Plasma NT1-tau and Aß42 correlate with age and cognitive function in two large Down syndrome cohorts.

INTRODUCTION: People with Down syndrome (DS) often develop Alzheimer's disease (AD). Here, we asked whether ultrasensitive plasma immunoassays for a tau N-terminal fragment (NT1-tau) and Aß isoforms predict cognitive impairment. METHODS: Plasma NT1-tau, Aß37 , Aß40 , and Aß42 levels were measured in a longitudinal discovery cohort (N = 85 participants, 220 samples) and a cross-sectional validation cohort (N = 239). We developed linear models and predicted values in the validation cohort. RESULTS: Discovery cohort linear mixed models for NT1-tau, Aß42 , and Aß37:42 were significant for age; there was no main effect of time. In cross-sectional models, NT1-tau increased and Aß42 decreased with age. NT1-tau predicted cognitive and functional scores. The discovery cohort linear model for NT1-tau predicted levels in the validation cohort. DISCUSSION: NT1-tau correlates with age and worse cognition in DS. Further validation of NT1-tau and other plasma biomarkers of AD neuropathology in DS cohorts is important for clinical utility.

An ultra-sensitive immunoassay detects and quantifies soluble Aß oligomers in human plasma.

INTRODUCTION: Evidence strongly suggests that soluble oligomers of amyloid beta protein (oAß) help initiate the pathogenic cascade of Alzheimer's disease (AD). To date, there have been no validated assays specific for detecting and quantifying oAß in human blood. METHODS: We developed an ultrasensitive oAß immunoassay using a novel capture antibody (71A1) with N-terminal antibody 3D6 for detection that specifically quantifies soluble oAß in the human brain, cerebrospinal fluid (CSF), and plasma. RESULTS: Two new antibodies (71A1; 1G5) are oAß-selective, label Aß plaques in non-fixed AD brain sections, and potently neutralize the synaptotoxicity of AD brain-derived oAß. The 71A1/3D6 assay showed excellent dilution linearity in CSF and plasma without matrix effects, good spike recovery, and specific immunodepletion. DISCUSSION: We have created a sensitive, high throughput, and inexpensive method to quantify synaptotoxic oAß in human plasma for analyzing large cohorts of aged and AD subjects to assess the dynamics of this key pathogenic species and response to therapy.

Target engagement in an alzheimer trial: Crenezumab lowers amyloid ß oligomers in cerebrospinal fluid.

OBJECTIVE: Oligomeric forms of amyloid ß protein (oAß) are believed to be principally responsible for neurotoxicity in Alzheimer disease (AD), but it is not known whether anti-Aß antibodies are capable of lowering oAß levels in humans. METHODS: We developed an ultrasensitive immunoassay and used it to measure oAß in cerebrospinal fluid (CSF) from 104 AD subjects participating in the ABBY and BLAZE phase 2 trials of the anti-Aß antibody crenezumab. Patients received subcutaneous (SC) crenezumab (300mg) or placebo every 2 weeks, or intravenous (IV) crenezumab (15mg/kg) or placebo every 4 weeks for 68 weeks. Ninety-eight of the 104 patients had measurable baseline oAß levels, and these were compared to levels at week 69 in placebo (n = 28), SC (n = 35), and IV (n = 35) treated patients. RESULTS: Among those receiving crenezumab, 89% of SC and 86% of IV patients had lower levels of oAß at week 69 versus baseline. The difference in the proportion of patients with decreasing levels was significant for both treatment arms: p = 0.0035 for SC and p = 0.01 for IV crenezumab versus placebo. The median percentage change was -48% in the SC arm and -43% in the IV arm. No systematic change was observed in the placebo group, with a median change of -13% and equivalent portions with negative and positive change. INTERPRETATION: Crenezumab lowered CSF oAß levels in the large majority of treated patients tested. These results support engagement of the principal pathobiological target in AD and identify CSF oAß as a novel pharmacodynamic biomarker for use in trials of anti-Aß agents. ANN NEUROL 2019;86:215-224.

Secreted amyloid ß-proteins in a cell culture model include N-terminally extended peptides that impair synaptic plasticity.

Evidence for a central role of amyloid ß-protein (Aß) in the genesis of Alzheimer's disease (AD) has led to advanced human trials of Aß-lowering agents. The "amyloid hypothesis" of AD postulates deleterious effects of small, soluble forms of Aß on synaptic form and function. Because selectively targeting synaptotoxic forms of soluble Aß could be therapeutically advantageous, it is important to understand the full range of soluble Aß derivatives. We previously described a Chinese hamster ovary (CHO) cell line (7PA2 cells) that stably expresses mutant human amyloid precursor protein (APP). Here, we extend this work by purifying an sodium dodecyl sulfate (SDS)-stable, ∼8 kDa Aß species from the 7PA2 medium. Mass spectrometry confirmed its identity as a noncovalently bonded Aß40 homodimer that impaired hippocampal long-term potentiation (LTP) in vivo. We further report the detection of Aß-containing fragments of APP in the 7PA2 medium that extend N-terminal from Asp1 of Aß. These N-terminally extended Aß-containing monomeric fragments are distinct from soluble Aß oligomers formed from Aß1-40/42 monomers and are bioactive synaptotoxins secreted by 7PA2 cells. Importantly, decreasing ß-secretase processing of APP elevated these alternative synaptotoxic APP fragments. We conclude that certain synaptotoxic Aß-containing species can arise from APP processing events N-terminal to the classical ß-secretase cleavage site.

Plasma NT1-tau and Aß 42 correlate with age and cognitive function in two large Down syndrome cohorts.

Introduction: People with Down syndrome (DS) often develop Alzheimer disease (AD). Here we asked whether ultrasensitive plasma immunoassays for a tau N-terminal fragment (NT1-tau) and Aß isoforms predict cognitive impairment. Methods: Plasma NT1-tau, Aß 37 , Aß 40 , and Aß 42 levels were measured in a longitudinal discovery cohort (N = 85 participants, 220 samples) and a cross-sectional validation cohort (N = 239). We developed linear models and predicted values in the validation cohort. Results: Linear mixed models for NT1-tau, Aß 42, and Aß 37:42 were significant for age, there was no main effect of time in the discovery cohort. In cross-sectional models, NT1-tau and Aß 42 increased with age. NT1-tau predicted DLD scores. The discovery cohort linear model for NT1-tau predicted NT1-tau levels in the validation cohort. Discussion: NT1-tau correlates with age and worse cognition in DS. Further validation of NT1-tau and other plasma biomarkers of AD neuropathology in DS cohorts is important for clinical utility.

Dynamic analysis of amyloid ß-protein in behaving mice reveals opposing changes in ISF versus parenchymal Aß during age-related plaque formation.

Growing evidence supports the hypothesis that soluble, diffusible forms of the amyloid ß-peptide (Aß) are pathogenically important in Alzheimer's disease (AD) and thus have both diagnostic and therapeutic salience. To learn more about the dynamics of soluble Aß economy in vivo, we used microdialysis to sample the brain interstitial fluid (ISF), which contains the most soluble Aß species in brain at steady state, in >40 wake, behaving APP transgenic mice before and during the process of Aß plaque formation (age 3-28 months). Diffusible forms of Aß, especially Aß(42), declined significantly in ISF as mice underwent progressive parenchymal deposition of Aß. Moreover, radiolabeled Aß administered at physiological concentrations into ISF revealed a striking difference in the fate of soluble Aß in plaque-rich (vs plaque-free) mice: it clears more rapidly from the ISF and becomes more associated with the TBS-extractable pool, suggesting that cerebral amyloid deposits can rapidly sequester soluble Aß from the ISF. Likewise, acute γ-secretase inhibition in plaque-free mice showed a marked decline of Aß(38), Aß(40), and Aß(42), whereas in plaque-rich mice, Aß(42) declined significantly less. These results suggest that most of the Aß(42) that populates the ISF in plaque-rich mice is derived not from new Aß biosynthesis but rather from the large reservoir of less soluble Aß(42) in brain parenchyma. Together, these and other findings herein illuminate the in vivo dynamics of soluble Aß during the development of AD-type neuropathology and after γ-secretase inhibition and help explain the apparent paradox that CSF Aß(42) levels fall as humans develop AD.

Complement component C3 and complement receptor type 3 contribute to the phagocytosis and clearance of fibrillar Aß by microglia.

Complement components and their receptors are found within and around amyloid ß (Aß) cerebral plaques in Alzheimer's disease (AD). Microglia defend against pathogens through phagocytosis via complement component C3 and/or engagement of C3 cleavage product iC3b with complement receptor type 3 (CR3, Mac-1). Here, we provide direct evidence that C3 and Mac-1 mediate, in part, phagocytosis and clearance of fibrillar amyloid-ß (fAß) by murine microglia in vitro and in vivo. Microglia took up not only synthetic fAß(42) but also amyloid cores from patients with AD, transporting them to lysosomes in vitro. Fibrillar Aß(42) uptake was significantly attenuated by the deficiency or knockdown of C3 or Mac-1 and scavenger receptor class A ligands. In addition, C3 or Mac-1 knockdown combined with a scavenger receptor ligand, fucoidan, further attenuated fibrillar Aß(42) uptake by N9 microglia. Fluorescent fibrillar Aß(42) microinjected cortically was significantly higher in C3 and Mac-1 knockout mice compared with wild-type mice 5 days after surgery, indicating reduced clearance in vivo. Together, these results demonstrate that C3 and Mac-1 are involved in phagocytosis and clearance of fAß by microglia, providing support for a potential beneficial role for microglia and the complement system in AD pathogenesis. © 2012 Wiley Periodicals, Inc.

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.

Evaluating the Safety and Efficacy of Crenezumab vs Placebo in Adults With Early Alzheimer Disease: Two Phase 3 Randomized Placebo-Controlled Trials.

Importance: Alzheimer disease (AD), a neurodegenerative disease characterized by ß-amyloid plaques and τ tangles in the brain, represents an unmet medical need with no fully approved therapeutics to modify disease progression. Objective: To investigate the safety and efficacy of crenezumab, a humanized monoclonal immunoglobulin G4 antibody targeting ß-amyloid oligomers, in participants with prodromal to mild (early) AD. Design, Setting, and Participants: Two phase 3 multicenter randomized double-blind placebo-controlled parallel-group efficacy and safety studies of crenezumab in participants with early AD, CREAD and CREAD2, were initiated in 2016 and 2017, respectively, and were designed to evaluate the efficacy and safety of crenezumab in participants with early AD. CREAD (194 sites in 30 countries) and CREAD2 (209 sites in 27 countries) were global multicenter studies. A total of 3736 and 3664 participants were screened in CREAD and CREAD2, respectively. A total of 3736 and 3664 participants were screened in CREAD and CREAD2, respectively. Both trials enrolled individuals aged 50 to 85 years with early AD. Participants with some comorbidities and evidence of cerebral infarction or more than 4 microbleeds or areas of leptomeningeal hemosiderosis on magnetic resonance imaging were excluded. After 2923 and 2858 were excluded, respectively, 813 participants in CREAD and 806 in CREAD2 were randomly assigned in a 1:1 ratio to either placebo or crenezumab. In the final analysis, there were 409 participants in the placebo group and 404 in the crenezumab group in CREAD and 399 in the placebo group and 407 in the crenezumab group in CREAD2. Data were analyzed up until January 2019 and August 2019, respectively. Interventions: Participants received placebo or 60 mg/kg crenezumab intravenously every 4 weeks for up to 100 weeks. Main Outcomes and Measures: The primary outcome was change from baseline to week 105 in Clinical Dementia Rating-Sum of Boxes (CDR-SB) score. Results: There were 813 participants in CREAD (mean [SD] age, 70.7 [8.2] years; 483 female and 330 male) and 806 in CREAD2 (mean [SD] age, 70.9 [7.7] years; 456 female and 350 male). Baseline characteristics were balanced between both groups. The between-group difference in mean change from baseline in CDR-SB score (placebo minus crenezumab) was -0.17 (95% CI, -0.86 to 0.53; P = .63) at week 105 in the CREAD study (88 placebo; 86 crenezumab). Compared with previous trials, no new safety signals were identified, and amyloid-related imaging abnormalities with edema were rare, mild, and transient. No meaningful changes in AD biomarkers were observed. Both studies were discontinued following a preplanned interim analysis indicating that CREAD was unlikely to meet the primary end point. Conclusions and Relevance: Crenezumab was well tolerated but did not reduce clinical decline in participants with early AD. Trial Registration: ClinicalTrials.gov Identifiers: CREAD, NCT02670083; CREAD2, NCT03114657.

Aph-1 associates directly with full-length and C-terminal fragments of gamma-secretase substrates.

Gamma-secretase is a ubiquitous, multiprotein enzyme composed of presenilin, nicastrin, Aph-1, and Pen-2. It mediates the intramembrane proteolysis of many type 1 proteins, plays an essential role in numerous signaling pathways, and helps drive the pathogenesis of Alzheimer disease by excising the hydrophobic, aggregation-prone amyloid beta-peptide from the beta-amyloid precursor protein. A central unresolved question is how its many substrates bind and enter the gamma-secretase complex. Here, we provide evidence that both the beta-amyloid precursor protein holoprotein and its C-terminal fragments, the immediate substrates of gamma-secretase, can associate with Aph-1 at overexpressed as well as endogenous protein levels. This association was observed using bi-directional co-immunoprecipitation in multiple systems and detergent conditions, and an beta-amyloid precursor protein-Aph-1 complex was specifically isolated following in situ cross-linking in living cells. In addition, another endogenous canonical gamma-substrate, Jagged, showed association of both its full-length and C-terminal fragment forms with Aph-1. We were also able to demonstrate that this interaction with substrates was conserved across the multiple isoforms of Aph-1 (beta, alphaS, and alphaL), as they were all able to bind beta-amyloid precursor protein with similar affinity. Finally, two highly conserved intramembrane histidines (His-171 and His-197) within Aph-1, which were recently shown to be important for gamma-secretase activity, are required for efficient binding of substrates. Taken together, our data suggest a dominant role for Aph-1 in interacting with gamma-secretase substrates prior to their processing by the proteolytic complex.

Pink1 Parkinson mutations, the Cdc37/Hsp90 chaperones and Parkin all influence the maturation or subcellular distribution of Pink1.

Mutations in the ubiquitously expressed gene PTEN-induced kinase 1 (Pink1) cause autosomal recessive Parkinson's disease. Pink1 encodes a putative serine/threonine kinase with an N-terminal mitochondrial targeting sequence. The mechanism that leads to selective degeneration of dopaminergic neurons via Pink1 mutations is unknown. A full-length pre-protein (66 kDa) and an N-terminally truncated mature form (55 kDa) have been described in human brain. Here, we report that the endogenous 66 kDa and 55 kDa Pink1 forms in cultured cells are not exclusive to mitochondria but also occur in cytosolic and microsome-rich fractions. Pink1 66 kDa is the predominant isoform in cultured cells. Using unbiased analyses of immunoisolated Pink1 complexes by mass spectrometry, co-immunoprecipitation and Hsp90 inhibitor studies, we identify Pink1 as a novel Cdc37/Hsp90 client kinase. This chaperone system influences both the subcellular distribution and the 66/55 kDa protein ratio of Pink1. PD-causing Pink1 mutations decrease whereas Parkin expression increases the Pink1 66/55 kDa protein ratio, biochemically linking Pink1 and Parkin and highlighting the potential relevance of this ratio for PD pathogenesis. Finally, we document the influence of Parkin on Pink1 subcellular distribution, providing further evidence for a common pathogenic pathway in recessive PD.

Plasma N-terminal tau fragment levels predict future cognitive decline and neurodegeneration in healthy elderly individuals.

The availability of blood-based assays detecting Alzheimer's disease (AD) pathology should greatly accelerate AD therapeutic development and improve clinical care. This is especially true for markers that capture the risk of decline in pre-symptomatic stages of AD, as this would allow one to focus interventions on participants maximally at risk and at a stage prior to widespread synapse loss and neurodegeneration. Here we quantify plasma concentrations of an N-terminal fragment of tau (NT1) in a large, well-characterized cohort of clinically normal elderly who were followed longitudinally. Plasma NT1 levels at study entry (when all participants were unimpaired) were highly predictive of future cognitive decline, pathological tau accumulation, neurodegeneration, and transition to a diagnosis of MCI/AD. These predictive effects were particularly strong in participants with even modestly elevated brain ß-amyloid burden at study entry, suggesting plasma NT1 levels capture very early cognitive, pathologic and neurodegenerative changes along the AD trajectory.

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.

Decoding the synaptic dysfunction of bioactive human AD brain soluble Aß to inspire novel therapeutic avenues for Alzheimer's disease.

Pathologic, biochemical and genetic evidence indicates that accumulation and aggregation of amyloid ß-proteins (Aß) is a critical factor in the pathogenesis of Alzheimer's disease (AD). Several therapeutic interventions attempting to lower Aß have failed to ameliorate cognitive decline in patients with clinical AD significantly, but most such approaches target only one or two facets of Aß production/clearance/toxicity and do not consider the heterogeneity of human Aß species. As synaptic dysfunction may be among the earliest deficits in AD, we used hippocampal long-term potentiation (LTP) as a sensitive indicator of the early neurotoxic effects of Aß species. Here we confirmed prior findings that soluble Aß oligomers, much more than fibrillar amyloid plaque cores or Aß monomers, disrupt synaptic function. Interestingly, not all (84%) human AD brain extracts are able to inhibit LTP and the degree of LTP impairment by AD brain extracts does not correlate with Aß levels detected by standard ELISAs. Bioactive AD brain extracts also induce neurotoxicity in iPSC-derived human neurons. Shorter forms of Aß (including Aß1-37, Aß1-38, Aß1-39), pre-Aß APP fragments (- 30 to - 1) and N-terminally extended Aßs (- 30 to + 40) each showed much less synaptotoxicity than longer Aßs (Aß1-42 - Aß1-46). We found that antibodies which target the N-terminus, not the C-terminus, efficiently rescued Aß oligomer-impaired LTP and oligomer-facilitated LTD. Our data suggest that preventing soluble Aß oligomer formation and targeting their N-terminal residues with antibodies could be an attractive combined therapeutic approach.

The effects of oxidative stress on parkin and other E3 ligases.

Autosomal recessive mutations within the Parkin gene are associated with degeneration of the substantia nigra and locus coeruleus and an inherited form of Parkinson's disease (PD). As loss-of-function mutations in parkin are responsible for a familial variant of PD, conditions that affect wild-type parkin are likely to be associated with increased risk of idiopathic disease. Previous studies uncovered a unique vulnerability of the parkin protein to dopamine (DA)-induced aggregation and inactivation. In this study, we compared several proteins that share structural elements or ubiquitinating activity with parkin. We report that oxidative stress in several cell lines and primary neurons induces the aggregation of parkin into high molecular weight species, at least a portion of which are self-associated homo-multimers. While parkin was preferentially affected by excess DA, each of the E3 proteins tested were made more insoluble by oxidative stress, and they varied in degree of susceptibility (e.g. parkin > HHARI congruent with CHIP > c-Cbl > E6AP). These conditions of oxidative stress were also associated with decreased parkin E3 ligase activity. Similar to recently conducted studies on alpha-synuclein processing, both macroautophagy and the proteasome participate in parkin degradation, with the proteasome playing the predominant role for normal parkin turnover and macroautophagy being more important in the degradation of aggregated parkin. These data further highlight the selective vulnerability of parkin to DA-induced modifications, demonstrating for the first time the ability of both endogenous and ectopically expressed parkin to transition into an insoluble state in part through self-association and oligomer formation.

Soluble Aß oligomers are rapidly sequestered from brain ISF in vivo and bind GM1 ganglioside on cellular membranes.

Soluble Aß oligomers contribute importantly to synaptotoxicity in Alzheimer's disease, but their dynamics in vivo remain unclear. Here, we found that soluble Aß oligomers were sequestered from brain interstitial fluid onto brain membranes much more rapidly than nontoxic monomers and were recovered in part as bound to GM1 ganglioside on membranes. Aß oligomers bound strongly to GM1 ganglioside, and blocking the sialic acid residue on GM1 decreased oligomer-mediated LTP impairment in mouse hippocampal slices. In a hAPP transgenic mouse model, substantial levels of GM1-bound Aß42 were recovered from brain membrane fractions. We also detected GM1-bound Aß in human CSF, and its levels correlated with Aß42, suggesting its potential as a biomarker of Aß-related membrane dysfunction. Together, these findings highlight a mechanism whereby hydrophobic Aß oligomers become sequestered onto GM1 ganglioside and presumably other lipids on neuronal membranes, where they may induce progressive functional and structural changes.

gamma-Secretase substrate selectivity can be modulated directly via interaction with a nucleotide-binding site.

gamma-Secretase is an unusual protease with an intramembrane catalytic site that cleaves many type I membrane proteins, including the amyloid beta-protein (Abeta) precursor (APP) and the Notch receptor. Genetic and biochemical studies have identified four membrane proteins as components of gamma-secretase: heterodimeric presenilin composed of its N- and C-terminal fragments, nicastrin, Aph-1, and Pen-2. Here we demonstrated that certain compounds, including protein kinase inhibitors and their derivatives, act directly on purified gamma-secretase to selectively block cleavage of APP- but not Notch-based substrates. Moreover, ATP activated the generation of the APP intracellular domain and Abeta, but not the generation of the Notch intracellular domain by the purified protease complex, and was a direct competitor of the APP-selective inhibitors, as were other nucleotides. In accord, purified gamma-secretase bound specifically to an ATP-linked resin. Finally, a photoactivable ATP analog specifically labeled presenilin 1-C-terminal fragments in purified gamma-secretase preparations; the labeling was blocked by ATP itself and APP-selective gamma-secretase inhibitors. We concluded that a nucleotide-binding site exists within gamma-secretase, and certain compounds that bind to this site can specifically modulate the generation of Abeta while sparing Notch. Drugs targeting the gamma-secretase nucleotide-binding site represent an attractive strategy for safely treating Alzheimer disease.

Gamma-secretase is a membrane protein complex comprised of presenilin, nicastrin, Aph-1, and Pen-2.

gamma-Secretase catalyzes the intramembrane proteolysis of Notch, beta-amyloid precursor protein, and other substrates as part of a new signaling paradigm and as a key step in the pathogenesis of Alzheimer's 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 gamma-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 gamma-secretase activity. These findings suggest that the four membrane proteins comprise the limiting components of gamma-secretase and coassemble to form the active enzyme in mammalian cells.

Complex N-linked glycosylated nicastrin associates with active gamma-secretase and undergoes tight cellular regulation.

The intramembranous proteolysis of Notch and the amyloid precursor protein by gamma-secretase exemplifies an unusual and newly recognized mechanism of signal transduction in multicellular organisms. Here, we show that only a form of nicastrin (NCT) containing N-linked complex oligosaccharides is present in active gamma-secretase complexes. Overexpression of NCT does not generate more of this mature protein, a phenomenon analogous to the strictly regulated formation of mature presenilin heterodimers from immature holoprotein. The absence of presenilin severely limits the maturation of NCT, yet combined overexpression of both proteins does not increase respective mature types. Taken together, our findings describe unusual regulatory features of this key signaling protease: the association of NCT with gamma-secretase is tightly regulated via glycosylation; at least one other cofactor exists; the least abundant member of the complex becomes limiting; and the cofactor that serves this role may vary by cell type.