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Bad"prin/n

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Background:0 0 Parkinson’s" disease" (PD)" is" the" second" most"common"neurodegenera/ve"disease" in" the"United"States." "The"onset"of"PD"is"characterized"by"the"death"of"dopamine"neurons"in" the" substan/a"nigra." " In" order" to" understand" the"molecular"mechanisms"of"PD,"it"is"desirable"to"culture"a"dopamine"cell"line"to"serve"as"an" in#vitro"model"of"the"disease." "In"the"1990’s,"our"laboratory"developed"a"dopaminergic"cell"line"derived"from"fetal"rat" mesencephalic" dopamine" neurons" immortalized" with" the"SV40"large"T"An/gen.""These"were"named"N27"cells."The"original"N27" cell" line" was" a" heterogeneous" mixture" of" cell" types"expressing"highly"variable" levels"of" tyrosine"hydroxylase" (TH),"a"primary" marker" for" the" dopamine" neuron" phenotype," our"objec/ve"was"to"culture"and"isolate"single"N27"cells"with"a"high"level" of" expression" of" TH," and" other" markers" of" authen/c"dopamine"neurons"to"create"a"more"homogenous"cell"line."

Fig.01:0Substan/a"nigra"of"control"(leW)"and"substan/a"nigra"of"a"Parkinson’s"pa/ent"(right)."Note"that"the"substan/a"nigra"of"the"Parkinson’s"pa/ent"is"diminished"and"has"lost"its"dark"brown"coloring."This"

is"characterized"by"the"death"of"dopamine"neurons."

ABSTRACT0 METHODS0

N270Cell0SelecEon:0N27"cells"were"first"plated"at"a"very"low"density"in"a"10Ycm"dish."AWer"a"few"days"of"growth,"the"cells"began"to"form"colonies."Twelve"of" these" colonies" were" then" selected" and" plated"into"a"12Ywell"plate"for"expansion."AWer"expanding"the" culture" in" duplicate," cells" were" treated" with"differen/a/ng" agents" or" were" maintained" as""undifferen/ated"cells.0

Immunohistochemistry:" The" cultures" were" fixed"with" 4%" Paraformaldehyde" for" 30" minute," then"rinsed"with"PBS." Cells"were" incubated"with" rabbit"an/Ytyrosine"hydroxylase"an/body"(1:200"dilu/on,"PelYFreez)" overnight" at" room" temperature," and"with" goat" an/Yrabbit" Alexa" 488" (1:200" dilu/on,"Invitrogen)" in" the" dark" for" two" hours" at" room"temperature." Finally," cells" were" rinsed" with" PBS"again," and" checked" for" immunostaining" under" a"fluorescent"microscope."

Western0 BloJng:0 The" cells" were" dissected" and"homogenized" in" dissocia/on" buffer"with" protease"inhibitors." Protein" concentra/on" was" determined"by" the" BCA" method." " 100" µg" of" protein" was"separated"on"12%"MiniYProtean"TGX"gel" (BioYRad)"and"transferred"to"a"nitrocellulose"membrane."Blot"was"probed"with"an/bodies"to"TH"(1:2,000,"Sigma)"and"βYac/n"(1:4000,"Sigma)."Blots"were" incubated"with" HRPYconjugated" secondary" an/body"(1:10,000;"Jackson"Immuno"Research),"followed"by"chemiluminescent"detec/on.0

RESULTS0

SUMMARY0

• The" N27" cell" line" is" a" heterogeneous" mixture" of" cell"types" expressing" highly" variable" levels" of" tyrosine"hydroxylase" (TH)," a" primary" marker" for" the" dopamine"neuron"phenotype."

• A" subclone" of"N27" cells" contains" small" clusters" of" cells"expressing"high" levels"of"TH."Upon"differen/a/on," these"TH" posi/ve" N27" cells" change" their" morphology" and"become"neuronal"shaped"with"long"neurites"

• The"process"of"enriching"and" isola/ng"single"cell"clones"expressing"high"amounts"of"TH."

FUTURE00RESEARCH0

• Con/nue"clonal"selec/on"• Isola/on"of"other"dopamine"markers,"DAT"

• Response"to"dopamine"neuron"toxins,"6YOHDA"and"MPP+"

• Transplant"cells"into"Parkinsonian"animal"models"

ACKNOWLEDGMENTS0Curt0Freed0Laboratory0Brent"Fitzwalter,"PhD"Candidate"

Breanna"Symmes,"PhD"(PostYdoc)"

Esteban"Lucero,"Graduate"Student"

Mary"Wang,"PRA"Kim"Bjugstad,"PhD"

Lu"Gao,"PhD"

Augustana0College0Ian"Harrington,"PhD"

Shara"Stough,"PhD"

Kevin"Geedey,"PhD"

Stephanie"Fuhr,"MS"

Wartburg0West0Program0Bonita"and"Nelson"Bock"

This research has been made possible by support from the Wartburg West Program, and by a grant from The Walter and Lucienne Driskill Foundation."

Div."of"Clinical"Pharmacology,"Dept"of"Medicine,"Neuroscience"Program,"Gates"Center"for"Stem"Cell"Biology,"and"Human"Medical"Gene/cs"and"Genomics"Program,"University"of"Colorado"Denver,"Aurora,"CO"80045"

*Neuroscience"and"Biology"Programs,"Augustana"College,"Rock"Island,"IL"61201"

Marcela"Fitzpatrick*,"Megan"Petersohn*,"Wenbo"Zhou,"PhD.,"Curt"Freed,"MD."Purifying0a0dopamine0cell0line0to0improve0a0cell0culture0model0of0Parkinson’s0disease"

Fig.0 4:0 (a)" N27" cells" five" day" postYdifferen/a/on," showing" uniform"neuronal" morphology" with" phase"contrast" (b)" This" image" represents"a" good" dopamine" phenotype"(elongated," spindle" shape)" and"posi/ve" TH" staining." Although" the"well" is" confluent," there" is" only" a"‘hot" spot’" of" TH" fluorescence." (c)"Although" these" cells" were"differen/ated," they" were" s/ll"dividing"slowly"into"what"appeared"to"be"sister"pairs."Small"numbers"of"these"sister"pairs"show"very"bright"TH" staining" of" green"fluorescence."It" is" expected" that" once" cells" are"differen/ated" they" should" stop"dividing," however" since" this" cell"line" is" immortalized"differen/a/ng"agents" do" not" inhibit" the" process"of"cellular"division."

Low"density"pla/ng"

Forma/on"of"colonies"

Culture"expansion"

Differen/ate/Freeze"

Immunostain"for"tyrosine"

hydroxylase"(TH)"

Fig02:00(a)"Original"heterogeneous"N27"cell"line"In#Vitro,"1994."

"(b)"Rat"embryo"aWer"15"days"of"gesta/on."N27"cells"are"derived"from"an"E15"fetal"rat"midbrain,"as"shown"below."

4a)0DifferenEated0Cell0Morphology0

3a)0UndifferenEated0Cell0Morphology0

4b)0DifferenEated0TH0Staining0

Fig.0 3:0 (a)" Undifferen/ated" N27"cells"aWer"five"days"of"growth."(b)"We" iden/fied" Clone" 3"which" had"small" clusters" of" cells" exhibi/ng"very" bright" TH" posi/ve" staining,"even" in"undifferen/ated"cultures,"as" demonstrated" by" green"fluorescence.""

N270CELL0SELECTION0

The" star/ng" N27" cell" line" was" a" mixed"popula/on" of" cells," and" immunostaining" for"tyrosine" hydroxylase" (TH)" revealed" a" few"clones"of"dividing" cells" that"had"high" levels"of"TH"and"a"good"neuronal"phenotype.""Western"Blosng"has"also"revealed"posi/ve"TH"presence;" these" results"were" faint" due" to" low"protein" concentra/on" (results" not" shown)."Further"immunohistochemical"experiments"are"underway.""

4c)0DifferenEated0TH0Staining0

3b)0UndifferenEated0TH0Staining0

Clone030

Clone030

N270Cell0Line0

N270Cell0Line0

Clone030

htp://jackkruse.com/theYdopamineYrxYgoodYchoicesYorYbad/"

2a)0Original0N270cell0line0

2b)0E150Rat0Embryo0

htp://www.cram.com/flashcards/neuroanatomyYlabY4YnotesYonYstructuresYinYmesencephalonY2342316"

Upon" observa/on" of" TH" staining"under"a"fluorescent"microscope,"we"discovered" that" Clone" 3" expressed"the" highest" dopamine" neuron"marker," TH." Bright" TH" fluorescence"and" elongated," neuronalYshaped"cells" can" be" observed" in" figures" 3"and"4."We" performed" a" series" of"experiments"trying"to"enrich"the"TH"posi/ve" cells" from" Clone" 3." This"enrichment"and" isola/on"process" is"s/ll"ongoing.""

CONCLUSION0Clonal"selec/on"can"improve"the"N27"dopamine"cell"line."

*Photos"of"cells"only"represent"only"about"1%"of"culture."

ISOLATION0OF0CLONE030