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.

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.

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.

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 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.

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.

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.

Activity-dependent isolation of the presenilin- gamma -secretase complex reveals nicastrin and a gamma substrate.

Presenilin heterodimers apparently contain the active site of gamma-secretase, a polytopic aspartyl protease involved in the transmembrane processing of both the Notch receptor and the amyloid-beta precursor protein. Although critical to embryonic development and the pathogenesis of Alzheimer's disease, this protease is difficult to characterize, primarily because it is a multicomponent complex of integral membrane proteins. Here the functional gamma-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 (beta-catenin, calsenilin, and presenilin-associated protein) did not bind. Moreover, anti-nicastrin antibodies immunoprecipitated gamma-secretase activity from detergent-solubilized microsomes. Unexpectedly, C83, the major endogenous amyloid-beta precursor protein substrate of gamma-secretase, was also quantitatively associated with the complex. These results provide direct biochemical evidence that nicastrin is a member of the active gamma-secretase complex, indicate that beta-catenin, calsenilin, and presenilin-associated protein are not required for gamma activity, and suggest an unprecedented mechanism of substrate-protease interaction.

Notch and the amyloid precursor protein are cleaved by similar gamma-secretase(s).

Gamma-secretase is an intramembrane-cleaving protease whose substrates include Notch and the amyloid precursor protein (APP). On the basis of initial genetic and pharmacologic data, the gamma-secretase activity responsible for cleavage of both proteins appears to be identical. However, apparent differences in the cleavage site and in sequence specificity raise questions about the degree of similarity between Notch and APP gamma-like proteolysis. In an effort to resolve this issue directly, we established an in vitro gamma-secretase activity assay that cleaves both APP- and Notch-based substrates, C100Flag and N100Flag. Analysis with specific gamma-secretase inhibitors, dominant-negative gamma-secretase preparations, and antibody co-immunoprecipitations all demonstrated identical cleavage of these substrates. Most importantly, we found that these substrates prevented cleavage of each other, indicating that the same gamma-secretase complex can cleave either protein. Finally, we provide evidence that both substrates are cut at two distinct regions in the transmembrane domain. These data resolve some of the apparent conflicts and strongly indicate that Notch and APP are proteolyzed by the same enzyme(s).

Detergent-dependent dissociation of active gamma-secretase reveals an interaction between Pen-2 and PS1-NTF and offers a model for subunit organization within the complex.

Gamma-secretase is a member of a new class of proteases with an intramembrane catalytic site and cleaves numerous type I membrane proteins, including the amyloid beta-protein precursor (APP) and the Notch receptor. Biochemical and genetic studies have identified four membrane proteins as components of gamma-secretase: a heterodimeric form of presenilin (PS), composed of its N- and C-terminal fragments (PS-NTF and PS-CTF, respectively), a highly glycosylated, mature form of nicastrin (NCT), Aph-1, and Pen-2. However, it is unclear how these components interact physically with each other and assemble into functional complexes. We and others recently found that Aph-1 interacts with a less glycosylated, immature form of nicastrin as an intermediate toward full assembly of gamma-secretase. Here we show that (1) the detergent dodecyl beta-d-maltoside (DDM) mediates the dissociation and inactivation of active gamma-secretase in a concentration-dependent manner, (2) DDM-dependent dissociation of the active gamma-secretase complex generates two major inactive complexes (Pen-2-PS1-NTF and mNCT-Aph-1) and two minor inactive complexes (mNCT-Aph1-PS1-CTF and PS1-NTF-PS1-CTF), and (3) Pen-2 can also associate with the PS holoprotein in complexes devoid of NCT and Aph-1. Taken together, our results demonstrate that Pen-2 interacts with PS-NTF within active gamma-secretase and offer a model for how the components of active gamma-secretase interact physically with each other.

Assembly of the gamma-secretase complex involves early formation of an intermediate subcomplex of Aph-1 and nicastrin.

The gamma-secretase complex is an unusual multimeric protease responsible for the intramembrane cleavage of a variety of type 1 transmembrane proteins, including the beta-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 gamma-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 gamma-secretase assembly through the stabilization and perhaps glycosylation of nicastrin and by scaffolding nicastrin to the immature gamma-secretase complex, and 2) that presenilin, and later Pen-2, bind to this intermediate during the formation of the mature protease.

Purification and characterization of the human gamma-secretase complex.

Gamma-secretase is a member of an unusual class of proteases with intramembrane catalytic sites. This enzyme cleaves many type I membrane proteins, including the amyloid beta-protein (Abeta) precursor (APP) and the Notch receptor. Biochemical and genetic studies have identified four membrane proteins as components of gamma-secretase: heterodimeric presenilin (PS) composed of its N- and C-terminal fragments (PS-NTF/CTF), a mature glycosylated form of nicastrin (NCT), Aph-1, and Pen-2. Recent data from studies in Drosophila, mammalian, and yeast cells suggest that PS, NCT, Aph-1, and Pen-2 are necessary and sufficient to reconstitute gamma-secretase activity. However, many unresolved issues, in particular the possibility of other structural or regulatory components, would be resolved by actually purifying the enzyme. Here, we report a detailed, multistep purification procedure for active gamma-secretase and an initial characterization of the purified protease. Extensive mass spectrometry of the purified proteins strongly suggests that PS-NTF/CTF, mNCT, Aph-1, and Pen-2 are the components of active gamma-secretase. Using the purified gamma-secretase, we describe factors that modulate the production of specific Abeta species: (1) phosphatidylcholine and sphingomyelin dramatically improve activity without changing cleavage specificity within an APP substrate; (2) increasing CHAPSO concentrations from 0.1 to 0.25% yields a approximately 100% increase in Abeta42 production; (3) exposure of an APP-based recombinant substrate to 0.5% SDS modulates cleavage specificity from a disease-mimicking pattern (high Abeta42/43) to a physiological pattern (high Abeta40); and (4) sulindac sulfide directly and preferentially decreases Abeta42 cleavage within the purified complex. Taken together, our results define a procedure for purifying active gamma-secretase and suggest that the lipid-mediated conformation of both enzyme and substrate regulate the production of the potentially neurotoxic Abeta42 and Abeta43 peptides.