Daniela Schlatzer

Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo

Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes. These cells are abundant in demyelinated regions of patients with multiple sclerosis, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention. To discover therapeutic compounds for enhancing myelination from endogenous oligodendrocyte progenitor cells, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitor cells. Here we show seven drugs function at nanomolar doses selectively to enhance the generation of mature oligodendrocytes from progenitor cells in vitro. Two drugs, miconazole and clobetasol, are effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increases the number of new oligodendrocytes and enhances remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in an experimental autoimmune encephalomyelitis mouse model of chronic progressive multiple sclerosis results in striking reversal of disease severity. Immune response assays show that miconazole functions directly as a remyelinating drug with no effect on the immune system, whereas clobetasol is a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies show that miconazole and clobetasol function in oligodendrocyte progenitor cells through mitogen-activated protein kinase and glucocorticoid receptor signalling, respectively. Furthermore, both drugs enhance the generation of human oligodendrocytes from human oligodendrocyte progenitor cells in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally modified derivatives, to enhance remyelination in patients.

Novel Urinary Protein Biomarkers Predicting the Development of Microalbuminuria and Renal Function Decline in Type 1 Diabetes

OBJECTIVE To define a panel of novel protein biomarkers of renal disease. RESEARCH DESIGN AND METHODS Adults with type 1 diabetes in the Coronary Artery Calcification in Type 1 Diabetes study who were initially free of renal complications (n = 465) were followed for development of micro- or macroalbuminuria (MA) and early renal function decline (ERFD, annual decline in estimated glomerular filtration rate of ≥3.3%). The label-free proteomic discovery phase was conducted in 13 patients who progressed to MA by the 6-year visit and 11 control subjects, and four proteins (Tamm-Horsfall glycoprotein, α-1 acid glycoprotein, clusterin, and progranulin) identified in the discovery phase were measured by enzyme-linked immunosorbent assay in 74 subjects: group A, normal renal function (n = 35); group B, ERFD without MA (n = 15); group C, MA without ERFD (n = 16); and group D, both ERFD and MA (n = 8). RESULTS In the label-free analysis, a model of progression to MA was built using 252 peptides, yielding an area under the curve (AUC) of 84.7 ± 5.3%. In the validation study, ordinal logistic regression was used to predict development of ERFD, MA, or both. A panel including Tamm-Horsfall glycoprotein (odds ratio 2.9, 95% CI 1.3–6.2, P = 0.008), progranulin (1.9, 0.8–4.5, P = 0.16), clusterin (0.6, 0.3–1.1, P = 0.09), and α-1 acid glycoprotein (1.6, 0.7–3.7, P = 0.27) improved the AUC from 0.841 to 0.889. CONCLUSIONS A panel of four novel protein biomarkers predicted early renal damage in type 1 diabetes. These findings require further validation in other populations for prediction of renal complications and treatment monitoring.

Human Biomarker Discovery and Predictive Models for Disease Progression for Idiopathic Pneumonia Syndrome Following Allogeneic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (SCT) is the only curative therapy for many malignant and nonmalignant conditions. Idiopathic pneumonia syndrome (IPS) is a frequently fatal complication that limits successful outcomes. Preclinical models suggest that IPS represents an immune mediated attack on the lung involving elements of both the adaptive and the innate immune system. However, the etiology of IPS in humans is less well understood. To explore the disease pathway and uncover potential biomarkers of disease, we performed two separate label-free, proteomics experiments defining the plasma protein profiles of allogeneic SCT patients with IPS. Samples obtained from SCT recipients without complications served as controls. The initial discovery study, intended to explore the disease pathway in humans, identified a set of 81 IPS-associated proteins. These data revealed similarities between the known IPS pathways in mice and the condition in humans, in particular in the acute phase response. In addition, pattern recognition pathways were judged to be significant as a function of development of IPS, and from this pathway we chose the lipopolysaccaharide-binding protein (LBP) protein as a candidate molecular diagnostic for IPS, and verified its increase as a function of disease using an ELISA assay. In a separately designed study, we identified protein-based classifiers that could predict, at day 0 of SCT, patients who: 1) progress to IPS and 2) respond to cytokine neutralization therapy. Using cross-validation strategies, we built highly predictive classifier models of both disease progression and therapeutic response. In sum, data generated in this report confirm previous clinical and experimental findings, provide new insights into the pathophysiology of IPS, identify potential molecular classifiers of the condition, and uncover a set of markers potentially of interest for patient stratification as a basis for individualized therapy.

Urinary Protein Profiles in a Rat Model for Diabetic Complications

Diabetes mellitus is estimated to affect ∼24 million people in the United States and more than 150 million people worldwide. There are numerous end organ complications of diabetes, the onset of which can be delayed by early diagnosis and treatment. Although assays for diabetes are well founded, tests for its complications lack sufficient specificity and sensitivity to adequately guide these treatment options. In our study, we employed a streptozotocin-induced rat model of diabetes to determine changes in urinary protein profiles that occur during the initial response to the attendant hyperglycemia (e.g. the first two months) with the goal of developing a reliable and reproducible method of analyzing multiple urine samples as well as providing clues to early markers of disease progression. After filtration and buffer exchange, urinary proteins were digested with a specific protease, and the relative amounts of several thousand peptides were compared across rat urine samples representing various times after administration of drug or sham control. Extensive data analysis, including imputation of missing values and normalization of all data was followed by ANOVA analysis to discover peptides that were significantly changing as a function of time, treatment and interaction of the two variables. The data demonstrated significant differences in protein abundance in urine before observable pathophysiological changes occur in this animal model and as function of the measured variables. These included decreases in relative abundance of major urinary protein precursor and increases in pro-alpha collagen, the expression of which is known to be regulated by circulating levels of insulin and/or glucose. Peptides from these proteins represent potential biomarkers, which can be used to stage urogenital complications from diabetes. The expression changes of a pro-alpha 1 collagen peptide was also confirmed via selected reaction monitoring.

Activation of tumor suppressor protein PP2A inhibits KRAS-driven tumor growth

Targeted cancer therapies, which act on specific cancer-associated molecular targets, are predominantly inhibitors of oncogenic kinases. While these drugs have achieved some clinical success, the inactivation of kinase signaling via stimulation of endogenous phosphatases has received minimal attention as an alternative targeted approach. Here, we have demonstrated that activation of the tumor suppressor protein phosphatase 2A (PP2A), a negative regulator of multiple oncogenic signaling proteins, is a promising therapeutic approach for the treatment of cancers. Our group previously developed a series of orally bioavailable small molecule activators of PP2A, termed SMAPs. We now report that SMAP treatment inhibited the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models. Mechanistically, we found that SMAPs act by binding to the PP2A A&agr; scaffold subunit to drive conformational changes in PP2A. These results show that PP2A can be activated in cancer cells to inhibit proliferation. Our strategy of reactivating endogenous PP2A may be applicable to the treatment of other diseases and represents an advancement toward the development of small molecule activators of tumor suppressor proteins.

Proteomics of Skin Proteins in Psoriasis: From Discovery and Verification in a Mouse Model to Confirmation in Humans

Herein, we demonstrate the efficacy of an unbiased proteomics screening approach for studying protein expression changes in the KC-Tie2 psoriasis mouse model, identifying multiple protein expression changes in the mouse and validating these changes in human psoriasis. KC-Tie2 mouse skin samples (n = 3) were compared with littermate controls (n = 3) using gel-based fractionation followed by label-free protein expression analysis. 5482 peptides mapping to 1281 proteins were identified and quantitated: 105 proteins exhibited fold-changes ≥2.0 including: stefin A1 (average fold change of 342.4 and an average p = 0.0082; cystatin A, human ortholog); slc25a5 (average fold change of 46.2 and an average p = 0.0318); serpinb3b (average fold change of 35.6 and an average p = 0.0345; serpinB1, human ortholog); and kallikrein related peptidase 6 (average fold change of 4.7 and an average p = 0.2474; KLK6). We independently confirmed mouse gene expression-based increases of selected genes including serpinb3b (17.4-fold, p < 0.0001), KLK6 (9-fold, p = 0.002), stefin A1 (7.3-fold; p < 0.001), and slc25A5 (1.5-fold; p = 0.05) using qRT-PCR on a second cohort of animals (n = 8). Parallel LC/MS/MS analyses on these same samples verified protein-level increases of 1.3-fold (slc25a5; p < 0.05), 29,000-fold (stefinA1; p < 0.01), 322-fold (KLK6; p < 0.0001) between KC-Tie2 and control mice. To underscore the utility and translatability of our combined approach, we analyzed gene and protein expression levels in psoriasis patient skin and primary keratinocytes versus healthy controls. Increases in gene expression for slc25a5 (1.8-fold), cystatin A (3-fold), KLK6 (5.8-fold), and serpinB1 (76-fold; all p < 0.05) were observed between healthy controls and involved lesional psoriasis skin and primary psoriasis keratinocytes. Moreover, slc25a5, cystatin A, KLK6, and serpinB1 protein were all increased in lesional psoriasis skin compared with normal skin. These results highlight the usefulness of preclinical disease models using readily-available mouse skin and demonstrate the utility of proteomic approaches for identifying novel peptides/proteins that are differentially regulated in psoriasis that could serve as sources of auto-antigens or provide novel therapeutic targets for the development of new anti-psoriatic treatments.

Human β-defensin 3 induces STAT1 phosphorylation, tyrosine phosphatase activity, and cytokine synthesis in T cells

The AMP hBD‐3 stimulates numerous immune effector functions in myeloid cells and keratinocytes, predominantly through the MAPK signaling cascade. In contrast, hBD‐3 was reported to neutralize the activation of T cells by antagonizing MAPK signaling initiated by SDF‐1α through CXCR4. With the use of complementary proteomic and immunochemical approaches, we investigated possible stimulatory effects of hBD‐3 on T cells and demonstrate that hBD‐3 induces STAT1 tyrosine phosphorylation within 5 min yet is unable to induce MAPK activation. Inclusion of a PTPase inhibitor increased hBD‐3‐induced phosphorylation dramatically, suggesting that hBD‐3 also stimulates PTPase activity concurrently. The increase in PTPase activity was confirmed by demonstrating that hBD‐3 suppresses IFN‐γ‐induced STAT1 tyrosine phosphorylation but not STAT1 serine and ERK1/2 threonine phosphorylation and stimulates the translocation of SHP‐2 into the nucleus within 15 min. The signaling pathways initiated by hBD‐3 may lead to the observed enhancement of distinct T cell effector functions during TCR activation, such as the increase in IL‐2 and IL‐10, but not IFN‐γ secretion. Thus, hBD‐3 initiates distinct lineage‐specific signaling cascades in various cells involved in host defense and induces a concurrent tyrosine kinase and tyrosine phosphatase signaling cascade that may activate simultaneously the targeted T cells and inhibit their response to other immune mediators. Furthermore, these results suggest that this evolutionarily conserved peptide, which exhibits a broad spectrum of antimicrobial and immunomodulatory activities, serves to integrate innate and adaptive immunity.

Plasma proteome analysis reveals overlapping, yet distinct mechanisms of immune activation in chronic HCV and HIV infections.

Background:HIV infection contributes to accelerated rates of progression of liver fibrosis during hepatitis C virus (HCV) infection, and HCV liver disease contributes to mortality during HIV infection. Although mechanisms underlying these interactions are not well known, soluble and cellular markers of immune activation associate with disease progression during both infections. Methods:We identified proteins varying in expression across the plasma proteomes of subjects with untreated HIV infection, untreated HCV infection with low aspartate transaminase/platelet ratio index, untreated HCV infection with high aspartate transaminase/platelet ratio index, HIV–HCV coinfection, and controls. We examined correlations between dysregulated proteins and markers of immune activation to uncover biomarkers specific to disease states. Results:We observed the anticipated higher frequencies of HLA-DR+CD38+CD4 and CD8 T cells, higher serum soluble CD14 levels, and higher serum interleukin-6 levels for HCV- and HIV-infected groups compared with controls. Plasma proteome analysis identified 2297 peptides mapping to 227 proteins, and quantitative analysis of peptide intensity identified significant changes in 85 proteins across the 5 groups. Abundance for 7 of these proteins was validated by enzyme-linked immunosorbent assay. Forty-three of these proteins correlated with markers of immune activation, including at least 2 proteins that may directly drive T-cell activation. As a functional validation, we tested the enzymatic pathway product (lysophosphatidic acid, LPA) of one such protein, ecotonucleotide pyrophosphatase/phosphodiesterase-2, for ability to activate T cells in vitro. LPA activated T cells to express CD38 and HLA-DR. Conclusions:These data indicate that elevated levels of ecotonucleotide pyrophosphatase/phosphodiesterase-2 and LPA during advanced HCV disease may play a role in exacerbating immune activation during HCV–HIV coinfection.

Proteomic and bioinformatics profile of paired human alveolar macrophages and peripheral blood monocytes

Little is known about proteomic differences between pluripotent human peripheral blood monocytes (MN) and their terminally‐differentiated pulmonary counterparts, alveolar macrophages (AM). To better characterize these cell populations, we performed a label‐free shotgun proteomics assessment of matched AM and MN preparations from eight healthy volunteers. With an FDR of less than 0.45%, we identified 1754 proteins within AM and 1445 from MN. Comparison of the two proteomes revealed that 1239 of the proteins found in AM were shared with MN, whereas 206 proteins were uniquely identified in MN and 515 were unique to AM. Molecular and cellular functions, protein classes, development associations, and membership in physiological systems and canonical pathways were identified among the detected proteins. Analysis of biologic processes represented by these proteomes indicated that MN were most prominently enriched for proteins involved in cellular movement and immune cell trafficking. In contrast, AM were enriched for proteins involved in protein trafficking, molecular transport, and cellular assembly and organization. These findings provide a baseline proteomic resource for further studies aimed at better understanding of the functional differences between MN and AM in both health and disease.

Tissue Specific Dysregulated Protein Subnetworks in Type 2 Diabetic Bladder Urothelium and Detrusor Muscle

Diabetes mellitus is well known to cause bladder dysfunction; however, the molecular mechanisms governing this process and the effects on individual tissue elements within the bladder are poorly understood, particularly in type 2 diabetes. A shotgun proteomics approach was applied to identify proteins differentially expressed between type 2 diabetic (TallyHo) and control (SWR/J) mice in the bladder smooth muscle and urothelium, separately. We were able to identify 1760 nonredundant proteins from the detrusor smooth muscle and 3169 nonredundant proteins from urothelium. Pathway and network analysis of significantly dysregulated proteins was conducted to investigate the molecular processes associated with diabetes. This pinpointed ERK1/2 signaling as a key regulatory node in the diabetes-induced pathophysiology for both tissue types. The detrusor muscle samples showed diabetes-induced increased tissue remodeling-type events such as Actin Cytoskeleton Signaling and Signaling by Rho Family GTPases. The diabetic urothelium samples exhibited oxidative stress responses, as seen in the suppression of protein expression for key players in the NRF2-Mediated Oxidative Stress Response pathway. These results suggest that diabetes induced elevated inflammatory responses, oxidative stress, and tissue remodeling are involved in the development of tissue specific diabetic bladder dysfunctions. Validation of signaling dysregulation as a function of diabetes was performed using Western blotting. These data illustrated changes in ERK1/2 phosphorylation as a function of diabetes, with significant decreases in diabetes-associated phosphorylation in urothelium, but the opposite effect in detrusor muscle. These data highlight the importance of understanding tissue specific effects of disease process in understanding pathophysiology in complex disease and pave the way for future studies to better understand important molecular targets in reversing bladder dysfunction.