Spinal Cord Transcriptome Analysis Using Suppression Subtractive Hybridization and Mirror Orientation Selection

Comparison of cDNA libraries derived from the spinal cord with those derived from the visual cortex by means of forward and reverse subtractive hybridization resulted in the cataloguing of 60 genes differentially expressed in the spinal cord. 1. The differentially expressed genes represent a mixture of novel and known sequences with known and unknown protein products. 2. The possibility that the subtraction process was simply overwhelmed by background sequences was significantly reduced by several observations including comparisons between suppression subtractive hybridization (SSH) and mirror orientation selection (MOS). 3. Nearly half of all genes up-regulated in the spinal cord are of myelin origin. 4. Twenty-five percent of all up-regulated clones in the spinal cord versus the visual cortex are for proteolipid protein. 5. Ten percent of all up-regulated clones in spinal cord versus visual cortex are for ferretin heavy chain, which is known to be produced in oligodendroglial cells in the CNS. 6. Two of the up-regulated sequences, proteolipid protein and N-myc down-regulated gene 4, are identified with genes known to directly affect neuron survival. 7. Two of the up-regulated genes, ferritin and transferrin, are indirectly associated with apoptosis through their ability to sequester iron and reduce free radical formation.     

Role of Normalization in the Elimination of Abundant Myelin Sequences in Spinal Cord cDNA Libraries Produced by Suppression Subtractive Hybridization

Spinal cord libraries subtracted against visual cortex using suppression subtractive hybridization SSH are dominated by abundant gene sequences derived from myelin elements. We compared our subtracted library results of three of these abundant sequences to published expressed sequence tag libraries that are not normalized and not subtracted and presumed representatives of murine spinal cord mRNA abundance. We show that: all three abundant sequences, myelin basic protein (Mbp), proteolipid protein (Plp1) and Ferretin heavy chain (Fth1) are highly expressed in spinal cord when this structure is compared to visual cortex; myelin basic protein is represented in our subtracted libraries but at a low frequency, whereas Plp1 and Fth1 represent nearly one-third of all sequences in these libraries; mirror orientation selection, a procedure designed to reduce background sequences, generates libraries very similar in abundance to SSH; proteolipid protein can be reduced in these libraries by adding Plp1 sequences to the driver in the SSH procedure and also by subtracting Plp1 directly from tester and driver. We conclude that adequate normalization is essential to reduce the presence of abundant sequences in SSH libraries.     

Immunocytochemical localization of carbonic anhydrase in the spinal cords of normal and mutant (shiverer) adult mice with comparisons among fixation methods

The peroxidase-antiperoxidase technique was used for immunocytochemical localization of carbonic anhydrase in the mouse spinal cord to detect whether this antigen was normally present in myelinated fibers, in oligodendrocytes in both white and gray matter, and in astrocytes, and to determine where the carbonic anhydrase might be localized in the spinal cords of dysmyelinating mutant (shiverer) mice. The most favorable methods for treating tissue were: 1) immersion in formalin-ethanol-acetic acid followed by paraffin embedding, or 2) light fixation with paraformaldehyde and preparation of vibratome sections. Carnoy's solution, followed by paraffin embedding, extracted myelin from the tissue, while aqueous aldehydes, when used before paraffin embedding, reduced staining everywhere except at sites of compact myelin. The latter conclusion was based, in part, on the almost complete loss of this antigen from the shiverer cord, where compact myelin is known to be virtually absent but where membrane-bound carbonic anhydrase was demonstrated enzymatically. When the optimal methods were used with normal mouse cords, carbonic anhydrase was found throughout the white matter columns and in the oligodendrocytes in gray and white matter. The staining of the white matter was attributed to myelinated fibers because of the similarity in distribution to both a histological myelin stain and the immunocytochemical staining for myelin basic protein. In the mutant mice the oligodendrocyte cell bodies and processes, which were stained in all areas of the spinal cord, were particularly numerous at the periphery of the sections. In contrast to the oligodendrocytes, the fibrous astrocytes appeared to lack carbonic anhydrase, or to have lower than detectable levels, since the astrocyte marker, glial fibrillary acidic protein, had a very different distribution from that of carbonic anhydrase. Even finer localization was obtained in vibratome sections, where the antibody against carbonic anhydrase permitted visualization of the processes connecting oligodendrocytes to myelinated fibers in the normal adult spinal cord.     

Upregulation of Vascular Endothelial Growth Factor Receptor-3 in the Spinal Cord of Lewis Rats with Experimental Autoimmune Encephalomyelitis

We investigated the spatiotemporal expression of vascular endothelial growth factor receptor–3 (VEGFR-3) in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. VEGFR-3 mRNA and protein were constitutively expressed in gray matter neurons and in a few white matter astrocytes. Induction of VEGFR-3 occurred predominantly in perivascular infiltrated macrophages in the spinal cord white matter during the inductive phase of EAE. VEGFR-3 expression was also induced in activated microglial cells in the gray and white matter, mainly in the peak phase. In addition, reactive astrocytes in the white matter, but not in the gray matter, expressed VEGFR-3 as disease severity increased. These data suggest that VEGFR-3 is involved in the recruitment of monocytic macrophages and in glial reactions during EAE.     

Comparison of sequences of cDNA clones obtained from oligo-capping cDNA libraries with those from unigene.

We compared in detail the characteristics of the sequences of the cDNA clones obtained by the oligo-capping method (oligo-capping clones) with that of the sequences in the UniGene database. To compare the completeness of the sequences, three new variables, "fullness-proportion of clones" (the ratio of complete clones to total clones in a library), "fullness-proportion of genes" (the ratio of complete genes to total genes in a library), and "fullness-proportion of database" (the ratio of complete genes to total genes in a database sampled from a library), were defined. The fullness-proportion of clones of oligo-capping clones was 57.3%, 2.2 times larger than that of UniGene (25.9%). The fullness-proportion of genes of oligo-capping clones was 41.8%, 2.4 times larger than that of UniGene (17.8%). When gene length was restricted to > or = 1.5 kb, the fullness-proportion of genes of oligo-capping clones was four times larger than that of UniGene. The fullness-proportion of database of oligo-capping clones was approximately the same as that of UniGene. By simulating the clone redundancy, this coincidence was found to be due to the large redundancy of the UniGene database. Consequently, the cDNA sequence database of oligo-capping clones enabled high throughput selection of full-length cDNA clones.     

DEVELOPMENTAL AND AGING CHANGES IN PROTEIN CONCENTRATION AND 2'',3''-CYCLIC NUCLEOSIDEMONOPHOSPHATE PHOSPHODIESTERASE ACTIVITY (EC 3.1.4.16) IN HUMAN CEREBRAL WHITE AND GRAY MATTER AND SPINAL CORD

Abstract— The concentration of protein as assayed by the Lowry method and the specific activity of 2′.3’-cyclic nucleosidemonophosphate phosphodiesterase (CNP), an enzyme characteristic of the myelin sheath, were determined in human CNS tissues obtained at autopsy from subjects ranging in age from 26 weeks gestation to 83 y. CNP activity in cerebral white matter samples was very low until approx 2 months of age when it increased rapidly, reaching near-adult levels by 2 y of age. CNP activity in adult (15–60 y) cerebral white matter was 8.1 ± 1.0 μmol/min/mg protein (mean ±s.d. ). The protein concentration of cerebral white matter increased from 64 mg/g wet tissue at 26 weeks gestation to adult levels (118.5 ± 10.0 mg/g wet tissue) by 16–18 months. CNP activity in cerebral gray matter was initially very low and showed only a small increase during development to adult values of approx 1.4 μmol/min/mg protein. In spinal cord, adult values (3.7 ± 0.56 μmol/min/mg protein) were found shortly after birth. The increase in CNP activity to near-adult values occurred earlier in cross-sections of cervical spinal cord than in cerebral white matter. The increase in spinal cord protein concentration showed a similar trend (adult values = 103.1 ± 9.5 mg/g wet tissue). The white matter protein concentration decreased significantly with age over the 15–83 y interval examined but the CNP specific activity in white matter did not. The protein concentration of the 61–83 y group was 8% lower than that of the 15–60 y group. The spinal cord protein concentration decreased significantly and the spinal cord CNP specific acitivity increased significantly with increasing time between death and sample freezing. The sex of the individual had no significant effect on any of the variables examined. The developmental curves obtained for these tissues are consistent with the hypothesis that CNP is an intrinsic myelin component in human CNS myelin. The marked increase in CNP activity in white matter coincides with the period of rapid myelin deposition as determined by other parameters. CNP activity may be useful as an index of myelination in human CNS tissues.     

猫腰髓白质年龄相关的形态学变化

以青年成年猫(1-3龄,2-2．5 kg)和老年猫(12龄,3-3．5kg)L6段脊髓白质为研究对象,用 神经丝蛋白(NF)免疫染色显示神经纤维,用改良的Holzer结晶紫染色显示所有胶质细胞并用成年动物Golgi 法显示其形态,用胶质纤维酸性蛋白(GFAP)免疫染色显示星形胶质细胞。光镜下对青年猫与老年猫腰髓白质 中神经纤维和胶质细胞进行形态学观察和定量研究。与青年猫相比,老年猫腰髓白质中的神经纤维密度显著下 降(P相似文献   

The composition of the myelin proteins of the central nervous system

Abstract— The amino acid composition of human, monkey and bovine centrum ovale myelin, of bovine optic nerve myelin, and of bovine spinal cord white matter myelin has been determined. In general, the amino acid patterns of the centrum ovale myelin of these species and the optic nerve myelin are identical. Differences are noted when these are compared to the spinal cord white matter myelin. It is shown that the amino acid composition of myelin cannot be duplicated by any combination of the Folch–Lees proteolipid protein and the basic protein fraction of myelin. It is necessary to postulate the existence of a third protein fraction that is rich in dicarboxylic amino acids.     

Age-related myelin dynamics revealed by increased oligodendrogenesis and short internodes

Aging is associated with many functional and morphological central nervous system changes. It is important to distinguish between changes created by normal aging and those caused by disease. In the present study we characterized myelin changes within the murine rubrospinal tract and found that internode lengths significantly decrease as a function of age which suggests active remyelination. We also analyzed the proliferation, distribution and phenotypic fate of dividing cells with Bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU). The data reveal a decrease in glial cell proliferation from 1 to 6, 14 and 21 months of age in gray matter 4 weeks post-BrdU injections. However, we found an increase in gliogenesis at 21st  month in white matter of the spinal cord. Half of newly generated cells expressed NG2. Most cells were positive for the early oligodendrocyte marker Olig2 and a few also expressed CC1. Very few cells ever became positive for the astrocytic markers S100β or GFAP. These data demonstrate ongoing oligodendrogenesis and myelinogenesis as a function of age in the spinal cord.     

Isolation and characterization of axolemma-enriched fractions from discrete areas of bovine CNS

Myelinated axons were isolated by flotation from bovine pons, middle cerebellar peduncle, cervical spinal cord and three regions of the subcortical white matter. The myelinated axons were osmotically and mechanically shocked, followed by fractionation on a linear 15% sucrose to 45% sucrose density gradient. Axolemma-enriched fractions (AEF) found in the 28% to 32% sucrose region of the gradient from brainstem and cord white matter had high acetylcholinesterase (AChE) while little or nil AChE activity was found in corresponding AEF derived from the subcortical white matter. Morphologically, the subcortical white matter from all regions contained a heterogeneous population of well-myelinated to thinly myelinated axons, while brainstem and cord regions contained a more homogeneous population of well-myelinated axons. Histochemical analysis of AChE localized this enzyme to axonal elements. The AEF derived from any white matter source had similar polypeptide compositions. AEF derived from subcortical white matter contained two-fold more myelin basic protein and a three-fold greater content of 2 3 cyclic nucleotide 3 phosphodiesterase (CNP) compared with AEF derived from well myelinated white matter. We conclude that the purity of the AEF is related to the degree of myelination of the white matter from which the AEF is derived. Homogeneously well myelinated white matter (pons, cerebellar peduncle, cervical spinal cord) yields the highest purity AEF, as judged by the low CNP and myelin basic protein content and highest enrichment in AChE specific activity.     

Meylin types with different protein components in the same species

Abstract— Myelin was isolated from bovine optic nerve, cerebral white matter, spinal cord white matter and peripheral nerve (intradural spinal roots). The freeze-dried myelin completely dissolved in phenol-formic acid-water (14:3:3, w/v/v), and acrylamide gel electrophoresis of the myelin proteins was performed with this solvent. Qualitative and quantitative differences were observed in the myelin proteins from the various regions of the CNS. Myelin of peripheral nerve contained proteins that are apparently unique to it and which are not found in the myelin of the CNS.     

Pnn and SR family proteins are differentially expressed in mouse central nervous system

Pinin (pnn) is an SR-related protein that is ubiquitously expressed in most cell types and functions in regulating pre-mRNA splicing and mRNA export. Previously, we demonstrated that pnn is expressed in all tissues during mouse embryonic development with highest levels of expression in the central nervous system (CNS). Here we show that pnn and other SR proteins including SC35 are differentially expressed in the adult mouse CNS, displaying cell type-specific distribution patterns. Immunohistochemical analysis of whole-brain sections showed that levels of pnn and SR proteins expression were very low or nonexistent in the corpus callosum and white matter of cerebellum and spinal cord. Double-immunostaining with antibodies specific to neuron or glial cells showed that most astrocytes and microglia expressed neither pnn nor SR proteins. In contrast, oligodendrocytes and neurons expressed moderate and high levels, respectively, of both pnn and SR proteins. These results suggest that astrocytes are unique among cell types of neuroblast origin in terms of expression SR family proteins. Our results pave the way for future studies of the functional roles of pnn and SR family proteins in adults.     

大鼠脊髓胸段平面少突胶质细胞分布和形态学差异的研究

目的：观察大鼠脊髓胸段（T8-T10）平面中少突胶质细胞在白质和灰质中分布和形态学差异。方法：应用免疫荧光组织化学方法,利用少突胶质细胞特异性标志物一抗大鼠Nogo-A分子单克隆抗体,观察大鼠脊髓胸段平面白质和灰质中少突胶质细胞分布和形态学差异。结果：Nogo—A免疫阳性标记主要集中在少突胶质细胞的胞体、突起及其形成的髓鞘。在冠状切面中,白质中的少突胶质细胞广泛分布,而灰质中少突胶质细胞主要分布于神经元的周围;白质中少突胶质细胞胞体较灰质中少突胶质细胞的胞体大,且白质中少突胶质细胞突起及形成的髓鞘结构较灰质中明显。在矢状切面中,白质中少突胶质细胞多成”串珠状”排列,而灰质中少突胶质细胞则紧贴神经元。在脊髓近端背根结结构中,可以观察到少突胶质细胞形成的轴突呈”蜂窝状”结构。结论：应用抗大鼠Nogo—A分子单克隆抗体的免疫荧光组织化学染色方法能够较好展示少突胶质细胞分布特点和形态学差异,与少突胶质细胞类别（束内细胞,卫星细胞）和功能特点相适应,为进一步研究生理和病理条件下,少突胶质细胞的机能奠定基础。     

Errors in CGAP xProfiler and cDNA DGED: the importance of library parsing and gene selection algorithms

Background  

The Cancer Genome Anatomy Project (CGAP) xProfiler and cDNA Digital Gene Expression Displayer (DGED) have been made available to the scientific community over a decade ago and since then were used widely to find genes which are differentially expressed between cancer and normal tissues. The tissue types are usually chosen according to the ontology hierarchy developed by NCBI. The xProfiler uses an internally available flat file database to determine the presence or absence of genes in the chosen libraries, while cDNA DGED uses the publicly available UniGene Expression and Gene relational databases to count the sequences found for each gene in the presented libraries.     

Membrane lipids of human peripheral nerve and spinal cord.

Major membrane lipids were determined in specimens of human peripheral nerve (cauda equina) and spinal cord of 10 subjects aged 20-70 years. The same lipids were also assayed in myelin from the same tissues isolated with two different procedures and in myelin of cauda equina from 3 subjects aged 17-91 years isolated with a third method. The concentrations (mean and standard deviation) of phospholipids were 90 +/- 11 and 96 +/- 9 nmol/g fresh weight; of cholesterol 70 +/- 15 and 101 +/- 16; of cerebroside 19 +/- 3 and 41 +/- 7; of sulfatide 10 +/- 1 and 11 +/- l; and of gangliosides 0.80 +/- 0.08 and 0.40 +/- 0.05 N in cauda equina and spinal cord, respectively. The proportion of ethanolamine phosphoglyceride was lower and that of sphingomyelin higher in cauda equina than in spinal cord. The myelin of peripheral nerve and spinal cord contained almost the same proportions of lipids as the whole tissue. The protein-bound sialic acid content was 3-fold higher than the lipid-bound sialic acid content in cauda myelin. The fatty acid patterns of choline, ethanolamine, inositol and serine phosphoglycerides of spinal cord and its myelin, were very similar to those of cerebral white matter, while the phosphoglycerides of cauda equina had higher proportions of monoenoic acids and lower proportions of polyunsaturated fatty acids. The fatty acid patterns of sphingomyelin, cerebroside and sulfatide of spinal cord were similar to those of cerebral white matter, while those of cauda equina contained significantly more saturated fatty acids. This suggests that the lipid and fatty acid compositions of peripheral nerve are particularly suitable for the formation of a tightly packed myelin membrane which can be a powerful shield against infections and other injuries.     

LOCALIZATION OF THE THY-1 ANTIGEN IN RAT BRAIN AND SPINAL CORD BY IMMUNOFLUORESCENCE

Abstract— The Thy-1 antigen of rat brain is a membrane glycoprotein of molecular weight 17,500. It was localized in sections of brain and spinal cord by indirect immunofluorescence using rabbit antisera raised against purified Thy-1 and fluorescein conjugated purified sheep F(ab')₂, anti-(rabbit IgG) antibody fragments. The specificity of the anti-(Thy-1) sera was tested by a quantitative indirect radioactive binding assay which is particularly useful for ascertaining the specificity of reagents used in immunohistochemical studies. Purified Thy-1 was used to absorb the anti-(Thy-1) sera for controls in the immunofluorescence experiments. Strong specific fluorescence was found throughout the gray matter of brain and spinal cord with lesser amounts in white matter. The nuclei of all neural cells and also myelin lacked fluorescence. Some of the large neurons contained weak cytoplasmic fluorescence, but the majority of the immunofluorescence was located in the neuropil of the brain and spinal cord. There was an indication that Thy-1 was associated with synaptic knobs due to its presence in synaptic glomeruli and its granular appearance around some neurons. An additional association with glial membranes could not be excluded.