Gel electrophoretic distinction between Congo Red nonreactive beta-amyloid (1-42) and beta-amyloid (1-40).

Congo-Red nonreactive beta-amyloid (1-42) exhibits in gel electrophoresis (pH 8.82, 0.01 M ionic strength, 2 degree C) a surface charge density larger than that of the corresponding peptide of length (1-40), and a size indistinguishable from that of (1-40).

Gel electrophoretic distinction between toxic and nontoxic forms of beta-amyloid (1-40).

The in vitro toxicity of synthetic beta-amyloid (1-40) correlates with its binding to Congo red (CR). Potentially, therefore, CR binding to the beta-amyloid containing neuritic plaques in Alzheimer's disease could be used diagnostically. Using polyacrylamide under nondenaturing conditions, the present study shows that both CR binding and nonbinding synthetic beta-amyloid exhibits multiple charge-isomeric and size-isomeric species. The CR binding species exhibit values of free electrophoretic mobility, related to the surface charge density of the protein, which are less than those of the CR non-binding species within 95% confidence limits. Since surface net charge and solubility are correlated, the decreased solubility of the CR binding species may be responsible for the relative abundance and CR binding of beta-amyloid in the neuritic plaques of Alzheimer patients.

Beta-amyloid induced increase in choline flux across PC12 cell membranes.

Beta-amyloid peptide is the main constituent of senile plaques and is implicated in the pathogenesis of Alzheimer's disease. It has been shown to be both neurotoxic and neurotrophic in vivo, and its effects have been suggested to be mediated in part by alterations in membrane transport. In the present study, we investigated the effect of beta-amyloid (1-40) on choline transport in cultured PC12 cells. We found that exposure to 46 or 92 microM beta-amyloid (1-40) increased [14C]choline flux in PC12 cells in a concentration-dependent manner, whereas exposure to reverse sequence beta-amyloid (40-1) had no effect. If there is a similar effect in vivo, the increased beta-amyloid dependent permeability to choline could lead to depletion of cellular choline stores and could contribute to the selective vulnerability of cholinergic neurons in Alzheimer's disease.

Simultaneous assessment of conformation and aggregation of beta-amyloid peptide using electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry was used to study conformation and aggregation of the synthetic beta-amyloid peptide, residues 1-40 (betaA4), as a function of concentration and sample aging. All mass spectra showed a major envelope of peaks corresponding to charge states of 7-3 of the monomeric form of betaA4. In addition, weaker envelopes of peaks corresponding to charge states of dimeric, trimeric, and tetrameric betaA4 species were seen under gentle ionization conditions. The average charge state of the envelope associated with the monomeric form decreased by ca. 0.5 z as samples were aged, indicating that the relatively open form (likely random coil) of the peptide was modified into the more compact form (likely beta-sheet) as a function of sample aging. The aggregate forms became weaker and ultimately were absent both in the more dilute solutions and in aged aliquots of the concentrated sample. These aggregates were interpreted as assemblies of the random coil form. We interpret our inability to see an ion envelope that can be associated with aggregates of the beta-sheet form to be a consequence of the presumed very compact nature of this form. A model for the formation of betaA4 fibrils is proposed and discussed.

Predicting the in vitro toxicity of synthetic beta-amyloid (1-40).

The in vitro toxicity of synthetic beta-amyloid (betaA4) is variable and unpredictable, limiting its use as a research tool. This study describes a method using Congo red (CR) to predict the in vitro toxicity of betaA4 solutions. Histopathologically, CR is used to stain the neuritic, betaA4-containing plaques, one of the hallmarks of Alzheimer's disease. In this study, synthetic betaA4 solutions were incubated with CR at a molar ratio of 1.0:2.5. The solutions were centrifuged and the absorbance of the supernatants were measured. Predictions of nontoxicity correlated with absorbance readings near zero. Toxicity was evaluated relative to control cells (vehicle only), using a hemocytometer to count PC-12 cells that excluded trypan blue. The positive predictive value of the test was 78% and the negative predictive value was 100%. To use this test, the toxic concentration(s) of betaA4 must first be established empirically. Then, the CR test can be used to evaluate the potential toxicity of betaA4 solutions at similar concentrations. Thus, this test can be used under a variety of laboratory circumstances.

Effects of chronic beta-amyloid treatment on fatty acid incorporation into rat brain.

The present study evaluated the effects of chronic A beta administration on radio-labeled plasma fatty acid incorporation in rat brain. A beta was chronically infused intraventricularly via an osmotic minipump, for 1 week, at a concentration of 460 microM. After the infusion, fatty acid incorporation was quantified using an in vivo method developed in this laboratory. Three radiolabeled fatty acids were separately infused IV in awake animals. Biochemical analyses of fatty acid incorporation and histology for A beta showed no differences between control (vehicle infusion only) and experimental groups. However, in vitro tests on the cytotoxicity of A beta showed that it caused significant cell death relative to controls (PC-12 cells). The lack of effect of infused A beta on radiolabeled fatty acid incorporation is discussed.

Distribution and synaptology of glossopharyngeal afferent nerve terminals in the nucleus of the solitary tract of the hamster.

The distribution and synaptology of the afferent fibers of the glossopharyngeal nerve (IXN) in the hamster were studied by using horseradish peroxidase (HRP) histochemistry visualized with light and electron microscopy. Crystals of HRP were applied to the trunk of IXN in the vicinity of the petrosal ganglion. The densest IXN afferent label was distributed within the nucleus of the solitary tract (nst), just caudal to but overlapping with the area of termination of the facial nerve. Labeled IXN fibers extended rostrally to the principal trigeminal nucleus and caudally to the cervical spinal cord. There was significant labeling within the spinal trigeminal complex; the area postrema and the medullary reticular formation contained some labeled fibers. Ultrastructurally, the synaptic arrangements of anterogradely labeled IXN fibers were examined in the nst. Quantitative measures were taken of the area, maximum diameter, perimeter, and vesicles of labeled endings and the length of their synaptic junctions with dendritic processes. These endings were compared to comparable endings in control material and to published descriptions of VIIth nerve afferent terminals in the hamster nst. The synaptic relations of IXN afferent endings were predominantly with dendritic spines and shafts. The majority (86.6%) of IXN afferent endings were with dendritic processes that were not in apparent contact with other, unlabeled processes. Only 13.4% of IXN synaptic relationships were with dendritic processes that were also contacted by unlabeled vesicle-containing processes. This is in contrast to 31.2% of facial nerve afferent endings in the nst which make synaptic contact with such processes. There were more direct synaptic contacts between facial endings and unlabeled vesicle-containing processes (26.1%) than between IXN endings and unlabeled vesicle-containing processes (1.3%). Thus, unlike the glomerular-like endings of the gustatory fibers of the VIIth nerve, less complex relations appeared to characterize IXN synapses in the nst. These differences were related to the differential physiology of gustatory fibers in the VIIth nerve and IXN.

Taste reactivity in the hamster.

Taste reactivity, which was first described in the rat, consists of ingestive and aversive response components, the latter seen mostly to bitter-tasting stimuli. The present experiment characterized the hamster's taste reactivity to an array of stimuli (sugars: 1 M sucrose, d-fructose and d-glucose; sodium salts: 1 M NaCl, Na2SO4 and NaNO3; acids: 30 mM HCl, tartaric acid and citric acid; bitter-tasting stimuli: 100 mM quinine hydrochloride and nicotine sulfate and 10 mM denatonium benzoate). These 12 stimuli were chosen to represent 3 examples each of stimuli that taste sweet, salty, sour, or bitter to humans; they were presented in random order via an intraoral fistula, one stimulus each day per animal (n = 10). Infusions of 0.6 ml were delivered over a 1-min period from a syringe pump. Orofacial and somatic motor responses were recorded on videotape for later analysis and were also coded online into a computer. Ingestive responses included forward and lateral tongue protrusions and aversive responses included gaping, chin rubbing, forelimb flailing, fluid rejection, increased locomotion, and aversive posturing. Each stimulus group produced a characteristic pattern of these behaviors, with sugars eliciting only ingestive behaviors and the bitter stimuli evoking predominantly aversive responses. Both sodium salts and acids produced ingestive responses, as seen previously in the rat, although these stimuli also elicited aversive behaviors in the hamster, including apes. The patterns of responses were characterized using multivariate procedures; the stimuli fell into distinct groups that were separated primarily along an hedonic dimension.