STATEMENT OF RESEARCH PLAN
My research will focus on the mechanisms by which neurons repair organelles damaged during glutamate receptor-based neurotransmission, and how genetic mutations interfere with this process. Mitochondria are damaged during synaptic activity, and mitochondrial dysfunction is an early symptom of both early- and late-onset cases of Alzheimer's disease (AD), an expanding health care issue that robs patients of their memory and eventually takes their lives. This deficit has been thoroughly documented at the functional and morphological levels. Cells containing significant damage to mitochondria display low levels of ATP, increased production of reactive oxygen specie, and a diminished capacity to maintain the plasma membrane potential. The latter allows intracellular calcium levels to rise, and the neuron eventually dies. However, it is unknown if the mitochondria is a causative factor or merely represents an end-stage deficit in the disease.
Results from a recent study provide evidence supporting the hypothesis that mitochondrial dysfunction resulting from mtDNA damage is a key factor in the development of the late-onset, sporadic form of AD. Many mutations in mtDNA obtained from these patients occur near the PL region, an important regulatory region for replication of the mitochondrial genome (Coskun et al., 2004). This damage blocks mtDNA replication and therefore mitochondrial biogenesis. In support of this possibility, there is a 50 percent reduction in mtDNA copy number in samples from AD patients in comparison to age-matched controls. Mitochondria constantly undergo fission and fusion, are replicated throughout the life of the cell, and removed by autophagocytosis. However, unlike the nucleus, a mitochondrion possesses multiple copies of its DNA, perhaps 4 or 5. So, damage to one copy does not necessarily compromise the organelle, if it can be removed and disposed. However, if a mitochondrion does not get rid of this DNA, it may accrue more damaged copies, and ultimately lose the ability to synthesize the encoded proteins of the respiratory chain. The mitochondrial theory of aging states that enlarged and functionally disabled mitochondria gradually displace normal ones during senescence. These changes are most pronounced in post-mitotic cells such as neurons. The large size suggests that fission is impaired, which would limit mitochondrial turnover. As more mitochondria suffer oxidative damage and accumulate damaged DNA, cellular energy production decreases while reactive oxygen specie formation increases. Ultimately, the neuron dies. While the Coskun paper did not determine how mtDNA is damaged, oxidative stress is the most likely culprit.
A similar scenario could underlie the etiology of early-onset, Familial forms of AD. Mitochondria have few DNA repair mechanisms, thus cells, especially post-mitotic neurons, may have evolved an alternate strategy to deal with these insults, deleting the entire copy of the damaged mtDNA. Perhaps individual copies of mtDNA are targeted for disposal through association with the mitochondrial permeability transition pore. The oxidative stress that follows mtDNA damage would trigger the opening of this pore, and this portion of the mitochondrion, including the damaged DNA, could subsequently be removed by fission.
One function of the APP may be to retrieve DNA, lipids and proteins damaged following oxidative stress, and direct these to the endosomal/lysosomal system for recycling or disposal. Such processing of mitochondrial fragments has not been described in the literature, although autophagocytosis of this organelle has been reported for many models of neurodegeneration. However, the APP is involved in intracellular trafficking, and associates with the mitochondrial permeability transition pore, intriguingly, in a non-glycosylated form. My unpublished results demonstrate that vesicles are formed and bud from mitochondria within minutes of glutamate receptor stimulation. Thus, the APP could serve to guide the useful components of these vesicles through the ER and Golgi as the protein is glycosylated, while damaged proteins, lipids, and DNA would be sorted and targeted to lysosomes for disposal. Alternatively, the initial cleavage of the APP by the alpha- and beta-secretase could target these vesicles for recycling or degradation. Thus, mutations that effect the processing of the APP could alter vesicular transport or targeting, allowing damaged mtDNA to accumulate in mitochondria and eventually kill the neuron, as described in the preceding discussion.
Although I have not obtained evidence for vesicular transport from the mitochondria to the ER, I have many electron micrographs that show intimate associations between the two. A similar set of images is available at the Atlas of Ultrastructural Neurocytology website ( see images 6-14). Figures 1.1.2.7 and 1.1.2.8 are most relevant, and nicely show the stacked subsurface (subplasmalemmal) cisterns formed by narrowing of regular cisterns of granular endoplasmic reticulum, and how they associate with individual mitochondrion. Additionally, similar associations occur between the ER and plasma membrane. Thus, the APP is not functionally constrained to mitochondria in the model I propose.
I will develop a research program to test the hypothesis that the APP and presenilins direct vesicular trafficking of mitochondrial components to recycling and degradative compartments within the cell. I previously demonstrated that glutamate receptor stimulation generates reactive oxygen specie (ROS) that damage mitochondrial components, and leads to a rapid reorganization of subcellular organelles within neurons (Leski et al., 2002). It is proposed that this reorganization forms a sorting assembly that returns functional material to mitochondria while removing damaged proteins, lipids and DNA. This hypothesis will be tested in three specific aims: Specific aim number one will demonstrate that glutamate receptor activity leads to increased mutations in promoter regions of mtDNA. Specific aim number two will prove that irreparable mtDNA is targeted to vacuoles and ultimately disposal. Specific aim number three will demonstrate that the ER, Golgi and lysosomes recycle mitochondrial components following glutamate receptor activity, and that the APP guides vesicles through this network. In addition, a fourth specific aim will develop a cell culture assay to identify novel drug candidates. Successful completion of these specific aims will characterize a novel mitochondrial recycling system, demonstrate that mutations in the APP adversely affect mitochondria recovery following glutamate receptor activity, and identify new compounds that may potentially delay or abolish the progression of AD.
These and other questions will be addressed using primary cultures of rat neurons and possibly human stem cells. Cerebellar granule cells will be generated from mice which express the mutant protein of interest (many such models are now commercially available), and treated with kainate under conditions that selectively activate AMPA-type ionotropic glutamate receptors. Damage to sub-cellular organelles will be assessed using a combination of confocal, epifluorescent and electron microscopy, while standard biochemical techniques will be used to measure damage to mtDNA and individual proteins and enzymes. A fluorescent assay can be developed to screen candidate drugs to treat AD patients. Using such an approach, approximately 100 compounds can be tested per week, with relatively little manpower. At the same time, assays can be developed in collaboration with other investigators to screen drugs for Parkinson's disease, amyotrophic lateral sclerosis, and other neurodegenerative diseases. As candidate drugs are identified, they will be subjected to a secondary screening process using an appropriate animal model.
In summary, my research will characterize a novel mitochondrial recycling process involving proteins that are instrumental in the progression of Alzheimer’s as well as other diseases. Through the basic research proposed herein, I hope to design drugs to treat Alzheimer's as well as other neurodegenerative diseases. This research will identify unique processes that are target for therapies, and will facilitate the work of other investigators.
Outline the preceding essay.
My research will focus on the mechanisms by which neurons repair organelles damaged during glutamate receptor-based neurotransmission, and how genetic mutations interfere with this process. Results from a recent study provide evidence supporting the hypothesis that mitochondrial dysfunction resulting from mtDNA damage is a key factor in the development of the late-onset, sporadic form of AD. A similar scenario could underlie the etiology of early-onset, Familial forms of AD. One function of the APP may be to retrieve DNA, lipids and proteins damaged following oxidative stress, and direct these to the endosomal/lysosomal system for recycling or disposal. Although I have not obtained evidence for vesicular transport from the mitochondria to the ER, I have many electron micrographs that show intimate associations between the two. I will develop a research program to test the hypothesis that the APP and presenilins direct vesicular trafficking of mitochondrial components to recycling and degradative compartments within the cell. These and other questions will be addressed using primary cultures of rat neurons and possibly human stem cells. In summary, my research will characterize a novel mitochondrial recycling process involving proteins that are instrumental in the progression of Alzheimer’s as well as other diseases.
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