Wouter David Bruinzeel

Recombinant expression, purification and dimerization of the neurotrophic growth factor Artemin for in vitro and in vivo use.

Artemin (ARTN) is a neurotrophic growth factor of the GDNF ligand family that signals through the specific GFRα-3 coreceptor/cRet tyrosine kinase-mediated signaling cascade. Its expression and signaling action in adults are restricted to nociceptive sensory neurons in the dorsal root ganglia. Consequently, Artemin supports survival and growth of sensory neurons and has been studied as a possible treatment for neuropathic pain paradigms. In this paper, we describe the development of an efficient method for the recombinant bacterial production of large quantities of highly pure, biologically active ARTN for in vitro and in vivo studies. Using Escherichia coli expression of an NH(2)-terminal SUMO-Artemin fusion protein and subsequent refolding from inclusion bodies followed by cleavage of the SUMO fusion part, mature Artemin with a native NH(2)-terminal amino acid sequence was obtained at high purity (>99%). Experiments using the reducing agent dithiothreitol (DTT) demonstrated that the intermolecular disulphide bridge in the cysteine knot is dispensable for dimerization of stable ARTN monomers. Our production method could facilitate in vitro and in vivo experimentation for the possible development of Artemin as a therapeutic agent for neuropathic pain.

Investigation of signalling cascades induced by neurotrophic synaptolepis factor K7 reveals a critical role for novel PKCε.

This study elucidates signalling cascades involved in the neurotrophic effects induced by an active compound of Synaptolepis kirkii, a plant that is used against snakebites and for treatment of epilepsy. The active compound of this plant, synaptolepis factor K7 (K7), is suggested to exert anti-tumoral and neurotrophic actions via modulation of PKC. In SH-SY5Y cells synthesis of the neuronal marker growth-associated protein 43 was increased upon 48h treatment with K7. Immunofluorescent staining of neurites revealed an increased neurite formation by synaptolepis factor K7. Short-term signal transduction events were followed at the level of extracellular-regulated kinase phosphorylation. Extracellular-regulated kinase (ERK) phosphorylation was transiently increased upon stimulation with synaptolepis factor K7 (300nM) with a maximal effect at 30min. Use of the general PKC inhibitor bisindolylmaleimide I blocked the K7-induced ERK phosphorylation suggesting involvement of PKC. Conversely, inhibition of conventional PKCs, α, β and γ by treatment with Go6976 did not inhibit ERK phosphorylation up to 1μM. Use of a specific-PKCε translocation inhibitor peptide or RNAi-mediated knockdown of PKC-epsilon (ε) abolished the K7-induced ERK phosphorylation implicating PKCε in K7 function. This was confirmed by the observed increase in PKCε translocation and autophosphorylation induced by the compound. These data show that synaptolepis factor K7 induces neuronal differentiation of SH-SY5Y cells concomitant with a transient increase in ERK phosphorylation that is mediated by activation of PKCε.