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The Journals of Gerontology, Series A: Biological Sciences and Medical Sciences, 2024, Vol. 79, No. 2 This report will summarize the talks presented at the Third Annual Symposium of the MAC.

The meeting also hosted a panel discussion for researchers of all career stages on how to prepare for the next step in their career, including advice on finding postdoctoral or faculty positions, transitions between positions, negotiation strategies, interviewing candidates, managerial concerns for principal investigators, and establishing a niche in a new environment.

Immune Changes in the Lung

To begin the symposium, Dr. Scott Budinger, a Professor from Northwestern University, presented his studies on SARS-CoV2 pneumonia, with a special focus on the role of alveolar macrophages. To determine what differentiates SARS-CoV2 pneumonia from other types of pneumonia, his group collected bronchoalveolar lavage (BAL) samples from patients with respiratory failure due to various types of pneumonia and analyzed them using flow cytometry and bulk and single-cell RNA sequencing (1,2). His group found that alveolar macrophages from patients with SARS-CoV2 harbor or are infected with the virus. Macrophages infected with the virus had a distinct transcriptomic profile that suggested a response to IFNγ and the release of chemokines involved in the recruitment of T cells and monocyte-derived alveolar macrophages. These findings informed a model for SARS-CoV2 pneumonia where persistent alveolar inflammation is driven by self-sustaining circuits between activated T cells releasing IFNγ and tissue resident and monocyte-derived alveolar macrophages harboring virus. This inflammatory signaling circuit persists until the virus is cleared (3). They further suggest that alveolar macrophages harboring the virus may carry the virus across the lung, via a recently recognized mechanism involving the movement of macrophages through the pores of Khon, overcoming the sparse expression of ACE2 in the alveolus. This model explains the prolonged clinical course observed in patients with SARS-CoV2 pneumonia relative to patients with pneumonia secondary to other pathogens.

Dr. Luisa Morales-Nebreda from Northwestern University continued the discussion of the lung's response to viral infection. Severe viral pneumonia can result in disruption of the alveolar epithelial and capillary membranes, leading to hyperfiltration of inflammatory molecules, alveolar flooding, and refractory hypoxemia (4). Aside from their well-established role in mediating homeostasis of peripheral immune tolerance, regulatory T (Treg) cells orchestrate tissue inflammation resolution and promote tissue regeneration (5). As with humans, aged mice are more susceptible to influenza-induced mortality and have greater levels of lung inflammation. Using heterochronic adoptive Treg cell transfer experiments, Dr. Morales-Nebreda demonstrated that aged Tregs exhibit a cell-autonomous impairment in their ability to resolve lung injury and promote tissue repair following influenza pneumonia. Transcriptional profiling of aged Treg cells demonstrated both a loss of reparative function and a gain of maladaptive proinflammatory programs with age. Specifically, aged Treg cells expressed lower levels of pro-reparative molecules (eg, amphiregulin and IL-10), while upregulating proinflammatory transcription factors and cytokines (eg, Tbx21, Rorγt, Ifn-γ, . Notably, the epigenomic analysis showed that the prorecovery transcriptional response to influenza pneumonia was regulated by DNA methylation, suggesting that age-specific cumulative changes in methylation patterning can alter a Treg cell's tissue-protective function.

Metabolic Programs Affected by Aging

The second keynote talk of the day, given by Dr. Malene Hansen of the Buck Institute for Research on Aging, highlighted the importance of autophagy for healthy aging. Autophagy is a cellular recycling process that normally declines with aging and age-related disorders (6), and is required for enhanced lifespan paradigms such as dietary restriction or fasting regimens (7). Recent work in the Hansen laboratory has focused on the directionality of the autophagy process-cargo to be degraded through autophagic means is picked up in the cell periphery and sequestered into double-membrane structures called autophagosomes. These are transported to and fuse with acidic lysosomes which cluster near the nucleus for the degradation of the cargo. Dr. Hansen showed that the localization of autophagosomes is dependent on the phosphorylation of the autophagy protein LC3B/Atg8, which influences the directionality of autophagosome movement along microtubules in polarized neurons (8). This study, along with others, has inspired the investigation of potential noncanonical functions for LC3B/Atg8 in autophagy outside of lysosomal degradation, for example in secretion. Specifically, Dr. Hansen discussed an unpublished study done in the nematode Caenorhabditis elegans, where knockdown of early-acting autophagy genes in neurons extended lifespan, improved proteostasis, and also increased the formation of exopheres, which are large vesicles extruded from neurons. Interestingly, these phenotypes were dependent on the autophagy gene ATG-16.2 and could be rescued by overexpression of full-length ATG-16.2, but not ATG-16.2 lacking a functional WD40 domain. Atg16 is a key protein important for the conjugation of LCB3/Atg8 to intracellular membranes, and its WD40 domain is required for noncanonical functions for autophagy in mammalian cells; this study, therefore, highlights the possibility that noncanonical functions may be relevant to longevity (https://www.biorxiv.org/ content/10.1101/2022.12.12.520171v1).

Dr. Charles Najt of the University of Minnesota presented a model of lipid metabolism that is also dependent on autophagy-related mechanisms. One means by which organisms respond to changes in nutrient availability is through the storage or release of lipids from lipid droplets, which must be transported to the mitochondria for utilization (9). The previously held theory of this transport mechanism was that of a direct transfer model-mitochondria are positioned to directly accept fatty acids by attachment to the surface of lipid droplets (10). However, Dr. Najt and his group have proposed a new model for lipid transport in which fatty acids are released from the lysosome to the outside of the cell and then taken up again either into lipid droplets or mitochondria not attached to the lipid droplet surface. To validate these findings, they developed a protocol to isolate mitochondria from different locations in the cell, including cytosolic mitochondria and lipid droplet (ie, peri-droplet) mitochondria. Using stable isotope labeling, they found that fatty acids were primarily taken up by cytosolic mitochondria, not the lipid droplet-associated mitochondria. Cytosolic mitochondria were also found to have increased expression of fatty-acid import proteins, and increased activity of electron transport complexes I, II, and IV. Thus, Dr. Najt proposed that there are distinct differences in function between mitochondria of unique cellular sublocalizations. This work was recently published in Cell Reports (11).

The importance of proper communication between organelles was further discussed by Dr. Hua Bai, an Associate Professor at Iowa State University, who presented work on the crosstalk between mitochondria and the peroxisome. The peroxisome, too, is involved with lipid anabolism, fatty acid catabolism, and reactive oxygen species (ROS) metabolism, and its dysfunction can lead to elevated oxidative and endoplasmic reticulum (ER) stress, dysregulated lipid metabolism, mitochondrial dysfunction, and apoptosis (12). With aging, the number of peroxisomes in the cell is unchanged, yet the import of matrix enzymes into the peroxisome, a process dependent on the Pex5 receptor, is impaired. In aged Drosophila, impaired peroxisomal import results in the induction of the cytokines Upd3 (fly homolog of mammalian IL-6), culminating in arrhythmia (13). Normal peroxisome biogenesis requires the recycling of Pex5 from the membrane back to the cytosol through its ubiquitination, but this turnover is also decreased with aging. To counter these effects, Dr. Bai found that overexpression of Pex5 in the Drosophila oenocytes (liver-like tissue) led to a rescue of matrix enzyme import into peroxisomes, downregulation of Upd3, preserved cardiac function, and ultimately longer survival. Dr. Bai also demonstrated that the peroxisome can influence mitochondrial fission, where a knockdown of Pex5 led to impaired fission as evidenced by enlarged and less functional mitochondria. The phenotype could be rescued by supplementing flies with plasmalogen, an ether phospholipid that is synthesized in peroxisomes the levels of which are decreased with age, illustrating how proper peroxisome function is tied to mitochondrial function.

Inhibition of mTORC1 by rapamycin has been repeatedly shown to delay age-related pathologies and extend lifespan in diverse model systems (14). Dr. Adam Konopka, an Assistant Professor from the University of Wisconsin-Madison, presented a high-level overview of the Everolimus Aging Study (EVERLAST), the first NIA-funded clinical trial testing whether mTORC1 inhibition by the rapalog everolimus can safely improve multiple physiological and molecular hallmarks of aging in humans. In this double-blinded, phase II trial, the investigative team will use a "double-dummy design" to randomize (1:1:1) insulin-resistant, older adults (n = 72; 55-80 years) to 1 of 3 groups: (1) once daily 0.5 mg everolimus and once weekly placebo, (2) a once daily placebo and once weekly 5 mg everolimus, and (3) a once daily placebo and once weekly placebo. They plan to perform deep phenotyping to determine if mTORC1 inhibition by everolimus can improve or preserve multiple measures of physiological function, including metabolic function (peripheral insulin sensitivity, glucose tolerance, glycemic variability, and hepatic insulin sensitivity), cognitive function (micro and macro vessel cerebral blood flow, learning, and memory), cardiac function (fractional shortening, E/A ratio, ejection fraction, etc.), and physical function (VO 2 max, maximal knee extensor power and strength, body composition). In addition, multi-omics of blood and/or skeletal muscle biopsy samples will be performed to evaluate mechanisms by which everolimus alters several biological hallmarks of aging and determine if everolimus can partially restore the biology of aging toward a young healthy control group (n = 14; 18-35 years old). Finally, EVERLAST will explore if mTORC1 inhibition by everolimus alters the senescence-associated secretory phenotype (SASP) and proposed biomarkers of aging in saliva, urine, blood, and/or skeletal muscle. By the completion of this trial, they aim to understand if the potent geroprotective effects of mTORC1 inhibition can be safely exploited for the treatment and prevention of age-related diseases in humans and inform on the design of a multisite, phase III clinical trial.

Senescence and Stem Cells

Muscle stem cells, also known as satellite cells, are responsible for tissue growth and maturation, as well as repair following injury. In the adult, these stem cells exist in a quiescent state. Upon injury they rapidly exit quiescence, activate, proliferate, and differentiate to repair the muscle fiber. A subset of the satellite cells return to quiescence to repopulate the stem cell population (15). Dr. Susan Eliazer, an Assistant Professor at the University of North Dakota, showed that Wnt4 from the muscle fiber niche is required for maintaining the satellite cells in a quiescent state. Genetic deletion of Wnt4 in muscle fibers caused an increase in the number of stem cells, and the stem cells were in an activated state at tissue homeostasis. She also demonstrated that injury in the muscle tissue after the loss of Wnt4 led to a more rapid repair response by the stem cells. Wnt4 in the muscle fibers activated RhoA in the quiescent satellite cells, which maintained the mechanoproperties of the cell. Deletion of Wnt4 caused changes to the stem cell morphology, leading to a more rounded, softer shape that allowed for movement of the stem cells out of their normal location in the niche, into the interstitial space between the muscle fibers (16). With age there is an increase in Rho activity and an increase in the stiffness of stem cells, leading to an impaired ability of aged stem cells to repair injured tissue that is improved following Wnt4 deletion in aged mice.

Accumulation of senescent cells with aging is also tied to the secretion of inflammatory cytokines, proteases, and other immune modulators, called the SASP (17). Development of the SASP is dependent on mitochondria (18), which are dysfunctional in senescent cells as illustrated by increased ROS, and decreased membrane potential, energy production, and fission (19). Under stress conditions, proapoptotic proteins form pores in the membranes of the mitochondria, resulting in the release of mitochondrial DNA (mtDNA) into the cytosol (20,21), which binds to cGAS, toll-like receptor 9 (TLR9), or proteins of the inflammasome, activating pro-inflammatory pathways. Dr. Stella Victorelli, a postdoctoral fellow from the Mayo Clinic, showed that a subset of mitochondria in senescent cells undergo mitochondrial outer membrane permeabilization (MOMP) without inducing cell death, a process known as minority MOMP (miMOMP). During miMOMP, BAX/BAK pores form in the outer mitochondrial membrane, allowing the release of mtDNA into the cytosol. Senescent cells were shown to exhibit BAX/BAK-dependent leakage of mtDNA into the cytosol, which colocalizes with mitochondrial transcription factor A (TFAM). Pharmacological inhibition of BAX or genetic deletion in senescent cells reduced cytosolic mtDNA and the SASP in vitro and in sub-lethally irradiated mice. Importantly, aged mice treated with a BAX inhibitor showed improved balance, grip strength, lower frailty index, and less age-related bone loss, demonstrating that amelioration of the miMOMP-driven SASP can help mitigate the development of aging-associated deficits.

Sex-based and Generational Influences on Aging

Organisms have both acute and chronic defenses against stress, characterized by either rapid, anabolic responses or more dormant, catabolic responses respectively. Acute responses may also create a preconditioned state that provides advanced protection against subsequent stresses and may even be passed on to offspring. Dr. Veena Prahlad, an Associate Professor from The University of Iowa, presented work on the ability of heat shock factors in C. elegans to provide such kinds of cephalic mechanisms of protection, that is, protection prior to a perturbation (22,23). Following heat shock, adult animals were protected from protein aggregation by the activation of heat shock factor 1 (HSF-1). Offspring from stressed mothers, in turn, were more likely to survive their own heat shock stress and demonstrated decreased protein aggregation. Dr. Prahlad sought to identify the means of passing on this protective mechanism between generations and found that HSF-1 in germ cells was able to recruit MET-2 to place repressive H3K9me2 marks on its own target genes, including the insulin receptor daf-2, following heat stress. When these marks were passed on to progeny, the offspring preferentially decreased insulin-like signaling as a means of thermotolerance, instead of utilizing a chaperone-mediated mechanism. This allowed for a means of transferring information regarding stress exposure and responses between parents and offspring through epigenetic memory (24). For poorly understood reasons, stress responses are activated at widely varying thresholds even between genetically identical individuals. The increase in H3K9me2 at HSF-1 target genes following initial stress exposure provides one mechanism by which the threshold of stress can be set based on prior experience.

Additional work on the 2-way relationship between parents and progeny has led to the question of whether it is maternal physiology that affects the determination of fetal sex, or vice versa. As an example of the former, the Trivers-Willard hypothesis states that the sex ratio of offspring tends to be biased toward males when the condition of the parents is good, but that there is a bias toward female offspring when the condition of the parents is poor due to the fact that females are more likely to continue reproducing and males are likely to be outcompeted (25). To the point of the former instance in which fetal sex may affect maternal physiology in an example of fetal drive (26), recent work has shown that the risk of conditions such as diabetes and pre-eclampsia is associated with fetal sex (higher with males), as well as changes that occur in breast milk or circumference (27,28). Dr. Colby Vorland of Indiana University-Bloomington presented an ongoing study conducted by his group utilizing dairy cows, a model with a number of advantages including a similar gestational period to humans, typically single offspring births, and easier manipulation of fetal sex through the use of sex-sorted semen than other species. Dr. Vorland and colleagues were thus able to causally test the influence of fetal sex on maternal physiology by randomizing the assignment of fetal sex via sex-sorted semen implantation and measuring glucose-related outcomes during gestation. Future results will include physiological readouts such as glucose tolerance in order to provide insight into the effect of sex determination on maternal health.

To examine the larger differences seen between males and females with aging, Dr. Rong Yuan from Southern Illinois University, presented his work identifying genes associated with longevity using quantitative trait locus mapping. One such example was IGF1, which is known to regulate female sexual maturation and longevity in mice (29). The evolutionary theory of aging suggests that there is a tradeoff between reproduction and aging in females, such that females who become sexually mature later in life will have a longer lifespan. This led Dr. Yuan to examine if there is an inverse relationship between IGF1 levels and lifespan, and indeed he found that females have longer maximum lifespans with lower IGF1 levels, while the opposite was true of males, who had higher levels of IGF with longer maximum lifespan (30)(31)(32). When they performed genome-wide association studies for genes involved with age of reproductive maturation, they identified NRIP1, which is essential for reproductive ability and may also be involved with autophagy, senescence, breast cancer, and inflammatory diseases (29,(33)(34)(35)(36). Deletion of Nrip1 leads to increased lifespan in females only, once again illustrating how female reproduction and lifespan are interconnected (36).

Conclusion

The 2022 MAC symposium showcased the strong aging biology research programs from a variety of institutions across the Midwest. Speakers represented the University of Iowa, Iowa State University, Northwestern University, University of North Dakota, University of Wisconsin-Madison, University of Minnesota, Mayo Clinic, Indiana University, Southern Illinois University, and the Buck Institute. In addition, 24 posters were presented virtually from these universities, centers, and institutes, and others including representation from almost all MAC constituent institutions. As evidenced by the diverse areas of aging biology covered and the deep roster of early career researchers affiliated, the MAC is poised to make substantial contributions to our understanding of aging biology and potentially begin to address some of the root mechanisms and develop treatments for chronic age-related diseases. The success of this year's symposium also builds on previous symposia and meetings in highlighting the growing array of affiliated institutions, with 15 and counting, providing evidence that aging research thrives even in regions of the country that have traditionally received less research infrastructure investment (37,38). Clearly, the MAC is home to exceptionally talented investigators and cutting-edge aging biology research.