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GRADUATE COLLEGE DEFENSE NOTICE
Anatomy and Neurobiology Graduate Program
The following dissertation is open to those in the University community.
Martin Hruska
Advisor: Dr. Rae Nishi, PhD
"Activation of Alpha 7 Subunit Containing
Nicotinic Acetylcholine Receptors Mediates Cell Death
of Neurons In the Avian Ciliary Ganglion”
Thursday, October 18, 2007
1:00 PM
HSRF 400
ABSTRACT
Programmed cell death is 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 a7-nAChRs specific antagonists, a-bungarotoxin or MLA. Since a7-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 a7-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 abtx that is tethered to
the cell membrane via the glycosylphosphatidylinositol anchor (GPIabtx) in the ciliary ganglion neurons inhibits the increases in [Ca2+]i
induced by nicotine through a7-nAChRs specifically. This cell-autonomous inhibition of a7-nAChRs prevents cell death of ciliary and
choroid neurons. For this to happen, GPIabtx 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, a7-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 abtx 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 a7-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 a7-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 a7-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 a7-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 a7-nAChRs during the development of the nervous system.