GPI-anchor signal sequence influences PrPC sorting, shedding and signalling, and impacts on different pathomechanistic aspects of prion disease in mice

The cellular prion protein (PrPC) is a cell surface glycoprotein attached to the membrane by a glycosylphosphatidylinositol (GPI)-anchor and plays a critical role in transmissible, neurodegenerative and fatal prion diseases. Alterations in membrane attachment influence PrPC-associated signaling, and the development of prion disease, yet our knowledge of the role of the GPI-anchor in localization, processing, and function of PrPC in vivo is limited We exchanged the PrPC GPI-anchor signal sequence of for that of Thy-1 (PrPCGPIThy-1) in cells and mice. We show that this modifies the GPI-anchor composition, which then lacks sialic acid, and that PrPCGPIThy-1 is preferentially localized in axons and is less prone to proteolytic shedding when compared to PrPC. Interestingly, after prion infection, mice expressing PrPCGPIThy-1 show a significant delay to terminal disease, a decrease of microglia/astrocyte activation, and altered MAPK signaling when compared to wild-type mice. Our results are the first to demonstrate in vivo, that the GPI-anchor signal sequence plays a fundamental role in the GPI-anchor composition, dictating the subcellular localization of a given protein and, in the case of PrPC, influencing the development of prion disease

Structural and mechanistic aspects influencing the ADAM10-mediated shedding of the prion protein

Background: Proteolytic processing of the prion protein (PrPC) by endogenous proteases generates bioactive membrane-bound and soluble fragments which may help to explain the pleiotropic roles of this protein in the nervous system and in brain diseases. Shedding of almost full-length PrPC into the extracellular space by the metalloprotease ADAM10 is of peculiar relevance since soluble PrP stimulates axonal outgrowth and is protective in neurodegenerative conditions such as Alzheimer’s and prion disease. However, molecular determinates and mechanisms regulating the shedding of PrP are entirely unknown. Methods: We produced an antibody recognizing the neo-epitope of shed PrP generated by ADAM10 in biological samples and used it to study structural and mechanistic aspects affecting the shedding. For this, we investigated genetically modified cellular and murine models by biochemical and morphological approaches. Results: We show that the novel antibody specifically detects shed PrP in cell culture supernatants and murine brain. We demonstrate that ADAM10 is the exclusive sheddase of PrPC in the nervous system and reveal that the glycosylation state and type of membrane-anchorage of PrPC severely affect its shedding. Furthermore, we provide evidence that PrP shedding can be modulated by pharmacological inhibition and stimulation and present data suggesting that shedding is a relevant part of a compensatory network ensuring PrPC homeostasis of the cell. Conclusions: With the new antibody, our study introduces a new tool to reliably investigate PrP-shedding. In addition, this study provides novel and important insight into the regulation of this cleavage event, which is likely to be relevant for diagnostic and therapeutic approaches even beyond neurodegeneration

Late term tolerance in head neck cancer patients irradiated in the IMRT era

BACKGROUND: The aim was to quantify severe transient and persisting late term effects in our single institution head neck cancer (HNC) cohort treated with curatively intended intensity modulated radiation therapy (IMRT). Hypothesis was if a 2-year follow up (FU) is sufficient to estimate the long term tolerance in HNC irradiated in the IMRT era. METHODS: Between 01/2002-8/2012, 707/1211 (58%) consecutively treated IMRT patients met the inclusion criteria of a FU time >12 months and loco-regional disease control (LRC). 45% presented with loco-regionally advanced disease; 55% were referred for curative definitive IMRT (66Gy-72Gy in 30--35 fractions), 45% underwent postoperative IMRT (60-66Gy in 30--33 fractions). Systemic concomitant therapy was administered in 85%. Highly consistent treatment procedures were performed with respect to contouring processes, dose constraints, radiation schedules, and the use of systemic therapy. Grade 3/4 late term effects were prospectively assessed and analyzed with respect to subgroups at particular risk for specific late effects. RESULTS: Mean/median FU of the cohort was 41/35 months (15--124). 13% of the patients (92/707) experienced any grade 3/4 late effects (101 events in 92/707 patients), 81% in the first 12 months after radiation. 4% of all developed persisting late grade 3/4 effects (25 events in 25/707 patients). CONCLUSIONS: IMRT led to a high late term tolerance in loco-regionally disease free HNC patients. The onset of any G3/4 effects showed a plateau at 2 years. The question of the cervical vessel tolerance in disease free long time survivors is still open and currently under evaluation at our institution

Safety and acceptability of an organic light-emitting diode sleep mask as a potential therapy for retinal disease

Purpose The purpose of the study was to study the effect of an organic light-emitting diode sleep mask on daytime alertness, wellbeing, and retinal structure/function in healthy volunteers and in diabetic macular oedema (DMO). Patients and methods Healthy volunteers in two groups, 18–30 yrs (A), 50–70 yrs (B) and people with DMO (C) wore masks (504 nm wavelength; 80 cd/m2 luminance; ≤8 h) nightly for 3 months followed by a 1-month recovery period. Changes from baseline were measured for (means): psychomotor vigilance task (PVT) (number of lapses (NL), response time (RT)), sleep, depression, psychological wellbeing (PW), visual acuity, contrast sensitivity, colour, electrophysiology, microperimetry, and retinal thickness on OCT. Results Of 60 participants, 16 (27%) withdrew, 8 (13%) before month 1, due to sleep disturbances and mask intolerance. About 36/55 (65%) who continued beyond month 1 reported ≥1 adverse event. At month 3 mean PVT worsened in Group A (RT (7.65%, P<0.001), NL (43.3%, P=0.005)) and mean PW worsened in all groups (A 28.0%, P=0.01, B 21.2%, P=0.03, C 12.8%, P<0.05). No other clinically significant safety signal was detected. Cysts reduced/resolved in the OCT subfield of maximal pathology in 67% Group C eyes. Thinning was greater at 3 and 4 months for greater baseline thickness (central subfield P<0.001, maximal P<0.05). Conclusion Sleep masks showed no major safety signal apart from a small impairment of daytime alertness and a moderate effect on wellbeing. Masks were acceptable apart from in some healthy participants. Preliminary data suggest a beneficial effect on retinal thickness in DMO. This novel therapeutic approach is ready for large clinical trials

Fundamental differences in patterns of retinal ageing between primates and mice

Photoreceptors have high metabolic demands and age rapidly, undermining visual function. We base our understanding mainly on ageing mice where elevated inflammation, extracellular deposition, including that of amyloid beta, and rod and cone photoreceptor loss occur, but cones are not lost in ageing primate although their function declines, revealing that primate and mouse age differently. We examine ageing primate retinae and show elevated stress but low inflammation. However, aged primates have a >70% reduction in adenosine triphosphate (ATP) and a decrease in cytochrome c oxidase. There is a shift in cone mitochondrial positioning and glycolytic activity increases. Bruch’s membrane thickens but unlike in mice, amyloid beta is absent. Hence, reduced ATP may explain cone functional decline in ageing but their retained presence offers the possibility of functional restoration if they can be fuelled appropriately to restore cellular function. This is important because as humans we largely depend on cone function to see and are rarely fully dark adapted. Presence of limited aged inflammation and amyloid beta deposition question some of the therapeutic approaches taken to resolve problems of retinal ageing in humans and the possible lack of success in clinical trials in macular degeneration that have targeted inflammatory agents

Male bisexual arousal: A matter of curiosity?

Conflicting evidence exists regarding whether bisexual-identified men are sexually aroused to both men and women. We hypothesized that a distinct characteristic, level of curiosity about sexually diverse acts, distinguishes bisexual-identified men with and without bisexual arousal. Study 1 assessed men's (n = 277) sexual arousal via pupil dilation to male and female sexual stimuli. Bisexual men were, on average, higher in their sexual curiosity than other men. Despite this general difference, only bisexual-identified men with elevated sexual curiosity showed bisexual arousal. Those lower in curiosity had responses resembling those of homosexual men. Study 2 assessed men's (n = 72) sexual arousal via genital responses and replicated findings of Study 1. Study 3 provided information on the validity on our measure of sexual curiosity by relating it to general curiosity and sexual sensation seeking (n = 83). Based on their sexual arousal and personality, at least two groups of men identify as bisexual

Endogenous VEGF Is Required for Visual Function: Evidence for a Survival Role on Müller Cells and Photoreceptors

Vascular endothelial growth factor (VEGF) is well known for its role in normal and pathologic neovascularization. However, a growing body of evidence indicates that VEGF also acts on non-vascular cells, both developmentally as well as in the adult. In light of the widespread use of systemic and intraocular anti-VEGF therapies for the treatment of angiogenesis associated with tumor growth and wet macular degeneration, systematic investigation of the role of VEGF in the adult retina is critical.Using immunohistochemistry and Lac-Z reporter mouse lines, we report that VEGF is produced by various cells in the adult mouse retina and that VEGFR2, the primary signaling receptor, is also widely expressed, with strong expression by Müller cells and photoreceptors. Systemic neutralization of VEGF was accomplished in mice by adenoviral expression of sFlt1. After 14 days of VEGF neutralization, there was no effect on the inner and outer retina vasculature, but a significant increase in apoptosis of cells in the inner and outer nuclear layers. By four weeks, the increase in neural cell death was associated with reduced thickness of the inner and outer nuclear layers and a decline in retinal function as measured by electroretinograms. siRNA-based suppression of VEGF expression in a Müller cell line in vitro supports the existence of an autocrine role for VEGF in Müller cell survival. Similarly, the addition of exogenous VEGF to freshly isolated photoreceptor cells and outer-nuclear-layer explants demonstrated VEGF to be highly neuroprotective.These results indicate an important role for endogenous VEGF in the maintenance and function of adult retina neuronal cells and indicate that anti-VEGF therapies should be administered with caution

Understanding the retinal basis of vision across species

The vertebrate retina first evolved some 500 million years ago in ancestral marine chordates. Since then, the eyes of different species have been tuned to best support their unique visuoecological lifestyles. Visual specializations in eye designs, large-scale inhomogeneities across the retinal surface and local circuit motifs mean that all species' retinas are unique. Computational theories, such as the efficient coding hypothesis, have come a long way towards an explanation of the basic features of retinal organization and function; however, they cannot explain the full extent of retinal diversity within and across species. To build a truly general understanding of vertebrate vision and the retina's computational purpose, it is therefore important to more quantitatively relate different species' retinal functions to their specific natural environments and behavioural requirements. Ultimately, the goal of such efforts should be to build up to a more general theory of vision