INTRODUCTION

The overarching goal of the Longer Life Foundation Longevity Research Program (LLF-LRP) at Washington University in St. Louis (WashU) is to conduct and stimulate new leading-edge research that supports LLF’s mission to “identify factors that either predict the mortality and morbidity of selected populations or influence improvements in longevity, health, and wellness.”

During the last few years, LLF-LRP has focused on the mechanisms involved in age-related sarcopenia and cardiometabolic diseases, specifically pre-diabetes/diabetes and atherosclerotic vascular disease, which reduce quality of life and are the leading causes of death, and evaluated how nutrition affects these processes.

The current aims of LLF-LRP are.

• Aim 1: Evaluate the effect of high protein intake and amino acids on factors involved in atherogenesis (mTOR signaling to autophagy/mitophagy in circulating monocytes/macrophages and platelets, endothelial cell biology).
• Aim 2: Strengthen existing and establish new collaborations with the goal of generating preliminary data for grants that support research studies that serve LLF’s mission.

PLANS

During the next year, we will continue to conduct the research we proposed in Aim 1 (evaluate the effect of high protein intake and amino acids on factors involved in atherogenesis) and will continue networking and career development activities. We also plan to apply for an NIH U19 award (PAR-22-213, Complex Multi-Component Projects in Aging Research) to evaluate new putative therapeutic strategies to improve cardiometabolic and physical function in older adults that will use preliminary data from studies that were supported by LLF-LRP. In addition, we plan to design experiments and initiate preliminary studies to evaluate whether increasing dietary protein intake during chemotherapy can mitigate the adverse effects of chemotherapy on heart and skeletal muscle cells. About 10 million people with cancer globally receive chemotherapy every year and that number is projected to increase sharply within the next decade. Although chemotherapy is highly effective in destroying tumor cells, commonly used chemotherapy agents such as anthracyclines can also damage healthy cells, including both cardiac and skeletal myocytes, which can lead to cardiac and skeletal muscle atrophy and dysfunction and increases risk of cardiac events and physical frailty.

It has been demonstrated that transcription factor TFEB is necessary and sufficient to cause anthracycline-related myocyte damage. TFEB is a downstream target of mTOR and becomes inactive when phosphorylated by mTOR. Dietary protein, through the subsequent rise in circulating amino acids, is a potent stimulator of mTOR signaling in myocytes.

Therefore, it stands to reason that high protein intake during chemotherapy will inactivate TFEB and prevent the adverse effects of chemotherapy on cardiac and skeletal myocytes. This research direction represents a logical evolution of the work we have conducted over the past few years and is a direct result of Dr. Mittendorfer’s networking and her vision to broaden the LLF-LRP’s scope and impact as proposed in Specific Aim 2. It also accommodates a change in personnel that occurred during the current funding cycle. First, Dr. Babak Razani, who was a basic science and physician collaborator on the LLF-LRP award, recently moved to the University of Pittsburgh. In the meantime, Dr. Mittendorfer has begun collaborating with Dr. Ali Javaheri, a former LLF P&F awardee, and like Dr. Razani, a cardiologist and a basic science, physician investigator with research focus on heart failure and cardiotoxicity. After Dr. Razani’s departure, Dr. Javaheri has provided expertise in clinical medicine and basic science research. His involvement in the LLF-LRP has stimulated new research ideas concerning the impact of dietary protein on skeletal muscle and cardiometabolic function in selected populations (i.e., patients with cancer).

Second, Dr. Mittendorfer has been recruited to MU in Columbia, Missouri as the NextGen Professor of Nutrition and Exercise Physiology, Director of the Clinical and Translational Science Unit, and Senior Associate Dean for Research at the MU School of Medicine. A major mission of Dr. Mittendorfer’s new position will be to strengthen and expand the already existing partnerships between WashU and MU to help investigators at both institutions to maximally leverage the resources at both locations. It is therefore anticipated that Dr. Mittendorfer will continue as an investigator on the LLF-LRP even after her relocation to MU later this year (without pay; her salary will be covered by funds at MU) and Dr. Javaheri will take on additional responsibilities and transition into the role of Multi-PI of the LLF-LRP. Dr. Javaheri’s involvement in the LLF-LRP will benefit the LLF-LRP, because of his research and clinical expertise and research focus that aligns with the LLF-LRP. In addition, he is well integrated into the WashU research community and has many connections. Furthermore, he has demonstrated great willingness to collaborate, support trainees, and further the mission of the LLF-LRP. Being part of the LLF-LRP leadership team will also serve as a catalyst for Dr. Javaheri’s career progression. These plans have been discussed with the LLF scientific review committee and will ensure a highly productive continuation of the LLF-LRP and will retain the longstanding research focus of the LLF-LRP.

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ABSTRACT

 Multiple myeloma is one the most common hematologic malignancies, accounting for approximately 13% of all hematologic malignancies and 1% of overall cancer. Despite advances in treatments, myeloma is still incurable and the lack of reliable biomarkers to predict its development is a critical barrier. Myeloma is always preceded by a premalignant phase, called monoclonal gammopathy of undetermined significance (MGUS), and can then progress to smoldering multiple myeloma (SMM) and/or malignant myeloma.

Although survival of myeloma patients has improved with new treatments, most patients suffer fatal relapse. Long non-coding RNAs (lncRNAs), which are longer than 200 base pairs, have important regulatory functions by binding to proteins, and have proven to play roles in promoting cancer. Due to tissue specificity of lncRNAs, they show promise as prognostic and diagnostic biomarkers for myeloma.

This project aims to address the critical gap in understanding the malignant evolution of myeloma by identifying and characterizing the mechanisms of lncRNAs for use as biomarkers for prognosis of disease progression. Thereby, we compared single­cell RNA sequencing data from plasma and B cells from a publicly available dataset of normal (n=11), MGUS (n=?), SMM (n=6), and myeloma (n=12) patient samples and a validation cohort of 18 myeloma patient samples from the Multiple Myeloma Research Foundation’s CoMMpass Study. We identified six differentially expressed lncRNAs comparing MGUS to SMM samples, 14 lncRNAs comparing SMM to myeloma, and 19 lncRNAs comparing normal to myeloma, which we term Multiple Myeloma Progression-associated lncRNAs (MMPals).

We focused on the top most differentially expressed lncRNA, MMPal1, also known as NEAT1. We detected little to no MMPal1 expression in normal samples, and saw an increase of expression in MGUS patient samples to myeloma samples. Silencing MMPal1 expression with silencer RNAs shows a decrease in proliferation and viability.

To determine if MMPal1 is associated with drug resistance, we treated cells with melphalan, a chemotherapy drug used as the conditioning agent in autologous stem cell transplantation. Melphalan-sensitive MM.1S cells showed less MMPal1 expression when compared to melphalan resistant U266B1 cells. Next, we assessed if MMPal1 binds to Chromobox 4 (CBX4) protein, due to its similar cellular location in nuclear speckles, epigenetic regulation, and known binding to lncRNAs. We conducted RNA immunoprecipitation and individual-nucleotide resolution cross-linking immunoprecipitation (iCLIP) qPCR to determine that indeed CBX4 binds to MMPal1. We show that MMPal1-CBX4 interaction only occurs in melphalan-resistant cells when treated with melphalan and not in melphalan-sensitive cells.

More recently, to determine clinical importance, we used multiplexed Fluorescent RNA In situ Hybridization (mFISH) to detect MMPal1 in cells and patient samples. Additionally, we created locked nucleic acid antisense oligonucleotides (LNA ASOs) targeting MMPal1 and saw a decrease in viability and increase in cytotoxicity and apoptosis with decreased MMPal1 expression. Our preliminary data serves as strong rationale for our hypothesis that MMPal1 binding to CBX4 plays a role as a master epigenetic regulator to promote myeloma progression and that lncRNAs can be utilized as biomarkers for prognosis of myeloma disease progression.

Our hypothesis will be tested in three specific aims.

• Aim 1 will assess interaction of MMPa/1 RNA and CBX4 protein in patient and melphalan-treated cells. We hypothesize that MMPal1 expression increases and interacts with CBX4 in melphalan-treated cells. To date, we have optimized detection of MMPal1 expression in myeloma cells and patient bone marrow aspirate samples using mFISH. Next, we will use mFISH combined with immunohistochemistry to assess MMPal1 RNA and CBX4 protein expression simultaneously in cells treated with and without melphalan and in myeloma patient samples.
• Aim 2 will identify CBX4-lncRNA interactions and their clinical importance in We discovered binding of MMPal1 RNA to CBX4 protein and hypothesize that other RNAs may also bind to CBX4 thereby promoting myeloma. Thus, we will conduct CBX4 iCLIP sequencing to identify all bound RNAs targets in vivo.
• Aim 3. MMPal1 CRISPR and CBX4 knockdown, overexpression, or LNA ASOs, will be assessed to determine cell viability, cytotoxicity, and apoptosis using the ApoTox-Glo Triplex Assay. RNA will be isolated from respective cells for sequencing to identify gene regulation.

Overall, this proposal will be the first to assess the MMPal1-CBX4 interaction in understanding multiple myeloma progression. Further, this proposal will fill a knowledge gap for the clinical significance of lncRNAs and have translational impact by evaluating lncRNAs as diagnostics and therapies to improve survival and longevity.

To read the full progress report, click here.

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ABSTRACT

Across a lifetime, neurons must retain a remarkable level of plasticity that facilitates learning, memory, and behavior. As animals encounter new sensory stimuli and learn complex behaviors, these experiences trigger changes in the activation of state of neurons in brains. In turn, increased neuronal activity induces the transcription of thousands of genes, the products of which dynamically modify the cells and circuits of the brain.

Neuronal activity-induced transcription is, however, a costly and risky endeavor. During transcription, the DNA is cut, unwound, and eventually resealed in a process that has the potential to create permanent mutations. How, then, do animals balance the benefits of elevated neuronal activity for plasticity with the risks it poses to the stability of their genetic code?

The goal of this proposal is to identify the molecular mechanisms that protect neuronal genomes from damage during periods of heightened neuronal activity. The proposal has two aims:

·       Aim 1: we will use mouse models to identify the burden of mutations that accrue during aging at activity-induced genes in different types of brain cells. These studies will identify the cell types most susceptible to damage and will highlight gene candidates with high levels of damage that may contribute to age-associated cognitive decline.

·       Aim 2: we will develop a platform for scalable, loss-of-function studies in human neurons to identify the protective factors that suppress damage and transcriptional dysfunction across long human lifespans.

Together, our work will provide foundational knowledge of how diverse neuronal cell types maintain transcriptional control and genome stability with age and how these genome control mechanisms go awry in aging and degenerative disease.

To read the full progress report, click here.

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ABSTRACT

Sarcopenia – the age-linked, involuntary decline of skeletal muscle mass and function – substantially contributes to frailty and diminished quality of life in the elderly. Decreased mitochondrial content and compromised mitochondrial function have long been linked to the development and progression of sarcopenia. Despite this strong link, few pharmacological strategies exist to bolster skeletal muscle mitochondria, creating a need for novel therapeutic approaches to maintain mitochondrial content and function in aging patients.

Our data suggest that modulating mitochondrial protein phosphorylation may be a powerful way to control mitochondrial content. Our recent data suggest that phosphorylation is a key regulator of mitophagy – the autophagy-dependent turnover of mitochondria. We found that genetic disruption of a mitochondrial phosphatase, Pptc7, causes the accumulation of phosphorylation on the mitophagy receptor Bnip3, which promotes Bnip3 overexpression. This Bnip3 overexpression leads to fewer mitochondria and substantially compromises mitochondrial function. Importantly, Bnip3 overexpression is strongly linked to the onset of skeletal muscle atrophy in both mice and humans, with Bnip3 recently classified as a bona fide atrogene. Our data suggest that dysregulated protein phosphorylation promotes Bnip3 overexpression, leading to skeletal muscle atrophy in mice and humans.

The goal of this proposal is to characterize the extent to which Bnip3 overexpression drives excessive mitophagy and skeletal muscle pathology, as well as how its phosphorylation at two specific serines modulates these phenotypes. Importantly, our data suggest that inhibiting specific kinases can promote Bnip3 turnover through disruption of this phosphorylation-based regulation. As kinases are pharmacologically tractable and have had significant clinical impact, these studies may reveal a novel therapeutic strategy to thwart the development and progression of sarcopenia.

To read the full progress report, click here.

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ABSTRACT

The epidemic of invasive Staphylococcus aureus infections poses a significant public health and economic burden. Invasive S. aureus infections may result in a spectrum of disease including uncomplicated bacteremia, osteoarticular infections, and often deadly endovascular infections. To date, data on origin of isolates causing invasive infections among people who use drugs is lacking, and it is unknown if these strains originate from skin colonization or the drugs themselves. Additionally, there is no reliable way to prognosticate which individuals will have mild disease courses or who will succumb to more devastating and often fatal outcomes.

In this research, we will leverage prior work funded by the Longer Life Foundation which has resulted in the accrual of a rich biospecimen bank including staphylococcal isolates from 100 patients, and associated patient sera, to investigate both pathogen and patient factors related to disease severity and outcomes.

LAY SUMMARY

The bacterium Staphylococcus aureus causes a wide range of infections with varying disease. While some patients may develop few to no symptoms as a result of infection, others have rapid often fatal disease courses. We propose to use previously collected patient samples to determine if bacterial genetics and/or patient metabolites produced as a result of infection can predict who will have a mild illness vs. who might have a fatal infection.

To read the full progress report, click here.

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ABSTRACT

The overarching goal of this proposal is to enhance both survival and quality of life after acute respiratory distress syndrome (ARDS), a major cause of mortality. Infectious causes of ARDS include pneumonia, and sterile causes include aspiration and ischemia-reperfusion injury after lung transplantation. However, the immune-mediated changes that incite and propagate acute lung injury (the pathological correlate of ARDS) are largely unknown. We and others have reported that circulating levels of complement proteins – a component of our innate immune response – are associated with improved survival during critical illness due to ARDS.

In the first year of this grant, we showed that C3 is critical for protection against stress-induced epithelial cell death using both in vitro and in vivo approaches. These observations hold true in both infectious as well as sterile injury, suggesting that the cytoprotective effects of C3 are not entirely dependent on pathogen clearance. Additionally, we demonstrated that an intracellular protein – complement Factor B – is necessary for the cytoprotective effects of C3. Data generated from this grant’s first year show that the absence of C3 in airway epithelial cells results in decreased mitochondrial function. What remains unclear is if the cytoprotective effects of C3 are due to the impaired clearance of mitochondria in the absence of C3 (i.e., impaired mitophagy).

To address this question, we need to first answer if damaged mitochondria promote complement activation, and subsequently evaluate whether complement facilitates mitophagy in airway epithelial cells. Thus, our specific aims for the second year of funding remain the same:

• Aim 1: dissect pathways by which mitochondria released from the lung during injury activate complement, and
• Aim 2: assess how complement modulates mitochondrial respiration and homeostasis during lung epithelial injury.

As part of Aim 1, we will address if mitochondria derived from human lung epithelial cells activate complement, and will identify the predominant pathways by which they activate the complement cascade (i.e., classical, lectin, or alternative). As part of Aim 2, we will assess how complement modulates cellular mitochondrial activity during lung epithelial injury. Utilizing live-cell imaging and flow cytometry of human lung epithelial cells, we will determine how the absence of intracellular complement proteins affects mitochondrial function and mitophagy in the setting of sterile (i.e., oxidative stress, acid) and infectious (i.e., bacteria-induced) injury. This approach will then be extrapolated to ex vivo models of lung injury using human lung tissue. Hence, through this proposal, we will decipher the mechanisms by which prognosticators of ARDS disease progression—identified by us and others—promote lung epithelial function, with the ultimate goal of mitigating severe lung disease.

To read the full progress report, click here.

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ABSTRACT

The long-term goal of these pilot studies is to determine whether the senescent brain promotes brain tumor pathogenesis and whether we can enhance the length and quality of life in older patients with brain tumors. Glioblastoma (GBM) is the most common and aggressive brain tumor. Despite surgery, radiation, and temozolomide chemotherapy, most GBMs recur within six months.

GBM is a disease of aging., and its incidence increases dramatically after age 60. However, very little research has focused on the age-dependent microenvironmental factors that promote their growth and recurrence. Our central hypothesis is that senescence – either from the aging brain or treatments themselves – promotes tumorigenesis and recurrence. Using genetically modified mice, and repurposing therapies known to be safe in humans and that have senolytic properties, we will test the causal effects of the senescent brain microenvironment on tumor pathogenesis. Pertinent to the mission of the Longer Life Foundation, we will chart a path to clinical translation and deeper mechanistic studies, so that this work will improve the longevity and quality of life of patients at risk for, or who have brain tumors.

LAY SUMMARY

Brain tumors increase in frequency with age. To date, all approved therapies target the tumor cells themselves. We are taking a different approach, that is, treating the aging brain. We hypothesize that the aging brain environment helps cause these tumors to grow. We will test whether senescent cells play a causative role in the development of brain tumors. We will also test whether therapies that target senescent cells in the brain can help prolong the survival of animal models of disease. If successful, this work can be brought rapidly to the clinic and will change the way we treat and prevent cancer.

To read the full progress report, click here. 

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ABSTRACT

The U.S. population is aging, and patients lose almost 50% of their renal function between their 20s and age 70. Nearly 15% of Americans suffer from chronic kidney disease (CKD) and age-related kidney dysfunction is an important contributor, along with hypertension and diabetes. Progressive CKD leading to end-stage kidney disease (ESKD) places a huge economic burden on the healthcare system and imparts significant mortality and morbidity on patients. Those with CKD who do not progress to ESKD still have an increased risk of cardiovascular disease and death from other causes (e.g., COVID-19). Metabolic dysfunction is a common feature of age-related dysfunction and aging-associated diseases (e.g., Alzheimer’s disease, Parkinson’s disease).

Most studies have focused on the role of mitochondria in metabolic dysfunction, but the overlooked peroxisome also plays an important role, particularly with fatty acid oxidation (FAO). The proximal tubule, highly metabolically active due to its reabsorptive capacity, prefers FAO to generate the required ATP to support its functions.

This proposal investigates the role of peroxisomal FAO in kidney aging and tests the hypothesis that impaired peroxisomal FAO contributes to the pathophysiology of kidney aging. The proposal has two aims:

• Aim 1: The first aim will determine how peroxisomal FAO affects the aging of primary proximal tubule-enriched (PT) cells by multiple passages in culture, an in vitro model that has similar molecular signatures to in vivo Primary PT cells generated from mice lacking tubular ACOX1, the rate-limiting enzyme for peroxisomal FAO, will be compared to PT cells from floxed control mice. Both male and female mice will be used to assess sex-specific differences, and some mice will be fed a high-fat diet prior to generation of primary PT cells.
• The second aim will investigate how drugs known to promote FAO, including SGLT2 inhibitors, alter the aging response of primary PT cells and whether the response is dependent upon intact peroxisomal FAO (i.e., ACOX1).

Concurrent with these studies, we will start the process of aging mice with and without tubular ACOX1. Based on data from this pilot project, we will treat the mice in the final three to four months with the drugs (identified in Aim 2) deemed most promising to slow the aging-related tubular injury. This pilot project will generate the necessary preliminary data for an R01 grant to the National Institute of Aging.

To read the full Progress Report, please click here.

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The gut microbiome (GM) contains a vast diversity of microbial taxa that interact with the host, and these interactions are increasingly implicated in neurodegeneration. Notably, altered intestinal microbial compositions that correlate with gut and brain inflammatory indicators have been reported in individuals with Alzheimer’s disease (AD) and in murine models of AD. We have found that these changes in GM composition can occur even before symptoms of AD start, and that the inclusion of taxa significantly enriched during preclinical AD status improved the classification accuracy of machine learning (ML) models for preclinical AD (Ferreiro et al. 2023; accepted to Science Translational Medicine). However, despite evidence that the GM is linked to AD progression, a mechanistic understanding of how it influences neurodegeneration, especially during preclinical AD, is still missing.

We propose studying the host gut transcriptional state of AD patients in conjunction with their GM will provide a more mechanistic understanding of AD pathology. Host mRNA in stool samples, referred to as the “exfoliome,” can serve as non-invasive biomarkers for inflammatory conditions in the gut, such as enteropathy and inflammatory bowel disease (IBD). Despite such potential, the exfoliome of AD patients has not been characterized.

Here, we propose to test the hypothesis that changes to the host transcriptome that are reflective of AD can be studied through the host exfoliome, and that these discriminatory transcripts, in conjunction with GM markers, will improve classification accuracy of ML models. By interrogating ~100 banked stool samples from healthy, preclinical, and symptomatic AD patients, we propose the following three aims:

• Aim 1: Characterize the exfoliome of adults in different stages of AD
• Aim 2: Explore the correlation of the host exfoliome with classical AD biomarkers, and
• Aim 3: Test whether the inclusion of host transcripts associated with AD, as well as GM compositional and functional data can improve ML models for AD diagnosis.

This proposal seeks to improve our systems-level understanding of the gut-brain-axis in AD and to identify novel biomarkers for AD diagnosis through stool, establishing a foundation for cost-effective and noninvasive measures for AD diagnosis.

LAY SUMMARY

There is a growing body of evidence highlighting the role of the gut microbiome (GM) and inflammation in neurogenerative conditions such as Alzheimer’s disease (AD). For example, the GM composition of symptomatic AD individuals is significantly different from that of healthy individuals. However, we do not understand how this difference in GM composition affects the host.

Up until recently, the only way to study the host gut was through invasive tissue sampling. Here we propose a novel, non-invasive approach to study the effects of the GM on the host by sequencing host mRNA in stool, the exfoliome, representing the host gut response to the GM. By sequencing the mRNA found in healthy, preclinical, and symptomatic AD patient’s stools and integrating it with already existing GM composition data, we can ascertain whether the changes in the GM are reflected by the host and how these changes are related to AD progression.

Our proposed noninvasive and inexpensive method will enable us to achieve a systems-level understanding of the gut-brain axis in AD and to identify novel biomarkers for AD diagnosis from patient stool.

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ABSTRACT

The long-term goal of these pilot studies is to determine whether the senescent brain promotes brain tumor pathogenesis and whether we can enhance the length and quality of life in older patients with brain tumors. Glioblastoma (GBM) is the most common and aggressive brain tumor. Despite surgery, radiation, and temozolomide chemotherapy, most GBMs recur within six months.

GBM is a disease of aging., and its incidence increases dramatically after age 60. However, very little research has focused on the age-dependent microenvironmental factors that promote their growth and recurrence. Our central hypothesis is that senescence – either from the aging brain or treatments themselves – promotes tumorigenesis and recurrence. Using genetically modified mice, and repurposing therapies known to be safe in humans and that have senolytic properties, we will test the causal effects of the senescent brain microenvironment on tumor pathogenesis. Pertinent to the mission of the Longer Life Foundation, we will chart a path to clinical translation and deeper mechanistic studies, so that this work will improve the longevity and quality of life of patients at risk for, or who have brain tumors.

LAY SUMMARY

Brain tumors increase in frequency with age. To date, all approved therapies target the tumor cells themselves. We are taking a different approach, that is, treating the aging brain. We hypothesize that the aging brain environment helps cause these tumors to grow. We will test whether senescent cells play a causative role in the development of brain tumors. We will also test whether therapies that target senescent cells in the brain can help prolong the survival of animal models of disease. If successful, this work can be brought rapidly to the clinic and will change the way we treat and prevent cancer.

The post Glioblastoma and the Senescent Brain Microenvironment appeared first on Longer Life Foundation.