Neuronal apoptosis in excitotoxicity : involvement of nitric oxide and cytoskeletal damage

Abstract

In the nervous system apoptosis may play an important role in the development of several neurodegenerative diseases. The overall objective of the present work is to investigate at the cellular and molecular levels, the mechanisms by which neurons are signaled to undergo apoptotic cell death after exposure to various effectors, such as nitric oxide (NO) and its byproducts, glutamate, and colchicine. These toxic agents mimic conditions of certain diseases and some of the downstream events directly involved in the apoptotic pathway(s) are reported here.

Since cytoskeletal alterations are among the most common neuropathological features observed in a variety of neurodegenerative disorders, in the first paper we investigated the relationship between cytoskeletal changes induced by colchicine and the onset of neuronal apoptosis. Colchicine, a microtubule disrupting agent, induces apoptosis in cerebellar granule cells (CGCs) through the rapid breakdown of the cytoskeleton as indicated by the protective effect of taxol. In fact, taxol stabilises microtubules and prevents nuclear fragmentation. Colchicine induces damage of tau protein and of (alpha-, beta-tubulins, followed by intracellular Ca2+ changes, altered gene expression and other downstream events in CGCs. Cytoskeletal alterations are also present in neuronal damage elicited by excitatory amino acids. Therefore, we explored the possibility that cytoskeletal elements could be involved in NO-induced apoptosis. Besides nitric oxide's physiological functions, it has been shown that NO can mediate, at least in part, glutamate toxicity through the formation of peroxynitrite.

In the second paper we studied the type of cell death which occurred in response to N-methyl-D-aspartate (NMDA) or NO-donors, such as S-nitrosocysteine (SNOC), 3-morpholinosydnonimine (SIN-1) and peroxymtrite in cortical cell cultures (CCCs). NO or its products induces either apoptotic or necrotic cell death depending on the intensity of the insult. Pretreatment with superoxide dismutase (SOD) and catalase to scavenge superoxide anion and hydrogen peroxide partially prevented the apoptotic process.

To further understand the downstream events of NO-toxicity, in the third paper a novel mechanism to explain the neuronal demise elicited by NO-donors is reported. This involves NO-mediated release of NMDA-receptor agonists with a subsequent activation of NMDA- receptor channels and intracellular calcium overload of CGCs. An early dissolution of actin filaments, followed by a breakdown of microtubules and nuclear lamins, precedes the appearance of typical apoptotic features. NMDA-receptor antagonists or blockers, such as dizocilpine (MK-801) and Mg2+respectively, prevented cytoskeletal alterations and apoptosis in CGCs.

In parallel, in the fourth paper we investigated the potential toxicity of glutamate in CGCs. Similar to our findings with NO toxicity, depending on the dose, glutamate induced either apoptotic or necrotic cell death. Our attempts to define the mechanism(s) of cell death have focused on the mitochondrial membrane potential of neurons injured with different doses of glutamate. The distinction between the two kinds of cell death is even accompanied by different mitochondrial activity: necrosis induces a drastic drop of the mitochondrial fimction and of ATP stores, but some mitochondrial activity and ATP production are required for the occurrence of CGC apoptosis.

Finally, in the fifth paper we studied the effect of bcl-2 overexpression on NO-induced apoptosis in GT1-7 cells. Overexpression of bcl-2 prevented the formation of apoptotic nuclei, DNA laddering and PARP cleavage during NO-treatment of GT1-7 cells. The understanding of the protective effect of bcl-2 is not yet clear, but the presence of PARP cleavage may suggest the involvement of proteolytic events.