overnight with primary antibody, and detected by species-specific fluorescein isothiocyanate- or rhodamine-conjugated secondary antibodies (1:100, Santa Cruz).

To generate the fusion peptides TAT-D6 and TAT Scr, a sequence containing the minimal translocation domain of the HIV-1 protein TAT (amino acids 47–57, YGRKKRRQRR) was inserted in-frame next to the N terminus of the WNK1-D6 peptide or scramble peptides (20 Alanines). The fusion construct was inserted into pET-28a, expressed, and purified using standard recombinant techniques.

4.4. Microscopes

4.10. RhoA activity assay

Fluorescence images were taken with a Leica SP5 confocal microscope or taken with the Nikon digital camera DXM1200 (Nikon, Japan) attached to a Nikon Eclipse E600 microscope (Nikon).

Active RhoA was determined using the GST-Rhotekin binding domain as described previously (Xu et al., 2009). Briefly, cells plate on GST-Nogo66 or GST for 60 min then washed with ice-cold phosphatebuffered saline and lysed in RIPA (50 mM Tris, pH 7.2, 1% Triton X100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl2, plus protease inhibitors). Clarified cell lysates were incubated with GST-Rhotekin binding domain (30 μg) bound to beads at 4 °C for 60 min. The beads were then washed 4 times in buffer B (Tris buffer containing 1% Triton X-100, 150 mM NaCl, 10 mM MgCl2, plus protease inhibitors) at 4 °C. Bound RhoA proteins were detected by Western blot using a monoclonal antibody against RhoA (1:1000, Santa Cruz).

4.5. Fluorescence images analysis To measure the membrane area of differential OPCs, images were analyzed with Image J software, which can calculate per cell MBP or GFP positive area. Converted canned color images of oligodendrocytes to grayscale followed in this order: Image → Type → 8-bit. Threshold of new image of cells using manual settings followed in this order: Image → Adjust → Threshold; play with sliders to include all of cell in red and click ‘Apply’; measure the cell area: Analyze → Analyze Particles. The data window listed the area (in pix) for each cell. For NG2 positive or MBP positive cell number analysis, cells from 10 random fields/well were analyzed with three replicated wells for individual treatment in three independent experiments. The percentage of NG2 positive or MBP positive cells was acquired by counting the number of NG2 positive or MBP positive cells compared with DAPI positive total cells per field of view. 4.6. Inhibition of WNK1 expression by RNA interference Rat WNK1 DNA sequences were selected for designing candidate small hairpin RNAs. The siRNA sequences are: WNK1si and GCAACA GGATGATATCGAA. These WNK1si sequences were constructed into

4.11. Statistics All data were analyzed with a Student's t-test or one-way ANOVA and statistics were performed using GraphPad Prism 5 statistics software. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mcn.2015.03.003. Acknowledgements This work was supported by Innovation Program of Shanghai Municipal Education Commission (12YZ016), National Natural Science Foundation of China (81171190, 31470053 and 81370031) and Project supported by Key Laboratory of Exercise and Health Sciences (Shanghai University of Sport), Ministry of Education.

142

Z.-H. Zhang et al. / Molecular and Cellular Neuroscience 65 (2015) 135–142

References Baer, A.S., Syed, Y.A., Kang, S.U., Mitteregger, D., Vig, R., Ffrench-Constant, C., Franklin, R.J., Altmann, F., Lubec, G., Kotter, M.R., 2009. Myelin-mediated inhibition of oligodendrocyte precursor differentiation can be overcome by pharmacological modulation of Fyn-RhoA and protein kinase C signalling. Brain 132, 465–481. Blakemore, W.F., Franklin, R.J., 2008. Remyelination in experimental models of toxininduced demyelination. Curr. Top. Microbiol. Immunol. 318, 193–212. Boulanger, J.J., Messier, C., 2014. From precursors to myelinating oligodendrocytes: contribution of intrinsic and extrinsic factors to white matter plasticity in the adult brain. Neuroscience 269, 343–366. Caillava, C., Vandenbosch, R., Jablonska, B., Deboux, C., Spigoni, G., Gallo, V., Malgrange, B., Baron-Van Evercooren, A., 2011. Cdk2 loss accelerates precursor differentiation and remyelination in the adult central nervous system. J. Cell Biol. 193, 397–407. Deloulme, J.C., Raponi, E., Gentil, B.J., Bertacchi, N., Marks, A., Labourdette, G., Baudier, J., 2004. Nuclear expression of S100B in oligodendrocyte progenitor cells correlates with differentiation toward the oligodendroglial lineage and modulates oligodendrocytes maturation. Mol. Cell. Neurosci. 27, 453–465. Dubois-Dalcq, M., Ffrench-Constant, C., Franklin, R.J., 2005. Enhancing central nervous system remyelination in multiple sclerosis. Neuron 48, 9–12. Fancy, S.P., Chan, J.R., Baranzini, S.E., Franklin, R.J., Rowitch, D.H., 2011. Myelin regeneration: a recapitulation of development? Annu. Rev. Neurosci. 34, 21–43. Franklin, R.J., 2002. Why does remyelination fail in multiple sclerosis? Nature reviews. Neuroscience 3, 705–714. Franklin, R.J., Blakemore, W.F., 1997. To what extent is oligodendrocyte progenitor migration a limiting factor in the remyelination of multiple sclerosis lesions? Mult. Scler. 3, 84–87. Guo, Q., Li, S., Su, B., 2007. Expression of oligodendrocyte myelin glycoprotein and its receptor NgR after the injury of rat central nervous system. Neurosci. Lett. 422, 103–108. Huang, J.Y., Wang, Y.X., Gu, W.L., Fu, S.L., Li, Y., Huang, L.D., Zhao, Z., Hang, Q., Zhu, H.Q., Lu, P.H., 2012. Expression and function of myelin-associated proteins and their common receptor NgR on oligodendrocyte progenitor cells. Brain Res. 1437, 1–15. Jeffery, N.D., Blakemore, W.F., 1997. Locomotor deficits induced by experimental spinal cord demyelination are abolished by spontaneous remyelination. Brain 120 (Pt 1), 27–37. Keirstead, H.S., Levine, J.M., Blakemore, W.F., 1998. Response of the oligodendrocyte progenitor cell population (defined by NG2 labelling) to demyelination of the adult spinal cord. Glia 22, 161–170. Kotter, M.R., Li, W.W., Zhao, C., Franklin, R.J., 2006. Myelin impairs CNS remyelination by inhibiting oligodendrocyte precursor cell differentiation. J. Neurosci. 26, 328–332. Kuhlmann, T., Miron, V., Cui, Q., Wegner, C., Antel, J., Bruck, W., 2008. Differentiation block of oligodendroglial progenitor cells as a cause for remyelination failure in chronic multiple sclerosis. Brain 131, 1749–1758. Kwon, H.S., Nakaya, N., Abu-Asab, M., Kim, H.S., Tomarev, S.I., 2014. Myocilin is involved in NgR1/Lingo-1-mediated oligodendrocyte differentiation and myelination of the optic nerve. J. Neurosci. 34, 5539–5551.

Lee, X., Yang, Z., Shao, Z., Rosenberg, S.S., Levesque, M., Pepinsky, R.B., Qiu, M., Miller, R.H., Chan, J.R., Mi, S., 2007. NGF regulates the expression of axonal LINGO-1 to inhibit oligodendrocyte differentiation and myelination. J. Neurosci. 27, 220–225. Lee, X., Shao, Z., Sheng, G., Pepinsky, B., Mi, S., 2014. LINGO-1 regulates oligodendrocyte differentiation by inhibiting ErbB2 translocation and activation in lipid rafts. Mol. Cell. Neurosci. 60, 36–42. McFarland, H.F., Frank, J.A., Albert, P.S., Smith, M.E., Martin, R., Harris, J.O., Patronas, N., Maloni, H., McFarlin, D.E., 1992. Using gadolinium-enhanced magnetic resonance imaging lesions to monitor disease activity in multiple sclerosis. Ann. Neurol. 32, 758–766. Mi, S., Lee, X., Shao, Z., Thill, G., Ji, B., Relton, J., Levesque, M., Allaire, N., Perrin, S., Sands, B., Crowell, T., Cate, R.L., McCoy, J.M., Pepinsky, R.B., 2004. LINGO-1 is a component of the Nogo-66 receptor/p75 signaling complex. Nat. Neurosci. 7, 221–228. Mi, S., Miller, R.H., Lee, X., Scott, M.L., Shulag-Morskaya, S., Shao, Z., Chang, J., Thill, G., Levesque, M., Zhang, M., Hession, C., Sah, D., Trapp, B., He, Z., Jung, V., McCoy, J.M., Pepinsky, R.B., 2005. LINGO-1 negatively regulates myelination by oligodendrocytes. Nat. Neurosci. 8, 745–751. Mi, S., Hu, B., Hahm, K., Luo, Y., Kam Hui, E.S., Yuan, Q., Wong, W.M., Wang, L., Su, H., Chu, T.H., Guo, J., Zhang, W., So, K.F., Pepinsky, B., Shao, Z., Graff, C., Garber, E., Jung, V., Wu, E.X., Wu, W., 2007. LINGO-1 antagonist promotes spinal cord remyelination and axonal integrity in MOG-induced experimental autoimmune encephalomyelitis. Nat. Med. 13, 1228–1233. Redwine, J.M., Armstrong, R.C., 1998. In vivo proliferation of oligodendrocyte progenitors expressing PDGFalphaR during early remyelination. J. Neurobiol. 37, 413–428. Shields, S.A., Gilson, J.M., Blakemore, W.F., Franklin, R.J., 1999. Remyelination occurs as extensively but more slowly in old rats compared to young rats following gliotoxininduced CNS demyelination. Glia 28, 77–83. Sim, F.J., Zhao, C., Penderis, J., Franklin, R.J., 2002. The age-related decrease in CNS remyelination efficiency is attributable to an impairment of both oligodendrocyte progenitor recruitment and differentiation. J. Neurosci. 22, 2451–2459. Woodruff, R.H., Fruttiger, M., Richardson, W.D., Franklin, R.J., 2004. Platelet-derived growth factor regulates oligodendrocyte progenitor numbers in adult CNS and their response following CNS demyelination. Mol. Cell. Neurosci. 25, 252–262. Xu, X., He, C., Zhang, Z., Chen, Y., 2006. MKLP1 requires specific domains for its dendritic targeting. J. Cell Sci. 119, 452–458. Xu, X.H., Zhou, J.F., Li, T.Z., Zhang, Z.H., Shan, L., Xiang, Z.H., Yu, Z.W., Zhang, W.D., He, C., 2009. Polygalasaponin G promotes neurite outgrowth of cultured neuron on myelin. Neurosci. Lett. 460, 41–46. Xu, X.H., Deng, C.Y., Liu, Y., He, M., Peng, J., Wang, T., Yuan, L., Zheng, Z.S., Blackshear, P.J., Luo, Z.G., 2014. MARCKS regulates membrane targeting of Rab10 vesicles to promote axon development. Cell Res. 24, 576–594. Zhang, Z., Xu, X., Zhang, Y., Zhou, J., Yu, Z., He, C., 2009. LINGO-1 interacts with WNK1 to regulate nogo-induced inhibition of neurite extension. J. Biol. Chem. 284, 15717–15728. Zhang, Z., Xu, X., Xiang, Z., Yu, Z., Feng, J., He, C., 2013. LINGO-1 receptor promotes neuronal apoptosis by inhibiting WNK3 kinase activity. J. Biol. Chem. 288, 12152–12160.