Download Amanda Henke - USD Biology

BDNF and Parkinson’s Disease
March 26th, 2010
What is Parkinson’s Disease?





Progressive loss of
dopaminergic
neurons in the
substantia nigra
Reduction in SN and
striatal DA
Increase in glial cells
in the SN
Neuromelanin (DA
pigment) loss
Lewy bodies
Diagnosis


Clinical features: Bradykinesia, resting tremors, muscle rigidity, loss of
postural reflexes, flexed posture, and the freezing phenomenon
– Parkinsonism diagnosis with 2 symptoms
Parkinsonisms:
– Primary: Parkinson’s disease (PD) – most common
 asymmetrical onset of motor symptoms
 rest tremor
 Substantial clinical response to levodopa therapy
– Secondary: drug-induced or postencephalitic parkinsonism
– Parkinson-plus syndromes - w/ other neurological features, i.e.
progressive supranuclear palsy and multiple system atrophy
– heredodegenerative disorders – parkinsonism features in a heritable
degenerative disorder (juvenile Huntington or Wilson disease)
Fahn and Sulzer, 2004
Neurotrophin Hypothesis in
Neurodegenerative (ND) disorders

Neurotrophins promote: development, heath,
survival of neurons
–

BDNF: synaptic plasticity, neuronal survival and
differentiation
Studies suggest BDNF disruption in:
–
–
–
–
Huntington’s
Alzheimer’s
Multiple Sclerosis
Parkinson’s
BREIF Overview of Parkinson’s &
BDNF Research…

Postmortem studies of PD patients: reduced levels of BDNF in
the SCN- substantia nigra pars compacta (Mogi
et al., 1999;
BDNF
Parain et al., 1999; Howells et al., 2000; Chauhan et al. 2001)

BDNF promotes survival & differentiation mesencephalic DA
neurons in culture (Hyman et al., 1999; Feng et al., 1999)

BDNF protects from toxic insults (Murer et al., 2001)

BDNF+/- mice have decreased striatal DA and impaired
behavioral responses (Dluzen et al., 2001, 2002)

trkB partial deletion – decreased TH, formation of α-synuclein
deposits (von Bohlen Und Halbach et al., 2005)
Normal BDNF Expression

DA neurons normally co-express BDNF in:
–
–
–

Substantia Nigra
Ventral Tegmental Area
Frontal cortex
DA neuron depletion  Decrease in BDNF
(trophic support)?
Exogenous BDNF Replacement


Goal: increase BDNF to preserve DA neurons and
improve disease symptoms
Problems:
–
–
–
–
–
Large molecular size (~28 kDa)
trkB wide distribution – no targeted effects
Carrier molecules: stem cells, viral vectors, biomaterials
Unknown treatment length for protection, BDNF delivery
rate, BDNF pharmokinetics
BDNF overexpression in animal models  seizures
Experimental therapeutic strategies for
restoring BDNF in ND diseases
Zuccato and
Cattaneo, 2009
Brain-Derived Neurotrophic Factor Is
Required for the Establishment of Proper
Number of Dopaminergic Neurons in the
Substantia Nigra Pars Compacta
Baquet et al., 2005.
Journal of
Neuroscience. 25(26):
6251-6259.
Aim of Study


Investigate the link between reduced BDNF
in the substantia nigra and deterioration of
dopamergic neurons in PD patients.
Create a conditional knock-out, as BDNF-/mice die.
Cre-Lox recombination
Wnt-1
promoter
R26R Cre
(BDNFneo)
LacZ
Resulting Mice
BDNF-/- BDNF+/-
Wnt-1:R26R
Wnt-BDNFKO
BDNFneo/lox+
Heterozygous
for BDNF
BDNF+/+
Wildtype
BDNF
Figure 1: BDNF Expression
Characterization
What is TH?
Kreek, et al. 2002
Figure 2: Expression of Cre in
midbrain BDNF-expressing neurons
Figure 3: Reduced BDNF protein leads to motor
deficits and reduced striatal TH in Wnt-BDNFKO
KO HT WT
KO
HT
WT
Figure 4: Wnt-BDNFKO Mice have reduced TH
expression in the SNC, but not the VTA
Anterior
Posterior
KO HT WT
Figure 5: No change in NeuN, CB, CR
NeuN
CB
CR
Conclusions

Selective BDNF deletion from the midbrain &
hindbrain show:
–
–
–

reduced TH (differentiated DA neurons)
reduction in striatal DA
display early PD phenotype
More evidence for a link between BDNF and
PD?
Protective Effects of Neurotrophic
Factor-Secreting Cells in a 6-OHDA
Rat Model of Parkinson Disease
Sadan et al., 2009.
Stem Cells and
Development.
18(8):1179-90.
Aim of study

Induce MSC to differentiate into neurotrophic
factor secreting astrocytes
–
–

Safe & efficient protocol
Increase NTF secretion
Study effects NTF (BDNF and GDNF) in:
–
–
–
behavior
dopamine levels/neurons in striatum
in vivo tracking of transplanted cells
Definition of a Stem Cell
1. make identical copies of
themselves for long
periods of time (longterm self-renewal)
2. give rise to mature cell
types that have
characteristic
morphologies (shapes)
and specialized
functions
8-cell stage
Why use stem cells for ND therapy?
1.
2.
Replacement of degenerated cells
Improve the environment of diseased neural
tissue – i.e. release neuroprotective factors


3.
Factors already secreted by stem cells
Specific gene introduction to stem cells for
secretion
Stem cells to induce/enhance neurogenesis
to mimic native stem cell populations
Obstacles in stem cell therapy






Immune (graft) rejection
Transplantation procedure
Risk of tumor development
Ethical issues
Matched donor
Fate assessment after therapy
Types of Stem Cells

Embryonic Stem Cells (ESC) – totipotent
–
–
–

Adult stem cells – many types, multipotent
–

Ethical issues
Tumorigenic
Non-autologous source
Different properties
induced Pluripotent stem cells
–
–
–
–
Autologous source
Unlimited differentiation
Tumorigenic
Lentivirus vectors for induction – dangerous mutations

Safer method = piggyBac
Bone marrow stem cells
Hematopoietic stem cell
N
e
u
r
o
n
G
l
i
a
Neural
Opposition to idea of MSC
transdifferentiation to neuronal cells



Observations of extending neurites mistaken
for cell-cell contacts
‘neural’ makers could have different roles in
MSC
Yet, recent reports suggest a subpopulation
of MSC originate from the neural crest
–
likely that at least of subset of the MSCs may
have a neural predisposition.
MSC advantages


Differentiate to DA neurons, astrocytes,
oligodendrocytes
Paracrine effect
–




Secrete soluble trophic factors (BDNF, VEGF, GDNF)
Cytokine secretion to inhibit lymphocyte proliferation
Migratory behavior
Neurogenesis – seen in stroke model and transplant
to dentate gyrus of hippocampus, attributed to NTF
secretion
Genetic manipulations to overexpress genes or
program cells
MSC induction to NF-SC
Human
Mesenchymal
Stem Cells
Passaged 1218 days
SPN
L-glutamate
dbcAMP
IBMX
N2
hEGR
hbFGF
PDGF
HRG1-β1
hbFGF
Media
replaced 72
hrs later
Neurotrophic
factor secreting
cells
Figure 1: Confirmation of neurotrophic
factor secretion.
In vitro model of Parkinson’s
Serum-free
media (control)
MSC
Culture
supernatant
(control)
Serum-free media
NTF-SC
Culture
supernatant
(contains NTF)
Serum-free media
+ 1h
32-160 μM
6-OHDA
6-OHDA



6-hydroxydopamine – selectively neurotoxic
for DA neurons
drug redistributes DA from synaptic vesicles
Oxidized DA = DA-quinone reacts w/ DA
uptake transporter
Figure 2: NTF-SC/MSC protect neuroblastoma
cells against 6-OHDA toxicity
• MSC and NTF-SC
groups were
statistically
significant @ 32,
48, and 72μM 6OHDA.
• No statistical
difference b/t MSC
& NTF-SC
• @ 160μM, NTF-SC
were statistically
different from
others
Figure 3: Behavioral tests after stem
cell transplant in 6-OHDA treated rats
Control
PBS
MSC
NTFSC
Treated
% Dopamine in lesioned compared to control
side
Cellular transplantation inhibited 6OHDA-induced dopamine depletion
100
*
90
80
70
60
50
40
30
20
10
0
Control
MSC
NTF-SC
Conclusions and Future Directions

NTF-SC could
–
–
–



increase production/ secretion of BDNF & GDNF
Attenuate 6-OHDA-induced behavior
Increase striatal dopamine
Autotransplantation of rat-derived MSC and induced
NTF-SC
Transplantation later and at a site further from the
lesion
Treatment for PD
Baquet et al., 2005 S Fig 1
Baquet et al., 2005 S Fig 2
Baquet et al., 2005 S Fig 2
Sudan et al, 2009 S Fig 1
S Fig 2
S Fig 3
Gene’s associated with early onset PD





α-synuclein UCHL1 (ubiquitin carboxy-terminal
hydrolase L1)
Parkin – ubiquitin E3 ligase that prepares proteins
for degradation
DJ1: a parkin associated protein involved with
oxidative stress
PINK1: Phosphatase and tensin homolog–INduced
Kinase; putative serine threonine kinase
possible pathogenic mechanisms?
PD Etiology

10% of cases: genes
–
–
–

α-synuclein
Parkin
DJ-1
90% of cases – unknown
–
–
Age
Environment (toxic exposure, drug use)