Uroš Logonder

Department of Biochemistry and Molecular Biology
Jozef Stefan Institute
Jamova 39,
SI-1000 Ljubljana
Slovenia
tel.: +386 1 477 35 44
fax: +386 1 477 39 84

email: uros.logonder{afna}ijs.si

Education:
Currently finishing PhD thesis: "Interaction of a neurotoxic phospholipase A₂ with presynaptic membrane" (Interakcija nevrotoksične fosfolipaze A₂ s presinaptično membrano).

B. Sc. (2003), University of Ljubljana, Faculty of Chemistry and Chemical Technology, Ljubljana, Slovenia.
Diploma thesis: "Subcellular localisation and an attempt to isolate the 25 kDa ammodytoxin receptor from the porcine cerebral cortex" (Subcelularna lokalizacija in poskus izolacije amoditoksinskega receptorja z maso 25 kDa v prašičji možganski skorji).

Abstract from the PhD thesis:
Snake toxins are a rich source of secreted phospholipases A₂ (sPLA₂), amongst which some are presynaptically neurotoxic (β-neurotoxic). Ammodytoxin (Atx) is β-neurotoxic sPLA₂ from a venom of the most dangerous of European vipers, the long-nosed viper (Vipera ammodytes ammodytes). As the principal toxin of this snake’s crude venom, it is primarily responsible for the neurotoxic signs and symptoms seen in many victims of snake-bite. Its mode of action is expressed as a disruption of neuromuscular transmission at the motor end-plates.

Atx acts presynaptically at the neuromuscular (NM) junctions and is probably internalised into nerve terminals of motoneurons. It seems that internalisation occurs via vesicular structures where a binding to M-type sPLA₂ receptor (R180) is not likely to be involved, although it is present on the presynaptic membrane of motoneurons. β-neurotoxic action of Atx is most probably a consequence of its intracellular actions. Neurosecretion can possibly be affected by the binding of Atx to calmodulin in the cytosol of the nerve terminal. Both proteins were shown to interact with each other under similar conditions. Besides R180 there must be additional unknown receptors for Atx on the presynaptic membrane that are capable of concentrating Atx in the presynaptic region. Therefore, they are of critical importance for the expression of toxin's β-neurotoxicity.

Atx causes a depletion of synaptic vesicles (SVs) from motor nerve terminals. However, the reduced number of SVs near active zones is likely not to be of crucial importance to achieve the complete blockade of indirectly elicited twitch responses of muscle. The absence of Ω-shaped profiles from the terminal boutons suggests that the recycling of SVs is not blocked at the stage of retrieval of SVs from axolemma by endocytosis. We conclude that Atx induces loss of coupling between action potential-induced depolarisation of the nerve terminal and evoked transmitter release. Endocytosis could be blocked at later stages as a secondary event of Atx-induced neuromuscular paralysis. Atx causes no disaggregation of axonal neurofilaments. On the other hand, nerve terminal mitochondria are swollen and damaged. Therefore, the reason for neurotoxic action of Atx could be a fall in ATP levels and de-energisation of the terminal. Last but not least, Atx could only have an influence over the activity-dependent pool of SVs. It does not affect the spontaneously recycling vesicles that are responsible for miniature end-plate potentials (MEPPs) as neither frequency nor amplitude of the MEPPs are changed after Atx action on the NM preparation.

Abstract from the diploma thesis:
Phospholipases A2 (PLA2s) are enzymes that catalyze hydrolysis of the sn -2 ester bond of glycerophospholipids. Secreted PLA2s are present in many mammalian tissues and excretions as well as in the venoms of different organisms, especially snakes. Besides their enzymatic activity, they also exhibit a wide variety of pharmacological effects, including neurotoxicity, miotoxicity and anticoagulant activity. Secreted PLA2s have to bind to high-affinity membrane receptors to exert some of these effects, including an irreversible blockade of acetylcholine release at the neuromuscular junction. Ammodytoxins (Atxs) are presynaptically neurotoxic group IIA PLA2s from the venom of the long-nosed viper ( Vipera ammodytes ammodytes ). In the porcine brain cortex, two receptors for Atxs were discovered, named R25 and R180, with molecular masses 25 and 180 kDa respectively. While R180 is located on the plasma membrane, R25 is believed to be an intracellular membrane-bound receptor.
In this work, we performed a detailed subcellular localization of R25 in the porcine brain cortex, thereby employing the following methods: subfractionation of the porcine brain cortex homogenate on discontinuous sucrose-density gradients, determination of specific enzyme activities in each of the subfractions and detection of AtxC-binding proteins in subfractions by silver staining and affinity labeling with 125I-AtxC. Based on the high correlation between the distribution of the mitochondrial marker and R25 in subfractions, we confirm that R25 is most likely localized in mitochondria.
In addition, we attempted to isolate R25 in a homogenous form. We prepared AtxC-affinity column using Sepharose 4B gel. On the affinity column, we succeeded in specifically retaining a protein with the molecular mass of approximately 25 kDa from a detergent extract of the R25-richest subfraction (P2d). Low capacity of the affinity column and inactivation of the receptor during the procedure unabled the molecular identification of the protein. Using AtxC-affinity chromatography, we also analyzed the extracts of the remaining ten membrane subfractions, and determined a subcellular distribution of the already known Atx-binding proteins: R180, Grp78, PDI and 14-3-3 (gamma and epsilon isoforms). In the P2 fraction, we detected a potentially new acceptor for AtxC with a molecular mass of about 21 kDa.

Research Interests:
Uroš is currently working on Atx-binding receptors from Torpedo marmorata electric organ.
At the moment, he is involved in studies of subcellular localization of gold-labelled AtxA mutant in vivo (mice).

Awards and Fellowships:
Zois scholarship for exceptionally gifted students (1994-2003)
The Preseren prize for students of the Faculty of Chemistry and Chemical Technology, University of Ljubljana (2003)
Member of the Slovenian Biochemical Society (since 2004)

Bibliography: COBISS or PubMed