Somatodendritic Plasma Membrane Research Articles

Transcytosis and trans-synaptic retention by postsynaptic ErbB4 underlie axonal accumulation of NRG3.

Neuregulins (NRGs) are EGF-like ligands associated with cognitive disorders. Unprocessed proNRG3 is cleaved by BACE1 to generate the mature membrane-bound NRG3 ligand, but the subcellular site of proNRG3 cleavage, mechanisms underlying its transport into axons, and presynaptic accumulation remain unknown. Using an optogenetic proNRG3 cleavage reporter (LA143-NRG3), we investigate the spatial-temporal dynamics of NRG3 processing and sorting in neurons. In dark conditions, unprocessed LA143-NRG3 is retained in the trans-Golgi network but, upon photoactivation, is cleaved by BACE1 and released from the TGN. Mature NRG3 then emerges on the somatodendritic plasma membrane from where it is re-endocytosed and anterogradely transported on Rab4+ vesicles into axons via transcytosis. By contrast, the BACE1 substrate APP is sorted into axons on Rab11+ vesicles. Lastly, by a mechanism we denote "trans-synaptic retention," NRG3 accumulates at presynaptic terminals by stable interaction with its receptor ErbB4 on postsynaptic GABAergic interneurons. We propose that trans-synaptic retention may account for polarized expression of other neuronal transmembrane ligands and receptors.

Trafficking of Kv2.1 Channels to the Axon Initial Segment by a Novel Nonconventional Secretory Pathway.

Kv2.1 is a major delayed-rectifier voltage-gated potassium channel widely expressed in neurons of the CNS. Kv2.1 localizes in high-density cell-surface clusters in the soma and proximal dendrites as well as in the axon initial segment (AIS). Given the crucial roles of both of these compartments in integrating signal input and then generating output, this localization of Kv2.1 is ideal for regulating the overall excitability of neurons. Here we used fluorescence recovery after photobleaching imaging, mutagenesis, and pharmacological interventions to investigate the molecular mechanisms that control the localization of Kv2.1 in these two different membrane compartments in cultured rat hippocampal neurons of mixed sex. Our data uncover a unique ability of Kv2.1 channels to use two molecularly distinct trafficking pathways to accomplish this. Somatodendritic Kv2.1 channels are targeted by the conventional secretory pathway, whereas axonal Kv2.1 channels are targeted by a nonconventional trafficking pathway independent of the Golgi apparatus. We further identified a new AIS trafficking motif in the C-terminus of Kv2.1, and show that putative phosphorylation sites in this region are critical for the restricted and clustered localization in the AIS. These results indicate that neurons can regulate the expression and clustering of Kv2.1 in different membrane domains independently by using two distinct localization mechanisms, which would allow neurons to precisely control local membrane excitability.SIGNIFICANCE STATEMENT Our study uncovered a novel mechanism that targets the Kv2.1 voltage-gated potassium channel to two distinct trafficking pathways and two distinct subcellular destinations: the somatodendritic plasma membrane and that of the axon initial segment. We also identified a distinct motif, including putative phosphorylation sites, that is important for the AIS localization. This raises the possibility that the destination of a channel protein can be dynamically regulated via changes in post-translational modification, which would impact the excitability of specific membrane compartments.

The SNARE Protein Syntaxin 3 Confers Specificity for Polarized Axonal Trafficking in Neurons.

Cell polarity and precise subcellular protein localization are pivotal to neuronal function. The SNARE machinery underlies intracellular membrane fusion events, but its role in neuronal polarity and selective protein targeting remain unclear. Here we report that syntaxin 3 is involved in orchestrating polarized trafficking in cultured rat hippocampal neurons. We show that syntaxin 3 localizes to the axonal plasma membrane, particularly to axonal tips, whereas syntaxin 4 localizes to the somatodendritic plasma membrane. Disruption of a conserved N-terminal targeting motif, which causes mislocalization of syntaxin 3, results in coincident mistargeting of the axonal cargos neuron-glia cell adhesion molecule (NgCAM) and neurexin, but not transferrin receptor, a somatodendritic cargo. Similarly, RNAi-mediated knockdown of endogenous syntaxin 3 leads to partial mistargeting of NgCAM, demonstrating that syntaxin 3 plays an important role in its targeting. Additionally, overexpression of syntaxin 3 results in increased axonal growth. Our findings suggest an important role for syntaxin 3 in maintaining neuronal polarity and in the critical task of selective trafficking of membrane protein to axons.

Transport along the dendritic endoplasmic reticulum mediates the trafficking of GABAB receptors.

In neurons, secretory organelles within the cell body are complemented by the dendritic endoplasmic reticulum (ER) and Golgi outposts (GOPs), whose role in neurotransmitter receptor trafficking is poorly understood. γ-aminobutyric acid (GABA) type B metabotropic receptors (GABABRs) regulate the efficacy of synaptic transmission throughout the brain. Their plasma membrane availability is controlled by mechanisms involving an ER retention motif and assembly-dependent ER export. Thus, they constitute an ideal molecular model to study ER trafficking, but the extent to which the dendritic ER participates in GABABR biosynthesis has not been thoroughly explored. Here, we show that GABAB1 localizes preferentially to the ER in dendrites and moves long distances within this compartment. Not only diffusion but also microtubule and dynein-dependent mechanisms control dendritic ER transport. GABABRs insert throughout the somatodendritic plasma membrane but dendritic post-ER carriers containing GABABRs do not fuse selectively with GOPs. This study furthers our understanding of the spatial selectivity of neurotransmitter receptors for dendritic organelles.

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.

Selective Participation of Somatodendritic HCN Channels in Inhibitory But Not Excitatory Synaptic Integration in Neurons of the Subthalamic Nucleus

The activity patterns of subthalamic nucleus (STN) neurons are intimately linked to motor function and dysfunction and arise through the complex interaction of intrinsic properties and inhibitory and excitatory synaptic inputs. In many neurons, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play key roles in intrinsic excitability and synaptic integration both under normal conditions and in disease states. However, in STN neurons, which strongly express HCN channels, their roles remain relatively obscure. To address this deficit, complementary molecular and cellular electrophysiological, imaging, and computational approaches were applied to the rat STN. Molecular profiling demonstrated that individual STN neurons express mRNA encoding several HCN subunits, with HCN2 and 3 being the most abundant. Light and electron microscopic analysis showed that HCN2 subunits are strongly expressed and distributed throughout the somatodendritic plasma membrane. Voltage-, current-, and dynamic-clamp analysis, two-photon Ca(2+) imaging, and computational modeling revealed that HCN channels are activated by GABA(A) receptor-mediated inputs and thus limit synaptic hyperpolarization and deinactivation of low-voltage-activated Ca(2+) channels. Although HCN channels also limited the temporal summation of EPSPs, generated through two-photon uncaging of glutamate, this action was largely shunted by GABAergic inhibition that was necessary for HCN channel activation. Together the data demonstrate that HCN channels in STN neurons selectively counteract GABA(A) receptor-mediated inhibition arising from the globus pallidus and thus promote single-spike activity rather than rebound burst firing.

Rapid Delivery of Internalized Signaling Receptors to the Somatodendritic Surface by Sequence-Specific Local Insertion

The recycling pathway is a major route for delivering signaling receptors to the somatodendritic plasma membrane. We investigated the cell biological basis for the remarkable selectivity and speed of this process. We focused on the mu-opioid neuropeptide receptor and the beta(2)-adrenergic catecholamine receptor, two seven-transmembrane signaling receptors that traverse the recycling pathway efficiently after ligand-induced endocytosis and localize at steady state throughout the postsynaptic surface. Rapid recycling of each receptor in dissociated neuronal cultures was mediated by a receptor-specific cytoplasmic sorting sequence. Total internal reflection fluorescence microscopy imaging revealed that both sequences drive recycling via discrete vesicular fusion events in the cell body and dendritic shaft. Both sequences promoted recycling via "transient"-type events characterized by nearly immediate lateral spread of receptors after vesicular insertion resembling receptor insertion events observed previously in non-neural cells. The sequences differed in their abilities to produce distinct "persistent"-type events at which inserted receptors lingered for a variable time period before lateral spread. Both types of insertion event generated a uniform distribution of receptors in the somatodendritic plasma membrane when imaged over a 1 min interval, but persistent events uniquely generated a punctate surface distribution over a 10 s interval. These results establish sequence-directed recycling of signaling receptors in CNS neurons and show that this mechanism has the ability to generate receptor-specific patterns of local surface distribution on a timescale overlapping that of rapid physiological signaling.

Endogenous SNAP-25 Regulates Native Voltage-gated Calcium Channels in Glutamatergic Neurons

In addition to its primary role as a fundamental component of the SNARE complex, SNAP-25 also modulates voltage-gated calcium channels (VGCCs) in various overexpression systems. Although these studies suggest a potential negative regulatory role of SNAP-25 on VGCC activity, the effects of endogenous SNAP-25 on native VGCC function in neurons are unclear. In the present study, we investigated the VGCC properties of cultured glutamatergic and GABAergic rat hippocampal neurons. Glutamatergic currents were dominated by P/Q-type channels, whereas GABAergic cells had a dominant L-type component. Also, glutamatergic VGCC current densities were significantly lower with enhanced inactivation rates and shifts in the voltage dependence of activation and inactivation curves compared with GABAergic cells. Silencing endogenous SNAP-25 in glutamatergic neurons did not alter P/Q-type channel expression or localization but led to increased VGCC current density without changes in the VGCC subtype proportions. Isolation of the P/Q-type component indicated that increased current in the absence of SNAP-25 was correlated with a large depolarizing shift in the voltage dependence of inactivation. Overexpressing SNAP-25 in GABAergic neurons reduced current density without affecting the VGCC subtype proportion. Accordingly, VGCC current densities in glutamatergic neurons from Snap-25(+/-) mice were significantly elevated compared with wild type glutamatergic neurons. Overall, this study demonstrates that endogenous SNAP-25 negatively regulates native VGCCs in glutamatergic neurons which could have important implications for neurological diseases associated with altered SNAP-25 expression.

Receptor Watching Enabled

Yudowski et al. report that they have been able to observe insertion of single receptor-containing vesicles into the plasma membrane of cultured hippocampal neurons from rats. The authors accomplished this by expressing in the cells a modified form of the β 2 -adrenergic receptor (β 2 AR) in which the extracellular end of the receptor was tagged with a variant of the green fluorescent protein (GFP). The fluorescence of this particular GFP protein is quenched when exposed to the acidic environment of endocytic vesicles but increases when exposed to culture medium of pH 7.4 at the cell surface. The vesicle fusion events appeared to represent recycling of internalized receptors to the cell surface because they were detected only after exposure of the cells to the β 2 AR agonist isoproterenol (which causes receptor internalization and subsequent reinsertion in the plasma membrane). Two distinct modes of insertion were detected: In one, spots of membrane insertion were visible transiently for an average duration of 340 ms, whereas another class of event showed persistence of about 31 s. Receptors in both categories subsequently appeared to disperse laterally within the membrane with a rate of 3 x 10 –10 square centimeters per second. The β 2 AR stimulates adenylyl cyclase, thus causing accumulation of the second messenger cyclic AMP (adenosine 3′,5′-monophosphate) and activation of protein kinase A (PKA). In cells previously stimulated with isoproterenol for 10 min, inhibition of PKA or removal of the isoproterenol signal rapidly (within the next minute) increased the frequency of the transient insertion events. The more stable insertion events were not affected by inhibition of PKA, but their frequency decreased if the receptor agonist was removed. The functional consequences of these distinct modes of insertion are not yet understood, but the authors propose that they are important for homeostatic regulation of signaling by β 2 ARs and emphasize that trafficking events that modulate receptor abundance may be subject to complex regulation like that controlling neurotransmitter release. G. A. Yudowski, M. A. Puthenveedu, M. von Zastrow, Distinct modes of regulated receptor insertion to the somatodendritic plasma membrane. Nat. Neurosci. 9 , 622-627 (2006). [Online Journal]

Two ways for insertion

When inserting surface receptors into the somatodendritic plasma membrane, neurons have a choice. Their decision determines how long receptors linger at the point of insertion, as shown by Guillermo Yudowski, Manojkumar Puthenveedu, and Mark von Zastrow (University of California, San Francisco, CA

Distinct modes of regulated receptor insertion to the somatodendritic plasma membrane

Many neural signaling receptors are regulated by endocytosis, but little is known about receptor insertion into the plasma membrane. Time-lapse imaging of the beta2 adrenergic receptor expressed in cultured rat hippocampal neurons, using pH-sensitive green fluorescent protein tagging and total internal reflection fluorescence microscopy, resolved distinct vesicular fusion events mediating receptor insertion into the somatodendritic plasma membrane. A 'transient' insertion mode resulted in rapid lateral dispersion of receptors immediately after insertion. A 'persistent' insertion mode resulted in the retention of inserted receptors in surface-accessible domains, which were relatively immobile for a prolonged 'wait' period before dispersing laterally. Distinct insertion modes were oppositely regulated by receptor activation and by mechanisms differing in their dependence on the signaling effector cyclic AMP-dependent protein kinase. These results reveal a new mechanism for homeostatic regulation of postsynaptic signaling and a 'kiss-and-wait' mode of regulated membrane protein insertion in neurons.

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.

Similar diffusibility of membrane proteins across the axon–soma and dendrite–soma boundaries revealed by a novel FRAP technique

In polarized mature neurons, the asymmetrical distribution of proteins between axonal and somatodendritic plasma membrane (PM) domains may be maintained by a diffusion barrier at the axon–soma boundary. At the boundary, a complex containing membrane-associated and cytoskeletal proteins is formed, anchoring axonal membrane proteins and indirectly hindering the diffusion of other membrane proteins. We examined the latter case, i.e., secondary diffusion impedance by comparing the mobility of fluorescently labeled membrane proteins within the axon–soma and dendrite–soma boundaries. We performed fluorescence recovery after photobleaching (FRAP) experiments using mature cultured hippocampal neurons that had been labeled specifically at their PMs with fluorescent proteins (FPs). The maturation of these neurons was confirmed by immunolocalization with Ankyrin-G, which is thought to participate in the creation of the diffusion barrier at the axon–soma boundary. We developed a wide-field microscope equipped with a device (digital micromirror device) composed of 1024 × 768 binary mirrors at the field-stop, allowing free control of the illumination area and intensity. After the FPs in peripheral processes were photobleached, nonbleached FPs diffused into all the processes at equivalent speeds. These results indicate that the secondary diffusion barrier to exogenously overexpressed membrane proteins is not specific to the axon–soma boundary.

Uncovering multiple axonal targeting pathways in hippocampal neurons.

Neuronal polarity is, at least in part, mediated by the differential sorting of membrane proteins to distinct domains, such as axons and somata/dendrites. We investigated the pathways underlying the subcellular targeting of NgCAM, a cell adhesion molecule residing on the axonal plasma membrane. Following transport of NgCAM kinetically, surprisingly we observed a transient appearance of NgCAM on the somatodendritic plasma membrane. Down-regulation of endocytosis resulted in loss of axonal accumulation of NgCAM, indicating that the axonal localization of NgCAM was dependent on endocytosis. Our data suggest the existence of a dendrite-to-axon transcytotic pathway to achieve axonal accumulation. NgCAM mutants with a point mutation in a crucial cytoplasmic tail motif (YRSL) are unable to access the transcytotic route. Instead, they were found to travel to the axon on a direct route. Therefore, our results suggest that multiple distinct pathways operate in hippocampal neurons to achieve axonal accumulation of membrane proteins.

Neuronal polarity: controlling the sorting and diffusion of membrane components.

Neuronal polarity: controlling the sorting and diffusion of membrane components.

Clusters of GABAA receptors on cultured hippocampal cells correlate only partially with functional synapses

We describe a method to label gamma-aminobutyric acid (GABA)A receptors on the surface of living hippocampal neurons in primary culture, and we compare the distribution of receptors with that of active synapses. To visualize GABAA receptors, the affinity-purified antibody beta3(1-13), recognizing the extracellular N-termini of the GABAA receptor beta2- and beta3-subunits, was used in combination with fluorescent secondary antibodies. The beta2- and beta3-subunits belong to the predominant GABAA receptor subunits in the hippocampus. As expected for aggregates of GABAA receptors in the somato-dendritic plasma membrane, a patchy staining pattern similar to that seen by labelling neurons after fixation was obtained. An antiserum recognizing an intracellular epitope of GABAA receptor beta3-subunits did not label the receptors in living neurons. Whole-cell recordings of GABA-evoked Cl - currents were not affected after decorating GABAA receptors with antibody beta3(1-13). Combining the staining of GABAA receptors with the labelling of active presynaptic terminals with the fluorescent dyes FM1-43 or FM4-64, consistently resulted in the detection of GABAA receptor clusters that were not located at active synapses. These amounted to approximately 50% of all labelled GABAA receptor clusters. GABAA receptor clusters that were not associated with active presynaptic terminals partially colocalized with the synaptic vesicle marker protein sv2, while another fraction had no presynaptic counterpart at all. These findings suggest the presence of presynaptically silent GABAergic synapses in cultured hippocampal neurons. They also indicate that for the maintenance of GABAA receptor aggregates, the release of GABA from an opposing active terminal is not essential.

A diffusion barrier maintains distribution of membrane proteins in polarized neurons.

The asymmetric distribution of proteins to distinct domains in the plasma membrane is crucial to the function of many polarized cells. In epithelia, distinct apical and basolateral surfaces are maintained by tight junctions that prevent diffusion of proteins and lipids between the two domains. Polarized neurons maintain axonal and somatodendritic plasma membrane domains without an obvious physical barrier. Indeed, the artificial lipid Dil encounters no diffusion barrier at the presumptive domain boundary, the axon hillock. By measuring the lateral mobility of membrane proteins using optical tweezers, we show here that some membrane proteins exhibit markedly reduced mobility in the initial segment of the axon. Disruption of F-actin and low levels of dimethyl sulphoxide (DMSO) abolish this diffusion barrier and lead to redistribution of membrane markers that had previously been polarized. Immobilization in the initial segment may reflect, at least in part, differential tethering to cytoskeletal components. Therefore, the ability to maintain a polarized distribution of membrane proteins depends on a specialized domain at the initial segment of the axon, which restricts lateral mobility and serves as a new type of diffusion barrier that acts in the absence of cell-cell contact.

Polarized distribution of the viral glycoproteins of vesicular stomatitis, fowl plague and Semliki Forest viruses in hippocampal neurons in culture: a light and electron microscopic study

We have shown previously using immunofluorescence microscopy that upon injection of polarized hippocampal cells in culture with vesicular stomatitis virus (VSV) and fowl plague virus (FPV) the VSV glycoprotein is delivered to the plasma membrane of the dendrites and of the cell body whereas the FPV hemagglutinin is transported to the axonal surface ( Cell, 62 (1990) 63–72). In this work electron microscopy of infected rat hippocampal neurons showed that VSV progeny budded from the plasma membrane of the dendrites and the cell body. The location of the budding virions corresponded to the distribution of the VSV glycoprotein which was detected over the somatodendritic plasma membrane by immunoelectron microscopy. In contrast, no FPV formation was seen in the infected neurons although the FPV hemagglutinin was localized to the axonal surface by immunoelectron microscopy. In Semliki Forest virus (SFV) infected hippocampal cells we observed that the viral glycoproteins were exclusivelly present in the dendrites and cell body but not in axons.