Jennifer Lee - US grants

SES-0623695
Jennifer Lee
Jody Agius
University of California, Irvine

This research studies the incorporation trajectories of the Mexican origin middle-class, an often neglected segment of the U.S. Mexican population. A number of scholars contend that Mexican immigrants and their children face limited prospects for successful economic and socio-cultural incorporation into the middle-class, and that Mexican immigrants and their children are prime candidates for downward mobility into a minority underclass culture. On the other hand, immigrants who achieve upward mobility are expected to incorporate into the white middle-class. However, some scholars have recently theorized that upwardly mobile immigrants and their children, like Mexicans, may incorporate into a minority middle-class culture. The goal of this study is to examine the trajectories of upwardly mobile Mexicans to determine whether they are incorporating as middle-class whites or as middle-class minorities. The research studies three distinct measures of incorporation: patterns of giving back to coethnics; participation in voluntary associations among the Mexican middle-class; and racial/ethnic identification. To answer the research questions at hand, the study draws on multiple methods: in-depth structured interviews with members of the Mexican-origin middle-class; participant and nonparticipant observation of a Latina professional organization; and analysis of the National Survey of Family and Households.

This study contributes to both the immigration and race/ethnicity literatures in a number of important ways. First, the research remedies the shortcoming in Portes and Zhou's (1993) theory of segmented assimilation, and also tests Neckerman, Carter, and Lee's (1999) theory of the minority culture of mobility that proposes that immigrants and their children may take multiple pathways into the middle-class. Second, the research examines whether the Mexican-origin middle-class displays patterns of giving back that mirror the collectivist orientation exhibited by African Americans, or whether they more closely resemble the individualistic orientation exhibited by middle-class whites. Third, the research goes beyond the study of second-generation adolescents and focuses on adults. Finally, the study moves beyond the traditional binary black/white paradigm by examining the racial/ethnic identity options and constraints among Mexicans in the United States, who identity neither as simply black nor white. More broadly, the study will provide insight into the economic and sociocultural incorporation processes of one and a half, second, and later generations of the Mexican origin middle-class population. The study has relevance for public policy since it offers an in-depth understanding of a population that is often touted as unassimalable, vulnerable to downward mobility, and likely to remain poor for generations to come.

We have carried out detailed investigations of membrane interactions and amyloid formation of alpha-syn that have provided residue-specific information and molecular insights into the mechanism of aggregation. Due to the complexity of the amyloid problem, the tools with which we attack have included molecular biology, steady-state and time-resolved fluorescence spectroscopy, nuclear magnetic resonance spectroscopy, electron microscopy, neutron reflectometry, and mass spectrometry. We are developing a chemical understanding in how specific phospholipids modulate protein structure, membrane binding, and aggregation propensity through different studies summarized below. In relating to the complex cellular lipid compositions, we are focusing towards understanding the effects of bilayer fluidity and phase state by changes in acyl chain length as well as chain saturation. Specifically, we have studied the effect of phosphatidylcholine (PC) membrane fluidity on the conformation and aggregation propensity of the physiologically relevant N-terminally acetylated (N-acetyl) alpha-syn. Using CD spectroscopy, we show that N-acetyl alpha-syn transitions from alpha-helical to disordered at the lipid melting temperature. We found that this fluidity sensing is a robust characteristic, unaffected by acyl chain length and preserved in its homologs beta- and gamma-syn. Interestingly, both N-acetyl alpha-syn membrane binding and amyloid formation trended with lipid order with gel-phase vesicles shortening aggregation kinetics and promoting fibril formation compared with fluid membranes. Furthermore, we found that acetylation enhances binding to PC micelles and small unilamellar vesicles with high curvature. Cholesterol concentration dependence results confirmed that the exposure of hydrocarbon chains (i.e. packing defects) is essential for binding to zwitterionic gel membranes. Collectively, our in vitro results suggest that N-acetyl alpha-syn localizes to highly curved, ordered membranes inside a cell. We propose that age-related changes in membrane fluidity can promote the formation of amyloid fibrils, insoluble materials associated with PD. To understand how membrane remodeling by alpha-syn affects amyloid formation, we have studied the alpha-syn aggregation process in the presence of phosphatidylglycerol (PG) micellar tubules, which were the first reported example of membrane tubulation by alpha-syn. Aggregation kinetics, beta-sheet content, and macroscopic protein-lipid structures were observed by Thioflavin T fluorescence, circular dichroism spectroscopy and transmission electron microscopy, respectively. Collectively, the presence of PG micellar tubules formed at a stochiometric ratio was found to stimulate alpha-syn fibril formation. Moreover, transmission electron microscopy and solid-state nuclear magnetic resonance spectroscopy revealed the co-assembly of PG and alpha-syn into fibril structures. However, isolated micellar tubules do not form fibrils by themselves, suggesting an important role of free alpha-syn monomers during amyloid formation. In contrast, fibrils did not form in the presence of excess PG lipids, where most of the alpha-syn molecules are in a membrane-bound alpha-helical form. Our results provide new mechanistic insights into how membrane tubules modulate alpha-syn amyloid formation and support a pivotal role of proteinlipid interaction in the dysfunction of alpha-syn.

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate excitatory signaling in the central nervous system. Upon ligand binding to the extracellular domain of iGluRs local conformational changes ensue and this motion is translated to the transmembrane domain inducing channel opening. We have used an isolated ligand binding domain (LBD), GluR2-S1S2J (GluR2), as a model system to study the protein-ligand complex. Using time-resolved fluorescence and anisotropy measurements, we characterized the excited state properties and local mobility of Trp residues in the isolated LBD, GluR2. Specifically, we determined that the widely used and structurally characterized antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX) acts as an efficient fluorescence energy transfer (FET) acceptor for Trp. Consistent with crystallographic data, our results indicate that the four native tryptophans are within Forsters radius (33 angstroms ) of the bound ligand. Additionally, we demonstrate the broader value of this technique by identifying an original FET ligand, 3-nitrotyrosine (3NY) for GluR2 (24 angstroms , apparent dissociation constant, Kd of approximately 170 micromolar). Estimated average donor-acceptor (Trp-to-ligand) distances extracted from tryptophan excited-state decays are similar for both ligands (24 angstroms) suggesting that 3NY binds in the structurally characterized ligand-binding cleft. Interestingly, we observe multiple rate components for 3NY suggestive of ligand-protein complex structural heterogeneity. However, due to the presence of multiple Trp donors we are only able to estimate average DNQX/3NY distances to the Trp residues. While the distance extracted (24 angstroms) from our analysis on DNQX-Trp GluR2 are on the order of the crystallographically determined distances (12-20 angstroms), they are somewhat longer. This may be due to the current limit of resolvable rates (tau of 0.5 ns) which would correspond to donor-acceptor distances shorter than those estimated for Trp-DNQX or it is possible that it is attributable to unfavorable orientations of the Trp and DNQX transition dipoles in the folded protein. Alternatively, our longer distances may simply reflect dynamical properties of the protein in solution. While DNQX is a well-known complex, its spectroscopic properties and potential application as a sensitive reporter for ligand binding to GluR2 have been overlooked previously. We have described a DNQX competition assay that represents a significant improvement over the fluorescence assays currently in use in that this is a turn-on sensor, eliminating false positive observations inherent to quenching events resulting from photobleaching. The use of FET competitor ligands and intrinsic Trp fluorescence should allow for the future identification of novel ligands for not only for GluR2 but also for other proteins when DNQX or structural analogs (i.e. CNQX and other nitrated aromatic compounds) are employed.

Although recent work points to a genetic component to PD involving the accumulation and deposit of a neuronal protein, alpha-syn, the sporadic form of the disease is far more common and possibly connected to environmental factors that promote oxidative stress and aberrant redox-active metal metabolism. For example, selective accumulation of fibrils in dopaminergic neurons in PD has been attributed to the presence of easily oxidizable catechols that stimulate protein cross-links, as well as to increased iron concentration in Lewy bodies and copper in cerebrospinal fluid of PD patients. Furthermore, metal-enhanced oxidative oligomerization has been observed for alpha-syn in vitro, and, specific metal-protein interactions have been proposed to be critical in other neurodegenerative diseases involving amyloidogenic biomolecules such as amyloid beta-peptide (Alzheimers disease), prion protein (spongiform encephalopathies), and superoxide dismutase (amyotrophic lateral sclerosis). A difficult issue to resolve is whether metal ions perturb protein structures and thereby alter functions, or whether metal-protein complexes directly participate in the production of reactive oxygen species, or whether both mechanisms are at work. Copper(II) Enhances Membrane-bound alpha-Synuclein Helix Formation We have examined the effect of copper(II) on alpha-syn/lipid interactions and to test whether the phospholipid vesicles can modulate the copper(II) binding properties. Copper(II) is known to coordinate to soluble alpha-syn within the first four residues (MDVF) through the N-terminal amine and backbone amide chelation (Jackson MS, Lee JC Inorg. Chem. 2009, 48, 9303-9307). Importantly, the N-terminal region is also anticipated to interact electrostatically with cellular membranes through seven imperfect amino acid repeats (KXKEGV) in the primary amino acid sequence. We show that copper(II) binding to the N-terminus of alpha-syn affects its helical propensity and moreover, our results indicate that the copper(II) binding affinity of F4W alpha-syn is enhanced when the protein is membrane-bound. Interestingly, this work demonstrates that N-terminal membrane association is a dynamic process at least in regards to the copper-binding site penetrating the vesicle surface (Lucas HR, Lee JC, Metallomics 2011, 3, 280-283). Role of Asp2 in the Primary Copper (II) Binding Site in alpha-Synuclein Tryptophan fluorescence measurements involving mutant alpha-synucleins (F4W and F4W/H50S) and comparisons to synthetic N-terminal peptides suggest that the primary copper(II) binding site is localized in the first four residues. Moreover, the alpha-amino terminus is required for Cu(II) binding. Using site-directed mutagenesis (D2N/F4W and D2E/F4W), we are probing the nature of the copper(II)-D2 interaction showing that D2 is an unlikely copper(II) ligand. Insights on the molecular details of copper coordination environment also are gained through circular dichroism analyses and the use of synthetic peptide models. Coordination Properties of Cu(I) Bound alpha-Synuclein In prior work, structural information on copper coordination environment was obtained through X-ray absorption spectroscopy (Lucas, HR, DeBeer, S, Hong, M-S, Lee JC, J. Am. Chem. Soc. 2010, 132, 6636-6637). Particularly, we have characterized the local conformational changes in both soluble and fibrillar Cu-alpha-syn forms using Cu K-edge and extended X-ray fine structure absorption spectroscopy and have shown that there is Cu(I)/dioxygen reactivity during amyloid formation. Current work is focused on studying directly the ability of alpha-syn to bind copper(I), the physiologically relevant metal oxidation state.

Pmel17 fibrils serve as the structural scaffolding required for melanin deposition in human skin and eyes. Melanin is synthesized in melanosomes, organelles related to both endosomes and lysosomes, and stored in melanocytes, cells responsible for pigmentation. While the melanosome maturation process has been shown to involve four distinct stages that have been characterized in detail at the ultrastructural level by transmission electron microscopy (TEM), the molecular nature of the intralumenal Pmel17 fibrils during each of these stages is not known. Moreover, which polypeptide domain solely or partly constitutes the amyloid core of the Pmel17 filaments also remains to be defined. We have begun to study the repeat domain (RPT, residues 315-444), an essential luminal polypeptide region of Pmel17, as a model system. To mimic the changing acidic pH conditions of the maturing melanosome, we measured RPT amyloid formation kinetics as a function of solution pH. Since tryptophan emission is highly sensitive to solvent polarity, local conformational changes, and protein-protein interactions, we exploited the only intrinsic tryptophan (Trp423) as a site-specific fluorescent probe of amyloid structure and aggregation kinetics. We find that Trp423 is exquisitely sensitive to soluble and fibrillar RPT conformation with spectral properties (intensity and mean wavelength) exhibiting distinct temporal changes under the various solution conditions examined. Complementary techniques, TEM and circular dichroism spectroscopy, also are employed to characterize fibrillar ultrastructures and secondary structural content, respectively.

DESCRIPTION (provided by applicant): Young women have much lower rates of cardiovascular disease (CVD, including stroke) than men. However, as midlife women transition to post-menopause, they lose this 'cardiovascular protection,'and CVD is most common in post-menopause than any other stage of a woman's lifespan. Metabolic Syndrome (MetS) is a clustering of 5 metabolic abnormalities and is a major predictor of CVD and type 2 diabetes. MetS is clinically diagnosed as having any, and at least, 3 of the 5 components. MetS occurrence increases during the menopausal transition (MT). Reasons for this are unclear;however this may be due to androgen excess, relative to estrogen, during the MT. The proposal's goal is to establish basic aspects of how the constellations of the MetS components evolve during the course of the MT, a key 5- to 10-year biological stage in a woman's lifespan. In turn, this is intended to identify customized ways of preventing MetS early and related CVD and diabetes, with effective intervention strategies during the MT. The proposal incorporates a shift in our thinking of menopause and sex hormones in midlife women, namely, that the increase in MetS occurrence may be due more to androgen gain (and less to estrogen loss), in the MT. Our starting hypothesis is that mapping the constellations of MetS components, and the number of MetS components satisfied, in the midlife will provide a window into bridging the MT, its changing sex hormones, and loss of CV protection in women. If correct, this would shift our clinical focus to individualize hormone strategies against characteristic MetS constellations and related CVD and diabetes in midlife and early post-menopausal women. Aim 1. To characterize the constellations of MetS components satisfied over time in women, of 5 race/ethnicities, who develop MetS as they undergo the MT. Aim 2. To determine the hormonal and inflammatory factors that predict the course of MetS constellations in midlife women as they undergo the MT. We propose an efficient study that analyzes unique, existing longitudinal data from the largest U.S. study of the MT, the Study of Women Across the Nation (SWAN), a multi-ethnic cohort of 3302 women. Our long-term objective is to prevent the dramatic increase in CVD in older women by implementing, in midlife, individualized preventative strategies. Both our aims bear directly on this wider objective. These would impact 60+ million midlife and older U.S. women. PUBLIC HEALTH RELEVANCE: The Metabolic Syndrome (MetS) is the major predictor for both CVD and type 2 diabetes, and can manifest as having one of several constellations of at least 3 of 5 risk components, such as high blood pressure and large waist girth. MetS occurs more often in women undergoing the menopausal transition, a key 5- to 10-year biological stage in a woman's lifespan. This study seeks to enhance significantly our understanding of how women in their 40s and early 50s, over time, develop MetS and hormonal changes and other factors during the menopausal transition that contribute to their increased risk of MetS. With this better understanding, we intend to ultimately prevent MetS in women who are high risk due to identifiable and modifiable characteristics of their transition to post-menopause. This potentially impacts tens of millions of midlife women and their families who face a nearly 1 in 3 chance of developing MetS in their remaining lifetime.

Mutations in the GBA1 gene, encoding the enzyme glucocerebrosidase (GCase), cause the lysosomal storage disorder, Gaucher disease (GD), and are associated with the development of Parkinsons disease (PD) and other Lewy body disorders. Interestingly, GBA1 variants are the most common genetic risk factor associated with PD. While clinical studies argue a strong case towards a link between GBA1 mutations and the development of PD, mechanistic insights have been lacking. Recent research suggests a relationship between GCase and the PD-related amyloid-forming protein, alpha-synuclein; however, the specific molecular mechanisms responsible for association remain elusive. While the effects of GCase on alpha-synuclein homeostasis are the subject of considerable work, a role for alpha-synuclein in enzyme function has not been established. Such information could have further implications and indicate other mechanisms responsible for the increased PD risk. In prior work, we showed that alpha-synuclein and GCase interact selectively under lysosomal conditions, and proposed that this newly identified interaction might influence cellular levels of alpha-synuclein by either promoting protein degradation and/or inhibiting aggregation. We now extend our study to consider how membranes would modulate this complex formation. Lipids function not only as the substrate for GCase in intralysosomal vesicles, but also a surface for alpha-synuclein conformation alteration. For the first time, we demonstrate that the two proteins associate both on and off the membrane with comparable affinity, with apparent dissociation constants in the micromolar range. Using site-specific fluorescence and Forster energy transfer probes, we mapped the protein-enzyme interacting regions on unilamellar vesicles. Our data suggest that on the membrane surface, the GCase-alpha-synuclein interaction involves a larger alpha-synuclein region compared to that found in solution. Binding of alpha-synuclein to the membrane is critical for complex formation. In the absence of the N-terminal membrane binding domain, alpha-synuclein and GCase do not associate on the membrane, whereas in solution, this complex stays intact. Due to the likelihood of electrostatic repulsion between the negatively charged membrane surface and the acidic C-terminal tail, we suggest that at least some of the N-terminal membrane-binding residues, 1-95, are required to anchor alpha-synuclein to the membrane, thereby promoting and/or stabilizing its binding to GCase at the C-terminal. Despite the current interest in defining the role of GCase in PD, our study is the first to investigate whether alpha-synuclein has a direct effect on GCase function. We show that alpha-synuclein is a potent GCase inhibitor (an apparent IC50 in the submicromolar range) only when it adopts an alpha-helical conformation, supporting the concept that not only is membrane binding critical in modulating activity, but it also affects the specific conformational change of alpha-synuclein upon lipid and enzyme association. The observed mixed mode of inhibition suggests that at the membrane interface, alpha-synuclein binding influences both substrate accessibility and turnover. This study supports the notion that GCase deficiency can contribute to the pathogenesis of the synucleinopathies. The observed interplay between alpha-synuclein-GCase association and activity can be a potential self-perpetuating mechanism connecting enzyme deficiency and the accumulation of alpha-synuclein and substrate. This link between membrane-bound alpha-synuclein and GCase could be one of many possible mechanisms that connect GD and PD. However, since only a minority of GD patients or carriers develops PD, other pathological factors are likely involved. Nevertheless, it is now possible to search for proteins and other molecules that could modulate this alpha-synuclein-GCase interaction and to evaluate their effects on enzyme activity.

DESCRIPTION (provided by applicant): This K08 proposal describes a 5-year training program for Dr. Jennifer Lee. In this program, Dr. Lee will acquire laboratory and research skills that are essential for her success in academic medicine to enable her to obtain independent NIH funding in pediatric neuroprotection research. Candidate and Career Development Plan: Dr. Jennifer Lee, a pediatric anesthesiologist and pediatrician with formal training in pediatric critial care medicine, has studied cerebrovascular autoregulation in experimental neonatal models under the mentorship of Dr. Raymond Koehler since 2006. More recently, she extended her work to evaluate autoregulation in a model of pediatric hypoxic-asphyxic (HA) cardiac arrest, therapeutic hypothermia, and rewarming. She has translated her laboratory work to patient care by leading several clinical studies involving autoregulation monitoring and neurologic outcomes in neonates with hypoxic-ischemic encephalopathy (HIE), children resuscitated from cardiac arrest, and children undergoing neurosurgery. Thus far, her laboratory research has focused primarily on physiologic experiments of vascular reactivity. However, she has not had the opportunity to take formal coursework or study the biochemical, cellular, and mechanistic effects of therapeutic hypothermia and rewarming after HA brain injury. To continue her development as a clinician-scientist, Dr. Lee intends to study neuronal and oligodendrocyte cell death pathways, the unfolded protein response (UPR) that occurs with endoplasmic reticulum stress, oxidative stress, inflammation, and cortical injury after delayed hypothermia and different rates of rewarming in a clinically relevant model of HA cardiac arrest. This work will enable her to obtain more advanced technical skills that will make her more competitive in basic science research. Her coursework is timed to parallel her laboratory skill development and to address each Aim in her proposal. With the proposed career development and research plans, Dr. Lee will learn the skills necessary to become a successful clinician-scientist with independent NIH funding. Environment: The Johns Hopkins University (JHU) Department of Anesthesiology and Critical Care Medicine is well equipped to support Dr. Lee's research and career development with the K08 award. The Department is currently supporting her with 75% protected, non-clinical time for research. With the K08 award, she will continue to have this protected time. Her co-mentors, Dr. Raymond Koehler and Dr. Lee Martin, are world- renowned experts in cerebrovascular physiology, neuroprotection after hypoxic-ischemic injury, cell death mechanisms, and cellular neuroscience; they have long histories of NIH funding and successful mentoring of clinician-scientists. Dr. Lee also has an experienced, multidisciplinary advisory committee of experts in neuronal cell death mechanisms, hypoxic brain injuries, therapeutic hypothermia/rewarming, and pediatric resuscitation medicine to guide and support her research and career development. All equipment necessary to carry out the proposed experiments is accessible within her department. She will have the full support of her mentors, laboratory manager, and technicians to learn and perform the laboratory techniques outlined in the research plan. JHU has provided Dr. Lee with an environment that will ensure her success in the K08 program. Research Project: Despite the use of therapeutic hypothermia, neurologic morbidity remains high in neonatal HIE and after pediatric cardiac arrest. Experimental models of HA cardiac arrest have shown that therapeutic hypothermia decreases neuronal death from necrosis. However, preliminary laboratory data and clinical studies suggest that rewarming from hypothermia may shift neuronal and oligodendrocyte cell death from necrotic to apoptotic pathways and increase oxidative stress and inflammation, thus raising the risk of secondary brain injury. Moreover, preliminary data indicate that activation of the UPR from endoplasmic reticulum stress after a hypoxic insult may promote apoptosis in the white matter yet be neuroprotective in other regions of the brain. Aim 1 will determine whether rewarming and different rates of rewarming increase cortical neuroapoptosis, oligodendrocyte apoptosis, oxidative stress, inflammatory markers, seizures, and cortical injury after HA injury. Aim 2 will determine if caspase-3 inhibition prevents neuronal and oligodendrocyte cell death during rewarming, thus providing an adjuvant neuroprotective therapy. Aim 3 will determine if UPR activation from rewarming after HA injury induces white matter apoptosis but is neuroprotective in other anatomic regions; and whether modulation of the UPR response influences neuroprotection. Findings from this project will have important implications for pediatric and neonatal resuscitation guidelines. They will also provide a basis for further investigations of adjunct therapies to reduce the adverse effects of rewarming after pediatric cardiac arrest and HIE.

SES-1558986
Karthick Ramakrishnan
University of California-Riverside

Asian Americans are the fastest-growing racial group in the United States, and there are important gaps in our understanding of their social and civic integration. In addition, Asian Americans exhibit significant socioeconomic diversity, making them ideal for answering larger theoretical questions on immigrant incorporation in the United States, as it is shaped by differences in national origin, residential concentration, and educational and labor market outcomes. This project will build a data resource for public release in June 2017 that will have widespread utility among scholars in social science disciplines, most notably in sociology, political science, and public policy, as well for journalists and bloggers versed in social science data and trends. The survey of Asian Americans will provide important data for questions related to immigrant incorporation, attitudes on economic inequality and race-relations, and civic and political engagement. In addition to providing comparisons within the Asian American racial category, we will include cross-racial comparisons (to African Americans, Hispanics/Latinos, and non-Hispanic whites) using the same instrument and mode of interview. This project advances the mission of the National Science Foundation to promote the progress of science, particularly the social sciences, as well as to advance national health, prosperity, and welfare. In particular, this project has relevance in terms of broader impacts as specified by Congress in 2010: Asian Americans have a disproportionately large number of high-skilled workers in STEM fields. Understanding their experiences, attitudes, opportunities, and barriers, will be critical to ensuring the economic competitiveness of the United States and the development of a globally competitive STEM workforce

This survey of Asian Americans will: 1) be contemporaneous with the 2016 General Social Survey (GSS) and American National Election Study (ANES), 2) replicate key questions from those surveys, and 3) build on the pioneering efforts of past surveys of Asian Americans. The project produces a dataset for public release in June 2017 through ICPSR (Inter-university Consortium for
Political and Social Research) that will have widespread utility among scholars in social science disciplines as well for journalists and bloggers versed in social science data and trends. The survey will provide data to address questions related to immigrant incorporation, attitudes on economic inequality and race-relations, and civic and political engagement. In addition to providing comparisons within the Asian American racial category (samples of approximately 400 adults each among major national origin groups), it will include cross-racial comparisons using the same instrument and mode of interview (approximately 400 adults each among whites, Latinos, and African Americans).

A growing number of studies focus on Asian Americans. However, existing surveys tend to be limited to a small number of national origin groups or to a relatively specific geographic area. More general studies such as the GSS and ANES do not have the requisite sample size or language support to accurately capture the experiences and attitudes of Asian Americans. The primary innovation of the 2016 National Asian American Survey is to replicate questions on important general surveys, specifically the GSS and the ANES, with a large sample of Asian Americans in a well-designed study. With this commitment to question replication, the 2016 National Asian American Survey will allow researchers, for the first time, to compare Asian Americans to the U.S. public on key social and political measures, and to make important comparisons within the racial group.

Project summary: We propose to establish an integrated MR imaging and analysis platform to examine hypoxic-ischemic (HI) brain injury in a neonatal piglet model, and to develop novel MRI markers that characterizes the evolving cellular pathology during injury progression. While MRI has been used extensively in HI, image interpretation and predictive accuracy of the conventional MRI markers, such as T1 and T2-weighted MRI or diffusion MRI (dMRI), leave much to be desired. In this project, we will develop microstructural MRI markers using the diffusion-time (td) dependent dMRI, which potentially improves the sensitivity and specificity of identifying cellular injury in HI. td-dMRI will be achieved by measuring water diffusivity at varying td's, using an oscillating gradient spin-echo (OGSE) dMRI sequence, to determine the td-dependency, which reflects the cell morphology. In a mouse model of neonatal HI, we have demonstrated that td-dMRI is sensitive to small microstructural changes in cells and subcellular organelles during early injury, and such microstructural details are not accessible by conventional dMRI. Here we will use a clinically-relevant piglet model of whole-brain HI, which exhibits well-defined phenotypes of gray and white matter injury that corresponds to human full-term newborns with birth hypoxia. Development of MRI markers in this model and investigation of the neuropathological substrates of the new MRI markers will have a high clinical impact. Clinical translation of td- dMRI, however, is challenging due to the gradient system on clinical scanners that limits the attainable td and detectable microstructural resolution. We will develop novel OGSE sequences to address the gradient limitation and evaluate the clinical potentials of td-dMRI using the piglet model. The study will be performed on 3T human scanners, and therefore, the MRI techniques will be readily translatable to clinical realm. We hypothesize that td-dMRI is sensitive to acute swelling of neurons and organelles after HI, and that early dMRI measures are predictive of long-term neuropathologic and neurologic outcomes. In Aim 1, we will build a piglet MRI platform with multi-metric MRI markers, including volumetric measures, high-order dMRI (DTI, DKI, tractography), magnetic transfer imaging, along with td-dMRI measures. We will also establish atlases of the developing piglet brains and atlas-based image analysis to achieve automated quantification of the multi-metric MRI data. In Aim 2, we will investigate the utility of td-dMRI in detecting microstructural injury during acute and subacute HI (6hrs - 7days), and explore the correlations between the early MRI markers with cellular and subcellular organelle pathology. In Aim 3, we will perform multimetric MRI to follow the injury progression in piglets over 30 days of recovery after HI, and evaluate the relations between early neuronal injury and white matter injury in the connecting tracts, as well as the long-term functional outcome with neurobehavioral tests. The MRI markers developed in this study will potentially improve the diagnosis and prognosis in clinical neonatal HI, which may lead to accurate and noninvasive evaluations of adjuvant therapies in these neonates.

We are developing Raman microspectroscopy for characterizing amyloid formation of alpha-syn, which is implicated in Parkinsons disease. This direct spectroscopic method reports on intrinsic molecular vibrations such as protein amide bonds, which arise from coupled vibrational modes of the polypeptide backbone. The position and widths of the amide bands depend on the peptide-bond angles and hydrogen-bonding patterns, and therefore, inform on protein secondary structure as well as local environment. Conformations of alpha-helix, beta-sheet, or random coil exhibit characteristic peak maxima, making quantification of structural compositions possible. To demonstrate the utility of Raman spectroscopy as an effective probe of amyloid structure, we examined the effects of pH and ionic strength as well as four PD-related mutations (A30P, E46K, G51D, and A53T) on alpha-syn fibrils. Raman spectral differences were observed in the amide-I, amide-III, and fingerprint regions, indicating secondary structure and tertiary contacts are influenced by pH and to a lesser extent by NaCl. Faster aggregation times appear to facilitate unique fibril structure as determined by the highly reproducible amide-I band widths, linking aggregation propensity and fibril polymorphism. Importantly, Raman spectroscopy revealed molecular-level perturbations of fibril conformation by the PD-related mutations that are not apparent through TEM or limited proteolysis. The amide-III band was found to be particularly sensitive, with G51D exhibiting the most distinctive features, followed by A53T and E46K. Relating to a cellular environment, our data would suggest that fibril polymorphs can be formed in different cellular compartments and potentially result in distinct phenotypes. In expanding our studies, we have coupled the Raman spectrometer with an inverted microscope, which yields both chemical and spatial information within macroscopic amyloid aggregates. Importantly, for the first time we directly compare intrinsic vibrations in pathological versus functional amyloids, further developing our understanding of structural features that define these two amyloid classes. Specifically, we investigated three pathological and two functional amyloids. For pathological amyloids, we chose to study (1) N-terminal acetylated alpha-syn, which is implicated in Parkinsons disease, (2) amyloid beta (1-40), a peptide important in the development of Alzheimers disease and one of the best characterized amyloids to date, and (3) apolipoprotein C-III, an apolipoprotein linked to cardiovascular disease. While previous studies have shown fibrillar alpha-synuclein and Abeta(1-40) form parallel in-register beta-sheets, there is no reported high-resolution structure of apolipoprotein C-III amyloid, which has an atypical ribbonlike morphology, in contrast to the other straight filaments. We also studied two functional amyloids: (1) the repeat domain (residues 315 to 444) of Pmel17, which is important in melanin biosynthesis and suggested to also form parallel in-register -sheet fibrils that are highly pH-dependent and reversible and (2) the prion-domain (residues 218 to 289) of the fungal protein Het-s, which forms a well-defined and highly uniform beta-solenoid conformation. As spontaneous Raman spectra have never been collected for apolipoprotein C-III or repeat domain of Pmel17, our work expanded the use of vibrational spectroscopy for amyloid characterization.

Neonatal hypoxic-ischemic encephalopathy (HIE) from birth asphyxia causes persistent and severe neurologic disabilities, even in patients who receive therapeutic hypothermia. We found in clinical studies that white matter injury on MRI persists after hypothermic treatment. Thus, hypothermia is not fully protective. White matter injury is a prominent yet understudied component of the neurologic disabilities observed in neonates who receive hypothermia for HIE. Therapeutic adjuncts that protect the white matter might reduce the risk of permanent neurologic injury in HIE. Hypoxia-ischemia (HI) in neonatal pig, which has human-like white matter tracts, produces brain damage similar to that of full-term human newborns with HIE, including the white matter injuries observed in clinical studies. Our model includes clinically relevant whole-body hypothermia, rewarming at 0.5°C/h, sedation, continuous hemodynamic monitoring, ventilator support, and correction of blood gas and electrolyte abnormalities to mimic clinical neonatal intensive care. Preliminary data suggest that insufficient proteasome function mediates persistent white matter injury after HI and hypothermia. We postulate that white matter proteasome insufficiency causes a failure to clear oxidatively damaged proteins, causing oligodendrocyte apoptosis, potential disruption of oligodendrocyte precursor maturation, myelin and axonal injury, and white matter volume loss after HI and hypothermia. We will elucidate the proteasome?s role in white matter injury after whole-body HI and overnight hypothermia in neonatal swine. White matter injury and oligodendrocyte biology will be studied with neuropathology (including oligodendrocyte precursor maturation, stereology, cell death, and electron microscopy) and biochemistry (including protein post-translational modification and proteasome composition and activity) through 1 month recovery after HI. T- maze neurocognitive behavior testing with neuropathology correlation will provide a functional outcome. We developed new methods to genetically modulate proteasome activity in distinct, targeted regions of white matter in neonatal pig forebrain using virus-mediated enforced expression of a proteasome activator subunit or proteasome inhibition with short hairpin small interfering RNA. We will also use a small molecule proteasome inhibitor to determine whether proteasome inhibition aggravates white matter injury. Moreover, we will test the potential of the drug oleuropein to protect white matter. Oleuropein is a readily bioavailable compound with proteasome activating properties and few clinical side effects. An intravenous oleuropein dosing regimen will be used that protects oligodendrocytes and myelin, increases proteasome expression, and promotes clearance of oxidized proteins after HI and hypothermia. We will identify whether oleuropein acts on the standard proteasome or the immunoproteasome. Cultured human oligodendrocyte experiments will validate the proteasome as a therapeutic target and oleuropein?s actions after oxygen glucose deprivation. This project will advance the neonatal HI and cell biology fields by investigating novel mechanisms by which proteasome insufficiency mediates hypothermia-resistant injury in white matter. We will discover whether proteasome activation is a relevant therapeutic adjunct to hypothermia to protect white matter and improve neurologic outcomes in HIE.