INVESTIGATION OF AUTOPHAGY IN HUNTINGTON'S DISEASE USING A MUTANT HUNTINGTIN KNOCK-IN
STRIATAL CELL LINE
Pine Crest SchoolLauren Houle Florida Atlantic University
Background- Huntington’s Disease (HD)
Huntington’s disease (HD) A fatal neurodegenerative
disorder causing neuronal death in the brain
Affects approximately 400,000 – 700,000 people worldwide
Causes accelerated physical, emotional, and cognitive decline Loss of memory Loss of learning ability Inability to control
voluntary movements Psychological issues
http://dailydiseasesanddisorders.tumblr.com/post/6040194582/huntingtons-diseaseImage:
Upper brain- Cross section of a HD brain with decreased brain size as a result of neuronal death, cortical degeneration, enlarged lateral ventriclesLower Brain- Cross section of a normal brain for comparison
HD Brain
Normal Brain
Background- Htt and mHtt The Huntingtin Protein (Htt)
Normal Htt gene has less than ~36 glutamine trinucleotide repeats
Mutant form (mHtt) is responsible for HD mHtt gene has over ~36 trinucleotide
(CAG) glutamine repeats Has many essential functions
Vesicular trafficking, early brain development However, the numerous, exact functions of
the Htt protein remain largely unknown
Cell lines used in project STHdhQ111
HD length- 111 glutamine repeats in polyglutamine tract
STHdhQ7 Normal length- 7 glutamine repeats in
polyglutamine tract
http://www.hinsdale86.org/staff/kgabric/Disease13/Huntington's_Disease/
Image:Top- Normal Htt protein with typical glutamine repeat lengthBottom- Mutant Htt protein with elongated glutamine tract
Background- Autophagy
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3272585/ Autophagy
Intracellular degradation system induced significantly under starvation conditions Degrades unnecessary/dysfunctional cytoplasmic constituents via autophagosomes
and lysosomes Important role in recycling organelles and proteins, replenishing energy, and
providing the cell with vital nutrients Role of Htt in autophagy
Vesicular trafficking in autophagy partly regulated by Htt Anterograde and retrograde motion of autophagosomes and lysosomes
along cytoskeletal fibers assisted by kinesin, dynein, and Htt (Colin E et al.) Impaired autophagy has detrimental consequences
Can cause cellular dysfunction that leads to neuronal death associated with HD
Purpose Understand HD cellular dysfunction on the molecular
level
Elucidate the relationship between the huntingtin protein and vesicular trafficking
Significance of lysosomal positioning and premature autophagosome-lysosome fusion in HD pathogenesis Poüs C et al., Korolchuk VI et al.
Investigate ways to correct localization abnormalities
Hypotheses HD STHdhQ111 Cells
Lesser quantities of autophagosomes under starvation Premature fusion of the autophagosomes and lysosomes
Aggregated lysosomes and autophagosomes localized to the region immediately surrounding the nucleus (perinuclear region)
Inhibited vesicular movement from mHtt interference will hinder travel away from nucleus, causing perinuclear clustering
More lysosomal redirection to the periphery when transfected with fHtt23Q
Lysosomal dispersion when microtubule deacetylation blocked More acetylation= more interactions/activity of motor proteins
Normal STHdhQ7 Cells Lysosomes spread out evenly between periphery and perinuclear
region Greater autophagosomal quantities
No signs of premature fusion More lysosomal redirection to the perinuclear region when transfected
with fHtt145Q No significant differences due to blocking deacetylation of microtubules
MethodsCell Culture Transgenic homozygous STHdhQ7/Q7 or HD
homozygous STHdhQ111/Q111 knock-in mouse cells Cultured in 33°C DMEM (Dulbecco’s Modification
of Eagle’s Medium) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.
Serum Starvation (To Induce Autophagy) Cells deprived of complete media Cells washed with partial media combined with
HBSS (Hank’s buffered saline solution) 2 hour incubationBlocking microtubule deacetylation Cells exposed to Trichostatin A (TSA) overnightTransfection Transiently transfected with pcDNA3.1-full length
Htt23Q or pcDNA3.1-full length Htt145Q and EGFP
Lipofectamine 2000 method fHtt23Q, fHtt145Q, and EGFP genes segments on
separate plasmids.
ImmunofluorescenceTargeted proteins for immunostaining:
LAMP1- Membranous lysosomal protein LC3- Membranous autophagosomal protein Actin- Cytoskeletal actin filament protein Tubulin- Cytoskeletal microtubule protein
1. Fixation in 3.7% paraformaldehyde and 5% sucrose in PBS or methanol and blocking/permeabilization using blocking buffer for 30 minutes 2. Incubation with primary antibodies that bind to target proteins (LAMP1, LC3, Actin, Tubulin) 3. Incubation with secondary antibodies with fluorescent markers
Alexa Fluor 488- green, Alexa Fluor 594- red 4. Prolong® Gold Antifade reagent with DAPI applied directly for blue nuclear fluorescence Modified immunofluorescence protocol for transfected cells
48 hours post-transfection- Incubation with 50nM LysoTracker Red DND-99 at 33°C for 30 minutes Wash with DMEM + Penicillin/streptomycin Fixation in 4% paraformaldehyde solution Rinse and mount on slides with Prolong® Gold Antifade reagent with DAPI
Methods Distance and Intensity
Quantification AxioVision, ImageJ, and ZEN
imaging software Calculated area, localization,
and fluorescent light intensity values
Modified clustering index employed by Falcón-Pérez et al. Ratio of average light intensity
of the perinuclear region to average light intensity of the entire cytosolic region
Value of Ratio
Significance of Value
Ratio < 1 Increased localization to the periphery
Ratio = 1 Even distribution throughout cell
Ratio > 1 Increased perinuclear localization
Lysosomal Accumulation
STHdhQ7-LAMP1-DAPI STHdhQ111-LAMP1-DAPI
Q7-LAMP1-DAPI cells with more evenly distributed lysosomes (red)
and less perinuclear lysosomal localization relative to Q111.
Q111-LAMP1-DAPI cells with aggregated lysosomes (red) and increased perinuclear lysosomal localization compared with Q7.
Blue- NucleusRed- Lysosomes
Control STHdhQ111 cells demonstrate higher lysosomal accumulation in the perinuclear region compared to STHdhQ7 cells as shown in the overall average clustering index value. The high index value (~3.6) of STHdhQ111 cells relative to the index value (~2.2) of STHdhQ7 cells indicates increased localization of lysosomes in the perinuclear region in model HD cells.
Value of Ratio
Significance of Index Value
Ratio < 1 Increased localization to the periphery
Ratio = 1 Even distribution throughout cell
Ratio > 1 Increased perinuclear localization
Htt and Lysosomal DistributionBlue- NucleusRed- LysosomesGreen- Transfected Neuron
STH
dhQ
7-CT
L
STH
dhQ
7-fH
tt14
5Q
STH
dhQ
111-
CTL
STH
dhQ
111-
fHtt
23Q
STHdhQ7 control cell showing little lysosomal perinuclear localization.
STHdhQ7+fHtt145Q cell showing increased lysosomal perinuclear localization.
STHdhQ111 control cell showing increased lysosomal perinuclear localization.
STHdhQ111+fHtt23Q cells showing increased lysosomal peripheral migration.
Expression of fHtt23Q in STHdhQ111 cells promoted the migration and relocation of lysosomes out to the periphery for more even lysosomal distribution.Expression of fHtt145Q in STHdhQ7 cells produced no significant change in lysosomal localization, likely due to low plasmid transfection efficiency for fHtt145Q. Increased perinuclear lysosomal aggregation post-transfection was expected and later observed.
HBSS Starvation and Autophagosomes
Blue- NucleusGreen- Autophagosomes
Control STHdhQ111 cells show higher basal levels of autophagosomes than do STHdhQ7 cells.
Under starvation, STHdhQ111 cells have reduced levels of autophagosomes compared with STHdhQ7 cells with HBSS and STHdhQ111 cells without HBSS.
No observed difference in autophagosomal localization.
- Starvation
+ Starvation
STH
dhQ
7+LC
3ST
Hdh
Q11
1+L
C3
Quantification of the Average Number of Autophagosomes per Cell for Each Cell Line
Higher basal levels of LC3 puncta (autophagosomes) in control STHdhQ111 cells than in control STHdhQ7 cells
The number of autophagosomes per cell in the starved STHdhQ111 cells was significantly less than the number of autophagosomes per cell in the control STHdhQ111 cells (due to premature autophagosome-lysosome fusion)
A higher number of autophagosomes per cell is expected for the starved STHdhQ7 cells compared with the non-starved STHdhQ7 cells. However, slight error occurred from the high confluency of the cell culture of non-starved STHdhQ7 cells mimicking starvation conditions.
No significant changes or differences in autophagosomal localization between the control and HBSS starvation STHdhQ7 and STHdhQ111 cells.
TSA and Lysosomal LocalizationQ
7-LA
MP1
-Tub
ulin
Q11
1-LA
MP1
-Tub
ulin
Blue- NucleusRed- LysosomesGreen- Microtubules
STHdhQ111+TSA cells show signs of increased lysosomal relocation to the periphery and more even lysosomal distribution compared to control STHdhQ111 cells. STHdhQ7 cells experienced no significant change in lysosomal localization in response to TSA exposure.
- TSA + TSA
STHdhQ111+TSA cells have less lysosomal perinuclear localization compared to control STHdhQ111 cells. This indicated that blocking microtubule deacetylation/indirectly promoting microtubule acetylation with TSA encourages anterograde lysosomal motion. STHdhQ111+TSA cells imitate healthy STHdhQ7 cell lysosomal patterns.No significant difference between STHdhQ7+TSA and control STHdhQ7 cells.
Results (summarized) Lysosomes accumulate in the perinuclear region of STHdhQ111 cells but
remain evenly distributed in STHdhQ7 cells Impaired vesicular trafficking in model HD cells Important because the abnormal positioning of HD lysosomes changes the functioning of the
lysosomes
Expression of Htt promotes even lysosomal distribution in model HD STHdhQ111 cells transiently transfected with fHtt23Q and EGFP Direct relationship between huntingtin protein and lysosomal positioning Correlation between expression of normal length huntingtin and more even lysosomal
distribution
Autophagosomal quantities differ between STHdhQ111 and STHdhQ7 cell lines and vary in response to serum starvation Rapid premature fusion of autophagosomes with lysosomes= characteristic of impaired
autophagy Possibly responsible for defects in autophagy leading to decreased cellular health of HD cells
Blocking deacetylation of microtubules causes lysosomal dispersion to cellular periphery in STHdhQ111 cells TSA inhibits histone deacetylases Decreased deacetylation leads to Increased interactions between motor proteins and
microtubules which altogether lead to increased vesicular (lysosomal) transport
Discussion Significant lysosomal localization difference between cell lines
indicates importance of Htt in coordinating lysosomal positioning Lysosomal positioning affects:
Lysosomal functioning Regulation of autophagy Disruptions in localization lead to abnormal regulation of cell functions,
potentially causing the cell death abundant in neurodegeneration Mode of HD pathogenesis via lysosomal localization and premature
autophagosome-lysosome fusion Correlation between mHtt expression, premature fusion, and perinuclear
lysosomal localization These impairments cause neurodegeneration via severe cellular dysfunction
and impairment of crucial intracellular processes
Potential for autophagy-based therapeutic strategy Blocking deacetylation as a mode of localization correction (HDAC inhibitors
like TSA)
Future Research Trials with transfection of STHdhQ7 cells with fHtt145Q
Higher plasmid transfection efficiency Autophagosomal amount quantification to further establish
relationship between mHtt and fusion
New design for blocking microtubule deacetylation TSA inhibits many classes of histone deacetylases (in nucleus
and mitochondria as well as in microtubules) Ensure changes in lysosomal dispersion occurred from blocking
microtubule deacetylation alone
F-actin remodeling Cytoskeletal filaments important in aggresome formation and
degradation of misfolded proteins Acetylated and deacetylated cortactin abundance to assess F-
actin branching Western blots
Acknowledgments Dr. Jianning Wei, Ph.D.
Mentor, Associate Professor of Biomedical Sciences at Florida Atlantic University
Ms. Christine Erie Graduate student, Florida Atlantic
University Ms. Jennifer Gordinier
Research Coordinator, Pine Crest School
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