Introduction to the Toxins Special Issue on Botulinum Toxins: New Uses in the Treatment of Diseases.

Studies on animals and humans have amply demonstrated the therapeutic efficacy of botulinum neurotoxins (BoNTs) in many pathologies [...].

Cyclin D3 deficiency promotes a slower, more oxidative skeletal muscle phenotype and ameliorates pathophysiology in the mdx mouse model of Duchenne muscular dystrophy.

We previously reported that cyclin D3-null mice display a shift toward the slow, oxidative phenotype in skeletal muscle, improved exercise endurance, and increased energy expenditure. Here, we explored the role of cyclin D3 in the physiologic response of skeletal muscle to external stimuli and in a model of muscle degenerative disease. We show that cyclin D3-null mice exhibit a further transition from glycolytic to oxidative muscle fiber type in response to voluntary exercise and an improved response to fasting. Since fast glycolytic fibers are known to be more susceptible to degeneration in Duchenne muscular dystrophy (DMD), we examined the effects of cyclin D3 inactivation on skeletal muscle phenotype in the mdx mouse model of DMD. Compared with control mdx mice, cyclin D3-deficient mdx mice display a higher proportion of slower and more oxidative myofibers, reduced muscle degenerative/regenerative processes, and reduced myofiber size variability, indicating an attenuation of dystrophic histopathology. Furthermore, mdx muscles lacking cyclin D3 exhibit reduced fatigability during repeated electrical stimulations. Notably, cyclin D3-null mdx mice show enhanced performance during recurrent trials of endurance treadmill exercise, and post-exercise muscle damage results decreased while the regenerative capacity is boosted. In addition, muscles from exercised cyclin D3-deficient mdx mice display increased oxidative capacity and increased mRNA expression of genes involved in the regulation of oxidative metabolism and the response to oxidative stress. Altogether, our findings indicate that depletion of cyclin D3 confers benefits to dystrophic muscle, suggesting that cyclin D3 inhibition may represent a promising therapeutic strategy against DMD.

Xeomin®, a Commercial Formulation of Botulinum Neurotoxin Type A, Promotes Regeneration in a Preclinical Model of Spinal Cord Injury.

Xeomin® is a commercial formulation of botulinum neurotoxin type A (BoNT/A) clinically authorized for treating neurological disorders, such as blepharospasm, cervical dystonia, limb spasticity, and sialorrhea. We have previously demonstrated that spinal injection of laboratory purified 150 kDa BoNT/A in paraplegic mice, after undergoing traumatic spinal cord injury (SCI), was able to reduce excitotoxic phenomena, glial scar, inflammation, and the development of neuropathic pain and facilitate regeneration and motor recovery. In the present study, as proof of concept in view of a possible clinical application, we studied the efficacy of Xeomin® in the same preclinical SCI model in which we highlighted the positive effects of lab-purified BoNT/A. Data comparison shows that Xeomin® induces similar pharmacological and therapeutic effects, albeit with less efficacy, to lab-purified BoNT/A. This difference, which can be improved by adjusting the dose, can be attributable to the different formulation and pharmacodynamics. Although the mechanism by which Xeomin® and laboratory purified BoNT/A induce functional improvement in paraplegic mice is still far from being understood, these results open a possible new scenario in treatment of SCI and are a stimulus for further research.

Botulinum Neurotoxins beyond Neurons: Interplay with Glial Cells.

In recent years, numerous studies have highlighted the significant use of botulinum neurotoxins (BoNTs) in the human therapy of various motor and autonomic disorders. The therapeutic action is exerted with the selective cleavage of specific sites of the SNARE's protein complex, which plays a key role in the vesicular neuroexocytosis which is responsible for neural transmission. The primary target of the BoNTs' action is the peripheral neuromuscular junction (NMJ), where, by blocking cholinergic neurons releasing acetylcholine (ACh), they interfere with neural transmission. A great deal of experimental evidence has demonstrated that BoNTs are also effective in blocking the release of other neurotransmitters or neuromodulators, such as glutamate, substance-P, and CGRP, and they can interfere with the function of glial cells, both at the peripheral and central level. The purpose of this review is to provide an update on the available experimental data from animal models that suggest or confirm the direct interactions between BoNTs and glial cells. From the data collected, it appears evident that, through mechanisms that are not yet fully understood, BoNTs can block the activation of spinal glial cells and their subsequent release of pro-inflammatory factors. BoNTs are also able to promote peripheral regeneration processes after nerve injury by stimulating the proliferation of Schwann cells. The data will be discussed in consideration of the possible therapeutic implications of the use of BoNTs on those pathological conditions where the contribution of glial cell activation is fundamental, such as in peripheral and central neuropathies.

Botulinum Neurotoxins in Central Nervous System: An Overview from Animal Models to Human Therapy.

Botulinum neurotoxins (BoNTs) are potent inhibitors of synaptic vesicle fusion and transmitter release. The natural target of BoNTs is the peripheral neuromuscular junction (NMJ) where, by blocking the release of acetylcholine (ACh), they functionally denervate muscles and alter muscle tone. This leads them to be an excellent drug for the therapy of muscle hyperactivity disorders, such as dystonia, spasticity, and many other movement disorders. BoNTs are also effective in inhibiting both the release of ACh at sites other than NMJ and the release of neurotransmitters other than ACh. Furthermore, much evidence shows that BoNTs can act not only on the peripheral nervous system (PNS), but also on the central nervous system (CNS). Under this view, central changes may result either from sensory input from the PNS, from retrograde transport of BoNTs, or from direct injection of BoNTs into the CNS. The aim of this review is to give an update on available data, both from animal models or human studies, which suggest or confirm central alterations induced by peripheral or central BoNTs treatment. The data will be discussed with particular attention to the possible therapeutic applications to pathological conditions and degenerative diseases of the CNS.

Introduction to the Toxins Special Issue on Botulinum Neurotoxins in the Nervous System: Future Challenges for Novel Indications.

Botulinum toxins (BoNTs) are a true wonder of nature [...].

Revealing the Therapeutic Potential of Botulinum Neurotoxin Type A in Counteracting Paralysis and Neuropathic Pain in Spinally Injured Mice.

Botulinum neurotoxin type A (BoNT/A) is a major therapeutic agent that has been proven to be a successful treatment for different neurological disorders, with emerging novel therapeutic indications each year. BoNT/A exerts its action by blocking SNARE complex formation and vesicle release through the specific cleavage of SNAP-25 protein; the toxin is able to block the release of pro-inflammatory molecules for months after its administration. Here we demonstrate the extraordinary capacity of BoNT/A to neutralize the complete paralysis and pain insensitivity induced in a murine model of severe spinal cord injury (SCI). We show that the toxin, spinally administered within one hour from spinal trauma, exerts a long-lasting proteolytic action, up to 60 days after its administration, and induces a complete recovery of muscle and motor function. BoNT/A modulates SCI-induced neuroglia hyperreactivity, facilitating axonal restoration, and preventing secondary cells death and damage. Moreover, we demonstrate that BoNT/A affects SCI-induced neuropathic pain after moderate spinal contusion, confirming its anti-nociceptive action in this kind of pain, as well. Our results provide the intriguing and real possibility to identify in BoNT/A a therapeutic tool in counteracting SCI-induced detrimental effects. Because of the well-documented BoNT/A pharmacology, safety, and toxicity, these findings strongly encourage clinical translation.

Botulinum Toxin and Neuronal Regeneration after Traumatic Injury of Central and Peripheral Nervous System.

Botulinum neurotoxins (BoNTs) are toxins produced by the bacteria Clostridiumbotulinum, the causing agent for botulism, in different serotypes, seven of which (A-G) are well characterized, while others, such as H or FA, are still debated. BoNTs exert their action by blocking SNARE (soluble N-ethylmale-imide-sensitive factor-attachment protein receptors) complex formation and vesicle release from the neuronal terminal through the specific cleavage of SNARE proteins. The action of BoNTs at the neuromuscular junction has been extensively investigated and knowledge gained in this field has set the foundation for the use of these toxins in a variety of human pathologies characterized by excessive muscle contractions. In parallel, BoNTs became a cosmetic drug due to its power to ward off facial wrinkles following the activity of the mimic muscles. Successively, BoNTs became therapeutic agents that have proven to be successful in the treatment of different neurological disorders, with new indications emerging or being approved each year. In particular, BoNT/A became the treatment of excellence not only for muscle hyperactivity conditions, such as dystonia and spasticity, but also to reduce pain in a series of painful states, such as neuropathic pain, lumbar and myofascial pain, and to treat various dysfunctions of the urinary bladder. This review summarizes recent experimental findings on the potential efficacy of BoNTs in favoring nerve regeneration after traumatic injury in the peripheral nervous system, such as the injury of peripheral nerves, like sciatic nerve, and in the central nervous system, such as spinal cord injury.

Lack of cyclin D3 induces skeletal muscle fiber-type shifting, increased endurance performance and hypermetabolism.

The mitogen-induced D-type cyclins (D1, D2 and D3) are regulatory subunits of the cyclin-dependent kinases CDK4 and CDK6 that drive progression through the G1 phase of the cell cycle. In skeletal muscle, cyclin D3 plays a unique function in controlling the proliferation/differentiation balance of myogenic progenitor cells. Here, we show that cyclin D3 also performs a novel function, regulating muscle fiber type-specific gene expression. Mice lacking cyclin D3 display an increased number of myofibers with higher oxidative capacity in fast-twitch muscle groups, primarily composed of myofibers that utilize glycolytic metabolism. The remodeling of myofibers toward a slower, more oxidative phenotype is accompanied by enhanced running endurance and increased energy expenditure and fatty acid oxidation. In addition, gene expression profiling of cyclin D3-/- muscle reveals the upregulation of genes encoding proteins involved in the regulation of contractile function and metabolic markers specifically expressed in slow-twitch and fast-oxidative myofibers, many of which are targets of MEF2 and/or NFAT transcription factors. Furthermore, cyclin D3 can repress the calcineurin- or MEF2-dependent activation of a slow fiber-specific promoter in cultured muscle cells. These data suggest that cyclin D3 regulates muscle fiber type phenotype, and consequently whole body metabolism, by antagonizing the activity of MEF2 and/or NFAT.

Botulinum Toxin B Affects Neuropathic Pain but Not Functional Recovery after Peripheral Nerve Injury in a Mouse Model.

Clinical use of neurotoxins from Clostridium botulinum is well established and is continuously expanding, including in treatment of pain conditions. Background: The serotype A (BoNT/A) has been widely investigated, and current data demonstrate that it induces analgesia and modulates nociceptive processing initiated by inflammation or nerve injury. Given that data concerning the serotype B (BoNT/B) are limited, the aim of the present study was to verify if also BoNT/B is able not only to counteract neuropathic pain, but also to interfere with inflammatory and regenerative processes associated with the nerve injury. Methods: As model of neuropathic pain, chronic constriction injury (CCI) of the sciatic nerve was performed in CD1 male mice. Mice were intraplantarly injected with saline (control) or BoNT/B (5 or 7.5 pg/mouse) into the injured hindpaw. For comparison, another mouse group was injected with BoNT/A (15 pg/mouse). Mechanical allodynia and functional recovery of the injured paw was followed for 101 days. Spinal cords and sciatic nerves were collected at day 7 for immunohistochemistry. Results and Conclusions: The results of this study show that BoNT/B is a powerful biological molecule that, similarly to BoNT/A, can reduce neuropathic pain over a long period of time. However, the analgesic effects are not associated with an improvement in functional recovery, clearly highlighting an important difference between the two serotypes for the treatment of this chronic pain state.

Utrophin up-regulation by artificial transcription factors induces muscle rescue and impacts the neuromuscular junction in mdx mice.

Up-regulation of the dystrophin-related gene utrophin represents a promising therapeutic strategy for the treatment of Duchenne Muscular Dystrophy (DMD). In order to re-program the utrophin expression level in muscle, we engineered artificial zinc finger transcription factors (ZF-ATFs) that target the utrophin 'A' promoter. We have previously shown that the ZF-ATF "Jazz", either by transgenic manipulation or by systemic adeno-associated viral delivery, induces significant rescue of muscle function in dystrophic "mdx" mice. We present the full characterization of an upgraded version of Jazz gene named "JZif1" designed to minimize any possible host immune response. JZif1 was engineered on the Zif268 gene-backbone using selective amino acid substitutions to address JZif1 to the utrophin 'A' promoter. Here, we show that JZif1 induces remarkable amelioration of the pathological phenotype in mdx mice. To investigate the molecular mechanisms underlying Jazz and JZif1 induced muscle functional rescue, we focused on utrophin related pathways. Coherently with utrophin subcellular localization and role in neuromuscular junction (NMJ) plasticity, we found that our ZF-ATFs positively impact the NMJ. We report on ZF-ATF effects on post-synaptic membranes in myogenic cell line, as well as in wild type and mdx mice. These results candidate our ZF-ATFs as novel therapeutic molecules for DMD treatment.

Botulinum neurotoxin A promotes functional recovery after peripheral nerve injury by increasing regeneration of myelinated fibers.

The injection of safe doses of botulinum neurotoxin A (BoNT/A) have been reported to be useful for the treatment of neuropathic pain, but it is still unknown how functional recovery is induced after peripheral nerve injury. We evaluated the effects of intranerve application of BoNT/A, on regeneration and sensorimotor functional recovery in partial and complete peripheral nerve injuries in the mouse. After sciatic nerve crush (SNC) and intranerve delivery of BoNT/A (15pg), axonal regeneration was measured by nerve pinch test at different days. Regeneration of myelinated and unmyelinated fibers was assessed by immunohistochemical double labeling for NF200/GAP43 and CGRP/GAP43. S100 was used as Schwann cells marker. Medial footpad skin reinnervation was assessed by PGP staining. Motor functions were assessed by means of nerve conduction tests. In other mice groups, nerve conduction tests were performed also after chronic constriction injury (CCI) of the sciatic nerve and intraplantar injection of BoNT/A (15pg). In SNC mice, BoNT/A increased the rate of axonal regeneration. The advantage of regrowing myelinated axons after BoNT/A injection was evidenced by longer NF200+ nerve profiles and confirmed by nerve histology. We observed also a higher expression of S100 in the distal portion of BoNT/A-injected regenerated nerves. In CCI mice, BoNT/A induced an increase in reinnervation of gastrocnemius and plantar muscles. These results show that a low dose of BoNT/A, insufficient to produce muscular dysfunction, conversely speeds up sensorimotor recovery by stimulating myelinated axonal regeneration, and points out its application as a multipotent treatment for peripheral neuropathies.

Dataset of botulinum toxin A influence on interleukins under neuropathy.

Our data show that botulinum toxin A (BoNT/A) didn't influence motor functions in naïve and CCI-exposed rats, but diminished the neuropathic pain-related behavior. The results indicate that BoNT/A administration diminished the spinal Iba-1 positive cells activation and, in parallel, downregulated IL-1beta. Moreover, we observed that in DRG the protein level of pronociceptive factors (IL-1beta and IL-18) decreased and antinociceptive (IL-10 and IL-1RA) factors increased. Additionally, our behavioral analysis shows that chronic minocycline treatment together with a single BoNT/A injection in CCI-exposed rats has beneficial analgesic effects (M. Zychowska, E. Rojewska, W. Makuch, S. Luvisetto, F. Pavone, S. Marinelli, B. Przewlocka, J. Mika, 2016) [1].

Participation of pro- and anti-nociceptive interleukins in botulinum toxin A-induced analgesia in a rat model of neuropathic pain.

Botulinum neurotoxin serotype A (BoNT/A) shows antinociceptive properties, and its clinical applications in pain therapy are continuously increasing. BoNT/A specifically cleaves SNAP-25, which results in the formation of a non-functional SNARE complex, thereby potently inhibiting the release of neurotransmitters and neuropeptides, including those involved in nociception. The aim of the present study was to determine the effects of BoNT/A (300pg/paw) on pain-related behavior and the levels of glial markers and interleukins in the spinal cord and dorsal root ganglia (DRG) after chronic constriction injury (CCI) to the sciatic nerve in rats. Glial activity was also examined after repeated intraperitoneal injection of minocycline combined with a single BoNT/A injection. Our results show that a single intraplantar BoNT/A injection did not influence motor function but strongly diminished pain-related behaviors in naïve and CCI-exposed rats. Additionally, microglial inhibition using minocycline enhanced the analgesic effects of BoNT/A. Western blotting results suggested that CCI induces the upregulation of the pronociceptive proteins IL-18, IL-6 and IL-1ß in the ipsilateral lumbar spinal cord and DRG, but no changes in the levels of the antinociceptive proteins IL-18BP, IL-1RA and IL-10 were observed. Interestingly, BoNT/A injection suppressed the CCI-induced upregulation of IL-18 and IL-1ß in the spinal cord and/or DRG and increased the levels of IL-10 and IL-1RA in the DRG. In summary, our results suggest that BoNT/A significantly attenuates pain-related behavior and microglial activation and restores the neuroimmune balance in a CCI model by decreasing the levels of pronociceptive factors (IL-1ß and IL-18) and increasing the levels of antinociceptive factors (IL-10 and IL-1RA) in the spinal cord and DRG.

17beta-estradiol counteracts neuropathic pain: a behavioural, immunohistochemical, and proteomic investigation on sex-related differences in mice.

Sex differences play a role in pain sensitivity, efficacy of analgesic drugs and prevalence of neuropathic pain, even if the underlying mechanisms are far from being understood. We demonstrate that male and female mice react differently to structural and functional changes induced by sciatic nerve ligature, used as model of neuropathic pain. Male mice show a gradual decrease of allodynia and a complete recovery while, in females, allodynia and gliosis are still present four months after neuropathy induction. Administration of 17ß-estradiol is able to significantly attenuate this difference, reducing allodynia and inducing a complete recovery also in female mice. Parallel to pain attenuation, 17ß-estradiol treated-mice show a functional improvement of the injured limb, a faster regenerative process of the peripheral nerve and a decreased neuropathy-induced gliosis. These results indicate beneficial effects of 17ß-estradiol on neuropathic pain and neuronal regeneration and focuses on the importance of considering gonadal hormones also in clinical studies.

Pathways Implicated in Tadalafil Amelioration of Duchenne Muscular Dystrophy.

Numerous therapeutic approaches for Duchenne and Becker Muscular Dystrophy (DMD and BMD), the most common X-linked muscle degenerative disease, have been proposed. So far, the only one showing a clear beneficial effect is the use of corticosteroids. Recent evidence indicates an improvement of dystrophic cardiac and skeletal muscles in the presence of sustained cGMP levels secondary to a blocking of their degradation by phosphodiesterase five (PDE5). Due to these data, we performed a study to investigate the effect of the specific PDE5 inhibitor, tadalafil, on dystrophic skeletal muscle function. Chronic pharmacological treatment with tadalafil has been carried out in mdx mice. Behavioral and physiological tests, as well as histological and biochemical analyses, confirmed the efficacy of the therapy. We then performed a microarray-based genomic analysis to assess the pattern of gene expression in muscle samples obtained from the different cohorts of animals treated with tadalafil. This scrutiny allowed us to identify several classes of modulated genes. Our results show that PDE5 inhibition can ameliorate dystrophy by acting at different levels. Tadalafil can lead to (1) increased lipid metabolism; (2) a switch towards slow oxidative fibers driven by the up-regulation of PGC-1α; (3) an increased protein synthesis efficiency; (4) a better actin network organization at Z-disk.

Altered cerebellum development and impaired motor coordination in mice lacking the Btg1 gene: Involvement of cyclin D1.

Cerebellar granule neurons develop postnatally from cerebellar granule precursors (GCPs), which are located in the external granule layer (EGL) where they massively proliferate. Thereafter, GCPs become postmitotic, migrate inward to form the internal granule layer (IGL), further differentiate and form synapses with Purkinje cell dendrites. We previously showed that the Btg family gene, Tis21/Btg2, is required for normal GCP migration. Here we investigated the role in cerebellar development of the related gene, Btg1, which regulates stem cell quiescence in adult neurogenic niches, and is expressed in the cerebellum. Knockout of Btg1 in mice caused a major increase of the proliferation of the GCPs in the EGL, whose thickness increased, remaining hyperplastic even after postnatal day 14, when the EGL is normally reduced to a few GCP layers. This was accompanied by a slight decrease of differentiation and migration of the GCPs and increase of apoptosis. The GCPs of double Btg1/Tis21-null mice presented combined major defects of proliferation and migration outside the EGL, indicating that each gene plays unique and crucial roles in cerebellar development. Remarkably, these developmental defects lead to a permanent increase of the adult cerebellar volume in Btg1-null and double mutant mice, and to impairment in all mutants, including Tis21-null, of the cerebellum-dependent motor coordination. Gain- and loss-of-function strategies in a GCP cell line revealed that Btg1 regulates the proliferation of GCPs selectively through cyclin D1. Thus, Btg1 plays a critical role for cerebellar maturation and function.

Botulinum Toxin Type a as a Therapeutic Agent against Headache and Related Disorders.

Botulinum neurotoxin A (BoNT/A) is a toxin produced by the naturally-occurring Clostridium botulinum that causes botulism. The potential of BoNT/A as a useful medical intervention was discovered by scientists developing a vaccine to protect against botulism. They found that, when injected into a muscle, BoNT/A causes a flaccid paralysis. Following this discovery, BoNT/A has been used for many years in the treatment of conditions of pathological muscle hyperactivity, like dystonias and spasticities. In parallel, the toxin has become a "glamour" drug due to its power to ward off facial wrinkles, particularly frontal, due to the activity of the mimic muscles. After the discovery that the drug also appeared to have a preventive effect on headache, scientists spent many efforts to study the potentially-therapeutic action of BoNT/A against pain. BoNT/A is effective at reducing pain in a number of disease states, including cervical dystonia, neuropathic pain, lower back pain, spasticity, myofascial pain and bladder pain. In 2010, regulatory approval for the treatment of chronic migraine with BoNT/A was given, notwithstanding the fact that the mechanism of action is still not completely elucidated. In the present review, we summarize experimental evidence that may help to clarify the mechanisms of action of BoNT/A in relation to the alleviation of headache pain, with particular emphasis on preclinical studies, both in animals and humans. Moreover, we summarize the latest clinical trials that show evidence on headache conditions that may obtain benefits from therapy with BoNT/A.

Effects of age-related loss of P/Q-type calcium channels in a mice model of peripheral nerve injury.

We analyzed the role of P/Q-type calcium channels in sciatic nerve regeneration after lesion induced by chronic constriction injury (CCI) in heterozygous null mutant mice lacking the CaV2.1α1 subunit of these channels (Cacna1a+/-). Compared with wild type, Cacna1a+/- mice showed an initial reduction of the CCI-induced allodynia, indicating a reduced pain perception, but they also evidenced a lack of recovery over time, with atrophy of the injured hindpaw still present 3 months after CCI when wild-type mice fully recovered. In parallel, Cacna1a+/- mice exhibited an early onset of age-dependent loss of P/Q-type channels, which can be responsible for the lack of functional recovery. Moreover, Cacna1a+/- mice showed an early age-dependent reduction of muscular strength, as well as of Schwann cells proliferation and sciatic nerve remyelination. This study demonstrates the important role played by P/Q-type channels in recovery from nerve injury and has important implications for the knowledge of age-related processes.

Analgesic effects of botulinum neurotoxin type A in a model of allyl isothiocyanate- and capsaicin-induced pain in mice.

We evaluate analgesic effects of BoNT/A in relation to the two main transient receptor potentials (TRP), the vanilloid 1 (TRPV1) and the ankyrin 1 (TRPA1), having a role in migraine pain. BoNT/A (15 pg/mouse) was injected in the inner side of the medial part of hindlimb thigh of mice, where the superficial branch of femoral artery is located. We chosen this vascular structure because it is similar to other vascular structures, such as the temporal superficial artery, whose perivascular nociceptive fibres probably contributes to migraine pain. After an interval, ranging from 7 to 30 days, capsaicin (agonist of TRPV1) or allyl isothiocyanate (AITC; agonist of TRPA1) were injected in the same region previously treated with BoNT/A and nocifensive response to chemicals-induced pain was recorded. In absence of BoNT/A, capsaicin and AITC induced extensive nocifensive response, with a markedly different temporal profile: capsaicin induced maximal pain during the first 5 min, while AITC induced maximal pain at 15-30 min after injection. Pretreatment with BoNT/A markedly reduced both the capsaicin- and AITC-induced pain for at least 21 days. These data suggest a long lasting analgesic effect of BoNT/A exerted via prevention of responsiveness of TRPV1 and TRPA1 toward their respective agonists.