Theme 5 Human cell biology and pathology.

Background: The delay from onset of the first symptoms to a definite ALS diagnosis depends also on the elusiveness of the initial clinical manifestations. The lack of disease-specific biomarkers to detect early pathology when ALS is supposed complicates the situation. This latency reduces the therapeutic time frame, in which neuron-rescuing strategies exert their greatest chance to work. Various biomarkers are currently promised, but none of them are specific enough to allow monitoring of disease progression. This, as well as the heterogeneity of the disease concerning clinical onset pattern and survival rates, makes difficult the correct stratification of patients into clinical trials, masking the potential positive outcome in some patients.Objective: Our main objective is to establish and test an early diagnostic tool based on microscopic immune cell monitoring of ALS patients' blood samples by using the Toponome Imaging System (TIS).Methods: TIS is based on automatically controlled microscopic device involving conjugated dye-tag incubation, protein-tag-dye-imaging, and tag-dye bleaching (1). This leads to the collection of at least 21 cycle images of fixated peripheral blood mononuclear cells (PBMCs) isolated from freshly drawn blood of ALS patients and healthy "control" donors. Resulting data sets contain combinatorial molecular information about the spatial protein network, called toponome. The PBMC toponome architectures are quantitatively analyzed as a threshold-binary code with 1 = protein is present and 0 = protein is absent.Results: Preliminary screening data of PBMCs from 4 ALS patients reveal a subpopulation of lymphocytes expressing a specific surface protein pattern, called "ALS toponome". These aberrant T cells could not be found in blood samples of controls. We observe that the number of these cells correlate with the ALS progression rate of patients, supporting the conclusion that these cells may be causal for the disease.Discussion and conclusion: Although these findings open up a potential strategy to detect early ALS disease and to monitor disease progression, a statistical analysis with many more patients, as well as data based differentiation to other neurodegenerative diseases, is mandatory. A clinical trial initiated by our faceALS foundation with at least 60 patients classified in three subsets (1. control, 2. ALS, and 3. Multiple Sclerosis (MS)) and in close cooperation with leading ALS centres in Germany is still in progress. The detection of specific and/or aberrant immune cells in blood samples of ALS patients may provide a key to understand disease onset and progression, could be used for the "staging" of disease, and contribute to effective therapy options.

Large molecular systems landscape uncovers T cell trapping in human skin cancer.

Immune surveillance of tumour cells is an important function of CD8 T lymphocytes, which has failed in cancer for reasons still unknown in many respect but mainly related to cellular processes in the tumour microenvironment. Applying imaging cycler microscopy to analyse the immune contexture in a human skin cancer we could identify and map 7,000 distinct cell surface-associated multi-protein assemblies. The resulting combinatorial geometry-based high-functional resolution led to discovery of a mechanism of T cell trapping in the epidermis, which involves SPIKE, a network of suprabasal keratinocyte projections piercing and interconnecting CD8 T cells. It appears initiated by clusters of infrabasal T and dendritic cells connected via cell projections across a fractured basal lamina to suprabasal keratinocytes and T lymphocytes.

Next-generation biomarkers based on 100-parameter functional super-resolution microscopy TIS.

Functional super-resolution (fSR) microscopy is based on the automated toponome imaging system (TIS). fSR-TIS provides insight into the myriad of different cellular functionalities by direct imaging of large subcellular protein networks in morphologically intact cells and tissues, referred to as the toponome. By cyclical fluorescence imaging of at least 100 molecular cell components, fSR-TIS overcomes the spectral limitations of fluorescence microscopy, which is the essential condition for the detection of protein network structures in situ/in vivo. The resulting data sets precisely discriminate between cell types, subcellular structures, cell states and diseases (fSR). With up to 16 bits per protein, the power of combinatorial molecular discrimination (PCMD) is at least 2(100) per subcellular data point. It provides the dimensionality necessary to uncover thousands of distinct protein clusters including their subcellular hierarchies controlling protein network topology and function in the one cell or tissue section. Here we review the technology and findings showing that functional protein networks of the cell surface in different cancers encompass the same hierarchical and spatial coding principle, but express cancer-specific toponome codes within that scheme (referred to as TIS codes). Findings suggest that TIS codes, extracted from large-scale toponome data, have the potential to be next-generation biomarkers because of their cell type and disease specificity. This is functionally substantiated by the observation that blocking toponome-specific lead proteins results in disassembly of molecular networks and loss of function.

Toponome mapping in prostate cancer: detection of 2000 cell surface protein clusters in a single tissue section and cell type specific annotation by using a three symbol code.

The toponome imaging technology MELC/TIS was applied to analyze prostate cancer tissue. By cyclical imaging procedures, we detected 2100 cell surface protein clusters in a single tissue section. This study provides the whole data set, a new kind of high dimensional data space, solely based on the structure-bound architecture of an in situ protein network, a putative fraction of the tissue code of prostate cancer. It is visualized as a colored mosaic composed of distinct protein clusters, together forming a motif expressed exclusively on the cell surface of neoplastic cells in prostate acini. Cell type specific expression of this motif, found in this preliminary study, suggests that high-throughput toponome analyses of a larger number of cases will provide insight into disease specific protein networks.

Inhibitory modulation of the mitochondrial permeability transition by minocycline.

The semi-synthetic tetracycline derivative minocycline exerts neuroprotective properties in various animal models of neurodegenerative disorders. Although anti-inflammatory and anti-apoptotic effects are reported to contribute to the neuroprotective action, the exact molecular mechanisms underlying the beneficial properties of minocycline remain to be clarified. We analyzed the effects of minocycline in a cell culture model of neuronal damage and in single-channel measurements on isolated mitoplasts. Treatment of neuron-enriched cortical cultures with rotenone, a high affinity inhibitor of the mitochondrial complex I, resulted in a deregulation of the intracellular Ca2+-dynamics, as recorded by live cell imaging. Minocycline (100 microM) and cyclosporin A (2 microM), a known inhibitor of the mitochondrial permeability transition pore, decreased the rotenone-induced Ca2+-deregulation by 60.9% and 37.6%, respectively. Investigations of the mitochondrial permeability transition pore by patch-clamp techniques revealed for the first time a dose-dependent reduction of the open probability by minocycline (IC(50)=190 nM). Additionally, we provide evidence for the high antioxidant potential of MC in our model. In conclusion, the present data substantiate the beneficial properties of minocycline as promising neuroprotectant by its inhibitory activity on the mitochondrial permeability transition pore.

Relationship between GABAergic interneurons migration and early neocortical network activity.

Available evidence converges to suggest that during the early development of the cerebral cortex, the emergence of the spontaneous network activity chronologically overlap with the end of the cell migration period in the developing cortex. We approached the functional regulation of neuronal migration in a culture model of neocortical networks, using time lapses to detect migratory movements, calcium-imaging to assess the activity of migratory neurons, and immunocytochemical methods to identify the migratory cells retrospectively. In cell cultures, early physiological development and cell migration are reproduced at a local network level, thus allowing the study of the interrelationships between cell migration and network development independent of the topographical complexity. Neurons migrate at least until 12 days in vitro and GABAergic neurons migrate faster compared with non-GABAergic neurons. A decline of migratory activity was coincident with the development of spontaneous synchronous network activity. Migrating interneurons did not participate in synchronous network activity, but interneurons that ended cell migration during observation time frequently engaged in synchronous activity within less than an hour. Application of GABA(A) and ionotropic glutamate receptor antagonists significantly increased the number of migrating GABAergic neurons without changing the dynamics of the migratory movements. Thus, neurotransmitters released by early network activity might favor the termination of neuronal migration. These results reinforce the idea that network activity plays an important role in the development of late-born GABAergic cells.

Minocycline, a possible neuroprotective agent in Leber's hereditary optic neuropathy (LHON): studies of cybrid cells bearing 11,778 mutation.

Leber's hereditary optic neuropathy (LHON) is a retinal neurodegenerative disorder caused by mitochondrial DNA point mutations. Complex I of the respiratory chain affected by the mutation results in a decrease in ATP and an increase of reactive oxygen species production. Evaluating the efficacy of minocycline in LHON, the drug increased the survival of cybrid cells in contrast to the parental cells after thapsigargin-induced calcium overload. Similar protection was observed by treatment with cyclosporine A, a blocker of the mitochondrial permeability transition pore (mPTP). Ratiometric Ca(2+) imaging reveals that acetylcholine/thapsigargin triggered elevation of the cytosolic calcium concentration is alleviated by minocycline and cyclosporine A. The mitochondrial membrane potential of LHON cybrids was significantly conserved and the active-caspase-3/procaspase-3 ratio was decreased in both treatments. Our observations show that minocycline inhibits permeability transition induced by thapsigargin in addition to its antioxidant effects. In relation with its high safety profile, these results would suggest minocycline as a promising neuroprotective agent in LHON.