Catherine Mytilineou - US grants

Dopamine neurons from the midbrain of fetal rats and/or newborn dogs will be grown in explant cultures. The growth, development and survival of the dopamine neurons will be monitored by measuring tyrosine hydroxylase activity, dopamine content and 3H-dopamine uptake, as well as evaluating the catecholamine histofluorescence and ultrastructural morphology of the dopamine neurons. We will examine whether the in vitro growth, maturation and survival of the dopamine neurons is influenced by the intracellular levels of neurotransmitter or by the presence in the same culture of brain areas that in vivo provide afferent input or are targets of the dopamine neurons. We will also grow the dopamine neurons in serum free medium and study the effects of hormones, such as glucocorticoids and thyroxine, on their growth and development. Finally we will study the regenerative capacity of the dopamine neurons at different stages of the in vitro development and examine whether regeneration is affected by the presence of hormones in the medium or by increased firing through depolarization of the neuronal membrane. The results of this study will provide information on the mechanisms that control and/or modulate growth and development of dopamine containing neurons. This information could be helpful in our efforts to understand parkinsonism, a disease involving a selective destruction of the midbrain dopaminergic neurons.

Parkinsonism is a common neurological disorder, particularly in the elderly population. Attempts to use brain transplants as a therapy for Parkinson's disease have been both promising and disappointing. There is relatively small improvement and a gradual deterioration of the beneficial effects after surgery, believed to be due to poor survival of the grafted dopaminergic neurons. My laboratory (Mytilineou et al., 1992) and others (Reynolds and Weiss, 1992) have demonstrated that CNS precursor cells can survive in an arrested phase both in vitro and in vivo and can be stimulated to re-enter the mitotic cycle when treated with epidermal growth factor (EGF). Our recent preliminary studies have indicated that neuronal precursors, already determined to become dopaminergic neurons, can be stimulated by growth factors to continue proliferating in vitro. We propose to study this process of neuronal precursor proliferation as an alternative way to prepare DA neurons for transplantation. Specifically, we have shown that basic fibroblast growth factor (bFGF) stimulates the proliferation of stem cells from the ventral mesencephalon of embryonic day 12 rat brain which differentiate into dopaminergic neurons. Highly homogeneous dopaminergic colonies, consisting of a large number of DA neurons, can be formed by this treatment. When the cell culture conditions do not promote adhesion and differentiation of neuronal precursors, bFGF treatment causes continuous proliferation of the precursor cells, which form cellular spheres in suspension. In our studies, colonies of dividing precursors will be generated in suspension and their differentiation will be studied in vitro and in vivo. The in vivo studies will determine the survival and differentiation of these precursors, when transplanted into the DA-depleted striatum of adult rats, by immunocytochemical analysis with antibodies to tyrosine hydroxylase. The biochemical and functional recovery that may result from these transplants will be examined by HPLC analysis of catecholamine levels in the graftcontaining striata and by comparing the rotational behavior in response to apomorphine or amphetamine before and after transplantation. The ability to maintain, in storage, cells appropriate for brain cell replacement therapy will be a significant advance for brain transplantation in parkinsonian patients and a host of other neurological impairments, where such a therapy is presently either attempted or contemplated.

The study of mitochondrial respiratory activity, such as complex I activity, in Parkinson's disease (PD) is an exceptionally active research area that has attracted numerous investigators. Our proposal is novel, however, because it is based on 3 new observations: (l) The deficiency in mitochondrial respiration in PD is expressed by human skin fibroblasts, representing a cell type that undergoes active cell division in culture to provide a continuing supply of cells for experimentation; (2) A deficit in mitochondrial respiration can be induced in normal cells by exposure to L-dopa, an agent widely used in the treatment of idiopathic PD; (3) Ascorbate, an antioxidant, enhances mitochondrial respiratory activity in control fibroblasts and blocks the decline in activity induced by exposure to L-dopa. These observations open a new window into (a) the effect of oxidative stress in mitochondrial function and (b) the significance and regulation of complex I activity. The proposed new studies will include: Comparison of mitochondrial respiratory activity in PD with other neurological disorders and appropriate controls (Aim l); further characterization of the defect in PD (Aim 2); assessment of cellular and mitochondrial antioxidant defenses in control and PD fibroblasts (Aim 3); evaluation of susceptibility of mitochondrial respiration to oxidative stress in PD and control fibroblasts, as well as in mesencephalic cell cultures containing dopaminergic neurons (Aim 4); and evaluation of antioxidant treatment for improvement of mitochondrial respiration in fibroblasts and neuronal cell cultures (Aim 5). Our studies will provide basic information on the possible causes of deficits in mitochondrial respiration in PD and on the effects of antioxidants on mitochondrial function.