Massive accumulation of luminal protease-deficient axonal lysosomes at Alzheimer's disease amyloid plaques.

Through a comprehensive analysis of organellar markers in mouse models of Alzheimer's disease, we document a massive accumulation of lysosome-like organelles at amyloid plaques and establish that the majority of these organelles reside within swollen axons that contact the amyloid deposits. This close spatial relationship between axonal lysosome accumulation and extracellular amyloid aggregates was observed from the earliest stages of ß-amyloid deposition. Notably, we discovered that lysosomes that accumulate in such axons are lacking in multiple soluble luminal proteases and thus are predicted to be unable to efficiently degrade proteinaceous cargos. Of relevance to Alzheimer's disease, ß-secretase (BACE1), the protein that initiates amyloidogenic processing of the amyloid precursor protein and which is a substrate for these proteases, builds up at these sites. Furthermore, through a comparison between the axonal lysosome accumulations at amyloid plaques and neuronal lysosomes of the wild-type brain, we identified a similar, naturally occurring population of lysosome-like organelles in neuronal processes that is also defined by its low luminal protease content. In conjunction with emerging evidence that the lysosomal maturation of endosomes and autophagosomes is coupled to their retrograde transport, our results suggest that extracellular ß-amyloid deposits cause a local impairment in the retrograde axonal transport of lysosome precursors, leading to their accumulation and a blockade in their further maturation. This study both advances understanding of Alzheimer's disease brain pathology and provides new insights into the subcellular organization of neuronal lysosomes that may have broader relevance to other neurodegenerative diseases with a lysosomal component to their pathology.

Depression is associated with recurrent chest pain with or without coronary artery disease: A prospective cohort study in the emergency department.

BACKGROUND: Only a small fraction of acute chest pain in the emergency department (ED) is due to obstructive coronary artery disease (CAD). ED chest pain remains associated with high rates of recidivism, often in the presence of nonobstructive CAD. Psychological states such as depression, anxiety, and elevation of perceived stress may account for this finding. The objective of the study was to determine whether psychological states predict recurrent chest pain (RCP). METHODS: We conducted a prospective cohort study of low- to moderate-cardiac risk ED patients admitted to the Yale Chest Pain Center with acute chest pain. Depression, anxiety, and perceived stress were assessed in each patient using multistudy-validated screening scales: Patient Health Questionnaire (PHQ8), Clinical Anxiety Scale (CAS), and Perceived Stress Scale (PSS), respectively. All patients ruled out for infarction underwent appropriate cardiac stress testing. Primary outcome was RCP at 30 days evaluated by phone follow-up and medical record. The relationship between each psychological scale and RCP was evaluated using ordinal logistic regressions, controlling for known sociodemographic and cardiac risk factors. Depression (PHQ8≥10), anxiety (CAS≥30), and perceived stress (PSS≥15) were considered positive. RESULTS: Between August 2013 and May 2015, 985 patients were screened at the Yale Chest Pain Center. Of 500 enrolled patients, 483 patients had complete data and 365 (76%) patients completed follow-up. Thirty-six percent (n=131) had RCP within 1 month. On multivariable regression models, depression (odds ratio [OR]=2.11, 95% CI 1.18-3.79) was a significant independent predictor of 30-day chest pain recurrence after adjustment, whereas PSS (OR=0.96, 95% CI 0.60-1.53) and anxiety (OR=1.59, 95% CI 0.80-3.20) were not. Similarly, there was a direct relationship between psychometric evaluation of depression (via PHQ8) and the frequency of chest pain. CONCLUSIONS: Depression is independently associated with RCP regardless of significant cardiac ischemia on stress testing. Identification and targeted interventions may curtail recidivism with RCP.

Selective high-level expression of epsin 3 in gastric parietal cells, where it is localized at endocytic sites of apical canaliculi.

Epsin is a ubiquitin-binding endocytic adaptor, which is highly concentrated at clathrin-coated pits and coordinates acquisition of bilayer curvature with coat recruitment and cargo selection. Epsin is encoded by three distinct genes in mammals. Epsin 1 and 2 have broad tissue distribution with high-level expression in the brain. In contrast, epsin 3 was reported to be expressed primarily in immature keratinocytes. Here, we show that epsin 3 is selectively expressed at high levels in the stomach (including the majority of gastric cancers), where it is concentrated in parietal cells. In these cells, epsin 3 is enriched and colocalized with clathrin around apical canaliculi, the sites that control acidification of the stomach lumen via the exo-endocytosis of vesicles containing the H/K ATPase. Deletion of the epsin 3 gene in mice did not result in obvious pathological phenotypes in either the stomach or other organs, possibly because of overlapping functions of the other two epsins. However, levels of EHD1 and EHD2, two membrane tubulating proteins with a role in endocytic recycling, were elevated in epsin 3 knock-out stomachs, pointing to a functional interplay of epsin 3 with EHD proteins in the endocytic pathway of parietal cells. We suggest that epsin 3 cooperates with other bilayer binding proteins with curvature sensing/generating properties in the specialized traffic and membrane remodeling processes typical of gastric parietal cells.

The need for establishing best practices and gold standards in psychedelic medicine.

Psychedelic substances are under investigation in several drug development programs. Controlled clinical trials are providing evidence for safe and effective use of psychedelic therapies for treating mental health conditions. With the anticipated FDA approval of MDMA-assisted therapy for posttraumatic stress disorder in 2023 and psilocybin therapy for depression disorders soon after, now is the time for the medical community to become informed on best practices and to actively participate in developing standards of care for these new treatments. Given the emergence of numerous drug sponsors and other companies developing therapeutic modalities for combination with psychedelic medications, it is essential that the medical professional field is at the forefront of communicating unbiased information related to safety and effectiveness. Gold standards have long been a part of medicine and serve to distinguish treatments and assessments as the highest quality by which all others can be compared to. For a treatment to be established as a gold standard, several factors are considered including the quantity and quality of the supporting data, the rigor of trials, and the safety and efficacy compared to other treatments. In this article, we review the origins of psychedelic-assisted therapy (PAT), minimum requirements for safe use of psychedelics, criteria for gold standards in mental health, and the nuances regarding how to establish gold standards in psychedelic medicine and guide clinical decision making.

A role of the Lowe syndrome protein OCRL in early steps of the endocytic pathway.

Mutations in the inositol 5-phosphatase OCRL are responsible for Lowe syndrome, whose manifestations include mental retardation and renal Fanconi syndrome. OCRL has been implicated in membrane trafficking, but disease mechanisms remain unclear. We show that OCRL visits late-stage, endocytic clathrin-coated pits and binds the Rab5 effector APPL1 on peripheral early endosomes. The interaction with APPL1, which is mediated by the ASH-RhoGAP-like domains of OCRL and is abolished by disease mutations, provides a link to protein networks implicated in the reabsorptive function of the kidney and in the trafficking and signaling of growth factor receptors in the brain. Crystallographic studies reveal a role of the ASH-RhoGAP-like domains in positioning the phosphatase domain at the membrane interface and a clathrin box protruding from the RhoGAP-like domain. Our results support a role of OCRL in the early endocytic pathway, consistent with the predominant localization of its preferred substrates, PI(4,5)P(2) and PI(3,4,5)P(3), at the cell surface.

Selective saturation of slow endocytosis at a giant glutamatergic central synapse lacking dynamin 1.

Exocytosis of synaptic vesicles is rapidly followed by compensatory plasma membrane endocytosis. The efficiency of endocytosis varies with experimental conditions, but the molecular basis for this control remains poorly understood. Here, the function of dynamin 1, the neuron-specific member of a family of GTPases implicated in vesicle fission, was investigated with high temporal resolution via membrane capacitance measurements at the calyx of Held, a giant glutamatergic synapse. Endocytosis at dynamin 1 KO calyces was the same as in wild type after weak stimuli, consistent with the nearly normal ultrastructure of mutant synapses. However, following stronger stimuli, the speed of slow endocytosis, but not of other forms of endocytosis, failed to scale with the increased endocytic load. Thus, high level expression of dynamin 1 is essential to allow the slow, clathrin-mediated endocytosis, which accounts for the bulk of the endocytic response, to operate efficiently over a wide range of activity.

Cell- and stimulus-dependent heterogeneity of synaptic vesicle endocytic recycling mechanisms revealed by studies of dynamin 1-null neurons.

Mice lacking expression of dynamin 1, a GTPase implicated in the fission reaction of synaptic vesicle endocytosis, fail to thrive and exhibit severe activity-dependent endocytic defects at their synapses. Here, we have used electron tomography to investigate the massive increase in clathrin-coated pit abundance that is selectively observed at a subset of synapses in dynamin 1 KO primary neuron cultures under conditions of spontaneous network activity. This increase, leading to branched tubular plasma membrane invaginations capped by clathrin-coated buds, occurs selectively at inhibitory synapses. A similar massive increase of clathrin-coated profiles (in this case, of clathrin-coated vesicles) is observed at inhibitory synapses of neurons that lack expression of synaptojanin 1, a phosphoinositide phosphatase involved in clathrin-coated vesicle uncoating. Thus, although excitatory synapses are largely spared under these conditions, inhibitory synapses are uniquely sensitive to perturbation of endocytic proteins, probably as a result of their higher levels of tonic activity leading to a buildup of clathrin-coated intermediates in these synapses. In contrast, the predominant endocytic structures observed at the majority of dynamin 1 KO synapses after acute stimulation are endosome-like intermediates that originate by a dynamin 1-independent form of endocytosis. These findings reveal a striking heterogeneity in the mode of synaptic vesicle recycling in different synapses and functional states.

All known patient mutations in the ASH-RhoGAP domains of OCRL affect targeting and APPL1 binding.

Mutations in the inositol 5-phosphatase OCRL are responsible for Lowe syndrome, an X-linked disorder characterized by bilateral cataracts, mental retardation, neonatal hypotonia, and renal Fanconi syndrome, and for Dent disease, another X-linked condition characterized by kidney reabsorption defects. We have previously described an interaction of OCRL with the endocytic adaptor APPL1 that links OCRL to protein networks involved in the disease phenotype. Here, we provide new evidence showing that among the interactions which target OCRL to membranes of the endocytic pathway, binding to APPL1 is the only one abolished by all known disease-causing missense mutations in the ASH-RhoGAP domains of the protein. Furthermore, we demonstrate that APPL1 and rab5 independently contribute to recruit OCRL to enlarged endosomes induced by the expression of constitutively active Rab5. Thus, binding to APPL1 helps localize OCRL at specific cellular sites, and disruption of this interaction may play a role in disease.

Influence of society for academic emergency medicine grant mechanisms on postaward academic productivity.

OBJECTIVES: The Society for Academic Emergency Medicine (SAEM) provides research training grants, but the future productivity of award recipients and nonrecipients is unclear. The study objective was to assess the association of the two SAEM research training mechanisms with scholarly productivity and rates of subsequent funding between nonrecipients and recipients. A secondary goal was to evaluate the productivity metrics for fellows trained at the Institutional Research Training Grant (IRTG) programs. METHODS: The authors surveyed all 2002 through 2011 Research Training Grant (RTG; n = 64) and Institutional Research Training Grant (IRTG; n = 38) applicants. RTG outcomes were federal funding as a principal investigator (PI) or co-PI using National Institutes of Health RePORTER and scholarly productivity using PubMed. IRTG outcomes were SAEM-approved research fellowships and National Heart, Lung and Blood Institute K12 training awards. Sites applying for or receiving the IRTG multiple times were only counted once. Relative risks (RRs) with 95% confidence intervals (CIs) were calculated. RESULTS: Over 10 years, nine of 64 (14%) RTG and 10 of 38 (26%) IRTG applications were funded (two sites received multiple awards). Federal funding was obtained by seven of nine (78%) RTG recipients and 22 of 55 (40%) RTG nonrecipients (RR = 1.94, 95% CI = 1.21 to 3.13). All nine (100%, 95% CI = 72% to 100%) of RTG recipients had at least one manuscript, compared to 48 of the 55 (87%, 95% CI = 76% to 95%) nonrecipients. All nine (100%, 95% CI = 72% to 100%) RTG recipients remained in academics versus 44 of 55 (80%, 95% CI = 67% to 90%) nonrecipients. For the IRTG, four of seven awardees (57%, 95% CI = 18% to 90%) versus 0 of the 16 (0%, 95% CI = 0 to 17%) nonrecipients received National Heart, Lung and Blood Institute K12 awards (RR = 19.1, 95% CI = 1.16 to 314.0). Additionally, five of seven (71%, 95% CI = 29% to 96%) institutions became SAEM-approved fellowships compared to one of 16 (6%, 95% CI = 0 to 30%) nonrecipients (RR = 11.4, 95% CI = 1.61 to 80.7). CONCLUSIONS: SAEM RTG recipients were more likely to obtain federal funding postaward than nonrecipients. IRTG recipients were more likely to develop successful research training programs than nonrecipients.

A role of OCRL in clathrin-coated pit dynamics and uncoating revealed by studies of Lowe syndrome cells.

Mutations in the inositol 5-phosphatase OCRL cause Lowe syndrome and Dent's disease. Although OCRL, a direct clathrin interactor, is recruited to late-stage clathrin-coated pits, clinical manifestations have been primarily attributed to intracellular sorting defects. Here we show that OCRL loss in Lowe syndrome patient fibroblasts impacts clathrin-mediated endocytosis and results in an endocytic defect. These cells exhibit an accumulation of clathrin-coated vesicles and an increase in U-shaped clathrin-coated pits, which may result from sequestration of coat components on uncoated vesicles. Endocytic vesicles that fail to lose their coat nucleate the majority of the numerous actin comets present in patient cells. SNX9, an adaptor that couples late-stage endocytic coated pits to actin polymerization and which we found to bind OCRL directly, remains associated with such vesicles. These results indicate that OCRL acts as an uncoating factor and that defects in clathrin-mediated endocytosis likely contribute to pathology in patients with OCRL mutations.

Role of dynamin, synaptojanin, and endophilin in podocyte foot processes.

Podocytes are specialized cells that play an integral role in the renal glomerular filtration barrier via their foot processes. The foot processes form a highly organized structure, the disruption of which causes nephrotic syndrome. Interestingly, several similarities have been observed between mechanisms that govern podocyte organization and mechanisms that mediate neuronal synapse development. Dynamin, synaptojanin, and endophilin are functional partners in synaptic vesicle recycling via interconnected actions in clathrin-mediated endocytosis and actin dynamics in neurons. A role of dynamin in the maintenance of the kidney filtration barrier via an action on the actin cytoskeleton of podocytes was suggested. Here we used a conditional double-KO of dynamin 1 (Dnm1) and Dnm2 in mouse podocytes to confirm dynamin's role in podocyte foot process maintenance. In addition, we demonstrated that while synaptojanin 1 (Synj1) KO mice and endophilin 1 (Sh3gl2), endophilin 2 (Sh3gl1), and endophilin 3 (Sh3gl3) triple-KO mice had grossly normal embryonic development, these mutants failed to establish a normal filtration barrier and exhibited severe proteinuria due to abnormal podocyte foot process formation. These results strongly implicate a protein network that functions at the interface between endocytosis and actin at neuronal synapses in the formation and maintenance of the kidney glomerular filtration barrier.

Recruitment of endophilin to clathrin-coated pit necks is required for efficient vesicle uncoating after fission.

Endophilin is a membrane-binding protein with curvature-generating and -sensing properties that participates in clathrin-dependent endocytosis of synaptic vesicle membranes. Endophilin also binds the GTPase dynamin and the phosphoinositide phosphatase synaptojanin and is thought to coordinate constriction of coated pits with membrane fission (via dynamin) and subsequent uncoating (via synaptojanin). We show that although synaptojanin is recruited by endophilin at bud necks before fission, the knockout of all three mouse endophilins results in the accumulation of clathrin-coated vesicles, but not of clathrin-coated pits, at synapses. The absence of endophilin impairs but does not abolish synaptic transmission and results in perinatal lethality, whereas partial endophilin absence causes severe neurological defects, including epilepsy and neurodegeneration. Our data support a model in which endophilin recruitment to coated pit necks, because of its curvature-sensing properties, primes vesicle buds for subsequent uncoating after membrane fission, without being critically required for the fission reaction itself.

Overlapping role of dynamin isoforms in synaptic vesicle endocytosis.

The existence of neuron-specific endocytic protein isoforms raises questions about their importance for specialized neuronal functions. Dynamin, a GTPase implicated in the fission reaction of endocytosis, is encoded by three genes, two of which, dynamin 1 and 3, are highly expressed in neurons. We show that dynamin 3, thought to play a predominantly postsynaptic role, has a major presynaptic function. Although lack of dynamin 3 does not produce an overt phenotype in mice, it worsens the dynamin 1 KO phenotype, leading to perinatal lethality and a more severe defect in activity-dependent synaptic vesicle endocytosis. Thus, dynamin 1 and 3, which together account for the overwhelming majority of brain dynamin, cooperate in supporting optimal rates of synaptic vesicle endocytosis. Persistence of synaptic transmission in their absence indicates that if dynamin plays essential functions in neurons, such functions can be achieved by the very low levels of dynamin 2.

Coordinated actions of actin and BAR proteins upstream of dynamin at endocytic clathrin-coated pits.

The GTPase dynamin, a key player in endocytic membrane fission, interacts with numerous proteins that regulate actin dynamics and generate/sense membrane curvature. To determine the functional relationship between these proteins and dynamin, we have analyzed endocytic intermediates that accumulate in cells that lack dynamin (derived from dynamin 1 and 2 double conditional knockout mice). In these cells, actin-nucleating proteins, actin, and BAR domain proteins accumulate at the base of arrested endocytic clathrin-coated pits, where they support the growth of dynamic long tubular necks. These results, which we show reflect the sequence of events in wild-type cells, demonstrate a concerted action of these proteins prior to, and independent of, dynamin and emphasize similarities between clathrin-mediated endocytosis in yeast and higher eukaryotes. Our data also demonstrate that the relationship between dynamin and actin is intimately connected to dynamin's endocytic role and that dynamin terminates a powerful actin- and BAR protein-dependent tubulating activity.

A selective activity-dependent requirement for dynamin 1 in synaptic vesicle endocytosis.

Dynamin 1 is a neuron-specific guanosine triphosphatase thought to be critically required for the fission reaction of synaptic vesicle endocytosis. Unexpectedly, mice lacking dynamin 1 were able to form functional synapses, even though their postnatal viability was limited. However, during spontaneous network activity, branched, tubular plasma membrane invaginations accumulated, capped by clathrin-coated pits, in synapses of dynamin 1-knockout mice. Synaptic vesicle endocytosis was severely impaired during strong exogenous stimulation but resumed efficiently when the stimulus was terminated. Thus, dynamin 1-independent mechanisms can support limited synaptic vesicle endocytosis, but dynamin 1 is needed during high levels of neuronal activity.