Molecular Implications of Monoamine Neurochemistry

1. α-DBH-Saporin antibodies

2. Dopamine Knock-Out Mice (Palmiter)

3. Pet-1 Knockout Mice (Deneris)

HouptCell and Molecular Neuroscience Fall 2012

Saporin

Ribosome inactivating protein (RIP), similar to ricin.

Very stable, resistant to denaturation and proteolysis.

Enzyme cleaves ribosomal RNA of ribosome large subunit; shuts down protein synthesis.

Has to be inside the cell to have toxic effect.

Conjugate to antibodies which bind extracellular protein that will be endocytosed.

Vesicular Recycling

http://web.squ.edu.om/med-Lib/MED_CD/E_CDs/anesthesia/site/content/v02/020499r00.htm

some transportedback to cell body

some transportedback to cell body

DBH is an integral membrane protein in vesicleDA converted to NE inside vesicles

DA

DBH

VMAT H+

DBH

DBH

DBH

DBH

DBH

DBH

DBH

NE

NE

NE NE

Saporin-conjugated antibodiescell death

http://www.atsbio.com

Control α-DBH-Saporin

Locus Coeruleus(NE)

α-DBH-Saporin kills NE cells

Control α-DBH-Saporin

α-DBH-Saporin spares DA cells and 5HT cells

Substantia Nigra (DA)

Dorsal Raphe(5HT)

Total 4-h intake of pelleted rat food following subcutaneous injection of saline (1 ml/kg), 2DG (200 mg/kg), or insulin (2 units/kg) in SAP control and DSAP-immunolesioned rats. DSAP rats did not increase their food intake in response to either 2DG-induced glucoprivation or to hypoglycemic doses of insulin.

Hudson & Ritter 2004

α-DBH-Saporin injection into hypothalamus blocks feeding in response to hypoglycemiasaporin alone has no effect

Transcription Factors (proteins) act at Promoter Elements (DNA) to Enhance/Repress Gene Expression

Tissue-Speci#c Transcription Factors act at Promoter Elements to Select Gene Expression

Dopamine and Pet-1 Knock-Out Mice

Eukaryotic Gene Regulation

1. RNA polymerase only works with transcription initiation complex bound to gene.

2. Gene expresion is controlled by upstream promoter elements and transcription factors (i.e. not by blockade of the RNA polymerase.)

1. Transcription initiation complex

5’ 3’TATA Box Transcription

start

25 bp

Almost all eukaryotic genes have TATA boxes just before transcription start.

Eukaryotic RNA polymerase only binds to DNA after transcription factors bind to the TATA box.

TATAAAAATATTTT

2. Transcription Factor Binding

Genes are turned off and on by binding of transcription factors to promoter control elements.

e.g. estrogen receptor in presence of estrogen binds to estrogen response element

2. Transcription Factor Binding

3’

ACGTCAnnnTGACCT

5’ Estrogen responsive gene e.g. breast cancer gene

5’ 3’TATAERE

Another Estrogen responsive gene e.g. bone density gene

TATA

Estrogenresponse element

ERE

TF = protein that binds DNA at response element to regulate transcription

E estrogen receptor= protein that binds to ERE

Estrogen responsive gene

5’ 3’TATAERE

E

mRNA

inactive estrogen receptor

5’ 3’TATAERE

E estrogen &active estrogen receptor

Estrogen-responsive gene expression(good for bones, but too much could cause breast

cancer)

ETamoxifen displaces estrogen,

inactivates receptor

T

5’ 3’TATAEREDecreased Estrogen-responsive gene expression

(#ghts cancer, but could cause weak bones)

Determination of Neuronal PhenotypeThere must be tissue-speci#c transcription factors that drive expression of each catecholaminergic enzyme gene

th

dbh

pnmt

TH?

DBH??

PNMT???

KVETNANSKY 2009,

Determination of Neuronal Phenotype

TH

dbh

th

pnmt

DA

DBH

dbh

th

pnmt

TH

NE

PNMTdbh

th

pnmt

DBH

TH

E

Dopamine Cell NorEpi Cell Epi Cell

Dopamine Knock-Out Mouse:Knock-Out TH gene from all cells in body -> no Dopamine

dbh

th

pnmt

DA

DBH

dbh

th

pnmt

PNMTdbh

th

pnmt

DBH

TH TH

NE E

Dopamine Cell NorEpi Cell Epi Cell

TH

No Dopamine No NorEpi No Epi

Solution?

dbh

pnmt

th

dbh-th

DBH

dbh

pnmt

th

PNMT

dbh-th

TH

DBH

dbh

pnmt

th

dbh-th

TH

Dopamine Knock-Out Mouse:Knock-Out TH gene from all cells in bodyKnock-In Transgene with DBH promoter and TH coding regionTH only expressed in cells that also express DBH (e.g. NorEpi & Epi cells)

NE E

Dopamine Cell NorEpi Cell Epi Cell

No Dopamine

normal TH gene

disrupted TH gene

DBH-TH gene

TH -/- mice die in utero

Zhou and Palmiter 1995

wildtype DA -/-

TH Immunoreactivity in Substantia Nigra and Locus Ceruleus of DA -/- mice

Zhou and Palmiter 1995

Zhou and Palmiter 1995

Zhou and Palmiter 1995

DD mice did not increase locomotor behavior in response to repeated AMPH. A, DD mice (n =12) were given L-dopa (50 mg/kg) at time 0, AMPH (2 mg/kg) at 24, 26, and 42 hr after L-dopa (filled arrowheads), and a control injection of PBS 44 hr after L-dopa (open arrowhead). The black bars indicate when room lights were off. The increased activity at 30 hr after L-dopa (white bar) is characteristic of DD mice (see Results). B, WT mice (n = 3) were treated as described for DD mice.

wildtype

DA -/-

L-DOPA

AMPH AMPH AMPH

Cannon & Palmiter 2003

Dopamine Knockout Mice do not Respond to Amphetamine

Serotonergic Gene Expression in theDorsal and Median Raphe Expression of TPH, 5HT1aR, 5HTT, etc.

de#nes 5HT-ergic phenotype of cell

There must be a common promoter element that responds to tissue-speci#c transcription factor to drive parallel expression of 5HT genes

tryptophan hydroxylase (5HT synthesis)

5HT transporter (5HT reuptake)

5HT 1A receptor (5HT autoreceptor)

TPH

5HT1aR

5HTT

?

?

?

5HTT

1aR

TPH5HT

tph

5htt

5ht1ar

pet-1

Serotonergic Gene Expression in theDorsal and Median Raphe

TPH

5HT1aR

5HTT

PET-1

PET-1 as Serotonergic Transcription Factor

PET-1 is colocalized with serotonin cells during development and adulthood.

Serotonergic genes expressed in serotonin cells have PET-1 binding site in promoter.

PET-1 is thought to mediate serotonergic phenotype during differentiation.

PET-1 5HTT

PET-1 & TPH

Hendricks et al. 1999

Serotonergic Gene Expression in theDorsal and Median Raphe

tph

5htt

5ht1ar

pet-1

PET-RE

PET-1

PET-RE

PET-RE

TPH

5HT1aR

5HTT

Pet-1 response element in 5HT genes?

Knock-Out Pet-1 gene -> no serotonergic cells

tph

5htt

5ht1ar

pet-1

PET-RE

PET-RE

PET-RE

PET-1

TPH

5HT1aR

5HTT

no 5HT

Pet-1 KO

Hendricks 2003

Wildtype Resident Intruder Test

Resident

Intruder

Pet-1 Knockout

Resident

Intruder

Serotonergic Gene Expression in theDorsal and Median Raphe

tph

5htt

5ht1ar

pet-1

PET-RE

???

PET-1

PET-RE

PET-RE

TPH

5HT1aR

5HTT

What are the upstream transcription factors or differentiation factors that make serotonergic cells express PET-1?