Douglas Rosene - US grants

The objective of this research program is to investigate the anatomy of the primate limbic system in the rhesus monkey. The importance of investigating the structure and organization of this system derives from clinical reports that damage in humans causes serious deficits in memory and other cognitive functions. Results from laboratory investigations of non-human primates confirm the importance of the limbic system in these deficits. Our rationale for pursuing these investigations in the monkey is that the phylogenetically ancient structures of the limbic system have not been philogenetically stagnant but have, in concert with the expanding primate neocortex, undergone a progressive development. Previous results have shown that this has manifested itself anatomically in direct connections from the limbic system to the neocortex that are unique to the primate and afford potential modes for participation in "higher cortical functions" often thought to be the exclusive domain of the neocortex. We propose to continue this research program by investigating: 1) the topography and collateralization of cortical and subcortical efferents of the hippocampal formation; 2) the topography and collateralization of cortical and subcortical efferents of the amygdala; 3) the topography and collateralization of basal forebrain efferents to the thalamus, limbic system and neocortex; 4) the connections of the medial frontal cortex with the limbic system and thalamus; 5) the topography of afferent and efferent connections of the entorhinal, prorhinal and perirhinal cortices; and 6) the morphology and intrinsic connections of the hippocampal formation. These investigations will use standard histological, histochemical and Golgi impregnation procedures to identify the architectonics and morphology of these areas. Axonal connections will be studied using axon tracing techniques that rely upon the normal physiological process of axonal transport: injections of 3H-labeled amino acids will be made and autoradiographic procedures used to identify the sites of anterograde transport and injections of either horseradish peroxidase or fluorescent dyes will be made to identify the retrogradely labeled neurons.

The Scientific Core is responsible for handling the overall aspects of tissue preparation and distribution once monkeys have completed the behavioral studies as well as conducting the final MRI scans on all subjects and preparing and archiving DMA samples. The specific aims of this core are as follows: 1) To conduct MRI scans on all monkeys before perfusion to enable quantitative analyses of the brain using T1 and T2 anatomical scans, Diffusion Tensor MRI, Magnetization Transfer and MR Spectroscopy. 2) To collect and store serum, whole blood for genomic DMA and CSF samples and to measure blood pressure and electrocardiograms. 3) To conduct all perfusions of monkeys, distribute tissue samples to the Projects and cut serial whole hemisphere sections from one hemisphere of each of the subjects. Two whole hemisphere series will be stained and all other series will be saved for study by the Projects. 4) To arrange for postmortem necropsies on all subjects and send the results to the Animal Records Review Committee of the Animal core which will ensure that there are no occult diseases that could confound our observations on the relationship of age-related changes in the brain to changes in cognitive function. 5) To maintain the Active Status Database tracking all monkeys in the Program as well as all tissue samples, blood chemistries and necropsy data. In addition samples for the Projects will be blind coded to prevent experimenter bias.

An analytical microdensitometer will be acquired to quantitatively measure a variety of reaction products visualized in microscopic sections of biological tissues. The machine is to be applied to a number of projects: 1) Changes in enzyme levels in ovarian cells as a result of exposure to tetrahydrocannabinol: 2) The process of degeneration in pericytes of diabetics; 3) Transmitter related enzymes in the central nervous system of monkeys; 4) The relationship of behavioral change to lesion-induced alterations in transmitter related enzymes in the central nervous system of monkeys; 5) Changes in the superior cervical ganglion as a result of exposure to testosterone or estradiol; and 6) Changes in the motor unit as a result of age or lesion. One of the exciting developments in instrumentation for biological research is the ability to couple microscopes to computers to allow easy quantitative analysis of microscopic images. All projects are critically dependent upon the densitometer as an investigative tool and will exploit the machine's ability to measure product on sections at various wavelengths with a great degree of accuracy and reproducibility. The availability of this instrument will allow several excellent investigators to greatly strengthen their research programs.

The Scientific Core will oversee several general aspects of data collection and analysis. First, during the course of the experiments the Scientific Core will arrange for MRI scans to be taken before each animal enters the experiments, 6 months throughout the protocol and finally just before the animals are killed. In this way we will be able to follow the development of some aspects of cerebrovascular disease including the appearance of larger infarcts, edema or small but clinically silent strokes as well as any larger strokes. In addition PET scans will be conducted on a subset of the animals pre-operatively and at 6 months post-operatively and then at 12 months intervals until the animal is killed. These PET scans will provide a direct measure of blood flow that can be related both to the development of changes seen in the MRI scans and in behavior. Second, the Scientific Core will be responsible for collecting all tissues at the time the animal are killed. At that time the animals will be anesthetized and perfused through the heart with ice cold Kreb's buffer during which time the brain, eyes and cerebral blood vessels will be removed. In all cases the brain will be blocked, in situ, in the coronal stereotactic plane and one hemisphere will be flash frozen at -70C while the other will be immersion fixed in 4% buffered paraformaldehyde. The Scientific Core will be responsible for cutting the immersion fixed hemisphere and providing series of sections to Projects 2 and 3. For the eyes, both will be slit at the limbus and one will be immersion fixed in 4% paraformaldehyde while the other will be immersion fixed in 2% paraformaldehyde/2% glutaraldehyde and both will be transferred to Project 4. Finally the cerebral blood vessels will be removed and transferred to project 2 for further analysis. The last responsibility of the Scientific Core will be to maintain a database into which all of the clinical data from the Animal Core, all of the MRI and PET data from the Scientific Core and all of the individual project data will be entered. This databases will be designed in such a way as to facilitate later transfer of some or all of the data to the Main frame for subsequent analysis.

Behavioral studies of the aging rhesus monkey clearly demonstrate significant age-related functional changes that are likely to reflect cortical dysfunction. Yet observations from this program have demonstrated that there is no overt loss of neurons with age in either the cortex or the hippocampal formation nor is there a major loss of synapses. Thus it appears that neuron and synapse loss is not a major factor in age-related cortical dysfunction. In contrast, quantitative analysis of MRI images demonstrated a loss of subcortical white matter and EM studies by ourselves and colleagues have demonstrated an age- related increase in dystrophic myelin. This latter observation could reflect a primary damage to myelin or a response to changes in the neuron and its axon. Using PET imaging we have observed an age- related reduction in cortical blood flow and metabolism and subsequent analysis of brain tissue with markers for the oxidative enzyme cytochrome oxidase revealed an age-related reduction in these markers of neuronal metabolism. Based upon these findings, the overall objective of this project is to look at the cortical neuron to determine the type and extent of age-related changes that may underlie the age-related cognitive dysfunction that occurs in monkeys over 20 years of age. These studies will focus on the hippocampus/entorhinal cortex, area 17 of the visual system and area 46 of the prefrontal cortex. The specific experiments that will be conducted are: 1) To use histochemical and in situ hybridization methods to determine the extent of age-related changes in markers of oxidative metabolism. 2) To use on-the-slide receptor autoradiography to determine the extent and distribution of changes in selected neurotransmitter receptors. 3) To use the in vitro slice preparation for a neurophysiological investigation of synaptic responses, long term potentiation, membrane properties and neurotransmitter receptor responsivity to selected agonists and antagonists. In the course of these experiments neurons will be filled with biocytin and the extent of their dendritic tree analyzed morphologically. 4) To use electronmicroscopic methods to quantify the type and number of synapses in selected layers of areas 17, 46 and the entorhinal cortex. 5) To use electronmicroscopic methods to quantify the number of axons and their morphology in the fornix, anterior commissure and perforant path. 6) To use the optical disector and fractionator to estimate neuron number and determine if there is neuronal loss in selected areas. Together, these studies will establish the extent and generality of age-related changes in the function and connectivity of cortical neurons.

The Scientific Resource core is responsible for carrying out a variety of scientific procedures to provide both data and tissue samples to the various projects and to perform overall data management and statistical analysis. The specific functions of this core are as follows: 1) To assign animals in a balance fashion to the three different perfusion groups to ensure an equal distribution of age animals that are cognitively impaired and those that are relatively spared. 2) To conduct MCI and PET scans on all monkeys before perfusion to enable quantitative volumetric and metabolic analyses to be conducted. 3) To conduct all perfusions of monkeys both in Boston and if necessary at Yerkes. 4) To prepare and distribute appropriate fresh or frozen tissue samples from hemisphere of each animal to all of the projects. 5) To cut serial whole hemisphere sections from the other hemisphere of all of the animals and prepare four archival series of stained sections (myelin, thionin, H&E and AChE) for use by all the projects. 6) To conduct and monitor the results of postmortem autopsies on all animals to ensure that there are no occult diseases that could confound our observations on the relationship of age- related changes in the brain to changes in cognitive function. 7) To maintain an integrated database of health records and other data extract from the Yerkes Animal Records System, experimental manipulations conducted by the program and the outcome variables of the different projects and to perform overall statistical analyses of these variables to identify major factors contributing to age-related cognitive dysfunction. 8) To encourage and make available to other investigators both brain tissue and other tissues from our animals that are not needed for the specific aims of this project.

This is a multi-disciplinary investigation of the neurobiological changes that occur in the brain during normal aging. It consists of 3 cores and 4 projects that use the rhesus monkeys as an experimental model of normal human aging. To ate our studies have identified a pattern of age-related cognitive impairments in memory and executive function that first appear in late middle age (15 to 19) and become increasingly prevalent and severe from 20 years of age to the maximal life span of around 35. Examinations of the brains of cognitively tested monkeys reveals that neurons are not lost from the neocortex of hippocampus with age. But we have observed loss of white matter volume, morphological and receptors and reductions in markers of oxidative metabolism. All of these are sublethal which are the critical changes underlying the cognitive decline and what mechanisms produce these changes. But we have recently observed activated astrocytes and microglia in aged monkeys where they are most prominent deficits and what mechanisms produce these changes. But we have recently have observed activated astrocytes and microglia in aged monkeys where they are most prominent in white matter. Since activated glia can produce free radicals and inflammatory mediators that damage neurons, oligodendroglia and myelin, we will investigate our hypothesis that such inflammatory insult leads to sublethal damage to neurons and myelin, compromising cerebral function and causing cognitive declined. For these studies we will behaviorally test young adults 5 to 1o years of age, middle aged monkeys 15 to 19 years old and elderly monkeys over 20 and examine their brains using physiological, biochemical and morphological methods. One goal will be to identify those monkeys that are most cognitively impaired and those that are successfully aging in order to identify the constellation of changes that most strongly predict severe age-related cognitive decline. A second goal will be identify in the late middle aged monkeys those changes that occur first and may constitute precipitating events that initiate a cascade of neuropathological changes. These studies will allow us to account for age-related pathology in the brain and the resulting cognitive decline. In addition they will allow us to designing experimental and therapeutic interventions.

GABA receptor; protein deficiency; mother /embryo /fetus nutrition; developmental neurobiology; neuroanatomy; hippocampus; granule cell; entorhinal cortex; interneurons; neurotrophic factors; apoptosis; dietary proteins; stress; stereotaxic techniques; laboratory rat; immunocytochemistry; nutrition related tag; autoradiography;

RE: Lay abstract Proposal IBN-9982889

This is a study to determine if two different regions of the brain, each of which have been separately implicated in memory function, actually work with each other to subserve certain aspects of memory. Work in humans from imaging studies suggest this possibility, but the only way to assess this with any degree of confidence, and to have the ability to understand the nature of the relationship, is to perform an experiment in an animal model. In this case, the rhesus monkey, which has a brain that is structured in a very similar manner to that of humans, is the species of choice. Monkeys will undergo selective damage to these two parts of the brain, one in the temporal lobe, and the other in the frontal lobe. For each structure, the damage will be made on one side of the brain, leaving the other side intact. We will then "disconnect" the two remaining structures to see if this produces a change in how the animals learn and remember. If a significant change occurs, we will have obtained initial evidence in determining that the two different structures participate together in learning and memory function. This study could have important implications in the assessment of memory function in certain disease states and could have eventual bearing on memory rehabilitation efforts.

[unreadable] DESCRIPTION (provided by applicant): The rhesus monkey is a model of normal human aging that enables cognitive testing to be followed by optimal preservation of brain tissue for multidisciplinary studies. The proposed studies will test the hypothesis that age-related cognitive decline results from a cascade of mild degenerative changes beginning in early middle age with inflammation and damage in white matter that leads to functional changes in axons and progresses to functional and structural changes in neurons. We will study three cohorts of monkeys to test various aspects of this hypothesis. The first cohort of 36 monkeys will be used in a cross-sectional study and consist of subjects covering the adult life span from 5 to 30 years old. After behavioral testing, MRI scans will be followed by in vivo neurophysiology and a new two stage perfusion fixation to provide fresh and fixed tissue to identify brain changes underlying cognitive decline. A second cohort of 6 middle aged monkeys will be treated like cohort 1 except they will be perfusion fixed with mixed aldehydes for electron microscopy to supplement existing samples and allow identification of the ultrastructural changes that are the first changes to appear in middle age in association with cognitive decline. A third cohort of 12 early middle aged monkeys (ages 13-15) will be followed longitudinally for 4+ years with repeated behavioral testing, MRI scans of the brain, and samples of blood and CSF. As the prevalence of cognitive impairment is low at 13-15 but high by 20, behavioral data will identify the cognitive profile and rate of decline of individual monkeys. Longitudinal MRIs will reveal concurrent changes in the in vivo brain structure, and CSF and blood samples will allow biomarkers to be followed. For cohorts 1 and 3, perfusion fixation will be preceded by in vivo neurophysiological assessment of compound action potentials. Then the two stage perfusion will follow to allow immediate collection of unfixed samples from one hemisphere before the remainder of the brain is fixed. Fresh tissue will be used for in vitro neurophysiology of synaptic currents and action potentials, for single cell PCR, and for biochemical and molecular studies of underlying mechanisms. Fixed tissue will be used for anatomical studies, including stereological studies of neuron numbers and immunocytochemical studies of inflammation and other degenerative processes. [unreadable] [unreadable] REVIEW OF INDIVIDUAL COMPONENETS [unreadable] [unreadable] CORE A: ADMINISTRATIVE AND DATA MANAGEMENT CORE; Dr. Douglas Rosene (CL) [unreadable] [unreadable] DESCRIPTION (provided by applicant): The Administrative Core is responsible for the overall scientific direction and financial and administrative management of this Program Project. To accomplish this the core will handle all orders and maintain appropriate financial records for all of the Cores and Projects. It will also provide administrative and clerical support to the Cores and Projects in preparation of manuscripts and the annual progress reports. The core will also organize biweekly meetings of the Program Project staff (all project and core leaders and their coinvestigators) at which scientific direction and progress will be discussed and decisions about priorities or new directions made and will also organize meetings of the External Advisory Committee (outside scientists selected to provide expertise in the areas we are investigating) in years two and four of the Program. These will be one day, off campus meetings at which the Program Project staff will present summaries of the their work to the advisors in the morning and then the afternoon will be devoted to round table discussion with the advisors. This group meeting will allow us to get their input on the overall scientific direction of this Program. Finally, the core will also provide statistical support for all of the Cores and Projects and will maintain the Archival Program Database of Program Project Data. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): In age-related neurodegenerative disorders like Alzheimer's disease, the loss of cortical neurons is the likely cause of progressive cognitive impairments. In contrast, in normal aging, the cause of the relatively mild cognitive impairments that develops remains unclear as cortical neurons are not lost. However, cortical neurons have been shown to become dysfunctional in a number of ways ranging from deterioration of myelinated axons that interconnect cortical areas to changes in action potential generation at the soma. A critical functional component of cortical information processing is the microcolumn, a vertical array of neurons that are tightly interconnected and that work together to process fundamental information. The classic example is the orientation column of the visual cortex. Accumulating evidence suggests that age-related changes in microcolumnar organization may be an important marker of age-related cortical dysfunction. Age-related alterations in microcolumns will be addressed using archival brain material available from a study of rhesus monkeys in which all animals are behaviorally tested to characterize cognitive status and the brains are harvested for neurobiological study. The first aim is to acquire whole brain photomontages to quantitatively assess microcolumnar structure throughout the entire cerebral cortex of both male and female rhesus monkeys that cover the entire adult life span. This will identify regions where the greatest age-related disruptions in microcolumns occur and where those changes are most strongly related to cognitive impairments. This will test the hypothesis that regional alterations in microcolumnar structure and associated cortical dysfunction account for age-related cognitive impairments. Based on the identification of most affected cortical areas, Aim 2 will utilize immunohistochemical methods to label intracellular cytoskeletal elements of dendrites of cortical neurons. These will be analyzed to test the hypothesis that alterations in dendritic structure are associated with the disruption of microcolumnar architecture. Similarly, Aim 3 will utilize NeuN immunohistochemistry to uniquely separate neurons from glia allowing for separate analysis of glia changes. This aim will test the hypothesis that disruptions in glial distribution are associated with age-related disruption of microcolumns. For both dendrites and glia, cross correlation methods will be used to quantify the relationship to microcolumn changes and for all three aims multivariate methods will assess the relationship with cognitive impairments. These data will generate testable mechanistic hypotheses regarding the causes of microcolumnar dysfunction and will provide insight into the basis of age-related cortical dysfunction and cognitive impairment. Future directions for this study will include analysis of the small but functionally significant population of GABAergic neurons and the distribution of intercellular adhesion molecules that bind the cortex together. PUBLIC HEALTH RELEVANCE: In normal aging, cognitive dysfunction occurs without the loss of cortical neurons yet evidence indicates disruption of the architecture of vertical arrays of cortical neurons that are organized as microcolumns. These microcolumns are a fundamental computational unit of the cerebral cortex, and their age-related degradation correlates with age-related cognitive impairment. These will be studied using advanced quantitative methods and compared with changes in dendritic structure and glia cells to determine the processes underlying age-related cognitive impairments. [unreadable] [unreadable] [unreadable]

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

In this project the PIs proposes to investigate the structure of the cerebral cortex with noninvasive diffusion-sensitive Magnetic Resonance imaging (MRI). At macroscopic scale, the human cerebral cortex consists of an estimated 50 or so discrete areas of distinct structure identifiable only at the microscopic level but of immense importance as each corresponds to a specialized function. These fundamental divisions, however, are impossible to discern with contemporary MRI. Recently, one of the PIs made a dramatic progress in MRI of the fiber pathways of cerebral white matter using diffusion spectrum MRI or DSI to obtain a large-scale fiber connectivity of the human brain, or the connectome. The aim of this collaborative project is to extend DSI from white matter into the gray matter by developing and validating a diffusion MRI methodology to image the intrinsic structure and connectivity of discrete cortical gray matter areas. Undergraduate, doctoral and post-doctoral level students will receive interdisciplinary training in their interactions with the other two collaborative labs. In this way students will be trained in the cross-disciplinary methods of this project, obtaining hands on familiarity with the ideas and methods of modern neuroscience, neuroimaging, and concepts of condensed matter physics. Additionally, these studies will be of interest to the scientific media and public. These studies offer an opportunity to present neuroscience, its goals, methods, and uses, to a broad general audience, both nationally and internationally. The present proposal mixes diverse disciplines and so offers a fertile opportunity for student education. Under the auspices of the NSF Distinguished Teacher Scholar (DTS) Award, co-PI Stanley's research group will continue to work intensely with high school students and with college non-science majors through designing hands-on activities that encourage the student to ask questions and better understand the activities of a practicing scientist.

DESCRIPTION (provided by applicant): We will use the rhesus monkey as a model of normal aging as it is free from neurodegenerative disease but does show age-related cognitive declines in cognitive function that begin by 13 years of age (humans ~ 39) and accelerate after 20 (humans > 60). Examining the neural bases of this cognitive aging, we found that cortical neurons are not lost but instead, major pathology occurs in myelinated fibers of the white matter as seen in the electron microscope (EM) and in MR imaging. Though the relationship of MRI markers to EM markers is unknown, both are strong predictors of cognitive aging. Importantly the causes of myelin damage are unknown. One hypothesis is that damage is caused by age-related oxidative stress. A novel corollary hypothesis is that this damage is magnified by age-related reductions in myelin repair. We will explore both hypotheses in two cohorts of behaviorally tested male and female monkeys that range from young adults (~5) to the elderly (>20). The first cohort consists of 24 monkeys that will be behaviorally tested and their brains analyzed for this study. The second consists of archived tissue samples and associated data from 31 monkeys used in prior studies. Data on these 55 monkeys will include cognition, MRI scans, BrdU labeling, CSF samples and serum samples. In Aim 1, we will quantify myelin pathology in brain using immunohistochemistry with an antibody to damaged myelin (EP) and assess its relationship to other pathology (e.g. lipid peroxidation) using multilevel immunofluroescence. To identify other potentially causal factors, samples of CSF, serum and brain will be analyzed biochemically with cytokine arrays. In Aim 2, multi-label immunofluorescence with BrdU and markers of mature and immature oligodendroglia will assess the capacity to maintain and repair myelin. In Aim 3, sections processed with EP will be co-registered to MRI scans that yield measures of white matter volume and myelin pathology (e.g. fractional anisotropy) to validate how well these MRI measures reflect tissue constituents. Finally, all measures will be analyzed to determine which best predict cognitive aging. This will enable future studies to target experimental treatments at specific processes of myelin damage and use validated MRI measures to follow their effects.

DESCRIPTION (provided by applicant): Abstract Approximately 800,000 individuals suffer a stroke each year in the United States. Development of Glial Growth Factor 2 (GGF2) for promotion of stroke recovery is significant because of the lack of therapeutics to treat stroke. GGF2 also has a very wide treatment window (days) because it is not dependent upon neuroprotection. Additionally, as GGF2 is already in clinical trials for another indication, barries to manufacturing, formulation and toxicology have either been eliminated, or greatly reduced. The innovative aspect of this application comes from leveraging the existing development work that has been completed with GGF2 for another indication to efficiently enable an IND for stroke. Expanding on a significant body of preclinical data, GGF2 dosing will be optimized in a rat middle cerebral artery occlusion (MCAO) stroke model. Studies will explore dose frequency, extended dose duration and determine permanence of effects. Additional toxicology will be performed to enable the specific dose regimen proposed for stroke. GGF2 efficacy will then be confirmed in an established non-human primate model of cortical ischemia using a gyrencephalic rhesus monkey model. This model emphasizes neurorecovery rather than lesion volume reduction. Dosing will begin 24 hours after ischemia and behavioral improvements will be followed for at least 8 weeks in both rat and monkey studies. Following completion of studies in rhesus monkey an IND will be filed with the FDA.

A better understanding of the relationship between brain structure and function is an integral component of the on-going efforts aimed at developing a better understanding of the human mind. Fundamental research is required to accelerate the development of new technologies for neuroscience and near engineering in order to address important societal needs with respect to the development of new ways to treat, prevent, and cure brain disorders. In this larger context, this collaborative project will extend methods of statistical physics to bridge from microscopic neurobiological observations of neurons, axons and dendrites to the mesoscopic images of brain organization seen in diffusion MRI images of the entire primate brain. A particular focus will be to address the question of how the processes of the brain might exploit this special architecture for the representation and processing of information, and in particular, how this regular structure might support time-coding and synchronization of information across the brain.

Joining a physics laboratory, a neurobiology laboratory, and an MRI laboratory, this team will investigates the hypothesis that brain connectivity is geometrically organized, with connectivity generally aligned with the axes of a curved, but essentially orthogonal coordinate system or 3D grid. The idea that the brain of all species with bilateral symmetry is based on an orthogonal plan is not new. It has been recognized in embryology and evolutionary biology for nearly 100 years and more recently has been validated in detail in studies of gene expression. Preliminary studies have suggested that this orthogonal motif pervades the structure of the brain, and particularly connectivity, from macroscopic down to a cellular level. In this interdisciplinary project, the investigators will quantify this phenomenon by looking at structural data from both diffusion MRI and advanced methods of 3D light microscopy and then apply the ideas and tools of condensed matter physics to characterize the structure and circuits of the brain as organized matter. As a first example, having observed 3 orthogonal fiber directions at each point in the brain that vary smoothly, it is natural to model this as a liquid crystal with a deformation energy and temperature. Then, one can investigate its scaling in the brain, and transitions such as those from white matter to gray matter. Functionally, we hypothesize that this rectilinear grid, may provide a new mechanism for neural activity to be temporally correlated, owing to its extremely high degeneracy of path lengths and transmission delays, which we will model as a directed percolation.

ABSTRACT Normal aging occurs in the absence of frank neurodegenerative disease but nevertheless is characterized by impairments in learning, memory and executive function. The rhesus monkey is a valuable model of normal aging as it does not suffer from Alzheimer's Disease (AD) or other neurodegenerative conditions. Yet, the monkey still exhibits patterns of cognitive decline similar to those observed in humans. While cognitive aging was long believed to be due to neuron loss, careful stereologic investigations have confirmed that neurons are not lost. Instead, with age, white matter volume decreases as myelin pathology, in the form of splitting and ballooning of the sheaths, increases. We have previously found that both loss of white matter volume and damage to myelinated fibers correlate with age-related cognitive impairment, but the cause of this pathology is not known. In searching for possible causes, the well documented age-related increases in oxidative stress and inflammation are prime candidates. Several studies from this lab have demonstrated that microglial activation and phagocytosis increase with age and correlate with cognitive decline but it is not clear if this is a response to the pathology or a causative factor. Our recent pilot studies have revealed infiltration of peripheral T cells into the brain white matter raising the possibility that they may cause or exacerbate pathology. In Aim 1, we propose to explore this issue using cryopreserved brain tissue sections collected from 50 behaviorally tested rhesus monkeys of both sexes that range in age from 5 to over 30 years old (human equivalent of 15 to about 90 years of age). Sections will be processed with immunohistochemistry to identify T cells and their subtypes, which will be quantified with stereology by location, frequency and T cell subtype. These results will be correlated with myelin damage by using the recently described SCoRe method (spectral confocal reflectance microscopy). Aim 2 will utilize 6 new behaviorally tested monkeys (3 young and 3 old) from which fresh tissue will be harvested to assess, ex vivo, the function of parenchymal T cells. This will be done by performing RNA sequencing on sorted CD3+ T cells from brain white matter, choroid plexus, and blood to compare differences in RNA phenotype across these environments. Understanding the extent to which T cells from the periphery mirror those that infiltrate the brain may provide novel peripheral biomarkers to assess brain aging. Additionally, we will isolate T cells, stabilize them in culture, and assess their myelin reactivity by exposing them to myelin antigens. All results will be analyzed relative to age, myelin pathology and the severity of cognitive impairment. In addition to an overall increase in parenchymal T cells with age, we expect to identify the inflammatory profiles likely to exacerbate myelin damage. Together, these data should provide insight into a possible role of T cells in age- related myelin pathology and cognitive impairment and provide a basis for a follow-up R01 that could uncover for experimental and therapeutic targets to ameliorate cognitive decline.

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.

SUMMARY / ABSTRACT In humans, severe cognitive impairment with neuron loss occurs in Alzheimer's Disease (AD) but cognitive decline also occurs in normal healthy aging without neurodegeneration. The rhesus monkey is a valuable model of normal healthy aging as it does not develop the AD pathology but does develop age-related cognitive impairments (Herndon et al, 1997; Moore et al, 2006) which parallel human impairments (Fjell & Walhovd, 2010; Moss et al, 2007). In searching for the cause of cognitive aging, we and others have shown that neurons are not lost with age (Peters et al, 1996; Morrison & Hof 1997) but instead myelin pathology develops, white matter is lost and both correlate with cognitive decline ?(Makris 2007; Wisco et al, 2008; Bowley et al., 2010; Peters & Kemper, 2012). Importantly, normal aging in humans without AD also show neuron preservation with white matter damage (Freeman et al, 2008; Guttmann et al., 1998; Marner et al., 2003; Raz 2003). Possible causes of myelin pathology are inflammation and oxidative stress that damage myelin or dysfunction of oligodendrocytes that maintain it. Phagocytosis may also be a factor as improper clearance of myelin by microglia inhibits normal remyelination (Lampron et al. 2015). Indeed, we found that microglial phagocytic activation increases with age and correlates with cognitive decline (Shobin et al., 2017). Here we propose three aims to study behaviorally tested aging monkeys of both sexes from 5 to over 30 years of age from three sources: Normal aging (NA-1) monkeys acquired and tested for this project (N=25), data and archived tissue samples from normal aging (NA- 2) monkeys studied here over the past decade (N=50), monkeys from the NIA intramural calorie restriction (CR) study (n=30, 17 brains on hand plus12 - 15 expected over 5 years). Aim 1 will use fresh frozen NA-1&2 tissue to assess gene expression in white matter and fresh NA-1 samples to isolate oligodendroglia and microglia for RNA expression. It will also use fixed tissue from NA-1&2 to verify the cellular correlates of these effects. Aim 2 will use fresh NA-1 samples of cingulate cortex as a model system to investigate with in vitro slice neurophysiology how myelin pathology affects neuronal signaling and follow-up with immunohistochemistry (IHC) and high resolution confocal microscopy on fixed slices to quantify effects on spines and synapses as well as synaptic pruning by microglia. Aim 3 will compare CR monkeys to NA-1&2 to determine if CR decreases inflammation and protects against both myelin pathology and cognitive decline. To this end, monkeys at NIA are currently being tested on our cognitive testing battery, and upon their death we receive one fixed hemisphere and fresh frozen frontal slabs from the other. In fresh frozen tissue, we will evaluate gene expression with microarray and targeted qPCR. In fixed tissue we will use IHC to assess myelin damage and markers of oligodendroglia and microglia function. These studies will provide new insights into mechanisms of age-related myelin pathology and determine if CR reduces myelin pathology and cognitive decline of normal aging.

ABSTRACT The rhesus monkey shows age-related cognitive decline similar to humans. Surprisingly both microscopic and MRI studies show that neurons and gray matter are not lost in the aging monkey brain. Instead, there is a progressive loss of white matter and accumulation of myelin damage that both predict age-related cognitive decline. Yet it is unknown whether myelin damage results from intrinsic changes in myelin-producing oligodendroglia or from interactions with microglia that normally clear myelin debris and facilitate remyelination. Data published by us and others show that microglia in the white matter become more inflammatory and phagocytically active with age, but lose phagocytic efficacy. These data correlate with myelin damage and cognitive decline. Here we propose experiments to test the hypothesis that functional changes in microglia impair oligodendroglial function and underlie age-related myelin pathology and cognitive impairment. The experiments will use tissue from the prefrontal cortex of 25 cognitively tested monkeys of both sexes that cover the adult life span from 5 years old to nearly 30 years old (human equivalent of 15 ? 90). This will be supplemented with archived and cryopreserved tissue from 50 monkeys behaviorally tested over the previous decade, providing unprecedented statistical power. In all cases, after cognitive testing, fresh brain tissue is harvested for use in novel in vitro and ex vivo assays while and the remainder of the brain is fixed for histopathological analyses. Studies proposed here will evaluate mechanisms underlying age-related changes in the role of oligodendroglia and microglia in the development of age-related myelin pathology according to 3 aims. In Aim 1, we will use primary cultures to assess OPC differentiation, immunohistochemistry (IHC) to quantify the ratio of OPCs to mature myelinating oligodendrocytes (OL) and spectral confocal reflectance (SCoRe) microscopy to quantify percent myelinated fibers and internode length. Functional effects of myelin pathology on the action potential will be assessed with in vitro slice electrophysiology followed by electron microscopy to validate the SCoRe measures. This will test the hypothesis that there is age-related impairment in the capacity of OPCs to differentiate into OLs leading to impaired myelin sheath maintenance. In Aim 2, we will use an ex vivo slice culture to assess phagocytic capacity by challenging microglia with myelin debris from cognitively impaired aged subjects and we will use IHC to assess microglial phagocytosis using markers for lysosomes and phagocytosed myelin. This will test the hypothesis that phagocytic functionality is impaired in aging as myelin debris is less efficiently cleared, which correlates with impaired OPC/OL differentiation and myelin sheath maintenance. In Aim 3, we will examine gene expression changes in the frontal white matter to identify transcriptional changes associated with myelin pathology and follow this up with cell-specific PCR to quantify transcriptional changes in microglia and oligodendroglia. Impact: Together, these aims will provide unique and novel mechanistic insights into of the role of oligodendroglia and microglia in age-related myelin pathology and cognitive impairment.