Anthony J. Windebank - US grants

myelination; lead; toxicology; lead poisoning; environmental contamination; tissue /cell culture; autoradiography; electron microscopy;

The use of platinum compounds is increasing in the treatment of cancer. Cisplatin, carboplatin and oxaliplatin produce a dose-related and dose-limiting sensory neuropathy. We have demonstrated that cisplatin binds to neuronal DNA, activates DMA-damage recognition pathways, initiates aberrant cell cycle entry, and induces apoptosis in vitro and in vivo. We now propose to test the hypothesis that platinum compounds produce neuronal injury by a common mechanism that involves separate nuclear (n-DNA) and mitochondrial DNA (mt-DNA) binding followed by synergistic activation of parallel death pathways. A new approach to measuring mt-DNA platination has been developed using inhibition of the polymerase chain reaction. We will determine whether the number of platinum adducts in mt-DNA of DRG neurons is sufficient to prevent replication and transcription of the mitochondrial genome. Function of respiratory chain components will be measured. Inability to repair Pt-DNA lesions in mt-DNA would result in attrition of mitochondria and chronic neuronal death explaining the clinical phenomenon of "coasting" or progression of neuropathy after drug cessation. We will use DRG neurons from Bax and cyclin D1 knockout mice to determine whether platinum-induced inhibition of mitochondrial function is sufficient to cause neuronal death. We will use the mitochondrial DNA synthesis inhibitor dideoxycytidine (ddC) to determine whether inhibition of mitochondrial DNA replication is independently sufficient to cause cell death. The mitochondrial genome will be protected by selectively increasing mitochondrial glutathione. The modifier and catalytic subunits ofglutamate cysteine ligase (GCL) andglutathione synthetase (GS) willbe targeted to mitochondria in an adeno-viral vector to reduce formation of mt-DNA adducts. Both nerve growth factor (NGF) and pigment epithelium derived growth factor (PEDF) have been demonstrated to partially protect DRG from cisplatin-induced apoptosis. We will determine whether a combination of therapeutic strategies that block n-DNA induced apoptosis and protect mt-DNA promote long-term survival of cisplatin treated DRG in vitro. If the combination strategy is effective,we will test it in an animal model by developing methods to provide long-term delivery of growth factors and viral targeting of GCL and GS to DRG in vivo. The goal is to develop a mechanism-based therapy that will prevent the major dose-limiting side effect of the platinum compounds.

DESCRIPTION (provided by applicant): Spinal cord axons have the capacity to regenerate following injury. However, functional improvement following spinal cord injury (SCI) in patients and in experimental animal models has been elusive. We have brought together a new research group combining expertise in polymer-based tissue engineering, cellular and molecular neurobiology, spine surgery, neurosurgery, and spinal cord injury. We have developed a series of novel biodegradable polymer implants for use in the treatment of SCI. Pilot studies of the implant in the rat transected spinal cord model demonstrated the potential for promoting axon regeneration. Implants were well-tolerated in the spinal cord and were loaded with Schwann cells that survive. During three months after implantation, there was axon growth throughout the length of the graft. We hypothesize that the implant can serve as a scaffold to support axon growth across a gap, as a source of supporting cells, and as a vehicle for controlled local delivery of agents that promote regeneration. We now propose to systematically manipulate the structural, cellular and molecular environment of the regenerating cord. In the first aim, we will study the degradation characteristics and biocompatibility of two polymers; poly (lactic-coglycolic)acid (PLGA) and poly(caprolactone fumarate) (PCLF). We will use computer-aided design to generate the three-dimensional structure of the scaffold and then determine whether vacuum molding or free-form fabrication (micro-printing) produces the best architecture. In the second aim we will examine the effect of scaffold geometry on regeneration by testing PLGA and PCLF scaffolds with varying diameter channels. The number and direction of axons regenerating through the scaffolds will be measured. In the third aim we will compare the ability of two cell types (primary Schwann cells and a Schwann cell line;SpL201) to support regeneration and to act as a source of biomolecules that promote regeneration. In the fourth aim we will examine the role of the biodegradable polymer as a delivery vehicle for therapeutic agents. Chondritinase-ABC will be used as a model protein. It is an enzyme that enhances axonal regeneration in the cord. Delivery of active enzyme after encapsulation in microspheres or in the graft will be compared and the effect of enzyme delivery in the regenerating cord will be assessed. Imaging with Micro-CT and MR microscopy will be combined with histological and functional assessments to measure success in promoting regeneration.

DESCRIPTION (provided by applicant): Loss of brain function is arguably the most dreaded consequence of getting old. We propose to test the hypothesis that restoration of youthful levels of mechano growth factor (MGF) will arrest the decline in adult neurogenesis that is linked to loss of brain function in old individuals. We will take advantage of an inducible gene expression system that utilizes control elements from the lac operon of E. coli to induce or repress transgene expression in the mouse. We will ask two questions: 1) if we induce transgenic MGF expression before endogenous MGF levels have started to decline in the mouse, can we prevent neuronal attrition in areas supplied by adult neurogenesis? And, 2) if we induce MGF when neurogenesis is already moderately or severely impaired, can we arrest neuronal attrition so that no further damage takes place or even repair the damage done up to that point? Mechano growth factor (MGF) is a non-hormonal form of insulin-like growth factor-1 (IGF-1), one of the most important postnatal hormones controlling growth in mammals. Like IGF-1, which peaks in adolescence when growth peaks and declines to low levels in old individuals, MGF is expressed in juvenile tissue at a much higher level than in tissue from old animals. MGF was discovered in adult muscle stem cells as a factor that stimulates proliferation after muscle stretch, stress, or damage. We have discovered that mechano growth factor is also expressed in adult neural stem cells (NSCs), where we think it might play a similar role. We propose that MGF is a critical juvenile protective factor that maintains brain function throughout life by stimulating the production of new neurons to replace those that have worn-out, become damaged, or died. The link between MGF, which is a non-secreted form of IGF-1, and the ability of stem cells to proliferate sheds new light on how the insulin/IGF system might regulate aging and longevity. Previous studies have shown very clearly that changes in the level of the insulin/IGF receptor and/or the activity of its associated signal transduction cascade can shorten or lengthen lifespan in a number of experimental animal species. Because MGF is not a hormone, but an isoform of IGF-1 that remains in the cell in which it is synthesized, it can have direct effects on cellular events that are independent of insulin/IGF signaling. In stem cells, the effect of insulin/IGF system on proliferation could be a combination of MGF intracellular activities and the activities of the signaling pathway. This could be one way the stem cell theory of aging and the known effects of the insulin/IGF-1 system on lifespan intersect. Loss of brain function is arguably the most dreaded consequence of getting old. We propose to test the hypothesis that restoration of youthful levels of mechano growth factor (MGF), a non-hormonal form of insulin-like growth factor (IGF)-1, will arrest the decline in adult neurogenesis that is linked to loss of brain function in old individuals. We will test this hypothesis in vivo, taking advantage of an inducible expression system our lab has developed that will allow us to flip the expression of MGF on and off at will.

Contact PD/PI: Khosla, Sundeep NRSA-Training-001 (253) A new generation of researchers is needed who can participate in and lead multidisciplinary teams that understand the process of translation from discovery to application to improvements in health. The long-term objective of TL1-supported programs within the Mayo Clinic Center for Clinical and Translational Science (CCaTS) is to train tomorrow's workforce of team-based, translational biomedical researchers at predoctoral levels. Three Specific Aims are proposed: 1) Recruit a diverse group of students at multiple career levels and integrate learning across these learner groups through a variety of programs. 2) Implement learning strategies for these students using novel curricula based on the R4 approach (the Right education at the Right time to the Right learner with the Right method), using novel approaches including case-based learning; team-based learning; task-oriented learning; and immersion in internships within industry, regulatory agencies, start-ups, and organizations that succeed through teamwork. 3) Rigorously evaluate outcomes and disseminate successful models, so that these novel programs can be continuously improved and the CTSA Consortium and others can benefit from these experiences. Ten slots are requested to support 3 different experiences: 1) The PhD Program in Clinical and Translational Science, an innovative PhD track developed de novo to accelerate the education of leaders in translational team research; 2) the Master's Degree in Clinical and Translational Science for medical students to prepare future physicians for careers in clinical and translational science; 3) short-term medical school research experiences to introduce students to the clinical and translational research process, and to motivate them to pursue further research training. An extensive framework for the TL1 Core that emphasizes diversity has been successfully built over the last 9 years. We will build upon this considerable experience, retaining those elements of proven benefit and implementing enhancements in the next funding cycle. Highlights of these enhancements include expanded training opportunities in team science, entrepreneurship, and the science of Translation and Regulatory Science; expanded opportunities for experiential learning on the Mayo campus, in partner institutions, and in novel extramural environments; enhanced ability to tailor didactic curricula to individual needs; and increased leveraging of institutional strengths in growth areas within the institution. Established collaborations with partner institutions, including the University of Minnesota CTSI and the University of Puerto Rico, will be strengthened, and rigorous evaluation systems that use innovative and comprehensive metrics to track individual and program progress and outcomes will be developed further. Successful practices will be disseminated through CTSA networks and national leadership. Project Summary/Abstract Page 1401 Contact PD/PI: Khosla, Sundeep NRSA-Training-001 (253) J. NRSA TRAINING CORE: Bibliography and References Cited 1. Staff NP, Runge BK, Windebank AJ. Breaking down translation barriers: investigator's perspective. Sci Transl Med. 2014;6(252):252cm257. 2. Pierret C, Sonju JD, Leicester JE, Hoody M, LaBounty TJ, Frimannsdottir KR, Ekker SC. Improvement in student science proficiency through InSciEd out. Zebrafish. 2012;9(4):155-168. PMCID: 3529492. 3. Greenberg AJ, McCormick J, Tapia CJ, Windebank AJ. Translating gene transfer: a stalled effort. Clin Transl Sci. 2011;4(4):279-281. PMCID: 3170101. 4. Juskewitch JE, Tapia CJ, Windebank AJ. Lessons from the Salk polio vaccine: methods for and risks of rapid translation. Clin Transl Sci. 2010;3(4):182-185. PMCID: 2928990. References Cited Page 1402