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Generation of midbrain dopaminergic neurons in vivo and in vitro: the role of Neurogenin2

Andersson, Elin LU (2005) In Lund University Faculty of Medicine Doctoral Dissertation Series
Abstract (Swedish)
Popular Abstract in Swedish

I hjärnan finns många olika typer av nervceller. De använder sig av olika signalsubstanser, kallade neurotransmittorer, för att kommunicera med andra nervceller. En viss typ av nervceller använder neurotransmittorn dopamin. Dopaminceller finns på många ställen i hjärnan men de flesta ligger i mellanhjärnan i ett par olika cellgrupper som var och en skickar signaler till sina specifika områden i andra delar av hjärnan. En av dessa cellgrupper kallas substantia nigra och signalerar till ställen som styr en människas motorik. Hos patienter med Parkinsons sjukdom, dör cellerna i denna grupp och då försvinner även dopaminsignalerna till de delar som styr motoriska förmågor. Därför har... (More)
Popular Abstract in Swedish

I hjärnan finns många olika typer av nervceller. De använder sig av olika signalsubstanser, kallade neurotransmittorer, för att kommunicera med andra nervceller. En viss typ av nervceller använder neurotransmittorn dopamin. Dopaminceller finns på många ställen i hjärnan men de flesta ligger i mellanhjärnan i ett par olika cellgrupper som var och en skickar signaler till sina specifika områden i andra delar av hjärnan. En av dessa cellgrupper kallas substantia nigra och signalerar till ställen som styr en människas motorik. Hos patienter med Parkinsons sjukdom, dör cellerna i denna grupp och då försvinner även dopaminsignalerna till de delar som styr motoriska förmågor. Därför har Parkinson-patienter typiska symptom, som problem med motoriken och svårighet att sätta igång rörelser. För att lindra dessa symptom kan Parkinsonpatienter ta medicin som ska ersätta dopaminet. Man har också testat andra behandlingsmetoder som går ut på att ersätta dopamincellerna inuti hjärnan. Genom att ta dopaminceller från fostervävnad och transplantera till hjärnan har man lyckats återskapa dopaminsignalleringen utan mediciner. Tyvärr kan denna teknik ännu inte tillämpas på många patienter eftersom det är svårt att få tag på tillräckligt mycket vävnad. Man har därför börjat undersöka hur man kan generera dopaminceller på annat sätt. En metod är att använda stamceller, celler som kan förökas i kultur och som kan utvecklas till vilka sorters celler som helst. För att få stamcellerna att bli dopaminceller så måste man veta vad det är som gör att just den sortens nervceller bildas. Vi måste förstå vilka de bakomliggande faktorerna är som styr cellutvecklingen mot dopaminceller.



I min avhandling har jag undersökt vilka signaler och gener som är viktiga för att dopaminceller



ska bildas. För att ta reda på det har jag tittat på dopaminceller under fosterutvecklingen. I mina studier använde jag möss som en modell för vad som händer i människan. Jag fann att en gen, Neurogenin2, var påslagen (uttryckt) i precis de celler som skulle bli dopaminceller hos mössfoster. När jag sedan undersökte muterade möss där denna gen var borttagen såg jag att dopamincellerna i mellanhjärnan inte bildades som de skulle. Detta visar att Neurogenin2 är viktig för bildandet av dopaminceller. Jag försökte också påverka odlade stamceller att utvecklas till dopaminceller genom att se till att Neurogenin2 uttrycktes i cellerna. När jag uttryckte Neurogenin2 tillsammans med en annan gen, Nurr1, som också är viktig för att det ska bli dopaminceller, gav det bättre resultat än att använda dem var och en för sig och jag såg att det bildades dopaminceller i cellkulturerna.



Med resultaten som presenteras i den här avhandlingen har vi kommit ännu en bit på väg för att veta hur dopaminceller genereras. Mina resultat kan användas bl.a för att identifiera celler som



ska bli dopaminceller. I ett längre perspektiv kan mina resultat bidra till att man kan generera



dopaminceller från stamceller och därmed ge de patienter som lider av Parkinsons sjukdom en alternativ behandling. (Less)
Abstract
Parkinsons disease (PD) is a neurodegenerative disorder where dopaminergic neurons of the substantia nigra (SNc) in the mesencephalon are progressively eliminated. The ensuing loss of dopaminergic innervation of the basal ganglia manifests itself as severe motor deficits in PD patients. Clinical trials have shown that cell replacement therapy, where dopaminergic neuroblasts derived from fetal ventral mesencephalon (VM) are transplanted to the striatum, may be an alternative to pharmacological treatment of PD patients. The limited access and ethical concerns with using fetal tissue have prompted the use of stem cells as a renewable and limitless source of dopaminergic neurons. However, the mechanisms of specification of mesDA neurons in... (More)
Parkinsons disease (PD) is a neurodegenerative disorder where dopaminergic neurons of the substantia nigra (SNc) in the mesencephalon are progressively eliminated. The ensuing loss of dopaminergic innervation of the basal ganglia manifests itself as severe motor deficits in PD patients. Clinical trials have shown that cell replacement therapy, where dopaminergic neuroblasts derived from fetal ventral mesencephalon (VM) are transplanted to the striatum, may be an alternative to pharmacological treatment of PD patients. The limited access and ethical concerns with using fetal tissue have prompted the use of stem cells as a renewable and limitless source of dopaminergic neurons. However, the mechanisms of specification of mesDA neurons in vivo need to be elucidated for identification and generation of mesencephalic dopaminergic (mesDA) neurons from stem cells in vitro.



In this thesis I have identified expression of the proneural gene Neurogenin2 (Ngn2) in a restricted pattern in the embryonic VM during mesDA neurogenesis. The protein was expressed in the progenitor population in the ventricular zone but not in mature neurons in the mantle zone. When isolating the Ngn2-expressing cells and their direct descendants by FACS from an Ngn2-GFP-KI mouse, I found that the Ngn2-GFP-positive cell fraction contained dopaminergic neurons, in contrast to Ngn2-GFP-negative cells. This shows that Ngn2 label early mesDA neuron precursors. Furthermore, when I analysed the Ngn2 knockout mutants, I found that they displayed an early loss of mesDA neurons that was partially maintained at postnatal stages, showing that Ngn2 has a role in the generation of the mesDA neurons. No other neuronal subtype in the VM was affected suggesting that this role for Ngn2 is specific for the mesDA neurons.



Using embryonic mouse tissue obtained at the stage of mesDA genesis, I was able to generate cultures of neural stem and progenitor cells, so called neurosphere cultures, that were neurogenic and maintained a ventral midbrain character over several passages. Although the neurospheres did not spontaneously give rise to dopaminergic neurons when differentiated, TH-positive cells were detected when Nurr1 was over-expressed in the cultures. The frequency with which this occurred, and the morphology of the TH-positive cells, differed from the results obtained when over-expressing Nurr1 in forebrain-derived expanded cells. This suggests that neurosphere expanded cells derived from VM specifically contain progenitors that can generate dopaminergic neurons under certain conditions. When over- expressing Ngn2 together with Nurr1 TH-positive cells were generated that displayed a mature neuronal morphology. Furthermore, I found that they expressed other dopaminergic markers which were not seen when either Nurr1 or Ngn2 were over-expressed alone. This suggests that Nurr1 and Ngn2 interact to specify a more mature dopaminergic phenotype.



The results in this thesis have identified a new cellular marker of mesDA progenitors in the developing embryo and also provided new insight into the development of mesDA neurons. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Professor Perlmann, Thomas, Karolinska Institute, Stockholm
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Medicine (human and vertebrates), Medicin (människa och djur), cell replacement, Parkinson?s, dopamine, neuronal specification, development, mesencephalon, TH, neuronal differentiation, proneural genes, transcription factors, bHLH, neurospheres, stem cell, progenitor cell
in
Lund University Faculty of Medicine Doctoral Dissertation Series
pages
176 pages
publisher
Department of Experimental Medical Science, Lund Univeristy
defense location
Segerfalksalen, BMC A10, Wallenberg Neurocentrum Sölvegatan 17
defense date
2005-12-17 09:30
ISSN
1652-8220
ISBN
91-85439-09-6
language
English
LU publication?
yes
id
50f4a257-1308-4247-a822-83d0ea4ad56d (old id 545931)
date added to LUP
2007-09-07 15:36:50
date last changed
2016-09-19 08:44:52
@phdthesis{50f4a257-1308-4247-a822-83d0ea4ad56d,
  abstract     = {Parkinsons disease (PD) is a neurodegenerative disorder where dopaminergic neurons of the substantia nigra (SNc) in the mesencephalon are progressively eliminated. The ensuing loss of dopaminergic innervation of the basal ganglia manifests itself as severe motor deficits in PD patients. Clinical trials have shown that cell replacement therapy, where dopaminergic neuroblasts derived from fetal ventral mesencephalon (VM) are transplanted to the striatum, may be an alternative to pharmacological treatment of PD patients. The limited access and ethical concerns with using fetal tissue have prompted the use of stem cells as a renewable and limitless source of dopaminergic neurons. However, the mechanisms of specification of mesDA neurons in vivo need to be elucidated for identification and generation of mesencephalic dopaminergic (mesDA) neurons from stem cells in vitro.<br/><br>
<br/><br>
In this thesis I have identified expression of the proneural gene Neurogenin2 (Ngn2) in a restricted pattern in the embryonic VM during mesDA neurogenesis. The protein was expressed in the progenitor population in the ventricular zone but not in mature neurons in the mantle zone. When isolating the Ngn2-expressing cells and their direct descendants by FACS from an Ngn2-GFP-KI mouse, I found that the Ngn2-GFP-positive cell fraction contained dopaminergic neurons, in contrast to Ngn2-GFP-negative cells. This shows that Ngn2 label early mesDA neuron precursors. Furthermore, when I analysed the Ngn2 knockout mutants, I found that they displayed an early loss of mesDA neurons that was partially maintained at postnatal stages, showing that Ngn2 has a role in the generation of the mesDA neurons. No other neuronal subtype in the VM was affected suggesting that this role for Ngn2 is specific for the mesDA neurons.<br/><br>
<br/><br>
Using embryonic mouse tissue obtained at the stage of mesDA genesis, I was able to generate cultures of neural stem and progenitor cells, so called neurosphere cultures, that were neurogenic and maintained a ventral midbrain character over several passages. Although the neurospheres did not spontaneously give rise to dopaminergic neurons when differentiated, TH-positive cells were detected when Nurr1 was over-expressed in the cultures. The frequency with which this occurred, and the morphology of the TH-positive cells, differed from the results obtained when over-expressing Nurr1 in forebrain-derived expanded cells. This suggests that neurosphere expanded cells derived from VM specifically contain progenitors that can generate dopaminergic neurons under certain conditions. When over- expressing Ngn2 together with Nurr1 TH-positive cells were generated that displayed a mature neuronal morphology. Furthermore, I found that they expressed other dopaminergic markers which were not seen when either Nurr1 or Ngn2 were over-expressed alone. This suggests that Nurr1 and Ngn2 interact to specify a more mature dopaminergic phenotype.<br/><br>
<br/><br>
The results in this thesis have identified a new cellular marker of mesDA progenitors in the developing embryo and also provided new insight into the development of mesDA neurons.},
  author       = {Andersson, Elin},
  isbn         = {91-85439-09-6},
  issn         = {1652-8220},
  keyword      = {Medicine (human and vertebrates),Medicin (människa och djur),cell replacement,Parkinson?s,dopamine,neuronal specification,development,mesencephalon,TH,neuronal differentiation,proneural genes,transcription factors,bHLH,neurospheres,stem cell,progenitor cell},
  language     = {eng},
  pages        = {176},
  publisher    = {Department of Experimental Medical Science, Lund Univeristy},
  school       = {Lund University},
  series       = {Lund University Faculty of Medicine Doctoral Dissertation Series},
  title        = {Generation of midbrain dopaminergic neurons in vivo and in vitro: the role of Neurogenin2},
  year         = {2005},
}