Methodological Pitfalls of Investigating Lipid Rafts in the Brain: What Are We Still Missing?

The purpose of this review is to succinctly examine the methodologies used in lipid raft research in the brain and to highlight the drawbacks of some investigative approaches. Lipid rafts are biochemically and biophysically different from the bulk membrane. A specific lipid environment within membrane domains provides a harbor for distinct raftophilic proteins, all of which in concert create a specialized platform orchestrating various cellular processes. Studying lipid rafts has proved to be arduous due to their elusive nature, mobility, and constant dynamic reorganization to meet the cellular needs. Studying neuronal lipid rafts is particularly cumbersome due to the immensely complex regional molecular architecture of the central nervous system. Biochemical fractionation, performed with or without detergents, is still the most widely used method to isolate lipid rafts. However, the differences in solubilization when various detergents are used has exposed a dire need to find more reliable methods to study particular rafts. Biochemical methods need to be complemented with other approaches such as live-cell microscopy, imaging mass spectrometry, and the development of specific non-invasive fluorescent probes to obtain a more complete image of raft dynamics and to study the spatio-temporal expression of rafts in live cells.

Start Me Up: How Can Surrounding Gangliosides Affect Sodium-Potassium ATPase Activity and Steer towards Pathological Ion Imbalance in Neurons?

Gangliosides, amphiphilic glycosphingolipids, tend to associate laterally with other membrane constituents and undergo extensive interactions with membrane proteins in cis or trans configurations. Studies of human diseases resulting from mutations in the ganglioside biosynthesis pathway and research on transgenic mice with the same mutations implicate gangliosides in the pathogenesis of epilepsy. Gangliosides are reported to affect the activity of the Na+/K+-ATPase, the ubiquitously expressed plasma membrane pump responsible for the stabilization of the resting membrane potential by hyperpolarization, firing up the action potential and ion homeostasis. Impaired Na+/K+-ATPase activity has also been hypothesized to cause seizures by several mechanisms. In this review we present different epileptic phenotypes that are caused by impaired activity of Na+/K+-ATPase or changed membrane ganglioside composition. We further discuss how gangliosides may influence Na+/K+-ATPase activity by acting as lipid sorting machinery providing the optimal stage for Na+/K+-ATPase function. By establishing a distinct lipid environment, together with other membrane lipids, gangliosides possibly modulate Na+/K+-ATPase activity and aid in "starting up" and "turning off" this vital pump. Therefore, structural changes of neuronal membranes caused by altered ganglioside composition can be a contributing factor leading to aberrant Na+/K+-ATPase activity and ion imbalance priming neurons for pathological firing.

Gangliosidome of a Human Hippocampus in Temporal Lobe Epilepsy Resolved by High-Resolution Tandem Mass Spectrometry.

In this study, we developed a high-resolution tandem mass spectrometry (HR MS) approach to assess presumed changes in gangliosidome of a human hippocampus affected by temporal lobe epilepsy (TLE) in comparison with a normal hippocampus. Gangliosides, membrane glycolipids, are particularly diverse and abundant in the human brain, and participate in ion transport and modulation of neuronal excitability. Changes in structural ganglioside pattern potentially linked to TLE molecular pathogenesis have not been explored in detail. Aiming to characterize TLE-specific gangliosidome, we analyzed the native gangliosides purified from a human hippocampal tissue sample affected by TLE and a control hippocampus using HR MS. Marked differences of ganglioside expression were shown in TLE vs. control, particularly with respect to the sialylation degree of components, discovered as a characteristic feature of TLE. Another major finding is the occurrence of tetrasialofucogangliosides in TLE and species modified by either O-acetylation or CH3COO-. Structural analysis by higher-energy collisional dissociation (HCD) MS/MS gave rise to fragmentation patterns implying that the GQ1b (d18:1/18:0) isomer is specifically associated with TLE. Further investigation in a larger sample is needed in order to confirm the discovery of ganglioside structures specifically expressed in human TLE and to provide information on the probable role of gangliosides in the molecular events underlying seizures.

Plasma Membrane Calcium ATPase-Neuroplastin Complexes Are Selectively Stabilized in GM1-Containing Lipid Rafts.

The recent identification of plasma membrane (Ca2+)-ATPase (PMCA)-Neuroplastin (Np) complexes has renewed attention on cell regulation of cytosolic calcium extrusion, which is of particular relevance in neurons. Here, we tested the hypothesis that PMCA-Neuroplastin complexes exist in specific ganglioside-containing rafts, which could affect calcium homeostasis. We analyzed the abundance of all four PMCA paralogs (PMCA1-4) and Neuroplastin isoforms (Np65 and Np55) in lipid rafts and bulk membrane fractions from GM2/GD2 synthase-deficient mouse brains. In these fractions, we found altered distribution of Np65/Np55 and selected PMCA isoforms, namely PMCA1 and 2. Cell surface staining and confocal microscopy identified GM1 as the main complex ganglioside co-localizing with Neuroplastin in cultured hippocampal neurons. Furthermore, blocking GM1 with a specific antibody resulted in delayed calcium restoration of electrically evoked calcium transients in the soma of hippocampal neurons. The content and composition of all ganglioside species were unchanged in Neuroplastin-deficient mouse brains. Therefore, we conclude that altered composition or disorganization of ganglioside-containing rafts results in changed regulation of calcium signals in neurons. We propose that GM1 could be a key sphingolipid for ensuring proper location of the PMCA-Neuroplastin complexes into rafts in order to participate in the regulation of neuronal calcium homeostasis.

Neuroplastin in human cognition: review of literature and future perspectives.

Synaptic glycoprotein neuroplastin is involved in synaptic plasticity and complex molecular events underlying learning and memory. Studies in mice and rats suggest that neuroplastin is essential for cognition, as it is needed for long-term potentiation and associative memory formation. Recently, it was found that some of the effects of neuroplastin are related to regulation of calcium homeostasis through interactions with plasma membrane calcium ATPases. Neuroplastin is increasingly seen as a key factor in complex brain functions, but studies in humans remain scarce. Here we summarize present knowledge about neuroplastin in human tissues and argue its genetic association with cortical thickness, intelligence, schizophrenia, and autism; specific immunolocalization depicting hippocampal trisynaptic pathway; potential role in tissue compensatory response in neurodegeneration; and high, almost housekeeping, level of spatio-temporal gene expression in the human brain. We also propose that neuroplastin acts as a housekeeper of neuroplasticity, and that it may be considered as an important novel cognition-related molecule in humans. Several promising directions for future investigations are suggested, which may complete our understanding of neuroplastin actions in molecular basis of human cognition.

Who's in, who's out? Re-evaluation of lipid raft residents.

Lipid rafts, membrane microdomains enriched with (glyco)sphingolipids, cholesterol, and select proteins, act as cellular signalosomes. Various methods have been used to separate lipid rafts from bulk (non-raft) membranes, but most often, non-ionic detergent Triton X-100 has been used in their isolation. However, Triton X-100 is a reported disruptor of lipid rafts. Histological evidence confirmed raft disruption by Triton X-100, but remarkably revealed raft stability to treatment with a related polyethylene oxide detergent, Brij O20. We report isolation of detergent-resistant membranes from mouse brain using Brij O20 and its use to determine the distribution of major mammalian brain gangliosides, GM1, GD1a, GD1b and GT1b. A different distribution of gangliosides-classically used as a raft marker-was discovered using Brij O20 versus Triton X-100. Immunohistochemistry and imaging mass spectrometry confirm the results. Use of Brij O20 results in a distinctive membrane distribution of gangliosides that is not all lipid raft associated, but depends on the ganglioside structure. This is the first report of a significant proportion of gangliosides outside raft domains. We also determined the distribution of proteins functionally related to neuroplasticity and known to be affected by ganglioside environment, glutamate receptor subunit 2, amyloid precursor protein and neuroplastin and report the lipid raft populations of these proteins in mouse brain tissue. This work will enable more accurate lipid raft analysis with respect to glycosphingolipid and membrane protein composition and lead to improved resolution of lipid-protein interactions within biological membranes.

Neuronal Signaling by Thy-1 in Nanodomains With Specific Ganglioside Composition: Shall We Open the Door to a New Complexity?

Thy-1 is a small membrane glycoprotein and member of the immunoglobulin superfamily of cell adhesion molecules. It is abundantly expressed in many cell types including neurons and is anchored to the outer membrane leaflet via a glycosyl phosphatidylinositol tail. Thy-1 displays a number of interesting properties such as fast lateral diffusion, which allows it to get in and out of membrane nanodomains with different lipid composition. Thy-1 displays a broad expression in different cell types and plays confirmed roles in cell development, adhesion and differentiation. Here, we explored the functions of Thy-1 in neuronal signaling, initiated by extracellular binding of αVß3 integrin, may strongly dependent on the lipid content of the cell membrane. Also, we assort literature suggesting the association of Thy-1 with specific components of lipid rafts such as sialic acid containing glycosphingolipids, called gangliosides. Furthermore, we argue that Thy-1 positioning in nanodomains may be influenced by gangliosides. We propose that the traditional conception of Thy-1 localization in rafts should be reconsidered and evaluated in detail based on the potential diversity of neuronal nanodomains.

Hippocampal expression of cell-adhesion glycoprotein neuroplastin is altered in Alzheimer's disease.

Cell-adhesion glycoprotein neuroplastin (Np) is involved in the regulation of synaptic plasticity and balancing hippocampal excitatory/inhibitory inputs which aids in the process of associative memory formation and learning. Our recent findings show that neuroplastin expression in the adult human hippocampus is specifically associated with major hippocampal excitatory pathways and is related to neuronal calcium regulation. Here, we investigated the hippocampal expression of brain-specific neuroplastin isoform (Np65), its relationship with amyloid and tau pathology in Alzheimer's disease (AD), and potential involvement of neuroplastin in tissue response during the disease progression. Np65 expression and localization was analysed in six human hippocampi with confirmed AD neuropathology, and six age-/gender-matched control hippocampi by imunohistochemistry. In AD cases with shorter disease duration, the Np65 immunoreactivity was significantly increased in the dentate gyrus (DG), Cornu Ammonis 2/3 (CA2/3), and subiculum, with the highest level of Np expression being located on the dendrites of granule cells and subicular pyramidal neurons. Changes in the expression of neuroplastin in AD hippocampal areas seem to be related to the progression of disease. Our study suggests that cell-adhesion protein neuroplastin is involved in tissue reorganization and is a potential molecular marker of plasticity response in the early neurodegeneration process of AD.

Neuroplastin deletion in glutamatergic neurons impairs selective brain functions and calcium regulation: implication for cognitive deterioration.

The cell adhesion molecule neuroplastin (Np) is a novel candidate to influence human intelligence. Np-deficient mice display complex cognitive deficits and reduced levels of Plasma Membrane Ca2+ ATPases (PMCAs), an essential regulator of the intracellular Ca2+ concentration ([iCa2+]) and neuronal activity. We show abundant expression and conserved cellular and molecular features of Np in glutamatergic neurons in human hippocampal-cortical pathways as characterized for the rodent brain. In Nptn lox/loxEmx1Cre mice, glutamatergic neuron-selective Np ablation resulted in behavioral deficits indicating hippocampal, striatal, and sensorimotor dysfunction paralleled by highly altered activities in hippocampal CA1 area, sensorimotor cortex layers I-III/IV, and the striatal sensorimotor domain detected by single-photon emission computed tomography. Altered hippocampal and cortical activities correlated with reduction of distinct PMCA paralogs in Nptn lox/loxEmx1Cre mice and increased [iCa2+] in cultured mutant neurons. Human and rodent Np enhanced the post-transcriptional expression of and co-localized with PMCA paralogs in the plasma membrane of transfected cells. Our results indicate Np as essential for PMCA expression in glutamatergic neurons allowing proper [iCa2+] regulation and normal circuit activity. Neuron-type-specific Np ablation empowers the investigation of circuit-coded learning and memory and identification of causal mechanisms leading to cognitive deterioration.