Marco Pirazzini

University of Padua | UNIPD · Department of Biomedical Sciences - DSB

Background and Purpose Botulinum neurotoxin type A1 (BoNT/A) is one of the most potent neurotoxins known. At the same time, it is also one of the safest therapeutic agents used for the treatment of several human disorders and in aesthetic medicine. Notwithstanding great effectiveness, strategies to accelerate the onset and prolong BoNT/A action wou...

Tetanus neurotoxin (TeNT) causes spastic paralysis by inhibiting neurotransmission in spinal inhibitory interneurons. TeNT binds to the neuromuscular junction, leading to its internalisation into motor neurons and subsequent transcytosis into interneurons. While the extracellular matrix proteins nidogens are essential for TeNT binding, the molecula...

Background and Purpose Botulinum Neurotoxin type A1 (BoNT/A) is one of the most potent neurotoxins known. At the same time, it is also one of the safest therapeutic agents used for the treatment of several human disorders and in aesthetic medicine. Notwithstanding great effectiveness, strategies to accelerate the onset and prolong BoNT/A action wou...

In this study, we characterize Designed Ankyrin Repeat Proteins (DARPins) as investigative tools to probe botulinum neurotoxin A1 (BoNT/A1) structure and function. We identify DARPin-F5 that completely blocks SNAP25 substrate cleavage by BoNT/A1 in vitro. X-ray crystallography reveals that DARPin-F5 inhibits BoNT/A1 activity by interacting with a s...

Overt muscle activity and impaired spinal locomotor control hampering coordinated movement is a hallmark of spasticity and movement disorders like dystonia. While botulinum toxin A (BoNT-A) standard therapy alleviates mentioned symptoms presumably due to its peripheral neuromuscular actions alone, the aim of present study was to examine for the fir...

Joensuu and colleagues have recently shown that botulinum neurotoxin (BoNT) type A exploits a heterotrimeric complex in the presynaptic membrane to bind to and enter neurons using a Trojan horse-like mechanism. Similar processes may be relevant to the neuronal entry of different botulinum toxin serotypes and other neuropathogens.

Background and purpose: Recent preclinical studies suggest that botulinum toxin type A (BoNT-A) reduces abnormal muscle tone following its traffic to second order spinal synapses. However, possible consequences of such actions on spinal neuromotor control that coordinates skilled movement have not been characterized. Therefore, we studied the centr...

Cephalic tetanus (CT) is a severe form of tetanus that follows head wounds and the intoxication of cranial nerves by tetanus neurotoxin (TeNT). Hallmarks of CT are cerebral palsy, which anticipates the typical spastic paralysis of tetanus, and rapid evolution of cardiorespiratory deficit even without generalized tetanus. How TeNT causes this unexpe...

People bitten by Alpine vipers are usually treated with antivenom antisera to prevent the noxious consequences caused by the injected venom. However, this treatment suffers from a number of drawbacks and additional therapies are necessary. The venoms of Vipera ammodytes and of Vipera aspis are neurotoxic and cause muscle paralysis by inducing neuro...

Polyglutamine expansion in the androgen receptor (AR) causes spinobulbar muscular atrophy (SBMA). Skeletal muscle is a primary site of toxicity; however, the current understanding of the early pathological processes that occur and how they unfold during disease progression remains limited. Using transgenic and knock-in mice and patient-derived musc...

Skeletal muscle is the most abundant tissue in the body and requires high levels of energy to function properly. Skeletal muscle allows voluntary movement and body posture, which require different types of fiber, innervation, energy, and metabolism. Here, we summarize the contribution received at the time of publication of this Introductory Issue f...

Regeneration of the neuromuscular junction (NMJ) leverages on extensive exchange of factors released from motor axon terminals (MATs), muscle fibers and perisynaptic Schwann cells (PSCs), among which hydrogen peroxide (H 2 O 2 ) is a major pro-regenerative signal. To identify critical determinants of NMJ remodeling in response to injury, we perform...

In this study, we characterized Designed Ankyrin Repat Proteins (DARPins) as investigative tools to probe botulinum neurotoxin (BoNT) function and as potential antidotes for botulism. We selected DARPins against the catalytic domain of BoNT/A1 and characterized them by biochemical, biophysical and structural studies in combination with functional a...

The mannose receptor (CD206) is an endocytic receptor expressed by selected innate immune cells and nonvascular endothelium, which plays a critical role in both homeostasis and pathogen recognition. Although its involvement in the development of several diseases and viral infections is well established, molecular tools able to both provide insight...

Immunotherapy is deemed one of the most powerful therapeutic approaches to treat cancer. However, limited response and tumor specificity are still major challenges to address. Herein, mannosylated polycations targeting mannose receptor- are developed as vectors for plasmid DNA (pDNA)-based vaccines to improve selective delivery of genetic material...

Tetanus and botulinum neurotoxins cause the neuroparalytic syndromes of tetanus and botulism, respectively, by delivering inside different types of neurons, metalloproteases specifically cleaving the SNARE proteins that are essential for the release of neurotransmitters. Research on their mechanism of action is intensively carried out in order to d...

Tetanus and Botulinum type B neurotoxins are bacterial metalloproteases that specifically cleave the vesicle-associated membrane protein VAMP at an identical peptide bond, resulting in inhibition of neuroexocytosis. The minute amounts of these neurotoxins commonly used in experimental animals are not detectable, nor is detection of their VAMP subst...

We used α-Latrotoxin (α-LTx), the main neurotoxic component of the black widow spider venom, which causes degeneration of the neuromuscular junction (NMJ) followed by a rapid and complete regeneration, as a molecular tool to identify by RNA transcriptomics factors contributing to the structural and functional recovery of the NMJ. We found that Uroc...

Polyglutamine (polyQ)-expanded androgen receptor (AR) causes spinobulbar muscular atrophy. Skeletal muscle is a primary site of polyQ-expanded AR toxicity, however it remains to be established what the early pathological processes are and how they unfold during disease progression. Using transgenic, knock-in SBMA mice and patient-derived muscle bio...

Schwann cells (SCs) are fundamental components of the peripheral nervous system (PNS) of all vertebrates and play essential roles in development, maintenance, function, and regeneration of peripheral nerves. There are distinct populations of SCs including: (1) myelinating SCs that ensheath axons by a specialized plasma membrane, called myelin, whic...

Tetanus neurotoxin (TeNT) is a protein exotoxin produced by Clostridium tetani that causes the deadly spastic neuroparalysis of tetanus. It consists of a metalloprotease light chain and of a heavy chain linked via a disulphide bond. TeNT binds to the neuromuscular junction (NMJ) and it is retro-axonally transported into vesicular compartments to th...

Human monoclonal antibodies were used here to study the mechanism of neuron intoxication by tetanus neurotoxin and to evaluate them as a safe preventive and therapeutic substitute of hyperimmune sera for tetanus in mice. By screening memory B cells of immune donors, we selected two monoclonal antibodies specific for tetanus neurotoxin with exceptio...

Human monoclonal antibodies were used here to study the mechanism of neuron intoxication by tetanus neurotoxin protein toxins and as a safe preventive and therapeutic substitute of hyperimmune sera. By screening memory B cells of immune donors, we selected two monoclonal antibodies specific for tetanus neurotoxin with exceptionally high neutralizin...

Botulinum neurotoxins (BoNTs) produced by soil bacterium Clostridium botulinum are cause of botulism and listed as biohazard agents, thus rapid screening assays are needed for taking the correct countermeasures in a timely fashion. The gold standard method relies on the mouse lethality assay with a lengthy analysis time, i.e., 2-5 days, hindering t...

Tetanus is a deadly but preventable disease caused by a protein neurotoxin produced by Clostridium tetani. Spores of C. tetani may contaminate a necrotic wound and germinate into a vegetative bacterium that releases a toxin, termed tetanus neurotoxin (TeNT). TeNT enters the general circulation, binds to peripheral motor neurons and sensory neurons,...

Botulinum neurotoxins (BoNTs) are a growing family of bacterial protein toxins that cause a generalized flaccid paralysis of botulism by inactivating neurotransmitter release at peripheral nerve terminals. They are the most potent toxins known thanks to the marvel of their protein design, which underlines their mechanism of action. Their unique bio...

Botulinum neurotoxins (BoNTs) are Janus‐faced biological agents. They are the most poisonous substances known and the causative agents of botulism, a deadly neuroparalytic syndrome of animals and humans. Owing to their potency, BoNTs have the potential to be used as biological weapons. At the same time, they are effective therapeutics for a variety...

Eubacterium tarantellae was originally cultivated from the brain of fish affected by twirling movements. Here, we present the draft genome sequence of E. tarantellae DSM 3997, which consists of 3,982,316 bp. Most protein-coding genes in this strain are similar to genes of Clostridium bacteria, supporting the renaming of E. tarantellae as Clostridiu...

Botulinum neurotoxins (BoNTs) are a growing family of bacterial protein toxins that cause botulism, a rare but often fatal animal and human disease. They are the most potent toxins known owing to their molecular architecture, which underlies their mechanism of action. BoNTs target peripheral nerve terminals by a unique mode of binding and enter int...

Polyglutamine (polyQ) expansions in the androgen receptor (AR) gene cause spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease characterized by lower motor neuron (MN) loss and skeletal muscle atrophy, with an unknown mechanism. We generated new mouse models of SBMA for constitutive and inducible expression of mutant AR and performed...

Objective: To test whether the signaling axis CXCL12α-CXCR4 is activated upon crush/cut of the sciatic nerve and to test the activity of NUCC-390, a new CXCR4 agonist, in promoting nerve recovery from damage. Methods: The sciatic nerve was either crushed or cut. Expression and localization of CXCL12α and CXCR4 were evaluated by imaging with spec...

Background: Skeletal muscle is a plastic tissue that can adapt to different stimuli. It is well established that Mammalian Target of Rapamycin Complex 1 (mTORC1) signalling is a key modulator in mediating increases in skeletal muscle mass and function. However, the role of mTORC1 signalling in adult skeletal muscle homeostasis is still not well de...

The activation of the G-protein coupled receptor CXCR4 by its ligand CXCL12α is involved in a large variety of physiological and pathological processes, including the growth of B cells precursors and of motor axons, autoimmune diseases, stem cell migration, inflammation, and several neurodegenerative conditions. Recently, we demonstrated that CXCL1...

A large number of bacterial toxins consist of active and cell binding protomers linked by an inter‐chain disulfide bridge. The largest family of such disulfide bridged exotoxins is that of the Clostridial neurotoxins that consist of two chains and comprise the tetanus neurotoxins (TeNT) causing tetanus and the botulinum neurotoxins (BoNT) causing b...

Spinal and bulbar muscular atrophy (SBMA) is caused by polyglutamine (polyQ) expansions in the androgen receptor (AR) gene. Although clinical and experimental evidence highlight a primary role for skeletal muscle in the onset, progression, and outcome of disease, the pathophysiological and molecular processes underlying SBMA muscle atrophy are poor...

Botulinum neurotoxin serotypes A and B are successfully used to treat a variety of human diseases characterized by hyperactive peripheral nerve terminals. However, a number of patients are primary resistant to these pharmaceuticals, without having antitoxin‐neutralizing antibodies. A straightforward explanation of this phenomenon posits that mutati...

Cerebellar Granule Neurons (CGN) from post-natal rodents have been widely used as a model to study neuronal development, physiology and pathology. CGN cultured in vitro maintain the same features displayed in vivo by mature cerebellar granule cells, including the development of a dense neuritic network, neuronal activity, neurotransmitter release a...

Vertebrate neuromuscular junctions (NMJs) have been conceived as tripartite synapses composed of motor neuron, Schwann cell, and muscle fiber. Recent work has shown the presence of sympathetic neurons in the immediate vicinity of NMJs and experimental and clinical findings suggest that this plays an eminent role in adult NMJ biology. The present st...

Medically relevant cases of snakebite in Europe are predominately caused by European vipers of the genus Vipera. Systemic envenoming by European vipers can cause severe pathology in humans and different clinical manifestations are associated with different members of this genus. The most representative vipers in Europe are V. aspis and V. berus and...

The presence of botulinum neurotoxin-producing Clostridia (BPC) in food sources is a public health concern. In favorable environmental conditions, BPC can produce botulinum neurotoxins (BoNTs) outside or inside the vertebrate host, leading to intoxications or toxico-infectious forms of botulism, respectively. BPC in food are almost invariably detec...

Botulinum neurotoxins (BoNTs), the etiological agents of botulism, are the deadliest toxins known to humans. Yet, thanks to their biological and toxicological features, BoNTs have become sophisticated tools to study neuronal physiology and valuable therapeutics for an increasing number of human disorders. BoNTs are produced by multiple bacteria of...

Neuromuscular junction (NMJ) is the specialized chemical synapse that mediates the transmission of the electrical impulse running along motor neuron axons to skeletal muscle fibers. NMJ is the best characterized chemical synapse and its study along many years of research has provided most of the general knowledge of synapse development, structure a...

The neuromuscular junction (NMJ) is the specialized synapse by which peripheral motor neurons innervate muscle fibers and control skeletal muscle contraction. The NMJ is the target of several xenobiotics, including chemicals, plant, animal and bacterial toxins, as well as of autoantibodies raised against NMJ antigens. Depending on their biochemical...

The Genome Aggregation Database presently contains >120,000 human genomes. We searched in this database for the presence of mutations at the sites of tetanus (TeNT) and botulinum neurotoxins (BoNTs) cleavages of the three SNARE proteins: VAMP, SNAP-25 and Syntaxin. These mutations could account for some of the BoNT/A resistant patients. At the same...

Botulinum (BoNTs) and tetanus (TeNT) neurotoxins are the most toxic substances known and form the growing family of Clostridial neurotoxins (CNT), the etiologic agents of botulism and tetanus. CNT are composed of a metalloprotease light chain (L), linked via a disulfide bond to a heavy chain (H). H mediates the binding to nerve terminals and the me...

Botulinum neurotoxin serotype C (BoNT/C) is a neuroparalytic toxin associated with outbreaks of animal botulism, particularly in birds, and is the only BoNT known to cleave two different SNARE proteins, SNAP-25 and syntaxin. BoNT/C was shown to be a good substitute for BoNT/A1 in human dystonia therapy because of its long lasting effects and absenc...

The cleavage of SNAP-25 by BoNT/C mutants in CGNs does not increase by prolonging the incubation time to 24 hours. CGNs were treated as in Fig 1 but incubation was prolonged to 24 hours. The cleavage of syntaxin-1A/1B and SNAP-25 was assayed by western blot using two antibodies recognizing both the intact and the cleaved forms of the proteins. (TIF...

BoNT/C mutants display a different cytotoxic effect on cultured neurons. CGNs were treated as in Fig 2 but incubation was prolonged to 24 hours. Neurons were then fixed and stained with an antibody against cleaved SNAP-25 (SNAP-25c, in red) and neurofilament-200 (NF200, in green). Cytotoxicity was evaluated following the appearance of varicosities...

SDS-PAGE analysis of the different BoNT/C toxins used in the study. From left to right, 250 nanograms of either native BoNT/C-wt, or recombinant BoNT/C-wt, BoNT/C α-51 or BoNT/C α-3W were loaded in a 12% gel under reducing conditions and revealed by Coomassie staining. The extent of hydrolytic activation of full-length BoNT/C by E. coli proteases w...

Mutations conferring to BoNT/C specificity for syntaxins. Space-filling representation of BoNT/C LC (PDB entry 2QN0) with highlighted triple mutations for syntaxin selectivity [32]: S51T/R52N/N53P (BoNT/C α-51) in green and L200W/M221W/I226W (BoNT/C 0078-3W) in red. Blue spot shows the metalloprotease active site. (TIF)

SNAP-25 cleaved by BoNT/C is recognized by an antibody raised against SNAP-25 cleaved by BoNT/A1. CGNs were treated with BoNT/A1 (0.1 nM) or BoNT/C-wt (0.1 nM) in normal culture medium at 37°C for 3 hours. Thereafter cells were fixed and stained with an antibody raised against SNAP-25 segment 185–197 (red) [37], corresponding to the C-terminus gene...

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The neuromuscular junction has retained through evolution the capacity to regenerate after damage, but little is known on the inter-cellular signals involved in its functional recovery from trauma, autoimmune attacks, or neurotoxins. We report here that CXCL12α, also abbreviated as stromal-derived factor-1 (SDF-1), is produced specifically by peris...

We investigated the effects of S1P3 deficiency on the age-related atrophy, decline of force and regenerative capacity of soleus muscle from 23-month-old male mice. During ageing, in old wild type mice, soleus mass and muscle fiber cross-sectional area (CSA) were reduced by about 26% and 24%, respectively, compared to the adult muscle. By contrast,...