Nicastrin, a presenilin-interacting protein, contains an aminopeptidase/transferrin receptor superfamily domain

Nicastrin, a protein implicated in Alzheimer's disease, has a domain that is found in the aminopeptidase/transferrin receptor superfamily. In nicastrin, this domain might possess catalytic activity (as observed with aminopeptidases) or it could serve merely as a binding domain (with analogy to the transferrin receptors) for the beta-amyloid precursor protein.     

The Nicastrin ectodomain adopts a highly thermostable structure

Nicastrin is a type I transmembrane glycoprotein, which is part of the high molecular weight γ-secretase complex. γ-Secretase is one of the key players associated with the generation of Alzheimer's disease pathology, since it liberates the neurotoxic amyloid β-peptide. Four proteins Nicastrin, anterior pharynx-defective-1 (Aph-1), presenilin enhancer-2 (Pen-2) and Presenilin are essential to form the active γ-secretase complex. Recently it has been shown, that Nicastrin has a key function in stabilizing the mature γ-secretase complex and may also be involved in substrate recognition. So far no structural data for the Nicastrin ectodomain or any other γ-secretase component are available. We therefore used Circular Dichroism (CD) spectroscopy to demonstrate that Nicastrin, similar to its homologues, the Streptomyces griseus aminopeptidase (SGAP) and the transferrin receptor (TfR), adopts a thermostable secondary structure. Furthermore, the Nicastrin ectodomain has an exceptionally high propensity to refold after thermal denaturation. These findings provide evidence to further support the hypothesis that Nicastrin may share evolutionary conserved properties with the aminopeptidase and the transferrin receptor family.     

Nicastrin is a resident lysosomal membrane protein

Nicastrin has been recently identified as part of the gamma-secretase complex that includes presenilin and other proteins. It is involved in the degradation of amyloid precursor protein to produce beta-amyloid peptides which are believed to be central to the pathophysiology of Alzheimer's disease. Previous reports have localized presenilin and nicastrin to the endoplasmic reticulum. However, during a proteomics-based characterization of lysosomal membrane proteins, a major spot observed on silver-stained IEF/SDS-PAGE gels was identified by mass spectrometric sequencing as nicastrin. Its M(r) corresponded to the reported mature M(r) for nicastrin, indicating that it is stable in the lysosomal environment. Furthermore, protease protection assays confirmed that nicastrin is contained in the outer lysosomal membrane, rather than in an internalized vesicle awaiting degradation, and that it is properly orientated with its amino-terminus facing the lysosomal lumen with its carboxyl-terminus facing the cytosol. We conclude that nicastrin is a resident lysosomal membrane protein.     

Role of the proteasome in Alzheimer's disease

The proteasome is a multicatalytic complex involved in the degradation of polyubiquitinated proteins. Here we review the clues of a possible involvement of the proteasome in Alzheimer's disease neuropathology. Thus, we discuss the fact that the proteasome modulates the intracellular concentrations of presenilins 1 and 2. These two proteins, when mutated, appear responsible for most of early onset forms of Alzheimer's disease and this is thought to be due to the exacerbation of the pathogenic pathway of the maturation of the beta-amyloid precursor protein. Controlling presenilins concentrations could have drastic repercussions on cell physiology as suggested by the fact that proteasome inhibitors drastically potentiate the 'normal' or 'pathogenic' presenilins phenotype related with betaAPP processing. The possibility of considering the proteasome as a potential target for therapeutic intervention in Alzheimer's disease is discussed.     

Nicastrin, presenilin, APH-1, and PEN-2 form active gamma-secretase complexes in mitochondria

Mitochondria are central in the regulation of cell death. Apart from providing the cell with ATP, mitochondria also harbor several death factors that are released upon apoptotic stimuli. Alterations in mitochondrial functions, increased oxidative stress, and neurons dying by apoptosis have been detected in Alzheimer's disease patients. These findings suggest that mitochondria may trigger the abnormal onset of neuronal cell death in Alzheimer's disease. We previously reported that presenilin 1 (PS1), which is often mutated in familial forms of Alzheimer's disease, is located in mitochondria and hypothesized that presenilin mutations may sensitize cells to apoptotic stimuli at the mitochondrial level. Presenilin forms an active gamma-secretase complex together with Nicastrin (NCT), APH-1, and PEN-2, which among other substrates cleaves the beta-amyloid precursor protein (beta-APP) generating the amyloid beta-peptide and the beta-APP intracellular domain. Here we have identified dual targeting sequences (for endoplasmic reticulum and mitochondria) in NCT and showed expression of NCT in mitochondria by immunoelectron microscopy. We also showed that NCT together with APH-1, PEN-2, and PS1 form a high molecular weight complex located in mitochondria. gamma-secretase activity in isolated mitochondria was demonstrated using C83 (alpha-secretase-cleaved C-terminal 83-residue beta-APP fragment from BD8 cells lacking presenilin and thus gamma-secretase activity) or recombinant C100-Flag (C-terminal 100-residue beta-APP fragment) as substrates. Both systems generated an APP intracellular domain, and the activity was inhibited by the gamma-secretase inhibitors l-685,458 or Compound E. This novel localization of NCT, PS1, APH-1, and PEN-2 expands the role and importance of gamma-secretase activity to mitochondria.     

Gamma-secretase exists on the plasma membrane as an intact complex that accepts substrates and effects intramembrane cleavage

Research on Alzheimer's disease led to the identification of a novel proteolytic mechanism in all metazoans, the presenilin/gamma-secretase complex. This unique intramembrane-cleaving aspartyl protease is required for the normal processing of Notch, Jagged, beta-amyloid precursor protein (APP), E-cadherin, and many other receptor-like proteins. We recently provided indirect evidence of gamma-secretase activity at the cell surface in HeLa cells following inhibition of receptor-mediated endocytosis. Here, we directly identify and isolate gamma-secretase as an intact complex (Presenilin, Nicastrin, Aph-1, and Pen-2) from the plasma membrane, both in overexpressing cell lines and endogenously. Inhibition of its proteolytic activity allowed cell surface gamma-secretase to be captured in association with its plasma membrane-localized APP substrates (C83 and C99). Moreover, non-denaturing isolation of the intact enzyme complex revealed that cell surface gamma-secretase can specifically generate amyloid beta-protein from an APP substrate and similarly cleave a Notch substrate. These data directly establish the proteolytic function of gamma-secretase on the plasma membrane, independent of a hypothesized substrate trafficking role. We conclude that presenilin/gamma-secretase exists as a mature complex at the cell surface, where it interacts with and can cleave its substrates, consistent with an essential function in processing many adhesion molecules and receptors required for cell-cell interaction or intercellular signaling.     

Transition-state analogue inhibitors of gamma-secretase bind directly to presenilin-1

The beta-amyloid precursor protein (beta-APP), which is involved in the pathogenesis of Alzheimer's disease, and the Notch receptor, which is responsible for critical signalling events during development, both undergo unusual proteolysis within their transmembrane domains by unknown gamma-secretases. Here we show that an affinity reagent designed to interact with the active site of gamma-secretase binds directly and specifically to heterodimeric forms of presenilins, polytopic proteins that are mutated in hereditary Alzheimer's and are known mediators of gamma-secretase cleavage of both beta-APP and Notch. These results provide evidence that heterodimeric presenilins contain the active site of gamma-secretase, and validate presenilins as principal targets for the design of drugs to treat and prevent Alzheimer's disease.     

Prevention of beta-amyloid neurotoxicity by blockade of the ubiquitin-proteasome proteolytic pathway

In many neurodegenerative disorders, such as Alzheimer's disease, inclusions containing ubiquitinated proteins have been found in the brain, suggesting a pathophysiological role for ubiquitin-mediated proteasomal degradation of neuronal proteins. Here we show for the first time that the beta-amyloid fragment 1-40, which in micromolar levels causes the death of cortical neurons, also induces the ubiquitination of several neuronal proteins. Prevention of ubiquitination and inhibition of proteasome activity block the neurotoxic effect of beta-amyloid. These data suggest that beta-amyloid neurotoxicity may cause toxicity through the activation of protein degradation via the ubiquitin-proteasome pathway. These findings suggest possible new pharmacological targets for the prophylaxis and/or treatment of Alzheimer's disease and possibly for other related neurodegenerative disorders.     

beta -Amyloid peptide-induced apoptosis regulated by a novel protein containing a g protein activation module

Degeneration of neurons in Alzheimer's disease is mediated by beta-amyloid peptide by diverse mechanisms, which include a putative apoptotic component stimulated by unidentified signaling events. This report describes a novel beta-amyloid peptide-binding protein (denoted BBP) containing a G protein-coupling module. BBP is one member of a family of three proteins containing this conserved structure. The BBP subtype bound human beta-amyloid peptide in vitro with high affinity and specificity. Expression of BBP in cell culture induced caspase-dependent vulnerability to beta-amyloid peptide toxicity. Expression of a signaling-deficient dominant negative BBP mutant suppressed sensitivity of human Ntera-2 neurons to beta-amyloid peptide mediated toxicity. These findings suggest that BBP is a target of neurotoxic beta-amyloid peptide and provide new insight into the molecular pathophysiology of Alzheimer's disease.     

Cholesterol, statins and dementia

PURPOSE OF REVIEW: Advances in cholesterol biology suggest that cholesterol metabolism modulates beta-amyloid production, and that pharmaceuticals that inhibit cholesterol metabolism might be valuable in therapy of Alzheimer's disease. Although the genetics and cell biology continue to support the link between cholesterol and Alzheimer's disease, recent clinical studies suggest that the animal studies might not directly translate to clinical studies in humans. RECENT FINDINGS: This review will highlight advances in genetics, cell biology and clinical sciences investigating the relationship between cholesterol and Alzheimer's disease. SUMMARY: Cholesterol, its catabolites and proteins that regulate cholesterol levels all modulate processing of amyloid precursor protein. Statins hold promise in therapy of Alzheimer's disease, but the current data are more consistent with a model of statins that act as neuroprotective agents rather than inhibitors of beta-amyloid production.     

Overexpression of Rab11 or constitutively active Rab11 does not affect sAPPalpha and Abeta secretions by wild-type and Swedish mutated betaAPP-expressing HEK293 cells

Presenilins 1 and 2 are two homologous proteins which, when mutated, appear responsible for most of the early-onset familial forms of Alzheimer's disease. Among various functional aspects, presenilins appear to behave as chaperoning partners of a series of proteins including the beta-amyloid precursor protein. Recently, presenilins were shown to interact with Rab11, a GTPase involved in intracellular transport. This suggested that Rab11-presenilin interaction could influence the routing of betaAPP and thereby modulate its maturation. In this context, we examined whether overexpression of Rab11 or its constitutively active mutant Rab11Q70L could affect betaAPP maturation in human HEK293 cells. We show here that the overexpression of both Rab11-related proteins does not modify the recovery of secreted sAPPalpha or Abeta in HEK293 cells expressing wild-type betaAPP or betaAPP harboring the Swedish double mutation. These data indicate that Rab11 does not influence betaAPP processing in HEK293 cells. However, it does not preclude the possibility for Rab11 to modulate other presenilin-mediated functions in human cells.     

Pore-forming scissors? A first structural glimpse of γ-secretase

Amyloid plaques, which are composed of amyloid-beta peptide (Abeta), signify Alzheimer's disease pathology. Secretases generate Abeta by processing the beta-amyloid precursor protein. gamma-Secretase, a complex comprising four different proteins, liberates Abeta from its precursor by intramembrane proteolysis. The first impression of the shape of gamma-secretase has recently been revealed by electron microscopy. It indicates a spherical transmembrane particle with an interior chamber that, presumably, accommodates its catalytic residues, and two openings that might be exit sites for the cleavage products.     

Identification of synaptic plasma membrane proteins co-precipitated with fibrillar beta-amyloid peptide

The beta-amyloid peptide that is overproduced in Alzheimer's disease rapidly forms fibrils, which are able to interact with various molecular partners. This study aimed to identify abundant synaptosomal proteins binding to the fibrillar beta-amyloid (fAbeta) 1-42. Triton X-100-soluble proteins were extracted from the rat synaptic plasma membrane fraction. Interacting proteins were isolated by co-precipitation with fAbeta, or with fibrillar crystallin as a negative control. Protein identification was accomplished (1) by separating the tryptically digested peptides of the protein pellet by one-dimensional reversed-phase HPLC and analysing them using an ion-trap mass spectrometer with electrospray ionization; and (2) by subjecting the precipitated proteins to gel electrophoretic fractionation, in-gel tryptic digestion and to matrix-assisted laser desorption/ionization time-of-flight mass measurements and post-source decay analysis. Six different synaptosomal proteins co-precipitated with fAbeta were identified by both methods: vacuolar proton-pump ATP synthase, glyceraldehyde-3-phosphate dehydrogenase, synapsins I and II, beta-tubulin and 2',3'-cyclic nucleotide 3'-phosphodiesterase. Most of these proteins have already been associated with Alzheimer's disease, and the biological and pathophysiological significance of their interaction with fAbeta is discussed.     

Presenilins mutated at Asp-257 or Asp-385 restore Pen-2 expression and Nicastrin glycosylation but remain catalytically inactive in the absence of wild type Presenilin

The Presenilins are part of the gamma-secretase complex that is involved in the regulated intramembrane proteolysis of amyloid precursor protein and other type I integral membrane proteins. Nicastrin, Pen-2, and Aph1 are the other proteins of this complex. The Presenilins probably contribute the catalytic activity to the protease complex. However, several investigators reported normal Abeta-peptide generation in cells expressing Presenilins mutated at the putative catalytic site residue Asp-257, contradicting this hypothesis. Because endogenously expressed wild type Presenilin could contribute to residual gamma-secretase activity in these experiments, we have reinvestigated the problem by expressing mutated Presenilins in a Presenilin-negative cell line. We confirm that Presenilins with mutated Asp residues are catalytically inactive. Unexpectedly, these mutated Presenilins are still partially processed into amino- and carboxyl-terminal fragments by a "Presenilinase"-like activity. They are also able to rescue Pen-2 expression and Nicastrin glycosylation in Presenilin-negative cells and become incorporated into large approximately 440-kDa complexes as assessed by blue native gel electrophoresis. Our study demonstrates that the catalytic activity of Presenilin and its other functions in the generation, stabilization, and transport of the gamma-secretase complex can be separated and extends the concept that Presenilins are multifunctional proteins.     

Profiling proteins related to amyloid deposited brain of Tg2576 mice

Alzheimer's disease (AD) is an age-related neurodegenerative disorder that is characterized by the extracellular deposition of beta-amyloid and intracellular hyperphosphorylation of tau in the cortex and hippocampus of the brain. These characterizations are caused by abnormal expression, modification and deposition of certain proteins. Post-translational modifications of proteins including oxidation and nitration might be involved in the pathogenesis of AD. In this study, AD-related proteins were identified in the cortex of Tg2576 mice used as a model for studying AD. Tg2576 mice express high levels of the Swedish mutated form of human beta-amyloid precursor protein (APP) and generated high levels of beta-amyloid in the brains. Using Western blotting and two-dimensional electrophoresis, proteins with differences in expression, oxidation and nitration in the cortex of Tg2576 mice brains were compared to littermate mice brains used as a control. The proteins with different expression levels were identified using matrix-assisted laser desorption/ionization-time of flight and liquid chromatography-tandem mass spectrometry analyses. As a result, 12 proteins were identified among 37 different proteins using the PDQuest program. Furthermore, two proteins, laminin receptor and alpha-enolase, were more susceptible to oxidative modification in the brains of Tg2576 mice compared to those of littermates. Similarly, alpha-enolase, calpain 12, and Atp5b were more modified by nitration in brains of Tg2576 mice than those of littermates. Taken together, these proteins and their modifications may play an important role in the plaque deposition of Tg2576 mice brains.     

Akt/GSK3beta serine/threonine kinases: evidence for a signalling pathway mediated by familial Alzheimer's disease mutations

Although Alzheimer's disease pathologically affects the brain, familial Alzheimer's disease associated mutations of beta-amyloid precursor protein and presenilin are ubiquitously expressed and therefore aberrant intracellular signals, separate from but similar to, the brain may be expected. Here, we report selective down regulation of the serine/threonine kinase, Akt/PKB, concurrent with elevated endogenous GSK3beta kinase activity in familial Alzheimer's disease beta-amyloid precursor protein expressing human embryonic kidney (HEK) and familial Alzheimer's disease presenilin lymphoblast cells. Further, familial Alzheimer's disease presenilin in the human lymphoblast was associated with beta-catenin destabilization. Moreover, limited immunohistochemistry analysis reveals Akt/PKB in a subset of neurofibrillary tangles where GSK3beta and tau have been reported to co-localize, suggesting a possible Akt/GSK3beta and tau interaction in vivo. Our data suggest that familial Alzheimer's disease mutants of beta-amyloid precursor protein and presenilin signal, at least in part, through the Akt/GSKbeta pathway and that Akt/GSK3beta-mediated signalling may contribute to the underlying Alzheimer's disease pathogenesis induced by familial Alzheimer's disease mutants.     

Expression of the human beta-amyloid protein of Alzheimer's disease specifically in the brains of transgenic mice

The human beta-amyloid protein is deposited in senile plaques and in the cerebro-vasculature of people with Alzheimer's disease and Down's syndrome. The precise role of beta-amyloid in Alzheimer's disease pathology is presently unknown. To study the properties of beta-amyloid in vivo, we generated transgenic mice that harbor the gene for the carboxyl-terminal 100 amino acids of the human amyloid precursor protein, beginning with the beta-amyloid region, under control of the JC viral early region promoter. The mRNA is expressed exclusively in brain tissue. Further, we demonstrate increased levels of beta-amyloid immunoreactivity on fixed brain tissue. These animals will be useful as a model to study beta-amyloid deposition and its consequences.     

Inhibition of beta-amyloid peptide aggregation and neurotoxicity by alpha-d-mannosylglycerate, a natural extremolyte

The aggregation of soluble beta-amyloid (Abeta) peptide into oligomers/fibrils is one of the key pathological features in Alzheimer's disease (AD). The use of naturally occurring small molecules for inhibiting protein aggregation has recently attracted many interests due to their effectiveness for treating protein folding diseases such as AD, Parkinson's, Huntington's disease, and other amyloidosis diseases. alpha-d-Mannosylglycerate (MG), a natural extremolyte identified in microorganisms growing under extremely high temperatures up to 100 degrees C, had been shown to protect proteins against various stress conditions such as heat, freezing, thawing, and drying. Here, we report the effectiveness of MG on the suppression of Alzheimer's Abeta aggregation and neurotoxicity to human neuroblastoma cells. According to our study--carried out by using thioflavin-T induced fluorescence, atomic force microscopy, and cell viability assay--MG had significant inhibitory effect against Abeta amyloid formation and could reduce the toxicity of amyloid aggregates to human neuroblastoma cells while MG itself was innocuous to cells. On the other hand, the structural analogs of MG such as alpha-d-mannosylglyceramide, mannose, methylmannoside, glycerol, showed negligible effect on Abeta aggregate formation. The results suggest that MG could be a potential drug candidate for treating Alzheimer's disease.