Research sigma xi presentation 2015

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Investigation of Autophagy in Huntington's Disease using a Mutant Huntingtin Knock-In Striatal Cell Line

Investigation of Autophagy in Huntington's Disease using a Mutant Huntingtin Knock-In Striatal Cell Line

Pine Crest SchoolLauren Houle

Florida Atlantic University

Background- Huntingtons Disease (HD)Huntingtons disease (HD)A fatal neurodegenerative disorder causing neuronal death in the brainAffects approximately 400,000 700,000 people worldwideCauses accelerated physical, emotional, and cognitive declineLoss of memoryLoss of learning abilityInability to control voluntary movementsPsychological 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 comparisonHD BrainNormal Brain

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Background- Htt and mHttThe Huntingtin Protein (Htt)Normal Htt gene has less than ~36 glutamine trinucleotide repeatsMutant form (mHtt) is responsible for HDmHtt gene has over ~36 trinucleotide (CAG) glutamine repeatsHas many essential functionsVesicular trafficking, early brain developmentHowever, the numerous, exact functions of the Htt protein remain largely unknown

Cell lines used in projectSTHdhQ111HD length- 111 glutamine repeats in polyglutamine tractSTHdhQ7Normal 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 length

Bottom- 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 conditionsDegrades unnecessary/dysfunctional cytoplasmic constituents via autophagosomes and lysosomesImportant role in recycling organelles and proteins, replenishing energy, and providing the cell with vital nutrients

Role of Htt in autophagyVesicular trafficking in autophagy partly regulated by HttAnterograde and retrograde motion of autophagosomes and lysosomes along cytoskeletal fibers assisted by kinesin, dynein, and Htt (Colin E et al.)

Impaired autophagy has detrimental consequencesCan cause cellular dysfunction that leads to neuronal death associated with HD

Image: Pathway of AutophagyA membrane (Phagopore) begins to elongate around specific cytoplasmic constituents (cargo)Membrane encloses this cargo to form the autophagosome

Impaired autohpagy-> inability to coordinate cellular functions properly neuronal deathAutophagosomes travel around the cell on cytoskeletal actin filaments and microtubules to locate lysosomesAutophagosomes fuse with lysosomes and are degraded along with their cargo

AutophagyThe intracellular degradation system that breaks down damaged or superfluous cellular componentsInduced significantly under starvation conditions Striatal cells with HD exhibit signs of inefficient, altered autophagy as a result of the production of mHtt Consequences of impaired autophagyLack of energy and nutrientsInability to dispose of excess/faulty proteins and organelles

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PurposeUnderstand 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 pathogenesisPos C et al., Korolchuk VI et al.

Investigate ways to correct localization abnormalities

HypothesesHD STHdhQ111 CellsLesser quantities of autophagosomes under starvationPremature fusion of the autophagosomes and lysosomesAggregated 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 clusteringMore lysosomal redirection to the periphery when transfected with fHtt23QLysosomal dispersion when microtubule deacetylation blockedMore acetylation= more interactions/activity of motor proteinsNormal STHdhQ7 CellsLysosomes spread out evenly between periphery and perinuclear regionGreater autophagosomal quantitiesNo signs of premature fusionMore lysosomal redirection to the perinuclear region when transfected with fHtt145QNo significant differences due to blocking deacetylation of microtubules

Methods

Cell Culture Transgenic homozygous STHdhQ7/Q7 or HD homozygous STHdhQ111/Q111 knock-in mouse cells Cultured in 33C DMEM (Dulbeccos Modification of Eagles 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 (Hanks buffered saline solution)2 hour incubationBlocking microtubule deacetylationCells exposed to Trichostatin A (TSA) overnightTransfectionTransiently transfected with pcDNA3.1-full length Htt23Q or pcDNA3.1-full length Htt145Q and EGFPLipofectamine 2000 methodfHtt23Q, 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 33C 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

The purpose of the immunofluorescence is to mark the lysosomes, autophagosomes, actin filaments, and microtubules with red or green fluorescence so as to view their positions relative to each other in the cell. DAPI staining requires only a drop of Prolong Gold Antifade Serum on the coverslip when mounting to make the nucleus fluoresce blue.

Step 1 Fixation for LC3: For LC3 staining, cells were instead fixed with methanol and blocked with blocking buffer

Primary Antibody Incubation Dilution: 1:500 Dilution in 1% NGS, 1% BSA in PBS bufferSecondary Antibody Incubation Dilution: 1:2000 Dilution in 1% NGS, 1% BSA in PBS buffer

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MethodsDistance and Intensity QuantificationAxioVision, ImageJ, and ZEN imaging software Calculated area, localization, and fluorescent light intensity valuesModified clustering index employed by Falcn-Prez et al.Ratio of average light intensity of the perinuclear region to average light intensity of the entire cytosolic region

Value of RatioSignificance of ValueRatio < 1Increased localization to the peripheryRatio = 1Even distribution throughout cellRatio > 1Increased perinuclear localization

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Lysosomal AccumulationSTHdhQ7-LAMP1-DAPISTHdhQ111-LAMP1-DAPIQ7-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 RatioSignificance of Index ValueRatio < 1Increased localization to the peripheryRatio = 1Even distribution throughout cellRatio > 1Increased perinuclear localization

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Htt and Lysosomal DistributionBlue- NucleusRed- LysosomesGreen- Transfected Neuron

STHdhQ7-CTLSTHdhQ7-fHtt145QSTHdhQ111-CTLSTHdhQ111-fHtt23QSTHdhQ7 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.

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HBSS Starvation and AutophagosomesBlue- 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+ StarvationSTHdhQ7+LC3STHdhQ111+LC3

Quantification of the Average Number of Autophagosomes per Cell for Each Cell LineHigher basal levels of LC3 puncta (autophagosomes) in control STHdhQ111 cells than in control STHdhQ7 cells The number of auto