Overexpression of glial cell line-derived neurotrophic factor induces genes regulating migration and differentiation of neuronal progenitor cells

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Abstract

The glial cell line-derived neurotrophic factor (GDNF) is involved in the development and maintenance of neural tissues. Mutations in components of its signaling pathway lead to severe migration deficits of neuronal crest stem cells, tumor formation, or ablation of the urinary system. In animal models of Parkinson's disease, GDNF has been recognized to be neuroprotective and to improve motor function when delivered into the cerebral ventricles or into the substantia nigra. Here, we characterize the network of 43 genes induced by GDNF overproduction of neuronal progenitor cells (ST14A), which mainly regulate migration and differentiation of neuronal progenitor cells. GDNF down-regulates doublecortin, Paf-ah1b (Lis1), dynamin, and α-tubulin, which are involved in neocortical lamination and cytoskeletal reorganization. Axonal guidance depends on cell-surface molecules and extracellular matrix proteins. Laminin, Mpl3, Alcam, Bin1, Id1, Id2, Id3, neuregulin1, the ephrinB2-receptor, neuritin, focal adhesion kinase (FAK), Tc10, Pdpk1, clusterin, GTP-cyclooxygenase1, and follistatin are genes up-regulated by GDNF overexpression. Moreover, we found four key enzymes of the cholesterol-synthesis pathway to be down-regulated leading to decreased farnesyl-pyrophospate production. Many proteins are anchored by farnesyl-derivates at the cell membrane. The identification of these GDNF-regulated genes may open new opportunities for directly influencing differentiation and developmental processes of neurons.

Introduction

The glial cell line-derived neurotrophic factor (GDNF) is vital to the development and maintenance of neural tissues. It is a distant member of the TGFβ-superfamily, which comprises an expanding list of multifunctional proteins serving as regulators of cell proliferation and differentiation [1]. Recently, the neuronal cell adhesion molecule (NCAM) has been identified as a receptor for GDNF. Functional assays of Schwann cell migration and axon growth of central nervous system (CNS) neurons suggest physiological significance for this newly discovered pathway [2]. However, the effector proteins of GDNF signaling still remain to be elucidated. In this paper, we highlight new genes, which are influenced by GDNF signaling.

In normal development, GDNF promotes survival of sympathetic, parasympathetic [3], and spinal motorneurons [4]. It even promotes regeneration of spinal motor neurons after spinal cord injury [5]. In Hirschsprung's disease, several mutations of signaling components of the known GDNF-signaling pathway were found to cause migration deficits of enteric ganglia [6]. GDNF was first identified as a survival factor for dopaminergic neurons of the midbrain [7]. These neurons degenerate in Parkinson's disease (PD), and treatment of animal models and PD patients with exogenous GDNF promotes functional recovery [8], [9]. However, GDNF is a large molecule that must be delivered directly to the brain rather than given peripherally [10]. When this is done, it can support the survival and outgrowth of dopaminergic neurons following transplantation [11]. Additionally, GDNF added to cell suspensions of embryonic ventral mesencephalic tissue improves the survival of dopaminergic neurons following grafting into the striatum [12]. Intermittent injections of GDNF near intrastriatally transplanted, fetal nigral cell suspension grafts have similar effects on survival and neurite outgrowth of transplanted dopaminergic neurons [13]. How these effects precisely arise and which genes and pathways are involved is still unknown.

Another strategy for the delivery of trophic molecules to transplanted dopaminergic neurons is to cograft cells that endogenously produce trophic factors [14]. Several studies have taken advantage of neuronal stem or progenitor cells for this purpose [15], [16]. One of the best-characterized progenitor cell lines used for transplantation and differentiation studies in rodents is the ST14A cell line, originally established by Cattaneo et al. [17]. These researchers isolated striatal progenitor cells from embryonic day 14 rats and immortalized the cells by retroviral transfection of a temperature-sensitive mutant of the SV40 large T-antigen (SV40-T). ST14A cells remain properties of neural progenitor cells, including the expression of nestin and the capability to differentiate into MAP2-positive cells [18]. The temperature-dependent oncogene expression allows controlled growth of mature or neurotrophin-producing ST14A cells in vitro [19], [20] and in vivo, thereby supplying a tool for in vitro expansion (33°C) and in vivo differentiation (39°C) [21]. The inactivation of the SV40-T at 39°C corresponds to the brain temperature of the grafted rodents. Several studies have shown that the transplantation of progenitor cells into embryonic or adult brain leads to a differentiation into mature neurons and glial cells [17], [22]. However, significant improvement of the neurological deficits in the PD model was not seen. On the other hand, ST14A cells can be easily modified genetically and therefore used as vehicle for endogenous overproduction of neurotrophins [19], [20]. In addition, the effects of endogenous overproduction of neurotrophic factors on survival, migration, and differentiation processes can easily be investigated with these cells.

In two recent studies, we showed that the overproduction of ciliary neurotrophic factor (CNTF) leads to an improved stress response of neuronal progenitor cells during the early stages of differentiation [19], [20]. Here, we identified 43 genes exclusively involved in GDNF-dependent signaling, which are directly or indirectly involved in the differentiation and migration of neuronal progenitor cells. The knowledge of these GDNF-regulated genes will open new possibilities for directly influencing differentiation and developmental processes, enabling the treatment of neurodegenerative diseases by modulating precisely these new effectors.

Section snippets

Cell culture

ST14A cells were cultured as monolayers in Dulbecco's MEM-Glutamax I (DMEM) supplemented with 50 IU/ml penicillin, 60 μg/ml streptomycin and 10% inactivated fetal bovine serum (FBS) (all solutions from GibcoBRL, Life Technologies) in humidified atmosphere of 33°C and 39°C, respectively [21].

Cloning of the rat GDNF

Total RNA was extracted from rat brain according to standard protocols. After reverse transcription (Superscript™, GibcoBRL), an aliquot of the resulting cDNA was PCR-amplified with rat GDNF-specific primers

Results

Our goal was to identify genes that are exclusively regulated by GDNF. We used our previous expression data from native and CNTF-transfected ST14A cells and compared those with the expression values of the GDNF-overexpressing cells [20]. The signal values were normalized to the expression of native ST14A cells at each timepoint and at 33°C. Expression changes of native, CNTF-, and GDNF-transfected ST14A cells were then compared. By doing so, we gained information about temperature-dependent

Discussion

In this study, we investigated the effects of GDNF overexpression in striatal precursor cells with special emphasis on functional pathways and common mechanisms of GDNF signaling during neuronal development and differentiation. For this purpose, we primarily compared the gene expression changes of GDNF-transfected ST14A cells during the early stages of temperature-induced differentiation with the result of our recent study in non-transfected and in CNTF-transfected ST14A cells [20]. By this

Acknowledgements

We thank K. Biedermann, N. Deinet and J. Kropp for their technical support and Prof. Lary C. Walker (Yerkes Institute, Emory Univerity, Atlanta, USA) as well as Prof. Mart Saarma (Institute of Biotechnology, University of Helsinki, Finland) for helpful comments on the manuscript. The work was supported by the Bundesministerium für Bildung und Forschung (NBL3 FKZ01-ZZ-0108).

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