Mitochondrial immobilization mediated by syntaphilin facilitates survival of demyelinated axons

Abstract

Axonal degeneration is a primary cause of permanent neurological disability in individuals with the CNS demyelinating disease multiple sclerosis. Dysfunction of axonal mitochondria and imbalanced energy demand and supply are implicated in degeneration of chronically demyelinated axons. The purpose of this study was to define the roles of mitochondrial volume and distribution in axonal degeneration following acute CNS demyelination. We show that the axonal mitochondrial volume increase following acute demyelination of WT CNS axons does not occur in demyelinated axons deficient in syntaphilin, an axonal molecule that immobilizes stationary mitochondria to microtubules. These findings were supported by time-lapse imaging of WT and syntaphilin-deficient axons in vitro. When demyelinated, axons deficient in syntaphilin degenerate at a significantly greater rate than WT axons, and this degeneration can be rescued by reducing axonal electrical activity with the Na + channel blocker flecainide. These results support the concept that syntaphilin-mediated immobilization of mitochondria to microtubules is required for the volume increase of axonal mitochondria following acute demyelination and protects against axonal degeneration in the CNS.A xonal loss is the major cause of permanent neurological disability in individuals with primary diseases of myelin (1). Axonal degeneration associated with myelin diseases has been well studied in the immune-mediated CNS demyelinating disease multiple sclerosis. Two mechanisms are responsible for the degeneration of demyelinated axons in multiple sclerosis brains. First, axons are transected in acute multiple sclerosis lesions (2) and axonal injury correlates with the number of infiltrating peripheral immune cells (3, 4). Edema associated with breakdown of the blood-brain barrier and toxic substances secreted by inflammatory immune cells are thought to transect the acutely demyelinated axon (5). Second, chronically demyelinated axons degenerate as a result of loss of trophic support provided by oligodendrocytes and myelin (6, 7). In addition to increasing the speed of nerve transmission, oligodendrocytes and myelin provide axons with trophic support that is essential for their longterm survival (8-10). Although most demyelinated axons compensate for loss of oligodendrocytes/myelin, additional insults to axonal mitochondria impair axonal metabolism, increase axonal Ca 2+ , and promote progressive disruption of mitochondrial function (11,12). Changes in axonal mitochondria have been described in chronically demyelinated axons in postmortem multiple sclerosis tissue, and include decreased expression of nuclear-encoded mitochondrial genes (13), reduced mitochondrial respiration (14), and genetic alterations in mitochondrial DNA (15). A vicious cycle of reduced ATP production and increased axonal Ca 2+ results in degeneration of the chronically demyelinated axon.Myelinated axons contain two populations of mitochondria. Most axonal mitochondria do not appear to translocate and are located at...

Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy

“…Mitochondria undergo continuous fission and fusion cycles 26 , are mobile and trafficked along the neurite processes 21 . Defects in mitochondrial trafficking and dynamics which are not purely numerical or structural can be easily missed by this approach, although, by looking at the number of mitochondria in an anatomically defined space such as presynapse one can interpret if the transport of mitochondria may be altered 39 . A second disadvantage is that it is performed only in a small portion of the brain, therefore circuitry specific defects in mitochondrial distribution may be missed.…”

“…Earlier SBFSEM analysis was greatly hindered due to lower resolution, however newer sample preparation techniques involving enhanced staining methods 40 have considerably improved the resolution to an extent that even a single synaptic vesicle can be easily resolved. At this resolution ultrastructural defects in mitochondria such as vacuolation, membrane disruption, and loss of cristae can be easily observed, and the distribution of defective mitochondria within cells can be determined 38,39 . The high resolution of this technique provides a major advantage over light microscopy where the resolution is limited to ~200 nm in XY axis and ~500 nm in Z-axis.…”

“…Multiple studies since then have established this technique as a major tool in reconstruction analysis of neuronal circuitry 34 . Furthermore, for many smaller scale projects, it provides reconstruction analysis to identify cellular organelles 27,[35][36][37][38][39] . Since, the acquired images are derived from low voltage back scatter electrons, new staining protocols which combine different known heavy metal staining techniques were developed to increase the resolution 40 .…”

“…In this paper, we provide a protocol for utilizing 3D electron microscopy imaging and volumetric analysis of brain mitochondria based on methods that have previously been used by us and others 38,39,41 . The tissue post-processing methods used were as previously described by Deerinck et al 40 .…”

Mitochondrial Function and Dynamics Imaged In Vivo

“…Using knockout mice, Ohno et al recently identifi ed that syntaphilin is necessary to increase mitochondrial content in demyelinated axons in vivo and that its absence leads to an increase in axonal beading and degeneration [ 91 ].…”