Anna Fasano

Conformation of bovine myelin basic protein purified with bound lipids.

Abstract The basic protein of myelin (called MBP) is an extrinsic protein of the myelin membrane. Its structure and function are still unknown. MBP has been extensively studied in its water-soluble form, but it is also known in a detergent-soluble form, which is purified with endogenous myelin lipids and should correspond to the native form of the protein in the membrane. In order to acquire insight into the structure of MBP, we have carried out circular dichroism (CD) experiments on the protein both in the lipid-free and in the lipid-bound form. Our data clearly show that lipid-free MBP is mainly disordered with only a small amount having α-helix and β-sheet motifs. On the other hand, the lipid-bound form of MBP appears to have a consistent amount of ordered secondary structure. Theoretical predictions, made using different computational methods, substantially confirm the tendency of the protein to assume an ordered secondary structure in accordance with our CD results.

Interferon-beta inhibits the expression of metalloproteinases in rat glial cell cultures: implications for multiple sclerosis pathogenesis and treatment

Matrix metalloproteinases (MMPs) have been identified as mediators of brain injury in multiple sclerosis (MS) and it has recently been reported that treatment of MS patients with interferon-beta (IFN-b) reduces MMP-9 serum levels and in vitro release from monocytes. We investigated whether IFN-b is able to modulate the expression of MMPs in glial cell cultures. Rat microglial and astrocyte cultures were treated with different doses of IFN-b, then activated by exposure to LPS. In another set of experiments cells were simultaneously activated with LPS and treated with IFN-b. C ulture supernatants collected from astrocytes and microglia were subjected to zymography for the assessment of MMP-2 and MMP-9. Increased amounts of MMP-9 and MMP-2 were observed in supernatants from LPS-treated astrocytes in comparison with supernatants from nontreated control cells. MMP-9 also increased in LPS-treated microglia. The treatment of astrocytes and microglia with IFN-b inhibited dose-dependently the expression of both MMP-2 and MMP-9 in LPS-treated astrocytes and of MMP-9 in LPS-treated microglia. These results demonstrate a modulating effect of IFN-b on the release of MMPs from C NS cells. This effect represents an additional mechanism by which IFN-b may decrease the development of new C NS lesions in the course of MS.

Inhibitory Effect of Polyunsaturated Fatty Acids on MMP-9 Release from Microglial Cells—Implications for Complementary Multiple Sclerosis Treatment

We investigated whether polyunsaturated fatty acids (PUFA), which might be a useful complementary therapy among patients with multiple sclerosis (MS), are able to modulate matrix metalloproteinase (MMP) production in microglial cultures. MMPs are myelinotoxic factors. Primary cultures of rat microglia were treated with different doses of omega-3 (ω-3) PUFA or purified fish oil, containing a mixture of ω-3 and ω-6 PUFA, and simultaneously activated by exposure to lipopolysaccharide (LPS). Culture supernatants were subjected to zymography and Western blot analysis for the assessment of MMP-2 and MMP-9 levels. Increased amounts of MMP-9, but not of the constitutively expressed MMP-2, were observed in supernatants from LPS-treated microglia in comparison with non-treated control cells. The treatment with both ω-3 PUFA and fish oil dose-dependently inhibited the LPS-induced production of MMP-9. Our results suggest that a low fat diet supplemented with ω-3 PUFA may become recommended for the well being of MS patients under therapy.

Multilamellar packing of myelin modeled by lipid-bound MBP.

Membrane compaction and adhesion at the major dense line (cytoplasmic apposition) of myelin, particularly in the central nervous system (CNS), is typically attributed to myelin basic protein (MBP). To explore the role of MBP in myelin membrane adhesion, we attempted to reconstitute the major dense line of myelin from purified lipid‐bound MBP, which is a detergent‐soluble form of MBP that retains the binding of all the myelin lipids. Removal of detergent by long‐term dialysis yielded a precipitate, which, when analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and thin‐layer chromatography, contained MBP that was still associated with myelin lipids, but in different proportions than in the native membrane. Comparison of lipid composition among isolated myelin, MBP‐free myelin lipids, and lipid‐bound MBP aggregates showed that the lipid‐bound form of the protein was specifically enriched in phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, phosphatidylinositol, and phosphatidylserine. Electron microscopy and x‐ray diffraction demonstrated that the lipid‐MBP complexes formed multilayers having periods of 70–85 Å, which correspond in width to individual myelin membranes. By contrast, the lipids alone assembled as multilayers having a period of ∼40 Å. Thus, the detergent‐soluble form of MBP, which is bound to lipids, might serve as a simple model for the cytoplasmic apposition of myelin. J. Neurosci. Res. 59:513–521, 2000

Small Angle X-Ray Scattering from Lipid-Bound Myelin Basic Protein in Solution

The structure of myelin basic protein (MBP), purified from the myelin sheath in both lipid-free (LF-MBP) and lipid-bound (LB-MBP) forms, was investigated in solution by small angle x-ray scattering. The water-soluble LF-MBP, extracted at pH < 3.0 from defatted brain, is the classical preparation of MBP, commonly regarded as an intrinsically unfolded protein. LB-MBP is a lipoprotein-detergent complex extracted from myelin with its native lipidic environment at pH > 7.0. Under all conditions, the scattering from the two protein forms was different, indicating different molecular shapes. For the LB-MBP, well-defined scattering curves were obtained, suggesting that the protein had a unique, compact (but not globular) structure. Furthermore, these data were compatible with earlier results from molecular modeling calculations on the MBP structure which have been refined by us. In contrast, the LF-MBP data were in accordance with the expected open-coil conformation. The results represent the first direct structural information from x-ray scattering measurements on MBP in its native lipidic environment in solution.

Impact of Manganese Neurotoxicity on MMP-9 Production and Superoxide Dismutase Activity in Rat Primary Astrocytes. Effect of Resveratrol and Therapeutical Implications for the Treatment of CNS Diseases

Manganese (Mn) is an environmental contaminant and its overexposure contributes to the pathophysiological processes of numerous disorders of the central nervous system in humans with mechanisms of action not completely understood. Activation of astrocytes and the subsequent release of neurotoxic factors have been implicated to contribute to neurodegeneration. Here, we assessed the molecular basis of the effects of Mn on modulation of matrix metalloproteinases-2 (MMP-2) and -9 (MMP-9) in rat astrocyte cultures. Primary cultures of rat astrocytes were exposed to different doses of MnCl2. Culture supernatants and cell lysates were used for the detection of MMP-2 and MMP-9 levels and mRNA expression, respectively. The exposure of astrocytes to MnCl2 induced the levels and expression of MMP-9 in a dose-dependent manner. The addition of resveratrol (RSV) inhibited both levels and expression of MMP-9 in astrocytes, whereas N-acetylcysteine (NAC) and quercetin (QRC) were ineffective in inhibiting MMP-9. As a possible mechanism of Mn-induced MMP-9, we determined intracellular redox state in Mn-treated astrocytes by assessing superoxide dismutase (SOD) activity and intracellular reactive oxygen species (ROS) and found a significant increase of ROS and a decrease of SOD activity. RSV, NAC, and QRC restored the redox state. The study of the mitogen-activated protein kinase signaling pathway demonstrated that MMP-9 transcription is mainly regulated by extracellular-regulated protein kinases (ERK). Pretreatment with RSV significantly reduced ERK activation suggesting that its ability to counteract MMP-9 overexpression is due not only to a general redox balance phenomenon but also to the modulation of ERK signaling pathway.

Cationic liposomes target sites of acute neuroinflammation in experimental autoimmune encephalomyelitis.

The binding selectivity of charged liposomes to the spinal cord of rats affected by experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, was investigated. Positively and negatively charged liposomes were injected into the tail vein of rats, and blood/brain barrier (BBB) targeting was determined by confocal microscopy as a function of the temporal evolution of the inflammatory response. Accumulation in spinal cord endoneural vessels was observed for cationic, but not for anionic, liposomes, and only in EAE but not in healthy rats. The overall binding efficacy paralleled the severity of the clinical score, but targeting was observed already before clinical manifestation of inflammation. Preferential binding of positively charged liposomes in the course of acute EAE can be ascribed to subtle changes of BBB morphology and charge distribution in a similar way as for the binding of cationic particles to proliferating vasculature in chronic inflammation and angiogenesis. Our findings suggest that vascular changes related to increased binding affinity for cationic particles are very early events within the inflammatory reaction in acute EAE. Investigation of cationic vascular targeting can help to shed further light on these occurrences, and, potentially, new diagnostic and therapeutic options may become available. In neuroinflammatory diseases, cationic colloidal carrier particles may enable intervention at affected BBB by an approach which is independent from permeability increase.

An X-Ray Absorption Spectroscopy Study of the Zinc Environment in Langmuir-Blodgett Phospholipid Multilayers

For the first time x-ray absorption spectroscopy was used to investigate the Zn environment in Langmuir-Blodgett multilayers. The multilayers were taken as a model of the multilamellar structure of the myelin sheath, the membrane surrounding the nerve axon, which plays a crucial role for signal transduction along the axon. The layers were assembled from the phospholipid dilauroylphosphatidic acid, both in the presence and in the absence of myelin basic protein. The analysis of the extended x-ray absorption fine structure and of the near edge regions of the x-ray absorption spectra at the Zn K-edge provided an accurate description of the local structure showing that the Zn ions are bound to the heads of the phospholipid molecules. The myelin basic protein induces a distortion on the Zn local environment due to a steric constraint but does not substitute the phosphate headgroups. These findings represent an important step in understanding the interplay among myelin basic protein, Zn, and the lipids of the myelin sheath.

Purification of bovine P2 myelin protein with bound lipids.

THE P2 protein is a neuritogenic, small basic protein present in PNS myelin. It belongs to the family of the cytoplasmic lipid-binding proteins and can be incorporated in lipidic bilayers. P2 has been purified and crystallized only in the lipid-free form. Here we show that the P2 protein can be purified with bound lipids by applying to PNS myelin the same procedure that as used to purify lipid-bound myelin basic protein from CNS myelin. SDS-PAGE showed a single band of 16. 5 kDa, and TLC showed the presence of most of the myelin lipids associated with the protein. Lipid-bound P2 revealed different circular dichroism spectra from the corresponding lipid-free form, indicating that lipids influence P2 conformation

Crystals of P2 myelin protein in lipid-bound form.

The P2 protein of peripheral nervous system myelin induces experimental allergic neuritis in rats, a model of Guillain-Barré syndrome in humans. Previous purification procedures have used acid extraction to obtain the protein in lipid-free form (LF-P2). Here, we have purified the P2 protein in lipid-bound form (LB-P2) by extracting myelin with the detergent CHAPS, followed by Cu(2+)-affinity column chromatography. All myelin lipids were present in the preparation as shown by high-performance thin-layer chromatography and mass spectrometry. The LB-P2 preparation, which differs from LF-P2 in solubility and in the secondary-structure composition, was dialyzed to remove unbound lipids and excess detergent and crystallized using the hanging-drop vapor diffusion technique. Crystals of lipid-bound P2 appeared usually very reproducibly within 2 weeks at pH 5.7 in polyethylene glycol 6000 (PEG6000) at concentrations of 20-30% (w/v), and larger crystals were obtained by additional sitting-drop crystallization. X-ray diffraction showed reflections up to 2.7A. The crystallization conditions (25-30% PEG6000, pH 5.0) and the unit cell dimensions (a = 94.5A, b = 94.5A, c=74.2A, alpha = beta = 90 degrees, gamma = 120 degrees ) of LB-P2 were different from those earlier described for LF-P2 (10% PEG4000, pH 3, and unit cell dimensions a = 91.8A, b = 99.5A, c = 56.5A, alpha = beta = gamma = 90.0 degrees ). It is important that P2 has been crystallized with specifically bound lipids; therefore, solving this new crystal structure will reveal details of this proteins behavior and role in the myelin sheath.