The Science

2012-2013

Neurolipid Highlights 2012/2013

Platelet activating factors in depression and coronary artery disease: a potential biomarker related to inflammatory mechanisms and neurodegeneration.

Highlight by Graham Mazereeuw

Visualization and Phospholipid Identification (VaLID): An online integrated search engine capable of identifying and visualizing glycerophospholipids with given mass over charge

Highlight by Alexandre P. Blanchard and Graeme McDowell



 

 

Platelet activating factors in depression and coronary artery disease: a potential biomarker related to inflammatory mechanisms and neurodegeneration.

 

Highlight by Graham Mazereeuw

 


Up to 50% of coronary artery disease (CAD) patients will experience a depressive episode after a cardiac event, often for prolonged periods of time. Depression in CAD patients is of high clinical importance as it doubles the risk of acute ischemic events and is linked with accelerated cognitive decline and the transition to dementia. Unfortunately, antidepressant pharmacotherapies have only modest effects in CAD patients and novel approaches are limited by a poor understanding of underlying mechanisms. In a novel hypothesis to explain this relationship, we recently proposed that the platelet activating factor (PAF) family of alkylacylglycerophosphocholines (AAGPCs) may be associated with the onset and/or persistence of a depressive episode in CAD patients due to their association with CAD and with proposed mechanisms for depression. Emerging preclinical evidence supports this by indicating that disrupted lipid metabolism may be a converging point for several pathophysiological processes active in both depression and CAD. PAFs and other AAGPCs are remodelled at the cellular membrane in response to multiple extracellular inputs such as inflammatory activity, oxidative stress, and platelet reactivity, all of which are present in depressed CAD patients. PAFs also mediate cerebrovascular pathology and neurodegenerative processes that are commonly observed in patients with prolonged depression. Currently, PAFs represent unexplored potential mechanistic markers of depression in CAD. As such, elucidation of their role may lead to a better understanding of its underlying mechanisms and the potential identification of novel therapeutic targets.

 

posted 16 Dec 2013
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References:
Original research paper

Mazereeuw G, Herrmann N, Bennett SAL, Swardfager W, Xu H, Valenzuela N, Fai S, Lanctôt KL, (2013) Platelet activating factors in depression and coronary artery disease: a potential biomarker related to inflammatory mechanisms and neurodegeneration. Neurosci Biobeh Rev 37:1611-1621. doi: 10.1016/j.neubiorev.2013.06.010.
Pubmed 23800745

 


 

 

Visualization and Phospholipid Identification (VaLID): An online integrated search engine capable of identifying and visualizing glycerophospholipids with given mass over charge.

 

Highlight by Alexandre P. Blanchard and Graeme McDowell

 


Lipids are intriguing cell constituents.  Despite species composition being in constant flux, tight regulation maintains vital integrity of plasma membrane and organelle boundaries.  Glycerophospholipids are key players in cellular signaling mechanisms.  There are nine major classes of glycerophospholipids, each represented by a rich diversity of multiple species.  Aberrant lipid metabolisms are associated with the major brain injuries and diseases such as, stroke, epilepsy, Alzheimer Disease, and Parkinson’s Disease.  Because each species has distinct chemical, physical, and biological proprieties, identification is crucial to elucidating discrete roles in neurodegenerative disease.  Where genomics and proteomics capitalize on sequence-based signatures, glycerophospholipids lack easily definable molecular fingerprints.  While advances in detecting lipid fragmentation patterns by mass spectrometry is enabling lipid identification at the molecular level, many m/z values are not represented in existing prediction engines.  To address this gap, we created Visualization and Phospholipid Identification (VaLID), a freely accessible, web-based application that includes a search engine link to an exhaustive phospholipid database (CDP-DG, PA, PC, PE, PG, PGP, PPA and PS) and multiple visualization features.  VaLID’s search engine considers the user-defined m/z and MS conditions and returns all theoretically possible species. Visualization algorithms produce multiple chemical structure files for each species.  Curated lipids detected by the Canadian Institutes of Health Research Training Program in Neurodegenerative Lipidomics (CTPNL) are provided as high-resolution structures.  The 3D models give the opportunity to see the species in a more biologically realistic representation.  VaLID has the overarching goal of facilitating the identification of the lipid species, understanding the research more deeply and assisting in its dissemination.

 

posted 5 February 2013
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References:
Original research paper

Blanchard AP†, McDowell GSV†, Valenzuela N†, Xu H, Gelbard S, Bertrand M, Slater GW, Figeys D*, Fai S*, Bennett SAL* (2013) Visualization and Phospholipid Identification (VaLID): An online integrated search engine capable of identifying and visualizing glycerophospholipids with given mass. Bioinformatics, 29: 284-285, †all authors contributed equally, *joint corresponding authors
Pubmed 23162086

2011-2012

Neurolipid Highlights 2011/2012

Progressive Dopaminergic Cell Loss With Unilateral-to-Bilateral Progression in a Genetic Model of Parkinson’s Disease

Highlight by Dr. Maxime W Rousseaux

 


 

Progressive Dopaminergic Cell Loss With Unilateral-to-Bilateral Progression in a Genetic Model of Parkinson’s Disease.

 

Highlight by Dr. Maxime W Rousseaux

 


Parkinson’s disease (PD) affects over 100,000 Canadians and is thought to increase in the future given rising life expectancies. Nevertheless, its pathogenesis is poorly understood and current therapies only focus on symptomatic relief. Thus, a strong focus in the field has been to generate better animal models of the disease in efforts to better understand its underlying pathogenic mechanism. In this study, we identified a subset of mice lacking the early-onset PD gene DJ-1, when crossed to a pure mouse strain, exhibit several cardinal features of PD. First, a subset of the DJ-1 null animals on this strain (termed DJ1-C57 affected) demonstrates unilateral degeneration at the level of the substantia nigra pars compacta (a key locus of degeneration in PD). Second, this unilateral degeneration progresses to bilateral degeneration as the mice age, a feature prominent in human PD. Third, these parkinsonian mice demonstrate relative sparing of the neighboring VTA dopaminergic neuron cluster, a feature also present in human PD. Fourth, young DJ1-C57 affected animals demonstrate indices of early compensation including exuberant fiber staining and excess post-synaptic activity, a feature known to occur in early human PD that disappears with age. Fifth, aged DJ1-C57 mice not only demonstrate degeneration of dopaminergic neurons in the substantia nigra, but also in another nucleus pathologically affected in PD: the Locus Ceruleus. Sixth, aged DJ1-C57 mice demonstrate mild motor impairment when tasked with motor behavior tests. Lastly, in efforts to identify how only a subset of DJ-1 null mice on this background exhibit this phenotype, we performed exome sequencing in affected versus unaffected mice and identified five candidate exon polymorphisms that may account for this phenotype together with DJ-1 loss of function. It is in this way, using these histopathological and behavioral hallmarks in these mice, that we can likely devise better disease-modifying therapies.

 

posted 10 October 2012
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References:
Original research paper

Rousseaux MW, Marcogliese PC, Qu D, Hewitt SJ, Seang S, Kim RH, Slack RS, Schlossmacher MG, Lagace DC, Mak TW, Park DS (2012) Progressive dopaminergic cell loss with unilateral-to-bilateral progression in a genetic model of Parkinson disease. Proc Natl Acad Sci U S A 109:15918-15923.
Pubmed 23019375

2010-2011

Neurolipid Highlights 2010/2011

Arylsulfatase A deficiency causes seminolipid accumulation and a lysosomal storage disorder in Sertoli cells

Highlight by Dr. Hongbin Xu

Improved recovery and identification of membrane proteins from rat hepatic cells using a centrifugal proteomic reactor

Highlight by Dr. Hu Zhou

The essential role of phospholipase D for protecting against the toxic effects of C16 PAF

Highlight by Dr. Michael Kennedy

Lyso-form fragment ions facilitate the determination of stereospecificity of diacyl glycerophospholipids

Highlight by David Hou and Sarah Gelbard

Digitizing a neurosphere - a hybrid visualization model

Highlight by Nico Valenzuela

Pannexin 2 palmitoylation and neural stem cell modulation

Highlight by Dr. Leigh Anne Swayne



lyso-form fragment ions glycerophospholipids

Arylsulfatase A deficiency causes seminolipid accumulation and a lysosomal storage disorder in Sertoli cells

Highlight by Dr. Hongbin Xu


Male infertility occurs in 9% of couples, frequently with unknown causes, leading to excessive use of assisted reproductive technology (ART) despite the possible negative health consequences on the children born.  Research to find bio-indices of male fertility is greatly needed.  Seminolipid (aka SGG), present at a high level in mammalian male germ cells, is essential for their development in the testis, as shown in experimental animals.  SGG on mature sperm is also important for fertilization.  We have recently shown that homeostasis (balanced levels) of SGG in testes, as regulated by Sertoli cells, is relevant for sperm production and sperm fertilizing ability, especially in older mice (equivalent to 40 years old in men).  Sertoli cells are oblong somatic cells spanning the tubules in the testis, where sperm are produced.  Tight junctions at the basal part of the tubules exist between adjacent Sertoli cells, forming a barrier that excludes blood components from entering the compartment above.  Developing germ cells are in this compartment and nourished by nutrients secreted from Sertoli cells.  Sertoli cells also 'clean up' germ cells that have undergone apoptosis, which occurs at a high rate in normal testis.  Certain molecules of apoptotic cells, when accumulated are toxic to Sertoli cells, and need to be removed.  SGG is one such molecule that has to be degraded by arylsulfatase A in Sertoli cell lysosomes.  The failure of this degradation leads to 'lysosomal storage disorder' in Sertoli cells and subsequently a disability to support germ cell development. 

 

posted 24 November 2011
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References:
Original research paper

Xu H, Kongmanas K, Kadunganattil S, Smith CE, Rupar T, Goto-Inoue N, Hermo L, Faull KF, Tanphaichitr N (2011). Arylsulfatase A deficiency causes seminolipid accumulation and a lysosomal storage disorder in Sertoli cells. J Lipid Res. 2011 Dec;52(12):2187-97. Epub 2011 Oct 2.
Pubmed 21965315


lyso-form fragment ions glycerophospholipids

Improved Recovery and Identification of Membrane Proteins from Rat Hepatic Cells using a Centrifugal Proteomic Reactor

Highlight by Dr. Hu Zhou


Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis due to their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and HPLC-ESI-MS/MS. The performance of this approach was illustrated for the study of the proteome of ER/Golgi microsomal membranes in rat hepatic cells.  The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63% and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signalling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure/function studies of membrane proteins involved in lipid and lipoprotein metabolism.

 

posted 8 September 2011
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References:
Original research paper
Zhou H, Wang F, Yao Z, Wang Y, Ning Z, Hou W, Wright TG, Sundaram M, Zhong S, Figeys D (2011). Improved Recovery and Identification of Membrane Proteins from Rat Hepatic Cells using a Centrifugal Proteomic Reactor. Mol Cell Proteomics. [Epub ahead of print]
Pubmed 21749988

 

lyso-form fragment ions glycerophospholipids

The essential role of phospholipase D for protecting against the toxic effects of C16 PAF

Highlight by Dr. Michael Kennedy

Increasing evidence suggests that the cognitive impairments associated with neurodegenerative diseases, such as Alzheimer's disease, is partially a result of changes in the way our brains metabolize specific types of lipids. Although the extent and the reason behind these changes in lipid metabolism are not fully known, a lipid known as C16:0 platelet activating factor (C16 PAF) has been found to be relatively elevated in patients with Alzheimer's disease compared to healthy individuals.  This finding has led to a number of studies that have demonstrated the toxic nature of C16 PAF towards neurons. The current study has expanded upon this knowledge and has provided insight into potential pathways mediating the toxic effects of this lipid and provides potential therapeutic targets for the treatment of Alzheimer’s disease. The difficult task of identifying the cellular mechanisms underlying the toxic effects of C16 PAF was accomplished by exploiting the conserved biology between baker's yeast and humans. Although lacking a brain, yeast have proved to be a robust tool for identifying key pathways mediating a number of biological processes from cancer to aging. In particular, we were able to determine an essential role for phospholipase D (PLD), known as Spo14 in yeast, for protection against the toxic effects of C16 PAF. These results were extended to neurons and suggest that therapeutic strategies aimed at enhancing PLD activity may be beneficial for reducing the cognitive decline observed in Alzheimer's disease. A further benefit of this work was the identification of a novel regulator of PLD activity in yeast. Future studies should help to clarify whether a functionally similar mechanism controls PLD activity in neurons and the relevance of such a mechanism in Alzheimer’s disease.

 

posted 6 May 2011
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References:
Original research paper
Kennedy MA, Kabani N, Lambert J-P, Swayne LA, Ahmed F, Figeys D, Bennett SAL, Bryan J, Baetz K (2011). Srf1 is a novel regulator of phospholipase D activity and is essential to buffer the toxic effects of C16:0 platelet activating factor. PLOS Genetics. 7: e1001299. doi:10.1371/journal.pgen.1001299
Pubmed 21347278


lyso-form fragment ions glycerophospholipids

Lyso-form fragment ions facilitate the determination of stereospecificity of diacyl glycerophospholipids

Highlight by David (Weimin) Hou and Sarah Gelbard

In this work we reported the development of a novel methodology that uses MS2 recorded on a hybrid quadrupole time-of-flight mass spectrometer to determine the stereospecificity of diacyl glycerophospholipids based on the lyso-form fragment ions, attributed to the neutral loss of fatty acyl moieties. The fragmentation patterns of a variety of diacyl glycerophospholipid standards were first fully examined over a wide range of collision energy. We observed that lyso-form fragment ions corresponding to the neutral loss of fatty acyl moieties attached to the sn2 position as free fatty acids and as ketenes exhibited consistently higher intensity than their counter part ions due to the neutral loss of fatty acyl moieties attached to the sn1 position. We concluded that an empirical fragmentation rule can be used to precisely determine the stereospecificity of diacyl glycerophospholipids, primarily on the basis of relative abundance of the lyso-form fragment ions.

Combining the novel methodology reported in this work with the currently widely practiced mass spectrometric techniques such as multiple precursor ion scans, fatty acyl scans, and multidimensional mass spectrometry-based shotgun lipidomics should enable a reliable and convenient platform for comprehensive glycerophospholipid profiling in the context of neurodegeneration using human samples and murine models of disease.

 

posted 11 Jan 2011
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References:

Original research paper
Hou W, Zhou H, Khalil MB, Seebun D, Bennett SA, Figeys D (2011). Lyso-form fragment ions facilitate the determination of stereospecificity of diacyl glycerophospholipids. Rapid Commun Mass Spectrom. 25(1):205-17. doi: 10.1002/rcm.4846.

Pubmed 21157865

 

neural stem cells

Digitizing a neurosphere: a hybrid visualization methodology

Highlight by Nico Valenzuela

Understanding the full impact of 3-dimensional (3D) topography and cellular positioning within neural stem cell cultures is constrained by the use of prevailing 2-dimensional (2D) representations. Microscopy images gathered from the serial cryosectioning of neurospheres are limited in that they present spatial information about cell and protein location along one view plane making comprehensive evaluation of the intact sphere as a 3D microstructure difficult.

Employing a combination of graphic editing and 3D modeling software, researchers with the Neurodegenerative Lipidomics Training Program have developed a hybrid visualization methodology for the digital re-assembly of 2D epifluorescent images into complete 3D culture topography. Key steps to a digital reconstruction of the neurosphere included alignment of digitized serial sections, creation of progenitor cell maps, establishment of a cell typology to simulate the variety of progenitor cell types present in the neurosphere, assembly of the 2D maps in three dimensions, locating the progenitor cell typologies in 3D and a final process of rendering and compositing the completed digital model into images for analysis and presentation. A detailed protocol of these operations is presented in the web-based Journal of Visualized Experiments.   

In order to preserve accurate morphology of the digitized serial sections whose positions during thaw-mounting onto microscope lines were affected during the process of image gathering, researchers carefully re-aligned each of the cryosections paying careful attention to cytoarchitecture and topographic detail. This process of alignment was followed by detailed construction of maps locating progenitor cell position and protein expression for each of the section images.

Parallel to this work, a progenitor cell typology used to simulate the variety of cell types present in the neurosphere was created using 3D modeling software. Images built during the earlier stages of cell mapping were imported along with the progenitor cell typology into a final modeling space where each 2D map was reassembled in three dimensions. Points of each map were given corresponding 3D coordinates as markers with which to assign the cells of the typology. The resultant model was rendered into a series of final images for evaluation and presentation, providing points of analysis previously unattainable through traditional serial sectioning alone.  

 

posted 18 Oct 2010
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References:

Original research paper
Imbeault S, Valenzuela N, Fai S, Bennett SA (2010). Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology. J Vis Exp.46. http://www.jove.com/index/details.stp?id=2483, doi: 10.3791/2483.

 

pannexin 2 palmitoylation and neural stem cell modulation

Pannexin 2 palmitoylation and neural stem cell modulation

Highlight by Dr. Leigh Anne Swayne

Back in 2008, little was known about central nervous system-enriched Panx2 – that was until Leigh Anne Swayne and Catherine Sorbara from the Bennett lab got on the case. In the hippocampus, a key site for postnatal neurogenesis, we initially observed that Panx2 levels increased during the peak period of granule cell neurogenesis (postnatal day 7).  Prior to this no physiological function had yet been ascribed to Panx2 beyond reports that, upon heterologous expression in Xenopus oocytes, Panx2 physically interacts with Panx1 to reduce current amplitude. Extremely intrigued by the potential association between a peak in expression coinciding with a peak in neurogenesis - our original aim was to determine whether Panx2 is expressed by postnatal neural stem and progenitor cells (NPCs), and, if so, to characterize this expression and determine whether Panx2 plays a role in regulating neurogenesis.

 

Using both primary NPC cultures and Neuro2a (N2a) cells to provide functional insight, we showed that Panx2 protein expression and lipid post-translational modification (S-palmitoylation) modulates the timing of NPC commitment to a neuronal lineage. By western blotting of NPCs cultured as neurospheres, we uncovered an NPC-specific Panx2 species that is not appreciably expressed in mature brain (hippocampus and/or cortex).  Over the course of a series of biochemical experiments designed to uncover the posttranslational modification responsible for the NPC-specific species, we observed that boiling in the presence of reducing agents, and hydroxylamine treatment significantly reduced the immunoreactivity of the NPC-specific band.  Both of these effects suggested palmitoylation of NPC-specific Panx2.  Using fluorescent-labelled (BODIPY) hexadecanoic acid, we performed metabolic labeling experiments followed by Panx2 immunoprecipitation, with and without subsequent hydroxylamine treatment to confirm Panx2 palmitoylation by measuring fluorescence intensity of immunoprecipitates with a microplate reader. Finally, we observed that reducing Panx2 expression using an shRNA approach accelerated morphological and biochemical indices of differentiation, suggesting to us that the presence of Panx2, in some way, contributes to maintaining NPC ‘stem-ness’.  These results have significant implications for unravelling the complex biological mechanisms involved in regulating the pool of NPCs in the postnatal hippocampus and were published in the Journal of Biological Chemistry

 

posted 13 Jan 2011
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References:

Original research paper
Swayne LA, Sorbara CD, Bennett SAL (2010) Pannexin 2 is expressed in hippocampal neural progenitor cells and modulates neuronal commitment. J Biol Chem, 285: 24977-24986