Jaime Grutzendler - US grants

DESCRIPTION (provided by applicant): Vascular cognitive impairment (VCI) is a heterogeneous entity that has been classically linked with ischemic disease. Furthermore, there is considerable evidence indicating that chronic cerebral hypoperfusion is an early feature of Alzheimer's disease (AD) and cardiovascular risk factors increase the incidence of both vascular cognitive impairment and AD. This has led many to speculate that chronic cerebral hypoperfusion could play important roles both in AD and VCI and could serve as a mechanistic link between these two entities. This proposal will address fundamental questions about the role of cerebral hypoperfusion in the development of age-related cognitive decline. Specifically, it will focus on its effects in the stability of synaptic connections, building up on preliminary observations we have made using time-lapse two-photon microscopy of neuronal structures in living mice. We have observed that dendritic spines the main sites of excitatory synaptic connections undergo remarkable destabilization in the presence of cerebral hypoperfusion in mouse models. This occurs in the absence of stroke, suggesting that hypoperfusion below the threshold for ischemia could be an underestimated mechanism of pathology. This proposal will systematically test mechanisms of neuronal circuit injury in cerebral hypoperfusion, examine their interactions with AD pathology and probe potential therapeutic strategies. Specifically, it will test the hypothesis that chronic cerebral hypoperfusion in the absence of ischemia induces synaptic destabilization and enhances the synaptotoxic effects of A[unreadable]-amyloid and neuroinflammation eventually leading to neurodegeneration. To test this hypothesis, we will use animal models of global and focal cerebral hypoperfusion, in vivo two-photon microscopy imaging of neuronal structures, amyloid plaques and microglia as well as histological and biochemical methods. In aim 1, we will further characterize the effects of chronic cerebral hypoperfusion on synaptic stability. In aim 2, we will examine the effects of chronic cerebral hypoperfusion on A[unreadable]-amyloid deposition and synaptotoxicity. In aim 3, we will investigate the role of neuroinflammation in hypoperfusion- induced synaptic disruption. Together, these studies will greatly advance our understanding of complex mechanisms through which CCH interacts with neurons, a-amyloid and microglia eventually leading to neuronal circuit injury and dementia. .

DESCRIPTION (provided by applicant): Microvascular occlusion by emboli occurs spontaneously throughout life and is common in a variety of disease processes. Although these small vessel occlusions may produce little acute symptomatology, their cumulative effect is likely to lead to organ dysfunction. This is especially relevant in the brain where micro-occlusions may eventually lead to cognitive impairment. The current view is that microvascular emboli are normally cleared by hemodynamic forces and the fibrinolytic system. However, we recently discovered an alternative cellular mechanism that effectively removes from the cerebral microvasculature emboli composed of virtually any substance including those not susceptible to fibrinolysis such as atheromatous cholesterol fragments. Clearance occurs by a previously unknown process of microvascular plasticity involving the engulfment of entire emboli by endothelial membrane projections and their subsequent translocation into the perivascular parenchyma leading to rapid reestablishment of blood flow and vessel sparing. In aging mice, the rate of embolus extravasation is severely delayed. Although the molecular control of the extravasation mechanism is likely to be complex, pathways involved in mechanotransduction, vascular plasticity, cytoskeletal dynamics, remodeling of endothial junctions and extracellular matrix are likely to play a critical role and may be affected in aging. We hypothesize that alterations in these molecular pathways can significantly change the efficiency of embolus extravasation, impacting the viability of occluded microvessels and surrounding tissue. To test this, our research program will investigate the molecular control of the extravasation process by using mutant mice and novel methods for focal vascular drug delivery. The precise effects of these manipulations on the dynamics of endothelial and perivascular cells will be characterized at high spatial and temporal resolution by multicolor two photon imaging in live mice and confocal and transmission electron microscopy in fixed tissues. Furthermore, we will test selected molecules for their ability to rescue the delayed extravasation phenotype in aging. To increase the throughput of our molecular and cellular analysis, we will develop a new model of microvascular embolization in zebrafish and take advantage of its versatility for pharmacological and genetic manipulations as well as in vivo imaging. Finally, we will examine in mice and zebrafish, the consequences of altering the efficiency of the extravasation process on the viability of blood vessels and surrounding parenchyma. Together, these translational experiments will provide a framework for understanding the cellular and molecular basis of this critical mechanism of microvascular clearance while suggesting targets for therapeutic intervention. Our results could have important implications in vascular biology, systemic embolic disorders, stroke and dementia. PUBLIC HEALTH RELEVANCE: Occlusion of microvessels in various organs is likely to occur frequently throughout life. The cumulative effect of these occlusions may lead to organ damage. In the brain, this may be the basis for age related cognitive decline and dementia. We have discovered a physiological mechanism that efficiently eliminates virtually any type of material occluding these small blood vessels. Alterations in the efficiency of this mechanism could have critical implications in a variety of diseases. The goal of our proposal is to develop an innovative approach using molecular manipulations in mice, zebrafish and live microscopy to discover molecular pathways that control this novel mechanism and to determine its importance in salvaging blood vessels and other cells in a variety of organs including the brain.

DESCRIPTION (provided by applicant): This proposal requests funds to purchase a Zeiss LSM 710 Laser Scanning Confocal Microscope for a group of 8 NIH funded major users and 5 minor users in the Department of Neurology, the Alzheimer's Center and the Neuroscience Institute at Northwestern University Feinberg School of Medicine. These investigators are conducting studies of cerebral cortex development and connectivity, synaptic plasticity, stem cell biology, cellular mechanisms of dementia, multiple sclerosis pathogenesis, molecular basis of hearing and somatic sensation and retinal physiology. Their projects are heavily reliant on confocal microscopy for optical sectioning of thick tissues, co-localization and spectral separation of multiple fluorophores in mouse and postmortem human tissue. Additional applications include live imaging of retina and cochlea organotypic preparations as well as brain slices. At present, there are two confocal microscopes available to the entire research community at large on the Medical School campus of Northwestern University. These microscopes are so heavily used that reservations for sessions longer than 2 hours are frequently required weeks ahead. The upright Zeiss LSM 710 is a state-of-the-art instrument with outstanding sensitivity and sophisticated spectral imaging capabilities for separation of multiple fluorophores and tissue auto-fluorescence. The new microscope will be housed in a location central to the 8 investigators. Training, administration and billing will be managed by the Cell Imaging Core which is directed by a full-time imaging specialist at the rank of Research Associate Professor. An advisory committee will be formed to oversee the management and equitable operation of the equipment. The University has committed funds to refurbish a room for the microscope, purchase service contracts and help maintain the equipment through the Cell Imaging core. This new system would greatly relieve the current usage on the existing microscopes and allow this group of investigators sufficient confocal access to expedite completion of their NIH-funded projects and to advance their research in directions previously unfeasible.

DESCRIPTION (provided by applicant): Brain metastases are a leading cause of mortality in cancer patients and there is currently no effective therapy that can prevent them. The search for therapies is limited by our poor understanding of the mechanisms of cancer cell brain invasion. We have recently discovered a novel mechanism of vascular plasticity that leads to the extravasation of emboli in the cerebral microcirculation. This process, which can clear any kind of material from the microvasculature, involves the engulfment of entire emboli by endothelial membrane projections and their subsequent translocation into the perivascular parenchyma. Our preliminary data shows that cancer cells undergo a similar process of microvascular engulfment. The goal of this proposal is to probe several potential roles of this mechanism in the metastatic process: a) cancer cells may co-opt this mechanism for the purpose of crossing the endothelial barrier and seeding the brain. This will be tested by using methods we have developed for transcranial two-photon imaging, confocal and electron microscopy. We will visualize at high spatial-temporal resolution the process of metastatic invasion in individual cerebral capillaries and determine if endothelial engulfment is required for cancer cell transvasation. b) the enveloping mechanism may help cancer cells remain insulated within microvessels promoting their latency. Using live and fixed tissue imaging and proliferation markers we will determine if cancer cells can remain dormant and viable for a long-term after microvessel engulfment. c) the engulfment process may serve as a surveillance mechanism by trapping and killing tumor cells within the vasculature. If the outcome of endothelial engulfment is death of malignant cells, we will monitor a variety of markers to characterize the death mechanism. Finally, we will examine several molecular pathways for their ability to modulate endothelial engulfment and will determine if this significantly impacts cancer cell transvasation, latency or survival. Together, these experiments will establish the importance of this mechanism of microvascular plasticity in the process of tumor invasion. Our results could suggest new targets for the prevention of brain metastasis.

DESCRIPTION (provided by applicant): Late onset cognitive decline is likely to be caused by a mixture of pathological processes. Clinical, neuropathological and epidemiological studies suggest that late onset cognitive decline is likely to be linked with microvascular factors. Microvascular pathology has been less studied as a potential mechanism of cognitive decline, perhaps because of difficulties in investigating it in humans due to a lack of tools for imaging microvessels at high resolution in vivo. Thus, there is still no clear understanding as to how the potential synergism between vascular and Alzheimer's pathologies might occur. We have previously discovered a novel mechanism of microvascular recanalization, termed angiophagy, involving the engulfment of emboli by the endothelium followed by their translocation through the vessel wall into the perivascular space leading to flow reestablishment. We propose the novel hypothesis that this mechanism of recanalization plays a critical role in the interactions between microvascular and Alzheimer's pathologies. To test these hypothesis we have developed sophisticated and sensitive experimental methods, combining an Alzheimer's mouse model with our fluorescent microembolization technique, high-resolution in vivo and fixed tissue imaging of emboli and vessels, and fluorescent nanoparticle labeling of clots for long term-tracking in fixed tissues. These set of experiments will greatly improve our understanding of the potential interactions between microvascular occlusion and cerebral amyloid angiopathy a potential critical link between these prevalent conditions. Our proposed work will determine whether there is a vicious cycle between microvascular abnormalities and AD pathology, providing critical novel avenues for therapy development.

DESCRIPTION (provided by applicant): Myelin is a fundamental component of mature neural networks that is affected in a large number of pathological conditions of the central nervous system (CNS). Critical for advancing knowledge about these conditions would be a better in vivo understanding of how oligodendrocytes and their respective myelin sheaths develop, are maintained throughout life and respond to injury. We have developed a new technique that allows high resolution label-less in vivo imaging of myelinated axons. This technique takes advantage of the high refractive index of lipid rich multilayered myelin and is based on multispectral confocal reflectance (MCORE) microscopy. Using MCORE we have obtained for the first time long- term images of the dynamics of cortical myelin on the cellular scale in a livig animal. Our preliminary data shows this technique as well as fluorescence imaging are a powerful set of tools that in combination provide a wealth of information about fine structural changes in oligodendrocytes and myelin in vivo. We demonstrate the feasibility to track the development of myelin pathology in a dysmyelinating mutant mouse and determine the temporal dynamics of demyelination after single oligodendrocyte ablation. We propose to use this powerful technique in combination with in vivo two-photon and confocal fluorescence imaging to address three fundamental questions concerning the plasticity and regeneration of neocortical myelin. First, we will determine the long-term in vivo dynamics of individual oligodendrocytes and myelinated axons from birth to death. Second, we will investigate how neuronal activity influences myelin formation and stability on individually active axons in vivo. Finally we will stuy the process of demyelination and remyelination after single oligodendrocyte ablation and determine how repeated ablation and neuronal activity influences the temporal dynamics of remyelination. Together these experiments will provide novel insight into the fundamental plasticity and regeneration capabilities of myelin and oligodendrocytes in relation to axonal activity throughout life.

DESCRIPTION (provided by applicant): The development, plasticity, and stability of dendrites and dendritic spines are defective in autism, mental retardation, stroke, and psychiatric diseases. Mutations or reduced levels of heterotrimeric laminin extracellular matrix proteins are associated with these human brain disorders. We provide evidence that neuron-specific ablation of the laminin alpha5 subunit in mice increases spine densities, destabilizes dendrite branches, and compromises normal synaptic transmission and animal behavior. We propose to elucidate the mechanisms by which laminin alpha5 and a new putative laminin alpha5 receptor we have discovered regulate dendrite and dendritic spine development and function. We will use complementary in vivo imaging, electrophysiological, biochemical, and genetic approaches to achieve the following aims: Aim 1. Determine how laminin alpha5 regulates development, plasticity, and function of dendrites, dendritic spines, and synapses. Our data strongly suggest that laminin alpha5 controls dendrite branch and dendritic spine dynamics. We will use transcranial two-photon microscopy of dendrites in the somatosensory cortex, alone and in combination with sensory input manipulation, to reveal how the loss of laminin alpha5 impacts branch and spine dynamics during development and activity-driven plasticity. We will also use electron microscopy and whole cell recording to test the hypothesis that laminin alpha5 regulates synaptic transmission by controlling the structure, transmission properties, and plasticity of individual synapses. Aim 2. Elucidate the composition, origin, and timing of function of alpha5-containing laminins in dendrite and spine development. We do not know which laminin beta and gamma chains partner with laminin alpha5, where they are produced, or when they act. We will use biochemical and genetic knockout approaches to identify laminin beta and gamma chains that associate with laminin alpha5 in neurons to regulate dendrite and spine development. We will also inactivate laminin alpha5 in specific cell types using inducible Cre transgenes to determine where and when laminin alpha5 is required to regulate dendrite and dendritic spine development. Aim 3. Characterize SIRPalpha function in laminin alpha5-mediated dendrite and dendritic spine development. We have shown that the integrin alpha3beta1 receptor for laminin alpha5 mediates dendrite branch stability, but our genetic analysis indicates that other receptors are essential to mediate the effects of laminin alpha5 on dendritic spine development. Our data strongly suggest that the Signal Regulatory Protein alpha (SIRPalpha) transmembrane receptor serves as a novel laminin alpha5 receptor in the control of spine development. We will use cell adhesion assays and in vitro binding assays with purified proteins to identify which domains in SIRPalpha and alpha5-laminins mediate these interactions. We will test how excitatory neuron-specific ablation of SIRPalpha function alone or in combination with integrin alpha3beta1 affects dendrite and spine development and synaptic function and plasticity.

DESCRIPTION (provided by applicant): The brain has exceptionally high energetic demand mainly to support synaptic transmission and action potential propagation. In the absence of blood flow, brain function is disrupted within seconds and neuronal damage occurs within minutes. Thus a microvascular network that precisely meets local metabolic demand is neurovascular coupling mechanisms have evolved to precisely match micro-regional blood flow to the fluctuating local oxygen consumption. While the larger vascular structures of the brain form during embryonic stages, the microvasculature, the site of most oxygen diffusion, develops between the end of the embryonic period and the first postnatal month in mice. There is, however, limited knowledge about the processes involved in the postnatal maturation of the microvascular network and the interactions between microvessels, astrocytes and pericytes (required for neurovascular coupling. required. Furthermore, neuro-glio-vascular unit -NGVU) we have recently performed the first comprehensive in vivo imaging study of microvascular plasticity from birth to death. We found, that the cortical microvasculature in neonatal mice develops through short-distance sprouting and concomitant regression. Perturbation of this process during a critical neonatal period, leads to irreversible reduction in microvascular density causing susceptibility to hypoxia and synaptic loss. We hypothesize that disruption in the neonatal development of the microvascular network and NGVU lead to metabolic demand/supply imbalance resulting in lifelong neural dysfunction and pathology. We will use sophisticated in vivo imaging tools, environmental and molecular manipulations to: 1) Determine the precise sequence of cellular interactions leading to the formation of a structurally and functionally mature NGVU. 2) Characterize cellular, molecular and environmental factors that disrupt the postnatal development of the NGVU. 3) Determine the functional and structural consequences of abnormal NGVU development. Our studies will improve understanding of mechanisms of neurovascular development that could have implications in the pathogenesis of developmental disorders, neurodegeneration and age-related cognitive decline.

DESCRIPTION (provided by applicant): The Role of Astrocytes in Myelin Maintenance and Regeneration Myelin is a fundamental component of mature neural networks that is affected in a large number of pathological conditions of the central nervous system (CNS). Several of these debilitating diseases are thought to be related to astrocyte malfunction so a better in vivo understanding of how astrocytes contribute to the maintenance of mature myelin sheaths and oligodendrocytes is crucial. We have developed a new technique that allows high resolution label-free in vivo imaging of myelinated axons. This technique takes advantage of the high refractive index of lipid rich multilayered myelin and is based on spectral confocal reflectance (SCoRe) microscopy. Using SCoRe we have obtained for the first time long-term images of the dynamics of cortical myelin on the cellular scale in a living animal. Our preliminary data shows this technique as well as fluorescence imaging of astrocytes, oligodendrocytes and axons are a powerful set of tools that in combination provide a wealth of information about fine structural dynamics of these structures in vivo. We demonstrate the feasibility to track the development of myelin pathology in a dysmyelinating mutant mouse and determine the temporal dynamics of demyelination after single oligodendrocyte and astrocyte ablation. We propose to use these powerful techniques to address three fundamental questions concerning the role astrocytes play in myelin maintenance and regeneration. First, we will determine whether astrocyte presence is necessary for myelin and oligodendrocyte structural maintenance in vivo. Next we will use single cell ablation techniques to determine if astrocytes are required for or alter the temporal dynamics of oligodendrocyte remyelination in vivo. Finally we will determine how manipulation of candidate molecules in single astrocytes influences local myelin structural maintenance and regeneration in vivo. Together these experiments will provide novel insight into the fundamental in vivo roles played by astrocytes on the maintenance and regeneration capabilities of myelin and oligodendrocytes.

PROJECT SUMMARY: Delirium is a clinical syndrome characterized by acute and reversible disturbance in mental function that occurs in the elderly following metabolic abnormalities, surgery or infection. Individuals with Alzheimer's disease (AD) are more prone to developing delirium and despite resolution of the initial acute symptoms, frequently suffer an acceleration in the expected rate of cognitive decline with poor long-term outcomes. The precise mechanisms responsible for the exacerbation of the chronic neurodegenerative processes are poorly understood, significantly impeding the development of therapeutic interventions. This application aims to explore complex neuro-immune interactions, and identify mechanisms underlying progressive post-delirium cognitive decline. Specifically, we will explore newly discovered neuroprotective functions of microglia and test the hypothesis that such functions become impaired during systemic infection/inflammation, leading to exacerbation of neurodegeneration. We hypothesize that molecular manipulation of key cellular targets during acute systemic inflammation will preserve the neuroprotective microglia functions, reduce neurodegeneration and improve long-term cognitive outcomes. We have developed a sophisticated set of tools to test these hypotheses in vivo, including longitudinal high- resolution optical imaging of amyloid plaques, microglia and neurons, as well as calcium imaging, molecular/pharmacological manipulations and behavioral phenotyping. This project will significantly improve our understanding of the role of microglia in AD and has the potential to uncover novel therapeutic targets for the prevention of post-delirium progressive cognitive decline.

SUMMARY Thromboembolic occlusions of the microvasculature are implicated in many acute ischemic conditions including stroke and myocardial infarction and may be partly responsible for the ?no-reflow? phenomenon. The fibrinolytic system and hemodynamic washout are considered the principal mechanisms for removing occlusive thromboemboli in all vascular beds, however we have shown that they have a high failure rate at the microvascular level. This may be partly due to a mechanism that we discovered and termed ?angiophagy?, whereby endothelial lamellipodia extensively envelop occluding emboli, trapping them within the vascular lumen, markedly reducing hemodynamic washout and limiting access to plasma fibrinolytic enzymes. In conditions such as stroke, it is likely that the early stage of thromboembolus engulfment is highly detrimental as it prevents distal microvascular recanalization following spontaneous reopening of large occluded vessels or after tissue plasminogen activator administration or mechanical thrombectomy. We hypothesize that pharmacologically preventing or delaying the early engulfment stages of angiophagy, can improve thromboembolic washout, and microvascular flow and viability, leading to better post-ischemic outcomes. We aim to discover signaling pathways that regulate the various stages of endothelial plasticity involved in this process, with the goal of identifying potential therapeutic targets. We will use an innovative multidisciplinary approach to elucidate these mechanisms including mutant mice, pharmacological manipulations and high resolution intravital imaging of occluded microvessels. Additionally, we will test our candidate drugs in a translational model of transient ischemic stroke. These studies are likely to advance our understanding of mechanisms of microvascular occlusion and recanalization and could identify novel targets to prevent the no- reflow phenomenon in stroke and other ischemic conditions.

ABSTRACT Endothelial cells (ECs), astrocytes and pericytes are integral components of the neurovascular unit (NVU) and play critical roles in blood brain barrier (BBB) formation and maintenance. These cells express uptake and efflux membrane transporters that regulate CNS penetration of molecules, therapeutic drugs and toxins. The function of the BBB and transporters is likely disrupted in many neurological disorders. Thus, development of tools to study NVU cells and their properties of BBB selective transport in vivo is a key priority in translational neuroscience research. We have discovered a unique set of small molecules that can be selectively transported into the cytoplasm of either ECs, pericytes or astrocytes with exquisite affinity and specificity. We have evidence that these molecules enter cells through membrane transporters, selectively expressed in each cell type. We hypothesize that these molecules could be adapted as probes for intravital animal and human imaging and also for cell-specific delivery of drugs. In this proposal, we aim to identify the precise mechanisms of probe membrane transport in vivo; establish the feasibility of using these molecules as cell-specific drug carriers or as radiopharmaceuticals for human imaging with positron emission tomography (PET) and finally test their in vivo properties in models of Alzheimer's disease and stroke. This project will improve our understanding of molecular transport mechanisms across the BBB and may transform the ability to selectively image and pharmacologically modulate cells of the NVU in health and disease.

ABSTRACT The overall goal of the Yale ADRC Research Education Component (REC) is to recruit and promote the careers of a cohort of junior investigators from a diverse scientific and cultural backgrounds that will pursue rigorous, innovative and high impact biological, translational, and clinical research and will become future leaders in the Alzheimer?s disease and related dementia fields. Our application builds upon a successful track record of Yale and the ADRC in promoting the careers of junior investigators with broad interest in research relevant to the dementia field. To achieve our goals, the REC will closely interact and coordinate educational activities with all other ADRC Resource cores including the community outreach core. Furthermore, in order to encourage investigators to explore the mechanistic heterogeneity of dementia including systemic and aging factors, we will closely link our REC educational and networking activities with those of the Yale Older Americans Independence Pepper Center. The ADRC REC will provide personalized mentorship programs and comprehensive and interdisciplinary didactic activities and courses addressing key research, leadership, teaching and grant writing skills. In addition, the REC will provide various levels of financial support commensurate with the investigators career stage and will offer relevant technical training and priority access to all ADRC research cores. To recruit an expanded group of investigators, including junior faculty, clinical/research fellows and graduate students, we will have three categories of REC investigators: ADRC Scholars will receive financial support to protect their research time and will have priority access to the Center Cores; Small REC Awardees will receive support for pilot work that will allow them to apply for future ADRC Scholar funding; REC Affiliates will have access to all the resources of the ADRC and participate in didactic, career development and academic activities. Overall, the newly organized REC will serve as a hub for all educational, didactic and career development activities aimed at promoting the development of future leaders that will bridge clinical and basic sciences to improve clinical outcomes and quality of life of individuals with dementing disorders.

Not required for this Supplement RFA