Swedish Mutant Amyloid Precursor Protein Research Articles

The Prenylflavonoid Xanthohumol Reduces Alzheimer-Like Changes and Modulates Multiple Pathogenic Molecular Pathways in the Neuro2a/APPswe Cell Model of AD.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that has proved refractory to drug treatment. Given evidence of neuroprotection in animal models of ischemic stroke, we assessed the prenylflavonoid xanthohumol from the Common Hop (Humulus lupulus L.) for therapeutic potential in murine neuroblastoma N2a cells stably expressing human Swedish mutant amyloid precursor protein (N2a/APP), a well-characterized cellular model of AD. The ELISA and Western-blot analysis revealed that xanthohumol (Xn) inhibited Aβ accumulation and APP processing, and that Xn ameliorated tau hyperphosphorylation via PP2A, GSK3β pathways in N2a/APP cells. The amelioration of tau hyperphosphorylation by Xn was also validated on HEK293/Tau cells, another cell line with tau hyperphosphorylation. Proteomic analysis (2D-DIGE-coupled MS) revealed a total of 30 differentially expressed lysate proteins in N2a/APP vs. wild-type (WT) N2a cells (N2a/WT), and a total of 21 differentially expressed proteins in lysates of N2a/APP cells in the presence or absence of Xn. Generally, these 51 differential proteins could be classified into seven main categories according to their functions, including: endoplasmic reticulum (ER) stress-associated proteins; oxidative stress-associated proteins; proteasome-associated proteins; ATPase and metabolism-associated proteins; cytoskeleton-associated proteins; molecular chaperones-associated proteins, and others. We used Western-blot analysis to validate Xn-associated changes of some key proteins in several biological/pathogenic processes. Taken together, we show that Xn reduces AD-related changes in stably transfected N2a/APP cells. The underlying mechanisms involve modulation of multiple pathogenic pathways, including those involved in ER stress, oxidative stress, proteasome molecular systems, and the neuronal cytoskeleton. These results suggest Xn may have potential for the treatment of AD and/or neuropathologically related neurodegenerative diseases.

Dipentylammonium Binds to the Sigma-1 Receptor and Protects Against Glutamate Toxicity, Attenuates Dopamine Toxicity and Potentiates Neurite Outgrowth in Various Cultured Cell Lines.

Alzheimer's disease is a neurodegenerative disease that affects 44 million people worldwide, costing the world $605 billion to care for those affected not taking into account the physical and psychological costs for those who care for Alzheimer's patients. Dipentylammonium is a simple amine, which is structurally similar to a number of other identified sigma-1 receptor ligands with high affinities such as (2R-trans)-2butyl-5-heptylpyrrolidine, stearylamine and dodecylamine. This study investigates whether dipentylammonium is able to provide neuroprotective effects similar to those of sigma-1 receptor agonists such as PRE-084. Here we identify dipentylammonium as a sigma-1 receptor ligand with nanomolar affinity. We have found that micromolar concentrations of dipentylammonium protect from glutamate toxicity and prevent NFκB activation in HT-22 cells. Micromolar concentrations of dipentylammonium also protect stably expressing amyloid precursor protein Swedish mutant (APP/Swe) Neuro2A cells from toxicity induced by 150μM dopamine, suggesting that dipentylammonium may be useful for the treatment of Parkinsonian symptoms in Alzheimer's patients which are often associated with a more rapid deterioration of cognitive and physical ability. Finally, we found that low micromolar concentrations of dipentylammonium could out preform known sigma-1 receptor agonist PRE-084 in potentiating neurite outgrowth in Neuro2A cells, further suggesting that dipentylammonium has a potential use in the treatment of neurodegenerative diseases and could be acting through the sigma-1 receptor.

Methyl Salicylate Lactoside Protects Neurons Ameliorating Cognitive Disorder Through Inhibiting Amyloid Beta-Induced Neuroinflammatory Response in Alzheimer's Disease.

Neuroinflammatory reactions mediated by microglia and astrocytes have been shown to play a key role in early progression of Alzheimer’s disease (AD). Increased evidences have demonstrated that neurons exacerbate local inflammatory reactions by producing inflammatory mediators and act as an important participant in the pathogenesis of AD. Methyl salicylate lactoside (MSL) is an isolated natural product that is part of a class of novel non-steroidal anti-inflammatory drugs (NSAID). In our previous studies, we demonstrated that MSL exhibited therapeutic effects on arthritis-induced mice and suppressed the activation of glial cells. In the current study, we investigated the effects of MSL on cognitive function and neuronal protection induced by amyloid-beta peptides (Aβ) and explored potential underlying mechanisms involved. Amyloid precursor protein (APP) and presenilin 1 (PS1) double transgenic mice were used to evaluate the effects of MSL through behavioral testing and neuronal degenerative changes. In addition, copper-injured APP Swedish mutation overexpressing SH-SY5Y cells were used to determine the transduction of cyclooxygenase (COX) and mitogen-activated protein kinase (MAPK) pathways. Our results indicated that at an early stage, MSL treatment ameliorated cognitive impairment and neurodegeneration in APP/PS1 mice. Moreover, in an in vitro AD model, MSL treatment protected injured cells by increasing cell viability, improving mitochondrial dysfunction, and decreasing oxidative damage. In addition, MSL inhibited the phosphorylated level of c-Jun N-terminal kinase (JNK) and p38 MAPK, and suppressed the expression of COX-1/2. As a novel NSAIDs and used for the treatment in early stage of AD, MSL clearly demonstrated cognitive preservation by protecting neurons via a pleiotropic anti-inflammatory effect in the context of AD-associated deficits. Therefore, early treatment of anti-inflammatory therapy may be an effective strategy for treating AD.

Catalpol Inhibits Amyloid-β Generation Through Promoting α-Cleavage of APP in Swedish Mutant APP Overexpressed N2a Cells.

Amyloid-β (Aβ) peptides play a crucial role in the pathogenesis of Alzheimer’s disease (AD), due to its neurotoxicity. Thus, blocking Aβ generation and aggregation in the brain has been realized as an efficient way for the prevention of AD. The natural product catalpol, isolated from Rehmannia glutinosa, has shown neuroprotective activities through inhibiting soluble Aβ production, degrading Aβ peptide, and attenuating Aβ toxicity and neuroinflammatory responses. In the present study, we aimed to evaluate whether catalpol reduce Aβ generation associated with regulating amyloid precursor protein (APP) proteolytic processing. By using Swedish mutant APP overexpressed N2a (SweAPP N2a) cells treated with catalpol, we found that catalpol was not able to reduce the expression levels of β-secretase (BACE-1) and γ-secretase (PS1, APH-1, PEN-2 and Nicastrin). By contrast, catalpol had a significant promotion effect on the expression of α-secretase (ADAM10) and its proteolytic products, sAPPα and C83, suggesting that catalpol reduced the production of Aβ might be involved in non-amyloidogenic APP pathway. In addition, we confirmed that the extracellular signal-related kinase/cAMP-response element binding protein (ERK/CREB) signaling pathways were responsible for the up-regulation of ADAM10 in catalpol-treated SweAPP N2a cells. The present data, for the first time, have demonstrated that the effect of catalpol on the inhibiting Aβ generation might be closely related to α-cleavage of APP processing.

CRISPR/Cas9 Mediated Disruption of the Swedish APP Allele as a Therapeutic Approach for Early-Onset Alzheimer’s Disease

The APPswe (Swedish) mutation in the amyloid precursor protein (APP) gene causes dominantly inherited Alzheimer’s disease (AD) as a result of increased β-secretase cleavage of the amyloid-β (Aβ) precursor protein. This leads to abnormally high Aβ levels, not only in brain but also in peripheral tissues of mutation carriers. Here, we selectively disrupted the human mutant APPSW allele using CRISPR. By applying CRISPR/Cas9 from Streptococcus pyogenes, we generated allele-specific deletions of either APPSW or APPWT. As measured by ELISA, conditioned media of targeted patient-derived fibroblasts displayed an approximate 60% reduction in secreted Aβ. Next, coding sequences for the APPSW-specific guide RNA (gRNA) and Cas9 were packaged into separate adeno-associated viral (AAV) vectors. Site-specific indel formation was achieved both in primary neurons isolated from APPSW transgenic mouse embryos (Tg2576) and after co-injection of these vectors into hippocampus of adult mice. Taken together, we here present proof-of-concept data that CRISPR/Cas9 can selectively disrupt the APPSW allele both ex vivo and in vivo—and thereby decrease pathogenic Aβ. Hence, this system may have the potential to be developed as a tool for gene therapy against AD caused by APPswe and other point mutations associated with increased Aβ.

Epigenetic modification of Nrf2 by sulforaphane increases the antioxidative and anti-inflammatory capacity in a cellular model of Alzheimer's disease

Sulforaphane was reported to exert neuroprotective effects via upregulating expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and has received increasing attention as an alternative candidate for treatment of Alzheimer's disease (AD). However, the mechanism to account for Nrf2 upregulation by sulforaphane in AD remains unknown. Herein, we found that sulforaphane upregulated Nrf2 expression and promoted Nrf2 nuclear translocation via decreasing DNA methylation levels of the Nrf2 promoter in mouse neuroblastoma N2a cells stably expressing human Swedish mutant amyloid precursor protein (N2a/APPswe cells), a cellular model of AD. Furthermore, sulforaphane (1.25 and 2.5 μM) decreased the levels of amyloid β 1-40 (Aβ1-40) (21.7% and 33.4% decrease for intracellular Aβ1-40; 22.0% and 30.2% decrease in culture medium), Aβ1-42 (26.4% and 42.9% decrease for intracellular Aβ1-42; 25.8% and 43.8% decrease in culture medium), reactive oxygen species (15.0% and 28.5% decrease), and malondialdehyde (MDA) (34.4% and 39.2% decrease) and increased superoxide dismutase (SOD) (60.0% and 89.3% increase) activity in N2a/APPswe cells. Sulforaphane also decreased the levels of pro-inflammatory cytokines interleukin 1β (IL-1β) (16.5% and 33.6% decrease) and IL-6 (15.6% and 26.1% decrease) and reduced phosphorylated nuclear factor-κB (NF-κB) p65 (19.2% and 32.2% decrease), cyclooxygenase-2 (COX-2) (20.5% and 28.6% decrease), and iNOS protein (40.2% and 54.7% decrease) expression levels in N2a/APPswe cells. Our study suggested that sulforaphane upregulated the expression of Nrf2 and promoted the nuclear translocation of Nrf2 by decreasing DNA demethylation levels of the Nrf2 promoter, thus leading to antioxidative and anti-inflammatory effects in a cellular model of AD.

The Isoquinoline Alkaloid Dauricine Targets Multiple Molecular Pathways to Ameliorate Alzheimer-Like Pathological Changes In Vitro.

Alzheimer's disease (AD), the most common neurodegenerative disease, has no effective treatment. Dauricine (DAU), a benzyl tetrahydroisoquinoline alkaloid isolated from the root of Menispermum dauricum DC, reportedly has neuroprotective effects in cerebral ischemia. Here, we investigated the effects of DAU on N2a cells stably transfected with Swedish mutant amyloid precursor protein (N2a/APP), an AD-like cell model. ELISA and Western blot analysis revealed that DAU inhibited APP processing and reduced Aβ accumulation. In addition, DAU ameliorated tau hyperphosphorylation via PP2A, p35/25, and CDK5 pathways in N2a/APP cells. The amelioration of tau hyperphosphorylation by DAU was also validated in HEK293/Tau cells, another cell line with tau hyperphosphorylation. Proteomic analysis revealed 85 differentially expressed proteins in the lysates between the wild-type N2a cells (N2a/WT) and the N2a/APP cells in the presence or absence of DAU; these were classified into 6 main categories according to their functions: endoplasmic reticulum (ER) stress-associated proteins, oxidative stress-associated proteins, cytoskeleton proteins, molecular chaperones, mitochondrial respiration and metabolism-related proteins, and signaling proteins. Taken together, we demonstrated that DAU treatment reduces AD-like pathology, thereby suggesting that DAU has potential therapeutic utility in AD.

Total flavonoid extract from Dracoephalum moldavica L. attenuates β-amyloid-induced toxicity through anti-amyloidogenesic and neurotrophic pathways

AimsAlzheimer's disease (AD) is an incurable neurodegenerative disorder characterized by global cognitive impairment that involves accumulation of amyloid-beta peptides (Aβ) in the brain. Herbal approaches can be used as alternative medicines to slow the progression of AD. This study aimed to determine the beneficial effects and potential underlying mechanisms of total flavonoid extract from Dracoephalum moldavica L. (TFDM) for attenuating Alzheimer-related deficits induced by Aβ. Main methodsWe used amyloid precursor protein (APP) and presenilin 1 (PS1) double transgenic mice and copper-injured APP Swedish mutation overexpressing SH-SY5Y cells to evaluate the beneficial effects of TFDM. Further, identifying the mechanisms of action was conducted on anti-amyloidogenic and neurotrophic transductions. Key findingsOur results indicated that TFDM treatment ameliorated cognitive impairments and neurodegeneration and improved the antioxidant defense system in APP/PS1 mice. TFDM also reduced Aβ burden by relieving Aβ deposition, decreasing insoluble Aβ levels, and inhibiting β-amyloidogenic processing pathway involving downregulation of β-secretase and β-C-terminal fragment in the brain. In the in vitro model of AD, TFDM treatment protected injured cells, and combined with the beneficial effects of decreasing APP levels, lowered Aβ1–42 and regulated the redox imbalance. Moreover, TFDM preserved the extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor pathway both in vitro and in vivo. SignificanceIn conclusion, TFDM clearly demonstrated neuroprotective effects by restoring the anti-amyloidogenic and neurotrophic transductions in the context of AD-associated deficits. These findings indicate the potential use of herb-based substances as supplements or potential alternative supplements for attenuating the progression of AD.

The Effects of Extracellular Serum Concentration on APP Processing in Npc1-Deficient APP-Overexpressing N2a Cells.

Amyloid precursor protein (APP) is cleaved by a set of proteases including α-/β-/γ- and recently identified η-secretases, generating C-terminal fragments (CTFs) of varying lengths and amyloid β (Aβ) peptides, which are considered to play a pivotal role in Alzheimer's disease (AD) pathogenesis. Cellular cholesterol content/distribution can regulate the production/clearance of APP metabolites and hence modify AD pathology. To determine the functional relation between endosomal-lysosomal (EL) cholesterol sequestration and APP metabolism, we used our recently developed mouse N2a-ANPC cells that overexpress Swedish mutant human APP in the absence of cholesterol-trafficking Niemann-Pick type C1 (Npc1) protein. Here, we report that neither increased levels nor EL cholesterol sequestration altered APP holoprotein levels but caused the intracellular accumulation of APP α-/β-/η-CTFs and Aβ1-40/42 peptides. The levels of APP-cleaved products increased as a function of extracellular serum concentration in N2a-ANPC cells, which are more vulnerable to death than the control cells. Additionally, we show that pH of the lysosomal vesicles in N2a-ANPC cells shifted to a less acidic range with increasing serum concentrations, thus making them less efficient functionally. Interestingly, the addition of cholesterol to the culture media not only increased the levels of cellular cholesterol and APP-cleaved products but also rendered the cells more vulnerable to toxicity. Collectively, our results suggest that extracellular cholesterol concentration in serum under conditions of Npc1 deficiency can influence intracellular cholesterol content/distribution and lysosomal efficacy, triggering the accumulation of toxic APP-cleaved products, eventually leading to cell death.

Mitochondrial dysfunction in the APP/PSEN1 mouse model of Alzheimer’s disease and a novel protective role for ascorbate

Mitochondrial dysfunction is elevated in very early stages of Alzheimer’s disease and exacerbates oxidative stress, which contributes to disease pathology. Mitochondria were isolated from 4-month-old wild-type mice, transgenic mice carrying the APPSWE and PSEN1dE9 mutations, mice with decreased brain and mitochondrial ascorbate (vitamin C) via heterozygous knockout of the sodium dependent vitamin C transporter (SVCT2+/-) and transgenic APP/PSEN1 mice with heterozygous SVCT2 expression. Mitochondrial isolates from SVCT2+/- mice were observed to consume less oxygen using high-resolution respirometry, and also exhibited decreased mitochondrial membrane potential compared to wild type isolates. Conversely, isolates from young (4 months) APP/PSEN1 mice consumed more oxygen, and exhibited an increase in mitochondrial membrane potential, but had a significantly lower ATP/ADP ratio compared to wild type isolates. Greater levels of reactive oxygen species were also produced in mitochondria isolated from both APP/PSEN1 and SVCT2+/- mice compared to wild type isolates. Acute administration of ascorbate to mitochondria isolated from wild-type mice increased oxygen consumption compared with untreated mitochondria suggesting ascorbate may support energy production. This study suggests that both presence of amyloid and ascorbate deficiency can contribute to mitochondrial dysfunction, even at an early, prodromal stage of Alzheimer’s disease, although occurring via different pathways. Ascorbate may, therefore, provide a useful preventative strategy against neurodegenerative disease, particularly in populations most at risk for Alzheimer’s disease in which stores are often depleted through mitochondrial dysfunction and elevated oxidative stress.

Low-Density Lipoprotein Receptor-Related Protein-1 (LRP1) C4408R Mutant Promotes Amyloid Precursor Protein (APP) α-Cleavage in Vitro.

Previous studies have demonstrated that the low-density lipoprotein receptor-related protein-1 (LRP1) plays conflicting roles in Alzheimer's disease (AD) pathogenesis, clearing β-amyloid (Aβ) from the brain while also enhancing APP endocytosis and resultant amyloidogenic processing. We have recently discovered that co-expression of mutant LRP1 C-terminal domain (LRP1-CT C4408R) with Swedish mutant amyloid precursor protein (APPswe) in Chinese hamster ovary (CHO) cells decreases Aβ production, while also increasing sAPPα and APP α-C-terminal fragment (α-CTF), compared with CHO cells expressing APPswe alone. Surprisingly, the location of this mutation on LRP1 corresponded with the α-secretase cleavage site of APP. Further experimentation confirmed that in CHO cells expressing APPswe or wild-type APP (APPwt), co-expression of LRP1-CT C4408R decreases Aβ and increases sAPPα and α-CTF compared with co-expression of wild-type LRP1-CT. In addition, LRP1-CT C4408R enhanced the unglycosylated form of LRP1-CT and reduced APP endocytosis as determined by flow cytometry. This finding identifies a point mutation in LRP1 which slows LRP1-CT-mediated APP endocytosis and amyloidogenic processing, while enhancing APP α-secretase cleavage, thus demonstrating a potential novel target for slowing AD pathogenesis.

Mitochondria are devoid of amyloid β-protein (Aβ)-producing secretases: Evidence for unlikely occurrence within mitochondria of Aβ generation from amyloid precursor protein

Mitochondrial dysfunction is implicated in the pathological mechanism of Alzheimer's disease (AD). Amyloid β-protein (Aβ), which plays a central role in AD pathogenesis, is reported to accumulate within mitochondria. However, a question remains as to whether Aβ is generated locally from amyloid precursor protein (APP) within mitochondria. We investigated this issue by analyzing the expression patterns of APP, APP-processing secretases, and APP metabolites in mitochondria separated from human neuroblastoma SH-SY5Y cells and those expressing Swedish mutant APP. APP, BACE1, and PEN-2 protein levels were significantly lower in crude mitochondria than microsome fractions while those of ADAM10 and the other γ-secretase complex components (presenilin 1, nicastrin, and APH-1) were comparable between fractions. The crude mitochondrial fraction containing substantial levels of cathepsin D, a lysosomal marker, was further separated via iodixanol gradient centrifugation to obtain mitochondria- and lysosome-enriched fractions. Mature APP, BACE1, and all γ-secretase complex components (in particular, presenilin 1 and PEN-2) were scarcely present in the mitochondria-enriched fraction, compared to the lysosome-enriched fraction. Moreover, expression of the β-C-terminal fragment (β-CTF) of APP was markedly low in the mitochondria-enriched fraction. Additionally, immunocytochemical analysis showed very little co-localization between presenilin 1 and Tom20, a marker protein of mitochondria. In view of the particularly low expression levels of BACE1, γ-secretase complex proteins, and β-CTF in mitochondria, we propose that it is unlikely that Aβ generation from APP occurs locally within this organelle.

Vulnerability of primary neurons derived from Tg2576 Alzheimer mice to oxygen and glucose deprivation: role of intraneuronal amyloid-β accumulation and astrocytes.

ABSTRACTMicrovascular dysfunction is considered an integral part of Alzheimer disease (AD) pathogenesis, but the possible relationship between amyloid pathology, microvascular dysfunction and cell death is still unclear. In order to investigate the influence of intraneuronal amyloid-β (Aβ) accumulation on vulnerability to hypoxia, we isolated primary cortical neurons from Tg2576 (carrying the amyloid precursor protein APPSwe mutation) and wild-type fetal mice. We first demonstrated that neurons isolated from Tg2576 newborn mice show an increase in VEGFa mRNA expression and a decrease in the expression of the two VEGF receptors, Flt1 and Kdr, compared with wild-type cells. Moreover, APPSwe primary neurons displayed higher spontaneous and glutamate-induced cell death. We then deprived the cultures of oxygen and glucose (OGD) as an in vitro model of hypoxia. After OGD, APPSwe neurons display higher levels of cell death in terms of percentage of pyknotic/fragmented nuclei and mitochondrial depolarization, accompanied by an increase in the intraneuronal Aβ content. To explore the influence of intraneuronal Aβ peptide accumulation, we used the γ-secretase inhibitor LY450139, which showed that the reduction of the intracellular amyloid fully protects APPSwe neurons from OGD-induced degeneration. Conditioned medium from OGD-exposed APPSwe or wild-type astrocytes protected APPswe neurons but not wild-type neurons, during OGD. In conclusion, the presence of the mutated human APP gene, leading to the intracellular accumulation of APP and Aβ fragments, worsens OGD toxicity. Protection of APPSwe neurons can be obtained either using a γ-secretase inhibitor or astrocyte conditioned medium.

Early hippocampal hyperexcitability in PS2APP mice: role of mutant PS2 and APP

Alterations of brain network activity are observable in Alzheimer's disease (AD) together with the occurrence of mild cognitive impairment, before overt pathology. However, in humans as well in AD mouse models, identification of early biomarkers of network dysfunction is still at its beginning. We performed in vivo recordings of local field potential activity in the dentate gyrus of PS2APP mice expressing the human amyloid precursor protein (APP) Swedish mutation and the presenilin-2 (PS2) N141I. From a frequency-domain analysis, we uncovered network hyper-synchronicity as early as 3 months, when intracellular accumulation of amyloid beta was also observable. In addition, at 6 months of age, we identified network hyperactivity in the beta/gamma frequency bands, along with increased theta-beta and theta-gamma phase-amplitude cross-frequency coupling, in coincidence with the histopathological traits of the disease. Although hyperactivity and hypersynchronicity were respectively detected in mice expressing the PS2-N141I or the APP Swedish mutant alone, the increase in cross-frequency coupling specifically characterized the 6-month-old PS2APP mice, just before the surge of the cognitive decline.

Anthocyanins protect from complex I inhibition and APPswe mutation through modulation of the mitochondrial fission/fusion pathways

Anthocyanins are a distinguished class of flavonoids with powerful free radical-scavenging activity that have been suggested as chemotherapeutic agents for the prevention of Alzheimer disease (AD). In this study, we examined the ability of nutraceutical Medox rich in purified cyanidin 3-O-glucoside (C3G), 3-O-b-glucosides and delphinidin 3-O-glucoside (D3G) to counteract mitochondrial deficiency induced by complex I inhibition and/or amyloid-β peptide (Aβ) induced toxicity. SH-SY5Y neuroblastoma cells were stably transfected with APP Swedish K670N/M671L double mutation (APPswe) or with the empty vector and treated with rotenone. We report that Medox treatment improves the metabolic activity and maintains cell integrity in both cell lines. At the mitochondrial level, APPswe and rotenone induced mitochondrial fragmentation, an effect that was counteracted by Medox through the modulation of fission and fusion proteins, resulting in a reshaped mitochondrial network. Although Medox was unable to fully neutralise the effects of rotenone on ATP levels and mitochondrial membrane potential, it was able to prevent rotenone-induced cytotoxicity.Our findings suggest that Medox anthocyanins, on top of their antioxidant capacity, ameliorate mitochondrial dysfunction generated by Aβ overproduction or by chemical inhibition of mitochondrial complex I via stabilization of the fusion/fission processes. Modulation of the mitochondrial network has been suggested as a novel therapeutic approach in diseases involving mitochondrial dysfunction and oxidative stress. Hence, increasing the understanding of how anthocyanins influence mitochondrial dynamics in a neurodegenerative context, could be of future therapeutic value.

Increased Epileptiform EEG Activity and Decreased Seizure Threshold in Arctic APP Transgenic Mouse Model of Alzheimer's Disease.

Several Alzheimer model mice carrying transgenic amyloid precursor protein (APP) with the Swedish mutation have been reported to exhibit spontaneous seizures and/or increased epileptiform EEG activity. The primary cause for the epilepsy phenotype is still under debate. In contrast to mice with APPswe mutation that develop extracellular amyloid plaques, mice with APP Arctic mutation (E693G) have no bias toward β-secretase cleavage and display intracellular amyloid deposits but not plaques. We conducted a systematic long-term video-EEG recording in three two-week sessions on 21 APParc and 11 wild-type control mice between 3.5 and 8 months of age. Spontaneous seizures were not detected more often in APParc mice than in their wild-type control mice. Long (1 - 5 s) epileptiform discharges were occasionally detected in both APParc and wild-type mice, but short (0.5 - <1 s) epileptiform discharges were more common in APParc mice than in wild-types. However, they were far less frequent than in 6 APPswe/PS1dE9 mice recorded in parallel. In pentylenetetrazole test for seizure susceptibility, APParc mice displayed a shorter latency to sharp-wave discharges than wildtype mice but no increase in seizure duration. These data speak for a relatively mild epilepsy phenotype in APParc mice compared to APPswe mice despite even higher extent of APP overexpression. Thus extracellular amyloid plaques or increased β-secretase cleavage products appear important for the epilepsy phenotype in APPswe mice.

567. CRISPR-Cas9 Mediated Gene Editing in a Monogenic Form of Alzheimer's Disease

BackgroundIndividuals heterozygous for the Swedish mutation of the amyloid precursor protein (APPswe) display an increased β-secretase cleavage leading to higher Aβ levels - both in brain and peripheral tissues, such as fibroblasts and plasma. The mutation is a double base change adjacent to each other and has a dominant effect. We hypothesize that the CRISPR system would selectively disrupt the mutated allele without affecting the wild-type allele. Therefore we set out to use the CRISPR system to disrupt expression of mutant APP to counteract Aβ overproduction in Swedish patient fibroblasts.MethodsHuman APPswe fibroblasts and non-mutated control fibroblasts from subjects of the same family (n=3 for both mutated and control cell lines) were cultured in DMEM medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Approximately one million cells were used for transfection with the SpCas9 plasmid together with different guide RNAs (gRNAs) designed to bind either the mutated or non-mutated site with the mutation in the gRNA recognition sequence. Four days after transfection, the GFP positive cells were sorted by FACS, enabling separation of Cas9-transfected from non-transfected cells. DNA was extracted from one fraction of the sorted cells, followed by Sanger sequencing and targeted deep sequencing. Another fraction of the cells was further cultured until they were confluent, after which they were kept for 48 h in FBS free media. ELISAs specific for Aβ40 and Aβ42 were used to measure peptide levels in conditioned media from the various cell populations.ResultsSanger sequencing was performed on cells that had been successfully transfected with CRISPR plasmids. On such cells, both the APPswe mutant and wt alleles could be disrupted with gRNAs designed against the mutated and non-mutated sites, respectively. Moreover, these effects appeared to be highly specific as assayed by deep sequencing as we did not find any random mutations on the wt allele with the gRNA targeting the mutated site or vice versa. Most of the genomic changes were deletions, but there were some insertions as well. ELISA measurements indicated robustly decreased levels of both Aβ40 and Aβ42 in conditioned media from both mutated and wt cells treated with the respective gRNAs. Shortening the gRNA led to decreased on-target effectivity.ConclusionsWith the CRISPR/Cas 9 system we could selectively disrupt both the mutated and wt site in fibroblasts from subjects with and without the APPswe mutation. The adjacent double base change created a unique opportunity to selectively disrupt the mutant allele with one of our gRNAs. Furthermore, we could demonstrate a decreased generation of Aβ40 and Aβ42 in the conditioned media from cells treated with the specific gRNAs. This study provides the first experimental evidence that the CRISPR/Cas9 method could be used to develop a novel treatment strategy against familial forms of Alzheimer's disease caused by dominant mutations.

The mouse that trolled (again).

We welcome the opportunity to respond to the commentaries on our paper—The Mouse that Trolled—by Hardy1, Sarnoff2, and Cordova and Feldman3. Their comments are academic criticism in the very best sense.We also take the opportunity to update on recent legal actions, whichwe had not predicted.This opportunity enriches our narrative history of the patenting of the APPswe mutation for early onset Alzheimer’s disease, and we hope the continued saga is of interest.

Autophagic and lysosomal defects in human tauopathies: analysis of post-mortem brain from patients with familial Alzheimer disease, corticobasal degeneration and progressive supranuclear palsy.

IntroductionThe accumulation of insoluble proteins within neurons and glia cells is a pathological hallmark of several neurodegenerative diseases. Abnormal aggregation of the microtubule-associated protein tau characterizes the neuropathology of tauopathies, such as Alzheimer disease (AD), corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP). An impairment of the lysosomal degradation pathway called macroautophagy, hereafter referred to as autophagy, could contribute to the accumulation of aggregated proteins. The role of autophagy in neurodegeneration has been intensively studied in the context of AD but there are few studies in other tauopathies and it is not known if defects in autophagy is a general feature of tauopathies. In the present study, we analysed autophagic and lysosomal markers in human post-mortem brain samples from patients with early-onset familial AD (FAD) with the APP Swedish mutation (APPswe), CBD and PSP and control individuals.ResultsFAD, CBD and PSP patients displayed an increase in LC3-positive vesicles in frontal cortex, indicating an accumulation of autophagic vesicles. Moreover, using double-immunohistochemistry and in situ proximity ligation assay, we observed colocalization of hyperphosphorylated tau with the autophagy marker LC3 in FAD, CBD and PSP patients but not in control individuals. Increased levels of the lysosomal marker LAMP1 was detected in FAD and CBD, and in addition Cathepsin D was diffusely spread in the cytoplasm in all tauopathies suggesting an impaired lysosomal integrity.ConclusionTaken together, our results indicate an accumulation of autophagic and lysosomal markers in human brain tissue from patients with primary tauopathies (CBD and PSP) as well as FAD, suggesting a defect of the autophagosome-lysosome pathway that may contribute to the development of tau pathology.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0292-9) contains supplementary material, which is available to authorized users.

Generation of aggregation prone N-terminally truncated amyloid β peptides by meprin β depends on the sequence specificity at the cleavage site.

BackgroundThe metalloprotease meprin β cleaves the Alzheimer’s Disease (AD) relevant amyloid precursor protein (APP) as a β-secretase reminiscent of BACE-1, however, predominantly generating N-terminally truncated Aβ2-x variants.ResultsHerein, we observed increased endogenous sAPPα levels in the brains of meprin β knock-out (ko) mice compared to wild-type controls. We further analyzed the cellular interaction of APP and meprin β and found that cleavage of APP by meprin β occurs prior to endocytosis. The N-terminally truncated Aβ2-40 variant shows increased aggregation propensity compared to Aβ1-40 and acts even as a seed for Aβ1-40 aggregation. Additionally, we observed that different APP mutants affect the catalytic properties of meprin β and that, interestingly, meprin β is unable to generate N-terminally truncated Aβ peptides from Swedish mutant APP (APPswe).ConclusionConcluding, we propose that meprin β may be involved in the generation of N-terminally truncated Aβ2-x peptides of APP, but acts independently from BACE-1.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0084-5) contains supplementary material, which is available to authorized users.