First in-human evaluation of [1-13C]pyruvate in D2O for hyperpolarized MRI of the brain: A safety and feasibility study.

PURPOSE: To investigate the safety and value of hyperpolarized (HP) MRI of [1-13C]pyruvate in healthy volunteers using deuterium oxide (D2O) as a solvent. METHODS: Healthy volunteers (n = 5), were injected with HP [1-13C]pyruvate dissolved in D2O and imaged with a metabolite-specific 3D dual-echo dynamic EPI sequence at 3T at one site (Site 1). Volunteers were monitored following the procedure to assess safety. Image characteristics, including SNR, were compared to data acquired in a separate cohort using water as a solvent (n = 5) at another site (Site 2). The apparent spin-lattice relaxation time (T1) of [1-13C]pyruvate was determined both in vitro and in vivo from a mono-exponential fit to the image intensity at each time point of our dynamic data. RESULTS: All volunteers completed the study safely and reported no adverse effects. The use of D2O increased the T1 of [1-13C]pyruvate from 66.5 ± 1.6 s to 92.1 ± 5.1 s in vitro, which resulted in an increase in signal by a factor of 1.46 ± 0.03 at the time of injection (90 s after dissolution). The use of D2O also increased the apparent relaxation time of [1-13C]pyruvate by a factor of 1.4 ± 0.2 in vivo. After adjusting for inter-site SNR differences, the use of D2O was shown to increase image SNR by a factor of 2.6 ± 0.2 in humans. CONCLUSIONS: HP [1-13C]pyruvate in D2O is safe for human imaging and provides an increase in T1 and SNR that may improve image quality.

The Iron Curtain: Macrophages at the Interface of Systemic and Microenvironmental Iron Metabolism and Immune Response in Cancer.

Macrophages fulfill central functions in systemic iron metabolism and immune response. Infiltration and polarization of macrophages in the tumor microenvironment is associated with differential cancer prognosis. Distinct metabolic iron and immune phenotypes in tumor associated macrophages have been observed in most cancers. While this prompts the hypothesis that macroenvironmental manifestations of dysfunctional iron metabolism have direct associations with microenvironmental tumor immune response, these functional connections are still emerging. We review our current understanding of the role of macrophages in systemic and microenvironmental immune response and iron metabolism and discuss these functions in the context of cancer and immunometabolic precision therapy approaches. Accumulation of tumor associated macrophages with distinct iron pathologies at the invasive tumor front suggests an "Iron Curtain" presenting as an innate functional interface between systemic and microenvironmental iron metabolism and immune response that can be harnessed therapeutically to further our goal of treating and eliminating cancer.

CTLA-4 blockade drives loss of Treg stability in glycolysis-low tumours.

Limiting metabolic competition in the tumour microenvironment may increase the effectiveness of immunotherapy. Owing to its crucial role in the glucose metabolism of activated T cells, CD28 signalling has been proposed as a metabolic biosensor of T cells1. By contrast, the engagement of CTLA-4 has been shown to downregulate T cell glycolysis1. Here we investigate the effect of CTLA-4 blockade on the metabolic fitness of intra-tumour T cells in relation to the glycolytic capacity of tumour cells. We found that CTLA-4 blockade promotes metabolic fitness and the infiltration of immune cells, especially in glycolysis-low tumours. Accordingly, treatment with anti-CTLA-4 antibodies improved the therapeutic outcomes of mice bearing glycolysis-defective tumours. Notably, tumour-specific CD8+ T cell responses correlated with phenotypic and functional destabilization of tumour-infiltrating regulatory T (Treg) cells towards IFNγ- and TNF-producing cells in glycolysis-defective tumours. By mimicking the highly and poorly glycolytic tumour microenvironments in vitro, we show that the effect of CTLA-4 blockade on the destabilization of Treg cells is dependent on Treg cell glycolysis and CD28 signalling. These findings indicate that decreasing tumour competition for glucose may facilitate the therapeutic activity of CTLA-4 blockade, thus supporting its combination with inhibitors of tumour glycolysis. Moreover, these results reveal a mechanism by which anti-CTLA-4 treatment interferes with Treg cell function in the presence of glucose.

Multiparametric classification of sub-acute ischemic stroke recovery with ultrafast diffusion, 23 Na, and MPIO-labeled stem cell MRI at 21.1 T.

MRI leverages multiple modes of contrast to characterize stroke. High-magnetic-field systems enhance the performance of these MRI measurements. Previously, we have demonstrated that individually sodium and stem cell tracking metrics are enhanced at ultrahigh field in a rat model of stroke, and we have developed robust single-scan diffusion-weighted imaging approaches that utilize spatiotemporal encoding (SPEN) of the apparent diffusion coefficient (ADC) for these challenging field strengths. Here, we performed a multiparametric study of middle cerebral artery occlusion (MCAO) biomarker evolution focusing on comparison of these MRI biomarkers for stroke assessment during sub-acute recovery in rat MCAO models at 21.1 T. T2 -weighted MRI was used as the benchmark for identification of the ischemic lesion over the course of the study. The number of MPIO-induced voids measured by gradient-recalled echo, the SPEN measurement of ADC, and 23 Na MRI values were determined in the ischemic area and contralateral hemisphere, and relative performances for stroke classification were compared by receiver operator characteristic analysis. These measurements were associated with unique time-dependent trajectories during stroke recovery that changed the sensitivity and specificity for stroke monitoring during its evolution. Advantages and limitations of these contrasts, and the use of ultrahigh field for multiparametric stroke assessment, are discussed.

Nestin+NG2+ Cells Form a Reserve Stem Cell Population in the Mouse Prostate.

In the prostate, stem and progenitor cell regenerative capacities have been ascribed to both basal and luminal epithelial cells. Here, we show that a rare subset of mesenchymal cells in the prostate are epithelial-primed Nestin-expressing cells (EPNECs) that can generate self-renewing prostate organoids with bipotential capacity. Upon transplantation, these EPNECs can form prostate gland tissue grafts at the clonal level. Lineage-tracing analyses show that cells marked by Nestin or NG2 transgenic mice contribute to prostate epithelium during organogenesis. In the adult, modest contributions in repeated rounds of regression and regeneration are observed, whereas prostate epithelial cells derived from Nestin/NG2-marked cells are dramatically increased after severe irradiation-induced organ damage. These results indicate that Nestin/NG2 expression marks a novel radioresistant prostate stem cell that is active during development and displays reserve stem cell activity for tissue maintenance.

Author Correction: Adrenergic nerve degeneration in bone marrow drives aging of the hematopoietic stem cell niche.

In the version of this article originally published, the key for Fig. 4c was incorrect. The symbols for 'Sham' and 'Den' were reversed. The error has been corrected in the PDF and HTML versions of the manuscript.

Imaging endogenous macrophage iron deposits reveals a metabolic biomarker of polarized tumor macrophage infiltration and response to CSF1R breast cancer immunotherapy.

Iron deposits are a phenotypic trait of tumor-associated macrophages (TAMs). Histological iron imaging and contrast-agent free magnetic resonance imaging (MRI) can detect these deposits, but their presence  in human cancer, and correlation with immunotherapeutic response is largely untested. Here, primarily using these iron imaging approaches, we evaluated the spatial distribution of polarized macrophage populations containing high endogenous levels of iron in preclinical murine models and human breast cancer, and used them as metabolic biomarkers to correlate TAM infiltration with response to immunotherapy in preclinical trials. Macrophage-targeted inhibition of the colony stimulating factor 1 receptor (CSF1R) by immunotherapy was confirmed to inhibit macrophage accumulation and slow mammary tumor growth in mouse models while also reducing hemosiderin iron-laden TAM accumulation as measured by both iron histology and in vivo iron MRI (FeMRI). Spatial profiling of TAM iron deposit infiltration defined regions of maximal accumulation and response to the CSF1R inhibitor, and revealed differences between microenvironments of human cancer according to levels of polarized macrophage iron accumulation in stromal margins. We therefore demonstrate that iron deposition serves as an endogenous metabolic imaging biomarker of TAM infiltration in breast cancer that has high translational potential for evaluation of immunotherapeutic response.

Quantification of Nanoparticle Enhancement in Polarized Breast Tumor Macrophage Deposits by Spatial Analysis of MRI and Histological Iron Contrast Using Computer Vision.

Magnetic resonance imaging applications utilizing nanoparticle agents for polarized macrophage detection are conventionally analyzed according to iron-dependent parameters averaged over large regions of interest (ROI). However, contributions from macrophage iron deposits are usually obscured in these analyses due to their lower spatial frequency and smaller population size compared with the bulk of the tumor tissue. We hypothesized that, by addressing MRI and histological pixel contrast heterogeneity using computer vision image analysis approaches rather than statistical ROI distribution averages, we could enhance our ability to characterize deposits of polarized tumor-associated macrophages (TAMs). We tested this approach using in vivo iron MRI (FeMRI) and histological detection of macrophage iron in control and ultrasmall superparamagnetic iron oxide (USPIO) enhanced mouse models of breast cancer. Automated spatial profiling of the number and size of iron-containing macrophage deposits according to localized high-iron FeMRI or Prussian blue pixel clustering performed better than using distribution averages to evaluate the effects of contrast agent injections. This analysis was extended to characterize subpixel contributions to the localized FeMRI measurements with histology that confirmed the association of endogenous and nanoparticle-enhanced iron deposits with macrophages in vascular regions and further allowed us to define the polarization status of the macrophage iron deposits detected by MRI. These imaging studies demonstrate that characterization of TAMs in breast cancer models can be improved by focusing on spatial distributions of iron deposits rather than ROI averages and indicate that nanoparticle uptake is dependent on the polarization status of the macrophage populations. These findings have broad implications for nanoparticle-enhanced biomedical imaging especially in cancer.

Adrenergic nerve degeneration in bone marrow drives aging of the hematopoietic stem cell niche.

Aging of hematopoietic stem cells (HSCs) is associated with a decline in their regenerative capacity and multilineage differentiation potential, contributing to the development of blood disorders. The bone marrow microenvironment has recently been suggested to influence HSC aging, but the underlying mechanisms remain largely unknown. Here we show that HSC aging critically depends on bone marrow innervation by the sympathetic nervous system (SNS), as loss of SNS nerves or adrenoreceptor ß3 signaling in the bone marrow microenvironment of young mice led to premature HSC aging, as evidenced by appearance of HSC phenotypes reminiscent of physiological aging. Strikingly, supplementation of a sympathomimetic acting selectively on adrenoreceptor ß3 to old mice significantly rejuvenated the in vivo function of aged HSCs, suggesting that the preservation or restitution of bone marrow SNS innervation during aging may hold the potential for new HSC rejuvenation strategies.

Iron imaging reveals tumor and metastasis macrophage hemosiderin deposits in breast cancer.

Iron-deposition is a metabolic biomarker of macrophages in both normal and pathological situations, but the presence of iron in tumor and metastasis-associated macrophages is not known. Here we mapped and quantified hemosiderin-laden macrophage (HLM) deposits in murine models of metastatic breast cancer using iron and macrophage histology, and in vivo MRI. Iron MRI detected high-iron pixel clusters in mammary tumors, lung metastasis, and brain metastasis as well as liver and spleen tissue known to contain the HLMs. Iron histology showed these regions to contain clustered macrophages identified by their common iron status and tissue-intrinsic association with other phenotypic macrophage markers. The in vivo MRI and ex vivo histological images were further processed to determine the frequencies and sizes of the iron deposits, and measure the number of HLMs in each deposit to estimate the in vivo MRI sensitivity for these cells. Hemosiderin accumulation is a macrophage biomarker and intrinsic contrast source for cellular MRI associated with the innate function of macrophages in iron metabolism systemically, and in metastatic cancer.

Iron deposition is associated with differential macrophage infiltration and therapeutic response to iron chelation in prostate cancer.

Immune cells such as macrophages are drivers and biomarkers of most cancers. Scoring macrophage infiltration in tumor tissue provides a prognostic assessment that is correlated with disease outcome and therapeutic response, but generally requires invasive biopsy. Routine detection of hemosiderin iron aggregates in macrophages in other settings histologically and in vivo by MRI suggests that similar assessments in cancer can bridge a gap in our ability to assess tumor macrophage infiltration. Quantitative histological and in vivo MRI assessments of non-heme cellular iron revealed that preclinical prostate tumor models could be differentiated according to hemosiderin iron accumulation-both in tumors and systemically. Monitoring cellular iron levels during "off-label" administration of the FDA-approved iron chelator deferiprone evidenced significant reductions in tumor size without extensive perturbation to these iron deposits. Spatial profiling of the iron-laden infiltrates further demonstrated that higher numbers of infiltrating macrophage iron deposits was associated with lower anti-tumor chelation therapy response. Imaging macrophages according to their innate iron status provides a new phenotypic window into the immune tumor landscape and reveals a prognostic biomarker associated with macrophage infiltration and therapeutic outcome.

Metabolic plasticity of metastatic breast cancer cells: adaptation to changes in the microenvironment.

Cancer cells adapt their metabolism during tumorigenesis. We studied two isogenic breast cancer cells lines (highly metastatic 4T1; nonmetastatic 67NR) to identify differences in their glucose and glutamine metabolism in response to metabolic and environmental stress. Dynamic magnetic resonance spectroscopy of (13)C-isotopomers showed that 4T1 cells have higher glycolytic and tricarboxylic acid (TCA) cycle flux than 67NR cells and readily switch between glycolysis and oxidative phosphorylation (OXPHOS) in response to different extracellular environments. OXPHOS activity increased with metastatic potential in isogenic cell lines derived from the same primary breast cancer: 4T1 > 4T07 and 168FARN (local micrometastasis only) > 67NR. We observed a restricted TCA cycle flux at the succinate dehydrogenase step in 67NR cells (but not in 4T1 cells), leading to succinate accumulation and hindering OXPHOS. In the four isogenic cell lines, environmental stresses modulated succinate dehydrogenase subunit A expression according to metastatic potential. Moreover, glucose-derived lactate production was more glutamine dependent in cell lines with higher metastatic potential. These studies show clear differences in TCA cycle metabolism between 4T1 and 67NR breast cancer cells. They indicate that metastases-forming 4T1 cells are more adept at adjusting their metabolism in response to environmental stress than isogenic, nonmetastatic 67NR cells. We suggest that the metabolic plasticity and adaptability are more important to the metastatic breast cancer phenotype than rapid cell proliferation alone, which could 1) provide a new biomarker for early detection of this phenotype, possibly at the time of diagnosis, and 2) lead to new treatment strategies of metastatic breast cancer by targeting mitochondrial metabolism.

Cardio-chemical exchange saturation transfer magnetic resonance imaging reveals molecular signatures of endogenous fibrosis and exogenous contrast media.

BACKGROUND: Application of emerging molecular MRI techniques, including chemical exchange saturation transfer (CEST)-MRI, to cardiac imaging is desirable; however, conventional methods are poorly suited for cardiac imaging, particularly in small animals with rapid heart rates. We developed a CEST-encoded steady state and retrospectively gated cardiac cine imaging sequence in which the presence of fibrosis or paraCEST contrast agents was directly encoded into the steady-state myocardial signal intensity (cardioCEST). METHODS AND RESULTS: Development of cardioCEST: A CEST-encoded cardiac cine MRI sequence was implemented on a 9.4T small animal scanner. CardioCEST of fibrosis was serially performed by acquisition of a series of CEST-encoded cine images at multiple offset frequencies in mice (n=7) after surgically induced myocardial infarction. Scar formation was quantified using a spectral modeling approach and confirmed with histological staining. Separately, circulatory redistribution kinetics of the paramagnetic CEST agent Eu-HPDO3A were probed in mice using cardioCEST imaging, revealing rapid myocardial redistribution, and washout within 30 minutes (n=6). Manipulation of vascular tone resulted in heightened peak CEST contrast in the heart, but did not alter redistribution kinetics (n=6). At 28 days after myocardial infarction (n=3), CEST contrast kinetics in infarct zone tissue were altered, demonstrating gradual accumulation of Eu-HPDO3A in the increased extracellular space. CONCLUSIONS: cardioCEST MRI enables in vivo imaging of myocardial fibrosis using endogenous contrast mechanisms, and of exogenously delivered paraCEST agents, and can enable multiplexed imaging of multiple molecular targets at high-resolution coupled with conventional cardiac MRI scans.

Ultrafast in vivo diffusion imaging of stroke at 21.1 T by spatiotemporal encoding.

PURPOSE: This study quantifies in vivo ischemic stroke brain injuries in rats using ultrahigh-field single-scan MRI methods to assess variations in apparent diffusion coefficients (ADCs). METHODS: Magnitude and diffusion-weighted spatiotemporally encoded imaging sequences were implemented on a 21.1 T imaging system, and compared with spin-echo and echo-planar imaging diffusion-weighted imaging strategies. ADC maps were calculated and used to evaluate the sequences according to the statistical comparisons of the ipsilateral and contralateral ADC measurements at 24, 48, and 72 h poststroke. RESULTS: Susceptibility artifacts resulting from normative anatomy and pathological stroke conditions were particularly intense at 21.1 T. These artifacts strongly distorted single-shot diffusion-weighted echo-planar imaging experiments, but were reduced in four-segment interleaved echo-planar imaging acquisitions. By contrast, nonsegmented diffusion-weighted spatiotemporally encoded images were largely immune to field-dependent artifacts. Effects of stroke were apparent in both magnitude images and ADC maps of all sequences. When stroke recovery was followed by ADC variations, spatiotemporally encoded, echo-planar imaging, and spin-echo acquisitions revealed statistically significant increase in ADCs. CONCLUSIONS: Consideration of experiment duration, image quality, and mapped ADC values provided by spatiotemporally encoded demonstrates that this single-shot acquisition is a method of choice for high-throughput, ultrahigh-field in vivo stroke quantification.

Area per lipid and cholesterol interactions in membranes from separated local-field (13)C NMR spectroscopy.

Investigations of lipid membranes using NMR spectroscopy generally require isotopic labeling, often precluding structural studies of complex lipid systems. Solid-state (13)C magic-angle spinning NMR spectroscopy at natural isotopic abundance gives site-specific structural information that can aid in the characterization of complex biomembranes. Using the separated local-field experiment DROSS, we resolved (13)C-(1)H residual dipolar couplings that were interpreted with a statistical mean-torque model. Liquid-disordered and liquid-ordered phases were characterized according to membrane thickness and average cross-sectional area per lipid. Knowledge of such structural parameters is vital for molecular dynamics simulations, and provides information about the balance of forces in membrane lipid bilayers. Experiments were conducted with both phosphatidylcholine (dimyristoylphosphatidylcholine (DMPC) and palmitoyloleoylphosphatidylcholine (POPC)) and egg-yolk sphingomyelin (EYSM) lipids, and allowed us to extract segmental order parameters from the (13)C-(1)H residual dipolar couplings. Order parameters were used to calculate membrane structural quantities, including the area per lipid and bilayer thickness. Relative to POPC, EYSM is more ordered in the ld phase and experiences less structural perturbation upon adding 50% cholesterol to form the lo phase. The loss of configurational entropy is smaller for EYSM than for POPC, thus favoring its interaction with cholesterol in raftlike lipid systems. Our studies show that solid-state (13)C NMR spectroscopy is applicable to investigations of complex lipids and makes it possible to obtain structural parameters for biomembrane systems where isotope labeling may be prohibitive.

Hyperpolarized functional magnetic resonance of murine skeletal muscle enabled by multiple tracer-paradigm synchronizations.

Measuring metabolism's time- and space-dependent responses upon stimulation lies at the core of functional magnetic resonance imaging. While focusing on water's sole resonance, further insight could arise from monitoring the temporal responses arising from the metabolites themselves, in what is known as functional magnetic resonance spectroscopy. Performing these measurements in real time, however, is severely challenged by the short functional timescales and low concentrations of natural metabolites. Dissolution dynamic nuclear polarization is an emerging technique that can potentially alleviate this, as it provides a massive sensitivity enhancement allowing one to probe low-concentration tracers and products in a single-scan. Still, conventional implementations of this hyperpolarization approach are not immediately amenable to the repeated acquisitions needed in real-time functional settings. This work proposes a strategy for functional magnetic resonance of hyperpolarized metabolites that bypasses this limitation, and enables the observation of real-time metabolic changes through the synchronization of stimuli-triggered, multiple-bolus injections of the metabolic tracer 13C1-pyruvate. This new approach is demonstrated with paradigms tailored to reveal in vivo thresholds of murine hind-limb skeletal muscle activation, involving the conversion of 13C1-pyruvate to 13C1-lactate and 13C1-alanine. These functional hind-limb studies revealed that graded skeletal muscle stimulation causes commensurate increases in glycolytic metabolism in a frequency- and amplitude-dependent fashion, that can be monitored on the seconds/minutes timescale using dissolution dynamic nuclear polarization. Spectroscopic imaging further allowed the in vivo visualization of uptake, transformation and distribution of the tracer and products, in fast-twitch glycolytic and in slow-twitch oxidative muscle fiber groups. While these studies open vistas in time and sensitivity for metabolic functional magnetic resonance studies in muscle, the simplicity of our approach makes this technique amenable to a wide range of functional metabolic tracer studies.

Referenceless reconstruction of spatiotemporally encoded imaging data: principles and applications to real-time MRI.

PURPOSE: Ultrafast sequences based on "Hybrid" spatiotemporal encoding (SPEN) replace echo-planar imaging's phase encoding "blips," while retaining a k-space readout acquisition. Hardware imperfections during acquisition may lead to ghosts and striped artifacts along the SPEN dimension; akin to echo-planar imaging's Nyquist ghosts, but weaker. A referenceless method to eliminate these artifacts in Hybrid SPEN is demonstrated. THEORY AND METHODS: Owing to its encoding in direct space, rather than reciprocal space, undersampling in SPEN does not generate an echo-planar-imaging-like aliasing, but instead lowers the spatial resolution. Hybrid SPEN data can be split into two undersampled signals: a reference one comprised of the odd-echos, and an even-echo set that has to be "corrected" for consistency with the former. A simple way of implementing such a correction that enables a joint high-resolution reconstruction is proposed. RESULTS: The referenceless algorithm is demonstrated with various examples, including oblique scans, large in vivo datasets from real-time dynamic contrast-enhanced perfusion experiments, and human brain imaging. CONCLUSIONS: The referenceless correction enables robust single-scan imaging under changing conditions-such as patient motion and changes in shimming over time-without the need of ancillary navigators. This opens new options for real-time MRI and interactive scanning.

In vivo magnetic resonance of hyperpolarized [(13)C1]pyruvate: metabolic dynamics in stimulated muscle.

The metabolic status of muscle changes according to the energetic demands of the organism. Two key regulators of these changes include exercise and insulin, with exercise eliciting catabolic expenditure within seconds and insulin enabling anabolic energy investment over minutes to hours. This study explores the potential of time-resolved hyperpolarized dynamic (13)C spectroscopy to characterize the in vivo metabolic phenotype of muscle during functional and biochemical insulin-induced stimulation of muscle. Using [(13)C1]pyruvic acid as a tracer, we find that despite the different time scales of these forms of stimulation, increases in pyruvate label transport and consumption and concomitant increases in initial rates of the tracer metabolism to lactate were observed for both stimuli. By contrast, rates of tracer metabolism to labeled alanine increased incrementally for insulin but remained unchanged following exercise-like muscle stimulation. Kinetic analysis revealed that branching of the hyperpolarized [(13)C]pyruvate tracer between lactate and alanine provides significant tissue-specific biomarkers that distinguish between anabolic and catabolic fates in vivo according to the routing of metabolites between glycolytic and amino acid pathways.

Solid-state ¹³C NMR reveals annealing of raft-like membranes containing cholesterol by the intrinsically disordered protein α-Synuclein.

Misfolding and aggregation of the intrinsically disordered protein α-Synuclein (αS) in Lewy body plaques are characteristic markers of late-stage Parkinson's disease. It is well established that membrane binding is initiated at the N-terminus of the protein and affects biasing of conformational ensembles of αS. However, little is understood about the effect of αS on the membrane lipid bilayer. One hypothesis is that intrinsically disordered αS alters the structural properties of the membrane, thereby stabilizing the bilayer against fusion. Here, we used two-dimensional (13)C separated local-field NMR to study interaction of the wild-type α-Synuclein (wt-αS) or its N-terminal (1-25) amino acid sequence (N-αS) with a cholesterol-enriched ternary membrane system. This lipid bilayer mimics cellular raft-like domains in the brain that are proposed to be involved in neuronal membrane fusion. The two-dimensional dipolar-recoupling pulse sequence DROSS (dipolar recoupling on-axis with scaling and shape preservation) was implemented to measure isotropic (13)C chemical shifts and (13)C-(1)H residual dipolar couplings under magic-angle spinning. Site-specific changes in NMR chemical shifts and segmental order parameters indicate that both wt-αS and N-αS bind to the membrane interface and change lipid packing within raft-like membranes. Mean-torque modeling of (13)C-(1)H NMR order parameters shows that αS induces a remarkable thinning of the bilayer (≈6Å), accompanied by an increase in phospholipid cross-sectional area (≈10Å(2)). This perturbation is characterized as membrane annealing and entails structural remodeling of the raft-like liquid-ordered phase. We propose this process is implicated in regulation of synaptic membrane fusion that may be altered by aggregation of αS in Parkinson's disease.