Modulation of γ-Secretase Reduces β-Amyloid Deposition in a Transgenic Mouse Model of Alzheimer's Disease

SummaryAlzheimer's disease (AD) is characterized pathologically by the abundance of senile plaques and neurofibrillary tangles in the brain. We synthesized over 1200 novel gamma-secretase modulator (GSM) compounds that reduced Aβ42 levels without inhibiting epsilon-site cleavage of APP and Notch, the generation of the APP and Notch intracellular domains, respectively. These compounds also reduced Aβ40 levels while concomitantly elevating levels of Aβ38 and Aβ37. Immobilization of a potent GSM onto an agarose matrix quantitatively recovered Pen-2 and to a lesser degree PS-1 NTFs from cellular extracts. Moreover, oral administration (once daily) of another potent GSM to Tg 2576 transgenic AD mice displayed dose-responsive lowering of plasma and brain Aβ42; chronic daily administration led to significant reductions in both diffuse and neuritic plaques. These effects were observed in the absence of Notch-related changes (e.g., intestinal proliferation of goblet cells), which are commonly associated with repeated exposure to functional gamma-secretase inhibitors (GSIs)

Increased Expression of PS1 Is Sufficient to Elevate the Level and Activity of γ-Secretase In Vivo

Increase in the generation and deposition of amyloid-β (Aβ) plays a central role in the development of Alzheimer's Disease (AD). Elevation of the activity of γ-secretase, a key enzyme required for the generation for Aβ, can thus be a potential risk factor in AD. However, it is not known whether γ-secretase can be upregulated in vivo. While in vitro studies showed that expression of all four components of γ-secretase (Nicastrin, Presenilin, Pen-2 and Aph-1) are required for upregulation of γ-secretase, it remains to be established as to whether this is true in vivo. To investigate whether overexpressing a single component of the γ-secretase complex is sufficient to elevate its level and activity in the brain, we analyzed transgenic mice expressing either wild type or familial AD (fAD) associated mutant PS1. In contrast to cell culture studies, overexpression of either wild type or mutant PS1 is sufficient to increase levels of Nicastrin and Pen-2, and elevate the level of active γ-secretase complex, enzymatic activity of γ-secretase and the deposition of Aβ in brains of mice. Importantly, γ-secretase comprised of mutant PS1 is less active than that of wild type PS1-containing γ-secretase; however, γ-secretase comprised of mutant PS1 cleaves at the Aβ42 site of APP-CTFs more efficiently than at the Aβ40 site, resulting in greater accumulation of Aβ deposits in the brain. Our data suggest that whereas fAD-linked PS1 mutants cause early onset disease, upregulation of PS1/γ-secretase activity may be a risk factor for late onset sporadic AD

Imaging and Functional Analysis of γ‑Secretase and Substrate in a Proteolipobead System with an Activity-Based Probe

Investigation of intramembranal protease catalysis demands the generation of intact biomembrane assemblies with structural integrity and lateral mobility. Here, we report the development of a microsphere supported-biomembrane platform enabling characterization of γ-secretase and substrate within proteolipobead assemblies via microscopy and flow cytometry. The active enzyme loading levels were tracked using an activity-based probe, with the biomembranes delineated by carbocyanine lipid reporters. Proteolipobeads formed from HeLa proteoliposomes gave rise to homogeneous distributions of active γ-secretase within supported biomembranes with native-like fluidity. The substrate loading into supported biomembranes was detergent-dependent, as evidenced by even colocalization of substrate and lipid tracers in confocal 3D imaging of individual proteolipobeads. Moreover, the loading level was tunable with bulk substrate concentration. γ-Secretase substrate cleavage and its inhibition within γ-secretase proteolipobeads were observed. This platform offers a means to visualize enzyme and substrate loading, activity, and inhibition in a controllable biomembrane microenvironment

Increased Expression of <i>PS1</i> Is Sufficient to Elevate the Level and Activity of γ-Secretase <i>In Vivo</i>

Increase in the generation and deposition of amyloid-β (Aβ) plays a central role in the development of Alzheimer's Disease (AD). Elevation of the activity of γ-secretase, a key enzyme required for the generation for Aβ, can thus be a potential risk factor in AD. However, it is not known whether γ-secretase can be upregulated in vivo. While in vitro studies showed that expression of all four components of γ-secretase (Nicastrin, Presenilin, Pen-2 and Aph-1) are required for upregulation of γ-secretase, it remains to be established as to whether this is true in vivo. To investigate whether overexpressing a single component of the γ-secretase complex is sufficient to elevate its level and activity in the brain, we analyzed transgenic mice expressing either wild type or familial AD (fAD) associated mutant PS1. In contrast to cell culture studies, overexpression of either wild type or mutant PS1 is sufficient to increase levels of Nicastrin and Pen-2, and elevate the level of active γ-secretase complex, enzymatic activity of γ-secretase and the deposition of Aβ in brains of mice. Importantly, γ-secretase comprised of mutant PS1 is less active than that of wild type PS1-containing γ-secretase; however, γ-secretase comprised of mutant PS1 cleaves at the Aβ42 site of APP-CTFs more efficiently than at the Aβ40 site, resulting in greater accumulation of Aβ deposits in the brain. Our data suggest that whereas fAD-linked PS1 mutants cause early onset disease, upregulation of PS1/γ-secretase activity may be a risk factor for late onset sporadic AD.</p

Dendritic Spine Anomalies and PTEN Alterations in a Mouse Model of VPA-induced Autism Spectrum Disorder

Mounting evidence suggests that the etiology of autism spectrum disorders (ASDs) is profoundly influenced by exposure to environmental factors, although the precise molecular and cellular links remain ill-defined. In this study, we examined how exposure to valproic acid (VPA) during pregnancy is associated with an increased incidence of Asp. A mouse model was established by injecting VPA at embryonic day 13, and its behavioral phenotypes including impaired social interaction, increased repetitive behaviors and decreased nociception were observed at postnatal days 21-42. VPA-treated mice showed dysregulation of synaptic structure in cortical neurons, including a reduced proportion of filopodium-type and stubby spines and increased proportions of thin and mushroom-type spines, along with a decreased spine head size. We also found that VPA-treatment led to decreased expression of phosphate and tensin homolog (PTEN) and increased levels of p-AKT protein in the hippocampus and cortex. Our data suggest that there is a correlation between VPA exposure and dysregulation of PTEN with ASD-like behavioral and neuroanatomical changes, and this may be a potential mechanism of VPA-induced ASD. (C) 2017 The Authors. Published by Elsevier Ltd.OAIID:RECH_ACHV_DSTSH_NO:T201713479RECH_ACHV_FG:RR00200001ADJUST_YN:EMP_ID:A079472CITE_RATE:4.897DEPT_NM:의과학과EMAIL:[email protected]_YN:YY

Increase of γ-secretase in primary neurons and non-neuronal cells derived from <i>PS1</i> mice.

<p>(A) Protein extracts (20 µg each) from primary neuronal and non-neuronal cultures derived from brains of <i>PS1</i> and non-transgenic control mice were immunoblotted with anti-sera specific to Nct, PS1-CTF, β-Tubulin III or Actin. (B) Quantification of Nct signals in protein blot of primary neuronal and non-neuronal cell extracts by Image J program. The signal density was normalized using Actin signals derived from the same blot.</p

Increase in generation and deposition Aβ in the brains of <i>APP^swe;PS1</i> mice.

<p>(A) ELISA analysis of Aβ40 peptides in the protein extracts of brains of <i>PS1</i> and non-transgenic control mice. The data were average +/− SEM from 5 mice for each genotype. (B) Sagittal brain sections (10 µm) of hippocampus area of 22-months old <i>APP^swe;PS1</i> and <i>APP^swe</i> female mice. The Aβ plaques were visualized by immunostaining with antibodies specific to ubiquitin and Aβ peptides (6E10). (C) Quantitative analysis of the levels of Aβ aggregation in the brains of <i>APP^swe;PS1</i> and <i>APP^swe</i> mice at 22 months of age by filter trap assay. (D) Quantification of the signals of Aβ aggregations in the filter trap assay using Image J program. (E) Analysis of Aβ deposition using unbiased stereology in the hippocampus of 22-months old <i>APP^swe;PS1</i> (n = 6) and <i>APP^swe</i> (n = 10) female mice.</p

Increase of γ-secretase activity in brains of <i>PS1</i> mice.

<p>(A) Protein extracts (20 µg each) from brains of <i>PS1</i> transgenic (lanes 1–3) and non-transgenic mice (lanes 4–6) were immunoblotted with anti-sera specific to Nct, PS1-CTF, PEN2, PS2-CTF, or Actin. (B) Quantification of signals of Nct, Pen-2, and PS2-CTF in protein blots by Image J program. The signal density was normalized using Actin signals derived from the same blot. (C) In vitro γ-secretase assay of brain extracts of wild type, <i>PS1</i> transgenic, and <i>Nct^+/−</i> mice. The data were average from 4 samples. (D) The CHAPSO solubilized membrane proteins from wild type and <i>PS1</i> transgenic mice were incubated with Compound 4 followed by precipitation with streptavidin-coupled beads. Solubilized membranes before ligand addition (Input: 10% of total), captured fraction (Capture<i>:</i> 20% of total) of the affinity ligand and the corresponding supernatants after capture (Unbound: 7.5% of total) were analyzed by Western blot using antisera specific to Nct, PS1-CTF, PS1-NTF, or BACE1. (E) Quantification of signals of Nct levels from in protein blots by Image J program. The signal density was normalized using BACE1 signals derived from the same blot.</p

Overexpression of FAD linked <i>PS1</i> mutant<i>s</i> increases the level of γ-secretase in the brain.

<p>(A) Protein extracts (20 µg each) from brains of <i>PS1ΔE9</i> (lanes 1, 2), <i>PS1-A246E</i> (lanes 3, 4), <i>PS1wt</i> (lanes 5, 6) and non-transgenic mice (lanes 7–9) were immunoblotted with anti-sera specific to Nct, PS1-NTF, PS1-CTF, PEN2, SOD-1 or Actin. (B) Quantification of signals of Nct, PEN2 and SOD-1 in protein blots of mutant and wild type <i>PS1</i> and non-transgenic control mice by Image J program. The signal density was normalized using Actin signal derived from the same blot. (C) <i>In vitro</i> γ-secretase assay of Aβ40 cleavage in brain extracts of <i>PS1ΔE9</i>, <i>PS1-A246E</i>, <i>PS1^wt</i> and non-transgenic control mice. The data represent averages +/- SEM from 4 mice for each genotype. (D) <i>In vitro</i> γ-secretase assay of Aβ42 cleavage in brain extracts of <i>PS1ΔE9</i>, <i>PS1-A246E</i>, <i>PS1^wt</i> and non-transgenic control mice. The data represent averages +/− SEM from 4 mice for each genotype. (E) The ratio of Aβ40 and Aβ42 cleavage activity in brain extracts of <i>PS1ΔE9</i>, <i>PS1-A246E</i>, <i>PS1^wt</i> and non-transgenic control mice. (F) Sagittal brain sections (10 µm) of 22-months old <i>APP^swe;PS1ΔE9</i> female mice. The Aβ plaques were visualized by immunostaining with antibodies specific to ubiquitin and Aβ peptides (6E10). (G) Analysis of Aβ deposition using unbiased stereology in brain sections of 22-months old <i>APP^swe;PS1ΔE9</i> (n = 5), <i>APP^swe;PS1</i> (n = 6), and <i>APP^swe</i> (n = 10) female mice. The data represent averages +/− SEM from each genotype.</p