Flavia Antonucci

Long-Distance Retrograde Effects of Botulinum Neurotoxin A

Botulinum neurotoxins (designated BoNT/A–BoNT/G) are bacterial enzymes that block neurotransmitter release by cleaving essential components of the vesicle fusion machinery. BoNT/A, which cleaves SNAP-25 (synaptosomal-associated protein of 25 kDa), is extensively exploited in clinical medicine to treat neuromuscular pathologies, facial wrinkles, and various types of pain. It is widely assumed that BoNT/A remains at the synaptic terminal and its effects are confined to the injection site. Here we demonstrate that catalytically active BoNT/A is retrogradely transported by central neurons and motoneurons and is then transcytosed to afferent synapses, in which it cleaves SNAP-25. SNAP-25 cleavage by BoNT/A was observed in the contralateral hemisphere after unilateral BoNT/A delivery to the hippocampus. Appearance of cleaved SNAP-25 resulted in blockade of hippocampal activity in the untreated hemisphere. Injections of BoNT/A into the optic tectum led to the appearance of BoNT/A-truncated SNAP-25 in synaptic terminals within the retina. Cleaved SNAP-25 also appeared in the facial nucleus after injection of the toxin into rat whisker muscles. Experiments excluded passive spread of the toxin and demonstrated axonal migration and neuronal transcytosis of BoNT/A. These findings reveal a novel pathway of BoNT/A trafficking in neurons and have important implications for the clinical uses of this neurotoxin.

Microvesicles released from microglia stimulate synaptic activity via enhanced sphingolipid metabolism

Microvesicles (MVs) released into the brain microenvironment are emerging as a novel way of cell‐to‐cell communication. We have recently shown that microglia, the immune cells of the brain, shed MVs upon activation but their possible role in microglia‐to‐neuron communication has never been explored. To investigate whether MVs affect neurotransmission, we analysed spontaneous release of glutamate in neurons exposed to MVs and found a dose‐dependent increase in miniature excitatory postsynaptic current (mEPSC) frequency without changes in mEPSC amplitude. Paired‐pulse recording analysis of evoked neurotransmission showed that MVs mainly act at the presynaptic site, by increasing release probability. In line with the enhancement of excitatory transmission in vitro, injection of MVs into the rat visual cortex caused an acute increase in the amplitude of field potentials evoked by visual stimuli. Stimulation of synaptic activity occurred via enhanced sphingolipid metabolism. Indeed, MVs promoted ceramide and sphingosine production in neurons, while the increase of excitatory transmission induced by MVs was prevented by pharmacological or genetic inhibition of sphingosine synthesis. These data identify microglia‐derived MVs as a new mechanism by which microglia influence synaptic activity and highlight the involvement of neuronal sphingosine in this microglia‐to‐neuron signalling pathway.

Phenotypic Changes, Signaling Pathway, and Functional Correlates of GPR17-expressing Neural Precursor Cells during Oligodendrocyte Differentiation

The developing and mature central nervous system contains neural precursor cells expressing the proteoglycan NG2. Some of these cells continuously differentiate to myelin-forming oligodendrocytes; knowledge of the destiny of NG2+ precursors would benefit from the characterization of new key functional players. In this respect, the G protein-coupled membrane receptor GPR17 has recently emerged as a new timer of oligodendrogliogenesis. Here, we used purified oligodendrocyte precursor cells (OPCs) to fully define the immunophenotype of the GPR17-expressing cells during OPC differentiation, unveil its native signaling pathway, and assess the functional consequences of GPR17 activation by its putative endogenous ligands, uracil nucleotides and cysteinyl leukotrienes (cysLTs). GPR17 presence was restricted to very early differentiation stages and completely segregated from that of mature myelin. Specifically, GPR17 decorated two subsets of slowly proliferating NG2+ OPCs: (i) morphologically immature cells expressing other early proteins like Olig2 and PDGF receptor-α, and (ii) ramified preoligodendrocytes already expressing more mature factors, like O4 and O1. Thus, GPR17 is a new marker of these transition stages. In OPCs, GPR17 activation by either uracil nucleotides or cysLTs resulted in potent inhibition of intracellular cAMP formation. This effect was counteracted by GPR17 antagonists and receptor silencing with siRNAs. Finally, uracil nucleotides promoted and GPR17 inhibition, by either antagonists or siRNAs, impaired the normal program of OPC differentiation. These data have implications for the in vivo behavior of NG2+ OPCs and point to uracil nucleotides and cysLTs as main extrinsic local regulators of these cells under physiological conditions and during myelin repair.

Evidence for Anterograde Transport and Transcytosis of Botulinum Neurotoxin A (BoNT/A)

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.

Antiepileptic Effects of Botulinum Neurotoxin E

Experimental studies suggest that the delivery of antiepileptic agents into the seizure focus might be of potential utility for the treatment of focal-onset epilepsies. Botulinum neurotoxin E (BoNT/E) causes a prolonged inhibition of neurotransmitter release after its specific cleavage of the synaptic protein synaptosomal-associated protein of 25 kDa (SNAP-25). Here, we show that BoNT/E injected into the rat hippocampus inhibits glutamate release and blocks spike activity of pyramidal neurons. BoNT/E effects persist for at least 3 weeks, as determined by immunodetection of cleaved SNAP-25 and loss of intact SNAP-25. The delivery of BoNT/E to the rat hippocampus dramatically reduces both focal and generalized kainic acid-induced seizures as documented by behavioral and electrographic analysis. BoNT/E treatment also prevents neuronal loss and long-term cognitive deficits associated with kainic acid seizures. Moreover, BoNT/E-injected rats require 50% more electrical stimulations to reach stage 5 of kindling, thus indicating a delayed epileptogenesis. We conclude that BoNT/E delivery to the hippocampus is both antiictal and antiepileptogenic in experimental models of epilepsy.

A reappraisal of the central effects of botulinum neurotoxin type A: by what mechanism?

Botulinum neurotoxin A (BoNT/A) is a metalloprotease that enters peripheral motor nerve terminals and blocks the release of acetylcholine via the specific cleavage of the synaptosomal‐associated protein of 25‐kDa. Localized injections of BoNT/A are widely employed in clinical neurology to treat several human diseases characterized by muscle hyperactivity. It is generally assumed that the effects of BoNT/A remain localized to the injection site. However, several neurophysiological studies have provided evidence for central effects of BoNT/A, raising the issue of how these actions arise. Here we review these data and discuss the possibility that retrograde axonal transport of catalytically active BoNT/A may explain at least some of its effects at the level of central circuits.

Active endocannabinoids are secreted on extracellular membrane vesicles

Endocannabinoids primarily influence neuronal synaptic communication within the nervous system. To exert their function, endocannabinoids need to travel across the intercellular space. However, how hydrophobic endocannabinoids cross cell membranes and move extracellularly remains an unresolved problem. Here, we show that endocannabinoids are secreted through extracellular membrane vesicles produced by microglial cells. We demonstrate that microglial extracellular vesicles carry on their surface N‐arachidonoylethanolamine (AEA), which is able to stimulate type‐1 cannabinoid receptors (CB1), and inhibit presynaptic transmission, in target GABAergic neurons. This is the first demonstration of a functional role of extracellular vesicular transport of endocannabinoids.

Botulinum neurotoxin E (BoNT/E) reduces CA1 neuron loss and granule cell dispersion, with no effects on chronic seizures, in a mouse model of temporal lobe epilepsy.

Mesial temporal lobe epilepsy (MTLE) is often the result of an early insult that induces a reorganization in hippocampal circuitry leading, after a latent period, to chronic epilepsy. Hippocampal rearrangements during the latent phase include neuronal loss, axonal and dendritic plasticity, neurogenesis, and cell repositioning, but the role of these changes in epilepsy development is unclear. Here we have tested whether administration of the synaptic blocker botulinum neurotoxin E (BoNT/E) interferes with development of spontaneous seizures and histopathological changes following an episode of status epilepticus (SE). SE was induced by unilateral intrahippocampal injection of kainic acid in mice and BoNT/E was delivered to the same hippocampus 3 h later. We found that treatment with BoNT/E prolonged the duration of the latent period but did not block the occurrence of spontaneous seizures. At the histopathological level, BoNT/E reduced loss of CA1 pyramidal neurons and dispersion of dentate granule cells. Downregulation of reelin expression along the hippocampal fissure was also suppressed by BoNT/E treatment. Our findings indicate that administration of BoNT/E after SE inhibits specific morphological changes in hippocampal circuitry but not the development of spontaneous seizures. This indicates a dissociation between certain anatomical modifications and establishment of chronic epilepsy in MTLE.

Epileptiform Activity and Cognitive Deficits in SNAP-25+/− Mice are Normalized by Antiepileptic Drugs

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a protein that participates in the regulation of synaptic vesicle exocytosis through the formation of the soluble NSF attachment protein receptor complex and modulates voltage-gated calcium channels activity. The Snap25 gene has been associated with schizophrenia, attention deficit hyperactivity disorder, and bipolar disorder, and lower levels of SNAP-25 have been described in patients with schizophrenia. We used SNAP-25 heterozygous (SNAP-25(+/-)) mice to investigate at which extent the reduction of the protein levels affects neuronal network function and mouse behavior. As interactions of genotype with the specific laboratory conditions may impact behavioral results, the study was performed through a multilaboratory study in which behavioral tests were replicated in at least 2 of 3 distinct European laboratories. Reductions of SNAP-25 levels were associated with a moderate hyperactivity, which disappeared in the adult animals, and with impaired associative learning and memory. Electroencephalographic recordings revealed the occurrence of frequent spikes, suggesting a diffuse network hyperexcitability. Consistently, SNAP-25(+/-) mice displayed higher susceptibility to kainate-induced seizures, paralleled by degeneration of hilar neurons. Notably, both EEG profile and cognitive defects were improved by antiepileptic drugs. These results indicate that reduction of SNAP-25 expression is associated to generation of epileptiform discharges and cognitive dysfunctions, which can be effectively treated by antiepileptic drugs.

Leucine-Rich Repeat Kinase 2 Binds to Neuronal Vesicles through Protein Interactions Mediated by Its C-Terminal WD40 Domain

ABSTRACT Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinsons disease (PD). LRRK2 is a complex protein that consists of multiple domains, including predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function.