Downregulation MIWI-piRNA regulates the migration of Schwann cells in peripheral nerve injury

Downregulation MIWI-piRNA regulates the migration of Schwann cells in peripheral nerve injury

Biochemical and Biophysical Research Communications 519 (2019) 605e612 Contents lists available at ScienceDirect Biochemical and Biophysical Researc...

3MB Sizes 0 Downloads 55 Views

Biochemical and Biophysical Research Communications 519 (2019) 605e612

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc

Downregulation MIWI-piRNA regulates the migration of Schwann cells in peripheral nerve injury Eun Jung Sohn*, Young Rae Jo, Hwan Tae Park Peripheral Neuropathy Research Center, Department of Molecular Neuroscience, College of Medicine, Dong-A University, Dongdaesin-Dong, Seo-Gu, Busan, 602-714, South Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 September 2019 Accepted 4 September 2019 Available online 17 September 2019

Although MIWI (PIWI in humans) regulates spermatogenesis and translation machinery, its role in peripheral nerve injury is poorly understood. In this study, we characterized the expression profiles of MIWI after sciatic nerve injury. The results revealed that MIWI was downregulated after sciatic nerve injury. MIWI was colocalized with S100 (a Schwan cell marker), and TOM20 (a mitochondrial marker) on uncut nerves, while some MIWI was also colocalized with myelin protein zero (a myelin marker) on injured nerves. Immunofluorescence revealed that some MIWI was colocalized with SOX10 in the nuclear compartment following nerve injury. MIWI depletion by MIWI siRNA resulted in the reduction of EGR2. To characterize the expression of PIWI interacting RNA (piRNA) during sciatic nerve injury, microarray-based piRNA was conducted. The results revealed that 3447 piRNAs were upregulated, while 4117 piRNAs were downregulated after nerve transection. Interestingly, piR 009614 downregulated the mRNA level of MBP and enhanced the migration of RT-4 Schwann cells. Together, our results suggest that the MIWI-piRNA complex may play a role in Schwann cell responses to nerve injury. © 2019 Elsevier Inc. All rights reserved.

Keywords: MIWI PiRNA Wallerian degeneration

1. Introduction After peripheral nerve injury, distal myelin and axons undergo Wallerian degeneration. During this degeneration, there is enhanced proliferation of Schwann cells, which move to the injury site, surround axons, and contribute to extensive morphological and biochemical changes within the distal stumps of injured nerves for the purpose of nerve regeneration [1]. During differentiation, immature Schwann cells express myelin-related proteins such as myelin protein zero (MPZ), myelin-associated glycoprotein (MAG), and transcription factors such as early growth response 2 (EGR2 or Krox20) to differentiate or induce myelination [2]; while following nerve injury, myelin genes and EGR2 are reduced [2]. PIWI (p-element induced wimpy testis) as an argonaute protein was first identified for its destructive effect on the testis [3,4]. There are four PIWI proteins in humans: PIWIL1, PIWIL2, PIWIL3, and PIWIL4. The Piwi family is associated with PIWI-interacting RNAs

* Corresponding author. Peripheral Neuropathy Research Center, Department of Molecular Neuroscience, College of Medicine, Dong-A University, DongdaesinDong, Seo-Gu, Busan, 602-714, South Korea. Tel.: þ82 051 240 2723; fax: þ82 051 247 3318. E-mail address: [email protected] (E.J. Sohn). https://doi.org/10.1016/j.bbrc.2019.09.008 0006-291X/© 2019 Elsevier Inc. All rights reserved.

(piRNAs), which are 24e32 nucleotides long and are members of a class of small RNAs. PIWI functions in germline maintenance and transposon repression [5], proliferation and differentiation of germline stem cells [6]. In mice, there are three PIWI-group homologs: MIWI (PIWIL1), MILI (PIWIL2), and MIWI2 (PIWIL4). MIWI null mice have early spermatogenic defects [7] and regulated translation machinery [8], while MILI and MIWI2 regulate transposon silencing in fetal gonocytes [7,8]. Accumulating evidence suggests that the PIWI-piRNA complex plays a role in various biological functions in somatic development [9], pancreatic beta cell memory [10], and cancer development [11]. It was recently reported that the MIWI-piRNA pathway involves axons of adult sensory neurons [12]. Nevertheless, the underlying mechanism of MIWI in sciatic nerve injury has not been clearly elucidated. In the present study, for the first time, we characterized the expression of MIWI in Schwann cells after sciatic nerve injury, and provided evidence that 7564 piRNAs were differentially expressed after sciatic nerve injury. Overall, our results suggest that the MIWI-PiRNA plays an important role in the Schwann cell responses implicated in demyelination and nerve regeneration after sciatic nerve injury.

606

E.J. Sohn et al. / Biochemical and Biophysical Research Communications 519 (2019) 605e612

RT-4 Schwann cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Waltham, MA, USA) containing 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA), 100 IU/ml penicillin, and 100 mg/ml streptomycin (Sigma, St. Louis, MO, USA) at 37  C in a 5% CO2 humidified atmosphere.

buffered saline, pH 7), the blots were incubated with primary antibodies against MIWI (1:500; Abclonal, MA, USA), myelin protein zero (MPZ) (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), EGR2 (1:1000; Abcam, Cambride, UK) and actin (1:1000; Sigma-Aldrich, St. Louis, MO, USA). After washing three times, the blot was incubated with secondary antibodies including horseradish peroxidase conjugated anti-mouse or anti-rabbit (GE Healthcare). The signals were visualized using the enhanced chemiluminescence system (ECL Advance kit; GE Healthcare) and analyzed using a LAS image analysis system (Fujifilm, Tokyo, Japan).

2.2. Animals

2.6. Double immunofluorescent staining

Surgical procedures involving C57BL/6 mice were approved by the Dong-A University Committee on Animal Research and conducted accordingly. Briefly, 8-week-old female C57BL/6 mice were anesthetized and injured by cutting using fine iris scissors (Fine Science Tools, Foster City, CA, USA). The injured nerve sections of distal segments were collected following nerve injury.

The sections were blocked with blocking buffer [2% bovine serum albumin (BSA) and 0.2% Triton-X100 in phosphate-buffered saline (PBS)] for 1 h at room temperature. The slides were incubated with primary antibodies against MIWI (1:500; Abclonal), S100 (a Swann cell marker, 1:100; Sigma-Aldrich), TOM20 (a mitochondria marker, 1:500; Santa Cruz Biotechnology), and MPZ (a myelin protein maker, 1:500; Santa Cruz Biotechnology) overnight at 4  C. After washing, a mixture of 488- and cy3-conjugated secondary antibodies (Abcam, Cambridge, MA, USA) was added and incubated for 1 h at room temperature. Hoechst stain (Thermo Fisher Scientific) was used to visualize the nuclei. Images of stained sections were examined using ZEISS ApopTome 2 fluorescence microscopes (Zeiss, Jena, Germany).

2. Material and methods 2.1. Cell culture

2.3. The piRNA profiling Total RNA from nerve transections (days 0 and 7) was quantitated using the NanoDrop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA). The Arraystar HG19 piRNA array (Arraystar, Rockville, MD, USA) was also used in this study [13]. 2.4. Reverse-time quantitative polymerase chain reaction (RTqPCR) Total RNA from the sciatic nerves after sciatic nerve injury was extracted using TRIzol reagent (Life Technologies, Carlsbad, CA, USA). To synthesize cDNA, a cDNA transcription kit (Promega, Madison, WI, USA) was used according to the manufacturer's instructions. Quantitative reverse transcription PCR (RT-qPCR) was conducted using the SYBR Green Premixure (Applied Biosystems, Foster City, CA, USA) with an Applied Biosystems real-time PCR system. The following primers were used to determine the expression of MIWI: mouse MIWI forward primer-CTCAAGTCAGT CGGGAGAGGTTAC and reserve primer-TGTCCCTGGAAGAGGGTTC TGAAG, mouse Glyceraldehye 3-phosphate dehydrogenase (Gapdh) forward primer -CAGGTTGTCTCCTGCGACTT and reverse primer-CCCTGTTGCTGTAGCCGTAT. Mouse Myelin basic protein (MBP) forward primer-TACCCTGGCTAAAGCAGAGC and reverse primer-GAGGTGGTGTTCGAGGTGTCAA. To determine the expression of piRNA, cDNA was synthesized using a TaqMan miRNA Reverse Transcription kit (Thermo Fisher Scientific). RT-qPCR was conducted using the SYBR Green Premixure (Applied Biosystems) with primers of piRNAs and U6 (Applied Biosystems) using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems). U6 was used as internal control. The following primer was used for piRNAs: piR-14384; GCAATTGAGGGCTGACCAC, piR-69959;GACACTTCGGTCTGGCCTGC, piR52954; CAGACTTTATCTGGAATCTG, piR-52274; GCAGGAATATGTC GAGGACTG.

2.7. Transfection For siRNA transfection, RT-4 Schwann cells were transfected with control or MIWI short interfering RNA (siRNA; Bioneer, Korea) using Inteferin transfection reagent (Polyplus-transfection Inc., New York, NY, USA). For piRNA transfection, control and piR 009614 (DQ685207) single stranded RNAs were synthesized by Bioneer (Korea). The sequence of the piR 009614 (DQ685207) was 50 - UGCAAUUGAGGGCUGACCACAGGUAG -30 . The sequence of control was 50 GUGUAACACGUCUAUACGCCCA- 30 . Control or piR 009614 (DQ685207) was transfected into RT-4 cells using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. 2.8. Wound healing assay 100 nM control or piR 009614 was transfected into RT-4 cells. After 24 h transfection, a sterile 200 mL tip was used to generate a linear wound and the cells were incubated for 16 h. To visualize the wound area, the cells were fixed with 4% of paraformaldehyde and stained with 1% crystal violet. 2.9. Statistical analysis The data were presented as the means ± the standard error of the mean (SEM). Student's t-test in GraphPad Prism software (GraphPad Software Inc. USA) was used for statistical analyses.

2.5. Western blot analysis

3. Results

Sciatic nerves after sciatic nerve injury were harvested and the lysates were isolated by homogenization with a Tissuelyser LT (Qiagen, San Diego, CA, USA). After centrifugation, the supernatant was collected. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate proteins, which were transferred to a nitrocellulose membrane (GE Healthcare, Chicago, IL, USA). After blocking with 5% nonfat dry milk in TBST (Tris-

3.1. Expression of MIWI after sciatic nerve injury To investigate whether the expression of MIWI changes after sciatic nerve injury, RT-qPCR analysis was performed after axonal injury. Fig. 1A shows that the mRNA level of MIWI was reduced from day 1 after axotomy, peaked on days 4e7, and then gradually increased until day 14. Myelin basic protein (MBP) mRNA was

E.J. Sohn et al. / Biochemical and Biophysical Research Communications 519 (2019) 605e612

607

Fig. 1. Expression of MIWI after sciatic nerve injury. (A) A real-time quantitative polymerase chain reaction (RT-qPCR) analysis of MIWI expression in nerve transections (days 0, 1, 4, 7, and 14 post-injury). Gapdh was used to normalize the data. * p < 0.05, *** p < 0.001 compared to control. (B) Western blot analysis of MIWI expression in nerve transections (days 0, 1, 4, 7, and 14 post-injury). (C) Immunofluorescence staining of myelin protein zero (MPZ) and MIWI in nerve stumps after nerve injury (days 0, 4, and 7 post-injury). Scale bar: 20 mm. (D) Effect of MIWI on EGR2 expression in MIWI depleted RT-4 cells by western blotting assay.

measured as a positive control (Fig. 1A). In a similar manner, western blotting revealed that MIWI and EGR2 were reduced at day 4 after nerve injury (Fig. 1B). Previously, it was reported that EGR2 was reduced after nerve injury [14]. To further identify the changes

in MIWI immunoreactivity after nerve injury, we performed immunohistochemistry with MIWI and MPZ which is one of myelin related proteins. MIWI staining decreased noticeably 7 days after axotomy, which paralleled the results of RT-qPCR and western

608

E.J. Sohn et al. / Biochemical and Biophysical Research Communications 519 (2019) 605e612

Fig. 2. Localization of MIWI after sciatic nerve injury. (A, B) Immunofluorescence analysis of MIWI (red) with S100 (green, A), TOM 20 (green, B) in normal control (upper panel) or sciatic nerve injury (lower panel). (C) Immunofluorescence analysis of MIWI (green) with MPZ (red) after sciatic nerve injury. Cell nuclei were stained with Hochest dye (blue). Scale bar: 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

E.J. Sohn et al. / Biochemical and Biophysical Research Communications 519 (2019) 605e612

blotting (Fig. 1C). Western blotting assay showed that silencing of MIWI by siRNA in RT-4 Schwann cells reduced the expression of EGR2 (Fig. 1D). 3.2. Localization of MIWI in Schwann cells after nerve injury To confirm that MIWI was localized in Schwann cells after nerve injury, we conducted a double-labeling immunofluorescence assay. The results revealed that MIWI immunoreactivity was colocalized with S100 (a Schwann cell marker) on uninjured nerves, but MIWI did not colocalize with S100 on injured nerves (Fig. 2A). Previous study has reported that PIWI is localized in mitochondria [15]. To determine whether MIWI was localized in mitochondria,

609

immunofluorescence localization was conducted using antibodies to MIWI and TOM20 (a mitochondrial marker) in both control (uncut) and injured nerves on day 4 after transection. The results revealed that MIWI was colocalized with TOM20 in the control (uncut) but was not colocalized with TOM20 after injury (Fig. 2B). Notably, the immunofluorescence assay revealed that some MIWI was colocalized with MBP after sciatic nerve injury (Fig. 2C). 3.3. Localization of MIWI in in the cytoplasmic compartment as well as in the nuclear compartment MIWI reportedly exists in the cytoplasmic compartment as well as in the nuclear compartment [16]. To determine the location of

Fig. 3. Nuclear localization of MIWI following sciatic nerve injury. (A, B) Immunofluorescence assay of MIWI (red) with SOX 10 (green, A), or ED-1 (green, macrophage marker, B) after sciatic nerve injury. Cell nuclei were stained with Hochest dye (blue). Scale bar: 20 mm(A), 50 mm(B). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

610

E.J. Sohn et al. / Biochemical and Biophysical Research Communications 519 (2019) 605e612

MIWI after sciatic nerve injury, we conducted immunofluorescence localization with MIWI and SOX10, which is a nuclear protein of Schwann cells. Fig. 3A shows that some MIWI was colocalized with SOX10 in the nucleus of control nerves (Fig. 3A, upper panel), and following nerve injury, some MIWI was also colocalized with SOX10 in the nucleus (Fig. 3A, lower panel, arrow). Overall, our results

indicate that some MIWI is localized in the nucleus following nerve injury. Macrophages have reportedly infiltrated the nerve injury site during Wallerian degeneration [17]. To determine whether MIWI localized with macrophages following injury, an immunofluorescence assay was conducted with ED1, a macrophage marker. As

Fig. 4. Differentially expressed piRNAs after sciatic nerve injury. (A) Hierarchical clustering analysis based on the expression levels of piRNAs on day 7 after sciatic nerve injury. (B) Scatter plot of piRNA expression. (C) Validation of piRNAs by RT-qPCR. * p < 0.05, ** p < 0.01,compared to control. (D) Effect of piR 009614 on MBP mRNA in piR 009614 transfected RT-4 cells. Data represent means ± SEM. ***p < 0.001 vs untreated control. (E) Wound healing assay was carried out to see migratory ability of control or piR 009614 transfected RT-4 cells. After 24 h transfection with control or piR 009614 in RT-4 cells, a wound was generated with a plastic tip in control or piR 009614 transfected RT-4 cells. After 16 h of incubation, the migration of cells in the scratched area was measured. Bar graphs represent quantification of migrated cells. Data are presented as means ± SEM of triplicate samples. *p < 0.05 versus untreated control.

E.J. Sohn et al. / Biochemical and Biophysical Research Communications 519 (2019) 605e612

shown in Fig. 3B, MIWI did not colocalize with ED1 on day 4 after nerve injury. 3.4. The expression of piRNA after sciatic nerve injury To determine the expression of piRNA after sciatic nerve injury, we conducted a microarray assay based on piRNA, with RNA from nerves on day 7 after sciatic nerve injury. Fig. 4A shows hierarchical clustering of most of the differentially regulated piRNAs. A scatter plot was used to determine the variation of piRNA expression between injured and control nerves (Fig. 4B). We found 7564 piRNAs differentially expressed after injury (transection day 7) compared to the uninjured control (fold change > 2.0). Among these, 3447 piRNAs were upregulated, while 4117 piRNAs were downregulated (Supplementary data 1, and 2). The top 10 up- or downdifferentially expressed piRNAs after sciatic nerve injury are listed in Tables 1 and 2. RT-qPCR was used to confirm these results for four piRNAs (piR 14384, piR 69959, piR 52953, and piR 52274). Overall, the results revealed that the levels of piR 14384, piR 69959, piR 52953, and piR 52274 were enhanced after nerve injury (Fig. 4C). To see biological function of piRNAs, we selected upregulated piR 009614 (DQ685207) based microarray. Our results showed that piR 009614 transfected RT-4 Schwann cells reduced the mRNA expression of MBP by RT-qPCR (Fig. 4D). Wound-healing assay showed that piR 009614 transfected RT-4 cells significantly enhanced cell migration compared to control (Fig. 4E). 4. Discussions Studies of piRNAs and PIWI have focused on somatic tissues, mouse hematopoietic and human cancer cells, and a few studies have also studied neurons. In the present study, we found that the MIWI was reduced after sciatic nerve injury, and that piRNAs were abundantly expressed after nerve injury, suggesting that MIWI and piRNA may play important roles in neural degeneration. During Wallerian degeneration, myelin breaks down and myelin-related proteins such as MBP and MPZ are downregulated [14]. To the best of our knowledge, we have shown for the first time that the expression of MIWI was dramatically altered in sciatic nerve injury. A previous study reported that MIWI exists in nerves, and that depletion of MIWI enhances axonal regrowth of dorsal root ganglion [12]; but the role of MIWI in sciatic nerve injury remains unknown. In the present study, we found that MIWI expression is downregulated following sciatic nerve injury, and some MIWI is partially colocalized with MPZ after injury. Our data therefore suggest that MIWI is associated with sciatic nerve injury. However, further studies are needed to determine the exact mechanism of MIWI in Wallerian degeneration. Extensive evidence has shown that MIWI shuttles between the nucleus and cytoplasm. For example, MIWI exists in both the cytoplasm and nucleus during spermatogenesis [16]. A previous

Table 1 List of the top ten upregulated piRNA after nerve injury. piRNA acc

Name

Fold change

DQ685207 DQ546282 DQ692043 DQ721466 DQ561831 DQ721862 DQ705635 DQ713219 DQ691712 DQ709017

piR-mmu-14384 piR-mmu-69959 piR-mmu-21530 piR-mmu-52953 piR-mmu-52274 piR-mmu-53454 piR-mmu-36128 piR-mmu-44237 piR-mmu-21207 piR-mmu-39733

426.9617442 213.1268726 89.9569786 53.9671277 30.1785587 27.5560367 27.4973141 25.0483749 24.4195194 24.3699097

611

Table 2 List of the top ten down regulated piRNA after nerve injury. piRNA acc

Name

Fold change

DQ710106 DQ690718 DQ695661 DQ686894 DQ540412 DQ566768 DQ721702 DQ688975 DQ568899 "DQ564504,DQ728199"

piR-mmu-40423 piR-mmu-20208 piR-mmu-25456 piR-mmu-16143 piR-mmu-5751 piR-mmu-63299 piR-mmu-53172 piR-mmu-18357 piR-mmu-65279 piR-mmu-61117

184.5093 54.1418 37.59353 35.00443 34.38506 31.09835 30.31267 29.96057 26.94181 26.51237

study also reported that SOX10 as a transcription factor plays an important role in Schwann cell differentiation after injury [18]. Following nerve injury, mature Schwann cells dedifferentiate into immature Schwann cells for nerve regeneration [19]. Our results showed that MIWI was colocalized with S100, which is a Schwann cell marker in the cytoplasm of normal nerves. Additionally, MIWI in both normal and injured nerves also colocalized with SOX10, implying that MIWI was localized in the nucleus. Our results suggest the possibility that MIWI may involve in Schwann cell dedifferentiation after injury. The piRNAs are approximately 24e32 nucleotides in length and interact with PIWI, and PIWI and piRNA exist predominantly in the gonads as well as the sexual reproductive cycle of the protists [20]. PIWI and the piRNA complex may play roles in spermatogenesis and epigenetic regulation of transposon silencing [16,21,22]. MIWI also forms a complex with piRNA in axoplasms of sciatic nerves [12]. In neurons, depletion of piRNAs in the cytoplasm of mouse hippocampal neurons results in a significant reduction of dendrite spine areas [12], and piRNA suppresses axon regeneration in C. elegans [23]. Several studies have shown that PIWI and piRNA are potential prognostic biomarkers in several diseases such as breast cancer [24] and colorectal cancer [25]. Although a previous study reported finding 18 piRNA based on the deep sequence of small RNAs in rat sciatic nerves after crush injury [12], in the present study, we first conducted microarray analyses using piRNAs with sciatic nerves after injury. Our results revealed that the expressions of 7564 piRNAs were altered (3447 upregulated and 4117 downregulated; > 2-fold) compared with control nerves. There are accumulating evidences that Schwann cell migration is an important for the peripheral nerve regeneration after nerve injury [26e28]. After nerve damage, Schwann cells dedifferentiate by regulating MBP and MAG for nerve regeneration [29]. In the present study, piR 009614 enhanced migration of Schwann cells and downregulated the expression of MBP. Our data imply that piRNAs may contribute Schwann cell migration after nerve injury. Taken together, our data indicate that expression of MIWI was inhibited following nerve injury. After nerve injury, some of the MIWI was colocalized with MPZ. Furthermore, 7564 piRNAs were differentially expressed following sciatic nerve injury. Thus, our results imply that MIWI and piRNA in sciatic nerve injury may contribute to Schwann cell responses to axonal injury, such as dedifferentiation into repair cells or demyelinating Schwann cells. Conflicts of interest The author(s) declare(s) that there is no conflict of interest. Acknowledgements This was supported by the National Research Foundation of Korea (NRF) grant funded by Korea government (MOE) (No.

612

E.J. Sohn et al. / Biochemical and Biophysical Research Communications 519 (2019) 605e612

2018R1D1A1B07043762) and Korea government (MSIT) (No. 2016R1A5A2007009). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.09.008. References [1] Y.J. Son, W.J. Thompson, Schwann cell processes guide regeneration of peripheral axons, Neuron 14 (1995) 125e132. [2] J. Svaren, D. Meijer, The molecular machinery of myelin gene transcription in Schwann cells, Glia 56 (2008) 1541e1551. [3] D.N. Cox, A. Chao, J. Baker, L. Chang, D. Qiao, H. Lin, A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell selfrenewal, Genes Dev. 12 (1998) 3715e3727. [4] D.N. Cox, A. Chao, H. Lin, Piwi encodes a nucleoplasmic factor whose activity modulates the number and division rate of germline stem cells, Development 127 (2000) 503e514. [5] S. Houwing, L.M. Kamminga, E. Berezikov, D. Cronembold, A. Girard, H. van den Elst, D.V. Filippov, H. Blaser, E. Raz, C.B. Moens, R.H. Plasterk, G.J. Hannon, B.W. Draper, R.F. Ketting, A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in Zebrafish, Cell 129 (2007) 69e82. [6] E.Y. Yakushev, E.A. Mikhaleva, Y.A. Abramov, O.A. Sokolova, I.M. Zyrianova, V.A. Gvozdev, M.S. Klenov, [The role of Piwi nuclear localization in the differentiation and proliferation of germline stem cells], Mol. Biol. (Mosk) 50 (2016) 713e720. [7] W. Deng, H. Lin, miwi, a murine homolog of piwi, encodes a cytoplasmic protein essential for spermatogenesis, Dev. Cell 2 (2002) 819e830. [8] S.T. Grivna, B. Pyhtila, H. Lin, MIWI associates with translational machinery and PIWI-interacting RNAs (piRNAs) in regulating spermatogenesis, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 13415e13420. [9] R.J. Ross, M.M. Weiner, H. Lin, PIWI proteins and PIWI-interacting RNAs in the soma, Nature 505 (2014) 353e359. [10] P. Rajasethupathy, I. Antonov, R. Sheridan, S. Frey, C. Sander, T. Tuschl, E.R. Kandel, A role for neuronal piRNAs in the epigenetic control of memoryrelated synaptic plasticity, Cell 149 (2012) 693e707. [11] M. Litwin, A. Szczepanska-Buda, A. Piotrowska, P. Dziegiel, W. Witkiewicz, The meaning of PIWI proteins in cancer development, Oncol. Lett. 13 (2017) 3354e3362. [12] M. Phay, H.H. Kim, S. Yoo, Analysis of piRNA-like small non-coding RNAs present in axons of adult sensory neurons, Mol. Neurobiol. 55 (2018) 483e494. [13] A. Dharap, V.P. Nakka, R. Vemuganti, Altered expression of PIWI RNA in the rat

brain after transient focal ischemia, Stroke 42 (2011) 1105e1109. [14] N. Tricaud, H.T. Park, Wallerian demyelination: chronicle of a cellular cataclysm, Cell. Mol. Life Sci. 74 (2017) 4049e4057. [15] C. Kwon, H. Tak, M. Rho, H.R. Chang, Y.H. Kim, K.T. Kim, C. Balch, E.K. Lee, S. Nam, Detection of PIWI and piRNAs in the mitochondria of mammalian cancer cells, Biochem. Biophys. Res. Commun. 446 (2014) 218e223. [16] E. Beyret, H. Lin, Pinpointing the expression of piRNAs and function of the PIWI protein subfamily during spermatogenesis in the mouse, Dev. Biol. 355 (2011) 215e226. [17] J.A. Stratton, A. Holmes, N.L. Rosin, S. Sinha, M. Vohra, N.E. Burma, T. Trang, R. Midha, J. Biernaskie, Macrophages regulate schwann cell maturation after nerve injury, Cell Rep. 24 (2018) 2561e2572, e2566. [18] S. Britsch, D.E. Goerich, D. Riethmacher, R.I. Peirano, M. Rossner, K.A. Nave, C. Birchmeier, M. Wegner, The transcription factor Sox10 is a key regulator of peripheral glial development, Genes Dev. 15 (2001) 66e78. [19] K.R. Jessen, R. Mirsky, Negative regulation of myelination: relevance for development, injury, and demyelinating disease, Glia 56 (2008) 1552e1565. [20] K. Mochizuki, N.A. Fine, T. Fujisawa, M.A. Gorovsky, Analysis of a piwi-related gene implicates small RNAs in genome rearrangement in tetrahymena, Cell 110 (2002) 689e699. [21] H.J. Ferreira, H. Heyn, X. Garcia del Muro, A. Vidal, S. Larriba, C. Munoz, A. Villanueva, M. Esteller, Epigenetic loss of the PIWI/piRNA machinery in human testicular tumorigenesis, Epigenetics 9 (2014) 113e118. [22] S. Bamezai, V.P. Rawat, C. Buske, Concise review: the Piwi-piRNA axis: pivotal beyond transposon silencing, Stem Cells 30 (2012) 2603e2611. [23] K.W. Kim, N.H. Tang, M.G. Andrusiak, Z. Wu, A.D. Chisholm, Y. Jin, A neuronal piRNA pathway inhibits axon regeneration in C. elegans, Neuron 97 (2018) 511e519, e516. [24] P. Krishnan, S. Ghosh, K. Graham, J.R. Mackey, O. Kovalchuk, S. Damaraju, Piwi-interacting RNAs and PIWI genes as novel prognostic markers for breast cancer, Oncotarget 7 (2016) 37944e37956. [25] W. Weng, N. Liu, Y. Toiyama, M. Kusunoki, T. Nagasaka, T. Fujiwara, Q. Wei, H. Qin, H. Lin, Y. Ma, A. Goel, Novel evidence for a PIWI-interacting RNA (piRNA) as an oncogenic mediator of disease progression, and a potential prognostic biomarker in colorectal cancer, Mol. Cancer 17 (2018) 16. [26] K. Torigoe, H.F. Tanaka, A. Takahashi, A. Awaya, K. Hashimoto, Basic behavior of migratory Schwann cells in peripheral nerve regeneration, Exp. Neurol. 137 (1996) 301e308. [27] D.J. Bryan, K.K. Wang, C. Summerhayes, Migration of schwann cells in peripheral-nerve regeneration, J. Reconstr. Microsurg. 15 (1999) 591e596. [28] E.J. Sohn, H.T. Park, MicroRNA mediated regulation of schwann cell migration and proliferation in peripheral nerve injury, BioMed Res. Int. 2018 (2018), 8198365. [29] S.Y. Chew, R. Mi, A. Hoke, K.W. Leong, The effect of the alignment of electrospun fibrous scaffolds on Schwann cell maturation, Biomaterials 29 (2008) 653e661.