Itzhak Fischer, PhD - US grants

Microtubule Associated protein 2 (MAP2), is a complex protein, which co-purifies with microtubules of the neuronal cytoskeleton, and seems to have an important role in the organization of microtubules and their interactions with other proteins and organelles. MAP2 is of particular interest in brain because of its abundance, its specific localization in dendrites, and the regulation of its expression during brain development and neuronal differentiation in culture. The molecular mechanism of MAP2 expression in brain and in PC12 cells, will be studied at the level of protein systhesis, mRNA and the genes that code for this protein. For that purpose, MAP2 cDNA will be isolated, by screening lambda gt11 brain libraries with highly specific polyclonal and monoclonal antibodies. The clone identity will be confirmed by hybrid selection, and by probing RNA derived from different tissues and cells lines with the cDNA. Initial observations showed a significant induction of MAP2 levels during treatment of PC12 cells with NGF. The mechanism of this induction will be analyzed by the temporal correlation between the rate of MAP2 synthesis (by pulse labeling), the translational activity of MAP2 mRNA (by in vitro translation), and the steady state levels of the mRNA (by Northern blots). Similar techniques will be used to follow the developmental regulation of MAP2 isotypes (MAP2a and MAP2b) in rat brain; specifically, whether these different isotypes are the product of precursor processing, or translation of two distinct transcripts. The genetic complexity of MAP2 will be examined by Southern blots, to determine whether the MAP2 gene is part of a family that shares structural and functional domains. Finally, the appearance and localization of MAP2 mRNA, measured by in situ hybridization, will be compared to the immunocytochemical distribution of MAP2 in dendrites, thus, testing the hypothesis that polysomes located near dendritic spines may be engaged in local protein synthesis. The objective of this research is to understand the regulation of cytoskeletal proteins during neuronal differentiation, and the role they play in the morphological abnormalities associated with many neurological disorders, particularly, in relation to dendrite dystrophy associated with mental retardation.

9319693 Nowycky Neurons and endocrine cells release chemical messengers to communicate with other cells. The triggering signals for release begin with the entry of calcium from the extracellular environment. Calcium ions appear to act on many proteins in a coordinated fashion for the release process to occur. In this grant proposal, we will use real- time measurements of release from single cells to study the intracellular responses to calcium entry which are necessary for release.***

MAP1B is a prominent microtubule associated protein whose expression is developmentally regulated, showing the highest levels in growing axons during early postnatal brain development. Our interest in the expression of MAPs in the nervous system has been related to the role of cytoskeletal proteins during neuronal differentiation, but the experiments that we propose will also address fundamental problems concerning the regulatory elements that control the expression of other brain- specific genes. Specific questions addressed in this proposal include: What are the mechanisms that regulate MAP1B expression? What are the regulatory elements that control the cell and stage-specificity during transcription of the MAP1B gene and its response to physiological signals (e.g., NGF, cAMP)? What is the role of MAP1B during axonal growth in neurons? The first set of experiments utilizes nuclear run-on assays to confirm that the control of MAP1B expression is exerted at the transcriptional level. Specific regulatory mechanisms can then be identified from studies of the structure and function of MAP1B promoter. We hypothesize that, as with other brain-specific genes, the basic elements of the MAP1B promoter are defined within a relatively small region upstream from the initiation site of transcription. In preliminary experiments we have already isolated genomic clones that contain part of the MAP1B promoter and determined the initiation site of transcription. Additional distal elements will be identified by nuclease hypersensitivity assays of neuronal cells. Our approach is to follow a set of experiments that are based on in vitro assays of chimeric constructs in which regulatory elements are linked to a reporter CAT gene for functional analysis of MAP1B promoter in both neuronal and non-neuronal cells. These experiments will be followed by physical mapping of specific regulatory regions by footprinting, methylation interference, and gel shift assays. Additional mapping experiments using nuclease hypersensitivity assays will be directed at more distal regulatory elements and help to design promoter constructs that can be studied in vivo with transgenic animals. Finally, the requirement for MAP1B during axonal differentiation will be examined by suppression of its expression in cultured primary neurons using antisense oligonucleotides and by microinjection of MAP1B peptides and antibodies prepared against selected regions of the molecule. These strategies can be extended to study promoters of other MAPs, for which, thus far, no promoters have been described, and to isolate specific transcription factors that control these genes. These studies will also become the basis for in vivo work and development of experimental tools for manipulation of gene expression in transgenic animals. Ultimately, the characterization of these genes and their promoters will provide an understanding of the molecular pathways of neuronal differentiation during development of the nervous system and in response to changes in the environment.

The devastating effects of spinal cord injury are due to the death of neurons and to the failure of the axons of surviving neurons to regenerate through the inhospitable environment created by the injury. The proposed experiments will test whether novel cellular and molecular strategies of repair will promote regeneration leading to locomotor and sensory recovery in well-characterized models of spinal cord injury in adult rats. In preliminary studies we have prepared retrovirus and recombinant adenovirus constructs of neurotrophins and shown that intraspinal transplants of fibroblasts genetically modified to express BDNF promote regeneration of rubrospinal axons that contribute to locomotor recovery. We have also used intraspinal injections of plasmid constructs and recombinant adenovirus to administer genes to spinal and supraspinal neurons that can enhance their survival and regeneration after axotomy. In the present experiments we will use as transplants multipotential neural stem cells isolated from embryonic rat spinal cord. These cells are very promising because of their capacities for self- renewal, differentiation into neurons and glia and genetic modification. We will genetically modify these cells to express neurotrophin factors BDNF and NT3 or adhesion protein L1, and in addition deliver the antiapoptotic gene Bcl-2 by plasmid injections or adenovirus. We propose that this combination of treatments will enhance neuron survival and axon regeneration and promote the recovery of locomotor and sensory function as measured by quantitative tests. In the first series of experiments we will test the idea that engineered neural stem cells transplanted into a unilateral cervical hemisection lesion will integrate with the injured host and supply factors that will rescue axotomized spinal and supraspinal neurons, promote regeneration of their axons and enhance recovery. In the second series of experiments we will test the idea that the best strategy of combined treatments will stimulate regeneration of descending pathways and recovery of hindlimb function after spinal cord transection, a model for complete spinal cord injury in humans in which results of anatomical and behavioral studies are unambiguous. The results of these experiments will contribute to developing an effective strategy for promoting neuron survival and axon regeneration that will enhance functional recovery after spinal cord injury.

DESCRIPTION (provided by applicant): This proposal is focused on application of recent advances in stem cell biology to a potential therapy for spinal cord injury. The proposed experiments will utilize mesenchymal stem cells derived from human bone marrow (marrow stromal cells, hMSCs), examine their phenotype following transplantation into spinal cord and test their ability to promote regeneration and recovery of function. Human MSCs are easy to obtain from a patient under local anesthesia, provide a renewal population and can be used for autologous grafting. Our preliminary data show that hMSCs grafted into spinal cord lesions survive, integrate well and appear to be permissive for axonal growth, and thus are excellent candidates for use in spinal core repair strategies. The full potential of hMSCs to differentiate is not well understood, but there is growing evidence that adult cells from bone marrow have remarkable plasticity and can assume diverse phenotypes dependent on the environment that they encounter. We reasoned that by transplanting hMSCs cells into spinal cord we can combine a systematic and direct examination of their fate in vivo with a detailed analysis of their therapeutic effects in neuronal rescue, axon regeneration and functional recovery in a well characterized model of spinal cord injury. Previous studies and our preliminary data suggest that hMSCs produce a variety of cytokines and neurotrophic factors that support survival and regeneration. The proposed experiments will examine the behavior of untreated and treated hMSCs in normal spinal cord (AIM 1) and in a well-characterized injury model of a unilateral C4 lesioned spinal cord (AIM 2). These cells will be analyzed with respect to their survival, migration and phenotype as well as their ability to rescue axotomized neurons (in the Red Nucleus), promote axon regeneration (of corticospinal and rubrospinal tracts) and restore function (using cylinder, rope and grid walking, reaching and patch removal tests). The therapeutic potential of different batches of hMSCs will then be correlated to their expression profile of secretory factors. In Aim 3 we will study the ability of grafted hMSCs to deliver therapeutic genes (BDNF and NT3) to the injured spinal cord and the effects of the genetic modification on the phenotypic properties of these cells and the potential for repair and restoration of function. Taken together, these experiments will provide a through preclinical analysis of the properties and therapeutic potential of hMSCs in the injured CNS.

DESCRIPTION (provided by applicant): Bone marrow stromal cells (MSC) have shown great promise in improving outcome after spinal cord injury. There is, however, no efficient method to deliver them to the injury site. This is a small and focused research proposal that aims to develop and optimize protocols for delivering MSC via a Lumbar Puncture (LP) in a rat model of spinal contusion. LP delivery of MSC is a minimally invasive procedure that can be easily translated into human patients. Delivery of MSC via LP places cells into the cerebrospinal fluid (CSF), away from the hostile environment of the contused cord, and yet the circulating CSF offers MSC an opportunity to home into the injured site. Preliminary data and our published experience demonstrates that LP delivery is a feasible proposition. First, this project proposes to determine optimal protocols for performing LP transplantation of MSC for spinal cord contusion. Next, it uses these optimal protocols and compares their functional outcome with the standard method of direct parenchymal transplantation. Finally, it explores the possibility of performing autologous MSC transplantation (using syngeneic transplantation to mimic the autologous model) without any immunosuppression. Long-term cell survival experiments are also proposed to demonstrate lack of neoplastic potential and other toxicities with MSC transplantation into the central nervous system. All protocols will be validated by performing detailed histological and behavioral assessments. It is hoped that the results generated from this project will produce an innovative methodology for delivering stem cells in a minimally invasive way. This novel technique will lead to significant breakthroughs in translating basic stem cell research into the clinic and will facilitate earlier clinical trials because of the minimally invasive nature of this technique.

DESCRIPTION (provided by applicant): Bone marrow stromal cells (MSC) have shown great promise in improving outcome after spinal cord injury. There is, however, no efficient method to deliver them to the injury site. This is a small and focused research proposal that aims to develop and optimize protocols for delivering MSC via a Lumbar Puncture (LP) in a rat model of spinal contusion. LP delivery of MSC is a minimally invasive procedure that can be easily translated into human patients. Delivery of MSC via LP places cells into the cerebrospinal fluid (CSF), away from the hostile environment of the contused cord, and yet the circulating CSF offers MSC an opportunity to home into the injured site. Preliminary data and our published experience demonstrates that LP delivery is a feasible proposition. First, this project proposes to determine optimal protocols for performing LP transplantation of MSC for spinal cord contusion. Next, it uses these optimal protocols and compares their functional outcome with the standard method of direct parenchymal transplantation. Finally, it explores the possibility of performing autologous MSC transplantation (using syngeneic transplantation to mimic the autologous model) without any immunosuppression. Long-term cell survival experiments are also proposed to demonstrate lack of neoplastic potential and other toxicities with MSC transplantation into the central nervous system. All protocols will be validated by performing detailed histological and behavioral assessments. It is hoped that the results generated from this project will produce an innovative methodology for delivering stem cells in a minimally invasive way. This novel technique will lead to significant breakthroughs in translating basic stem cell research into the clinic and will facilitate earlier clinical trials because of the minimally invasive nature of this technique.

Despite progress in elucidating neural stem cell properties and using them for transplantation, the challenges of neuronal cell replacement following CNS injury remain difficult to resolve. This proposal will use transplants of neuronal and glial restricted progenitors (NRP/GRP) to reconnect the disrupted sensory system after acute and chronic spinal cord injury (SCI). The experiments are designed to build neuronal relays across a dorsal column lesion reconnecting the denervated dorsal column nucleus (DCN). The proposal addresses important issues related to application of stem cell biology to CNS injury, including how to generate graft derived neurons, direct their axon growth, overcome the inhibitory environment of the injury, and, most importantiy, how to form functional synapses with the denervated target. We will continue our studies that demonstrated the principles of relay formation with NRP/GRP transplants by testing specific hypotheses for improving the functional aspects of the relay and advance this strategy to chronic SCI. In Aim 1, we will test the role of synaptic density and activity-induced plasticity in producing stable and functional synaptic connections at the DCN target. We propose that a) synaptic activity with target neurons will improve through summation of the evoked potentials at the target site by increasing synaptic density (more axons) and reducing inhibition of perineuronal nets at the DCN, and b) synapse stability will improve with time, with task-specific activity, and with stimulation of the sensory relay. In Aim 2 we will test the hypothesis that denervated targets can be reconnected and retrained to correctly interpret sensory information at a chronic injury stage. Specifically, we will test a) how to promote host sensory axons to grow into the site of a chronic injury, and b) how to promote connectivity at the target, where the DCN has been subject to prolonged deafferentation. To achieve connectivity in the chronic injury, we will use neurotrophins, chondroitinase (delivered by novel viral vectors), and exercise/training protocols. This relay model allows us to go beyond the current focus on regeneration, exploit the advantages of embryonic neurons produced by neural stem cells, and examine the steps needed to restore connectivity and improve function in a well-defined system.

DESCRIPTION (provided by applicant): Spinal cord injury (SCI) affects approximately 10,000 individuals in the United States every year. SCI occurs most commonly in young adults, leaving them seriously disabled for the remainder of their lives. Apart from paralysis, patients of SCI suffer from additional disabilities including bladder, bowel and sexual dysfunction, and neuropathic pain syndromes. Several potentially useful therapeutic strategies have emerged over the last decade including the use of scaffolds and bridges, delivery of neurotrophic factors, other therapeutic peptides and use of stem cells to promote neuronal regeneration and functional recovery. However, none of the current strategies have shown enough effect to move to clinical trials and no major efforts have been undertaken to test a combination of these strategies, which can potentially be synergistic, and lead to greater therapeutic effect. Therefore, a need exists to develop a multifunctional construct which can integrate multiple, promising therapeutic strategies. This project brings together the disciplines of biomaterial engineering, neurobiology, basic neuroscience and neurosurgery in an attempt to develop a multi-disciplinary solution to the complex problem of spinal cord injury. We believe that the proposed system holds a number of benefits over previously described hydrogels, cellular and neurotrophin delivery systems in the CNS. Notably, the hydrogel is injectable and its properties can be readily tuned to match the compliance of host tissues, deliver therapeutic factors at tailored rates, and deliver cells to the injury site. In this case, we are delivering neural stem cells (NPC) to the site of spinal cord injury (SCI). These cells have been shown to survive and differentiate into neurons and glia and the hydrogel matrix can act as a scaffold that will include growth factors to further survival and differentiation of NPCs. We hypothesize that localized, sustained, simultaneous delivery of multiple therapeutic proteins into the CNS along with an injectable polymeric-cellular scaffold creates a synergistic effect by synchronously modulating the injured environment and activating different signaling pathways. By engineering this injectable hydrogel and cellular based scaffold to mimic the host tissues we can create a novel platform technology with applications in treatment of SCI and other tissue engineering applications. All the design parameters will be tested and validated using in-vitro bioassays and in-vivo experiments using rodent models of spinal cord injury.PUBLIC HEALTH RELEVANCE: Spinal cord injury (SCI) affects approximately 10,000 individuals in the United States every year. SCI occurs most commonly in young adults, leaving them seriously disabled for the remainder of their lives. We propose to develop a novel, injectable scaffold containing neural precursor cells and neurotrophic factors and hypothesize that localized, sustained, simultaneous delivery of multiple therapeutic proteins into the CNS along with an injectable polymeric-cellular scaffold creates a synergistic effect by synchronously modulating the injured environment and activating different signaling pathways.

DESCRIPTION (provided by applicant): The BioForce Nano eNabler 4.6 is a molecular printer that can be used to produce patterned molecular substrates on a variety of surfaces, including 3D surfaces. The nanoscale precision and versatility to deposit practically any biomaterial onto a large variety of surfaces is makes the Nano eNabler unique. In the experiments proposed here, the Nano eNabler will be used to address questions regarding the response of neurons to signaling proteins encountered during development and regeneration. The questions will be approached from different angles as a diverse group of researchers from the fields of cellular neuroscience, stem cell biology with focus on neurodegenerative disorders, and bioengineering/material science will investigate mechanisms of cell differentiation and process and synapse formation in vitro. Ultimately, the results obtained in these studies will be used to design in vivo treatment strategies that lead to improved regeneration in the adult injured or diseased CNS. Experiments of all users will involve the printing of single molecules and complex multicomponent patterns of molecules onto physiological or bioengineered substrates to investigate underlying signaling mechanisms implicated in (1) neuron differentiation and neuronal subtype specification, (2) axon dynamics during development (growth cone motility, axon extension, guidance and branching), (3) dendritic arbor formation, (4) synapse formation and plasticity (using the neuromuscular synapse as a model system), and (5) stem cell/neuron responses to inhibitory molecule combination resembling the adult injured CNS environment. In addition, the Nano eNabler will be used to screen components for incorporation in 3D hydrogel scaffolds that will serve as multifunctional platforms allowing the delivery of cells and therapeutic factors to the injury site. )