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Genetics strongly implicate the amyloid β-peptide (Aβ) in the pathogenesis of Alzheimer's disease. Dominant missense mutation in the presenilins and the amyloid precursor protein (APP) cause early-onset familial Alzheimer's disease (FAD). As presenilin is the catalytic component of the γ-secretase protease complex that produces Aβ from APP, mutation of the enzyme or substrate that produce Aβ leads to FAD. However, the mechanism by which presenilin mutations cause FAD has been controversial, with gain of function and loss of function offered as binary choices. This overview will instead present the case that presenilins are dysfunctional in FAD. γ-Secretase is a multi-functional enzyme that proteolyzes the APP transmembrane domain in a complex and processive manner. Reduction in a specific function-the carboxypeptidase trimming of initially formed long Aβ peptides containing most of the transmembrane domain to shorter secreted forms-is an emerging common feature of FAD-mutant γ-secretase complexes.

Early-onset familial Alzheimer's disease (FAD) is pathologically and clinically similar to the more common late-onset sporadic form of the disease. The study of rare genetic mutations that cause FAD should provide insight into the pathogenesis of sporadic Alzheimer's disease. FAD mutations have only been found in the substrate (amyloid precursor protein, APP) and protease (γ-secretase) that produces the amyloid-β peptide (Aβ). The secreted, aggregation-prone 42-residue Aβ peptide (Aβ42) has long been considered the pathogenic entity in Alzheimer's disease. However, recent understanding of the complexity of the processing of APP by γ-secretase and the effects of FAD mutations on this processing suggest other forms of Aβ as potentially pathogenic., (© 2019 Elsevier Inc. All rights reserved.)

Summary: The National Institute of Environmental Health Sciences (NIEHS) introduces a new translational research framework that builds upon previous biomedical models to create a more comprehensive and integrated environmental health paradigm. The framework was developed as a graphical construct that illustrates the complexity of designing, implementing, and tracking translational research in environmental health. We conceptualize translational research as a series of concentric rings and nodes, defining "translation" as movement either from one ring to another or between nodes on a ring. A "Fundamental Questions" ring expands upon the research described in other frameworks as "basic" to include three interrelated concepts critical to basic science research: research questions, experimental settings, and organisms. This feature enables us to capture more granularity and thus facilitates an approach for categorizing translational research and its growth over time. We anticipate that the framework will help researchers develop compelling long-term translational research stories and accelerate public health impacts by clearly mapping out opportunities for collaborations. By using this paradigm, researchers everywhere will be better positioned to design research programs, identify research partners based on cross-disciplinary research needs, identify stakeholders who are likely to use the research for environmental decision-making and intervention, and track progress toward common goals. https://doi.org/10.1289/EHP3657.

Amyloid plaques, a neuropathological hallmark of Alzheimer's disease, are largely composed of amyloid β (Aβ) peptide, derived from cleavage of amyloid precursor protein (APP) by β- and γ-secretases. The endosome is increasingly recognized as an important crossroad for APP and these secretases, with major implications for APP processing and amyloidogenesis. Among various post-translational modifications affecting APP accumulation, ubiquitination of cytodomain lysines may represent a key signal controlling APP endosomal sorting. Here, we show that substitution of APP C-terminal lysines with arginine disrupts APP ubiquitination and that an increase in the number of substituted lysines tends to increase APP metabolism. An APP mutant lacking all C-terminal lysines underwent the most pronounced increase in processing, leading to accumulation of both secreted and intracellular Aβ40. Artificial APP ubiquitination with rapalog-mediated proximity inducers reduced Aβ40 generation. A lack of APP C-terminal lysines caused APP redistribution from endosomal intraluminal vesicles (ILVs) to the endosomal limiting membrane, with a subsequent decrease in APP C-terminal fragment (CTF) content in secreted exosomes, but had minimal effects on APP lysosomal degradation. Both the increases in secreted and intracellular Aβ40 were abolished by depletion of presenilin 2 (PSEN2), recently shown to be enriched on the endosomal limiting membrane compared with PSEN1. Our findings demonstrate that ubiquitin can act as a signal at five cytodomain-located lysines for endosomal sorting of APP. They further suggest that disruption of APP endosomal sorting reduces its sequestration in ILVs and results in PSEN2-mediated processing of a larger pool of APP-CTF on the endosomal membrane., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

4-Methylcyclohexanemethanol (MCHM) is a flotation reagent used in fine coal beneficiation. On January 9, 2014, crude MCHM, a mixture containing predominantly MCHM, was inadvertently released into the Elk River, a municipal water source that serves about 300,000 people in the Charleston, WV area, resulting in temporary contamination of 15 percent of the state's tap water and causing significant dermal exposure. The current studies were undertaken to determine whether crude MCHM or MCHM has the potential to produce dermal irritancy and/or sensitization. BALB/c female mice were treated daily for 3 consecutive days by direct epicutaneous application of 25 μL of various concentrations of crude MCHM or MCHM to the dorsum of each ear. A mouse ear-swelling test was used to determine irritancy potential and was undertaken in combination with the standardized Local Lymph Node Assay (LLNA) to determine skin sensitizing potential. MCHM was found to produce skin irritation at concentrations above 20% and did not produce sensitization. Crude MCHM also produced irritation, although weaker, and in addition was found to be a weak to moderate skin sensitizer. The results are discussed in terms of potential human health hazard., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Evaluating the impact of federally funded research with a broad, methodical, and objective approach is important to ensure that public funds advance the mission of federal agencies., Objectives: We aimed to develop a methodical approach that would yield a broad assessment of National Toxicology Program's (NTP's) effectiveness across multiple sectors and demonstrate the utility of the approach through a case study., Methods: A conceptual model was developed with defined activities, outputs (products), and outcomes (proximal, intermediate, distal) and applied retrospectively to NTP's research on hexavalent chromium (CrVI). Proximal outcomes were measured by counting views of and requests for NTP's products by external stakeholders. Intermediate outcomes were measured by bibliometric analysis. Distal outcomes were assessed through Web and LexisNexis searches for documents related to legislation or regulation changes., Results: The approach identified awareness of NTP's work on CrVI by external stakeholders (proximal outcome) and citations of NTP's research in scientific publications, reports, congressional testimonies, and legal and policy documents (intermediate outcome). NTP's research was key to the nation's first-ever drinking water standard for CrVI adopted by California in 2014 (distal outcome). By applying this approach to a case study, the utility and limitations of the approach were identified, including challenges to evaluating the outcomes of a research program., Conclusions: This study identified a broad and objective approach for assessing NTP's effectiveness, including methodological needs for more thorough and efficient impact assessments in the future. Citation: Xie Y, Holmgren S, Andrews DMK, Wolfe MS. 2017. Evaluating the impact of the U.S. National Toxicology Program: a case study on hexavalent chromium. Environ Health Perspect 125:181-188; http://dx.doi.org/10.1289/EHP21., Competing Interests: The authors declare they have no actual or potential competing financial interests.

Proteolysis within the membrane is catalyzed by a diverse family of proteases immersed within the hydrophobic environment of cellular membranes. These ubiquitous intramembrane-cleaving proteases (I-CLiPs) hydrolyze the transmembrane domains of a large variety of membrane-embedded proteins to facilitate signaling events essential to normal biological functions found in all forms of life. The importance of this unique class of enzyme is highlighted by its central involvement in a variety of human pathologies, including Alzheimer's disease (AD), Parkinson's disease, cancer, and the virulence of a number of viral, bacterial, and fungal pathogens. I-CLiPs therefore represent promising targets for the therapeutic treatment of numerous diseases. The key to understanding the normal biological function of I-CLiPs and capitalizing on their therapeutic potential is through a thorough understanding of the complex catalytic mechanisms that govern this unusual class of enzyme. This is an intrinsically difficult endeavor, given that these enzymes and their substrates reside within lipid membranes, making any in vitro assay technically challenging to design and execute. Here, we describe several in vitro enzymatic assays for the study of the AD-associated γ-secretase protease, which have aided the development of potent γ-secretase-targeting compounds as candidate therapeutics. These assays have also been applied in various forms for the study of other I-CLiPs, providing valuable mechanistic insights into some of the functional similarities and differences between several members of this fascinating family of proteases., (© 2017 Elsevier Inc. All rights reserved.)

The amyloid β-peptide (Aβ) of Alzheimer's disease (AD) is generated by proteolysis within the transmembrane domain (TMD) of a C-terminal fragment of the amyloid β protein-precursor (APP CTFβ) by the γ-secretase complex. This processing produces Aβ ranging from 38 to 49 residues in length. Evidence suggests that this spectrum of Aβ peptides is the result of successive γ-secretase cleavages, with endoproteolysis first occurring at the ε sites to generate Aβ48 or Aβ49, followed by C-terminal trimming mostly every three residues along two product lines to generate shorter, secreted forms of Aβ: the primary Aβ49-46-43-40 line and a minor Aβ48-45-42-38 line. The major secreted Aβ species are Aβ40 and Aβ42, and an increased proportion of the longer, aggregation-prone Aβ42 compared to Aβ40 is widely thought to be important in AD pathogenesis. We examined TMD substrate determinants of the specificity and efficiency of ε site endoproteolysis and carboxypeptidase trimming of CTFβ by γ-secretase. We determined that the C-terminal negative charge of the intermediate Aβ49 does not play a role in its trimming by γ-secretase. Peptidomimetic probes suggest that γ-secretase has S1', S2', and S3' pockets, through which trimming by tripeptides may be determined. However, deletion of residues around the ε sites demonstrates that a depth of three residues within the TMD is not a determinant of the location of endoproteolytic ε cleavage of CTFβ. We also show that instability of the CTFβ TMD helix near the ε site significantly increases endoproteolysis, and that helical instability near the carboxypeptidase cleavage sites facilitates C-terminal trimming by γ-secretase. In addition, we found that CTFβ dimers are not endoproteolyzed by γ-secretase. These results support a model in which initial interaction of the array of residues along the undimerized single helical TMD of substrates dictates the site of initial ε cleavage and that helix unwinding is essential for both endoproteolysis and carboxypeptidase trimming.

γ-secretase is responsible for the proteolysis of amyloid precursor protein (APP) into short, aggregation-prone amyloid-beta (Aβ) peptides, which are centrally implicated in the pathogenesis of Alzheimer's disease (AD). Despite considerable interest in developing γ-secretase targeting therapeutics for the treatment of AD, the precise mechanism by which γ-secretase produces Aβ has remained elusive. Herein, we demonstrate that γ-secretase catalysis is driven by the stabilization of an enzyme-substrate scission complex via three distinct amino-acid-binding pockets in the enzyme's active site, providing the mechanism by which γ-secretase preferentially cleaves APP in three amino acid increments. Substrate occupancy of these three pockets occurs after initial substrate binding but precedes catalysis, suggesting a conformational change in substrate may be required for cleavage. We uncover and exploit substrate cleavage preferences dictated by these three pockets to investigate the mechanism by which familial Alzheimer's disease mutations within APP increase the production of pathogenic Aβ species., Competing Interests: The authors declare that no competing interests exist.

There is high demand in environmental health for adoption of a structured process that evaluates and integrates evidence while making decisions and recommendations transparent. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) framework holds promise to address this demand. For over a decade, GRADE has been applied successfully to areas of clinical medicine, public health, and health policy, but experience with GRADE in environmental and occupational health is just beginning. Environmental and occupational health questions focus on understanding whether an exposure is a potential health hazard or risk, assessing the exposure to understand the extent and magnitude of risk, and exploring interventions to mitigate exposure or risk. Although GRADE offers many advantages, including its flexibility and methodological rigor, there are features of the different sources of evidence used in environmental and occupational health that will require further consideration to assess the need for method refinement. An issue that requires particular attention is the evaluation and integration of evidence from human, animal, in vitro, and in silico (computer modeling) studies when determining whether an environmental factor represents a potential health hazard or risk. Assessment of the hazard of exposures can produce analyses for use in the GRADE evidence-to-decision (EtD) framework to inform risk-management decisions about removing harmful exposures or mitigating risks. The EtD framework allows for grading the strength of the recommendations based on judgments of the certainty in the evidence (also known as quality of the evidence), as well as other factors that inform recommendations such as social values and preferences, resource implications, and benefits. GRADE represents an untapped opportunity for environmental and occupational health to make evidence-based recommendations in a systematic and transparent manner. The objectives of this article are to provide an overview of GRADE, discuss GRADE's applicability to environmental health, and identify priority areas for method assessment and development., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Mutations in the presenilins that cause familial Alzheimer's disease alter the activity of these proteases to increase generation of an aggregation-prone isoform of the amyloid β-peptide (Aβ). How these mutations do so has been unclear. Sannerud et al. now show that regulation of subcellular localization plays a central role, advancing our understanding of the cell biology of Alzheimer's disease., (Copyright © 2016 Elsevier Inc. All rights reserved.)

γ-Secretase is one of two proteases directly involved in the production of the amyloid β-peptide (Aβ), which is pathogenic in Alzheimer's disease. Inhibition of γ-secretase to suppress the production of Aβ should not block processing of one of its alternative substrates, Notch1 receptors, as interference with Notch1 signaling leads to severe toxic effects. In the course of our studies to identify γ-secretase inhibitors with selectivity for APP over Notch, 1 [3-(benzyl(isopropyl)amino)-1-(naphthalen-2-yl)propan-1-one] was found to inhibit γ-secretase-mediated Aβ production without interfering with γ-secretase-mediated Notch processing in purified enzyme assays. As 1 is chemically unstable, efforts to increase the stability of this compound led to the identification of 2 [naphthalene-2-carboxylic acid benzyl-isopropyl-amide] which showed similar biological activity to compound 1. Synthesis and evaluation of a series of amide analogs resulted in benzofuranyl amide analogs that showed promising Notch-sparing γ-secretase inhibitory effects. This class of compounds may serve as a novel lead series for further study in the development of γ-secretase inhibitors., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In search for novel lead compounds as γ-secretase inhibitors, analogs of aminopyrido[2,3-d]pyrimidin-7-ones (I) were synthesized and evaluated for inhibitory effects on amyloid-β-peptide production and cleavage of the Notch1 receptor mediated by γ-secretase. Selected pyridopyrimidines, such as 1, 8, 9, 10, 11 and 16 are γ-secretase inhibitors that did not have an effect on Notch1 receptor processing., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

One therapeutic approach for Alzheimer's disease is to inhibit the cleavage of the amyloid precursor protein (APP) by γ-secretase. At the beginning of a series of studies from our laboratories, a series of novel γ-amino alcohols (1) were found to possess γ-secretase inhibitory activity and Notch-sparing effects. A new one-pot synthesis of γ-amino alcohols and the structure-activity relationship (SAR) of these analogs will be discussed., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

γ-Secretase is an intramembrane-cleaving protease that processes many type-I integral membrane proteins within the lipid bilayer, an event preceded by shedding of most of the substrate's ectodomain by α- or β-secretases. The mechanism by which γ-secretase selectively recognizes and recruits ectodomain-shed substrates for catalysis remains unclear. In contrast to previous reports that substrate is actively recruited for catalysis when its remaining short ectodomain interacts with the nicastrin component of γ-secretase, we find that substrate ectodomain is entirely dispensable for cleavage. Instead, γ-secretase-substrate binding is driven by an apparent tight-binding interaction derived from substrate transmembrane domain, a mechanism in stark contrast to rhomboid--another family of intramembrane-cleaving proteases. Disruption of the nicastrin fold allows for more efficient cleavage of substrates retaining longer ectodomains, indicating that nicastrin actively excludes larger substrates through steric hindrance, thus serving as a molecular gatekeeper for substrate binding and catalysis.

Two proteolytic enzymes, β- and γ-secretases, work together to produce the amyloid β-peptide of Alzheimer's disease. New evidence suggests that these proteases directly interact and compounds that disrupt this interaction reduce amyloid β-peptide levels without directly blocking either enzyme's solo activity.

The biological underpinnings linking stress to Alzheimer's disease (AD) risk are poorly understood. We investigated how corticotrophin releasing factor (CRF), a critical stress response mediator, influences amyloid-β (Aβ) production. In cells, CRF treatment increases Aβ production and triggers CRF receptor 1 (CRFR1) and γ-secretase internalization. Co-immunoprecipitation studies establish that γ-secretase associates with CRFR1; this is mediated by β-arrestin binding motifs. Additionally, CRFR1 and γ-secretase co-localize in lipid raft fractions, with increased γ-secretase accumulation upon CRF treatment. CRF treatment also increases γ-secretase activity in vitro, revealing a second, receptor-independent mechanism of action. CRF is the first endogenous neuropeptide that can be shown to directly modulate γ-secretase activity. Unexpectedly, CRFR1 antagonists also increased Aβ. These data collectively link CRF to increased Aβ through γ-secretase and provide mechanistic insight into how stress may increase AD risk. They also suggest that direct targeting of CRF might be necessary to effectively modulate this pathway for therapeutic benefit in AD, as CRFR1 antagonists increase Aβ and in some cases preferentially increase Aβ42 via complex effects on γ-secretase., (© 2015 The Authors.)

During synthesis, purification, and especially storage, antibody-drug conjugates (ADCs) may be exposed to various types of light. Several of the drugs commonly conjugated to antibodies contain photosensitive functional groups. Exposure to light could generate an excited state of the drug that subsequently triggers drug and/or protein degradation. To mimic and study photoinduced ADC degradation, we designed a model ADC in which the monoclonal antibody (mAb) trastuzumab was treated with the amine-reactive probe eosin-5-isothiocyanate to yield an antibody-eosin conjugate (T-EO). Photoinduced degradation was monitored by size exclusion chromatography (SEC), dynamic light scattering (DLS), SDS-PAGE under reducing and nonreducing conditions, and MS/MS analysis. SEC analysis of the model ADC showed the formation of higher molecular weight species directly following a 20 W-hr/m(2) exposure of UVA light. DLS analysis of these samples showed the formation of larger soluble particles, and precipitate was observed 24 h post light exposure. These results were not seen in control samples of the model ADC that were shielded from light. Furthermore, these results were not seen in control samples containing mAb alone, suggesting that aggregation was the result of light exposure of the conjugate. Importantly, when eosin-5-isothiocyanate was added separately to solutions containing mAb (i.e., without conjugation), the extent of photoinduced aggregation was substantially less, indicating that the conjugation of the photosensitizer to the mAb specifically promoted photoinduced aggregation. Reducing and nonreducing SDS-PAGE suggested that photoinduced interchain covalent cross-linking occurred through a mechanism other than disulfide formation. Using peptide mapping and MS/MS analysis, we identified key peptides in the T-EO sequence that undergo photodegradation. Finally, we also show that cross-linking products formed in as little as 1 h of exposure to ambient light. These findings suggest that precautions should be taken to ensure minimal exposure to light during the synthesis, purification, and storage of ADCs containing photosensitive drugs.

Background: A cognitive-behavioural therapy (CBT) programme designed for post-traumatic stress disorder (PTSD) in people with severe mental illness, including breathing retraining, education and cognitive restructuring, was shown to be more effective than usual services., Aims: To evaluate the incremental benefit of adding cognitive restructuring to the breathing retraining and education components of the CBT programme (trial registration: clinicaltrials.gov identifier: NCT00494650)., Method: In all, 201 people with severe mental illness and PTSD were randomised to 12- to 16-session CBT or a 3-session brief treatment programme (breathing retraining and education). The primary outcome was PTSD symptom severity. Secondary outcomes were PTSD diagnosis, other symptoms, functioning and quality of life., Results: There was greater improvement in PTSD symptoms and functioning in the CBT group than in the brief treatment group, with both groups improving on other outcomes and effects maintained 1-year post-treatment., Conclusions: Cognitive restructuring has a significant impact beyond breathing retraining and education in the CBT programme, reducing PTSD symptoms and improving functioning in people with severe mental illness., (© The Royal College of Psychiatrists 2015.)

To promote and maintain health in agricultural and food systems, appropriate criteria are needed for the description and assessment of the health of soils, plants, animals, humans and ecosystems. Here we identify the concept of resilience as a universally applicable and fundamentally important criterion of health in all relevant areas of agriculture. We discuss definitions of resilience for soils, plants, animals, humans and ecosystems, and explore ways in which resilience can be applied as a criterion of health in different agricultural contexts. We show how and why resilience can be seen as a key criterion of health. Based on this, we discuss how resilience can be used as a link between soil, plant, animal, human and ecosystem health. Finally, we highlight four key areas for future research on resilience in agriculture, namely spatial and temporal scaling of resilience; effects of diversity; the role of networks for resilience; and stakeholder involvement., (© 2013 Society of Chemical Industry.)

The presenilin-containing γ-secretase complex produces the amyloid β-peptide (Aβ) through intramembrane proteolysis, and >100 presenilin mutations are associated with familial early-onset Alzheimer disease (AD). The question of whether these mutations result in AD through a gain or a loss of function remains highly controversial. Mutations in presenilins increase ratios of 42- to 40-residue Aβ critical to pathogenesis, but other Aβs of 38-49 residues are also formed by γ-secretase. Evidence in cells suggests the protease first cleaves substrate within the transmembrane domain at the ϵ site to form 48- or 49-residue Aβ. Subsequent cleavage almost every three residues from the C terminus is thought to occur along two pathways toward shorter secreted forms of Aβ: Aβ49 → Aβ46 → Aβ43 → Aβ40 and Aβ48 → Aβ45 → Aβ42 → Aβ38. Here we show that the addition of synthetic long Aβ peptides (Aβ45-49) directly into purified preparations of γ-secretase leads to the formation of Aβ40 and Aβ42 whether the protease complex is detergent-solubilized or reconstituted into lipid vesicles, and the ratios of products Aβ42 to Aβ40 follow a pattern consistent with the dual-pathway hypothesis. Kinetic analysis of five different AD-causing mutations in presenilin-1 revealed that all result in drastic reduction of normal carboxypeptidase function. Altered trimming of long Aβ peptides to Aβ40 and Aβ42 by mutant proteases occurs at multiple levels, independent of the effects on initial endoproteolysis at the ϵ site, all conspiring to increase the critical Aβ42/Aβ40 ratio implicated in AD pathogenesis. Taken together, these results suggest that specific reduction of carboxypeptidase function of γ-secretase leads to the gain of toxic Aβ42/Aβ40., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The 19-transmembrane, multisubunit γ-secretase complex generates the amyloid β-peptide (Aβ) of Alzheimer's disease (AD) by an unusual intramembrane proteolysis of the β-amyloid precursor protein. The complex, which similarly processes many other type 1 transmembrane substrates, is composed of presenilin, Aph1, nicastrin, and presenilin enhancer (Pen-2), all of which are necessary for proper complex maturation and enzymatic activity. Obtaining a high-resolution atomic structure of the intact complex would greatly aid the rational design of compounds to modulate activity but is a very difficult task. A complementary method is to generate structures for each individual subunit to allow one to build a model of the entire complex. Here, we describe a method by which recombinant human Pen-2 can be purified from bacteria to > 95% purity at milligram quantities per liter, utilizing a maltose binding protein tag to both increase solubility and facilitate purification. Expressing the same construct in mammalian cells, we show that the large N-terminal maltose binding protein tag on Pen-2 still permits incorporation into the complex and subsequent presenilin-1 endoproteolysis, nicastrin glycosylation and proteolytic activity. These new methods provide valuable tools to study the structure and function of Pen-2 and the γ-secretase complex., (© 2014 International Society for Neurochemistry.)

The intramembrane protease γ-secretase is a key player in signaling and Alzheimer's disease, but its structural features have remained obscure. A structure reported recently reveals a horseshoe-shaped arrangement of 19 transmembrane helices and an extracellular domain positioned for substrate recognition. This advance bodes well for a finer resolution before long., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The 19-transmembrane γ-secretase complex generates the amyloid β-peptide of Alzheimer's disease by intramembrane proteolysis of the β-amyloid precursor protein. This complex is comprised of presenilin, Aph1, nicastrin, and Pen-2. The exact function and mechanism of the highly conserved Pen-2 subunit remain poorly understood. Using systematic mutagenesis, we confirm and extend our understanding of which key regions and specific residues play roles in various aspects of γ-secretase function, including maturation, localization, and activity, but not processivity. In general, mutations (1) within the first half of transmembrane domain (TMD) 1 of Pen-2 decreased PS1 endoproteolysis and γ-secretase proteolytic activity, (2) within the second half of TMD1 increased proteolytic activity, (3) within the cytosolic loop region decreased proteolytic activity, (4) within TMD2 decreased PS1 endoproteolysis, (5) within the first half of TMD2 decreased proteolytic activity, and (6) within C-terminal residues decreased proteolytic activity. Specific mutational effects included N33A in TMD1 causing an increase in γ-secretase complexes at the cell surface and a modest decrease in stability and the previously unreported I53A mutation in the loop region reducing stability 10-fold and proteolytic activity by half. In addition, we confirm that minor PS1 endoproteolysis can occur in the complete absence of Pen-2. Together, these data suggest that rather than solely being a catalyst for γ-secretase endoproteolysis, Pen-2 may also stabilize the complex prior to PS1 endoproteolysis, allowing time for full assembly and proper trafficking.

Background: Systematic-review methodologies provide objectivity and transparency to the process of collecting and synthesizing scientific evidence in reaching conclusions on specific research questions. There is increasing interest in applying these procedures to address environmental health questions., Objectives: The goal was to develop a systematic-review framework to address environmental health questions by extending approaches developed for clinical medicine to handle the breadth of data relevant to environmental health sciences (e.g., human, animal, and mechanistic studies)., Methods: The Office of Health Assessment and Translation (OHAT) adapted guidance from authorities on systematic-review and sought advice during development of the OHAT Approach through consultation with technical experts in systematic review and human health assessments, as well as scientific advisory groups and the public. The method was refined by considering expert and public comments and through application to case studies., Results and Discussion: Here we present a seven-step framework for systematic review and evidence integration for reaching hazard identification conclusions: 1) problem formulation and protocol development, 2) search for and select studies for inclusion, 3) extract data from studies, 4) assess the quality or risk of bias of individual studies, 5) rate the confidence in the body of evidence, 6) translate the confidence ratings into levels of evidence, and 7) integrate the information from different evidence streams (human, animal, and "other relevant data" including mechanistic or in vitro studies) to develop hazard identification conclusions., Conclusion: The principles of systematic review can be successfully applied to environmental health questions to provide greater objectivity and transparency to the process of developing conclusions.

The targeting of structural features in mRNA with specificity remains a great chemical challenge. A hairpin structure near exon 10 in the pre-mRNA encoding the tau protein controls its splicing, and dementia-causing mutations that disrupt this structure increase exon 10 splicing. We previously reported the discovery of small molecules, mitoxantrone (MTX) and analogs, which bind to the tau RNA hairpin structure and the design of bipartite antisense oligonucleotides (ASOs) that simultaneously bind to the discontinuous sequences that flank this hairpin. Herein we report the synthesis of a bipartite ASO conjugated to MTX using the tau RNA hairpin and flanking sequences as a template. A set of six MTX analogs, each containing a linker-azide, and a set of ten bipartite ASOs, each containing a branched linker-alkyne, were synthesized and tested in combinatorial fashion for their ability to conjugate in the presence or absence of template RNA. A single template-dependent MTX-ASO conjugate was identified from among the 60 reaction mixtures, demonstrating that the MTX and ASO precursors could simultaneously bind the RNA template and allow proper positioning of azide and alkyne for 1,3-cycloaddition. While the MTX-ASO conjugate proved too cytotoxic for cell-based assays, the conjugate inhibited tau exon 10 splicing under cell-free conditions more effectively than MTX or bipartite ASO alone., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Considerable evidence, particularly from genetics, points to the aggregation-prone amyloid β-peptide as a pathogenic entity in Alzheimer's disease. Hence, the proteases that produce this peptide from its precursor protein have been prime targets for the development of potential therapeutics. One of these proteases, γ-secretase, has been a particular focus. Many inhibitors and modulators of this membrane-embedded protease complex have been identified, with some brought into late-stage clinical trials, where they have spectacularly failed. The reasons for these failures will be discussed, along with recent findings on the mechanism of γ-secretase and of Alzheimer-causing mutations that may suggest new strategies for targeting this enzyme.

Brain deposition of the amyloid beta-protein (A β ) and tau are characteristic features in Alzheimer's disease (AD). Mutations in the A β precursor protein (APP) and a protease involved in A β production from APP strongly argue for a pathogenic role of A β in AD, while mutations in tau are associated with related disorders collectively called frontotemporal lobar degeneration (FTLD). Despite intense effort, therapeutic strategies that target A β or tau have not yet yielded medications, suggesting that alternative approaches should be pursued. In recent years, our laboratory has studied the role of mRNA in AD and FTLD, specifically those encoding tau and the A β -producing protease BACE1. As many FTLD-causing tau mutations destabilize a hairpin structure that regulates RNA splicing, we have targeted this structure with small molecules, antisense oligonucleotides, and small molecule-antisense conjugates. We have also discovered that microRNA interaction with the 3'-untranslated region of tau regulates tau expression. Regarding BACE1, we found that alternative splicing leads to inactive splice isoforms and antisense oligonucleotides shift splicing toward these inactive isoforms to decrease A β production. In addition, a G-quadruplex structure in the BACE1 mRNA plays a role in splice regulation. The prospects for targeting tau and BACE1 mRNAs as therapeutic strategies will be discussed.

Tau pathologically aggregates in Alzheimer's disease, and evidence suggests that reducing tau expression may be safe and beneficial for the prevention or treatment of this disease. We sought to examine the role of the 3'-untranslated region (3'-UTR) of human tau mRNA in regulating tau expression. Tau expresses two 3'-UTR isoforms, long and short, as a result of alternative polyadenylation. Using luciferase reporter constructs, we found that expression from these isoforms is differentially controlled in human neuroblastoma cell lines M17D and SH-SY5Y. Several microRNAs were computationally identified as candidates that might bind the long, but not short, tau 3'-UTR isoform. A hit from a screen of candidates, miR-34a, was subsequently shown to repress the expression of endogenous tau protein in M17D cells. Conversely, inhibition of endogenously expressed miR-34 family members leads to increased endogenous tau expression. In addition, through an unbiased screen of fragments of the human tau 3'-UTR using a luciferase reporter assay, we identified several other regions in the long tau 3'-UTR isoform that contain regulatory cis-elements. Improved understanding of the regulation of tau expression by its 3'-UTR may ultimately lead to the development of novel therapeutic strategies for the treatment of Alzheimer's disease and other tauopathies. mRNA 3'-untranslated regions (3'-UTR) often regulate transcript stability or translation. Despite the centrality of the tau protein in Alzheimer's and other neurodegenerative diseases, the human tau 3'-UTR has been little studied. This report identifies regions of the tau 3'-UTR that influence expression and shows that microRNA (miR)-34a targets this 3'-UTR to lower expression, which is considered an important therapeutic goal., (© 2013 International Society for Neurochemistry.)

Presenilin is the catalytic component of the γ-secretase complex, a membrane-embedded aspartyl protease that plays a central role in biology and in the pathogenesis of Alzheimer's disease. Upon assembly with its three protein cofactors (nicastrin, Aph-1 and Pen-2), presenilin undergoes autoproteolysis into two subunits, each of which contributes one of the catalytic aspartates to the active site. A family of presenilin homologs, including signal peptide peptidase, possess proteolytic activity without the need for other protein factors, and these simpler intramembrane aspartyl proteases have given insight into the action of presenilin within the γ-secretase complex. Cellular and molecular studies support a nine-transmembrane topology for presenilins and their homologs, and small-molecule inhibitors and cysteine scanning with crosslinking have suggested certain presenilin residues and regions that contribute to substrate recognition and handling. Identification of partial complexes has also offered clues to protein-protein interactions within the γ-secretase complex. Biophysical methods have allowed 3D views of the γ-secretase complex and presenilins. Most recently, the crystal structure of a microbial presenilin homolog has confirmed a nine-transmembrane topology and intramembranous location and proximity of the two conserved and essential aspartates. The crystal structure also provides a platform for the formulation of specific hypotheses regarding substrate interaction and catalysis as well as the pathogenic mechanism of Alzheimer-causing presenilin mutations. This article is part of a Special Issue entitled: Intramembrane Proteases., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Approximately 15% of human genetic diseases are estimated to involve dysregulation of alternative pre-mRNA splicing. Antisense molecules designed to alter these and other splicing events typically target continuous linear sequences of the message. Here, we show that a structural feature in a pre-mRNA can be targeted by bipartite antisense molecules designed to hybridize with the discontinuous elements that flank the structure and thereby alter splicing. We targeted a hairpin structure at the boundary between exon 10 and intron 10 of the pre-mRNA of tau. Mutations in this region that are associated with certain forms of frontotemporal dementia, destabilize the hairpin to cause increased inclusion of exon 10. Via electrophoretic mobility shift and RNase protection assays, we demonstrate that bipartite antisense molecules designed to simultaneously interact with the available sequences that immediately flank the tau pre-mRNA hairpin do indeed bind to this structured region. Moreover, these agents inhibit exon 10 splicing and reverse the effect of destabilizing disease-causing mutations, in both in vitro splicing assays and cell culture. This general bipartite antisense strategy could be employed to modulate other splicing events that are regulated by RNA secondary structure.

γ-Secretase is a membrane-embedded protease complex with presenilin as the catalytic component. Cleavage within the transmembrane domain of the amyloid β-protein precursor (APP) by γ-secretase produces the C-terminus of the amyloid β-peptide (Aβ), a proteolytic product prone to aggregation and strongly linked to Alzheimer's disease (AD). Presenilin mutations are associated with early-onset AD, but their pathogenic mechanisms are unclear. One hypothesis is that these mutations cause AD through a toxic gain of function, changing γ-secretase activity to increase the proportion of 42-residue Aβ over the more soluble 40-residue form. A competing hypothesis is that the mutations cause AD through a loss of function, by reducing γ-secretase activity. However, γ-secretase apparently has two types of activities, an endoproteolytic function that first cuts APP to generate a 48/49-residue form of Aβ, and a carboxypeptidase activity that processively trims these longer Aβ intermediates approximately every three residues to form shorter, secreted forms. Recent studies suggest a resolution of the gain-of-function vs. loss-of-function debate: presenilin mutations may increase the proportion of longer, more aggregation-prone Aβ by specifically decreasing the trimming activity of γ-secretase. That is, the reduction of this particular proteolytic function of presenilin, not its endoproteolytic activity, may lead to the neurotoxic gain of function.

β-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is the transmembrane aspartyl protease that catalyzes the first cleavage step in the proteolysis of the APP to the amyloid β-protein (Aβ), a process involved in the pathogenesis of Alzheimer disease. BACE1 pre-mRNA undergoes complex alternative splicing, the regulation of which is not well understood. We identified a G-rich sequence within exon 3 of BACE1 involved in controlling splice site selection. Mutation of the G-rich sequence decreased use of the normal 5' splice site of exon 3, which leads to full-length and proteolytically active BACE1, and increased use of an alternative splice site, which leads to a shorter, essentially inactive isoform. Nuclease protection assays, nuclear magnetic resonance, and circular dichroism spectroscopy revealed that this sequence folds into a G-quadruplex structure. Several proteins were identified as capable of binding to the G-rich sequence, and one of these, heterogeneous nuclear ribonucleoprotein H, was found to regulate BACE1 exon 3 alternative splicing and in a manner dependent on the G-rich sequence. Knockdown of heterogeneous nuclear ribonucleoprotein H led to a decrease in the full-length BACE1 mRNA isoform as well as a decrease in Aβ production from APP, suggesting new possibilities for therapeutic approaches to Alzheimer's disease., (© 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

The 19-transmembrane multisubunit γ-secretase complex generates the amyloid β-peptide (Aβ) of Alzheimer's disease (AD) by intramembrane proteolysis of the β-amyloid precursor protein (APP). Despite substantial advances in elucidating how this protein complex functions, the effect of the local membrane lipid microenvironment on γ-secretase cleavage of substrates is still poorly understood. Using detergent-free proteoliposomes to reconstitute purified human γ-secretase, we examined the effects of fatty acyl (FA) chain length, saturation and double-bond isomerization, and membrane lipid polar headgroups on γ-secretase function. We analyzed γ-secretase activity and processivity [i.e., sequential cleavages in the APP transmembrane domain that convert longer Aβ species (e.g., Aβ(46)) into shorter ones (e.g., Aβ(40))] by quantifying the APP intracellular domain (AICD) and various Aβ peptides, including via a bicine/urea gel system that detects multiple Aβ lengths. These assays revealed several trends. (1) Switching from a cis to a trans isomer of a monounsaturated FA chain in phosphatidylcholine (PC) increased γ-activity, did not affect Aβ(42):Aβ(40) ratios, but decreased the ratio of long (≥42) versus short (≤41) Aβ peptides. (2) Increasing the FA carbon chain length (14, 16, 18, and 20) increased γ-activity, reduced longer Aβ species, and reduced the Aβ(42):Aβ(40) ratio. (3) Shifting the position of the double bond in 18:1(Δ9-cis) PC to the Δ6 position substantially reduced activity. (4) Gangliosides increased γ-activity but decreased processivity, thus elevating the Aβ(42):Aβ(40) ratio. (5) Phosphatidylserine decreased γ-activity but increased processivity. (6) Phosphatidylinositol strongly inhibited γ-activity. Overall, our results show that subtle changes in membrane lipid composition can greatly influence γ-secretase activity and processivity, suggesting that relatively small changes in lipid membrane composition may affect the risk of AD at least as much as presenilin or APP mutations do.

γ-Secretase is a protease complex responsible for cutting the transmembrane domain of the amyloid β-protein precursor (APP) to form the amyloid β-protein (Aβ), an aggregation-prone product that accumulates in the brain in Alzheimer's disease. As evidence suggests that Aβ is critical to Alzheimer pathogenesis, γ-secretase is considered a key target for the development of disease-modifying therapeutics. The protease complex cuts many other substrates, and some of these proteolytic events are part of signaling pathways or other important cellular functions. Among these, proteolysis of the Notch receptor is essential for signaling that is involved in a number of cell-fate determinations. Many inhibitors of γ-secretase have been identified, but it is clear that drug candidates for Alzheimer's disease should have minimal effects on the Notch signaling pathway, as serious safety issues have arisen with nonselective inhibitors. Two types of promising candidates that target this protease complex have emerged: the so-called "Notch-sparing" γ-secretase inhibitors, which block cleavage of APP selectively over that of Notch, and γ-secretase modulators, which shift the proportion of Aβ peptides produced in favor of shorter, less aggregation-prone species. The current status and prospects for these two general types of candidates will be discussed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The abnormal deposition of proteins in and around neurons is a common pathological feature of many neurodegenerative diseases. Among these pathological proteins, the microtubule-associated protein tau forms intraneuronal filaments in a spectrum of neurological disorders. The discovery that dominant mutations in the MAPT gene encoding tau are associated with familial frontotemporal dementia strongly supports abnormal tau protein as directly involved in disease pathogenesis. This and other evidence suggest that tau is a worthwhile target for the prevention or treatment of tau-associated neurodegenerative diseases, collectively called tauopathies. However, it is critical to understand the normal biological roles of tau, the specific molecular events that induce tau to become neurotoxic, the biochemical nature of pathogenic tau, the means by which pathogenic tau exerts neurotoxicity, and how tau pathology propagates. Based on known differences between normal and abnormal tau, a number of approaches have been taken toward the discovery of potential therapeutics. Key questions still remain open, such as the nature of the connection between the amyloid- β protein of Alzheimer's disease and tau pathology. Answers to these questions should help better understand the nature of tauopathies and may also reveal new therapeutic targets and strategies.

Presenilins were first discovered as sites of missense mutations responsible for early-onset Alzheimer disease (AD). The encoded multipass membrane proteins were subsequently found to be the catalytic components of γ-secretases, membrane-embedded aspartyl protease complexes responsible for generating the carboxyl terminus of the amyloid β-protein (Aβ) from the amyloid protein precursor (APP). The protease complex also cleaves a variety of other type I integral membrane proteins, most notably the Notch receptor, signaling from which is involved in many cell differentiation events. Although γ-secretase is a top target for developing disease-modifying AD therapeutics, interference with Notch signaling should be avoided. Compounds that alter Aβ production by γ-secretase without affecting Notch proteolysis and signaling have been identified and are currently at various stages in the drug development pipeline.

γ-Secretase is a membrane embedded aspartyl protease complex with presenilin as the catalytic component. Along with β-secretase, this enzyme produces the amyloid β-protein of Alzheimer's disease (AD) from the amyloid β-protein precursor. Because of its key role in the pathogenesis of AD, γ-secretase has been a prime target for drug discovery, and many inhibitors of this protease have been developed. The therapeutic potential of these inhibitors is virtually negated by the fact that γ-secretase is an essential part of the Notch signaling pathway, rendering the compounds unacceptably toxic upon chronic exposure. However, these compounds have served as useful chemical tools for biological investigations. In contrast, γ-secretase modulators continue to be of keen interest as possible AD therapeutics. These modulators either shift amyloid β-protein production to shorter, less pathogenic peptides or inhibit the proteolysis of amyloid β-protein precursor selectively compared to that of Notch. The various chemical types of inhibitors and modulators will be discussed, along with their use as probes for basic biology and their potential as AD therapeutics., (© 2011 The Author. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

DISC1 (Disrupted in schizophrenia-1) plays essential roles in neuronal proliferation, neuronal migration and axon guidance and has been implicated in schizophrenia and related psychiatric disorders. DISC1 forms a functional complex with nuclear distribution element-like protein-1 (NDEL1), a key component that regulates microtubule organization during cell division and neuronal migration. DISC1 polymorphisms at the binding interface of DISC1-NDEL1 complex have been implicated in schizophrenia. However, it is unknown how schizophrenia risk polymorphisms perturb its interaction with NDEL1 and how they change the inherent biochemical properties of DISC1. Here, we characterize the oligomerization and binding property of DISC1 and its natural schizophrenia risk variant, S704C. Our results show that DISC1 forms octamers via dimers as building blocks and directly interacts with tetramers of NDEL1. The schizophrenia risk variant S704C affects the formation of octamers of DISC1 and exhibits higher-order self-oligomerization. However, the observed formation of new oligomeric species did not influence its binding with NDEL1. These results suggest that the improper oligomeric assembly of DISC1-S704C may underlie the observed phenotypic variation due to the polymorphism.

The amyloid β-peptide (Aβ), strongly implicated in the pathogenesis of Alzheimer's disease (AD), is produced from the amyloid β-protein precursor (APP) through consecutive proteolysis by β- and γ-secretases. The latter protease contains presenilin as the catalytic component of a membrane-embedded aspartyl protease complex. Missense mutations in presenilin are associated with early-onset familial AD, and these mutations generally both decrease Aβ production and increase the ratio of the aggregation-prone 42-residue form (Aβ42) to the 40-residue form (Aβ40). The connection between these two effects is not understood. Besides Aβ40 and Aβ42, γ-secretase produces a range of Aβ peptides, the result of initial cutting at the ε site to form Aβ48 or Aβ49 and subsequent trimming every three or four residues. Thus, γ-secretase displays both overall proteolytic activity (ε cutting) and processivity (trimming) toward its substrate APP. Here we tested whether a decrease in total activity correlates with decreased processivity using wild-type and AD-mutant presenilin-containing protease complexes. Changes in pH, temperature, and salt concentration that reduced the overall activity of the wild-type enzyme did not consistently result in increased proportions of longer Aβ peptides. Low salt concentrations and acidic pH were notable exceptions that subtly alter the proportion of individual Aβ peptides, suggesting that the charged state of certain residues may influence processivity. Five different AD mutant complexes, representing a broad range of effects on overall activity, Aβ42:Aβ40 ratios, and ages of disease onset, were also tested, revealing again that changes in total activity and processivity can be dissociated. Factors that control initial proteolysis of APP at the ε site apparently differ significantly from factors affecting subsequent trimming and the distribution of Aβ peptides.