Biomedical subjects

Harald Hirling

Publications and source records attributed to Harald Hirling.

15 recordsLinked to original sources

PubMed2006

Phosphorylation of glutamate receptor interacting protein 1 regulates surface expression of glutamate receptors.

The number of synaptic alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors (AMPARs) controls the strength of excitatory transmission. AMPARs cycle between internal endosomal compartments and the plasma membrane. Interactions between the AMPAR subunit GluR2, glutamate receptor interacting protein 1 (GRIP1), and the endosomal protein NEEP21 are essential for correct GluR2 recycling. Here we show that an about 85-kDa protein kinase phosphorylates GRIP1 on serine 917. This kinase is present in NEEP21 immunocomplexes and is activated in okadaic acid-treated neurons. Pulldown assays and atomic force microscopy indicate that phosphorylated GRIP shows reduced binding to NEEP21. AMPA or N-methyl-D-aspartate stimulation of hippocampal neurons induces delayed phosphorylation of the same serine 917. A wild type carboxy-terminal GRIP1 fragment expressed in hippocampal neurons interferes with GluR2 surface expression. On the contrary, a S917D mutant fragment does not interfere with GluR2 surface expression. Likewise, coexpression of GluR2 together with full-length wild type GRIP1 enhances GluR2 surface expression in fibroblasts, whereas full-length GRIP1-S917D had no effect. This indicates that this serine residue is implicated in AMPAR cycling. Our results identify an important regulatory mechanism in the trafficking of AMPAR subunits between internal compartments and the plasma membrane.

Animals↗

PubMed2006

Intracellular trafficking of interleukin-1 receptor I requires Tollip.

Interleukin-1 receptor (IL-1RI) is a master regulator of inflammation and innate immunity. When triggered by IL-1beta, IL-1RI aggregates with IL-1R-associated protein (IL-1RAcP) and forms a membrane proximal signalosome that potently activates downstream signaling cascades. IL-1beta also rapidly triggers endocytosis of IL-1RI. Although internalization of IL-1RI significantly impacts signaling, very little is known about trafficking of IL-1RI and therefore about precisely how endocytosis modulates the overall cellular response to IL-1beta. Upon internalization, activated receptors are often sorted through endosomes and delivered to lysosomes for degradation. This is a highly regulated process that requires ubiquitination of cargo proteins as well as protein-sorting complexes that specifically recognize ubiquitinated cargo. Here, we show that IL-1beta induces ubiquitination of IL-1RI and that via these attached ubiquitin groups, IL-1RI interacts with the ubiquitin-binding protein Tollip. By using an assay to follow trafficking of IL-1RI from the cell surface to late endosomes and lysosomes, we demonstrate that Tollip is required for sorting of IL-1RI at late endosomes. In Tollip-deficient cells and cells expressing only mutated Tollip (incapable of binding IL-1RI and ubiquitin), IL-1RI accumulates on late endosomes and is not efficiently degraded. Furthermore, we show that IL-1RI interacts with Tom1, an ubiquitin-, clathrin-, and Tollip-binding protein, and that Tom1 knockdown also results in the accumulation of IL-1RI at late endosomes. Our findings suggest that Tollip functions as an endosomal adaptor linking IL-1RI, via Tom1, to the endosomal degradation machinery.

Animals↗

PubMed2006

Rescuing defective vesicular trafficking protects against alpha-synuclein toxicity in cellular and animal models of Parkinson's disease.

Studies in yeast are providing critical insights into the mechanisms of neurodegeneration in Parkinson's disease (PD). A recent study shows that disruption of vesicular trafficking between the endoplasmic reticulum (ER) and the Golgi, caused by the overexpression and/or aggregation of alpha-synuclein, is linked to degeneration of dopamine neurons. Overexpression of proteins that are known to enhance ER-to-Golgi transport rescue defective trafficking in yeast, worm, fly, and cellular models of PD.

Animals↗

PubMed2006

Plasticity of astrocytic coverage and glutamate transporter expression in adult mouse cortex.

Astrocytes play a major role in the removal of glutamate from the extracellular compartment. This clearance limits the glutamate receptor activation and affects the synaptic response. This function of the astrocyte is dependent on its positioning around the synapse, as well as on the level of expression of its high-affinity glutamate transporters, GLT1 and GLAST. Using Western blot analysis and serial section electron microscopy, we studied how a change in sensory activity affected these parameters in the adult cortex. Using mice, we found that 24 h of whisker stimulation elicited a 2-fold increase in the expression of GLT1 and GLAST in the corresponding cortical column of the barrel cortex. This returns to basal levels 4 d after the stimulation was stopped, whereas the expression of the neuronal glutamate transporter EAAC1 remained unaltered throughout. Ultrastructural analysis from the same region showed that sensory stimulation also causes a significant increase in the astrocytic envelopment of excitatory synapses on dendritic spines. We conclude that a period of modified neuronal activity and synaptic release of glutamate leads to an increased astrocytic coverage of the bouton-spine interface and an increase in glutamate transporter expression in astrocytic processes.

Amino Acid Transport System X-AG↗

PubMed2005

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.

Animals↗

PubMed2005

NSF interaction is important for direct insertion of GluR2 at synaptic sites.

Here, we use a cell surface thrombin cleavage assay to investigate directly the role of NSF in the surface delivery and synaptic accumulation of alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) receptors. In cultured hippocampal neurons, the GluR2 subunit (which specifically interacts with NSF) inserts rapidly into the plasma membrane from intracellular compartments and accumulates in synaptic sites. In contrast, surface accumulation of GluR3 (a subunit that does not interact with NSF) or a GluR2 mutant defective in NSF binding (DeltaA849-Q853) occurs initially at extrasynaptic sites and is kinetically slower than wild-type GluR2. Introducing a binding site for NSF into GluR3 (GluR3NSF) generates a subunit that behaves like GluR2 in terms of kinetics and site of surface insertion. These data suggest that the NSF interaction is necessary for rapid incorporation of AMPA receptor subunits into synapses and is sufficient to confer this property on GluR3.

Animals↗

PubMed2005

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.

Animals↗

PubMed2005

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.

Animals↗

PubMed2004

Crucial role of neuron-enriched endosomal protein of 21 kDa in sorting between degradation and recycling of internalized G-protein-coupled receptors.

Recycling of endocytosed G-protein-coupled receptors involves a series of molecular events through early and recycling endosomes. The purpose of this work was to study the role of neuron-enriched endosomal protein of 21 kDa (NEEP21) in the recycling process of neurotensin receptors-1 and -2. Here we showed that suppression of NEEP21 expression does not modify the internalization rate of both receptors but strongly inhibited the recycling of the neurotensin receptor-2. In contrast, overexpression of NEEP21 changes the behavior of the neurotensin receptor-1 from a non-recycling to a recycling state. Recycling of the neurotensin receptor-2 involves both the phosphatidylinositol 3-kinase and the recycling endosome pathways, whereas recycling of the neurotensin receptor-1 induced by overexpression of NEEP21 only occurs by the phosphatidylinositol 3-kinase-dependent pathway. Taken together, these results confirm the essential role of NEEP21 in the recycling mechanism and show that this protein acts at the level of early endosomes to promote sorting of receptors toward a recycling pathway.

Animals↗

PubMed2004

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.

Animals↗

PubMed2004

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.

Animals↗

PubMed2003

Morphological and molecular heterogeneity in release sites of single neurons.

We have previously shown that labelling intensities for synaptic proteins vary strongly among synaptic boutons. Here we addressed the questions as to whether there are heterogeneous levels of integral membrane synaptic vesicle proteins at distinct active release sites of single neurons and if these sites possess the ultrastructural features of synapses. By double-immunostaining with specific antibodies against synaptophysin, synaptotagmin I, VAMP1 and VAMP2, we identified different relative levels of these integral membrane proteins of synaptic vesicles in comparison to boutons of the same rat cortical neuron. This heterogeneity could also be observed between the two isoforms VAMP1 and VAMP2. By studying pairs of these proteins implicated in neurotransmitter release, including both VAMP isoforms, we also show that the sites that contained predominantly one protein were nevertheless functional, as they internalized and released FM1-43 upon potassium stimulation. Using electron microscopy, we show that these active sites could have either synaptic specializations, or the features of vesicle-containing varicosities without a postsynaptic target. Different varicosities of the same neuron showed different intensities for synaptic vesicle proteins; some varicosities were capable of internalizing and releasing FM1-43, while others were silent. These results show that integral membrane synaptic vesicle proteins are differentially distributed among functional release sites of the same neuron.

Animals↗

PubMed2002

Developmental and spatial expression pattern of syntaxin 13 in the mouse central nervous system.

Vesicular transport involves SNARE (soluble- N-ethylmaleimide-sensitive-factor-attachment-protein-receptor) proteins on transport vesicles and on target membranes. Syntaxin 13 is a SNARE enriched in brain, associated with recycling endosomes; its overexpression in PC12 cells promotes neurite outgrowth. This suggests an important role for receptor recycling during neuronal differentiation. Here we describe the spatiotemporal pattern of syntaxin 13 expression during mouse brain development. During early embryogenesis (E12-E15), it was found in the forebrain ventricular zone and in primary motor and sensory neurons in the brainstem, spinal cord and sensory ganglia. In the forebrain at E15, syntaxin 13 was not detected in neuroblasts in the intermediate zone of the embryonic hemispheric wall, while there was labeling in cortical neurons in deeper layers starting at E15-18, and progressively in later-generated neurons up to layer II around P6. Syntaxin 13 reached maximal expression in all brain divisions at about P7, followed by a decrease, with heterogeneous neuron populations displaying various staining intensities in adult brain. While usually restricted to the soma of neurons, we transiently detected syntaxin 13 in dendrites of pyramidal neurons during the first postnatal week. In conclusion, the developmentally regulated syntaxin 13 expression in various neuronal populations is consistent with its involvement in endocytic trafficking and neurite outgrowth.

Aging↗

PubMed2002

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.

Androstadienes↗

PubMed2002

Pancreatic beta-cell protein granuphilin binds Rab3 and Munc-18 and controls exocytosis.

Granuphilin/Slp-4 is a member of the synaptotagmin-like protein family expressed in pancreatic beta-cells and in the pituitary gland. We show by confocal microscopy that both granuphilin-a and -b colocalize with insulin-containing secretory granules positioned at the periphery of pancreatic beta-cells. Overexpression of granuphilins in insulin-secreting cell lines caused a profound inhibition of stimulus-induced exocytosis. Granuphilins were found to bind to two components of the secretory machinery of pancreatic beta-cells, the small GTP-binding protein Rab3 and the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-binding protein Munc-18. The interaction with Rab3 occurred only with the GTP-bound form of the protein and was prevented by a point mutation in the effector domain of the GTPase. Structure-function studies using granuphilin-b mutants revealed that complete loss of Rab3 binding is associated with a reduction in the capacity to inhibit exocytosis. However, the granuphilin/Rab3 complex alone is not sufficient to mediate the decrease of exocytosis, suggesting the existence of additional binding partners. Taken together, our observations indicate that granuphilins play an important role in pancreatic beta-cell exocytosis. In view of the postulated role of Munc-18 in secretory vesicle docking, our data suggest that granuphilins may also be involved in this process.

Animals↗