Detection of Mitochondria-Associated Endoplasmic Reticulum Membrane Tightness Using Inducible FRET Biosensors

Summary

We describe a method for measuring FRET values of inducible FRET biosensors targeting the ER (CFP-tagged) or mitochondria (YFP-tagged) in vitro to assess mitochondria-associated ER membranes (MAMs) tightness, which influences Aβ generation, and this method can be used for initial screening of synthetic or natural MAM-modulating agents as potential AD therapeutics.

Abstract

Multiple studies have demonstrated that the number, length, and gap width (thickness) of ER-mitochondria contacts, or mitochondria-associated membranes (MAMs), influence their biological roles. Our previous work showed that the stabilization of tight MAMs, characterized by a gap width of approximately 7 nm, leads to an increase in amyloid β (Aβ) levels, whereas the presence of loose MAMs, with a gap width of around 40 nm, reduces Aβ production in a three-dimensional (3D) neural model of Alzheimer's disease (AD). To investigate the effects of MAMs with different gap widths, ER- and mitochondria-targeted FRET (Förster Resonance Energy Transfer) biosensors-ER-CFP (cyan fluorescent protein) and Mito-YFP (yellow fluorescent protein), respectively-were developed to quantify tight MAMs (<10 nm gap width) in contrast to loose or non-MAMs. FRET occurs when the donor fluorophore (CFP) and the acceptor fluorophore (YFP) are within 10 nm of each other, making this system suitable for assessing MAM proximity. This protocol outlines the use of spectral ratiometric FRET to measure the extent of tight MAM formation.

Introduction

Mitochondria-associated endoplasmic reticulum contacts (MERCs), which are isolated biochemically as mitochondria-associated ER membranes (MAMs), are implicated in several neurodegenerative disorders, including Alzheimer's disease (AD)¹. The recently proposed "MAM hypothesis" suggests that MAMs play a central role in the generation of amyloid β (Aβ), thereby initiating the pathogenic cascade of AD. This cascade includes the formation of neurofibrillary tangles (NFTs), calcium dyshomeostasis, and neuroinflammation². MAMs consist of cholesterol-rich, lipid raft-like microdomains in the endoplasmic reti....

Protocol

The reagents and the equipment used in this study are listed in the Table of Materials.

1.Transfection

1. Transfect N2A_APP cells with MAM-Tav2A expression plasmid (14-18 h).
   1. Before beginning, plate 1 × 10⁶ N2A_APPcellsper well of 6-well plates containing glass coverslips and maintain the cells in the growth media for 12-16 h.
   2. Transfect 1-3 μg MAM-Tav2A plasmid (Supplementary File 1) using Lipofectamine transfection reagent following the manufacturer's protocol.
   3. After 12-16 h of transfect....

Results

Ratiometric FRET analysis of FACS-enriched N2A_APP cells expressing MAM-Tav2A demonstrated a dose-dependent increase in Aβ40 levels following rapamycin treatment⁸. Rapamycin acts by inhibiting the mammalian target of rapamycin (mTOR) signaling pathway, which in turn downregulates TREM2 (Triggering Receptor Expressed on Myeloid Cells 2) expression in microglia, leading to reduced Aβ plaque clearance in 5XFAD mouse models of Alzheimer's disease

Discussion

The modulation of MAMs is an emerging area of research with potential implications for various disorders, including cancer, metabolic disorders, and neurodegenerative diseases¹³. Many pharmacological agents, including the dynein-antagonist LDC-3/Dynarrestin, antidiabetic drug metformin, and anti-cancer agent sulforaphane, disrupt MAMs and are currently under pre-clinical and clinical trials for cancer and metabolic disorders. These pharmacological agents modulate the ER-mitochondria contacts or MA.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BSA	Fisher Scientific	501781532
Confocal microscope	Olympus FV3000RS (Tokyo, Japan)	N/A
DMEM with GlutaMAX supplement	Gibco/Thermo Fisher Scientific	10564011
EDTA	Life Technologies	41116134
Falcon 6 Well Plates	VWR International	41122107
GAPDH Polyclonal Antibody	Thermo Fisher Scientific	PA1-988
Gelatin	VWR International	9000-70-8
Geneticin (G418 Sulfate)	Gibco/Thermo Fisher Scientific	10131035
GFP monoclonal Antibody	Invitrogen	MA5-15256
Graphpad Prism N/A	Prism 9, version 9.5.0	N/A
Heparin	Sigma-Aldrich	H0200000
ImageJ Software	ImageJ 1.53a	N/A
Inverted confocal microscope	Nikon (C2 Eclipse Ti2)	N/A
Lipofectamine2000	Invitrogen	11668027
MS Excel	Microsoft Excel, version 2302	N/A
NaCl	Fisher Scientific	7647145
NuPAGE 4–12% Bis-Tris gel	Invitrogen	NP0321BOX
Penicillin/Streptomycin/Amphotericin B	Lonza	17-745E
Photoshop	Adobe Photoshop CC 20.0.10	N/A
Tris-HCL, pH 7.6	Boston BioProducts	42000000
Triton X-100	Sigma-Aldrich	T8787
Trypsin-EDTA	Gibco/Thermo Fisher Scientific	25200056
Tween 20	Fisher Scientific	501657287