Julia Teister

Glaucoma related Proteomic Alterations in Human Retina Samples

Glaucoma related proteomic changes have been documented in cell and animal models. However, proteomic studies investigating on human retina samples are still rare. In the present work, retina samples of glaucoma and non-glaucoma control donors have been examined by a state-of-the-art mass spectrometry (MS) workflow to uncover glaucoma related proteomic changes. More than 600 proteins could be identified with high confidence (FDR < 1%) in human retina samples. Distinct proteomic changes have been observed in 10% of proteins encircling mitochondrial and nucleus species. Numerous proteins showed a significant glaucoma related level change (p < 0.05) or distinct tendency of alteration (p < 0.1). Candidates were documented to be involved in cellular development, stress and cell death. Increase of stress related proteins and decrease of new glaucoma related candidates, ADP/ATP translocase 3 (ANT3), PC4 and SRFS1-interacting protein 1 (DFS70) and methyl-CpG-binding protein 2 (MeCp2) could be documented by MS. Moreover, candidates could be validated by Accurate Inclusion Mass Screening (AIMS) and immunostaining and supported for the retinal ganglion cell layer (GCL) by laser capture microdissection (LCM) in porcine and human eye cryosections. The workflow allowed a detailed view into the human retina proteome highlighting new molecular players ANT3, DFS70 and MeCp2 associated to glaucoma.

Intravitreal injection of β-crystallin B2 improves retinal ganglion cell survival in an experimental animal model of glaucoma

Purpose of this study was to investigate firstly specific proteomic changes within the retina in the course of an animal glaucoma model and to identify secondly new approaches for neuroprotective, therapeutic options in glaucoma by addressing those specific changes. Intraocular pressure was elevated through cauterization of episcleral veins in adult Sprague Dawley rats. Molecular and morphological changes were surveyed using mass spectrometry, optical coherence tomography as well as immunohistochemical cross section- and flat mount stainings. By quantifying more than 1500 retinal proteins, it was found that the HspB5 protein and numerous beta-crystallins showed a uniform and unique shifting expression pattern as a result of different periods of elevated IOP exposure. Crystallins showed a significant downregulation (p<0.05) after 3 weeks of elevated IOP and an upregulation after 7 weeks. Counteracting those typical changes, an intravitreal injection of β-crystallin B2 at the time of IOP elevation was found to reduce retinal ganglion cell loss (p<0.05), decrease of the retinal nerve fiber layer (p<0.05) and impairment of the optic nerve. Ultimately, proteomic data revealed that β-crystallin B2 might influence calcium-depended cell signaling pathways with severe effect on apoptosis and gene regulation. In this context especially annexin A5, calcium-transporting ATPase 1 and various histone proteins seem to play a major role.

Decelerated neurodegeneration after intravitreal injection of α-synuclein antibodies in a glaucoma animal model

Although elevated intraocular pressure (IOP) remains the major risk factor in glaucoma, neurodegenerative processes continue despite effective IOP lowering. Altered α-synuclein antibody (Abs) levels have been reported to play a crucial role. This study aimed at identifying whether α-synuclein Abs are capable to decelerate neuronal decay while providing insights into proteomic changes. Four groups of Sprague Dawley rats received episcleral vein occlusion: (1) CTRL, no intravitreal injection, n = 6, (2) CTRL IgG, intravitreal injection of unspecific IgG, n = 5, (3) Buffer, intravitreal injection of buffer, n = 6, (4), α-synuclein Ab, intravitreal injection of α-synuclein Ab, n = 5. IOP and retinal nerve fiber layer thickness (RNFLT) were monitored and immunohistochemistry, microarray and proteomic analysis were performed. RNFLT was reduced in CTRL, CTRL IgG and Buffer group (all p < 0.01) and α-synuclein Ab group (p = 0.17). Axon and RGC density showed an increased neurodegeneration in CTRL, CTRL IgG and Buffer group (all p < 0.01) and increased neuronal survival in α-synuclein Ab group (p = 0.38 and 0.06, respectively) compared with fellow eyes. Proteomic analysis revealed alterations of cofilin 1 and superoxide dismutase 1 expression. This data indicate that α-synuclein Ab might indirectly modulate the actin cytoskeleton organization and negatively regulate apoptotic processes via cofilin 1 and superoxide dismutase 1.

The Small Heat Shock Protein α-Crystallin B Shows Neuroprotective Properties in a Glaucoma Animal Model

Glaucoma is a neurodegenerative disease that leads to irreversible retinal ganglion cell (RGC) loss and is one of the main causes of blindness worldwide. The pathogenesis of glaucoma remains unclear, and novel approaches for neuroprotective treatments are urgently needed. Previous studies have revealed significant down-regulation of α-crystallin B as an initial reaction to elevated intraocular pressure (IOP), followed by a clear but delayed up-regulation, suggesting that this small heat-shock protein plays a pathophysiological role in the disease. This study analyzed the neuroprotective effect of α-crystallin B in an experimental animal model of glaucoma. Significant IOP elevation induced by episcleral vein cauterization resulted in a considerable impairment of the RGCs and the retinal nerve fiber layer. An intravitreal injection of α-crystallin B at the time of the IOP increase was able to rescue the RGCs, as measured in a functional photopic electroretinogram, retinal nerve fiber layer thickness, and RGC counts. Mass-spectrometry-based proteomics and antibody-microarray measurements indicated that a α-crystallin injection distinctly up-regulated all of the subclasses (α, β, and γ) of the crystallin protein family. The creation of an interactive protein network revealed clear correlations between individual proteins, which showed a regulatory shift resulting from the crystallin injection. The neuroprotective properties of α-crystallin B further demonstrate the potential importance of crystallin proteins in developing therapeutic options for glaucoma.

Peripapillary fluorescence lifetime reveals age-dependent changes using fluorescence lifetime imaging ophthalmoscopy in rats

&NA; Many fundus diseases accompany fundus autofluorescence change. Fluorescence lifetime imaging ophthalmoscope (FLIO) is a latest technique in imaging fundus autofluorescence. With FLIO, the fundus fluorescence lifetime (FLT) is recorded topographically, assisting to diagnose and monitor multiple fundus diseases. The purpose of this study was to evaluate the repeatability of FLT using FLIO on adult rats and to analyze the age‐dependency of the peripapillary FLT of the fundus in a short spectral channel (498–560 nm) and a long spectral channel (560–720 nm). Sprague Dawley rats (n of eyes = 10) were used for repeatability experiments. Age‐dependent changes were investigated in young (two months old, n of eyes = 20) and old (eight months old, n of eyes = 10) rats. Repeatability experiments showed highly corresponding data for all segments in both spectral channel, with higher repeatability in the short spectral channel. FLT decreased significantly in all areas in the short (young: 991 ± 29 ps; old: 547 ± 42 ps) and long (young: 382 ± 28 ps; old: 261 ± 16 ps) spectral channels, indicating an overall metabolic change of the fundus in old animals. FLT of veins increased in the short spectral channel (young: 385 ± 43 ps; old: 424 ± 25 ps) and no change was observed in the long spectral channel (young: 274 ± 9 ps; old: 269 ± 24 ps). FLIO represents a highly repeatable and sensitive method to detect changes of the FLT in aged eyes for monitoring the degeneration of the rodent retinae. HighlightsFLIO is highly repeatable in the short spectral channel.Rodent fundus fluorescence lifetime is sensitive to aging.FLIO can be used as a imaging tool for monitoring the fundus.

Correlation of Crystallin Expression and RGC Susceptibility in Experimental Glaucoma Rats of Different Ages

ABSTRACT Purpose: Glaucoma is one of the leading causes of blindness worldwide with age being an important risk factor. However, the pathogenesis remains poorly understood. Aim of this study was to focus on age-dependent molecular changes in an experimental animal model of glaucoma. Methods: Intraocular pressure was elevated in Sprague-Dawley rats aged 3, 14, and 47 weeks for a period of 7 weeks by episcleral vein cauterization. Ganglion cell loss was monitored by an immunohistochemical staining of the Brain-specific homeobox/POU (Pit-1, Oct-2, Unc-86) domain protein 3A positive cells in retinal flat-mounts and spectral-domain optical coherence tomography measuring the retinal nerve fiber layer thickness. Molecular protein alterations were analyzed using a comprehensive mass spectrometric proteomics approach of the retina and vitreous body. Results: While juvenile animals did not show a significant loss of retinal ganglion cells due to intraocular pressure elevation, adolescent animals showed a decrease up to 26% (p < 0.05). A shift of retinal crystallin protein expression levels within all protein-family subclasses (α, β, γ) could be observed in the youngest animal group (p < 0.05), while the upregulation of crystallin proteins in older animals was less striking. In addition, numerous crystallin proteins were also detected in the vitreous body. Conclusion: These results provide insights of a potential correlation of age-related glaucomatous damage and the absence of crystallin proteins in the retina.