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.

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.

"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.

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.

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.

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.

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.

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.

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.

Lysosomes to the rescue: Anterograde axonal lysosome transport and neuronal proteostasis.

Niemann-Pick is a lysosomal storage disease caused by loss of the lysosomal cholesterol exporter NPC1 and leads to axon degeneration. Roney et al. report that immature autophagosomes accumulate in axons because cholesterol-laden lysosomes in the soma are not transported to the axon for autophagosome fusion and maturation because they aberrantly sequester non-functioning kinesin-1.

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.

Degradation of dendritic cargos requires Rab7-dependent transport to somatic lysosomes.

Neurons are large and long lived, creating high needs for regulating protein turnover. Disturbances in proteostasis lead to aggregates and cellular stress. We characterized the behavior of the short-lived dendritic membrane proteins Nsg1 and Nsg2 to determine whether these proteins are degraded locally in dendrites or centrally in the soma. We discovered a spatial heterogeneity of endolysosomal compartments in dendrites. Early EEA1-positive and late Rab7-positive endosomes are found throughout dendrites, whereas the density of degradative LAMP1- and cathepsin (Cat) B/D-positive lysosomes decreases steeply past the proximal segment. Unlike in fibroblasts, we found that the majority of dendritic Rab7 late endosomes (LEs) do not contain LAMP1 and that a large proportion of LAMP1 compartments do not contain CatB/D. Second, Rab7 activity is required to mobilize distal predegradative LEs for transport to the soma and terminal degradation. We conclude that the majority of dendritic LAMP1 endosomes are not degradative lysosomes and that terminal degradation of dendritic cargos such as Nsg1, Nsg2, and DNER requires Rab7-dependent transport in LEs to somatic lysosomes.

Transcytosis of TrkA leads to diversification of dendritic signaling endosomes.

The development of the peripheral nervous system relies on long-distance signaling from target organs back to the soma. In sympathetic neurons, this long-distance signaling is mediated by target derived Nerve Growth Factor (NGF) interacting with its axonal receptor, TrkA. This ligand receptor complex internalizes into what is commonly referred to as the signaling endosome which is transported retrogradely to the soma and dendrites to mediate survival signaling and synapse formation, respectively. The molecular identity of signaling endosomes in dendrites has not yet been determined. Here, we perform a detailed analysis of TrkA endosomal compartments and trafficking patterns. We find that signaling endosomes are not uniform but molecularly diversified into Rab7 (late endosome) and Rab11 (recycling endosome) populations in axons and dendrites in vitro and in the soma in vivo. Surprisingly, TrkA-NGF signaling endosomes in dendrites undergo dynamic trafficking events, including putative fusion and fission. Overall, we find that signaling endosomes do not remain as a singular endosomal subtype but instead exist in multiple populations that undergo dynamic endosomal trafficking events. These dynamic events might drive functional diversification of the signaling endosome.

The endosomal neuronal proteins Nsg1/NEEP21 and Nsg2/P19 are itinerant, not resident proteins of dendritic endosomes.

Membrane traffic critically regulates most aspects of neuronal function. Neurons express many neuronal-specific proteins that regulate membrane traffic, including the poorly understood small transmembrane proteins neural-specific gene 1 and 2 (Nsg1/NEEP21 and Nsg2/P19). Nsg1 has been implicated in regulating endosomal recycling and sorting of several important neuronal receptors. Nsg2 is largely unstudied. At steady-state, Nsg1 and Nsg2 only partially co-localize with known endosomal compartments, and it was suggested that they mark a neuronal-specific endosome. Since Nsg1 localizes to and functions in the dendritic endosome, we set out to discover how Nsg1 and Nsg2 localization to endosomes is regulated in primary rat hippocampal neurons, using quadruple immunolocalization against endogenous proteins, live imaging of dendritic endosomes, and interference approaches against the endosomal regulators Rab5 and Rab7. In contrast to previous conclusions, we now show that Nsg1 and Nsg2 are not resident endosomal proteins, but traffic rapidly from the cell surface to lysosomes and have a half-life of less than two hours. Their partial co-localization with canonical endosomal markers thus reflects their rapid flux towards degradation rather than specific targeting to a singular compartment. These findings will require rethinking of how this class of endosomal proteins regulates trafficking of much longer-lived receptors.

The related neuronal endosomal proteins NEEP21 (Nsg1) and P19 (Nsg2) have divergent expression profiles in vivo.

Endosomal maturation and transport constitutes a complex trafficking system present in all cell types. Neurons have adapted their endosomal system to meet their unique and complex needs. These adaptations include repurposing existing proteins to diversify endocytosis and trafficking, as well as preferential expression of certain regulators more highly in neurons than other cell types. These neuronal regulators include the family of Neuron-Specific Gene family members (Nsg), NEEP21 (Nsg1), and P19 (Nsg2). NEEP21/Nsg1 plays a role in the trafficking of multiple receptors, including the cell adhesion molecule L1/NgCAM, the neurotransmitter receptor GluA2, and ß-APP. Recently, we showed that NEEP2/Nsg1 and P19/Nsg2 are not expressed in all neuronal cell types in vitro. However, it is not known where and when NEEP21/Nsg1 and P19/Nsg2 are expressed in vivo, and whether both proteins are always coexpressed. Here, we show that NEEP21/Nsg1 and P19/Nsg2 are present in both overlapping and distinct cell populations in the hippocampus, neocortex, and cerebellum during development. NEEP21/Nsg1 and P19/Nsg2 levels are highest during embryonic development, and expression persists in the juvenile mouse brain. In particular, a subset of layer V cortical neurons retains relatively high expression of both NEEP21/Nsg1 and P19/Nsg2 at postnatal day 16 as well as in the CA1-3 regions of the hippocampus. In the cerebellum, NEEP21/Nsg1 expression becomes largely restricted to Purkinje neurons in adulthood whereas P19/Nsg2 expression strikingly disappears from the cerebellum with age. This divergent and restricted expression likely reflects differential needs for this class of trafficking regulators in different neurons during different stages of maturation.

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.

Ctip2-, Satb2-, Prox1-, and GAD65-Expressing Neurons in Rat Cultures: Preponderance of Single- and Double-Positive Cells, and Cell Type-Specific Expression of Neuron-Specific Gene Family Members, Nsg-1 (NEEP21) and Nsg-2 (P19).

The brain consists of many distinct neuronal cell types, but which cell types are present in widely used primary cultures of embryonic rodent brain is often not known. We characterized how abundantly four cell type markers (Ctip2, Satb2, Prox1, GAD65) were represented in cultured rat neurons, how easily neurons expressing different markers can be transfected with commonly used plasmids, and whether neuronal-enriched endosomal proteins Nsg-1 (NEEP21) and Nsg-2 (P19) are ubiquitously expressed in all types of cultured neurons. We found that cultured neurons stably maintain cell type identities that are reflective of cell types in vivo. This includes neurons maintaining simultaneous expression of two transcription factors, such as Ctip2+/Satb2+ or Prox1+/Ctip2+ double-positive cells, which have also been described in vivo. Secondly, we established the superior efficiency of CAG promoters for both Lipofectamine-mediated transfection as well as for electroporation. Thirdly, we discovered that Nsg-1 and Nsg-2 were not expressed equally in all neurons: whereas high levels of both Nsg-1 and Nsg-2 were found in Satb2-, Ctip2-, and GAD65-positive neurons, Prox1-positive neurons in hippocampal cultures expressed low levels of both. Our findings thus highlight the importance of identifying neuronal cell types for doing cell biology in cultured neurons: Keeping track of neuronal cell type might uncover effects in assays that might otherwise be masked by the mixture of responsive and non-responsive neurons in the dish.

Adapting for endocytosis: roles for endocytic sorting adaptors in directing neural development.

Proper cortical development depends on the orchestrated actions of a multitude of guidance receptors and adhesion molecules and their downstream signaling. The levels of these receptors on the surface and their precise locations can greatly affect guidance outcomes. Trafficking of receptors to a particular surface locale and removal by endocytosis thus feed crucially into the final guidance outcomes. In addition, endocytosis of receptors can affect downstream signaling (both quantitatively and qualitatively) and regulated endocytosis of guidance receptors is thus an important component of ensuring proper neural development. We will discuss the cell biology of regulated endocytosis and the impact on neural development. We focus our discussion on endocytic accessory proteins (EAPs) (such as numb and disabled) and how they regulate endocytosis and subsequent post-endocytic trafficking of their cognate receptors (such as Notch, TrkB, ß-APP, VLDLR, and ApoER2).