Elevated glial brain-derived neurotrophic factor in Parkinson's diseased nigra.

We show the cellular distribution of immunoreactivity (IR) for brain-derived-neurotrophic-factor (BDNF), neurotrophin-3 (NT-3) and tyrosine kinase receptors TRKB and TRKC in idiopathic Parkinson's disease (IPD) and controls at post-mortem. In both groups, nigral neurons, astrocytes, ramified and amoeboid microglia expressed all antigens. Caudate-putamen neurons expressed all antigens except BDNF with similar distribution between groups. In IPD nigra, increased numbers of BDNF-IR and, less frequently, NT-3-IR ramified glia surrounded fragmented neurons, accompanied by BDNF-IR in surrounding neuropil. Amoeboid microglia were abundant only in IPD nigral scars. In IPD, glia might up-regulate neurotrophins in response to signals released from failing nigral neurons.

Endogenous brain-derived neurotrophic factor and neurotrophin-3 antagonistically regulate survival of axotomized corticospinal neurons in vivo.

Neuronal growth factors regulate the survival of neurons by their survival and death-promoting activity on distinct populations of neurons. The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) promote neuronal survival via tyrosine kinase (Trk) receptors, whereas NGF and BDNF can also induce apoptosis in developing neurons through p75(NTR) receptors in the absence of their respective Trk receptors. Using mutant mice and inactivation of neurotrophins and their receptors with antibodies in rats, we show that endogenous NT-3 induces death of adult BDNF-dependent, axotomized corticospinal neurons (CSNs). When NT-3 is neutralized, the neurons survive even without BDNF, suggesting complete antagonism. Whereas virtually all unlesioned and axotomized CSNs express both trkB and trkC mRNA, p75 is barely detectable in unlesioned CSNs but strongly upregulated in axotomized CSNs by day 3 after lesion, the time point when cell death occurs. Blocking either cortical TrkC or p75(NTR) receptors alone prevents death, indicating that the opposing actions of NT-3 and BDNF require their respective Trk receptors, but induction of death depends on p75(NTR) cosignaling. The results show that neuronal survival can be regulated antagonistically by neurotrophins and that neurotrophins can induce neuronal death in the adult mammalian CNS. We further present evidence that signaling of tyrosine kinase receptors of the trk family can be crucially involved in the promotion of neuronal death in vivo.

Chicken macrophage stimulating protein is a ligand of the receptor protein-tyrosine kinase Sea.

Affinity chromatography, employing the extracellular domain of the Sea receptor, was used to enrich Sea-binding proteins from chicken serum. One isolated protein bound both a Sea-immunoglobulin fusion protein and an antisera raised against murine macrophage stimulating protein. Amino-terminal sequencing of the dual-reactive protein yielded sequences which were identical to the predicted alpha and beta subunits of chicken macrophage stimulating protein. The partially purified chicken macrophage stimulating protein caused autophosphorylation of the Sea receptor. Previous work showed that recombinant expression of fully activatible human or mouse macrophage stimulating protein required a specific Cys to Ala substitution (Wahl, R. C., Costigan, V. J., Batac, J. P., Chen, K., Cam, L., Courchesne, P. L., Patterson, S. D. Zhang, K., and Pacifici, R. E. (1997) J. Biol. Chem. 272, 1-4). Therefore, we expressed both the wild type and the specific Cys to Ala form of chicken macrophage stimulating protein as recombinant proteins. After proteolytic activation, only conditioned media from COS cells transfected with the C665A chicken macrophage stimulating protein, but not from wild type chicken macrophage-stimulating protein, or control vector, was detected by the Sea-immunoglobulin fusion protein in Western blotting experiments. Conditioned media containing the C665A chicken macrophage-stimulating protein readily caused Sea phosphorylation, while conditioned media containing the wild type chicken macrophage-stimulating protein was only effective at inducing receptor phosphorylation at high concentrations. In addition to receptor phosphorylation, the C665A chicken macrophage-stimulating protein induced phosphorylation of Shc, Erk1, and Erk 2. We conclude that macrophage-stimulating protein is a ligand of the Sea receptor protein-tyrosine kinase.

Biochemical, biophysical, and pharmacological characterization of bacterially expressed human agouti-related protein.

The agouti-related protein gene (Agrp) is a novel gene implicated in the control of feeding behavior. The hypothalamic expression of Agrp is regulated by leptin, and overexpression of Agrp in transgenic animals results in obesity and diabetes. By analogy with the known actions of agouti, these data suggest a role for the Agrp gene product in the regulation of melanocortin receptors expressed in the central nervous system. The availability of recombinant, highly purified protein is required to fully address this potential interaction. A nearly full-length form of AGRP (MKd5-AGRP) was expressed in the cytosolic or soluble fraction of Escherichia coli and appeared as large intermolecular disulfide-bonded aggregates. Following oxidation, refolding, and purification, this protein was soluble, and eluted as a single symmetric peak on RP-HPLC. Circular dichroism studies indicated that the purified protein contains primarily random coil and beta-sheet secondary structure. Sedimentation velocity studies at neutral pH demonstrated that MKd5-AGRP is monomeric at low micromolar concentrations. Mobility shifts observed using SDS-PAGE under reducing and nonreducing conditions for bacterially expressed and mammalian expressed AGRP were identical, an indication of a similar disulfide structure. The purification to homogeneity of a second, truncated form of AGRP (Md65-AGRP) which was expressed in the insoluble or inclusion body fraction is also described. Both forms act as competitive antagonists of alpha-melanocyte stimulating hormone (alpha-MSH) at melanocortin-3 (MC-3) and melanocortin-4 receptors (MC-4). The demonstration that AGRP is an endogenous antagonist with respect to these receptors is a unique mechanism within the central nervous system, and has important implications in the control of feeding.

Human leptin receptor. Determination of disulfide structure and N-glycosylation sites of the extracellular domain.

The leptin receptor (OB-R) is a member of the class I cytokine receptor family and mediates the weight regulatory effects of its ligand through interaction with cytoplasmic kinases. The extracellular domain of this receptor is comprised of two immunoglobulin-like and cytokine-receptor homology domains each and type III fibronectin domains. The extracellular domain of human leptin receptor was expressed in and purified from Chinese hamster ovary cells and was found to contain extensive N-glycosylation (approximately 36% of the total protein). The purified protein had a molecular weight of approximately 145,000 and exhibited ligand binding ability as evidenced by formation of ligand-receptor complex, followed by chemical cross-linking. The determined disulfide motif of the soluble leptin receptor contained several distinct cystine knots as well as 10 free cysteines. The N-glycosylation analysis revealed that Asn624 of the WSXWS motif (residues 622-626) within the C-terminal cytokine receptor homology domain was glycosylated, indicating that this region is solvent-exposed. On the other hand, the N-terminal WSXWS motif was not glycosylated.

Neural membrane protein 35 (NMP35): a novel member of a gene family which is highly expressed in the adult nervous system.

We have identified and isolated a cDNA that codes for the novel protein NMP35 (neural membrane protein 35) using RNA arbitrarily primed PCR (RAP-PCR) for differential display in the developing rat sciatic nerve. NMP35 is predominantly found in the adult nervous system where both transcripts and protein are strongly upregulated during postnatal development. In situ hybridization studies show that NMP35 mRNA is widely distributed in the brain and spinal cord with a neuronal expression pattern. Database comparisons reveal that NMP35 shares significant homologies with the rat glutamate-binding protein (GBP), the Drosophila NMDARA1, and two orphan C. elegans genes. Comparative analysis of NMP35 and GBP expression indicates that they are similarly regulated during development and display regionally overlapping cellular patterns. We conclude that NMP35 and GBP are members of a gene family which is likely to play a role in the development and the maintenance of the nervous system.

Expression of growth factors and their receptors in the postnatal rat cochlea.

RT-PCR was used to assay for growth factors and receptors from seven different protein families in cochlea tissues of the juvenile rat. There was a broad representation of the growth factor families in all the cochlea tissues examined, though the organ of Corti and stria vascularis expressed a greater variety than the spiral ganglion. This broad expression suggests that a variety of known growth factors play significant roles in the development, maintenance, and repair of the inner ear. The results of this survey serve as a basis for the design of future in vitro experiments that will address the ability of growth factors to protect hair cells from damage and to evoke a repair-regeneration response by injured hair cells.

Cloning and characterization of a specific receptor for mouse oncostatin M.

Oncostatin M (OSM) is a member of a family of cytokines that includes ciliary neurotrophic factor, interleukin-6, interleukin-11, cardiotrophin-1, and leukemia inhibitory factor (LIF). The receptors for these cytokines consist of a common signaling subunit, gp130, to which other subunits are added to modify ligand specificity. We report here the isolation and characterization of a cDNA encoding a subunit of the mouse OSM receptor. In NIH 3T3 cells (which endogenously express gp130, LIF receptor beta [LIFRbeta], and the protein product, c12, of the cDNA described here), mouse LIF, human LIF, and human OSM signaled through receptors containing the LIFRbeta and gp130 but not through the mouse OSM receptor. Mouse OSM, however, signaled only through a c12-gp130 complex; it did not use the LIF receptor. Binding studies demonstrated that mouse OSM associated directly with either the c12 protein or gp130. These data highlight the species-specific differences in receptor utilization and signal transduction between mouse and human OSM. In mouse cells, only mouse OSM is capable of activating the mouse OSM receptor; human OSM instead activates the LIF receptor. Therefore, these data suggest that all previous studies with human OSM in mouse systems did not elucidate the biology of OSM but, rather, reflected the biological actions of LIF.

Interactions between brain-derived neurotrophic factor and the TRKB receptor. Identification of two ligand binding domains in soluble TRKB by affinity separation and chemical cross-linking.

The extracellular domain of the human neurotrophin TRKB receptor expressed in Chinese hamster ovary cells is a highly glycosylated protein, possessing binding ability for brain-derived neurotrophic factor (BDNF). Two distinct ligand binding domains of TRKB were isolated from proteolytic digests of the receptor by affinity separation on immobilized BDNF. One of these domains consists of amino acid residues 103-181 and contains both the third leucine-rich motif and the second cysteine cluster domain. The second domain is close to the second immunoglobulin-like domain (amino acid residues 342-394). Each of these two domains can bind BDNF independently. Disulfide linkages present in the first domain are necessary for BDNF binding, probably because of preservation of the native conformation. To study the second domain in greater detail, a truncated form of TRKB containing the second immunoglobulin-like domain (residues 248-398) was expressed in Escherichia coli. This domain was cross-linked to BDNF through a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide coupling reaction. Several synthetic peptides corresponding to amino acid residues 343-379 were able to bind immobilized BDNF. Amino acid substitution and cross-linking analysis indicated that amino acids Phe347, Asp354, and Tyr361 are intimately involved in BDNF binding. These results, obtained from a variety of experimental techniques, highlight the importance of two distinct regions of the extracellular domain of the TRKB receptor in binding BDNF.

Neurotrophins and time: different roles for TrkB signaling in hippocampal long-term potentiation.

We examined the role of TrkB ligands in hippocampal long-term potentiation (LTP) using function-blocking TrkB antiserum (Ab) and Trk-IgG fusion proteins. Incubation of hippocampal slices with TrkB Ab had no effect on basal synaptic transmission, short-term plasticity, or LTP induced by several trains of tetanic stimulation. The TrkB Ab-treated slices, however, showed significant deficits in LTP induced by either theta-burst stimulation (TBS) or "pairing." Slices exposed to the same number of inducing stimuli, delivered either as TBS or as a single 100 Hz epoch, only exhibited TrkB-sensitive LTP when TBS was used, indicating that the temporal pattern of stimulation determines the neurotrophin dependence. The late phase of LTP (2-3 hr) was also significantly impaired in slices pretreated with TrkB Ab or a TrkB-IgG. The application of a TrkB-IgG 30 min after LTP induction caused previously potentiated synaptic transmission to return to baseline levels, indicating that TrkB ligands are required to maintain LTP for up to 1 hr after induction. Taken together, these results indicate that both the temporal patterns of synaptic activity and the different temporal phases of synaptic enhancement are important in determining the neurotrophin dependence of plasticity in the hippocampus.

BDNF/NT4-5 receptor TrkB and cadherin participate in cell-cell adhesion.

The neurotrophin receptor TrkB plays a key role in promoting cell survival and differentiation in the nervous system. Two adhesive motifs in the extracellular domain of TrkB have been proposed based on its predicted secondary structure. To investigate the potential adhesive function of trkB, a full length trkB cDNA was stably transfected into NIH 3T3 cells and TrkB-expressing clones isolated. Transfectant clones producing different levels of TrkB protein were subjected to a homotypic aggregation assay. Results showed that parental cells were non-adhesive during the assay while TrkB-expressing cells displayed varying degrees of aggregation depending on the amount of TrkB protein expressed. The observed adhesion was Ca(2+)-, Mg(2+)-, and temperature-dependent, characteristics shared by the cadherin family of adhesion molecules. The transfected cell lines also expressed cadherin in proportion to TrkB expression and both molecules were required for cell adhesion. Double immunofluorescence staining studies showed that TrkB was colocalized with cadherin and catenin at cell-cell contact sites. Whether TrkB and cadherin mediate adhesion separately or synergistically remains to be determined.

Expression of DA11, a neuronal-injury-induced fatty acid binding protein, coincides with axon growth and neuronal differentiation during central nervous system development.

DA11 is the first fatty acid binding protein (FABP) for which gene expression has been shown to be upregulated following neuronal injury in the adult peripheral nervous system. To understand better the potential regulatory role(s) of this unique FABP in axonal growth and neuronal differentiation, we undertook a temporal and spatial study of DA11 gene expression in the developing rat central nervous system (CNS). Transient upregulation of DA11 mRNA and protein levels in CNS tissues were quantified by Northern blot hybridization and Western immunoblot analyses at different developmental ages. Homogenates of embryonic and neonatal cerebral cortex, cerebellum, brain stem, and hippocampal tissues contained 100-fold more DA11 mRNA and protein than corresponding adult tissues. Significant increase in DA11 mRNA was observed as early as embryonic day (E) 14 in cerebral cortex and cerebellum and E19 in brain stem and hippocampus. Postnatal levels of DA11 remained elevated through postnatal day (P) 10 in cerebral cortex, P14 in brain stem and hippocampus, and P20 in cerebellum. Localization of DA11-like immunoreactivity to specific CNS tissues, cell types, and intracellular compartments at P9 revealed a spatial pattern of neuronal expression different than that reported for other FABPs. DA11 protein was detected in the nucleus, cytoplasm, axons, and dendrites of differentiating neurons in cerebral cortex, hippocampus, cerebellum, brain stem, spinal cord, and olfactory bulb. The strong association of DA11 gene expression with development throughout the CNS suggests that this unique FABP plays an important role in axonal growth and neuronal differentiation in many different neuronal populations.

Expression of brain-derived neurotrophic factor protein in the adult rat central nervous system.

We have generated and characterized a multi-functional polyclonal anti-brain-derived neurotrophic factor antibody. Western blot analysis, dorsal root ganglion neurite outgrowth and dorsal root ganglion neuron survival assays showed that this antibody specifically recognized brain-derived neurotrophic factor and not the other neurotrophins. Furthermore, it was capable of blocking the functional effects of brain-derived neurotrophic factor. Using this antibody, we examined the expression of brain-derived neurotrophic factor in adult rat brains by immunohistochemistry. We found distinct brain-derived neurotrophic factor immunoreactivity in several structures of the brain. These included the neocortex, piriform cortex, amygdaloid complex, hippocampal formation, claustrum, some thalamic and hypothalamic nuclei, the substantia nigra and some brainstem structures. In contrast to brain-derived neurotrophic factor messenger RNA expression, brain-derived neurotrophic factor immunoreactivity was also found in the lateral septum, bed nucleus of the stria teminalis, medial preoptic nucleus, olivery pretectal nucleus, lateral paragigantocellular nucleus and the dorsal horn of the spinal cord. In normal adult rat brains, there was little or no staining in the CA1 region or the granule cell layer of the dentate gyrus of the hippocampus. However, kainate treatments greatly increased brain-derived neurotrophic factor immunoreactivity in the pyramidal cells of the CA1 region, as well as in the dentate gyrus, CA2 and CA3 hippocampal regions. We present evidence for both the subcellular localization and anterograde transport of endogenous brain-derived neurotrophic factor in the central nervous system. The detection of brain-derived neurotrophic factor protein in several discrete regions of the adult brain, and brain-derived neurotrophic factor's dramatic up-regulation following kainate treatment, strongly supports a role of brain-derived neurotrophic factor in the maintenance of adult neurons and synapses. Since several populations of neurons lost during neurodegenerative diseases synthesize brain-derived neurotrophic factor protein, modulation of brain-derived neurotrophic factor levels may be clinically beneficial. The antibody described in this paper will be helpful in determining more precisely the functional activities of brain-derived neurotrophic factor in the adult.

Immunocytochemical localization of TrkB in the central nervous system of the adult rat.

The TrkB family of transmembrane proteins serve as receptors for brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-4/5, and possibly NT-3, three members of the neurotrophin family of neurotrophic factors. In order to understand the potential roles played by these receptors, we have examined the distribution of the TrkB receptor proteins in the adult rat brain by using immunohistochemistry. Several different antisera, directed against either synthetic peptides corresponding to different regions of TrkB or a recombinant fusion protein comprising part of the extracellular domain, were generated. Each of these antisera was directed to epitopes found on all known TrkB isoforms (both the tyrosine kinase-possessing isoform and the truncated kinase-lacking isoforms). In addition, a commercially available antibody to the intracellular domain of TrkB was also used. Widespread and distinct staining was observed on the surface of neuronal cell bodies, axons, and dendrites in many structures, including the cerebral cortex, hippocampus, dentate gyrus, striatum, septal nuclei, substantia nigra, cerebellar Purkinje cells, brainstem and spinal motor neurons, and brainstem sensory nuclei. Staining was also observed in the pia matter, on a subpopulation of ependymal cells lining the cerebral ventricle wall, and other nonneuronal cells. The expression pattern of TrkB receptor protein suggests that TrkB plays a broad role in the central nervous system. In addition, the detection of TrkB immunoreactivity on cell bodies and dendrites is consistent with recent models suggesting that neurotrophins may be derived from presynaptic and/or autocrine sources in addition to the classical postsynaptic target.

Changing patterns of expression and subcellular localization of TrkB in the developing visual system.

Neurotrophins play important roles in the survival, differentiation, and maintenance of CNS neurons. To begin to investigate specific roles for these factors in the mammalian visual system, we have examined the cellular localization of the neurotrophin receptor trkB within the developing cerebral cortex and thalamus of the ferret using extracellular domain-specific antibodies. At prenatal ages (gestation is 41 d), trkB-immunostained fibers were observed in the internal capsule and as two distinct fascicles within the intermediate zone of the cerebral cortex. The staining of these fiber tracts declined with increasing age, whereas soma and dendrite staining of cortical neurons was first evident in early postnatal life and increased during subsequent development. Staining of subplate neurons [by prenatal day 5 (P5)] was followed by staining of cortical layer 5 neurons (at P10). By P31, trkB immunoreactivity was particularly prominent in layers 3 and 5 but was absent from subplate neurons. Staining included cells, especially pyramidal neurons, in all cortical layers by P45, and this pattern was maintained into adulthood. The optic tract and fibers within the lateral geniculate nucleus (LGN) were also strongly trkB immunoreactive at prenatal ages. Cellular staining of a subset of LGN neurons, those within the C-layers and perigeniculate nucleus, was apparent by P10 and maintained until P45, when the adult pattern of highly trkB-immunoreactive neurons in all layers of the LGN first appeared. The pattern of trkB immunoreactivity suggests that specific subsets of cortical and thalamic neurons may respond to neurotrophins such as brain-derived neurotrophic factor and/or NT-4/5 at discrete developmental times and locations. The appearance of trkB on axon fibers early in development and then on cell bodies and dendritic processes later is consistent with roles for both long-range and local, including autocrine and/or paracrine, delivery of neurotrophins in cell survival and maturation.

Identification and characterization of a cDNA and the structural gene encoding the mouse epithelial membrane protein-1.

The PMP22/EMP/MP20 gene family includes four closely related proteins, peripheral myelin protein-22 (PMP22), epithelial membrane protein-1 (EMP-1), epithelial membrane protein-2 (EMP-2), and epithelial membrane protein-3 (EMP-3), which share amino acid identities ranging from 33 to 43%. In addition, the lens-specific membrane protein MP20 represents a more distant relative. Functionally, this family of proteins is likely to play important roles in the control of cell proliferation, cell differentiation, and cell death. In particular, mutations affecting the PMP22 gene are responsible for various hereditary peripheral neuropathies in humans and mice. We report the isolation and characterization of a mouse EMP-1 cDNA and the corresponding emp-1 gene. Mouse EMP-1 displays 93% amino acid identity to rat EMP-1 and 39% identity to mouse PMP22. The cDNA-predicted EMP-1 protein contains four putative membrane-associated domains and can be N-linked glycosylated in vitro. EMP-1 is encoded by a single-copy gene with the positions of introns exactly conserved between emp-1 and PMP22, corroborating the hypothesis that both genes belong to the same family. Computer-predicted structural domains of EMP-1 are partially mirrored by the exon/intron structure of emp-1. Most interestingly, exon 4, which covers the potential second transmembrane domain, a small intracellular loop, and half of the third transmembrane domain, encodes the most highly conserved regions between the EMP-1 and PMP22 proteins and is also remarkably conserved in the MP20 gene, indicating some shared functional significance for this module in the PMP22/EMP/MP20 family.

Comparison of neurotrophin regulation of human and rat neuropeptide Y (NPY) neurons: induction of NPY production in aggregate cultures derived from rat but not from human fetal brains.

Previous studies established that brain-derived neurotrophic factor (BDNF) induces neuropeptide Y (NPY) production and accumulation of NPY-mRNA in cultures of rat fetal brain tissues. In this study, we addressed the question: Are cultured human NPY neurons regulated by BDNF and/or by another member of the neurotrophin (NT) family of growth factors? Using aggregate cultures derived from human fetal cortical hemispheres, we assessed the effect of BDNF on NPY production varying the following experimental conditions: fetal and culture age; medium composition (with and without serum), dose and duration of exposure to BDNF, and neurotrophin species tested (BDNF, NT-4/5, NT-3 or NGF). Under none of these conditions did BDNF, NT-4/5, NT-3 or NGF induce an increase in NPY production. This was in contrast to forskolin + phorbol 12 myristate 13-acetate (PMA) which were highly effective in inducing NPY production, verifying that expression of NPY is a regulated process in these cultures. None of these neurotrophins enhanced the response to forskolin + PMA. By comparison, using aggregate cultures derived from rat fetal cortices, BDNF and NT-4/5 were equipotent in inducing NPY production but NT-3 and NGF were essentially ineffective. Moreover, the effects of BDNF or NT-4/5 and forskolin + PMA on NPY production were additive, indicating the involvement of distinct intracellular signalling pathways. Western blot analyses of human- and rat-derived aggregates indicated the presence of full-length Trk receptors which are tyrosine-phosphorylated in response to either BDNF, NT-4/5 or NT-3. Primary cultures of astrocytes (rat as well as human) were devoid of a functional TrkB receptor, strongly suggesting a neuronal expression of TrkB in the aggregates. Thus, a functional TrkB receptor is expressed by both the human and rat aggregates, but only the rat aggregates responded to BDNF or NT-4/5. These results are consistent with a difference in a post TrkB-receptor event(s) mediating BDNF action in the cultured human and rat fetal NPY neurons.

Fatty acid binding protein is induced in neurons of the dorsal root ganglia after peripheral nerve injury.

Peripheral nerve trauma induces the expression of genes presumed to be involved in the process of nerve degeneration and repair. In the present study, an in vivo paradigm was employed to identify molecules which may have important roles in these processes. A cDNA library was constructed with RNA extracted from rat dorsal root ganglia (DRG) 3 days after a sciatic nerve crush. After differential hybridization to this library, several cDNAs were identified that encoded mRNAs that were upregulated in the DRG ipsilateral to the crush injury, as opposed to the contralateral or naive DRG. Approximately 0.15% of all the clones screened were found to be induced. This report presents the types of induced sequences identified and characterizes one of them, DA11. The 0.7 kb DA11 full length cDNA clone contains a 405 nucleotide open reading frame that encodes a putative protein of 15.2 kDa (135 amino acid residues) and is a member of the family of fatty acid binding proteins (FABP). The DA11 protein differs by one amino acid residue from the sequence of the C-FAPB protein and by eight residues from the sequence of mal1, proteins found in rat and mouse skin, respectively. Northern and Western blot analyses showed that the DA11 mRNA and protein were induced in the injured DRG. Furthermore, studies using antibodies generated against DA11 found that the DA11-like immunoreactivity was more pronounced in the nuclei of neurons located in the DRG ipsilateral to the sciatic cut than those located in the contralateral DRG. The induction of DA11 mRNA and protein in DRG neurons suggests, for the first time, the involvement of a neuronal FABP in the process of degeneration and repair in the nervous system.

Epithelial membrane protein-1, peripheral myelin protein 22, and lens membrane protein 20 define a novel gene family.

Peripheral myelin protein 22 (PMP22) is expressed in many tissues but mainly by Schwann cells as a component of compact myelin of the peripheral nervous system (PNS). Mutations affecting PMP22 are associated with hereditary motor and sensory neuropathies. Although these phenotypes are restricted to the PNS, PMP22 is thought to play a dual role in myelin formation and in cell proliferation. We describe the cloning and characterization of epithelial membrane protein-1 (EMP-1), a putative four-transmembrane protein of 160 amino acids with 40% amino acid identity to PMP22. EMP-1 and PMP22 are co-expressed in most tissues but with differences in relative expression levels. EMP-1 is most prominently found in the gastrointestinal tract, skin, lung, and brain but not in liver. In the corpus gastricum, EMP-1 protein can be detected in epithelial cells of the gastric pit and isthmus of the gastric gland in a pattern consistent with plasma membrane association. EMP-1 and PMP22 mRNA levels are inversely regulated in the degenerating rat sciatic nerve after injury and by growth arrest in NIH 3T3 fibroblasts. The discovery of EMP-1 as the second member of a novel gene family led to the identification of the lens-specific membrane protein 20 (MP20) as a third but distant relative. The proteins of this family are likely to serve similar functions possibly related to cell proliferation and differentiation in a variety of cell types.

Purification and identification of brain-derived neurotrophic factor from human serum.

Brain-derived neurotrophic factor (BDNF), a 27-kDa noncovalently linked homodimer with subunits of approximately 13.5 kDa as viewed by SDS-PAGE, is thought to be primarily produced in the central nervous system. We report here the isolation of BDNF from pooled normal human sera, using a two-step purification process followed by SDS-PAGE, transfer to a polyvinylidene difluoride membrane, and subsequent identification of the protein by sequence analysis of the appropriate band(s) from the membrane. The level of BDNF in pooled human sera was estimated to be approximately 15 ng/ml as determined by an enzyme-linked immunosorbant assay. The average for six individuals was 18.9 +/- 5.7 ng/ml. There is an approximately 200-fold increase in the levels of BDNF in serum relative to plasma. Results from experiments using differential centrifugation suggest that the source of this increase is due to release from platelets. The presence of high levels of BDNF in serum suggests a role for this neurotrophin either in nerve repair at sites of injured tissue or in nonneuronal functions.