8/11/2019 Copy of 140605 - PsychFest - Lipid Droplets in Hepatocytes

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Hepatic Phenotypes in Isolated HttQ111/Q7 Hepatocytes

1 BehavioralNeuroscienceProgram, Departmentof Psychology, Western Washington University.

AJ Keefe 1 1

Abstract

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Background

Funding provided by: Huntington Society of Canada

Huntingtons Disease (HD) is an autosomal dominant genetic disorder caused by an expanded poly-

glutamine repeat within the Huntingtin gene. This disorder causes broad neurodegeneration but is

most pronounced in a subcortical brain structure called the striatum. While the striatum and other

related neural circuits have received a great deal of research, studies examining whole body pathol-

ogy have been largely ignored. Due to the nearly ubiquitous expression of Huntingtin in the human

body, examining the location of the protein within hepatocytes will be vital to characterizing hepat-

ic phenotypes in HD. Hepatocytes were isolated from three month old Q111 HD mouse model mice

via portal vein perfusions and plated onto gelatin coated cover slides. Immunocytochemistry was

used to identify the location of the Huntingtin protein and 4',6-diamidino-2-phenylindole (DAPI)

staining was used to visualize nuclei. Mutant mice displayed larger lipid bodies versus wildtype-

 Thedamage caused by Huntingtons Diseaseultimately stems from the mutation ofa gene that is later translat-

ed into a toxic protein.H untingtin (HTT) has been found to interact with over 100other proteins,but theprim a-

ry roleof HTT is disputed.The protein is expressed in many peripheral organs such as thekidney and liver,sug-

alterations in HDpatients that arenot associated with neuronal function such as immunopathology,weight

loss,cancer resistance, and dysfunctional methionine metabolism.The latter is a well established diagnostic

could helpelucidate theproteins fundamental activity and even support a theory ofcell autonomous neurode-

generation.

 Thehuntingtin protin has been found to act as transcriptional regulator for a multitudeof genes.Using

which preceded any transcriptional dysregulation in thebrain. This data suggests that theearliest pathological

such as thestriatum.

protein serves within thesecells.The protein is commonly found ubiquitously distributed throughout thecyto-

plasm with no clear subcellular localization.It is important to notethat the distribution ofHTT is unique from

thedistribution of theaggregates ofhuntingtin. Theaggregated form ofHTT is theresult proteolytic activity,

and does not sharehomology to the original protein.This research reveals that even full length wild-type

and not aggregation.In addition to HTT localization,nuclei were analyzed for shapeand size,and polynucleated

frequency.Nuclei shape and sizeis an indicator of diseases such as cancer or laminopathies and can even be

altered by lipid catabolism.Lipid droplets wereanalyzed for size and density as lipid metabolism is an impera-

tivefunction of hepatocytes,and any metabolic perturbations between genotypes would contributegreatly to

theunderstandingofHDpathology.

Huntingtin location within the secretory pathway

Huntingtin aggregate antibody

Regulated Secretory Secretion

Retrograde

Golgi-to-ER

 Transport

Cis-Golgi Reticulum  Trans-Golgi ReticulumEndoplasmic Reticulum

Proteins are

synthesized

directly into

the ER lumen

Constitutive Secretion

Lysosome & Endosome Sorting

111 Hepatocytes

Big Questions

Each step in this pathway is marked with unique proteins that serve unique cellular functions!

- Why does Huntingtin appe

using additional antibodies

- Will other cells show this st

 The next step in this study

raised under a 60% (high fa

fat diet (15%). The liver orch

could materialize in many w

location may be subsequen

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Retrograde Transport

Extracellular

Matrix

- Why do HD hepatocytes h

 Top:Control imagerepresenting theimmunocy-tochemical protocol minus theprimary antibody.

 This effectively measures theproportion of stainingthat is dueto theprimary antibodyversus artifacts and fluorescent static.Note the

fluorescencein thenuclei. This is likely duetooverlappingabsorption and emission spectrabetween theFITCand DAPI wavelengths.Due tothis noise,intranuclear HTT staining could not beaccessed

but did not display an increase in number of lipid droplets per cell. Nuclei did not display any

significant trends.

 This research has revealedof huntingtin staining in Qmunocytochemical analyduces a diffuse and nonsption of HTT. Although the

be a phenotype unique tostaining pattern suggestsin some kind of vesicuar t

 The following are antibodsidered for colocalization

Lamp1-“Lysozomeassociated memb

Lysosomes areresponsiblefor digest

carbohydrates and nucleic acids.

Atg3-“Autophagocytosis associated p

autophagocytosis This process is resp

mic proteins to thelysosomes for deg

RAB5A -“Ras-related protein 5A”mark

somes sort proteins and lipids for de

membrane,or to thetrans-Golgi netw

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Lipid droplet size

Distribution ofLipid Droplet Size