Commentary on Some Recent Theses Relevant to Combating Aging: October 2015

Characterization of Salivary Microbiome in Patients with Pancreatic Cancer

Pedro Torres, PhD, San Diego State University

Clinical manifestations of pancreatic cancer often do not occur until the cancer has undergone metastasis, resulting in a very low survival rate. In this study, we investigated whether salivary bacterial profiles might provide useful biomarkers for early detection of pancreatic cancer. Using high-throughput bacterial small subunit ribosomal RNA (16S rRNA) gene sequencing, we characterized the salivary microbiota of patients with pancreatic cancer and compared them to healthy patients, and patients with digestive and non-digestive diseases. A total of 146 patients were enrolled at the Univeristy of California San Diego Moores Cancer Center, and saliva, buccal swabs, and patient demographic data were collected from each patient. Of these, we analyzed the salivary microbiome of 108 patients—eight diagnosed with pancreatic cancer, 13 with pancreatic disease, 53 with digestive disease (including cancer), 12 with a non-digestive disease (including non-digestive cancer), and 22 non-disease (healthy) controls. 16S rRNA genes were amplified directly from salivary DNA extractions and subject to high-throughput sequencing (HTS). After sequence quality assessment and sequencing depth normalization, we found significant differences in the absolute abundances of several bacterial species, including Porphyromonas, Haemophilus, and Campylobacter, between patients with pancreatic cancer and patients in other categories. In particular, we found the ratio of Leptotrichia to Porphyromonas bacteria was consistently higher in pancreatic cancer patients compared to pancreatic disease, other digestive diseases, and healthy controls. This ratio also discriminated between pancreatic cancer and other diseases commonly misdiagnosed as pancreatic cancer. Our results suggest that culture-independent analysis of salivary bacteria holds promise for early detection of pancreatic cancer.

Comment: The commensal microbiome within a human body has enormous potential as a source of clinical information, as well as already better-appreciated opportunities for therapeutic intervention. ¹¹ The intense competitive equilibrium between diverse microbes implies that even small deviations from homeostasis can, over a period of time, result in distinctive (if prima facie unpredictable) distortions of the species balance; thus, the microbiome may be thought of as a “living record” of recent biological history, amplifying consistent but otherwise undetectably tiny variations in the biochemical composition of the body. Interrogation of this record has been challenging, in no small part due to the historical requirement for culturing to produce enough members of a given species to identify. Because many microbial species cannot be cultured at all, and many more react with varying enthusiasm to any given culture environment, this has imposed a severe distortion on any data so gathered. Modern ultra-sensitive, high-throughput sequencing techniques are beginning to overcome this obstacle by eliminating the culture step and drawing genetic material directly from a primary patient sample. Of course microbial variation around the world is quite significant, and so it was wise of the authors to draw their patient cohort from a single geographic area—such tests are quite likely to need to be calibrated to the specific region in which they are applied. Nonetheless, this study is a truly impressive proof of principle for the general field. Given its non-invasive sampling requirements and comparatively straightforward analytical method, we are optimistic that replication and expansion in much larger patient cohorts will soon follow.

Cross-Linked Hyaluronic Acid Hydrogel Networks Designed As Mechanical Actuators

Pushkar Varde, PhD, Syracuse University

Bioengineers are in constant pursuit of solutions to problems facing the medical and pharmaceutical fields by designing biomaterials that closely mimic the target natural systems. A unique collection of polymers, known as polymeric actuators, have been devised with the ability to convert an external stimulus to a change in shape, size, or permeability. The current options within polymeric biomaterials with multi-functionality include matrices that are biocompatible, biodegradable, quick transitioning/shape changing, and mechanically tunable. These properties have been harnessed for application, such as stents, valves, semi-permeable membranes, and dynamic cell culture substrates. For such applications, quick and uniform actuator response that does not need to be sustained for more than a few hours is desired. However, there exist other areas of biomedical applications, such as wound closure/healing and nerve regeneration, where polymeric actuators have been under-utilized. These applications, however, call for a polymer system that can actuate at controlled slow speeds and sustain this actuation for several days. At present, there is a lack of such slow-actuating polymer systems. Each year, over 50,000 peripheral nerve repair procedures are performed (National Center for Health Statistics 1995). The total annual costs in United States alone exceed $7 billion (American Paralysis Association 1997). The treatment of a nerve transection is dependent on the size of the injury gap. Similarly, the extent of regeneration and re-innervation in the peripheral nervous system (PNS) is also governed by the size of the gap. For a smaller gap (<10 mm), the surgeon can pull the severed nerve ends closer and suture them to repair the injury. For larger gaps, autologous nerve transplant is the gold standard treatment despite the inherent disadvantages. Over the past decades, biomaterial researchers have tested several polymeric nerve conduits as an alternative to autologous nerve grafts. However, none have been able to match the success rates of autologous grafts. There is a lack of an effective biomaterial solution to the problem of a large gap nerve injury. For many years, there has been a hypothesis that nervous tissue can be successfully elongated via application of an external mechanical force alone, which could be used to treat peripheral nerve gap injuries. Mechanical actuation studies have been shown to produce successful stretch growth in individual axons and axonal bundles. This phenomenon is at play in nature during embryonic growth and development of the body of organisms to adulthood. Applying tensional forces at appropriate rates (<100 μm/hr) causes sustained axonal stretch growth. The solution we propose in this work is a biomaterial that can be programmed to perform the function of a mechanical actuator at rates suitable for axonal stretch growth. We designed, fabricated, and characterized a novel hyaluronic acid (HA)-based hydrogel that shrinks over time along a pre-defined axis, thereby providing the source for tension that could be used for sustained axonal stretch growth. The shear thinning property of HA enabled us to test if we could store a retractive stress in a rapidly cross-linked network under shear flow and then release this stress controllably and achieve shrinkage of the network scaffold along one desired axis. We investigated two strategies to achieve this goal. The retractive stress trapped in the cross-linked network was released either by manipulating the main backbone HA chains or by selectively breaking the cross-links. The shrinkage rates obtained were within the range of stretching rates that have successfully stretched neuronal cells. We also confirmed that the material's cytocompatibility was unaffected by the chemical modifications to which HA was subjected. This polymer system is a novel addition to the existing polymeric actuators and is a step toward filling the void of a slow, long-term actuating polymer.

Comment: Lesions above a critical size in the peripheral nervous system or spinal cord following traumatic injury or infection remain one of the most intractable medical challenges, resulting in many tens to hundreds of thousands of cases of permanent paralysis worldwide per year despite extensive research into the mechanisms that limit regeneration across the injury site. Here, the natural capacity of axons to elongate under consistent mild tension during development is exploited in developing a biomaterial suitable to induce directional axonal regrowth in adults. This elegant method has the potential to function effectively, even in the absence of a full understanding of the biochemical cues that normally direct the process, and we eagerly look forward to data on its application in vivo. As the authors note, such materials are also likely to be useful in assisting the closure and resolution of other poorly healing large tissue defects, such as diabetic ulcers, perhaps in conjunction with appropriate stem cell therapy. ¹²

Extended Treatment Window Exists for Improving Sensorimotor Recovery Using Anti-Nogo-A Immunotherapy in Adult Rats with Chronic Stroke Deficits

Katherine Podraza, PhD, Loyola University Chicago

Stroke-related death ranks as the fourth most common cause of mortality in the United States. Current therapeutic options following stroke are limited. One potential therapy involves the use of anti-Nogo-A immunotherapy post-stroke. Our laboratory has shown that the administration of this novel treatment results in sensorimotor recovery in adult and aged rats. This therapy has been shown to be efficacious when applied immediately and at 24 hr and 1 week post-stroke in rats with mild to moderate sensorimotor deficits. In addition, recent results suggest that administration of anti-Nogo-A immunotherapy up to 9 weeks after stroke still induces functional recovery and neuroanatomical plasticity in adult rats with mild to moderate sensorimotor deficits. However, whether and how long anti-Nogo-A immunotherapy is efficacious when administered in the chronic stroke phase in rats with moderate to very severe sensorimotor deficits is unknown. In the present project, we sought to investigate the effectiveness of anti-Nogo-A immunotherapy as a therapeutic intervention to improve sensorimotor recovery when applied to chronic stroke-impaired adult rats with moderate to very severe sensorimotor deficits. Prior to testing the efficacy of anti-Nogo-A immunotherapy, we set out to evaluate the spontaneous motor recovery profiles in adult rats after stroke. We found that animals displayed different recovery profiles based on initial deficit severity after stroke, and overall all spontaneous recovery occurred within the first 4 weeks post-stroke. Therefore, the neurological deficit in the stroke animals had plateaued by the time anti-Nogo-A immunotherapy was given at 9 and 25 weeks post-stroke. We found that animals with moderate to very severe deficits treated with anti-Nogo-A immunotherapy at 9 or 25 weeks post-stroke showed improved sensorimotor recovery at both time points. However, the improved sensorimotor function was not associated with dendritic changes in either the perilesional or contralesional cortex. Our finding of improved sensorimotor recovery even when treatment was started 25 weeks post-stroke demonstrates the promising therapeutic potential for anti-Nogo-A immunotherapy to treat stroke sufferers long after the initial ischemic damage has taken place.

Comment: Most injuries to the body resolve naturally over a period of days to weeks, largely by recycling of the damaged tissue en masse accompanied by its replacement with new healthy tissue. For reasons that are not fully understood, this process is much less effective in the central nervous system (CNS); consequently, therapies for CNS injuries focus preferentially on halting the progression of damage. In the case of ischemic stroke, the standard of care involves the use of tissue plasminogen activator (tPA), a drug that dissolves the clot causing ischemia and thus reduces the area of tissue that suffers irreparable hypoxic insult. Although highly effective in reducing post-stroke disability when given as early as possible, tPA must be administered within a time window of at most 4.5 hr from onset and carries a risk of aggravating the patient's condition through increased non-specific bleeding. Widespread unawareness of stroke symptoms and misdiagnosis of the condition by emergency responders means that even in developed nations only a few percent of stroke patients ever receive tPA. There remains a desperate need for a more practical therapeutic option. Nogo-A is a potent neurite outgrowth inhibitor specific to the CNS, playing a critical role during development in limiting the growth of neural tissue and implicated in the failure of neural regeneration in adults. This study demonstrates that blocking Nogo-A activity through immunotherapy can dramatically improve the prognosis after an ischemic event, even when performed many weeks later. While the absence of detectable dendritic changes means that the mechanism of action involved is quite unclear, the need to translate this work to the clinic by accelerated means ¹³ is certainly not.

Japan's Aging Society: The Crisis of Care and the Hope of Prevention

Shana Ricart, PhD, University of Chicago

In an attempt to drive down national spending on a generous and comprehensive Long-term Care Insurance (LTCI) system (Kaigo Hoken), available to all persons aged 65 and over, Japan's Ministry of Health, Labor, and Welfare has promoted gerontological research into old-age disease prevention and health promotion, the development of prevention programs and services among local municipal governments, and established national policies organized around the prevention of the need for care and enrollment in LTCI. Through an ethnographic analysis of the newly formed Care-Prevention System (Kaigo Yobo Seido), I explore how health, self, and society are experienced and understood by its proponents and participants, including gerontologists, government employees, program managers, and seniors. Care prevention, as the name implies, is inextricably linked with the concept and practice of care in Japan today. The nation has been described as facing an Aging Society Crisis (shoshi korei ka shakai mondai), a crisis of an excess of care and the inability to support the demand. During this signal moment of health care policy change (the transition of policy focus from the provisioning of care to that of forestalling the need for care), the concept of “healthy aging” and how it can be achieved has gained precedence.

Healthy aging in Japan's care-prevention initiative is associated with continued activity, namely social engagement with others. If one remains active—mentally, physically, and especially socially—then one can remain healthy until the last days of one's life. In the ontology of old age forming here, what is stimulated through activity is a kind of vital energy latent within all humans. This vital energy emerges from within and is exchanged through human-social relations and with the surrounding living environment. The prevention of care in old age depends upon the stimulation of this latent desire for active living and sociality. Thus, the care-prevention movement is readily involved in community building, aiming to form ideal societies that are marked by the balanced flow of individuals, commerce, and nature, forming a kind of self-perpetuating organic whole. If we follow the ontology of old age care prevention being popularized in Japan today, once one is no longer able to stay active, one can no longer participate in sociality, and one loses the basis of one's humanity—active social interrelations. The senior in need of care is figured as a non-human thing more than a person, as the treatment of the in-need-of-care senior and concern attributed to him or her focuses on fostering the senior's smooth interactions with the surrounding living environments (promoting barrier-free living) and incorporating assistive technologies into their care regiment.

Comment: The traditional perception of aging as an immutable, inevitable process has motivated a general strategy of ever-improving palliative care throughout the field of geriatric medicine. While the desire to reduce suffering is of course laudable, this approach is sadly self-defeating. Any therapy that preserves the lives of the seriously ill without remedying their illness is doomed to ever-increasing costs, ¹⁴ costs that in many nations have now begun to edge out other forms of spending, including research into genuinely curative treatments. Although public perceptions of the aging process are finally beginning to change, as a result of recent achievements in research and the establishment of well-funded private organizations explicitly dedicated to intervention in aging, ¹⁵ most governments remain trapped in the 20^th century mindset. It is perhaps unsurprising that Japan, the country with the highest average life expectancy worldwide, is one of the first to officially recognize the need to take measures not just to make the lives of the elderly more bearable but to actively prevent the decline that results in a need for long-term care and dependency. The care prevention system comprises three key components—the detection of frail seniors, the implementation of programs to delay frailty (at present principally by encouraging physical and mental exercise and preventing malnutrition), and the evidence-based evaluation and development of those programs. Although its current methods are similar to those being applied in other nations, the combination of an explicit focus on the prevention of age-related disease before the onset of severe pathology and a systematic search for better techniques for achieving the same gives us considerable hope that Japan will become a leader in the design and validation of genuinely effective prophylactic medicine against the aging process itself. Meanwhile, the emphasis on keeping seniors engaged with society is itself likely to help raise awareness of the suffering caused by age-related disease.

Myocardin-Related Transcription Factor A Regulates Conversion of Progenitors to Beige Adipocytes

Chendi Li, PhD, Boston University

Thermogenic brown adipose tissue generates heat via the mitochondrial uncoupling protein-1 (UCP-1), increases whole-body energy expenditure, and may protect against obesity and metabolic disorders. White adipocytes store excess energy in the form of triglycerides. UCP-1–positive adipocytes develop within white adipose tissue (beige or brite adipocytes) in response to exposure to cold or β3 adrenergic agonists. It is known that beige adipocytes arise from a distinct lineage from that of brown adipocytes, but the developmental origin of the beige adipocytes is still unclear. Signaling pathways that control beige adipocyte determination and formation are essentially unknown. Here, we identified a novel signaling pathway that regulates the lineage specification of beige adipocytes. Bone morphogenetic protein 7 (BMP7), a known brown adipogenesis inducer, suppresses Rho-GTPase kinase (ROCK) and depolymerizes F-actin (filamentous actin) into G-actin (globular actin) in mesenchymal stem cells. G-actin regulates myocardin-related transcription factor A (MRTFA), which co-transactivates serum response factor (SRF) and promotes smooth muscle cell differentiation in various organs. Subcutaneous white adipose tissue from MRTFA ^−/− mice had enhanced accumulation of UCP-1⁺ adipocytes and elevated levels of brown-selective proteins. Compared with wild-type (WT) controls, MRTFA ^−/− mice exhibited improved metabolic profiles and were protected from diet-induced obesity and insulin resistance, suggesting that the beige adipocytes are physiologically functional. Compared to WT mice, stromal vascular cells from MRTFA ^−/− mice expressed higher levels of distinct beige progenitor markers and reduced levels of smooth muscle markers. Our studies demonstrate a novel ROCK–actin–MRTFA/SRF pathway that contributes to the development of beige adipocytes.

Comment: Obesity contributes in varying degree to a huge raft of cardiovascular, ¹⁶ metabolic, and other diseases, and its global prevalence continues to increase despite efforts to treat it through public education programs. Such programs are founded on a common perception of obesity as essentially a failure of self-control, aggravated by the large differences between the ancestral and contemporary food environments, but nonetheless resolvable simply through changes of habit. Although such a resolution is theoretically possible for a majority of sufferers, we are very skeptical that it is the most facile option, even for those without a specific genetic predisposition to weight gain. Rather than seeking to have a large fraction of the population engage in constant daily battle with their evolved drives, we would much prefer a biomedical solution able to stack the odds back in the patient's favor. In this context we have previously speculated on the therapeutic potential of brown adipocytes, specialized cells (actually more closely related to skeletal muscle than to white adipocytes) found primarily in neonates and hibernating species, which evolved to “waste” calories through constitutively inefficient mitochondrial respiration. Although brown adipocytes are found in adult mammals in very small numbers, and although previous research in mice has succeeded in expanding the population by various means, this represents a significant deviation from normal body composition. The long-term safety of brown adiposes is unclear; in particular, a greater risk of atherosclerotic plaque rupture has been observed in the context of elevated brown adipose populations. The more recently identified beige adipocyte population may represent a safer avenue of intervention, given that the activity of these cells is non-constitutive and inherently sensitive to metabolic state. By identifying a key pathway in the natural regulation of beige adipocyte development, this study brings us that much closer to a genuinely effective and safe “diet pill.”

Regulated Protein Aggregation: How It Takes TIA-1 to Tangle

Tara Vanderweyde, PhD, Boston University

The eukaryotic stress response involves translational suppression of non-housekeeping proteins and the sequestration of unnecessary mRNA transcripts into stress granules (SGs). This process is dependent on mRNA binding proteins (RBPs), such as T cell intracellular antigen (TIA-1). RBPs interact with unnecessary mRNA transcripts through prion and polyglutamine-like domains, and their aggregation mirrors proteins linked to neurodegenerative diseases. Recent advances in molecular genetics emphasize the importance of SG biology in disease by associating multiple RBPs linked to SGs with neurodegenerative disease. The major difference between SG proteins and aggregation-prone proteins in neurodegeneration is that aggregation of SGs is transient and rapidly reverses when the stress is removed. In contrast, aggregates associated with disease are stable and accumulate over time.

This study identifies over-abundant SGs as a novel pathology in Alzheimer's disease and related tauopathies. The data suggest that TIA-1 is intimately linked to tau pathogenesis, acting as a modifier of tau aggregation and associated toxicity. TIA-1 is present in a protein complex with tau protein, including hyper-phosphorylated and misfolded tau. The expression of WT or P301L mutant tau increases the formation and size of TIA-1–positive SGs, and the localization and dynamics of these SGs are altered. Conversely, the expression of TIA-1 increases the formation and stabilization of phospho- and misfolded tau inclusions, as well as visible alterations in microtubule morphology, perhaps reflecting a loss of tau function. The data further show that co-expression of TIA-1 and tau leads to dendrite shortening, increases in caspase cleavage, and apoptosis in primary neurons, suggesting that an interaction between TIA-1 and tau results in neurotoxicity. This toxicity is SG-dependent and is rescued by microtubule stabilizing drugs.

The results of this thesis research suggest that the aggregation of tau may proceed through the SG pathway, with SG formation accelerating the pathophysiology of tau aggregation. These studies propose that these tau aggregates serve as a nidus for further accelerated aggregation of SGs, leading to formation of long-lived pathological SGs.

Comment: Transient molecular damage occurs continually as a side effect of metabolism. In young individuals, this damage is swiftly remediated, allowing the system to reset to its healthy state. Species resistant to a particular form of aging tend to achieve this by better controlling levels of such intermediates. ¹⁷ However, and especially under conditions of high transient damage flux, some small proportion of such events fail to resolve before further less reversible processes take effect to convert the damage to a persistent form that is difficult or impossible for the body to remove by itself. The conversion of glycated amino acid residues and similar damage ¹⁸ to stable intermolecular cross-links is one canonical example, as is the formation of tangles and plaques through the aggregation of certain protein oligomers. The accumulation of such persistent damage is the defining characteristic of aging; when it results in an impairment of the processes responsible for removal of transient damage, or otherwise elevates the average load thereof, it leads inevitably to a vicious cycle of accelerating degeneration. This study identifies an exceptionally strong candidate for the transient damage type preceding the stable protein aggregates associated particularly with many neurodegenerative diseases, the initial pathogenesis of which has remained unclear despite enormous research efforts. The key role of a single protein raises the prospect—admittedly speculative—of engineering TIA-1 to reduce its affinity for pathogenic tau species without altering its normal role in stress granule (SG) biology. Even if this proves impossible, derangements of SG biology appear likely to be valuable as very early biomarkers of neurodegeneration.