The cloning and characterisation of link1: A LIM-domain containing protein kinase

This thesis describes the isolation and cloning of a novel mouse gene, named mLimkl, which exhibits high homology to the human LIMK gene. mLimkl represents a single copy gene and maps to the distal end of mouse chromosome 5. Northern blot analysis showed preferential expression of a 3.5kb message in adult spinal cord and brain. In situ hybridisation studies confirmed high expression levels in the nervous system, particularly in the spinal cord and the cranial nerves and dorsal root ganglia. The amino acid sequence reveals two features which place mLimkl into a novel class of protein kinases. Firstly, although mLimkl contains all motifs found in catalytic kinase domains, amino acids previously described to be diagnostic of either serine/threonine- or tyrosine-kinases are not present. It is demonstrated that mLimkl-fusion protein can autophosphorylate on serine, tyrosine and threonine residues in vitro, and mutation of residue D460 within the IHRDL motif abolishes kinase activity. Secondly, mLimkl has two tandem LIM-domains in the amino-terminal region. These zinc-finger like domains can mediate protein-protein interactions and have been described in transcription factors and cytoskeletal proteins. The combination of LIM- and kinase domains may provide a novel route by which intracellular signaling can be integrated

Astrocytes derived from glial-restricted precursors promote spinal cord repair

BACKGROUND: Transplantation of embryonic stem or neural progenitor cells is an attractive strategy for repair of the injured central nervous system. Transplantation of these cells alone to acute spinal cord injuries has not, however, resulted in robust axon regeneration beyond the sites of injury. This may be due to progenitors differentiating to cell types that support axon growth poorly and/or their inability to modify the inhibitory environment of adult central nervous system (CNS) injuries. We reasoned therefore that pre-differentiation of embryonic neural precursors to astrocytes, which are thought to support axon growth in the injured immature CNS, would be more beneficial for CNS repair. RESULTS: Transplantation of astrocytes derived from embryonic glial-restricted precursors (GRPs) promoted robust axon growth and restoration of locomotor function after acute transection injuries of the adult rat spinal cord. Transplantation of GRP-derived astrocytes (GDAs) into dorsal column injuries promoted growth of over 60% of ascending dorsal column axons into the centers of the lesions, with 66% of these axons extending beyond the injury sites. Grid-walk analysis of GDA-transplanted rats with rubrospinal tract injuries revealed significant improvements in locomotor function. GDA transplantation also induced a striking realignment of injured tissue, suppressed initial scarring and rescued axotomized CNS neurons with cut axons from atrophy. In sharp contrast, undifferentiated GRPs failed to suppress scar formation or support axon growth and locomotor recovery. CONCLUSION: Pre-differentiation of glial precursors into GDAs before transplantation into spinal cord injuries leads to significantly improved outcomes over precursor cell transplantation, providing both a novel strategy and a highly effective new cell type for repairing CNS injuries

Transplantation of Specific Human Astrocytes Promotes Functional Recovery after Spinal Cord Injury

Repairing trauma to the central nervous system by replacement of glial support cells is an increasingly attractive therapeutic strategy. We have focused on the less-studied replacement of astrocytes, the major support cell in the central nervous system, by generating astrocytes from embryonic human glial precursor cells using two different astrocyte differentiation inducing factors. The resulting astrocytes differed in expression of multiple proteins thought to either promote or inhibit central nervous system homeostasis and regeneration. When transplanted into acute transection injuries of the adult rat spinal cord, astrocytes generated by exposing human glial precursor cells to bone morphogenetic protein promoted significant recovery of volitional foot placement, axonal growth and notably robust increases in neuronal survival in multiple spinal cord laminae. In marked contrast, human glial precursor cells and astrocytes generated from these cells by exposure to ciliary neurotrophic factor both failed to promote significant behavioral recovery or similarly robust neuronal survival and support of axon growth at sites of injury. Our studies thus demonstrate functional differences between human astrocyte populations and suggest that pre-differentiation of precursor cells into a specific astrocyte subtype is required to optimize astrocyte replacement therapies. To our knowledge, this study is the first to show functional differences in ability to promote repair of the injured adult central nervous system between two distinct subtypes of human astrocytes derived from a common fetal glial precursor population. These findings are consistent with our previous studies of transplanting specific subtypes of rodent glial precursor derived astrocytes into sites of spinal cord injury, and indicate a remarkable conservation from rat to human of functional differences between astrocyte subtypes. In addition, our studies provide a specific population of human astrocytes that appears to be particularly suitable for further development towards clinical application in treating the traumatically injured or diseased human central nervous system

Targeting Neuronal Function to Restore Motor Control Following Contusion Spinal Cord Injury

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Neuroscience Graduate Program, 2017.The central nervous system (CNS) is a unique tissue that is incapable of major repair after injury. Many CNS disorders have similar injury sequelae including neuronal death, loss of trophic support due to astrocytic activation, and myelin damage or loss. Very few therapeutics have successfully reversed the injury process, and there remains a large unmet need for treatments of CNS injury. Due to the conserved processes in CNS traumas, we chose to study the effects of our therapeutics in contusive spinal cord injury (SCI) as a model of both traumatic injury, and in its later stages,neurodegeneration. In our first study, study we look at the effects of the potassium channel blocker 4- aminopyridine (4AP) on acute spinal cord injury. 4AP has been used safely and effectively, long-term, in patients with multiple sclerosis, and has moderate success in patients with chronic SCI. Recently, our collaborators have shown 4AP to be a fast and effect means of treating peripheral nerve injury, and here we present data showing it to be as effective in CNS injury. Not only do we see rapid recovery of motor control, but we see reductions in lesion volume, and a greater preservation of neurons and oligodendrocytes in the 4AP treated animals compared to saline treated controls. This safe, well-tolerated, and FDA-approved drug may be a critical component of care for future patients with SCI. In our second study, we repeat and expand upon work that we have previously published by utilizing iPSC-derived astrocytes. We have previously shown that a specific population of astrocytes derived from embryonic tissue is capable of restoring motor function to animals with hemisection spinal cord injuries, as well as the more diffuse 6- hydroxydopamine injury that models Parkinson’s disease. Since then, we have attempted to use IPS-derived astrocytes, which will be more clinically relevant, and have found that there are differences between cell lines that translate to different rates of recovery. Furthermore, by switching to a contusion spinal cord injury, we have a more clinically relevant model to better understand how these cells may interact in a human patient with spinal cord injury. Taken together, both studies indicate that surviving neurons are sufficient to restore motor behavior. In both instances, treatment is started after the animal is completely paralyzed, and in both instances we see animals recover the ability to walk beyond the saline controls. We see no evidence of neurogenesis in either experiment, and therefore conclude that recovery must come from existing neurons. Using two different approaches to treat spinal cord injury, we think we may be able to target different aspects of the disease. By better understanding the mechanisms of both treatments, we may be able to apply them to other CNS insults, and eventually even to neurodegenerative diseases, where there is no acute trauma, but protecting long term health of the brain and spinal cord may be sufficient to slowing the progression of the disease

Astrocyte-based Approaches to Therapeutic Interventions for CNS Diseases

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pathology & Laboratory Medicine, 2013.Due to the limited regenerative capability of adult central nervous system (CNS), patients with traumatic injuries or neurodegenerative diseases must endure the long term debilitating consequences of these pathological conditions. Taking advantage of the neuroregeneration promoting properties of glial cells, we have developed a cell therapy for CNS diseases using a specific type of astrocyte. These astrocytes, referred to as glial-derived astrocytes by BMP induction (GDAsBMP), are derived in vitro from rodent embryonic glial restricted precursors (GRPs). Upon transplantation, GDAsBMP significantly promote axonal regeneration, cell survival and motor function recovery in a rat model of hemi-section spinal cord injury. To further evaluate the clinical potential of this novel, multimodal cell therapeutic, we proceed in a three-pronged strategy: 1) to identify the regeneration promoting factors produced by GDAsBMP for future use to enhance SCI therapies; 2) to test the usefulness of GDABMP transplants for neurodegenerative diseases of the CNS, and 3) to provide a more accessible source of GDAsBMP-like astrocytes by directed differentiation from human embryonic stem cells (hESCs). Using differential expression analysis between GRPs and GDAs, we have revealed a list of candidate factors that can modulate the pathological microenvironment in CNS to encourage regeneration. Among these candidates, we identified the novel function of a secreted factor, periostin, in promoting neurite outgrowth. Periostin is selectively expressed by GDAsBMP and essential for GDABMP-mediated axonal regeneration after transection injury to the spinal cord. Recombinant periostin overcame adverse effect of inhibitory substrates enriched at the site of CNS lesion and promotes neurite extension in cultured neurons. Furthermore, we also revealed that GDAsBMP produce significantly higher amount of anti-oxidants than other glial cells and can protect neurons from oxidative insults, which are common causes of neurodegenerative diseases. This anti-oxidant property contributes to the therapeutic efficacy of GDAsBMP in rescuing histological and behavioral deficits in a 6-hydroxydopamine model of Parkinson’s disease. Lastly, we successfully derived human astrocytes from human pluripotent stem cells (hESC and iPSC) through an intermediate neural progenitor stage. These hESC-derived astrocytes recapitulate characteristics of GDAsBMP with respect to neuronal survival and axonal outgrowth promoting effects. These results reveal the multimodal therapeutic activities of GDAsBMP and provide clinical applicable strategies for future CNS disorder therapies

The Study of astrocyte development and function using rat embryonic spinal cord derived glial restricted precursors

Thesis (Ph. D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pharmacology and Physiology, 2008.The purpose of this thesis is to study the regulation of astrocyte differentiation and to study the function of astrocytes and their interaction with neurons. We isolated glial restricted precursors (GRP) from the rat embryonic neural tube and used this cell type to examine factors that influence the differentiation of astrocytes from GRPs. We demonstrated that endothelin3-mediated signaling was one modulator of astrocyte generation. Endothelin3 induced the generation of an astrocyte precursor cell but not of mature astrocytes from GRP cells. Additionally, endothelin3 blocked the generation of oligodendrocytes, the other potential cell fate of GRP cells. The suppression of the oligodendrocyte lineage pathway by endothelin3 was accompanied by the down-regulation of oligodendrocyte lineage transcription factors. One of these transcription factors, Olig2 was actively degraded by endothelin3 through a post-translational pathway. Olig2 was found to block astrocyte precursor induction when over-expressed in GRP cells. In addition to the effects of endothelin exposure, the intracellular redox state also affected astrocyte generation from GRP cells. More oxidized GRP cells differentiated into astrocytes more readily than reduced GRPs. Moreover, GRP cells dissected from different regions of the rat embryonic neural tube bad different redox states and demonstrated a varied potential to give rise to astrocytes. We also studied the properties of two populations of astrocytes derived from the same GRP cells in terms of the interactions of astrocytes with neurons. These two groups of astrocytes differed in the expression of Olig2, production of axon growth inhibitors, and the ability to support neuronal growth. Olig2 was also a critical regulator of astrocytic phenotype, since over-expression or knock-down of Olig2 changed the properties of either kind of astrocyte. Our work provides new insights into the early stages of astrocyte development and mechanisms underlying roles of astrocytes in injuries, which will enhance the ability to use astrocytes and their precursors in cell transplantation therapies for tissue repair

Astrocytes derived from glial-restricted precursors promote spinal cord repair

Abstract Background Transplantation of embryonic stem or neural progenitor cells is an attractive strategy for repair of the injured central nervous system. Transplantation of these cells alone to acute spinal cord injuries has not, however, resulted in robust axon regeneration beyond the sites of injury. This may be due to progenitors differentiating to cell types that support axon growth poorly and/or their inability to modify the inhibitory environment of adult central nervous system (CNS) injuries. We reasoned therefore that pre-differentiation of embryonic neural precursors to astrocytes, which are thought to support axon growth in the injured immature CNS, would be more beneficial for CNS repair. Results Transplantation of astrocytes derived from embryonic glial-restricted precursors (GRPs) promoted robust axon growth and restoration of locomotor function after acute transection injuries of the adult rat spinal cord. Transplantation of GRP-derived astrocytes (GDAs) into dorsal column injuries promoted growth of over 60% of ascending dorsal column axons into the centers of the lesions, with 66% of these axons extending beyond the injury sites. Grid-walk analysis of GDA-transplanted rats with rubrospinal tract injuries revealed significant improvements in locomotor function. GDA transplantation also induced a striking realignment of injured tissue, suppressed initial scarring and rescued axotomized CNS neurons with cut axons from atrophy. In sharp contrast, undifferentiated GRPs failed to suppress scar formation or support axon growth and locomotor recovery. Conclusion Pre-differentiation of glial precursors into GDAs before transplantation into spinal cord injuries leads to significantly improved outcomes over precursor cell transplantation, providing both a novel strategy and a highly effective new cell type for repairing CNS injuries.</p