RESUMO
The myelin-associated glycoprotein (MAG) is an integral membrane glycoprotein that is located in the periaxonal membrane of myelin-forming Schwann cells. On the basis of this localization, it has been hypothesized that MAG plays a structural role in (a) forming and maintaining contact between myelinating Schwann cells and the axon (the 12-14-nm periaxonal space) and (b) maintaining the Schwann cell periaxonal cytoplasmic collar of myelinated fibers. To test this hypothesis, we have determined the immunocytochemical localization of MAG in the L4 ventral roots from 11-mo-old quaking mice. These roots display various stages in the association of remyelinating Schwann cells with axons, and abnormalities including loss of the Schwann cell periaxonal cytoplasmic collar and dilation of the periaxonal space of myelinated fibers. Therefore, this mutant provides distinct opportunities to observe the relationships between MAG and (a) the formation of the periaxonal space during remyelination and (b) the maintenance of the periaxonal space and Schwann cell periaxonal cytoplasmic collar in myelinated fibers. During association of remyelinating Schwann cells and axons, MAG was detected in Schwann cell adaxonal membranes that apposed the axolemma by 12-14 nm. Schwann cell plasma membranes separated from the axolemma by distances greater than 12-14 nm did not react with MAG antiserum. MAG was present in adaxonal Schwann cell membranes that apposed the axolemma by 12-14 nm but only partially surrounded the axon and, therefore, may be actively involved in the ensheathment of axons by remyelinating Schwann cells. To test the dual role of MAG in maintaining the periaxonal space and Schwann cell periaxonal cytoplasmic collar of myelinated fibers, we determined the immunocytochemical localization of MAG in myelinated quaking fibers that displayed pathological alterations of these structures. Where Schwann cell periaxonal membranes were not stained by MAG antiserum, the cytoplasmic side of the periaxonal membrane was "fused" with the cytoplasmic side of the inner compact myelin lamella and formed a major dense line. This loss of MAG and the Schwann cell periaxonal cytoplasmic collar usually resulted in enlargement of the 12-14-nm periaxonal space and ruffling of the apposing axolemma. In myelinated fibers, there was a strict correlation between the presence of MAG in the Schwann cell periaxonal membrane and (a) maintenance of the 12-14-nm periaxonal space, and (b) presence of the Schwann cell periaxonal cytoplasmic collar.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Axônios/ultraestrutura , Camundongos Quaking/fisiologia , Proteínas da Mielina/fisiologia , Bainha de Mielina/ultraestrutura , Células de Schwann/ultraestrutura , Animais , Técnicas Imunoenzimáticas , Camundongos , Microscopia Eletrônica , Glicoproteína Associada a Mielina , Células de Schwann/citologia , Medula Espinal/citologia , Medula Espinal/ultraestruturaRESUMO
Quaking is an autosomal recessive hypo/dysmyelinating mutant mouse which has a 1-Mbp deletion on chromosome 17. The mutation exhibits pleiotrophy and does not include genes encoding characterized myelin proteins. The levels of the 67-kD isoform of the myelin-associated glycoprotein (S-MAG) relative to those of the 72-kD isoform (L-MAG) are increased in the quaking CNS, but not in other dysmyelinating mutants. Abnormal expression of MAG isoforms in quaking may result from altered transcription of the MAG gene or from abnormal sorting, transport, or targeting of L-MAG or S-MAG. To test these hypotheses, we have determined the distribution of L-MAG and S-MAG in cervical spinal cord of 7-, 14-, 21-, 28-, and 35-d-old quaking mice. In 7-d-old quaking and control spinal cord, L- and S-MAG was detectable in periaxonal regions of myelinated fibers and in the perinuclear cytoplasm of oligodendrocytes. Between 7 and 35 d, L-MAG was removed from the periaxonal membrane of quaking but not control mice. Compared to control mice, a significant increase in MAG labeling of endosomes occurred within oligodendrocyte cytoplasm of 35-d-old quaking mice. S-MAG remained in periaxonal membranes of both quaking and control mice. Analysis of the cytoplasmic domain of L-MAG identifies amino acid motifs at tyrosine 35 and tyrosine 65 which meet the criteria for "tyrosine internalization signals" that direct transmembrane glycoproteins into the endocytic pathway. These results establish that L-MAG is selectively removed from the periaxonal membrane of CNS-myelinated fibers by receptor-mediated endocytosis. The loss of L-MAG from quaking periaxonal membranes results from increased endocytosis of L-MAG and possibly a decrease in L-MAG production.
Assuntos
Endocitose/fisiologia , Camundongos Quaking/fisiologia , Bainha de Mielina/química , Glicoproteína Associada a Mielina/análise , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Axônios/química , Axônios/ultraestrutura , Imuno-Histoquímica , Isomerismo , Camundongos , Microscopia Confocal , Dados de Sequência Molecular , Proteína Proteolipídica de Mielina/análise , Proteína Proteolipídica de Mielina/imunologia , Glicoproteína Associada a Mielina/imunologia , Glicoproteína Associada a Mielina/metabolismo , Medula Espinal/química , Frações Subcelulares/químicaRESUMO
Myelin of considerable purity may be isolated from small (minimum 1 mg wet weight) samples of central nervous tissue, using a 4-step centrifugation procedure. The separation of myelin proteins by micro-linear gradient polyacrylamide gel electrophoresis yields similar results to those obtained by macro-scale (homogeneous) gel systems. These techniques have been employed for a preliminary study of the regional composition of myelin fractions from the Quaking mouse.
Assuntos
Química Encefálica , Camundongos Quaking/fisiologia , Bainha de Mielina/análise , Medula Espinal/análise , Animais , Centrifugação/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Camundongos , Microquímica/métodos , Proteínas da Mielina/análise , Proteolipídeos/análiseRESUMO
The quaking(viable) (qkv) mutation, a spontaneous deletion of a multigenic region encompassing roughly 1 Mb at 5.9 cM on the proximal end of mouse chromosome 17, causes severe trembling in all homozygous animals and infertility in all homozygous males. Physiologically, quaking mice exhibit dysmyelination and postmeiotic spermatogenic arrest. Molecular defects in Qkv mice occur in the affected tissues, indicating the primary causes of these pathologies are cell autonomous. However, because both the reproductive and neurological defects are in immune-privileged sites and because some similar pathologies at both sites have been shown to be immune mediated, we tested whether the immune system participates secondarily in manifestation of Qkv phenotypes. The qkv mutation was bred into a severe combined immune-deficient mouse line (SCID; devoid of mature B and T cells) and penetrance of the neurological and the male sterile phenotypes was measured. Results showed that neither defect was ameliorated in the immune-deficient background. We conclude that the Qkv pathologies do not likely involve a B- or T-cell-dependent response against these immune-privileged sites.
Assuntos
Camundongos Quaking/genética , Camundongos Quaking/imunologia , Camundongos SCID/genética , Camundongos SCID/imunologia , Animais , Sequência de Bases , DNA Complementar/genética , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/imunologia , Feminino , Infertilidade Masculina/genética , Infertilidade Masculina/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Quaking/fisiologia , Camundongos SCID/fisiologia , Fenótipo , Gravidez , Reprodução/genética , Reprodução/imunologia , Espermatogênese/genética , Espermatogênese/imunologiaRESUMO
Fragments of normal embryonic cerebellum were transplanted into adult Quaking mice to examine using peroxidase-antiperoxidase immunocytochemistry the development of genetically normal tissue in an abnormal host environment. The Quaking mouse animals used as hosts are characterized by defective myelin associated glycoprotein. Normal characteristic expression and distribution of neurofilaments was observed in the cerebellar grafts. Nonphosphorylated epitopes of neurofilaments were seen in Purkinje cell bodies and dendrites. The phosphorylated epitopes of neurofilaments were observed in basket cell axons. Phosphorylated epitopes were also present in numerous myelinated axons, which were probably fibers from deep cerebellar nuclei. These data support the notion that neurogenesis with alternative connections can occur in transplants. Staining patterns with myelin associated glycoprotein and myelin basic protein also suggest that normal myelination occurs in grafts transplanted to the brains of Quaking mice.
Assuntos
Cerebelo/transplante , Camundongos Quaking/fisiologia , Animais , Cerebelo/análise , Cerebelo/fisiologia , Sobrevivência de Enxerto , Imuno-Histoquímica , Proteínas de Filamentos Intermediários/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neurofilamentos , Transplante HomólogoRESUMO
The effect of 5-hydroxytryptophan (5-HTP) on brain stem auditory evoked response (BAER) amplitude in Quaking (qk) and normal littermate mice was examined. Administration of 5-hydroxytryptophan (5-HTP) (75 mg/kg, i.p.) to normal mice increased the amplitude of BAER peaks I, II, and III but had no effect on peak IV. In qk mice, however, 5-HTP did not affect the amplitude of any BAER peaks. Our data indicate that although 5-HTP increased BAER amplitude in normal mice, it failed to modulate BAER in qk mice. These findings are consistent with the possibility that 5-HTP receptor sites associated with myelin basic protein may be reduced in the myelin-deficient mutant qk mice.
Assuntos
5-Hidroxitriptofano/farmacologia , Tronco Encefálico/efeitos dos fármacos , Potenciais Evocados Auditivos/efeitos dos fármacos , Camundongos Quaking/fisiologia , Bainha de Mielina/fisiologia , Animais , Camundongos , Camundongos Quaking/genéticaRESUMO
Myelin basic protein (MBP) was quantified using a RIA technique in the spinal cord, cerebellum, diencephalon plus brainstem region and cerebral hemispheres of two dysmyelinating murine mutants, quaking (qk) and jimpy (jp) mice. Comparison was made with normal control values. The whole life-span has been investigated: ie, ages ranging from 0 to 26 days for the jp, O to one year for the qk, and prenatal stage to three years for the control animals. Assays in the mutants at early ages were rendered feasible by the use of marker genes, which has allowed the diagnosis of the mutation at birth, 12 days before the expression of their typical tremor phenotype. Special care was given to the period of early myelinogenesis in order to clarify the dysynchrony between the various parts of the central nervous system. In normal mice, MBP was already detected in the brain of 19-day-old embryos. During development, rapid accumulation of MBP first occurred in the spinal cord then in the diencephalon, the brainstem, the cerebellum, and finally in the cerebral hemispheres. In the 25-day-old jimpy mutant, levels of MBP were found dramatically decreased, never exceeding 6% of the normal controls in any of the areas investigated. The situation for the quaking mouse was quite different. This mutant could be investigated up to one year old. At that age, a high discrepancy was observed between the values found in the brain and in the spinal cord (respectively, 10% and 35%) compared to normal controls. In both mutants, not only were the levels of MBP decreased, but also its appearance during development was delayed. Nevertheless, in both mutants the caudo-rostral timing of myelination as assayed by MBP levels was maintained. Furthermore, the later myelination occurred, the stronger weas the deficit in MBP. Interestingly, in the quaking mutant, the specific plasticity of the spinal cord was exemplified by its ability to reduce constantly, even at an advanced age, its initial deficit of MBP.