RESUMO
Dopaminergic neurons of the substantia nigra exist in a persistent state of vulnerability resulting from high baseline oxidative stress, high-energy demand, and broad unmyelinated axonal arborisations. Impairments in the storage of dopamine compound this stress because of cytosolic reactions that transform the vital neurotransmitter into an endogenous neurotoxicant, and this toxicity is thought to contribute to the dopamine neuron degeneration that occurs Parkinson's disease. We have previously identified synaptic vesicle glycoprotein 2C (SV2C) as a modifier of vesicular dopamine function, demonstrating that genetic ablation of SV2C in mice results in decreased dopamine content and evoked dopamine release in the striatum. Here, we adapted a previously published in vitro assay utilising false fluorescent neurotransmitter 206 (FFN206) to visualise how SV2C regulates vesicular dopamine dynamics and determined that SV2C promotes the uptake and retention of FFN206 within vesicles. In addition, we present data indicating that SV2C enhances the retention of dopamine in the vesicular compartment with radiolabelled dopamine in vesicles isolated from immortalised cells and from mouse brain. Further, we demonstrate that SV2C enhances the ability of vesicles to store the neurotoxicant 1-methyl-4-phenylpyridinium (MPP+) and that genetic ablation of SV2C results in enhanced 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced vulnerability in mice. Together, these findings suggest that SV2C functions to enhance vesicular storage of dopamine and neurotoxicants and helps maintain the integrity of dopaminergic neurons.
Assuntos
Dopamina , Neurônios Dopaminérgicos , Glicoproteínas de Membrana , Proteínas do Tecido Nervoso , Vesículas Sinápticas , Animais , Humanos , Camundongos , Corpo Estriado/metabolismo , Corpo Estriado/efeitos dos fármacos , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/efeitos dos fármacosRESUMO
OBJECTIVES: The mature botulinum neurotoxin (BoNT) is a long peptide chain consisting of a light chain (L) and a heavy chain (H) linked by a disulfide bond, where the heavy chain is divided into a translocation domain and an acceptor binding domain (Hc). In this study, we further explored the biology activity and characteristics of recombinant L-HN fragment (EL-HN) composed of the L and HN domains of BoNT/E in vivo and in vitro. METHODS: Neurotoxicity of L-HN fragments from botulinum neurotoxins was assessed in mice. Cleavage of dichain EL-HN in vitro and in neuro-2a cells was assessed and compared with that of single chain EL-HN. Interaction of HN domain and the receptor synaptic vesicle glycoprotein 2C (SV2C) was explored in vitro and in neuro-2a cells only expressing SV2C. RESULTS: We found that the 50% mouse lethal dose of the nicked dichain EL-HN fragment (EL-HN-DC) was 0.5 µg and its neurotoxicity was the highest among the L-HN's of the four serotypes of BoNT (A/B/E/F). The cleavage efficiency of EL-HN-DC toward synaptosome associated protein 25 (SNAP25) in vitro was 3-fold higher than that of the single chain at the cellular level, and showed 200-fold higher animal toxicity. The EL-HN-DC fragment might enter neuro-2a cells via binding to SV2C to efficiently cleave SNAP25. CONCLUSIONS: The EL-HN fragment showed good biological activities in vivo and in vitro, and could be used as a drug screening model and to further explore the molecular mechanism of its transmembrane transport.
Assuntos
Toxinas Botulínicas Tipo A , Camundongos , Animais , Toxinas Botulínicas Tipo A/toxicidade , Toxinas Botulínicas Tipo A/química , Toxinas Botulínicas Tipo A/genética , Sorogrupo , BiologiaRESUMO
Botulinum neurotoxin subtype A4 (BoNT/A4) is ~1000-fold less potent than BoNT/A1. This study addresses the basis for low BoNT/A4 potency. Utilizing BoNT/A1-A4 and BoNT/A4-A1 Light Chain-Heavy Chain (LC-HC) chimeras, HC-A4 was responsible for low BoNT/A4 potency. Earlier studies showed BoNT/A1-receptor binding domain (Hcc) bound a ß-strand peptide (556-564) and glycan-N559 within Luminal Domain 4 (LD4) of SV2C, the BoNT/A protein receptor. Relative to BoNT/A1, the Hcc of BoNT/A4 possesses two amino acid variants (D1141 and N1142) within the ß-peptide binding interface and one amino acid variant (R1292) located near the SV2C glycan-N559. Introduction of BoNT/A4 ß-strand peptide variant (D1141 and N1142) into BoNT/A1 reduced toxin potency 30-fold, and additional introduction of the BoNT/A4 glycan-N559 variant (D1141, N1142, and R1292) further reduced toxin potency to approach BoNT/A4. While introduction of BoNT/A1 glycan-N559 variant (G1292) into BoNT/A4 did not alter toxin potency, additional introduction of BoNT/A1 ß-strand peptide variants (G1141, S1142, and G1292) resulted in potency approaching BoNT/A1 potency. Thus, outcomes from these functional and modeling studies indicate that in rodent models, disruption of Hcc -SV2C ß-peptide and -glycan-N559 interactions mediate low BoNT/A4 potency, while in human motor neurons, disruption of Hcc-SV2C ß-peptide alone mediates low BoNT/A4 potency, which link to a species-specific variation at SV2C563.
Assuntos
Aminoácidos , Humanos , Ligação Proteica , Domínios ProteicosRESUMO
BACKGROUND: The synaptic vesicle glycoprotein 2 (SV2) family is essential to the synaptic machinery involved in neurotransmission and vesicle recycling. The isoforms SV2A, SV2B and SV2C are implicated in neurological diseases such as epilepsy, Alzheimer's and Parkinson's disease. Suitable cell systems for studying regulation of these proteins are essential. Here we present gene expression data of SV2A, SV2B and SV2C in two human neuroblastoma cell lines after differentiation. METHODS: Human neuroblastoma cell lines SiMa and IMR-32 were treated for seven days with growth supplements (B-27 and N-2), all-trans-retinoic acid (ATRA) or vasoactive intestinal peptide (VIP) and gene expression levels of SV2 and neuronal targets were analyzed. RESULTS: The two cell lines reacted differently to the treatments, and only one of the three SV2 isoforms was affected at a time. SV2B and choline O-acetyltransferase (CHAT) expression was changed in concert after growth supplement treatment, decreasing in SiMa cells while increasing in IMR-32. ATRA treatment resulted in no detected changes in SV2 expression in either cell line while VIP increased both SV2C and dopamine transporter (DAT) in IMR-32 cells. CONCLUSION: The synergistic expression patterns between SV2B and CHAT as well as between SV2C and DAT mirror the connectivity between these targets found in disease models and knock-out animals, although here no genetic alteration was made. These cell lines and differentiation treatments could possibly be used to study SV2 regulation and function.
Assuntos
Diferenciação Celular/genética , Regulação Neoplásica da Expressão Gênica , Glicoproteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Neuroblastoma/genética , Neuroblastoma/patologia , Sítios de Ligação , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Tretinoína/farmacologia , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
This study aimed to explore gene expression changes in the inferior colliculus (IC) after single-sided deafness (SSD). Forty 8-week-old female Sprague-Dawley rats were used. Twenty rats underwent right-side cochlear ablation, and IC tissues were harvested after 2 weeks (SSD 2-week group). Twenty rats underwent a sham operation and were sacrificed after 2 weeks (control group). Both sides of the IC were analyzed using a gene expression array. Pathway analyses were performed on genes that were differentially expressed compared with their levels in the control group. The expression levels of genes involved in the candidate pathways were confirmed using reverse transcription polymerase chain reaction (RT-PCR). Among the genes with ≥ 1.5-fold changes in expression levels and P < 0.05, there were 7 and 9 genes with increased and decreased expression, respectively, in the ipsilateral IC and 10 and 12 genes with increased and decreased expression, respectively, in the contralateral IC. The pathway analysis did not identify significantly related pathway. In the bilateral analysis, a total of 14 genes were ≥ 1.3-fold downregulated in both the ipsilateral and contralateral IC in the SSD 2-week group compared with their expression in the control group. Pathway analyses of these 14 genes included 7 genes, namely, amine compound solute carrier (Slc)5a7; Slc18a3; Slc6a5; synaptic vesicle glycoprotein 2C (Sv2c); S100 calcium binding protein A10 (S100a10); a gene with sequence similarity to family 111, member A (Fam111a); and peripherin (Prph), that were related to the acetylcholine neurotransmitter release cycle, SLC transporters, and the neurotransmitter release cycle pathways. RT-PCR showed reduced expression of Slc5a7, Sv2c, and Prph in the contralateral IC and Slc18a3 and Slc6a5 in the ipsilateral IC of the SSD 2-week group compared with that in the control group.
Assuntos
Cóclea/cirurgia , Perfilação da Expressão Gênica , Colículos Inferiores/metabolismo , Animais , Limiar Auditivo , Feminino , Perda Auditiva/genética , Perda Auditiva/fisiopatologia , Colículos Inferiores/cirurgia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Members of the synaptic vesicle glycoprotein 2 (SV2) family of proteins are involved in synaptic function throughout the brain. The ubiquitously expressed SV2A has been widely implicated in epilepsy, although SV2C with its restricted basal ganglia distribution is poorly characterized. SV2C is emerging as a potentially relevant protein in Parkinson disease (PD), because it is a genetic modifier of sensitivity to l-DOPA and of nicotine neuroprotection in PD. Here we identify SV2C as a mediator of dopamine homeostasis and report that disrupted expression of SV2C within the basal ganglia is a pathological feature of PD. Genetic deletion of SV2C leads to reduced dopamine release in the dorsal striatum as measured by fast-scan cyclic voltammetry, reduced striatal dopamine content, disrupted α-synuclein expression, deficits in motor function, and alterations in neurochemical effects of nicotine. Furthermore, SV2C expression is dramatically altered in postmortem brain tissue from PD cases but not in Alzheimer disease, progressive supranuclear palsy, or multiple system atrophy. This disruption was paralleled in mice overexpressing mutated α-synuclein. These data establish SV2C as a mediator of dopamine neuron function and suggest that SV2C disruption is a unique feature of PD that likely contributes to dopaminergic dysfunction.
Assuntos
Dopamina/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Doença de Parkinson/metabolismo , Vesículas Sinápticas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Gânglios da Base/metabolismo , Biomarcadores , Modelos Animais de Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Feminino , Deleção de Genes , Expressão Gênica , Humanos , Locomoção , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Nicotina/metabolismo , Nicotina/farmacologia , Doença de Parkinson/genética , Doença de Parkinson/fisiopatologia , Ligação Proteica , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
The synaptic vesicle protein 2 (SV2) is involved in synaptic vesicle trafficking. The SV2A isoform is the most studied and its implication in epilepsy therapy led to the development of the first SV2A PET radiotracer [18F]UCB-H. The objective of this study was to evaluate in vivo, using microPET in rats, the specificity of [18F]UCB-H for SV2 isoform A in comparison with the other two isoforms (B and C) through a blocking assay. Twenty Sprague Dawley rats were pre-treated either with the vehicle, or with specific competitors against SV2A (levetiracetam), SV2B (UCB5203) and SV2C (UCB0949). The distribution volume (Vt, Logan plot, t* 15 min) was obtained with a population-based input function. The Vt analysis for the entire brain showed statistically significant differences between the levetiracetam group and the other groups (p < 0.001), but also between the vehicle and the SV2B group (p < 0.05). An in-depth Vt analysis conducted for eight relevant brain structures confirmed the statistically significant differences between the levetiracetam group and the other groups (p < 0.001) and highlighted the superior and the inferior colliculi along with the cortex as regions also displaying statistically significant differences between the vehicle and SV2B groups (p < 0.05). These results emphasize the in vivo specificity of [18F]UCB-H for SV2A against SV2B and SV2C, confirming that [18F]UCB-H is a suitable radiotracer for in vivo imaging of the SV2A proteins with PET.
Assuntos
Encéfalo/diagnóstico por imagem , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Piridinas/metabolismo , Pirrolidinonas/metabolismo , Animais , Encéfalo/metabolismo , Levetiracetam/administração & dosagem , Levetiracetam/farmacologia , Imageamento por Ressonância Magnética , Masculino , Modelos Animais , Estrutura Molecular , Tomografia por Emissão de Pósitrons , Piridinas/química , Pirrolidinonas/química , Ratos , Ratos Sprague-Dawley , Sensibilidade e EspecificidadeRESUMO
Objective: Individual differences were observed in the clinical efficacy of Botulinum toxin A (BoNT-A) in the treatment of the primary Meige syndrome. Our study aimed to explore the potential associations between the clinical efficacy of BoNT-A in the treatment of the primary Meige syndrome and variants of SNAP25, SV2C and ST3GAL2, which are involving in the translocation of the BoNT-A in vivo. Methods: Patients with the primary Meige syndrome treated with BoNT-A were enrolled. Clinical efficacy was evaluated by the maximum improvement rate of motor symptoms and the duration of efficacy. Variants of SNAP25, SV2C and ST3GAL2 were obtained by Sanger sequencing. Another cohort diagnosed with primary cervical dystonia was also enrolled in the replication stage. Results: Among the 104 primary Meige syndrome patients, 80 patients (76.9%) had a good efficacy (the maximum improvement rate of motor symptoms ≥30%) and 24 (23. 1%) had a poor (the maximum improvement rate of motor symptoms <30%). As to the duration of efficacy, 52 patients (50.0%) had a long duration of efficacy (≥4 months), and 52 (50.0%) had a short (<4 months). In terms of primary Meige syndrome, SNAP25 rs6104571 was found associating with the maximum improvement rate of motor symptoms (Genotype: P = 0.02, OR = 0.26; Allele: P = 0.013, OR = 0.29), and SV2C rs31244 was found associating with the duration of efficacy (Genotype: P = 0.024, OR = 0.13; Allele: P = 0.012, OR = 0.13). Besides, we also conducted the association analyses between the variants and BoNT-A-related adverse reactions. Although, there was no statistical difference between the allele of SV2C rs31244 and BoNT-A-related adverse reactions, there was a trend (P = 0.077, OR = 2.56). In the replication stage, we included 39 patients with primary cervical dystonia to further expanding the samples' size. Among the 39 primary cervical dystonia patients, 25 patients (64.1%) had a good efficacy (the maximum improvement rate of motor symptoms ≥50%) and 14 (35.9%) had a poor (the maximum improvement rate of motor symptoms <50%). As to the duration of efficacy, 32 patients (82.1%) had a long duration of efficacy (≥6 months), and 7 (17.9%) had a short (<6 months). Integrating primary Meige syndrome and primary cervical dystonia, SV2C rs31244 was still found associating with the duration of efficacy (Genotype: P = 0.002, OR = 0. 23; Allele: P = 0.001, OR = 0. 25). Conclusion: In our study, SNAP25 rs6104571 was associated with the maximum improvement rate of motor symptoms in patients with primary Meige syndrome treated with BoNT-A, and patients carrying this variant had a lower improvement rate of motor symptoms. SV2C rs31244 was associated with duration of treatment in patients with primary Meige syndrome treated with BoNT-A and patients carrying this variant had a shorter duration of treatment. Patients with primary Meige syndrome carrying SV2C rs31244 G allele have an increase likelihood of BoNT-A-related adverse reactions. Involving 39 patients with primary cervical dystonia, the results further verify that SV2C rs31244 was associated with duration of treatment and patients carrying this variant had a shorter duration of treatment.
RESUMO
BACKGROUND: The synaptic vesicle glycoprotein 2 (SV2) has been implicated in synaptic function throughout the brain. Accumulating evidence investigated that SV2C contributed to dopamine release and the disrupted expression of SV2C was considered to be a unique feature of PD that may facilitate dopaminergic neuron dysfunction. OBJECTIVE: This study aimed to examine the relationship between the SV2C rs1423099 single nucleotide polymorphism and sporadic Parkinson's disease (PD) in the Chinese Han population. MATERIALS AND METHODS: This study enrolled 351 patients with sporadic PD and 240 normal controls in Chinese Han population. Peripheral blood DNA was extracted by DNA extraction kits and the rs1423099 genotype was analyzed by Agena MassARRAY DNA mass spectrometry. The differences in genotype and allele distribution frequencies between PD patients and control groups were compared using chi-squared tests or Fisher's exact tests. RESULTS: No statistical difference was revealed in age and sex distribution between the cases and control groups, and the distribution of genotype and allele frequencies was consistent with the Hardy-Weinberg equilibrium test. In SV2C rs1423099 dominant model, the frequency of the CC/CT genotype was significantly higher in the PD group compared to the control group (OR = 4.065,95% CI: 2.801-10.870, p = 0.002). Nevertheless, in the recessive model, CC or CT/TT genotypes have no statistical difference in the two groups (p = 0.09). Additionally, in allelic analysis, the C allele was investigated to increase the risk of PD (OR = 1.346, 95% CI: 1.036-1.745, p = 0.026); Furthermore, subgroup analysis suggested that those carrying the C allele in the male subgroup were at a higher risk to afflicted with PD (OR = 1.637, 95% CI: 1.147-2.336, p = 0.006). CONCLUSION: SV2C rs1423099 single nucleotide polymorphism was associated with sporadic Parkinson's disease in the Chinese Han population, particularly in males.
Assuntos
Doença de Parkinson , Polimorfismo de Nucleotídeo Único , Humanos , Masculino , Estudos de Casos e Controles , China , Frequência do Gene , Predisposição Genética para Doença/genética , Genótipo , Glicoproteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Doença de Parkinson/genética , FemininoRESUMO
Noradrenaline (NE) plays an integral role in shaping behavioral outcomes including anxiety/depression, fear, learning and memory, attention and shifting behavior, sleep-wake state, pain, and addiction. However, it is unclear whether dysregulation of NE release is a cause or a consequence of maladaptive orientations of these behaviors, many of which associated with psychiatric disorders. To address this question, we used a unique genetic model in which the brain-specific vesicular monoamine transporter-2 (VMAT2) gene expression was removed in NE-positive neurons disabling NE release in the entire brain. We engineered VMAT2 gene splicing and NE depletion by crossing floxed VMAT2 mice with mice expressing the Cre-recombinase under the dopamine ß-hydroxylase (DBH) gene promotor. In this study, we performed a comprehensive behavioral and transcriptomic characterization of the VMAT2DBHcre KO mice to evaluate the role of central NE in behavioral modulations. We demonstrated that NE depletion induces anxiolytic and antidepressant-like effects, improves contextual fear memory, alters shifting behavior, decreases the locomotor response to amphetamine, and induces deeper sleep during the non-rapid eye movement (NREM) phase. In contrast, NE depletion did not affect spatial learning and memory, working memory, response to cocaine, and the architecture of the sleep-wake cycle. Finally, we used this model to identify genes that could be up- or down-regulated in the absence of NE release. We found an up-regulation of the synaptic vesicle glycoprotein 2c (SV2c) gene expression in several brain regions, including the locus coeruleus (LC), and were able to validate this up-regulation as a marker of vulnerability to chronic social defeat. The NE system is a complex and challenging system involved in many behavioral orientations given it brain wide distribution. In our study, we unraveled specific role of NE neurotransmission in multiple behavior and link it to molecular underpinning, opening future direction to understand NE role in health and disease.
Assuntos
Encéfalo , Transcriptoma , Camundongos , Animais , Encéfalo/metabolismo , Norepinefrina/metabolismo , Depressão/metabolismo , Antidepressivos/farmacologiaRESUMO
Botulinum neurotoxin A1 (BoNT/A1) is the most potent natural poison in human. BoNT/A1 recognize the luminal domain of SV2A (LD-SV2A) and its glycosylation at position N573 (N573g) or the luminal domain of SV2C (LD-SV2C) and its glycosylation at position N559 (N559g) to bind neural membrane. Our computational data suggest that the N-glycan at position 480 (N480g) in the luminal domain of SV2C (LD-SV2C) indirectly enhanced the contacts of the neurotoxin surface with the second N-glycan at position 559 (N559g) by acting as a shield to prevent N559g to interact with residues of LD-SV2C. The absence of an N-glycan homologous to N480g in LD-SV2A leads to a decrease of the binding of N573g to the surface of BoNT/A1. Concerning the intermolecular interactions between BoNT/A and the protein part of LD-SV2A or LD-SV2C, we showed that the high affinity of the neurotoxin for binding LD-SV2C are mediated by a better compaction of its F557-F562 part provided by a π-π network mediated by residues F547, F552, F557 and F562 coupled with the presence of two aromatic residues at position 563 and 564 that optimize the binding of BoNT/A1 via cation-pi and CH-pi interaction. Finally, in addition to the well-known ganglioside binding site which accommodates a ganglioside on the surface of BoNT/A1, we identified a structure we coined the ganglioside binding loop defined by the sequence 1253-HQFNNIAK-1260 that is conserved across all subtypes of BoNT/A and is predicted to has a high affinity to interact with gangliosides. These data solved the puzzle generated by mutational studies that could be only partially understood with crystallographic data that lack both a biologically relevant membrane environment and a full glycosylation of SV2.
Assuntos
Gangliosídeos , Neurotoxinas , Humanos , Sorogrupo , Ligação Proteica , Sítios de Ligação , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismoRESUMO
The synaptic vesicle glycoprotein 2C (SV2C) is an undercharacterized protein with enriched expression in phylogenetically old brain regions. Its precise role within the brain is unclear, though various lines of evidence suggest that SV2C is involved in the function of synaptic vesicles through the regulation of vesicular trafficking, calcium-induced exocytosis, or synaptotagmin function. SV2C has been linked to multiple neurological disorders, including Parkinson's disease and psychiatric conditions. SV2C is expressed in various cell types-primarily dopaminergic, GABAergic, and cholinergic cells. In mice, it is most highly expressed in nuclei within the basal ganglia, though it is unknown if this pattern of expression is consistent across species. Here, we use a custom SV2C-specific antiserum to describe localization within the brain of mouse, nonhuman primate, and human, including cell-type localization. We found that the immunoreactivity with this antiserum is consistent with previously-published antibodies, and confirmed localization of SV2C in the basal ganglia of rodent, rhesus macaque, and human. We observed strongest expression of SV2C in the substantia nigra, ventral tegmental area, dorsal striatum, pallidum, and nucleus accumbens of each species. Further, we demonstrate colocalization between SV2C and markers of dopaminergic, GABAergic, and cholinergic neurons within these brain regions. SV2C has been increasingly linked to dopamine and basal ganglia function. These antisera will be an important resource moving forward in our understanding of the role of SV2C in vesicle dynamics and neurological disease.
Assuntos
Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Animais , Gânglios da Base/metabolismo , Encéfalo/metabolismo , Neurônios Colinérgicos/metabolismo , Neurônios Dopaminérgicos/metabolismo , Neurônios GABAérgicos/metabolismo , Perfilação da Expressão Gênica/métodos , Células HEK293 , Humanos , Soros Imunes/imunologia , Imuno-Histoquímica/métodos , Macaca , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Doença de Parkinson/metabolismo , Vesículas Sinápticas/metabolismo , Transcriptoma/genéticaRESUMO
BACKGROUND: Botulinum neurotoxin type A (BoNT/A) is emerging as a treatment modality for Raynaud's phenomenon (RP). However, the mechanism of the role of BoNT/A in antagonizing the constriction of arteriola in RP remains unclear. MATERIALS AND METHODS: We tested the constriction of arteriole diameter and the distribution of adrenergic receptors on the rat cremaster modle. Moreover, we measured the secretion of norepinephrine (NE), protein level changes and related receptors on cultured rat superior cervical ganglia neurons(SCGNs), a model of sympathetic neuron. RESULTS: Based on our results, the inhibition of arteriole vasoconstriction was increased with increasing doses of BoNT/A. BoNT/A, prazosin, and BQ123 treatment can result in significant inhibition of arteriole vasoconstriction with the same electrical stimulation. The inhibition effect of prazosin was equivalent to BoNT/A, while BQ123 has a synergistic effect with BoNT/A. After treating SCGNs using BoNT/A for 30 min, the decrease in fluorescence intensity of FM1-43 slowed down which was correlated with the doses of BoNT/A. Furthermore, release of NE in the supernatant was significantly decreased as measured by enzyme-linked immunosorbent assay, 24 h after a high dose of BoNT/A (25 µ/mL). Cleaved-SNAP-25 was detected by Western blotting 24 h following BoNT/A (50 µ/mL) treatment. Moreover, receptor SV2C, GM1, and FGFR3 were detected on sympathetic neurons, similarly to cholinergic neurons. CONCLUSION: Our study showed that BoNT/A could significantly inhibit electrical stimulation-induced arteriole vasoconstriction through the sympathetic pathway. The mechanism was similar to the cholinergic one, in which the vesicle release of sympathetic neurons could be inhibited by cleavage of SNAP-25. The end result was blocked vesicle fusion with the presynaptic membrane after BoNT/A treatment, inhibiting the release of the NE.
Assuntos
Toxinas Botulínicas Tipo A/uso terapêutico , Doença de Raynaud/tratamento farmacológico , Animais , Arteríolas/efeitos dos fármacos , Arteríolas/fisiologia , Toxinas Botulínicas Tipo A/farmacologia , Relação Dose-Resposta a Droga , Masculino , Norepinefrina/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos alfa 1/análise , Proteína 25 Associada a Sinaptossoma/fisiologiaRESUMO
Synaptic vesicle 2 C (SV2C) is a novel isoform belonging to the synaptic vesicle 2 (SV2) protein superfamily; a family of proteins known to have roles in vesicle trafficking, exocytosis and neurotransmission. In humans, SV2C is expressed in evolutionarily older brain regions, and is a known receptor for botulinum neurotoxin/A (BoNT/A), controlling glucose-evoked granule recruitment and regulating dopamine release, thus serving as a potential target molecule in the treatment of psychosis. In addition, recent researches have shown that SV2C regulates hypertension and accelerates venous thromboembolism (VTE) and coagulation pathways and may play roles in several non-nervous system diseases. In terms of regulation, SV2C is positively regulated by both alendronate and statins. As SV2C may provide a potential novel therapeutic target for psychosis and other diseases, this article reviews the progress made thus far in understanding the structure, distribution, function and regulation of SV2C.
Assuntos
Proteínas do Tecido Nervoso , Sinapses/metabolismo , Animais , Humanos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Transporte ProteicoRESUMO
Botulinum neurotoxin A causes botulism but is also used for medical and cosmetic applications. A detailed molecular understanding of BoNT/A--host receptor interactions is therefore fundamental for improving current clinical applications and for developing new medical strategies targeting human disorders. Towards this end, we recently solved an X-ray crystal structure of BoNT/A1 in complex with its neuronal protein receptor SV2C. Based on our findings, we discuss the potential implications for BoNT/A function.
Assuntos
Toxinas Botulínicas Tipo A/química , Animais , Toxinas Botulínicas Tipo A/metabolismo , Cristalografia por Raios X , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Estrutura Molecular , Neurônios/metabolismo , Ligação ProteicaRESUMO
INTRODUCTION: Botulinum neurotoxin type A (BoNTA) is one of seven serotypes produced by Clostridium botulinum (types A thru G) and is the serotype most widely used to treat both cosmetic and medical conditions. Potency for botulinum toxin preparations is expressed in mouse LD50 units. There is a need to develop a non-animal based replacement for this potency assay. METHODS: An in vitro potency assay measuring BoNTA activity has been developed that addresses both BoNTA heavy chain binding to its cell receptor SV2C and BoNTA light chain enzymatic activity in cleaving SNAP-25, an intracellular protein essential in neurotransmitter release. This bifunctional assay utilizes a 96 well microtiter format and well defined reagents. Assay characterization determined that the relative standard deviation for intermediate precision was less than 10%. RESULTS: The assay standard curve covers the range of BoNTA concentrations from 0.0624 to 32 ng/mL. Specificity was demonstrated with purified BoNTA heavy chain which inhibited the activity in a dose dependent manner. A correlation between this bifunctional assay and the mouse LD50 potency assay was demonstrated.