Transcellular degradation of axonal mitochondria.

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.

Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death.

Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs. In cultured RGCs, DLK signaling is both necessary and sufficient for cell death. DLK undergoes robust posttranscriptional up-regulation in response to axonal injury in vitro and in vivo. Using a conditional knockout approach, we confirmed that DLK is required for RGC JNK activation and cell death in a rodent model of optic neuropathy. In addition, tozasertib, a small molecule protein kinase inhibitor with activity against DLK, protects RGCs from cell death in rodent glaucoma and traumatic optic neuropathy models. Together, our results establish a previously undescribed drug/drug target combination in glaucoma, identify an early marker of RGC injury, and provide a starting point for the development of more specific neuroprotective DLK inhibitors for the treatment of glaucoma, nonglaucomatous forms of optic neuropathy, and perhaps other CNS neurodegenerations.

Myelination transition zone astrocytes are constitutively phagocytic and have synuclein dependent reactivity in glaucoma.

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 up-regulate 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.

A conserved motif at the C terminus of sororin is required for sister chromatid cohesion.

Sororin is a positive regulator of sister chromatid cohesion that interacts with the cohesin complex. Sororin is required for the increased stability of the cohesin complex on chromatin following DNA replication and sister chromatid cohesion during G(2). The mechanism by which sororin ensures cohesion is currently unknown. Because the primary sequence of sororin does not contain any previously characterized structural or functional motifs, we have undertaken a structure-function analysis of the sororin protein. Using a series of mutant derivatives of sororin, we show that the ability of sororin to bind to chromatin is separable from both its role in sister chromatid cohesion and its interaction with the cohesin complex. We also show that derivatives of sororin with deletions or mutations in the conserved C terminus fail to rescue the loss-of-cohesion phenotype caused by sororin RNAi and that these mutations also abrogate the association of sororin with the cohesin complex. Our data suggest that the interaction of the highly conserved motif at the C terminus of sororin with the cohesin complex is critical to its ability to mediate sister chromatid cohesion.

Repetitive mild traumatic brain injury with impact acceleration in the mouse: Multifocal axonopathy, neuroinflammation, and neurodegeneration in the visual system.

Repetitive mild traumatic brain injury (mTBI) is implicated in chronic neurological illness. The development of animal models of repetitive mTBI in mice is essential for exploring mechanisms of these chronic diseases, including genetic vulnerability by using transgenic backgrounds. In this study, the rat model of impact acceleration (IA) was redesigned for the mouse cranium and used in two clinically relevant repetitive mTBI paradigms. We first determined, by using increments of weight dropped from 1m that the 40g weight was most representative of mTBI and was not associated with fractures, brain contusions, anoxic-ischemic injury, mortality, or significant neurological impairments. Quantitative evaluation of traumatic axonal injury (TAI) in the optic nerve/tract, cerebellum and corpus callosum confirmed that weight increase produced a graded injury. We next evaluated two novel repetitive mTBI paradigms (1 time per day or 3 times per day at days 0, 1, 3, and 7) and compared the resulting TAI, neuronal cell death, and neuroinflammation to single hit mTBI at sub-acute (7days) and chronic time points (10weeks) post-injury. Both single and repetitive mTBI caused TAI in the optic nerve/tract, cerebellum, corticospinal tract, lateral lemniscus and corpus callosum. Reactive microglia with phagocytic phenotypes were present at injury sites. Severity of axonal injury corresponded to impact load and frequency in the optic nerve/tract and cerebellum. Both single and repeat injury protocols were associated with retinal ganglion cell loss and optic nerve degeneration; these outcomes correlated with impact load and number/frequency. No phosphorylated tau immunoreactivity was detected in the brains of animals subjected to repetitive mTBI. Our findings establish a new model of repetitive mTBI model featured by TAI in discrete CNS tracts, especially the visual system and cerebellum. Injury in retina and optic nerve provides a sensitive measure of severity of mTBI, thus enabling further studies on mechanisms and experimental therapeutics. Our model can also be useful in exploring mechanisms of chronic neurological disease caused by repetitive mTBI in wild-type and transgenic mice.

Evidence for accelerated tauopathy in the retina of transgenic P301S tau mice exposed to repetitive mild traumatic brain injury.

Chronic traumatic encephalopathy (CTE) is associated with repetitive mild traumatic brain injury (mTBI) in the context of contact and collision sports, but not all exposed individuals develop this condition. In addition, experiments in animal models in several laboratories have shown that non-transgenic mice do not develop tauopathy after exposure to repetitive mTBI schedules. It is thus reasonable to assume that genetic factors may play an etiological role in the development of CTE. More than 40 mutations in the tau gene are known to confer proneness to aggregation and are thought to cause neurodegenerative diseases including frontotemporal degeneration (FTD). Transgenic mice harboring these mutations can be used to ask the question whether repetitive mTBI can accelerate onset and course of tauopathy or worsen the outcomes of transgenic disease. In this study, we exposed mice harboring the tau P301S transgene associated with FTD to repetitive mTBI schedules by impact acceleration (IA) that we have previously characterized. We explored the progression of tauopathy in the retina and neocortex based on density of neuronal profiles loaded with tau pS422, a marker of advanced tau hyperphosphorylation. We found that the density of tau pS422 (+) retinal ganglion cells (RGCs) increased twenty fold with one mTBI hit, a little over fifty fold with four mTBI hits and sixty fold with 12 mTBI hits. The severity of mTBI burden (number of hits) was a significant factor in tauopathy outcome. On the other hand, we found no association between repetitive mTBI and density of pS422 (+) neuronal profiles in neocortex, a region that is not featured by significant TAI in our repetitive mTBI model. We observed similar, but less prominent, trends in tauopathy-prone transgenic mice harboring all 6 isoforms of wild-type human tau without mouse tau. Our findings indicate that repetitive mTBI accelerates tauopathy under diverse genetic conditions predisposing to tau aggregation and suggest a vulnerability-stress model in understanding some cases of acquired neurodegenerative disease after repetitive mTBI.