2015 Pilot Award
Aileen Anderson, PhD
Inhibition of neutrophil infiltration to improve donor human neural stem cell-driven motor and sensory function after spinal cord injury
The microenvironment after spinal cord injury (SCI) may play a critical role in the potential for transplantedhuman neural stem cells (hNSC) to mediate functional recovery. We have identified evidence for a multiphasiccellular response after SCI that includes an early (1 day post-injury (dpi)) polymorphonuclear leukocyte (PMN or neutrophil) invasion and a surprising biphasic (7dpi & 60dpi) response by macrophages (MAC)/microglia inrodents. As the first predominant immune cells to infiltrate the injured spinal cord, PMN reside at the injury epicenter and synthesize many factors including complement proteins, cytokines, proteases and reactive oxygen species (ROS); these factors and their associated pathways are interconnected to affect cell death, fate/migration and function. We hypothesize that PMN modulate the migration and lineage selection of transplanted hNSC because, unlike subacute (9dpi) and chronic (30dpi) transplantation paradigms, acute/immediate (0dpi) transplantation of hNSC results in cell migration toward the injury epicenter as well as predominant astrocytic differentiation (>90%). Critically, this shift in migration and fate is correlated with the failure of hNSC transplants to produce functional locomotor recovery. Furthermore, conditioned media (CM) collected from PMN, but not MAC, promotes hNSC migration and astrocytic lineage differentiation in vitro. In this study we test whether the depletion of PMN via a PMN-specific Ly6G antibody can prevent hNSC astrogliosis and restore motor/sensory function after SCI in response to hNSC transplantation. The proposed work will provide new insight into the role of controlling transplantation timing and modification of the inflammatory microenvironment in the ongoing development of translational stem cell therapies, and could yield insight into the control and responsiveness of endogenous NSC populations after SCI. These studies will lay the foundation for development of strategies to control neural stem cell fate and migration in a therapeutic context.
Daniela Bota MD, PhD
An old drug with a new potential use: N-acetylcysteine preclinical testing as a treatment for chemotherapy-related cognitive impairment
This application focuses on the preclinical development of a pharmacological intervention for the prevention and treatment of chemotherapy-related cognitive impairments (CRCI). Millions of people are diagnosed with cancer every year, and more than 60% of these now survive to 20 years, unfortunately with severely diminished quality of life due to treatment-induced cognitive impairments. CRCI also influences the decisions of oncologists, and fear of CRCI constrains the doses and duration of chemotherapeutic regimens required for arresting tumors, with a diminished chance for cancer patients to achieve remission. Notably, research focused on therapeutic interventions for CRCI is still in its infancy, and clinical trials for medical treatments targeted at CRCI are currently unavailable. This proposal concentrates on preventing and reversing the cognitive impairments caused by cisplatin, which is a widely used cancer drug in stage III and IV ovarian cancer women. This patient population develops CRCI consistently during and after cisplatin-based chemotherapy. The PI identified that the cognitive deficits caused by cisplatin seem to result from the loss of excitatory synapses and dendritic spines that anchor them, as well as from injury to adult and developing neurons. Furthermore, this neuronal toxicity derives from mitochondrial damage and increased free radical production. N-acetylcysteine is a precursor of glutathione, the most important intracellular antioxidant. N-acetylcysteine is able to cross the blood-brain barrier, and has proven efficacy in both human and rodent models of neurodegenerative disorders. Oral N-acetylcysteine is a relatively cheap, convenient, safe drug, which was studied in multiple clinical trials in combination with cisplatin and has shown some promising results in preventing cisplatin-induced ototoxicity and peripheral neuropathy. N-acetylcysteine prevents cancer recurrence in colon cancer patients and its addition to chemotherapy does not decrease the anti-cancer effect of other drugs. Our preliminary data suggest that N-acetylcysteine prevents free radical production and ameliorates the apoptotic cellular death and mitigates the dendritic spine loss in cultured hippocampal neurons. Two specific aims are proposed: Aim 1. To determine if in vivo administration of the antioxidant N-acetylcysteine prevents synaptic loss and cell death of CA1 and CA3 hippocampal neurons provoked by a cisplatin regimen that recapitulates the cisplatin chemotherapy administered to women diagnosed with stage III-IV ovarian cancer. Aim 2. To determine if in vivo administration of the antioxidant N-acetylcysteine prevents the learning and memory deficits provoked by a cisplatin regimen that recapitulates the clinical treatment for ovarian cancer. The final aim of this project is to generate the data needed for a successful resubmission of the PIs R01 grant which proposes to conduct a clinical trial of the identified and pre-clinically-tested treatment paradigm.
Lisa Flanagan, PhD
Stem Cell Scaffolds to Treat Brain Trauma
Every year 800,000 people in the United States suffer a debilitating stroke. Stem cell transplantation stands as a promising treatment for those battling to regain function after enduring such a medical calamity. An appropriate scaffold implanted in conjunction with neural stem/progenitor cells (NSPCs) can promote proper survival and function of transplanted cells and may improve stem cell-mediated recovery in the central nervous system (CNS). Since the CNS consists of soft tissue, scaffolds injected as a liquid that polymerize in vivo can form a tight apposition with the necrosed area in the brain and promote recovery without further damaging surrounding tissue. Injectable scaffolds must be non-toxic, have tuned degradation rates, and their mechanical properties should match that of the native tissue to provide an optimal niche for transplanted NSPCs. The development of transplantation scaffolds with appropriate mechanical properties is critical because stem cell fate is strongly influenced by mechanotransduction, which describes the process by which cells convert mechanical stimuli into intracellular signals. Our preliminary work with NSPCs in 2-dimensional (2D) cultures has illustrated that cell differentiation is strongly affected by mechanical stimuli such as stretch and substrate stiffness, and this effect is likely mediated by integrin binding. Natural materials such as fibrin and hyaluronic acid (HA) are good choices as scaffolds for stem cell transplantation, but each material alone may lack the optimal physical characteristics for stem cell-based CNS repair. The proposed work in an in vivo model of stroke will investigate NSPC behavior within combinations of these scaffolds in order to synergistically take advantage of their individual material properties. Emphasis will be placed on optimizing the mechanical properties and degradation rates of the scaffolds for NSPC transplantation and integration into the infarct cavity of the stroke-insulted brain.
David Fruman, PhD
Efficiency and selectivity of a novel drug combination in aggressive Lymphoma
The goal of this proposal is to evaluate the efficacy and selectivity of ABT-199 in combination with HMG-CoA-reductase inhibitors (statins) in preclinical models. ABT-199 (venetoclax) is a small molecule inhibitor of BCL-2, a key pro-survival protein that is highly expressed in many leukemias and lymphomas. Statins are commonly used to control plasma cholesterol levels and are among the most widely prescribed medications worldwide. However, statins are also known to have anti-cancer potential. We have observed potent synergy of statins combined with ABT-199 in human cell lines derived from diffuse large B cell lymphoma (DLBCL). This synergy is also seen in murine B lymphoma cells derived from a genetically engineered mouse model. Using a BH3 profiling assay, we observed that simvastatin increases mitochondrial priming, correlating with its ability to synergize with ABT-199. Mechanistic studies support the hypothesis that statins prime lymphoma cells for apoptosis by blocking prenylation pathways downstream of mevalonate production by HMG-CoA-reductase. To extend these findings to an in vivo preclinical model, the first Aim of this project is to test combinations of ABT-199 and simvastatin in mice transplanted with B cell lymphoma driven by BCL-2 and MYC. We will assess differences in lymphoma outgrowth and mouse survival, and correlate with a pharmacodynamic marker of statin action. The second Aim is to assess the selectivity of the drug combination for lymphoma cells versus normal lymphocytes. We will measure the impact of drug treatments on normal lymphocytes in the in vivo mouse lymphoma models. In addition, we will obtain peripheral blood mononuclear cells from healthy human volunteers and assess viability of lymphocytes subsets after treatment with ABT-199, statins, or drug combinations. Together these experiments will establish efficacy and tumor selectivity of ABT-199 plus statins, providing proof-of-concept to support future clinical trials in aggressive lymphomas.
Harrison Lin, PhD
Chronic implantation of the auditory nerve: a successor to the cochlear implant
The cochlear implant is arguably the most successful present-day implantable neural prosthesis. In best-case conditions, it can bring speech recognition to profoundly deaf people. Nevertheless, even the most successful cochlear implant users cannot understand speech in noisy environments, are unable to localize sound sources, and lack pitch perception. The lack of pitch perception, in turn, greatly limits music appreciation and recognition of the emotional content of speech and precludes understanding of tonal language. These limitations result from the remote position of a conventional cochlear implant, separated by a bony wall from the target neural elements. We recently have demonstrated the feasibility of an alternate mode of prosthetic auditory stimulation: a penetrating electrode array that lies in intimate contact with auditory nerve fibers. By every measure possible in short-term (<24 hours) tests in anesthetized animals, the penetrating "intraneural" array out-performed a conventional cochlear implant, in some instances dramatically. However, the device that we tested in short-term experiments would not be suitable for long-term (decades-long) clinical application, and, importantly, auditory data from chronic intraneural implantation of the cochlear nerve in animal studies is still lacking. We propose now to obtain these data by implanting a commercially-available, chronically-implantable electrode array in the auditory nerves of cats. We would test the device first in short-term experiments intended to yield baseline values and then would evaluate three-month implantations, monitoring long-term stability of responses and any anatomical changes. Results from these three-month implantations of an off-the-shelf device would serve as pilot results to develop and test a device suitable for human use. These initial chronic animal studies are an essential step toward translating intraneural stimulation to a practical, next-generation human auditory prosthesis.
Hartmut Luecke, PhD
Understanding how lead compounds reactivate p53 cancer mutant function using novel biochemical and biophysical techniques
The tumor suppressor p53 is an important cell cycle regulating transcription factor and is the most mutated gene in human cancers. Most of these p53 cancer mutations are single amino acid substitution in the DNA-binding domain (DBD) that cause a destabilization of the p53 DBD. The most common of these p53 DBD missense mutations is the p53R175H mutation. A pharmaceutical which can restore endogenous p53 function to p53R175H or any of these single amino acid mutations could have an enormous impact on our treatment of cancer. Using an R175H protein-based chemical library screen and a cancer cell cultures validation, we have discovered the copper thiosemicarbazone, NSC635448, which is able to stabilize the R175H DBD and induce R175H-dependent cell cycle arrest in osteosarcoma cells. We are looking to gain important preliminary results on the restoration of mutant p53 function with NSC635448 in order to position ourselves to secure extramural funding. Using several biophysical techniques we plan to: (1)Use Biacore, electrospray ionization mass spectroscopy, and X-ray crystallography to gain a more definitive understanding of the binding of copper thiosemicarbazones to mutant p53 DBD (2)Validate that copper thiosemicarbazones actually restore function to mutant p53 within cancer cells using live-cell spatiotemporal tracking of mutant p53 activity and (3)Evaluate copper thiosemicarbazone toxicity in non-cancerous cells containing wild-type p53. The overarching goal of this project is to understand in detail how copper thiosemicarbazones restore function to mutant p53 and gain preclinical evidence for
copper thiosemicarbazones use as a mutant p53-targeting therapeutic.
Wayne Poon, PhD
Genetics of Sporadic AD: The Identification of Endophenotypes as Potential Diagnostic Biomarkers
Recent genome wide association studies (GWAS) have identified several single nucleotide polymorphisms (SNPs) that increase one’s risk of developing sporadic, late-onset Alzheimer’s disease (AD), the most prevalent cause of dementia. Individually, each inherited SNP modifies risk modestly and suggests that sporadic AD is a complex disease involving both genetic and environmental risk factors. Many of these SNPs are linked to genes common to biological pathways including endocytosis and immune function. One of the major neuropathological hallmarks of AD, β-amyloid, accumulates due to over-production of the toxic protein, the inability to clear the peptide sufficiently, acquisition of prion-like propagation, or a combination. The failure of clinical drug trials to date is likely due in part to the complex etiology of AD that may involve several biological processes that are unlikely to be targeted by a single compound. Furthermore, this complex etiology impacts the development of a diagnostic AD biomarker. The aim of this proposal is to identify AD risk endophenotypes that are diagnostic of increased number of polymorphisms within either the endocytic or the inflammatory pathway. Understanding the mechanism by which these SNPs contribute to AD risk will identify novel therapeutic targets. Our data suggest that the accumulation of SNPs for the endocytic pathway leads to increased hippocampal plaque burden. In preliminary studies, endocytic-pathway dysregulation results in increased Aβ production whereas immune function genes may determine the effectiveness of microglia to properly clear amyloid from the brain. The translational significance of these studies is that genetics may predict peripheral endophenotypes that that can in turn be used to develop personalized therapies targeting specific biological mechanisms.
Jing Yang MD, PhD
Evaluation of Retinal Progenitor Cells (RPCs) as Therapeutic Candidate for Retinopathy of Prematurity (ROP)
Retinopathy of prematurity (ROP) is a leading cause of childhood blindness that severely affects quality of life. It is associated with abnormal retinal vascular development and occurs only in premature infants. ROP is characterized by pathological angiogenesis, or neovascularization (NV), of the retina. The few treatment option that are currently available are associated with frequent unfavorable outcomes. Stem cell therapy, through its proven potential in tissue preservation/regeneration, provides an attractive strategy for treating ROP and thereby restoring vision. The proposed pilot study is designed to test the hypothesis that intravitreal injection of retinal progenitor cells (RPC) will result in improved visual outcomes in an established animal model of ROP. The oxygen induced retinopathy (OIR) rat model closely resembles the pathology present in human preterm infants with severe ROP. The newborn pups will be placed into a controlled oxygen chamber that changes oxygen level from 50% to 10% every 24 hours for 14 days. 2ul of rat RPCs or sham (vehicle only) will be injected into the vitreous cavity at the end of this induction period. Contralateral eyes will serve as untreated controls; at least 5 animals of each group will be evaluated in order to generate statistically meaningful data. Behavioral testing and histological evaluation will be performed on age-matched control animals (room air adapted rats) for comparison to cell/sham-injected animals. Functional analysis of retinal preservation, including optomotor response and ERG, will be performed at day 18. Histopathology and morphometric analysis will be performed to confirm retinal preservation. Given the PI's extensive preclinical work experience, including successful prior IND submission, performance of this pilot study should be relatively straightforward. The data generated by this study would provide Proof of Concept in support of a future large translational research grant application, e.g., CIRM early translational (ET) or disease team (DT) grant.