Parkinsonian serum carries complement-dependent toxicity for rat mesencephalic dopaminergic neurons in culture.

The presence of antibodies recognizing specific epitopes of dopaminergic neurons in serum of patients suffering of Parkinson's Disease (PD) as well as their capability to induce neuronal damage was investigated utilizing serum-free dissociated mesencephalic-striatal co-cultures. High affinity dopamine (DA) and GABA uptakes were assessed as specific, functional markers of dopaminergic and GABAergic cell viability, respectively. Heat-inactivated serum samples from 18 and 13 patients suffering from idiopathic and vascular parkinsonism, respectively and from 18 neurologic controls, were added to co-cultures on day 4 in vitro. Twenty four hours later, reconstituted rabbit complement was added for 60 min and uptake parameters as well as immunocytochemical staining for tyrosine hydroxylase (TH)-containing cells were subsequently assessed. DA, but not GABA, uptake was significantly decreased only when complement was added to cultures containing serum samples from 14 out of 18 patients with idiopathic parkinsonism and 3 out of 13 patients with vascular parkinsonism (Fisher test, P < 0.01). Complement addition to cultures containing serum samples from seropositive parkinsonian patients significantly reduced immunocytochemical staining of TH-containing cells. Seropositive and seronegative patients did not differ in demographic and clinical features. These results suggest that a complement-dependent humoral immune response occurs mainly in idiopathic parkinsonian patients, but its clinical relevance remains to be established.

Interaction of ganglioside GM1 with the B subunit of cholera toxin modulates intracellular free calcium in sensory neurons.

The B subunit of cholera toxin, which binds specifically to GM1 ganglioside on cell surfaces, has previously been shown to modulate intracellular calcium levels and growth in several cell types. To explore a role for such changes in calcium in the growth regulatory function of cell-associated GM1 in neurons, dissociated neurons from chicken embryonic day 8 dorsal root ganglia were exposed to the B subunit. To enhance sensitivity to B subunit, some neurons were also enriched with added GM1 (100 microM) and then exposed to B subunit. Incubation of naive cultures with 1 microgram/ml of the B subunit was sufficient to produce modest increases in intracellular free calcium above basal levels in a minor percentage of cells for at least 5 min, as measured by fura-2 fluorescence imaging. Pretreatment of the cells with GM1 for 48 hr increased even further the elevations in intracellular free calcium and the percentage of responding neurons observed after B subunit exposure. These increases in intracellular calcium required the presence of external Ca2+, but were not inhibited by calcium channel blockers. Such changes in calcium were accompanied by fine alterations in morphology affecting mostly the branching of neurites and were more pronounced in the presence of GM1. However, the morphological changes did not result in altered neurofilament protein expression. Immunogold electron microscopy using anti-choleragenoid depicted extensive aggregations of immunoreactive gold particles on neuronal surfaces, which were more extensive in cells treated with GM1. The results demonstrate that cell incorporated GM1 may modulate calcium fluxes, perhaps accounting for the growth regulatory functions of GM1 in both neuronal and other cell types.

Nerve growth factor transcriptional control of c-fos promoter transfected in cultured spinal sensory neurons.

High efficiency gene transfer (greater than 90%) in chicken dorsal root ganglion neurons has been obtained by DNA calcium phosphate co-precipitation, hence providing an important tool to study control of gene expression in primary neurons. Transfection with c-fos promoter sequences linked to the chloramphenicol acetyltransferase reporter gene showed that the serum responsive element functions as a strong transcriptional enhancer. Transcription from this element is developmentally regulated, and mediates the genetic response to nerve growth factor (NGF) in developing avian sensory neurons. Furthermore, NGF exerts a negative effect on transcription from the cyclic AMP responsive element, thereby supporting the involvement of tyrosine kinase activation by NGF in primary sensory neurons.

Basic fibroblast growth factor promotes the survival and development of mesencephalic neurons in culture.

Neuronotrophic effects of basic fibroblast growth factor (bFGF) have been reported for some central nervous system neurons. Here we report that also rat mesencephalic neurons are responsive to bFGF. bFGF produces a significant increase in the number of neurite-bearing cells, as well as in the degree of their fiber network. The present findings also provide the first evidence that bFGF can affect, in a concentration-dependent manner, at least two defined CNS neuronal populations, i.e., dopaminergic and gabaergic neurons. This effect was quantified by assessing the specific [3H]dopamine and [gamma-14C]aminobutyric acid uptakes with time in culture. Stimulation of uptake was more pronounced for dopaminergic neurons, suggesting a relative specificity in the actions of bFGF. These effects of bFGF were completely blocked by affinity-purified polyclonal antibodies. The possibility that bFGF plays a key role in normal nervous system development or function is discussed.