VDAC in Retinal Health and Disease.

The retina, a tissue of the central nervous system, is vital for vision as its photoreceptors capture light and transform it into electrical signals, which are further processed before they are sent to the brain to be interpreted as images. The retina is unique in that it is continuously exposed to light and has the highest metabolic rate and demand for energy amongst all the tissues in the body. Consequently, the retina is very susceptible to oxidative stress. VDAC, a pore in the outer membrane of mitochondria, shuttles metabolites between mitochondria and the cytosol and normally protects cells from oxidative damage, but when a cell's integrity is greatly compromised it initiates cell death. There are three isoforms of VDAC, and existing evidence indicates that all three are expressed in the retina. However, their precise localization and function in each cell type is unknown. It appears that most retinal cells express substantial amounts of VDAC2 and VDAC3, presumably to protect them from oxidative stress. Photoreceptors express VDAC2, HK2, and PKM2-key proteins in the Warburg pathway that also protect these cells. Consistent with its role in initiating cell death, VDAC is overexpressed in the retinal degenerative diseases retinitis pigmentosa, age related macular degeneration (AMD), and glaucoma. Treatment with antioxidants or inhibiting VDAC oligomerization reduced its expression and improved cell survival. Thus, VDAC may be a promising therapeutic candidate for the treatment of these diseases.

Detection and verification of neurodegeneration after traumatic brain injury in the mouse: Immunohistochemical staining for amyloid precursor protein.

Previous studies of human traumatic brain injury (TBI) have shown diffuse axonal injury as varicosities or spheroids in white matter (WM) bundles when using immunoperoxidase-ABC staining with 22C11, a mouse monoclonal antibody against amyloid precursor protein (APP). These findings have been interpreted as TBI-induced axonal pathology. In a mouse model of TBI however, when we used immunofluorescent staining with 22C11, as opposed to immunoperoxidase staining, we did not observe varicosities or spheroids. To explore this discrepancy, we performed immunofluorescent staining with Y188, an APP knockout-validated rabbit monoclonal that shows baseline immunoreactivity in neurons and oligodendrocytes of non-injured mice, with some arranged-like varicosities. In gray matter after injury, Y188 intensely stained axonal blebs. In WM, we encountered large patches of heavily stained puncta, heterogeneous in size. Scattered axonal blebs were also identified among these Y188-stained puncta. To assess the neuronal origin of Y188 staining after TBI we made use of transgenic mice with fluorescently labeled neurons and axons. A close correlation was observed between Y188-stained axonal blebs and fluorescently labeled neuronal cell bodies/axons. By contrast, no correlation was observed between Y188-stained puncta and fluorescent axons in WM, suggesting that these puncta in WM did not originate from axons, and casting further doubt on the nature of previous reports with 22C11. As such, we strongly recommend Y188 as a biomarker for detecting damaged neurons and axons after TBI. With Y188, stained axonal blebs likely represent acute axonal truncations that may lead to death of the parent neurons. Y188-stained puncta in WM may indicate damaged oligodendrocytes, whose death and clearance can result in secondary demyelination and Wallerian degeneration of axons. We also provide evidence suggesting that 22C11-stained varicosities or spheroids previously reported in TBI patients might be showing damaged oligodendrocytes, due to a cross-reaction between the ABC kit and upregulated endogenous biotin.

Concussion increases CA1 activity during prolonged inactivity in a familiar environment.

Although hippocampal damage plays a key role in impairments after concussion, differences in hippocampal information processing during recovery are unknown. Micro-endoscopic calcium imaging was performed before and after primary blast injury in freely behaving mice in two environments: their familiar home cage and a novel open field. Results show that after concussion CA1 activity increased in the familiar environment in which animals were awake and mostly immobile but was unaltered in a novel environment which the animals actively and constantly explored. As awake immobility parallels cognitive rest, a common treatment for patients, the results imply that prolonged cognitive rest may unwittingly impede concussion recovery.

Primary blast injury causes cognitive impairments and hippocampal circuit alterations.

Blast-induced traumatic brain injury (bTBI) and its long term consequences are a major health concern among veterans. Despite recent work enhancing our knowledge about bTBI, very little is known about the contribution of the blast wave alone to the observed sequelae. Herein, we isolated its contribution in a mouse model by constraining the animals' heads during exposure to a shockwave (primary blast). Our results show that exposure to primary blast alone results in changes in hippocampus-dependent behaviors that correspond with electrophysiological changes in area CA1 and are accompanied by reactive gliosis. Specifically, five days after exposure, behavior in an open field and performance in a spatial object recognition (SOR) task were significantly different from sham. Network electrophysiology, also performed five days after injury, demonstrated a significant decrease in excitability and increase in inhibitory tone. Immunohistochemistry for GFAP and Iba1 performed ten days after injury showed a significant increase in staining. Interestingly, a threefold increase in the impulse of the primary blast wave did not exacerbate these measures. However, we observed a significant reduction in the contribution of the NMDA receptors to the field EPSP at the highest blast exposure level. Our results emphasize the need to account for the effects of primary blast loading when studying the sequelae of bTBI.

Lack of mGluR6-related cascade elements leads to retrograde trans-synaptic effects on rod photoreceptor synapses via matrix-associated proteins.

Heterotrimeric G-proteins couple metabotropic receptors to downstream effectors. In retinal ON bipolar cells, Go couples the metabotropic receptor mGluR6 to the TRPM1 channel and closes it in the dark, thus hyperpolarizing the cell. Light, via GTPase-activating proteins, deactivates Go , opens TRPM1 and depolarizes the cell. Go comprises Gαo1 , Gß3 and Gγ13; all are necessary for efficient coupling. In addition, Gß3 contributes to trafficking of certain cascade proteins and to maintaining the synaptic structure. The goal of this study was to determine the role of Gαo1 in maintaining the cascade and synaptic integrity. Using mice lacking Gαo1 , we quantified the immunostaining of certain mGluR6-related components. Deleting Gαo1 greatly reduced staining for Gß3, Gγ13, Gß5, RGS11, RGS7 and R9AP. Deletion of Gαo1 did not affect mGluR6, TRPM1 or PCP2. In addition, deleting Gαo1 reduced the number of rod bipolar dendrites that invaginate the rod terminal, similar to the effect seen in the absence of mGluR6, Gß3 or the matrix-associated proteins, pikachurin, dystroglycan and dystrophin, which are localized presynaptically to the rod bipolar cell. We therefore tested mice lacking mGluR6, Gαo1 and Gß3 for expression of these matrix-associated proteins. In all three genotypes, staining intensity for these proteins was lower than in wild type, suggesting a retrograde trans-synaptic effect. We propose that the mGluR6 macromolecular complex is connected to the presynaptic rod terminal via a protein chain that includes the matrix-associated proteins. When a component of the macromolecular chain is missing, the chain may fall apart and loosen the dendritic tip adherence within the invagination.

Differential function of Gγ13 in rod bipolar and ON cone bipolar cells.

Heterotrimeric G-proteins (comprising Gα and Gßγ subunits) are critical for coupling of metabotropic receptors to their downstream effectors. In the retina, glutamate released from photoreceptors in the dark activates metabotropic glutamate receptor 6 (mGluR6) receptors in ON bipolar cells; this leads to activation of Go , closure of transient receptor potential melastatin 1 channels and hyperpolarization of these cells. Go comprises Gαo , Gß3 and a Gγ. The best Gγ candidate is Gγ13, although functional data to support this are lacking. Thus, we tested Gγ13 function by generating Gng13(-/-) knockout (KO) mice, recording electroretinograms (ERG) and performing immunocytochemical staining. The amplitude of scotopic ERG b-waves in KO mice was lower than in wild-type (WT) mice. Furthermore, in both KO and WT mice, the ERG b-wave decreased with age; this decrease was much more pronounced in KO mice. By contrast, the photopic ERG b-waves in KO mice were hardly affected at any age. In KO mice retinas, immunostaining for Gß3 and for the GTPase activating proteins RGS7, RGS11, R9AP and Gß5 decreased significantly in rod bipolar cells but not in ON cone bipolar cells. Staining for Gαo and certain other cascade elements decreased only slightly. Analysis of our ON bipolar cDNA library showed that these cells express mRNAs for Gγ5, Gγ10 and Gγ11. Quantitative RT-PCR of retinal cDNA showed greater values for these transcripts in retinas of KO mice, although the difference was not significant. Our results suggest that Gγ13 contributes to mGluR6 signalling in rod bipolar cells more than in ON cone bipolar cells, and that this contribution includes both coupling the receptor and maintaining a stable localization of the mGluR6-related cascade elements.

PDE9A is expressed in the inner retina and contributes to the normal shape of the photopic ERG waveform.

The ubiquitous second messenger cGMP is synthesized by guanylyl cyclase and hydrolyzed by phosphodiesterase (PDE). cGMP mediates numerous signaling pathways in multiple tissues. In the retina, cGMP regulates signaling in nearly every cell class including photoreceptors, bipolar cells, amacrine cells, and ganglion cells. In order to understand the specific role of cGMP and its regulating enzymes in different cell types, it is first necessary to localize these components and dissect their influence on the circuits. Here we tested the contribution of PDE9A to retinal processing by recording the electroretinograms (ERG) of PDE9A (™/™) (KO) mice and by localizing the enzyme. We found that while the scotopic ERG of KO was the same as that of wild type (WT) in both amplitude and kinetics, the photopic ERG was greatly affected. The greatest effect was on the recovery of the b-wave; the falling phase and the b-wave duration were significantly longer in the KO mice for all photopic stimuli (UV, green, or saturating white flashes). The rising phase was slower in KO than in WT for UV and green stimuli. For certain stimuli, amplitudes of both the a- and b-waves were smaller than in WT. Using Lac-Z expression in KO retinas as a reporter for PDE9A expression pattern, we found that PDE9A is localized to GABA-positive and GABA-negative amacrine cells, and likely also to certain types of ganglion cells. Our results indicate that PDE9A, by controlling the level of cGMP, modulates inhibitory processes within the cone pathway. We speculate that these circuits involve NO/cGMP signaling pathways.

Localization of Cacna1s to ON bipolar dendritic tips requires mGluR6-related cascade elements.

PURPOSE: L-type voltage gated calcium channels in retina localize primarily at the presynaptic active zones of photoreceptors and bipolar cells where they modulate glutamate release. However, the pore forming subunit Cacna1s of certain L-type channels is also expressed postsynaptically at the tips of ON bipolar cell dendrites where it colocalizes with mGluR6, but has an unknown function. At these dendritic tips, the components of the mGluR6 signaling cascade cluster together in a macromolecular complex, and each one's localization often depends on that of the others. Thus, we explored if Cacna1s is part of the mGluR6 complex. METHODS: We determined Cacna1s expression by PCR using an ON bipolar library, by Western blotting, and by standard immunohistochemistry. RESULTS: The PCR amplification confirmed expression of the transcript in ON bipolar cells, and Western blotting showed the expected bands. Immunostaining for Cacna1s was stronger in the dendritic tips of rod bipolar cells than in those of ON cone bipolar cells. This staining severely decreased in mice missing various mGluR6 cascade elements (Grm6(-/-), Gnao1(-/-), Gnb3(-/-), Gng13(-/-), and Trpm1(-/-)). During development, the ratio of the number of Cacna1s puncta to the number of presynaptic ribbons followed a sigmoidal pattern, rising rapidly from P13 to P17. The mGluR6 expression preceded that of Cacna1s and RGS11. CONCLUSIONS: Our results show that the localization and stability of Cacna1s depend on the expression of mGluR6 and its cascade components, and they suggest that Cacna1s is part of the mGluR6 complex. We hypothesize that Cacna1s contributes to light adaptation by permeating calcium.

Kir2.4 surface expression and basal current are affected by heterotrimeric G-proteins.

Kir2.4, a strongly rectifying potassium channel that is localized to neurons and is especially abundant in retina, was fished with yeast two-hybrid screen using a constitutively active Gαo1. Here, we wished to determine whether and how Gαo affects this channel. Using transfected HEK 293 cells and retinal tissue, we showed that Kir2.4 interacts with Gαo, and this interaction is stronger with the GDP-bound form of Gαo. Using two-electrode voltage clamp, we recorded from oocytes that were injected with Kir2.4 mRNA and a combination of G-protein subunit mRNAs. We found that the wild type and the inactive mutant of Gαo reduce the Kir2.4 basal current, whereas the active mutant has little effect. Other pertussis-sensitive Gα subunits also reduce this current, whereas Gαs increases it. Gßγ increases the current, whereas m-phosducin, which binds Gßγ without affecting the state of Gα, reduces it. We then tested the effect of G-protein subunits on the surface expression of the channel fused to cerulean by imaging the plasma membranes of the oocytes. We found that the surface expression is affected, with effects paralleling those seen with the basal current. This suggests that the observed effects on the current are mainly indirect and are due to surface expression. Similar results were obtained in transfected HEK cells. Moreover, we show that in retinal ON bipolar cells lacking Gß3, localization of Kir2.4 in the dendritic tips is reduced. We conclude that Gßγ targets Kir2.4 to the plasma membrane, and Gαo slows this down by binding Gßγ.

Effects of inhibition of neuronal nitric oxide synthase on basal retinal blood flow regulation.

Nitric oxide (NO) has been observed to regulate blood flow under basal and stimulated conditions in the retina. Recent evidence suggests that NO produced by neuronal nitric oxide synthase (nNOS) may regulate blood flow in addition to that produced by endothelial nitric oxide synthase (eNOS). The objective of the current study was to investigate the contribution of NO produced by nNOS in the regulation of basal retinal blood flow. A non-specific NOS inhibitor N (G)-nitro-l-arginine methyl ester (l-NAME) and the specific nNOS inhibitors 1-(2-trifluoromethylphenyl) imidazole (TRIM) and (4S)-N-(4-amino-5 [aminoethyl] aminopentyl)-N-nitroguanidine (AAAN) were injected into the vitreous (intravitreal) of Long-Evans rats. Vessel diameters, velocities and volumetric blood flow rates (VBF) in the retinal circulation were determined prior to and in 30-min intervals for 4-4.5h after injection. In addition, the basal amount of nNOS in the rat retina was quantified using a specific enzyme linked immunoassay (ELISA). Treatment with l-NAME and TRIM significantly decreased diameters and VBF. Compared with saline, treatment with l-NAME and TRIM produced a significant (p<0.001) decrease of approximately 12-17% in vessel diameters. Treatment with AAAN significantly decreased vessel diameters and venous VBF. Compared with saline AAAN produced a significant decrease of approximately 7% in arterial (p<0.001) and 5% in venous (p=0.011) diameters, respectively. The amount of nNOS in the rat retina was 0.17+/- 0.0147 pmol mg(-1) of dry retina. The results suggest that though inhibition of nNOS decreases basal diameters, constant VBF is maintained in the retinal circulation.

Computational modeling of factor Xa inhibition by immobilized tissue factor pathway inhibitor.

Coating surfaces of implanted devices with anticoagulants can reduce thrombosis and studies using a recombinant form of endogenous tissue factor pathway inhibitor (rTFPI) are promising. The anticoagulant function of immobilized rTFPI is thought to occur primarily by its inhibition of plasma clotting factor Xa (FXa); however the kinetics of this reaction at a surface are as yet unknown. To better understand the surface inhibition reaction under flow conditions, a theoretical model was developed delineating the roles of mass transport and reaction kinetics for an in vitro parallel plate device used in prior experimental studies [Hall et al., J. Biomech. Eng. 120:484-490, 1998]. As a first approximation, the kinetics of inhibition of FXa by rTFPI reported for static, homogeneous systems was considered. The unsteady convection-diffusion equation was solved for different wall-shear rates and inlet concentrations of FXa using the computational fluid dynamics software CFD-ACE (ESI Software Group). The results show that the heterogeneous inhibition reaction is diffusion controlled prior to saturation of the rTFPI. The experimental results compare favorably with the model at the lower shear rates (100-400 s(-1)). At higher shear rates (>400 s(-1)) the theoretical results follow the same trend as the experimental results but show a greater inhibition of FXa, implying an effect of flow or shear on the inhibition reaction.