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 the state of neurons in the brain. 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. In our first aim, we will identify new mechanisms that repair activity-induced DNA damage, with an eye towards future work assessing how these protective mechanisms change with age in mouse models. In our second aim, we will 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 mutations that may contribute to age-associated cognitive decline. 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.

Lay Summary: 
Our aging population is expected to develop increased incidences of neurodegenerative disease and dementia, but the molecular mechanisms that underlie these complex processes remain poorly understood. This proposal aims to understand how neuronal activity-dependent gene expression and DNA damage repair can regulate brain aging by leveraging a newly identified, activity-inducible protein complex, NPAS4:NuA4. Our work will shed light on how changing levels of activity in the brain influence the accumulation of DNA damage across neuronal genomes as we age and the consequences of this damage for brain function. These experiments will lay critical groundwork for designing targeted strategies to slow or reverse decline in the neuronal cell types most susceptible to
age-dependent diseases.

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ABSTRACT

Doxorubicin (Dox) is a commonly used chemotherapeutic agent that has adverse effects on skeletal and cardiac muscle. Dox cardiotoxicity is initially characterized by skeletal and cardiac muscle atrophy and cardiac fibrosis. Dox-induced heart and skeletal muscle toxicity can progress to contractile dysfunction, sarcopenia, heart failure, and ultimately physical frailty – a phenotype of accelerated aging – and premature mortality. The immediate goal of this proposal is to determine how a range of dietary interventions can mitigate Dox toxicity and in so doing to identify biological mechanisms that can be therapeutically exploited. By understanding the mechanisms of accelerated aging and frailty, we can identify ways of intervening to mitigate the process. Accomplishing this objective will help us understand how to prevent physical frailty long-term. As an example, in a series of experiments published recently in Cell Metabolism, my laboratory discovered that sustained alternate-day fasting (ADF) in mice provokes Dox cardiotoxicity. In new studies in the first year of our Longer Life Foundation Program Award, we identified that Dox combined with ADF leads to the expansion of brown adipose tissue and increases in a secreted axonal guidance protein, SLIT2. By leveraging human samples from the Penn Heart Failure Study and aptamer proteomics, we identified that SLIT2 is the top biomarker that distinguishes Dox cardiomyopathy from other cardiomyopathies, and we have now further shown that SLIT2 is necessary and sufficient for Dox cardiotoxicity. This work, which was one specific aim of our proposal, is presently under consideration at Cell.

Having exploited our observation that fasting potentiates Dox toxicity to identify a novel biomarker and therapeutic (SLIT2), we currently propose to identify a dietary regimen that can mitigate Dox toxicity. In new preliminary studies, we randomized mice to standard high carbohydrate chow or a high protein, or high-fat diet. In these studies, we find that a high-fat diet mitigates Dox cardiotoxicity while a high-protein diet exacerbates it. These findings are surprising because a high-protein diet is currently considered the standard dietary approach to prevent muscle loss, albeit with limited scientific evidence for efficacy. Dox-treated mice fed a high-protein diet also exhibited reduced exercise capacity compared with Dox-treated mice fed a high-fat diet. In an analogous manner to what we have accomplished with fasting – namely, we used our fasting model to identify a translational disease mechanism and biomarker – we now propose to a) identify how high-fat feeding attenuates Dox cardiotoxicity, b) identify mechanisms of cardiac muscle toxicity induced by high protein intake, and c) evaluate the effect of a high protein diet on skeletal muscle mass and contractile function in Dox-treated mice.

Lay Summary:
Chemotherapy is a common treatment for cancer, but it can worsen other age-related health problems, such as heart disease, muscle loss, and weakness. In the first year of our Longer Life Foundation Award, we showed that intermittent fasting increases the side effects of chemotherapy in mice. This research has helped us identify potential new treatments to reduce these side effects. In the second and third years, we will study if other dietary approaches, like high-protein or high-fat diets, can change the side effects of chemotherapy. We will also explore new methods to help manage and reduce these side effects. These studies have enormous potential relevance not only to humans receiving chemotherapy but also to the selection of dietary macronutrients during aging and muscle loss.

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ABSTRACT

Non-communicable diseases (NCDs) are the primary cause of death and disability globally, accounting for 71% of the 41 million deaths annually. Among NCDs, hypertension (HTN) is a significant challenge in the United States, disproportionately affecting older, low-income, and minority communities. In St. Louis, HTN prevalence increased from 23% in 2007 to 36% in 2016, with a higher incidence in Black populations (44%) compared to white populations (31%).

In response, the US Surgeon General called for research on the impact of social determinants of health (SDOH)—the social and environmental conditions that affect health and well-being—on HTN development and management. Frameworks such as Healthy People 2023 emphasize healthcare accessibility and neighborhood and built environment factors as key health domains. However, these factors are often studied in isolation, highlighting the need for tools to comprehensively assess the multiple SDOH influencing HTN. To address this need, this project aims to establish a geospatial model to assess social and environmental risk factors associated with NCDs, focusing on HTN in Greater St. Louis. The model will merge objective and perceived data from two domains: healthcare accessibility and neighborhood and built environment. This will elucidate the complex associations between HTN and SDOH factors, serving as a scalable model for other health issues. The project will be guided by two concurrent aims: Aim 1 is to utilize a novel geospatial SDOH model to identify geographic disparities in HTN, healthcare accessibility, and neighborhood and built
environment factors in St. Louis. This model will integrate health and demographic data from approximately 1.5 million EMR patients from the BJC Network, GIS-modeled health accessibility (e.g., travel time estimates), outdoor air pollution, noise, and light exposure data for all EMR participants, and perceived health accessibility and environmental exposure data from a subset of 300 participants. Aim 2 is to utilize the geospatial SDOH model to evaluate the association between HTN and objective and perceived healthcare accessibility and neighborhood and built environment SDOH factors. This will serve as a “proof of concept” evaluation using advanced statistical models to assess the relationship between HTN and both objective measures (e.g., proximity to diagnosis locations, pharmacy locations, and geographical neighborhood characteristics) and perceived measures of healthcare access.

Additionally, to explore the interaction between environmental exposures and healthcare accessibility. We anticipate that objective and perceived measures of SDOH will
vary geographically, representing potential disparities in health services accessibility. We also expect that participant-reported accessibility barriers (e.g., travel costs, time, availability) will vary by objective measures (e.g., distance, travel time). In the Exploratory Aim, we expect that objective and perceived measures of accessibility will be associated with HTN. Overall, results will provide novel information regarding the local geographic drivers of HTN health services accessibility across diverse populations in St. Louis, MO. This information will support the future development of interventions aiming to reduce disparities related to HTN accessibility and outcomes.

Lay Summary
This project aims to establish a geospatial model to assess social and environmental risk factors associated with hypertension (HTN) in Greater St. Louis, merging objective and perceived data on healthcare accessibility and neighborhood factors. Guided by two aims, the study will identify geographic disparities in HTN and evaluate the association between HTN and both objective and perceived SDOH factors, including the interaction between environmental exposures and healthcare accessibility. The findings will inform the development of targeted interventions to reduce disparities in HTN accessibility and outcomes across diverse populations in St. Louis.

The post Investigating Geographic Disparities in Social Determinants of Health and Hypertension in the Greater St. Louis Area appeared first on Longer Life Foundation.

ABSTRACT

It is well known that muscle dysfunction and impaired exercise tolerance are common co-morbidities of aging and disease. Muscle dysfunction commonly manifests as reduced skeletal muscle mass and disrupted mitochondrial energy metabolism, which can have detrimental effects on patient mobility and independence, as well as increase the risk of debilitating falls, the need for long-term care, and mortality. Delaying or preventing muscle dysfunction in aged and diseased populations would improve patient quality of life and lessen disease severity. Several studies have focused on improving muscle health by targeting either skeletal muscle mass loss or impaired mitochondrial metabolism.

Our lab recently identified a gene (S1P) that controls both skeletal muscle mass and mitochondrial metabolism. Furthermore, we found that a mutation in this gene is linked to exercise impairment in a young, female patient (PMID: 31070020). Moreover, our recent report shows that deletion of S1P in skeletal muscle increases muscle mass in young and aged mice and that S1P inhibits mitochondrial metabolism in muscle (PMID: 37002930). This proposal seeks to expand our knowledge of S1P and at the same time to expand the field’s knowledge of the physiology and pathophysiology of aging through Aim 1 – Determine how S1P inhibits muscle mitochondrial metabolism; Aim 2 – Examine how S1P decreases muscle mass; and Aim 3 – Determine whether S1P depletion improves mobility during aging.

The Aims above will be examined using our S1P skeletal muscle-specific knockout mouse strain and an S1P small molecule inhibitor. This strategy will enhance the experimental rigor by using both genetic and pharmacologic approaches and provide proof of concept evidence for a novel small molecule therapeutic approach to maintain muscle mass and prevent mitochondrial dysfunction in aging and disease. Because decreased muscle mass and impaired mitochondrial metabolism are hallmarks of many disease states (i.e., muscular dystrophy, obesity, chronic kidney disease, etc), findings from this proposal may be applicable to several chronic and debilitating human diseases.

Lay Summary  
Muscle dysfunction and impaired exercise tolerance are common co-morbidities of aging and disease; however, the molecular mechanisms responsible for muscle dysfunction in these populations are not clearly understood — meaning that therapeutics for effectively combating and potentially preventing muscle dysfunction are lacking. Our lab has identified a molecular player that controls both muscle function and exercise tolerance. This proposal will expand our understanding of this player’s role in muscle and exercise biology and provide essential pre-clinical data necessary to justify subsequent studies to target this gene for the treatment for sarcopenia and exercise intolerance.

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ABSTRACT

Aging-related pathologies lead to an abbreviated health- and lifespan. Cellular senescence, which is an irreversible cessation of the cell cycle, occurs during aging and with associated metabolic dysfunction. The p16INK4a protein is a key regulator of the cell cycle and its activation leads to a block of the cell cycle and proliferation. While exercise exerts an anti-senescent effect, whether progressive resistance training has this effect and whether the mechanism involves p16INK4a remains undetermined. Furthermore, while targeted pharmacogenetic methods to ablate p16INK4a expressing cells improve lifespan and metabolic dysfunction, the anti-senescent effects of genetically deleting p16INK4a specifically in adipose tissue stem cells remains untested.

The work proposed in this application is based on findings that p16INK4a expression is increased in adipose and adipose tissue stem cells during aging and its associated metabolic dysfunction. To test this association, we will study whether or not an anti-senescent effect occurs with resistance exercise in concert with decreased adipose and adipose stem cell p16INK4a expression. Furthermore, we will test the effects of p16INK4a genetic deletion in adipose tissue stem cells on metabolic dysfunction across the lifespan. Overall, the findings from the proposed studies should enhance our understanding of the relationship between cellular senescence and aging-related metabolic dysfunction.

Lay Summary
Aging contributes to many pathologies including metabolic disease and the dysfunction of fat tissue. Typically, in a healthy fat pad, fat stem cells grow, proliferate and differentiate into mature fat cells. However, during aging, these fat stem cells cease to grow and proliferate, a process termed cellular senescence. Since cellular senescence is a symptom of aging, targeting senescence could be an attractive therapeutic avenue for treating the deleterious effects of aging. To this end, we will study the effects of progressive resistance exercise training and of the protein named p16INK4a on senescence in fat tissue and in fat-tissue-resident stem cells of mice. In Specific Aim 1 of this proposal, we will characterize the effects of progressive resistance training on cellular senescence and p16INK4a expression in adipose tissues and adipose tissue stem cells of wild-type mice. In Specific Aim 2, we will characterize the effects of genetically deleting p16INK4a specifically in mice’s fat stem cells over their lifespan. Overall, our proposed studies will provide novel mechanistic insight that could potentially be used to develop new countermeasures for aging and improve metabolic function and health span for the life span.

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ABSTRACT

Neurodegenerative conditions including Alzheimer’s disease (AD) are challenging to study due to a lack of tractable models that reproduce the onset, progression, and sporadic nature of the diseases. Most human studies rely on neurons generated from induced pluripotent stem cells (iPSCs) through a process that strips them of their
age-associated epigenetic and cellular characteristics. Consequently, neurons differentiated from iPSCs show characteristics of fetal neurons, limiting their ability to capture cellular properties intrinsic to aged neurons. We have developed a novel and highly efficient method of generating human neurons via microRNA-induced direct
conversion of skin fibroblasts. This approach retains the age-associated epigenetic signatures of the original donor cells and natively exhibits disease-associated cellular changes in multiple neurodegenerative conditions including AD. The microRNA direct conversion system allows neurons generated from donors of different ages to be compared with those from patients with various stages of AD. Ultimately, we aim to understand aging as distinct from disease, to provide personalized medical predictions to determine an individual’s risk of developing AD, as well as create a platform for drug discovery using age-appropriate human cells.

This proposal focuses on the behavior of the microtubule-binding protein tau and the impact of microtubule changes on autophagosomes and mitochondrial resilience as a function of advancing age and sporadic AD. Tau dysregulation and accumulation of tau “tangles” are required for the development of sporadic AD, and both aging and AD have shown perturbations to the health and function of autophagosomes and mitochondria. We hypothesize that advanced age increases axonal accumulation of tau, resulting in an age-related decline in cellular transport. We posit that these cytoskeletal changes become pathologic in at-risk individuals when combined with age-related mitochondrial fragility and decreased autophagosome maturation. These concepts will be tested by evaluating the motility, function, and distribution of mitochondria and autophagosomes in directly converted neurons derived from healthy young and old individuals using live-cell imaging and confocal microscopy. Physiologic age-related changes will be contrasted with those observed in neurons from donors with sporadic late-onset AD. Neurons will additionally be treated with siRNA targeting tau to establish the role of tau during aging and disease. Our findings will provide fundamental insights into cytoskeletal dynamics and the resulting behavior of neuronal organelles in healthy aging and AD.

Ultimately, this work will guide future pharmaceutical advances by providing a neuronal model for drug testing to assist with both anti-aging medications and targeted AD therapeutics. Our next steps include testing small molecule activators of mitochondrial fusion as well as autophagy enhancers to determine if these chemicals can improve the survival of AD patient neurons. In the future, we hope that individualized assessment of patient-derived microRNA-induced neurons may be used to predict neurodegenerative disease risk in patients.

Lay Summary
Aging poses the biggest risk for developing Alzheimer’s disease and other neurodegenerative diseases. It is difficult to study how aging affects the human brain because there are not good models of Alzheimer’s disease. The brains of animals do not age the same way as human brains, and animals do not develop Alzheimer’s disease. Human brain cells (neurons) can be grown in a dish, but traditional culture methods create cells that act like infant neurons and not like adult neurons.

This project uses a new technique that converts human skin cells into neurons that keep the age-related markers of the person who provided the skin sample. This allows us to compare living neurons from many types of people including young people, old people, and people with Alzheimer’s disease. The focus of this project is on structures inside the cell that support its internal organization and shape, as well as mitochondria which supply the cell with energy, and autophagosomes which act as cellular trash collectors. We predict that structures inside the cell become more fragile with age and that this results in the breakdown of normal movement and debris collection. When these processes go wrong, certain at-risk cells may sicken and develop signs of Alzheimer’s disease. By examining living cells under a microscope from people of different ages and disease severity, we hope to clarify the role that these changes play in the risk of developing Alzheimer’s disease as people age.

The post Evaluation of Neuronal Changes Associated with Healthy Aging and Alzheimer’s Disease in Directly Converted Human Neurons appeared first on Longer Life Foundation.

ABSTRACT

BRCA1 plays an essential role in the error-free repair of DNA double-strand breaks. Loss-of-function mutation in BRCA1 increases the lifetime risk for breast and ovarian cancers by ~77% and ~44%, respectively. BRCA1 mutation typically results in triple-negative breast cancers and high-grade serous ovarian carcinoma, two highly aggressive malignancies with limited therapeutic options. These cancers therefore significantly reduce lifespan and quality of life. Notably, efforts in the past decade have uncovered multiple ways by which the risk for these cancers can be effectively mitigated. Prophylactic surgery, which includes bilateral mastectomy and salpingo-oophorectomy, has been shown to significantly reduce the risk of cancer development in carriers with pathogenic BRCA1 mutation. In addition, chemopreventive and hormonal therapy options have also been shown to be effective. However, there is a knowledge gap on which BRCA1 mutation(s) will invariably result in cancer development. This knowledge is critical to assess which individuals would truly benefit from surgical or other preventative measures. As per CinVar, there are a total of 14,662 germline mutations reported in the BRCA1 gene. Amongst these ~36% of mutations belong to the group of conflicting classification, uncertain significance, and likely pathogenic categories. Hence, we urgently need a viable approach to accurately predict the significance of the germline BRCA1 mutations. To address this knowledge gap, we propose to develop a high-content screening assay to predict the pathological relevance of germline BRCA1 mutations. The screening approach has been designed to predict the pathogenicity in an unbiased manner and it overcomes the limitations of CRISPR-based strategies that have been previously used. The pathogenic mutation identified in the screen will be functionally characterized to assess how the variant impacts the double-strand break and replication fork repair functions of BRCA1. Findings from this work will lay a strong foundation for a biomarker platform that can robustly predict which BRCA1 mutation carriers should take the available cancer risk-reducing measures, thereby delaying or even preventing the onset of aggressive cancers. This assessment will potentially result in a longer lifespan for BRCA1 mutation carriers. Furthermore, the feasibility and simplicity of our proposed approach would be amenable to screening cancer-predisposing mutations in multiple other genes involved in error-free repair of DNA double-strand breaks.

Summary and description in lay language
Mutations in the BRCA1 gene contribute to approximately 70% of triple-negative breast cancers and 50% of high-grade serous ovarian carcinoma. These two cancer types are highly aggressive and significantly reduce life span. In addition, the current therapeutic measures are associated with severe side effects that hamper quality of life. Fortunately, there are multiple approaches that can significantly reduce the risk of BRCA1 mutation cancer development. This entails surgical removal of breast and fallopian tissue, hormonal therapy, and other drug regimens. However, there are a total of 14,662 reported mutations in the BRCA1 gene. Some of these mutations likely predispose to cancer, but it is unclear which ones do. Therefore, it is difficult to predict which individuals would benefit from the preventative measures. In this proposal, we aim to develop a robust assay that can accurately predict which BRCA1 mutations are cancer-predisposing. This knowledge will be imperative to inform the carriers of BRCA1 mutations to take appropriate prevention measures in a timely manner. This information will help delay or even prevent cancer onsets in individuals with BRCA1 mutations, enabling them to live a longer and healthier life.

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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.

The post PROGRESS REPORT (July 2023) LRP appeared first on Longer Life Foundation.

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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