A Proteomics Approach to Identify Candidate Proteins Secreted by Müller Glia that Protect Ganglion Cells in the Retina.

The retinal Müller glial cells, can enhance the survival and activity of neurons, especially of retinal ganglion cells (RGCs), which are the neurons affected in diseases such as glaucoma, diabetes, and retinal ischemia. It has been demonstrated that Müller glia release neurotrophic factors that support RGC survival, yet many of these factors remain to be elucidated. To define these neurotrophic factors, a quantitative proteomic approach was adopted aiming at identifying neuroprotective proteins. First, the conditioned medium from porcine Müller cells cultured in vitro under three different conditions were isolated and these conditioned media were tested for their capacity to promote survival of primary adult RGCs in culture. Mass spectrometry was used to identify and quantify proteins in the conditioned medium, and osteopontin (SPP1), clusterin (CLU), and basigin (BSG) were selected as candidate neuroprotective factors. SPP1 and BSG significantly enhance RGC survival in vitro, indicating that the survival-promoting activity of the Müller cell secretome is multifactorial, and that SPP1 and BSG contribute to this activity. Thus, the quantitative proteomics strategy identify proteins secreted by Müller glia that are potentially novel neuroprotectants, and it may also serve to identify other bioactive proteins or molecular markers.

Proteomic Landscape of Patient-Derived CD4+ T Cells in Recent-Onset Type 1 Diabetes.

The pathophysiology underlying the autoimmune disease type 1 diabetes (T1D) is poorly understood. Obtaining an accurate proteomic profile of the T helper cell population is essential for understanding the pathogenesis of T1D. Here, we performed in-depth proteomic profiling of peripheral CD4+ T cells in a pediatric cohort to identify cellular signatures associated with the onset of T1D. Using only 250 000 CD4+ T cells per patient, isolated from biobanked PBMC samples, we identified nearly 6000 proteins using deep-proteome profiling with LC-MS/MS data-independent acquisition. Our analysis revealed an inflammatory signature in patients with T1D; this signature is characterized by circulating mediators of neutrophils, platelets, and the complement system. This signature likely reflects the inflammatory extracellular milieu, which suggests that activation of the innate immune system plays an important role in disease onset. Our results emphasize the potential value of using high-resolution LC-MS/MS to investigate limited quantities of biobanked samples to identify disease-relevant proteomic patterns. Proteomic profiles of 114 individuals have been deposited in a comprehensive portable repository serving as a unique resource for CD4+ T cell expression in the context of both health and T1D disease.

The Proteome of Native Adult Müller Glial Cells From Murine Retina.

To date, the proteomic profiling of Müller cells, the dominant macroglia of the retina, has been hampered because of the absence of suitable enrichment methods. We established a novel protocol to isolate native, intact Müller cells from adult murine retinae at excellent purity which retain in situ morphology and are well suited for proteomic analyses. Two different strategies of sample preparation - an in StageTips (iST) and a subcellular fractionation approach including cell surface protein profiling were used for quantitative liquid chromatography-mass spectrometry (LC-MSMS) comparing Müller cell-enriched to depleted neuronal fractions. Pathway enrichment analyses on both data sets enabled us to identify Müller cell-specific functions which included focal adhesion kinase signaling, signal transduction mediated by calcium as second messenger, transmembrane neurotransmitter transport and antioxidant activity. Pathways associated with RNA processing, cellular respiration and phototransduction were enriched in the neuronal subpopulation. Proteomic results were validated for selected Müller cell genes by quantitative real time PCR, confirming the high expression levels of numerous members of the angiogenic and anti-inflammatory annexins and antioxidant enzymes (e.g. paraoxonase 2, peroxiredoxin 1, 4 and 6). Finally, the significant enrichment of antioxidant proteins in Müller cells was confirmed by measurements on vital retinal cells using the oxidative stress indicator CM-H2DCFDA. In contrast to photoreceptors or bipolar cells, Müller cells were most efficiently protected against H2O2-induced reactive oxygen species formation, which is in line with the protein repertoire identified in the proteomic profiling. Our novel approach to isolate intact glial cells from adult retina in combination with proteomic profiling enabled the identification of novel Müller glia specific proteins, which were validated as markers and for their functional impact in glial physiology. This provides the basis to allow the discovery of novel glial specializations and will enable us to elucidate the role of Müller cells in retinal pathologies - a topic still controversially discussed.

Proteome Dynamics in Biobanked Horse Peripheral Blood Derived Lymphocytes (PBL) with Induced Autoimmune Uveitis.

Equine recurrent uveitis is the only spontaneous model for recurrent autoimmune uveitis in humans, where T cells target retinal proteins. Differences between normal and autoaggressive lymphocytes were identified in this study by analyzing peripheral blood derived lymphocytes (PBL) proteomes from the same case with interphotoreceptor retinoid binding protein induced uveitis sampled before (Day 0), during (Day 15), and after uveitic attack (Day 23). Relative protein abundances of PBL were investigated in a quantitative, label-free differential proteome analysis in cells that were kept frozen for 14 years since the initial experiment. Quantitative data could be acquired for 2632 proteins at all three time points. Profound changes (≥2-fold change) in PBL protein abundance were observed when comparing Day 0 with 15, representing acute inflammation (1070 regulated proteins) and Day 0 with 23 (cessation; 1571 regulated). Significant differences applied to proteins with functions in integrin signaling during active uveitis, involving "Erk and pi-3 kinase are necessary for collagen binding in corneal epithelia," "integrins in angiogenesis," and "integrin-linked kinase signaling" pathways. In contrast, at cessation of uveitic attack, significantly changed proteins belonged to pathways of "nongenotropic androgen signaling," "classical complement pathway," and "Amb2 integrin signaling." Several members of respective pathways were earlier shown to be changed in naturally occurring uveitis, underscoring the significance of these findings here and proofing the value of the induced model in mimicking spontaneous autoimmune uveitis. All MS data have been deposited to the ProteomeXchange consortium via the PRIDE partner repository (dataset identifier PXD005580).

ASL mRNA-LNP Therapeutic for the Treatment of Argininosuccinic Aciduria Enables Survival Benefit in a Mouse Model.

Argininosuccinic aciduria (ASA) is a metabolic disorder caused by a deficiency in argininosuccinate lyase (ASL), which cleaves argininosuccinic acid to arginine and fumarate in the urea cycle. ASL deficiency (ASLD) leads to hepatocyte dysfunction, hyperammonemia, encephalopathy, and respiratory alkalosis. Here we describe a novel therapeutic approach for treating ASA, based on nucleoside-modified messenger RNA (modRNA) formulated in lipid nanoparticles (LNP). To optimize ASL-encoding mRNA, we modified its cap, 5' and 3' untranslated regions, coding sequence, and the poly(A) tail. We tested multiple optimizations of the formulated mRNA in human cells and wild-type C57BL/6 mice. The ASL protein showed robust expression in vitro and in vivo and a favorable safety profile, with low cytokine and chemokine secretion even upon administration of increasing doses of ASL mRNA-LNP. In the ASLNeo/Neo mouse model of ASLD, intravenous administration of the lead therapeutic candidate LNP-ASL CDS2 drastically improved the survival of the mice. When administered twice a week lower doses partially protected and 3 mg/kg LNP-ASL CDS2 fully protected the mice. These results demonstrate the considerable potential of LNP-formulated, modified ASL-encoding mRNA as an effective alternative to AAV-based approaches for the treatment of ASA.