Collapse of late endosomal pH elicits a rapid Rab7 response via the V-ATPase and RILP.

Endosomal-lysosomal trafficking is accompanied by the acidification of endosomal compartments by the H+-V-ATPase to reach low lysosomal pH. Disruption of the correct pH impairs lysosomal function and the balance of protein synthesis and degradation (proteostasis). Here, we treated mammalian cells with the small dipeptide LLOMe, which is known to permeabilize lysosomal membranes, and find that LLOMe also impacts late endosomes (LEs) by neutralizing their pH without causing membrane permeabilization. We show that LLOMe leads to hyperactivation of Rab7 (herein referring to Rab7a), and disruption of tubulation and mannose-6-phosphate receptor (CI-M6PR; also known as IGF2R) recycling on pH-neutralized LEs. pH neutralization (NH4Cl) and expression of Rab7 hyperactive mutants alone can both phenocopy the alterations in tubulation and CI-M6PR trafficking. Mechanistically, pH neutralization increases the assembly of the V1G1 subunit (encoded by ATP6V1G1) of the V-ATPase on endosomal membranes, which stabilizes GTP-bound Rab7 via RILP, a known interactor of Rab7 and V1G1. We propose a novel pathway by which V-ATPase and RILP modulate LE pH and Rab7 activation in concert. This pathway might broadly contribute to pH control during physiologic endosomal maturation or starvation and during pathologic pH neutralization, which occurs via lysosomotropic compounds and in disease states.

Disruption of Golgi markers by two RILP-directed shRNAs in neurons: A new role for RILP or a neuron-specific off-target phenotype?

In neurons, degradation of dendritic cargos requires RAB7 and dynein-mediated retrograde transport to somatic lysosomes. To test if the dynein adapter RAB-interacting lysosomal protein (RILP) mediated the recruitment of dynein to late endosomes for retrograde transport in dendrites, we obtained several knockdown reagents previously validated in non-neuronal cells. Striking endosomal phenotypes elicited by one shRILP plasmid were not reproduced by another one. Furthermore, we discovered a profound depletion of Golgi/TGN markers for both shRILP plasmids. This Golgi disruption was only observed in neurons and could not be rescued by re-expression of RILP. This Golgi phenotype was also not found in neurons treated with siRILP or gRILP/Cas9. Lastly, we tested if a different RAB protein that interacts with RILP, namely the Golgi-associated RAB34, might be responsible for the loss of Golgi markers. Expression of a dominant-negative RAB34 did indeed cause changes in Golgi staining in a small subset of neurons but manifested as fragmentation rather than loss of staining. Unlike in non-neuronal cells, interference with RAB34 did not cause dispersal of lysosomes in neurons. Based on multiple lines of experimentation, we conclude that the neuronal Golgi phenotype observed with shRILP is likely off-target in this cell type specifically. Any observed disruptions of endosomal trafficking caused by shRILP in neurons might thus be downstream of Golgi disruption. It would be interesting to identify the actual target for this neuronal Golgi phenotype. Cell type-specific off-target phenotypes therefore likely occur in neurons, necessitating revalidation of reagents that were previously validated in other cell types.

Dynein Is Required for Rab7-Dependent Endosome Maturation, Retrograde Dendritic Transport, and Degradation.

In all cell types, endocytosed cargo is transported along a set of endosomal compartments, which are linked maturationally from early endosomes (EEs) via late endosomes (LEs) to lysosomes. Lysosomes are critical for degradation of proteins that enter through endocytic as well as autophagic pathways. Rab7 is the master regulator of early-to-late endosome maturation, motility, and fusion with lysosomes. We previously showed that most degradative lysosomes are localized in the soma and in the first 25 µm of the dendrite and that bulk degradation of dendritic membrane proteins occurs in/near the soma. Dendritic late endosomes therefore move retrogradely in a Rab7-dependent manner for fusion with somatic lysosomes. We now used cultured E18 rat hippocampal neurons of both sexes to determine which microtubule motor is responsible for degradative flux of late endosomes. Based on multiple approaches (inhibiting dynein/dynactin itself or inhibiting dynein recruitment to endosomes by expressing the C-terminus of the Rab7 effector, RILP), we now demonstrate that net retrograde flux of late endosomes in dendrites is supported by dynein. Inhibition of dynein also delays maturation of somatic endosomes, as evidenced by excessive accumulation of Rab7. In addition, degradation of dendritic cargos is inhibited. Our results also suggest that GDP-GTP cycling of Rab7 appears necessary not only for endosomal maturation but also for fusion with lysosomes subsequent to arrival in the soma. In conclusion, Rab7-dependent dynein/dynactin recruitment to dendritic endosomes plays multifaceted roles in dendritic endosome maturation as well as retrograde transport of late endosomes to sustain normal degradative flux.SIGNIFICANCE STATEMENT Lysosomes are critical for degradation of membrane and extracellular proteins that enter through endocytosis. Lysosomes are also the endpoint of autophagy and thus responsible for protein and organelle homeostasis. Endosomal-lysosomal dysfunction is linked to neurodegeneration and aging. We identify roles in dendrites for two proteins with links to human diseases, Rab7 and dynein. Our previous work identified a process that requires directional retrograde transport in dendrites, namely, efficient degradation of short-lived membrane proteins. Based on multiple approaches, we demonstrate that Rab7-dependent recruitment of dynein motors supports net retrograde transport to lysosomes and is needed for endosome maturation. Our data also suggest that GDP-GTP cycling of Rab7 is required for fusion with lysosomes and degradation, subsequent to arrival in the soma.

Spatial regulation of endosomes in growing dendrites.

Many membrane proteins are highly enriched in either dendrites or axons. This non-uniform distribution is a critical feature of neuronal polarity and underlies neuronal function. The molecular mechanisms responsible for polarized distribution of membrane proteins has been studied for some time and many answers have emerged. A less well studied feature of neurons is that organelles are also frequently non-uniformly distributed. For instance, EEA1-positive early endosomes are somatodendritic whereas synaptic vesicles are axonal. In addition, some organelles are present in both axons and dendrites, but not distributed uniformly along the processes. One well known example are lysosomes which are abundant in the soma and proximal dendrite, but sparse in the distal dendrite and the distal axon. The mechanisms that determine the spatial distribution of organelles along dendrites are only starting to be studied. In this review, we will discuss the cell biological mechanisms of how the distribution of diverse sets of endosomes along the proximal-distal axis of dendrites might be regulated. In particular, we will focus on the regulation of bulk homeostatic mechanisms as opposed to local regulation. We posit that immature dendrites regulate organelle motility differently from mature dendrites in order to spatially organize dendrite growth, branching and sculpting.

Nestin Selectively Facilitates the Phosphorylation of the Lissencephaly-Linked Protein Doublecortin (DCX) by cdk5/p35 to Regulate Growth Cone Morphology and Sema3a Sensitivity in Developing Neurons.

Nestin, an intermediate filament protein widely used as a marker of neural progenitors, was recently found to be expressed transiently in developing cortical neurons in culture and in developing mouse cortex. In young cortical cultures, nestin regulates axonal growth cone morphology. In addition, nestin, which is known to bind the neuronal cdk5/p35 kinase, affects responses to axon guidance cues upstream of cdk5, specifically, to Sema3a. Changes in growth cone morphology require rearrangements of cytoskeletal networks, and changes in microtubules and actin filaments are well studied. In contrast, the roles of intermediate filament proteins in this process are poorly understood, even in cultured neurons. Here, we investigate the molecular mechanism by which nestin affects growth cone morphology and Sema3a sensitivity. We find that nestin selectively facilitates the phosphorylation of the lissencephaly-linked protein doublecortin (DCX) by cdk5/p35, but the phosphorylation of other cdk5 substrates is not affected by nestin. We uncover that this substrate selectivity is based on the ability of nestin to interact with DCX, but not with other cdk5 substrates. Nestin thus creates a selective scaffold for DCX with activated cdk5/p35. Last, we use cortical cultures derived from Dcx KO mice to show that the effects of nestin on growth cone morphology and on Sema3a sensitivity are DCX-dependent, thus suggesting a functional role for the DCX-nestin complex in neurons. We propose that nestin changes growth cone behavior by regulating the intracellular kinase signaling environment in developing neurons. The sex of animal subjects is unknown.SIGNIFICANCE STATEMENT Nestin, an intermediate filament protein highly expressed in neural progenitors, was recently identified in developing neurons where it regulates growth cone morphology and responsiveness to the guidance cue Sema3a. Changes in growth cone morphology require rearrangements of cytoskeletal networks, but the roles of intermediate filaments in this process are poorly understood. We now report that nestin selectively facilitates phosphorylation of the lissencephaly-linked doublecortin (DCX) by cdk5/p35, but the phosphorylation of other cdk5 substrates is not affected. This substrate selectivity is based on preferential scaffolding of DCX, cdk5, and p35 by nestin. Additionally, we demonstrate a functional role for the DCX-nestin complex in neurons. We propose that nestin changes growth cone behavior by regulating intracellular kinase signaling in developing neurons.

A dominant dendrite phenotype caused by the disease-associated G253D mutation in doublecortin (DCX) is not due to its endocytosis defect.

Doublecortin (DCX) is a protein needed for cortical development, and DCX mutations cause cortical malformations in humans. The microtubule-binding activity of DCX is well-described and is important for its function, such as supporting neuronal migration and dendrite growth during development. Previous work showed that microtubule binding is not sufficient for DCX-mediated promotion of dendrite growth and that domains in DCX's C terminus are also required. The more C-terminal regions of DCX bind several other proteins, including the adhesion receptor neurofascin and clathrin adaptors. We recently identified a role for DCX in endocytosis of neurofascin. The disease-associated DCX-G253D mutant protein is known to be deficient in binding neurofascin, and we now asked if disruption of neurofascin endocytosis underlies the DCX-G253D-associated pathology. We first demonstrated that DCX functions in endocytosis as a complex with both the clathrin adaptor AP-2 and neurofascin: disrupting either clathrin adaptor binding (DCX-ALPA) or neurofascin binding (DCX-G253D) decreased neurofascin endocytosis in primary neurons. We then investigated a known function for DCX, namely, increasing dendrite growth in cultured neurons. Surprisingly, we found that the DCX-ALPA and DCX-G253D mutants yield distinct dendrite phenotypes. Unlike DCX-ALPA, DCX-G253D caused a dominant-negative dendrite growth phenotype. The endocytosis defect of DCX-G253D thus was separable from its detrimental effects on dendrite growth. We recently identified Dcx-R59H as a dominant allele and can now classify Dcx-G253D as a second Dcx allele that acts dominantly to cause pathology, but does so via a different mechanism.

Different Doublecortin (DCX) Patient Alleles Show Distinct Phenotypes in Cultured Neurons: EVIDENCE FOR DIVERGENT LOSS-OF-FUNCTION AND "OFF-PATHWAY" CELLULAR MECHANISMS.

Doublecortin on the X-chromosome (DCX) is a neuronal microtubule-binding protein with a multitude of roles in neurodevelopment. In humans, DCX is a major genetic locus for X-linked lissencephaly. The best studied defects are in neuronal migration during corticogenesis and in the hippocampus, as well as axon and dendrite growth defects. Much effort has been directed at understanding the molecular and cellular bases of DCX-linked lissencephaly. The focus has been in particular on defects in microtubule assembly and bundling, using knock-out mice and expression of WT and mutant Dcx in non-neuronal cells. Dcx also binds other proteins besides microtubules, such as spinophilin (abbreviated spn; gene name Ppp1r9b protein phosphatase 1 regulatory subunit 9b) and the clathrin adaptors AP-1 and AP-2. Even though many non-sense and missense mutations of Dcx are known, their molecular and cellular defects are still only incompletely understood. It is also largely unknown how neurons are affected by expression of DCX patient alleles. We have now characterized several patient DCX alleles (DCX-R89G, DCX-R59H, DCX-246X, DCX-272X, and DCX-303X) using a gain-of-function dendrite growth assay in cultured rat neurons in combination with the determination of molecular binding activities and subcellular localization in non-neuronal and neuronal cells. First, we find that several mutants (Dcx-R89G and Dcx-272X) were loss-of-function alleles (as had been postulated) but surprisingly acted via different cellular mechanisms. Second, one allele (Dcx-R59H) formed cytoplasmic aggregates, which contained Hspa1B (heat shock protein 1B hsp70) and ubiquitinated proteins, trapped other cytoskeletal proteins, including spinophilin, and led to increased autophagy. This allele could thus be categorized as "off-pathway"/possibly neomorph. Our findings thus suggested that distinct DCX alleles caused dysfunction by different mechanisms.

Maturational conversion of dendritic early endosomes and their roles in L1-mediated axon growth.

The function of endosomes is intricately linked to cellular function in all cell types, including neurons. Intriguingly, neurons express cell type-specific proteins that localize to endosomes, but little is known about how these neuronal proteins interface with canonical endosomes and ubiquitously expressed endosomal components, such as EEA1 (Early Endosomal Antigen 1). NEEP21 (Neuronal Early Endosomal Protein 21 kDa) localizes to somatodendritic endosomes, and downregulation of NEEP21 perturbs the correct trafficking of multiple receptors, including glutamate receptors (GluA2) during LTP and amyloidogenic processing of ßAPP. Our own work implicated NEEP21 in correct trafficking of the axonal cell adhesion molecule L1/neuron-glia cell adhesion molecule (NgCAM). NEEP21 dynamically localizes with EEA1-positive early endosomes but is also found in EEA1-negative endosomes. Live imaging reveals that NEEP21-positive, EEA1-negative endosomes arise as a consequence of maturational conversion of EEA1/NEEP21 double-positive endosomes. Interfering with EEA1 function causes missorting of L1/NgCAM, axon outgrowth defects on the L1 substrate, and disturbance of NEEP21 localization. Last, we uncover evidence that functional interference with NEEP21 reduces axon and dendrite growth of primary rat hippocampal neurons on L1 substrate but not on N-cadherin substrate, thus implicating endosomal trafficking through somatodendritic early endosomes in L1-mediated axon growth.

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.

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

T-type calcium channels play essential roles in regulating neuronal excitability and network oscillations in the brain. Mutations in the gene encoding Cav3.2 T-type Ca(2+) channels, CACNA1H, have been found in association with various forms of idiopathic generalized epilepsy. We and others have found that these mutations may influence neuronal excitability either by altering the biophysical properties of the channels or by increasing their surface expression. The goals of the present study were to investigate the excitability of neurons expressing Cav3.2 with the epilepsy mutation, C456S, and to elucidate the mechanisms by which it influences neuronal properties. We found that expression of the recombinant C456S channels substantially increased the excitability of cultured neurons by increasing the spontaneous firing rate and reducing the threshold for rebound burst firing. Additionally, we found that molecular determinants in the I-II loop (the region in which most childhood absence epilepsy-associated mutations are found) substantially increase the surface expression of T-channels but do not alter the relative distribution of channels into dendrites of cultured hippocampal neurons. Finally, we discovered that expression of C456S channels promoted dendritic growth and arborization. These effects were reversed to normal by either the absence epilepsy drug ethosuximide or a novel T-channel blocker, TTA-P2. As Ca(2+)-regulated transcription factors also increase dendritic development, we tested a transactivator trap assay and found that the C456S variant can induce changes in gene transcription. Taken together, our findings suggest that gain-of-function mutations in Cav3.2 T-type Ca(2+) channels increase seizure susceptibility by directly altering neuronal electrical properties and indirectly by changing gene expression.

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.

Endosomal Transport to Lysosomes and the Trans-Golgi Network in Neurons and Other Cells: Visualizing Maturational Flux.

High-level microscopy enables the comprehensive study of dynamic intracellular processes. Here we describe a toolkit of combinatorial approaches for fixed cell imaging and live cell imaging to investigate the interactions along the trans-Golgi network (TGN)-endosome-lysosome transport axis, which underlie the maturation of endosomal compartments and degradative flux. For fixed cell approaches, we specifically highlight how choices of permeabilization conditions, antibody selection, and antibody multiplexing affect interpretation of results. For live cell approaches, we emphasize the use of sensors that read out pH and degradative capacity in combination with endosomal identity for elucidating dynamic compartment changes.

"Disruption of Golgi markers by two RILP-directed shRNAs in neurons: a new role for RILP or a neuron-specific off-target phenotype?"

In neurons, degradation of dendritic cargos requires RAB7 and dynein-mediated retrograde transport to somatic lysosomes. In order to test if the dynein adaptor RILP (RAB-interacting lysosomal protein) mediated the recruitment of dynein to late endosomes for retrograde transport in dendrites, we obtained several knockdown reagents which had been previously validated in non-neuronal cells. We found that striking endosomal phenotypes elicited by one shRILP plasmid were not reproduced by another one. Furthermore, we discovered a profound depletion of Golgi/TGN markers for both shRILP plasmids. This Golgi disruption was only observed in neurons and could not be rescued by re-expression of RILP. This Golgi phenotype was also not found in neurons treated with siRILP or gRILP/Cas9. Lastly, we tested if a different RAB protein that interacts with RILP, namely the Golgi-associated RAB34, might be responsible for the loss of Golgi markers. Expression of a dominant-negative RAB34 did indeed cause changes in Golgi staining in a small subset of neurons but manifested as fragmentation rather than loss of markers. Unlike in non-neuronal cells, interference with RAB34 did not cause dispersal of lysosomes in neurons. Based on multiple lines of experimentation, we conclude that the neuronal Golgi phenotype observed with shRILP is likely off-target in this cell type specifically. Any observed disruptions of endosomal trafficking caused by shRILP in neurons might thus be downstream of Golgi disruption. Different approaches will be needed to test if RILP is required for late endosomal transport in dendrites. Cell type-specific off-target phenotypes therefore likely occur in neurons, making it prudent to re-validate reagents that were previously validated in other cell types.

Quantifying Single and Dual Channel Live Imaging Data: Kymograph Analysis of Organelle Motility in Neurons.

Live imaging is commonly used to study dynamic processes in cells. Many labs carrying out live imaging in neurons use kymographs as a tool. Kymographs display time-dependent microscope data (time-lapsed images) in two-dimensional representations showing position vs. time. Extraction of quantitative data from kymographs, often done manually, is time-consuming and not standardized across labs. We describe here our recent methodology for quantitatively analyzing single color kymographs. We discuss the challenges and solutions of reliably extracting quantifiable data from single-channel kymographs. When acquiring in two fluorescent channels, the challenge becomes analyzing two objects that may co-traffic together. One must carefully examine the kymographs from both channels and decide which tracks are the same or try to identify the coincident tracks from an overlay of the two channels. This process is laborious and time consuming. The difficulty in finding an available tool for such analysis has led us to create a program to do so, called KymoMerge. KymoMerge semi-automates the process of identifying co-located tracks in multi-channel kymographs and produces a co-localized output kymograph that can be analyzed further. We describe our analysis, caveats, and challenges of two-color imaging using KymoMerge.

Expected and unexpected roles for dynein regulation of dendritic late endosomes.

Dendrites differ from axons in multiple ways, including the presence of minus-end out microtubules intermixed with the more conventional plus-end out microtubules. The mixed microtubule polarity makes regulation of directional transport in dendrites a challenge. Dynein can in principle be a retrograde and anterograde motor in dendrites. We show in our recent paper that dynein supports bi-directional transport of late endosomes in dendrites. We also show that overexpression of the RAB7 effector RILP which recruits dynein to late endosomes imparts retrograde bias onto late endosomes. Inhibition of dynein leads to a decrease in bi-directional motility of late endosomes, an expected result. Unexpectedly, inhibition of dynein also impairs endosome maturation as evidenced by increased association of GTP-RAB7 with late endosomes. Ultimately, dynein inhibition causes degradation defects of short-lived dendritic receptors and stunted dendrite morphologies. Much more work is required to fully understand how endosomal pathways are regulated in time and space in dendrites. Given the prevalence of neurological disorders where endosome-lysosome functions are impaired, this is a topic of great translational relevance.

Dynamics and distribution of endosomes and lysosomes in dendrites.

All cells are filled with membrane-bound organelles which are responsible for the synthesis and transport as well as degradation of membrane proteins. The localization of these organelles inside cells is highly regulated. The regulation of organelle positioning has been widely studied in many cell types. In neurons, organelle positioning and its regulation is of particular interest because of the enormous size of neurons and the high spatial heterogeneity of different functional domains, such as axons, proximal and distal portions of dendrites, and synapses. We will discuss new discoveries with regard to the dynamic positioning of endosomes and lysosomes between soma and along dendrites. Just as the "how" of dynamic endosome/lysosome positioning is still being investigated, the "why" is also being explored. An exciting possibility is that synaptic activity influences organelle behaviors. We will discuss what is currently known about the how and the why of endosome/lysosome dynamics in dendrites.

Global loss of Neuron-specific gene 1 causes alterations in motor coordination, increased anxiety, and diurnal hyperactivity in male mice.

The Neuron-specific gene family (NSG1-3) consists of small endolysosomal proteins that are critical for trafficking multiple receptors and signaling molecules in neurons. NSG1 has been shown to play a critical role in AMPAR recycling from endosomes to plasma membrane during synaptic plasticity. However, to date nothing is known about whether NSG1 is required for normal behavior at an organismal level. Here we performed a battery of behavioral tests to determine whether loss of NSG1 would affect motor, cognitive, and/or affective behaviors, as well as circadian-related activity. Consistent with unique cerebellar expression of NSG1 among family members, we found that NSG1 was obligatory for motor coordination but not for gross motor function or learning. NSG1 knockout (KO) also altered performance across other behavioral modalities including anxiety-related and diurnal activity paradigms. Surprisingly, NSG1 KO did not cause significant impairments across all tasks within a given modality, but had specific effects within each modality. For instance, we found increases in anxiety-related behaviors in tasks with multiple stressors (e.g., elevation and exposure), but not those with a single main stressor (e.g., exposure). Interestingly, NSG1 KO animals displayed a significant increase in locomotor activity during subjective daytime, suggesting a possible impact on diurnal activity rhythms or vigilance. Surprisingly, loss of NSG1 had no effect on hippocampal-dependent learning despite previous studies showing deficits in CA1 long-term potentiation. Together, these findings do not support a role of NSG1 in hippocampal-dependent learning, but support a role in mediating proper neuronal function across amygdalar and cerebellar circuits.

Alterations of EHD1/EHD4 protein levels interfere with L1/NgCAM endocytosis in neurons and disrupt axonal targeting.

Axon growth is regulated by many proteins, including adhesion molecules, which need to be trafficked correctly to axons. The adhesion molecule L1/neuron-glia cell adhesion molecule (NgCAM) travels to axons via an endocytosis-dependent pathway (transcytosis), traversing somatodendritic endosomes. The Eps15 homology domain (EHD) family proteins (EHD1-EHD4) play important roles in endosomal recycling and possibly in endocytosis. We investigated whether EHD1 regulates L1/NgCAM trafficking in neurons. Both short hairpin-mediated downregulation and overexpression of EHD1 led to dendritic mistargeting of NgCAM. Downregulation of EHD1 showed increased endosomal accumulation of NgCAM, whereas, surprisingly, overexpression of EHD1 led to impairment of L1/NgCAM internalization in neurons but not in fibroblasts. Transferrin internalization, however, was unaffected. At longer overexpression times of EHD1, NgCAM endocytosis returned to normal, suggesting rapid upregulation of compensatory endocytic pathways. EHD1 is capable of hetero-oligomerization, and an endogenous complex of EHD1 and EHD4 was identified previously. We therefore tested whether short-term overexpression of other EHD family members showed a similar endocytosis defect. Expression of EHD4, but not of EHD3, also caused a defect in L1/NgCAM endocytosis. Oligomerization of EHD1 was required to cause NgCAM endocytosis defects, and simultaneous expression of EHD1 and EHD4 rescued NgCAM endocytosis. Therefore, balanced levels of EHD1-EHD4 are important for NgCAM endocytosis in neurons. Our data suggest that EHD1 plays roles in both endosomal recycling and a specialized endocytosis pathway in neurons used by NgCAM. We propose that EHD1 and EHD4 act as hetero-oligomeric complexes in this pathway.

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.