Fast and Efficient Postsynthetic DNA Labeling in Cells by Means of Strain-Promoted Sydnone-Alkyne Cycloadditions

Fast and efficient: DNA strands, modified with the novel bioorthogonal reporters sydnones, undergo fast and efficient labeling with cyclooctynes and have the potential to become essential tools for imaging DNA and possibly RNA in cells. Sydnones are highly stable mesoionic 1,3-dipoles that react with cyclooctynes through strain-promoted sydnone-alkyne cycloaddition (SPSAC). Although sydnones have been shown to be valuable bioorthogonal chemical reporters for the labeling of proteins and complex glycans, nucleic acids have not yet been tagged by SPSAC. Evaluation of SPSAC kinetics with model substrates showed fast reactions with cyclooctyne probes (up to k=0.59 M$^{-1}$ s$^{-1}$), and two different sydnones were effectively incorporated into both 2’-deoxyuridines at position 5, and 7-deaza-2’-deoxyadenosines at position 7. These modified nucleosides were synthetically incorporated into single-stranded DNAs, which were successfully postsynthetically labeled with cyclooctyne probes both in vitro and in cells. These results show that sydnones are versatile bioorthogonal tags and have the premise to become essential tools for tracking DNA and potentially RNA in living cells

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

Effects of ranolazine on astrocytes and neurons in primary culture

Ranolazine (Rn) is an antianginal agent used for the treatment of chronic angina pectoris when angina is not adequately controlled by other drugs. Rn also acts in the central nervous system and it has been proposed for the treatment of pain and epileptic disorders. Under the hypothesis that ranolazine could act as a neuroprotective drug, we studied its effects on astrocytes and neurons in primary culture. We incubated rat astrocytes and neurons in primary cultures for 24 hours with Rn (10−7, 10−6 and 10−5 M). Cell viability and proliferation were measured using trypan blue exclusion assay, MTT conversion assay and LDH release assay. Apoptosis was determined by Caspase 3 activity assay. The effects of Rn on proinflammatory mediators IL-β and TNF-α was determined by ELISA technique, and protein expression levels of Smac/Diablo, PPAR-γ, Mn-SOD and Cu/Zn-SOD by western blot technique. In cultured astrocytes, Rn significantly increased cell viability and proliferation at any concentration tested, and decreased LDH leakage, Smac/Diablo expression and Caspase 3 activity indicating less cell death. Rn also increased anti-inflammatory PPAR-γ protein expression and reduced pro-inflammatory proteins IL-1 β and TNFα levels. Furthermore, antioxidant proteins Cu/Zn-SOD and Mn-SOD significantly increased after Rn addition in cultured astrocytes. Conversely, Rn did not exert any effect on cultured neurons. In conclusion, Rn could act as a neuroprotective drug in the central nervous system by promoting astrocyte viability, preventing necrosis and apoptosis, inhibiting inflammatory phenomena and inducing anti-inflammatory and antioxidant agents

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