Aβ Mediated Diminution of MTT Reduction—An Artefact of Single Cell Culture?

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) reduction assay is a frequently used and easily reproducible method to measure beta-amyloid (Aβ) toxicity in different types of single cell culture. To our knowledge, the influence of Aβ on MTT reduction has never been tested in more complex tissue. Initially, we reproduced the disturbed MTT reduction in neuron and astroglia primary cell cultures from rats as well as in the BV2 microglia cell line, utilizing four different Aβ species, namely freshly dissolved Aβ (25-35), fibrillar Aβ (1-40), oligomeric Aβ (1-42) and oligomeric Aβ (1-40). In contrast to the findings in single cell cultures, none of these Aβ species altered MTT reduction in rat organotypic hippocampal slice cultures (OHC). Moreover, application of Aβ to acutely isolated hippocampal slices from adult rats and in vivo intracerebroventricular injection of Aβ also did not influence the MTT reduction in the respective tissue. Failure of Aβ penetration into the tissue cannot explain the differences between single cells and the more complex brain tissue. Thus electrophysiological investigations disclosed an impairment of long-term potentiation (LTP) in the CA1 region of hippocampal slices from rat by application of oligomeric Aβ (1-40), but not by freshly dissolved Aβ (25-35) or fibrillar Aβ (1-40). In conclusion, the experiments revealed a glaring discrepancy between single cell cultures and complex brain tissue regarding the effect of different Aβ species on MTT reduction. Particularly, the differential effect of oligomeric versus other Aβ forms on LTP was not reflected in the MTT reduction assay. This may indicate that the Aβ oligomer effect on synaptic function reflected by LTP impairment precedes changes in formazane formation rate or that cells embedded in a more natural environment in the tissue are less susceptible to damage by Aβ, raising cautions against the consideration of single cell MTT reduction activity as a reliable assay in Alzheimer's drug discovery studies

Amyloid-Beta Induced Changes in Vesicular Transport of BDNF in Hippocampal Neurons

The neurotrophin brain derived neurotrophic factor (BDNF) is an important growth factor in the CNS. Deficits in transport of this secretory protein could underlie neurodegenerative diseases. Investigation of disease-related changes in BDNF transport might provide insights into the cellular mechanism underlying, for example, Alzheimer’s disease (AD). To analyze the role of BDNF transport in AD, live cell imaging of fluorescently labeled BDNF was performed in hippocampal neurons of different AD model systems. BDNF and APP colocalized with low incidence in vesicular structures. Anterograde as well as retrograde transport of BDNF vesicles was reduced and these effects were mediated by factors released from hippocampal neurons into the extracellular medium. Transport of BDNF was altered at a very early time point after onset of human APP expression or after acute amyloid-beta(1-42) treatment, while the activity-dependent release of BDNF remained unaffected. Taken together, extracellular cleavage products of APP induced rapid changes in anterograde and retrograde transport of BDNF-containing vesicles while release of BDNF was unaffected by transgenic expression of mutated APP. These early transport deficits might lead to permanently impaired brain functions in the adult brain

Influence of Aβ on MTT reduction, PI uptake and GFAP expression of OHC.

<p>A) Time dependent MTT reduction activity of OHC. Numbers indicate the time after MTT application in minutes B) Influence of glutamate, freshly dissolved Aβ (25-35), fibrillar Aβ (1-40) and oligomeric Aβ (1-42) on MTT reduction activity of OHC under different conditions. Application of 10 µM Aβ for 3–6 days did not diminish the MTT reduction of OHC under different conditions; application of glutamate (15 µM) significantly reduced the MTT reduction, compared to control; the dashed line indicates the control level; * = p≤0.05, Mann–Whitney U-test, n≥12 per group C) PI staining of Aβ and glutamate treated OHCs. Application of 10 µM freshly dissolved Aβ (25-35) and 10 µM fibrillar Aβ (1-40) into the medium for 3 days did not cause cell death. Application of glutamate (15 µM) induced cell death D) Immunostaining of cross sections against fibrillar Aβ (1-40) revealed the presence of Aβ in the slice E) GFAP and DAPI staining of oligomeric Aβ (1-42) treated and control slice. Aβ (1-42) caused an activation of astroglia within the OHC, indicated by an increased GFAP expression.</p

Influence of Aβ on MTT reduction activity of OHC and single cells, generated from OHC.

<p>A) Aβ (25-35) 1 µM was applied to the slice for 3 days. The MTT assay was done 2 hours after the preparation of the single cells out of the slice. In this case, 1 µM Aβ did not diminish the MTT reduction of OHC and single cells; B) 1 µM Aβ was applied to the slices and single cells after the preparation for 2 days. In this case, Aβ (25-35) 1 µM significantly reduced the MTT reduction of single cells, compared to control; * = p≤0.05, Mann–Whitney U-test, n = 10 per group.</p

Influence of Aβ on MTT reduction of single cell cultures.

<p>A) Influence of Aβ on MTT reduction of neuron and microglia single cell cultures. When applied to cell cultures for 3 days, at 1 µM all Aβ species diminished the MTT reduction significantly in both cell types. The dashed line indicates the control level; * = p≤0.05, Mann–Whitney U-test, n = 10 per group B) Concentration dependent influence of Aβ on MTT reduction activity of astroglia single cell culture. When applied to cell culture for 3 days, any Aβ species diminished the MTT reduction significantly, compared to control. Congo red (2 µM) completely reverses the Aβ effect; Aβ (25-35) diminished the MTT reduction in NB medium, normally used for cultivation of OHC; the dashed line indicates the control level; * = p≤0.05, Mann–Whitney U-test, n = 10 per group C) Electron microscopic images (EMI) revealed that freshly dissolved Aβ (25-35) did not form aggregates. Moreover, EMI conformed the needle like structure of fibrillar Aβ (1-40) and the smaller, spherical shape of oligomeric Aβ (1-40) and oligomeric Aβ (1-42).</p

Influence of Aβ on LTP and MTT reduction of acute isolated slices.

<p>A) Influence of freshly dissolved Aβ (25-35), oligomeric Aβ (1-40) and fibrillar Aβ (1-40) on LTP of acute hippocampal slices. Oligomeric Aβ (1-40) significantly reduced the LTP, compared to control potentiation. Freshly dissolved Aβ (25-35) and fibrillar Aβ (1-40) did not effect the LTP; * = p≤0.05 ANOVA with repeated measures; The bar indicates the time of Aβ application. Tetanus was applied at time point 0; Analogue traces represent typical recordings of single experiments taken 20 minutes before tetanization (1), and 240 minutes after tetanization (2). B) Aβ treated acute slices did not differ from control slices in their MTT reduction activity. C) Influence of Aβ on MTT reduction activity of <i>ex vivo</i> slices. Injection of freshly dissolved Aβ (25-35) and oligomeric Aβ (1-42) for 3 days did not diminish the MTT reduction of the <i>ex vivo</i> slices, compared to untreated animals and the reverse control protein Aβ (35-25). D) Immunostaining of cross sections against Aβ revealed the presence of oligomeric Aβ (1-42) in the hippocampus.</p

Mechanism of amyloid plaque formation suggests an intracellular basis of Aβ pathogenicity

The formation of extracellular amyloid plaques is a common patho-biochemical event underlying several debilitating human conditions, including Alzheimer’s disease (AD). Considerable evidence implies that AD damage arises primarily from small oligomeric amyloid forms of Aβ peptide, but the precise mechanism of pathogenicity remains to be established. Using a cell culture system that reproducibly leads to the formation of Alzheimer’s Aβ amyloid plaques, we show here that the formation of a single amyloid plaque represents a template-dependent process that critically involves the presence of endocytosis- or phagocytosis-competent cells. Internalized Aβ peptide becomes sorted to multivesicular bodies where fibrils grow out, thus penetrating the vesicular membrane. Upon plaque formation, cells undergo cell death and intracellular amyloid structures become released into the extracellular space. These data imply a mechanism where the pathogenic activity of Aβ is attributed, at least in part, to intracellular aggregates