Transport along the dendritic endoplasmic reticulum mediates the trafficking of GABAB receptors.

In neurons, secretory organelles within the cell body are complemented by the dendritic endoplasmic reticulum (ER) and Golgi outposts (GOPs), whose role in neurotransmitter receptor trafficking is poorly understood. γ-aminobutyric acid (GABA) type B metabotropic receptors (GABABRs) regulate the efficacy of synaptic transmission throughout the brain. Their plasma membrane availability is controlled by mechanisms involving an ER retention motif and assembly-dependent ER export. Thus, they constitute an ideal molecular model to study ER trafficking, but the extent to which the dendritic ER participates in GABABR biosynthesis has not been thoroughly explored. Here, we show that GABAB1 localizes preferentially to the ER in dendrites and moves long distances within this compartment. Not only diffusion but also microtubule and dynein-dependent mechanisms control dendritic ER transport. GABABRs insert throughout the somatodendritic plasma membrane but dendritic post-ER carriers containing GABABRs do not fuse selectively with GOPs. This study furthers our understanding of the spatial selectivity of neurotransmitter receptors for dendritic organelles.

Developmental shaping of dendritic arbors in Drosophila relies on tightly regulated intra-neuronal activity of protein kinase A (PKA).

Dendrites develop morphologies characterized by multiple levels of complexity that involve neuron type specific dendritic length and particular spatial distribution. How this is developmentally regulated and in particular which signaling molecules are crucial in the process is still not understood. Using Drosophila class IV dendritic arborization (da) neurons we test in vivo the effects of cell-autonomous dose-dependent changes in the activity levels of the cAMP-dependent Protein Kinase A (PKA) on the formation of complex dendritic arbors. We find that genetic manipulations of the PKA activity levels affect profoundly the arbor complexity with strongest impact on distal branches. Both decreasing and increasing PKA activity result in a reduced complexity of the arbors, as reflected in decreased dendritic length and number of branching points, suggesting an inverted U-shape response to PKA. The phenotypes are accompanied by changes in organelle distribution: Golgi outposts and early endosomes in distal dendritic branches are reduced in PKA mutants. By using Rab5 dominant negative we find that PKA interacts genetically with the early endosomal pathway. We test if the possible relationship between PKA and organelles may be the result of phosphorylation of the microtubule motor dynein components or Rab5. We find that Drosophila cytoplasmic dynein components are direct PKA phosphorylation targets in vitro, but not in vivo, thus pointing to a different putative in vivo target. Our data argue that tightly controlled dose-dependent intra-neuronal PKA activity levels are critical in determining the dendritic arbor complexity, one of the possible ways being through the regulation of organelle distribution.

The organization and function of the Golgi apparatus in dendrite development and neurological disorders.

Dendrites are specialized neuronal compartments that sense, integrate and transfer information in the neural network. Their development is tightly controlled and abnormal dendrite morphogenesis is strongly linked to neurological disorders. While dendritic morphology ranges from relatively simple to extremely complex for a specified neuron, either requires a functional secretory pathway to continually replenish proteins and lipids to meet dendritic growth demands. The Golgi apparatus occupies the center of the secretory pathway and is regulating posttranslational modifications, sorting, transport, and signal transduction, as well as acting as a non-centrosomal microtubule organization center. The neuronal Golgi apparatus shares common features with Golgi in other eukaryotic cell types but also forms distinct structures known as Golgi outposts that specifically localize in dendrites. However, the organization and function of Golgi in dendrite development and its impact on neurological disorders is just emerging and so far lacks a systematic summary. We describe the organization of the Golgi apparatus in neurons, review the current understanding of Golgi function in dendritic morphogenesis, and discuss the current challenges and future directions.

Live Imaging of Golgi Outposts in Drosophila Dendritic Arbors.

Neurons are polarized cells whose polarity and morphology rely on the robust localization of cellular organelles and cargo to axons or dendrites. Developing neurons require an active secretory pathway, which includes the endoplasmic reticulum and Golgi apparatus, to supply membrane and proteins to growing dendrites and axons. In some neurons, a subset of the Golgi called Golgi "outposts" localize to dendrites and contribute to local secretory networks. The movement and positioning of Golgi outposts have been correlated with dendrite branch growth and stabilization as the dendritic arbor is established. Live imaging is essential to capture the dynamic nature of these organelles. Here we outline a protocol to image and quantify Golgi outposts in peripheral sensory neurons in live, intact Drosophila larvae.

Microglia reactivity entails microtubule remodeling from acentrosomal to centrosomal arrays.

Microglia reactivity entails a large-scale remodeling of cellular geometry, but the behavior of the microtubule cytoskeleton during these changes remains unexplored. Here we show that activated microglia provide an example of microtubule reorganization from a non-centrosomal array of parallel and stable microtubules to a radial array of more dynamic microtubules. While in the homeostatic state, microglia nucleate microtubules at Golgi outposts, and activating signaling induces recruitment of nucleating material nearby the centrosome, a process inhibited by microtubule stabilization. Our results demonstrate that a hallmark of microglia reactivity is a striking remodeling of the microtubule cytoskeleton and suggest that while pericentrosomal microtubule nucleation may serve as a distinct marker of microglia activation, inhibition of microtubule dynamics may provide a different strategy to reduce microglia reactivity in inflammatory disease.

Organelle mapping in dendrites of human iPSC-derived neurons reveals dynamic functional dendritic Golgi structures.

Secretory pathways within dendrites of neurons have been proposed for local transport of newly synthesized proteins. However, little is known about the dynamics of the local secretory system and whether the organelles are transient or stable structures. Here, we quantify the spatial and dynamic behavior of dendritic Golgi and endosomes during differentiation of human neurons generated from induced pluripotent stem cells (iPSCs). In early neuronal development, before and during migration, the entire Golgi apparatus transiently translocates from the soma into dendrites. In mature neurons, dynamic Golgi elements, containing cis and trans cisternae, are transported from the soma along dendrites, in an actin-dependent process. Dendritic Golgi outposts are dynamic and display bidirectional movement. Similar structures were observed in cerebral organoids. Using the retention using selective hooks (RUSH) system, Golgi resident proteins are transported efficiently into Golgi outposts from the endoplasmic reticulum. This study reveals dynamic, functional Golgi structures in dendrites and a spatial map for investigating dendrite trafficking in human neurons.

Fringe-positive Golgi outposts unite temporal Furin 2 convertase activity and spatial Delta signal to promote dendritic branch retraction.

Golgi outposts (GOPs) in dendrites are known for their role in promoting branch extension, but whether GOPs have other functions is unclear. We found that terminal branches of Drosophila class IV dendritic arborization (C4da) neurons actively grow during the early third-instar (E3) larval stage but retract in the late third (L3) stage. Interestingly, the Fringe (Fng) glycosyltransferase localizes increasingly at GOPs in distal dendritic regions through the E3 to the L3 stage. Expression of the endopeptidase Furin 2 (Fur2), which proteolyzes and inactivates Fng, decreases from E3 to L3 in C4da neurons, thereby increasing Fng-positive GOPs in dendrites. The epidermal Delta ligand and neuronal Notch receptor, the substrate for Fng-mediated O-glycosylation, also negatively regulate dendrite growth. Fng inhibits actin dynamics in dendrites, linking dendritic branch retraction to suppression of the C4da-mediated thermal nociception response in late larval stages. Thus, Fng-positive GOPs function in dendrite retraction, which would add another function to the repertoire of GOPs in dendrite arborization.

Sunday Driver Mediates Multi-Compartment Golgi Outposts Defects Induced by Amyloid Precursor Protein.

Golgi defects including Golgi fragmentation are pathological features of Alzheimer's disease (AD). As a pathogenic factor in AD, amyloid precursor protein (APP) induces Golgi fragmentation in the soma. However, how APP regulates Golgi outposts (GOs) in dendrites remains unclear. Given that APP resides in and affects the movements of GOs, and in particular, reverses the distribution of multi-compartment GOs (mcGOs), we investigated the regulatory mechanism of mcGO movements in the Drosophila larvae. Knockdown experiments showed that the bidirectional mcGO movements were cooperatively controlled by the dynein heavy chain (Dhc) and kinesin heavy chain subunits. Notably, only Dhc mediated APP's regulation of mcGO movements. Furthermore, by loss-of-function screening, the adaptor protein Sunday driver (Syd) was identified to mediate the APP-induced alteration of the direction of mcGO movements and dendritic defects. Collectively, by elucidating a model of bidirectional mcGO movements, we revealed the mechanism by which APP regulates the direction of mcGO movements. Our study therefore provides new insights into AD pathogenesis.

Local Secretory Trafficking Pathways in Neurons and the Role of Dendritic Golgi Outposts in Different Cell Models.

A fundamental characteristic of neurons is the relationship between the architecture of the polarized neuron and synaptic transmission between neurons. Intracellular membrane trafficking is paramount to establish and maintain neuronal structure; perturbation in trafficking results in defects in neurodevelopment and neurological disorders. Given the physical distance from the cell body to the distal sites of the axon and dendrites, transport of newly synthesized membrane proteins from the central cell body to their functional destination at remote, distal sites represents a conundrum. With the identification of secretory organelles in dendrites, including endoplasmic reticulum (ER) and Golgi outposts (GOs), recent studies have proposed local protein synthesis and trafficking distinct from the conventional anterograde transport pathways of the cell body. A variety of different model organisms, including Drosophila, zebrafish, and rodents, have been used to probe the organization and function of the local neuronal secretory network. Here, we review the evidence for local secretory trafficking pathways in dendrites in a variety of cell-based neuronal systems and discuss both the similarities and differences in the organization and role of the local secretory organelles, especially the GOs. In addition, we identify the gaps in the current knowledge and the potential advances using human induced pluripotent stem cells (iPSCs) in defining local membrane protein trafficking in human neurons and in understanding the molecular basis of neurological diseases.

C9orf72-Associated Arginine-Rich Dipeptide Repeat Proteins Reduce the Number of Golgi Outposts and Dendritic Branches in Drosophila Neurons.

Altered dendritic morphology is frequently observed in various neurological disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the cellular and molecular basis underlying these pathogenic dendritic abnormalities remains largely unclear. In this study, we investigated dendritic morphological defects caused by dipeptide repeat protein (DPR) toxicity associated with G4C2 expansion mutation of C9orf72 (the leading genetic cause of ALS and FTD) in Drosophila neurons and characterized the underlying pathogenic mechanisms. Among the five DPRs produced by repeat-associated non-ATG translation of G4C2 repeats, we found that arginine-rich DPRs (PR and GR) led to the most significant reduction in dendritic branches and plasma membrane (PM) supply in Class IV dendritic arborization (C4 da) neurons. Furthermore, expression of PR and GR reduced the number of Golgi outposts (GOPs) in dendrites. In Drosophila brains, expression of PR, but not GR, led to a significant reduction in the mRNA level of CrebA, a transcription factor regulating the formation of GOPs. Overexpressing CrebA in PR-expressing C4 da neurons mitigated PM supply defects and restored the number of GOPs, but the number of dendritic branches remained unchanged, suggesting that other molecules besides CrebA may be involved in dendritic branching. Taken together, our results provide valuable insight into the understanding of dendritic pathology associated with C9-ALS/FTD.

Golgi Outpost Synthesis Impaired by Toxic Polyglutamine Proteins Contributes to Dendritic Pathology in Neurons.

Dendrite aberration is a common feature of neurodegenerative diseases caused by protein toxicity, but the underlying mechanisms remain largely elusive. Here, we show that nuclear polyglutamine (polyQ) toxicity resulted in defective terminal dendrite elongation accompanied by a loss of Golgi outposts (GOPs) and a decreased supply of plasma membrane (PM) in Drosophila class IV dendritic arborization (da) (C4 da) neurons. mRNA sequencing revealed that genes downregulated by polyQ proteins included many secretory pathway-related genes, including COPII genes regulating GOP synthesis. Transcription factor enrichment analysis identified CREB3L1/CrebA, which regulates COPII gene expression. CrebA overexpression in C4 da neurons restores the dysregulation of COPII genes, GOP synthesis, and PM supply. Chromatin immunoprecipitation (ChIP)-PCR revealed that CrebA expression is regulated by CREB-binding protein (CBP), which is sequestered by polyQ proteins. Furthermore, co-overexpression of CrebA and Rac1 synergistically restores the polyQ-induced dendrite pathology. Collectively, our results suggest that GOPs impaired by polyQ proteins contribute to dendrite pathology through the CBP-CrebA-COPII pathway.

Assembly, Secretory Pathway Trafficking, and Surface Delivery of Kainate Receptors Is Regulated by Neuronal Activity.

Ionotropic glutamate receptor (iGluR) trafficking and function underpin excitatory synaptic transmission and plasticity and shape neuronal networks. It is well established that the transcription, translation, and endocytosis/recycling of iGluRs are all regulated by neuronal activity, but much less is known about the activity dependence of iGluR transport through the secretory pathway. Here, we use the kainate receptor subunit GluK2 as a model iGluR cargo to show that the assembly, early secretory pathway trafficking, and surface delivery of iGluRs are all controlled by neuronal activity. We show that the delivery of de novo kainate receptors is differentially regulated by modulation of GluK2 Q/R editing, PKC phosphorylation, and PDZ ligand interactions. These findings reveal that, in addition to short-term regulation of iGluRs by recycling/endocytosis and long-term modulation by altered transcription/translation, the trafficking of iGluRs through the secretory pathway is under tight activity-dependent control to determine the numbers and properties of surface-expressed iGluRs.

Imaging Golgi Outposts in Fixed and Living Neurons.

Here we describe the use of confocal microscopy in combination with antibodies specific to Golgi proteins to visualize dendritic Golgi outposts (GOPs) in cultured hippocampal pyramidal neurons. We also describe the use of spinning disk confocal microscopy, in combination with ectopically expressed glycosyltransferases fused to GFP variants, to visualize GOPs in living neurons.

Membrane Trafficking in Neuronal Development: Ins and Outs of Neural Connectivity.

During development, neurons progress through rapid yet stereotypical shape changes to achieve proper neuronal connectivity. This morphological progression requires carefully orchestrated plasma membrane expansion, insertion of membrane components including receptors for extracellular cues into the plasma membrane and removal and trafficking of membrane materials and proteins to specific locations. This review outlines the cellular machinery of membrane trafficking that play an integral role in neuronal cell shape change and function from initial neurite formation to pathway navigation and synaptogenesis.

Diversifying the secretory routes in neurons.

Nervous system homeostasis and synaptic function need dedicated mechanisms to locally regulate the molecular composition of the neuronal plasma membrane and allow the development, maintenance and plastic modification of the neuronal morphology. The cytoskeleton and intracellular trafficking lies at the core of all these processes. In most mammalian cells, the Golgi apparatus (GA) is at the center of the biosynthetic pathway, located in the proximity of the microtubule-organizing center. In addition to this central localization, the somatic GA in neurons is complemented by satellite Golgi outposts (GOPs) in dendrites, which are essential for dendritic morphogenesis and are emerging like local stations of membranes trafficking to synapses. Largely, GOPs participation in post-ER trafficking has been determined by imaging the transport of the exogenous protein VSVG. Here we review the diversity of neuronal cargoes that traffic through GOPs and the assortment of different biosynthetic routes to synapses. We also analyze the recent advances in understanding the role of cytoskeleton and Golgi matrix proteins in the biogenesis of GOPs and how the diversity of secretory routes can be generated.