Neuronal integration in an abuttingretinas culture system

PURPOSE. Limited integration is consistently observed between subretinal transplants and host retinas. In the current study, an in vitro model system for studying connections forming between two abutting retinas was developed. METHODS. Neuroretinas were dissected from normal wild-type (WT) mice and green fluorescent protein (GFP) transgenic mice (obtained at postnatal days [P]0, P5, or P60), as well as from adult rd mice. Pieces from two different retinas (WT-WT, GFP-WT, GFP-rd) were placed side-by-side (contacting each other at the margins) or overlapping each other in organ cultures for 7 or 12 days. The abutting retinal pieces derived from animals of the same age (P5-P5; P60-P60) or of different ages (P0-P60; P5-P60). Retinal cells and fibers were visualized in wholemount preparations and in cross sections by immunocytochemistry using antibodies against neurofilament (NFϩ), neuronal nitric oxide synthase (NOSϩ), and protein kinase C (PKCϩ) and by GFP fluorescence (GFPϩ). RESULTS. In side-by-side pairs (WT-WT, GFP-WT), numerous horizontal cell fibers (NFϩ) and amacrine cell fibers (NOSϩ) crossed the interface between the two pieces, forming continuous plexiform layers. In overlapping pairs, NFϩ, NOSϩ, and PKCϩ fibers displayed parallel plexiform layers, and no crossover of fibers was observed in any of the pair combinations examined (WT-WT, GFP-WT, GFP-rd). Some integration was seen only in small areas where the structure of both retinal pieces was disrupted at the interface. CONCLUSIONS. The results demonstrate the ability of neurites to extend between abutting retinas and to make appropriate target choices when they are placed side-by-side. However, this ability is limited when they overlap each other, similar to that observed in subretinal transplantation. (Invest Ophthalmol Vis Sci. 2003;44:4936 -4946 ). Prompted by the problem of poor graft-host integration, we developed a modified culture system in which the outgrowth of fibers between two retinal pieces could be analyzed. The system consists of two abutting retinal pieces, placed overlapping each other, which is analogous to the in vivo situation of subretinal transplantation, or side by side. Using specific neuronal markers, we examined in wholemount preparations and in transverse sections, whether neuronal fibers can extend from one retinal piece into the abutting piece. Pairs were formed using retinal pieces derived from 5-day-old (P5) mice and cultured for 7 days, thus encompassing a time window (P5-P12) during which, in normal mouse development, substantial outgrowth of retinal cell processes occurs within the synaptic layers, and synaptic maturation is initiated. 11,12 MATERIALS AND METHODS Animals and Tissue Culture Preparation The experiments were conducted with the approval of the local animal experimentation and ethics committee. Animals were handled according to the guidelines on care and use of experimental animals set forth by the Government Committee on Animal Experimentation at the University of Lund and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The organ culture condition has been described in detail. 13 Retinas were dissected from normal mice (C57BL/6), from GFP mice (harboring a transgene consisting of enhanced GFP [EGFP] cDNA under the control of a chicken ␤-actin promoter and a cytomegalovirus enhancer), After the superior and nasal cornea were marked, the eyes were enucleated under sterile conditions and transferred to a dish containing serum-free medium (R16; Invitrogen-Gibco, Gaithersburg, MD). 15 Retinas were dissected from the retinal pigment epithelium (RPE) and from hyaloid vessels. Each retina was cut under fresh medium into four pieces along the superior-inferior and the nasal-temporal axes (Fig

SNAREs Interact with Retinal Degeneration Slow and Rod Outer Segment Membrane Protein-1 during Conventional and Unconventional Outer Segment Targeting

The authors would like to thank Mr. Marc Banworth, Mr. Justin Burnett, and Ms. Jamie Watson for their technical assistance, Drs. Muayyad Al-Ubaidi and David Sherry for their comments on the manuscript, and Drs. Roger Janz, Roderick McInnes, Neeraj Agarwal, Vadim Arshavsky, Robert Molday and Anand Swaroop for the provision of reagents as indicated in the text.Mutations in the photoreceptor protein peripherin-2 (also known as RDS) cause severe retinal degeneration. RDS and its homolog ROM-1 (rod outer segment protein 1) are synthesized in the inner segment and then trafficked into the outer segment where they function in tetramers and covalently linked larger complexes. Our goal is to identify binding partners of RDS and ROM-1 that may be involved in their biosynthetic pathway or in their function in the photoreceptor outer segment (OS). Here we utilize several methods including mass spectrometry after affinity purification, in vitro co-expression followed by pull-down, in vivo pull-down from mouse retinas, and proximity ligation assay to identify and confirm the SNARE proteins Syntaxin 3B and SNAP-25 as novel binding partners of RDS and ROM-1. We show that both covalently linked and non-covalently linked RDS complexes interact with Syntaxin 3B. RDS in the mouse is trafficked from the inner segment to the outer segment by both conventional (i.e., Golgi dependent) and unconventional secretory pathways, and RDS from both pathways interacts with Syntaxin3B. Syntaxin 3B and SNAP-25 are enriched in the inner segment (compared to the outer segment) suggesting that the interaction with RDS/ROM-1 occurs in the inner segment. Syntaxin 3B and SNAP-25 are involved in mediating fusion of vesicles carrying other outer segment proteins during outer segment targeting, so could be involved in the trafficking of RDS/ROM-1.Yeshttp://www.plosone.org/static/editorial#pee

Studies into The Mechanisms of Neurodegeneration and Neuroprotection in Rd1 Mouse Retina.

Retinitis Pigmentosa (RP) is a group of uncured retinal diseases with the world wide incidence of about 1/3500. In spite of the huge efforts in past years, the mechanisms underlying this type of hereditary blindness has not been elucidated and nor any effective treatment available. The rd1 mouse strain carries a mutation in ?-subunit of phosphodiesterase gene which hydrolyzes the cGMP and is needed for normal visual phototransduction. Once this enzyme is impaired, the cGMP gated channels remain in the open state. This raise the intracellular Ca2+ beyond physiological level and this is believed to be the main cause for photoreceptor cell death in rd1 mouse retina. Since this mutation is homologous to some form of human blindness, the rd1 mouse retina is considered as an ideal model for studying retina degeneration in human. The genes which are differentially expressed at a crucial time point of the degenerative process in the rd1 mouse retina could increase our current understanding of pathways involved in photoreceptor cell demise. We applied a microarray analysis to perform a global screening of rd1 mouse retina and the age matched wild type counterparts at post natal day 11. At this age the decision for the death of rd1 photoreceptors has been made but cells are mostly not yet eliminated. In the first part of the study (microarray analysis) we identified 328 up regulated and 110 downregulated genes. Gene ontology analysis was used to categorize these genes. Further studies showed that calpain activity is much higher in rd1 photoreceptor and might be involved in photoreceptor cell death. Our data also showed that mRNA and protein level of three members of novel PKCs ?, ? and µ were increased in rd1. Moreover, PKC ?, ? are more phosphorylated in rd1 retina. The changes are partly located in photoreceptor layer and therefore they could very well be related to the rd1 induced cell death. In this work we also aimed at understanding the molecular events which take place following application of a combination of neurotrophic factors namely CNTF+ BDNF in rd1 retina explants. Following the above mentioned treatment, endogenous BDNF, CNTF, GFAP, p-ERK, p-Akt levels were increased and FGF2 level was decreased. Rd1 treated explants compared to untreated counterparts showed less tunnel positive cells. The observed changes suggest that CNTF+BDNF stimulates survival signaling pathways which might creates a prospective treatment for RP patients

Calpain activation and apoptosis in motor neurons of cultured adult mouse spinal cord

Calpain, a Ca2+-dependent cysteine protease, has been implicated in neuronal apoptosis following spinal cord injury. In this study, activation of calpain was investigated in motor neurons of adult spinal cord slices from the mouse, using a cell-permeable fluorogenic calpain substrate and Western blotting. Calpain was rapidly activated in the motor neurons of excised spinal cord slices and calpain activity was observed both in the cytoplasm and the nuclei. In these neurons, nuclear and chromatin condensation were pronounced. Both calpain inhibitor VI and EGTA (ethyleneglycol-bis(beta-aminoethyl ether) N´,N´,N´,N´-tetraacetic acid) inhibited calpain activation and subsequent appearance of apoptotic nuclei. In contrast, the general caspase inhibitor Z-VAD.fmk had no effect. Calpain activation was also observed in the slices by Western blotting using an antibody to 150-kD calpain-cleaved a-fodrin fragment. These results show that calpain is rapidly activated in injured motor neurons and imply that this activation could be responsible for execution of caspase-independent apoptosis in injured adult motor neuron

Up-regulation and increased phosphorylation of protein kinase C (PKC) delta, mu and theta in the degenerating rd1 mouse retina.

The rd1 mouse serves as a model for inherited photoreceptor degeneration: retinitis pigmentosa. Microarray techniques were employed to compare the transcriptomes of rd1 and congenic wild-type retinas at postnatal day 11, when degenerative processes have started but most photoreceptors are still present. Of the several genes that were differentially expressed, focus was put on those associated with the protein kinase C (PKC) signaling pathway, in particular PKCδ, μ and θ. Microarray identified these as being up-regulated in the rd1 retina, which was confirmed by QRT-PCR. Western blotting and immunostaining, using antibodies against either total or phosphorylated variants of the PKC isoforms, revealed increased expression and phosphorylation of PKCδ, μ and θ in the rd1 retina at the protein level as well. Our results suggest that these PKC isoforms are involved in rd1 degeneration

Thyroid-beta2 and the retinoid RAR-alpha, RXR-gamma and ROR-beta2 receptor mRNAs; expression profiles in mouse retina, retinal explants and neocortex.

In neonatal retinal explants cultured long-term green cones are missing. Recently it was reported that thyroid hormone beta2 receptors (TR-beta2) are essential for these green cones to differentiate. Therefore transcript level of these receptors was investigated in our mouse retinal explants. However, thyroid receptors function as heterodimers with retinoid receptors (RR); so the fate of selected RRs was similarly analyzed using semi-quantitative RT-PCR. Loss of TR-beta2 and RR (RXR-gamma and ROR-beta2) mRNAs was observed after culturing the neonatal retina for 12 days. This indicates that these proteins are involved in determination of green cone identity. In addition, levels of the selected RR transcripts are differentially affected by short- or long-term culture. In the latter case an attached retinal pigment epithelium seems to play a protective role. Furthermore, divergent diurnal peaks of RR mRNAs are present in young as well as aged mouse retina and neocortex. This data might be relevant in the context of human ageing disorders

Thyroid-beta2 and the retinoid RAR-alpha, RXR-gamma and ROR-beta2 receptor mRNAs; expression profiles in mouse retina, retinal explants and neocortex

In neonatal retinal explants cultured long-term green cones are missing. Recently it was reported that thyroid hormone beta2 receptors (TR-beta2) are essential for these green cones to differentiate. Therefore transcript level of these receptors was investigated in our mouse retinal explants. However, thyroid receptors function as heterodimers with retinoid receptors (RR); so the fate of selected RRs was similarly analyzed using semi-quantitative RT-PCR. Loss of TR-beta2 and RR (RXR-gamma and ROR-beta2) mRNAs was observed after culturing the neonatal retina for 12 days. This indicates that these proteins are involved in determination of green cone identity. In addition, levels of the selected RR transcripts are differentially affected by short- or long-term culture. In the latter case an attached retinal pigment epithelium seems to play a protective role. Furthermore, divergent diurnal peaks of RR mRNAs are present in young as well as aged mouse retina and neocortex. This data might be relevant in the contextof human ageing disorders

CNTF plus BDNF treatment and neuroprotective pathways in the rd1 mouse retina

The rd1 mouse is a relevant model for studying the mechanisms of photoreceptor degeneration in retinitis pigmentosa. Treatment with ciliary neurotrophic factor (CNTF) in combination with brain derived neurotrophic factor (BDNF) is known to rescue photoreceptors in cultured rd1 retinal explants. To shed light on the underlying mechanisms, we studied the effects of 9 days (starting at postnatal day 2) in vitro CNTF+BDNF treatment on the endogenous production of CNTF, BDNF, fibroblast growth factor 2 (FGF2), or the activation of extracellular signal-regulated kinase (ERK), Akt and CAMP-response-element-binding protein (CREB) in retinal explants. In rd1 explants, CNTF+BDNF decreased the number of TUNEL-positive photoreceptors. The treatment also increased endogenous rd1 levels of CNTF and BDNF, but lowered the level of FGF2 expression in rd1 explants. When wild-type explants were treated, endogenous CNTF was similarly increased, while BDNF and FGF2 levels remained unaffected. In addition, treatment of rd1 retinas strongly increased the phosphorylation of ERK, Akt and CREB. In treated wild-type explants, the same parameters were either unchanged (ERK) or decreased (Akt and CREB). The results suggest a role for Akt, ERK and CREB in conveying the neuroprotective effect of CNTF+BDNF treatment in rd1 retinal explants. (c) 2006 Elsevier B.V. All rights reserved

Neuronal integration in an abutting-retinas culture system

PURPOSE: Limited integration is consistently observed between subretinal transplants and host retinas. In the current study, an in vitro model system for studying connections forming between two abutting retinas was developed.METHODS: Neuroretinas were dissected from normal wild-type (WT) mice and green fluorescent protein (GFP) transgenic mice (obtained at postnatal days [P]0, P5, or P60), as well as from adult rd mice. Pieces from two different retinas (WT-WT, GFP-WT, GFP-rd) were placed side-by-side (contacting each other at the margins) or overlapping each other in organ cultures for 7 or 12 days. The abutting retinal pieces derived from animals of the same age (P5-P5; P60-P60) or of different ages (P0-P60; P5-P60). Retinal cells and fibers were visualized in wholemount preparations and in cross sections by immunocytochemistry using antibodies against neurofilament (NF+), neuronal nitric oxide synthase (NOS+), and protein kinase C (PKC+) and by GFP fluorescence (GFP+).RESULTS: In side-by-side pairs (WT-WT, GFP-WT), numerous horizontal cell fibers (NF+) and amacrine cell fibers (NOS+) crossed the interface between the two pieces, forming continuous plexiform layers. In overlapping pairs, NF+, NOS+, and PKC+ fibers displayed parallel plexiform layers, and no crossover of fibers was observed in any of the pair combinations examined (WT-WT, GFP-WT, GFP-rd). Some integration was seen only in small areas where the structure of both retinal pieces was disrupted at the interface.CONCLUSIONS: The results demonstrate the ability of neurites to extend between abutting retinas and to make appropriate target choices when they are placed side-by-side. However, this ability is limited when they overlap each other, similar to that observed in subretinal transplantation