Ischemic injury and faulty gene transcripts in the brain

Abstract

The brain has the highest metabolic rate of all organs and depends predominantly on oxidative metabolism as a source of energy. Oxidative metabolism generates reactive oxygen species, which can damage all cellular components, including protein, lipids and nucleic acids. The processes of DNA repair normally remove spontaneous gene damage with few errors. However, cerebral ischemia followed by reperfusion leads to elevated oxidative stress and damage to genes in brain tissue despite a functional mechanism of DNA repair. These critical events occur at the same time as the expression of immediate early genes, the products of which trans-activate late effector genes that are important for sustaining neuronal viability. These findings open the possibility of applying genetic tools to identify molecular mechanisms of gene repair and to derive new therapies for stroke and brain injury.

Fig. 1

Schematic illustration of the formation of DNA and RNA lesions. Hydroxyl radicals can attack nearby DNA at several places, as indicated by the arrows. Some lesions resulting from the attack are listed in Fig. 2. Numbers represent the position of attack by reactive oxygen species. (1) attack on the base which produces base modifications. (2) attack on the glycosylic bond which produces sites without base (AP sites). (3) attack on the phosphate bond (P), which produces DNA strand breaks, either (3a) with 3′ hydroxyl ends (3′-OH) or (3b) with 3′ phosphate ends (3′-PO₄). (4) attack on the ribose which produces 3′ phosphoglycolate ends (3′-PG).

Fig. 2

Nucleic acid modifications identified in the brain after oxidative stress. (a) 8-hydroxyguanine [Oh⁸G], the hydrogen on the C-8 carbon of the guanine base (also termed 8-oxo-7,8-dihydroguanine) is replaced with a hydroxyl group. (b) 2,6 diamino-4-hydroxy-5-N-methylformamidopyrimidine (FapyGua), an open ring results from a broken bond between the C-8 and C-9 carbons in the guanine base. (c) 8-hydroxyadenosine (Oh⁸A), the hydrogen on the C-8 carbon of the adenine base is replaced with a hydroxyl group. (d) 5-hydroxycytosine (Oh⁵C), the hydrogen on the C-5 carbon of the cytosine base is replaced with a hydroxyl group. (e) AP site in DNA. (f) and (g) DNA breaks with two different termini. Most of these lesions have been reported in brain injury models, including the forebrain ischemia—reperfusion (experimental cardiac arrest) model, the focal cerebral ischemia model (experimental stroke), the seizure model and the traumatic brain injury model,,,,-.

Fig. 3

The two distinct base-excision-repair pathways in mammalian cells. To repair a modified base, the glycosylic bond linking the deoxyribose (dR) and the damaged base (G*) is excised via glycosylase (1) to generate an abasic (AP)-site intermediate. The damaged base can also be released spontaneously, without enzyme. The DNA backbone 5′ to the AP site is cleaved by AP endonuclease in Step 2, to generate a single-stranded break with 3′ hydroxyl and 5′-deoxyribose phosphate (dRP) termini that can be repaired by one of two pathways. In the classical pathway, dRP is removed by DNA polymerase-β (DNA pol-β; 3a) followed filling of the one-nucleotide gap (4a) and DNA ligation (5). The alternative pathway (3b and 4b), which occurs in the absence of functional DNA pol-β, involves repair synthesis by proliferating cell nuclear antigen (PCNA), DNA pol-δ or -ε and flip endonuclease (FEN). AP endonuclease, DNA pol-β and DNA ligase I form a multiprotein complex in which DNA ligase I can be replaced by DNA ligase III plus complementary factor XRCC1.

Fig. 4

Proposed mechanism by which ODLs in c-fos could delay trans-activation of late genes by producing faulty gene expression without repair (thin red arrows). Repair processes (green arrows) will enhance the expression of intact mRNA (Ref. 25). Abbreviations: ROS, reactive oxygen species; NGF, nerve growth factor.