1. Developmental Biology
  2. Neuroscience

Extracellular Pgk1 enhances neurite outgrowth of motoneurons through Nogo66/NgR-independent targeting of NogoA

  1. Cheng Yung Lin
  2. Chia Lun Wu
  3. Kok Zhi Lee
  4. You Jei Chen
  5. Po Hsiang Zhang
  6. Chia Yu Chang
  7. Horng Jyh Harn
  8. Shinn Zong Lin  Is a corresponding author
  9. Huai Jen Tsai  Is a corresponding author
  1. Mackay Medical College, Taiwan
  2. National Taiwan University, Taiwan
  3. Buddhist Tzu Chi Medical Foundation, Taiwan
  4. Buddhist Tzu Chi General Hospital, Taiwan
  5. Buddhist Tzu Chi General Hospital and Tzu Chi University, Taiwan
https://doi.org/10.7554/eLife.49175
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Cite this article
  1. Cheng Yung Lin
  2. Chia Lun Wu
  3. Kok Zhi Lee
  4. You Jei Chen
  5. Po Hsiang Zhang
  6. Chia Yu Chang
  7. Horng Jyh Harn
  8. Shinn Zong Lin
  9. Huai Jen Tsai
(2019)
Extracellular Pgk1 enhances neurite outgrowth of motoneurons through Nogo66/NgR-independent targeting of NogoA
eLife 8:e49175.
https://doi.org/10.7554/eLife.49175
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Abstract

NogoA inhibits neurite outgrowth of motoneurons (NOM) through interaction with its receptors, Nogo66/NgR. Inhibition of Nogo receptors rescues NOM, but not to the extent exhibited by NogoA-knockout mice, suggesting the presence of other pathways. We found that NogoA-overexpressing muscle cells reduced phosphoglycerate kinase 1 (Pgk1) secretion, resulting in inhibiting NOM. Apart from its glycolytic role and independent of the Nogo66 pathway, extracellular Pgk1 stimulated NOM by triggering a reduction of p-Cofilin-S3, a growth cone collapse marker, through decreasing a novel Rac1-GTP/p-Pak1-T423/p-P38-T180/p-MK2-T334/p-Limk1-S323/p-Cofilin-S3 molecular pathway. Not only did supplementary Pgk1 enhance NOM in defective cells, but injection of Pgk1 rescued denervation in muscle-specific NogoA-overexpression of zebrafish and an Amyotrophic Lateral Sclerosis mouse model, SOD1 G93A. Thus, Pgk1 secreted from muscle is detrimental to motoneuron neurite outgrowth and maintenance.

https://doi.org/10.7554/eLife.49175.001

Introduction

The role of NogoA in limiting axonal fiber growth and regeneration following neuronal injury is well known (Delekate et al., 2011). NogoA inhibits neurite outgrowth of motoneurons (NOM) via Nogo66 and NiGΔ20 domains (GrandPré et al., 2000). Pharmaceutical and genetic strategies to ablate known receptors interacting with Nogo66 domain (Fournier et al., 2001; Atwal et al., 2008; Nakamura et al., 2011) have not successfully reversed inhibited NOM to the extent exhibited by NogoA-knockout mice (Simonen et al., 2003), suggesting the presence of other pathways.

In Amyotrophic Lateral Sclerosis (ALS) patients, overexpression of NogoA in muscle was positively correlated with the loss of motor endplates, and as the presence of NogoA in muscle cells increases, denervation of endplates also increases (Bruneteau et al., 2015). It has also been reported that patients whose NogoA was overexpressed in muscle cells and who were diagnosed with lower motor neuron syndrome (LMNS) also had a higher likelihood of progressing to ALS symptoms (Pradat et al., 2007). In a mouse, NogoA could be detected in the muscles of embryos at E15 and P1 stages, but not in the muscles in adults (Wang et al., 2002). Interestingly, a significant increase of NogoA was found in the muscle fibers of SOD1 G86R mice and ALS patients (Dupuis et al., 2002; Jokic et al., 2005). NogoA-knockout in SOD1 G86R mice (G86R/NogoA-/-) showed longer survival time and increased survival rate (Jokic et al., 2006). Moreover, when NogoA was overexpressed in the muscle of mice, morphological changes of neuromuscular junction (NMJ) were also observed (Jokic et al., 2006). More recently, in a zebrafish model, overexpression of Rtn4al (human NogoA homolog) in muscle exhibited an ALS-like phenotype (Lin et al., 2019). This line of evidence suggests that overexpression of NogoA/Rtn4a in muscle cells may contribute to the etiology of ALS-like disease at an early stage, even though NogoA could not be detected in the blood (Harel et al., 2009). However, the molecular mechanism that would explain the association between increased NogoA in muscle cells and the occurrence of neurodegeneration remains largely unclear. Therefore, it is a plausible hypothesis that the inhibitory effect of NogoA-overexpressed muscle cells on NOM may be mediated through some secretory myokine released by muscle cells.

Therefore, to gain a better understanding of NogoA overexpression in muscle relative to the effects of myokine secretion, we first collected conditional medium (CM) from cultured Sol8 myogenic cells in which NogoA is overexpressed (Sol8-NogoA CM). Next, we incubated mouse motoneuron hybrid NSC34 cells in Sol8-NogoA CM and found appreciable inhibition of NOM. Then, we found that the amount of secreted phosphoglycerate kinase 1 (Pgk1) was dramatically reduced in Sol8-NogoA CM compared to control Sol8 CM. Upon addition of Pgk1, NOM derived from NSC34 cells cultured in Sol8-NogoA CM was rescued. Furthermore, we found that extracellular Pgk1 (ePgk1) reduces the phosphorylation of Pak1/P38/MK2/Limk1/Cofilin axis, which, in turn, enhances the NOM in a manner functionally independent from its intracellular, canonical role as a supplier of energy. Therefore, we conclude that reduced secretion of Pgk1 from NogoA-overexpressed muscle cells inhibits NOM promises a paradigm shift that will inspire new thinking about therapeutic targets against failure NOM and denervated NMJ.

Results

Supplementary addition of Pgk1 improves neurite outgrowth of NSC34 cells cultured in CM obtained from cultured myoblast cells expressing Sol8-NogoA

The inhibition of NOM by muscle-specific NogoA might result from inducing a canonical Nogo66/NgR or Nogo66/PirB signaling pathway or from influencing the secretion of myokines. To define this molecular mechanism, we employed mouse Sol8 myoblasts and established a Doxycycline (Dox)-inducible cell line stably harboring the Sol8-vector only (Sol8-vector) and the Sol8 vector with NogoA insert (Sol8-NogoA) (Figure 1—figure supplement 1). After induction, the CM obtained from cultured myoblast cells harboring Sol8-vector was directly used to culture motoneuron cell line NSC34. After culturing, motoneurons exhibited extended neurites, while motoneurons incubated with Sol8-NogoA CM failed to exhibit neurite outgrowth (Figure 1—figure supplement 1). Importantly, NogoA was not detected in the CM cultured by Sol8-NogoA cells after Dox induction for 48 hr (Figure 1—figure supplement 1C). This line of evidence suggested that the component(s) contained in CM from cultured Sol8-NogoA play(s) a role in NOM inhibition, but not through NogoA contained in medium. Moreover, unlike neurons treated with Sol8-vector, Western blot analysis demonstrated that the signal intensity of phosphorylated Cofilin at S3 (p-Cofilin-S3), which is a hallmark of growth cone collapse in neuronal cells, as well as a marker of reduced axonal actin dynamics in ALS patients with depleted and mutated C9ORF72 (Heredia et al., 2006; Sivadasan et al., 2016), was upregulated in neurons treated with Sol8-NogoA CM (Figure 1—figure supplement 1). Collectively, this line of evidence suggested that the component(s) contained in CM from cultured Sol8-NogoA play(s) a role in NOM inhibition.

We further employed 2D electrophoresis, followed by LC MS/MS, to analyze the total proteins content in CM. Among these examined proteins, we found that the level of Pgk1 protein contained in Sol8-NogoA CM was significantly reduced compared to that in Sol8-vector CM (see Figure 1—figure supplement 2). Furthermore, the degree of p-Cofilin expression in NSC34 cells cultured in CM from Pgk1-overexpressed Sol8-NogoA cells was significantly reduced compared with that in NSC34 cells cultured in CM from Sol8-NogoA cells. Therefore, Pgk1 was chosen for further study to confirm its potential role in NOM. Additionally, we used Western blot analysis to detect the protein level of Pgk1 in the sera of transgenic SOD1 G93A mice, in which NogoA was overexpressed in muscle (Figure 1—figure supplement 3), the results of which were consistent with those reported by Bros-Facer et al. (2014). We found that the amount of Pgk1 in the sera of SOD1 G93A mice was significantly reduced compared with that in the sera of WT mice (Figure 1—figure supplement 3). Collectively, based on the above results provided in vitro and in vivo evidence, we chose Pgk1 for further study to confirm its potential role in NOM.

We added Pgk1 directly into Sol8-NogoA CM and found that p-Cofilin-S3 in NSC34 cultured with Sol8-NogoA CM plus Pgk1 was lower than that of cells cultured with Sol8-NogoA CM (Figure 1A). Importantly, supplementary Pgk1 could reduce p-Cofilin-S3 to then restore NOM of NSC34 cells, even though they had been cultured in Sol8-NogoA CM, indicating that stalled NOM of NSC34 cells cultured in Sol8-NogoA CM could be rescued upon the addition of Pgk1.

Figure 1 with 4 supplements see all
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Supplementary addition of Pgk1 in culture medium promotes neurite outgrowth developed from NSC34 cells.

(A) Neurite outgrowth of motoneurons developed from NSC 34 motoneuron cells cultured in either Sol8-vector CM or Sol8-NogoA CM with and without Pgk1 addition. (B) Neurite outgrowth and p-Cofilin-S3 expression in NSC34 cells cultured in Sol8-vector CM without Pgk1. Rabbit IgG (control) and Pgk1 antibody were separately added into Sol8-vector CM. (C) Neurite outgrowth of motoneurons developed from NSC34 cells cultured in differentiation media (DM) with or without Pgk1 addition. (A-C) Right panels: Western blot analysis of total Cofilin and p-Cofilin-S3 contained in NSC34 cells. Statistical analysis used Student’s t-test (***, p<0.001; **, p<0.01). (D–E) The patterns of neurite length distribution. Cell number with various lengths of neurites of NSC 34 cells cultured by (D) CM obtained from Sol8-vector, Sol8-NogoA with or without Pgk1 addition, Sol8-vector with IgG and with Pgk1 antibody addition, as indicated, and by (E) DM with or without containing Pgk1 was determined (120 cells per experimental condition).

https://doi.org/10.7554/eLife.49175.002

To further confirm whether Pgk1 secreted by muscle cells could enhance NOM, we added Pgk1-specific antibody to Sol8-vector CM and then cultured NSC34 cells. In response, NOM was totally inhibited and p-Cofilin-S3 was higher compared to those observed in the control groups, such as Sol8-vector CM and Sol8-vector CM plus rabbit IgG (Figure 1B). Thus, we suggest that Pgk1 is secreted by myoblasts and that it exists in muscle cell CM to play an important role in enhancing NOM.

Next, to determine if Pgk1 alone is sufficient to induce NOM and neuronal differentiation, we switched to low trophic factor differentiation media (DM). Under the same condition, Pgk1 was able to extend NOM and downregulate p-Cofilin-S3 (Figure 1C). We also found that the effect of adding Pgk1 on the downregulation of p-Cofilin-S3 was dose-dependent from 0, 11, 33, 66 and 99 ng/ml (Figure 1—figure supplement 4). Moreover, when NSC34 cells were treated with Pgk1 at 33 ng/ml for 0, 8, 16, 24 and 48 hr, we found that the effect of Pgk1 on the downregulation of p-Cofilin-S3 was dose-dependent from 8 through 24 hr (Figure 1—figure supplement 4). However, we noticed that p-Cofilin-S3 was unexpectedly increased if cells were treated with Pgk1 for 48 hr. The doses of Pgk1 we examined did not cause any negative effect on cell growth and survival. However, based on the above results and to ensure that we did not seriously affect cell physiology, we treated NSC34 cells with the minimal concentration (33 ng/ml of Pgk1 for 24 hr) throughout the entire study. Interestingly, the addition Pgk1 at that concentration not only rescued the number of neurite-bearing cells, but also enhanced neurite length of NSC34 cells cultured in Sol8-NogoA CM and DM (Figure 1D–E).

Supplementary addition of Pgk1 can enhance the maturation of NSC34 cells cultured in Sol8-NogoA CM

To further learn if these restored neurites in NSC34 cells could differentiate into mature neurons, we employed antibody combined with fluorescence staining to specifically detect Syn1, which is a marker labeling neuronal synaptic vesicles in cell terminals (Fletcher et al., 1991), and maturation markers, such as MAP2, GAP43 and ChAT (Maier et al., 2013). Based on the distribution by immunofluorescence staining against Syn1 within NSC34 cells cultured in CM, the percentages of NSC34 cells displaying the Syn1 signal in the growth cones terminal were 43 ± 4.78, 13 ± 1.63 and 45 ± 4.55% in Sol8-vector CM, Sol8-NogoA CM and Sol8-NogoA CM plus Pgk1, respectively (Figure 2A–D). The number of neurons with Syn1 signal at the end of growth cones could be restored to that cultured in Sol8-vector CM after Pgk1 was added in Sol8-NogoA CM.

Figure 2
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Supplementary addition of Pgk1 enables more NSC34 cells cultured in Sol8-NogoA CM to differentiate into mature motor neurons.

Immunofluorescence staining of NSC34 cells cultured in (A) Sol8-vector CM, (B) Sol8-NogoA CM, and (C) Sol8-NogoA CM plus Pgk1. DAPI was labeled by blue fluorescent signal to mark nucleus, and acetyl-tubulin was labeled by green signal, while Syn1 was labeled by red signal. The growth cone of NSC34 cells was marked with white arrowheads. Scale bar, 20 μm. (D) In total, 110 to 120 cells were randomly selected from each culture group, and the number of Syn1-positive cells with growth cone was counted as a percentage. (E) Western blotting analysis was performed to quantify the expression levels of MAP2, GAP43, Syn1 and ChAT from three treatment groups. α-tubulin served as internal control. The relative value of each examined protein was used for comparison among the three groups when the value obtained from the Sol8-vector CM group was normalized as 1. Data were averaged from three independent experiments. Statistical analysis used Student’s t-test (***, significant difference at p<0.001; **, p<0.01; *, p<0.05).

https://doi.org/10.7554/eLife.49175.007

We next examined the expression levels of functional proteins, such as MAP2, GAP43, Syn1 and ChAT, in NSC34 cells. The expressions of MAP2, GAP43, Syn1 and ChAT were all increased when Pgk1 was added to the Sol8-NogoA CM/NSC34 culture (Figure 2E), suggesting that Pgk1 addition could rescue weak cell differentiation caused by culturing in Sol8-NogoA CM. Taken together, we conclude that exogenous addition of Pgk1 enables NSC34 cells cultured in Sol8-NogoA CM to improve differentiation, maturation, and neurite growth.

Reduced protein level of p-Cofilin-S3, as mediated by ePgk1, is independent of glycolysis

Since Pgk1 is a key enzyme for glycolysis, it is necessary to determine whether reduced p-Cofilin-S3 and resultant enhancement of NOM by addition Pgk1 could be attributed to glycolytic effect. Neither the absence nor presence of ePgk1 resulted in any significant difference in glycolytic function of neurons (Figure 3A). Even in the low glycolytic condition induced by glycolysis inhibitor 3-(3-Pyridinyl)−1-(4-pyridinyl)−2-propen-1-one (3PO) (Figure 3B), in which p-Cofilin-S3 was insignificant compared to the untreated group (Figure 3C, lanes 1 vs. 3), ePgk1 still downregulated p-Cofilin-S3 (Figure 3C, lanes 3 vs. 4). Moreover, addition of Pgk1 mutants, Pgk1-T378P and Pgk1-D375N, both of which lost their catalytic activities (Lay et al., 2002; Chiarelli et al., 2012) in DM, could still reduce p-Cofilin-S3 (Figure 3D), suggesting ePgk1 promotes NOM independent of glycolysis.

Figure 3
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Enhancement of neurite outgrowth, as mediated by supplementary addition of Pgk1, is independent of metabolic glycolysis.

(A) Analysis of glycolytic function in NSC34 cells cultured in DM with or without the addition of Pgk1 or in (B) glycolysis inhibitor 3PO. (C) Western blot analysis of total Cofilin, p-Cofilin-S3, and protein control marker (α-tubulin) of NSC34 cells cultured in conditions, as indicated. (D) Western blot analysis of total Cofilin, phosphorylated Cofilin (p-Cofilin-S3), and protein control marker (α-tubulin) of NSC34 cells cultured in DM containing Pgk1 and its catalytic mutants, Pgk1-T378P and Pgk1-D375N. All of the above data were averaged from three independent experiments.

https://doi.org/10.7554/eLife.49175.008

Specific knockout of Pgk1 in zebrafish muscle cells causes defective development of motoneurons

To confirm the role of Pgk1 secreted from muscle cells regulates motoneuron development in vivo, we employed the CRISPR/Cas9 system to knock out Pgk1 in zebrafish embryos. We used muscle-specific α-actin promoter to drive overexpression of Cas9 in muscle cells in order to exclusively knock out the pgk1 gene. Since turbo-red fluorescent protein (tRFP) was engineered to fuse with Cas9 and P2A peptide, it served as a reporter to reflect the overexpression of Cas9 (Figure 4A). Compared to control Tg(mnx:GFP) embryos at 30 hpf (Figure 4C), the tRFP signal was observed in the muscle of pZα-Cas9-injected embryos, indicating that Cas9 was overexpressed in certain muscle cells (Figure 4D). Nevertheless, motoneurons were normally developed, which suggests that overexpression of Cas9 in muscle cells had no effect on development. However, when Tg(mnx:GFP) embryos were coinjected with pZα-Cas9 and pgk1 sgRNA, which inhibits the production of Pgk1 in muscle cells (Figure 4—figure supplement 1), defective motoneurons were observed (Figure 4E), suggesting that the reduction of Pgk1 in muscle cells is followed by impairment of NOM. In a parallel experiment, by conditional knockout of phosphoglycerate mutase 2 (pgam2) in muscle cells (Figure 4—figure supplement 1), a downstream enzyme of Pgk1 in the glycolytic pathway, we again confirmed that the glycolysis pathway in muscle did not affect NOM (Figure 4F). Collectively, these results suggested that defects in NOM result from a decrease in Pgk1 secretion in muscle cells. To prove the consistency of this result in vivo, we constructed a plasmid containing Pgk1 fused with P2A peptide and tRFP (Figure 4B). After injecting this construct into zebrafish embryos, RFP exhibited specifically in muscle cells. When these RFP-expressing cells were sorted out and examined by Western blot analysis, the results demonstrated that Pgk1 was overexpressed (Figure 4—figure supplement 1). Meanwhile, motoneuron axons exhibited ectopic growth (Figure 4G). As suggested by quantification of the axonal extension phenotype shown in Figure 4—figure supplement 2, the increase of Pgk1 in muscle cells enhances NOM.

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Muscle-specific overexpression of Pgk1 enhances NOM in zebrafish embryos.

(A) Diagram of plasmid pZα-Cas9. The Cas9-P2A-tRFP cassette is driven by zebrafish muscle-specific α-actin promoter. The fusion protein Cas9-P2A-tRFP expressed in muscle cells is digested into Cas9, which is bound by transferred sgRNA, resulting in silencing the target gene in muscle cells. (B) Diagram of plasmid pZα-Pgk1. Pgk1 is specifically overexpressed in muscle cells. (C–G) Injection of different materials, as indicated, into embryos from transgenic line Tg(mnx:GFP) and observation of fluorescent signals expressed in embryos at 30-hpf. GFP-labeled motor neurons observed under confocal microscopy. (C’–G’) Location of RFP-labeled muscle cells in which Cas9 and/or Pgk1 is overexpressed. (C’’–G’’) Two fluorescent signals were merged. Numbers shown in the lower right corner were the number of phenotypes out of total examined embryos. (C–C”) Untreated embryos served as the control group. (D–D”) Injection of pZα-Cas9. NOM was not affected. (E–E”) Injection pZα-Cas9 combined with pgk1 sgRNA. The length of NOM became shorter (white arrows). (F–F”) Injection of pZα-Cas9 combined with pgam2 sgRNA (served as negative control). The NOM was not affected. (G–G”) Injection of pZα-Pgk1. The NOM became increasingly ectopic toward the muscle cells in which Pgk1 was overexpressed (white arrowheads).

https://doi.org/10.7554/eLife.49175.009

ePgk1 reduces the p-Cofilin-S3 expressed in NSC34 cells by decreasing the phosphorylation of Limk1 at S323

We next sought to clarify the molecular pathway that allows addition of Pgk1 to promote neuronal differentiation and NOM of NSC34 cells. The binding of Nogo66 domain to Nogo receptor (NgR) induces an acute intracellular signaling response to inhibit NOM through (i) increasing p-Cofilin-S3 (Ohashi et al., 2000; Manetti, 2012) by phosphorylating Limk1 (p-Limk1-T508) in the Rho/ROCK pathway, (ii) increasing phosphorylated EGFR (p-EGFR-Y1173) (Koprivica et al., 2005), or (iii) downregulating AKT phosphorylation (p-AKT-S473) (Wang et al., 2011). Here, we demonstrated there was no significant changes of p-ROCK2-Y256, p-EGFR-Y1173 or p-AKT-S473 expressions among NSC34 cells cultured in Sol8-vector CM, Sol8-NogoA CM and Sol8-NogoA CM plus Pgk1 (Figure 5—figure supplement 1). Surprisingly, Sol8-NogoA CM plus Pgk1 did reduce p-Cofilin-S3 via p-Limk1-S323, but not p-Limk1-T508 (Figure 5A), suggesting that the ePgk1-mediated pathway is independent of the Nogo66/NgR/ROCK2-Y256/Limk1-T508 axis.

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Pgk1 reduces the protein level of p-Cofilin-S3 through the decrease of phosphorylated Limk1 at S323 in NSC34 cells.

(A) The expression levels of p-Limk1-S323, -T508, Limk1, p-Cofilin-S3 and total Cofilin in NSC34 cells treated with condition, as indicated. (B) Phosphorylated (p-Cofilin-S3) and total Cofilin contained in cells were examined by Western blot analysis. NCS34 cells cultured in DM were introduced separately with a plasmid of pCS2+, pCS2-Limk1-flag (expressing normal Limk1), pCS2-Limk1-S323A-flag (expressing S323A-mutated Limk1), and pCS2-Limk1-T508V-flag (expressing T508V-mutated Limk1). (C) Plasmids encoding overexpression of Limk1-, Limk1-S323A- and Limk1-T508V-flag fusion proteins were separately introduced into NSC34 cells cultured in DM with or without Pgk1. (D) Expression of P38/MK2 and Rac1/Pak1 markers of NSC34 cells cultured in condition, as indicated. (E) Detection of P38/MK2 and Limk-S323/Cofilin signaling pathway markers of NSC34 cells introduced with Pak1-overexpressing plasmid and cultured in DM with or without Pgk1. α-tubulin served as an internal control. Relative value represents the ratio of phosphorylated form value over total protein values. All data were averaged from three independent experiments with statistical analysis by Student’s t-test (***, significant difference at p<0.001; **, p<0.01; *, p<0.05).

https://doi.org/10.7554/eLife.49175.012

The overexpression of Limk1, Limk1-S323A and Limk1-T508V in NSC34 cells cultured in DM resulted in increasing the basal level of intracellular p-Cofilin-S3 (Figure 5B). When we treated cells overexpressing Limk1, Limk1-S323A, and Limk1-T508V with Pgk1, the cells overexpressing Limk1 still responded to Pgk1, as evidenced by decreased p-Cofilin-S3 (Figure 5C). Overexpression of Limk1-S323A, but not Limk1-T508V, abolished Pgk1-mediated p-Cofilin-S3 downregulation (Figure 5C), again confirming that Pgk1 decreased p-Cofilin-S3 through Limk1-S323, but not Limk1-T508.

The signaling pathway underlying the involvement of ePgk1-mediated reduction of p-Cofilin-S3

We further identified upstream kinases, including P38/MK2 and Rac1/Pak1 (Figure 5D), along with their phosphorylated sites, using Sol8-vector CM and Sol8-NogoA CM in the absence and presence of Pgk1. Pak1 overexpression confirmed this series of signaling transduction pathway, consistent with our results using Sol8-vector CM and Sol8-NogoA CM (Figure 5E). On the other hand, when NSC34 cells were cultured in DM plus a Pak1 inhibitor, FRAX597, we found that the intracellular p-Pak1 was reduced and that the levels of p-P38-T180, p-MK2-T334, p-Limk-S323 and p-Cofilin-S3 were also reduced (Figure 5—figure supplement 2), suggesting that P38, MK2, Limk, and Cofilin are all downstream effectors of Pak1. In sum, ePgk1 triggers a reduction in p-Cofilin-S3, in turn promoting NOM through decreasing a novel p-Pak1-T423/p-P38-T180/p-MK2-T334/p-Limk1-S323 axis via reducing Rac1-GTP activity in neuronal cells.

To confirm whether Pgk1 involved regulating pathway is independent of activation of NgR receptor by NogoA. First, we determined whether the p-Cofilin expression is different between GST-Nogo66 addition and GST-Nogo66 plus Pgk1 addition into NSC34 cultured in DM. Compared to the control group in which GST was added in DM, NSC34 cells cultured in GST-Nogo66-added DM exhibited the increase of p-ROCK2-Y256 and p-EGFR-Y1173 expression and the decrease of p-Akt-S473 (Figure 5—figure supplement 3A), suggesting GST-Nogo66 addition did activate NgR response pathway. Interestingly, the increased p-ROCK2-Y256 and p-EGFR-Y1173 and the decreased p-Akt-S473 remained unchanged in NSC34 cultured in GST-Nogo66 with Pgk1 addition (Figure 5—figure supplement 3A), suggesting the addition of Pgk1 did not affect Nogo66/NgR pathway. However, compared to GST-Nogo66-added cells, the GST-Nogo66 plus Pgk1-treated NSC34 cells exhibited the reduction of p-Cpfilin-S3 expression through decreasing p-Limk-S323 but not through p-Limk-T508 (Figure 5—figure supplement 3B), suggesting the reduced expression of p-Cofilin is due to the presence of Pgk1 through pathway other than Nogo66/NgR interaction.

Second, we determined whether the p-Cofilin-S3 expression is still reduced in NSC34 by addition of Pgk1 into NSC34 cultured in DM when NgR receptor is blocked by NAP2, a NgR receptor antagonist peptide. The results showed that addition of NAP2 in culture led to the reduction of ROCK2 expression in NSC34 cells, which was consistent with the result reported by Sun et al. (2016) indicating that NAP2 is able to block NgR receptor (Figure 5—figure supplement 3C). However, unlike the increased ROCK2 in NSC34 cultured in NAP2 addition, the ROCK2 expression remained unchanged in NSC34 cells cultured in NAP2 plus Pgk1 addition (Figure 5—figure supplement 3C). On contrast, the expression of p-Pak1-T423/p-P38-T180/p-MK2-T334/p-Limk1-S323/p-Cofiln-S3 axis was reduced (Figure 5—figure supplement 3D).

Taken together, we suggest that the reduction of p-Cofilin mediated by ePgk1 is not through the Nogo66/NgR interaction in neuronal cells since ePgk1 addition can be still functional to reduce p-Pak1/p-P38/p-MK2/p-Limk1-S323/p-Cofiln axis without the presence of NgR receptor of neuronal cells. In sum, ePgk1 triggers a reduction in p-Cofilin-S3, in turn promoting NOM through decreasing a novel p-Pak1-T423/p-P38-T180/p-MK2-T334/p-Limk1-S323 axis via reducing Rac1-GTP activity in neuronal cells. And, the molecular pathway ePgk1 involved in NOM is independent of the Nogo66/NgR interaction.

Intramuscular injection of Pgk1 is able to rescue NMJ denervation caused by Rtn4al-overexpression transgenic zebrafish and SOD1 G93A transgenic mice

Muscle-specific NogoA-overexpression has been shown to disassemble NMJ in animal models, exhibiting muscle atrophy and reduced movement (Jokic et al., 2006; Lin et al., 2019). Noting the colocalization of the presynaptic markers synaptic vesicle glycoprotein 2A (SV2) and Neurofilament-H (NF-H), as well as the postsynaptic marker acetylcholine receptor labeled with α-Bungarotoxin (α-BTX), we asked if ePgk1 could re-establish NMJ integrity after its disassembly by NogoA overexpression. While we observed denervation at NMJ after Rtn4al/NogoA induction in adult zebrafish (Figure 6—figure supplement 1), as evidenced by reduced colocalization of presynaptic and postsynaptic markers, this defect saw much improvement by addition of Pgk1, but not GFP control, through intramuscular injection (Figure 6A–G), suggesting ePgk1 supports NMJ integrity. Although adult zebrafish muscle injected with Pgk1 did exhibit delayed NMJ denervation, it could still retain its normal biological activity for two weeks after a single shot (Figure 6—figure supplement 2).

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Supplementary addition of Pgk1 rescues denervation caused by NogoA-overexpression in the muscle cells of zebrafish, as well as ALS mouse model.

​ NMJ phenotype of transgenic zebrafish Tg(Zα:TetON-Rtn4al) harboring Rtn4al (NogoA homolog) cDNA driven by a Dox-inducible muscle-specific α-actin promoter after intramuscular injection of (A–C) GFP protein control and (D–F) Pgk1. Axons were labeled by synaptic vesicle glycoprotein 2A (SV2) in green, while postsynaptic receptors were labeled by α-Bungarotoxin (α-BTX) in red. (G) Statistical analysis of the number of colocalized axons and postsynaptic receptors in muscle of NogoA-overexpression zebrafish ALS-like model using Student’s t-test (***, p<0.001). NMJ phenotype of ALS mouse model harboring human SOD1 G93A after intramuscular injection of (H–J) PBS and (K–M) Pgk1 in the gastrocnemius muscle of the right hind leg. Neurofilament-H (NF-H) and SV2 labeled by green fluorescent signal were used to detect the axon terminal of motoneurons, while α-BTX labeled by red fluorescence signal was used to detect the acetylcholine receptor on motor endplates. (N) Statistical analysis of the number of innervated NMJ among PBS-injected WT mouse, PBS-injected SOD1 G93A mouse and Pgk1-injected SOD1 G93A mouse.​ Statistical analysis used Student’s t-test (***, significant difference at p<0.001).

https://doi.org/10.7554/eLife.49175.016

We then used an ALS cell line and mouse model to determine any beneficial effects of Pgk1 supplementation. We first differentiated motor neurons derived from human induced pluripotent stem cells (iPSCs) harboring human SOD1 mutant (G85R) (Figure 6—figure supplement 3) and then added Pgk1. Pgk1 addition reduced p-Cofilin through decreasing p-Limk1-S323 (Figure 6—figure supplement 4), as noted above, indicating that ePgk1 induces the same signal transduction pathway as that determined from the mouse cell line and human motor neurons.

Next, to continue exploring this question in vivo, we performed intramuscular injection of Pgk1 into the gastrocnemius muscle of the right hind leg of 60-day-old transgenic SOD1 G93A mice, followed by another injection every 15 days until mice were 120 days old. We quantified the proportion of innervated NMJ in the gastrocnemius muscle of the right hind leg of mice. Compared to the control group, the proportion of innervated NMJ of WT mice injected with PBS (WT/PBS), which was normalized as 100%, the proportion of innervated NMJ of SOD1 G93A/PBS was 13 ± 0.02%, indicating that the signal of motor neuron axon and axon terminal (NF-H/SV2) was significantly reduced. However, the proportion of innervated NMJ of SOD1 G93A mice injected with Pgk1 (SOD1 G93A/Pgk1) was 57 ± 0.08%, suggesting that supplementary addition of Pgk1 could rescue NMJ denervation (Figure 6H–N).

Furthermore, we examined the muscle contraction ability of hind leg in 130-day-old mice. In the WT/PBS group, the muscle contraction of both hind legs was normal, exhibiting strong movement. In contrast, in the SOD1 G93A/PBS group, muscle contraction of both hind legs was extremely poor (Figure 6—videos 1–3; Figure 6—figure supplement 5). Nevertheless, in the SOD1 G93A/Pgk1 group, muscle contraction of the left hind leg was as poor as that of the SOD1 G93A/PBS mice. Interestingly, muscle contraction of Pgk1-injected right hind leg remained functional, exhibiting a superior movement (Figure 6—videos 4–6). Exercise capability value of the SOD1 G93A/Pgk1 group was significantly higher than that of the SOD1 G93A/PBS group (Figure 6—figure supplement 5). Additionally, we found that the proportion of innervated NMJ in the gastrocnemius muscle of the Pgk1-injected right hind leg was increased compared to that of the left hind leg. Taken together, it is suggested that the Pgk1-injected right hind leg of SOD1 G93A mice could maintain some normal motor neurons to innervate muscle contraction.

Our collective findings reveal a novel target against the inhibition of neuron maturation and NOM by overexpression of NogoA through a Nogo66-independent pathway. NogoA has been known to inhibit neuron regeneration via Nogo66 and NiGΔ20 domains. To date, only Nogo66-interacting receptors were found. In this study, we revealed that overexpression of NogoA in muscle inhibits NOM through decreasing Pgk1 secretion (ePgk1), but without inducing the canonical Nogo66/NgR pathway. Here, we demonstrated that the addition of Pgk1 to media could promote NOM by reducing p-Cofilin-S3 through decreasing the phosphorylation of the Pak1/P38/MK2/Limk1-S323 signaling pathway via decreased Rac1-GTP (Figure 7). Whether NogoA also affects the secretion of other trophic factors to inhibit neuronal maturation and maintain NMJ integrity remains a topic for further study.

Figure 7
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Diagrams used to illustrate the extracellular Pgk1-mediated signal pathway proposed by this study to demonstrate how Pgk1 secreted from NogoA-overexpressed muscle cells affects the neurite outgrowth of motor neurons.

Left panel illustrates the small amount of NogoA presenting in muscle cells slightly reduces the amount of Pgk1 secreted from these muscle cells, resulting in strong inhibition of the Rac1-GTP/p-Pak1-T423/p-P38-T180/p-MK2-T334/p-Limk1-S323 axis within motor neurons, which, in turn, decreases the degree of p-Cofilin-S3. Consequently, the neurite outgrowth of motor neurons is developed. Right panel illustrates that overexpression of NogoA in muscle cells greatly reduces the amount of Pgk1 secreted from these muscle cells, resulting in weak inhibition of the Rac1-GTP/p-Pak1-T423/p-P38-T180/p-MK2-T334/p-Limk1-S323 axis within motor neurons, which, in turn, increases the degree of p-Cofilin-S3. Consequently, the neurite outgrowth of motor neurons is inhibited.

https://doi.org/10.7554/eLife.49175.028

Discussion

Overexpression of NogoA in cells affects the secretion of proteins

The expression of NogoA can generally affect the secretion of proteins. For example, the amount of insulin secreted from beta cells is increased in NogoA-deficient mice (Bonal et al., 2013). In PC12 cells, Zhong et al. (2015) demonstrated that silencing NogoA results in reducing the secretion of inflammatory factors, such as tumor necrosis factor-alpha and interleukin-6 . On the other hand, overexpression of NogoA increases Aβ42 secretion in rat cerebral cortical neurons (Xiao et al., 2012). In this study, when NSC34 motoneuron cells were cultured in CM obtained from cultured Sol8 myoblast cells, which harbor a NogoA-overexpression plasmid, we found that NOM was compromised. Furthermore, we demonstrated that Pgk1 is a key factor contributing to the locomotive properties of neurons in terms of NOM, neurite-bearing cell number and the decrease of p-Cofilin in neuron cells. This finding further demonstrates that improper crosstalk between muscle and nerve tissues impedes the NOM. Although Pgk1 addition could rescue failure NOM induced by culturing NSC34 cells in Sol8-NogoA CM, we noticed that the efficacy of enhancement mediated by Pgk1 alone could barely reach levels as high as those of NSC34 cells cultured in CM from Sol8-vector, suggesting that Pgk1 may not be the only secreted protein involved in enhancing synaptic growth.

Enhancement of NOM is a noncanonical function of ePgk1

To address the reduced secretion of Pgk1 from muscle cells by NogoA, as well as inhibition of the neuronal intracellular Rac1 pathway by ePgk1, we turn to the literature. First, Lay et al. (2000) found that Pgk1 secreted from tumor cells exhibits disulfide reductase activity, resulting in the release of angiostatin through reducing the disulfide bond of plasmin. Wang et al. (2007) also reported that CXCR4 could positively regulate the expression and secretion of Pgk1 in cancer cells. Overexpression of Pgk1 causes an increase of angiostatin, which, in turn, reduces the secretion of vascular endothelial growth factor and interleukin-8. At metastatic sites, a high level of CXCL12 reduces Pgk1 expression, resulting in reduced angiostatin function, which activates angiogenesis. Interestingly, when Rtn4a/NogoA is overexpressed in the somite boundary cells of zebrafish embryos, Lin et al. (2017) found that the expression of Cxcr4a is decreased. Aase et al. (2007) and Yi et al. (2011) reported that angiomotin (Amot), a cell membrane receptor of angiostatin, negatively controls Rac1 activity in endothelial and epithelial cells. Based on this convincing evidence from the literature, we speculate that NogoA/Rtn4a-overexpressed muscle cells may cause a decrease of Cxcr4a expression, resulting in reducing of Pgk1 secretion. The reduced ePgk1 may then cause a decrease in extracellular proteins, such as angiostatin, which, in turn, would increase the activity of Rac1, finally causing a physiological microenvironment for motor neurons that would not favor neurite outgrowth.

Boyd et al. (2017) reported the intracellular, bioenergetic function of Pgk1 in neuron cells, while we described the effect of Pgk1 secreted from muscle cells on NOM through a bioenergetic- and Nogo66-independent pathway. As Boyd et al. (2017) reported, when Pgk1 is insufficiently expressed, its participation in the glycolysis pathway is not effective, resulting in a reduced production of ATP, which, in turn, causes impaired neurological function. Thus, they concluded that Pgk1 is expressed in motor neurons, but also has a bioenergetics function. However, since the effect of pgk1-MO and pgk1 mRNA was not studied in the context of expression in muscle tissue, it remains unclear if the motor neuron phenotype results from the direct effect of Pgk1 within neurons or from indirect effect by defective function of surrounding cells or tissues. Therefore, in this study, we employed a muscle-specific promoter combined with the CRISPR/Cas9 system to specifically knock down Pgk1 in muscle cells, and we found that NOM was inhibited in adjacent nerve cells. Interestingly, when we used the muscle promoter to specifically overexpress Pgk1 in muscle cells, the failed NOM induced by NogoA/Rtn4al overexpression in muscle cells could be rescued. Additionally, we demonstrated that addition of Pgk1 in CM and DM had no impact on the efficiency of intracellular glycolysis in cultured NSC34 cells. In fact, the addition of Pgk1 enables the enhancement of NOM in NSC34 cells, even under reduced glycolytic condition. Additionally, when Pgk1 was ubiquitously knocked down, as Boyd et al. (2017) reported, the total amount of Pgk1 was reduced, including the small amount of secreted Pgk1 from muscle cells, resulting in a phenotype similar to what we observed in this study. Based on this line of evidence, we demonstrated that ePgk1 enhances NOM in a manner functionally independent of its intracellular, canonical role as a supplier of energy, as described by Boyd et al. (2017).

Potential application of ePgk1

Thus, we have identified a Pgk1/p-Limk1-S323/p-Cofilin-S3 axis in which secreted Pgk1 emerges as an extracellular trigger in the NOM. As illustrated in Figure 6, the following cascade of events takes place. ePgk1 decreases p-Limk1 at S323, which, in turn, results in the decrease of p-Cofilin-S3 and, finally, the rescue of NOM inhibited by culturing in Sol8-NogoA CM. In the process of investigating this novel signaling pathway, we found that overexpression of NogoA in muscle cells resulted in altering the amount of some secreted proteins in muscle cells. For example, the amount of secreted Pgk1 is reduced, which, in turn, inhibits the NOM. Meanwhile, we know that NogoA, as marker of morbidity, overexpresses in the muscle cells of ALS patients at an early stage (Pradat et al., 2007). Because NogoA does not contain a signal peptide (Chen et al., 2000), it is barely detectable in patients’ blood (Harel et al., 2009). Therefore, we have detected a property of Pgk1 in serum that may serve as an indicator of incipient ALS. More recently, Meininger et al. (2017) reported that treatment with Ozanezumab, a humanized monoclonal antibody against NogoA, showed no significant improvement for ALS patients in phase two trial, suggesting that NogoA may not be an effective therapeutic target. Moving away from this target, we proposed in the present study that secreted protein from NogoA-overexpressed muscle cells is a highly promising alternative therapeutic target. More specifically, we showed that the reduced amount of secreted Pgk1 causes neurodegeneration and that the restorative effect of Pgk1 on NOM does not arise through the Nogo66/NgR/ROCK/Limk1-T508 pathway. Although intercellular Nogo66/NgR interaction is blocked by NogoA antibody, it may not change the fact that NogoA is still overexpressed within muscle cells, resulting in the decrease of Pgk1 secretion. Meanwhile, Pgk1 deficiency is inherited as an X-linked recessive genetic trait. It is caused by mutated Pgk1, the folding and stability of which are altered (Chiarelli et al., 2012; Pey et al., 2014). Pgk1 deficiency is accompanied by suffering from muscle lesions, motor neuron defect, neurological dysfunction and myopathy (Valentini et al., 2013; Matsumaru et al., 2017), as well as susceptibility to Parkinson’s disease (Sakaue et al., 2017). Therefore, it can be reasonably speculated that the abnormal secretion of Pgk1 may affect neuronal development, which supports our finding that Pgk1 secreted from muscle cells plays a novel role in NOM and neuronal development.

Materials and methods

Establishment of stable cell lines harboring Sol8-vector and Sol8-NogoA

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The lentivirus vectors pAS4.1w.Pbsd-aOn and pAS4.1w.Ppuro-aOn, purchased from the National RNAi Core Facility Platform, Academia Sinica, Taiwan, were used to establish Sol8 myoblasts (RRID:CVCL_6449), which express transgene conditionally. The Sol8-vector was a plasmid pAS4.1w.Ppuro-aOn, while Sol8-NogoA was a plasmid pAS4.1w.Ppuro-aOn with an insert of NogoA cDNA, pAS4.1w.Ppuro-aOn-NogoA. Expression plasmids pCMVΔR8.91 and pMD.G, and lentivirus vector were transfected into cells by Lipofectamine 2000 (Invitrogen) with a 0.9:0.1:1 ratio, respectively, according to the manufacturer’s protocol. The culture medium containing infected virus particles of HEK293T cells (RRID:CVCL_0063, confirmed negative for mycoplasma contamination) was collected at 24 and 48 hr post-transfection after spinning down at 1250 rpm at room temperature. When Sol8 cells grew up to 40% confluency, the supernatant was saved and transferred to Sol8 myoblasts with addition of Polybrene (Sigma) at the concentration of 8 μg/ml for 24 hr. After incubation, the medium was replaced by a fresh growth medium containing Puromycin at the concentration of 4 μg/ml.

NSC34 cells were cultured in conditioned medium

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After cells reached at 90% confluency, medium was replaced by horse serum (HS)-containing differentiation medium DMEM (2.5% HS, 100 units/ml Penicillin and 0.1 mg Streptomycin) containing Dox at 1 μg/ml. After 24 hr incubation, medium was again replaced by FBS-containing DMEM containing Dox. After another 24 hr incubation, the conditioned medium of Sol8 myoblasts was collected and centrifuged at 1000 rpm. The saved supernatant was used to culture NSC34 cells (Cashman et al., 1992) until they reached at 60% confluency. Medium was then replaced by fresh FBS-containing DMEM containing Dox and continuously cultured for 24 hr. The conditioned media of cultured Sol8 myoblasts was collected by centrifugation and used to culture NSC34 cells for 48 hr. Then, cell lysate was obtained for Western blot analysis.

In vitro platform of NOM derived from NSC34 cells

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NSC34 cells were seeded in 10% FBS DMEM medium onto six-well plates at cell density of 3 × 104 per well. After 24 hr incubation, culture medium was replaced by 2.5% FBS DMEM medium to stimulate differentiation of NSC34. After another 24 hr incubation, medium was exchanged for Sol8-conditioned medium and incubated for 96 hr. Finally, cells were fixed with 4% PFA overnight. Neurites extending from each cell were captured by microscopy (Leica) under 200 times magnification and labeled by Neurolucida Neuron Tracing Software 9.0 (Neurolucida 9.0). For each image, 40 cells cultured in either CM or DM were randomly chosen to measure the length of neurites. Data were obtained from three independent trials (120 cells in total). To profile neurite length distribution, we grouped the neurite length into several groups, namely every 25 intervals under 50 µm, every 50 intervals under 200 µm, and 201–300 and 301–600 µm, and calculated the number of cells in each range. To count the number of neurite-bearing cells, we calculated the number of cells with neurites at least 50 μm in length in each range.

Frozen section and immunostaining of zebrafish, mice and NSC34 cells

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The procedures of frozen section and immunostaining of zebrafish and mice tissues and cells were previously described by Lin et al. (2019), except primary antibodies, including anti-Syn1 (RRID:AB_2200097; 1:250), anti-neurofilament H (NF-H) (RRID:AB_91202; 1:500) and anti-acetyl-tubulin (RRID:AB_297928; 1:500), and secondary antibodies, including anti-rabbit Cy2 (RRID:AB_827264; 1:200) and Alexa Fluor 488-conjugated Goat anti-mouse IgG (RRID:AB_2576208; 1:500), were used. The patterns were observed under a Laser Scanning Confocal Fluorescence Microscope (Zeiss). Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (Sigma).

Two-dimensional (2D) SDS-PAGE and LC/MS-MS analyses

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Total proteins of CM from cultured muscle cells in Sol8-NogoA and Sol8-vector were separately collected. Before total proteins were analyzed on 2D SDS-PAGE, Cibacron Blue F3G-A and ammonium sulfate precipitation were performed to remove albumin contained in CM (Colantonio et al., 2005), while remaining proteins were mixed with acetone, precipitated by centrifugation, and dissolved in rehydration buffer (8 M urea, 2% NP-40, and 100 mM DTT). The strips were subjected to rehydration overnight and isoelectric focusing. After isoelectric focusing, the strips were removed and equilibrated. The equilibrated strips were run on the gel as previously described by Sanchez et al. (1999). Gels were stained by Coomassie blue and dried between two sheets of cellophane. Protein spots were excised and subjected to in-gel digestion. The procedures of in-gel digestion and LC/MS-MS analyses were done as previously described in Fu et al. (2012). First, we employed 2D electrophoresis to analyze total protein content in CM. Next, we selected a total of 20 spots at random, all displaying more or less intensity, and we then made a comparison between the Sol8-NogoA CM protein pattern and that of Sol8-vector CM. Following LC MS/MS, 42 candidate proteins were identified (see Figure 1—figure supplement 2). We picked up 10 proteins reduced in Sol8-NogoA CM, cloned their cDNAs, and performed genetic engineering to overexpress these genes individually in Sol8-NogoA cells. We also picked up 10 proteins enhanced in Sol8-NogoA CM, determined their cDNAs, and designed the corresponding siRNAs to knock down their expression individually in Sol8-NogoA cells. All corresponding CMs were collected to culture NSC34 cells separately. After 48 hr in culture, the degree of p-Cofilin expression in NSC34 cells was quantified, and the genes exhibiting lower expression of p-Cofilin were further selected.

Zebrafish husbandry and microscopy observation

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Zebrafish (Danio rerio) wild-type AB strain (RRID:ZIRC_ZL1) and transgenic lines Tg(mnx1:GFP) (RRID:ZIRC_ZL1163) (Flanagan-Steet et al., 2005), purchased from ZIRC, and Tg(Zα:TetON-Rtn4al) (Lin et al., 2019) were used. Production and stage identification of embryos followed the description by Lin et al. (2019).

Ethics statement

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The Mackay Medical College Institutional Animal Care and Use Committee (IACUC) reviewed and approved the protocol described below (MMC-A1060009).

Using CRISPR/Cas9 system to perform knockdown experiments

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The sgRNAs of pgk1 (TGGACGTGAAAGGAAAGC) and pgam2 (CCTGGAGGAGGCGAAACG) were subcloned into plasmid pDR274 and transcribed into mRNAs in vitro by the mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies). The linearized plasmid pZα-Cas9, in which Cas9 was driven by the muscle-specific alpha-actin promoter of zebrafish, was coinjected separately with pgk1 and pgam2 (control) sgRNAs into zebrafish embryos, and phenotypes were observed at 30 hpf.

Plasmid construction

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The coding region of Limk1 cDNA (NM_010717.3) fused with FLAG-tag was inserted into pCS2+ to generate pCS2-Limk1-flag. Mutated forms of Limk1, such as Limk1 S323A and T508V, were obtained by PCR and inserted into pCS2+ to generate pCS2-Limk1-S323A-flag and pCS2-Limk1-T508V-flag, respectively. Similarly, Pak1 cDNA (NM_011035.2) with FLAG-tag was inserted into pcDNA3 to generate pcDNA3-Pak1-flag. Additionally, zebrafish pgk1 (NM_213387.1) fused with P2A-RFP and Cas9 fused with P2A-RFP were synthesized and engineered with zebrafish muscle-specific α-actin promoter to generate pZα-Pgk1 and pZα-Cas9 for zebrafish embryo microinjection.

Fluorescence-activated cell sorting (FACS)

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Procedure of the dissociation of zebrafish embryonic cells was followed the previous study reported by Lin et al. (2017) with some modifications. Briefly, the WT embryos and embryos injected with pZα-Cas9, pZα-Cas9 plus pgk1 sgRNA, pZα-Cas9 plus pgam2 sgRNA and pZα-Pgk1 at 30 hpf were incubated with trypsin (Sigma) for 20 min at room temperature. After treatment, embryos were shattered by gently pipetting, followed by separating cells completely from tissues. Then, the RFP-expressing cells were sorted and collected by Cell Sorters (BD FACSAria III).

Protein expression and purification

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Protein purification followed the procedures described by Fu et al. (2017). Plasmids expressing FLAG-fusion proteins were transfected into the HEK293T cell line. Transfected cells were lysed using Pierce IP lysis buffer (ThermoFisher Scientific; TFS) with protease inhibitor cocktail (Roche). After cell debris was removed by centrifugation, anti-FLAG M2 affinity gel beads were added to cell extracts and incubated for 16 hr at 4°C. Beads-FLAG-protein complex was eluted by incubation with 3X FLAG peptide for one hr. The FLAG-fusion proteins eluate was restored and used for the following experiment. Plasmids pGEX-GST and pGEX-GST-Nogo66 were used to express recombinant proteins using 0.1 mM Isopropyl β-D-1-thiogalactopyranoside induction for 1 hr at 37°C in an Escherichia coli BL21 (ATCC BAA-1025TM) expression system and purified by Glutathione resin (Clontech).

Western blot analysis

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Total proteins extracted from embryos were analyzed on a 10% SDS-PAGE followed by Western blot analysis according to the procedures described by Lin et al. (2017). The antibodies against NogoA (RRID:AB_650319; 1:500), Cofilin (RRID:AB_10622000; 1:1000), phosphorylated Cofilin at S3 (RRID:AB_2080597; 1:1000), Rho-associated protein kinase 2 (ROCK2) (RRID:AB_10829468; 1:2000), phosphorylated ROCK2 at Y256 (RRID:AB_2182301; 1:2000), Epidermal growth factor receptor (EGFR) (RRID:AB_2246311; 1:500), phosphorylated EGFR at Y1173 (RRID:AB_331795; 1:2000), Akt (Protein kinase B; PKB) (RRID:AB_329827; 1:1000), phosphorylated Akt at S473 (RRID:AB_2315049; 1:1000), LIM domain kinase 1 (Limk1) (RRID:AB_648350; 1:500), phosphorylated Limk1 at S323 (RRID:AB_2136940; 1:1000), phosphorylated Limk1 at T508 (RRID:AB_2136943; 1:500), Phosphoglycerate kinase 1 (Pgk1) (RRID:AB_2161220; 1:2000) (RRID:AB_2268000 for zebrafish; 1:1000), Phosphoglycerate mutase 2 (Pgam2) (RRID:AB_1951200; 1:1000), Ras-related C3 botulinum toxin substrate 1 (Rac1) (RRID:AB_2721173; 1:500), p21-activated kinase 1 (Pak1) (RRID:AB_330222; 1:1000), phosphorylated Pak1 at T423 (RRID:AB_330220; 1:1000), P38 mitogen-activated protein kinases (P38) (RRID:AB_330713; 1:1000), phosphorylated P38 at T180 (RRID:AB_331641; 1:1000), MAP kinase-activated protein kinase 2 (MK2) (RRID:AB_10694238; 1:1000), phosphorylated MK2 at T334 (RRID:AB_490936; 1:1000), Growth Associated Protein 43 (GAP43) (RRID:AB_443303; 1:1000), Choline acetyltransferase (ChAT) (RRID:AB_2244867; 1:1000), Microtubule-associated protein 2 (MAP2) (RRID:AB_2138153; 1:1000), α-tubulin (RRID:AB_477579; 1:5000), Myc (RRID:AB_439680; 1:2000), Flag (RRID:AB_446355; 1:5000), rabbit anti-sheep-HRP (RRID:AB_656968; 1:5000), goat anti-mouse-HRP (RRID:AB_955439; 1:5000) and goat anti-rabbit-HRP (RRID:AB_631746; 1:5000) were used.

Glycolysis stress test assay

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Seahorse XF glycolysis stress test kit (Seahorse Bioscience, USA) was used to measure the extracellular acidification rate (ECAR; mpH/min), also called the H+ production rate, of NSC34 cells. Briefly, NSC34 cells were seeded in a 10% FBS DMEM medium onto SeaHorse XF 24-well plates at a cell density of 2.5 × 105 per well. After 24 hr incubation, culture medium was replaced by a 2.5% FBS DMEM medium to initiate cell differentiation. Then, NSC34 cells were treated with either Pgk1 (Bio-Techne R and D Systems; BT) at 33 ng/ml or 3PO (SA) at 3 M for two days. When medium was replaced by glucose-free Seahorse 24-well XF Cell Culture, cells were continuously incubated in a non-CO2 incubator at 37°C for 30 min before assay. To measure the ECAR of the surrounding media, glycolytic flux, such as basal glycolysis, glycolytic capacity and glycolytic reserve, was analyzed by the sequential addition of 10 mM glucose, 1 M Oligomycin and 50 mM 2-deoxyglucose in an XF24 flux analyzer.

Medium for culturing iPS cell lines

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The generation of healthy- and ALS-iPS cells from peripheral blood mononuclear cells of hSOD1 G85R was approved under the Institutional Review Board of Hualien Tzu Chi General Hospital, Hualien, Taiwan (IRB-105–131-A). Briefly, iPS cells were established with Cytotune-ips 2.0 Sendai Reprogramming kit (TFS) and cultured in Essential eight medium (TFS) on 1.0% hES qualified Matrigel (Becton Dickinson)-coated cell culture dishes. The cells were passaged per 3–5 days using Accutase (Merck-Millipore), mechanically scraped, and then reseeded at a 1:5 to 1:10 ratio. The culture medium was refreshed daily.

Motor neuron induction and maturation

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When cell confluence was reached at 80% on the culture dish, cells were treated with Accutase for 2–5 min and scraped. The cell clumps were dissociated into 200–300 mm clusters and transferred to noncoated Petri dishes for 48 hr for embryoid body (EB) formation. The EBs were applied by the modified BiSF neural induction method, according to the methods described previously (Chen et al., 2015). The differentiation medium within the first two days was Essential 6 (TFS). Subsequently, on D2 of differentiation, the cell culture medium was changed to DMEM-F12 supplied with 1% N2 supplement (TFS), 1 mM NEAAs and 2 mM glutamate. Neuron-inducing factors, including 3 µM CHIR99021 (SA), 10 µM SB431542 (SA) and 10 ng/ml recombinant human FGF-2 (R and D Systems) were added on D2 for 2 days. On D4, the neural induction medium was removed, and the neurospheres were cultured in neurobasal medium (TFS) with 1% N2 supplement. Motor neuron patterning factors including 0.5 µM retinoic acid (RA, SA), 0.5 µM purmorphamine and 0.5 µM Smoothened Agonist (SAG, SA) were added from D4 for 5 days, and then reduced dosage of motor neuron patterning factors (0.1 µM RA and 0.25 µM SAG) were added for another 5 days. After complete motor neuron patterning, the cells were dissociated into small clumps by Accutase and seeded on 1% Matrigel (TFS)-coated cell culture dishes for neural maturation. The motor neuron progenitors (MNPs) proliferated in neurobasal medium with 2% B27 supplement (TFS), 10 ng/mL brain-derived neurotrophic factor (BT), 10 ng/ml Glial Cell Line-derived Neurotrophic Factor (BT) and 1 µM Dibutyryl-cAMP (dbcAMP, SA). During the passaging of MNPs with Accutase, providing 10 µM Y27632 (SA) or RevitaCell Supplement (TFS) effectively attenuated dissociation-triggered cell death.

Intramuscular injection of Pgk1

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We directly injected Pgk1 (2 μg) into muscle of adult fish from transgenic line Tg(Zα:TetON-Rtn4al), followed by Dox immersion for one week when fish exhibited motorneuron degeneration. The percentage of overlapping signals between SV2 and α-BTX were calculated. We also performed intramuscular injection of Pgk1 into the gastrocnemius muscle of the right hind leg of six 60-day-old (at P60) transgenic SOD1 G93A mice (RRID:IMSR_JAX:002726). Each time, a volume of 80 µl Pgk1 dissolved in PBS solution in a concentration of 375 µg/ml was injected. On three of these frozen longitudinal sections, we performed immunofluorescence staining to quantify the proportion of innervated NMJ in the gastrocnemius muscle of the right hind leg at P75. The remaining three were continuously given Pgk1 injections every 15 days until mice were 120 days old. Six (three for NMJ and three for exercise studies) wild-type mice injected with 80 µl PBS (WT/PBS) into the right leg served as the sham group, and six (three for NMJ and three for exercise studies) SOD1 G93A mice injected with PBS (SOD1 G93A/PBS) served as the negative control group. When the SOD1 G93A/PBS and SOD1 G93A/Pgk1 mice were 130 days old, their movement was recorded by video, and exercise capability of both hind legs was evaluated by quantifying the number of leg contractions within one min. Two legs of each mouse were counted separately. The exercise capability value of each mouse was calculated from the increased fold(s) in the number of contractions between injected right leg versus untreated left leg. Data of each group were averaged from three mouse samples and presented as mean ±S.D.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

References

Decision letter

  1. Didier Y Stainier
    Senior Editor; Max Planck Institute for Heart and Lung Research, Germany
  2. Fadel Tissir
    Reviewing Editor; Université Catholique de Louvain, Belgium
  3. Fadel Tissir
    Reviewer; Université Catholique de Louvain, Belgium
  4. Libing Zhou
    Reviewer; Guangdong-Hongkong-Macau Institute of CNS Regeneration, China

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your work entitled "Extracellular Pgk1 enhances neurite outgrowth of motoneurons through Nogo66-independent targeting of NogoA" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Fadel Tissir as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Senior Editor.

While the three reviewers found the work interesting, there are issues that need to be addressed by additional experiments. These include the validation and appropriate controls for Pgk1 Crispr/Cas experiments (two reviewers), the confirmation that overexpression of NogoA affects Pgk1 secretion in vivo (all reviewers), and the claim that Pgk1 function is NgR independent (two reviewers).

Addressing these points will require more than the typical 2 months for a standard eLife revision. We would be happy to re-consider the manuscript should you consider to revise along the suggestions of reviewers appended below.

Reviewer #1:

In this manuscript, Chen Yung Lin, Huai Jen Tsai and colleagues report that Pgk1 promotes neurites outgrowth of motor neurons independently of its glycolytic function and of Nogo66. They used downregulation, upregulation, and rescue experiments in cells (myoblasts, and NSC34 derived motor neuron cell-like cells), as well as zebrafish and mouse; and showed that:

1) Conditioned medium of myoblast cells overexpressing NogoA inhibits motor neuron neurites outgrowth

2) Analysis of the conditioned medium by 2D electrophoresis and LC MS shows that overexpression of NogoA reduces the level of secreted Pgk1.

3) Addition of Pgk1 restores neurites growth, decreases the amount of p-cofilin via pLimk-S323, and promotes neuronal differentiation and maturation.

4) Pgk1 blocking antibody has the opposite effect

5) The effect of Pgk1 on neurites growth in independent of its glycolytic role as Pgk1 mutants that have no catalytic activity are still capable to reduce p-cofilin levels and induce changes in neurites growth

6) CrispR/Cas-mediated inactivation of Pgk1 in fish muscle cells inhibits neurites outgrowth while upregulation of Pgk1 promotes (ectopic) growth.

7) Intramuscular injection of Pgk1 recues the neuromuscular junction defects in Rtn4al transgenic Fish and SOD1 transgenic mice.

Overall, the work is of high quality. The results are compelling, and virtually all possible controls have been done.

Points to address:

1) The text contains too many typographical and grammatical errors that make the manuscript difficult to understand sometimes. The text has to be edited to facilitate the reading and clarify the message.

2) Were all the "spots" identified by 2D electrophoresis subjected to MS analysis, and is Pgk1 the only protein identified by LC-MS? If not, why this one has been selected?

3) The validation of Cripr/cas9-mediated inactivation/upregulation of Pgk1 in zebrafish was done by monitoring the expression of the RFP reporter. While changes in RFP expression are obvious, this does not tell much about Pgk1 levels in the KO and transgenic Fish compared to endogenous expression. Western blot analysis would be helpful. Also, could the authors rule out any off-target effect as injection Cas9 alone (driven by the muscle specific α actin promoter, but in absence guide RNA) seems to produce some effects on motor axons (compare D-D" to C-C")?

Reviewer #2:

In the manuscript by Lin, et al., the authors identifiy Pgk1 as a novel secreted molecule that can stimulate neurite outgrowth. They further show that the secretion of Pgk1 is reduced when NogoA is expressed in the secreting cells, identify a potential mechanism by which NogoA reduces axon outgrowth independent of its interactions with Nogo receptors.

While the identification of this novel role for Pgk1 is very interesting, there are a number of issues throughout the study that are of major concern, including a lack of important controls. In particular are the following points:

1) Throughout the paper, there is inadequate information about "N"s in experiments, and inappropriate statistical analysis for multiple comparisons.

2) There is no indication of Pgk1 dose or timecourse throughout the entire study. Is the dose used physiologically relevant? Is there a dose dependent response?

3) Does the NogoA-Pgk1 secretion mechanism also function in primary muscle cells and motor neurons in culture? Is there evidence for NogoA over-expression inducing a reduction in Pgk1 expression in zebrafish and in mouse in vivo?

4) At the end of the Results section, the authors mention anecdotally that there is a motor behavior phenotype and provide videos. However, there is no quantification and no description in the Materials and methods section to indicate any type of quantitative assessment of motor behavior. How many mice were used in these experiments?

5) The authors conclude that Pgk1 functions independent of NgR. However, their only evidence for this is the lack of activation of downstream signaling cascades, and there are no positive controls included in these experiments. This is very indirect, and does not allow the authors to conclude Pgk1 function is independent of NgR.

6) How NogoA reduces the secretion of Pgk1 from muscle cells and how extracellular Pgk1 inhibits the intracellular Rac1-PAK pathway is unclear and needs additional investigation and/or discussion.

Additional comments on the specific Figures:

Figure 1:

The authors need to confirm that there is no NOGO-A in the conditioned media

The specificity of the Pgk1 Ab used in Figure 1B needs to be confirmed

Figure 2:

It is unclear how well the markers used (MAP2, ChAT, GAP43, SYN1) can actually reflect a differentiated status in NSC34 cells. These cells have very small processes, and therefore are unlikely to represent a truly differentiated state. In subsection “Supplementary addition of Pgk1 can enhance the maturation of NSC34 cells cultured in Sol8-NogoA CM”, the authors state "The number of neurons with Syn1 signal at the end of synapse could be restored to that cultured in Sol8-vector…". It is unlikely that these are synapses, they look more like growth cones and don't contact "postsynaptic" structures

Figure 4:

In zebrafish experiments, the authors use CRISPR/Cas9 system to knockout Pgk1 and Pgam2 from muscle cells. There needs to be data validating that sgRNAs targeting Pgk1 and Pgam2 are effective in reducing Pgk1 and Pgam2 expression, respectively. Similarly, in the muscle-specific over expression paradigm, the authors need to show that Pgk1 expression is increased.

While the authors provide information on the penetrance of the phenotypes, they do not quantify the extent of axonal extension in any meaningful way.

Figure 5:

The authors conclude that the effect of Pgk1 is NgR-independent due to the lack of p-ROCK2-Y256, p-EGFR-Y1173 and p-Akt-S473 (Figure 5—figure supplement 1). However, there is no positive control in this experiment. The authors should show that they are able to detect changes in these pathways in response to NgR activation by NOGO-A.

Figure 6:

Because the authors did not provide information on the dose-dependency and timecourse of Pgk1 in their in vitro experiments, it is unclear why they chose the specific dose and timing regimens for their in vivo experiments. Fish were injected with 2ug Pgk1, then placed in Dox+ water for 1 week. How long is the injected Pgk1 biologically active?

There is a discrepancy between what is in the Results section and the Materials and methods section with respect to the experiments done in mice in vivo. The authors indicate that mice were injected with Pkg1 once every 15 days between P60 and P120. However, in the Materials and methods section (546-548) they indicate tissue sections were taken at P75. Is the data presented in Figure 6 H-N from P75 or P120 mice?

The authors should provide images and other metrics (neurite outgrowth, expression of motor neuron specific genes) from their iPS-SOD G85R motor neuron cultures so the degree of differentiation these iPS cells can be assessed.

- Figure 6—figure supplement 2 panel B: Why is there no change in the levels of p-Cofilin in response to Pgk1 in the control cells? In Figure 1A, this same treatment causes a ~3-fold decrease p-Cofilin levels.

Reviewer #3:

In this manuscript, the authors found: 1) Overexpression of NogoA in a muscle cell line Sol8 reduced the secretion of Pgk1 detected from the condition medium; 2) Neurite outgrowth of NSC34 cells was inhibited by Sol8-NogoA CM (conditioned medium) and could be rescued by additional Pgk1; 3) Sol8-NogoA CM increased p-Cofilin S3, while extracellular Pgk1 decreased Rac1-GTP/p-Pak1-T423/p-P38-T180/p-MK2-T334/p-Limk1-S323/p-Cofilin-S3 pathway; 4) Conditional KO of Pgk1 in zebrafish muscle cells impaired motoneuron axon growth and the increase of Pgk1 in muscles cells resulted in ectopic growth of motoneuron axons; 5) Intramuscular injection of Pgk1 fostered motoneuon axon regrowth and NMJ formation in Rtn4al-overexpressing transgenic zebrafish and SOD1 transgenic mice. This study revealed a new indirect mechanism how NogoA in the muscle affected neurite growth of motoneurons. The finding is interesting and helpful for understanding the probable mechanisms in steering axon growth. However, there are some issues needed to be addressed.

1) Nogo-A is a transmembrane protein, it could exert its effects by direct interacting with its receptors on the neurons, or it can work indirectly through affecting other molecule expression or secretion in the muscle. The authors used CM from NogoA-overexpressing cells to treat neurons, which supports the indirect effect. Given NogoA is a transmembrane protein, it is not likely present in the CM. Without the co-culture experiment of muscle cells and motor neurons, it's inappropriate to exclude the possibility that NogoA in the muscle could also affects neurite growth through a direct ligand/receptor interaction. Therefore, the conclusion "In this study, we revealed that overexpression of NogoA in muscle inhibits NOM through decreasing Pgk1 secretion (ePgk1), but without inducing the canonical Nogo66/NgR pathway."(subsection “Intramuscular injection of Pgk1 is able to rescue NMJ denervation caused by Rtn4al-overexpression transgenic zebrafish and SOD1 G93A transgenic mice”) is not accurate.

2) Is there any evidence in vivo that NogoA upregulation in the muscle affected its Pgk1 production, i.e. the NogoA and Pgk1 levels in the SOD1 mutant mice or Rtn4al-overexpressing transgenic zebrafish? Or at least confirm whether overexpressing NogoA in the muscle of normal adult mice using viral vector will also affect Pgk1 secretion.

3) The authors seemed to confuse with neurite and synapse: subsection “in vitro platform of neurite outgrowth of motor neurons derived from NSC34 cells”; "Synapses extending from each cell were captured by microscope.…"; "calculate the length of synapses…"

4) In Figure 1—figure supplement 2, the authors need quantify the Pgk1 amount in the CM. In addition, there is no information about the source and the concentration of ePgk1 added into the culture. Whether is it comparable to the amount in the CM?

5) In the method of neurite outgrowth (subsection “in vitro platform of neurite outgrowth of motor neurons derived from NSC34 cells”), it says: "In each image, 40~50 400 cells were randomly chosen to calculate the length of synapses and the number of neurite-bearing cells, the synapses of which were at least 50 μm in length.". If only cells with neurite length longer than 50 μm were chosen, why the average neurite lengths in some groups of Figure 1E are shorter than 50 μm?

https://doi.org/10.7554/eLife.49175.030

Author response

Reviewer #1:

Points to address:

1) The text contains too many typographical and grammatical errors that make the manuscript difficult to understand sometimes. The text has to be edited to facilitate the reading and clarify the message.

I apologize for having made so many mistakes. I have asked an expert to edit this manuscript.

2) Were all the "spots" identified by 2D electrophoresis subjected to MS analysis, and is Pgk1 the only protein identified by LC-MS? If not, why this one has been selected?

In the revised Material and Methods section, we described in more detail how Pgk1 was selected for this study, as follows: “First, we employed 2D electrophoresis to analyze total protein content in CM. Next, we selected a total of 20 spots at random, all displaying more or less intensity, and we then made a comparison between the Sol8-NogoA CM protein pattern and that of Sol8-vector CM. Following LC MS/MS, 42 candidate proteins were identified (see Figure 1—figure supplement 2). We picked up 10 proteins reduced in Sol8-NogoA CM, cloned their cDNAs, and performed genetic engineering to overexpress these genes individually in Sol8-NogoA cells. We also picked up 10 proteins enhanced in Sol8-NogoA CM, determined their cDNAs, and designed the corresponding siRNAs to knock down their expression individually in Sol8-NogoA cells. All corresponding CMs were collected to culture NSC34 cells separately. After 48 hr in culture, the degree of p-Cofilin expression in NSC34 cells was quantified, and the genes exhibiting lower expression of p-Cofilin were further selected.”

The Results section has been revised as follows: “We further employed 2D electrophoresis, followed by LC MS/MS, to analyze the total protein content in CM. Among these examined proteins, we found that the level of Pgk1 protein contained in Sol8-NogoA CM was significantly reduced compared to that in Sol8-vector CM (see Figure 1—figure supplement 2). Furthermore, the degree of p-Cofilin expression in NSC34 cells cultured in CM from Pgk1-overexpressed Sol8-NogoA cells was significantly reduced compared with that in NSC34 cells cultured in CM from Sol8-NogoA cells. Therefore, Pgk1 was chosen for further study to confirm its potential role in NOM.”

3) The validation of Cripr/cas9-mediated inactivation/upregulation of Pgk1 in zebrafish was done by monitoring the expression of the RFP reporter. While changes in RFP expression are obvious, this does not tell much about Pgk1 levels in the KO and transgenic Fish compared to endogenous expression. Western blot analysis would be helpful.

In response to your suggestion, we added Crispr/cas9-mediated inactivation and upregulation of Pgk1 in zebrafish muscle cells and highlighted in blue the relevant changes in the Results section, as follows: “We used muscle-specific α-actin promoter to drive overexpression of Cas9 in muscle cells in order to exclusively knock out the Pgk1 gene. […] As suggested by quantification of the axonal extension phenotype shown in Figure 4—figure supplement 2, the increase of Pgk1 in muscle cells enhances NOM.”

Also, could the authors rule out any off-target effect as injection Cas9 alone (driven by the muscle specific α actin promoter, but in absence guide RNA) seems to produce some effects on motor axons (compare D-D" to C-C")?

In a previous manuscript, our image of the Cas9-injected embryo seemed to reflect delayed development. To confirm this result, we repeated this experiment and found that almost Cas9-injected embryos did not exhibit defective motoneurons except that only very low percentage of injected embryos exhibited delayed development. Therefore, to avoid misinterpretation, we have substituted a new image in the revised Figure 4D.

When we injected either Cas9 alone or pZα-Cas9, off-target effect might have have been occasionally found. For example, sometimes few injected embryos seemed to exhibit developmental delay. However, we noticed that this defect is commonly observed in zebrafish embryos injected with exogenous plasmid DNA molecules and thus not specific to pZα-Cas9 injection. It should be noted that plasmid pZα-Cas9 used in this study was designed for overexpression of muscle-specific Cas9 since Cas9 was driven by muscle-specific α-actin promoter. Therefore, we believe that injection of pZα-Cas9 might pose less risk of interfering with the development of other tissues. For example, the same phenotype of motor axon defect was found in all defective embryos (65% of 56 injected embryos) injected with pZα-Cas9 plus Pgk1 sgRNA. At the same time, however, no phenotype was found in more than 96% of 50 embryos injected with pZα-Cas9 plus Pgam2 sgRNA. This line of evidence suggested that the off-target effect caused by pZα-Cas9 hardly occurred in our study and, therefore, did not represent that the phenotypes we described here were due to off-target effect.

More recently, Yang et al. (2018) published a paper entitled “Generation of Cas9 transgenic zebrafish and their application in establishing an ERV-deficient animal model” (Biotechnol Lett, 40:1507–1518), in which they used Elongation Factor 1 (Elf1α) promoter to drive the overexpression of Cas9 around the whole body. They could inhibit target gene by injection sgRNA in F2 generation because they found that Cas9 overexpression alone did not affect the growth of zebrafish. I am suggesting that this evidence tends to support our use of Cas9 in this study.

Reviewer #2:

In the manuscript by Lin, et al., the authors identifiy Pgk1 as a novel secreted molecule that can stimulate neurite outgrowth. They further show that the secretion of Pgk1 is reduced when NogoA is expressed in the secreting cells, identify a potential mechanism by which NogoA reduces axon outgrowth independent of its interactions with Nogo receptors.

While the identification of this novel role for Pgk1 is very interesting, there are a number of issues throughout the study that are of major concern, including a lack of important controls. In particular are the following points:

1) Throughout the paper, there is inadequate information about "N"s in experiments, and inappropriate statistical analysis for multiple comparisons.

Thank you for pointing out these mistakes. In the revised manuscript, we present adequate percentages of experimental data among the total number of each “N” in all relevant sections and figure legends. Statistical analysis was also appropriately presented in each figure. Especially, we readjusted the representation of Western blot data, e.g., the relative quantification of phosphorylated proteins. We defined the expression level of control cell group, such as Sol8-vector CM or DM, as 1, and then we normalized it to the expression levels of experimental cell groups. Therefore, we deleted the p-value of the control group versus other experimental groups since there was no standard deviation (nonparametric) in the control group. Student t-test analysis was performed only for groups with standard deviation, such as the Sol8-NogoA CM group compared to the Sol8-NogoA CM + Pgk1 group. Statistical analyses were revised in Figures 1A, 1B, 1C, 2E, 5A, 5C, 5D and Figure 5—figure supplement 1D.

2) There is no indication of Pgk1 dose or timecourse throughout the entire study. Is there a dose dependent response?

We have performed a new experiment in which different doses of Pgk1 (0-, 11-, 33-, 66-, and 99-ng/ml) were added to NSC34 cells cultured in differentiation medium. As shown in the following Figure A, the effect of Pgk1 on the downregulation of p-Cofilin-S3 was dose-dependent (see new Figure 1—figure supplement 4). Moreover, we performed a time course experiment, in which NSC34 cells were treated with Pgk1 (33 ng/ml) for 0, 8, 16, 24 and 48 hrs. As shown in the following Figure B, the effect of Pgk1 on the downregulation of p-Cofilin-S3 was time-dependent from 8 through 24 hr (see new Figure 1—figure supplement 4). However, we noticed that p-Cofilin-S3 was unexpectedly increased if cells were treated with Pgk1 for 48 hr. The doses of Pgk1 we examined did not cause any negative effect on cell growth and survival. However, based on the above results and to ensure that we did not seriously affect cell physiology, we treated NSC34 cells with the minimal concentration (33-ng/ml of Pgk1 for 24 hr) throughout the entire study.

We added the above new data in the Results section as follows: “Next, to determine if Pgk1 alone is sufficient to induce NOM and neuronal differentiation, we switched to low trophic factor differentiation media (DM). […] Interestingly, the addition Pgk1 at that concentration not only rescued the number of neurite-bearing cells, but also enhanced neurite length of NSC34 cells cultured in Sol8-NogoA CM and DM (Figure 1D-E).”

Is the dose used physiologically relevant?

The Pgk1 dose (33 ng) used in this study was physiologically relevant and based on the concentration from what we learned in our preliminary investigation. Specifically, when we collected conditioned medium (CM) from culturing Sol8 cells transfected with Sol8-vector and Sol8-NogoA individually, we quantified Pgk1 contained in CM by the relative quantification method using commercially available standard Pgk1 protein. The result showed that the Pgk1 concentration was 31.80 ng/ml in Sol8-vector CM, while it was 14.97 ng/ml in Sol8-NogoA CM (see Author response image 1). Based on these data, we chose 33 ng/ml of Pgk1, which aligns with the result above for Sol8-vector CM, and decided to add it into DM for subsequent experiments in this study. Additionally, since the concentration of commercially available Pgk1 stock was 440 ng/ul, it was more convenient to make a 40X dilution to obtain final concentration of 11 ng/ul, followed by taking a volume of 3 ul to perform experiments. However, the actual effective concentration of Pgk1 in vivoneeds to be further analyzed.

Author response image 1
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3) Does the NogoA-Pgk1 secretion mechanism also function in primary muscle cells and motor neurons in culture? Is there evidence for NogoA over-expression inducing a reduction in Pgk1 expression in zebrafish and in mouse in vivo?

In response to your question, we performed a new experiment, in which we extracted blood samples from zebrafish and mouse and detected the expression level of Pgk1 in sera. Since it is very hard to extract blood from zebrafish and since its serum is very limited, we preferred to use WT and SOD1 G93A mice. The result demonstrated that the Pgk1 level in sera of SOD1 G93A mice at 90 days old was significantly lower than that found in the sera of WT mice at same age (see Figure 1—figure supplement 3). We also demonstrated that NogoA was overexpressed in the muscle of SOD1 G93A mice at 90 days old (see Figure 1—figure supplement 3A), which was consistent with the result reported by Bros-Facer et al. (2014). The above data deomonstrated in mouse in vivo system is strongly supportive for our finding that overexpression of NogoA in muscle cells results in a reduction in Pgk1 secretion.

As results shown above, we have demonstrated the NogoA-Pgk1 secretion mechanism functions in vivo, we, therefore, did not study further whether NogoA-Pgk1 secretion mechanism also function in primary muscle cells and motor neurons in culture.

We added the above results in the Results section and revised as follows: “We further employed 2D electrophoresis, followed by LC MS/MS, to analyze the total protein content in CM. […] Collectively, based on the above results provided in vitro and in vivo evidence, we chose Pgk1 for further study to confirm its potential role in NOM.”

4) At the end of the Results section, the authors mention anecdotally that there is a motor behavior phenotype and provide videos. However, there is no quantification and no description in the Materials and methods section to indicate any type of quantitative assessment of motor behavior. How many mice were used in these experiments?

We added more detail about motor behavior in the revised manuscript. In the Materials and methods section, we added the following text: “We also performed intramuscular injection of Pgk1 into the gastrocnemius muscle of the right hind leg of six 60-day-old (at P60) transgenic SOD1 G93A mice. […] The exercise capability value of each mouse was calculated from the increased fold(s) in the number of contractions between injected right leg versus untreated left leg. Data of each group were averaged from three mouse samples and presented as mean ± S.D.”

We also added some description in the Results section, as follows: “In the WT/PBS group, the muscle contraction of both hind legs was normal, exhibiting strong movement. […] Taken together, it is suggested that the Pgk1-injected right hind leg of SOD1 G93A mice could maintain some normal motor neurons to innervate muscle contraction.”

5) The authors conclude that Pgk1 functions independent of NgR. However, their only evidence for this is the lack of activation of downstream signaling cascades, and there are no positive controls included in these experiments. This is very indirect, and does not allow the authors to conclude Pgk1 function is independent of NgR.

In response to your question, we performed several new experiments to confirm whether the involvement of Pgk1 in pathway regulation was independent of NgR receptor activation by NogoA. Two study approaches were designed. First, we determined whether p-Cofilin expression was different between GST-Nogo66 addition and GST-Nogo66 plus Pgk1 addition to NSC34 cells cultured in differentiation medium (DM). Compared to the control group in which GST(100 ng/ml) was added in DM, NSC34 cells cultured in GST-Nogo66 (100 ng/ml) added to DM exhibited an increase of p-ROCK2 and p-EGFR expression and a decrease of p-Akt (please see Figure 5—figure supplement 3A), suggesting that the addition of GST-Nogo66 activates the NgR response pathway. Interestingly, the increased p-ROCK2 and p-EGFR and decreased p-Akt remained unchanged in NSC34 cells cultured in GST-Nogo66 plus Pgk1 addition (please see Figure 5—figure supplement 3A), suggesting that the addition Pgk1 did not affect the Nogo66/NgR pathway. However, compared to GST-Nogo66-treated cells, the GST-Nogo66 plus Pgk1-treated NSC34 cells exhibited reduced p-Cofilin expression through decreasing p-Limk-S323, but not through p-Limk-T508 (please see Figure 5—figure supplement 3B), suggesting that the reduced expression of p-Cofilin results from the presence of Pgk1 through some pathway other than Nogo66/NgR interaction.

Second, we determined whether p-Cofilin expression remained in a reduced state in NSC34 cells by the addition of Pgk1 into NSC34 cells cultured in DM when NgR receptor was blocked by NAP2 (NgR receptor antagonist peptide; 10 uM) (Sun et al., 2016). The addition of NAP2 in the culture caused a reduction of ROCK2 expression in NSC34 cells, a result consistent with that reported by Sun et al., 2016, indicating that NAP2 could block the NgR receptor (New Figure 5—figure supplement 3C). However, unlike increased ROCK2 in NSC34 cells cultured in NAP2 addition, ROCK2 expression remained unchanged in NSC34 cells cultured in NAP2 plus Pgk1 addition (New Figure 5—figure supplement 3D). In contrast, the expression of p-Pak1/p-P38/p-MK2/p-Limk1-S323/p-Cofiln axis was reduced (New Figure 5—figure supplement 3D).

Taken together, we suggest that the reduction of p-Cofilin mediated by ePgk1 does not occur through Nogo66/NgR interaction in neuronal cells since ePgk1 addition can still functionally reduce the p-Pak1/p-P38/p-MK2/p-Limk1-S323/ p-Cofiln axis in the absence of NgR receptor of neuronal cells.

Based on above new data, it should strengthen our conclusion that ePgk1 reduces the expression of p-Cpfilin in NSC34 cells to enhance neurite outgrowth of motor neurons is independent of Nogo66/NgR pathway. We added these new data in the Results section under the second paragraph of subtitle entitled The signaling pathway underlying the involvement of ePgk1-mediated reduction of p-Cofilin-S3 as follows:

“To confirm whether Pgk1 involved regulating pathway is independent of activation of NgR receptor by NogoA. […]And, the molecular pathway ePgk1 involved in NOM is independent of the Nogo66/NgR interaction.”

6) How NogoA reduces the secretion of Pgk1 from muscle cells and how extracellular Pgk1 inhibits the intracellular Rac1-PAK pathway is unclear and needs additional investigation and/or discussion.

In response to your question, we added some statements in the Discussion as follows: “To address the reduced secretion of Pgk1 from muscle cells by NogoA, as well as inhibition of the neuronal intracellular Rac1 pathway by ePgk1, we turn to the literature. […] The reduced ePgk1 may then cause a decrease in extracellular proteins, such as angiostatin, which, in turn, would increase the activity of Rac1, finally causing a physiological microenvironment for motor neurons that would not favor neurite outgrowth.”

Additional comments on the specific Figures:

Figure 1:

The authors need to confirm that there is no NOGO-A in the conditioned media

In response of this question, we confirmed the absence of NogoA in the condition medium cultured by Sol8-NogoA cells. We added these data in the Results section as follows: “Importantly, NogoA was not detected in the CM cultured by Sol8-NogoA cells after Dox induction for 48 hr (Figure 1—figure supplement 1C). This line of evidence suggested that the component(s) contained in CM from cultured Sol8-NogoA play(s) a role in NOM inhibition, but not through NogoA contained in medium”.

The specificity of the Pgk1 Ab used in Figure 1B needs to be confirmed

We have increased the amount of total proteins from 4 to 20 ug for Western blot analysis. Similar to the previous 4 ug data the result demonstrated that only one sharp positive band was exclusively shown on the gel when Pgk1 antibody (Abcam: ab38007) was used, suggesting that the Pgk1 antibody used in this study presents sufficiently high specificity to detect Pgk1. (Please see newly added Figure 1—figure supplement 2B shown).

Figure 2:

It is unclear how well the markers used (MAP2, ChAT, GAP43, SYN1) can actually reflect a differentiated status in NSC34 cells. These cells have very small processes, and therefore are unlikely to represent a truly differentiated state.

We followed a paper published in Neurochemistry International (2013, 62: 1029-38) by Maier et al., entitled “Differentiated NSC34 motoneuron-like cells as experimental model for cholinergic neurodegeneration.” The authors employed markers of MAP2, ChAT, GAP43, and SYN1 to define differentiation of NSC34 cells and stated that the increased expression of these proteins served as indicators of NSC34 differentiation. Although the expression of these proteins is unlikely to represent a truly differentiated state, as you pointed out, we used the intracellular markers MAP2, ChAT, and GAP43, as well as Syn1-labeling signal in the growth cones’ terminus, as a guidepost in determining whether NSC34 cells further develop toward differentiation and maturation or whether these cells maintain a proliferative state when cultured in Sol8-vector CM, Sol8-NogoA CM and Sol8-NogoA CM plus Pgk1.

In subsection “Supplementary addition of Pgk1 can enhance the maturation of NSC34 cells cultured in Sol8-NogoA CM”, the authors state "The number of neurons with Syn1 signal at the end of synapse could be restored to that cultured in Sol8-vector…". It is unlikely that these are synapses, they look more like growth cones and don't contact "postsynaptic" structures

Thank you for pointing out this mistake. We corrected it as follows: “….The number of neurons with Syn1 signal at the end of growth cones could be restored to that cultured in Sol8-vector…”

Figure 4:

In zebrafish experiments, the authors use CRISPR/Cas9 system to knockout Pgk1 and Pgam2 from muscle cells. There needs to be data validating that sgRNAs targeting Pgk1 and Pgam2 are effective in reducing Pgk1 and Pgam2 expression, respectively. Similarly, in the muscle-specific over expression paradigm, the authors need to show that Pgk1 expression is increased.

In response to your suggestion, we added Crispr/cas9-mediated inactivation and upregulation of Pgk1 in zebrafish muscle cells in the Results section, as follows: “We used muscle-specific α-actin promoter to drive overexpression of Cas9 in muscle cells in order to exclusively knock out the Pgk1 gene. […] As suggested by quantification of the axonal extension phenotype shown in Figure 4—figure supplement 2, the increase of Pgk1 in muscle cells enhances NOM.”

While the authors provide information on the penetrance of the phenotypes, they do not quantify the extent of axonal extension in any meaningful way.

Thank you for your suggestion. In the revised manuscript, we quantified the percentage of axonal extension phenotype, presented a new Figure 4—figure supplement 2, and added these data in the Results section as follows: “When these RFP-expressing cells were sorted and examined by Western blot analysis, the results demonstrated that Pgk1 was overexpressed (Figure 4—figure supplement 1). Meanwhile, motoneuron axons exhibited ectopic growth (Figure 4G). As suggested by quantification of the axonal extension phenotype shown in Figure 4—figure supplement 2, the increase of Pgk1 in muscle cells enhances NOM.”

Figure 5:

The authors conclude that the effect of Pgk1 is NgR-independent due to the lack of p-ROCK2-Y256, p-EGFR-Y1173 and p-Akt-S473 (Figure 5—figure supplement 1). However, there is no positive control in this experiment. The authors should show that they are able to detect changes in these pathways in response to NgR activation by NOGO-A.

In response your question, we designed a positive control, in which Nogo66 fused with GST (GST-Nogo66) was used as a positive control, to determine whether p-Cofilin expression was any different between GST-Nogo66 addition and GST-Nogo66 plus Pgk1 addition to NSC34 cells cultured in DM. The results were shown in new Figure 5 and Figure 5—figure supplement 3A-B.

Compared to the control group in which GST(100 ng/ml) was added to DM, NSC34 cells cultured in GST-Nogo66 (100 ng/ml) added to DM exhibited increased p-ROCK2-Y256 and p-EGFR-Y1173 expression and decreased p-Akt-S473 expression (please see figures related to #5 above, as well as new Figure 5—figure supplement 3A), suggesting that GST-Nogo66 addition activated the NgR response pathway (served as positive control). Interestingly, the increased p-ROCK2-Y256/p-EGFR-Y1173 and decreased p-Akt-S473 remained unchanged in NSC34 cells cultured in GST-Nogo66 with Pgk1 addition (see Figure 5—figure supplement 3A), suggesting that the addition of Pgk1 did not affect the Nogo66/NgR pathway. However, compared to GST-Nogo66-treated NSC34 cells, the GST-Nogo66 plus Pgk1-treated NSC34 cells exhibited a reduction of p-Cofilin-S3 expression through a decrease in p-Limk-S323, but not through p-Limk-T508 (see figures shown for #5 and new Figure 5—figure supplement 3B), suggesting that the reduced expression of p-Cofilin results from the presence of Pgk1 through a pathway other than Nogo66/NgR interaction. Based on this line of evidence, we conclude that the effect of ePgk1 on the reduction of p-Cofilin in neuronal cells is NgR-independent.

Figure 6:

Because the authors did not provide information on the dose-dependency and timecourse of Pgk1 in their in vitro experiments, it is unclear why they chose the specific dose and timing regimens for their in vivo experiments.

In response to your question, we performed a new experiment in which different doses of Pgk1 (0-, 11-, 33-, 66-, and 99-ng/ml) were added to NSC34 cells cultured in differentiation medium. As shown in the following Figure A, the effect of Pgk1 on the downregulation of p-Cofilin-S3 was dose-dependent (see new Figure 1—figure supplement 4). Moreover, we performed a time course experiment, in which NSC34 cells were treated with Pgk1 (33 ng/ml) for 0, 8, 16, 24 and 48 hrs. As shown in the following Figure B, the effect of Pgk1 on the downregulation of p-Cofilin-S3 was time-dependent from 8 through 24 hr (see new Figure 1—figure supplement 4). However, we noticed that p-Cofilin-S3 was unexpectedly increased if cells were treated with Pgk1 for 48 hr. The doses of Pgk1 we examined did not cause any negative effect on cell growth and survival. However, based on the above results and to ensure that we did not seriously affect cell physiology, we treated NSC34 cells with the minimal concentration (33-ng/ml of Pgk1 for 24 hr) throughout the entire study.

We added the above new data in the Results section as follows: “Next, to determine if Pgk1 alone is sufficient to induce NOM and neuronal differentiation, we switched to low trophic factor differentiation media (DM). […] Interestingly, the addition Pgk1 at that concentration not only rescued the number of neurite-bearing cells, but also enhanced neurite length of NSC34 cells cultured in Sol8-NogoA CM and DM (Figure 1D-E).”

Fish were injected with 2ug Pgk1, then placed in Dox+ water for 1 week. How long is the injected Pgk1 biologically active?

In response to your question, we performed a new experiment, in which we injected 2 ug of GFP (control group) or Pgk1 (experimental group) into transgenic zebrafish line Tg(TetON-Rtn4al), followed by immersing zebrafish in water containing Dox, to induce overexpression of Rtn4al/NogoA in muscle. In the control group, as immersion time increased from one, two and three weeks, the degree of NMJ denervation also increased. However, compared with the GFP-injected control group, delay in NMJ denervation was still observed in the Pgk1-injected group at two weeks after injection. Nevertheless, no significant difference was observed in the extent of NMJ denervation between control and experimental groups at three weeks after injection. This line of evidence suggested that adult zebrafish muscle injected Pgk1 (2 ug) was able to retain its biological activity for two weeks post-injection. We added this information in the Results and attached a new figure in the revised version.

We added these data in the Results section as follows: “While we observed denervation at NMJ after Rtn4al/NogoA induction in adult zebrafish (Figure 6—figure supplement 1), as evidenced by reduced colocalization of presynaptic and postsynaptic markers, this defect saw much improvement by addition of Pgk1, but not GFP control, through intramuscular injection (Figure 6A-G), suggesting ePgk1 supports NMJ integrity. Although adult zebrafish muscle injected with Pgk1 did exhibit delayed NMJ denervation, it could still retain its normal biological activity for two weeks after a single shot (Figure 6—figure supplement 2).”

There is a discrepancy between what is in the Results section and the Materials and methods section with respect to the experiments done in mice in vivo. The authors indicate that mice were injected with Pkg1 once every 15 days between P60 and P120. However, in the Materials and methods section (546-548) they indicate tissue sections were taken at P75. Is the data presented in Figure 6 H-N from P75 or P120 mice?

We apologize for this. The mice shown in Figures 6 H-N were injected with Pgk1 on the right hind leg at P60, followed by performing frozen section and immunofluorescence staining to examine the innervated NMJ at P75. This was what we described in the Materials and methods section (546-548). While mice shown in the video were injected with Pgk1 on the right hind leg at P60, another Pgk1 injection was continuously administered every 15 days up to P120. When they were at P130, we recorded their movement by video (Figure 6—video 1-6) and evaluated their muscle contraction capability (Figure 6—figure supplement 5).

To make this description clear, we added more detail in the Materials and methods section as follows: “We also performed intramuscular injection of Pgk1 into the gastrocnemius muscle of the right hind leg of six 60-day-old (at P60) transgenic SOD1 G93A mice. […] Data of each group were averaged from three mouse samples and presented as mean ± S.D. “

We also added some description in the Results section, as follows: “In the WT/PBS group, the muscle contraction of both hind legs was normal, exhibiting strong movement. […] Taken together, it is suggested that the Pgk1-injected right hind leg of SOD1 G93A mice could maintain some normal motor neurons to innervate muscle contraction.”

The authors should provide images and other metrics (neurite outgrowth, expression of motor neuron specific genes) from their iPS-SOD G85R motor neuron cultures so the degree of differentiation these iPS cells can be assessed.

In response to your suggestion, we provided more detailed information and added new images in new Figure 6—figure supplement 3. As shown in this figure, after 14 days of motor neuron induction, the iPSC-SOD1G85R cells expressed the pluripotency-specific markers Oct4, Nanog and SSEA4 and then began to differentiate motor neurons. After 15 days of motor neuron differentiation, more than 90% of induced cells expressed the neural stem cell-specific markers sox1 and N-cadherin, as well as the motor neuron precursor-specific markers Oligo2 and Islet1. After 27 days of motor neuron differentiation, more than 90% of induced cells expressed motor neuron-specific protein HB9 and nerve fiber protein neurofilament. Therefore, our results demonstrated the highly efficient differentiation of motor neurons from iPSCs harboring a G256C point mutation (G85R on peptide sequence) on human SOD1 gene in an ALS patient.

- Figure 6—figure supplement 2 panel B: Why is there no change in the levels of p-Cofilin in response to Pgk1 in the control cells? In Figure 1A, this same treatment causes a ~3-fold decrease p-Cofilin levels.

We apologize for this mistake. We repeated this experiment and replaced this figure by a new one. (see new Figure 6—figure supplement 4B)

Reviewer #3:

[…] This study revealed a new indirect mechanism how NogoA in the muscle affected neurite growth of motoneurons. The finding is interesting and helpful for understanding the probable mechanisms in steering axon growth. However, there are some issues needed to be addressed.

1) Nogo-A is a transmembrane protein, it could exert its effects by direct interacting with its receptors on the neurons, or it can work indirectly through affecting other molecule expression or secretion in the muscle. The authors used CM from NogoA-overexpressing cells to treat neurons, which supports the indirect effect. Given NogoA is a transmembrane protein, it is not likely present in the CM. Without the co-culture experiment of muscle cells and motor neurons, it's inappropriate to exclude the possibility that NogoA in the muscle could also affects neurite growth through a direct ligand/receptor interaction. Therefore, the conclusion "In this study, we revealed that overexpression of NogoA in muscle inhibits NOM through decreasing Pgk1 secretion (ePgk1), but without inducing the canonical Nogo66/NgR pathway."(subsection “Intramuscular injection of Pgk1 is able to rescue NMJ denervation caused by Rtn4al-overexpression transgenic zebrafish and SOD1 G93A transgenic mice”) is not accurate.

We believe our conclusion is accurate. In this study, NSC34 cells were treated with CM from cultured NogoA-overexpressing muscle cells, but no NSC34 cells were directly cultured with NogoA-overexpressing muscle cells. Furthermore, we could not detect NogoA protein in CM (see Figure 1—figure supplement 1C). Thus, it is reasonable to rule out the possibility that NogoA on the muscle cell membrane interacts directly with NgR receptor on the neuronal cells through ligand/receptor interaction to enhance neurite growth, as you speculated.

Moreover, we designed two new study strategies to support our hypothesis. First, we determined whether p-Cofilin expression was different between GST-Nogo66 addition and GST-Nogo66 plus Pgk1 addition to NSC34 cells cultured in differentiation medium (DM). Compared to the control group in which GST (100 ng/ml) was added in DM, NSC34 cells cultured in GST-Nogo66 (100 ng/ml) added to DM exhibited an increase of p-ROCK2 and p-EGFR expression and a decrease of p-Akt (please see Figure 5—figure supplement 3A), suggesting that the addition of GST-Nogo66 activates the NgR response pathway (served as positive control). Interestingly, the increased p-ROCK2 and p-EGFR and decreased p-Akt remained unchanged in NSC34 cells cultured in GST-Nogo66 plus Pgk1 addition (please see Figure 5—figure supplement 3A), suggesting that the addition of Pgk1 did not affect the Nogo66/NgR pathway. However, compared to GST-Nogo66-treated cells, the GST-Nogo66 plus Pgk1-treated NSC34 cells exhibited reduced p-Cofilin expression through decreasing p-Limk-S323, but not through p-Limk-T508 (please see Figure 5—figure supplemental 3B), suggesting that the reduced expression of p-Cofilin results from the presence of Pgk1 through some pathway other than Nogo66/NgR interaction.

Second, we determined whether p-Cofilin expression remained in a reduced state in NSC34 cells by the addition of Pgk1 to NSC34 cells cultured in DM when NgR receptor was blocked by NAP2 (NgR receptor antagonist peptide; 10 uM) (Sun et al., 2016). The addition of NAP2 in the culture caused the reduction of ROCK2 expression in NSC34 cells, a result consistent with that reported by Sun et al., 2016, indicating that NAP2 could block the NgR receptor. However, unlike increased ROCK2 in NSC34 cells cultured in NAP2 addition, ROCK2 expression remained unchanged in NSC34 cells cultured in NAP2 plus Pgk1 addition (New Figure 5—figure supplement 3C). In contrast, the expression of p-Pak1/p-P38/p-MK2/p-Limk1-S323/p-Cofilin axis was reduced (New Figure 5—figure supplement 3D).

Taken together, we suggest that the reduction of p-Cofilin mediated by ePgk1 does not occur through Nogo66/NgR interaction in neuronal cells since ePgk1 addition can still functionally reduce the p-Pak1/p-P38/p-MK2/p-Limk1-S323/p-Cofilin axis in the absence of NgR receptor of neuronal cells. In sum, ePgk1 triggers a reduction in p-Cofilin-S3, in turn promoting NOM through decreasing a novel p-Pak1-T423/p-P38-T180/p-MK2-T334/p-Limk1-S323 axis via reducing Rac1-GTP activity in neuronal cells. Therefore, we conclude that 1) a cross-tissue mediator, i.e., Pgk1 secreted from muscle cells, was found; (2) this extracellular Pgk1 enhances neurite growth of motor neurons through a novel signal pathway which is distinct from the well-known NogoA(Nogo66)/NgR pathway between muscle and neuronal cells or among neuronal cells; (3) overexpression of NogoA in muscle cells causes the decrease of secreted Pgk1, resulting in the increase of p-Cofilin in neuronal cells, which, in turn, inhibits neurite outgrowth of motor neurons, suggesting that neurite outgrowth of motor neurons can also be negatively regulated by reducing the amount of secreted Pgk1 from NogoA-overexpressing muscle cells; and, finally, (4) the effect of ePgk1 on neuronal cells is NgR-independent.

2) Is there any evidence in vivo that NogoA upregulation in the muscle affected its Pgk1 production, i.e. the NogoA and Pgk1 levels in the SOD1 mutant mice or Rtn4al-overexpressing transgenic zebrafish? Or at least confirm whether overexpressing NogoA in the muscle of normal adult mice using viral vector will also affect Pgk1 secretion.

In response to your question, we performed a new experiment, in which blood samples from 90-day-old WT and SOD1 G93A mice were extracted to detect the expression level of Pgk1 in sera. The Pgk1 level in SOD1 G93A mice sera was significantly lower than that in the sera of WT mice (see Figure 1—figure supplement 3). In addition, NogoA was overexpressed in the muscle of SOD1 G93A mice, which was consistent with the result reported by Bros-Facer et al., 2014. These results provide in vivo evidence that NogoA upregulation in muscle cells negatively affects the secretion of Pgk1.

We added the above results in the Results section and revised as follows: “We further employed 2D electrophoresis, followed by LC MS/MS, to analyze the total protein content in CM. Among these examined proteins, we found that the level of Pgk1 protein contained in Sol8-NogoA CM was significantly reduced compared to that in Sol8-vector CM (Figure 1—figure supplement 2). […] Collectively, the above results provided both in vitro and in vivo evidence of the reduction of Pgk1 secretion in NogoA-overexpressed muscle; therefore, we chose Pgk1 for further study to confirm its potential role in NOM.”

3) The authors seemed to confuse with neurite and synapse: subsection “in vitro platform of neurite outgrowth of motor neurons derived from NSC34 cells”; "Synapses extending from each cell were captured by microscope.…"; "calculate the length of synapses…"

Thank you for pointing out this mistake. We made the following change: “Neurites extending from each cell were captured by microscopy (Leica) under 200 times magnification and labeled by Neurolucida Neuron Tracing Software 9.0 (Neurolucida 9.0). […] To count the number of neurite-bearing cells, we calculated the number of cells with neurites at least 50 μm in length in each range.”

4) In Figure 1—figure supplement 2, the authors need quantify the Pgk1 amount in the CM. In addition, there is no information about the source and the concentration of ePgk1 added into the culture. Whether is it comparable to the amount in the CM?

In response to your question we quantified the amount of Pgk1 in CM. When we collected conditioned medium (CM) from culturing Sol8 cells transfected with Sol8-vector and Sol8-NogoA individually, we quantified Pgk1 contained in CM by the relative quantification method using commercially available standard Pgk1 protein. Results showed that the Pgk1 concentration was 31.80 ng/ml in Sol8-vector CM, while it was 14.97 ng/ml in Sol8-NogoA CM. Based on these data, we chose 33 ng/ml of Pgk1, a choice which aligns well with the result above for Sol8-vector CM, and decided to add it into DM for subsequent experiments in this study. Additionally, since the concentration of commercially available Pgk1 stock was 440 ng/ul, it was more convenient to make a 40X dilution to obtain a final concentration of 11 ng/ul, followed by taking a volume of 3 ul to perform experiments.

5) In the method of neurite outgrowth (subsection “in vitro platform of neurite outgrowth of motor neurons derived from NSC34 cells”), it says: "In each image, 40~50 400 cells were randomly chosen to calculate the length of synapses and the number of neurite-bearing cells, the synapses of which were at least 50 μm in length.". If only cells with neurite length longer than 50 μm were chosen, why the average neurite lengths in some groups of Figure 1E are shorter than 50 μm?

We apologize for the confusion. To make this description clear, we profiled the neurite length distribution of each group. Specifically, after we measured the length of neurites of all 120 examined cells in each group, we grouped their size into several groups, such as every 25 intervals under 50 um, every 50 intervals under 200 um, 201-300 and 301-600 um. To know the number of neurite-bearing cells, we marked a line on the figures to indicate the neurite length which was more than 50 μm (see Figures 1D-E).

In the revised manuscript, we added more detailed information to the Materials and methods section, as follows: “Neurites extending from each cell were captured by microscopy (Leica) under 200 times magnification and labeled by Neurolucida Neuron Tracing Software 9.0 (Neurolucida 9.0). For each image, 40 cells cultured in either CM or DM were randomly chosen to measure the length of neurites. Data were obtained from three independent trials (120 cells in total). To profile neurite length distribution, we grouped neurite length into several groups, namely every 25 intervals under 50 um, every 50 intervals under 200 um, and 201-300 and 301-600 um, and calculated the number of cells in each range. To count the number of neurite-bearing cells, we calculated the number of cells with neurites at least 50 μm in length in each range.”

In the Results section, we added the following: “Interestingly, the addition Pgk1 at that concentration not only rescued the number of neurite-bearing cells, but also enhanced neurite length of NSC34 cells cultured in Sol8-NogoA CM and DM (Figure 1D-E).”

https://doi.org/10.7554/eLife.49175.031

Article and author information

Author details

  1. Cheng Yung Lin

    Institute of Biomedical Sciences, Mackay Medical College, New Taipei City, Taiwan
    Contribution
    Data curation, Formal analysis, Investigation, Writing—original draft
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9570-0218

  2. Chia Lun Wu

    Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan
    Contribution
    Data curation, Formal analysis, Investigation, Methodology, Writing—original draft
    Competing interests
    No competing interests declared

  3. Kok Zhi Lee

    Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan
    Contribution
    Data curation, Investigation, Methodology, Writing—original draft
    Competing interests
    No competing interests declared

  4. You Jei Chen

    Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan
    Contribution
    Data curation, Investigation, Methodology
    Competing interests
    No competing interests declared

  5. Po Hsiang Zhang

    Institute of Biomedical Sciences, Mackay Medical College, New Taipei City, Taiwan
    Contribution
    Data curation, Investigation, Methodology
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9745-0213

  6. Chia Yu Chang

    1. Bioinnovation Center, Buddhist Tzu Chi Medical Foundation, Hualien City, Taiwan
    2. Department of Medical Research and Neuroscience Center, Buddhist Tzu Chi General Hospital, Hualien City, Taiwan
    Contribution
    Data curation, Methodology
    Competing interests
    No competing interests declared

  7. Horng Jyh Harn

    1. Bioinnovation Center, Buddhist Tzu Chi Medical Foundation, Hualien City, Taiwan
    2. Department of Pathology, Buddhist Tzu Chi General Hospital and Tzu Chi University, Hualien City, Taiwan
    Contribution
    Resources, Methodology
    Competing interests
    No competing interests declared

  8. Shinn Zong Lin

    1. Bioinnovation Center, Buddhist Tzu Chi Medical Foundation, Hualien City, Taiwan
    2. Department of Neurosurgery, Buddhist Tzu Chi General Hospital, Hualien City, Taiwan
    Contribution
    Resources, Funding acquisition
    For correspondence
    shinnzong@yahoo.com.twn
    Competing interests
    No competing interests declared

  9. Huai Jen Tsai

    Institute of Biomedical Sciences, Mackay Medical College, New Taipei City, Taiwan
    Contribution
    Formal analysis, Investigation, Resources, Supervision, Funding acquisition, Writing—review and editing
    For correspondence
    1. hjtsai@ntu.edu.tw
    2. hjtsai@mmc.edu.tw
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8242-4939

Funding

Ministry of Science and Technology, Taiwan (107-2311-B-715-001)

  • Huai Jen Tsai

Ministry of Science and Technology, Taiwan (107--2314-B-303-003)

  • Shinn Zong Lin

Liver Disease Prevention and Treatment Research Foundation, Taiwan

  • Cheng Yung Lin
  • Huai Jen Tsai

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was supported by Prof. Jin-Chuan Sheu, Dr. Hsiao-Ching Nien and Mr. Spencer Lee, the Liver Disease Prevention and Treatment Research Foundation, Taiwan. We thank TC3 and TC5 Technology Commons, College of Life Science, NTU, for providing the FACS, Microscope and 2D PAGE equipments. We also thank Biomedical Instrumental Center, Mackay Medical College, for providing the SeaHorse XF 24 for Glycolysis Stress Test Assay. We also thank Prof. Hsinyu Lee, College of Life Science, NTU, for providing plasmids pCMVΔR8.91 and pMD.G. We also thank Dr. Neil Cashman, University of Toronto, for providing NSC34 cell line.

Ethics

Animal experimentation: The Mackay Medical College Institutional Animal Care and Use Committee (IACUC) reviewed and approved the protocol described below (MMC-A1060009).

Senior Editor

  1. Didier Y Stainier, Max Planck Institute for Heart and Lung Research, Germany

Reviewing Editor

  1. Fadel Tissir, Université Catholique de Louvain, Belgium

Reviewers

  1. Fadel Tissir, Université Catholique de Louvain, Belgium
  2. Libing Zhou, Guangdong-Hongkong-Macau Institute of CNS Regeneration, China

Version history

  1. Received: June 8, 2019
  2. Accepted: July 5, 2019
  3. Version of Record published: July 30, 2019 (version 1)
  4. Version of Record updated: August 7, 2019 (version 2)
  5. Version of Record updated: September 11, 2019 (version 3)

Copyright

© 2019, Lin et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Cheng Yung Lin
  2. Chia Lun Wu
  3. Kok Zhi Lee
  4. You Jei Chen
  5. Po Hsiang Zhang
  6. Chia Yu Chang
  7. Horng Jyh Harn
  8. Shinn Zong Lin
  9. Huai Jen Tsai
(2019)
Extracellular Pgk1 enhances neurite outgrowth of motoneurons through Nogo66/NgR-independent targeting of NogoA
eLife 8:e49175.
https://doi.org/10.7554/eLife.49175

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