Age- and glaucoma-induced changes to the ocular glymphatic system.

The ocular glymphatic system supports bidirectional fluid transport along the optic nerve, thereby removes metabolic wastes including amyloid-ß. To better understand this biological process, we examined the distributions of intravitreally and intracisternally infused tracers in full-length optic nerves from different age groups of mice. Aging was linked to globally impaired ocular glymphatic fluid transport, similar to what has seen previously in the brain. Aging also reduced the pupillary responsiveness to light stimulation and abolished light-induced facilitation in anterograde ocular glymphatic flow. In contrast to normal aging, in the DBA/2 J model of glaucoma, we found a pathological increase of glymphatic fluid transport to the anterior optic nerve that was associated with dilation of the perivascular spaces. Thus, aging and glaucoma have fundamentally different effects on ocular glymphatic fluid transport. Manipulation of glymphatic fluid transport might therefore present a new target for the treatment of glaucoma.

Early immune responses are independent of RGC dysfunction in glaucoma with complement component C3 being protective.

Various immune response pathways are altered during early, predegenerative stages of glaucoma; however, whether the early immune responses occur secondarily to or independently of neuronal dysfunction is unclear. To investigate this relationship, we used the Wlds allele, which protects from axon dysfunction. We demonstrate that DBA/2J.Wlds mice develop high intraocular pressure (IOP) but are protected from retinal ganglion cell (RGC) dysfunction and neuroglial changes that otherwise occur early in DBA/2J glaucoma. Despite this, immune pathways are still altered in DBA/2J.Wlds mice. This suggests that immune changes are not secondary to RGC dysfunction or altered neuroglial interactions, but may be directly induced by the increased strain imposed by high IOP. One early immune response following IOP elevation is up-regulation of complement C3 in astrocytes of DBA/2J and DBA/2J.Wlds mice. Unexpectedly, because the disruption of other complement components, such as C1Q, is protective in glaucoma, C3 deficiency significantly increased the number of DBA/2J eyes with nerve damage and RGC loss at an early time point after IOP elevation. Transcriptional profiling of C3-deficient cultured astrocytes implicated EGFR signaling as a hub in C3-dependent responses. Treatment with AG1478, an EGFR inhibitor, also significantly increased the number of DBA/2J eyes with glaucoma at the same early time point. These findings suggest that C3 protects from early glaucomatous damage, a process that may involve EGFR signaling and other immune responses in the optic nerve head. Therefore, therapies that target specific components of the complement cascade, rather than global inhibition, may be more applicable for treating human glaucoma.

KLF9 and JNK3 Interact to Suppress Axon Regeneration in the Adult CNS.

Neurons in the adult mammalian CNS decrease in intrinsic axon growth capacity during development in concert with changes in Krüppel-like transcription factors (KLFs). KLFs regulate axon growth in CNS neurons including retinal ganglion cells (RGCs). Here, we found that knock-down of KLF9, an axon growth suppressor that is normally upregulated 250-fold in RGC development, promotes long-distance optic nerve regeneration in adult rats of both sexes. We identified a novel binding partner, MAPK10/JNK3 kinase, and found that JNK3 (c-Jun N-terminal kinase 3) is critical for KLF9's axon-growth-suppressive activity. Interfering with a JNK3-binding domain or mutating two newly discovered serine phosphorylation acceptor sites, Ser106 and Ser110, effectively abolished KLF9's neurite growth suppression in vitro and promoted axon regeneration in vivo These findings demonstrate a novel, physiologic role for the interaction of KLF9 and JNK3 in regenerative failure in the optic nerve and suggest new therapeutic strategies to promote axon regeneration in the adult CNS.SIGNIFICANCE STATEMENT Injured CNS nerves fail to regenerate spontaneously. Promoting intrinsic axon growth capacity has been a major challenge in the field. Here, we demonstrate that knocking down Krüppel-like transcription factor 9 (KLF9) via shRNA promotes long-distance axon regeneration after optic nerve injury and uncover a novel and important KLF9-JNK3 interaction that contributes to axon growth suppression in vitro and regenerative failure in vivo These studies suggest potential therapeutic approaches to promote axon regeneration in injury and other degenerative diseases in the adult CNS.

Trabecular meshwork morphogenesis: A comparative analysis of wildtype and anterior segment dysgenesis mouse models.

The trabecular meshwork (TM), a tissue residing in the iridocorneal angle of the eye, is the primary site of aqueous humor outflow and often develops abnormally in children with anterior segment dysgenesis (ASD). However, the cellular mechanisms underlying both normal and pathophysiological TM formation are poorly understood. Here, we improve the characterization of TM development via morphological and molecular analyses. We first assessed the TM of wild-type C57BL/6J mice at multiple time points throughout development (E15.5-P21). The morphology of TM cells, rate of cell division, presence of apoptotic cell death, and age of onset of an established TM marker (αSMA) were each assessed in the developing iridocorneal angle. We discovered that TM cells are identifiable histologically at P1, which coincided with both the onset of αSMA expression and a significant decrease in TM precursor cell proliferation. Significant apoptotic cell death was not detected during TM development. These findings were then used to assess two mouse models of ASD. Jag1 and Bmp4 heterozygous null mice display ASD phenotypes in the adult, including TM hypoplasia and corneal adherence to the iris. We further discovered that both mutants exhibited similar patterns of developmental TM dysgenesis at P1, P5, and P10. Our data indicate that P1 is an important time point in TM development and that TM dysgenesis in Jag1 and Bmp4 heterozygous null mice likely results from impaired TM cell migration and/or differentiation.

Role of SARM1 and DR6 in retinal ganglion cell axonal and somal degeneration following axonal injury.

Optic neuropathies such as glaucoma are characterized by the degeneration of retinal ganglion cells (RGCs) and the irreversible loss of vision. In these diseases, focal axon injury triggers a propagating axon degeneration and, eventually, cell death. Previous work by us and others identified dual leucine zipper kinase (DLK) and JUN N-terminal kinase (JNK) as key mediators of somal cell death signaling in RGCs following axonal injury. Moreover, others have shown that activation of the DLK/JNK pathway contributes to distal axonal degeneration in some neuronal subtypes and that this activation is dependent on the adaptor protein, sterile alpha and TIR motif containing 1 (SARM1). Given that SARM1 acts upstream of DLK/JNK signaling in axon degeneration, we tested whether SARM1 plays a similar role in RGC somal apoptosis in response to optic nerve injury. Using the mouse optic nerve crush (ONC) model, our results show that SARM1 is critical for RGC axonal degeneration and that axons rescued by SARM1 deficiency are electrophysiologically active. Genetic deletion of SARM1 did not, however, prevent DLK/JNK pathway activation in RGC somas nor did it prevent or delay RGC cell death. These results highlight the importance of SARM1 in RGC axon degeneration and suggest that somal activation of the DLK/JNK pathway is activated by an as-yet-unidentified SARM1-independent signal.

Phospholipid flippase ATP8A2 is required for normal visual and auditory function and photoreceptor and spiral ganglion cell survival.

ATP8A2 is a P4-ATPase that is highly expressed in the retina, brain, spinal cord and testes. In the retina, ATP8A2 is localized in photoreceptors where it uses ATP to transport phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the exoplasmic to the cytoplasmic leaflet of membranes. Although mutations in ATP8A2 have been reported to cause mental retardation in humans and degeneration of spinal motor neurons in mice, the role of ATP8A2 in sensory systems has not been investigated. We have analyzed the retina and cochlea of ATP8A2-deficient mice to determine the role of ATP8A2 in visual and auditory systems. ATP8A2-deficient mice have shortened photoreceptor outer segments, a reduction in photoresponses and decreased photoreceptor viability. The ultrastructure and phagocytosis of the photoreceptor outer segment appeared normal, but the PS and PE compositions were altered and the rhodopsin content was decreased. The auditory brainstem response threshold was significantly higher and degeneration of spiral ganglion cells was apparent. Our studies indicate that ATP8A2 plays a crucial role in photoreceptor and spiral ganglion cell function and survival by maintaining phospholipid composition and contributing to vesicle trafficking.

Quantitative measurement of retinal ganglion cell populations via histology-based random forest classification.

The inner surface of the retina contains a complex mixture of neurons, glia, and vasculature, including retinal ganglion cells (RGCs), the final output neurons of the retina and primary neurons that are damaged in several blinding diseases. The goal of the current work was two-fold: to assess the feasibility of using computer-assisted detection of nuclei and random forest classification to automate the quantification of RGCs in hematoxylin/eosin (H&E)-stained retinal whole-mounts; and if possible, to use the approach to examine how nuclear size influences disease susceptibility among RGC populations. To achieve this, data from RetFM-J, a semi-automated ImageJ-based module that detects, counts, and collects quantitative data on nuclei of H&E-stained whole-mounted retinas, were used in conjunction with a manually curated set of images to train a random forest classifier. To test performance, computer-derived outputs were compared to previously published features of several well-characterized mouse models of ophthalmic disease and their controls: normal C57BL/6J mice; Jun-sufficient and Jun-deficient mice subjected to controlled optic nerve crush (CONC); and DBA/2J mice with naturally occurring glaucoma. The result of these efforts was development of RetFM-Class, a command-line-based tool that uses data output from RetFM-J to perform random forest classification of cell type. Comparative testing revealed that manual and automated classifications by RetFM-Class correlated well, with 83.2% classification accuracy for RGCs. Automated characterization of C57BL/6J retinas predicted 54,642 RGCs per normal retina, and identified a 48.3% Jun-dependent loss of cells at 35 days post CONC and a 71.2% loss of RGCs among 16-month-old DBA/2J mice with glaucoma. Output from automated analyses was used to compare nuclear area among large numbers of RGCs from DBA/2J mice (n = 127,361). In aged DBA/2J mice with glaucoma, RetFM-Class detected a decrease in median and mean nucleus size of cells classified into the RGC category, as did an independent confirmation study using manual measurements of nuclear area demarcated by BRN3A-immunoreactivity. In conclusion, we have demonstrated that histology-based random forest classification is feasible and can be utilized to study RGCs in a high-throughput fashion. Despite having some limitations, this approach demonstrated a significant association between the size of the RGC nucleus and the DBA/2J form of glaucoma.

Notch2 regulates BMP signaling and epithelial morphogenesis in the ciliary body of the mouse eye.

The ciliary body (CB) of the mammalian eye is responsible for secreting aqueous humor to maintain intraocular pressure, which is elevated in the eyes of glaucoma patients. It contains a folded two-layered epithelial structure comprising the nonpigmented inner ciliary epithelium (ICE), the pigmented outer ciliary epithelium (OCE), and the underlying stroma. Although the CB has an important function in the eye, its morphogenesis remains poorly studied. In this study, we show that conditional inactivation of the Jagged 1 (Jag1)-Notch2 signaling pathway in the developing CB abolishes its morphogenesis. Notch2 is expressed in the OCE of the CB, whereas Jag1 is expressed in the ICE. Conditional inactivation of Jag1 in the ICE or Notch2 in the OCE disrupts CB morphogenesis, but neither affects the specification of the CB region. Notch2 signaling in the OCE is required for promoting cell proliferation and maintaining bone morphogenetic protein (BMP) signaling, both of which have been suggested to be important for CB morphogenesis. Although Notch and BMP signaling pathways are known to cross-talk via the interaction between their downstream transcriptional factors, this study suggests that Notch2 maintains BMP signaling in the OCE possibly by repressing expression of secreted BMP inhibitors. Based on our findings, we propose that Jag1-Notch2 signaling controls CB morphogenesis at least in part by regulating cell proliferation and BMP signaling.

Using genetic mouse models to gain insight into glaucoma: Past results and future possibilities.

While all forms of glaucoma are characterized by a specific pattern of retinal ganglion cell death, they are clinically divided into several distinct subclasses, including normal tension glaucoma, primary open angle glaucoma, congenital glaucoma, and secondary glaucoma. For each type of glaucoma there are likely numerous molecular pathways that control susceptibility to the disease. Given this complexity, a single animal model will never precisely model all aspects of all the different types of human glaucoma. Therefore, multiple animal models have been utilized to study glaucoma but more are needed. Because of the powerful genetic tools available to use in the laboratory mouse, it has proven to be a highly useful mammalian system for studying the pathophysiology of human disease. The similarity between human and mouse eyes coupled with the ability to use a combination of advanced cell biological and genetic tools in mice have led to a large increase in the number of studies using mice to model specific glaucoma phenotypes. Over the last decade, numerous new mouse models and genetic tools have emerged, providing important insight into the cell biology and genetics of glaucoma. In this review, we describe available mouse genetic models that can be used to study glaucoma-relevant disease/pathobiology. Furthermore, we discuss how these models have been used to gain insights into ocular hypertension (a major risk factor for glaucoma) and glaucomatous retinal ganglion cell death. Finally, the potential for developing new mouse models and using advanced genetic tools and resources for studying glaucoma are discussed.

Mutations in a P-type ATPase gene cause axonal degeneration.

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.

Transcription factors SOX4 and SOX11 function redundantly to regulate the development of mouse retinal ganglion cells.

SOX family proteins belong to the high-mobility-group (HMG) domain-containing transcription factors, and function as key players to regulate embryonic development and cell fate determination. The highly related group C Sox genes Sox4 and Sox11 are widely expressed in the development of mouse retina and share a similar expression pattern with each other in this process. Here, to investigate the roles of Sox4 and Sox11 in the retinal development, Sox4, Sox11, and Sox4/Sox11 conditional knock-out (CKO) mice with deletion of Sox4, Sox11, and Sox4/Sox11 in retinas were generated. Our studies demonstrated that targeted disruption of Sox4 or Sox11 in retinas caused a moderate reduction of generation of RGCs. However, a complete loss of RGCs was observed in Sox4/Sox11-null retinas, suggesting the two genes play similar roles in the development of RGCs. Our further analysis confirms that Sox4 and Sox11 function redundantly to regulate the generation of RGCs at early embryonic stages as well as the survival of RGCs at late embryonic stages. In addition, we demonstrated that loss of Math5 impairs the expression of Sox4 and Sox11 in the ganglion cell layer while deletion of Brn3b has no effect on the expression of Sox4 and Sox11. Taken together, these findings elucidate SoxC genes as essential contributors to maintain the survival of RGCs, and imply their intermediate position between Math5 and Brn3b in the genetic hierarchy of RGC development.

DLK-dependent signaling is important for somal but not axonal degeneration of retinal ganglion cells following axonal injury.

Injury to retinal ganglion cell (RGC) axons triggers rapid activation of Jun N-terminal kinase (JNK) signaling, a major prodeath pathway in injured RGCs. Of the multiple kinases that can activate JNK, dual leucine kinase (Dlk) is known to regulate both apoptosis and Wallerian degeneration triggered by axonal insult. Here we tested the importance of Dlk in regulating somal and axonal degeneration of RGCs following axonal injury. Removal of DLK from the developing optic cup did not grossly affect developmental RGC death or inner plexiform layer organization. In the adult, Dlk deficiency significantly delayed axonal-injury induced RGC death. The activation of JUN was also attenuated in Dlk deficient retinas. Dlk deficiency attenuated the activation of the somal pool of JNK but did not prevent activation of the axonal pool of JNK after axonal injury, indicating that JNK activation in different cellular compartments of an RGC following axonal injury is regulated by distinct upstream kinases. In contrast to its robust influence on somal degeneration, Dlk deficiency did not alter RGC axonal degeneration after axonal injury as assessed using physiological readouts of optic nerve function.

Tumor necrosis factor alpha has an early protective effect on retinal ganglion cells after optic nerve crush.

BACKGROUND: 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. METHODS: RGC death was induced by optic nerve crush in mice. The role of TNFα in this process was examined by quantitative PCR of Tnfα gene expression, and quantification of cell loss in Tnfα (-/-) mice or in wild-type animals receiving an intraocular injection of exongenous TNFα either before or after crush. Signaling pathways downstream of TNFα were examined by immunolabeling for JUN protein accumulation or activation of EGFP expression in NFκB reporter mice. RESULTS: Optic nerve crush caused a modest increase in Tnfα gene expression, with kinetics similar to the activation of both macroglia and microglia. A pre-injection of TNFα attenuated ganglion cell loss after crush, while ganglion cell loss was more severe in Tnfα (-/-) mice. Conversely, over the long term, a single exposure to TNFα induced extrinsic apoptosis in RGCs. Müller cells responded to exogenous TNFα by accumulating JUN and activating NFκB. CONCLUSION: Early after optic nerve crush, TNFα appears to have a protective role for RGCs, which may be mediated through Müller cells.

Focal damage to macaque photoreceptors produces persistent visual loss.

Insertion of light-gated channels into inner retina neurons restores neural light responses, light evoked potentials, visual optomotor responses and visually-guided maze behavior in mice blinded by retinal degeneration. This method of vision restoration bypasses damaged outer retina, providing stimulation directly to retinal ganglion cells in inner retina. The approach is similar to that of electronic visual protheses, but may offer some advantages, such as avoidance of complex surgery and direct targeting of many thousands of neurons. However, the promise of this technique for restoring human vision remains uncertain because rodent animal models, in which it has been largely developed, are not ideal for evaluating visual perception. On the other hand, psychophysical vision studies in macaque can be used to evaluate different approaches to vision restoration in humans. Furthermore, it has not been possible to test vision restoration in macaques, the optimal model for human-like vision, because there has been no macaque model of outer retina degeneration. In this study, we describe development of a macaque model of photoreceptor degeneration that can in future studies be used to test restoration of perception by visual prostheses. Our results show that perceptual deficits caused by focal light damage are restricted to locations at which photoreceptors are damaged, that optical coherence tomography (OCT) can be used to track such lesions, and that adaptive optics retinal imaging, which we recently used for in vivo recording of ganglion cell function, can be used in future studies to examine these lesions.

IL1A enhances TNF-induced retinal ganglion cell death.

Glaucoma is a neurodegenerative disease that leads to the death of retinal ganglion cells (RGCs). A growing body of literature suggests a role for neuroinflammation in RGC death after glaucoma-relevant insults. For instance, it was shown that deficiency of three proinflammatory cytokines, complement component 1, subcomponent q ( C1q ), interleukin 1 alpha ( Il1a ), and tumor necrosis factor ( Tnf ), resulted in near complete protection of RGCs after two glaucoma-relevant insults, optic nerve injury and ocular hypertension. While TNF and C1Q have been extensively investigated in glaucoma-relevant model systems, the role of IL1A in RGC is not as well defined. Thus, we investigated the direct neurotoxicity of IL1A on RGCs in vivo. Intravitreal injection of IL1A did not result in RGC death at either 14 days or 12 weeks after insult. Consistent with previous studies, TNF injection did not result in significant RGC loss at 14 days but did after 12 weeks. Interestingly, IL1A+TNF resulted in a relatively rapid RGC death, driving significant RGC loss two weeks after injection. JUN activation and SARM1 have been implicated in RGC death in glaucoma and after cytokine insult. Using mice deficient in JUN or SARM1, we show RGC loss after IL1A+TNF insult is JUN-independent and SARM1-dependent. Furthermore, RNA-seq analysis showed that RGC death by SARM1 deficiency does not stop the neuroinflammatory response to IL1A+TNF. These findings indicate that IL1A can potentiate TNF-induced RGC death after combined insult is likely driven by a SARM1-dependent RGC intrinsic signaling pathway.

Neuronal protein phosphatase 1ß regulates glutamate release, cortical myelination, node of Ranvier formation, and action potential propagation in the optic nerve.

Precise regulation of protein phosphorylation is critical for many cellular processes, and dysfunction in this process has been linked to various neurological disorders and diseases. Protein phosphatase 1 (PP1) is a ubiquitously expressed serine/threonine phosphatase with three major isoforms, (α, ß, γ) and hundreds of known substrates. Previously, we reported that PP1α and PP1γ are essential for the known role of PP1 in synaptic physiology and learning/memory, while PP1ß displayed a surprising opposing function. De novo mutations in PP1ß cause neurodevelopmental disorders in humans, but the mechanisms involved are currently unknown. A Cre-Lox system was used to delete PP1ß specifically in neurons in order to study its effects on developing mice. These animals fail to survive to 3 postnatal weeks, and exhibit deficits in cortical myelination and glutamate release. There was defective compound action potential (CAP) propagation in the optic nerve of the null mice, which was traced to a deficit in the formation of nodes of Ranvier. Finally, it was found that phosphorylation of the PP1ß-specific substrate, myosin light chain 2 (MLC2), is significantly enhanced in PP1ß null optic nerves. Several novel important in vivo roles of PP1ß in neurons were discovered, and these data will aid future investigations in delineating the mechanisms by which de novo mutations in PP1ß lead to intellectual and developmental delays in patients.

Microglia Depletion leads to Increased Susceptibility to Ocular Hypertension-Dependent Glaucoma.

In recent years, microglia have been highlighted for playing integral roles in neurodegenerative diseases, like glaucoma. To better understand the role of microglia during chronic ocular hypertension, we depleted microglia from aged (9-12 months old) DBA/2J (D2) mice, which exhibit age-related increases in intraocular pressure, using a dietary CSF1R antagonist, PLX5622. Retinal ganglion cell (RGC) somas were counted, and optic nerve cross-sections stained and assessed for glaucomatous damage. Sustained administration of dietary PLX5622 significantly reduced the numbers of retinal microglia. Dietary PLX5622 did not lead to changes in intraocular pressure in D2 or normotensive DBA/2J-Gpnmb+ (D2-Gpnmb+) control mice. While PLX5622-treated D2-Gpnmb+ did not develop optic nerve damage, PLX5622-treated D2 mice showed a significant increase in moderate-to-severe optic nerve damage compared to D2 mice fed a control diet. In conclusion, global reduction of microglia exacerbated glaucomatous neurodegeneration in D2 mice suggesting microglia play an overall beneficial role in protecting from ocular hypertension associated RGC loss.

The Usher 1B protein, MYO7A, is required for normal localization and function of the visual retinoid cycle enzyme, RPE65.

Mutations in the MYO7A gene cause a deaf-blindness disorder, known as Usher syndrome 1B.  In the retina, the majority of MYO7A is in the retinal pigmented epithelium (RPE), where many of the reactions of the visual retinoid cycle take place.  We have observed that the retinas of Myo7a-mutant mice are resistant to acute light damage. In exploring the basis of this resistance, we found that Myo7a-mutant mice have lower levels of RPE65, the RPE isomerase that has a key role in the retinoid cycle.  We show for the first time that RPE65 normally undergoes a light-dependent translocation to become more concentrated in the central region of the RPE cells.  This translocation requires MYO7A, so that, in Myo7a-mutant mice, RPE65 is partly mislocalized in the light.  RPE65 is degraded more quickly in Myo7a-mutant mice, perhaps due to its mislocalization, providing a plausible explanation for its lower levels.  Following a 50-60% photobleach, Myo7a-mutant retinas exhibited increased all-trans-retinyl ester levels during the initial stages of dark recovery, consistent with a deficiency in RPE65 activity.  Lastly, MYO7A and RPE65 were co-immunoprecipitated from RPE cell lysate by antibodies against either of the proteins, and the two proteins were partly colocalized, suggesting a direct or indirect interaction.  Together, the results support a role for MYO7A in the translocation of RPE65, illustrating the involvement of a molecular motor in the spatiotemporal organization of the retinoid cycle in vision.

JUN regulates early transcriptional responses to axonal injury in retinal ganglion cells.

The AP1 family transcription factor JUN is an important molecule in the neuronal response to injury. In retinal ganglion cells (RGCs), JUN is upregulated soon after axonal injury and disrupting JUN activity delays RGC death. JUN is known to participate in the control of many different injury response pathways in neurons, including pathways controlling cell death and axonal regeneration. The role of JUN in regulating genes involved in cell death, ER stress, and regeneration was tested to determine the overall importance of JUN in regulating RGC response to axonal injury. Genes from each of these pathways were transcriptionally controlled following axonal injury and Jun deficiency altered the expression of many of these genes. The differentially expressed genes included, Atf3, Ddit3, Ecel1, Gadd45α, Gal, Hrk, Pten, Socs3, and Sprr1a. Two of these genes, Hrk and Atf3, were tested for importance in RGC death using null alleles of each gene. Disruption of the prodeath Bcl2 family member Hrk did not affect the rate or amount of RGC death after axonal trauma. Deficiency in the ATF/CREB family transcription factor Atf3 did lessen the amount of RGC death after injury, though it did not provide long term protection to RGCs. Since JUN's dimerization partner determines its transcriptional targets, the expression of several candidate AP1 family members were examined. Multiple AP1 family members were induced by axonal injury and had a different expression profile in Jun deficient retinas compared to wildtype retinas (Fosl1, Fosl2 and Jund). Overall, JUN appears to play a multifaceted role in regulating RGC response to axonal injury.

BCL2L1 (BCL-X) promotes survival of adult and developing retinal ganglion cells.

The Bcl-2 family is responsible for regulating cell death pathways in neurons during development, after injury and in disease. The activation of the pro-death family member BAX is often the final step before cell death in neurons. Pro-survival family members such as BCL-X (BCL2L1) act to inhibit BAX activation. Overexpression studies have suggested that BCL-X could play an important physiological role in mediating neuronal viability. Loss-of-function studies performed in vivo have implicated BCL-X as a mediator of neuronal survival during the early stages of neurodevelopment. To assess whether BCL-X is needed to promote the survival of neurons in the central nervous system throughout life, Bcl-x was conditionally removed from the optic cup or throughout the adult mouse. During development BCL-X was required for the survival of differentiating retinal ganglion cells (RGCs) leading up to their normal window of developmental death. Despite its expression in adult RGCs, BCL-X was not required for maintaining RGC viability in adult retinas. However, the loss of BCL-X in adult RGCs did significantly increase the rate of death of RGCs after axonal injury. Thus, in developing and injured RGCs there appears to be an active cell survival program preventing neuronal death.