Pascal Steiner

Glial glutamate transporters mediate a functional metabolic crosstalk between neurons and astrocytes in the mouse developing cortex.

Neuron-glia interactions are essential for synaptic function, and glial glutamate (re)uptake plays a key role at glutamatergic synapses. In knockout mice, for either glial glutamate transporters, GLAST or GLT-1, a classical metabolic response to synaptic activation (i.e., enhancement of glucose utilization) is decreased at an early functional stage in the somatosensory barrel cortex following activation of whiskers. Investigation in vitro demonstrates that glial glutamate transport represents a critical step for triggering enhanced glucose utilization, but also lactate release from astrocytes through a mechanism involving changes in intracellular Na(+) concentration. These data suggest that a metabolic crosstalk takes place between neurons and astrocytes in the developing cortex, which would be regulated by synaptic activity and mediated by glial glutamate transporters.

Interactions between NEEP21, GRIP1 and GluR2 regulate sorting and recycling of the glutamate receptor subunit GluR2.

Trafficking of AMPA‐type glutamate receptors (AMPAR) between endosomes and the postsynaptic plasma membrane of neurons plays a central role in the control of synaptic strength associated with learning and memory. The molecular mechanisms of its regulation remain poorly understood, however. Here we show by biochemical and atomic force microscopy analyses that NEEP21, a neuronal endosomal protein necessary for receptor recycling including AMPAR, is associated with the scaffolding protein GRIP1 and the AMPAR subunit GluR2. Moreover, the interaction between NEEP21 and GRIP1 is regulated by neuronal activity. Expression of a NEEP21 fragment containing the GRIP1‐binding site decreases surface GluR2 levels and delays recycling of internalized GluR2, which accumulates in early endosomes and lysosomes. Infusion of this fragment into pyramidal neurons of hippocampal slices induces inward rectification of AMPAR‐mediated synaptic responses, suggesting decreased GluR2 expression at synapses. These results indicate that NEEP21–GRIP1 binding is crucial for GluR2‐AMPAR sorting through endosomes and their recruitment to the plasma membrane, providing a first molecular mechanism to differentially regulate AMPAR subunit cycling in internal compartments.

Modulation of receptor cycling by neuron-enriched endosomal protein of 21 kD

Although correct cycling of neuronal membrane proteins is essential for neurite outgrowth and synaptic plasticity, neuron-specific proteins of the implicated endosomes have not been characterized. Here we show that a previously cloned, developmentally regulated, neuronal protein of unknown function binds to syntaxin 13. We propose to name this protein neuron-enriched endosomal protein of 21 kD (NEEP21), because it is colocalized with transferrin receptors, internalized transferrin (Tf), and Rab4. In PC12 cells, NEEP21 overexpression accelerates Tf internalization and recycling, whereas its down-regulation strongly delays Tf recycling. In primary neurons, NEEP21 is localized to the somatodendritic compartment, and, upon N-methyl-d-aspartate (NMDA) stimulation, the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor subunit GluR2 is internalized into NEEP21-positive endosomes. NEEP21 down-regulation retards recycling of GluR1 to the cell surface after NMDA stimulation of hippocampal neurons. In summary, NEEP21 is a neuronal protein that is localized to the early endosomal pathway and is necessary for correct receptor recycling in neurons.

Interactions between synaptic vesicle fusion proteins explored by atomic force microscopy

Measuring the biophysical properties of macromolecular complexes at work is a major challenge of modern biology. The protein complex composed of vesicle-associated membrane protein 2, synaptosomal-associated protein of 25 kDa, and syntaxin 1 [soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) complex] is essential for docking and fusion of neurotransmitter-filled synaptic vesicles with the presynaptic membrane. To better understand the fusion mechanisms, we reconstituted the synaptic SNARE complex in the imaging chamber of an atomic force microscope and measured the interaction forces between its components. Each protein was tested against the two others, taken either individually or as binary complexes. This approach allowed us to determine specific interaction forces and dissociation kinetics of the SNAREs and led us to propose a sequence of interactions. A theoretical model based on our measurements suggests that a minimum of four complexes is probably necessary for fusion to occur. We also showed that the regulatory protein neuronal Sec1 injected into the atomic force microscope chamber prevented the complex formation. Finally, we measured the effect of tetanus toxin protease on the SNARE complex and its activity by on-line registration during tetanus toxin injection. These experiments provide a basis for the functional study of protein microdomains and also suggest opportunities for sensitive screening of drugs that can modulate protein–protein interactions.

Reticulon 1-C/neuroendocrine-specific protein-C interacts with SNARE proteins

Reticulons are proteins of neuroendocrine cells localized primarily to the endoplasmic reticulum membrane. Despite their implication in cellular processes like apoptosis or axonal regeneration, their intracellular molecular function is still largely unknown. Here, we show that reticulon 1‐C can be detected in a protein complex of 150–200 kDa, and that a number of soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) proteins, i.e. syntaxin 1, syntaxin 7, syntaxin 13 and VAMP2, can be co‐immunoprecipitated with reticulon 1‐C. Moreover, it localizes to a nocodazole‐sensitive, but calreticulin‐negative domain of the endoplasmic reticulum. Finally, overexpression in PC12 cells of a reticulon 1‐C fragment which binds to SNAREs, significantly enhances human growth hormone secretion. These results suggest that reticulons are involved in vesicle trafficking events, including regulated exocytosis.

Differential endocytic sorting of p75NTR and TrkA in response to NGF: a role for late endosomes in TrkA trafficking

NGF binds to two receptors, p75NTR and TrkA. The endosomal trafficking of receptors is of emerging importance for the understanding of their signaling. We compared the endocytic trafficking of the two NGF receptors in PC12 cells. Both p75NTR and TrkA were internalized in response to NGF and colocalized with early endosomes. However, surprisingly, the subsequent endosomal trafficking paths of both NGF receptors diverged: whereas p75NTR recycled back to the surface, TrkA moved to late endosomes and underwent lysosomal degradation. By performing subcellular fractionations of NGF stimulated PC12 cells, tyrosine-phosphorylated TrkA was recovered in fractions corresponding to late endosomes. This implicates these organelles as novel endosomal NGF signaling platforms. Furthermore, the trafficking of NGF receptors could be manipulated by pharmacological means. Disrupting p75NTR recycling diminished TrkA activation in response to low concentrations of NGF, demonstrating a functional role for the recycling of p75NTR.

Syntaxin 13 is a developmentally regulated SNARE involved in neurite outgrowth and endosomal trafficking.

In addition to its role in exocytosis, SNAP‐25 is essential for axonal outgrowth. In order to identify SNARE proteins involved in neurite growth we have used SNAP‐25 antibodies to affinity‐purify protein complexes enriched in developing rat brain membrane extracts. We have identified a complex between SNAP‐25 and syntaxin 13 predominantly present in brain at embryonic or early postnatal stages. We show that syntaxin 13 is developmentally regulated with a decrease in adult brain. In differentiated neuroendocrine PC12 cells as well as primary cortical neurons the protein is localized to a punctated and tubular staining in the perinuclear region and along processes with high levels in the central region of growth cones. Carboxy‐terminally tagged syntaxin 13 was also detected on the plasma membrane by in vivo surface‐labelling where it colocalized with SNAP‐25. Syntaxin 13 has recently been shown to be implicated in early endosomal trafficking. In our study, colocalization with internalized transferrin in the cell body and along neurites confirmed endosomal location in both compartments. Finally, overexpression of full‐length syntaxin 13 enhanced neurite outgrowth in NGF‐stimulated PC12 cells, whilst it had no effect on regulated secretion. The data suggest that a syntaxin 13‐dependent endocytic trafficking step plays a limiting role in membrane expansion during neuronal development.

Regulated exocytosis of an H+/myo-inositol symporter at synapses and growth cones

Phosphoinositides, synthesized from myo‐inositol, play a critical role in the development of growth cones and in synaptic activity. As neurons cannot synthesize inositol, they take it up from the extracellular milieu. Here, we demonstrate that, in brain and PC12 cells, the recently identified H+/myo‐inositol symporter HMIT is present in intracellular vesicles that are distinct from synaptic and dense‐core vesicles. We further show that HMIT can be triggered to appear on the cell surface following cell depolarization, activation of protein kinase C or increased intracellular calcium concentrations. HMIT cell surface expression takes place preferentially in regions of nerve growth and at varicosities and leads to increased myo‐inositol uptake. The symporter is then endocytosed in a dynamin‐dependent manner and becomes available for a subsequent cycle of stimulated exocytosis. HMIT is thus expressed in a vesicular compartment involved in activity‐dependent regulation of myo‐inositol uptake in neurons. This may be essential for sustained signaling and vesicular traffic activities in growth cones and at synapses.

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

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.

Overexpression of neuronal Sec1 enhances axonal branching in hippocampal neurons.

The soluble N-ethylmaleimide-sensitive factor-attached protein receptor (SNARE) proteins syntaxin 1 and synaptosomal-associated protein-25 have been implicated in axonal outgrowth. Neuronal Sec1 (nSec1), also called murine unc18a (Munc18a), is a syntaxin 1-binding protein involved in the regulation of SNARE complex formation in synaptic vesicle membrane fusion. Here we analysed whether nSec1/Munc18a is involved in neurite formation. nSec1/Munc18a expressed under the control of an inducible promoter in differentiated PC12 cells as well as in hippocampal neurons appears first in the cell body, and at later times after induction along neurites and in growth cones. It is localised to distinct tubular and punctated structures. In addition, exogenous nSec1/Munc18a inhibited regulated secretion in PC12 cells. Overexpression in PC12 cells of nSec1/Munc18a or its homologue Munc18b, reduced the total length of neurites. This effect was enhanced with nSec1-T574A, a mutant that lacks a cyclin-dependent kinase 5 phosphorylation site and displays an increased binding to syntaxin 1. In contrast, in hippocampal neurons the total length of all primary neurites and branches was increased upon transfection of nSec1/Munc18a. Detailed morphometric analysis revealed that this was a consequence of an increased number of axonal side branches, while the average lengths in primary neurites and of side branches were not affected. From these results we suggest that nSec1/Munc18a is involved in the regulation of SNARE complex-dependent membrane fusion events implicated in the ramification of axonal processes in neurons.