Human Myelin-associated Glycoprotein Research Articles

Long-Term Disease Prevention with a Gene Therapy Targeting Oligodendrocytes in a Mouse Model of Adrenomyeloneuropathy.

Adrenomyeloneuropathy (AMN) is a late-onset axonopathy of spinal cord tracts caused by mutations of the ABCD1 gene that encodes adrenoleukodystrophy protein (ALDP), a peroxisomal transporter of very long-chain fatty acids (VLCFA). Disturbed metabolic interaction between oligodendrocytes (OL) and axons is suspected to play a major role in AMN axonopathy. To develop a vector targeting OL, the human ABCD1 gene driven by a short 0.3 kb part of the human myelin-associated glycoprotein (MAG) promoter was packaged into an adeno-associated viral serotype 9 (rAAV9). An intravenous injection of this vector on postnatal day 10 in Abcd1-/- mice, a model of AMN, allowed a near normal motor performance to persist for 24 months, while age-matched untreated mice developed major defects of balance and motricity. Three weeks postvector, 50-54% of spinal cord white matter OL was expressing human ALDP (hALDP) at the cervical level, and only 6-7% after 24 months. In addition, 29-32% of cervical spinal cord astrocytes at 3 weeks and 16-19% at 24 months also expressed ALDP. C26:0-lysoPC, a sensitive VLCFA marker of AMN, was lower by 41% and 50%, respectively, in the spinal cord and brain of vector-treated compared with untreated mice. In a nonhuman primate, the intrathecal injection of the rAAV9-MAG vector induced abundant ALDP expression at 3 weeks in spinal cord OL (43%, 29%, and 26% at cervical, thoracic, and lumbar levels) and cerebellum OL (35%). In addition, 33-41% of spinal cord astrocytes expressed hALDP, and 27% of cerebellar astrocytes. To our knowledge, OL targeting had not been obtained before in primates with other vectors or promoters. The current results thus provide a robust proof-of-concept not only for the gene therapy of AMN but also for other central nervous system diseases, where the targeting of OL with the rAAV9-MAG vector may be of interest.

Recombinant Human Myelin-Associated Glycoprotein Promoter Drives Selective AAV-Mediated Transgene Expression in Oligodendrocytes.

Leukodystrophies are hereditary central white matter disorders caused by oligodendrocyte dysfunction. Recent clinical trials for some of these devastating neurological conditions have employed an ex vivo gene therapy approach that showed improved endpoints because cross-correction of affected myelin-forming cells occurred following secretion of therapeutic proteins by transduced autologous grafts. However, direct gene transfer to oligodendrocytes is required for the majority of leukodystrophies with underlying mutations in genes encoding non-secreted oligodendroglial proteins. Recombinant adeno-associated viral (AAV) vectors are versatile tools for gene transfer to the central nervous system (CNS) and proof-of-concept studies in rodents have shown that the use of cellular promoters is sufficient to target AAV-mediated transgene expression to glia. The potential of this strategy has not been exploited. The major caveat of the AAV system is its limited packaging capacity of ~5 kb, providing the rationale for identifying small yet selective recombinant promoters. Here, we characterize the human myelin associated glycoprotein (MAG) promoter for reliable targeting of AAV-mediated transgene expression to oligodendrocytes in vivo. A homology screen revealed highly conserved genomic regions among mammalian species upstream of the transcription start site. Recombinant AAV expression cassettes carrying the cDNA encoding enhanced green fluorescent protein (GFP) driven by truncated versions of the recombinant MAG promoter (2.2, 1.5 and 0.3 kb in size) were packaged as cy5 vectors and delivered into the dorsal striatum of mice. At 3 weeks post-injection, oligodendrocytes, neurons and astrocytes expressing the reporter were quantified by immunohistochemical staining. Our results revealed that both 2.2 and 1.5 kb MAG promoters targeted more than 95% of transgene expression to oligodendrocytes. Even the short 0.3 kb fragment conveyed high oligodendroglial specific transgene expression (>90%) in vivo. Moreover, cy5-MAG2.2-GFP delivery to the neonate CNS resulted in selective GFP expression in oligodendrocytes for at least 8 months. Broadly, the characterization of the extremely short yet oligodendrocyte-specific human MAG promoter may facilitate modeling neurological diseases caused by oligodendrocyte pathology and has translational relevance for leukodystrophy gene therapy.

Myelin-associated glycoprotein-related neuropathy associated with psoriasis: a case report

IntroductionPsoriasis vulgaris is a common inflammatory disease of the skin, and myelin-associated glycoprotein-related neuropathy is a chronic sensory-predominant polyneuropathy. Although both of these diseases are considered autoimmune diseases, psoriasis with concomitant myelin-associated glycoprotein-related neuropathy is very rare. Here, we report a case of myelin-associated glycoprotein-related neuropathy associated with psoriasis.Case presentationA 66-year-old Japanese man, having experienced sternocostoclavicular pain for ten years, was admitted to our hospital because of gait disturbance and numbness of the limbs. Our patient had normal cranial nerve function and normal limb muscle strength. His vibratory and position sense was severely impaired and his touch, temperature and pinprick sensations were mildly disturbed in a glove and stocking distribution. A myelin-associated glycoprotein western blot analysis showed the presence of a 91 to 94kDa band using purified human myelin-associated glycoprotein antigen. His skin lesions were moderately pruritic and Auspitz’s sign was positive. Our patient also showed osteitis of his clavicle and manubrium. We diagnosed our patient with myelin-associated glycoprotein-related neuropathy associated with psoriatic arthritis. Five days after intravenous immunoglobulin therapy, his deep sensory impairment began to improve and his sternocostoclavicular pain diminished dramatically.ConclusionsBecause myelin-associated glycoprotein-related neuropathy and psoriatic arthritis are both considered autoimmune diseases, we conclude that intravenous immunoglobulin therapy is very effective for patients with an association of these diseases.

Generation and characterization of antibodies to sulfated glucuronyl glycolipids in Lewis rats

Antibodies to sulfated glucuronyl glycolipids (SGGLs) have been reported in sera of patients with peripheral neuropathies including patients with IgM gammopathy. However, the role of anti-SGGL antibodies in the pathogenesis of neuropathy remains unclear. In order to study the role of antibodies to SGGLs in the pathogenesis of neuropathy, Lewis female rats were injected with purified SGPG mixed with keyhole limpet hemocyanin (KLH) and emulsified with equal amount of complete Freund's adjuvant. High titer anti-SGPG antibodies were detected by ELISA in sera of all rats inoculated with SGPG. All anti-SGPG antibodies cross-reacted with human myelin-associated glycoprotein (MAG). None of the sensitized rats exhibited clinical signs of neuropathy. Histological examination showed that there was no demyelination or axonal damage in peripheral nerves. Our data demonstrate that SGPG is a highly immunogenic glycolipid but high titer antibodies against it do not produce an experimental autoimmune neuropathy in Lewis rats.

Expression of glycosylated recombinant human myelin-associated glycoprotein on a neuroblastoma cell line and its reactivity with HNK-1 but not human anti-MAG antibodies

A correctly glycosylated myelin-associated glycoprotein (MAG) must express the carbohydrate epitope HNK-1, which is the target antigen for IgM antibodies in some patients with neuropathy. We transfected a human MAG cDNA clone into the neuroblastoma cell line SK-N-SH and verified by immunoblot the expression of the HNK-1 epitope on the recombinant molecule. By the same method and by indirect immunofluorescence we did not find any reactivity of human anti-MAG IgM antibodies with glycosylated recombinant MAG and transfected neuroblastoma cells. These findings suggest that the mere presence of the HNK-1 epitope is probably not sufficient for MAG to be recognized by human antibodies and that other factors such as the concentration or fine structure of this epitope in MAG, which mostly depend on the cellular context, may be also critical for this reactivity.

Characterization of myelin-associated glycoprotein (MAG) proteolysis in the human central nervous system.

Purified human central nervous system myelin contains an endogenous cysteine protease which degrades the 100-kDa myelin-associated glycoprotein into a slightly smaller 90-kDa derivative called dMAG, and which has been implicated in demyelinating diseases. The native proteolytic site in human MAG was determined in order to characterize this cysteine protease in humans further. This was accomplished by identifying the carboxy-terminus of purified dMAG. The results of these experiments, in conjunction with peptidolysis assays of myelin, demonstrated that the enzyme which proteolyses MAG is extracellular and has cathepsin L-like specificity. Furthermore, it was shown that this cathepsin L-like activity potentially was regulated by the endogenous extracellular inhibitor cystatin C.

Development of myelination in the bovine fetal brain: an immunohistochemical study.

In this study, the development of myelination in the fetal bovine brain (age-range: between 1-2 and 8-9 months) was examined applying: 1. Immunohistochemical staining methods and antibodies against bovine proteolipid protein (PLP), synthetic tridecapeptide of bovine PLP, human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG); and 2. Using the Luxol fast-blue (LFB) technique.

Generation and characterization of anti-sulfoglucuronosyl paragloboside monoclonal antibody NGR50 and its immunoreactivity with peripheral nerve.

Sulfoglucuronosyl paragloboside (SGPG) is a member of the sulfated glucuronic acid-containing glycolipid (SGGL) family found primarily in peripheral nerves. These glycolipids contain the HNK-1 carbohydrate epitope and are recognized by monoclonal IgM from patients with chronic demyelinating neuropathy and paraproteinemia. Recent studies indicate that SGGLs may serve as ligands for selectins, amphoterin, and laminin, suggesting that these glycolipids may play an important role in cellular adhesion. To elucidate the biological function of these glycolipids, we produced a murine monoclonal antibody (mAb) and studied its antigenic specificity. Using an enzyme-linked immunosorbent assay (ELISA), we found that the mAb designated as NGR50 belonged to the IgG2a subclass, and that the minimal titer (2 SD above the mean optical density value of control) of this mAb was 1:640, with 20 ng of purified SGPG as the antigen. Thin-layer chromatography (TLC) immunoblotting revealed that this mAb reacted specifically with SGPG and sulfoglucuronosyl lactosaminyl paragloboside (SGLPG), which is a structural analogue of the former, but not with other glycolipids. Desulfated derivates of SGPG and SGLPG did not react with mAb NGR50. Western blot analysis showed crossreactivity with human myelin-associated glycoprotein (MAG), but not with rat MAG or rat glycoprotein P0. Unlike anti-HNK-1 monoclonal antibody, however, NGR50 reacted only weakly with several proteins in the 20-30-kD regions, including human P0, suggesting that mAb50 has a different fine specificity as an anti-HNK-1 antibody. Immunocytochemical study of rat sciatic nerve using mAb NGR50 revealed positive staining at the outer surface of the myelin sheath and Schwann cells, as well as in the intervening connective tissues. Faint staining was also visible at the axolemmal-myelin interface; however, compact myelin was not stained.

Sensitization of Lewis rats with sulfoglucuronosyl paragloboside: Electrophysiological and immunological studies of an animal model of peripheral neuropathy

Antibodies against sulfoglucuronosyl glycosphingolipids (SGGLs) are known to be present in sera of patients with chronic polyneuropathy associated with IgM paraproteinemia. We recently studied rats sensitized with sulfoglucuronosyl paragloboside (SGPG), a major SGGL species, emulsified with keyhold limpet hemocyanin and Freund's adjuvant. The titer of the IgM class antibodies against SGPG increased up to 1:1,600, while that of the IgG class increased up to 1:800 2 weeks after sensitization. The antibodies showed a high degree of antigenic specificity; no cross-reactivity with other brain glycolipids could be detected. They, however, reacted with human myelin-associated glycoprotein (MAG) by Western blot analysis, but not with rat MAG. These animal models showed minor but clear clinical signs of neuropathy, consisting of mild tail muscle tone loss and walking disabilities. Electrophysiological examination of the sciatic nerves revealed nerve conduction abnormalities which consisted of conduction block and mild decrease in conduction velocity. Thus, our results support the concept that anti-SGPG antibodies may play an important pathogenetic role in this type of chronic neuropathy. © 1996 Wiley-Liss, Inc.

Sensitization of Lewis rats with sulfoglucuronosyl paragloboside: electrophysiological and immunological studies of an animal model of peripheral neuropathy.

Antibodies against sulfoglucuronosyl glycosphingolipids (SGGLs) are known to be present in sera of patients with chronic polyneuropathy associated with IgM paraproteinemia. We recently studied rats sensitized with sulfoglucuronosyl paragloboside (SGPG), a major SGGL species, emulsified with keyhold limpet hemocyanin and Freund's adjuvant. The titer of the IgM class antibodies against SGPG increased up to 1:1,600, while that of the IgG class increased up to 1:800 2 weeks after sensitization. The antibodies showed a high degree of antigenic specificity; no cross-reactivity with other brain glycolipids could be detected. They, however, reacted with human myelin-associated glycoprotein (MAG) by Western blot analysis, but not with rat MAG. These animal models showed minor but clear clinical signs of neuropathy, consisting of mild tail muscle tone loss and walking disabilities. Electrophysiological examination of the sciatic nerves revealed nerve conduction abnormalities which consisted of conduction block and mild decrease in conduction velocity. Thus, our results support the concept that anti-SGPG antibodies may play an important pathogenetic role in this type of chronic neuropathy.

Biosynthesis and regulation of expression of the HNK-1 epitope on myelin-associated glycoprotein in a transfected cell model system.

The HNK-1 antibody recognizes a carbohydrate epitope expressed by many cell adhesion molecules in the nervous system that has been proposed to be an important adhesive determinant. This epitope is particularly prominent on the myelin-associated glycoprotein (MAG) and is related to the antigenic target in an autoimmune mediated demyelinating neuropathy. Elucidation of the mechanisms underlying the biosynthesis and regulation of expression of the HNK-1 epitope is therefore likely to have important functional and clinical implications. In order to investigate its biosynthesis and the regulation of its expression, we have expressed both human and rat MAG in several different cell lines by retroviral infection. These studies indicate that the cellular milieu determines whether the HNK-1 epitope is expressed on the MAG polypeptide and provide an explanation for the significant variation in HNK-1 levels that has been noted in different species. Using a transfected human neuroblastoma line, we have determined that this epitope is present on the fourth and/or fifth immunoglobulin-like domain of rat MAG and that it is added intracellularly, probably in the trans Golgi. Finally we have found that expression of the HNK-1 epitope is increased by activation of different second messenger systems, providing direct evidence that its expression can be regulated independently from that of the MAG polypeptide.

Identification of the Glycosylated Sequons of Human Myelin-Associated Glycoprotein

Myelin-associated glycoprotein (MAG) is a neural cell adhesion molecule expressing the L2/HNK-1 carbohydrate epitope. MAG is heavily glycosylated containinq 30% carbohydrate by weight. In this study, human MAG glycopeptides were isolated and sequenced. Of the 9 MAG sequons 7 were glycosylated and 1 was partially glycosylated at Asn106. Asn332 which was not recovered in the glycopeptide fractions was probably not glycosylated. Furthermore, preliminary data indicate that all NAG glycosylated sequons miqht bear the L2/HNK-1 epitope.

Experimental paraprotein neuropathy, demyelination by passive transfer of human IgM anti‐myelin‐associated glycoprotein

Circulating monoclonal IgM antibodies that react with myelin-associated glycoprotein are strongly associated with a specific type of human peripheral nerve demyelination. There has been great interest in this syndrome because, if the paraprotein could be shown to cause the demyelination, then it would be the first proven example of antibody-mediated demyelination in humans. Systemic transfusion of chickens with monoclonal IgM antibody isolated from one of these patients produced peripheral demyelination highly characteristic of the human syndrome. The experimental lesion consists of segmental demyelination and remyelination with minimal inflammation, specific antibody bound to myelin, and widening of the myelin lamellae. In the experimental model, antibody is concentrated in specialized myelin structures, the nodes of Ranvier, and Schmidt-Lanterman incisures, suggesting that myelin-associated glycoprotein may be the antigenic target in vivo. This demonstration that human myelin-associated glycoprotein antibodies cause demyelination in vivo is the final information needed to prove that this type of human demyelination is antibody mediated. This strengthens the proposition that nerve antibodies, present in other human neurological syndromes, may also cause disease.

The T‐lymphocyte response against myelin‐associated glycoprotein and myelin basic protein in patients with multiple sclerosis

In addition to myelin basic protein (MBP), other minor components of myelin, such as myelin-associated glycoprotein (MAG), may be important autoantigens in MS. To determine whether MAG might be involved in an autoimmune reaction in MS, we screened peripheral blood lymphocytes from MS patients and normal subjects for their sensitization to human MBP and MAG antigen using a [3H]thymidine incorporation assay. We recorded three patterns in MS patients: (1) patients who responded neither to MAG nor to MBP (4/11); (2) patients who responded to both MBP and MAG (5/11); and (3) patients who gave an exclusive response to MAG (2/11). The 10 healthy controls did not respond to either MAG or MBP. That some individuals with MS expressed a specific response against MAG and that more than 50% of MS patients expressed a sensitization to MAG suggest that MAG may have a role in the pathogenesis of MS.

Axons induce differentiation of neurofibroma Schwann-like cells

Neurofibromatosis type 1 (NF-1, von Recklinghausen's disease) is characterized by the focal accumulation of Schwann-like cells (SLC) to form subcutaneous and plexiform neurofibromas and schwannomas. The aim of the present study was to determine whether NF-SLC are competent to differentiate in the presence of axons. Five dermal neurofibromas from five patients with NF-type 1 were enzymatically dissociated and the resultant cells were co-cultured with fetal rat dorsal root ganglion neurons. The cultures were studied by indirect immunofluorescence microscopy using antibodies against galactocerebroside (galC), P0 glycoprotein, human nerve growth factor receptor (NGFR) and human myelin-associated glycoprotein (MAG). SLC were strongly NGFR+ but galC- and MAG-SLC for the 2 weeks of coculture. After 3 weeks in vitro, SLC-NGFR was down-regulated but some of the spindle shaped cells had become galC+. MAG-SLC first appeared after 5 weeks in vitro but P0 glycoprotein was never detected when studied up to 6 weeks. Our data demonstrate that axons induce SLC to down-regulate surface NGFR and to express some myelin components in a qualitatively normal fashion.

The Epitope(s) Recognized by HNK‐1 Antibody and IgM Paraprotein in Neuropathy Is Present on Several N‐Linked Oligosaccharide Structures on Human P0 and Myelin‐Associated Glycoprotein

The mouse monoclonal antibody HNK-1 and the human monoclonal IgM antibody present in patients with polyneuropathy both recognize carbohydrate epitope(s) on human myelin-associated glycoprotein and P0. In the present study, the oligosaccharide structures that bear the antibody epitope(s) were investigated. The extracellular derivative of myelin-associated glycoprotein (dMAG) was purified by immunoaffinity chromatography. P0 was electroeluted from gel slices. Western blot analysis of whole glycoproteins demonstrated that the epitopes for HNK-1 and the human monoclonal IgM antibody were different. The glycopeptides obtained by proteolysis of purified dMAG and P0 were separated and characterized by affinity chromatography on concanavalin A-Sepharose. Both dMAG and P0 displayed heterogeneity in their oligosaccharide structures, i.e., they both contained mainly tri- and tetraantennary oligosaccharides (approximately 80%), although biantennary (10%) and high-mannose and/or hybrid (10%) oligosaccharides were present. The human monoclonal IgM antibody epitope was present on all types of isolated oligosaccharide structures from either dMAG and P0. The HNK-1 epitope was present on all types of oligosaccharide structures of dMAG, whereas it was present only on tri- and tetraantennary structures of P0.

Molecular cloning of human myelin‐associated glycoprotein

The nucleotide sequence for human myelin-associated glycoprotein (MAG) and its deduced amino acid sequence, obtained by analysis of two overlapping cDNA clones isolated from a human brain cDNA library, is presented and compared to that reported for rat MAG. The sequence provides an open reading frame of 1,878 nucleotides encoding a peptide of 626 amino acids with a calculated molecular weight of 69.1 kD. It is 89% homologous in nucleotide sequence to the large isoform of rat MAG, with 95% homology in the amino acid sequence. It contains 9 potential glycosylation sites, one more than in rat, and shares other key features with rat MAG, including 5 immunoglobulin-like regions of internal homology, an RGD sequence, and potential phosphorylation sites. Its structure appears to be highly conserved in evolution, possibly suggesting a close interdependence between its structure and function. The human gene is located on the proximal long arm of chromosome 19 (19q12----q13.2).

CDNA cloning and amino acid sequence for human myelin-associated glycoprotein

cDNA clones of human myelin-associated glycoprotein were isolated and analyzed. The combination of the two overlapping cDNA clones covered the full coding region and the complete amino acid sequence was deduced. In rat and mouse, expression of the two forms of mRNA is developmentally regulated; the mRNA without exon 12 portion is expressed mainly in the actively myelinating stage of development. Although the cDNA library used here was prepared from adult human brain poly(A) + RNA, all five clones obtained corresponded to the mRNA without exon 12 portion.

Antibodies to Myelin-Associated Glycoprotein (MAG) in the cerebrospinal fluid of multiple sclerosis patients

Cerebrospinal fluids (CSF) and sera from patients with multiple sclerosis (MS), other neurological disease (ONDs) and healthy controls were tested for antibodies to myelin-associated glycoprotein (MAG) by several different assays. Using a very sensitive, solid-phase radioimmunoassay with radioiodinated protein A, a statistically significant elevation of anti-MAG antibodies was detected in MS CSFs in comparison to those from ONDs and healthy controls. The antibodies reacted with human MAG, but not with rat MAG, and appeared to be directed towards carbohydrate dterminants in the glycoprotein. The CSFs from high IgG producers had significantly greater anti-MAG antibody levels than those from low IgG producers, even though the assays were done on CSF samples that had been normalized to the same IgG concentration, the elevated antibodies were not detected when the same samples were tested with a liquid-phase radioimmunoassay or an enzyme-linked immunosorbent assay, and the antibodies in the MS CSF also could not be detected by Western blotting. An elevated level of antibodies was not found in sera from MS patients by any of the assays, possibly because these samples gave higher and more variable background. The results suggest that there is a low level of humoral immunity to MAG in MS patients that can only be detected by the most sensitive assays. This weak immune response to MAG may be secondary to the demyelinating process, but could play a role in the progression of the disease.

Analysis of the feline immune response to human myelin-associated glycoprotein

Elucidation of the pathogenesis of demyelinating peripheral neuropathy associated with myelin-associated glycoprotein (MAG) binding IgM paraproteins requires an in vivo animal model of the syndrome. Multiple immunizations of cats with MAG in Freund's adjuvant did not produce an antibody response but four immunizations with MAG-iscom (Morein, B. et al. (1984) Nature, 308: 457–460) did induce IgM antibodies which bound to human MAG and cat peripheral nerve myelin. Despite the presence of antibody for a 13-month period, no neuropathy developed. At necropsy, the peripheral nerves were ultrastructurally normal and no antibody was detectable in the endoneurium. A competitive ELISA indicated that the cat and human IgM antibodies recognized different epitopes.