Alleviation of extensive visual pathway dysfunction by a remyelinating drug in a chronic mouse model of multiple sclerosis.

Visual deficits are among the most prevalent symptoms in patients with multiple sclerosis (MS). To understand deficits in the visual pathway during MS and potential treatment effects, we used experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model of MS. The afferent visual pathway was assessed in vivo using optical coherence tomography (OCT), electroretinography (ERG), and visually evoked cortical potentials (VEPs). Inflammation, demyelination, and neurodegeneration were examined by immunohistochemistry ex vivo. In addition, an immunomodulatory, remyelinating agent, the estrogen receptor ß ligand chloroindazole (IndCl), was tested for its therapeutic potential in the visual pathway. EAE produced functional deficits in visual system electrophysiology, including suppression of ERG and VEP waveform amplitudes and increased signal latencies. Therapeutic IndCl rescued overall visual system latency by VEP but had little impact on amplitude or ERG findings relative to vehicle. Faster VEP conduction in IndCl-treated mice was associated with enhanced myelin basic protein signal in all visual system structures examined. IndCl preserved retinal ganglion cells (RGCs) and oligodendrocyte density in the prechiasmatic white matter, but similar retinal nerve fiber layer thinning by OCT was noted in vehicle and IndCl-treated mice. Although IndCl differentially attenuated leukocyte and astrocyte staining signal throughout the structures analyzed, axolemmal varicosities were observed in all visual fiber tracts of mice with EAE irrespective of treatment, suggesting impaired axonal energy homeostasis. These data support incomplete functional recovery of VEP amplitude with IndCl, as fiber tracts displayed persistent axon pathology despite remyelination-induced decreases in latencies, evidenced by reduced optic nerve g-ratio in IndCl-treated mice. Although additional studies are required, these findings demonstrate the dynamics of visual pathway dysfunction and disability during EAE, along with the importance of early treatment to mitigate EAE-induced axon damage.

Mispositioned Neurokinin-1 Receptor-Expressing Neurons Underlie Heat Hyperalgesia in Disabled-1 Mutant Mice.

Reelin (Reln) and Disabled-1 (Dab1) participate in the Reln-signaling pathway and when either is deleted, mutant mice have the same spinally mediated behavioral abnormalities, increased sensitivity to noxious heat and a profound loss in mechanical sensitivity. Both Reln and Dab1 are highly expressed in dorsal horn areas that receive and convey nociceptive information, Laminae I-II, lateral Lamina V, and the lateral spinal nucleus (LSN). Lamina I contains both projection neurons and interneurons that express Neurokinin-1 receptors (NK1Rs) and they transmit information about noxious heat both within the dorsal horn and to the brain. Here, we ask whether the increased heat nociception in Reln and dab1 mutants is due to incorrectly positioned dorsal horn neurons that express NK1Rs. We found more NK1R-expressing neurons in Reln-/- and dab1-/- Laminae I-II than in their respective wild-type mice, and some NK1R neurons co-expressed Dab1 and the transcription factor Lmx1b, confirming their excitatory phenotype. Importantly, heat stimulation in dab1-/- mice induced Fos in incorrectly positioned NK1R neurons in Laminae I-II. Next, we asked whether these ectopically placed and noxious-heat responsive NK1R neurons participated in pain behavior. Ablation of the superficial NK1Rs with an intrathecal injection of a substance P analog conjugated to the toxin saporin (SSP-SAP) eliminated the thermal hypersensitivity of dab1-/- mice, without altering their mechanical insensitivity. These results suggest that ectopically positioned NK1R-expressing neurons underlie the heat hyperalgesia of Reelin-signaling pathway mutants, but do not contribute to their profound mechanical insensitivity.

Insights into the structure and molecular topography of the fatty acylated domain of synaptotagmin-1.

Abundant attention has focused on synaptotagmin's C2 domains, but less is known about the structure and function of its other regions. Here, we synthesized the N-acetylated, C-end amidated and Cys-palmitated peptide (VLTCCFCICK KCLFKKKNKK K) which includes the fatty acylated cysteine residues in the membrane-affiliated domain of synaptotagmin-1. Fourier-transform infrared spectrometry indicated that this peptide's conformation is influenced by environmental polarity. In artificial bilayer membranes, this peptide exhibited abundant ß-structure. Electron microscopy revealed that this peptide also promoted the stacking of liposome membranes. Together these results suggest that the fatty acylated region of synaptotagmin-1 is likely to adopt ß-structure in biological membranes. This preference for ß-structure versus α-helix has functional implications for the role of synaptotagmin-1 in synaptic vesicle exocytosis.

The Absence of DHHC3 Affects Primary and Latent Herpes Simplex Virus 1 Infection.

UL20, an essential herpes simplex virus 1 (HSV-1) protein, is involved in cytoplasmic envelopment of virions and virus egress. We reported recently that UL20 can bind to a host protein encoded by the zinc finger DHHC-type containing 3 (ZDHHC3) gene (also known as Golgi-specific DHHC zinc finger protein [GODZ]). Here, we show for the first time that HSV-1 replication is compromised in murine embryonic fibroblasts (MEFs) isolated from GODZ-/- mice. The absence of GODZ resulted in blocking palmitoylation of UL20 and altered localization and expression of UL20 and glycoprotein K (gK); the expression of gB and gC; and the localization and expression of tegument and capsid proteins within HSV-1-infected MEFs. Electron microscopy revealed that the absence of GODZ limited the maturation of virions at multiple steps and affected the localization of virus and endoplasmic reticulum morphology. Virus replication in the eyes of ocularly HSV-1-infected GODZ-/- mice was significantly lower than in HSV-1-infected wild-type (WT) mice. The levels of UL20, gK, and gB transcripts in the corneas of HSV-1-infected GODZ-/- mice on day 5 postinfection were markedly lower than in WT mice, whereas only UL20 transcripts were reduced in trigeminal ganglia (TG). In addition, HSV-1-infected GODZ-/- mice showed notably lower levels of corneal scarring, and HSV-1 latency reactivation was also reduced. Thus, normal HSV-1 infectivity and viral pathogenesis are critically dependent on GODZ-mediated palmitoylation of viral UL20.IMPORTANCE HSV-1 infection is widespread. Ocular infection can cause corneal blindness; however, approximately 70 to 90% of American adults exposed to the virus show no clinical symptoms. In this study, we show for the first time that the absence of a zinc finger protein called GODZ affects primary and latent infection, as well as reactivation, in ocularly infected mice. The reduced virus infectivity is due to the absence of the GODZ interaction with HSV-1 UL20. These results strongly suggest that binding of UL20 to GODZ promotes virus infectivity in vitro and viral pathogenesis in vivo.

Stimulation of synaptoneurosome glutamate release by monomeric and fibrillated α-synuclein.

The α-synuclein protein exists in vivo in a variety of covalently modified and aggregated forms associated with Parkinson's disease (PD) pathology. However, the specific proteoform structures involved with neuropathological disease mechanisms are not clearly defined. Since α-synuclein plays a role in presynaptic neurotransmitter release, an in vitro enzyme-based assay was developed to measure glutamate release from mouse forebrain synaptoneurosomes (SNs) enriched in synaptic endings. Glutamate measurements utilizing SNs from various mouse genotypes (WT, over-expressers, knock-outs) suggested a concentration dependence of α-synuclein on calcium/depolarization-dependent presynaptic glutamate release from forebrain terminals. In vitro reconstitution experiments with recombinant human α-synuclein proteoforms including monomers and aggregated forms (fibrils, oligomers) produced further evidence of this functional impact. Notably, brief exogenous applications of fibrillated forms of α-synuclein enhanced SN glutamate release but monomeric forms did not, suggesting preferential membrane penetration and toxicity by the aggregated forms. However, when applied to brain tissue sections just prior to homogenization, both monomeric and fibrillated forms stimulated glutamate release. Immuno-gold and transmission electron microscopy (TEM) detected exogenous fibrillated α-synuclein associated with numerous SN membranous structures including synaptic terminals. Western blots and immuno-gold TEM were consistent with SN internalization of α-synuclein. Additional studies revealed no evidence of gross disruption of SN membrane integrity or glutamate transporter function by exogenous α-synuclein. Overall excitotoxicity, due to enhanced glutamate release in the face of either overexpressed monomeric α-synuclein or extrasynaptic exposure to fibrillated α-synuclein, should be considered as a potential neuropathological pathway during the progression of PD and other synucleinopathies. © 2017 Wiley Periodicals, Inc.

Myeloid ATG16L1 Facilitates Host-Bacteria Interactions in Maintaining Intestinal Homeostasis.

Intact ATG16L1 plays an essential role in Paneth cell function and intestinal homeostasis. However, the functional consequences of ATG16L1 deficiency in myeloid cells, particularly macrophages, are not fully characterized. We generated mice with Atg16l1 deficiency in myeloid and dendritic cells and showed that mice with myeloid Atg16l1 deficiency had exacerbated colitis in two acute and one chronic model of colitis with increased proinflammatory to anti-inflammatory macrophage ratios, production of proinflammatory cytokines, and numbers of IgA-coated intestinal microbes. Mechanistic analyses using primary murine macrophages showed that Atg16l1 deficiency led to increased reactive oxygen species production, impaired mitophagy, reduced microbial killing, impaired processing of MHC class II Ags, and altered intracellular trafficking to the lysosomal compartments. Increased production of reactive oxygen species and reduced microbial killing may be general features of the myeloid compartment, as they were also observed in Atg16l1-deficient primary murine neutrophils. A missense polymorphism (Thr300Ala) in the essential autophagy gene ATG16L1 is associated with Crohn disease (CD). Previous studies showed that this polymorphism leads to enhanced cleavage of ATG16L1 T300A protein and thus reduced autophagy. Similar findings were shown in primary human macrophages from controls and a population of CD patients carrying the Atg16l1 T300A risk variant and who were controlled for NOD2 CD-associated variants. This study revealed that ATG16L1 deficiency led to alterations in macrophage function that contribute to the severity of CD.

Novel Disabled-1-expressing neurons identified in adult brain and spinal cord.

Components of the Reelin-signaling pathway are highly expressed in embryos and regulate neuronal positioning, whereas these molecules are expressed at low levels in adults and modulate synaptic plasticity. Reelin binds to Apolipoprotein E receptor 2 and Very-low-density lipoprotein receptors, triggers the phosphorylation of Disabled-1 (Dab1), and initiates downstream signaling. The expression of Dab1 marks neurons that potentially respond to Reelin, yet phosphorylated Dab1 is difficult to detect due to its rapid ubiquitination and degradation. Here we used adult mice with a lacZ gene inserted into the dab1 locus to first verify the coexpression of ß-galactosidase (ß-gal) in established Dab1-immunoreactive neurons and then identify novel Dab1-expressing neurons. Both cerebellar Purkinje cells and spinal sympathetic preganglionic neurons have coincident Dab1 protein and ß-gal expression in dab1(lacZ/+) mice. Adult pyramidal neurons in cortical layers II-III and V are labeled with Dab1 and/or ß-gal and are inverted in the dab1(lacZ/lacZ) neocortex, but not in the somatosensory barrel fields. Novel Dab1 expression was identified in GABAergic medial septum/diagonal band projection neurons, cerebellar Golgi interneurons, and small neurons in the deep cerebellar nuclei. Adult somatic motor neurons also express Dab1 and show ventromedial positioning errors in dab1-null mice. These findings suggest that: (i) Reelin regulates the somatosensory barrel cortex differently than other neocortical areas, (ii) most Dab1 medial septum/diagonal band neurons are probably GABAergic projection neurons, and (iii) positioning errors in adult mutant Dab1-labeled neurons vary from subtle to extensive.

Distribution and chemical speciation of arsenic in ancient human hair using synchrotron radiation.

Pre-Columbian populations that inhabited the Tarapacá mid river valley in the Atacama Desert in Chile during the Middle Horizon and Late Intermediate Period (AD 500-1450) show patterns of chronic poisoning due to exposure to geogenic arsenic. Exposure of these people to arsenic was assessed using synchrotron-based elemental X-ray fluorescence mapping, X-ray absorption spectroscopy, X-ray diffraction and Fourier transform infrared spectromicroscopy measurements on ancient human hair. These combined techniques of high sensitivity and specificity enabled the discrimination between endogenous and exogenous processes that has been an analytical challenge for archeological studies and criminal investigations in which hair is used as a proxy of premortem metabolism. The high concentration of arsenic mainly in the form of inorganic As(III) and As(V) detected in the hair suggests chronic arsenicism through ingestion of As-polluted water rather than external contamination by the deposition of heavy metals due to metallophilic soil microbes or diffusion of arsenic from the soil. A decrease in arsenic concentration from the proximal to the distal end of the hair shaft analyzed may indicate a change in the diet due to mobility, though chemical or microbiologically induced processes during burial cannot be entirely ruled out.

Modulation of mouse rod response decay by rhodopsin kinase and recoverin.

Light isomerizes 11-cis-retinal in a retinal rod and produces an active form of rhodopsin (Rh*) that binds to the G-protein transducin and activates the phototransduction cascade. Rh* is turned off by phosphorylation by rhodopsin kinase [G-protein-coupled receptor kinase 1 (GRK1)] and subsequent binding of arrestin. To evaluate the role of GRK1 in rod light response decay, we have generated the transgenic mouse RKS561L in which GRK1, which is normally present at only 2-3% of rhodopsin, is overexpressed by ∼12-fold. Overexpression of GRK1 increases the rate of Rh* phosphorylation and reduces the exponential decay constant of the response (τ(REC)) and the limiting time constant (τ(D)) both by ∼30%; these decreases are highly significant. Similar decreases are produced in Rv(-/-) rods, in which the GRK1-binding protein recoverin has been genetically deleted. These changes in response decay are produced by acceleration of light-activated phosphodiesterase (PDE*) decay rather than Rh* decay, because light-activated PDE* decay remains rate limiting for response decay in both RKS561L and Rv(-/-) rods. A model incorporating an effect of GRK1 on light-activated PDE* decay rate can satisfactorily account for the changes in response amplitude and waveform. Modulation of response decay in background light is nearly eliminated by deletion of recoverin. Our experiments indicate that rhodopsin kinase and recoverin, in addition to their well-known role in regulating the turning off of Rh*, can also modulate the decay of light-activated PDE*, and the effects of these proteins on light-activated PDE* decay may be responsible for the quickening of response recovery in background light.

Effect of the ILE86TER mutation in the γ subunit of cGMP phosphodiesterase (PDE6) on rod photoreceptor signaling.

The light-dependent decrease in cyclic guanosine monophosphate (cGMP) in the rod outer segment is produced by a phosphodiesterase (PDE6), consisting of catalytic α and ß subunits and two inhibitory γ subunits. The molecular mechanism of PDE6γ regulation of the catalytic subunits is uncertain. To study this mechanism in vivo, we introduced a modified Pde6g gene for PDE6γ into a line of Pde6g(tm1)/Pde6g(tm1) mice that do not express PDE6γ. The resulting ILE86TER mice have a PDE6γ that lacks the two final carboxyl-terminal Ile(86) and Ile(87) residues, a mutation previously shown in vitro to reduce inhibition by PDE6γ. ILE86TER rods showed a decreased sensitivity and rate of activation, probably the result of a decreased level of expression of PDE6 in ILE86TER rods. More importantly, they showed a decreased rate of decay of the photoresponse, consistent with decreased inhibition of PDE6 α and ß by PDE6γ. Furthermore, ILE86TER rods had a higher rate of spontaneous activation of PDE6 than WT rods. Circulating current in ILE86TER rods that also lacked both guanylyl cyclase activating proteins (GCAPs) could be increased several fold by perfusion with 100µM of the PDE6 inhibitor 3-isobutyl-1-methylxanthine (IBMX), consistent with a higher rate of dark PDE6 activity in the mutant photoreceptors. In contrast, IBMX had little effect on the circulating current of WT rods, unlike previous results from amphibians. Our results show for the first time that the Ile(86) and Ile(87) residues are necessary for normal inhibition of PDE6 catalytic activity in vivo, and that increased basal activity of PDE can be partially compensated by GCAP-dependent regulation of guanylyl cyclase.

Lis1 reduction causes tangential migratory errors in mouse spinal cord.

Mutations in human LIS1 cause abnormal neuronal migration and a smooth brain phenotype known as lissencephaly. Lis1+/− (Pafah1b1) mice show defective lamination in the cerebral cortex and hippocampal formation, whereas homozygous mutations result in embryonic lethality. Given that Lis1 is highly expressed in embryonic neurons, we hypothesized that sympathetic and parasympathetic preganglionic neurons (SPNs and PPNs) would exhibit migratory defects in Lis1+/− mice. The initial radial migration of SPNs and PPNs that occurs together with somatic motor neurons appeared unaffected in Lis1+/− mice. The subsequent dorsally directed tangential migration, however, was aberrant in a subset of these neurons. At all embryonic ages analyzed, the distribution of SPNs and PPNs in Lis1+/− mice was elongated dorsoventrally compared with Lis1+/+ mice. Individual cell bodies of ectopic preganglionic neurons were found in the ventral spinal cord with their leading processes oriented along their dorsal migratory trajectory. By birth, Lis1+/− SPNs and PPNs were separated into distinct groups, those that were correctly, and those incorrectly positioned in the intermediate horn. As mispositioned SPNs and PPNs still were detected in P30 Lis1+/− mice, we conclude that these neurons ceased migration prematurely. Additionally, we found that a dorsally located group of somatic motor neurons in the lumbar spinal cord, the retrodorsolateral nucleus, showed delayed migration in Lis1+/− mice. These results suggest that Lis1 is required for the dorsally directed tangential migration of many sympathetic and parasympathetic preganglionic neurons and a subset of somatic motor neurons.

Function of the asparagine 74 residue of the inhibitory γ-subunit of retinal rod cGMP-phophodiesterase (PDE) in vivo.

The inhibitory subunit of rod cyclic guanosine monophosphate (cGMP) phosphodiesterase, PDE6γ, is a major component of rod transduction and is required to support photoreceptor integrity. The N74A allele of PDE6γ has previously been shown in experiments carried out in vitro to reduce the regulatory inhibition on the PDE6 catalytic core subunits, PDE6αß. This should, in intact rods, lead to an increase in basal (dark) PDE6 activity producing a state equivalent to light adaptation in the rods and we have examined this possibility using ERG and suction-electrode measurements. The murine opsin promoter was used to drive the expression of a mutant N74A and a wild-type PDE6γ control transgene in the photoreceptors of +/Pde6g(tm1) mice. This transgenic line was crossed with Pde6g(tm1)/Pde6g(tm1) mice to generate animals able to synthesize only the transgenic mutant PDE6γ. We find that the N74A mutation did not produce a significant decrease in circulating current, a decrease in sensitivity or affect the kinetics of the light response, all hallmarks of the light-adapted state. In an in vitro assay of the PDE purified from the N74A transgenic mice and control mice we could find no increase in basal activity of the mutant PDE6. Both the results from the physiology and the biochemistry experiments are consistent with the interpretation that the mutation causes a much milder phenotype in vivo than was predicted from observations made using a cell-free assay system. The in vivo regulation of PDE6γ on PDE6αß may be more dynamic and context-dependent than was replicated in vitro.

Night blindness and the mechanism of constitutive signaling of mutant G90D rhodopsin.

The G90D rhodopsin mutation is known to produce congenital night blindness in humans. This mutation produces a similar condition in mice, because rods of animals heterozygous (D+) or homozygous (D+/+) for this mutation have decreased dark current and sensitivity, reduced Ca(2+), and accelerated values of tau(REC) and tau(D), similar to light-adapted wild-type (WT) rods. Our experiments indicate that G90D pigment activates the cascade, producing an equivalent background light of approximately 130 Rh* rod(-1) for D+ and 890 Rh* rod(-1) for D+/+. The active species of the G90D pigment could be unregenerated G90D opsin or G90D rhodopsin, either spontaneously activated (as Rh*) or in some other form. Addition of 11-cis-retinal in lipid vesicles, which produces regeneration of both WT and G90D opsin in intact rods and ROS membranes, had no effect on the waveform or sensitivity of dark-adapted G90D responses, indicating that the active species is not G90D opsin. The noise spectra of dark-adapted G90D and WT rods are similar, and the G90D noise variance is much less than of a WT rod exposed to background light of about the same intensity as the G90D equivalent light, indicating that Rh* is not the active species. We hypothesize that G90D rhodopsin undergoes spontaneous changes in molecular conformation which activate the transduction cascade with low gain. Our experiments provide the first indication that a mutant form of the rhodopsin molecule bound to its 11-cis-chromophore can stimulate the visual cascade spontaneously at a rate large enough to produce visual dysfunction.

Functional rescue of degenerating photoreceptors in mice homozygous for a hypomorphic cGMP phosphodiesterase 6 b allele (Pde6bH620Q).

PURPOSE: Approximately 8% of autosomal recessive retinitis pigmentosa (RP) cases worldwide are due to defects in rod-specific phosphodiesterase PDE6, a tetramer consisting of catalytic (PDE6alpha and PDE6beta) and two regulatory (PDE6gamma) subunits. In mice homozygous for a nonsense Pde6b(rd1) allele, absence of PDE6 activity is associated with retinal disease similar to humans. Although studied for 80 years, the rapid degeneration Pde6b(rd1) phenotype has limited analyses and therapeutic modeling. Moreover, this model does not represent human RP involving PDE6B missense mutations. In the current study the mouse missense allele, Pde6b(H620Q) was characterized further. METHODS: Photoreceptor degeneration in Pde6b(H620Q) homozygotes was documented by histochemistry, whereas PDE6beta expression and activity were monitored by immunoblotting and cGMP assays. To measure changes in rod physiology, electroretinograms and intracellular Ca(2+) recording were performed. To test the effectiveness of gene therapy, Opsin::Pde6b lentivirus was subretinally injected into Pde6b(H620Q) homozygotes. RESULTS: Within 3 weeks of birth, the Pde6b(H620Q) homozygotes displayed relatively normal photoreceptors, but by 7 weeks degeneration was largely complete. Before degeneration, PDE6beta expression and PDE6 activity were reduced. Although light-/dark-adapted total cGMP levels appeared normal, Pde6b(H620Q) homozygotes exhibited depressed rod function and elevated outer segment Ca(2+). Transduction with Opsin::Pde6b lentivirus resulted in histologic and functional rescue of photoreceptors. CONCLUSIONS: Pde6b(H620Q) homozygous mice exhibit a hypomorphic phenotype with partial PDE6 activity that may result in an increased Ca(2+) to promote photoreceptor death. As degeneration in Pde6b(H620Q) mutants is slower than in Pde6b(rd1) mice and can be suppressed by Pde6b transduction, this Pde6b(H620Q) model may provide an alternate means to explore new treatments of RP.

GAP-independent termination of photoreceptor light response by excess gamma subunit of the cGMP-phosphodiesterase.

We have generated a mouse with rod photoreceptors overexpressing the gamma inhibitory subunit (PDE6gamma) of the photoreceptor G-protein effector cGMP phosphodiesterase (PDE6). PDE6gamma overexpression decreases the rate of rise of the rod response at dim intensities, indicating a reduction in the gain of transduction that may be the result of cytoplasmic PDE6gamma binding to activated transducin alpha GTP (Talpha-GTP) before the Talpha-GTP binds to endogenous PDE6gamma. Excess PDE6gamma also produces a marked acceleration in the falling phase of the light response and more rapid recovery of sensitivity and circulating current after prolonged light exposure. These effects are not mediated by accelerating GTP hydrolysis through the GAP (GTPase activating protein) complex, because the decay of the light response is also accelerated in rods that overexpress PDE6gamma but lack RGS9. Our results show that the PDE6gamma binding sites of PDE6 alpha and beta are accessible to excess (presumably cytoplasmic) PDE6gamma in the light, once endogenous PDE6gamma has been displaced from its binding site by Talpha-GTP. They also suggest that in the presence of Talpha-GTP, the PDE6gamma remains attached to the rest of the PDE6 molecule, but after conversion of Talpha-GTP to Talpha-GDP, the PDE6gamma may dissociate from the PDE6 and exchange with a cytoplasmic pool. This pool may exist even in wild-type rods and may explain the decay of rod photoresponses in the presence of nonhydrolyzable analogs of GTP.

Opsin activation of transduction in the rods of dark-reared Rpe65 knockout mice.

Rpe65 knockout mice (Rpe65-/-) are unable to synthesize the visual pigment chromophore 11-cis retinal; however, if these animals are reared in complete darkness, the rod photoreceptors accumulate a small amount of 9-cis retinal and its corresponding visual pigment isorhodopsin. Suction-electrode recording of single rods from dark-reared Rpe65-/- mice showed that the rods were about 400 times less sensitive than wild-type control rods and that the maximum responses were much smaller in amplitude. Spectral sensitivity measurements indicated that Rpe65-/- rod responses were generated by isorhodopsin rather than rhodopsin. Sensitivity and pigment concentration were compared in the same mice by measuring light responses from rods of one eye and pigment concentration from the retina of the other eye. Retinas had 11-35% of the normal pigment level, but the rods were of the order of 20-30 times less sensitive than could be accounted for by the loss in quantum catch. This extra desensitization must be caused by opsin-dependent activation of the visual cascade, which leads to a state equivalent to light adaptation in the dark-adapted rod. By comparing the sensitivity of dark-reared Rpe65-/- rods to that produced in normal rods by background light, we estimate that Rpe65-/- opsin is of the order of 2.5x10(-5) as efficient in activating transduction as photoactivated rhodopsin (Rh*) in WT mice. Dark-reared Rpe65-/- rods are less desensitized than rods from cyclic light-reared Rpe65-/- mice, have about 50% more photocurrent and degenerate at a slower rate. Retinas sectioned after 9 months in darkness show a larger number of photoreceptor nuclei in dark-reared animals than in cyclic light-reared animals, though both have fewer nuclei than in cyclic light-reared wild-type retinas. Both also have shorter outer segments and a lower free-Ca2+ concentration. These experiments provide the first quantitative measurement of opsin activation in physiologically responding mammalian rods.

Light-induced Ca2+ release in the visible cones of the zebrafish.

We used suction-pipette recording and fluo-4 fluorescence to study light-induced Ca2+ release from the visible double cones of zebrafish. In Ringer, light produces a slow decrease in fluorescence which can be fitted by the sum of two decaying exponentials with time constants of 0.5 and 3.8 s. In 0Ca2+-0Na+ solution, for which fluxes of Ca2+ across the outer segment plasma membrane are greatly reduced, light produces a slow increase in fluorescence. Both the decrease and increase are delayed after incorporation of the Ca2+ chelator BAPTA, indicating that both are produced by a change in Ca2+. If the Ca2+ pool is first released by bright light in 0Ca2+-0Na+ solution and the cone returned to Ringer, the time course of Ca2+ decline is much faster than in Ringer without previous light exposure. This indicates that the time constants of 0.5 and 3.8 s actually reflect a sum of Na+/Ca2+-K+ exchange and light-induced release of Ca2+. The Ca2+ released by light appears to come from at least two sites, the first comprising 66% of the total pool and half-released by bleaching 4.8% of the pigment. Release of the remaining Ca2+ from the second site requires the bleaching of nearly all of the pigment. If, after release, the cone is maintained in darkness, a substantial fraction of the Ca2+ returns to the release pool even in the absence of pigment regeneration. The light-induced release of Ca2+ can produce a modulation of the dark current as large as 0.75 pA independently of the normal transduction cascade, though the rise time of the current is considerably slower than the normal light response. These experiments show that Ca2+ can be released within the cone outer segment by light intensities within the physiological range of photopic vision. The role this Ca2+ release plays remains unresolved.

Light stimulates a transducin-independent increase of cytoplasmic Ca2+ and suppression of current in cones from the zebrafish mutant nof.

Transducins couple visual pigments to cGMP hydrolysis, the only recognized phototransduction pathway in vertebrate photoreceptors. Here we describe a zebrafish mutant, no optokinetic response f(w21) (nof), with a nonsense mutation in the gene encoding the alpha subunit of cone transducin. Retinal morphology and levels of phototransduction enzymes are normal in nof retinas, but cone transducin is undetectable. Dark current in nof cones is also normal, but it is insensitive to moderate intensity light. The nof cones do respond, however, to bright light. These responses are produced by a light-stimulated, but transducin-independent, release of Ca2+ into the cone cytoplasm. Thus, in addition to stimulating transducin, light also independently induces release of Ca2+ into the photoreceptor cytoplasm.

BetaB1-crystallin: identification of a candidate ciliary body uveitis antigen.

PURPOSE: Perineuclear anti-neutrophil cytoplasmic antibody (pANCA), a marker antibody present in 12% of patients with anterior uveitis, recognizes cytoplasmic antigens in the nonpigmented ciliary body epithelium, a probable site of immunologic reactivity in this inflammatory disease. In this study, a recombinantly isolated pANCA monoclonal antibody was used to identify the corresponding antigenic target(s) in the ciliary body. METHODS: Proteins from microdissected eye bank ocular ciliary body tissue were used to identify the corresponding ANCA antigen. Parallel two-dimensional protein gels were used for simultaneous identification of candidate antigenic protein spots by Western blot analysis and as a source of material for proteomic analysis. Multiple independent methods including Western blot analysis, confocal microscopy, and RT-PCR were used to provide additional characterization of the candidate protein. RESULTS: Proteomic analysis suggested that beta B1 (betaB1)-crystallin is the primary ciliary body antigen. The presence of betaB1-crystallin in the human ciliary body was confirmed by Western blot with a betaB1 specific anti-peptide antibody. Confocal microscopy revealed colocalization of the antigenic reactivity of both anti-betaB1 antibody and monoclonal pANCA. RT-PCR confirmed the presence of betaB1-crystallin RNA in the ciliary body tissues. CONCLUSIONS: This study identified betaB1-crystallin as a new cytoplasmic ciliary body antigenic target of a marker autoantibody associated with uveitis. This characterization of betaB1-crystallin outside the lens raises questions about its extralenticular expression, intracellular role, and potential target of inflammation in uveitis.

P2 purinergic receptor-coupled signaling in the rabbit ciliary body epithelium.

PURPOSE: To identify and characterize P2 purinergic receptors and their signaling pathways in the epithelial cells of the rabbit ciliary body. METHODS: Real-time fluorescence ratio imaging of the intact fura-2-loaded nonpigmented ciliary body epithelial (NPE) cells of rabbit were used to record changes in the intracellular free calcium concentration ([Ca(2+)](i)), in response to a number of purinergic agonists and antagonists. The effects of some of these drugs on the inositol phosphate (IP) levels in ciliary processes were also examined. RESULTS: Adenosine diphosphate (ADP), adenosine triphosphate (ATP), and uridine triphosphate (UTP) dose dependently increased the [Ca(2+)](i) and IP levels. The [Ca(2+)](i) increases induced by ADP and UTP were distinguishable, both kinetically and pharmacologically. The effect of ADP on [Ca(2+)](i) was mimicked by a number of P2Y(1)-selective agonists, and was blocked by three P2Y(1)-receptor-specific antagonists. The [Ca(2+)](i) increases elicited by ADP (or its analogs) and UTP were additive. CONCLUSIONS: Rabbit ciliary body epithelium possesses both P2Y(1) and P2Y(2) metabotropic purinergic receptor subtypes, which differentially use the IP(3)/Ca(2+) second-messenger pathway.