Lipid Raft Microdomains are Altered in Aged-Associated Neuropathologies
Raquel Marin*a, Ana Canerina-Amaroa, Ricardo Puertas Avendañoa, Jenni Rodriguez-Hernandeza, Miriam Gonzalez-Gomeza, Ibrahim Gonzalez-Marreroa, David Quinto-Alemanyb, Fátima Mesa-Herrerab, Mario Díazb
aLaboratory of Cellular Neurobiology, Department of Basic Medical Sciences, Medicine, Faculty of Health Sciences, University of La Laguna, Tenerife, Spain; bLaboratory of Membrane Physiology and Biophysics, Department of Animal Biology, Edaphology and Geology, University of La Laguna, Tenerife, Spain.
Lipid rafts are plasma membrane microstructures characterized by a particular lipid composition, rich in cholesterol, sphingolipids and saturated fatty acids. This composition confers to these microdomains distinct physico-chemical properties, including higher structural order and different viscosity. Furthermore, lipid rafts are the preferential anchoring site of multiple proteins involved in signal transduction, synapsis, cell-cell communication and neuroprotection. Recent research work has evidenced that lipid rafts show molecular alterations associated with the most common age-related neuropathologies, Alzheimer’s disease (AD) and Parkinson disease (PD), even at early asymptomatic stages. We and others have characterized that these changes include variations in the lipid composition, thereby inducing modifications in the peroxidability index and fluidity, and protein / lipid rearrangements that contribute to neuropathological development. In particular, it has been reported that lipid rafts are key factors in amyloidogenesis, aberrant protein aggregation in AD, PD and other synucleopathies. In addition, adjustments in the associations of raft-integrated proteins in signaling platforms may induce toxic cell signaling that contribute to pathological intracellular responses. Furthermore, detailed lipid profile analysis of these microenvironments in the frontal cortex of subjects at the first stages of AD (ADI/II) and incidental PD shows that profound molecular changes are observed in cortical areas since the beginning of these pathologies, suggesting that lipid raft alterations are an early common even in age-associated neurodegenerative diseases. Indeed, lipid changes are accompanied by shifting the dynamic of some of the main hallmarks of either AD or PD, such as the amyloid precursor protein, the beta-secretase (BACE) involved in amyloid beta processing in AD, and alpha synuclein aggregation in the case of PD. Overall, these findings signify that lipid and protein changes in lipid raft microdomains may be used as early predictors of these brain diseases, as potential tools of early diagnosis.
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Lipid rafts are distinct plasma membrane subdomains that have been characterized in a plethora of different cell types and tissues, including neurons. These liquid-ordered regions are resistant to non-ionic detergents, and have been traditionally extracted by flotation in 1% to 30% linear sucrose gradients, where they distribute in the upper part of the gradient. Figure 1 shows a schematic representation of lipid raft separation on a sucrose step gradient. The particular lipid composition of lipid raft microenvironments, rich in distinct lipid classes including cholesterol, sphingolipids and saturated fatty acid facilitates the compartmentalization of specific subsets of proteins. These raft-integrated proteins form clusters in highly dynamic signaling platforms (signalosomes) that significantly contribute to numerous physiological functions. Increasing data have evidenced the importance of preserving lipid raft homeostasis and balance for correct neuronal functioning. In this sense, alterations in the molecular composition of these microdomains correlate with different neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson disease (PD), Prion diseases and amyotrophic lateral sclerosis (ALS), among others. Indeed, lipid rafts appear to contribute to amyloid beta (Ab) processing and senile plaque formation, alpha synuclein (a-syn) aggregation and prion protein (PrP) interactions (1).
Figure 1. Schematic representation of the abbreviated protocol to isolate lipid raft fractions in non-ionic detergent sucrose (1% to 35%) gradients. Following ultracentrifugation (200,000g for 16-20 h), lipid raft content is distributed in the upper fraction (fraction 1) whereas non-raft membrane material remains in the pellet (Fraction 6).
Paradoxically, these microstructures are also the preferential site for triggering neuroprotective signaling responses against different injuries. In this sense, lipid rafts are a preferential target of a plethora of proteins that anchor to specific lipid moieties present in these microstructures, such as glycosylphosphatidylionositol (GPI), or are modified by palmitoylation or myristoylation to provide protein stability. Clustering of lipid/protein in lipid rafts are required for the binding of different extracellular ligands that modulate numerous neuronal processes involved in synaptic function, neuronal plasticity, cell adhesion, neurotrophism, neuroprotection, axon growth and guidance, and exo/endocytosis (2).
Changes in the lipid matrix of lipid rafts are already observed at early stages of, both, AD and PD, and include a drastic reduction in cholesterol and long-chain polyunsaturated fatty acids (PUFA) content. Consequently, there is an increase in membrane packing, compressibility and viscosity, enhancing neuropathological progression.
Our recent evidence has demonstrated that a direct consequence of the pathological lipid changes in lipid raft microstructures, in particular at early stages of AD (ADI/II) and incidental PD (iPD), is the physiological impairment of signalosomes integrated in these microdomains. These variations include aberrant protein aggregation and multimeric formation that may lead to toxic intracellular signaling responses. Thus, some studies have shown that palmitoylation of the amyloid precursor protein (APP) is required for its trafficking to lipid rafts, where it associates with scaffolding protein flotillin 1 to regulate Ab formation. This is also accompanied by a translocation of the b-secretase (BACE) to lipid rafts, thereby favoring a close interaction with APP, contributing to AD progression. Interestingly, APP/BACE convergence in lipid rafts was correlated with a higher microviscosity and molecular order of these microstructures in cortical brain areas even at ADI/II stages whereas cerebellar microdomains from the same subjects did not reproduce these results. Thus, a plausible scenario is that clustering of amyloidogenic configurations with lipid raft components may provoke microstructure destabilization and amyloidogenesis. Moreover, other amyloidopathologies such as PD and Prion protein diseases appear to involve binding of the hallmarks of these diseases to specific lipid classes found in lipid rafts, such as GM1 and PUFA which modulate multimeric aggregation and behavior of these proteins. Furthermore, PUFA deficit appears to be the key point for lipid raft imbalance and impairment. These fatty acids are preferentially distributed in the inner leaflet of the plasma membrane, and are crucial to keep homeoviscosity and peroxidation capability within the plasma membrane. In this sense, a significant deficit in these PUFA, docosahexaenoic acid (DHA) and arachidonic acid (AA) in particular, has been detected in the cortical areas of AD, PD and Lewy body dementia (LBD), a phenomenon that may profoundly affect the physiological biophysical properties of lipid raft microenvironments and, consequently, lipid raft-protein dynamics. Figure 2 illustrates a schematic representation of the proposed events associated with lipid raft impairment and neuropathology progression.
Figure 2. A proposed succession of events associating early lipid raft impairment with progressive neuropathology progression. Lipid raft alterations may appear at asymptomatic early stages of AD (I/II) and incidental PD (iPD). This impairment includes decrease levels of cholesterol, docosahexaenoic acid (DHA) and arachidonic acid (AA) that may consequently provoke an increase in the viscosity, molecular order and peroxidation of the raft microenvironment. These lipid changes also induce aberrant protein aggregations of anatomopathological markers of these neurodegenerative diseases, thereby promoting the formation of insoluble pathological aggregates, such as amyloid plaques and Lewy bodies. Moreover, protein rearrangements may trigger toxic intracellular signaling modifying healthy responses. These events may ultimately set in motion the vicious circle of neurotoxicity, thereby accelerating the neuropathology development.
Overall, on the basis of these observations we can speculate that a main early cause of lipid raft anomalies may be due to the depletion of phospholipid unsaturation that unbalances the proportion of saturated/unsaturated fatty acids. This phenomenon may increase the lipoperoxidation rate, thus increasing the generation of lipid peroxides, which are activators of distinct toxic cellular responses and disruptors or structural biomarkers.
The fact that significant lipid raft molecular changes are observed at asymptomatic stages of the most common age-related neurodegenerative diseases suggest that early lipid raft impairment may be a powerful predictor of neuropathology. In this sense, there is still a lack of accurate diagnostic tools in preclinical AD and PD. Presently, non-invasive biomarkers validated for AD include the determination in the cerebrospinal fluid (CSF) the levels of Ab1-42, total tau and phospho-tau. Similarly, CSF biomarkers for PD diagnosis include these molecules together with a-syn. However, these molecular identities are not conclusive for early stages of these diseases. Therefore, a major challenge is the characterization of novel multiparametric detectors in at-risk individuals of this sort of pathologies. In this sense, detection of lipid raft alterations may be a novel strategic approach to reach this goal (3). As previously mentioned, apart from lipid derangement, significant perturbations in the levels, location and associations of proteins integrated in lipid rafts have been detected in asymptomatic phases of, both, AD and PD. Therefore, it is plausible that perturbations of lipid raft molecular machinery may form part of ethiopathological events. In support of this, extracellular vesicles containing raft material are found in peripheral fluids, such as CSF, urine and serum. Thus, we suggest that analysis of lipoperoxidative damage-associated metabolites, and enzymes involved in lipid degradation (sphingomyelinases and phospholipases), together with changes in the dynamics of raft-associated proteins may provide valid clues from the initial signs of these neuropathologies. In this sense, phospholipase 2 (PLA2) activity, and levels of F(2)-isoprostanes are increased in CSF of AD subjects, in parallel with a reduction in the levels of free phospholipids. Moreover, aberrant assemblies of raft-related proteins observed in CSF may mirror some of the neurotoxic anomalies occurring in the cortex prior to pathological onset. Also, abnormal post-transductional modifications of some raft proteins detected at the peripheral level may predict early development of the disease. As an example, dephosphorylation of the voltage-dependent anion channel (VDAC) integrated in lipid rafts has been associated with AD progression. Furthermore, S-nitrosylation and carbonylation has been correlated with neurotoxicity. In this order of ideas, BACE1 has been shown dually regulated by S-nitrosylation and oxidative stress.
On the basis of these relevant data, we are tempted to hypothesize that future biomarkers for these age-related diseases may include multivariate lipid and protein markers found in lipid raft microdomains, and other membrane nanoscale structures that may alter their interplay as predictors of neuropathological events. Further characterization of lipid raft impairment at early-onset of these diseases may be required to provide a potential source of novel neuronal membrane biomarkers. No doubt that these studies will pave the way for more reliable tools for early recognition of these diseases.
- Marin R, Fabelo N, Fernández-Echevarría C, Canerina-Amaro A, Rodríguez-Barreto D, Quinto-Alemany D, Mesa-Herrera F, Díaz M. 2016 Lipid Raft Alterations in Aged-Associated Neuropathologies. Curr Alzheimer Res. 2016;13(9):973-84
- Marin R 2011 Signalosomes in the brain: relevance in the development of certain neuropathologies such as Alzheimer’s disease. Front Physiol. 2011 Jun 3;2:23. doi: 10.3389/fphys.2011.00023
- Marin R, Rojo JA, Fabelo N, Fernandez CE, Diaz M. 2013 Lipid raft disarrangement as a result of neuropathological progresses: a novel strategy for early diagnosis? Neuroscience. 2013 Aug 15;245:26-39. doi: 10.1016/j.neuroscience.2013.04.025
Acknowledgements: This study was supported by grant SAF2014-52582-R from Ministerio de Economía y Competitividad (Spain). ACA holds a research fellowship from ACISII, and FM from Fundación La Caixa-CajaCanarias (Spain).
Raquel Marin, Ph.D.
Dept of Basic Medical Sciences
Section Medicine, Faculty of Health Sciences
University of La Laguna
La Laguna 38320, Tenerife, Spain