Spatial-Temporal Mapping Reveals the Golgi as the Major Processing Site for the Pathogenic Swedish APP Mutation: Familial APP Mutant Shifts the Major APP Processing Site.

Alzheimer's disease is associated with increased levels of amyloid beta (Aß) generated by sequential intracellular cleavage of amyloid precursor protein (APP) by membrane-bound secretases. However, the spatial and temporal APP cleavage events along the trafficking pathways are poorly defined. Here, we use the Retention Using Selective Hooks (RUSH) to compare in real time the anterograde trafficking and temporal cleavage events of wild-type APP (APPwt) with the pathogenic Swedish APP (APPswe) and the disease-protective Icelandic APP (APPice). The analyses revealed differences in the trafficking profiles and processing between APPwt and the APP familial mutations. While APPwt was predominantly processed by the ß-secretase, BACE1, following Golgi transport to the early endosomes, the transit of APPswe through the Golgi was prolonged and associated with enhanced amyloidogenic APP processing and Aß secretion. A 20°C block in cargo exit from the Golgi confirmed ß- and γ-secretase processing of APPswe in the Golgi. Inhibition of the ß-secretase, BACE1, restored APPswe anterograde trafficking profile to that of APPwt. APPice was transported rapidly through the Golgi to the early endosomes with low levels of Aß production. This study has revealed different intracellular locations for the preferential cleavage of APPwt and APPswe and Aß production, and the Golgi as the major processing site for APPswe, findings relevant to understand the molecular basis of Alzheimer's disease.

Advances in the cell biology of the trafficking and processing of amyloid precursor protein: impact of familial Alzheimer's disease mutations.

The production of neurotoxic amyloid-ß peptides (Aß) is central to the initiation and progression of Alzheimer's disease (AD) and involves sequential cleavage of the amyloid precursor protein (APP) by ß- and γ-secretases. APP and the secretases are transmembrane proteins and their co-localisation in the same membrane-bound sub-compartment is necessary for APP cleavage. The intracellular trafficking of APP and the ß-secretase, BACE1, is critical in regulating APP processing and Aß production and has been studied in several cellular systems. Here, we summarise the intracellular distribution and transport of APP and its secretases, and the intracellular location for APP cleavage in non-polarised cells and neuronal models. In addition, we review recent advances on the potential impact of familial AD mutations on APP trafficking and processing. This is critical information in understanding the molecular mechanisms of AD progression and in supporting the development of novel strategies for clinical treatment.

Segregation of the membrane cargoes, BACE1 and amyloid precursor protein (APP) throughout the Golgi apparatus.

The intracellular trafficking of ß-site amyloid precursor protein (APP) cleaving enzyme (BACE1) and APP regulates amyloid-ß production. Our previous work demonstrated that newly synthesized BACE1 and APP are segregated into distinct trafficking pathways from the trans-Golgi network (TGN), and that alterations in their trafficking lead to an increase in Aß production in non-neuronal and neuronal cells. However, it is not known whether BACE1 and APP are transported through the Golgi stacks together and sorted at the TGN or segregated prior to arrival at the TGN. To address this question, we have used high-resolution Airyscan technology followed by Huygens deconvolution to quantify the overlap of BACE1 and APP in Golgi subcompartments in HeLa cells and primary neurons. Here, we show that APP and BACE1 are segregated, on exit from the endoplasmic reticulum and in the cis-Golgi and throughout the Golgi stack. In contrast, the transferrin receptor, which exits the TGN in AP-1 mediated transport carriers as for BACE1, colocalizes with BACE1, but not APP, throughout the Golgi stack. The segregation of APP and BACE1 is independent of the Golgi ribbon structure and the cytoplasmic domain of the cargo. Overall, our findings reveal the segregation of different membrane cargoes early in the secretory pathway, a finding relevant to the regulation of APP processing events.

Amyloid ß production along the neuronal secretory pathway: Dangerous liaisons in the Golgi?

ß-amyloid peptides (Aß) are generated in intracellular compartments of neurons and secreted to form cytotoxic fibrils and plaques. Dysfunctional membrane trafficking contributes to aberrant Aß production and Alzheimer's disease. Endosomes represent one of the major sites for Aß production and recently the Golgi has re-emerged also as a major location for amyloid precursor protein (APP) processing and Aß production. Based on recent findings, here we propose that APP processing in the Golgi is finely tuned by segregating newly-synthesised APP and the ß-secretase BACE1 within the Golgi and into distinct trans-Golgi network transport pathways. We hypothesise that there are multiple mechanisms responsible for segregating APP and BACE1 during transit through the Golgi, and that perturbation in Golgi morphology associated with Alzheimer's disease, and or changes in cholesterol metabolism associated with Alzheimer's disease risk factors, may lead to a loss of partitioning and enhanced Aß production.

The role of microtubules in secretory protein transport.

Microtubules are part of the dynamic cytoskeleton network and composed of tubulin dimers. They are the main tracks used in cells to organize organelle positioning and trafficking of cargos. In this Review, we compile recent findings on the involvement of microtubules in anterograde protein transport. First, we highlight the importance of microtubules in organelle positioning. Second, we discuss the involvement of microtubules within different trafficking steps, in particular between the endoplasmic reticulum and the Golgi complex, traffic through the Golgi complex itself and in post-Golgi processes. A large number of studies have assessed the involvement of microtubules in transport of cargo from the Golgi complex to the cell surface. We focus here on the role of kinesin motor proteins and protein interactions in post-Golgi transport, as well as the impact of tubulin post-translational modifications. Last, in light of recent findings, we highlight the role microtubules have in exocytosis, the final step of secretory protein transport, occurring close to focal adhesions.

Functional Assessment of Genetic Variants with Outcomes Adapted to Clinical Decision-Making.

Understanding the medical effect of an ever-growing number of human variants detected is a long term challenge in genetic counseling. Functional assays, based on in vitro or in vivo evaluations of the variant effects, provide essential information, but they require robust statistical validation, as well as adapted outputs, to be implemented in the clinical decision-making process. Here, we assessed 25 pathogenic and 15 neutral missense variants of the BRCA1 breast/ovarian cancer susceptibility gene in four BRCA1 functional assays. Next, we developed a novel approach that refines the variant ranking in these functional assays. Lastly, we developed a computational system that provides a probabilistic classification of variants, adapted to clinical interpretation. Using this system, the best functional assay exhibits a variant classification accuracy estimated at 93%. Additional theoretical simulations highlight the benefit of this ready-to-use system in the classification of variants after functional assessment, which should facilitate the consideration of functional evidences in the decision-making process after genetic testing. Finally, we demonstrate the versatility of the system with the classification of siRNAs tested for human cell growth inhibition in high throughput screening.

Microtubule-independent secretion requires functional maturation of Golgi elements.

The Golgi complex is responsible for processing and sorting of secretory cargos. Microtubules are known to accelerate the transport of proteins from the endoplasmic reticulum (ER) to the Golgi complex and from the Golgi to the plasma membrane. However, whether post-Golgi transport strictly requires microtubules is still unclear. Using the retention using selective hooks (RUSH) system to synchronize the trafficking of cargos, we show that anterograde transport of tumor necrosis factor (TNF) is strongly reduced without microtubules. We show that two populations of Golgi elements co-exist in these cells. A centrally located and giantin-positive Golgi complex that sustains trafficking, and newly formed peripheral Golgi mini-stacks that accumulate cargos in cells without microtubules. Using a genome-edited GFP-giantin cell line, we observe that the trafficking-competent Golgi population corresponds to the pre-existing population that was present before removal of microtubules. All Golgi elements support trafficking after long-term depletion of microtubules and after relocation of Golgi proteins to the ER after treatment with Brefeldin A. Our results demonstrate that functional maturation of Golgi elements is needed to ensure post-Golgi trafficking, and that microtubule-driven post-Golgi transport is not strictly required.

Yeast cells reveal the misfolding and the cellular mislocalization of the human BRCA1 protein.

Understanding the effect of an ever-growing number of human variants detected by genome sequencing is a medical challenge. The yeast Saccharomyces cerevisiae model has held attention for its capacity to monitor the functional impact of missense mutations found in human genes, including the BRCA1 breast and ovarian cancer susceptibility gene. When expressed in yeast, the wild-type full-length BRCA1 protein forms a single nuclear aggregate and induces a growth inhibition. Both events are modified by pathogenic mutations of BRCA1. However, the biological processes behind these events in yeast remain to be determined. Here, we show that the BRCA1 nuclear aggregation and the growth inhibition are sensitive to misfolding effects induced by missense mutations. Moreover, misfolding mutations impair the nuclear targeting of BRCA1 in yeast cells and in a human cell line. In conclusion, we establish a connection between misfolding and nuclear transport impairment, and we illustrate that yeast is a suitable model to decipher the effect of misfolding mutations.

DNA methylation associated with polycomb repression in retinoic acid receptor ß silencing.

Retinoic acid receptor ß 2 (RARß2) is a tumor suppressor gene whose loss of expression is recurrent in prostate cancers. Here we studied the epigenetic mechanisms leading to its stable silencing. First, we characterized all RARß isoforms in 6 human tumor cell lines (prostate DU145, LNCaP, PC3, lung A549, breast Hs578T, and colon HCT116) by RT-PCR and Western blot. We excluded loss of heterozygosity (2D-FISH) and loss of RARa expression, an upstream regulator, as origin of RARß2 silencing. All data concluded to an epigenetic silencing. In agreement, a DNA methylation inhibitor restored its expression. Second RARß2 loss of expression was found associated with different epigenetic profiles in LNCaP and DU145 cells. According to bisulfite sequencing and ChIP analysis, we observed heavy methylation (97%) of the RARß2 promoter with repressive histone mark H3K9me3 in LNCaP. While DNA methylation and polycomb repression are described to be mutually exclusive at CpG-rich promoters, we observed that in DU145, moderate DNA methylation (36%) and H3K9me3 mark were present concomitantly with H3K27me3, a signature of polycomb repression. In summary, we provide new insights on how the RARß2 promoter is silenced, reveal the existence of two distinct repressive chromatin profiles at the same locus, and support a polycomb-mediated epigenetic repression process in prostate cancer.

Long-term live cell imaging during differentiation of human iPSC-derived neurons.

Live-cell imaging is crucial to appreciate the dynamics and the complexity of cellular interaction processes. However, live-cell imaging of human neurons is challenging due to neuronal sensitivity. Here, we describe a long-term live-cell imaging protocol for neurons derived from human induced pluripotent stem cells. By using an IncuCyte live-cell imaging system, we have obtained information on neuronal dynamics during the different stages of neurogenesis. The protocol has also been developed to monitor the dynamics of the neuronal intracellular organelles. For complete details on the use and execution of this protocol, please refer to Wang et al.1.

Quantification of Golgi Entry and Exit Kinetics of Protein Cargoes.

The Golgi apparatus is a pivotal secretory organelle in membrane trafficking, a hub responsible for posttranslational modifications, sorting, and trafficking of newly synthetized proteins received from the endoplasmic reticulum (ER). Different protein cargoes have been shown to travel through the Golgi stacks with different kinetics. Dysregulated transport and altered residency time of cargoes in the Golgi can impair their functionality. To study the anterograde trafficking of specific protein cargoes, innovative molecular methods have been developed to synchronize the traffic of selected cargoes from the ER in live cells. These methods of synchronization now provide the ability to quantify the Golgi entry and exit kinetics of defined cargo. In this chapter, we describe a quantitative, accurate, and semiautomated protocol to image and quantify the anterograde trafficking of individual cargo traversing the Golgi. This protocol, using free software, is compatible with different synchronization techniques, and can be used for a range of applications, such as comparing the Golgi kinetics of (1) different cargoes, (2) wild-type cargo vs mutated cargo, (3) the same cargo under different Golgi conditions, and (4) cargoes in drug screening platforms. The method can also be applied to study the localization and transit of a cargo through different organelles other than the Golgi apparatus.

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.

Retention Using Selective Hooks-Synchronized Secretion to Measure Local Exocytosis.

Proteins destined to be exposed to the extracellular space enter the secretory pathway at the level of the endoplasmic reticulum. Proteins are then transported to the Golgi apparatus and addressed to their destination compartment, such as the plasma membrane for exocytic cargos. Exocytosis constitutes the last step of the anterograde transport of secretory cargos. Exocytic vesicles fuse with the plasma membrane, releasing soluble proteins to the extracellular milieu and transmembrane proteins to the plasma membrane. In order to monitor local exocytosis of cargos, we describe in this chapter how to perform synchronization of the anterograde transport of an exocytic cargo of interest using the retention using selective hooks (RUSH) assay in combination with selective protein immobilization (SPI). SPI is based on the coating of coverslips with anti-green fluorescent protein (GFP) antibodies, which capture the GFP-tagged RUSH cargos once exposed to the cell surface after its release by the addition of biotin.

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.

Distinct anterograde trafficking pathways of BACE1 and amyloid precursor protein from the TGN and the regulation of amyloid-ß production.

Processing of amyloid precursor protein (APP) by the ß-secretase BACE1 is the initial step of the amyloidogenic pathway to generate amyloid-ß (Aß). Although newly synthesized BACE1 and APP are transported along the secretory pathway, it is not known whether BACE1 and APP share the same post-Golgi trafficking pathways or are partitioned into different transport routes. Here we demonstrate that BACE1 exits the Golgi in HeLa cells and primary neurons by a pathway distinct from the trafficking pathway for APP. By using the Retention Using Selective Hooks system, we show that BACE1 is transported from the trans-Golgi network to the plasma membrane in an AP-1- and Arf1/4-dependent manner. Subsequently, BACE1 is endocytosed to early and recycling endosomes. Perturbation of BACE1 post-Golgi trafficking results in an increase in BACE1 cleavage of APP and increased production of both Aß40 and Aß42. These findings reveal that Golgi exit of BACE1 and APP in primary neurons is tightly regulated, resulting in their segregation along different transport routes, which limits APP processing.

RAB6 and microtubules restrict protein secretion to focal adhesions.

To ensure their homeostasis and sustain differentiated functions, cells continuously transport diverse cargos to various cell compartments and in particular to the cell surface. Secreted proteins are transported along intracellular routes from the endoplasmic reticulum through the Golgi complex before reaching the plasma membrane along microtubule tracks. Using a synchronized secretion assay, we report here that exocytosis does not occur randomly at the cell surface but on localized hotspots juxtaposed to focal adhesions. Although microtubules are involved, the RAB6-dependent machinery plays an essential role. We observed that, irrespective of the transported cargos, most post-Golgi carriers are positive for RAB6 and that its inactivation leads to a broad reduction of protein secretion. RAB6 may thus be a general regulator of post-Golgi secretion.