Beth L. Ostaszewski

Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity.

Accumulation of the amyloid-β protein (Aβ) in the cerebral cortex is an early and invariant event in the pathogenesis of Alzheimers disease. The final step in the generation of Aβ from the β-amyloid precursor protein is an apparently intramembranous proteolysis by the elusive γ-secretase(s). The most common cause of familial Alzheimers disease is mutation of the genes encoding presenilins 1 and 2, which alters γ-secretase activity to increase the production of the highly amyloidogenic Aβ42 isoform. Moreover, deletion of presenilin-1 in mice greatly reduces γ-secretase activity, indicating that presenilin-1 mediates most of this proteolytic event. Here we report that mutation of either of two conserved transmembrane (TM) aspartate residues in presenilin-1, Aspu2009257 (in TM6) and Aspu2009385 (in TM7), substantially reduces Aβ production and increases the amounts of the carboxy-terminal fragments of β-amyloid precursor protein that are the substrates of γ-secretase. We observed these effects in three different cell lines as well as in cell-free microsomes. Either of the Asp → Ala mutations also prevented the normal endoproteolysis of presenilin-1 in the TM6 → TM7 cytoplasmic loop. In a functional presenilin-1 variant (carrying a deletion in exon 9) that is associated with familial Alzheimers disease and which does not require this cleavage, the Aspu2009385 → Ala mutation still inhibited γ-secretase activity. Our results indicate that the two transmembrane aspartate residues are critical for both presenilin-1 endoproteolysis and γ-secretase activity, and suggest that presenilin 1 is either a unique diaspartyl cofactor for γ-secretase or is itself γ-secretase, an autoactivated intramembranous aspartyl protease.

γ-Secretase is a membrane protein complex comprised of presenilin, nicastrin, aph-1, and pen-2

γ-Secretase catalyzes the intramembrane proteolysis of Notch, β-amyloid precursor protein, and other substrates as part of a new signaling paradigm and as a key step in the pathogenesis of Alzheimers disease. This unusual protease has eluded identification, though evidence suggests that the presenilin heterodimer comprises the catalytic site and that a highly glycosylated form of nicastrin associates with it. The formation of presenilin heterodimers from the holoprotein is tightly gated by unknown limiting cellular factors. Here we show that Aph-1 and Pen-2, two recently identified membrane proteins genetically linked to γ-secretase, associate directly with presenilin and nicastrin in the active protease complex. Coexpression of all four proteins leads to marked increases in presenilin heterodimers, full glycosylation of nicastrin, and enhanced γ-secretase activity. These findings suggest that the four membrane proteins comprise the limiting components of γ-secretase and coassemble to form the active enzyme in mammalian cells.

Dopamine covalently modifies and functionally inactivates parkin

Inherited mutations in PARK2, the gene encoding parkin, cause selective degeneration of catecholaminergic neurons in the substantia nigra and locus coeruleus of the brainstem, resulting in early-onset parkinsonism. But the role of parkin in common, sporadic forms of Parkinson disease remains unclear. Here we report that the neurotransmitter dopamine covalently modifies parkin in living dopaminergic cells, a process that increases parkin insolubility and inactivates its E3 ubiquitin ligase function. In the brains of individuals with sporadic Parkinson disease, we observed decreases in parkin solubility consistent with its functional inactivation. Using a new biochemical method, we detected catechol-modified parkin in the substantia nigra but not other regions of normal human brain. These findings show a vulnerability of parkin to modification by dopamine, the principal transmitter lost in Parkinson disease, suggesting a mechanism for the progressive loss of parkin function in dopaminergic neurons during aging and sporadic Parkinson disease.

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

The β-amyloid precursor protein (β-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 γ-secretases. Here we show that an affinity reagent designed to interact with the active site of γ-secretase binds directly and specifically to heterodimeric forms of presenilins, polytopic proteins that are mutated in hereditary Alzheimer’s and are known mediators of γ-secretase cleavage of both β-APP and Notch. These results provide evidence that heterodimeric presenilins contain the active site of γ-secretase, and validate presenilins as principal targets for the design of drugs to treat and prevent Alzheimer’s disease.

Mutagenesis identifies new signals for β-amyloid precursor protein endocytosis, turnover, and the generation of secreted fragments, including Aβ42

It has long been assumed that the C-terminal motif, NPXY, is the internalization signal for β-amyloid precursor protein (APP) and that the NPXY tyrosine (Tyr743 by APP751 numbering, Tyr682 in APP695) is required for APP endocytosis. To evaluate this tenet and to identify the specific amino acids subserving APP endocytosis, we mutated all tyrosines in the APP cytoplasmic domain and amino acids within the sequence GYENPTY (amino acids 737–743). Stable cell lines expressing these mutations were assessed for APP endocytosis, secretion, and turnover. Normal APP endocytosis was observed for cells expressing Y709A, G737A, and Y743A mutations. However, Y738A, N740A, and P741A or the double mutation of Y738A/P741A significantly impaired APP internalization to a level similar to that observed for cells lacking nearly the entire APP cytoplasmic domain (ΔC), arguing that the dominant signal for APP endocytosis is the tetrapeptide YENP. Although not an APP internalization signal, Tyr743 regulates rapid APP turnover because half-life increased by 50% with the Y743A mutation alone. Secretion of the APP-derived proteolytic fragment, Aβ, was tightly correlated with APP internalization, such that Aβ secretion was unchanged for cells having normal APP endocytosis but significantly decreased for endocytosis-deficient cell lines. Remarkably, secretion of the Aβ42 isoform was also reduced in parallel with endocytosis from internalization-deficient cell lines, suggesting an important role for APP endocytosis in the secretion of this highly pathogenic Aβ species.

Activity-dependent isolation of the presenilin– γ-secretase complex reveals nicastrin and a γ substrate

Presenilin heterodimers apparently contain the active site of γ-secretase, a polytopic aspartyl protease involved in the transmembrane processing of both the Notch receptor and the amyloid-β precursor protein. Although critical to embryonic development and the pathogenesis of Alzheimers disease, this protease is difficult to characterize, primarily because it is a multicomponent complex of integral membrane proteins. Here the functional γ-secretase complex was isolated by using an immobilized active site-directed inhibitor of the protease. Presenilin heterodimers and nicastrin bound specifically to this inhibitor under conditions tightly correlating with protease activity, whereas several other presenilin-interacting proteins (β-catenin, calsenilin, and presenilin-associated protein) did not bind. Moreover, anti-nicastrin antibodies immunoprecipitated γ-secretase activity from detergent-solubilized microsomes. Unexpectedly, C83, the major endogenous amyloid-β precursor protein substrate of γ-secretase, was also quantitatively associated with the complex. These results provide direct biochemical evidence that nicastrin is a member of the active γ-secretase complex, indicate that β-catenin, calsenilin, and presenilin-associated protein are not required for γ activity, and suggest an unprecedented mechanism of substrate–protease interaction.

Amyloid beta-peptide is transported on lipoproteins and albumin in human plasma.

The amyloid β-peptide (Aβ) is the major constituent of neuritic plaques in Alzheimers disease and occurs as a soluble 40-42-residue peptide in cerebrospinal fluid and blood of both normal and AD subjects. It is unclear whether Aβ, once it is secreted by cells, remains free in biological fluids or is associated with other proteins and thus transported and metabolized with them. Such knowledge of the normal fate of Aβ is a prerequisite for understanding the changes that may lead to the pathological aggregation of soluble Aβ in vivo, the possible influence of certain extracellular proteins, particularly apolipoprotein E, on plaque formation, and the pharmacology of putative Aβ-lowering drugs. To address the question of Aβ distribution in human biological fluids, we incubated fresh human plasma from 38 subjects with physiological concentrations (0.5-0.7 nM) of radioiodinated Aβ1-40 and seven plasma samples with Aβ1-42. Lipoproteins and lipid-free proteins were separated and analyzed for bound iodinated Aβ1-40. We found that up to 5% of Aβ added to plasma is bound to selected lipoproteins: very low density, low density, and high density, but not lipoprotein(a). The large majority (≈89%), however, is bound to albumin, and very little Aβ is free. Aβ distribution in plasma was not significantly influenced by apolipoprotein E genotype. We conclude that Aβ is normally bound to and transported by albumin and specific lipoproteins in human plasma under physiological conditions.

Pink1 forms a multiprotein complex with Miro and Milton, linking Pink1 function to mitochondrial trafficking.

Recessive mutations in Pink1 lead to a selective degeneration of dopaminergic neurons in the substantia nigra that is characteristic of Parkinson disease. Pink1 is a kinase that is targeted in part to mitochondria, and loss of Pink1 function can alter mitochondrial morphology and dynamics, thus supporting a link between mitochondrial dysfunction and Parkinson disease etiology. Here, we report the unbiased identification and confirmation of a mitochondrial multiprotein complex that contains Pink1, the atypical GTPase Miro, and the adaptor protein Milton. Our screen also identified an interaction between Pink1 and Mitofilin. Based on previously established functions for Miro and Milton in the trafficking of mitochondria along microtubules, we postulate here a role for Pink1 in mitochondrial trafficking. Using subcellular fractionation, we show that the overexpression of Miro and Milton, both of which are known to reside at the outer mitochondrial membrane, increases the mitochondrial Pink1 pool, suggesting a function of Pink1 at the outer membrane. Further, we document that Pink1 expressed without a mitochondrial targeting sequence can still be targeted to a mitochondria-enriched subcellular fraction via Miro and Milton. The latter finding is important for the interpretation of a previously reported protective effect of Pink1 expressed without a mitochondrial targeting sequence. Finally, we find that Miro and Milton expression suppresses altered mitochondrial morphology induced by loss of Pink1 function in cell culture. Our findings suggest that Pink1 functions in the trafficking of mitochondria in cells.

Transition-state analogue inhibitors of γ-secretase bind directlyto presenilin-1

The β-amyloid precursor protein (β-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 γ-secretases. Here we show that an affinity reagent designed to interact with the active site of γ-secretase binds directly and specifically to heterodimeric forms of presenilins, polytopic proteins that are mutated in hereditary Alzheimer’s and are known mediators of γ-secretase cleavage of both β-APP and Notch. These results provide evidence that heterodimeric presenilins contain the active site of γ-secretase, and validate presenilins as principal targets for the design of drugs to treat and prevent Alzheimer’s disease.

Assembly of the γ-Secretase Complex Involves Early Formation of an Intermediate Subcomplex of Aph-1 and Nicastrin

The γ-secretase complex is an unusual multimeric protease responsible for the intramembrane cleavage of a variety of type 1 transmembrane proteins, including the β-amyloid precursor protein and Notch. Genetic and biochemical data have revealed that this protease consists of the presenilin heterodimer, a highly glycosylated form of nicastrin, and the recently identified gene products, Aph-1 and Pen-2. Whereas current evidence supports the notion that presenilin comprises the active site of the protease and that the other three components are members of the active complex required for proteolytic activity, the individual roles of the three co-factors remain unclear. Here, we demonstrate that endogenous Aph-1 interacts with an immature species of nicastrin, forming a stable intermediate early in the assembly of the γ-secretase complex, prior to the addition of presenilin and Pen-2. Our data suggest 1) that Aph-1 is involved in the early stages of γ-secretase assembly through the stabilization and perhaps glycosylation of nicastrin and by scaffolding nicastrin to the immature γ-secretase complex, and 2) that presenilin, and later Pen-2, bind to this intermediate during the formation of the mature protease.