Golgi morphology and function are dependent on an intact microtubule and actin cytoskeleton. Myosin VI, an unusual actin-based motor protein moving towards the minus ends of actin filaments, has been localized to the Golgi complex at the light and electron microscopic level. Myosin VI is present in purified Golgi membranes as a peripheral membrane protein, targeted by its globular tail domain. To investigate the function of myosin VI at the Golgi complex, immortal fibroblastic cell lines of Snell's waltzer mice lacking myosin VI were established. In these cell lines, where myosin VI is absent, the Golgi complex is reduced in size by approximately 40% compared with wild-type cells. Furthermore, protein secretion of a reporter protein from Snell's waltzer cells is also reduced by 40% compared with wild-type cells. Rescue experiments showed that fully functional myosin VI was able to restore Golgi complex morphology and protein secretion in Snell's waltzer cells to the same level as that observed in wild-type cells.

Glaucoma is an optic neuropathy that is characterized by the loss of retinal ganglion cells (RGCs) initiated by damage to axons in the optic nerve. The degeneration and death of RGCs has been thought to occur in two waves. The first is axogenic, caused by direct insult to the axon. The second is somatic, and is thought to be caused by the production of inflammatory cytokines from the activated retinal innate immune cells. One of the cytokines consistently linked to glaucoma and RGC damage has been TNFα. Despite strong evidence implicating this protein in neurodegeneration, a direct injection of TNFα does not mimic the rapid loss of RGCs observed after acute optic nerve trauma or exposure to excitotoxins. This suggests that our understanding of TNFα signaling is incomplete.

Some pathological manifestations of diabetes in the eye include retinopathy, cataracts and elevated intraocular pressure (IOP). Loss of retinal ganglion cells (RGCs) in non-proliferative stages of diabetic retinopathy and small increases in IOP in diabetic patients has raised the possibility that diabetes affects the development and progression of ocular hypertension and glaucoma. The Ins2Akita mutation is known to cause diabetes and retinopathy on a C57BL/6J (B6) background by as early as 3 months of age. Here, the impact of the Akita mutation on glaucoma was assessed using DBA/2J (D2) mice, a widely used mouse model of ocular hypertension induced glaucoma. In D2.Ins2Akita/+ mice, the contribution of diabetes to vascular permeability, IOP elevation, RGC loss, and glaucoma development was assessed. D2.Ins2Akita/+ mice developed a severe diabetic nephropathy and early mortality between 6-8 months of age. This agrees with previous reports showing that the D2 background is more susceptible to diabetes than the B6 background. In addition, D2.Ins2Akita/+ mice had vascular leakage, astrocyte reactivity and a significant increase in IOP. However no RGC loss and no anterograde axonal transport dysfunction were found at 8.5 months of age. Therefore, our data show that despite severe diabetes and an increased IOP compared to controls, RGCs do not lose axon transport or degenerate. This may be due to a DBA/2J-specific genetic modifier(s) that could provide novel and important avenues for developing new therapies for diabetic retinopathy and possibly glaucoma.

Mutations in collagen, type IV, alpha 1 (COL4A1), a major component of basement membranes, cause multisystem disorders in humans and mice. In the eye, these include anterior segment dysgenesis, optic nerve hypoplasia and retinal vascular tortuosity. Here we investigate the retinal pathology in mice carrying dominant-negative Col4a1 mutations. To this end, we examined retinas longitudinally in vivo using fluorescein angiography, funduscopy and optical coherence tomography. We assessed retinal function by electroretinography and studied the retinal ultrastructural pathology. Retinal examinations revealed serous chorioretinopathy, retinal hemorrhages, fibrosis or signs of pathogenic angiogenesis with chorioretinal anastomosis in up to approximately 90% of Col4a1 mutant eyes depending on age and the specific mutation. To identify the cell-type responsible for pathogenesis we generated a conditional Col4a1 mutation and determined that primary vascular defects underlie Col4a1-associated retinopathy. We also found focal activation of Müller cells and increased expression of pro-angiogenic factors in retinas from Col4a1(+/Δex41)mice. Together, our findings suggest that patients with COL4A1 and COL4A2 mutations may be at elevated risk of retinal hemorrhages and that retinal examinations may be useful for identifying patients with COL4A1 and COL4A2 mutations who are also at elevated risk of hemorrhagic strokes.

We have observed that the commonly used ketamine/xylazine anesthesia mix can induce a focally severe and permanent corneal opacity. The purpose of this study was to establish the clinical and histological features of this deleterious side effect, its sensitivity with respect to age and anesthesia protocol, and approaches for avoiding it.

Glaucoma is a neurodegenerative disease characterized by the apoptotic death of retinal ganglion cells (RGCs). The primary insult to RGCs in glaucoma is thought to occur to their axons as they exit the eye in the optic nerve head. However, pathological signaling pathways that exert central roles in triggering RGC death following axonal injury remain unidentified. It is likely that the first changes to occur following axonal injury are signal relay events that transduce the injury signal from the axon to the cell body. Here we focus on the c-Jun N-terminal kinase (JNK1-3) family, a signaling pathway implicated in axonal injury signaling and neurodegenerative apoptosis, and likely to function as a central node in axonal injury-induced RGC death. We show that JNK signaling is activated immediately after axonal injury in RGC axons at the site of injury. Following its early activation, sustained JNK signaling is observed in axonally-injured RGCs in the form of JUN phosphorylation and upregulation. Using mice lacking specific Jnk isoforms, we show that Jnk2 and Jnk3 are the isoforms activated in injured axons. Combined deficiency of Jnk2 and Jnk3 provides robust long-term protection against axonal injury-induced RGC death and prevents downregulation of the RGC marker, BRN3B, and phosphorylation of JUN. Finally, using Jun deficient mice, we show that JUN-dependent pathways are important for axonal injury-induced RGC death. Together these data demonstrate that JNK signaling is the major early pathway triggering RGC death after axonal injury and may directly link axon injury to transcriptional activity that controls RGC death.

Using a variety of double and triple labeling techniques, we have reevaluated the death of retinal neurons in a mouse model of hereditary glaucoma. Cell-specific markers and total neuron counts revealed no cell loss in any retinal neurons other than the ganglion cells. Within the limits of our ability to define cell types, no group of ganglion cells was especially vulnerable or resistant to degeneration. Retrograde labeling and neurofilament staining showed that axonal atrophy, dendritic remodeling, and somal shrinkage (at least of the largest cell types) precedes ganglion cell death in this glaucoma model. Regions of cell death or survival radiated from the optic nerve head in fan-shaped sectors. Collectively, the data suggest axon damage at the optic nerve head as an early lesion, and damage to axon bundles would cause this pattern of degeneration. However, the architecture of the mouse eye seems to preclude a commonly postulated source of mechanical damage within the nerve head.

Here, we use a mouse model (DBA/2J) to readdress the location of insult(s) to retinal ganglion cells (RGCs) in glaucoma. We localize an early sign of axon damage to an astrocyte-rich region of the optic nerve just posterior to the retina, analogous to the lamina cribrosa. In this region, a network of astrocytes associates intimately with RGC axons. Using BAX-deficient DBA/2J mice, which retain all of their RGCs, we provide experimental evidence for an insult within or very close to the lamina in the optic nerve. We show that proximal axon segments attached to their cell bodies survive to the proximity of the lamina. In contrast, axon segments in the lamina and behind the eye degenerate. Finally, the Wld(s) allele, which is known to protect against insults to axons, strongly protects against DBA/2J glaucoma and preserves RGC activity as measured by pattern electroretinography. These experiments provide strong evidence for a local insult to axons in the optic nerve.

In addition to needing acute emergency management, blast-mediated traumatic brain injury (TBI) is also a chronic disorder with delayed-onset symptoms that manifest and progress over time. While the immediate consequences of acute blast injuries are readily apparent, chronic sequelae are harder to recognize. Indeed, the identification of individuals with mild-TBI or TBI-induced symptoms is greatly impaired in large part due to the lack of objective and robust biomarkers. The purpose of this study was to address these need by identifying candidates for serum-based biomarkers of blast TBI, and also to identify unique or differentially regulated protein expression in the thalamus in C57BL/6J mice exposed to blast using high throughput qualitative screens of protein expression. To identify thalamic proteins differentially or uniquely associated with blast exposure, we utilized an antibody-based affinity-capture strategy (referred to as "proteomics-based analysis of depletomes"; PAD) to deplete thalamic lysates from blast-treated mice of endogenous thalamic proteins also found in control mice. Analysis of this "depletome" detected 75 unique proteins, many with associations to the myelin sheath. To identify blast-associated proteins eliciting production of circulating autoantibodies, serum antibodies of blast-treated mice were immobilized, and their immunogens subsequently identified by proteomic analysis of proteins specifically captured following incubation with thalamic lysates (a variant of a strategy referred to as "proteomics-based expression library screening"; PELS). This analysis identified 46 blast-associated immunogenic proteins, including 6 shared in common with the PAD analysis (ALDOA, PHKB, HBA-A1, DPYSL2, SYN1, and CKB). These proteins and their autoantibodies are appropriate for further consideration as biomarkers of blast-mediated TBI.

Proliferative vitreoretinopathy (PVR) is characterized by the growth and contraction of cellular membranes within the vitreous cavity and on both surfaces of the retina, resulting in recurrent retinal detachments and poor visual outcomes. Proinflammatory cytokines like tumor necrosis factor alpha (TNFα) have been associated with PVR and the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells. Cigarette smoke is the only known modifiable risk factor for PVR, but the mechanisms are unclear. The purpose of this study was to examine the impact of cigarette smoke on the proinflammatory TNFα/NF-κB/Snail pathway in RPE cells to better understand the mechanisms through which cigarette smoke increases the risk of PVR. Human ARPE-19 cells were exposed to cigarette smoke extract (CSE), for 4 to 24-hours and TNFα, Snail, IL-6, IL-8, and α-SMA levels were analyzed by qPCR and/or Western blot. The severity of PVR formation was assessed in a murine model of PVR after intravitreal injection of ARPE-19 cells pre-treated with CSE or not. Fundus imaging, OCT imaging, and histologic analysis 4 weeks after injection were used to examine PVR severity. ARPE-19 cells exposed to CSE expressed higher levels of TNFα, SNAIL, IL6 and IL8 mRNA as well as SNAIL, Vimentin and α-SMA protein. Inhibition of TNFα and NF-κB pathways blocked the effect of CSE. In vivo, intravitreal injection of ARPE-19 cells treated with CSE resulted in more severe PVR compared to mice injected with untreated RPE cells. These studies suggest that the TNFα pathway is involved in the mechanism whereby cigarette smoke increases PVR. Further investigation into the role of TNFα/NF-κB/Snail in driving PVR and pharmacological targeting of these pathways in disease are warranted.

Ocular tissues of mice have been studied in many ways using replication-deficient species C type 5 adenovirus (Ad5) as a tool for manipulating gene expression. Whereas refinements to injection protocols and tropism have led to several advances in targeting cells of interest, there remains a relative lack of information concerning how Ad5 may influence other ocular cell types capable of confounding experimental interpretation. Here, a slit lamp is used to thoroughly photodocument the sequelae of intraocular Ad5 injections over time in mice, with attention to potentially confounding indices of inflammation.

Endothelin (EDN, also known as ET) signaling has been suggested to be an important mediator of retinal ganglion cell (RGC) death in glaucoma. Antagonism of EDN receptors (EDNRA and EDNRB, also known as ET-A and ET-B) prevented RGC death in mouse models of chronic ocular hypertension, and intravitreal injection of EDN ligand was sufficient to drive RGC death. However, it remains unclear which cell types EDN ligands directly affect to elicit RGC death. Multiple cell types in the retina and optic nerve express EDNRA and EDNRB and thus could respond to EDN ligands in the context of glaucoma. Here, we systematically deleted Edn receptors from specific cell types to identify the critical EDN receptor mediating RGC death in vivo. Deletion of both Ednra and Ednrb from retinal neurons (including RGCs) and macroglia did not prevent RGC loss after exposure to EDN1 ligands, suggesting EDN1 ligands cause RGC death via an indirect mechanism involving a secondary cell type. Deletion of Ednra from the full body, and then specifically from vascular mural cells, prevented EDN1-induced vasoconstriction and RGC death. Together, these data suggest EDN ligands cause RGC death via a mechanism initiated by vascular mural cells. It is possible RGC death is a consequence of vascular mural cell-induced vasoconstriction and its pathological sequelae. These results highlight the potential importance of neurovascular dysfunction in glaucoma.

Despite popularity of optical coherence tomography (OCT) in glaucoma studies, it's unclear how well OCT-derived metrics compare to traditional measures of retinal ganglion cell (RGC) abundance. Here, Diversity Outbred (J:DO) mice are used to directly compare ganglion cell complex (GCC) thickness measured by OCT to metrics of retinal anatomy measured ex vivo with retinal wholemounts and optic nerve histology.

Anterior chamber depth (ACD) is a quantitative trait associated with primary angle closure glaucoma (PACG). Although ACD is highly heritable, known genetic variations explain a small fraction of the phenotypic variability. The purpose of this study was to identify additional ACD-influencing loci using strains of mice. Cohorts of 86 N2 and 111 F2 mice were generated from crosses between recombinant inbred BXD24/TyJ and wild-derived CAST/EiJ mice. Using anterior chamber optical coherence tomography, mice were phenotyped at 10-12 weeks of age, genotyped based on 93 genome-wide SNPs, and subjected to quantitative trait locus (QTL) analysis. In an analysis of ACD among all mice, six loci passed the significance threshold of p = 0.05 and persisted after multiple regression analysis. These were on chromosomes 6, 7, 11, 12, 15 and 17 (named Acdq6, Acdq7, Acdq11, Acdq12, Acdq15, and Acdq17, respectively). Our findings demonstrate a quantitative multi-genic pattern of ACD inheritance in mice and identify six previously unrecognized ACD-influencing loci. We have taken a unique approach to studying the anterior chamber depth phenotype by using mice as genetic tool to examine this continuously distributed trait.

The R345W mutation in EFEMP1 causes malattia leventinese, an autosomal dominant eye disease with pathogenesis similar to an early-onset age-related macular degeneration. In mice, Efemp1R345W does not cause detectable degeneration but small subretinal deposits do accumulate. The purpose of this study was to determine whether there were abnormal responses to light at this presymptomatic stage in Efemp1R345W mice.

Neuronal loss and axonal degeneration are important pathological features of many neurodegenerative diseases. The molecular mechanisms underlying the majority of axonal degeneration conditions remain unknown. To better understand axonal degeneration, we studied a mouse mutant wabbler-lethal (wl). Wabbler-lethal (wl) mutant mice develop progressive ataxia with pronounced neurodegeneration in the central and peripheral nervous system. Previous studies have led to a debate as to whether myelinopathy or axonopathy is the primary cause of neurodegeneration observed in wl mice. Here we provide clear evidence that wabbler-lethal mutants develop an axonopathy, and that this axonopathy is modulated by Wld(s) and Bax mutations. In addition, we have identified the gene harboring the disease-causing mutations as Atp8a2. We studied three wl alleles and found that all result from mutations in the Atp8a2 gene. Our analysis shows that ATP8A2 possesses phosphatidylserine translocase activity and is involved in localization of phosphatidylserine to the inner leaflet of the plasma membrane. Atp8a2 is widely expressed in the brain, spinal cord, and retina. We assessed two of the mutant alleles of Atp8a2 and found they are both nonfunctional for the phosphatidylserine translocase activity. Thus, our data demonstrate for the first time that mutation of a mammalian phosphatidylserine translocase causes axon degeneration and neurodegenerative disease.

Optic nerve injury is an important pathological component in neurodegenerative diseases such as traumatic optic neuropathies and glaucoma. The molecular signaling pathway(s) critical for retinal ganglion cell (RGC) death after axonal insult, however, is/are not fully defined. RGC death after axonal injury is known to occur by BAX-dependent apoptosis. Two transcription factors JUN (the canonical target of JNK) and DDIT3 (CHOP; a key mediator of the endoplasmic reticulum stress response) are known to be important apoptotic signaling molecules after axonal injury, including in RGCs. However, neither Jun nor Ddit3 deficiency provide complete protection to RGCs after injury. Since Jun and Ddit3 are important apoptotic signaling molecules, we sought to determine if their combined deficiency might provide additive protection to RGCs after axonal injury.

Heterozygous Bmp4 mutations in humans and mice cause severe ocular anterior segment dysgenesis (ASD). Abnormalities include pupil displacement, corneal opacity, iridocorneal adhesions, and variable intraocular pressure, as well as some retinal and vascular defects. It is presently not known what source of BMP4 is responsible for these defects, as BMP4 is expressed in several developing ocular and surrounding tissues. In particular, BMP4 is expressed in the ciliary margins of the optic cup which give rise to anterior segment structures such as the ciliary body and iris, making it a good candidate for the required source of BMP4 for anterior segment development. Here, we test whether ciliary margin-derived BMP4 is required for ocular development using two different conditional knockout approaches. In addition, we compared the conditional deletion phenotypes with Bmp4 heterozygous null mice. Morphological, molecular, and functional assays were performed on adult mutant mice, including histology, immunohistochemistry, in vivo imaging, and intraocular pressure measurements. Surprisingly, in contrast to Bmp4 heterozygous mutants, our analyses revealed that the anterior and posterior segments of Bmp4 conditional knockouts developed normally. These results indicate that ciliary margin-derived BMP4 does not have a major role in ocular development, although subtle alterations could not be ruled out. Furthermore, we demonstrated that the anterior and posterior phenotypes observed in Bmp4 heterozygous animals showed a strong propensity to co-occur, suggesting a common, non-cell autonomous source for these defects.

Latanoprost is a common glaucoma medication. Here, we study longitudinal effects of sustained latanoprost treatment on intraocular pressure (IOP) in C57BL/6J mice, as well as two potential side-effects, changes in iris pigmentation and central corneal thickness (CCT). Male C57BL/6J mice were treated daily for 16 weeks with latanoprost. Control mice were treated on the same schedule with the preservative used with latanoprost, benzalkonium chloride (BAK), or handled, without ocular treatments. IOP and CCT were studied at pre-treatment, 2 "early" time points, and 2 "late" time points; slit-lamp analysis performed at a late time point; and expression of corneal and iridial candidate genes analyzed at the end of the experiment. Latanoprost lowered IOP short, but not long-term. Sustained application of BAK consistently resulted in significant corneal thinning, whereas sustained treatment with latanoprost resulted in smaller and less consistent changes. Neither treatment affected iris pigmentation, corneal matrix metalloprotease expression or iridial pigment-related genes expression. In summary, latanoprost initially lowered IOP in C57BL/6J mice, but became less effective with sustained treatment, likely due to physiological adaptation. These results identify a new resource for studying changes in responsiveness associated with long-term treatment with latanoprost and highlight detrimental effects of commonly used preservative BAK.

The Shack-Hartmann wavefront sensor (SHWS) spots upon which ocular aberration measurements depend have poor quality in mice due to light reflected from multiple retinal layers. We have designed and implemented a SHWS that can favor light from a specific retinal layer and measured monochromatic aberrations in 20 eyes from 10 anesthetized C57BL/6J mice. Using this instrument, we show that mice are myopic, not hyperopic as is frequently reported. We have also measured longitudinal chromatic aberration (LCA) of the mouse eye and found that it follows predictions of the water-filled schematic mouse eye. Results indicate that the optical quality of the mouse eye assessed by measurement of its aberrations is remarkably good, better for retinal imaging than the human eye. The dilated mouse eye has a much larger numerical aperture (NA) than that of the dilated human eye (0.5 NA vs. 0.2 NA), but it has a similar amount of root mean square (RMS) higher order aberrations compared to the dilated human eye. These measurements predict that adaptive optics based on this method of wavefront sensing will provide improvements in retinal image quality and potentially two times higher lateral resolution than that in the human eye.