Prefibrillar amyloid aggregates could be generic toxins in higher organisms

Abstract

More than 40 human diseases are associated with fibrillar deposits of specific peptides or proteins in tissue. Amyloid fibrils, or their precursors, can be highly toxic to cells, suggesting their key role in disease pathogenesis. Proteins not associated with any disease are able to form oligomers and amyloid assemblies in vitro displaying structures and cytotoxicity comparable with those of aggregates of disease-related polypeptides. In isolated cells, such toxicity has been shown to result from increased membrane permeability with disruption of ion homeostasis and oxidative stress. Here we microinjected into the nucleus basalis magnocellularis of rat brains aggregates of an Src homology 3 domain and the N-terminal domain of the prokaryotic HypF, neither of which is associated with amyloid disease. Prefibrillar aggregates of both proteins, but not their mature fibrils or soluble monomers, impaired cholinergic neuron viability in a dose-dependent manner similar to that seen in cell cultures. Contrary to the situation with cultured cells, however, under our experimental conditions, cell stress in tissue is not followed by a comparable level of cell death, a result that is very likely to reflect the presence of protective mechanisms reducing aggregate toxicity. These findings support the hypothesis that neurodegenerative disorders result primarily from a generic cell dysfunction caused by early misfolded species in the aggregation process.

Figure 1.

Confocal microscopy images of the NBM from rats at the sites of microinjection. The presence of prefibrillar (A) and fibrillar (B) aggregates of HypF-N labeled with the fluorescent dye Texas Red is shown (10× magnification in each case). Images were taken 24 h after injection. For technical details, see Materials and Methods. Right panels show atomic force microscopy images of the two types of injected aggregates [kindly provided by A. Relini (University of Genoa, Genoa, Italy) and obtained as reported previously (Relini et al., 2004)].

Figure 2.

Microglia activation resulting from microinjection of aggregates of HypF-N and PI₃SH3 into the NBM of rat brains. Activation was detected by MHC class II immunoreactivity imaged by light microscopy, using the monoclonal antibody OX-6, followed by staining with DAB, as described in Materials and Methods. A, Micrographs (10× magnification) of representative regions of tissue slices 7 d after injection of PBS, 5000 ng of fibrillar aggregates, or 0.0002 or 5000 ng of HypF-N prefibrillar aggregates (top left to bottom right, respectively). B, Similar micrographs taken 7 d after injection of 5000 ng of fibrillar and 5000 ng of prefibrillar aggregates of PI₃SH3. C, Quantification of microglia activation 7 d after the injections (5 sections per animal). Rats were injected with PBS, 5000 ng of native HypF-N, 5000 ng of fibrillar HypF-N, or with 5000, 500, and 0.0002 ng of prefibrillar HypF-N (n = 6 for each type of injection). Data are reported as total OX-6-positive area in pixels of microglia activation after injection. Statistical analysis of the mean was performed using one-way ANOVA, followed by the Newman–Keuls multiple comparison test (F = 16.13; p < 0.05). D, The same as in C but after the injection of PBS or 5000 ng of native, prefibrillar, or fibrillar PI₃SH3. Statistical analysis of the mean was performed as in C. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus PBS. For details, see Materials and Methods. Scale bar, 60 μm

Figure 3.

Disappearance of ChAT-positive neurons in the NBM injected with HypF-N or PI₃SH3. Cholinergic neurons were visualized by immunohistochemistry as specified in Materials and Methods. A, Representative photomicrographs (20× magnification) obtained after injection of 1.0 μl aliquots of PBS, 5.0 μg of HypF-N fibrillar aggregates, and 0.010 ng and 5.0 μg of HypF-N prefibrillar aggregates (top left to bottom right, respectively). ChAT immunoreactivity was localized in intensely labeled NBM neurons located at the border between the internal capsule and the globus pallidus. B, The same as in A but taken after microinjection of 5.0 μg of fibrillar and 5.0 μg of prefibrillar PI₃SH3. C, D, Quantification of ChAT immunoreactivity in the NBM 7 d after the injections (5 sections per animal). Rats were injected with PBS, 5.0 μg of native HypF-N, 5.0 μg of fibrillar HypF-N, varying quantities of prefibrillar HypF-N, 5.0 μg of native PI₃SH3, 5.0 μg of fibrillar PI₃SH3, or 5.0 μg of prefibrillar PI₃SH3 (n = 6 for each type of injection). The total neuronal count in the NBM injected with PBS (190 ± 1.5, mean ± SD) was taken as 100%. C, The number of ChAT-positive cells in the NBM injected with 1.0 μl of PBS containing 5.0 μg (500 μm), 0.50 μg (50 μm), 10.0 ng (1.00 μm), 1.0 ng (100 nm), 0.100 ng (10.0 nm), and 0.010 ng (1.0 nm) of prefibrillar HypF-N was reduced by 77% (p < 0.001), 75% (p < 0.001), 60% (p < 0.001), 50% (p < 0.001), 17% (p < 0.01), and 3% (p < 0.05), respectively, relative to the number of ChAT-positive neurons found in the NBM of the contralateral hemisphere injected with PBS. (Molar concentrations are those of the monomeric HypF-N.) D, The number of ChAT-positive cells in the NBM injected with 5.0 μg of native, fibrillar, or prefibrillar PI₃SH3 was reduced by ∼5% (p > 0.05), 20% (p < 0.01), and 60% (p < 0.001), respectively, with respect to the number of positive neurons found in the NBM of the contralateral hemisphere injected with PBS. (Molar concentrations are those of the monomeric PI₃SH3.) Statistical analysis on the mean was performed by one-way ANOVA, followed by the Newman–Keuls multiple comparison test (F = 53.03; p < 0.05). ∗∗p < 0.01 and ∗∗∗p < 0.001 versus PBS. For details, see Materials and Methods. Scale bar, 30 μm. E, The same data as in C plotted as the concentration of injected HypF-N prefibrillar aggregates against the percentage of ChAT-immunopositive neurons (open squares). The percentages of viable cultured cells (as determined by the MTT test) exposed to differing concentrations of prefibrillar HypF-N (open circles) domain (Bucciantini et al., 2002), Aβ₄₀ (filled squares), or Aβ₄₂ (filled circles) peptides (Fezoui et al., 2000) are also reported.

Figure 4.

Reduction in the levels of AChE in the site of microinjection of HypF-N and PI₃SH3 aggregates in the NBM. AChE was visualized by histochemistry and DAB staining. Representative photomicrographs (10× magnification) obtained after injection of 1.0 μl aliquots of PBS, 0.01 ng of HypF-N fibrillar aggregates, 5.0 μg of fibrillar, and of 5.0 μg of prefibrillar PI₃SH3. The arrows indicate the AChE-positive fibers. For additional details, see Materials and Methods. Scale bar, 60 μm.

Figure 5.

Total number of neurons in the NBM 7 d after microinjecting HypF-N or PI₃SH3 aggregates. A, Number of neurons in the NBM injected with PBS (top left), monomeric (top right), prefibrillar (bottom right), or fibrillar (bottom left) HypF-N as determined by quantifying the cells stained with an anti-NeuN antibody (10× magnification in each image). ∗∗p < 0.01 versus PBS. B, Microinjection of large amounts of HypF-N or PI₃SH3 prefibrillar aggregates causes more extensive DNA fragmentation than does injection of very low amounts of prefibrillar aggregates of HypF-N or of high amounts of PI₃SH3 fibrillar aggregates as shown by TUNEL staining (40× magnification in each case). Additional details are given in Materials and Methods.