Fusion of diphtheria toxin and urotensin II produces a neurotoxin selective for cholinergic neurons in the rat mesopontine tegmentum

S. D. Clark , H. L. Alderson, P. Winn, M. P. Latimer, H. P. Nothacker, O. Civelli

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

Urotensin II is a neuropeptide first isolated from fish and later found in mammals: where it has potent cardiovascular, endocrine and behavioral effects. In rat brain the urotensin II receptor (UII-R) is predominately expressed in the cholinergic neurons of the pedunculopontine (PPTg) and laterodorsal tegmental nuclei. Typically, the function of the PPTg has been examined using excitotoxins, destroying both cholinergic and non-cholinergic neurons, which confounds interpretation. We took advantage of UII-R's unique expression profile, by combining UII with diphtheria toxin, to engineer a toxin specific for cholinergic neurons of the PPTg. In vitro, two different toxin constructs were shown to selectively activate UII-R (average EC50 ≈ 30 nmol/L; calcium mobility assay) and to be 10 000-fold more toxic to UII-R expressing CHO cells, than wildtype cells (average LD50 ≈ 2 nmol/L; cell viability). In vivo, pressure injection into the PPTg of rats, resulted in specific loss of choline transporter and NADPH diaphorase positive neurons known to express the UII-R. The lesions developed over time, resulting in the loss of over 80% of cholinergic neurons at 21 days, with little damage to surrounding neurons. This is the first highly selective molecular tool for the depletion of mesopontine cholinergic neurons. The toxin will help to functionally dissect the pedunculopontine and laterodorsal tegmental nuclei, and advance the understanding of the functions of these structures.

Original languageEnglish
Pages (from-to)112-120
Number of pages9
JournalJournal of Neurochemistry
Volume102
Issue number1
DOIs
Publication statusPublished - 1 Jul 2007

Fingerprint

Diphtheria Toxin
Cholinergic Neurons
Neurotoxins
Cholinergic Agents
Neurons
Rats
Fusion reactions
Pedunculopontine Tegmental Nucleus
NADPH Dehydrogenase
CHO Cells
Poisons
Lethal Dose 50
Neuropeptides
Mammals
Cell Survival
Fishes
Calcium
Pressure
Injections
urotensin II

Keywords

  • acetylcholine
  • fusion toxin
  • laterodorsal tegmental nucleus
  • pedunculopontine tegmental nucleus
  • cholinergic neurons
  • carrier protein
  • animal experiment
  • diphtheria toxin
  • protein engineering

Cite this

Clark , S. D. ; Alderson, H. L. ; Winn, P. ; Latimer, M. P. ; Nothacker, H. P. ; Civelli, O. / Fusion of diphtheria toxin and urotensin II produces a neurotoxin selective for cholinergic neurons in the rat mesopontine tegmentum. In: Journal of Neurochemistry. 2007 ; Vol. 102, No. 1. pp. 112-120.
@article{8e675c2d3e964375912529e7d67749ee,
title = "Fusion of diphtheria toxin and urotensin II produces a neurotoxin selective for cholinergic neurons in the rat mesopontine tegmentum",
abstract = "Urotensin II is a neuropeptide first isolated from fish and later found in mammals: where it has potent cardiovascular, endocrine and behavioral effects. In rat brain the urotensin II receptor (UII-R) is predominately expressed in the cholinergic neurons of the pedunculopontine (PPTg) and laterodorsal tegmental nuclei. Typically, the function of the PPTg has been examined using excitotoxins, destroying both cholinergic and non-cholinergic neurons, which confounds interpretation. We took advantage of UII-R's unique expression profile, by combining UII with diphtheria toxin, to engineer a toxin specific for cholinergic neurons of the PPTg. In vitro, two different toxin constructs were shown to selectively activate UII-R (average EC50 ≈ 30 nmol/L; calcium mobility assay) and to be 10 000-fold more toxic to UII-R expressing CHO cells, than wildtype cells (average LD50 ≈ 2 nmol/L; cell viability). In vivo, pressure injection into the PPTg of rats, resulted in specific loss of choline transporter and NADPH diaphorase positive neurons known to express the UII-R. The lesions developed over time, resulting in the loss of over 80{\%} of cholinergic neurons at 21 days, with little damage to surrounding neurons. This is the first highly selective molecular tool for the depletion of mesopontine cholinergic neurons. The toxin will help to functionally dissect the pedunculopontine and laterodorsal tegmental nuclei, and advance the understanding of the functions of these structures.",
keywords = "acetylcholine, fusion toxin, laterodorsal tegmental nucleus, pedunculopontine tegmental nucleus, cholinergic neurons, carrier protein, animal experiment, diphtheria toxin, protein engineering",
author = "Clark, {S. D.} and Alderson, {H. L.} and P. Winn and Latimer, {M. P.} and Nothacker, {H. P.} and O. Civelli",
year = "2007",
month = "7",
day = "1",
doi = "10.1111/j.1471-4159.2007.04529.x",
language = "English",
volume = "102",
pages = "112--120",
journal = "Journal of Neurochemistry",
issn = "0022-3042",
number = "1",

}

Fusion of diphtheria toxin and urotensin II produces a neurotoxin selective for cholinergic neurons in the rat mesopontine tegmentum. / Clark , S. D. ; Alderson, H. L.; Winn, P.; Latimer, M. P.; Nothacker, H. P.; Civelli, O.

In: Journal of Neurochemistry, Vol. 102, No. 1, 01.07.2007, p. 112-120.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Fusion of diphtheria toxin and urotensin II produces a neurotoxin selective for cholinergic neurons in the rat mesopontine tegmentum

AU - Clark , S. D.

AU - Alderson, H. L.

AU - Winn, P.

AU - Latimer, M. P.

AU - Nothacker, H. P.

AU - Civelli, O.

PY - 2007/7/1

Y1 - 2007/7/1

N2 - Urotensin II is a neuropeptide first isolated from fish and later found in mammals: where it has potent cardiovascular, endocrine and behavioral effects. In rat brain the urotensin II receptor (UII-R) is predominately expressed in the cholinergic neurons of the pedunculopontine (PPTg) and laterodorsal tegmental nuclei. Typically, the function of the PPTg has been examined using excitotoxins, destroying both cholinergic and non-cholinergic neurons, which confounds interpretation. We took advantage of UII-R's unique expression profile, by combining UII with diphtheria toxin, to engineer a toxin specific for cholinergic neurons of the PPTg. In vitro, two different toxin constructs were shown to selectively activate UII-R (average EC50 ≈ 30 nmol/L; calcium mobility assay) and to be 10 000-fold more toxic to UII-R expressing CHO cells, than wildtype cells (average LD50 ≈ 2 nmol/L; cell viability). In vivo, pressure injection into the PPTg of rats, resulted in specific loss of choline transporter and NADPH diaphorase positive neurons known to express the UII-R. The lesions developed over time, resulting in the loss of over 80% of cholinergic neurons at 21 days, with little damage to surrounding neurons. This is the first highly selective molecular tool for the depletion of mesopontine cholinergic neurons. The toxin will help to functionally dissect the pedunculopontine and laterodorsal tegmental nuclei, and advance the understanding of the functions of these structures.

AB - Urotensin II is a neuropeptide first isolated from fish and later found in mammals: where it has potent cardiovascular, endocrine and behavioral effects. In rat brain the urotensin II receptor (UII-R) is predominately expressed in the cholinergic neurons of the pedunculopontine (PPTg) and laterodorsal tegmental nuclei. Typically, the function of the PPTg has been examined using excitotoxins, destroying both cholinergic and non-cholinergic neurons, which confounds interpretation. We took advantage of UII-R's unique expression profile, by combining UII with diphtheria toxin, to engineer a toxin specific for cholinergic neurons of the PPTg. In vitro, two different toxin constructs were shown to selectively activate UII-R (average EC50 ≈ 30 nmol/L; calcium mobility assay) and to be 10 000-fold more toxic to UII-R expressing CHO cells, than wildtype cells (average LD50 ≈ 2 nmol/L; cell viability). In vivo, pressure injection into the PPTg of rats, resulted in specific loss of choline transporter and NADPH diaphorase positive neurons known to express the UII-R. The lesions developed over time, resulting in the loss of over 80% of cholinergic neurons at 21 days, with little damage to surrounding neurons. This is the first highly selective molecular tool for the depletion of mesopontine cholinergic neurons. The toxin will help to functionally dissect the pedunculopontine and laterodorsal tegmental nuclei, and advance the understanding of the functions of these structures.

KW - acetylcholine

KW - fusion toxin

KW - laterodorsal tegmental nucleus

KW - pedunculopontine tegmental nucleus

KW - cholinergic neurons

KW - carrier protein

KW - animal experiment

KW - diphtheria toxin

KW - protein engineering

UR - http://www.scopus.com/inward/record.url?scp=34250197937&partnerID=8YFLogxK

U2 - 10.1111/j.1471-4159.2007.04529.x

DO - 10.1111/j.1471-4159.2007.04529.x

M3 - Article

VL - 102

SP - 112

EP - 120

JO - Journal of Neurochemistry

JF - Journal of Neurochemistry

SN - 0022-3042

IS - 1

ER -