Daniel A. Kirschner

pH-dependent structural transitions of Alzheimer amyloid peptides.

To understand the molecular interactions leading to the assembly of beta/44 protein into the hallmark fibrils of Alzheimers disease (AD), we have examined the ability of synthetic peptides that correspond to the beta/A4 extracellular sequence to form fibrils over the range of pH 3-10. Peptides included the sequences 1-28, 19-28, 17-28, 15-28, 13-28, 11-28, and 9-28 of beta/A4. The model fibrils were compared with isolated amyloid with respect to morphology, conformation, tinctorial properties, and stability under denaturing conditions. Electron microscopy, Fourier-transform infrared (FT-IR) spectroscopy, and x-ray diffraction revealed that the ionization states of the amino acid sidechains appeared to be a crucial feature in fibril formation. This was reflected by the ability of several peptides to undergo fibril assembly and disassembly as a function of pH. Comparisons between different beta/A4 sequences demonstrated that the fibrillar structure representative of AD amyloid was dependent upon electrostatic interactions, likely involving His-13 and Asp-23, and hydrophobic interactions between uncharged sidechains contained within residues 17-21. The results also indicated an exclusively beta-sheet conformation for the synthetic (and possibly AD fibrils) in contrast to certain other (e.g., systemic) amyloids.

Structure of beta-crystallite assemblies formed by Alzheimer beta-amyloid protein analogues: analysis by x-ray diffraction

To elucidate the relation between amyloid fibril formation in Alzheimer disease and the primary structure of the beta/A4 protein, which is the major component of the amyloid, we have been investigating the ability of peptides sharing sequences with beta/A4 to form fibrils in vitro. In previous studies we focused on the macroscopic morphology of the assemblies formed by synthetic peptides corresponding in sequence to different regions of this protein. In the present study we analyze the x-ray diffraction patterns obtained from these assemblies. All specimens showed wide angle reflections that could be indexed by an orthogonal lattice of beta-crystallites having unit cell dimensions a = 9.4 A, b = 7 A, and c = 10 A, where a refers to hydrogen bonding direction, b to polypeptide chain direction, and c to intersheet direction. Given the amino acid sequence of beta/A4 as NH2-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAT-COOH, we found that, based on their orientation and assembly, the analogues could be classified into three groups: Group A, residues 19-28, 13-28, 12-28, 11-28, 9-28, 1-28, 1-38, 1-40, 6-25, 11-25 and 34-42; Group B, residues 18-28, 17-28, and 15-28; and Group C, residues 22-35 and 26-33. For Groups A and C, the sharpest reflections were (h00), indicating that the assemblies were fibrillar, i.e., elongated in a single direction. Lateral alignment of the crystallites in Group A account for its cross-beta pattern, in which the hydrogen bonding (H-bonding) direction is the fiber (rotation) axis. By comparison, the beta-crystallites of Group C had no preferential orientation, thus giving circular scattering. For Group B, the sharpest reflections were (h0l) on the meridian, indicating that the assemblies were plate-like, i.e., extended in two directions. A series of equatorial Bragg reflections having a 40 A period indicated regular stacking of the plates, and the rotation axis was normal to the surface of the plates. Of the Group A peptides, the analogues 11-28 and 6-25 showed intensity maxima on the equator as well as on higher layer lines, indicating that the beta-crystallites are highly ordered relative to one another in the axial, H-bonding direction. This sampling of the layer lines by a larger period (60 A) suggests that the beta-crystallites are arrayed either in cylindrical or small restricted crystalline lattices. Consistent with its electron microscopic images, we modeled the structure as a tube with five or six f,-crystallites constituting the wall and with the individual crystallite, which either rotates freely or is restricted, made of five or fewer beta-pleated sheets. For the Group B peptides, the electron density projection along the b-axis was calculated from the observed intensities using phase combinations from fl-keratin.Amino acid side-chain positions were apparent and, when refined as 4-A-diameter spheres, led to a substantial decrease in the R-factors.For peptide 18-28 the electron density peaks, which are thought to correspond to side chains, were centered 3.3 A from the peptide backbone, whereas for peptides 17-28 and 15-28, these peaks were centered 1 A or more further from the backbone. Peaks having high electron density faced peaks having lower density, suggesting a favorable stereochemical arrangement of the residues. Thus, our analysis of the fiber x-ray patterns from beta/A4 peptides shows the organization of the beta-crystallites that form the wall of the amyloid fibrils as well as possible side-chain interactions.

Membrane interactions in nerve myelin: II. Determination of surface charge from biochemical data

In our accompanying paper (Inouye and Kirschner, 1988) we calculated the surface charge density at the extracellular surfaces in peripheral and central nervous system (PNS; CNS) myelins from observations on the dependency of the width of the extracellular space on pH and ionic strength. Here, we have determined the surface charge density of the membrane surfaces in myelin from its chemical composition and the localization of some of its molecular components. We then analyzed the attractive and repulsive forces between the apposed surfaces and calculated equilibrium periods for comparison with the measured values. The biochemical model accounts for the observed isoelectric range of the myelin period and, with the surface charge reduced (possibly by divalent cation binding or a space charge approximation), the model also accounts for the dependency of period on pH above the isoelectric range. At the extracellular (and cytoplasmic) surfaces the contribution of lipid (with pI approximately 2) to the net surface charge is about the same in both PNS and CNS myelin, whereas the contribution of protein depends on which ones are exposed at the two surfaces. The protein conformation and localization modulate the surface charge of the lipid, resulting in positively-charged cytoplasmic surfaces (pI approximately 9) and negatively-charged extracellular surfaces (pI approximately 2-4). The net negative charge at the extracellular surface is due in CNS myelin to lipid, and in PNS myelin to both lipid and (PO) glycoprotein. The net positive charge at the cytoplasmic surface is due in CNS myelin mostly to basic protein, and in PNS myelin to PO glycoprotein and basic protein. The invariance of the cytoplasmic packing may be due to specific short-range interactions. Our models demonstrate how the particular myelin proteins and their localization and conformation can account for the differences in inter-membrane interactions in CNS and PNS myelins.

Membrane interactions in nerve myelin. I. Determination of surface charge from effects of pH and ionic strength on period.

We have used x-ray diffraction to study the interactions between myelin membranes in the sciatic nerve (PNS) and optic nerve (CNS) as a function of pH (2-10) and ionic strength (0-0.18). The period of myelin was found to change in a systematic manner with pH and ionic strength. PNS periods ranged from 165 to 250 A or more, while CNS periods ranged from 150 to 230 A. The native periods were observed only near physiological ionic strength at neutral or alkaline pH. The smallest periods were observed in the pH range 2.5-4 for PNS myelin and pH 2.5-5 for CNS myelin. The minimum period was also observed for PNS myelin after prolonged incubation in distilled water. At pH 4, within these acidic pH ranges, myelin period increased slightly with ionic strength; however, above these ranges, the period increased with pH and decreased with ionic strength. Electron density profiles calculated at different pH and ionic strength showed that the major structural alteration underlying the changes in period was in the width of the aqueous space at the extracellular apposition of membranes; the width of the cytoplasmic space was virtually constant. Assuming that the equilibrium myelin periods are determined by a balance of nonspecific forces/i.e., the electrostatic repulsion force and the van der Walls attractive force, as well as the short-range repulsion force (hydration force, or steric stabilization), then values in the period-dependency curve can be used to define the isoelectric pH and exclusion length of the membrane. The exclusion length, which is related to the minimum period at isoelectric pH, was used to calculate the electrostatic repulsion force given the other forces. The electrostatic repulsion was then used to calculate the surface potential, which in turn was used to calculate the surface charge density (at different pH and ionic strength). We found the negative surface charge increases with pH at constant ionic strength and with ionic strength at constant pH. We suggest that the former is due to deprotonation of the ionizable groups on the surface while the latter is due to ion binding. Interpretation of our data in terms of the chemical composition of myelin is given in the accompanying paper (Inouye and Kirschner, 1988). We also calculated the total potential energy functions for the different equilibrium periods and found that the energy minima became shallower and broader with increasing membrane separation. Finally, it was difficult to account directly for certain structural transitions from a balance of nonspecific forces. Such transitions included the abrupt appearance of the native period at alkaline pH and physiological ionic strength and the discontinuous compaction after prolonged treatment in distilled water. Possibly, in PNS myelin conformational modification of PO glycoprotein occurs under these conditions. The invariance of the cytoplasmic space suggests the presence of specific short-range interactions between surfaces at this apposition.

Ganglioside localization on myelinated nerve fibres by cholera toxin binding

SummaryGM1 ganglioside has been localized on the surfaces of myelinated, peripheral nerve fibres by using immunofluorescence to detect cholera toxin receptors. Unfixed, mouse sciatic nerves were teased into individual, intact fibres in order to expose their extracellular surfaces. Cholera toxin binding sites were abundant at all nodes of Ranvier; they were scarce on the internodal fibre surfaces. The nodal receptors were resistant to various degradative enzymes, including trypsin and proteinase K. Proteases did not unmask receptors on the internodal surfaces. Exogenous GM1 successfully competed for the toxin binding sites on the fibres. From this evidence and the specificity of cholera toxin binding, we conclude that GM1 ganglioside is abundantly present on the membrane surfaces of peripheral nodes of Ranvier and is not present on the internodal Schwann cell surfaces in an appreciable amount. The patterns of fluorescence within the node suggest that the axon and Schwann cell structures are sites where GM1 is localized.Treatment of the teased fibres withVibrio cholerae neuraminidase, which is known to reduce polysialogangliosides to the monosialoganglioside Gm1, induced cholera toxin binding on the internodal Schwann cell surfaces. The induced receptors, as well as their precursors, were resistant to trypsin and proteinase K. We conclude that the internodal Schwann cell surface is rich in an unidentified polysialoganglioside(s) that can be converted to GM1 by neuraminidase.

Membrane structure in isolated and intact myelins

The biochemical composition of myelin and the topology of its constituent lipids and proteins are typically studied using membranes that have been isolated from whole, intact tissue using procedures involving hypotonic shock and sucrose density gradient centrifugation. To what extent, however, are the structure and intermembrane interactions of isolated myelin similar to those of intact myelin? We have previously reported that intact and isolated myelins do not always show identical myelin periods, indicating a difference in membrane-membrane interactions. The present study addresses the possibility that this is due to altered membrane structure. Because x-ray scattering from isolated myelin sometimes consists of overlapping Bragg reflections or is continuous, we developed nonlinear least squares procedures for analyzing the total intensity distribution after film scaling, background subtraction, and Lorentz correction. We calculated electron density profiles of isolated myelin for comparison with membrane profiles from intact myelin. The change in the width of the extracellular space and the relative invariance of the cytoplasmic space as a function of pH and ionic strength that we previously found for intact nerve was largely paralleled by isolated myelin. There were two exceptions: isolated CNS myelin was resistant to swelling under all conditions, and isolated PNS myelin in hypotonic saline showed indefinite swelling at the extracellular apposition. However, electron density profiles of isolated myelins, calculated to 30 A resolution, did not show any major change in structure compared with intact myelin that could account for the differences in interactions.

A Survey of Neurological Mutant Mice. pp 99–109

The lipids of white matter and peripheral nerve from mutant mice with known myelin deficiencies were analyzed by one- and two-dimensional high-performance thin-layer chromatography and quantitated by

A survey of neurological mutant mice. II: Lipid composition of myelinated tissue in possible myelin mutants

The lipids of white matter and peripheral nerve from neurological mutant mice with possible myelin abnormalities were analyzed by thin-layer chromatography and quantitated by densitometry. Eight mutants had major abnormalities in the central nervous system (CNS) and/or peripheral nervous system (PNS) tissues examined (optic nerve, and trigeminal and sciatic nerves). In the optic nerve of axJ/axJ, there were increases of 20-30% in the levels of the major phospholipids; peripheral nerve was normal. In bc3J/bc3J CNS, the major phospholipids and cholesterol were increased by 25-40%; the PNS was normal. In myd/myd CNS, there were increases of about 20% in the levels of both forms of cerebrosides and in the major phospholipids; in the PNS the lipids were normal. ot/ot CNS had 20-40% reductions of all the glycolipids and minor alterations in some of the phospholipids and cholesterol; the PNS had 20% losses of both forms of cerebrosides. In the PNS of ji/ji, there were decreases of 10-40% among the glycolipids and of 15-25% in three of the major phospholipids; the CNS was virtually normal. In the PNS of dtJ/dtJ, vb/vb and wr/wr, almost all lipids were significantly decreased. The CNS of dtJ/dtJ and vb/vb were normal; wr/wr had minor reductions of certain glycolipids and phospholipids. Six mutants had relatively minor lipid abnormalities in their myelinated tissues. In cr/cr PNS, there were elevated levels of the cerebrosides and major phospholipids; the CNS was virtually normal. In db/db CNS and PNS, there were reduced levels of the nonhydroxy forms of cerebroside and sulfatide. The major change in htr/htr was the elevation of all the glycolipids in the CNS. In the CNS of Lc/+, nonhydroxy cerebroside was reduced. In shm/shm PNS, nonhydroxy sulfatide was elevated and there were small decreases in some of the phospholipids. wl/wl CNS showed decreases among most of the glycolipids. Mutants homozygous for du, mto, spa and tg had virtually normal lipid levels in both the optic and peripheral nerves. Cholesterol ester, lysophospholipids and other unusual lipid species were not detected in any of the mutants. The plasmalogen forms of ethanolamine and choline phosphatides were at normal levels in all mutants that otherwise had significant alterations among their lipids. Although many alterations in lipid composition were found in these mutants, the changes were moderate compared to the classical myelin mutants and indicate that none of the mutants are severely myelin-deficient.(ABSTRACT TRUNCATED AT 400 WORDS)

Radial component of CNS myelin: Junctional subunit structure and supramolecular assembly

SummaryThe radial component is a structural specialization within CNS myelin that is believed to stabilize the apposition of membranes in the internode. Previous observations on thin sections and freeze-fracture replicas show that this junctional complex consists of linear, particulate strands that run parallel to the nerve fibre axis and radially through the myelin sheath, but details on its molecular organization are lacking. The objective of our current study was to gain further insight into its arrangement and composition by examining its fine-structure and incidence in: myelin with known deficits in protein composition (e.g.,shiverer, transgenicshiverer, myelin deficient andjimpy mutant mice); isolated CNS myelin, which has been shown by X-ray diffraction to be more stable than intact CNS myelin; and human white matter, in which this junctional complex has not yet been described. Our results confirm the localization and general appearance of the radial component as previously reported. In addition, we found that: (1) the radial component occurs abundantly in human CNS myelin where it has a complex subunit structure; (2) the constituent junctional unit of this structure is organized as a pair of globular domains (each ∼40 Å diameter) at the extracellular apposition which is linked by ∼15 Å diameter filaments extending through the bilayer to ∼25 Å globular domains in the adjacent cytoplasmic apposition; (3) the radial component is present with apparently normal structure in the sparse, compact myelin of murine mutants containing either different amounts of MBP or no PLP which indicates that neither of these proteins is necessary for junctional integrity; (4) the radial component is present in purified CNS myelin membranes which may account for the stability of these membranes; and (5) the radial component is structurally resistant to Triton, which suggests a method for its further biochemical characterization. Finally, from an analysis of images from tilted transverse and longitudinal sections, we haye reconstructed a model of its three-dimensional, supramolecular organization

Myelin Membrane from Adrenoleukodystrophy Brain White Matter—Biochemical Properties

Abstract: Adrenoleukodystrophy (ALD) is an X‐linked progressive neurological disorder characterized by the accumulation of saturated very‐long‐chain fatty acids (C24 to C30) in lipids, especially cholesterol esters of tne brain white matter and adrenal cortex. In the present study we have investigated the localization of accumulated cholesterol esters in brain white matter. During isolation of purified myelin membrane from regions of active demyelination, significant enrichment in cholesterol ester was found in two fractions, mainly in a low‐density floating fraction and to a lesser degree in the purified myelin preparation. The fatty acid composition of cholesterol esters from both the ALD floating and myelin fractions was enriched approximately 10‐fold in saturated very‐long‐chain fatty acids (≥C24) compared with control preparations.