Nobuhisa Aoyagi

GSK3β Activity Modifies the Localization and Function of Presenilin 1

Presenilin 1, a causative gene product of familial Alzheimer disease, has been reported to be localized mainly in the endoplasmic reticulum and Golgi membranes. However, endogenous Presenilin 1 also localizes at the plasma membrane as a biologically active molecule. Presenilin 1 interacts with N-cadherin/β-catenin to form a trimeric complex at the synaptic site through its loop domain, whose serine residues (serine 353 and 357) can be phosphorylated by glycogen synthase kinase 3β. Here, we demonstrate that cell-surface expression of Presenilin 1/γ-secretase is enhanced by N-cadherin-based cell-cell contact. Physical interaction between Presenilin 1 and N-cadherin/β-catenin plays an important role in this process. Glycogen synthase kinase 3β-mediated phosphorylation of Presenilin 1 reduces its binding to N-cadherin, thereby down-regulating its cell-surface expression. Moreover, reduction of the Presenilin 1·N-cadherin·β-catenin complex formation leads to an impaired activation of contact-mediated phosphatidylinositol 3-kinase/Akt cell survival signaling. Furthermore, phosphorylation of Presenilin 1 hinders ϵ-cleavage of N-cadherin, whereas ϵ-cleavage of APP remained unchanged. This is the first report that clarifies the regulatory mechanism of Presenilin 1/γ-secretase with respect to its subcellular distribution and its differential substrate cleavage. Because the cleavage of various membrane proteins by Presenilin 1/γ-cleavage is involved in cellular signaling, glycogen synthase kinase 3β-mediated phosphorylation of Presenilin 1 should be deeply associated with signaling functions. Our findings indicate that the abnormal activation of glycogen synthase kinase 3β can reduce neuronal viability and synaptic plasticity via modulating Presenilin 1/N-cadherin/β-catenin interaction and thus have important implications in the pathophysiology of Alzheimer disease.

N-cadherin Regulates p38 MAPK Signaling via Association with JNK-associated Leucine Zipper Protein IMPLICATIONS FOR NEURODEGENERATION IN ALZHEIMER DISEASE

Synaptic loss, which strongly correlates with the decline of cognitive function, is one of the pathological hallmarks of Alzheimer disease. N-cadherin is a cell adhesion molecule essential for synaptic contact and is involved in the intracellular signaling pathway at the synapse. Here we report that the functional disruption of N-cadherin-mediated cell contact activated p38 MAPK in murine primary neurons, followed by neuronal death. We further observed that treatment with Aβ42 decreased cellular N-cadherin expression through NMDA receptors accompanied by increased phosphorylation of both p38 MAPK and Tau in murine primary neurons. Moreover, expression levels of phosphorylated p38 MAPK were negatively correlated with that of N-cadherin in human brains. Proteomic analysis of human brains identified a novel interaction between N-cadherin and JNK-associated leucine zipper protein (JLP), a scaffolding protein involved in the p38 MAPK signaling pathway. We demonstrated that N-cadherin expression had an inhibitory effect on JLP-mediated p38 MAPK signal activation by decreasing the interaction between JLP and p38 MAPK in COS7 cells. Also, this study demonstrated a novel physical and functional association between N-cadherin and p38 MAPK and suggested neuroprotective roles of cadherin-based synaptic contact. The dissociation of N-cadherin-mediated synaptic contact by Aβ may underlie the pathological basis of neurodegeneration such as neuronal death, synaptic loss, and Tau phosphorylation in Alzheimer disease brain.

Amyloid β inhibits ectodomain shedding of N-cadherin via down-regulation of cell-surface NMDA receptor

Dysfunction in the synapse is recognized as an early and the primary pathological process in Alzheimers disease (AD). N-cadherin, an essential adhesion molecule for excitatory synaptic contact, forms a complex with presenilin 1 (PS1) and beta-catenin in the synaptic membrane. N-cadherin is sequentially cleaved by ADAM10 and PS1/gamma-secretase, producing a cytoplasmic fragment, N-cadherin C-terminal fragment (Ncad/CTF2) after NMDA receptor stimulation [Marambaud P, Wen PH, Dutt A, Shioi J, Takashima A, Siman R, Robakis NK (2003) A CBP binding transcriptional repressor produced by the PS1/epsilon-cleavage of N-cadherin is inhibited by PS1 FAD mutations. Cell 114:635-645; Reiss K, Maretzky T, Ludwig A, Tousseyn T, de Strooper B, Hartmann D, Saftig P (2005) ADAM10 cleavage of N-cadherin and regulation of cell-cell adhesion and beta-catenin nuclear signalling. EMBO J 24:1762]. Ncad/CTF2 translocates to the nucleus together with beta-catenin to enhance beta-catenin nuclear signaling [Uemura K, Kihara T, Kuzuya A, Okawa K, Nishimoto T, Bito H, Ninomiya H, Sugimoto H, Kinoshita A, Shimohama S (2006a) Activity-dependent regulation of beta-catenin via epsilon-cleavage of N-cadherin. Biochem Biophys Res Commun 345:951-958]. To examine whether an impairment of N-cadherin metabolism is involved in AD pathogenesis, we investigated the effect of amyloid beta peptide (Abeta) treatment on sequential N-cadherin cleavage. Here, we demonstrate that both synthetic and cell-derived Abeta species inhibit ectodomain shedding of mouse N-cadherin. Inhibition of N-cadherin cleavage by Abeta treatment was suggested to be mediated by the enhanced endocytosis of NMDA receptor, resulting in reduced turnover of N-cadherin. Since both N-cadherin and beta-catenin are essential for synaptic plasticity, impairment of N-cadherin cleavage caused by Abeta may underlie the synapse toxicity involved in AD pathogenesis.

Presenilin 1 is involved in the maturation of β-site amyloid precursor protein-cleaving enzyme 1 (BACE1)

One of the pathologic hallmarks of Alzheimers disease is the excessive deposition of β‐amyloid peptides (Aβ) in senile plaques. Aβ is generated when β‐amyloid precursor protein (APP) is cleaved sequentially by β‐secretase, identified as β‐site APP‐cleaving enzyme 1 (BACE1), and γ‐secretase, a putative enzymatic complex containing presenilin 1 (PS1). However, functional interaction between PS1 and BACE1 has never been known. In addition to this classical role in the generation of Aβ peptides, it has also been proposed that PS1 affects the intracellular trafficking and maturation of selected membrane proteins. We show that the levels of exogenous and endogenous mature BACE1 expressed in presenilin‐deficient mouse embryonic fibroblasts (PS−/−MEFs) were reduced significantly compared to those in wild‐type MEFs. Moreover, the levels of mature BACE1 were increased in human neuroblastoma cell line, SH‐SY5Y, stably expressing wild‐type PS1, compared to native cells. Conversely, the maturation of BACE1 was compromised under the stable expression of dominant–negative mutant PS1 overexpression. Immunoprecipitation assay showed that PS1 preferably interacts with proBACE1 rather than mature BACE1, indicating that PS1 can be directly involved in the maturation process of BACE1. Further, endogenous PS1 was immunoprecipitated with endogenous BACE1 in SH‐SY5Y cells and mouse brain tissue. We conclude that PS1 is directly involved in the maturation of BACE1, thus possibly functioning as a regulator of both β‐ and γ‐secretase in Aβ generation.

N-cadherin-based adhesion enhances Aβ release and decreases Aβ42/40 ratio

In neurons, Presenilin 1(PS1)/γ‐secretase is located at the synapses, bound to N‐cadherin. We have previously reported that N‐cadherin‐mediated cell–cell contact promotes cell‐surface expression of PS1/γ‐secretase. We postulated that N‐cadherin‐mediated trafficking of PS1 might impact synaptic PS1‐amyloid precursor protein interactions and Aβ generation. In the present report, we evaluate the effect of N‐cadherin‐based contacts on Aβ production. We demonstrate that stable expression of N‐cadherin in Chinese hamster ovary cells, expressing the Swedish mutant of human amyloid precursor protein leads to enhanced secretion of Aβ in the medium. Moreover, N‐cadherin expression decreased Aβ42/40 ratio. The effect of N‐cadherin expression on Aβ production was accompanied by the enhanced accessibility of PS1/γ‐secretase to amyloid precursor protein as well as a conformational change of PS1, as demonstrated by the fluorescence lifetime imaging technique. These results indicate that N‐cadherin‐mediated synaptic adhesion may modulate Aβ secretion as well as the Aβ42/40 ratio via PS1/N‐cadherin interactions.

PI3K inhibition causes the accumulation of ubiquitinated presenilin 1 without affecting the proteasome activity.

gamma-Secretase is an enzymatic complex, composed of presenilin 1 (PS1), nicastrin, pen-2, and aph-1, and is responsible for the intramembranous cleavage of various type-I membrane proteins. The level of each component is tightly regulated in a cell via proteasomal degradation. On the other hand, it has previously been reported that PS1/gamma-secretase is involved in the activation of phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway. PI3K is inhibited in Alzheimers disease (AD) brain, whereas the effects of PI3K inhibition on the metabolism of PS1/gamma-secretase have not been elucidated. Here, we demonstrate that the treatment of neurons with PI3K inhibitors leads to increased levels of PS1/gamma-secretase components through an inhibitory effect on their degradation. Moreover, PI3K inhibition accelerated ubiquitination of PS1. We further show the evidence that the PS1 ubiquitination after PI3K inhibition is represented by the multiple mono-ubiquitination, instead of poly-ubiquitination. Accordingly, treatment of cells with PI3K inhibitor led to a differential intracellular redistribution of PS1 from the one observed after the proteasomal inhibition. These results suggest that PI3K inhibition may trigger the multiple mono-ubiquitination of PS1, which precludes the degradation of PS1/gamma-secretase through the proteasomal pathway. Since PS1/gamma-secretase is deeply involved in the production of Abeta protein, a deeper knowledge into its metabolism could contribute to a better elucidation of AD pathogenesis.

Analysis of N-cadherin interacting proteins in Alzheimer's diesase

and 3 6 0.25 year old) were used in this study. Animals were randomly assigned to 2 groups as follows: Group 1 (n1⁄4 6), normal chow and group 2 (n 1⁄4 6), chow supplemented with 2% cholesterol . Cholesterol-treated animals and their matched controls were euthanized 12 weeks later. Results: Cholesterol-enrichded diets inhibit active Akt and m-TOR. Inhibition of Akt is associated with increased levels of GSK3ab, an enzyme that increases bamyloid and phosphorylated tau production. Inhibition of mTOR also causes tau hyperphosphorylation. Conclusions: Our results suggest that cholesterol-enriched diets cause AD-like pathology by mechanisms involving an Akt/mTOR signaling pathway.

Nicotinic receptor stimulation is protective for dopaminergic neuron

Epidemiological studies reported that paraquat, a hervicide, is one of the environmental risk factors of Parkinson disease. Paraquat induced apoptosis in PC12 cells. The endoplasmic reticulum (ER) stress was found to be involved in this paraquat -induced apoptosis because upregulation of CHOP and activation of Jun-N-terminal kinase (JNK), ER stress activated substrates, were observed. Upregulation of mRNA of BiP, an ER-specific chaperone, and splicing of the mRNA of the X box binding protein were observed, indicating the occurrence of the unfolded protein response (UPR). Prominent production of reactive oxygen species (ROS) was observed after paraquat exposure. Pre-incubation of phytoestrogens such as resveratrol prevalent in the red wine and genistein derived from soybean reduced the paraquat -induced ROS. Resvertrol reduced the UPR and increased the viability of PC12 cells was increased. In contrast, genistein was less effective to prevent the ER stress and the paraquat-induced cell death.