Elsevier

Experimental Neurology

Volume 214, Issue 1, November 2008, Pages 69-77
Experimental Neurology

Striatal-enriched protein tyrosine phosphatase regulates dopaminergic neuronal development via extracellular signal-regulated kinase signaling

https://doi.org/10.1016/j.expneurol.2008.07.014Get rights and content

Abstract

The striatal-enriched protein tyrosine phosphatase (STEP) is highly expressed within dopaminoceptive neurons, suggesting the possibility that STEP may interact with dopaminergic signaling. We have previously shown that signaling through dopamine D2 receptor (D2R)-mediated extracellular signal-regulated kinase (ERK) activation plays a critical role in mesencephalic dopaminergic neuronal development. Here, we investigate the role of STEP in D2R-mediated ERK signaling, especially in dopaminergic neuronal development. Analyses of developmental expression of STEP and tyrosine hydroxylase (TH) in mouse brain demonstrate that STEP- and TH-positive cells are co-localized in the substantia nigra compacta of brains of postnatal 8-day-old mice, displaying STEP expression in dopaminergic neurons at this stage. Stereological analysis demonstrates a dynamic change in the number of STEP-expressing cells from midbrain to striatum during development in WT mice and significantly decreased number of STEP-expressing cells in mice lacking D2R (D2R−/− mice). The knockdown of STEP expression by treatment with oligomeric STEP siRNA significantly decreased the number of mesencephalic TH cells and inhibited D2R-mediated development of dopaminergic neurons on primary mesencephalic culture from WT mice, but not in primary cultures from D2R−/− mice. Furthermore, knockdown of STEP expression perturbed D2R-mediated ERK signaling in dopaminergic neuronal cells from WT mice, but not from D2R−/− mice. These results suggest that STEP is an important mediator in the dopamine D2R-mediated activation of ERK signaling and in the regulation of dopaminergic neuronal development.

Introduction

Protein tyrosine phosphorylation plays a central role in numerous neuronal processes (Sun and Tonks, 1994; Van Vactor, 1998). Tyrosine phosphorylation has been implicated in axonal navigation, growth cone elongation, synapse formation, cell–cell or cell–extracellular matrix interactions, and differentiation (Winslow et al., 1995, Maness et al., 1988, Qu et al., 1990, Atashi et al., 1992, Stoker, 2001). To regulate these processes, a delicate balance exists between the level of phosphorylation caused by protein tyrosine kinases (PTK) and the opposing actions of protein tyrosine phosphatases (PTP) (Tonks and Neel, 1996, Tonks and Neel, 2001, Denu et al., 1996). Investigating whether PTPs are regulated by specific neurotransmitter systems in the brain is an important step in understanding the underlying signaling pathways.

The striatal-enriched protein tyrosine phosphatase (STEP) is primarily expressed within the basal ganglia as well as within dopaminoceptive neurons of the central nervous system (Lombroso et al., 1991, Lombroso et al., 1993). Alternative splicing produces the STEP family members, STEP61 and STEP46. Both isoforms include a catalytic domain that can dephosphorylate tyrosine and a kinase interactive motif (KIM) domain (Lombroso et al., 1991, Lombroso et al., 1993, Boulanger et al., 1995, Bult et al., 1996). The 16-amino-acid KIM region is also conserved in several related protein tyrosine phosphatases, including PTP-SL, HePTP, and PTP-ER (Pulido et al., 1998, Saxena et al., 1999, Tarrega et al., 2002). This domain is crucial for the association of these phosphatases with extracellular signal-related kinase1/2 (ERK1/2), which plays an essential role in many important biological processes such as the stress response, cell proliferation, apoptosis, and tumorigenesis (Munoz et al., 2003, Paul et al., 2003, Ip and Davis, 1998, Fiore et al., 1993, Ortiz et al., 1995).

The presence of STEP within dopaminoceptive neurons of the CNS suggested that STEP may be regulated by dopaminergic signaling. Dopamine D1 receptors have been reported to mediate the phosphorylation of STEP via a cAMP-dependent protein kinase (PKA)-dependent pathway (Paul et al., 2000). However, the role of STEP in dopaminergic neurotransmission via ERK modulation has not yet been explored.

Dopamine D2 receptor (D2R) can activate ERK signaling (Choi et al., 1999, Kim et al., 2004, Kim et al., 2006), and it has recently been reported that D2R can regulate the development of dopaminergic neurons via ERK and Nurr1 activation in mouse primary mesencephalic cultures (Kim et al., 2006). The dopamine D2R is abundantly expressed not only in striatum but also in midbrain (Baik et al., 1995), and since D2R-mediated signaling also exploits the ERK pathway (Choi et al., 1999, Kim et al., 2004, Kim et al., 2006), STEP may play an important role in D2R-mediated ERK signaling and corresponding physiological responses.

In the present study, we investigated the role of STEP in D2R-mediated ERK signaling, especially in dopaminergic neuronal development. We found that STEP is expressed in dopaminergic neurons in early developmental stage and assessed how knockdown of STEP affects D2R-mediated ERK signaling and D2R-mediated dopaminergic neuronal development.

Section snippets

Animal preparation and mesencephalic neuronal cell culture

Genotypes of D2R−/− mice and WT littermates purchased from Jax® Mice and Services (The Jackson laboratory, Maine, USA) were identified by PCR as described in the manufacturer's instructions (Kelly et al., 1997). WT and D2R−/− mice used in the initial phase of this study were originated from the breeding of heterozygous D2R−/− mice described by Baik et al., (Baik et al., 1995) and were identified by Southern hybridization analyses as described previously (An et al., 2004, Baik et al., 1995).

Developmental expression of STEP in mouse brain

We analyzed the developmental expression of STEP in midbrain, striatum, and hippocampus from embryonic to adult stages compared to the expression levels of tyrosine hydroxylase (TH). As shown in Fig. 1A, STEP expression was restricted to mouse midbrain during the embryonic stage, gradually increasing in the striatum while decreasing in midbrain during adult stages. The STEP expression profile was comparable to that of TH during development in midbrain and striatum. Furthermore, the

Discussion

It is now largely accepted that ERK activation contributes to different physiological responses in neuronal cells such as neuronal cell death and development as well as synaptic plasticity and that modulating ERK activity in the CNS can result in different neurophysiological responses (Chang and Karin, 2001, Sweatt, 2004, Thomas and Huganir, 2004). In addition, ERK activation can be also regulated by various neurotransmitter systems, which can be complex but is finely tuned depending on the

Acknowledgments

This work was supported by a research grant (grant no. M103KV010015-07K2201-01510) from the Brain Research Center of the 21st Century Frontier Research Program and by basic research grant from KOSEF (Grant No. R01-2004-000-10671-0), funded by the Research Program of the Korean Ministry of Science and Technology. Dr. S.Y. Kim, Y.N. Kim, and H.J. Lee were the recipient of a Brain Korea 21 Program Grant from the Korean Ministry of Education.

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These authors contributed equally to this work.

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