The Regulation of Mitophagy in a Cellular Model of Huntington’s Disease
Nikolle Lambrinos1, Matthew Sacino2, Jianning Wei, Ph.D.2
1Honors Thesis Undergraduate Program,
Department of Biological Sciences, Charles E. Schmidt College of Science
2Charles E. Schmidt College of Medicine
Florida Atlantic University, Boca Raton, Florida, 33431
Experimental Design
Abstract
Huntington’s disease (HD) is an inherited, autosomal dominant
neurodegenerative disorder that is currently incurable. The
accumulation of damaged mitochondria within neurons is one
factor thought to play a role in HD pathogenesis, given that it leads
to adverse effects on neuron physiology. In healthy individuals, the
mitophagy, or “mitochondrial autophagy”, pathway regularly
degrades the damaged mitochondria. Recent studies indicate that
the mitophagy pathway is impaired in HD. In the present study, we
investigated the molecular mechanisms underlying impaired
mitophagy in a cellular model of HD using plasmid transfection,
Western blot, and immunofluorescence techniques. Our current
data suggest that the mutant huntingtin protein (mHtt) may
interfere with the proper clearance of the damaged
mitochondria, an important process in the Parkin-mediated
mitophagy pathway. An understanding of such molecular
pathways that are altered by mHtt expression is important for the
discovery of novel drug targets for HD treatment.
Aim 1 – Analyze mitochondrial fragmentation in neuronal cell lines
derived from healthy and HD transgenic mice.
Establish healthy
and HD neuronal
cell lines
Stain
mitochondria
with:
Aim 2 – Determine the mechanism of Parkin-mediated mitophagy
under stress-induced (w/CCCP treatment) and stress-free
conditions.
COXIV: mitochondrial
fragmentation
Mitotracker Red CMXRos:
Δψm
Background
Fluorescence microscopy reveals fragmented mitochondria
in HD neuronal cells
Normal
htt-EGFP
mCherry-Parkin
merged
n-httQ103
CTRL
Cytosolic Parkin translocates to the mitochondria in
normal (Q23) and HD (Q145) PC12 neuronal cells in
response to CCCP treatment
n-httQ103
+CCCP
(24hr)
Ub
merged
publications.nigms.nih.gov
Q23
+CCCP
(90’)
damaged
mitochondrion
Future
uture Work
Figure 3. Confocal microscopy reveals a decrease in Tom20 (blue) in
n-httQ25 cells in response to 24 hour CCCP treatment. The mitochondria
of n-httQ25 cells disappear in response to CCCP treatment, while the
mitochondria of n-httQ103 cells form clusters. Cells co-transfected with httEGFP (green) and mCherry-Parkin (red). Scale bar = 10 µm.
Parkin
Q145
CTRL
A.
Q23
CCCP
0hr 1hr
3hr
Q145
7hr
0hr
1hr
3hr
7hr
84 kDa
Mfn1
43 kDa
beta-Actin
B.
% Mfn1/beta-Actin in PC12 cells
with and without CCCP treatment
Quantify mitochondrial
ubiquitination levels.
References
Q145
+CCCP
(90’)
1.  Zuccato, C., M. Valenza, et al. (2010). "Molecular Mechanisms and Potential
Therapeutical Targets in Huntington's Disease." Physiological Reviews 90(3):
905-981.
2.  Arias, E., A. M. Cuervo, et al. (2010). "Cargo recognition failure is responsible for
inefficient autophagy in Huntington's disease." Nature Neuroscience 13(5): 567-578.
3.  Steffan, J. S. (2010). "Does Huntingtin play a role in selective macroautophagy?" Cell
cycle (Georgetown, Tex.) 9(17): 3401-3413.
lysosome
Nature Reviews Molecular Cell Biology
HYPOTHESIS
We hypothesize that mitophagy is impaired in HD neuronal
cells, ultimately leading to the accumulation of damaged
mitochondria.
POSTER TEMPLATE BY:
Determine if mHtt forms
aggregates with Parkin
near or on the
mitochondrial membrane.
ubiquitin
•  Ubiquitin tagging mediates the
degradation of the damaged
mitochondria by the lysosomes.
www.PosterPresentations.com
Analyze downstream
targets in the Parkinmediated mitophagy
pathway within our
neuronal cell lines.
Mitofusin-1 (Mfn1) levels decrease in response to
CCCP treatment in normal (Q23) neuronal cells, but
remain the same in HD (Q145) neuronal cells
•  Parkin = E3-ubiquitin ligase
•  Mitophagy is induced when
cytosolic Parkin ubiquitinates
the proteins on the outer
mitochondrial membrane under
stress conditions.
•  Levels of Mfn1 (a mitochondrial outer membrane protein
responsible for the fusion of the mitochondria) decrease when
exposed to CCCP; this indicates the occurrence of mitophagy in
normal PC12 cells, but not in PC12 cells expressing mHtt.
n-httQ25
+CCCP
(24hr)
Figure 1. Immunofluorescent analysis of mitochondrial morphology in normal
and HD neuronal cell lines via confocal microscopy. The mitochondria in normal
cells (left panel) appear elongated and tubular in structure. The mitochondria in HD
cells (right panel) appear short and fragmented. Nuclei stained with DAPI (blue);
mitochondria stained with COXIV (green). Scale bar = 20 µm.
EYFP-Parkin
•  There is an accumulation of fragmented (damaged)
mitochondria in HD neuronal cells, but not in healthy neuronal
cells.
•  Mitochondria (Tom20) levels decrease in normal HeLa cells in
response to CCCP treatment; Tom20 levels generally remain
the same in HeLa cells expressing mHtt, suggesting impaired
mitophagy during exposure to CCCP.
n-httQ25
CTRL
Q23
CTRL
Parkin-mediated mitophagy
Mitochondria levels decrease in
normal (n-httQ25) HeLa cells in response
to CCCP treatment
HD
•  The mitochondria of healthy neuronal cells exhibit elongated
and tubular morphology, while the mitochondria of HD neuronal
cells appear fragmented.
•  Cytosolic Parkin appears to translocate to the mitochondria and
ubiquitinate the outer membrane upon stress (CCCP treatment)
in both PC12 inducible neuronal cell lines (Q23 and Q145).
Tom20
Tom20
mitochondrion
Immunofluorescence
Preliminary Results
The HTT gene
•  Vital for neuronal
function
•  Mutation: “CAG”
repeat expansion
•  CAG = Glutamine (Q)
•  Strong correlation between mHtt expression and the
accumulation of damaged and fragmented mitochondria
within HD neuronal cells [1,2,3].
Perform Western blot
to analyze Parkin
expression/ protein
ubiquitination levels
Isolate
mitochondria
Transfect cells w/
Parkin-YFP &
HA-Ub plasmids
COXIV
DAPI
Overview of Huntington’s disease (HD)
•  Autosomal dominant
•  Mutation in the Huntingtin gene (HTT) results in HD phenotype
•  motor control, cognitive function, psychological stability
•  Basal ganglia = highly vulnerable
•  30,000 affected nationwide
•  Currently incurable
Discussion
Figure 2. Subcellular localization of Parkin in Q23 and Q145 PC12 cells, as
determined by confocal microscopy. Mitochondria stained with anti-Tom20 antibody
(blue). Parkin is completely translocated to the mitochondria in both normal (Q23) and
HD (Q145) neuronal cells in response to 90 minutes of CCCP treatment, as shown in
the merged images. Cells transfected with EYFP-Parkin (green). Scale bar = 10 µm.
Figure 4. Distribution of Mfn1 (84 kDa) and beta-Actin (43 kDa) in PC12
neuronal cells. A) Western blot analysis of Mfn1 expression when exposed
to CCCP for 0, 1, 3, and 7 hours. B) Qualitative analysis of Mfn1 expression
in PC12 cells exposed to CCCP for 0 and 7 hours.
Acknowledgements
We thank the FAU Honors Thesis program, Dr. Evelyn Frazier, Dr. John Nambu, Ramon GarciaAreas, and fellow Honors peers for their support. We also thank the 2012-2013 FAU Undergraduate
Research Grant and the NINDS grant R15NS066339 for funding our project.