The deposition of the amyloid beta (Abeta) peptide in neuritic plaques plays a critical role in the pathogenesis of Alzheimer's disease (AD). Abeta is generated through the proteolysis of amyloid precursor protein (APP) by the sequential actions of beta- and gamma-secretases. Although recent evidence has unveiled much about the biochemical identity and characteristics of gamma-secretase, the mechanism regulating endogenous gamma-secretase activity remains elusive. To identify possible extracellular signals and associated signaling cascades that could regulate APP proteolysis by gamma-secretase activity, we have developed a cell-based reporter gene assay by stably cotransfecting HEK293 cells with the Gal4-driven luciferase reporter gene and the Gal4/VP16-tagged C-terminal fragment of APP (C99-GV), the immediate substrate of gamma-secretase. The cleavage of C99-GV by gamma-secretase releases the transcription factor that activates luciferase expression, providing a quantitative measurement of gamma-secretase activity. Using this reporter assay, we have demonstrated that interferon-gamma, interleukin-1beta, and tumor necrosis factor-alpha can specifically stimulate gamma-secretase activity, concomitant with increased production of Abeta and the intracellular domain of APP (AICD). The gamma-secretase-dependent cleavage of Notch is also enhanced upon the stimulation of these cytokines. The cytokine-enhanced gamma-secretase activity can be suppressed by a potent inhibitor of c-Jun N-terminal kinase (JNK). Furthermore, cells transfected with dominant-positive MEKK1, one of the most potent activators of the JNK cascade, exhibit increased gamma-secretase activity, suggesting that the JNK-dependent mitogen-activated protein kinase pathway could mediate the cytokine-elicited regulation of gamma-secretase. Our studies provide direct evidence that cytokine-elicited signaling cascades control Abeta production by modulating gamma-secretase activity.

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.

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.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood, accounting for 8% to 10% of all pediatric malignancies. 1 This tumor arises from primitive neuroepithelial cells of the neural crest. 2 The behavior of NB is markedly heterogeneous, ranging from highly undifferentiated tumors with very poor outcomes to welldifferentiated benign ganglioneuroma or NB that may spontaneously regress with favorable prognosis. 3 Half of all patients with newly diagnosed NB are in a high-risk subset with poor overall survival despite intensive therapy. Therefore, it is important to develop useful prognostic tools and to understand NB pathogenesis to help design improved NB therapies.Glycosylation is regulated spatiotemporally during development of the nervous system. 4 Altered carbohydrate structures on tumors are often associated with tumor metastasis and progression. Tumor-associated carbohydrate epitopes commonly found in cancers include GM2,