Examining the neurobehavioral and toxic effects of an amino-terminal fragment of ApoE4 in zebrafish

Alzheimer’s disease (AD) is a progressive, fatal disorder neurodegenerative disease that is the most common cause of dementia. In addition to advancing age, there are known genetic risk factors associated with AD. Of the genetic risk factors identified, the 34 kDa protein, apolipoprotein (Apo) E4, is of significance importance as APOE4 carriers account for 65-80% of all AD cases. Although ApoE4 plays a normal role in lipoprotein transport, how it contributes to AD pathogenesis is currently unknown. Recent data from our lab suggests that, in vitro, a 151 amino-terminal fragment of ApoE4 (nApoE41-151) can traffic to the nucleus leading to toxicity and expression of inflammatory genes in BV2 microglia cells. The goal of this proposal is to expand those findings in vivo, by assessing the mechanisms by which this fragment may induce toxicity, developmental abnormalities, and behavior deficits in a model system consisting of zebrafish. Experiments described in Aim 1 will rigorously test the hypothesis that nApoE41-151 may increase mortality following exogenous treatment in 48 hours post-fertilization (hpf) zebrafish embryos. Parallel experiments will also assess whether trafficking of nApoE41-151 to the nucleus occurs within the nervous system by fluorescence confocal microscopy. In addition, an assessment of developmental abnormalities will be undertaken based on well-defined empirically derived criteria. We will determine if nApoE41-151 leads to toxicity or deformation of any other organ system including the cardiovascular system. Finally, examination for tau pathology following treatment with this fragment could provide a link between ApoE4 and a signature molecular pathology found in the human AD brain. Experiments outlined in Aim 2 will determine what sublethal effects the nApoE41-151 fragment may have on juvenile zebrafish by examining motor behavior as well as memory integrity. Behavior tests for memory and motor functions (assessed by tail flicking and T-maze) will be assessed following treatment of nApoE41-151. Finally, in Aim 3. we will determine the potential deleterious effects of endogenously generated the nApoE41-151 in zebrafish. In this Aim, we propose to generate a new zebrafish strain that expresses nApoE41-151 to determine whether low-level chronic expression leads to similar developmental and behavioral impairments as exogenous treatment. To accomplish this Aim, we will employ a Tol2 transposase method whereby the nApoE41-151 fragment will be expressed. The goal of these experiments will be to generate a permanent transgenic zebrafish line that can be bred in perpetuity in order to fully study the developmental effects in embryos and young juveniles but also continuing in adult zebrafish if warranted Overall, we are proposing an in vivo model system to extend our in vitro findings and to obtain more robust, reliable data that can be extrapolated to an intact organism including humans. Zebrafish are emerging as an effective in vivo model system to study AD for many reasons including the fact that their neuroanatomic and neurochemical pathways share strong similarities with the human brain. In addition, they exhibit a relatively simple nervous system and optical transparency of embryos permit for easy analyses of organ systems and brain development. To strengthen our zebrafish studies, we will also take advantage of the relative ease to express human transgenes in zebrafish to assess in a more physiological milieu the potential long-term effects of expressing this amino-terminal fragment of ApoE4. Using such a model, we hypothesize that the nApoE41- 151 fragment will demonstrate significant developmental abnormalities, motor dysfunction, and memory impairments that will further support a novel role of this fragment in the etiology associated with AD.