Mutations in the cytoplasmic domain of P0 reveal a role for PKC-mediated phosphorylation in adhesion and myelination.

Mutations in P0 (MPZ), the major myelin protein of the peripheral nervous system, cause the inherited demyelinating neuropathy Charcot-Marie-Tooth disease type 1B. P0 is a member of the immunoglobulin superfamily and functions as a homophilic adhesion molecule. We now show that point mutations in the cytoplasmic domain that modify a PKC target motif (RSTK) or an adjacent serine residue abolish P0 adhesion function and can cause peripheral neuropathy in humans. Consistent with these data, PKCalpha along with the PKC binding protein RACK1 are immunoprecipitated with wild-type P0, and inhibition of PKC activity abolishes P0-mediated adhesion. Point mutations in the RSTK target site that abolish adhesion do not alter the association of PKC with P0; however, deletion of a 14 amino acid region, which includes the RSTK motif, does abolish the association. Thus, the interaction of PKCalpha with the cytoplasmic domain of P0 is independent of specific target residues but is dependent on a nearby sequence. We conclude that PKC-mediated phosphorylation of specific residues within the cytoplasmic domain of P0 is necessary for P0-mediated adhesion, and alteration of this process can cause demyelinating neuropathy in humans.

DM-GRASP, a novel immunoglobulin superfamily axonal surface protein that supports neurite extension.

We have identified a 95 kd cell surface protein, DM-GRASP, that is expressed on a restricted population of axons. Its expression begins early in chick embryogenesis, and within the spinal cord it is localized to axons in the dorsal funiculus, midline floorplate cells, and motoneurons. Antibodies to DM-GRASP impair neurite extension on axons, and purified DM-GRASP supports neurite extension from chick sensory neurons. We have cloned and sequenced the cDNA corresponding to this protein and find that it is a new member of the immunoglobulin superfamily of adhesion molecules. Consequently we have named this protein DM-GRASP, since it is an immunoglobulin-like restricted axonal surface protein that is expressed in the dorsal funiculus and ventral midline of the chick spinal cord.

Differential regulation of the 2',3'-cyclic nucleotide 3'-phosphodiesterase gene during oligodendrocyte development.

The two major isoforms of 2',3'-cyclic nucleotide phosphodiesterase (CNP), 48 and 46 kDa, have recently been shown to be produced from a single gene by alternative splicing. In addition, messenger RNA encoding the larger isoform is transcribed from a separate promoter, approximately 1 kb upstream from that encoding the smaller isoform. We have investigated the expression of these two CNP isoforms and have found that they are differentially expressed during the process of oligodendrocyte maturation. In oligodendrocyte precursors, only the mRNA encoding the larger protein is found. At the time of oligodendrocyte differentiation, however, both CNP mRNAs are induced. These patterns of CNP expression are likely due to stage-specific transcriptional regulation of the two CNP promoters during the process of oligodendrocyte differentiation.

Proteolipid protein is necessary in peripheral as well as central myelin.

Alternative products of the proteolipid protein gene (PLP), proteolipid protein (PLP) and DM20, are major components of compact myelin in the central nervous system, but quantitatively minor constituents of Schwann cells. A family with a null allele of PLP has a less severe CNS phenotype than those with other types of PLP mutations. Moreover, individuals with PLP null mutations have a demyelinating peripheral neuropathy, not seen with other PLP mutations of humans or animals. Direct analysis of normal peripheral nerve demonstrates that PLP is localized to compact myelin. This and the clinical and pathologic observations of the PLP null phenotype indicate that PLP/DM20 is necessary for proper myelin function both in the central and peripheral nervous systems.

Double-stranded RNA unwinding and modifying activity is detected ubiquitously in primary tissues and cell lines.

A double-stranded RNA unwinding and modifying activity was found to be present in a wide range of tissues and cell types. The level of activity did not vary significantly with respect to the state of cell differentiation, cell cycle, or transformation. Thus, the unwinding and modifying activity, localized in the nucleus in somatic cells and capable of converting many adenosine residues to inosine, appears to be one of the housekeeping genes.

Organization of the mouse GP42/Basigin gene: a member of the Ig superfamily.

We have mapped and sequenced the GP42/Basigin gene isolated from a Balb/C mouse genomic library. The genomic organization and upstream, putative regulatory, regions of this gene have not been previously reported. Our data show that exon 5 of the GP42/Basigin gene encodes the carboxy proximal half of the second Ig-like domain, the highly conserved transmembrane region and a portion of the cytoplasmic tail. This inclusion of Ig-like and other functional domains in a single exon is unusual. Splice junction analysis indicates that two reported alternate GP42/Basigin cDNA isoforms are likely due to cloning artifacts. In addition, we find that GP42/Basigin is polymorphic in mice. Our data also support the proposal that the transmembrane domain and portions of the cytoplasmic region of GP42/Basigin have been evolutionarily conserved.

Modulation of cell-mediated immunity prolongs adenovirus-mediated transgene 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. Adenovirus-mediated gene transfer was limited, however, by demyelination and Wallerian degeneration at the site of virus injection, as well as by attenuation of viral transgene expression over time. In our current work we have optimized adenoviral vector-mediated transgene 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 rats, decreases thereafter, and there is minimal associated tissue injury. In contrast, few lacZ-expressing Schwann cells are found in nerve of adult animals 10 days after injection, probably owing to immune clearance of virus-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 beta2-microglobulin-deficient mice (beta2M4-/-), which have a genetic defect in cellular immunity, or of adult mice treated with the immunosuppressing agent FK506. In addition, adenovirus-infected Schwann cells cocultured with axons in vitro, in the absence of a host immune response, ensheathe axons and express lacZ for at least 8 weeks. These data thus demonstrate that lacZ transgene 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.

Heterozygous P0 knockout mice develop a peripheral neuropathy that resembles chronic inflammatory demyelinating polyneuropathy (CIDP).

Demyelinating peripheral neuropathies are clinically divided into inherited and acquired types. Inherited demyelinating neuropathies are caused by mutations in genes expressed by myelinating Schwann cells, whereas acquired ones, including chronic inflammatory demyelinating polyneuropathy (CIDP), are probably caused by autoimmune mechanisms. We find that heterozygous P0 knockout (P0+/-) mice develop a neuropathy that resembles CIDP. By one year of age, P0+/- mice develop severe, asymmetric slowing of motor nerves, with temporal dispersion or conduction block, which are features of acquired demyelinating neuropathies including CIDP. Moreover, morphological analysis of affected nerves reveals severe and selective demyelination of motor fibers, focal regions of demyelination, and inflammatory cells. These data suggest that immune-mediated mechanisms may contribute to the pathogenesis of the neuropathy in P0+/- mice.

Molecular cloning and characterization of the purE operon of Escherichia coli.

The purE operon of Escherichia coli has been cloned and localized to a 1.7-kb HpaI fragment. The operon has been further characterized by subcloning into the lac fusion vector, pMC1403, and by the construction of BAL 31-generated deletions. The purE regulation region has been identified by assay of beta-galactosidase produced by pur-lac fusion plasmids and by RNA polymerase binding to end-labelled restriction fragments. Two purE promoters have been identified; one strong that is regulated by purines, the other weaker which is not regulated. The latter may be internal to the purE1 structural gene.

The molecular pathogenesis of Pelizaeus-Merzbacher disease.

In 1885, Pelizaeus described 5 boys in a single family with nystagmus, spastic quadriparesis, ataxia, and delay in cognitive development. In 1910, Merzbacher reexamined this family, which then included 14 affected individuals, including 2 girls, and found that all affected family members shared a common female ancestor. Also, he noted that the disease was passed exclusively through the female line without male-to-male transmission. Pathological analysis of brain tissue from one affected individual showed that most of the central white matter lacked histochemical staining for myelin, although there were occasional small regions of preserved myelin, giving the sections a "tigroid" appearance. The description of this family provides the clinical, genetic, and pathological basis for Pelizaeus-Merzbacher disease (PMD): an X-linked disorder of myelination classically characterized by nystagmus, spastic quadriparesis, ataxia, and cognitive delay in early childhood.

Complicated hereditary spastic paraparesis with cerebral white matter lesions.

A family is described with 5 males in a single generation affected with a previously undescribed complicated form of hereditary spastic paraparesis (HSP). The disease is characterized by speech difficulties, lower limb spasticity and hyper-reflexia, mental retardation, cerebellar ataxia, and tremor. The disease starts in the first decade of life and progresses for 3 to 6 years before stabilizing. Magnetic resonance imaging (MRI) of the brain demonstrates bilateral posterior periventricular white matter lesions. Visual evoked responses are markedly prolonged, but electromyography (EMG) and nerve conduction velocity studies are normal. Three of the 4 living affected members of this pedigree exhibit red-green color vision defects. The presentation of a new complicated hereditary spastic paraparesis syndrome in this pedigree extends our understanding of the variability and heterogenity of this syndrome and suggests an approach for the evaluation of similar families in future genetic studies.

Analysis of clinical variation seen in patients with 18q terminal deletions.

Twenty-six patients with deletions of 18q were analyzed at the clinical and molecular levels in an attempt to delineate regions of chromosome 18 important to the 18q- syndrome phenotype. Molecular cytogenetic analysis was carried out using fluorescence in situ hybridization (FISH), and deletions ranging from 18q21.1-qter to 18q22.3-qter were detected. The parental origin of the deletions was determined by the analysis of inheritance of microsatellite markers. No correlation between size, parental origin, or severity of the resulting phenotype was found. The results suggest that a critical region for 18q- syndrome lies in the most distal portion of 18q and that it confers susceptibility for the various clinical manifestations of the 18q- syndrome when present in one copy.

Oculodentodigital dysplasia syndrome associated with abnormal cerebral white matter.

Oculodentodigital dysplasia (ODDD) syndrome is an uncommon inherited disorder with eye and facial abnormalities, syndactyly, and defects in tooth enamel. Some of the previously reported patients with ODDD syndrome also manifested spastic quadriparesis. We describe a patient with sporadic ODDD syndrome referred for evaluation of progressive spastic paraparesis. Magnetic resonance imaging of the brain demonstrated abnormal white matter, which suggests an explanation for the observed spastic paraparesis.

Neuropsychiatry of 18q- syndrome.

Our understanding of neuropsychiatric abnormalities in patients with deletions of the long arm of chromosome 18 (18q- syndrome) is based mainly on sporadic case reports. We characterized the neuropsychiatric phenotype in 27 patients across a wide age range (2-47 years) with breakpoints ranging from 18q22.3-18q21.2. Adaptive behavior scores (Vineland Composite) were significantly higher in females than in males (62 +/- 5 vs. 43 +/- 3). Intelligence ranged from borderline to severely deficient (IQ, 73- < 40), with academic achievement similarly impaired. Performance in specific neuropsychological functions, including attention, novel problem solving, memory, language, visuomotor integration, and fine motor dexterity, was consistently in the moderately-to-severely impaired range. Behavioral problems were common in both sexes, including aggressivity, hyperactivity, and temper tantrums. Contrary to the few previous reports, we found no evidence of psychosis in any patients. In a subset of patients selected on the basis of no prior knowledge of behavioral problems, 1 of 16 patients (6%) had autism, as defined by the Autistic Diagnostic Interview--Revised (ADI-R) [Lord et al., 1994: J Autism Dev Disord 24:659-685]. Thus, the prevalence of autism in 18q- syndrome is probably no greater than that in other developmental disabilities with a similar level of cognitive impairment. In contrast to what has been believed since 18q- was first described 30 years ago, we found no relationship between chromosome deletion size and any measure of cognition or behavior; nor were there any correlations between any of these measures with the presence or absence of abnormalities on MRI or somatosensory-evoked potentials.

The single lamprey neurofilament subunit (NF-180) lacks multiphosphorylation repeats and is expressed selectively in projection neurons.

The lamprey is considered the most primitive living vertebrate and its neurofilaments (NFs) are unique in being homopolymers of a single 180 kDa subunit (NF-180). Previous immunologic studies have suggested that the sidearm of NF-180 is highly phosphorylated selectively in the largest diameter axons. We report in this study the isolation and characterization of cDNA clones encoding the NF-180 lamprey protein. In situ hybridization with digoxigenin-labeled cRNA revealed NF-180 message exclusively in neurons with long axons, such as reticulospinal neurons and cranial motor neurons. The core of NF-180 was similar in structure to those of mammalian neurofilaments, but surprisingly, the carboxy sidearm lacked the multiphosphorylation repeats characteristic of higher vertebrate and invertebrate neurofilaments. Overall there was a paucity of potential phosphorylation sites in the NF-180 carboxy-terminus compared to NF-M and NF-H of mammals, fish and squid. This, along with the highly acidic nature of the NF-180 sidearm, makes it unlikely that phosphorylation of sidearm residues regulates interfilament spacing and axon diameter through global electrostatic repulsion of the carboxy-terminus away from the filament backbone. Furthermore, the expression of a single neurofilament subunit in the lamprey that is most similar to the NF-M of higher vertebrates suggests that all three mammalian neurofilament subunits evolved from a single NF-M-like precursor.

Regulation of myelin-specific gene expression. Relevance to CMT1.

Schwann cells, the myelinating cells of the peripheral nervous system, are derived from the neural crest. Once neural crest cells are committed to the Schwann cell fate, they can take on one of two phenotypes to become myelinating or nonmyelinating Schwann cells, a decision that is determined by interactions with axons. The critical step in the differentiation of myelinating Schwann cells is the establishment of a one-to-one relationship with axons, the so-called "promyelinating" stage of Schwann cell development. The transition from the promyelinating to the myelinating stage of development is then accompanied by a number of significant changes in the pattern of gene expression, including the activation of a set of genes encoding myelin structural proteins and lipid biosynthetic enzymes, and the inactivation of a set of genes expressed only in immature or nonmyelinating Schwann cells. These changes are regulated mainly at the transcriptional level and also require continuous interaction between Schwann cells and their axons. Two transcription factors, Krox 20 (EGR2) and Oct 6 (SCIP/Tst1), are necessary for the transition from the promyelinating to the myelinating stage of Schwann cell development. Krox 20, expressed in myelinating but not promyelinating Schwann cells, is absolutely required for this transition, and myelination cannot occur in its absence. Oct 6, expressed mainly in promyelinating Schwann cells and then down-regulated before myelination, is necessary for the correct timing of this transition, since myelination is delayed in its absence. Neither Krox 20 nor Oct 6, however, is required for the initial activation of myelin gene expression. Although the mechanisms of Krox 20 and Oct 6 action during myelination are not known, mutation in Krox 20 has been shown to cause CMT1, further implicating this protein in the pathogenesis of this disease. Identifying the molecular mechanisms of Krox 20 and Oct 6 action will thus be important both for understanding myelination and for designing future treatments for CMT1. Point mutlations in the genes encoding the myelin proteins PMP22 and P0 cause CMT1A without a gene duplication and CMT1B, respectively. Although the clinical and pathological phenotypes of CMT1A and CMT1B are similar, their molecular pathogenesis is quite different. Point mutations in PMP22 alter the trafficking of the protein, so that it accumulates in the endoplasmic reticulum (ER) and intermediate compartment (IC). Mutant PMP22 also sequesters its normal counterpart in the ER, further reducing the amount of PMP22 available for myelin synthesis at the membrane, and accounting, at least in part, for its severe effect on myelination. Mutant PMP22 probably also activates an ER-to-nucleus signal transduction pathway associated with misfolded proteins, which may account for the decrease of myelin gene expression in Schwann cells in Trembler mutant mice. In contrast, absence of expression of the homotypic adhesion molecule, P0, in mice in which the gene has been inactivated, produces a unique pattern of Schwann cell gene expression, demonstrating that P0 plays a regulatory as well as a structural role in myelination. Whether this role is direct, through a P0-mediated adhesion pathway, or indirect, through adhesion pathways mediated by cadherins or integrins, however, remains to be determined. The molecular mechanisms underlying dysmyelination in CMT1 are thus complex, with pleitropic effects on Schwann cell physiology that are determined both by the type of mutation and the protein mutated. Identifying these molecular mechanisms, however, are important both for understanding myelination and for designing future treatments for CMT1. Although demyelination is the hallmark of CMT1, the clinical signs and symptoms of this disease are probably produced by axonal degeneration, not demyelination. (ABSTRACT TRUNCATED)

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 SCOD mice, which have a genetic defect in both cellular and humoral immunity, of adult beta 2 microglobulin-deficient mice (beta 2 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.

Correlation between weakness and axonal loss in patients with CMT1A.

We have developed a protocol to measure the progression of disability in patients with Charcot Marie Tooth (CMT) disease, particularly CMT1 over a several year period. Because CMT1 is a chronic disease, the natural history of changes occurring in such a brief period are not well understood, making clinical trials for CMT1 patients difficult to evaluate. We hypothesize that weakness in CMT1 correlates with axonal loss secondary to the abnormalities in Schwann cell myelin gene expression, which cause the disease. To test this hypothesis, we elected to carefully evaluate CMT patients by various modalities to measure strength, sensory loss, and axonal loss and demyelination and to compare these modalities to determine whether they correlated with findings on clinical examination. As suspected, patient weakness correlates more with secondary axonal loss than with demyelination, even though the primary abnormality in CMT1 is demyelination.

The absence of myelin P0 protein produces a novel molecular phenotype in Schwann cells.

In order to better understand the pathogenesis of demyelination in P0 knockout (P0-/-) mice, we analyzed the myelin gene expression and the localization of myelin proteins in P0 null mouse sciatic nerve. We have demonstrated that the severe demyelinating neuropathy of P0-knockout mouse is associated with changes in the program of myelin gene expression. Some changes in myelin gene expression occur early, others occur during adulthood. We also provide evidence that the absence of P0 is associated with changes in the localization of specific paranodal proteins in the peripheral nerve. These data suggest that P0 plays an important role, either directly or indirectly, in the program of Schwann cell gene expression and in the specific distribution of peripheral myelin proteins. Furthermore, myelin gene dysregulation and improper localization of paranodal proteins may account, in part, for the pathogenesis of demyelination in P0-knockout mice, as well as in human demyelinating peripheral neuropathy associated with mutations in the P0 gene.