Marianne C. Cilluffo

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 2.5 × 10−5 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.

GAP-Independent Termination of Photoreceptor Light Response by Excess γ Subunit of the cGMP-Phosphodiesterase

We have generated a mouse with rod photoreceptors overexpressing the γ inhibitory subunit (PDE6γ) of the photoreceptor G-protein effector cGMP phosphodiesterase (PDE6). PDE6γ 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 PDE6γ binding to activated transducin α GTP (Tα-GTP) before the Tα-GTP binds to endogenous PDE6γ. Excess PDE6γ 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 PDE6γ but lack RGS9. Our results show that the PDE6γ binding sites of PDE6 α and β are accessible to excess (presumably cytoplasmic) PDE6γ in the light, once endogenous PDE6γ has been displaced from its binding site by Tα-GTP. They also suggest that in the presence of Tα-GTP, the PDE6γ remains attached to the rest of the PDE6 molecule, but after conversion of Tα-GTP to Tα-GDP, the PDE6γ 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.

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.

Replacing the rod with the cone transducin α subunit decreases sensitivity and accelerates response decay

Cone vision is less sensitive than rod vision. Much of this difference can be attributed to the photoreceptors themselves, but the reason why the cones are less sensitive is still unknown. Recent recordings indicate that one important factor may be a difference in the rate of activation of cone transduction; that is, the rising phase of the cone response per bleached rhodopsin molecule (Rh*) has a smaller slope than the rising phase of the rod response per Rh*, perhaps because some step between Rh* and activation of the phosphodiesterase 6 (PDE6) effector molecule occurs with less gain. Since rods and cones have different G‐protein α subunits, and since this subunit (Tα) plays a key role both in the interaction of G‐protein with Rh* and the activation of PDE6, we investigated the mechanism of the amplification difference by expressing cone Tα in rod Tα‐knockout rods to produce so‐called GNAT2C mice. We show that rods in GNAT2C mice have decreased sensitivity and a rate of activation half that of wild‐type (WT) mouse rods. Furthermore, GNAT2C responses recover more rapidly than WT responses with kinetic parameters resembling those of native mouse cones. Our results show for the first time that part of the difference in sensitivity and response kinetics between rods and cones may be the result of a difference in the G‐protein α subunit. They also indicate more generally that the molecular nature of G‐protein α may play an important role in the kinetics of G‐protein cascades for metabotropic receptors throughout the body.

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 (τREC) and the limiting time constant (τD) 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.

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 Ca2+, and accelerated values of τREC and τD, similar to light-adapted wild-type (WT) rods. Our experiments indicate that G90D pigment activates the cascade, producing an equivalent background light of ∼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.

Removal of phosphorylation sites of γ subunit of phosphodiesterase 6 alters rod light response

The phosphodiesterase 6 γ (PDE6γ) inhibitory subunit of the rod PDE6 effector enzyme plays a central role in the turning on and off of the visual transduction cascade, since binding of PDE6γ to the transducin α subunit (Tα) initiates the hydrolysis of the second messenger cGMP, and PDE6γ in association with RGS9‐1 and the other GAP complex proteins (Gβ5, R9AP) accelerates the conversion of TαGTP to TαGDP, the rate‐limiting step in the decay of the rod light response. Several studies have shown that PDE6γ can be phosphorylated at two threonines, T22 and T35, and have proposed that phosphorylation plays some role in the physiology of the rod. We have examined this possibility by constructing mice in which T22 and/or T35 were replaced with alanines. Our results show that T35A rod responses rise and decay more slowly and are less sensitive to light than wild‐type (WT). T22A responses show no significant difference in initial time course with WT but decay more rapidly, especially at dimmer intensities. When the T22A mutation is added to T35A, double mutant rods no longer showed the prolonged decay of T35A rods but remained slower than WT in initial time course. Our experiments suggest that the polycationic domain of PDE6γ containing these two phosphorylation sites can influence the rate of PDE6 activation and deactivation and raise the possibility that phosphorylation or dephosphorylation of PDE6γ could modify the time course of transduction, thereby influencing the wave form of the light response.

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.

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 β‐galactosidase (β‐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 β‐gal expression in dab1lacZ/+ mice. Adult pyramidal neurons in cortical layers II–III and V are labeled with Dab1 and/or β‐gal and are inverted in the dab1lacZ/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.

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.