Trafficking Of Complexes Research Articles

Pharmacologic Stabilization of Retromer Rescues Endosomal Pathology Induced by Defects in the Alzheimer’s gene SORL1

AbstractBackgroundDefects in the Sortilin‐related receptor 1 gene, SORL1, are highly pathogenic Alzheimer’s disease (AD), on par with other causal AD genes. SORL1 is a key regulator of endosomal trafficking in neurons, and loss of SORL1 function disrupts trafficking pathways that are vital for neuronal health. SORL1 has been found to interact with retromer, a trafficking complex that is a ‘master conductor’ of endosomal outflow. Here we rely on retromer pharmacological chaperones that stabilize retromer in vitro, and thus enhance retromer function in cells to test the hypothesis that they will correct AD‐associated neuronal pathologies.MethodWe used an isogenic series of human induced pluripotent cell lines either deficient in SORL1 expression (SORL1 KO or SORL1+/−) or harboring a one copy of a missense variant linked to increased risk for AD (SORL1Var). We treated neurons derived from these hiPSCs with the established retromer chaperone, TPT‐260, and tested whether they corrected indicators of AD’s defining endosomal, amyloid, and tau pathologies.ResultWe observed that TPT‐260 treatment was able to correct all of these pathological indicators, however the degree of rescue of some of the phenotypes was dependent one whether the neurons harbor at least one functional copy of SORL1 or whether they are fully deficient.ConclusionMore than just validating that SORL1‐retromer acts a trafficking functional unit in neurons, our work sheds new light on how SORL1‐retromer is linked to AD’s cardinal pathologies and illuminates how this pathway can be therapeutically harnessed.

Proteomic identification and structural basis for the interaction between sorting nexin SNX17 and PDLIM family proteins

The sorting nexin SNX17 controls endosomal recycling of transmembrane cargo proteins including integrins, the amyloid precursor protein, and lipoprotein receptors. This requires association with the Commander trafficking complex and depends on the C terminus of SNX17 through unknown mechanisms. Using proteomics, we find that the SNX17 C terminus is sufficient for Commander interaction and also associates with members of the PDZ and LIM domain (PDLIM) family. SNX17 contains a type III PDZ binding motif that binds specifically to the PDLIM proteins. The structure of the PDLIM7 PDZ domain bound to the SNX17 C terminus reveals an unconventional perpendicular peptide interaction mediated by electrostatic contacts and a uniquely conserved proline-containing loop sequence in the PDLIM protein family. Our results define the mechanism of SNX17-PDLIM interaction and suggest that the PDLIM proteins may play a role in regulating the activity of SNX17 in conjunction with Commander and actin-rich endosomal trafficking domains.

A calpain-6/YAP axis in sarcoma stem cells that drives the outgrowth of tumors and metastases

Sarcomas include cancer stem cells, but how these cells contribute to local and metastatic relapse is largely unknown. We previously showed the pro-tumor functions of calpain-6 in sarcoma stem cells. Here, we use an osteosarcoma cell model, osteosarcoma tissues and transcriptomic data from human tumors to study gene patterns associated with calpain-6 expression or suppression. Calpain-6 modulates the expression of Hippo pathway genes and stabilizes the hippo effector YAP. It also modulates the vesicular trafficking of β-catenin degradation complexes. Calpain-6 expression is associated with genes of the G2M phase of the cell cycle, supports G2M-related YAP activities and up-regulated genes controlling mitosis in sarcoma stem cells and tissues. In mouse models of bone sarcoma, most tumor cells expressed calpain-6 during the early steps of tumor out-growth. YAP inhibition prevented the neoformation of primary tumors and metastases but had no effect on already developed tumors. It could even accelerate lung metastasis associated with large bone tumors by affecting tumor-associated inflammation in the host tissues. Our results highlight a specific mechanism involving YAP transcriptional activity in cancer stem cells that is crucial during the early steps of tumor and metastasis outgrowth and that could be targeted to prevent sarcoma relapse.

HIV‐1 Nef hijacks both exocytic and endocytic pathways of host intracellular trafficking through differential regulation of Rab GTPases

HIV-1 Nef regulates several cellular functions in an infected cell which results in viral persistence and AIDS pathogenesis. The currently understood molecular mechanism(s) underlying Nef-dependent cellular function(s) are unable to explain how events are coordinately regulated in the host cell. Intracellular membranous trafficking maintains cellular homeostasis and is regulated by Rab GTPases - a member of the Ras superfamily. In the current study, we tried to decipher the role of Nef on the Rab GTPases-dependent complex and vesicular trafficking. Expression profiling of Rabs in Nef-expressing cells showed that Nef differentially regulates the expression of individual Rabs in a cell-specific manner. Further analysis of Rabs in HIV-1NL4-3 or ΔNef infected cells demonstrated that the Nef protein is responsible for variation in Rabs expression. Using a panel of competitive peptide inhibitors against Nef, we identified the critical domain of HIV-1 Nef involved in modulation of Rabs expression. The molecular function of Nef-mediated upregulation of Rab5 and Rab7 and downregulation of Rab11 increased the transport of SERINC5 from the cell surface to the lysosomal compartment. Moreover, the Nef-dependent increase in Rab27 expression assists exosome release. Reversal of Rabs expression using competitive inhibitors against Nef and manipulation of Rabs expression reduced viral release and infectivity of progeny virions. This study demonstrates that Nef differentially regulates the expression of Rab proteins in HIV-1 infected cells to hijack the host intracellular trafficking, which augments viral replication and HIV-1 pathogenesis. Our study emphasized the indispensable role of HIV-1 protein Nef on various aspects of the intracellular trafficking regulated by Rabs GTPases, which explained how HIV-1 Nef may hijack membrane trafficking pathways in infected cells.

The Exocyst Is Required for CD36 Fatty Acid Translocase Trafficking and Free Fatty Acid Uptake in Skeletal Muscle Cells.

In obesity, chronic membrane-localization of CD36 free fatty acid (FFA) translocase, but not other FFA transporters, enhances FFA uptake and intracellular lipid accumulation. This ectopic lipid accumulation promotes insulin resistance by inhibiting insulin-induced GLUT4 glucose transporter trafficking and glucose uptake. GLUT4 and CD36 cell surface delivery is triggered by insulin- and contraction-induced signaling, which share conserved downstream effectors. While we have gathered detailed knowledge on GLUT4 trafficking, the mechanisms regulating CD36 membrane delivery and subsequent FFA uptake in skeletal muscle are not fully understood. The exocyst trafficking complex facilitates the docking of membrane-bound vesicles, a process underlying the controlled surface delivery of fuel transporters. The exocyst regulates insulin-induced glucose uptake via GLUT4 membrane trafficking in adipocytes and skeletal muscle cells and plays a role in lipid uptake in adipocytes. Based on the high degree of conservation of the GLUT4 and CD36 trafficking mechanisms in adipose and skeletal muscle tissue, we hypothesized that the exocyst also contributes to lipid uptake in skeletal muscle and acts through the targeted plasma membrane delivery of CD36 in response to insulin and contraction. Here, we show that the exocyst complex is necessary for insulin- and contraction-induced CD36 membrane trafficking and FFA uptake in muscle cells.

Fatty acid translocase: a culprit of lipid metabolism dysfunction in disease.

Dysregulation of lipid deposition into and mobilization from white adipose tissue (WAT) underlies various diseases. Long-chain fatty acids (LCFA) and cholesterol trafficking in and out of adipocytes is a process relying on transporters shuttling lipids from the plasma membrane (PM) to lipid droplets (LD). CD36 is the fatty acid translocase (FAT) that transports LCFA and cholesterol across the PM. Interactions of CD36 with proteins PHB1, ANX2, and CAV1 mediate intercellular lipid transport between adipocytes, hematopoietic, epithelial, and endothelial cells. Intracellularly, the FAT complex has been found to regulate LCFA trafficking between the PM and LD. This process is regulated by CD36 glycosylation and S-acylation, as well as by post-translational modifications of PHB1 and ANX2, which determine both protein–protein interactions and the cellular localization of the complex. Changes in extracellular and intracellular LCFA levels have been found to induce the post-translational modifications and the function of the FAT complex in lipid uptake and mobilization. The role of the CD36/PHB1/ANX2 complex may span beyond lipid trafficking. The requirement of PHB1 for mitochondrial oxidative metabolism in brown adipocytes has been revealed. Cancer cells which take advantage of lipids mobilized by adipocytes and oxidized in leukocytes are indirectly affected by the function of FAT complex in other tissues. The direct importance of CD36 interaction with PHB1/and ANX2 in cancer cells remains to be established. This review highlights the multifaceted roles of the FAT complex in systemic lipid trafficking and discuss it as a potential target in metabolic disease and cancer.

203-LB: The Role of the Exocyst Trafficking Complex in CD36 Fatty Acid Translocase Trafficking in Skeletal Muscle Cells

Skeletal muscle free fatty acid (FFA) uptake correlates with the amount of fatty acid translocase CD36 on the cell surface. In obesity, chronic membrane-localization of CD36, but not other FFA transporters, augments FFA uptake and intracellular lipid accumulation. This ectopic lipid accumulation then causes insulin resistance (IR) by inhibiting GLUT4 glucose transporter trafficking and glucose uptake in response to insulin. GLUT4 and CD36 cell-surface delivery is stimulated by insulin and contraction signaling, sharing conserved downstream effectors. The eight-protein exocyst complex is responsible for the targeted docking of membrane-bound vesicles, a process underlying cell-surface delivery of fuel transporters. The exocyst indeed regulates insulin-induced glucose uptake via GLUT4 trafficking to the membrane in adipocytes and skeletal muscle. In addition, it controls lipid uptake in adipocytes, but we do not know which of the FFA-transporting proteins the exocyst targets during this process. Based on the high degree of conservation of the fuel transporter trafficking mechanisms in adipose and skeletal muscle tissue, we hypothesized that the exocyst contributes to lipid uptake in skeletal muscle via the targeted trafficking of CD36. Our data shows that the exocyst plays a role in FFA uptake in skeletal muscle cells. Using an L6 GLUT4myc myoblast model we demonstrate that the exocyst is recruited to CD36 vesicles upon insulin and contraction signaling. We also show that disrupted exocyst activity leads to an impaired CD36 surface delivery and decreased FFA uptake in skeletal myoblasts. We are the first to demonstrate that the exocyst is necessary for insulin- and contraction-induced CD36 membrane trafficking and FFA metabolism in muscle cells. Further studies will continue to investigate the mechanism of exocyst-regulated FFA uptake and lipid metabolism in skeletal muscle to help our understanding of ectopic lipid accumulation leading to IR. Disclosure N. K. Nakamura: None. D. S. Tokunaga: None. H. Y. J. Ha: None. N. Polgar: None.

Host succinate inhibits influenza virus infection through succinylation and nuclear retention of the viral nucleoprotein

Influenza virus infection causes considerable morbidity and mortality, but current therapies have limited efficacy. We hypothesized that investigating the metabolic signaling during infection may help to design innovative antiviral approaches. Using bronchoalveolar lavages of infected mice, we here demonstrate that influenza virus induces a major reprogramming of lung metabolism. We focused on mitochondria‐derived succinate that accumulated both in the respiratory fluids of virus‐challenged mice and of patients with influenza pneumonia. Notably, succinate displays a potent antiviral activity in vitro as it inhibits the multiplication of influenza A/H1N1 and A/H3N2 strains and strongly decreases virus‐triggered metabolic perturbations and inflammatory responses. Moreover, mice receiving succinate intranasally showed reduced viral loads in lungs and increased survival compared to control animals. The antiviral mechanism involves a succinate‐dependent posttranslational modification, that is, succinylation, of the viral nucleoprotein at the highly conserved K87 residue. Succinylation of viral nucleoprotein altered its electrostatic interactions with viral RNA and further impaired the trafficking of viral ribonucleoprotein complexes. The finding that succinate efficiently disrupts the influenza replication cycle opens up new avenues for improved treatment of influenza pneumonia.

Abstract 452: Classification And Effect Of Correctors On ABCG8 Sitosterolemia Associated Cytosolic Mutants

Background: Sitosterolemia is a rare form of Familial Hypercholesterolemia (FH) which is unique from other forms of FH due to the accumulation of phytosterols in the plasma and tissues. Sitosterolemia is autosomal recessive and caused by mutations in either the ABCG5 or ABCG8 gene. ABCG5 and ABCG8 form a heterodimer (ABCG5/ABCG8) that functions at the apical surface of hepatocytes and enterocytes to promote cholesterol and phytosterol excretion. Small molecule modulators classified as either correctors or potentiators have therapeutic benefit when used to treat mutants of ABCB4 (PFIC3) or ABCC7 (Cystic Fibrous) that have impaired folding, stability, or activity. There are over 40 missense mutations in ABCG5/ABCG8 which have been clinically confirmed in patients with Sitosterolemia. Methods: We developed a classification system for ABCG5 and ABCG8 mutations based on the underlying molecular defect for ABCG5/ABCG8 dysfunction (maturation, trafficking, activity, etc.). We used site directed mutagenesis to introduce ABCG8 missense mutations located in the N-terminal, cytosolic domain of human ABCG8. Normal and mutant ABCG5/ABCG8 constructs were transiently transfected into a human hepatocyte cell line, Huh-7, and analyzed for heterodimerization, trafficking beyond the ER, and localization at the cell surface. Results: Of the ABCG8 cytosolic mutants studied, R184H, L196Q, L228P, and R263Q failed to traffic beyond the ER to form a stable heterodimer (class II) while P231T, T400K, N409D, N409I, and P415H were trafficking competent. Conclusions: Forty four percent (4 of 9) of cytosolic, ABCG8 mutations that cause Sitosterolemia are due to failures in complex formation and trafficking beyond the ER. Fifty six (5 of 9) percent were trafficking competent, indicating a stable ABCG5/G8 complex, but loss of activity due to impaired ATP hydrolysis or substrate binding. ABCG8 mutants may be potentially restored by proteostatic regulators and/or potentiators & correctors that have shown to be effective in disease-causing mutations in other ABC transporters.

Investigating Phosphorylation of the Cardiac Potassium Channel hERG using MALDI‐TOF Mass Spectrometry

Mutations in the voltage‐gated cardiac potassium channel proteins hERG and KvLQT1 are known to be associated with a cardiac condition called Long QT Syndrome (LQTS), which can result in arrhythmia, fainting, and/or sudden cardiac death. Previous research from the Darling lab suggests that hERG and KvLQT1, alpha subunits of two distinct ion channels, are capable of direct protein‐protein interactions, which can be regulated by PKA‐mediated phosphorylation.. They also found that phospho‐null mutants of hERG do not interact with KvLQT1 to the same extent as wild‐type hERG, leading to the hypothesis that the phosphorylation state of hERG dominates the regulation of interactions between hERG and KvLQT1. Thus, understanding the phosphorylation pattern of wild‐type hERG would help us characterize these protein‐protein interactions as well as increase our knowledge about the complex biogenesis and trafficking of hERG. In order to elucidate this baseline phosphorylation state, the goal of this project is to use matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF MS) to characterize which of the four known PKA‐phosphorylation sites of hERG are in fact modified when the protein is synthesized in heterologous model cells. In order to do this, the project aims to determine whether MALDI‐TOF MS can sensitively and reproducibly detect phosphorylation patterns in hERG that is expressed in model cell systems.

Zinc transporters ZIPT-2.4 and ZIPT-15 are required for normal C. elegans fecundity.

The requirement of zinc for the development and maturation of germ lines and reproductive systems is deeply conserved across evolution. The nematode Caenorhabditis elegans offers a tractable platform to study the complex system of distributing zinc to the germ line. We investigated several zinc importers to investigate how zinc transporters play a role in the reproductive system in nematodes, as well as establish a platform to study zinc transporter biology in germline and reproductive development. Previous high throughput transcriptional datasets as well as phylogenetic analysis identified several putative zinc transporters that have a function in reproduction in worms. Phenotypic analysis of CRISPR-generated knockouts and tags included characterization of offspring output, gonad development, and protein localization. Light and immunofluorescence microscopy allowed for visualization of physiological and molecular effects of zinc transporter mutations. Disruption of two zinc transporters, ZIPT-2.4 and ZIPT-15, was shown to lead to defects in reproductive output. A mutation in zipt-2.4 has subtle effects on reproduction, while a mutation in zipt-15 has a clear impact on gonad and germline development that translates into a more pronounced defect in fecundity. Both transporters have germline expression, as well as additional expression in other cell types. Two ZIP-family zinc transporter orthologs of human ZIP6/10 and ZIP1/2/3 proteins are important for full reproductive fecundity and participate in development of the gonad. Notably, these zinc transporters are present in gut and reproductive tissues in addition to the germ line, consistent with a complex zinc trafficking network important for reproductive success.

Brainstem BBSome Regulates Glucose Metabolism and High Fat Diet Sensitivity

Bardet‐Biedl syndrome (BBS) proteins form a protein trafficking complex (BBSome) that regulates the composition of membrane proteins and contributes to neuron function. Patients harboring mutations in any BBS gene results in BBSome dysfunction and the development of metabolic complications including obesity and type 2 diabetes. Studies from our lab and others indicate that hypothalamic BBSome is a critical regulator of metabolic homeostasis, however the role of the BBSome in the brainstem is unknown. To determine whether hindbrain BBSome contributes to metabolic regulation, we crossed Phox2bCre mice with BBS1fl/fl mice (Phox2bCre/BBS1fl/fl) yielding BBS1 deletion specifically in brainstem nuclei. BBS1 deletion exclusively in hindbrain nuclei was validated by breeding these mice on a tdTomato Cre reporter background. Male Phox2bCre/BBS1fl/fl mice maintained on standard chow diet did not exhibit energy imbalance as evident by comparable body weight (37.2±2.8g vs. 35.8±4.4g), body composition (fat 20.8±2.8% vs 15.6±4.1%, lean 60.6±2.5% vs 64.2±3.0%), and food intake (3.5±0.4g/day vs 3.8±0.5g/day) relative to litter‐mate controls at 20 weeks of age. Interestingly, hindbrain BBSome disruption impairs glucose clearance and induces insulin resistance suggested by glucose (AUC 36,323±3027 vs 26,129±1972) and insulin (AUC 11,749±1678 vs 7,737±1559) tolerance tests. Strikingly, Phox2bCre/BBS1fl/fl male mice exhibit resistance to diet‐induced obesity indicated by decreased body weight (45.0±1.8g vs. 54.1±1.3g) when maintained on a high fat/high sugar diet independent of body composition (fat 42.5±1.6% vs 40.1±2.1%, lean 45.5±0.7 vs 48.4±1.5%) and food consumption (2.7±0.2g/day vs 3.0±0.1g/day). Both Phox2bCre/BBS1fl/fl and littermate controls developed diabetes when exposed to an obesogenic diet as indicated by glucose (AUC 63,645±4438 vs 62,477±2654) and insulin (AUC 14,303±3446 vs 16,406±2160) tolerance tests. These findings suggest that brainstem BBSome critically regulates glucose metabolism, but not energy balance, in mice maintained on a standard diet regimen. Furthermore, our studies indicate that hindbrain BBSome contributes to the development of diet‐induced obesity without effecting glucose handling and food intake.

Delineating the Neuropathology of Lysosomal Storage Diseases Using Patient-Derived Induced Pluripotent Stem Cells.

Lysosomal storage diseases (LSDs) are inherited metabolic diseases caused by deficiency of lysosomal enzymes, essential for the normal development of the brain and other organs. Approximately two-thirds of the patients suffering from LSD exhibit neurological deficits and impose an escalating challenge to the medical and scientific field. The advent of induced pluripotent stem cell (iPSC) technology has aided researchers in efficiently generating functional neuronal and non-neuronal cells through directed differentiation protocols, as well as in decoding the cellular, subcellular, and molecular defects associated with LSDs using two-dimensional cultures and cerebral organoid models. This review highlights the information assembled from patient-derived iPSCs on neurodevelopmental and neuropathological defects identified in LSDs. Multiple studies have identified neural progenitor cell migration and differentiation defects, substrate accumulation, axon growth and myelination defects, impaired calcium homeostasis, and altered electrophysiological properties, using patient-derived iPSCs. In addition, these studies have also uncovered defective lysosomes, mitochondria, endoplasmic reticulum, Golgi complex, autophagy and vesicle trafficking and signaling pathways, oxidative stress, neuroinflammation, blood-brain barrier dysfunction, neurodegeneration, gliosis, and altered transcriptomes in LSDs. The review also discusses the therapeutic applications such as drug discovery, repurposing of drugs, synergistic effects of drugs, targeted molecular therapies, gene therapy, and transplantation applications of mutation-corrected lines identified using patient-derived iPSCs for different LSDs.

Perspectives on Mechanisms Supporting Neuronal Polarity From Small Animals to Humans.

Axon-dendrite formation is a crucial milestone in the life history of neurons. During this process, historically referred as “the establishment of polarity,” newborn neurons undergo biochemical, morphological and functional transformations to generate the axonal and dendritic domains, which are the basis of neuronal wiring and connectivity. Since the implementation of primary cultures of rat hippocampal neurons by Gary Banker and Max Cowan in 1977, the community of neurobiologists has made significant achievements in decoding signals that trigger axo-dendritic specification. External and internal cues able to switch on/off signaling pathways controlling gene expression, protein stability, the assembly of the polarity complex (i.e., PAR3-PAR6-aPKC), cytoskeleton remodeling and vesicle trafficking contribute to shape the morphology of neurons. Currently, the culture of hippocampal neurons coexists with alternative model systems to study neuronal polarization in several species, from single-cell to whole-organisms. For instance, in vivo approaches using C. elegans and D. melanogaster, as well as in situ imaging in rodents, have refined our knowledge by incorporating new variables in the polarity equation, such as the influence of the tissue, glia-neuron interactions and three-dimensional development. Nowadays, we have the unique opportunity of studying neurons differentiated from human induced pluripotent stem cells (hiPSCs), and test hypotheses previously originated in small animals and propose new ones perhaps specific for humans. Thus, this article will attempt to review critical mechanisms controlling polarization compiled over decades, highlighting points to be considered in new experimental systems, such as hiPSC neurons and human brain organoids.

A family of models in support of realistic drug interdiction location decision‐making

AbstractLong‐standing federal drug‐control policy aims to reduce the flow of narcotics into the USA, in part by intercepting cocaine shipments en route from South American production regions to North American consumer markets. Drug interdiction efforts operate over a large geographic area, containing complex drug trafficking networks in a dynamic environment. The extant interdiction models in the operations research and location science literature do not realistically model the objectives of and constraints on the interdiction forces, and therefore counterdrug organizations do not employ those models in their decision‐making processes. This article presents three new models built on the maximal covering location problem (MCLP): the maximal covering location problem for interdiction (MCLP‐I), multiple‐demand maximal covering location problem (MD‐MCLP), and multiple‐type maximal covering location problem (MT‐MCLP). These are novel formulations that permit multiple types of demands and facilities to be covered, and the utility of these formulations is demonstrated in the context of counterdrug operations. Optimal interdiction locations are determined within the geography of the Central American transit zone, using a coupled GIS and optimization framework. The results identify the optimal interdiction locations for known or estimated drug shipments and can constrain those optimal locations by differentiating among drug traffickers, the types of interdiction resources, and agency jurisdictions. This research both demonstrates the flexibility in designing alternative interdiction scenarios and presents novel covering models that may be extended to other application areas and operational contexts.

Organization of Presynaptic Autophagy-Related Processes.

Brain synapses pose special challenges on the quality control of their protein machineries as they are far away from the neuronal soma, display a high potential for plastic adaptation and have a high energy demand to fulfill their physiological tasks. This applies in particular to the presynaptic part where neurotransmitter is released from synaptic vesicles, which in turn have to be recycled and refilled in a complex membrane trafficking cycle. Pathways to remove outdated and damaged proteins include the ubiquitin-proteasome system acting in the cytoplasm as well as membrane-associated endolysosomal and the autophagy systems. Here we focus on the latter systems and review what is known about the spatial organization of autophagy and endolysomal processes within the presynapse. We provide an inventory of which components of these degradative systems were found to be present in presynaptic boutons and where they might be anchored to the presynaptic apparatus. We identify three presynaptic structures reported to interact with known constituents of membrane-based protein-degradation pathways and therefore may serve as docking stations. These are (i) scaffolding proteins of the cytomatrix at the active zone, such as Bassoon or Clarinet, (ii) the endocytic machinery localized mainly at the peri-active zone, and (iii) synaptic vesicles. Finally, we sketch scenarios, how presynaptic autophagic cargos are tagged and recruited and which cellular mechanisms may govern membrane-associated protein turnover in the presynapse.

Activation of the CaMKII-Sarm1-ASK1-p38 MAP kinase pathway protects against axon degeneration caused by loss of mitochondria.

Mitochondrial defects are tightly linked to axon degeneration, yet the underlying cellular mechanisms remain poorly understood. In Caenorhabditis elegans, PVQ axons that lack mitochondria degenerate spontaneously with age. Using an unbiased genetic screen, we found that cell-specific activation of CaMKII/UNC-43 suppresses axon degeneration due to loss of mitochondria. Unexpectedly, CaMKII/UNC-43 activates the conserved Sarm1/TIR-1-ASK1/NSY-1-p38 MAPK pathway and eventually the transcription factor CEBP-1 to protect against degeneration. In addition, we show that disrupting a trafficking complex composed of calsyntenin/CASY-1, Mint/LIN-10, and kinesin suppresses axon degeneration. Further analysis indicates that disruption of this trafficking complex activates the CaMKII-Sarm1-MAPK pathway through L-type voltage-gated calcium channels. Our findings identify CaMKII as a pivot point between mitochondrial defects and axon degeneration, describe how it is regulated, and uncover a surprising neuroprotective role for the Sarm1-p38 MAPK pathway in this context.

Effectiveness of an 8-week Curriculum in Changing Medical Student Self-assessment of Knowledge and Confidence toward Sex Trafficking

ABSTRACT Although trafficked persons frequently present to healthcare systems, physicians and physician trainees often lack training on identification and care of this patient population. Currently, there is no mandatory or standard training on human trafficking during one’s medical education. A pilot 8-week curriculum focusing on sex trafficking, designed and implemented at a regional medical school, aims to provide medical students with the appropriate information and training to understand the complexity of sex trafficking, and enhance their role as members of the healthcare team in combating this issue. Self-assessments of the participants’ attitudes and knowledge prior to and after taking the curriculum showed a level of increased knowledge and confidence in identifying and managing trafficked individuals presenting in the healthcare setting. Findings support the implementation of such a curriculum, and its benefits to counter trafficking through early medical education. The practical contribution of this article is laying the groundwork for a sex trafficking curriculum implemented into medical education, in an effort to counter the issue of human trafficking by destigmatizing and providing knowledge to medical students.

The Alzheimer\u2019s gene SORL1 is a regulator of endosomal traffic and recycling in human neurons

BackgroundLoss of the Sortilin-related receptor 1 (SORL1) gene seems to act as a causal event for Alzheimer’s disease (AD). Recent studies have established that loss of SORL1, as well as mutations in autosomal dominant AD genes APP and PSEN1/2, pathogenically converge by swelling early endosomes, AD’s cytopathological hallmark. Acting together with the retromer trafficking complex, SORL1 has been shown to regulate the recycling of the amyloid precursor protein (APP) out of the endosome, contributing to endosomal swelling and to APP misprocessing. We hypothesized that SORL1 plays a broader role in neuronal endosomal recycling and used human induced pluripotent stem cell-derived neurons (hiPSC-Ns) to test this hypothesis. We examined endosomal recycling of three transmembrane proteins linked to AD pathophysiology: APP, the BDNF receptor Tropomyosin-related kinase B (TRKB), and the glutamate receptor subunit AMPA1 (GLUA1).MethodsWe used isogenic hiPSCs engineered to have SORL1 depleted or to have enhanced SORL1 expression. We differentiated neurons from these cell lines and mapped the trafficking of APP, TRKB and GLUA1 within the endosomal network using confocal microscopy. We also performed cell surface recycling and lysosomal degradation assays to assess the functionality of the endosomal network in both SORL1-depleted and -overexpressing neurons. The functional impact of GLUA1 recycling was determined by measuring synaptic activity. Finally, we analyzed alterations in gene expression in SORL1-depleted neurons using RNA sequencing.ResultsWe find that as with APP, endosomal trafficking of GLUA1 and TRKB is impaired by loss of SORL1. We show that trafficking of all three cargoes to late endosomes and lysosomes is affected by manipulating SORL1 expression. We also show that depletion of SORL1 significantly impacts the endosomal recycling pathway for APP and GLUA1 at the level of the recycling endosome and trafficking to the cell surface. This has a functional effect on neuronal activity as shown by multi-electrode array (MEA). Conversely, increased SORL1 expression enhances endosomal recycling for APP and GLUA1. Our unbiased transcriptomic data further support SORL1’s role in endosomal recycling. We observe altered expression networks that regulate cell surface trafficking and neurotrophic signaling in SORL1-depleted neurons.ConclusionCollectively, and together with other recent observations, these findings suggest that one role for SORL1 is to contribute to endosomal degradation and recycling pathways in neurons, a conclusion that has both pathogenic and therapeutic implications for Alzheimer’s disease.

Cell Biology Methods to Study Recombinant Proteins in Seeds.

Seeds are an attractive platform for the production of recombinant proteins because of their excellent storage properties and their well-developed endomembrane system, which allows accumulation of the product within specialized storage organelles. Due to the presence of these additional organelles and the resulting complexity of intracellular protein trafficking it is interesting to investigate the transport and storage of a recombinant protein within seed tissues, its interactions with endogenous reserve proteins and its impact on the ultrastructure of the endomembrane system. Possible approaches include sequential extraction procedures, subcellular fractionation and 2D as well as 3D electron microscopy techniques such as electron tomography (ET) and serial block face scanning electron microscopy (SBF-SEM), which are described and discussed in this chapter.