Thomas P. Flanigan

Ganglioside GM1 Does Not Initiate, but Enhances Neurite Regeneration of Nerve Growth Factor‐Dependent Sensory Neurones

Abstract: An enzyme‐linked immunoadsorbent assay (ELISA) for neurofilament protein was utilised to quantify the effect of exogenous ganglioside on neurite regeneration in cultures of dorsal root ganglion neurones. In contrast to nerve growth factor (NGF), ganglioside GM1 (100 μg/ml) failed to support neuronal survival and neurite regeneration as quantified by the ELISA assay and confirmed by morphological criteria. However, the simultaneous presence of GM1 (100 μg/ml) and NGF (0.5–5 ng/ml) throughout a 5‐day period of culture resulted in an enhancement of previously reported NGF‐induced increases in the expression of neurofilament protein. Further, the addition of GM1 (0–200 μg/ml) at 48 h in vitro to cultures initially established in the presence of 5 ng/ml NGF substantially increased the subsequent expression of neurofilament protein, this response being both independent of and not potentiated by NGF. The results in the present system suggest that GM1 cannot initiate a programme of neurite regeneration; however, GM1 can enhance this process with the response being secondary to the effect of NGF.

Choline acetyltransferase messenger RNA expression in developing and adult rat brain: regulation by nerve growth factor

The polymerase chain reaction (PCR) was used to develop a method for detection and relative quantification of the choline acetyltransferase (ChAT) mRNA in neonatal and adult rat CNS. Oligonucleotide primers derived from a porcine ChAT cDNA sequence were used in coupled reverse transcriptase (RT)-PCR to amplify a cDNA sequence of 206 bp which arises in a cycle- and RNA-dependent manner and which hybridizes with both an internal oligonucleotide and a ChAT cDNA probe. ChAT mRNA was detected in spinal cord, septal area, striatum, cortex and hippocampus but not in cerebellum and cardiac or skeletal muscle. In the septal area, relative quantitative evaluation of ChAT mRNA levels by RT-PCR indicates that this transcript is developmentally regulated and increased following intracerebral administration of nerve growth factor (NGF) to both neonatal and young adult rats. This suggests that the increases of ChAT activity observed in basal forebrain during development or after NGF administration are, at least in part, associated with an increase in corresponding levels of mRNA.

Factors controlling the expression of the NGF receptor in PC12 cells

An enzyme-linked immunoadsorbent assay (ELISA) has been used to study the relative expression of the nerve growth factor receptor (NGF-R) in PC12 cells. Both nerve growth factor (NGF) and fibroblast growth factor (FGF) were found to induce time-dependent increases in receptor expression. The former response was half-maximal at approximately 2 ng/ml and maximal at 25-50 ng/ml. The induction of NGF-R expression by NGF was associated with an increase in the relative level of a 3.8 kb mRNA species encoding this protein. Whereas the induction of the NGF-R by NGF and FGF were both fully inhibited by cholera toxin and cordycepin, the responses were differentially inhibited by the kinase inhibitor K-252a.

Quantitative Evaluation of Neurite Outgrowth in Cultures of Human Foetal Brain and Dorsal Root Ganglion Cells Using an Enzyme‐Linked Immunoadsorbent Assay for Human Neurofilament Protein

Abstract: An enzyme‐linked immunoadsorbent assay has been developed to evaluate comparative levels of neurofilament protein in developing primary cultures of human foetal dorsal root ganglion and brain tissue. The quantitative parameters of the assay, relating linearity of response with varying levels of neurofilament protein, were verified by comparing the relative binding of human species‐specific (BF10) and cross‐species‐reactive (RT97) monoclonal antibodies to mixtures of human and baboon spinal cord homogenates that had been passively adsorbed onto microtitre wells. In human neural cultures, the localisation of neurofilament protein to growing neurites was determined by indirect immunofluorescence staining with anti‐neurofilament antibodies and, using the immunoadsorbent assay, a time‐dependent increase in the level of neurofilament protein was detected that correlated with the morphological time course of neurite development. In the case of dorsal root ganglion cells over 6 days in vitro, a seven‐ to ninefold greater increase in neurofilament protein levels was observed in cultures treated with nerve growth factor when compared with control unstimulated preparations. The quantitative responsiveness of dorsal root ganglion neurones to nerve growth factor detected by the neurofilament assay indicates its potential usefulness in the identification and analysis of neurotrophic and neurotoxic factors or cellular interactions operating in vitro.

Neural cell adhesion molecule (N-CAM) expression during cardiac development in the rat

Neural cell adhesion molecule (N-CAM) expression was examined in the rat heart using immunohistochemical and immunochemical techniques. N-CAM immunoreactivity was displayed by myocardial cells from embryonic day E12 and by cardiac nerves when first identified at day E18. Myocardial immunostaining increased up until about postnatal day 1 and then declined rapidly thereafter whereas neural immunoreactivity persisted in the adult. N-CAM cardiac isoforms also exhibited developmental changes from the main embryonic moieties (105 and 145 kDa) to the principal postnatal (125 and 155 kDa) and adult isoforms (125 kDa). Cardiac N-CAM expression is therefore subject to temporal regulation and may modulate cellular interactions in the developing heart.

Molecular specificity of ganglioside effects on neurite regeneration of sensory neurons in vitro

Highly purified preparations of individual gangliosides have been tested for their ability to modulate the survival and morphological differentiation of embryonic chick dorsal root ganglion neurons. When added at 48 h to established cultures of nerve growth factor (NGF)-dependent neurons, all ganglioside species tested increased the expression of neurofilament protein. Poly- and trisialogangliosides were more effective than di- or monosialogangliosides. In contrast, neither NGF nor an antiserum against NGF influenced neurofilament protein expression over this period of culture. In addition, ganglioside-induced expression of neurofilament protein was not inhibited by the anti-NGF serum.

Cell survival characteristics and choline acetyltransferase activity in motor neurone-enriched cultures from chick embryo spinal cord.

Abstract: The effect of muscle extract on cell survival and choline acetyltransferase (ChAT) activity in cultures of enriched cholinergic neurones from 7‐day chick embryo spinal cord was examined. When neurones were grown on hydrated collagen gels, considerable cell survival and ChAT activity were obtained even in the absence of tissue extract. These parameters were stimulated twofold in the presence of skeletal muscle extract but not liver or skin extracts. The cholinergic neurotrophic activity was found to be heat‐ and trypsin‐sensitive, non‐dialysable, and to act in the virtual absence of glial cells. These data are consistent with a retrogradely acting motor neurone trophic activity.

The effect of nerve growth factor and its antibodies on neurofilament protein expression in primary cultures of sensory and spinal neurons

An enzyme-linked immunoadsorbent assay has been used to quantify the binding of an antineurofilament monoclonal antibody, RT97, to primary cultures of embryonic chick dorsal root ganglion and spinal cord tissue. In the case of dorsal root ganglion cells a dose-dependent relationship between nerve growth factor (NGF) concentration and the level of RT97 binding was observed. This response could be antagonised by an anti-serum directed against NGF. In contrast, neither NGF nor the antiserum against NGF influenced the binding of RT97 in primary cultures initiated from chick spinal cord.

Identification of cell-surface antigens present exclusively on a sub-population of astrocytes in human foetal brain cultures ☆

We have examined two monoclonal antibodies (McAbs; coded MI/N1 and 308) raised to human neuroblastoma cells for cell-type-specific reactivity in cultures of human neural tissues and in frozen sections of intact primate spinal cord. In dual-label immunofluorescence assays using established cell-type antigenic markers as positive controls, the reactivity patterns obtained with both McAbs MI/N1 and 308 were consistent with the detection of astrocyte-specific cell-surface antigens. No reactivity of the antibodies with other human neural cell-types, or with human muscle cells was detected. In cultures of human foetal brain a sub-population of astrocytic cells remained unlabeled by antibodies MI/N1 and 308. The significance of the latter observation has not yet been defined but may represent a developmental or functional division within the astrocytic cell lineage.

Effects of amyotrophic lateral sclerosis serum on cultured chick spinal neurons.

We used a quantitative immunoassay to examine the effects of human serum and immunoglobulins on neurofilament protein expression in cultures of chick spinal neurons. Compared with cultures grown in the presence of serum from healthy controls or patients with other neurologic disorders, ALS serum lowered the level of neurofilament proteins. Effects were similar with or without muscle-derived neurotrophic factors; there was no specificity for motor neurons. No neurotoxic activity was found in immunoglobulin fractions, and there was no evidence of circulating antibodies that might neutralize muscle-derived neurotrophic factors or induce cytolysis of spinal neurons.