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; 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)

BDNF

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

(BDNFneo) LacZR26R Cre

Resulting Mice

Wnt-BDNFKO BDNFneo/lox+

Heterozygous for BDNF

Wildtype BDNF

BDNF-/- BDNF+/- BDNF+/+

Wnt-1:R26R

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 (long-term 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. Replacement of degenerated cells

2. Improve the environment of diseased neural tissue – i.e. release neuroprotective factors Factors already secreted by stem cells Specific gene introduction to stem cells for

secretion

3. 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– Ethical issues– Tumorigenic– Non-autologous source

Adult stem cells – many types, multipotent– Different properties

induced Pluripotent stem cells– Autologous source– Unlimited differentiation– Tumorigenic– Lentivirus vectors for induction – dangerous mutations

Safer method = piggyBac

Hematopoietic stem cell

Bone marrow stem cells

Neural

Neuron

Gl ia

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

SPN L-glutamate

N2hEGRhbFGF

MSC induction to NF-SC

Human Mesenchymal

Stem Cells

dbcAMP IBMX

PDGFHRG1-β1hbFGF

Neurotrophic factor secreting

cells

Passaged 12-18 days

Media replaced 72 hrs later

Figure 1: Confirmation of neurotrophic factor secretion.

In vitro model of Parkinson’s

Serum-free

media (control)

Culture supernatant

(control)Serum-free media

Culture supernatant

(contains NTF)

MSC

Serum-free media

NTF-SC

32-160 μM 6-OHDA

+ 1h

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 6-OHDA.

• 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 Treated

PBS

MSC

NTF-SC

Cellular transplantation inhibited 6-OHDA-induced dopamine depletion

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Control MSC NTF-SC% D

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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)