A 2-Substituted 8-Hydroxyquinoline Stimulates Neural Stem Cell Proliferation by Modulating ROS Signalling.

TL;DR: The results indicate that DMAMQ can stimulate neurogenesis via the Nox signalling pathway, which may provide therapeutic benefit in treating dementias of various types by replenishing neurones using the brain’s own reserves.

Abstract: Eight-hydroxyquinolines (8HQs) are a class of compounds that have been identified as potential therapeutics for a number of neurodegenerative diseases. Understanding the influence of structural modifications to the 8HQ scaffold on cellular behaviour will aid the identification of compounds that might be effective in treating dementias. In this study, we describe the action of 2-[(dimethylamino)methyl]-8-hydroxyquinoline (DMAMQ) on adult murine neural stem cells (NSCs) cultured in vitro. Treatment of NSCs with DMAMQ resulted in enhanced self-renewal and increased neurite outgrowth. Concurrent with the positive growth effects was an increase in intracellular reactive oxygen species, with the growth being inhibited by inactivation of the NADPH oxidase (Nox) enzyme family. Our results indicate that DMAMQ can stimulate neurogenesis via the Nox signalling pathway, which may provide therapeutic benefit in treating dementias of various types by replenishing neurones using the brain's own reserves. The narrow concentration range over which these effects were observed, however, suggests that there may exist only a small therapeutic window for neuro-regenerative applications.

Summary

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.

Figures

Figure 1. Structure of DMAMQ (2-[(dimethylamino)methyl]-8-hydroxyquinoline).

Figure 6. DMAMQ mediates its effects through stimulation of NADPH oxidase to increase intracellular ROS production. (a) Intracellular ROS increases in response to DMAMQ treatment in proliferating NSCs treated with DMAMQ in the low μM range, as measured by DCF fluorescence. (b) Co-treatment with DMAMQ and the NOX inhibitor, DPI, prevents the DMAMQ ROS burst, suggesting the ROS increase is associated with cellular signaling involving NADPH oxidase. One-way ANOVA, n = 4, *p < 0.05, **p < 0.01 (significantly different from untreated control).

Figure 7. Nox inhibition with DPI blocks DMAMQ induced growth. (a) Representative colonys grown in the presence or absence of Nox family inhibitor DPI and DMAMQ. Scale bar, 100 μm. (b) Total volume of growth at 21 days calculated from the number of colonies formed (c) and the diameter of the colonies (d). Two-way ANOVA, n = 3, *p < 0.05, **p < 0.01.

Figure 4. Effects of DMAMQ on NSC differentiation. (a) Immunofluorescence staining demonstrates that NSCs treated with DMAMQ throughout 7 days differentiation retain their capacity to differentiate and are multipotent. Green, NF-L (neuronal marker); Red, GFAP (astrocytic marker); Blue, DAPI (nuclear stain); Scale bar = 20 μm. (b) Example western blot images of cells either differentiated after 24 hours priming with DMAMQ or differentiated in the presence of DMAMQ. Densitometric quantification of western blot intensity for (c) nestin, (d) NF-L and (e) GFAP relative to the total-protein (coomassie) loading control. No differences are observed in lineage preference as a result of DMAMQ exposure either before or during differentiation; one-way ANOVA n = 5.

Figure 3. DMAMQ reduces the non-proliferative population of NSCs and increases NSC growth. (a) Flow cytometry analysis of Ki67 proliferation marker expression in NSCs after 24 hours treatment with 2.5 µM DMAMQ. Both populations were gated identically using the unstained background control populations shown in grey behind the Ki67 negative (blue) and Ki67 positive (red) populations. (b) Quantification of the percentage increase in Ki67 expression from the profiles in a; Students t-test, p = 0.025, n = 4. (c) Quantification of βgalactosidase staining following three days incubation with 2.5 μM DMAMQ, change is expressed relative to control cells; Students t-test, n = 4. (d) Flow cytometry profiles of

Figure 2. DMAMQ promotes the proliferation of NSCs. (a) Representative field of view showing neurosphere morphology with and without 2.5 µM DMAMQ treatment for 21 days. Scale bar = 35 µm. (b) Colony volume measurement of increasing DMAMQ doses. Dissociated cells (single cells) were seeded at equal density with/without DMAMQ and the growth of neurospheres present at 21 days post plating measured (change in volume is expressed relative to the no DMAMQ control; one-way ANOVA, n = 3). Neurosphere volume at 2.5 µM DMAMQ is increased both because of (c) increased colony formation (Students t-test, n = 3) and (d) a shift in the size of the neurospheres towards larger diameters (with significant differences being obtained for diameters in the 30–59 μm and 90–199 μm range; two-way ANOVA, p < 0.001, n = 3).

Full text

1
A 2-substituted 8-hydroxyquinoline stimulates neural stem cell proliferation by
modulating ROS signalling
Cathryn L. Haigh,
1,
* Carolin Tumpach,
2
Steven J. Collins,
1
and Simon C. Drew
2,
*
1
Department of Medicine, Royal Melbourne Hospital, The University of Melbourne, Victoria
3010, Australia,
2
The Florey Department of Neuroscience and Mental Health, The University
of Melbourne, Victoria 3010, Australia.
* To whom correspondence should be addressed: sdrew@unimelb.edu.au (+61 3 9035 8684),
chaigh@unimelb.edu.au (+61 3 8344 1952)
Running Title:
8HQ stimulation of NSC proliferation
Keywords:
8-hydroxyquinoline, neural stem cells, neurosphere, proliferation, NADPH oxidase, reactive
oxygen species, 2-[(dimethylamino)methyl]-8-hydroxyquinoline, DMAMQ, PBT2

2
Abstract
Eight-hydroxyquinolines (8HQs) are a class of compounds that have been identified as
potential therapeutics for a number of neurodegenerative diseases. Understanding the
influence of structural modifications to the 8HQ scaffold on cellular behaviour will aid the
identification of compounds that might be effective in treating dementias. In this study, we
describe the action of 2-[(dimethylamino)methyl]-8-hydroxyquinoline (DMAMQ) on adult
murine neural stem cells (NSCs) cultured in vitro. Treatment of NSCs with DMAMQ
resulted in enhanced self-renewal and increased neurite outgrowth. Concurrent with the
positive growth effects was an increase in intra-cellular reactive oxygen species, with the
growth being inhibited by inactivation of the NADPH oxidase (Nox) enzyme family. Our
results indicate that DMAMQ can stimulate neurogenesis via the Nox signalling pathway,
which may provide therapeutic benefit in treating dementias of various types by replenishing
 The narrow concentration range over which these
effects were observed, however, suggests there may exist only a small therapeutic window for
neuro-regenerative applications.

3
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
]. In
rodent models of Alzheimer's disease (AD), production of beta-amyloid, one hallmark of AD,
changes the growth and differentiation of these cells [
3
,
4
,
5
,
6
,
7
]. Neurogenesis has been
extensively linked with both learning and forgetting [
8
,
9
,
10
]; therefore, changes in these
processes might contribute significantly to the cognitive changes that occur during
neurodegenerative diseases, including AD. 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. Side-chain modifications to the 8HQ backbone may lead to
functional differences in vivo, with applications of substituted 8HQs as anti-microbial agents
[
11
,
12
], anti-cancer agents [
13
,
14
], epigenetic modulators [
15
,
16
], dementia treatments
[
17
,
18
,
19
,
20
], artificial nucleobases [
21
] and medical imaging agents [
22
,
23
]. Moreover,
biosynthesis of substituted 8HQs has been identified in mammals (eg. xanthurenic acid [
24
]),
bacteria (eg. quinolobactin [
25
]) and insects (eg. 2-carboxy-8HQ [
26
]). In dementia
applications, studies utilising 5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline
reported an increased neurite number following treatment of cultured PC12 cells and a
restoration of hippocampal dendritic spine density (but not number) in transgenic mouse
model of AD [18].

4
In the current study, we sought to further understand the action of this class of 8HQ by
treating adult murine NSC cultures with 2-[(dimethylamino)methyl]-8-hydroxyquinoline
(DMAMQ; Figure 1). We specifically assessed the ability of these cells to self-renew,
essential for maintaining their numbers in the brain throughout life, and to differentiate into
new CNS lineage cells. Our findings show that whilst neurogenesis is unchanged, NSCs
treated with DMAMQ demonstrate a dose-dependent increase in NSC proliferation and
increased neurite outgrowth during neurogenesis that was signalled by increased production
of reactive oxygen species (ROS) signalling intermediates by the NADPH oxidase (Nox)
enzyme family. These effects were only observed within a narrow concentration range,
suggesting a small therapeutic window for neuro-regenerative applications.

5
Materials and Methods:
Synthesis
2-[(dimethylamino)methyl]-8-hydroxyquinoline (DMAMQ) was synthesised as described
previously [
27
].
Cell culture
Murine NSCs were harvested from the brains of 6-8 week old Balb/c mice and grown without
modification as neurospheres in liquid culture as described previously [
28
].
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

Frequently asked questions

Q1. What are the contributions mentioned in the paper "A 2-substituted 8-hydroxyquinoline stimulates neural stem cell proliferation by modulating ros signalling" ?

In this paper, the effect of substituted 8-hydroxyquinolines ( 8HQs ) on NSC proliferation and neurite outgrowth was investigated. 

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.