The striking differences between the clinical symptoms of tetanus and botulism have been ascribed to the different fate of the parental neurotoxins once internalised in motor neurons. Tetanus toxin (TeNT) is known to undergo transcytosis into inhibitory interneurons and block the release of inhibitory neurotransmitters in the spinal cord, causing a spastic paralysis. In contrast, botulinum neurotoxins (BoNTs) block acetylcholine release at the neuromuscular junction, therefore inducing a flaccid paralysis. Whilst overt experimental evidence supports the sorting of TeNT to the axonal retrograde transport pathway, recent findings challenge the established view that BoNT trafficking is restricted to the neuromuscular junction by highlighting central effects caused by these neurotoxins. These results suggest a more complex scenario whereby BoNTs also engage long-range trafficking mechanisms. However, the intracellular pathways underlying this process remain unclear. We sought to fill this gap by using primary motor neurons either in mass culture or differentiated in microfluidic devices to directly monitor the endocytosis and axonal transport of full length BoNT/A and BoNT/E and their recombinant binding fragments. We show that BoNT/A and BoNT/E are internalised by spinal cord motor neurons and undergo fast axonal retrograde transport. BoNT/A and BoNT/E are internalised in non-acidic axonal carriers that partially overlap with those containing TeNT, following a process that is largely independent of stimulated synaptic vesicle endo-exocytosis. Following intramuscular injection in vivo, BoNT/A and TeNT displayed central effects with a similar time course. Central actions paralleled the peripheral spastic paralysis for TeNT, but lagged behind the onset of flaccid paralysis for BoNT/A. These results suggest that the fast axonal retrograde transport compartment is composed of multifunctional trafficking organelles orchestrating the simultaneous transfer of diverse cargoes from nerve terminals to the soma, and represents a general gateway for the delivery of virulence factors and pathogens to the central nervous system. Botulinum neurotoxins are the most toxic molecules known to mankind, and as a result, are currently listed among the top bio-threats. However, their ability to bind specifically to neurons and their inhibitory effects on regulated secretion prompted their clinical use in pathologies characterised by increased muscular tone, such as dystonia and various forms of spasticity, or abnormal secretion, such as drooling and excessive sweating, to cite a few. As a consequence, botulinum neurotoxin A, which is the serotype most commonly used in human therapy, has become the treatment of choice for an ever-expanding number of pathological and non-pathological (e.g. cosmetic) conditions. All current indications show that the systemic effects and toxicity of botulinum neurotoxin A are minimised by the specific route of administration (local injection) and the low diffusion of this molecule in tissues. However, recent reports suggest that in contrast to this common belief, botulinum neurotoxin A is able to reach distal sites in the body and may have previously unanticipated effects in the central nervous system. In this study, we demonstrate that botulinum neurotoxin A and E enter alternative endocytic pathway(s) in addition to synaptic vesicle recycling, and undergo long-range transport in a non degradative compartment in spinal cord motor neurons. Our results show that axonal retrograde transport is a common pathway for the dissemination in the central nervous system of pathogens and virulence factors important for human and animal health.

Botulinum neurotoxin type A (BoNT/A) is a metalloprotease that blocks synaptic transmission via the cleavage of SNAP-25 (synaptosomal-associated protein of 25 kDa). BoNT/A is successfully used in clinical neurology for the treatment of several neuromuscular pathologies and pain syndromes. Despite its widespread use, relatively little is known on BoNT/A intracellular trafficking in neurons. Using the visual pathway as a model system, here we show that catalytically active BoNT/A is capable of undergoing anterograde axonal transport and transcytosis. Following BoNT/A injection into the rat eye, significant levels of BoNT/A-cleaved SNAP-25 appeared in the retinorecipient layers of the superior colliculus (SC). Anterograde propagation of BoNT/A effects required axonal transport, ruling out a systemic spread of the toxin. Cleaved SNAP-25 was present in presynaptic structures of the tectum, but retinal terminals were devoid of the immunoreactivity, indicative of transcytosis. Experiments based on sequential administration of BoNT/A and BoNT/E showed a persistent catalytic activity of BoNT/A in tectal cells following its injection into the retina. Our findings demonstrate that catalytically active BoNT/A is anterogradely transported from the eye to the SC and transcytosed to tectal synapses. These data are important for a more complete understanding of the mechanisms of action of BoNT/A.

Early life experiences can affect brain development, contributing to shape interindividual differences in stress vulnerability and anxiety-like behavior. In rodents, high levels of maternal care have long-lasting positive effects on the behavior of the offspring and stress response; post-weaning rearing in an enriched environment (EE) or massage counteract the negative effects of maternal separation or prenatal stressors. We recently found that insulin-like growth factor 1 (IGF-1) is a key mediator of early EE or massage on brain development. Whether early enrichment of experience can induce long-lasting effects on anxiety-like behavior and whether IGF-1 is involved in these effects is not known. We assessed anxiety-like behavior by means of the elevated plus maze in control adult rats and in adult rats subjected to early EE or to massage. We found that both EE and massage reduced adult anxiety-like behavior. Early IGF-1 systemic injections in rat pups reared in standard condition mimic the effects of EE and massage, reducing anxiety-like behavior in the adult; blocking early IGF-1 action in massaged and EE animals prevents massage and EE effects. In EE and IGF-1-treated animals, we assessed the hippocampal expression of glucocorticoid receptors (GRs) at postnatal day 12 (P12) and P60, finding a significantly higher GR expression at P60 for both treatments. These results suggest that IGF-1 could be involved in mediating the long-lasting effects of early life experiences on vulnerability/resilience to stress in adults.