Quantitative Assessment of Morphological Changes in Lipid Droplets and Lipid-Mito Interactions with Aging in Brown Adipose.

The physical characteristics of brown adipose tissue (BAT) are defined by the presence of multilocular lipid droplets (LD) within the brown adipocytes and a high abundance of iron-containing mitochondria, which give it its characteristic color. Normal mitochondrial function is, in part, regulated by organelle-to-organelle contacts. Particularly, the contact sites that mediate mitochondria-LD interactions are thought to have various physiological roles, such as the synthesis and metabolism of lipids. Aging is associated with mitochondrial dysfunction, and previous studies show that there are changes in mitochondrial structure and proteins that modulate organelle contact sites. However, how mitochondria-LD interactions change with aging has yet to be fully clarified. Therefore, we sought to define age-related changes in LD morphology and mitochondria-lipid interactions in BAT. We examined the three-dimensional morphology of mitochondria and LDs in young (3-month) and aged (2-year) murine BAT using serial block face-scanning electron microscopy and the Amira program for segmentation, analysis, and quantification. Analysis showed reductions in LD volume, area, and perimeter in aged samples compared to young samples. Additionally, we observed changes in LD appearance and type in aged samples compared to young samples. Notably, we found differences in mitochondrial interactions with LDs, which could implicate that these contacts may be important for energetics in aging. Upon further investigation, we also found changes in mitochondrial and cristae structure for mitochondria interacting with LD lipids. Overall, these data define the nature of LD morphology and organelle-organelle contacts during aging and provide insight into LD contact site changes that interconnect biogerontology and mitochondrial functionality, metabolism, and bioactivity in aged BAT.

A review of undergraduate research programs aimed at underrepresented students.

It is well-understood that the science, technology, engineering, and mathematics (STEM) fields have unique challenges that discourage recruiting and retaining underrepresented minorities. Research programs aimed at undergraduates have arisen as a critical mechanism for fostering innovation and addressing the challenges faced by underrepresented minorities. Here, we review various undergraduate research programs designed to provide exposure to undergraduates, with a focus on underrepresented minorities in STEM disciplines. We provide insight into selected programs' objectives, key features, potential limitations, and outcomes. We also offer recommendations for future improvements of each research program, particularly in the context of mentorship. These programs range from broad-reaching initiatives (e.g., Leadership Alliance) to more specific programs targeting underrepresented students. By offering a nuanced understanding of each program's structure, we seek to provide a brief overview of the landscape of diversity-focused STEM initiatives and a guide on how to run a research program effectively.

Ablation of Sam50 is associated with fragmentation and alterations in metabolism in murine and human myotubes.

The sorting and assembly machinery (SAM) Complex is responsible for assembling ß-barrel proteins in the mitochondrial membrane. Comprising three subunits, Sam35, Sam37, and Sam50, the SAM complex connects the inner and outer mitochondrial membranes by interacting with the mitochondrial contact site and cristae organizing system complex. Sam50, in particular, stabilizes the mitochondrial intermembrane space bridging (MIB) complex, which is crucial for protein transport, respiratory chain complex assembly, and regulation of cristae integrity. While the role of Sam50 in mitochondrial structure and metabolism in skeletal muscle remains unclear, this study aims to investigate its impact. Serial block-face-scanning electron microscopy and computer-assisted 3D renderings were employed to compare mitochondrial structure and networking in Sam50-deficient myotubes from mice and humans with wild-type (WT) myotubes. Furthermore, autophagosome 3D structure was assessed in human myotubes. Mitochondrial metabolic phenotypes were assessed using Gas Chromatography-Mass Spectrometry-based metabolomics to explore differential changes in WT and Sam50-deficient myotubes. The results revealed increased mitochondrial fragmentation and autophagosome formation in Sam50-deficient myotubes compared to controls. Metabolomic analysis indicated elevated metabolism of propanoate and several amino acids, including ß-Alanine, phenylalanine, and tyrosine, along with increased amino acid and fatty acid metabolism in Sam50-deficient myotubes. Furthermore, impairment of oxidative capacity was observed upon Sam50 ablation in both murine and human myotubes, as measured with the XF24 Seahorse Analyzer. Collectively, these findings support the critical role of Sam50 in establishing and maintaining mitochondrial integrity, cristae structure, and mitochondrial metabolism. By elucidating the impact of Sam50-deficiency, this study enhances our understanding of mitochondrial function in skeletal muscle.

Metabolite accumulation from oral NMN supplementation drives aging-specific kidney inflammation.

The mitochondrial-rich renal tubule cells are key regulators of blood homeostasis via excretion and reabsorption of metabolic waste. With age, tubules are subject to increasing mitochondrial dysfunction and declining nicotinamide adenine dinucleotide (NAD+) levels, both hampering ATP production efficiency. We tested two mitochondrial interventions in young (6-mo) and aged (26-mo) adult male mice: elamipretide (ELAM), a tetrapeptide in clinical trials that improves mitochondrial structure and function, and nicotinamide mononucleotide (NMN), an NAD+ intermediate and commercially available oral supplement. Kidneys were analyzed from young and aged mice after eight weeks of treatment with ELAM (3 mg/kg/day), NMN (300 mg/kg/day), or from aged mice treated with the two interventions combined (ELAM+NMN). We hypothesized that combining pharmacologic treatments to ameliorate mitochondrial dysfunction and boost NAD+ levels, would more effectively reduce kidney aging than either intervention alone. Unexpectedly, in aged kidneys, NMN increased expression of genetic markers of inflammation (IL-1-beta; and Ccl2) and tubule injury (Kim-1). Metabolomics of endpoint sera showed that NMN-treated aged mice had higher circulating levels of uremic toxins than either aged controls or young NMN-treated mice. ELAM+NMN-treated aged mice accumulated uremic toxins like NMN-only aged mice, but reduced IL-1-beta; and Ccl2 kidney mRNA. This suggests that pre-existing mitochondrial dysfunction in aged kidney underlies susceptibility to inflammatory signaling with NMN supplementation in aged, but not young, mice. These findings demonstrate age and tissue dependent effects on downstream metabolic accumulation from NMN and highlight the need for targeted analysis of aged kidneys to assess the safety of anti-aging supplements in older populations.

Running a successful STEMM summer program: A week-by-week guide.

While some established undergraduate summer programs are effective across many institutions, these programs may only be available to some principal investigators or may not fully address the diverse needs of incoming undergraduates. This article outlines a 10-week science, technology, engineering, mathematics, and medicine (STEMM) education program designed to prepare undergraduate students for graduate school through a unique model incorporating mentoring dyads and triads, cultural exchanges, and diverse activities while emphasizing critical thinking, research skills, and cultural sensitivity. Specifically, we offer a straightforward and adaptable guide that we have used for mentoring undergraduate students in a laboratory focused on mitochondria and microscopy, but which may be customized for other disciplines. Key components include self-guided projects, journal clubs, various weekly activities such as mindfulness training and laboratory techniques, and a focus on individual and cultural expression. Beyond this unique format, this 10-week program also seeks to offer an intensive research program that emulates graduate-level experiences, offering an immersive environment for personal and professional development, which has led to numerous achievements for past students, including publications and award-winning posters.

TREM1 disrupts myeloid bioenergetics and cognitive function in aging and Alzheimer disease mouse models.

Human genetics implicate defective myeloid responses in the development of late-onset Alzheimer disease. A decline in peripheral and brain myeloid metabolism, triggering maladaptive immune responses, is a feature of aging. The role of TREM1, a pro-inflammatory factor, in neurodegenerative diseases is unclear. Here we show that Trem1 deficiency prevents age-dependent changes in myeloid metabolism, inflammation and hippocampal memory function in mice. Trem1 deficiency rescues age-associated declines in ribose 5-phosphate. In vitro, Trem1-deficient microglia are resistant to amyloid-ß42 oligomer-induced bioenergetic changes, suggesting that amyloid-ß42 oligomer stimulation disrupts homeostatic microglial metabolism and immune function via TREM1. In the 5XFAD mouse model, Trem1 haploinsufficiency prevents spatial memory loss, preserves homeostatic microglial morphology, and reduces neuritic dystrophy and changes in the disease-associated microglial transcriptomic signature. In aging APPSwe mice, Trem1 deficiency prevents hippocampal memory decline while restoring synaptic mitochondrial function and cerebral glucose uptake. In postmortem Alzheimer disease brain, TREM1 colocalizes with Iba1+ cells around amyloid plaques and its expression is associated with Alzheimer disease clinical and neuropathological severity. Our results suggest that TREM1 promotes cognitive decline in aging and in the context of amyloid pathology.

Mitochondria in disease: changes in shapes and dynamics.

Mitochondrial structure often determines the function of these highly dynamic, multifunctional, eukaryotic organelles, which are essential for maintaining cellular health. The dynamic nature of mitochondria is apparent in descriptions of different mitochondrial shapes [e.g., donuts, megamitochondria (MGs), and nanotunnels] and crista dynamics. This review explores the significance of dynamic alterations in mitochondrial morphology and regulators of mitochondrial and cristae shape. We focus on studies across tissue types and also describe new microscopy techniques for detecting mitochondrial morphologies both in vivo and in vitro that can improve understanding of mitochondrial structure. We highlight the potential therapeutic benefits of regulating mitochondrial morphology and discuss prospective avenues to restore mitochondrial bioenergetics to manage diseases related to mitochondrial dysfunction.

Speaking up for the invisible minority: First-generation students in higher education.

A first-generation college student is typically defined as a student whose biological parent(s) or guardian(s) never attended college or who started but did not finish college. However, "first-generation" can represent diverse family education situations. The first-generation student community is a multifaceted, and intersectional group of individuals who frequently lack educational/financial resources to succeed and, consequently, require supportive environments with rigorous mentorship. However, first-generation students often do not make their identity as first-generation students known to others due to several psychosocial and academic factors. Therefore, they are often "invisible minorities" in higher education. In this paper, we describe the diverse family situations of first-generation students, further define "first-generation," and suggest five actions that first-generation trainees at the undergraduate/graduate stages can engage in to succeed in an academic climate. We also provide suggestions for mentors to accommodate first-generation students' unique experiences and equip them with tools to deliver intentional mentoring practices. We hope that this paper will help promote first-generation student success throughout the academic pipeline.

A guide to establishing, implementing, and optimizing diversity, equity, inclusion, and accessibility (DEIA) committees.

Diversity, equity, inclusion, and accessibility (DEIA) efforts are increasingly recognized as critical for the success of academic institutions. These efforts are facilitated mainly through the formation of dedicated DEIA committees. DEIA committees enhance professional development and create a more inclusive environment, which benefits all members of the institution. Although leadership and faculty membership have recognized the importance and necessity of DEIA, the roles of DEIA committees may be more ambiguous. Although leadership and faculty may seek to support DEIA at their institutions, they may not always fully understand the necessity of these committees or how to successfully create a committee, foster and promote its success, and sustain its impact. Thus, here, we offer a background rationale and guide for strategically setting up DEIA committees for success and impact within an academic institution with applicability to scientific societies.

Human Heart Failure Alters Mitochondria and Fiber 3D Structure Triggering Metabolic Shifts.

This study, utilizing SBF-SEM, reveals structural alterations in mitochondria and myofibrils in human heart failure (HF). Mitochondria in HF show changes in structure, while myofibrils exhibit increased cross-sectional area and branching. Metabolomic and lipidomic analyses indicate concomitant dysregulation in key pathways. The findings underscore the need for personalized treatments considering individualized structural changes in HF.

Latent transforming growth factor beta binding protein 4: A regulator of mitochondrial function in acute kidney injury.

Recently, latent transforming growth factor beta binding protein 4 (LTBP4) was implicated in the pathogenesis of renal damage through its modulation of mitochondrial dynamics. The seminal article written by Su et al. entitled "LTBP4 (Latent Transforming Growth Factor Beta Binding Protein 4) Protects Against Renal Fibrosis via Mitochondrial and Vascular Impacts" uncovers LTBP4's renoprotective role against acute kidney injury via modulating mitochondrial dynamics. Recently, LTBP4 has emerged as a driver in the mitochondrial-dependent modulation of age-related organ pathologies. This article aims to expand our understanding of LTBP4's diverse roles in these diseases in the context of these recent findings.

A Minimal Kynurenine Pathway Was Preserved for Rhodoquinone but Not for De Novo NAD+ Biosynthesis in Parasitic Worms: The Essential Role of NAD+ Rescue Pathways.

Aims: To determine the role of the kynurenine (KYN) pathway in rhodoquinone (RQ) and de novo NAD+ biosynthesis and whether NAD+ rescue pathways are essential in parasitic worms (helminths). Results: We demonstrate that RQ, the key electron transporter used by helminths under hypoxia, derives from the tryptophan (Trp) catabolism even in the presence of a minimal KYN pathway. We show that of the KYN pathway genes only the kynureninase and tryptophan/indoleamine dioxygenases are essential for RQ biosynthesis. Metabolic labeling with Trp revealed that the lack of the formamidase and kynurenine monooxygenase genes did not preclude RQ biosynthesis in the flatworm Mesocestoides corti. In contrast, a minimal KYN pathway prevented de novo NAD+ biosynthesis, as revealed by metabolic labeling in M. corti, which also lacks the 3-hydroxyanthranilate 3,4-dioxygenase gene. Our results indicate that most helminths depend solely on NAD+ rescue pathways, and some lineages rely exclusively on the nicotinamide salvage pathway. Importantly, the inhibition of the NAD+ recycling enzyme nicotinamide phosphoribosyltransferase with FK866 led cultured M. corti to death. Innovation: We use comparative genomics of more than 100 hundred helminth genomes, metabolic labeling, HPLC-mass spectrometry targeted metabolomics, and enzyme inhibitors to define pathways that lead to RQ and NAD+ biosynthesis in helminths. We identified the essential enzymes of these pathways in helminth lineages, revealing new potential pharmacological targets for helminthiasis. Conclusion: Our results demonstrate that a minimal KYN pathway was evolutionary maintained for RQ and not for de novo NAD+ biosynthesis in helminths and shed light on the essentiality of NAD+ rescue pathways in helminths.

A Caenorhabditis elegans model of adenylosuccinate lyase deficiency reveals neuromuscular and reproductive phenotypes of distinct etiology.

Inborn errors of purine metabolism are rare syndromes with an array of complex phenotypes in humans. One such disorder, adenylosuccinate lyase deficiency (ASLD), is caused by a decrease in the activity of the bi-functional purine biosynthetic enzyme adenylosuccinate lyase (ADSL). Mutations in human ADSL cause epilepsy, muscle ataxia, and autistic-like symptoms. Although the genetic basis of ASLD is known, the molecular mechanisms driving phenotypic outcome are not. Here, we characterize neuromuscular and reproductive phenotypes associated with a deficiency of adsl-1 in Caenorhabditis elegans. We demonstrate that adsl-1 function contributes to regulation of spontaneous locomotion, that adsl-1 functions acutely for proper mobility, and that aspects of adsl-1-related dysfunction are reversible. Using pharmacological supplementation, we correlate phenotypes with distinct metabolic perturbations. The neuromuscular defect correlates with accumulation of a purine biosynthetic intermediate whereas reproductive deficiencies can be ameliorated by purine supplementation, indicating differing molecular mechanisms behind the phenotypes. Because purine metabolism is highly conserved in metazoans, we suggest that similar separable metabolic perturbations result in the varied symptoms in the human disorder and that a dual-approach therapeutic strategy may be beneficial.

Juneteenth in STEMM and the barriers to equitable science.

We are 52 Black scientists. Here, we establish the context of Juneteenth in STEMM and discuss the barriers Black scientists face, the struggles they endure, and the lack of recognition they receive. We review racism's history in science and provide institutional-level solutions to reduce the burdens on Black scientists.

Ablation of Sam50 is associated with fragmentation and alterations in metabolism in murine and human myotubes.

The Sorting and Assembly Machinery (SAM) Complex is responsible for assembling ß-barrel proteins in the mitochondrial membrane. Comprising three subunits, Sam35, Sam37, and Sam50, the SAM complex connects the inner and outer mitochondrial membranes by interacting with the mitochondrial contact site and cristae organizing system (MICOS) complex. Sam50, in particular, stabilizes the mitochondrial intermembrane space bridging (MIB) complex, which is crucial for protein transport, respiratory chain complex assembly, and regulation of cristae integrity. While the role of Sam50 in mitochondrial structure and metabolism in skeletal muscle remains unclear, this study aims to investigate its impact. Serial block-face-scanning electron microscopy (SBF-SEM) and computer-assisted 3D renderings were employed to compare mitochondrial structure and networking in Sam50-deficient myotubes from mice and humans with wild-type (WT) myotubes. Furthermore, autophagosome 3D structure was assessed in human myotubes. Mitochondrial metabolic phenotypes were assessed using Gas Chromatography-Mass Spectrometry-based metabolomics to explore differential changes in WT and Sam50-deficient myotubes. The results revealed increased mitochondrial fragmentation and autophagosome formation in Sam50-deficient myotubes compared to controls. Metabolomic analysis indicated elevated metabolism of propanoate and several amino acids, including ß-Alanine, phenylalanine, and tyrosine, along with increased amino acid and fatty acid metabolism in Sam50-deficient myotubes. Furthermore, impairment of oxidative capacity was observed upon Sam50 ablation in both murine and human myotubes, as measured with the XF24 Seahorse Analyzer. Collectively, these findings support the critical role of Sam50 in establishing and maintaining mitochondrial integrity, cristae structure, and mitochondrial metabolism. By elucidating the impact of Sam50-deficiency, this study enhances our understanding of mitochondrial function in skeletal muscle.

Using mass spectrometry imaging to visualize age-related subcellular disruption.

Metabolic homeostasis balances the production and consumption of energetic molecules to maintain active, healthy cells. Cellular stress, which disrupts metabolism and leads to the loss of cellular homeostasis, is important in age-related diseases. We focus here on the role of organelle dysfunction in age-related diseases, including the roles of energy deficiencies, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, changes in metabolic flux in aging (e.g., Ca2+ and nicotinamide adenine dinucleotide), and alterations in the endoplasmic reticulum-mitochondria contact sites that regulate the trafficking of metabolites. Tools for single-cell resolution of metabolite pools and metabolic flux in animal models of aging and age-related diseases are urgently needed. High-resolution mass spectrometry imaging (MSI) provides a revolutionary approach for capturing the metabolic states of individual cells and cellular interactions without the dissociation of tissues. mass spectrometry imaging can be a powerful tool to elucidate the role of stress-induced cellular dysfunction in aging.

NADK-mediated de novo NADP(H) synthesis is a metabolic adaptation essential for breast cancer metastasis.

Metabolic reprogramming and metabolic plasticity allow cancer cells to fine-tune their metabolism and adapt to the ever-changing environments of the metastatic cascade, for which lipid metabolism and oxidative stress are of particular importance. NADPH is a central co-factor for both lipid and redox homeostasis, suggesting that cancer cells may require larger pools of NADPH to efficiently metastasize. NADPH is recycled through reduction of NADP+ by several enzymatic systems in cells; however, de novo NADP+ is synthesized only through one known enzymatic reaction, catalyzed by NAD+ kinase (NADK). Here, we show that NADK is upregulated in metastatic breast cancer cells enabling de novo production of NADP(H) and the expansion of the NADP(H) pools thereby increasing the ability of these cells to adapt to the challenges of the metastatic cascade and efficiently metastasize. Mechanistically, we found that metastatic signals lead to a histone H3.3 variant-mediated epigenetic regulation of the NADK promoter, resulting in increased NADK levels in cells with metastatic ability. Together, our work presents a previously uncharacterized role for NADK and de novo NADP(H) production as a contributor to breast cancer progression and suggests that NADK constitutes an important and much needed therapeutic target for metastatic breast cancers.

3D Mitochondrial Structure in Aging Human Skeletal Muscle: Insights into MFN-2 Mediated Changes.

Sarcopenia is an age-related loss of skeletal muscle, characterized by loss of mass, strength, endurance, and oxidative capacity during aging. Notably, bioenergetics and protein turnover studies have shown that mitochondria mediate this decline in function. Although mitochondrial aging is associated with decreased mitochondrial capacity, the three-dimensional (3D) mitochondrial structure associated with morphological changes in skeletal muscle during aging still requires further elucidation. Although exercise has been the only therapy to mitigate sarcopenia, the mechanisms that govern these changes remain unclear. We hypothesized that aging causes structural remodeling of mitochondrial 3D architecture representative of dysfunction, and this effect is mitigated by exercise. We used serial block-face scanning electron microscopy to image human skeletal tissue samples, followed by manual contour tracing using Amira software for 3D reconstruction and subsequent analysis of mitochondria. We then applied a rigorous in vitro and in vivo exercise regimen during aging. We found that mitochondria became less complex with age. Specifically, mitochondria lost surface area, complexity, and perimeter, indicating age-related declines in ATP synthesis and interaction capacity. Concomitantly, muscle area, exercise capacity, and mitochondrial dynamic proteins showed age-related losses. Exercise stimulation restored mitofusin 2 (MFN2), which we show is required for mitochondrial structure. Furthermore, we show that this pathway is evolutionarily conserved with Marf, the MFN2 ortholog in Drosophila, as Marf knockdown alters mitochondrial morphology and leads to the downregulation of genes regulating mitochondrial processes. Our results define age-related structural changes in mitochondria and further suggest that exercise may mitigate age-related structural decline through modulation of mitofusins.

NAD precursors cycle between host tissues and the gut microbiome.

Nicotinamide adenine dinucleotide (NAD) is an essential redox cofactor in mammals and microbes. Here we use isotope tracing to investigate the precursors supporting NAD synthesis in the gut microbiome of mice. We find that dietary NAD precursors are absorbed in the proximal part of the gastrointestinal tract and not available to microbes in the distal gut. Instead, circulating host nicotinamide enters the gut lumen and supports microbial NAD synthesis. The microbiome converts host-derived nicotinamide into nicotinic acid, which is used for NAD synthesis in host tissues and maintains circulating nicotinic acid levels even in the absence of dietary consumption. Moreover, the main route from oral nicotinamide riboside, a widely used nutraceutical, to host NAD is via conversion into nicotinic acid by the gut microbiome. Thus, we establish the capacity for circulating host micronutrients to feed the gut microbiome, and in turn be transformed in a manner that enhances host metabolic flexibility.