Scientiae forum / models and speculations pathways for axonal targeting of membrane proteins

The distribution of proteins to the correct domains of neurons is crucial for neuronal functioning. Here I discuss possible mechanisms underlying sorting to the axonal plasma membrane which differ with respect to the steps providing selectivity for axonal vs. somatodendritic cargo. Selectivity can be executed at one or more than one step, including sorting into distinct carriers in the Golgi, preferential transport along axonal microtubules, fusion with the plasma membrane, and importantly retrieval by endocytosis. A protein might in fact be sorted multiple times at different places to achieve axonal localization.     

AP-4: autophagy-four mislocalized proteins in axons

Neurons are highly polarized cells composed of two distinct domains, the axon and the somatodendritic domain. Although AMPA-type glutamate receptors, which mediate fast excitatory neurotransmission in the vertebrate CNS, are preferentially expressed in the somatodendritic domain, the molecular mechanisms underlying such polarized distribution have remained elusive. We recently demonstrated that adaptor protein complex-4 (AP-4) binds to transmembrane AMPA receptor regulatory proteins (TARPs), thereby mediating the selective trafficking of AMPA receptors to the somatodendritic domain; genetic disruption of AP-4 (AP-4beta(-/-)), results in the mislocalization of TARPs and AMPA receptors in the axons. Similarly, low-density lipoprotein receptors and delta2 glutamate receptors are mislocalized in axons, while other cargos, such as NMDA receptors and metabotropic glutamate receptors, are properly excluded from AP-4beta(-/-) axons. These findings indicate that there exist AP-4-dependent and -independent sorting mechanisms. Unexpectedly, mislocalized AMPA receptors do not reach the cell surface and accumulate in autophagosomes in the bulging portions of AP-4beta(-/-) axons. Several lines of evidence indicate that mislocalized AMPA receptors activate the autophagic pathway. Since increased autophagy and axonal swelling are suggested to occur in various neuronal disorders, further studies using AP-4beta(-/-) mice are warranted to understand the mechanisms regulating autophagy in axons.     

Neuronal polarity.

The axonal and somatodendritic domains of neurons differ in their cytoskeletal and membrane composition, complement of organelles, and capacity for macromolecular synthesis. Recently there has been progress in elucidating the cellular mechanisms that underlie the establishment and maintenance of neuronal polarity, including microtubule organization and the sorting, transport, and anchoring of membrane proteins.     

The somatodendritic endosomal regulator NEEP21 facilitates axonal targeting of L1/NgCAM

Correct targeting of proteins to axons and dendrites is crucial for neuronal function. We showed previously that axonal accumulation of the cell adhesion molecule L1/neuron-glia cell adhesion molecule (NgCAM) depends on endocytosis (Wisco, D., E.D. Anderson, M.C. Chang, C. Norden, T. Boiko, H. Folsch, and B. Winckler. 2003. J. Cell Biol. 162:1317-1328). Two endocytosis-dependent pathways to the axon have been proposed: transcytosis and selective retrieval/retention. We show here that axonal accumulation of L1/NgCAM occurs via nondegradative somatodendritic endosomes and subsequent anterograde axonal transport, which is consistent with transcytosis. Additionally, we identify the neuronal-specific endosomal protein NEEP21 (neuron-enriched endosomal protein of 21 kD) as a regulator of L1/NgCAM sorting in somatodendritic endosomes. Down-regulation of NEEP21 leads to missorting of L1/NgCAM to the somatodendritic surface as well as to lysosomes. Importantly, the axonal accumulation of endogenous L1 in young neurons is also sensitive to NEEP21 depletion. We propose that small endosomal carriers derived from somatodendritic recycling endosomes can serve to redistribute a distinct set of membrane proteins from dendrites to axons.     

Neuronal polarity and trafficking

Among the most morphologically complex cells, neurons are masters of membrane specialization. Nowhere is this more striking than in the division of cellular labor between the axon and the dendrites. In morphology, signaling properties, cytoskeletal organization, and physiological function, axons and dendrites (or more properly, the somatodendritic compartment) are radically different. Such polarization of neurons into domains specialized for either receiving (dendrites) or transmitting (axons) cellular signals provides the underpinning for all neural circuitry. The initial specification of axonal and dendritic identity occurs early in neuronal life, persists for decades, and is manifested by the presence of very different sets of cell surface proteins. Yet, how neuronal polarity is established, how distinct axonal and somatodendritic domains are maintained, and how integral membrane proteins are directed to dendrites or accumulate in axons remain enduring and formidable questions in neuronal cell biology.     

Delta/notch-like epidermal growth factor (EGF)-related receptor, a novel EGF-like repeat-containing protein targeted to dendrites of developing and adult central nervous system neurons

We have identified a novel epidermal growth factor (EGF)-like repeat-containing single-pass transmembrane protein that is specifically expressed in the developing and mature central nervous system. Sequence analysis revealed that the 10 EGF-like repeats in the extracellular domain are closely related to those of the developmentally important receptor Notch and its ligand Delta. We thus named the molecule Delta/Notch-like EGF-related receptor (DNER). DNER protein is strongly expressed in several types of post-mitotic neurons, including cortical and hippocampal pyramidal neurons, cerebellar granule cells, and Purkinje cells. DNER protein is localized to the dendritic plasma membrane and endosomes and is excluded from the axons, even when overexpressed. The tyrosine-based sorting motif in the cytoplasmic domain is required for dendritic targeting of DNER. Direct in vivo binding of DNER to the coat-associated protein complex AP-1 strongly suggests that DNER undergoes AP-1-dependent sorting to the somatodendritic compartments from the trans-Golgi network and subsequent passage through the endosomal system.     

The beta-amyloid domain is essential for axonal sorting of amyloid precursor protein.

We have analysed the axonal sorting signals of amyloid precursor protein (APP). Wild-type and mutant versions of human APP were expressed in hippocampal neurons using the Semliki forest virus system. We show that wild-type APP and mutations implicated in Alzheimer's disease and another brain beta-amyloidosis are sorted to the axon. By analysis of deletion mutants we found that the membrane-inserted APP ectodomain but not the cytoplasmic tail is required for axonal sorting. Systematic deletions of the APP ectodomain identified two regions required for axonal delivery: one encoded by exons 11-15 in the carbohydrate domain, the other encoded by exons 16-17 in the juxtamembraneous beta-amyloid domain. Treatment of the cells with the N-glycosylation inhibitor tunicamycin induced missorting of wild-type APP, supporting the importance of glycosylation in axonal sorting of APP. The data revealed a hierarchy of sorting signals on APP: the beta-amyloid-dependent membrane proximal signal was the major contributor to axonal sorting, while N-glycosylation had a weaker effect. Furthermore, recessive somatodendritic signals, most likely in the cytoplasmic tail, directed the protein to the dendrites when the ectodomain was deleted. Analysis of detergent solubility of APP and another axonally delivered protein, hemagglutinin, demonstrated that only hemagglutinin formed CHAPS-insoluble complexes, suggesting distinct mechanisms of axonal sorting for these two proteins. This study is the first delineation of sorting requirements of an axonally targeted protein in polarized neurons and indicates that the beta-amyloid domain plays a major role in axonal delivery of APP.     

Autophagy researchers

Neurons are highly polarized cells composed of two distinct domains, the axon and the somatodendritic domain. Although AMPA-type glutamate receptors, which mediate fast excitatory neurotransmission in the vertebrate CNS, are preferentially expressed in the somatodendritic domain, the molecular mechanisms underlying such polarized distribution have remained elusive. We recently demonstrated that adaptor protein complex-4 (AP-4) binds to transmembrane AMPA receptor regulatory proteins (TARPs), thereby mediating the selective trafficking of AMPA receptors to the somatodendritic domain; genetic disruption of AP-4 (AP-4β^–/–), results in the mislocalization of TARPs and AMPA receptors in the axons. Similarly, low-density lipoprotein receptors and δ2 glutamate receptors are mislocalized in axons, while other cargos, such as NMDA receptors and metabotropic glutamate receptors, are properly excluded from AP-4β^–/– axons. These findings indicate that there exist AP-4-dependent and -independent sorting mechanisms. Unexpectedly, mislocalized AMPA receptors do not reach the cell surface and accumulate in autophagosomes in the bulging portions of AP-4β^–/– axons. Several lines of evidence indicate that mislocalized AMPA receptors activate the autophagic pathway. Since increased autophagy and axonal swelling are suggested to occur in various neuronal disorders, further studies using AP-4β^–/– mice are warranted to understand the mechanisms regulating autophagy in axons.

Addendum to: Matsuda S, Miura E, Matsuda K, Kakegawa W, Kohda K, Watanabe M, Yuzaki M. Accumulation of AMPA receptors in autophagosomes in neuronal axons lacking adaptor protein AP-4. Neuron 2008; 57:730-45.     

Accumulation of AMPA receptors in autophagosomes in neuronal axons lacking adaptor protein AP-4

AP-4 is a member of the adaptor protein complexes, which control vesicular trafficking of membrane proteins. Although AP-4 has been suggested to contribute to basolateral sorting in epithelial cells, its function in neurons is unknown. Here, we show that disruption of the gene encoding the beta subunit of AP-4 resulted in increased accumulation of axonal autophagosomes, which contained AMPA receptors and transmembrane AMPA receptor regulatory proteins (TARPs), in axons of hippocampal neurons and cerebellar Purkinje cells both in vitro and in vivo. AP-4 indirectly associated with the AMPA receptor via TARPs, and the specific disruption of the interaction between AP-4 and TARPs caused the mislocalization of endogenous AMPA receptors in axons of wild-type neurons. These results indicate that AP-4 may regulate proper somatodendritic-specific distribution of its cargo proteins, including AMPA receptor-TARP complexes and the autophagic pathway in neurons.     

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.     

Polarized Traffic of LRP1 Involves AP1B and SNX17 Operating on Y-dependent Sorting Motifs in Different Pathways

Low-density lipoprotein receptor–related protein 1 (LRP1) is an endocytic recycling receptor with two cytoplasmic tyrosine-based basolateral sorting signals. Here we show that during biosynthetic trafficking LRP1 uses AP1B adaptor complex to move from a post-TGN recycling endosome (RE) to the basolateral membrane. Then it recycles basolaterally from the basolateral sorting endosome (BSE) involving recognition by sorting nexin 17 (SNX17). In the biosynthetic pathway, Y₂₉ but not N₂₆ from a proximal NPXY directs LRP1 basolateral sorting from the TGN. A N₂₆A mutant revealed that this NPXY motif recognized by SNX17 is required for the receptor's exit from BSE. An endocytic Y₆₃ATL₆₆ motif also functions in basolateral recycling, in concert with an additional endocytic motif (LL_86,87), by preventing LRP1 entry into the transcytotic apical pathway. All this sorting information operates similarly in hippocampal neurons to mediate LRP1 somatodendritic distribution regardless of the absence of AP1B in neurons. LRP1 basolateral distribution results then from spatially and temporally segregation steps mediated by recognition of distinct tyrosine-based motifs. We also demonstrate a novel function of SNX17 in basolateral/somatodendritic recycling from a different compartment than AP1B endosomes.     

Axonal transport and neuronal transcytosis of trophic factors, tracers, and pathogens

Neurons can specifically internalize macromolecules, such as trophic factors, lectins, toxins, and other pathogens. Upon internalization in terminals, proteins can move retrogradely along axons, or, upon internalization at somatodendritic domains, they can move into an anterograde axonal transport pathway. Release of internalized proteins from neurons after either retrograde or anterograde axonal transport results in transcytosis and trafficking of proteins across multiple synapses. Recent studies of binding properties of several such proteins suggest that pathogens and lectins may utilize existing transport machineries designed for trafficking of trophic factors. Specific pathways may protect trophic factors, pathogens, and toxins from degradation after internalization and may target the trophic or pathogenic cargo for transcytosis after either retrograde or anterograde transport along axons. Elucidating the molecular mechanisms of sorting steps and transport pathways will further our understanding of trophic signaling and could be relevant for an understanding and possible treatment of neurological diseases such as rabies, Alzheimer's disease, and prion encephalopathies. At present, our knowledge is remarkably sparse about the types of receptors used by pathogens for trafficking, the signals that sort trophins or pathogens into recycling or degradation pathways, and the mechanisms that regulate their release from somatodendritic domains or axon terminals. This review intends to draw attention to potential convergences and parallels in trafficking of trophic and pathogenic proteins. It discusses axonal transport/trafficking mechanisms that may help to understand and eventually treat neurological diseases by targeted drug delivery.     

The sorting nexin, DSH3PX1, connects the axonal guidance receptor, Dscam, to the actin cytoskeleton

Dock, an adaptor protein that functions in Drosophila axonal guidance, consists of three tandem Src homology 3 (SH3) domains preceding an SH2 domain. To develop a better understanding of axonal guidance at the molecular level, we used the SH2 domain of Dock to purify a protein complex from fly S2 cells. Five proteins were obtained in pure form from this protein complex. The largest protein in the complex was identified as Dscam (Down syndrome cell adhesion molecule), which was subsequently shown to play a key role in directing neurons of the fly embryo to correct positions within the nervous system (Schmucker, D., Clemens, J. C., Shu, H., Worby, C. A., Xiao, J., Muda, M., Dixon, J. E., and Zipursky, S. L. (2000) Cell 101, 671-684). The smallest protein in this complex (p63) has now been identified. We have named p63 DSH3PX1 because it appears to be the Drosophila orthologue of the human protein known as SH3PX1. DSH3PX1 is comprised of an NH(2)-terminal SH3 domain, an internal PHOX homology (PX) domain, and a carboxyl-terminal coiled-coil region. Because of its PX domain, DSH3PX1 is considered to be a member of a growing family of proteins known collectively as sorting nexins, some of which have been shown to be involved in vesicular trafficking. We demonstrate that DSH3PX1 immunoprecipitates with Dock and Dscam from S2 cell extracts. The domains responsible for the in vitro interaction between DSH3PX1 and Dock were also identified. We further show that DSH3PX1 interacts with the Drosophila orthologue of Wasp, a protein component of actin polymerization machinery, and that DSH3PX1 co-immunoprecipitates with AP-50, the clathrin-coat adapter protein. This evidence places DSH3PX1 in a complex linking cell surface receptors like Dscam to proteins involved in cytoskeletal rearrangements and/or receptor trafficking.     

Phosphatidylinositol-4-kinase type II alpha contains an AP-3-sorting motif and a kinase domain that are both required for endosome traffic

The adaptor complex 3 (AP-3) targets membrane proteins from endosomes to lysosomes, lysosome-related organelles and synaptic vesicles. Phosphatidylinositol-4-kinase type II alpha (PI4KIIalpha) is one of several proteins possessing catalytic domains that regulate AP-3-dependent sorting. Here we present evidence that PI4KIIalpha uniquely behaves both as a membrane protein cargo as well as an enzymatic regulator of adaptor function. In fact, AP-3 and PI4KIIalpha form a complex that requires a dileucine-sorting motif present in PI4KIIalpha. Mutagenesis of either the PI4KIIalpha-sorting motif or its kinase-active site indicates that both are necessary to interact with AP-3 and properly localize PI4KIIalpha to LAMP-1-positive endosomes. Similarly, both the kinase activity and the sorting signal present in PI4KIIalpha are necessary to rescue endosomal PI4KIIalpha siRNA-induced mutant phenotypes. We propose a mechanism whereby adaptors use canonical sorting motifs to selectively recruit a regulatory enzymatic activity to restricted membrane domains.     

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.     

Recycling endosomes in neuronal membrane traffic

Neurons are highly polarized cells with axonal and somatodendritic membrane surfaces that spatially separate signal-sending from signal-receiving membrane domains. As found in many other cell types, different populations of endosomes are involved in the sorting of synaptic and other membrane cargo in neurons. The exact source of the membrane for neurite extension and process remodelling during neuronal differentiation has remained uncertain, and we do not know exactly how polarized sorting of neuronal membrane proteins is achieved. In the present article, we will provide a brief overview of endosomes and their putative or proven functions in fibroblasts, epithelial cells and neurons. On the basis of insights from non-neuronal cell types and recent studies on the function of recycling endosomes during synaptic plasticity-induced membrane remodelling, we postulate a speculative model regarding the role of recycling endosomes in neuronal differentiation.     

Transcytosis of the polymeric immunoglobulin receptor in cultured hippocampal neurons

BACKGROUND: A wide variety of proteins are transported across epithelial cells by vesicular carriers. This process, transcytosis, is used to generate cell surface polarity and to transport macromolecules between the luminal and serosal sides of the epithelial layer. The polymeric immunoglobulin receptor is a well-characterized transcytotic molecule in epithelia. It binds to its ligand, polymeric immunoglobulin, at the basolateral surface, and the receptor-ligand complex is transcytosed to the apical surface, where the ligand is released. Our previous studies have shown that hippocampal neurons may employ mechanisms similar to those of epithelial cells to sort proteins to two plasma membrane domains. The machinery used for axonal delivery recognizes proteins that are targeted apically in epithelia, whereas basolaterally destined proteins are delivered to the dendrites. It has not been clear, however, whether transcytosis occurs in neurons. RESULTS: We report expression of the polymeric immunoglobulin receptor in cultured hippocampal neurons, using a Semliki Forest Virus expression system, and show by immunofluorescence microscopy that the newly synthesized receptor is targeted from the Golgi complex predominantly to the dendrites - only about 20% of the infected neurons display axonal immunofluorescence. Addition of ligand leads to significant redistribution of the receptor to the axons, shown by an approximately three-fold increase in axonal immunoreactivity with the anti-receptor antibodies. CONCLUSIONS: Our results suggest that a transcytotic route, analogous to that in epithelia, exists in neurons, where it transports proteins from the somatodendritic to the axonal domain. Cultured neurons expressing the polymeric immunoglobulin receptor offer an experimental system that should be useful for further characterization of this novel neuronal pathway at the molecular and functional level.     

Axon/dendrite targeting of metabotropic glutamate receptors by their cytoplasmic carboxy-terminal domains

The subcellular targeting of neurotransmitter receptors is vital in controlling polarized information flow in the brain. We show here that metabotropic glutamate receptors are differentially targeted when expressed from defective viral vectors in cultured hippocampal neurons; mGluR1a and mGluR2 are targeted to dendrites and excluded from axons, whereas mGluR7 is targeted to axons and dendrites. Chimeras and deletions revealed that axon exclusion of mGluR2 versus axon targeting of mGluR7 is mediated by their 60 amino acid C-terminal cytoplasmic domains. Addition of the mGluR7 C-terminal sequence to mGluR2 or to the unrelated somatodendritic protein telencephalin (tln) induced axon targeting, indicating dominance of the axonal signal. These mGluR sorting signals represent novel plasma membrane axon/dendrite targeting signals.     

Myosin VI is required for sorting of AP-1B-dependent cargo to the basolateral domain in polarized MDCK cells

In polarized epithelial cells, newly synthesized membrane proteins are delivered on specific pathways to either the apical or basolateral domains, depending on the sorting motifs present in these proteins. Because myosin VI has been shown to facilitate secretory traffic in nonpolarized cells, we investigated its role in biosynthetic trafficking pathways in polarized MDCK cells. We observed that a specific splice isoform of myosin VI with no insert in the tail domain is required for the polarized transport of tyrosine motif containing basolateral membrane proteins. Sorting of other basolateral or apical cargo, however, does not involve myosin VI. Site-directed mutagenesis indicates that a functional complex consisting of myosin VI, optineurin, and probably the GTPase Rab8 plays a role in the basolateral delivery of membrane proteins, whose sorting is mediated by the clathrin adaptor protein complex (AP) AP-1B. Our results suggest that myosin VI is a crucial component in the AP-1B-dependent biosynthetic sorting pathway to the basolateral surface in polarized epithelial cells.     

AP-1A and AP-3A lysosomal sorting functions

Heterotetrameric adaptor-protein complexes AP-1A and AP-3A mediate protein sorting in post-Golgi vesicular transport. AP-1A and AP-3A have been localized to the trans -Golgi network, indicating a function in protein sorting at this compartment. AP-3A appears to mediate trans -Golgi network-to-lysosome and also endosome-to-lysosome protein sorting. AP-1A is thought to be required for both trans -Golgi network-to-endosome transport and endosome-to- trans -Golgi network transport. However, the recent discovery of a role for monomeric GGA (Golgi localized γ-ear containing, ARF binding protein) adaptor proteins in trans -Golgi network to endosome protein transport has brought into question the long-discussed trans -Golgi network-to-endosome sorting function of AP-1A. Murine cytomegalovirus gp48 contains an unusual di-leucine-based lysosome sorting signal motif and mediates lysosomal sorting of gp48/major histocompatibility complex class I receptor complexes, preventing exposure of major histocompatibility complex class I at the plasma membrane. We analyzed lysosomal sorting of gp48/major histocompatibility complex class I receptor complexes in cell lines deficient for AP-1A, AP-3A and both, to determine their sorting functions. We find that AP1-A and AP3-A mediate distinct and sequential steps in the lysosomal sorting. Both sorting functions are required to prevent MHC class I exposure at the plasma membrane at steady-state.