Genetics of photoreceptor degeneration and regeneration in zebrafish

Zebrafish are unique in that they provide a useful model system for studying two critically important problems in retinal neurobiology, the mechanisms responsible for triggering photoreceptor cell death and the innate stem cell–mediated regenerative response elicited by this death. In this review we highlight recent seminal findings in these two fields. We first focus on zebrafish as a model for studying photoreceptor degeneration. We summarize the genes currently known to cause photoreceptor degeneration, and we describe the phenotype of a few zebrafish mutants in detail, highlighting the usefulness of this model for studying this process. In the second section, we discuss the several different experimental paradigms that are available to study regeneration in the teleost retina. A model outlining the sequence of gene expression starting from the dedifferentiation of Müller glia to the formation of rod and cone precursors is presented.     

The absence of melanopsin alters retinal clock function and dopamine regulation by light

The retinal circadian clock is crucial for optimal regulation of retinal physiology and function, yet its cellular location in mammals is still controversial. We used laser microdissection to investigate the circadian profiles and phase relations of clock gene expression and Period gene induction by light in the isolated outer (rods/cones) and inner (inner nuclear and ganglion cell layers) regions in wild-type and melanopsin-knockout (Opn ₄ ^?/? ) mouse retinas. In the wild-type mouse, all clock genes are rhythmically expressed in the photoreceptor layer but not in the inner retina. For clock genes that are rhythmic in both retinal compartments, the circadian profiles are out of phase. These results are consistent with the view that photoreceptors are a potential site of circadian rhythm generation. In mice lacking melanopsin, we found an unexpected loss of clock gene rhythms and of the photic induction of Per1-Per2 mRNAs only in the outer retina. Since melanopsin ganglion cells are known to provide a feed-back signalling pathway for photic information to dopaminergic cells, we further examined dopamine (DA) synthesis in Opn ₄ ^?/? mice. The lack of melanopsin prevented the light-dependent increase of tyrosine hydroxylase (TH) mRNA and of DA and, in constant darkness, led to comparatively high levels of both components. These results suggest that melanopsin is required for molecular clock function and DA regulation in the retina, and that Period gene induction by light is mediated by a melanopsin-dependent, DA-driven signal acting on retinal photoreceptors.     

Effects of barium on separately recorded fast and slow PIII responses in bullfrog retina

Investigation of Ba2+ effects on fast and slow PIII responses in isolated bullfrog retina revealed that Ba2+ suppressed slow PIII completely with little effect on fast PIII. A light-induced [K+]0 decrease in the photoreceptor layer was observed in spite of Ba2+ perfusion, indicating the suppressive action of Ba2+ on the K+ conductance of the Müller cell membrane.     

Calcium binding, structural stability and guanylate cyclase activation in GCAP1 variants associated with human cone dystrophy

Guanylate cyclase activating protein 1 (GCAP1) is a neuronal Ca²⁺ sensor (NCS) that regulates the activation of rod outer segment guanylate cyclases (ROS-GCs) in photoreceptors. In this study, we investigated the Ca²⁺-induced effects on the conformation and the thermal stability of four GCAP1 variants associated with hereditary human cone dystrophies. Ca²⁺ binding stabilized the conformation of all the GCAP1 variants independent of myristoylation. The myristoylated wild-type GCAP1 was found to have the highest Ca²⁺ affinity and thermal stability, whereas all the mutants showed decreased Ca²⁺ affinity and significantly lower thermal stability in both apo and Ca²⁺-loaded forms. No apparent cooperativity of Ca²⁺ binding was detected for any variant. Finally, the nonmyristoylated mutants were still capable of activating ROS-GC1, but the measured cyclase activity was shifted toward high, nonphysiological Ca²⁺ concentrations. Thus, we conclude that distorted Ca²⁺-sensor properties could lead to cone dysfunction.     

Realism without tears II: The structuralist legacy of sensory physiology

This paper examines the implications of the Doctrine of Specific Nerve Energies for contemporary philosophy and psychology. Part I analyzed Johannes Peter Müller’s canonical formulation of the Doctrine, arguing that it follows from empirical results combined with methodological principles. Here, I argue that these methodological principles remain valid in psychology today, consequently, any naturalistic philosophy of perception must accept the Doctrine’s skeptical conclusion, that the qualities of our perceptual experience are not determined by, and thus do not reveal the nature of, their causes in the world. Nevertheless, this does not mean that we must be global skeptics; rather, I argue that contemporary epistemology of perception should embrace Müller’s own response to the Doctrine: epistemic structural realism. As articulated by Müller’s student, Helmholtz, structural realism follows from the Doctrine once we recognize that active exploration constitutes part of the mechanism that determines perceptual experience, a view congenial to contemporary theories of embodied perception in cognitive science. Structural realists in philosophy of science should likewise heed the lessons of the Doctrine, as it played a critical part in the early history of their view, and may still serve a constructive role as exemplar today.     

Light-dependent phosphorylation of Bardet–Biedl syndrome 5 in photoreceptor cells modulates its interaction with arrestin1

Arrestins are dynamic proteins that move between cell compartments triggered by stimulation of G-protein-coupled receptors. Even more dynamically in vertebrate photoreceptors, arrestin1 (Arr1) moves between the inner and outer segments according to the light conditions. Previous studies have shown that the light-driven translocation of Arr1 in rod photoreceptors is initiated by rhodopsin through a phospholipase C/protein kinase C (PKC) signaling cascade. The purpose of this study is to identify the PKC substrate that regulates the translocation of Arr1. Mass spectrometry was used to identify the primary phosphorylated proteins in extracts prepared from PKC-stimulated mouse eye cups, confirming the finding with in vitro phosphorylation assays. Our results show that Bardet–Biedl syndrome 5 (BBS5) is the principal protein phosphorylated either by phorbol ester stimulation or by light stimulation of PKC. Via immunoprecipitation of BBS5 in rod outer segments, Arr1 was pulled down; phosphorylation of BBS5 reduced this co-precipitation of Arr1. Immunofluorescence and immunoelectron microscopy showed that BBS5 principally localizes along the axonemes of rods and cones, but also in photoreceptor inner segments, and synaptic regions. Our principal findings in this study are threefold. First, we demonstrate that BBS5 is post-translationally regulated by phosphorylation via PKC, an event that is triggered by light in photoreceptor cells. Second, we find a direct interaction between BBS5 and Arr1, an interaction that is modulated by phosphorylation of BBS5. Finally, we show that BBS5 is distributed along the photoreceptor axoneme, co-localizing with Arr1 in the dark. These findings suggest a role for BBS5 in regulating light-dependent translocation of Arr1 and a model describing its role in Arr1 translocation is proposed.     

Realism without tears I: Müller’s Doctrine of Specific Nerve Energies

The Doctrine of Specific Nerve Energies has been and continues to be enormously influential in the physiology, psychology, and philosophy of perception. In simple terms, the Doctrine states that we directly perceive in the first instance the activity of our nerves, rather than properties in the external world. The canonical early statement of the Doctrine by the physiologist Johannes Peter Müller had profound influence on both the philosophy and psychology of the 19^th and early 20^th centuries, especially as reformulated and transmitted by Müller’s student Helmholtz. A common assumption of historical and ongoing debate about the Doctrine has been its supposedly idealist or skeptical implications. What is not commonly recognized is that Müller himself advanced a realist interpretation along lines that would be recognized today as a form of epistemic structural realism. This paper analyzes Müller’s structuralist epistemology in detail and reconstructs his articulation and defense of the Doctrine of Specific Nerve Energies in its canonical form. Part II argues for the continued importance of the Doctrine and its structuralist interpretation for contemporary psychology, philosophy of perception, and history of philosophy of science.     

Soluble factors and the development of rod photoreceptors

Photoreceptors are the most abundant cell type in the vertebrate neural retina. Like the other retinal neurons and the Müller glia, they arise from a population of precursor cells that are multipotent and intrinsic to the retina. Approximately 10 years ago, several studies demonstrated that retinal precursor cells (RPCs) are competent to respond to environmental factors that promote cell type determination and differentiation. Since those studies, significant effort has been directed at identifying the molecular nature of these environmental signals and understanding the precise mechanisms they employ to drive RPCs towards the different retinal fates. In this review, we describe the recent progress toward understanding how environmental factors influence the development of vertebrate rod photoreceptors.     

Defective germinal center B-cell response and reduced arthritic pathology in microRNA-29a-deficient mice

MicroRNA (miR) are short non-coding RNA sequences of 19–24 nucleotides that regulate gene expression by binding to mRNA target sequences. The miR-29 family of miR (miR-29a, b-1, b-2 and c) is a key player in T-cell differentiation and effector function, with deficiency causing thymic involution and a more inflammatory T-cell profile. However, the relative roles of different miR-29 family members in these processes have not been dissected. We studied the immunological role of the individual members of the miR-29 family using mice deficient for miR-29a/b-1 or miR-29b-2/c in homeostasis and during collagen-induced arthritis. We found a definitive hierarchy of immunological function, with the strong phenotype of miR-29a-deficiency in thymic involution and T-cell activation being reduced or absent in miR-29c-deficient mice. Strikingly, despite elevating the Th1 and Th17 responses, loss of miR-29a conferred near-complete protection from collagen-induced arthritis (CIA), with profound defects in B-cell proliferation and antibody production. Our results identify the hierarchical structure of the miR-29 family in T-cell biology, and identify miR-29a in B cells as a potential therapeutic target in arthritis.     

Sensitivity and response kinetics alter during suppression-recovery in cone photoreceptors

Threshold and response amplitude of cone photoreceptors were measured during progression from flicker-induced suppression to recovery. Increases in sensitivity closely paralleled increases in amplitude. Recovered responses exhibited faster kinetics than suppressed responses. The idea that recovery from suppression is a manifestation of the shift in the stimulus-response relationship which occurs with light adaptation is supported.     

Cone monochromacy and a reversed Purkinje shift in the gerbil

Summary Retinal spectral sensitivity of the gerbil (Meriones unguiculatus) shows that this animal has a unique photoreceptor complement: there is only a single class of cone and the spectrum for these receptors is peak shifted toward the short wavelengths relative to the spectra obtained from rods.     

A di-arginine ER retention signal regulates trafficking of HCN1 channels from the early secretory pathway to the plasma membrane

Hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels carry I_h, which contributes to neuronal excitability and signal transmission in the nervous system. Controlling the trafficking of HCN1 is an important aspect of its regulation, yet the details of this process are poorly understood. Here, we investigated how the C-terminus of HCN1 regulates trafficking by testing for its ability to redirect the localization of a non-targeted reporter in transgenic Xenopus laevis photoreceptors. We found that HCN1 contains an ER localization signal and through a series of deletion constructs, identified the responsible di-arginine ER retention signal. This signal is located in the intrinsically disordered region of the C-terminus of HCN1. To test the function of the ER retention signal in intact channels, we expressed wild type and mutant HCN1 in HEK293 cells and found this signal negatively regulates surface expression of HCN1. In summary, we report a new mode of regulating HCN1 trafficking: through the use of a di-arginine ER retention signal that monitors processing of the channel in the early secretory pathway.     

<Emphasis Type="Italic">C</Emphasis>-<Emphasis Type="Italic">Raf</Emphasis> deficiency leads to hearing loss and increased noise susceptibility

The family of RAF kinases transduces extracellular information to the nucleus, and their activation is crucial for cellular regulation on many levels, ranging from embryonic development to carcinogenesis. B-RAF and C-RAF modulate neurogenesis and neuritogenesis during chicken inner ear development. C-RAF deficiency in humans is associated with deafness in the rare genetic insulin-like growth factor 1 (IGF-1), Noonan and Leopard syndromes. In this study, we show that RAF kinases are expressed in the developing inner ear and in adult mouse cochlea. A homozygous C-Raf deletion in mice caused profound deafness with no evident cellular aberrations except for a remarkable reduction of the K⁺ channel Kir4.1 expression, a trait that suffices as a cause of deafness. To explore the role of C-Raf in cellular protection and repair, heterozygous C-Raf ^+/? mice were exposed to noise. A reduced C-RAF level negatively affected hearing preservation in response to noise through mechanisms involving the activation of JNK and an exacerbated apoptotic response. Taken together, these results strongly support a role for C-RAF in hearing protection.     

Ultrastructure and biochemistry of the pineal organ in deep-sea lanternfishes (Myctophidae)

Summary Pineal structural and biochemical adaptations in lanternfishes included: 1) few photoreceptor outer segment discs; 2) conventional synapses between photoreceptors and pineal neurons; and 3) low levels (0–60 pg/pineal) of serotonin compared to those (>1.0 ng/pineal) in the goldfish pineal organ. These findings suggest reduced photosensory and/or neuroendocrine functions in these deep-sea fishes.     

Ultrastructure and biochemistry of the pineal organ in deep-sea lanternfishes (Myctophidae)

Pineal structural and biochemical adaptations in lanternfishes included: 1) few photoreceptor outer segment discs; 2) conventional synapses between photoreceptors and pineal neurons; and 3) low levels (0-60 pg/pineal) of serotonin compared to those (greater than 1.0 ng/pineal) in the goldfish pineal organ. These findings suggest reduced photosensory and/or neuroendocrine functions in these deep-sea fishes.     

Complement factor H in host defense and immune evasion

Complement is the major humoral component of the innate immune system. It recognizes pathogen- and damage-associated molecular patterns, and initiates the immune response in coordination with innate and adaptive immunity. When activated, the complement system unleashes powerful cytotoxic and inflammatory mechanisms, and thus its tight control is crucial to prevent damage to host tissues and allow restoration of immune homeostasis. Factor H is the major soluble inhibitor of complement, where its binding to self markers (i.e., particular glycan structures) prevents complement activation and amplification on host surfaces. Not surprisingly, mutations and polymorphisms that affect recognition of self by factor H are associated with diseases of complement dysregulation, such as age-related macular degeneration and atypical haemolytic uremic syndrome. In addition, pathogens (i.e., non-self) and cancer cells (i.e., altered-self) can hijack factor H to evade the immune response. Here we review recent (and not so recent) literature on the structure and function of factor H, including the emerging roles of this protein in the pathophysiology of infectious diseases and cancer.     

Zur Wirkung von Strahlenschutzstoffen an Thymuszellen

Summary Lymphocytes of the thymus can be protected against early pycnotic degeneration by several SH-compounds. Protective and non-protective SH-compounds induce the same reaction in the non-irradiated mouse, weight loss caused by cell migration, stimulation of the reticulum. A direct relationship between the activity of the reticulum and the radioresistance of lymphocytes was not found.     

Sequential inactivation of Rho GTPases and Lim kinase by Pseudomonas aeruginosa toxins ExoS and ExoT leads to endothelial monolayer breakdown

Pseudomonas aeruginosa is a major human opportunistic pathogen and one of the most important causal agents of bacteremia. For non-blood-borne infection, bacterial dissemination requires the crossing of the vascular endothelium, the main barrier between blood and the surrounding tissues. Here, we investigated the effects of P. aeruginosa type 3 secretion effectors, namely ExoS, ExoT, and ExoY, on regulators of actin cytoskeleton dynamics in primary endothelial cells. ExoS and ExoT similarly affected the Lim kinase-cofilin pathway, thereby promoting actin filament severing. Cofilin activation was also observed in a mouse model of P. aeruginosa-induced acute pneumonia. Rho, Rac, and Cdc42 GTPases were sequentially inactivated, leading to inhibition of membrane ruffling, filopodia, and stress fiber collapse, and focal adhesion disruption. At the end of the process, ExoS and ExoT produced a dramatic retraction in all primary endothelial cell types tested and thus a rupture of the endothelial monolayer. ExoY alone had no effect in this context. Cell retraction could be counteracted by overexpression of actin cytoskeleton regulators. In addition, our data suggest that moesin is neither a direct exotoxin target nor an important player in this process. We conclude that any action leading to inhibition of actin filament breakdown will improve the barrier function of the endothelium during P. aeruginosa infection.