Commentary on Some Recent Theses Relevant to Combating Aging: June 2014

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

–7 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.

Brain–Machine Interface for Reaching: Accounting for Target Size, Multiple Motor Plans, and Bimanual Coordination

Peter Ifft, PhD, Duke University

Brain-machine interfaces (BMIs) offer the potential to assist millions of people worldwide suffering from immobility due to loss of limbs, paralysis, and neurodegenerative diseases. BMIs function by decoding neural activity from intact cortical brain regions in order to control external devices in real time. While there has been exciting progress in the field over the past 15 years, the vast majority of the work has focused on restoring of motor function of a single limb. In the work presented in this thesis, I first investigate the expanded role of the primary sensory (S1) and motor (M1) cortex during reaching movements. By varying target size during reaching movements, I discovered the cortical correlates of the speed–accuracy tradeoff known as Fitts' law. Similarly, I analyzed cortical motor processing during tasks where the motor plan is quickly reprogrammed. In each study, I found that parameters relevant to the reach, such as target size or alternative movement plans, could be extracted by neural decoders in addition to simple kinematic parameters, such as velocity and position. As such, future BMI functionality could expand to account for relevant sensory information and reliably decode intended reach trajectories, even amidst transiently considered alternatives.

The second portion of my thesis work was the successful development of the first bimanual brain–machine interface. To reach this goal, I expanded the neural recordings system to enable bilateral, multi-site recordings from approximately 500 neurons simultaneously. In addition, I upgraded the experiment to feature a realistic virtual reality end effector, customized primate chair, and eye tracking system. Third, I modified the tuning function of the unscented Kalman filter (UKF) to conjointly represent both arms in a single 4D model. As a result of widespread cortical plasticity in M1, S1, the supplementary motor area (SMA), and the posterior parietal cortex (PPC), the bimanual BMI enabled rhesus monkeys to simultaneously control two virtual limbs without any movement of their own body. I demonstrated the efficacy of the bimanual BMI in both a subject with prior task training using joysticks and a subject naïve to the task altogether, which simulates a common clinical scenario. The neural decoding algorithm was selected as a result of a methodical comparison between various neural decoders and decoder settings. Last, I introduced a two-stage switching model with a classify step and predict step that was designed and tested to generalize decoding strategies to include both unimanual and bimanual movements.

Comment: Although the gold standard for the restoration of function entails the replacement of failing tissue like-for-like with new, ex vivo–engineered or in situ–regenerated⁸ tissue, these efforts continue to be slowed by our limited understanding of developmental processes and the myriad challenges of working safely with biological systems.^9,10 Consequently, we can reasonably expect prosthetics to remain therapeutically important for the foreseeable future. The utility of a prosthetic, however, depends critically on how effectively it replaces the function of the original tissue—particularly, on how well it is connected to the nervous system, and hence how readily the brain is able to integrate it as a “natural” body part. Pre-clinical work in animal models has provided reassuring evidence that the brain is receptive to such integrations, even in quite unnatural scenarios where the prosthetic adds to the existing body rather than acting as a replacement. Thus, a large part of the remaining challenge is on the software side—finding algorithms to process neural firing patterns and reliably extract the operational intent behind them. This thesis expands significantly on the prior state of the art in that field, representing both a convincing proof of principle for the multi-limb prosthetics required by the most severely injured patients and a demonstration of the feasibility of the approach in the typical clinical scenario where the system cannot be trained in the context of normal healthy function. Once sufficient information can be obtained non-invasively, similar software will find an even broader use in guiding the operation of external mobility aids, compensating directly for the decline in kinetic parameters and gait quality observed in the elderly.¹¹

Function of Plasma Membrane V-ATPases in Breast Tumor Cell Invasion

Joseph Capecci, PhD, Tufts University

The vacuolar H⁺ adenosine triphosphatases (ATPases) (V-ATPases) are a family of adenosine triphosphate (ATP)-driven proton pumps that couple ATP hydrolysis with translocation of protons across membranes. V-ATPases are expressed in intracellular compartments, such as endosomes and lysosomes, where they participate in processes such as membrane trafficking and protein degradation. They are also present in the plasma membrane of specialized cells, such as osteoclasts and renal cells, where they function in bone resorption and urinary acidification, respectively. Previous studies have implicated V-ATPases in human cancer cell invasion. The a subunit, which controls cellular targeting of V-ATPases, is expressed as four isoforms in mammalian cells (a1–a4). The a3 and a4 isoforms target V-ATPases to the plasma membrane of osteoclasts and renal intercalated cells, respectively. Prior work from our laboratory comparing a subunit expression in human breast cancer cell lines has shown that both a3 and a4 are highly expressed in highly invasive MDA-MB231 cells compared to poorly invasive MCF7 cells and that knockdown of either isoform using isoform-specific siRNAs significantly inhibits invasion of MDA-MB231 cells. To further examine whether expression of particular a subunit isoforms is critical to invasiveness of breast tumor cells, two closely related cell lines have been examined. MCF10a is a non-invasive, immortalized, human breast epithelial cell line, and MCF10CA1a is a highly invasive, H-Ras-transformed derivative of the MCF10a cell line that has been selected for its ability to form metastases in mice. We find that inhibition of V-ATPase activity by concanamycin reduced in vitro invasion of MCF10CA1a cells, but not the parental MCF10a cells. MCF10CA1a cells expressed higher levels of the a3 isoform and higher levels of plasma membrane V-ATPases relative to MCF10a cells, and knockdown of a3 (but not other isoforms) using isoform-specific siRNAs inhibits invasion of MCF10CA1a cells. Importantly, overexpression of the a3 isoform in the parental MCF10a cells significantly increased both the level of plasma membrane V-ATPases and in vitro invasion. To determine whether expression of V-ATPases at the plasma membrane is important in breast cancer cell invasion, we employed an inhibitory antibody that selectively blocks plasma membrane V-ATPase activity. Inhibition of plasma membrane V-ATPases is shown to significantly reduce invasion of MDA-MB231 cells. These studies suggest that human breast tumor cells employ particular a subunit isoforms to target V-ATPases to the plasma membrane, where they aid in tumor cell invasion.

Comment: A sometimes underappreciated obstacle to cancer's efforts to spread throughout the body is the extracellular matrix—the web of collagen and other proteins that binds cells together. Failing to break through this restrictive net greatly restricts the cancer's proliferation, reducing its opportunity for mutation and its risk of acquiring other highly lethal phenotypes, while affording additional time for diagnosis and treatment. The matrix metalloproteinases (MMPs) are a family of enzymes expressed under tight control throughout the body, distinguished by their ability to efficiently degrade matrix proteins. Direct inhibition of the MMPs frequently associated with metastasis (such as MMP-9, the level of which is sufficiently elevated in some cancers to be detectable by breath testing¹²) has thus far proven therapeutically ineffective for reasons that remain unclear. Still, their dependence on acidic pH for optimal function suggests an indirect approach by preventing the development of such an environment. The finding in this work that an inhibitory antibody to the V-ATPases is effective in blocking metastatic invasion is particularly promising, as such contact therapeutics are relatively difficult for cancerous cells to evolve resistance to (typically by intracellular degradation or export from the cell). Although off-target toxicity in the kidneys is a concern, the off-target effect of reduced bone resorption might actually be beneficial in patients with impaired bone mass due to aging or the side effects of some other medications.

In Vivo Characterization of CLR01, an Aggregation and Toxicity Inhibitor, with an Alzheimer's Disease Focus

Aida Attar, PhD, University of California, Los Angeles

Aberrant protein self-assembly underlies over 30 human diseases called amyloidoses, for which there are no cures. In these diseases, particular proteins misfold and self-assemble into toxic oligomers that disrupt cellular function and proceed to form insoluble amyloid fibrils that deposit in specific tissues. A promising strategy for preventing and treating amyloidoses is inhibition or modulation of the self-assembly process to disrupt the formation of the toxic oligomers. In practice, this has proven immensely difficult because the oligomer structures are unknown, are metastable, and do not have distinct binding sites.

In this dissertation, three primary studies are presented that evaluate and characterize a small molecule, CLR01, which utilizes a novel strategy circumventing these challenges and has been found to be efficacious as an aggregation and toxicity inhibitor in vitro and in vivo. In the first study, CLR01 was evaluated for its ability to rescue synaptic toxicity in cell culture and brain slices. Additionally, it was tested in a transgenic mouse model of Alzheimer's disease (AD) for its ability to reduce the pathological hallmarks of AD—amyloid plaques and neurofibrillary tangles. This study found positive results in all domains tested—a rescue from amyloid β-protein (Aβ)-induced depletion of synaptic spine density, a rescue of Aβ-induced disruption of basal synaptic transmission and long-term potentiation, and reduction of brain Aβ, hyperphosphorylated tau, and microglial burden. CLR01 also showed low propensity for causing metabolic toxicity or drug–drug interaction, indicating favorable drug-like characteristics.

In the second study, CLR01's safety and pharmacological profile were characterized in mice. CLR01 was found not to disrupt normal protein assembly, to have a high safety margin in mice, and to penetrate the blood–brain barrier (BBB) at 1%–3%. Interestingly, brain levels of CLR01 remained stable for 72 hr following administration, despite rapid clearance from the plasma. These results suggest a large safety margin for CLR01 and a pharmacokinetic profile that allows reaching high levels in the brain by administering relatively low doses.

The third study delineates a detailed optimization of behavioral testing of mice for detection of memory deficits using the Barnes maze and validates for the first time memory deficits in a triple-transgenic mouse model of AD at the youngest age described in the literature. The study provides a framework for analysis of CLR01's influence on learning and memory deficits in this triple transgenic model. Additionally, the study provides specific and detailed guidelines for optimizing both the performance and the analysis of the Barnes maze in a manner that increases the likelihood of detecting subtle changes in future studies using mouse models of AD. The work described in this dissertation provides a strong foundation supporting formal pre-clinical development of CLR01 as a promising disease-modifying therapeutic drug for AD.

Comment: CLR01 is one of a class of so-called “molecular tweezers”—a chain of aromatic rings modified in symmetrical positions with cross-ring bonds to create consistent curvature, forming a crescent. The hydrophobic core of the molecule wraps around the butylene moiety within lysine side-chains in disordered peptides, while its two phosphates interact with the lysine's ε-amino group and with the peptide backbone. This interaction effectively blocks the relatively weak electrostatic and hydrophobic interactions that permit nucleation and amyloid fibril formation, and, given the ubiquity of lysine residues, the effect is observed regardless of the particular amyloid species involved. Even highly stable pre-formed Aβ fibrils have been shown to gradually dissociate in the sustained presence of CLR01 and related compounds,¹³ resulting ultimately in their clearance from the tissue. Combining this agent with other methods for interfering with fibril formation¹⁴ or enhanced lysosomal degradation¹⁵ clearly offers potential synergy. At the same time, the tweezers' binding affinity is too weak to interfere with the folding and stability of normal proteins, which depends on much stronger and more coordinated bonding tuned by evolution. Consequently, molecular tweezers appear remarkably safe and may even be suited to long-term prophylactic administration in healthy individuals to prevent future amyloidoses. Although future advances in cell therapy will be required to repair the damage already inflicted on aged brains and bodies by decades of amyloid accumulation, this work offers considerable hope that a general prophylactic may be within reach.

Investigating the Mechanisms of Reprogramming and Optimizing the Generation of Potentially Therapeutically Useful Induced Pluripotent Stem Cell Derivatives

Jason Awe, PhD, University of California, Los Angeles

Human induced pluripotent stem cells (hiPSCs), derived from easily obtainable skin cells, possess enormous opportunity for autologous cellular treatment therapies, gene correction, and disease modeling without worries of ethical constraints associated with human embryonic stem cells (hESCs). Although lentivirus-based reprogramming remains as one of the most popular methods for reprogramming, potentially oncogenic viral integrations in random locations throughout the genome along with non-human antigens associated with the reprogramming process thwart the clinical applications of these hiPSCs. To address these concerns, we derived a hiPSC line void of any exogenous reprogramming factors and differentiated these hiPSCs into clinically relevant cell derivatives. In addition, to maintaining clinical relevance, we implemented a methodology to clean our hiPSCs from non-human antigens to allow for current good manufacturing practice conditions that could help set the standard for human clinical trials with our factor-free hiPSCs. The field of stem cell reprogramming has rapidly advanced, and a new technique involving mRNA-based reprogramming was introduced that we found to be difficult to reproduce due to an innate immune response–based degradation of mRNA when introduced into the cell. To solve this problem, a small chemical compound was utilized that blocked important aspects of the innate immune response to single stranded mRNA that yielded robust and uniform expression of a key reprogramming factor. This stabilization could be important in increasing mRNA-based reprogramming efficiency of hiPSC derivation. Another challenge in the hiPSC field is investigating nuanced potential differences manifested in transcriptional, epigenetic, immunological, and differentiation potentials between hESCs and hiPSCs. To help and potentially solve this problem and allow for more complete and faithful reprogramming to a hESC state, global microarray transcriptional analysis of oocyte cytoplasm was utilized to find eight putative novel shared reprogramming factors across multiple species. These factors have identifiable roles in opening up chromatin that can allow reprogramming factors to better access reprogramming loci that could confer the known reprogramming advantage that somatic cell nuclear transfer–based reprogramming maintains over current direct reprogramming approaches. To address the recently observed immunogenicity issues of iPSCs, we studied the expression of two normally fetally associated genes implicated in an iPSC-specific immune response. We found high line-to-line variation between both hESC and hiPSC lines across different levels of differentiation and confirmed that current differentiation protocols derive cell types with a fetal phenotype as opposed to the adult phenotype needed for clinical applications as indicated by aberrant expression of specific fetal genes. Taken altogether, we hope these studies allow for more robust, reproducible, and clinically relevant hiPSCs that more closely resemble hESCs and maintain full ability to differentiate into clinically relevant cell types that can be used for potential human clinical trials for disease and cell replacement therapy.

Comment: Pathologies in which lost cells are inadequately replenished are common in aging, as a result of intrinsic stem cell dysfunction (due for example to epigenetic¹⁶ or telomeric¹⁷ damage), alterations to the systemic environment (e.g., the senescence-associated secretory phenotype¹⁸), or a combination of both (e.g., microvascular rarefaction¹⁹). They are also a frequent side effect of cytotoxic medicine. Regenerative medicine seeks to treat such conditions by introducing healthy new stem or progenitor cells, either in isolation or as part of a tissue-engineered construct grown ex vivo. Such treatments can be performed with allogeneic cells, but doing so requires long-term immunosuppression—which is physiologically taxing—or a method for inducing permanent tolerance to an entire panel of foreign antigens, which cannot yet be reliably achieved. (Modifying the transplanted cells to avoid stimulating immune attack may help²⁰ but seems less likely to offer a permanent solution.) Consequently, the phenomenon of induced pluripotency—in which experimental manipulation causes a fully differentiated cell to revert to an earlier developmental state with greater proliferative potential—has been of great interest since its discovery in 2006. Refinements in the field have brought iPSCs steadily closer to the clinic, but progress continues to be slowed by safety concerns related to the vectors used to induce pluripotency, as well as the lack of a clear explanation or remedy for the subtle but detectable differences in gene expression between iPSCs and ESCs. The chromatin-modifying factors identified in this study should resolve some of the confusion over the latter, and its findings overall form a valuable contribution to this advancing field.

Tracing Human Cancer Evolution with Hypermutable DNA

Kamila Naxerova, PhD, Harvard University

Metastasis is the main cause of cancer morbidity and mortality. Despite its clinical significance, several fundamental questions about the metastatic process in humans remain unsolved. Does metastasis occur early or late in cancer progression? Do metastases emanate directly from the primary tumor or give rise to each other? How does heterogeneity in the primary tumor relate to the genetic composition of secondary lesions? Addressing these questions in representative patient populations is crucial, but has been difficult so far. Here we present a simple, scalable PCR assay that enables the tracing of tumor lineage in patient tissue specimens. Our methodology relies on somatic variation in highly mutable polyguanine (poly-G) repeats located in non-coding genomic regions. We show that poly-G mutations are present in a variety of human cancers. Using colon carcinoma as an example, we demonstrate an association between patient age at diagnosis and tumor mutational burden, suggesting that poly-G variants accumulate during normal division in colonic stem cells. We further show that poorly differentiated colon carcinomas have fewer mutations than well-differentiated tumors, possibly indicating a shorter mitotic history of the founder cell in these cancers. We collect multiple spatially separated samples from primary carcinomas and their metastases and use poly-G fingerprints to build well-supported phylogenetic trees that illuminate each patient's path of progression. Our results imply that levels of intra-tumor heterogeneity vary significantly among patients. Our approach can generate reliable lineage information in large numbers of patients with minimal expenditure of time and cost. It can be used in its own right to study tumor evolution, or as an efficient screening tool to select samples for deeper analysis by next-generation sequencing. Further development and successful application of targeted cancer therapies will vitally depend on an accurate understanding of clonal architecture in human tumors. The mitotic history of a neoplasm, as captured by neutral lineage markers, can provide an important backdrop on which to project the distribution of hundreds of therapeutically relevant mutations.

Comment: Highly repetitive DNA sequences are subject to the greatest rates of mutation during normal meiosis, principally because the energy barrier to slippage of the replication machinery is low. Polyguanine repeats (GGG…) are the most mutable of all, undergoing deletion or insertion mutations at a frequency of around 10⁻⁴ per cell per generation in mammals. By sampling multiple poly-G tracts per cell, it can become possible to reliably identify all the daughters descended from a single parent, even after considerable time and/or phenotypic differentiation, and without dependency on the delivery or retention of any labelling agent. The method has recently been used to build lineage maps of murine development, similar to (if necessarily less deterministic than) the Caenorhabditis elegans cellular atlas. Turning the same technique on the development of cancer, as this study has shown feasible, promises to shed light on very fundamental but as yet poorly quantified parameters of early cancer development and metastatic transformation. The cheapness and scalability of the approach makes it particularly well suited for large-scale research efforts of the sort that will be required to identify common patterns of development in a condition as diverse as cancer. Meanwhile, new biomarkers such as intra-tumor poly-G heterogeneity seem likely to have immediate clinical application. Finally, it seems plausible that the distribution of poly-G fingerprints in the most differentiated cells of a tissue, monitored over a span of years to decades, might yield useful information about the health and diversity of the supporting stem and progenitor cells with very minimal invasiveness. Such records will also help in understanding the impact of long-term infections such as cytomegalovirus (CMV)²¹ and human immunodeficiency virus (HIV) on the hematopoietic compartment.

Vertebral Implantation of NELL-1 Enhances Bone Formation in Osteoporotic Sheep

Michael Chiang, MS, University of California, Los Angeles

Background: Vertebral compression fractures related to osteoporosis greatly afflict the aging population. The most commonly used therapy today is balloon kyphoplasty, a procedure that involves the inflation of a balloon and injection of cement such as polymethyl methacrylate. However, this treatment is far from ideal as it is associated with significant side effects, including local toxicity and rare systemic complications. NELL-1, a secreted osteoinductive factor that possesses both pro-osteogenic and anti-resorptive properties, is a promising candidate for an alternative to current treatment modalities. The use of NELL-1 in balloon kyphoplasty might improve post-surgical outcome for over 10 million osteoporotic American who are susceptible to vertebral compression fractures. The present study utilizes the pro-osteogenic properties of recombinant human NELL-1 (rhNELL-1) in lumbar spine balloon kyphoplasty using an osteoporotic sheep model.

Methods: Osteoporosis in n=8 sheep was induced through ovariectomy, dietary depletion of calcium and vitamin D, and steroid administration. After osteoporotic induction was confirmed by dual-energy X-ray absorptiometry (DXA), balloon kyphoplasty was performed in n=6 sheep with significant osteopenia. Sheep were randomly implanted with the control vehicle, comprised of hyaluronic acid (HA), hydroxyapatite, and β-tricalcium phosphate (β-TCP), or the treatment material of rhNELL-1 protein lyophilized onto β-TCP mixed with HA. Analysis of lumbar spine healing was performed by radiographic, histologic, and biomechanical testing.

Results: rhNELL-1 treatment significantly increased lumbar spine bone formation, as determined by bone mineral density, fractional bone volume, and mean cortical width by micro-computed tomography. Histological analysis revealed a significant increase in bone area as well as osteoblast number and decrease in osteoclast number around the implant site. Biomechanical properties of trabecular bone were analyzed using Finite Element Analysis (FEA), demonstrating that rhNELL-1-treatment resulted in a significantly more stress-resistant composition.

Conclusion: Our findings suggest rhNELL-1–based balloon kyphoplasty successfully improved cortical and cancellous bone regeneration in the lumbar spine of osteoporotic sheep. Thus, rhNELL-1–based balloon kyphoplasty represents a potential new anabolic, anti-resorptive local therapy for treating osteoporotic bone loss and/or fractures.

Comment: Osteoporosis is a progressive loss of bone mineral density, with an incidence that increases with age as part of a cluster of interrelated diseases known as the frailty syndrome. Although the condition manifests systemically, particular anatomical locations are associated with high risk of “fragility fracture”; these include the vertebrae, neck of the femur, and the wrist, all of which can break during a normal fall from standing height in the presence of advanced osteoporosis. These falls become increasingly common in the presence of sarcopenia, reduced sensory acuity, and other features of aging. The ability to robustly repair such fractures— present in healthy young patients, but lacking in the elderly—is critical to preventing a loss of instrumental physical function. In frail patients, such a loss, especially combined with a fear of further falls, can all too easily lead to irreversible decline.²² This work introduces a novel method to improve the outcomes of fracture repair in osteoporotic patients, and the relatively intensive and multi-factored osteoporotic regimen used as a model provides additional confidence that NELL-1 could also provide benefits in human senile osteoporosis.

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