Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1998-10-09
2001-09-04
Priebe, Scott D. (Department: 1632)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S320100, C435S325000, C435S368000, C424S093210, C514S04400A, C536S023100, C536S023500
Reexamination Certificate
active
06284539
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention discloses a method for generating dopaminergic cells from a culture of mammalian CNS stem cells, methods for treating a patient with a neurological disorder, such as Parkinson's Disease, methods of screening for compounds which generate or increase the number of dopaminergic cells in a culture of mammalian CNS stem cells and methods for discovering genes involved in generating dopaminergic cells.
2. Description of the Related Art
A. Development of Dopaminergic Neurons
Generating the enormous cellular complexity of the mammalian central nervous system requires an intricate developmental program whose molecular mechanisms are only now beginning to be understood. This developmental program produces countless cell populations that are distinct in their morphology, function and/or biochemical characteristics. One such population has its cell bodies in the region of the ventral midbrain (VM) known as the substantia nigra pars compacta and projects to the striatum. These neurons are distinguished biochemically by the fact that they secrete dopamine as a neurotransmitter and thus express at high levels the enzyme tyrosine hydroxylase (TH) which catalyzes the rate-limiting step in the biosynthesis of dopamine
43
.
Dopaminergic neurons are of enormous clinical significance because it is these cells that progressively lose function in patients with Parkinson's Disease (PD)
57
. The progressive loss of nigral dopaminergic function interferes with the normal working of the neuronal circuitry subserving motor control so that patients with PD show characteristic motor disturbances that typically consist of akinesia, rigidity and rest tremor but may also include pain, impaired olfaction, alterations of personality and depression
49
. Due to the potential clinical significance of such studies, much experimentation has been directed toward understanding the molecular mechanisms by which dopaminergic neurons develop.
One of the hallmarks of early embryonic development is the formation of the neural plate, a region of specialized ectoderm lying along the dorsal midline of the embryo. This neural plate subsequently develops a central depression and ultimately closes on itself to form the neural tube. The cells lining the interior of the tube then proliferate, differentiate and migrate to appropriate locations in the central nervous system. In the case of the cells destined to form the substantia nigra, mitotic precursor cells are generated near the midbrain/hindbrain junction. These cells then migrate radially to their ultimate position in the VM, where they differentiate into the various cells types found in the substantia nigra, the ventral tegmentum and the retrorubral nuclei
43
.
The differentiation of the VM appears to be regulated in large part by inductive signals from the floor plate, a band of tissue that comprises the ventral midline of the developing mesencephalon. Removal of the floor plate results in the loss of dopaminergic neurons, among other cell types
21,47
. Conversely, replacement of a previously extirpated floor plate tube with an exogenous floor plate, though not with other cellular structures, restores the lost cellular phenotypes
68
. Similarly, addition of a supernumerary floor plate induces the ectopic formation of dopaminergic neurons
68
.
Recent work has identified several genes that appear to play central roles in the differentiation of dopaminergic neurons in the VM. Several of these are described below while another, Nurr1, forms the subject of this application and will be discussed in a later section. Each of these genes is expressed in an appropriate temporal and spatial pattern to affect the differentiation of CNS stem cells into dopaminergic neurons in vivo. In addition, when each gene has been subjected to targeted disruption in mice, the resultant phenotypes have all shown distortions of normal VM patterning including aberrant differentiation of dopaminergic neurons.
One molecular signal responsible for the ability of the floor plate to influence the formation of dopaminergic neurons appears to be the expression of the Shh gene by floor plate cells. Adding an Shh gene product to embryonic VM explants increases the number of dopaminergic cells in the explants
20,63
. Another soluble protein shown to have some activity in promoting a dopaminergic fate choice is fibroblast growth factor 8 (FGF-8). Dopaminergic neurons were induced to form in ectopic locations when embryonic midbrain explants were exposed to latex beads coated with FGF-8. This effect, however, was blocked by antibodies to Sonic Hedgehog, indicating that in the absence of Shh functions
69
, FGF-8 alone could not direct precursor cells to adopt a dopaminergic fate.
As shown by the experiments described above, the Shh signaling cascade, perhaps augmented by FGF-8, appears to play a key role in inducing the formation of dopaminergic neurons. However, distinct from prior methods using these two genes, we have developed other methods to increase the number of dopaminergic cells present in CNS stem cell cultures by activating an endogenous transcriptional regulator, Nurr1, rather than by utilizing soluble proteins like Sonic Hedgehog or FGF-8.
B. Other Soluble Factors can Promote the Dopaminergic Phenotype
Interleukin-1 has been reported to increase the number of cells expressing tyrosine hydroxylase (TH) when applied to a culture of VM tissue, an effect that was potentiated by interleukin-11, leukemia inhibitory factor (LIF), and glial cell line-derived neurotrophic factor (GDNF) as well as by mesencephalic membrane fragments and striatal culture-conditioned media
31
. Similarly, co-culture of VM precursor cells with VM neurons is also reported to increase the number of dopaminergic cells
48
. In cultures of the IMR-32 neuroblastoma cells line, exposure to basic fibroblast growth factor (bFGF) caused these tumor-derived cells to express TH, an effect that was potentiated by ciliary neurotropic factor (CNTF)
50
. bFGF has also been claimed to promote an increase in TH-positive neurons from mixed cultures of VM precursors and neurons
34
, as has ascorbic acid
24
. Similarly, while platelet-derived growth factor was found to increase the number of neurons derived from VM CNS stem cell cultures, it did not promote an increase in dopaminergic neurons
23
.
Other factors have been shown to enhance the survival of dopaminergic neurons. For example, brain derived neurotrophic factor (BDNF), neurotrophin 4/5, glial derived neurotrophic factor (GDNF) and neurturin have been shown to promote the survival of dopaminergic neurons from rat or human midbrain. Both epidermal growth factor (EGF) and bFGF have been shown to enhance the survival of dopaminergic neurons although this appears to be due to the proliferative effect that these proteins have on glia rather than a direct action on neurons. Cytokines such as transforming growth factor-&agr; (TGF-&agr;), platelet derived growth factor, interleukin-1b, interleukin-6 and insulin-like growth factor-I have also been shown to promote the survival of dopaminergic neurons while insulin like growth factor II has been reported to increase neurite outgrowth from these cells. Non-proteinaceous factors such as GM1 ganglioside and cyclic AMP also increase dopaminergic neuron survival (reviewed in references 43 and 61).
In addition to the single factors described above, several complex mixtures or culture conditions have been shown to increase the survival of postmitotic dopaminergic neurons. These conditions include co-culturing VM neurons with striatal astrocytes or striatal cells or exposing VM neuronal cultures to striatal extract or striatal membranes. Similarly, conditioned media from cortical astrocytes, the B49 glial cell line, the R33 neural retina glial cell line, the mesencephalic glial cell line Mes42 and the JS1 Schwannoma cell line have all been reported to increase the survival of dopaminergic neurons as has an uncharacterized 14 kDa protein upregulated by retinoic acid (reviewed
Bowen David C.
Johe Karl K.
Bell, Boyd & Lloyd LL
NeuralStem Biopharmaceuticals, Ltd.
Paras, Jr. Peter
Priebe Scott D.
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