UMEÅ UNIVERSITY MEDICAL DISSERTATIONS
New Series No 1257 – ISSN 0346-6612 – ISBN 978-91-7264-767-1
From Dopamine Nerve Fiber Formation to
Astrocytes
Franziska Marschinke
Umeå 2009
Department of Integrative Medical Biology,
Section for Histology and Cell Biology,
Umeå University, Umeå, Sweden
Department of Integrative Medical Biology
Section for Histology and Cell Biology
Umeå University
Cover illustration: Photograph of TH-positive neurons from E14 CD47 wild type
cultures and vimentin-positive astroglia from E18 spinal cord cultures.
Copyright  Franziska Marschinke 2009
ISSN: 0346-6612
ISBN: 978-91-7264-767-1
New Series No: 1257
Printed in Sweden by Arkitektkopia, Umeå 2009
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To my family
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5
Omnium rerum principia parva sunt
Everything has a small beginning.
Marcus Tullius Cicero
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Table of contents
Abbreviations
Abstract
List of publications
Introduction
Parkinson’s Disease
Dopamine, dopaminergic neurons and dopamine receptors
The Basal Ganglia
Treatments strategies in Parkinson’s disease
Development of the dopaminergic nervous system
Neurotoxins for creating animal PD models
Neuroinflammation
Tumor necrosis factor alpha (TNFα) and its receptors
CD47 and Sirpα
Extracellular matrix molecules – Proteoglycans
Nerve fiber formation
Aims of the thesis
Materials and methods
Animals
Dissection of the ventral mesencephalon, frontal cortex, and spinal cord
Primary organotypic tissue cultures
Culture medium
Immunohistochemistry
Image analysis
Measurements and cell counts
Statistics
Results and discussion
Non-glial-associated versus glial-associated nerve fiber outgrowth
Age at plating influences nerve fiber growth patterns
Astroglia affects the presence of non-glial-associated TH-positive growth
Effects of inhibiting proteoglycans
Effects of tumor necrosis factor α
Microglia in VM cultures treated with TNFα
TNFα and its receptors
Blocking TNFR1 or TNFR2 during TNFα treatment
Blocking TNFR1 or TNFR2 without TNFα treatment
Blocking endogenous TNFα
Long-distance nerve fiber outgrowth in CD47-/-mice
Concluding remarks
Acknowledgement
References
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Abbreviations
6-OHDA
β-xyloside
Ara-C
ChABC
Chondroitin sulfate PGs
CNS
CRL
DA
DAPI
DIV
DMEM
E
ECM
ESC
FCS
GABA
GAG
GLAST
GP
GPe
GPi
KO
L-dopa
MPP+
MPTP
P
PBS
PD
PGs
ROS
Sirpα
SN
SNpc
SNpr
STN
SVZ
SY
TH
TNFα
TNFR1
TNFR2
TSP
VM
VTA
WT
6-hydroxydopamine
Methyl-umbelliferyl-β-D-xyloside
Cytosine β-D-arabinofuranoside
Chondroitinase ABC
CSPGs
Central nervous system
Crown-rump length
Dopamine
4´,6-diamidino-2-pheylindole
Days in vitro
Dulbecco´s modified Eagle´s medium
Embryonic day
Extracellular matrix
Embryonic stem cells
Fetal calf serum
Gamma-aminobutyric acid
Glycosaminoglycan
Glia glutamate transporter
Globus palladus
Globus palladus externa
Globus palladus interna
Knockout
3,4-dihydroxy-L-phenylalanine
1-methyl-4-phenylpyridinium
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
Postnatal
Phosphate buffer saline
Parkinson’s disease
Proteoglycans
Reactive oxygen species
Signal regulatory protein alpha
Substantia nigra
Substantia nigra pars compacta
Substantia nigra pars reticulata
Subthalamic nucleus
Subventricular zone
Synapthophysin
Tyrosine Hydroxylase
Tumor necrosis factor alpha
Tumor necrosis factor alpha receptor 1
Tumor necrosis factor alpha receptor 2
Thrombospondin
Ventral mesencephalon
Ventral tegmental area
Wildtype
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Abstract
Parkinson’s disease (PD) is a progressive neurodegenerative disease and
characterized by the loss of dopaminergic (DA) neurons in the substantia nigra in the
midbrain. The causes of the disease are still unknown. The most commonly used
treatment is administration of L-DOPA, however, another possible treatment strategy is
to transplant DA neurons to the striatum of PD patients to substitute the loss of neurons.
Clinical trials have demonstrated beneficial effects from transplantation, but one
obstacle with the grafting trials has been the variable outcome, where limited graft
reinnervation of the host brain is one important issue to solve. To improve and control
the graft DA nerve fiber outgrowth organotypic tissue cultures can be utilized. Cultures
of fetal ventral mesencephalon (VM) have been used to investigate astrocytic migration
and dopamine nerve fiber formations at different time points and under varying
conditions to study how to control nerve fiber formation. The early appearing DA nerve
fibers as revealed by tyrosine hydroxylase (TH) –immunoreactivity, form their fibers in
the absence of glial cell bodies, are not persistent over time, and is called non-glialassociated TH-positive nerve fiber outgrowth. A monolayer of astrocytes guides a
second persistent subpopulation of nerve fibers, the glial-associated TH-positive nerve
fiber formation. Investigations of the interactions between the astrocytic migration and
nerve fiber formations were made. In embryonic (E) day 14 VM cultures the mitosis of
the astrocytes was inhibited with the antimitotic agent β-D-arabinofuranoside. The
results revealed decreased astrocytic migration, reduced glial-associated TH-positive
outgrowth, and enhanced presence of the non-glial-associated TH-positive outgrowth in
the cultures. Thus, astrocytes affect both the non-glial- and the glial-associated growths
by either its absence or presence, respectively. The astrocytes synthesize proteoglycans.
Therefore the nerve fiber formation was studied in VM or spinal cord cultures treated
with the proteoglycan blockers chondroitinase ABC (ChABC), which degrades the
proteoglycans, or methyl-umbelliferyl-β-D-xyloside (β-xyloside), which blocks the
proteoglycan synthesis. β-xyloside inhibited the migration of the astrocytes and the
outgrowth of the glial-associated TH-positive nerve fibers in both VM and spinal cord
cultures, whereas ChABC treatment had no effect in E14 VM or spinal cord cultures.
E18 VM and spinal cord cultures were evaluated to investigate how the different
developmental stages influence astrocytes and the two nerve fiber formations after 14
DIV. No nerve fiber formation was found in E18 VM cultures, while the non-glial9
associated nerve fiber outgrowth was obvious as long and robust fibers in E18 spinal
cord cultures. The astrocytic migration was similar in VM and spinal cord cultures. βxyloside and ChABC did not affect nerve fiber growth but astrocytic migration in E18
VM cultures, while no effects was found in the spinal cord cultures. However, the
neuronal migration found in control cultures was abolished in both VM and spinal cord
cultures after both ChABC and β-xyloside.
Neuroinflammation plays a critical role in the development of PD. Increased
levels of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) are
observed in postmortem PD brains and the levels of TNFα receptors on circulating Tlymphocytes in cerebrospinal fluid of PD patients are increased. The effects of TNFα
were studied on E14 VM cultures. The outgrowth of the non-glial-associated THpositive nerve fibers was inhibited while it stimulated astrocytic migration and glialassociated TH-positive nerve fiber outgrowth at an early treatment time point.
Furthermore, blocking the endogenous levels of TNFα resulted in cell death of the THpositive neurons. Furthermore, cultures of E14 mice with gene deletion for the protein
CD47 were investigated. CD47 is expressed in all tissues and serves as a ligand for the
signal regulatory protein (SIRP) α, which promotes e.g migration and synaptogenesis.
CD47-/- cultures displayed massive and long non-glial-associated TH-positive nerve
fiber outgrowth despite a normal astrocytic migration and the presence of glialassociated TH-positive nerve fiber outgrowth. For the first time, it was observed that the
non-glial-guided TH-positive nerve fiber outgrowth did not degenerate after 14 DIV.
Taken together, there is an interaction between astrocytes and TH-positive nerve fiber
formations. Both nerve fiber formations seem to have their task during the development
of the DA system.
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List of publications
This thesis is based on the following papers, which are referred to in the text by their
roman numerals:
I.
af Bjerkén, S., Marschinke, F., Strömberg, I.
Inhibition of astrocytes promotes long-distance growing nerve fibers in
ventral mesencephalic cultures.
International Journal of Developmental Neuroscience. 2008
Nov;26(7):683-91.
II.
Marschinke, F. and Strömberg, I.
Degradation of proteoglycans affects astrocytes and neurite formation in
organotypic tissue cultures
Manuscript
III.
Marschinke, F. and Strömberg, I.
Dual effects of TNFα on nerve fiber formation from ventral
mesencephalic organotypic tissue cultures.
Brain research, 2008 Jun 18;1215:30-9.
IV.
Marschinke, F., Oldenborg, P-A and Strömberg, I.
CD47 - Sirpα interactions affects long-distance growing nerve fibers from
cultured ventral mesencephalic dopamine neurons.
Manuscript
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Introduction
Parkinson’s Disease
Parkinson’s disease (PD), a disease with symptoms of “involuntary tremulous
motion, with lessened muscular power, in parts not in action and even when
supported, with a propensity to bend the trunk forwards, and to pass from a walking to
a running pace, the senses and intellects being injured” was first mentioned and
described in 1817 in “An Essay on the Shaking Palsy” by the English physician
James Parkinson and was therefore named after him.
Parkinson’s
disease
is
the
second
most
common
of
age-related
neurodegenerative diseases. Around 1% of the world’s population in the age of 65-75,
and 4 -5% in the age above 85 are affected by PD (Fahn, 2003). Sporadic PD
develops in 95% of the patients, who have a disease onset at 70 years. In contrast,
patients with heritable/familial PD develop the symptoms much earlier with a disease
onset before the age of 50 (Farrer, 2006; Tanner, 2003). The cardinal symptoms of PD
are bradykinesia (slowness of the movement), rigidity (stiffness, increased muscle
tone) and resting tremor. The typical PD patient is characterized by a shuffling gait, a
flexed posture, and impaired balance. Other symptoms may include speaking in a
weak monotonic voice, an empty facial expression, and difficulties in swallowing and
writing. Most patients develop sleeping problems, depression, loss of appetite, and
dementia. As a result of their drug administration, some PD patients develop
dyskinesia (involuntary movements) (Ahlskog and Muenter, 2001).
The main feature of PD is a slowly progressing degeneration of midbrain
dopaminergic (DA) neurons, which project from the substantia nigra pars compacta to
the striatum. The typical symptoms of PD i.e. the loss of movement control appears
when approximately 80% of the DA neurons in the substantia nigra pars compacta
have degenerated (Fearnley and Lees, 1991; Marsden, 1990). In the striatum, a greater
loss of DA nerve fibers is found in the putamen than in the caudate nucleus of PD
patients, and within the putamen the caudal portions are more affected than rostral
portions (Kish et al., 1988; Nyberg et al., 1983). Other neurons and nuclei, besides the
dopaminergic neurons in the substantia nigra, like noradrenergic neurons in the locus
coeruleus, serotonergic neurons in the raphe nuclei, and cholinergic neurons in the
nucleus basalis are involved in the pathogenesis of PD (Braak et al., 2003; Chan-
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Palay and Asan, 1989; Jellinger and Paulus, 1992; Mann and Yates, 1983; Nakano
and Hirano, 1984; Zarow et al., 2003).
The etiology of PD and especially what causes the loss of DA neurons is still
unknown. Several theories for sporadic PD have been discussed. The most important
factor in sporadic PD seems to be age, but environmental toxins, neuroinflammation,
oxidative stress, mitochondrial dysfunction, drugs, and head trauma are also important
factors. In familial PD, mutations in several genes enhance the severity of the disease.
In addition, gene variances are also seen in sporadic PD.
Intracytoplasmic eosinophilic inclusions called Lewy bodies are observed in
DA neurons in the substantia nigra and glial cells in sporadic patients but also in some
cases of familial PD patients. Lewy bodies consist mainly of the protein alphasynuclein, which is encoded by the causative gene of familial PD 1 (familial
PD/PARK1) (for review see (Pankratz and Foroud, 2004)). Enriched alpha-synuclein
has the tendency to aggregate under certain condition, which leads to a deficiency in
the mitochondrial complex 1 in cells and oxidative stress (Ischiropoulos and
Beckman, 2003; Jenner and Olanow, 1996; Sherer et al., 2003). Causative mutation in
familial PD have been found in several genes like PARK1 (alpha-synclein), PARK2
(parkin), PARK4 (triplication of alpha-synuclein), PARK5 (UCHL1), PARK6
(PINK1 = PTEN-induced kinase 1), PARK7 (DJ-1) and PARK8 (LRRK2 = leucinerich repeat kinase 2) (for review see (Mizuno et al., 2006)).
In both the substantia nigra and the locus coeruleus, neurons bear the polymer
pigment neuromelanin, which is the main iron deposit in DA neurons (for review see
(Zecca et al., 2006)). The function of neuromelanin is not clear, however, it has been
suggested to be a waste product in the metabolism of catecholamine or to be involved
in the formation of Lewy bodies (for review see (Zecca et al., 2006)).
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Fig 1. Typical appearance of a PD patient.
The Basal Ganglia
The basal ganglia is a subcortical motor system in the brain. It includes four
major nuclei: striatum, globus palladus (GP), subthalamic nucleus (STN) and
substantia nigra (SN). The major output, the nucleus striatum, consists of the caudate
nucleus, putamen, and the nucleus accumbens. The cerebral cortex, thalamus, and the
substantia nigra form the main input to the striatum. Medium spiny neurons, with
large dendritic trees, form the largest population (around 95%) of the striatal neurons
(Graybiel and Ragsdale, 1978). They utilize γ-aminobutric acid (GABA) as a
neurotransmitter and are classified into two groups, depending on which neuropeptide
is co-localized. Other neurons found in the striatum besides the medium spiny
neurons are large aspiny neurons (around 1-2% of all neurons), which use
acetylcholine as a neurotransmitter, and small aspiny cells, which use GABA as
neurotransmitter. The GP includes internal (GPi) and external (GPe) segments and the
ventral pallidum. The substantia nigra is divided into two segments, the dense core of
dopaminergic neurons containing pars compacta (SNpc) and the more sparse cellular
pars reticulata (SNpr).
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Two striatopallidal output pathways are known, the direct and the indirect
(Albin et al., 1989; Alexander et al., 1986; Crossman, 1989; Delong, 1990). The
direct pathway projects from the striatum directly to GPi and SNpr and is represented
by the GABA neurons that express DA receptor 1 (D1), dynorphin, and substance P.
The indirect pathway projects via GPe and STN to GPi and SNpr. These neurons
express the DA receptor 2 (D2) and enkephalin. Both pathways balance the motor
control of the basal ganglia. Included in the basal ganglia is the nigrostriatal pathway
where dopaminergic neurons project their axons from SNpc to the striatum. In PD,
there is an imbalance of the direct and indirect pathways because of the loss of the
dopaminergic neurons in the SNpc, a loss of activity in the direct pathway and an
increase in activity in the indirect pathway, which leads to a reduced activity in the
thalamic output. Decreased DA input from the SNpc to the striatum leads to a reduced
excitatory input to the striatal D1 receptor-carrying neurons and results in an inhibited
GABA output in the direct way to the GPi/SNpr. Simultaneously, the D2 receptors
expressing neurons in the striatum become less inhibited through the reduced DA
input from the SNpc, which results in an increased GABA activity from the striatum
to the GPe in the indirect way. The neurons in the GPe receive an increased inhibitory
GABA input, and neuronal projection from GPe releases a decreased amount of
GABA to the STN. Thus, inhibited GABA input to the STN, results in increased
glutamatergic input to the SNpr. Taken together, reduced inhibitory GABA input
from the direct pathway and increased release of glutamate from the indirect pathway
leads to increased GABA output to the thalamus. In the thalamus the increased
GABA activity inhibits the glutamate release to the cortex, which results in reduced
motor activity (see figure 2).
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Fig 2. Diagram of the basal ganglia model.
Dopamine, dopaminergic neurons and dopamine receptors
Arvid Carlsson and his colleagues characterized dopamine (DA) in 1952 and
he was awarded the Nobel Prize in 2000 for the discovery. The neurotransmitter
dopamine is a member of the catecholamine family and a precursor of noradrenalin
and adrenaline. Tyrosine hydroxylase (TH) catalyzes the conversion of the amino acid
tyrosine to DOPA (dihydroxyphenylalanine), which is further converted to dopamine
via dopa decarboxylase (L-amino acid decarboxylase). Dopamine is packed into
vesicles in neurons and released by synaptic activity. Tyrosine hydroxylaseexpression is commonly used as a molecular marker for the detection of DA neurons.
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Tyrosine → Dopa → Dopamine → Noradrenaline → Adrenaline
TH
AADC
DBH
PNMT
TH = tyrosine Hydroxylase; AADC = dopa decarboxylase; DBH = dopamine-β-hydroxylase
PNMT = phenylethanolamine-N-methyltransferase
Fig 3. Pathway for dopamine synthesis.
Dopaminergic
neurons
are
present
in
four
brain
systems,
the
tuberoinfundibular, the mesolimbic and the mesocortical pathways, which are
involved in cognitive and rewarding behavior and the nigro/mesocortical pathway,
which is involved in voluntary motor movements (Fuxe et al., 1970; Lindvall et al.,
1984). In humans, approximately 590,000 DA neurons are found in the ventral
tegmental area (VTA) and SN in the first four decades of life. In a rat VTA and SN
around 45,000 DA neurons exist (Vernier et al., 2004).
Dopamine receptors are G-protein coupled and, to date, five DA receptors are
known so far. They are classified into two families, D1-like and D2-like receptors.
The D1 and D5 receptor belong to D1-like receptor family. D2a, D2b, D3, and D4
receptors belong to the D2-like receptor family. D1 receptors stimulate adenylate
cyclase whereas D2 receptors inhibit adenylate cyclase (Kebabian and Greengard,
1971; Kebabian and Calne, 1979; Spano et al., 1978).
Treatment strategies in Parkinson’s disease
After the discovery that dopamine is involved in the pathogenesis of
Parkinson’s disease, researchers focused their interest on the dopamine precursor
levodopa (L-DOPA) as a possible treatment for PD (Birkmayer and Hornykiewicz,
1961; Carlsson et al., 1958; Cotzias et al., 1967). Since then L-DOPA is the most
common treatment of PD patients. The drug is taken orally and in combination with a
peripheral dopa-decarboxylase inhibitor (benzerazid, carbidopa) to increase the
efficiency of L-DOPA. L-DOPA crosses the blood-brain-barrier, unlike dopamine,
and is converted to dopamine by intact, surviving dopamine neurons and released
17
from their nerve terminals. Other neurons, like the serotonergic neurons, may also be
involved in the conversion of L-DOPA (Arai et al., 1995). With long-term treatment
patients develop side effects from L-DOPA administration like L-DOPA-induced
dyskinesia and/or on/off symptoms (Ahlskog and Muenter, 2001; Granerus, 1978).
Dopamine agonists acting via postsynaptic dopamine receptors, inhibitors of
monoamine oxidase B and catechol-O-methyl transferase, both of which block the
metabolism of dopamine, are also used in the treatment against the parkinsonian
symptoms.
Deep brain stimulation is used in parkinsonian patients as a surgical
intervention. High frequently stimulating electrodes are implanted in the subthalamic
nucleus or the GPi to change neuronal activity in the basal ganglia. The treatment can
reduce tremor, bradykinesia, rigidity, and L-DOPA, induced dyskinesia (Benabid et al.,
1991; Benabid et al., 1998; Kumar et al., 1998a; Kumar et al., 1998b; Putzke et al.,
2003). However, neither drugs nor the deep brain stimulation can cure PD, i.e. restore
the nigrostriatal pathway or alter the natural history of the disease.
An alternative therapy to drug administration and deep brain stimulation in PD
is cell replacement. The aim with cell replacement is to restore the degenerated
nigrostriatal pathway. The perfect cell would be a dopaminergic neuron, which
produces and releases dopamine, forms long axons and interacts with appropriate host
neurons and cells in the degenerating brain. Therefore, one possibly therapy is
transplantation of tissue from the fetal ventral mesencephalon (VM) into the striatum.
Studies done in 1979 by Perlow and coworkers and by Björklund and Stenevi
demonstrated a therapeutic benefit in rodents (Björklund and Stenevi, 1979; Perlow et
al., 1979). Behavioral motor deficits could be compensated when fetal rat VM was
grafted into unilateral 6-hydroxydopamine (6-OHDA) lesioned rats (Björklund and
Stenevi, 1979; Perlow et al., 1979). Later, several studies in rodents demonstrated that
the grafted VM innervated the striatum in a target specific manner, showed long term
survival, released dopamine, formed synapses with the host neurons, and, had a
normalized striatal firing rate (Fisher et al., 1991; Freed et al., 1980; Freund et al.,
1985; Jaeger, 1985; Mahalik et al., 1985; Rose et al., 1985; Strömberg et al., 1988;
Strömberg and Bickford, 1996; Van Horne et al., 1990; Wictorin et al., 1992;
Zetterström et al., 1986). Studies performed by Strömberg et al. and Brundin et al. in
1986 showed that xenografts of human fetal VM in immunosuppressed 6-OHDA
lesioned rodents survived and exerted functional effects on motor behavior (Brundin
18
et al., 1986; Strömberg et al., 1986). The documentation that human grafts worked in
rodents became the starting point for clinical trials such as transplantation of fetal
human VM to immunosuppressed parkinsonian patients (Lindvall et al., 1989). Later,
several clinical trials showed graft survival for up to 18 years and improved motor
function in the absence of medication including reduced time spent in “off” state
(Kordower et al., 2008; Li et al., 2008; Mendez et al., 2008). In successful cases,
transplantation patients could stop their medication. Postmortem studies demonstrated
that dopamine neurons from the graft survived and reinnervated the putamen
(Kordower et al., 1998). Unfortunately, the outcome of transplantation in PD patients
demonstrated a variable degree of success. One problem is the immune response of
the host brain, since inflammation was observed around the transplant (Kordower et
al., 1995). Other problems, besides poor survival of dopamine neurons and
incomplete reinnervation of the dopamine neuron depleted areas of the host brain, is
graft-induced dyskinesia (Freed et al., 2001; Hagell et al., 2002), risk of infection,
ectopic graft placement, cell sources, and of course ethical issues arising from the use
of human fetal cells. It is important to investigate further the role of enhanced neuritic
formation, including neuroprotection and neurotrophic factors.
Lentiviral vectors are able to transduce cell types such as neurons, astrocytes
and adult neuronal stem cells in the brain and are used for gene therapy (Jakobsson
and Lundberg, 2006). In PD gene therapy it has been observed that the overexpression
of TH leads to production of DA in the transformed cells. Neuroprotection of the
dying dopaminergic neurons has been provided by overexpression of trophic factors,
like glial cell line-derived neurotrophic factor (for review see (Jakobsson and
Lundberg, 2006)). Unfortunately, overexpression of glial cell line-derived
neurotrophic factor is harmful in rodents and leads to side effects like downregulation of the TH expression, sprouting of dopaminergic fibers in inappropriate
areas and altered DA levels in the striatum.
Great hope for a novel development of the transplant strategy is based on
grafting embryonic/adult stem cells and neuronal progenitor cells into the striatum of
PD patients. Again, the ideal situation would be that stem cells differentiate in a large
number of functional dopaminergic neurons, which connect with the host brain. After
a complex culture procedure, stem cells can display the dopaminergic phenotype (for
review see (Riaz and Bradford, 2005)). Several exogenous factors like epidermal
growth factor, brain derived neurotrophic factor, basic fibroblast growth factor make
19
the embryonic stem cells (ESC) differentiate to dopaminergic neurons. Animal
transplant studies of ESC demonstrated long-term survival of the transplant and
improved motor behavior (Englund et al., 2002; Rodriguez-Gomez et al., 2007).
Unfortunately not all studies could reproduce these effects. Thus, the problems with
stem cell transplant that remain to be solved are poor survival, insufficient
differentiation into dopaminergic neurons, poor innervation, and development of
tumors.
Another strategy to achieve recovery of the nigrostriatal pathway is the use of
neural stem cells generated in the subventricular zone (SVZ), the hippocampus, the
hindbrain and the forebrain (Conti et al., 2006). In an animal model for PD improved
motor function was demonstrated after transplantation of dopamine expressing
midbrain stem/progenitor cells (Hovakimyan et al., 2006). Furthermore, increased
neurogenesis in the SVZ was followed by a migration of dopaminergic neurons into
the striatum after striatal DA depletion in adult rat (Aponso et al., 2008).
Development of the dopaminergic nervous system
Dopaminergic neurons of the VM are found in the SNpc, VTA and the
retrorubral field (RrF). Dahlström and Fuxe mapped dopamine cell groups in 1964
(Dahlström and Fuxe, 1964). They named the dopamine neurons in the
mesencephalon A8 (RrF), A9 (SNpc) and A10 (VTA). The newly born DA precursor
cells express TH at embryonic day (E) 10.5 in mice (Riddle and Pollock, 2003; Smidt
et al., 2003). The peak of RrF and SN DA neurogenesis is between E11 and E12 in
mice, while DA neurons from VTA peak somewhat later, at around E12- E13 (Bayer
et al., 1995; Marti et al., 2002). A similar time course of the DA system development
occurs in rat. At E12.5, the first DA expression is observed and one day later DA
nerve fibers reach the striatum (Gates et al., 2006; Seiger and Olson, 1973). By
postnatal week 3, the DA neurons have reached their fully development, their adult
morphology and functionality (Voorn et al., 1988).
20
Neurotoxins for creating animal PD models
Young drug abusers in the 1980´s in California developed symptoms similar
to PD after consuming synthetic heroin (Davis et al., 1979; Langston et al., 1983).
These symptoms were induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP), which is a side product of synthetic heroin. MPTP is a highly potent
neurotoxin, which rapidly crosses the blood- brain barrier after administration. Today
MPTP is a commonly used drug for inducing PD-like symptoms in animals. It is
metabolized by the enzyme monoamine B oxidase and is further oxidized to the actual
toxin 1-methyl-4-phenylpyridinium (MPP+) (Burns et al., 1983; Elsworth et al., 1987;
Jenner et al., 1984; Langston et al., 1984; Rios et al., 1995). MPP+ is taken up by
preferentially DA transporters of A9 nigro-striatal DA neurons and accumulates
within the inner mitochondrial membrane and inhibits the mitochondrial complex 1.
This results in the interruption of electron transport, release of reactive oxygen species
(ROS) and depletion of ATP. ROS triggers an apoptotic pathway especially in A9 DA
neurons, leading to degeneration of DA nerve terminals and consequently decreased
level of DA in the striatum and the appearance of PD-like symptoms. Increased
sensitivity to MPTP is found in humans and monkeys. MPTP is also used in nonprimate animals like mice, to produce parkinsonian symptoms, even though the
typical Lewy body inclusions are not observed. However, MPTP infusion via minipumps in mice causes the appearance of alpha-synuclein and ubiquitin-positive
aggregates resembling the Lewy bodies, whose presence correspond to parkinsonianlike symptoms (Fornai et al., 2005). Rats do not show such a high susceptibility to
MPTP.
Recently, rotenone has been used in animals to induce PD-like symptoms of
hypokinesia and rigidity. It is isolated from roots of certain plants and is the active
compound of many insecticides. It inhibits the mitochondrial complex 1 and causes
through oxidative stress, ROS formation and ATP depletion resulting in highly
selective degeneration of A9 nigro-striatal DA neurons, and the development of PD
symptoms (Betarbet et al., 2000).
The classical neurotoxin used to create a PD animal model is 6hydroxydopamine (6-OHDA). It is specific for catecholaminergic neurons and has
been most frequently used as a hemiparkisonian model in rodents (Kostrzewa and
21
Jacobowitz, 1974; Tranzer and Thoenen, 1967; Ungerstedt, 1968). This neurotoxin is
an unstable compound, which cannot cross the blood-brain barrier. 6-OHDA is taken
up via catecholamine transporter systems including DA receptors where it is easily
oxidized to produce ROS and hence induce neuronal cell death (Kostrzewa and
Jacobowitz, 1974).
Neuroinflammation
Neuroinflammation has been suggested to be one of the causes for neuronal
death in neurodegenerative diseases. The inflammatory process includes changes in
the levels of neuroprotective/ neurotoxic cytokines, and increased number of activated
microglia. Microglia, which constitute 20% of the total glial cell population in the
brain are involved in the immune response in the central nervous system (CNS) and
they can move towards injured tissue, plaques, and infectious agents. They undergo
morphological changes in response to injury in the brain. Resting microglia cells
appear in a ramified form, and possess branched processes, which can move around
and survey the surroundings, while the cell body is small and motionless. When
needed, they are able to phagocytose debris and then change their morphology to an
ameboid form. Microglia cells are neuroprotective, due to the production of cytokines
but also neurotoxic due to secretion of neurotoxic proinflammatory cytokines
(Sawada et al., 2008).
In postmortem PD brains, elevated levels of cytokines have been observed in
the cerebrospinal fluid and activated microglia in the brain parenchyma (Boka et al.,
1994; Mogi et al., 1994; Nagatsu, 2002; Sawada et al., 2006). The cytokines include
interleukins (IL)-1β, IL-2, IL-4, and IL-6, tumor necrosis factor alpha (TNFα) and
several transforming growth factors, like TGFα and TGFβ 1 and 2 (Mogi et al., 1994;
Mogi et al., 1996). These data have been confirmed in several animal models. Mogi et
al. showed increased levels of IL-1β especially in the striatum of MPTP treated mice
and increased levels of TNFα was observed in SN and striatum in the
hemiparkinsonian rat model (Mogi et al., 1998; Mogi and Nagatsu, 1999). In a study
by McGeer et al. the level of major histocompatibility complex class II antigen22
positive reactive microglia in the SN of PD patients was increased (McGeer et al.,
1988). Other studies suggest that activated microglia in the putamen of PD patients
produce TNFα and IL-6 (Imamura et al., 2003).
Increased levels of cytokines lead to oxidative stress, followed by a
proapoptotic and/or necrotic environment and a cascade of death signals. Intracellular
accumulation of reactive oxygen and nitrogen species within the cells induces
oxidative stress. Mitochondria are known to be involved in the production of ROS
and TNFα promotes overproduction of ROS in mitochondria (Goossens et al., 1995).
Besides the role of microglia, activated astrocytes and oligodendrocytes take part in
the inflammatory process. For instances, astrocytes regulate the glutamate uptake in
the CNS. Activation of astrocytes takes place through chemical or physical damage
and is characterized by the up-regulation of the glial fibrially acidic protein and a gapjunction protein called Connexin 43 (Haupt et al., 2007). Oligodendrocyte activation
is distinguished by secretion of inflammatory molecules, like nitric oxide, cytokines,
and by the up-regulation of chondroitin sulfate proteoglycans (Rhodes et al., 2006).
Thus, neuroinflammation plays an important role in PD and most likely affects the
outcome of grafting DA-rich tissues to the PD patient.
Tumor necrosis factor alpha (TNFα) and its receptors
In December 1968, a cytotoxic factor was first mentioned in the literature.
Granger et al. and Ruddle and Waksman contemporaneously published reports about
a cytotoxic factor produced by lymphocytes and named it lymphotoxin (Kolb and
Granger, 1968; Ruddle and Waksman, 1968). In 1975, another cytotoxic factor
produced by macrophages was found and named tumor necrosis factor (TNF)
(Carswell et al., 1975). A similarity between both factors was their ability to kill
mouse fibrosarcoma L-929 cells. Today it is known that this cytotoxic factor is a
TNF-family cytokine. TNFα and TNFβ are members of the TNF-family.
TNFα is involved in the response of the immune system and low levels are
produced by macrophages and neurons (Breder et al., 1993). TNF receptor 1 (TNFR1)
and TNF receptor 2 (TNFR2) are the two known receptors that bind TNFα. TNFR1 is
23
expressed in many cell types including nigral dopaminergic neurons (Aloe and Fiore,
1997; Boka et al., 1994; Carvey et al., 2005; Ling et al., 1998; McGuire et al., 2001;
Tartaglia et al., 1993) and is known as the “death receptor”. TNFα binds to TNFR1,
which results in a cascade that activates the apoptotic pathway. TNFR2 is expressed
by immune cells, including microglia but also in dopaminergic, hippocampal and
cortical neurons and activates inflammation and promotes survival through a cascade
of proteins (Bernardino et al., 2005; Dopp et al., 1997; Heldmann et al., 2005;
Marchetti et al., 2004; McGuire et al., 2001; Rao et al., 1995; Rothe et al., 1994;
Rothe et al., 1995).
TNFα is mostly secreted by activated microglia and is neurotoxic at a high
concentration. In animal studies both neurotoxic as well as a neuroprotective effects
of TNFα has been reported (Gemma et al., 2007; McGuire et al., 2001). A neurotoxic
effect is found for fetal VM DA neurons in a dose-dependent manner (McGuire et al.,
2001). In TNFα deficient mice, the neurotoxin MPTP is less effective on DA neurons
(Ferger et al., 2004). Neuroprotective effects of TNFα are found on injured DA
neurons in VM cultures (Shinpo et al., 1999). It seems, that TNFα, depending on
receptor activation, can give signals to mitochondrial toxicity, ROS production, and
apoptosis, but also to cell survival, proliferation, and differentiation.
CD47 and Sirpα
CD47, also called integrin-associated protein (IAP), plays a role in the
neuronal development (Murata et al., 2006; Numakawa et al., 2004). CD47 is a
member of the transmembrane immunoglobin (Ig) superfamily, possessing an Ig-Vlike extracellular region, five putative transmembrane domains, and a short
cytoplasmic tail. There are different isoforms of CD47 depending on the length of its
cytoplasmic tail; the most predominant is isoform 2. The second most abundant
isoform, isoform 4 has the longest cytoplasmic tail, which is expressed on neurons.
It is known that among other factors, integrins are involved in the formation,
maintenance, and function of synapses (Einheber et al., 1996; Ronn et al., 2000). The
function of CD47 has been well studied in non-neuronal cell populations. CD47
24
participates in the migration and activation of leucocytes in response to bacterial
infection (Verdrengh et al., 1999). Effects in the CNS have also been documented in
CD47 deficient mice; a reduction of memory retention and long-term potentiation has
been observed (Chang et al., 1999). Furthermore, overexpressing CD47, neurons
displayed enhanced dendritic outgrowth and up-regulation of synaptic proteins
(Numakawa et al., 2004).
The extracellular region of CD47 is a ligand for the transmembrane protein
signal regulatory protein alpha (Sirpα). Sirpα, also known as BIT, SHPS-1, p84,
MFR or CD172a, was first identified in neurons and later also found in cells outside
the brain (Jiang et al., 1999; Seiffert et al., 1999). The interaction between CD47 and
Sirpα in non-neuronal cells can among other things inhibit phagocytosis of immune
cells (Oldenborg et al., 2000). Both CD47 and Sirpα are found in synapse-rich
regions in the cerebellum and hippocampus (Mi et al., 2000; Ohnishi et al., 2005).
Interestingly, CD47 adenovirus transfection in cultured cerebral cortical neurons
induces apoptosis (Koshimizu et al., 2002).
CD47 serves also as a receptor for thrombospondin (TSP). TSPs are large
extracellular matrix proteins, which are involved in cell-cell and cell-matrix
interactions by binding membrane receptors, other cellular matrix proteins or
cytokines (Adams and Tucker, 2000; Adams, 2001; Bornstein, 2001; Lawler, 2000).
Five TSP members belong to the TSP family, TSP1-TSP5. TSP-1 and TSP-2 are
expressed in the brain but only TSP-1 binds to CD47 (Gao and Frazier, 1994).
Immature astrocytes produce TSP-1 and TSP-2 during brain development, and both
promote the development of synapses (Christopherson et al., 2005). However, the
interaction between CD47 and Sirpα and TSP-1 is not fully understood in the brain.
Extracellular matrix molecules - Proteoglycans
Neuronal growth is promoted and regulated by neurotrophic factors and
nondiffusible molecules, including extra cellular matrix molecules (ECM) and cell
adhesion molecules. Collagens, adhesive glycoproteins, and proteoglycans (PGs) are
part of the ECM family. The structure of PGs involves a core protein and
25
glycosaminoglycan (GAG) side chains attached to the core protein (see fig 4.). They
are located on the surface within the membrane and in the extracellular space
(Margolis et al., 1975). Proteoglycans participate in several cellular functions like
neuronal survival, adhesion, determination and migration, axonal growth and
guidance, synapse formation and glial differentiation during the development of the
nervous system (see reviews (Margolis and Margolis, 1993; Oohira et al., 1994; Small
et al., 1996)). Proteoglycans are divided into four major families based on the GAG
side chains they contain; keratan sulfate, hyaluronic acid, heparan sulfate plus
heparin, and chondroitin sulfate plus dermatan sulfate. In embryonic brains, the
chondroitin sulfate PGs (CSPGs) are highly expressed and have a primary role in
axonal guidance and axon pathfinding during development (Bandtlow and
Zimmermann, 2000; Bovolenta and Fernaud-Espinosa, 2000).
In the injured adult CNS, PGs fail to enhance axon guidance and axons are not
able to regenerate. This results in many neurons undergoing necrotic/apoptotic
processes. The glial scar, formed after the injury, becomes a barrier for growing axons
(Rhodes and Fawcett, 2004; Rhodes et al., 2006; Silver and Miller, 2004). One group
of PGs that mediate this inhibitory effect is the CSPGs, especially via their GAG
chains. The enzyme chondroitinase ABC (ChABC) catalyzes hydrolysis of the GAG
chains from the core protein without altering the core protein structure (Yamagata et
al., 1968). Thus, ChABC treatment promotes axon re-growth both in vivo and in vitro
(Bradbury et al., 2002; Fidler et al., 1999; McKeon et al., 1995; Moon et al., 2001).
ChABC treatment results in functional recovery of injured spinal cord neurons
(Bradbury et al., 2002).
The compound methyl-umbelliferyl-β-D-xyloside (β-xyloside) can be used to
inhibit
the
effects
of
the
CSPGs.
β-xyloside
blocks
the
activity
of
galactosyltransferase, which results in an insertion of xylosyl-serine in the core
protein. The altered GAG chains are unable to bind to the core protein, causing an
increased amount of nonbound GAG chains in the cytoplasm of the cell. As a result of
this, the PGs are never released (Niederost et al., 1999; Schwartz, 1977).
26
Fig 4. PGs consist of a core protein and GAG side chains.
Nerve fiber formation
Fetal VM tissue is used for grafting to the striatum of PD patients to improve
graft function it is important to control the graft outgrowth. Results obtained from
organotypic tissues culture studies can be used to improve reinnervation in the host
brain, and survival of the graft. In organotypic tissues cultures of fetal VM two
temporally different dopaminergic nerve fiber formations are observed; one formed in
the absence of glial cells and the other in close association of glial cells (Johansson
and Strömberg, 2002). In the cultures, the non-glial-associated TH-positive nerve
fiber formation appears after some days but does not persist over time. The later
appearing TH-positive nerve fiber formation is formed after 5-7 days and is more
persistent.
A8, A9 and A10 dopaminergic neurons are included in the VM tissue when
dissecting for transplants or tissue cultures (Dahlström and Fuxe, 1964). In
organotypic tissue cultures is demonstrated that A9 DA neurons participate in
27
producing both the non-glial-associated as well as the glial-associated nerve fibers as
revealed by aldehyde dehydrogenase A1-immunreactivity (Berglof et al., 2007).
VM tissue
slice
Glial-associated TH-positive outgrowth
Non-glial-associated TH-positive outgrowth
Fig. 5. The drawing displays the non-glial associated and the glial-associated TH-positive outgrowth
seen in VM cultures.
Taken together, the organotypic tissue culture technique is a useful tool to study nerve
fiber formation from fetal VM tissue because one can easily monitor the interaction
between nerve fibers and astrocytes while controlling the environmental conditions.
28
Aims of this thesis
•
To characterize and compare the interactions between astroglia and neuronal
outgrowth in ventral mesencephalic slice cultures, in respect to:
-
Fetal tissue age at plating
-
Effects of an inflammatory environment
-
CD47 gene deletion
• To compare ventral mesencephalic and spinal cord cultures concerning astrocytic
migration and the neuronal nerve fiber formations
29
Material and methods
Animals
Female Sprague-Dawley rats were used and tissue cultures were generated
from fetuses from the same strain (Paper I, II, III). In paper IV, CD47 wildtype and
knockout mice were used. Generation of CD47 knockout mice has been described
previously (Lindberg et al., 1996). The mice generated from the Balb/C strain and
backcrossed for 16 or more generations (Jackson Laboratory, Bar Harbor, ME). Mice
mating occurred over one night.
A gentle palpation of the abdomen of the rats was performed to determine the
developmental stage of the fetuses. Pregnant rats and mice were deeply anaesthetized
using 4% isofluran (Baxter Medical) the neck was dislocated and the fetuses were
obtained. Embryonic day (E) 12 rat fetuses had a crown-rump length (CRL) of 6-7
mm (paper I), E14 rat CRL = 12-13 mm (paper I, II, III), E18 rat CRL = 25-28 mm
(paper I, II) or E14 mouse CRL = 10 mm (paper IV). All animals used were kept in a
room with controlled temperature and a 12h/12h day and night rhythm. The animals
had free access to food pellets and water ad libitum. All experiments were approved
by the local ethics committee.
Dissection of the ventral mesencephalon, frontal cortex, and spinal
cord
Ventral mesencephalon (VM) was dissected from rat and mouse fetuses. In
Paper I, the frontal cortex was dissected to investigate interactions between VM tissue
and frontal cortex and the spinal cord was dissected from fetuses (paper II). Under a
microscope and sterile conditions the VM, the frontal cortex, and the spinal cord from
rat fetuses were dissected and transferred to Dulbecco’s modified Eagle’s medium
(DMEM; Gibco). The butterfly-shaped VM tissue and the spinal cord were sliced
with a tissues chopper in 300 µm coronal sections and every slice was cut in the
midline. One such piece was used for each culture. Fetal frontal cortex was dissected
30
bilaterally and cut into small pieces. E14 VM from CD47 wildtype and knockout
fetuses were dissected and chopped in the same way as the rat fetuses. In all papers
the “roller drum” culture technique was used (Gähwiler et al., 1997; Stoppini et al.,
1991) (see figure 6).
Fig 6. For culturing fetal VM the “roller drum” method was used in all papers. The chopped VM piece
was transferred into a plasma/thrombin clot, plated on a coverslip, placed into a 15 ml tube and placed
in a “roller drum” in an incubator.
Primary organotypic tissue cultures
The advantage of using the “roller drum” technique is that the constant flow of
medium over the slice culture minimizes direct contact with cell debris and other
waste products since the medium flushes over the culture rather than the culture,
being placed within the medium. Each tissue piece was transferred onto a 10x24 mm
poly-D-lysine (Sigma-Aldrich) coated glass coverslip. Chicken plasma (SigmaAldrich) and thrombin (Sigma-Aldrich) clot was used to ensure attachment of the
tissue piece to the coverslip. After drying of the clot for 20 min the coverslip was
inserted in a 15 ml Falcon tube containing 0.9 ml medium. All culture tubes were
placed in an incubator with a temperature of 37 °C and a CO2 flow of 5% in a “roller
drum” turning with a speed of 0.5 rpm. Fresh medium was provided twice a week.
VM (paper I-IV) and spinal cord (Paper II) was cultured as single pieces, and VM
was also co-cultured with frontal cortex (paper I).
31
Spinal cord
Cut in
half
Fig 7. The dissected spinal cord pieces were placed into a plasma/thrombin clot and transferred into a
15 ml tube.
Culture medium
The culture medium contained 55% DMEM, 32,5% Hanks´ balanced salt
solution (Gibco), 10% fetal bovine serum (Gibco), 1.5% glucose (Gibco) and 1%
Hepes (Gibco). After mixing the medium it was filtered through a sterile filter with a
pore size of 0.22 µm. The medium was stored at -20°C and thawed and heated to
37°C. At plating, a final concentration of 1% antibiotics (10.000 units/ml penicillin,
10 mg/ml streptomycin, 25 µg/ml amphotericin; Gibco) was added to the medium.
Medium was changed twice a week. Antibiotics were excluded from the first medium
change.
In paper III, 40 ng/ml tumor necrosis factor alpha (TNFα; R&D Systems) was
added to the medium between days 4-7 in vitro or between days 11-14 in vitro to
study the effect of TNFα specifically on the non-glial versus the glial-associated DA
nerve fiber outgrowth, respectively. In addition, cultures received TNFα plus
antibodies against TNFR1 (2.5 µg or 5.0 µg; Santa Cruz Biotechnology) or TNFR2
(2.5 µg or 5.0 µg; Santa Cruz Biotechnology) treatment with or without TNFα the
32
medium between days 4-7. Furthermore, neutralizing antibodies against TNFα (0.15
µg/ml; Serotec Oxford, UK) alone was added to the medium during days 4-7 in vitro.
In paper I the antimiotic agent cytosine β-D-arabinofuranoside (Ara-C; SigmaAldrich) was added to the medium to the concentrations 0.5, 1, 2, and 5 µM.
Antibodies against CD47 (mAB mIAP301; rat anti mouse; originating from Dr. F.P.
Lindberg, Washington University School of Medicine, St Louis, MO), Sirpα (mAB
P84; rat anti mouse; originating from Dr. C. Lagenaur, University of Pittsburgh, PA)
and TSP-1 (mAb-4; Clone A6.1; mouse monoclonal; Lab Vision USA) at a
concentration of 2.5 µg/ml were added to the CD47 WT culture medium in paper IV.
The CD47, Sirpα and TSP-1 antibodies were purified from hybridoma supernatants
by ammonium sulfate precipitation and affinity chromatography using Protein A or G
High Trap columns (Amersham Bioscience, Piscataway, NJ). In paper II, rat cultures
received 1.5 mM β-xyloside (Sigma-Aldrich) or 0.1 U/ml ChABC (Sigma-Aldrich)
for all medium changes.
Paper
Type of tissue
Treatment
Concentration
I
II
VM
VM, spinal cord
III
VM
Ara-C
ChABC,
β-xyloside
TNFα,
Antibodies
against
TNFR1,
TNFR2,
TNFα
0.5, 1, 2, 5 µM
0.1 U/ml
1.5 mM
40 ng/ml
IV
VM
2.5 or 5 µg/ml
2.5 or 5 µg/ml
0.15 µg/ml
Antibodies
against Sirpα,
TSP-1, CD47
Treatment
time
from day 2
all the time
all the time
between
days 4-7 or
11-14
4-7
4-7
4-7
DIV
7
14
14
7 or 14
14
2.5 µg/ml
Tab 1. Illustration of the treatments in paper I-IV.
33
all the time
Immunohistochemistry
Organotypic cultures were fixed in 2% paraformaldhyde in 0.1 M phosphate
buffer for 1 hour. The cultures were rinsed 3 times for 10 minutes in 0.1 M phospate
buffered saline (PBS) before and after antibody incubation. Incubation in primary
antibodies was performed for 48-72 hours at 4°C in a humid chamber followed by 1
hour incubation in secondary antibodies at room temperature. Primary antibodies
against TH, β-tubulin, S100, vimentin, Ki67, Iba1, GLAST and synaptophysin were
used. Incubation in 5% goat serum (Sigma-Aldrich) was performed for 15 minutes at
room temperature to block non-specific bindings. The serum was then gently tipped
off after incubation and cultures were incubated in antibodies against TH. For
visualizing cell nuclei, DAPI staining was performed for 10 minutes at room
temperature. All antibodies, except Ki67 were diluted in 1% triton-X-100 in 0.1 M
PBS. Cultures stained for Ki67 were preincubated with 0.3% triton-X-100 in 0.1 M
PBS and the antibody was diluted in PBS. All cultures were mounted on glass slides
with 90% glycerol in PBS. For antibody and dilutions used see table 2.
Primary
Type
Dilution
Source
Paper
1/300
1/300
II, III
I, IV
2.5 or 5 µg/ml
DiaSorin
Pel-Freez, USA
Novocastra
Laboratries
Wako
Chemicals
Santa Cruz
Biotechnology
Santa Cruz
Biotechnology
1/400
Dako
III
1/200
Sigma-Aldrich
I, II
1/200
1/100
Sigma-Aldrich
Abcam
I
I
antibodies
TH
TH
Ki67
Iba 1
TNFR1
TNFR2
S100
β-tubulin
Vimentin
Vimentin
Mouse
monoclonal
Rabbit anti rat
Mouse
monoclonal
Rabbit
polyclonal
Goat
polyclonal
Rabbit
polyclonal
Rabbit
polyclonal
Mouse
monoclonal
Mouse
monoclonal
Rabbit anti calf
1/150
1/1000
2.5 or 5 µg/ml
34
III
III
III
III
Vimentin
GLAST
Synaptophysin
Chicken
polyclonal
1/200
Guinea pig anti
rat
1/4000
Mouse
monoclonal
1/1000
Abcam
ChemiconMillipore
II, IV
Millipore
Originating
IV
IV
I
from Dr. F.P.
Lindberg, St
CD47
Rat anti mouse
2.5 µg/ml
Louis, MO
Originating
IV
from Dr. C.
Lagenaur,
University of
Sirpα
TSP-1
Secondary
antibodies
A594
A594
A488
A488
A488
A488
Rat anti mouse
2.5 µg/ml
Pittsburgh, PA
Mouse
2.5 µg/ml
Lab Vision
IV
Source
Molecular
Probes
Molecular
Probes
Molecular
Probes
Molecular
Probes
Molecular
Probes
Molecular
probes
Paper
monoclonal
Type
Goat anti
mouse
Goat anti
rabbit
Goat anti
mouse
Goat anti
rabbit
Goat anti
guinea pig
Goat anti
chicken
Dilution
1/500
1/500
1/200
1/500
1/500
1/200
Tab 2. Primary and secondary antibodies used in paper I-IV.
35
I, II, III
I, III, IV
I, III, IV
I, III
I
II, IV
Image analysis
Measurements and cell counts
In all papers (I-IV), astrocytic migration and nerve fiber outgrowth from fetal
VM and spinal cord cultures were measured using a scale mounted in one ocular of
the microscope. In each culture, astrocytic migration was measured from the
periphery of the tissue slice, in four perpendicularly placed directions, to the distal
end to which the astrocytes migrated. The mean values for each parameter and culture
were used for statistical analysis. Nerve fiber outgrowth was measured for both the
non-glial- and the glial-associated nerve fibers. Three to four measurements were
made from the periphery of the tissue slice to the distal end that the nerve fibers
reached per culture and type of growth. In paper III, all Iba-1 positive cells were
counted within a standardized frame mounted in one ocular of the microscope at a
magnification of 20 x.
Statistics
All cultures were analyzed blind coded. Statistical analyses were performed
using one- or two-way analysis of variance (ANOVA) followed by Fisher’s post hoc
(paper 1II) analysis and Bonferoni post hoc analysis (paper I, II, IV) or using
Student’s t-test (paper I and IV). All data are expressed as group means ± SEM.
Statistically significant was determined when p<0.05*, p<0.01**, p<0.001***.
36
Results and discussion
This thesis is focused on the two timely and morphologically separated nerve
fiber outgrowths from organotypic tissue cultures and how astrocytes affect the nerve
fiber formations. The aims are to optimize the conditions for dopaminergic implanted
transplants into the parkinsonian brain and to improve graft function by controlling
grafted nerve fiber outgrowth.
In organotypic VM tissue cultures two different kinds of TH-positive nerve
fiber outgrowth have been described (Berglof et al., 2007; Johansson and Strömberg,
2002). An early dopaminergic nerve fiber formation, with long fibers, but not
persistent over time, appears in VM cultures at 3-5 DIV without any glia present. The
second, later appearing nerve fiber formation grows and extends their fibers onto a
monolayer of astroglial cells. The astrocytes migrate from the tissue slice and form a
monolayer surrounding the tissue slice. At the distal end of the outgrowth, a mesh-like
nerve fiber network is formed. This outgrowth is persistent over time. Since, the
initially formed nerve fibers occur in the absence of astroglia and the later formed
wave of nerve fibers is always attached to the astroglia these are described as nonglial-associated and glial-associated growth, respectively.
Non-glial-associated versus glial-associated nerve fiber outgrowth
E14 and E18 cultures from VM and spinal cord were chosen for comparison
of TH/β-tubulin -positive nerve fiber formations from two different tissue sources
(paper II). The cultures were analyzed using TH- or β-tubulin- and vimentinimmunohistochemistry after 14 DIV. In E14 VM as well as in spinal cord cultures the
same outgrowth patterns were observed. Both culture types displayed the non-glialand the glial-associated TH/β-tubulin-positive nerve fiber formations. In E14 spinal
cord cultures the non-glial-associated outgrowth appeared very massive and reached
longer distances compared to VM cultures, where the non-glial-associated THpositive formation appeared more dotted and degenerative. The glial-associated THpositive outgrowth grew around 400 µm from the tissue slice in a close association
with the astrocytes in E14 VM cultures. The nerve fiber growth patterns in E18 VM
37
and spinal cord cultures were changed compare to E14 cultures. Non-glial THpositive nerve fiber outgrowth was not obtained in the E18 VM cultures while in
spinal cord, β-tubulin immunohistochemistry displayed long abundant nerve fibers. In
both, E18 VM and spinal cord cultures neurons migrated from the tissue slice onto the
monolayer of astrocytes (paper I, II).
The non-glial-associated nerve fibers have a great capacity for long-distance
growth, often several millimeters (Johansson and Strömberg, 2003). Therefore, it
would be useful to learn how to control the non-glial-associated nerve fiber growth to
be used in transplantation, since graft-derived innervation of the host brain reaches
typically a maximum of 1 mm. The glial-associated TH-positive nerve fiber
outgrowth, on the other hand, survives for long-term while the growth has terminated
at approximately two weeks (Johansson and Strömberg, 2003). Interestingly, the
distance that the glial-associated nerve fibers reach is similar to that found in the
grafted VM tissue. Therefore, to overcome the short-distance innervation from grafted
VM tissue into the parkinsonian brain, multiple transplants are needed to cover the
DA-degenerated areas (Nikkhah et al., 1994). This may cause the DA “hot spots” that
may participate in the development of graft-induced dyskinesia (Steece-Collier et al.,
2003). To summarize the earlier and the present findings organotypic tissue cultures
produce non-glial- and glial-associated growths. These appear to be a general
phenomenon for neuronal tissues since the same feature was observed both in VM
and spinal cord tissue cultures.
The age at plating influences nerve fiber growth patterns
The influence of the developmental stage of the tissue at plating on formation
of the non-glial-associated TH-positive growth was studied. Thus, VM from E12,
E14, and E18 was dissected and cultured in a “roller drum”, and studied at 7 DIV
using TH-, vimentin-, or GLAST-immunohistochemistry, and staining nuclei by
DAPI (paper I). GLAST was used as a marker for radial glia (Shibata et al., 1997). In
E12 VM cultures, the non-glial-associated TH-positive nerve fiber outgrowth was
detected, and appeared together with thin vimentin- and GLAST-positive fibers in the
38
absence of cell bodies as confirmed by DAPI negativity (paper I, fig. 3a, 5d-f). These
fibers were sometimes seen in high density. The non-glial-associated TH-positive
nerve fiber formation was also observed in E14 cultures but then with a dotted
degenerating appearance, while E18 VM cultures did neither display this early nerve
fiber formation nor the GLAST-positive fibers.
The glial-associated TH-positive nerve fiber formation was more or less
absent, or had barely started to grow in E12 cultures at the time point for evaluation
and the neurons were numerous and small in size (paper I, fig. 5c). Instead, in E14
VM cultures these nerve fibers grew onto a monolayer of astrocytes. However, in E18
cultures TH-positive neurons migrated several hundred micrometers away from the
tissue slice. Measurements of the astrocytic migration showed that the astrocytes in
E18 cultures reached the longest migration distance compared to cultures of E12 and
E14 (paper I, fig.4). However, at E18 this long migration did not promote nerve fibers
to grow.
The appearance of the two types of TH-positive nerve fiber outgrowths seems
to be dependent on different developmental stages. The non-glial-associated nerve
fiber formation and the GLAST/vimentin-positive fibers might act as pathfinders and
guide the astrocytes and the glial-associated nerve fiber formation. Radial glia has
been considered to be a scaffold for neuronal migration (Hidalgo et al., 1995; Hidalgo
and Booth, 2000; Shults et al., 1990).
The non-glial-associated TH-positive nerve fiber outgrowth innervated the
cortex when E14 VM and E14 cortex were co-cultured (paper I, fig. 6), while the
glial-associated TH-positive nerve fiber outgrowth targeted the lateral ganglion
eminence (Johansson and Strömberg, 2003). Thus, each type of nerve fiber targeted
different areas. Further, attempts have been made to distinguish if the two different
TH-positive nerve fiber formations are derived from either A9 or A10 DA neurons,
since both types are included in the dissected and cultured VM (Berglof et al., 2007).
However, the TH-positive nerve fiber outgrowths could not be correlated to A9 and
A10 dopaminergic neurons, but as revealed by their preferred target growth, the nonglial-associated TH-positive nerve fibers is suggested to be A10 rather than A9 (paper
I).
39
Astroglia affects the presence of non-glial-associated TH-positive
growth
Fetal astroglia play a role in guidance and support for neuronal development.
In paper I, astrocytic migration was reduced with the antimitotic agent Ara-C to
observe the effects of the two subtypes of TH-positive nerve fiber formations. E14 rat
VM “roller drum” cultures were treated with a dose-response of Ara-C for 7 DIV
(paper I, fig 1). The migration of vimentin-positive astrocytes was measured from the
periphery of the tissue slice to the distal end to which they migrated. High Ara-C
concentration of 2 µM and 5 µM led to poor survival of the neurons, and a low
concentration of 0.5 µM Ara-C had no effect on the astrocytes. The optimal working
concentration of 1 µM was selected for further investigations. Using vimentinimmunohistochemistry no difference was seen in the distance that the astroglia
migrated in control and treated cultures at day 5, but a significant reduction of
migration in Ara-C-treated compared to control cultures was found at 7 and 14 DIV
(paper I, fig. 2). The glial-associated TH-positive nerve fiber outgrowth reached
similar length as the control cultures at 5 or 7 DIV. However, decreased astrocytic
migration by Ara-C treatment significantly reduced the glial-associated TH-positive
nerve fiber formation compared to control cultures at 14 DIV (paper I fig. 3b, c, e).
The effect of blocking astroglia proliferation resulted in a higher frequency of
cultures displaying the non-glial-associated TH-positive nerve outgrowth compared to
control cultures at 5 and 14 DIV. Although, the length of the nerve fibers increased
over time, no difference was noticed comparing Ara-C treated and control cultures.
Therefore, the present results strengthened previous studies that are a direct
interaction between astrocytes and the two kinds of TH-positive nerve fiber subtypes
(Berglof et al., 2007). Astroglia promotes the long-lasting TH-positive nerve fiber
formation probably through releasing molecules such as ECM and PGs.
40
Effects of inhibiting proteoglycans
In paper I the effect of inhibiting astroglia proliferation was demonstrated.
Further investigations were made to study the consequences of degrading PG
synthesis for the astrocytes and the two nerve fiber outgrowths. PGs are involved in
the neuronal development. PG synthesis can be inhibited by the compound βxyloside, which degrades the action of the PGs and GAG chains are never released
from the astrocytes (Niederost et al., 1999). The enzyme ChABC cleaves the GAG
chains from the core protein resulting in free active extracellular GAG chains
(Yamagata et al., 1968). β-xyloside and ChABC are used after spinal cord injuries to
promote neuronal regeneration. These two compounds might be helpful to increase
neuronal outgrowth in VM grafts.
In paper II VM and spinal cord “roller drum” cultures from E14 and E18
fetuses were treated with 1.5 mM β-xyloside or 0.1 U/ml ChABC for 14 DIV and
evaluated using either antibodies against TH or β-tubulin and vimentin. In β-xylosidetreated E14 VM cultures, the astrocytes migrated significantly shorter distances from
the tissues slice compared to control cultures and E14 VM ChABC-treated cultures
(paper II, fig. 3a). The measured distance of the migrated vimentin-positive astrocytes
in ChABC-treated VM E14 cultures did not differ from control cultures (paper II,
fig.3a). E14 spinal cord cultures demonstrated the same effect as that seen in E14 VM
cultures. The astrocytic migration of β-xyloside treated spinal cord cultures were
inhibited compared to control cultures while spinal cord ChABC treated cultures
reached around the same migration distance compared to control cultures (paper II,
fig. 3b)
When studied nerve fiber formations, similar results were achieved for both
E14 VM and spinal cord cultures. The non-glial-associated nerve fiber outgrowth was
inhibited in β-xyloside treated cultures (paper II, fig. 2a, 2b). The reduced astrocytic
migration seen after β-xyloside treatment consequently affected the glial-associated
outgrowth, which also was reduced and often appeared with a dotted
immunoreactivity pattern (paper II, fig. 2c, f, i). Instead, ChABC treatment did not
affect the length of the non-glial-associated or the glial-associated nerve fiber
formation in VM or spinal cord cultures (paper II, fig. 2a-c, e, h).
41
In both E14 VM and spinal cord systems degrading of PGs synthesis (βxyloside treatment) inhibited the astrocytic migration while the presence of free GAG
chains (ChABC treatment) did not affect the migration. Inhibiting PG synthesis
affected the nerve fiber formations by a reduced outgrowth and often a dotted TH/βtubulin immunohistochemical reactivity was observed in the culture slices. Thus, both
systems were sensitive to β-xyloside treatment. Therefore, PGs are necessary in the
early development of the VM and the spinal cord while active GAG chains /core
proteins play the main part. The core protein and GAG chains separately, are thought
to have their own effect in promoting or inhibiting neurite growth (Carbonetto and
Cochard, 1987; Oohira et al., 1994; Snow et al., 1990).
Since, investigations were made about astrocytic migration at different
developmental stages (paper I), VM and spinal cord cultures from E18 fetuses were
studied for the effect of degrading PGs at later stages. Non-glial-associated THpositive nerve fiber outgrowth was not detected in none of the E18 VM cultures while
it was seen in E18 spinal cord cultures (paper II, fig. 4a, 4d, 4g). The fibers in E18
spinal cord control and β-xyloside treated cultures appeared often abundant and
massive. In ChABC treated cultures the non-glial-associated β-tubulin nerve fiber
outgrowth was detected in a few cultures with only single fibers. The long nerve fiber
formation is a common feature and seems to be even more potent and long-lasting in
spinal cord than in VM cultures.
The astrocytic migration in E18 VM β-xyloside treated cultures was reduced
compared to control and ChABC treated cultures while it did not affect the astrocytic
migration in E18 spinal cord cultures. ChABC treatment did not affect the distance
that the astrocytes migrated in E18 VM and spinal cord cultures (paper II, fig. 3 c,
3d,). Furthermore, the neurons were inhibited to migrate from the tissue slice and
follow the astrocytes as seen in control cultures (paper II, fig. 4a-f).
Degrading PGs or cleaving of the GAG chains of the PGs has a variable effect
on different developmental stages in VM and spinal cord cultures. Free GAG chains/
core proteins are not enough to support the migration of the neurons such as the intact
PGs are. PGs are known to be involved in controlling the migration of fetal neurons
(Snow et al., 1990). In adult nerve tissue ChABC treatment promotes neuronal
regeneration (Moon et al., 2001; Nakamae et al., 2009). This study supports others
studies showing the importance and the developmental switch of the function of PGs
42
in fetal tissue compared to adult, although the present results suggest a switch already
between E14 and E18 (Mark et al., 1989; Mark et al., 1990).
Effects of tumor necrosis factor α
Neuroinflammation is involved in the development of PD. TNFα is one of the
up-regulated proinflammatory cytokines in PD brains (Boka et al., 1994). It is
released by activated astrocytes and activated microglia. Hence, VM grafts are
transplanted into a neuroinflammatory environment in the brain, which might
influence the survival of the transplant and its ability to reinnervate the host.
Investigation of the effects of these proinflammatory cytokines on glia cells and the
two DA nerve fiber populations were made by adding TNFα (40 ng/ml) to the
medium of E14 VM “roller drum” cultures between days 4-7 (early time point) or 1114 (late time point) to affect either the non- glial- or the glial-associated TH-positive
growth, respectively (paper III). The cultures were processed for TH- and S100immunohistochemistry after 7 or 19 DIV. Interestingly, TNFα had a stimulatory
effect on astroglia migration at the early treatment time point. The astrocytic
migration reached significantly longer distances from the tissue slice compared to
control cultures (paper III, fig. 2b). The same effect was observed for the glialassociated TH-positive nerve fibers and the early TNFα treatment enhanced the
length of their outgrowth compared control cultures (paper III, fig. 1b, 2a). However,
the proliferation of the astrocytes was not enhanced in the early treated cultures (paper
III, fig. 6). The astrocytic migration and the outgrowth of the glial-associated nerve
fiber formation were not affected in cultures treated at the late time point and
evaluated at 19 DIV compared to control cultures (paper III, fig. 1b, 2a, 2b).
The non-glial-associated TH-positive nerve fiber outgrowth was prevented to
TNFα. This effect was observed after the early TNFα treatment resulting in a reduced
number of cultures displaying the nerve fibers. None of the cultures, that received the
late TNFα treatment, displayed the non-glial-associated nerve fiber formation (Paper
III, tab.1). This finding is interesting concerning the fact that PD brains often show a
high level of inflammation. Therefore our work suggests that grafting into a PD brain
43
with a neuroinflammatory environment is toxic for non-glial-associated nerve fiber
formation, which might act as a pathfinder for guiding the transplant and support the
migration of the astrocytes and the glial-associated nerve fiber outgrowth. At the same
time, early TNFα treatment promoted astrocytic migration and the glial-associated
TH-positive nerve fiber formation. Similar results have been observed in a study on
E15 midbrain DA neurons where TNFα treatment induced cell death while a
subpopulation of DA neurons were not affected (McGuire et al., 2001). Early TNFα
treatment in 6-OHDA lesioned rats reduced the loss of TH-positive neurons in the
striatum suggesting that TNFα may have protective properties (Gemma et al., 2007).
Thus, TNFα may exert dual effects.
Microglia in VM cultures treated with TNFα
Microglia phagocytose debris but also produce many toxic compounds. The behavior
of microglia was investigated using the pan-microglia marker Iba1 in VM cultures.
The early TNFα treatment (days 4-7) did not affect the number Iba-1-positive cells.
By contrast, TNFα treatment at a later time point (days 11-14) caused an increased
amount of microglia compared to controls (paper III, fig. 4a). A decreased amount of
OX-6-positive microglia after early (1-7 days) TNFα treatment was observed in a 6OHDA lesioned brain (Gemma et al., 2007). Taken, together, it appears that the time
point with TNFα treatment seems to play an important role in the activation and the
amount of microglia. Interestingly, not only the number of microglia was changed but
also their morphology. The morphological change of the microglia might be based on
the fetal developmental stages of the microglia and be independent of the TNFα
treatment. The role of microglia in a developing brain is not clear, while in neonatal
and adult brain the inflammatory properties of microglia through TNFα activation
and release have been well studied (Jonakait, 2007).
44
TNFα and its receptors
Blocking TNFR1 or TNFR2 during TNFα treatment
The effects found in TNFα treated VM cultures were further investigated by
adding antibodies either against TNFR1 or TNFR2 to TNFα treated cultures (paper
III). The antibodies were added to the medium at two different concentrations, a low
dose (2.5 µg/ml) or a high dose (5 µg/ml) between days 4-7 simultaneously with
TNFα treatment. At 7 DIV, the treated cultures were examined using TH- and S100immunohistochemistry. Treatment with TNFα together with antibodies against
TNFR1 in both doses enhanced the number of cultures displaying the non-glialassociated TH-positive nerve fiber outgrowth compared to TNFα treated cultures.
Adding TNFR2 antibodies with a high dose to TNFα treated cultures resulted in a
reduced number of cultures showing the non-glial-associated TH-positive outgrowth
compared with cultures treated with TNFα alone. In cultures treated with TNFα plus
a low dose of TNFR2 antibodies, 50% of these cultures demonstrated the non-glialassociated TH-positive nerve fiber formation (paper III, fig.5).
The astrocytic migration and the glial-associated TH-positive outgrowth were
reduced when blocking either TNFR1 or TNFR2 along with TNFα compared to
TNFα treatment alone but within the same range as compared to control cultures. In
TNFR2 + TNFα treated cultures the number of cultures displaying the glialassociated TH-positive outgrowth was reduced (paper III, fig. 4b).
Blocking TNFR1 or TNFR2 without TNFα treatment
Antibodies against TNFR1 or TNFR2 in two different doses, a low dose (2.5
µg/ml) or a high dose (5 µg/ml) were added to the medium of E14 VM cultures
between days 4-7. TH and S100 immunohistochemistry was used for investigation at
7 DIV. The non-glial-associated TH-positive outgrowth was seen in cultures treated
either with antibodies against TNFR1 or TNFR2 independent of which doses of the
antibodies were used. The length of this nerve fiber formation in treated cultures was
similar to that in control cultures.
Both concentrations of either antibodies against TNFR1 or TNFR2 resulted in
a decreased number of cultures displaying the glial-associated TH-positive nerve fiber
outgrowth and a reduced length of the outgrowth compared to early TNFα treated
45
cultures (paper III, fig. 4b) and these nerve fibers did not look healthy. They appeared
as dots rather than intact nerve fibers. Adding TNFR1 or TNFR2 antibodies led to a
reduced migration of the astrocytes compared to cultures treated with TNFα but
reached around the same distance as measured in control cultures (paper III, fig. 4b).
Blocking endogenous TNFα
By adding antibodies against TNFα, the endogenous TNFα level was
neutralized in VM E14 cultures, which resulted in no TH-positive outgrowth and
reduced astrocytic migration compared to TNFα treated cultures (paper III, fig. 4b).
Taken together, both TNFR1 and TNFR2 are needed for TNFα to exert its
effect. The effect could not be recovered by adding TNFα to the medium when one
receptor was blocked. In this study TNFα had neuroprotective and neurotoxic effects
on the different kinds of TH-positive nerve fiber. TNFα is necessary for the
development and the outgrowth of TH-positive nerve fibers and both TNFRs are
expressed on DA neurons (McGuire et al., 2001). In E12.5 midbrain cultures, the
number on DA neurons was enhanced after TNFα treatment (Doherthy, 2007). There
is a switch when TNFα induces neurotoxicity to DA neurons. One is a matter of the
developmental stage of the DA neurons and the other is dose-dependent TNFα
treatment. TNFα treatment of E16 and E18 midbrain cultures decreased the number
of DA neurons (Doherthy, 2007). The same effect was observed on mesencephalic
DA neurons from E15 cultures where TNFα induces neurotoxicity in a dose
dependent manner (McGuire et al., 2001). Interestingly, a DA subpopulation was not
affected by the TNFα treatment (McGuire et al., 2001). TNFα is needed for the cells
in a low doses but it is toxic at a later developmental stage and in high doses.
Concerning the fact that VM grafts are often transplanted into a TNFα enriched
environment, and the grafted neurons are at an early developmental stage, our studies
suggest that the environment for the grafts might not be as toxic as previously
suggested.
46
Long-distance nerve fiber outgrowth in CD47-/- mice
The optimal conditions for a VM graft would be, apart from survival and
functionality, that the graft, and especially the long-distance nerve fiber formation
does not degenerate and is resistant to a neuroinflammatory environment. To further
study this issue one interesting strategy is a mouse model with a knockout for the
CD47 gene. The cross talk between CD47 and Sirpα is important for migration and
synptogenesis (Matozaki et al., 2009). The VM of E14 mice was dissected and
processed for TH- and vimentin-immunohistochemistry after 7 and 14 DIV in a
“roller drum” (paper IV). At 7 DIV, no differences were seen in CD47-/- and CD47+/+
cultures with respect to astrocytic migration or the two waves of TH-positive nerve
fiber formation (paper IV, fig. 1a,b, g-i). However, after 14 DIV the CD47-/- cultures
displayed massive, viable, and extremely long non-glial-associated TH-positive nerve
fibers, while these nerve fibers degenerated in CD47+/+ (paper IV, fig 1c, f, g). The
nerve fiber outgrowth in CD47-/- cultures grew 1 mm longer than in CD47+/+ cultures.
Interestingly, the migration of the astrocytes and the length of glial-associated THpositive outgrowth did not differ in cultures from CD47-/- compared to CD47+/+
cultures (paper IV, fig. 1 h, i). For the first time, it was observed that the non-glialassociated TH-positive nerve fiber formation did not degenerate in the cultures when
astrocytes had migrated. The interaction between astrocytes and the two waves of THpositive nerve fiber formations in CD47 knockout mice follows the same growth
pattern as seen in CD47+/+ mice. The long-distance outgrowth reflects great potential
to improve the length of DA neuronal growth and gives great hope for future grafting
experiments.
To investigate through which receptor CD47 influenced the long-distance
nerve fiber formation, antibodies against Sirpα or TSP-1 were added to the medium
of wild type cultures for 14 DIV. The cultures receiving Sirpα-antibodies displayed
similar distribution of nerve fibers and astrocytes as observed in CD47-/- cultures
(paper IV, fig. 2b). The long non-glial-associated TH-positive nerve fiber formation
reached nearly the same length as found in CD47-/- cultures with a similarly high
density (paper IV, fig. 2d). Blocking TSP-1 also promote the nerve fiber formation
while the long massive appearance was not obtained (paper IV, fig. 2c, d). The
47
astrocytic migration and the length of glial-associated TH-positive nerve fiber
formation were not affected by blocking Sirpα or TSP-1 (paper IV, fig. 2e, f).
Cultures from CD47+/+ and CD47
-/-
mice were examined by TH- and
synapthophysin-(SY) immunohistochemistry after 14 DIV. SY is the most abundant
protein of the synaptic vesicle, it is expressed throughout most nerve terminals and is
correlated with synaptogenesis. High density of SY- immunoreactivity was found in
the tissue slices and in the area where the non-glial-associated TH-positive fibers
grew in both kinds of cultures. SY and TH-positive fibers did not show colocalization (paper IV, fig. 2g). However, in CD47-/- cultures an enhanced SYpositive immunoreactivity was observed on cells migrating from the tissue slice
compared to CD47+/+ cultures (paper IV, fig. 2h, i).
The non-glial-associated TH-positive fiber formation is usually degenerating
in VM cultures over time but not in CD47 gene deleted VM cultures (Berglof et al.,
2007). In fetal VM cultures the lack of CD47 promotes long, viable, and massive
nerve fiber formation and the expression of synaptophysin. The role of CD47 in the
brain is not well understood and the results are controversial. On one hand CD47
overexpression promotes dendritic outgrowth and up-regulation of the synaptic
proteins but on the other hand CD47 adenovirus infection induced cell death in
cultured cerebral cortical neurons (Koshimizu et al., 2002; Numakawa et al., 2004).
Furthermore, Sirpα is involved in synapse formation (Umemori and Sanes, 2008).
These results suggest that a cross talk between CD47 and Sirpα inhibits long nerve
fiber formation while inactivation of CD47 supports the presence of synaptoactive
molecules via action through other receptors (Umemori and Sanes, 2008). This
manipulating CD47 levels in graft tissue might be a condition that needs to examine
in future grafting experiments.
48
Concluding remarks
♦ The formation of the long-distance growing non-glial-associated and the glialassociated growths appears to be a general phenomenon that applies to most
neurons, and their presence are depending on the age of the tissue at plating, i.e.
the non-glial-associated tissue is more persistent in cultures from earlier fetal
stages. In cultures from older stages (E18) neurons tend to migrate instead of
forming nerve fibers.
♦ The presence of the long non-glial associated and the glial-associated outgrowths
are dependent on the presence of astroglia such that radial glia are present along
the non-glial-associated growth and the glial-associated growth is strictly growing
onto a monolayer of astrocytes. The direct correlation with nerve fiber formation
and the presence of astrocytes can be demonstrated by inhibiting the migration of
astrocytes using a mitosis inhibitor.
♦ PGs are important for the nerve fiber formation, and inhibiting PG synthesis
reduces astrocytic migration and glial-associated outgrowth from both E14 VM
and spinal cord cultures, whereas degrading the PGs does not change these
parameters. The PGs promote astrocytic migration in E14 tissue cultures and
neurons in E18 cultures.
♦ The proinflammatory cytokine TNFα exerts toxic effects to the non-glialassociated nerve fiber formation while it stimulates astrocytic migration and the
glial-associated nerve fiber outgrowth in VM cultures at an early treatment.
Blocking endogenous TNFα results in severe cell death of TH-positive neurons.
Thus, TNFα has dual effects on nerve fiber formation.
♦ The non-glial-associated growth is persistent simultaneously with the presence of
glial-associated growth and migrating astrocytes in the absence of CD47. The
presence of robust non-glial- and glial-associated nerve fibers is correlated with the
presence of synaptophysin, a synaptoactive molecule.
49
Taken together, this thesis has demonstrated an interaction between astroglia and
nerve fiber formations at different ages, the switch of the necessity of PGs and the
dual effect of TNFα. The long non-glial-associated nerve fiber formation is common
in cultured nerve tissue and might be controlled through CD47/Sirpα cross talk.
50
Acknowledgement
Big thanks to my supervisor Ingrid Strömberg for being a great mentor, for your help,
support, patience, encouragement and believing in me. Thanks for creating a nice lab
atmosphere, taking us to good conferences at so many nice places and thanks that your doors
are always open.
Thanks to Janove Sehlin, Per Lindström, Gerd Larsson-Nyrén, Barbo Borgström, Anita
Dreyer-Perkiömäki, Göran Dahlström, Ann-Charlotte Idahl, Gunilla Forsgren, EvaMaj Lindström, Eva Boström, Eva Hallin, Karin Grabbe, Doris Johansson (special
thanks for your help with all my mice mating), Tomas Jonsson (Thanks for helping me
handling the rats, especially one rat) Mats Högström and all the people from IMB for your
help, support and making IMB a nice working environment. Thanks Per-Arne Oldenborg
for collaboration and coming up with German words and polish jokes.
Thanks to all the lab girls, Mattias and Anders for making the lab a nice, relaxed working
place and for your help, support, talks and all the fun and the good tasting fikas. Thanks Sara
a.k.a. Api for forcing me to scratching your arms and back, giving me a water bottle and a
lock for my first IKSU visit, being happy and teaching me a lot of things. Elisabet, thanks for
coming up with Swedish grammar roles, trying to go to IKSU with me in the morning and for
being a nice office friend. Anna R., you and me = fika kompisar! Even though you fika
sometimes hemligt. Thanks for being happy, positive, funny…I just say thumbs up! Nina, the
lab sunshine, always there, helpful, kind, good –humored and jaja…learning fast…from who?
The master? Me? Btw vegetables are not dangerous. Anna N. it is very nice to share the
office with you. Thanks for being always happy, positive and listening to all my talking. And
akta stora skyltar på äpplen! Mattias, thanks for being a nice office mate and ja it is your
fault I started with running and now I have to run Lidingöloppet. Anders, you made the best
pictures, hang in there with us girls. Thanks to Maria, Åsa and Ing-Marie for good cakes,
nice talks and being there for me. Thanks to Heather, Martin, Micke and all the other “old”
histologen for help, support and fun.
Kristina, durch Dich hab ich gemerkt...ich bin wirklich ein Plani ;-). Ich bin so froh, dass wir
uns getroffen haben und Du mich jeden Samstag ne Runde um den See scheuchst! Du bist
echt ein Sonnenschein!
Grazie mille to my favorite Italian Barbara for everything, you fill my life with happiness,
pleasure and joy.
51
Alexandra, dank Dir für unsere netten Küchentischpsychologierunden und dass Du immer
für mich da bist.
Thank you Gautam, for being my friend, I had so much fun with you, even when you not
keep my IKSU plans and what about the garden plans?!! Thanks for helping me with my
scientific problems (MR. Western) and with my English. You are a great guy!
Uta, Dir danke ich besonders für die super Einführung ins Umeå Leben (inklusive unseren
traditionellen samstägigen Stadtgängen, “The swan” und “TopModel”) ohne Dich wäre ich
nicht hier geblieben.
Elke und Ruth, vielen Dank, dass Ihr mir die Umeå Zeit so verschönt habt. Schade, dass wir
so weit weg voneinander wohnen.
Gracias Ana for being you, believing in me. I had so much fun with you! And I am very glad
I unwrapped my package.
Katja und Silvia - meine beste Wochenend-Reisegruppe! Ich danke Euch für unsere super
Trips, die wir gemacht haben und noch machen werden, für das was wir zusammen erlebt
haben. Danke auch, dass Ihr immer für mich da seid, immer ein offenes Ohr habt und für
aufmunternde Worte bei langen Telefonaten aus Spanien/Schweiz.
Vielen Dank auch an die Berliner Gang u.a. Thordis, Saskia, Grobi, Aniko und Ina. Schön,
dass Ihr mich alle mal besucht habt und vor allem, dass Ihr es immer eingerichtet habt mich
zu treffen, wenn ich auf Kurzbesuch in Berlin war.
Meine lieben Frankfurter Mädels will ich natürlich auch danken. Romy, Mareen, Kathleen
und Christine & Co danke, dass Ihr mich immer für mich da seid trotz der großen
Entfernung und Euch immer Zeit nehmt, wenn ich in Ffo. bin.
Thanks to everyone I forgot to mention for a nice and good time in Umeå.
Ganz großen Dank an meine liebe Familie für die Unterstützung, Gedanken, Hilfe und was
ihr sonst noch alles für mich getan habt, ohne Euch hätte ich das nicht geschafft. Danke!!!
Och stor tack till min lilla svenski Micke, du är bäst! Tack till din familj.
52
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