Artificial intelligence-based analysis of time-lapse images of sphere formation and process of plate adhesion and spread of pancreatic cancer cells.

Background: Most pancreatic cancers are pancreatic ductal adenocarcinomas (PDAC). Spherical morphology formed in three-dimensional (3D) cultures and the effects of anticancer drugs differ between epithelial and mesenchymal PDAC cell lines. In the human pancreas, cancer cells form 3D tumors, migrate to adjacent tissues, and metastasize to other organs. However, no effective methods exist to examine the ability of the tumor mass to migrate to surrounding tissues in vitro. We used spheres formed in 3D culture to investigate whether the migratory ability of tumors of PDAC cell lines, including epithelial and mesenchymal cell lines, varies. Methods: Sphere formation and adhesion and spread on culture plates were examined by artificial intelligence-based analysis of time-lapse imaging using five epithelial and three mesenchymal PDAC cell lines. Fused and non-fused areas of the sphere surface during sphere formation on low-attachment plates, the adhesion area to normal culture plates, and the sphere area maintaining its original form during adhesion to plates were measured. Results: Immunocytochemical staining confirmed that E-cadherin was highly expressed in epithelial PDAC spheres, as was vimentin in mesenchymal PDAC spheres, in 2D culture. When forming spheres using low-attachment plates, most epithelial PDAC cell lines initially showed decreased sphere area, and then the covering cells fused to form a smooth surface on the sphere. Mesenchymal PANC-1 and MIA PaCa-2 cells showed little reduction in sphere area and few areas of sphere surface fusion. When formed PDAC spheres were seeded onto normal culture plates, spheres of epithelial PK-8 cells-which have the highest E-cadherin expression, form numerous cysts, and have smooth sphere surfaces-did not adhere to normal plates even after 60 h, and epithelial PK45-P and T3M-4 spheres hardly adhered. Conversely, the area of adhesion and spread of mesenchymal PANC-1 and KP4 cell spheres on normal plates markedly increased from early on, forming large areas of attachment to plates. Conclusion: Seeding spheres formed in 3D culture onto culture plates can clarify differences in tumor migration potential to surrounding areas. The masses formed by each PDAC cell line varied in migratory ability, with mesenchymal PDAC masses being more migratory than epithelial PDAC masses.

Accelerated telomere shortening in adrenal zona reticularis in patients with prolonged critical illness.

Background: The number of patients with prolonged critical illness (PCI) has been increasing in many countries, and the adrenal gland plays an important role in maintaining homeostasis during PCI. Chronic disease burden is reportedly associated with shorter telomere lengths in human tissues. Telomere shortening in human somatic cells is largely dependent on cell divisions, and critically short telomeres lead to cellular dysfunction and aging. However, the association between PCI and telomere lengths in human adrenal cells is poorly understood. In this study, we investigated this association to assess whether the burden of PCI could accelerate the aging process in adrenal cells. Methods: Adrenocortical tissues from patients who died after PCI usually show a diffuse pattern of intracellular cholesterol ester depletion (i.e., lipid depletion). This study examined near-normal adrenal glands obtained from autopsied patients who died suddenly (control group) and lipid-depleted adrenal glands obtained from autopsied patients who died after PCI (PCI group). The control group included 7 men aged 80 to 94 years (mean age: 85.3 years) and 7 women aged 84 to 94 years (mean age: 87.7 years). The PCI group included 10 men aged 71 to 88 years (mean age: 78.8 years) and 8 women aged 77 to 95 years (mean age: 85.6 years). By using quantitative fluorescence in situ hybridization, relative telomere lengths (RTLs) were determined in the parenchymal cells of the three adrenocortical zones (zona glomerulosa, zona fasciculata, and zona reticularis [ZR]) and in the chromaffin cells of the medulla. The number of adrenal parenchymal cells was determined by immunohistochemistry and digital image analysis. Results: RTLs in ZR cells were significantly shorter in the PCI group than in the control group for both men and women (P = 0.0001 for men and P = 0.0012 for women). However, RTLs in the remaining three types of adrenal cells did not differ between the control and PCI groups for both men and women. The number of ZR cells was higher in the PCI group than in the control group for both men and women (P < 0.0001 for both men and women). The proportion of the number of ZR cells to the total number of adrenocortical parenchymal cells was also higher in the PCI group than in the control group (P < 0.0001 for both men and women). The Ki-67 proliferation index in ZR cells was higher in the PCI group than in the control group (P = 0.0039 for men and P = 0.0063 for women). Conclusions: This study demonstrated ZR cell-specific telomere shortening in patients with adrenal lipid depletion who died after PCI. Our results suggest that the reactive proliferation of ZR cells accelerates the telomere shortening and aging process in ZR cells in these patients. The results of our study may contribute to the understanding of adrenal aging during PCI.

Multiple cystic sphere formation from PK-8 cells in three-dimensional culture.

Three-dimensional (3D) culture of cancer cells mimics the in vivo environment. Recently, we reported that pancreatic ductal adenocarcinoma (PDAC) cell lines with epithelial and mesenchymal features formed differently shaped spheres in 3D culture. However, only PK-8 cells, the epithelial PDAC cell line with the highest E-cadherin expression among the eight PDAC cell lines, formed multiple cystic spheres in 3D culture. Optical coherence tomography revealed interconnected cysts inside the spheres. A weak inter-cellular adhesion, individual cell degeneration, necrosis, and secretory granules in the cytoplasm were observed in the PK-8 spheres using electron microscopy. The expression of MUC1, MUC5AC, and amylase was increased in PK-8 cells in the 3D culture compared with that in 2D culture. These findings suggest that highly E-cadherin-expressing epithelial PK-8 cells form multiple cystic spheres, which may be promoted by enhanced mucin and amylase synthesis in 3D culture.

Gp130-Mediated STAT3 Activation Contributes to the Aggressiveness of Pancreatic Cancer through H19 Long Non-Coding RNA Expression.

Signaling pathways involving signal transducer and activator of transcription 3 (STAT3) play key roles in the aggressiveness of pancreatic ductal adenocarcinoma (PDAC), including their tumorigenesis, invasion, and metastasis. Cancer stem cells (CSCs) have been correlated with PDAC aggressiveness, and activation of STAT3 is involved in the regulation of CSC properties. Here, we investigated the involvement of interleukin-6 (IL-6) or the leukemia inhibitory factor (LIF)/glycoprotein 130 (gp130)/STAT3 pathway and their role in pancreatic CSCs. In PDAC CSC-like cells formed by culturing on a low attachment plate, autocrine/paracrine IL-6 or LIF contributes to gp130/STAT3 pathway activation. Using a gp130 inhibitor, we determined that the gp130/STAT3 pathway contributes to the maintenance of stemness features, the expression of membrane-type 1 matrix metalloproteinase (MT1-MMP), and the invasion of PDAC CSC-like cells. The gp130/STAT3 pathway also modulates the transforming growth factor (TGF)-ß1/Smad pathway required for epithelial-mesenchymal transition induction through regulation of TGFß-RII expression in PDAC CSC-like cells. Furthermore, chromatin immunoprecipitation assays revealed that p-STAT3 can access the active promoter region of H19 to influence this metastasis-related long non-coding RNA and contribute to its transcription in PDAC CSC-like cells. Therefore, the autocrine/paracrine IL-6 or LIF/gp130/STAT3 pathway in PDAC CSC-like cells may eventually facilitate invasion and metastasis, two hallmarks of malignancy. We propose that inhibition of the gp130/STAT3 pathway provides a promising strategy for targeting CSCs for the treatment of PDAC.

Epithelial and Mesenchymal Features of Pancreatic Ductal Adenocarcinoma Cell Lines in Two- and Three-Dimensional Cultures.

Pancreatic ductal adenocarcinoma (PDAC) is an intractable cancer that is difficult to diagnose early, and there is no cure other than surgery. PDAC is classified as an adenocarcinoma that has limited effective anticancer drug and molecular-targeted therapies compared to adenocarcinoma found in other organs. A large number of cancer cell lines have been established from patients with PDAC that have different genetic abnormalities, including four driver genes; however, little is known about the differences in biological behaviors among these cell lines. Recent studies have shown that PDAC cell lines can be divided into epithelial and mesenchymal cell lines. In 3D cultures, morphological and functional differences between epithelial and mesenchymal PDAC cell lines were observed as well as the drug effects of different anticancer drugs. These effects included gemcitabine causing an increased growth inhibition of epithelial PDAC cells, while nab-paclitaxel caused greater mesenchymal PDAC cell inhibition. Thus, examining the characteristics of epithelial or mesenchymal PDAC cells with stromal cells using a 3D co-culture may lead to the development of new anticancer drugs.

Morphofunctional analysis of human pancreatic cancer cell lines in 2- and 3-dimensional cultures.

Genetic, transcriptional, and morphological differences have been reported in pancreatic ductal adenocarcinoma (PDAC) cases. We recently found that epithelial or mesenchymal features were enhanced in three-dimensional (3D) cultures compared to two-dimensional (2D) cultures. In this study, we examined the differences in the morphological and functional characteristics of eight PDAC cell lines in 2D and 3D cultures. Most PDAC cells showed similar pleomorphic morphologies in 2D culture. Under 3D culture, PDAC cells with high E-cadherin and low vimentin expression levels (epithelial) formed small round spheres encircled with flat lining cells, whereas those with high vimentin and low E-cadherin expression levels (mesenchymal) formed large grape-like spheres without lining cells and were highly proliferative. In 3D culture, gemcitabine was more effective for the spheres formed by PDAC cells with epithelial features, while abraxane was more effective on those with mesenchymal features. The expression levels of drug transporters were highest PDAC cells with high vimentin expression levels. These findings indicate that PDAC cells possess various levels of epithelial and mesenchymal characteristics. The 3D-culture method is useful for investigating the diversity of PDAC cell lines and may play important roles in the development of personalized early diagnostic methods and anticancer drugs for PDAC.

Polyvinyl alcohol increased growth, migration, invasion, and sphere size in the PK-8 pancreatic ductal adenocarcinoma cell line.

Polyvinyl alcohol (PVA) is a water-soluble synthetic polymer used in eye drops, embolization particles, and artificial cartilage. It has also been shown to cause expansion of functional multipotent self-renewing hematopoietic stem cells under serum-free conditions. In this study, we examined the effects of PVA on human pancreatic ductal adenocarcinoma (PDAC) cell lines using 2-dimensional (2D) and 3D-cultures with serum-free medium. In the 2D-culture, PVA-treatment induced an aggregated colony-like appearance in PDAC cells. It increased the growth of PK-8 cells in a dose-dependent manner as well as significantly increasing migration and invasion abilities. qRT-PCR showed an increase in α2 integrin and a decrease in matrix metalloprotease levels in PVA-treated PK-8 cells. Through qRT-PCR analysis, ß1 integrin expression at the mRNA level was found to be decreased; however, it was unaltered at the protein level when assessed using FACS analysis. PVA further induced mesenchymal to epithelial transition-like alterations, including increased E-cadherin and decreased Vimentin and N-cadherin expression. Four cancer stem cell (CSC) markers were higher in PVA-treated PK-8 cells compared to controls. In 3D-culture, PVA-treated PK-8 cells showed a rod-like appearance with larger sphere size and higher growth ability. qRT-PCR showed that CSC markers did not increase and 2 of 4 drug transporters had decreased in PVA-treated PK-8 cells. These findings suggest that PVA increases the growth, migration, invasion, and sphere size of PK-8 cells, but does not increase the proportion of pancreatic CSCs under 3D-culture conditions with serum-free medium.

FGFR4 Inhibitor BLU9931 Attenuates Pancreatic Cancer Cell Proliferation and Invasion While Inducing Senescence: Evidence for Senolytic Therapy Potential in Pancreatic Cancer.

Fibroblast growth factor receptor 4 (FGFR4), one of four tyrosine kinase receptors for FGFs, is involved in diverse cellular processes. Activation of FGF19/FGFR4 signaling is closely associated with cancer development and progression. In this study, we examined the expression and roles of FGF19/FGFR4 signaling in human pancreatic ductal adenocarcinoma (PDAC). In human PDAC cases, FGFR4 expression positively correlated with larger primary tumors and more advanced stages. Among eight PDAC cell lines, FGFR4 was expressed at the highest levels in PK-1 cells, in which single-nucleotide polymorphism G388R in FGFR4 was detected. For inhibition of autocrine/paracrine FGF19/FGFR4 signaling, we used BLU9931, a highly selective FGFR4 inhibitor. Inhibition of signal transduction through ERK, AKT, and STAT3 pathways by BLU9931 reduced proliferation in FGF19/FGFR4 signaling-activated PDAC cells. By contrast, BLU9931 did not alter stemness features, including stemness marker expression, anticancer drug resistance, and sphere-forming ability. However, BLU9931 inhibited cell invasion, in part, by downregulating membrane-type matrix metalloproteinase-1 in FGF19/FGFR4 signaling-activated PDAC cells. Furthermore, downregulation of SIRT1 and SIRT6 by BLU9931 contributed to senescence induction, priming these cells for quercetin-induced death, a process termed senolysis. Thus, we propose that BLU9931 is a promising therapeutic agent in FGFR4-positive PDAC, especially when combined with senolysis (195/200).

Characterization of the metastatic potential of the floating cell component of MIA PaCa-2, a human pancreatic cancer cell line.

In pancreatic cancer, morphologically and functionally heterogeneous cancer cells reside within the same patient. The heterogeneity is believed to promote metastasis and resistance to chemoradiotherapy. MIA PaCa-2, an established human pancreatic ductal adenocarcinoma (PDAC) cell line, contains round and spindle-shaped adherent cells, as well as, round floating cells. In this study, we aimed to assess if the floating cells might have greater metastatic potential and/or be more resistant to drug-induced apoptosis compared to adherent cells. Time-lapse analysis revealed that the two types of adherent cells transformed bilaterally, and some of the adherent, round cells converted to floating cells. Flow cytometry and electron microscopy showed that approximately 90% of the floating cells were viable. qRT-PCR analysis revealed that floating cells expressed lower levels of integrins and ATP-binding cassette (ABC) transporters than adherent cells. In contrast, except for vimentin, floating cells expressed more epithelial to mesenchymal transition markers than adherent cells. Floating cells included a larger population of G2/M-phase cells, and migration assays revealed a decreased migration ability by floating cells relative to adherent cells. A cell aggregation assay showed that the aggregative properties of the floating cells were lower than those of the adherent cells. In 3D culture, spheres derived from floating cells were more sensitive to anti-cancer drugs, including gemcitabine, 5-FU, and abraxane, than those derived from adherent cells. Expression levels of stemness markers in the spheres derived from floating cells were lower than those derived from adherent cells. Morphological characterization of human PDAC cell lines may help to clarify the series of alterations cancer cells undergo during the metastatic process and may contribute to the development of new PDAC diagnostics and more patient-specific treatments for those with PDAC.

Correlation Between Telomere Attrition of Zona Fasciculata and Adrenal Weight Reduction in Older Men.

CONTEXT: Although numerous theories are reported on sex differences in longevity, the underlying biological mechanisms remain unknown. We previously reported that telomere length in the zona reticularis cells of the human adrenal cortex was significantly longer in older than that in younger subjects. However, we could not evaluate sex differences in the telomere lengths. OBJECTIVE: To compare the telomere lengths of adrenocortical and adrenal medullar cells between men and women from infancy through older adulthood. METHODS: Adrenal glands of 30 male (aged 0 to 100 years) and 25 female (aged 0 to 104 years) autopsied subjects were retrieved from autopsy files. Using quantitative fluorescence in situ hybridization, relative telomere lengths were determined in the parenchymal cells of the 3 adrenocortical zones and medulla. Age-related changes in the weight of adrenal glands were also investigated. MAIN RESULTS: Older male subjects (aged 65 years or older) had significantly shorter telomere lengths in zona fasciculata (ZF) cells compared to the corresponding female subjects. In men, older subjects exhibited a significant age-related reduction in adrenal weight; however, no age-related changes in adrenal weight were detected in women. CONCLUSION: Telomere attrition of ZF cells was correlated with adrenal weight reduction in older men but not in older women, suggesting a decreased number of ZF cells in older men. This may help us understand the possible biological mechanisms of sex difference in longevity of humans.

Ganglioside GM2, highly expressed in the MIA PaCa-2 pancreatic ductal adenocarcinoma cell line, is correlated with growth, invasion, and advanced stage.

Gangliosides, a group of glycosphingolipids, are known to be cell surface markers and functional factors in several cancers. However, the association between gangliosides and pancreatic ductal adenocarcinoma (PDAC) has not been well elucidated. In this study, we examined the expression and roles of ganglioside GM2 in PDAC. GM2+ cells showed a higher growth rate than GM2- cells in the adherent condition. When GM2- and GM2+ cells were cultured three-dimensionally, almost all cells in the spheres expressed GM2, including cancer stem cell (CSC)-like cells. A glycolipid synthesis inhibitor reduced GM2 expression and TGF-ß1 signaling in these CSC-like cells, presumably by inhibiting the interaction between GM2 and TGFß RII and suppressing invasion. Furthermore, suppression of GM2 expression by MAPK inhibition also reduced TGF-ß1 signaling and suppressed invasion. GM2+ cells formed larger subcutaneous tumors at a high incidence in nude mice than did GM2- cells. In PDAC cases, GM2 expression was significantly associated with younger age, larger tumor size, advanced stage and higher histological grade. These findings suggest that GM2 could be used as a novel diagnostic and therapeutic target for PDAC.

Enhanced morphological and functional differences of pancreatic cancer with epithelial or mesenchymal characteristics in 3D culture.

Pancreatic cancer, composed of heterogeneous cancer cells, alters epithelial to mesenchymal features during growth and metastasis. In this study, we aimed to characterize pancreatic ductal adenocarcinoma (PDAC) cells showing epithelial or mesenchymal features in 3D culture. In 3D culture, PK-1 cells had high E-cadherin and low vimentin expression and exhibited a round-like appearance encircled by flat cells. PANC-1 cells had high vimentin and low E-cadherin expression and formed grape-like spheres. PK-1 cells had secretary granules and many microvilli, desmosomes, and adherens junctions, while PANC-1 cells had few microvilli, adherens junction, and no desmosomes. Cytokeratin 7, trypsin, CA19-9, and E-cadherin were highly expressed in PK-1 cells but not in PANC-1 cells. Ki-67 was diffusely expressed in PANC-1 spheres but was restricted to the peripheral flat cells of PK-1 spheres. PANC-1 and PK-1 cells were positive for transforming growth factor (TGF) ß receptor II and phosphorylated smad2/3, but PK-1 cells were smad4 negative. Taken together, 3D culture enhanced morphofunctional differences of PDAC cells showing epithelial or mesenchymal characteristics, and epithelial phenotype maintenance may be due to the ineffectiveness of the TGF- ß pathway. Clarification of heterogeneity using 3D culture may be useful for development of individualized diagnostic and therapeutic methods in patients with PDAC.

Comparison of functional glycans between cancer stem cells and normal stem cells.

Cancer stem cells (CSCs) are a small group of cells within a tumor that preserve stemness and enhance regrowth of cancer cells. CSCs have important implications in resistance to conventional therapies and tumor relapse, although their detailed properties remain unknown. Thus, CSCs represent promising targets to improve cancer treatment. So far, a number of cell surface markers containing glycans have been exploited to identify and isolate CSCs. Cell surface glycans are well-known markers for specific cell types and also play important cellular roles, such as regulation of cell signaling. In normal stem cells, including embryonic and tissue stem cells, glycan markers in an undifferentiated state have been identified. These markers are mostly known to regulate signaling pathways required for maintenance of stemness. In contrast, CSC-specific glycans have not been well characterized yet. In this review, we summarize functional commonalities between CSCs and normal stem cells in glycan-mediated signaling pathways. Identification of CSC-specific glycans may lead to early diagnosis and radical treatment of cancer.

Fetal bovine serum enlarges the size of human pancreatic cancer spheres accompanied by an increase in the expression of cancer stem cell markers.

Pancreatic ductal adenocarcinoma (PDAC) is a major histological type of pancreatic cancer and remains one of the most lethal cancers with a high mortality rate owing to its aggressive growth, high metastatic rate, and recurrence. Recent studies on cancer stem cells (CSCs) have suggested that the aggressive growth, high metastatic rate, and recurrence might be caused by the ability of CSCs to self-renew, differentiate, and drive tumorigenesis. Thus, CSCs are expected to be a therapeutic target for PDAC. Sphere forming assay of cancer cells, including PDAC cells, is commonly performed using epidermal growth factor and fibroblast growth factor-2 containing serum-free medium to identify and isolate the enriched CSCs. Recently, we observed that PDAC spheres cultured in fetal bovine serum containing medium are morphologically similar to spheres cultured in the growth factor containing medium. In this study, we cultured two PDAC cell lines, PANC-1 and PK-1, in growth factor containing serum-free medium or serum containing medium, and compared the morphology of the spheres formed in detail by electron microscopy and examined the expression of major CSC marker genes. Both the PDAC cells formed larger spheres in the serum containing medium than in the growth factor containing medium. PK-1 cells formed more morphologically differentiated spheres, with peripheral flat lining cells, in the serum containing medium. Expression levels of most of the CSC markers were higher in the spheres of the two PDAC cells in both the culture mediums than in the cells cultured under adherent conditions. The expression levels of CSC markers in PDAC spheres cultured in the growth factor containing medium were not necessarily higher than that in the spheres cultured in the serum containing medium. These findings suggest that sphere forming assay using serum containing medium, by which large PDAC spheres with enriched CSCs are formed, may be useful for deciphering the characteristics of CSCs and for developing anti-CSC therapies for PDAC.

Up-regulation of NSP3 by Oligomeric Aß Accelerates Neuronal Death Through Cas-independent Rap1A Activation.

ß-Amyloid (Aß) plays an important role in the early pathogenesis of Alzheimer's disease (AD). In vitro studies have demonstrated that Aß oligomers induce hippocampal and neocortical neuronal death. However the neurotoxic mechanisms by which soluble Aß oligomers cause neuronal damage and death remain to be fully elucidated. To this end, we analyzed the gene expression profile of rat cerebral cortical neurons treated with Aß oligomers in vitro. Aß treatment induced the expression of novel SH2-containing protein 3 (NSP3), an adaptor molecule interacting with Cas family proteins. NSP3 expression was upregulated not only in oligomeric-Aß-treated cultured neurons but also in the neocortex of aged Tg2576 mice. NSP3 overexpression in cultured cortical neurons accelerated neuronal death. The C-terminal region of NSP3 unbound to a Cas protein was necessary for the NSP3-induced acceleration of neuronal death, as was Cas-independent Rap1A activation downstream of NSP3. Moreover, NSP3 RNAi knockdown partially rescued Aß-oligomer-treated neurons. These results indicate that NSP3 upregulation by soluble Aß oligomers may accelerate neuronal death via Cas-independent Rap1A activation, implicating NSP3 in the pathogenesis of AD.

The role of calsyntenin-3 in dystrophic neurite formation in Alzheimer's disease brain.

ß-Amyloid (Aß) oligomers may play an important role in the early pathogenesis of Alzheimer's disease: cognitive impairment caused by synaptic dysfunction. Dystrophic neurites surrounding Aß plaques, another pathological feature of Alzheimer's disease, are plaque-associated neuritic alterations preceding the appearance of synaptic loss. In the present review, we focus on the mechanism of dystrophic neurite formation by Aß oligomers, and discuss the neurotoxic role of Aß-induced calsyntenin-3 in mediating dystrophic neurite formation.

Kinesin light chain-1 isoform E does not interact with calsyntenin-1.

ß-Amyloid is generated by the sequential cleavage of amyloid precursor protein. Calsyntenin-1 and kinesin light chain-1 splice variant E (KLC1-E) have been proposed to regulate ß-amyloid production from amyloid precursor protein. Vesicles containing calsyntenin-1 are transported from the Golgi apparatus to axons by interaction between calsyntenin-1 and KLC1 in their C-terminal regions. However, it is unclear whether KLC1 isoform E influences the interaction between KLC1 and calsyntenin-1, resulting in the impaired axonal transport of calsyntenin-1 vesicles. Here, we show that KLC1-E does not interact with calsyntenin-1 using a pull-down assay, coimmunoprecipitation, and immunocytochemistry. These findings suggest that KLC1-E enrichment may impair the axonal transport of calsyntenin-1 vesicles.

Calsyntenin-3 C-terminal fragment accumulates in dystrophic neurites surrounding aß plaques in tg2576 mouse and Alzheimer disease brains: its neurotoxic role in mediating dystrophic neurite formation.

Dystrophic neurites surrounding ß-amyloid (Aß) plaques precede neuronal death in Alzheimer disease. These neuritic alterations may be one of the initial stages for synaptic loss and dysfunction. However, intracellular pathways that cause local disruption of neuronal processes by Aß remain to be fully elucidated. The identification of Aß-induced genes that mediate neuritic pathology would provide considerable insight into the mechanisms of Alzheimer's disease. Previously, we reported that selective up-regulation of calsyntenin-3 (Cst-3) by Aß and accumulation of neurotoxic Cst-3 in dystrophic neurites surrounding Aß plaques may lead to local disruption of these neurites. Like amyloid precursor protein, Cst-3 undergoes two-step proteolytic processing: the primary cleavage with α-secretase generates an N-terminal ectodomain and a C-terminal fragment (CTF). The CTF is subsequently cleaved into p3 peptide and an intracellular domain via γ-secretase. It would be interesting to know whether accumulated Cst-3 in dystrophic neurites surrounding Aß plaques is the full-length version or a CTF. Herein, we show that the CTF but not full-length Cst-3 accumulated in dystrophic neurites surrounding Aß plaques in Tg2576 mouse and Alzheimer disease brains. In vitro experiments with Cst-3 fragments have revealed that only the CTF resulted in acceleration of neuronal death. These results indicate that accumulation of the neurotoxic CTF in neurites surrounding Aß plaques may lead to local disruption of neuronal processes and development of dystrophic neurites.

MAP1B 1-126 interacts with tubulin isoforms and induces neurite outgrowth and neuronal death of cultured cortical neurons.

Previously we reported that overexpression of MAP1B containing N-terminal 126 amino acids promoted neuronal death. Here, we identified α-, ß-, and ßIII-tubulins as proteins interacting with MAP1B 1-126 by two-hybrid and pull-down assays. Transfection experiments indicated that MAP1B 1-126 interacts with microtubules, but to a much lesser extent than two previously reported microtubule-binding domains. Overexpression of MAP1B 1-126 induced both neurite extension and neuronal death, suggesting that MAP1B 1-126 could be involved in neuronal degeneration and aberrant sprouting.