Laboratory of Molecular Neurosurgery

Director: Professor Dr. med. Guido Nikkhah, PhD

Group Leader: Dr. Máté Döouml;ssy, PhD

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research

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Our research investigates the mechanisms of functional and behavioural restoration in neurodegenerative diseases such as Parkinson's (PD) or Huntington's diseases (HD). We are currently working on stem cell- or progenitor cell-based therapies both in the animal model and in human trials. Genetically modifying cells, applying new cell culture protocols and transplantation techniques as well as assessment of cognitive and motor behaviour are the main features of our research work.

Methodology

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Funding

We would like to acknowledge the following agencies and institutions for their generous support:

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Scientific background information

1. Parkinson's disease
2. Huntington's disease

1. Parkinson's disease
The symptomatology of Parkinson's disease (PD) comprises the well known motor symptoms (e.g. akinesia and rigidity) but as we have learnt, they are not due to simple motor dysfunction but to deficits in higher order programming of behaviour which is so far not fully understood. PD is linked to a hypofunctional dopamine (DA) system, mainly located in the nigrostriatal pathway and the development of symptomatology of PD-patients is generally considered to be due to a certain degree of DA loss. Due to strong side effects of the standard symptomatic drug therapy (L-DOPA), new approaches of treatment of PD are needed. Thus, transplantation of embryonic DAergic progenitor cells in PD has become a hallmark in the field of brain repair, based on its progress both in basic science research as well as in clinical trials (Bjoerklund et al., 2003). The underlying concept is the replacement of degenerated cells by the implantation or activation of neural precursor cells to repopulate damaged brain areas and, thereby, to replace lost neuronal and/or glial cells and to restore brain function. Studies in rodent and non-human primate models of Parkinson's disease have demonstrated that intrastriatal grafts of fetal mesencephalic tissue, rich in dopamine neurons, reinnervate the striatum and ameliorate motor symptoms (Bjoerklund et al., 2000; Nikkhah et al., 2001). In spite of very promising preliminary results these approaches rise a number of unresolved problems. They range from difficulties to obtain appropriate amounts of fetal tissue (one transplantation requires 6-8 fetuses) to the ethical controversies (Brundin et al., 2001; Dunnett and Bjoerklund , 2001). As an alternative, neural precursor cells can be isolated from the embryonic and adult CNS (Lie et al., 2004). They comprise at least two different classes of distinct cell populations, i.e. stem and progenitor cells (Alvarez-Buylla and Garcia-Verdugo, 2002; Gage, 2002; Rietze et al., 2001) and can be enriched in cell culture. For cell replacement therapies such a stem cell pool would provide for sufficient time for cell characterization and preoperative cell banking. Additionally, it has been demonstrated that human neural stem cells are capable of self-renewal and differentiation into specific neuronal and glial cell progeny that can repopulate and functionally integrate into damaged host brain areas (Fallon et al., 2000; Nunes et al., 2003; Palmer et al., 2001). Most recently, an astrocyte ribbon was identified in the adult human brain that could form clonal neurospheres in cell culture given rise to TuJ1-positive neurons as well as astrocytes and oligodendrocytes (Sanai et al., 2004).
There are at least four types of stem cells that can lead to neuronal progeny: neural stem cells, non-neural adult or fetal stem cells (bone marrow or skin cells), embryonic stem cells, and nuclear transfer or parthenogenetic stem cells (Bjoerklund et al., 2003). However, ethical constraints and immunological and safety concerns currently limit the use of the two latter options, and, in addition, transdifferentiated cells derived from non-neuronal tissue may express tyrosine hydroxylase (Jiang et al., 2002), but their restorative capacity to reverse dopamine-deficient syndromes in animal models of PD has not been demonstrated yet. In contrast, neural stem cells derived from the developing or adult brain can be propagated and differentiated into functional DA neurons, as shown for mouse and rat brain tissue (Studer et al., 1998; Daadi and Weiss, 2001).
Interestingly, cell culture conditions like low oxygen and higher concentrations of ascorbic acid can significantly increase the proliferation and DA differentiation of rat neural precursors cells in vitro (Studer et al., 2000; Yan et al., 2001). Whether and under which conditions human neural stem cells can be directed in a similar manner, and turned into a self renewing source of functional DA neurons, provides for a considerable scientific challenge, both for the better understanding of stem cell biology as well as the promotion of stem-cell based therapy for PD.

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2. Huntington's disease
Huntington's disease ("Chorea Huntington") begins at any age but, preferentially around 30-40 years, i.e. it affects young adults, in full professional activity who often have young children to raise. Exitus due to disease is inevitable in 10 to 20 years, the patients die - often by cachexia - in postural rigidity and severe dementia. The gene responsible for the disease is located on the short arm of chromosome 4 and the molecular defect consist in a multiplication of a CAG- sequence repeat in a gene (IT15) coding for a protein called "huntingtin". Its role is currently still unknown, but a connection with apoptosis is suspected (Brouillet and Al, Medicine/ 2000 Sciences).
HD is relatively rare. The clinically identified patients' prevalence approximates 10 for 100.000 which represent nevertheless almost 6000 patients in France. The social consequences of the disease are however definitely higher than one might think. Because, for each patient, one has to count additionally an average of 5 people with a risk of 50% and of 11 people with a risk 25% according to epidemiological evaluations. Furthermore, if the drama lived by the couple and the families is taken into account, the tragedy created by HD concerns, in our country, more than 100,000 people who require a psychological, or even psychiatric and social support.
HD is caused by a loss of neurons in the striatum which was identified by Meynert more than a century ago. The caudate nucleus and the putamen undergo a severe atrophy up to 60% of there normal forms. The loss involves especially the type II Golgi-neurons ("medium spiny") which form the main part of the striatal efferences and represent 95% of striatal neurons. Vonsattel and colleagues could therefore define "grades" of striatal atrophy which correspond clearly to the clinical stages of the disease. Nowadays, these grades based on the anatomo-clinical correlation form the basis for the classification in HD. Studies of functional imaging carried out in the last few years by recording the cerebral blood flow and by analysis of cerebral activity in PET confirm a primary and early functional loss of the striatum in HD, well before other cerebral regions are affected. One can see indeed a bilateral hypometabolism of the caudate nucleus whatever imaging used, PET or SPECT technique. This hypometabolism is pronounced since, according to the series and the stage of the patients, it goes from 37 to 91% and seems to progress by 7% each year (Kremer et al., Neurology 1999). This topographical characteristic permits to hope for clinical effectiveness of a substitutive treatment restricted to the striatum.
The role of the loss of striatal neurons for the clinical symptomatology during the course of the disease is the subject of this study. Studies investigating imaging and neuropsychological analysis show a positive correlation between the striatal dysfunction and cognitive deficits (for a review see Brandt, 1991). A comparison of neuropsychological deficits observed among patients affected by HD with those having dementia associated with Parkinson's disease on one side and Alzheimer's disease on the other side favors also a close connection between the subcortical lesions (striatal) and dementia in HD. In contrary to Alzheimer's disease, which is primarily a cortical atrophy, HD is not defined by the triad aphasia-apraxia-agnosia but by a much more diffuse disorder characterized in particular by mnesic and executive deficits. In addition, deficits in attention, difficulties in using acquired information and a slowness of mental processing are typical. The progressive destruction of the caudate nucleus and of the putamen seems to disconnect the prefrontal cortex from the striatum, stopping at the same time the exchange of information between these two structures and therefore leads to a frontal syndrome without a primary affection of the prefrontal cortex itself.
In summary, all the neuropathological data, the imaging and the neuropsychological findings argue a primary and probably long lasting isolated defect of the striatum in patients with HD. It appears therefore valuable to replace, at this stage, the affected neurons by healthy fetal neurons.

2.1 Fetal neuronal graft - a therapeutic hope based on many experimental studies
Until now a valid treatment for Huntington's disease is still not available. Nevertheless, numerous experimental studies in mouse, rat and non-human primates have shown that it is possible to reverse clinical deficits caused by neurodegenerative lesions by the implantation of fetal striatal neurons. These studies were reported extensively and discussed in several recent reviews (Freeman et al., Cell transplant 1995; Shannon and Kordower, Cell transplant 1996; Dunnett, Neuropathol Appl Neurobiol 1999; Lindvall, Mov Disorders 1999, Peschanski et al. 2004). Their conclusions confirm those presented in a review published by Peschanski et al. (1995) which had served as the basis of the clinical pilot-study carried out in Créteil. We will thus not point out these data here.
Two important original articles published in 1998, however, supplemented these studies by showing what can be obtained in the best experimental models of the disease available today in non-human primates. The group of Steve Dunnett (Kendall and Al, Nature Medicine 1998) used an excitotoxic lesion model in the marmoset monkey to study in terms of bradykinesia and dyskinesia the effects of a striatal lesion and those of a restoration by allograft of homologous fetal neurons. By the anatomical reconstruction (shown experimentally) of the cortico-pallidal circuits which relay in the striatum, the neuronal graft allowed complete recovery of the signs induced by the lesion. The group of Philippe Hantraye (Palfi et Al, Nature Medicine 1998) used a model with bilateral striatal lesion in the macaque induced by a systemic injection of 3-nitropropionic acid, an irreversible inhibitor of the mitochondrial succinat dehydrogenase. They studied primarily the cognitive behavior of these animals and showed that a frontal syndrome (loss of strategic adaptation shown in the Object Retrieval Detour Task) caused by the bilateral lesion was completely reversed within months following the implantation of the fetal neurons.
In addition, Dunnett et al. have shown beneficial effects of allografts of homologous fetal neurons in transgenic mice that express the mutated huntingtin gene; a murin model which is considered as the first genotypic model for the human disease (Dunnett et al., Exp Neurol 1998).
In summary, the experimental work demonstrates a considerable therapeutic capacity of grafts with homologous fetal neurons in animal models of the HD.

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