Rescue of a lysosomal storage disorder caused by Grn loss of function with a brain penetrant progranulin biologic.

GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn-/- mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn-/- brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN-a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn-/- phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn-/- CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.

Lipid transporter TMEM24/C2CD2L is a Ca2+-regulated component of ER-plasma membrane contacts in mammalian neurons.

Close appositions between the endoplasmic reticulum (ER) and the plasma membrane (PM) are a general feature of all cells and are abundant in neurons. A function of these appositions is lipid transport between the two adjacent bilayers via tethering proteins that also contain lipid transport modules. However, little is known about the properties and dynamics of these proteins in neurons. Here we focused on TMEM24/C2CD2L, an ER-localized SMP domain containing phospholipid transporter expressed at high levels in the brain, previously shown to be a component of ER-PM contacts in pancreatic ß-cells. TMEM24 is enriched in neurons versus glial cells and its levels increase in parallel with neuronal differentiation. It populates ER-PM contacts in resting neurons, but elevations of cytosolic Ca2+ mediated by experimental manipulations or spontaneous activity induce its transient redistribution throughout the entire ER. Dissociation of TMEM24 from the plasma membrane is mediated by phosphorylation of an array of sites in the C-terminal region of the protein. These sites are only partially conserved in C2CD2, the paralogue of TMEM24 primarily expressed in nonneuronal tissues, which correspondingly display a much lower sensitivity to Ca2+ elevations. ER-PM contacts in neurons are also sites where Kv2 (the major delayed rectifier K+ channels in brain) and other PM and ER ion channels are concentrated, raising the possibility of a regulatory feedback mechanism between neuronal excitability and lipid exchange between the ER and the PM.

Osmotherapy for malignant cerebral edema in a phase 2 prospective, double blind, randomized, placebo-controlled study of IV glibenclamide.

BACKGROUND/OBJECTIVE: Malignant edema can be a life-threatening complication of large hemispheric infarction (LHI), and is often treated with osmotherapy. In this exploratory analysis of data from the GAMES-RP study, we hypothesized that patients receiving osmotherapy had symptomatic cerebral edema, and that treatment with intravenous (IV) glibenclamide would modify osmotherapy use as compared with placebo. METHODS: GAMES-RP was a phase 2 multi-center prospective, double blind, randomized, placebo-controlled study in LHI. Patients were randomized to IV glibenclamide (e.g. IV glyburide) or placebo. Cerebral edema therapies included osmotherapy and/or decompressive craniectomy at the discretion of the treating team. Total bolus osmotherapy dosing was quantified by "osmolar load". Radiographic edema was defined by dichotomizing midline shift at 24 h. Clinical changes were defined as any increase in NIHSS1a. RESULTS: Osmotherapy was administered to 40 of the 77 patients at a median of 39 [27-55] h after stroke onset. The median baseline DWI lesion volume was significantly larger in the osmotherapy treated group (167 [146-211] mL v. 139 [112-170] mL; P=0.046). Adjudicated malignant edema (75% v. 16%; P<0.001) was more common in the osmotherapy treated group. There were no differences in the proportion of patients receiving osmotherapy or the median total osmolar load between treatment arms. Most patients (76%) had a decrease in consciousness (NIHSS item 1A ≥1) on the day they began receiving osmotherapy. CONCLUSIONS: In the GAMES-RP trial, osmolar therapies were most often administered in response to clinical symptoms of decreased consciousness. However, the optimal timing of administration and impact on outcome after LHI have yet to be defined.

The impact of avocado intake on anthropometric measures among Hispanic/Latino children and adolescents: A cluster randomized controlled trial.

BACKGROUND/OBJECTIVE: While the association between avocado consumption and low metabolic risk has been shown in some studies conducted in adults, little is known about the potential effects of avocado consumption on health outcomes in children and adolescents. Thus, we investigated the impact of two levels of avocado allotment, plus a standard nutrition education, on measures of adiposity in children and adolescents (<18 years old). METHODS: Children (aged 5-12, n = 58) and adolescents (aged 13-17, n = 32) in seventy-two families that self-identified as Hispanic, with at least 3 members over the age of 5 that resided in the same home, were free of severe chronic disease, and not on specific diets, were randomized to one of two levels of avocado allotment plus bi-weekly nutrition education sessions. Low allotment families received 3 avocados per week, while high allotment families received 14 avocados per week for 6 months. We performed an intention-to-treat analysis, using unpaired, 2-sided t-tests to test the mean changes in anthropometric measures of adiposity (body mass index (BMI), waist circumference, hip circumference, and weight) between children and adolescents from high and low allotment families after the 6-month intervention. RESULTS: At six months, there were no significant differences in body mass index, waist circumference, hip circumference, or waist circumference to weight ratio by avocado allotment group. In children, there was a significant difference in weight (difference in means: 1.10, 95% CI: 0.09, 2.10, p-value = 0.03) and waist circumference to height ratio (difference in means: 0.27, 95% CI: 0.12, 0.41, p-value <0.01) between the avocado allotment groups at six months, but these did not remain significant after sensitivity analyses including per-protocol analyses. In adolescents only, there was a significant reduction in waist to hip circumference ratio in the high allotment group compared to the low allotment group after 6 months (difference in means: -0.05, 95% CI: -0.08, 0.00, p-value = 0.04) that persisted after multiple sensitivity analyses. CONCLUSIONS: Different levels of avocado availability among children and adolescents does not appear to result in significant changes in anthropometric measures. Further study is needed to determine whether avocado consumption promotes metabolic health in this age group.

Angiotensin II in liver transplantation (AngLT-1): protocol of a randomised, double-blind, placebo-controlled trial.

INTRODUCTION: Catecholamine vasopressors such as norepinephrine are the standard drugs used to maintain mean arterial pressure during liver transplantation. At high doses, catecholamines may impair organ perfusion. Angiotensin II is a peptide vasoconstrictor that may improve renal perfusion pressure and glomerular filtration rate, a haemodynamic profile that could reduce acute kidney injury. Angiotensin II is approved for vasodilatory shock but has not been rigorously evaluated for treatment of hypotension during liver transplantation. The objective is to assess the efficacy of angiotensin II as a second-line vasopressor infusion during liver transplantation. This trial will establish the efficacy of angiotensin II in decreasing the dose of norepinephrine to maintain adequate blood pressure. Completion of this study will allow design of a follow-up, multicentre trial powered to detect a reduction of organ injury in liver transplantation. METHODS AND ANALYSIS: This is a double-blind, randomised clinical trial. Eligible subjects are adults with a Model for End-Stage Liver Disease Sodium Score ≥25 undergoing deceased donor liver transplantation. Subjects are randomised 1:1 to receive angiotensin II or saline placebo as the second-line vasopressor infusion. The study drug infusion is initiated on reaching a norepinephrine dose of 0.05 µg kg-1 min-1 and titrated per protocol. The primary outcome is the dose of norepinephrine required to maintain a mean arterial pressure ≥65 mm Hg. Secondary outcomes include vasopressin or epinephrine requirement and duration of hypotension. Safety outcomes include incidence of thromboembolism within 48 hours of the end of surgery and severe hypertension. An intention-to-treat analysis will be performed for all randomised subjects receiving the study drug. The total dose of norepinephrine will be compared between the two arms by a one-tailed Mann-Whitney U test. ETHICS AND DISSEMINATION: The trial protocol was approved by the local Institutional Review Board (#20-30948). Results will be posted on ClinicalTrials.gov and published in a peer-reviewed journal. TRIAL REGISTRATION NUMBER: ClinicalTrials.govNCT04901169.

Evidence for involvement of GNB1L in autism.

Structural variations in the chromosome 22q11.2 region mediated by nonallelic homologous recombination result in 22q11.2 deletion (del22q11.2) and 22q11.2 duplication (dup22q11.2) syndromes. The majority of del22q11.2 cases have facial and cardiac malformations, immunologic impairments, specific cognitive profile and increased risk for schizophrenia and autism spectrum disorders (ASDs). The phenotype of dup22q11.2 is frequently without physical features but includes the spectrum of neurocognitive abnormalities. Although there is substantial evidence that haploinsufficiency for TBX1 plays a role in the physical features of del22q11.2, it is not known which gene(s) in the critical 1.5 Mb region are responsible for the observed spectrum of behavioral phenotypes. We identified an individual with a balanced translocation 46,XY,t(1;22)(p36.1;q11.2) and a behavioral phenotype characterized by cognitive impairment, autism, and schizophrenia in the absence of congenital malformations. Using somatic cell hybrids and comparative genomic hybridization (CGH) we mapped the chromosome-22 breakpoint within intron 7 of the GNB1L gene. Copy number evaluations and direct DNA sequencing of GNB1L in 271 schizophrenia and 513 autism cases revealed dup22q11.2 in two families with autism and private GNB1L missense variants in conserved residues in three families (P = 0.036). The identified missense variants affect residues in the WD40 repeat domains and are predicted to have deleterious effects on the protein. Prior studies provided evidence that GNB1L may have a role in schizophrenia. Our findings support involvement of GNB1L in ASDs as well.

Social Determinants of Digital Health Adoption: Pilot Cross-sectional Survey.

BACKGROUND: Interest in and funding for digital health interventions have rapidly grown in recent years. Despite the increasing familiarity with mobile health from regulatory bodies, providers, and patients, overarching research on digital health adoption has been primarily limited to morbidity-specific and non-US samples. Consequently, there is a limited understanding of what personal factors hold statistically significant relationships with digital health uptake. Moreover, this limits digital health communities' knowledge of equity along digital health use patterns. OBJECTIVE: This study aims to identify the social determinants of digital health tool adoption in Georgia. METHODS: Web-based survey respondents in Georgia 18 years or older were recruited from mTurk to answer primarily closed-ended questions within the following domains: participant demographics and health consumption background, telehealth, digital health education, prescription management tools, digital mental health services, and doctor finder tools. Participants spent around 15 to 20 minutes on a survey to provide demographic and personal health care consumption data. This data was analyzed with multivariate linear and logistic regressions to identify which of these determinants, if any, held statistically significant relationships with the total number of digital health tool categories adopted and which of these determinants had absolute relationships with specific categories. RESULTS: A total of 362 respondents completed the survey. Private insurance, residence in an urban area, having a primary care provider, fewer urgent emergency room (ER) visits, more ER visits leading to inpatient stays, and chronic condition presence were significantly associated with the number of digital health tool categories adopted. The separate logistic regressions exhibited substantial variability, with 3.5 statistically significant predictors per model, on average. Age, federal poverty level, number of primary care provider visits in the past 12 months, number of nonurgent ER visits in the past 12 months, number of urgent ER visits in the past 12 months, number of ER visits leading to inpatient stays in the past 12 months, race, gender, ethnicity, insurance, education, residential area, access to the internet, difficulty accessing health care, usual source of care, status of primary care provider, and status of chronic condition all had at least one statistically significant relationship with the use of a specific digital health category. CONCLUSIONS: The results demonstrate that persons who are socioeconomically disadvantaged may not adopt digital health tools at disproportionately higher rates. Instead, digital health tools may be adopted along social determinants of health, providing strong evidence for the digital health divide. The variability of digital health adoption necessitates investing in and building a common framework to increase mobile health access. With a common framework and a paradigm shift in the design, evaluation, and implementation strategies around digital health, disparities can be further mitigated and addressed. This likely will begin with a coordinated effort to determine barriers to adopting digital health solutions.

Constant Light and Frequent Schedule Changes Do Not Impact Resistance to Parasites in Monarch Butterflies.

Organisms have evolved internal biological clocks to regulate their activities based on external environmental cues, such as light, temperature, and food. Environmental disruption of these rhythms, such as caused by constant light or frequent light schedule changes, has been shown to impair development, reduce survival, and increase infection susceptibility and disease progression in numerous organisms. However, the precise role of the biological clock in host-parasite interactions is understudied and has focused on unnatural host-parasite combinations in lab-adapted inbred models. Here, we use the natural interaction between monarch butterflies (Danaus plexippus) and their virulent protozoan parasite, Ophryocystis elektroscirrha, to investigate the effects of constant light and frequent light schedule changes on development, survival, and parasite susceptibility. We show that constant light exposure slows the monarchs' rate of development but does not increase susceptibility to parasitic infection. Furthermore, frequent schedule changes decrease parasite growth, but have no effect on egg-to-adult survival of infected monarchs. Interestingly, these conditions are usually disruptive to the biological clock, but do not significantly impact the clock of monarch larvae. These unexpected findings show that constant light and frequent schedule changes can uncouple host and parasite performance and highlight how natural relationships are needed to expand our understanding of clocks in host-parasite interactions.

Lipid transport by TMEM24 at ER-plasma membrane contacts regulates pulsatile insulin secretion.

Insulin is released by ß cells in pulses regulated by calcium and phosphoinositide signaling. Here, we describe how transmembrane protein 24 (TMEM24) helps coordinate these signaling events. We showed that TMEM24 is an endoplasmic reticulum (ER)-anchored membrane protein whose reversible localization to ER-plasma membrane (PM) contacts is governed by phosphorylation and dephosphorylation in response to oscillations in cytosolic calcium. A lipid-binding module in TMEM24 transports the phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] precursor phosphatidylinositol between bilayers, allowing replenishment of PI(4,5)P2 hydrolyzed during signaling. In the absence of TMEM24, calcium oscillations are abolished, leading to a defect in triggered insulin release. Our findings implicate direct lipid transport between the ER and the PM in the control of insulin secretion, a process impaired in patients with type II diabetes.

Epsin deficiency impairs endocytosis by stalling the actin-dependent invagination of endocytic clathrin-coated pits.

Epsin is an evolutionarily conserved endocytic clathrin adaptor whose most critical function(s) in clathrin coat dynamics remain(s) elusive. To elucidate such function(s), we generated embryonic fibroblasts from conditional epsin triple KO mice. Triple KO cells displayed a dramatic cell division defect. Additionally, a robust impairment in clathrin-mediated endocytosis was observed, with an accumulation of early and U-shaped pits. This defect correlated with a perturbation of the coupling between the clathrin coat and the actin cytoskeleton, which we confirmed in a cell-free assay of endocytosis. Our results indicate that a key evolutionary conserved function of epsin, in addition to other roles that include, as we show here, a low affinity interaction with SNAREs, is to help generate the force that leads to invagination and then fission of clathrin-coated pits.

Legionella pneumophila infection of Drosophila S2 cells induces only minor changes in mitochondrial dynamics.

During infection of cells by Legionella pneumophila, the bacterium secretes a large number of effector proteins into the host cell cytoplasm, allowing it to alter many cellular processes and make the vacuole and the host cell into more hospitable environments for bacterial replication. One major change induced by infection is the recruitment of ER-derived vesicles to the surface of the vacuole, where they fuse with the vacuole membrane and prevent it from becoming an acidified, degradative compartment. However, the recruitment of mitochondria to the region of the vacuole has also been suggested by ultrastructural studies. In order to test this idea in a controlled and quantitative experimental system, and to lay the groundwork for a genome-wide screen for factors involved in mitochondrial recruitment, we examined the behavior of mitochondria during the early stages of Legionella pneumophila infection of Drosophila S2 cells. We found that the density of mitochondria near vacuoles formed by infection with wild type Legionella was not different from that found in dotA(-) mutant-infected cells during the first 4 hours after infection. We then examined 4 parameters of mitochondrial motility in infected cells: velocity of movement, duty cycle of movement, directional persistence and net direction. In the 4 hours following infection, most of these measures were indistinguishable between wild type and dotA(-).infection. However, wild type Legionella did induce a modest shift in the velocity distribution toward faster movement compared dotA(-) infection, and a small downward shift in the duty cycle distribution. In addition, wild type infection produced mitochondrial movement that was biased in the direction of the bacterial vacuole relative to dotA-, although not enough to cause a significant accumulation within 10 um of the vacuole. We conclude that in this host cell, mitochondria are not strongly recruited to the vacuole, nor is their motility dramatically affected.