Chan Choo Yap

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.

Ankyrin-dependent and -independent mechanisms orchestrate axonal compartmentalization of L1 family members neurofascin and L1/neuron-glia cell adhesion molecule.

Axonal initial segments (IS) and nodes of Ranvier are functionally important membrane subdomains in which the clustering of electrogenic channels enables action potential initiation and propagation. In addition, the initial segment contributes to neuronal polarity by serving as a diffusion barrier. To study the mechanisms of axonal compartmentalization, we focused on two L1 family of cell adhesion molecules (L1-CAMs) [L1/neuron–glia cell adhesion molecule (L1/NgCAM) and neurofascin (NF)] and two neuronal ankyrins (ankB and ankG). NF and ankG accumulate specifically at the initial segment, whereas L1/NgCAM and ankB are expressed along the entire lengths of axons. We find that L1/NgCAM and NF show distinct modes of steady-state accumulation during axon outgrowth in cultured hippocampal neurons. Despite their different steady-state localizations, both L1/NgCAM and NF show slow diffusion and low detergent extractability specifically in the initial segment but fast diffusion and high detergent extractability in the distal axon. We propose that L1-CAMs do not strongly bind ankB in the distal axon because of spatial regulation of ankyrin affinity by phosphorylation. NF, conversely, is initially enriched in an ankyrin-independent manner in the axon generally and accumulates progressively in the initial segment attributable to preferential binding to ankG. Our results suggest that NF and L1/NgCAM accumulate in the axon by an ankyrin-independent pathway, but retention at the IS requires ankyrin binding.

Harnessing the Power of the Endosome to Regulate Neural Development

Endocytosis and endosomal trafficking play a multitude of roles in cellular function beyond regulating entry of essential nutrients. In this review, we discuss the cell biological principles of endosomal trafficking, the neuronal adaptations to endosomal organization, and the role of endosomal trafficking in neural development. In particular, we consider how cell fate decisions, polarity, migration, and axon outgrowth and guidance are influenced by five endosomal tricks: dynamic modulation of receptor levels by endocytosis and recycling, cargo-specific responses via cargo-specific endocytic regulators, cell-type-specific endocytic regulation, ligand-specific endocytic regulation, and endosomal regulation of ligand processing and trafficking.

Chapter 7 Compartmentalizing the Neuronal Plasma Membrane: From Axon Initial Segments to Synapses

Many membrane proteins localize to restricted domains in neurons, such as axons, dendrites, synapses, or axon initial segments. The exquisite subcellular compartmentalization of adhesion molecules, growth factor receptors, signaling receptors, voltage-gated and ligand-gated channels, and others underlies the complex functioning of neurons and ultimately vectorial propagation of signaling in neuronal circuits. This chapter discusses the cellular mechanisms for compartmentalizing the neuronal plasma membrane. Among the mechanisms contributing to protein segregation in the membrane are sorting and targeting in the Golgi/TGN, endocytosis, recycling, and degradation, and control of membrane protein diffusion. The molecular underpinnings of these cellular mechanisms are reviewed in the first part. The second part discusses the contribution of each cellular mechanism to targeting proteins to axons and dendrites, to synapses, to axon initial segments, and to Nodes of Ranvier. For most, if not all proteins and locations, all four mechanisms are in effect and additively contribute to the precise localization of membrane proteins in neurons. Since disruption of proper protein distribution results in defects in neuronal function and can lead to neurodegenerative diseases, a full understanding of the cellular mechanisms of plasma membrane compartmentalization is an important goal for the future.

Pathway selection to the axon depends on multiple targeting signals in NgCAM

Similar to most differentiated cells, both neurons and epithelial cells elaborate distinct plasma membrane domains that contain different membrane proteins. We have previously shown that the axonal cell-adhesion molecule L1/NgCAM accumulates on the axonal surface by an indirect transcytotic pathway via somatodendritic endosomes. MDCK epithelial cells similarly traffic NgCAM to the apical surface by transcytosis. In this study, we map the signals in NgCAM required for routing via the multi-step transcytotic pathway. We identify both a previously mapped tyrosine-based signal as a sufficient somatodendritic targeting signal, as well as a novel axonal targeting signal in the cytoplasmic tail of NgCAM. The axonal signal is glycine and serine rich, but only the glycine residues are required for activity. The somatodendritic signal is cis-dominant and needs to be inactivated in order for the axonal signal to be executed. Additionally, we show that the axonal cytoplasmic signal promotes apical targeting in MDCK cells. Transcytosis of NgCAM to the axon thus requires the sequential regulated execution of multiple targeting signals.

Mechanisms by which a CACNA1H mutation in epilepsy patients increases seizure susceptibility

Mutations in the Cav3.2 T‐type Ca2+ channel were found in patients with idiopathic generalized epilepsies, yet the mechanisms by which these mutations increase neuronal excitability and susceptibility to seizures remain to be determined. Using electrophysiological and transfection methods, we validate in cultured hippocampal neurons the hypothesis that an epilepsy mutation increases neuronal excitability. Mutations in the I–II loop of the channel increase trafficking to the plasma membrane without altering trafficking into dendrites. Mutations also enhance dendritic arborization. Additionally, we provide the first evidence that Cav3.2 can signal to Ca2+‐regulated transcription factors, which are known to play important roles in neuronal development and gene expression.

Neuronal early endosomes require EHD1 for L1/NgCAM trafficking.

In neurons, the endosomal system is essential for membrane receptor trafficking to dendrites and axons and thereby participates in various neuronal functions, such as neurite outgrowth and synaptic plasticity. A multitude of regulators coordinates trafficking through endosomes, but most of them have not been studied in detail in neurons. In non-neuronal cells, EHD1 (Eps15 homology-domain containing protein 1) functions in the recycling endosome and is required for endosome-to-plasma membrane transport of multiple cargos. In this study, we analyze the role of EHD1 in neurons. In particular, we investigate whether EHD1 is required for polarized trafficking of the dendritically targeted transferrin and the axonal adhesion molecule L1/NgCAM (neuron–glia cell adhesion molecule) and, if so, in what compartment it is required. We find that endosomal recycling of both L1/NgCAM and transferrin is impaired when EHD1 is downregulated. We show that EHD1 colocalizes with L1/NgCAM and transferrin mostly in EEA1 (early endosome antigen 1)-positive early endosomes and less extensively with recycling endosomes. Using live imaging, we observe that EHD1 is stably associated with endosomal membranes during their maturation into EEA1-positive compartments and often persists on them longer than EEA1. Finally, we show that downregulation of EHD1 causes a delay of L1/NgCAM in exiting EEA1-positive endosomes, resulting in impaired targeting of L1/NgCAM to the axonal membrane. We conclude that, in neurons, EHD1 functions in early endosomes rather than (or possibly in addition to) recycling endosomes. These findings point to the existence of neuronal adaptations of the endosomal system.

Doublecortin (Dcx) family proteins regulate filamentous actin structure in developing neurons

Doublecortin (Dcx) is the causative gene for X-linked lissencephaly, which encodes a microtubule-binding protein. Axon tracts are abnormal in both affected individuals and in animal models. To determine the reason for the axon tract defect, we performed a semiquantitative proteomic analysis of the corpus callosum in mice mutant for Dcx. In axons from mice mutant for Dcx, widespread differences are found in actin-associated proteins as compared with wild-type axons. Decreases in actin-binding proteins α-actinin-1 and α-actinin-4 and actin-related protein 2/3 complex subunit 3 (Arp3), are correlated with dysregulation in the distribution of filamentous actin (F-actin) in the mutant neurons with increased F-actin around the cell body and decreased F-actin in the neurites and growth cones. The actin distribution defect can be rescued by full-length Dcx and further enhanced by Dcx S297A, the unphosphorylatable mutant, but not with the truncation mutant of Dcx missing the C-terminal S/P-rich domain. Thus, the C-terminal region of Dcx dynamically regulates formation of F-actin features in developing neurons, likely through interaction with spinophilin, but not through α-actinin-4 or Arp3. We show with that the phenotype of Dcx/Doublecortin-like kinase 1 deficiency is consistent with actin defect, as these axons are selectively deficient in axon guidance, but not elongation.

Endocytosis and Endosomes at the Crossroads of Regulating Trafficking of Axon Outgrowth-Modifying Receptors

In neurons, many receptors must be localized correctly to axons or dendrites for proper function. During development, receptors for nerve growth and guidance are targeted to axons and localized to growth cones where receptor activation by ligands results in promotion or inhibition of axon growth. Signaling outcomes downstream of ligand binding are determined by the location, levels and residence times of receptors on the neuronal plasma membrane. Therefore, the mechanisms controlling the trafficking of these receptors are crucial to the proper wiring of circuits. Membrane proteins accumulate on the axonal surface by multiple routes, including polarized sorting in the trans Golgi network, sorting in endosomes and removal by endocytosis. Endosomes also play important roles in the signaling pathways for both growth‐promoting and ‐inhibiting molecules: signaling endosomes derived from endocytosis are important for signaling from growth cones to cell bodies. Growth‐promoting neurotrophins and growth‐inhibiting Nogo‐A can use EHD4/Pincher‐dependent endocytosis at the growth cone for their respective retrograde signaling. In addition to retrograde transport of endosomes, anterograde transport to axons in endosomes also occurs for several receptors, including the axon outgrowth‐promoting cell adhesion molecule L1/NgCAM and TrkA. L1/NgCAM also depends on EHD4/Pincher‐dependent endocytosis for its axonal polarization. In this review, we will focus on receptors whose trafficking has been reported to be modulated by the EHD4/Pincher family of endosomal regulators, namely L1/NgCAM, Trk and Nogo‐A. We will first summarize the pathways underlying the axonal transport of these proteins and then discuss the potential roles of EHD4/Pincher in mediating their endocytosis.

Doublecortin (DCX) mediates endocytosis of neurofascin independently of microtubule-binding

Doublecortin on X chromosome (DCX) is one of two major genetic loci underlying human lissencephaly, a neurodevelopmental disorder with defects in neuronal migration and axon outgrowth. DCX is a microtubule-binding protein, and much work has focused on its microtubule-associated functions. DCX has other reported binding partners, including the cell adhesion molecule neurofascin, but the functional significance of the DCX–neurofascin interaction is not understood. Neurofascin localizes strongly to the axon initial segment in mature neurons, where it plays a role in assembling and maintaining other axon initial segment components. During development, neurofascin likely plays additional roles in axon guidance and in GABAergic synaptogenesis. We show here that DCX can modulate the surface distribution of neurofascin in developing cultured rat neurons and thereby the relative extent of accumulation between the axon initial segment and soma and dendrites. Mechanistically, DCX acts via increasing endocytosis of neurofascin from soma and dendrites. Surprisingly, DCX increases neurofascin endocytosis apparently independently of its microtubule-binding activity. We additionally show that the patient allele DCXG253D still binds microtubules but is deficient in promoting neurofascin endocytosis. We propose that DCX acts as an endocytic adaptor for neurofascin to fine-tune its surface distribution during neuronal development.