Autologous Peripheral Nerve Grafts to the Brain for the Treatment of Parkinson\u27s Disease

Parkinson’s disease (PD) is a disorder of the nervous system that causes problems with movement (motor symptoms) as well as other problems such as mood disorders, cognitive changes, sleep disorders, constipation, pain, and other non-motor symptoms. The severity of PD symptoms worsens over time as the disease progresses, and while there are treatments for the motor and some non-motor symptoms there is no known cure for PD. Thus there is a high demand for therapies to slow the progressive neurodegeneration observed in PD. Two clinical trials at the University of Kentucky College of Medicine (NCT02369003, NCT01833364) are currently underway that aim to develop a disease-modifying therapy that slows the progression of PD. These clinical trials are evaluating the safety and feasibility of an autologous peripheral nerve graft to the substantia nigra in combination with Deep Brain Stimulation (DBS) for the treatment of PD. By grafting peripheral nerve tissue to the Substantia Nigra, the researchers aim to introduce peripheral nerve tissue, which is capable of functional regeneration after injury, to the degenerating Substantia Nigra of patients with PD. The central hypothesis of these clinical trials is that the grafted tissue will slow degeneration of the target brain region through neural repair actions of Schwann cells as well as other pro-regenerative features of the peripheral nerve tissue. This dissertation details analysis of the peripheral nerve tissue used in the above clinical trials with respect to tissue composition and gene expression, both of injury-naive human peripheral nerve as well as the post-conditioning injury nerve tissue used in the grafting procedure. RNA-seq analysis of sural nerve tissue pre and post-conditioning show significant changes in gene expression corresponding with transdifferentiation of Schwann cells from a myelinating to a repair phenotype, release of growth factors, activation of macrophages and other immune cells, and an increase in anti-apoptotic and neuroprotective gene transcripts. These results reveal in vivo gene expression changes involved in the human peripheral nerve injury repair process, which has relevance beyond this clinical trial to the fields of Schwann cell biology and peripheral nerve repair. To assess the neurobiology of the graft post-implantation we developed an animal model of the grafting procedure, termed Neuro-Avatars, which feature human graft tissue implanted into athymic nude rats. Survival and infiltration of human graft cells into the host brain were shown using immunohistochemistry of Human Nuclear Antigen. Surgical methods and outcomes from the ongoing development of this animal model are reported. To connect the results of these laboratory studies to the clinical trial we compared the severity of motor symptoms before surgery to one year post-surgery in patients who received the analyzed graft tissue. Motor symptom severity was assessed using the Unified Parkinson’s Disease Rating Scale Part III. Finally, the implications and future directions of this research is discussed. In summary, this dissertation advances the translational science cycle by using clinical trial findings and samples to answer basic science questions that will in turn guide future clinical trial design

RNA Sequencing of Human Peripheral Nerve in Response to Injury: Distinctive Analysis of the Nerve Repair Pathways

The development of regenerative therapies for central nervous system diseases can likely benefit from an understanding of the peripheral nervous system repair process, particularly in identifying potential gene pathways involved in human nerve repair. This study employed RNA sequencing (RNA-seq) technology to analyze the whole transcriptome profile of the human peripheral nerve in response to an injury. The distal sural nerve was exposed, completely transected, and a 1 to 2 cm section of nerve fascicles was collected for RNA-seq from six participants with Parkinson\u27s disease, ranging in age between 53 and 70 yr. Two weeks after the initial injury, another section of the nerve fascicles of the distal and pre-degenerated stump of the nerve was dissected and processed for RNA-seq studies. An initial analysis between the pre-lesion status and the postinjury gene expression revealed 3,641 genes that were significantly differentially expressed. In addition, the results support a clear transdifferentiation process that occurred by the end of the 2-wk postinjury. Gene ontology (GO) and hierarchical clustering were used to identify the major signaling pathways affected by the injury. In contrast to previous nonclinical studies, important changes were observed in molecular pathways related to antiapoptotic signaling, neurotrophic factor processes, cell motility, and immune cell chemotactic signaling. The results of our current study provide new insights regarding the essential interactions of different molecular pathways that drive neuronal repair and axonal regeneration in humans

Increased O-GlcNAc levels correlate with decreased O-GlcNAcase levels in Alzheimer disease brain

The potential role of the posttranslational modification of proteins with O-linked N-acetyl-β-D-glucosamine (O-GlcNAc) in the pathogenesis of Alzheimer disease (AD) has been studied extensively, yet the exact function of O-GlcNAc in AD remains elusive. O-GlcNAc cycling is facilitated by only two highly conserved enzymes: O-GlcNAc transferase (OGT) catalyzes the addition, while O-GlcNAcase (OGA) catalyzes the removal of GlcNAc from proteins. Studies analyzing global O-GlcNAc levels in AD brain have produced inconsistent results and the reasons for altered O-GlcNAcylation in AD are still poorly understood. In this study, we show a 1.2-fold increase in cytosolic protein O-GlcNAc modification in AD brain when compared to age-matched controls. Interestingly, O-GlcNAc changes seem to be attributable to differential modification of a few individual proteins. While our finding of augmented O-GlcNAcylation concurs with some reports, it is contrary to others demonstrating decreased O-GlcNAc levels in AD brain. These conflicting results emphasize the need for further studies providing conclusive evidence on the subject of O-GlcNAcylation in AD. We further demonstrate that, while OGT protein levels are unaffected in AD, OGA protein levels are significantly decreased to 75% of those in control samples. In addition, augmented protein O-GlcNAc modification correlates to decreased OGA protein levels in AD subjects. While OGA inhibitors are already being tested for AD treatment, our results provide a strong indication that the general subject of O-GlcNAcylation and specifically its regulation by OGA and OGT in AD need further investigation to conclusively elucidate its potential role in AD pathogenesis and treatment