Changes in neuronal properties induced by neurotropic infections

Abstract: The nervous system can be the target for various bacterial and viral infectious agents. Certain bacterial toxins have been found to impair specific proteins, such as proteins involved in transmitter release and in the regulation of the cytoskeleton. Viral infections can also cause specific disturbances in neuronal function, but less is known about their actions on the cellular level. The aim of the present study was to obtain further knowledge about the cellular actions of two types of neurotropic agents, clostridial toxins and enveloped RNA viruses, on central nervous system (CNS) neurons. As an experimental model, hippocampal neurons in primary culture were used. The neurons were subjected to toxin treatment or viral infection, and changes in cellular protein content were monitored by immunolabelling. The actions on synaptic transmission, or on ionic currents, were eximined by patch clamp whole-cell recordings, and relative changes of intracellular Ca2+ concentration with Ca2+ imaging. Certain presynaptic and viral proteins were overexpressed using the Semliki forest virus (SFV) vector. The following conclusions can be drawn from the present study: 1. Incubation of cultured neurons with tetanus toxin (TeTx), which cleaves the SNARE protein synaptobrevin, was found to block synaptic transmission completely. Botulinum neurotoxin A (BoNT/A) which cleaves the C- terminal of SNAP-25, on the other hand, caused only a partial inhibition of the synaptic response even at high doses. This type of synaptic block could be overcome by high frequency stimulation. When full-length SNAP-25 was overexpressed using the SFV vector, synaptic transmission was inhibited in a similar manner as after TeTx treatment, i.e. the inhibition could not be overcome by repetitive stimulation. Thus, it seems that the C-terminal of SNAP-25 plays a specific role in setting the level of transmitter release. 2. Incubation with large Clostridial Cytotoxins (LCTs), which inactivate GTPases of the Ras (Rap, Rai, R- Ras, Ras) and Rho families (Rho, Rac, Cdc42), inhibited synaptic transmission and modified the activity- dependent modulation. To examine the possible involvement of Rai, a dominant negative mutant of this GTPase, was overexpressed using the SFV vector. The Ral-overexpressing neurons exhibited an abnormal activitydependent facilitation of the synaptic response. These results provide evidence for an involvement of Ras- related non-Rab GTPases, including Rai, in presynaptic regulation. 3. Infections of cultures with mumps virus (RW) or a neuroadapted strain of influenza A virus (WSN/33), were found to affect the neuronal Ca2+ homeostasis. Mumps virus reduced voltage-dependent Ca2+ currents to the same degree in infected and non-infected neurons, probably due to a disturbed interaction between glial cells and neurons. Influenza A virus reduced Ca2+ currents in infected neurons at an early time-point, presumably due to a direct effect on Ca2+ channels. Later during infection Ca2+ Currents were also reduced in non- infected neurons. At this late time-point, a substantial fraction of the cells in culture had died due to infection, and the surviving neurons also showed an increased cytosolic Ca2+ concentration. 4. The influenza nucleoprotein (NP) is known to interact with actin. When NP was overexpressed with the SFV vector, it was targeted to dendritic spines, where it colocalized with actinin. This targeting to the postsynaptic element did not affect synaptic transmission. In summary, the study has provided further insight into the actions of clostridial toxins on presynaptic function, and shown that infection with enveloped RNA viruses affect somatic Ca2+ homeostasis by direct and indirect mechanisms. It has also shown that the SFV vector is a useful tool for analysis of synaptic function.