Neural Development and Apoptosis

Head of Laboratory: Huseyin Mehmet PhD

Scientific Staff:

• Grisha Pirianov PhD (Wellcome Trust funded)

Graduate Students:

• Nigel Kennea MB Bchir. (Wellcome Trust funded)
• Adeline Lesay MSc (CLIMB funded)

Aims

Our research aim is to understand the molecular basis of neural cell loss following brain injury in new-born infants. By combining the manipulation of apoptotic signalling pathways with a stem cell-based therapy approach, in the longer term we aim to reduce the very high prevalence of neurodevelopmental impairment in infants born at less than 30 weeks gestation.

Background

Cerebral palsy has an incidence of 1.5 - 3 per 1000 live births and cerebral hypoxic-ischaemic injury (HII) at or around the time of birth is responsible for a large number of these cases. Although the precise mechanisms of neuronal loss are not fully understood, recent neuropathological studies in animal models and in human infants have indicated that following cerebral HII, a significant proportion of brain cells die by apoptosis.

The pattern of damage to the developing central nervous system (CNS) appears to depend in part on gestational age. Term infants tend to develop discrete parasaggital or basal ganglia pathology, with a significant involvement of grey matter. However, the risk of cerebral palsy is much greater after pre-term birth and, despite improvements in perinatal care, the prevalence of cerebral palsy has increased over the last two decades mainly due to an increase in the number of survivors of premature birth. Recent studies in our institution using magnetic resonance imaging (MRI) have shown that more than 50% of infants born before 30 weeks gestation have brain abnormalities at birth and this figure rises to more than 90% by the time they reach term corrected age.

In pre-term brain injury characteristically leads to widespread loss of white matter with apparent secondary disruption of cortical development and growth. Oligodendrocyte precursors (OP) seem particularly sensitive to damage in the pre-term brain and this may explain the predisposition towards white matter loss. We have preliminary evidence suggesting that these deaths are mediated by inflammatory cytokines, including those that belonging to the tumour necrosis factor (TNF) superfamily of death receptors.

The information obtained from our work will further our understanding of the molecular mechanisms of apoptosis resulting from brain injury and may facilitate the design of effective cerebroprotective strategies to combat cerebral palsy in the perinatal period.

CURRENT RESEARCH PROJECTS

Death Receptor signalling in the oligodendrocyte lineage

Death-inducing cytokines and their receptors have been implicated in cerebral hypoxic-ischaemic injury to both the developing and adult brain. Our aim is to study whether modulation of these receptors can improve the outcome of certain types of brain injury (i.e. hypoxia ischaemia). We are characterising the expression of Fas and other "death receptors" (TRAILR, TNFR, p75NTR) as well as the components of the Death Inducing Signalling Complex (DISC) throughout oligodendrocyte development. Once the molecular characterisation is achieved we will proceed to study the functional relevance of these receptors and the modulation of the apoptotic pathways they trigger.

Inflammatory cytokines and neonatal brain injury

Infection and inflammation in the fetomaternal unit is a leading cause of extremely preterm birth and is associated with a high incidence of cerebral damage. However, neither the mechanism of this effect nor the nature of the associated inflammatory responses have been determined. We have recently found that there is a strong correlation between the presence of inflammatory cytokines in cord blood and the severity of brain injury in pre-term infants. The goal of this project is to further investigate the relationship between inflammatory cytokines and neuronal cell death. Specifically, we will determine the effects of TNF, IL1 and IL6 on the viability and differentiation of oligodendrocytes and their precursors.

The molecular basis of apoptosis in maple syrup urine disease

Maple Syrup Urine Disease (MSUD) is an inborn error of metabolism that can result in progressive neurodegeneration. This disorder is caused by a deficiency in the mitochondrial enzyme, branched chain amino acid dehydrogenase that results in the accumulation of the branched chain keto acids (BCKA). Recently, we have found that these metabolites trigger apoptosis in neural cells. The current project aims first to elucidate the signalling pathways in BCKA induced apoptosis, then to determine the mitochondrial mechanism of reduced cell respiration in BCKA treated cells. Finally, we aim to verify that these mechanisms occur in vivo using fibroblasts from MSUD patients.

Stress activated protein kinase activation following injury to the developing brain

Cerebral injury at or around the time of birth is a major cause of death and neurological impairment in human infants. The aim of this project is to investigate the role of defined stress activated kinases (SAPKs) in neural cell death following cerebral injury in the developing brain. Specifically, this research project will investigate changes in the cerebral expression and activity of defined SAPKs, (JNK-1, JNK-2, JNK-3 and p38) following hypoxia-ischaemia and determine whether the use of SAPK inhibitors can reduce SAPK activity, the phosphorylation of the downstream targets (e.g. c-Jun and ATF-2) and neural cell loss.

Identification of a novel brain-specific zinc finger gene

Neural apoptosis is tightly regulated by the controlled activation or repression of specific transcription factors. A number of such transcription factors which belong to the immediate-early Cys2His2 zinc finger class have been shown to modulate apoptotic transcriptional events following hypoxic-ischaemic neuronal injury. We have recently identified a novel brain-specific zinc finger gene (Znfx5) belonging to this family which also contains the baculoviral inhibition of apoptosis protein (BIR) repeat domain found in genes known to suppress apoptosis. The research aims to further define the DNA sequence and neural expression pattern of Znfx5 in the developing and adult brain and to determine its relevance to programmed cell death during neural development and to apoptosis following cerebral hypoxic-ischaemic injury.

Targets of nitric oxide in neuronal cells

In this study we aim to identify the targets of nitric oxide in neuronal cells. Currently we are working on PC12 cells, a rat phaeochromocytoma cell line that are differentiated with NGF and are used as a model of sympathetic neurones. In previous work defined redox states of nitric oxide have been shown to trigger apoptosis or necrosis depending on the charge. In our current work we will use different nitric oxide donors, releasing NO., NO- or NO+, labelled with 15N. Subcellular fractionation and 15N NMR will then be used to identify specific molecular targets of nitric oxide.

Circulating mesenchymal stem cells as potential therapy for newborn brain injury

Transplantation of newly discovered human fetal mesenchymal stem cells (MSC) may provide a therapy capable of cellular repair in newborn brain injury, thereby improving outcome for children with cerebral palsy. The objectives of this study are to define optimal culture conditions for MSC growth and neural differentiation, and to explore the factors mediating their proliferation, differentiation and apoptosis. The most effective stage of brain development for graft survival will then be determined and finally, MSC transplantation into a model of injury to the developing brain will be performed.

Selected Publications

Khan, S., Kayahara, M., Joashi, U., Mazarakis, N.D., Sarraf, C., Edwards, A.D., Hughes, M.N. and Mehmet, H. (1997) Differential induction of apoptosis in Swiss 3T3 cells by nitric oxide and the nitrosonium cation. J. Cell Sci., 110, 2315-2322.

Edwards, A.D., Yue, X., Cox, P., Hope, P.L., Azzopardi, D.V., Squier, M.V. and Mehmet, H. (1997) Apoptosis in the brains of infants suffering intrauterine cerebral injury. Ped Res. 42 (5), 684-689.

Mehmet, H., Yue, X., Penrice, J., Cady, E., Wyatt, J.S., Sarraf, C., Squier, M.V. and Edwards, A.D. (1998) Relation of impaired energy metabolism to apoptosis and necrosis following transient cerebral hypoxia-ischaemia. Cell Death Diff., 5, 321-329.

BaidenAmissah, K., Joashi, U; Blumberg, R., Mehmet, H., Edwards, A.D. and Cox, P.M. (1998) Expression of amyloid precursor protein (beta-APP) in the neonatal brain following hypoxic ischaemic injury. Neuropath. Applied Neurobiol., 24 (5): 346-352.

Joashi, U.C., Greenwood, K., Taylor, D.L., Kozma, M., Mazarakis, N.D., Edwards, A.D. and Mehmet, H. (1999) Poly(ADP ribose) polymerase cleavage precedes neuronal death in the hippocampus and cerebellum following injury to the developing rat forebrain. Eur. J. Neurosci., 11 (1) : 91-100.

Blumberg, R.M., Taylor, D.L., Yue, X., Aguan, K., Mackenzie, J., Cady, E.B., Weiner, C.P., Mehmet, H. and Edwards, A.D. (1999) Increased nitric oxide synthesis is not involved in delayed cerebral energy failure following focal hypoxic-ischaemic injury to the developing brain. Ped. Res., 46 (2), 224-231.

Felderhoff-Mueser,U., Taylor, D.L., Greenwood, K., Joashi, U.C., Kozma, M., Stibenz, D., Edwards, A.D. and Mehmet, H. (2000) Fas/CD95/APO-1 can function as a death receptor for neuronal cells in vitro and in vivo and is upregulated following cerebral hypoxic-ischemic injury to the developing rat brain. Brain Pathol. 10, 17-29.

Jouvet, P., Rustin, P., Taylor, D.L., Felderhoff-Mueser, U., Pocock, J., Joashi, U., Mazarakis, N.D., Sarraf, C., Greenwood, K., Kozma, M., Edwards, A.D. and Mehmet, H. (2000) Branched chain amino acids induce apoptosis in glial cells without mitochondrial membrane depolarisation or cytochrome c release: implications for neurological impairment associated with maple syrup urine disease. Mol. Biol. Cell. 11 (5), 1919-1932.

Mehmet, H. (2000) Caspases find a new place to hide. Nature, 403 (6765), 29-30.

Mehmet H. (2001) Stroke treatment enters the Fas lane. Cell Death Differ. 8(7), 659-661.

For enquiries about research opportunities please contact Huseyin Mehmet by e-mail.