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.
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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.
Enhanced Phagocytic Capacity Endows Chondrogenic Progenitor Cells with a Novel Scavenger Function Within Injured Cartilage
Cheng Zhou, PhD, University of Iowa
Articular cartilage underwent serious joint injuries seldom repair spontaneously and might progress to post-traumatic osteoarthritis. This is majorly because articular cartilage's unique properties that lack blood and nerve supply intrinsically. This peculiar structure, in addition, generates an unfavorable environment for certain phagocytes (macrophages, monocytes, neutrophils, etc.) to infiltrate to cartilage to scavenge debris from cartilage matrix and cell caused from joint injuries. Therefore, physiological and functional regeneration of damaged cartilage is urgently needed and several clinical techniques have been developed, including microfracture, autograft transplantation, autologous chondrocytes implantation.
We previously identified highly migratory cells emerged and repopulated in cartilage damaged surface after ∼10 days of artificial cartilage injury. These cells were later named chondrogenic progenitor cells (CPCs) due to their enhanced potential of chondrogenic differentiation. However, this important finding contrasts the conventional theory that cartilage harbors only one cell type, chondrocytes. Here we hypothesize that CPCs are a distinct cell type in cartilage, and more importantly, one of CPCs' crucial natures is to phagocytose debris more effectively than chondrocytes.
To test these, we first harvested CPCs from cartilage surfaces, chondrocytes, synovial cells (synoviocytes and synovial fluid cells) for microarray assay to evaluate the closeness among these joint cells on whole gene expression level. Quantitative PCR were then conducted to verify gene expression of certain functional interests. Moreover, debris from cell and extracellular matrix were generated and incubated with CPCs and chondrocytes to compare their phagocytic capacity via multiple experimental assessments.
In confocal microscopy examination, the emergence of CPCs could be clearly observed after cartilage injury. Aside from their distinguishable morphology compared to chondrocyte, CPCs possess several vital properties including highly migratory, chemotactic, clonogenic. Microarray data revealed that CPCs, from gene expression profile, are distinctively isolated from chondrocytes and are more akin to synovial cells. Additionally, the series of phagocytosis related experiments showed that CPCs are dramatically superior to chondrocytes in engulfing debris, along with enhanced lysosomal activities indicating the following debris degradation.
Taken all these data together, CPCs, activated by cartilage injury, emerged and migrated to damaged sites. They are a distinct cell type residing in cartilage apart from chondrocytes. Their enhanced capacity to sustainably phagocytose and clear debris provides a novel insight for cartilage regeneration and prevention of osteoarthritis.
Comment:
Affecting over 30 million adults in the United States, osteoarthritis is a leading contributor to disability and loss of independence—particularly among the elderly, for whom exercise is an essential defense against the degenerative spiral of frailty.
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While the suffering caused by the condition can be somewhat mitigated with a range of medications and physical therapies, there is still no effective cure. Thankfully, however, that situation seems poised to change in the very near future. Although long suspected to play a role, evidence obtained in the last few years has firmly implicated cellular senescence as a key driver of cartilage degeneration in several models of inflammatory joint disease. Introducing senescent chondrocytes into healthy joints (much as occurs following traumatic injury, which triggers senescence in a proportion of the affected cells) has now been shown to replicate multiple features of arthritic disease, while the elimination of such cells leads to remarkably rapid regeneration. The latter finding was unexpected; elements of the senescence-associated secretory phenotype (SASP) could very plausibly lead to the observed pathology, but the present understanding of cartilage biology suggested that any recovery would be slow at best. Resolving that puzzle, this work characterizes a previously unappreciated population of cartilage-resident cells capable of turning over damaged matrix far more efficiently than conventional chondrocytes. Assuming that this function is suppressed by the SASP—which does not seem unreasonable, since chronic inflammatory stimulation frequently causes cells to downregulate their inducible response to the same signals–then their reemergence after the removal of their senescent neighbors would readily explain the swift recovery seen in mouse models. Unity Biotechnology, a company dedicated to the development of senolytic drugs (a new class of agents that selectively kill senescent cells), is currently running an exploratory study to verify the relationship between senescent cell loads and pathology in human osteoarthritis patients. Once that formality is completed, an interventional trial is expected to follow almost immediately. Even better, there is good reason to hope that the drug involved will be effective against senescent cells elsewhere in the body–raising the possibility of off-label use against a broad range of age-related diseases.
Examining Nuclear Mitochondria to Determine Their Role in Cell Biology
Usman Hameedi, MS, Icahn School of Medicine at Mount Sinai
In 1965, Brandes et al. observed nuclear mitochondria (mitochondria trapped within the nuclear envelope) in L 1210 Mus musculus lymphocytic leukemia cells. My experimental goal was to interrogate the biological function of nuclear mitochondria. I hypothesized that the nucleus can serve as a vanguard for mitochondria and allow cells to replenish cytosolic mitochondria. I identified nuclear mitochondria in COS-7, A549, A375, SK-MEL-28, and H1299 cell lines through the use of epi-fluorescent and confocal imaging. I also developed methods to image nuclear mitochondria 3-dimensionally. I observed that uncoupling reagents and overexpression of DRP1 can induce fragmentation in nuclear mitochondria. I also utilized Parkin overexpression to label dysfunctional nuclear mitochondria. Future experiments will aim to specifically label nuclear mitochondria for the purpose of cell biology studies. My initial findings provide a broad foundation for future studies on the characterization and significance of nuclear mitochondria.
Comment:
The appearance of intact mitochondria within cellular nuclei was, in fact, first reported somewhat earlier than 1965: a single instance was documented in 1958,
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with a more robust replication—across several cancerous cell lines, plus regenerating amphibian liver cells—following in 1960,
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although neither publication was widely noted. Of course, the nuclear envelope breaks down and reforms during mitosis; thus, it was parsimonious to suggest that mitochondria occasionally become trapped during its reassembly, or indeed that their appearance in nuclei was simply an experimental artifact. Both arguments lost some credibility in 1976, when the phenomenon was observed repeatedly in diseased cardiomyocytes (which are of course effectively postmitotic, although such populations are not entirely immune to aberrant cell cycle reentry
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)—yet not once in healthy tissue examined by the same researchers—and more so in the 1990s, when essentially the same result was independently replicated by two other groups. By 2001, a relatively straightforward rat model (involving chronic alcohol intoxication combined with a catalase inhibitor) had been identified; this too gained little recognition, despite its authors' quite reasonable suggestion that the phenomenon may be a short-lived late stage of apoptosis. It does seem unlikely to be a coincidence that virtually all lines where such mitochondria are seen are also cancerous. While almost certainly detrimental to healthy cells, nuclear-localized mitochondria could provide certain unique benefits to a growing neoplasm—most obviously as a fount of mutagenic reactive oxygen species, but also potentially by providing access to the slightly different translational machinery (tRNAs, etc.) of the mitochondrial genome. Much more work is needed to establish what role nuclear-localized mitochondria play in biology—if, for example, there is any connection to the preferential accumulation of dysfunctional mitochondrial genomes with aging. Hopefully, the more modern techniques employed in this thesis to characterize them will help to lend credibility to a long-neglected puzzle. In the age of “omics” and systems biology, it is somewhat daunting to think that our understanding of cellular processes could still contain such a substantial gap. Thankfully, an exhaustive model is not required to master the repair of age-related damage, especially when conducted at the tissue level
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—an achievement that will grant us all the time needed to appreciate even the most exotic elements of our physiology.
Lysosomal Reacidification by Degradation of Poly(dl-lactide-co-glycolide) Nanoparticles in a Lipotoxic Cardiomyopathy Model
Frederick Zasadny, MS, University of Iowa
Lipotoxic cardiomyopathy increases the risk of heart failure in obese patients by adversely altering heart structure and function via toxic lipid specie mediated cellular stress and cell death. Increased fatty acid uptake and esterification in cardiomyocytes increases toxic lipid intermediates. These cardiotoxic lipid species such as diacylglycerol have recently been shown to deacidify lysosomes in cardiomyocytes by activating protein kinase C βII mediated NADPH oxidase 2 generation of superoxide that inhibits proton pumps on lysosomal membranes by S-nitrosylation. Autophagy, a lysosome-dependent cellular survival process, is impaired upon cardiomyocyte lipid-overload due to inhibition of pH-dependent proteolytic autophagosome degradation in the lysosome. Subsequent accumulation of autophagic vesicles heightens cardiomyocyte sensitization to additional stresses of ischemia-reperfusion or ER dysfunction, culminating in impaired cardiac metabolic flexibility leading to cell death. Low cardiomyocyte regenerative capacity calls for strategies to preserve cell number in states of increased stress, such as lipid-induced impairment of autophagy. Lysosome-targeted reacidifying devices can provide an effective means to restore autophagic flux.
In this thesis, a therapeutic strategy utilizing poly(dl-lactide-co-glycolide) (PLGA) nanoparticle degradation to reacidify lysosomes and revert cardiotoxic lipid species-induced blockade in autophagic flux in cardiomyocytes is presented. Endocytosed PLGA acidic nanoparticles were designed to rapidly degrade and release acidic monomers in lysosomes to restore pH-dependent phosphatase and cathepsin L activity in cardiomyocytes with acute lipotoxicity. Optimized pre-palmitate treatment periods demonstrated that PLGA nanoparticles with polyethylenimine cationic surface coatings provide an effective restoration of autophagic flux in the presence of lipid-overload modeled by acute palmitate treatment in cardiomyocytes.
Comment:
Lysosomal recycling of damaged or surplus macromolecules is an essential part of cellular metabolism, and dysfunctions of this system are associated with the progression of several serious age-related diseases. In many cases, the blame appears to lie with one or more toxic, highly recalcitrant molecular species—the bisretinoid A2E in macular degeneration, oxidized cholesterol derivatives in atherosclerosis, and so forth—but the evidence that these species are the root cause of pathology, let alone necessary and sufficient for its persistence, is generally still only circumstantial. An alternative hypothesis suggests that the primary defect in such maladies is a failure of lysosomal pH maintenance, most likely caused by oxidative damage to their membranes, which then serves to create the conditions under which toxic species can accumulate. Of course, the two are not mutually exclusive–both factors may well contribute (and indeed synergize) to varying degrees in different tissues. PLGA is a highly biocompatible, FDA-approved polymer of glycolic and lactic acid; importantly, both monomers have pKa values below the healthy lysosomal pH of 4.5–5.0, and thus have the potential to counter any pathological increase in that pH. This work presents an intriguing proof of principle for the use of PLGA nanoparticles to directly restore the acidity of failing lysosomes, which might itself suffice to break the vicious cycle of pH dysregulation and junk accumulation—and at the very least, should be a useful adjunct to those forthcoming therapies, which introduce novel hydrolytic enzymes to degrade recalcitrant toxins.
Mechanism of Aggregation of Oxidation-Mimicking Mutants of Human γD-Crystallin
Evgeny Serebryany, PhD, Massachusetts Institute of Technology
Protein misfolding and aggregation is a ubiquitous challenge in biology and medicine. Among its many manifestations is age-onset cataract disease, the leading cause of vision loss. Cataracts arise from increased light scattering in the eye lens due to aggregation of the lens crystallins, misfolded because of various covalent modifications accrued over a lifetime. Such modifications include oxidation of Trp residues to more hydrophilic residues following exposure to ultraviolet light. Structural features of the aggregation precursors, the aggregated state itself, and the mechanisms of aggregation are not well understood.
This thesis focuses on human γD crystallin, a β-sheet rich, topologically complex two-domain protein that is abundant in the lens nucleus, associated with cataract by both genetic and proteomic data, and representative of the larger γ-crystallin family. To mimic oxidative damage, we substituted its four conserved buried Trp residues, one at a time, with more hydrophilic amino acids and studied the mutants' aggregation propensity. Effects on aggregation were strongly position-dependent, with only Trp 42 and Trp 130 substitutions showing substantial aggregation under non-denaturing conditions. The aggregates were neither covalent nor amyloid, showed little exposure of hydrophobic surfaces, and formed without broad denaturation. They were, however, highly redox-sensitive. Computational modeling suggested, and experiments confirmed, that a non-native disulfide bond, between Cys32 and Cys41, was required for the aggregation of W42Q/R mutants; this bond matches that suggested by a recent proteomic study of cataractous lenses. Modeling the partially misfolded intermediate resulting from this disulfide bond allowed us to propose a molecular mechanism of aggregation via N-terminal hairpin extrusion followed by β-sheet completion with the C-terminal domain.
To test whether mutant or damaged polypeptides may template aggregation of wild-type or undamaged ones in a prion-like manner, we examined aggregation in WT/mutant mixtures. Surprisingly, the result was “inverse prion” behavior: WT γD crystallin specifically promoted aggregation of its W42Q/R mutants without detectably coaggregating with them. Isolated WT N-terminal domain carrying the same Cys32Cys41 disulfide templated mutant aggregation, but did not escape coaggregation. Interaction between the two domains may account for both the templating activity and the escape, leading to this unexpected aggregation mechanism.
Comment:
Damage to certain long-lived proteins accumulates as a result of random molecular events over the entire lifespan. Unlike the highly specific reactions of biochemistry, this stochastic process can, in principle, generate a huge spectrum of different unnatural modifications
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—a considerable obstacle for the design of therapies to comprehensively repair that damage. However, that well-established premise does not rule out the possibility that some clinically relevant forms of damage are of a more homogenous nature. Such cases are clear low-hanging fruit for those seeking to engineer longer, healthier lives. Excluding a minor contribution from congenital mutations, cataract formation shows a sharply age-related onset—with prevalence less than 5% in those under 50, yet close to 50% by age 75. This sudden deterioration is due, in part, to the unusual biology of the lens cells, which discard virtually all normal organelles (including even the nucleus) during development, and are thus unable to perform anything, but the most elementary metabolic functions. To compensate for that deficiency, lens cells are densely packed with the chaperone α-crystallin, which robustly inhibits the pathological aggregation of other species. Of course, in the absence of protein synthesis and thus in contrast to other typical chaperones,
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the pool of α-crystallin is not a renewable resource. Its saturation is very probably a necessary prerequisite for the formation of clinical cataract. This dissertation identifies a single unnatural disulfide bond as a driver for the aggregation of γD-crystallin, one of the three major crystallin proteins required for transparency of the lens, and a major (although by no means sole) contributor to aggregates sampled from mature cataractous lenses. That finding raises the possibility that a small molecule, or appropriately retargeted enzymatic reductase, could be employed topically to dissolve such aggregates in situ, and conceivably also modify one or both of the residues involved to block future reversion. Since all unstable proteins in the lens compete for the finite chaperone activity provided predominantly by α-crystallin, the depletion of one misfolded species should also improve the situation with regard to others, even if they themselves prove less inherently tractable, and thus mitigate not only visual deterioration but also the various downstream consequences
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of persistent cataracts.
Nondestructive Viscoelasticity Microscopy: A Spectroscopic Approach Using Dual Brillouin/Raman Scattering Processes
Zhaokai Meng, PhD, Texas A&M University
The tremendous progress in life sciences and medicine has been greatly facilitated by the development of new imaging modalities. The elastic properties of molecules, subcellular and cellular structures play a crucial role in many areas of biology and medicine. Tissue elasticity has recently been recognized as a critical regulator of cell behavior, with clear roles in embryogenesis, tissue morphogenesis and stem cell differentiation, as well as contributing to pathologies such as tumor progression, coronary artery disease and tissue scarring. This dissertation is focused on developing a novel instrumentation to image viscoelastic properties of cells and tissues using Brillouin microspectroscopy. Following design, construction and optimizations that maximize the signal quality, we obtained the highest resolution Brillouin imaging system in a confocal backscattering arrangement suitable for bio-imaging applications. Furthermore, a powerful combination of Brillouin and Raman spectroscopies has yielded a confocal microscope capable of performing simultaneous mechanical and chemical imaging in a non-invasive and non-contact manner.
The novel instrument was optimized and validated for several biomedical applications. For example, we demonstrated that Brillouin spectroscopy is capable of performing in-vivo measurements of the mechanical properties of artificial biocompatible materials such as photocrosslinkable gelatin methacrylate (GelMA). With the assistance of animal models of human congenital muscular dystrophies, we show that Brillouin spectroscopy can serve as a unique diagnosis tool, which can detect differences in muscle elasticity even between very similar muscular dystrophy genotypes. We have also demonstrated that Brillouin spectroscopy is an invaluable approach in developmental biology since it is capable of making non-destructive imaging of an embryo's elasticity during its development process, which is crucial to understand the formation of many essential organs such as bone and brain.
In summary, we have developed a novel instrument for biomedical imaging sensing, which is compatible with other microscopic imaging modalities and is specific to local elasticity. Numerous applications of this new technology have been explored, and the instrument's performance was validated for several systems.
Comment:
At the atomic level, and at biologically relevant temperatures, even the most rigid materials are far from stationary; individual atoms vibrate constantly as a consequence of their nonzero thermal energy. In macromolecules with regular structures these motions are approximately harmonic, and collectively adopt a highly coordinated pattern sometimes dubbed a “material wave.” Such waves can be most easily understood by introducing the concept of quasiparticles (in this case called phonons, due to their role in sound propagation), which closely approximate the true behavior of the system, while eliminating the difficulties of modeling a highly interdependent many-body quantum ensemble. Brillouin scattering describes the phenomenon observed when photons interact with such a material, by either absorbing or creating a phonon, and in the process undergo a predictable change in energy. (Raman scattering differs in that the incident photon interacts with a “real” molecule, or chemical bond, rather than a quasiparticle.) The energy of the quasiparticle, and thus the Brillouin shift induced as a result of the interaction, is directly proportional to the rigidity of the material in which it occurs; thus, the method can be used to interrogate the emergent property of elasticity on the smallest scales without the need to mechanically manipulate a sample. Brillouin scattering has been used extensively in materials science since its discovery almost a century ago; however, its application in biology has only just begun. Many tissues in the human body become stiffer as we age, primarily as a result of random molecular crosslinking by sugars and other reactive species, and this deterioration plays a role in several pathologies, including the most visually evident sign of aging, the wrinkling of the skin, but rather more critically also the loss of elasticity within the cardiovascular system, which contributes to hypertension and consequent vascular disease. Similarly, alterations to the mechanical properties of the extracellular matrix are heavily implicated in tumorigenesis,
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although in this case, the causal relationship is most likely bidirectional. Following the robust demonstration of utility in this dissertation, we would anticipate rapid developments in biological Brillouin spectroscopy over the next few years—especially since the wavelengths involved fall conveniently within the near-infrared window, where light has its maximal penetrance through tissue—and since the technique combines so readily with its far better-known relative, Raman spectroscopy.
T Cell-Intrinsic PHD Proteins Regulate Pulmonary Immunity
David Clever, PhD, Ohio State University
Local immunity is an important feature of metastatic sites. Circulating tumor cells must evade secondary site immune responses for successful metastasis. The lung is a common metastatic site for numerous cancer types including malignant melanoma. While the diffuse pulmonary vascular architecture contributes to metastatic seeding, we hypothesized that organ-specific immunoregulatory mechanisms establish the lung as an immunologically permissive niche for tumor colonization.
T lymphocytes play a critical role in coordinating organ-specific immune responses. Pulmonary T cell responses are restrained despite continuous exposure to innocuous foreign antigens. Excessive T cell effector activity within the pulmonary environment can result in adverse immune-mediated pathology. Thus, T cells must possess an intrinsic mechanism to sense their entry into the lungs and subsequently suppress responses against harmless self and foreign antigens. Consequently, however, such mechanisms might also repress T cell responses against infiltrating metastatic tumor cells.
In the lung parenchyma T cells are exposed to localized concentrations of molecular oxygen (O2) as much as two to threefold higher than other lymphoid and non-lymphoid tissues. The prolyl-hydroxylase (PHD) family of proteins forms the cellular oxygen sensing machinery. We hypothesized that oxygen sensing by T cell-intrinsic PHD proteins coordinates an immunosuppressive program in the lung. Utilizing a mouse model with a T cell-specific deletion of all three PHD proteins (PHD-tKO), we found that T cell-intrinsic oxygen sensing is required to prevent mild autoimmune inflammation of the lungs. PHD proteins enable environmental oxygen to limit pulmonary type helper (Th)-1 responses, promote induction of CD4+-regulatory T (Treg) cells, and restrain CD8+ T cell effector differentiation and function in the steady state and following exposure to innocuous environmental antigens.
Consequently, T cell-intrinsic PHD proteins establish the lung as an immunologically favorable metastatic niche and powerfully license colonization by circulating tumor cells. Tumor infiltration is accompanied by PHD protein-dependent induction of pulmonary Treg cells and suppression of IFN-γ-dependent tumor clearance. Strikingly, T cell-intrinsic deletion or pharmacological inhibition of PHD proteins limits tumor colonization of the lung. Thus, the PHD proteins represent a novel therapeutic target to enhance anti-tumor T cell-mediated immunity.
Adoptive cell transfer immunotherapy (ACT) is an emerging therapeutic strategy that harnesses the power of tumor-specific T cells to mediate extensive tumor regression. Pharmacologic inhibition of PHD proteins using the small molecule DMOG promotes the effector capacity of tumor-specific CD4+ and CD8+ T cells. Importantly, following transfer into tumor bearing hosts, DMOG treated tumor-specific T cells mediated superior tumor regression at multiple sites of disease compared to control treated cells.
Collectively, our results provide the first demonstration of an oxygen-dependent immunoregulatory program in the lung. We identify a non-redundant role for the oxygen-sensing PHD proteins in T cell biology. We also identify an immunological basis for preferential hematogenous metastasis of cancer cells to the lung and importantly elucidate a novel targetable pathway to enhance the efficacy of immune-based therapy by modulating the activity of the PHD proteins.
Comment:
Primary lung cancer remains the second most prevalent form of the disease, despite a steady decline in cases since the early 1990s (precipitated, in large part, by sharply falling cigarette consumption from the 1960s onward). Unfortunately, that decline has had little impact on the rate of secondary pulmonary involvement; the lung is consistently still found among the top three metastatic sites across virtually all common cancers. Of course, this is not greatly surprising! The unique environment of the lung, where the vast surface area for gas exchange exposes the blood to a constant flux of environmental antigens, necessitates a mechanism to prevent excessive immune reactions. However, the nature of that mechanism has until now remained obscure, frustrating efforts to prevent its subversion by neoplastic cells. This fascinating thesis highlights levels of molecular oxygen as a key regulator of pulmonary T cell activity. While the therapeutic approach suggested herein—engineering adoptively transferred cells to be “oxygen-agnostic”—is certainly one reasonable strategy, we note that a rather simpler approach might also prove effective. It is now increasingly straightforward to maintain blood oxygen and CO2 levels through extracorporeal membrane oxygenation, in which the patient's deoxygenated blood is subjected to artificial gas exchange outside the body. Placing a patient on such a system in conjunction with a hypoxic air supply (sealed and filtered to eliminate, as far as possible, environmental contamination) might suffice to engage the full effector function of their own, unmodified T lymphocytes, and thus purge the lung of its malignant invaders. Notably, that technique does not obviously require any novel pharmaceutical or cellular entity, not even tumor-specific T cells, and thus has the potential to move through the regulatory system at a considerably less glacial pace than that to which oncologists have become sadly accustomed. Indeed the intensity of such a therapy could seemingly be controlled in near real time simply by adjusting the respiratory gas mixture, thus reducing the risk of a run-away immune reaction and further contributing to patient safety.
