Martin Glösmann

A dual role for autophagy in a murine model of lung cancer

Autophagy is a mechanism by which starving cells can control their energy requirements and metabolic states, thus facilitating the survival of cells in stressful environments, in particular in the pathogenesis of cancer. Here we report that tissue-specific inactivation of Atg5, essential for the formation of autophagosomes, markedly impairs the progression of KRas(G12D)-driven lung cancer, resulting in a significant survival advantage of tumour-bearing mice. Autophagy-defective lung cancers exhibit impaired mitochondrial energy homoeostasis, oxidative stress and a constitutively active DNA damage response. Genetic deletion of the tumour suppressor p53 reinstates cancer progression of autophagy-deficient tumours. Although there is improved survival, the onset of Atg5-mutant KRas(G12D)-driven lung tumours is markedly accelerated. Mechanistically, increased oncogenesis maps to regulatory T cells. These results demonstrate that, in KRas(G12D)-driven lung cancer, Atg5-regulated autophagy accelerates tumour progression; however, autophagy also represses early oncogenesis, suggesting a link between deregulated autophagy and regulatory T cell controlled anticancer immunity.

Bat Eyes Have Ultraviolet-Sensitive Cone Photoreceptors

Mammalian retinae have rod photoreceptors for night vision and cone photoreceptors for daylight and colour vision. For colour discrimination, most mammals possess two cone populations with two visual pigments (opsins) that have absorption maxima at short wavelengths (blue or ultraviolet light) and long wavelengths (green or red light). Microchiropteran bats, which use echolocation to navigate and forage in complete darkness, have long been considered to have pure rod retinae. Here we use opsin immunohistochemistry to show that two phyllostomid microbats, Glossophaga soricina and Carollia perspicillata, possess a significant population of cones and express two cone opsins, a shortwave-sensitive (S) opsin and a longwave-sensitive (L) opsin. A substantial population of cones expresses S opsin exclusively, whereas the other cones mostly coexpress L and S opsin. S opsin gene analysis suggests ultraviolet (UV, wavelengths <400 nm) sensitivity, and corneal electroretinogram recordings reveal an elevated sensitivity to UV light which is mediated by an S cone visual pigment. Therefore bats have retained the ancestral UV tuning of the S cone pigment. We conclude that bats have the prerequisite for daylight vision, dichromatic colour vision, and UV vision. For bats, the UV-sensitive cones may be advantageous for visual orientation at twilight, predator avoidance, and detection of UV-reflecting flowers for those that feed on nectar.

Thyroid Hormone Controls Cone Opsin Expression in the Retina of Adult Rodents

Mammalian retinas display an astonishing diversity in the spatial arrangement of their spectral cone photoreceptors, probably in adaptation to different visual environments. Opsin expression patterns like the dorsoventral gradients of short-wave-sensitive (S) and middle- to long-wave-sensitive (M) cone opsin found in many species are established early in development and thought to be stable thereafter throughout life. In mouse early development, thyroid hormone (TH), through its receptor TRβ2, is an important regulator of cone spectral identity. However, the role of TH in the maintenance of the mature cone photoreceptor pattern is unclear. We here show that TH also controls adult cone opsin expression. Methimazole-induced suppression of serum TH in adult mice and rats yielded no changes in cone numbers but reversibly altered cone patterns by activating the expression of S-cone opsin and repressing the expression of M-cone opsin. Furthermore, treatment of athyroid Pax8−/− mice with TH restored a wild-type pattern of cone opsin expression that reverted back to the mutant S-opsin-dominated pattern after termination of treatment. No evidence for cone death or the generation of new cones from retinal progenitors was found in retinas that shifted opsin expression patterns. Together, this suggests that opsin expression in terminally differentiated mammalian cones remains subject to control by TH, a finding that is in contradiction to previous work and challenges the current view that opsin identity in mature mammalian cones is fixed by permanent gene silencing.

Cone photoreceptors and potential UV vision in a subterranean insectivore, the European mole.

We have examined the presence, the distribution, and the opsin identity of photoreceptor types in the retina of the European mole, Talpa europaea, a subterranean insectivore with regressed morphology of the visual system. Cones and rods were identified using opsin antisera, and their topographies determined from flat-mounted retinas. The retina (total area 0.75 mm(2)) contains about 100,000 photoreceptors, 10-12% of which are cones. Rod density is low (theoretical maximum 127,000 mm(-2)). Cone density peaks in central retina (17,750 mm(-2)). Similar to most mammals, two cone opsins, shortwave-sensitive (S) and middle-to-long-wave-sensitive (M), are present. Cone distribution shows a dorsoventral gradient with higher S cone numbers in ventral retina. Coexpression of S and M opsin occurs in more than 30% of the cones. Partial sequencing of the S opsin gene strongly supports UV sensitivity of the mole S cone photopigment. Amino acids that spectrally tune the S opsin are identical in T. europaea and in mammals with known UV cone photosensitivity. The lens transmits light down to 300 nm. Together, our data suggest that photopic vision and UV sensitivity of a cone pigment play a functional role in the European mole.

Developmental Changes of Cone Opsin Expression but Not Retinal Morphology in the Hypothyroid Pax8 Knockout Mouse

PURPOSE The effects of postnatal hypothyroidism on retinal development and spatial patterning of cone opsin expression were studied in Pax8-deficient mice. Pax8(-/-) mice are incapable of synthesizing thyroxine and serve as a model for congenital hypothyroidism. METHODS Pax8(-/-), Pax8(+/-), and Pax8(+/+) littermates were studied. Serum thyroid hormone levels, body weight, and eye size were measured. Retinal cell-type-specific antibodies were used on frozen sections to examine the postnatal development of the major retinal cell classes and of retinal structure. The expression of short-wavelength-sensitive (S) and middle-to-long-wavelength-sensitive (M) cone opsins was assessed with opsin antibodies on retinal sections and whole retinas. The pattern of S opsin mRNA was assessed by in situ hybridization. RESULTS In Pax8(-/-) mice, S opsin was upregulated in all cones, whereas M opsin was downregulated throughout the retina, the wild-type dorsoventral gradients of S and M opsin expression were absent. Otherwise, Pax8(-/-) mice showed no overt mutant phenotype in eye size, gross retinal anatomy, and the time-course of structural differentiation of retinal photoreceptors, horizontal cells, bipolars, amacrines, ganglion cells, and Müller glia cells. CONCLUSIONS Pax8(-/-) mice show a pattern of cone opsin expression that differs substantially from the wild-type pattern, but exhibit no apparent alterations in general retinal development. The finding that a postnatal decrease in serum thyroid hormone yields changes in postnatal cone opsin expression is consistent with a ligand-dependent role of thyroid hormone receptor beta2 in S opsin repression and M opsin activation.

Retinal photoreceptors of two subterranean tuco-tuco species (Rodentia, Ctenomys): morphology, topography, and spectral sensitivity.

Traditionally, vision was thought to be useless for animals living in dark underground habitats, but recent studies in a range of subterranean rodent species have shown a large diversity of eye features, from small subcutaneous eyes to normal‐sized functional eyes. We analyzed the retinal photoreceptors in the subterranean hystricomorph rodents Ctenomys talarum and Ctenomys magellanicus to elucidate whether adaptation was to their near‐lightless burrows or rather to their occasional diurnal surface activity. Both species had normally developed eyes. Overall photoreceptor densities were comparatively low (95,000–150,000/mm2 in C. magellanicus, 110,000–200,000/mm2 in C. talarum), and cone proportions were rather high (10–31% and 14–31%, respectively). The majority of cones expressed the middle‐to‐longwave‐sensitive (L) opsin, and a 6–16% minority expressed the shortwave‐sensitive (S) opsin. In both species the densities of L and S cones were higher in ventral than in dorsal retina. In both species the tuning‐relevant amino acids of the S opsin indicate sensitivity in the near UV rather than the blue/violet range. Photopic spectral electroretinograms were recorded. Unexpectedly, their sensitivity profiles were best fitted by the linear summation of three visual pigment templates with λmax at 370 nm (S pigment, UV), at 510 nm (L pigment), and at 450 nm (an as‐yet unexplained mechanism). Avoiding predators and selecting food during the brief aboveground excursions may have exerted pressure to retain robust cone‐based vision in Ctenomys. UV tuning of the S cone pigment is shared with a number of other hystricomorphs. J. Comp. Neurol. 518:4001–4015, 2010.

A mouse-like retinal cone phenotype in the Syrian hamster: S opsin coexpressed with M opsin in a common cone photoreceptor

Previous immunocytochemical, physiological, and molecular studies have reported that the Syrian hamster lacks a shortwave-sensitive (S) cone photopigment but retains circadian responses to ultraviolet (UV) light. Using opsin antibodies and a sensitive detection protocol, we here show that S opsin immunoreactivity colocalizes with M opsin immunoreactivity in a common type of cone photoreceptor. S opsin signal within individual cone outer segments is low and continuously decreases from the ventral to dorsal retina. Only double-labeled cones were found. During development, S opsin expression precedes that of M opsin, but there is no indication of transdifferentiation. Our results imply that in the Syrian hamster low levels of S opsin colocalize with M opsin in a common cone phenotype. We suggest that, similar to other murid rodents, the S pigment absorbs maximally in the UV range, and thus may contribute to mediating the circadian response of the Syrian hamster to UV light.

Histomorphometry in rodents.

Bone histomorphometry remains an important tool to study the pathophysiology of bone disease and the cellular mechanism by which treatments work. Here, we review the methods for embedding, sectioning, staining, and analysis of bone sections in rodents.

Cav1.4 IT mouse as model for vision impairment in human congenital stationary night blindness type 2

Mutations in the CACNA1F gene encoding the Cav1.4 Ca2+ channel are associated with X-linked congenital stationary night blindness type 2 (CSNB2). Despite the increasing knowledge about the functional behavior of mutated channels in heterologous systems, the pathophysiological mechanisms that result in vision impairment remain to be elucidated. This work provides a thorough functional characterization of the novel IT mouse line that harbors the gain-of-function mutation I745T reported in a New Zealand CSNB2 family.1 Electroretinographic recordings in IT mice permitted a direct comparison with human data. Our data supported the hypothesis that a hyperpolarizing shift in the voltage-dependence of channel activation—as seen in the IT gain-of-function mutant2—may reduce the dynamic range of photoreceptor activity. Morphologically, the retinal outer nuclear layer in adult IT mutants was reduced in size and cone outer segments appeared shorter. The organization of the outer plexiform layer was disrupted, and synaptic structures of photoreceptors had a variable, partly immature, appearance. The associated visual deficiency was substantiated in behavioral paradigms. The IT mouse line serves as a specific model for the functional phenotype of human CSNB2 patients with gain-of-function mutations and may help to further understand the dysfunction in CSNB.

Melanin Pigmentation in Rat Eyes: In Vivo Imaging by Polarization-Sensitive Optical Coherence Tomography and Comparison to Histology.

PURPOSE The purpose of this study was to demonstrate polarization-sensitive optical coherence tomography (PS-OCT) for imaging pigmented structures in the posterior eye segments of albino and pigmented rats and to correlate depolarization contrast of the retinal pigment epithelium (RPE) and choroid in in vivo PS-OCT to melanin pigmentation detected in postmortem histologic serial sections. METHODS In vivo three-dimensional PS-OCT imaging was performed in adult albino and pigmented rat eyes at 70-kHz A-line rate. Degree of polarization uniformity (DOPU) fundus maps and radial DOPU profiles were generated. Postmortem histomorphologic analysis was performed in order to investigate melanin pigmentation of the RPE and choroid. Fundus pigmentation maps were extracted from histologic serial sections. Pigmentation profiles were correlated to DOPU profiles of the same eyes. RESULTS Strong depolarization was found in the RPE/choroid complex of pigmented rats, whereas the same structures exhibited uniform polarization in albino rats. The difference between the depolarization characteristics between albino and pigmented animals was statistically significant. In the fundus pigmentation maps, optical pigment density was zero in albino rat eyes. In pigmented rat eyes, a strong negative correlation between optical pigment density and DOPU was observed. CONCLUSIONS This in vivo and ex vivo investigation of posterior rat eyes indicates that melanin is the cause of depolarization in retinal PS-OCT images. It further demonstrates that melanin pigmentation in the RPE and choroid can be quantified via depolarization imaging and therefore suggests that PS-OCT is a useful tool for the noninvasive quantitative assessment of pigmentary changes in vision-threatening diseases such as age-related macular degeneration.