A large-scale conformational change couples membrane recruitment to cargo binding in the AP2 clathrin adaptor complex.

The AP2 adaptor complex (alpha, beta2, sigma2, and mu2 subunits) crosslinks the endocytic clathrin scaffold to PtdIns4,5P(2)-containing membranes and transmembrane protein cargo. In the "locked" cytosolic form, AP2's binding sites for the two endocytic motifs, YxxPhi on the C-terminal domain of mu2 (C-mu2) and [ED]xxxL[LI] on sigma2, are blocked by parts of beta2. Using protein crystallography, we show that AP2 undergoes a large conformational change in which C-mu2 relocates to an orthogonal face of the complex, simultaneously unblocking both cargo-binding sites; the previously unstructured mu2 linker becomes helical and binds back onto the complex. This structural rearrangement results in AP2's four PtdIns4,5P(2)- and two endocytic motif-binding sites becoming coplanar, facilitating their simultaneous interaction with PtdIns4,5P(2)/cargo-containing membranes. Using a range of biophysical techniques, we show that the endocytic cargo binding of AP2 is driven by its interaction with PtdIns4,5P(2)-containing membranes.

Improved mammalian retromer cryo-EM structures reveal a new assembly interface.

Retromer (VPS26/VPS35/VPS29 subunits) assembles with multiple sorting nexin proteins on membranes to mediate endosomal recycling of transmembrane protein cargoes. Retromer has been implicated in other cellular processes, including mitochondrial homeostasis, nutrient sensing, autophagy, and fission events. Mechanisms for mammalian retromer assembly remain undefined, and retromer engages multiple sorting nexin proteins to sort cargoes to different destinations. Published structures demonstrate mammalian retromer forms oligomers in vitro, but several structures were poorly resolved. We report here improved retromer oligomer structures using single-particle cryo-EM by combining data collected from tilted specimens with multiple advancements in data processing, including using a 3D starting model for enhanced automated particle picking in RELION. We used a retromer mutant (3KE retromer) that breaks VPS35-mediated interfaces to determine a structure of a new assembly interface formed by the VPS26A and VPS35 N-termini. The interface reveals how an N-terminal VPS26A arrestin saddle can link retromer chains by engaging a neighboring VPS35 N- terminus, on the opposite side from the well-characterized C-VPS26/N-VPS35 interaction observed within heterotrimers. The new interaction interface exhibits substantial buried surface area (∼7000 Å2) and further suggests that metazoan retromer may serve as an adaptable scaffold.

Molecular basis for the sorting of the SNARE VAMP7 into endocytic clathrin-coated vesicles by the ArfGAP Hrb.

SNAREs provide the specificity and energy for the fusion of vesicles with their target membrane, but how they are sorted into the appropriate vesicles on post-Golgi trafficking pathways is largely unknown. We demonstrate that the clathrin-mediated endocytosis of the SNARE VAMP7 is directly mediated by Hrb, a clathrin adaptor and ArfGAP. Hrb wraps 20 residues of its unstructured C-terminal tail around the folded VAMP7 longin domain, demonstrating that unstructured regions of clathrin adaptors can select cargo. Disrupting this interaction by mutation of the VAMP7 longin domain or depletion of Hrb causes VAMP7 to accumulate on the cell's surface. However, the SNARE helix of VAMP7 binds back onto its longin domain, outcompeting Hrb for binding to the same groove and suggesting that Hrb-mediated endocytosis of VAMP7 occurs only when VAMP7 is incorporated into a cis-SNARE complex. These results elucidate the mechanism of retrieval of a postfusion SNARE complex in clathrin-coated vesicles.

Beyond body condition: Experimental evidence that plasma metabolites improve nutritional state measurements in a free-living seabird.

The ability to efficiently measure the health and nutritional status of wild populations in situ is a valuable tool, as many methods of evaluating animal physiology do not occur in real-time, limiting the possibilities for direct intervention. This study investigates the use of blood plasma metabolite concentrations, measured via point-of-care devices or a simple plate reader assay, as indicators of nutritional state in free-living seabirds. We experimentally manipulated the energy expenditure of wild black-legged kittiwakes on Middleton Island, Alaska, and measured the plasma concentrations of glucose, cholesterol, B-hydroxybutyrate, and triglycerides throughout the breeding season, along with measures of body condition (size-corrected mass [SCM] and muscle depth). Supplemental feeding improved the nutritional state of kittiwakes by increasing feeding rate (higher glucose and triglycerides, lower cholesterol), and flight-handicapping caused a slight nutritional decline (lower glucose and triglycerides, higher cholesterol and B-hydroxybutyrate). Glucose and triglycerides were the best indicators of nutritional state when used alongside SCM, and improved upon commonly used metrics for measuring individual condition (i.e. SCM or mass alone). Metabolite concentrations varied across the breeding period, suggesting that the pre-laying stage, when feeding rates tend to be lower, was the most nutritionally challenging period for kittiwakes (low glucose, high cholesterol). Muscle depth also varied by treatment and breeding stage, but differed from other nutritional indices, suggesting that muscle depth is an indicator of exercise and activity level rather than nutrition. Here we demonstrate potential for the use of blood plasma metabolites measured via point-of-care devices as proxies for evaluating individual health, population health, and environmental food availability.

Unveiling the cryo-EM structure of retromer.

Retromer (VPS26/VPS35/VPS29) is a highly conserved eukaryotic protein complex that localizes to endosomes to sort transmembrane protein cargoes into vesicles and elongated tubules. Retromer mediates retrieval pathways from endosomes to the trans-Golgi network in all eukaryotes and further facilitates recycling pathways to the plasma membrane in metazoans. In cells, retromer engages multiple partners to orchestrate the formation of tubulovesicular structures, including sorting nexin (SNX) proteins, cargo adaptors, GTPases, regulators, and actin remodeling proteins. Retromer-mediated pathways are especially important for sorting cargoes required for neuronal maintenance, which links retromer loss or mutations to multiple human brain diseases and disorders. Structural and biochemical studies have long contributed to the understanding of retromer biology, but recent advances in cryo-electron microscopy and cryo-electron tomography have further uncovered exciting new snapshots of reconstituted retromer structures. These new structures reveal retromer assembles into an arch-shaped scaffold and suggest the scaffold may be flexible and adaptable in cells. Interactions with cargo adaptors, particularly SNXs, likely orient the scaffold with respect to phosphatidylinositol-3-phosphate (PtdIns3P)-enriched membranes. Pharmacological small molecule chaperones have further been shown to stabilize retromer in cultured cell and mouse models, but mechanisms by which these molecules bind remain unknown. This review will emphasize recent structural and biophysical advances in understanding retromer structure as the field moves towards a molecular view of retromer assembly and regulation on membranes.

Structure and evolution of ENTH and VHS/ENTH-like domains in tepsin.

Tepsin is currently the only accessory trafficking protein identified in adaptor-related protein 4 (AP4)-coated vesicles originating at the trans-Golgi network (TGN). The molecular basis for interactions between AP4 subunits and motifs in the tepsin C-terminus have been characterized, but the biological role of tepsin remains unknown. We determined X-ray crystal structures of the tepsin epsin N-terminal homology (ENTH) and VHS/ENTH-like domains. Our data reveal unexpected structural features that suggest key functional differences between these and similar domains in other trafficking proteins. The tepsin ENTH domain lacks helix0, helix8 and a lipid binding pocket found in epsin1/2/3. These results explain why tepsin requires AP4 for its membrane recruitment and further suggest ENTH domains cannot be defined solely as lipid binding modules. The VHS domain lacks helix8 and thus contains fewer helices than other VHS domains. Structural data explain biochemical and biophysical evidence that tepsin VHS does not mediate known VHS functions, including recognition of dileucine-based cargo motifs or ubiquitin. Structural comparisons indicate the domains are very similar to each other, and phylogenetic analysis reveals their evolutionary pattern within the domain superfamily. Phylogenetics and comparative genomics further show tepsin within a monophyletic clade that diverged away from epsins early in evolutionary history (~1500 million years ago). Together, these data provide the first detailed molecular view of tepsin and suggest tepsin structure and function diverged away from other epsins. More broadly, these data highlight the challenges inherent in classifying and understanding protein function based only on sequence and structure.

Molecular Basis for the Interaction Between AP4 ß4 and its Accessory Protein, Tepsin.

The adaptor protein 4 (AP4) complex (ϵ/ß4/µ4/σ4 subunits) forms a non-clathrin coat on vesicles departing the trans-Golgi network. AP4 biology remains poorly understood, in stark contrast to the wealth of molecular data available for the related clathrin adaptors AP1 and AP2. AP4 is important for human health because mutations in any AP4 subunit cause severe neurological problems, including intellectual disability and progressive spastic para- or tetraplegias. We have used a range of structural, biochemical and biophysical approaches to determine the molecular basis for how the AP4 ß4 C-terminal appendage domain interacts with tepsin, the only known AP4 accessory protein. We show that tepsin harbors a hydrophobic sequence, LFxG[M/L]x[L/V], in its unstructured C-terminus, which binds directly and specifically to the C-terminal ß4 appendage domain. Using nuclear magnetic resonance chemical shift mapping, we define the binding site on the ß4 appendage by identifying residues on the surface whose signals are perturbed upon titration with tepsin. Point mutations in either the tepsin LFxG[M/L]x[L/V] sequence or in its cognate binding site on ß4 abolish in vitro binding. In cells, the same point mutations greatly reduce the amount of tepsin that interacts with AP4. However, they do not abolish the binding between tepsin and AP4 completely, suggesting the existence of additional interaction sites between AP4 and tepsin. These data provide one of the first detailed mechanistic glimpses at AP4 coat assembly and should provide an entry point for probing the role of AP4-coated vesicles in cell biology, and especially in neuronal function.

What is the optimal duration of human chorionic gonadotrophin surveillance following evacuation of a molar pregnancy? A retrospective analysis on over 20,000 consecutive patients.

OBJECTIVE: To quantify the risk of developing post-molar gestational trophoblastic neoplasia (pGTN) beyond the first normal human chorionic gonadotrophin (hCG) in women who have had a complete (CHM) or partial molar pregnancy (PHM) and to re-evaluate the current UK Hydatidiform mole hCG surveillance guidelines. METHODS: The Charing Cross Hospital Trophoblast Disease Centre database was screened to identify all registered cases of hydatidiform mole (HM) between 1980 and 2009. RESULTS: We identified 20,144 cases of HM, comprising 8400 CHM, 9586 PHM, and 2158 cases of unclassified hydatidiform mole (UHM). Twenty-nine cases (20 CHM, 3 PHM and 6 UHM) developed pGTN after the first normal hCG. For CHM the risk of pGTN at the point of hCG normalisation was 1 in 406, and fell rapidly in the first six months of monitoring. For PHM the risk of pGTN at the point of hCG normalisation was 1 in 3195. Women with CHM where hCG normalisation occurred beyond 56days after uterine evacuation of molar tissue were found to have a 3.8-fold higher risk of pGTN. CONCLUSIONS: Our results show that pGTN can occur after hCG normalisation following PHM but the risk is extremely low. Women with CHM have a comparatively higher risk of pGTN after hCG normalisation. Those with CHM where hCG normalises within 56days have a lower risk of pGTN. We have revised the current UK hCG surveillance protocol for PHM to a single additional confirmatory normal urine hCG measurement one month after first normalisation. The protocol for CHM remains unchanged.

Evaluation of a Preoperative Optimization Protocol for Primary Hip and Knee Arthroplasty Patients.

BACKGROUND: Preoperative optimization of risk factors has been suggested as a strategy to improve the value of total joint arthroplasty (TJA) care. We assessed the implementation of a TJA preoperative optimization protocol and its impact on length of hospital stay, discharge destination, 90-day readmissions, and hospital direct variable costs. METHODS: This retrospective cohort study included adults undergoing primary elective TJA from 07/2015-09/2016 at an urban tertiary care hospital. Post-implementation patients were preoperatively screened for 19 risk factors; results and recommended interventions were reported to surgeons, who had the option to postpone or continue surgery as scheduled. Metrics from hospital administrative databases were compared between post-implementation (02/2016-09/2016) and pre-implementation cohorts (07/2015-11/2015). RESULTS: The 314 post-implementation patients were slightly younger compared to the 351 pre-implementation patients (64.2 years vs 65.8 years, P = .02) and a higher percentage of patients had diabetes (18% vs 5.1%, P < .001). Of the 98% of post-implementation patients screened, 74% had at least 1 risk factor identified. Obstructive sleep apnea was the most common risk factor (52%), followed by depression (22%) and obesity (body mass index > 40 kg/m2 or 35-40 kg/m2 with comorbidities) (13%). Forty-six patients (20%) did not follow through with the recommended optimization before undergoing elective surgery. The post-implementation cohort had shorter average length of hospital stay (1.9 days vs 2.2 days, P < .001) and lower average total direct variable costs excluding implants ($5409 vs $5852, P < .001). There was no difference in patients discharged home (90% vs 89%, P = .53) or 90-day readmissions (4.1% vs 4.3%, P = .93). CONCLUSION: In our experience, the majority of elective TJA patients have modifiable risk factors, indicating opportunity for preoperative intervention. Our evidence-based preoperative optimization program resulted in higher value care, demonstrated by similar outcomes with lower resource utilization.

Near infrared (NIR) strong field ionization and imaging of C60 sputtered molecules: overcoming matrix effects and improving sensitivity.

Strong field ionization (SFI) was applied for the secondary neutral mass spectrometry (SNMS) of patterned rubrene films, mouse brain sections, and Botryococcus braunii algal cell colonies. Molecular ions of rubrene, cholesterol, C31 diene/triene, and three wax monoesters were detected, representing some of the largest organic molecules ever ionized intact by a laser post-ionization experiment. In rubrene, the SFI SNMS molecular ion signal was ~4 times higher than in the corresponding secondary-ion mass spectroscopy (SIMS) analysis. In the biological samples, the achieved signal improvements varied among molecules and sampling locations, with SFI SNMS, in some cases, revealing analytes made completely undetectable by the influence of matrix effects in SIMS.

Savoring mental imagery: Electrocortical effects and association with depression.

The ability to focus on and increase positive emotion in response to mental imagery may play a key role in emotional wellbeing. Moreover, deficits in this ability might underlie emotional disorders such as depression. Here, we set out to determine whether people could use savoring to upregulate subjective and electrocortical response to mental imagery of previously viewed positive and neutral pictures, and whether this would be negatively affected by depression. On each trial, participants (N = 49) viewed a positive or neutral picture, prior to simply re-imagining the previously presented picture ("view") or re-imagining the picture while savoring it ("savor"). Results showed that savoring increased electrocortical and subjective response to imagined stimuli; however, this effect was only evident at the electrocortical level when controlling for depression. Moreover, depression moderated electrocortical findings, such that individuals who were more depressed showed a reduced effect of savoring on neural response to mental imagery. Results are in line with recent work that has shown the benefits of positive affect treatment for depression, to suggest that deficits in savoring mental imagery may play a role in the development and/or maintenance of depression.

Tepsin binds LC3B to promote ATG9A trafficking and delivery.

Tepsin is an established accessory protein found in Adaptor Protein 4 (AP-4) coated vesicles, but the biological role of tepsin remains unknown. AP-4 vesicles originate at the trans-Golgi network (TGN) and target the delivery of ATG9A, a scramblase required for autophagosome biogenesis, to the cell periphery. Using in silico methods, we identified a putative LC3-Interacting Region (LIR) motif in tepsin. Biochemical experiments using purified recombinant proteins indicate tepsin directly binds LC3B preferentially over other members of the mammalian ATG8 family. Calorimetry and structural modeling data indicate this interaction occurs with micromolar affinity using the established LC3B LIR docking site. Loss of tepsin in cultured cells dysregulates ATG9A export from the TGN as well as ATG9A distribution at the cell periphery. Tepsin depletion in a mRFP-GFP-LC3B HeLa reporter cell line using siRNA knockdown increases autophagosome volume and number, but does not appear to affect flux through the autophagic pathway. Reintroduction of wild-type tepsin partially rescues ATG9A cargo trafficking defects. In contrast, reintroducing tepsin with a mutated LIR motif or missing N-terminus drives diffuse ATG9A subcellular distribution. Together, these data suggest roles for tepsin in cargo export from the TGN; ensuring delivery of ATG9A-positive vesicles; and in overall maintenance of autophagosome structure.

Implementation of a Risk Reduction Protocol in Youth Violence Research.

This article presents data from the Growing up with Media study related to the implementation of a risk reduction protocol that resulted in three groups of youth: low-risk youth (no flags), youth flagged because of violence involvement and not clinically referred; and flagged youth who were referred to a team clinician due to additional risk considerations. Data is from 3,979 U.S. youth 14-15 years of age recruited through social media between October 2018-August 2019. Four in ten youth were flagged for review. Findings suggest that this methodology of identifying and reviewing cases appears to be working as intended: Not only did referred youth have more flags than non-referred youth, but post-hoc analyses suggested that these youth also had higher rates of psychosocial problems (e.g., non-victimization adversity, substance use and depressed mood). The implementation of a risk reduction protocol such as the one described in this article adds a layer of human subject protection beyond the more standard list of websites and hotlines provided to all participants in most studies. This protocol leads the way for future studies to recreate a similar process to address concerning responses collected from survey research.

Parkinson's Disease Associated with G2019S LRRK2 Mutations without Lewy Body Pathology.

BACKGROUND: The G2019S leucine-rich repeat kinase 2 (LRRK2) gene mutation is an important and commonly found genetic determinant of Parkinson's disease (PD). The neuropathological findings associated with this mutation have thus far been varied but are most often associated with Lewy body (LB) pathology. OBJECTIVE: Describe a case of clinical Parkinson's disease with levodopa responsiveness found to have LRRK2 mutations and the absence of Lewy bodies. METHOD: We present an 89-year-old man with a 10-year history of slowly progressive parkinsonism suspected to be secondary to Parkinson's disease. RESULTS: Neuropathological evaluation revealed nigral degeneration without Lewy bodies or Lewy neurites, but there were frequent tau-immunopositive neurites and astrocytes in the putamen and substantia nigra, neocortical glial tau positive astrocytes associated with aging-related tau astrogliopathy (ARTAG), as well as neurofibrillary tangles, beta amyloid plaques, and amyloid angiopathy typical of advanced Alzheimer's disease. G2019S LRRK2 homozygous mutations were found. CONCLUSION: This case illustrates that levodopa-responsive clinical PD caused by G2019S LRRK2 mutations can occur without Lewy bodies.

Clinical Characteristics and Outcomes in Young-Onset Multiple System Atrophy.

BACKGROUND: Young-onset multiple system atrophy (YOMSA) is defined as the onset of multiple system atrophy (MSA) before the age of 40 years old. YOMSA is rare and there is much uncertainty of the phenotype and natural history in patients with YOMSA. OBJECTIVE: The objective is to evaluate the characteristics and disease course of patients with YOMSA. METHODS: We retrospectively reviewed medical records of patients with MSA who were evaluated at all Mayo Clinic sites from 1998 to 2021. We identified patients with YOMSA and evaluated clinical characteristics, autonomic function testing results, and disease course. RESULTS: Of 1496 patients with a diagnosis of clinically probable or clinically established MSA, 20 patients had YOMSA. The median age of onset was 39.1 (interquartile range [IQR] = 37.1, 40.1) years; 13 patients (65%) were male. MSA-parkinsonism was the most common subtype (65%). The median duration of symptom onset to YOMSA diagnosis was 4.9 (IQR = 3.7, 9) years. At the time of medical record review, 17 patients were deceased with a median survival of 8.3 (IQR = 7, 10.9) years. Univariate analysis showed that initial onset of autonomic failure predicted unfavorable survival (hazard ratio = 2.89, P = 0.04) compared to those who presented with motor impairment only at onset. At the time of YOMSA diagnosis, composite autonomic severity score was available in 19 patients with a median of 5 (IQR = 4, 6.5). CONCLUSIONS: YOMSA resembles MSA in most aspects including phenotype and prognosis, although the diagnosis is usually delayed. The presence of autonomic failure at symptom onset may be a poor predictor for survival.