Ashley Lloyd
May 26, 2012
Exploring Caspase-Independent Apoptosis of Type I Spiral Ganglion Neurons
Background
As life expectancy of humans has increased, so has the prevalence of age-related hearing loss. Age-related hearing loss has been associated with the death of Type I Spiral Ganglion Neurons (SGNs) due to glutamate ototoxicity.1 Once hair cells and SGNs die, they are unable to be regenerated; therefore, studying the mechanisms behind cell death in the auditory system is necessary in hopes of discovering preventative measures against destroying cochlear cells. Previous studies have shown that too much glutamate induces cochlear tissue damage.2 Glutamate is the neurotransmitter released from inner hair cells at synapses with Type I SGNs.1
In previous experiments, cochlear cells of wild-type adult guinea pigs were exposed to a high dosage of glutamate (20mM) for 2 hours and fixed 6 hours later. Cochlear cells were then analyzed using immunocytochemistry techniques to look for the prevalence of Apoptosis Inducing Factor (AIF) and Caspase-3, which are both proteins involved in different apoptotic cascades observed in other parts of the central nervous system. The results of the immunocytochemistry showed AIF in the cytoplasm and nucleus of Type I SGNs. Caspase-3 was not shown to be present in Type I SGNs. Nuclear vacuolations were also observed in some cells that had AIF localized in the nucleus, a morphological indication of the start of cell death.2
AIF induces apoptosis in a caspase-independent manner.2 Once released from the mitochondria, AIF has a nuclear localization signal which causes translocation into the nucleus when nuclear import proteins bind. The nuclear localization signal of AIF is composed of a cluster of positively charged amino acids, specifically the 445KLGRRRV451 peptide found on the surface of the protein. Once in the nucleus, AIF binds to DNA, acting to condense and damage DNA, ultimately causing the death of Type I SGNs.3
Hypothesis
If the AIF gene is manipulated causing a point mutation in the nuclear localization signal, then it is hypothesized that translocation of AIF into the nucleus could be prevented, ultimately preventing the caspase-independent apoptotic death cascade in Type I SGNs that occur after exposure to high concentrations of glutamate.
Experimental Design
This experiment will use wild-type mice as the control group and transgenic mice with a point mutation in the nuclear localization signal of AIF as the experimental group. A 20mM glutamate solution will be perfused through a hole in the scala tympani and drained through the scala vestibuli of the mice for 2 hours. The mice will be left for another 6 hours before killing them and fixing cochlear cells to maintain morphology. Immunocytochemistry is used to visualize the localization of AIF. Primary antibodies against AIF will be added, followed by secondary antibodies containing a fluorescent tag. The cellular localization of AIF will be observed using confocal microscopy. Additionally, 4, 6-Diamidino-2-phenylindole (DAPI) will be used to stain DNA to observe the nucleus using confocal microscopy. Images of AIF and DAPI expression will be overlapped in order to show if AIF is localized to the nucleus.
Expected Results
If transgenic mice are grown with a point mutation in the peptide sequence necessary for nuclear localization by replacing a positively charged amino acid with a neutral or negatively charged amino acid, the conformation of the nuclear import signal will be slightly different. Ideally, conformation change would prevent nuclear import proteins from binding AIF and prevent translocation of AIF to the nuclei of the SGNs without altering other functions of AIF. It is expected that if performed correctly, the control group of wild-type mice will show AIF localized to both the cytoplasm and the nucleus of the Type I SGN, whereas the transgenic mice will not show AIF localization in the nucleus. If there is no AIF in the nucleus, it is also expected that nuclear vacuolation will not be observed in these cells.
This hypothesis will be proven incorrect if AIF is observed in the nuclei of the Type I SGNs in the experimental transgenic mouse group. It will also be proven incorrect if nuclear vacuolation is observed in the experimental group. If Type I SGNs show signs of death in the experimental group, it suggests a pathway other than caspase-independent apoptosis may be going on.
Lastly, it should be noted that it is understood that AIF has multiple functions in many different cells besides SGNs. Even within the SGNs, AIF may be necessary for functions other than cell death when it is found in locations other than the nucleus. The goal of this experiment is not to cure glutamate ototoxicity using a genetic mutation, but rather to show with more certainty that apoptosis occurring in Type I SGNs is caspase-independent. This information can then be used to explore pharmacological methods of locally preventing AIF from entering the nucleus in Type I SGNs as a means of preventing age-related deafness due to too much glutamate exposure.
References:
1. Perez, Philip, Jianxin Bao. “Why do hair cells and spiral ganglion neurons in the cochlea die during aging?” Aging and Disease, 2.3 (2011): 231–241.
2. Lu, Haitao, Xiang Wang, Wenyan Sun, Yao Hu, and Shusheng Gong. "New Insights into Glutamate Ototoxicity in Cochlear Hair Cells and Spiral Ganglion Neurons." Acta Oto-Laryngologica, 130.12 (2010): 1316-1323.
3. Irina F. Sevrioukova. “Apoptosis-Inducing Factor: Structure, Function, and Redox Regulation.” Antioxidants & Redox Signaling, 14.12 (2011): 2545-2579.