RNAi-mediated suppression of vimentin or glial fibrillary acidic protein prevents the establishment of Müller glial cell hypertrophy in progressive retinal degeneration.

Gliosis is a complex process comprising upregulation of intermediate filament (IF) proteins, particularly glial fibrillary acidic protein (GFAP) and vimentin, changes in glial cell morphology (hypertrophy) and increased deposition of inhibitory extracellular matrix molecules. Gliosis is common to numerous pathologies and can have deleterious effects on tissue function and regeneration. The role of IFs in gliosis is controversial, but a key hypothesized function is the stabilization of glial cell hypertrophy. Here, we developed RNAi approaches to examine the role of GFAP and vimentin in vivo in a murine model of inherited retinal degeneration, the Rhodopsin knockout (Rho-/- ) mouse. Specifically, we sought to examine the role of these IFs in the establishment of Müller glial hypertrophy during progressive degeneration, as opposed to (more commonly assessed) acute injury. Prevention of Gfap upregulation had a significant effect on the morphology of reactive Müller glia cells in vivo and, more strikingly, the reduction of Vimentin expression almost completely prevented these cells from undergoing degeneration-associated hypertrophy. Moreover, and in contrast to studies in knockout mice, simultaneous suppression of both GFAP and vimentin expression led to severe changes in the cytoarchitecture of the retina, in both diseased and wild-type eyes. These data demonstrate a crucial role for Vimentin, as well as GFAP, in the establishment of glial hypertrophy and support the further exploration of RNAi-mediated knockdown of vimentin as a potential therapeutic approach for modulating scar formation in the degenerating retina.

Müller Glia Reactivity and Development of Gliosis in Response to Pathological Conditions.

Within the mammalian retina, both Müller glia and astrocytes display reactivity in response to many forms of retinal injury and disease in a process termed gliosis. Reactive gliosis is a complex process that is considered to represent a cellular response to protect the retina from further damage and to promote its repair following pathological insult. It includes morphological, biochemical and physiological changes, which may vary depending on the type and degree of the initial injury. Not only does gliosis have numerous triggers, but also there is a great degree of heterogeneity in the glial response, creating multiple levels of complexity. For these reasons, understanding the process of glial scar formation and how this process differs in different pathological conditions and finding strategies to circumvent these barriers represent major challenges to the advancement of many ocular therapies.

Hyperactivity and memory/learning deficits evoked by developmental exposure to nicotine and/or ethanol are mitigated by cAMP and cGMP signaling cascades activation.

Pregnant smoking women are frequently episodic drinkers. Here, we investigated whether ethanol exposure restricted to the brain growth spurt period when combined with chronic developmental exposure to nicotine aggravates memory/learning deficits and hyperactivity, and associated cAMP and cGMP signaling disruption. To further investigate the role of these signaling cascades, we verified whether vinpocetine (a phosphodiesterase inhibitor) ameliorates the neurochemical and behavioral outcomes. Swiss mice had free access to nicotine (NIC, 50 µg/ml) or water to drink during gestation and until the 8th postnatal day (PN8). Ethanol (ETOH, 5 g/kg, i.p.) or saline were injected in the pups every other day from PN2 to PN8. At PN30, animals either received vinpocetine (20 mg/kg, i.p.) or vehicle before being tested in the step-down passive avoidance or open field. Memory/learning was impaired in NIC, ETOH and NIC + ETOH mice, and vinpocetine mitigated ETOH- and NIC + ETOH-induced deficits. Locomotor hyperactivity identified in ETOH and NIC + ETOH mice was ameliorated by vinpocetine. While cyclic nucleotides levels in cerebral cortex and hippocampus were reduced by NIC, ETOH and NIC + ETOH, this outcome was more consistent in the latter group. As observed for behavior, vinpocetine normalized NIC + ETOH nucleotides levels. pCREB levels were also increased in response to vinpocetine, with stronger effects in the NIC + ETOH group. Exposure to both drugs of abuse worsens behavioral and neurochemical disruption. These findings and the amelioration of deleterious effects by vinpocetine support the idea that cAMP and cGMP signaling contribute to nicotine- and ethanol-induced hyperactivity and memory/learning deficits.

Transplanted Donor- or Stem Cell-Derived Cone Photoreceptors Can Both Integrate and Undergo Material Transfer in an Environment-Dependent Manner.

Human vision relies heavily upon cone photoreceptors, and their loss results in permanent visual impairment. Transplantation of healthy photoreceptors can restore visual function in models of inherited blindness, a process previously understood to arise by donor cell integration within the host retina. However, we and others recently demonstrated that donor rod photoreceptors engage in material transfer with host photoreceptors, leading to the host cells acquiring proteins otherwise expressed only by donor cells. We sought to determine whether stem cell- and donor-derived cones undergo integration and/or material transfer. We find that material transfer accounts for a significant proportion of rescued cells following cone transplantation into non-degenerative hosts. Strikingly, however, substantial numbers of cones integrated into the Nrl-/- and Prph2rd2/rd2, but not Nrl-/-;RPE65R91W/R91W, murine models of retinal degeneration. This confirms the occurrence of photoreceptor integration in certain models of retinal degeneration and demonstrates the importance of the host environment in determining transplantation outcome.

Rescue of mutant rhodopsin traffic by metformin-induced AMPK activation accelerates photoreceptor degeneration.

Protein misfolding caused by inherited mutations leads to loss of protein function and potentially toxic 'gain of function', such as the dominant P23H rhodopsin mutation that causes retinitis pigmentosa (RP). Here, we tested whether the AMPK activator metformin could affect the P23H rhodopsin synthesis and folding. In cell models, metformin treatment improved P23H rhodopsin folding and traffic. In animal models of P23H RP, metformin treatment successfully enhanced P23H traffic to the rod outer segment, but this led to reduced photoreceptor function and increased photoreceptor cell death. The metformin-rescued P23H rhodopsin was still intrinsically unstable and led to increased structural instability of the rod outer segments. These data suggest that improving the traffic of misfolding rhodopsin mutants is unlikely to be a practical therapy, because of their intrinsic instability and long half-life in the outer segment, but also highlights the potential of altering translation through AMPK to improve protein function in other protein misfolding diseases.

Gliosis Can Impede Integration Following Photoreceptor Transplantation into the Diseased Retina.

Retinal degenerations leading to the loss of photoreceptor (PR) cells are a major cause of vision impairment and untreatable blindness. There are few clinical treatments and none can reverse the loss of vision. With the rapid advances in stem cell biology and techniques in cell transplantation, PR replacement by transplantation represents a broad treatment strategy applicable to many types of degeneration. The number of donor cells that integrate into the recipient retina determines transplantation success, yet the degenerating retinae presents a number of barriers that can impede effective integration. Here, we briefly review recent advances in the field of PR transplantation. We then describe how different aspects of gliosis may impact on cell integration efficiency.

Müller glia activation in response to inherited retinal degeneration is highly varied and disease-specific.

Despite different aetiologies, most inherited retinal disorders culminate in photoreceptor loss, which induces concomitant changes in the neural retina, one of the most striking being reactive gliosis by Müller cells. It is typically assumed that photoreceptor loss leads to an upregulation of glial fibrilliary acidic protein (Gfap) and other intermediate filament proteins, together with other gliosis-related changes, including loss of integrity of the outer limiting membrane (OLM) and deposition of proteoglycans. However, this is based on a mix of both injury-induced and genetic causes of photoreceptor loss. There are very few longitudinal studies of gliosis in the retina and none comparing these changes across models over time. Here, we present a comprehensive spatiotemporal assessment of features of gliosis in the degenerating murine retina that involves Müller glia. Specifically, we assessed Gfap, vimentin and chondroitin sulphate proteoglycan (CSPG) levels and outer limiting membrane (OLM) integrity over time in four murine models of inherited photoreceptor degeneration that encompass a range of disease severities (Crb1rd8/rd8, Prph2+/Δ307, Rho-/-, Pde6brd1/rd1). These features underwent very different changes, depending upon the disease-causing mutation, and that these changes are not correlated with disease severity. Intermediate filament expression did indeed increase with disease progression in Crb1rd8/rd8 and Prph2+/Δ307, but decreased in the Prph2+/Δ307 and Pde6brd1/rd1 models. CSPG deposition usually, but not always, followed the trends in intermediate filament expression. The OLM adherens junctions underwent significant remodelling in all models, but with differences in the composition of the resulting junctions; in Rho-/- mice, the adherens junctions maintained the typical rod-Müller glia interactions, while in the Pde6brd1/rd1 model they formed predominantly between Müller cells in late stage of degeneration. Together, these results show that gliosis and its associated processes are variable and disease-dependent.

Photoreceptor replacement therapy: challenges presented by the diseased recipient retinal environment.

Vision loss caused by the death of photoreceptors is the leading cause of irreversible blindness in the developed world. Rapid advances in stem cell biology and techniques in cell transplantation have made photoreceptor replacement by transplantation a very plausible therapeutic strategy. These advances include the demonstration of restoration of vision following photoreceptor transplantation and the generation of transplantable populations of donor cells from stem cells. In this review, we present a brief overview of the recent progress in photoreceptor transplantation. We then consider in more detail some of the challenges presented by the degenerating retinal environment that must play host to these transplanted cells, how these may influence transplanted photoreceptor cell integration and survival, and some of the progress in developing strategies to circumnavigate these issues.

Influence of combined therapy with rosuvastatin and amitriptyline on the oxidation-reduction status in rats.

The aim of the present study was to assess the impact of combined therapy with rosuvastatin (10 mg/kg) and amitriptyline (10 mg/kg) on oxidation-reduction status in the blood of rats. After 2-week application of drugs alone or their combination, the activity of glutathione peroxidase (GPX), glutathione reductase (GR) and total antioxidant status (TAS) were determined. It was noticed that combined therapy with rosuvastatin and amitriptyline significantly increased the activity of GPX in comparison to the group receiving only rosuvastatin and decreased the activity of GR in comparison to groups receiving only rosuvastatin or amitriptyline. However, the activity of these enzymes as a result of combined therapy was placed in the level of the control groups. Our studies indicated that the combined therapy with both drugs caused an increase of TAS compared to the groups of animals receiving only one of these drugs. The results indicate on the oxidation-reduction balance and increasing the antioxidant status in rats treated with rosuvastatin and amitriptyline.

Glutamatergic modulation of synaptic-like vesicle recycling in mechanosensory lanceolate nerve terminals of mammalian hair follicles.

Our aim in the present study was to determine whether a glutamatergic modulatory system involving synaptic-like vesicles (SLVs) is present in the lanceolate ending of the mouse and rat hair follicle and, if so, to assess its similarity to that of the rat muscle spindle annulospiral ending we have described previously. Both types of endings are formed by the peripheral sensory terminals of primary mechanosensory dorsal root ganglion cells, so the presence of such a system in the lanceolate ending would provide support for our hypothesis that it is a general property of fundamental importance to the regulation of the responsiveness of the broad class of primary mechanosensory endings. We show not only that an SLV-based system is present in lanceolate endings, but also that there are clear parallels between its operation in the two types of mechanosensory endings. In particular, we demonstrate that, as in the muscle spindle: (i) FM1-43 labels the sensory terminals of the lanceolate ending, rather than the closely associated accessory (glial) cells; (ii) the dye enters and leaves the terminals primarily by SLV recycling; (iii) the dye does not block the electrical response to mechanical stimulation, in contrast to its effect on the hair cell and dorsal root ganglion cells in culture; (iv) SLV recycling is Ca(2+) sensitive; and (v) the sensory terminals are enriched in glutamate. Thus, in the lanceolate sensory ending SLV recycling is itself regulated, at least in part, by glutamate acting through a phospholipase D-coupled metabotropic glutamate receptor.

Combined exposure to tobacco smoke and ethanol in adolescent mice elicits memory and learning deficits both during exposure and withdrawal.

INTRODUCTION: Adolescents often associate tobacco smoking and consumption of alcoholic beverages. In spite of that, little is known about the neurobehavioral consequences of the dual exposure in the adolescent brain. In the present work, we assessed the effects of tobacco smoke and/or ethanol exposure during adolescence on memory/learning. METHODS: From postnatal day 30 to 45 (PN30-45), male and female Swiss mice were exposed to tobacco smoke (SMK-generated from research cigarettes type 3R4F, whole body exposure, 8hr/day) and/or ethanol (ETOH-25% solution, 2g/kg intraperitoneally injected every other day) as follows: (a) SMK+ETOH exposure; (b) SMK exposure; (c) ETOH exposure; (d) Control. Memory/learning was evaluated during exposure (PN44-45) and during short- (PN49-50) and long-standing withdrawal (PN74-75). At each timepoint, mice were trained and tested in a step-down passive avoidance task (0.3 mA, 3 s footshock). Two retention tests were carried out in each animal, one at 3hr after training to measure short-term memory and another at 24hr to measure long-term memory. RESULTS: During exposure, the short-term memory was impaired in all groups and the long-term memory was impaired in SMK and SMK+ETOH. During the short-standing withdrawal, a significant impairment was observed only in long-term memory of the male SMK+ETOH mice. At long-standing withdrawal, there were no significant differences between groups. CONCLUSIONS: Tobacco smoke and ethanol exposures during adolescence of mice negatively affect learning/memory performance. Deficits that were still present during SMK+ETOH short-standing withdrawal suggest that the combined exposure elicits a worsened memory/learning outcome and that males are more susceptible.