Subcellular Fractions Of Rat Brain Research Articles

Importance of a Novel Oxidative Mechanism for Elimination of Brain Cholesterol: TURNOVER OF CHOLESTEROL AND 24(S)-HYDROXYCHOLESTEROL IN RAT BRAIN AS MEASURED WITH 18O2TECHNIQUES IN VIVO AND IN VITRO

The brain is the most cholesterol-rich organ in the body. Brain cholesterol is characterized by a very low turnover with very little exchange with lipoproteins in the circulation. Very recently we showed that there is a continuous age-dependent flux of 24(S)-hydroxycholesterol from the human brain into the circulation (Lütjohann, D., Breuer, O., Ahlborg, G., Nennesmo, I., Sidén, A., Diczfalusy, U., and Björkhem, I. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9799-9804). Here we measured the rate of synthesis of cholesterol as well as the conversion of cholesterol into 24(S)-hydroxycholesterol in rat brain in vivo with use of an 18O2 inhalation technique and mass isotopomer distribution analysis. Cholesterol synthesis was found to correspond to 0.03 +/- 0.01% of the pool per h. Conversion of cholesterol into 24(S)-hydroxycholesterol was of a similar magnitude, about 0.02% of the pool per h. Brain microsomes converted endogenous cholesterol into 24(S)-hydroxycholesterol at a similar rate when incubated in the presence of NADPH. When incubated with whole homogenate and subcellular fractions of rat brain, there was no significant conversion of tritium-labeled 24-hydroxycholesterol into more polar products. Plasma from 18O2-exposed rats contained 24(S)-hydroxycholesterol with an enrichment of 18O similar to that in 24(S)-hydroxycholesterol in the brain. The results suggest that the present 24(S)-hydroxylase mediated mechanism is most important for elimination of cholesterol from the brain of rats. There is a slow conversion of brain cholesterol into 24(S)-hydroxycholesterol with a rapid turnover of the small pool of the latter oxysterol due to leakage to the circulation (half-life of brain 24(S)-hydroxycholesterol is about 0.5 days as compared with 2-4 months for brain cholesterol). It is evident that the 24(S)-hydroxylation greatly facilitates transfer of cholesterol over the blood-brain barrier and that this hydroxylation may be critical for cholesterol homeostasis in the brain.

Enzymatic synthesis of oleamide (cis-9, 10-octadecenoamide), an endogenous sleep-inducing lipid, by rat brain microsomes.

The enzymatic synthesis of a novel sleep-inducing lipid, oleamide (cis-9,10-octadecenoamide), was studied using rat brain subcellular fractions as enzyme sources. We found that oleamide was formed from oleic acid and ammonia on incubation with a brain homogenate. The enzyme activity catalyzing the formation of oleamide from oleic acid and ammonia was highest in the microsomal fraction among the subcellular fractions. Boiled microsomes did not exhibit appreciable enzyme activity. These results strongly suggest that oleamide can be synthesized enzymatically in the brains of stimulated animals.

Ganglioside long-chain base composition of rat brain subcellular fractions after chronic ethanol administration

Rats of two different ages (2 and 7 months) were treated with an ethanol-containing liquid diet for 24 days and changes of the ceramide composition of gangliosides were studied in the brain synaptosomal, microsomal and myelin fractions. Greater differences were observed in the younger age, where ethanol treatment caused a significant increase of C20: 1 LCB in GM1 ganglioside of synaptosomes and microsomes and in GDla of myelin.

Subcellular localization of ryanodine receptors in rat brain

Subcellular fractions of rat brain were used to determine the subcellular localization of ryanodine receptors. [ 3H]Ryanodine binding in purified cortical, cerebellar and hippocampal synaptosomes was up to 3.6-fold higher compared with mitochondrial fractions. The density of sites ( B max) in hippocampal mossy fiber synaptosomes (249 fmol/mg protein) was 3.7-fold greater than in cortical synaptosomes (67 fmol/mg protein) and binding affinity was approximately 2-fold greater in the former ( K D, 6.1 nM) than the latter ( K D, 3.1 nM). At single sub-saturating concentrations of [ 3H]ryanodine, binding was 1.6-fold higher in mossy fibers compared with total hippocampal synaptosomes. [ 3H]Ryanodine binding sites were distributed similarly in subfractions of cortical synaptosomes and microsomes from discontinuous sucrose density gradients. An enrichment of sites was found in the lightest fractions containing the lowest activities of plasma membrane (5′-nucleotidase) and endoplasmic reticulum (glucose 6-phosphatase) enzyme markers when data for microsomal and synaptosomal subfractions were expressed as activity/binding per mg protein and when data for synaptosomal subfractions were expressed as a percentage of total activity/binding in collected fractions. Thus, ryanodine receptors appear to be concentrated in presynaptic terminals where they may play a major role in neurotransmitter release, and appear to be localized either in a specialized endoplasmic reticulum subcompartment or a distinct subcellular organelle.

The Vo Sector of the V-ATPase, Synaptobrevin, and Synaptophysin Are Associated on Synaptic Vesicles in a Triton X-100-resistant, Freeze-thawing Sensitive, Complex

Anti-synaptobrevin 2 immunoprecipitates obtained from freshly prepared Triton X-100 extracts of rat synaptosomes contained, in addition to synaptophysin, a 10-kDa band, which we identified by peptide sequencing and Western blotting as the c subunit of the vacuolar proton pump (V-ATPase) also called ductin or mediatophore. Ac39 and Ac116, two other transmembrane subunits of the V0 sector of the V-ATPase, were also found by Western blotting to be enriched in the immunoprecipitates. None of these V-ATPase subunits, or synaptophysin, was present in anti-synaptobrevin 2 immunoprecipitates obtained from frozen-thawed Triton X-100 extracts, which were greatly enriched, instead, in SNAP-25 and syntaxin 1. Accordingly, V-ATPase subunit c was found in anti-synaptophysin immunoprecipitates. Thus, the two complexes appear to be mutually exclusive. Subcellular fractionation of rat brain demonstrated that V-ATPase subunit c is localized with synaptobrevin 2 and synaptophysin in synaptic vesicles. The coprecipitation of V-ATPase subunit c with the synaptobrevin-synaptophysin complex suggests that this interaction may play a role in recruiting the proton pump into synaptic vesicles. Freeze-thawing, which involves a mild denaturing step, may produce a conformational change which dissociates the complex and mimics a change which occurs in vivo as a prerequisite to SNARE complex formation.

Stimulus-dependent Phosphorylation of MacMARCKS, a Protein Kinase C Substrate, in Nerve Termini and PC12 Cells

MacMARCKS (also known as myristoylated alanine-rich C kinase substrate (MARCKS)-related protein) is a member of the MARCKS family of protein kinase C substrates, which binds Ca2+/calmodulin in a phosphorylation-dependent manner. Immunoprecipitation demonstrated that MacMARCKS is present in both PC12 cells and in neurons. Upon depolarization of PC12 cells with 60 mM KCl, MacMARCKS phosphorylation increased 4-fold over basal levels in a Ca(2+)-dependent manner. By immunofluorescence microscopy, MacMARCKS was colocalized in PC12 cells to neurite tips with the synaptic vesicle membrane protein synaptophysin and to vesicles in the perinuclear region. Subcellular fractionation demonstrated that MacMARCKS associates tightly with membranes in PC12 cells. In Percoll-purified rat cerebrocortical synaptosomes, depolarization with 60 mM KCl in the presence of exogenous Ca2+ transiently increased MacMARCKS phosphorylation, whereas phorbol ester promoted a sustained increase in MacMARCKS phosphorylation. Subcellular fractionation of rat brain indicated that MacMARCKS was present in both soluble and particulate fractions; particulate MacMARCKS was associated with both small vesicles and highly purified synaptic vesicles. These results are consistent with a role for MacMARCKS in integrating Ca(2+)-calmodulin and protein kinase C-dependent signals in the regulation of neurosecretion.

Subcellular distribution of phosphorylase kinase in rat brain. Association of the enzyme with mitochondria and membranes

The evaluation of glycogen phosphorylase kinase in rat brain subcellular fractions was undertaken in order to get further insight into the association of this kinase with specific neuronal cell compartments. The enzyme was found to be primarily soluble, but considerable latent specific activities were observed in particulate fractions, especially in microsomes, mitochondria and synaptosomes, which could be unmasked by treatment with Triton X-100. The submitochondrial and subsynaptic distribution patterns of phosphorylase kinase revealed high overt activity in the mitochondrial intermembrane space and high latent activities in mitochondrial membranes, and synaptic vesicles, membranes and mitochondria. The Ca 2+-dependency of soluble phosphorylase kinase was similar to that of microsomal enzyme but higher than that of other particulate enzyme forms. Mitochondrial phosphorylase kinase showed a higher pH 6.8:8.2 activity ratio than the soluble and the microsomal enzyme. The rate of inactivation of cytosolic phosphorylase kinase by proteinase K was higher than that of microsomal and mitochondrial enzymes. Antibodies against rabbit skeletal muscle phosphorylase kinase effectively inhibited both cytosolic and microsomal enzyme but failed to significantly affect the kinase activity present in intact mitochondria and intermembrane space. Western blotting with anti-phosphorylase kinase showed that rat brain mitochondria exhibited a significantly lower immunoreactivity compared to soluble cytosol. In conclusion, the presence of phosphorylase kinase activity in a variety of particulate fractions of rat brain suggests a multiplicity of actions of this kinase in neuronal tissues.

Purification and characterization of a fatty acid-activated protein kinase (PKN) from rat testis.

PKN, a novel protein kinase with a catalytic domain homologous to that of the protein kinase C (PKC) family and unique N-terminal leucine-zipper-like sequences, was identified by molecular cloning from a human hippocampus cDNA library [Mukai and Ono (1994) Biochem. Biophys. Res. Commun. 199, 897-904]. Recently we partially purified recombinant PKN from COS7 cells transfected with the cDNA construct encoding human PKN, and demonstrated that the recombinant PKN was activated by unsaturated fatty acids and limited proteolysis [Mukai, Kitagawa, Shibata et al. (1994) Biochem. Biophys. Res. Commun. 204, 348-356]. The present work has focused on the further purification and characterization of PKN from native rat tissue. Immunochemical measurement revealed that PKN was found in every tissue, and was especially abundant in testis, spleen and brain; subcellular fractionation of rat brain showed that half of the PKN was localized in the soluble cytosolic fraction. PKN was purified approx. 8000-fold to apparent homogeneity from the cytosolic fraction of rat testis by DEAE-cellulose chromatography, ammonium sulphate fractionation and chromatography on butyl-Sepharose, heparin-Sepharose, Mono Q and protamine-CH-Sepharose. The enzyme migrates as a band of apparent molecular mass 120 kDa. Using serine-containing peptides based on the pseudosubstrate sequence of PKC-delta as phosphate acceptors, the kinase activity was stimulated several-fold by 40 microM unsaturated fatty acids or by detergents such as 0.04% sodium deoxycholate and 0.004% SDS. In the absence of modifiers, protamine sulphate, myelin basic protein and synthetic peptides based on the pseudosubstrate site of PKCs or ribosomal S6 protein were good substrates for phosphorylation by the kinase. In the presence of 40 microM arachidonic acid the kinase activity of PKN for these phosphate acceptors was increased 2-18-fold. The autophosphorylation activity of purified PKN was partially inhibited by pretreatment with alkaline phosphatase. These properties appear to distinguish PKN from many protein kinases isolated previously.

P145, a major Grb2-binding protein in brain, is co-localized with dynamin in nerve terminals where it undergoes activity-dependent dephosphorylation.

Three major rat brain proteins were recently found to bind the SH3 domains of Grb2: synapsin I, dynamin, and a novel 145-kDa protein (p145) (McPherson, P. S., Czernik, A. J., Chilcote, T. J., Onofri, F., Benfenati, F., Greengard, P., Schlessinger, J., and De Camilli, P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6486-6490). We have now used antibodies raised against p145 which had been purified by Grb2 affinity chromatography and SDS-polyacrylamide gel electrophoresis to characterize this protein. p145 is neuron-specific and co-distributes with dynamin in subcellular fractions of rat brain. Both proteins are partially recovered in soluble and particulate fractions. By immunofluorescence, p145 is closely co-localized with dynamin, which we show here to be highly concentrated in nerve terminals. In contrast to synapsin I, which is highly enriched in a purified synaptic vesicle fraction, both p145 and dynamin are de-enriched in this fraction. However, both proteins are found on membranes which are immunoisolated with antibodies directed against the synaptic vesicle membrane protein synaptophysin, suggesting that dynamin and p145 are localized in part on organelles which represent intermediate stages in the reformation of synapatic vesicles during recycling. Finally, we have determined that p145, like dynamin, is a phosphoprotein which undergoes dephosphorylation in response to nerve terminal depolarization. These findings suggest that p145 participates with dynamin in synaptic vesicle endocytosis and recycling.

Localization of RC3 (neurogranin) in rat brain subcellular fractions

Previous studies have shown that RC3 (neurogranin) is a postsynaptic, protein kinase C (PKC)/calmodulin-binding substrate that accumulates throughout the perikaryal and dendritic cytoplasm and is often closely associated with the postsynaptic density (PSD) in dendritic spines of neostriatal neurons. Here Western immunoblotting studies of rat brain subcellular fractions confirm that RC3 is predominantly a cytosolic protein but is found in lower amounts in membrane-enriched microsomes and synaptosomes. Solubilization of synaptosomes suggests that RC3 may only be loosely associated with the PSD.

Age‐Related Changes in the Ceramide Composition of the Major Gangliosides Present in Rat Brain Subcellular Fractions Enriched in Plasma Membranes of Neuronal and Myelin Origin

Age-related changes of the ceramide composition of gangliosides were studied in the synaptosomal and myelin fractions from rat brain, carrying plasma membranes of neuronal and glial origin, respectively. The five major gangliosides (GM1, GD1a, GD1b, GT1b, and GQ1b) present in these fractions were separated and quantitated by normal-phase HPLC. Each ganglioside was then fractionated by reverse-phase HPLC into the molecular species carrying a single long-chain base (LCB). The largely preponderant LCBs in the synaptosomal and myelin fractions were the C18:1 and C20:1. The content of C20:1 LCB, generally low at 1 month, increased with age in all analyzed gangliosides and in all subcellular fractions and was greater in the "b series" than in the "a series" gangliosides. Remarkably, GM1 was the only ganglioside where the proportion of LCB 20:1 was higher in the synaptosomal fraction than in the myelin fraction. The fatty acid composition of the C18:1 or C20:1 LCB species of the different gangliosides in the synaptosomal and myelin fractions did not undergo appreciable changes with age. Stearic acid was largely predominant in all the gangliosides of the synaptosomal fraction, more in the C18:1 than in the C20:1 LCB species (80-90% vs. 60-70%). The gangliosides of the myelin fraction were characterized by a lower content of 18:0 and a much higher content of 16:0 and 18:1 fatty acids than those of the synaptosomal fraction. Thus, the ceramide composition is different in the gangliosides of neuronal and myelin origin and appears to be subjected to an age-related control.

Antibodies raised against a peptide corresponding to a Gs alpha domain reveal a vimentin-associated protein.

In an attempt to localize the guanine nucleotide binding protein Gs alpha by immunocytochemistry, we synthetized peptides corresponding to several stretches of residues deduced from the published cDNA sequence of Gs alpha and raised antibodies against them. Among the peptides, one corresponding to residues 367-381 elicited antibodies that immunocytochemically detected, at the optical level, what appeared to be vimentin in several cells and tissues. Studies at the ultrastructural level confirmed this observation and also showed weak staining of some areas of plasma membranes of glial and nerve cells. Analysis by Western blots of rat brain subcellular fractions indicated that: (a) the protein stained by the anti-peptide antibodies was associated with the cytoskeleton; and (b) it was not vimentin but a protein of higher molecular weight, 65 KD. We accordingly suggest that the Gs alpha-derived peptide elicited two types of antibodies, one recognizing Gs alpha in fixed tissues, the other recognizing an epitope located in a vimentin-associated protein. This study emphasizes the caution that is needed when conclusions are drawn on the basis of immunocytochemical studies using antipeptide antibodies.

Brain cytochrome P450 and testosterone metabolism by rat brain subcellular fractions: Presence of cytochrome P450 3A immunoreactive protein in rat brain mitochondria

The hydroxylation of testosterone by rat brain subcellular fractions has been studied using an HPLC method with an enhanced resolution for the separation of testosterone and its monohydroxy derivatives. Although the analysis time is longer than that reported for earlier methods, a baseline separation was obtained between all hydroxytestosterones, excepting 6α-hydroxytestosterone and 15β-hydroxytestosterone, which were separated using a second chromatography system. This separation was important as rat brain microsomes metabolized testosterone to 15α-, 6β-, 15β-, 16β-, 2β-, 1β-hydroxytestosterone and androstenedione. Testosterone metabolism was found to be linear with time and protein concentration. The rat brain mitochondrial fraction metabolized testosterone to androstenedione. Small amounts of immunoreactive bands comigrating with purified cytochromes P450j, P450b, and P450p were detected by Western blot analysis in rat brain microsomes, while only an immunoreactive protein related to cytochrome P450p was found in the mitochondrial fractions. Immunoinhibition studies showed that BEA33, a monoclonal antibody to cytochrome P450b and simultaneously recognizing cytochromes P450e and P450a, was able to inhibit the metabolism of testosterone to the 1β-, 15α-, 2β-, and 6α-hydroxylated metabolites, whereas polyclonal anticytochrome P450p did not inhibit the formation of the 6β-hydroxytestosterone by rat brain microsomes. The metabolism of testosterone by rat brain microsomal or mitochondrial fractions was refractory to induction by 3-methylcholanthrene or pregnenolone-16α-carbonitrile. Thus, in the brain multiple isozymes of cytochrome P450 are constitutively expressed in different subcellular fractions, which suggests that brain cytochrome P450 may play an important role in the metabolism of endogenous compounds. The significance and role of cytochrome P450p-related protein in the rat brain mitochondrial fraction are yet to be determined.

Differential coupling of opioid binding sites to guanosine triphosphate binding regulatory proteins in subcellular fractions of rat brain.

In this study, we present evidence for the occurrence of mu, delta, and kappa opioid binding sites in synaptic plasma membranes (SPM) and microsomes of rat brain. Binding to all three opioid classes was inhibited by 5'-guanylylimidodiphosphate (Gpp[NH]p) in SPM, while microsomal sites proved to be insensitive to this GTP analog. Sensitivity was restored upon solubilization of microsomes with digitonin, suggesting that opioid receptors are physically separated from G proteins in this fraction. Modulation of microsomal binding by Na+ and Mn++ was greater than that of SPM. Pertussis toxin-catalyzed adenosine diphosphate (ADP) ribosylation revealed the presence of G proteins with alpha-subunit molecular weights of 40 kDa in both subcellular fractions. Basal low Km GTPase activity in SPM was greater than in microsomes. Etorphine elicited a concentration-dependent stimulation of guanosine triphosphatase (GTPase) activity in SPMs but not in microsomes, indicating functional coupling of opioid receptors to G protein in the former and an uncoupling in the latter. Microsomes from 3-day-old rat brain contained more mu opioid sites and they were more sensitive to Gpp(NH)p inhibition than those in adults. These results are consistent with the hypothesis that opioid binding sites in adult microsomes are internalized and G protein uncoupled, while those in neonates are newly synthesized, coupled receptors.

Protein kinase C and Ca 2+/calmodulin-dependent protein kinase II phosphorylate a novel 58-kDa protein in synaptic vesicles

A monoclonal antibody was made using the spleen cells of a mouse immunized with chick synaptic membranes and designated as mAb 1D12. It immunoprecipitated 25% of the ω-conotoxin binding protein but no dihydropyridine binding protein solubilized from chick brain membranes. By immunoblotting, a polypeptide of 58-kDa was identified as the antigen of this antibody in chick, rat, rabbit and guinea pig brain. Immunohistochemical observation indicated the immunoreactivity of mAb 1D12 to be localized in the synaptic regions of central and peripheral neurons. In peripheral organs, there was additional staining in the distal portions of nerve fibers. Immunoelectron microscopy showed immunoreactivity to be located in synaptic vesicle and presynaptic plasma membranes. In the subcellular fractionation of rat brain, 58-kDa protein was recovered in the fractions of synaptic vesicles and plasma membranes but not soluble proteins. This protein could be extracted from membranes by Triton X-100 but treatment with EDTA, acid, base or high salt failed to have such effect. Solubilized 58-kDa protein of rat brain was purified by immunoaffinity chromatography using mAb 1D12. Both protein kinase C and Ca 2+/calmodulin-dependent protein kinase II (CaM kinase II) phosphorylated purified 58-kDa protein, and maxima of 0.47 and 0.94 mol of phosphates, respectively, were incorporated per mol of 58-kDa protein. 58-kDa protein was not phosphorylated by either cAMP-dependent of cGMP-dependent protein kinase. When present in membranes, it was also phosphorylated by protein kinase C and CaM kinase II. Possible involvement of 58-kDa protein in the protein kinase C and CaM kinase II-mediated regulation of synaptic transmission in central and peripheral neurons is discussed.

Dystrophin in central nervous system: a developmental, regional distribution and subcellular localization study

Dystrophin, the protein encoded by the Duchenne muscular dystrophy gene has been shown to be expressed in central nervous system. In the present study, polyclonal antibodies raised against 3 fusion proteins constructed from different structural domains of dystrophin were used to identify dystrophin in protein extracts from rat and mdx mouse brain. The developmental expression of the protein, its regional distribution in rat brain and its localization in rat brain subcellular fractions were also examined. We found that dystrophin or a ‘dystrophin-related protein’ is expressed in mdx mouse brain. Dystrophin is detectable at very early stages of rat brain development and is expressed in all adult brain regions examined, although quantitative regional differences were found. Subcellular distribution analysis indicates that dystrophin is absent in mitochondrial and synaptic vesicle-enriched fractions but is recovered in the synaptic plasma membrane fraction.

3H]Ryanodine binding sites in rat brain demonstrated by membrane binding and autoradiography

High affinity [ 3H]ryanodine binding sites were characterized in P1 (crude nuclear), P2 (mitochondrial/synaptosomal) and P3 (microsomal) subcellular fractions of rat brain. Binding in each of the fractions was highest at 37°C and pH 8–9, optimal in the presence of 100 μM Ca 2+, 550 μM ATP and 1.0 M KCl, and increased linearly as a function of protein. Saturation analyses revealed a single class of binding sites with mean K D values (nM) of 8.9, 1.6 and 5.7 and B max values (fmol/mg protein) of 122, 69 and 106 for the P1, P2 and P3 fractions, respectively. The levels of [ 3H]ryanodine binding in P1 and P2 fractions of 4 brain regions were fairly uniform while those in P3 fractions were 5-fold greater in cerebral cortex than in the other areas examined. By autoradiography, a high concentration of [ 3H]ryanodine binding sites was seen in the dentate gyrus and CA3 subregions of the hippocampus. The results suggest that [ 3H]ryanodine binding sites, perhaps similar to [ 3H]ryanodine receptors in muscle, are associated with various subcellular structures and are heterogeneously distributed in the CNS.

The possibility of participation of different protein kinases in the regulation of degradation of Glp 6[ 125I]Tyr 8SP 6–11 with different endopeptidases in subcellular fractions of rat brain

1. 1. Nuclear fraction of cortex and synaptosomal fraction of hippocampus of rat brain were preincubated with ATP and different, “second messengers” as well as some “classical” neurotransmitters and then incubated with GIp 6[ 125I]Tyr 8SP 6–11 (JP). 2. 2. It was found that different neurotransmitters and especially “second messengers” might be involved in the activation or inhibition of peptidase I and II. 3. 3. Activation of different protein kinases results in substantial modulation of degradation of JP (SP C-terminal fragment). 4. 4. The data confirm that products of one peptidase might be inhibitors of the second peptidase acting on JP, although the second enzyme might be affected additionally in conditions activating different protein kinase systems.

Histamine increases ornithine decarboxylase activity in different neonatal rat brain subcellular fractions

The subcellular localization of ornithine decarboxylase activity was investigated in control or histamine-treated 6-day-old rat brains. Ornithine decarboxylase activity was located mainly in the cytosolic fraction (75%), whereas significant lower activity was observed in the crude nuclear (7%), crude mitochondrial (15%) and crude microsomal (3%) fractions. Cytosolic and nuclear ornithine decarboxylase activity were increased after treatment with histamine (35 and 400%, respectively). Histamine did not affect ODC activity in crude mitochondrial and crude microsomal fractions. The present findings suggest that the major part of ODC activity in the neonatal rat brain is located in the cytosolic fraction and are the first showing an induction of nuclear ODC activity by a neuromodulator.

A diverse set of developmentally regulated proteoglycans is expressed in the rat central nervous system

Cellular interactions in neural development are influenced by various extracellular proteins, many of which bind glycosaminoglycans or proteoglycans. Precise functions of nervous system proteoglycans remain unknown, in part because neural proteoglycan composition is poorly understood. In this study, 25 putative proteoglycan core proteins were identified in subcellular fractions of rat brain. Levels of many of these varied considerably during development. Membrane-associated proteoglycans included two heparan sulfate proteoglycans (cores of 50 and 59 kd) that are covalently linked to glycosyl-phosphatidylinositol lipid, as well as several that appear to aggregate either with themselves or with copurifying proteins. These data indicate that brain proteoglycans exhibit the abundance, structural diversity, and developmental regulation that would be anticipated for molecules with diverse developmental functions.