The role of ASK1 in selective striatal lesion formation induced by neuronal injury

Dept. of Medical Science/박사Apoptosis signal-regulating kinase-1 (ASK1), an early signaling element in the cell death pathway, has been suggested to participate in the pathology of neurodegenerative diseases, which may be associated with environmental factors that impact the diseases. The systemic administration of 3-nitropropionic acid (3-NP) facilitates the development of selected striatal lesions and it remains unclear whether specific neurons are selectively targeted in 3-NP infused striatal degeneration. Although not entirely elucidated, the mechanisms of neurotoxicity induced by 3-NP have been shown to include the exhaustion of adenosine triphosphate, mitochondrial membrane depolarization, dysregulation of intracellular calcium homeostasis, calpain activation, and the release of pro-apoptotic proteins from mitochondria. The present study is to characterize the regulation of BDNF in each cortical and striatal subregion. This study investigates that mild and chronic exposure of mitochondrial toxin can modulate the C1q level both in cortex and striatum via regulation of TGF-beta from astrocyte. Consequently we investigate how the BDNF is dominantly depleted in striatum, and eventually whether striatal lesion is established in involving in ASK1 pathway.The results of the present work show an alteration of ASK1 pathway molecules, TGF-beta, C1q level, and BDNF level as a final standard to striatal degeneration. By ASK1 down-regulation, improvement in each molecules containing behavioral impairment was evaluated in 3NP- infused mice and 3-NP treated primary neuronal cells. We propose the hypothesis that (1) ASK1 overexpression by systemic infusion of 3-NP promotes the formation of selective striatal lesions, and this occurs apart from just ROS generation. (2) ASK1 may differentially regulate C1q secretion level via active TGF-beta in each brain subregion of cortex and striatum, consequently involved in axon degeneration of corticostriatal projection neuron. When brain is mildly and chronically exposed to mitochondrial toxin, presynaptic neuron (in cortical neuron) degrades first, and then postsynaptic neuron of striatal MSN neuron withers as a consequence of it.Consolidating these results, we suggest that the increased ASK1 is linked to regulation of TGF-beta secreted in astrocytes, and differential C1q expression in neurons triggered by TGF-beta leads degradation of cortical projection and depletion of BDNF in striatal neuron in mice brains systemically infused with 3-NP.ope

Pharmacological Inhibition of Sonic Hedgehog Signaling Suppresses Tumor Development in a Murine Model of Intrahepatic Cholangiocarcinoma

Cholangiocarcinoma (CCC) is the second most primary liver cancer with an aggressive biological behavior, and its incidence increases steadily. An aberrant up-regulation of the sonic hedgehog signaling pathway has been reported in a variety of hepatic diseases including hepatic inflammation, fibrosis, as well as cancer. In this study, we determined the effect of a sonic hedgehog inhibitor, vismodegib, on the development of CCC. Through database analyses, we found sonic hedgehog signaling was up-regulated in human CCC, based on overexpression of its target genes, GLI1 and GLI2. Further, human CCC cells were highly sensitive to the treatment with vismodegib in vitro. Based on the data, we investigated the in vivo anti-cancer efficacy of vismodegib in CCC employing a murine model of CCC developed by hydrodynamic tail vein injection method. In the murine model, CCC induced by constitutively active forms of TAZ and PI3K exhibited up-regulated sonic hedgehog signaling. Treatment of vismodegib significantly suppressed tumor development in the murine CCC model, based on comparison of gross morphologies and liver weight/body weight. It is expected that pharmacological inhibition of sonic hedgehog signaling would be an effective molecular target therapy for CCC.ope

Identification of Antimicrobial Peptide Hexamers against Oral Pathogens through Rapid Screening of a Synthetic Combinatorial Peptide Library

A positional scanning synthetic peptide combinatorial library (PS-SCL) was screened in order to identify antimicrobial peptides against the cariogenic oral bacteria, Streptococcus mutans. Activity against Streptococcus gordonii and Aggregatibacter actinomycetemcomitans was also examined. The library was comprised of six sub-libraries with the format O(1-6)XXXXX-NH2, where O represents one of 19 amino acids (excluding cysteine) and X represents equimolar mixture of these. Each sub-library was tested for antimicrobial activity against S. mutans and evaluated for antimicrobial activity against S. gordonii and A. actinomycetemcomitans. The effect of peptides was observed using transmission electron microscopy (TEM). Two semi-mixture peptides, RXXXXN-NH2 (pep-1) and WXXXXN-NH2 (pep-2), and one positioned peptide, RRRWRN-NH2 (pep-3), were identified. Pep-1 and pep-2 showed significant antimicrobial activity against Gram positive bacteria (S. mutans and S. gordonii), but not against Gram negative bacteria (A. actinomycetemcomitans). However, pep-3 showed very low antimicrobial activity against all three bacteria. Pep-3 did not form an amphiphilic alpha-helix, which is a required structure for most antimicrobial peptides. Pep-1 and pep-2 were able to disrupt the membrane of S. mutans. Small libraries of biochemically-constrained peptides can be used to generate antimicrobial peptides against S. mutans and other oral microbes. Peptides derived from such libraries may be candidate antimicrobial agents for the treatment of oral microorganisms.ope

Expression of Apurinic/apyrimidinic Endonuclease and Neuronal Apoptosis in the Striatum after Treatment of 3-Nitropropionic Acid in Mice

BACKGROUND: 3-Nitroporpionic acid (3-NP) is an irreVersible inhibitor of succinate dehydrogenase in mitochondria and can induce apoptosis-like cell death in the striatum. It has been reported that oxidative stress plays a role in the 3-NP induced neuronal damage. 3-NP induced striatal damage is implicated in the pathogenesis of several neurological diseases, such as chronic neurodegenerative diseases and stroke. The DNA repair enzyme, apurinic/apyrimidinic endonuclease (APE), is a multifunctional protein in the DNA base excision repair (BER) pathway. To clarify the relationship between APE and neuronal cell death associated with the apoptosis in the striatum was induced by 3-NP in vivo. METHODS: After intra-striatal injection of 3-NP, expression of the APE protein and mRNA were evaluated by Western blot, immunohistochemistry, RT-PCR and DNA fragmentation patterns. Oxidative DNA damage was investigated by detection of oxidized DNA, AP site and superoxide. RESULTS: Expression levels of APE was rapidly reduced as early as 1hr after injection of 3-NP. DNA fragmentation was observed 24 hours after 3-NP treatment but not 4 hours. APE gene expression was increased to 1hr after 3-NP treatment. The number of AP sites were reduced and the reduction of APE proteins were blocked by a superoxide scavenger, MnTBAP-treatment. CONCLUSIONS: These results suggest that the reduction of APE is the preceding event of DNA fragmentation that causes apoptosis and a decrease of APE may be induced by ROS after 3-NP treatment.ope

Comparison of liver oncogenic potential among human RAS isoforms.

Mutation in one of three RAS genes (i.e., HRAS, KRAS, and NRAS) leading to constitutive activation of RAS signaling pathways is considered a key oncogenic event in human carcinogenesis. Whether activated RAS isoforms possess different oncogenic potentials remains an unresolved question. Here, we compared oncogenic properties among RAS isoforms using liver-specific transgenesis in mice. Hydrodynamic transfection was performed using transposons expressing short hairpin RNA downregulating p53 and an activated RAS isoform, and livers were harvested at 23 days after gene delivery. No differences were found in the hepatocarcinogenic potential among RAS isoforms, as determined by both gross examination of livers and liver weight per body weight ratio (LW/BW) of mice expressing HRASQ61L, KRAS4BG12V and NRASQ61K. However, the tumorigenic potential differed significantly between KRAS splicing variants. The LW/BW ratio in KRAS4AG12V mice was significantly lower than in KRAS4BG12V mice (p < 0.001), and KRAS4AG12V mice lived significantly longer than KRRAS4BG12V mice (p < 0.0001). Notably, tumors from KRAS4AG12V mice displayed higher expression of the p16INK4A tumor suppressor when compared with KRAS4BG12V tumors. Forced overexpression of p16INK4A significantly reduced tumor growth in KRAS4BG12V mice, suggesting that upregulation of p16INK4A by KRAS4AG12V presumably delays tumor development driven by the latter oncogene.ope

Breaking a Paradigm in Treatment of Acute Ischemic Stroke

Stroke is the second leading cause of death in Korea and responsible for serious long-term disability. Although rt-PA is an approved drug, only 1-3% of stroke patients are receiving the treatment because of its limited 3 hour time window. In this review, we will overview the basic molecular mechanism of acute ischemic stroke and current stroke treatment then discuss about current issue in Korea. Basic concept of stroke treatment is focused on saving the ischemic penumbra; area that is at risk of cell death but potentially salvageable. Apoptosis are believed to play critical roles in ischemic damage, especially in the penumbral zone. Apoptosis is an active form of cell death as a strategy to preserve genomic stability from DNA damage. If DNA damage is overwhelming the capacity of DNA repair, cells that harbor DNA damage are removed from the population by death. Execution of apoptosis is preceded rapidly by activation of complex signal pathways like caspase-dependent and caspase-independent pathways. By ‘turning off’ the death signal which is activated by massive DNA damage, initiation of apoptosis can be stopped and this concept can be a new therapeutic target of acute ischemic stroke. Reperfusion therapy and neuroprotective agent are two main streams for treatment of acute ischemic stroke. Although, many clinical trials have been done, rt-PA is the only FDA approved medical therapy for patients who can be treated within 3 hours of stroke onset. Emerging endovascular mechanical reperfusion device (clot retrieval device or MERCI) is approved by FDA and its results of recent clinical trials are promising. Neuroprotective agents were successful in animal models of ischemia, they have been unsuccessful in human trials. Clinical trials of NXY-059, one of promising neuroprotective agents, fail to widen the therapeutic window. So far, treatments of acute ischemic stroke have been based on epidemiological approach, not molecular pathophysiology. Introduction of neurocritical care, which will provide more professional, true meaning of treatment, is urgent. And paradigm of treatment of acute ischemic stroke should be focused on repairing damaged neuron by ischemia, not preventing further damage by reperfusion or neuroprotective agentope

Overexpressed Apurinic/Apyrimidinic Endonuclease/Redox Effector Factor-1 Using Adenoviral Vector Inhibits Induction of Neuronal Cell Death after Transient Ischemic Stroke in Mice

Background: Despite the correlation between changes in the levels of apurinic/apyrimidinic endonuclease/redox effector factor-1 (APE/Ref-1), the DNA base excision repair protein, and ischemic neuronal damage, no studies have addressed the question of whether the overexpression or restoration of APE/Ref-1 may prevent ischemic neuronal cell death in vivo. Therefore, we investigated whether increasing APE/Ref-1 may inhibit the loss of APE/Ref-1 and DNA fragmentation, and prevent neuronal cell death after cerebral ischemia/reperfusion (I/R). Methods: Adult male C57BL/6J mice were subjected to intraluminal suture occlusion of the middle cerebral artery for 1 hour followed by reperfusion. Pre-ischemic treatment of the adenoviral vector harboring an entire APE/Ref-1 gene sequence was introduced intrastriatally. Immunohistochemistry assays in AP sites, and TUNEL were performed. Results: APE/Ref-1 was overexpressed by using adenoviral-vector-mediated APE/Ref-1 in striatum. Pre-ischemic administration of the Adenoviral vector harboring an APE/Ref-1 gene, by 4 or 24 hours after reperfusion, significantly decreased AP site and 8-OHdG, and subsequent inhibited the induction of apoptotic DNA fragmentation at 24 hours after I/R. Conclusion: The overexpression of APE/Ref-1 can prevent induction of apoptotic cell death after I/R. Using adenoviral-vector-mediated APE/Ref-1 could represent a molecular target for prevention of ischemic neuronal cell death.ope

Let7a involves in neural stem cell differentiation relating with TLX level

Neural stem cells (NSCs) have the potential for differentiation into neurons known as a groundbreaking therapeutic solution for central nervous system (CNS) diseases. To resolve the therapeutic efficiency of NSCs, recent researchers have focused on the study on microRNA's role in CNS. Some micro RNAs have been reported significant functions in NSC self-renewal and differentiation through the post-transcriptional regulation of neurogenesis genes. MicroRNA-Let7a (Let7a) has known as the regulator of diverse cellular mechanisms including cell differentiation and proliferation. In present study, we investigated whether Let7a regulates NSC differentiation by targeting the nuclear receptor TLX, which is an essential regulator of NSC self-renewal, proliferation and differentiation. We performed the following experiments: western blot analysis, TaqMan assay, RT-PCR, and immunocytochemistry to confirm the alteration of NSCs. Our data showed that let7a play important roles in controlling NSC fate determination. Thus, manipulating Let-7A and TLX could be a novel strategy to enhance the efficiency of NSC's neuronal differentiation for CNS disorders.ope

Tracing of Noradrenergic Neuronal Circuitry and Functional Recovery after Permanent Focal Cerebral Ischemia in Mice

Background: Cardiovascular autonomic imbalances after stroke may be responsible for dysfunction of noradrenergic (NA) system in brain. However, exact locations or extent of NA-circuitry damage after stroke and its functional implications are remaining to be elucidated. Methods: Adult male C57BL/6J mice were subjected to permanent focal cerebral ischemia (pFCI) by intraluminal suture occlusion of middle cerebral artery. Recombinant adenoviral vector introducing wheat germ agglutinin (WGA)-cDNA was injected into locus coelureus (LC) using stereotaxic method. Behavioral and physiological tests were executed and immunohistochemistry for BrdU, tyrosine hydroxylase (TH), and WGA were performed at the same time. Results: Colocalization of GFP, TH, and WGA and restrictive expression of WGAmRNA in LC showed that WGA protein was endogenously synthesized in NA-cells of LC. Within 6 months after pFCI onset, the extensively damaged NA-circuitry was observed in thalamic nuclei, lateral hypothalamic area, hippocampus, and amygdaloid nuclei in ipsilateral side. The damaged NA-circuits were partly reorganized over time in the area of previous lesion with the gradual recovery of autonomic dysfunction and neurobehavioral deficits. Many migrated BrdU-immunopositive cells were detected in the lesioned thalamus and hypothalamus. Conclusion: In this study, we successfully visualized NA-circuitries in mice by using transsynaptic tracing with PRSWGA adenovirus. It was also confirmed the close relationships between the functional recovery and the reorganization of damaged NA-circuits. Moreover, this study demonstrated that the process of reorganization may involve axonal sprouting of intact NA-projections on newly proliferated and migrated neurons to the infarct area.ope

Transgenic mouse model expressing P53(R172H), luciferase, EGFP, and KRAS(G12D) in a single open reading frame for live imaging of tumor

Genetically engineered mouse cancer models allow tumors to be imaged in vivo via co-expression of a reporter gene with a tumor-initiating gene. However, differential transcriptional and translational regulation between the tumor-initiating gene and the reporter gene can result in inconsistency between the actual tumor size and the size indicated by the imaging assay. To overcome this limitation, we developed a transgenic mouse in which two oncogenes, encoding P53(R172H) and KRAS(G12D), are expressed together with two reporter genes, encoding enhanced green fluorescent protein (EGFP) and firefly luciferase, in a single open reading frame following Cre-mediated DNA excision. Systemic administration of adenovirus encoding Cre to these mice induced specific transgene expression in the liver. Repeated bioluminescence imaging of the mice revealed a continuous increase in the bioluminescent signal over time. A strong correlation was found between the bioluminescent signal and actual tumor size. Interestingly, all liver tumors induced by P53(R172H) and KRAS(G12D) in the model were hepatocellular adenomas. The mouse model was also used to trace cell proliferation in the epidermis via live fluorescence imaging. We anticipate that the transgenic mouse model will be useful for imaging tumor development in vivo and for investigating the oncogenic collaboration between P53(R172H) and KRAS(G12D).ope