Lalli, Giovanna; (2002) The binding fragment of tetanus neurotoxin: A probe to study neuronal endocytosis and retrogade transport. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The Clostridial Neurotoxin (CNT) family is composed of Tetanus Neurotoxin (TeNT) and seven serotypes of Botulinum Neurotoxins (BoNT). These toxins specifically target neuronal cells, leading to an irreversible block of neurotransmitter release. CNTs trafficking can be exploited to investigate the organisation of neuronal endocytic pathways, which are still poorly understood. We have expressed and functionally characterised recombinant carboxy-terminal binding domains (HC) of TeNT and BoNT/A, /B and /E. These fragments can fully mimic the specific binding and internalisation of the respective holotoxins and provide useful tools for the analysis of neuronal trafficking. We have used TeNT HC to study the peculiar intracellular route that distinguishes TeNT from the other CNT family members. In fact, BoNTs act at the presynaptic terminal, whereas TeNT undergoes retrograde transport in the motor neuron and subsequent transcytosis in the adjacent interneuron. Very little is known about the organisation and the dynamics of this special transport route that could be exploited not only by pathogens, but also by physiological ligands for their biological functions. TeNT HC was used to characterise a putative TeNT protein receptor and to visualise for the first time axonal retrograde transport in living motor neurons. This experimental system provides a novel assay for the study of retrograde axonal transport and identifies two main types of retrograde carriers requiring both actin microfilaments and microtubules for efficient progression. TeNT HC compartments lack markers of the classical endocytic pathway and are not acidified during axonal transport. Importantly, TeNT HC and Nerve Growth Factor (NGF) share retrograde carriers, which are characterised by the presence of the neurotrophin receptor p75NTR. Altogether, these findings reveal the existence of a novel endocytic route to the neuronal cell body that escapes lysosomal targeting and that could be shared by physiological ligands such as NGF.

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