Pannexin 1 Modulates Angiogenic Activities of Human Endothelial Colony-Forming Cells Through IGF-1 Mechanism and Is a Marker of Senescence.

BACKGROUND: We examined the role of Panxs (pannexins) in human endothelial progenitor cell (EPC) senescence. METHODS: Young and replication-induced senescent endothelial colony-forming cells (ECFCs) derived from human circulating EPCs were used to examine cellular activities and senescence-associated indicators after transfection of short interference RNA specific to Panx1 or lentivirus-mediated Panx1 overexpression. Hind limb ischemia mice were used as in vivo angiogenesis model. Protein and phospho-kinase arrays were used to determine underlying mechanisms. RESULTS: Panx1 was the predominant Panx isoform in human ECFCs and upregulated in both replication-induced senescent ECFCs and circulating EPCs from aged mice and humans. Cellular activities of the young ECFCs were enhanced by Panx1 downregulation but attenuated by its upregulation. In addition, reduction of Panx1 in the senescent ECFCs could rejuvenate cellular activities with reduced senescence-associated indicators, including senescence-associated ß-galactosidase activity, p16INK4a (cyclin-dependent kinase inhibitor 2A), p21 (cyclin-dependent kinase inhibitor 1), acetyl-p53 (tumor protein P53), and phospho-histone H2A.X (histone family member X). In mouse ischemic hind limbs injected senescent ECFCs, blood perfusion ratio, salvaged limb outcome, and capillary density were all improved by Panx1 knockdown. IGF-1 (insulin-like growth factor 1) was significantly increased in the supernatant from senescent ECFCs after Panx1 knockdown. The enhanced activities and paracrine effects of Panx1 knockdown senescent ECFCs were completely inhibited by anti-IGF-1 antibodies. FAK (focal adhesion kinase), ERK (extracellular signal-regulated kinase), and STAT3 (signal transducer and activator of transcription 3) were activated in senescent ECFCs with Panx1 knockdown, in which the intracellular calcium level was reduced, and the activation was inhibited by supplemented calcium. The increased IGF-1 in Panx1-knockdown ECFCs was abrogated, respectively, by inhibitors of FAK (PF562271), ERK (U0126), and STAT3 (NSC74859) and supplemented calcium. CONCLUSIONS: Panx1 expression is upregulated in human ECFCs/EPCs with replication-induced senescence and during aging. Angiogenic potential of senescent ECFCs is improved by Panx1 reduction through increased IGF-1 production via activation of the FAK-ERK axis following calcium influx reduction. Our findings provide new strategies to evaluate EPC activities and rejuvenate senescent EPCs for therapeutic angiogenesis.

Dual-Specificity Phosphatase 15 (DUSP15) Modulates Notch Signaling by Enhancing the Stability of Notch Protein.

Dual-specificity phosphatases (DUSPs) comprise a unique group of enzymes that dephosphorylate signaling proteins at both phospho-serine/threonine and phospho-tyrosine residues. Since Notch signaling is an essential pathway for neuronal cell fate determination and development that is also upregulated in Alzheimer's disease tissues, we sought to explore whether and how DUSPs may impact Notch processing. Our results show that overexpression of DUSP15 concomitantly and dose-dependently increased the steady-state levels of recombinant Notch (extracellular domain-truncated Notch, NotchΔE) protein and its cleaved product, Notch intracellular domain (NICD). The overall ratio of NotchΔE to NICD was unchanged by overexpression of DUSP15, suggesting that the effect is independent of γ-secretase. Interestingly, overexpression of DUSP15 also dose-dependently increased phosphorylated ERK1/2. Phosphorylated ERK1/2 is known to be positively correlated with Notch protein level, and we found that DUSP15-mediated regulation of Notch was dependent on ERK1/2 activity. Together, our findings reveal the existence of a previously unidentified DUSP15-ERK1/2-Notch signaling axis, which could potentially play a role in neuronal differentiation and neurological disease.

Activation of Aryl Hydrocarbon Receptor by Kynurenine Impairs Progression and Metastasis of Neuroblastoma.

Neuroblastoma is the most common malignant disease of infancy, and amplification of the MYCN oncogene is closely associated with poor prognosis. Recently, expression of MYCN was shown to be inversely correlated with aryl hydrocarbon receptor (AHR) expression in neuroblastoma, and overexpression of AHR downregulated MYCN expression, promoting cell differentiation. Therefore, we further investigated the potential of AHR to serve as a prognostic indicator or a therapeutic target in neuroblastoma. First, the clinical significance of AHR in neuroblastoma was examined. Positive AHR immunostaining strongly correlated with differentiated histology of neuroblastoma and predicted better survival for patients. The mouse xenograft model showed that overexpression of AHR significantly suppressed neuroblastoma tumor growth. In addition, activation of AHR by the endogenous ligand kynurenine inhibited cell proliferation and promoted cell differentiation in vitro and in vivo. kynurenine treatment also upregulated the expression of KISS1, a tumor metastasis suppressor, and attenuated metastasis in the xenograft model. Finally, analysis of KISS1 levels in neuroblastoma patient tumors using the R2: Genomics Analysis and Visualization Platform revealed that KISS1 expression positively correlated with AHR, and high KISS1 expression predicted better survival for patients. In conclusion, our results indicate that AHR is a novel prognostic biomarker for neuroblastoma, and that overexpression or activation of AHR offers a new therapeutic possibility for patients with neuroblastoma. SIGNIFICANCE: These findings show that AHR may function as a tumor suppressor in childhood neuroblastoma, potentially influencing the aetiologic and therapeutic targeting of the disease.

Novel Endogenous Ligands of Aryl Hydrocarbon Receptor Mediate Neural Development and Differentiation of Neuroblastoma.

Aryl hydrocarbon receptor (AHR) signaling has been suggested to play roles in various physiological functions independent of its xenobiotic activity, including cell cycle regulation, immune response, and embryonic development. Several endogenous ligands were also identified by high-throughput screening techniques. However, the mechanism by which these molecules mediate AHR signaling in certain functions is still elusive. In this study, we investigated the possible pathway through which AHR and its endogenous ligands regulate neural development. We first identified two neuroactive steroids, 3α,5α-tetrahydrocorticosterone and 3α,5ß-tetrahydrocorticosterone (5α- and 5ß-THB), as novel AHR endogenous ligands through the use of an ultrasensitive dioxin-like compound bioassay and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS). We then treated zebrafish embryos with 5α- and 5ß-THB, which enhance the expression of neurogenesis marker HuC. Furthermore, 5α- and 5ß-THB both enhanced the expression of myelinating glial cell markers, sex determining region Y-box 10 (Sox10), and myelin-associated proteins myelin basic protein (Mbp) and improved the mobility of zebrafish larvae via the Ahr2 pathway. These results indicated that AHR mediates zebrafish neurogenesis and gliogenesis, especially the differentiation of oligodendrocyte or Schwann cells. Additionally, we showed that these molecules may induce neuroblastoma (NB) cell differentiation suggesting therapeutic potential of 5α- and 5ß-THB in NB treatment. In summary, our results reveal that 5α- and 5ß-THB are endogenous ligands of AHR and have therapeutic potential for NB treatment. By the interaction with THB, AHR signaling regulates various aspects of neural development.

KC21 Peptide Inhibits Angiogenesis and Attenuates Hypoxia-Induced Retinopathy.

Desmogleins (Dsg2) are the major components of desmosomes. Dsg2 has five extracellular tandem cadherin domains (EC1-EC5) for cell-cell interaction. We had previously confirmed the Dsg2 antibody and its epitope (named KC21) derived from EC2 domain suppressing epithelial-mesenchymal transition and invasion in human cancer cell lines. Here, we screened six peptide fragments derived from EC2 domain and found that KR20, the parental peptide of KC21, was the most potent one on suppressing endothelial colony-forming cell (ECFC) tube-like structure formation. KC21 peptide also attenuated migration but did not disrupt viability and proliferation of ECFCs, consistent with the function to inhibit VEGF-mediated activation of p38 MAPK but not AKT and ERK. Animal studies showed that KC21 peptides suppressed capillary growth in Matrigel implant assay and inhibited oxygen-induced retinal neovascularization. The effects were comparable to bevacizumab (Bev). In conclusion, KC21 peptide is an angiogenic inhibitor potentially useful for treating angiogenesis-related diseases.

Quantitative Measurement of γ-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms.

We have developed a pair of cell-based reporter gene assays to quantitatively measure γ-secretase cleavage of distinct substrates. This manuscript describes procedures that may be used to monitor γ-secretase-mediated cleavage of either APP-C99 or Notch, using a Gal4 promoter-driven firefly luciferase reporter system. These assays were established by stably co-transfecting HEK293 cells with the Gal4-driven luciferase reporter gene and either the Gal4/VP16-tagged C-terminal fragment of APP (APP-C99; CG cells), or the Gal4/VP16-tagged Notch-ΔE (NΔE; NG cells). Using these reporter assays in parallel, we have demonstrated that an ErbB2 inhibitor, CL-387,785, can preferentially suppress γ-secretase cleavage of APP-C99 in CG cells, but not NΔE in NG cells. The differential responses exhibited by the CG and NG cells, when treated with CL-387,785, represent a preferred characteristic for γ-secretase modulators, and these responses are in stark contrast to the pan-inhibition of γ-secretase induced by DAPT. Our studies provide direct evidence that γ-secretase activities toward different substrates can be differentiated in a cellular context. These new assays may therefore be useful tools in drug discovery for improved AD therapies.

VEGF expression correlates with neuronal differentiation and predicts a favorable prognosis in patients with neuroblastoma.

Neuroblastoma (NB) is a childhood cancer with a low survival rate and great metastatic potential. Vascular endothelial growth factor (VEGF), an angiogenesis factor, has been found to be involved in CRT-related neuronal differentiation of NB cells. In this study, we further confirmed the role VEGF in NB through mouse xenograft model and clinical analysis from NB patients. In xenograft experiments, CRT overexpression effectively inhibited the tumor growth. In addition, the mRNA and protein levels of VEGF and differentiation marker GAP-43 were upregulated by induced CRT expression. However, no significant correlation between the expression level of VEGF and microvessel density was observed in human NB tumors, suggesting a novel mechanism of VEGF participating in NB tumorigenesis through an angiogenesis-independent pathway. In NB patients' samples, mRNA expression levels of CRT and VEGF were positively correlated. Furthermore, positive VEGF expression by immunostaining of NB tumors was found to correlate well with histological grade of differentiation and predicted a favorable prognosis. In conclusion, our findings suggest that VEGF is a favorable prognostic factor of NB and might affect NB tumor behavior through CRT-driven neuronal differentiation rather than angiogenesis that might shed light on a novel therapeutic strategy to improve the outcome of NB.

Critical Roles of Dual-Specificity Phosphatases in Neuronal Proteostasis and Neurological Diseases.

Protein homeostasis or proteostasis is a fundamental cellular property that encompasses the dynamic balancing of processes in the proteostasis network (PN). Such processes include protein synthesis, folding, and degradation in both non-stressed and stressful conditions. The role of the PN in neurodegenerative disease is well-documented, where it is known to respond to changes in protein folding states or toxic gain-of-function protein aggregation. Dual-specificity phosphatases have recently emerged as important participants in maintaining balance within the PN, acting through modulation of cellular signaling pathways that are involved in neurodegeneration. In this review, we will summarize recent findings describing the roles of dual-specificity phosphatases in neurodegeneration and offer perspectives on future therapeutic directions.

ErbB2 regulates autophagic flux to modulate the proteostasis of APP-CTFs in Alzheimer's disease.

Proteolytic processing of amyloid precursor protein (APP) C-terminal fragments (CTFs) by γ-secretase underlies the pathogenesis of Alzheimer's disease (AD). An RNA interference screen using APP-CTF [99-residue CTF (C99)]- and Notch-specific γ-secretase interaction assays identified a unique ErbB2-centered signaling network that was predicted to preferentially govern the proteostasis of APP-C99. Consistently, significantly elevated levels of ErbB2 were confirmed in the hippocampus of human AD brains. We then found that ErbB2 effectively suppressed autophagic flux by physically dissociating Beclin-1 from the Vps34-Vps15 complex independent of its kinase activity. Down-regulation of ErbB2 by CL-387,785 decreased the levels of C99 and secreted amyloid-ß in cellular, zebrafish, and mouse models of AD, through the activation of autophagy. Oral administration of an ErbB2-targeted CL-387,785 for 3 wk significantly improves the cognitive functions of APP/presenilin-1 (PS1) transgenic mice. This work unveils a noncanonical function of ErbB2 in modulating autophagy and establishes ErbB2 as a therapeutic target for AD.

Calreticulin Regulates VEGF-A in Neuroblastoma Cells.

Calreticulin (CRT) has been previously correlated with the differentiation of neuroblastoma (NB), implying a favorable prognostic factor. Vascular endothelial growth factor (VEGF) has been reported to participate in the behavior of NB. This study investigated the association of CRT and VEGF-A in NB cells. The expressions of VEGF-A and HIF-1α, with overexpression or knockdown of CRT, were measured in three NB cells (SH-SY5Y, SK-N-DZ, and stNB-V1). An inducible CRT NB cell line and knockdown CRT stable cell lines were also established. The impacts of CRT overexpression on NB cell apoptosis, proliferation, and differentiation were also evaluated. We further examined the role of VEGF-A in the NB cell differentiation via VEGF receptor blockade. Constitutive overexpression of CRT led to NB cell differentiation without proliferation. Thus, an inducible CRT stNB-V1 cell line was generated by a tetracycline-regulated gene system. CRT overexpression increased VEGF-A and HIF-1α messenger RNA (mRNA) expressions in SH-SY5Y, SK-N-DZ, and stNB-V1 cells. CRT overexpression also enhanced VEGF-A protein expression and secretion level in conditioned media in different NB cell lines. Knockdown of CRT decreased VEGF-A and HIF-1α mRNA expressions and lowered VEGF-A protein expression and secretion level in conditioned media in different NB cell lines. We further demonstrated that NB cell apoptosis was not affected by CRT overexpression in stNB-V1 cells. Nevertheless, overexpression of CRT suppressed cell proliferation and enhanced cell differentiation in stNB-V1 cells, whereas blockage of VEGFR-1 markedly suppressed the expression of neuron-specific markers including GAP43, NSE2, and NFH, as well as TrkA, a molecular marker indicative of NB cell differentiation. Our findings suggest that VEGF-A is involved in CRT-related neuronal differentiation in NB. Our work may provide important information for developing a new therapeutic strategy to improve the outcome of NB patients.

Discovery of small molecular (D)-leucinamides as potent, Notch-sparing γ-secretase modulators.

Structural optimization of the prior lead 3 led to the small molecular (D)-leucinamides with potent modulating activity and Notch-sparing selectivity on the proteolytic processing of amyloid-ß precursor proteins. The N-(R)-epoxypropyl analog 10c exhibited potent γ-secretase modulation compared to DAPT and showed substantial substrate selection for APP cleavage over Notch cleavage, while N-(2-fluoro)benzyl analog 10e showed the most potent γ-secretase inhibition with dull selectivity. The exceptional suppression of ERK-mediated activation suggested that these potent γ-secretase modulators may adapt an alternative pathway to prominently induce the differential inhibition of C99 cleavage by γ-secretase.

Calreticulin activates ß1 integrin via fucosylation by fucosyltransferase 1 in J82 human bladder cancer cells.

Fucosylation regulates various pathological events in cells. We reported that different levels of CRT (calreticulin) affect the cell adhesion and metastasis of bladder cancer. However, the precise mechanism of tumour metastasis regulated by CRT remains unclear. Using a DNA array, we identified FUT1 (fucosyltransferase 1) as a gene regulated by CRT expression levels. CRT regulated cell adhesion through α1,2-linked fucosylation of ß1 integrin and this modification was catalysed by FUT1. To clarify the roles for FUT1 in bladder cancer, we transfected the human FUT1 gene into CRT-RNAi stable cell lines. FUT1 overexpression in CRT-RNAi cells resulted in increased levels of ß1 integrin fucosylation and rescued cell adhesion to type-I collagen. Treatment with UEA-1 (Ulex europaeus agglutinin-1), a lectin that recognizes FUT1-modified glycosylation structures, did not affect cell adhesion. In contrast, a FUT1-specific fucosidase diminished the activation of ß1 integrin. These results indicated that α1,2-fucosylation of ß1 integrin was not involved in integrin-collagen interaction, but promoted ß1 integrin activation. Moreover, we demonstrated that CRT regulated FUT1 mRNA degradation at the 3'-UTR. In conclusion, the results of the present study suggest that CRT stabilized FUT1 mRNA, thereby leading to an increase in fucosylation of ß1 integrin. Furthermore, increased fucosylation levels activate ß1 integrin, rather than directly modifying the integrin-binding sites.

Aryl hydrocarbon receptor downregulates MYCN expression and promotes cell differentiation of neuroblastoma.

Neuroblastoma (NB) is the most common malignant disease of infancy. MYCN amplification is a prognostic factor for NB and is a sign of highly malignant disease and poor patient prognosis. In this study, we aimed to investigate novel MYCN-related genes and assess how they affect NB cell behavior. The different gene expression found in 10 MYCN amplification NB tumors and 10 tumors with normal MYCN copy number were analyzed using tissue oligonucleotide microarrays. Ingenuity Pathway Analysis was subsequently performed to identify the potential genes involved in MYCN regulation pathways. Aryl hydrocarbon receptor (AHR), a receptor for dioxin-like compounds, was found to be inversely correlated with MYCN expression in NB tissues. This correlation was confirmed in a further 14 human NB samples. Moreover, AHR expression in NB tumors was found to correlate highly with histological grade of differentiation. In vitro studies revealed that AHR overexpression in NB cells induced spontaneous cell differentiation. In addition, it was found that ectopic expression of AHR suppressed MYCN promoter activity resulting in downregulation of MYCN expression. The suppression effect of AHR on the transcription of MYCN was compensated for by E2F1 overexpression, indicating that E2F1 is involved in the AHR-regulating MYCN pathway. Furthermore, AHR shRNA promotes the expression of E2F1 and MYCN in NB cells. These findings suggest that AHR is one of the upstream regulators of MYCN. Through the modulation of E2F1, AHR regulates MYCN gene expression, which may in turn affect NB differentiation.

Ligand-dependent activation of EphA4 signaling regulates the proteolysis of amyloid precursor protein through a Lyn-mediated pathway.

Alzheimer's disease is the most common dementia afflicting the elderly in modern society. This disease arises from the neurotoxicity elicited by abnormal aggregates of amyloid-ß (Aß) protein. Such aggregates form through the cleavage of amyloid precursor protein (APP) by ß-secretase and the subsequent proteolysis of the APP C-terminal fragment (APP-ßCTF or C99) by γ-secretase to yield Aß and APP intracellular domain (AICD). Recent evidence suggests that C99 and AICD may exert harmful effects on cells, suggesting that the proteolytic products of APP, including Aß, C99, and AICD, could play a pivotal role in neuronal viability. Here, we demonstrate that ligand-activated EphA4 signaling governs the proteostasis of C99, AICD, and Aß, without significantly affecting γ-secretase activity. EphA4 induced accumulation of C99 and AICD through a Lyn-dependent pathway; activation of this pathway triggered phosphorylation of EphA4, resulting in positive feedback of C99 and AICD proteostasis. Inhibition of EphA4 by dasatinib, a receptor tyrosine kinase inhibitor, effectively suppressed C99 and AICD accumulation. Furthermore, EphA4 signaling controlled C99 and AICD proteolysis through the ubiquitin-proteasome system. In conclusion, we have identified an EphA4-Lyn pathway that is essential for the metabolism of APP and its proteolytic derivatives, thereby providing novel pharmacological targets for the development of anti-Aß therapeutics for AD.

Presenilin-1 regulates the expression of p62 to govern p62-dependent tau degradation.

Mutations in presenilin-1 (PS1) are tightly associated with early-onset familial Alzheimer's disease (FAD), which is characterized by extracellular amyloid plaques and the accumulation of intracellular Tau. In addition to being the catalytic subunit of γ-secretase, PS1 has been shown to regulate diverse cellular functions independent of its proteolytic activity. We found that cells deficient in PS1 exhibit reduced levels of p62 protein, a cargo-receptor shuttling Tau for degradation. The downregulation of PS1 led to a significant decrease in both the protein and mRNA transcript of p62, concomitant with attenuated p62 promoter activity. This PS1-dependent regulation of p62 expression was mediated through an Akt/AP-1 pathway independent of the proteolytic activity of PS1/γ-secretase. This p62-mediated Tau degradation was significantly impaired in PS1-deficient cells, which can be rescued by ectopic expression of either p62 or wild-type PS1 but not mutant PS1 containing FAD-linked mutations. Our study suggests a novel function for PS1 in modulating p62 expression to control the proteostasis of Tau.

Establishment of a cell-free bioassay for detecting dioxin-like compounds.

Dioxins are byproducts from incomplete combustion processes and belong to a group of mostly toxic chemicals known as persistent organic pollutants, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is considered to be the most toxic species of all dioxin-like compounds. Analytical chemical processes are employed to determine the specific dioxin content in environmental samples. However, cost-ineffectiveness and excess time consumption limit their routine utilization. The aryl hydrocarbon receptor (AhR) is the major TCDD receptor. Upon binding to dioxin, the AhR dissociates from Hsp90 and other cofactors. TCDD-bound AhR subsequently translocates to the nucleus and interacts with the AhR nuclear translocator (Arnt) to induce signal transduction. Here, we describe a highly sensitive and cost-effective alternative assay based on detecting stability of bioluminescence signals. We generated cells that stably co-express Renilla luciferase tagged-AhR (AhR-RL), Ah receptor-interacting protein (AIP), p23 and yellow fluorescent protein-tagged Arnt (Arnt-YFP) (AAPA cells) for detection of dioxin-like compounds. Treatment with 3-methylcholanthrene (3MC), AhR agonist, enhanced the interaction between AhR and Arnt and avoided proteosomal degradation. In addition, treatment with 3MC or TCDD stabilized Renilla luminescence from AhR-RL of AAPA cell-free extracts in a concentration-dependent manner. The TCDD detection limit in this cell-free system was as low as 10(-18 )M. These results highlight the potential of AAPAA cell-free extracts to detect dioxin-like pollutants.

Retinoic acid-elicited RARα/RXRα signaling attenuates Aß production by directly inhibiting γ-secretase-mediated cleavage of amyloid precursor protein.

Retinoic acid (RA)-elicited signaling has been shown to play critical roles in development, organogenesis, and the immune response. RA regulates expression of Alzheimer's disease (AD)-related genes and attenuates amyloid pathology in a transgenic mouse model. In this study, we investigated whether RA can suppress the production of amyloid-ß (Aß) through direct inhibition of γ-secretase activity. We report that RA treatment of cells results in significant inhibition of γ-secretase-mediated processing of the amyloid precursor protein C-terminal fragment APP-C99, compared with DMSO-treated controls. RA-elicited signaling was found to significantly increase accumulation of APP-C99 and decrease production of secreted Aß40. In addition, RA-induced inhibition of γ-secretase activity was found to be mediated through significant activation of extracellular signal-regulated kinases (ERK1/2). Treatment of cells with the specific ERK inhibitor PD98059 completely abolished RA-mediated inhibition of γ-secretase. Consistent with these findings, RA was observed to inhibit secretase-mediated proteolysis of full-length APP. Finally, we have established that RA inhibits γ-secretase through nuclear retinoic acid receptor-α (RARα) and retinoid X receptor-α (RXRα). Our findings provide a new mechanistic explanation for the neuroprotective role of RA in AD pathology and add to the previous data showing the importance of RA signaling as a target for AD therapy.

Microgrooved patterns enhanced PC12 cell growth, orientation, neurite elongation, and neuritogenesis.

Understanding neurite outgrowth, orientation, and migration is important for the design of biomaterials that interface with the neuronal tissue. Micropatterns can significantly influence neurite outgrowth, neurite length, orientation, extracellular matrix expression, neuron differentiation, and migrating velocity. We analyzed the neuritogenesis and neurite outgrowth of PC12 cells in three-dimensional Si wafer with various micropatterns fabricated using photolithography and etching techniques. When nerve growth factor was added into culture medium, PC12 cells started to grow neurites. Extended neurites were significantly affected by different patterns and displayed higher growth-associated protein-43 expression. Cellular performance including growth rate, bipolar phenotype elongation, neurite extension, and growth-associated protein-43 expression of the PC12 cells with a differentiated character are higher on a grooved substrate. However, the grooved pattern can restrict the motility of PC12 cells and decrease the velocity of cellular movement. The average of the number of neurites per cell is the highest on the pillar substrate, but their neurite length is the shortest. In contrast, actin and lamimin expression, motion track, angular deviation, and movement velocity of PC12 cells are most excellent on the planar Si wafer. These findings confirmed that topographical features can cooperatively act with nerve growth factor, signaling the regulation of the formation of neurites.

Establishment of a bioluminescence-based bioassay for the detection of dioxin-like compounds.

Dioxin and dioxin-like compounds are among the most prevalent and toxic environmental pollutants. At present, analytical chemical techniques are considered the gold standard for detection of dioxins. Here, we describe a highly sensitive and cost-effective alternative, based on bioluminescence and bioluminescence resonance energy transfer (BRET). Upon binding to dioxin, aryl hydrocarbon receptor (AHR) dissociates from HSP90 and subsequently translocates to the nucleus, where it interacts with AHR nuclear translocator (ARNT). We generated cell lines that stably co-express a fusion protein of AHR and Renilla luciferase (AHR-RL) and either HSP90 or ARNT tagged with yellow fluorescent protein (HSP90-YFP or ARNT-YFP). The fluorescent signals of YFP are activated by the emission of RL while the interactions between AHR and HSP90 (or ARNT) were monitored. Application of 3-methylcholanthrene, the AHR agonist, enhances BRET signals in cells co-expressing AHR-RL, AIP-His, P23-His and ARNT-YFP (AAPA cells), while suppressing BRET signals in cells co-expressing AHR-RL, AIP-His, P23-His and HSP90-YFP (AAPH cells). In addition, dioxin treatment reduced Renilla luminescence in AAPH cells in a concentration-dependent manner, due to the degradation of AHR. Intriguingly, the detection limit for dioxin in our AHR degradation assay was as low as 10(-17) M. This work highlights the potential of AHR-RL degradation assays to detect dioxin-like pollutants.

Autophagy: a double-edged sword in Alzheimer's disease.

Autophagy is a major protein degradation pathway that is essential for stress-induced and constitutive protein turnover. Accumulated evidence has demonstrated that amyloid-beta (A beta) protein can be generated in autophagic vacuoles, promoting its extracellular deposition in neuritic plaques as the pathological hallmark of Alzheimer's disease (AD). The molecular machinery for A beta generation, including APP, APP-C99 and beta-/gamma-secretases, are all enriched in autophagic vacuoles. The induction of autophagy can be vividly observed in the brain at early stages of sporadic AD and in an AD transgenic mouse model. Accumulated evidence has also demonstrated a neuroprotective role of autophagy in mediating the degradation of aggregated proteins that are causative of various neurodegenerative diseases. Autophagy is thus widely regarded as an intracellular hub for the removal of the detrimental A beta peptides and Tau aggregates. Nonetheless, compelling data also reveal an unfavorable function of autophagy in facilitating the production of intracellular A beta. The two faces of autophagy on the homeostasis of A beta place it in a very unique and intriguing position in AD pathogenesis. This article briefly summarizes seminal discoveries that are shedding new light on the critical and unique roles of autophagy in AD and potential therapeutic approaches against autophagy-elicited AD.