RESUMO
In animals infected with a transmissible spongiform encephalopathy, or prion disease, conformational isomers (known as PrPSc proteins) of the wild-type, host-encoded cellular prion protein (PrPc) accumulate. The infectious agents, prions, are composed mainly of these conformational isomers, with distinct prion isolates or strains being associated with different PrPSc conformations and patterns of glycosylation. Here we show that two different human PrPSc types, seen in clinically distinct subtypes of classical Creutzfeldt-Jakob disease, can be interconverted in vitro by altering their metal-ion occupancy. The dependence of PrPSc conformation on the binding of copper and zinc represents a new mechanism for post-translational modification of PrP and for the generation of multiple prion strains, with widespread implications for both the molecular classification and the pathogenesis of prion diseases in humans and animals.
Assuntos
Cobre/metabolismo , Síndrome de Creutzfeldt-Jakob/metabolismo , Proteínas PrPC/química , Proteínas PrPSc/química , Conformação Proteica , Zinco/metabolismo , Sítios de Ligação , Encéfalo/metabolismo , Cobre/farmacologia , Síndrome de Creutzfeldt-Jakob/classificação , Endopeptidase K , Humanos , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Conformação Proteica/efeitos dos fármacos , Zinco/farmacologiaRESUMO
Transgenic mice expressing human prion protein in the absence of endogenous mouse prion protein faithfully replicate human prions. These models reproduce all of the key features of human disease, including long clinically silent incubation periods prior to fatal neurodegeneration with neuropathological phenotypes that mirror human prion strain diversity. Critical contributions to our understanding of human prion disease pathogenesis and aetiology have only been possible through the use of transgenic mice. These models have provided the basis for the conformational selection model of prion transmission barriers and have causally linked bovine spongiform encephalopathy with variant Creutzfeldt-Jakob disease. In the future these models will be essential for evaluating newly identified potentially zoonotic prion strains, for validating effective methods of prion decontamination and for developing effective therapeutic treatments for human prion disease.
Assuntos
Animais Geneticamente Modificados/fisiologia , Doenças Priônicas/genética , Doenças Priônicas/patologia , Animais , Bovinos , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Fenótipo , Príons/classificação , Príons/genética , Príons/fisiologiaRESUMO
The largest kindred with inherited prion disease P102L, historically Gerstmann-Sträussler-Scheinker syndrome, originates from central England, with émigrés now resident in various parts of the English-speaking world. We have collected data from 84 patients in the large UK kindred and numerous small unrelated pedigrees to investigate phenotypic heterogeneity and modifying factors. This collection represents by far the largest series of P102L patients so far reported. Microsatellite and genealogical analyses of eight separate European kindreds support multiple distinct mutational events at a cytosine-phosphate diester-guanidine dinucleotide mutation hot spot. All of the smaller P102L kindreds were linked to polymorphic human prion protein gene codon 129M and were not connected by genealogy or microsatellite haplotype background to the large kindred or each other. While many present with classical Gerstmann-Sträussler-Scheinker syndrome, a slowly progressive cerebellar ataxia with later onset cognitive impairment, there is remarkable heterogeneity. A subset of patients present with prominent cognitive and psychiatric features and some have met diagnostic criteria for sporadic Creutzfeldt-Jakob disease. We show that polymorphic human prion protein gene codon 129 modifies age at onset: the earliest eight clinical onsets were all MM homozygotes and overall age at onset was 7 years earlier for MM compared with MV heterozygotes (P = 0.02). Unexpectedly, apolipoprotein E4 carriers have a delayed age of onset by 10 years (P = 0.02). We found a preponderance of female patients compared with males (54 females versus 30 males, P = 0.01), which probably relates to ascertainment bias. However, these modifiers had no impact on a semi-quantitative pathological phenotype in 10 autopsied patients. These data allow an appreciation of the range of clinical phenotype, modern imaging and molecular investigation and should inform genetic counselling of at-risk individuals, with the identification of two genetic modifiers.
Assuntos
Doença de Gerstmann-Straussler-Scheinker/genética , Mutação Puntual , Príons/genética , Adulto , Idade de Início , Idoso , Encéfalo/patologia , Eletrocardiografia , Eletromiografia , Inglaterra , Europa (Continente) , Feminino , Genealogia e Heráldica , Testes Genéticos , Doença de Gerstmann-Straussler-Scheinker/diagnóstico , Haplótipos , Heterozigoto , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Tomografia Computadorizada por Raios XRESUMO
The occurrence of new variant Creutzfeldt-Jakob disease and the experimental confirmation that it is caused by the same prion strain as BSE has dramatically highlighted the need for a precise understanding of the molecular basis of prion propagation. The molecular basis of prion-strain diversity, previously a major challenge to the protein-only model, is now becoming clearer. The conformational change thought to be central to prion propagation, from a predominantly alpha-helical fold to one predominantly comprising beta-structure, can now be reproduced in vitro, and the ability of beta-PrP to form fibrillar aggregates provides a plausible molecular mechanism for prion propagation. These and other advances in the fundamental biology of prion propagation are leading to prion diseases becoming arguably the best understood of the neurodegenerative conditions and strategies for the development of rational therapeutics are becoming clearer.
Assuntos
Príons/genética , Animais , Bovinos , Síndrome de Creutzfeldt-Jakob/genética , Síndrome de Creutzfeldt-Jakob/transmissão , Encefalopatia Espongiforme Bovina/genética , Encefalopatia Espongiforme Bovina/transmissão , Variação Genética , HumanosAssuntos
Doenças em Gêmeos/epidemiologia , Doenças Priônicas/epidemiologia , Gêmeos Monozigóticos , Idade de Início , Encéfalo/metabolismo , Encéfalo/patologia , Síndrome de Creutzfeldt-Jakob/metabolismo , Doenças em Gêmeos/genética , Doenças em Gêmeos/patologia , Meio Ambiente , Feminino , Humanos , Pessoa de Meia-Idade , Linhagem , Proteínas PrPSc/metabolismo , Doenças Priônicas/genética , Doenças Priônicas/patologia , Proteínas Priônicas , Príons/genética , Análise de Sequência de DNARESUMO
BACKGROUND: Disease-related prion protein (PrP(Sc)) is readily detectable in lymphoreticular tissues in variant Creutzfeldt-Jakob disease (vCJD), but not in other forms of human prion disease. This distinctive pathogenesis, with the unknown population prevalence of asymptomatic vCJD infection, has led to significant concerns that secondary transmission of vCJD prions will occur through a wide range of surgical procedures. To date PrP(Sc):prion infectivity ratios have not been determined in vCJD, and it is unknown whether vCJD prions are similar to experimental rodent prions, where PrP(Sc) concentration typically reflects infectious prion titre. AIM: To investigate prion infectivity in vCJD tissue containing barely detectable levels of PrP(Sc). METHODS: Transgenic mice expressing only human PrP (Tg(HuPrP129M(+/+)Prnp(o/o))-35 and Tg(HuPrP129M(+/+)Prnp(o/o))-45 mice) were inoculated with brain or rectal tissue from a previously characterised patient with vCJD. These tissues contain the maximum and minimum levels of detectable PrP(Sc) that have been observed in vCJD. RESULTS: Efficient transmission of prion infection was observed in transgenic mice inoculated with vCJD rectal tissue containing PrP(Sc) at a concentration of 10(4.7)-fold lower than that in vCJD brain. CONCLUSIONS: These data confirm the potential risks for secondary transmission of vCJD prions via gastrointestinal procedures and support the use of PrP(Sc) as a quantitative marker of prion infectivity in vCJD tissues.
Assuntos
Encéfalo , Síndrome de Creutzfeldt-Jakob/transmissão , Príons/análise , Reto , Animais , Encéfalo/metabolismo , Síndrome de Creutzfeldt-Jakob/metabolismo , Humanos , Immunoblotting/métodos , Imuno-Histoquímica/métodos , Camundongos , Camundongos Transgênicos , Proteínas PrPC/análise , Proteínas PrPSc/análise , Reto/metabolismoRESUMO
BACKGROUND: Human prion diseases have sporadic, acquired and inherited etiologies and show considerable phenotypic heterogeneity. An individual inherited prion disease offers an opportunity to study the determinants of this clinicopathologic heterogeneity among individuals with the same causal mutation. METHODS: We report clinical and pathologic data from three families with different 5-octapeptide repeat insertion (5-OPRI) mutations of the prion protein gene (PRNP), extending the reported phenotypic range of this mutation. RESULTS: The proband of a South African family presented with a rapidly progressive dementia and atypical pathology associated with kuru-like prion protein plaques. The original mutation in this family probably occurred on a PRNP allele encoding a 1-octapeptide repeat deletion polymorphism. This has not been previously reported as a precursor allele in over 30 other OPRI mutation kindreds. An English family with a genetically distinct mutation but identical protein product showed clinical onsets that varied 30 years between father and daughter, an effect that may be explained by their genotypes at PRNP codon 129. A patient from Northern Ireland with a phenotype of sporadic Creutzfeldt-Jakob disease presenting with visual disturbance was unexpectedly found to have a 5-OPRI. CONCLUSIONS: When these cases were combined with the existing world literature, the mean age at onset for patients with 5-octapeptide repeat insertion (5-OPRI) was significantly later than that for patients with 6-OPRI, but both mutations exhibit a similar powerful disease modifying effect of PRNP codon 129.
Assuntos
Predisposição Genética para Doença/genética , Mutação/genética , Doenças Priônicas/genética , Príons/genética , Sequências Repetitivas de Aminoácidos/genética , Adulto , Códon/genética , Análise Mutacional de DNA , Progressão da Doença , Feminino , Testes Genéticos , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Irlanda do Norte , Linhagem , Fenótipo , Polimorfismo Genético/genética , Doenças Priônicas/etnologia , Doenças Priônicas/metabolismo , África do SulRESUMO
Using high-resolution PAGE and Western-blotting techniques the lectin Ricinus communis agglutinin-1 (RCA-1) was tested for its ability to recognize laminin subunits from the mouse Engelbreth-Holm-Swarm (EHS) tumour and from bovine cardiac and skeletal muscle. Biotinylated RCA-1 recognized both the A and B chains of purified EHS-tumour laminin with a sensitivity comparable to anti-(EHS laminin) antibodies. In cardiac and skeletal muscle RCA-1 also recognized the B chains of laminin, together with a approximately 330 kDa RCA-1-binding glycoprotein that was undetectable in smooth muscle. This glycoprotein was not recognized by antibodies raised to laminin from the EHS tumour. Purification of the 330 kDa binding glycoprotein from skeletal muscle, using ion-exchange and lectin-affinity chromatography, revealed that in its native form, this glycoprotein is disulphide-bonded to the B chains of laminin. The demonstrated properties of the approximately 330 kDa RCA-1-binding glycoprotein are identical to those reported for the variant M chain of merosin which is known to replace the A chain in laminin from the extrasynaptic regions of skeletal muscle. These results establish that biotinylated RCA-1 can recognize A-, B- and M-chain subunits of laminin isoforms, and that, when used in conjunction with other techniques, they provide a useful method for the assignment of laminin heavy chains.
Assuntos
Laminina/análise , Lectinas , Plantas Tóxicas , Ricinus communis , Animais , Western Blotting , Bovinos , Eletroforese em Gel de Poliacrilamida , Camundongos , Sondas Moleculares , Músculos/química , Lectinas de Plantas , Sarcoma Experimental/químicaRESUMO
Apamin, a peptide neurotoxin from bee venom, blocks small conductance Ca(2+)-activated K+ channels in central synapses and peripheral tissues. Using 125I-apamin, single classes of high affinity binding sites (Kd 1-3 pM) were identified on plasma membranes from rat, rabbit, guinea pig, and bovine brain and from rabbit, guinea pig, and bovine liver. Binding was sensitive to scyllatoxin, dequalinium, gallamine, and d-tubocurarine but not to charybdotoxin, toxin I, or mast cell degranulating peptide. In contrast, saturable binding of 125I-apamin to rat liver plasma membranes was virtually undetectable, thereby providing a correlation with the ability to measure apamin-sensitive Ca(2+)-activated potassium currents in rabbit and guinea pig hepatocytes but not in rat hepatocytes. In agreement with membrane binding studies, homobifunctional cross-linkers identified apparently identical 33-kDa 125I-apamin binding polypeptides on brain plasma membranes from all species and analogous but distinct polypeptides on plasma membranes from rabbit, guinea pig, and bovine liver. None of these affinity-labeled polypeptides were detectable on plasma membranes from rat liver. Affinity labeling was abolished on both liver and brain membranes by apamin, scyllatoxin, dequalinium, gallamine, and d-tubocurarine. These results indicate that comparable approximately 30-kDa polypeptides may fulfill equivalent functional roles within putative subtypes of apamin-sensitive small conductance Ca(2+)-activated K+ channels.
Assuntos
Apamina/metabolismo , Cálcio/metabolismo , Canais de Potássio/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Bovinos , Membrana Celular/metabolismo , Cobaias , Fígado/metabolismo , Fígado/ultraestrutura , Potenciais da Membrana , Bloqueadores Neuromusculares/farmacologia , Neurotoxinas/farmacologia , Peptídeos/metabolismo , Coelhos , RatosRESUMO
125I-Apamin and photolabile derivatives of the toxin have been used to investigate the binding properties and subunit composition of small conductance Ca2+-activated potassium channels (SK(Ca) channels) expressed on plasma membranes from rat brain, rabbit liver, or rat pheochromocytoma (PC12) cells. On all preparations, 125I-apamin recognized single classes of acceptor binding sites with similar high affinity (Kd approximately 3-6 pM). Gallamine, however, was found to readily discriminate between 125I-apamin acceptors present in these preparations, showing a maximal approx nine-fold difference in affinity for acceptors expressed by rabbit liver or PC12 cells. Affinity-labeling patterns revealed the expression of different hetero-oligomeric combinations of high (86 or 59 kDa) and low (33 or 30 kDa) molecular mass 125I-apamin-binding polypeptides, consistent with pharmacological differences. Alternative expression of either 86- or 59-kDa polypeptides appeared to be the most important factor influencing gallamine's affinity for SK(Ca) channel subtypes. Both high- and low-molecular-mass polypeptides are integral membrane proteins, the latter being glycosylated in a tissue-specific manner.
Assuntos
Canais de Potássio Cálcio-Ativados , Canais de Potássio/química , Marcadores de Afinidade , Animais , Apamina/metabolismo , Química Encefálica , Cálcio/farmacologia , Trietiodeto de Galamina/metabolismo , Ativação do Canal Iônico , Fígado/química , Células PC12 , Canais de Potássio/classificação , Canais de Potássio/metabolismo , Coelhos , Ratos , Canais de Potássio Ativados por Cálcio de Condutância BaixaRESUMO
Taicatoxin, isolated from the venom of the Australian taipan snake Oxyuranus scutellatus, has been previously regarded as a specific blocker of high threshold Ca2+ channels in heart. Here we show that taicatoxin (in contrast to a range of other Ca2+ channel blockers) interacts with apamin-sensitive, small conductance, Ca2+-activated potassium channels on both chromaffin cells and in the brain. Taicatoxin displays high affinity recognition of 125I-apamin acceptor-binding sites, present on rat synaptosomal membranes (Ki = 1.45 +/- 0.22 nM) and also specifically blocks affinity-labeling of a 33-kDa 125I-apamin-binding polypeptide on rat brain membranes. Taicatoxin (50 nM) completely blocks apamin-sensitive after-hyperpolarizing slow tail K+ currents generated in rat chromaffin cells (mean block 97 +/- 3%, n = 12) while only partially reducing total voltage-dependent Ca2+ currents (mean block 12 +/- 4%, n = 6). In view of these findings, the use of taicatoxin as a specific ligand for Ca2+ channels should now be reconsidered.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Venenos Elapídicos/farmacologia , Bloqueadores dos Canais de Potássio , Canais de Potássio Cálcio-Ativados , Canais de Potássio , Animais , Apamina/metabolismo , Encéfalo/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Células Cromafins/efeitos dos fármacos , Células Cromafins/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Peso Molecular , Ratos , Canais de Potássio Ativados por Cálcio de Condutância BaixaRESUMO
The structure of apamin-sensitive Ca(2+)-activated K+ channels has been investigated using high-affinity, photolabile azidoaryl derivatives of 125I-[alpha-formyl-Cys1]apamin and 125I-[epsilon-formyl-Lys4]-apamin. Labeling patterns suggest that similar structural constraints are required for labeling analogous polypeptides associated with distinct channel subtypes. When photoprobes are coupled at the epsilon-amino-Lys4 position of apamin, comparable low molecular mass (approximately 30 kDa) polypeptides are efficiently labeled on either brain or liver plasma membranes, irrespective of the structure of the photoprobe. However, when photoprobes are coupled at the alpha-amino-Cys1 position of apamin, the pattern of labeling on both brain and liver plasma membranes varies, depending upon the length of the spacer arm incorporated into the photoprobe. Spacer arms of approximately 8-9 A efficiently label only high molecular mass polypeptides (86, 59 kDa), accompanied by weak, variable labeling of a 44-kDa component. A shorter spacer arm (5.7 A) results in feeble labeling of 86- and 59-kDa polypeptides and barely detectable labeling of 44- and approximately 30-kDa polypeptides. In contrast, a long spacer arm (12.8 A) efficiently labels only approximately 30-kDa polypeptides. These findings point to close similarities in the topography of the 125I-apamin binding site present on pharmacologically distinct subtypes of apamin-sensitive Ca2+-activated K+ channels and indicates that heterooligomeric association of high and low molecular mass polypeptide subunits may be a general structural feature of members belonging to this family of K+ channels.
Assuntos
Apamina/análogos & derivados , Apamina/metabolismo , Córtex Cerebral/metabolismo , Canais de Potássio/metabolismo , Animais , Apamina/isolamento & purificação , Autorradiografia , Membrana Celular/metabolismo , Reagentes de Ligações Cruzadas , Radioisótopos do Iodo , Cinética , Fígado/metabolismo , Substâncias Macromoleculares , Fotólise , Canais de Potássio/química , Canais de Potássio/fisiologia , Coelhos , Ensaio Radioligante , Ratos , Membranas Sinápticas/metabolismoRESUMO
Botulinum neurotoxins (types A and B), which are microbial proteins consisting of two disulfide-linked chains, inhibit specifically and with high potency the release of acetylcholine from peripheral nerve terminals. As a prerequisite for a long-term development of effective treatments for botulism, the internalization and inhibitory action of the toxin and its constituent chains were examined by electrophysiological methods at identified synapses in Aplysia preparations that allow both intracellular and bath application of the neurotoxins. Intracellular recordings from cholinergic cells of the buccal ganglion demonstrated that extra- or intracellular application of low doses of botulinum neurotoxin results in a specific blockade of evoked transmitter release, without changing the quantal size; an intraneuronal site of action has thus been established. In contrast, release from noncholinergic neurons of cerebral ganglion was prevented by the neurotoxin only after injection into the cell. Purified preparations of the individual renatured chains, shown to be nontoxic in a mouse bioassay, failed to affect acetylcholine release when applied extra- or intracellularly. However, inhibition of release was observed after intracellular administration of both chains or when the light chain was injected and the heavy chain was bath-applied. These findings show that both chains are required on the cytosolic side of the neuronal plasma membrane for expression of toxicity and that the cholinergic specificity of the neurotoxin is attributable to its heavy chain, which mediates targeting and subsequent neuronal uptake.
Assuntos
Toxinas Botulínicas/farmacologia , Neurônios/efeitos dos fármacos , Neurotransmissores/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Acetilcolina/metabolismo , Animais , Aplysia , Toxinas Botulínicas/metabolismo , Citoplasma , Técnicas In Vitro , Substâncias Macromoleculares , Neurônios/metabolismo , Taxa Secretória/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Variant Creutzfeldt-Jakob disease (vCJD) has a pathogenesis distinct from other forms of human prion disease: disease-related prion protein (PrP(Sc)) is readily detectable in lymphoreticular tissues. Quantitation of risk of secondary transmission, and targeting of risk reduction strategies, is limited by lack of knowledge about relative prion titres in these and other peripheral tissues, the unknown prevalence of preclinical vCJD, and a transmission barrier which limits the sensitivity of bioassay. We aimed to improve immunoblotting methods for high sensitivity detection of PrP(Sc) to investigate the distribution of PrP(Sc) in a range of vCJD tissues. METHODS: We obtained tissues at necropsy from four patients with neuropathologically confirmed vCJD and from individuals without neurological disease. Tissues were analysed by sodium phosphotungstic acid precipitation of PrP(Sc) and western blotting using high sensitivity enhanced chemiluminescence. FINDINGS: We could reliably detect PrP(Sc) in the equivalent of 50 nL 10% vCJD brain homogenate, with a maximum limit of detection equivalent to 5 nl. PrP(Sc) could be detected in tissue homogenates when present at concentrations 10(4)-10(5) fold lower than those reported in brain. Tonsil, spleen, and lymph node were uniformly positive for PrP(Sc) at concentrations in the range of 0.1-15% of those found in brain: the highest concentrations were consistently seen in tonsil. PrP(Sc) was readily detected in the retina and proximal optic nerve of vCJD eye at levels of 2.5 and 25%, respectively of those found in brain. Other peripheral tissues studied were negative for PrP(Sc) with the exception of low concentrations in rectum, adrenal gland, and thymus from a single patient with vCJD. vCJD appendix and blood (Buffy coat fraction) were negative for PrP(Sc) at this level of assay sensitivity. INTERPRETATION: We have developed a highly sensitive immunoblot method for detection of PrP(Sc) in vCJD tissues that can be used to provide an upper limit on PrP(Sc) concentrations in peripheral tissues, including blood, to inform risk assessment models. Rectal and other gastrointestinal tissues should be further investigated to assess risk of iatrogenic transmission via biopsy instruments. Ophthalmic surgical instruments used in procedures involving optic nerve and the posterior segment of the eye, in particular the retina, might represent a potential risk for iatrogenic transmission of vCJD. Tonsil is the tissue of choice for diagnostic biopsy and for population screening of surgical tissues to assess prevalence of preclinical vCJD infection within the UK and other populations.
Assuntos
Síndrome de Creutzfeldt-Jakob/metabolismo , Proteínas PrPSc/análise , Animais , Western Blotting/métodos , Síndrome de Creutzfeldt-Jakob/epidemiologia , Síndrome de Creutzfeldt-Jakob/patologia , Síndrome de Creutzfeldt-Jakob/transmissão , Humanos , Doença Iatrogênica , Medições Luminescentes , Tonsila Palatina/química , Ácido Fosfotúngstico , Proteínas PrPSc/isolamento & purificação , Prevalência , Fatores de Risco , Sensibilidade e Especificidade , Instrumentos Cirúrgicos , Distribuição Tecidual , Reino Unido/epidemiologiaRESUMO
1. The contribution of a proteolytic fragment (H2L) of botulinum neurotoxin type A (comprised of the aminoterminal region of the heavy-chain disulphide-linked to the light chain) to inhibition of neurotransmitter release was investigated, using central cholinergic synapses of Aplysia, rodent nerve-diaphragm preparations and cerebrocortical synaptosomes. 2. No reduction in neurotransmitter release was observed following external application to these preparations of highly purified H2L or after intracellular injection into Aplysia neurons. 3. The lack of activity was not the result of alteration in the light chain of H2L during preparation of the latter because (a) renaturation of this light chain with intact heavy chain produced a toxic di-chain form and (b) simultaneous application of heavy chain and light chain from H2L inhibited transmitter release in Aplysia. 4. Bath application of H2L and heavy chain together inhibited release of transmitter; however, at the neuromuscular junction the potency of this mixture was much lower than that of native toxin. A similar blockade resulted when heavy chain was applied intracellularly and H2L added to the bath, demonstrating that H2L is taken up into cholinergic neurons of Aplysia. This uptake is shown to be mediated by the amino-terminal moiety of heavy chain (H2), because bath application of light chain plus H2 led to a decrease in acetylcholine release from a neuron that had been injected with heavy chain. 5. A role within the neuron is implicated for a carboxy-terminal portion of heavy chain (H1) since intracellular injection of light chain and H2 did not affect transmitter release. Although the situation is unclear in mammalian nerves, these collective findings indicate that blockade of transmitter release in Aplysia neurons requires the intracellular presence of light chain and H1 (by inference), whilst H2 contributes to the internalization step.
Assuntos
Toxinas Botulínicas/toxicidade , Neurotoxinas/toxicidade , Neurotransmissores/metabolismo , Fragmentos de Peptídeos/toxicidade , Acetilcolina/metabolismo , Animais , Aplysia , Transmissão SinápticaRESUMO
1. The effects on the release of transmitter by botulinum neurotoxins (BoNT; types A, B, E), tetanus toxin (TeTx), constituent chains or fragments were studied on identified cholinergic and non-cholinergic synapses in Aplysia. 2. Cholinergic synapses in the buccal ganglion were found to be greater than 100 fold more sensitive to extracellular application of BoNT than to TeTx whereas in non-cholinergic synapses of the cerebral ganglion the potencies of the toxins were reversed. When intracellularly applied TeTx and BoNT were found nearly equipotent. This disparity in the susceptibilities of BoNT and TeTx to inhibit transmission was attributed to differences in the toxin's acceptors or uptake systems in the two neurone types. 3. Micro-injection into cholinergic neurones of the isolated renatured toxins' chains showed that both light and heavy chains of BoNT are intracellularly required whereas the light chain of TeTx alone is sufficient. 4. The heavy chain of BoNT as well as that of TeTx were found to mediate internalization of active moieties via its amino-terminal half. Furthermore the heavy chain of one toxin could internalize the light chain of the other.
Assuntos
Aplysia/citologia , Toxinas Botulínicas/farmacologia , Fibras Colinérgicas , Neurotransmissores/antagonistas & inibidores , Sinapses/metabolismo , Toxina Tetânica/farmacologia , Animais , Toxinas Botulínicas/química , Neurotransmissores/metabolismo , Toxina Tetânica/químicaRESUMO
The neuroparalytic activities of botulinum neurotoxin type A (BoNT A), tetanus toxin (TeTx), or homologous and heterologous combinations of their constituent polypeptides were examined at cholinergic and non-cholinergic synapses of Aplysia californica. When applied extracellularly, BoNT A or a mixture of its heavy (HC) and light (LC) chains were far more potent in blocking transmitter release at cholinergic than non-cholinergic synapses. The reverse was true for TeTx or a mixture its constituent chains. Such selectivity was assigned to differences in neuronal targetting and uptake of the neurotoxins since both exhibited similar potencies when injected directly into the cell body of either cell type. When bath-applied, heterologous combinations of the toxins' HC and LC appeared as effective as the parent neurotoxins from whence each HC was derived. Moreover, targetting/internalization was attributable to the analogous N-terminal moieties, H2 and beta 2, of the HC from BoNT A and TeTx. Thus, it may be postulated that the latter regions possess two functional domains, one being distinct and responsible for the divergent neuronal specificity, whereas the other serves a common role in translocating the LC of either toxin. Also, it was shown that the C-terminal portion of the HC of TeTx is unable to play the intracellular role of its counterpart in BoNT A.
Assuntos
Toxinas Botulínicas/farmacologia , Neuroglia/efeitos dos fármacos , Neurotransmissores/antagonistas & inibidores , Toxina Tetânica/farmacologia , Animais , Aplysia , Toxinas Botulínicas/química , Toxina Tetânica/químicaRESUMO
1. The abilities of botulinum neurotoxins, types A and B (single and two-chain forms) to inactivate an intraneuronal component required for transmitter release were quantified in a phrenic-nerve-diaphragm preparation, cerebrocortical synaptosomes or the buccal ganglion of Aplysia californica and compared with the mouse toxicity assay. 2. Homogeneous preparations of the individually renatured polypeptide chains of both toxin types showed low residual toxicity in the whole animal and had no effect on neurotransmission in all three systems, when tested singly. 3. Mixtures of individually renatured heavy chain, from type A or B, and either light chain proved very effective in blocking the evoked release of acetylcholine when bath-applied to the buccal ganglion of Aplysia whilst they were relatively inactive on mammalian nerve terminals, indicating a less efficient uptake of the polypeptides in the latter. 4. When renatured together, the homologous, but not the heterologous, chains of each toxin type yielded toxic, disulphide-linked two-chain species. 5. A role for the heavy chain alone in acceptor recognition and membrane translocation was implicated by the blockade of acetylcholine release produced when light chain was applied to a ganglion of Aplysia previously bathed in heavy chain and washed extensively. No blockade was observed when the order of application of the two chains was reversed. 6. These findings are discussed in the context of the intracellular requirement for both the constituent toxin chains for toxicity, and in the apparent need for these chains to be linked via a disulphide bond for uptake in rodents but not in Aplysia.
Assuntos
Acetilcolina/metabolismo , Toxinas Botulínicas/farmacologia , Junção Neuromuscular/fisiologia , Neurônios/fisiologia , Neurotoxinas , Norepinefrina/metabolismo , Sinaptossomos/metabolismo , Animais , Aplysia , Toxinas Botulínicas/isolamento & purificação , Toxinas Botulínicas/toxicidade , Córtex Cerebral/fisiologia , Técnicas In Vitro , Cinética , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos BALB C , Contração Muscular/efeitos dos fármacos , Junção Neuromuscular/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Nervo Frênico/fisiologia , Ratos , Sinaptossomos/efeitos dos fármacosRESUMO
1. A large-scale purification procedure has been developed for Clostridium botulinum type F neurotoxin. Commencing with 160 litres of bacterial culture, 101 mg of purified type F neurotoxin with a specific toxicity of 2 x 10(7) mouse LD50 (median lethal dose).mg-1 were obtained. 2. Purified type F neurotoxin was labelled to high specific radioactivity (900-1360 Ci/mmol) without loss of biological activity using a chloramine-T procedure. Of the two neurotoxin subunits, the heavy chain was preferentially radiolabelled. 3. Radiolabelled type F neurotoxin displayed specific saturable binding to rat synaptosomes. At least two pools of acceptors were evident: a low content of high-affinity acceptors sites [KD approximately 0.15 nM; Bmax (maximal binding) 20 fmol/mg] and a larger pool of lower-affinity sites (KD greater than 20 nM; Bmax greater than 700 fmol/mg). Both pools of acceptors were sensitive to trypsin and neuraminidase treatment, which suggests that protein and sialic acid residues are components of the synaptosomal acceptors. 4. Experiments investigating competition among botulinum neurotoxin types A, B, E and F for acceptors on rat brain synaptosomes showed that type F neurotoxin binds to acceptor molecules which are completely distinct from those of the other three neurotoxins.