Dynamic changes in the localization of synapse associated proteins during development and differentiation of the mammalian retina

We have examined the developmental distribution and differential localization of presynaptic terminal associated proteins in the mammalian retina. We have used antibodies specific for synaptic vesicle associated proteins Synaptotagmin, Rab 3A, Synaptophysin and Synaptobrevin, and presynaptic terminal membrane associated proteins SNAP-25 and Syntaxin, to characterize their spatio-temporal distribution during retinal differentiation and in the mature retina;The vertebrate retina has a laminar organization consisting of three cellular layers separated by two synaptic layers, the inner piexiform layer and the outer plexiform layer. In general, immunoreactivity for presynaptic terminal associated proteins was first observed in cellular layers, and as differentiation progressed, immunoreactivity was localized to the synaptic or plexiform layers. However, there were distinct differences in the relative intensities and localization of patterns of immunoreactivity for these proteins;This analysis revealed a temporal difference in the onset of detectable immunoreactivity for Synaptotagmin and Rab 3A compared with Synaptophysin and Synaptobrevin, suggesting they may have additional roles in vesicle trafficking during neural development. Immunoreactivity for presynaptic terminal membrane associated protein SNAP-25 is at relatively high levels in cholinergic amacrine cells during their differentiation, but not at maturity. This transient expression of high levels of SNAP-25 may contribute to the functional role these cells play in propagation of spontaneous retinal activity. Spatial and temporal differences in localization of SNARE complex proteins (Synaptophysin, SNAP-25 and Syntaxin), were also observed during development and at maturity. Detectable SNAP-25- and Syntaxin-immunoreactivity preceded Synaptobrevin-immunoreactivity during differentiation of synaptic layers. In addition, immunoreactivity for each protein had a distinct pattern of differential intensities within the inner plexiform or synaptic layer during development and in the mature retina, and differential localization in the OPL;This analysis was the first to systematically characterize the distribution of multiple presynaptic terminal associated proteins, including proteins of the SNARE complex, during development of an organized tissue like the retina. The dynamic, differential patterns of immunoreactivity for these proteins suggests several of them may have additional roles in the development of the nervous system

Changes in Retinal Function and Morphology Are Early Clinical Signs of Disease in Cattle with Bovine Spongiform Encephalopathy

Bovine spongiform encephalopathy (BSE) belongs to a group of fatal, transmissible protein misfolding diseases known as transmissible spongiform encephalopathies (TSEs). All TSEs are caused by accumulation of misfolded prion protein (PrPSc) throughout the central nervous system (CNS), which results in neuronal loss and ultimately death. Like other protein misfolding diseases including Parkinson’s disease and Alzheimer’s disease, TSEs are generally not diagnosed until the onset of disease after the appearance of unequivocal clinical signs. As such, identification of the earliest clinical signs of disease may facilitate diagnosis. The retina is the most accessible part of the central nervous system, and retinal pathology in TSE affected animals has been previously reported. Here we describe antemortem changes in retinal function and morphology that are detectable in BSE inoculated animals several months (up to 11 months) prior to the appearance of any other signs of clinical disease. We also demonstrate that differences in the severity of these clinical signs reflect the amount of PrPSc accumulation in the retina and the resulting inflammatory response of the tissue. These results are the earliest reported clinical signs associated with TSE infection and provide a basis for understanding the pathology and evaluating therapeutic interventions

Characterization of the retinal proteome during rod photoreceptor genesis

<p>Abstract</p> <p>Background</p> <p>The process of rod photoreceptor genesis, cell fate determination and differentiation is complex and multi-factorial. Previous studies have defined a model of photoreceptor differentiation that relies on intrinsic changes within the presumptive photoreceptor cells as well as changes in surrounding tissue that are extrinsic to the cell. We have used a proteomics approach to identify proteins that are dynamically expressed in the mouse retina during rod genesis and differentiation.</p> <p>Findings</p> <p>A series of six developmental ages from E13 to P5 were used to define changes in retinal protein expression during rod photoreceptor genesis and early differentiation. Retinal proteins were separated by isoelectric focus point and molecular weight. Gels were analyzed for changes in protein spot intensity across developmental time. Protein spots that peaked in expression at E17, P0 and P5 were picked from gels for identification. There were 239 spots that were picked for identification based on their dynamic expression during the developmental period of maximal rod photoreceptor genesis and differentiation. Of the 239 spots, 60 of them were reliably identified and represented a single protein. Ten proteins were represented by multiple spots, suggesting they were post-translationally modified. Of the 42 unique dynamically expressed proteins identified, 16 had been previously reported to be associated with the developing retina.</p> <p>Conclusions</p> <p>Our results represent the first proteomics study of the developing mouse retina that includes prenatal development. We identified 26 dynamically expressed proteins in the developing mouse retina whose expression had not been previously associated with retinal development.</p

Clinical and Pathologic Features of H-Type Bovine Spongiform Encephalopathy Associated with E211K Prion Protein Polymorphism

The majority of bovine spongiform encephalopathy (BSE) cases have been ascribed to the classical form of the disease. H-type and L-type BSE cases have atypical molecular profiles compared to classical BSE and are thought to arise spontaneously. However, one case of H-type BSE was associated with a heritable E211K mutation in the prion protein gene. The purpose of this study was to describe transmission of this unique isolate of H-type BSE when inoculated into a calf of the same genotype by the intracranial route. Electroretinograms were used to demonstrate preclinical deficits in retinal function, and optical coherence tomography was used to demonstrate an antemortem decrease in retinal thickness. The calf rapidly progressed to clinical disease (9.4 months) and was necropsied. Widespread distribution of abnormal prion protein was demonstrated within neural tissues by western blot and immunohistochemistry. While this isolate is categorized as BSE-H due to a higher molecular mass of the unglycosylated PrPSc isoform, a strong labeling of all 3 PrPSc bands with monoclonal antibodies 6H4 and P4, and a second unglycosylated band at approximately 14 kDa when developed with antibodies that bind in the C-terminal region, it is unique from other described cases of BSE-H because of an additional band 23 kDa demonstrated on western blots of the cerebellum. This work demonstrates that this isolate is transmissible, has a BSE-H phenotype when transmitted to cattle with the K211 polymorphism, and has molecular features that distinguish it from other cases of BSE-H described in the literature

Using Evolutionary Conserved Modules in Gene Networks as a Strategy to Leverage High Throughput Gene Expression Queries

Background: Large-scale gene expression studies have not yielded the expected insight into genetic networks that control complex processes. These anticipated discoveries have been limited not by technology, but by a lack of effective strategies to investigate the data in a manageable and meaningful way. Previous work suggests that using a pre-determined seednetwork of gene relationships to query large-scale expression datasets is an effective way to generate candidate genes for further study and network expansion or enrichment. Based on the evolutionary conservation of gene relationships, we test the hypothesis that a seed network derived from studies of retinal cell determination in the fly, Drosophila melanogaster, will be an effective way to identify novel candidate genes for their role in mouse retinal development. Methodology/Principal Findings: Our results demonstrate that a number of gene relationships regulating retinal cell differentiation in the fly are identifiable as pairwise correlations between genes from developing mouse retina. In addition, we demonstrate that our extracted seed-network of correlated mouse genes is an effective tool for querying datasets and provides a context to generate hypotheses. Our query identified 46 genes correlated with our extracted seed-network members. Approximately 54% of these candidates had been previously linked to the developing brain and 33% had been previously linked to the developing retina. Five of six candidate genes investigated further were validated by experiments examining spatial and temporal protein expression in the developing retina. Conclusions/Significance: We present an effective strategy for pursuing a systems biology approach that utilizes an evolutionary comparative framework between two model organisms, fly and mouse. Future implementation of this strategy will be useful to determine the extent of network conservation, not just gene conservation, between species and will facilitate the use of prior biological knowledge to develop rational systems-based hypotheses

A comparison of classical and H-type bovine spongiform encephalopathy associated with E211K prion protein polymorphism in wild type and EK211 cattle following intracranial inoculation

In 2006, a case of H-type bovine spongiform encephalopathy (BSE-H) was diagnosed in a cow that was associated with a heritable polymorphism in the bovine prion protein gene (PRNP) resulting in a lysine for glutamine amino acid substitution at codon 211 (called E211K) of the prion protein. Although the prevalence of this polymorphism is low, cattle carrying the K211 allele may be predisposed to rapid onset of BSE-H when exposed or to the potential development of a genetic BSE. This study was conducted to better understand the relationship between the K211 polymorphism and its effect on BSE phenotype. BSE-H from the US 2006 case was inoculated intracranially (IC) in one PRNP wild type (EE211) calf and one EK211 calf. In addition, one wild type calf and one EK211 calf were inoculated IC with brain homogenate from a US 2003 classical BSE case. All cattle developed clinical disease. The survival times of the E211K BSE-H inoculated EK211 calf (10 months) was shorter than the wild type calf (18 months). This genotype effect was not observed in classical BSE inoculated cattle (both 26 months). Significant changes in retinal function were observed in H-type BSE challenged cattle only. Cattle challenged with the same inoculum showed similar severity and neuroanatomical distribution of vacuolation and disease-associated prion protein deposition in the brain, though differences in neuropathology were observed between E211K BSE-H and classical BSE inoculated animals. Western blot results for brain tissue from challenged animals were consistent with the inoculum strains. This study demonstrates that the phenotype of E211K BSE-H remains stable when transmitted to cattle without the K211 polymorphism, and exhibits a number of features that differ from classical BSE in both wild type and heterozygous EK211 animals

Clinical and Pathologic Features of H-Type Bovine Spongiform Encephalopathy Associated with E211K Prion Protein Polymorphism

The majority of bovine spongiform encephalopathy (BSE) cases have been ascribed to the classical form of the disease. H-type and L-type BSE cases have atypical molecular profiles compared to classical BSE and are thought to arise spontaneously. However, one case of H-type BSE was associated with a heritable E211K mutation in the prion protein gene. The purpose of this study was to describe transmission of this unique isolate of H-type BSE when inoculated into a calf of the same genotype by the intracranial route. Electroretinograms were used to demonstrate preclinical deficits in retinal function, and optical coherence tomography was used to demonstrate an antemortem decrease in retinal thickness. The calf rapidly progressed to clinical disease (9.4 months) and was necropsied. Widespread distribution of abnormal prion protein was demonstrated within neural tissues by western blot and immunohistochemistry. While this isolate is categorized as BSE-H due to a higher molecular mass of the unglycosylated PrPSc isoform, a strong labeling of all 3 PrPSc bands with monoclonal antibodies 6H4 and P4, and a second unglycosylated band at approximately 14 kDa when developed with antibodies that bind in the C-terminal region, it is unique from other described cases of BSE-H because of an additional band 23 kDa demonstrated on western blots of the cerebellum. This work demonstrates that this isolate is transmissible, has a BSE-H phenotype when transmitted to cattle with the K211 polymorphism, and has molecular features that distinguish it from other cases of BSE-H described in the literature.This article is published as Greenlee, Justin J., Jodi D. Smith, M. Heather West Greenlee, and Eric M. Nicholson. "Clinical and pathologic features of H-type bovine spongiform encephalopathy associated with E211K prion protein polymorphism." PloS one 7, no. 6 (2012): e38678. doi:10.1371/journal.pone.0038678. Posted with permission.</p