Ischemia-induced apoptosis in the mammalian brain crucially correlates with longlasting phosphorylation of c-Jun, suppresion of ATF-2 an expression of the death-inducing ligands CD95-Ligand an TRAIL. [original]

Ana Martin-Villalba

Dr. med.

Ischemia-induced apoptosis in the mammalian brain crucially correlates with long-

lasting phosphorylation of c-Jun, suppression of ATF-2 and expression of the death-

inducing ligands CD95-Ligand and TRAIL.

Geboren am 13.11.1971 in Madrid

Reifeprüfung am 19.7.1989 in Murcia, Spanien

Studiengang der Fachrichtung Medizin vom WS 1989 bis SS 1995

Physikum an der Universität Murcia, Spanien

Klinishes Studium in Murcia, Spanien

Praktisches Jahr in Leeds, England und Murcia, Spanien

Staatsexamen am 7.1995 an der Universität Murcia, Spanien

Promotionsfach: Physiologie

Doktorvater: Prof. Dr. med. T. Herdegen

Neuronal apoptosis plays an important role in the pathogenesis of cerebral ischemia, but the

underlying mechanisms remain to be defined. Neuronal programmmed cell death was studied

in an established rat model of transient focal ischemia that resulted in a defined pattern of

ischemic brain damage. The expression of the death-inducing ligands (DILs) CD95-Ligand,

TRAIL and TNF-α, and of the transcription factor c-Jun in its active, phosphorylated form,

was examined in the apoptotic compartments of the postischemic brain. Between 3 h and 5

days following ishcmeia, CD95-Ligand, TRAIL and TNF-α were upregulated and neuronal

c-Jun was phosphorylated at residue serine-73. Treatment of human neuron-derived

neuroblastoma cells with recombinant TRAIL, TNF-α or an agonistic CD95 antibody caused

death of the majority of the neurons, with TRAIL being the most potent cytotoxic factor. This

suggests that the neuronal expression of TRAIL, CD95-L and TNF-α mediates ischemic cell

death in the mammalian brain.

JNK/SAPK control the activity of c-Jun and ATF-2 and the transcription of c-Jun. Their

activation is a downstream event of the DIL-induced cascades. Recent data indicate that the

promoter of the CD95-L gene is activated in response to stimulation of AP-1 activity by the

JNK/SAPK pathway. I found that JNK/SAPK activity increased significantly in the

postischemic brain. Also, the constitutively expressed ATF-2 protein disappeared in the

apoptotic compartments. Moreover, ATF-2 suppression paralleled the long-lasting

phosphorylation of c-Jun and the expression of CD95-L. Thus, the results of the present study

indicate the existence of a positive autoregulatory loop between DILs and the transcription

factor c-Jun with JNK/SAPK as intermediates. The self-sustaining of this loop may be

facilitated by the suppression of the constitutive transcription factor ATF-2, a putative

competitor of c-Jun for JNK/SAPK.

Application of the immunosuppressant FK506 shortly after onset of ischemia reduced brain

damage and prevented the occurrence of apoptotic nuclei. Also, FK506 prevented the

upregulation of DILs, the suppression of ATF-2 and the phosphorylation of c-Jun –but not its

expression– in the postischemic brain. In neuroblastoma cells, FK506 counteracted both the

doxorubicin-mediated cell death and the subsequent upregulation of DILs and c-jun mRNA. I

hypothesize that this drug may neuroprotect either by preventing the calcium-mediated

activation of JNK/SAPK, or by activating the protein kinase C, or by increasing the

mitochondrial membrane potential. The exact steps blocked by FK506 remain to be defined.

The discovery of the antiapoptotic actions of FK506 following brain ischemia opens new

possibilities for the treatment of stroke.