Method Article

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

DOI:

10.3791/67914

⸱

August 7th, 2026

 ,  , 
1Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Henry and Allison McCance Center for Brain Health, Department of Neurology, Massachusetts General Hospital, Harvard Medical School,

In This Article

Summary

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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 reticulum and the outer mitochondrial membrane (OMM), linked by specific tethering proteins that contribute to both their structural and functional diversity3. The stabilization of ER-mitochondria contacts influences various cellular processes, including ER calcium signaling and apoptosis4,5. It is estimated that 5%-20% of mitochondria form physical contacts with the ER to establish MAMs6. Several studies have indicated that the number, length, and gap width (thickness) between the ER and mitochondria at MAM sites are important determinants of their functional roles. Cryo-electron microscopy and in vivo analyses of mouse liver have demonstrated that the metabolic state of hepatocytes modulates both the thickness (ranging from 14 nm to 20 nm) and the length (ranging from 145 nm to 270 nm) of MAMs7.

MAMs are also emerging as potential therapeutic targets for various disorders, including cancer and metabolic disorders. MAM thickness or gap width regulates cell survival. The constitutive MAM stabilizers, MAM 1X and MAM 9X, designed to stabilize tight (6 nm ± 1 nm gap width) and loose MAMs (24 nm ± 3 nm gap width), respectively, demonstrated that the stabilization of tight MAMs dramatically increased Aβ generation in a novel 3D neural culture model of AD. In contrast, the stabilization of the loose MAMs had no effect8. Thus, converting the stabilization of tight MAMs to loose MAMs may be beneficial to lower amyloid pathology. The structural and functional complexities and the lack of precise quantitative analysis of the degree of MAM stabilization make targeting MAMs for drug discovery challenging. A novel live-cell imaging and kymography-based analysis of the axonal velocity of the ER-bound mitochondria demonstrated that the axonal velocity of mitochondria may be used as a metric to quantitatively measure MAM stabilization because tighter contact with the ER significantly reduced the mitochondrial axonal velocity compared to free mitochondria8,9. Using an inducible stabilizer of MAMs in hAPP-expressing Neuro 2A (N2AAPP) cells8, we found that tightening MAM gap widths increases Aβ generation in a dose-dependent manner8. The inducible MAM stabilizers are expression plasmids encoding the CFP (cyan fluorescence protein)-epitope-tagged ER-targeting and YFP (yellow fluorescence protein)-epitope-tagged mitochondria-targeting biosensors fused to the rapamycin-inducible FK506 rapamycin binding (FRB) or 12-kDa FK506-binding protein (FKBP) domains. The biosensors were denoted as ER-CFP and Mito-YFP, respectively. The CFP is a donor fluorophore that transfers energy to the acceptor fluorophore YFP when they are separated by >10 nm. Cells (N2AAPP) expressing the inducible biosensors increased Aβ generation upon rapamycin treatment8, suggesting that increasing tight MAMs increased Aβ generation. These methods will be beneficial for screening small molecule modulators of MAMs and their effective concentration required to achieve the stabilization of an optimal level of tight MAMs that will convert pathogenic ("Aβ increasing") to therapeutic ("Aβ lowering") MAMs without completely destabilizing the ER and mitochondria contacts, important for cell survival.

Experimentalmodel
This study used Neuro2A(N2A)cells constitutively expressing APP751 (N2AAPP). These cells release detectable levels of Aβ into the conditioned media (CM) compared to naïve N2A cells. N2AAPP cells were maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamate supplemented with 200 µg/mL G418, as described previously10.

Expression plasmid
An expression plasmid was generated by fusing the YFP (yellow fluorescent protein)-tagged mitochondrial targeting sequence of the mammalian A-kinase anchor protein 1 [AKAP1(34-63)] and CFP (cyan fluorescent protein)-tagged ER-targeting sequence of phosphatidylinositol-3-phosphatase Sac1 phosphatase [Sac1(521-587)] with a self-cleaving Tav2A sequence encoding the EGRGSLLTCGDVEENPGP peptide sequence, denoted as pYFP-AKAP1-Tav2A-Sac1-CFP8. The Tav2A-fused expression plasmid is also flanked with nucleotide sequences encoding the FK506 rapamycin binding (FRB) domain [EMWHEGLEEASRLYFGERNVKGMFEVLEPL HAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNV (K2095P)DLTQAWDLYYHVFRRISKQ] or the 12-kDa FK506-binding protein (FKBP) domain (MGVEKQVIRPGNGPKPAPGQTVTVHCTGFGKD GDLSQKFWSTKDEGQKPFSFQIGKGAVIKGWDEGVIGMQ IGEVARLRCSSDYAYGAGGFPAWGIQPNSVLDFEIEVLSVQ). The fusion plasmid FRB-YFP-AKAP1(34-63)-Tav2A-Sac1(521-587)-CFP-FKBP, denoted here as MAM-Tav2A, was generated to obtain equimolar expression levels of FRB-YFP-AKAP1(34-63) and Sac1(521-587)-CFP-FKBP forthe ratiometric FRET analysis. The Sac1(521-587)-CFP-FKBP, denoted as ER-CFP, is the donor fluorophore, whereas Mito-YFP-FRB, denoted as, Mito-YFP, is the acceptor fluorophore that generates FRET when separated by ~10 nm8. Thus, the FRET-based biosensors detect tight MAMs (6-10 nm, gap width). The FRB and FKBP were introduced to induce the tightening of MAM gap widths upon the addition of rapamycin in a dose-dependent manner that could be quantified by measuring the increased FRET signal8.

Protocol

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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

1.Transfection

  1. Transfect N2AAPP cells with MAM-Tav2A expression plasmid (14-18 h).
    1. Before beginning, plate 1 × 106 N2AAPPcellsper 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 transfection, perform drug treatment.
      NOTE: Always have cells transfected with an expression plasmid encoding only the donor (ER-CFP) or the acceptor (Mito-YFP) to be used as the FRET background.

2. Drug treatment

  1. Treat the MAM-Tav2A-expressing N2AAPP cells with Rapamycin (Rapa) or Rapalog (Log) (3-6 h).
    1. Before treatment, prepare stock solutions (10 mM) of Rapa and Log.
    2. Change the media with fresh media containing 0 nM, 100 nM, or 250 nM Rapa or Log.
    3. After 3 h of treatment, add 3.3% paraformaldehyde (PFA) to fix the cells.
    4. Prepare the slides for fluorescence confocal microscopy by placing one drop of antifade reagent.
    5. Mount the coverslips over the droplet and gently press with the forceps.
      NOTE: The glass coverslips are fragile and require caution. As an alternative to glass coverslips, use glass bottom plates. The major shortcoming of using glass-bottom plates is that the antifade reagent cannot be used, which makes imaging difficult due to bleaching of the fluorophores. DO NOT use antifade reagent containing DAPI (4′,6-diamidino-2-phenylindole) because its blue fluorescence will interfere with FRET between CFP and YFP.

3. Confocal microscopy for Fluorescence Resonance Energy Transfer (FRET)

  1. Place the slides under an inverted microscope and adjust the focus of the microscope until the cells become visible.
  2. Excite the fluorophore using a 440 nm laser and capture images using 468-503 nm emission for the donor CFP (FKBP-CFP-ER) or 525-565 nm for the acceptor YFP (FRB-YFP-Mito).
  3. Adjust the signal intensity using the built-in fluorescent filter until the background signal has dissipated (should be nearly solid black).
    NOTE: A confocal microscope was used to capture fluorescent images using NIS Element AR software. 60x magnification was used at a resolution of 512 pixels.
  4. Save the images as .nds files.
  5. Perform spectral ratiometric FRET analysis.

4. Ratiometric FRET analysis (6 - 12 h)

NOTE: Fluorescence emission from the donor ER-CFP (468-503 nm) and the acceptor Mito-YFP (525-565 nm) was detected using a confocal microscope. ImageJ software was used to quantify the average pixel fluorescence intensity following background subtraction. The ratio of YFP to CFP emission intensity was calculated and used as the readout for spectral FRET analysis.

  1. Open Image J (Fiji).
  2. Either drag the .nds file onto Image J, or press File -> Open and select the Image.
  3. On the following screen, select the Split Channels box.
  4. Click on the C = 0 panel.
  5. Click on Analyze -> Measure.
    NOTE: Write down the Mean Value for later calculation.
  6. Click on Process -> Math -> Subtract.
  7. Enter the Mean Value.
  8. Repeat step 4 for the C = 1 panel.
  9. Click on the Freehand selection tool.
  10. Circle a cell of interest, then press Ctrl+T to record it to the ROI.
  11. Click on Show All at the bottom of the ROI to show what you have already selected.
  12. Select all selected cells and click on Measure, Record these values into Excel.
  13. Click on the C = 1 panel and click on Show All.
  14. Select all cells and click on Measure again, and record these values into a separate column in Excel.
  15. In Excel, create a function to put the values of the donor signal over the recipient (C0/C1). This is the FRET ratio.

Results

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Ratiometric FRET analysis of FACS-enriched N2AAPP 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 disease11. The rapamycin analog AP21967, commonly referred to as rapalog (Log), exhibits low affinity for mTOR and is therefore relatively non-toxic to cells. However, it retains the ability to bind the FRB domain with the same affinity as rapamycin, due to a compensatory cavity-forming mutation (K2095P) introduced into the FRB domain12.

Rapa and Log treatment increased the FRET ratio (Figure 1). The treatment exhibited little or no effect on the levels of APP or other MAM proteins, such as IP3R3 or VDAC1. Thus, increasing tight (<10 nm) MAMs increased Aβ generation in vitro8. Notably, the FRET assays performed in naïve N2A cells also generated similar results.

Diagram of MAM-Tav2A interaction showing FRET analysis; protein bands and bar charts of Rivastigmine effect.
Figure 1: Tightening MAM gap widths increased Aβ generation in vitro. (A) Schematic of the inducible MAM stabilizer CFP-FRB-ER-Tav2A-Mito-FKBP-YFP (MAM-Tav2A) that undergoes self-cleavage, generating equimolar levels of the donor (CFP-FRB-ER or ER-CFP) and the acceptor (Mito-FKBP-YFP or Mito-YFP), and promotes FRET/FLIM when attached to the endogenous tight MAMs or upon enhancing the tight MAM formation by inducing the FRB and FKBP interaction with Rapamycin (Rapa) or its analog Rapalog (Log). (B) IB of cells expressing MAM-Tav2A (Tav2A) showing equimolar expression of the CFP-ER (blue box) and Mito-YFP (yellow box) after the self-cleavage of Tav2A. (C) Confocal images of DMSO (Veh) or Rapa (250 nM) treated cells overexpressing MAM-Tav2A. (D,E). Ratiometric FRET analysis of cells treated without (Veh) or with 100 nM rapamycin (Rapa) (D) or rapalog (Log) (E). FRET values are presented as ratios of intensities between channel 2 (465-500 nm) and channel 1 (525-555 nm). For every FRET analysis, 40-50 cells were randomly selected to generate ROIs on a cell-by-cell basis. One-way ANOVA. ***p < 0.0001. (F) Representative Western blot image of MAM-Tav2A-expressing N2AAPP cells after 24-h treatment with Rivastigmine (Riv: 0 or 50 µM). MAM-Tav2A generated equimolar levels of the donor (CFP-ER) and acceptor (Mito-YFP). (G) Quantitation of the FRET ratio between the donor and the acceptor of vehicle-treated (0 µM) or Riv-treated (50 µM). (H) Aβ ELISA (Wako) of the conditioned media (CM) of vehicle-treated (0 µM) or Riv-treated (50 µM) cells showed a significant reduction of Aβ40 (pM) levels after 50 µM Riv-treatment. n = 6, p < 0.001. (I) FRET (ratio) and Aβ (pM) values are tabulated. The figure have been modified from Zellmer et al8. Please click here to view a larger version of this figure.

Supplementary File 1: MAM-Tav2A plasmid sequence file. The DNA sequence of the expression plasmid encoding the ER-targeting Sac1 sequence (521FLALPIIMVVAFSMCIICLLMA GDTWTETLAYVLFWGVASIGTFFIILYNGKDFVDAPRLVQ KEKID587) linked to FKBP and CFP sequence and the mitochondria-targeting AKAP1 sequence (34SSHDEQQVEAGAVQLRADPAIKEPLPVEDV63) linked to FRB and YFP sequence tethered with the self-cleaving Tav2A sequence (EGRGSLLTCGD VEENPGP). Please click here to download this file.

Discussion

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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 MAM contact widths by regulating the interactions between MAM-tethering proteins such as PTPIP51, VAPB, VDAC1, PACS2, MFN2, VDAC1, and IP3R314,15,16,17,18,19,20. The sigma-1 receptor (S1R) antagonist (NE-100) downregulated MAMs and dramatically reduced axonal Aβ levels in a 3D neuronal model of AD21. A recent report demonstrated that the loss of S1R increased loose MAMs and decreased tight MAMs, suggesting S1R as a therapeutic target to modulate MAM tightness8,9.

Live FRET (fluorescence resonance energy transfer) imaging of neurons derived from Alzheimer's disease (AD) transgenic rats revealed a significant disruption in tight MAMs (<10 nm), with no observable change in loose MAMs (~20 nm), when compared to those in wild-type controls1. Inducible FRET/FLIM-based MAM stabilizers incorporating rapamycin (Rapa)/rapalog (Log)-inducible FRB and FKBP dimerization domains, expressed in N2AAPP cells, demonstrated that Rapa/Log treatment enhanced MAM tightness and increased Aβ40 production in a dose-dependent manner8,9. This provided the first proof of concept that narrowing the MAM gap width promotes amyloidogenic processing. The FDA-approved anti-amnestic drug rivastigmine, currently under preclinical investigation for mild to moderate AD22,23, significantly reduced Aβ40 levels (p < 0.001) without altering the expression of VDAC1 or BACE1, or affecting the colocalization frequency between CFP-ER and Mito-YFP8,9. Rivastigmine appears to influence MAM stabilization by modulating the levels of the MAM-anchoring protein mitofusin-2 (MFN2)24. This finding aligns with other studies reporting that rivastigmine reduces Aβ levels in cultured neurons and AD mouse models (3xTg)25,26. As MAM gap width regulates various cellular processes essential for cell survival27, targeted modulation of MAM stability may offer a more effective therapeutic strategy against Aβ accumulation than complete destabilization of MAMs, which could lead to unintended adverse effects.

A major limitation of the method is that the ratiometric FRET technique relies on the equimolar expression of the donor and the acceptor. Co-expression of the expression plasmids encoding the donor, or the acceptor will produce inconsistent results. Using a self-cleaving Tav2A connecting the donor and the acceptor biosensors ensures equimolar expression of the biosensors.However, the expression levels of the biosensors were checked using Western blot analysis prior to FRET analysis.

Rapamycin (Rapa) may not be ideal for assessing the effects of MAM tightening on Aβ production, as it inhibits the mammalian target of rapamycin (mTOR) signaling pathway, leading to downregulation of TREM2 (Triggering Receptor Expressed on Myeloid Cells 2) in microglia and consequently reducing Aβ plaque clearance in Alzheimer's disease (AD) mouse models (5XFAD)11. As an alternative, the rapamycin analog AP21967, commonly referred to as rapalog (Log), may be used. Log exhibits low binding affinity for mTOR, making it relatively non-toxic to cells. However, it retains the ability to bind the FRB domain with similar affinity to rapamycin, due to the introduction of a compensatory cavity-forming mutation (K2095P) in the FRB domain12.

The described method has high significance. Despite the evidence of several pre-clinical studies showing promising therapeutic effects of synthetic or natural small-molecule modulators of MAMs in cancer and metabolic disorders, no clinical trials have yet been undertaken. One of the primary reasons for this is the lack of a reliable method that can quantitatively measure MAM stability, a prerequisite for developing effective therapies. Quantification of MAM's structure-function relationship requires a complete view of the cell, which cannot be achieved for mature neurons by the traditional TEM or other high-resolution microscopies, but can be acquired with low magnification28. Thus, traditional techniques like TEM, cryo-TEM, or Scanning Electron Microscopies (SEM) may detect cellular structures at the nanoscale level but have limitations in measuring the degree of MAM stabilization because the highly dynamic nature of mitochondria29 and the ER30,31 makes MAM structures transient32,33,34. To investigate the MAM's structure function relationship, measuring the axonal velocity of mitochondria connected to the ER via tight or loose MAMs employing a live-cell imaging and kymography analysis has been proposed8,9.This is consistent with reports demonstrating decreased axonal transport of mitochondria in cortical neurons from AD brains35 and differentiated AD cybrid cells36. Despite the high quantitative accuracy, the live-cell imaging technique may not be useful for high-throughput drug screening. In contrast, FRET/FLIM analysis can be applied as a faster quantitative technique than the existing live-cell or high-resolution microscopy in the future to identify small-molecule therapeutics for AD.

The FRET protocol described here has been demonstrated to be applicable specifically in the context of artificially induced linkage between the ER and mitochondria. Its simplicity enables efficient assessment of ER-mitochondria contact stability and investigation of the resulting effects on amyloid pathology, which constitutes the primary objective of this approach. We aim to further utilize this method to screen a library of potential MAM-modulating small molecules within a recently developed three-dimensional (3D) AD drug screening platform37. The results will be immensely useful in selecting potential anti-Aβ small-molecule MAM modulators13 in vitro that can be extended to in vivo or clinical studies in the future.

Acknowledgements

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

We sincerely thank Dr. Rudolph E. Tanzi, Professor at Massachusetts General Hospital (MGH), for his insightful comments on the manuscript. We thank Dr. Masato Maesako, Assistant Professor at MGH, for sharing his expertise in FLIM assays. We thank Dr. Gyorgy Hajnoczky, Professor, Thomas Jefferson University, Philadelphia, for generously providing us with expression plasmids encoding ER-CFP or Mito-YFP. This study was supported by the Cure Alzheimer's Fund to RB.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
BSAFisher Scientific501781532
Confocal microscope Olympus FV3000RS (Tokyo, Japan)N/A
DMEM with GlutaMAX supplementGibco/Thermo Fisher Scientific10564011
EDTA  Life Technologies41116134
Falcon 6 Well Plates VWR International41122107
GAPDH Polyclonal AntibodyThermo Fisher ScientificPA1-988
Gelatin VWR International9000-70-8
Geneticin (G418 Sulfate)Gibco/Thermo Fisher Scientific10131035
GFP monoclonal AntibodyInvitrogenMA5-15256
Graphpad Prism N/APrism 9, version 9.5.0N/A
HeparinSigma-Aldrich H0200000
ImageJ Software ImageJ 1.53aN/A
Inverted confocal microscope Nikon (C2 Eclipse Ti2)N/A
Lipofectamine2000Invitrogen11668027
MS Excel  Microsoft Excel, version 2302N/A
NaCl  Fisher Scientific7647145
NuPAGE 4–12% Bis-Tris gel  InvitrogenNP0321BOX
Penicillin/Streptomycin/Amphotericin B Lonza 17-745E
PhotoshopAdobe Photoshop CC 20.0.10 N/A
Tris-HCL, pH 7.6  Boston BioProducts42000000
Triton X-100  Sigma-AldrichT8787
Trypsin-EDTAGibco/Thermo Fisher Scientific25200056
Tween 20Fisher Scientific501657287

References

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,
  1. Martino Adami, P. V., et al. Perturbed mitochondria-ER contacts in live neurons that model the amyloid pathology of Alzheimer's disease. J Cell Sci. 132 (20), 229906(2019).
  2. Area-Gomez, E., Schon, E. A. On the pathogenesis of Alzheimer's disease: The MAM hypothesis. FASEB J. 31 (3), 864-867 (2017).
  3. Fujimoto, M., Hayashi, T. New insights into the role of mitochondria-associated endoplasmic reticulum membrane. Int Rev Cell Mol Biol. 292, 73-117 (2011).
  4. Prudent, J., et al. MAPL SUMOylation of Drp1 stabilizes an ER/mitochondrial platform required for cell death. Mol Cell. 59 (6), 941-955 (2015).
  5. Carpio, M. A., et al. controls apoptosis by Ca2+ through ER-mitochondrial contact sites. Cell Rep. 34 (10), 108827(2021).
  6. Degechisa, S. T., Dabi, Y. T., Gizaw, S. T. The mitochondrial associated endoplasmic reticulum membranes: A platform for the pathogenesis of inflammation-mediated metabolic diseases. Immun Inflamm Dis. 10 (7), e647-e658 (2022).
  7. Csordás, G., Weaver, D., Hajnóczky, G. Endoplasmic reticulum-mitochondrial contactology: Structure and signaling functions. Trends Cell Biol. 28 (7), 523-540 (2018).
  8. Zellmer, J. C., et al. Stabilization of mitochondria-associated endoplasmic reticulum membranes regulates Aβ generation in a three-dimensional neural model of Alzheimer's disease. Alzheimers Dement. 21 (2), e14417(2024).
  9. Zellmer, J. C., Lomoio, S., Tanzi, R. E., Bhattacharyya, R. Quantitative analysis of mitochondria-associated endoplasmic reticulum membrane (MAM) stabilization in a neural model of Alzheimer's disease (AD). J Vis Exp. (215), e66129(2025).
  10. Bhattacharyya, R., et al. The neuronal-specific isoform of BIN1 regulates beta-secretase cleavage of APP and Abeta generation in a RIN3-dependent manner. Sci Rep. 12 (1), 3486-3500 (2022).
  11. Shi, Q., et al. Microglial mTOR activation upregulates Trem2 and enhances β-amyloid plaque clearance in the 5XFAD Alzheimer's disease model. J Neurosci. 42 (27), 5294-5313 (2022).
  12. Wilmington, S. R., Matouschek, A. An inducible system for rapid degradation of specific cellular proteins using proteasome adaptors. PLoS One. 11 (4), e0152679(2016).
  13. Magalhaes Rebelo, A. P., et al. Chemical modulation of mitochondria-endoplasmic reticulum contact sites. Cells. 9 (7), 1637-1655 (2020).
  14. De Vos, K. J., et al. VAPB interacts with the mitochondrial protein PTPIP51 to regulate calcium homeostasis. Hum Mol Genet. 21 (6), 1299-1311 (2012).
  15. Yang, M., et al. Mitochondria-associated ER membranes: The origin site of autophagy. Front Cell Dev Biol. 8, 595(2020).
  16. Filadi, R., et al. Mitofusin 2 ablation increases endoplasmic reticulum-mitochondria coupling. Proc Natl Acad Sci U S A. 112 (17), E2174-E2181 (2015).
  17. Filadi, R., et al. On the role of Mitofusin 2 in endoplasmic reticulum-mitochondria tethering. Proc Natl Acad Sci U S A. 114 (12), E2266-E2267 (2017).
  18. Xie, Q., et al. ABT737 reverses cisplatin resistance by regulating ER-mitochondria Ca2+ transduction in human ovarian cancer cells. Int J Oncol. 49 (6), 2507-2519 (2016).
  19. Tsunoda, T., et al. Inositol 1,4,5-trisphosphate receptor type 1 modulates the acquisition of cisplatin resistance in bladder cancer cell lines. Oncogene. 24 (8), 1396-1402 (2005).
  20. Tubbs, E., et al. Sulforaphane improves disrupted ER-mitochondria interactions and suppresses exaggerated hepatic glucose production. Mol Cell Endocrinol. 461, 205-214 (2018).
  21. Bhattacharyya, R., et al. Axonal generation of amyloid-beta from palmitoylated APP in mitochondria-associated endoplasmic reticulum membranes. Cell Rep. 35 (7), 109134(2021).
  22. Alhazmi, H. A., Albratty, M. An update on the novel and approved drugs for Alzheimer disease. Saudi Pharm J. 30 (12), 1755-1764 (2022).
  23. Rountree, S. D., et al. Effectiveness of antidementia drugs in delaying Alzheimer's disease progression. Alzheimers Dement. 9 (3), 338-345 (2013).
  24. Hadwen, J., et al. Transcriptomic RNAseq drug screen in cerebrocortical cultures: Toward novel neurogenetic disease therapies. Hum Mol Genet. 27 (18), 3206-3217 (2018).
  25. Ray, B., et al. Rivastigmine modifies the α-secretase pathway and potentially early Alzheimer's disease. Transl Psychiatry. 10 (1), 47(2020).
  26. Bailey, J. A., Ray, B., Greig, N. H., Lahiri, D. K. Rivastigmine lowers Aβ and increases sAPPα levels, which parallel elevated synaptic markers and metabolic activity in degenerating primary rat neurons. PLoS One. 6 (7), e21954(2011).
  27. Ziegler, D. V., Martin, N., Bernard, D. Cellular senescence links mitochondria-ER contacts and aging. Commun Biol. 4 (1), 1323(2021).
  28. Neikirk, K., et al. Call to action to properly utilize electron microscopy to measure organelles to monitor disease. Eur J Cell Biol. 102 (4), 151365(2023).
  29. Liu, X., et al. Mitochondrial 'kiss-and-run': Interplay between mitochondrial motility and fusion-fission dynamics. EMBO J. 28 (20), 3074-3089 (2009).
  30. Szymanski, J., et al. Interaction of mitochondria with the endoplasmic reticulum and plasma membrane in calcium homeostasis, lipid trafficking and mitochondrial structure. Int J Mol Sci. 18 (7), ijms18071576(2017).
  31. Tikhomirova, M. S., et al. A role for endoplasmic reticulum dynamics in the cellular distribution of microtubules. Proc Natl Acad Sci U S A. 119 (15), e2104309119(2022).
  32. Stacchiotti, A., et al. Perspective: Mitochondria-ER contacts in metabolic cellular stress assessed by microscopy. Cells. 8 (1), 8010005(2018).
  33. Cieri, D., et al. SPLICS: A split green fluorescent protein-based contact site sensor for narrow and wide heterotypic organelle juxtaposition. Cell Death Differ. 25 (6), 1131-1145 (2018).
  34. Giacomello, M., Pellegrini, L. The coming of age of the mitochondria-ER contact: A matter of thickness. Cell Death Differ. 23 (9), 1417-1427 (2016).
  35. Dai, J., et al. Impaired axonal transport of cortical neurons in Alzheimer's disease is associated with neuropathological changes. Brain Res. 948 (1), 138-144 (2002).
  36. Trimmer, P. A., Borland, M. K. Differentiated Alzheimer's disease transmitochondrial cybrid cell lines exhibit reduced organelle movement. Antioxid Redox Signal. 7 (9-10), 1101-1109 (2005).
  37. Quinti, L., et al. Neurotherapeutics in the Era of Translational. Smith, R. A., Kaspar, B. K., Svendsen, C. N. , Academic Press. 311-331 (2021).

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Tags

Biology
Video Coming Soon

Related Articles