Yong-Ping Liu

The role of ataxin 10 in the pathogenesis of spinocerebellar ataxia type 10

Background: Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant disorder characterized by cerebellar ataxia and seizures. SCA10 is caused by an expansion of an ATTCT pentanucleotide repeat in intron 9 of the ataxin 10 (ATXN10) gene encoding an approximately 55-kd protein of unknown function. However, how this mutation leads to SCA10 is unknown. Methods: In an effort to understand the pathogenic mechanism of SCA10, the authors conducted a series of experiments to address the effect of repeat expansion on the transcription and RNA processing of the ATXN10 gene. In addition, we generated Sca10 (mouse ataxin 10 homolog)–null mice and addressed the role of Sca10 gene dosage on the cerebellum. Results: Mutant ATXN10 allele is transcribed at the normal level, and the pre-mRNA containing an expanded repeat is processed normally in patient-derived cells. Sca10-null mice exhibited embryonic lethality. Heterozygous mutants were overtly normal and did not develop SCA10 phenotype Conclusion: A simple gain of function or loss of function of ATXN10 is unlikely to be the major pathogenic mechanism contributing to the spinocerebellar ataxia type 10 phenotype.

The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3’-P and 5’-OH, are processed by mammalian polynucleotide kinase 3’-phosphatase (PNKP), a bifunctional enzyme with 3’-phosphatase and 5’-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14–41 to 55–82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP’s 3’ phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3’-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients’ brain. Finally, long amplicon quantitative PCR analysis of human MJD patients’ brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.

Inactivation of PNKP by Mutant ATXN3 Triggers Apoptosis by Activating the DNA Damage-Response Pathway in SCA3

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an untreatable autosomal dominant neurodegenerative disease, and the most common such inherited ataxia worldwide. The mutation in SCA3 is the expansion of a polymorphic CAG tri-nucleotide repeat sequence in the C-terminal coding region of the ATXN3 gene at chromosomal locus 14q32.1. The mutant ATXN3 protein encoding expanded glutamine (polyQ) sequences interacts with multiple proteins in vivo, and is deposited as aggregates in the SCA3 brain. A large body of literature suggests that the loss of function of the native ATNX3-interacting proteins that are deposited in the polyQ aggregates contributes to cellular toxicity, systemic neurodegeneration and the pathogenic mechanism in SCA3. Nonetheless, a significant understanding of the disease etiology of SCA3, the molecular mechanism by which the polyQ expansions in the mutant ATXN3 induce neurodegeneration in SCA3 has remained elusive. In the present study, we show that the essential DNA strand break repair enzyme PNKP (polynucleotide kinase 3’-phosphatase) interacts with, and is inactivated by, the mutant ATXN3, resulting in inefficient DNA repair, persistent accumulation of DNA damage/strand breaks, and subsequent chronic activation of the DNA damage-response ataxia telangiectasia-mutated (ATM) signaling pathway in SCA3. We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-δ pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3. Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible approach to treatment.

Vasoactive intestinal peptide re-balances TREM-1/TREM-2 ratio in acute lung injury

Vasoactive intestinal peptide (VIP) is one of the most plentiful neuropeptides in the lung and it has anti-inflammatory effects in the respiratory system. Triggering receptors expressed on myeloid cells-1 (TREM-1) and triggering receptors expressed on myeloid cells-2 (TREM-2) regulate immune responses to lipopolysaccharide (LPS). In the present study, we tested the expressions of TREM-1 and TREM-2 in various pulmonary cell lines and/or tissue using an animal model of LPS-induced acute lung injury (ALI), and determined the effects of VIP on expression of the TREM-1 and TREM-2 in lung tissues and cells from ALI mice. We found 1) expression of the TREM-1 mRNA from lung tissues of ALI was significantly increased, whereas the expression of TREM-2 mRNA was decreased in these tissues; 2) TREM-1 mRNA was only expressed in macrophages, while TREM-2 mRNA was detected in HBECs, lung fibroblasts, lung adenocarcinoma cells and macrophages; 3) the ratio of TREM-1 mRNA to TREM-2 mRNA was increased in LPS-induced lung tissues and macrophages; 4) VIP inhibited expression of the TREM-1 mRNA in a time- and dose-dependent manner in lung cells from LPS-induced ALI mice; however, it increased expression of the TREM-2 mRNA. As a result of these effects, VIP normalized the ratio of TREM-1 to TREM-2 mRNA in these cells. Our results suggest that VIP might exert its anti-inflammatory effect through a mechanism involved in regulation of expression of the TREM-1 and TREM-2 in LPS-induced ALI.

Calcitonin gene-related peptide promotes the wound healing of human bronchial epithelial cells via PKC and MAPK pathways

Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide derived from the calcitonin gene. CGRP is widely distributed in the central and peripheral neuronal systems. In the lung, CGRP could modulate dendritic cell function, stimulate proliferation of alveolar epithelial cells and mediate lung injury in mice. In this study, we investigated the effect of CGRP on the wound healing of human bronchial epithelial cells (HBECs) in vitro. The results showed that CGRP accelerated the recovery of wound area of monolayer HBECs in a dose-dependent manner. CGRP inhibited the lipopolysaccharide-induced apoptosis in HBECs. The percentage of S phase and G2/M phase was increased in HBECs after CGRP treatment. CGRP upregulated the expression of Ki67 in a dose-dependent manner. Some pathway inhibitors were used to investigate the signal pathway in which CGRP was involved. We found out that PKC pathway inhibitor (H-7) and MAPK pathway inhibitor (PD98059) could partially attenuate the effect of CGRP, which indicated that CGRP might promote the wound healing of HBECs via PKC and/or MAPK dependent pathway by accelerating migration and proliferation, and inhibiting apoptosis.

Ethanol suppresses PGC-1α expression by interfering with the cAMP-CREB pathway in neuronal cells.

Alcohol intoxication results in neuronal apoptosis, neurodegeneration and manifest with impaired balance, loss of muscle coordination and behavioral changes. One of the early events of alcohol intoxication is mitochondrial (Mt) dysfunction and disruption of intracellular redox homeostasis. The mechanisms by which alcohol causes Mt dysfunction, disrupts cellular redox homeostasis and triggers neurodegeneration remains to be further investigated. Proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) plays critical roles in regulating Mt biogenesis and respiration, cellular antioxidant defense mechanism, and maintenance of neuronal integrity and function. In this study, we sought to investigate whether alcohol causes Mt dysfunction and triggers neurodegeneration by suppressing PGC-1α expression. We report that ethanol suppresses PGC-1α expression, and impairs mitochondrial function and enhances cellular toxicity in cultured neuronal cell line and also in human fetal brain neural stem cell-derived primary neurons. Moreover, we report that cells over-expressing exogenous PGC-1α or treated with Rolipram, a selective phosphodiesterase-4 inhibitor, ameliorate alcohol-induced cellular toxicity. Further analysis show that ethanol decreases steady-state intracellular cAMP levels, and thus depletes phosphorylation of cAMP-response element binding protein (p-CREB), the key transcription factor that regulates transcription of PGC-1α gene. Accordingly, we found PGC-1α promoter activity and transcription was dramatically repressed in neuronal cells when exposed to ethanol, suggesting that ethanol blunts cAMP→CREB signaling pathway to interfere with the transcription of PGC-1α. Ethanol-mediated decrease in PGC-1α activity results in the disruption of Mt respiration and function and higher cellular toxicity. This study might lead to potential therapeutic intervention to ameliorate alcohol-induced apoptosis and/or neurodegeneration by targeting PGC-1α.

Soluble Epoxide Hydrolase Inhibitor Attenuates Lipopolysaccharide-Induced Acute Lung Injury and Improves Survival in Mice

ABSTRACT Acute lung injury (ALI) is characterized by rapid alveolar injury, vascular leakage, lung inflammation, neutrophil accumulation, and induced cytokines production leading to lung edema. The mortality rate of patients suffering from ALI remains high. Epoxyeicosatrienoic acids (EETs) are cytochrome P450-dependent derivatives of polyunsaturated fatty acid with antihypertensive, profibrinolytic, and anti-inflammatory functions. EETs are rapidly hydrated by soluble epoxide hydrolase (sEH) to their less potent diols. The aim of this study was to investigate the role of sEH inhibitor trifluoromethoxyphenyl propionylpiperidin urea (TPPU) and EETs in lipopolysaccharide (LPS)-induced ALI of mice. Our studies revealed that inhibition of sEH with TPPU attenuated the morphological changes in mice, decreased the neutrophil infiltration to the lung, pro-inflammatory cytokine levels (IL-1&bgr; and TNF-&agr;) in serum and bronchoalveolar lavage fluid (BALF), and alveolar capillary leakage (lung wet/dry ratio and total protein concentration in BALF). TPPU improved the survival rate of LPS-induced ALI. In addition, in vitro experiments revealed that both TPPU and EETs (11,12-EET and 14,15-EET) suppressed the expression of IL-1&bgr; and TNF-&agr;, and LDH release in RAW264.7 cells. These results indicate that EETs play a role in dampening LPS-induced acute lung inflammation, and suggest that sEH could be a valuable candidate for the treatment of ALI.

Blocking triggering receptor expressed on myeloid cells-1 attenuates lipopolysaccharide-induced acute lung injury via inhibiting NLRP3 inflammasome activation.

Acute lung injury (ALI) is associated with high mortality and uncontrolled inflammation plays a critical role in ALI. TREM-1 is an amplifier of inflammatory response, and is involved in the pathogenesis of many infectious diseases. NLRP3 inflammasome is a member of NLRs family that contributes to ALI. However, the effect of TREM-1 on NLRP3 inflammasome and ALI is still unknown. This study aimed to determine the effect of TREM-1 modulation on LPS-induced ALI and activation of the NLRP3 inflammasome. We showed that LR12, a TREM-1 antagonist peptide, significantly improved survival of mice after lethal doses of LPS. LR12 also attenuated inflammation and lung tissue damage by reducing histopathologic changes, infiltration of the macrophage and neutrophil into the lung, and production of the pro-inflammatory cytokine, and oxidative stress. LR12 decreased expression of the NLRP3, pro-caspase-1 and pro-IL-1β, and inhibited priming of the NLRP3 inflammasome by inhibiting NF-κB. LR12 also reduced the expression of NLRP3 and caspase-1 p10 protein, and secretion of the IL-1β, inhibited activation of the NLRP3 inflammasome by decreasing ROS. For the first time, these data show that TREM-1 aggravates inflammation in ALI by activating NLRP3 inflammasome, and blocking TREM-1 may be a potential therapeutic approach for ALI.

Vasoactive intestinal peptide suppresses macrophage-mediated inflammation by downregulating interleukin-17A expression via PKA- and PKC-dependent pathways.

Interleukin (IL)‐17A is a pro‐inflammatory cytokine that markedly enhances inflammatory responses in the lungs by recruiting neutrophils and interacting with other pro‐inflammatory mediators. Reducing the expression of IL‐17A could attenuate inflammation in the lungs. However, whether VIP exerts its anti‐inflammatory effects by regulating the expression of IL‐17A has remained unclear. Here, we show that there is a remarkable increase of IL‐17A in bronchoalveolar lavage fluid (BALF) and lung tissue of mice with acute lung injury (ALI). Moreover, lipopolysaccharides (LPS) stimulated elevated expression of IL‐17A, which was evident by the enhanced levels of mRNA and protein observed. Furthermore, we also found that VIP inhibited LPS‐mediated IL‐17A expression in a time‐ and dose‐dependent manner in an in vitro model of ALI and that this process might be mediated via the phosphokinase A (PKA) and phosphokinase C (PKC) pathways. Taken together, our results demonstrated that VIP might be an effective protector during ALI by suppressing IL‐17A expression.

Vasoactive intestinal peptide overexpression mediated by lentivirus attenuates lipopolysaccharide-induced acute lung injury in mice by inhibiting inflammation

HIGHLIGHTSWe firstly found intratracheal injection of Lenti‐VIP for 7days significantly increased intrapulmonary VIP content in mice.VIP alleviates ALI induced by LPS in vivo.VIP inhibits pro‐inflammatory TNF‐&agr; expression in murine macrophages stimulated by LPS via PKC and PKA pathways. ABSTRACT Vasoactive intestinal peptide (VIP) is one of the most abundant neuropeptides in the lungs with various biological characters. We have reported that VIP inhibited the expressions of TREM‐1 and IL‐17A, which are involved in the initiation and amplification of inflammation in acute lung injury (ALI). However, the overall effect of VIP on ALI remains unknown. The aim of this study is to investigate the therapeutic effect of VIP mediated by lentivirus (Lenti‐VIP) on lipopolysaccharide (LPS)‐induced murine ALI. We found that the expression of intrapulmonary VIP peaked at day7 after the intratracheal injection of Lenti‐VIP. Lenti‐VIP increased the respiratory rate, lung compliance, and tidal volume, while decreased airway resistance in ALI mice, detected by Buxco system. Lenti‐VIP significantly reduced inflammatory cell infiltration and maintained the integrity of the alveolar septa. Lenti‐VIP also remarkably decreased the total protein level, the number of neutrophil and lactate dehydrogenase activity in the bronchoalveolar lavage fluid of LPS‐induced ALI mice. In addition, Lenti‐VIP down‐regulated pro‐inflammatory tumor necrosis factor (TNF)‐&agr; mRNA and protein expression, while up‐regulated anti‐inflammatory interleukin‐10 mRNA and protein expression in lungs of ALI mice. Furthermore, we observed that VIP reduced the TNF‐&agr; expression in murine macrophages under LPS stimulation through protein kinase C and protein kinase A pathways. Together, our findings show that in vivo administration of lentivirus expressing VIP exerts a potent therapeutic effect on LPS‐induced ALI in mice via inhibiting inflammation.