Stephanie Cologna (Presenter)
University of Illinois at Chicago
Bio: Stephanie received her B.S. (Chemistry) from the University of Arizona in 2005. She then pursued graduate studies under the guidance of Professor David H. Russell at Texas A&M University where she worked on novel electrophoretic separation methodologies coupled with mass spectrometry for proteomics. In 2010, Stephanie began her postdoctoral training under the dual mentorship of Drs. Forbes D. Porter and Alfred L. Yergey. As a postdoctoral fellow, she worked on protein biomarker identification in cerebrospinal fluid from patients with genetic disorders related to cholesterol homeostasis. In fall 2015, Stephanie joined the faculty in the Department of Chemistry at UIC where her group works on mass spectrometry-based lipidomic and proteomic studies of genetic, neurodegenerative disorders.
Authorship: (1) Fernando Tobias, (2) Matthew T. Olson, (1) Stephanie M. Cologna
1) University of Illinois at Chicago 2) Mayo Clinic
Niemann-Pick Disease, type C1 (NPC1) is a fatal, genetic, neurodegenerative disease that is characterized by the accumulation of unesterified cholesterol and sphingolipids in the endo/lysosomal system. A hallmark of NPC1 includes progressive neurodegeneration of the cerebellum. In this study, we performed mass spectrometry imaging on cerebellar tissue from control and mutant NPC1 mice. Additionally, we developed an algorithmic platform to analyze technical and biological replicate similarity. Finally, our LC-MS studies have been used to confirm potential lipid alterations in NPC1. The results of these studies will be presented.
Niemann-Pick Disease, type C1 (NPC1) is an autosomal recessive, neurodegenerative disorder characterized by progressive cerebellar neuron loss. Downstream of the genetic defect, NPC1 biochemically results in improper trafficking of cholesterol and glycosphingolipids thereby accumulating in the endo/lysosomal system. The phenotypic spectrum of NPC is broad, typical clinical presentations include ataxia, liver dysfunction and seizures among others. The is currently no FDA-approved therapy for NPC1. This study aims at mapping lipids in the mouse model of NPC1 in an effort to understand the role of lipid signaling and neuron death and potential reveal targets for new therapies. To carry this out, we use mass spectrometry imaging and validate with traditional LC-MS methods.
Control (Npc1+/+) and mutant (Npc1-/-) mice were obtained from heterozygous breeding in accordance with an UIC-approved animal protocol. Brain tissue from seven-week-old animals was flash frozen and cryosectioned to 16 µm. Sections were thaw-mounted and washed in cold ammonium acetate buffer followed by vacuum drying. Matrix application was performed using an artistic air brush and the matrix used was 9-aminoacridine. Images were collected in positive and negative ion mode using a model 4800 MALDI-TOF/TOF Proteomics Analyzer (Sciex). LC-MS studies were carried out on a model 6545 ESI-Q-TOF (Agilent). Cerebellar tissue lysates were processed for lipid extraction using the Folch or MTBE method. Lipid assignments were made using accurate mass measurements and tandem MS when possible. Newly developed bioinformatics was performed using R-programming.
The NPC1 null mouse model recapitulates the human disease. Animals up to about one month of age are asymptomatic. Tremors and ataxia are observed from five to seven weeks and the model is fatal by 12 weeks of age. The loss of cerebellar purkinje neurons occurs in a rostral to caudal manner as the disease progresses with general retention of purkinje neurons in lobule X. In this study, we performed MALDI-MS imaging on control and mutant seven week animals. Given the known glycosphingolipid storage and increase from previous studies, we first sought to map GM1, GM2 and GM3. Indeed, we observe accumulation of various GM species, particularly GM2 and GM3 in lobule X. Interestingly, GM3 (d18:1/18:0) and GM3 (d18:1/20:0) localize to two different regions within lobule X. Conversely, GM2 (d18:1/18:0) and GM2 (d18:1/20:0) have similar regional accumulation in lobule X.
A second goal of our work was to develop an informatic platform to analyze multiple biological and technical replicate analyses. Doing so provides the ability to understand variability in the method and also can indicate previously undetected differential lipids with spatial information. In total, six control and five mutant animals were analyzed for a total of 13-17 replicate images obtained in both ionization modes. Centroided spectra were processed for peak peaking, clustering and normalize to unit vectors. Normalized spectra were evaluated for spectral similarity and similar spectral were used to obtain consensus spectra. Consensus spectra analysis of the whole cerebellum indicated differences in GM2 and GM3 species as observed from manual analysis. With a focus only on lobule X, we were able to identify lipids that may be altered in NPC1 such as cardiolipins and smaller gangliosides. These observations are currently being validated using LC-MS technologies.
Conclusions & Discussion
We have performed a MALDI-MS imaging study in NPC1. In addition to identifying previously known lipids which accumulate in NPC1, we have also obtained cerebellar spatial distributions. Using our newly developed algorithm for data processing, we have generated consensus spectra for replicate imaging analysis and identified potentially new lipid biomarkers in NPC1.
References & Acknowledgements:
Olson, M.T. et al., J Am Soc Mass Spectrom. 22, 969-975 (2011)
McDonnell et al., J Prot Res., 7, 3619–3627 (2008)
|Grants||yes||Ara Parseghian Medical Research Foundation, UIC|
|Board Member||yes||American Society for Mass Spectrometry - Digital Communications|
IP Royalty: no
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