Hélène Boudin

Constitutive activation drives compartment-selective endocytosis and axonal targeting of type 1 cannabinoid receptors.

The type 1 cannabinoid receptor (CB1R) is one of the most abundant G-protein-coupled receptors (GPCRs) in the brain, predominantly localized to axons of GABAergic neurons. Like several other neuronal GPCRs, CB1R displays significant in vitro constitutive activity (i.e., spontaneous activation in the absence of ligand). However, a clear biological role for constitutive GPCR activity is still lacking. This question was addressed by studying the consequences of constitutive activation on the intracellular trafficking of endogenous or transfected CB1Rs in cultured hippocampal neurons using optical and electron microscopy. We found that constitutive activity results in a permanent cycle of endocytosis and recycling, which is restricted to the somatodendritic compartment. Thus, CB1Rs are continuously removed by endocytosis from the plasma membrane in the somatodendritic compartment but not in axons, where CB1Rs accumulate on surface. Blocking constitutive activity by short-term incubation with inverse agonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM281) results in sequestration of recycled CB1Rs on the somatodendritic plasma membrane. Long-term inhibition of endocytosis by cotransfection of dominant-negative proteins results in impaired axonal polarization of surface-bound CB1Rs. Kinetic analysis shows that the majority of newly synthesized CB1Rs arrive first to the somatodendritic plasma membrane, from where they are rapidly removed by AM281-sensitive constitutive endocytosis before being delivered to axons. Thus, constitutive-activity driven somatodendritic endocytosis is required for the proper axonal targeting of CB1R, representing a novel, conformation-dependent targeting mechanism for axonal GPCRs.

CELLULAR DISTRIBUTION OF NEUROTENSIN RECEPTORS IN RAT BRAIN : IMMUNOHISTOCHEMICAL STUDY USING AN ANTIPEPTIDE ANTIBODY AGAINST THE CLONED HIGH AFFINITY RECEPTOR

Receptors for the neuropeptide, neurotensin, were localized by immunohistochemistry in the rat brain by using an antibody raised against a sequence of the third intracellular loop of the cloned high affinity receptor. Selective receptor immunostaining was observed throughout the brain and brainstem. This immunostaining was totally prevented by preadsorbing the antibody with the immunogenic peptide. The regional distribution of the immunoreactivity conformed for the most part to that of [3H]‐ or [125I]‐neurotensin binding sites previously identified by autoradiography. Thus, the highest levels of immunostaining were observed in the islands of Calleja, diagonal band of Broca, magnocellular preoptic nucleus, pre‐ and parasubiculum, suprachiasmatic nucleus, anterodorsal nucleus of the thalamus, substantia nigra, ventral tegmental area, pontine nuclei and dorsal motor nucleus of the vagus, all of which had previously been documented to contain high densities of neurotensin binding sites. There were, however, a number of regions reportedly endowed with neurotensin binding sites, including the central amygdaloid nucleus, periaqueductal gray, outer layer of the superior colliculus and dorsal tegmental nucleus, which showed no or divergent patterns of immunostaining, suggesting that they might be expressing a molecularly distinct form of the receptor. At the cellular level, neurotensin receptor immunoreactivity was predominantly associated with perikarya and dendrites in some regions (e.g., in the basal forebrain, ventral midbrain, pons and rostral medulla) and with axons and axon terminals in others (e.g., in the lateral septum, bed nucleus of the stria terminalis, neostriatum, paraventricular nucleus of the thalamus and nucleus of the solitary tract). These data indicate that neurotensin may act both post‐ and presynaptically in the central nervous system and confirm that some of its effects are exerted on projection neurons. There were also areas, such as the cerebral cortex, nucleus accumbens and para‐ and periventricular nucleus of the hypothalamus, which contained both immunoreactive perikarya/dendrites and axon terminals, consistent with either a joint association of the receptor with afferent and efferent elements or its presence on interneurons. Taken together, these results also suggest that the neurotensin high affinity receptor protein is associated with a neuronal population that is more extensive than originally surmised from in situ hybridization studies.

The chemokine SDF-1 differentially regulates axonal elongation and branching in hippocampal neurons.

Recent data have shown that the chemokine SDF-1 plays a critical role in several aspects of brain development such as cell migration and axon pathfinding. However, its potential function in the generation of axons and dendrites is poorly characterized. In order to better understand the role of SDF-1 in the development of central neurons, we studied the cellular distribution of the SDF-1 receptor CXCR4 by immunocytochemistry of developing hippocampal neurons and tested the effect of SDF-1 in process patterning at the early stages of neuronal development. We found that CXCR4 immunoreactivity undergoes a striking redistribution during development. At the early stages, from day 2 to day 4 in culture, CXCR4 is particularly concentrated at the leading edge of growing neurites. As the cells mature, staining declines at the tip of the processes and becomes more broadly distributed along axons and, to a lesser extent, dendrites. SDF-1 stimulation of neurons at day 1-2 in culture triggers several effects on neuronal morphogenesis. SDF-1 reduces growth cone number and axonal outgrowth but stimulates axonal branching. These latter two effects are not observed in other neurites. This study unravels a new role for SDF-1/CXCR4 in specifying hippocampal neuron morphology by regulating axonal patterning at an early stage of neuronal development.

The Signaling Adaptor Protein CD3ζ Is a Negative Regulator of Dendrite Development in Young Neurons

A novel idea is emergxsing that a large molecular repertoire is common to the nervous and immune systems, which might reflect the existence of novel neuronal functions for immune molecules in the brain. Here, we show that the transmembrane adaptor signaling protein CD3zeta, first described in the immune system, has a previously uncharacterized role in regulating neuronal development. Biochemical and immunohistochemical analyses of the rat brain and cultured neurons showed that CD3zeta is mainly expressed in neurons. Distribution of CD3zeta in developing cultured hippocampal neurons, as determined by immunofluorescence, indicates that CD3zeta is preferentially associated with the somatodendritic compartment as soon as the dendrites initiate their differentiation. At this stage, CD3zeta was selectively concentrated at dendritic filopodia and growth cones, actin-rich structures involved in neurite growth and patterning. siRNA-mediated knockdown of CD3zeta in cultured neurons or overexpression of a loss-of-function CD3zeta mutant lacking the tyrosine phosphorylation sites in the immunoreceptor tyrosine-based activation motifs (ITAMs) increased dendritic arborization. Conversely, activation of endogenous CD3zeta by a CD3zeta antibody reduced the size of the dendritic arbor. Altogether, our findings reveal a novel role for CD3zeta in the nervous system, suggesting its contribution to dendrite development through ITAM-based mechanisms.

High‐affinity neurotensin receptors in the rat nucleus accumbens: Subcellular targeting and relation to endogenous ligand

Neurotensin is present in selective mesolimbic dopaminergic projections to the nucleus accumbens (NAc) shell but also is synthesized locally in this region and in the motor‐associated NAc core. We examined the electron microscopic immunolabeling of the high‐affinity neurotensin receptor (NTR) and neurotensin in these subdivisions of rat NAc to determine the sites for receptor activation and potential regional differences in distribution. Throughout the NAc, NTR immunoreactivity was localized discretely within both neurons and glia. NTR‐labeled neuronal profiles were mainly axons and axon terminals with diverse synaptic structures, which resembled dopaminergic and glutamatergic afferents, as well as collaterals of inhibitory projection neurons. These terminals had a significantly higher numerical density in the NAc core than in the shell but were prevalent in both regions, suggesting involvement in both motor and limbic functions. In each region, neurotensin was detected in a few NTR‐immunoreactive axon terminals and in terminals that formed symmetric, inhibitory type synapses with NTR‐labeled somata and dendrites. The NTR labeling, however, was not seen within these synapses and, instead, was localized to segments of dendritic and glial plasma membranes often near excitatory type synapses. Neuronal NTR immunoreactivity also was associated with cytoplasmic tubulovesicles and nuclear membranes. Our results suggests that, in the NAc shell and core, NTR is targeted mainly to presynaptic sites, playing a role in the regulated secretion and/or retrograde signaling in diverse, neurotransmitter‐specific neurons. The findings also support a volume mode of neurotensin actions, specifically affecting excitatory transmission through activation of not only axonal but also dendritic and glial NTR. J. Comp. Neurol. 435:142–155, 2001.

AUF1 and Hu proteins in the developing rat brain: Implication in the proliferation and differentiation of neural progenitors

Posttranscriptional events such as RNA stabilization are important for cell differentiation, but little is known about the impact of AU‐rich binding proteins (AUBPs) on the fate of neural cells. Expression of destabilizing AUBPs such as AUF1 and neuronal‐specific stabilizing proteins such as HuB, HuC and HuD was therefore analyzed in the developing central nervous system. Real‐time RT‐PCR indicated a specific developmental pattern in the postnatal cerebellum, with a progressive down‐regulation of AUF1 from P1, whereas HuB was strongly up‐regulated at about P7. These changes were accompanied by a progressive increase in AUF1p45 and the disappearance of one HuB isoform from P15, suggesting particular roles for these AUBPs in the developing cerebellum. AUF1 was detected in the three main cerebellar layers, whereas Hu proteins were found only in postmitotic neurons. A role for Hu proteins in the early stages of neuronal differentiation is further supported by arrest of cell proliferation following induction of HuB or HuD expression in a neural stem cell line. The decrease in nestin expression suggest that HuD, but not HuB, favors the transition of neural progenitors into early neuroblasts, but other factors are most probably required for their full differentiation into neurons, insofar as GAP‐43 was not detected in HuD‐transfected cells. These data suggest critical roles for HuB at the very earliest stages of neuronal differentiation, such as cell cycle exit, and HuD might also be involved in the transition of neural progenitors into early neuroblasts. Taken together, the present results strengthen the importance of AUBPs in brain ontogenesis.

Dendrite-selective redistribution of the chemokine receptor CXCR4 following agonist stimulation

The chemokine SDF-1 is a secreted protein that plays a critical role in several aspects of neuron development through interaction with its unique receptor CXCR4. A key mechanism that controls neuron responsiveness to extracellular signals during neuronal growth is receptor endocytosis. Since we previously reported that SDF-1 regulates axon development without affecting the other neurites, we asked whether this could correlate with a compartment-selective trafficking of CXCR4. We thus studied CXCR4 behavior upon SDF-1 exposure in rat hippocampus slices and in transfected neuron cultures. A massive agonist-induced redistribution of CXCR4 in endosomes was observed in dendrites whereas no modification was evidenced in axons. Our data suggest that CXCR4 trafficking may play a role in mediating selective effects of SDF-1 on distinct neuronal membrane subdomains.

In vivo regulation of neurotensin receptors following long-term pharmacological blockade with a specific receptor antagonist

Adaptive changes in brain neurotensin (NT) receptors were investigated in rats after repeated administration of SR 48692, a potent and selective non-peptide NT receptor antagonist. Administration of SR 48692 (1 mg/kg i.p.) for 15 days did not alter NT content in the brain but highly enhanced the expression of NT receptor mRNA as shown by quantitative in situ hybridization. The increase of the signal was observed in numerous areas of the brain, such as the anterior cingulate, perirhinal and retrosplenial cortices, the suprachiasmatic nucleus, the ventral tegmental area, the substantia nigra and the posterior cortical nucleus of the amygdaloid complex. Moreover, the SR 48692 treatment induced the expression of NT receptor mRNA in several nuclei of the diencephalon where it could not be detected in basal conditions. Immunoblot analysis with a specific antibody directed against the rat cloned NT receptor revealed an important increase in NT receptor protein in the brain of SR 48692-treated rats, correlating well with the increase in NT receptor mRNA levels. Surprisingly, the number and the affinity constant of NT binding sites determined on brain membrane homogenates remained unchanged after SR 48692 treatment, even after membrane permeabilization with low concentrations of digitonin. These results suggest that chronic treatment with a specific NT antagonist induces an up-regulation of NT receptors at the level of mRNA and protein. Moreover, they indicate that after a chronic treatment with SR 48692, the number of NT binding sites remains stable in contrast to what is observed after 5-day treatment or with central monoaminergic receptor following their long-term blockade.

The immune molecule CD3zeta and its downstream effectors ZAP-70/Syk mediate ephrin signaling in neurons to regulate early neuritogenesis.

J. Neurochem. (2011) 119, 708–722.

Impaired spatial memory in mice lacking CD3ζ is associated with altered NMDA and AMPA receptors signaling independent of T-cell deficiency

The immunoreceptor-associated protein CD3ζ is known for its role in immunity and has also been implicated in neuronal development and synaptic plasticity. However, the mechanism by which CD3ζ regulates synaptic transmission remains unclear. In this study, we showed that mice lacking CD3ζ exhibited defects in spatial learning and memory as examined by the Barnes maze and object location memory tasks. Given that peripheral T cells have been shown to support cognitive functions and neural plasticity, we generated CD3ζ−/− mice in which the peripheral T cells were repopulated to a normal level by syngeneic bone marrow transplantation. Using this approach, we showed that T-cell replenishment in CD3ζ−/− mice did not restore spatial memory defects, suggesting that the cognitive deficits in CD3ζ−/− mice were most likely mediated through a T-cell-independent mechanism. In support of this idea, we showed that CD3ζ proteins were localized to glutamatergic postsynaptic sites, where they interacted with the NMDAR subunit GluN2A. Loss of CD3ζ in brain decreased GluN2A-PSD95 association and GluN2A synaptic localization. This effect was accompanied by a reduced interaction of GluN2A with the key NMDAR downstream signaling protein calcium/calmodulin-dependent protein kinase II (CaMKII). Using the glycine-induced, NMDA-dependent form of chemical long-term potentiation (LTP) in cultured cortical neurons, we showed that CD3ζ was required for activity-dependent CaMKII autophosphorylation and for the synaptic recruitment of the AMPAR subunit GluA1. Together, these results support the model that the procognitive function of CD3ζ may be mediated through its involvement in the NMDAR downstream signaling pathway leading to CaMKII-dependent LTP induction.