Commentary on Some Recent Theses Relevant to Combating Aging: February 2016

Abstract

In this column, we continue the series, begun in issue 10(1), of surveys highlighting a small selection of recently completed doctoral theses with particular relevance to the fields covered by Rejuvenation Research.^{1

–11} While it has become common for thesis work to appear in the general academic literature, it remains valuable to scan the thesis databases for important advances that one might otherwise overlook.

Analysis of Genomic Rearrangements in Cancer from High-Throughput Sequencing Data

Tracy Ballinger, PhD, University of California, Santa Cruz

In the last century, cancer has become increasingly prevalent and is the second largest killer in the United States, estimated to afflict one in four people during their life. Despite our long history with cancer and our herculean efforts to thwart the disease, in many cases we still do not understand the underlying causes or have successful treatments. In my graduate work, I have developed two approaches to the study of cancer genomics and applied them to the whole genome sequencing data of cancer patients from The Cancer Genome Atlas (TCGA). In collaboration with Dr. Ewing, I built a pipeline to detect retrotransposon insertions from paired-end high-throughput sequencing data and found somatic retrotransposon insertions in one-fifth of cancer patients. My second novel contribution to the study of cancer genomics is the development of the copy number ancestral variation graph (CN-AVG) pipeline, a method for reconstructing the evolutionary history of a single tumor by predicting the order of structural mutations such as deletions, duplications, and inversions. The CN-AVG theory was developed by Drs. Haussler, Zerbino, and Paten and samples potential evolutionary histories for a tumor using Markov Chain Monte Carlo sampling. I contributed to the development of this method by testing its accuracy and limitations on simulated evolutionary histories. I found that the ability to reconstruct a history decays exponentially with increased breakpoint reuse, but that we can estimate how accurately we reconstruct a mutation event using the likelihood scores of the events. I further designed novel techniques for the application of CN-AVG to whole genome sequencing data from actual patients and applied these techniques to search for evolutionary patterns in glioblastoma multiforme using sequencing data from TCGA. My results show patterns of two-hit deletions, as we would expect, and amplifications occurring over several mutational events. I also find that the CN-AVG method frequently makes use of whole chromosome copy number changes followed by localized deletions, a bias that could be mitigated through modifying the cost function for an evolutionary history.

Comment: Our ability to understand the development of cancer, and particularly to predict its response to therapy, has historically been limited by the presence of considerable genetic heterogeneity within any given tumor. This heterogeneity obscures the early key events in the pathological process, especially because few cancers are detected before having the opportunity to go through many hundreds of cellular generations. Recent developments in high-throughput sequencing are now beginning to overcome the former obstacle, notably revealing that heterogeneity (at least at the exome level; disordered methylation plays a role that may be just as important as actual genomic mutations) correlates independently with adverse clinical outcomes and usually increases in response to cytotoxic therapy. Some researchers have now begun to argue that therapeutic combinations should be designed to minimize the rate of subclonal evolution within a tumor, and the consequently increased risk of developing resistance, rather than for synergistically enhanced lethality. Efficient sequencing of a mature tumor alone, however, can do little to reveal the sequence of mutational events that initiated disease, whereas this thesis tackles that issue directly. Methods such as CN-AVG will ultimately reveal which specific mutational events predispose an early cancer to subsequent genetic and phenotypic changes. In conjunction with detailed subclonal sequencing, this will enable clinicians to assess far more accurately the risk posed by a given tumor. In the longer term, those seeking to enhance the natural defenses of the genome with additional engineered error-catching mechanisms will find these models useful to identify the most critical sites to monitor and protect.

Focused Ultrasound Mediated Blood–Brain Barrier Opening in Non-Human Primates: Safety, Efficacy, and Drug Delivery

Matthew Downs, PhD, Columbia University

The blood–brain barrier (BBB) is physiologically essential for brain homeostasis. While it protects the brain from noxious agents, it prevents almost all currently available drugs from crossing to the parenchyma. This greatly hinders drug delivery for the treatment of neurological diseases and disorders such as Parkinson's, Alzheimer's, and Huntington's, as well as the development of drugs for the treatment of such diseases. Current drug delivery techniques to the brain are either invasive and target specific or non-invasive with low special specificity. Neither group of techniques is optimal for long-term treatment of patients with neurological diseases or disorders. Focused ultrasound coupled with intravenous administration of microbubbles (FUS) has been proven as an effective technique to open the BBB selectively and non-invasively in multiple in vivo models, including non-human primates (NHP). Although this technique has promising potential for clinical outpatient procedures, as well as a powerful tool in the lab, the safety and potential neurological effects of this technique need to be investigated further. This thesis focuses on validating the safety and efficacy of using the FUS technique to open the BBB in NHP as well as the ability of the technique to facility drug delivery. First, a longitudinal study of repeatedly applying the FUS technique targeting the basal ganglia region in four NHP was conducted to determine any potential long-term adverse side effects over a duration of 4–20 months. The safety of the technique was evaluated using both magnetic resonance imaging (MRI) as well as behavioral testing. Results demonstrated that repeated application of the FUS technique to the basal ganglia in NHP did not generate permanent side effects, nor did it induce a permanent opening of the BBB in the targeted region. The second study investigated the potential of the FUS technique as a method to deliver drugs, such as a low dose of haloperidol, to the basal ganglia in NHP and mice to elicit pharmacodynamical effects on responses to behavioral tasks. After opening the BBB in the basal ganglia of mice and NHP, a low dose of haloperidol was successfully delivered, generating significant changes in baseline motor responses to behavioral tasks. Domperidone was also successfully delivered to the caudate of NHP after opening the BBB and induced transient hemilateral neglect. In the final section of this thesis, the safety and efficacy of the FUS technique was evaluated in fully alert NHP. The FUS technique was successful in generating BBB opening volumes larger on average than those of the BBB opening volumes in anesthetized experiments. Safety results through MRI verification as well as behavioral testing during application of the technique demonstrated that the FUS technique did not generate adverse neurological effects. Conversely, the FUS technique was found to induce slight positive effects on the response of the NHP to the behavioral task. Collectively, the work presented in this thesis demonstrates the safety and effectiveness of the FUS technique to open the BBB and deliver neuroactive drugs in NHP.

Comment: The BBB is formed by specialized endothelial cells lining those capillaries that supply the central nervous system. The tight junctions between these cells are among the most robust in any living tissue, permitting free passage only to water, some gases, and fat-soluble molecules. For all other substances, the barrier is traversable in either direction only via substrate-specific active transport, ensuring a highly regulated extracellular milieu within the brain. However, such isolation comes at a price; we have suspected for some time that this extraordinary defense may be a double-edged sword. Aging is characterized in large part by the accumulation of rare biochemical side-products that by virtue of their atypical structures are unlikely to benefit from active transport out of the brain, and thus seem doomed to build up to a particularly serious extent in the cerebral space. Furthermore, the BBB is impassable even to our own antibodies; consequently, any such deposits are essentially invisible to the adaptive immune system, which alone might otherwise be able to identify and remove them over the life course. The notion of opening transient holes in the barrier for therapeutic reasons is not a new one, but has always been regarded with considerable wariness given the very obvious risk of side effects. Reports over the last several years have indicated that focused ultrasound can reproducibly and transiently open such breaches, and preliminary evidence indicates beneficial effects in rodent models of Alzheimer's disease—with or without the addition of antibodies engineered specifically to target amyloid plaques, implying that the sheer isolation posed by the barrier could indeed be a significant factor in the development of pathology. However, translation of these findings to patients remains mired in safety concerns. The first clinical application of the technique did not take place until November of last year, and even then was permitted largely because the patient in question suffered with a malignant brain tumor. Thus, we are delighted by the results of this work, which demonstrates remarkable long-term safety of the FUS technique in large NHP under various conditions and over a span of months to years. This data will greatly facilitate the testing of FUS as a therapy in its own right for conditions such as Alzheimer's, as well as its use to facilitate the delivery of drugs and stem cell therapies to the brain.

Hippocampal Neurogenesis-Mediated Forgetting in Adult Mice

Aijing Gao, MSc, University of Toronto

New neurons are continuously generated in the dentate gyrus of the hippocampus throughout adulthood. These new neurons gradually integrate into hippocampal circuitry and likely support the formation of new memories. Once sufficiently mature, however, as they integrate into existing circuits, they may also disrupt pre-existing memories stored in those circuits. Recently, we found artificially elevated levels of adult neurogenesis through voluntary running exercise or p53 ablation after memory formation promoted degradation in contextual fear memories and spatial memories. Besides, mice with elevated neurogenesis levels immediately after training or after a 4-week delay had different freezing responses to a fearful context. Only increasing levels of neurogenesis immediately but not after a long delay after training induced forgetting, suggesting that as memories age, they become less vulnerable to neurogenesis-mediated forgetting, possibly due to a reduced involvement of the hippocampus in the retrieval of memories over time.

Comment: Ramon y Cajal once proclaimed that in adults, “the nerve paths are something fixed and immutable: Everything may die, nothing may be regenerated.” Despite preliminary evidence in rodents uncovered as early as the 1960s, the production of new neuron-like cells in the mature human brain was considered impossible or at least irrelevant by most researchers until two decades later. In the 1980s, concrete evidence of adult neurogenesis with a definite functional role was discovered in songbirds—where it underpins seasonal acquisition of new calls—sparking a dramatic increase in scientific interest. It is now well established that there are at least three depots of neurogenic activity in the adult brain, each with distinct progenitor cell populations and migratory properties, and factors are known that both promote and impair this process (respectively, exercise and age-related changes to the systemic environment). More recently, translational researchers have begun exploring the potential for this endogenous neurogenic activity to be co-opted to repair damage to the central nervous system caused by ischemic and neurodegenerative pathologies.¹² One concern raised in response to such work has been that the introduction of new neurons might disrupt extant cognitive pathways, causing degradation of existing memories or even of personality. This study illustrates exactly that phenomenon, but simultaneously offers reassurance that well-established memories are much more stable against degradation than those formed in the immediate past. A clear understanding of the cognitive impact of neurogenic therapy will be needed to determine the optimal post-treatment care for recovering patients. Conversely, in some cases, enhanced forgetting is precisely what is desired; exercise is an established treatment for post-traumatic stress disorder (PTSD), and these findings suggest that initiating such therapy as soon as possible after the traumatic event will yield the greatest benefits.

Oncolytic Vaccinia Virus for the Treatment of Peritoneal Carcinomatosis: Combination with Chemotherapy or Targeted Radiotherapy

Kathryn Ottolino-Perry, PhD, University of Toronto

Oncolytic viruses (OVs) represent a novel class of cancer biotherapeutics currently under clinical development for treatment of a range of malignancies. OVs are ideal candidates for treatment of peritoneal carcinomatosis (PC) due to their high tumor specificity, excellent safety profile, and suitability for peritoneal delivery. OVs are unlikely to be delivered alone but rather as part of a combination therapy regimen. The research presented in this thesis provides two examples of novel combination treatment strategies for improving oncolytic vaccinia virus (VV) treatment of colorectal PC. Delivered as a monotherapy, vvDD efficacy was associated with mice that had well-vascularized and highly proliferative tumours. When combined with the first-line colorectal cancer chemotherapy drugs oxaliplatin and irinotecan (CPT-11), we observed synergistic improvements in in vitro tumor cell death and improved in vivo survival in tumor-bearing mice (with CPT-11). Synergy was attributed to sensitization of cells to drug treatment due to VV-induced S-phase arrest. In addition to combinations with chemotherapy, we examined the use of VV-mediated somatostatin receptor (SSTR) gene transfer using the radiolabeled somatostatin analogue ¹⁷⁷Lu-DOTATOC. vvDD–SSTR led to tumor-specific accumulation of ¹⁷⁷Lu-DOTATOC with minimal effects on normal tissue biodistribution in a xenograft model of colorectal PC. Radiovirotherapy was well tolerated and significantly improved survival over monotherapy. Our findings could have important implications for the design of future VV clinical trials and the treatment of colorectal PC. The proposed mechanism of synergy between vvDD and CPT-11 suggests that this combination therapy could represent a treatment option for patients with chemoresistant disease. Radiovirotherapy has wider potential applicability; any VV-susceptible tumor could be made a target of SSTR-directed therapy. Continued investigation of the mechanisms of interaction between these treatment modalities will provide a better understanding of potential improvements to therapy delivery as well as the clinical setting in which they have the greatest impact.

Comment: Radiotherapy remains a popular treatment option for many cancers, particularly because its effectiveness is not tightly bound to the existence of a specific metabolic path or receptor in the neoplastic cells. However, irradiation of sites within the body using an external source always entails exposing intervening healthy tissue to radiation and the ensuing potential for secondary carcinogenesis. Although this danger can be reduced by using multiple beams converging on a single point (such that the intensity at the focus is many times greater than along the path of any individual beam), it cannot be entirely eliminated. Furthermore, external beam radiotherapy relies on knowing the precise location and size of the target(s), making it unsuitable for highly disseminated or hard-to-image tumors. This thesis develops the innovative, if little-studied, alternative known as radiovirotherapy, in which a tumor-specific virus is employed to sensitize cancer cells to the subsequent injection of a (relatively stable, to minimize exposure of the vasculature) radioisotope. Previously, this has generally been achieved by forcing expression of a channel protein (the sodium iodide symporter) that efficiently takes up a particular radioisotope from the circulation, concentrating it in the transformed cells. Although the specific mechanism used here is different, the end result is the same; the isotope is concentrated in close proximity to cancerous cells wherever they may reside in the body. Notably, except in the most extremely metastatic cases, this ensures that even cells that avoid infection by the virus still receive an effective dose of radiation delivered by their less-evasive neighbors. Like the author, we anticipate that future cancer treatment will rely increasingly heavily on therapeutic combinations using a range of qualitatively different mechanisms to destroy even highly heterogeneous and swiftly evolving tumors efficiently.

The Mechanism of Ageism: The Relationship Between Perceived Ageism and Depressive Symptoms in Later Life

Hyejin Kim, PhD, Florida State University

Extending the human life span has long been a desire of human beings. It seems that the wish has been actualized. However, simply living a long life does not always seem to be a blessing. Older adults report a lower level of psychological well-being and quality of life and even have a higher rate of suicide compared to the general population. Furthermore, as older adults are likely to experience the death of a loved one, deteriorating health, retirement, and changing relationships with others, older adults have a high risk of depression. Social work has a long history of providing social services to older adults. In particular, social work has provided older adults with a variety of services to enhance their quality of life and mental health status. Because ageism is one form of discrimination that social work has long been concerned with, this study focuses on the relationship between perceived ageism and depressive symptoms among older adults. Therefore, the purpose of this study is to examine the relationship between perceived ageism and depressive symptoms among older adults and to investigate the mechanism linking perceived ageism to late-life depression. Three primary effects were examined: (1) the direct effect of perceived ageism on depressive symptoms in later life, (2) the mediating effects of self-perception of aging and purpose in life on the relationship between perceived ageism and late-life depression, and (3) the moderating effect of perceived ageism on the association between religiosity and depression among older adults. The stress process model was used to provide an overall idea to explain the three types of the effects related to the mechanism of ageism. Cooley's looking-glass self, Beck's cognitive theory of depression, and Levy's stress embodiment theory were used to explain the mediating effect of self-perception of aging, and Beck's cognitive theory of depression was applied again to clarify the mediating effect of purpose in life. The stress buffer theory and the life span theory of control indicated the moderating effect of perceived ageism on the relationship between religiosity and depression. Secondary analysis of existing data was conducted using the Health and Retirement Study (HRS) datasets, a longitudinal panel study of the US population ages 50 and over. The sample of this study was 3991 participants, who were older adults aged 65 and over and whose answers were available for the study analyses. Descriptive and preliminary analyses were performed to examine roughly the relationships between variables. With regard to the direct effect of perceived ageism on depressive symptoms, multiple regression analyses were performed controlling for significant variables. With regard to the mediating effects of self-perception of aging and purpose in life, structural equation modeling (SEM) analyses using structural regression models were conducted. In respect to the moderating effect of perceived ageism on the relationship between religiosity and depression, SEM analyses with multiple groups were performed. On the basis of the results of the several different analyses, a direct relationship between perceived ageism and late-life depression was found. That is, older adults who perceive ageism are likely to have a higher level of depressive symptoms than their counterparts. This direct relationship between perceived ageism and late depressive symptoms among older adults, however, was not detected after controlling for self-perception of aging and purpose in life, indicating the full mediation effects of self-perception of aging and purpose in life. That is, older adults who perceive ageism are likely to have a negative self-perception of aging, and this negative view of their own aging is likely to increase depressive symptoms. Additionally, older adults who have a negative view of their aging are likely to have a negative view of their future, and this lower purpose in life also increases depressive symptoms. The strength of the effect of religiosity on depression did not differ between the perceived ageism and the not-perceived ageism groups, indicating no moderating effect of perceived ageism on the relationship between religiosity and depression. This study contributes to the existing body of knowledge by providing information about the relationship between perceived ageism and depression and the mechanism of this relationship. Also, this study re-examined and supported established theories in the context of perceived ageism. Additionally, the current study suggests the necessity of anti-ageism policies and social work services and describes possible ways of providing such social policies and social work services at the micro, mezzo, and macro levels. The results of this study imply that more efforts and resources are necessary to reduce ageism and its negative effect on depression among older adults, and these efforts will eventually contribute to making a more just, better society.

Comment: The term “ageism” was coined in 1969 to describe discrimination against individuals on the basis of their actual or perceived age. It manifests most commonly in the form of (generally negative) age stereotypes, which prime individuals to draw different conclusions from the same evidence based on the extent to which the subject fits the stereotype. For example, technological ineptitude in a young adult is attributed to a lack of relevant education, whereas in a senior the same trait is blamed on an intrinsic lack of learning capacity or interest in the topic. Similarly, it is assumed that a young person exhibiting a physical disability such as a limp will soon recover, whereas an elder with exactly the same symptom is likely to be considered “beyond help” (even though most “inevitable” physical decline is in fact preventable with sufficient exercise¹³). The fatalism expressed by the majority of the population regarding age-related decline blocks correction of these stereotypes, and the phenomenon still receives very little attention as compared to racism, sexism, and other better known and more readily disproved forms of prejudice. The psychological effects of prejudicial treatment are well established and, as found in this study, are often mediated substantially by the extent to which the individual has internalized—“believes”—the negative assertions involved, although their sheer frequency is, of course, also significant. Lifelong deaf elders perform better on tests of memory than those with normal hearing, a result attributed to their greatly reduced exposure to ageist remarks. The overlap between the negative effects of internalized prejudice and the symptoms of genuine age-related cognitive decline¹⁴ makes ageism a particularly insidious and self-fulfilling prophecy. Increased public education on the distinction between aging per se and the numerous disorders that correlate with it may help to break this vicious cycle, but we fear the real breakthrough will only occur once biomedical progress begins to render those disorders effectively treatable—a prospect that is indeed foreseeable.¹⁵

Vascular Smooth Muscle: A Target for Treatment of Aging-Induced Aortic Stiffness

Yuan Gao, PhD, Boston University

Cardiovascular disease is the leading cause of human death worldwide. Currently, the prevalence of cardiovascular disease and health care costs associated with its onset continue to increase in both developed and developing societies. Concordant with the need to improve preventative measures is the imperative to develop more effective and efficient remedies for incident cardiovascular pathologies. Increased aortic stiffness with aging has recently emerged as an early, independent, and consistent physiological predictor of cardiovascular disease and represents an attractive target for possible therapeutic options. The success of any biomedical strategy in this regard is incumbent upon comprehension of biological processes and mechanical properties attributable to constituent components within the aortic wall. This dissertation tested the hypothesis that aging-induced changes to smooth muscle maintenance of biomechanical homeostasis within the aorta lead to undesirable increases in stiffness, correlative with increased risk of negative cardiovascular outcomes. Conventionally, mechanical studies and models have identified extracellular matrix as the primary determinant of changes in stiffness, but new research presented here shows that this may not be true. In viable ex vivo preparations of aortic tissue, roughly half of the maximal elastic modulus results from alpha-agonist activation of smooth muscle cells. Investigation of the biochemical interactions that characterize this effect revealed a link between aging and decreased expression of Src, a kinase involved in numerous signaling pathways governing cellular growth and survival, as well as defective regulation of focal adhesions between the smooth muscle cells and extracellular matrix. These findings were integrated into a model of aortic contractility and stiffness that establishes an aging-impaired regulatory complex comprising focal adhesions and non-muscle actin cytoskeleton in vascular smooth muscle cells. A better understanding of the mechanisms underlying this model may motivate the design of potential therapeutics, deliverable to previously overlooked target sites within aortic smooth muscle, and ultimately novel treatments for aging-induced cardiovascular disease.

Comment: The pressure waves generated by each contraction of the heart place considerable stress on the vascular system—stress that is mitigated by the elasticity of the vessel walls. This elasticity declines during aging, particularly in large vessels such as the aorta, and as a result the finer and more fragile peripheral vasculature is subjected to a greater proportion of the force of each heartbeat. This phenomenon is strongly suspected to contribute to increased inflammation and leakage of the smaller vessels, factors in the development of multiple age-related diseases both in and beyond the cardiovascular system. The loss of elasticity has classically been attributed to alterations to the extracellular matrix, particularly the formation of stable sugar-induced cross-links between collagen fibers, with less well understood components thought to arise from the mechanical fraying of elastin and from mineralization.¹⁶ This interesting thesis highlights, however, that a significant fraction of the defect may instead be attributed to excessive (or excessively sustained) contraction of the smooth muscle cells incorporated in the vessel walls. Whether this will prove directly therapeutically useful is not yet clear. Simply inhibiting the contraction is unlikely to help, because it is the dynamic response of these cells to changes in pressure that contributes to the vessel's functional elasticity. Indeed, such an intervention seems unfortunately likely to transfer the bulk of the damage back to the then effectively “weakened” central vessels—hardly a desirable outcome. While a long-term solution to vascular stiffening will certainly need to address the accumulated damage to the matrix, the development and evaluation of that solution will benefit greatly from a clearer picture of all the factors contributing to the problem. We will be particularly interested to discover the extent to which the regulatory defect identified can be traced back to age-related changes to the systemic environment,¹⁷ downstream of long-lived damage accumulating elsewhere in the body—and thus anticipated to self-correct naturally once that damage is repaired.

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