It is generally accepted that healthy cells degrade their own mitochondria. Here, we report that retinal ganglion cell axons of WT mice shed mitochondria at the optic nerve head (ONH), and that these mitochondria are internalized and degraded by adjacent astrocytes. EM demonstrates that mitochondria are shed through formation of large protrusions that originate from otherwise healthy axons. A virally introduced tandem fluorophore protein reporter of acidified mitochondria reveals that acidified axonal mitochondria originating from the retinal ganglion cell are associated with lysosomes within columns of astrocytes in the ONH. According to this reporter, a greater proportion of retinal ganglion cell mitochondria are degraded at the ONH than in the ganglion cell soma. Consistently, analyses of degrading DNA reveal extensive mtDNA degradation within the optic nerve astrocytes, some of which comes from retinal ganglion cell axons.Together, these results demonstrate that surprisingly large proportions of retinal ganglion cell axonal mitochondria are normally degraded by the astrocytes of the ONH. This transcellular degradation of mitochondria, or transmitophagy, likely occurs elsewhere in the CNS, because structurally similar accumulations of degrading mitochondria are also found along neurites in superficial layers of the cerebral cortex. Thus, the general assumption that neurons or other cells necessarily degrade their own mitochondria should be reconsidered.mitophagy | phagocytosis T he number, half-life, and morphology of mitochondria vary widely across cell types and are regulated by both intrinsic and extrinsic mechanisms. Mitochondria number is controlled through regulated production (1) and degradation (2), as well as by the regulated fusion and fission of existing mitochondria (3). Damaged mitochondria are removed by mitophagy, a subtype of autophagy that involves the enwrapping of mitochondria in autophagosomes that subsequently fuse with lysosomes to become autophagolysosomes (2). Implicit in the categorization of mitophagy as a subtype of autophagy is the assumption that each cell degrades its own mitochondria.Recently, we described a phenomenon at the optic nerve head (ONH) of WT mice, where evulsions originating from otherwise intact axons are engulfed and degraded by resident phagocytic astrocytes (4). Serial section-based 3D reconstructions obtained through serial block-face scanning electron microscopy (SBEM) revealed that the protrusions on axons and the evulsions near axons were common throughout the ONH in both the glial lamina, where retinal ganglion cell axons are unmyelinated, and in the adjacent myelination transition zone (MTZ). The axonal protrusions and evulsions were, on average, larger than the mean diameter of axons and contained membrane-bound organelles of unknown identity. Results Axonal Protrusions and Evulsions Within the ONH Contain Mitochondria.To determine the identity of the membranous material contained within the axonal evulsions at the ONH, a 3-mo-old WT C57BL/6J mouse was an...

Optic nerve head (ONH) astrocytes have been proposed to play both protective and deleterious roles in glaucoma. We now show that, within the postlaminar ONH myelination transition zone (MTZ), there are astrocytes that normally express Mac-2 (also known as Lgals3 or galectin-3), a gene typically expressed only in phagocytic cells. Surprisingly, even in healthy mice, MTZ and other ONH astrocytes constitutive internalize large axonal evulsions that contain whole organelles. In mouse glaucoma models, MTZ astrocytes further upregulate Mac-2 expression. During glaucomatous degeneration, there are dystrophic processes in the retina and optic nerve, including the MTZ, which contain protease resistant γ-synuclein. The increased Mac-2 expression by MTZ astrocytes during glaucoma likely depends on this γ-synuclein, as mice lacking γ-synuclein fail to up-regulate Mac-2 at the MTZ after elevation of intraocular pressure. These results suggest the possibility that a newly discovered normal degradative pathway for axons might contribute to glaucomatous neurodegeneration.DBA/2J mice | retinal ganglion cell | Sncg G laucoma, a neurodegenerative disorder that kills retinal ganglion cells (RGCs), affects more than 60 million people and is the second leading cause of blindness worldwide (1). In glaucomatous retinas, both astrocytes and Müller glia increase their reactivity (2, 3). Within the orbital portion of the optic nerve, astrocytes increase in number and reactivity (4). Within the optic nerve head (ONH), astrocytes also increase reactivity in glaucoma animal models and in the human disease (5). These ONH astrocytes are of particular interest as they enwrap axons at the location where damage would account for the arcuate vision field loss characteristic of glaucoma.Here, we identify a spatially discrete population of astrocytes within the ONH, those at the myelination transition zone (MTZ), which express the phagocytosis-related gene Mac-2. Surprisingly, astrocytes throughout the ONH including the MTZ phagocytose large axonal evulsions even in unaffected mice. Mac-2 expression is increased in optic nerve astrocytes upon injury and at the MTZ in two mouse models of glaucoma. In glaucomatous mice, there are protease resistant forms of γ-synuclein, including at the MTZ. Further, mice lacking γ-synuclein fail to up-regulate Mac-2 at the MTZ in response to increased intraocular pressure (IOP). These results suggest the possibility that failure to properly clear axon-derived material at the MTZ, including γ-synuclein, may contribute to axon loss in glaucoma.

Progressive rod-cone degeneration (PRCD) is a small protein residing in the light-sensitive disc membranes of the photoreceptor outer segment. Until now, the function of PRCD has remained enigmatic despite multiple demonstrations that its mutations cause blindness in humans and dogs. Here, we generated a PRCD knockout mouse and observed a striking defect in disc morphogenesis, whereby newly forming discs do not properly flatten. This leads to the budding of disc-derived vesicles, specifically at the site of disc morphogenesis, which accumulate in the interphotoreceptor matrix. The defect in nascent disc flattening only minimally alters the photoreceptor outer segment architecture beyond the site of new disc formation and does not affect the abundance of outer segment proteins and the photoreceptor’s ability to generate responses to light. Interestingly, the retinal pigment epithelium, responsible for normal phagocytosis of shed outer segment material, lacks the capacity to clear the disc-derived vesicles. This deficiency is partially compensated by a unique pattern of microglial migration to the site of disc formation where they actively phagocytize vesicles. However, the microglial response is insufficient to prevent vesicular accumulation and photoreceptors of PRCD knockout mice undergo slow, progressive degeneration. Taken together, these data show that the function of PRCD is to keep evaginating membranes of new discs tightly apposed to each other, which is essential for the high fidelity of photoreceptor disc morphogenesis and photoreceptor survival.

Abnormal structure and function of astrocytes have been observed within the lamina cribrosa region of the optic nerve head (ONH) in glaucomatous neurodegeneration. Glutamate excitotoxicity-mediated mitochondrial alteration has been implicated in experimental glaucoma. However, the relationships among glutamate excitotoxicity, mitochondrial alteration and ONH astrocytes in the pathogenesis of glaucoma remain unknown. We found that functional N-methyl-D-aspartate (NMDA) receptors (NRs) are present in human ONH astrocytes and that glaucomatous human ONH astrocytes have increased expression levels of NRs and the glutamate aspartate transporter. Glaucomatous human ONH astrocytes exhibit mitochondrial fission that is linked to increased expression of dynamin-related protein 1 (DRP1) and its phosphorylation at Serine 616. In BAC ALDH1L1 eGFP or Thy1-CFP transgenic mice, NMDA treatment induced axon loss as well as hypertrophic morphology and mitochondrial fission in astrocytes of the glial lamina. In human ONH astrocytes, NMDA treatment in vitro triggered mitochondrial fission by decreasing mitochondrial length and number, thereby reducing mitochondrial volume density. However, blocking excitotoxicity by memantine (MEM) prevented these alterations by increasing mitochondrial length, number and volume density. In glaucomatous D2 mice, blocking excitotoxicity by MEM inhibited the morphological alteration as well as increased mitochondrial number and volume density in astrocytes of the glial lamina. However, blocking excitotoxicity decreased autophagosome/autolysosome volume density in both astrocytes and axons in the glial lamina of glaucomatous D2 mice. These findings provide evidence that blocking excitotoxicity prevents ONH astrocyte dysfunction in glaucomatous neurodegeneration by increasing mitochondrial fission, increasing mitochondrial volume density and length, and decreasing autophagosome/autolysosome formation.

Oligodendrocytes can adapt to increases in axon diameter through the addition of membrane wraps to myelin segments. Here, we report that myelin segments can also decrease their length in response to optic nerve (ON) shortening during Xenopus laevis metamorphic remodeling. EM-based analyses revealed that myelin segment shortening is accomplished by focal myelin-axon detachments and protrusions from otherwise intact myelin segments. Astrocyte processes remove these focal myelin dystrophies using known phagocytic machinery, including the opsonin milk fat globule-EGF factor 8 (Mfge8) and the downstream effector ras-related C3 botulinum toxin substrate 1 (Rac1). By the end of metamorphic nerve shortening, one-quarter of all myelin in the ON is enwrapped or internalized by astrocytes. As opposed to the removal of degenerating myelin by macrophages, which is usually associated with axonal pathologies, astrocytes selectively remove large amounts of myelin without damaging axons during this developmental remodeling event.thyroid hormone | glia | lipid droplet | Mfge8 M yelin exists as regularly spaced segments that enable fast and efficient transfer of information across long distances through saltatory propagation of action potentials between nodes of Ranvier (1). The number, length, and thickness of individual segments vary with species and nervous system region (2). The proper thickness and length of myelin segments are likely established, at least in part, during the myelination process itself, which involves the dynamic elongation, shortening, and removal of individual segments (3, 4). However, once established, some myelin segments must be modified further to accommodate axonal growth. Developmental increases in axon diameter are coupled to the addition of membrane wraps to myelin segments, thereby maintaining a near-linear relationship between axon caliber and myelin thickness (5, 6). In the peripheral nervous system, Schwann cell myelin segments can also elongate in proportion to nerve length, increasing internodal distances by as much as a factor of four (3, 7). The regulation of myelin on axons is essential for the proper function of the vertebrate nervous system, because both hypomyelination and hypermyelination lead to neuropathy (8). However, the mechanisms involved in myelin segment plasticity have remained poorly understood, in part, because of the difficulty in studying a process that occurs in mammals during a protracted period as the animals mature (2).During metamorphic remodeling of the head in Xenopus laevis, the optic nerve (ON) and its associated axons rapidly shorten in length (9). A description of this process (10) noted abnormally folded myelin on axons and membranous material inside glial cells, suggesting that myelin might be remodeling, thereby providing a model system in which to study myelin plasticity. The current study set out to determine how myelin segments remodel during ON shortening. Results The ON and Its Myelinated Axons Widen and Shorten During X. laevisMetamorphosis. The head of ...