Date of Award
Doctor of Philosophy (PhD)
Programmed cell death is a widespread phenomenon in the developing nervous system. During early development, neurons are initially produced in excess and up to 70% of them are eliminated in later stages of development, during a period of synapse formation with their targets. However, the mechanisms that initiate the death of neurons are not clear. In the avian ciliary ganglion, neurons go through the period of target-dependent cell loss between E8 and E14; however, almost all neurons in the ganglion are prevented from dying by the chronic in ovo treatment with α7-nAChRs specific antagonists, α- bungarotoxin or MLA. Since α7-nAChRs are implicated in the cell death of ciliary ganglion neurons, I tested whether the activation of these receptors directly on the ciliary ganglion neurons facilitates cell death by inducing large increases in intracellular Ca2+. I found that the ciliary ganglion neurons are heterogeneous with respect to their surface α7-nAChR density and, as a result, activation of these receptors by nicotine leads to large increases in [Ca2+]i in some neurons but not in others. Furthermore, immature E8 neurons exhibit slower rates of Ca2+ decay after nicotine stimulation than E13 neurons, suggesting that E8 neurons do not clear [Ca2+]i efficiently and could be more susceptible to Ca2+ overload. Expressing the αbtx that is tethered to the cell membrane via the glycosylphosphatidylinositol anchor (GPIαbtx) in the ciliary ganglion neurons inhibits the increases in [Ca2+]i induced by nicotine through α7-nAChRs specifically. This cellautonomous inhibition of α7-nAChRs prevents cell death of ciliary and choroid neurons. For this to happen, GPIαbtx must be expressed in neurons; the expression of this construct in the surrounding non-neural tissue does not prevent neuronal loss in the ciliary ganglion. Later in development, α7-nAChRs are prevented from inducing cell death by the chicken PSCA molecule that is significantly upregulated in the ciliary ganglion between E8 and E15. The chicken PSCA is neuronal specific molecule that belongs to the Ly-6/neurotoxin superfamily that includes αbtx and lynx1 and compared to other tissues, it is highly expressed in the ciliary ganglion. The expression of the PSCA mRNA in tissues correlates with the expression of α7-nAChR mRNA, suggesting that PSCA modulates the signaling via these receptors. In fact, overexpressing the PSCA in the ciliary ganglion neurons prevents nicotine-induced increases in [Ca2+]i through α7- nAChRs. Misexpressing the PSCA in E8 ciliary ganglion prevents choroid but not ciliary neurons from dying. Therefore afferent inputs can induce cell death by activation of α7- nAChRs in the developing ciliary ganglion by increasing the [Ca2+]i over the threshold for cell death. Upregulation of endogenous prototoxins, such as PSCA, opposes the large increases in [Ca2+]i via α7-nAChRs and prevents these channels from facilitating cell death after the final numbers of neurons have been established. These results indicate that the control of cell death is more complex than originally proposed by the neurotrophic hypothesis and present the mechanism by which cell death in the developing ciliary ganglion is regulated, thus, further highlighting the importance of non-traditional roles of α7-nAChRs during the development of the nervous system.
Hruska, Martin, "Activation of Alpha7 Subunit Containing Nicotinic Acetylcholine Receptors Mediates Cell Death of Neurons in the Avian Ciliary Ganglion " (2008). Graduate College Dissertations and Theses. 112.