WO2011038366A1

WO2011038366A1 - Dopaminergic selective factors

Info

Publication number: WO2011038366A1
Application number: PCT/US2010/050438
Authority: WO
Inventors: John W. Commissiong
Original assignee: Amarantus Therapeutics, Inc.
Priority date: 2009-09-25
Filing date: 2010-09-27
Publication date: 2011-03-31

Abstract

Disclosed herein are compositions and methods comprising dopaminergic selective factors and their use. One method provided herein allows for the selection of a subtype of dopaminergic neuronal cell. Other methods provided herein allow for the determination and/or selection of potential therapeutic agents which can increase the growth and/or survival of dopaminergic neuronal cells, thus providing a mechanism for discovery of new therapeutics for diseases affecting neuronal function.

Description

DOPAMINERGIC SELECTIVE FACTORS

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/246,009; filed September 25, 2009which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

[0001] In the development of neuroprotective drugs to treat Parkinson's disease (PD), there is an assumption that the dopaminergic (DA) neuronal population in the substantia nigra (SNc) is

homogeneous. However, in terms of morphology, response to dopamine agonists and antagonists (Bannon et al., (1983) Pharmacol. Rev. 23:53-69, 1983), level of expression of tyrosine hydroxylase (TH) (Weiss- Wunder et al, (1991) J Comp Neurol 303 : 478-488), expression of the calcium binding protein calbinin-28 (Neuhoff et al., (2002) JNeurosci 22: 1290-1302), the pattern of firing of action potentials (Bunney et al., (1991) Synapse 9: 79-94), and especially their response to neuroprotective agents, nigral DA neurons are highly heterogeneous. Anecdotal evidence from recent clinical trials of GDNF for PD, and IGF-1 for Lou Gehrig's disease (ALS) suggests that a sub-population of patients in each clinical trial may have benefitted from the treatments. No serious attempt has yet been made to understand if the response of PD patients to treatment with neurotrophic factors could depend on the composition of the patient's residual DA neurons. We have constructed dose-response curves of percentage survival of DA neurons versus concentration of astrocyte conditioned medium (ACM) prepared from astrocyte cell lines (Panchision et al. (1998) JMol Neurosci 1 1 : 209-221). The elegant work of Bushong et al. (Bushong et al, (2002) JNeurosci 22: 183-192) and others

(Benarroch, (2005) Mayo Clin Proc 80: 1326-1338), based on 3-D confocal imaging of astrocytes has established that astrocytes exist in non-overlapping, microanatomical domains (Bushong et al., (2002) J Neurosci 22: 183-192). Their neuroprotective action on adjacent DA neurons is therefore local (Petrova et al, (2004) Prog Brain Res 146: 168-183).

[0002] Thus, using patterns of neuroprotection on DA neurons, we describe herein methods and compositions which can be used to diagnose, prognose, treat, and/or prevent diseases which impair neurological function, such as Parkinson's Disease (PD).

SUMMARY OF THE INVENTION

[0003] Disclosed herein is a method for selecting a population of a subtype of dopaminergic neuronal (DAergic) cell, comprising culturing a population DAergic cells in astrocyte- derived conditioned media for a sufficient amount of time to allow neuronal cell protection, and selecting at least one DAergic cell subtype that proliferates in said media.

[0004] Also disclosed herein is a method for identifying a candidate agent useful in supporting the growth and/or survival of DAergic neurons, comprising the steps of 1) contacting a candidate pharmacological agent with a population of DAergic cells in astrocyte- derived conditioned media, wherein in the absence of the candidate pharmacological agent, a baseline amount of growth or survival of the DAergic cell subtypes is observed; and 2) determining a test amount of growth or survival of the DAergic cell subtypes in the presence of the pharmacological agent as a measure of the effect of the pharmacological agent on the growth or survival of the DAergic cell subtypes, wherein a test amount of growth or survival of the DAergic cell subtypes that is greater than the baseline amount indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent that is useful in supporting the growth and/or survival of DAergic neurons.

[0005] Further disclosed herein is a method for identifying a candidate agent useful in supporting the growth and/or survival of a subtype cell of a DAergic neuron, comprising: 1) isolating at least one subtype of a DAergic cell by culturing a population of neuronal cells in astrocyte- derived conditioned media; 2) contacting a candidate pharmacological agent with the subtype of a DAergic cell in (a), wherein in the absence of the candidate pharmacological agent, results in a baseline amount of growth or survival of the subtype; 3) determining a test amount of growth or survival of the subtype in the presence of the pharmacological agent as a measure of the effect of the pharmacological agent on the growth or survival of the subtype, wherein a test amount of growth or survival of the subtype that is greater than the baseline amount indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent that is useful in supporting the growth and/or survival of the subtype. In any of the methods described herein, a subtype of cell can be a unipoloar cell, a multipolar cell, a pyramidal cell a goblet cell or a claw cell. Astrocyte-derived conditioned media can be from the ventral mesencephalon, cerebral cortex or hippocampus cell lines.

[0006] Further described herein is a kit comprising an astrocyte-derived conditioned media; at least one cell selected from a ventral mesencephalon, cerebral cortex, hippocampus and striatum; and instructions for culturing a subtype of a DAergic cell.

INCORPORATION BY REFERENCE

[0007] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0009] Figure 1 shows selective actions of four ACM on five DA morphological sub-types in a nigral cell culture. DETAILED DESCRIPTION OF THE INVENTION

[0010] Current approaches to determine the effectiveness of treatment for diseases which affect neurological function, for example Parkinson's Disease (PD), utilize traditional randomized, placebo control versus drug treated groups (Lang et al. (2006) Ann Neurol 59: 459-466), however, our methodology would allow all patients in a test population to be treated with the drug and utilize the methodology herein to determine the effectiveness of the treatment. Thus, disclosed herein are compositions and methods for detecting neuroprotective molecules which are DA-selective, as well as treating diseases which affect neurological function. These neuroprotective molecules can be effective on multiple morphological subtypes of DA neurons. Additionally, patterns of specificity for astrocyte conditioned medium (ACM) can be utilized to diagnose, prognose, and/or used to develop treatment for patients.

[0011] In experiments described herein, we select astrocyte cell lines that secrete DA-selective neuroprotective molecules and test their protective action on each of the six morphological sub-types of DA neurons. These data sets can reveal a pattern of specificity of each ACM, for each DA sub-type, if such a pattern exists. These data could confirm that our present approach to treatment is correct. They could also alert us to the need to target different sub-populations of PD patients with different neurotrophic factors if these molecules are to be successful in the clinic as drugs (Lang et al. (2006) Ann Neurol 59: 459-466: Felice et al. (2008) Neurology 47: S93-S95). Furthermore, upon

characterization of the level of protection provided to mixed and/or substantially pure neuronal cell subtype populations, the ACM can be used to determine the effects of test compounds on the growth and/or protection of dopaminergic neuronal cells (DAergic cells).

[0012] Sub-types of DA neurons in the SNc respond differently to DA-selective neurotrophic factors in ACM. We hypothesize that the response of PD patients to DA-selective neuroprotective factors can depend on the composition of each patient's DA neuronal population. The methods described herein can utilize any sub-type of dopaminergic neuronal cell, including, but not limited to unipolar cells, bipolar cells, multipolar cells, pyramidal cells, goblet cells and claw cells. This hypothesis is tested in rat, nigral neuronal cultures using ten ACM that contain DA-selective neuroprotective molecules like mesencephalic astrocyte-derived neurotrophic factor (MANF). The neuroprotective effect of each ACM, on each of the six sub-types of DA neurons is analyzed. The data sets indicate whether the current approach to treating PD patients with neurotrophic factors in clinical trials is optimal or not.

[0013] The growth, survival, and differentiation of neurons in the peripheral and central nervous systems (PNS and CNS, respectively) are dependent, in part, on target- derived, paracrine, and autocrine neurotrophic factors. Conversely, the lack of neurotrophic factors is thought to play a role in the etiology of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease). In neuronal cell cultures, neurotrophic support is provided by co- culturing with astrocytes or by providing conditioned medium (CM) prepared from astrocytes. Astrocytes of ventral mesencephalic origin exert much greater efficacy in promoting the survival of ventral, mesencephalic dopaminergic neurons, compared with astrocytes from other regions of the CNS, such as the neostriatum and cerebral cortex. In chronic, mesencephalic cultures of 21 days in vitro (DIV) or longer, the percentage of dopaminergic neurons increases from 20% to 60%, coincident with proliferation of a monolayer of astrocytes. In contrast, in conditions in which the proliferation of astrocytes was inhibited, dopaminergic, but not GABAergic neurons, were almost eliminated from the cultures by 5 DIV. These results demonstrate the importance of homotypically- derived astrocytes for the survival and development of adjacent dopaminergic neurons, and suggest that mesencephalic astrocytes are a likely source of a physiological, paracrine neurotrophic factor for mesencephalic dopaminergic neurons.

[0014] The repeated demonstration that astrocytes secrete molecules that promote neuronal survival has made astrocytes a focus in the search for therapeutics to treat neurodegenerative diseases. Many laboratories have attempted to isolate astrocyte-derived neurotrophic factors, but have been hindered by a major technical problem: serum is an essential component of the medium for the optimal growth of primary astrocytes in culture, yet the presence of serum interferes with the subsequent purification of factors secreted into the conditioned medium. Astrocyte-derived conditioned medium (ACM) production and characterization has been described (see, e.g., US Patent Application 12/535,029). Cells utilized herein (either isolated populations or those used to produce ACM) can be derived from any relevant neuronal tissue (e.g., the ventral mesencephalon, cerebral cortex, hippocampus, striatum, etc.).

[0015] One of the embodiments disclosed herein is a method of selecting a population of a subtype of a dopaminergic neuronal (DAergic) cell. For instance, the methods described herein can be utilized to select for unipolar cells, bipolar cells, multipolar cells, goblet cells, pyramidal cells or claw cells.

Particular experiments describing the methodology are provided below. However, the experiments are particular examples. Generally, the methods described herein allow for the isolation of a desired subtype of DAergic cell by culturing a population of cells comprising the subtype in the presence astrocyte- derived conditioned medium (ACM). ACM comprises one or more components which facilitate the growth, survival, and/or proliferation of a desired subtype. Cells are cultured for a sufficient time in ACM - for example, 6 hours, 12 hours, 28 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, 62 hours, 68 hours, or more - to allow for the proliferation of a selected subtype (see, e.g., FIG. 1). For example, described herein are several specific ACMs. Culturing of a population containing ACM #2 (described in the examples below), results in a proliferation (or protection) of unipolar cells. Thus, in a non-limiting example, using the methods described herein, a user could selectively grow unipolar cells (or another desired type) from a mixed-cell culture using a culture system comprising ACM#2. This methodology can be utilized to proliferate any desired subtype (or mix of subtypes) from a culture comprising that subtype (or subtypes).

[0016] In its several aspects, the present disclosure usefully provides methods for screening for agents which can be used to treat or prevent the effects of diseases which affect neurological function; for the testing of such therapies; for the development of drugs targeting pathways involved in diseases affecting neurological function; for the identification of new anti-neurological disease therapeutic targets; and for the screening of patients (e.g., as an adjunct for mammosraphy). In general, such methodology comprises exposing dopaminergic neuronal cells in ACM to a test compound to determine whether the test compound increases the growth and/or survival of the cells. Cell populations so tested can be mixed populations (e.g., more than one subtype) or can be substantially pure subtypes (e.g., unipolar cells, multipolar cells, etc.). The invention can be used as a model to test patients' disease sensitivity to known therapies; and as a model for identification of new therapeutic targets.

[0017] For example, various potential drug candidates, e.g., small molecules, siRNA, peptides, hormones, etc., can be screened to determine if they can modulate any of the targets described herein or pathways of the targets described herein. Those drug candidates that are able to module the targets or target pathways can be further assayed to determine if they can be effective as therapeutic agents, or if they can lead to other therapeutic agents or targets, or whether a mechanism of action can be determined based on their activity. Agents to be analyzed for potential therapeutic value can be any compound, small molecule, protein, lipid, carbohydrate, nucleic acid or other agent appropriate for therapeutic use. Isolated cells of a target population can be exposed to libraries of potential therapeutic agents (e.g., antibody libraries, small molecule libraries) to determine effects in the systems described herein.

[0018] The present disclosure also provides for kits useful for culturing dopaminergic neuronal cells. Kits can contain an astrocyte-derived conditioned medium of the present invention. Kits typically contain frozen, preserved, live or other dopaminergic neuronal cells. Kits can also contain instructions for culturing a mixed population of dopaminergic neuronal cells and/or an isolated subtype of such cells (e.g., goblet cells, pyramidal cells, unipolar cells, bipolar cells, multipolar cells, or claw cells).

[0019] The methods and compositions of the present disclosure can be used in the diagnosis, prognosis, treatment and/or prevention of diseases which affect neurological function. Such diseases include acute and chronic disorders arising from any cause. Non-limiting examples of such disorders include ischemia, stroke (ischemic and hemorrhagic), neurodegenerative disorders, drenoleukodystrophy, Alexander's disease, Alper's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease), ataxia telangiectasia, Batten disease, bovine spongiform encephalopathy, Canavan disease, Cockayne syndrome, corticobasal degeneration, Creutzfeldt- Jakob disease familial fatal insomnia, frontotemporal lobar degeneration, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, Lewy body dementia, neuroborreliosis, machado- Joseph disease (spinocerebellar ataxia type 3), multiple system atrophy, multiple sclerosis, narcolepsy, Nieman Pick disease, Parkinson's disease, Pelizaeus-Merzbacher disease, Pick's disease, primary lateral sclerosis, prion diseases, progressive supranuclear palsy, Refsum's disease, Sandhoff disease, Schilder's disease, subacute combined degeneration of the spinal cord, Spielmeyer-Vogt-Sjogren-Batten disease, Spinocerebellar ataxia, spinal muscular atrophy, Steele-Richardson-Olszewski disease, tabes dorsalis, toxic

encephalopathy. These disorders are provided for illustrative purposes only and are not meant to limit the application of the compositions or methods of the present invention in any manner.

[0020] The following examples are to illustrate the invention. They are not meant to limit the invention in any way. [0021] EXAMPLE 1

[0022] We sought to discover a scientific basis for dividing PD patients into sub-groups, based on their response to DA-selective neurotrophic factors in clinical trials. The results could help to change how clinical trials for neurotrophic factors are designed. Instead of the traditional randomized, placebo control versus drug-treated group, all patients will be treated with the drug. During the trial, patients are separated into responders versus non-responders.

[0023] Nigral Cell Culture

[0024] Pregnant, E14 rats are killed by exposure to CO₂ gas, a laparotomy is done and the uterine sacs collected in cold DPBS, pH 7.4, minus Ca²⁺ and Mg²⁺. The fetuses are transferred to cold HBSS, pH 7.4 (Invitrogen) in 9 cm petridishes, and the brains removed intact. A 1.0 mm³ piece of tissue is dissected from the roof of the mesencephalic flexure (Shimoda et al. (1992) Brain Res 586: 319-331), pooled in cold, oxygenated HBSS, digested with papain (Sigma Chemical Co, 10 U/ml, 37°C, 15 min), washed, triturated, and centrifuged at 1000 rpm for 5 min. The medium is aspirated, and the cells resuspended in fresh DMEM/F-12 with supplements (Takeshima et al. (1996) J Neurosci Meth 67: 27-41).

[0025] Cell Viability Assay

[0026] A two-color, fluorescence assay (Invitrogen-Molecular Probes, Cat. No. L3224) is used to test cell viability. The two dyes used, ethidium homodimer (3.8 mM) and calcein-AM (2.0 mM) emit red

(dead cells) and green (live cells) light, respectively, in response to excitation with the filter set for fluorescein (490-505 nm) (Takeshima et al. (1994) J Neurosci 14: 4769-4779). The cells are incubated in the dyes for 15 min at room temperature, and examined with a Nikon Microphot-FXA fluorescence microscope.

[0027] Microislands

[0028] The cells are resuspended at a final density of 5.0 xl0⁵/ml. A 25 ul droplet of the cell suspension (1.25 x 10⁴ cells) is plated in glass, 8-well chamber slides (Nunc, Inc., IL) coated with poly-D-lysine. Each MI occupies an area of 12.5 mm², for a final density of 1.0 x 10⁵/cm². The cultures are incubated for 4 h at 37°C for cell attachment, then 375 μΐ of growth medium is added per well. The entirety of the medium is changed after the first 12 h, and 50% every second day thereafter.

[0029] Immunocytochemistry

[0030] The medium is aspirated and the cultures fixed with 4% paraformaldehyde in PBS for 10 min, then permeabilized using cold (-20°C) 1% CH₃COOH/95% EtOH for 20 min. Non-specific binding is blocked with 4% donkey serum (Chemicon) in 0.05% BSA-PBS for 30 min. Primary rabbit anti-TH (1 :750, Pel Freeze); rabbit anti-GABA (1 :800, Sigma); rabbit anti-5HT (1 :800, Sigma); mouse anti-MAP2 (1 :750, Sigma); mouse anti-GFAP (1 :250, Sigma) are applied to the wells, and the slides incubated at 4°C for 16-24 hr. After washing with PBS, donkey anti-mouse IgG-FITC (Chemicon), and donkey anti-rabbit IgG-CY3 (Chemicon), secondary antibodies are applied, and the slides incubated for 1 hr at room temperature. After washing with PBS, the chamber walls are removed, and the slides prepared for microscopic analysis. Alternatively, TH and MAP2 are identified using the biotinylated, Ni-enhanced, diaminobenzidine (DAB) reaction product (ABC kits; Vector Laboratories).

[0031] Imaging and Data Collection

[0032] Data are collected using a Nikon E-800 Microphot Fluorescence Microscope and Northern Eclipse Software (Empix Imaging). Four fields: Upper-L, Upper-R, Lower-R and Lower-L per well are analyzed, centred on the middle of the MI. Neurons are counted manually by tagging. Data are logged and transferred to an ExCel spread sheet.

[0033] Statistics

[0034] The data are analyzed using a one-way analysis of variance, ANOVA, followed by post hoc testing using the Student-Newman-Keuls test for comparison of multiple pairs of data, as appropriate. In cases of failure of the normality test, the Kruskal- Wallis equal variance test, that tests one-way analysis of variance on ranks will be used, followed by the Student-Newman-Keuls test.

[0035] Experiment #1: Percentage of Each Dopaminergic Sub-Type in the Nigral Culture

[0036] Four, 8-well chamber slides are plated with 25 ul MI droplets of cell suspensions. Slides #land #3 (N=16) are the untreated control, and slides #2 and #4 (N=16) are treated with 25% v/v VMCLl-ACM on days 0, 2 and 4. On day 5, the cultures are fixed and stained to visualize TH and MAP2. Data are collected from four fields per MI, equal to 64 fields each from the control and experimental groups. Each DA sub-type is tagged with a different color, and the data exported to an ExCel spread sheet. Two data sets are collected: 1) The number of each DA sub-type per field; 2) The number of each DA sub-type per MI.

[0037] Experiment #2: Selective Neuroprotection of Dopaminergic Neuronal Sub- Types

[0038] ACM is prepared from 10 cell lines that secrete DA-selective neuroprotective factors. Twelve, 8- well chamber slides are plated with 25 ul MI droplets of the nigral cell suspension, 12.5 mm² per MI, 5.0 x 10⁵ cells/ml, 1.25 x 10⁴ cells/droplet, 1.0 x 10⁵ cells/cm². Slides #1 and 12, coded A and L respectively, are the negative, untreated and positive, (treated with 25% v/v VMCLl-ACM) controls. Each of slides #2-1 1, coded B-K is treated with one of the 10 ACM at 10% v/v on days 0, 2 and 4, and stained on day 5. The Mis are analyzed as described above. Sixty (60) DA neurons, per sub-type, per well, are analyzed, permitting a robust statistical analysis to detect differences in the neuroprotective action of the ACM on the DA sub-types. Experiment #3: A Specific Concentration of ACM that Protects a Single DA Sub- Type

[0039] Three DA sub-types and three ACM will be selected for further analysis, based on the results of Experiment #2. Five concentrations of each ACM will be tested: 1.0, 2.5, 5.0, 10 and 25 v/v. Plating, treatment protocols, data collection and analysis are done as described for Experiment #2. Where a specific concentration of ACM selectively protects a specific DA sub-type. Effects of four separate ACM (#2, #3, #4 and #5) on five DA morphological subtypes is determined (FIG. 1). As can be seen in Figure 1, ACM #2 protects Unipolar, Multipolar and Goblet sub-types, but not Bipolars or Pyramidals. None of the four ACM tested affected Pyramidals and Goblets. The actions of the four ACM tested on the multipolar sub-type mirror exactly their effects on the total DA population.

[0040] Interpretation of Data

[0041] At least one concentration of ACM will likely selectively protect one specific DA sub-type. In the SNc, astrocyte-neuron interactions could range from highly discrete and specific, to very complex. The concepts presented herein help to explain past failures of neurotrophic factors in clinical trials and can help to mitigate failure in future trials.

[0042] Experiment #4: Testing compounds to determine neuroprotectivity on mixed populations, or subtypes, of DAergic cells

[0043] In this experiment, different test compounds are added to dopaminergic neuronal cell populations (DAergic cells) in ACM to determine whether the compounds support the growth and/or survival of the cells. Cells are cultured in the presence of ACM and in the presence of one or more test compounds (e.g., proteins, small molecules, antibodies). As one control, cells are cultured in ACM alone and, as a second control, cells are cultured in ACM and the presence of a known stimulator of growth and/or survival of the cells. Substances to be tested are added to wells containing the cells (as described above). Cultures containing test compounds which show an increase in the amount of growth and/or survival of the dopaminergic neuronal cell population that is greater than the baseline amount (i.e., compared to the cells in ACM alone) are selected for further analysis of neuroprotective effect. These experiments are performed on both mixed populations of DAergic cells (i.e., mixed populations) and isolated subtype DAergic cells (i.e., substantially pure populations of subtypes such as unipolar cells, multipolar cells, bipolar cells, pyramidal cells, goblet cells, and claw cells). Where isolated subtype populations are utilized.

[0044] The laboratory has developed the tools needed to apply this knowledge to a drug development program, including: a proprietary method to immortalize type-1 astrocytes (Panchision et al. (1998) J Mol Neurosci 1 1 : 209-221); a DA-enriched nigral cell culture (20% of DA neurons) (Shimoda et al. (1992) Brain Res 586: 319-331); a MI plating method [12]; a library of 88 astrocyte cell lines; a method to identify cell lines that secrete DA-selective neurotrophic factors, and a drug candidate, MANF, for PD.

[0045] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method for selecting a population of a subtype of dopaminergic neuronal (DAergic) cell, comprising culturing a population DAergic cells in astrocyte- derived conditioned media for a sufficient amount of time to allow neuronal cell protection, and selecting at least one DAergic cell subtype that proliferates in said media.

2. A method for identifying a candidate agent useful in supporting the growth and/or survival of DAergic neurons, comprising:

a) contacting a candidate pharmacological agent with a population of DAergic cells in astrocyte-derived conditioned media, wherein in the absence of the candidate pharmacological agent, a baseline amount of growth or survival of the DAergic cell subtypes is observed;

b) determining a test amount of growth or survival of the DAergic cell subtypes in the presence of the pharmacological agent as a measure of the effect of the pharmacological agent on the growth or survival of the DAergic cell subtypes, wherein a test amount of growth or survival of the DAergic cell subtypes that is greater than the baseline amount indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent that is useful in supporting the growth and/or survival of DAergic neurons.

3. A method for identifying a candidate agent useful in supporting the growth and/or survival of a subtype cell of a DAergic neuron, comprising:

a) isolating at least one subtype of a DAergic cell by culturing a population of neuronal cells in astrocyte-derived conditioned media;

b) contacting a candidate pharmacological agent with the subtype of a DAergic cell in (a), wherein in the absence of the candidate pharmacological agent, results in a baseline amount of growth or survival of the subtype;

c) determining a test amount of growth or survival of the subtype in the presence of the pharmacological agent as a measure of the effect of the pharmacological agent on the growth or survival of the subtype, wherein a test amount of growth or survival of the subtype that is greater than the baseline amount indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent that is useful in supporting the growth and/or survival of the subtype.

4. A kit comprising an astrocyte-derived conditioned media; at least one cell selected from a ventral mesencephalon, cerebral cortex, hippocampus and striatum; and instructions for culturmg a subtype of a DAergic cell.

5. The method of claims 1-4, wherein the subtype is selected from a group consisting of a unipolar, bipolar, multipolar, pyramidal, goblet and claw cell.

6. The method of claims 1-4, wherein said astrocyte-derived conditioned media is from ventral mesencephalon, cerebral cortex and hippocampus cell lines.

7. The method of claim 1-4, wherein said astrocyte-derived conditioned media is from ventral mesencephalon.