Taisuke Tomita

APP Processing and Synaptic Function

A large body of evidence has implicated Abeta peptides and other derivatives of the amyloid precursor protein (APP) as central to the pathogenesis of Alzheimers disease (AD). However, the functional relationship of APP and its proteolytic derivatives to neuronal electrophysiology is not known. Here, we show that neuronal activity modulates the formation and secretion of Abeta peptides in hippocampal slice neurons that overexpress APP. In turn, Abeta selectively depresses excitatory synaptic transmission onto neurons that overexpress APP, as well as nearby neurons that do not. This depression depends on NMDA-R activity and can be reversed by blockade of neuronal activity. Synaptic depression from excessive Abeta could contribute to cognitive decline during early AD. In addition, we propose that activity-dependent modulation of endogenous Abeta production may normally participate in a negative feedback that could keep neuronal hyperactivity in check. Disruption of this feedback system could contribute to disease progression in AD.

AMPAR Removal Underlies Aβ-Induced Synaptic Depression and Dendritic Spine Loss

Beta amyloid (Abeta), a peptide generated from the amyloid precursor protein (APP) by neurons, is widely believed to underlie the pathophysiology of Alzheimers disease. Recent studies indicate that this peptide can drive loss of surface AMPA and NMDA type glutamate receptors. We now show that Abeta employs signaling pathways of long-term depression (LTD) to drive endocytosis of synaptic AMPA receptors. Synaptic removal of AMPA receptors is necessary and sufficient to produce loss of dendritic spines and synaptic NMDA responses. Our studies indicate the central role played by AMPA receptor trafficking in Abeta-induced modification of synaptic structure and function.

The role of presenilin cofactors in the gamma-secretase complex.

Mutations in presenilin genes account for the majority of the cases of the familial form of Alzheimers disease (FAD). Presenilin is essential for γ-secretase activity, a proteolytic activity involved in intramembrane cleavage of Notch and β-amyloid precursor protein (βAPP). Cleavage of βAPP by FAD mutant presenilin results in the overproduction of highly amyloidogenic amyloid β42 peptides. γ-Secretase activity requires the formation of a stable, high-molecular-mass protein complex that, in addition to the endoproteolysed fragmented form of presenilin, contains essential cofactors including nicastrin, APH-1 (refs 15–18) and PEN-2 (refs 16, 19). However, the role of each protein in complex formation and the generation of enzymatic activity is unclear. Here we show that Drosophila APH-1 (Aph-1) increases the stability of Drosophila presenilin (Psn) holoprotein in the complex. Depletion of PEN-2 by RNA interference prevents endoproteolysis of presenilin and promotes stabilization of the holoprotein in both Drosophila and mammalian cells, including primary neurons. Co-expression of Drosophila Pen-2 with Aph-1 and nicastrin increases the formation of Psn fragments as well as γ-secretase activity. Thus, APH-1 stabilizes the presenilin holoprotein in the complex, whereas PEN-2 is required for endoproteolytic processing of presenilin and conferring γ-secretase activity to the complex.

Effects of PS1 Deficiency on Membrane Protein Trafficking in Neurons

We have examined the trafficking and metabolism of the beta-amyloid precursor protein (APP), an APP homolog (APLP1), and TrkB in neurons that lack PS1. We report that PS1-deficient neurons fail to secrete Abeta, and that the rate of appearance of soluble APP derivatives in the conditioned medium is increased. Remarkably, carboxyl-terminal fragments (CTFs) derived from APP and APLP1 accumulate in PS1-deficient neurons. Hence, PS1 plays a role in promoting intramembrane cleavage and/or degradation of membrane-bound CTFs. Moreover, the maturation of TrkB and BDNF-inducible TrkB autophosphorylation is severely compromised in neurons lacking PS1. We conclude that PS1 plays an essential role in modulating trafficking and metabolism of a selected set of membrane and secretory proteins in neurons.

A loss of function mutation of presenilin-2 interferes with amyloid beta-peptide production and notch signaling.

Presenilin-1 (PS1) facilitates γ-secretase cleavage of the β-amyloid precursor protein and the intramembraneous cleavage of Notch1. Although Alzheimer’s disease-associated mutations in the homologous presenilin (PS2) gene elevate amyloid β-peptide (Aβ42) production like PS1 mutations, here we demonstrate that a gene ablation of PS2 (unlike that of PS1) in mice does not result in a severe phenotype resembling that of Notch-ablated animals. To investigate the amyloidogenic function of PS2 more directly, we mutagenized a conserved aspartate at position 366 to alanine, because the corresponding residue of PS1 is known to be required for its amyloidogenic function. Cells expressing the PS2 D366A mutation exhibit significant deficits in proteolytic processing of β-amyloid precursor protein indicating a defect in γ-secretase activity. The reduced γ-secretase activity results in the almost complete inhibition of Aβ and p3 production in cells stably expressing PS2 D366A, whereas cells overexpressing the wild-type PS2 cDNA produce robust levels of Aβ and p3. Using highly sensitive in vivo assays, we demonstrate that the PS2 D366A mutation not only blocks γ-secretase activity but also inactivates PS2 activity in Notch signaling by inhibiting the proteolytic release of the cytoplasmic Notch1 domain. These data suggest that PS2 is functionally involved in Aβ production and Notch signaling by facilitating similar proteolytic cleavages.

Presenilins mediate a dual intramembranous γ-secretase cleavage of Notch-1

Following ectodomain shedding, Notch‐1 undergoes presenilin (PS)‐dependent constitutive intramembranous endoproteolysis at site‐3. This cleavage is similar to the PS‐dependent γ‐secretase cleavage of the β‐amyloid precursor protein (βAPP). However, topological differences in cleavage resulting in amyloid β‐peptide (Aβ) or the Notch‐1 intracellular domain (NICD) indicated independent mechanisms of proteolytic cleavage. We now demonstrate the secretion of an N‐terminal Notch‐1 Aβ‐like fragment (Nβ). Analysis of Nβ by MALDI‐TOF MS revealed that Nβ is cleaved at a novel site (site‐4, S4) near the middle of the transmembrane domain. Like the corresponding cleavage of βAPP at position 40 and 42 of the Aβ domain, S4 cleavage is PS dependent. The precision of this cleavage is affected by familial Alzheimers disease‐associated PS1 mutations similar to the pathological endoproteolysis of βAPP. Considering these similarities between intramembranous processing of Notch and βAPP, we conclude that these proteins are cleaved by a common mechanism utilizing the same protease, i.e. PS/γ‐secretase.

A presenilin dimer at the core of the gamma-secretase enzyme: insights from parallel analysis of Notch 1 and APP proteolysis.

Notch receptors and the amyloid precursor protein are type I membrane proteins that are proteolytically cleaved within their transmembrane domains by a presenilin (PS)-dependent γ-secretase activity. In both proteins, two peptide bonds are hydrolyzed: one near the inner leaflet and the other in the middle of the transmembrane domain. Under saturating conditions the substrates compete with each other for proteolysis, but not for binding to PS. At least some Alzheimers disease-causing PS mutations reside in proteins possessing low catalytic activity. We demonstrate (i) that differentially tagged PS molecules coimmunoprecipitate, and (ii) that PS N-terminal fragment dimers exist by using a photoaffinity probe based on a transition state analog γ-secretase inhibitor. We propose that γ-secretase contains a PS dimer in its catalytic core, that binding of substrate is at a site separate from the active site, and that substrate is cleaved at the interface of two PS molecules.

Sulindac Sulfide Is a Noncompetitive γ-Secretase Inhibitor That Preferentially Reduces Aβ42 Generation

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been known to reduce risk for Alzheimers disease. In addition to the anti-inflammatory effects of NSAIDs to block cylooxygenase, it has been shown recently that a subset of NSAIDs selectively inhibits the secretion of highly amyloidogenic Aβ42 from cultured cells, although the molecular target(s) of NSAIDs in reducing the activity of γ-secretase for Aβ42 generation (γ42-secretase) still remain unknown. Here we show that sulindac sulfide (SSide) directly acts on γ-secretase and preferentially inhibits the γ42-secretase activity derived from the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate-solubilized membrane fractions of HeLa cells, in an in vitroγ-secretase assay using recombinant amyloid β precursor protein C100 as a substrate. SSide also inhibits activities for the generation of Aβ40 as well as for Notch intracellular domain at higher concentrations. Notably, SSide displayed linear noncompetitive inhibition profiles for γ42-secretase in vitro. Our data suggest that SSide is a direct inhibitor of γ-secretase that preferentially affects the γ42-secretase activity.

Amyloid Precursor Proteins Inhibit Heme Oxygenase Activity and Augment Neurotoxicity in Alzheimer's Disease

Amyloid precursor protein (APP) generates the beta-amyloid peptide, postulated to participate in the neurotoxicity of Alzheimers disease. We report that APP and APLP bind to heme oxygenase (HO), an enzyme whose product, bilirubin, is antioxidant and neuroprotective. The binding of APP inhibits HO activity, and APP with mutations linked to the familial Alzheimers disease (FAD) provides substantially greater inhibition of HO activity than wild-type APP. Cortical cultures from transgenic mice expressing Swedish mutant APP have greatly reduced bilirubin levels, establishing that mutant APP inhibits HO activity in vivo. Oxidative neurotoxicity is markedly greater in cerebral cortical cultures from APP Swedish mutant transgenic mice than wild-type cultures. These findings indicate that augmented neurotoxicity caused by APP-HO interactions may contribute to neuronal cell death in Alzheimers disease.

C-terminal Fragment of Presenilin Is the Molecular Target of a Dipeptidic γ-Secretase-specific Inhibitor DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-Butyl Ester)

γ-Secretase is a multimeric membrane protein complex composed of presenilin (PS), nicastrin, Aph-1 and, Pen-2 that is responsible for the intramembrane proteolysis of various type I transmembrane proteins, including amyloid β-precursor protein and Notch. The direct labeling of PS polypeptides by transition-state analogue γ-secretase inhibitors suggested that PS represents the catalytic center of γ-secretase. Here we show that one of the major γ-secretase inhibitors of dipeptidic type, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), targets the C-terminal fragment of PS, especially the transmembrane domain 7 or more C-terminal region, by designing and synthesizing DAP-BpB (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-(S)-phenylglycine-4-(4-(8-biotinamido)octylamino)benzoyl)benzyl)methylamide), a photoactivable DAPT derivative. We also found that DAP-BpB selectively binds to the high molecular weight γ-secretase complex in an activity-dependent manner. Photolabeling of PS by DAP-BpB is completely blocked by DAPT or its structural relatives (e.g. Compound E) as well as by arylsulfonamides. In contrast, transition-state analogue inhibitor L-685,458 or α-helical peptidic inhibitor attenuated the photolabeling of PS1 only at higher concentrations. These data illustrate the DAPT binding site as a novel functional domain within the PS C-terminal fragment that is distinct from the catalytic site or the substrate binding site.