Autophagy is essential for oligodendrocyte differentiation, survival, and proper myelination.

Deficient myelination, the spiral wrapping of highly specialized membrane around axons, causes severe neurological disorders. Maturation of oligodendrocyte progenitor cells (OPC) to myelinating oligodendrocytes (OL), the sole providers of central nervous system (CNS) myelin, is tightly regulated and involves extensive morphological changes. Here, we present evidence that autophagy, the targeted isolation of cytoplasm and organelles by the double-membrane autophagosome for lysosomal degradation, is essential for OPC/OL differentiation, survival, and proper myelin development. A marked increase in autophagic activity coincides with OL differentiation, with OL processes having the greatest increase in autophagic flux. Multiple lines of evidence indicate that autophagosomes form in developing myelin sheathes before trafficking from myelin to the OL soma. Mice with conditional OPC/OL-specific deletion of the essential autophagy gene Atg5 beginning on postnatal Day 5 develop a rapid tremor and die around postnatal Day 12. Further analysis revealed apoptotic death of OPCs, reduced differentiation, and reduced myelination. Surviving Atg5-/- OLs failed to produce proper myelin structure. In vitro, pharmacological inhibition of autophagy in OPC/dorsal root ganglion (DRG) co-cultures blocked myelination, producing OLs surrounded by many short processes. Conversely, autophagy stimulation enhanced myelination. These results implicate autophagy as a key regulator of OPC survival, maturation, and proper myelination. Autophagy may provide an attractive target to promote both OL survival and subsequent myelin repair after injury.

Preservation, Sectioning, and Staining of Schwann Cell Cultures for Transmission Electron Microscopy Analysis.

The transmission electron microscope (TEM) enables a unique and valuable examination of cellular and extracellular elements in tissue in situ, in cultured cells, or in pellets derived from suspensions of cells or other materials such as nanoparticles. Here we focus on the preparation of cultured Schwann cells or Schwann cell-containing dorsal root ganglion cultures. To gain as life-like as possible views of the cellular details, it is imperative to achieve excellent preservation of the cellular structure. The steps in the preparation of cultures described in this chapter represent the results of many years of accumulated TEM images to find the best methods of preservation for Schwann cells, myelin, and basal lamina components. All the materials required are listed. The methods for fixing, dehydrating, and embedding a culture are described. Choosing an area in the culture to view, scoring it, cutting it out of the resin-embedded culture, mounting it appropriately for enface or cross-sectioning, and performing the semi-thin and thin sectioning are detailed. Explaining the way in which the sections are then stained for TEM completes the Methods section. Preservation of cultured Schwann cells and their myelin sheaths can be outstanding due to the direct and rapid but careful addition of the fixative solution to the culture dish.

Ligand binding and membrane insertion compete with oligomerization of the BclXL apoptotic repressor.

B-cell lymphoma extra large (BclXL) apoptotic repressor plays a central role in determining the fate of cells to live or die during physiological processes such as embryonic development and tissue homeostasis. Herein, using a myriad of biophysical techniques, we provide evidence that ligand binding and membrane insertion compete with oligomerization of BclXL in solution. Of particular importance is the observation that such oligomerization is driven by the intermolecular binding of its C-terminal transmembrane (TM) domain to the canonical hydrophobic groove in a domain-swapped trans fashion, whereby the TM domain of one monomer occupies the canonical hydrophobic groove within the other monomer and vice versa. Binding of BH3 ligands to the canonical hydrophobic groove displaces the TM domain in a competitive manner, allowing BclXL to dissociate into monomers upon hetero-association. Remarkably, spontaneous insertion of BclXL into DMPC/DHPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dihexanoyl-sn-glycero-3-phosphocholine) bicelles results in a dramatic conformational change such that it can no longer recognize the BH3 ligands in what has come to be known as the "hit-and-run" mechanism. Collectively, our data suggest that oligomerization of a key apoptotic repressor serves as an allosteric switch that fine-tunes its ligand binding and membrane insertion pertinent to the regulation of apoptotic machinery.

Ultrastructural study of the primary olfactory pathway in Macaca fascicularis.

Olfactory ensheathing glial cells (OEGs) interact with a wide repertoire of cell types and support extension of olfactory axons (OAs) within the olfactory pathway. OEGs are thought to exclude OAs from contact with all other cells between the olfactory epithelium and the glomerulus of the olfactory bulb. These properties have lead to testing to determine whether OEGs support axonal growth following transplantation. The cellular interactions of transplanted OEGs will probably resemble those that occur within the normal pathway where interactions between OEGs and fibroblasts are prominent. No previous primate studies have focused on these interactions, knowledge of which is important if clinical application is envisioned. We describe the detailed intercellular interactions of OAs with supporting cells throughout the olfactory epithelium, the lamina propria, the fila olfactoria, and the olfactory nerve layer by using transmission electron microscopy in adult Macaca fascicularis. Patterns of OEG ensheathment and variations of the endo- and perineurium formed by olfactory nerve fibroblasts are described. OAs mainly interacted with horizontal basal cells, OEGs, and astrocytes. At both transitional ends of the pathway seamless intercellular interactions were observed, and fibroblast processes were absent. Perineurial cells produced surface basal lamina; however, endoneurial, epineurial, and meningeal fibroblasts did not. Perineurial cells contained intermediate filaments and were distinct from other fibroblasts and meningeal cells. OAs had direct contacts with astrocytes near the glia limitans. The properties of OEGs differed depending on whether astrocytic or fibroblastic processes were present. This indicates the importance of the cellular milieu in the structure and function of OEGs in primates.

cAMP and Schwann cells promote axonal growth and functional recovery after spinal cord injury.

Central neurons regenerate axons if a permissive environment is provided; after spinal cord injury, however, inhibitory molecules are present that make the local environment nonpermissive. A promising new strategy for inducing neurons to overcome inhibitory signals is to activate cAMP signaling. Here we show that cAMP levels fall in the rostral spinal cord, sensorimotor cortex and brainstem after spinal cord contusion. Inhibition of cAMP hydrolysis by the phosphodiesterase IV inhibitor rolipram prevents this decrease and when combined with Schwann cell grafts promotes significant supraspinal and proprioceptive axon sparing and myelination. Furthermore, combining rolipram with an injection of db-cAMP near the graft not only prevents the drop in cAMP levels but increases them above those in uninjured controls. This further enhances axonal sparing and myelination, promotes growth of serotonergic fibers into and beyond grafts, and significantly improves locomotion. These findings show that cAMP levels are key for protection, growth and myelination of injured CNS axons in vivo and recovery of function.

Basic fibroblast growth factor promotes neuronal survival but not behavioral recovery in the transected and Schwann cell implanted rat thoracic spinal cord.

It was investigated whether the addition of basic fibroblast growth factor (FGF-2) enhances the efficacy of a Schwann cell (SC) bridge to repair the transected spinal cord by assessing tissue sparing and neuronal survival near the graft-cord interfaces, axonal regeneration and myelination in the graft, and behavioral recovery up to 12 weeks post-grafting. Experimental animals received a bridge of SCs within fibrin containing 1 microg of FGF-2; control animals received a SC implant without FGF-2. Sparing of tissue in a 2.5-mm-long segment near the graft-cord borders was 69% in the rostral and 52% in the caudal cord at 6 weeks post-grafting, not significantly different from the control group. With FGF-2, survival of NeuN-positive cells was increased in the rostral cord: 24.4%, 20.4%, and 17.2% of the number of positive cells in the uninjured cord compared to 13.5%, 9.1%, and 8.9% in controls at 3, 6, and 12 weeks post-grafting, respectively. Similarly, in the caudal cord, survival of NeuN-positive cells was increased with FGF-2: 19.3%, 16.8%, and 14.5% compared to 10.8%, 5.6%, and 6.1% in controls. The staining intensity of glial fibrillary acidic protein was significantly higher at the interfaces of both cord stumps at 3 weeks with SC/FGF-2 grafts; chondroitin sulfate proteoglycan (CS-56) staining was more intense in the rostral cord but only at 6 weeks. Blood vessels in the FGF-2 grafts were larger and less regular in shape than those in control grafts. Axonal growth into the bridge was not improved by the addition of FGF-2. Retrogradely traced neurons were not found rostral to the implant, indicating that axons had not grown a few mm into the caudal spinal tissue. Recovery of hind limb function was similar in both groups. Despite the neuroprotective effects of FGF-2, improved effects on axonal regeneration and functional recovery were not observed.

Schwann cell but not olfactory ensheathing glia transplants improve hindlimb locomotor performance in the moderately contused adult rat thoracic spinal cord.

Cultured adult rat Schwann cells (SCs) or olfactory ensheathing glia (OEG), or both, were transplanted in the adult Fischer rat thoracic (T9) spinal cord 1 week after a moderate contusion (10 gm, 12.5 mm, NYU impactor). Rats received either a total of 2 x 10(6) cells suspended in culture medium or culture medium only (controls). At 12 weeks after injury, all grafted animals exhibited diminished cavitation. Although in medium-injected rats 33% of spinal tissue within a 5-mm-long segment of cord centered at the injury site was spared, significantly more tissue was spared in SC (51%), OEG (43%), and SC/OEG (44%) grafted animals. All three types of glial grafts were filled with axons, primarily of spinal origin. SC grafts contained more myelinated axons than SC/OEG and OEG grafts. Both types of SC-containing grafts expressed more intense staining for glial fibrillary acidic protein and chondroitin sulfate proteoglycan compared with OEG-only grafts. Retrograde tracing demonstrated that the number of propriospinal and brainstem axons reaching 5-6 mm beyond the grafted area was significantly higher with SC and SC/OEG grafts but not with OEG-only grafts compared with controls. Corticospinal fibers terminated closer to the lesion epicenter in all grafted animals than in controls. With SC-only grafts, a modest but statistically significant improvement in hindlimb locomotor performance was detected at 8-11 weeks after injury. Thus, in addition to this functional improvement, our results show that an SC graft is more effective in promoting axonal sparing/regeneration than an SC/OEG or OEG graft in the moderately contused adult rat thoracic spinal cord.

Purified adult ensheathing glia fail to myelinate axons under culture conditions that enable Schwann cells to form myelin.

Several studies have suggested that olfactory ensheathing glia (EG) can form Schwann cell (SC)-like myelin. Because of possible misinterpretation attributable to contaminating SCs, the capacity of EG to produce myelin needs to be explored further. Therefore, we compared the abilities of adult EG, purified by immunopanning with p75 antibody, and adult SCs to produce myelin when cocultured with purified dorsal root ganglion neurons (DRGNs) in serum-free and serum-containing media. In both media formulations, the number of myelin sheaths in SC/DRGN cultures was far higher than in EG/DRGN cultures; the number of sheaths in EG/DRGN cultures was equal to that in purified DRGN cultures without added cells. The latter result demonstrates that myelination by a few SCs remaining in purified DRGN cultures may occur, suggesting that myelin in EG/DRGN cultures could be SC myelin. Striking differences in the relationship of EG and SC processes to axons were observed. Whereas SCs displayed relatively short, thick processes that engulfed axons in small bundles or in individual cytoplasmic furrows and segregated larger axons into one-to-one relationships, EG extended flattened sheets that partitioned or only partially encircled fascicles of axons, sometimes spanning the entire culture. SCs exhibited behavior typical of SCs in peripheral nerves, whereas EG exhibited characteristics resembling those of EG in olfactory nerves. In sum, p75-selected EG from adult animals did not exhibit an SC-like relationship to axons and did not form myelin.