Robia G. Pautler, Ph.D. - US grants

The objectives of this project are to develop MRI techniques for monitoring of brain function. This work was begun by development of a perfusion MRI technique that relies on arterial spin labeling of endogenous water as a perfusion tracer. Over the past five years, this class of perfusion MRI technique has been established as useful for measuring regional blood flow in the brain during rest, task activation, and in disease states in both humans and animal models. This technique makes quantitative measurements of regional blood flow with the spatial and temporal resolution of MRI in a variety of tissues. Further development of this class of techniques is being carried out with experiments designed to extend strategies for arterial spin labeling, make use of the labeled water as a probe of water extraction into tissue, and make it routine to acquire rapid three dimensional images in the mouse brain. Furthermore, when applied to the brain, this class of perfusion imaging technique should be useful for brain mapping studies and, in particular, forms a complement to BOLD techniques. Presently, all functional MRI techniques monitor the hemodynamic consequences of neuronal activation rather than a direct effect of neuronal activity. The cascade of events that lead to neuronal activation are release of neurotransmitter, depolarization, influx of calcium, release of neurotransmitter, and so on. Hemodynamic changes occur as a response of the brain to maintain homeostasis. A more direct MRI measure of neuronal activation might be very useful. Preliminary results indicate that Mn2~ might be an excellent contrast agent to probe calcium influx associated with neuronal activity in animal models. In addition, Mn2~ appears to act as an anterograde neuronal tracer once it enters neurons. Both of these exciting results open the possibility of extending functional MR techniques for the brain.

The Center has provision for the use of its resources for pilot and feasibility experiments to be carried out without the need for the request to be reviewed by the Scientific Advisory Committee. These experiments are usually of short duration and carried out for the purpose of obtaining a very limited set of data. These resources are allocated at the discretion of the Director. The NMR Center has assisted several groups in this regard: (i) obtain high resolution MR images of the rat brain for the purpose of preparing an atlas of the rat brain for use with PET data, (ii) obtain high-resolution MR images of an isolated human heart specimen for modeling biomechanics of the heart, (iii) obtain high-resolution MR images of a fixed human brain, (iv) obtain additional data on cerebral perfusion maps of a rat model with subarachnoid hemorrhage to complete a study that was carried out the previous year, and (v) obtain apparent diffusion coefficient (ADC) maps and perfusion maps of the rat brain in a photothrobolytic stroke model. Allocation of a limited amount of NMR Center's time on the scanners in a timely manner serves a very useful purpose of providing the investigators data which are sometimes required with minimal delay. In the past, a number of investigators have used this Director's discretionary time for obtaining pilot data for applying for grant support, for carrying out feasibility studies which were used in designing new experiments that were subsequently carried out in the NMR Center as regular projects, and for tying up loose ends of previosly carried out experiments in the Center.

DESCRIPTION (provided by applicant): It is possible to tract trace neuronal pathways in vivo in the central nervous system (CMS) utilizing a technique that we developed, Manganese Enhanced Magnetic Resonance Imaging (MEMRI) tract tracing. Manganese ion, Mn2+, is a calcium analogue and can enter neurons through calcium (Ca2+) channels. Furthermore, Mn2+ is transported along microtubules via fast axonal transport and is also paramagnetic, rendering it MRI detectable in spin-lattice (H)-weighted MRI images. It is therefore possible to utilize MRI to repeatedly measure dynamic changes in signal intensity, relfective of fast axonal transport of Mn2+ ion, within the same animal before and during disease progression. Axonal transport deficits have been observed in flies and cultured rodent neurons exposed to excess amyloid precursor protein (APR) or amyloid-beta, but neither the molecular basis of the transport deficit nor the temporal relationship of the transport deficit and the acquisition of Alzheimer's Disease (AD) are known. We hypothesize that: 1) Normal aging mice will exhibit declines in axonal transport rates throughout the brain as aging ensues 2) The presence of excess APR causes a reduction in axonal transport prior to the formation of neuritic plaques and 3) Impairment in the sequestering of Mn2+ into the endoplasmic reticulum results in the observed early decline in axonal transport rates. We plan to test our hypotheses through the following Specific Aims: Aim 1: We will determine longitudinally the in vivo axonal transport rates of Mn2+ ion in the CMS in control mice and an APR overexpressing mouse model of AD (TG 2576) as the animals age; Aim 2: We will resolve the mechanism of this axonal transport deficit by determining which steps in Mn2+ uptake and transport are affected in normal and TG 2576 mice as aging ensues.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is an age-related, neurodegenerative disease that is one of the leading causes of dementia afflicting 1% of people under the age of 60 to more than 40% of people over the age of 85. The symptoms of this disease are typified by memory loss and a progressive decline of cognitive abilities. This disease is characterized by the extracellular deposition of amyloid-beta (Ab) aggregates known as plaques that are surrounded by dystrophic neurites and activated glial cells as well as intracellular neurofibrillary tangles that are comprised of hyperphosphorylated tau protein aggregates. As detailed in the February 2006 Alzheimer's Research Forum, one of the most critical needs in AD research is the identification of biomarkers that can not only predict disease but also monitor responses to treatment. In this project, we first propose to utilize a novel MRI methodology, Manganese Enhanced MRI (MEMRI) that we helped develop to delineate the role of AB on in vivo axonal transport rates and pre-synaptic release and post-synaptic uptake mechanisms in mouse models of AD. Second, we will test MEMRI as an in vivo neuroimaging diagnostic to assess and potentially detect very early on responses to treatment in mouse models of AD. Third, we will determine if Ab1- 40, Ab1-42 or the combination of the two species contributes to in vivo changes in axonal transport and pre- synaptic release and post-synaptic uptake mechanisms in mouse models of AD.

DESCRIPTION (provided by applicant): Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of liver diseases, ranging from simple steatosis (fat in the liver) to non-alcoholic steatohepatitis (NASH) to cirrhosis. According to the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), it is the most common form of chronic liver disease and with the risk factors of obesity, diabetes, and metabolic syndrome on the rise, the prevalence of NAFLD is also expected to increase. While the role of oxidative stress in NAFLD is accepted, what is not known is the effectiveness of reducing oxidative stress in preclinical models of NAFLD. Without this knowledge, we cannot determine the utility of novel classes of high- potency antioxidants as potential therapeutics for NAFLD damage. To develop antioxidants for NAFLD that exhibit potent antioxidant activity, that do not pass radicals on to other molecules and that can be targeted, we have turned to nanotechnology. These nano-antioxidants, polyethylene glycolated hydrophilic carbon clusters (PEG-HCCs), are based on carbon nano structures that have conjugated ring structures so that they act as terminal oxidant acceptors or radical sponges. We have also demonstrated that these PEG-HCCs can be targeted to specific cell types (manuscript in preparation). Our long term goal is to understand the mechanisms of reactive oxygen species (ROS) in NAFLD pathology in order to develop effective therapeutics that can prevent and potentially reverse damage due to oxidative stress. Our objective is to determine the effect of reducing ROS through high-efficiency novel antioxidant compounds on the development of NAFLD pathology by in vivo imaging, biochemistry and histopathology in mouse models of NAFLD. Our central hypothesis is that reducing ROS will improve liver function, reduce liver damage and oxidative stress in mouse models of NAFLD. The rationale for the proposed work is that a mechanistic understanding of oxidative stress on NAFLD pathologies will lay the foundation for viable therapeutic approaches. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because we are conducting research that will advance our understanding of the role of oxidative stress in the development of non-alcoholic fatty liver disease (NAFLD). As a result, the proposed work is relevant to the component of NIH's mission of developing novel and innovative methods to treat NAFLD, a disease that is on the rise and has significant health consequences.

DESCRIPTION (provided by applicant): Duchenne muscular dystrophy (DMD) is the most devastating type of muscular dystrophy, with an incidence of 1 in every 3500 males. DMD is an X-linked, muscle-wasting disease caused by mutations in the cytoskeletal protein dystrophin.. Young DMD patients experience muscle damage that is followed by regeneration; however, as the disease progresses regeneration is impeded and muscle fibers are progressively replaced by connective tissue and fatty deposits. Profound muscle weakness results in decreased mobility by 10 to 12 year of age and eventually death by the age of 20 to 30 due to respiratory and/or cardiac failure. While there is currently no treatment for the disease, many different therapeutic approaches for DMD are entering clinical trials. Based on the trajectory of the pathogenic phenotype precise and reliable non-invasive measures that follow the temporal progression of the dystrophic process in children are needed., The goal of this proposal is to develop and validate a novel MRI technique (manganese enhanced MRI, MEMRI) as a noninvasive imaging procedure to assess skeletal muscle function in muscular dystrophy. The Specific Aims of the proposed research are to: 1) establish the biodistribution of Mn2+ within skeletal muscle after intravenous injection, and 2) establish a robust method using MEMRI for assessment of structural/functional changes in dystrophic skeletal muscle. If successful, the proposed research will provide a robust non- invasive method currently not employed for assessing both structure and function of skeletal muscle in DMD, which will be critical in evaluating the beneficial effects of these therapies.

ABSTRACT/PROJECT SUMMARY Alzheimer?s disease (AD) is characterized by hyperphosphorylated tau protein which causes impaired axonal transport in neurons. In recent years, multiple researchers have determined that oxidative stress appears to be a significant early event in the pathogenesis of AD in preclinical AD models and in AD patients. We have shown that reducing oxidative stress through quenching of mitochondrial reactive oxygen species (ROS) results in significant improvements in AD pathology in mice. Unfortunately, targeting oxidative stress to date has not been efficacious clinically in mitigating AD pathology. Dietary antioxidants are low potency, require high doses, are potentially toxic at those doses and have not proven clinically effective. There is thus an urgent clinical need to develop potent, stable intracellular antioxidants for the treatment of AD. Polyethylene glycol (PEG)-conjugated hydrophilic carbon clusters (PEG-HCCs) are novel nanoparticle antioxidants that convert superoxide to O2 faster than many single-active-site catalysts. These are also stable, soluble, and non-toxic at moderately high concentrations. We have shown that PEG-HCCs enter cells and preferentially accumulate at or near mitochondria. We hypothesize that PEG-HCCs inhibit action of IL-6 via ROS quenching, which then prevents increased activity of p35 and cdk5, resulting in a normalization of hyperphosphorylated tau and a reduction in AD pathology. The PEG-HCC technology has been awarded multiple patents. Acelerox has obtained the international license to the relevant patents for all fields of use and is set to fully explore and develop the unique properties of this novel nanomaterial in treating Alzheimer?s disease. In this proposal we seek to conduct key animal experiments to establish proof of principle of this novel therapy in animal models of AD. In Aim 1, we will Investigate repeated intranasal administration as a potential route of delivery of PEG-HCCs. In Aim 2, we will perform a proof-of-concept experiment to establish efficacy of PEG-HCC in mouse model of tauopathy. In summary, we will evaluate a novel class of nano-antioxidants, the PEG-HCCs, to effectively reduce ROS in a tau model of AD. We are in the unique position to use these highly active, bioavailable, and selective antioxidant nanomaterials to test the hypothesis that oxidative stress is a viable therapeutic option in treating AD and also begin to assess the mechanisms of improvement in a pre-clinical system. The extensive data generated using these nanoparticles suggest that they are taken up by cells and targeted to mitochondria, the primary source of antioxidants and appear to be well tolerated.

The requested equipment is a hardware upgrade for the BCM Bruker 9.4T/20 cm bore small animal MRI that includes: 1) updating the electronics from Avance I to Avance IIIHD; 2) updating the microgradients to allow for optimal imaging in mouse models and to interface with the new electronics and 3) to update the coils to interface with the upgraded electronics. The upgraded electronics will replace the Avance I electronics, which are out of date and starting to fail. This 9.4T MRI is now part of the Advanced Technology Core (ATC) at Baylor College of Medicine. BCM has turned to this model to centralize all advanced technologies to promote ease of access, multiple collaborations and a means to institute College support for all Advanced Technologies at BCM. This upgrade is essential for multiple reasons: 1) The current hardware (Avance I) for the BCM 9.4T is not ?state of the art? and is in fact three generations behind the current platform (Avance III-HD); 2) Bruker is halting production of replacement parts for the Avance I electronics; 3) The current hardware is starting to fail as evidenced by line artifacts in the images acquired on this magnet. 4) The latest version of the software, Paravision 6.0, that runs the MRI only interfaces with Avance II or higher electronics. Because we are currently operating on Avance I electronics, we are not able to use the latest version of the software. 5) In December 2015, we had some of the hardware fail and the system was down until February 2016, until Bruker could first help us identify the problem and then locate the Bruker site with the appropriate replacement parts. Again, the Avance I replacement parts are becoming fewer and far between. In this case, the replacement parts were located in France and it took several weeks to receive them. For this application, we have identified 17 PIs as major users as well as an additional 5 minor users specific to the BCM 9.4T MRI. We estimate that both major and minor users at the moment use the MRI at about 93% of its capacity, which also gives us opportunity to expand and allow for PIs to collect preliminary data for new projects and grant applications. 

Alzheimer?s disease (AD) is a progressive neurodegenerative disorder characterized by the neuropathological accumulation of amyloid beta (Ab) plaques and neurofibrillary tangles comprised of hyperphosphorylated tau. Recent data from multiple groups support that given enough time, Ab and tau spreads throughout the brain in a well-defined manner along brain networks, very similar to the way a prion protein travels throughout the brain. Notably, the olfactory system has been reported to be one of the first systems affected in AD. Our past work has focused on using Manganese Enhanced MRI (MEMRI) to assess axonal transport in the olfactory system in mouse models of AD. Indeed, we have reported that axonal transport deficits in the olfactory system are detectable prior to the development of learning and memory deficits and well before plaque formation. These data are consistent with the idea that AD pathology spreads throughout the brain, beginning with the olfactory system. Additionally, our prior work has also focused on the effects of reducing oxidative stress in mouse models of AD. When we reduced oxidative stress by overexpressing superoxide dismutase 2 (SOD-2) in mouse models of AD, we observed a complete recovery in learning and memory deficits, a complete recovery in axonal transport deficits in the olfactory system as well as an over 50% reduction in Ab plaque formation. Although oxidative stress has been identified as a significant player in the development of AD, efforts to reduce oxidative stress with antioxidants has met with limited success. Some of the reasons for this are thought to include the inability to target sufficient quantities of administered antioxidants to the appropriate regions within the brain. Additionally, clinically available antioxidants have poor solubility and do not readily enter cells. Thus, we have turned to nanotechnology and have been working with nano-antioxidants that are much more potent than clinically available antioxidants, are non-toxic and readily enter cells. We therefore hypothesize that protecting olfactory and adjoining structures with intranasally administered nano-antioxidants (PEG-HCCs) will slow down the progression of AD as assessed with behavioral assays, MEMRI, resting state fMRI (rs-fMRI) as well as 31P measurements and histology. We will also incorporate machine learning, specifically, a probabilistic graphical model, to determine the interactions of the readouts in Aims 1 and 2 in mouse models of AD and controls with and without treatment with PEG-HCCs. We also propose to incorporate machine learning to predict 1) the degree to which superoxide levels should be reduced to improve AD pathology and 2) which stages of AD (e.g. pre vs post plaque) are beyond rescue. Completion of this highly innovative project will have significant impact towards future AD therapeutic strategies.