Yung-Feng Liao

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-β (Aβ) 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 Aβ generation, including APP, APP-C99 and β-/γ-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 Aβ peptides and Tau aggregates. Nonetheless, compelling data also reveal an unfavorable function of autophagy in facilitating the production of intracellular Aβ. The two faces of autophagy on the homeostasis of Aβ 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.

Silencing of miR-124 induces neuroblastoma SK-N-SH cell differentiation, cell cycle arrest and apoptosis through promoting AHR.

Neuroblastoma is the most common extracranial solid tumor in children. We investigate whether miR‐124, the abundant neuronal miRNA, plays a pivotal role in neuroblastoma. Knockdown of miR‐124 promotes neuroblastoma SK‐N‐SH cell differentiation, cell cycle arrest and apoptosis. Further miR‐124 is predicted to target aryl hydrocarbon receptor (AHR) which may promote neuroblastoma cell differentiation. We validate that miR‐124 may suppress the expression of AHR by targeting its 3′‐UTR. These results suggest that miR‐124 could serve as a potential therapeutic target of neuroblastoma.

Tumor Necrosis Factor-α–elicited Stimulation of γ-Secretase Is Mediated by c-Jun N-terminal Kinase-dependent Phosphorylation of Presenilin and Nicastrin

Gamma-secretase is a multiprotein complex composed of presenilin (PS), nicastrin (NCT), Aph-1, and Pen-2, and it catalyzes the final proteolytic step in the processing of amyloid precursor protein to generate amyloid-beta. Our previous results showed that tumor necrosis factor-alpha (TNF-alpha) can potently stimulate gamma-secretase activity through a c-Jun N-terminal kinase (JNK)-dependent pathway. Here, we demonstrate that TNF-alpha triggers JNK-dependent serine/threonine phosphorylation of PS1 and NCT to stimulate gamma-secretase activity. Blocking of JNK activity with a potent JNK inhibitor (SP600125) reduces TNF-alpha-triggered phosphorylation of PS1 and NCT. Consistent with this, we show that activated JNKs can be copurified with gamma-secretase complexes and that active recombinant JNK2 can promote the phosphorylation of PS1 and NCT in vitro. Using site-directed mutagenesis and a synthetic peptide, we clearly show that the Ser(319)Thr(320) motif in PS1 is an important JNK phosphorylation site that is critical for the TNF-alpha-elicited regulation of gamma-secretase. This JNK phosphorylation of PS1 at Ser(319)Thr(320) enhances the stability of the PS1 C-terminal fragment that is necessary for gamma-secretase activity. Together, our findings strongly suggest that JNK is a critical intracellular mediator of TNF-alpha-elicited regulation of gamma-secretase and governs the pivotal step in the assembly of functional gamma-secretase.

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.

Notch1 Expression Predicts an Unfavorable Prognosis and Serves as a Therapeutic Target of Patients with Neuroblastoma

Purpose: Notch signaling has been implicated to play a critical role in the tumorigenesis of neuroblastoma (NB) and can modulate calreticulin (CRT) expression that strongly correlates with tumor differentiation and favorable prognosis of NB. We thus sought to determine how Notch regulates CRT expression and affects NB tumor behavior. Experimental Design: The Notch-dependent regulation of CRT expression in cultured NB cells was analyzed by confocal microscopy and Western blotting. Notch1 protein expression in 85 NB tumors was examined by immunohistochemistry and correlated with the clinicopathologic/biological characters of NB patients. The progression of NB tumors in response to attenuated Notch signaling was examined by using a xenograft mouse model. Results: We showed that CRT is essential for the neuronal differentiation of NB cells elicited by inhibition of Notch signaling. This effect was mediated by a c-Jun-NH2-kinase–dependent pathway. Furthermore, NB tumors with elevated Notch1 protein expression were strongly correlated with advanced tumor stages, MYCN amplification, an undifferentiated histology, as well as a low CRT expression level. Most importantly, the opposing effect between Notch1 and CRT could reciprocally affect the survival of NB patients. The administration of a γ-secretase inhibitor into a xenograft mouse model of NB significantly suppressed the tumor progression. Conclusions: Our findings provide the first evidence that a c-Jun-NH2-kinase-CRT–dependent pathway is essential for the neuronal differentiation elicited by Notch signaling blockade and that Notch1 and CRT can synergistically predict the clinical outcomes of NB patients. The present data suggest that Notch signaling could be a therapeutic target for NB. Clin Cancer Res; 16(17); 4411–20. ©2010 AACR.

Characterization of Neuroblastic Tumors Using 18F-FDOPA PET

Neuroblastic tumors are childhood neoplasms that possess amino acid decarboxylase (AADC) activity and can theoretically be imaged by 18F-fluorodihydroxyphenylalanine (18F-FDOPA) PET, a new diagnostic tool for neuroendocrine tumors. In this study, we explored the accuracy and clinical role of 18F-FDOPA PET in neuroblastic tumors. Methods: From 2008 to 2011, patients with tissue-proven neuroblastic tumors receiving 18F-FDOPA PET at initial diagnosis or during follow-ups were enrolled. The sensitivity and specificity of 18F-FDOPA PET were compared with those of 123I-metaiodobenzylguanidine (123I-MIBG) scintigraphy and 18F-FDG PET, using tumor histology as the standard. The maximum standardized uptake value and tumor-to-liver uptake ratio on 18F-FDOPA PET were measured and correlated with AADC messenger RNA level in tumor tissue. Results: Fifty tumors from 34 patients, including 42 neuroblastic tumors and 8 lesions without viable tumor cells, were eligible for analysis. 18F-FDOPA PET successfully detected neuroblastic tumors of different histologic types in various anatomic sites, at a sensitivity of 97.6% (87.4%–99.9%) and a specificity of 87.5% (47.3%–99.7%). In tumors with concomitant studies, 18F-FDOPA PET demonstrated a higher sensitivity than 123I-MIBG scintigraphy (n = 18; P = 0.0455) or 18F-FDG PET (n = 46; P = 0.0455). Among the 18 tumors with concomitant 123I-MIBG scans, 4 tumors with viable cells were 123I-MIBG–negative but were successfully detected by 18F-FDOPA PET. The tumor uptake of 18F-FDOPA significantly correlated with AADC expression (n = 15 nonhepatic tumors; maximum standardized uptake value, P = 0.0002; tumor-to-liver uptake ratio, P < 0.0001). Conclusion: 18F-FDOPA PET showed high sensitivity and specificity in detecting and tracking neuroblastic tumors in this preliminary study with a small cohort of patients and might be complementary to 123I-MIBG scintigraphy and 18F-FDG PET. By correlating with AADC expression, 18F-FDOPA PET might serve as a useful imaging tool for the functional assessment of neuroblastic tumors.

Identification of GRP75 as an Independent Favorable Prognostic Marker of Neuroblastoma by a Proteomics Analysis

Purpose: Neuroblastoma (NB) is a heterogeneous neoplasm. Detailed biological discrimination is critical for the effective treatment of this disease. Because the tumor behavior of NB is closely associated with the histologic state of differentiation, we thus aimed to identify novel differentiation-associated markers of NB with prognostic implication. Experimental Design: A human NB cell line SH-SY5Y was used as a model system to explore potential biomarkers for the differentiation of NB by proteomic analyses. Seventy-two NB tumor tissues were subsequently investigated by immunohistochemistry to validate the correlations between the expression of a novel prognostic marker, various clinicopathologic and biological factors, and patient survival. Results: Using two-dimensional differential gel electrophoresis, we found a total of 24 spots of proteins in SH-SY5Y cells whose expression was enhanced following differentiation. Glucose-regulated protein 75 (GRP75) was unambiguously identified as one of the five proteins that were dramatically up-regulated following differentiation. Immunohistochemical analyses of 72 NB tumor tissues further revealed that positive GRP75 immunostaining is strongly correlated with differentiated histologies (P < 0.001), mass-screened tumors (P = 0.016), and early clinical stages (P < 0.001) but inversely correlated with MYCN amplification (P = 0.010). Univariate and multivariate survival analyses showed that GRP75 expression is an independent favorable prognostic factor. Conclusions: The present findings clearly showed that our proteomics-based novel experimental paradigm could be a powerful tool to uncover novel biomarkers associated with the differentiation of NB. Our data also substantiate an essential role of GRP75 in the differentiation of NB.

B4GALNT3 Expression Predicts a Favorable Prognosis and Suppresses Cell Migration and Invasion via β1 Integrin Signaling in Neuroblastoma

β1,4-N-acetylgalactosaminyltransferase III (B4GALNT3) promotes the formation of GalNAcβ1,4GlcNAc (LacdiNAc or LDN). Drosophila β1,4-N-acetylgalactosaminyltransferase A (B4GALNTA) contributes to the synthesis of LDN, which helps regulate neuronal development. In this study, we investigated the expression and role of B4GALNT3 in human neuroblastoma (NB). We used IHC analysis to examine 87 NB tumors, and we identified correlations between B4GALNT3 expression and clinicopathologic factors, including patient survival. Effects of recombinant B4GALNT3 on cell behavior and signaling were studied in SK-N-SH and SH-SY5Y NB cells. Increased expression of B4GALNT3 in NB tumors correlated with a favorable histologic profile (P < 0.001, χ² test) and early clinical staging (P = 0.041, χ² test) and was a favorable prognostic factor for survival as evaluated by univariate and multivariate analyses. Reexpression of B4GALNT3 in SK-N-SH and SH-SY5Y cells suppressed cell proliferation, colony formation, migration, and invasion. Moreover, B4GALNT3 increased the LacdiNAc modification of β₁ integrin, leading to decreased phosphorylation of focal adhesion kinase (FAK), Src, paxillin, Akt, and ERK1/2. B4GALNT3-mediated suppression of cell migration and invasion were substantially reversed by concomitant expression of constitutively active Akt or MEK. We conclude that B4GALNT3 predicts a favorable prognosis for NB and suppresses the malignant phenotype via decreasing β₁ integrin signaling.

The evolutionarily conserved interaction between LC3 and p62 selectively mediates autophagy-dependent degradation of mutant huntingtin.

Mammalian p62/sequestosome-1 protein binds to both LC3, the mammalian homologue of yeast Atg8, and polyubiquitinated cargo proteins destined to undergo autophagy-mediated degradation. We previously identified a cargo receptor-binding domain in Atg8 that is essential for its interaction with the cargo receptor Atg19 in selective autophagic processes in yeast. We, thus, sought to determine whether this interaction is evolutionally conserved from yeast to mammals. Using an amino acid replacement approach, we demonstrate that cells expressing mutant LC3 (LC3-K30D, LC3-K51A, or LC3-L53A) all exhibit defective lipidation of LC3, a disrupted LC3–p62 interaction, and impaired autophagic degradation of p62, suggesting that the p62-binding site of LC3 is localized within an evolutionarily conserved domain. Importantly, whereas cells expressing these LC3 mutants exhibited similar overall autophagic activity comparable to that of cells expressing wild-type LC3, autophagy-mediated clearance of the aggregation-prone mutant Huntingtin was defective in the mutant-expressing cells. Together, these results suggest that p62 directly binds to the evolutionarily conserved cargo receptor-binding domain of Atg8/LC3 and selectively mediates the clearance of mutant Huntingtin.

Sodium selenite inhibits γ-secretase activity through activation of ERK

Previous studies have demonstrated that the ERK MAPK acts as a negative regulator of gamma-secretase. Here, we demonstrate that the activation of ERK MAPK pathway by sodium selenite can inhibit endogenous gamma-secretase activity. Consistently, the gamma-secretase-mediated production of amyloid-beta (Abeta) was dramatically attenuated by sodium selenite in a temporal manner. To substantiate the functional role of ERK MAPK in the regulation of gamma-secretase, we demonstrate that cells transfected with the wild-type MEK1 and a constitutively active mutant of MEK1 also displayed a significant attenuation of gamma-secretase activity. The active purified ERK1/2 can significantly reduce the gamma-secretase-mediated processing of C99, possibly through inducing alterations in the phosphorylation of both nicastrin and presenilin-1. Together, our data suggest that the selenite-elicited ERK activation could effectively reduce Abeta production, supporting that selenium compounds could represent a novel class of nutrient supplements to slow down the progression of Alzheimers disease.