Lipid metabolism in myelinating glial cells: lessons from human inherited disorders and mouse models.

The integrity of central and peripheral nervous system myelin is affected in numerous lipid metabolism disorders. This vulnerability was so far mostly attributed to the extraordinarily high level of lipid synthesis that is required for the formation of myelin, and to the relative autonomy in lipid synthesis of myelinating glial cells because of blood barriers shielding the nervous system from circulating lipids. Recent insights from analysis of inherited lipid disorders, especially those with prevailing lipid depletion and from mouse models with glia-specific disruption of lipid metabolism, shed new light on this issue. The particular lipid composition of myelin, the transport of lipid-associated myelin proteins, and the necessity for timely assembly of the myelin sheath all contribute to the observed vulnerability of myelin to perturbed lipid metabolism. Furthermore, the uptake of external lipids may also play a role in the formation of myelin membranes. In addition to an improved understanding of basic myelin biology, these data provide a foundation for future therapeutic interventions aiming at preserving glial cell integrity in metabolic disorders.

Consequences of modified lipid and glucose metabolism on peripheral nervous system structure and function

284 million people worldwide suffered from type 2 diabetes mellitus (T2DM) in 2010, which will, in approximately half of them, lead to the development of diabetic peripheral neuropathy (DPN). Although DPN is the most common complication of diabetes mellitus and the leading cause of non-traumatic amputations its pathophysiology is still poorly understood. To get more insight into the molecular mechanism underlying DPN in T2DM, I used a rodent model of T2DM, the db/db mice.¦ln vivo electrophysiological recordings of diabetic animals indicated that in addition to reduced nerve conduction velocity db/db mice also present increased nerve excitability. Further ex vivo evaluation of the electrophysiological properties of db/db nerves clearly established a presence of the peripheral nerve hyperexcitability (PNH) phenotype in diabetic animals. Using pharmacological inhibitors we demonstrated that PNH is mostly mediated by the decreased activity of Kv1 channels. ln agreement with these data 1 observed that the diabetic condition led to a reduced presence of the Kv1.2 subunits in juxtaparanodal regions of db/db peripheral nerves whereas its mANA and protein expression levels were not affected. Lmportantly, I confirmed a loss of juxtaparanodal Kv1.2 subunits in nerve biopsies from type 2 diabetic patients. Together these observations indicate that the type 2 diabetic condition leads to potassium-channel mediated changes of nerve excitability thus identifying them as potential drug targets to treat sorne of the DPN related symptoms.¦Schwann cells ensheath and isolate peripheral axons by the production of myelin, which consists of lipids and proteins in a ratio of 2:1. Peripheral myelin protein 2 (= P2, Pmp2 or FABP8) was originally described as one of the most abundant myelin proteins in the peripheral nervous system. P2, which is a member of the fatty acid binding protein (FABP) family, is a 14.8 kDa cytosolic protein expressed on the cytoplasmic side of compact myelin membranes. As indicated by their name, the principal role of FABPs is thought to be the binding and transport of fatty acids.¦To study its role in myelinating glial cells I have recently generated a complete P2 knockout mouse model (P2-/-). I confirmed the loss of P2 in the sciatic nerve of P2-/- mice at the mRNA and protein level. Electrophysiological analysis of the adult (P56) mutant mice revealed a mild but significant reduction in the motor nerve conduction velocity. lnterestingly, this functional change was not accompanied by any detectable alterations in general myelin structure. However, I have observed significant alterations in the mRNA expression level of other FABPs, predominantly FABP9, in the PNS of P2-/- mice as compared to age-matched P2+/+ mice indicating a role of P2 in the glial myelin lipid metabolism.¦Le diabète de type 2 touche 284 million de personnes dans le monde en 2010 et son évolution conduit dans la moitié des cas à une neuropathie périphérique diabétique. Bien que la neuropathie périphérique soit la complication la plus courante du diabète pouvant conduire jusqu'à l'amputation, sa physiopathologie est aujourd'hui encore mal comprise. Dans le but d'améliorer les connaissances moléculaires expliquant les mécanismes de la neuropathie liée au diabète de type 2, j'ai utilisé un modèle murin du diabète de type 2, les souris db/db.¦ln vivo, les enregistrements éléctrophysiologiques des animaux diabétiques montrent qu'en plus d'une diminution de la vitesse de conduction nerveuse, les souris db/db présentent également une augmentation de l'excitabilité nerveuse. Des mesures menées Ex­ vivo ont montré l'existence d'un phénotype d'hyperexcitabilité sur les nerfs périphériques isolés d'animaux diabétiques. Grâce à l'utilisation d'inhibiteurs pharmacologiques, nous avons pu démontrer que l'hyperexcitabilité démontrée était due à une réduction d'activité des canaux Kv1. En accord avec ces données, j'ai observé qu'une situation de diabète conduisait à une diminution des canaux Kv1.2 aux régions juxta-paranodales des nerfs périphériques db/db, alors que l'expression du transcrit et de la protéine restait stable. J'ai également confirmé l'absence de canaux Kv1.2 aux juxta-paranoeuds de biopsies de nerfs de patients diabétiques. L'ensemble de ces observations montrent que les nerfs périphériques chez les patients atteints de diabète de type 2 est due à une diminution des canaux potassiques rapides juxtaparanodaux les identifiant ainsi comme des cibles thérapeutiques potentielles.¦Les cellules de Schwann enveloppent et isolent les axones périphériques d'une membrane spécialisée, la myéline, composée de deux fois plus de lipides que de protéines. La protéine P2 (Pmp2 "peripheral myelin protein 2" ou FABP8 "fatty acid binding protein") est l'une des protéines les plus abondantes au système nerveux périphérique. P2 appartient à la famille de protéines FABP liant et transportant les acides gras et est une protéine cytosolique de 14,8 kDa exprimée du côté cytoplasmique de la myéline compacte.¦Afin d'étudier le rôle de P2 dans les cellules de Schwann myélinisantes, j'ai généré une souris knockout (P2-/-). Après avoir validé l'absence de transcrit et de protéine P2 dans les nerfs sciatiques P2-/-, des mesures électrophysiologiques ont montré une réduction modérée mais significative de la vitesse de conduction du nerf moteur périphérique. Il est important de noter que ces changements fonctionnels n'ont pas pu être associés à quelconque changement dans la structure de la myéline. Cependant, j'ai observé dans les nerfs périphériques P2-/-, une altération significative du niveau d'expression d'ARNm d'autres FABPs et en particulier FABP9. Ce dernier résultat démontre l'importance du rôle de la protéine P2 dans le métabolisme lipidique de la myéline.

Polymer-induced tubulation in lipid vesicles

A mechanism of extraction of tubular membranes from a lipid vesicle is presented. A concentration gradient of anchoring amphiphilic polymers generates tubes from budlike vesicle protrusions. We explain this mechanism in the framework of the Canham-Helfrich model. The energy profile is analytically calculated and a tube with a fixed length, corresponding to an energy minimum, is obtained in a certain regime of parameters. Further, using a phase-field model, we corroborate these results numerically. We obtain the growth of tubes when a polymer source is added, and the budlike shape after removal of the polymer source, in accordance with recent experimental results.

Membrane-destabilizing activity of pH-responsive cationic lysine-based surfactants: role of charge position and alkyl chain length

Many strategies for treating diseases require the delivery of drugs into the cell cytoplasm following internalization within endosomal vesicles. Thus, compounds triggered by low pH to disrupt membranes and release endosomal contents into the cytosol are of particular interest. Here, we report novel cationic lysine-based surfactants (hydrochloride salts of N¿- and N¿-acyl lysine methyl ester) that differ in the position of the positive charge and the length of the alkyl chain. Amino acid-based surfactants could be promising novel biomaterials in drug delivery systems, given their biocompatible properties and low cytotoxic potential. We examined their ability to disrupt the cell membrane in a range of pH values, concentrations and incubation times, using a standard hemolysis assay as a model of endosomal membranes. Furthermore, we addressed the mechanism of surfactant-mediated membrane destabilization, including the effects of each surfactant on erythrocyte morphology as a function of pH. We found that only surfactants with the positive charge on the ¿-amino group of lysine showed pH-sensitive hemolytic activity and improved kinetics within the endosomal pH range, indicating that the positive charge position is critical for pH-responsive behavior. Moreover, our results showed that an increase in the alkyl chain length from 14 to 16 carbon atoms was associated with a lower ability to disrupt cell membranes. Knowledge on modulating surfactant-lipid bilayer interactions may help us to develop more efficient biocompatible amino acid-based drug delivery devices.

Actively maintained lipid nanodomains in biomembranes

Lipid rafts, defined as domains rich in cholesterol and sphingolipids, are involved in many important plasma membrane functions. Recent studies suggest that the way cells handle membrane cholesterol is fundamental in the formation of such lateral heterogeneities. We propose to model the plasma membrane as a nonequilibrium phase-separating system where cholesterol is dynamically incorporated and released. The model shows how cellular regulation of membrane cholesterol may determine the nanoscale lipid organization when the lipid mixture is close to a phase separation boundary, providing a plausible mechanism for raft formation in vivo.

Nonequilibrium patterns and shape fluctuations in reactive membranes

A simple kinetic model of a two-component deformable and reactive bilayer is presented. The two differently shaped components are interconverted by a nonequilibrium reaction, and a phenomenological coupling between local composition and curvature is proposed. When the two components are not miscible, linear stability analysis predicts, and numerical simulations show, the formation of stationary nonequilibrium composition/curvature patterns whose typical size is determined by the reactive process. For miscible components, a linearization of the dynamic equations is performed in order to evaluate the correlation function for shape fluctuations from which the behavior of these systems in micropipet aspiration experiments can be predicted.

Generation of dynamic structures in nonequilibrium reactive membranes

We present a nonequlibrium approach for the study of a flexible bilayer whose two components induce distinct curvatures. In turn, the two components are interconverted by an externally promoted reaction. Phase separation of the two species in the surface results in the growth of domains characterized by different local composition and curvature modulations. This domain growth is limited by the effective mixing due to the interconversion reaction, leading to a finite characteristic domain size. In addition to these effects, first introduced in our earlier work [ Phys. Rev. E 71 051906 (2005)], the important new feature is the assumption that the reactive process actively affects the local curvature of the bilayer. Specifically, we suggest that a force energetically activated by external sources causes a modification of the shape of the membrane at the reaction site. Our results show the appearance of a rich and robust dynamical phenomenology that includes the generation of traveling and/or oscillatory patterns. Linear stability analysis, amplitude equations, and numerical simulations of the model kinetic equations confirm the occurrence of these spatiotemporal behaviors in nonequilibrium reactive bilayers.

The V-ATPase proteolipid cylinder promotes the lipid-mixing stage of SNARE-dependent fusion of yeast vacuoles.

The V-ATPase V(0) sector associates with the peripheral V(1) sector to form a proton pump. V(0) alone has an additional function, facilitating membrane fusion in the endocytic and late exocytic pathways. V(0) contains a hexameric proteolipid cylinder, which might support fusion as proposed in proteinaceous pore models. To test this, we randomly mutagenized proteolipids. We recovered alleles that preserve proton translocation, normal SNARE activation and trans-SNARE pairing but that impair lipid and content mixing. Critical residues were found in all subunits of the proteolipid ring. They concentrate within the bilayer, close to the ring subunit interfaces. The fusion-impairing proteolipid substitutions stabilize the interaction of V(0) with V(1). Deletion of the vacuolar v-SNARE Nyv1 has the same effect, suggesting that both types of mutations similarly alter the conformation of V(0). Also covalent linkage of subunits in the proteolipid cylinder blocks vacuole fusion. We propose that a SNARE-dependent conformational change in V(0) proteolipids might stimulate fusion by creating a hydrophobic crevice that promotes lipid reorientation and formation of a lipidic fusion pore.

Heat shock response in photosynthetic organisms: membrane and lipid connections.

The ability of photosynthetic organisms to adapt to increases in environmental temperatures is becoming more important with climate change. Heat stress is known to induce heat-shock proteins (HSPs) many of which act as chaperones. Traditionally, it has been thought that protein denaturation acts as a trigger for HSP induction. However, increasing evidence has shown that many stress events cause HSP induction without commensurate protein denaturation. This has led to the membrane sensor hypothesis where the membrane's physical and structural properties play an initiating role in the heat shock response. In this review, we discuss heat-induced modulation of the membrane's physical state and changes to these properties which can be brought about by interaction with HSPs. Heat stress also leads to changes in lipid-based signaling cascades and alterations in calcium transport and availability. Such observations emphasize the importance of membranes and their lipids in the heat shock response and provide a new perspective for guiding further studies into the mechanisms that mediate cellular and organismal responses to heat stress.

Grape epicatechin conjugates prevent erythrocyte membrane protein oxidation

Epicatechin conjugates obtained from grape have shown antioxidant activity in various systems. However, how these conjugates exert their antioxidant benefits has not been widely studied. We assessed the activity of epicatechin and epicatechin conjugates on the erythrocyte membrane in the presence and absence of a peroxyl radical initiator, to increase our understanding of their mechanisms. Thus, we studied cell membrane fluidity by fluorescence anisotropy measurements, morphology of erythrocytes by scanning electron microscopy, and finally, red cell membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our data showed that incubation of red cells in the presence of epicatechin derivatives altered membrane fluidity and erythrocyte morphology but not the membrane protein pattern. The presence in the medium of the peroxyl radical initiator 2,2′-azobis(amidinopropane) dihydrochloride (AAPH) resulted in membrane disruptions at all levels analyzed, causing changes in membrane fluidity, cell morphology, and protein degradation. The presence of antioxidants avoided protein oxidation, indicating that the interaction of epicatechin conjugates with the lipid bilayer might reduce the accessibility of AAPH to membranes, which could explain in part the inhibitory ability of these compounds against hemolysis induced by peroxidative insult.

Lipid mobilization and gluconeogenesis in plants: Do glyoxylate cycle enzyme activities constitute a real cycle? A hypothesis

Glyoxysomes are specialized peroxisomes present in various plant organs such as germinating cotyledons or senescing leaves. They are the site of beta-oxidation and of the glyoxylate cycle. These consecutive pathways are essential to the maintenance of gluconeogenesis initiated by the degradation of reserve or structural lipids. In contrast to mitochondrial beta-oxidation, which is prevalent in animal cells, glyoxysomal beta-oxidation and the glyoxylate cycle have no direct access to the mitochondrial respiratory chain because of the impermeability of the glyoxysomal membrane to the reduced cofactors. The necessity of NAD(+) regeneration can conceivably be fulfilled by membrane redox chains and/or by transmembrane shuttles. Experimental evidence based on the active metabolic roles of higher plant glyoxysomes and yeast peroxisomes suggests the coexistence of two mechanisms, namely a reductase/peroxidase membrane redox chain and a malate/aspartate shuttle susceptible to transfer electrons to the mitochondrial ATP generating system. Such a model interconnects beta-oxidation, the glyoxylate cycle, the respiratory chain and gluconeogenesis in such a way that glyoxysomal malate dehydrogenase is an essential and exclusive component of beta-oxidation (NAD(+) regeneration). Consequently, the classical view of the glyoxylate cycle is superseded by a tentative reactional scheme deprived of cyclic character.

Anestésicos locais: interação com membranas de eritrócitos de sangue humano, estudada por ressonância magnética nuclear de ¹H e 31P

The literature carries many theories about the mechanism of action of local anesthetics (LA). We can highlight those focusing the direct effect of LA on the sodium channel protein and the ones that consider the interaction of anesthetic molecules with the lipid membrane phase. The interaction between local anesthetics and human erythrocyte membranes has been studied by ¹H and 31P nuclear magnetic resonance spectroscopy. It was found that lidocaine (LDC) and benzocaine (BZC) bind to the membranes, increase the mobility of the protons of the phospholipid's acyl chains, and decrease the mobility and/or change the structure of the polar head groups. The results indicate that lidocaine molecules are inserted across the polar and liquid interface of the membrane, establishing both electrostatic (charged form) and hydrophobic (neutral form) interactions. Benzocaine locates itself a little deeper in the bilayer, between the interfacial glycerol region and the hydrophobic core. These changes in mobility or conformation of membrane lipids could affect the Na+-channel protein insertion in the bilayer, stabilizing it in the inactivated state, thus causing anesthesia.

Dual Action of BPC194: A Membrane Active Peptide Killing Bacterial Cells

Membrane active peptides can perturb the lipid bilayer in several ways, such as poration and fusion of the target cell membrane, and thereby efficiently kill bacterial cells. We probe here the mechanistic basis of membrane poration and fusion caused by membrane-active, antimicrobial peptides. We show that the cyclic antimicrobial peptide, BPC194, inhibits growth of Gram-negative bacteria and ruptures the outer and inner membrane at the onset of killing, suggesting that not just poration is taking place at the cell envelope. To simplify the system and to better understand the mechanism of action, we performed Förster resonance energy transfer and cryogenic transmission electron microscopy studies in model membranes and show that the BPC194 causes fusion of vesicles. The fusogenic action is accompanied by leakage as probed by dual-color fluorescence burst analysis at a single liposome level. Atomistic molecular dynamics simulations reveal how the peptides are able to simultaneously perturb the membrane towards porated and fused states. We show that the cyclic antimicrobial peptides trigger both fusion and pore formation and that such large membrane perturbations have a similar mechanistic basis

Myoglobins: the link between discoloration and lipid oxidation in muscle and meat

Aerobic metabolism changes rapidly to glycolysis post-mortem resulting in a pH-decrease during the transformation of muscle in to meat affecting ligand binding and redox potential of the heme iron in myoglobin, the meat pigment. The "inorganic chemistry" of meat involves (i) redox-cycling between iron(II), iron(III), and iron(IV)/protein radicals; (ii) ligand exchange processes; and (iii) spin-equilibra with a change in coordination number for the heme iron. In addition to the function of myoglobin for oxygen storage, new physiological roles of myoglobin are currently being discovered, which notably find close parallels in the processes in fresh meat and nitrite-cured meat products. Myoglobin may be characterized as a bioreactor for small molecules like O2, NO, CO, CO2, H2O, and HNO with importance in bio-regulation and in protection against oxidative stress in vivo otherwise affecting lipids in membranes. Many of these processes may be recognised as colour changes in fresh meat and cured meat products under different atmospheric conditions, and could also be instructive for teaching purposes.

Double-bilayer: a new phase formed by lysophospholipids and the corresponding fatty acid

The product of catalytic activity of the enzyme phospholipase A2, which resembles the core unit of animal toxins, on phospholipids is a 1:1 mixture of lysolipid and fatty acid. This mixture was studied by time-resolved simultaneous small- and wide angle x-ray diffraction over the temperature range from 23 to 53.5ºC. An unusually large lamellar structure was observed, with d = 11 nm, contradicting the complex functional dimer model between lysolipid and fatty acid. It can be explained by formation of a "double-bilayer", a new phase consisting of two different bilayers, one formed by lysophospholipid and other by fatty acid, bound together by head group interactions. Its strucutre was confirmed by simulations of the X-ray scattering pattern.