A 2-Substituted 8-Hydroxyquinoline Stimulates Neural Stem Cell Proliferation by Modulating ROS Signalling.
Summary (2 min read)
Introduction
- Endogenous neural stem cells (NSCs) are an as-yet untapped resource in combatting dementia.
- Low levels of NSCs persist in the brain throughout adult life, maintaining the ability to self-replicate and to differentiate into mature brain cells.
- During dementia, neurogenesis (the ability to form new neurones) is seen to become dysregulated [1,2].
- Compounds that modulate or normalise the functions of NSCs represent a prime target for alleviating the symptoms associated with, or delaying the course of, neurodegenerative diseases.
- Unbiased chemical screening of substituted 8-hydroxyquinolines (8HQs) and a range of investigations in yeast, nematodes, mice and humans have highlighted the variable mechanisms by they may act.
DMAMQ treatments
- DMAMQ was included in the culture medium at the concentrations indicated.
- For all assays, the compound was added as a single treatment at the start of the assay and was not replenished when the media was changed.
Neural Colony Forming Assay (NCFA)
- The NCFA has been described previously [29].
- For each independent experiment over 50 colonies were measured.
- As stated above, DMAMQ was included in the matrix once only at the start of the incubation.
Neurite outgrowth assay
- Neurite outgrowth was measured using a neurite outgrowth staining kit (Merck-Millipore) as per the manufacturer's instructions with the following modifications.
- One hundred thousand cells per condition were incubated in high FGF (20 ng/mL), no EGF, growth media before seeding 100,000 cells per well insert (1 μm pore size) in differentiation medium with or without test compound.
- Neurite outgrowth was permitted for two days before assay.
Dichlorofluorescein (DCF) intracellular ROS assay
- The DCF assay has been described previously [30].
- Fluorescence intensity was measured every 5 min for 12 hours using a FLUOstar Optima (BMG Labtech) fitted with 490 nm excitation and 520 nm emission filters and initial rates were calculated using tangents to the curve.
MTS metabolism assay
- Five µL of One Solution MTS reagent per 100 µL media was added to test and media only background control conditions, and incubated under normal culture conditions for 90 min.
- The cells were stained using a Cell Signaling Technologies' β-galactosidase staining kit as per the manufacturer's instructions.
- To solubilise the stain for quantitation by spectrometry, the staining solution was removed from the wells and replaced with 350 μL of DMSO.
- The plate was warmed with agitation at 50C for 1 hour before 100 μL of dissolved dye was transferred to three wells of a 96-well plate for triplicate reads.
Ki67 flow cytometry
- Ki67 expression in proliferating cells was quantified using the Muse Ki67 Proliferation Kit as per the manufacturer's instructions.
- For the final analysis the number of unstained cells gated as positive was subtracted from the stained cells and percentage change was calculated from the control cells.
- Cell cycle phase analysis by DNA content was determined using the Muse Cell Cycle Analysis Kit as per the manufacturer's instructions.
PAGE and Western Blotting
- Polyacryalmide gel electrophoresis and western blotting was conducted as described previously [32].
- Relative densitometric comparisons were normalised for total protein loading as shown in Coomassie stained images.
Calcium assay
- Cells were seeded in 96-well plates as above.
- At the beginning of the assay 50 μl of culture media was removed from each well and replaced with calcium assay working buffer as per manufacturer's instructions .
- Readings were taken using 488 nm excitation and 530 nm emission filters in a FluoSTAR Optima (BMG Labtech) every 60 seconds for 1 hour from addition.
- Statistical analyses Statistical analyses were carried out using GraphPad Prism 5 statistical software.
- Graphs show the mean and standard error of the mean (SEM) of n independent experiments unless otherwise stated.
Results and Discussion
- Herein the authors investigated the potential for DMAMQ to enhance the regenerative capacity of NSCs cultured in vitro.
- The ability of DMAMQ to stimulate neurite outgrowth during differentiation was assessed using a colourimetric neurite staining protocol and showed that, over two days of differentiation, DMAMQ-treated cells displayed ca. 10% more neurite outgrowth than was observed for control cells .
- When DPI and DMAMQ were co-incubated in the mediamatrix (single treatment at the start of the assay) the growth of the spheres was stunted, with fewer colonies formed and decreased diameter at the 2.5 μM treatment concentrations of DMAMQ.
- Whilst their results concur with previous literature that increased ROS increases NSC growth and suggest that modulation of Nox to increase ROS signalling initiates such growth, the data presented cannot exclude intrinsic ROS production by DMAMQ that mimics, but does not outcompete inhibition of, this pathway.
- Increased neurogenesis may represent a compensatory response by the NSCs within the brain to replenish lost neurones.
Conclusions
- This study demonstrates that DMAMQ modulates NSC growth and neurite outgrowth during differentiation when cultured in vitro by stimulating ROS production and Nox signalling.
- This suggests that DMAMQ and similar 8HQs may have neuro-regenerative potential in vivo; however, the very narrow concentration range in which DMAMQ modulates NSC growth presents some challenges for therapeutic administration.
- In particular, the potential benefits of replenishing damaged brain tissue must be balanced against the possibility of overstimulating neurogenesis and the propensity for undesirable “off-target” effects.
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Citations
64 citations
Cites background from "A 2-Substituted 8-Hydroxyquinoline ..."
...Indeed, studies using a homologous terdentate 8HQ resulted in a significant increase in pGSK 3β only at cytotoxic concentrations (Haigh et al., 2016)....
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...…in the presence of the biological reductant such as ascorbate, the dominant ternary metal complexes can produce as many hydroxyl radicals as Cu(Aβ1−x) in vitro (Mital et al., 2016) and ROS production can be observed following addition of such 8HQs to neural stem cell cultures (Haigh et al., 2016)....
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..., 2016) and ROS production can be observed following addition of such 8HQs to neural stem cell cultures (Haigh et al., 2016)....
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59 citations
52 citations
Cites background from "A 2-Substituted 8-Hydroxyquinoline ..."
...Interestingly, the generation of ROS can be detected by adding such ligands to the culture of neural stem cells [324], in contrast to the founding principle of therapeutic chelation therapy [325]....
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35 citations
31 citations
References
116 citations
"A 2-Substituted 8-Hydroxyquinoline ..." refers background in this paper
...functions of heart muscle [50, 51] and systemic immune responses [37, 52], whilst acute and chronic Nox activity is associated with neuroinflammation [53]....
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112 citations
"A 2-Substituted 8-Hydroxyquinoline ..." refers background in this paper
...Some of these considerations may explain why clioquinol and 5,7-dichoro-DMAMQ have not shown evidence of clinical benefit in treating AD [55]....
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108 citations
"A 2-Substituted 8-Hydroxyquinoline ..." refers background in this paper
...cium mobilisation are also linked with redox signalling [36] and increased cytosolic Ca can directly activate some Nox...
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105 citations
"A 2-Substituted 8-Hydroxyquinoline ..." refers background in this paper
...Redox signalling pathways have been linked with NSC growth and an increased ROS burst has been linked with induction of proliferation [38,39,40]....
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101 citations
"A 2-Substituted 8-Hydroxyquinoline ..." refers result in this paper
...Inactivation (by phosphorylation of Ser9) or pharmacological inhibition of GSK-3 leads to both increased proliferation and enhanced neurogenesis in neural stem cell cultures [33, 34] and this phosphorylation has previously been reported in some studies using the 5,7-dichlorinated analogue of DMAMQ [18]; however, we did not observe any change in the phosphorylation status of GSK-3b following treatment with concentrations of DMAMQ that induced NSC growth (Fig....
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Q2. What are the future works in "A 2-substituted 8-hydroxyquinoline stimulates neural stem cell proliferation by modulating ros signalling" ?
In particular, the potential benefits of replenishing damaged brain tissue must be balanced against the possibility of overstimulating neurogenesis and the propensity for undesirable “ off-target ” effects.