Kengo Uemura

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.

Phenylpiperidine‐type γ‐secretase modulators target the transmembrane domain 1 of presenilin 1

Amyloid‐β peptide ending at the 42nd residue (Aβ42) is implicated in the pathogenesis of Alzheimers disease (AD). Small compounds that exhibit selective lowering effects on Aβ42 production are termed γ‐secretase modulators (GSMs) and are deemed as promising therapeutic agents against AD, although the molecular target as well as the mechanism of action remains controversial. Here, we show that a phenylpiperidine‐type compound GSM‐1 directly targets the transmembrane domain (TMD) 1 of presenilin 1 (PS1) by photoaffinity labelling experiments combined with limited digestion. Binding of GSM‐1 affected the structure of the initial substrate binding and the catalytic sites of the γ‐secretase, thereby decreasing production of Aβ42, possibly by enhancing its conversion to Aβ38. These data indicate an allosteric action of GSM‐1 by directly binding to the TMD1 of PS1, pinpointing the target structure of the phenylpiperidine‐type GSMs.

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.

Nicotinic receptor stimulation protects nigral dopaminergic neurons in rotenone-induced Parkinson's disease models.

Parkinsons disease (PD) is the second most common neurodegenerative disease and is characterized by dopaminergic (DA) neuronal cell loss in the substantia nigra. Although the entire pathogenesis of PD is still unclear, both environmental and genetic factors contribute to neurodegeneration. Epidemiologic studies show that prevalence of PD is lower in smokers than in nonsmokers,. Nicotine, a releaser of dopamine from DA neurons, is one of the candidates of antiparkinson agents in tobacco. To assess the protective effect of nicotine against rotenone‐induced DA neuronal cell toxicity, we examined the neuroprotective effects of nicotine in rotenone‐induced PD models in vivo and in vitro. We observed that simultaneous subcutaneous administration of nicotine inhibited both motor deficits and DA neuronal cell loss in the substantia nigra of rotenone‐treated mice. Next, we analyzed the molecular mechanisms of DA neuroprotective effect of nicotine against rotenone‐induced toxicity with primary DA neuronal culture. We found that DA neuroprotective effects of nicotine were inhibited by dihydro‐β‐erythroidine (DHβE), α‐bungarotoxin (αBuTx), and/or PI3K‐Akt/PKB (protein serine/threonine kinase B) inhibitors, demonstrating that rotenone‐toxicity on DA neurons are inhibited via activation of α4β2 or α7 nAChRs‐PI3K‐Akt/PKB pathway or pathways. These results suggest that the rotenone mouse model may be useful for assessing candidate antiparkinson agents, and that nAChR (nicotinic acetylcholine receptor) stimulation can protect DA neurons against degeneration.

Chronic cerebral hypoperfusion accelerates amyloid β deposition in APPSwInd transgenic mice

Chronic cerebral ischemia may accelerate clinicopathological changes in Alzheimers disease. We have examined whether chronic cerebral hypoperfusion accelerates amyloid beta deposition in amyloid protein precursor transgenic (APP-Tg) mouse. At 5, 8, and 11 months of age, C57Bl/6J male mice overexpressing a mutant form of the human APP bearing the both Swedish (K670N/M671L) and the Indiana (V717F) mutations (APPSwInd) and their litterrmates were subjected to either sham operation or bilateral carotid artery stenosis (BCAS) using microcoils with an internal diameter of 0.18 mm (short-period group). One month after the sham operation or BCAS, these animals were examined by immunohistochemistry for glial fibrillary acidic protein, amyloid beta(1-40) (Abeta(1-40)), amyloid beta(1-42) (Abeta(1-42)), as well as Western blotting and filter assay for Abeta. Another batch of the littermates of APPSwInd mice were subjected to either sham operation or BCAS at 3 months and were examined in the same manner after survival for 9 months (long-period group). In the BCAS-treated group, the white matter was rarefied and astroglia was proliferated. Amyloid beta(1-40) immunoreactivity was found in a few axons in the white matter after BCAS, whereas Abeta(1-42) was accumulated in the scattered cortical neurons and the axons at ages of 6 months and thereafter in the short- and long-period groups. In the neuropil, both Abeta(1-40) and Abeta(1-42) were deposited in the sham-operated and BCAS-treated mice at ages of 9 and 12 months. There were no differences between the short-period group at ages of 12 months and the long-period group. Filter assay showed an increase of Abeta fibrils in the extracellular enriched fraction. Taken together, chronic cerebral hypoperfusion increased Abeta fibrils and induced Abeta deposition in the intracellular compartment and, therefore, may accelerate the pathological changes of Alzheimers disease.

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.

Allosteric Modulation of PS1/γ-Secretase Conformation Correlates with Amyloid β42/40 Ratio

Background Presenilin 1(PS1) is the catalytic subunit of γ-secretase, the enzyme responsible for the Aβ C-terminal cleavage site, which results in the production of Aβ peptides of various lengths. Production of longer forms of the Aβ peptide occur in patients with autosomal dominant Alzheimer disease (AD) due to mutations in presenilin. Many modulators of γ-secretase function have been described. We hypothesize that these modulators act by a common mechanism by allosterically modifying the structure of presenilin. Methodology/Principal Findings To test this hypothesis we generated a genetically encoded GFP-PS1-RFP (G-PS1-R) FRET probe that allows monitoring of the conformation of the PS1 molecule in its native environment in live cells. We show that G-PS1-R can be incorporated into the γ-secretase complex, reconstituting its activity in PS1/2 deficient cells. Using Förster resonance energy transfer (FRET)-based approaches we show that various pharmacological and genetic manipulations that target either γ-secretase components (PS1, Pen2, Aph1) or γ-secretase substrate (amyloid precursor protein, APP) and are known to change Aβ42 production are associated with a consistent conformational change in PS1. Conclusions/Significance These results strongly support the hypothesis that allosteric changes in PS1 conformation underlie changes in the Aβ42/40 ratio. Direct measurement of physiological and pathological changes in the conformation of PS1/γ-secretase may provide insight into molecular mechanism of Aβ42 generation, which could be exploited therapeutically.

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.

Optineurin suppression causes neuronal cell death via NF‐κB pathway

Mutations in more than 10 genes are reported to cause familial amyotrophic lateral sclerosis (ALS). Among these genes, optineurin (OPTN) is virtually the only gene that is considered to cause classical ALS by a loss‐of‐function mutation. Wild‐type optineurin (OPTNWT) suppresses nuclear factor‐kappa B (NF‐κB) activity, but the ALS‐causing mutant OPTN is unable to suppress NF‐κB activity. Therefore, we knocked down OPTN in neuronal cells and examined the resulting NF‐κB activity and phenotype. First, we confirmed the loss of the endogenous OPTN expression after siRNA treatment and found that NF‐κB activity was increased in OPTN‐knockdown cells. Next, we found that OPTN knockdown caused neuronal cell death. Then, overexpression of OPTNWT or OPTNE50K with intact NF‐κB‐suppressive activity, but not overexpression of ALS‐related OPTN mutants, suppressed the neuronal death induced by OPTN knockdown. This neuronal cell death was inhibited by withaferin A, which selectively inhibits NF‐κB activation. Lastly, involvement of the mitochondrial proapoptotic pathway was suggested for neuronal death induced by OPTN knockdown. Taken together, these results indicate that inappropriate NF‐κB activation is the pathogenic mechanism underlying OPTN mutation‐related ALS.

Distinct Dendritic Spine and Nuclear Phases of Calcineurin Activation after Exposure to Amyloid-β Revealed by a Novel Fluorescence Resonance Energy Transfer Assay

Calcineurin (CaN) activation is critically involved in the regulation of spine morphology in response to oligomeric amyloid-β (Aβ) as well as in synaptic plasticity in normal memory, but no existing techniques can monitor the spatiotemporal pattern of CaN activity. Here, we use a spectral fluorescence resonance energy transfer approach to monitor CaN activation dynamics in real time with subcellular resolution. When oligomeric Aβ derived from Tg2576 murine transgenic neurons or human AD brains were applied to wild-type murine primary cortical neurons, we observe a dynamic progression of CaN activation within minutes, first in dendritic spines, and then in the cytoplasm and, in hours, in the nucleus. CaN activation in spines leads to rapid but reversible morphological changes in spines and in postsynaptic proteins; longer exposure leads to NFAT (nuclear factor of activated T-cells) translocation to the nucleus and frank spine loss. These results provide a framework for understanding the role of calcineurin in synaptic alterations associated with AD pathogenesis.