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Background The aim of the present study was to establish a competing risk nomogram to predict parotid gland cancer-specific mortality (PGC-SM). Methods Seven thousand nine hundred and sixty-two patients extracted from SEER database were randomly categorized into training and validation sets. The competing risk model was used to identify factors associated with PGC-SM. The nomogram was evaluated via concordance indexes (C-indexes), calibration plots, and decision curve analysis (DCA). Results Male, elderly, white, widowed, larger tumor, no surgery, advanced tumor grade, lymph node (LN) metastasis, adenocarcinoma (ADC), and higher TNM stage were associated with higher incidence of PGC-SM. Calibration plots showed that the nomogram was well calibrated. C-indexes for nomogram were 0.84 (95% CI: 0.81-0.86) and 0.84 (95% CI: 0.82-0.86) in training and validation sets, respectively. DCA demonstrated the clinical usefulness of nomogram. Conclusions The competing risk nomogram shows high performance in predicting PGC-SM, which might enable clinicians formulate suitable treatment protocols for patients with parotid gland carcinoma (PGC).

As the most common malignancy, lung cancer is characterised by high rates of occurrence and mortality. Although circular RNAs (circRNAs) are known to act as important regulators in cancer, their role in lung cancer remains poorly understood. In this study, circ_GRHPR expression was found to be significantly upregulated in the serum of five patients with non‐small cell lung cancer (NSCLC), compared to that in healthy controls. It is expressed at high levels in NSCLC cell lines, as revealed by qRT‐PCR analysis. Functionally, we demonstrated that circ_GRHPR promotes NSCLC proliferation and invasion in vitro and in vivo by cell proliferation, transwell, cell cycle, and tumour‐forming assays. Mechanistically, RNA pull‐down and RNA immunoprecipitation assays showed that circ_GRHPR interacts with the RNA‐binding protein poly(rC)‐binding protein 2 (PCBP2) and regulates its subcellular localisation by forming the circ_GRHPR/PCBP2 complex, localizing PCBP2 mainly in the cytoplasm and reducing the proportion found in the nucleus. Furthermore, we demonstrated that four‐and‐a‐half LIM‐only protein 3 (FHL3) is a tumour‐stimulating factor in NSCLC that interacts with and is influenced by PCBP2. Circ_GRHPR increased FHL3 expression in the nucleus of NSCLC cells by decreasing PCBP2 expression therein and promoting the proliferation and invasion of NSCLC cells. Therefore, our study identified that circ_GRHPR promotes NSCLC proliferation and invasion, providing a possible explanation for its mechanism of action.

A visible-light promoted radical cascade reaction of N-propargylanilines with sodium sulfinates as sulfonyl radical precursors was developed under metal-free conditions. This mild protocol represents a speedy and efficient method for synthesizing 3-sulfonylquinolines via the formation of C–S and C–C bonds in one step.

Hybridization is an important technique and is widely used in fish genetic breeding; this technique prevents variety degeneration and improves variety. In this research, we obtained a hybrid lineage (WR-F1-F4) by interspecific hybridization derived from female white crucian carp (Carassius cuvieri, WCC) and male red crucian carp (Carassius auratus red var., RCC), and a new type of improved fish (WR-II) was obtained by back-crossing between the female hybrid (WR-F1) and the male WCC. We investigated the morphological traits, chromosomal numbers, DNA content, gonadal development, sperm shape and 5S rDNA in the hybrid lineage and WR-II. The results showed that all the assessed morphological traits were not significantly different between the progenies of the hybrid lineage and their original parents, but the WR-II showed smaller head and higher body compared with its original parents; the hybrid lineage and WR-II were diploid with 100 chromosomes; all the tested males and females of the hybrid lineage and WR-II had normal gonadal development and produced mature sperm and egg, respectively; the 5S rDNA of the hybrid lineage and WR-II inherited the characteristics of their parents and had some mutant bases or chimeric sequences, which revealed heredity, variability and heterozygosity in the hybrid progenies; finally, the 5S rDNA of the hybrid lineage could be highly inherited from the second generation (WR-F2) to the next generations. This study will be beneficial for the establishment and application of hybrid lineages in genetic breeding.

Inflammation is associated with skeletal muscle dysfunction and atrophy in patients with chronic obstructive pulmonary disease (COPD). Theophylline has an anti-inflammatory role in COPD. However, the effects of theophylline on inflammation in skeletal muscle in COPD have rarely been reported. The aims of this study were to explore whether theophylline has an anti-inflammatory effect on skeletal muscle in a mouse model of emphysema and to investigate the molecular mechanism underlying this effect. In mice, cigarette smoke (CS) exposure for 28 wk resulted in atrophy of the gastrocnemius muscle. Histone deacetylase 2 (HDAC2) and nuclear factor-κBp65 (NF-κBp65) mRNA and protein levels were significantly decreased and increased, respectively, in gastrocnemius muscle. This effect was revered by aminophylline. The exposure of murine skeletal muscle C2C12 cells to CS extract (CSE) significantly increased IL-8 and TNF-α levels as well as NF-κBp65 mRNA and protein levels and NF-κBp65 activity. This effect was reversed by theophylline. HDAC2 knockdown enhanced the activity of NF-κBp65 and increased IL-8 and TNF-α levels in C2C12 cells. CSE significantly increased the interaction of HDAC2 with NF-κBp65 in C2C12 cells. These data suggest that theophylline has an anti-inflammatory effect on skeletal muscle in a mouse model of emphysema by upregulating HDAC2 expression and decreasing NF-κBp65 activation.

Aim: To explore the expression profiles of long noncoding RNAs (lncRNAs) and identify novel lncRNAs as biomarkers for early diagnosis and therapy of hepatocellular carcinoma (HCC). Materials & methods: Expression profiles of lncRNAs and mRNAs in five paired HCC and adjacent normal tissues were obtained by RNA sequencing. Eight lncRNAs, including two novel liver-specific lncRNAs (NONHSAT059247.2 and NONHSAT013897.2), were validated in another 74 pairs of HCC and adjacent normal tissues by quantitative reverse transcription PCR. Results: The results of quantitative reverse transcription PCR showed that NONHSAT252133.1, NONHSAT112116.2 and NONHSAT242657.1 were significantly upregulated in HCC tissues, whereas NONHSAT169790.1, NONHSAT059247.2 and NONHSAT013897.2 were significantly downregulated. Two liver-specific lncRNAs demonstrated excellent diagnostic performance: NONHSAT059247.2 (area under the curve = 0.941, 95% CI: 0.902–0.979, p < 0.0001), NONHSAT013897.2 (area under the curve = 0.944, 95% CI: 0.906–0.983, p < 0.0001). Conclusion: The liver-specific lncRNAs NONHSAT059247.2 and NONHSAT013897.2, may provide new biomarkers for the future study on diagnosis, therapy and mechanisms of HCC.

Hepatocellular carcinoma (HCC) is a highly malignant tumor. In this study, we sought to identify a novel biomarker for HCC by analyzing transcriptome and clinical data. The R software was used to analyze the differentially expressed genes (DEGs) in the datasets GSE74656 and GSE84598 downloaded from the Gene Expression Omnibus database, followed by a functional annotation. A total of 138 shared DEGs were screened from two datasets. They were mainly enriched in the “Metabolic pathways” pathway (Padj = 8.21E-08) and involved in the carboxylic acid metabolic process (Padj = 0.0004). The top 10 hub genes were found by protein-protein interaction analysis and were upregulated in HCC tissues compared to normal tissues in The Cancer Genome Atlas database. Survival analysis distinguished 8 hub genes CENPE, SPDL1, Hyaluronan-mediated motility receptor, Rac GTPase activating protein 1, Thyroid hormone receptor interactor 13, cytoskeleton-associated protein (CKAP) 2, CKAP5, and Integrin subunit beta 3 binding protein (ITGB3BP) were considered as prognostic hub genes. Multivariate cox regression analysis indicated that all the prognostic hub genes were independent prognostic factors for HCC. Furthermore, the receiver operating characteristic curve revealed that the 8-hub genes model had better prediction performance for overall survival compared to the T stage (p = 0.008) and significantly improved the prediction value of the T stage (p = 0.002). The Human Protein Atlas showed that the protein expression of ITGB3BP was upregulated in HCC, so the expression of ITGB3BP was further verified in our cohort. The results showed that ITGB3BP was upregulated in HCC tissues and was significantly associated with lymph node metastasis.

The white crucian carp (Carassius auratus cuvieri, WCC) not only is one of the most economically important fish in Asia, characterized by strong reproductive ability and rapid growth rates, but also represents a good germplasm to produce hybrid progenies with heterosis. Gene knockout technique provides a safe and acceptant way for fish breeding. Achieving gene knockout in WCC and its hybrid progeny will be of great importance for both genetic studies and hybridization breeding. Tyrosinase (TYR) is a key enzyme in melanin synthesis. Depletion of tyr in zebrafish and mice results in mosaic pigmentation or total albinism. Here, we successfully used CRISPR-Cas9 to target tyr in WCC and its hybrid progeny (WR) derived from the cross of WCC (♀) and red crucian carp (Carassius auratus red var., RCC, ♂). The level of TYR protein was significantly reduced in mutant WCC. Both the mutant WCC and the mutant WR showed different degrees of melanin reduction compared with the wild-type sibling control fish, resulting from different mutation efficiency ranging from 60% to 90%. In addition, the transcriptional expression profiles of a series of pivotal pigment synthesis genes, i.e. tyrp1, mitfa, mitfb, dct and sox10, were down-regulated in tyr-CRISPR WCC, which ultimately caused a reduction in melanin synthesis. These results demonstrated that tyr plays a key role in melanin synthesis in WCC and WR, and CRISPR-Cas9 is an effective tool for modifying the genome of economical fish. Furthermore, the tyr-CRISPR models could be valuable in understanding fundamental mechanisms of pigment formation in non-model fish.

Hybridization can combine the genomes of different strains or species, which leads to changes of genotype and phenotype in the hybrids. In this study, we aimed to investigate the genetic variations of hybrids (WR-F1 and WR-F2) derived from the intraspecific hybridization of white crucian carp (Carassius auratus cuvieri, WCC, ♀) and red crucian carp (Carassius auratus red var., RCC, ♂). Here, we compared the orthologous genes in the liver transcriptomes of hybrids with those of WCC and RCC, and classified the orthologous genes into eight gene patterns within three categories (chimera, mutant, and biparental origin genes). The results revealed 19.04%, 4.17% chimeric genes and 6.90%, 5.05% mutations of orthologous genes in WR-F1 and WR-F2 respectively. Seventeen of twenty-three characterized genes (77%) were confirmed to be the chimeras at the genomic DNA level. The GO classification discovered that some chimeric and mutant genes were related to metabolic process, immune system and developmental process in WR-F1. Our results provide the new evidence that hybridization can combine the parental genomes, leading to changes in the genotype of the resultant hybrids. This is the first report on the formation of chimeric genes from fish intraspecific hybridization, which is potentially interesting from the context of both evolution and the genetic breeding of fish.

Background Penicillium marneffei (P. marneffei) is a thermally dimorphic fungus pathogen that causes fatal infection. Alveolar macrophages are innate immune cells that have critical roles in protection against pulmonary fungal pathogens and the macrophage polarization state has the potential to be a deciding factor in disease progression or resolution. The aim of this study was to investigate mouse alveolar macrophage polarization states during P. marneffei infection. Results We used enzyme-linked immunosorbent (ELISA) assays, quantitative real-time PCR (qRT-PCR), and Griess, arginase activity to evaluate the phenotypic markers of alveolar macrophages from BALB/C mice infected with P. marneffei. We then treated alveolar macrophages from infected mice with P. marneffei cytoplasmic yeast antigen (CYA) and investigated alveolar macrophage phenotypic markers in order to identify macrophage polarization in response to P. marneffei antigens. Our results showed: i) P. marneffei infection significantly enhanced the expression of classically activated macrophage (M1)-phenotypic markers (inducible nitric oxide synthase [iNOS] mRNA, nitric oxide [NO], interleukin-12 [IL-12], tumor necrosis factor-alpha [TNF-α]) and alternatively activated macrophage (M2a)-phenotypic markers (arginase1 [Arg1] mRNA, urea) during the second week post-infection. This significantly decreased during the fourth week post-infection. ii) During P. marneffei infection, CYA stimulation also significantly enhanced the expression of M1 and M2a-phenotypic markers, consistent with the results for P. marneffei infection and CYA stimulation preferentially induced M1 subtype. Conclusions The data from the current study demonstrated that alveolar macrophage M1/M2a subtypes were present in host defense against acute P. marneffei infection and that CYA could mimic P. marneffei to induce a host immune response with enhanced M1 subtype. This could be useful for investigating the enhancement of host anti-P. marneffei immune responses and to provide novel ideas for prevention of P. marneffei-infection. Electronic supplementary material The online version of this article (doi:10.1186/s12866-017-1086-3) contains supplementary material, which is available to authorized users.

Summary: Brown adipose tissue (BAT) is a thermogenic organ that maintains body temperature and energy homeostasis. Transcriptional regulation plays an important role in the program of brown adipogenesis. However, it remains unclear how the transcriptional events are controlled in this program. In this study, we analyze an SENP2 BAT conditional knockout mouse model and find that SENP2-mediated de-SUMOylation is essential for BAT development. SENP2 catalyzes de-SUMOylation of cAMP response element-binding protein (CREB) to suppress Necdin expression, which induces brown adipocyte differentiation and brown adipogenesis. Mechanistically, we find that SUMOylation enhances CREB interaction with serine/threonine protein phosphatase 2A (PP2A) to de-phosphorylate CREB, which activates Necdin transcription. SENP2 deficiency enhances the expression of Necdin to inhibit brown adipocyte differentiation. Therefore, we reveal a crucial role of SENP2-mediated de-SUMOylation of CREB in suppression of Necdin expression during brown adipose development and brown adipogenesis. : Liang et al. show that SENP2 suppresses Necdin expression, leading to brown adipocyte differentiation, which is essential for brown adipose tissue (BAT) adipogenesis. Keywords: SUMOylation, SENP2, brown adipogenesis, Necdin, CREB

Title of data- Sequences of P. marneffei strains using PCR and the sequence of PCR product. Description of data- The GXHCBR P. marneffei strains were identified by gold standard DNA sequencing of the fungal ITS region using PCR and the sequence of PCR product. (DOCX 12 kb)

Title of data-raw data. Description of data- The raw data of Fig. 4, Fig. 5 and Fig. 6, please see Tables 1, 2, and 3, respectively. (DOCX 27 kb)

GABARAPL1/GEC1 is an early estrogen-induced gene which encodes a protein highly conserved from C. elegans to humans. Overexpressed GABARAPL1 interacts with GABAA or kappa opioid receptors, associates with autophagic vesicles, and inhibits breast cancer cell proliferation. However, the function of endogenous GABARAPL1 has not been extensively studied. We hypothesized that GABARAPL1 is required for maintaining normal autophagic flux, and plays an important role in regulating cellular bioenergetics and metabolism. To test this hypothesis, we knocked down GABARAPL1 expression in the breast cancer MDA-MB-436 cell line by shRNA. Decreased expression of GABARAPL1 activated procancer responses of the MDA-MB-436 cells including increased proliferation, colony formation, and invasion. In addition, cells with decreased expression of GABARAPL1 exhibited attenuated autophagic flux and a decreased number of lysosomes. Moreover, decreased GABARAPL1 expression led to cellular bioenergetic changes including increased basal oxygen consumption rate, increased intracellular ATP, increased total glutathione, and an accumulation of damaged mitochondria. Taken together, our results demonstrate that GABARAPL1 plays an important role in cell proliferation, invasion, and autophagic flux, as well as in mitochondrial homeostasis and cellular metabolic programs.

How cellular metabolic activities regulate autophagy and determine the susceptibility to oxidative stress and ultimately cell death in neuronal cells is not well understood. An important example of oxidative stress is 4-hydroxynonenal (HNE), which is a lipid peroxidation product that is formed during oxidative stress, and accumulates in neurodegenerative diseases causing damage. The accumulation of toxic oxidation products such as HNE, is a prevalent feature of neurodegenerative diseases, and can promote organelle and protein damage leading to induction of autophagy. In this study, we used differentiated SH-SY5Y neuroblastoma cells to investigate the mechanisms and regulation of cellular susceptibility to HNE toxicity and the relationship to cellular metabolism. We found that autophagy is immediately stimulated by HNE at a sublethal concentration. Within the same time frame, HNE induces concentration dependent CASP3/caspase 3 activation and cell death. Interestingly, both basal and HNE-activated autophagy, were regulated by glucose metabolism. Inhibition of glucose metabolism by 2-deoxyglucose (2DG), at a concentration that inhibited autophagic flux, further exacerbated CASP3 activation and cell death in response to HNE. Cell death was attenuated by the pan-caspase inhibitor Z-VAD-FMK. Specific inhibition of glycolysis using koningic acid, a GAPDH inhibitor, inhibited autophagic flux and exacerbated HNE-induced cell death similarly to 2DG. The effects of 2DG on autophagy and HNE-induced cell death could not be reversed by addition of mannose, suggesting an ER stress-independent mechanism. 2DG decreased LAMP1 and increased BCL2 levels suggesting that its effects on autophagy may be mediated by more than one mechanism. Furthermore, 2DG decreased cellular ATP, and 2DG and HNE combined treatment decreased mitochondrial membrane potential. We conclude that glucose-dependent autophagy serves as a protective mechanism in response to HNE.

// Dongmei Huang 1, * , Zhiying Ma 1, * , Yili He 1 , Ying Xiao 1 , Honglin Luo 1 , Qiuli Liang 1 , Xiaoning Zhong 1 , Jing Bai 1 and Zhiyi He 1 1 Department of Respiratory Medicine, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, 530021, China * These authors have contributed equally to this work Correspondence to: Zhiyi He, email: zhiyi-river@163.com Keywords: cigarette smoke exposure, muscle inflammation, histone deacetylase 2, nuclear factor-κB Received: December 02, 2016 Accepted: April 27, 2017 Published: May 23, 2017 ABSTRACT Long-term cigarette smoke induces lung inflammatory injury and chronic obstructive pulmonary disease (COPD), associated with skeletal muscle inflammation. This study aimed at investigating how cigarette smoke promotes skeletal muscle inflammation and its molecular pathogenesis. Mice were exposed to air or cigarette smoke for 12 or 24 weeks, and C2C12 cells were stimulated with cigarette smoke extract (CSE). The mass and function, myotube formation, inflammatory cytokine production, histone deacetylase 2 (HDAC2) and nuclear factor-κB (NF-κB) p65 expression were detected in the gastrocnemius muscles of mice and C2C12 cells. In comparison with the control mice, cigarette smoke significantly damaged the lung and reduced the gastrocnemius muscle mass and body weights in mice. Cigarette smoke significantly down-regulated myosin heavy chain (MHC)-IIβ and HDAC2 expression, but enhanced NF-κBp65, keratinocyte chemoattractant (KC) and tumor necrosis factor (TNF)-α expression in the gastrocnemius muscles. CSE stimulation significantly inhibited the myotube formation, MyoD and HDAC2 expression, but enhanced NF-κBp65 expression, KC and TNF-α production in C2C12 cells, which were enhanced by HDAC2 knockdown and abrogated by a NF-κB inhibitor. CSE significantly inhibited the interaction of HDAC2 with NF-κBp65, and increased the levels of acetyl-NF-κBp65 in C2C12 cells. These data indicated that cigarette smoke inhibited HDAC2 expression and its interaction with NF-κBp65 to stimulate inflammation, contributing to the pathogenesis of COPD-related skeletal muscle atrophy in mice.

We have previously demonstrated that calmodulin (CaM) binds directly to c-FLIP(L) in a Ca(2+)-dependent manner. Deletion of the CaM-binding region (amino acid 197-213) results in reduced CaM binding, and increased Fas-mediated apoptosis and decreased tumorigenesis of cholangiocarcinoma cells. The present studies were designed to identify the precise amino acids between 197 and 213 that are responsible for CaM/FLIP binding, and their roles in mediating the anti-apoptotic function of c-FLIP(L). Sequence analysis of the CaM-binding region at 197-213 predicted three unique positively charged residues at 204, 207 and 209, which might be responsible for the CaM/FLIP binding. A point mutation at H204 of c-FLIP(L) was found to markedly reduce CaM binding, whereas point mutation at R207 or K209 did not affect c-FLIP(L) binding to CaM. Decreased CaM/FLIP binding was confirmed in cholangiocarcinoma cells overexpressing the H204 c-FLIP(L) mutant. Reduced CaM binding by the H204 mutant resulted in increased sensitivity to Fas-mediated apoptosis and inhibited tumor growth in mice compared with wild-type c-FLIP(L). Death-inducing signaling complex (DISC) analysis showed that the reduced CaM binding to H204 mutant resulted in less c-FLIP(L) recruited into the DISC. Concurrently, increased caspase 8 was recruited to the DISC, which resulted in increased cleavage and activation of caspase 8, activation of downstream caspase 3 and increased apoptosis. Therefore, these results demonstrate that the H204 residue is responsible for c-FLIP(L) binding to CaM, which mediates the anti-apoptotic function of c-FLIP(L), most likely through affecting recruitment of caspase 8 into the DISC and thus caspase 8 activation. These studies further characterized CaM/FLIP interaction and its function in regulating Fas-mediated apoptosis and tumorigenesis, which may provide new therapeutic targets for cancer therapy.

Metal ions are essential as well as toxic to the cell. The mechanism of metal-induced toxicity is not well established. Here, for the first time we studied two essential nutritional elements, copper and manganese, for their apoptotic effects in yeast Saccharomyces cerevisiae. Although beneficial at subtoxic levels, we demonstrated that at moderately toxic levels, both metals induce extensive apoptosis in yeast cells. At even higher concentrations, necrosis takes over. Furthermore, we investigated the molecular pathways mediating Cu- and Mn-mediated apoptotic action. Mitochondria-defective yeast exhibit a much reduced apoptotic marker expression and better survival under Cu and Mn stress, indicating mitochondria are involved in both Cu- and Mn-induced apoptosis. Reactive oxygen species (ROS) are generated in high amounts in Cu- but not in Mn-induced cell death, and Cu toxicity can be alleviated by overexpression of superoxide dismutase 2, suggesting ROS mediate Cu but not Mn toxicity. Yeast metacaspase Yca1p is not involved in Cu-induced apoptosis, although it plays an important role in the Mn-induced process. A genetic screen identified Cpr3p, a yeast cyclophilin D homologue, as mediating the Cu-induced apoptotic program. Cpr3p mutant seems to eliminate Cu-induced apoptosis without affecting ROS production, while leaving necrosis intact. These results may provide important insight into a detailed understanding at the molecular and cellular level of metal toxicity and metal accumulation diseases.

Excessive nitric oxide (NO) production is known to damage mitochondrial proteins and the autophagy repair pathway and so can potentially contribute to neurotoxicity. Accordingly, we hypothesized that protection against protein damage from reactive oxygen and nitrogen species (ROS/RNS) under conditions of low oxygen by the autophagy pathway in neurons would be impaired by NO and enhance bioenergetic dysfunction. Rat primary cortical neurons maintained the same basal cellular respiration in hypoxia as normoxia, whereas NO exposed cells exhibited a gradual decrease in mitochondrial respiration in hypoxia. Upon reoxygenation, the respiration in NO treated cells did not recover to pre-hypoxic levels. Hypoxia-reoxygenation in the presence of NO was associated with inhibition of autophagy and the inability to recovery during reoxygenation was exacerbated by the inhibitor of autophagy, 3-methyladenine. The effects of hypoxia could be recapitulated by inhibiting glycolytic flux under normoxic conditions. Under both normoxic and hypoxic conditions NO exposure induced immediate stimulation of glycolysis but prolonged NO exposure, associated with irreversible inhibition of mitochondrial respiration in hypoxia, inhibited glycolysis. Importantly, we found that NO inhibited basal respiration under normoxic conditions only when glucose was absent from the media or glycolysis was inhibited by 2-deoxy-D-glucose, revealing a novel NO-dependent mechanism for the inhibition of mitochondrial respiration which is modulated by glycolysis. Taken together these data suggest an oxygen-dependent interaction between mitochondrial respiration, glycolysis and autophagy in protecting neuronal cells exposed to NO. Importantly, they indicate that mitochondrial dysfunction is intimately linked to a failure of glycolytic flux induced by exposure to NO. In addition, these studies provide new insights into understanding how autophagy and NO may play an interactive role in neuroinflammation-induced cellular damage which is pertinent to our understanding of the pathology neurodegenerative diseases in which excessive NO is generated.

Parkinson's disease is a neurodegenerative movement disorder. The histopathology of Parkinson's disease comprises proteinaceous inclusions known as Lewy bodies, which contains aggregated α-synuclein. Cathepsin D (CD) is a lysosomal protease previously demonstrated to cleave α-synuclein and decrease its toxicity in both cell lines and mouse brains in vivo. Here, we show that pharmacological inhibition of CD, or introduction of catalytically inactive mutant CD, resulted in decreased CD activity and increased cathepsin B activity, suggesting a possible compensatory response to inhibition of CD activity. However, this increased cathepsin B activity was not sufficient to maintain α-synuclein degradation, as evidenced by the accumulation of endogenous α-synuclein. Interestingly, the levels of LC3, LAMP1, and LAMP2, proteins involved in autophagy-lysosomal activities, as well as total lysosomal mass as assessed by LysoTracker flow cytometry, were unchanged. Neither autophagic flux nor proteasomal activities differs between cells over-expressing wild-type versus mutant CD. These observations point to a critical regulatory role for that endogenous CD activity in dopaminergic cells in α-synuclein homeostasis which cannot be compensated for by increased Cathepsin B. These data support the potential need to enhance CD function in order to attenuate α-synuclein accumulation as a therapeutic strategy against development of synucleinopathy. Insufficiencies in the autophagy-lysosome pathway may contribute to neurodegenerative disease pathogenesis and progression. We found that a catalytically inactive mutant cathepsin D (D295N CD) decreases endogenous CD activity, similar to inhibition of endogenous CD activity by the addition of CD inhibitor, PepA, leads to accumulation of α-synuclein, without significant changes of overall autophagy activities. As accumulation of α-synuclein is a main pathological feature of Parkinson's disease (PD), this study further demonstrate a crucial role of CD activities in attenuation of synucleinopathies such as PD.

Background Huntington's disease is caused by aggregation of mutant huntingtin (mHtt) protein containing more than a 36 polyQ repeat. Upregulation of macroautophagy was suggested as a neuroprotective strategy to degrade mutant huntingtin. However, macroautophagy initiation has been shown to be highly efficient in neurons whereas lysosomal activities are rate limiting. The role of the lysosomal and other proteases in Huntington is not clear. Some studies suggest that certain protease activities may contribute to toxicity whereas others are consistent with protection. These discrepancies may be due to a number of mechanisms including distinct effects of the specific intermediate digestion products of mutant huntingtin generated by different proteases. These observations suggested a critical need to investigate the consequence of upregulation of individual lysosomal enzyme in mutant huntingtin accumulation and toxicity. Results In this study, we used molecular approaches to enhance lysosomal protease activities and examined their effects on mutant huntingtin level and toxicity. We found that enhanced expression of lysosomal cathepsins D and B resulted in their increased enzymatic activities and reduced both full-length and fragmented huntingtin in transfected HEK cells. Furthermore, enhanced expression of cathepsin D or B protected against mutant huntingtin toxicity in primary neurons, and their neuroprotection is dependent on macroautophagy. Conclusions These observations demonstrate a neuroprotective effect of enhancing lysosomal cathepsins in reducing mutant huntingtin level and toxicity in transfected cells. They highlight the potential importance of neuroprotection mediated by cathepsin D or B through macroautophagy.

Neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, are a group of age-dependent, progressive disorders that exhibit prominent neuronal death. Alzheimer’s disease and Parkinson’s disease are mainly sporadic, whereas Huntington’s disease is entirely genetic. Studies on human postmortem brains highlighted the possible involvement of apoptosis and autophagy in neuron death in the diseases. Studies using genetically engineered mouse models confirmed contributions of key apoptosis genes in disease progression in these experimental systems. In addition, mouse models confirmed that neurotoxins may accelerate and exacerbate disease progression. A better understanding of neuron death mechanisms in these diseases will help design better treatment strategies.

α-synuclein (α-syn) is a main component of Lewy bodies (LB) that occur in many neurodegenerative diseases, including Parkinson's disease (PD), dementia with LB (DLB) and multi-system atrophy. α-syn mutations or amplifications are responsible for a subset of autosomal dominant familial PD cases, and overexpression causes neurodegeneration and motor disturbances in animals. To investigate mechanisms for α-syn accumulation and toxicity, we studied a mouse model of lysosomal enzyme cathepsin D (CD) deficiency, and found extensive accumulation of endogenous α-syn in neurons without overabundance of α-syn mRNA. In addition to impaired macroautophagy, CD deficiency reduced proteasome activity, suggesting an essential role for lysosomal CD function in regulating multiple proteolytic pathways that are important for α-syn metabolism. Conversely, CD overexpression reduces α-syn aggregation and is neuroprotective against α-syn overexpression-induced cell death in vitro. In a C. elegans model, CD deficiency exacerbates α-syn accumulation while its overexpression is protective against α-syn-induced dopaminergic neurodegeneration. Mutated CD with diminished enzymatic activity or overexpression of cathepsins B (CB) or L (CL) is not protective in the worm model, indicating a unique requirement for enzymatically active CD. Our data identify a conserved CD function in α-syn degradation and identify CD as a novel target for LB disease therapeutics.

Apoptosis is a highly regulated cellular suicide program crucial for metazoan development. Yeast counterparts of central metazoan apoptotic regulators, such as metacaspase Yca1p, have been identified. In spite of the importance of Yca1p in yeast apoptotic process, many other factors such as Aif1p, orthologs of EndoG, AMID and cyclophilin D play important roles in caspase-independent apoptotic pathways. This review summarized recent progress about studies of various intrinsic and extrinsic apoptotic stimuli that may induce yeast cell death via caspase-independent apoptosis.

Apoptosis-inducing factor (AIF) and AIF-homologous mitochondrion-associated inducer of death (AMID) are both mitochondrial flavoproteins that trigger caspase-independent apoptosis. Phylogenetic analysis suggests that these two proteins evolutionarily diverge back from their common prokaryote ancestor. Compared with AIF, the proapoptotic nature of AMID and its mode of action are much less clarified. Here, we show that overexpression of yeast AMID homologue internal NADH dehydrogenase (NDI1), but not external NADH dehydrogenase (NDE1), can cause apoptosis-like cell death, and this effect can be repressed by increased respiration on glucose-limited media. This result indicates that the regulatory network of energy metabolism, in particular the cross-talk between mitochondria and the rest of the cell, is involved in Ndi1p-induced yeast cell apoptosis. The apoptotic effect of NDI1 overexpression is associated with increased production of reactive oxygen species (ROS) in mitochondria. In addition, NDI1 overexpression in sod2 background causes cell lethality in both fermentable and semifermentable media. Interruption of certain components in the electron transport chain can suppress the growth inhibition from Ndi1p overexpression. We finally show that disruption of NDI1 or NDE1 decreases ROS production and elongates the chronological life span of yeast, accompanied by the loss of survival fitness. Implication of these findings for Ndi1p-induced apoptosis is discussed.

Reoxygenation Maximal Reserve Capacity O C R ( p m o l/ m in /μ g p tn ) Control DADS 50μM DetaNO 150μM DetaNO+DADS A Natural H2S Donor Found in Garlic Attenuates Ischemia-Reperfusion Damage in Neurons by Protecting Mitochondrial Respiration Gloria A. Benavides1,2, Qiuli Liang 1,2, Jianhua Zhang1,2, Victor M. Darley-Usmar1,2 1Center for Free Radical Biology, 2Department of Pathology. University of Alabama at Birmingham