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

Works Cited1. "Autophagy." Sigma-Aldrich. Sigma-Aldrich, 2010. Web. 1 Dec. 2013. <http://www.sigmaaldrich.com/life-science/cell-biology/cell-biology-products.html?TablePage=1048994>. 2. Bhuwania R, Castro-Castro A, Linder S. Microtubule acetylation regulates dynamics of KIF1C-powered vesicles and contact of microtubule plus ends with podosomes. Eur J Cell Biol. 2014 Aug 2. pii: S0171-9335(14)00102-2.3. Caviston JP, Zajac AL, Tokito M, Holzbaur EL. Huntingtin coordinates the dynein-mediated dynamic positioning of endosomes and lysosomes. Mol Biol Cell. 2011 Feb 15;22(4):478-92. 4. "CH00095." Coriell Institute for Medical Research. Coriell Institute, 2009. Web. 20 Jan. 2014. <http://ccr.coriell.org/Sections/Search/Sample_Detail.aspx?Ref=CH00095>. 5. "CH00097." Coriell Institute for Medical Research. Coriell Institute, 2009. Web. 20 Jan. 2014. <http://ccr.coriell.org/Sections/Search/Sample_Detail.aspx?Ref=CH00097>. 6. Chicurel, Marina. "Autophagy in the Pathogenesis and Treatment of Huntington’s Disease." Hdfoundation.org. Hereditary Disease Foundation, 1-2 Oct. 2005. Web. 2 Dec. 2013. <http://www.hdfoundation.org/PDF/Autophagy_WS_Rpt-Oct05.pdf>. 7. Colin E, Zala D, Liot G, Rangone H, Borrell-Pagès M, Li X, Saudou F, Humbert S. Huntingtin phosphorylation acts as a molecular switch for anterograde/retrograde transport in neurons. EMBO J. Aug 6, 2008; 27(15): 2124-2134.8. Dehay B, Martinez-Vicente M, Caldwell GA, Caldwell KA, Yue Z, Cookson MR, Klein C, Vila M, Bezard E. Lysosomal impairment in Parkinson's disease. Mov Disord. 2013 Jun;28(6):725-32.9. Dompierre JP, Godin JD, Bénédicte CC, Cordeliéres FP, King SJ, Humbert S, Saudou F. Histone Deacetylase 6 Inhibition Compensates for the Transport Deficit in Huntington's Disease by Increasing Tubulin Acetylation. The Journal of Neuroscience, 28 March 2007, 27(13): 3571-3583.10. Falcón-Pérez JM, Nazarian R, Sabatti C, Dell’Angelica EC. Distribution and dynamics of Lamp1-containing endocytic organelles in fibroblasts deficient in BLOC-3. J Cell Sci. 2005 Nov 15;118(Pt 22):5243-55.11. Gauthier LR, Charrin BC, et al. Huntingtin Controls Neurotrophic Support and Survival of Neurons by Enhancing BDNF Vesicular Transport along Microtubules. Cell. 2004 Jul 9;118(1):127-38.12. "Huntington's Disease (HD)." Lundbeck. Lundbeck, 7 May 2013. Web. 17 Jan. 2014. <http://www.lundbeck.com/us/our-commitment/disease-information/huntingtons-disease-hd>.13. "Huntington’s Disease Comparisons." HOPES. Stanford University, 26 June 2010. Web. 12 Apr. 2013. <http://web.stanford.edu/group/hopes/cgi-bin/wordpress/2010/06/huntingtons-disease-comparisons/>.14. "Intel International Science and Engineering Fair: International Rules and Guidelines 2014." Intel ISEF Rules & Guidelines 2014. Society for Science & the Public, 2014. Web. 28 Nov. 2013. <http://member.societyforscience.org/document.doc?id=398>. 15. Koga H, Martinez-Vicente M, Arias E, Kaushik S, and Cuervo AM, et al. Constitutive Upregulation of Chaperone-mediated Autophagy in Huntington’s Disease. J Neurosci. 2011 Dec 14;31(50):18492-505.

16. Korolchuk VI, Saiki S, Lichtenberg M, Siddiqi FH, Roberts EA, Imarisio S, Jahreiss L, Sarkar S, Futter M, Menzies FM, O’Kane CJ, Deretic V, Rubinsztein DC. Lysosomal positioning coordinates cellular nutrient responses. Nature Cell Biology 13, 453–460 (2011).17. Leon R, Bhagavatula N, Ulukpo O, McCollum M, Wei J. BimEL as a Possible Molecular Link between Proteasome Dysfunction and Cell Death Induced by Mutant Huntingtin. Eur J Neurosci. 2010 Jun;31(11):1915-25.18. "Lipofectamine® 2000." Life Technologies. Life Technologies Corporation, 11 July 2006. Web. 3 Dec. 2013. <http://www.lifetechnologies.com/us/en/home/references/protocols/cell-culture/transfection-protocol/lipofectamine-2000.html>. 19. Lodish, Harvey. "Section 19.3- Kinesin, Dynein, and Intracellular Transport." Molecular Cell Biology. 4th ed. New York City: W. H. Freeman and, 2000. N. pag. Kinesin, Dynein, and Intracellular Transport. U.S. National Library of Medicine, 2000. Web. 30 Nov. 2013. <http://www.ncbi.nlm.nih.gov/books/NBK21710/>. 20. Martinez-Vicente M, Talloczy Z, Wong E, Tang G, Koga H, Kaushik S, Arias E, Cuervo AM, Sulzer D, Harris S, de Vries R. Cargo Recognition Failure is Responsible for Inefficient Autophagy in Huntington’s disease. Nat Neurosci. May 2010; 13(5): 567–56.21. Mizushima N. Autophagy: Process and Function. Genes Dev. 2007 Nov 15;21(22):2861-73.22. Mizushima N, Yoshimorim T, Levine B. Methods in Mammalian Autophagy Research. Cell. Feb 5, 2010; 140(3): 313–326.23. Orr ME, Oddo S. Autophagic/lysosomal dysfunction in Alzheimer’s disease. Alzheimer's Research & Therapy 2013, 5:53.24. Orr A, Li S, Wang C, Li H, Wang J, Rong J, Xu X, Li X, Mastroberardino P, Greenamyre JT. N-Terminal Mutant Huntingtin Associates with Mitochondria and Impairs Mitochondrial Trafficking. J Neurosci. Mar 12, 2008; 28(11): 2783–2792.25. Pipathsouk, A. "About Autophagy." HOPES. Stanford University, 26 June 2011. Web. 1 Dec. 2013. <http://www.stanford.edu/group/hopes/cgi-bin/wordpress/2011/06/about-autophagy>. 26. Poüs C, Codogno P. Lysosome positioning coordinates mTORC1 activity and autophagy. Nature Cell Biology 13, 342–344 (2011)27. Stenerson, M. "The Basics of Huntington’s Disease." HOPES. Stanford University, 26 June 2011. Web. 28 Jan. 2014. <https://www.stanford.edu/group/hopes/cgi-bin/wordpress/2011/06/the-basics-of-huntingtons-disease-text-and-audio/>.28. Wang C, Klionsky DJ, et al. The Molecular Mechanism of Autophagy. Mol Med. 2003 Mar-Apr; 9(3-4): 65–76.29. Wu L, Zhou XF. Huntingtin associated protein 1 and its functions. Cell Adh Migr. 2009 Jan-Mar; 3(1): 71–76.30. Zuccato C, et al. Loss of Huntingtin-mediated BDNF Gene Transcription in Huntington's Disease. Science. 2001 Jul 20;293(5529):493-8.31. Zuccato C, Cattaneo E, et al. Role of Brain-derived Neurotrophic Factor in Huntington’s Disease. Prog Neurobiol. 2007 Apr;81(5-6):294-330.