Sorting Out Presenilins in Alzheimer's Disease.

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 ß-peptide (Aß). 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.

γ-Secretase: a horseshoe structure brings good luck.

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.

Giving Alzheimer's the old one-two.

A dual goal for treating Alzheimer's disease (AD) is to decrease deposition of neurotoxic amyloid beta-peptide in the brain and to boost repair of damaged neurons. Donmez et al. (2010) now show that SIRT1 may mediate both processes by deacetylating the transcription factor retinoic acid receptor beta, a potential new therapeutic target for AD.

Presenilin/γ-Secretase Activity Is Located in Acidic Compartments of Live Neurons.

Presenilin (PSEN)/γ-secretase is a protease complex responsible for the proteolytic processing of numerous substrates. These substrates include the amyloid precursor protein (APP), the cleavage of which by γ-secretase results in the production of ß-amyloid (Aß) peptides. However, exactly where within the neuron γ-secretase processes APP C99 to generate Aß and APP intracellular domain (AICD) is still not fully understood. Here, we employ novel Förster resonance energy transfer (FRET)-based multiplexed imaging assays to directly "visualize" the subcellular compartment(s) in which γ-secretase primarily cleaves C99 in mouse cortex primary neurons (from both male and female embryos). Our results demonstrate that γ-secretase processes C99 mainly in LysoTracker-positive low-pH compartments. Using a new immunostaining protocol which distinguishes Aß from C99, we also show that intracellular Aß is significantly accumulated in the same subcellular loci. Furthermore, we found functional correlation between the endo-lysosomal pH and cellular γ-secretase activity. Taken together, our findings are consistent with Aß being produced from C99 by γ-secretase within acidic compartments such as lysosomes and late endosomes in living neurons.SIGNIFICANCE STATEMENT Alzheimer's disease (AD) genetics and histopathology highlight the importance of amyloid precursor protein (APP) processing by γ-secretase in pathogenesis. For the first time, this study has enabled us to directly "visualize" that γ-secretase processes C99 mainly in acidic compartments such as late endosomes and lysosomes in live neurons. Furthermore, we uncovered that intracellular ß-amyloid (Aß) is significantly accumulated in the same subcellular loci. Emerging evidence proposes the great importance of the endo-lysosomal pathway in mechanisms of misfolded proteins propagation (e.g., Tau, α-Syn). Therefore, the predominant processing of C99 and enrichment of Aß in late endosomes and lysosomes may be critical events in the molecular cascade leading to AD.

Verteporfin is a substrate-selective γ-secretase inhibitor that binds the amyloid precursor protein transmembrane domain.

This work reports substrate-selective inhibition of a protease with broad substrate specificity based on direct binding of a small-molecule inhibitor to the substrate. The target for these studies was γ-secretase protease, which cleaves dozens of different single-span membrane protein substrates, including both the C99 domain of the human amyloid precursor protein and the Notch receptor. Substrate-specific inhibition of C99 cleavage is desirable to reduce production of the amyloid-ß polypeptide without inhibiting Notch cleavage, a major source of toxicity associated with broad specificity γ-secretase inhibitors. In order to identify a C99-selective inhibitors of the human γ-secretase, we conducted an NMR-based screen of FDA-approved drugs against C99 in model membranes. From this screen, we identified the small-molecule verteporfin with these properties. We observed that verteporfin formed a direct 1:1 complex with C99, with a KD of 15-47 µM (depending on the membrane mimetic used), and that it did not bind the transmembrane domain of the Notch-1 receptor. Biochemical assays showed that direct binding of verteporfin to C99 inhibits γ-secretase cleavage of C99 with IC50 values in the range of 15-164 µM, while Notch-1 cleavage was inhibited only at higher concentrations, and likely via a mechanism that does not involve binding to Notch-1. This work documents a robust NMR-based approach to discovery of small-molecule binders to single-span membrane proteins and confirmed that it is possible to inhibit γ-secretase in a substrate-specific manner.

Understanding Reactive Sulfur Species through P/S Synergy.

We investigated the differential oxidative and nucleophilic chemistry of reactive sulfur and oxygen anions (SSNO-, SNO-, NO2-, S42-, and HS-) using the simple reducing electrophile PPh2Cl. In the case of SSNO- reacting with PPh2Cl, a complex mixture of mono and diphosphorus products is formed exclusively in the P(V) oxidation state. We found that the phosphine stoichiometry dictates selectivity for oxidation to P=S/P=O products or transformation to P2 species. Interestingly, only chalcogen atoms are incorporated into the phosphorus products and, instead, nitrogen is released in the form of NO gas. Finally, we demonstrate that more reducing anions (S42- and HS-) also react with PPh2Cl with P=S bond formation as a key reaction driving force.

Newborn hearing screening methodology impacts the timing of diagnosis for auditory neuropathy spectrum disorder.

INTRODUCTION: Auditory Neuropathy Spectrum Disorder (ANSD) accounts for 10 % to 15 % of pediatric hearing loss. In most cases, otoacoustic emissions (OAE) are present as the outer hair cell function is normal, and the auditory brainstem response (ABR) is abnormal. Newborn hearing screen (NBHS) is completed using OAE or ABR depending on the institution. Because OAEs are often present in ANSD, NBHS done solely with OAE can miss and delay diagnosis of patients with ANSD. OBJECTIVES: To assess whether NBHS methodology impacts the age of diagnosis of ANSD. METHODS: This is a retrospective study of patients, 0-18 years of age, diagnosed with ANSD at two tertiary pediatric hospitals from 1/01/2010 to 12/31/2018 after referral from NBHS performed in the community. Data recorded included patient demographics, method of NBHS, NICU stay, and age at ANSD diagnosis. RESULTS: 264 patients were diagnosed with ANSD. Of those, 123 (46.6 %) were female, and 141 (53.4 %) were male. Ninety-seven (36.8 %) were admitted to NICU and the mean stay was 6.98 weeks (STD = 10.7; CI = 4.8-9.1). The majority (244, 92.4 %) of patients had NBHS with ABR, and 20 (7.5 %) had NBHS with OAE. Patients screened with ABR were diagnosed with ANSD earlier than those who screened with OAE, with a mean age of 14.1 versus 27.3 weeks (p = 0.0397, CI = 15.2-39.3). Among those screened with ABR, median age at diagnosis was 4 months for NICU infants and 2.5 months for infants with no history of NICU stay over 5 days. In comparison, median diagnosis age was 8 months for non-NICU infants screened with OAEs. CONCLUSION: Patients with ANSD who had NBHS with ABR were diagnosed earlier than those with OAE. Our data suggest that universal screening with ABR may facilitate earlier diagnosis of ANSD and earlier evaluation for aural rehabilitation, especially in high-risk cohorts such as NICU patients. Further research is needed into factors that contribute to earlier diagnosis among patients screened with ABR.

Identification of the Aß37/42 peptide ratio in CSF as an improved Aß biomarker for Alzheimer's disease.

INTRODUCTION: Identifying CSF-based biomarkers for the ß-amyloidosis that initiates Alzheimer's disease (AD) could provide inexpensive and dynamic tests to distinguish AD from normal aging and predict future cognitive decline. METHODS: We developed immunoassays specifically detecting all C-terminal variants of secreted amyloid ß-protein and identified a novel biomarker, the Aß 37/42 ratio, that outperforms the canonical Aß42/40 ratio as a means to evaluate the γ-secretase activity and brain Aß accumulation. RESULTS: We show that Aß 37/42 can distinguish physiological and pathological status in (1) presenilin-1 mutant vs wild-type cultured cells, (2) AD vs control brain tissue, and (3) AD versus cognitively normal (CN) subjects in CSF, where 37/42 (AUC 0.9622) outperformed 42/40 (AUC 0.8651) in distinguishing CN from AD. DISCUSSION: We conclude that the Aß 37/42 ratio sensitively detects presenilin/γ-secretase dysfunction and better distinguishes CN from AD than Aß42/40 in CSF. Measuring this novel ratio alongside promising phospho-tau analytes may provide highly discriminatory fluid biomarkers for AD.

Unraveling the complexity of γ-secretase.

γ-Secretase was initially defined as a proteolytic activity that cleaves within the transmembrane of the amyloid precursor protein (APP) to produce the amyloid ß-peptide of Alzheimer's disease. The discovery of mutations in APP and the presenilins associated with familial Alzheimer's disease and their effects on APP processing dovetailed with pharmacological studies on γ-secretase, leading to the revelation that presenilins are unprecedented membrane-embedded aspartyl proteases. Other members of what became known as the γ-secretase complex were subsequently identified. In parallel with these advances, connections between presenilins and Notch receptors essential to metazoan development became evident, resulting in the concurrent realization that γ-secretase also carries out intramembrane proteolysis of Notch as part of its signaling mechanism. Substantial progress has been made toward elucidating how γ-secretase carries out complex processing of transmembrane domains, how it goes awry in familial Alzheimer's disease, the scope of its substrates, and the atomic details of its structure. Critical questions remain for future study, toward further unraveling the complexity of this unique membrane-embedded proteolytic machine and its roles in biology and disease.

Familial Alzheimer's disease mutations in amyloid protein precursor alter proteolysis by γ-secretase to increase amyloid ß-peptides of ≥45 residues.

Production of amyloid ß-protein (Aß) is carried out by the membrane-embedded γ-secretase complex. Mutations in the transmembrane domain of amyloid ß-protein precursor (APP) associated with early-onset familial Alzheimer's disease (FAD) can alter the ratio of aggregation-prone 42-residue Aß (Aß42) to 40-residue Aß (Aß40). However, APP substrate is proteolyzed processively by γ-secretase along two pathways: Aß49→Aß46→Aß43→Aß40 and Aß48→Aß45→Aß42→Aß38. Effects of FAD mutations on each proteolytic step are unknown, largely due to difficulties in detecting and quantifying longer Aß peptides. To address this, we carried out systematic and quantitative analyses of all tri- and tetrapeptide coproducts from proteolysis of wild-type and 14 FAD-mutant APP substrates by purified γ-secretase. These small peptides, including FAD-mutant forms, were detected by tandem mass spectrometry and quantified by establishing concentration curves for each of 32 standards. APP intracellular domain (AICD) coproducts were quantified by immunoblot, and the ratio of AICD products corresponding to Aß48 and Aß49 was determined by mass spectrometry. Levels of individual Aß peptides were determined by subtracting levels of peptide coproducts associated with degradation from those associated with production. This method was validated for Aß40 and Aß42 by specific ELISAs and production of equimolar levels of Aß and AICD. Not all mutant substrates led to increased Aß42/40. However, all 14 disease-causing mutations led to inefficient processing of longer forms of Aß ≥ 45 residues. In addition, the effects of certain mutations provided insight into the mechanism of processive proteolysis: intermediate Aß peptides apparently remain bound for subsequent trimming and are not released and reassociated.

Hydrophilic loop 1 of Presenilin-1 and the APP GxxxG transmembrane motif regulate γ-secretase function in generating Alzheimer-causing Aß peptides.

γ-Secretase is responsible for the proteolysis of amyloid precursor protein (APP) into amyloid-beta (Aß) peptides, which are centrally implicated in the pathogenesis of Alzheimer's disease (AD). The biochemical mechanism of how processing by γ-secretase is regulated, especially as regards the interaction between enzyme and substrate, remains largely unknown. Here, mutagenesis reveals that the hydrophilic loop-1 (HL-1) of presenilin-1 (PS1) is critical for both γ-secretase step-wise cleavages (processivity) and its allosteric modulation by heterocyclic γ-modulatory compounds. Systematic mutagenesis of HL-1, including all of its familial AD mutations and additional engineered variants, and quantification of the resultant Aß products show that HL-1 is necessary for proper sequential γ-secretase processivity. We identify Y106, L113, and Y115 in HL-1 as key targets for heterocyclic γ-secretase modulators (GSMs) to stimulate processing of pathogenic Aß peptides. Further, we confirm that the GxxxG domain in the APP transmembrane region functions as a critical substrate motif for γ-secretase processivity: a G29A substitution in APP-C99 mimics the beneficial effects of GSMs. Together, these findings provide a molecular basis for the structural regulation of γ-processivity by enzyme and substrate, facilitating the rational design of new GSMs that lower AD-initiating amyloidogenic Aß peptides.

Mechanism of Tripeptide Trimming of Amyloid ß-Peptide 49 by γ-Secretase.

The membrane-embedded γ-secretase complex processively cleaves within the transmembrane domain of amyloid precursor protein (APP) to produce 37-to-43-residue amyloid ß-peptides (Aß) of Alzheimer's disease (AD). Despite its importance in pathogenesis, the mechanism of processive proteolysis by γ-secretase remains poorly understood. Here, mass spectrometry and Western blotting were used to quantify the efficiency of tripeptide trimming of wild-type (WT) and familial AD (FAD) mutant Aß49. In comparison to WT Aß49, the efficiency of tripeptide trimming was similar for the I45F, A42T, and V46F Aß49 FAD mutants but substantially diminished for the I45T and T48P mutants. In parallel with biochemical experiments, all-atom simulations using a novel peptide Gaussian accelerated molecular dynamics (Pep-GaMD) method were applied to investigate the tripeptide trimming of Aß49 by γ-secretase. The starting structure was the active γ-secretase bound to Aß49 and APP intracellular domain (AICD), as generated from our previous study that captured the activation of γ-secretase for the initial endoproteolytic cleavage of APP (Bhattarai, A., ACS Cent. Sci. 2020, 6, 969-983). Pep-GaMD simulations captured remarkable structural rearrangements of both the enzyme and substrate, in which hydrogen-bonded catalytic aspartates and water became poised for tripeptide trimming of Aß49 to Aß46. These structural changes required a positively charged N-terminus of endoproteolytic coproduct AICD, which could dissociate during conformational rearrangements of the protease and Aß49. The simulation findings were highly consistent with biochemical experimental data. Taken together, our complementary biochemical experiments and Pep-GaMD simulations have enabled elucidation of the mechanism of tripeptide trimming of Aß49 by γ-secretase.

Principles of mRNA control by human PUM proteins elucidated from multimodal experiments and integrative data analysis.

The human PUF-family proteins, PUM1 and PUM2, posttranscriptionally regulate gene expression by binding to a PUM recognition element (PRE) in the 3'-UTR of target mRNAs. Hundreds of PUM1/2 targets have been identified from changes in steady-state RNA levels; however, prior studies could not differentiate between the contributions of changes in transcription and RNA decay rates. We applied metabolic labeling to measure changes in RNA turnover in response to depletion of PUM1/2, showing that human PUM proteins regulate expression almost exclusively by changing RNA stability. We also applied an in vitro selection workflow to precisely identify the binding preferences of PUM1 and PUM2. By integrating our results with prior knowledge, we developed a "rulebook" of key contextual features that differentiate functional versus nonfunctional PREs, allowing us to train machine learning models that accurately predict the functional regulation of RNA targets by the human PUM proteins.

Discovery of aryl aminothiazole γ-secretase modulators with novel effects on amyloid ß-peptide production.

A series of analogs based on a prototype aryl aminothiazole γ-secretase modulator (GSM) were synthesized and tested for their effects on the profile of 37-to-42-residue amyloid ß-peptides (Aß), generated through processive proteolysis of precursor protein substrate by γ-secretase. Certain substitutions on the terminal aryl D ring resulted in an altered profile of Aß production compared to that seen with the parent molecule. Small structural changes led to concentration-dependent increases in Aß37 and Aß38 production without parallel decreases in their precursors Aß40 and Aß42, respectively. The new compounds therefore apparently also stimulate carboxypeptidase trimming of Aß peptides ≥ 43 residues, providing novel chemical tools for mechanistic studies of processive proteolysis by γ-secretase.

ERß Regulation of Gonadotropin Responses during Folliculogenesis.

Gonadotropins are essential for regulating ovarian development, steroidogenesis, and gametogenesis. While follicle stimulating hormone (FSH) promotes the development of ovarian follicles, luteinizing hormone (LH) regulates preovulatory maturation of oocytes, ovulation, and formation of corpus luteum. Cognate receptors of FSH and LH are G-protein coupled receptors that predominantly signal through cAMP-dependent and cAMP-independent mechanisms that activate protein kinases. Subsequent vital steps in response to gonadotropins are mediated through activation or inhibition of transcription factors required for follicular gene expression. Estrogen receptors, classical ligand-activated transcriptional regulators, play crucial roles in regulating gonadotropin secretion from the hypothalamic-pituitary axis as well as gonadotropin function in the target organs. In this review, we discuss the role of estrogen receptor ß (ERß) regulating gonadotropin response during folliculogenesis. Ovarian follicles in Erß knockout (ErßKO) mutant female mice and rats cannot develop beyond the antral state, lack oocyte maturation, and fail to ovulate. Theca cells (TCs) in ovarian follicles express LH receptor, whereas granulosa cells (GCs) express both FSH receptor (FSHR) and LH receptor (LHCGR). As oocytes do not express the gonadotropin receptors, the somatic cells play a crucial role during gonadotropin induced oocyte maturation. Somatic cells also express high levels of estrogen receptors; while TCs express ERα and are involved in steroidogenesis, GCs express ERß and are involved in both steroidogenesis and folliculogenesis. GCs are the primary site of ERß-regulated gene expression. We observed that a subset of gonadotropin-induced genes in GCs, which are essential for ovarian follicle development, oocyte maturation and ovulation, are dependent on ERß. Thus, ERß plays a vital role in regulating the gonadotropin responses in ovary.

Probing Mechanisms and Therapeutic Potential of γ-Secretase in Alzheimer's Disease.

The membrane-embedded γ-secretase complex carries out hydrolysis within the lipid bilayer in proteolyzing nearly 150 different membrane protein substrates. Among these substrates, the amyloid precursor protein (APP) has been the most studied, as generation of aggregation-prone amyloid ß-protein (Aß) is a defining feature of Alzheimer's disease (AD). Mutations in APP and in presenilin, the catalytic component of γ-secretase, cause familial AD, strong evidence for a pathogenic role of Aß. Substrate-based chemical probes-synthetic peptides and peptidomimetics-have been critical to unraveling the complexity of γ-secretase, and small drug-like inhibitors and modulators of γ-secretase activity have been essential for exploring the potential of the protease as a therapeutic target for Alzheimer's disease. Such chemical probes and therapeutic prototypes will be reviewed here, with concluding commentary on the future directions in the study of this biologically important protease complex and the translation of basic findings into therapeutics.

Targeting γ-Secretase for Familial Alzheimer's Disease.

Familial Alzheimer's disease (FAD) is a rare early-onset genetic form of a common dementia of old age. Striking in middle age, FAD is caused by missense mutations in three genes: APP (encoding the amyloid precursor protein) and PSEN1 and PSEN2 (encoding presenilin-1 and presenilin-2). APP is proteolytically processed successively by ß-secretase and γ-secretase to produce the amyloid ß-peptide (Aß). Presenilin is the catalytic component of γ-secretase, a membrane-embedded aspartyl protease complex that cleaves APP within its single transmembrane domain to produce Aß of varying lengths. Thus, all FAD mutations are found in the substrate and the enzyme that produce Aß. The 42-residue variant Aß42 has been the primary focus of Alzheimer drug discovery for over two decades, as this particular peptide is highly prone to aggregation, is the major protein deposited in the characteristic cerebral plaques of Alzheimer's disease, and is proportionately elevated in FAD. Despite extensive efforts, all agents targeting Aß and Aß42 have failed in the clinic, including γ-secretase inhibitors, leading to questioning of the amyloid hypothesis of Alzheimer pathogenesis. However, processing of the APP transmembrane domain by γ-secretase is complex, involving initial endoproteolysis followed by successive carboxypeptidase trimming steps to secreted Aß peptides such as Aß42. Recent findings reveal that FAD mutations in PSEN1 and in APP result in deficient trimming of initially formed long Aß peptides. A logical drug discovery strategy for FAD could therefore involve the search for compounds that rescue this deficient carboxypeptidase activity. The rare early-onset FAD arguably presents a simpler path to developing effective therapeutics compared to the much more complex heterogeneous sporadic Alzheimer's disease.

Defluorinative Functionalization of Pd(II) Fluoroalkyl Complexes.

When subjected to arylboranes, anionic trifluoromethyl and difluorobenzyl palladium(II) complexes undergo fluoride abstraction followed by 1,1-migratory insertion. The resulting intermediate fluoroalkyl species can be induced to undergo a subsequent transmetalation and reductive elimination from either an in situ formed fluoroboronate (FB(Ar3)-) or an exogenous boronic acid/ester (ArB(OR)2) and nucleophilic activator, representing a net defluorinative arylation reaction. The latter method enabled a structurally diverse substrate scope to be prepared from either an isolated palladium-CF3 complex, or from Pd(PPh3)4 and other commercially available reagents.

Design of Substrate Transmembrane Mimetics as Structural Probes for γ-Secretase.

γ-Secretase is a membrane-embedded aspartyl protease complex central in biology and medicine. How this enzyme recognizes transmembrane substrates and catalyzes hydrolysis in the lipid bilayer is unclear. Inhibitors that mimic the entire substrate transmembrane domain and engage the active site should provide important tools for structural biology, yielding insight into substrate gating and trapping the protease in the active state. Here, we report transmembrane peptidomimetic inhibitors of the γ-secretase complex that contain an N-terminal helical peptide region that engages a substrate docking exosite and a C-terminal transition-state analog moiety targeted to the active site. Both regions are required for stoichiometric inhibition of γ-secretase. Moreover, enzyme inhibition kinetics and photoaffinity probe displacement experiments demonstrate that both the docking exosite and the active site are engaged by the bipartite inhibitors. The solution conformations of these potent transmembrane-mimetic inhibitors are similar to those of bound natural substrates, suggesting these probes are preorganized for high-affinity binding and should allow visualization of the active γ-secretase complex, poised for intramembrane proteolysis, by cryo-electron microscopy.

Epidemiology and Diagnosis of Gastroparesis in the United States: A Population-based Study.

GOALS: We aimed to establish the epidemiological characteristics and documentation of diagnostic workup for gastroparesis (GP). BACKGROUND: No study has used a national database to evaluate the prevalence, demographics, and associated comorbid conditions of GP, and document rates of proper diagnosis. MATERIALS AND METHODS: This was a cross-sectional population-based study using the Explorys Platform to determine the prevalence of GP in a large and diverse population highly representative of the US population and to examine the diagnostic approach of GP. Data collected were individual characteristics from electronic medical records (EMRs) included age, ethnicity/race, sex, diagnostic report for esophagogastroduodenoscopy (EGD) and gastric emptying study (GES). RESULTS: A total of 43,827,910 medical records were surveyed (1999 to 2014), of which 69,950 had a diagnosis of GP, yielding an overall prevalence of 0.16%. We identified 249,930 EMRs with type 1 diabetes mellitus (T1DM), and 2,940,280 EMR's with type 2 diabetes mellitus (T2DM), of which 11,470 (4.59%) and 38,670 (1.31%) EMR's had concurrent GP, respectively. The remainder 19,810 EMRs with a diagnosis of GP were classified as having idiopathic GP. In all three subgroups, women and Caucasians had the highest prevalence of GP. The diagnosis of GP was confirmed by both GES and EGD in 9,950 of patients (14.22%). For patients with T1DM, T2DM, or idiopathic GP, GP was confirmed by both diagnostic tests in 16.8%, 14.0%, and 13.2%, respectively. CONCLUSIONS: Our estimated rates of prevalence of GP in T1DM and T2DM indicate that GP is not a common clinical complication in these populations. Majority of EMRs that indicated a diagnosis of GP did not include any documentation of definitive diagnostic testing (EGD and/or GES).