EphB3 receptors function as dependence receptors to mediate oligodendrocyte cell death following contusive spinal cord injury.

We demonstrate that EphB3 receptors mediate oligodendrocyte (OL) cell death in the injured spinal cord through dependence receptor mechanism. OLs in the adult spinal cord express EphB3 as well as other members of the Eph receptor family. Spinal cord injury (SCI) is associated with tissue damage, cellular loss and disturbances in EphB3-ephrinB3 protein balance acutely (days) after the initial impact creating an environment for a dependence receptor-mediated cell death to occur. Genetic ablation of EphB3 promotes OL survival associated with increased expression of myelin basic protein and improved locomotor function in mice after SCI. Moreover, administration of its ephrinB3 ligand to the spinal cord after injury also promotes OL survival. Our in vivo findings are supported by in vitro studies showing that ephrinB3 administration promotes the survival of both oligodendroglial progenitor cells and mature OLs cultured under pro-apoptotic conditions. In conclusion, the present study demonstrates a novel dependence receptor role of EphB3 in OL cell death after SCI, and supports further development of ephrinB3-based therapies to promote recovery.

EphrinB3 blocks EphB3 dependence receptor functions to prevent cell death following traumatic brain injury.

Eph receptor tyrosine kinases and their membrane-bound ligands, ephrins, have a variety of roles in the developing and adult central nervous system that require direct cell-cell interactions; including regulating axon path finding, cell proliferation, migration and synaptic plasticity. Recently, we identified a novel pro-survival role for ephrins in the adult subventricular zone, where ephrinB3 blocks Eph-mediated cell death during adult neurogenesis. Here, we examined whether EphB3 mediates cell death in the adult forebrain following traumatic brain injury and whether ephrinB3 infusion could limit this effect. We show that EphB3 co-labels with microtubule-associated protein 2-positive neurons in the adult cortex and is closely associated with ephrinB3 ligand, which is reduced following controlled cortical impact (CCI) injury. In the complete absence of EphB3 (EphB3(-/-)), we observed reduced terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL), and functional improvements in motor deficits after CCI injury as compared with wild-type and ephrinB3(-/-) mice. We also demonstrated that EphB3 exhibits dependence receptor characteristics as it is cleaved by caspases and induces cell death, which is not observed in the presence of ephrinB3. Following trauma, infusion of pre-clustered ephrinB3-Fc molecules (eB3-Fc) into the contralateral ventricle reduced cortical infarct volume and TUNEL staining in the cortex, dentate gyrus and CA3 hippocampus of wild-type and ephrinB3(-/-) mice, but not EphB3(-/-) mice. Similarly, application of eB3-Fc improved motor functions after CCI injury. We conclude that EphB3 mediates cell death in the adult cortex through a novel dependence receptor-mediated cell death mechanism in the injured adult cortex and is attenuated following ephrinB3 stimulation.

Polyomavirus EGFP-pseudocapsids: analysis of model particles for introduction of proteins and peptides into mammalian cells.

A vector for preparation of mouse polyomavirus capsid-like particles for transfer of foreign peptides or proteins into cells was constructed. Model pseudocapsids carrying EGFP fused with the C-terminal part of the VP3 minor protein (EGFP-VLPs) have been prepared and analysed for their ability to be internalised and processed by mouse cells and to activate mouse and human dendritic cells (DC) in vitro. EGFP-VLPs entered mouse epithelial cells, fibroblasts and human and mouse DC efficiently and were processed by both, lysosomes and proteasomes. Surprisingly, they did not induce upregulation of DC co-stimulation molecules or maturation markers in vitro; however, they did induce interleukin 12 secretion.

Caveolae are involved in the trafficking of mouse polyomavirus virions and artificial VP1 pseudocapsids toward cell nuclei.

Electron and confocal microscopy were used to observe the entry and the movement of polyomavirus virions and artificial virus-like particles (VP1 pseudocapsids) in mouse fibroblasts and epithelial cells. No visible differences in adsorption and internalization of virions and VP1 pseudocapsids ("empty" or containing DNA) were observed. Viral particles entered cells internalized in smooth monopinocytic vesicles, often in the proximity of larger, caveola-like invaginations. Both "empty" vesicles derived from caveolae and vesicles containing viral particles were stained with the anti-caveolin-1 antibody, and the two types of vesicles often fused in the cytoplasm. Colocalization of VP1 with caveolin-1 was observed during viral particle movement from the plasma membrane throughout the cytoplasm to the perinuclear area. Empty vesicles and vesicles with viral particles moved predominantly along microfilaments. Particle movement was accompanied by transient disorganization of actin stress fibers. Microfilaments decorated by the VP1 immunofluorescent signal could be seen as concentric curves, apparently along membrane structures that probably represent endoplasmic reticulum. Colocalization of VP1 with tubulin was mostly observed in areas close to the cell nuclei and on mitotic tubulin structures. By 3 h postinfection, a strong signal of the VP1 (but no viral particles) had accumulated in the proximity of nuclei, around the outer nuclear membrane. However, the vast majority of VP1 pseudocapsids did not enter the nuclei.

Regulation of Trk receptors following contusion of the rat spinal cord.

Neurotrophins function through high-affinity tyrosine kinase (Trk) receptors to promote growth and survival of cells in the injured nervous system. To investigate the role of Trk receptors in the adult nervous system, we examined TrkA, TrkB, and TrkC mRNA expression in spinal cord and brain after spinal contusion. At 1 day postinjury, all Trk receptor transcripts were down regulated at and around the site of injury, a situation that persisted through the first week. By 42 days, Trk expression was absent only within the cavity. In addition, truncated TrkB expression was substantially increased in ependymal cells and astrocytes surrounding the lesion cavity of chronically injured spinal cords. Rostral and caudal to the injury site, TrkA, TrkB, and TrkC mRNA expression did not differ from that of uninjured control spinal cords. Furthermore, no changes were observed in TrkB or TrkC expression in the axotomized corticospinal and rubrospinal neurons. These studies suggest that loss of Trk receptors at the injury site may contribute to the early progressive cellular loss in injured spinal cords, while increased presence of truncated TrkB receptors in the chronic injured spinal cord may sequester and restrict BDNF availability to support axonal regeneration and neuronal survival. The persistence of Trk receptors on supraspinal neurons suggests that neurotrophin application can support growth and survival in the acute and chronic injury states.

Production of polyomavirus structural protein VP1 in yeast cells and its interaction with cell structures.

The gene for mouse polyomavirus major structural protein VP1 was expressed in Saccharomyces cerevisiae from the inducible GAL7 promoter. VP1 pseudocapsids were purified from cell lysates. Their subpopulation contained fragments of host DNA, which, in contrast to those of VP1 pseudocapsids produced in insect cells, did not assemble with cellular histones into pseudonucleocores. VP1 pseudocapsids accumulated in the yeast cell nuclei. A strong interaction of VP1 with tubulin fibres of the mitotic spindle was observed. The fibres of spindles were larger in diameter, apparently due to tight VP1 binding. Substantial growth inhibition of yeast cells producing VP1 was observed.

BDNF regulates eating behavior and locomotor activity in mice.

Brain-derived neurotrophic factor (BDNF) was studied initially for its role in sensory neuron development. Ablation of this gene in mice leads to death shortly after birth, and abnormalities have been found in both the peripheral and central nervous systems. BDNF and its tyrosine kinase receptor, TrkB, are expressed in hypothalamic nuclei associated with satiety and locomotor activity. In heterozygous mice, BDNF gene expression is reduced and we find that all heterozygous mice exhibit abnormalities in eating behavior or locomotor activity. We also observe this phenotype in independently derived inbred and hybrid BDNF mutant strains. Infusion with BDNF or NT4/5 can transiently reverse the eating behavior and obesity. Thus, we identify a novel non-neurotrophic function for neurotrophins and indicate a role in behavior that is remarkably sensitive to alterations in BDNF activity.

Loss of brain-derived neurotrophic factor-dependent neural crest-derived sensory neurons in neurotrophin-4 mutant mice.

Peripheral ganglion neurons confer sensory information including touch, pain, temperature, and proprioception. Sensory modality is linked to specific neurotrophin (NTF) requirements. NT-3 supports survival of neurons that differentiate primarily into proprioceptors whereas nerve growth factor and brain-derived neurotrophic factor (BDNF) support subpopulations that transmit nociception and mechanoreception, respectively. We examined sensory neurons of gene-targeted mouse mutants at the NT-4, BDNF, NT-3, and TrkA loci. We show that NT-4 functions early in gangliogenesis, upstream of BDNF. In the absence of NT-4 function, BDNF-dependent, TrkB-expressing neurons fail to appear. The results are consistent with the model that precursor cells intended to become BDNF-dependent mechanoreceptors instead differentiate into NT-3-dependent proprioceptive neurons.

NT4/5 mutant mice have deficiency in gustatory papillae and taste bud formation.

Neurotrophins are key determinants for controlling the survival of peripheral neurons during development. Brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT4/5) exert their action through a common trkB receptor but independently support gustatory sensory neurons. To assess the role of NT4/5 during development, we examined the postnatal development and maintenance of fungiform taste buds in mice carrying a deletion of NT4/5. The absence of NT4/5 results in embryonic deficits in gustatory innervation and a reduced number of fungiform papillae at birth. No degenerative deficits of fungiform papillae were observed for the first 3 weeks of postnatal development. However, these remaining fungiform papillae were smaller in appearance and many did not contain taste pores. By postnatal day 60, there was 63% decrease in the number of fungiform papillae, and remaining papillae were smaller in size or modified into filiform-like spines. These papillae had either no taste bud or a taste bud with a reduced number of taste cells compared to controls. These findings demonstrate that the NT4/5 gene functions in the maintenance of fungiform gustatory papillae and raises the possibility for an earlier role in development.

Mice lacking NT-3, and its receptor TrkC, exhibit profound deficiencies in CNS glial cells.

Neurotrophin-3 (NT-3) and its receptor TrkC are known to be important for neuronal survival. More recently, NT-3 has been implicated as playing a role in oligodendrocyte (OL) proliferation and survival in vitro. Examination of NT-3 and TrkC knockout mice revealed a reduction in NT-3-dependent neurons. To date, no study has examined alterations in glial cell populations in these knockout mice. In this report, we demonstrate a decline in OL progenitor cell numbers within the CNS of NT-3 and TrkC knockout mice. We also observed that immature and mature OL-specific markers were attenuated in the NT-3 and TrkC knockout animals. Deficiencies in other CNS glial cells, including astrocytes and ameboid microglia, were also observed. The subventricular zone (SVZ), a highly proliferative region for progenitor glial cells, was reduced in size. Furthermore, a nuclear-specific stain revealed a decline in the numbers of pyknotic nuclei in and around the SVZ of the knockout mice. These data will support an in vivo NT-3-dependent mechanism for the normal development of CNS glial cells.

Inhibition of dopamine and choline acetyltransferase concentrations in rat CNS neurons by rat alpha 1- and alpha 2-macroglobulins.

Previous studies have implicated human alpha-2-macroglobulin (alpha2M) as a potential regulator of neuronal development and function. Rat alpha-1-macroglobulin (alpha1M) and acute-phase alpha-2-macroglobulin (alpha2M) are murine homologues of human alpha2M. In this report, we tested the effect of intracranially infused serotonin-activated rat alpha1M (5HT-alpha1M) on the concentration of dopamine (DA) in the corpus striatum in vivo and the effect of 5HT-activated rat alpha1M and alpha2M on the choline acetyltransferase (ChAT) activity upon embryonic basal forebrain neurons in culture. The results show that direct infusion of 0.65 nmole rat 5HT-alpha1M into the adult rat corpus striatum produced a consistent attenuation upon striatal DA concentrations. This decrease was particularly prominent at 5-7 days post-infusion. In addition, rat 5HT-alpha1M and rat 5HT-alpha2M, like human 5HT-alpha2M, all significantly inhibited ChAT activity of embryonic rat cerebral cortex neurons. Although normal human alpha2M and rat alpha2M were either marginally or insignificantly inhibitory in this preparation, normal rat alpha1M dose-dependently inhibited ChAT activity. These results demonstrate that monoamine-activated alpha-macroglobulins from rat depress dopaminergic and cholinergic neurotransmitter systems in the CNS, and this suggests a potential regulatory role of these alpha-macroglobulins in neurotransmitter metabolism.

Absence of sensory neurons before target innervation in brain-derived neurotrophic factor-, neurotrophin 3-, and TrkC-deficient embryonic mice.

Gene-targeting experiments of Trk receptors and neurotrophins has confirmed the expectation that embryonic sensory and sympathetic neurons require neurotrophin function for survival. They have further revealed correlation between a specific neurotrophin requirement and eventual sensory modality. We have analyzed embryonic and neonatal mice with mutations in the BDNF, neurotrophin 3 (NT-3), and TrkC genes. Our data confirm an unexpectedly high proportion of sensory neuron losses in NT-3 (>70%), BDNF (>20%), and TrkC (>30%) mutants, which encompass populations thought to be NGF-dependent. Direct comparison of TrkC and NT-3 mutants indicates that only a subset of the NT-3-dependent neurons also requires TrkC. The observed losses in our TrkC mutant, which is null for all proteins encoded by the gene, are more severe than those previously reported for the kinase-negative TrkC mutation, implicating additional and important functions for the truncated receptors. Our data further indicate that mature NGF-requiring neurons undergo precocious and transitory requirements for NT-3 and/or BDNF. We suggest that neurotrophins may function in creating early heterogeneity that would enable ganglia to compensate for diverse modality requirements before the period of naturally occurring death.

Lactation associated with acidophilic pituitary adenoma, pheochromocytoma, and cystic endometrial hyperplasia in two goats.

Two unbred adult female goats were examined for persistent, inappropriate lactation. Prostaglandin F2 alpha treatment was ineffective in relieving the condition. Over 5 months, 1 goat developed evidence of CNS disease; this same goat had persistently high serum prolactin concentrations. At necropsy, both goats had an acidophilic adenoma of the pars distalis, a condition that, to our knowledge, has not previously been reported in goats. In addition, both goats had pheochromocytomas and cystic endometrial hyperplasia, conditions that are rarely reported in small ruminants. The inciting cause of inappropriate lactation in goats can be difficult to determine. Pituitary adenomas should be suspected when treatment with prostaglandins is unsuccessful, signs of CNS disease develop, or persistently high serum prolactin concentrations are detected.

Leptospira interrogans serovar grippotyphosa infection in dogs.

Leptospirosis attributed to infection with serovar grippotyphosa was diagnosed in 11 dogs. In naturally and experimentally infected dogs, a stereotypic serologic response to infection with Leptospira serovar grippotyphosa was detected. Although the highest serum antibody titers developed against serovar grippotyphosa, most dogs also had lower titers against serovars bratislava and pomona. Acute renal failure was evident in 10 dogs. One dog died prior to initiation of treatment; the remaining 10 dogs were treated with antibiotics and fluids. Two dogs were euthanatized, 2 dogs recovered without clinical or biochemical evidence of residual renal dysfunction, and 6 dogs recovered but had varying degrees of renal insufficiency. Hepatic involvement appeared to be a minor component of the disease in these dogs. Our results indicate that Leptospira serovar grippotyphosa infection is an important problem in dogs and should be considered when evaluating a dog with renal failure.

Monoamine-activated alpha 2-macroglobulin inhibits choline acetyltransferase of embryonic basal forebrain neurons and reversal of the inhibition by NGF and BDNF but not NT-3.

Monoamine-activated alpha 2-macroglobulin (alpha 2M) has recently been shown to inhibit the growth and survival of cholinergic neurons of the basal forebrain (Liebl and Koo: J Neurosci Res 35:170-182, 1993). The mechanism of this inhibitory effect is believed to involve the regulation of growth factor activities by alpha 2M. The objectives of this study are to determine whether monoamine-activated alpha 2M can inhibit choline acetyltransferase (ChAT) activity of cholinergic basal forebrain neurons, and whether some common neurotrophins in the CNS can reverse the inhibition. This study demonstrates that both methylamine-activated alpha 2M (MA-alpha 2M) and serotonin-activated alpha 2M (5HT-alpha 2M) can dose-dependently suppress the expression of normal basal levels of ChAT activity in embryonic rat basal forebrain cells in vitro, while normal alpha 2M has little or no effect. As little as 0.35 microM monoamine-activated alpha 2M can suppress the ChAT activity, whereas either nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF), but not neurotrophin-3 (NT-3), stimulates ChAT expression of these cells. The addition of either NGF or BDNF to the alpha 2M-suppressed cells can increase ChAT activity back to its normal levels, while NT-3 can not. These results demonstrate that (1) monoamine-activated alpha 2M is a potent non-cytotoxic inhibitor of the ChAT activity in cholinergic basal forebrain neurons, and (2) NGF and BDNF are capable of not only stimulating the ChAT activity but can also specifically reverse the alpha 2M inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonin-activated alpha 2-macroglobulin inhibits neurite outgrowth and survival of embryonic sensory and cerebral cortical neurons.

Methylamine-modified alpha-2-macroglobulin (MA-alpha 2M) has been recently shown to inhibit the biological activity of beta-nerve growth factor (NGF) in promoting neurite outgrowth by embryonic dorsal root ganglia in culture (Koo PH, Liebl DJ, J Neurosci Res 31:678-692, 1992). The objectives of this study are to determine whether alpha 2M can also be modified by larger aromatic biogenic amines such as 5-hydroxytryptamine (5HT; serotonin), the nature of interaction between NGF and 5HT-modified alpha-2-M (5HT-alpha 2M), and the effect of 5HT-alpha 2M on the neurite extension and the growth of embryonic sensory and cholinergic neurons in 2 disparate animal species (chicken and rats). This study demonstrates that each mole of alpha 2M can combine with 15.2 +/- 1.8 moles of 5HT, in which up to 4.5 +/- 0.4 moles may be covalently bonded. As determined by gel filtration and polyacrylamide gel electrophoresis studies, both 5HT-alpha 2M and normal alpha 2M combine noncovalently with NGF, but 5HT-alpha 2M by comparison can combine with NGF somewhat more effectively. In contrast to normal alpha 2M, 5HT-alpha 2M at concentrations greater than about 0.17 microM exerts a dose-dependent inhibition on the NGF-stimulated neurite outgrowth by embryonic dorsal root ganglia and dissociated cells in culture, and the inhibitory effect can be overcome by higher NGF concentrations. Both 5HT-alpha 2M and MA-alpha 2M at 1.0 microM inhibit neurite extension by embryonic rat cerebral cortical cells and seriously damage these cells in culture. Such neurite-inhibitory activity, however, can only be partially blocked by extraneously added NGF alone. Normal alpha 2M (at 1.0 microM) and 5HT (at 188 microM), on the other hand, under the identical conditions produce very little or no effect on the normal cellular and axonal growth of these cells. We conclude that alpha 2M can potentially interact with nucleophilic monoamines, including neurotransmitters, to form inhibitory complexes which may inhibit/regulate NGF-promoted neurite outgrowth and neuronal survival. In addition, higher concentrations of such complexes can seriously damage certain CNS neurons which do not depend solely on NGF for survival.

Comparative binding of neurotrophins (NT-3, CNTF and NGF) and various cytokines to alpha 2-macroglobulin.

All the nine common cytokines in this study (including NT-3, IGF-1, CNTF and TGF-alpha) bind noncovalently, yet with different specificities and to different degrees, with both normal alpha 2-macroglobulins (alpha 2M) and monoamine-modified alpha 2M. The binding of NGF is by far the most efficient and is least affected by cationic proteins. The binding of NT-3 is slightly affected by cationic proteins but is completely blocked by NGF. The binding of TGF-alpha, TGF-beta 1, CNTF, and IL-6 is severely blocked by cationic proteins/NGF. We conclude that NGF and NT-3 appear to bind specifically in significant quantities to the same alpha 2M sites; but the other cytokines by comparison bind minimally, and primarily or entirely use nonspecific molecular interactions in their binding to alpha 2M.

Follicular cystic ovaries and cystic endometrial hyperplasia in a bitch.

Treatment of cystic ovaries in a bitch was unsuccessful with human chorionic gonadotropin, but aspiration of the follicles at laparotomy was successful. Follicular aspiration is described, with special emphasis on preventing bursal adhesions. Aspiration of persistent follicles is a simple treatment for cystic ovarian disease that is nonresponsive to parenterally administered hormones. The persistent estrus associated with cystic follicles is a primary cause of cystic endometrial hyperplasia (CEH) and severe cases of CEH are associated with infertility. Rapid intervention (surgical or hormonal) will limit the severity of CEH and, therefore, decrease infertility in breeding bitches.

Inhibition of nerve growth factor-stimulated neurite outgrowth by methylamine-modified alpha 2-macroglobulin.

alpha 2-Macroglobulin (alpha 2M) is a rather ubiquitous protein in extracellular spaces of mammals. It is an inhibitor of endopeptidases, can be modified by aliphatic amines, and combines with a number of hormones/cytokines such as beta-nerve growth factor (NGF) [Koo PH, Stach RW (1989): J Neurosci Res 22:247]. The objective of this study is to compare the NGF-binding properties of methylamine-modified human alpha 2M (MA-alpha 2M) versus normal alpha 2M and their effects on the biological activity of NGF and neurite extension by embryonic chicken dorsal root ganglia. As determined by gel filtration, polyacrylamide gel electrophoresis, and equilibrium binding studies, these two forms of alpha 2M are similar in their binding affinities, with MA-alpha 2M binding about twice as much NGF as normal alpha 2M. Both normal alpha 2M and MA-alpha 2M combine noncovalently with NGF, and prior modification of alpha 2M is unnecessary for the binding to occur. In contrast to normal alpha 2M, MA-alpha 2M potently inhibits the biological activity of NGF and exerts a dose-dependent inhibition on the NGF-stimulated neurite outgrowth by embryonic chicken dorsal root ganglia in culture. The inhibitory effect of MA-alpha 2M can be overcome by higher NGF concentrations, but is irreversible at lower NGF concentrations. Trypsin-modified alpha 2M combines covalently and noncovalently with more NGF than normal alpha 2M but has very little neurite inhibitory activity. The mechanism of inhibition by MA-alpha 2M is discussed.