Structural analysis of Alzheimer's beta(1-40) amyloid: protofilament assembly of tubular fibrils.

Detailed structural studies of amyloid fibrils can elucidate the way in which their constituent polypeptides are folded and self-assemble, and exert their neurotoxic effects in Alzheimer's disease (AD). We have previously reported that when aqueous solutions of the N-terminal hydrophilic peptides of AD beta-amyloid (A beta) are gradually dried in a 2-Tesla magnetic field, they form highly oriented fibrils that are well suited to x-ray fiber diffraction. The longer, more physiologically relevant sequences such as A beta(1-40) have not been amenable to such analysis, owing to their strong propensity to polymerize and aggregate before orientation is achieved. In seeking an efficient and inexpensive method for rapid screening of conditions that could lead to improved orientation of fibrils assembled from the longer peptides, we report here that the birefringence of a small drop of peptide solution can supply information related to the cooperative packing of amyloid fibers and their capacity for magnetic orientation. The samples were examined by electron microscopy (negative and positive staining) and x-ray diffraction. Negative staining showed a mixture of straight and twisted fibers. The average width of both types was approximately 70 A, and the helical pitch of the latter was approximately 460 A. Cross sections of plastic-embedded samples showed a approximately 60-A-wide tubular structure. X-ray diffraction from these samples indicated a cross-beta fiber pattern, characterized by a strong meridional reflection at 4.74 A and a broad equatorial reflection at 8.9 A. Modeling studies suggested that tilted arrays of beta-strands constitute tubular, 30-A-diameter protofilaments, and that three to five of these protofilaments constitute the A beta fiber. This type of structure--a multimeric array of protofilaments organized as a tubular fibril--resembles that formed by the shorter A beta fragments (e.g., A beta(6-25), A beta(11-25), A beta(1-28)), suggesting a common structural motif in AD amyloid fibril organization.

In vitro amyloid fibril formation by synthetic peptides corresponding to the amino terminus of apoSAA isoforms from amyloid-susceptible and amyloid-resistant mice.

Specific proteins of the apolipoprotein serum amyloid (apoSAA) family that are synthesized in large quantities during the acute, early phase of inflammation can serve as the proteinaceous precursors for amyloid fibrils. To model fibrillogenesis in such inflammatory diseases, we have used electron microscopy and X-ray diffraction to examine the structures formed by synthetic peptides corresponding in sequence to the 11 amino-terminal amino acids of murine apoSAA1, apoSAAcej, and apoSAA2 and to the 15 amino-terminal amino acids of apoSAA2. This region is reported to be the major fibrillogenic determinant of apoSAA isoforms. Both in 1 mM Tris buffer and in 35% acetonitrile, 0.1% trifluoracetic acid (ACN/TFA), all of the peptides formed macromolecular assemblies consisting of twisted, approximately 40- to 60-A-thick ribbons, which varied in width from around 40-70 A (for 11-mer apoSAA2 in Tris) up to 900 A (for the other peptides). X-ray diffraction patterns recorded from lyophilized peptides, vapor-hydrated samples, and solubilized/dried samples showed hydrogen bonding and intersheet reflections typical of a beta-pleated sheet conformation. The coherent lengths measured from the breadths of the X-ray reflections indicated that with hydration the growth of the assemblies in the intersheet stacking direction was comparable to that in the hydrogen-bonding direction, and analysis of oriented samples showed that the beta-strands were oriented perpendicular to both the long axis and the face of the assemblies. These X-ray results are consistent with the ribbon- or plate-like morphology of the individual aggregates and emphasize the polymorphic nature of amyloidogenic peptides. Our findings demonstrate that X-ray diffraction measurements on vapor-hydrated or solubilized/dried versus lyophilized, amyloidogenic peptides are a good indicator of their fibrillogenic potential. For example, from the highest to the lowest potential, the peptides examined here were ranked as: Abeta1-28 > Abeta1-40 > apoSAA1 approximately apoSAAcej > apoSAA2 > Abeta17-42. Experiments in which the three different 11-mer apoSAA isoforms were solubilized in ACN/TFA and then combined as binary mixtures showed that the ribbon morphology was not affected but that the extent of hydrogen bonding in the assemblies was substantially reduced. Our observations on the in vitro assembly of apoSAA analogs emphasize that amyloid fibril formation and morphology depend on primary sequence, length of polypeptide chain, the presence of additional fibrillogenic polypeptides, and solvent conditions.

An immunochemical, ultrastructural, and developmental characterization of the horizontal basal cells of rat olfactory epithelium.

The olfactory epithelium, which retains a capacity for neurogenesis throughout life, contains two categories of basal cells, dark/horizontal and light/globose, neither of which is fully characterized with respect to their function during the processes of neurogenesis and epithelial reconstitution after injury. The aim of this study was to define the potential biological role(s) of dark/horizontal basal cells (D/HBCs) in the epithelium by performing immunochemical, electron microscopic, and developmental analyses of this cell population. The D/HBCs express several specific immunochemical characteristics, which include the rat homologues of human cytokeratins 5 and 14, which were identified on the basis of staining with subunit-specific monoclonal antibodies and two-dimensional immunoblot analysis of the immunoreactive proteins. Indeed, the D/HBCs are the only cells in the olfactory mucosa that express these specific cytokeratins. The D/HBCs also express an alpha-galactose or alpha-N-acetyl galactosamine moiety to which the I beta 4 isolectin from Bandeiraea simplicifolia binds. Moreover, the D/HBCs are heavily labeled by two different antibodies against the EGF receptor and by a monoclonal antibody that binds to phosphotyrosine. These characteristics are also common to the basal cells of respiratory epithelium. The electron microscopic analysis of the basal region of the olfactory epithelium and the light microscopic immunofluorescence observations demonstrate that the D/HBCs provide a bridge between the basal processes of some sustentacular cells and the basal lamina. The most striking ultrastructural feature of the D/HBCs is their enfolding of virtually all bundles of olfactory axons within tunnels formed where D/HBCs arch over the basal lamina. The intimacy of the arrangement between D/HBCs and olfactory axons suggests that signals may pass from axons to D/HBCs or vice-versa. With respect to the development of D/HBCs, cells that express cytokeratins 5 and 14 and the EGF receptor first appear near the boundary with respiratory epithelium late in development, but do not extend throughout the olfactory epithelium until the middle of the first postnatal week. Taken together, the present findings and previously published data suggest that D/HBCs help to maintain the structural integrity of the olfactory epithelium, participate in its recovery from injury, and may also function to signal the status of the neuronal population of the epithelium.

Histopathology of olfactory mucosa in Kallmann's syndrome.

Olfactory mucosa was harvested by intranasal biopsy from a man with Kallmann's syndrome in whom the absence of the olfactory bulbs was documented by magnetic resonance imaging. On electron microscopic examination, several pathologic changes were evident in the olfactory mucosa. First, most olfactory neurons lacked cilia (ie, were morphologically immature). Second, the fila olfactoria had fewer than the normal number of axons, and a large proportion of them were apparently undergoing electron lucent degeneration. Finally, neuromatous collections of axons were seen superficial to the basement membrane in the epithelium. Similar changes have been observed in the mucosa of experimentally bulbectomized rodents. Accordingly, a constellation of pathologic changes--axonal degeneration, neuronal immaturity, and the formation of intraepithelial neuromas--seems to be characteristic of olfactory mucosa that cannot innervate the olfactory bulb in both humans and animals. On the basis of our observations, it is worth investigating the status of the olfactory bulb in other forms of human anosmia in which similar morphological changes are observed in the mucosa, such as persistent posttraumatic anosmia and isolated congenital anosmia.

Olfactory sensory neurons are trophically dependent on the olfactory bulb for their prolonged survival.

In most neural systems, developing neurons are trophically dependent on contact with their synaptic target for their survival and for some features of their differentiation. However, in the olfactory system, it is unclear whether or not the survival and differentiation of olfactory sensory neurons depend on contact with the olfactory bulb (normally the sole synaptic target for these neurons). In order to address this issue, we examined neuronal life-span and differentiation in adult rats subjected to unilateral olfactory bulb ablation at least 1 month prior to use. Life-span of a newly generated cohort of olfactory neurons was determined by labeling them at their "birth" via the incorporation of 3H-thymidine. In the absence of the bulb, neurons are continually produced at a twofold greater rate. However, the epithelium on the ablated side is thinner, indicating that average neuronal life-span must be reduced in the targetless epithelium. Indeed, nearly 90% of the labeled neurons disappear from the bulbectomized side between 5 d and 2 weeks of neuronal age. Moreover, on electron microscopic examination, olfactory axons are degenerating in large numbers on the ablated side. Since labeled neurons migrate apically through the width of the epithelium during this same period, it appears that most, if not all, neurons on the ablated side have a life-span on the order of 2 weeks or less. In contrast, there is a more moderate degree of neuronal loss on the unoperated side of the same animals during the first 2 weeks after tracer injection, and that occurs while the neurons are concentrated in the deeper half of the epithelium, suggesting that there is a preexisting population of neurons in the control epithelium that does not die during this period. Likewise, degenerating axons are much less frequent on the unoperated side. We conclude that life-span is significantly shorter for olfactory neurons born in the targetless epithelium and that olfactory neurons are trophically dependent on the presence of the bulb for their prolonged survival. Neuronal differentiation in the absence of the bulb was assessed according to ultrastructural criteria and the pattern of protein expression using antisera to the growth associated protein GAP-43 and the olfactory marker protein. By both measures, most neurons in the epithelium on the bulbectomized side, but not all, are immature.(ABSTRACT TRUNCATED AT 400 WORDS)