DAF-16/FOXO and HLH-30/TFEB comprise a cooperative regulatory axis controlling tubular lysosome induction in C. elegans.

Although life expectancy has increased, longer lifespans do not always align with prolonged healthspans and, as a result, the occurrence of age-related degenerative diseases continues to increase. Thus, biomedical research has been shifting focus to strategies that enhance both lifespan and healthspan concurrently. Two major transcription factors that have been heavily studied in the context of aging and longevity are DAF-16/FOXO and HLH-30/TFEB; however, how these two factors coordinate to promote longevity is still not fully understood. In this study, we reveal a new facet of their cooperation that supports healthier aging in C. elegans. Namely, we demonstrate that the combinatorial effect of daf-16 and hlh-30 is required to trigger robust lysosomal tubulation, which contributes to systemic health benefits in late age by enhancing cross-tissue proteostasis mechanisms. Remarkably, this change in lysosomal morphology can be artificially induced via overexpression of SVIP, a previously characterized tubular lysosome stimulator, even when one of the key transcription factors, DAF-16, is absent. This adds to growing evidence that SVIP could be utilized to employ tubular lysosome activity in adverse conditions or disease states. Mechanistically, intestinal overexpression of SVIP leads to nuclear accumulation of HLH-30 in gut and non-gut tissues and triggers global gene expression changes that promotes systemic health benefits. Collectively, our work reveals a new cellular process that is under the control of DAF-16 and HLH-30 and provides further insight into how these two transcription factors may be exerting their pro-health effects.

The Montana Interfacility Blood Network: A Novel Lifesaving "Hand-off" for the Optimal Care of Rural Patients.

Purpose: The state of Montana encompasses and defines rural health care as it is known in the United States (US) today. This vast area is punctuated by pockets of health care availability with varying access to blood products for transfusion. Furthermore, timely transport is frequently challenged by weather that may limit air transportation options, resulting in multiple hours in ground transport to definitive care. Patients and Methods: The Montana State Trauma Care Committee (MT-STCC) developed the Montana Interfacility Blood Network (MT-IBN) to ensure blood availability in geographically distanced cases where patients may otherwise not survive. The index case that led to the formal development of the MT-IBN is described, followed by a second case illustrating the IBN process. Results: This process and development manuscript details the innovative efforts of MT-STCC to develop this fledgling idea unique to rural US health care. We review guidelines that have been developed to define broad aspects of the MT-IBN including the reason to share resources, proper packaging, paperwork necessary for transfer, and how to provide resources directly to the patient. Finally, we describe implementation within the state. Conclusion: The MT-IBN was developed by MT-STCC to facilitate the hand-off of lifesaving blood to patients being transported by ground to definitive care in Montana without having to stop at an intermediary facility. This has already led to lives saved in areas that are limited in blood availability due to rurality.

Restricting bioenergetic efficiency enhances longevity and mitochondrial redox capacity in Drosophila melanogaster.

Mitochondria are essential for survival and as such, impairments in organelle homeostasis significantly accelerate age-related morbidity and mortality. Here, we determined the contribution of bioenergetic efficiency to life span and health span in Drosophila melanogaster utilizing the mitochondrial uncoupler BAM15. Life span was determined in flies fed a normal diet (ND) or high fat diet (HFD) supplemented with vehicle or BAM15. Locomotor function was determined by negative geotaxis assay in middle-aged flies fed vehicle or BAM15 under ND or HFD conditions. Redox capacity (high-resolution respirometry/fluorometry), citrate synthase (enzyme activity), mtDNA content (qPCR), gene expression (qPCR), and protein expression (western blot) were assessed in flight muscle homogenates of middle-aged flies fed vehicle or BAM15 ND. The molar ratio of H2 O2 and O2 (H2 O2 :O2 ) in a defined respiratory state was calculated as a measure of redox balance. BAM15 extended life span by 9% on ND and 25% on HFD and improved locomotor activity by 125% on ND and 53% on HFD. Additionally, BAM15 enhanced oxidative phosphorylation capacity supported by pyruvate + malate, proline, and glycerol 3-phosphate. Concurrently, BAM15 enhanced the mitochondrial H2 O2 production rate, reverse electron flow from mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) to Complex I, mGPDH, and Complex I without altering the H2 O2 :O2 ratio. BAM15 upregulated transcriptional signatures associated with mitochondrial function and fitness as well as antioxidant defense. BAM15-mediated restriction of bioenergetic efficiency prolongs life span and health span in Drosophila fed a ND or HFD. Improvements in life span and health span in ND were supported by synergistic enhancement of muscular redox capacity.

Modular Splicing Is Linked to Evolution in the Synapse-Specificity Molecule Kirrel3.

Kirrel3 is a cell-adhesion molecule that instructs the formation of specific synapses during brain development in mouse and Kirrel3 variants may be risk factors for autism and intellectual disabilities in humans. Kirrel3 is predicted to undergo alternative splicing but brain isoforms have not been studied. Here, we present the first in-depth characterization of Kirrel3 isoform diversity in brain using targeted, long-read mRNA sequencing of mouse hippocampus. We identified 19 isoforms with predicted transmembrane and secreted forms and show that even rare isoforms generate detectable protein in the brain. We also analyzed publicly-available long-read mRNA databases from human brain tissue and found 11 Kirrel3 isoforms that, similar to mouse, encode transmembrane and secreted forms. In mice and humans, Kirrel3 diversity arises from alternative, independent use of protein-domain coding exons and alternative early translation-stop signals. Intriguingly, the alternatively spliced exons appear at branch points in the chordate phylogenetic tree, including one exon only found in humans and their closest living relatives, the great apes. Together, these results validate a simple pipeline for analyzing isoform diversity in genes with low expression and suggest that Kirrel3 function is fine-tuned by alternative splicing and may play a role in brain evolution.

Tubular lysosome induction couples animal starvation to healthy aging.

Dietary restriction promotes longevity in several species via autophagy activation. However, changes to lysosomes underlying this effect remain unclear. Here using the nematode Caenorhabditis elegans, we show that the induction of autophagic tubular lysosomes (TLs), which occurs upon dietary restriction or mechanistic target of rapamycin inhibition, is a critical event linking reduced food intake to lifespan extension. We find that starvation induces TLs not only in affected individuals but also in well-fed descendants, and the presence of gut TLs in well-fed progeny is predictive of enhanced lifespan. Furthermore, we demonstrate that expression of Drosophila small VCP-interacting protein, a TL activator in flies, artificially induces TLs in well-fed worms and improves C. elegans health in old age. These findings identify TLs as a new class of lysosomes that couples starvation to healthy aging.

A familial natural short sleep mutation promotes healthy aging and extends lifespan in Drosophila.

Sleep loss typically imposes negative effects on animal health. However, humans with a rare genetic mutation in the dec2 gene (dec2P384R) present an exception; these individuals sleep less without the usual effects associated with sleep deprivation. Thus, it has been suggested that the dec2P384R mutation activates compensatory mechanisms that allows these individuals to thrive with less sleep. To test this directly, we used a Drosophila model to study the effects of the dec2P384R mutation on animal health. Expression of human dec2P384R in fly sleep neurons was sufficient to mimic the short sleep phenotype and, remarkably, dec2P384R mutants lived significantly longer with improved health despite sleeping less. The improved physiological effects were enabled, in part, by enhanced mitochondrial fitness and upregulation of multiple stress response pathways. Moreover, we provide evidence that upregulation of pro-health pathways also contributes to the short sleep phenotype, and this phenomenon may extend to other pro-longevity models.

VCP promotes tTAF-target gene expression and spermatocyte differentiation by downregulating mono-ubiquitylated H2A.

Valosin-containing protein (VCP) binds and extracts ubiquitylated cargo to regulate protein homeostasis. VCP has been studied primarily in aging and disease contexts, but it also affects germline development. However, the precise molecular functions of VCP in the germline, particularly in males, are poorly understood. Using the Drosophila male germline as a model system, we find that VCP translocates from the cytosol to the nucleus as germ cells transition into the meiotic spermatocyte stage. Importantly, nuclear translocation of VCP appears to be one crucial event stimulated by testis-specific TBP-associated factors (tTAFs) to drive spermatocyte differentiation. VCP promotes the expression of several tTAF-target genes, and VCP knockdown, like tTAF loss of function, causes cells to arrest in early meiotic stages. At a molecular level, VCP activity supports spermatocyte gene expression by downregulating a repressive histone modification, mono-ubiquitylated H2A (H2Aub), during meiosis. Remarkably, experimentally blocking H2Aub in VCP-RNAi testes is sufficient to overcome the meiotic-arrest phenotype and to promote development through the spermatocyte stage. Collectively, our data highlight VCP as a downstream effector of tTAFs that downregulates H2Aub to facilitate meiotic progression.

Rbp4-Gal4, a germline driver that activates in meiosis, reveals functions for VCP in spermatid development.

Valosin-containing protein (VCP) is a versatile and ubiquitously expressed AAA+ ATPase that regulates multiple stages of Drosophila spermatogenesis. While VCP has documented roles in mitotic spermatogonia and meiotic spermatocytes, it is also highly expressed in post-meiotic spermatids, suggesting potential late-stage developmental functions as well. However, tools to assess late-stage activities of pleiotropic spermatogenesis genes such as VCP are lacking. Available germline-specific Gal4 drivers activate in stem cells or spermatogonia; consequently, knocking down VCP using one of these drivers disrupts or blocks early germ-cell development, precluding analysis of VCP in later stages. A Gal4 driver that activates later in development, such as at the meiotic spermatocyte stage, may permit functional analyses of VCP and other factors in post-meiotic stages. Here, we describe a germline-specific Gal4 driver, Rbp4-Gal4, which drives transgene expression beginning in the early spermatocyte stage. We find that Rbp4-Gal4-driven knockdown of VCP causes defects in spermatid chromatin condensation and individualization without affecting earlier developmental stages. Interestingly, the defect in chromatin condensation appears linked to errors in the histone-to-protamine transition, a key event in spermatid development. Overall, our study reveals roles for VCP in spermatid development and establishes a powerful tool to dissect the functions of pleiotropic spermatogenesis genes.

Comparison of activity, structure, and dynamics of SF-1 and LRH-1 complexed with small molecule modulators.

Steroidogenic factor-1 (SF-1) is a phospholipid-sensing nuclear receptor expressed in the adrenal glands, gonads, and hypothalamus which controls steroidogenesis and metabolism. There is significant therapeutic interest in SF-1 because of its oncogenic properties in adrenocortical cancer. Synthetic modulators are attractive for targeting SF-1 for clinical and laboratory purposes due to the poor pharmaceutical properties of its native phospholipid ligands. While small molecule agonists targeting SF-1 have been synthesized, no crystal structures have been reported of SF-1 in complexes with synthetic compounds. This has prevented the establishment of structure-activity relationships that would enable better characterization of ligand-mediated activation and improvement in current chemical scaffolds. Here, we compare the effects of small molecules in SF-1 and its close homolog, liver receptor homolog-1 (LRH-1), and identify several molecules that specifically activate LRH-1. We also report the first crystal structure of SF-1 in complex with a synthetic agonist that displays low nanomolar affinity and potency for SF-1. We use this structure to explore the mechanistic basis for small molecule agonism of SF-1, especially compared to LRH-1, and uncover unique signaling pathways that drive LRH-1 specificity. Molecular dynamics simulations reveal differences in protein dynamics at the pocket mouth as well as ligand-mediated allosteric communication from this region to the coactivator binding interface. Our studies, therefore, shed important insight into the allostery driving SF-1 activity and show potential for modulation of LRH-1 over SF-1.

A familial natural short sleep mutation promotes healthy aging and extends lifespan in Drosophila.

Sleep loss typically imposes negative effects on animal health. However, humans with a rare genetic mutation in the dec2 gene ( dec2 P384R ) present an exception; these individuals sleep less without the usual effects associated with sleep deprivation. Thus, it has been suggested that the dec2 P384R mutation activates compensatory mechanisms that allows these individuals to thrive with less sleep. To test this directly, we used a Drosophila model to study the effects of the dec2 P384R mutation on animal health. Expression of human dec2 P384R in fly sleep neurons was sufficient to mimic the short sleep phenotype and, remarkably, dec2 P384R mutants lived significantly longer with improved health despite sleeping less. The improved physiological effects were enabled, in part, by enhanced mitochondrial fitness and upregulation of multiple stress response pathways. Moreover, we provide evidence that upregulation of pro-health pathways also contributes to the short sleep phenotype, and this phenomenon may extend to other pro-longevity models.

Use of the Esophageal Sponge in Directing Food Reintroduction in Eosinophilic Esophagitis.

BACKGROUND & AIMS: Dietary therapy is successful in eosinophilic esophagitis (EoE) but requires multiple upper endoscopies. The aim of this study was to determine if food reintroduction in EoE can be directed by minimally-invasive esophageal sponge cytology. METHODS: In this prospective non-blinded trial, 22 responders to 6-food elimination diets underwent sequential food reintroduction guided by esophageal sponge cytology. Foods were reintroduced followed by unsedated esophageal sponge cytology assessment. A food trigger was defined by sponge cytology peak eosinophil count of ≥15 eos/high-powered field (hpf). Symptoms (EoE symptom activity index [EEsAI]), endoscopic score (EoE endoscopic reference score [EREFS]), and biopsy histology (peak eosinophil count) were collected pre-dietary therapy and post-dietary therapy, and then 4 weeks post food reintroduction. RESULTS: The EEsAI and EREFS were similar post-dietary therapy to post-food reintroduction: 12.0 (interquartile range [IQR], 0.0-27.0) vs 16.5 (IQR, 9.0-28.8) (P = .265) and 1.5 (IQR, 0.2-3.0) vs 1.0 (IQR, 0.0-2.0) (P = .185). However, the peak eosinophil count was increased post-food reintroduction compared with post-dietary therapy: 20.0 (IQR, 5.0-51.5) vs 2.0 (IQR, 1.0-4.0) (P < .001), suggesting a failure of identification of all food triggers. The peak eosinophil count was lower post-food reintroduction compared with pre-dietary therapy: 20.0 (IQR, 5.0-51.5) vs 52.0 (IQR, 30.8-76.2) (P = .008). At the post food reintroduction evaluation, sponge cytology and biopsy histology were in agreement in 59% (13/22) of cases using a cutoff of <15 eos/hpf and 68% (15/22) of cases using a cutoff of <6 eos/hpf. CONCLUSIONS: In the first study to evaluate a non-endoscopic technique in the clinical management of EoE, the esophageal sponge was moderately successful at guiding food reintroduction in EoE dietary responders in the outpatient setting. CLINICALTRIALS: gov, Number NCT02599558.

Public Attitudes toward Pharmacogenomic Testing and Establishing a Statewide Pharmacogenomics Database in the State of Minnesota.

The clinical adoption and implementation of pharmacogenomics (PGx) beyond academic medical centers remains slow, restricting the general population from benefitting from this important component of personalized medicine. As an initial step in the statewide initiative of PGx implementation in Minnesota, we engaged community members and assessed attitudes towards PGx testing and acceptability of establishing a secure statewide PGx database for clinical and research use among Minnesota residents. Data was collected from 808 adult attendees at the 2021 Minnesota State Fair through an electronic survey. Eighty-four percent of respondents felt comfortable getting a PGx test for clinical care. Most respondents trusted health professionals (78.2%) and researchers (73.0%) to keep their PGx data private. The majority expressed their support and interest in participating in a statewide PGx database for clinical and research use (64-72%). Higher acceptability of the statewide PGx database was associated with younger age, higher education, higher health literacy, having health insurance, and prior genetic testing. The study sample representing Minnesota residents expressed high acceptability of receiving PGx testing and willingness to participate in PGx data sharing for clinical and research use. Community support and engagement are needed to advance PGx implementation and research on the state scale.

Development and Validation of the Minnesota Assessment of Pharmacogenomic Literacy (MAPL).

Ensuring that patients have an adequate understanding of pharmacogenomic (PGx) test results is a critical component of implementing precision medicine into clinical care. However, no PGx-specific validated literacy assessment has yet been developed. To address this need, we developed and validated the Minnesota Assessment of Pharmacogenomic Literacy (MAPLTM). Foundational work included a scoping review of patient and general public attitudes and experiences with pharmacogenomic testing, three focus groups, readability assessments, and review by experts and members of the general public. This resulted in a 15-item assessment designed to assess knowledge in four domains: underlying concepts, limitations, benefits, and privacy. For validation, 646 participants completed the MAPL as a part of a larger survey about pharmacogenomic research and statewide PGx implementation. Two items were deemed to be "too easy" and dropped. The remaining 13 items were retained in the final MAPL with good internal reliability (Cronbach's alpha = 0.75). Confirmatory factor analysis validated the four-domain construct of MAPL and suggested good model performance and high internal validity. The estimated coefficient loadings across 13 questions on the corresponding domains are all positive and statistically significant (p < 0.05). The MAPL covers multiple knowledge domains of specific relevance to PGx and is a useful tool for clinical and research settings where quantitative assessment of PGx literacy is of value.

Branching Off: New Insight Into Lysosomes as Tubular Organelles.

Lysosomes are acidic, membrane-bound organelles that play essential roles in cellular quality control, metabolism, and signaling. The lysosomes of a cell are commonly depicted as vesicular organelles. Yet, lysosomes in fact show a high degree of ultrastructural heterogeneity. In some biological contexts, lysosome membranes naturally transform into tubular, non-vesicular morphologies. Though the purpose and regulation of tubular lysosomes has been historically understudied, emerging evidence suggests that tubular lysosomes may carry out unique activities, both degradative and non-degradative, that are critical to cell behavior, function, and viability. Here, we discuss recent advances in understanding the biological significance of tubular lysosomes in cellular physiology, and we highlight a growing number of examples that indicate the centrality of this special class of lysosomes to health and disease.

A meiotic switch in lysosome activity supports spermatocyte development in young flies but collapses with age.

Gamete development ultimately influences animal fertility. Identifying mechanisms that direct gametogenesis, and how they deteriorate with age, may inform ways to combat infertility. Recently, we found that lysosomes acidify during oocyte maturation in Caenorhabditis elegans, suggesting that a meiotic switch in lysosome activity promotes female germ-cell health. Using Drosophila melanogaster, we report that lysosomes likewise acidify in male germ cells during meiosis. Inhibiting lysosomes in young-male testes causes E-cadherin accumulation and loss of germ-cell partitioning membranes. Notably, analogous changes occur naturally during aging; in older testes, a reduction in lysosome acidity precedes E-cadherin accumulation and membrane dissolution, suggesting one potential cause of age-related spermatocyte abnormalities. Consistent with lysosomes governing the production of mature sperm, germ cells with homozygous-null mutations in lysosome-acidifying machinery fail to survive through meiosis. Thus, lysosome activation is entrained to meiotic progression in developing sperm, as in oocytes, and lysosomal dysfunction may instigate male reproductive aging.

Reproductive tradeoffs govern sexually dimorphic tubular lysosome induction in Caenorhabditis elegans.

Sex-specific differences in animal behavior commonly reflect unique reproductive interests. In the nematode Caenorhabditis elegans, hermaphrodites can reproduce without a mate and thus prioritize feeding to satisfy the high energetic costs of reproduction. However, males, which must mate to reproduce, sacrifice feeding to prioritize mate-searching behavior. Here, we demonstrate that these behavioral differences influence sexual dimorphism at the organelle level; young males raised on a rich food source show constitutive induction of gut tubular lysosomes, a non-canonical lysosome morphology that forms in the gut of hermaphrodites when food is limited or as animals age. We found that constitutive induction of gut tubular lysosomes in males results from self-imposed dietary restriction through DAF-7/TGFß, which promotes exploratory behavior. In contrast, age-dependent induction of gut tubular lysosomes in hermaphrodites is stimulated by self-fertilization activity. Thus, separate reproductive tradeoffs influence tubular lysosome induction in each sex, potentially supporting different requirements for reproductive success.

Differential Modulation of Nuclear Receptor LRH-1 through Targeting Buried and Surface Regions of the Binding Pocket.

Liver receptor homologue-1 (LRH-1) is a phospholipid-sensing nuclear receptor that has shown promise as a target for alleviating intestinal inflammation and metabolic dysregulation in the liver. LRH-1 contains a large ligand-binding pocket, but generating synthetic modulators has been challenging. We have had recent success generating potent and efficacious agonists through two distinct strategies. We targeted residues deep within the pocket to enhance compound binding and residues at the mouth of the pocket to mimic interactions made by phospholipids. Here, we unite these two designs into one molecule to synthesize the most potent LRH-1 agonist to date. Through a combination of global transcriptomic, biochemical, and structural studies, we show that selective modulation can be driven through contacting deep versus surface polar regions in the pocket. While deep pocket contacts convey high affinity, contacts with the pocket mouth dominate allostery and provide a phospholipid-like transcriptional response in cultured cells.

Degradative tubular lysosomes link pexophagy to starvation and early aging in C. elegans.

Organelle-specific autophagy directs degradation of eukaryotic organelles under certain conditions. Like other organelles, peroxisomes are subject to autophagic turnover at lysosomes. However, peroxisome autophagy (pexophagy) has yet to be analyzed in a live-animal system, limiting knowledge on its regulation during an animal's life. Here, we generated a tandem-fluorophore reporter that enabled real-time tracking of pexophagy in live Caenorhabditis elegans. We observed that pexophagy occurred at a population of non-canonical, tubular lysosomes specifically during starvation and aging. Remarkably, in these contexts, tubular lysosomes were the predominant type of lysosome in the intestine, transforming from vesicles. Though we found that peroxisomes were largely eliminated in early adulthood, they appeared restored in new generations. We identified peroxisomal genes that regulated age-dependent peroxisome loss and demonstrated that modifying this process altered animal lifespan. These findings reveal new facets of peroxisome homeostasis relevant to aging and challenge the prevailing perception of lysosome homogeneity in autophagy.Abbreviations: GFP: green fluorescent protein; SKL: serine-lysine-leucine peroxisome signal sequence; spin: spinster; TLs: tubular lysosomes.

Localization of the ubiquitin ligase Dma1 to the fission yeast contractile ring is modulated by phosphorylation.

The timing of cytokinesis relative to other mitotic events in the fission yeast Schizosaccharomyces pombe is controlled by the septation initiation network (SIN). During a mitotic checkpoint, the SIN is inhibited by the E3 ubiquitin ligase Dma1 to prevent chromosome mis-segregation. Dma1 dynamically localizes to spindle pole bodies (SPBs) and the contractile ring (CR) during mitosis, though its role at the CR is unknown. Here, we examined whether Dma1 phosphorylation affects its localization or function. We found that preventing Dma1 phosphorylation by substituting the six phosphosites with alanines diminished its CR localization but did not affect its mitotic checkpoint function. These studies reinforce the conclusion that Dma1 localization to the SPB is key to its role in the mitotic checkpoint.

Phosphorylation in the intrinsically disordered region of F-BAR protein Imp2 regulates its contractile ring recruitment.

The F-BAR protein Imp2 is an important contributor to cytokinesis in the fission yeast Schizosaccharomyces pombe. Because cell cycle-regulated phosphorylation of the central intrinsically disordered region (IDR) of the Imp2 paralog Cdc15 controls Cdc15 oligomerization state, localization and ability to bind protein partners, we investigated whether Imp2 is similarly phosphoregulated. We found that Imp2 is endogenously phosphorylated on 28 sites within its IDR, with the bulk of phosphorylation being constitutive. In vitro, the casein kinase 1 (CK1) isoforms Hhp1 and Hhp2 can phosphorylate 17 sites, and Cdk1 (also known as Cdc2) can phosphorylate the remaining 11 sites. Mutations that prevent Cdk1 phosphorylation result in precocious Imp2 recruitment to the cell division site, and mutations designed to mimic these phosphorylation events delay Imp2 accumulation at the contractile ring (CR). Mutations that eliminate CK1 phosphorylation sites allow CR sliding, and phosphomimetic substitutions at these sites reduce Imp2 protein levels and slow CR constriction. Thus, like Cdc15, the Imp2 IDR is phosphorylated at many sites by multiple kinases. In contrast to Cdc15, for which phosphorylation plays a major cell cycle regulatory role, Imp2 phosphorylation is primarily constitutive, with milder effects on localization and function. This article has an associated First Person interview with the first author of the paper.