Phenotypic clustering in MPZ mutations.

Myelin protein zero (MPZ) is a member of the immunoglobulin gene superfamily with single extracellular, transmembrane and cytoplasmic domains. Homotypic interactions between extracellular domains of MPZ adhere adjacent myelin wraps to each other. MPZ is also necessary for myelin compaction since mice which lack MPZ develop severe dysmyelinating neuropathies in which compaction is dramatically disrupted. MPZ mutations in humans cause the inherited demyelinating neuropathy CMT1B. Some mutations cause the severe neuropathies of infancy designated as Dejerine-Sottas disease, while others cause a 'classical' Charcot-Marie-Tooth (CMT) disease Type 1B (CMT1B) phenotype with normal early milestones but development of disability during the first two decades of life. Still other mutations cause a neuropathy that presents in adults, with normal nerve conduction velocities, designated as a 'CMT2' form of CMT1B. To correlate the phenotype of patients with MPZ mutations with their genotype, we identified and evaluated 13 patients from 12 different families with eight different MPZ mutations. In addition, we re-analysed the clinical data from 64 cases of CMT1B from the literature. Contrary to our expectations, we found that most patients presented with either an early onset neuropathy with signs and symptoms prior to the onset of walking or a late onset neuropathy with signs and symptoms at around age 40 years. Only occasional patients presented with a 'classical' CMT phenotype. Correlation of specific MPZ mutations with their phenotypes demonstrated that addition of either a charged amino acid or altering a cysteine residue in the extracellular domain caused a severe early onset neuropathy. Severe neuropathy was also caused by truncation of the cytoplasmic domain or alteration of an evolutionarily conserved amino acid. Taken together, these data suggest that early onset neuropathy is caused by MPZ mutations that significantly disrupt the tertiary structure of MPZ and thus interfere with MPZ-mediated adhesion and myelin compaction. In contrast, late onset neuropathy is caused by mutations that more subtly alter myelin structure and which probably disrupt Schwann cell-axonal interactions.

Increased survival and function of SOD1 mice after glial cell-derived neurotrophic factor gene therapy.

Amyotrophic lateral sclerosis (ALS) is caused by a progressive degeneration of motor neurons. The cause of sporadic ALS is not known, but 1-2% of all cases are familial and caused by mutations in the copper-zinc superoxide dismutase (SOD1) gene. Transgenic SOD1 mice serve as a transgenic mouse model for these cases. Glial cell-derived neurotrophic factor (GDNF) has a potent trophic effect on motor neurons. Clinical trials in which growth factors have been systemically administered to ALS patients have not been effective, owing in part to the short half-life of these factors and their low concentrations at target sites. Gene transfer of therapeutic factors to motor neurons and/or their target cells, such as muscle, may overcome these problems. Previously, we and others have shown that intramuscularly administered adenovirus vector (AVR) results in foreign gene expression not only in muscle cells, but also in relevant motor neurons in the spinal cord, because of retrograde axonal transport. In this study we utilized an AVR to introduce GDNF into muscles of neonatal SOD1 mice. We showed that AVR-mediated GDNF expression delayed the onset of disease by 7 +/- 8 days (mean +/- SD), prolonged survival by 17 +/- 10 days, and delayed the decline in motor functions (as determined on a rotating rod) by 7-14 days. These results demonstrate that gene delivery to muscle and motor neurons has the potential to treat devastating neurodegenerative diseases such as ALS.

Overcoming Cellular Immunity to Prolong Adenoviral-Mediated Gene Expression in Sciatic Nerve.

In a previous report, we demonstrated that a first generation (E1- and E3-deleted) recombinant adenovirus can transduce expression of the E. coli lacZ gene into Schwann cells, both in vitro and in vivo, suggesting that this method might be useful for future therapy of peripheral neuropathy, including CMT1. Adenoviral-mediated gene transfer was limited, however, by demyelination and Wallerian degeneration at the site of virus injection, as well as by attenuation of viral gene expression over time. In our current work we have optimized adenoviral-mediated gene expression after intraneural injection into sciatic nerve. Using an improved injection protocol, peak expression of lacZ occurs between 10 and 14 days after injection of 2-week-old animals, decreases thereafter, and there is minimal associated tissue injury. In contrast, very few adenoviral-infected Schwann cells are found in nerves of adult animals 10 days after injection, probably due to immune clearance of viral-infected cells. Consistent with this notion, high levels of lacZ are found in sciatic nerve 30 days after injection of adult SCID mice, which have a genetic defect in both cellular and humoral immunity, of adult ß2 microglobulin-deficient mice (ß2 M -/-), which have a genetic defect in cellular immunity, or of adult mice treated with the immunosuppressing agent FK506. In addition, adenoviral-infected Schwann cells co-cultured with axons in vitro, in the absence of a host immune response, ensheath axons and express lacZ for at least 8 weeks. These data thus demonstrate that expression of first generation recombinant adenovirus in sciatic nerve in adult mice, as in other tissues, is limited mainly by the host cellular immune response to the virus, which can be overcome by attenuation of host cell-mediated immunity. Adenoviral vectors might thus be used to modulate Schwann cell gene expression in patients with peripheral neuropathy after appropriate immunosuppression.