Dataset Information

CRISPR Transcriptional Activation Analysis Unmasks an Occult ?-Secretase Processivity Defect in Familial Alzheimer's Disease Skin Fibroblasts.

ABSTRACT: Mutations in presenilin (PSEN) 1 and 2, which encode components of the ?-secretase (GS) complex, cause familial Alzheimer's disease (FAD). It is hypothesized that altered GS-mediated processing of the amyloid precursor protein (APP) to the A?42 fragment, which is accumulated in diseased brain, may be pathogenic. Here, we describe an in vitro model system that enables the facile analysis of neuronal disease mechanisms in non-neuronal patient cells using CRISPR gene activation of endogenous disease-relevant genes. In FAD patient-derived fibroblast cultures, CRISPR activation of APP or BACE unmasked an occult processivity defect in downstream GS-mediated carboxypeptidase cleavage of APP, ultimately leading to higher A?42 levels. These data suggest that, selectively in neurons, relatively high levels of BACE1 activity lead to substrate pressure on FAD-mutant GS complexes, promoting CNS A?42 accumulation. Our results introduce an additional platform for analysis of neurological disease.

SUBMITTER: Inoue K

PROVIDER: S-EPMC5704930 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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CRISPR Transcriptional Activation Analysis Unmasks an Occult γ-Secretase Processivity Defect in Familial Alzheimer's Disease Skin Fibroblasts.

Inoue Keiichi K Oliveira Luis M A LMA Abeliovich Asa A

Cell reports 20171101 7

Mutations in presenilin (PSEN) 1 and 2, which encode components of the γ-secretase (GS) complex, cause familial Alzheimer's disease (FAD). It is hypothesized that altered GS-mediated processing of the amyloid precursor protein (APP) to the Aβ42 fragment, which is accumulated in diseased brain, may be pathogenic. Here, we describe an in vitro model system that enables the facile analysis of neuronal disease mechanisms in non-neuronal patient cells using CRISPR gene activation of endogenous diseas ...[more]

PMID: 29141208

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Project description:The 19-transmembrane multisubunit ?-secretase complex generates the amyloid ?-peptide (A?) of Alzheimer's disease (AD) by intramembrane proteolysis of the ?-amyloid precursor protein (APP). Despite substantial advances in elucidating how this protein complex functions, the effect of the local membrane lipid microenvironment on ?-secretase cleavage of substrates is still poorly understood. Using detergent-free proteoliposomes to reconstitute purified human ?-secretase, we examined the effects of fatty acyl (FA) chain length, saturation and double-bond isomerization, and membrane lipid polar headgroups on ?-secretase function. We analyzed ?-secretase activity and processivity [i.e., sequential cleavages in the APP transmembrane domain that convert longer A? species (e.g., A?(46)) into shorter ones (e.g., A?(40))] by quantifying the APP intracellular domain (AICD) and various A? peptides, including via a bicine/urea gel system that detects multiple A? lengths. These assays revealed several trends. (1) Switching from a cis to a trans isomer of a monounsaturated FA chain in phosphatidylcholine (PC) increased ?-activity, did not affect A?(42):A?(40) ratios, but decreased the ratio of long (?42) versus short (?41) A? peptides. (2) Increasing the FA carbon chain length (14, 16, 18, and 20) increased ?-activity, reduced longer A? species, and reduced the A?(42):A?(40) ratio. (3) Shifting the position of the double bond in 18:1(?9-cis) PC to the ?6 position substantially reduced activity. (4) Gangliosides increased ?-activity but decreased processivity, thus elevating the A?(42):A?(40) ratio. (5) Phosphatidylserine decreased ?-activity but increased processivity. (6) Phosphatidylinositol strongly inhibited ?-activity. Overall, our results show that subtle changes in membrane lipid composition can greatly influence ?-secretase activity and processivity, suggesting that relatively small changes in lipid membrane composition may affect the risk of AD at least as much as presenilin or APP mutations do.

Dataset Information

CRISPR Transcriptional Activation Analysis Unmasks an Occult ?-Secretase Processivity Defect in Familial Alzheimer's Disease Skin Fibroblasts.

Publications

CRISPR Transcriptional Activation Analysis Unmasks an Occult γ-Secretase Processivity Defect in Familial Alzheimer's Disease Skin Fibroblasts.

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