Factor H's Control of Complement Activation Emerges as a Significant and Promising Therapeutic Target for Alzheimer's Disease Treatment.

The complement is a component of the innate immune system designed to fight infections and tissue- or age-related damages. Complement activation creates an inflammatory microenvironment, which enhances cell death. Excessive complement inflammatory activity has been linked to alterations in the structure and functions of the blood-brain barrier, contributing to a poor prognosis for Alzheimer's disease (AD). In the AD preclinical phase, individuals are often clinically asymptomatic despite evidence of AD neuropathology coupled with heightened inflammation. Considering the involvement of the complement system in the risk of developing AD, we hypothesize that inhibiting complement activation could reduce this inflammatory period observed even before clinical signs, thereby slowing down the onset/progression of AD. To validate our hypothesis, we injected complement inhibitor factor H into the brain of APP/PS1 AD mice at early or late stages of this pathology. Our results showed that the injection of factor H had effects on both the onset and progression of AD by reducing proinflammatory IL6, TNF-α, IL1ß, MAC and amyloid beta levels. This reduction was associated with an increase in VGLUT1 and Psd95 synaptic transmission in the hippocampal region, leading to an improvement in cognitive functions. This study invites a reconsideration of factor H's therapeutic potential for AD treatment.

3D imaging and morphometric descriptors of vascular networks on optically cleared organs.

The vascular system is a multi-scale network whose functionality depends on its structure, and for which structural alterations can be linked to pathological shifts. Though biologists use multiple 3D imaging techniques to visualize vascular networks, the 3D image processing methodologies remain sources of biases, and the extraction of quantitative morphometric descriptors remains flawed. The article, first, reviews the current 3D image processing methodologies, and morphometric descriptors of vascular network images mainly obtained by light-sheet microscopy on optically cleared organs, found in the literature. Second, it proposes operator-independent segmentation and skeletonization methodologies using the freeware ImageJ. Third, it gives more extractable network-level (density, connectivity, fractal dimension) and segment-level (length, diameter, tortuosity) 3D morphometric descriptors and how to statistically analyze them. Thus, it can serve as a guideline for biologists using 3D imaging techniques of vascular networks, allowing the production of more comparable studies in the future.

Concomitant Retinal Alterations in Neuronal Activity and TNFα Pathway Are Detectable during the Pre-Symptomatic Stage in a Mouse Model of Alzheimer's Disease.

The pre-symptomatic stage of Alzheimer's disease (AD) is associated with increased amyloid-ß (Aß) precursor protein (APP) processing and Aß accumulation in the retina and hippocampus. Because neuronal dysfunctions are among the earliest AD-related alterations, we asked whether they are already detectable in the retina during the pre-symptomatic stage in a APPswePS1dE9 (APP/PS1) mouse model. The age chosen for the study (3-4 months) corresponds to the pre-symptomatic stage because no retinal Aß was detected, in spite of the presence of ßCTF (the first cleavage product of APP). We observed an increase in ERG amplitudes in APP/PS1 mice in comparison to the controls, which indicated an increased retinal neuron activity. These functional changes coincided with an increased expression of retinal TNFα and its receptors type-1 (TNFR1). Consistently, the IkB expression increased in APP/PS1 mice with a greater proportion of the phosphorylated protein (P-IkB) over total IkB, pointing to the putative involvement of the NFkB pathway. Because TNFα plays a crucial role in the control of neuronal excitability, it is likely that, as in the hippocampus, TNFα signaling via the TNFR1/NFkB pathway may be also involved in early, AD-associated, retinal neuron hyperexcitability. These results further demonstrate the interest of the retina for early disease detection with a potential to assess future therapeutic strategies.

Meteorin Is a Novel Therapeutic Target for Wet Age-Related Macular Degeneration.

The aim of this study was to evaluate the potential anti-angiogenic effect of MTRN (meteorin) in the laser-induced CNV rat model and explore its mechanisms of action. MTRN, thrompospondin-1, glial cell markers (GFAP, vimentin), and phalloidin were immuno-stained in non-human primate flat-mounted retinas and human retina cross sections. The effect of MTRN at different doses and time points was evaluated on laser-induced CNV at 14 days using in vivo fluorescein angiography and ex vivo quantification of CNV. A pan transcriptomic analysis of the retina and the RPE/choroid complex was used to explore MTRN effects mechanisms. In human retina, MTRN is enriched in the macula, expressed in and secreted by glial cells, and located in photoreceptor cells, including in nuclear bodies. Intravitreal MTRN administered preventively reduced CNV angiographic scores and CNV size in a dose-dependent manner. The highest dose, administered at day 7, also reduced CNV. MTRN, which is regulated by mineralocorticoid receptor modulators in the rat retina, regulates pathways associated with angiogenesis, oxidative stress, and neuroprotection. MTRN is a potential novel therapeutic candidate protein for wet AMD.

Mechanisms of FH Protection Against Neovascular AMD.

A common allele (402H) of the complement factor H (FH) gene is the major risk factor for age-related macular degeneration (AMD), the leading cause of blindness in the elderly population. Development and progression of AMD involves vascular and inflammatory components partly by deregulation of the alternative pathway of the complement system (AP). The loss of central vision results from atrophy and/or from abnormal neovascularization arising from the choroid. The functional link between FH, the main inhibitor of AP, and choroidal neovascularization (CNV) in AMD remains unclear. In a murine model of CNV used as a model for neovascular AMD (nAMD), intraocular human recombinant FH (recFH) reduced CNV as efficiently as currently used anti-VEGF (vascular endothelial growth factor) antibody, decreasing deposition of C3 cleavage fragments, membrane attack complex (MAC), and microglia/macrophage recruitment markers in the CNV lesion site. In sharp contrast, recFH carrying the H402 risk variant had no effect on CNV indicating a causal link to disease etiology. Only the recFH NTal region (recFH1-7), containing the CCPs1-4 C3-convertase inhibition domains and the CCP7 binding domain, exerted all differential biological effects. The CTal region (recFH7-20) containing the CCP7 and CCPs19-20 binding domains was antiangiogenic but did not reduce the microglia/macrophage recruitment. The antiangiogenic effect of both recFH1-20 and recFH-CCP7-20 resulted from thrombospondin-1 (TSP-1) upregulation independently of the C3 cleavage fragments generation. This study provides insight on the mechanistic role of FH in nAMD and invites to reconsider its therapeutic potential.

Brain-Immune Interactions and Neuroinflammation After Traumatic Brain Injury.

Traumatic brain injury (TBI) is the principal cause of death and disability in children and young adults. Clinical and preclinical research efforts have been carried out to understand the acute, life-threatening pathophysiological events happening after TBI. In the past few years, however, it was recognized that TBI causes significant morbidity weeks, months, or years after the initial injury, thereby contributing substantially to the overall burden of TBI and the decrease of life expectancy in these patients. Long-lasting sequels of TBI include cognitive decline/dementia, sensory-motor dysfunction, and psychiatric disorders, and most important for patients is the need for socio-economic rehabilitation affecting their quality of life. Cerebrovascular alterations have been described during the first week after TBI for direct consequence development of neuroinflammatory process in relation to brain edema. Within the brain-immune interactions, the complement system, which is a family of blood and cell surface proteins, participates in the pathophysiology process. In fact, the complement system is part of the primary defense and clearance component of innate and adaptive immune response. In this review, the complement activation after TBI will be described in relation to the activation of the microglia and astrocytes as well as the blood-brain barrier dysfunction during the first week after the injury. Considering the neuroinflammatory activity as a causal element of neurological handicaps, some major parallel lines of complement activity in multiple sclerosis and Alzheimer pathologies with regard to cognitive impairment will be discussed for chronic TBI. A better understanding of the role of complement activation could facilitate the development of new therapeutic approaches for TBI.

CFH exerts anti-oxidant effects on retinal pigment epithelial cells independently from protecting against membrane attack complex.

Age Related Macular Degeneration (AMD) is the first cause of social blindness in people aged over 65 leading to atrophy of retinal pigment epithelial cells (RPE), photoreceptors and choroids, eventually associated with choroidal neovascularization. Accumulation of undigested cellular debris within RPE cells or under the RPE (Drusen), oxidative stress and inflammatory mediators contribute to the RPE cell death. The major risk to develop AMD is the Y402H polymorphism of complement factor H (CFH). CFH interacting with oxidized phospholipids on the RPE membrane modulates the functions of these cells, but the exact role of CFH in RPE cell death and survival remain poorly understood. The aim of this study was to analyze the potential protective mechanism of CFH on RPE cells submitted to oxidative stress. Upon exposure to oxidized lipids 4-HNE (4-hydroxy-2-nonenal) derived from photoreceptors, both the human RPE cell line ARPE-19 and RPE cells derived from human induced pluripotent stem cells were protected from death only in the presence of the full length human recombinant CFH in the culture medium. This protective effect was independent from the membrane attack complex (MAC) formation. CFH maintained RPE cells tight junctions' structure and regulated the caspase dependent apoptosis process. These results demonstrated the CFH anti-oxidative stress functions independently of its capacity to inhibit MAC formation.

AßPP-induced UPR Transcriptomic Signature of Glial Cells to Oxidative Stress as an Adaptive Mechanism to Preserve Cell Function and Survival.

BACKGROUND: Alzheimer's disease (AD) and age-related macular degeneration (AMD) present similarities, particularly with respect to oxidative stress, including production of 4-Hydroxy-2- nonenal (HNE). AMD has been named the AD in the eye. The Müller cells (MC) function as a principal glia of the retina and maintain water/potassium, glutamate homeostasis and redox status. Any MC dysfunction results in retinal neurodegeneration. OBJECTIVES: We investigated the effects of HNE in human MC. RESULTS: HNE induced an increase of the reactive oxygen species associated with mitochondrial dysfunction and apoptosis. HNE induced endoplasmic reticulum (ER) stress (upregulation of GRP78/Bip, and the proapoptotic factor, CHOP). HNE also impaired expression of genes controlling potassium homeostasis (KCNJ10), glutamate detoxification (GS), and the visual cycle (RLBP1). MC adaptive response to HNE included upregulation of amyloid-ß protein precursor (AßPP). To determine the role of AßPP, we overexpressed AßPP in MC. Overexpression of AßPP induced strong antioxidant and anti-ER stress (PERK downregulation and GADD34 upregulation) responses accompanied by activation of the prosurvival branch of the unfolded protein response. It was also associated with upregulation of major genes involved in MC-controlled retinal homeostasis (KCNJ10, GS, and RLBP1) and protection against HNE-induced apoptosis. Therefore, AßPP is an ER and oxidative stress responsive molecule, and is able to stimulate the transcription of major genes involved in MC functions impaired by HNE. CONCLUSION: Our study suggests that targeting oxidative and ER stress might be a potential therapeutic strategy against glia impairment in AMD and AD, in light of the common features between the two pathologies.

Amyloidosis in Retinal Neurodegenerative Diseases.

As a part of the central nervous system, the retina may reflect both physiological processes and abnormalities related to pathologies that affect the brain. Amyloidosis due to the accumulation of amyloid-beta (Aß) was initially regarded as a specific and exclusive characteristic of neurodegenerative alterations seen in the brain of Alzheimer's disease (AD) patients. More recently, it was discovered that amyloidosis-related alterations, similar to those seen in the brain of Alzheimer's patients, also occur in the retina. Remarkably, these alterations were identified not only in primary retinal pathologies, such as age-related macular degeneration (AMD) and glaucoma, but also in the retinas of Alzheimer's patients. In this review, we first briefly discuss the biogenesis of Aß, a peptide involved in amyloidosis. We then discuss some pathological aspects (synaptic dysfunction, mitochondrial failure, glial activation, and vascular abnormalities) related to the neurotoxic effects of Aß. We finally highlight common features shared by AD, AMD, and glaucoma in the context of Aß amyloidosis and further discuss why the retina, due to the transparency of the eye, can be considered as a "window" to the brain.

Amyloid Precursor-Like Protein 2 deletion-induced retinal synaptopathy related to congenital stationary night blindness: structural, functional and molecular characteristics.

BACKGROUND: Amyloid precursor protein knockout mice (APP-KO) have impaired differentiation of amacrine and horizontal cells. APP is part of a gene family and its paralogue amyloid precursor-like protein 2 (APLP2) has both shared as well as distinct expression patterns to APP, including in the retina. Given the impact of APP in the retina we investigated how APLP2 expression affected the retina using APLP2 knockout mice (APLP2-KO). RESULTS: Using histology, morphometric analysis with noninvasive imaging technique and electron microscopy, we showed that APLP2-KO retina displayed abnormal formation of the outer synaptic layer, accompanied with greatly impaired photoreceptor ribbon synapses in adults. Moreover, APLP2-KO displayed a significant decease in ON-bipolar, rod bipolar and type 2 OFF-cone bipolar cells (36, 21 and 63 %, respectively). Reduction of the number of bipolar cells was accompanied with disrupted dendrites, reduced expression of metabotropic glutamate receptor 6 at the dendritic tips and alteration of axon terminals in the OFF laminae of the inner plexiform layer. In contrast, the APP-KO photoreceptor ribbon synapses and bipolar cells were intact. The APLP2-KO retina displayed numerous phenotypic similarities with the congenital stationary night blindness, a non-progressive retinal degeneration disease characterized by the loss of night vision. The pathological phenotypes in the APLP2-KO mouse correlated to altered transcription of genes involved in pre- and postsynatic structure/function, including CACNA1F, GRM6, TRMP1 and Gα0, and a normal scotopic a-wave electroretinogram amplitude, markedly reduced scotopic electroretinogram b-wave and modestly reduced photopic cone response. This confirmed the impaired function of the photoreceptor ribbon synapses and retinal bipolar cells, as is also observed in congenital stationary night blindness. Since congenital stationary night blindness present at birth, we extended our analysis to retinal differentiation and showed impaired differentiation of different bipolar cell subtypes and an altered temporal sequence of development from OFF to ON laminae in the inner plexiform layer. This was associated with the altered expression patterns of bipolar cell generation and differentiation factors, including MATH3, CHX10, VSX1 and OTX2. CONCLUSIONS: These findings demonstrate that APLP2 couples retina development and synaptic genes and present the first evidence that APLP2 expression may be linked to synaptic disease.

Distinct effects of inflammation on gliosis, osmohomeostasis, and vascular integrity during amyloid beta-induced retinal degeneration.

In normal retinas, amyloid-ß (Aß) accumulates in the subretinal space, at the interface of the retinal pigment epithelium, and the photoreceptor outer segments. However, the molecular and cellular effects of subretinal Aß remain inadequately elucidated. We previously showed that subretinal injection of Aß(1-42) induces retinal inflammation, followed by photoreceptor cell death. The retinal Müller glial (RMG) cells, which are the principal retinal glial cells, are metabolically coupled to photoreceptors. Their role in the maintenance of retinal water/potassium and glutamate homeostasis makes them important players in photoreceptor survival. This study investigated the effects of subretinal Aß(1-42) on RMG cells and of Aß(1-42)-induced inflammation on retinal homeostasis. RMG cell gliosis (upregulation of GFAP, vimentin, and nestin) on day 1 postinjection and a proinflammatory phenotype were the first signs of retinal alteration induced by Aß(1-42). On day 3, we detected modifications in the protein expression patterns of cyclooxygenase 2 (COX-2), glutamine synthetase (GS), Kir4.1 [the inwardly rectifying potassium (Kir) channel], and aquaporin (AQP)-4 water channels in RMG cells and of the photoreceptor-associated AQP-1. The integrity of the blood-retina barrier was compromised and retinal edema developed. Aß(1-42) induced endoplasmic reticulum stress associated with sustained upregulation of the proapoptotic factors of the unfolded protein response and persistent photoreceptor apoptosis. Indomethacin treatment decreased inflammation and reversed the Aß(1-42)-induced gliosis and modifications in the expression patterns of COX-2, Kir4.1, and AQP-1, but not of AQP-4 or GS. Nor did it improve edema. Our study pinpoints the adaptive response to Aß of specific RMG cell functions.

Amyloid precursor protein is required for normal function of the rod and cone pathways in the mouse retina.

Amyloid precursor protein (APP) is a transmembrane glycoprotein frequently studied for its role in Alzheimer's disease. Our recent study in APP knockout (KO) mice identified an important role for APP in modulating normal neuronal development in the retina. However the role APP plays in the adult retina and whether it is required for vision is unknown. In this study we evaluated the role of APP in retinal function and morphology comparing adult wildtype (WT) and APP-KO mice. APP was expressed on neuronal cells of the inner retina, including horizontal, cone bipolar, amacrine and ganglion cells in WT mice. The function of the retina was assessed using the electroretinogram and although the rod photoreceptor responses were similar in APP-KO and WT mice, the post-photoreceptor, inner retinal responses of both the rod and cone pathways were reduced in APP-KO mice. These changes in inner retinal function did not translate to a substantial change in visual acuity as assessed using the optokinetic response or to changes in the gross cellular structure of the retina. These findings indicate that APP is not required for basic visual function, but that it is involved in modulating inner retinal circuitry.

CCR2/CCL2-mediated inflammation protects photoreceptor cells from amyloid-ß-induced apoptosis.

Age-related macular degeneration is characterized by the formation of drusen containing amyloid-ß (Aß) and the degeneration of photoreceptors. To explore the largely unknown role of Aß in the retina, we investigated the effects on photoreceptors of the oligomeric form of Aß(1-42). Subretinal injection of the Aß peptide induced misplaced expression of recoverin and synaptophysin in the photoreceptors, oxidative stress in their inner and outer segments, and finally apoptosis. Aß did not induce cell death in purified photoreceptor cell cultures, but did so in retinal cell cultures, thereby suggesting that the cellular environment plays a role in Aß-induced photoreceptor apoptosis. Subretinal injection of Aß was followed by activation and migration of microglial cells and then by photoreceptor apoptosis. Microglial cells phagocytosed rhodopsin-containing debris and Aß in the subretinal space. Quantitative RT-PCR allowed us to identify a specific gene expression profile associated with the Aß-induced progression of retinal degeneration and consistent with oxidative stress, inflammation, and an apoptotic program. The gene most highly upregulated in Aß-injected retinas was that for the chemokine CCL2, and its absence or that of its cognate receptor CCR2 greatly reduced migration of activated microglial cells to the site of retinal injury and profoundly worsened photoreceptor degeneration and disorganization of the retinal pigment epithelium in Aß-injected retinas. Our study pinpoints the roles of Aß and of CCL2/CCR2 axis-dependent inflammation in photoreceptor apoptosis.

APP involvement in retinogenesis of mice.

Very few studies have examined expression and function of amyloid precursor protein (APP) in the retina. We showed that APP mRNA and protein are expressed according to the different waves of retinal differentiation. Depletion of App led to an absence of amacrine cells, a 50% increase in the number of horizontal cells and alteration of the synapses. The retinas of adult APP(-/-) mice showed only half as many glycinergic amacrine cells as wild-type retinas. We identified Ptf1a, which plays a role in controlling both amacrine and horizontal cell fates, as a downstream effector of APP. The observation of a similar phenotype in sorLA knockout mice, a major regulator of APP processing, suggests that regulation of APP functions via sorLA controls the determination of amacrine and horizontal cell fate. These findings provide novel insights that indicate that APP plays an important role in retinal differentiation.

Amyloid-beta(1-42) alters structure and function of retinal pigmented epithelial cells.

Age-related macular degeneration (AMD) is characterized by the formation of drusen, extracellular deposits associated with atrophy of the retinal pigmented epithelium (RPE), disturbance of the transepithelial barrier and photoreceptor death. Amyloid-beta (Abeta) is present in drusen but its role during AMD remains unknown. This study investigated the in vitro and in vivo effects of the oligomeric form of Abeta(1-42) - OAbeta(1-42) - on RPE and found that it reduced mitochondrial redox potential and increased the production of reactive oxygen species, but did not induce apoptosis in RPE cell cultures. It also disorganized the actin cytoskeleton and halved occludin expression, markedly decreasing attachment capacity and abolishing the selectivity of RPE cell transepithelial permeability. Antioxidant pretreatment partially reversed the effects of OAbeta(1-42) on mitochondrial redox potential and transepithelial permeability. Subretinally injected OAbeta(1-42) induced pigmentation loss and RPE hypertrophy but not RPE cell apoptosis in C57BL/6 J mice. Rapid OAbeta(1-42)-induced disorganization of cytoskeletal actin filaments was accompanied by decreased RPE expression of the tight junction proteins occludin and zonula occludens-1 and of the visual cycle proteins cellular retinaldehyde-binding protein and RPE65. The number of photoreceptors decreased by half within a few days. Our study pinpoints the role of Abeta in RPE alterations and dysfunctions leading to retinal degeneration and suggests that targeting Abeta may help develop selective methods for treating diseases involving retinal degeneration, such as AMD.

Impact of melatonin receptors on pCREB and clock-gene protein levels in the murine retina.

In several mammalian species, the retina is capable of synthesizing melatonin and contains an autonomous circadian clock that relies on interlocking transcriptional/translational feedback loops involving several clock genes, such as Per1 and Cry2. Our previous investigations have shown remarkable differences in retinae of melatonin-deficient (C57BL) and melatonin-proficient (C3H) mice with regard to the protein levels of PER1, CRY2, and phosphorylated (p) cyclic AMP response element binding protein (CREB). To elucidate the melatonin receptor type possibly responsible for these differences, we have performed immunocytochemical analyses for PER1, CRY2, and pCREB in retinae of melatonin-proficient wild type (WT) mice and mice with targeted deletions of the MT1 receptor (MelaaBB) or the MT1 and MT2 receptors (Melaabb) at four different time points. Immunoreactions for PER1, CRY2 and pCREB were localized to the nuclei of cells in the inner nuclear layer (INL) and ganglion cell layer (GC) of all strains. Surprisingly, in MelaaBB and Melaabb the day/night rhythm of pCREB, PER1, and CRY2 levels was not abolished, but the maxima and minima of PER1 were 180 degrees out of phase as compared to the WT. These data suggest that MT1 and MT2 melatonin receptors are not necessary to maintain rhythmic changes in clock-gene protein levels in the murine retina, but, as shown for PER1, appear to be involved in internal synchronization.

Clock gene expression in the retina of melatonin-proficient (C3H) and melatonin-deficient (C57BL) mice.

In several mammalian species, the retina contains an autonomous circadian clock and is capable of synthesizing melatonin. The function of circadian clocks depends on interlocking transcriptional/translational feedback loops involving several clock genes. Here we investigated the expression of two clock genes (Per1, Cry2) and the level of phosphorylated (p) cyclic AMP response element binding protein (CREB) in retinae of melatonin-deficient (C57BL) with an intact retina and melatonin-proficient (C3H) mice with degenerated outer nuclear layer. RNase protection assay and in situ hybridization revealed in both strains a rhythm in transcript levels for Per1 with a peak at zeitgeber time (ZT) 08, but not for Cry2. Immunoreactions for PER1, CRY2 and pCREB were localized to the nuclei of cells in the inner nuclear layer (INL) and ganglion cell layer (GC) of both strains and to the outer nuclear layer of C57BL. In C3H, protein levels of PER1 and CRY2 followed a clear day/night rhythm in the INL and the GC with a peak at the end of the day (ZT14). pCREB levels peaked at the beginning of the day. Noteably, in melatonin-deficient C57BL mice, protein levels of PER1, CRY2 and pCREB did not show significant changes over a 16L/8D cycle. These data suggest that melatonin influences PER1 and CRY2 protein levels via post-transcriptional mechanisms and also plays a role in rhythmic regulation of pCREB levels in the mammalian retina.

Melatoninergic differentiation of retinal photoreceptors: activation of the chicken hydroxyindole-O-methyltransferase promoter requires a homeodomain-binding element that interacts with Otx2.

The gene encoding the last enzyme of the melatonin-synthesis pathway, hydroxyindole-O-methyltransferase (HIOMT), is selectively expressed in retinal photoreceptors and pineal cells. Here, we analysed the promoter of the chicken HIOMT gene and we found that a homeodomain-binding element located in the proximal region of this promoter was essential for its activation in primary cultures of embryonic chicken retinal cells. This homeodomain-regulatory element interacted with a protein expressed in the chicken retina and pineal gland, which was recognized by an anti-Otx2 antiserum. Recombinant Otx2 expressed in vitro was able to bind this DNA element and to directly transactivate the chicken HIOMT promoter. This promoter was also transactivated by another member of the Otx family, Otx5, but the amplitude of stimulation was lower than with Otx2. The spatio-temporal pattern of Otx2 expression was compatible with a possible role of this transcription factor in HIOMT gene activation. In adult chicken, Otx2 mRNA was found to be present in those two tissues that express HIOMT: the retina and the pineal gland. During development, a burst of Otx2 mRNA closely matched the timing of HIOMT gene activation in these two tissues. In the pineal, Otx2 immunolabelling was specifically localized in the nuclei of photoreceptor cells. In the neural retina, Otx2 immunoreactivity brightly decorated the photoreceptor nuclei and extended more faintly to the outer half of the inner nuclear layer. Together, the data support a role of Otx2 in the onset of HIOMT expression in developing chicken photoreceptors.

Immunocytochemical demonstration of day/night changes of clock gene protein levels in the murine adrenal gland: differences between melatonin-proficient (C3H) and melatonin-deficient (C57BL) mice.

The circadian system comprises several peripheral oscillators and a central rhythm generator that, in mammals, is located in the suprachiasmatic nucleus of the hypothalamus. Expression of clock genes is a characteristic feature of the central rhythm generator and the peripheral oscillators. With regard to the rhythmic production of glucocorticoids, the adrenal gland can be considered as peripheral oscillator, but little is known about clock gene expression in this tissue. Therefore, the present study investigates the levels of three clock gene proteins PER1, BMAL1 and CRY2 in the murine adrenal cortex and medulla at seven different time points of a 12-hr light/12-hr dark cycle. To determine a potential role of melatonin we compared the patterns of clock gene proteins in the adrenal gland of melatonin-proficient mice (C3H) with those of melatonin-deficient mice (C57BL). In C3H mice, both, the adrenal cortex and medulla displayed day/night variation in PER1-, CRY2- and BMAL1-protein levels. PER1 and CRY2 peaked in the middle of the light phase, whereas BMAL1 peaked in the dark phase. This pattern was also observed in the adrenal medulla of C57BL, but in the adrenal cortex of C57BL clock gene protein levels did not change with time and were consistently lower than in C3H mice. These results support the hypothesis that the adrenal gland is a peripheral oscillator and raise the possibility that melatonin may be involved in the control of clock gene protein levels in the adrenal cortex of mice.