Tara L. Moore - US grants

DESCRIPTION (provided by applicant): Stroke, primarily a disease-state observed in late middle age and early senescence, results in cortical injury that frequently affects motor activity of the extremities. Given the number of individuals that experience a stroke each year there is a critical need to understand the neurobiological basis of functional recovery in the aging brain. The development of a non-human primate model of cortical ischemia with older animals with small, focal unilateral lesions in the area of the motor cortex that controls the upper extremity provides a unique opportunity to not only develop coordination and strength tools to assess impairment and recovery, but also to establish the extent of cortical reorganization in this age group. In the current proposal we plan to use rhesus monkeys to develop a model of cortical ischemia and reorganization that allows for the quantifiable assessment of motor function recovery in young and middle-aged animals. Three newly developed wrist/digit coordination and strength tasks will be used with monkeys that have small, focal unilateral lesions in the area of the motor cortex that controls the forearm, wrist and digits. Once the monkey reaches asymptotic levels of performance, a unilateral lesion will be made in the area of the motor cortex identified as controlling the digits, hand and forearm. The lesion will produce impairment in the use of the contralateral hand. Monkeys will be retested post-operatively. Following completion of testing a subset of monkeys will receive one final intense training session and then will be perfused. Brains will be processed for cFos activation to quantify the expression of the c-fos protein, a marker of immediate early gene activation and cortical activity. This will allow us to conduct a global survey for differentially activated cortical and subcortical regions in each animal resulting from the final testing session. A second group of animals will undergo a second surgery that will consist of electrophysiological techniques used to identify the areas of reorganization of the cortical representation of the forearm, wrist and digits. A second small, focal lesion will then be made in this area of re-organization in order to establish that this area was in fact facilitating the functional recovery observed during post-operative testing. These animals will be re-tested on all tasks for four weeks to establish the presence and extent of a motor deficit similar in magnitude to the deficit recorded after the first surgery. This proposal will develop a model of stroke that allows for the quantitative assessment of hand/digit motor performance following a localized cortical stroke and can be used in future studies to quantify the efficacy of therapeutic treatments aimed at facilitating recovery of motor function. PUBLIC HEALTH RELEVANCE: Stroke, primarily a disease-state observed in late middle age and early senescence, results in cortical injury that frequently affects motor activity of the extremities. Given the number of individuals that experience a stroke each year there is a critical need to understand the neurobiological basis of functional recovery in the aging brain. This grant proposes the development of a rhesus monkey model of stroke using young and middle-aged animals that involves creating focal unilateral damage in the area of the motor cortex that controls the forearm, hand and digits. It will provide a unique opportunity to assess impairment and spontaneous recovery of hand/digit coordination and strength and for the assessment of the areas of cortical reorganization that may be responsible for recovery. Finally, the establishment of this model and the acquisition of extensive baseline data, will allow for a future RO1 application to investigate various pharmaceutical and occupational therapeutic interventions in a middle-aged and aged non-human primates with motor functions and cortical organization most like those of humans.

DESCRIPTION (provided by applicant): Approximately 750,000 Americans experience a new or recurrent stroke each year, and 80% of these experience impairment of motor function of the extremities. Partial recovery of motor function occurs even without pharmacological interventions. Clinical studies and animal models suggest that this recovery results from adaptive plasticity and reorganization in intact cortical areas. At the cellular level, reorganizaton following ischemic injury has been found to correlate with dendritic remodeling, increased levels of presynaptic growth- associated proteins and synaptogenesis in peri-infarct regions. Though the precise mechanisms promoting axonal growth and synaptogenesis are unclear, the relationship between these markers of plasticity and recovery provides compelling evidence for investigating plasticity as a target for therapeutic intervention. The therapeutic agent, inosine, stimulates axonal growth and has been shown to enhance functional recovery in rodent models of stroke. Following unilateral stroke, inosine enhances the ability of neurons in the undamaged hemisphere to extend axon collaterals into brainstem and spinal cord areas that have lost normal innervation. This rewiring is accompanied by improved use of an impaired limb. Inosine is a naturally occurring purine nucleoside that crosses the cell membrane and activates Mst3b, a protein kinase that plays a central role in the cell-signaling pathway through which trophic factors stimulate axonal growth. The plasticity enhancing properties of inosine are currently being tested clinically in patients with multiple sclerosis and Parkinson's Disease (Parkinson's Disease Study Group, 2011; Markowitz et al, 2009). The goal of this proposal is to use our rhesus monkey model of cortical ischemic stroke developed with R21 AG-028680 to explore the efficacy of inosine in the recovery of motor function following cortical ischemia in a gyrencephalic animal with brain structure and fine motor dexterity highly similar to humans. ! PUBLIC HEALTH RELEVANCE: Brain damage from stroke commonly results in permanent disability.While a great deal of research has focused on limiting damage through neuroprotective strategies, it has provided limited benefits, as treatments must be administered within hours of onset. Consequently, alternative strategies to improve recovery of function in the weeks and months following stroke are needed and this proposal seeks to assess the efficacy of inosine on recovery of function and associated brain plasticity in rhesus monkeys.

Abstract/Project Summary In this proposal, we outline our strategy to advance a novel preclinical therapy for the treatment of cortical injury. Our preliminary data in rodents and monkeys indicate that delivery of exosomes derived from bone marrow mesenchymal stem cells (MSCs) aid in functional recovery after cortical injury, likely through enhancement of cortical plasticity. We have gathered in vivo data showing that rats injected with exosomes derived from MSCs score better on behavioral tests for weeks after stroke than animals injected with vehicle control. In keeping with STAIR recommendations that treatments be tested in gyrencephalic animals, such as monkeys, we have begun testing exosomes derived from bone marrow MSCs in our non-human primate model (NHP) of cortical injury. Following injury to primary motor cortex, we administered exosomes and tested recovery in three monkeys. All three monkeys demonstrated functional recovery of fine motor function of the hand that represented a return to pre-injury type of grasp. It is important to note that our preliminary results with only 3 monkeys show an unprecedented level of recovery ? monkeys given exosomes showed a pattern and magnitude of fine motor recovery equivalent to a more complete, full recovery of individual digits. This is rarely observed in human patients post-stroke or TBI. These data show the feasibility of this treatment in monkeys and the importance of continued investigation of exosomes as a neuroregenerative treatment. We have previously submitted an expanded version of this proposal twice to NINDS as an R01 application. However, based on reviewer comments requesting that data on the role of exosomes in recovery of function in our model be obtained prior to launching a full-scale study, we now seek support for additional monkeys in order to produce this pilot data. Following the completion of the studies outlined in this proposal, we feel we would then be in a better position to submit a competitive R01 application in the future to conduct an extensive study of exosomes as a novel treatment in our NHP model of cortical injury. Accordingly, we propose two aims. In Aim 1, we will assess the functional recovery after exosome delivery in male and female aged monkeys. In Aim 2, we will measure the effects of exosomes on cortical plasticity by characterizing synaptic and network mechanisms underlying plasticity. This will be the first study to administer exosomes in a NHP model of cortical injury to investigate their role in mediating recovery of function and cortical plasticity. The study is highly translational, and the data gathered in this study will take a step toward developing a safe and translatable neurorestorative therapy.

ABSTRACT: Injury to the brain due to stroke or trauma is a leading cause of disability. While research has focused on reducing stroke related brain damage by neuroprotection such as immediate treatment with tissue plasminogen activator (tPA), the benefits are limited. As an alternative, neurorestorative therapies facilitate plasticity and recovery of function but have been less extensively investigated. Over the past 5 years with NIH and industry funding we have used our monkey model of cortical injury that impairs fine motor function of one hand to test four neurorestorative treatments. We found that all four significantly enhance recovery of function following injury. These were: 1) Cell Therapy with human umbilical tissue-derived cells (hUTC) which do not differentiate into new neurons but instead release a variety of growth factors that stimulate endogenous plasticity; 2) Treatment using exosomes derived from bone marrow mesenchymal stem cells that provide an enriched and modifiable source of the same growth factors as the hUTCs; 3) Purine Nucleoside Treatment with inosine which stimulates axonal growth in culture and promotes corticospinal tract sprouting; 4) Treatment with Glial Growth Factor 2 (GGF2), a neuregulin which increases axonal sprouting and synaptic density and may stimulate myelination. While untreated monkeys developed compensatory movements similar to that observed in human stroke patients, our treated monkeys returned to pre-injury levels of fine motor function. Interestingly, while each treatment resulted in a greater recovery than typically observed in other animal models of injury or stroke, the extent and timing of recovery differed across treatments suggesting different mechanisms. Pilot work suggests that recovery may be the result of reduction of post-injury inflammation and oxidative damage, thereby limiting the evolution of the lesion. Another process may be axonal and synaptic reorganization in surviving motor cortices or spinal cord. There is also evidence of remyelination and increased density of mature oligodendrocytes in peri-infarct regions of recovered subjects. Here we propose to investigate these issues using a unique resource of archived cryopreserved tissue sections from cortex and spinal cord of 29 monkeys that had cortical damage followed 24 hours later by one of the treatments or vehicle control. In cortex and spinal cord gray and white matter we will use c-fos immunohistochemistry to identify in treated subjects compared to controls cortical areas differentially activated during a final behavioral testing session of the impaired hand. Second, in adjacent sections we will quantify markers of axonal and synaptic plasticity (GAP43 & synaptophysin). Third, we will also quantify markers of inflammation and oxidative damage (4HNE, 8OHG) as well as activated microglia and astrocytes. Fourth, we will quantify myelination and both proliferating oligodendroglia precursor cells and myelinating oligodendroglia. This offers a unique opportunity to conduct a comprehensive investigation of the loci in cortex and spinal cord that are associated with recovery and to identify the underlying neurobiological processes modulated by 4 distinct neurorestorative treatments.

Abstract The efficacy of exosomes harvested from mesenchymal stromal cells (MSCs) to enhance recovery has been demonstrated in rodent models of stroke (1-3). Building on this, we showed following cortical injury in monkeys that exosomes harvested from monkey MSCs facilitate recovery within 3-5 weeks(5). Here we propose to now conduct an analysis of the time course and mechanisms underlying exosome-mediated recovery. In previous studies, we harvested brain tissue at 14 weeks post-injury, at which time markers of acute inflammation and repair processes have stabilized. Additionally, our recent findings show a marked difference in the time course of recovery, with exosome-treated monkeys exhibiting full recovery by 3-5 weeks post-injury and untreated animals reaching a plateau in recovery by 8-12 weeks. To better understand the effects on both inflammation and plasticity, we now propose to harvest brains at 4 and 8 weeks post-injury while collecting blood and CSF at multiple time points to investigate temporal changes in biomarkers that underlie recovery. We hypothesize that exosomes will limit acute damage after cortical injury by acting on microglia and other brain cells to promote a switch from pro-inflammatory to anti-inflammatory phenotypes, and transition to a restorative microenvironment. We will test this hypothesis by comparing the recovery of function with and without the administration of exosomes following unilateral cortical injury limited to the hand representation. Following treatment, monkeys will be tested on our motor tasks for either 4 or 8 weeks to assess recovery. After completing testing, monkeys will undergo multi-dimensional diffusion MRI to assess the microstructure of the brain. Brains will then be harvested in order to comprehensively investigate the effect of exosomes on injury-related inflammation and repair processes in several ways. We will assess changes in molecular markers of inflammation, myelin damage, and repair in blood, CSF, and brain tissue and test the effects of exosomes on microglia in vitro. Blood and CSF and brain tissue lysates will be used for ELISA quantification of inflammatory and trophic markers and associated changes in gene expression will be assessed with qPCR. We will also use immunohistochemistry to quantify markers of microglia (LN3, P2YR12, IBA1) and synaptic (VGAT and VGLUTs) and neurite (GAP-43, MAP2) remodeling, and label-free spectral confocal reflectance (SCoRe) microscopy to assess myelin integrity. Then to mechanistically determine the action of exosomes, we will assess the direct effects of exosomes in in vitro, using oxygen glucose deprivation in acute brain slices. In addition, we will assess the effects of exosomes on injury-related changes in neuronal morphology, excitability and signaling by comparing the neurophysiological properties of pyramidal neurons using whole-cell patch-clamp recordings and intracellular filling in acute brain slices. Finally, we will conduct a proteomics analysis of exosomes used in this study to understand the content of exosomes. This project will identify molecules mediating exosome action and generate testable hypotheses for interventions to enhance recovery of function in humans with cortical injury.

ABSTRACT Normal aging is characterized by deficits in cognition particularly in the domains of memory (1-6) and executive function.(1, 2, 7-11) Evidence suggests these changes are not a consequence of widespread cortical neuronal loss, but rather are attributable to alteration and/or loss of white matter myelin, axons, synapses and dendrites, and changes in oxidative metabolism and inflammation.(12-18) Further there is growing evidence that accumulation of pathological Tau and A-Beta are related to age-related cognitive decline, even in the absence of Alzheimer?s Disease and Related Dementias (ADRD). Thus, one potential therapy, mesenchymal stem cells (MSCs), have recently received attention as a possible intervention in aging,(19-22) as they are known to suppress inflammation and facilitate tissue repair and remyelination. (19, 23, 24) Further, we have demonstrated that human umbilical- derived cells, and MSC-derived extracellular vesicles (EVs), the active product of MSCs, reduce inflammation and enhance recovery of motor function in non-human primates (NHP) and rodents following cortical injury(25, 26) and promote axonal growth and myelination in vitro.(27-37) Similarly, EVs have been shown to improve cognitive deficits in diabetic rats and APP/PS1 mice (model of Alzheimer?s disease).(24, 38, 39) Whether these EV-mediated beneficial effects can be applied to normal aging and age-related neuropathology in primates is largely unknown. However, our pilot data show that administration of MSC-EVs in aged female rhesus monkeys decreases A-Beta deposition. Based on this evidence and our data showing EV-mediated enhancement of recovery after cortical injury in NHPs, we build upon on our experience over the past three decades (NIH-NIA Program Project AG00001-34) characterizing cognitive function in the rhesus monkey across the life-span and age-related changes in the brain (40, 41) to assess the impact of MSC-EVs in our rhesus monkey model of aging. Specifically, we will investigate the efficacy of EVs to slow or reverse age-related cognitive decline and reduce markers of inflammation, myelin atrophy, and tau and A-Beta deposition. We will then quantify the effects of EVs on reducing age-related synaptic dysfunction and changes in electrophysiological properties of neurons. Finally, we will conduct an in-depth proteomic analysis of treatment and endogenous EVs to establish the profile of the active biological cargo load responsible for treatment efficacy. This longitudinal, multi-disciplinary study will shed light on the relationships of neuroinflammatory pathways, myelin damage and ADRD like pathology and cognitive decline associated with normal aging, and test the efficacy of EVs in ameliorating these age-related deficits in neural structure and function.