Akira Kuzuya

Characterization of sequential N-cadherin cleavage by ADAM10 and PS1

N-cadherin is essential for excitatory synaptic contact in the hippocampus. At the sites of synaptic contact, it forms a complex with Presenilin 1(PS1) and beta-catenin. N-cadherin is cleaved by ADAM10 in response to NMDA receptor stimulation, producing a membrane fragment Ncad/CTF1 in neurons. NMDA receptor stimulation also enhances PS1/gamma-secretase-mediated cleavage of N-cadherin. To characterize the regulatory mechanisms of the ADAM10 and PS1-mediated cleavages, we first identified the precise cleavage sites of N-cadherin by ADAM10 and PS1/gamma-secretase by producing cleavage-deficient N-cadherin mutants. Next, we found that ectodomain shedding of N-cadherin by ADAM10 is a primary regulatory step in response to calcium influx, and that it is required for the subsequent PS1/gamma-secretase-mediated epsilon-cleavage of N-cadherin, which is a constitutive process to yield a cytoplasmic fragment, Ncad/CTF2. Since N-cadherin is essential for the structure and function of synapses including the long-term potentiation, those proteolytic events of N-cadherin should affect the adhesive behavior of the synapses, thereby taking part in learning and memory.

Exercise Is More Effective than Diet Control in Preventing High Fat Diet-induced β-Amyloid Deposition and Memory Deficit in Amyloid Precursor Protein Transgenic Mice

Background: Exercise and diet control are fundamental approaches to metabolic conditions caused by high fat diet (HFD). Results: HFD-induced memory deficit and Aβ deposition were more ameliorated in the exercise- than in the diet control-induced mice. Conclusion: Exercise was more effective than diet control in preventing HFD-induced AD development. Significance: Exercise has the highest priority in the prevention of AD. Accumulating evidence suggests that some dietary patterns, specifically high fat diet (HFD), increase the risk of developing sporadic Alzheimer disease (AD). Thus, interventions targeting HFD-induced metabolic dysfunctions may be effective in preventing the development of AD. We previously demonstrated that amyloid precursor protein (APP)-overexpressing transgenic mice fed HFD showed worsening of cognitive function when compared with control APP mice on normal diet. Moreover, we reported that voluntary exercise ameliorates HFD-induced memory impairment and β-amyloid (Aβ) deposition. In the present study, we conducted diet control to ameliorate the metabolic abnormality caused by HFD on APP transgenic mice and compared the effect of diet control on cognitive function with that of voluntary exercise as well as that of combined (diet control plus exercise) treatment. Surprisingly, we found that exercise was more effective than diet control, although both exercise and diet control ameliorated HFD-induced memory deficit and Aβ deposition. The production of Aβ was not different between the exercise- and the diet control-treated mice. On the other hand, exercise specifically strengthened the activity of neprilysin, the Aβ-degrading enzyme, the level of which was significantly correlated with that of deposited Aβ in our mice. Notably, the effect of the combination treatment (exercise and diet control) on memory and amyloid pathology was not significantly different from that of exercise alone. These studies provide solid evidence that exercise is a useful intervention to rescue HFD-induced aggravation of cognitive decline in transgenic model mice of AD.

Environmental enrichment ameliorated high-fat diet-induced Aβ deposition and memory deficit in APP transgenic mice.

The pathogenesis of Alzheimers disease (AD) is tightly associated with metabolic dysfunctions. In particular, a potential link between type 2 diabetes (T2DM) and AD has been suggested epidemiologically, clinically, and experimentally, and some studies have suggested that exercise or dietary intervention reduces risk of cognitive decline. However, there is little solid molecular evidence for the effective intervention of metabolic dysfunctions for prevention of AD. In the present study, we established the AD model mice with diabetic conditions through high-fat diet (HFD) to examine the effect of environmental enrichment (EE) on HFD-induced AD pathophysiology. Here, we demonstrated that HFD markedly deteriorated memory impairment and increased β-amyloid (Aβ) oligomers as well as Aβ deposition in amyloid precursor protein (APP) transgenic mice, which was reversed by exposure to an enriched environment for 10 weeks, despite the continuation of HFD. These studies provide solid evidence that EE is a useful intervention to ameliorate behavioral changes and AD pathology in HFD-induced aggravation of AD symptoms in APP transgenic mice.

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.

Presenilin 1 is involved in maturation and trafficking of N-cadherin to the plasma membrane

One pathological characteristic of Alzheimers disease (AD) is extensive synapse loss. Presenilin 1 (PS1) is linked to the pathogenesis of early onset familial Alzheimers disease (FAD) and is localized at the synapse, where it binds N‐cadherin and modulates its adhesive activity. To elucidate the role of the PS1/N‐cadherin interaction in synaptic contact, we established SH‐SY5Y cells stably expressing wild‐type (wt) PS1 and dominant‐negative (D385A) PS1. We show that the formation of cadherin‐based cell–cell contact among SH‐SY5Y cells stably expressing D385A PS1 was suppressed. Conversely, wt PS1 cells exhibited enhanced cell–cell contact and colony formation. Suppression of cell–cell contact in D385A cells was accompanied by an alteration in N‐cadherin subcellular localization; N‐cadherin was retained mainly in the endoplasmic reticulum (ER) and cell surface expression was reduced. We conclude that PS1 is essential for efficient trafficking of N‐cadherin from the ER to the plasma membrane. PS1‐mediated delivery of N‐cadherin to the plasma membrane is important for N‐cadherin to exert its physiological function, and it may control the state of cell–cell contact.

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.

Presenilin 1 mediates retinoic acid-induced differentiation of SH-SY5Y cells through facilitation of Wnt signaling.

Presenilin 1 interacts with β‐catenin, an essential component of the Wnt signaling pathway. To elucidate the role of presenilin 1‐β‐catenin interaction in neuronal differentiation, we established SH‐SY5Y cells stably expressing wild‐type presenilin 1, P117L mutant presenilin 1, which is linked to the early‐onset familial form of Alzheimers disease, and D385A mutant presenilin 1, which has no aspartyl proteinase activity. We demonstrate that SH‐SY5Y cells stably expressing D385A mutant presenilin 1 failed to differentiate in response to retinoic acid treatment. Retinoic acid caused an increase in nuclear β‐catenin levels in SH‐SY5Y cells, which was followed by an increase in cyclin D1 protein levels. Abnormal cellular accumulation of β‐catenin was observed in D385A mutant transfected cells, whereas nuclear β‐catenin and cellular cyclin D1 levels failed to increase. Conversely, SH‐SY5Y cells expressing the P117L mutant differentiated normally and showed increased nuclear β‐catenin and cellular cyclin D1 levels. These findings suggest that neuronal differentiation of SH‐SY5Y cells involves the Wnt signaling pathway and that presenilin 1 plays a crucial role in Wnt signal transduction by regulating the nuclear translocation of β‐catenin.

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.

Protein Trafficking and Alzheimers Disease

Mutations in presenilin 1 (PS1) cause early-onset familial Alzheimer;s disease (FAD). Although FAD accounts for less than 5% of all cases of Alzheimer;s disease (AD), extensive analyses of PS1 function have elucidated an important neuronal mechanism underling AD pathogenesis. PS1 is considered to be an essential component of gamma-secretase, which cleaves amyloid precursor protein (APP) at the transmembrane region and releases amyloid beta (Abeta) peptide. In addition to this well-documented function, a growing amount of evidence suggests that PS1 is involved in the intracellular trafficking of selected membrane proteins (i.e. APP, nicastrin, trkB, telencephalin). Recently, we have also shown that PS1 is involved in the trafficking of N-cadherin from the endoplasmic reticulum to the plasma membrane via the microtubule network. N-cadherin is localized at the synaptic junctional complex, providing an adhesive force across the synaptic cleft, and the its regulation is crucial for the neuron to exert its specific function, i.e. synaptic activity. In a mature neuron, polarized targeting of proteins from the cell body to the axonal and dendritic processes is essential for its proper function, especially, for the maintenance of synaptic function. Alterations in protein transport caused by a dysfunction in PS1 could lead to a disturbance in synaptic transmission and finally to neurodegeneration. This article will review the current knowledge of PS1 function in protein trafficking and discuss its potential role in AD pathogenesis.