Regulation of adult neurogenesis in Huntington
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Regulation of adult neurogenesis in Huntington's disease [Elektronische Ressource] : the role of TGF-beta1 signaling in the neurogenic niche / Mahesh Kandasamy

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129 Pages
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Regulation of adult neurogenesis in Huntington’s disease: The role of TGF-beta1 signaling in the neurogenic niche DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER NATURWISSENSCHAFTEN (DR. RER. NAT.) DER FAKULTÄT FÜR BIOLOGIE UND VORKLINISCHE MEDIZIN DER UNIVERSITÄT REGENSBURG vorgelegt von Mahesh Kandasamy aus Dharmapuri, India 2010 Das Promotionsgesuch wurde eingereicht am: 24.09.2010 Die Arbeit wurde angeleitet von: Prof. Dr. Inga Neumann und Prof. Dr. Ludwig Aigner in der Klinik und Poliklinik für Neurologie der Universität Regensburg Table of Contents 1. Introduction .......................................................................................................................... 1 1.1. Adult neurogenesis ................. 1 1.2. History of adult neurogenesis ................................ 2 1.3. Stem cells in the adult brain ................................................................... 3 1.4. Hippocampal neurogenesis ..... 4 1.5. Neurogenesis in the SVZ-RMS-OB system .............................................. 5 1.6. Regulation of adult neurogenesis............................................................ 7 1.7. Regulation of neurogenesis by signaling molecules .............................................................. 8 1.8. Transforming growth factors ...................................

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Regulation of adult neurogenesis in Huntington’s disease:
The role of TGF-beta1 signaling in the neurogenic niche




DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES
DER NATURWISSENSCHAFTEN (DR. RER. NAT.)
DER FAKULTÄT FÜR BIOLOGIE UND VORKLINISCHE MEDIZIN
DER UNIVERSITÄT REGENSBURG



vorgelegt von
Mahesh Kandasamy
aus
Dharmapuri, India






2010

Das Promotionsgesuch wurde eingereicht am: 24.09.2010
Die Arbeit wurde angeleitet von: Prof. Dr. Inga Neumann und Prof. Dr. Ludwig Aigner
in der Klinik und Poliklinik für Neurologie der Universität Regensburg

































Table of Contents

1. Introduction .......................................................................................................................... 1
1.1. Adult neurogenesis ................. 1
1.2. History of adult neurogenesis ................................ 2
1.3. Stem cells in the adult brain ................................................................... 3
1.4. Hippocampal neurogenesis ..... 4
1.5. Neurogenesis in the SVZ-RMS-OB system .............................................. 5
1.6. Regulation of adult neurogenesis............................................................ 7
1.7. Regulation of neurogenesis by signaling molecules .............................................................. 8
1.8. Transforming growth factors ................................... 9
1.8.1. The TGF-beta signaling pathway ...................................................... 10
1.8.2. TGF-beta expression in the normal and pathological brain .............................................. 11
1.8.3. TGF-beta signal transduction in the brain ........................................ 12
1.8.4. Roles of TGF-beta in the brain.......................................................... 13
1.8.5. Regulation of adult neurogenesis by TGF-beta1 .............................. 13
1.8.6. Enhanced TGF-beta 1 level and impaired neurogenesis in neurodegenerative disorders 14
1.9. Huntington’s disease............................................................................................................. 15
1.9.1. Clinical aspects of Huntington’ s disease ......... 15
1.9.2. History of Huntington’s disease ....................... 16
1.9.3. Epidemiology of Huntington’s disease ............................................................................. 17
1.9.4. Localization and function of the physiological Huntingtin protein ... 18
1.9.5. Expanded CAG repeats in the huntingtin gene................................................................. 19
1.9.6. Neuropathological hallmarks of HD .................................................................................. 20
1.9.7. Experimental models of Huntington’s disease . 21
1.9.7.1. Acute models for Huntington’s disease......... 21
1.9.7.2. Transgenic models of Huntington’s disease ................................................................. 21
1.9.8. Neurogenesis in Huntington’s disease ............ 22

2. Aim of the study ................................................................................................................ 23

3. Material and Methods....... 24

3.1. Materials .............................................................................................................................. 24
3.1.1. Expendable materials ....................................... 24
3.1.2. Chemicals for in vivo immunological procedure 25
3.1.3. Chemicals for Western blot .............................................................................................. 26
3.1.4. Cell culture medias ........................................... 26
3.1.5. Other reagents for cell culture.......................................................................................... 27
3.1.6. Buffer, solutions and stock solutions............... 28
3.1.7. Primary antibodies ........................................................................................................... 31
3.1.8. Secondary antibodies ...... 32
3.1.7. Devices............................................................................................................................. 32
3.1.8. Software ........................... 33

3.2. Methods............................................................................................................................... 34
3.2.1. Animals....... ...................... 34
3.2.2. Intracerebroventricular infusions of TGF-beta1................................ 34
3.2.3. BrdU labelling................................................................................................................... 35
3.2.4. Tissue processing and immunohistochemistry 35
3.2.5. Counting procedures........ 37
3.2.6. Western Blotting............................................................................................................... 39
3.2.7. Neural stem and progenitor cells culture ......... 40
3.2.8. Cell cycle analysis ............ 41
3.2.9. Immunocytochemistry ...................................................................................................... 41
3.3. Statistics ............................................................. 42

4. Results ................................................................................................. 44
4.1. TGF-beta1 signaling components are expressed throughout the adult rat brain.................. 44
4.2. Table 1-Semiquantitative measurement of immunoreactivity of TGF-betaRII, TGF-betaRI and
pSmad2 in the adult rat brain ............................................................................................. 49
4.3. pSmad2 is predominantly present in postmitotic cells in the hippocampus of the adult
brain. .................................................................... 51
4.4. Induced over-expression of TGF-beta1 in the hippocampus reduces cell proliferation but
promotes neuronal differentiation and survival.. .................................. 54
4.5. Elevated levels of TGF-beta1 provokes expression of pSmad2 in neural stem and
progenitor cells .................................................................................................................... 57
4.6. Regulation of hippocampal neurogenesis in tgHD rats ........................ 61
4.7. Hippocampal cell proliferation in tgHD rats gets progressively impaired between 8 and 12
months of age....................................................................................................................... 61
4.8. Impaired survival of newly generated cells and reduced neuronal density was mediated by
reduced CREB signaling in tgHD rat hippocampus. ............................. 61
4.9. Increased quiescence of newly generated cells in tgHD dentate gyrus ................................ 65
4.10. Neuroblast proliferation compensate s stem cell quiescence in tgHD dentate gyrus........... 67
4.11. Reduced cell proliferation in tgHD animals correlates with increased TGF-beta1 signaling
in hippocampal stem cells.................................................................................................... 70



5. Discussion .......................................................................................................................... 77

7. Bibliography ....................... 88

Acknowledgements ............................................................................................................ 118



List of abbreviations
-MEM Alpha Modified Eagle Media
AD Alzheimer's disease
ALK Activin like kinase
BDNF Brain Derived Neurotrophic Factor
Bmi-1 B lymphoma Mo-MLV insertion region-1
BMP-2 Bone Morphogenetic Protein 2
BMP-4 Bone Morphogenetic Protein 4
bp Base pair
BSA Bovine Serum Albumine
BrdU 5-bromo-2-deoxyuridine
BT Biotinylated
CAG Cytosine, Adenine and Guanine
CBP CREB-binding protein
CNTF Ciliary neurotrophic factor
Cor Cortex
CNS Central Nervous System
CREB cAMP response element-binding
CSF Cerebrospinal fluid
ACSF Artificial Cerebrospinal fluid
DAPI 4’,6-Diamidino-2-phenylindole
DG Dentate Gyrus
DCX Doublecortin
DNA Deoxyribonucleic acid
DNI Dystrophic Neuritic Inclusions
DMEM Dulbecco’s Modified Eagle Media
EDTA Ethylenediaminetetraacetic acid
EGF Epidermal Growth Factor
ES cell Embryonic Stem cell
EPO Erythropoietin
FBS Fetal Bovine Serum
FGF Fibroblast Growth Factor
FSGB Fish Skin Gelatin Buffer
GABA Gama Aminobutyric acid
GCL Granule Cells Layer
G-CSF Granulocyte colony-stimulating factor
GDF Growth differentiation factors
GFAP Glial Fibrillary Acidic Protein
GS-Domain Glycin-Serine domain
IGF-1 Insulin-like Growth Factor 1
h Hour
HC Hippocampus
HD Huntington’s disease
Hes Hairy and enhancer of split
HGF Hepatocyte growth factor
HIP1 Huntingtin Interacting Protein1
HAP1 Huntingtin-associated protein 1
HPA Hypothalamic-pituitary-adrenal axis
HRP Horseradish peroxidase
HSCs Hematopoietic Stem Cells
HTT Huntingtin Gene
IT15 Interesting Transcript 15
LAP Latency Associated Protein
LTBP Latent TGF-beta-binding proteins
Map2ab Microtubule-associated Protein 2 Isoform a and
b
MBP Myelin Basic Protein
MSC Mesenchymal stem cells
min Minute
MH Mad Homology
NB Neurobasal
NGF Nerve Growth Factor
NI Neuronal Inclusion
NPCs Neural Progenitor Cells
NSCs Neural Stem Cells
NeuN Neuronal Nuclei
NMDA N-methyl-D-aspartic acid
OB Olfactory Bulb
Olig 1 Oligodendrocyte transcription factor1
Olig 2 Oligodendrocyte transcription factor2
PBS Phosphate Buffered Saline
PCNA Proliferating Cell Nuclear Antigen
pCREB Phospho cAMP response element-binding
PD Parkinson's Disease
PFA Paraformaldehyde
PMFS Phenylmethylsulfonyl fluoride
PNS Peripheral nervous system
PRL Prolactin
pSmad phospho mothers against decapentaplegic
(MAD) and the Caenorhabditis elegans protein
SMA
RA Retinoic Acid
RMS Rostra Migratory Stream
SD Standard Deviation
SDS Sodium Dodecyl Sulfate
SEL Subependymal layer
SF Scatter Factor
SGZ Subgranular Zone
Shh Sonic Hedgehog
SMI94 Antibody against Myelin Basic Protein
Sox2 SRY (sex determining region Y)-box 2
SSC Saline Sodium Citrate
Smad mothers against decapentaplegic (MAD) and the
Caenorhabditis elegans protein SMA
SVZ Subventricular Zone
Str Striatum
Stat3 Signal transducer and activator of transcription 3
TBS Tris Buffered Saline
TgHD rats Transgenic HD rats
TGF-beta1 Trasnforming growth factor beta1
TGF-beta2 Trasnforming growth factor beta2
TGF-beta3 Trasnforming growth factor beta3
TGF-bR1 Type 1 receptor Trasnforming growth factor beta
TGF-bR2 Type 2 receptor Trasnforming growth factor beta
TGF-bR3 Type 3 receptor Trasnforming growth factor beta
VEGF Vascular Endothelial Growth Factor
WT Wild Type
WB Western blot 1

1. Introduction
1.1. Adult neurogenesis
The renowned Spanish neuroanatomist Ramón y Cajal stated that “Once
development was ended, the founts of growth and regeneration of the axons and
dendrites dried up irrevocably. In adult centres, the nerve paths are something fixed
1and immutable: everything may die, nothing may be regenerated” . Therefore, it has
been believed that no new neurons are generated in the adult brain and most of the
common central nervous system (CNS) pathologies accompanied by neuronal loss
cannot be restored. Amongst them are well known ones: Parkinson's disease
accompanied by the degeneration of dopaminergic neurons in the substantia nigra,
Alzheimer's disease with a neuronal loss in the cerebral cortex and certain
subcortical regions and stroke where a certain brain area lacks oxygen supply
followed by neuronal death. According to the above dogma, the vast majority of
2, 3neurons in the mammalian brain are generated during embryonic development .
This statement stands true for most of the regions of the adult brain. However, this
doctrine ended in 1965 when newly generated neurons were found in two specific
regions of the adult brain: the subgranular zone (SGZ) in the dentate gyrus (DG)
generates new granular neurons in granule cell layer (GCL) of the hippocampus and
the subventricular zone (SVZ) of the lateral ventricle wall that gives rise to new cells
that migrate along the rostral migratory stream (RMS) to become neurons in the
4, 5olfactory bulb (Fig.1. 1). 2



Fig.1.1. Neurogenic niches in the adult human and in the rat brain.
The dentate gyrus of the hippocampus and the subventricular zone are known to
produce new neurons in the adult brain. From the subventricular zone of the lateral
ventricle cells migrate via the rostral migratory stream to the olfactory bulb, where
they differentiate into mature neurons and integrate. (Figure extracted from
www.pubs.niaaa.nih.gov/publications/arh27-2/IMAGES/Page198.gif)


1.2. History of adult neurogenesis
A study suggested in 1912 by a Canadian scientist Ezra Allen, is considered
for the preliminary document of mitotic activity in cells of the adult rodent central
6nervous system . Although there were some occasional reports on mitotic cells in
7the brain of adult mammals there were no convincing methods to prove that these
new cells would differentiate into neurons and be functionally integrated. Joseph
Altman and Gopal Das proposed the concept of persistent neurogenesis in the adult
3brain in 1965 where they used tritiated (H ) thymidine and autoradiography to
8suggest the production of new neurons in the hippocampus . Later on, it was
demonstrated through autoradiography and electron microscopy that the newborn
5, 9neurons in the hippocampus were structurally integrated . Many advanced
3techniques emerged in the 1990s. Instead of H thymidine, bromodeoxyuridine
10-15(BrdU) and retroviral labelling were used to monitor the newly dividing cells .