The endosomal protein NEEP21 regulates AMPA receptor-mediated synaptic transmission and plasticity in the hippocampus

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Abstract

The neuron-enriched endosomal protein 21 (NEEP21) has recently been implicated in the regulation of AMPA receptor (AMPAR) trafficking and proposed to participate in the control of synaptic strength. We tested here this possibility at CA3–CA1 synapses in hippocampal slice cultures using antisense-mediated down-regulation of NEEP21 expression or transfection of a fragment of the cytosolic domain of NEEP21. We found that NEEP21 suppression or expression of the dominant-negative fragment reduced spontaneous and evoked AMPAR-mediated synaptic currents without affecting presynaptic properties. The effect specifically resulted from a reduction of currents mediated by AMPA as opposed to NMDA receptors. Blockade of endocytosis, using a peptide interfering with dynamin, revealed a progressive increase of AMPAR responses due to receptor accumulation in control cells, but not following NEEP21 suppression or expression of the fragment. Also, the enhanced receptor cycling induced by bath application of NMDA resulted in a depression that was enhanced following interference with NEEP21 function. Finally, LTP induction, which involves expression of new synaptic receptors, was abolished in NEEP21-depleted cells or cells expressing the dominant-negative fragment. Together, we conclude that NEEP21 contributes to the regulation of synaptic transmission and plasticity in slice cultures by affecting the recycling and targeting of AMPA receptors to the synapse.

Introduction

Activity-induced changes in the strength of synaptic communication are crucial for the establishment of proper neuronal communication during development and for experience-dependent plasticity and learning mechanisms. In the hippocampus, glutamatergic AMPARs have been shown to play a critical role in these processes. This has been proposed to occur through modifications of receptor channel properties (Barria et al., 1997, Benke et al., 1998), but increasing evidence indicates that activity-regulated incorporation or removal of AMPAR from synaptic structures also directly participates in the control of synaptic efficacy (Barry and Ziff, 2002, Lu et al., 2001, Malinow and Malenka, 2002, Sheng and Lee, 2001, Shi et al., 1999, Song and Huganir, 2002). An important question therefore is how AMPAR targeting and sorting at the synapse are regulated.

AMPA receptors form hetero-oligomeric complexes of different combinations of the subunits GluR1 to GluR4. In order to maintain synaptic receptor levels constant without changing synaptic efficacy, AMPARs constitutively cycle with equal rates of endocytosis and exocytosis (Turrigiano, 2000). These mechanisms are however also regulated by activity in a subunit-specific manner. In mature neurons, GluR1 has been shown to be predominantly involved in the activity-dependent synaptic delivery of AMPAR associated with LTP induction (Hayashi et al., 2000, Meng et al., 2003, Passafaro et al., 2001, Shi et al., 2001). AMPAR trafficking is therefore probably controlled by complex interactions between individual AMPAR subunits and different groups of intracellular proteins associated either with the postsynaptic density (Dong et al., 1997, Kim et al., 2001, Osten et al., 2000, Perez et al., 2001, Terashima et al., 2004) or endosomal compartments (Park et al., 2004). Recently, Steiner et al. (2002) identified a new neuron-specific protein, Neuronal Enriched Endosomal Protein of 21 kDa (NEEP21), that they found to be only present in early endosomes, that formed a protein complex with the endosomal SNARE protein syntaxin 13, was co-localized with internalized AMPARs, and interfered with correct AMPAR trafficking. They thus proposed that NEEP21 could be one of the possible candidate proteins involved in the regulation of AMPAR recycling. We tested here this possibility by analyzing whether NEEP21 could participate in the control of synaptic strength and plasticity. The results obtained using an antisense-mediated approach to downregulate the expression of NEEP21 or by expression of the cytosolic domain of the protein indicate that NEEP21 is indeed a major regulator of AMPAR expression at the synapse.

Section snippets

NEEP21 expression in control and transfected CA1 neurons

NEEP21 is an early endosomal protein that is strongly expressed in hippocampal organotypic slice cultures during the first 3 weeks after slice preparation. As illustrated in Fig. 1A, the expression pattern of NEEP21 shows a slight developmental down-regulation similar to that reported in dissociated neuron cultures. In dissociated hippocampal neurons, immunocytochemical analyses of NEEP21 show a punctuate staining that co-localizes with endosomes. Staining does not overlap with synaptophysin,

Discussion

Membrane receptors undergo a continuous cycling process that involves internalization, transport to early endosomes, and sorting towards either degradation pathways or recycling back to the plasma membrane (Horton and Ehlers, 2004). This process has recently been shown to contribute to mechanisms of synaptic plasticity (Park et al., 2004). In this study, we identified NEEP21, an endosomal protein, as an important molecule for the regulation of expression of AMPA receptors at synaptic membranes.

Slices culture, cDNA constructs, and transfection

Hippocampal organotypic slice cultures were prepared as described (Stoppini et al., 1991) and maintained for 11–12 days in culture before use. Cultures were transfected using a biolistic approach (Gene gun, BioRad) according to the manufacturer's instructions. Different constructs were tested including a pcDNA3.1-EGFP-NEEP21-antisense (Steiner et al., 2002), a control pcDNA3.1-EGFP construct, or an EGFP-NEEP21 construct coding for amino acids 129–164 of the cytoplasmic domain of NEEP21

Acknowledgments

We thank M. Moosmayer and L. Parisi-Jourdain for excellent technical help. This work was supported by grants 31-56852.99, 3100A0-105721, and 3100AO-100834/1 from the Swiss Science Foundation, Novartis Foundation, and Leenaards Foundation Award (1907/ep).

References (33)

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