Cellular and molecular mechanisms of bilirubin induced neural cell apoptosis and respective therapeutic interventions

by Bhatia, Inderjeet

(Uncorrected OCR) Abstract of thesis entitled

"Cellular and molecular mechanisms of bilirubin induced neural cell apoptosis and respective therapeutic interventions"

submitted by

Inderj eet Bhatia

For the degree of Doctor of Philosophy at the University of Hong Kong

in August 2004

Severe hyperbilirubinemia in newborn babies continues to be a cause of profound neurological deficit. In order to delineate the cellular and molecular mechanisms underlying such pathology and pave the way for developing therapeutic interventions for affected babies, a mouse model of bilirubin-induced neurotoxicity has been developed and used in the current study for defining the key events mediating the neural cell apoptosis induced by bilirubin in vivo

Immunoblotting analysis revealed time-dependent increase in Fas death-domain receptor protein, in postnatal mouse brain within an hour following acute bilirubin insult, without concomitant alteration in its cognate ligand, Fas L. Cleavage of caspase-8 paralleled Fas increase, suggestive of recruitment of Fas-activation mediated pro-apoptosis pathway, which was consistent with the well-defined tempo of sequential caspase-9 and caspase-3 activations. Immunohistochemistry for Bax and cytochrome c and flow cytometric evaluation of L\W m were employed to delineate whether the observed caspase-9 activation was

mediated indirectly through caspase-8 activating Bel family of molecules or direct through mitochondrial perturbation by bilirubin. Indeed both Bax and cytochrome c were found increased in early hours while mitochondrial depolarization was induced by bilirubin insult. These changes occurred temporally after the up regulation of Fas and caspase-8, further strengthening that mitochondrial regulation was involved.

To establish the functional role of such observed molecular changes in bilirubin neurotoxicity, a caspase-8 specific inhibitor (z-IETD-fmk) and a broad spectrum caspase inhibitor (B- D-fmk) were used. Activation of caspase-3, 8 and 9 into their active forms was inhibited. Administration of these inhibitors showed a moderate amelioration of bilirubin-induced injury by about 50%. Such inhibition of apoptosis did not result in increase in necrOSIS as evidenced by combined TUNEL/Propidium Iodide staining evaluation. Blocking the mitochondrial permeability transition by Cyelosporin A (CsA) resulted in more significant neuroprotection (over 70%) than either of the two caspase inhibitors. It could also prevent L\Wm depolarization induced by bilirubin and inhibit the activation of caspase-8, -9 and -3.

The current study provides solid evidence of crucial involvement of Fas-mediated, and mitochondrial amplified apoptosis in neonatal brains subjected to bilirubin insult. That general caspase-inhibitor effected only moderate reduction of the apoptotic death supports the notion that bilirubin-induced apoptosis is both caspase-dependent and caspase-independent. This conceptual advancement in understanding the mechanisms of neuronal apoptosis in the immature brain should be pivotal for guiding development of appropriately timed and targeted therapies for rescuing neural cells from bilirubin insult. Our

experimental findings also agree well with evolving concepts in the field of apoptosis and serve to reinforce the need to base structured designs of therapeutic interventions on sound cellular and molecular understandings.