Young Bin Im

MicroRNA 486 is a potentially novel target for the treatment of spinal cord injury

MicroRNAs have been shown to effectively regulate gene expression at the translational level. Recently, we identified novel microRNAs that were upregulated in a mouse model of spinal cord injury. Among those, we have focused on microRNA 486, which directly represses NeuroD6 expression through a conserved sequence in its untranslated region. We correlated the overexpression of microRNA 486 in motor neurons with a poor outcome due to progressive neurodegeneration and a pathophysiology that is mediated by reactive oxygen species. The expression of microRNA 486 was induced by reactive oxygen species that were produced by inflammatory factors, and reactive oxygen species were accumulated in response to the knockdown of NeuroD6, which enhances the downregulation of glutathione peroxidase 3 and thioredoxin-like 1 after traumatic spinal cord injury. NeuroD6 directly bound to regulatory regions of thioredoxin-like 1 and glutathione peroxidase 3 in motor neurons and activated their expression, which promoted reactive oxygen species scavenging. Moreover, knocking down microRNA 486 induced the expression of NeuroD6, which effectively ameliorated the spinal cord injury and allowed the mice to recover motor function. The infusion of exogenic NeuroD6 in spinal cord injury lesions effectively blocked apoptosis by reactivating thioredoxin-like 1 and glutathione peroxidase 3, which was accompanied by a recovery of motor function. Collectively, these findings have identified a novel microRNA in spinal cord injury lesions called microRNA 486, demonstrating a new role for NeuroD6 in neuroprotection, and suggest a potential therapeutic target for spinal cord injuries.

Silencing of miR20a is crucial for Ngn1-mediated neuroprotection in injured spinal cord.

MicroRNAs (miRNAs) compose a relatively new discipline in biomedical research, and many physiological processes in disease have been associated with changes in miRNA expression. Several studies report that miRNAs participate in biological processes such as the control of secondary injury in several disease models. Recently, we identified novel miRNAs that were abnormally up-regulated in a traumatic spinal cord injury (SCI). In the current study, we focused on miR20a, which causes continuing motor neuron degeneration when overexpressed in SCI lesions. Blocking miR20a in SCI animals led to neural cell survival and eventual neurogenesis with rescued expression of the key target gene, neurogenin 1 (Ngn1). Infusion of siNgn1 resulted in functional deficit in the hindlimbs caused by aggressive secondary injury and actively enhanced the inflammation involved in secondary injury progression. The events involving miR20a underlie motor neuron and myelin destruction and pathophysiology and ultimately block regeneration in injured spinal cords. Inhibition of miR20a expression effectively induced definitive motor neuron survival and neurogenesis, and SCI animals showed improved functional deficit. In this study, we showed that abnormal expression of miR20a induces secondary injury, which suggests that miR20a could be a potential target for therapeutic intervention following SCI.

miR23b ameliorates neuropathic pain in spinal cord by silencing NADPH oxidase 4.

AIMS Neuropathic pain is a well-known type of chronic pain caused by damage to the nervous system. Until recently, researchers have been primarily focused on identifying the cellular or chemical sources of neuropathic pain or have approached neuropathic pain via the basis of biological study. We investigated whether mmu-mir-23b (miR23b) infusion can alleviate pain by compensating for the abnormally downregulated miR23b by reducing the expression of its target gene, NADPH oxidase 4 (NOX4), a reactive oxygen species (ROS) family member overexpressed in neuropathic pain. RESULTS Ectopic miR23b expression effectively downregulated NOX4 and was normalized to GAD65/67 expression. Moreover, the animals with neuropathic pain showed significant improvements in the paw withdrawal thresholds following miR23b infusion. Normalizing miR23b expression in tissue lesions caused by neuropathic pain induction reduced inflammatory mediator expression and increased the level of several ROS scavengers. Moreover, GABAergic neurons coexpressed suboptimal levels of miR23b and elevated NOX4/ROS after pain induction at the cellular level. MiR23b protects GABAergic neurons against ROS/p38/JNK-mediated apoptotic death. By evaluating the functional behavior of the mice receiving pain/miR23b, normal/anti-miR23b, or anti-miR23b/si-NOX4, the positive role of miR23b and the negative role of NOX4 in neuropathic pain were confirmed. INNOVATION AND CONCLUSION Based on this study, we conclude that miR23b plays a crucial role in the amelioration of neuropathic pain in the injured spinal cord by inactivating its target gene, NOX4, and protecting GABAergic neurons from cell death. We finally suggest that miR23b may provide attractive diagnostic and therapeutic resources for effective pain modulation in neuropathic pain.

MBD6 is a direct target of Oct4 and controls the stemness and differentiation of adipose tissue-derived stem cells

Argonaute 2 (Ago2) is a pivotal regulator of cell fate in adult stem cells. Its expression is significantly downregulated in late passages of cells, concomitant with a prominent increase in Ago2 cytosolic localization in single cells. Nuclear localization of Ago2 is crucial for the survival, proliferation, and differentiation of hATSCs (human adipose tissue-derived stem cells), mediated by the specific binding of the regulatory regions of functional genes, which positively or negatively altered gene expression. Ago2 targets genes that control stemness, reactive oxygen species scavenging, and microRNA expression, all of which are crucial for hATSC survival and self-renewal. Ago2 promotes cell proliferation and self-renewal by activating the expression of octamer-binding transcription factor 4 (Oct4). We confirmed the direct regulation of Oct4 activity by Ago2, as indicated by the results of the ChIP analysis. Methyl-CpG-binding protein 6 (MBD6) was detected as an Oct4 regulatory gene. As predicted, knockdown of MBD6 expression attenuated cell proliferation and eventually induced cell death. We hypothesized that MBD6 functions downstream of Oct4 in the regulation of stemness-related genes, cell proliferation, self-renewal activity, and survival. MBD6 also promoted cell transdifferentiation into neural and endodermal β-cells while significantly attenuating differentiation into the mesodermal lineage. We demonstrate that MBD6 is regulated by Ago2 via an interaction with Oct4, which alters self-renewal and gene expression in hATSCs. MBD6 was promoted cell proliferation through a novel set of signal mediators that may influence differentiation by repressing MBD2 and MBD3, which are possibly recruited by germ cell nuclear factor (GCNF).

Evaluation of Th1/Th2-Related Immune Response against Recombinant Proteins of Brucella abortus Infection in Mice.

Brucellosis is a zoonotic disease caused by Brucella, a genus of gram-negative bacteria. Cytokines have key roles in the activation of innate and acquired immunities. Despite several research attempts to reveal the immune responses, the mechanism of Brucella infection remains unclear. Therefore, immune responses were analyzed in mice immunized with nine recombinant proteins. Cytokine production profiles were analyzed in the RAW 264.7 cells and naive splenocytes after stimulation with three recombinant proteins, metal-dependent hydrolase (r0628), bacterioferritin (rBfr), and thiamine transporter substrate-binding protein (rTbpA). Immune responses were analyzed by ELISA and ELISpot assay after immunization with proteins in mice. The production levels of NO, TNF-α, and IL-6 were time-dependently increased after having been stimulated with proteins in the RAW 264.7 cells. In naive splenocytes, the production of IFN-γ and IL-2 was increased after stimulation with the proteins. It was concluded that two recombinant proteins, r0628 and rTbpA, showed strong immunogenicity that was induced with Th1-related cytokines IFN-γ, IL-2, and TNF-α more than Th2-related cytokines IL-6, IL-4, and IL-5 in vitro. Conversely, a humoral immune response was activated by increasing the number of antigen-secreting cells specifically. Furthermore, these could be candidate diagnosis antigens for better understanding of brucellosis.

Argonaute2 Regulation for K+ Channel-Mediated Human Adipose Tissue-Derived Stromal Cells Self-Renewal and Survival in Nucleus

Argonaute2 (Ago2) is a well-known factor that has intrinsic endonuclease activity and is a part of the fundamental gene regulatory machinery. Recently, we showed that nuclear Ago2 regulates voltage-gated potassium (Kv) channels and that Ago2/Kv1.3 has crucial functions in the self-renewal and cell de-aging processes in adipose tissue-derived stromal cells (ATSCs). In the nucleus, Ago2 bound to the promoter regions of calcium-activated potassium channel 3, potassium channel subfamily K member 1 (KCNK1), and voltage-gated potassium channel 2, and the expression of these genes was significantly upregulated at the level of transcription. We detected an active K+ channel that plays a critical role in Ago2-mediated ATSC self-renewal through the control of membrane potential during cell self-renewal and differentiation. Among the several regulatory subunits of voltage-dependent K+ (Kv) channels, Kv1.3 and Kv1.5 have been shown to impact tissue differentiation and cell growth in cultured ATSCs following their direct binding to the regulatory region of the Kv channel gene. In ATSCs, interference with Ago2 or K+ channel gene expression or treatment with tetraethylammonium significantly downregulated stemness gene expression, induced cell cycle arrest, and inhibited the ability of cells to transdifferentiate into neurons or β-cells via Oct4 knockdown. Blockage of the K+ channel significantly induced protein kinase C (PKC) α, β, and δ phosphorylation and negatively affected Ago2 and Oct4 expression. This K+ channel blockage also resulted in the upregulation of p53 and p21 expression and the inactivation of mitogen-activated protein kinase (MEK), extracellular signal-regulated kinase 1/2 (ERK 1/2), AKT, β-catenin, and STAT3. Our results suggest that the nuclear Ago2 regulation of the K+ channel or stemness-related gene expression plays a critical role in adult stem cell self-renewal and differentiation.

Crucial Role of Nuclear Ago2 for hUCB-MSCs Differentiation and Self-Renewal via Stemness Control

AIMS Argonaute2 (Ago2) has intrinsic endonuclease activity in microRNA processing that plays a fundamental role in gene regulation. In this study, we demonstrate novel functions and molecular mechanisms of nuclear Ago2 in the self-renewal and plasticity of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs). RESULTS Nuclear Ago2 binds to a set of regulatory genes, including Ago2 itself, Oct4, Sox2, Nanog, GATA, STAT3, and β-catenin, that potentially target fundamental functions of stem cells. Direct regulation of the stemness genes by nuclear Ago2 was also crucial for cell self-renewal, survival, and differentiation into various types of tissues or cells, including neural cells and β-cells. Moreover, regulation of Oct4 by Ago2 directly controls the stem cell plasticity-determining signal mediators JAK2/STAT3 and Wnt5A/β-catenin and positively regulates cell proliferation and differentiation via blockage of ROS generation and P38/JNK inactivation. Nuclear Ago2 or stemness expression lead increased stem cell identity and decreased differentiation into a mesodermal lineage but also led to increased neural differentiation and β-cell differentiation in hUCB-MSCs. Nuclear Ago2-mediated stemness expression in hUCB-MSCs is also involved in cell survival, helping cells escape apoptotic cell death via inactivation of P38/JNK, caspase-3, and Bax. INNOVATION AND CONCLUSION This study reveals that nuclear Ago2 globally controls stem cell self-renewal and differentiation through direct regulation of stemness genes and important signal mediator activation following inactivation of ROS/P38/JNK and activation of the JAK/STAT3 and Wnt/ β-catenin signal pathways.

Expression of cytokine and apoptosis-related genes in bovine peripheral blood mononuclear cells stimulated with Brucella abortus recombinant proteins

Brucellosis is a clinically and economically important disease. Therefore, eradication programs of the disease have been implemented in several countries. One hurdle in these programs is the detection of infected animals at the early stage. Although the protein antigens as diagnostic antigens have recently received attention, the exact mechanisms at the beginning of immune responses are not yet known. Therefore, genes encoding five B. abortus cellular proteins were cloned and the expressed recombinant proteins were purified. The expression of several cytokine genes (IL-1β, IL-4, IL-6, IL-12p40, IFN-γ, TNF-α, and iNOS) was analyzed in bovine peripheral blood mononuclear cells (bPBMC) after stimulation with the recombinant proteins. Three apoptosis-related genes, Bax, Bcl-2, and TLR4, were also included in the analysis to find out the adverse effects of the proteins to the cells. Each protein induced different patterns of cytokine expression depending on the stimulation time and antigen dose. Expression of IL-6, IL-12p40, and IFN-γ was induced with all of the proteins while IL-1β, IL-4, TNF-α, and iNOS gene expression was not. Expression of apoptosis-related genes was not altered except TLR4. These results suggest that the cellular antigens of B. abortus induce both humoral and cellular immunity via the production of IL-6, IL-12p40, and IFN-γ in bPBMC without exerting any adverse effects on the cells.

Novel, small molecule induced GABA-hATSCs for targeting of neuropathic pain.

Recent study showed that ROS has a crucial function during neuropathic pain development and maintenance. In this study, we suggest that a small, novel molecule, CMB-1078, can effectively induce GABAergic neuronal differentiation from human adipose tissue-derived stromal cells (hATSCs; GABA-hATSCs), which play a key role in ameliorating neuropathic pain caused by spinal cord injury. Compared to control hATSCs, the engraftment of GABA-hATSCs into animals with neuropathic pain significantly reduced secondary injury, including inflammation, GABAergic neuronal degeneration, and the circulation or propagation of proinflammatory factors cyclooxygenase2 (COX2), interlukin-1 β (IL-1β), NADPH oxidase 2 (NOX 2), NADPH oxidase 4 (NOX 4) and tumor necrosis factor α (TNFα) into the lesion. At the protein level, we also demonstrated that GABA-hATSCs engrafted into animals with neuropathic pain increased glutamic acid decarboxylase 65 (GAD65) and glutamic acid decarboxylase 67 (GAD67) expression levels. In addition, we evaluated functional pain behavior in the GABA-hATSCs- or control hATSCs-engrafted animal group, the pain in the PBS-infused animal group, and healthy animals by measuring mechanical and heat sensitivity. The pain plus GABA-hATSCs-engrafted animal groups showed paw withdrawal thresholds (PWTs) that gradually improved. In contrast, the mice with neuropathic pain did not show improved PWT. Further, the control hATSCs-engrafted animal showed attenuated PWTs. Finally, we suggest that the molecular function of GABA-hATSCs in neuropathic pain may provide potential therapeutic tools for the treatment of pain by controlling the pathology of neuropathic pain through neuroprotection and regeneration.

Cytokines production and toll-like receptors expression in human leukemic monocyte cells, THP-1, stimulated with Brucella abortus cellular antigens

A zoonotic pathogen, Brucella spp. is the causative agent of brucellosis, which results in abortion and loss in milk production in domestic animals, and undulant fever, osteoarticular pain and splenomegaly in humans. Due to the capability of the bacteria to modulate the host cell functions and survive in macrophages, early detection and eradication of the intracellular bacteria has received significant attention. Moreover, understanding the immunological alterations in Brucella infection is crucial to help develop control measures. Cytokines and toll-like receptors (TLRs) are some of the major compounds that play important roles in modulating the innate immunity and acquired immunity in host after infection. In this study, therefore, human leukemic monocyte cells (THP-1 cells) were stimulated with five Brucella abortus cellular components: outer membrane protein 10 (OMP10), outer membrane protein 19 (OMP19), thiamine transporter substrate-binding protein (TbpA), arginase (RocF), and malate dehydrogenase (Mdh). Post stimulation, the cytokine productions and TLR expressions in the cells were evaluated at different time points (12 h and 24 h), and analyzed using ELISA and real time RT-PCR, respectively. In the production of cytokines, it was observed that the production of TNF-α and IL-6 was highly induced in THP-1 cells stimulated with five recombinant protein antigens. Also, TLR8 was induced in a time-dependent manner after stimulation with two recombinant proteins, rOMP19 and rMdh, until 24 h. These results suggest that the two B. abortus antigens, rOMP19 and rMdh, might be involved in TLR8 signaling pathway in THP-1 cells in a time-dependent manner. These two proteins are therefore potentially effective antigen candidates which would help to provide better understandings of the immune responses after Brucella infection.