DMS as an orthogonal separation to LC/ESI/MS/MS for quantifying isomeric cerebrosides in plasma and cerebrospinal fluid.

Figure 1. Chemical structures of isomeric GlcCer and GalCer. Shown are structures of GlcCer(d18:1/16:0) and GalCer(d18:1/16:0). Both have the same Cer(d18:1/16:0) core structure to which a sugar molecule, glucose or galactose, is attached via β-glycosidic linkage. The only structural difference between them is the 4’-hydroxyl group of the sugar moiety (shaded) being either in the equatorial position (GlcCer) or axial position (GalCer).

Glucosylceramides (GlcCers) and galactosylceramides (GalCers) are structurally related lipids that are important for mammalian cells. However, their presence in the body is tissue-specific, and problems with the body’s ability to maintain the right balance of theses lipids can lead to devastating but drastically different diseases. For example, mutations in GBA gene with deficient glucosylcerebrosidase lead to build up of GlcCer in cells and the production of toxic glucosylsphingosine. Individuals with mutations of GBA gene also present higher risk of developing Parkinson’s disease. On the other hand, people with mutations in the GALC gene develop Krabbe disease due to cell’s inability to clear GalCer and the production of toxic galactosylsphingosine. Thus, specific profiling and quantification of GlcCer and GalCer are essential for biomarker discovery and drug screening. The specific quantification of GlcCer and GalCer is, however, particularly challenging due to their virtually identical structures (Figure 1). To address this challenge, we developed a new liquid chromatography mass spectrometry (LC/MS)-based method using differential ion mobility spectrometry (DMS) to allow rapid and reproducible separation and quantification of structurally very similar GlcCer and GalCer in a single run (Figure 2). We have shown that our method is capable of quantifying specific GlcCer and GalCer in human plasma and cerebrospinal fluid (CSF). As human CSF is a scarce and precious resource, particularly from disease-specific biobanks, this method facilitates measurement in the applied neurological research setting.

Figure 2. Profiling and quantification of GlcCer and GalCer in human plasma (Left panel) and CSF (Right panel) using our method.

Xu H, Boucher FR, Nguyen TT, Taylor G.P, Tomlinson JJ, Ortega RA, Simons B, Schlossmacher MG, Saunders-Pullman R, Shaw W, and Bennet SAL.. (2019) DMS as an orthogonal separation to LC/ESI/MS/MS for quantifying isomeric cerebrosides in plasma and cerebrospinal fluid. J Lipid Res 60: 200-211. doi: 10.1194/jlr.D089797[download]

Dysregulated lipid metabolism and its role in α-Synucleinopathy in Parkinson’s Disease.

Parkinson’s disease (PD) is projected to affect up to 9 million people worldwide by 2030, with the ultimate cause behind 90% of these cases remaining a black box. While the pathomechanisms underlying Parkinson’s disease have still not been elucidated, recent research is pointing more and more towards the involvement of lipids in the development and progression of PD, including cytotoxic interactions with the protein α-synuclein (the accumulation of which is the major pathological hallmark of PD) and dysregulation of lipid pathways. Furthermore, mutations in genes encoding for enzymes involved in lipid metabolism have been identified as risk factors for PD, while a number of studies employing lipidomics have shown PD-specific lipid alterations in both brain and plasma of patients. Here we review the current and recent knowledge about the physiological and pathophysiological aspects of lipids and their dysregulation in PD while highlighting conflicting and incongruous findings, as well as gaps in knowledge. As lipid changes appear to precede other molecular changes, identifying these could lead to earlier diagnosis, the ability to stratify patients with other dementias, as well as the development of strategies to impede neurodegeneration. Aside from toxic lipid changes, we also discuss potentially protective lipids, the modulation of whose levels could be employed as a novel therapeutic strategy. Continuing to explore PD-associated changes in lipids will in the future lead to the characterization of PD at the molecular level and allow for the development of disease-mechanism associated biomarkers and cause-directed treatment focusing on lipid metabolism.

Alecu I, Bennett SAL. (2019). Frontiers in Neuroscience. 13:328. doi: 10.3389/fnins.2019.00328. [download]

Connexins and pannexins in neuronal development and adult neurogenesis

In neurodegenerative disorders, specific cell lineages (i.e., neurons or glia) and subtypes (i.e., neurons expressing certain transmitters) are deleted. Little can be done to replace damaged brain cells or restore cellular connectivity in the weeks that follow a stroke or over the course of a progressive neurodegenerative disorder such as Alzheimer Disease. The adult human brain supports neural stem and progenitor cells - cells capable of producing new neurons and glia that functionally integrate into the existing circuitry. Mobilizing or introducing these populations into damaged tissue represents a novel, potentially transformative, approach to brain repair. Yet injury-induced neurogenesis can also result in the integration of inappropriate neuronal subtypes into existing brain circuitry (i.e., inhibitory neurons replacing excitatory neurons). The presence of these "ectopic" neurons can precipitate further neurological impairment. Clearly, brain repair strategies will depend upon a comprehensive understanding of how appropriate cell lineages are effectively generated in degenerating tissue. Connexin and pannexin proteins are the structural units of gap junction channels that bridge two cells allowing direct exchange of material when the bridge opens and two cells become one. We review here compelling evidence implicating the passage of ions, metabolites, and second messengers between cells via gap junctions or through open hemichannels in non-junctional membranes in the control of neural stem and progenitor cell proliferation, survival, and commitment to either a glial or neuronal lineage.

Swayne LA*, Bennett SAL (2016) "Connexins and pannexins in neuronal development and adult neurogenesis." BMC Cell Biology 2016, 17(Suppl 1):10 DOI 10.1186/s12860-016-0089-5 *Corresponding author [download] [neurogenesis movie]

Preparation of gap junctions in membrane microdomains for immunoprecipitation and mass spectrometry interactome analysis.

As we develop more complex biochemical and analytical techniques for identifying protein interaction networks of hub proteins, it is becoming increasingly clear that these hubs are critical platforms for intracellular signaling cascades. Progress in this field has been limited by several technical challenges, namely that the cellular environment is too complex to properly identify protein interactions of less abundant proteins. In addition, the lipid environment of each subcellular organelle presents a unique complement of structural lipids that must be appropriately solubilized to enable integral membrane proteins to be liberated from their lipid environments, without de-stabilizing any associated protein complexes. Here, we present a step-by-step protocol for isolating membrane microdomains from liver tissue, to facilitate the identification of gap junction protein interactomes by immunoprecipitation and mass spectrometry. We provide technical notes for adapting this protocol to other integral membrane proteins, tissues, and cell culture models. These procedures are valuable tools for enrichment and purification of less abundant membrane proteins, and greatly facilitate the identification of integral membrane protein signaling interactomes.

Fowler S*, Akins M, Bennett SAL* (2016) "Preparation of gap junctions in membrane microdomains for immunoprecipitation and mass spectrometry interactome analysis." In Gap Junctions Protocols, edited by Mathieu Vinken and Scott Johnstone, 1147: 113-132, New York, NY: Springer Press. *Joint corresponding authors [download]

A TgCRND8 Mouse Model of Alzheimer's Disease Exhibits Sexual Dimorphisms in Behavioral Indices of Cognitive Reserve

Alzheimer's disease (AD) is sexually dimorphic in that while more men succumb to a prodromal form of the disease yet females more readily convert to late onset AD. Due to an enhanced cognitive reserve, it seems that males are more resistant to toxic AD pathologies in the brain than females. Cognitive reserve is defined as the ability adapt to different cognitive strategies using alternative brain networks to cope with progressively toxic pathologies. Our lab has recently reported and characterized a transgenic mouse model that mimics this effect. These mice are transgenically altered to give them a genetic susceptibility to the human pathologies that afflict AD patients. In comparing male and female mice, we have found that while both are impaired relative to sex-matched control mice in a learning and memory task, female mice are more impaired than males. This was due to the ability of males to adopt different cognitive strategies in the face of AD pathology while females could not which demonstrates that males have greater cognitive reserve than females in this mouse model. We have also found that this is not the influence of greater AD pathology in females, further demonstrating the common relationship with the human condition. This research is crucial to further understanding the relationship between AD cognitive decline and sex. It provides an ideal model to discover and target sex-specific processes involved the susceptibility to AD pathology.

Granger MW, Franko B, Taylor MW, Messier C, St George-Hyslop P, Bennett SAL (2016) A TgCRND8 mouse model of Alzheimer's disease exhibits sexual dimorphisms in behavioral indices of cognitive reserve, J Alz Dis, 51:757-773 doi: 10.3233/JAD-150587 [download]