Alzheimer's disease linked Aß42 exerts product feedback inhibition on γ-secretase impairing downstream cell signaling.

Amyloid ß (Aß) peptides accumulating in the brain are proposed to trigger Alzheimer's disease (AD). However, molecular cascades underlying their toxicity are poorly defined. Here, we explored a novel hypothesis for Aß42 toxicity that arises from its proven affinity for γ-secretases. We hypothesized that the reported increases in Aß42, particularly in the endolysosomal compartment, promote the establishment of a product feedback inhibitory mechanism on γ-secretases, and thereby impair downstream signaling events. We conducted kinetic analyses of γ-secretase activity in cell-free systems in the presence of Aß, as well as cell-based and ex vivo assays in neuronal cell lines, neurons, and brain synaptosomes to assess the impact of Aß on γ-secretases. We show that human Aß42 peptides, but neither murine Aß42 nor human Aß17-42 (p3), inhibit γ-secretases and trigger accumulation of unprocessed substrates in neurons, including C-terminal fragments (CTFs) of APP, p75, and pan-cadherin. Moreover, Aß42 treatment dysregulated cellular homeostasis, as shown by the induction of p75-dependent neuronal death in two distinct cellular systems. Our findings raise the possibility that pathological elevations in Aß42 contribute to cellular toxicity via the γ-secretase inhibition, and provide a novel conceptual framework to address Aß toxicity in the context of γ-secretase-dependent homeostatic signaling.

Alzheimer's disease linked Aß42 exerts product feedback inhibition on γ-secretase impairing downstream cell signaling.

Amyloid ß (Aß) peptides accumulating in the brain are proposed to trigger Alzheimer's disease (AD). However, molecular cascades underlying their toxicity are poorly defined. Here, we explored a novel hypothesis for Aß42 toxicity that arises from its proven affinity for γ-secretases. We hypothesized that the reported increases in Aß42, particularly in the endolysosomal compartment, promote the establishment of a product feedback inhibitory mechanism on γ-secretases, and thereby impair downstream signaling events. We show that human Aß42 peptides, but neither murine Aß42 nor human Aß17-42 (p3), inhibit γ-secretases and trigger accumulation of unprocessed substrates in neurons, including C-terminal fragments (CTFs) of APP, p75 and pan-cadherin. Moreover, Aß42 treatment dysregulated cellular homeostasis, as shown by the induction of p75-dependent neuronal death in two distinct cellular systems. Our findings raise the possibility that pathological elevations in Aß42 contribute to cellular toxicity via the γ-secretase inhibition, and provide a novel conceptual framework to address Aß toxicity in the context of γ-secretase-dependent homeostatic signaling.

Enzyme-substrate interface targeting by imidazole-based γ-secretase modulators activates γ-secretase and stabilizes its interaction with APP.

Alzheimer's disease (AD) pathogenesis has been linked to the accumulation of longer, aggregation-prone amyloid ß (Aß) peptides in the brain. Γ-secretases generate Aß peptides from the amyloid precursor protein (APP). Γ-secretase modulators (GSMs) promote the generation of shorter, less-amyloidogenic Aßs and have therapeutic potential. However, poorly defined drug-target interactions and mechanisms of action have hampered their therapeutic development. Here, we investigate the interactions between the imidazole-based GSM and its target γ-secretase-APP using experimental and in silico approaches. We map the GSM binding site to the enzyme-substrate interface, define a drug-binding mode that is consistent with functional and structural data, and provide molecular insights into the underlying mechanisms of action. In this respect, our analyses show that occupancy of a γ-secretase (sub)pocket, mediating binding of the modulator's imidazole moiety, is sufficient to trigger allosteric rearrangements in γ-secretase as well as stabilize enzyme-substrate interactions. Together, these findings may facilitate the rational design of new modulators of γ-secretase with improved pharmacological properties.

Aß profiles generated by Alzheimer's disease causing PSEN1 variants determine the pathogenicity of the mutation and predict age at disease onset.

Familial Alzheimer's disease (FAD), caused by mutations in Presenilin (PSEN1/2) and Amyloid Precursor Protein (APP) genes, is associated with an early age at onset (AAO) of symptoms. AAO is relatively consistent within families and between carriers of the same mutations, but differs markedly between individuals carrying different mutations. Gaining a mechanistic understanding of why certain mutations manifest several decades earlier than others is extremely important in elucidating the foundations of pathogenesis and AAO. Pathogenic mutations affect the protease (PSEN/γ-secretase) and the substrate (APP) that generate amyloid ß (Aß) peptides. Altered Aß metabolism has long been associated with AD pathogenesis, with absolute or relative increases in Aß42 levels most commonly implicated in the disease development. However, analyses addressing the relationships between these Aß42 increments and AAO are inconsistent. Here, we investigated this central aspect of AD pathophysiology via comprehensive analysis of 25 FAD-linked Aß profiles. Hypothesis- and data-driven approaches demonstrate linear correlations between mutation-driven alterations in Aß profiles and AAO. In addition, our studies show that the Aß (37 + 38 + 40) / (42 + 43) ratio offers predictive value in the assessment of 'unclear' PSEN1 variants. Of note, the analysis of PSEN1 variants presenting additionally with spastic paraparesis, indicates that a different mechanism underlies the aetiology of this distinct clinical phenotype. This study thus delivers valuable assays for fundamental, clinical and genetic research as well as supports therapeutic interventions aimed at shifting Aß profiles towards shorter Aß peptides.

Exploring the origins of nucleation.

An approach called deep mutational scanning is improving our understanding of amyloid beta aggregation.

Extracellular interface between APP and Nicastrin regulates Aß length and response to γ-secretase modulators.

γ-Secretase complexes (GSECs) are multimeric membrane proteases involved in a variety of physiological processes and linked to Alzheimer's disease (AD). Presenilin (PSEN, catalytic subunit), Nicastrin (NCT), Presenilin Enhancer 2 (PEN-2), and Anterior Pharynx Defective 1 (APH1) are the essential subunits of GSECs. Mutations in PSEN and the Amyloid Precursor Protein (APP) cause early-onset AD GSECs successively cut APP to generate amyloid-ß (Aß) peptides of various lengths. AD-causing mutations destabilize GSEC-APP/Aßn interactions and thus enhance the production of longer Aßs, which elicit neurotoxic events underlying pathogenesis. Here, we investigated the molecular strategies that anchor GSEC and APP/Aßn during the sequential proteolysis. Our studies reveal that a direct interaction between NCT ectodomain and APPC99 influences the stability of GSEC-Aßn assemblies and thereby modulates Aß length. The data suggest a potential link between single-nucleotide variants in NCSTN and AD risk. Furthermore, our work indicates that an extracellular interface between the protease (NCT, PSEN) and the substrate (APP) represents the target for compounds (GSMs) modulating Aß length. Our findings may guide future rationale-based drug discovery efforts.

microRNA diagnostic panel for Alzheimer's disease and epigenetic trade-off between neurodegeneration and cancer.

microRNAs (miRNAs) have been extensively studied as potential biomarkers for Alzheimer's disease (AD). Their profiles have been analyzed in blood, cerebrospinal fluid (CSF) and brain tissue. However, due to the high variability between the reported data, stemming from the lack of methodological standardization and the heterogeneity of AD, the most promising miRNA biomarker candidates have not been selected. Our literature review shows that out of 137 miRNAs found to be altered in AD blood, 36 have been replicated in at least one independent study, and out of 166 miRNAs reported as differential in AD CSF, 13 have been repeatedly found. Only 3 miRNAs have been consistently reported as altered in three analyzed specimens: blood, CSF and the brain (hsa-miR-146a, hsa-miR-125b, hsa-miR-135a). Nonetheless, all 36 repeatedly differential miRNAs in AD blood are promising as components of the diagnostic panel. Given their predicted functions, such miRNA panel may report multiple pathways contributing to AD pathology, enabling the design of personalized therapies. In addition, the analysis revealed that the miRNAs dysregulated in AD overlap highly with miRNAs implicated in cancer. However, the directions of the miRNA changes are usually opposite in cancer and AD, indicative of an epigenetic trade-off between the two diseases.

Novel interaction between Alzheimer's disease-related protein presenilin 1 and glutamate transporter 1.

Neuronal hyperactivity is one of the earliest events observed in Alzheimer's disease (AD). Moreover, alterations in the expression of glutamate transporters have been reported to exacerbate amyloid pathology and cognitive deficits in transgenic AD mouse models. However, the molecular links between these pathophysiological changes remain largely unknown. Here, we report novel interaction between presenilin 1 (PS1), the catalytic component of the amyloid precursor protein-processing enzyme, γ-secretase, and a major glutamate transporter-1 (GLT-1). Our data demonstrate that the interaction occurs between PS1 and GLT-1 expressed at their endogenous levels in vivo and in vitro, takes place in both neurons and astrocytes, and is independent of the PS1 autoproteolysis and γ-secretase activity. This intriguing discovery may shed light on the molecular crosstalk between the proteins linked to the maintenance of glutamate homeostasis and Aß pathology.

Selective plane illumination microscopy (SPIM) with time-domain fluorescence lifetime imaging microscopy (FLIM) for volumetric measurement of cleared mouse brain samples.

We have developed an imaging technique which combines selective plane illumination microscopy with time-domain fluorescence lifetime imaging microscopy (SPIM-FLIM) for three-dimensional volumetric imaging of cleared mouse brains with micro- to mesoscopic resolution. The main features of the microscope include a wavelength-adjustable pulsed laser source (Ti:sapphire) (near-infrared) laser, a BiBO frequency-doubling photonic crystal, a liquid chamber, an electrically focus-tunable lens, a cuvette based sample holder, and an air (dry) objective lens. The performance of the system was evaluated with a lifetime reference dye and micro-bead phantom measurements. Intensity and lifetime maps of three-dimensional human embryonic kidney (HEK) cell culture samples and cleared mouse brain samples expressing green fluorescent protein (GFP) (donor only) and green and red fluorescent protein [positive Förster (fluorescence) resonance energy transfer] were acquired. The results show that the SPIM-FLIM system can be used for sample sizes ranging from single cells to whole mouse organs and can serve as a powerful tool for medical and biological research.

Oxidant/Antioxidant Imbalance in Alzheimer's Disease: Therapeutic and Diagnostic Prospects.

Alzheimer's disease (AD) is the most common cause of dementia and a great socioeconomic burden in the aging society. Compelling evidence demonstrates that molecular change characteristics for AD, such as oxidative stress and amyloid ß (Aß) oligomerization, precede by decades the onset of clinical dementia and that the disease represents a biological and clinical continuum of stages, from asymptomatic to severely impaired. Nevertheless, the sequence of the early molecular alterations and the interplay between them are incompletely understood. This review presents current knowledge about the oxidative stress-induced impairments and compromised oxidative stress defense mechanisms in AD brain and the cross-talk between various pathophysiological insults, with the focus on excessive reactive oxygen species (ROS) generation and Aß overproduction at the early stages of the disease. Prospects for AD therapies targeting oxidant/antioxidant imbalance are being discussed, as well as for the development of novel oxidative stress-related, blood-based biomarkers for early, noninvasive AD diagnostics.

Dynamic Nature of presenilin1/γ-Secretase: Implication for Alzheimer's Disease Pathogenesis.

Presenilin 1 (PS1) is a catalytic component of the γ-secretase complex, responsible for the intramembraneous cleavage of more than 90 type I transmembrane proteins, including Alzheimer's disease (AD)-related amyloid precursor protein (APP). The γ-secretase-mediated cleavage of the APP C-terminal membrane stub leads to the production of various amyloid ß (Aß) species. The assembly of Aß into neurotoxic oligomers, which causes synaptic dysfunction and neurodegeneration, is influenced by the relative ratio of the longer (Aß42/43) to shorter Aß (Aß40) peptides. The ratio of Aß42 to Aß40 depends on the conformation and activity of the PS1/γ-secretase enzymatic complex. The latter exists in a dynamic equilibrium of the so called "closed" and "open" conformational states, as determined by the Förster resonance energy transfer (FRET)-based PS1 conformation assay. Here we review several factors that can allosterically influence conformational status of the enzyme, and hence the production of Aß peptides. These include genetic variations in PS1, APP and other γ-secretase components, environmental stressors implicated in AD pathogenesis and pharmacological agents. Since "closed" PS1 conformation is the common outcome of many AD-related insults, the novel assays monitoring PS1 conformation in live/intact cells in vivo and in vitro might be utilized for diagnostic purposes and for validation of the potential therapeutic approaches.

Dynamic presenilin 1 and synaptotagmin 1 interaction modulates exocytosis and amyloid ß production.

BACKGROUND: Alzheimer's disease (AD)-linked protein, presenilin 1 (PS1), is present at the synapse, and the knock-out of presenilin in mice leads to synaptic dysfunction. On the other hand, synaptic activity was shown to influence PS1-dependent generation of distinct amyloid ß (Aß) species. However, the precise nature of these regulations remains unclear. The current study reveals novel role of PS1 at the synapse, and deciphers how PS1 and synaptic vesicle-associated protein, synaptotagmin 1 (Syt1) modulate each other functions in neurons via direct activity-triggered interaction. Additionally, the therapeutic potential of fostering PS1-Syt1 binding is investigated as a synapse-specific strategy for AD prevention. METHODS: PS1-based cell-permeable peptide targeting PS1-Syt1 binding site was designed to inhibit PS1-Syt1 interaction in neurons. PS1 conformation, synaptic vesicle exocytosis and trafficking were assayed by fluorescence lifetime imaging microscopy (FLIM), glutamate release/synaptopHluorin assay, and fluorescence recovery after photobleaching, respectively. Syt1 level and interaction with PS1 in control and sporadic AD brains were determined by immunohistochemistry and FLIM. AAV-mediated delivery of Syt1 into mouse hippocampi was used to investigate the therapeutic potential of strengthening PS1-Syt1 binding in vivo. Statistical significance was determined using two-tailed unpaired Student's t-test, Mann-Whitney's U-test or two-way ANOVA followed by a Bonferroni's post-test. RESULTS: We demonstrate that targeted inhibition of the PS1-Syt1 binding in neurons, without changing the proteins' expression level, triggers "pathogenic" conformational shift of PS1, and consequent increase in the Aß42/40 ratio. Moreover, our data indicate that PS1, by binding directly to Syt1, regulates synaptic vesicle trafficking and facilitates exocytosis and neurotransmitter release. Analysis of human brain tissue revealed that not only Syt1 levels but also interactions between remaining Syt1 and PS1 are diminished in sporadic AD. On the other hand, overexpression of Syt1 in mouse hippocampi was found to potentiate PS1-Syt1 binding and promote "protective" PS1 conformation. CONCLUSIONS: The study reports novel functions of PS1 and Syt1 at the synapse, and demonstrates the importance of PS1-Syt1 binding for exocytosis and safeguarding PS1 conformation. It suggests that reduction in the Syt1 level and PS1-Syt1 interactions in AD brain may present molecular underpinning of the pathogenic PS1 conformation, increased Aß42/40 ratio, and impaired exocytosis.

Interrelationship between Changes in the Amyloid ß 42/40 Ratio and Presenilin 1 Conformation.

The ratio of the longer (i.e., Aß42/Aß43) to shorter (i.e. Aß40) species is a critical factor determining amyloid fibril formation, neurotoxicity and progression of the amyloid pathology in Alzheimer's disease. The relative levels of the different Aß species are affected by activity and conformation of the γ-secretase complex catalytic component - presenilin 1 (PS1). The enzyme exists in a dynamic equilibrium of the conformational states, with so-called "close" conformation associated with the shift of the γ-secretase cleavage towards the production of longer, neurotoxic Aß species. In the current study, fluorescence lifetime imaging microscopy, spectral Förster resonance energy transfer, calcium imaging and cytotoxicity assays were utilized to explore reciprocal link between the Aß42 and Aß40 peptides present at various ratios and PS1 conformation in primary neurons. We report that exposure to Aß peptides at a relatively high ratio of Aß42/40 causes conformational change within the PS1 subdomain architecture towards the pathogenic "closed" state. Mechanistically, the Aß42/40 peptides present at the relatively high ratio increase intracellular calcium levels, which were shown to trigger pathogenic PS1 conformation. This indicates that there is a reciprocal crosstalk between the extracellular Aß peptides and PS1 conformation within a neuron, with Aß40 showing some protective effect. The pathogenic shift within the PS1 domain architecture may further shift the production of Aß peptides towards the longer, neurotoxic Aß species. These findings link elevated calcium, Aß42 and PS1/γ-secretase conformation, and offer possible mechanistic explanation of the impending exacerbation of the amyloid pathology.

Collagen Q--a potential target for autoantibodies in myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disorder caused by autoantibodies targeting proteins expressed at the neuromuscular junction (NMJ). In most cases the targets are acetylcholine receptor (AChR), muscle-specific tyrosine kinase (MuSK), or occasionally low-density lipoprotein receptor-related protein 4 (LRP4), but there is still a group of patients, often called seronegative MG (SNMG), with unknown antibody targets. One potential target is collagen Q (COLQ), which is restricted to the NMJ and is crucial for anchoring the NMJ-specific form of acetylcholinesterase (AChE). 415 serum samples with a clinical diagnosis of MG and 43 control samples were screened for the presence of COLQ autoantibodies using a cell-based assay (CBA) with HEK293 cells overexpressing COLQ at the cell surface. COLQ antibodies were detected in 12/415 MG sera and in one/43 control samples. Five of the COLQ-Ab+individuals were also positive for AChR-Abs and 2 for MuSK-Abs. Although the COLQ antibodies were only present at low frequency, and did not differ significantly from the small control cohort, further studies could address whether they modify the clinical presentation or the benefits of anti-cholinesterase therapy.