Gerald A. Schwarting

Peptide growth factor control of olfactory neurogenesis and neuron survival in vitro: Roles of EGF and TGF-βs

The olfactory epithelium (OE) is unique in the mammalian nervous system as a site of continual neurogenesis. Though many studies have described this process in vivo and olfactory neurogenesis can be demonstrated in vitro, the specific factors that modulate this process have not been defined. Noting the common ectodermal origin and structural similarity between the OE and epidermis, peptide factors known to modulate epidermal differentiation were tested in OE cultures. Our results demonstrate that EGF acts as a mitogen for the basal cells that give rise to olfactory neurons and that transforming growth factor-beta s (TGF-beta s) promote neurogenesis. Using a neutralizing antibody, we show that it is likely that the endogenous neurogenic factor is TGF-beta 2, or a very closely related factor.

Sulfated glucuronic acid-containing glycoconjugates are temporally and spatially regulated antigens in the developing mammalian nervous system☆

Monoclonal antibody 4F4, which was raised against a cell suspension of embryonic rat forebrain, reacts with acidic glycolipids and several high-molecular-weight glycoproteins in rodent brain. The major reactive glycolipid is maximally expressed at Embryonic Day 15 (E15) and is no longer detectable at Postnatal Day 14 (P14) in the rat. 4F4 antibody reacts with a glucuronic acid- and sulfate-containing lipid isolated from human sciatic nerve as well as with lipids from mouse and rat embryonic brain tissue. Although the glycolipid disappears postnatally, the immunoreactive glycoproteins continue to be expressed in brain until adulthood. Both sciatic nerve and embryonic brain glycolipids are hydrolyzed by glucuronidase/sulfatase treatment but are insensitive to all other glycosidases tested. In addition, the observed 4F4 reactivity with extracted glycolipids, glycoproteins, and tissue sections of embryonic brain is identical to the reactivity demonstrated by HNK-1 antibodies. Immunocytochemical studies in developing brain showed stage-specific distribution of this carbohydrate antigen. At E10 in the mouse, immunoreactivity is associated with the mantle layer of the neural tube. At E15 in the cortex, the most intense staining is associated with the molecular layer and the subplate, and weaker staining is seen in the intermediate zone and cortical plate, suggesting that the antigen is highly concentrated on postmigratory cells in the embryonic nervous system.

Sulfoglucuronyl-Neolacto Series of Glycolipids in Peripheral Nerves Reacting with HNK-1 Antibody

Abstract: Novel sulfoglucuronyl‐neolacto series of glycolipids were detected in peripheral nerves of various species by TLC followed by immunostaining with HNK‐1 antibody. The major antigenic glycolipid, sulfoglucuronylneolactote‐traosylceramide, previously described in human nerves, was shown to be also present in the sciatic nerves of various species including rodents. A second slower migrating antigenic glycolipid present in the sciatic nerves of human and dog was isolated and purified. It was characterized by chemical and enzymatic degradation, sugar analysis after permethylation, and gas liquid chromatography‐mass spectrometry techniques as well as by fast atom bombardment‐mass spectrometry, as 3‐sulfogIucuronylneolactohexaosylceramide. During postnatal development of the rat sciatic and trigeminal nerves the concentration of these antigenic glycolipids increased with age.

Gonadotropin-Releasing Hormone Containing Neurons and Olfactory Fibers During Development: From Lamprey to Mammals

Gonadotropin releasing-hormone (GnRH) regulates the hypothalamo-pituitary-gonadal axis in all vertebrates. The vast majority of GnRH neurons are thought to be derived from progenitor cells in medial olfactory placodes. Several antibodies and lectins that recognize cell surface carbohydrates have been useful for delineating the migratory pathway from the olfactory placodes and vomeronasal organ, through the nasal compartment, and across the cribriform plate into the brain. In rats, alpha-galactosyl-linked glycoconjugates (immunoreactive with the CC2 monoclonal antibody) are expressed on fibers along the GnRH migration pathway and approximately 10% of the GnRH neuronal population. In lamprey, the alpha-galactosyl binding lectin, Grifonia simplicifolia-I (GS-1), identifies cells and fibers of the developing olfactory system. In contrast to the CC2 immunoreactive GnRH neurons in rats, the GS-1 does not label a subpopulation of presumptive GnRH neurons in lamprey. Results from these and other experiments suggest that GnRH neurons in developing lamprey do not originate within the olfactory placode, but rather within proliferative zones of the diencephalon. However, the overlap of olfactory- and GnRH-containing fibers from prolarval stages to metamorphosis, suggest that olfactory stimuli may play a major role in the regulation of GnRH secretion in lamprey throughout life. By contrast, olfactory fibers are directly relevant to the migration of GnRH neurons from the olfactory placodes in mammalian species. Primary interactions between olfactory fibers and GnRH neurons are likely transient in mammals, and so in later life olfactory modulation of GnRH secretion is likely to be indirect.

A unique neuronal glycolipid defines rostrocaudal compartmentalization in the accessory olfactory system of rats

A monoclonal antibody, CC6, reacts with a complex glycolipid whose expression in the rat is restricted to the olfactory system. Structural analysis reveals that the glycolipid contains two alpha-fucose branches; one each at internal and external beta-galactose residues. Immunocytochemistry demonstrates in rat embryos that CC6 glycolipid expression is restricted to a subset of neurons in the vomeronasal organ (VNO) and their corresponding axon projections in the caudal accessory olfactory bulb (AOB). This pattern of expression in the accessory olfactory system is the converse of the pattern revealed by a previously characterized antiglycolipid antibody that reacts with VNO neurons projecting to the rostral AOB. The CC6-reactive glycolipid is also expressed on a subset of neurons in the main olfactory epithelium. Postnatally, axons from these CC6 positive sensory neurons converge to form a limited number of axon bundles running longitudinally through the nerve layer of the main olfactory bulb. These data provide further evidence that groups of vomeronasal and olfactory neurons expressing unique surface molecules project axons that terminate in selected targets in the AOB and OB, respectively.

A monoclonal antibody, WCC4, recognizes a developmentally regulated ganglioside containing α-galactose and α-fucose present in the rat nervous system

Abstract A monoclonal antibody, WCC4, raised against PC12 cells, recognizes a ganglioside which is present in low concentrations in the postnatal rat nervous system. The antigen is also present in the adrenal and kidney, as determined immunohistochemically, but is not detectable in liver or spleen. A neutral glycosphingolipid is also immunoreactive. In the present report, the chemical characterization of this ganglioside, isolated from PC12 cells, and the anatomical distribution of the antigens recognized by the WCC4 antibody are described. By enzymatic cleavage of terminal saccharide moieties, the ganglioside is identified as α-galactosyl, (α-fucosyl) G MI . The ganglioside increases in concentration postnatally to day 35 (P35) and is present in a slightly diminished concentration in the adult. Immunohistochemical studies revealed that this glycolipid is also present on neuronal cell soma throughout the cerebrum, cerebellum and spinal cord. It is expressed in highest concentration in the molecular layer of the dentate gyrus and is also present in the olfactory bulb, the molecular layer of the hippocampus, the piriform cortex, the olfactory tubercle and the entorhinal cortex. The dentate molecular layer receives most of its innervation from neurons in the entorhinal cortex, and gangliosides are known to have an effect on plasticity following entorhinal cortical lesions. Therefore, the WCC4 antibody should prove to be a useful tool for the study of the role of endogenous gangliosides in this region of the nervous system.

Differential laminin isoform expression in the developing rat olfactory system

Members of the laminin family influence mammalian cells in a variety of ways, mediating adhesion, proliferation, migration, and growth of neuronal processes. Specific laminin domains act through a number of cellular interaction sites to mediate these activities. In the developing olfactory system, axons grow from the olfactory epithelium to synaptic sites in the olfactory bulb a matrix rich in laminins and known mediators of laminin-axon interactions include integrins and a galectin-1/glycoconjugate adhesion system. Using biochemistry, immunocytochemistry, and in situ hybridization, we identified alpha 2, alpha 3, beta 1, beta 2 and gamma 1 laminin isoforms in the late embryonic and neonatal rat olfactory system. However, alpha 1-containing laminin could not be detected in association with olfactory neurons. Immunocytochemistry revealed that beta 2 laminin is preferentially expressed in the ventral and lateral nerve layer of the olfactory bulb and in the main olfactory axon tracks, but is undetectable in the accessory system during embryonic and early postnatal development. In contrast, beta 1 and gamma 1 laminins are evenly distributed throughout the olfactory bulb and in both the main and accessory olfactory axon tracks. The differential localization of laminin chains in vivo is likely to have functional significance for the development and maintenance of the olfactory system.

Production and Purification of Antibody to Bovine White Matter Proteolipid Apoprotein

Circulating antibody to the bovine white matter proteolipid apoprotein was detected in rabbits 1 month after a single injection of the water‐soluble form of the apoprotein. By double immunodiffusion, the antiserum reacted specifically with the delipidated proteolipid apoprotein and the crude proteolipid fraction containing complex lipids; after exposure of the proteolipid apoprotein to sodium dodecyl sulfate (SDS), no reactivity was observed. The antiserum did not react with other myelin components, i.e., basic protein, cerebroside or GM1 ganglioside, nor was there reactivity with non‐neural proteolipids. The anti‐apoprotein antibody was purified by affinity chromatography. The antibody‐antigen interaction is apparently very hydrophobic, since elution of the antibody from the affinity column requires buffer containing 0.5% Triton X‐100‐4 M‐urea.

Differential hormonal modulation of brain antigens recognized by the AB-2 monoclonal antibody

The expression of monoclonal antibody AB-2 immunoreactivity is age- and sex-dependent in radial glia of developing rat hypothalamus and is regulated by prenatal exposure to gonadal steroids. In the present study, several proteins were recognized by AB-2 and were distributed selectively in subcellular fractions from neonatal hypothalamus (HYP), remaining forebrain (FB), and brainstem regions. Immunoblots revealed polypeptide bands in 3 major molecular weight classes: one at approximately 195 kDa in the cytosolic compartment; and two doublets at 220 kDa and 340 kDa in both microsomal and crude mitochondrial membrane fractions. The 220 kDa and 340 kDa doublets were also Triton-insoluble, suggesting a cytoskeletal association. The 195 kDa-AB-2-immunoreactive band was present in both Triton-soluble and insoluble fractions. AB-2 also recognized several acidic glycolipids extracted from postnatal rat brain regions on immunoblots following high performance thin layer chromatography. One of the bands from postnatal rat brain extracts migrated similarly to purified bovine brain sulfatide, which was also immunoreactive with AB-2. AB-2 immunoreactivity with proteins, polar lipids, and sulfatide suggests that the epitope is a carbohydrate present in multiple cellular compartments. AB-2 recognized the same molecular bands in males and females. Testosterone treatment selectively decreased the level of the 195 kDa AB-2-immunoreactive polypeptide. The 195 kDa AB-2-immunoreactive polypeptide possibly acts in radial glia in the determination of sexually dimorphic neurons in the preoptic area/hypothalamus.

Characterization of SSEA-1 glycolipids from the brain of a patient with fucosidosis.

Neutral glycolipids from the brain of a patient with Fucosidosis were analyzed and two complex glycolipids containing five and eight sugars were isolated from the cortical grey matter. These two glycolipids reacted with antibodies recognizing the SSEA-1 [Lex(X)] carbohydrate determinant. SSEA-1 glycolipids are normally expressed in human embryonic brain but are found in only small amounts in postnatal human brain. The accumulation of the two SSEA-1 glycolipids in Fucosidosis brain thus represents a defect which affects the normal developmentally regulated decrease in postnatal, expression of these glycolipids, and may be a contributing factor in the abnormal brain development associated with the disease. Chemical characterization of the two isolated glycolipids by gas chromatographic and mass spectrometric analyses has identified the two glycolipids as lacto-N-fucopentaosylceramide (III) and difucosyl-neolactonorhexaosylceramide.