Retinal ganglion cells adapt to ionic stress in experimental glaucoma

IntroductionIdentification of early adaptive and maladaptive neuronal stress responses is an important step in developing targeted neuroprotective therapies for degenerative disease. In glaucoma, retinal ganglion cells (RGCs) and their axons undergo progressive degeneration resulting from stress driven by sensitivity to intraocular pressure (IOP). Despite therapies that can effectively manage IOP many patients progress to vision loss, necessitating development of neuronal-based therapies. Evidence from experimental models of glaucoma indicates that early in the disease RGCs experience altered excitability and are challenged with dysregulated potassium (K+) homeostasis. Previously we demonstrated that certain RGC types have distinct excitability profiles and thresholds for depolarization block, which are associated with sensitivity to extracellular K+.MethodsHere, we used our inducible mouse model of glaucoma to investigate how RGC sensitivity to K+ changes with exposure to elevated IOP.ResultsIn controls, conditions of increased K+ enhanced membrane depolarization, reduced action potential generation, and widened action potentials. Consistent with our previous work, 4 weeks of IOP elevation diminished RGC light-and current-evoked responses. Compared to controls, we found that IOP elevation reduced the effects of increased K+ on depolarization block threshold, with IOP-exposed cells maintaining greater excitability. Finally, IOP elevation did not alter axon initial segment dimensions, suggesting that structural plasticity alone cannot explain decreased K+ sensitivity.DiscussionThus, in response to prolonged IOP elevation RGCs undergo an adaptive process that reduces sensitivity to changes in K+ while diminishing excitability. These experiments give insight into the RGC response to IOP stress and lay the groundwork for mechanistic investigation into targets for neuroprotective therapy

Development of X-43A Mach 10 Leading Edges

The nose leading edge of the Hyper-X Mach 10 vehicle was orginally anticipated to reach temperatures near 4000 F at the leading-edge stagnation line. A SiC coated carbon/carbon (C/C) leading-edge material will not survive that extreme temperature for even a short duration single flight. To identify a suitable leading edge for the Mach 10 vehicle, arc-jet testing was performed on thirteen leading-edge segments fabricated from different material systems to evaluate their performance in a simulated flight environment. Hf, Zr, Si, and Ir based materials, in most cases as a coating on C/C, were included in the evaluation. Afterwards, MER, Tucson, AZ was selected as the supplier of the flight vehicle leading edges. The nose and the vertical and horizontal tail leading edges were fabricated out of a 3:1 biased high thermal conductivity C/C. The leading edges were coated with a three layer coating comprised of a SiC conversion of the top surface of the C/C, followed by a chemical vapor deposited layer of SiC, followed by a thin chemical vapor deposited layer of HfC. This paper will describe the fabrication of the Mach 10 C/C leading edges and the testing performed to validate performance

Ccl5 Mediates Proper Wiring of Feedforward and Lateral Inhibition Pathways in the Inner Retina

The β-chemokine Ccl5 and its receptors are constitutively expressed in neurons of the murine inner retina. Here, we examined the functional and structural significance of this constitutive Ccl5 signaling on retinal development. We compared outcomes of electrophysiology, ocular imaging and retinal morphology in wild-type mice (WT) and mice with Ccl5 deficiency (Ccl5-/-). Assessment of retinal structure by ocular coherence tomography and histology revealed slight thinning of the inner plexiform layer (IPL) and inner nuclear layer (INL) in Ccl5-/- mice, compared to WT (p < 0.01). Assessment of postnatal timepoints important for development of the INL (P7 and P10) revealed Ccl5-dependent alterations in the pattern and timing of apoptotic pruning. Morphological analyses of major inner retinal cell types in WT, Ccl5-/-, gustducingfp and gustducingfp/Ccl5-/- mice revealed Ccl5-dependent reduction in GNAT3 expression in rod bipolar cells as well as a displacement of their terminals from the IPL into the GCL. RGC dendritic organization and amacrine cell morphology in the IPL was similarly disorganized in Ccl5-/- mice. Examination of the intrinsic electrophysiological properties of RGCs revealed higher spontaneous activity in Ccl5-/- mice that was characterized by higher spiking frequency and a more depolarized resting potential. This hyperactive phenotype could be negated by current clamp and correlated with both membrane resistance and soma area. Overall, our findings identify Ccl5 signaling as a mediator of inner retinal circuitry during development of the murine retina. The apparent role of Ccl5 in retinal development further supports chemokines as trophic modulators of CNS development and function that extends far beyond the inflammatory contexts in which they were first characterized

Divergent Roles of Clock Genes in Retinal and Suprachiasmatic Nucleus Circadian Oscillators

The retina is both a sensory organ and a self-sustained circadian clock. Gene targeting studies have revealed that mammalian circadian clocks generate molecular circadian rhythms through coupled transcription/translation feedback loops which involve 6 core clock genes, namely Period (Per) 1 and 2, Cryptochrome (Cry) 1 and 2, Clock, and Bmal1 and that the roles of individual clock genes in rhythms generation are tissue-specific. However, the mechanisms of molecular circadian rhythms in the mammalian retina are incompletely understood and the extent to which retinal neural clocks share mechanisms with the suprachiasmatic nucleus (SCN), the central neural clock, is unclear. In the present study, we examined the rhythmic amplitude and period of real-time bioluminescence rhythms in explants of retina from Per1-, Per2-, Per3-, Cry1-, Cry2-, and Clock-deficient mice that carried transgenic PERIOD2::LUCIFERASE (PER2::LUC) or Period1::luciferase (Per1::luc) circadian reporters. Per1-, Cry1- and Clock-deficient retinal and SCN explants showed weakened or disrupted rhythms, with stronger effects in retina compared to SCN. Per2, Per3, and Cry2 were individually dispensable for sustained rhythms in both tissues. Retinal and SCN explants from double knockouts of Cry1 and Cry2 were arrhythmic. Gene effects on period were divergent with reduction in the number of Per1 alleles shortening circadian period in retina, but lengthening it in SCN, and knockout of Per3 substantially shortening retinal clock period, but leaving SCN unaffected. Thus, the retinal neural clock has a unique pattern of clock gene dependence at the tissue level that it is similar in pattern, but more severe in degree, than the SCN neural clock, with divergent clock gene regulation of rhythmic period

Astrocyte Networks as Therapeutic Targets in Glaucomatous Neurodegeneration

Astrocytes are intimately involved in the response to neurodegenerative stress and have become an attractive target for the development of neuroprotective therapies. However, studies often focus on astrocytes as single-cell units. Astrocytes are densely interconnected by gap junctions that are composed primarily of the protein connexin-43 (Cx43) and can function as a broader network of cells. Such networks contribute to a number of important processes, including metabolite distribution and extracellular ionic buffering, and are likely to play an important role in the progression of neurodegenerative disease. This review will focus on the pro-degenerative and pro-survival influence of astrocyte Cx43 in disease progression, with a focus on the roles of gap junctions and hemichannels in the spread of degenerative stress. Finally, we will highlight the specific evidence for targeting these networks in the treatment of glaucomatous neurodegeneration and other optic neuropathies

Periods of retinal <i>Per1::luc</i> or PER2::LUC rhythms in mice of different genotypes.

<p>Letters indicate significant difference with aP values are for comparisons to wild-type mice. A minimum of three animals were sampled for each genotype.</p

Neutral sphingomyelinase inhibition promotes local and network degeneration in vitro and in vivo

Abstract Background Cell-to-cell communication is vital for tissues to respond, adapt, and thrive in the prevailing milieu. Several mechanisms mediate intercellular signaling, including tunneling nanotubes, gap junctions, and extracellular vesicles (EV). Depending on local and systemic conditions, EVs may contain cargoes that promote survival, neuroprotection, or pathology. Our understanding of pathologic intercellular signaling has been bolstered by disease models using neurons derived from human pluripotent stems cells (hPSC). Methods Here, we used hPSC-derived retinal ganglion cells (hRGC) and the mouse visual system to investigate the influence of modulating EV generation on intercellular trafficking and cell survival. We probed the impact of EV modulation on cell survival by decreasing the catabolism of sphingomyelin into ceramide through inhibition of neutral sphingomyelinase (nSMase), using GW4869. We assayed for cell survival in vitro by probing for annexin A5, phosphatidylserine, viable mitochondria, and mitochondrial reactive oxygen species. In vivo, we performed intraocular injections of GW4869 and measured RGC and superior colliculus neuron density and RGC anterograde axon transport. Results Following twenty-four hours of dosing hRGCs with GW4869, we found that inhibition of nSMase decreased ceramide and enhanced GM1 ganglioside accumulation. This inhibition also reduced the density of small EVs, increased the density of large EVs, and enriched the pro-apoptotic protein, annexin A5. Reducing nSMase activity increased hRGC apoptosis initiation due to enhanced density and uptake of apoptotic particles, as identified by the annexin A5 binding phospholipid, phosphatidylserine. We assayed intercellular trafficking of mitochondria by developing a coculture system of GW4869-treated and naïve hRGCs. In treated cells, inhibition of nSMase reduced the number of viable mitochondria, while driving mitochondrial reactive oxygen species not only in treated, but also in naive hRGCs added in coculture. In mice, 20 days following a single intravitreal injection of GW4869, we found a significant loss of RGCs and their axonal recipient neurons in the superior colliculus. This followed a more dramatic reduction in anterograde RGC axon transport to the colliculus. Conclusion Overall, our data suggest that perturbing the physiologic catabolism of sphingomyelin by inhibiting nSMase reorganizes plasma membrane associated sphingolipids, alters the profile of neuron-generated EVs, and promotes neurodegeneration in vitro and in vivo by shifting the balance of pro-survival versus -degenerative EVs. Video Abstrac