Familial Alzheimer's Disease Mutations in Presenilin Generate Amyloidogenic Ab Peptide Seeds

Recently it was proposed that the familial Alzheimer’s disease (FAD) causing presenilin (PSEN) mutations PSEN1-L435F and PSEN1-C410Y do not support the generation of Aβ-peptides from the amyloid precursor protein (APP). This challenges the amyloid hypothesis and disagrees with previous work showing that PSEN1 FAD causing mutations generate invariably long Aβ and seed amyloid. We contrast here the proteolytic activities of these mutant PSEN alleles with the complete loss-of-function PSEN1-D257A allele. We find residual carboxy- and endo-peptidase γ-secretase activities, similar to the formerly characterized PSEN1-R278I. We conclude that the PSEN1-L435F and -C410Y mutations are extreme examples of the previously proposed “dysfunction” of γ-secretase that characterizes FAD-associated PSEN. This Matters Arising paper is in response to Xia et al. (2015), published in Neuron. See also the response by Xia et al. (2016), published in this issue

Cerebral Aβ deposition precedes reduced cerebrospinal fluid and serum Aβ42/Aβ40 ratios in the AppNL−F/NL−F knock-in mouse model of Alzheimer’s disease

Background: Aβ42/Aβ40 ratios in cerebrospinal fluid (CSF) and blood are reduced in preclinical Alzheimer’s disease (AD), but their temporal and correlative relationship with cerebral Aβ pathology at this early disease stage is not well understood. In the present study, we aim to investigate such relationships using App knock-in mouse models of preclinical AD. / Methods: CSF, serum, and brain tissue were collected from 3- to 18-month-old AppNL−F/NL−F knock-in mice (n = 48) and 2–18-month-old AppNL/NL knock-in mice (n = 35). The concentrations of Aβ42 and Aβ40 in CSF and serum were measured using Single molecule array (Simoa) immunoassays. Cerebral Aβ plaque burden was assessed in brain tissue sections by immunohistochemistry and thioflavin S staining. Furthermore, the concentrations of Aβ42 in soluble and insoluble fractions prepared from cortical tissue homogenates were measured using an electrochemiluminescence immunoassay. / Results: In AppNL−F/NL−F knock-in mice, Aβ42/Aβ40 ratios in CSF and serum were significantly reduced from 12 and 16 months of age, respectively. The initial reduction of these biomarkers coincided with cerebral Aβ pathology, in which a more widespread Aβ plaque burden and increased levels of Aβ42 in the brain were observed from approximately 12 months of age. Accordingly, in the whole study population, Aβ42/Aβ40 ratios in CSF and serum showed a negative hyperbolic association with cerebral Aβ plaque burden as well as the levels of both soluble and insoluble Aβ42 in the brain. These associations tended to be stronger for the measures in CSF compared with serum. In contrast, no alterations in the investigated fluid biomarkers or apparent cerebral Aβ plaque pathology were found in AppNL/NL knock-in mice during the observation time. / Conclusions: Our findings suggest a temporal sequence of events in AppNL−F/NL−F knock-in mice, in which initial deposition of Aβ aggregates in the brain is followed by a decline of the Aβ42/Aβ40 ratio in CSF and serum once the cerebral Aβ pathology becomes significant. Our results also indicate that the investigated biomarkers were somewhat more strongly associated with measures of cerebral Aβ pathology when assessed in CSF compared with serum

Aberrant Excitatory–Inhibitory Synaptic Mechanisms in Entorhinal Cortex Microcircuits During the Pathogenesis of Alzheimer’s Disease

Synaptic dysfunction is widely proposed as an initial insult leading to the neurodegeneration observed in Alzheimer’s disease (AD). We hypothesize that the initial insult originates in the lateral entorhinal cortex (LEC) due to deficits in key interneuronal functions and synaptic signaling mechanisms, in particular, Wnt (Wingless/integrated). To investigate this hypothesis, we utilized the first knock-in mouse model of AD (App^{NL/NL-F}, expressing a mutant form of human amyloid-β (Aβ) precursor protein. This model shows an age-dependent accumulation of Aβ, neuroinflammation, and neurodegeneration. Prior to the typical AD pathology, we showed a decrease in canonical Wnt signaling activity first affecting the LEC in combination with synaptic hyperexcitation and severely disrupted excitatory–inhibitory inputs onto principal cells. This synaptic imbalance was consistent with a reduction in the number of parvalbumin-containing (PV) interneurons, and a reduction in the somatic inhibitory axon terminals in the LEC compared with other cortical regions. However, targeting GABA_{A} receptors on PV cells using allosteric modulators, diazepam, zolpidem, or a nonbenzodiazepine, L-838,417 (modulator of α2/3 subunit-containing GABAA receptors), restored the excitatory–inhibitory imbalance observed at principal cells in the LEC. These data support our hypothesis, providing a rationale for targeting the synaptic imbalance in the LEC for early stage therapeutic intervention to prevent neurodegeneration in AD

A fully automated home cage for long-term continuous phenotyping of mouse cognition and behavior

Automated home-cage monitoring systems present a valuable tool for comprehensive phenotyping of natural behaviors. However, current systems often involve complex training routines, water or food restriction, and probe a limited range of behaviors. Here, we present a fully automated home-cage monitoring system for cognitive and behavioral phenotyping in mice. The system incorporates T-maze alternation, novel object recognition, and object-in-place recognition tests combined with monitoring of locomotion, drinking, and quiescence patterns, all carried out over long periods. Mice learn the tasks rapidly without any need for water or food restrictions. Behavioral characterization employs a deep convolutional neural network image analysis. We show that combined statistical properties of multiple behaviors can be used to discriminate between mice with hippocampal, medial entorhinal, and sham lesions and predict the genotype of an Alzheimer's disease mouse model with high accuracy. This technology may enable large-scale behavioral screening for genes and neural circuits underlying spatial memory and other cognitive processes

Calpain-mediated degradation of p35 to p25 in postmortem human and rat brains

AbstractTau in Alzheimer neurofibrillary tangles has been shown to be hyperphosphorylated and CDK5, GSK3, MAP kinase and SAP kinases are the candidate kinases for the phosphorylation of tau. Recently, it was reported that the conversion of p35, the activator of CDK5, to p25 was upregulated in Alzheimer’s disease (AD) brains, and that p35 is cleaved to yield p25 by calpain. Here we show that p35 is rapidly cleaved to p25 in rat and human brains within a short postmortem delay and that the conversion of p35 to p25 is partially dependent on calpain activity. Immunoblot analysis of brains prepared from patients with AD or age-matched control individuals with a short postmortem delay revealed no specific increase in the levels of p25 in AD brains, whereas the levels of active form of calpain were increased in AD brains compared to the those in controls. These observations suggest that the conversion of p35 to p25 is a postmortem degradation event and may not be upregulated in AD brains

APP mouse models for Alzheimer's disease preclinical studies

Animal models of human diseases that accurately recapitulate clinical pathology are indispensable for understanding molecular mechanisms and advancing preclinical studies. The Alzheimer's disease (AD) research community has historically used first-generation transgenic (Tg) mouse models that overexpress proteins linked to familial AD (FAD), mutant amyloid precursor protein (APP), or APP and presenilin (PS). These mice exhibit AD pathology, but the overexpression paradigm may cause additional phenotypes unrelated to AD Second-generation mouse models contain humanized sequences and clinical mutations in the endogenous mouse App gene. These mice show Aβ accumulation without phenotypes related to overexpression but are not yet a clinical recapitulation of human AD In this review, we evaluate different APP mouse models of AD, and review recent studies using the second-generation mice. We advise AD researchers to consider the comparative strengths and limitations of each model against the scientific and therapeutic goal of a prospective preclinical study

Contribution of GABAergic interneurons to amyloid-β plaque pathology in an APP knock-in mouse model

The amyloid-β (Aβ) peptide, the primary constituent of amyloid plaques found in Alzheimer’s disease (AD) brains, is derived from sequential proteolytic processing of the Amyloid Precursor Protein (APP). However, the contribution of different cell types to Aβ deposition has not yet been examined in an in vivo, non-overexpression system. Here, we show that endogenous APP is highly expressed in a heterogeneous subset of GABAergic interneurons throughout various laminae of the hippocampus, suggesting that these cells may have a profound contribution to AD plaque pathology. We then characterized the laminar distribution of amyloid burden in the hippocampus of an APP knock-in mouse model of AD. To examine the contribution of GABAergic interneurons to plaque pathology, we blocked Aβ production specifically in these cells using a cell type-specific knock-out of BACE1. We found that during early stages of plaque deposition, interneurons contribute to approximately 30% of the total plaque load in the hippocampus. The greatest contribution to plaque load (75%) occurs in the stratum pyramidale of CA1, where plaques in human AD cases are most prevalent and where pyramidal cell bodies and synaptic boutons from perisomatic-targeting interneurons are located. These findings reveal a crucial role of GABAergic interneurons in the pathology of AD. Our study also highlights the necessity of using APP knock-in models to correctly evaluate the cellular contribution to amyloid burden since APP overexpressing transgenic models drive expression in cell types according to the promoter and integration site and not according to physiologically relevant expression mechanisms

Epigenetic repression of Wnt receptors in AD: a role for Sirtuin2-induced H4K16ac deacetylation of Frizzled1 and Frizzled7 promoters

Growing evidence supports a role for deficient Wnt signalling in Alzheimer’s disease (AD). First, the Wnt antagonist DKK1 is elevated in AD brains and is required for amyloid-β-induced synapse loss. Second, LRP6 Wnt co-receptor is required for synapse integrity and three variants of this receptor are linked to late-onset AD. However, the expression/role of other Wnt signalling components remain poorly explored in AD. Wnt receptors Frizzled1 (Fzd1), Fzd5, Fzd7 and Fzd9 are of interest due to their role in synapse formation/plasticity. Our analyses showed reduced FZD1 and FZD7 mRNA levels in the hippocampus of human early AD stages and in the hAPPNLGF/NLGF mouse model. This transcriptional downregulation was accompanied by reduced levels of the pro-transcriptional histone mark H4K16ac and a concomitant increase of its deacetylase Sirt2 at Fzd1 and Fzd7 promoters in AD. In vitro and in vivo inhibition of Sirt2 rescued Fzd1 and Fzd7 mRNA expression and H4K16ac levels at their promoters. In addition, we showed that Sirt2 recruitment to Fzd1 and Fzd7 promoters is dependent on FoxO1 activity in AD, thus acting as a co-repressor. Finally, we found reduced levels of SIRT2 inhibitory phosphorylation in nuclear samples from human early AD stages with a concomitant increase in the SIRT2 phosphatase PP2C. This results in hyperactive nuclear Sirt2 and favours Fzd1 and Fzd7 repression in AD. Collectively, our findings define a novel role for nuclear hyperactivated SIRT2 in repressing Fzd1 and Fzd7 expression via H4K16ac deacetylation in AD. We propose SIRT2 as an attractive target to ameliorate AD pathology

Calpain Cleavage Prediction Using Multiple Kernel Learning

Calpain, an intracellular -dependent cysteine protease, is known to play a role in a wide range of metabolic pathways through limited proteolysis of its substrates. However, only a limited number of these substrates are currently known, with the exact mechanism of substrate recognition and cleavage by calpain still largely unknown. While previous research has successfully applied standard machine-learning algorithms to accurately predict substrate cleavage by other similar types of proteases, their approach does not extend well to calpain, possibly due to its particular mode of proteolytic action and limited amount of experimental data. Through the use of Multiple Kernel Learning, a recent extension to the classic Support Vector Machine framework, we were able to train complex models based on rich, heterogeneous feature sets, leading to significantly improved prediction quality (6% over highest AUC score produced by state-of-the-art methods). In addition to producing a stronger machine-learning model for the prediction of calpain cleavage, we were able to highlight the importance and role of each feature of substrate sequences in defining specificity: primary sequence, secondary structure and solvent accessibility. Most notably, we showed there existed significant specificity differences across calpain sub-types, despite previous assumption to the contrary. Prediction accuracy was further successfully validated using, as an unbiased test set, mutated sequences of calpastatin (endogenous inhibitor of calpain) modified to no longer block calpain's proteolytic action. An online implementation of our prediction tool is available at http://calpain.org

Subtle behavioral changes and increased prefrontal-hippocampal network synchronicity in APPNL-G-F mice before prominent plaque deposition

Amyloid-β (Aβ) peptides occur in the brains of patients with Alzheimer's disease (AD), but their role in functional impairment is still debated. High levels of APP and APP fragments in mice that overexpress APP might confound their use in preclinical research. We examined the occurrence of behavioral, cognitive and neuroimaging changes in APPNL-G-F knock-in mice that display Aβ42 amyloidosis in the absence of APP overexpression. Female APPNL-G-F mice (carrying Swedish, Iberian and Arctic APP mutations) were compared to APPNL mice (APP Swedish) at 3, 7 and 10 months. Mice were subjected to a test battery that referred to clinical AD symptoms, comprising cage activity, open field, elevated plus maze, social preference and novelty test, and spatial learning, reversal learning and spatial reference memory performance. Our assessment confirmed that behavior at these early ages was largely unaffected in these mice in accordance with previous reports, with some subtle behavioral changes, mainly in social and anxiety-related test performance. Resting-state functional MRI (rsfMRI) assessed connectivity between hippocampal and prefrontal regions with an established role in flexibility, learning and memory. Increased prefrontal-hippocampal network synchronicity was found in 3-month-old APPNL-G-F mice. These functional changes occurred before prominent amyloid plaque deposition