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To elucidate effective and long-lasting neuroprotective strategies, we analysed a combination of mitochondrial protection and neurotrophic support in two well-defined animal models of neurodegeneration, traumatic lesion of optic nerve and complete 6-hydroxydopamine (6-OHDA) lesion of nigrostriatal pathway. Neuroprotection by BclX(L), Glial cell line-derived neurotrophic factor (GDNF) or BclX(L) plus GDNF co-expression were studied at 2 weeks and at 6-8 weeks after lesions. In both lesion paradigms, the efficacy of this combination approach significantly differed depending on post-lesion time. We show that BclX(L) expression is more important for neuronal survival in the early phase after lesions, whereas GDNF-mediated neuroprotection becomes more prominent in the advanced state of neurodegeneration. BclX(L) expression was not sufficient to finally inhibit degeneration of deafferentiated central nervous system neurons. Long-lasting GDNF-mediated neuroprotection depended on BclX(L) co-expression in the traumatic lesion paradigm, but was independent of BclX(L) in the 6-OHDA lesion model. The results demonstrate that neuroprotection studies in animal models of neurodegenerative diseases should generally be performed over extended periods of time in order to reveal the actual potency of a therapeutic approach.

Functional characterization of disease-related proteins, their splice variants and dominant negative mutants in the context of complex CNS tissues such as brain and retina is frequently assessed by in vivo gene transfer. For correct interpretation of results it is imperative that the protein under investigation is unambiguously detected in the transduced cell types and can be distinguished from any endogenously expressed physiological variants. Therefore the first systematic evaluation of epitope tags used to trace ectopically expressed proteins in the central nervous system is presented here. Substantial differences in the performances of various epitope tag-antibody combinations with respect to sensitivity, specificity and influence of the epitope tag on the fusion protein are elucidated. Epitope tags already established for protein detection in vitro and to some extent in vivo (c-Myc, HA and FLAG tags) were immunohistochemically detected with high sensitivity. However, detection of these tags revealed problems with background staining and we also document structural and functional influence of the tags on the fusion protein. In order to prevent such unwanted side-effects, epitope tags which have not yet been used for in vivo applications (IRS, EE and AU1 tags) were characterized in brain, retina and cultured neurons. While use of the IRS and EE tags was hindered by low sensitivity or specificity, optimal results were obtained with the AU1 epitope, which may develop into a standard tool for detection of ectopic protein expression in the central nervous system.

The Semliki Forest virus (SFV) system has been shown to be highly efficient in transduction of cell lines and primary cells. We employed a novel "noncytotoxic" SFV(PD) vector for transduction of primary ventral midbrain floor cultures in vitro and rat substantia nigra in vivo. Rapid protein expression was noted with preferential transduction of neuronal cells including the dopaminergic subpopulation. To examine the suitability of the SFV vector system for functional gene expression, SFV(PD) vectors encoding for antiapoptotic proteins Bcl-X(L) and XIAP were designed. Despite effective transgene expression, SFV(PD) vectors were unable to rescue dopaminergic neurons from MPP+-induced apoptosis. In vivo, virus injection into substantia nigra resulted in fast onset of transgene expression, but elicited an activation of microglia and an inflammation response. We conclude that the use of novel SFV(PD) vectors is currently limited by persistent neurotoxicity of the vector system. Although SFV(PD) vectors may be useful for protein localization studies in dopaminergic neurons, functional applications will require the development of even less cytopathic vector systems.

RNA interference is rapidly becoming a powerful tool for gene silencing in mammalian cells. Introduction of siRNA into primary cells, however, remains one of the major difficulties of this novel technique. Using cationic lipid-based transfection reagents satisfactory transfection results are observed in cell lines, but low transfection efficiency and cytotoxicity limit applications in primary cells, especially primary neurons. The application of "naked" siRNA has been previously used successfully in nematodes and mammals in vivo. We therefore evaluated the effects of non-cationic-lipid-based siRNA application to primary hippocampal neuron cultures. "Naked" siRNA was bound to the cell surface and was taken up into endosomes. No significant silencing effect of endogenous or reporter genes was observed, rather application of "naked" siRNA was accompanied by a moderate downregulation of metabolic activity in culture. We postulate that endosomal degradation of "naked" siRNA in neurons prevents the induction of significant RNAi-mediated mRNA-downregulation and is accompanied by a global impairment of the cell metabolism. Transfection methods circumventing the endosomal pathway therefore might prove useful for siRNA transduction of primary neurons.

Determination of matrix metalloproteinase-9 (MMP-9) in human cerebrospinal fluid (CSF) to study blood–brain barrier impairment and immune cell migration in inflammatory neurological diseases recently became a matter of major interest. Regularly, MMP-9 was determined qualitatively or semi-quantitatively by zymography (gelatin gel electrophoresis) or quantitatively by enzyme immunoassay (EIA). As yet, it was not possible by either method to detect MMP-9 in CSF of controls (patients without pathologically increased CSF parameters). We developed an ultrasensitive two-side enzyme-linked immunosorbent assay (ELISA) which allows for the first time to measure reliably MMP-9 concentrations in CSF of controls. This ELISA uses a monoclonal as capture and a polyclonal as detector antibody. The detection limit of the assay is below 10 pg/ml and the assay range is 15–2000 pg/ml. Intra-assay precision is 2.5% for low and 3.7% for high, inter-assay precision is 11% for low and 10.7% for high values, respectively. The determination of the MMP-9 concentration in 50 control CSF gave the following results: range, 22–146 pg/ml; median, 76 pg/ml. The measurement of native and recombinant MMP-9 was carried out with three commercially available ELISAs, most widely employed in MMP-9 research, and compared to the newly developed one. All ELISAs recognize recombinant MMP-9 by factors of 5–20 less sensitively than native MMP-9.

Although multiple sclerosis (MS) is considered primarily as a demyelinating disease, neuronal damage is abundant and correlates with the neurological deficit. Therefore, we investigated the frequency and characteristics of human T cells specific for synapsin-a neuronal protein highly conserved among species. Synapsin specific T cell responses were detected at a frequency similar to that of MBP specific T cells in MS patients, one patient with acute demyelinating encephalomyelitis (ADEM) and controls. Long-term T cell lines specific for synapsin exhibited a CD3(+), CD4(+), CD8(-) phenotype and produced high amounts of tumor-necrosis-factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) after antigen specific stimulation, whereas lymphotoxin (LT), interleukin-4 (IL-4) and interleukin-10 (IL-10) were detectable in smaller quantities.

Matrix metalloproteinase-9 (MMP-9) was investigated by enzyme-linked immunosorbent assay (ELISA) and zymography in 111 paired CSF and serum samples from patients with various neurological disorders. In 20 patients with blood-brain barrier (BBB) impairment but normal CSF cell count, elevated levels of MMP-9 were not observed by ELISA measurement. Another 11 patients characterized in the same way, exhibited only slightly increased MMP-9 levels. In contrast, in 12 patients with intact BBB but elevated CSF cell count, MMP-9 was increased too. It was shown by the more sensitive zymography that MMP-9 increased if CSF cell count exceeded five cells per microl. Spearman rank statistics revealed that MMP-9 concentration in CSF correlated with CSF cell count (r=0.755; P

Glatiramer acetate (GA), represents an established treatment of relapsing/remitting multiple sclerosis (MS). The mechanisms responsible for the effect of GA are not fully understood. We generated GA-, myelin basic protein (MBP)- and purified protein derivative (PPD)-specific T cell lines from three MS patients and one healthy donor. The GA-specific lines were CD3+, CD4+, CD8- and produced tumor-necrosis-factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-4 (IL-4), interleukin-6 (IL-6) and interleukin-10 (IL-10) after stimulation with GA in the presence of irradiated peripheral blood mononuclear cells. MBP-specific T cell lines showed an identical phenotype and secreted TNF-alpha, IFN-gamma, IL-4, IL-10, but not IL-6. Co-culture experiments demonstrated, that GA-specific T cell lines have the capability to suppress the proliferation of MBP-specific T cell lines.

Transmigration of human granulocytes across a basal lamina equivalent was studied in vitro. Transwell® inserts were coated with Matrigel®, a reconstituted basement membrane. Granulocytes (2×10 6 ) were applied to the upper chamber. As chemoattractant interleukin-8 (IL-8; 25 ng/ml) was added to the lower chamber. After 1 h of migration, cells were counted in the lower chamber. Specific hydroxamate inhibitors of MMPs (BB-3103, Ro 31-9790) or of serine proteases (Pefabloc®, leupeptin) were added at various concentrations to both chambers before the start of migration. Additional experiments were performed with α 2 -macroglobulin, a natural inhibitor of MMPs and a monoclonal antibody which specifically blocks the activity of MMP-9. Migration of granulocytes through Matrigel could not be reduced significantly by any of the MMP inhibitors. A dose-dependent impairment of transmigration was only found with Pefabloc, however, this substance also induced severe morphological changes of the cells. The other inhibitor of serine proteases, leupeptin, did not influence migration at all.

Adeno-associated- (AAV) based vectors are promising tools for gene therapy applications in several organs, including the brain, but are limited by their small genome size. Two short promoters, the human synapsin 1 gene promoter (hSYN) and the murine cytomegalovirus immediate early promoter (mCMV), were evaluated in bicistronic AAV-2 vectors for their expression profiles in cultured primary brain cells and in the rat brain. Whereas transgene expression from the hSYN promoter was exclusively neuronal, the murine CMV promoter targeted expression mainly to astrocytes in vitro and showed weak transgene expression in vivo in retinal and cortical neurons, but strong expression in thalamic neurons. We propose that neuron specific transgene expression in combination with enhanced transgene capacity will further substantially improve AAV based vector technology.