Pediatric optic neuritis: brain MRI abnormalities and risk of multiple sclerosis.

BACKGROUND: Optic neuritis is often the initial presentation of multiple sclerosis (MS). As established by the Optic Neuritis Treatment Trial, an abnormal baseline brain MRI is a strong predictor of MS after isolated optic neuritis in adults. However, the rate of conversion to MS after optic neuritis in children based upon brain MRI findings is unknown. METHODS: We reviewed the medical records of children (<18 years) presenting with optic neuritis between 1993 and 2004 at the Children's Hospital of Philadelphia. Children with a history of demyelinating disease or prior optic neuritis were excluded. Symptoms, ophthalmologic findings, MRI findings, and clinical outcomes were recorded. RESULTS: We identified 29 consecutive children with idiopathic optic neuritis. Eleven patients (38%) had white matter T2/FLAIR lesions in the brain (not including the optic nerves). Eighteen patients were followed for more than 24 months, and 3 of the 18 (17%) developed MS. All 3 patients had an abnormal brain MRI scan at their initial presentation of optic neuritis. None of the patients with a normal brain MRI scan at presentation developed MS over an average follow-up of 88.5 months. Patients with one or more white matter lesions on MRI were more likely to develop MS (3/7 vs 0/11, p = 0.04, Fisher exact test). CONCLUSIONS: Children with brain MRI abnormalities at the time of the diagnosis of optic neuritis have an increased risk of multiple sclerosis. Larger collaborative studies are needed to further define the prognosis for childhood optic neuritis.

Mega-corpus callosum, polymicrogyria, and psychomotor retardation: confirmation of a syndromic entity.

A mega-corpus callosum (CC) is not a common manifestation of neurological disease. Previous reports of patients with a constellation of findings including megalencephaly, perisylvian polymicrogyria, distinct facies, psychomotor retardation and mega-corpus callosum were designated as having megalencephaly, mega-corpus callosum, and complete lack of motor development [OMIM 603387; also referred to as megalencephaly-polymicrogyria-mega-corpus callosum (MEG-PMG-MegaCC)] syndrome. Three patients were initially reported with this syndrome, and a fourth was reported recently. Another case had similar findings in utero and upon autopsy. We present an additional patient who conforms to this phenotype; however, he is not megalencephalic, but has a normal head circumference in the setting of short stature. This patient is also noted to have abnormal saccades and mask-like facies. His motor function is more developed than in the other reported patients and was further improved by treatment with L-DOPA/carbidopa, which was started because of his extrapryramidal symptoms and signs which were associated with low cerebral spinal fluid (CSF) catecholamine levels.

Exon skipping mutations in collagen VI are common and are predictive for severity and inheritance.

Mutations in the genes encoding collagen VI (COL6A1, COL6A2, and COL6A3) cause Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), two related conditions of differing severity. BM is a relatively mild dominantly inherited disorder characterized by proximal weakness and distal joint contractures. UCMD was originally regarded as an exclusively autosomal recessive condition causing severe muscle weakness with proximal joint contractures and distal hyperlaxity. We and others have subsequently modified this model when we described UCMD patients with heterozygous in-frame deletions acting in a dominant-negative way. Here we report 10 unrelated patients with a UCMD clinical phenotype and de novo dominant negative heterozygous splice mutations in COL6A1, COL6A2, and COL6A3 and contrast our findings with four UCMD patients with recessively acting splice mutations and two BM patients with heterozygous splice mutations. We find that the location of the skipped exon relative to the molecular structure of the collagen chain strongly correlates with the clinical phenotype. Analysis by immunohistochemical staining of muscle biopsies and dermal fibroblast cultures, as well as immunoprecipitation to study protein biosynthesis and assembly, suggests different mechanisms each for exon skipping mutations underlying dominant UCMD, dominant BM, and recessive UCMD. We provide further evidence that de novo dominant mutations in severe UCMD occur relatively frequently in all three collagen VI chains and offer biochemical insight into genotype-phenotype correlations within the collagen VI-related disorders by showing that severity of the phenotype depends on the ability of mutant chains to be incorporated in the multimeric structure of collagen VI.

Neuregulin signaling through a PI3K/Akt/Bad pathway in Schwann cell survival.

beta-Neuregulin (betaNRG) is a potent Schwann cell survival factor that binds to and activates a heterodimeric ErbB2/ErbB3 receptor complex. We found that NRG receptor signaling rapidly activated phosphoinositide 3-kinase (PI3K) in serum-starved Schwann cells, while PI3K inhibitors markedly exacerbated apoptosis and completely blocked NRG-mediated rescue. NRG also rapidly signaled the phosphorylation of mitogen-activated protein kinase (MAPK) and the serine/threonine kinase Akt. The activation of Akt and MAPK in parallel pathways downstream from PI3K resulted in the phosphorylation of Bad at different serine residues. PI3K inhibitors that blocked NRG-mediated rescue also blocked the phosphorylation of Akt, MAPK, and Bad. However, selective inhibition of MEK-dependent Bad phosphorylation downstream from PI3K had no effect on NRG-mediated survival. Conversely, ectopic expression of wild-type Akt not only enhanced Bad phosphorylation but also enhanced autocrine- and NRG-mediated Schwann cell survival. Taken together, these results demonstrate that NRG receptor signaling through a PI3K/Akt/Bad pathway functions in Schwann cell survival.

Progressive spinal muscular atrophies.

Spinal muscular atrophy is the most common autosomal-recessive genetic disorder lethal to infants. It was first described in the 1890s. Since then our understanding of the disorder has progressed significantly. Progression of the disease is due to loss of anterior horn cells, thought to be caused by apoptosis. Diagnosis is based on the course of the illness, as well as certain changes seen on nerve and muscle biopsy and electrodiagnostic studies. More recently, our understanding of the genetics of this disorder has provided a noninvasive approach to diagnosis. This method of testing has its downside, but the quest for a more sensitive analysis is still underway. Even though our knowledge of this disease has come a long way since its first recognition, the therapies available to these children are still only supportive. Again, researchers eagerly look for new therapeutic interventions to allow for improved quality of life and an extended life span.

Signals for proinflammatory cytokine secretion by human Schwann cells.

Wallerian degeneration is a post-traumatic process of the peripheral nervous system whereby damaged axons and their surrounding myelin sheaths are phagocytosed by infiltrating leukocytes. Our studies indicate that Schwann cells could initiate the process of Wallerian degeneration by releasing proinflammatory cytokines involved in leukocyte recruitment and differentiation including IL-1beta, MCP-1, IL-8 and IL-6. A comparison of the secretory pattern between nerve explants and cultured Schwann cells showed that each cytokine was differentially regulated by growth factor deprivation or axonal membrane fragments. Since Wallerian-like degeneration occurs in a wide variety of peripheral neuropathies, Schwann cell-mediated cytokine production may play an important role in many disease processes.

Reciprocal Id expression and myelin gene regulation in Schwann cells.

Id proteins are thought to act as dominant negative antagonists of basic helix-loop-helix (bHLH) transcription factors that direct differentiation in various cell types. We found that Schwann cells express all four Id-family genes and that their transcript levels were reciprocally regulated in pairs during nerve maturation in vivo and cAMP-mediated differentiation in vitro. The rapid induction as part of the early response to axonal membranes and cytokines suggested that Id3 is involved in myelin gene repression. An inverse relationship between Id1/3 and myelin P0 expression was consistent with a role for these two Id proteins as inhibitors of differentiation, and Id1/3 proteins strongly repressed myelin gene promoter activity. Nuclear factors isolated from Schwann cells and intact sciatic nerves were found to bind three different HLH recognition sequences (E boxes) in the proximal region of the P0 promoter, and production of these DNA binding complexes was altered during differentiation. These data support the concept that Id proteins regulate myelin gene expression by controlling the formation of specific bHLH DNA binding complexes with different E-box preferences.

Schwann cell-conditioned medium promotes neuroblastoma survival and differentiation.

Neuroblastomas are histopathologically heterogeneous, ranging from immature malignant tumors to benign ganglioneuromas. The amount of Schwann cell stroma greatly increases with neuroblastoma differentiation, and these Schwann cells appear to be normal cells that infiltrate the tumor. To determine whether Schwann cells influence neuroblast differentiation, four human neuroblastoma cell lines were cultured in the presence or absence of human Schwann cell-conditioned medium for 7 days. Neuroblastoma cell survival, as determined by a colorimetric assay, more than doubled in three of the four neuroblastoma cell lines in the Schwann cell-conditioned medium. There was a corresponding reduction in apoptosis as measured by a nick-end labeling assay, with little change in mitotic rate. Schwann cell-conditioned medium induced extensive neurite outgrowth in all of the neuroblastoma cell lines, and these processes contained mature neurofilament in three of the cell lines. These results indicate that Schwann cells produce soluble substances capable of supporting survival and differentiation in neuroblastoma cell lines. This may represent a biological mechanism responsible for neuronal differentiation in stroma-rich neuroblastomas.

A role for Pak protein kinases in Schwann cell transformation.

Neurofibromatosis type 1 (NF1), a common autosomal dominant disorder caused by loss of the NF1 gene, is characterized clinically by neurofibromas and more rarely by neurofibrosarcomas. Neurofibromin, the protein encoded by NF1, possesses an intrinsic GTPase accelerating activity for the Ras proto-oncogene. Through this activity, it is a negative regulator of Ras. The Pak protein kinase is a candidate for a downstream signaling protein that may mediate Ras signals because it is activated by Rac and Cdc42, two small G proteins required for Ras signaling. Here, we use Pak mutants to explore the role of Pak in Ras signaling in Schwann cells, the cells affected in NF1. Whereas an activated Pak mutant does not transform cells, dominant negative Pak mutants are potent inhibitors of Ras transformation of rat Schwann cells and of a neurofibrosarcoma cell line from an NF1 patient. Although activated Pak stimulated jun-N-terminal kinase, inhibition of Ras transformation by dominant negative Pak did not require inhibition of jun-N-terminal kinase. Instead, the Pak mutants appeared to inhibit transformation by preventing Ras activation of the ERK/mitogen-activated protein kinase cascade. These results have implications for our understanding of NF1 because a neurofibrosarcoma cell line derived from a patient with NF1 was reverted by stable expression of the Pak dominant negative mutants.

Regulation of glucose transport in cultured Schwann cells.

Glucose is the major source of metabolic energy in the peripheral nerve. Energy derived from glucose is mostly utilized for axonal repolarization. One route by which glucose may reach the axon is by crossing the Schwann cells that initially surround the axons. Considering the ability of neurons to control many glial cell functions, we postulated that Schwann cell glucose transporters might be transiently regulated by axonal contact. Glucose transport was studied in a cultured, differentiated rat Schwann cell line stably expressing SV40 T antigen regulated by a synthetic mouse metallothionein promoter. 3[H]-2-deoxy-D-glucose uptake was measured in cultured cells in basal and in various experimental conditions. Glucose transporter gene expression was determined after RNA isolation from cultured cells through Northern and RNAse protection assay. In vitro, Schwann cells were found to express high-affinity, insulin-insensitive, facilitative glucose transporters and predominantly GLUT1 mRNA. Schwann cell 2-deoxyglucose uptake was increased by axolemmal membranes or forskolin but unchanged by elevated glucose levels. Regulation of Schwann cell glucose transporters by axolemma and their resistance to glucose-induced down-regulation suggest extrinsic rather than intrinsic regulation that might enhance Schwann cell vulnerability to glucotoxicity.

Characterization of insulin-like growth factor-I and its receptor and binding proteins in transected nerves and cultured Schwann cells.

The insulin-like growth factors (IGFs) are trophic factors whose growth-promoting actions are mediated via the IGF-I receptor and modulated by six IGF binding proteins (IGFBPs). In this study, we observed increased transcripts of both IGF-I and IGF-I receptor after rat sciatic nerve transection. Schwann cells (SCs) were the main source of IGF-I and IGFBP-5 immunoreactivity until 7 days after nerve transection, when invading macrophages in the distal nerve stumps were strongly IGF-I positive. In vitro, IGF-I promoted SC mitogenesis. Northern analysis revealed that SCs expressed IGF-I receptor and IGFBP-5. IGF-I treatment increased the intensity of IGFBP-5 without affecting gene expression. Des(1-3)IGF-I, an IGF-I analogue with low affinity for IGFBP, had no such effect. Incubation of recombinant human IGFBP-5 with SC conditioned media revealed IGF-I protection of IGFBP-5 from proteolysis, implying the presence of an IGFBP-5 protease in SC conditioned media. Collectively, these data support the concept that, in response to nerve injury, invading macrophages produce IGF-I and SC express the IGF-I receptor, to facilitate regeneration. This regenerative process may be augmented further by the ability of SC to secrete IGFBPs, which in turn may increase local IGF-I bioavailability.

The zinc finger transcription factor Zif268/Egr-1 is essential for Schwann cell expression of the p75 NGF receptor.

Nerve injury alters the function of Schwann cells from quiescent, myelin forming cells to proliferating cells that facilitate nerve repair. The transcription factor, Zif268, may be involved in transmitting injury-related signals since its expression is rapidly induced by nerve transection in vivo and without intervening protein synthesis by injury-related signals in vitro. Expression of the low-affinity p75 nerve growth factor receptor (NGFRp75) by Schwann cells after nerve injury closely correlated with the zif268 expression profile, and Zif268 transactivated the NGFRp75 promoter in transient transfection assays. Conversely, the NGFRp75 gene was not expressed when Zif268 protein was depleted by stable transfection of antisense cDNA. Moreover, nuclear proteins corresponding to Zif268 bound to the NGFRp75 promoter by Southwestern blotting, indicating that a direct interaction of Zif268 with the NGFR gene is required for its expression in Schwann cells.

The simian virus 40 large T antigen does not inhibit translation of the 14-kDa myelin basic protein mRNA in reticulocyte lysates or in transfected cells.

Viral T antigens are transcription factors that have been suspected of inhibiting expression of the myelin basic protein (MBP) mRNA at the translational level in vitro and in vivo. The effect of simian virus 40 (SV40) large T antigen (T-ag) was examined on the translation of the 14-kDa MBP mRNA in reticulocyte lysates and on MBP expression after transfection into cells that express SV40 T-ag. SV40 T-ag did not inhibit translation of 14-kDa MBP cRNAs in cell-free translations even at 30 microM (approximately 600 micrograms/ml) T-ag. Permanent transfection of COS-1 cells (which endogenously express SV40 T-ag) with the 14-kDa MBP cDNA resulted in the expression of the 14-kDa MBP as determined by western blot analysis. Permanent transfection of N20.1 cells, an oligodendrocyte line immortalized with a temperature-sensitive SV40 T-ag, with the 14-kDa MBP cDNA construct also resulted in the expression of the 14-kDa MBP under conditions in which the cells expressed functional SV40 T-ag. These results indicate that SV40 T-ag does not prevent expression of the MBP gene at the translational level and that in those immortalized oligodendrocyte lines that express MBP mRNA, but not MBP protein, some factor other than the SV40 large T-ag is responsible for the posttranscriptional regulation.

Purification and expansion of human Schwann cells in vitro.

The ability to culture cells from the human nervous system provides new insight into the pathophysiology of neurological diseases and could be crucial to the development of gene replacement therapies and neural transplantation. We report that the proliferation of human Schwann cells isolated from paediatric and adult nerves is sustained in vitro by recombinant glial growth factor. Agents that increase intracellular cyclic cAMP were also mitogenic towards Schwann cells but suppress growth of contaminating fibroblasts. As the lifespan of highly enriched cultures can be extended for up to twelve population doublings, large numbers of cells can be generated from nerve biopsies.

Partial structure and mapping of the human myelin P2 protein gene.

The myelin P2 protein, a 14,800-Da cytosolic protein found primarily in peripheral nerves, belongs to a family of fatty acid binding proteins. Although it is similar in amino acid sequence and tertiary structure to fatty acid binding proteins found in the liver, adipocytes, and intestine, its expression is limited to the nervous system. It is detected only in myelin-producing cells of the central and peripheral nervous systems, i.e., the oligodendrocytes and Schwann cells, respectively. As part of a program to understand the regulation of expression of this gene, to determine its function in myelin-producing cells, and to study its role in peripheral nerve disease, we have isolated and characterized overlapping human genomic clones encoding the P2 protein. We report here on the partial structure of this gene, and on its localization within the genome. By using a panel of human-hamster somatic cell hybrids and by in situ hybridization, we have mapped the human P2 gene to segment q21 on the long arm of chromosome 8. This result identifies the myelin P2 gene as a candidate gene for autosomal recessive Charcot-Marie-Tooth disease type 4A.

Sources of human Schwann cells and the influence of donor age.

We evaluated several tissues as possible sources for culturing human Schwann cells. The average cell yield (total cell number/mg of nerve fascicle) obtained from adult autopsy cases and transplant organ donors was similar (2 x 10(4) and 2.9 x 10(4), respectively), but significantly higher yields were obtained from dorsal roots of pediatric patients undergoing selective dorsal rhizotomy (6.1 x 10(4)). Fresh tissue was not essential since cells isolated from 0 to 20 h postmortem were equally viable. However, we found evidence that donor age affects the intrinsic growth rate of Schwann cells and perineurial fibroblasts in culture.

Advances in understanding the development of the nervous system.

Over the past several years remarkable progress has been made towards unraveling the complex mechanisms that regulate neuronal development. Because the end results of abnormalities in brain development are often developmental disabilities, it is timely to review several recent advances in the field of neurobiology. This review, with contributions from several co-authors, provides a synopsis of breakthroughs from the fields of embryology, cell biology, and molecular genetics that hold promise for exciting clinical application. This article is arranged to reflect the stages of normal development. Understanding the mechanisms underlying neuronal induction, regional specification, neuronal specification, migration, axonal growth, neurotrophic factors, and myelination should clarify the pathophysiology of numerous neurological disorders, and provide new insights into their treatment.