A Single Injection of rAAV-shmTOR in Peripheral Nerve Persistently Attenuates Nerve Injury-Induced Mechanical Allodynia.

Activation of mammalian target of rapamycin (mTOR) has been known as one of the contributing factors in nociceptive sensitization after peripheral injury. Its activation followed by the phosphorylation of downstream effectors causes hyperexcitability of primary sensory neurons in the dorsal root ganglion. We investigated whether a single injection of rAAV-shmTOR would effectively downregulate both complexes of mTOR in the long-term and glial activation as well. Male SD rats were categorized into shmTOR (n = 29), shCON (n = 23), SNI (n = 13), and Normal (n = 8) groups. Treatment groups were injected with rAAV-shmTOR or rAAV-shCON, respectively. DRG tissues and sciatic nerve were harvested for Western blot and immunohistochemical analyses. Peripheral sensitization was gradually attenuated in the shmTOR group, and it reached a peak on PID 21. Western blot analysis showed that both p-mTORC1 and p-mTORC2 were downregulated in the DRG compared to shCON and SNI groups. We also found decreased expression of phosphorylated p38 and microglial activation in the DRG. We first attempted a therapeutic strategy for neuropathic pain with a low dose of AAV injection by interfering with the mTOR signaling pathway, suggesting its potential application in pain treatment.

Negative Regulation of IKKε-Mediated IRF7 Phosphorylation by HSP70.

Immune reactions are controlled by the delicate spatiotemporal orchestration of multiple cells communicating by cytokines. Studies of cytokines that began with the discovery of IFN focused on positive regulatory mechanisms that induce secretion in response to harmful stimuli. However, there is a growing awareness that negative regulatory mechanisms that stop secretion of cytokines at specific times and spaces are also important for a successful immune reaction. Type I IFN is the primary cytokine in innate immunity. Although its induction is a prerequisite for the consequent adaptive immune reaction, its oversecretion can cause destructive tissue damage. IFN regulatory factor 7 (IRF7) is a master transcription factor of type I IFN, and multiple observations indicate the key role of IRF7 and the importance of its negative regulation. In this study, we found that the inducible heat shock protein 70 (HSP70) regulated the early type I IFN response by using mice knockout for HSP70. HSP70 dampened IRF7 activation; the inhibitory effect of HSP70 over IKKε-mediated IRF7 activation originated from simple competitive binding. This suggests the possibility of blocking the feed-forward loop between IRF7 and type I IFN in stress environments with increased expression of HSP70.

Correlative Light and Electron Microscopy for Nanoparticle-Cell Interaction and Protein Localization.

Various silica-based fluorescent nanoparticles ((Si-FNP)) with magnetic or metal cores represent a standard class of nanoparticles offering new opportunities for high-resolution cellular imaging and biomedicine applications, such as drug delivery. Their high solubility, homogeneity, biocompatibility, and chemical inertness Si-FNPs make them attractive probes for correlative light and electron microscopy (CLEM) studies, offering novel insights into nanoparticle-cell interactions in detail. In the present chapter, we present a procedure for imaging silica-based fluorescent magnetic core-shell nanoparticles (Si-FMNP) at the single-particle scale in cells. Our method facilitates the acquisition of information on the extracellular and intercellular distribution of nanoparticles and their various interactions with various cellular organelles when cells are cultured and electroporated by NPs. In addition, such information could facilitate the evaluation of the efficacy of nanocarriers designed for drug delivery.

CXCL10 production induced by high levels of IKKε in nasal airway epithelial cells in the setting of chronic inflammation.

The airway is the major entry route of pathogens due to continuous gas exchange with the environment. In particular, the nasal epithelial layer is the common site of airborne mucotropic virus infections. The inflammatory response to such infections must be tightly controlled due to its non-specific nature. Unrestrained inflammation breaks down the physiological mucosal defense system and leads to secondary bacterial or fungal infections. Chronic rhinosinusitis (CRS) is a prevalent inflammatory disease that compromises quality of life. In spite of its importance in the initiation of inflammation, the role of interferon signaling in nasal airway epithelial cells is largely unknown. We analyzed the expression of interferon signaling genes using clinical lavage specimens and nasal airway epithelial cells collected from CRS patients and controls. Basal expression of IFNAs, IKBKE, STAT1, and some CXC chemokines was elevated in samples from CRS patients. In subsequent in vitro studies, we found IKKε to be the key molecule and augmented CXCL10 secretion. Based on our findings and review of the literature, we hypothesized that high levels of IKKε might induce intractable inflammation via CXCL10. We tested the hypothesis in an animal model and found not only that IKKε induced severe eosinophilic inflammation with CXCL10 over-production, but also that inhibition of IKKε resolved the inflammation.

MicroRNA expression profiling of adult hippocampal neural stem cells upon cell death reveals an autophagic cell death-like pattern.

Adult hippocampal neural (HCN) stem cells promptly undergo irreversible autophagic cell death (ACD) if deprived of insulin in culture. Small, non-coding microRNAs (miRNA) play an important role in regulating biological processes, including proliferation and cell death. However, there have been no reports thus far regarding miRNA involvement in the induction of adult HCN stem cell death under insulin-deprived conditions, for which we performed a microarray-based analysis to examine the expression signature of miRNAs in adult rat HCN stem cells. Three independent specimens per culture condition either with or without insulin were prepared and a miRNA microarray analysis carried out. A total of 12 exhibited significantly altered expression levels upon cell death due to the absence of insulin when compared to HCN stem cells cultured with insulin present (cut-off limit; p < 0.05 and fold-change >1.3) The resulting volcano plot showed that, among these miRNAs, seven were upregulated and five were downregulated. The upregulated miRNAs were capable of modulating HCN stem cell death. Caspase-3 activity analysis, LC3 conversion, and TEM of autophagosome formation consistently suggested that ACD, not apoptosis, was most likely the mechanism affecting HCN cell death. As such, we have come to term these miRNAs, "HCN stem cell-specific autophagic cell death regulators." Taken together, our data suggest that the miRNA expression profile of HCN stem cells is altered during ACD occurring due to insulin deprivation and that differentially expressed miRNAs are involved in HCN stem cell viability. Detailed explorations of the underlying mechanisms regarding HCN stem cell viability modulation by these miRNAs would be beneficial in further understanding the physiological features of adult HCN stem cells and are currently being investigated.

Attenuation of natural killer cell functions by capsaicin through a direct and TRPV1-independent mechanism.

The assessment of the biological activity of capsaicin, the compound responsible for the spicy flavor of chili pepper, produced controversial results, showing either carcinogenicity or cancer prevention. The innate immune system plays a pivotal role in cancer pathology and prevention; yet, the effect of capsaicin on natural killer (NK) cells, which function in cancer surveillance, is unclear. This study found that capsaicin inhibited NK cell-mediated cytotoxicity and cytokine production (interferon-γ and tumor necrosis factor-α). Capsaicin impaired the cytotoxicity of NK cells, thereby inhibiting lysis of standard target cells and gastric cancer cells by modulating calcium mobilization in NK cells. Capsaicin also induced apoptosis in gastric cancer cells, but that effect required higher concentrations and longer exposure times than those required to trigger NK cell dysfunction. Furthermore, capsaicin inhibited the cytotoxicity of isolated NK cells and of an NK cell line, suggesting a direct effect on NK cells. Antagonists of transient receptor potential vanilloid subfamily member 1 (TRPV1), a cognate capsaicin receptor, or deficiency in TRPV1 expression failed to prevent the defects induced by capsaicin in NK cells expressing functional TRPV1. Thus, the mechanism of action of capsaicin on NK cells is largely independent of TRPV1. Taken together, capsaicin may have chemotherapeutic potential but may impair NK cell function, which plays a central role in tumor surveillance.

Suppression of stent-induced tissue hyperplasia in rats by using small interfering RNA to target matrix metalloproteinase-9.

BACKGROUND AND STUDY AIMS: We evaluated the efficacy of small interfering RNA (siRNA) in targeting matrix metalloproteinase (MMP-9) to suppress stent-induced tissue hyperplasia in a rat esophageal model. METHODS: The silencing effect of the candidate siRNA (termed (MMP-9 siRNA) was evaluated in 9 L rat glial cells. Four groups of rats (n = 10, each group) were used: Eso-S, stent insertion only, comparison; Eso-R, stent insertion plus treatment with MMP-9 siRNA complexed with Chol-R9 for delivery, experimental; Eso-P, stent insertion plus treatment with pCMV-luc complexed with Chol-R9, for confirmation of Chol-R9 delivery effect; and Eso-N, no stent insertion and no treatment, controls. All rats were sacrificed at 3 weeks. The therapeutic efficacy of the MMP-9 siRNA/Chol-R9 complex was assessed. RESULTS: The most potent MMP-9 siRNA was selected. Compared with the Eso-S group, the Eso-R group showed significantly less tissue hyperplasia with a lower percentage of granulation tissue and smaller granulation tissue area, and also significantly lower MMP-9 level. CONCLUSIONS: MMP-9 siRNA/Chol-R9 is effective for inhibiting stent-induced tissue hyperplasia in a rat esophageal model.

Multispecies-compatible antitumor effects of a cross-species small-interfering RNA against mammalian target of rapamycin.

Successful development of sequence-specific siRNA (small interfering RNA)-based drugs requires an siRNA design that functions consistently in different organisms. Utilizing the CAPSID program previously developed by our group, we here designed siRNAs against mammalian target of rapamycin (mTOR) that are entirely complementary among various species and investigated their multispecies-compatible gene-silencing properties. The mTOR siRNAs markedly reduced mTOR expression at both the mRNA and protein levels in human, mouse, and monkey cell lines. The reduction in mTOR expression resulted in inactivation of both mTOR complex I and II signaling pathways, as confirmed by reduced phosphorylation of p70S6K (70-kDa ribosomal protein S6 kinase), 4EBP1 (eIF4E-binding protein 1), and AKT, and nuclear accumulation of FOXO1 (forkhead box O1), with consequent cell-cycle arrest, proliferation inhibition, and autophagy activation. Moreover, interfering with mTOR activity in vivo using mTOR small-hairpin RNA-expressing recombinant adeno-associated virus led to significant antitumor effects in xenograft and allograft models. Thus, the present study demonstrates that cross-species siRNA successfully silences its target and readily produces multispecies-compatible phenotypic alterations-antitumor effects in the case of mTOR siRNA. Application of cross-species siRNA should greatly facilitate the development of siRNA-based therapeutic agents.

Integrative analysis of microRNA-mediated mitochondrial dysfunction in hippocampal neural progenitor cell death in relation with Alzheimer's disease

Adult hippocampal neurogenesis plays a pivotal role in maintaining cognitive brain function; however, this process diminishes with age, particularly in patients with neurodegenerative disorders. While small, non-coding microRNAs (miRNAs) are crucial for hippocampal neural stem (HCN) cell maintenance, their involvement in neurodegenerative disorders remains unclear. This study aims to elucidate the mechanisms through which miRNAs regulate HCN cell death and their potential involvement in neurodegenerative disorder. We performed a comprehensive microarray-based analysis to investigate changes in miRNA expression in insulin-deprived HCN cells, as an in vitro model for cognitive impairment. Remarkably, miR-150-3p, miR-323-5p, and miR-370-3p which increased significantly over time following insulin withdrawal, induced pronounced mitochondrial fission and dysfunction, ultimately leading to HCN cell death. Notably, these miRNAs collectively target the mitochondrial fusion protein OPA1, with miR-150-3p also targeting MFN2. Furthermore, data-driven analyses involving human subjects within the hippocampus and brain revealed significant reductions of OPA1 and MFN2 in the Alzheimer's disease (AD) patients. Our results indicate that miR-150-3p, miR-323-5p, and miR-370-3p contribute to deficits in hippocampal neurogenesis by modulating mitochondrial dynamics. Our findings provide a novel insight into the intricate connection between miRNAs and mitochondrial dynamics, shedding light on their potential involvement in conditions characterized by deficits in hippocampal neurogenesis, such as AD.

Antiviral effects of small interfering RNA simultaneously inducing RNA interference and type 1 interferon in coxsackievirus myocarditis.

Antiviral therapeutics are currently unavailable for treatment of coxsackievirus B3, which can cause life-threatening myocarditis. A modified small interfering RNA (siRNA) containing 5'-triphosphate, 3p-siRNA, was shown to induce RNA interference and interferon activation. We aimed to develop a potent antiviral treatment using CVB3-specific 3p-siRNA and to understand its underlying mechanisms. Virus-specific 3p-siRNA was superior to both conventional virus-specific siRNA with an empty hydroxyl group at the 5' end (OH-siRNA) and nonspecific 3p-siRNA in decreasing viral replication and subsequent cytotoxicity. A single administration of 3p-siRNA dramatically attenuated virus-associated pathological symptoms in mice with no signs of toxicity, and their body weights eventually reached the normal range. Myocardial inflammation and fibrosis were rare, and virus production was greatly reduced. A nonspecific 3p-siRNA showed relatively less protective effect under identical conditions, and a virus-specific OH-siRNA showed no protective effects. We confirmed that virus-specific 3p-siRNA simultaneously activated target-specific gene silencing and type I interferon signaling. We provide a clear proof of concept that coxsackievirus B3-specific 3p-siRNA has 2 distinct modes of action, which significantly enhance antiviral activities with minimal organ damage. This is the first direct demonstration of improved antiviral effects with an immunostimulatory virus-specific siRNA in coxsackievirus myocarditis, and this method could be applied to many virus-related diseases.

Characterization of infections of human leukocytes by non-polio enteroviruses.

To elucidate the detailed susceptibilities of leukocytes to clinically important non-polio enteroviruses (EVs), primary monocytes and various human leukocyte cell lines were infected with coxsackievirus A24 (CVA24), coxsackievirus B3 (CVB3), and enterovirus 70 (EV70). The permissiveness was then assessed by determining virus replication and resultant cytopathic effects. Different EVs varied markedly in their ability to infect leukocyte cell lines. CVB3 replicated effectively in leukocytes of B-cell, T-cell, and monocyte origin, CVA24 in leukocytes of B-cell and monocyte origin, and EV70 in leukocytes of monocyte origin. Primary monocytes, as well as monocyte-derived U-937 cells, were permissive to all three EVs. We observed a positive correlation between cytotoxicity and active virus replication, except in CVB3-infected monocytes. U-937 cells efficiently generated CVB3 progeny virus without severe cellular damage, including cell death. Moreover, infectivity on leukocytes was not absolutely associated with the availability of viral receptors. These findings suggest that the susceptibility of human leukocytes to non-polio EVs may be responsible for virus transport during the viremic phase, particularly to secondary target organs, and that active replication of CVB3 in all human leukocyte lineages leads to greater dissemination, in agreement with the ability of CVB to cause systemic diseases.

Evaluation of the incidence of esophageal complications associated with balloon dilation and their management in patients with malignant esophageal strictures.

OBJECTIVE: The objective of this study was to investigate the incidence of esophageal complications associated with balloon dilation and their management in patients with malignant esophageal strictures. MATERIALS AND METHODS: Fluoroscopically guided esophageal balloon dilation was performed in 89 patients with malignant esophageal strictures during a period of 15 years. Inclusion criteria were patients with unresected esophageal or gastric carcinoma showing short-segment stricture (≤4 cm) at the esophagogastric junction; patients who had previously received chemotherapy, radiation therapy, or both to manage malignant strictures; or patients who were scheduled for chemotherapy or radiation therapy to manage malignant strictures. Of these patients, 72 had esophageal cancer and 17 had stomach cancer. Esophageal rupture was categorized as intramural, transmural, or transmural with mediastinal leakage. RESULTS: A total of 120 procedures were performed, with each patient undergoing one to four procedures. Esophageal rupture occurred in 13 patients (15%): eight with intramural rupture, four with transmural rupture, and one with transmural rupture with mediastinal leakage. Improvements in dysphagia score were observed in 76 of 89 patients (85%) after balloon dilation. All esophageal ruptures were detected immediately after the procedure. Intramural and transmural ruptures were treated conservatively, whereas transmural rupture with mediastinal leakage was treated by temporary stent placement. CONCLUSION: The overall prevalence of esophageal rupture was 15%. All intramural and transmural ruptures were successfully managed conservatively, whereas transmural rupture with mediastinal leakage was treated by temporary stent placement. We found no relationship between rupture incidence and balloon diameter.

Polytetrafluoroethylene-covered retrievable expandable nitinol stents for malignant esophageal obstructions: factors influencing the outcome of 270 patients.

OBJECTIVE: The purpose of this study was to evaluate the clinical effectiveness of polytetrafluoroethylene (PTFE)-covered retrievable expandable nitinol stents in patients with malignant esophageal strictures and to identify prognostic factors associated with clinical outcomes. MATERIALS AND METHODS: From 2001 to 2010, 320 PTFE-covered stents were placed in 270 patients. Technical and clinical success, complications, survival, and stent patency were measures of clinical effectiveness. The relationships among complications and age, sex, stricture location, stricture length, chemotherapy alone, chemoradiotherapy, and malignancy source were examined. Independent prognostic factors of overall survival and stent patency were identified. RESULTS: Stent placement and removal were technically successful and tolerated without procedural complications, and 98% of patients achieved clinical success. The complication rate was 30%. Two removed stents exhibited covering membrane separation. Chemotherapy was associated with increased stent migration (p = 0.002). Stricture location and chemoradiotherapy were associated with esophagorespiratory fistula development (p = 0.033 and p < 0.001, respectively). Median and mean survival periods were 114 days (95% CI, 102-126 days) and 166 days (138-193 days). Chemotherapy and chemoradiotherapy were independent prognostic factors for survival (p = 0.050 and p = 0.032, respectively). The median and mean stent patency periods were 60 days (41-79 days) and 90 days (71-108 days). Chemoradiotherapy was the only independent prognostic factor for stent patency (p = 0.012). CONCLUSION: The PTFE-covered stents were clinically effective. Membrane degradation was not evident, although 0.7% of the patients experienced covering membrane separation. Chemotherapy was associated with increased migration and prolonged survival. Chemoradiotherapy was associated with increased esophagorespiratory fistula formation and decreased stent patency.

Microglial deactivation by adeno-associated virus expressing small-hairpin GCH1 has protective effects against neuropathic pain development in a spinothalamic tract-lesion model.

AIMS: Neuropathic pain after spinal cord injury is one of the most difficult clinical problems after the loss of mobility, and pharmacological or neuromodulation therapy showed limited efficacy. In this study, we examine the possibility of pain modulation by a recombinant adeno-associated virus (rAAV) encoding small-hairpin RNA against GCH1 (rAAV-shGCH1) in a spinal cord injury model in which neuropathic pain was induced by a spinothalamic tract (STT) lesion. METHODS: Micro-electric lesioning was used to damage the left STT in rats (n = 32), and either rAAV-shGCH1 (n = 19) or rAAV control (n = 6) was injected into the dorsal horn of the rats at the same time. On postoperative days 3, 7, and 14, we evaluated neuropathic pain using a behavioral test and microglial activation by immunohistochemical staining. RESULTS: A pain modulation effect of shGCH1 was observed from postoperative days 3 to 14. The mechanical withdrawal threshold was 13.0 ± 0.95 in the shGCH1 group, 4.3 ± 1.37 in the control group, and 3.49 ± 0.85 in sham on postoperative day 3 (p < 0.0001) and continued to postoperative day 14 (shGCH1 vs. control: 11.4 ± 1.1 vs. 2.05 ± 0.60, p < 0.001 and shGCH1 vs. sham: 11.4 ± 1.1 vs. 1.43 ± 0.54, p < 0.001). Immunohistochemical staining of the spinal cord dorsal horn showed deactivation of microglia in the shGCH1 group without any change of delayed pattern of astrocyte activation as in STT model. CONCLUSIONS: Neuropathic pain after spinal cord injury can be modulated bilaterally by deactivating microglial activation after a unilateral injection of rAAV-shGCH1 into the dorsal horn of a STT lesion spinal cord pain model. This new attempt would be another therapeutic approach for NP after SCI, which once happens; there is no clear curative options still now.

mTOR inhibition as a novel gene therapeutic strategy for diabetic retinopathy.

In addition to laser photocoagulation, therapeutic interventions for diabetic retinopathy (DR) have heretofore consisted of anti-VEGF drugs, which, besides drawbacks inherent to the treatments themselves, are limited in scope and may not fully address the condition's complex pathophysiology. This is because DR is a multifactorial condition, meaning a gene therapy focused on a target with broader effects, such as the mechanistic target of rapamycin (mTOR), may prove to be the solution in overcoming these concerns. Having previously demonstrated the potential of a mTOR-inhibiting shRNA packaged in a recombinant adeno-associated virus to address a variety of angiogenic retinal diseases, here we explore the effects of rAAV2-shmTOR-SD in a streptozotocin-induced diabetic mouse model. Delivered via intravitreal injection, the therapeutic efficacy of the virus vector upon early DR processes was examined. rAAV2-shmTOR-SD effectively transduced mouse retinas and therein downregulated mTOR expression, which was elevated in sham-treated and control shRNA-injected (rAAV2-shCon-SD) control groups. mTOR inhibition additionally led to marked reductions in pericyte loss, acellular capillary formation, vascular permeability, and retinal cell layer thinning, processes that contribute to DR progression. Immunohistochemistry showed that rAAV2-shmTOR-SD decreased ganglion cell loss and pathogenic Müller cell activation and proliferation, while also having anti-apoptotic activity, with these effects suggesting the therapeutic virus vector may be neuroprotective. Taken together, these results build upon our previous work to demonstrate the broad ability of rAAV2-shmTOR-SD to address aspects of DR pathophysiology further evidencing its potential as a human gene therapeutic strategy for DR.

AAV expressing an mTOR-inhibiting siRNA exhibits therapeutic potential in retinal vascular disorders by preserving endothelial integrity.

Expanding on previous demonstrations of the therapeutic effects of adeno-associated virus (AAV) carrying small-hairpin RNA (shRNA) in downregulating the mechanistic target of rapamycin (mTOR) in in vivo retinal vascular disorders, vascular endothelial growth factor (VEGF)-stimulated endothelial cells were treated with AAV2-shmTOR to examine the role of mTOR inhibition in retinal angiogenesis. AAV2-shmTOR exposure significantly reduced mTOR expression in human umbilical vein endothelial cells (HUVECs) and decreased downstream signaling cascades of mTOR complex 1 (mTORC1) and mTORC2 under VEGF treatment. Moreover, the angiogenic potential of VEGF was significantly inhibited by AAV2-shmTOR, which preserved endothelial integrity by maintaining tight junctions between HUVECs. These data thus support previous in vivo studies and provide evidence that AAV2-shmTOR induces therapeutic effects by inhibiting the neovascularization of endothelial cells.

An ex vivo model of coxsackievirus infection using multilayered human conjunctival epithelial cells.

BACKGROUND: The purpose of this study was to establish an ex vivo model of coxsackievirus infection since there seems to be no suitable disease model currently. METHODS: Human conjunctival epithelial cells (HCECs) were cultured for 2 weeks in a serum-free air-liquid interface system to produce a multilayered structure. The cells were infected with coxsackievirus A24 (CVA24). Histological changes were investigated by staining the cells with H&E and DAPI, and apoptosis was evaluated using the TUNEL technique. Virus replication was measured in HeLa cells infected with viral progeny from multilayered HCECs, after 1 and 3 days, using the TCID(50) method. RESULTS: Cultured HCECs formed multiple layers. The cells showed characteristics of conjunctival epithelial cells and goblet cells, being immunohistochemically positive for CK19 and MUC5AC, respectively. CVA24 replicated readily in cultured multilayered HCECs. A mild cytopathic effect was noted 1 day after viral inoculation. Cell damage was extensive at 3 days. TUNEL imaging confirmed that the cytopathology was attributable to apoptosis. The TCID(50) data from HeLa cells indicated that the virus was actively replicating at both 1 and 3 days after inoculation. CONCLUSIONS: This novel infection model may be useful in investigating the pathogenesis of acute hemorrhagic conjunctivitis and the effectiveness of antiviral treatments.

Optimal Ratio of Wnt3a Expression in Human Mesenchymal Stem Cells Promotes Axonal Regeneration in Spinal Cord Injured Rat Model.

OBJECTIVE: Through our previous clinical trials, the demonstrated therapeutic effects of MSC in chronic spinal cord injury (SCI) were found to be not sufficient. Therefore, the need to develop stem cell agent with enhanced efficacy is increased. We transplanted enhanced Wnt3asecreting human mesenchymal stem cells (hMSC) into injured spines at 6 weeks after SCI to improve axonal regeneration in a rat model of chronic SCI. We hypothesized that enhanced Wnt3a protein expression could augment neuro-regeneration after SCI. METHODS: Thirty-six Sprague-Dawley rats were injured using an Infinite Horizon (IH) impactor at the T9-10 vertebrae and separated into five groups : 1) phosphate-buffered saline injection (injury only group, n=7); 2) hMSC transplantation (MSC, n=7); 3) hMSC transfected with pLenti vector (without Wnt3a gene) transplantation (pLenti-MSC, n=7); 4) hMSC transfected with Wnt3a gene transplantation (Wnt3a-MSC, n=7); and 5) hMSC transfected with enhanced Wnt3a gene (1.7 fold Wnt3a mRNA expression) transplantation (1.7 Wnt3a-MSC, n=8). Six weeks after SCI, each 5×105 cells/15 µL at 2 points were injected using stereotactic and microsyringe pump. To evaluate functional recovery from SCI, rats underwent Basso-Beattie-Bresnahan (BBB) locomotor test on the first, second, and third days post-injury and then weekly for 14 weeks. Axonal regeneration was assessed using growth-associated protein 43 (GAP43), microtubule-associated protein 2 (MAP2), and neurofilament (NF) immunostaining. RESULTS: Fourteen weeks after injury (8 weeks after transplantation), BBB score of the 1.7 Wnt3a-MSC group (15.0±0.28) was significantly higher than that of the injury only (10.0±0.48), MSC (12.57±0.48), pLenti-MSC (12.42±0.48), and Wnt3a-MSC (13.71±0.61) groups (p<0.05). Immunostaining revealed increased expression of axonal regeneration markers GAP43, MAP2, and NF in the Wnt3a-MSC and 1.7 Wnt3a-MSC groups. CONCLUSION: Our results showed that enhanced gene expression of Wnt3a in hMSC can potentiate axonal regeneration and improve functional recovery in a rat model of chronic SCI.

miR-351-5p/Miro2 axis contributes to hippocampal neural progenitor cell death via unbalanced mitochondrial fission.

Adult hippocampal neurogenesis supports the structural and functional plasticity of the brain, while its decline is associated with neurodegeneration common in Alzheimer's disease (AD). Although the dysregulation of certain microRNAs (miRNAs) in AD have been observed, the effects of miRNAs on hippocampal neurogenesis are largely unknown. In this study, we demonstrated miR-351-5p as a causative factor in hippocampal neural progenitor cell death through modulation of the mitochondrial guanosine triphosphatase (GTPase), Miro2. Downregulation of Miro2 by siMiro2 induced cell death, similar to miR-351-5p, whereas ectopic Miro2 expression using an adenovirus abolished these effects. Excessively fragmented mitochondria and dysfunctional mitochondria were indexed by decreased mitochondrial potential, and increased reactive oxygen species were identified in miR-351-5p-induced cell death. Moreover, subsequent induction of mitophagy via Pink1 and Parkin was observed in the presence of miR-351-5p and siMiro2. The suppression of mitochondrial fission by Mdivi-1 completely inhibited cell death by miR-351-5p. miR-351-5p expression increased whereas the level of Miro2 decreased in the hippocampus of AD model mice, emulating expression in AD patients. Collectively, the data indicate the mitochondrial fission and accompanying mitophagy by miR-351-5p/Miro2 axis as critical in hippocampal neural progenitor cell death, and a potential therapeutic target in AD.

Gene therapeutic approach for inhibiting hepatitis C virus replication using a recombinant protein that controls interferon expression.

The hepatitis C virus (HCV) is a continuing threat to public health. The systemic administration of interferon alpha with ribavirin is the only currently approved treatment. However, this treatment is associated with a wide spectrum of systemic side effects that limits its effectiveness; thus, there is an urgent need for new treatment modalities. In this study, we describe a novel anti-HCV strategy employing a recombinant transcription factor that we have engineered in such a way that NS3/4a viral protease controls its intracellular localization, thereby restoring interferon secretion specifically in cells infected with HCV. Proof-of-concept experiments validated the strategy, showing that the recombinant transcription factor was triggered to stimulate interferon promoter by NS3/4A and remained inactive in cells without NS3/4a. Using an adenovirus-associated viral vector delivery system, we found that the recombinant transcription factor inhibited HCV replication effectively in vitro in cultured cells.