Efficacy of PARP inhibition in Pde6a mutant mouse models for retinitis pigmentosa depends on the quality and composition of individual human mutations.

Retinitis pigmentosa (RP), an inherited blinding disease, is caused by a variety of different mutations that affect retinal photoreceptor function and survival. So far there is neither effective treatment nor cure. We have previously shown that poly(ADP-ribose)polymerase (PARP) acts as a common and critical denominator of cell death in photoreceptors, qualifying it as a potential target for future therapeutic intervention. A significant fraction of RP-causing mutations affect the genes for the rod photoreceptor phosphodiesterase 6A (PDE6A) subunit, but it is not known whether they all engage the same death pathway. Analysing three homozygous point mutations (Pde6a R562W, D670G, and V685M) and one compound heterozygous Pde6a (V685M/R562W) mutation in mouse models that match human RP patients, we demonstrate excessive activation of PARP, which correlated in time with the progression of photoreceptor degeneration. The causal involvement of PARP activity in the neurodegenerative process was confirmed in organotypic retinal explant cultures treated with the PARP-selective inhibitor PJ34, using different treatment time-points and durations. Remarkably, the neuroprotective efficacy of PARP inhibition correlated inversely with the strength of the genetically induced insult, with the D670G mutant showing the best treatment effects. Our results highlight PARP as a target for neuroprotective interventions in RP caused by PDE6A mutations and are a first attempt towards personalized, genotype-matched therapy development for RP. In addition, for each of the different mutant situations, our work identifies windows of opportunity for an optimal treatment regimen for further in vivo experimentation and possibly clinical studies.

High fat diet-induced modifications in membrane lipid and mitochondrial-membrane protein signatures precede the development of hepatic insulin resistance in mice.

OBJECTIVE: Excess lipid intake has been implicated in the pathophysiology of hepatosteatosis and hepatic insulin resistance. Lipids constitute approximately 50% of the cell membrane mass, define membrane properties, and create microenvironments for membrane-proteins. In this study we aimed to resolve temporal alterations in membrane metabolite and protein signatures during high-fat diet (HF)-mediated development of hepatic insulin resistance. METHODS: We induced hepatosteatosis by feeding C3HeB/FeJ male mice an HF enriched with long-chain polyunsaturated C18:2n6 fatty acids for 7, 14, or 21 days. Longitudinal changes in hepatic insulin sensitivity were assessed via the euglycemic-hyperinsulinemic clamp, in membrane lipids via t-metabolomics- and membrane proteins via quantitative proteomics-analyses, and in hepatocyte morphology via electron microscopy. Data were compared to those of age- and litter-matched controls maintained on a low-fat diet. RESULTS: Excess long-chain polyunsaturated C18:2n6 intake for 7 days did not compromise hepatic insulin sensitivity, however, induced hepatosteatosis and modified major membrane lipid constituent signatures in liver, e.g. increased total unsaturated, long-chain fatty acid-containing acyl-carnitine or membrane-associated diacylglycerol moieties and decreased total short-chain acyl-carnitines, glycerophosphocholines, lysophosphatidylcholines, or sphingolipids. Hepatic insulin sensitivity tended to decrease within 14 days HF-exposure. Overt hepatic insulin resistance developed until day 21 of HF-intervention and was accompanied by morphological mitochondrial abnormalities and indications for oxidative stress in liver. HF-feeding progressively decreased the abundance of protein-components of all mitochondrial respiratory chain complexes, inner and outer mitochondrial membrane substrate transporters independent from the hepatocellular mitochondrial volume in liver. CONCLUSIONS: We assume HF-induced modifications in membrane lipid- and protein-signatures prior to and during changes in hepatic insulin action in liver alter membrane properties - in particular those of mitochondria which are highly abundant in hepatocytes. In turn, a progressive decrease in the abundance of mitochondrial membrane proteins throughout HF-exposure likely impacts on mitochondrial energy metabolism, substrate exchange across mitochondrial membranes, contributes to oxidative stress, mitochondrial damage, and the development of insulin resistance in liver.

In situ cell surface proteomics reveals differentially expressed membrane proteins in retinal pigment epithelial cells during autoimmune uveitis.

Retinal pigment epithelium (RPE) builds the outer blood-retinal barrier of the eye and plays an important role in pathogenesis of the sight threatening disease equine recurrent uveitis (ERU). ERU is a spontaneous autoimmune mediated inflammatory disease characterised by the breakdown of the outer blood-retinal barrier and an influx of autoaggressive T-cells into the inner eye. Therefore, identification of molecular mechanisms contributing to changed function of blood-retinal barrier in ERU is important for the understanding of pathophysiology. Cell surface proteins of RPE collected from healthy horses and horses with ERU were captured by in situ biotinylation and analysed with high resolution mass spectrometry coupled to liquid chromatography (LC-MS/MS) to identify differentially expressed proteins. With label free differential proteomics, a total of 27 differently expressed cell surface proteins in diseased RPE could be detected. Significant down-regulation of three very interesting proteins, synaptotagmin 1, basigin and collectrin was verified and further characterised. BIOLOGICAL SIGNIFICANCE: We applied an innovative and successful method to detect changes in the plasma cell surface proteome of RPE cells in a spontaneous inflammatory eye disease, serving as a valuable model for human autoimmune uveitis. We were able to identify 27 differentially expressed plasma cell membrane proteins, including synaptotagmin 1, basigin and collectrin, which play important roles in cell adhesion, transport and cell communication.

Novel in situ activity assays for the quantitative molecular analysis of neurodegenerative processes in the retina.

The mechanisms of neuronal cell death are still only poorly understood, which has hindered the advancement of therapies for many currently untreatable neurodegenerative diseases. This calls for the development of new methods which reveal critical molecular mechanisms of the celldeath machinery with both high sensitivity and cellular resolution. Using animal models for hereditary neurodegeneration in the retina, we have developed or adapted different biochemical assays to determine the enzymatic activities of calpain, poly-ADP-ribose-polymerase (PARP), and histone deacetylase (HDAC) directly and in situ. Additionally, the enzymatic activity of cGMP-dependent protein kinase (PKG) was assessed indirectly using in situ immunohistological techniques to detect PKG-activity-dependent products. Combining these assays with in situ cell death markers revealed close temporospatial correlations, suggesting causal connections between the PKG, HDAC, PARP and calpain activities and neuronal cell death. Using different pharmacological and genetic manipulations, causality could indeed be demonstrated. Surprisingly, the often dramatic rises in metabolic activities didnot match by corresponding increases in expression, highlighting the importance of analyses of protein activities at the cellular level. The above mentioned studies identified a number of metabolic processes previously unknownto be involved in inherited retinal degeneration. Comparing different animal retinal degeneration models uncovered striking similarities in enzymatic activities, suggesting a generality of the destructive pathways. Taken together, these findings provided a number of novel targets for neuroprotection and as such opened up new perspectives for the therapy of hereditary neurodegeneration in the retina and possibly other parts of the central nervous system.

Knockout of PARG110 confers resistance to cGMP-induced toxicity in mammalian photoreceptors.

Hereditary retinal degeneration (RD) relates to a heterogeneous group of blinding human diseases in which the light sensitive neurons of the retina, the photoreceptors, die. RD is currently untreatable and the underlying cellular mechanisms remain poorly understood. However, the activity of the enzyme poly-ADP-ribose polymerase-1 (PARP1) and excessive generation of poly-ADP-ribose (PAR) polymers in photoreceptor nuclei have been shown to be causally involved in RD. The activity of PARP1 is to a large extent governed by its functional antagonist, poly-ADP-glycohydrolase (PARG), which thus also may have a role in RD. To investigate this, we analyzed PARG expression in the retina of wild-type (wt) mice and in the rd1 mouse model for human RD, and detected increased PARG protein in a subset of degenerating rd1 photoreceptors. Knockout (KO) animals lacking the 110 kDa nuclear PARG isoform were furthermore analyzed, and their retinal morphology and function were indistinguishable from wild-type animals. Organotypic wt retinal explants can be experimentally treated to induce rd1-like photoreceptor death, but PARG110 KO retinal explants were unexpectedly highly resistant to such treatment. The resistance was associated with decreased PAR accumulation and low PARP activity, indicating that PARG110 may positively regulate PARP1, an event that therefore is absent in PARG110 KO tissue. Our study demonstrates a causal involvement of PARG110 in the process of photoreceptor degeneration. Contrasting its anticipated role as a functional antagonist, absence of PARG110 correlated with low PARP activity, suggesting that PARG110 and PARP1 act in a positive feedback loop, which is especially active under pathologic conditions. This in turn highlights both PARG110 and PARP1 as potential targets for neuroprotective treatments for RD.

Therapeutic targeting of naturally presented myeloperoxidase-derived HLA peptide ligands on myeloid leukemia cells by TCR-transgenic T cells.

T cells have been proven to be therapeutically effective in patients with relapsed leukemias, although target antigens on leukemic cells as well as T-cell receptors (TCRs), potentially recognizing those antigens, are mostly unknown. We have applied an immunopeptidomic approach and isolated human leukocyte antigen (HLA) ligands from primary leukemia cells. We identified a number of ligands derived from different genes that are restrictedly expressed in the hematopoietic system. We exemplarily selected myeloperoxidase (MPO) as a potential target and isolated a high-avidity TCR with specificity for a HLA-B*07:02-(HLA-B7)-restricted epitope of MPO in the single HLA-mismatched setting. T cells transgenic for this TCR demonstrated high peptide and antigen specificity as well as leukemia reactivity in vitro and in vivo. In contrast, no significant on- and off-target toxicity could be observed. In conclusion, we here demonstrate, exemplarily for MPO, that leukemia-derived HLA ligands can be selected for specific effector tool development to redirect T cells to be used for graft manipulation or adoptive T-cell therapies in diverse transplant settings. This approach can be extended to other HLA ligands and HLA molecules in order to provide better treatment options for this life-threatening disease.

How long does a photoreceptor cell take to die? Implications for the causative cell death mechanisms.

The duration of cell death may allow deducing the underlying degenerative mechanism. To find out how long a photoreceptor takes to die, we used the rd1 mouse model for retinal neurodegeneration, which is characterized by phosphodiesterase-6 (PDE6) dysfunction and photoreceptor death triggered by high cGMP levels. Based on cellular data on the progression of cGMP accumulation, cell death, and survival, we created a mathematical model to simulate the temporal development of the degeneration and the clearance of dead cells. Both cellular data and modelling suggested that at the level of the individual cell, the degenerative process was rather slow, taking around 80 h to complete. Organotypic retinal explant cultures derived from wild-type animals and exposed to the selective PDE6 inhibitor zaprinast, confirmed the surprisingly long duration of an individual photoreceptor cell's death. We briefly discuss the possibility to link different cell death stages and their temporal progression to specific enzymatic activities known to be causally connected to cell death. This in turn opens up new perspectives for the treatment of inherited retinal degeneration, both in terms of therapeutic targets and temporal windows-of-opportunity.

[Current therapeutic approaches in inherited retinal degeneration: from genes to chip]. / Therapieansätze für erbliche Netzhauterkrankungen: von den Genen bis zum Chip.

Different strategies for the treatment of inherited photoreceptor degeneration are currently being investigated, with each of these approaches facing specific challenges. Gene therapy, for instance, may be feasible only for genetically well-defined pathologies. However, inherited retinal disorders are genetically highly heterogeneous and early onset disorders may restrict the therapeutic window. The majority of currently developed molecular approaches aim at the reconstitution of physiologically important functions in RPE and photoreceptor. Neuroprotection attempts to prolong cell survival and proper function via sustained delivery systems that fulfil a long-term dynamic reservoir function for therapeutic neuroprotective compounds. Cell-based approaches include replacement strategies such as cell transplantation, the implantation of prosthetic devices or optogenetics. They aim at replacing lost neurosensory functions of the retina. This short review aims at providing an insight into current therapeutic strategies and future treatment options for retinal disorders. Pharmacological and nutritional support strategies are only briefly discussed as we focus here on molecular and prosthetic therapeutic approaches.

True blue: S-opsin is widely expressed in different animal species.

Colour vision in animals is an interesting, fascinating subject. In this study, we examined a wide variety of species for expression of S-opsin (blue sensitive) and M-/L-opsin (green-red sensitive) in retinal cones using two novel monoclonal antibodies specific for peptides from human opsins. Mouse, rat and hare did not express one of the investigated epitopes, but we could clearly prove existence of cones through peanut agglutinin labelling. Retinas of guinea pig, dog, wolf, marten, cat, roe deer, pig and horse were positive for S-opsin, but not for M-/L-opsin. Nevertheless all these species are clearly at least dichromats, because we could detect further S-opsin negative cones by labelling with cone arrestin specific antibody. In contrast, pheasant and char had M-/L-opsin positive cones, but no S-opsin expressing cones. Sheep, cattle, monkey, men, pigeon, duck and chicken were positive for both opsins. Visual acuity analyzed through density of retinal ganglion cells revealed least visual discrimination by horses and highest resolution in pheasant and pigeon. Most mammals studied are dichromats with visual perception similar to red-green blind people.

[Development and first results of fast and cost-effective examination methods for an ophthalmological screening within the National Cohort]. / Entwicklung und erste Anwendungserfahrungen einer schnellen und kostengünstigen ophthalmologischen Untersuchung für die Nationale Kohorte.

BACKGROUND: For an implemented ophthalmological screening within a German long-term cohort study (National Cohort) simple and effective methods for an examination of visual acuity and for non-mydriatic retina photografies should be evaluated. Furthermore standard operating-procedures (SOP) should be developed. METHODS: In the years 2011 and 2012 pinhole visual acuity measurements and automated retina photographies (DRS, CenterVue S. p. a., Padua, Italy) were made at three different epidemiological study centers within Germany. Furthermore, anterior segment images were taken by the camera. Standard operating procedures (SOP) regarding the ophthalmological screening were developed and evaluated within the study. The main question was whether it is possible to implement the screening methods within the National Cohort. Further main outcomes were quality and interpretability of the taken images. RESULTS: 457 subjects (914 eyes) were examined within the investigation. Median VA was 0.8 for right and left eyes (p > 0.42). Image quality of the photographies was good in 491 cases (54 %), fair in 239 cases (26 %) and bad in 179 cases (20 %). The usability of the images was without limitations in 686 cases (75 %), limited in 152 cases (17 %) and not given in 71 cases (8 %). Increasing age of the subjects was slightly correlated with decreasing image quality (r = 0.26) and decreasing image usability (r = 0.2). Anterior segment photographies were usable in 176 eyes (56 %). CONCLUSION: The developed screening method fulfilled the specifications of the National Cohort. The used pinhole visual acuity examination was fast and cheap. Image quality and usability of the retina photographies could be improved with prolonged pupil recovery times. The quality of the anterior segment images could not fulfill the expectations and were taken out of the further examinations of the ophthalmological screening. The written SOP showed good acceptance within the investigators' daily routine. The ophthalmological screening within the National Cohort generates information (e. g., pathologies of the vessels or of the retina) which are useful not only from an ophthalmological point of view.

Osteopontin inhibits osmotic swelling of retinal glial (Müller) cells by inducing release of VEGF.

Osmotic swelling of retinal neurons and glial cells is an important pathogenic factor of retinal edema formation. Here, we show that the neuroprotective factor osteopontin (OPN), which is released from retinal glial (Müller) cells after stimulation of the cells with glial cell line-derived neurotrophic factor (Del Río et al., 2011, Glia 59:821-832), inhibits the swelling of rat Müller cells induced by hypoosmotic exposure of retinal slices in the presence of barium ions and H2O2, respectively, and in slices of postischemic retinas. OPN did not inhibit the hypoosmotic swelling of bipolar cells in slices of control and postischemic retinas. The inhibitory effect of OPN on Müller cell swelling was dose-dependent, with a half-maximal effect at ∼0.6 ng/ml. The effect of OPN was abrogated in the presence of pharmacological blockers of vascular endothelial growth factor (VEGF) receptor-2, metabotropic glutamate receptors, and purinergic receptors (P2Y1, adenosine A1 receptors), as well as of a neutralizing anti-VEGF antibody. The data suggest that OPN induces the release of VEGF, glutamate, ATP, and adenosine from Müller cells. The effect of OPN was also prevented by blockers of voltage-gated sodium channels (tetrodotoxin), T-type voltage-gated calcium channels (kurtoxin), potassium channels (clofilium), and chloride channels 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). The swelling-inhibitory effect of OPN was dependent on intracellular calcium signaling, activation of phospholipase C and protein kinase C, and vesicular exocytosis of glutamate. In retinal slices, Müller glial cells display immunoreactivity of OPN. The data suggest that Müller cell-derived OPN has (in addition to the effects on photoreceptors and retinal neurons) autocrine effects. The neuroprotective effects of OPN may be in part mediated by the prevention of cytotoxic Müller cell swelling and the release of VEGF and adenosine from Müller cells.

Retinitis pigmentosa: rapid neurodegeneration is governed by slow cell death mechanisms.

For most neurodegenerative diseases the precise duration of an individual cell's death is unknown, which is an obstacle when counteractive measures are being considered. To address this, we used the rd1 mouse model for retinal neurodegeneration, characterized by phosphodiesterase-6 (PDE6) dysfunction and photoreceptor death triggered by high cyclic guanosine-mono-phosphate (cGMP) levels. Using cellular data on cGMP accumulation, cell death, and survival, we created mathematical models to simulate the temporal development of the degeneration. We validated model predictions using organotypic retinal explant cultures derived from wild-type animals and exposed to the selective PDE6 inhibitor zaprinast. Together, photoreceptor data and modeling for the first time delineated three major cell death phases in a complex neuronal tissue: (1) initiation, taking up to 36 h, (2) execution, lasting another 40 h, and finally (3) clearance, lasting about 7 h. Surprisingly, photoreceptor neurodegeneration was noticeably slower than necrosis or apoptosis, suggesting a different mechanism of death for these neurons.

From quantitative protein complex analysis to disease mechanism.

Interest in the field of cilia biology and cilia-associated diseases - ciliopathies - has strongly increased over the last few years. Proteomic technologies, especially protein complex analysis by affinity purification-based methods, have been used to decipher various basic but also disease-associated mechanisms. This review focusses on some selected recent studies using affinity purification-based protein complex analysis, thereby exemplifying the great possibilities this technology offers.

Neuroprotective strategies for the treatment of inherited photoreceptor degeneration.

Photoreceptor degeneration is the hallmark of several groups of inherited neurodegenerative diseases causing blindness in humans. These diseases are a major cause of visual handicap and to date no satisfactory treatment is available. Here, we briefly review different approaches for the treatment of photoreceptor degeneration, to then focus on neuroprotection. Up to date, translation of experimental neuroprotection into a clinical setting has faced major obstacles, which are in part due to an incomplete understanding of the regulation of pro-survival as well as neurodegenerative mechanisms. Previous approaches were often based on the hypothesis that photoreceptor cell death was governed by a single, apoptotic cell death mechanism. This perception has turned out too simple as recent work has demonstrated that photoreceptor cell death is governed by non-apoptotic mechanisms as well. Moreover, there is evidence, that several different destructive processes are executed in parallel. Briefly reviewing the complexity of degenerative mechanisms, this review discusses relevant pathways, options to target signaling cascades, final common denominators of cell death, and the interplay of events executing cell death. In particular, we focus on cGMP-signaling, epigenetic and proteolytic processes and the corresponding enzymatic activities that were recently shown to be causally related to retinal degeneration. Finally, we illustrate how a better understanding of destructive mechanisms may enable identification and validation of novel targets for neuroprotection, and allow development of next generation neuroprotective treatments as well as combination therapy.

Factores neurotróficos derivados de las células de Müller. En el camino hacia la terapia neuroprotectora en la retina / Müller cell-derived neurotrophic factors. A road to neuroprotective therapy in the retina

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Prevention of interferon-stimulated gene expression using microRNA-designed hairpins.

RNA interference allows selective gene silencing, and is widely used for functional analysis of individual genes in vertebrate cells and represents an attractive therapeutic option for treating central nervous system diseases. However, growing evidence exists that the expression of short hairpin RNAs (shRNAs) can trigger cellular immune response resulting in unspecific cellular phenotypes and severe side effects. We found that lentiviral vector (LV)-mediated expression of shRNAs in primary cortical cultures resulted in strong expression of the interferon-stimulated gene oligoadenylate synthetase 1 (Oas1), which was accompanied by accelerated apoptosis and substantial net neuron loss. Modification of the shRNA construct by implementing features of the naturally occurring microRNA-30 (miR-30) precursor avoided Oas1 induction in transduced primary cultures, whereby modification of the passenger strand seems to be a crucial feature to circumvent interferon-stimulated gene expression. This work represents the first experimental study showing that an miR-30-based shRNA construct prevents Oas1 pathway associated off-target effects, which we consider as an essential prerequisite for shRNA use in future gene therapeutic approaches.

Genetic mouse models for behavioral analysis through transgenic RNAi technology.

Pharmacological inhibitors and knockout mice have developed into routine tools to analyze the role of specific genes in behavior. Both strategies have limitations like the availability of inhibitors for only a subset of proteins and the large efforts required to construct specific mouse mutants. The recent emergence of RNA interference (RNAi)-mediated gene silencing provides a fast alternative that can be applied to any coding gene. We established an approach for the efficient generation of transgenic knockdown mice by targeted insertion of short hairpin (sh) RNA vectors into a defined genomic locus and studied the efficiency of gene silencing in the adult brain and the utility of such mice for behavioral analysis. We generated shRNA knockdown mice for the corticotropin-releasing hormone receptor type 1 (Crhr1), the leucine-rich repeat kinase 2 (Lrkk2) and the purinergic receptor P2X ligand-gated ion channel 7 (P2rx7) genes and show the ubiquitous expression of shRNA and efficient suppression of the target mRNA and protein in the brain of young and 11-month-old knockdown mice. Knockdown mice for the Crhr1 gene exhibited decreased anxiety-related behavior, an impaired stress response, and thereby recapitulate the phenotype of CRHR1 knockout mice. Our results show the feasibility of gene silencing in the adult brain and validate knockdown mice as new genetic models suitable for behavioral analysis.