Onset Familial Alzheimer's Disease Research Articles

The Use of Zebrafish in Transcriptome Analysis of the Early Effects of Mutations Causing Early Onset Familial Alzheimer's Disease and Other Inherited Neurodegenerative Conditions.

The degree to which non-human animals can be used to model Alzheimer's disease is a contentious issue, particularly as there is still widespread disagreement regarding the pathogenesis of this neurodegenerative dementia. The currently popular transgenic models are based on artificial expression of genes mutated in early onset forms of familial Alzheimer's disease (EOfAD). Uncertainty regarding the veracity of these models led us to focus on heterozygous, single mutations of endogenous genes (knock-in models) as these most closely resemble the genetic state of humans with EOfAD, and so incorporate the fewest assumptions regarding pathological mechanism. We have generated a number of lines of zebrafish bearing EOfAD-like and non-EOfAD-like mutations in genes equivalent to human PSEN1, PSEN2, and SORL1. To analyze the young adult brain transcriptomes of these mutants, we exploited the ability of zebrafish to produce very large families of simultaneous siblings composed of a variety of genotypes and raised in a uniform environment. This "intra-family" analysis strategy greatly reduced genetic and environmental "noise" thereby allowing detection of subtle changes in gene sets after bulk RNA sequencing of entire brains. Changes to oxidative phosphorylation were predicted for all EOfAD-like mutations in the three genes studied. Here we describe some of the analytical lessons learned in our program combining zebrafish genome editing with transcriptomics to understand the molecular pathologies of neurodegenerative disease.

Thy1-ApoE4/C/EBPβ double transgenic mice act as a sporadic model with Alzheimer's disease.

Early onset familial Alzheimer's disease (FAD) with APP, PS1/2 (presenilins) mutation accounts for only a small portion of AD cases, and most are late-onset sporadic. However, majority of AD mouse models are developed to mimic the genetic cause of human AD by overexpressing mutated forms of human APP, PS1/2, and/or Tau protein, though there is no Tau mutation in AD, and no single mouse model recapitulates all aspects of AD pathology. Here, we report Thy1-ApoE4/C/EBPβ double transgenic mouse model that demonstrates key AD pathologies in an age-dependent manner in absence of any human APP or PS1/2 mutation. Using the clinical diagnosis criteria, we show that this mouse model exhibits tempo-spatial features in AD patient brains, including progressive cognitive decline associated with brain atrophy, which is accompanied with extensive neuronal degeneration. Remarkably, the mice display gradual Aβ aggregation and neurofibrillary tangles formation in the brain validated by Aβ PET and Tau PET. Moreover, the mice reveal widespread neuroinflammation as shown in AD brains. Hence, Thy1-ApoE4/C/EBPβ mouse model acts as a sporadic AD mouse model, reconstituting the major AD pathologies.

Human 8-cell embryos enable efficient induction of disease-preventive mutations without off-target effect by cytosine base editor.

Approximately 140 million people worldwide are homozygous carriers of APOE4 (ε4), a strong genetic risk factor for late onset familial and sporadic Alzheimer's disease (AD), 91% of whom will develop AD at earlier age than heterozygous carriers and noncarriers. Susceptibility to AD could be reduced by targeted editing of APOE4, but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies. Here, we first screened eight cytosine base editor variants at four injection stages (from 1- to 8-cell stage), and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate (up to 100%) with the lowest bystander effects. In particular, 80% of AD-susceptible ε4 allele copies were converted to the AD-neutral ε3 allele in human ε4-carrying embryos. Stringent control measures combined with targeted deep sequencing, whole genome sequencing, and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells. Furthermore, base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage. Finally, we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia. Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos, a potential approach for reducing human susceptibility to AD or other genetic diseases.

PSEN1 M139L mutation in a Chinese pedigree with early‐onset familial Alzheimer's disease

AbstractBackgroundpresenilin 1(PSEN1) is the most common gene associated with familial Alzheimer's disease. we reported a case with M139L mutation of PSEN1 which was reported as pathogenic mutation in February 2019.Methodchose an early‐onset Alzheimer's patient (proband) and her family (I‐V generation) as the research object. clinical and imaging data of the proband and her family members were collected. High throughput second generation exon sequencing and sanger method was used to analyze the gene of the proband.Result1. Proband clinical characteristic: The proband (IV8) started to progressive memory loss, especially short‐term memory at 38 years old. She can't work because of memory problem. Now she lives on her own, can use mobile phone and APP, drive, remember familiar roads, but cannot shop, take bus to new place, handle own finances. The MMSE score was 27/30, and the MOCA score was 20/30 (delayed recall, time orientation, visuospatial disfunction, calculation) (Figure 1). Brain Magnetic Resonance Imaging suggested hippocampal atrophy (Figure 2). 2. Family phenotypic distribution (Figure 3): The onset age of Alzheimer's disease in this family was early, starting between 38 and 55 years old. The first and prominent clinical manifestations were memory loss, followed by orientation and executive function impairment. Movement disorder appears later or does not appear. It may have mild anxiety or depression. 3. Gene results (Figure 4): a heterozygotic mutation was found in the exon region of the PSEN1 gene: c.415A>T (adenine > thymine), resulting in an amino acid change of p.139l (methionine > leucine).ConclusionWe reported the early onset familial Alzheimer's disease caused by M139L mutation of PSEN1 in Han Chinese family which was helpful to enrich the clinical phenotypic characteristics of early onset familial Alzheimer's disease associated with PSEN1 mutation.

Gene mutations associated with early onset familial Alzheimer's disease in China: An overview and current status.

BackgroundMutations of three causative genes, namely presenilin 1 (PSEN1), presenilin 2 (PSEN2), and amyloid precursor protein (APP), have been identified as the major causes of early‐onset familial Alzheimer's disease (EOFAD). The prevalence of causative gene mutations in patients with EOFAD has been reported in previous studies worldwide but remains unclear in China. The patients with these known mutations always show considerable clinical phenotypic variability. However, to date, there have been no detailed descriptions of the clinical phenotypes associated with these Chinese EOFAD mutations. Thus, the aim of this study was to describe all of the known mutations in three EOFAD causative genes and genotype–phenotype correlations in Chinese patients with EOFAD.MethodWe systematically searched the PubMed, MEDLINE, CNKI, VIP, and WAN‐FANG databases to find Chinese EOFAD mutations in reports from inception through May 2020.ResultWe identified 31 studies reporting mutations of three causative genes in China. 10 mutations in APP gene, 27 mutations in PSEN1 gene and six mutations in PSEN2 were discovered in Chinese EOFAD. This review summarized all these probably pathogenic mutations as well as its clinical features. To the best of our knowledge, this is the first systemic review of causative gene mutations in patients with EOFAD in China.ConclusionThe analysis of the genetic and clinical phenotype correlations in this review supports the idea that the clinical phenotype might be influenced by specific genetic defects. It also suggests genetic testing and genotype–phenotype correlations are important for the accurate diagnosis and for understanding disease‐associated pathways and might also improve disease therapy and prevention.

Designed Helical Peptides as Functional Probes for γ-Secretase.

γ-Secretase is a membrane-embedded aspartyl protease complex with presenilin as the catalytic component that cleaves within the transmembrane domain (TMD) of >90 known substrates, including the amyloid precursor protein (APP) of Alzheimer's disease. Processing by γ-secretase of the APP TMD produces the amyloid β-peptide (Aβ), including the 42-residue variant (Aβ42) that pathologically deposits in the Alzheimer brain. Complex proteolysis of APP substrate by γ-secretase involves initial endoproteolysis and subsequent carboxypeptidase trimming, resulting in two pathways of Aβ production: Aβ49 → Aβ46 → Aβ43 → Aβ40 and Aβ48 → Aβ45 → Aβ42 → Aβ38. Dominant mutations in APP and presenilin cause early onset familial Alzheimer's disease (FAD). Understanding how γ-secretase processing of APP is altered in FAD is essential for elucidating pathogenic mechanisms in FAD and developing effective therapeutics. To improve our understanding, we designed synthetic APP-based TMD substrates as convenient functional probes for γ-secretase. Installation of the helix-inducing residue α-aminoisobutyric acid provided full TMD helical substrates while also facilitating their synthesis and increasing the solubility of these highly hydrophobic peptides. Through mass spectrometric analysis of proteolytic products, synthetic substrates were identified that were processed in a manner that reproduced physiological processing of APP substrates. Validation of these substrates was accomplished through mutational variants, including the installation of two natural APP FAD mutations. These FAD mutations also resulted in increased levels of formation of Aβ-like peptides corresponding to Aβ45 and longer, raising the question of whether the levels of such long Aβ peptides are indeed increased and might contribute to FAD pathogenesis.

P2‐047: EFFECT OF WEAK COMBINED MAGNETIC FIELDS ON SPATIAL MEMORY IN AN ANIMAL MODEL OF HERITABLE ALZHEIMER'S DISEASE

In this work the effect of the weak combined magnetic fields (MFs) was studied in vivo in heritable animal model of AD: well characterized mice transgenic for mutant APPswe and mutant presenilin 1 (PS1dE9) that cause early onset familial Alzheimer's disease (AD). Transgenic mice (Tg) and control C3H mice were exposed to the same MF at age of 8 months. Mice were also exposed by MF in two groups at the same time (Tg+MF and C3H+MF). A setup for generating a MFs consisted of two pairs of coaxial Helmhollz coils oriented along the geomagnetic field vector. A direct current was supplied to one of the pairs of coils to generate a constant component of MFs with induction of 42 mcT. An alternating current signal produced by a programmable sinusoidal current generator was fed to other pair of coils to create a variable component of MFs with induction of the amplitude of 80 nT. The current signal was the sum of two frequencies of 4.38 and 4.88 Hz. These frequencies correspond to the cyclotron frequencies of glutamic and aspartic acids. After exposure to the MFs, the mice were trained in a Morris water maze for 18 days (four trials per day). Spatial memory was tested on the following day after completion of training with the hidden platform removed. During the test period (60 s), occupancy time spent in each sector was recorded. The MFs improved the memory in Tg animals. The results of the factor analysis demonstrate that factors of the sector preference became statistically significant for Tg groups after MFs exposure. Thus, the data suggest that MFs can be applied for preventive purposes in a group of risk of AD.

Analysis of gene and imaging on a late onset Alzheimer's pedigree concomitant with presenilin 1 mutation

Objective To analyze the clinical manifestation, imaging data and genetics mutation variants of late onset familial Alzheimer's disease concomitant with a novel mutation of presenilin 1. Methods The clinical manifestations and auxiliary examination recordings of the pedigree were analyzed.DNA was extracted from peripheral blood samples of the proband and her sons.Mutational analysis was performed by the next-generation sequencing technology and the mutation event was confirmed by Sanger sequencing technology. Results Two patients of the family presenting as Alzheimer's dementia were late onset.MRI of the proband showed extensive cerebral microbleeds.The gene detection showed p. S289P mutation in the exon 8 of presenilin 1 of the proband. Conclusion Mutation of p. S289P in the presenilin 1 gene may contribute to late onset Alzheimer's disease accompanied by amyloid angiopathy. Key words: Alzheimer's disease; Presenilin 1; Cerebral microbleeds; Amyloid angiopathy

Aβ42/Aβ40 Ratios of Presenilin 1 Mutations Correlate with Clinical Onset of Alzheimer's Disease.

Missense mutations in presenilin 1 cause early onset familial Alzheimer's disease in a way that is not understood. Increased Aβ42/Aβ40 ratios are the most consistent biochemical phenotype of such mutations in cultured cells and in vivo, and are thus considered central to the amyloid hypothesis. Previously, an inverse relation has been observed between the Aβ42/Aβ40 ratio of such mutants and the clinical age of symptom onset in patients carrying the mutation. However, a recent extensive study by Sun et al. of assayed presenilin 1 mutants concluded that such a relationship is not evident. To reconcile the disagreement, three different clinical datasets were compared directly with the data by Sun et al. After considering data noise and measurement uncertainty, we find a clear and highly significant inverse correlation between the Aβ42/Aβ40 ratio and the clinical age of onset in all three datasets even without removing noisy single- and double-patient data. With these data removed, the correlation coefficients increase further. The probability that these relationships are coincidental are approximately 0.1%.

The antimicrobial protection hypothesis of Alzheimer's disease

ObjectiveWe explore here a novel model for amyloidogenesis in Alzheimer's disease (AD). This new perspective on AD amyloidosis seeks to provide a rational framework for incorporating recent and seemingly independent findings on the antimicrobial role of β-amyloid and emerging experimental, genetic, and epidemiological data, suggesting innate immune-mediated inflammation propagates AD neurodegeneration. BackgroundAD pathology is characterized by cerebral deposition of amyloid-β protein (Aβ) as β-amyloid. Genetic studies have confirmed the key role of Aβ in AD, revealing that mutation-mediated shifts in the peptides generation lead to early onset familial Alzheimer's disease. However, Aβ generation appears normal for the majority of AD patients, who lack early onset familial Alzheimer's disease mutations. In prevailing models of nonfamilial AD, individual genetics and age-associated changes in brain milieu promote an intrinsically abnormal propensity of Aβ for self-association. However, emerging findings are increasingly inconsistent with characterization of Aβ oligomerization as a nonphysiological and exclusively pathological activity. Recent studies suggest Aβ is an ancient, highly conserved effector molecule of innate immunity. Moreover, Aβ oligomerization and β-amyloid generation appear to be important innate immune pathways that mediate pathogen entrapment and protect against infection. New AD amyloidogenesis modelRecent findings on inflammation-mediated neurodegeneration and the role of Aβ in immunity have led to emergence of the “Antimicrobial Protection Hypothesis” of AD. In this model, β-amyloid deposition is an early innate immune response to genuine, or mistakenly perceived, immunochallenge. Aβ first entraps and neutralizes invading pathogens in β-amyloid. Aβ fibrillization drives neuroinflammatory pathways that help fight the infection and clear β-amyloid/pathogen deposits. In AD, chronic activation of this pathway leads to sustained inflammation and neurodegeneration. Mounting data link elevated brain microbe levels with AD. The Antimicrobial Protection Hypothesis reveals how increased brain microbial burden may directly exacerbate β-amyloid deposition, inflammation, and AD progression. Amyloid cascade hypothesisIn the antimicrobial protection model, the modality of Aβ's pathophysiology is shifted from abnormal stochastic behavior toward dysregulated innate immune response. However, β-amyloid deposition in AD still leads to neurodegeneration. Thus, the new model extends but remains broadly consistent with the Amyloid Cascade Hypothesis and overwhelming data showing the primacy of Aβ in AD pathology.

Lipid/Polydiacetylene Vesicle Composition Alters Mutant Beta-Amyloid Peptide Interaction

Alzheimer's disease (AD) is commonly identified by the formation of proteinaceous fibrillar aggregate deposits known as amyloids, and the presence of neuritic amyloid plaques containing aggregated β-amyloid (Aβ). Point mutations grouped in the hydrophobic core of Aβ (positions 21–23) are linked to familial forms of AD. Understanding Aβ peptide interactions with lipid surfaces will provide detailed information into how surface interactions can drive protein aggregation. We hypothesized changes in the lipid composition of a vesicle will result in varied mutant Aβ peptide aggregation morphology and kinetics, further modulating peptide-lipid interactions. To test our hypothesis we utilized a colorimetric, lipid vesicle-binding assay exposed to specific Aβ peptides. The assay integrates a lipid within a polydiacetylene (PDA) matrix forming a blue vesicle when polymerized by UV irradiation. Lipid/PDA vesicles are able to detect peptide-lipid interaction by quantitatively measuring a blue-to-red conversion as a percent colorimetric response (%CR). We investigated the interaction of Wild Type (Aβ1-40) and five mutant Aβ peptides (E22G Arctic, E22Q Dutch, A21G Flemish, D23N Iowa, and E22K Italian) with lipid/PDA vesicle systems comprised of total brain lipid extract (TBLE), dimyristoylphosphatideylcholine (DMPC), and dimyristoylphosphatidylglycerol (DMPG). Time-resolved absorbance measurements of the “blue” (630 nm) and “red” (490 nm) wavelengths were obtained. Statistical analysis (ANOVA and t-tests) was performed to test for significant differences between peptides among the lipid/PDA compositions, as well as compare each mutant Aβ peptide to Wild Type within the context of each lipid. The most peptide-lipid interaction was observed on the negatively charged DMPG/PDA vesicle system, and the least on the neutral DMPC/PDA system. Dutch and Flemish induced the smallest, while Arctic induced the largest %CR on all lipid/PDA vesicles, respectively. These studies illustrate the role of lipid composition and Aβ interactions in the onset of familial AD.

Genetic Modifiers of Age at Onset in Carriers of the G206A Mutation in PSEN1 With Familial Alzheimer Disease Among Caribbean Hispanics.

The present study identified potential genetic modifiers that may delay or accelerate age at onset of familial Alzheimer disease (AD) by examining age at onset in PSEN1 mutation carrier families, and further investigation of these modifiers may provide insight into the pathobiology of AD and potential therapeutic measures. To identify genetic variants that modify age at onset of AD. Using a subset of Caribbean Hispanic families that carry the PSEN1 p.G206A mutation, we performed a 2-stage genome study. The mutation carrier families from an ongoing genetic study served as a discovery set, and the cohort of those with LOAD served as a confirmation set. To identify candidate loci, we performed linkage analysis using 5 p.G206A carrier families (n = 56), and we also performed whole-exome association analysis using 31 p.G206A carriers from 26 families. To confirm the genetic modifiers identified from the p.G206A carrier families, we analyzed the GWAS data for 2888 elderly individuals with LOAD. All study participants were Caribbean Hispanics. Age at onset of AD. Linkage analysis of AD identified the strongest linkage support at 4q35 (LOD [logarithm of odds] score, 3.69), and the GWAS of age at onset identified variants on 1p13.1, 2q13, 4q25, and 17p11. In the confirmation stage, genewise analysis identified SNX25, PDLIM3, and 3 SH3 domain genes (SORBS2, SH3RF3, and NPHP1) to be significantly associated with LOAD. Subsequent allelic association analysis confirmed SNX25, PDLIM3, and SORBS2 as genetic modifiers of age at onset of EOAD and LOAD and provided modest support for SH3RF3 and NPHP1. Our 2-stage analysis revealed that SNX25, PDLIM3, and SORBS2 may serve as genetic modifiers of age at onset in both EOAD and LOAD.

Development of isotope labeling liquid chromatography mass spectrometry for mouse urine metabolomics: quantitative metabolomic study of transgenic mice related to Alzheimer's disease.

Because of a limited volume of urine that can be collected from a mouse, it is very difficult to apply the common strategy of using multiple analytical techniques to analyze the metabolites to increase the metabolome coverage for mouse urine metabolomics. We report an enabling method based on differential isotope labeling liquid chromatography mass spectrometry (LC-MS) for relative quantification of over 950 putative metabolites using 20 μL of urine as the starting material. The workflow involves aliquoting 10 μL of an individual urine sample for ¹²C-dansylation labeling that target amines and phenols. Another 10 μL of aliquot was taken from each sample to generate a pooled sample that was subjected to ¹³C-dansylation labeling. The ¹²C-labeled individual sample was mixed with an equal volume of the ¹³C-labeled pooled sample. The mixture was then analyzed by LC-MS to generate information on metabolite concentration differences among different individual samples. The interday repeatability for the LC-MS runs was assessed, and the median relative standard deviation over 4 days was 5.0%. This workflow was then applied to a metabolomic biomarker discovery study using urine samples obtained from the TgCRND8 mouse model of early onset familial Alzheimer's disease (FAD) throughout the course of their pathological deposition of beta amyloid (Aβ). It was showed that there was a distinct metabolomic separation between the AD prone mice and the wild type (control) group. As early as 15-17 weeks of age (presymptomatic), metabolomic differences were observed between the two groups, and after the age of 25 weeks the metabolomic alterations became more pronounced. The metabolomic changes at different ages corroborated well with the phenotype changes in this transgenic mice model. Several useful candidate biomarkers including methionine, desaminotyrosine, taurine, N1-acetylspermidine, and 5-hydroxyindoleacetic acid were identified. Some of them were found in previous metabolomics studies in human cerebrospinal fluid or blood samples. This work illustrates the utility of this isotope labeling LC-MS method for biomarker discovery using mouse urine metabolomics.

Complex Relationships between Substrate Sequence and Sensitivity to Alterations in γ-Secretase Processivity Induced by γ-Secretase Modulators

γ-Secretase catalyzes the final cleavage of the amyloid precursor protein (APP), resulting in the production of amyloid-β (Aβ) peptides with different carboxyl termini. Presenilin (PSEN) and amyloid precursor protein (APP) mutations linked to early onset familial Alzheimer’s disease modify the profile of Aβ isoforms generated, by altering both the initial γ-secretase cleavage site and subsequent processivity in a manner that leads to increased levels of the more amyloidogenic Aβ42 and in some circumstances Aβ43. Compounds termed γ-secretase modulators (GSMs) and inverse GSMs (iGSMs) can decrease and increase levels of Aβ42, respectively. As GSMs lower the level of production of pathogenic forms of long Aβ isoforms, they are of great interest as potential Alzheimer’s disease therapeutics. The factors that regulate GSM modulation are not fully understood; however, there is a growing body of evidence that supports the hypothesis that GSM activity is influenced by the amino acid sequence of the γ-secretase substrate. We have evaluated whether mutations near the luminal border of the transmembrane domain (TMD) of APP alter the ability of both acidic, nonsteroidal anti-inflammatory drug-derived carboxylate and nonacidic, phenylimidazole-derived classes of GSMs and iGSMs to modulate γ-secretase cleavage. Our data show that point mutations can dramatically reduce the sensitivity to modulation of cleavage by GSMs but have weaker effects on iGSM activity. These studies support the concept that the effect of GSMs may be substrate selective; for APP, it is dependent on the amino acid sequence of the substrate near the junction of the extracellular domain and luminal segment of the TMD.

Characterization and Molecular Profiling of PSEN1 Familial Alzheimer's Disease iPSC-Derived Neural Progenitors

Presenilin 1 (PSEN1) encodes the catalytic subunit of γ-secretase, and PSEN1 mutations are the most common cause of early onset familial Alzheimer's disease (FAD). In order to elucidate pathways downstream of PSEN1, we characterized neural progenitor cells (NPCs) derived from FAD mutant PSEN1 subjects. Thus, we generated induced pluripotent stem cells (iPSCs) from affected and unaffected individuals from two families carrying PSEN1 mutations. PSEN1 mutant fibroblasts, and NPCs produced greater ratios of Aβ42 to Aβ40 relative to their control counterparts, with the elevated ratio even more apparent in PSEN1 NPCs than in fibroblasts. Molecular profiling identified 14 genes differentially-regulated in PSEN1 NPCs relative to control NPCs. Five of these targets showed differential expression in late onset AD/Intermediate AD pathology brains. Therefore, in our PSEN1 iPSC model, we have reconstituted an essential feature in the molecular pathogenesis of FAD, increased generation of Aβ42/40, and have characterized novel expression changes.

Presenilins are novel substrates for TRAF6-mediated ubiquitination

Mutations in presenilins (PS1 and PS2) have been linked to the pathogenesis of early onset familial Alzheimer's disease. Presenilins function as the catalytic component of the γ-secretase protease complexes responsible for the cleavage of the amyloid precursor protein (APP), subsequent generation of amyloid-β and associated amyloid plaques in Alzheimer's disease. Biochemical and genetic studies have revealed that through interactions with several proteins, the presenilins are functionally involved in a range of cellular processes, including the regulation of intracellular calcium homeostasis. Our group has previously reported an association between presenilins and members of the tumour necrosis factor receptor-associated factor (TRAF) family of proteins. In this study we further investigated the association between TRAF6, an E3 ubiquitin ligase, and the presenilins. Here we show that the presenilin full-length holoproteins are novel substrates of TRAF6-mediated Lysine-63-linked ubiquitination. Interestingly, co-expression of catalytically active TRAF6 with the presenilins leads to decreased turnover of PS1 full-length holoprotein accompanying elevated presenilin protein levels. Similarly, while overexpression of TRAF6 increases presenilin holoprotein levels and ubiquitination in HEK293 cells, expression of catalytically deficient TRAF6 or TRAF6-deficiency leads to a reduction in presenilin protein levels and reduced PS1 ubiquitination. We also demonstrate that TRAF6 induces PS1 gene transcription in a JNK-dependent manner. Notably, we reveal that TRAF6-mediated ubiquitination of presenilin does not affect γ-secretase enzyme activity, but may regulate presenilin function in calcium signalling. Taken together, we propose that presenilins are novel substrates for TRAF6-mediated K63-linked ubiquitination and that ubiquitination of presenilins by TRAF6 increases presenilin holoprotein levels and in conditions in which TRAF6 ubiquitination of presenilins is reduced results in reduction of calcium release from the endoplasmic reticulum.

Involvement of presenilin holoprotein upregulation in calcium dyshomeostasis of Alzheimer's disease

Mutations in presenilins (PS1 and PS2) account for the vast majority of early onset familial Alzheimer's disease cases. Beside the well investigated role of presenilins as the catalytic unit in γ-secretase complex, their involvement in regulation of intracellular calcium homeostasis has recently come into more focus of Alzheimer's disease research. Here we report that the overexpression of PS1 full-length holoprotein forms, in particular familial Alzheimer's disease-causing forms of PS1, result in significantly attenuated calcium release from thapsigargin- and bradykinin-sensitive stores. Interestingly, treatment of HEK293 cells with γ-secretase inhibitors also leads to decreased amount of calcium release from endoplasmic reticulum (ER) accompanying elevated PS1 holoprotein levels. Similarly, the knockdown of PEN-2 which is associated with deficient PS1 endoproteolysis and accumulation of its holoprotein form also leads to decreased ER calcium release. Notably, we detected enhanced PS1 holoprotein levels also in postmortem brains of patients carrying familial Alzheimer's disease PS1 mutations. Taken together, the conditions in which the amount of full length PS1 holoprotein is increased result in reduction of calcium release from ER. Based on these results, we propose that the disturbed ER calcium homeostasis mediated by the elevation of PS1 holoprotein levels may be a contributing factor to the pathogenesis of Alzheimer's disease.

Abnormal cross‐talk between mutant presenilin 1 ( I143T, G384A ) and glycosphingolipid biosynthesis

Mutations in the presenilin 1 (PS1) gene are associated with early onset familial Alzheimer's disease (FAD). In this study, we found that the expression of mutant-PS1 in stable transfectants of SH-SY5Y neuroblastoma cells results in a reduction of the biosynthesis and steady-state levels of glucosylceramide. As an in vivo corroboration of these data, there was a significant reduction of brain glucosylceramide and gangliosides in an animal model of FAD. In mutant-PS1-transfectants (I143T, G384A), immunocytochemistry disclosed a remarkable reduction of glucosylceramide synthase (GlcT-1)-like immunoreactivity in the cells when compared with those of mock- and wild-PS1 transfectants. Immunoprecipitation of GlcT-1 protein from mutant-PS1 transfectants demonstrated a marked reduction in GlcT-1 protein, but there was no reduction in the levels of GlcT-1 mRNA. Both coprecipitation and γ-secretase inhibition experiments suggest that mutant-PS1 seems to form a complex with GlcT-1 protein and to be involved in GlcT-1 degradation, which was never found in other cell types. Thus, mutations in the PS1 gene result in profound glycosphingolipids abnormalities by abnormal molecular interaction with GlcT-1.

Val97Leu mutant presenilin‐1 induces tau hyperphosphorylation and spatial memory deficit in mice and the underlying mechanisms

Although the pathological role of presenilin-1 mutation in early onset familial Alzheimer's disease has been widely studied, few focused on how the presenilin-1 mutations result in memory impairment and tau hyperphosphorylation. In the present study, we expressed human Val97Leu mutant presenilin-1, which is reported in Chinese pedigrees by our group, in transgenic mice and found that the mutant presenilin-1 induced spatial memory deficit and tau hyperphosphorylation at PHF-1, pS199/202, pT231 and pS396 epitopes, but not at pS214 and pS422 epitopes. Pearson analysis showed that the memory deficit was only significantly correlated with tau phosphorylation level at PHF-1, pS199/202, pT231 and pS396 epitopes. Additionally, the hyperphosphorylated tau and tangle-like argentophilic structures were detected at CA3 and CA4, but not CA1, region of hippocampus, and we also found tangle-like structure and wizened degenerative neurons in frontal cortex. We demonstrated the tau hyperphosphorylation at the same epitopes in N2a cells expressing the mutant presenilin-1, which is caused by inhibition of phosphoinositol-3 kinase/Akt and activation of glycogen synthase kinase-3 specifically. Our data demonstrated that human Val97Leu mutant presenilin-1 causes spatial memory deficit in mice and increases tau phosphorylation level in glycogen synthase kinase-3-dependent manner.

Duplication of amyloid precursor protein (APP), but not prion protein (PRNP) gene is a significant cause of early onset dementia in a large UK series.

Amyloid precursor protein gene (APP) duplications have been identified in screens of selected probands with early onset familial Alzheimer's disease (FAD). A causal role for copy number variation (CNV) in the prion protein gene (PRNP) in prion dementias is not known. We aimed to determine the prevalence of copy number variation in APP and PRNP in a large referral series, test a screening method for detection of the same, and expand knowledge of clinical phenotype. We used a 3-tiered screening assay for APP and PRNP duplication (exonic real-time quantitative polymerase chain reaction [exon-qPCR], fluorescent microsatellite quantitative PCR [fm-q-PCR], and Illumina array [Illumina Inc., San Diego, CA, USA]) for analysis of a heterogeneous referral series comprising 1531 probands. Five of 1531 probands screened showed APP duplication, a similar prevalence to APP missense mutation. Real-time quantitative PCR and fluorescent microsatellite quantitative PCR were similar individually but are theoretically complementary; we used Illumina arrays as our reference assay. Two of 5 probands were from an autosomal dominant early onset Alzheimer's disease (familial Alzheimer's disease) pedigree. One extensive, noncontiguous duplication on chromosome 21 was consistent with an unbalanced translocation not including the Down's syndrome critical region. Seizures were prominent in the other typical APP duplications. A range of imaging, neuropsychological, cerebrospinal fluid, and pathological findings are reported that extend the known phenotype. APP but not PRNP duplication is a significant cause of early onset dementia in the UK. The recognized phenotype may be expanded to include the possibility of early seizures and apparently sporadic disease which, in part, may be due to different mutational mechanisms. The pros and cons of our screening method are discussed.