• To evaluate effects of Trametinib, an inhibitor of the ERK1/2 MAP kinase pathway, on protein translation, selective mRNA translation, and mTORC1 function.
• To evaluate effects of new candidate anti-aging drugs for effects on MAPK and mTORC1 function in mice.

Garcia has found in previous work that the male-specific anti-aging drug Canagliflozin resembled another male-specific anti-aging drug, 17aE2, with parallel effects on both the pERK and p38 MAPK pathways. The pERK pathway controls protein translation, while the p38 pathway helps to buffer responses to inflammation. Cana also resembled 17aE2 in its sex-specific effecs on the mTORC1 functional activity. The results, and in particular the sex-specific effects on lifespan and the ERK kinase pathway, imply that modification of protein translation may play an important role in the ability of these drugs to slow aging and prevent lethal disease at older ages.

Garcia has now begun a series of experiments on Trametinib, which inhibits the ERK MAP kinase pathway that modulates protein translation. He is interested in the idea that this drug could synergize for mTOR inhibitors to provide anti-aging effects by complementary action on the parallel pathways by which translation is initiated. His interest in this set of kinases has been stimulated by unpublished data, from another lab, showing that Trametinib can extend mouse lifespan, on its own and in combination with Rapamycin. His analysis of Trametinib effects, and the effects of a second MAPK-specific inhibitor, are the main thrust of his current Glenn-supported work.

His second goal for this study will be to assess cap-independent translation, mTORC action, and MAPK pathways in samples from mice treated with new drugs (loperamide, deguelin, antimycin A, tamoxifen, and fluoxetine) that have been proposed as potential anti-aging drugs based on their ability to induce cellular resistance to stress in vitro (a project initiated with Glenn Center funds in a prior year).

Recent publication: Jiang, E., A. Dinesh, S. Jadhav, R. A. Miller and G. G. Garcia. 2023. Canagliflozin shares common mTOR and MAPK signaling mechanisms with other lifespan extension treatments. Life Sciences 328:121904. PMID: 37406767 DOI: 10.1016/j.lfs.2023.121904.

• Characterization of adipose tissue in PTEN mutant mice.
• Evaluation of thermogenesis not dependent on UCP1 in mitochondria; and (3) Evaluation of macrophage polarization in multiple tissues to see if they mimic effects seen in fat tissue.

Dr. Li's work in the previous year led to her publication (GeroScience, 2023) showing changes in fat, fat-associated macrophages, muscle, and brain induced by a calorie-restricted diet, as well as by four drugs (rapamycin, acarbose, 17aE2, and canagliflozin) that postpone late life diseases and extend lifespan in mice. The results were parallel in nearly all respects to her earlier work, also funded by the Glenn Center, on mice whose longevity was due to genetic mutations, supporting the case that there are fundamental similarities linking all anti-aging interventions, whether these are caused by diet, genetic endowment, or drugs. A review of these studies has now been published (Miller, Li, and Garcia) in Aging Biology.

• Assessment of fat and fat-associated inflammatory cells in long-lived mice over-expressing PTEN.
• Evaluation of UCP1-independent modes of thermogenesis in fat tissue.
• Measurement of macrophage polarization in other organs, including lung, liver, and brain. The PTEN data have shown that many of the changes previously noted in Snell, GHRKO and PAPPA mutants, in CR mice, and in Rapa-treated mice are also seen in fat tissue of the PTEN stock.

Lastly, Dr. Li will continue her collaboration with Dr. Nikolovska-Coleska to evaluate the effects of her PAPPA inhibitor drug, P100.

Recent publication: Hager, M, P. Chang, M. Lee, C. M. Burns, S. J. Endicott, R. A. Miller, X. Li. 2023. Recapitulation of anti-aging phenotypes by global overexpression of PTEN in mice. GeroScience doi: 10.1007/s11357-023-01025-8. PMID: 38114855.

The Pletcher laboratory uses a variety of unconventional techniques to study how sensory neurons communicate information about nutrition, danger, and conspecifics to initiate rapid changes health and lifespan. Many of these changes occur in coordination with known behavioral outcomes, suggesting similarities in the underlying neural circuitry. We have shown that small groups of neurons and select neuropeptides, which are known to control neural states such as hunger and sexual reward, regulate lifespan. Our discoveries provoke the notion that aging, which has long been considered a process to which animals are passively exposed, may instead have much in common with complex behaviors. It is acutely malleable, susceptible to sensory influences, and strictly controlled by coordinated sets of neurons.

Armed with this perspective together, with novel technology for behavioral and lifespan analyses, we use funds from the Glenn Center to study specific mechanisms through which the brain regulates aging in response to specific motivations and, in so doing, to set the groundwork for a greater understanding of its means of command.

Recent research in our laboratory supported by the Glenn Medical Foundation has led to a greater understanding of how sensory information is processed and about the mechanisms through which the brain orchestrates appropriate behavioral and physiological responses throughout the animal to modulate aging. For example, we recently demonstrated that certain dietary nutrients, branched-chain amino acids and isoleucine in particular, are capable of specifying homeostatic hunger states in Drosophila (i.e., hunger that is driven by caloric deficit). These states appear to be encoded in the neural epigenome, and they slow aging. We are in the process of investigating whether aging is similarly modulated by other types of hunger drives, including, for example, hedonic hunger, which is stimulated by the pleasurable aspects of feeding rather than energetic needs.

Consistent with a long-running focus of our Glenn-funded research, serotonin signaling appears to be influential in the specification of neural states that slow aging. We have, for example, recently completed work demonstrating that a handful of neurons in a structure called the ellipsoid body (EB) of the Drosophila brain, called R2/R4 neurons, respond to serotonin and act as a rheostat in transducing sensory information to modulate lifespan. In response to certain sensory inputs, the R2/R4 rheostat modulates insulin production in a different part of the brain, which then influences physiology and health in the periphery.

Ongoing work is focused on investigating how the R2/R4d system responds to a diverse set of sensory inputs that control aging and on interrogating its neural and peripheral targets to determine whether these circuits recruit well known aging pathways or invoke aging mechanisms yet to be discovered.

This project uses the nematode Caenorhabditis elegans to investigate how environmental perception, stress signaling, and stress response modify aging. Because C. elegans is one of the simplest multicellular organisms widely studied, it allows for high throughput studies on healthspan and longevity in a genetically tractable model. The approach of this project is complementary to that of the Glenn project led by Scott Pletcher, and seeks to understand how organisms recognize relay signals in response to real or perceived stress, and subsequently modify their physiology. The eventual goal is to identify new approaches to improve healthspan and longevity through by altering stress-response pathways contained within all organisms.

• Cell non-autonomous regulation of aging. This project focuses on how organisms perceive stresses such as lack of nutrients, lack of oxygen, or presence of pathogens, and signal through the nervous system to modify physiology. By interrogating these pathways, which involve conserved signals such as serotonin and dopamine, we can understand how environmental perception leads to physiological changes and can potentially co-opt these signals to improve health.
• Small molecule lifespan extension: This project utilizes collaborative efforts to identify small molecule drugs that improve stress response in primary mouse cells by further interrogating hits from this work in a nematode context. The goal is to identify multiple candidates for lifespan extension and to use the genetics of C. elegans to discover the mechanisms and pathways that each drug uses to improve long-term health.
• Microbiome interplay with stress and longevity: This project tests how the bacteria that worms eat and colonize their intestines modify their health and longevity. Since C. elegans is usually cultured with a single bacteria species, we can modify the microbiome and diet together and learn about how nutrition and signaling from microbes affects the physiology of a host organism.

Nikolovska-Coleska's research program focuses on chemical genomics, discovery, and development of chemical probes for interrogation of biological processes in human diseases and validation of potential therapeutic targets. The group is applying an interdisciplinary approach combining biophysical and biochemical methods, structural biology, medicinal chemistry, and molecular and cellular biology to identify, develop, and characterize novel chemical tools and therapeutic strategies. Nikolovska-Coleska has previously made significant contributions in the discovery and development of small-molecule inhibitors targeting the apoptotic pathways, including Mcl-1 and Bfl-1, members of the Bcl-2 family of proteins, and targeting epigenetic regulators including histone methyltransferase DOT1L, and AF9 and ENL, members of the YEATS domain family of proteins.

This project has led to the identification of the first in class inhibitors of Pregnancy-associated plasma protein A (PAPP-A), a zinc metalloprotease that cleaves IGF-binding proteins (IGFBPs) and enhances the local bioavailability of Insulin-like growth factors (IGFs).

The group developed and optimized a novel homogenous fluorescence quenched resonance energy transfer (FRET) biochemical assay to measure PAPP-A proteolytic activity. Using this biochemical assay and applying high throughput screening strategy, we identified and validated several classes of small-molecule PAPP-A inhibitors. Followed by medicinal chemistry efforts, novel analogs were designed, synthesized, and characterized using multiple biochemical and functional assays for analyzing their potency in inhibiting PAPP-A cleavage of substrates, IGFBP4 and IGFBP5, and establishing the structure activity relationship (SAR).

For their functional characterization and evaluating their ability to modulate PAPP-A-dependent phenotypes in cells, we measured phosphorylation of IGFR in HEK293T cells upon stimulation with IGF1 in the presence of IGFBP-4 and PAPP-A. Consistent with the results obtained from biochemical and immunoblot enzymatic assays, PAPP-A inhibitors blocked IGF1 signaling in HEK293 cells in a dose-dependent fashion, as measured by phosphorylation of IGFR.

Ongoing work is focused on further optimization and characterization of small molecule PAPP-A inhibitors towards development of potent and selective compounds to be used as chemical tools in cellular and in in vivo models for elucidating the role of PAPP-A/IGF signaling axis in aging biology and human disease.

The primary focus of this project is determining mechanisms that regulate proteostasis in the context of aging and aging-associated diseases. Truttmann is particularly interested in Hsp70 family chaperones and their regulation by post-translational protein modifications (PTMs).

The group utilizes a multidisciplinary approach including molecular biology, genetics, neuroscience, and biochemistry in conjunction with several model systems (C. elegans, mouse models, cell lines, purified proteins) to elucidate the functional consequences of PTM-mediated chaperone regulation.

The overarching goal is to identify and characterize novel mechanisms of proteostasis control which attenuate toxicity in aging-associated disease models to ultimately develop targets for disease-modifying therapies.

The Kaczorowski laboratory focuses on genetic and cellular mechanisms that promote resilience to cognitive aging, Alzheimer's disease, and other age-related dementias. Using mice that model the genetic diversity and phenotypic variation of human populations, the group uses mouse and human datasets, based on genetics, omics, imaging, and behavior, to identify molecules and pathways that could be targeted to promote resistance to cognitive aging. Some of the ongoing studies in the lab also aim at examining the physiological changes occurring in hippocampus and memory functions over the course of aging, which will provide insight into the mechanism of preservation of cognitive function in aging.

• Assess learning and memory functions in aging mice.
• Investigate the neuropathological features including neuroinflammation and neurogenesis.
• investigate neurophysiological characteristics associated with age-related cognitive decline in UM HET3 mice.

Recently, the Kaczorowski lab performed a pilot study to evaluate the memory functions in UM-HET3 mice. Results showed that both contextual fear acquisition (CFA)/short-term memory and long-term contextual memory (CFM), were decreased in 28-month-old UM-HET3 mice compared to 15-month-old UM-HET3 mice (unpublished). However, to better understand the age of onset of brain aging and the cognitive decline, a cross-sectional study involving different ages has been proposed, as a foundation for our future work on interventions we hope will slow cognitive aging.

• Assessing memory functions in animals at five different age points, for mice aged 10, 16, or 22 months of age. These mice will be tested for associative learning and long-term memory status using contextual fear conditioning (CFC.)
• Investigating whether changes in the markers of gliosis or neurogenesis, and pre and postsynaptic markers associate with decline in memory functions with age.
• Evaluating the neurophysiological features associated with age-related cognitive decline in UM-HET3 mice. For this aim, patch clamp recording will be used to characterize the morphological and electrophysiological features of individual neurons. Lastly, correlation analysis will determine which, if any, of the biochemical, morphological, and electrophysiological endpoints associates with different degrees of cognitive decline in UM-HET3 mice.

Based on Seq-Scope, a state-of-the-art spatial transcriptomics technology originally developed in the Lee lab, the Glenn Center is championing collaborative aging research. Seq-Scope stands out with its unmatched microscopic resolution (0.5-1 μm), eclipsing other unbiased spatial transcriptomics methods. This innovative technology facilitates an in-depth examination of spatial transcriptomes.

In the first project, a joint effort with Dr. Susan Brooks, the focus is on muscle biology. Employing Seq-Scope, we undertook a study on normal mouse muscle, scrutinizing the spatial transcriptome of an entire longitudinal soleus muscle section. This meticulous high-resolution analysis revealed transcriptome phenotypes unique to specific myofiber types, non-myocytes (such as smooth muscle, fibroblasts, immune cells), and subcellular structures including mitochondria, nuclei, and neuromuscular junction (NMJ) structures. Further exploration of the NMJ area identified distinct domains for terminal and myelinating Schwann cells and post-synaptic myonuclei, pinpointing specific marker genes for each. This breakthrough signifies the potential for ultra-high-resolution spatial transcriptome analysis. The team also investigated normal and degenerating muscles from mice, pre and post denervation (at 0, 3, and 7 days). Preliminary results suggest the capability to characterize fiber type-specific responses to denervation, potentially enhancing our understanding of denervation responses linked to age-related muscle loss.

The second project delved into analyzing the mouse brain hippocampus, yielding convincing proof-of-concept data for the high-resolution characterization of WT brains. This included contrasting sections of GHRKO brains for differential expression analysis. Despite no significant anatomical or transcriptomic differences between WT and GHRKO brains, subtle variations were detected, warranting further study. These nuances might explain some of the slow-aging phenotypes observed in GHRKO mice.

Both the muscle and brain studies experienced setbacks due to Illumina discontinuing the HISEQ sequencing platform used for spatial decoding. We've recently determined that the NOVASEQ platform can be adapted for our procedures, and are gearing up to resume analyses.

Moreover, the core team, in collaboration with other departments, devised a new data pipeline from raw FASTQ sequence data to cell type mapping and gene expression atlases, seamlessly integrated into a web browser system for Glenn collaborators. This system is now operational for navigating datasets. The core is also advancing technologies to heighten molecular capture efficiency, broaden imaging areas, and incorporate capabilities for detecting non-mRNA entities such as proteins and epigenomic signatures. These technological enhancements are set to bolster ongoing aging research at the Glenn Center.

The group is interested in understanding the mechanisms by which communication between translation, quality control, and protein degradation pathways enables the cell to maintain protein homeostasis (proteostasis). Proteostasis collapse is a hallmark of aging and aging-associated neurodegenerative diseases. We apply fluorescence microscopy, molecular biology, and genetics approaches to determine the causes and consequences of proteostasis collapse in human cells. Understanding the mechanisms that underlie proteostasis collapse holds promise for the identification of novel therapeutic intervention strategies to improve health across the lifespan.

The Guo laboratory studies aging, regeneration, and age reversal in the "immortal" planarians, Schmidtea mediterranea. Aging in planarians shares remarkable similarities to mice and humans. Most tissues in planarians and mammals contain resident adult stem cells, which undergo self-renewal and differentiation. Old, differentiated cells die due to wear-and-tear, and will be replaced by newly differentiated cells from adult stem cells. We are never the same as us one month ago. The same is true for planarians. The difference is that we do not normally observe natural death of planarians. They will use funds from the Glenn Center to study the basic biology of aging in planarians, and how they manage to live an extremely long lifespan.

Recent research in our laboratory supported by the Glenn award has led to a greater understanding of two questions: Do planarians age? If planarians age, how do they live an "immortal" life? Recent work from the Guo lab showed that planarians lose fertility and motility and undergo neurodegeneration and sarcopenia within a year (Dai et al. 2023 biorxiv, under revision). In addition, this work showed that injury and regeneration of the lost tissues were able to restore lost neurons and muscles to proper proportions and rescue fertility and motility globally. Such regeneration induced rejuvenation mechanisms likely function at smaller scales during whole-body homeostasis states, which contribute to their extreme longevity.

These findings demonstrated that rejuvenation medicine can potentially reach all major cell types of the body and reverse biological age significantly to a much younger state. To understand how planarians age and rejuvenate will establish valuable resources for comparative analysis in aging and rejuvenation in mammals. Funds from the Glenn Center award gave Guo's group the freedom to investigate these uncharted territories.