Guénard V, Schweitzer B, Flechsig E, Hemmi S, Martini R, Suter U, Schachner M
Department of Neurobiology, Swiss Federal Institute of Technology, Zürich.
Glia. 1999 Jan 15;25(2):165-78.
Mutations in the gene encoding for the myelinating Schwann cell protein P0 have been linked to inherited peripheral neuropathies, including the Charcot-Marie-Tooth type 1B disease (CMT1B) and Dejerine-Sottas syndrome (DSS). Recently generated mice deficient in the P0 gene (P0-/- mice) resemble cases of CMT1B and DSS with impaired myelin dosage (Martini et al., 1995a). Potential approaches to treat such diseases include the introduction of the normal gene in the nerves of strongly affected patients. In the present study we used P0-/- mice to evaluate the efficiency of a replication-defective, E1-deleted adenovirus vector carrying the lacZ (Ad-RSV-lacZ) or P0 (Ad-RSV-P0) gene to infect abnormally myelinating Schwann cells. The Ad-RSV-lacZ vector suspension was injected into the left sciatic nerve ofPO-/- mice and the nerves examined for beta-galactosidase activity by X-gal histochemistry. Contralateral nerves injected with vehicle solution or non-injected served as controls. Beta-galactosidase activity was detected in nerves injected with the Ad-RSV-lacZ vector up to 2 weeks post-injection. Immunosuppressing the mice with FK506 to decrease the infiltration of activated T-cells in infected nerves lengthened beta-galactosidase activity to 8 weeks, the longest time point examined. Ultrastructural analysis indicated that X-gal crystals were present mostly in abnormally myelinating Schwann cells. These findings demonstrate that an adenovirus vector can successfully infect Schwann cells in P0-/- mice and expression can be maintained for several weeks. The Ad-RSV-P0 suspension was then injected in the sciatic nerve of immunosuppressed P0-/- mice. Two and four weeks post-injection both P0 mRNA and protein could be detected by in situ hybridization and Western blotting in some of the nerves. Furthermore, P0 protein expression was observed in myelin-like structures and onion bulb-like cells by immunohistochemistry. These results indicate that Schwann cells in P0-/- mice can be induced to produce P0 protein after gene transfer. Genetic repair of abnormal Schwann cells by using adenovirus vectors might be a possible technique to treat animal models of inherited peripheral neuropathies.
编码髓鞘形成雪旺细胞蛋白P0的基因突变与遗传性周围神经病有关，包括1B型夏科-马里-图斯病（CMT1B）和德热里纳-索塔斯综合征（DSS）。最近培育出的P0基因缺陷小鼠（P0-/-小鼠）类似于髓鞘量受损的CMT1B和DSS病例（Martini等人，1995a）。治疗此类疾病的潜在方法包括将正常基因导入病情严重患者的神经中。在本研究中，我们使用P0-/-小鼠来评估携带lacZ（Ad-RSV-lacZ）或P0（Ad-RSV-P0）基因的复制缺陷型、E1缺失腺病毒载体感染异常髓鞘形成雪旺细胞的效率。将Ad-RSV-lacZ载体悬液注入P0-/-小鼠的左侧坐骨神经，并通过X-gal组织化学检查神经中的β-半乳糖苷酶活性。注射赋形剂溶液或未注射的对侧神经作为对照。在注射Ad-RSV-lacZ载体的神经中，直至注射后2周都检测到了β-半乳糖苷酶活性。用FK506对小鼠进行免疫抑制以减少活化T细胞在受感染神经中的浸润，可将β-半乳糖苷酶活性延长至8周，这是检测的最长时间点。超微结构分析表明，X-gal晶体主要存在于异常髓鞘形成的雪旺细胞中。这些发现表明，腺病毒载体可以成功感染P0-/-小鼠中的雪旺细胞，并且表达可以维持数周。然后将Ad-RSV-P0悬液注射到免疫抑制的P0-/-小鼠的坐骨神经中。注射后2周和4周，通过原位杂交和蛋白质印迹法在一些神经中均可检测到P0 mRNA和蛋白质。此外，通过免疫组织化学在髓鞘样结构和洋葱球样细胞中观察到了P0蛋白表达。这些结果表明，基因转移后P0-/-小鼠中的雪旺细胞可被诱导产生P0蛋白。使用腺病毒载体对异常雪旺细胞进行基因修复可能是治疗遗传性周围神经病动物模型的一种可行技术。
