Rae Nishi

Purification and characterization of a trophic factor for embryonic peripheral neurons: Comparison with fibroblast growth factors

The validation of NGF as a physiologically important neurotrophic factor has led to intense efforts to identify novel polypeptide growth factors for neurons. We report here the details of a greater than 80,000-fold purification of a neurotrophic molecule, referred to as growth-promoting activity (GPA), from chicken sciatic nerves. The final product of the purification migrated as a protein band of 21.5 kd, its apparent pI was approximately 4.8, and the ED50 of the most active preparation was approximately 10 pg/ml. Amino acid sequence of a proteolytic digestion fragment of GPA revealed homology with the recently published sequences for rabbit and rat sciatic nerve CNTF. Thus this molecule may be the chicken form of CNTF. Analysis of the specificity of action of GPA showed that, in addition to E8 ciliary ganglion neurons, the factor was able to support short-term survival of E8 dorsal root ganglion and E12 sympathetic neurons. This range of specificities of biological action was also seen with both acidic and basic FGF in the presence of heparin. The biological activity of GPA differed from that of FGF in that it was not potentiated by heparin and did not stimulate mitogenesis in chick fibroblasts.

Temporal modulation of cytokine expression following focal cerebral ischemia in mice.

There is increasing evidence that the inflammatory response plays an important role in CNS ischemia. The murine model of focal ischemia, however, remains incompletely characterized. In this study we examined expression of several cytokines and the vascular adhesion molecule E-selectin, in order to characterize the molecular events following stroke in the C57BL/6J mouse. Using a multi-probe RNAse protection assay (RPA), mRNA for 19 cytokines was analyzed following permanent and transient occlusion of the middle cerebral artery in mice. In addition, samples from the same mice were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) to evaluate E-selectin mRNA expression levels. Several cytokine mRNAs showed a similar expression pattern in both permanent and transient CNS ischemia while others showed a temporal expression pattern that was dependent on the type of stroke. For both models, mRNA levels of TNFalpha rose early (4 h) followed by IL-6 (10-18 h) and a comparatively late increase (96 h) in TGFbeta1. IL-1alpha, IL-1beta and IL-1ra levels showed a model dependent shift in temporal expression. Reperfusion appeared to delay the induction of these cytokines. Temporal changes in cytokine mRNA expression in the mouse CNS occur following ischemic damage. Our findings demonstrate the utility and power of multi-probe RPA for evaluation of changes in cytokine mRNA levels. Moreover, this study is, to our knowledge the first to show temporal changes in cytokine mRNA in mouse cerebral ischemia, forming a basis for further exploration of the roles of these cytokines in modulating ischemic neuronal damage in this model.

Cloning, expression during development, and evidence for release of a trophic factor for ciliary ganglion neurons

Ciliary ganglion (CG) neurons undergo a period of cell death during development that may be regulated by the limited availability of trophic factor produced by their target tissues. We have previously reported the purification of a ciliary neurotrophic factor from adult chick sciatic nerve that we called growth promoting activity (GPA). Here we demonstrate that GPA can be purified and cloned from embryonic day 15 (E15) chick eyes, which contain all the target tissues of the CG. Our studies show the following: GPA mRNA is induced in embryonic chick eyes during the period of CG neuron cell death; GPA mRNA is expressed specifically in the layer of the eye that contains the targets of the CG and in primary cultures of smooth muscle cells isolated from the choroid layer of the eye; and biologically active GPA is released from cells transfected with a GPA cDNA.

Induction of somatostatin immunoreactivity in cultured ciliary ganglion neurons by activin in choroid cell-conditioned medium

We have previously shown that the expression of somatostatin-like immunoreactivity in cultured ciliary ganglion neurons is stimulated by a macromolecule found in choroid cell-conditioned medium (ChCM). Here, we present the following evidence that this somatostatin-stimulating activity (SSA) is activin: human recombinant activin induces somatostatin-like immunoreactivity in CG neurons; ChCM induces hemoglobin synthesis in K562 cells, a biological activity characteristic of activin; activin A-specific antibodies recognize a protein in ChCM; cultured choroid cells contain activin RNA; and SSA is inhibited by follistatin, a specific activin-binding protein. Thus, activin is likely to be a neurodifferentiation factor for CG neurons in vivo.

Activin A and follistatin expression in developing targets of ciliary ganglion neurons suggests a role in regulating neurotransmitter phenotype

The avian ciliary ganglion contains choroid neurons that innervate choroid vasculature and express somatostatin as well as ciliary neurons that innervate iris/ciliary body but do not express somatostatin. We have previously shown in culture that activin A induces somatostatin immunoreactivity in both neuron populations. We now show in vivo that both targets contain activin A; however, choroid expressed higher levels of activin A mRNA. In contrast, follistatin, an activin A inhibitor, was higher in iris/ciliary body. Iris cell-conditioned medium also contained an activity that inhibited activin A and could be depleted with anti-follistatin antibodies. These results suggest that development of somatostatin is limited to choroid neurons by differential expression of activin A and follistatin in ciliary ganglion targets.

Prostate stem cell antigen is an endogenous lynx1-like prototoxin that antagonizes alpha7-containing nicotinic receptors and prevents programmed cell death of parasympathetic neurons.

Vertebrate α-bungarotoxin-like molecules of the Ly-6 superfamily have been implicated as balancers of activity and survival in the adult nervous system. To determine whether a member of this family could be involved in the development of the avian ciliary ganglion, we identified 6 Gallus genes by their homology in structure to mouse lynx1 and lynx2. One of these genes, an ortholog of prostate stem cell antigen (psca), is barely detectable at embryonic day (E) 8, before neuronal cell loss in the ciliary ganglion, but increases >100-fold as the number of neurons begins to decline between E9 and E14. PSCA is highly expressed in chicken and mouse telencephalon and peripheral ganglia and correlates with expression of α7-containing nicotinic acetylcholine receptors (α7-nAChRs). Misexpressing PSCA before cell death in the ciliary ganglion blocks α7-nAChR activation by nicotine and rescues the choroid subpopulation from dying. Thus, PSCA, a molecule previously identified as a marker of prostate cancer, is a member of the Ly-6 neurotoxin-like family in the nervous system, and is likely to play a role as a modulator of α7 signaling-induced cell death during development.

Cellular distribution, subcellular localization and possible functions of basic and acidic fibroblast growth factors

The distribution in the rat nervous system of acidic and basic fibroblast growth factors (FGFs) was analysed by a combination of biochemical and anatomical methods. Acidic FGF (aFGF) was found to be present exclusively in specific neuronal populations, such as motor neurons and basal forebrain cholinergic neurons. Basic FGF (bFGF) was found in astrocytes and in neurons in hippocampal area CA2. Within labelled astrocytes and CA2-neurons, bFGF was detected in both the cytoplasm and the nucleus. The levels of intracellular bFGF were manipulated by antisense oligonucleotide treatment of cultures of developing neural crest cells. Results indicated that the amount of melanogenesis in the cultures is likely to be regulated by intracellular, possibly nuclear bFGF.

Differential localization and possible functions of aFGF and bFGF in the central and peripheral nervous systems.

We investigated the relative distribution of acidic and basic FGF (aFGF and bFGF) in the nervous system of the rat, using a combination of biological, biochemical, immunochemical, and immunohistochemical methods that can differentiate unambiguously between aFGF and bFGF. We found that different regions of the nervous system contained varying levels of aFGF and bFGF. In the central nervous system, bFGF was present nearly exclusively in astrocytes. Most neurons did not contain detectable amounts of bFGF immunoreactivity, with the notable exception of pyramidal cells in hippocampal area CA2. Interestingly, bFGF immunoreactivity was localized to the nucleus of both CA2 neurons and astrocytes. Astrocytes in vitro were also found to express bFGF, whereas cortical neurons in culture did not contain detectable amounts of bFGF. Transection of the optic nerve led to an approximately twofold increase of bFGF in the distal stump, which is consistent with the observation that bFGF is expressed by astrocytes. Transection of rat and chicken sciatic nerve resulted in a rapid and complete disappearance of aFGF from the distal nerve stump, suggesting that aFGF is present in axons projecting through the sciatic nerve. We observed, in agreement with this notion, that cultured sensory neurons contain reasonably high levels of FGF-like bioactivity. Similar levels of activity were found in developing sciatic nerve, suggesting that neuronal aFGF might be involved in regulating the development of the peripheral nervous system.

Cell-Autonomous Inhibition of α7-Containing Nicotinic Acetylcholine Receptors Prevents Death of Parasympathetic Neurons during Development

Neurotrophic molecules are key retrograde influences of cell survival in the developing nervous system, but other influences such as activity are also emerging as important factors. In the avian ciliary ganglion, half the neurons are eliminated between embryonic day 8 (E8) and E14, but it is not known how cell death is initiated. Because systemic application of α7-nicotinic acetylcholine receptor (nAChR) antagonists prevents this cell loss, we examined differences in receptor densities and responses of intracellular calcium to nicotine using the calcium-sensitive dye fura-2. In addition, we determined whether cell-autonomous inhibition of α7 activation in neurons prevented cell death. E8 neurons are heterogeneous with respect to α7-nAChR density, which leads to large increases in [Ca2+]i in some neurons; E8 neurons also exhibit a slower rate of Ca2+ decay after nicotinic stimulation than E13 neurons. Expressing α-bungarotoxin that is tethered to the membrane by a glycosylphosphatidylinositol linkage (GPIαbtx) in ciliary ganglion neurons with the retroviral vector RCASBP(A) blocks increases in intracellular calcium induced by nicotine through α7-nAChRs and prevents neurons from dying. Expression of GPIαbtx in surrounding non-neural tissues, but not in neurons, does not prevent cell loss. Furthermore, the GPIαbtx is not efficiently expressed in the accessory oculomotor neurons, eliminating preganglionic inputs as another site for action of the antagonist. These results support the hypothesis that cholinergic inputs facilitate cell death in the developing autonomic nervous system by activating α7-nAChRs, possibly by leading to increases in intracellular calcium that exceed the threshold for cell survival.

Expression of a chicken ciliary neurotrophic factor in targets of ciliary ganglion neurons during and after the cell-death phase

Ciliary ganglion (CG) neurons, like other neuronal populations, become dependent on their targets for survival during development. We have previously purified and cloned a secreted ciliary neurotrophic factor that was called growth‐promoting activity (GPA). We report here the expression and purification of a highly active form of recombinant GPA, the preparation of GPA‐specific polyclonal and monoclonal antibodies, and the use of these antibodies to investigate the cellular location and timing of GPA expression in tissues innervated by CG neurons. Virtually all of the trophic activity in extracts of embryonic eyes could be depleted by GPA‐specific antibodies. GPA‐like immunoreactivity was found in both targets of the CG: the arterial vasculature of the choroid layer and the ciliary body of the eye. In the choroid layer, GPA was localized to smooth muscle cells surrounding the choroid arteries. Staining in the choroid layer was first detectable at embryonic day (E) 10, or about 2 days after cell death has begun in the ganglion, then increased in intensity through E19. Quantification of trophic activity from whole eye extracts at various ages showed a small increase in activity observed between E9 and E12 and at least a 10‐fold increase between E12 and E18. The presence of GPA protein in target cells of CG neurons during the specific developmental period when these neurons undergo cell death is consistent with its proposed function as a target‐derived ciliary neurotrophic factor.