SUMMARYIn contrast to the situation in mammals and birds, neurons in the central nervous system (CNS) of fish-such as the retinal ganglion cells-are capable of regenerating their axons and restoring vision. Special properties of the glial cells and the neurons of the fish visual pathway appear to contribute to the success of axonal regeneration. The fish oligodendrocytes lack the axon growth inhibiting molecules that interfere with axonal extension in mammals. Instead, fish optic nerve oligodendrocytes support-at least in vitro-axonal elongation of fish as well as that of rat retinal axons. Moreover, the fish retinal ganglion cells re-express upon injury a set of growthassociated cell surface molecules and equip the regenerating axons throughout their path and up into their target, the tectum opticum with these molecules. This may indicate that the injured fish ganglion cells reactivate the cellular machinery necessary for axonal regrowth and pathfinding. Furthermore, the target itself provides positional marker molecules even in adult fish. These marker molecules are required to guide the regenerating axons back to their retinotopic home territory within the tCCtUm.

Disease progression in amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motoneurons (MN) and their axons, but is also influenced by neighboring cells such as astrocytes and microglial cells. The role of microglia in ALS is complex as it switches from an anti-inflammatory and neuroprotective phenotype in early disease to a proinflammatory and neurotoxic phenotype in later stages. Our previous studies in models of neurodegeneration identified rho kinase (ROCK) as a target, which can be manipulated to beneficially influence disease progression. Here, we examined the neuroprotective potential of the ROCK inhibitor Fasudil to target the central pathogenic features of ALS. Application of Fasudil to kainic acid-lesioned primary MN in vitro resulted in a strong prosurvival effect. In vivo, SOD1(G93A) mice benefited from oral treatment with Fasudil showing prolonged survival and improved motor function. These findings were correlated to an improved survival of motor neurons and a pronounced alteration of astroglial and microglial cell infiltration of the spinal cord under Fasudil treatment. Modeling a proinflammatory microglial phenotype by stimulation with LPS in vitro, Fasudil decreased the release of proinflammatory cytokines and chemokines TNFα, Il6, CCL2, CCL3, and CCL5 while CXCL1 release was only transiently suppressed. In sciatic nerve motor axons, neuromuscular junction remodeling processes were increased. In conclusion, we provide preclinical and neurobiological evidence that inhibition of ROCK by the clinically approved small molecule inhibitor Fasudil may be a novel therapeutic approach in ALS combining both neuroprotection and immunomodulation for the cure of this devastating disease.

BackgroundIn multiple sclerosis relapses refractory to intravenous corticosteroid therapy, plasma exchange is recommended. Immunoadsorption (IA) is regarded as an alternative therapy, but its efficacy and putative mechanism of action still needs to be established.MethodsWe prospectively treated 11 patients with multiple sclerosis who had optical neuritis and fulfilled the indications for apheresis therapy (Trial registration DE/CA25/00007080-00). In total, five IA treatments were performed using tryptophan-IA. Clinical activity (visual acuity, Expanded Disability Status Scale, Incapacity Status Scale), laboratory values and visual evoked potentials were measured before, during and after IA, with a follow-up of six months. Moreover, proteomic analyses were performed to analyze column-bound proteins as well as corresponding changes in patients’ sera.ResultsAfter the third IA, we detected an improvement of vision in eight of eleven patients, whom we termed responders. Amongst these, the mean visual acuity improved from 0.15 ± 0.12 at baseline to 0.47 ± 0.32 after the third IA (P = 0.0252) up to 0.89 ± 0.15 (P < 0.0001) at day 180 ± 10 after IA. Soluble interleukin-2 receptor decreased in responders (P = 0.03), whereas in non-responders it did not. Proteomic analyses of proteins adsorbed to IA columns revealed that several significant immunological proteins as well as central nervous system protein fragments, including myelin basic protein, had been removed by IA.ConclusionsIA was effective in the treatment of corticosteroid-refractory optic neuritis. IA influenced the humoral immune response. Strikingly, however, we found strong evidence that demyelination products and immunological mediators were also cleared from plasma by IA.

No abstract

Proteomics and cellomics clearly benefit from the molecular insights in cellular biochemical events that can be obtained by advanced quantitative microscopy techniques like fluorescence lifetime imaging microscopy and Fö rster resonance energy transfer imaging. The spectroscopic information detected at the molecular level can be combined with cellular morphological estimators, the analysis of cellular localization, and the identification of molecular or cellular subpopulations. This allows the creation of powerful assays to gain a detailed understanding of the molecular mechanisms underlying spatiotemporal cellular responses to chemical and physical stimuli. This work demonstrates that the high content offered by these techniques can be combined with the high throughput levels offered by automation of a fluorescence lifetime imaging microscope setup capable of unsupervised operation and image analysis. Systems and software dedicated to image cytometry for analysis and sorting represent important emerging tools for the field of proteomics, interactomics, and cellomics. These techniques could soon become readily available both to academia and the drug screening community by the application of new allsolid-state technologies that may results in cost-effective turnkey systems. Here the application of this screening technique to the investigation of intracellular ubiquitination levels of ␣-synuclein and its familial mutations that are causative for Parkinson disease is shown. The finding of statistically lower ubiquitination of the mutant ␣-synuclein forms supports a role for this modification in the mechanism of pathological protein aggregation. Molecular & Cellular Proteomics 6:1446 -1454, 2007.Above and beyond the isolation and identification of proteins, the field of proteomics faces the challenges of detecting protein cellular localization and quantifying molecular states such as protein conformations, protein-protein interactions, and post-translational modifications. In the past decade, Fö rster resonance energy transfer (FRET) 1 and fluorescence lifetime imaging microscopy (FLIM) have proven to be instrumental for the quantitative imaging of these biochemical states in single cells (1). Similarly the analysis of different cellular populations (cellomes) will also benefit from these imaging methods. Quantitative multiparametric microscopy is a very young field in which advances in liquid/sample handling robotics and information technology are gradually being integrated into automated microscopes (2, 3). These automated imaging systems merge the high content image information with the high throughput volumes provided by their automation and unsupervised operation.Screening techniques have now reached (ultra)high throughput levels, i.e. they are capable of performing more than 10 5 assays/day in microliter volumes. Such a high throughput is necessary for applications where (bio)chemical libraries are tested, e.g. for drug discovery and interactomics research (4). Although the advance in throughput scale is neces...

Background: To clarify whether circulating matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) can be used as serum markers of fibroproliferation in chronic liver diseases, we studied the expression of MMP-2 and MMP-9 in relation to TIMP-1 and TIMP-2 in peripheral blood mononuclear leukocytes (MNLs) and polymorphonuclear leukocytes (PMLs), and compared this expression to circulating concentrations and hepatic histology in patients with chronic active hepatitis C (CAH). Methods: Quantitative reverse transcription-PCR/ELISA assays were performed for MMP and TIMP RNA, and corresponding circulating protein concentrations were studied by ELISA in 20 healthy controls, 40 patients with CAH, and 20 patients with hepatitis C-induced cirrhosis (Ci). Results: MMP-2 mRNA was found almost exclusively in the liver, MMP-9 mRNA in leukocytes. TIMP RNA-equivalents were decreased in MNLs of CAH patients, but neither MMP-9 nor TIMP RNA expression showed any correlation to the extent of inflammation and fibrosis. MMP-2 and TIMP-1 protein concentrations were increased in Ci patients and showed a wide overlap in CAH patients and healthy controls. MMP-9 values were lower in CAH and Ci patients than in healthy controls. TIMP-2 values showed a wide overlap in all three groups. The MMP-2/TIMP-1 and MMP-9/TIMP-1 ratios were lower in Ci patients than in healthy controls; the MMP-2/TIMP-2 and MMP-9/TIMP-2 ratios were not different. Circulating TIMP-1 and the MMP-2/TIMP-1 ratio correlated to the inflammatory activity in liver biopsies, but only the circulating MMP-2/TIMP-1 ratio also correlated with the degree of fibrosis. Conclusions: Peripheral blood cell expression of MMP-2, MMP-9, and TIMP revealed no correlation with the circulating concentrations of these proteins. Only the circulating MMP-2/TIMP-1 ratio correlated to the histological degree of fibrosis in hepatitis C and should be further evaluated as a progression marker in patients with chronic liver disease.

Intracerebral haemorrhage still causes considerable disability and mortality. The studies on conservative and operative management are inconclusive, probably due to inexact volumetry of the haemorrhage. We investigated whether three-dimensional (3-D), voxel-based volumetry of the haemorrhage and its mass effect is feasible with routine computed tomography (CT) scans. The volumes of the haemorrhage, ventricles, midline shift, the intracranial volume and ventricular compression in CT scans of 12 patients with basal ganglia haemorrhage were determined with the 3-D slicer software. Indices of haemorrhage and intracranial or ventricular volume were calculated and correlated with the clinical data. The intended measures could be determined with an acceptable intra-individual variability. The 3-D volumetric data tended to correlate better with the clinical course than the conventionally assessed distance of midline shift and volume of haemorrhage. 3-D volumetry of intracranial haemorrhage and its mass effect is feasible with routine CT examination. Prospective studies should assess its value for clinical studies on intracranial space-occupying diseases.

Magnetic resonance spectroscopy (MRS) provides the unique ability to monitor several disease-related pathological processes via their characteristic metabolic markers in vivo. In the present study metabolic compositions were assessed every six months over the period of two years in 36 patients with Multiple Sclerosis (MS) including 21 relapsing-remitting (RR), 15 secondary progressive (SP) patients and 12 normal subjects. The concentrations of the main MRS-detectable metabolites N-acetylaspartate and N-acetylaspartylglutamate (tNAA), creatine and phosphocreatine (tCr), choline containing compounds (Cho), myo-Inositol (Ins), glutamine and glutamate (Glx) and their ratios were calculated in the normal appearing white matter (NAWM) and in selected non-enhancing white matter (WM) lesions. Association between metabolic concentrations in the NAWM and disability were investigated. Concentration of tNAA, a marker for neuroaxonal integrity, did not show any difference between the investigated groups. However, the patients with SPMS showed significant reduction of tNAA in the NAWM over the investigation period of two years indicating diffuse neuroaxonal loss during the disease course. Furthermore, we found a significant increase of Ins, Ins/tCr and Ins/tNAA in WM lesions independently from the course of the disease suggesting ongoing astrogliosis in silent-appearing WM lesions. Analyzing correlations between MRS metabolites in the NAWM and patients clinical status we found the positive correlation of Ins/tNAA with disability in patients with RRMS. In SPMS positive correlation of Cho with disability was found.