Adult Schwann Cells Research Articles

TFEB/3 Govern Repair Schwann Cell Generation and Function Following Peripheral Nerve Injury.

TFEB and TFE3 (TFEB/3), key regulators of lysosomal biogenesis and autophagy, play diverse roles depending on cell type. This study highlights a hitherto unrecognized role of TFEB/3 crucial for peripheral nerve repair. Specifically, they promote the generation of progenitor-like repair Schwann cells after axonal injury. In Schwann cell-specific TFEB/3 double knock-out mice of either sex, the TFEB/3 loss disrupts the transcriptomic reprogramming that is essential for the formation of repair Schwann cells. Consequently, mutant mice fail to populate the injured nerve with repair Schwann cells and exhibit defects in axon regrowth, target reinnervation, and functional recovery. TFEB/3 deficiency inhibits the expression of injury-responsive repair Schwann cell genes, despite the continued expression of c-jun, a previously identified regulator of repair Schwann cell function. TFEB/3 binding motifs are enriched in the enhancer regions of injury-responsive genes, suggesting their role in repair gene activation. Autophagy-dependent myelin breakdown is not impaired despite TFEB/3 deficiency. These findings underscore a unique role of TFEB/3 in adult Schwann cells that is required for proper peripheral nerve regeneration.

Manipulation of the Myc Interactome to Enhance Nerve Regeneration in a Murine Model.

This study was undertaken to explore manipulation of the Myc protein interactome, members of an oncogene group, in enhancing the intrinsic growth of injured peripheral adult postmitotic neurons and the nerves they supply. New approaches to enhance adult neuron growth properties are a key strategy in improving nerve regeneration. Expression and impact of Myc interactome members c-Myc, N-Myc, Mad1, and Max were evaluated within naive and "preconditioned" adult sensory neurons and Schwann cells (SCs), using siRNA and transfection of CRISPR/Cas9 or luciferase reporter in vitro. Morphological, behavioral, and electrophysiological indices of nerve regeneration were analyzed in vivo. c-Myc, N-Myc, Max, and Mad were expressed in adult sensory neurons and in partnering SCs. In vitro knockdown (KD) of either Mad1 or Max, competitive inhibitors of Myc, unleashed heightened neurite outgrowth in both naive uninjured or preconditioned adult neurons. In contrast, KD or inhibition of both isoforms of Myc was required to suppress growth. In SCs, Mad1 KD not only enhanced migratory behavior but also conditioned increased outgrowth in separately cultured adult sensory neurons. In vivo, local Mad1 KD improved electrophysiological, behavioral, and structural indices of nerve regeneration out to 60 days of follow-up. Members of the Myc interactome, specifically Mad1, are novel targets for improving nerve regeneration. Unleashing of Myc growth signaling through Mad1 KD enhances the regrowth of both peripheral neurons and SCs to facilitate better regrowth of nerves. ANN NEUROL 2024;96:216-230.

480-P: Synergistic Effects of Hyperglycemia and Dyslipidemia on Diabetic Neuropathy—High Glucose Aggravates Oxidized LDL-Induced Schwann Cell Death via Hyperactivation of TLR4

Diabetic neuropathy is a progressive complication of diabetes mellitus, with persistent pain and loss of sensory function. Oxidized LDL (oxLDL), an oxidized form of LDL, has been shown to play a significant role in the development of neuronal dysfunction. Circulating oxLDL is elevated in type 2 diabetes, leading to higher cardiovascular risk. However, the combined effects of hyperglycemia and oxLDL on the progression of diabetic neuropathy remain unknown. Here, we investigated the synergistic effects of high glucose on oxLDL-induced schwann cell death and its detailed mechanism. Immortalized adult mouse Schwann (IMS32) cells were treated with oxLDL (0, 150, 300 μg/mL) under normal glucose (NG, 5.5 mM) or high glucose (HG, 25 mM) conditions for 24 hours. Following treatment, we evaluated cell viability, gene, and protein expression of Toll-like receptor 4 (TLR4), a receptor for oxLDL. Under NG conditions, 300 μg/mL oxLDL, but not 150 μg/mL reduced the cell viability. Moreover, under HG conditions, both 150 μg/mL and 300 μg/mL oxLDL decreased the cell viability. Likewise, the gene and protein expression of TLR4 was synergistically upregulated by the combination of oxLDL and HG. We further evaluated the effects of TLR4 inhibition on the cell viability and apoptotic caspase-3 activity. The cells were pretreated with TAK-242, a TLR4 inhibitor, for 2 hours prior to 150 μg/mL of oxLDL treatment under NG or HG conditions. TAK-242 pretreatment attenuated the oxLDL-induced cell death in HG conditions. The caspase-3 activity was increased by oxLDL in HG conditions, but not in NG. Furthermore, the increased caspase-3 activity by oxLDL and HG was abolished by TAK-242. These results indicate that high glucose and oxLDL induce activation of TLR4, leading to exacerbated schwann cell apoptosis. The dysregulation of TLR4 signal in diabetes complicated with dyslipidemia may contribute to the pathogenesis of diabetic neuropathy. Disclosure W.Nihei: None. A.Kato: None. T.Himeno: None. M.Kondo: None. J.Nakamura: None. H.Kamiya: Research Support; Kissei Pharmaceutical Co., Ltd., Ono Pharmaceutical Co., Ltd., Eli Lilly Japan K.K., Merck & Co., Inc., Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Japan Tobacco Inc., Novo Nordisk, Taisho Pharmaceutical Holdings Co., Ltd., Speaker's Bureau; Sanofi, Novo Nordisk, Sumitomo Dainippon Pharma Co., Ltd., Boehringer Ingelheim Japan, Inc., Eli Lilly Japan K.K., Daiichi Sankyo, Ono Pharmaceutical Co., Ltd., Kissei Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Kowa Company, Ltd., Novartis Pharma K.K., Merck & Co., Inc., Sanwa Kagaku Kenkyusho, Otsuka Pharmaceutical Co., Ltd., Taisho Pharmaceutical Holdings Co., Ltd., Astellas Pharma Inc., Terumo Corporation, Fukuda Denshi, LifeScan Diabetes Institute, EA Pharma Co., Ltd, KAKEN PHARMACEUTICAL CO., LTD, AstraZeneca. K.Sango: None. K.Kato: None.

Culture Conditions for Human Induced Pluripotent Stem Cell-Derived Schwann Cells: A Two-Centre Study

Adult human Schwann cells represent a relevant tool for studying peripheral neuropathies and developing regenerative therapies to treat nerve damage. Primary adult human Schwann cells are, however, difficult to obtain and challenging to propagate in culture. One potential solution is to generate Schwann cells from human induced pluripotent stem cells (hiPSCs). Previously published protocols, however, in our hands did not deliver sufficient viable cell numbers of hiPSC-derived Schwann cells (hiPSC-SCs). We present here, two modified protocols from two collaborating laboratories that overcome these challenges. With this, we also identified the relevant parameters to be specifically considered in any proposed differentiation protocol. Furthermore, we are, to our knowledge, the first to directly compare hiPSC-SCs to primary adult human Schwann cells using immunocytochemistry and RT-qPCR. We conclude the type of coating to be important during the differentiation process from Schwann cell precursor cells or immature Schwann cells to definitive Schwann cells, as well as the amounts of glucose in the specific differentiation medium to be crucial for increasing its efficiency and the final yield of viable hiPSC-SCs. Our hiPSC-SCs further displayed high similarity to primary adult human Schwann cells.

Peroxisomes attenuate cytotoxicity of very long-chain fatty acids

One of the major functions of peroxisomes in mammals is oxidation of very long-chain fatty acids (VLCFAs). Genetic defects in peroxisomal β-oxidation result in the accumulation of VLCFAs and lead to a variety of health problems, such as demyelination of nervous tissues. However, the mechanisms by which VLCFAs cause tissue degeneration have not been fully elucidated. Recently, we found that the addition of small amounts of isopropanol can enhance the solubility of saturated VLCFAs in an aqueous medium. In this study, we characterized the biological effect of extracellular VLCFAs in peroxisome-deficient Chinese hamster ovary (CHO) cells, neural crest-derived pheochromocytoma cells (PC12), and immortalized adult Fischer rat Schwann cells (IFRS1) using this solubilizing technique. C20:0 FA was the most toxic of the C16–C26 FAs tested in all cells. The basis of the toxicity of C20:0 FA was apoptosis and was observed at 5 μM and 30 μM in peroxisome-deficient and wild-type CHO cells, respectively. The sensitivity of wild-type CHO cells to cytotoxic C20:0 FA was enhanced in the presence of a peroxisomal β-oxidation inhibitor. Further, a positive correlation was evident between cell toxicity and the extent of intracellular accumulation of toxic FA. These results suggest that peroxisomes are pivotal in the detoxification of apoptotic VLCFAs by preventing their accumulation.

463-P: Effects of Imeglimin, a New Oral Hyperglycemic Agent, on High Glucose and Low Glucose–Induced Cell Death and Mitochondrial Dysfunction in Schwann Cells

It is known that hyperglycemia-induced oxidative stress is a major cause of the pathogenesis of diabetic neuropathy, and hyperglycemia-induced mitochondrial ROS production is considered as one pivotal mechanism of increased oxidative stress. On the other hand, imeglimin is a new class of oral hypoglycemic agents called “glymines” containing tetrahydrotriazines, which targets mitochondrial dysfunction. We investigated the effects of imeglimin on cell death and mitochondrial dysfunction induced by high glucose and low glucose in Schwann cells. Immortalized adult mouse Schwann (IMS32) cells were cultured for 1 hour in the medium with normal glucose concentration of 5.5 mM, low glucose of 2.5 mM, and high glucose of 25 mM. Compared to normal glucose, not only high glucose but also low glucose decreased cell viability of IMS32 cells and increased mitochondrial oxidative stress. Mitochondrial membrane potential was also decreased either by high glucose or low glucose. In addition, mitochondrial oxygen consumption rate (OCR) was increased by low glucose as well as high glucose. Mitochondria isolated from IMS32 cells exposed to high glucose and low glucose for 1 hour exhibited increased activity of complex I. The expression of complex I protein was unchanged by high glucose for 1 hour. Imeglimin ameliorated the decrease in cell viability and improved the mitochondrial dysfunctions via inhibiting the increase of mitochondrial oxidative stress, OCR and the activity of complex I caused by high glucose and low glucose. These results indicate that hyperglycemia and hypoglycemia induce mitochondrial dysfunction in nerves, and that imeglimin may prevent against diabetic neuropathy by attenuation mitochondrial dysfunction and cell death in Schwann cells. Disclosure T. Himeno: None. J. Nakamura: None. H. Kamiya: Research Support; Abbott Japan Co., Ltd., Eli Lilly and Company, Japan Tobacco Inc., Kissei Pharmaceutical Co., Ltd., Kowa Company, Ltd., Merck & Co., Inc., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk, Ono Pharmaceutical Co., Ltd., Sanwa Kagaku Kenkyusho, Sumitomo Dainippon Pharma Co., Ltd., Taiho Pharmaceutical Co. Ltd., Takeda Pharmaceutical Company Limited, Terumo Corporation. Speaker's Bureau; Astellas Pharma Inc., AstraZeneca, Boehringer Ingelheim International GmbH, Daiichi Sankyo, Eli Lilly and Company, Kissei Pharmaceutical Co., Ltd., Kowa Company, Ltd., Merck & Co., Inc., Mitsubishi Tanabe Pharma Corporation, Novartis Pharma K.K., Novo Nordisk, Ono Pharmaceutical Co., Ltd., Sanofi K.K., Sanwa Kagaku Kenkyusho, Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Holdings Co., Ltd. K. Sango: None. K. Kato: None.

Incorporating fingolimod through poly(lactic‐co‐glycolic acid) nanoparticles in electrospun polyurethane/polycaprolactone/gelatin scaffold: An in vitro study for nerve tissue engineering

AbstractFingolimod as a useful drug in nerve regeneration is a promising candidate for promoting axonal regeneration by positively affecting neonatal and adult Schwann cells. Nerve tissue engineering show a novel practical approach by recruiting the scientific methods for creating neural extracellular matrix. In this study, fingolimod was incorporated in polylactic‐co‐glycolic acid and then, an electrospun scaffold composed of polyurethane (PU), polycaprolactone (PCL) and gelatin polymers containing different amount of encapsulated fingolimod (0.01%, 0.02%, and 0.03%) were fabricated. The morphology and microstructure of each scaffold were assessed by scanning electron microscopy (SEM). The physicochemical properties of scaffolds including Fourier transform infrared spectroscopy, mechanical properties, water contact angle, and degradation test were evaluated. MTT assay and also SEM were utilized for the investigation of cell viability and adhesion. The results showed that the mean fiber diameter of scaffold was increased from 151 ± 31 to 243 ± 68 nm followed by increase of fingolimod. Young's modulus was showed all scaffold had in the expected range for nerve tissues. Cell viability assessment proved that PU/PCL/Gel/0.01% fingolimod had a higher cell survival among other groups. The results suggest that PU/PCL/gelatin scaffolds loading from 0.01 fingolimod can be a suitable candidate for peripheral nerve regeneration.

205-OR: Docosahexaenoic Acid Suppresses Oxidative Stress-Induced Autophagy and Cell Death through the AMPK-Dependent Signaling Pathway in Immortalized Adult Rat Schwann (IFRS1) Cells

Autophagy is a catabolic process that removes damaged intracellular organelles components by lysosomal degradation, but it is also activated by oxidative stress. We previously reported that the docosahexaenoic acid (DHA) suppressed oxidative stress-induced autophagy and cell death in immortalized adult rat Schwann (IFRS1) cells. However, the detailed mechanism has not been clarified yet. Therefore, we aimed to elucidate the intracellular signal transduction pathway by which DHA suppresses oxidative stress-induced autophagy in IFRS1 cells. Treatment with tert-butyl hydroperoxide (tBHP) for 3 hours decreased cell viability measured by MTT assay in IFRS1 cells, while pretreatment with 10 μM DHA for 12 hours significantly protected tBHP-induced cytotoxicity. The signal transduction of autophagy was assessed by immunoblotting of LC3, p62, AMP-activated protein kinase (AMPK) phosphorylation and UNC51-like kinase (ULK1) phosphorylation in IFRS1 cells. 50 μM tBHP increased the LC3-II/LC3-I ratio, and AMPK phosphorylation, whereas tBHP decreased the p62 protein expression, indicating that autophagy was induced by tBHP. Pretreatment with 10 μM DHA for 12 hours significantly decreased the LC3-II/LC3-I ratio and AMPK phosphorylation that were increased by tBHP, whereas DHA increased the p62 protein expression that was decreased by tBHP. p70S6Kinase and 4E-BP phosphorylation was not changed either by tBHP or by DHA. Autophagosome and autolysosome were induced by 50 μM tBHP in IFRS1 cells, as demonstrated by DAPRed and DALGreen staining. DHA pretreatment significantly reduced the production of tBHP-induced autophagosome, but suppression of autolysosome was partial. These results suggest that DHA may prevent against diabetic neuropathy by attenuating oxidative stress-induced autophagy and cell death through the AMPK-dependent signaling pathway in Schwann cells. Disclosure Y. Tatsumi: Research Support; Self; Daiichi Sankyo, Johnson & Johnson, Kyowa Kirin Co., Ltd., Lilly Diabetes, MSD K. K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K. K., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co. J. Nakamura: Research Support; Self; Astellas Pharma Inc., Daiichi Sankyo Company, Limited, Eli Lilly Japan K. K., Japan Tobacco Inc., Novartis Pharma K. K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K. K., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Speaker’s Bureau; Self; Astellas Pharma Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Eli Lilly Japan K. K., Kowa Company, Ltd., Mitsubishi Tanabe Pharma Corporation, MSD K. K., Novartis Pharma K. K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. K. Sango: None. K. Kato: None. A. Kato: None. N. Niimi: None. H. Yako: None. T. Himeno: None. M. Kondo: None. S. Tsunekawa: None. Y. Kato: None. H. Kamiya: Speaker’s Bureau; Self; Eli Lilly Japan K. K., MDS Co., Ltd., Novartis Pharma K. K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K. K., Sumitomo Dainippon Pharma Co., Ltd. Funding Grant-in-Aid for Scientific Research (18K06763)

The Effects of Insulin on Immortalized Rat Schwann Cells, IFRS1.

Schwann cells play an important role in peripheral nerve function, and their dysfunction has been implicated in the pathogenesis of diabetic neuropathy and other demyelinating diseases. The physiological functions of insulin in Schwann cells remain unclear and therefore define the aim of this study. By using immortalized adult Fischer rat Schwann cells (IFRS1), we investigated the mechanism of the stimulating effects of insulin on the cell proliferation and expression of myelin proteins (myelin protein zero (MPZ) and myelin basic protein (MBP). The application of insulin to IFRS1 cells increased the proliferative activity and induced phosphorylation of Akt and ERK, but not P38-MAPK. The proliferative potential of insulin-stimulated IFRS1 was significantly suppressed by the addition of LY294002, a PI3 kinase inhibitor. The insulin-stimulated increase in MPZ expression was significantly suppressed by the addition of PD98059, a MEK inhibitor. Furthermore, insulin-increased MBP expression was significantly suppressed by the addition of LY294002. These findings suggest that both PI3-K/Akt and ERK/MEK pathways are involved in insulin-induced cell growth and upregulation of MPZ and MBP in IFRS1 Schwann cells.

Exendin-4 Promotes Schwann Cell Survival/Migration and Myelination In Vitro.

Besides its insulinotropic actions on pancreatic β cells, neuroprotective activities of glucagon-like peptide-1 (GLP-1) have attracted attention. The efficacy of a GLP-1 receptor (GLP-1R) agonist exendin-4 (Ex-4) for functional repair after sciatic nerve injury and amelioration of diabetic peripheral neuropathy (DPN) has been reported; however, the underlying mechanisms remain unclear. In this study, the bioactivities of Ex-4 on immortalized adult rat Schwann cells IFRS1 and adult rat dorsal root ganglion (DRG) neuron–IFRS1 co-culture system were investigated. Localization of GLP-1R in both DRG neurons and IFRS1 cells were confirmed using knockout-validated monoclonal Mab7F38 antibody. Treatment with 100 nM Ex-4 significantly enhanced survival/proliferation and migration of IFRS1 cells, as well as stimulated the movement of IFRS1 cells toward neurites emerging from DRG neuron cell bodies in the co-culture with the upregulation of myelin protein 22 and myelin protein zero. Because Ex-4 induced phosphorylation of serine/threonine-specific protein kinase AKT in these cells and its effects on DRG neurons and IFRS1 cells were attenuated by phosphatidyl inositol-3′-phosphate-kinase (PI3K) inhibitor LY294002, Ex-4 might act on both cells to activate PI3K/AKT signaling pathway, thereby promoting myelination in the co-culture. These findings imply the potential efficacy of Ex-4 toward DPN and other peripheral nerve lesions.

Schwann cell plasticity regulates neuroblastic tumor cell differentiation via epidermal growth factor-like protein 8

Adult Schwann cells (SCs) possess an inherent plastic potential. This plasticity allows SCs to acquire repair-specific functions essential for peripheral nerve regeneration. Here, we investigate whether stromal SCs in benign-behaving peripheral neuroblastic tumors adopt a similar cellular state. We profile ganglioneuromas and neuroblastomas, rich and poor in SC stroma, respectively, and peripheral nerves after injury, rich in repair SCs. Indeed, stromal SCs in ganglioneuromas and repair SCs share the expression of nerve repair-associated genes. Neuroblastoma cells, derived from aggressive tumors, respond to primary repair-related SCs and their secretome with increased neuronal differentiation and reduced proliferation. Within the pool of secreted stromal and repair SC factors, we identify EGFL8, a matricellular protein with so far undescribed function, to act as neuritogen and to rewire cellular signaling by activating kinases involved in neurogenesis. In summary, we report that human SCs undergo a similar adaptive response in two patho-physiologically distinct situations, peripheral nerve injury and tumor development.

Multipotent Schwann Cell Precursors Generate Steroid-Producing Cells of the Adrenal Cortex and Chromaffin Cells of the Adrenal Medulla

The adrenal gland is a key regulator during stress, and a high degree of plasticity is critical for sustaining homeostasis. Previously, we have shown that this is achieved in part through adult Nestin(+) progenitors located in both the adrenal cortex and medulla. To obtain a comprehensive characterization of these cells, we performed transcriptomics on isolated Nestin(+) cells from the two adrenal tissues. This analysis revealed that both populations displayed Schwann cell precursor properties suggesting a common neural crest origin, though the two populations showed distinct characteristics. Adrenomedullary Nestin(+) cells showed a commitment to the neural/glial lineage, while adrenocortical Nestin(+) cells showed a predestination to become steroid-producing cells. The data suggest that adult adrenal Schwann cell precursors might fulfill a role of coordinated structural tissue remodeling under stress answering the question why two completely different organs are united under one organ capsule.

Magnetic separation of peripheral nerve-resident cells underscores key molecular features of human Schwann cells and fibroblasts: an immunochemical and transcriptomics approach

Nerve-derived human Schwann cell (SC) cultures are irreplaceable models for basic and translational research but their use can be limited due to the risk of fibroblast overgrowth. Fibroblasts are an ill-defined population consisting of highly proliferative cells that, contrary to human SCs, do not undergo senescence in culture. We initiated this study by performing an exhaustive immunological and functional characterization of adult nerve-derived human SCs and fibroblasts to reveal their properties and optimize a protocol of magnetic-activated cell sorting (MACS) to separate them effectively both as viable and biologically competent cells. We next used immunofluorescence microscopy imaging, flow cytometry analysis and next generation RNA sequencing (RNA-seq) to unambiguously characterize the post-MACS cell products. High resolution transcriptome profiling revealed the identity of key lineage-specific transcripts and the clearly distinct neural crest and mesenchymal origin of human SCs and fibroblasts, respectively. Our analysis underscored a progenitor- or stem cell-like molecular phenotype in SCs and fibroblasts and the heterogeneity of the fibroblast populations. In addition, pathway analysis of RNA-seq data highlighted putative bidirectional networks of fibroblast-to-SC signaling that predict a complementary, yet seemingly independent contribution of SCs and fibroblasts to nerve regeneration. In sum, combining MACS with immunochemical and transcriptomics approaches provides an ideal workflow to exhaustively assess the identity, the stage of differentiation and functional features of highly purified cells from human peripheral nerve tissues.

Novel Glial Cell Functions: Extensive Potency, Stem Cell-Like Properties, and Participation in Regeneration and Transdifferentiation

Glial cells are the most abundant cells in both the peripheral and central nervous systems. During the past decade, a subpopulation of immature peripheral glial cells, namely, embryonic Schwann cell-precursors, have been found to perform important functions related to development. These cells have properties resembling those of the neural crest and, depending on their location in the body, can transform into several different cell types in peripheral tissues, including autonomic neurons. This review describes the multipotent properties of Schwann cell-precursors and their importance, together with innervation, during early development. The heterogeneity of Schwann cells, as revealed using single-cell transcriptomics, raises a question on whether some glial cells in the adult peripheral nervous system retain their stem cell-like properties. We also discuss how a deeper insight into the biology of both embryonic and adult Schwann cells might lead to an effective treatment of the damage of both neural and non-neural tissues, including the damage caused by neurodegenerative diseases. Furthermore, understanding the potential involvement of Schwann cells in the regulation of tumor development may reveal novel targets for cancer treatment.

551-P: Docosahexaenoic Acid Attenuates Oxidative Stress–Induced Autophagy and Cell Death in Immortalized Adult Rat Schwann (IFRS1) Cells

We previously reported that the docosahexaenoic acid (DHA) induced the antioxidant enzyme, heme oxygenase-1 (HO-1) through the nuclear factor erythroid 2-related factor 2 (Nrf2) and protected immortalized adult mouse Schwann (IMS32) cells from oxidative stress. Whereas autophagy is the process by which intracellular components are degraded by the lysosomes, but it also involved in stabilization of Nrf2 and transcriptional activation of Nrf2 target genes. Therefore, autophagy is thought to be closely related to oxidative stress, which is one major cause of diabetic neuropathy. However, the relationship between autophagy and oxidative stress-induced cell death in diabetic neuropathy has not been elucidated. Treatment with tert-butyl hydroperoxide (tBHP) for 3 hours decreased cell viability measured by MTT assay in immortalized adult rat Schwann (IFRS1) cells, while pretreatment with 10 μM DHA for 12 hours significantly protected tBHP-induced cytotoxicity. Autophagy was assessed by immunoblotting of LC3B and p62. 50 μM tBHP increased the LC3B-II/LC3B-I ratio and decreased the p62 expression in IFRS1 cells, indicating that autophagy was induced by tBHP. DHA pretreatment alleviated tBHP-induced autophagy. Autophagosome and autolysosome were induced by 50 μM tBHP in IFRS1 cells, as demonstrated by DAPRed and DALGreen staining. DHA pretreatment significantly reduced the production of tBHP-induced autophagosome and autolysosome. These results suggest that oxidative stress induces autophagy and promotes cell death in Schwann cells and that treatment with DHA may protect against diabetic neuropathy by attenuating oxidative stress-induced autophagy and cell death in Schwann cells. Disclosure Y. Tatsumi: None. A. Kato: None. K. Sango: None. T. Himeno: None. M. Kondo: None. S. Tsunekawa: None. Y. Kato: None. H. Kamiya: Speaker’s Bureau; Self; Astellas Pharma Inc., AstraZeneca K.K., Boehringer Ingelheim K.K., Daiichi Sankyo, Eli Lilly Japan K.K., Fukuda Denshi, Kissei Pharmaceutical Co., Ltd., Kowa Company, Ltd., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, MSD K.K., Novartis Pharma K.K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K.K., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. J. Nakamura: Research Support; Self; Astellas Pharma Inc., Boehlinger Ingelheim Japan Co., Ltd., Daiichi Sankyo, Eli Lilly Japan K.K., Japan Tobacco Inc., Kaken Pharmaceutical Co., Ltd., Kowa Company, Ltd., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, MSD K.K., Novartis Pharma K.K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Otsuka Pharmaceutical Co., Ltd., Pfizer Japan Inc., Sanofi K.K., Sanwa Kagaku Kenkyusho, Shionogi & Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker’s Bureau; Self; Abbott Japan Co., Ltd., ARKRAY, Astellas Pharma Inc., AstraZeneca K.K., Boehlinger Ingelheim Japan Co., Ltd.,, Daiichi Sankyo, Eli Lilly Japan K.K., Fukuda Denshi, Kissei Pharmaceutical Co., Ltd., Kowa Company, Ltd., Mitsubishi Tanabe Pharma Corporation, MSD K.K., Mylan, Novartis Pharma K.K., Novo Nordisk Pharma Ltd, Ono Pharmaceutical Co., Ltd., Sanofi, Sanwa Kagaku Kenkyusho, Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, Terumo Medical Corporation. K. Kato: None. Funding Japan Society for the Promotion of Science (18K06763)

Automated image analysis of stained cytospins to quantify Schwann cell purity and proliferation.

In response to injury, adult Schwann cells (SCs) re-enter the cell cycle, change their expression profile, and exert repair functions important for wound healing and the re-growth of axons. While this phenotypical instability of SCs is essential for nerve regeneration, it has also been implicated in cancer progression and de-myelinating neuropathies. Thus, SCs became an important research tool to study the molecular mechanisms involved in repair and disease and to identify targets for therapeutic intervention. A high purity of isolated SC cultures used for experimentation must be demonstrated to exclude that novel findings are derived from a contaminating fibroblasts population. In addition, information about the SC proliferation status is an important parameter to be determined in response to different treatments. The evaluation of SC purity and proliferation, however, usually depends on the time consuming, manual assessment of immunofluorescence stainings or comes with the sacrifice of a large amount of SCs for flow cytometry analysis. We here show that rat SC culture derived cytospins stained for SC marker SOX10, proliferation marker EdU, intermediate filament vimentin and DAPI allowed the determination of SC identity and proliferation by requiring only a small number of cells. Furthermore, the CellProfiler software was used to develop an automated image analysis pipeline that quantified SCs and proliferating SCs from the obtained immunofluorescence images. By comparing the results of total cell count, SC purity and SC proliferation rate between manual counting and the CellProfiler output, we demonstrated applicability and reliability of the established pipeline. In conclusion, we here combined the cytospin technique, a multi-colour immunofluorescence staining panel, and an automated image analysis pipeline to enable the quantification of SC purity and SC proliferation from small cell aliquots. This procedure represents a solid read-out to simplify and standardize the quantification of primary SC culture purity and proliferation.

Axon-dependent expression of YAP/TAZ mediates Schwann cell remyelination but not proliferation after nerve injury.

Previously we showed that YAP/TAZ promote not only proliferation but also differentiation of immature Schwann cells (SCs), thereby forming and maintaining the myelin sheath around peripheral axons (Grove et al., 2017). Here we show that YAP/TAZ are required for mature SCs to restore peripheral myelination, but not to proliferate, after nerve injury. We find that YAP/TAZ dramatically disappear from SCs of adult mice concurrent with axon degeneration after nerve injury. They reappear in SCs only if axons regenerate. YAP/TAZ ablation does not impair SC proliferation or transdifferentiation into growth promoting repair SCs. SCs lacking YAP/TAZ, however, fail to upregulate myelin-associated genes and completely fail to remyelinate regenerated axons. We also show that both YAP and TAZ are redundantly required for optimal remyelination. These findings suggest that axons regulate transcriptional activity of YAP/TAZ in adult SCs and that YAP/TAZ are essential for functional regeneration of peripheral nerve.

SAT-739 Membrane Progesterone Receptors (mPRs/PAQRs) Promote Proliferation, Migration and Neurotrophin Expression in Schwann Cell-like Adipose Stem Cells, Potentially Improving Peripheral Nerve Regeneration

Peripheral nerve injury is a problem affecting millions of people worldwide, causing significant disability and a reduction in the quality of life. The neurons of the peripheral nervous system are characterized by a significant regeneration capability. However, the functional recovery is unsatisfactory in most cases, making the identification of new strategies to improve the regenerative outcome a major medical need. Schwann cells (SCs), the main neuroglial cells of the peripheral nervous system, trans-differentiate after nerve injury towards a phenotype, known as the repair phenotype, that promotes nerve regeneration. Nonetheless, adult human SCs have poor expansion capability in vitro, undermining their potential clinical development. Several studies have shown that mesenchymal stem cells, especially when differentiated into a Schwann cell-like (SCL) phenotype, may represent an alternative to primary SCs, promoting pro-regenerative effects both in vitro and in vivo. Recently, membrane progesterone receptors (mPRs), members of the progestin and adipoQ receptor (PAQR) family, have been shown to be present and active in Schwann cells. Progesterone activates mPRs in SCs to promote cell migration and proliferation, change cell morphology, and modulate the expression of key SC differentiation factors. These findings suggest a possible role for mPRs in nerve repair. The goal of the present project is to study the role of mPRs in the promotion of pro-regenerative effects in mesenchymal adipose stem cells (ASC), both undifferentiated (uASC) and after differentiation toward the SCL phenotype (SCL-ASC). We first characterized uASC and SCL-ASC, confirming that the latter showed increased expression of SC markers like S100B, Sox10, GFAP, P0 and Krox20, alongside a more pronounced spindle-like shape leading to an increased aspect ratio. The differentiation protocol also increased mPR gene expression in SCL-ASC compared to uASC, especially mPRα (PAQR7) and mPRβ (PAQR8). Treatment with the specific mPR agonist Org OD 02-0 (02-0) caused cell elongation in SCL-ASC, mimicking what happens to SCs in vivo when they trans-differentiate towards the repair phenotype. 02-0 treatment also led to increased cell migration and cell proliferation in both uASC and SCL-ASC, consistent with a pro-regenerative role of mPRs. The effect was more rapid and evident in SCL-ASC. Lastly, we analyzed the effect of mPR activation on neutrophin production and release in uASC and SCL-ASC. A significant increase was observed in the expression and release of the brain derived neurotrophic factor (BDNF), whose role in promoting neurogenesis is well established. Our results support the hypothesis that mPRs have an important role in the modulation of uASC and SCL-ASC physiology and their activation may promote nerve regeneration.

A comparison of the use of adipose-derived and bone marrow-derived stem cells for peripheral nerve regeneration in vitro and in vivo

BackgroundTo date, it has repeatedly been demonstrated that infusing bone marrow-derived stem cells (BMSCs) into acellular nerve scaffolds can promote and support axon regeneration through a peripheral nerve defect. However, harvesting BMSCs is an invasive and painful process fraught with a low cellular yield.MethodsIn pursuit of alternative stem cell sources, we isolated stem cells from the inguinal subcutaneous adipose tissue of adult Sprague–Dawley rats (adipose-derived stem cells, ADSCs). We used a co-culture system that allows isolated adult mesenchymal stem cells (MSCs) and Schwann cells (SCs) to grow in the same culture medium but without direct cellular contact. We verified SC phenotype in vitro by cell marker analysis and used red fluorescent protein-tagged ADSCs to detect their fate after being injected into a chemically extracted acellular nerve allograft (CEANA). To compare the regenerative effects of CEANA containing either BMSCs or ADSCs with an autograft and CEANA only on the sciatic nerve defect in vivo, we performed histological and functional assessments up to 16 weeks after grafting.ResultsIn vitro, we observed reciprocal beneficial effects of ADSCs and SCs in the ADSC–SC co-culture system. Moreover, ADSCs were able to survive in CEANA for 5 days after in vitro implantation. Sixteen weeks after grafting, all results consistently showed that CEANA infused with BMSCs or ADSCs enhanced injured sciatic nerve repair compared to the acellular CEANA-only treatment. Furthermore, their beneficial effects on sciatic injury regeneration were comparable as histological and functional parameters evaluated showed no statistically significant differences. However, the autograft group was roundly superior to both the BMSC- or ADSC-loaded CEANA groups.ConclusionThe results of the present study show that ADSCs are a viable alternative stem cell source for treating sciatic nerve injury in lieu of BMSCs.

Adult tissue-derived neural crest-like stem cells: Sources, regulatory networks, and translational potential.

Neural crest (NC) cells are a multipotent stem cell population that give rise to a diverse array of cell types in the body, including peripheral neurons, Schwann cells (SC), craniofacial cartilage and bone, smooth muscle cells, and melanocytes. NC formation and differentiation into specific lineages takes place in response to a set of highly regulated signaling and transcriptional events within the neural plate border. Premigratory NC cells initially are contained within the dorsal neural tube from which they subsequently emigrate, migrating to often distant sites in the periphery. Following their migration and differentiation, some NC‐like cells persist in adult tissues in a nascent multipotent state, making them potential candidates for autologous cell therapy. This review discusses the gene regulatory network responsible for NC development and maintenance of multipotency. We summarize the genes and signaling pathways that have been implicated in the differentiation of a postmigratory NC into mature myelinating SC. We elaborate on the signals and transcription factors involved in the acquisition of immature SC fate, axonal sorting of unmyelinated neuronal axons, and finally the path toward mature myelinating SC, which envelope axons within myelin sheaths, facilitating electrical signal propagation. The gene regulatory events guiding development of SC in vivo provides insights into means for differentiating NC‐like cells from adult human tissues into functional SC, which have the potential to provide autologous cell sources for the treatment of demyelinating and neurodegenerative disorders.