NO nitrosylates heme-centers of proteins resulting in mitochondrial dysfunction and generation of additional free radicals. In addition, NO can disrupt heme-containing proteins and cause them to release heme into the intracellular environment. This would result in iron-mediated generation of oxidants, especially peroxynitrite, which nitrates tyrosine residues and disrupts protein structure and function. Massive neurofilament derangement and nitrotyrosine positive aggregates are found in the motor neurons of ALS patients and are hallmarks of the disease. NO can act on redox sites on receptors as well as modify proteins (phosphorylation and thiol sites etc.); this is a possible mechanism by which it may launch a redox-regulated signal transduction cascade.

Induction of the heme-metabolizing enzyme, heme oxygenase-1 (HO-1), has been linked to cellular resistance to heavy metals and other oxidants. HO-1 metabolizes heme generating the end products: CO, billirubin (both have been shown to have an anti-oxidant effects) and iron. HO-1 can exert a direct anti-oxidant effect by breaking down redox-active iron-containing heme groups. It can exert an indirect effect by releasing iron from the heme groups, which causes the upregulation of ferritin synthesis resulting in increased iron sequestration and elimination. Also, HO1 leads to increased iron elimination from the cell through other mechanisms. All this has the possible net effect of lowering the concentration of intracellular iron thereby protecting the cell from iron-mediated damage by peroxynitrite. In addition, iron released from heme groups may act on redox sensitive sites of proteins (IRP and others) either indirectly (by changing the redox milieu of the cell) or directly (iron is redox active) and thereby elicit a signal transduction cascade which ultimately turns on or off genes. There is much yet to be discovered about NO, HO1, iron metabolism and the redox regulation of gene expression.

Neurons are exposed to a lifetime of low levels of NO, during normal cell metabolism, and to high levels during pathological events. Certain neurodegenerative diseases or injury the cells' normal resistance mechanisms are overwhelmed or are defective. In the course of studying NO and cell death I found that under appropriate conditions the cells show a remarkable adaptation to high levels of NO. Specifically, I found that when cells were pretreated with low levels of NO they become resistant to toxic levels of NO (induced adaptive resistance).

Summary of Research Findings:

1. Immortalized motor neurons (NSC34 cells) and primary cells exhibit induced adaptive resistance to NO.
2. This phenomenon extends to other oxidants.
3. The amount of DNA damage is less in adapted NSC34 and in adapted primary neurons, which has implications for prevention of carcinogenesis.
4. The amount of intracellular nitrotyrosine, a quantifiable marker for NO-mediated damage, is significantly less in adapted NSC34 and in adapted primary neurons
5. These effects on nitrotyrosine formation and cell death are dependent on the expression and activity of HO1.
6. The HO1 expression and the induced adaptive resistance are cGMP independent
7. Neurons isolated from HO1 null mice show no adaptive resistance and have increased nitrotyrosine formation and DNA damage in response to NO.
8. Glial cells isolated from HO1 null cells have decreased NO resistance but this denouement is less striking than that in neurons (preliminary).
9. HO1 null cells have increased intracellular iron and decreased elimination of iron.
10. Other gene products (anti-oxidants and DNA repair enzymes) studied thus far do not appear to be involved (preliminary).
11. In NSC34 cells NO resistance correlates with higher % neurite outgrowth suggesting a possible link between adaptive resistance and cell differentiation.

Research Plan:

The overall goal of my laboratory will be to explore resistance to nitro-oxidative stress in CNS cells. The specific aims are to:

1. Determine if the adaptive resistance extends to other oxidants and other CNS cell types.
2. Determine which cellular targets of NO-mediated damage are protected by HO1 induction and induced adaptive resistance.
3. Characterize NO-mediated signal transduction pathways that induce HO1.
4. Characterize the NO-mediated increase of HO1 mRNA stability and/or transcriptional induction of HO1.
5. Determine what other genes are turned/off by HO1 induction and whether their induction/inhibition is necessary for the induced adaptive resistance.
6. Characterize the role of HO-1, HO-1-mediated heme metabolism and iron in induced adaptive resistance.
7. Characterize of the role of cytostasis and differentiation in NO resistance.
8. Eventually use whole animals for studies of induced adaptive resistance in the CNS.
9. Whole animal studies of induced recovery from spinal transection.
10. Study the influence of the low gravity/high radiation environment of space flight on resistance mechanisms to oxidative stress in the CNS.

Understanding mechanisms of induced adaptive resistance in the CNS has therapeutic potential to prevent carcinogenesis as well as preserve cells during early stages of disease (ALS etc.) and during injury (stroke, spinal cord transection). Elucidation of these mechanisms will also give us insights into stimulating regenerative processes in the CNS.