Scott R. Whittemore

The expression, localization and functional significance of β-nerve growth factor in the central nervous system

NGF and central catecholaminergic neuronal function ........................................................................................ 2.1, NGF-induced alteration in adrenergic CNS function ..................................................................................... 2.2. Retrograde transport of NGF in adrenergic pathways ................................................................................... 2.3. Summary ...........................................................................................................................................

Schwann cells genetically modified to secrete human BDNF promote enhanced axonal regrowth across transected adult rat spinal cord.

The infusion of BDNF and NT‐3 into Schwann cell (SC) grafts promotes regeneration of brainstem neurones into the grafts placed in adult rat spinal cord transected at T8 ( Xu et al. 1995b ). Here, we compared normal SCs with SCs genetically modified to secrete human BDNF, grafted as trails 5 mm long in the cord distal to a transection site and also deposited in the transection site, for their ability to stimulate supraspinal axonal regeneration beyond the injury. SCs were infected with the replication‐deficient retroviral vector pL(hBDNF)RNL encoding the human preproBDNF cDNA. The amounts of BDNF secreted (as detected by ELISA) were 23 and 5 ng/24 h per 106 cells for infected and normal SCs, respectively. Biological activity of the secreted BDNF was confirmed by retinal ganglion cell bioassay. The adult rat spinal cord was transected at T8. The use of Hoechst prelabelled SCs demonstrated that trails were maintained for a month. In controls, no SCs were grafted. One month after grafting, axons were present in SC trails. More 5‐HT‐positive and some DβH‐positive fibres were observed in the infected vs. normal SC trails. When Fast Blue was injected 5 mm below the transection site (at the end of the trail), as many as 135 retrogradely labelled neurones could be found in the brainstem, mostly in the reticular and raphe nuclei (normal SCs, up to 22, mostly in vestibular nuclei). Numerous neurones were labelled in the ventral hypothalamus (normal SCs, 0). Also, following Fast Blue injection, a mean of 138 labelled cells was present in dorsal root ganglia (normal SCs, 46) and spinal cord (39 vs. 32) rostral to the transection. No labelled spinal neurones rostral to the transection were seen when SCs were not transplanted. Thus, the transplantation of SCs secreting increased amounts of BDNF improved the regenerative response across a transection site in the thoracic cord. Moreover, the enhanced regeneration observed with infected SCs may be specific as the largest response was from neurones known to express trkB.

Expression of the receptors for the C5a anaphylatoxin, interleukin-8 and FMLP by human astrocytes and microglia

The expression of chemotactic receptors in the central nervous system is largely unexplored. In this study, we examined human astrocytes and microglia as well as the conditionally immortalized human astrocyte cell line HSC2 for expression of the C5a-anaphylatoxin receptor (C5aR), the interleukin-8 receptor (IL-8R) and the f-Met-Leu-Phe receptor (FMLPR). Using flow cytometry, indirect immunofluorescence and RT-PCR analysis, we demonstrated that astrocytes, microglia and HSC2 cells contain specific RNA and express surface protein for all three chemotactic receptors. These are the first studies to demonstrate definitively the expression of these chemotactic receptors astrocytes and microglia, thereby expanding the types of cells known to express chemotactic receptors. Moreover, these data suggest that these chemotactic receptors may play an important role in mediating the inflammatory response and perhaps other yet undescribed biological phenomena in the central nervous system.

Induction of Eph B3 after spinal cord injury.

Spinal cord injury (SCI) in adult rats initiates a cascade of events producing a nonpermissive environment for axonal regeneration. This nonfavorable environment could be due to the expression of repulsive factors. The Eph receptor protein tyrosine kinases and their respective ligands (ephrins) are families of molecules that play a major role in axonal pathfinding and target recognition during central nervous system (CNS) development. Their mechanism of action is mediated by repellent forces between receptor and ligand. The possible role that these molecules play after CNS trauma is unknown. We hypothesized that an increase in the expression of Eph proteins and/or ephrins may be one of the molecular cues that restrict axonal regeneration after SCI. Rats received a contusive SCI at T10 and in situ hybridization studies 7 days posttrauma demonstrated: (i) a marked up-regulation of Eph B3 mRNA in cells located in the white matter at the lesion epicenter, but not rostral or caudal to the injury site, and (ii) an increase in Eph B3 mRNA in neurons in the ventral horn and intermediate zone of the gray matter, rostral and caudal to the lesion. Immunohistochemical analyses localizing Eph B3 protein were consistent with the mRNA results. Colocalization studies performed in injured animals demonstrated increased Eph B3 expression in white matter astrocytes and motor neurons of the gray matter. These results suggest that Eph B3 may contribute to the unfavorable environment for axonal regeneration after SCI.

Physiological relevance and functional potential of central nervous system-derived cell lines.

Central nervous system (CNS)-derived neural cell lines have proven to be extremely useful for delineating mechanisms controlling such diverse phenomena as cell lineage choice and differentiation, synaptic maturation, neurotransmitter synthesis and release, and growth factor signalling. In addition, there has been hope that such lines might play pivotal roles in CNS gene therapy and repair. The ability of some neural cell lines to integrate normally into the CNS following transplantation and to express foreign, often corrective gene productsin situ might offer potential therapeutic approaches to certain neurodegenerative diseases. Five general strategies have evolved to develop neural cell lines: isolation and cloning of spontaneous or mutagenically induced malignancies, targeted oncogenesis in transgenic mice, somatic cell fusion, growth factor mediated expansion of CNS progenitor or stem cells, and retroviral transduction of neuroepithelial precursors. In this article, we detail recent progress in these areas, focusing on those cell lines that have enabled novel insight into the mechanisms controlling neuronal cell lineage choice and differentiation, both in vitro and in vivo.

Synergistic Activation of DNA Synthesis in Astrocytes by Fibroblast Growth Factors and Extracellular ATP

Abstract: The effects of extracellular ATP and polypeptide growth factors on DNA synthesis in primary cultures of rat astrocytes have been examined. It was found that ATP acts synergistically with either acidic or basic fibroblast growth factor to stimulate DNA synthesis. The specificity of this effect was demonstrated by the inability of ATP to potentiate DNA synthesis induced by platelet‐derived growth factor or epidermal growth factor. ATP appears to act via P2 purinergic receptors, because (a) it was more effective than adenosine and (b) the synergistic effect was observed with the hydrolysis‐resistant P2 agonists, ADPβS and ATPγS. The evidence suggests that extracellular ATP may be an important factor in regulating the extent of gliosis and, as such, may be involved in mechanisms of neural injury and repair.

Autocrine BDNF Secretion Enhances the Survival and Serotonergic Differentiation of Raphe Neuronal Precursor Cells Grafted into the Adult Rat CNS

RN46A cells are a temperature-sensitive neuronal cell line derived from the E13 rat raphe nucleus. RN46A cells grafted into the adult rat hippocampus and cerebral cortex do not survive beyond 2 weeks. Brain-derived neurotrophic factor (BDNF) regulates the in vitro survival and serotonergic phenotype of RN46A cells, and we hypothesized that expression of BDNF in RN46A cells would potentiate their survival and serotonin (5HT) expression in vivo. The gene encoding rat BDNF was transfected into RN46A cells and the clonal 46A-B14 cell line isolated, 46A-B14 cells synthesize and secrete biologically active BDNF in vitro and synthesize 5HT following partial membrane depolarization. Two weeks following 46A-B14 cell transplantation into the adult rat cortex and hippocampus, there is a threefold increase in survival of 46A-B14 cells compared to RN46A cells transfected with the vector alone. The grafted 46A-B14 cells immunohistochemically stain for BDNF and 5HT, while RN46A cells transfected with vector only are negative for both BDNF and 5HT. In addition, 46A-B14 cells attain more morphologically complex phenotypes, indicating enhanced neuronal differentiation. Autocrine secretion of BDNF by RN46A cells thus potentiates survival and can be used to deliver both BDNF and 5HT in vivo.

Altered acidic and basic fibroblast growth factor expression following spinal cord injury.

In normal spinal cord, acidic fibroblast growth factor (aFGF) immunoreactivity was localized in the cytoplasm of ventral motor neurons and sensory fibers in the dorsal columns. Basic FGF (bFGF) immunoreactivity was restricted to astrocyte nuclei and the cytoplasm of a few neurons in the intermediate gray matter. Spinal cord lesions resulted in complete destruction of the dorsal columns at T8. Two days postlesion, aFGF immunoreactivity was increased in ventral motor neurons and was now seen in intermediate gray matter neurons. Acidic FGF was not detected in the lesioned fasciculus gracilis at T4-5, but markedly increased in the fasciculus cuneatus. At L1-2, aFGF-immunoreactive fibers in the fasciculus gracilis also increased. This aFGF immunostaining was maintained 5 and 12 days postlesion. A lesion-induced loss of aFGF immunoreactivity in the nucleus gracilis suggests that aFGF is anterogradely transported in ascending sensory fibers. Two days postlesion, glial fibrillary acidic protein immunoreactivity increased at the lesion site, as well as at T4-5 and L1-2, with no change in bFGF staining. Five days postlesion, increased bFGF immunoreactivity appeared at the edge of the cystic cavity and the dorsal columns at T4-5 in both the nucleus and the cytoplasm of reactive astrocytes, and was increased at 12 days postlesion. The differential cellular, temporal, and spatial expression of aFGF and bFGF following spinal cord lesion suggest they subserve distinct roles in the response to CNS injury.

BDNF induction of tryptophan hydroxylase mRNA levels in the rat brain.

We have previously demonstrated an augmentation of serotonergic activity within various brain areas following infusion of brain‐derived neurotrophic factor (BDNF) into the midbrain near the periaqueductal gray and dorsal and median raphe nuclei (PAG/DR). However, the mechanism of this BDNF‐induced modulatory effect on serotonergic systems was unclear. The aim of the present work was to study the regulation of tryptophan hydroxylase (TPH) mRNA levels after chronic BDNF administration invivo. TPH mRNA levels were measured using a quantitative competitive reverse transcription polymerase chain reaction (RT‐PCR) assay. A significant increase in the expression of TPH mRNA (13‐fold) was found within the PAG/DR as early as 24 hr after onset of BDNF infusion and was sustained throughout the duration of infusion (11 days). This was accompanied by increased serotonin (5‐hydroxytryptamine, 5‐HT) levels and decreased nociceptive responsiveness assessed by tail‐flick latency. BDNF induction of TPH mRNA levels was also observed in a serotonergic cell line derived from raphe neurons, indicating that BDNF can directly regulate TPH mRNA levels. These results suggest that BDNF augments 5‐HT synthesis in vivo by directly enhancing steady‐state TPH mRNA levels, and subsequently leading to marked behavioral alterations. J. Neurosci. Res. 52:149–158, 1998. © 1998 Wiley‐Liss, Inc.

Transplants of neuronal cells bioengineered to synthesize GABA alleviate chronic neuropathic pain.

The use of cell lines utilized as biologic “minipumps” to provide antinociceptive molecules, such as GABA, in animal models of pain is a newly developing area in transplantation biology. The neuronal cell line, RN33B, derived from E13 brain stem raphe and immortalized with the SV40 temperature-sensitive allele of large T antigen (tsTag), was transfected with rat GAD67 cDNA (glutamate decarboxylase, the synthetic enzyme for GABA), and the GABAergic cell line, 33G10.17, was isolated. The 33G10.17 cells transfected with the GAD67 gene expressed GAD67 protein and synthesized low levels of GABA at permissive temperature (33°C), when the cells were proliferating, and increased GAD67 and GABA during differentiation at nonpermissive temperature (39°C) in vitro, because GAD67 protein expression was upregulated with differentiation. A control cell line, 33V1, transfected with the vector alone, contained no GAD67 or GABA at either temperature. These cell lines were used as grafts in a model of chronic neuropathic pain induced by unilateral chronic constriction injury (CCI) of the sciatic nerve. Pain-related behaviors, including cold and tactile allodynia and thermal and tactile hyperalgesia, were evaluated after CCI in the affected hind paw. When 33G10.17 and 33V1 cells were transplanted in the lumbar subarachnoid space of the spinal cord 1 week after CCI, they survived greater than 7 weeks on the pia mater around the spinal cord. Furthermore, the tactile and cold allodynia and tactile and thermal hyperalgesia induced by CCI was significantly reduced during the 2–7-week period after grafts of 33G10.17 cells. The maximal effect on chronic pain behaviors with the GABAergic grafts occurred 2–3 weeks after transplantation. Transplants of 33V1 control cells had no effect on the allodynia and hyperalgesia induced by CCI. These data suggest that a chronically applied, low local dose of GABA presumably supplied by transplanted cells near the spinal dorsal horn was able to reverse the development of chronic neuropathic pain following CCI. The use of neural cell lines that are able to deliver inhibitory neurotransmitters, such as GABA, in a model of chronic pain offers a novel approach to pain management.