The University of Kentucky Phase I Center of Research in central nervous system metabolism is supported by a $10.6 million, five-year award (P20 GM148326) from the National Institute of General Medical Sciences (NIGMS). The NIGMS is a part of the National Institutes of Health (NIH) that supports thematic, multidisciplinary Centers of Biomedical Research Excellence (COBRE) across the country through its Institutional Development Award (IDeA) program.
The central nervous system (CNS) has a very high requirement for energy which results in high metabolism rates. As such, CNS metabolism and neuronal excitability are interdependent, and disruptions of CNS metabolism are implicated in numerous neurological disease states, including Alzheimer’s Disease, Parkinson’s disease, epilepsy, ischemia, and traumatic CNS injury, but the mechanisms and underlying connections between CNS metabolism and disease are not well-defined. The University of Kentucky College of Medicine has made significant investments over the past decade in investigators with strong metabolic and metabolomics expertise and instrumentation to support research efforts, which has greatly enhanced existing strongholds in neuroscience, cancer, cardiovascular, diabetes, and obesity research. The goal of our multidisciplinary Center of Biomedical Research Excellence (COBRE) focused on CNS Metabolism (CNS-Met) is a strategically designed, sustainable framework that promotes leading-edge research focused on the role of metabolic mediators of brain function and disease, with an emphasis on encouraging research excellence from Junior Research Project Leaders. To accomplish these goals, we will meet four specific aims: (1) develop a critical mass of funded investigators with research programs directly related to the COBRE’s unifying theme; (2) provide strong team-based mentoring combining basic and clinical expertise; (3) recruit new investigators to the COBRE in multidisciplinary areas of neurologic dysfunction through pilot project grant and recruitment of junior Research Project Leaders; and (4) create synergy among research projects via critical links to strong research centers and core facilities at UK, including existing COBREs. Overall, the CNS-Met will create a critical mass of scientists who are skilled and well-equipped to foster a sustainable research center focused on CNS metabolism.
P20GM148326/GM/NIGMS NIH HHS/United States.
CNS-Met Metabolomics Core
The overarching goal of the CNS-Met Metabolomics Core is to make cutting-edge metabolomics and mitochondrial energetics techniques and analyses available to researchers. The core will assist in experimental design, run samples, train personnel, and analyze brain metabolism data. To achieve these goals, the core will Aim 1: Provide experimental design consultation and project-specific study planning. Proper experimental design and sample preparation will lay the foundation for a successful metabolomics experiment. Aim 2: Provide metabolomics expertise and training. The core will utilize integrated processes established by core leadership from experimental design through rapid and consistent sample acquisition, sample fixation, sample extraction, data reduction, statistical analysis, data analysis, and biological interpretation to ensure the success of researchers.
Facilities and Other Resources
The CNS-Met Metabolomics Core is housed on the first floor of the Biomedical and Biological Research Building (BBSRB) The Metabolomics Core occupies >900 sq ft in BBSRB on the first floor in a closed room with a fume hood, sink, explosion proof chemical cabinet, lab bench, and storage space. The CNS-Met Metabolomics Core consists of a set of combined instruments to provide analysis and interpretation services for a wide range of metabolites.
Agilent 5977B single quadrupole GCMS. This mass spectrometer is the most popular Agilent mass spectrometer on the market for analytics. It is routinely used for environmental, chemical, food, forensic, and pharmaceutical applications. Dr. Oliver Fiehn designed the Fiehn metabolomics library for this GCMS in 2009. The 5977B has a femtogram detection limit and is equipped with a 150-sample auto-sampler that is designed for high sample numbers. It is a workhorse instrument built to operate 24-hours/day with minimal downtime.
The workflow is based on the Fiehn metabolomics GCMS-method and routinely profiles 100-300 polar metabolites from central carbon metabolism (e.g. TCA cycle, pentose phosphate pathway, and glycolysis metabolites) with high confidence. This unit is especially useful to profile biofluids, such as cerebrospinal fluid and plasma, which contain abundant volatile small metabolites as well as amino acids. In addition to polar samples, this system will be utilized to analyze biomass after polar extraction (i.e. the insoluble pellet) that contains protein, DNA, RNA and glycogen. This biomass is hydrolyzed to monomeric forms using 2N hydrochloric acid (HCl) and analyzed by the 5977B. In addition to biomass analysis, protein derived glutamate is used to normalize the polar metabolites for improved rigor and reproducibility. The Agilent 5977B is a workhorse instrument designed for high sample numbers while providing outstanding consistency and reproducibility.
Agilent 6545 quadrupole time of flight LC/CE-QTOF. This high-end accurate-mass mass spectrometer is used for SIRM analysis, lipidomics and unknown-discovery metabolomics. The 6545 maintains femtogram sensitivity with a mass resolution of 40,000 FWHM. This detection range is perfectly suited for distinguishing between different isotopologues for entry-level SIRM experiments and initial identification of different lipid species. Coupled orthogonally to either a HPLC or CE, the 6545 can analyze metabolites ranging from 100 Da to 10,000 Da. This unit is currently used to assign over 50,000 polar mass features that correspond to over 1200 polar metabolites covering major central carbon metabolic pathways from glycolysis to nucleotide sugar biosynthesis. Further, we can define 13C isotopologues in many of these metabolites when enriched with stable tracer. The LC/CE-QTOF can process up to 12,000 samples per year. Based on current and project usage, the projected annual instrument usage for this unit is <50% of the available instrument time.
Seahorse Biosciences XFe Flux Analyzers: This instrument provides an advanced and high-throughput method for mitochondrial bioenergetics. The core has the 24-well (XFe24) and 96-well (XFe96) units. This format allows for analyses of mitochondrial function (respiration rates, glycolysis, pH shifts and ATP production/consumption) in isolated mitochondria, slice cultures or cell cultures with 4 additions of preloaded compounds (substrates, inhibitors, uncouplers, drugs, peptides etc.).
BIOTEK Cytation 5: Normalizes cell numbers in the XFe 96 Seahorse.
Oroboros O2K-Fluorometer: The Oroboros O2K-Fluorometer provides state-of-the-art, high resolution respirometry coupled with high resolution fluorometry using dual Clark-type oxygen electrode micro chambers that are coupled with fluorescent fiber optic probes. This allows for simultaneous measurement of mitochondrial or cellular respiration, membrane potential, Ca2+ cycling and ROS production in either isolated mitochondria (reported 1-5 ug/sample) or minced/ homogenized tissue without mitochondrial isolation. A principle benefit of the Oroboros is its ability to measure mitochondrial function with high fidelity in small tissue samples without having to isolate mitochondria directly.
Additional equipment. In addition to the mass spectrometers, the core also has a dedicated hood for sample prep with heat blocks and a dedicated sample prep mini-centrifuges. The core has a Labconco Centrivap vacuum centrifuge and freeze dryer hybrid dedicated to sample preparation for metabolomics analysis. The lab space has two sinks, bench space outside of the hood for sample preparation and the appropriate safety equipment (eye wash, fire extinguisher, etc.)
CNS-Met Pilot Project Grants
The NIH Center of Biomedical Research Excellence (COBRE) on CNS Metabolism at the University of Kentucky offers pilot project grants. These pilot grants are intended to assist investigators to generate sufficient data to be competitive for extramural funding in the research area of the role of CNS metabolic mediators of brain function and disease.
Pilot grants will be awarded for up to $50,000 for one year, direct costs only and must be spent by June 30, 2024. Applications are due by 5 p.m. on August 21, 2023.
Eligibility is limited to full-time regular, special, and clinical title faculty as well as full-time research faculty of the University of Kentucky. Junior faculty are encouraged to apply. Senior faculty with new projects of relevance to the CNS-Met are also eligible for pilot funding. Current and former key personnel on the CNS-Met COBRE are not eligible to apply.
- Pilot projects are solicited once a year.
IRB or IACUC Approvals
Applicants do not need IRB or IACUC approvals to submit a pilot grant application, but necessary IRB or IACUC protocols should be submitted as soon as possible upon notification of the preliminary approval of your application. (These approvals are required before CNS-Met will release the use of funds.) Funds may not be awarded if required IRB/IACUC approvals are not obtained in a timely manner consistent with the grant award period.
Please provide the following in order:
- NIH biosketch with other support
- Abstract including project title (one page)
- Research proposal (six pages)
- NIH budget and justification
- Supporting documents (optional)