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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.
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.
Emerging evidence suggests that the specialized Mitochondria-associated Endoplasmic Reticulum Contacts (MERCs), biochemically harvested as Mitochondria-Associated ER Membranes, often referred to as MAMs1,2 play a role in several neurodegenerative diseases, including AD3,4. 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 MAMs5,6,7. The recently coined MAM hypothesis posits that the increase of MAMs leads to enhanced Aβ production and the pathogenic cascade of AD, including neurofibrillary tangle (NFT) formation, calcium dyshomeostasis, and neuroinflammation3,8. About 5%-20% of mitochondria make physical contact with the ER to form MAMs9. The gap width of MAMs is determined by the smooth and rough ER (sER and rER, respectively). The variable gap width between sER-mitochondria (10-50 nm) and rER-mitochondria (50-80 nm) suggests that the gap width of MAMs has a long spectrum that ranges between tight (~10 nm) to loose (~80 nm)10,11,12,13. MAM gap width determines MAM functions, such as calcium homeostasis and lipid transport1,14. A recent report has shown that the MAMs formed between tightly (~10 nm) connected ER and mitochondria, called full MAMs, are apoptotic. In contrast, MAMs formed between loosely connected (~25 nm) ER and mitochondria, termed defective or medium MAMs, are anti-apoptotic14,15,16. Stabilization of MAMs with a gap width of 6 nm ± 1 nm increased Aβ generation from a novel 3-dimensional (3D) neural culture model of AD. In contrast, the stabilization of MAMs with a gap width 24 nm ± 3 nm has no effect on Aβ generation17. This finding suggests for the first time that regulating the degree of MAM stabilization, but not destabilizing MAMs, is the key to regulating Aβ generation. An attempt to completely destabilize MAMs may have unwanted consequences because MAMs maintain several cellular events critical for cell survival12.
The modulation of MAMs is an emerging area of research with potential implications for various disorders, including cancer, metabolic disorders, and neurodegenerative diseases18. Despite the availability of many MAM modulators, no major attempt has so far been taken to test their abilities to destabilize MAMs and lower AD pathology, primarily because the structural diversities of MAMs make them a highly complex system to target for drug discovery. But, the newly developed structural systems pharmacology, which considers the specific properties of the drug targets and their environment18,19 should overcome the difficulties and develop highly potent drugs targeting MAMs or MAM-associated proteins in AD. However, the search for an effective modulator of MAM stabilization requires methods to quantify the degree of MAM stabilization precisely. Traditional techniques like electron microscopy (EM) or super-resolution microscopy have limitations in determining MAM stabilization. Overcoming these challenges would likely require the development of novel, more dynamic imaging techniques or biochemical assays that can provide quantitative measures of MAM stabilization in living cells. Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) of primary neurons revealed that the ER tends to form a network around mitochondria likely to limit mitochondrial motility20,21. The disruption of mitochondrial transport systems, either retrograde, anterograde, or both, had a profound impact on synaptic and neuronal function22. Thus, the novel live-cell imaging and kymography-based analysis of axonal velocity of ER-bound mitochondria described here as a metric to quantitatively measure MAM stabilization will facilitate the identification of MAM modulator(s) that can switch the MAM stabilization threshold to one that maintains or possibly lowers as opposed to increases Aβ generation.
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 (APPSwe/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 available. ReN GA lines were obtained from Dr. Doo Y. Kim, Associate Professor, Massachusetts General Hospital (MGH)23,24,25.
Expression plasmids: AKP1 (34-63) and ER-targeting sequence of Ubc 6 (283-303) proteins linked directly with RFP (Mito-RFP-ER denoted as MAM 1X) or contain a 9 amino acid linker (Mito-9X-RFP-ER denoted as MAM 9X) designed to stabilize MAMs of 6 nm ± 1 nm or 24 nm + 3 nm gap widths, respectively15,26 (Figure 1A).
1. Electroporation
2. Live cell imaging
3. Post-processing (7 days)
NOTE: To analyze transport and generate kymographs, Fiji ImageJ macros were utilized. Vesicles that moved less than 0.1 mm/s were categorized as stationary. The frequency of particle movement was calculated by dividing the number of particles moving in a given direction (anterograde, retrograde) or not moving (stationary) by the total number of particles analyzed in the kymograph. The time each vesicle spent pausing or moving was calculated by averaging the percentage of time spent in each condition for all vesicles in each neuron analyzed. The frequency distribution for velocity and run length was calculated using only moving vesicles for each experimental condition. The analysis was performed on 100 mm axonal tracts for 3 min.
Live-cell imaging and kymographic analyses were performed to measure the motility of free mitochondria labeled with Mito-RFP or ER-bound mitochondria of tight (6 nm ± 1 nm) or loose (24 nm ± 3 nm) contact widths stabilized by MAM 1X or MAM 9X, respectively, in the longest neuronal process of each ReN GA (AD) or ReN (naïve) neuron which is at least 500 nm long, considering this as an axon (Figure 1 and Figure 2). Frequencies of movements (overall, ...
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...
We thank Dr. György Hajnóczky, Professor, Thomas Jefferson University, Philadelphia for generously providing us with expression plasmids encoding RFP-Mito, MAM 1X, MAM 9X, and MAM 18X. A special thanks to Dr. Lai Ding, Senior Imaging Scientist, Brigham and Women’s Hospital for helping us write the code for generating, tracking and measuring the kymograph data. This study was supported by the Cure Alzheimer's Fund to RB and NIH grant 5R01NS045860-20 to RET.
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 |
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