Keighley Reisenauer

Specific Aims
Leigh Syndrome (LS) is one of the most lethal mitochondrial diseases for infants and children. Between three
months and two years old, LS presents as symmetrical degeneration of the central nervous system and failure
to thrive [1, 7]. LS can present in a variety of ways, all of which will impact longevity. Mutations in SURF1
cause one of the most severe LS phenotypes, which results in death before age six [3]. SURF1, a nuclear
gene, encodes one of 13 subunits comprising cytochrome c oxidase (COX4) complex in mitochondria.
Mitochondria are essential for cellular bioenergetics by way of energy production in the form of ATP, through
the process of oxidative phosphorylation. This crucial task is executed by five mitochondrial multi-protein
complexes; the fourth (IV) complex is terminal in ATP production [2]. LS affects the brain so heavily because
cells/tissues with a high energy demand are hypersensitive to any decrease in mitochondrial function and
energy output. This can lead to cell death, lesions, and loss of motor function, hearing, and sight. The role of
SURF1 in neuron mitochondrial function, however, has not been fully explored.
Single point mutations in the SURF1 gene lead to a premature stop codon, which impairs mitochondrial energy
production. Long-term, I aim to understand how SURF1 mutations lead to cell death and, ultimately, nervous
system degeneration. My primary objective is to determine how loss of SURF1 in mitochondria impacts
neuronal function. It has been understood that energy demands vary across neuronal compartments with
regards to time [4]. Focusing on SURF1 and neuronal mitochondrial function, I will pursue the following three
specific aims:
Aim 1: Determine dynamics of SURF1 in neural cells
Hypothesis: Mutated SURF1 degradation will occur shortly after entering the cytosol and before reaching the
mitochondria.
Approach/Rationale: Use the CRISPR-Cas9 system to tag WT and mutant SURF1 with GFP in mitochondria.
Since mutant SURF1 is truncated and non-functional, I expect to see a decrease in functional localization
because I anticipate cytosolic degradation. By observing GFP location during degradation, the time it takes for
the cell to recognize mutated SURF1 can be extrapolated. Collect time point measurements in D.
melanogaster neural cells to observe SURF1 movement from nucleus to mitochondria. D. melanogaster has
been widely studied in neurodegeneration literature, including LS studies [6]. The fundamental neurological
pathways are conserved between species and, moreover, the SURF1 protein specifically is 46% homologous
[7].
Aim 2: Establish time of protein decay using SILAC quantitative proteomics
Hypothesis: SURF1 will decrease in abundance with age in LS patients and cells will show an increase of
cellular death effects over time.
Approach/Rationale: Use biopsy samples of normal and diseased neuronal cells from D. melanogaster to
study the age of onset and protein degradation of SURF1, as well as cellular death effects, like apoptosis. The
cells will be differentially cultured in media enriched with isotopically-labeled amino acids. This approach will
measure relative levels of SURF1 protein abundance across time, as well as the relative normal levels [5]. By
monitoring protein abundance, I intend to better understand SURF1 degradation in neuronal cells during LS
patient lifespan.
Aim 3: Understand how neurons die in mitochondrial diseases, like LS
Hypothesis: SURF1 mutations result in COX IV defects causing an imbalance of the proton gradient, thus an
increase of lethal ROS, leading to neuronal cell death.
Approach/Rationale: It is known that elevated levels of radical oxygen species (ROS) are linked to apoptosis,
and that neurodegeneration occurs in cells facing defects in respiratory chain function [8]. DCFDA is a known
chemical marker of increased ROS in mitochondria from live cells [9]. DCFDA acts as a fluorescent marker to
H2O2, a proportional downstream form of ROS. Biopsies from Aim 2 will be analyzed for ROS markers over
time to create a correlative timeline between SURF1 degradation and ROS increase over the course of LS.
The identification of neural cell mitochondrial function in response to SURF1 mutations will contribute to the
appreciation of how SURF1 affects mitochondrial function and neuronal cell death. This will further augment
the overall understanding of the nervous system degeneration typical to LS and mitochondrial diseases as a
whole.

Keighley Reisenauer
References
[1] "Leigh Syndrome." Genetics Home Reference: October 2011.
[2] Rak, Malgorzata, et. a. "Mitochondrial cytochrome c oxidase deficiency." Clinical Science: February 2016.
[3] "NINDS Leigh's Disease Information Page." National Institute of Neurological Disorders and Stroke: December 2011.
4] Zsurka, Gabor and Wolfram S. Kunz. Mitochondrial dysfunction and seizures: the neuronal energy crisis. Lancet Neuronal: 2015.
[5] Fierro-Monti, Ivo, et. al. A Novel Pulse-Chase SILAC Strategy Measures Changes in Protein Decay and Synthesis Rates Induced by Perturbation of
Proteostasis with an Hsp90 Inhibitor. Plos One: 2013.
[6] Jiebmann, Astrid and Werner Paulus. Drosophila melanogaster as a Model Organism of Brain Diseases. International Journal of Molecular
Sciences: February 2009.
[7] BLAST Data http://www.ncbi.nlm.nih.gov/homologene/2387
[8] Schon, Eric A. and Giovanni Manfredi. Neuronal degeneration and mitochondrial dysfunction. Journal of Clinical Investigation: February 2003.
[9] Esposti, Mauro Degli. Measuring mitochondrial reactive oxygen species. Science Direct: 2002.