Cytoskeleton disruption causes apoptotic degeneration of dentate granule cells in hippocampal slice cultures

Abstract

Colchicine, a potent microtubule-depolymerizing agent, is well known to selectively kill dentate granule cells in the hippocampal formation in vivo. Using organotypic cultures of rat entorhino-hippocampal slices, we confirmed that in vitro exposure to 1 μM and 10 μM of colchicine reproduced a specific degeneration of the granule cells after 24 h. Similar results were obtained with other types of microtubule-disrupting agents, i.e., nocodazole, vinblastine, and Taxol. Interestingly, the actin-depolymerizing agents cytochalasin D and latrunculin A also elicited selective neurotoxicity in the dentate gyrus without affecting survival of hippocampal pyramidal cells. The selective pattern of degeneration was observable 24 h after a brief treatment with the toxins as short as 5 min, but this delayed neuronal death was unlikely to be a result of excitotoxicity because it was virtually unaffected by glutamate receptor antagonists, tetrodotoxin, or extracellular Ca²⁺-free conditions. The damaged tissues contained a large number of TUNEL-positive neurons and exhibited an increased level in caspase-3-like activity, suggesting that cytoskeleton disruption triggers an apoptosis-like process in dentate granule cells. Thus, this study may provide a basis for understanding the distinctive mechanism that supports granule cell survival.

Introduction

The neuronal cytoskeleton is a complex meshwork consisting of microtubules, actin microfilaments, intermediate filaments, and other associated proteins. This intracellular system is responsible for determining neuronal morphology and for regulating transport and anchoring of cellular constituents (Hirokawa, 1994).

The granule cells of the dentate gyrus (DG) are the most abundant neurons in the hippocampal formation and possess the unusual properties of prolonged postnatal neurogenesis and limited lifetime (Altman and Das, 1965, Kaplan and Hinds, 1977, Eriksson et al., 1998). As a result, they undergo a continuous turnover over the period of weeks even in the adult brains (Gould et al., 1999). To date, very little is known about how these neurons survive for such a short lifetime and die thereafter.

The microtubule-disrupting agent colchicine is a well-known neurotoxin specific for DG granule cells in the hippocampal formation and hence has been widely used as an experimental tool for selective cell ablation in neurobiology and neurotoxicology (Goldschmidt and Steward, 1980, Heale et al., 1995). The toxin binds tightly to the β-tubulin subunit of the α/β-tubulin heterodimer, thereby decreasing the soluble tubulin pool and inhibiting microtubule assembly (Uppuluri et al., 1993). In spite of ubiquitous expression of tubulin proteins, however, it is puzzling why colchicine exerts such a cell-specific toxicity. Therefore, elucidating the action of colchicine on DG granule cells would help to clarify the cellular mechanisms responsible for their unique property.

The aim of this study is to determine whether other microtubule-disrupting agents also destroy DG neurons, whether actin-disrupting agents produce a similar pattern of neurotoxicity, and what is the mechanism underlying the characteristic toxicity. Here we report that all tested cytoskeleton disruptors cause highly selective, apoptosis-like death of DG granule cells in organotypic cultures of hippocampal slices, suggesting a critical role of cytoskeleton dynamics in the survival of DG neurons.