Welcome to the AD/PD™ 2022 Interactive Program
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THE CASE FOR LOW-LEVEL BACE1 INHIBITION FOR THE PREVENTION OF ALZHEIMER DISEASE
Abstract
Abstract Body
Alzheimer disease (AD) is the most common cause of dementia in older individuals (>65 years) and has a long presymptomatic phase. Preventive therapies for AD are not yet available, and potential disease- modifying therapies targeting amyloid-β plaques in symptomatic stages of AD have only just been approved in the United States. Small- molecule inhibitors of β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1; also known as β-secretase 1) reduce the production of amyloid- β peptide and are among the most advanced drug candidates for AD. However, to date all phase II and phase III clinical trials of BACE inhibitors were either concluded without benefit or discontinued owing to futility or the occurrence of adverse effects. Adverse effects included early, mild cognitive impairment that was associated with all but one inhibitor; preliminary results suggest that the cognitive effects are non- progressive and reversible. These discontinuations have raised questions regarding the suitability of BACE1 as a drug target for AD. In this Perspective, we discuss the status of BACE inhibitors and suggest ways in which the results of the discontinued trials can inform the development of future clinical trials of BACE inhibitors and related secretase modulators as preventative therapies. We also propose a series of experiments that should be performed to inform ‘go–no- go’ decisions in future trials with BACE inhibitors and consider the possibility that low levels of BACE1 inhibition could avoid adverse effects while achieving efficacy for AD prevention.
MECHANISM OF TRIPEPTIDE TRIMMING OF LONG AΒETA BY GAMMA-SECRETASE
Abstract
Aims
Production of amyloid β-peptide (Aβ) is carried out by the membrane-embedded γ-secretase complex. The C-terminal fragment of amyloid precursor protein (APP) is processively proteolyzed by γ-secretase along two pathways: Aβ49→Aβ46→Aβ43→Aβ40 and Aβ48→Aβ45→Aβ42→Aβ38. Using the structure of APP substrate crosslinked to inactive γ-secretase as a starting point, we recently reported Gaussian-accelerated molecular dynamics (GaMD) simulation modeling of endoproteolytic (ε) cleavage that leads to production of Aβ48 or Aβ49 and their corresponding APP intracellular domain (AICD) coproducts. Biochemical analysis of AICD fragments provided validation for this model. Despite this advance, little is known about structural mechanisms of subsequence tripeptide trimming of long Aβ intermediates by γ-secretase, which is deficient in familial Alzheimer’s disease (FAD).
Methods
Here we describe the development of a GaMD dynamic model for tripeptide trimming of Aβ49. In parallel, we designed APP transmembrane peptidomimetics as probes for cryoelectron microscopy (cryo-EM) to noncovalently trap active γ-secretase at the transition state of tripeptide trimming.
Results
We found that the presence of N-terminally charged AICD is required to set up Aβ49→Aβ46 trimming. The GaMD model correctly predicted the biochemical effects of five different APP FAD mutants on this trimming step. The cryo-EM structure of γ-secretase bound to a designed probe revealed the catalytic aspartates were coordinated with the transition state mimicking moiety as designed, and this structure was remarkably similar to that seen in the GaMD model.
Conclusions
This new model suggests structural mechanisms of how FAD mutations alter γ-secretase function and may facilitate structure-based design of new γ-secretase modulators.
SYNAPTIC ACCUMULATION OF APP-C99 IN DIFFERENT FORMS OF ALZHEIMER'S DISEASE
Abstract
Aims
Different groups recently shown APP-C99 accumulation in the brain of animal models of Alzheimer’s disease (AD) and of patients with sporadic (SAD) and autosomal-dominant AD (ADAD). The aim of this study was to determine whether APP-C99 accumulates in synapses in AD.
Methods
We isolated synaptosomal fractions from frozen frontal cortex (n=38) and temporal cortex (n=38) tissue blocks provided by the Neurological Tissue Brain Bank-IDIBAPS. We used postmortem brain samples of patients with SAD (n=10), ADAD (n=10), Down syndrome with AD (n=10) and healthy controls without AD changes (n=8). We performed Western Blot to confirm the presence of APP-C99 in synaptosomes. We measured APP-C99 and Aβ40 concentrations in brain homogenates and in synaptosomes across groups using commercially available immunoassays (APP-β-CTF:IBL America; APP-Amyloid-β (1-40) Aβ:IBL America)
Results
Our results showed that APP-C99 is increased in synapsis in frontal and temporal cortex. APP-C99 is increased in genetic forms of AD in frontal cortex and in all pathological conditions in temporal cortex. Concentrations of APP-C99 in homogenates and synaptosomes correlated with each other. Aβ40 levels in synaptosomes showed no differences between brain regions. Aβ40 concentrations in synaptosomes were significantly increased in SAD, ADAD and DS in temporal and frontal cortex. Interestingly, there is a positive correlation of APP-C99 and Aβ40 levels in synaptosomes in frontal and temporal cortex.
Conclusions
Our data reveal that APP-C99 accumulates in synapses in sporadic and genetic forms of AD suggesting a potential role of APP-C99 in the synaptic damage in AD. Therapies aimed at mitigating APP-C99 accumulation could be potentially beneficial in AD.
LOSS OF TACE ΑLPHA-SECRETASE ACTIVITY IN PRION DISEASES AND PATHOLOGICAL CONSEQUENCES
Abstract
Aims
The α-secretase TACE exerts protective functions in neurons by catalyzing the cleavage of several plasma membrane substrates, including TNFα receptors (TNFRs), the cellular prion protein PrPC, and the amyloid precursor protein (APP). We examined whether the dysregulation of TACE would contribute to prion diseases.
Methods
We exploit the 1C11 neuroectodermal cell line, primary neuronal cultures, and mice infected with prion.
Results
We show that pathogenic prions PrPSc cause a deficit of TACE activity in neurons. By overstimulating the kinase PDK1, PrPSc triggers the internalization of TACE. This diverts TACE activity away from (i) TNFRs, sensitizing neurons to TNFα-associated inflammation, (ii) PrPC, amplifying the production of PrPSc, and (iii) APP, leading to the accumulation of Aβ40/42 peptides. Aβ peptides do not impact prion replication and infectivity. They can deposit and form Aβ plaques in the mouse brain but only if a seed of trimers of Aβ is co-transmitted with PrPSc. Importantly, brain Aβ deposition accelerates death of prion-infected mice. By rescuing TACE cleavage activity toward TNFR, PrPC, and APP at the plasma membrane of prion-infected neurons, PDK1 inhibition prolongs the survival of prion-infected mice.
Conclusions
Our data stress that dysregulation of the PDK1-TACE pathway contributes to neurodegeneration in prion diseases. They further introduce seeds of Aβ trimers as being aggregative, propagative, and neurotoxic entities that cooperate with PrPSc for the formation of Aβ plaques. This would account for the occurrence of Aβ plaques and the onset of a mixed Aβ/PrPSc pathology in the brain of some iatrogenic Creutzfeldt-Jakob disease patients.
LONG-TERM POTENTIATION AND MEMORY IMPAIRMENT CAUSED BY EXTRACELLULAR AMYLOID-ΒETA AND TAU OLIGOMERS IS DEPENDENT UPON PRESYNAPTIC AMYLOID PRECURSOR PROTEIN
Abstract
Aims
The toxic effect of both extracellular Aß- and tau-oligomers on memory and its electrophysiological surrogate, long-term potentiation (LTP) is dependent upon the presence of amyloid precursor protein (APP), a transmembrane protein expressed at the synapse throughout life. Here, we dissected the relative role of pre-synaptic vs. post-synaptic APP at the CA3-CA1 hippocampal synapse in the Aß- and tau-oligomer-induced damage of memory and LTP.
Methods
We used a combination of gene editing, electrophysiological, behavioral and biochemical techniques.
Results
We found that APP knock out (KO) in the post-synaptic neuron did not alter the negative effects of Aß and tau oligomers on LTP and memory. By contrast, specific ablation of APP expression in the pre-synaptic neuron reduced LTP. Aß and tau oligomers did not induce further LTP deficits, suggesting that presynaptic APP mediates the synapto-toxic effect of Aß and tau. Interestingly, both Aß and tau rescued the LTP defect caused by pre-synaptic APP deletion. Further investigation demonstrated that pre-synaptic (but not post-synaptic) APP deletion causes exaggerated refilling after depletion of the readily-releasable pool that is dependent on intracellular calcium levels. We found a similar impairment in intracellular calcium homeostasis both in basal conditions and after activity in full APP-KO mice. Western blot analysis demonstrated a significant decrease in the levels of inositol 1,4,5‐trisphosphate receptor, ryanodine receptor and the calcium pump, SERCA3, in APP-KO mice, providing a possible link for how APP suppression elevates calcium in endoplasmic reticulum.
Conclusions
These data support the view that pre-synaptic APP is a key player in synaptic physiology and pathology.
PRE-RECORDED: IDENTIFICATION OF NOVEL IN VIVO SUBSTRATES OF THE BETA-SECRETASE-HOMOLOG BACE2: MAY THEY CONTRIBUTE TO THE SIDE EFFECTS OF BACE INHIBITORS?
Abstract
Aims
The beta-secretase BACE1 is a major drug target for Alzheimer’s disease, but several late-stage clinical trials with BACE-inhibitors were associated with adverse events, most notably cognitive worsening. The adverse events are assumed to be mechanism-based and to result from loss-of-cleavage of substrates of BACE1 or its homolog BACE2. Yet, little is known about physiological substrates and functions of BACE2 in vivo and no efficient pharmacodynamic assay exists for measuring BACE2 activity. Here, we identified in vivo-relevant BACE2 substrates and set up an assay for fast in vivo BACE2 activity measurement.
Methods
Plasma and CSF from wild-type and BACE2-deficient mice was analyzed proteomically by mass spectrometry. Substrates were validated using in vitro assays and primary cells.
Results
Membrane proteins with reduced ectodomain levels in body fluids from BACE2-deficient mice were identified and validated as new in vivo BACE2 substrates. One of them was used to set up a pharmacodynamic, sandwich immunoassay for BACE2 activity measurements in vivo. BACE inhibition reduced cleavage of the BACE2 substrate in vivo in a time- and dose-dependent manner – even after a single dose – and was superior to the current state-of-the-art assay, which relies on measurement of hair depigmentation after chronic inhibitor dosing.
Conclusions
This study identifies and functionally characterizes new in vivo-relevant BACE2 substrates and establishes a new and fast pharmacodynamic assay for measuring BACE2 activity in vivo. Application of this assay may help to understand and prevent the occurrence of side-effects observed with BACE inhibitors and may facilitate the development of BACE1-specific inhibitors.
PRE-RECORDED: EXOPEPTIDASES INVOLVED IN Aβ N-TERMINAL TRUNCATION: IDENTIFICATION, CONTRIBUTION TO AD NEUROPATHOLOGY AND BEHAVIORAL DEFECTS AND ALTERATIONS IN AD BRAINS
Abstract
Abstract Body
One of the main components of senile plaques in Alzheimer’s disease (AD)-affected brain is the Aβ peptide species harboring a pyroglutamate at position three (pE3-Aβ). Several studies indicated that pE3-Aβ is toxic, prone to aggregation and serves as a seed of Aβ aggregation. The cyclisation of the glutamate in position 3 requires prior removal of the Aβ N-terminal aspartyl residue to allow subsequent biotransformation. We have identified aminopeptidase A (APA)and dipeptidyl peptidase 4 (DDD4) as the main exopeptidases involved in an additional manner. First, we show by mass-spectrometry that human recombinant APA and DPP4 truncate synthetic Aβ1-40 to yield Aβ2-40 and Aβ3-40, respectively. We demonstrate that the pharmacological blockade of APA and DPP4 with theirs selective inhibitors RB150 and sitagliptin restores the density of mature spines and significantly reduced filopodia-like processes in hippocampal organotypic slices cultures virally transduced with the Swedish mutated Aβ-precursor protein (βAPP). Pharmacological reduction of enzymes activities and lowering of their expression by shRNA affect Aβ pE3-42- and Aβ1-42-positive plaques and expressions in 3xTg-AD mice brains. Further, we show that both APA and DPP4 inhibitors and specific shRNA partly alleviate learning and memory deficits observed in 3xTg-AD mice. Importantly, we demonstrate that, concomitantly to the occurrence of Aβ pE3-42-positive plaques, APA and DPP4 activity are augmented at early Braak stages in sporadic AD brains. Overall, our data indicate that APA and DPP4 are two key exopeptidases involved in Aβ N-terminal truncation and suggest the potential benefit of targeting this proteolytic activity to interfere with Aβ-related AD pathology.
PRE-RECORDED: THE PROBABILISTIC AMYLOID CASCADE HYPOTHESIS OF ALZHEIMER’S DISEASE
Abstract
Abstract Body
The current conceptualization of Alzheimer disease (AD) is driven by the amyloid hypothesis, a deterministic chain of events leading from amyloid and then tau deposition, to neurodegeneration and progressive cognitive impairment. This model fits autosomal dominant AD but is less applicable to sporadic AD. Owing to emerging information regarding the complex biology of AD and the challenges of developing amyloid-targeting drugs, the amyloid hypothesis needs to be reconsidered. We propose a probabilistic model of AD, in which amyloid is still a key player, but the pathogenic weight of amyloid and stochastic factors is inversely proportional to the penetrance of genetic factors. The model identifies three variants of AD: autosomal-dominant AD, APOE ɛ4-related sporadic AD and APOE ɛ4-unrelated sporadic AD. Together, these variants account for a large share of the neuropathological and clinical variability observed in people with AD. The implementation of this model in research might lead to a better understanding of disease pathophysiology, a revision of the current clinical taxonomy , and accelerated development of strategies to prevent and treat this disease.