Calretinin (CR) is a calcium binding protein that is found in subpopulations of brain neurons such as the substantia nigra (SN) and the ventral tegmental area. We recently demonstrated that the neurotoxicity induced in primary neuronal mesencephalic cultures by excitatory amino acids (EAA) was selective for tyrosine hydroxylase (TH)-containing neurons rather than calretinin-containing neurons (Isaacs et al., 1996). We are in the process of surveying other neuronal cultures (cortex, thalamus and olfactory bulb) to determine if this apparent neuroprotective status can be generalized to other neuronal cell types which also contain CR. Because CR and related proteins have been linked to neuroprotective functions in the SN, we decided to treat mesencephalic cultured neurons with L-DOPA, the precursor to the neurotransmitter dopamine, and a drug used to treat Parkinson's disease. We found that TH- containing neurons were very sensitive to low levels of L-DOPA in culture and over 60% were lost after a five day treatment. CR-containing neurons, in contrast were only marginally affected. Of most interest, however, was that when CR was colocalized with TH in dopaminergic neurons, these neurons were immune to the deleterious effects of L-DOPA. In an attempt to determine whether CR alone was serving a neuroprotective function in neurons due to its calcium binding capacity, we transfected PC12 cells with a plasmid which coded for a fusion protein consisting of green fluorescent protein and CR. In this manner we were able to monitor the morphology and number of the fluorescent cells (which also contained CR) after serum deprivation and treatment with calcium ionophores which would raise internal calcium levels to excitotoxic levels. We found no evidence of neuroprotection derived from the presence of CR in either treatment condition. We would suggest that either the calcium influx following calcium ionophore treatment of transfected PC 12 cells was too drastic to offer the cells protection via CR or that CR requires specific target proteins in order to be effective which are present in SN cells but not in transfected PC12 cells. Alternatively, CR has no relevance to neuroprotection and is only coincidentally linked with protection in the substantia nigra. We propose to further analyze the response of CR- containing cells (endogenous or transfected) to a variety of treatments to investigate these critical questions.
