Apoptosis in cultured hNT neurons

Programmed cell death (apoptosis) is an important mechanism shaping the siz e of different cell populations within the developing nervous system. In ou r study we used the NT2/D1 clone originally established from the Ntera 2 ce ll line to investigate the baseline levels of apoptosis in cultured postmit otic hNT (NT2-N) neurons previously treated for 3, 4 or 5 weeks with retino ic acid (RA) and compared it with apoptosis in NT2 precursors unexposed to RA. First, we examined whether different lengths of exposure to RA might af fect baseline apoptotic rate in differentiating hNT neurons. Second, we inv estigated whether cultured hNT neurons, previously shown to possess dopamin ergic characteristics, would be preferentially affected by apoptosis. Using the terminal deoxynucleotidyl transferase (tdt)-labeling technique we foun d that the postmitotic hNT neuronal cells exposed to RA demonstrated signif icantly higher numbers of apoptotic cells (12.5-15.8%) in comparison to rap idly dividing NT2 precursor cell line (3.6-4.4%) at both studied (I and 5 d ays in vitro, DIV) time points. Similar apoptotic nuclear morphology, inclu ding a variable extent of nuclear fragmentation was observed in all examine d hNT cultures. On the other hand, the incidence of apoptotic nuclei was ra re in cultures of NT2 precursors not subjected to RA treatment. Combined im munocytochemistry for tyrosine hydroxylase (TH) and Hoechst staining reveal ed dopaminergic I hNT neurons destined to die. Our double-labeling studies have demonstrated that only a subset of TH-positive hNT cells had condensed chromatin after 1 (approx. 15%) and 5 (approx. 20%) DIV. NT2 precursors we re not TH-positive. Collectively, our results demonstrated that exposure to differentiating agent RA triggers an apoptotic commitment in a subset of p ostmitotic hNT neurons. These results suggest that this cell line may serve as a model of neuronal development to test various pathogenic factors impl icated in the etiology of Parkinson's disease (PD), as well as to screen nu merous pharmacological treatments that may slow or prevent dopaminergic det erioration. (C) 2001 Elsevier Science B.V. All rights reserved.