Differential involvement of phosphatidylinositol 3-kinase and p42/p44 mitogen activated protein kinase pathways in brain-derived neurotrophic factor-induced trophic effects on cultured striatal neurons

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Abstract

Brain-derived neurotrophic factor (BDNF) is a potent trophic factor for striatal cells that promotes survival and/or differentiation of GABAergic neurons in vitro. In the present study, we show that the stimulation of cultured striatal cells with BDNF increased the phosphorylation of Akt and p42/p44. This effect was specifically blocked by inhibitors of phosphatidylinositol 3-kinase (PI3-K) pathways (LY294002 and wortmannin) or p42/p44 mitogen-activated protein (MAP) kinase (PD98059 and U0126). BDNF treatment induced an increase in the number of calbindin-positive neurons but not in the number of GABAergic or total cells. Furthermore, BDNF increased the degree of dendritic arborization, soma area and axon length of striatal neurons. However, PD98059 was more effective blocking BDNF effects on calbindin- than on GABA-positive neurons, whereas LY294002 inhibited morphological differentiation in both neuronal populations. Moreover, BDNF induced neuronal survival only through the activation of the PI3-K pathway.

Introduction

The striatum is the major afferent component of the basal ganglia where it participates in the regulation of motor and cognitive functions (Gerfen, 1992). The predominant neuronal type in the striatum is the medium-sized spiny neuron (MSN) which contains GABA as a primary neurotransmitter. Although these neurons are morphologically homogeneous, they can be subdivided depending on receptor and neurotransmitter expression, patch or matrix localization and projecting areas (Graybiel, 1990). The calcium binding protein, calbindin, is a specific marker for MSN. Calbindin transiently labels patch neurons at birth but later becomes a marker for a large subset of matrix neurons (Liu and Graybiel, 1992). Because GABA expression in MSN is earlier than that of calbindin (Lauder et al., 1986), the acquisition of the calbindin phenotype can be considered as a late stage of maturation of striatal GABAergic neurons.

Neurotrophic factors participate in the development, establishment and maintenance of striatal connectivity. The neurotrophin brain-derived neurotrophic factor (BDNF) regulates striatal neuron survival (Alcantara et al., 1997) and maturation (Jones et al., 1994) during development. Furthermore, at early stages as well as at the later ones, the BDNF receptor, TrkB, is localized in MSN (Merlio et al., 1992). The source of striatal BDNF can be intrinsic, because BDNF mRNA and protein are detected in the striatum Canals et al., 1998, Checa et al., 2000, Katoh-Semba et al., 1997, Yurek et al., 1998, or extrinsic by anterograde transport from the cortex or substantia nigra (Altar et al., 1997). Biological effects of BDNF on striatal cells in vitro comprise the induction of GABAergic neuron survival and/or differentiation Mizuno et al., 1994, Ventimiglia et al., 1995. In this context, BDNF regulates the expression of specific markers of MSN maturation such as DARPP-32 or calbindin proteins Ivkovic and Ehrlich, 1999, Ivkovic et al., 1997. BDNF is also a neuroprotective factor for excitotoxic-injured MSN Bemelmans et al., 1999, Gratacos et al., 2001a, Perez-Navarro et al., 1999, Perez-Navarro et al., 2000 or against hypoxic–ischemic insults Han and Holtzman, 2000, Larsson et al., 1999. As these striatal neurons degenerate in Huntington's disease, knowledge of intracellular signaling pathways mediating BDNF regulation of MSN phenotype and/or survival could help to develop new therapeutical strategies for this neurodegenerative disorder.

Binding of BDNF to its receptor, TrkB, activates multiple intracellular signal transduction pathways, such as p42/p44 mitogen-activated protein kinase (MAP) kinase, phosphatidylinositol 3-kinase (PI3-K) and phospholipase C-γ1 (PLC-γ1) Chao, 2003, Patapoutian and Reichardt, 2001 pathways. Signaling through PI3-K or p42/p44 MAP kinase pathways leads to different trophic effects. The most widespread paradigm is that the PI3-K pathway is involved in survival Hetman et al., 1999, Vaillant et al., 1999, while the p42/p44 MAP kinase pathway has been related to neuronal differentiation (Perron and Bixby, 1999). However, this pathway could also be involved in cell survival Feng et al., 1999, Parrizas et al., 1997. In the striatum, it has been described that the regulation of DARPP-32 expression in MSN in vitro requires the activation of the PI3-K pathway (Stroppolo et al., 2001), but the intracellular pathways that are responsible for the biological effects of BDNF are not yet well understood.

In the present study, we examine the intracellular pathways activated by BDNF in MSN in vitro and whether these pathways contribute to BDNF-induced striatal neuron survival and GABAergic neuron maturation to a calbindin phenotype.

Section snippets

BDNF induces the activation of PI3-K and p42/p44 MAP kinase pathways

To test whether BDNF was able to activate PI3-K and p42/p44 MAP kinase pathways in striatal cultures, cells were stimulated with BDNF (10 ng/ml) at 3 days in vivo (DIV), and the phosphorylation of Akt and p42/p44 was examined by Western blot at different time points. Our results show that Akt (Fig. 1A) and p42/p44 (Fig. 1B) were phosphorylated after BDNF treatment with a similar time course. Both proteins presented an enhancement of phosphorylation from 5 min to 1 h after BDNF treatment,

Discussion

Present results show that both PI3-K and p42/p44 MAP kinase pathways are involved in the effect of BDNF in the maturation of striatal GABAergic neurons in vitro. BDNF treatment favors the acquisition of the calbindin phenotype, because it increased the number of calbindin-positive cells without affecting GABAergic neurons and total cell number. This effect was blocked when specific inhibitors of the PI3-K (LY294002) or of the p42/p44 MAP kinase (PD98059) pathways were added to the cultures.

Cell culture

Animal handling procedures were approved by the Local Committee (99/1 University of Barcelona) and the Generalitat de Catalunya (1094/99), in accordance with the Directive 86/609/EU of the European Commission. Certified time-pregnant Sprague–Dawley dams (Iberfauna, Spain) were deeply anesthetized on gestational day 19, and fetuses were rapidly removed from the uterus, as previously described (Gratacos et al., 2001b). Cells were plated at a density of 50,000 cells/cm2 onto 24-well plates or

Acknowledgements

We are very grateful to Maria Teresa Muñoz for technical assistance. Financial support was obtained from CICYT (SAF-2002-0314; Ministerio de Ciencia y Tecnologı́a, Spain), Fundació La Caixa, Fundación Ramón Areces and Redes Temáticas de Investigación Cooperativa (G 03/167, Ministerio de Sanidad y Consumo). N.G. was a fellow of the University of Barcelona and E.G. of the Fundació La Marató de TV3.

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