Mitochondria-localized AMPK responds to local energetics and contributes to exercise and energetic stress-induced mitophagy

Mitochondria form a complex, interconnected reticulum that is maintained through coordination among biogenesis, dynamic fission, and fusion and mitophagy, which are initiated in response to various cues to maintain energetic homeostasis. These cellular events, which make up mitochondrial quality control, act with remarkable spatial precision, but what governs such spatial specificity is poorly understood. Herein, we demonstrate that specific isoforms of the cellular bioenergetic sensor, 5′ AMP-activated protein kinase (AMPKα1/α2/β2/γ1), are localized on the outer mitochondrial membrane, referred to as mitoAMPK, in various tissues in mice and humans. Activation of mitoAMPK varies across the reticulum in response to energetic stress, and inhibition of mitoAMPK activity attenuates exercise-induced mitophagy in skeletal muscle in vivo. Discovery of a mitochondrial pool of AMPK and its local importance for mitochondrial quality control underscores the complexity of sensing cellular energetics in vivo that has implications for targeting mitochondrial energetics for disease treatment

Energy Transfer Efficiency based on One- and Two-Photon FRET

We are investigating membrane-based sorting processes in polarized epithelial MDCK cells, which most likely involves membrane microdomains. We have postulated that proteins contained in these microdomains, cluster, and to prove this, we have internalized differently fluorophore labeled pIgA-R ligands in MDCK cells, stably transfected with polymeric IgA receptors (pIgA-R), from opposite plasma membranes. Our previous work showed that these receptor-ligand complexes colocalize in the apical recycling endosome (ARE), underneath the apical plasma membrane. Quantitative one-photon confocal and 2-photon (2-P) FRET microscopy allowed us to calculate energy transfer efficiency (E%). Unquenched donor levels where established based on a novel algorithm, which corrects the FRET contamination of acceptor bleed-through and donor crosstalk. Using different emission filters also confirmed the veracity of the algorithm. 2-P FRET allows the selection of a specific donor wavelength, which does not precipitate acceptor bleed-through, a clear advantage over 1-P confocal microscopy. Results show that E% is independent of acceptor levels, an indication of a clustered distribution, as in random distribution E% rises with increasing acceptor levels. However, E% decreases with increasing donor and donor:acceptor ratio levels, which we have termed donor geometric exclusion , where some donors in a cluster block others from interacting with an acceptor. We submit that this is a second indicator for a clustered pattern, because in a random, dispersed situation donors are not likely to be in close proximity to have such an effect. We have developed a model explaining this phenomenon

Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration

Levels of newly-synthesized RNA decline as a function of distance from nerve injury.

<p><b>A</b>, low-magnification micrograph of transected end showing newly-synthesized RNA (green) and ribosomes detected by anti-P antibody (red). Bar = 100 µm. <b>B</b>, BrU-RNA signal plotted as a function of distance from the transection. Each point represents the mean of 10 nerve fragments with standard errors. <b>C–H</b>, series of images of a single fiber from the transected end, distal to proximal, showing newly-synthesized RNA labeled by BrU (green) and F-actin (red). <b>C</b>, transected end with a high concentration of newly-synthesized BrU-RNA. <b>D</b>, first proximal Schwann-cell nucleus from the tip. <b>E</b>, first node of Ranvier proximal from the tip. <b>F</b>, second Schwann cell nucleus. <b>G</b>, second node of Ranvier. <b>H</b>, third node of Ranvier. Bar = 10 µm.</p

Most newly-synthesized axonal RNA is not mitochondrial.

<p>Cryosections of injured BrU-labeled (green) sciatic nerve fragments were stained for BrU <b>(A, D, G)</b> and a monoclonal antibody against the mitochondrial Complex IV Subunit I <b>(B, E, H)</b>. A paranodal axon is shown in <b>A–C</b> and nodes of Ranvier are shown in <b>D–I</b>. Mitochondria corresponding to empty spaces in A and D are designated by arrows. Bar = 5 µm.</p

Newly synthesized RNA is present in axons and bands of Cajal.

<p><b>A</b>, confocal plane including a BrU-labeled axon. The myelin is unlabeled. The external border of the myelin is the outer wrap of Schwann cell cytoplasm that includes bands of Cajal. <b>B</b>, stack of confocal planes with the plane shown in A as the midpoint, showing the spiraling bands of Cajal (arrows). <b>C, D, E</b>, projected cross-sections boxed in the stack shown in panel B showing the separation between newly-synthesized RNA in the axon and band of Cajal. Bar = 10 µm.</p

Actin depolymerization in injured sciatic nerves prevents transfer of RNA into axons.

<p><b>A, C, E, G, I</b>, representative confocal images of BrU labeling at nodes of Ranvier. Bar = 10 µm. <b>B, D, F, H, J</b>, quantification of BrU fluorescence from 10 or more line scans across perinodal regions normalized to the mean of each linescan. Error bars represent standard errors. <b>A and B</b>, control BrU labeling without Latrunculin A; <b>C and D</b>, 0.07 µg/ml Latrunculin A during BrU labeling; <b>E and F</b>, 0.2 µg/ml; <b>G and H</b>, 0.6 µg/ml; <b>I and J</b>, 1.8 µg/ml. <b>K</b>, absolute BrU fluorescence intensities for the 8 bins at each edge combined (n = 304), representing RNA in the outer Schwann cell wrap, and the 20 bins in the center of each linescan (n = 380), representing RNA in the axon, for the control untreated and highest latrunculin A concentration (1.8 µg/ml) nerves. Error bars represent standard errors.</p

Myosin-Va function is required for transfer of RNA from Schwann cells to axons.

<p>Longitudinal 10-µm sections of transected sciatic nerves from null (d-l) <i>Myo5a</i> mutant mice have reduced axoplasmic levels of newly synthesized RNA. <b>A and C</b>, null mutant; <b>B and D</b>, wild-type control. RNA labeled by BrU is shown in green, the paranodal marker Caspr in red. Panels C and D show higher magnification views of boxed regions in panels A and B respectively. Arrows, nodes of Ranvier; arrowheads, bands of Cajal (compare to arrows in Fig. 6). Micrographs are single optical sections from Z-stacks imaged with a laser scanning confocal microscope. Bar = 5 µm. <b>E</b>, linescan quantitation of abundance of BrU-labeled RNA across fibers from d-l mutant and wild-type control mice. Edges are the outer wraps of Schwann cells; center approximates the location of the axon. Intensity measurements were normalized to the mean of each linescan. Bars represent standard deviations. <b>F</b>, Absolute BrU fluorescence intensities in edges (as shown in <b>E</b>, 4 bins at each end combined; n = 160) and centers (10 central bins combined; n = 200). Error bars represent standard errors.</p

Newly-synthesized RNA is transferred from Schwann cells to axons after sciatic nerve transection.

<p><b>A–D</b>, experimental procedure. <b>E–F</b>, single confocal planes of fibers at nodes of Ranvier showing BrU incorporation (green) and F-actin (red). <b>G</b>, Axonal BrU fluorescence intensity plotted as a function of distance from the node of Ranvier for uninjured control (open circles) and injured (closed circles) nerves. Statistical significance at each distance between injured and uninjured nerves was determined by Student's t-test. Error bars represent standard errors. <b>H–I</b>, single confocal planes showing BrU labeling (green) of F-actin-rich (red) Schmidt-Lanterman incisures (arrows). Bars = 5 µm.</p

Most of the newly-synthesized RNA is produced by RNA Polymerase II.

<p><b>A–E</b>, injured control nerves without α-amanitin (<b>A </b><b>and </b><b>C)</b>, and injured nerves treated with α-amanitin during the BrU labeling period <b>(B and D)</b> were stained for BrU (green) and F-actin with phalloidin (red). <b>A and </b><b>B</b>, Schwann cell nuclei; <b>C</b> and <b>D</b>, nodes of Ranvier. Bar = 10 µm. <b>E</b>, BrU-RNA fluorescence intensities plotted as a function of distance from the node of Ranvier for controls without α-amanitin (circles) and nerves treated with 10 µg/ml α-amanitin (triangles). Statistical significance at each distance was determined by Student's t-test. Error bars represent standard errors. <b>F</b>, Neurofilament L (NF-L) mRNA is found in both Schwann cells and axons by <i>in situ</i> hybridization (red) and BrU-RNA (green). Arrows are pointing to axons. <b>G</b>, negative control NF-L sense probe. Bar = 5 µm.</p