Carboxypeptidase E Independently Changes Microtubule Glutamylation, Dendritic Branching, and Neuronal Migration.

Neuronal migration and dendritogenesis are dependent on dynamic changes to the microtubule (MT) network. Among various factors that regulate MT dynamics and stability, post-translational modifications (PTMs) of MTs play a critical role in conferring specificity of regulatory protein binding to MTs. Thus, it is important to understand the regulation of PTMs during brain development as multiple developmental processes are dependent on MTs. In this study, we identified that carboxypeptidase E (CPE) changes tubulin polyglutamylation, a major PTM in the brain, and we examine the impact of CPE-mediated changes to polyglutamylation on cortical neuron migration and dendrite morphology. We show, for the first time, that overexpression of CPE increases the level of polyglutamylated α-tubulin while knockdown decreases the level of polyglutamylation. We also demonstrate that CPE-mediated changes to polyglutamylation are dependent on the CPE zinc-binding motif and that this motif is necessary for CPE action on p150Glued localization. However, overexpression of a CPE mutant that does not increase MT glutamylation mimics the effects of overexpression of wild type CPE on dendrite branching. Furthermore, although overexpression of wild type CPE does not alter cortical neuron migration, overexpression of the mutant may act in a dominant-negative manner as it decreases the number of neurons that reach the cortical plate (CP), as we previously reported for CPE knockdown. Overall, our data suggest that CPE changes MT glutamylation and redistribution of p150Glued and that this function of CPE is independent of its role in shaping dendrite development but plays a partial role in regulating cortical neuron migration.

A role for BDNF- and NMDAR-induced lysosomal recruitment of mTORC1 in the regulation of neuronal mTORC1 activity.

Memory and long term potentiation require de novo protein synthesis. A key regulator of this process is mTORC1, a complex comprising the mTOR kinase. Growth factors activate mTORC1 via a pathway involving PI3-kinase, Akt, the TSC complex and the GTPase Rheb. In non-neuronal cells, translocation of mTORC1 to late endocytic compartments (LEs), where Rheb is enriched, is triggered by amino acids. However, the regulation of mTORC1 in neurons remains unclear. In mouse hippocampal neurons, we observed that BDNF and treatments activating NMDA receptors trigger a robust increase in mTORC1 activity. NMDA receptors activation induced a significant recruitment of mTOR onto lysosomes even in the absence of external amino acids, whereas mTORC1 was evenly distributed in neurons under resting conditions. NMDA receptor-induced mTOR translocation to LEs was partly dependent on the BDNF receptor TrkB, suggesting that BDNF contributes to the effect of NMDA receptors on mTORC1 translocation. In addition, the combination of Rheb overexpression and artificial mTORC1 targeting to LEs by means of a modified component of mTORC1 fused with a LE-targeting motif strongly activated mTOR. To gain spatial and temporal control over mTOR localization, we designed an optogenetic module based on light-sensitive dimerizers able to recruit mTOR on LEs. In cells expressing this optogenetic tool, mTOR was translocated to LEs upon photoactivation. In the absence of growth factor, this was not sufficient to activate mTORC1. In contrast, mTORC1 was potently activated by a combination of BDNF and photoactivation. The data demonstrate that two important triggers of synaptic plasticity, BDNF and NMDA receptors, synergistically power the two arms of the mTORC1 activation mechanism, i.e., mTORC1 translocation to LEs and Rheb activation. Moreover, they unmask a functional link between NMDA receptors and mTORC1 that could underlie the changes in the synaptic proteome associated with long-lasting changes in synaptic strength.

Optoproteomics elucidates the interactome of L-type amino acid transporter 3 (LAT3).

Membrane protein interactions are crucial for diverse biological processes. We report the application of genetic code expansion in combination with photo-crosslinking chemistry, as we termed "optoproteomics", to identify proteins interacting with the human L-type membrane amino acid transporter 3 (LAT3, also known as SLC43A1). The site-specifically incorporated photo-cross-linker p-azido-L-phenylalanine (AzF), which reacts with proteins in their proximity, enabled the capture of weak and transient partners of LAT3 in living cells. We identify 11 unique interacting proteins which are light-sensitive and 19 unique proteins that are site-specific, validating the approach and providing insights into the LAT3 protein-protein interaction network currently unavailable.

Cortical Neuron Migration and Dendrite Morphology are Regulated by Carboxypeptidase E.

Higher brain function relies on proper development of the cerebral cortex, including correct positioning of neurons and dendrite morphology. Disruptions in these processes may result in various neurocognitive disorders. Mutations in the CPE gene, which encodes carboxypeptidase E (CPE), have been linked to depression and intellectual disability. However, it remains unclear whether CPE is involved in early brain development and in turn contributes to the pathophysiology of neurocognitive disorders. Here, we investigate the effects of CPE knockdown on early brain development and explore the functional significance of the interaction between CPE and its binding partner p150Glued. We demonstrate that CPE is required for cortical neuron migration and dendrite arborization. Furthermore, we show that expression of CPE-C10 redistributes p150Glued from the centrosome and that disruption of CPE interaction with p150Glued leads to abnormal neuronal migration and dendrite morphology, suggesting that a complex between CPE and p150Glued is necessary for proper neurodevelopment.

Regulation of the Hippocampal Network by VGLUT3-Positive CCK- GABAergic Basket Cells.

Hippocampal interneurons release the inhibitory transmitter GABA to regulate excitation, rhythm generation and synaptic plasticity. A subpopulation of GABAergic basket cells co-expresses the GABA/glycine vesicular transporters (VIAAT) and the atypical type III vesicular glutamate transporter (VGLUT3); therefore, these cells have the ability to signal with both GABA and glutamate. GABAergic transmission by basket cells has been extensively characterized but nothing is known about the functional implications of VGLUT3-dependent glutamate released by these cells. Here, using VGLUT3-null mice we observed that the loss of VGLUT3 results in a metaplastic shift in synaptic plasticity at Shaeffer's collaterals - CA1 synapses and an altered theta oscillation. These changes were paralleled by the loss of a VGLUT3-dependent inhibition of GABAergic current in CA1 pyramidal layer. Therefore presynaptic type III metabotropic could be activated by glutamate released from VGLUT3-positive interneurons. This putative presynaptic heterologous feedback mechanism inhibits local GABAergic tone and regulates the hippocampal neuronal network.

Burst-Dependent Bidirectional Plasticity in the Cerebellum Is Driven by Presynaptic NMDA Receptors.

Numerous studies have shown that cerebellar function is related to the plasticity at the synapses between parallel fibers and Purkinje cells. How specific input patterns determine plasticity outcomes, as well as the biophysics underlying plasticity of these synapses, remain unclear. Here, we characterize the patterns of activity that lead to postsynaptically expressed LTP using both in vivo and in vitro experiments. Similar to the requirements of LTD, we find that high-frequency bursts are necessary to trigger LTP and that this burst-dependent plasticity depends on presynaptic NMDA receptors and nitric oxide (NO) signaling. We provide direct evidence for calcium entry through presynaptic NMDA receptors in a subpopulation of parallel fiber varicosities. Finally, we develop and experimentally verify a mechanistic plasticity model based on NO and calcium signaling. The model reproduces plasticity outcomes from data and predicts the effect of arbitrary patterns of synaptic inputs on Purkinje cells, thereby providing a unified description of plasticity.

Nitric oxide synthase 1 adaptor protein, a protein implicated in schizophrenia, controls radial migration of cortical neurons.

BACKGROUND: Where a neuron is positioned in the brain during development determines neuronal circuitry and information processing needed for normal brain function. When aberrations in this process occur, cognitive disorders may result. Patients diagnosed with schizophrenia have been reported to show altered neuronal connectivity and heterotopias. To elucidate pathways by which this process occurs and become aberrant, we have chosen to study the long isoform of nitric oxide synthase 1 adaptor protein (NOS1AP), a protein encoded by a susceptibility gene for schizophrenia. METHODS: To determine whether NOS1AP plays a role in cortical patterning, we knocked down or co-overexpressed NOS1AP and a green fluorescent protein or red fluorescent protein (TagRFP) reporter in neuronal progenitor cells of the embryonic rat neocortex using in utero electroporation. We analyzed sections of cortex (ventricular zone, intermediate zone, and cortical plate [CP]) containing green fluorescent protein or red fluorescent protein TagRFP positive cells and counted the percentage of positive cells that migrated to each region from at least three rats for each condition. RESULTS: NOS1AP overexpression disrupts neuronal migration, resulting in increased cells in intermediate zone and less cells in CP, and decreases dendritogenesis. Knockdown results in increased migration, with more cells reaching the CP. The phosphotyrosine binding region, but not the PDZ-binding motif, is necessary for NOS1AP function. Amino acids 181 to 307, which are sufficient for NOS1AP-mediated decreases in dendrite number, have no effect on migration. CONCLUSIONS: Our studies show for the first time a critical role for the schizophrenia-associated gene NOS1AP in cortical patterning, which may contribute to underlying pathophysiology seen in schizophrenia.

Cannabinoid-induced actomyosin contractility shapes neuronal morphology and growth.

Endocannabinoids are recently recognized regulators of brain development, but molecular effectors downstream of type-1 cannabinoid receptor (CB1R)-activation remain incompletely understood. We report atypical coupling of neuronal CB1Rs, after activation by endo- or exocannabinoids such as the marijuana component ∆(9)-tetrahydrocannabinol, to heterotrimeric G12/G13 proteins that triggers rapid and reversible non-muscle myosin II (NM II) dependent contraction of the actomyosin cytoskeleton, through a Rho-GTPase and Rho-associated kinase (ROCK). This induces rapid neuronal remodeling, such as retraction of neurites and axonal growth cones, elevated neuronal rigidity, and reshaping of somatodendritic morphology. Chronic pharmacological inhibition of NM II prevents cannabinoid-induced reduction of dendritic development in vitro and leads, similarly to blockade of endocannabinoid action, to excessive growth of corticofugal axons into the sub-ventricular zone in vivo. Our results suggest that CB1R can rapidly transform the neuronal cytoskeleton through actomyosin contractility, resulting in cellular remodeling events ultimately able to affect the brain architecture and wiring.

Activation-dependent plasticity of polarized GPCR distribution on the neuronal surface.

Directionality of information flow through neuronal networks is sustained at cellular level by polarized neurons. However, specific targeting or anchoring motifs responsible for polarized distribution on the neuronal surface have only been identified for a few neuronal G-protein-coupled receptors (GPCRs). Here, through mutational and pharmacological modifications of the conformational state of two model GPCRs, the axonal CB1R cannabinoid and the somatodendritic SSTR2 somatostatin receptors, we show important conformation-dependent variations in polarized distribution. The underlying mechanisms include lower efficiency of conformation-dependent GPCR endocytosis in axons, compared with dendrites, particularly at moderate activation levels, as well as endocytosis-dependent transcytotic delivery of GPCRs from the somatodendritic domain to distal axonal portions, shown by using compartmentalized microfluidic devices. Kinetic modeling predicted that GPCR distribution polarity is highly regulated by steady-state endocytosis, which is conformation dependent and is able to regulate the relative amount of GPCRs targeted to axons and that axonally polarized distribution is an intermediary phenotype that appears at moderate basal activation levels. Indeed, we experimentally show that gradual changes in basal activation-dependent endocytosis lead to highly correlated shifts of polarized GPCR distribution on the neuronal surface, which can even result in a fully reversed polarized distribution of naturally somatodendritic or axonal GPCRs. In conclusion, polarized distribution of neuronal GPCRs may have a pharmacologically controllable component, which, in the absence of dominant targeting motifs, could even represent the principal regulator of sub-neuronal distribution. Consequently, chronic modifications of basal GPCR activation by therapeutic or abused drugs may lead to previously unanticipated changes in brain function through perturbation of polarized GPCR distribution on the neuronal surface.

Activation-dependent subcellular distribution patterns of CB1 cannabinoid receptors in the rat forebrain.

Chronic cannabinoid exposure results in tolerance due to region-specific desensitization and down-regulation of CB1 cannabinoid receptors (CB1Rs). For most G-protein-coupled receptors, internalization closely follows rapid desensitization as an important component of long-term down-regulation. However, in vivo patterns of CB1R internalization are not known. Here we investigate the subcellular redistribution of CB1Rs in the rat forebrain following activation by agonist CP55 940 or inhibition by antagonist/inverse agonist AM251. At steady state, CB1Rs are mainly localized to the cell membrane of preterminal axon shafts and, to a lesser degree, to synaptic terminals. A high proportion of CB1Rs is also localized to somatodendritic endosomes. Inhibition of basal activation by acute AM251 administration decreases the number of cell bodies containing CB1R-immunoreactive endosomes, suggesting that CB1Rs are permanently activated and internalized at steady state. On the contrary, acute agonist treatment induces rapid and important increase of endosomal CB1R immunolabeling, likely due to internalization and retrograde transport of axonal CB1Rs. Repeated agonist treatment is necessary to significantly reduce initially high levels of axonal CB1R labeling, in addition to increasing somatodendritic endosomal CB1R labeling in cholecystokinin-positive interneurons. This redistribution displays important region-specific differences; it is most pronounced in the neocortex and hippocampus and absent in basal ganglia.

Axonal targeting of the 5-HT1B serotonin receptor relies on structure-specific constitutive activation.

By analogy to other axonal proteins, transcytotic delivery following spontaneous endocytosis from the somatodendritic membrane is expected to be essential for polarized distribution of axonal G-protein coupled receptors (GPCRs). However, possible contribution from constitutive activation, which may also result in constitutive GPCR endocytosis, is poorly known. Using two closely related but differentially distributed serotonin receptors, here we demonstrate higher constitutive activation and spontaneous endocytosis for the axonal 5-HT(1B) R, as compared to the somatodendritic 5-HT(1A) R, both in non-neuronal cells and neurons. Activation-dependent constitutive endocytosis is crucial for axonal targeting, because inverse-agonist treatment, which prevents constitutive activation, leads to atypical accumulation of newly synthesized 5-HT(1B) Rs on the somatodendritic plasma membrane. Using receptor chimeras composed of different domains from 5-HT(1A) R and 5-HT(1B) R, we show that the complete third intracellular loop of 5-HT(1B) R is necessary and sufficient for constitutive activation and efficient axonal targeting, both sensitive to inverse-agonist treatment. These results suggest that activation and targeting of 5-HT(1B) Rs are intimately interconnected in neurons.

NOS1AP regulates dendrite patterning of hippocampal neurons through a carboxypeptidase E-mediated pathway.

During neuronal development, neurons form elaborate dendritic arbors that receive signals from axons. Additional studies are needed to elucidate the factors regulating the establishment of dendritic patterns. Our work explored possible roles played by nitric oxide synthase 1 adaptor protein (NOS1AP; also known as C-terminal PDZ ligand of neuronal nitric oxide synthase or CAPON) in dendritic patterning of cultured hippocampal neurons. Here we report that the long isoform of NOS1AP (NOS1AP-L) plays a novel role in regulating dendrite outgrowth and branching. NOS1AP-L decreases dendrite number when overexpressed at any interval between day in vitro (DIV) 0 and DIV 12, and knockdown of NOS1AP-L results in increased dendrite number. In contrast, the short isoform of NOS1AP (NOS1AP-S) decreases dendrite number only when overexpressed during DIV 5-7. Using mutants of NOS1AP-L, we show that neither the PDZ-binding domain nor the PTB domain is necessary for the effects of NOS1AP-L. We have functionally narrowed the region of NOS1AP-L that mediates this effect to the middle amino acids 181-307, a region that is not present in NOS1AP-S. Furthermore, we performed a yeast two-hybrid screen and identified carboxypeptidase E (CPE) as a binding partner for the middle region of NOS1AP-L. Biochemical and cellular studies reveal that CPE mediates the effects of NOS1AP on dendrite morphology. Together, our results suggest that NOS1AP-L plays an important role in the initiation, outgrowth, and maintenance of dendrites during development.

Targeting of the 5-HT1A serotonin receptor to neuronal dendrites is mediated by Yif1B.

The 5-HT(1A) receptor (5-HT(1A)R) is the most extensively characterized serotonin (5-HT) receptor mainly because of its involvement in the mode of action of antidepressants. The 5-HT(1A)R is confined to the somatodendritic domain of central neurons, where it mediates serotonin-evoked hyperpolarization. Our previous studies underlined the role of the short 5-HT(1A)R C-terminal domain in receptor targeting to dendrites. We used this 17 aa region as bait in a yeast two-hybrid screen, and identified, for the first time, an intracellular protein interacting with the 5-HT(1A)R. This protein is homologous to the yeast Yif1p, previously implicated in vesicular trafficking between the endoplasmic reticulum (ER) and the Golgi apparatus, but not yet characterized in mammals. We confirmed 5-HT(1A)R-Yif1B interaction by glutathione S-transferase pull-down experiments using rat brain extracts and transfected cell lines. Yif1B is highly expressed in the brain, and specifically in raphe 5-HT(1A)R-expressing neurons. Colocalization of Yif1B and 5-HT(1A)R was observed in small vesicles involved in transient intracellular trafficking. Last, inhibition of endogenous expression of Yif1B in primary neuron cultures by small interfering RNA specifically prevented the addressing of 5-HT(1A)R to distal portions of the dendrites, without affecting other receptors, such as sst2A, P2X(2), and 5-HT(3A) receptors. Together, our results provide strong evidence that Yif1B is a member of the ER/Golgi trafficking machinery, which plays a key role in specific targeting of 5-HT(1A)R to the neuronal dendrites. This finding opens up new pathways for the study of 5-HT(1A)R regulation by partner proteins and for the development of novel antidepressant drugs.

Role of the C-terminal di-leucine motif of 5-HT1A and 5-HT1B serotonin receptors in plasma membrane targeting.

The 5-HT1A and 5-HT1B serotonin receptors exhibit different subcellular localizations in neurons. Evidence has been reported that the C-terminal domain is involved in the somato-dendritic and axonal targeting of 5-HT1AR and 5-HT1BR, respectively. Here we analyzed the consequences of the mutation of a di-leucine motif and palmitoylated cysteines within this domain. Replacement of I414-I415 by a di-alanine in 5-HT1AR led to endoplasmic reticulum (ER) sequestration of the corresponding mutant expressed in cell lines as well as in hippocampal neurons in culture. Furthermore, di-leucine-mutated receptors were unable to bind 5-HT1A agonists and presented a major deficit in their glycosylation state, suggesting that they are misfolded. By contrast, mutation of the di-leucine motif in the C-terminal domain of 5-HT1BR had no major consequence on its subcellular targeting. However, in the case of the 1ActB chimera (substitution of the C-terminal domain of the 5-HT1BR into 5-HT1AR), this mutation was also found to cause sequestration within the ER. Replacement of palmitoylated cysteines by serines had no consequence on either receptor type. These data indicate that the di-leucine motif of the 5-HT1AR and 5-HT1BR tails is implicated in proper folding of these receptors, which is necessary for their ER export.

Constitutive endocytic cycle of the CB1 cannabinoid receptor.

The CB1 cannabinoid receptor (CB1R) displays a significant level of ligand-independent (i.e. constitutive) activity, either when heterologously expressed in nonneuronal cells or in neurons where CB1Rs are endogenous. The present study investigates the consequences of constitutive activity on the intracellular trafficking of CB1R. When transfected in HEK-293 cells, CB1R is present at the plasma membrane, but a substantial proportion ( approximately 85%) of receptors is localized in intracellular vesicles. Detailed analysis of CB1-EGFP expressed in HEK-293 cells shows that the intracellular CB1R population is mostly of endocytic origin and that treatment with inverse agonist AM281 traps CB1R at the plasma membrane through a monensin-sensitive recycling pathway. Co-transfection with dominant positive or dominant negative mutants of the small GTPases Rab5 and Rab4, but not Rab11, profoundly modifies the steady-state and ligand-induced intracellular distribution of CB1R, indicating that constitutive endocytosis is Rab5-dependent, whereas constitutive recycling is mediated by Rab4. In conclusion, our results indicate that, due to its natural constitutive activity, CB1R permanently and constitutively cycles between plasma membrane and endosomes, leading to a predominantly intracellular localization at steady state.