Emerging structures and dynamic mechanisms of γ-secretase for Alzheimer's disease.

γ-Secretase, called "the proteasome of the membrane," is a membrane-embedded protease complex that cleaves 150+ peptide substrates with central roles in biology and medicine, including amyloid precursor protein and the Notch family of cell-surface receptors. Mutations in γ-secretase and amyloid precursor protein lead to early-onset familial Alzheimer's disease. γ-Secretase has thus served as a critical drug target for treating familial Alzheimer's disease and the more common late-onset Alzheimer's disease as well. However, critical gaps remain in understanding the mechanisms of processive proteolysis of substrates, the effects of familial Alzheimer's disease mutations, and allosteric modulation of substrate cleavage by γ-secretase. In this review, we focus on recent studies of structural dynamic mechanisms of γ-secretase. Different mechanisms, including the "Fit-Stay-Trim," "Sliding-Unwinding," and "Tilting-Unwinding," have been proposed for substrate proteolysis of amyloid precursor protein by γ-secretase based on all-atom molecular dynamics simulations. While an incorrect registry of the Notch1 substrate was identified in the cryo-electron microscopy structure of Notch1-bound γ-secretase, molecular dynamics simulations on a resolved model of Notch1-bound γ-secretase that was reconstructed using the amyloid precursor protein-bound γ-secretase as a template successfully captured γ-secretase activation for proper cleavages of both wildtype and mutant Notch, being consistent with biochemical experimental findings. The approach could be potentially applied to decipher the processing mechanisms of various substrates by γ-secretase. In addition, controversy over the effects of familial Alzheimer's disease mutations, particularly the issue of whether they stabilize or destabilize γ-secretase-substrate complexes, is discussed. Finally, an outlook is provided for future studies of γ-secretase, including pathways of substrate binding and product release, effects of modulators on familial Alzheimer's disease mutations of the γ-secretase-substrate complexes. Comprehensive understanding of the functional mechanisms of γ-secretase will greatly facilitate the rational design of effective drug molecules for treating familial Alzheimer's disease and perhaps Alzheimer's disease in general.

A metal-free strategy to construct fluoroalkyl-olefin linkages using fluoroalkanes.

We present a metal-free strategy to access fluoroalkyl-olefin linkages from fluoroalkane precursors and vinyl-pinacol boronic ester (BPin) reagents. This reaction sequence is templated by the boron reagent, which induces C-C bond formation upon oxidation. We developed this strategy into a one-pot synthetic protocol using RCF2H precursors directly with vinyl-BPin reagents in the presence of a Brønsted base, which tolerated oxygen- and nitrogen-containing heterocycles, and aryl halogens. We also found that HCF3 (HCF-23; a byproduct of the Teflon industry) and CH2F2 (HCF-32; a low-cost refrigerant) are amenable to this protocol, representing distinct strategies to generate RCF2H and RCF3 molecules. Finally, we demonstrate that the vinyldifluoromethylene products can be readily derivatized, representing an avenue for late-stage modification after installing the fluoroalkyl unit.

Familial Alzheimer mutations stabilize synaptotoxic γ-secretase-substrate complexes.

Mutations that cause familial Alzheimer's disease (FAD) are found in amyloid precursor protein (APP) and presenilin, the catalytic component of γ-secretase, that together produce amyloid ß-peptide (Aß). Nevertheless, whether Aß is the primary disease driver remains controversial. We report here that FAD mutations disrupt initial proteolytic events in the multistep processing of APP substrate C99 by γ-secretase. Cryoelectron microscopy reveals that a substrate mimetic traps γ-secretase during the transition state, and this structure aligns with activated enzyme-substrate complex captured by molecular dynamics simulations. In silico simulations and in cellulo fluorescence microscopy support stabilization of enzyme-substrate complexes by FAD mutations. Neuronal expression of C99 and/or presenilin-1 in Caenorhabditis elegans leads to synaptic loss only with FAD-mutant transgenes. Designed mutations that stabilize the enzyme-substrate complex and block Aß production likewise led to synaptic loss. Collectively, these findings implicate the stalled process-not the products-of γ-secretase cleavage of substrates in FAD pathogenesis.

Evolutionary flexibility and rigidity in the bacterial methylerythritol phosphate (MEP) pathway.

Terpenoids are a diverse class of compounds with wide-ranging uses including as industrial solvents, pharmaceuticals, and fragrances. Efforts to produce terpenoids sustainably by engineering microbes for fermentation are ongoing, but industrial production still largely relies on nonrenewable sources. The methylerythritol phosphate (MEP) pathway generates terpenoid precursor molecules and includes the enzyme Dxs and two iron-sulfur cluster enzymes: IspG and IspH. IspG and IspH are rate limiting-enzymes of the MEP pathway but are challenging for metabolic engineering because they require iron-sulfur cluster biogenesis and an ongoing supply of reducing equivalents to function. Therefore, identifying novel alternatives to IspG and IspH has been an on-going effort to aid in metabolic engineering of terpenoid biosynthesis. We report here an analysis of the evolutionary diversity of terpenoid biosynthesis strategies as a resource for exploration of alternative terpenoid biosynthesis pathways. Using comparative genomics, we surveyed a database of 4,400 diverse bacterial species and found that some may have evolved alternatives to the first enzyme in the pathway, Dxs making it evolutionarily flexible. In contrast, we found that IspG and IspH are evolutionarily rigid because we could not identify any species that appear to have enzymatic routes that circumvent these enzymes. The ever-growing repository of sequenced bacterial genomes has great potential to provide metabolic engineers with alternative metabolic pathway solutions. With the current state of knowledge, we found that enzymes IspG and IspH are evolutionarily indispensable which informs both metabolic engineering efforts and our understanding of the evolution of terpenoid biosynthesis pathways.

Molecular Dynamics Activation of γ-Secretase for Cleavage of the Notch1 Substrate.

γ-Secretase is an intramembrane aspartyl protease complex that cleaves the transmembrane domain of over 150 peptide substrates, including amyloid precursor protein (APP) and the Notch family of receptors, via two conserved aspartates D257 and D385 in the presenilin-1 (PS1) catalytic subunit. However, while the activation of γ-secretase for cleavage of APP has been widely studied, the cleavage of Notch by γ-secretase remains poorly explored. Here, we combined Gaussian accelerated molecular dynamics (GaMD) simulations and mass spectrometry (MS) analysis of proteolytic products to present the first dynamic models for cleavage of Notch by γ-secretase. MS showed that γ-secretase cleaved the WT Notch at Notch residue G34, while cleavage of the L36F mutant Notch occurred at Notch residue C33. Initially, we prepared our simulation systems starting from the cryoEM structure of Notch-bound γ-secretase (PDB: 6IDF) and failed to capture the proper cleavages of WT and L36F Notch by γ-secretase. We then discovered an incorrect registry of the Notch substrate in the PS1 active site through alignment of the experimental structure of Notch-bound (PDB: 6IDF) and APP-bound γ-secretase (PDB: 6IYC). Every residue of the APP substrate was systematically mutated to the corresponding Notch residue to prepare a resolved model of Notch-bound γ-secretase complexes. GaMD simulations of the resolved model successfully captured γ-secretase activation for proper cleavages of both WT and L36F mutant Notch. Our findings presented here provided mechanistic insights into the structural dynamics and enzyme-substrate interactions required for γ-secretase activation for cleavage of Notch and other substrates.

A neurodegeneration checkpoint mediated by REST protects against the onset of Alzheimer's disease.

Many aging individuals accumulate the pathology of Alzheimer's disease (AD) without evidence of cognitive decline. Here we describe an integrated neurodegeneration checkpoint response to early pathological changes that restricts further disease progression and preserves cognitive function. Checkpoint activation is mediated by the REST transcriptional repressor, which is induced in cognitively-intact aging humans and AD mouse models at the onset of amyloid ß-protein (Aß) deposition and tau accumulation. REST induction is mediated by the unfolded protein response together with ß-catenin signaling. A consequence of this response is the targeting of REST to genes involved in key pathogenic pathways, resulting in downregulation of gamma secretase, tau kinases, and pro-apoptotic proteins. Deletion of REST in the 3xTg and J20 AD mouse models accelerates Aß deposition and the accumulation of misfolded and phosphorylated tau, leading to neurodegeneration and cognitive decline. Conversely, viral-mediated overexpression of REST in the hippocampus suppresses Aß and tau pathology. Thus, REST mediates a neurodegeneration checkpoint response with multiple molecular targets that may protect against the onset of AD.

The genetics of aerotolerant growth in an alphaproteobacterium with a naturally reduced genome.

Reduced genome bacteria are genetically simplified systems that facilitate biological study and industrial use. The free-living alphaproteobacterium Zymomonas mobilis has a naturally reduced genome containing fewer than 2,000 protein-coding genes. Despite its small genome, Z. mobilis thrives in diverse conditions including the presence or absence of atmospheric oxygen. However, insufficient characterization of essential and conditionally essential genes has limited broader adoption of Z. mobilis as a model alphaproteobacterium. Here, we use genome-scale CRISPRi-seq (clustered regularly interspaced short palindromic repeats interference sequencing) to systematically identify and characterize Z. mobilis genes that are conditionally essential for aerotolerant or anaerobic growth or are generally essential across both conditions. Comparative genomics revealed that the essentiality of most "generally essential" genes was shared between Z. mobilis and other Alphaproteobacteria, validating Z. mobilis as a reduced genome model. Among conditionally essential genes, we found that the DNA repair gene, recJ, was critical only for aerobic growth but reduced the mutation rate under both conditions. Further, we show that genes encoding the F1FO ATP synthase and Rhodobacter nitrogen fixation (Rnf) respiratory complex are required for the anaerobic growth of Z. mobilis. Combining CRISPRi partial knockdowns with metabolomics and membrane potential measurements, we determined that the ATP synthase generates membrane potential that is consumed by Rnf to power downstream processes. Rnf knockdown strains accumulated isoprenoid biosynthesis intermediates, suggesting a key role for Rnf in powering essential biosynthetic reactions. Our work establishes Z. mobilis as a streamlined model for alphaproteobacterial genetics, has broad implications in bacterial energy coupling, and informs Z. mobilis genome manipulation for optimized production of valuable isoprenoid-based bioproducts. IMPORTANCE The inherent complexity of biological systems is a major barrier to our understanding of cellular physiology. Bacteria with markedly fewer genes than their close relatives, or reduced genome bacteria, are promising biological models with less complexity. Reduced genome bacteria can also have superior properties for industrial use, provided the reduction does not overly restrict strain robustness. Naturally reduced genome bacteria, such as the alphaproteobacterium Zymomonas mobilis, have fewer genes but remain environmentally robust. In this study, we show that Z. mobilis is a simplified genetic model for Alphaproteobacteria, a class with important impacts on the environment, human health, and industry. We also identify genes that are only required in the absence of atmospheric oxygen, uncovering players that maintain and utilize the cellular energy state. Our findings have broad implications for the genetics of Alphaproteobacteria and industrial use of Z. mobilis to create biofuels and bioproducts.

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.

Electronic Monitoring Of Mom's Schedule (eMOMSTM): Recruitment of pregnant populations with elevated BMI in a feasibility randomized controlled trial.

Underrepresentation of pregnant populations in randomized controlled trials of lifestyle change interventions is concerning due to high attrition and providers' limited clinical time. The purpose of this evaluative study was to assess intervention uptake of pregnant individuals enrolled in a three-arm feasibility randomized controlled trial, electronic Monitoring Of Mom's Schedule (eMOMSTM), examining lifestyle changes and lactation support alone, and in combination. Measures included: (1) participation and completion rates, and characteristics of intervention completers versus other eligible participants; and (2) provider experiences with screening and enrolling pregnant participants. Pregnant people with a pre-pregnancy body mass index ≥ 25 and < 35 kg/m2 were enrolled into the eMOMSTM trial between September 2019 - December 2020. Of the 44 consented participants, 35 were randomized, at a participation rate of 35%, and 26 completed the intervention, resulting in a completion rate of 74%. Intervention completers were slightly older and entered the study earlier in pregnancy compared to non-completers. Completers were more likely to be first-time mothers, resided in urban areas, had higher educational attainment, and were slightly more racially and ethnically diverse. A majority of providers reported willingness to participate, believed the study aligned with their organization's mission, and were satisfied with using iPads for screening. Lessons learned to guide recruitment success include use of: (1) designated research staff in combination with physician support; and (2) user-friendly technology to help mitigate time burden on physicians and their staff. Future work should focus on successful strategies to recruit/retain pregnant populations in clinical trials.

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.

Health change awareness and its association with weight loss following bariatric surgery.

OBJECTIVE: Patients' ability to judge health change over time has important clinical implications for treatment, but is understudied in longitudinal contexts with meaningful health change. We assess patients' awareness of health change for 5 years following bariatric surgery, and its association with weight loss. METHOD: Participants were part of the Longitudinal Assessment of Bariatric Surgery (N = 2,027). Perceived health change for each year was assessed by comparing it to self-reports of health on the SF-36 health survey. Participants were categorized as concordant when perceived and actual self-reported health change corresponded, and as discordant when they did not correspond. RESULTS: Year-to-year concordance between perceived and actual self-reported health change occurred less than 50% of the time. Discordance between perceived and actual health was associated with weight loss following surgery. Discordant-positive participants who perceived their health change as more positive than was warranted lost more weight post-surgery and thus had lower body mass index scores than concordant participants. Conversely, discordant-negative participants who perceived their health as worse than what was warranted lost less weight post-surgery and thus had higher body mass index scores. CONCLUSIONS: These results suggest that recollection of past health is generally poor and can be biased by salient factors during recall. Clinicians are advised to use caution when retrospective judgments of health are utilized. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Effects of presenilin-1 familial Alzheimer's disease mutations on γ-secretase activation for cleavage of amyloid precursor protein.

Presenilin-1 (PS1) is the catalytic subunit of γ-secretase which cleaves within the transmembrane domain of over 150 peptide substrates. Dominant missense mutations in PS1 cause early-onset familial Alzheimer's disease (FAD); however, the exact pathogenic mechanism remains unknown. Here we combined Gaussian accelerated molecular dynamics (GaMD) simulations and biochemical experiments to determine the effects of six representative PS1 FAD mutations (P117L, I143T, L166P, G384A, L435F, and L286V) on the enzyme-substrate interactions between γ-secretase and amyloid precursor protein (APP). Biochemical experiments showed that all six PS1 FAD mutations rendered γ-secretase less active for the endoproteolytic (ε) cleavage of APP. Distinct low-energy conformational states were identified from the free energy profiles of wildtype and PS1 FAD-mutant γ-secretase. The P117L and L286V FAD mutants could still sample the "Active" state for substrate cleavage, but with noticeably reduced conformational space compared with the wildtype. The other mutants hardly visited the "Active" state. The PS1 FAD mutants were found to reduce γ-secretase proteolytic activity by hindering APP residue L49 from proper orientation in the active site and/or disrupting the distance between the catalytic aspartates. Therefore, our findings provide mechanistic insights into how PS1 FAD mutations affect structural dynamics and enzyme-substrate interactions of γ-secretase and APP.

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.

A sputum bioassay for airway eosinophilia using an eosinophil peroxidase aptamer.

Eosinophils are granulocytes that play a significant role in the pathogenesis of asthma and other airway diseases. Directing patient treatment based on the level of eosinophilia has been shown to be extremely effective in reducing exacerbations and therefore has tremendous potential as a routine clinical test. Herein, we describe the in vitro selection and optimization of DNA aptamers that bind to eosinophil peroxidase (EPX), a protein biomarker unique to eosinophils. Fifteen rounds of magnetic bead aptamer selection were performed prior to high throughput DNA sequencing. The top 10 aptamer candidates were assessed for EPX binding using a mobility shift assay. This process identified a lead aptamer candidate termed EAP1-05 with low nanomolar affinity and high specificity for EPX over other common sputum proteins. This aptamer sequence was further optimized through truncation and used to develop an easy-to-use colourimetric pull-down assay that can detect EPX over a concentration range from 1 - 100 nM in processed sputum. Forty-six clinical samples were processed using a new sputum dispersal method, appropriate for a rapid assessment assay, that avoids centrifugation and lengthy processing times. The assay showed 89% sensitivity and 96% specificity to detect eosinophilia (compared to gold standard sputum cytometry), with results being produced in under an hour. This assay could allow for an easy assessment of eosinophil activity in the airway to guide anti-inflammatory therapy for several airway diseases.

RfaH Counter-Silences Inhibition of Transcript Elongation by H-NS-StpA Nucleoprotein Filaments in Pathogenic Escherichia coli.

Expression of virulence genes in pathogenic Escherichia coli is controlled in part by the transcription silencer H-NS and its paralogs (e.g., StpA), which sequester DNA in multi-kb nucleoprotein filaments to inhibit transcription initiation, elongation, or both. Some activators counter-silence initiation by displacing H-NS from promoters, but how H-NS inhibition of elongation is overcome is not understood. In uropathogenic E. coli (UPEC), elongation regulator RfaH aids expression of some H-NS-silenced pathogenicity operons (e.g., hlyCABD encoding hemolysin). RfaH associates with elongation complexes (ECs) via direct contacts to a transiently exposed, nontemplate DNA strand sequence called operon polarity suppressor (ops). RfaH-ops interactions establish long-lived RfaH-EC contacts that allow RfaH to recruit ribosomes to the nascent mRNA and to suppress transcriptional pausing and termination. Using ChIP-seq, we mapped the genome-scale distributions of RfaH, H-NS, StpA, RNA polymerase (RNAP), and σ70 in the UPEC strain CFT073. We identify eight RfaH-activated operons, all of which were bound by H-NS and StpA. Four are new additions to the RfaH regulon. Deletion of RfaH caused premature termination, whereas deletion of H-NS and StpA allowed elongation without RfaH. Thus, RfaH is an elongation counter-silencer of H-NS. Consistent with elongation counter-silencing, deletion of StpA alone decreased the effect of RfaH. StpA increases DNA bridging, which inhibits transcript elongation via topological constraints on RNAP. Residual RfaH effect when both H-NS and StpA were deleted was attributable to targeting of RfaH-regulated operons by a minor H-NS paralog, Hfp. These operons have evolved higher levels of H-NS-binding features, explaining minor-paralog targeting. IMPORTANCE Bacterial pathogens adapt to hosts and host defenses by reprogramming gene expression, including by H-NS counter-silencing. Counter-silencing turns on transcription initiation when regulators bind to promoters and rearrange repressive H-NS nucleoprotein filaments that ordinarily block transcription. The specialized NusG paralog RfaH also reprograms virulence genes but regulates transcription elongation. To understand how elongation regulators might affect genes silenced by H-NS, we mapped H-NS, StpA (an H-NS paralog), RfaH, σ70, and RNA polymerase (RNAP) locations on DNA in the uropathogenic E. coli strain CFT073. Although H-NS-StpA filaments bind only 18% of the CFT073 genome, all loci at which RfaH binds RNAP are also bound by H-NS-StpA and are silenced when RfaH is absent. Thus, RfaH represents a distinct class of counter-silencer that acts on elongating RNAP to enable transcription through repressive nucleoprotein filaments. Our findings define a new mechanism of elongation counter-silencing and explain how RfaH functions as a virulence regulator.

Nucleophilic strategies to construct -CF2- linkages using borazine-CF2Ar reagents.

Using a Lewis acid-quenched CF2Ph- reagent, we show C-C bond formation through nucleophilic addition reactions to prepare molecules containing internal -CF2- linkages. We demonstrate C(sp2)-C(sp3) coupling using both SNAr reactions and Pd-catalysis. Finally, C(sp3)-C(sp3) bonds are forged using operationally simple SN2 reactions that tolerate medicinally-relevant motifs.

In-Depth Characterization of Endo-Lysosomal Aß in Intact Neurons.

Amyloid-beta (Aß) peptides are produced within neurons. Some peptides are released into the brain parenchyma, while others are retained inside the neurons. However, the detection of intracellular Aß remains a challenge since antibodies against Aß capture Aß and its precursor proteins (i.e., APP and C99). To overcome this drawback, we recently developed 1) the C99 720-670 biosensor for recording γ-secretase activity and 2) a unique multiplexed immunostaining platform that enables the selective detection of intracellular Aß with subcellular resolution. Using these new assays, we showed that C99 is predominantly processed by γ-secretase in late endosomes and lysosomes, and intracellular Aß is enriched in the same subcellular loci in intact neurons. However, the detailed properties of Aß in the acidic compartments remain unclear. Here, we report using fluorescent lifetime imaging microscopy (FLIM) that intracellular Aß includes both long Aß intermediates bound to γ-secretase and short peptides dissociated from the protease complex. Surprisingly, our results also suggest that the dissociated Aß is bound to the glycoproteins on the inner membrane of lysosomes. Furthermore, we show striking cell-to-cell heterogeneity in intracellular Aß levels in primary neurons and APP transgenic mouse brains. These findings provide a basis for the further investigation of the role(s) of intracellular Aß and its relevance to Alzheimer's disease (AD).