The discovery of long-lived epithelial stem cells in the bulge region of the hair follicle led to the hypothesis that epidermal renewal and epidermal repair after wounding both depend on these cells. To determine whether bulge cells are necessary for epidermal renewal, here we have ablated these cells by targeting them with a suicide gene encoding herpes simplex virus thymidine kinase (HSV-TK) using a Keratin 1-15 (Krt1-15) promoter. We show that ablation leads to complete loss of hair follicles but survival of the epidermis. Through fate-mapping experiments, we find that stem cells in the hair follicle bulge do not normally contribute cells to the epidermis which is organized into epidermal proliferative units, as previously predicted. After epidermal injury, however, cells from the bulge are recruited into the epidermis and migrate in a linear manner toward the center of the wound, ultimately forming a marked radial pattern. Notably, although the bulge-derived cells acquire an epidermal phenotype, most are eliminated from the epidermis over several weeks, indicating that bulge stem cells respond rapidly to epidermal wounding by generating short-lived 'transient amplifying' cells responsible for acute wound repair. Our findings have implications for both gene therapy and developing treatments for wounds because it will be necessary to consider epidermal and hair follicle stem cells as distinct populations.

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...

S-nitrosylation by nitric oxide (NO) is a major mode of signaling to cellular proteins1, including prominent nuclear proteins such as HDAC22 and PARP13. The high reactivity of the NO group with protein thiols implies the existence of selective targeting mechanisms. Specificity of NO signaling is often achieved by the binding of NO synthase (NOS) to target proteins, either directly4 or through scaffolding proteins such as PSD-955 and CAPON6. As the three principal isoforms of NOS - neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) - are primarily non-nuclear, the mechanisms by which nuclear proteins are selectively nitrosylated have been elusive. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is physiologically nitrosylated at its Cys150 residue, conferring upon it the ability to bind to Siah1, which possesses a nuclear localization signal and conveys nitrosylated GAPDH (SNO-GAPDH) to the nucleus7. We now show that SNO-GAPDH physiologically transnitrosylates nuclear proteins, including the deacetylating enzyme SIRT1, histone deacetylase-2 (HDAC2), and DNA-activated protein kinase (DNA-PK). Our findings reveal a novel mechanism for targeted nitrosylation of nuclear proteins and suggest that protein-protein transfer of NO groups may be a general mechanism in cellular signal transduction.

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.G laucoma is the leading cause of irreversible blindness worldwide (1). It is a neurodegenerative disease in which vision loss is caused by the axonal injury and death of retinal ganglion cells (RGCs) (2), the projection neurons that process and transmit vision from the retina to the brain. Current therapies (i.e., surgery, laser, and eye drops) all act by lowering intraocular pressure (IOP). However, pressure reduction can be difficult to achieve, and even with significant pressure lowering, RGC loss can continue. Efforts have therefore been made to develop neuroprotective agents that would complement IOP-lowering therapies by directly inhibiting the RGC cell death process (3, 4). However, no neuroprotective agent has yet been approved for clinical use.Protein kinases provide attractive targets for the development of neuroprotective agents. A number of kinases, including cyclindependent kinases, death-associated protein kinases, JNK1-3, MAPKs, and glycogen synthase kinase-3β, are involved in neuronal cell death (5-12). An additional attraction is that protein kinases are readily druggable. The pharmacology and medicinal chemistry of kinase inhibitors are well-developed, with kinases now being the most important class of drug targets after G protein-coupled receptors (13). Although the primary clinical use of kinase inhibitors continues to be as antineoplastic agents, increasing attention is being paid to their use in other areas (14,15).To identify, in a comprehensive and unbiased manner, kinases that could serve as targets for neuroprotective glaucoma therapy, we screened the entire mouse kinome for kinases whose inhibition promotes RGC survival. For this screen, we develope...