Association of calnexin with mutant peripheral myelin protein-22 ex vivo: a basis for "gain-of-function" ER diseases.

Schwann cell-derived peripheral myelin protein-22 (PMP-22) when mutated or overexpressed causes heritable neuropathies with a previously unexplained "gain-of-function" endoplasmic reticulum (ER) retention phenotype. In wild-type sciatic nerves, PMP-22 associates in a specific, transient (t(1/2 ) approximately equal to 11 min), and oligosaccharide processing-dependent manner with the lectin chaperone calnexin (CNX), but not calreticulin nor BiP. In Trembler-J (Tr-J) sciatic nerves, prolonged association of mutant PMP-22 with CNX is found (t(1/2) > 60 min). In 293A cells overexpressing PMP-22(Tr-J), CNX and PMP-22 colocalize in large intracellular structures identified at the electron microscopy level as myelin-like figures with CNX localization in the structures dependent on PMP-22 glucosylation. Similar intracellular myelin-like figures were also present in Schwann cells of sciatic nerves from homozygous Trembler-J mice with no detectable activation of the stress response pathway as deduced from BiP and CHOP expression. Sequestration of CNX in intracellular myelin-like figures may be relevant to the autosomal dominant Charcot-Marie-Tooth-related neuropathies.

PMP22 carrying the trembler or trembler-J mutation is intracellularly retained in myelinating Schwann cells.

Missense mutations in the murine peripheral myelin protein-22 gene (Pmp22) underly the neuropathies in the trembler (Tr) and trembler-J (Tr-J) mice and in some humans with Charcot-Marie-Tooth disease. We have generated replication-defective adenoviruses containing epitope-tagged, wild-type-, Tr-, or Tr-J-PMP22 bicistronic with the Lac-Z reporter gene. These viruses were microinjected into the sciatic nerves of 10-day-old Sprague-Dawley rats and, later, analyzed by immunohistochemistry to determine the distribution of mutant protein in infected myelinating Schwann cells. We found that epitope-tagged, wild-type PMP22 is successfully transported to compact myelin, whereas the Tr and the Tr-J mutant proteins are retained in cytoplasmic compartment, colocalizing with the endoplasmic reticulum. These results provide in vivo evidence that the pathogenesis of the Tr and Tr-J mutations are most likely a function of abnormal retention within the endoplasmic reticulum of myelinating Schwann cells.

The anatomy and cell biology of peripheral myelin protein-22.

The gain of function phenotypes exhibited by the heterozygous Tr, Tr-J, and CMT1A mutations indicate that these mutations interfere with more than the function of a single PMP22 allele. The identification of proteins that interact with PMP22 and that are sensitive both to stoichiometry and the effects of the mutations could provide important leads to a unified hypothesis to explain the riddle of the PMP22-related neuropathies.

Intracerebral adenovirus-mediated p53 tumor suppressor gene therapy for experimental human glioma.

Malignant gliomas of astrocytic origin are good candidates for gene therapy because they have proven incurable with conventional treatments. Although mutation or inactivation of the p53 tumor suppressor gene occurs at early stages in gliomas and is associated with tumor progression, many tumors including high-grade glioblastoma multiforme carry a functionally intact p53 gene. To evaluate the effectiveness of p53-based therapy in glioma cells that contain endogenous wild-type p53, a clinically relevant model of malignant human glioma was established in athymic nu/nu mice. Intracerebral, rapidly growing tumors were produced by stereotactic injection of the human U87 MG glioma cell line that had been genetically modified for tracking purposes to express the Escherichia coli lacZ gene encoding beta-galactosidase. Overexpression of the p53 gene by adenovirus-mediated delivery into the tumor mass resulted in rapid cell death with the eradication of beta-galactosidase-expressing glioma cells through apoptosis. In long-term experiments, the survival of mice treated with the p53 adenoviral recombinant was significantly longer than that of mice that had received control adenoviral recombinant. During the observation period of 1 year, a complete cure was achieved in 27% of animals after a single injection of p53 adenoviral recombinant, and 38% of the animals were tumor free in the group receiving multiple injections of p53 adenoviral recombinant into a larger tumor mass. These experiments demonstrate that overexpression of p53 in gliomas, even in the presence of endogenous functional wildtype p53, leads to efficient elimination of tumor cells. These results point to the potential therapeutic usefulness of this approach for all astrocytic brain tumors.

Characterization of chronic bronchopulmonary Pseudomonas aeruginosa infection in resistant and susceptible inbred mouse strains.

Chronic bronchopulmonary Pseudomonas aeruginosa infection, initiated by intratracheal instillation of 1 to 2 x 10(5) colony-forming units of a mucoid strain of bacteria trapped in agar beads, was characterized in resistant BALB/c mice and susceptible C57BL/6 (B6) mice through 28 d postinfection. B6 mice experienced a more severe infection than BALB/c mice as evidenced by significantly higher mortality and significantly greater weight loss during the first 14 d. Furthermore, B6 mice had significantly higher numbers of bacteria in the lungs through 21 d after infection. Overall, only 22% of these hosts cleared the infection. In contrast, 67% of BALB/c mice cleared the infection. These differences between resistant and susceptible mice were found to correlate with histopathologic differences in the type of inflammation and the extent of tissue damage. An acute, predominantly neutrophilic inflammation and extensive tissue damage were apparent in the lungs of susceptible B6 mice, whereas chronic, granulomatous inflammation and little or no tissue damage were visible in resistant BALB/c mice. The finding of acute inflammation in the lungs of infected B6 mice was confirmed by fluorescence-activated cell sorter (FACS) analyses, which demonstrated that these mice had significantly greater proportions of polymorphonuclear neutrophils in the lungs on Days 7 and 14 after infection than did BALB/c mice. FACS analyses also revealed significant and similar increases in CD3(+) lung cells in both strains as the infection progressed. The CD4/CD8 ratio was significantly greater in BALB/c mice by 21 d after infection when the majority of these animals, but not B6 mice, had cleared the infection.

Temporal expression pattern of peripheral myelin protein 22 during in vivo and in vitro myelination.

Peripheral myelin protein 22 (PMP22) was initially described as a minor component of peripheral myelin. Mutations affecting the PMP22 gene cause demyelinating neuropathies, supporting a role for the protein in PNS myelination. Furthermore, PMP22 carries the L2/HNK-1 carbohydrate epitope suggesting an adhesion/recognition function. Despite advances in characterizing the PMP22 gene, the specific role(s) of the protein in myelin remains unknown. In this study we determined the temporal expression pattern of PMP22 in comparison to galactocerebroside (GalC) and myelin associated glycoprotein (MAG), early constituents of PNS myelin, and to protein zero (P0) and myelin basic protein (MBP), late components of myelin. In sciatic nerve lysates, PMP22 was detected at postnatal day 3, after MAG, but before MBP expression. The same results were obtained in cocultures of dorsal root ganglion neurons and Schwann cells (SCs). Low levels of PMP22 were found in early, anti-MAG and anti-GalC immunoreactive, myelinating cocultures. However, PMP22 could only be detected in the SC plasma membrane after basal lamina formation. In long-term myelinating cocultures PMP22 levels continued to increase and the protein was found in anti-P0 and anti-MBP immunoreactive myelin segments. Furthermore, PMP22, MBP, and P0 protein levels were greatly enhanced by progesterone treatment of the cocultures. The highest levels of PMP22 expression were associated with late stages of myelination; however the presence of the protein in nonmyelinating SCs and in SCs commencing myelination supports multiple roles for PMP22 in peripheral nerve biology.

Trigeminal nerve hypertrophy in chronic inflammatory demyelinating polyradiculoneuropathy.

An unusual clinical manifestation of nerve hypertrophy in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is described. A patient with a 13-year history of CIDP developed diplopia and proptosis. Imaging of the neuraxis showed marked bilateral trigeminal nerve hypertrophy and lumbosacral nerve root hypertrophy. Biopsy of the right infraorbital nerve revealed inflammatory infiltrates and extensive onion bulb formation, consistent with CIDP.

Surgical treatment of epilepsy in tuberous sclerosis: strategies and results in 18 patients.

BACKGROUND: Seizures in patients with tuberous sclerosis complex (TSC) are often intractable to antiepileptic medications and searching investigation may provide evidence that surgical treatment can be considered. OBJECTIVE: To review the results of investigation and surgical therapy, a treatment modality not generally considered in patients with medically refractory seizures and TSC. METHODS: We report 18 patients (9 male) with TSC who underwent surgical treatment of medically refractory epilepsy. Twelve patients had a well-localized epileptogenic lesion and were treated by lesionectomy or focal resection. Resections were: 7 frontal, 4 temporal, 1 frontotemporal, 1 occipital, and 1 frontoparietal. Four patients underwent more than one operation. Six patients had corpus callosotomy (CC). RESULTS: Follow-up ranged from 1 month to 47 years. Outcome of the patients treated by resection was excellent in 7 (5 were seizure-free and 2 had auras only), good in 1, fair in 3, and 1 was lost to follow-up. Best outcome was obtained in patients who had focal seizures and good imaging and EEG correlation, although they might have multiple seizure types, other imaging abnormalities, and multifocal or generalized EEG findings. When there was no such correlation, CC was found to be an option as five patients had at least some improvement and only one showed no change. CONCLUSION: Surgical treatment of patients with TSC and intractable epilepsy is most effective when a single tuber or epileptogenic area can be identified as the source of seizures and resected. This may be possible even when other tubers or diffuse EEG abnormalities are present. In patients with unlocalizable epileptic abnormalities, palliation may be obtained by CC.

Meningeal melanocytoma. Report of a case and a historical comparison.

Meningeal melanocytomas are rare tumors of the central nervous system that are found almost exclusively in the posterior fossa and spinal cord and whose natural history is poorly defined. In this report, the authors review the clinical presentation, radiological appearance, operative findings, and histological features in two cases of meningeal melanocytoma: one cranial and one spinal. Two women, aged 21 and 30 years, were admitted to the hospital 60 years apart: the first because of progressive paraplegia and the second because of slowly progressive hearing loss. The first patient had an extradural tumor that was treated by laminectomy, subtotal resection, and postoperative radiotherapy in 1936. Her symptoms recurred 16 years later and she underwent reoperation of the residual tumor, which was found to have an intradural component. The authors' patient, who presented 60 years later, underwent plain and enhanced computerized tomography and magnetic resonance imaging that demonstrated a large posterior fossa lesion indicative of either an acoustic neuroma or a meningioma. She underwent posterior fossa decompression but only partial excision of the tumor could be accomplished because vigorous bleeding limited the extent of the resection. Surgery was followed by radiotherapy. The residual tumor enlarged despite these measures and required repeated resection 6 months later. At the second operation the tumor was much less vascular, perhaps reflecting the effects of radiotherapy, and was removed almost entirely. The patient died 6 months later from an anticoagulant-related cerebellar hemorrhage. In both cases the lesions were jet black, and histological examination revealed melanin-containing hypercellular tumors with rare mitotic figures. Meningeal melanocytomas are being diagnosed with increased frequency in parallel with improvements in neuroimaging and clarification of histological features. Clinical presentation of patients with these tumors typically occurs in their fifth decade and women are affected twice as often as men. The posterior fossa lesions can mimic acoustic neuromas and meningiomas in location and radiological appearance; however, the internal auditory canal is normal. In the spine, meningeal melanocytomas present with the clinical features of myeloradiculopathy. Diagnosis is made intraoperatively from the gross, jet-black appearance of the tumor and from histological examination. Vascularity, size, and location may render complete resection unfeasible. Because of the tumor's propensity to recur, radiotherapy has been recommended but its role remains to be elucidated.

Common themes in peripheral neuropathy disease genes.

After a century of study, mutations in connexin32, peripheral myelin protein22, and protein zero are now known to culminate in the prototypical phenotype of Charcot-Marie-Tooth disease. Many of these mutations have been modeled in rodents and in tissue culture. Consequently, structure-function predictions for these mutations are now possible and detailed analyses of many of them are ongoing. Despite the marked differences in the functions of these three proteins, it is profitable to consider the many similarities between them, including the types of mutational mechanisms and their effects on myelin structure and function. Accordingly, the biology and genetics of Charcot-Marie-Tooth disease and other inherited peripheral neuropathies due to mutations in these proteins are reviewed.

Progressive myoclonus epilepsy in young adults with neuropathologic features of Alzheimer's disease.

Progressive myoclonus epilepsy (PME) may develop in adult life. We present two patients with PME appearing around the age of 30 years in whom the disorder represented a manifestation of Alzheimer's disease. This diagnosis must be considered in addition to possible Kufs' disease or myoclonic epilepsy with ragged red fibers (MERRF) when PME develops in young adults.

Neurons promote the translocation of peripheral myelin protein 22 into myelin.

Schwann cells express low levels of myelin proteins in the absence of neurons. When Schwann cells and neurons are cultured together the production of myelin proteins is elevated, and myelin is formed. For peripheral myelin protein 22 (PMP22), the exact amount of protein produced is critical, because peripheral neuropathies result from its underexpression or overexpression. In this study we examined the effect of neurons on Schwann cell PMP22 production in culture and in peripheral nerve using metabolic labeling and pulse-chase studies as well as immunocytochemistry. Most of the newly synthesized PMP22 in Schwann cells is rapidly degraded in the endoplasmic reticulum. Only a small proportion of the total PMP22 acquires complex glycosylation and accumulates in the Golgi compartment. This material is translocated to the Schwann cell membrane in detectable amounts only when axonal contact and myelination occur. Myelination does not, however, alter the rapid turnover of PMP22 in Schwann cells. PMP22 may therefore be a unique myelin protein in that axonal contact promotes its insertion into the Schwann cell membrane and myelin without altering its rapid turnover rate within the cell.

Upregulation of the endosomal-lysosomal pathway in the trembler-J neuropathy.

A nonconservative leucine to proline mutation in peripheral myelin protein 22 (PMP22) causes the Trembler-J (TrJ) neuropathy in mice and humans. The expression levels and localization of the PMP22 protein in the TrJ mouse have not been previously determined. The aim of our studies was to reevaluate the extent of myelin deficit in genotyped heterozygous and homozygous animals and to examine how the TrJ mutation alters the normal in vivo post-translational processing of PMP22. Morphological studies show evidence for primary dysmyelination and myelin instability in affected animals. As expected, Western blot analysis indicates that in adult heterozygous TrJ animals, the level of PMP22 is markedly decreased, similar to myelin basic protein and protein zero, whereas myelin-associated glycoprotein is largely unaffected. The decrease in myelin protein expression is associated with an increase in lysosomal biogenesis, suggestive of augmented endocytosis or autophagy. Double-immunolabeling experiments show the accumulation of PMP22 in endosomal/lysosomal structures of TrJ Schwann cells, and chloroquine treatment of nerve segments indicates that the degradation of protein zero, PMP22, and myelin basic protein is augmented in TrJ nerves. These studies suggest that the TrJ mutation alters myelin stability and that the mutant protein is likely degraded via the lysosomal pathway.

Ultrastructural distribution of PMP22 in Charcot-Marie-Tooth disease type 1A.

Peripheral Myelin Protein-22 (PMP22) is a membrane glycoprotein which represents up to 5% of total protein in myelin of peripheral nerves. Mutations affecting the PMP22 gene have been linked to the inherited peripheral neuropathies Charcot-Marie-Tooth disease type 1A (CMT1A; duplications and point mutations), Dejerine-Sottas syndrome (DSS; point mutations), and hereditary neuropathy with liability to pressure palsies (HNPP; deletions). In this study, we determined the ultrastructural distribution of PMP22 and other myelin proteins in normal human peripheral nervous system (PNS) nerves and in CMT1 patients with or without the CMT1A duplication on chromosome 17. Our results demonstrate that PMP22, P0 protein, and myelin basic protein are present in compact myelin of all patients examined. PMP22 was also present in the plasma membrane of Schwann cells of unmyelinated fibers and onion bulbs. Although the precise biological role of PMP22 remains to be discovered, our results support the hypothesis that this protein serves multiple functions in Schwann cells.

Molecular basis of common hereditary motor and sensory neuropathies in humans and in mouse models.

The Hereditary Motor and Sensory Neuropathies (HMSNs) are well known to be clinically, morphologically, and genetically heterogeneous. Yet, recent advances in the cellular and molecular biology of the peripheral nervous system coupled with remarkable progress in human and mouse genetics have provided a framework that has profoundly changed our understanding of the pathogenesis of these diseases. It now appears that most of the HMSNs are related to mutations affecting genes encoding Schwann cell proteins, specifically the Peripheral Myelin Protein PMP22, Myelin Protein Zero, and one of the gap junction proteins, connexin-32. Accordingly, these findings are discussed in the context of the clinical and pathologic features of the human HMSNs, but are interpreted in the context of basic research findings on the cellular and molecular biology of the peripheral nervous system derived from in vivo and in vitro studies in spontaneously-occurring and genetically engineered animal models for the HMSNs.

Molecular anatomy and genetics of myelin proteins in the peripheral nervous system.

Myelin contains a number of proteins, the major examples of which are protein zero (Po), P2 protein, peripheral myelin protein 22 (PMP22), myelin basic proteins (MBPs), myelin-associated glycoprotein (MAG) and the recently described connexin 32 (Cx32). This list is probably still incomplete. The localisation and possible functions of these proteins are reviewed. In the past few years a number of inherited demyelinating neuropathies in mice and the human have been shown to be due to mutations affecting the genes PMP22, Po and Cx32 so that it has become possible to characterise the molecular pathology of the majority of these disorders. This has provided important insights into the relationships between the structure of myelin and the function of its constituent proteins.

Peripheral myelin protein 22: facts and hypotheses.

Mutations affecting the peripheral myelin protein 22 (PMP22) gene are associated with inherited motor and sensory neuropathies in mouse (Trembler and Trembler-J) and human (Charcot-Marie-Tooth disease type 1A and Dejerine-Sottas syndrome). Although genetic studies have established a critical role of PMP22 in the formation and/or maintenance of myelin in the peripheral nervous system, the biological function of PMP22 in myelin and in non-myelin forming cells remains largely enigmatic. In this Mini-Review, we will summarize the current knowledge about PMP22 and discuss its hypothetical function(s) in a broad context.