Photoreceptor Compartment-Specific TULP1 Interactomes.

Photoreceptors are highly compartmentalized cells with large amounts of proteins synthesized in the inner segment (IS) and transported to the outer segment (OS) and synaptic terminal. Tulp1 is a photoreceptor-specific protein localized to the IS and synapse. In the absence of Tulp1, several OS-specific proteins are mislocalized and synaptic vesicle recycling is impaired. To better understand the involvement of Tulp1 in protein trafficking, our approach in the current study was to physically isolate Tulp1-containing photoreceptor compartments by serial tangential sectioning of retinas and to identify compartment-specific Tulp1 binding partners by immunoprecipitation followed by liquid chromatography tandem mass spectrometry. Our results indicate that Tulp1 has two distinct interactomes. We report the identification of: (1) an IS-specific interaction between Tulp1 and the motor protein Kinesin family member 3a (Kif3a), (2) a synaptic-specific interaction between Tulp1 and the scaffold protein Ribeye, and (3) an interaction between Tulp1 and the cytoskeletal protein microtubule-associated protein 1B (MAP1B) in both compartments. Immunolocalization studies in the wild-type retina indicate that Tulp1 and its binding partners co-localize to their respective compartments. Our observations are compatible with Tulp1 functioning in protein trafficking in multiple photoreceptor compartments, likely as an adapter molecule linking vesicles to molecular motors and the cytoskeletal scaffold.

The Retinol-Binding Protein Receptor 2 (Rbpr2) Is Required for Photoreceptor Survival and Visual Function in the Zebrafish.

Vitamin A/retinol (ROL) and its metabolites (retinoids) play critical roles in eye development and photoreception. Short-term dietary vitamin A deficiency (VAD) manifests clinically as night blindness, while prolonged VAD is known to cause retinal pigment epithelium (RPE) and photoreceptor degeneration. Therefore, sustained uptake of dietary vitamin A, for ocular retinoid production, is essential for photoreceptor health and visual function. The mechanisms influencing the uptake, storage, and supply of dietary vitamin A, for ocular retinoid production, however, are not fully understood. We investigated, in zebrafish, the physiological role of the retinol-binding protein receptor 2 (Rbpr2), for the uptake of dietary ROL, which is necessary for vision. NIH3T3 cells expressing zebrafish Rbpr2 showed plasma membrane localization patterns and were capable of ROL uptake from its bound form. Using whole-mount in situ hybridization, Rbpr2 was found to be expressed exclusively in the liver, intestine, and pancreas, of staged zebrafish larvae. At 5.5 days post fertilization, TALEN-generated rbpr2 mutants (rbpr2 -/- ) had smaller eyes and shorter OS lengths and showed loss of PNA (cones) and rhodopsin (rods) by immunofluorescence staining. Finally, tests for visual function using optokinetic response (OKR) showed no consistent OKR in rbpr2 -/- larval zebrafish. Our analysis, therefore, suggests that Rbpr2 is capable of ROL uptake and loss of this membrane receptor in zebrafish results in photoreceptor defects that adversely affect visual function.

A Novel Approach to Identify Photoreceptor Compartment-Specific Tulp1 Binding Partners.

Photoreceptors (PRs) are highly polarized and compartmentalized cells with large amounts of proteins synthesized in the inner segment (IS) and transported to the outer segment (OS) and synaptic terminal. The PR-specific protein, Tulp1, is localized to the IS and synapse and is hypothesized to be involved in protein trafficking. To better understand the molecular processes that regulate protein trafficking in PRs, we aimed to identify compartment-specific Tulp1 binding partners. Serial tangential sectioning of Long Evans rat retinas was utilized to isolate the IS and synaptic PR compartments. Tulp1 binding partners in each of these layers were identified using co-immunoprecipitation (co-IP) with Tulp1 antibodies. The co-IP eluates were separated by SDS-PAGE, trypsinized into peptide fragments, and proteins were identified by liquid chromatography tandem mass spectrometry. In the IS, potential Tulp1-binding partners included cytoskeletal scaffold proteins, protein trafficking molecules, as well as members of the phototransduction cascade. In the synaptic region, the majority of interacting proteins identified were cytoskeletal. A separate subset of proteins were identified in both the IS and synapse including chaperones and family members of the GTPase activating proteins. Tulp1 has two distinct PR compartment-specific interactomes. Our results support the hypothesis that Tulp1 is involved in the trafficking of proteins from the IS to the OS and the continuous membrane remodeling and vesicle cycling at the synaptic terminal.

Histopathological comparison of eyes from patients with autosomal recessive retinitis pigmentosa caused by novel EYS mutations.

To evaluate the retinal histopathology in donor eyes from patients with autosomal recessive retinitis pigmentosa (arRP) caused by EYS mutations. Eyes from a 72-year-old female (donor 1, family 1), a 91-year-old female (donor 2, family 2), and her 97-year-old sister (donor 3, family 2) were evaluated with macroscopic, scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) imaging. Age-similar normal eyes and an eye donated by donor 1's asymptomatic mother (donor 4, family 1) were used as controls. The perifovea and peripheral retina were processed for microscopy and immunocytochemistry with markers for cone and rod photoreceptor cells. DNA analysis revealed EYS mutations c.2259 + 1G > A and c.2620C > T (p.Q874X) in family 1, and c.4350_4356del (p.I1451Pfs*3) and c.2739-?_3244 + ?del in family 2. Imaging studies revealed the presence of bone spicule pigment in arRP donor retinas. Histology of all three affected donor eyes showed very thin retinas with little evidence of stratified nuclear layers in the periphery. In contrast, the perifovea displayed a prominent inner nuclear layer. Immunocytochemistry analysis demonstrated advanced retinal degenerative changes in all eyes, with near-total absence of rod photoreceptors. In addition, we found that the perifoveal cones were more preserved in retinas from the donor with the midsize genomic rearrangement (c.4350_4356del (p.I1451Pfs*3) and c.2739-?_3244 + ?del) than in retinas from the donors with the truncating (c.2259 + 1G > A and c.2620C > T (p.Q874X) mutations. Advanced retinal degenerative changes with near-total absence of rods and preservation of some perifoveal cones are observed in arRP donor retinas with EYS mutations.

Retinal histopathology in eyes from patients with autosomal dominant retinitis pigmentosa caused by rhodopsin mutations.

PURPOSE: To evaluate the histopathology in donor eyes from patients with autosomal dominant retinitis pigmentosa (ADRP) caused by p.P23H, p.P347T and p.P347L rhodopsin ( RHO ) gene mutations. METHODS: Eyes from a 72-year-old male (donor 1), an 83-year-old female (donor 2), an 80-year-old female (donor 3), and three age-similar normal eyes were examined macroscopically, by scanning laser ophthalmoscopy and optical coherence tomography imaging. Perifoveal and peripheral pieces were processed for microscopy and immunocytochemistry with markers for photoreceptor cells. RESULTS: DNA analysis revealed RHO mutations c.68C>A (p.P23H) in donor 1, c.1040C>T (p.P347L) in donor 2 and c.1039C>A (p.P347T) in donor 3. Histology of the ADRP eyes showed retinas with little evidence of stratified nuclear layers in the periphery and a prominent inner nuclear layer present in the perifoveal region in the p.P23H and p.P347T eyes, while it was severely atrophic in the p.P347L eye. The p.P23H and p.P347T mutations cause a profound loss of rods in both the periphery and perifovea, while the p.P347L mutation displays near complete absence of rods in both regions. All three rhodopsin mutations caused a profound loss of cones in the periphery. The p.P23H and p.P347T mutations led to the presence of highly disorganized cones in the perifovea. However, the p.P347L mutation led to near complete absence of cones also in the perifovea. CONCLUSIONS: Our results support clinical findings indicating that mutations affecting residue P347 develop more severe phenotypes than those affecting P23. Furthermore, our results indicate a more severe phenotype in the p.P347L retina as compared to the p.P347T retina.

Interaction of tubby-like protein-1 (Tulp1) and microtubule-associated protein (MAP) 1A and MAP1B in the mouse retina.

Tubby-like protein-1 (Tulp1) is a photoreceptor-specific protein involved in the transport of specific proteins from the inner segment (IS) to the outer segment (OS) in photoreceptor cells. Mutations in the human TULP1 gene cause an early onset form of retinitis pigmentosa. Our previous work has shown an association between Tulp1 and the microtubule-associated protein, MAP1B. An allele of Mtap1a, which encodes the MAP1A protein, significantly delays photoreceptor degeneration in Tulp1 mutant mice. MAP1 proteins are important in stabilizing microtubules in neuronal cells, but their role in photoreceptors remains obscure. To investigate the relationship between Tulp1 and MAP1 proteins, we performed western blots, immunoprecipitations (IP), immunohistochemistry and proximity ligand assays (PLA) in wild-type and tulp1-/- mouse retinas. Our IP experiments provide evidence that Tulp1 and MAP1B interact while PLA experiments localize their interaction to the outer nuclear layer and IS of photoreceptors. Although MAP1A and MAP1B protein levels are not affected in the tulp1-/- retina, they are no longer localized to the OS of photoreceptors. This may be the cause for disorganized OSs in tulp1-/- mice, and indicate that their transport to the OS is Tulp1-dependent.

Pharmacogenetics for genes associated with age-related macular degeneration in the Comparison of AMD Treatments Trials (CATT).

PURPOSE: To evaluate the pharmacogenetic relationship between genotypes of single nucleotide polymorphisms (SNPs) known to be associated with age-related macular degeneration (AMD) and response to treatment with ranibizumab (Lucentis; Genentech, South San Francisco, CA) or bevacizumab (Avastin; Genentech) for neovascular AMD. DESIGN: Clinical trial. PARTICIPANTS: Eight hundred thirty-four (73%) of 1149 patients participating in the Comparison of AMD Treatments Trials (CATT) were recruited through 43 CATT clinical centers. METHODS: Each patient was genotyped for SNPs rs1061170 (CFH), rs10490924 (ARMS2), rs11200638 (HTRA1), and rs2230199 (C3), using TaqMan SNP genotyping assays (Applied Biosystems, Foster City, CA). MAIN OUTCOMES MEASURES: Genotypic frequencies were compared with clinical measures of response to therapy at one year, including mean visual acuity (VA), mean change in VA, 15-letter or more increase in VA, retinal thickness, mean change in total foveal thickness, presence of fluid on OCT, presence of leakage on fluorescein angiography (FA), mean change in lesion size, and mean number of injections administered. Differences in response by genotype were evaluated with tests of linear trend calculated from logistic regression models for categorical outcomes and linear regression models for continuous outcomes. To adjust for multiple comparisons, P≤0.01 was considered statistically significant. RESULTS: No statistically significant differences in response by genotype were identified for any of the clinical measures studied. Specifically, there were no high-risk alleles that predicted final VA or change in VA, the degree of anatomic response (fluid on OCT or FA, retinal thickness, change in total foveal thickness, change in lesion size), or the number of injections. Furthermore, a stepwise analysis failed to show a significant epistatic interaction among the variants analyzed; that is, response did not vary by the number of risk alleles present. The lack of association was similar whether patients were treated with ranibizumab or bevacizumab or whether they received monthly or pro re nata dosing. CONCLUSIONS: Although specific alleles for CFH, ARMS2, HTRA1, and C3 may predict the development of AMD, they did not predict response to anti-vascular endothelial growth factor therapy.

Retinal deimination and PAD2 levels in retinas from donors with age-related macular degeneration (AMD).

Deimination is a form of protein posttranslational modification carried out by the peptidyl arginine deiminases (PADs) enzymes. PAD2 is the principal deiminase expressed in the retina. Elevated levels of PAD2 and protein deimination are present in a number of human neurological diseases, with or without ocular manifestation. To define the association of deimination with the pathogenesis of age-related macular degeneration (AMD), we studied protein deimination and PAD2 levels in retinas of AMD donor eyes compared to age-matched non-AMD retinas. Eyes from non-AMD and AMD donors were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde in phosphate buffer. Retina and retinal pigment epithelium (RPE) from donor eyes were processed for immunohistochemical detection and western blotting using antibodies to PAD2 and citrulline residues. The ganglion cell, inner plexiform, inner nuclear and outer nuclear layers were labeled by both PAD2 and citrulline antibodies. Changes in the localization of deiminated residues and PAD2 were evident as the retinal layers were remodeled coincident with photoreceptor degeneration in AMD retinas. Immunodetection of either PAD2 or citrulline residues could not be evaluated in the RPE layer due to the high autofluorescence levels in this layer. Interestingly, higher deimination immunoreactivity was detected in AMD retinal lysates. However, no significant changes in PAD2 were detected in the AMD and non-AMD retinas and RPE lysates. Our observations show increased levels of protein deimination but not PAD2 in AMD retinas and RPE, suggesting a reduced rate of turnover of deiminated proteins in these AMD retinas.

Immunocytochemical evidence of Tulp1-dependent outer segment protein transport pathways in photoreceptor cells.

Tulp1 is a protein of unknown function exclusive to rod and cone photoreceptor cells. Mutations in the gene cause autosomal recessive retinitis pigmentosa in humans and photoreceptor degeneration in mice. In tulp1-/- mice, rod and cone opsins are mislocalized, and rhodopsin-bearing extracellular vesicles accumulate around the inner segment, indicating that Tulp1 is involved in protein transport from the inner segment to the outer segment. To investigate this further, we sought to define which outer segment transport pathways are Tulp1-dependent. We used immunohistochemistry to examine the localization of outer segment proteins in tulp1-/- photoreceptors, prior to retinal degeneration. We also surveyed the condition of inner segment organelles and rhodopsin transport machinery proteins. Herein, we show that guanylate cyclase 1 and guanylate cyclase activating proteins 1 and 2 are mislocalized in the absence of Tulp1. Furthermore, arrestin does not translocate to the outer segment in response to light stimulation. Additionally, data from the tulp1-/- retina adds to the understanding of peripheral membrane protein transport, indicating that rhodopsin kinase and transducin do not co-transport in rhodopsin carrier vesicles and phosphodiesterase does not co-transport in guanylate cyclase carrier vesicles. These data implicate Tulp1 in the transport of selective integral membrane outer segment proteins and their associated proteins, specifically, the opsin and guanylate cyclase carrier pathways. The exact role of Tulp1 in outer segment protein transport remains elusive. However, without Tulp1, two rhodopsin transport machinery proteins exhibit abnormal distribution, Rab8 and Rab11, suggesting a role for Tulp1 in vesicular docking and fusion at the plasma membrane near the connecting cilium.

Assessing susceptibility to age-related macular degeneration with proteomic and genomic biomarkers.

Age-related macular degeneration (AMD) is a progressive disease and major cause of severe visual loss. Toward the discovery of tools for early identification of AMD susceptibility, we evaluated the combined predictive capability of proteomic and genomic AMD biomarkers. We quantified plasma carboxyethylpyrrole (CEP) oxidative protein modifications and CEP autoantibodies by ELISA in 916 AMD and 488 control donors. CEP adducts are uniquely generated from oxidation of docosahexaenoate-containing lipids that are abundant in the retina. Mean CEP adduct and autoantibody levels were found to be elevated in AMD plasma by approximately 60 and approximately 30%, respectively. The odds ratio for both CEP markers elevated was 3-fold greater or more in AMD than in control patients. Genotyping was performed for AMD risk polymorphisms associated with age-related maculopathy susceptibility 2 (ARMS2), high temperature requirement factor A1 (HTRA1), complement factor H, and complement C3, and the risk of AMD was predicted based on genotype alone or in combination with the CEP markers. The AMD risk predicted for those exhibiting elevated CEP markers and risk genotypes was 2-3-fold greater than the risk based on genotype alone. AMD donors carrying the ARMS2 and HTRA1 risk alleles were the most likely to exhibit elevated CEP markers. The results compellingly demonstrate higher mean CEP marker levels in AMD plasma over a broad age range. Receiver operating characteristic curves suggest that CEP markers alone can discriminate between AMD and control plasma donors with approximately 76% accuracy and in combination with genomic markers provide up to approximately 80% discrimination accuracy. Plasma CEP marker levels were altered slightly by several demographic and health factors that warrant further study. We conclude that CEP plasma biomarkers, particularly in combination with genomic markers, offer a potential early warning system for assessing susceptibility to this blinding, multifactorial disease.

Tubby-like protein 1 (Tulp1) is required for normal photoreceptor synaptic development.

Mutations in the photoreceptor-specific tubby-like protein 1 (TULP1) underlie a form of autosomal recessive retinitis pigmentosa in humans and photoreceptor degeneration in mice. In wild type (wt) mice, Tulp1 is localized to the photoreceptor inner segment, connecting cilium and synapse. To investigate the role of Tulp1 in the synapse, we examined the pre- and postsynaptic architecture in tulp1-/- mice. We used immunohistochemistry to examine tulp1-/- mice prior to retinal degeneration and made comparisons to wt littermates and rd10 mice. In the tulp1-/- synapse, the spatial relationship between the ribbon-associated proteins, Bassoon and Piccolo, are disrupted, and few intact ribbons are present. Furthermore, bipolar cell dendrites are stunted, most likely a direct consequence of the malformed photoreceptor synapses. Comparable abnormalities are not seen in rd10 mice. The association of early onset and severe photoreceptor degeneration, which is preceded by synaptic abnormalities, appears to represent a phenotype not previously described. Our new evidence indicates that Tulp1 is not only critical for photoreceptor function and survival, but is essential for the proper development of the photoreceptor synapse.

Proteomic and genomic biomarkers for age-related macular degeneration.

Toward early detection of susceptibility to age-related macular degeneration (AMD), we quantified plasma carboxyethylpyrrole (CEP) oxidative protein modifications and CEP autoantibodies by ELISA in 916 AMD and 488 control donors. Mean CEP adduct and autoantibody levels were elevated in AMD plasma by ∼60 and ∼30%, respectively, and the odds ratio for both CEP markers elevated was ∼3-fold greater in AMD than in control patients. Genotyping was performed for AMD risk polymorphisms associated with age-related maculopathy susceptibility 2 (ARMS2), high-temperature requirement factor A1 (HTRA1), complement factor H (CFH), and complement C3. The AMD risk predicted for those exhibiting elevated CEP markers and risk genotypes was 2- to 3-fold greater than the risk based on genotype alone. AMD donors carrying the ARMS2 and HTRA1 risk alleles were the most likely to exhibit elevated CEP markers. Receiver operating characteristic curves suggest that CEP markers alone can discriminate between AMD and control plasma donors with ∼76% accuracy and in combination with genomic markers, provide up to ∼80% discrimination accuracy. CEP plasma biomarkers, particularly in combination with genomic markers, offer a potential early warning system for predicting susceptibility to this blinding disease.

Evidence of complement dysregulation in outer retina of Stargardt disease donor eyes.

Stargardt macular degeneration (STGD) is a central blinding disease caused by loss of or dysfunctional ABCA4 transporter in both photoreceptors and retinal pigment epithelial (RPE) cells. Toxic bisretinoid-lipofuscin buildup in the RPE cells is a pathological hallmark of STGD patients and its mouse model, the Abca4-/-. These vitamin A-derived fluorophores have been shown to induce oxidative stress, stimulate complement activity, and cause chronic inflammation of the RPE. In vivo modulation of complement regulatory pathway in the STGD mouse model has partially rescued the STGD phenotype suggesting that complement attack on the RPE is an important etiologic factor in disease pathogenesis. While bisretinoid-dependent complement activation was further evidenced in cultured RPE cells, this pathway has never been investigated directly in the context of RPE from STGD donor eyes. In the current study, we evaluate the complement reactivity in postmortem donor eyes of clinically diagnosed STGD patients. All three STGD donor eyes RPE displayed strong immunoreactivity for an antibody specific to 4-Hydroxynonenal, a lipid peroxidation byproduct. Also, unlike the control eyes, all three STGD donor eyes showed significantly increased membrane attack complex deposition on the RPE cells. In STGD eyes, increased MAC accumulation was mirrored by elevated C3 fragments internalized by the RPE and inversely correlated with the levels of complement factor H, a major complement regulatory protein. Here, we report the first direct evidence of RPE complement dysregulation as a causative factor in developing Stargardt phenotype.

Geographic Atrophy: Confocal Scanning Laser Ophthalmoscopy, Histology, and Inflammation in the Region of Expanding Lesions.

Purpose: To describe the pathology of AMD in eyes with geographic atrophy (GA) using confocal scanning laser ophthalmoscopy (SLO) blue light autofluorescence (BAF), and near-infrared (IR) AF and to correlate it with the histology and immunohistochemistry analysis at the margins of the GA lesion. Methods: Enucleated, fixed eyes from seventeen donors with GA were imaged and analyzed by BAF-SLO, IRAF-SLO, and by fundus macroscopy (FM). Tissue from the margins of the GA lesions was cut and processed for resin embedding and histology or cryosectioning and fluorescence in the green and far-red channels, and immunohistochemistry to assess markers of inflammation. Isolated DNA from donors was genotyped for single nucleotide polymorphisms (SNPs) previously shown to be risk factors for the development and progression of AMD. Results: Around the leading edge of the GA lesions we observed hypertrophic RPE cells with cytoplasm filled with granules fluorescent both in the far-red and green-red channels; abundant microglia and macrophage; deposition of complement factor H (CFH) in Bruch's membrane (BM) and increased membrane attack complex (MAC) on RPE cells. Conclusions: Fluorescence imaging of cryosections of RPE cells around the leading edge of the GA lesions suggest that IRAF-SLO visualizes mostly melanin-related compounds. In addition, medium-size GA atrophy displayed the most significant changes in inflammation markers.

Genetic analysis of complement factor H related 5, CFHR5, in patients with age-related macular degeneration.

PURPOSE: To investigate the complement factor H related 5 (CFHR5) gene, encoding a member of the complement factor H family, for the presence of genetic polymorphisms or mutations associated with age-related macular degeneration (AMD). METHODS: We screened 639 unrelated patients with AMD and 663 age-matched normal controls using direct genomic sequencing of the ten coding exons, along with the immediately flanking intronic DNA. The pathologic impact of the identified sequence variants were analyzed by computational methods using PolyPhen and PMut algorithms. RESULTS: We identified five heterozygous sequence changes in CFHR5. Asp169Asp had a minor allele frequency of 0.001% in patients and 0.014% in controls (p<0.0001), while Arg356His had a minor allele frequency of 0.016% in patients and 0.007% in controls. Val379Leu, Met514Arg, and Cys568Ter were found only in normal controls. In silico analysis predicted Arg356His and Val379Leu to be neutral and benign. Met514Arg was predicted to be pathological and damaging to the function of the CFHR5 protein. CONCLUSIONS: No definitive pathogenic CFHR5 mutations have been found in any of 639 unrelated patients with AMD, indicating that sequence variations in CFHR5 do not play a major role in determining AMD susceptibility. However, our findings suggest a possible protective role for Asp169Asp. Further studies of different and larger populations of patient and control samples will be required to address this observation.

Sequence alterations in RX in patients with microphthalmia, anophthalmia, and coloboma.

PURPOSE: Microphthalmia, anophthalmia, and coloboma are ocular malformations with a significant genetic component. Rx is a homeobox gene expressed early in the developing retina and is important in retinal cell fate specification as well as stem cell proliferation. We screened a group of 24 patients with microphthalmia, coloboma, and/or anophthalmia for RX mutations. METHODS: We used standard PCR and automated sequencing techniques to amplify and sequence each of the three RX exons. Patients' charts were reviewed for clinical information. The pathologic impact of the identified sequence variant was analyzed by computational methods using PolyPhen and PMut algorithms. RESULTS: In addition to the polymorphisms we identified a single patient with coloboma having a heterozygous nucleotide change (g.197G>C) in the first exon that results in a missense mutation of arginine to threonine at amino acid position 66 (R66T). In silico analysis predicted R66T to be a deleterious mutation. CONCLUSIONS: Sequence variations in RX are uncommon in patients with congenital ocular malformations, but may play a role in disease pathogenesis. We observed a missense mutation in RX in a patient with a small, typical chorioretinal coloboma, and postulate that the mutation is responsible for the patient's phenotype.

Interaction between the photoreceptor-specific tubby-like protein 1 and the neuronal-specific GTPase dynamin-1.

PURPOSE: Tubby-like proteins (TULPs) are a family of four proteins, two of which have been linked to neurosensory disease phenotypes. TULP1 is a photoreceptor-specific protein that is mutated in retinitis pigmentosa, an inherited retinal disease characterized by the degeneration of rod and cone photoreceptor cells. To investigate the function of TULP1 in maintaining the health of photoreceptors, the authors sought the identification of interacting proteins. METHODS: Immunoprecipitation from retinal lysates, followed by liquid chromatography tandem mass spectrometry and in vitro binding assays, were used to identify TULP1 binding partners. RT-PCR was performed on total RNA from wild-type mouse retina to identify the Dynamin-1 isoform expressed in the retina. Immunocytochemistry was used to determine the localization of TULP1 and Dynamin-1 in photoreceptor cells. Electroretinography (ERG) and light microscopy were used to phenotype tulp1-/- mice at a young age. RESULTS: Immunoprecipitation from retinal lysate identified Dynamin-1 as a possible TULP1 binding partner. GST pull-down assays further supported an interaction between TULP1 and Dynamin-1. In photoreceptor cells, Dynamin-1 and TULP1 colocalized primarily to the outer plexiform layer, where photoreceptor terminals synapse on second-order neurons and, to a lesser extent, to the inner segments, where polarized protein translocation occurs. ERG analyses in young tulp1-/- mice indicated a decreased b-wave at ages when the retina retained a full complement of photoreceptor cells. CONCLUSIONS: These data indicated that TULP1 interacts with Dynamin-1 and suggested that TULP1 is involved in the vesicular trafficking of photoreceptor proteins, both at the nerve terminal during synaptic transmission and at the inner segment during protein translocation to the outer segment. These results also raised the possibility that normal synaptic function requires TULP1, and they motivate a closer look at synaptic architecture in the developing tulp1-/- retina.

Fibrin glue system for adjuvant brachytherapy of brain tumors with 188Re and 186Re-labeled microspheres.

Brain tumors such as glioblastoma reappear in their original location in almost 50% of cases. To prevent this recurrence, we developed a radiopharmaceutical system that consists of a gel applied immediately after surgical resection of a brain tumor to deliver local radiation booster doses. The gel, which strongly adheres to tissue in the treatment area, consists of fibrin glue containing the beta-emitters rhenium-188 and rhenium-186 in microsphere-bound form. Such microspheres can be prepared by short (2 h or less) neutron activation even in low neutron flux reactors, yielding a mixture of the two beta-emitters rhenium-188 (E(max)=2.1 MeV, half life=17 h) and rhenium-186 (E(max)=1.1 MeV, half life=90.6h). The dosimetry of this rhenium-188/rhenium-186 fibrin glue system was determined using gafchromic film measurements. The treatment efficacy of the radioactive fibrin glue was measured in a 9L-glioblastoma rat model. All animals receiving the non-radioactive fibrin glue died within 17+/-3 days, whereas 60% of the treated animals survived 36 days, the final length of the experiment. Control animals that were treated with the same amount of radioactive fibrin glue, but had not received a previous tumor cell injection, showed no toxic effects over one year. The beta-radiation emitting rhenium-188/rhenium-186-based gel thus provides an effective method of delivering high doses of local radiation to tumor tissue, particularly to wet areas where high adhesive strength and long-term radiation (with or without drug) delivery are needed.