Murine synaptosomal lipid raft protein and lipid composition are altered by expression of human apoE 3 and 4 and by increasing age.

Apolipoprotein E (apoE) 4 and aging are risk factors for Alzheimer's disease (AD). Mice expressing human apoE4 and aged wild-type mice show a similarity in the transbilayer distribution of cholesterol in synaptic plasma membranes (SPMs) but differ markedly compared with apoE3 mice and young mice. The largest changes in cholesterol distribution were observed in the SPM exofacial leaflet where there was a doubling of cholesterol. Lipid rafts are thought to be associated with the exofacial leaflet, and we proposed that lipid raft protein and lipid composition would be associated with apoE genotype and age. Lipid rafts were isolated from synaptosomes of different age groups (2, 12, 24 months) of mice expressing human apoE3 and apoE4. Lipid raft markers, alkaline phosphatase (ALP), flotillin-1, cholesterol and sphingomyelin (SM) were examined. Lipid rafts of young apoE4 mice were more similar to older mice as compared with young apoE3 mice in reductions in alkaline phosphatase activity and flotillin-1 abundance. Lipid raft cholesterol and sphingomyelin levels were not significantly different between the young apoE3 and apoE4 mice but cholesterol levels of lipid rafts did increase with age in both genotypes. Results of the present study demonstrate that the two risk factors for Alzheimer's disease, apoE4 genotype and increasing age have similar effects on brain lipid raft protein markers and these findings support the notion that the transbilayer distribution of cholesterol is associated with lipid raft function.

Starvation induces tau hyperphosphorylation in mouse brain: implications for Alzheimer's disease.

Hyperphosphorylated tau is the major component of paired helical filaments in neurofibrillary tangles found in Alzheimer's disease brains, and tau hyperphosphorylation is thought to be a critical event in the pathogenesis of this disease. The objective of this study was to reproduce tau hyperphosphorylation in an animal model by inducing hypoglycemia. Food deprivation of mice for 1 to 3 days progressively enhanced tau hyperphosphorylation in the hippocampus, to a lesser extent in the cerebral cortex, but the effect was least in the cerebellum, in correspondence with the regional selectivity of tauopathy in Alzheimer's disease. This hyperphosphorylation was reversible by refeeding for 1 day. We discuss possible mechanisms of this phenomenon, and propose the starved mouse as a simple model to study in vivo tau phosphorylation and dephosphorylation which are altered in Alzheimer's disease.

Immunohistochemical study of subclasses of olfactory nerve fibers and their projections to the olfactory bulb in the rabbit.

The organization of the olfactory nerve projection to the olfactory bulb was studied immunohistochemically in the rabbit by using monoclonal antibodies (MAbs). Out of 42 MAbs raised against the homogenate of the olfactory bulb, two types of MAbs that strongly stained the olfactory nerve fibers (axons of olfactory receptor cells) were selected and their staining patterns were analysed in detail. MAbs of one type (represented by MAb R2D5) specifically labeled all olfactory receptor cells in the nasal epithelium and all olfactory nerve fibers and their terminal portions in the bulb. The other type of MAbs (represented by MAb R4B12) recognized only a subgroup of olfactory nerve fibers. The R4B12-positive fibers were distributed over the ventrolateral areas but not in the dorsomedial areas of the epithelium. Similarly in the bulb, the R4B12-positive fibers terminated in the glomeruli in the ventrolateral and the caudal regions but not in the dorsomedial region. These results demonstrate for the first time the cellular heterogeneity among olfactory receptor neurons at the molecular level. The segregated distribution of the subtypes of olfactory receptor cell axons both in the epithelium and the bulb indicates a defined topographical organization of the olfactory nerve projection. These results also suggest a functional division between dorsomedial and ventrolateral areas both in the epithelium and the bulb.

Monoclonal antibody immunohistochemistry of adult rabbit olfactory structures.

Immunohistochemical staining patterns of two monoclonal antibodies produced against the rabbit olfactory bulb were studied in adult rabbit olfactory structures. One monoclonal antibody 112D5 (monoclonal antibody 2D5) stained all of the olfactory receptor cells, whereas the other 114G12 (monoclonal antibody 4G12) stained the upper two-thirds to three-fourths of the receptor cell layer. The negative region in the receptor cell layer was designated the deep compartment. Neither monoclonal antibody stained the supporting cells, basal cells, or Bowman's glands. Monoclonal antibody 2D5 stained the olfactory nerve layer and glomeruli in the olfactory bulb, whereas monoclonal antibody 4G12 stained the whole of the olfactory bulb, particularly the glomeruli and the mitral cells. The piriform cortex was unstained by monoclonal antibody 2D5 whereas the highest immunoreaction to monoclonal antibody 4G12 was in layer Ia. Immunoblot analysis revealed that the molecular weight values of monoclonal antibody 4G12 antigens in the olfactory epithelium were approx. 26,000. Thus, monoclonal antibody 4G12, specific to neurons, recognized an epitope different from the olfactory marker protein specific to the olfactory receptor neurons.

Projections of two subclasses of vomeronasal nerve fibers to the accessory olfactory bulb in the rabbit.

The organization of the projections of subclasses of vomeronasal nerve fibers to the accessory olfactory bulb was analysed using monoclonal antibodies generated against a homogenate of the rabbit olfactory bulb. Monoclonal antibody R2D5 labels all the somata of vomeronasal receptor cells in the vomeronasal organ as well as all their axons (vomeronasal nerve fibers). Another monoclonal antibody (R4B12), which has been shown to selectively bind and thus identify a subclass of olfactory nerve fibers, also labels a subclass of vomeronasal nerve fibers. The R4B12-positive subclass of vomeronasal nerve fibers project to the glomeruli in the rostrolateral part of the accessory olfactory bulb. The third monoclonal antibody (R5A10) recognizes a complementary subclass of vomeronasal nerve fibers projecting to the glomeruli in the caudomedial part of the accessory bulb. In contrast to the clearly segregated terminations in the accessory bulb, the two subclasses of vomeronasal nerve fibers are intermingled with each other in the vomeronasal nerve bundles. Retrograde labeling of vomeronasal receptor cell somata following injection of horseradish peroxidase within the rostrolateral (R4B12-positive) part of the accessory bulb indicates that vomeronasal receptor cells of this subtype are widely distributed in the vomeronasal sensory epithelium. These results demonstrate the heterogeneity of vomeronasal receptor cells and the specificity of projections arising from subclasses of vomeronasal nerve fibers to the accessory olfactory bulb.

A monoclonal antibody identifies a novel epitope surrounding a subpopulation of the mammalian central neurons.

A monoclonal antibody was obtained by immunizing mice with an extract of monkey brain. The monoclonal antibody 473 stained a small subpopulation of neurons in various regions of monkey and rat central nervous system. The perimeters of neuronal somata and the proximal parts of dendrites bound the antibody. Electron microscopic analysis showed that the immunoreactivity was associated with the outer surface of the cell. The immunoreactivity in the rat cerebral cortex appeared gradually during the second four weeks after birth. The antibody stained fetal cartilages but otherwise was specific to the nervous system. Experiments on the stability of the immunoreactivity to enzymatic and chemical treatments of the sections suggest that the antigen molecule is of proteoglycan nature.

Electron microscopic localization and expression on cultured cells of a novel surface-associated molecule in the rat central nervous system.

Subcellular localization and in vitro expression of a newly raised monoclonal antibody 374 antigen, which outlines a subset of neurons in various regions of the rat CNS, were studied. Electron microscopy revealed that the immunoreactivity is associated with the outer surfaces of neuronal subsets, the surfaces of glial cell processes facing such neurons and less frequently, with the Golgi apparatus and inner cell surfaces. The monoclonal antibody also stained part of the cytoplasm of a population of dendrites. In order to test whether the antigen originates from neurons, we examined the expression of the antigen in cultured cortical cells. Double immunofluorescence staining with neuron-specific enolase and glial fibrillary acidic protein revealed that the antigen is expressed on a subset of neuronal cells and to a lesser extent on glial cells. The antigen is considered to be expressed on the outer surface because the cells were stained by the monoclonal antibody irrespective of whether they were alive or fixed. The monoclonal antibody 374 antigen may be a novel neuronal surface molecule which plays a role in neuron-neuron or glial-neuron interactions in the CNS.

Identification of Drosophila indirect flight muscle myofibrillar proteins by means of two-dimensional electrophoresis.

When proteins of whole Drosophila thorax were analyzed by two-dimensional gel electrophoresis, 186 spots were detected by protein staining with Coomassie brilliant blue R-250. Two methods were developed to identify proteins which exist in indirect flight muscle (IFM) and its myofibrils. 1) A whole fly was freeze-dried in a dry ice-acetone mixture, and indirect flight muscle fibers were cleanly dissected out from the thorax. The muscle cells and the rest of the thorax were analyzed separately. The muscle contained 146 polypeptides, of which 12 were not detected elsewhere. 2) Flies were frozen in liquid nitrogen and shaken vigorously so that their thoraces broke off from heads and abdomens. The thoraces were separated from the rest by sieving and centrifugation. After homogenization of the thorax, myofibrils were prepared by centrifugation in a discontinuous sucrose density gradient. The myofibril fraction contained at least 20 proteins. There were two types of actin (II and III), myosin heavy chain, tropomyosin and paramyosin. Nine of the other myofibrillar proteins were specific to this muscle.

Developmental changes of chick cerebellar cortex revealed by monoclonal antibodies.

Immunohistochemistry studies of the embryonic and newly hatched chick cerebellum were performed with 13 monoclonal antibodies (MAbs) raised against the embryonic chick optic nerve and a MAb which binds to cell nuclei. Neural MAbs differentially stained Purkinje cells, the external granular layer, molecular layer, internal granular layer, climbing fibers, basket cell axons, Bergmann glia and Ramón y Cajal's ansiform fibers. At the different developmental stages each component responded to MAbs differently. For example, staining of Purkinje cells with MAbs 23C10, 82E10 and 94C2 appeared on day 11 of incubation and disappeared sequentially after day 18. These results reveal molecular heterogeneity not only in cerebellar neurons but also at various developmental stages.

Monoclonal antibodies demonstrate regional specificity in the spinal funiculi of the chick embryo.

To explore the extent and significance of chemical diversity of neurons, monoclonal antibodies were generated that bind to the chick embryonic central nervous system with varying degrees of regional specificity. Mice were immunized against a homogenate of optic nerves from 8-day chick embryos. Antibody was screened by an indirect fluorescence immunohistochemical method using frozen sections of embryonic neural tissues. From 3 fusion experiments, 58 lines of hybrydoma were cloned. Twenty-three monoclonal antibodies were studied in detail, and here reported, for their characteristic staining patterns in the spinal cord of 6-day embryos. The majority of the antibodies bound preferentially to different subregions of spinal funiculi, in which glial elements are still undeveloped. At least 6 subregions could be distinguished in the funiculi, probably corresponding to the developing spinal tracts. For 8 of the antibodies, immunoreactive polypeptides were identified in an electrophoretic analysis.

Glycosaminoglycan-related epitopes surrounding different subsets of mammalian central neurons.

Among a panel of monoclonal antibodies generated against monkey brain tissue, a class of antibodies was found to produce perineuronal staining of small subsets of mammalian central neurons. Three antibodies (MAbs 473, 376, 528) we report here define two different, though partially overlapping, neuronal subsets in the monkey neocortex. All 3 antibodies stain in addition certain chondrocytes. The neural immunoreactivities were lost, and the chondral immunoreactivities either lost or enhanced, after treatment of the sections with chondroitinase ABC. Independently, 3 other antibodies (MAbs 1B5, 9A2, 3B3) with established specificity to glycosaminoglycan epitopes also produced perineuronal staining of a related subset of central neurons. Immunoblot experiments with two of the antibodies revealed bands of high molecular weight. These findings indicate that certain glycosaminoglycans occur surrounding mammalian central neurons, and suggest that different neuronal subsets are associated with different combinations of proteoglycan epitopes.

Apolipoprotein E is found in astrocytes but not in microglia in the normal mouse brain.

Apolipoprotein E (apoE) is a known risk factor for Alzheimer's disease, but neither its roles in the pathogenesis nor its exact physiological functions in the brain is known. In order to study the apoE protein in the brains of normal mouse and transgenic mouse models of neurodegeneration, hamster monoclonal antibodies (MAbs) specific to mouse apoE were generated. N- and C-terminal fragments of mouse apoE protein were produced in E. coli as fusion proteins and used to immunize Armenian hamsters. Specificity of the antibodies was established by immunoblotting against sera and brain homogenates of wild type and apoE-deficient mice. MAb 884F11 was found most suitable for immunohistochemistry on 4% PFA-fixed brain tissues. The strongly positive structure in the normal brain was the astrocytes as identified by simultaneous staining for GFAP with lesser and regionally variable diffuse staining of the neuropil. GFAP-positive cells were variable in their content of apoE. ApoE immunoreactivity in the hippocampus and neocortex did not coincide with the tomato lectin binding, indicating that this apolipoprotein is not detectable in the microglial cells of the normal adult mouse brain.

A monoclonal antibody specific for a subpopulation of retinal bipolar cells in the frog and other vertebrates.

One monoclonal antibody 115A10 (MAb 5A10) specifically stained a subpopulation of retinal bipolar cells in various vertebrates. In the bullfrog retina, MAb 5A10 stained the large bipolar cells, but not the small bipolar cells. Labeling of the living bipolar cells was observed in isolated cell preparation of the frog retina. MAb 5A10 can serve as a cell-specific marker of the bipolar cells.

Different subsets of CNS neurons express different glycosaminoglycan epitopes on large perineuronal proteoglycans.

Proteoglycans are known to occur in the central nervous tissue, but their function is not well understood. We made a biochemical study of four perineuronal antigens on different subsets of rat brain neurons and found that three antigens recognized by antibodies against glycosaminoglycan epitopes were large proteoglycans. Molecular masses estimated by immunoblotting were 700 kDa for 473, 376 and 1B5 antigens and 600 kDa for the 374 antigen. Reactivities of the antigens on immunoblots to monoclonal antibodies (MAbs) 473 and 376 were lost, and that to MAb 1B5 uncovered, after chondroitinase ABC treatment as in the brain sections. The elution profiles from ion-exchange and gel-filtration column chromatography of 473, 376 and 1B5 antigens were quite similar, but those of 374 antigen were slightly different. The immunoaffinity-purified 473 antigen migrated at 700 kDa on sodium dodecylsulfate gel electrophoresis, and chondroitinase ABC treatment decreased the molecular mass to 600 kDa. The 473 antigen was recognized by MAbs 376 and 1B5 although these MAbs also reacted with 473-negative 700 kDa molecules. These results suggest that neuronal subset-specific glycosylation may occur on the 700 kDa proteoglycans, and that the glycosaminoglycan structures may be involved in the pericellular diversity of CNS neurons.

Subclasses of olfactory receptor cells and their segregated central projections demonstrated by a monoclonal antibody.

A library of monoclonal antibodies (MAbs) was generated against a homogenate of the rabbit olfactory bulb. One of them immunohistochemically distinguished a subgroup of olfactory nerves. Both in the olfactory bulb and the epithelium, this MAb labeled most olfactory receptor axons in the lateral but only a small fraction in the medial portion. These findings demonstrate a molecular heterogeneity among olfactory receptor cells and suggest a functional division between the lateral and the medial portions of the epithelium and the bulb.

Immunochemical identification of subgroups of vomeronasal nerve fibers and their segregated terminations in the accessory olfactory bulb.

Vomeronasal nerve (VNN) fibers and their terminations in the accessory olfactory bulb (AOB) were studied immunohistochemically using 3 monoclonal antibodies (MAbs). One MAb (R2D5) labeled all VNN fibers. Another MAb (R4B12) labeled a subgroup of the VNN fibers which terminated in the rostrolateral glomeruli in the AOB. The third MAb (R5A10) recognized a complementary subgroup of the VNN which terminated in the caudomedial portion of the AOB. These results for the first time show occurrence of subtypes in the VNN axons with segregated terminations in the AOB.

Four synaptic vesicle-specific proteins: identification by monoclonal antibodies and distribution in the nervous tissue and the adrenal medulla.

Synaptic vesicles from the guinea-pig cerebrum were isolated and administered to mice for the production of monoclonal antibodies (MAb). Four vesicle-associated proteins in the guinea-pig nervous tissue were specifically and differentially recognized by MAbs thus obtained. These proteins had molecular weights of 30,000, 36,000, 38,000 and 65,000 Da and were named SVPs (synaptic vesicle proteins) 30, 36, 38 and 65, respectively. Immunohistochemistry demonstrated that all SVPs were localized in the synaptic regions throughout the central nervous system and in the adrenal medulla. Nerve terminals in skeletal muscle, smooth muscle and sympathetic ganglion contained SVPs 36 and 38. Immunoelectron microscopy of the cerebellar cortex confirmed the localization of SVPs in the synaptic vesicles and the adjacent membranes of the presynaptic nerve terminals. Fractionation of the cerebral tissue and treatment with various agents showed that SVPs were localized in the synaptic vesicles and the synaptic plasma membrane and that SVPs were integrated within the membrane and liberated only after solubilization of the membrane.

Identification of a synaptic vesicle-specific 38,000-dalton protein by monoclonal antibodies.

Synaptic vesicles were purified from the guinea pig cerebrum by sucrose density gradient centrifugation, and monoclonal antibodies (MAbs) were produced against this vesicle fraction. Seven MAbs (171B5, 171E8, 174D12, 174H11, 177A2, 177H11 and 178D4) recognized a novel acidic protein of about 38,000 daltons which was specific to synaptic vesicles. In immunofluorescence microscopy, the staining pattern of these MAbs corresponded to the distribution of the synapses in the guinea pig central nervous system. These MAbs appeared to stain all synaptic regions, irrespective of their synaptic function or type of neurotransmitters. MAb 171B5 and 174H11 stained the rat, rabbit and bovine synapses similarly to the guinea pig. Two other MAbs (171E8 and 177H11) stained other mammals weakly but the remaining 3 MAbs reacted only with the guinea pig. In immunoelectron microscopy of both the cerebellar tissue and isolated vesicle fraction, these MAbs selectively labeled the synaptic vesicles but not other structures. Immunoblot analysis was performed on electrophoretically separated proteins in vesicle fraction and brain homogenate. All of 7 MAbs reacted with a band at a molecular weight of about 38,000 from the guinea pig. Isoelectric focussing disclosed that this protein was acidic (pI 4.5-5).

Cochlear histopathology of the mutant bustling mouse, BUS/Idr.

The inner ear of mutant bustling mice, BUS/Idr, was examined histopathologically. LM examinations revealed an age-dependent degeneration of the auditory organ of Corti in BUS homozygotes, but not heterozygotes. Cochlear base-to-apex gradient in severity of the degeneration was noted. First signs of degeneration were found in the outer hair cells of the cochlear basal turn at about 3 weeks of age, followed by degeneration of the spiral ganglion cells which occurred slowly. As examined by SEM, stereociliary derangements of both the inner and outer hair cells were apparent in homozygotes as early as after 10 days. No normal arrays of stereocilia were found in homozygotes examined at 10 days through 6 months. The results of immunohistochemical examinations suggest that the sensory cells of the Corti's organ of homozygotes are structurally once normally innervated. No significant difference was found in the expression of protooncogene c-mos in the CNS between BUS homozygotes and control mice. We propose that BUS mice be categorized as a member of the so-called "waltzer-shaker" mutants group.