Adaptive autophagy reprogramming in Schwann cells during peripheral demyelination.

The myelin sheath is an essential structure for the rapid transmission of electrical impulses through axons, and peripheral myelination is a well-programmed postnatal process of Schwann cells (SCs), the myelin-forming peripheral glia. SCs transdifferentiate into demyelinating SCs (DSCs) to remove the myelin sheath during Wallerian degeneration after axonal injury and demyelinating neuropathies, and macrophages are responsible for the degradation of myelin under both conditions. In this study, the mechanism by which DSCs acquire the ability of myelin exocytosis was investigated. Using serial ultrastructural evaluation, we found that autophagy-related gene 7-dependent formation of a "secretory phagophore (SP)" and tubular phagophore was necessary for exocytosis of large myelin chambers by DSCs. DSCs seemed to utilize myelin membranes for SP formation and employed p62/sequestosome-1 (p62) as an autophagy receptor for myelin excretion. In addition, the acquisition of the myelin exocytosis ability of DSCs was associated with the decrease in canonical autolysosomal flux and was demonstrated by p62 secretion. Finally, this SC demyelination mechanism appeared to also function in inflammatory demyelinating neuropathies. Our findings show a novel autophagy-mediated myelin clearance mechanism by DSCs in response to nerve damage.

COUP-TFII plays a role in cAMP-induced Schwann cell differentiation and in vitro myelination by up-regulating Krox20.

Schwann cells (SCs) are known to produce myelin for saltatory nerve conduction in the peripheral nervous system (PNS). Schwann cell differentiation and myelination processes are controlled by several transcription factors including Sox10, Oct6/Pou3f1, and Krox20/Egr2. Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII/NR2F2) is an orphan receptor that plays a role in the development and differentiation. However, the role of COUP-TFII in the transcriptional regulatory network of SC differentiation has not been fully identified yet. Thus, the objective of this study was to investigate the role and molecular hierarchy of COUP-TFII during cAMP-induced SC differentiation. Our results showed that dibutyryl-cAMP (db-cAMP) increased expression levels of COUP-TFII along with the expressions of Oct6, Krox20, and myelin-related genes known to be related to SC differentiation. Our mechanistic studies showed that COUP-TFII acted downstream of Hsp90/ErbB2/Gab1/ERK-AKT pathway during db-cAMP-induced SC differentiation. In addition, we found that COUP-TFII induced Krox20 expression by directly binding to Krox20-MSE8 as revealed by chromatin immunoprecipitation assay and promoter activity assay. In line with this, the expression of COUP-TFII was increased before up-regulation of Oct6, Krox20, and myelin-related genes in the sciatic nerves during early postnatal myelination period. Finally, COUP-TFII knockdown by COUP-TFII siRNA or via AAV-COUP-TFII shRNA in SCs inhibited db-cAMP-induced SC differentiation and in vitro myelination of sensory axons, respectively. Taken together, these findings indicate that COUP-TFII might be involved in postnatal myelination through induction of Krox20 in SCs. Our results present a new insight into the transcriptional regulatory mechanism in SC differentiation and myelination.

Regulation of the V-ATPase subunit ATP6V0D2 and its role in demyelination after peripheral nerve injury.

After peripheral nerve injury, demyelinating Schwann cells discharge myelin debris and macrophages execute myelin degradation, leading to demyelination of degenerating axons, which is essential for efficient nerve regeneration. In this study, we show that vacuolar-type proton ATPase subunit d2 (Atp6v0d2) is among the most highly upregulated genes in degenerating mouse sciatic nerves after nerve injury using microarray analysis. ATP6V0D2 is mostly expressed in macrophages of injured nerves. Atp6v0d2 knockout mice display delayed peripheral nerve demyelination and significantly attenuated myelin lipid digestion after nerve injury. However, macrophage recruitment and Schwann cell dedifferentiation are unaffected by loss of Atp6v0d2 expression. Taken together, these data demonstrate that ATP6V0D2 in macrophages is specifically required for demyelination during Wallerian degeneration.

Neural cell adhesion molecule 1 is a cellular target engaged plasma biomarker in demyelinating Charcot-Marie-Tooth disease.

BACKGROUND AND PURPOSE: Elevated plasma concentrations of neural cell adhesion molecule 1 (NCAM1) and p75 neurotrophin receptor (p75) in patients with peripheral neuropathy have been reported. This study aimed to determine the specificity of plasma concentration elevation of either NCAM1 or p75 in a subtype of Charcot-Marie-Tooth disease (CMT) and its correlation with pathologic nerve status and disease severity. METHODS: Blood samples were collected from 138 patients with inherited peripheral neuropathy and 51 healthy controls. Disease severity was measured using Charcot-Marie-Tooth Neuropathy Score version 2 (CMTNSv2), and plasma concentrations of NCAM1 and p75 were analyzed by enzyme-linked immunosorbent assay. Eight sural nerves from CMT patients were examined to determine the relation of histopathology and plasma NCAM1 levels. RESULTS: Plasma concentration of NCAM1, but not p75, was specifically increased in demyelinating subtypes of CMT (median = 7100 pg/mL, p < 0.001), including CMT1A, but not in axonal subtype (5964 pg/mL, p > 0.05), compared to the control (3859 pg/mL). CMT1A patients with mild or moderate severity (CMTNSv2 < 20) showed higher levels of plasma NCAM1 than healthy controls. Immunofluorescent NCAM1 staining for the sural nerves of CMT patients showed that NCAM1-positive onion bulb cells and possible demyelinating Schwann cells might be associated with the specific increase of plasma NCAM1 in demyelinating CMT. CONCLUSIONS: The plasma NCAM1 levels in demyelinating CMT might be a surrogate biomarker reflecting pathological Schwann cell status and disease progression.

Potential neuron-autonomous Purkinje cell degeneration by 2',3'-cyclic nucleotide 3'-phosphodiesterase promoter/Cre-mediated autophagy impairments.

Studies of neuroglial interaction largely depend on cell-specific gene knockout (KO) experiments using Cre recombinase. However, genes known as glial-specific genes have recently been reported to be expressed in neuroglial stem cells, leading to the possibility that a glia-specific Cre driver results in unwanted gene deletion in neurons, which may affect sound interpretation. 2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) is generally considered to be an oligodendrocyte (OL) marker. Accordingly, Cnp promoter-controlled Cre recombinase has been used to create OL-specific gene targeting mice. However, in this study, using Rosa26-tdTomato-reporter/Cnp-Cre mice, we found that many forebrain neurons and cerebellar Purkinje neurons belong to the lineages of Cnp-expressing neuroglial stem cells. To answer whether gene targeting by Cnp-Cre can induce neuron-autonomous defects, we conditionally deleted an essential autophagy gene, Atg7, in Cnp-Cre mice. The Cnp-Cre-mediated Atg7 KO mice showed extensive p62 inclusion in neurons, including cerebellar Purkinje neurons with extensive neurodegeneration. Furthermore, neuronal areas showing p62 inclusion in Cnp-Cre-mediated Atg7 KO mice overlapped with the neuronal lineage of Cnp-expressing neuroglial stem cells. Moreover, Cnp-Cre-mediated Atg7-KO mice did not develop critical defects in myelination. Our results demonstrate that a large population of central neurons are derived from Cnp-expressing neuroglial stem cells; thus, conditional gene targeting using the Cnp promoter, which is known to be OL-specific, can induce neuron-autonomous phenotypes.

Ceria-Zirconia nanoparticles reduce intracellular globotriaosylceramide accumulation and attenuate kidney injury by enhancing the autophagy flux in cellular and animal models of Fabry disease.

BACKGROUND: Fabry disease (FD) is a lysosome storage disease (LSD) characterized by significantly reduced intracellular autophagy function. This contributes to the progression of intracellular pathologic signaling and can lead to organ injury. Phospholipid-polyethyleneglycol-capped Ceria-Zirconia antioxidant nanoparticles (PEG-CZNPs) have been reported to enhance autophagy flux. We analyzed whether they suppress globotriaosylceramide (Gb3) accumulation by enhancing autophagy flux and thereby attenuate kidney injury in both cellular and animal models of FD. RESULTS: Gb3 was significantly increased in cultured human renal proximal tubular epithelial cells (HK-2) and human podocytes following the siRNA silencing of α galactosidase A (α-GLA). PEG-CZNPs effectively reduced the intracellular accumulation of Gb3 in both cell models of FD and improved both intracellular inflammation and apoptosis in the HK-2 cell model of FD. Moreover these particles attenuated pro fibrotic cytokines in the human podocyte model of FD. This effect was revealed through an improvement of the intracellular autophagy flux function and a reduction in reactive oxygen species (ROS). An FD animal model was generated in which 4-week-old male B6;129-Glatm1Kul/J mice were treated for 8 weeks with 10 mg/kg of PEG-CZNPs (twice weekly via intraperitoneal injection). Gb3 levels were reduced in the kidney tissues of these animals, and their podocyte characteristics and autophagy flux functions were preserved. CONCLUSIONS: PEG-CZNPs alleviate FD associated kidney injury by enhancing autophagy function and thus provide a foundation for the development of new drugs to treat of storage disease.

Antibodies Regulate Dual-Function Enzyme IYD to Induce Functional Synergy between Metabolism and Thermogenesis.

Iodotyrosine deiodinase (IYD) is a type of deiodinase enzyme that scavenges iodide from the thyroid gland. Previously, we showed that H3 Ab acts as an agonist on IYD to induce migration of cells to the heart and differentiate human stem cells into brown adipocyte-like cells. To continue this study, we investigated the dual function of IYD in hypothyroidism by blocking IYD and in thermogenesis by looking at the induction of brown adipocyte-like cells by treatment with H3 Ab in a mouse model. Surprisingly, our results suggest H3 Ab acts on IYD as both an antagonist and agonist to reduce T4 and increase core body temperature in the mouse model. Taken together, the data suggest IYD has a dual function that can regulate physiological metabolism and enhance thermogenesis.

Red-Emitting Latex Nanoparticles by Stepwise Entrapment of ß-Diketonate Europium Complexes.

The core-shell structure of poly(St-co-MAA) nanoparticles containing ß-diketonate Eu3+ complexes were synthesized by a step-wise process. The ß-diketonate Eu3+ complexes of Eu (TFTB)2(MAA)P(Oct)3 [europium (III); 4,4,4-Trifluoro-1-(2-thienyl)-1,3-butanedione = TFTB; trioctylphosphine = (P(Oct)3); methacrylic acid = MAA] were incorporated to poly(St-co-MAA). The poly(St-co-MAA) has highly monodispersed with a size of 300 nm, and surface charges of the poly(St-co-MAA) are near to neutral. The narrow particle size distribution was due to the constant ionic strength of the polymerization medium. The activated carboxylic acid of poly(St-co-MAA) further chelated with europium complex and polymerize between acrylic groups of poly(St-co-MAA) and Eu(TFTB)2(MAA)P(Oct)3. The Em spectra of europium complexes consist of multiple bands of Em at 585, 597, 612 and 650 nm, which are assigned to 5D0→7FJ (J = 0-3) transitions of Eu3+, respectively. The maximum Em peak is at 621 nm, which indicates a strong red Em characteristic associated with the electric dipole 5D0→7F2 transition of Eu3+ complexes. The cell-specific fluorescence of Eu(TFTB)2(MAA)P(Oct)3@poly(St-co-MAA) indicated endocytosis of Eu(TFTB)2(MAA)P(Oct)3@poly(St-co-MAA). There are fewer early apoptotic, late apoptotic and necrotic cells in each sample compared with live cells, regardless of the culture period. Eu(TFTB)2(MAA)P(Oct)3@poly(St-co-MAA) synthesized in this work can be excited in the full UV range with a maximum Em at 619 nm. Moreover, these particles can substitute red luminescent organic dyes for intracellular trafficking and cellular imaging agents.

Aconitate Decarboxylase 1 Deficiency Exacerbates Mouse Colitis Induced by Dextran Sodium Sulfate.

Ulcerative colitis is a complex inflammatory bowel disorder disease that can induce rectal and colonic dysfunction. Although the prevalence of IBD in Western countries is almost 0.5% of the general population, genetic causes are still not fully understood. In a recent discovery, itaconate was found to function as an immune-modulating metabolite in mammalian immune cells, wherein it is synthesized as an antimicrobial compound from the citric acid cycle intermediate cis-aconitic acid. However, the association between the Acod1 (Aconitate decarboxylase 1)-itaconate axis and ulcerative colitis has rarely been studied. To elucidate this, we established a DSS-induced colitis model with Acod1-deficient mice and then measured the mouse body weights, colon lengths, histological changes, and cytokines/chemokines in the colon. We first confirmed the upregulation of Acod1 RNA and protein expression levels in DSS-induced colitis. Then, we found that colitis symptoms, including weight loss, the disease activity index, and colon shortening, were worsened by the depletion of Acod1. In addition, the extent of intestinal epithelial barrier breakdown, the extent of immune cell infiltration, and the expression of proinflammatory cytokines and chemokines in Acod1-deficient mice were higher than those in wild-type mice. Finally, we confirmed that 4-octyl itaconate (4-OI) alleviated DSS-induced colitis in Acod1-deficient mice and decreased the expression of inflammatory cytokines and chemokines. To our knowledge, this study is the first to elucidate the role of the Acod1-itaconate axis in colitis. Our data clearly showed that Acod1 deletion resulted in severe DSS-induced colitis and substantial increases in inflammatory cytokine and chemokine levels. Our results suggest that Acod1 may normally play an important regulatory role in the pathogenesis of colitis, demonstrating the potential for novel therapies using 4-OI.

Comparison of Macrophage Immune Responses and Metabolic Reprogramming in Smooth and Rough Variant Infections of Mycobacterium mucogenicum.

Mycobacterium mucogenicum (Mmuc), a rapidly growing nontuberculous mycobacterium (NTM), can infect humans (posttraumatic wound infections and catheter-related sepsis). Similar to other NTM species, Mmuc exhibits colony morphologies of rough (Mmuc-R) and smooth (Mmuc-S) types. Although there are several case reports on Mmuc infection, no experimental evidence supports that the R-type is more virulent. In addition, the immune response and metabolic reprogramming of Mmuc have not been studied on the basis of morphological characteristics. Thus, a standard ATCC Mmuc strain and two clinical strains were analyzed, and macrophages were generated from mouse bone marrow. Cytokines and cell death were measured by ELISA and FACS, respectively. Mitochondrial respiration and glycolytic changes were measured by XF seahorse. Higher numbers of intracellular bacteria were found in Mmuc-R-infected macrophages than in Mmuc-S-infected macrophages. Additionally, Mmuc-R induced higher levels of the cytokines TNF-α, IL-6, IL-12p40, and IL-10 and induced more BMDM necrotic death. Furthermore, our metabolic data showed marked glycolytic and respiratory differences between the control and each type of Mmuc infection, and changes in these parameters significantly promoted glucose metabolism, extracellular acidification, and oxygen consumption in BMDMs. In conclusion, at least in the strains we tested, Mmuc-R is more virulent, induces a stronger immune response, and shifts bioenergetic metabolism more extensively than the S-type. This study is the first to report differential immune responses and metabolic reprogramming after Mmuc infection and might provide a fundamental basis for additional studies on Mmuc pathogenesis.

Trichinella Infection Ameliorated Vincristine-Induced Neuroinflammation in Mice.

Vincristine (VCR) is a chemotherapeutic agent widely used in treatment of malignancies. However, VCR has a limitation in use since it commonly causes a painful neuropathy (VCR-induced peripheral neuropathy, VIPN). Inflammatory cytokines secreted by immune cells such as macrophages can exacerbate allodynia and hyperalgesia, because inhibiting the inflammatory response is a treatment target for VIPN. In this study, we investigated whether Trichinella spiralis, a widely studied helminth for its immunomodulatory abilities, can alleviate VCR-induced allodynia. Von Frey test showed that T. spiralis infection improved mechanical allodynia at 10 days after VCR injection. We further observed whether the difference was due to mitigated axon degeneration, but no significant difference between the groups in axonal degeneration in sciatic nerves and intra-epidermal nerve fibers was found. Conversely, we observed that number of infiltrated macrophages was decreased in the sciatic nerves of the T. spiralis infected mice. Moreover, treatment of T. spiralis excretory-secretory products caused peritoneal macrophages to secrete decreased level of IL-1ß. This study suggests that T. spiralis can relieve VCR-induced mechanical allodynia by suppressing neuroinflammation and that application of controllable degree of helminth may prove beneficial for VIPN treatment.

The GATOR2-mTORC2 axis mediates Sestrin2-induced AKT Ser/Thr kinase activation.

Sestrins represent a family of stress-inducible proteins that prevent the progression of many age- and obesity-associated disorders. Endogenous Sestrins maintain insulin-dependent AKT Ser/Thr kinase (AKT) activation during high-fat diet-induced obesity, and overexpressed Sestrins activate AKT in various cell types, including liver and skeletal muscle cells. Although Sestrin-mediated AKT activation improves metabolic parameters, the mechanistic details underlying such improvement remain elusive. Here, we investigated how Sestrin2, the Sestrin homolog highly expressed in liver, induces strong AKT activation. We found that two known targets of Sestrin2, mTOR complex (mTORC) 1 and AMP-activated protein kinase, are not required for Sestrin2-induced AKT activation. Rather, phosphoinositol 3-kinase and mTORC2, kinases upstream of AKT, were essential for Sestrin2-induced AKT activation. Among these kinases, mTORC2 catalytic activity was strongly up-regulated upon Sestrin2 overexpression in an in vitro kinase assay, indicating that mTORC2 may represent the major link between Sestrin2 and AKT. As reported previously, Sestrin2 interacted with mTORC2; however, we found here that this interaction occurs indirectly through GATOR2, a pentameric protein complex that directly interacts with Sestrin2. Deleting or silencing WDR24 (WD repeat domain 24), the GATOR2 component essential for the Sestrin2-GATOR2 interaction, or WDR59, the GATOR2 component essential for the GATOR2-mTORC2 interaction, completely ablated Sestrin2-induced AKT activation. We also noted that Sestrin2 also directly binds to the pleckstrin homology domain of AKT and induces AKT translocation to the plasma membrane. These results uncover a signaling mechanism whereby Sestrin2 activates AKT through GATOR2 and mTORC2.

Scaffolding protein Gab2 is involved in postnatal development and lipopolysaccharide-induced activation of microglia in the mouse brain.

Grb2-associated-binding protein-2 (Gab2) is a member of the Gab/DOS family and functions as an adapter protein downstream of several growth factor signaling pathways. Gab2 is considered an Alzheimer's disease susceptibility gene. However, the role of Gab2 in the brain is still largely unknown. Herein, we report that Gab2 is involved in the postnatal development of microglia in mice. The Gab2 expression in the brain was detected at postnatal day 1 (P1) and increased until P14 but decreased thereafter. The tyrosine phosphorylation of Gab2 (pGab2) was also detected at P1 and increased until P14. Next, we focused on microglial development in Gab2 knockout and heterozygous mice. Although differences were not detected in the cytoplasmic area of Iba1-labeled microglia between Gab2(±) and Gab2(-/-) mice, the analysis of CD68 and cathepsin D (indicators of microglial lysosomal activation) immunolabeling within Iba1+ cells revealed significant underdevelopment of microglial lysosomes in Gab2(-/-) mice at P60. In addition to the developmental abnormality of microglia in Gab2(-/-) mice, lipopolysaccharide-induced lysosomal activation was selectively suppressed in Gab2(-/-) mice compared to that in Gab2(±) mice. Our findings suggest that Gab2 is involved not only in postnatal development but also in lysosomal activation of microglia, therefore Gab2 dysfunction in microglia might potentially contribute to the development of neurodegenerative diseases.

TGF-ß secreted by human umbilical cord blood-derived mesenchymal stem cells ameliorates atopic dermatitis by inhibiting secretion of TNF-α and IgE.

Human mesenchymal stem cells (MSCs) are promising therapeutics for autoimmune diseases due to their immunomodulatory effects. In particular, human umbilical cord blood-derived MSCs (hUCB-MSCs) have a prominent therapeutic effect on atopic dermatitis (AD). However, the underlying mechanism is unclear. This study investigated the role of transforming growth factor-beta (TGF-ß) in the therapeutic effect of hUCB-MSCs on AD. Small interfering RNA (siRNA)-mediated depletion of TGF-ß disrupted the therapeutic effect of hUCB-MSCs in a mouse model of AD by attenuating the beneficial changes in histopathology, mast cell infiltration, tumor necrosis factor-alpha (TNF-α) expression, and the serum IgE level. To confirm that hUCB-MSCs regulate secretion of TNF-α, we investigated whether they inhibit TNF-α secretion by activated LAD2 cells. Coculture with hUCB-MSCs significantly inhibited secretion of TNF-α by LAD2 cells. However, this effect was abolished by siRNA-mediated depletion of TGF-ß in hUCB-MSCs. TNF-α expression in activated LAD2 cells was regulated by the extracellular signal-related kinase signaling pathway and was suppressed by TGF-ß secreted from hUCB-MSCs. In addition, TGF-ß secreted by hUCB-MSCs inhibited maturation of B cells. Taken together, our findings suggest that TGF-ß plays a key role in the therapeutic effect of hUCB-MSCs on AD by regulating TNF-α in mast cells and maturation of B cells.

Finger drop sign as a new variant of acute motor axonal neuropathy.

We propose the finger drop sign as a new clinical variant of acute motor axonal neuropathy (AMAN) defined by immunological and radiological evidence. We identified eight consecutive patients who had AMAN. All of them developed prominent involvement of the finger extensors. We performed magnetic resonance imaging (MRI) of the extremity muscles and serological assays for antiganglioside antibodies and Campylobacter jejuni. Patients with AMAN showed characteristic and a markedly sustained weakness of the finger extensors with a distinctive pattern of the finger drop sign. Limb MRI revealed unevenly distributed abnormal signals in the muscles mainly innervated by the posterior interosseous nerve. All tested patients showed positivity for immunoglobulin G antibody against ganglioside complex of GM1 and phosphatidic acid. A pathophysiological understanding of this unique syndrome can provide further insight into antiganglioside-antibody-mediated axonal injury in Guillain-Barré syndrome.

Behind the pathology of macrophage-associated demyelination in inflammatory neuropathies: demyelinating Schwann cells.

In inflammatory peripheral demyelinating disorders, demyelination represents segmental demyelination in which the myelin sheath of a myelinating Schwann cell (SC) is completely removed by macrophages or a partial myelin degeneration in the paranode occurring due to autoantibodies attacking the node/paranode. For the segmental demyelination from living myelin-forming SCs, macrophages infiltrate within the endoneurium and insinuate between myelin lamellae and the cytoplasm of SCs, and the myelin is then removed via phagocytosis. During the macrophage invasion into the SC cytoplasm from the node of Ranvier and internodal areas, the attacked SCs do not remain quiescent but transdifferentiate into inflammatory demyelinating SCs (iDSCs), which exhibit unique demyelination pathologies, such as myelin uncompaction from Schmidt-Lanterman incisures with myelin lamellae degeneration. The longitudinal extension of this self-myelin clearance process of iDSCs into the nodal region is associated with the degeneration of nodal microvilli and paranodal loops, which provides a potential locus for macrophage infiltration. In addition to the nodal intrusion, macrophages appear to be able to invade fenestrated internodal plasma membrane or the degenerated outer mesaxon of iDSC. These SC demyelination morphologies indicate that the SC reprogramming to iDSCs may be a prerequisite for macrophage-mediated inflammatory demyelination. In contrast, paranodal demyelination caused by autoantibodies to nodal/paranodal antigens does not result in iDSC-dependent macrophage infiltration and subsequent segmental demyelination. In the context of inflammatory demyelination, the novel perspective of iDSCs provides an important viewpoint to understand the pathophysiology of demyelinating peripheral neuropathies and establish diagnostic and therapeutic strategies.

Involvement of the miR-363-5p/P2RX4 Axis in Regulating Schwann Cell Phenotype after Nerve Injury.

Although microRNAs (miRNAs or miRs) have been studied in the peripheral nervous system, their function in Schwann cells remains elusive. In this study, we performed a microRNA array analysis of cyclic adenosine monophosphate (cAMP)-induced differentiated primary Schwann cells. KEGG pathway enrichment analysis of the target genes showed that upregulated miRNAs (mR212-5p, miR335, miR20b-5p, miR146b-3p, and miR363-5p) were related to the calcium signaling pathway, regulation of actin cytoskeleton, retrograde endocannabinoid signaling, and central carbon metabolism in cancer. Several key factors, such as purinergic receptors (P2X), guanine nucleotide-binding protein G(olf) subunit alpha (GNAL), P2RX5, P2RX3, platelet-derived growth factor receptor alpha (PDGFRA), and inositol 1,4,5-trisphosphate receptor type 2 (ITPR2; calcium signaling pathway) are potential targets of miRNAs regulating cAMP. Our analysis revealed that miRNAs were differentially expressed in cAMP-treated Schwann cells; miRNA363-5p was upregulated and directly targeted the P2X purinoceptor 4 (P2RX4)-UTR, reducing the luciferase activity of P2RX4. The expression of miRNA363-5p was inhibited and the expression of P2RX4 was upregulated in sciatic nerve injury. In contrast, miRNA363-5p expression was upregulated and P2RX4 expression was downregulated during postnatal development. Of note, a P2RX4 antagonist counteracted myelin degradation after nerve injury and increased pERK and c-Jun expression. Interestingly, a P2RX4 antagonist increased the levels of miRNA363-5p. This study suggests that a double-negative feedback loop between miRNA363-5p and P2RX4 contributes to the dedifferentiation and migration of Schwann cells after nerve injury.

Discoidin Domain Receptor 2 Mediates Lysophosphatidic Acid-Induced Ovarian Cancer Aggressiveness.

Lysophosphatidic acid (LPA), a bioactive lipid produced extracellularly by autotaxin (ATX), has been known to induce various pathophysiological events, including cancer cell invasion and metastasis. Discoidin domain receptor 2 (DDR2) expression is upregulated in ovarian cancer tissues, and is closely associated with poor clinical outcomes in ovarian cancer patients. In the present study, we determined a critical role and signaling cascade for the expression of DDR2 in LPA-induced ovarian cancer cell invasion. We also found ectopic expression of ATX or stimulation of ovarian cancer cells with LPA-induced DDR2 expression. However, the silencing of DDR2 expression significantly inhibited ATX- and LPA-induced ovarian cancer cell invasion. In addition, treatment of the cells with pharmacological inhibitors of phosphoinositide 3-kinase (PI3K), Akt, and mTOR abrogated LPA-induced DDR2 expression. Moreover, we observed that HIF-1α, located downstream of the mTOR, is implicated in LPA-induced DDR2 expression and ovarian cancer cell invasion. Finally, we provide evidence that LPA-induced HIF-1α expression mediates Twist1 expression to upregulate DDR2 expression. Collectively, the present study demonstrates that ATX, and thereby LPA, induces DDR2 expression through the activation of the PI3K/Akt/mTOR/HIF-1α/Twist1 signaling axes, aggravating ovarian cancer cell invasion.

Loss-of-function of EBP50 is a new cause of hereditary peripheral neuropathy: EBP50 functions in peripheral nerve system.

Finding causative genetic mutations is important in the diagnosis and treatment of hereditary peripheral neuropathies. This study was conducted to find new genes involved in the pathophysiology of hereditary peripheral neuropathy. We identified a new mutation in the EBP50 gene, which is co-segregated with neuropathic phenotypes, including motor and sensory deficit in a family with Charcot-Marie-Tooth disease. EBP50 is known to be important for the formation of microvilli in epithelial cells, and the discovery of this gene mutation allowed us to study the function of EBP50 in the nervous system. EBP50 was strongly expressed in the nodal and paranodal regions of sciatic nerve fibers, where Schwann cell microvilli contact the axolemma, and at the growth tips of primary Schwann cells. In addition, EBP50 expression was decreased in mouse models of peripheral neuropathy. Knockout mice were used to study EBP50 function in the peripheral nervous system. Interestingly motor function deficit and abnormal histology of nerve fibers were observed in EBP50+/- heterozygous mice at 12 months of age, but not 3 months. in vitro studies using Schwann cells showed that NRG1-induced AKT activation and migration were significantly reduced in cells overexpressing the I325V mutant of EBP50 or cells with knocked-down EBP50 expression. In conclusion, we show for the first time that loss of function due to EBP50 gene deficiency or mutation can cause peripheral neuropathy.

Assessment of mitophagy in mt-Keima Drosophila revealed an essential role of the PINK1-Parkin pathway in mitophagy induction in vivo.

Mitophagy has been implicated in mitochondrial quality control and in various human diseases. However, the study of in vivo mitophagy remains limited. We previously explored in vivo mitophagy using a transgenic mouse expressing the mitochondria-targeted fluorescent protein Keima (mt-Keima). Here, we generated mt-Keima Drosophila to extend our efforts to study mitophagy in vivo. A series of experiments confirmed that mitophagy can be faithfully and quantitatively measured in mt-Keima Drosophila. We also showed that alterations in mitophagy upon environmental and genetic perturbation can be measured in mt-Keima Drosophila. Analysis of different tissues revealed a variation in basal mitophagy levels in Drosophila tissues. In addition, we found a significant increase in mitophagy levels during Drosophila embryogenesis. Importantly, loss-of-function genetic analysis demonstrated that the phosphatase and tensin homolog-induced putative kinase 1 (PINK1)-Parkin pathway is essential for the induction of mitophagy in vivo in response to hypoxic exposure and rotenone treatment. These studies showed that the mt-Keima Drosophila system is a useful tool for understanding the role and molecular mechanism of mitophagy in vivo. In addition, we demonstrated the essential role of the PINK1-Parkin pathway in mitophagy induction in response to mitochondrial dysfunction.-Kim, Y. Y., Um, J.-H., Yoon, J.-H., Kim, H., Lee, D.-Y., Lee, Y. J., Jee, H. J., Kim, Y. M., Jang, J. S., Jang, Y.-G., Chung, J., Park, H. T., Finkel, T., Koh, H., Yun, J. Assessment of mitophagy in mt-Keima Drosophila revealed an essential role of the PINK1-Parkin pathway in mitophagy induction in vivo.