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.

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.

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.

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.

Extracellular Tau Oligomers Induce Invasion of Endogenous Tau into the Somatodendritic Compartment and Axonal Transport Dysfunction.

Aggregates composed of the microtubule associated protein, tau, are a hallmark of Alzheimer's disease and non-Alzheimer's tauopathies. Extracellular tau can induce the accumulation and aggregation of intracellular tau, and tau pathology can be transmitted along neural networks over time. There are six splice variants of central nervous system tau, and various oligomeric and fibrillar forms are associated with neurodegeneration in vivo. The particular extracellular forms of tau capable of transferring tau pathology from neuron to neuron remain ill defined, however, as do the consequences of intracellular tau aggregation on neuronal physiology. The present study was undertaken to compare the effects of extracellular tau monomers, oligomers, and filaments comprising various tau isoforms on the behavior of cultured neurons. We found that 2N4R or 2N3R tau oligomers provoked aggregation of endogenous intracellular tau much more effectively than monomers or fibrils, or of oligomers made from other tau isoforms, and that a mixture of all six isoforms most potently provoked intracellular tau accumulation. These effects were associated with invasion of tau into the somatodendritic compartment. Finally, we observed that 2N4R oligomers perturbed fast axonal transport of membranous organelles along microtubules. Intracellular tau accumulation was often accompanied by increases in the run length, run time and instantaneous velocity of membranous cargo. This work indicates that extracellular tau oligomers can disrupt normal neuronal homeostasis by triggering axonal tau accumulation and loss of the polarized distribution of tau, and by impairing fast axonal transport.

mTOR and neuronal cell cycle reentry: How impaired brain insulin signaling promotes Alzheimer's disease.

A major obstacle to presymptomatic diagnosis and disease-modifying therapy for Alzheimer's disease (AD) is inadequate understanding of molecular mechanisms of AD pathogenesis. For example, impaired brain insulin signaling is an AD hallmark, but whether and how it might contribute to the synaptic dysfunction and neuron death that underlie memory and cognitive impairment has been mysterious. Neuron death in AD is often caused by cell cycle reentry (CCR) mediated by amyloid-ß oligomers (AßOs) and tau, the precursors of plaques and tangles. We now report that CCR results from AßO-induced activation of the protein kinase complex, mTORC1, at the plasma membrane and mTORC1-dependent tau phosphorylation, and that CCR can be prevented by insulin-stimulated activation of lysosomal mTORC1. AßOs were also shown previously to reduce neuronal insulin signaling. Our data therefore indicate that the decreased insulin signaling provoked by AßOs unleashes their toxic potential to cause neuronal CCR, and by extension, neuron death.

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.

Farnesylthiosalicylic acid inhibits mammalian target of rapamycin (mTOR) activity both in cells and in vitro by promoting dissociation of the mTOR-raptor complex.

The mammalian target of rapamycin (mTOR) functions with raptor and mLST8 in a signaling complex that controls rates of cell growth and proliferation. Recent results indicate that an inhibitor of the Ras signaling pathway, farnesylthiosalicylic acid (FTS), decreased phosphorylation of the mTOR effectors, PHAS-I and S6K1, in breast cancer cells. Here we show that incubating 293T cells with FTS produced a stable change in mTOR activity that could be measured in immune complex kinase assays using purified PHAS-I as substrate. Similarly, FTS decreased the PHAS-I kinase activity of mTOR when added to cell extracts or to immune complexes containing mTOR. Incubating either cells or extracts with FTS also decreased the amount of raptor that coimmunoprecipitated with mTOR, although having relatively little effect on the amount of mLST8 that coimmunoprecipitated. The concentration effect curves of FTS for inhibition of mTOR activity and for dissociation of the raptor-mTOR complex were almost identical. Caffeine, wortmannin, LY294002, and rapamycin-FKBP12 also markedly inhibited mTOR activity in vitro, but unlike FTS, none of the other mTOR inhibitors appreciably changed the amount of raptor associated with mTOR. Thus, our findings indicate that FTS represents a new type of mTOR inhibitor, which acts by dissociating the functional mTOR-raptor signaling complex.

Two motifs in the translational repressor PHAS-I required for efficient phosphorylation by mammalian target of rapamycin and for recognition by raptor.

Mammalian target of rapamycin (mTOR) is the central element of a signaling pathway involved in the control of mRNA translation and cell growth. The actions of mTOR are mediated in part through the phosphorylation of the eukaryotic initiation factor 4E-binding protein, PHAS-I. In vitro mTOR phosphorylates PHAS-I in sites that control PHAS-I binding to eukaryotic initiation factor 4E; however, whether mTOR directly phosphorylates PHAS-I in cells has been a point of debate. The Arg-Ala-Ile-Pro (RAIP motif) and Phe-Glu-Met-Asp-Ile (tor signaling motif) sequences found in the NH2- and COOH-terminal regions of PHAS-I, respectively, are required for the efficient phosphorylation of PHAS-I in cells. Here we show that mutations in either motif markedly decreased the phosphorylation of recombinant PHAS-I by mTOR in vitro. Wild-type PHAS-I, but none of the mutant proteins, was coimmunoprecipitated with hemagglutinin-tagged raptor, an mTOR-associated protein, after extracts of cells overexpressing raptor had been supplemented with recombinant PHAS-I proteins. Moreover, raptor overexpression enhanced the phosphorylation of wild-type PHAS-I by mTOR but not the phosphorylation of the mutant proteins. The results not only provide direct evidence that both the RAIP and tor signaling motifs are important for the phosphorylation by mTOR, possibly by allowing PHAS-I binding to raptor, but also support the view that mTOR phosphorylates PHAS-I in cells.

The rapamycin-binding domain governs substrate selectivity by the mammalian target of rapamycin.

The mammalian target of rapamycin (mTOR) is a Ser/Thr (S/T) protein kinase, which controls mRNA translation initiation by modulating phosphorylation of the translational regulators PHAS-I and p70(S6K). Here we show that in vitro mTOR is able to phosphorylate these two regulators at comparable rates. Both (S/T)P sites, such as Thr36, Thr45, and Thr69 in PHAS-I and the h(S/T)h site (where h is a hydrophobic amino acid) Thr389 in p70(S6K), were phosphorylated. Rapamycin-FKBP12 inhibited mTOR activity. Surprisingly, the extent of inhibition depended on the substrate. Moreover, mutating Ser2035 in the rapamycin-binding domain (FRB) not only decreased rapamycin sensitivity as expected but also dramatically affected the sites phosphorylated by mTOR. The results demonstrate that mutations in Ser2035 are not silent with respect to mTOR activity and implicate the FRB in substrate recognition. The findings also impose new limitations on interpreting results from experiments in which rapamycin and/or rapamycin-resistant forms of mTOR are used to investigate mTOR function in cells.