Jason D. Hinman, Ph.D. - US grants

DESCRIPTION (provided by applicant): Stroke is a leading cause of death and disability in the US. Of the 795,000 new strokes per year, approximately 25% of these strokes are termed small vessel strokes affecting brain white matter, producing significant disability and cognitive decline. The use of magnetic resonance imaging demonstrates that white matter strokes expand and patient's disability progresses, often while patients remain under care in the hospital. This lost therapeutic opportunity is due, in part, to a poor understanding of the molecular events that follow white matter stroke, particularly those that involve the unique cellular elements of brain white matter: the axoglial unit. Injury to white matter disrupts the molecular connection between the myelinating oligodendrocyte, the axon, and its associated neuronal cell body (the axoglial unit) resulting in progressive axonal degeneration and stroke expansion. Studies in this grant will employ a novel mouse model of white matter stroke to identify the cellular and molecular mechanisms of cell-cell adhesion and energy transfer within the axoglial unit that lead to progressive axonal degeneration and stroke expansion. In addition, the retrograde effects of white matter stroke on the proximal axonal segment of the neuronal cell body far from the site of injury will be determined. These goals reflect my immediate career objectives of achieving an improved understanding of the molecular events associated with white matter stroke and micro vascular disease of the brain. Over the long-term, I plan to use this knowledge to design new molecular therapeutics for the treatment of stroke, acting to reduce the burden of stroke and stroke-related disability through my research, therapeutic development, and academic leadership. This mentored award will provide specific advanced training in rodent stroke modeling, laser capture micro dissection, RNAseq exome sequencing, and in vivo gene manipulation strategies. This training will be conducted under the direction of Dr. S. Thomas Carmichael, a leader in translational stroke research and co-mentored by Dr. Jeffrey Saver, a world leader in clinical stroke science. A career development plan providing training in these molecular techniques and the strategies needed to translate bench findings into therapeutics will be acquired through regular meetings with these mentors, carefully selected coursework, and hands-on experience. The University of California Los Angeles has a large and active academic neurology department that is well-recognized for training clinician-scientists. The proposed work will also take advantage of the resources available at UCLA in scientific cores and through established collaborations within the Department of Neurology. The UCLA Department of Neurology is committed to the advancement of my academic career and will provide a structured and supportive environment for the early stage of my career.

Project Summary Alzheimer?s disease (AD) and cerebrovascular disease account for over 80% of dementia diagnoses. Clinical, pathologic, and neuroimaging data indicate there is a synergistic relationship between cerebrovascular disease and Alzheimer?s disease. This high degree of co-morbidity implies a neurobiologic link, yet no clear molecular relationship has been established between these two common age-related impairments. In this proposal, we provide preliminary data that directly link these pathologies together and seek to identify the role that a specific stroke-activated molecular process plays in potentiating tau aggregation. Using an approach developed by the Co-PI to perform layer-specific neuronal capture and analysis after stroke, we identified that subcortical ischemic axonal injury within white matter leads to up-regulation of the microtubule-associated regulatory kinase, Mark4 in layer 5 cortical projection neurons. Mark4 is found in association with neurofibrillary tangles, phosphorylates tau at Ser262, and acts as a gateway phosphorylation event for downstream tau phosphorylation events including those that precipitate tau aggregation. These findings indicate that subcortical axonal ischemic injury primes neuronal tau phosphorylation and suggests a two-hit hypothesis that can explain the common pathologic overlap between cerebrovascular injury and AD. Here, we hypothesize that subcortical stroke primes tau aggregation through Mark4 expression and increases neuronal sensitivity to the other major pathologic hallmark of AD: extracellular b-amyloid. To prove that Mark4 acts a link between cerebrovascular injury and dementia pathologies, we will determine the effect of subcortical white matter stroke in PS19 transgenic mice that accumulate tau aggregates (Aim 1a). We will also determine the additive effect of subcortical stroke on cognitive and motor behavior in PS19 mice. In Aim 1b, we will use a peptide inhibitor discovered in bacteria to block Mark4 activity in cortical neurons and determine if we can prevent stroke-induced tau phosphorylation and aggregation in vivo (Aim 1b). In support of our hypothesis that subcortical ischemic axonal injury primes neurons for tau aggregation, we will use a novel biosensor assay developed by the Co-PI. Preliminary data using this novel assay shows that b-amyloid increases the propensity of tau to aggregate. Here, we hypothesize that Mark4 activity further primes b-amyloid-mediated tau aggregation and will test this in human biosensor cells (Aim 2a) and cultured neurons and iPSC-derived cultured neurons (Aim 2b). Together, these studies will expand our understanding of common molecular pathways in neurodegenerative disease that contribute to cognitive impairment and identify a novel therapeutic approach to target tau accumulation in mixed dementia.

Project Summary Cerebral microvascular disease (CMD) causes white matter injury and is a major contributor of the vascular contributions to cognitive impairment and dementia (VCID), including as the most common co-morbidity to clinical Alzheimer?s Disease. Chronic vascular risk factors such as obesity accelerate the progression of CMD by primarily damaging brain endothelial cells. Risk factor-induced changes in cerebral endothelial cells contribute to an increased risk of dementia. The molecular changes in cerebral endothelial cells caused by chronic cerebrovascular risk factors remain unknown yet are critical to designing therapies to prevent and repair ischemic white matter lesions thereby lessening the burden of VCID. We propose that a central mechanism of CMD progression is dysregulated signaling in brain endothelial cells damaged by chronic vascular risk factors. Using endothelial cell-specific transcriptional profiling, we show that chronic endothelial injury resulting from obesity results in abnormal vascular expression of an interleukin/chemokine signaling pathway. This molecular pathway results in dysregulated vascular-oligodendrocyte progenitor cell (OPC) signaling. OPCs are a critical progenitor cell population in brain white matter that respond to injury and are responsible for remyelination. Preliminary data demonstrate that chronically injured endothelial cells up-regulate IL-17 receptor b (IL-17Rb) and its effector chemokine CXCL5. Though many inflammatory pathways may play a role in brain ischemia, we show that this is the major inflammatory pathway that is active in endothelial cells injured by this chronic vascular risk factor. Critically, we further demonstrate that endothelial expression of CXCL5 results in the chemotaxis of OPCs to the vasculature, limiting their ability to remyelinate after a focal white matter ischemic lesion. Using gain and loss of function studies at the in vitro, in vivo, and functional levels after stroke, we will dissect the molecular pathways involved in dysregulated vascular-OPC signaling and identify a role for chemokine signaling in regulating white matter injury underlying VCID. Studies in Aim 1 will use an in vitro conditioned medium paradigm to identify the precise signaling mechanisms in endothelial cells that promote CXCL5 expression while identifying the necessary receptors on OPCs that regulate migration and differentiation. In Aim 2, we will broadly determine the role of chemokine receptor activation on the ability of OPCs to differentiate and remyelinate after stroke using CXCR2 knockout and small molecule antagonism. Finally, we will show in Aim 3 that blocking the expression of CXCL5 in white matter endothelia can reduce cognitive and motor impairment associated with focal white matter stroke by promoting remyelination within the peri-infarct tissue adjacent to stroke. Together, these studies establish new molecular mechanisms for the vascular regulation of remyelination as critical to the pathogenesis of CMD and establish a new therapeutic target for VCID.

PROJECT SUMMARY/ABSTRACT The overall goal of this proposal is to establish that the focal region of low shear stress (0-4 dyne/cm2) immediately downstream or in the post-stenotic segment of ICAD is a marker of atherogenesis, providing a therapeutic target for anti-inflammatory or anti-thrombotic interventions. Our central hypothesis is that post- stenotic low shear stress associated with atherogenic endothelial pathophysiology provides a rational basis for precision medicine of ICAD. Our preliminary data on low shear stress in these regions within SAMMPRIS confirm the potential influential role of shear stress associated with endothelial pathophysiology recognized in systemic atherosclerosis, yet extended to the cerebral circulation for the first time. Our three independent specific aims leverage an ongoing, invaluable collaboration and the unmatched quality of the SAMMPRIS data archive. The Neurovascular Imaging Research Core at UCLA will conduct the prospective experiments to validate focal low shear stress measured on CTA CFD of MCA ICAD with detailed anatomical flow models created from the same source images, with co-registered flow measured on 4D MRA [SA-1]. This step enables us to use these validated flow models to directly observe flow vortices and adjacent low shear stress on particle image velocimetry under microscopy [SA-1]. These validated MCA flow models serve as a scaffold for endothelium, where the cell morphology, expression of VCAM-1 and platelet aggregation can be studied [SA- 2]. The clinical relevance of post-stenotic low shear stress (0-4 dyne/cm2) in these 50 MCA lesions will be corroborated via comparison with CTA CFD of the contralateral homologous segment [SA-3]. Associations of this clearly defined potential therapeutic target of post-stenotic low shear stress will be examined with respect to other clinical variables and subsequent neurological outcomes in SAMMPRIS [SA-3]. These observations will be similarly conducted across all 140 SAMMPRIS CTA CFD subjects to investigate the generalizability of non-invasive CTA CFD in other arterial lesion sites [SA-3]. All image post-processing, CTA CFD, 4D MRA, 3D printing and biological assays of endothelial pathophysiology will be conducted at UCLA, where we have pioneered this workflow. The collaboration and guidance of the WASID and SAMMPRIS trial leadership is an important element of this new approach to ICAD that employs the statistical expertise at Emory of these landmark trials and their detailed imaging and clinical analyses. Our extensive preliminary work reflecting collaborative expertise on a novel imaging and biological framework, coupled with intensive experience linking the SAMMPRIS imaging and clinical datasets, provide a logical extension of knowledge on atherogenic low shear stress into the cerebral circulation.