RESUMO
Axons and their synapses distal to an injury undergo rapid Wallerian degeneration, but axons in the C57BL/WldS mouse are protected. The degenerative and protective mechanisms are unknown. We identified the protective gene, which encodes an N-terminal fragment of ubiquitination factor E4B (Ube4b) fused to nicotinamide mononucleotide adenylyltransferase (Nmnat), and showed that it confers a dose-dependent block of Wallerian degeneration. Transected distal axons survived for two weeks, and neuromuscular junctions were also protected. Surprisingly, the Wld protein was located predominantly in the nucleus, indicating an indirect protective mechanism. Nmnat enzyme activity, but not NAD+ content, was increased fourfold in WldS tissues. Thus, axon protection is likely to be mediated by altered ubiquitination or pyridine nucleotide metabolism.
Assuntos
Axônios/metabolismo , Proteínas Fúngicas/genética , Junção Neuromuscular/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Proteínas Recombinantes de Fusão/genética , Proteínas de Saccharomyces cerevisiae , Traumatismos do Sistema Nervoso , Degeneração Walleriana/genética , Degeneração Walleriana/metabolismo , Potenciais de Ação/genética , Animais , Axônios/ultraestrutura , Sequência de Bases/fisiologia , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Sobrevivência Celular/genética , Proteínas Fúngicas/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Microscopia Eletrônica , Dados de Sequência Molecular , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/inervação , Músculo Esquelético/metabolismo , Mutação/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Sistema Nervoso/metabolismo , Sistema Nervoso/patologia , Junção Neuromuscular/ultraestrutura , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Nervo Isquiático/lesões , Nervo Isquiático/metabolismo , Nervo Isquiático/ultraestrutura , Transmissão Sináptica/genética , Vesículas Sinápticas/metabolismo , Enzimas de Conjugação de Ubiquitina , Degeneração Walleriana/fisiopatologiaRESUMO
Exons of three genes were identified within the 85-kilobase tandem triplication unit of the slow Wallerian degeneration mutant mouse, C57BL/Wld(S). Ubiquitin fusion degradation protein 2 (Ufd2) and a previously undescribed gene, D4Cole1e, span the proximal and distal boundaries of the repeat unit, respectively. They have the same chromosomal orientation and form a chimeric gene when brought together at the boundaries between adjacent repeat units in Wld(S). The chimeric mRNA is abundantly expressed in the nervous system and encodes an in-frame fusion protein consisting of the N-terminal 70 amino acids of Ufd2, the C-terminal 302 amino acids of D4Cole1e, and an aspartic acid formed at the junction. Antisera raised against synthetic peptides detect the expected 43-kDa protein specifically in Wld(S) brain. This expression pattern, together with the previously established role of ubiquitination in axon degeneration, makes the chimeric gene a promising candidate for Wld. The third gene altered by the triplication, Rbp7, is a novel member of the cellular retinoid-binding protein family and is highly expressed in white adipose tissue and mammary gland. The whole gene lies within the repeat unit leading to overexpression of the normal transcript in Wld(S) mice. However, it is undetectable on Northern blots of Wld(S) brain and seems unlikely to be the Wld gene. These data reveal both a candidate gene for Wld and the potential of the Wld(S) mutant for studies of ubiquitin and retinoid metabolism.
Assuntos
Proteínas Fúngicas/genética , Nicotinamida-Nucleotídeo Adenililtransferase , Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas de Ligação ao Retinol/genética , Proteínas de Saccharomyces cerevisiae , Degeneração Walleriana , Sequência de Aminoácidos , Animais , Sequência de Bases , Sistema Nervoso Central/metabolismo , DNA Complementar , Éxons , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Sistema Nervoso Periférico/metabolismo , RNA Mensageiro/genética , Proteínas Celulares de Ligação ao Retinol , Homologia de Sequência de Aminoácidos , Enzimas de Conjugação de UbiquitinaRESUMO
Kinesin and kinesin superfamily proteins are molecular motors involved in important intracellular functions such as organelle transport and cell division. They are microtubule-activated ATPases composed of a motor domain that binds to microtubules and a cargo-binding domain that binds to specific organelles. While searching for the slow Wallerian degeneration mutation (WldS) on distal mouse Chromosome (Chr) 4, we have identified a member of the kinesin superfamily whose predicted gene product has the N-terminal motor domain of Kif1b and a novel C-terminal cargo-binding domain homologous to Kif1a. Kif1b is responsible for the movement of mitochondria along the axon, but the novel isoform containing the alternative C-terminal domain is likely to have a different cargo-binding specificity. cDNA library screening and Northern blot analysis indicate that the alternatively spliced form of Kif1b containing the novel 3'end accounts for the most part of Kif1b expression. We also found more alternatively spliced exons that can give rise to heterogeneous transcripts. Therefore, alternative splicing, as well as multiple genes, may contribute to the selective movement of diverse organelles by anterograde axonal transport. Kif1b maps on distal mouse Chr 4, within the Wld genetic candidate interval, but outside the recently identified triplication. There is, however, no evidence that Kif1b is the Wld gene.
Assuntos
Processamento Alternativo , Encéfalo/metabolismo , Cinesinas/genética , Mitocôndrias/metabolismo , Mutação , Proteínas do Tecido Nervoso/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Mapeamento Cromossômico , Clonagem Molecular , Análise Mutacional de DNA , Cinesinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Isoformas de Proteínas/genética , Análise de Sequência , Homologia de Sequência de AminoácidosRESUMO
Wallerian degeneration is the degeneration of the distal stump of an injured axon. It normally occurs over a time course of around 24 hr but it is delayed in the slow Wallerian degeneration mutant mouse (C57BL/Wlds) for up to 3 weeks. The gene, which protects from rapid Wallerian degeneration, Wld, previously has been mapped to distal chromosome 4. This paper reports the fine genetic mapping of the Wld locus, the generation of a 1.4-Mb bacterial artificial chromosome and P1 artificial chromosome contig, and the identification of an 85-kb tandem triplication mapping within the candidate region. The mutation is unique to C57BL/Wlds among 36 strains tested and therefore is a strong candidate for the mutation that leads to delayed Wallerian degeneration. There are very few reports of tandem triplications in a vertebrate and no evidence for a mutation mechanism so this unusual mutation was characterized in more detail. Sequence analysis of the boundaries of the repeat unit revealed a minisatellite array at the distal boundary and a matching 8-bp sequence at the proximal boundary. This finding suggests that recombination between short homologous sequences ("illegitimate" or "nonhomologous" recombination) was involved in the rearrangement. In addition, a duplication allele was identified in two Wlds mice, indicating some instability in the repeat copy number and suggesting that the triplication arose from a duplication by unequal crossing over.
Assuntos
Família Multigênica , Sequências Repetitivas de Ácido Nucleico , Degeneração Walleriana/genética , Alelos , Animais , Sequência de Bases , Mapeamento Cromossômico , Troca Genética , Primers do DNA/genética , Rearranjo Gênico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase , Recombinação GenéticaAssuntos
Mapeamento Cromossômico , Camundongos Endogâmicos C57BL/genética , Muridae/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/genética , Receptores de Fator de Crescimento Neural/genética , Receptores do Fator de Necrose Tumoral/genética , Alelos , Animais , Antígenos CD , Sequência de Bases , Cruzamentos Genéticos , Primers do DNA , Marcadores Genéticos , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ratos , Superfamília Shaker de Canais de Potássio , Membro 9 da Superfamília de Receptores de Fatores de Necrose TumoralRESUMO
An experiment was designed to investigate aspects of the population dynamics of acquired immunity to Heligmosomoides polygyrus in laboratory mice. The influence of host strain (CBA or NIH), rate of exposure (5 or 40 L3/mouse/2 weeks) and diet (3 or 16% protein w/w) on the population dynamics of repeated infection and the response to a standard challenge infection were investigated. The time delay between the end of the period of repeated infection and the subsequent challenge (between 1 and 24 weeks) had no effect on worm recovery. The effects of both exposure and diet were significant and similar whether assessed on the basis of the dynamics of repeated infection or response to challenge: low rates of exposure and low dietary protein were both associated with low levels of acquired immunity. Mouse strain was the most important determinant of worm recovery after challenge, but had no significant effect on the degree to which parasite population growth was constrained by acquired immunity during repeated infection. It is suggested that both CBA and NIH mice raise immune responses which act on parasite survival, but that only NIH mice raise responses operative against larval establishment.
Assuntos
Infecções por Nematoides/imunologia , Nematospiroides dubius/imunologia , Fenômenos Fisiológicos da Nutrição Animal , Animais , Animais de Laboratório , Proteínas Alimentares/administração & dosagem , Imunidade Ativa/genética , Imunidade Ativa/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos CBA/parasitologia , Camundongos Endogâmicos/parasitologia , Infecções por Nematoides/genética , Infecções por Nematoides/parasitologia , Nematospiroides dubius/crescimento & desenvolvimento , Dinâmica PopulacionalRESUMO
Outbred MF1 mice were characterized with respect to their susceptibility to infection with Heligmosomoides polygyrus (Nematoda) on the basis of faecal egg counts after 8 weeks of repeated infection (50 larvae/week). Selective breeding for resistance and susceptibility was carried out over 3 generations. The H-2 type of a sample of the mice was determined, and antigen recognition assessed on the basis of Western blots against adult and larval H. polygyrus homogenate. The selective breeding programme yielded very strong evidence for the heritability of susceptibility to infection. The results were consistent with a model of single gene control with resistance dominant over susceptibility. The presence of the H-2k haplotype was significantly associated with susceptibility, as was the recognition of a 17 kDa antigen in blots against both larval and adult worm homogenate. The proportion of mice phenotypically susceptible to infection, the proportion bearing the H-2k haplotype, and the proportion recognizing the 17 kDa antigen, were all approximately 0.25.