Opposing functions for retromer and Rab11 in extracellular vesicle traffic at presynaptic terminals

Neuronal extracellular vesicles (EVs) play important roles in intercellular communication and pathogenic protein propagation in neurological disease. However, it remains unclear how cargoes are selectively packaged into neuronal EVs. Here, we show that loss of the endosomal retromer complex leads to accumulation of EV cargoes including amyloid precursor protein (APP), synaptotagmin-4 (Syt4), and neuroglian (Nrg) at Drosophila motor neuron presynaptic terminals, resulting in increased release of these cargoes in EVs. By systematically exploring known retromer-dependent trafficking mechanisms, we show that EV regulation is separable from several previously identified roles of neuronal retromer. Conversely, mutations in rab11 and rab4, regulators of endosome-plasma membrane recycling, cause reduced EV cargo levels, and rab11 suppresses cargo accumulation in retromer mutants. Thus, EV traffic reflects a balance between Rab4/Rab11 recycling and retromer-dependent removal from EV precursor compartments. Our data shed light on previous studies implicating Rab11 and retromer in competing pathways in Alzheimer’s disease, and suggest that misregulated EV traffic may be an underlying defect.     

Matriptase-2- and proprotein convertase-cleaved forms of hemojuvelin have different roles in the down-regulation of hepcidin expression

Hemojuvelin (HJV) is an important regulator of iron metabolism. Membrane-anchored HJV up-regulates expression of the iron regulatory hormone, hepcidin, through the bone morphogenic protein (BMP) signaling pathway by acting as a BMP co-receptor. HJV can be cleaved by the furin family of proprotein convertases, which releases a soluble form of HJV that suppresses BMP signaling and hepcidin expression by acting as a decoy that competes with membrane HJV for BMP ligands. Recent studies indicate that matriptase-2 binds and degrades HJV, leading to a decrease in cell surface HJV. In the present work, we show that matriptase-2 cleaves HJV at Arg(288), which produces one major soluble form of HJV. This shed form of HJV has decreased ability to bind BMP6 and does not suppress BMP6-induced hepcidin expression. These results suggest that the matriptase-2 and proprotein convertase-cleavage products have different roles in the regulation of hepcidin expression.     

ACSL4-dependent ferroptosis does not represent a tumor-suppressive mechanism but ACSL4 rather promotes liver cancer progression

Ferroptosis is a novel type of programmed cell death that differs from apoptosis in that it involves iron-dependent peroxidation of membrane phospholipids. Its role in a variety of human disorders, including cancer has been hypothesized in recent years. While it may function as an endogenous tumor suppressor in a variety of cancers, its role during initiation and progression of liver cancer, particularly hepatocellular carcinoma (HCC), is yet unknown. Because HCC is most commonly found in chronically injured livers, we utilized two well-established mouse models of chronic injury-dependent HCC formation: Treatment with streptozotocin and high-fat diet as metabolic injury model, as well as treatment with diethylnitrosamine and carbon tetrachloride as toxic injury model. We used mice with hepatocyte-specific deletion of Acsl4, a key mediator of ferroptosis, to explore the significance of ferroptotic cell death in hepatocytes, the cell type of origin for HCC. Surprisingly, preventing ferroptotic cell death in hepatocytes by deleting Acsl4 does not increase the formation of HCC. Furthermore, Acsl4-deficient livers display less fibrosis and proliferation, especially in the HCC model of toxic damage. Intriguingly, in this model, the absence of ACSL4-dependent processes such as ferroptosis significantly slow down the growth of HCC. These findings suggest that during HCC formation in a chronically injured liver, ferroptotic cell death is not an endogenous tumor-suppressive mechanism. Instead, we find that ACSL4-dependent processes have an unanticipated cancer-promoting effect during HCC formation, which is most likely due to aggravated liver damage as demonstrated by increased hepatic fibrosis. Previous studies suggested that ferroptosis might have beneficial effects for patients during HCC therapy. As a result, during HCC progression and therapy, ferroptosis may have both cancer-promoting and cancer-inhibitory effects, respectively.Subject terms: Cancer models, Cancer genetics, Cell death     

Gruffi: an algorithm for computational removal of stressed cells from brain organoid transcriptomic datasets

Organoids enable in vitro modeling of complex developmental processes and disease pathologies. Like most 3D cultures, organoids lack sufficient oxygen supply and therefore experience cellular stress. These negative effects are particularly prominent in complex models, such as brain organoids, and can affect lineage commitment. Here, we analyze brain organoid and fetal single‐cell RNA sequencing (scRNAseq) data from published and new datasets, totaling about 190,000 cells. We identify a unique stress signature in the data from all organoid samples, but not in fetal samples. We demonstrate that cell stress is limited to a defined subpopulation of cells that is unique to organoids and does not affect neuronal specification or maturation. We have developed a computational algorithm, Gruffi, which uses granular functional filtering to identify and remove stressed cells from any organoid scRNAseq dataset in an unbiased manner. We validated our method using six additional datasets from different organoid protocols and early brains, and show its usefulness to other organoid systems including retinal organoids. Our data show that the adverse effects of cell stress can be corrected by bioinformatic analysis for improved delineation of developmental trajectories and resemblance to in vivo data.     

The Rab11-family interacting proteins reveal selective interaction of mammalian recycling endosomes with the Toxoplasma parasitophorous vacuole in a Rab11- and Arf6-dependent manner

After mammalian cell invasion, the parasite Toxoplasma multiplies in a self-made membrane-bound compartment, the parasitophorous vacuole (PV). We previously showed that Toxoplasma interacts with many host cell organelles, especially from recycling pathways, and sequestrates Rab11A and Rab11B vesicles into the PV. Here, we examine the specificity of host Rab11 vesicle interaction with the PV by focusing on the recruitment of subpopulations of Rab11 vesicles characterized by different effectors, for example, Rab11-family interacting roteins (FIPs) or Arf6. Our quantitative microscopic analysis illustrates the presence of intra-PV vesicles with FIPs from class I (FIP1C, FIP2, FIP5) and class II (FIP3, FIP4) but to various degrees. The intra-PV delivery of vesicles with class I, but not class II, FIPs is dependent on Rab11 binding. Cell depletion of Rab11A results in a significant decrease in intra-PV FIP5, but not FIP3 vesicles. Class II FIPs also bind to Arf6, and we observe vesicles associated with FIP3-Rab11A or FIP3-Arf6 complexes concomitantly within the PV. Abolishing FIP3 binding to both Rab11 and Arf6 reduces the number of intra-PV FIP3 vesicles. These data point to a selective process of mammalian Rab11 vesicle recognition and scavenging mediated by Toxoplasma, suggesting that specific parasite PV proteins may be involved in these processes.     

Effect of clinician information sessions on diagnostic testing for Chagas disease

BackgroundChagas disease is a potentially life-threatening neglected disease of poverty that is endemic in continental Latin America. Caused by Trypanosoma cruzi (T. cruzi), it is one of six parasitic diseases in the United States targeted by the Centers for Disease Control as a public health problem in need of action. An estimated 300,000 people are infected with T. cruzi in the United States (US). Although its morbidity, mortality and economic burden are high, awareness of Chagas disease is lacking among many healthcare providers in the US. The purpose of this analysis is to determine if the number of diagnostic tests performed at a community health center serving an at-risk population for Chagas disease increased after information sessions. A secondary aim was to determine if there was a difference by provider type, i.e., nurse practitioner vs. physician, or by specialty in the number of patients screened.Methodology/Principal findingsWe conducted a retrospective data analysis of the number of Chagas serology tests performed at a community health center before and after information sessions for clinicians. A time series analysis was conducted focusing on the Adult and Family Medicine Departments at East Boston Neighborhood Health Center (EBNHC). Across all departments there were 1,957 T. cruzi tests performed before the sessions vs. 2,623 after the sessions. Interrupted time series analysis across departments indicated that testing volume was stable over time prior to the sessions (pre-period slope = +4.1 per month; p = 0.12), followed by an immediate shift after the session (+51.6; p = 0.03), while testing volume remained stable over time after the session (post-period slope = -6.0 per month; p = 0.11).Conclusion/SignificanceIn this study, Chagas testing increased after information sessions. Clinicians who began testing their patients for Chagas disease after learning of the importance of this intervention added an extra, potentially time-consuming task to their already busy workdays without external incentives or recognition.     

Bodians Protargol Method Applied to Other than Neurological Preparations

Some applications of the Bodian technic for other than neurological purposes are described. After fixation for 48 hours, in formalin, 5 ml., acetic acid, glacial, 5 ml., and 80% ethyl alcohol, 90 ml., the routine procedures are recommended, with the exception that the exposure to protargol for 24 hours and subsequent reduction should be repeated once. Gold toning may be o-mitted. With this method the argentaffin (chromaffin) cells of the digestive tract (rat, hamster), the alpha cells of the pancreatic islands (hamster) and the medullary cells of the suprarenal gland (hamster) are selectively impregnated. In the mammalian pituitary gland (rat, hamster) certain of the granulated chromophile cells are impregnated. In the rat only the basophiles appear to react with the silver.     

Motor neuropathy‐associated mutation impairs Seipin functions in neurotransmission

Gain‐of‐toxic‐function mutations in Seipin (Asparagine 88 to Serine (N88S) and Serine 90 to Leucine (S90L) mutations, both of which disrupt the N‐glycosylation) cause autosomal dominant motor neuron diseases. However, the mechanism of how these missense mutations lead to motor neuropathy is unclear. Here, we analyze the impact of disruption of N‐glycosylation of Seipin on synaptic transmission by over‐expressing mutant Seipin in cultured cortical neurons via lentiviral infection. Immunostaining shows that over‐expressed Seipin is partly colocalized with synaptic vesicle marker synaptophysin. Electrophysiological recordings reveal that the Seipin mutation significantly decreases the frequency, but not the amplitudes of miniature excitatory post‐synaptic currents and miniature inhibitory post‐synaptic currents. The amplitude of both evoked excitatory post‐synaptic currents and inhibitory post‐synaptic current is also compromised by mutant Seipin over‐expression. The readily releasable pool and vesicular release probability of synaptic vesicles are both altered in neurons over‐expressing Seipin‐N88S, whereas neither γ‐amino butyric acid (GABA) nor α‐Amino‐3‐hydroxy‐5‐methyl‐4‐ isoxazolepropionic acid (AMPA) induced whole cell currents are affected. Moreover, electron microscopy analysis reveals decreased number of morphologically docked synaptic vesicles in Seipin‐N88S‐expressing neurons. These data demonstrate that Seipin‐N88S mutation impairs synaptic neurotransmission, possibly by regulating the priming and docking of synaptic vesicles at the synapse.