Myelin-associated glycoprotein (Siglec-4) expression is progressively and selectively decreased in the brains of mice lacking complex gangliosides

Myelin-associated glycoprotein (MAG, Siglec-4) is a quantitatively minor membrane component expressed preferentially on the innermost myelin wrap, adjacent to the axon. It stabilizes myelin-axon interactions by binding to complementary ligands on the axolemma. MAG, a member of the Siglec family of sialic acid-binding lectins, binds specifically to gangliosides GD1a and GT1b, which are the major sialoglycoconjugates on mammalian axons. Mice with a disrupted Galgt1 gene lack UDP-GalNAc:GM3/GD3 N-acetylgalactosaminyltransferase (GM2/GD2 synthase) and fail to express complex brain gangliosides, including GD1a and GT1b, instead expressing a comparable amount of the simpler gangliosides GM3, GD3, and O-acetyl-GD3. Galgt1-null mice produce similar amounts of total myelin compared to wild-type mice, but as the mice age, they exhibit axon degeneration and dysmyelination with accompanying motor behavioral deficits. Here we report that Galgt1-null mice display progressive and selective loss of MAG from the brain. At 1.5 months of age, MAG expression was similar in Galgt1-null and wild-type mice. However, by 6 months of age MAG was decreased approximately 60% and at 12 months of age approximately 70% in Galgt1-null mice compared to wild-type littermates. Expression of the major myelin proteins (myelin basic protein and proteolipid protein) was not reduced in Galgt1-null mice compared to wild type. MAG mRNA expression was the same in 12-month-old Galgt1-null compared to wild-type mice, an age at which MAG protein expression was markedly reduced. We conclude that the maintenance of MAG protein levels depends on the presence of complex gangliosides, perhaps due to enhanced stability when MAG on myelin binds to its complementary ligands, GD1a and GT1b, on the apposing axon surface.     

Dendritic morphology and spine density is not altered in motor cortex and dentate granular cells in mice lacking the ganglioside biosynthetic gene B4galnt1 - A quantitative Golgi cox study

Gangliosides are characteristic plasma membrane constituents of vertebrate brain used as milestones of neuronal development. As neuronal morphology is a good indicator of neuronal differentiation, we analyzed how lack of the ganglioside biosynthetic gene Galgt1 whose product is critical for production of four major adult mammalian brain complex gangliosides (GM1, GD1a, GD1b and GT1b) affects neuronal maturation in vivo. To define maturation of cortical neurons in mice lacking B4galnt1 we performed a morphological analysis of Golgi-Cox impregnated pyramidal neurons in primary motor cortex and granular cells of dentate gyrus in 3, 21 and 150 days old B4galnt1-null and wild type mice. Quantitative analysis of basal dendritic tree on layer III pyramidal neurons in the motor cortex showed very immature dendritic picture in both mice at postnatal day 3. At postnatal day 21 both mice reached adult values in dendritic length, complexity and spine density. No quantitative differences were found between B4galnt1-null and wild type mice in pyramidal cells of motor cortex or granular cells of dentate gyrus at any examined age. In addition, the general structural and neuronal organization of all brain structures, qualitatively observed on Nissl and Golgi-Cox, were similar Our results demonstrate that neurons can develop normal dendritic complexity and length without presence of complex gangliosides in vivo. Therefore, behavioral differences observed in B4galnt1-null mice may be attributed to functional rather than morphological level of dendrites and spines of cortical pyramidal neurons.     

Loss of ABCA8B decreases myelination by reducing oligodendrocyte precursor cells in mice

The myelin sheath, which is wrapped around axons, is a lipid-enriched structure produced by mature oligodendrocytes. Disruption of the myelin sheath is observed in several neurological diseases, such as multiple sclerosis. A crucial component of myelin is sphingomyelin, levels of which can be increased by ABCA8, a member of the ATP-binding cassette transporter family. ABCA8 is highly expressed in the cerebellum, specifically in oligodendroglia. However, whether ABCA8 plays a role in myelination and mechanisms that would underlie this role remain unknown. Here, we found that the absence of Abca8b, a mouse ortholog of ABCA8, led to decreased numbers of cerebellar oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes in mice. We show that in oligodendrocytes, ABCA8 interacts with chondroitin sulfate proteoglycan 4 (CSPG4), a molecule essential for OPC proliferation, migration, and myelination. In the absence of Abca8b, localization of CSPG4 to the plasma membrane was decreased, contributing to reduced cerebellar CSPG4 expression. Cerebellar CSPG4+ OPCs were also diminished, leading to decreased mature myelinating oligodendrocyte numbers and cerebellar myelination levels in Abca8b^?/? mice. In addition, electron microscopy analyses showed that the number of nonmyelinated cerebellar axons was increased, whereas cerebellar myelin thickness (g-ratio), myelin sheath periodicity, and axonal diameter were all decreased, indicative of disordered myelin ultrastructure. In line with disrupted cerebellar myelination, Abca8b^?/? mice showed lower cerebellar conduction velocity and disturbed locomotion. In summary, ABCA8 modulates cerebellar myelination, in part through functional regulation of the ABCA8-interacting protein CSPG4. Our findings suggest that ABCA8 disruption may contribute to the pathophysiology of myelin disorders.     

Biosynthesis of the major brain gangliosides GD1a and GT1b

Gangliosides-sialylated glycosphingolipids-are the major glycoconjugates of nerve cells. The same four structures-GM1, GD1a, GD1b and GT1b-comprise the great majority of gangliosides in mammalian brains. They share a common tetrasaccharide core (Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1'Cer) with one or two sialic acids on the internal galactose and zero (GM1 and GD1b) or one (GD1a and GT1b) α2-3-linked sialic acid on the terminal galactose. Whereas the genes responsible for the sialylation of the internal galactose are known, those responsible for terminal sialylation have not been established in vivo. We report that St3gal2 and St3gal3 are responsible for nearly all the terminal sialylation of brain gangliosides in the mouse. When brain ganglioside expression was analyzed in adult St3gal1-, St3gal2-, St3gal3- and St3gal4-null mice, only St3gal2-null mice differed significantly from wild type, expressing half the normal amount of GD1a and GT1b. St3gal1/2-double-null mice were no different than St3gal2-single-null mice; however, St3gal2/3-double-null mice were >95% depleted in gangliosides GD1a and GT1b. Total ganglioside expression (lipid-bound sialic acid) in the brains of St3gal2/3-double-null mice was equivalent to that in wild-type mice, whereas total protein sialylation was reduced by half. St3gal2/3-double-null mice were small, weak and short lived. They were half the weight of wild-type mice at weaning and displayed early hindlimb dysreflexia. We conclude that the St3gal2 and St3gal3 gene products (ST3Gal-II and ST3Gal-III sialyltransferases) are largely responsible for ganglioside terminal α2-3 sialylation in the brain, synthesizing the major brain gangliosides GD1a and GT1b.     

Effects of deletion of macrophage ABCA7 on lipid metabolism and the development of atherosclerosis in the presence and absence of ABCA1

ABCA7, a close relative of ABCA1 which facilitates cholesterol efflux to lipid-poor apoproteins, has been implicated in macrophage lipid efflux and clearance of apoptotic cells in in vitro studies. In the current study, we investigated the in vivo effects of macrophage ABCA7 deficiency on lipid metabolism and atherosclerosis. Chimeras with dysfunctional ABCA7 in macrophages and other blood cells were generated by transplantation of bone marrow from ABCA7 knockout (KO) mice into irradiated low-density lipoprotein receptor (LDLr) KO mice. Unexpectedly, macrophage ABCA7 deficiency did not significantly affect atherosclerosis susceptibility of LDLr KO mice after 10 weeks Western-type diet feeding. However, ABCA7 deficiency was associated with 2-fold (p<0.05) higher macrophage ABCA1 mRNA expression levels. Combined disruption of ABCA1 and ABCA7 in bone-marrow-derived cells increased atherosclerotic lesion development (1.5-fold (p>0.05) as compared to wild type transplanted mice. However, single deletion of ABCA1 had a similar effect (1.8-fold, p<0.05). Macrophage foam cell accumulation in the peritoneal cavity was reduced in ABCA1/ABCA7 dKO transplanted animals as compared to single ABCA1 KO transplanted mice, which was associated with increased ABCG1 expression. Interestingly, spleens of ABCA1/ABCA7 double KO transplanted mice were significantly larger as compared to the other 3 groups and showed massive macrophage lipid accumulation, a reduction in CD3+ T-cells, and increased expression of key regulators of erythropoiesis. In conclusion, deletion of ABCA7 in bone marrow-derived cells does not affect atherogenesis in the arterial wall neither in the absence or presence of ABCA1. Interestingly, combined deletion of bone marrow ABCA1 and ABCA7 causes severe splenomegaly associated with cellular lipid accumulation, a reduction in splenic CD3+ T cells, and induced markers of erythropoeisis. Our data indicate that ABCA7 may play a role in T cell proliferation and erythropoeisis in spleen.     

Lipid content of brain, brain membrane lipid domains, and neurons from acid sphingomyelinase deficient mice

The cholesterol, sphingolipid, and glycerophospholipid content of total brain, of detergent-resistant membranes prepared from the total brain, and of cerebellar granule cells differentiated in culture from wild type (WT) and acid sphingomyelinase knockout (ASMKO) were studied. Brains derived from 7-month-old ASMKO animals showed a fivefold higher level of sphingomyelin and a significant increase in ganglioside content, mainly because of monosialogangliosides GM3 and GM2 accumulation, while the cholesterol and glycerophospholipid content was unchanged with respect to WT animals. An increase in sphingomyelin, but not in gangliosides, was also detected in cultured cerebellar granule neurons from ASMKO mice, indicating that ganglioside accumulation is not a direct consequence of the enzyme defect. When a detergent-resistant membrane fraction was prepared from ASMKO brains, we observed that a higher detergent-to-protein ratio was needed than in WT animals. This likely reflects a reduced fluidity in restricted membrane areas because of a higher enrichment in sphingolipids in the case of ASMKO brain.     

ApoA-I lipidation in primary mouse hepatocytes. Separate controls for phospholipid and cholesterol transfers

The liver is the major site of both apolipoprotein A-I (apoA-I) synthesis and ATP-binding cassette transporter A1 (ABCA1) expression. Here, we compare the lipidation with cholesterol and phospholipid of newly synthesized human apoA-I (hapoA-I) using adenoviral vector-mediated endogenous expression or exogenously added hapoA-I in wild type and ABCA1-null hepatocytes. Hepatocytes were labeled with [3H]cholesterol (delivered with LDL or methyl-beta-cyclodextrin), [3H]mevalonate, or [3H]choline. ABCA1 deficiency decreased apoA-I phospholipidation by 80%, but acquisition of de novo synthesized and exogenous cholesterol only decreased by 40-60%. The transfer of de novo synthesized cholesterol to apoA-I was decreased at all time points, but that of exogenously delivered cholesterol was independent of ABCA1 activity at the early time points. Progesterone does not affect apoA-I synthesis or its lipidation but inhibited the early phase of apoA-I cholesterol lipidation in both wild type and ABCA1-null hepatocytes. Fast protein liquid chromatography analysis of medium lipoproteins confirmed that with ABCA1 deficiency, the proportion of secreted high density lipoprotein-associated apoA-I and cholesterol decreased by about 50%. The very low density lipoprotein (VLDL)/LDL size fraction also contained a significant level of cholesterol in ABCA1 deficiency, consistent with the result of immunoprecipitations showing the presence of lipoproteins with both apoA-I and murine apoB. ApoA-I lipidation with newly synthesized cholesterol in ABCA1-null hepatocytes was significantly decreased by brefeldin A and monensin. In conclusion, we demonstrate that: (i) whereas most hepatic phospholipidation of apoA-I is mediated by ABCA1, acquisition of cholesterol depends on active transfer from intracellular compartments by ABCA1-dependent and -independent pathways, both sensitive to progesterone and (ii) there is separate regulation of phospholipid and cholesterol lipidation of apoA-I in hepatocytes.     

Pathologic and phenotypic alterations in a mouse expressing a connexin47 missense mutation that causes Pelizaeus-Merzbacher-like disease in humans

Gap junction channels are intercellular conduits that allow diffusional exchange of ions, second messengers, and metabolites. Human oligodendrocytes express the gap junction protein connexin47 (Cx47), which is encoded by the GJC2 gene. The autosomal recessive mutation hCx47M283T causes Pelizaeus-Merzbacher-like disease 1 (PMLD1), a progressive leukodystrophy characterized by hypomyelination, retarded motor development, nystagmus, and spasticity. We introduced the human missense mutation into the orthologous position of the mouse Gjc2 gene and inserted the mCx47M282T coding sequence into the mouse genome via homologous recombination in embryonic stem cells. Three-week-old homozygous Cx47M282T mice displayed impaired rotarod performance but unchanged open-field behavior. 10-15-day-old homozygous Cx47M282T and Cx47 null mice revealed a more than 80% reduction in the number of cells participating in glial networks after biocytin injections into oligodendrocytes in sections of corpus callosum. Homozygous expression of mCx47M282T resulted in reduced MBP expression and astrogliosis in the cerebellum of ten-day-old mice which could also be detected in Cx47 null mice of the same age. Three-month-old homozygous Cx47M282T mice exhibited neither altered open-field behavior nor impaired rotarod performance anymore. Adult mCx47M282T expressing mice did not show substantial myelin alterations, but homozygous Cx47M282T mice, additionally deprived of connexin32, which is also expressed in oligodendrocytes, died within six weeks after birth and displayed severe myelin defects accompanied by astrogliosis and activated microglia. These results strongly suggest that PMLD1 is caused by the loss of Cx47 channel function that results in impaired panglial coupling in white matter tissue.     

Attenuation of interleukin 2 signal in the spleen cells of complex ganglioside-lacking mice.

T cell development and function in complex ganglioside-lacking (GM2/GD2 synthase gene-disrupted) mice were analyzed. GM1, asialo-GM1, and GD1b were representative gangliosides expressed on T cells of the wild type mice and completely deleted on those of the mutant mice. The sizes and cell numbers of the mutant mice spleen and thymus were significantly reduced. Spleen cells from the mutant mice showed clearly reduced proliferation compared with the wild type when stimulated by interleukin 2 (IL-2) but not when treated with concanavalin A or anti-CD3 cross-linking. Expression levels of IL-2 receptor alpha, beta, and gamma were almost equivalent, and up-regulation of alpha chain after T cell activation was also similar between the mutant and wild type mice. Activation of JAK1, JAK3, and SAT5 after IL-2 treatment was reduced, and c-fos expression was delayed and reduced in the mutant spleen cells, suggesting that the IL-2 signal was attenuated in the mutant mice probably due to the modulation of IL-2 receptors by the lack of complex gangliosides.     

Morphological and morphometric studies of the dysmyelinating mutant,the Long Evans shaker rat

The Long Evans shaker (les) rat is a recently identified CNS myelin mutant with an autosomal recessive mode of inheritance. Although scattered myelin sheaths are present in some areas of the CNS, most notably the ventral spinal cord in the young neonatal rat, this myelin is gradually lost, and 8-12 weeks little myelin is present throughout the CNS. Despite this severe myelin deficiency, some mutants may live beyond one year of age. Rare, thin myelin sheaths that are present early in development lack myelin basic protein (MBP) and on ultrastructural examination are poorly compacted and lack a major dense line. Many oligodendrocytes develop an accumulation of vesicles and membranous bodies, but no abnormal cell death is observed. In the optic nerve, cell kinetic studies show an increase in proliferation at early time points in les, while total glial cell counts are also increased in les from 2 months of age. In situ hybridization studies demonstrate that the numbers of mature oligodendrocytes are similar to controls early in life and increase with time compared to controls. There is both a progressive astrocyte hypertrophy and microgliosis. While les has a mutation in the myelin basic protein (mbp) gene, it is dissimilar in both genotype and phenotype to the previously described mbp mouse mutants, shiverer (shi) and shiverermld. Unlike shi and its allele, where myelin increases with time and oligodendrocytes become ultrastructurally normal, les oligodendrocytes are permanently disabled, continue to demonstrate cytoplasmic abnormalities, and fail to produce myelin beyond the first weeks of life.     

Gemfibrozil,a Lipid-lowering Drug,Increases Myelin Genes in Human Oligodendrocytes via Peroxisome Proliferator-activated Receptor-β

An increase in CNS remyelination and a decrease in CNS inflammation are important steps to halt the progression of multiple sclerosis. Earlier studies have shown that gemfibrozil, a lipid-lowering drug, has anti-inflammatory properties. The current study identified another novel property of gemfibrozil in stimulating the expression of myelin-specific genes (myelin basic protein, myelin oligodendrocyte glycoprotein, 2′,3′-cyclic-nucleotide 3′-phosphodiesterase, and proteolipid protein (PLP)) in primary human oligodendrocytes, mixed glial cells, and spinal cord organotypic cultures. Although gemfibrozil is a known activator of peroxisome proliferator-activated receptor-α (PPAR-α), we were unable to detect PPAR-α in either gemfibrozil-treated or untreated human oligodendrocytes, and gemfibrozil increased the expression of myelin genes in oligodendrocytes isolated from both wild type and PPAR-α(−/−) mice. On the other hand, gemfibrozil markedly increased the expression of PPAR-β but not PPAR-γ. Consistently, antisense knockdown of PPAR-β, but not PPAR-γ, abrogated the stimulatory effect of gemfibrozil on myelin genes in human oligodendrocytes. Gemfibrozil also did not up-regulate myelin genes in oligodendroglia isolated from PPAR-β(−/−) mice. Chromatin immunoprecipitation analysis showed that gemfibrozil induced the recruitment of PPAR-β to the promoter of PLP and myelin oligodendrocyte glycoprotein genes in human oligodendrocytes. Furthermore, gemfibrozil treatment also led to the recruitment of PPAR-β to the PLP promoter in vivo in the spinal cord of experimental autoimmune encephalomyelitis mice and suppression of experimental autoimmune encephalomyelitis symptoms in PLP-T cell receptor transgenic mice. These results suggest that gemfibrozil stimulates the expression of myelin genes via PPAR-β and that gemfibrozil, a prescribed drug for humans, may find further therapeutic use in demyelinating diseases.     

Brain lipid composition in grey-lethal mutant mouse characterized by severe malignant osteopetrosis

The grey-lethal mouse (gl/gl) mutant most closely resembles the severe human malignant autosomal recessive OSTM1-dependent form of osteopetrosis that it has been described to be associated with neurological abnormalities. For this reason, we have analyzed the brain lipid composition (gangliosides, neutral glycosphingolipids, phospholipids and cholesterol), from gl/gl mice at different ages of development and compared with wild type mice. Both cholesterol and glycerophospholipid content and pattern in the gl/gl and control mice were very similar. In contrast, significant differences were observed in the content of several sphingolipids. Higher amount of the monosialogangliosides GM2 and GM3, and lower content of sphingomyelin, sulfatide and galactosylceramide were observed in the gl/gl brain with respect to controls. The low content of sphingomyelin, sulfatide and galactosylceramide is consistent with the immunohistochemical results showing that in the grey-lethal brain significant depletion and disorganization of the myelinated fibres is present, thus supporting the hypothesis that loss of function of the OSTM1 causes neuronal impairment and myelin deficit.     

Effect of the Jimpy Mutation on Expression of Myelin Proteins in Heterozygous and Hemizygous Mouse Brain

The levels of myelin basic protein, proteolipid protein, and 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37) in cerebral hemispheres of wild-type, heterozygous jp/+, and hemizygous jp/Y mice of different ages were determined by radioimmunoassay and immunoblotting. In jp/Y brain the level of myelin basic protein was 8% that of wild-type at all ages. All forms of the protein were reduced although the 21.5K Mr form was relatively spared at early ages compared to the 18.5K, 17K, and 14K Mr forms. The level of 2',3'-cyclic nucleotide 3'-phosphohydrolase was 8% that of wild-type at all ages, and proteolipid protein was undetectable at any age. These results are consistent with the hypothesis that the jimpy mutation blocks myelin morphogenesis subsequent to incorporation of 21.5K Mr myelin basic protein but prior to incorporation of proteolipid protein. In jp/+ brain the levels of the three proteins were reduced commensurately to 60-70% those of wild-type. The deficit was apparent as early as 10 days after birth and remained proportionately constant throughout development. These results suggest that in jp/+ mice, X-chromosome inactivation produces a mosaic population of functionally wild-type and functionally jimpy oligodendrocytes. The former elaborate normal amounts of myelin but do not completely compensate for the myelin deficit due to the latter.     

Down-regulation of polysialic acid is required for efficient myelin formation

Oligodendrocyte precursor cells modify the neural cell adhesion molecule (NCAM) by the attachment of polysialic acid (PSA). Upon further differentiation into mature myelinating oligodendrocytes, however, oligodendrocyte precursor cells down-regulate PSA synthesis. In order to address the question of whether this down-regulation is a necessary prerequisite for the myelination process, transgenic mice expressing the polysialyltransferase ST8SiaIV under the control of the proteolipid protein promoter were generated. In these mice, postnatal down-regulation of PSA in oligodendrocytes was abolished. Most NCAM-120, the characteristic NCAM isoform in oligodendrocytes, carried PSA in the transgenic mice at all stages of postnatal development. Polysialylated NCAM-120 partially co-localized with myelin basic protein and was present in purified myelin. The permanent expression of PSA-NCAM in oligodendrocytes led to a reduced myelin content in the forebrains of transgenic mice during the period of active myelination and in the adult animal. In situ hybridizations indicated a significant decrease in the number of mature oligodendrocytes in the forebrain. Thus, down-regulation of PSA during oligodendrocyte differentiation is a prerequisite for efficient myelination by mature oligodendrocytes. Furthermore, myelin of transgenic mice exhibited structural abnormalities like redundant myelin and axonal degeneration, indicating that the down-regulation of PSA is also necessary for myelin maintenance.     

Identification of Tmem10 as a Novel Late-stage Oligodendrocytes Marker for Detecting Hypomyelination

Oligodendrocytes ensheath axons to form compact insulating multilamellar structures known as myelin. Tmem10 is a novel type I transmembrane glycoprotein that is highly expressed in oligodendrocytes and whose biological function remains largely unknown. Furthermore, the expression pattern of Tmem10 remains a matter of some controversy. Given the inconsistency of its expression pattern and the lack of validated specific antibodies, Tmem10 is not widely accepted as a marker for mature oligodendrocytes. As a means to solve these problems and to aid future studies of oligodendrocyte-associated diseases, we have generated a highly specific Tmem10 antibody. Using this Tmem10 antibody, we clarify that Tmem10 protein is firstly expressed at 2 weeks in the postnatal mouse brain with age-related increase, only in the central nervous system (CNS). We also reveal that Tmem10 is expressed specifically in late stage oligodendrocytes and later than MAG, a late-stage myelin marker. Finally, we show that Tmem10 co-expresses with MOG- and MBP-positive myelin fibers and is dramatically reduced in a hypomyelination mouse model. In conclusion, our study demonstrates that Tmem10 can be used as a specific marker for myelinating oligodendrocytes and perhaps for the evaluation of myelination diseases, such as multiple sclerosis.     

Matrix metalloproteinase-9 deficiency leads to prolonged foreign body response in the brain associated with increased IL-1β levels and leakage of the blood-brain barrier

Matrix metalloproteinases (MMPs) are enzymes with specificity towards extracellular matrix (ECM) components. MMPs, especially MMP-9, have been shown to degrade components of the basal lamina and disrupt the blood-brain barrier (BBB) and thus, contribute to neuroinflammation. In the present study we examined the role of MMP-9 in the foreign body response in the brain. Millipore filters of mixed cellulose ester were implanted into the brain cortex of wild type and MMP-9-null mice for a period of 2 d to 8 wks and the response was analyzed by histology and immunohistochemistry. We observed enhanced and prolonged neuroinflammation in MMP-9-null mice, evidenced by persistence of neutrophils, macrophages/microglia, and reactive astrocytes up to 8 wks post-implantation. In addition, blood vessel density around implants was increased in MMP-9-null mice and detection of mouse serum albumin (MSA) indicated that vessels were leaky. Immunohistochemical and western blot analyses indicated that this defect was associated with the absence of tight junction proteins zonula occludens-1 (ZO-1) and ZO-2 from vessels in proximity to implants. Analysis of brain sections and brain protein extracts revealed that the levels of the pro-inflammatory cytokine interleukin-1β (IL-1β), which is a substrate for MMP-9, were significantly higher in MMP-9-null mice at 8wks post-implantation. Collectively, our studies suggest that increased levels of IL-1β and the delayed repair of BBB are associated with prolongation of the FBR in MMP-9-null mice.