Bernard Poulain

How do the Botulinum Neurotoxins block neurotransmitter release: from botulism to the molecular mechanism of action

An overall picture of the botulinum toxins is provided, linking the recent developments in their molecular and cellular aspects to the physiological actions. The pathophysiological aspects of botulinum poisoning (clinical symptoms and physical findings, synaptic effects at the motor and non-motor nerve terminals) and indirect consequences (muscle atrophy, transient formation of new end-plates) are described. The role of the botulinum toxin complex constituents in dissemination, selective binding of the neurotoxins to nerve terminals and intestinal epithelial cells, endocytosis, intracellular trafficking, translocation into the cytosol, and proteolytic attack of the neurotransmitter release machinery are analysed in a molecular and cellular perspective. [Received 23 October 2007; Accepted 12 December 2007]

Molecular Mechanism of Action of Botulinal Neurotoxins and the Synaptic Remodeling They Induce In Vivo at the Skeletal Neuromuscular Junction

Botulinal neurotoxins (BoNTs) have long been known to have potent and specific paralytic effects at the vertebrate neuromuscular junction (NMJ). Although they are the most toxic substances known, the serotype A is now being used for therapeutic purposes, mainly to treat involuntary muscle contractions, but also for a number of other medical applications (reviewed in ref. 1).

CHAPTER 19 – Attack of the nervous system by clostridial toxins: physical findings, cellular and molecular actions

This chapter summarizes the known pathophysiological and molecular actions on nerve terminals and nerve tissue of several potent toxins produced by Clostridium species. This includes the botulinum and tetanus neurotoxins, which are to date the best documented toxins acting on neurotransmitter release. These toxins can indiscriminately damage membranes from different cells, including those of neuronal origin. Among them, C. perfringens epsilon toxin has the fundamental structure of a pore-forming toxin, is cytotoxic for kidney epithelial cells, and also possesses specific neurotoxic activity. Other toxins have developed various internalization processes, therefore specifically modifying an intracellular target. Thus, several bacterial proteins like the large clostridial toxins have the ability to bind/enter various cell types that include those of neuronal origin. During their evolution, clostridial pathogens have developed a potent arsenal of noxious proteins thataffect the central and peripheral nervous system of various vertebrates. These commonly named neurotoxins specifically target some key functions, or cellular processes, of eukaryotic cells, which subsequently cause a wide array of life-threatening diseases in humans and animals. The specificity of these toxic proteins has enabled them to become useful tools to elucidate and characterize crucial processes for eukaryotic cells, which include neurotransmitter release, physiological signaling pathways, and constitutive cellular mechanisms. Additional studies are needed to completely identify their specific membrane-receptors, as well as comprehend the mechanisms and structures involved in toxin routing throughout the nervous system.

Synaptic Maturation at Cortical Projections to the Lateral Amygdala in a Mouse Model of Rett Syndrome

Rett syndrome (RTT) is a neuro-developmental disorder caused by loss of function of Mecp2 - methyl-CpG-binding protein 2 - an epigenetic factor controlling DNA transcription. In mice, removal of Mecp2 in the forebrain recapitulates most of behavioral deficits found in global Mecp2 deficient mice, including amygdala-related hyper-anxiety and lack of social interaction, pointing a role of Mecp2 in emotional learning. Yet very little is known about the establishment and maintenance of synaptic function in the adult amygdala and the role of Mecp2 in these processes. Here, we performed a longitudinal examination of synaptic properties at excitatory projections to principal cells of the lateral nucleus of the amygdala (LA) in Mecp2 mutant mice and their wild-type littermates. We first show that during animal life, Cortico-LA projections switch from a tonic to a phasic mode, whereas Thalamo-LA synapses are phasic at all ages. In parallel, we observed a specific elimination of Cortico-LA synapses and a decrease in their ability of generating presynaptic long term potentiation. In absence of Mecp2, both synaptic maturation and synaptic elimination were exaggerated albeit still specific to cortical projections. Surprisingly, associative LTP was unaffected at Mecp2 deficient synapses suggesting that synaptic maintenance rather than activity-dependent synaptic learning may be causal in RTT physiopathology. Finally, because the timing of synaptic evolution was preserved, we propose that some of the developmental effects of Mecp2 may be exerted within an endogenous program and restricted to synapses which maturate during animal life.

Stereotyped spatial patterns of functional synaptic connectivity in the cerebellar cortex

Motor coordination is supported by an array of highly organized heterogeneous modules in the cerebellum. How incoming sensorimotor information is channeled and communicated between these anatomical modules is still poorly understood. In this study, we used transgenic mice expressing GFP in specific subsets of Purkinje cells that allowed us to target a given set of cerebellar modules. Combining in vitro recordings and photostimulation, we identified stereotyped patterns of functional synaptic organization between the granule cell layer and its main targets, the Purkinje cells, Golgi cells and molecular layer interneurons. Each type of connection displayed position-specific patterns of granule cell synaptic inputs that do not strictly match with anatomical boundaries but connect distant cortical modules. Although these patterns can be adjusted by activity-dependent processes, they were found to be consistent and predictable between animals. Our results highlight the operational rules underlying communication between modules in the cerebellar cortex. DOI: http://dx.doi.org/10.7554/eLife.09862.001

Clostridial neurotoxins: from the cellular and molecular mode of action to their therapeutic use

Botulinum (BoNT) and tetanus (TeNT) neurotoxins are potent toxins responsible for flaccid and spastic paralysis, respectively. BoNTs are divided into types and subtypes according to their immunological properties and amino acid sequence variations, whereas only one type of TeNT has been characterized. BoNTs associate with nontoxic proteins to form large complexes that are resistant to acidic pH and protease degradation, unlike TeNT, which is produced as a unique protein. BoNTs and TeNT enter target neuronal cells by interacting with specific cell surface receptors. BoNTs proteolytically cleave soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins (SNAP-25, VAMP, or syntaxin) mainly at the cholinergic neuromuscular junctions impairing the evoked release of acetylcholine, whereas TeNT inhibits the neuroexocytosis in central inhibitory interneurons by cleavage of VAMP. BoNT-mediated muscle paralysis induces neuronal sprouting, which after various time periods, leads to a recovery of neurotransmission. BoNT/A, which has a long duration of activity, is used in numerous medical applications.

Frequency-dependent mobilization of heterogeneous pools of synaptic vesicles shapes presynaptic plasticity

The segregation of the readily releasable pool of synaptic vesicles (RRP) in sub-pools that are differentially poised for exocytosis shapes short-term plasticity. However, the frequency-dependent mobilization of these sub-pools is poorly understood. Using slice recordings and modeling of synaptic activity at cerebellar granule cell to Purkinje cell synapses of mice, we describe two sub-pools in the RRP that can be differentially recruited upon ultrafast changes in the stimulation frequency. We show that at low-frequency stimulations, a first sub-pool is gradually silenced, leading to full blockage of synaptic transmission. Conversely, a second pool of synaptic vesicles that cannot be released by a single stimulus is recruited within milliseconds by high-frequency stimulation and support an ultrafast recovery of neurotransmitter release after low-frequency depression. This frequency-dependent mobilization or silencing of sub-pools in the RRP in terminals of granule cells may play a role in the filtering of sensorimotor information in the cerebellum.

Botulinal Neurotoxins: Mode of Action on Neurotransmitter Release

Publisher Summary This chapter discusses the mode of action on neurotransmitter release by botulinal neurotoxins. Botulinal neurotoxins (BoNTs) are among the most potent biological agents known so far. Seven immunologically distinguishable forms of these neuroparalytic proteins, designated as types A, B, C 1 , D, E, F, and G, are produced by different strains of the anaerobic, spore-forming bacterium, Clostridium botulinum. These neurotoxins are synthesized as single-chain proteins. Depending on the physiology of the bacterium and the conditions of the bacterial culture, they may be cleaved into a di-chain protein. The possibility that BoNTs can affect presynaptic Ca 2+ entry and/or intraterminal Ca 2+ levels has been tested. The chapter describes a method in which microelectrodes is inserted in the perineurium of small preterminal nerve bundles to record electric signals related to membrane currents. It is found that BoNT type A or D poisoned endings display normal Ca 2+ currents.

Organotypic cultures of cerebellar slices as a model to investigate demyelinating disorders

ABSTRACT Introduction: Demyelinating disorders, characterized by a chronic or episodic destruction of the myelin sheath, are a leading cause of neurological disability in young adults in western countries. Studying the complex mechanisms involved in axon myelination, demyelination and remyelination requires an experimental model preserving the neuronal networks and neuro-glial interactions. Organotypic cerebellar slice cultures appear to be the best alternative to in vivo experiments and the most commonly used model for investigating etiology or novel therapeutic strategies in multiple sclerosis. Areas covered: This review gives an overview of slice culture techniques and focuses on the use of organotypic cerebellar slice cultures on semi-permeable membranes for studying many aspects of axon myelination and cerebellar functions. Expert opinion: Cerebellar slice cultures are probably the easiest way to faithfully reproduce all stages of axon myelination/demyelination/remyelination in a three-dimensional neuronal network. However, in the cerebellum, neurological disability in multiple sclerosis also results from channelopathies which induce changes in Purkinje cell excitability. Cerebellar cultures offer easy access to electrophysiological approaches which are largely untapped and we believe that these cultures might be of great interest when studying changes in neuronal excitability, axonal conduction or synaptic properties that likely occur during multiple sclerosis.

Inhibition promotes long-term potentiation at cerebellar excitatory synapses.

The ability of the cerebellar cortex to learn from experience ensures the accuracy of movements and reflex adaptation, processes which require long-term plasticity at granule cell (GC) to Purkinje neuron (PN) excitatory synapses. PNs also receive GABAergic inhibitory inputs via GCs activation of interneurons; despite the involvement of inhibition in motor learning, its role in long-term plasticity is poorly characterized. Here we reveal a functional coupling between ionotropic GABAA receptors and low threshold CaV3 calcium channels in PNs that sustains calcium influx and promotes long-term potentiation (LTP) at GC to PN synapses. High frequency stimulation induces LTP at GC to PN synapses and CaV3-mediated calcium influx provided that inhibition is intact; LTP is mGluR1, intracellular calcium store and CaV3 dependent. LTP is impaired in CaV3.1 knockout mice but it is nevertheless recovered by strengthening inhibitory transmission onto PNs; promoting a stronger hyperpolarization via GABAA receptor activation leads to an enhanced availability of an alternative Purkinje-expressed CaV3 isoform compensating for the lack of CaV3.1 and restoring LTP. Accordingly, a stronger hyperpolarization also restores CaV3-mediated calcium influx in PNs from CaV3.1 knockout mice. We conclude that by favoring CaV3 channels availability inhibition promotes LTP at cerebellar excitatory synapses.