The ADNP (Activity dependent neuroprotective protein) gene encodes a transcription factor that is essential for embryonic development and brain formation. Mutations within this gene cause a neurodevelopmental disorder known as Helsmoortel-Van Der Aa syndrome. ADNP is one of the most commonly mutated single genes associated with autism spectrum disorder. However, its role in neurodevelopment is unclear. Our goal in this study is to dissect the role of Adnp in neurogenesis using the mouse retina as a tractable model system. We hypothesized that Adnp might have a crucial role in retinal neurogenesis. We found that Adnp was consistently expressed in all progenitor cells throughout retinal development. Interestingly, Adnp expression was relatively high in differentiated cells and persisted in the adult retina. Adnp was found to regulate retinal size, possibly by controlling cell survival during retinal development. In the retina, Adnp was found to interact with the Chd4 chromodomain helicase protein and might therefore be involved in chromatin remodelling. On the other hand, it also interacted with Pogz – a zinc finger protein associated with heterochromatin and might have a specific role in neural gene regulation. Altogether, these findings indicate that Adnp plays a crucial role in retinal neurogenesis and identify possible neurodevelopmental mechanisms that might depend on Adnp.

Maternal exposure to viral and bacterial infection during pregnancy is associated with fetal dysmorphology and neurodevelopmental disorders including schizophrenia, cerebral palsy, autism and mental retardation. Previous studies in our laboratory using an established mouse model of endotoxin-induced fetal dysmorphology have led to the hypothesis that birth defects caused by infections during pregnancy are the result of fetal zinc deficiency resulting from the induction of a zinc-binding protein, metallothionein (MT) in the maternal liver as part of the maternal inflammatory response. Thus, we predicted that zinc deficiency would exacerbate the negative fetal outcomes caused by bacterial endotoxin lipopolysaccharide (LPS) and that zinc supplementation would protect against LPS-mediated teratogenicity. This premise was investigated herein and was extended to investigate underlying molecular mechanism, including the identification of markers of neurodevelopmental damage following LPS administration in early and late pregnancy, and to determine the influence of zinc treatment on any changes in expression of these markers. In Chapter 2 it was demonstrated that prenatal exposure to LPS on gestational day (GD) 8 resulted in the development of physical birth defects including exencephaly, microcephaly, cleft lip and or palate, and micrognathia in GD 18 fetuses. Dietary zinc supplementation throughout pregnancy was found to prevent the LPS-related abnormalities. Furthermore, low dietary zinc and LPS exposure were found to be synergistic on teratogenicity. In addition, an inverse linear relationship was observed between the concentration of zinc in the diet and teratogenicity with a reduction in the incidence of birth defects observed with increasing concentration of dietary zinc, a finding suggesting that even small increments of zinc above normal dietary intake are likely to have a beneficial impact on teratogenicity. Maternal infection during late pregnancy has also been linked with prenatal brain damage. A major causal link underpinning this relationship is thought to be the cytokines released following a maternal inflammatory response to infection. In Chapter 3, the presence of cytokines released in response to LPS given on GD 16 was demonstrated by an increased number of tumour necrosis factor-alpha (TNF-!)-reactive cells and astrogliosis accompanied by extensive apoptotic cell death in GD 18 fetal brain. Recently our laboratory has reported that dietary zinc supplementation throughout pregnancy, prevented impairments in object recognition memory in offspring from dams exposed to prenatal LPS on GD 8. The question arises as to whether zinc is protective against LPS-exposure in late pregnancy. In Chapter 3, it is further demonstrated that LPS-induced brain injury was prevented by concurrent zinc treatment at the time of LPS exposure. In Chapter 4, the expression of activity-dependent neuroprotective protein (ADNP) mRNA was identified as a marker of changes occurring in the fetus as a result of LPS exposure in early pregnancy. ADNP has been found to be essential for organogenesis and is a sensitive indicator of brain injury. Here it was demonstrated that LPS caused a rapid increase in embryonic ADNP expression, which was highly significant 24 hours after exposure. Whether the elevation in ADNP expression is in response to inflammatory damage or is induced by cytokines released by the maternal inflammatory response is not clear. However, a major finding of the study is that concomitant zinc treatment prevented the LPS-induced increase in ADNP activity. The mechanism of protection by zinc is presumed to be centred on preventing the fall in plasma zinc and associated fetal zinc deficiency caused by LPS induction of MT, but may also include MT-independent actions of zinc including prevention of apoptosis and oxidative damage, or enhance tissue repair processes. Taken together the findings in this thesis support earlier evidence that maternal MTmediated transient fetal zinc deficiency in early pregnancy underpins LPS-induced teratogenicity. This is the first study to demonstrate that this mechanism may also apply to LPS-induced neurodevelopmental damage in early and late pregnancy. However, further studies are warranted to discriminate between the influence of MT and that of other inflammatory reactants (e.g. cytokines) on LPS-mediated damage late in pregnancy. The major finding of the thesis is that zinc treatment (either given subcutaneously with LPS or as dietary zinc supplementation throughout pregnancy) prevents the negative fetal outcomes including neurodevelopmental damage caused by prenatal exposure to LPS. This finding highlights the importance of zinc nutrition in pregnancy and the benefits that might be gained as a potential prophylactic treatment to minimise fetal damage caused by infections during pregnancy. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

Maternal exposure to viral and bacterial infection during pregnancy is associated with fetal dysmorphology and neurodevelopmental disorders including schizophrenia, cerebral palsy, autism and mental retardation. Previous studies in our laboratory using an established mouse model of endotoxin-induced fetal dysmorphology have led to the hypothesis that birth defects caused by infections during pregnancy are the result of fetal zinc deficiency resulting from the induction of a zinc-binding protein, metallothionein (MT) in the maternal liver as part of the maternal inflammatory response. Thus, we predicted that zinc deficiency would exacerbate the negative fetal outcomes caused by bacterial endotoxin lipopolysaccharide (LPS) and that zinc supplementation would protect against LPS-mediated teratogenicity. This premise was investigated herein and was extended to investigate underlying molecular mechanism, including the identification of markers of neurodevelopmental damage following LPS administration in early and late pregnancy, and to determine the influence of zinc treatment on any changes in expression of these markers. In Chapter 2 it was demonstrated that prenatal exposure to LPS on gestational day (GD) 8 resulted in the development of physical birth defects including exencephaly, microcephaly, cleft lip and or palate, and micrognathia in GD 18 fetuses. Dietary zinc supplementation throughout pregnancy was found to prevent the LPS-related abnormalities. Furthermore, low dietary zinc and LPS exposure were found to be synergistic on teratogenicity. In addition, an inverse linear relationship was observed between the concentration of zinc in the diet and teratogenicity with a reduction in the incidence of birth defects observed with increasing concentration of dietary zinc, a finding suggesting that even small increments of zinc above normal dietary intake are likely to have a beneficial impact on teratogenicity. Maternal infection during late pregnancy has also been linked with prenatal brain damage. A major causal link underpinning this relationship is thought to be the cytokines released following a maternal inflammatory response to infection. In Chapter 3, the presence of cytokines released in response to LPS given on GD 16 was demonstrated by an increased number of tumour necrosis factor-alpha (TNF-!)-reactive cells and astrogliosis accompanied by extensive apoptotic cell death in GD 18 fetal brain. Recently our laboratory has reported that dietary zinc supplementation throughout pregnancy, prevented impairments in object recognition memory in offspring from dams exposed to prenatal LPS on GD 8. The question arises as to whether zinc is protective against LPS-exposure in late pregnancy. In Chapter 3, it is further demonstrated that LPS-induced brain injury was prevented by concurrent zinc treatment at the time of LPS exposure. In Chapter 4, the expression of activity-dependent neuroprotective protein (ADNP) mRNA was identified as a marker of changes occurring in the fetus as a result of LPS exposure in early pregnancy. ADNP has been found to be essential for organogenesis and is a sensitive indicator of brain injury. Here it was demonstrated that LPS caused a rapid increase in embryonic ADNP expression, which was highly significant 24 hours after exposure. Whether the elevation in ADNP expression is in response to inflammatory damage or is induced by cytokines released by the maternal inflammatory response is not clear. However, a major finding of the study is that concomitant zinc treatment prevented the LPS-induced increase in ADNP activity. The mechanism of protection by zinc is presumed to be centred on preventing the fall in plasma zinc and associated fetal zinc deficiency caused by LPS induction of MT, but may also include MT-independent actions of zinc including prevention of apoptosis and oxidative damage, or enhance tissue repair processes. Taken together the findings in this thesis support earlier evidence that maternal MTmediated transient fetal zinc deficiency in early pregnancy underpins LPS-induced teratogenicity. This is the first study to demonstrate that this mechanism may also apply to LPS-induced neurodevelopmental damage in early and late pregnancy. However, further studies are warranted to discriminate between the influence of MT and that of other inflammatory reactants (e.g. cytokines) on LPS-mediated damage late in pregnancy. The major finding of the thesis is that zinc treatment (either given subcutaneously with LPS or as dietary zinc supplementation throughout pregnancy) prevents the negative fetal outcomes including neurodevelopmental damage caused by prenatal exposure to LPS. This finding highlights the importance of zinc nutrition in pregnancy and the benefits that might be gained as a potential prophylactic treatment to minimise fetal damage caused by infections during pregnancy. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

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