Daniel Lüscher

Coincident pre- and postsynaptic activation induces dendritic filopodia via neurotrypsin-dependent agrin cleavage.

The synaptic serine protease neurotrypsin is essential for cognitive function, as its deficiency in humans results in severe mental retardation. Recently, we demonstrated the activity-dependent release of neurotrypsin from presynaptic terminals and proteolytical cleavage of agrin at the synapse. Here we show that the activity-dependent formation of dendritic filopodia is abolished in hippocampal neurons from neurotrypsin-deficient mice. Administration of the neurotrypsin-dependent 22 kDa fragment of agrin rescues the filopodial response. Detailed analyses indicated that presynaptic action potential firing is necessary for the release of neurotrypsin, whereas postsynaptic NMDA receptor activation is necessary for the neurotrypsin-dependent cleavage of agrin. This contingency characterizes the neurotrypsin-agrin system as a coincidence detector of pre- and postsynaptic activation. As the resulting dendritic filopodia are thought to represent precursors of synapses, the neurotrypsin-dependent cleavage of agrin at the synapse may be instrumental for a Hebbian organization and remodeling of synaptic circuits in the CNS.

Specific cleavage of agrin by neurotrypsin, a synaptic protease linked to mental retardation

The synaptic serine protease neurotrypsin is thought to be important for adaptive synaptic processes required for cognitive functions, because humans deficient in neurotrypsin suffer from severe mental retardation. In the present study, we describe the biochemical characterization of neurotrypsin and its so far unique substrate agrin. In cell culture experiment as well as in neurotrypsin‐deficient mice, we showed that agrin cleavage depends on neurotrypsin and occurs at two conserved sites. Neurotrypsin and agrin were expressed recombinantly, purified, and assayed in vitro. A catalytic efficiency of 1.3 × 104 M−1 • s−1 was determined. Neurotrypsin activity was shown to depend on calcium with an optimal activity in the pH range of 7–8.5. Mutagenesis analysis of the amino acids flanking the scissile bonds showed that cleavage is highly specific due to the unique substrate recognition pocket of neurotrypsin at the active site. The C‐termi‐nal agrin fragment released after cleavage has recently been identified as an inactivating ligand of the Na+/ K+‐ATPase at CNS synapses, and its binding has been demonstrated to regulate presynaptic excitability. Therefore, dysregulation of agrin processing is a good candidate for a pathogenetic mechanism underlying mental retardation. In turn, these results may also shed light on mechanisms involved in cognitive functions.—Reif, R., Sales, S., Hettwer, S., Dreier, B., Gisler, C., Wolfel, J., Luscher, D., Zurlinden, A., Stephan, A., Ahmed, S., Baici, A., Ledermann, B., Kunz, B., Sonderegger, P. Specific cleavage of agrin by neurotrypsin, a synaptic protease linked to mental retardation. FASEB J. 21, 3468–3478 (2007)

Specific proteolytic cleavage of agrin regulates maturation of the neuromuscular junction

During the initial stage of neuromuscular junction (NMJ) formation, nerve-derived agrin cooperates with muscle-autonomous mechanisms in the organization and stabilization of a plaque-like postsynaptic specialization at the site of nerve–muscle contact. Subsequent NMJ maturation to the characteristic pretzel-like appearance requires extensive structural reorganization. We found that the progress of plaque-to-pretzel maturation is regulated by agrin. Excessive cleavage of agrin via transgenic overexpression of an agrin-cleaving protease, neurotrypsin, in motoneurons resulted in excessive reorganizational activity of the NMJs, leading to rapid dispersal of the synaptic specialization. By contrast, expression of cleavage-resistant agrin in motoneurons slowed down NMJ remodeling and delayed NMJ maturation. Neurotrypsin, which is the sole agrin-cleaving protease in the CNS, was excluded as the physiological agrin-cleaving protease at the NMJ, because NMJ maturation was normal in neurotrypsin-deficient mice. Together, our analyses characterize agrin cleavage at its proteolytic α- and β-sites by an as-yet-unspecified protease as a regulatory access for relieving the agrin-dependent constraint on endplate reorganization during NMJ maturation.

Purification and enzymological characterization of murine neurotrypsin

An increasing number of studies indicate that serine proteases play an important role in structural plasticity associated with learning and memory formation. Neurotrypsin is a multidomain serine protease located at the presynaptic terminal of neurons. It is thought to be crucial for cognitive brain functions. A deletion in the neurotrypsin gene causes severe mental retardation in humans. For a biochemical characterization, we produced murine neurotrypsin recombinantly in a eukaryotic expression system using myeloma cells. From the culture medium we purified neurotrypsin using heparin-, hydrophobic interaction- and immobilized metal affinity chromatography. For an enzymological characterization two fragments of agrin containing the natural cleavages sites of neurotrypsin were used as substrates. The highest catalytic activity of neurotrypsin was observed in the pH range between 7.0 and 8.5. Calcium ions were required for neurotrypsin activity and an ionic strength exceeding 500 mM decreased substrate cleavage. Site-specific mutations of the amino acids flanking the scissile bonds showed that cleavage is highly specific and requires a basic amino acid preceded by a glutamate residue on the N-terminal side of the scissile bond. This sequence requirement argues for a unique substrate binding pocket of neurotrypsin. This observation was further substantiated by the fact that almost all tested serine protease inhibitors except dichloroisocoumarin and PMSF did not affect neurotrypsin activity.

Effects of hypericin and a chlorin based photosensitizer alone or in combination in squamous cell carcinoma cells in the dark

INTRODUCTION The toxic influence of photosensitizers in the dark is poorly investigated. In our study we used the photosensitizers liposomal meso-tetrahydroxyphenyl chlorin derivative (Foslipos(®)) and hypericin as well as their 1:1 combination on two different head and neck squamous cell carcinoma (HNSCC) cell lines (UMB-SCC 745 and UMB-SCC 969). MATERIALS AND METHODS We examined uptake, efflux and localization of the photosensitizers with confocal microscopy. Fluorescence quantification was measured with a micro-plate spectrometer. Special interest was given to effects on cell proliferation (BrdU proliferation assay), RNA quality (Bioanalyzer measurements) and DNA damage (comet assays) in the dark. RESULTS Foslipos(®) uptake was linear over time and its efflux was not achieved even after 24 h while uptake of hypericin reached a plateau after 5 h and was almost eliminated after 24 h. Localization of Foslipos(®) was organelle-unspecific. Hypericin was found mainly at membranes and in trans-golgi network. Foslipos(®) treated cells showed cell toxicity for the highest concentration (10 μg/mL). In contrast, hypericin was toxic for all concentrations (10-0.6 μg/mL). The photosensitizer combination was non-toxic for all concentrations (10-0.6 μg/mL). No changes in RNA quality were monitored. Initial DNA damage was found only in hypericin treated UMB-SCC 745, which recovered after 3h. No significant DNA damage was found for UMB-SCC 969. CONCLUSION Our data shows that the combinatorial application decrease photosensitizer toxicity, which can be advantageous in PDT treatments.

Photodynamic mechanisms induced by a combination of hypericin and a chlorin based-photosensitizer in head and neck squamous cell carcinoma cells.

The aim of this study was to elucidate photodynamic therapy (PDT) effects mediated by hypericin and a liposomal meso‐tetrahydroxyphenyl chlorin (mTHPC) derivative, with focus on their 1:1 mixture, on head and neck squamous cell carcinoma cell lines. Absorption, excitation and photobleaching were monitored using fluorescence spectrometry, showing the same spectral patterns for the mixture as measured for single photosensitizers. In the mixture mTHPC showed a prolonged photo‐stability. Singlet oxygen yield for light‐activated mTHPC was ΦΔ = 0.66, for hypericin ΦΔ = 0.25 and for the mixture ΦΔ = ~0.4. A linear increase of singlet oxygen yield for mTHPC and the mixture was found, whereas hypericin achieved saturation after 35 min. Reactive oxygen species fluorescence was only visible after hypericin and mixture‐induced PDT. Cell viability was also more affected with these two treatment options under the selected conditions. Examination of death pathways showed that hypericin‐mediated cell death was apoptotic, with mTHPC necrotic and the 1:1 mixture showed features of both. Changes in gene expression after PDT indicated strong up‐regulation of selected heat‐shock proteins. The application of photosensitizer mixtures with the features of reduced dark toxicity and combined apoptotic and necrotic cell death may be beneficial in clinical PDT. This will be the focus of our future investigations.

Zymogen activation of neurotrypsin and neurotrypsin-dependent agrin cleavage on the cell surface are enhanced by glycosaminoglycans

The serine peptidase neurotrypsin is stored in presynaptic nerve endings and secreted in an inactive zymogenic form by synaptic activity. After activation, which requires activity of postsynaptic NMDA (N-methyl-D-aspartate) receptors, neurotrypsin cleaves the heparan sulfate proteoglycan agrin at active synapses. The resulting C-terminal 22-kDa fragment of agrin induces dendritic filopodia, which are considered to be precursors of new synapses. In the present study, we investigated the role of GAGs (glycosaminoglycans) in the activation of neurotrypsin and neurotrypsin-dependent agrin cleavage. We found binding of neurotrypsin to the GAG side chains of agrin, which in turn enhanced the activation of neurotrypsin by proprotein convertases and resulted in enhanced agrin cleavage. A similar enhancement of neurotrypsin binding to agrin, neurotrypsin activation and agrin cleavage was induced by the four-amino-acid insert at the y splice site of agrin, which is crucial for the formation of a heparin-binding site. Non-agrin GAGs also contributed to binding and activation of neurotrypsin and, thereby, to agrin cleavage, albeit to a lesser extent. Binding of neurotrypsin to cell-surface glycans locally restricts its conversion from zymogen into active peptidase. This provides the molecular foundation for the local action of neurotrypsin at or in the vicinity of its site of synaptic secretion. By its local action at synapses with correlated pre- and post-synaptic activity, the neurotrypsin-agrin system fulfils the requirements for a mechanism serving experience-dependent modification of activated synapses, which is essential for adaptive structural reorganizations of neuronal circuits in the developing and/or adult brain.