Abstract
A Bioactive Scaffold of Aligned Peptide Amphiphile Nanofibers for Neural Regeneration
Eric Berns, PhD, Northwestern University
Innovative approaches are needed to regenerate the nervous system after damage or degeneration. This dissertation describes a scaffold comprised of aligned peptide amphiphile (PA) nanofibers engineered to direct neurite growth and cell migration. A series of PAs displaying the laminin-derived IKVAV epitope were designed for inclusion in aligned PA gels. Characterization of their nanostructures revealed that increased electrostatic repulsion from additional glutamic acid (Glu) residues disrupted β-sheet conformation, fiber formation, and fiber aggregation, leading to spherical micelle formation by PAs with the greatest number of Glu residues, which transformed into nanofibers by charge screening with calcium chloride. Enhancement of neurite growth was greatest in gels composed of IKVAV PAs with the greatest number of Glu residues, demonstrating that disruption of hydrophobic epitope aggregation via electrostatic repulsion is an effective strategy for increasing bioactivity. Another PA (TenC1 PA) was developed to promote neurite outgrowth and neuroblast migration in aligned gels. The TenC1 PA mimics the bioactivity of the extracellular matrix glycoprotein tenascin-C by displaying a peptide sequence from the protein. Aligned gels with TenC1 PA nanofibers promoted greater neurite length and production. Gel surfaces formed with TenC1 PA nanofibers increased independent cell migration of neural progenitor cells (NPCs). In addition to greater neurite lengths, neurons entrapped in aligned gels extended neurites that were highly aligned in the nanofiber direction. Aligned PA gels demonstrated several abilities critical for neuroregenerative applications; they were found to support electrophysiological maturation and synapse formation, differentiate NPCs into neurons, and promote directional migration of neural cells. As a strategy for treatment of chronic spinal cord injury, it was found that aligned gels could be formed in the spinal cord while simultaneously transplanting NPCs, which differentiate and grow processes in the direction of nanofibers. Last, to assess the potential of using aligned gels as a materials-based therapy that guides endogenous neuroblasts to brain regions with local neuronal loss, aligned PA gels formed in the brain demonstrated the ability to divert migrating neuroblasts from the rostral migratory stream to the neocortex. Gels incorporating TenC1 PA nanofibers were found to increase neuroblast migration along the PA track.
A Critical Role for Cortical Activation in Protection from Ischemic Stroke Damage
Melissa Davis, PhD, University of California, Irvine
Stroke is the leading cause for long-term disability and the number four cause of death in the United States. Despite a decrease in incidence, prevalence is on the rise due to increasing aging populations. The physiological, emotional, and economic effects of this disease are devastating in developed and undeveloped countries alike; in fact, stroke incidence in developing countries is still increasing and lack of advanced medical facilities means worse outcomes for patients. Nearly 90% of strokes are ischemic or are due to a blockage of blood flow to the brain. To date, despite intense clinical and pre-clinical efforts, only moderately effective treatment strategies are available. The literature suggests that although the intuitive solution is returning blood flow, the deprived area in ischemic stroke is not always correlated with positive outcome and can cause additional damage in some cases. Thus, reperfusion may be only a part of an ideal treatment strategy. This dissertation will present evidence demonstrating that complete protection from impending ischemic damage can be induced if sensory stimulation is delivered within the first 2 hr following ischemic onset in a rodent model. In clinical treatment, reperfusion is induced via pharmacological or mechanical thrombolysis without explicitly evoking activity and results in incomplete and inconsistent protection. In our model, reperfusion is also achieved via collateral vessels, but concomitantly with stimulation-induced cortical activity in the ischemic region resulting in complete protection. Evidence that increasing the volume of the cortex activated speed of recovery in protected animals is also presented. The results described so far were also replicated in an aged rodent model, further supporting the potential for translation of our findings to the more stroke-vulnerable aged population. We further demonstrate that evoked cortical activity eliminates ischemic damage in a temporary artery occlusion model that mimics blood flow return seen human patients with spontaneous or treatment-induced reperfusion. Evidence that evoked activity in one sensory cortex can protect from damage in an adjacent sensory cortex will be presented; blocking the spread of cortical activity from the auditory cortex to the somatosensory cortex by transecting the gray matter between cortices blocks protection. This transection does not, however, disrupt blood flow return, further supporting our hypothesis that cortical activity plays a critical role in protection and that reperfusion alone is an insufficient stroke treatment. If translational, the addition of sensory stimulation treatment to current reperfusion strategies might lead to outcomes more similar to the complete protection observed in our rodent model. Furthermore, stimulation treatment could provide an effective, non-invasive, low-cost treatment—one that could be implemented by untrained individuals even before arrival to medical facilities—making it of profound potential benefit in developing and undeveloped countries alike.
Advances and Challenges in Human Adeno-Associated Virus-Mediated Gene Transfer: Immunological Insights from a Mouse Model of Human Glycosylation
George Buchlis, PhD, University of Pennsylvania
Successful gene transfer for monogenic human disease can potentially provide a singularly administered, lifelong cure. Yet concerns remain over the safety and efficacy of gene transfer. Adeno-associated virus (AAV) is a commonly used gene transfer vector that is predominantly non-integrating, can transduce and persist in non-dividing cells, and is relatively non-inflammatory. AAVs have seen extensive pre-clinical success in animal models of Hemophilia B, with recent efficacy in the clinic. In both muscle-directed and liver-directed gene transfer of Factor IX (F.IX), multi-year expression of F.IX from AAV was observed in mice, dogs, and non-human primates. However, muscle-directed transfer to Hemophilia B human subjects resulted in sub-therapeutic circulating F.IX, and liver-directed transfer led to transient therapeutic F.IX plasma levels that were eliminated by a hepatocyte-clearing CD8 T cell response directed against the AAV capsid. Thus the challenge of maintaining long-term, clinically meaningful levels of F.IX from an AAV vector in human subjects remains.
The second chapter of this dissertation details a follow up study on the initial muscle-directed, AAV-F.IX trial. We now show 10-year F.IX expression in the muscle of a trial subject. This is the longest expression yet demonstrated in humans from a parenterally administered gene therapy vector. Although therapeutic levels were never achieved in this trial, the persistence of gene expression over a decade after vector administration is an important finding for the field of gene transfer.
Interestingly, AAV-F.IX delivery to the liver did result in efficacious levels of F.IX, but in the first human trial in liver, levels fell to baseline by 2 months post- delivery. The CD8 T cell response directed against the AAV capsid in these patients was not predicted in any pre-clinical animal studies. The third chapter of this dissertation investigates a uniquely human glycosylation mutation in the Cmah gene that potentially rendered humans more immunologically reactive. When modeled in mice, this mutation leads to enhanced T cell proliferation and activation in vitro, and to more robust T cell responses to viral challenges in vivo.
The goal of these investigations is to highlight the long-term potential of AAV-mediated gene transfer, while attempting to delineate the uniquely human immune mechanisms that that influence duration of expression and that were not predicted by extensive studies in other species.
Lysophosphatidic Acid Signaling via LPA5 Inhibits CD8+ T Cell Activation and Control of Tumor Progression
Shannon Oda, PhD, University of Colorado
CD8+ T lymphocytes are an important component of adaptive immunity, capable of specific identification of a foreign antigen, rapid proliferation, and cytotoxic activity to kill cells expressing the targeted antigen. T cell activation occurs via engagement of the T cell receptor (TCR) by its cognate antigen within the context of the major histocompatibility complex (MHC) molecule and is modulated by co-receptors that function to either support or inhibit activation. The adaptive immune system, and T cells in particular, are important for their ability to recognize and eliminate nascent tumors in cancer immunosurveillance and tumor rejection. However, multiple inhibitory mechanisms within the tumor microenvironment have been described that protect the tumor from T cells, including increased signaling of inhibitory co-receptors. Lysophosphatidic acid (LPA) is a lysophospholipid that is present at low nanomolar concentrations in the plasma of healthy individuals. It has been well documented that many cancers aberrantly produce LPA, and the increased level of LPA has been shown to be beneficial to the tumor, promoting tumorigenesis, invasion, metastases, and vascularization. However, the effects of elevated levels of LPA on the adaptive immune response have not been addressed. We demonstrate here that LPA inhibits CD8+ T cell activation in vitro and in vivo. Specifically, we show that CD8+ T cells express LPA receptor 5 (LPA5) that, in the presence of LPA, is able to inhibit TCR signaling and subsequent cell activation and proliferation. These data document that LPA is able to negatively regulate T cell activation and that LPA5 functions as an inhibitory T cell co-receptor. Finally, we have shown that transfer of LPA5-deficient T cells is able to abate tumor progression. We propose that lysophospholipid signaling is an additional protective mechanism of the tumor microenvironment to avoid anti-tumor CD8+ T cell immunity.
Pathogenesis of Neurodegenerative Diseases via Templated Recruitment
Ging Juo, PhD, University of Pennsylvania
A common feature of many neurodegenerative diseases is the deposition of filamentous protein aggregates in the central nervous system (CNS), including neurofibrillary tangles (NFTs) composed of tau protein, and Lewy bodies (LBs) consisting of α-synuclein (α-syn), which are the hallmark lesions of Alzheimer disease (AD) and Parkinson disease (PD), respectively. What causes the conversion of normally soluble proteins into insoluble fibrils has always been enigmatic, and cell models that recapitulate the abnormal accumulation of tau into NFT-like aggregates were lacking due to the high solubility of tau. Enlightened by in vitro studies showing nucleation-dependent fibrillization of tau, we tested the hypothesis that pre-formed tau fibrils (tau pffs) assembled from recombinant protein may act as seeds to nucleate the fibrillization of soluble tau in cultured cells. Indeed, we found that minute quantities of tau pffs internalized into cells over-expressing tau can rapidly recruit large amounts of endogenous tau into detergent-insoluble filamentous inclusions with properties very similar to NFTs. Moreover, the spontaneous uptake of tau pffs was shown to be mediated by endocytosis. Together with similar studies on tau and other disease-associated proteins, our study implicates cell-to-cell transmission of misfolded proteins through templated recruitment as a plausible mechanism for the onset and progression of CNS amyloidosis. Another mysterious phenomenon of neurodegenerative diseases is the frequent co-occurrence of different protein aggregates, such as NFTs and LBs. To test whether fibrillar α-syn can directly cross-seed tau into pathological aggregates, we used our recently developed synucleinopathy models in primary neurons and transgenic mice involving delivery of α-syn pffs. Intriguingly, we discovered two distinct strains of α-syn fibrils demonstrating a striking difference in the efficiency of cross-seeding tau pathology both in neurons and transgenic mice. Biochemical analyses indicated conformational differences between the two strains, thereby revealing the ability of a single molecule to assemble into more than one misfolded conformers. We speculate that the existence of conformationally diverse strains may be another shared feature of amyloid aggregates, accounting for the tremendous heterogeneity of neurodegenerative diseases with differential extent of concomitant pathologies and highly variable but sometimes overlapping clinical symptoms.
The Caenorhabditis elegans Life Span Machine and Its Application to the Temperature Scaling of Life Span
Nicholas Stroustrup, PhD, Harvard University
Life span results from the complex interaction between genetic, environmental, and stochastic factors, and therefore varies widely even among isogenic individuals. In Caenorhabditis elegans, the action of molecular mechanisms on aging can be inferred from their statistical effects on the distribution of life spans within populations. However, such investigations are hindered by limitations in the methods available for collecting life span data. To enable the rapid collection of survival curves at any desired statistical resolution, we developed an automated platform for determining the life spans of large populations of nematodes.
Our method combines high-throughput imaging with automated image analysis to generate a visual record of individual nematode deaths, from which survival curves are automatically constructed and visually validated. This approach produces results consistent with the manual method for several mutants in both standard and stressful environments, allowing rapid screening for genetic and environmental determinants of life span and enabling quantitative investigations into the statistical structure of aging.
We applied our method to study the effects of temperature on C. elegans aging across the range of 20°C to 36.5°C at fractional degree intervals. We found that the functional form of life span distributions appears invariant to temperature and is well-described across the entire temperature range by a Weibull model including a frailty correction. C. elegans' mean life span, however, appears not to follow a single scaling function of temperature. Rather, life span scales with temperature in a segmental manner, suggesting distinct physiological transitions occur around 30°C, 31.5°C, and 35°C. These findings imply a distinction between the molecular mechanisms that determine average life span, which differ between temperature ranges, and the overall stochastic behavior of mechanisms generating variation in life span, which appear temperature invariant.
We integrate these findings to propose a model of C. elegans aging where death occurs as a consequence of the first failure among vital components, all failing independently according to the same temporal distribution. Extreme value phenomena such as this can produce Weibull distributions across a diverse range of failure dynamics in underlying components, and thus might explain the shape of life span distributions and its conservation across a wide temperature range.