Innovativa_projekt_ALF_2010

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					Exploring a new source of autologous cells for transplantation in Parkinson’s
Disease

Project rationale
Novel source of cells for transplantation
Transplanted fetal dopaminergic neurons can survive in the Parkinson’s disease (PD) brain and
support functional recovery in open label trials. Hopefully stem cell-derived neurons can be
grafted and consistently alleviate symptoms in large numbers of patients in the future (Deierborg
et al., 2008). However, currently stem cell-derived dopamine neurons suitable for clinical
grafting trials do not exist. Poor graft survival, teratoma formation (grafts derived from
embryonic stem cells) and graft rejection (allografts) are all hurdles that have to be overcome.
In this proposal we address a novel source of cells that can be transplanted in attempts to restore
dopaminergic neurotransmission in PD. We will explore the capacity of adult skin-derived
melanocytes to generate DOPA and mitigate behavioral deficits when grafted into the striatum.

Why attempt DOPA replacement?
The rationale for developing a cell-based DOPA-replacement strategy is supported by gene
therapy experiments. In the striatum of dopamine–denervated rats, DOPA levels have been
successfully increased by intrastriatal injections of adenovirus vector encoding human tyrosine
hydroxylase (TH) (Corti et al., 1999). Over the past decade, several similar viral-vector mediated
DOPA delivery strategies have provided robust behavioral recovery in PD animal models
(Bjorklund and Kirik, 2009). This has led to clinical trials with viral-vector mediated delivery of
TH in PD. The gene therapy trials strongly suggest that local production of DOPA in the striatum
is a viable therapeutic strategy in PD. While the gene therapy approach to delivering DOPA
production-capacity to the striatum is promising, it also has safety concerns, e.g. regarding
inflammation. Moreover, the recent failure of the Ceregene phase II gene therapy trial with
Neurturin, suggests that it is not trivial to transduce sufficient numbers of cells in the human
striatum. Therefore alternate methods to deliver DOPA to the striatum would be valuable, and
autologous cell transplants might be one such approach.

Can transplanted melanocytes be a suitable source of DOPA?
In the nervous system, tyrosine hydroxylase catalyzes the conversion of tyrosine to DOPA. Skin
melanocytes are derived from the neural crest. They have the capacity of autocrine de novo
synthesis of L-DOPA catalyzed by the enzymes tyrosinase and tyrosine hydroxylase
(Schallreuter et al., 2003). They also exhibit some similarities to dopamine-producing neurons,
i.e. they can synthesize dopamine and display a dendritic morphology (Fig. 1).

                                             Furthermore, they can communicate with the
                                             surrounding tissue through transfer of vesicles
                                             called melanosomes, at specialized connections
                                             (Scott et al., 2002). Previous work has shown that
                                             DOPA-producing cells transplanted into the
                                             striatum in hemiparkinsonian rats can increase
                                             striatal dopamine levels and mitigate behavioral
                                             deficits (Horellou et al., 1990). In short, if grafted
                                             into the brain, melanocytes may have the capacity
to produce DOPA that can reverse dopamine deficiency in PD. Indeed, Fillmore et al recently
published a patent claiming that grafted melanocytes reverse motor asymmetry when grafted to
the striatum of hemiparkinsonian rats (Pat No.: US 2003/0022369).



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Do melanocytes exhibit specific advantages as a source of transplanted cells?
Melanocytes can easily be harvested, expanded and grafted back to the same patient as they were
derived from (autograft). There is no risk of tumor growth. For over 15 years, we have cultured
human adult melanocytes for transplantation treatments in several hundred patients without side-
effects (Olsson and Juhlin, 2002). Our technology is based on a safe approach to culture adult
melanocytes without exposure to foreign proteins, infectious or co-carcinogenic agents. We have
already successfully cultured melanocytes derived from PD patients (unpublished).

Hypothesis
We hypothesize that autografted melanocytes can act as a source of DOPA in the striatum of PD
patients and thereby relieve disease symptoms. We will address this hypothesis by examining the
capacity of human melanocytes to survive xenografting to the striatum and support behavioral
recovery in an immunosuppressed rat model of PD.

Experimental design
We will test our hypothesis by transplanting human melanocytes into the striatum of
hemiparkinsonian rats. We will harvest melanocytes from adult human (healthy volunteers) skin
biopsies and multiply and purify the cells using an established special culturing system (Olsson
and Juhlin, 2002). We will first investigate the behavioral effects of the melanocyte grafts and
examine if the treatment can mitigate the motor asymmetry following unilateral 6-OHDA
lesions. We will use well-established behavioral tests: i.e. drug-induced rotation and the cylinder
test. After completion of the behavioral testing, eight weeks after grafting, we will perfuse the
animals for immunohistochemical examination of the graft and the host brain response.

Grafting of melanocytes into the brain of hemiparkinsonsian rats
We will lesion the mesostriatal pathway by 6-OHDA in adult Sprague Dawley rats to generate
hemiparkinsonian rats (Lane et al., 2009). One week after the 6-OHDA lesion, we will perform
amphetmine-induced rotational behavior (Ungerstedt and Arbuthnott, 1970) to select animals
with a profound dopamine-denervation in the striatum (>6 turns/min over 90 min observation
time). One week later, we will graft the selected rats with either 100 000 or 500 000 melanocytes
into the centre of the striatum (Bregma: R:1.2, L:2.8, V:4.5). We will immunosuppress all rats
with Cyclosporine A (15 mg/kg i.p. Sandimmune, Novartis) from 1 day prior to grafting, daily
for 2 weeks after the transplantation, and then at 10 mg/kg per day. Seven weeks after grafting,
we will pulse the animals with the thymidine analogue BrdU (50 mg/kg, i.p.) to monitor if the
grafted cells are proliferating.

Behavioral analysis
To study if striatal grafts of human melanocytes can compensate for the dopamine loss, we will
examine the rats in motor asymmetry tests. The behavioral tests are identical to the behavioral
tests that have been shown to be relevant and sensitive when the concentration of L-DOPA is
elevated in the striatum by intrastriatal TH gene therapy (Kirik et al., 2002). This comparison
will allow us to validate our results by an established experimental therapy known to
significantly raise the L-DOPA concentration in the striatum. We will use the following tests:
    1) Amphetamine-induced rotation, before grafting (selection of complete lesion rats) and 8
        weeks after grafting.
    2) Apomorphine-induced rotation at 3, 5 and 7 weeks after grafting.
    3) Cylinder test (Tillerson et al., 2001) at 8 weeks after grafting to study the spontaneous
        forelimb use.



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The apomorphine-induced rotation test will be particularly important in our experiment. It
addresses whether dopamine receptor supersensitivity is normalized due to increased
dopaminergic neurotransmission as a result of graft-induced restoration of DOPA levels.

Immunohistochemistry of the grafted melanocytes and the host brain response
We will study the graft by immunohistochemistry and measure graft survival, tyrosine
immunoreactivity of the grafted cells (DOPA production), immunological host response in terms
of T-cell (CD-4/-8) and microglia/macrophage (CD11b, Iba-1) occurrence. We will characterize
the grafted melanocytes phenotypically using antibodies for melanocyte specific proteins such as
tyrosinase, tyrosinase-related-protein 1 (TYRP1), dopacrome tautomerase (DCT), Pmel 17 and
gp100 and markers for melanocyte-specifix cell-surface proteins. We will also assess the
proliferative status using endogenous markers (Ki-67, PCNA) and BrdU immunohistochemistry.

Justification of the applicant expertise to carry out proposed experiments
The principal investigator (Dr Brundin) has over 25 years experience of neural grafting and
restorative treatments for PD. Dr Olsson is an expert in melanocyte biology for the last 17 years
and has a novel, but established, technique to harvest human melanocytes and expand them in
vitro to pure and large quantities suitable for transplantation. Dr Deierborg is an expert in
neurorestorative research and has more than 10 years of in vivo model experience. Taken
together, we believe our team is eminently suited to perform the proposed research project.

References
Bjorklund, T., and Kirik, D. (2009). Scientific rationale for the development of gene therapy strategies for
         Parkinson's disease. Biochim Biophys Acta.
Corti, O., Sanchez-Capelo, A., Colin, P., Hanoun, N., Hamon, M., and Mallet, J. (1999). Long-term doxycycline-
         controlled expression of human tyrosine hydroxylase after direct adenovirus-mediated gene transfer to a rat
         model of Parkinson's disease. Proc Natl Acad Sci U S A 96(21), 12120-5.
Deierborg, T., Soulet, D., Roybon, L., Hall, V., and Brundin, P. (2008). Emerging restorative treatments for
         Parkinson's disease. Prog Neurobiol 85(4), 407-32.
Horellou, P., Brundin, P., Kalen, P., Mallet, J., and Bjorklund, A. (1990). In vivo release of dopa and dopamine from
         genetically engineered cells grafted to the denervated rat striatum. Neuron 5(4), 393-402.
Kirik, D., Georgievska, B., Burger, C., Winkler, C., Muzyczka, N., Mandel, R. J., and Bjorklund, A. (2002).
         Reversal of motor impairments in parkinsonian rats by continuous intrastriatal delivery of L-dopa using
         rAAV-mediated gene transfer. Proc Natl Acad Sci U S A 99(7), 4708-13.
Lane, E. L., Vercammen, L., Cenci, M. A., and Brundin, P. (2009). Priming for L-DOPA-induced abnormal
         involuntary movements increases the severity of amphetamine-induced dyskinesia in grafted rats. Exp
         Neurol.
Olsson, M. J., and Juhlin, L. (2002). Long-term follow-up of leucoderma patients treated with transplants of
         autologous cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension. Br J Dermatol
         147(5), 893-904.
Schallreuter, K. U., Kothari, S., Hasse, S., Kauser, S., Lindsey, N. J., Gibbons, N. C., Hibberts, N., and Wood, J. M.
         (2003). In situ and in vitro evidence for DCoH/HNF-1 alpha transcription of tyrosinase in human skin
         melanocytes. Biochem Biophys Res Commun 301(2), 610-6.
Scott, G., Leopardi, S., Printup, S., and Madden, B. C. (2002). Filopodia are conduits for melanosome transfer to
         keratinocytes. J Cell Sci 115(Pt 7), 1441-51.
Tillerson, J. L., Cohen, A. D., Philhower, J., Miller, G. W., Zigmond, M. J., and Schallert, T. (2001). Forced limb-
         use effects on the behavioral and neurochemical effects of 6-hydroxydopamine. J Neurosci 21(12), 4427-
         35.
Ungerstedt, U., and Arbuthnott, G. W. (1970). Quantitative recording of rotational behavior in rats after 6-hydroxy-
         dopamine lesions of the nigrostriatal dopamine system. Brain Res 24(3), 485-93.




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