Brian J. Bacskai - US grants

DESCRIPTION (provided by applicant): In vivo imaging of cellular and molecular structures in the intact brain provides a powerful tool for wideranging investigations in normal physiology, or in experimental models of disease processes. We have recently developed methods using a light microscope-based technique, multiphoton microscopy, to image microscopic structures in the brains of living transgenic mice over periods of months. Multiphoton microscopy utilizes a near-infrared laser for excitation of fluorophores deep within scattering tissue, with high spatial and temporal resolution. The spatial resolution of this imaging technique is about 1micrometer, several orders of magnitude better than other in vivo techniques, like PET, or MRI. In this application, we propose to develop new techniques that will provide important in rive readouts for biological imaging. This research will also lay the groundwork for development of contrast reagents suitable for use in human brain imaging with PET or MRI. We will develop, in Aim 1, techniques for high-resolution, in vivo imaging of structural reporters in the brain. We will investigate procedures to image individual neurons and microglia with high spatial resolution in the intact brain. In Aim 2, we propose to develop imaging techniques that exploit functional reporters in these living cells in the brain. Development of these molecular imaging techniques will build upon techniques accomplished in Aim 1. We have been using an experimental, transgenic mouse model of Alzheimer's disease that develops senile similar to those found in patients with Alzheimer's disease (AD). Our imaging techniques have allowed us to image the senile plaques in vivo in these mice with high spatial resolution. We will apply our new imaging techniques to this mouse model and address important questions that will provide insight into the pathophysiology of this disease. Our current techniques, however, rely on invasive procedures to gain access to the brain for imaging. In Aim 3, we will develop new techniques for non-invasive, in rive detection of senile plaques. New techniques using nearinfrared contrast reagents, and IR-sensitive detectors will allow non-invasive detection of plaques in the intact animal, and may also lead to clinically relevant diagnostic procedures for AD patients. In summary, the proposals outlined in this application will lead to generally applicable new techniques for cellular and molecular imaging in the intact brain.

DESCRIPTION (provided by applicant): Transgenic mouse models over-expressing APP develop senile plaques in an age-dependent fashion similar to those found in patients with Alzheimer's disease (AD). Recent evidence shows that active or passive immunotherapy dramatically pevents amyloid-beta deposition in transgenic mice. Clearance of existing amyloid-beta3 deposits present in Alzheimer's disease patients, in addition to prevention of new plaque formation, will be critical for an effective treatment. Observing senile plaques before and after treatment is the only direct way to measure clearance of existing deposits, but until recently this has not been possible. We have developed novel multiphoton microscopy techniques that allow longitudinal in vivo imaging of individual plaques. Using this approach, we demonstrated clearance of existing plaques in transgenic mice 3-5 days after a single application of antibodies to the cortex. In this application, we propose to test hypotheses about the mechanism of clearance. Aim 1 follows from our observation that clearance can also occur with addition of F(ab')2 fragments, suggesting that Fc-mediated mechanisms are not necessary. We propose a model whereby clearance results from a two-step process involving disaggregation of amyloid-13 deposits via direct biophysical interaction followed by active removal of the amyloid. Aim 2 asks whether systemic immunization, rather than topical application of antibody to cortex, will lead to clearance; if so, we will determine necessary titers and optimal epitopes. Aim 3 takes advantage of in vitro results from several laboratories demonstrating that amyloid-13 binding compounds prevent or reverse formation of amyloid fibrils. We will test whether they are disaggregating agents in vivo. The results will strongly impact the development of treatments aimed at removing senile plaques and the associated neurological damage in Alzheimer's disease.

Alzheimer's disease (AD) is a devastating neurological illness with no known cure, yet a central hypothesis implicating oxidative stress as a cause of the disease has been postulated for more than a decade. AD is characterized in post-mortem tissue by the presence of senile plaques that result from the progressive brain accumulation of amyloid-p (A(3)peptides;thus Ap is the principle therapeutic target for treating AD. There are numerous studies with anti-oxidant therapy, however none examine the AD-specific contribution quantitatively. Clinical trials with anti-oxidant therapy have have also shown limited efficacy. Our approach provides quantitative readouts of AD-specific oxidative stress to optimize an anti-oxidant treatment. While we have shown that oxidative stress results from the senile plaques of AD themselves, it is likely that other p species, such as small diffusible aggregates, oligpmers, or Ap derived diffusible ligands (ADDLs) are also a source of reactive oxygen species. This grant application proposes to identify aggregated and soluble Ap components that are sources of oxidative stress, and evaluate anti-oxidant treatments for protective activity both in vitro and in vivo using transgenic mouse models of AD. Our strength lies in the utilization of. sophisticated imaging techniques based on multiphoton microscopy that allow us to image senile plaques structurally and functionally in vitro and in vivo. Small diffusible aggregates of Ap like oligomers and ADDLs can be analyzed and characterized using high-throughput plate-reader assays or multiphoton fluorescence correlation spectroscopy (PCS). Anti-oxidants can be tested for their ability to reduce or prevent the oxidative stress resulting from these small toxic Ap species. In combination, these experimental paradigms will be used to screen potential anti-oxidants from both traditional and alternative sources to systematically evaluate whether compounds like ginkgo biloba extract, vitamin E, or grape seed extract are effective anti- oxidants for Alzheimer's disease treatment. The results will bridge the gap between the description of oxidative stress in Alzheimer's disease to direct determination of the anti-oxidant ability of natural and synthetic products that should hold promise for treatment of AD patients.

[unreadable] DESCRIPTION (provided by applicant): Non-invasive optical imaging capitalizing on the transmissive properties of the near-infrared spectrum is a rapidly evolving molecular imaging approach complementary to MRI, PET, or SPECT. Optical imaging is inexpensive and allows in vivo imaging of intact animals with spatial resolutions less than 1 mm. Significant strides in structural detection of tumors and functional imaging of blood flow, metabolism, and protease activities have recently been accomplished in both animal models and humans. The next generation of optical imaging approaches will depend on novel contrast reagents that specifically identify structures or functions within intact animals. We propose to accelerate the development of optical imaging techniques, as well as to generate novel optical contrast reagents for near infrared molecular imaging. We will modify an existing optical imaging system to optimize the sensitivity and spatial resolution with advances in both hardware and software for intracranial imaging of intact mice. We will also develop a suite of molecular imaging probes that will target specific intracranial structures, with applications for in vivo imaging for a broad range of neurological diseases. We will develop fluorescent probes for non-invasive measurement of grey matter, white matter, cerebral vasculature, and the pathological protein aggregates found in Alzheimer's disease. Successful implementation of these techniques will not only aid in the characterization of the natural history of structural alterations in the brain in a variety of animal models, but will serve as a quantitative end-point for evaluation and screening of therapeutics aimed at ameliorating the progression of disease in animal models. This proposal brings together a multidisciplinary team with a broad range of experience. Principal investigator Brian Bacskai at MGH has extensive experience in optical imaging, in vivo detection of neuropathology and biomedical engineering. The multidisciplinary team includes MIT chemist Timothy Swager, U. Pittsburgh Psychiatrist/Chemist William Klunk, MGH optical engineer David Boas, MGH Neuroanatomist Brad Hyman, and MGH Neuroscientist Steven Reeves. The combined efforts from this project will lead to a new arsenal of molecular imaging tools to exploit the newly emerging technology of NIR spectroscopy. [unreadable] [unreadable] [unreadable]

Multiphoton microscopy for in vivo neural imaging Abstract Alzheimer's disease (AD) is a debilitating neurodegenerative disorder that affects millions and is poised to bankrupt our healthcare system unless effective treatments can be translated to the clinic. AD is characterized by the presence of amyloid plaques, neurofibrillary tangles, and severe neurodegeneration. However, it is also clear that a range of other more subtle alterations occur in the brain that may be upstream of these drastic neuropathological alterations. Animal models of AD, though imperfect, have provided key insights into how some of these severe and subtle alterations occur in the brain, with many having observable clinical correlates. We have pioneered the use of multiphoton microscopy in the brains of these mouse models and have successfully monitored both subtle and severe alterations. With the increasing popularity of multiphoton microscopy, many optical advances have been made that overcome some of the limitations of early versions of laser scanning microscopes. We propose to adopt and extend some of these advances that include long wavelength excitation, adaptive optics, and wavefront shaping to turn our current microscope into a state of the art instrument for advanced brain imaging. We will apply these new techniques to target the pathophysiology of altered neuronal circuits in AD mouse models, and use a variety of techniques to understand the mechanism of the hyperexcitability of these networks. We will exploit three model networks in anesthesized or awake animals; spontaneous cortical activity, imaging of visual cortex during presentation of visual stimulus, and slow cortical oscillations (0.6Hz) that may relate to human sleep rhythms and consolidation of memories. We will focus on the role of specific subtypes of inhibitory interneurons by taking advantage of new Cre recombinase mouse models and bring to bear the power of targeted optogenetics and calcium imaging to define the mechanisms of the circuit dysfunctions. This platform will then allow us to evaluate candidate therapeutics aimed at restoring the altered network activity that should ultimately translate to clinical treatments for AD.

DESCRIPTION (provided by applicant): Multiphoton microscopy is a powerful technique for high resolution intravital imaging in deep tissue, with particularly exciting results obtained from brain imaging. We have successfully implemented and innovated in vivo brain imaging over the past 10 years, most notably in the study of Alzheimer's disease mouse models. These innovations, however, have extended to other brain imaging applications around the world where we have either directly or indirectly assisted in advancing the applications. Our application of multiphoton microscopy began with the first commercial multiphoton microscope, the Biorad 1024MP, which is still functional in the lab, but antiquated. There is no upgrade path for this instrument: it needs to be replaced. We are proposing to purchase a state-of-the-art multiphoton microscopy system to replace this instrument. The configuration of the microscope platform chosen is optimized for fast and deep in-vivo imaging. Using negative chirp optics to compensate for group velocity dispersion in the optical path, the proposed instrument allows optimized pulse-widths for deep tissue imaging with reduced average power. We have assembled a group of experienced, NIH-funded investigators with applications in a variety of neurological diseases that will take advantage of the capabilities of the instrument in an environment rich in experience and proven success. We have been using multiphoton microscopy to image structure and function in vivo on spatial scales as small as a single spine on an identified dendrite from a single neuron. We have developed approaches to image the pathology associated with Alzheimer's disease, neurons, glia, and the vasculature. We have also been using fluorescence lifetime imaging microscopy (FLIM) for sensitive detection of fluorescence resonance energy transfer (FRET), which can be used to reveal interactions between individual proteins. While FLIM has been largely relegated to in vitro or cell culture measurements, we aim to apply FLIM for quantitative imaging in populations of neurons and glia in vivo. The new microscope will be a vital resource for NIH funded imaging studies in mouse models of neurodegenerative diseases, stroke, and epilepsy. PUBLIC HEALTH RELEVANCE: We propose to replace an old multiphoton microscope with a newer more advanced model. We have been using multiphoton microscopy for 10 years to try to understand the pathophysiology in the brain of living mouse models of neurodegenerative disease, and a new microscope will ensure that we can continue our successful research programs.

? DESCRIPTION (provided by applicant): Alzheimer's disease is the leading cause of dementia in the elderly, and because the number of at risk individuals is rapidly increasing, AD represents a major health crisis. At this point, while a number of key insights and clinical tools have arisen there are still no effective treatments to prevent or reverse the disease. The genetics of AD have led to a simple and plausible hypothesis of disease progression: amyloid-ß, a known cytotoxic peptide that forms both small diffusible and large insoluble aggregates, leads to neurodegeneration and AD. This simple hypothesis has led to myriad studies using high concentrations of synthetic amyloid peptide that is almost impossible to work with from a chemical biology standpoint, and absolutely leads to cell toxicity in every cell based assay used. Indiscriminate use of synthetic amyloid preparations have confounded the field, for the most part hampering and not helping research progress in AD. It is clear now that the progression of disease is a much more complex process. AD is a multifactorial disease that must include a spectrum of cellular and molecular events that change over time. Therefore, while it is impossible to ignore that Aß is somehow central to the disease, there is a clear need to determine the sequence of events in physiologically relevant models that will identify age sensitive treatment strategies. There are other key tenets of disease progression that are central, but still not clearly defined. It is well established that oxidative stress, mitochondrial alterations, and calcium dyshomeostasis are key molecular components on the pathway to cell death. However, there is no consensus as to whether these events are related, causal, or reflective of the disease. Therefore, we aim to systematically evaluate the temporal sequence of these molecular and cellular events in the living brain of the most thoroughly characterized transgenic mouse models of AD to identify the contribution of these factors, the causality of these factors, and the appropriate timing of these factors in the course of the disease to inform multifactorial therapeutic strategies based on duration and extent of progression. We will test the hypothesis that Aß leads to neurodegeneration through a pathway involving calcium dyshomeostasis, ROS generation, and mitochondrial dysregulation. To test this hypothesis, we will develop tools to image each of these endpoints in the living brain of APP mice using multiphoton microscopy that in and of themselves will be broadly useful to the neuroscience community. Finally, we will explore interventions to identify therapeutic pathways that ultimately will lead to treatments for Alzheimer's disease in patients.

? DESCRIPTION (provided by applicant): The small vessels of the brain play key roles in both age-related vascular cognitive impairment and clearance of the ß-amyloid peptide (Aß). Cerebrovascular deposition of Aß as cerebral amyloid angiopathy (CAA) sets up a potentially self-reinforcing mechanistic loop in which CAA-related vascular injury and dysfunction lead to reduced Aß clearance and progressively worse Aß deposition, CAA, and Alzheimer disease pathology. We propose a systematic, multidisciplinary analysis of the mechanisms underlying Aß-related cerebrovascular injury, vascular dysfunction, and impaired perivascular clearance in human CAA and transgenic mouse models. Specific experiments, each designed to translate from mouse models to reliably diagnosed human CAA, focus on the effects of CAA on cerebral small vessel compliance, physiologic reactivity, and their relationship to focal brain lesions (SA1), the effects of altered physiology on Aß clearance and accumulation (SA2), and the effects of CAA on gene expression in cerebrovascular endothelium and smooth muscle (SA3). The proposal builds on the applicants' long record of successful mutual collaborations and their internationally recognized expertise and leadership in noninvasive detection and analysis of human CAA, real-time measurement of vascular structure and physiology in living transgenic mouse models, and molecular analysis of cerebrovascular gene expression in control and disease states. The proposal also builds on a wide range of cutting-edge methodologic advances such as ultrahigh-field functional MRI, serial molecular Aß imaging, intravital multiphoton microscopy, vasculomic analysis, and laser-capture microdissection of post-mortem tissue. Successful completion of the proposed highly translational experiments will determine Aß's vascular effects at the molecular, single-vessel, and whole-brain levels, establish their relevance to clinical disease, and yield entirely new and promising approaches to interrupting the vicious cycle of vascular injury and reduced Aß clearance key to the propagation of CAA and Alzheimer's disease.

Astrocytes are the most abundant cells in the central nervous system, however their role in health and disease remains a mystery. Astrocytes are very heterogeneous in structure and molecular profile. A single astrocyte creates a distinct non-overlapping territory that encompasses thousands of synapses. Their extensive branches and fine processes allow direct communication over long distances, as well as indirect communication through secretion of chemokines and cytokines. Astrocytes are also a significant component of the neurovascular unit as their endfeet processes terminate directly onto cerebral vessels, regulating cerebral blood flow according to metabolic demand. While in vitro models, including primary astrocytes and acute brain slices, have provided great insight into the physiology of astrocytes in health and disease scenarios, it is clear these preparations devoid of the complexity of the role of astrocytes in vivo. Thus, efforts to study astrocyte physiology should be directed as much as possible to the most physiologically relevant system: the intact living brain. We and others have demonstrated through in vivo imaging of intracellular calcium that astrocytes are dynamic players in the brain, and that the progression of Alzheimer's disease pathology alters their morphology and signaling characteristics. More specifically, we determined that in the presence of senile plaques, astrocytes in the brain have elevated intracellular calcium, exhibit hyperactive signaling, and can initiate spontaneous calcium waves. This allows astrocytes to respond to focal pathological insults with both focal and long range responses. These observations demonstrated that astrocytes respond to amyloid deposition with a change in function, but left several fundamental questions unanswered. Here, we wish to extend our previous observations and determine what the contribution of the altered calcium signaling is to the degenerative process that occurs in AD. The use of genetically encoded indicators, specifically expressed in astrocytes, along with in-vivo imaging will allow us to explore the effect of amyloid on astrocyte structure and function at the synapse and the neurovascular unit. We will also determine if the alterations depend on senile plaque deposition, or soluble oligomeric A? and whether the alterations are beneficial or detrimental to brain function. We propose experiments where astrocyte specific increases or decreases in calcium signaling will be evaluated in healthy and diseased brains. Finally, we will ask how clinically relevant manipulation of the amyloid cascade will affect calcium signaling in astrocytes and the degenerative process. These experiments will shed light on the role of astrocytes in the healthy and diseased brain, and will lead to new targets for therapeutic manipulation in Alzheimer's disease.

Sleep-wake disruptions and cognitive impairments are prevalent and disabling features of Alzheimer's disease (AD). AD patients exhibit profound sleep disturbances including disruption of non-rapid eye movement (NREM) sleep. A major restorative feature of NREM sleep, which is also associated with proper cognitive functioning, is slow-wave activity (SWA). Recent findings suggest a causal relationship between impaired generation of SWA during sleep and AD pathogenesis including extracellular accumulation of the amyloid-? (A?) peptide and neuronal dysfunction. While evidence indicates that cortical-thalamic loops regulate SWA, the exact cellular and molecular mechanisms for impaired SWA in AD are unknown. Thus, a need exists to characterize the cells and molecular mechanisms responsible for SWA generation to reduce the impairments in SWA and pathogenesis of AD. We propose studies that will elucidate the sleep state related mechanisms by which SWA protects against AD. Studies using AD animal models suggest that inhibitory neurotransmission is impaired during periods of SWA. The overall objective of this proposal is to identify and stimulate specific SWA modulating interneuronals to determine which cells restore SWA and mitigate AD-related pathology using an established AD mouse model. Herein, we propose to employ optogenetics and chemogenetics to control neuronal circuits aimed to restore SWA and slow AD progression. Thus, our findings will determine the cellular and molecular relationships between sleep and AD, with the targeting of interneurons during specific periods of sleep as a novel therapeutic approach.

Focal microvascular lesions ? such as microinfarcts and microhemorrhages ? are frequently observed in the brains of older individuals and are strongly associated with cognitive impairment and dementia. Yet, the underlying pathophysiological mechanisms and structural and functional consequences of these lesions are poorly understood. This proposal aims to unravel the mechanisms underlying microvascular lesion formation and their impact on local and remote vascular and neuronal networks, in the context of cerebral amyloid angiopathy (CAA). CAA is one of the most common age-related cerebral small vessel diseases, characterized by the accumulation of amyloid ? in small cortical arterioles. It has been suggested that vascular amyloid deposition involves a self-reinforcing cycle of vascular smooth muscle cell degeneration and impaired perivascular clearance of solutes (including amyloid) from the brain, which eventually results in the formation of microinfarcts and microhemorrhages. Although CAA has traditionally been considered a local disease resulting in focal lesions from individually affected cortical vessels, recent preliminary observations suggest profound alterations in surrounding vascular and neuronal networks, with more remote brain tissue injury as a result. The proposed research will address these network-wide effects of local vascular amyloid accumulation, by combining advanced neuroimaging and histopathology techniques in ex vivo human brains with cutting- edge optical imaging tools in living mice. The proposal builds on the applicants? international leadership in CAA, their strong background in post-mortem MRI and state-of-the-art histopathology techniques, and pioneering work in two-photon microscopy of mouse models with CAA. Combined with the world-class resources and international collaborations with experts in the field to perform advanced image processing, the proposed set of experiments will likely yield much needed answers regarding the mechanisms involved in cerebral small vessel disease. Novel insights resulting from this project may also yield promising new targets to prevent vascular cognitive impairment and dementia in the elderly.