Clostridial neurotoxins, including tetanus toxin and the seven serotypes of botulinum toxin (A-G), are produced as single chains and cleaved to generate toxins with two chains joined by a single disulphide bond (Fig. 1). The heavy chain (M(r) 100,000 (100K)) is responsible for specific binding to neuronal cells and cell penetration of the light chain (50K), which blocks neurotransmitter release. Several lines of evidence have recently suggested that clostridial neurotoxins could be zinc endopeptidases. Here we show that tetanus and botulinum toxins serotype B are zinc endopeptidases, the activation of which requires reduction of the interchain disulphide bond. The protease activity is localized on the light chain and is specific for synaptobrevin, an integral membrane protein of small synaptic vesicles. The rat synaptobrevin-2 isoform is cleaved by both neurotoxins at the same single site, the peptide bond Gln 76-Phe 77, but the isoform synaptobrevin-1, which has a valine at the corresponding position, is not cleaved. The blocking of neurotransmitter release of Aplysia neurons injected with tetanus toxin or botulinum toxins serotype B is substantially delayed by peptides containing the synaptobrevin-2 cleavage site. These results indicate that tetanus and botulinum B neurotoxins block neurotransmitter release by cleaving synaptobrevin-2, a protein that, on the basis of our results, seems to play a key part in neurotransmitter release.

Degenerative disorders of motor neurons include a range of progressive fatal diseases such as amyotrophic lateral sclerosis (ALS), spinal-bulbar muscular atrophy (SBMA), and spinal muscular atrophy (SMA). Although the causative genetic alterations are known for some cases, the molecular basis of many SMA and SBMA-like syndromes and most ALS cases is unknown. Here we show that missense point mutations in the cytoplasmic dynein heavy chain result in progressive motor neuron degeneration in heterozygous mice, and in homozygotes this is accompanied by the formation of Lewy-like inclusion bodies, thus resembling key features of human pathology. These mutations exclusively perturb neuron-specific functions of dynein.

Vesicular pathways coupling the neuromuscular junction with the motor neuron soma are essential for neuronal function and survival. To characterize the organelles responsible for this long-distance crosstalk, we developed a purification strategy based on a fragment of tetanus neurotoxin (TeNT H(C)) conjugated to paramagnetic beads. This approach enabled us to identify, among other factors, the small GTPase Rab7 as a functional marker of a specific pool of axonal retrograde carriers, which transport neurotrophins and their receptors. Furthermore, Rab5 is essential for an early step in TeNT H(C) sorting but is absent from axonally transported vesicles. Our data demonstrate that TeNT H(C) uses a retrograde transport pathway shared with p75(NTR), TrkB, and BDNF, which is strictly dependent on the activities of both Rab5 and Rab7. Therefore, Rab7 plays an essential role in axonal retrograde transport by controlling a vesicular compartment implicated in neurotrophin traffic.

ALS is a fatal neurodegenerative disease characterized by selective motor neuron death resulting in muscle paralysis. Mutations in superoxide dismutase 1 (SOD1) are responsible for a subset of familial cases of ALS. Although evidence from transgenic mice expressing human mutant SOD1 G93A suggests that axonal transport defects may contribute to ALS pathogenesis, our understanding of how these relate to disease progression remains unclear. Using an in vivo assay that allows the characterization of axonal transport in single axons in the intact sciatic nerve, we have identified clear axonal transport deficits in presymptomatic mutant mice. An impairment of axonal retrograde transport may therefore represent one of the earliest axonal pathologies in SOD1 G93A mice, which worsens at an early symptomatic stage. A deficit in axonal transport may therefore be a key pathogenic event in ALS and an early disease indicator of motor neuron degeneration.

No abstract

PtdIns4P is the major precursor for the synthesis of the multifunctional plasma membrane lipid, PtdIns(4,5)P2. Yet PtdIns4P also functions as a regulatory lipid in its own right, particularly at the Golgi apparatus. In the present study we define specific conditions that enable preservation of several organellar membranes for the immunocytochemical detection of PtdIns4P. We report distinct pools of this lipid in both Golgi and plasma membranes, which are synthesized by different PI4K (phosphatidylinositol 4-kinase) activities, and also the presence of PtdIns4P in cytoplasmic vesicles, which are not readily identifiable as PI4K containing trafficking intermediates. In addition, we present evidence that the majority of PtdIns4P resides in the plasma membrane, where it is metabolically distinct from the steady-state plasma membrane pool of PtdIns(4,5)P2.

Tetanus and botulinum neurotoxins are produced by Clostridia and cause the neuroparalytic syndromes of tetanus and botulism. Tetanus neurotoxin acts mainly at the CNS synapse, while the seven botulinum neurotoxins act peripherally. Clostridial neurotoxins share a similar mechanism of cell intoxication: they block the release of neurotransmitters. They are composed of two disulfide-linked polypeptide chains. The larger subunit is responsible for neurospecific binding and cell penetration. Reduction releases the smaller chain in the neuronal cytosol, where it displays its zinc-endopeptidase activity specific for protein components of the neuroexocytosis apparatus. Tetanus neurotoxin and botulinum neurotoxins B, D, F and G recognize specifically VAMP/ synaptobrevin. This integral protein of the synaptic vesicle membrane is cleaved at single peptide bonds, which differ for each neurotoxin. Botulinum A, and E neurotoxins recognize and cleave specifically SNAP-25, a protein of the presynaptic membrane, at two different sites within the carboxyl-terminus. Botulinum neurotoxin type C cleaves syntaxin, another protein of the nerve plasmalemma. These results indicate that VAMP, SNAP-25 and syntaxin play a central role in neuroexocytosis. These three proteins are conserved from yeast to humans and are essential in a variety of docking and fusion events in every cell. Tetanus and botulinum neurotoxins form a new group of zinc-endopeptidases with characteristic sequence, mode of zinc coordination, mechanism of activation and target recognition. They will be of great value in the unravelling of the mechanisms of exocytosis and endocytosis, as they are in the clinical treatment of dystonias.

Variants of UNC13A, a critical gene for synapse function, increase the risk of amyotrophic lateral sclerosis and frontotemporal dementia1–3, two related neurodegenerative diseases defined by mislocalization of the RNA-binding protein TDP-434,5. Here we show that TDP-43 depletion induces robust inclusion of a cryptic exon in UNC13A, resulting in nonsense-mediated decay and loss of UNC13A protein. Two common intronic UNC13A polymorphisms strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia risk overlap with TDP-43 binding sites. These polymorphisms potentiate cryptic exon inclusion, both in cultured cells and in brains and spinal cords from patients with these conditions. Our findings, which demonstrate a genetic link between loss of nuclear TDP-43 function and disease, reveal the mechanism by which UNC13A variants exacerbate the effects of decreased TDP-43 function. They further provide a promising therapeutic target for TDP-43 proteinopathies.