Quantitative Analysis of Mitochondria-Associated Endoplasmic Reticulum Membrane (MAM) Stabilization in a Neural Model of Alzheimer's Disease (AD)

Summary

Here, we describe measuring the axonal transport rate of constitutive stabilizers of mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) by increasing or maintaining neurotoxic β-amyloid (Aβ) generation from Alzheimer's disease (AD) neurons in real-time to serve as a direct and quantitative metric to measure MAM stabilization and aid the development of AD therapeutics.

Abstract

A method to quantitate the stabilization of Mitochondria-Associated endoplasmic reticulum Membranes (MAMs) in a 3-dimensional (3D) neural model of Alzheimer's disease (AD) is presented here. To begin, fresh human neuro progenitor ReN cells expressing β-amyloid precursor protein (APP) containing familial Alzheimer's disease (FAD) or naïve ReN cells are grown in thin (1:100) Matrigel-coated tissue culture plates. After the cells reach confluency, these are electroporated with expression plasmids encoding red fluorescence protein (RFP)-conjugated mitochondria-binding sequence of AKAP1(34-63) (Mito-RFP) that detects mitochondria or constitutive MAM stabilizers MAM 1X or MAM 9X that stabilize tight (6 nm ± 1 nm gap width) or loose (24 nm ± 3 nm gap width) MAMs, respectively. After 16-24 h, the cells are harvested and enriched by a fluorescence-activated cell sorter (FACS). An equal number of FACS-enriched cells are seeded in the 3-dimensional matrix (1:1 Matrigel) and allowed to differentiate into mature neurons for 10 days. Live cell images of the 10-day differentiated cells expressing the RFP-conjugated MAM stabilizers are captured under a fluorescent microscope equipped with a live-cell imaging culture chamber maintaining the CO₂ (5%), temperature (37 °C), and humidity (~90%). Toward this end, we performed live-cell imaging and kymographic analyses to measure the motility of free mitochondria labeled with Mito-RFP or ER-bound mitochondria of tight or loose gap widths stabilized by MAM 1X or MAM 9X, respectively, in the most extended neuronal process of each ReN GA neuron which is at least 500 nm long, considering these as axons.

Introduction

Emerging evidence suggests that the specialized Mitochondria-associated Endoplasmic Reticulum Contacts (MERCs), biochemically harvested as Mitochondria-Associated ER Membranes, often referred to as MAMs^1,2 play a role in several neurodegenerative diseases, including AD^3,4. These MAMs are composed of cholesterol-rich lipid raft-like microdomains in the ER and the outer membrane of mitochondria tethered by a series of proteins that create structural and functional diversities among the MAMs^5,....

Protocol

AD neural culture models: This study used neurons derived from human neural progenitor ReN cells [naïve ReN (Millipore)] or ReN cells expressing familial AD (fAD) mutations in the amyloid precursor protein (APP) gene (APP^Swe/Lon), ReN GA cells. ReN-GA three-dimensional (3D) culture system recapitulates AD pathology, namely Aβ oligomer- driven neurofibrillary tangles (NFTs) ^23,24. Naïve ReN cells are commercially.......

Discussion

Inhibition of sigma-1 receptor (S1R) downregulated MAM stabilization in the neuronal processes and dramatically reduced (~90%) Aβ generation from axons but not from soma of a three-dimensional (3D) culture system of human neural progenitor (ReN) cells expressing familial AD [FAD] mutations in the amyloid precursor protein [APP] gene (ReN GA)^23,24,25,27. RFP-labeled constitutive MA.......

Materials

Name	Company	Catalog Number	Comments
6 Well Glass Bottom Plate	Cellvis	P06-1.5H-N
B-27 Supplement (50X), serum free	Gibco/Thermo Fisher Scientific	17504044
bFGF	R&D System	233-FB
BSA	Fisher Scientific	501781532
Countess Cell Counting Chamber Slides	Invitrogen	C10283
DMEM/F12 with L-glutamine	Gibco/Thermo Fisher Scientific	11320-033
EDTA	Life Technologies	41116134
EGF	Sigma-Aldrich	92090408
Falcon 6 Well Plates	VWR International	41122107
GAPDH Polyclonal Antibody	Thermo Fisher Scientific	PA1-988
Gelatin	VWR International	9000-70-8
Graphpad Prism N/A	Prism 9, version 9.5.0	N/A
Heparin	Sigma-Aldrich	H0200000
ImageJ Software	ImageJ 1.53a	N/A
Matrigel Basement Membrane Matrix	Corning	356234
mCherry Polyclonal Antibody	Invitrogen	PA5-34974
MS Excel	Microsoft Excel, version 2302	N/A
Multi-array electrochemiluminescence assay kit	Meso Scale Diagnostics (MSD)	K15200E-2	V-PLEX Aβ Peptide Panel 1 (6E10) kit
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
Rat Neuron Nucleofector Kit	Lonza	VPG-1003
StemPro Accutase	Gibco	A1110501
Tris-HCL, pH 7.6	Boston BioProducts	42000000
Triton X-100	Sigma-Aldrich	T8787
Tween 20	Fisher Scientific	501657287