doi:10.1093/braincomms/fcz005
BRAIN COMMUNICATIONS 2019: Page 1 of 21
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BRAIN
AIN COMMUNICATIONS
Richard I. Tuxworth,1,† Matthew J. Taylor,1 Ane Martin Anduaga,2,‡ Alaa Hussien-Ali,3
Sotiroula Chatzimatthaiou,1 Joanne Longland,4 Adam M. Thompson,4 Sharif Almutiri,4,5
Pavlos Alifragis,3 Charalambos P. Kyriacou,2 Boris Kysela1,6,† and Zubair Ahmed4,†
†
These authors contributed equally to this work.
‡
Present address: Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, 02453, USA.
DNA double-strand breaks are a feature of many acute and long-term neurological disorders, including neurodegeneration, following neurotrauma and after stroke. Persistent activation of the DNA damage response in response to double-strand breaks contributes to neural dysfunction and pathology as it can force post-mitotic neurons to re-enter the cell cycle leading to senescence or
apoptosis. Mature, non-dividing neurons may tolerate low levels of DNA damage, in which case muting the DNA damage response
might be neuroprotective. Here, we show that attenuating the DNA damage response by targeting the meiotic recombination 11,
Rad50, Nijmegen breakage syndrome 1 complex, which is involved in double-strand break recognition, is neuroprotective in three
neurodegeneration models in Drosophila and prevents Ab1-42-induced loss of synapses in embryonic hippocampal neurons.
Attenuating the DNA damage response after optic nerve injury is also neuroprotective to retinal ganglion cells and promotes dramatic regeneration of their neurites both in vitro and in vivo. Dorsal root ganglion neurons similarly regenerate when the DNA
damage response is targeted in vitro and in vivo and this strategy also induces significant restoration of lost function after spinal
cord injury. We conclude that muting the DNA damage response in the nervous system is neuroprotective in multiple neurological
disorders. Our results point to new therapies to maintain or repair the nervous system.
1 Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham B15 2TT, UK
2 Department of Genetics & Genome Biology, University of Leicester, Leicester LE1 7RH, UK
3 Centre for Biomedical Science, Centre of Gene and Cell Therapy, School of Biological Sciences, Royal Holloway University of
London, Surrey TW20 0EX, UK
4 Neuroscience and Ophthalmology, College of Medical and Dental Sciences, Institute of Inflammation and Ageing, University of
Birmingham, Birmingham B15 2TT, UK
5 Applied Medical Science College, Shaqra University, Addawadmi, Riyadh, Saudi Arabia
6 Aston Medical School, Aston Medical Research Institute, Aston University, Aston Triangle, Birmingham B4 7ET, UK
Correspondence to: Zubair Ahmed, PhD
Neuroscience and Ophthalmology, College of Medical and Dental Sciences,
Institute of Inflammation and Ageing, University of Birmingham,
Room 386 Robert Aitken Institute of Clinical Research,
Edgbaston, Birmingham B15 2TT, UK
E-mail: z.ahmed.1@bham.ac.uk
Received May 23, 2019. Revised June 14, 2019. Accepted June 19, 2019. Advance Access publication July 2, 2019
C The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain.
V
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse,
distribution, and reproduction in any medium, provided the original work is properly cited.
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Attenuating the DNA damage response to
double-strand breaks restores function in
models of CNS neurodegeneration
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| BRAIN COMMUNICATIONS 2019: Page 2 of 21
R. I. Tuxworth et al.
Correspondence may also be addressed to: Richard I. Tuxworth, PhD,
Institute of Cancer and Genomic Sciences,
University of Birmingham, Birmingham B15 2TT, UK.
E-mail: r.i.tuxworth@bham.ac.uk
Correspondence may also be addressed to: Boris Kysela, PhD.
E-mail: b.kysela@aston.ac.uk
Keywords: DNA damage; neurodegeneration; neuroprotection; CNS trauma; spinal cord injury
Abbreviations: ATM ¼ ataxia telangiectasia mutated; cAMP ¼ cyclic adenosine monophosphate; CAP ¼ compound action poten-
Graphical Abstract
Introduction
DNA double-strand breaks are the most deleterious type
of DNA damage. In mitotically cycling cells, doublestrand breaks trigger the DNA damage response to arrest
the cell cycle in and mount repair via non-homologous
end-joining in G1 or G2 phases or homologous recombination in M and S phases (Mladenov et al., 2016).
Double-strand breaks are a feature of many acute and
long-term neurological disorders, including many forms
of neurodegeneration (Simpson et al., 2015; Merlo et al.,
2016), following neurotrauma (Kotipatruni et al., 2011)
and after stroke (Hayashi et al., 1998). Unrepaired double-strand breaks in neurons lead to persistent activation
of the DNA damage response which, in turn, is a trigger
for dysregulation of the cell cycle and aberrant re-entry
of neurons into G1 leading to neural dysfunction, apoptosis and senescence (Herrup et al., 2004). Re-entry of
neurons into the cell cycle and senescence are features of
Alzheimer’s disease (McShea et al., 1997; Nagy et al.,
1997; Fielder et al., 2017) whilst dysfunctional regulation
of the cell cycle and has also been documented in models
of cerebral ischaemia (Katchanov et al., 2001; Wen et al.,
2004) and in post-mortem brain samples of stroke
patients (Love, 2003).
If persistent activation of the DNA damage response is
a trigger for neuronal dysfunction, apoptosis and senescence then potentially muting the DNA damage response
would be neuroprotective. A key sensor and early processor of double-strand breaks is the MRN complex, comprising the Mre11, Rad50 and Nbs1/Nbn proteins
(Lamarche et al., 2010). Association of the MRN complex to double-strand breaks leads to recruitment and activation of the ataxia telangiectasia mutated (ATM)
kinase, which coordinates multiple arms of the DNA
damage response, including cell-cycle arrest, repair and
apoptosis (Shiloh and Ziv, 2013). ATM activation is particularly associated with non-homologous end-joining; in
contrast, the related kinase, ataxia telangiectasia and
Rad3-related (ATR) protein, regulates homologous recombination. Double-strand breaks are characterized by the
activation of sensor kinases, including DNA-protein
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tial; CNTF ¼ ciliary neurotrophic factor; DAPI ¼ 4’,6-diamidino-2-phenylindole; DC ¼ dorsal column; DRG ¼ dorsal root ganglion;
DRGN ¼ dorsal root ganglion neurons; GAP43 ¼ growth associated protein 43; GLMM ¼ generalized linear mixed models; IC50 ¼
half maximum inhibitory concentration; IgG ¼ immunoglobulin; lLNv ¼ large lateral neuron ventral; LMM ¼ linear mixed models;
Mre11 ¼ meiotic recombination 11; MRN complex ¼ meiotic recombination 11/Radiation 50/Nijmegen breakage syndrome 1 complex; Nbs1 ¼ Nijmegen breakage syndrome 1; ONC ¼ optic nerve crush; PAGE ¼ polyacrylamide gel electrophoresis; PBS ¼ phosphate buffered saline; PDF ¼ pigment dispersing factor; P/S ¼ penicillin/streptomycin; PTEN ¼ phosphatase and tensin homologue;
RGC ¼ retinal ganglion cells; Rad50 ¼ Radiation 50; SCI ¼ spinal cord injury; SDS ¼ sodium dodecyl sulphate; shRNA ¼ short
hairpin RNA; siCASP2 ¼ short interfering RNA to caspase-2; sLNv ¼ small lateral neuron ventral; UAS ¼ upstream activation
sequence
Attenuating DNA damage is neuroregenerative
Materials and methods
Drosophila stocks and breeding
For all Drosophila experiments except circadian analysis, expression of the neurodegeneration-associated transgenes was
restricted to adult neurons by use of an elavC155; GAL80ts
driver line. Drosophila was bred on standard yeast/agar
media in bottles at 18 C on a 12 h light/dark cycle until
eclosion. To induce expression, flies were shifted to and
maintained at 29 C, 70% humidity on a 12 h light/dark
cycle for the duration of the experiment. Virgin females of
the driver line were crossed to males of the control or experimental lines. The control used was an isogenic w1118
strain and all experimental lines were backcrossed over five
generations into this line. UAS-lines and the rad50EP1 allele
were followed during backcrossing by the presence of the
wþ transgene and the nbs alleles by PCR. All fly stocks
were obtained from the Vienna or Bloomington Drosophila
stock centres except for UAS-Ab1-42 (12-linker) (Speretta
et al., 2012), which was a kind gift of Dr. Damien
Crowther (University of Cambridge) and UAS-0N4R Tau
R406W (Wittmann et al., 2001), which was a kind gift of
Dr. Mel Feany (Harvard University).
Quantification of fly movement
Newly eclosed adult flies were separated into cohorts of
20 flies in vials and shifted to 29 C to begin expression
by climbing assay or movement tracking.
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Negative geotaxis climbing assays
To test climbing, flies were removed from the incubator
and allowed to acclimatize for 1 h to the room temperature (RT) and humidity. Each vial of flies was tipped
into an empty vial and left for 60 s to acclimatize. Flies
were tapped to the base and allowed to climb up for
30 s. The percentage of flies climbing above a line 2.5 cm
above the base was recorded as the mean of three
repeats. Groups of five vials were tested together. Flies
were transferred to fresh food vials after testing and
returned to the incubator. For the Htt.Q128 experiment,
second-order polynomials were fitted to the data by
non-linear regression in Prism 7 and compared by sumof-squares F-test. For the Ab1-42 experiment, data were
compared by two-way ANOVA. Significance in both
cases was set at P < 0.05.
Movement tracking with DART
Flies were housed individually in 65 5 mm locomotor
tubes (Trikinetics). Tubes were mounted in groups of 20
horizontally on custom platforms and movement recorded
from above via a Logitech C920 HD camera mounted on
a copy stand. The position of each fly was determined at
5 Hz and movement quantified using DART software running in MATLAB 2017a (Faville et al., 2015). Vibrational
stimulation was applied five times at 10-min intervals to
the flies via motors mounted to the underside of each platform and controlled by the DART software. The mean
speed of the population of 20 flies over the 120 s before
each stimulation, and the maximum amplitude of the
population response to stimulation were quantified by
DART. Sigmoidal trend lines were fitted to the data
by non-linear regression in Prism 7 and compared by sumof-squares F-test. A full description of the adaptation of
the DART circadian behaviour system to quantify the startle response of flies was published elsewhere (Taylor and
Tuxworth, 2019).
Circadian analysis
tim-Gal4 was used to express Ab1-42 in clock neurons.
Flies were maintained at 25 C throughout development
and into adulthood in bottles. Newly eclosed adult flies
were sorted for genotype and transferred to individual
65 5 mm locomotor tubes in DAM activity monitors
(Trikinetics) and maintained in 12 h light/12 h dark cycle
for 3 days then in 12 h dark/12 h dark for 10 days.
Circadian behaviour was analysed with both spectral analysis using CLEAN and cosinor as described previously
(Levine et al., 2002; Rosato and Kyriacou, 2006).
Activity in the first day of DD was discarded. Genotypes
were compared by ANOVA with a Tukey’s post hoc test.
Additional flies from the same breeding cohorts were
sorted for genotype but maintained in vials in 12 h light/
dark cycle to maintain circadian cohesion. After 10 days,
the brains were dissected at ZT0 (just before lights on)
and fixed and stained essentially as described (Dissel
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kinase catalytic subunit and regions of DNA damage
incorporating phosphorylated histone cH2Ax (Shrivastav
et al., 2008). Therefore, cH2Ax is commonly used to
monitor direct activation of the DNA damage response in
terms of double-strand breaks (Celeste et al., 2002;
Simpson et al., 2015; Milanese et al., 2018). ATM is
recruited at the site of lesions such as double-strand
breaks to phosphorylate in cis the histone H2Ax
(cH2Ax) (Celeste et al., 2002). cH2Ax marks the initiation of a nucleation process resulting in the formation
of characteristic cH2Axþ nuclear foci, which leads to
additional ATM recruitment at the damage site, thereby
escalating the kinase activity (Shiloh and Ziv, 2013). In
post-mitotic neurons, homologous recombination is unlikely
to feature in the repair of double-strand breaks as no sister
chromatid is available to act as a template for repair.
Given its position at the apex of the DNA damage response, the MRN complex is a potential target to attenuate
or mute the DNA damage response in neurons. Here, we
targeted the MRN complex using both genetic approaches
in Drosophila models of neurodegeneration and using
small-molecule inhibitors in two rat models of acute neurotrauma: ocular injury and spinal cord injury (SCI). We
identified that targeting of the MRN complex was neuroprotective in each scenario. This universality opens up new
possibilities for treating human neurological disorders.
BRAIN COMMUNICATIONS 2019: Page 3 of 21
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Drugs
Mirin and KU-60019 were both purchased from Tocris,
Bristol, UK. Mirin is a small-molecule inhibitor that blocks
the 30 and 50 exonuclease activity associated with Mre11
and prevents ATM activation in response to double-strand
breaks (Dupre et al., 2008; Stivers, 2008; Garner et al.,
2009). KU-60019 is a potent ATM kinase inhibitor and
has been used to inhibit migration and invasion of human
glioma cells in vitro (Golding et al., 2009).
Rat primary hippocampal neuron
culture
Primary cultures were prepared from dissected hippocampi of E18 Sprague-Dawley rat embryos as previously
described (Russell et al., 2012). Cells were plated at a
density of either 75 000 or 500 000 cells on poly-D-lysine
(Sigma, Poole, UK; 0.1 mg/ml in borate buffer, pH 8.5)
coated glass cover slips or six-well plates, respectively.
The plating medium was Dulbecco’s modified Eagle’s
medium supplemented with 5% foetal bovine serum,
penicillin/streptomycin (P/S) and 0.5 mM L-glutamine (all
from ThermoFisher). On the next day, the medium was
changed to Neurobasal medium supplemented with B27,
P/S and 0.5 mM L-glutamine (ThermoFisher). Cultures
were incubated at 37 C and 5% CO2 and were used between 18 and 21 days in vitro.
Rat primary dorsal root ganglion
neurons cultures
Primary adult rat dorsal root ganglion neurons (DRGN)
were prepared as described previously (Ahmed et al.,
2005). DRGN cells were cultured in Neurobasal-A
(ThermoFisher) at a plating density of 500 DRGN/well in
chamber slides (Beckton–Dickinson, Watford, UK) precoated with 100 lg/ml poly-D-lysine. In preliminary
experiments, the optimal concentration of mirin (100 lM)
and KU-60019 (10 lM) that promoted DRGN survival
and neurite outgrowth were determined. The positive
control was pre-optimized fibroblast growth factor-2
[Peprotech, London, UK; 10 ng/ml (Ahmed et al., 2005)].
Cells were cultured for 4 days in a humidified chamber at
37 C and 5% CO2.
Rat primary retinal cultures
Primary adult retinal cultures, containing enriched populations of retinal ganglion cells (RGC) were prepared as
described previously (Ahmed et al., 2006b). In brief, retinal cells were dissociated into single cell suspensions using
a Papain dissociation kit (Worthington Biochemicals, New
Jersey, USA). 125 103 retinal cells/well were cultured in
chamber slides (Beckton–Dickinson) pre-coated with
100 mg/ml poly-D-lysine. In preliminary experiments, the
optimal concentration of mirin (100 mM) and KU-60019
(10 mM) that promoted RGC survival and neurite outgrowth were determined. The positive control was preoptimized ciliary neurotrophic factor (CNTF) [Peprotech;
20 ng/ml (Douglas et al., 2009)]. Cells were cultured for
4 days in a humidified chamber at 37 C and 5% CO2.
Preparation of Ab oligomers and
neuronal treatment
Preparation of Ab peptides and treatment of neurons has
been previously described (Marsh et al., 2017). In brief,
Ab1-42 (Bachem) was prepared by dissolving the peptide
in dimethyl sulfoxide (1 mM). The reconstituted peptides
were diluted in 10 mM Tris, pH7.8 at a working concentration of 300 mM and stored in aliquots at 80 C.
Peptides were diluted to working concentration in full
Neurobasal medium after thawing and used immediately.
Neurons were incubated with 0.5 mM Ab1-42 or 0.5 mM
Ab1-42 þ 100 mM mirin for 24 h.
Immunocytochemistry
Adult fly brains were dissected in phosphate buffered saline (PBS) and fixed for 20 min in 4% formaldehyde in
PBS. After washing, brains were blocked in 1% bovine
serum albumin in PBS for 1 h then incubated with rat
anti-Elav (Developmental Studies Hybridoma Bank clone
7E8A1, 1:25) and mouse anti-phosphorylated pH2Av
(DSHB clone UNC93-5.2.1, 1:25) for 48 h at 4 C. Brains
were washed in PBS þ 0.3% triton X-100 for 2 h at RT
then incubated in cross-adsorbed Alexa-594 anti-rat and
Alexa-488 anti-mouse secondary antibodies (Jackson
ImmunoResearch, both 1:400) for 48 h at 4 C. After
washing as before, brains were mounted on bridge slides
in Vectashield (Vector Labs).
Hippocampal cells on coverslips following treatment
were rinsed once with pre-warmed (37 C) PBS and fixed
with 4% paraformaldehyde (TAAB, Peterborough, UK),
2% sucrose in PBS. Fixed neurons were washed with
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et al., 2004) except that the brains were incubated in primary antibodies for 4 days and in secondary antibody
overnight (both at 4 C). Primary antibodies used were:
mouse anti-PDF C7-s (1:50; Developmental Studies
Hybridoma Bank) and rabbit anti-Per1 (1:200; Santa
Cruz, CA, USA). Secondary antibodies were: Alexa-488
goat anti-mouse immunoglobulin (IgG) and cyanine 3
goat anti-rabbit IgG (both 1:100 dilution; ThermoFisher,
Leicester, UK). Brains were viewed using an Olympus
FV1000 confocal microscope. Perþ cells were counted
manually and their identities determined based on position. For statistical analysis, the PDFþ lLNv and sLNv
cells were considered together and, similarly, the PDFdorsal neuron 1, dorsal neuron 2 and lateral neuron
dorsal cells were pooled. At least seven brains were used
per genotype. Cell numbers were compared by Kruskal–
Wallis with a Dunn’s post hoc test.
R. I. Tuxworth et al.
Attenuating DNA damage is neuroregenerative
In vivo surgical procedures
Experiments were licensed by the UK Home Office and
all experimental protocols were approved by the
University of Birmingham’s Animal Welfare and Ethical
Review Board. All animal surgeries were carried out in
strict accordance to the guidelines of the UK Animals
Scientific Procedures Act, 1986 and the Revised European
Directive 1010/63/EU and conformed to the guidelines
and recommendation of the use of animals by the
Federation of the European Laboratory Animal Science
Associations (FELASA). Animals were housed in a standard facility and kept on a 12 h light–12 h dark cycle,
with a daytime luminance of 80 lux, fed and watered ad
libitum. For all in vivo experiments, adult female
Sprague-Dawley rats weighing 170–220 g (Charles River,
Margate, UK) were used. Rats were randomly assigned
to each experimental group with the investigators masked
to the treatment conditions.
Optic nerve crush injury
Rats
were
injected
subcutaneously
with
50 ml
Buprenorphine to provide analgesia prior to surgery and
anaesthetized using 5% of Isoflurane in 1.8 ml/l of O2
with body temperature and heart rate monitored throughout surgery. Optic nerve crush (ONC) was performed
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2 mm from the lamina cribrosa using watchmaker’s forceps (Berry et al., 1996; Ahmed et al., 2005).
In pilot dose-finding experiments, mirin and KU-60019
were intravitreally injected at 1, 2, 2.5, 5, 7.5 and 10 mg
(n ¼ 3 rats/group, two independent repeats), without damaging the lens, immediately after ONC and every other
day, or twice weekly or once every 7 days, in a final volume of 5 ml saline for 24 days (not shown). Rats were
then killed and retinae were dissected out, lysed in icecold lysis buffer, separated on 12% SDS-PAGE gels and
subjected to western blot detection of cH2Ax levels. We
determined that the dosing frequency of twice weekly
and 2.5 and 5 mg of mirin and KU-60019 optimally
reduced cH2Ax levels, respectively. Twice weekly intravitreal injections were sufficient in the eye since the vitreous may acts as a slow release gel due to its
composition, being made of mainly water, collagen type
II fibrils with glycosaminoglycans, hyaluronan and opticin. Optimal doses were then used for all experiments
described in this manuscript. Rats were killed in rising
concentrations of CO2 at 1 and 24 days after optic nerve
crush injury for western blot analyses or at 24 days after
ONC for determination of RGC survival and axon regeneration, as described below.
For the experiments reported in this manuscript, n ¼ 6
rats/group were used and assigned to: (1) Intact controls
(no surgery to detect baseline parameters); (2) ONC þ
vehicle (ONC followed by intravitreal injection of vehicle
solution; to detect surgery-induced changes); (3), ONC þ
mirin (ONC followed by intravitreal injection of 2.5 mg
of mirin, twice weekly; to monitor effects of inhibiting
Mre11); and (4), ONCþKU-60019 (ONC followed by
intravitreal injection of 5 mg of KU-60019; to monitor
effects of inhibiting ATM). FluoroGold was injected into
the proximal nerve stump 2 days before sacrifice and
whole retinal flatmounts were used to assess RGC survival in vivo, as described by us previously (Ahmed
et al., 2011). Each experiment was repeated on three independent occasions with a total n ¼ 18 rats/group/test.
Dorsal column crush injury
For the dorsal column (DC) lesion model, experiments
also comprised n ¼ 6 rats/group: (1), Sham controls
(Sham; to detect surgery-induced changes; partial laminectomy but no DC lesion); (2), DC transected controls þ
intrathecal injection of vehicle (PBS); (3), (DC þ vehicle;
to detect injury-mediated changes); (4), DC þ intrathecal
injection of mirin (DC þ mirin; to monitor effects of
inhibiting Mre11); (5)and DC þ intrathecal injection of
KU-60019 (DCþKU-60019; to monitor effects of inhibiting ATM). Each experiment was repeated on three independent occasions with a total n ¼ 18 rats/group/test.
Rats were injected subcutaneously with 50 ml
Buprenorphine to provide analgesia prior to surgery and
anaesthetized using 5% of Isoflurane in 1.8 ml/l of O2
with body temperature and heart rate monitored throughout surgery. For optic nerve crush injury, a supraorbital
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Tris buffered saline, permeabilized with 0.1% Tween-20
and 5% horse serum in Tris buffered saline for 45 min at
RT and incubated with primary antibody overnight at
4 C. Coverslips were mounted using the ProLong Gold
reagent (ThermoFisher). The antibodies used were: mouse
anti-H2Ax pSer319 (cH2Ax; JBW301; 1;1000 dilution;
Merck) and Alexa-568 anti-mouse IgG (ThermoFisher).
The cells were visualized on a spin disc confocal system
(CARV from Digital Imaging Solutions) with an EMCCD camera (Rolera/QI Cam 3500) mounted on an
Olympus X71 microscope, using a 100 fluoplan objective (NA 4.2). The microscope confocal system was supported by Image Pro 6.0 software.
DRGN and RGC were fixed in 4% paraformaldehyde,
washed in three changes of PBS before being subjected to
immunocytochemistry as described previously (Ahmed
et al., 2005; Ahmed et al., 2006b). Antibodies used were:
mouse anti-bIII tubulin (1:200 dilution; Sigma) to visualize neuronal cell soma and neurites and Alexa-488 goat
anti-mouse IgG (1:400 dilution; ThermoFisher). Slides
were then viewed with an epifluorescent Axioplan 2
microscope, equipped with an AxioCam HRc and running Axiovision Software (all from Zeiss, Hertfordshire,
UK). The proportion of DRGN with neurites and the
mean neurite length were calculated using Axiovision
Software by an investigator masked to the treatment conditions as previously described (Ahmed et al., 2005;
Ahmed et al., 2006b). All experiments were performed in
triplicate and repeated on three independent occasions.
BRAIN COMMUNICATIONS 2019: Page 5 of 21
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Knockdown of Mre11 using shRNA
in vivo
SMARTvector Lentiviral shRNAs to Mre11 (shMre11; cat
no. V3SR11242-240192989) and ATM (shATM; cat no.
V3SR11242-238270626) under the control of a
cytomegalovirus
promoter
were
purchased
from
Dharmacon and plasmid DNA was prepared from glycerol
stocks according to the manufacturer’s instructions. A control plasmid containing the cytomegalovirus promoter and
a non-targeting control (shControl; cat no. VSC11721) was
also purchased from Dharmacon. Plasmid DNA containing
shMre11, shATM and shControl were complexed with
in vivo-jetPEI (referred to as PEI from herein; Polyplus
Transfection, New York, USA) according to the manufacturer’s instructions and injected into the DRG immediately
after DC injury, as described by us previously (Jacques
et al., 2012; Almutiri et al., 2018). We used in vivo-jetPEI,
a non-viral vector, to deliver the shRNAs to DRGN
in vivo, since we have previously shown that it transduces
similar proportions of DRGN as adeno-associated virus 8
and that it does not require intra-DRG injection 1–2 week
prior to injury to ensure maximum transgene expression
(Almutiri et al., 2018). In a preliminary experiment, we
optimized the amount of plasmid DNA required to cause
maximum suppression of cH2Axþ foci in DRGN as 2 mg
for both shMre11 and shATM (not shown). Pre-optimized
plasmid DNA was then injected intra-DRG and animals
(n ¼ 6/group/test, three independent repeats, total n ¼ 18
rats/group/test) were killed after 28 days for immunohistochemistry to detect cH2Axþ or 6 weeks for electrophysiological and behavioural studies.
Immunohistochemistry
Tissue preparation for cryostat sectioning and immunohistochemistry were performed as described previously
(Surey et al., 2014). In brief, rats were intracardially perfused with 4% formaldehyde and optic nerves, eyes, L4/
L5 DRG and segments of T8 cord containing the DC injury sites were dissected out and post-fixed for 2 h at RT.
Tissues were then cryoprotected in a sucrose gradient
prior to mounting in optimal cutting temperature embedding medium (ThermoFisher) and frozen on dry ice.
Samples were then sectioned using a cryostat and immunohistochemistry was performed on sections from the
middle of the optic nerve, DRG or spinal cord as
described previously (Surey et al., 2014). Sections were
permeabilized in PBS containing 0.1% Triton X-100,
blocked in PBS þ 3% w/v bovine serum albumin þ
0.05% Tween-20 then stained with primary antibodies
overnight at 4 C. After washing in PBS, sections were
incubated with secondary antibodies for 1 h at RT then
washed further in PBS and mounted in Vectashield containing DAPI (Vector Laboratories). Primary antibodies
used were: mouse anti-H2Ax pSer139 (cH2Ax; JBW301;
1:400 dilution; Merck) and rabbit anti-neurofilament 200
(1:400
dilution;
Sigma);
mouse
anti-GAP43
(ThermoFisher; 1:400 dilution) was used to detect regenerating axons in the optic nerve and spinal cord.
Regenerating axons in the DC were detected using
GAP43 immunohistochemistry (Ahmed et al., 2014;
Almutiri et al., 2018; Farrukh et al., 2019) since Cholera
toxin B labelling in our hands did not label regenerating
axons by retrograde transport labelling in the rat (Ahmed
et al., 2014), despite others demonstrating successful
labelling (Neumann and Woolf, 1999; Neumann et al.,
2002). Secondary antibodies used were Alexa-488 goat
anti-mouse IgG and TexasRed goat anti-rabbit IgG (both
from ThermoFisher). Controls were included in each run
where the primary antibodies were omitted and these sections were used to set the background threshold prior to
image capture. Image capture and analysis was performed
by an investigator masked to the treatment conditions.
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approach was used to expose the optic nerve and crushed
with calibrated watchmaker’s forceps 2 mm from the lamina cribrosa of the eye (Berry et al., 1996). Animals were
immediately injected intravitreally and avoiding damaging
the lens with vehicle, mirin or KU-60019. Animals were
then allowed to survive for 24 days before assessment of
RGC survival and axon regeneration. For the DC injury
model, a partial T8 laminectomy was performed and the
DC were crushed bilaterally using calibrated watchmaker’s forceps as described (Surey et al., 2014; Almutiri
et al., 2018). The subarachnoid space was cannulated
with a polyethylene tube (PE-10; Beckton–Dickinson)
through the atlanto-occipital membrane as described by
others (Yaksh and Rudy, 1976). The catheter tip was
advanced 8 cm caudally to the L1 vertebra and the other
end of the catheter was sealed with a stainless-steel plug
and affixed to the upper back. Animals were injected immediately with vehicle (PBS), mirin or KU-60019 followed by a 10 ml PBS catheter flush. Injections were
repeated every 24 h and drugs and vehicle reagents were
delivered over 1 min time period using a Hamilton microlitre syringe (Hamilton Co, USA).
In a pilot experiment, mirin and KU-60019 were
injected as described above at 1, 2, 5, 10 and 15 mg
(n ¼ 3 rats/group, two independent repeats) in a final volume of 10 ml saline either daily, every other day or twice
weekly for 28 days (not shown). Rats were then killed
and the lesion site plus 5 mm either side were harvested,
lysed in ice-cold lysis buffer, separated on 12% SDSPAGE gels and subjected to western blot detection of
cH2Ax levels (Surey et al., 2014). We determined that
the amount of mirin and KU-60019 to optimally reduce
cH2Ax levels by intrathecal delivery was 5 and 10 mg, respectively, with a dosing frequency of every 24 h.
Optimal doses were then used for all experiments
described in this manuscript. Rats were killed in a rising
concentration of CO2 at either 28 days for immunohistochemistry and western blot analyses or 6 weeks for electrophysiology and functional tests.
R. I. Tuxworth et al.
Attenuating DNA damage is neuroregenerative
Quantification of axon regeneration
Axon regeneration in the spinal cord was quantified
according to previously published methods (Hata et al.,
2006). In brief, serial parasagittal sections of cords were
reconstructed by collecting all serial 50 mm-thick sections
(70–80 sections/animal; n ¼ 10 rats/treatment) and the
number of intersections of GAP43þ fibres through a
dorsoventral-orientated line was counted from 6 mm rostral to 4 mm caudal to the lesion site. Axon number was
calculated as a % of fibres seen 4 mm above the lesion,
where the DC was intact.
Six weeks after surgery or treatment, compound action
potentials (CAP) were recorded after vehicle, mirin and
KU-60019 treatment as previously described (Lo et al.,
2003; Hains et al., 2004; Almutiri et al., 2018). In brief,
with the experimenter masked to the treatment conditions, silver wire electrodes were used apply single-current
pulses (0.05 ms) through a stimulus isolation unit in
increments (0.2, 0.3, 0.6, 0.8, and 1.2 mA) at lumbar
(L)1-L2 and CAP recorded at cervical (C)4-C5 along the
surface of the midline spinal cord. CAP amplitudes were
calculated between the negative deflection after the stimulus artefact and the next peak of the wave. CAP area
was calculated by rectifying the negative component (fullwave rectification in Spike 2 software) and measuring its
area at the different stimulation intensities. The dorsal
half of the spinal cord was transected between stimulating and recording electrodes at the end of the experiment
to confirm that a CAP could not be detected.
Electrophysiology was analysed using Spike2 software
(Cambridge Electronic Design, Cambridge, UK) and representative processed data are shown for CAP traces.
Functional tests after DC injury
Functional testing after DC lesions was carried out as
described by previously (Fagoe et al., 2016; Almutiri
et al., 2018). In brief, animals [n ¼ 6 rats/group, three independent repeats (total n ¼ 18/group/test)] randomly
assigned and treatment status masked from the investigators, received training to master traversing the horizontal
ladder for 1 week before functional testing. Baseline
parameters for all functional tests were established 2–3
days before injury. Animals were then tested 2 days after
DC lesion þ treatment and then weekly for 6 weeks.
Experiments were performed by two observers blinded to
treatment with animals tested in the same order and at
the same time of day. Three individual trials were performed each time for each animal.
Horizontal ladder crossing test
This tests the animals’ locomotor function and is performed on a 0.9-m-long horizontal ladder with a diameter of 15.5 cm and randomly adjusted rungs with
| 7
variable gaps of 3.5–5.0 cm. Animals were assessed traversing the ladder with the total number of steps taken
to cross the ladder and the number of left and right rear
paw slips being recorded. The mean error rate was then
calculated by dividing the number of slips by the total
number of steps taken.
Tape removal test (sensory function)
The tape removal test determines touch perception from
the left hind paw. Animals were held with both hindpaws extended and the time it took for the animal to detect and remove a 15 15 mm piece of tape (Kip
Hochkrepp, Bocholt, Germany) was recorded and used to
calculate the mean sensing time.
Analysis of functional tests
The whole time-course of lesioned and sham-treated animals for the horizontal ladder crossing and mean tape
sensing/removal test was compared using generalized linear
mixed models (GLMM) or linear mixed models (LMM),
as described previously (Fagoe et al., 2016; Almutiri et al.,
2018). For the horizontal ladder test, we scored individual
steps as either a successful step or a slip and therefore the
data follows a binomial distribution. Data were compared
using binomial GLMM, with lesioned/sham (‘LESION’; set
to true in lesioned animals post-surgery, false otherwise)
and operated/unoperated (OPERATED; set to false before
surgery, true after surgery) as fixed factors, animals as a
random factors and time as a continuous covariate.
Binomial GLMMs were then fitted in R using package
lme4 with the glmer function using the following model
formulae (Fagoe et al., 2016):
outcome LESION * time þ OPERATED þ (time\animal)
(Model 1)
outcome LESION þ time þ OPERATED þ (time\animal)
(Model 2)
outcome LESION þ time þ OPERATED þ (1\animal)
(Model 3)
outcome time þ OPERATED þ (1\animal)
(Model 4)
Bracketed terms refer to the ‘random effects’ and account
for the presence of repeated measurements in estimation
of the effect size and significance of INT and LESION.
‘Outcome’ for binomial GLMMs is a two-column list of
counts of successes/fails per run as described in the individual models above. INT refers to the interaction term
of LESION over time (Model 1). P-values for INT were
then calculated, which represents differences in the evolution of outcomes over time, and LESION, the unconditional main effect of lesioning. Significance of specific
parameters in LMMs and GLMMs can be determined by
comparing a model containing the parameter of interest
to a reduced model without it. Thus, INT was assessed
by comparing Models 1 and 2, while LESION was
assessed by comparing Models 3 and 4. LESION,
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Electrophysiology
BRAIN COMMUNICATIONS 2019: Page 7 of 21
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| BRAIN COMMUNICATIONS 2019: Page 8 of 21
Analysis of pH2A positive foci
Drosophila brains
Adult fly brains were dissected in PBS and fixed and
stained as described above. 41 41 13.5 mm volumes
of the same region of the central brain were imaged at
16-bit depth for each brain using a 63 water immersion
N.A. 1.2 objective on a Zeiss LSM880 confocal microscope. The zoom was set at 3.2 and z-step at 0.45 mm
and Airy scan processing module used with super-resolution settings to improve spatial resolution post-collection. To display visualize brightly staining pH2Avþ foci
only, the lower intensity pan-nuclear staining was eliminated by manually resetting the black level threshold in
Fiji-3. The identical original and processed images are
displayed
together
in
Supplementary
Figures.
Supplementary Videos 1 and 2 were rendered in Fiji-3.
were thresholded and the mean integrated density of pixels/cell was recorded.
3. Western blot: Total protein was extracted from
pooled L4/L5 DRG pairs from n ¼ 3 rats/group (i.e. six
DRG) after DC þ vehicle, mirin and KU-60019 treatment
and western blots followed by subsequent densitometry
was performed as described by us previously and in brief
below (Ahmed et al., 2005). Experiments were repeated
on three independent occasions (total n ¼ 9 rats/group i.e.
18 DRG/group).
Western blots
Proteins were extracted from hippocampal, retinal or
DRG neuron cultures/tissues as described by us previously (Ahmed et al., 2005). In brief, cultures were treated
with ice-cold lysis buffer [20 mM HEPES pH 7.5, 1 mM
EDTA, 150 mM NaCl, 1% NP-40 and 1 mM dithiothreitol supplemented with protease (Roche, Welwyn Garden
City, UK) and phosphatase (ThermoFisher) inhibitor
cocktails)], protein concentration was determined by
Bradford Protein Assay and 15 mg of total protein separated on 12% Tris-glycine SDS-PAGE gels. Proteins were
transferred to nitrocellulose membranes, blocked with
2.5% non-fat dry milk in Tris buffered saline containing
0.05% Tween-20 for 90 min with agitation then incubated for 2 h with primary antibodies and for 1 h with
secondary antibodies diluted in 2.5% non-fat dry milk at
RT. For hippocampal neurons, proteins were detected
using the Odyssey imaging scanner (LI-COR). Primary
antibodies used were: mouse anti-Synapsin antibody
(Santa Cruz) and mouse anti-actin antibody (Sigma,
A5316). Secondary antibodies used were: Alexa-680 goat
anti-mouse IgG and Alexa-800 goat anti-rabbit IgG (New
England Biolabs). For DRGN cultures, membranes were
probed with primary anti-cH2Ax (pSer139 monoclonal
antibody; 1:400 dilution; Merck) and horseradish
peroxidase-labelled anti-mouse secondary antibody before
bands being detected using an enhanced chemiluminescence kit (GE Healthcare, Buckingham, UK). b-actin
(Sigma: 1:1000 dilution) was used as a protein loading
control for western blots.
Dorsal root ganglion neurons
Densitometry
1. Frequency distribution: Images of the entire DRG in
the middle three sections (n ¼ 160 images/DRG) of L4/L5
DRG from each animal (n ¼ 10) were captured by an investigator masked to the treatment conditions at 10
magnification using a Zeiss Axioplan 200 epifluorescent
microscope. Images were merged in Adobe Photoshop
using Photomerge and the frequency of cH2Axþ foci in
different diameter DRGNs was recorded (Jacques et al.,
2012).
2. Relative fluorescent staining intensity: the integrated
density of fluorescence was measured using ImageJ as
previously described (Surey et al., 2014). In brief, images
from the middle three DRG sections from n ¼ 10 rats
Western blots were scanned into Adobe Photoshop
(Adobe Systems, San Jose, CA, USA) keeping all scanning
parameters constant between blots. The integrated density
of bands was analysed using the built-in macros for gel
analysis in ImageJ as described by us previously (Ahmed
et al., 2006a).
Statistical analysis
All data are presented as means 6 standard error of the
mean (SEM). Comparison of means was performed using
one-way or two-way ANOVA using Prism 7 (San Diego,
CA, USA) or SPSS (Version 25, IBM, New York, USA).
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represents the overall effect of lesioning on the outcome,
while INT represents the difference in slope of the
outcome between the two groups, i.e. speed of recovery.
P-values for GLMMs were calculated by model comparison using parametric bootstrap for INT and LESION
against the null hypothesis that each parameter is zero,
using pbkrtest in R package. Between 1000 and 20 000
simulations were used.
For tape removal test, the time-courses of lesioned versus
sham were compared using LMMs with the R package
lme4 with the glmer function (Fagoe et al., 2016; Almutiri
et al., 2018). Model formulae were the same as for the
ladder crossing test above. Standard regression diagnostics
(quantile plots of the residuals versus the normal distribution, plots of residuals versus fitted values) were carried
out for the data fitted with LMMs. P-values for the INT
and LESION parameters of the LMMs were calculated by
model comparison using package pbkrtest in R, with the
Kenward–Roger method. For the tape sensing and removal
test, log of the withdrawal time was used as the data were
expected to follow an exponential distribution. Independent
sample T-tests were performed to determine statistical differences at individual time points.
R. I. Tuxworth et al.
Attenuating DNA damage is neuroregenerative
BRAIN COMMUNICATIONS 2019: Page 9 of 21
| 9
Tukey’s significant difference test followed ANOVA’s, as
appropriate. In Fig. 2A, a Kruskal–Wallis test with
Dunn’s post hoc was performed. Functional tests in rats
were analysed using R package (www.r-project.org) as
described above. Tests used for each experiment are
described in the corresponding methods. In all tests,
P < 0.05
was
considered
statistically
significant.
Supplementary Table 1 lists all tests performed, the comparisons made, and the associated P-values.
Data availability
The authors confirm that the data supporting the findings
of this study are available within the article and its
Supplementary material.
Results
Genetic targeting of the double-strand break-sensing
MRN complex is neuroprotective in Drosophila. Doublestrand breaks are a feature of early stage Alzheimer’s disease and correlate with reduced cognitive score (Simpson
et al., 2015). Double-strand breaks are also generated in
neurons in vivo exposed to Ab1-42 oligomers (Suberbielle
et al., 2013). Hence, we chose to target the MRN complex in a Drosophila model of Alzheimer’s model as
proof-of-concept. In this model, tandem Ab1-42 oligomers
for Alzheimer’s disease were expressed in, and secreted
from, adult post-mitotic neurons to replicate the extracellular deposition in Alzheimer’s disease (Speretta et al.,
2012). Expression was restricted to adult neurons only to
rule out confounding developmental phenotypes (see
Materials and Methods section for genetics). First, we
confirmed that double-strand breaks were generated in
neurons exposed to Ab1-42 oligomers, as predicted from
other studies and our own studies in rat hippocampal
neurons (Fig. 2). Activation and recruitment of ATM to
the site of double-strand breaks lead to multiple phosphorylation events, one of which is the phosphorylation
of histone cH2Ax at and around the site of the break,
resulting in the formation of characteristic cH2Axþ nuclear foci (Shrivastav et al., 2008). Therefore, antibodies
to cH2Ax (H2Av in Drosophila) are widely use to visualize the histone modifications surrounding a double-strand
break (Celeste et al., 2002; Simpson et al., 2015;
Milanese et al., 2018). Anti-pH2Av staining of control
brains revealed neurons with a single brightly staining
focus per nucleus corresponding to the nucleolus organizer region. In contrast, many of the Ab1-42-expressing
neurons had multiple, clustered foci (Fig. 1A,
Supplementary Fig. 1 and Video 1).
Next, we asked whether reducing nbs levels genetically
would be neuroprotective in Ab1-42 expressing flies.
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Figure 1 Targeting the MRN complex is neuroprotective in Drosophila models of neurodegeneration. (A) Expression of tAb1-42 in
adult neurons generates DNA double-strand breaks. Projections of central brain neurons stained with anti-pH2Av. Control neurons display a
single pH2Avþ focus per nucleus corresponding to the nucleolus organising region. Multiple neurons expressing Ab1-42 display large numbers of
clustered foci (arrowheads). (B) Flies were startled by tapping to the base of a vial and the negative geotaxis climbing response quantified as a
measure of neural output. Flies expressing (B) Ab1-42 or (C) Htt.Q128 in adult neurons show a rapid decline in climbing ability which is partially
suppressed in flies heterozygous for a null allele of nbs. Statistical comparisons in B by two-way ANOVA and in B by extra sum-of-squares F-test,
*** indicates P < 0.001. (D) When stimulated by vibration, Tau-expressing flies show a rapid decline in the startle response which is partially
suppressed in nbs1/þ flies. Dashed arrows indicate days of expression required for 50% decline in response determined from fitted lines;
comparison by two-tailed t-test. Scale bars in A ¼ 10 mm.
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| BRAIN COMMUNICATIONS 2019: Page 10 of 21
R. I. Tuxworth et al.
We chose to introduce one null allele of nbs to reduce
the gene dosage by 50% and then used the ability of flies
to climb after being startled as a measure of neural output. This negative geotaxis assay is widely used in
Drosophila neurodegeneration studies: climbing ability
declines rapidly and correlates with a reduced speed of
neural transmission (Kerr et al., 2011). In flies expressing
Ab1-42 oligomers, climbing ability decreased rapidly with
age yet this was partially suppressed in nbs/þ heterozygous flies (Fig. 1B and see Supplementary Video 2). The
toxicity of the less aggregative tAb1-42 construct lacking the
12 amino acid flexible linker was similar suppressed in
nbs1/þ heterozygous flies and a second null allele, nbs2/þ
showed a similar effect (Supplementary Fig. 2A and B).
To confirm that the neuroprotective effect was not
specific to Ab1-42 pathology, we used the same approach for flies expressing an expanded Htt protein
associated with Huntington’s disease (Htt.Q128)
(Romero et al., 2008) and saw a similar neuroprotective
effect in climbing assays (Fig. 1C). Again, to confirm
the suppression was not specific to climbing ability, we
switched to a tracking system capable of monitoring the
horizontal movement of flies continuously and
expressed the human 0N4R Tau carrying the R406W
point mutation associated with frontal temporal dementia with Parkinsonism (Wittmann et al., 2001). Tauexpressing flies moved at normal speed when unstimulated (not shown) but their escape response declined
rapidly with age. Again, this was partially suppressed in
nbs/þ flies (Fig. 1D) and this neuroprotective effect
was not due to a reduction in Tau levels nor to changes
in Tau phosphorylation in the brains of these flies
(Supplementary Fig. 2C). Finally, to ensure these protective effects were not specific to nbs, we depleted
rad50 in Htt.Q128-expressing flies using the same strategy of reducing the gene dosage with a null allele. Flies
with reduced rad50 (rad50/þ) partially suppressed the
decline in climbing ability in the Htt.Q128 flies
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Figure 2 Targeting the MRN complex protects against Alzheimer’s disease-relevant phenotypes. (A) Expression of Ab1-42 in clock
neurons leads to loss of Perþ cells. In nbs/þ flies, loss of cells is partially prevented (Kruskal–Wallis with Dunn’s post hoc test; P-values and n are
indicated). (B) The free-running circadian locomotor cycle of control flies is approximately 24 h but is considerably lengthened when Ab1-42 is
expressed in clock neurons indicating weaker rhythmicity in the circadian circuitry. The increase is suppressed in nbs1/þ flies (CLEAN spectral
analysis; comparisons by ANOVA with Tukey’s post hoc test; P-values and n are indicated). (C) Exposure of rat hippocampal neurons to Ab1-42
oligomers in vitro generate double-strand breaks that can be visualized by staining with anti-cH2Ax (red). DNA is visualized with DAPI (blue).
(D) Quantification of synapsin levels in hippocampal neurons by western blot. Exposure to Ab1-42 oligomers leads to loss of the pre-synaptic
protein, synapsin, which is reversed by the Mre11 inhibitor, mirin (mean 6 SEM; ANOVA with Tukey’s post hoc test; P-values and n are indicated).
Scale bars in C ¼ 10 mm.
Attenuating DNA damage is neuroregenerative
(Supplementary Fig. 2D). We also expressed expanded
HttQ128 in the developing eye under the control of
GMR-gal4, which generates a progressive degeneration
evidenced as a widespread loss of pigmentation after 6–
7 weeks (Romero et al., 2008). In rad50/þ flies, pigmentation is restored, indicating enhanced cellular survival (Supplementary Fig. 2E).
Targeting the MRN complex
suppresses neurodegenerationrelevant phenotypes
Mre11 or ATM inhibition prevents
apoptosis and stimulates
regeneration after neurotrauma
Double-strand breaks are a feature of acute neurological
disorders (Hayashi et al., 1998; Kotipatruni et al., 2011).
DNA strand breaks have been observed in RGC after optic
nerve injury (He et al., 2012) and neurons suffering from
ischaemia display genome fragility and fragmented DNA
both in vitro and in vivo (Yang et al., 2016). We used an
adult rat primary retinal culture system enriched in RGC,
| 11
where cells are grown in the presence of inhibitory CNS
myelin extracts to simulate a post-injury environment
(Ahmed et al., 2006b). As expected, we detected the presence of double-strand breaks with anti-cH2Ax antibodies
in the RGC (Fig. 3A and B). The RGC die rapidly by
apoptosis and, even if apoptosis is blocked with inhibitors,
they fail to regenerate neurites (Vigneswara et al., 2013).
We treated the RGC with mirin to attenuate the DNA
damage response. This blocked RGC apoptosis and dramatically stimulated neurite regrowth. Mirin treatment was
significantly more effective than a current positive control
treatment: CNTF (Fig. 3C and D). Since a key function of
the MRN complex is to recruit and activate ATM at double-strand breaks (Lee and Paull, 2005), and given that
Huntington’s disease-pathology is reduced by targeting
ATM (Lu et al., 2014), we asked whether inhibiting ATM
directly would also be neuroprotective to RGC. Treatment
with the highly selective ATM inhibitor, KU-60019, had
similarly dramatic effects on RGC survival and neurite outgrowth as mirin (Fig. 3C–F).
We extended our in vitro findings to an in vivo optic
nerve crush injury paradigm. We observed low levels of
cH2Ax in intact control rat retinae in vivo (Fig. 4A
and B). However, high levels of cH2Ax were observed
within 1 day and at 24 days after optic nerve crush injury, suggesting rapid activation of double-strand
breaks and persistence of high levels for the duration of
the experiment i.e. 24 days. Thus, the DNA damage response pathway was persistently activated in the retina
post-injury. We used a pre-optimized dosing regimen
for mirin and KU-60019 for 24 days after optic nerve
crush injury to attenuate the DNA damage response, as
demonstrated by significantly suppressed cH2Ax in
western blots (Fig. 4C) and subsequent densitometry
(Fig. 4D) and saw unprecedented RGC survival of 93%
and 91%, respectively, when compared with intact controls (Fig. 4E and F). Both mirin and KU-60019 also
promoted significant numbers of RGC to regenerate
their axons, which emerged from the lesion site and
grew for long distances (Fig. 4G and H). The numbers
of regenerating axons we see are, to the best of our
knowledge, unprecedented when compared with known
treatments and the axons also regenerate longer distances into the distal optic nerve than has been seen previously (Berry et al., 1996; Leon et al., 2000; Vigneswara
et al., 2013).
Mre11 and ATM inhibitors promote
DRGN survival and neurite
outgrowth in vitro and DC axon
regeneration after SCI in vivo
Double-strand breaks are also generated in spinal neurons
after SCI (Kotipatruni et al., 2011) and there is an urgent
need for effective therapies to treat SCI patients. Initially,
we tested our DNA damage response attenuation strategy
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Alzheimer’s disease patients commonly suffer disrupted
circadian behaviour patterns (Saeed and Abbott, 2017)
and a similar effect is observed in Ab-expressing flies
(Long et al., 2014). We asked whether reducing nbs
genetically would suppress this more directly diseaserelevant phenotype. Tandem Ab1-42 was expressed specifically in clock neurons under the control of tim-Gal4
and their survival studied with immunocytochemistry
(Renn et al., 1999). Ab-expression induced loss of some
Perþ neurons but this was partially suppressed in nbs/þ
flies (Fig. 2A). Consistent with the loss of clock neurons
(Renn et al., 1999), Ab-expressing flies exhibited significantly longer free-running circadian locomotor cycles than
controls but the normal 24 h periodicity was restored in
nbs/þ flies (Fig. 2B and Supplementary Fig. 2F).
We extended our findings to mammalian neurons by
examining synapse loss—a common early feature of neurodegenerative diseases (Gillingwater and Wishart, 2013).
Cultured primary hippocampal neurons were exposed to
Ab1-42 oligomers and stained with anti-cH2Ax antibodies.
As expected, the characteristic foci corresponding to double-strand breaks in the nuclei were induced by exposure
to Ab1-42 (Fig. 2C). Synapsin levels were significantly
reduced 24 h after addition of Ab oligomers (Fig. 2D)
suggesting that one route to synapse loss in Alzheimer’s
disease might be via Ab-induced double-strand breaks
triggering the DNA damage response. Mirin, a small-molecule Mre11 exonuclease inhibitor (Dupre et al., 2008),
prevented synapsin loss (Fig. 2D), indicating that targeting the MRN complex can also protect mammalian neurons
from
changes associated
with
early-stage
neurodegeneration.
BRAIN COMMUNICATIONS 2019: Page 11 of 21
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R. I. Tuxworth et al.
of inhibitory CNS myelin extracts. (A) Western blot and (B) subsequent densitometry to show attenuated levels of anti-cH2Ax after
treatment with mirin and KU-60019. (C) Mirin or KU-60019 significantly enhanced RGC survival. (D) Representative images from RGC treated
with vehicle, CNTF (positive control), mirin and KU-60019. Mirin or KU-60019 treatment (E) increased the mean RGC neurite length and (F) %
RGC with neurites. n ¼ 3 wells/treatment, three independent repeats (total n ¼ 9 wells/condition). AU ¼ arbitrary units. Comparisons in B, C, E
and F by one-way ANOVA with Dunnett’s post hoc test. Scale bars in D ¼ 100 mm.
using a similar in vitro culture model to the retina in
which DRGN containing double-strand breaks are cultured from adult rats and grown in the presence of inhibitory CNS myelin extracts (Supplementary Fig. 3A).
Again, mirin and KU-60019 treatment stimulated significant DRGN survival and promoted neurite outgrowth
(Supplementary Fig. 3B–E).
To generate a SCI in vivo, we surgically injured the
ascending long tract axons of the dorsal funiculus in
adult rats by DC crush injury (Surey et al., 2014) and
mirin or KU-60019 inhibitors were administered through
an intrathecal catheter. None of the animals showed adverse effects to SCI and all animals were included for
analysis. Double-strand breaks formed in DRGN of all
diameters after DC þ vehicle treatment, as predicted
from previous studies (Kotipatruni et al., 2011) (Fig. 5A
and B). However, quantification of the intensity of
cH2Axþ was significantly reduced in DC þ mirin and
DCþKU-60019-treated animals (Fig. 5C). Similarly, western blot and subsequent quantification for cH2Ax levels
showed significant attenuation in DC þ mirin and
DCþKU-60019-treated rats, indicating partial suppression
of the DNA damage response (Fig. 5D and E).
We next investigated if suppression of DNA damage response by mirin and KU-60019 promotes DC axon regeneration after injury in rats. Little or no GAP43þ
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Figure 3 Inhibition of Mre11 prevents RGC apoptosis and stimulates neurite outgrowth after 4 days in culture in the presence
Attenuating DNA damage is neuroregenerative
BRAIN COMMUNICATIONS 2019: Page 13 of 21
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crush injury. (A) Western blot and (B) subsequent densitometry to show phosphorylation of H2Ax (cH2Ax) as a marker of DNA damage
after optic nerve crush injury (n ¼ 6 retinae/time point, three independent repeats (total n ¼ 18 retinae/time point)). (C) Western blot and (D)
densitometry to show that mirin and KU-60019 significantly suppress optic nerve crush injury-induced cH2Ax levels (n ¼ 6 retinae/time point,
three independent repeats (total n ¼ 18 retinae/condition). (E) Representative images and (F) quantification of FluoroGold backfilled RGC in
retinal wholemounts to demonstrate that mirin and KU-60019 significantly enhanced RGC survival at 24 days after optic nerve injury (n ¼ 6
retinae/time point, three independent repeats (total n ¼ 18 retinae/condition). (G) Representative images and (H), quantification to show that
mirin and KU-60019 significantly enhanced RGC axon regeneration as detected by GAP43 immunoreactivity (n ¼ 6 nerves/condition, three
independent repeats (total n ¼ 18 nerves/condition). AU ¼ arbitrary units. Comparisons in B, D, F and H by one-way ANOVA with Dunnett’s
post hoc test. Scale bars in E ¼ 50mm and in G ¼ 200 mm.
immunoreactivity was detected in DC þ vehicle-treated
rats whilst a large cavity was present at the lesion site (#)
(Fig. 6A). Quantification of the % of axons in DC þ vehicle-treated rats falls rapidly to 0% at the lesion site
(i.e. 0 mm) and remains at 0% for all distances quantified
(Fig. 6B). In DC þ mirin and DCþKU-60019-treated
rats, significant GAP43þ immunoreactivity was present
beyond the lesion cavity and in the rostral segment of
the spinal cord (Fig. 6A and B). Quantification of the %
of axons demonstrated significantly enhanced numbers at
all distances when compared to DC þ vehicle-treated
rats, with DCþKU-60019 treatment regenerating slightly
greater numbers of GAP43þ axons at all distances rostral
to the lesion (Fig. 6B). For example, DCþKU-60019
treatment regenerated 47 6 8%, 35 6 6%, 25 6 4% and
18 6 3% of axons at 0, 2, 4 and 6 mm rostral to the
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Figure 4 Inhibition of Mre11 and ATM prevents RGC apoptosis and stimulates axon regeneration at 24 days after optic nerve
14
| BRAIN COMMUNICATIONS 2019: Page 14 of 21
R. I. Tuxworth et al.
lesion site, compared with DC þ vehicle-treated rats
(Fig. 6B).
Mre11 and ATM inhibitors restore
function after SCI in vivo
We employed electrophysiology and simple functional
tests (Almutiri et al., 2018) to quantify recovery from the
injury after mirin or KU-60019 treatment. In DC þ vehicle-treated rats, the normal CAP trace across the lesion
site in Sham controls was abolished, whilst treatment
with either mirin and KU-60019 restored 50–55% of the
CAP trace (Fig. 7A) and the CAP amplitude (Fig. 7B)
observed in sham-treated animals. As expected, dorsal
hemisection between the stimulating and the recording
electrodes at the end of the experiment abolished CAP
traces in all animals (if a CAP trace was observed), confirming that the experiment was technically successful
(Fig. 7A). The CAP area was also significantly improved
in mirin and KU60019-treated rats compared with vehicle
groups (Fig. 7C). The improved electrophysiological function translated into very dramatic improvements in
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Figure 5 Inhibition of Mre11 and ATM reduces the number of DRGN with double-strand breaks and the levels of cH2Ax after
DC injury in vivo. (A) Representative images from DC þ vehicle, DC þ mirin and DCþKU-60019-treated sections of rat DRGN (n ¼ 6 rats/
group, three independent repeats; total n ¼ 18 rats/group) demonstrating cH2Ax (green) localization in the nucleus of DRGN (red). Images are
counterstained with DAPI (blue) to demarcate the nucleus. (B) Quantification of the frequency of cH2Axþ DRGN sorted by different soma size.
(C) Quantification of the mean pixel intensity/DRGN in DC þ vehicle, DC þ mirin and DCþKU-60019-treated DRGN. (D) Western blot and
(E) subsequent densitometry to demonstrate reduction of cH2Ax protein levels in DRGN after treatment with mirin and ATM inhibitors. AU ¼
arbitrary units. Comparisons in C and E by one-way ANOVA with Dunnett’s post hoc test. n ¼ 6 rats/treatment, three independent repeats (total
n ¼ 18 rats/treatment). Scale bars in A ¼ 50 mm, insets in A ¼ 10 mm.
Attenuating DNA damage is neuroregenerative
inhibitors promote significant axon regeneration at 6
weeks after SCI. (A) GAP43þ (green) immunoreactivity was
largely absent in DC þ vehicle-treated groups (Blue ¼ DAPIþ
nuclei). However, in DC þ mirin and DCþKU-60019-treated rats,
enhanced GAP43þ immunoreactivity was present in the rostral
segment of the spinal cord in beyond the lesion site (#) where a
large cavity remained. (B) Quantification of the % of axons at
different distances rostral to the lesion site showed significantly
enhanced numbers of GAP43þ regenerating axons at all distances
tested after treatment with DC þ mirin and DCþKU-60019.
(n ¼ 10 nerves/condition). Scale bars in A ¼ 200 mm. Comparisons
in B by one-way ANOVA with Dunnet’s post hoc test (DC þ vehicle
versus DCþKU-60019).
sensory and locomotor function. We used a tape sensingand-removal test and a horizontal ladder-crossing test to
quantify recovery of sensory and motor function, respectively (Almutiri et al., 2018). In sham-treated animals,
there is a small initial decline in mean sensing/removal
time (Fig. 7D) and mean ratio of slips to total steps
(Fig. 7E) due to the effects of anaesthetic but these are
restored to baseline levels within 3 weeks. Vehicle-treated
groups displayed significantly impaired sensory and
motor function that failed to return to baseline levels
even after 6 weeks. In contrast, mirin or KU-60019treated groups both showed dramatically improved sensing times and ladder-crossing ability 2 days after surgery
and after 3 weeks were indistinguishable from the shamtreated control. These results demonstrate that either
| 15
Mre11 or ATM inhibition is able to promote functional
recovery after SCI in vivo, which we believe to be at unprecedented levels.
Finally, we confirmed the results obtained with the
inhibitors using specifically designed shRNA plasmids targeting Mre11 (shMre11) and ATM (shATM). These
shRNA plasmids were delivered using a non-viral gene
delivery vector, in vivo-JetPEI (PEI), which we have
shown to be as efficient as AAV8 in transducing DRGN
after Intra-DRG injection in vivo and does not invoke an
off-target immune response (Almutiri et al., 2018). We
initially demonstrated significant knockdown of Mre11
and ATM mRNA (84% compared with DC þ PEIshControl) in vivo using in vivo-JetPEI (Supplementary
Fig. 4A). The intensity and the number of cH2Axþ pixels/cell as a measure of double-strand breaks, were also
significantly attenuated after DC þ PEI-shMre11 and DC
þ PEI-shATM treatment indicating that knockdown of
Mre11 and ATM reduces double-strand breaks
(Supplementary Fig. 4B and C). Electrophysiology across
the lesion site after treatment with shMre11 and shATM
demonstrated similar improvements in CAP amplitude
(Supplementary Fig. 4D) and CAP area (Supplementary
Fig. 4E) as observed after treatment with mirin and KU60019. The mean sensing and removal time
(Supplementary Fig. 4F) and mean ratio of slips to total
steps (Supplementary Fig. 4G) in the sensory and locomotor tests were also similarly improved, with animals
also being indistinguishable from sham-treated animals by
3 weeks, when compared with mirin and KU-60019 treatment. These results demonstrate that knockdown of
Mre11 and ATM as well as mirin and KU-60019 treatment to inhibit the DNA damage response is functionally
beneficial after SCI.
Discussion
Double-strand breaks occur in both long-term and acute
forms of neurological disease, including Alzheimer’s,
Parkinson’s and Huntington’s diseases, amyotrophic lateral
sclerosis, post-cerebral ischaemia and following SCI: potentially they are a universal feature. If double-strand breaks
accumulate in neurological conditions, attenuating the
DNA damage response as a therapeutic strategy logically
should enhance disease rather than suppress it. Here, we
demonstrate the opposite is true: inhibiting apical components of the DNA damage response to mute the response
to double-strand breaks is beneficial in models of neurodegeneration and results in unprecedented survival, axon regeneration and recovery of function after neurotrauma.
Attenuating the DNA damage
response in neurodegenerative
disease
The incidence of double-strand breaks in the brains of
Alzheimer’s disease patients has only recently been
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Figure 6 Intrathecal injection of Mre11 and ATM
BRAIN COMMUNICATIONS 2019: Page 15 of 21
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| BRAIN COMMUNICATIONS 2019: Page 16 of 21
R. I. Tuxworth et al.
processed CAP traces from representative sham controls, DC þ vehicle, DC þ mirin and DCþKU-60019-treated rats showing some recovery
of dorsum cord potentials in mirin and KU-60019-treated rats. Dorsal hemisection between the stimulating and the recording electrodes at the
end of the experiment ablated CAP traces in all animals demonstrating technical success of the experiment. (B) Negative CAP amplitudes were
significantly attenuated in DC þ vehicle-treated rats but were significantly improved in DC þ mirin and DCþKU-60019-treated rats [P < 0.0001,
one-way ANOVA (main effect)]. (C) Mean CAP area at different stimulation intensities were significantly attenuated in DC þ vehicle-treated rats
but improved significantly in DC þ mirin and DCþKU-60019-treated rats (P < 0.0001, one-way ANOVA [main effect)]. (D) Mean tape sensing
and removal time are restored to normal three weeks after treatment with mirin and KU-60019 (P < 0.0001, independent sample t-test, no post
hoc test (DC þ vehicle versus DC þ mirin/DCþKU-60019 at 3 weeks) whilst a significant deficit remains in DC þ vehicle-treated rats
(#P < 0.00012, generalized linear mixed models over the whole 6 weeks). (E) Mean error ratio to show the number of slips versus total number
of steps in the horizontal ladder walking test also returns to normal 3 weeks after treatment with mirin and KU-60019 (P < 0.0001, independent
sample t-test, no post hoc test [DC þ vehicle versus DC þ mirin/DCþKU-60019 at 3 weeks)], with a deficit remaining in DC þ vehicle-treated
rats (##P < 0.00011, linear mixed models over the whole 6 weeks). n ¼ 6 rats/treatment/test, three independent repeats (total n ¼ 18 rats/
treatment/test).
quantified. Double-strand breaks accumulate in earlystage Alzheimer’s disease and correlate with reduced cognitive scores (Simpson et al., 2015). It is plausible that
these un-repaired double-strand breaks trigger the cellcycle re-entry phenomena in Alzheimer’s disease brains
described in the 1990s (McShea et al., 1997; Nagy et al.,
1997) and the increase in senescence identified more recently (He et al., 2013; Wei et al., 2016). Consistent
with cell cycle re-entry affecting neural health and acting
as trigger for apoptosis, inhibitors of cell-cycle progression have shown some efficacy in cerebral ischaemia
models (Osuga et al., 2000) and have been suggested as
therapies in neurodegeneration [reviewed in Kruman and
Schwartz (2008)]. Similarly caffeine, a non-specific inhibitor of ATM, is neuroprotective against etoposide-induced
DNA damage to neurons in vitro (Kruman et al., 2004)
and reducing ATM gene dosage is neuroprotective in a
mouse model of Huntington’s disease (Lu et al., 2014).
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Figure 7 Intrathecal injection of Mre11 and ATM inhibitors promotes significant functional repair after SCI. (A) Spike 2 software
Attenuating DNA damage is neuroregenerative
Attenuating the DNA damage
response in optic nerve injury
Clinically, the optic nerve crush injury model is directly
relevant to glaucoma and other eye diseases where RGC
death occurs such as ischaemic optic neuropathy. For example, we previously evaluated the effect of an siRNA to
caspase-2 (siCASP2) in protecting RGC from death in the
same model used in this article (Ahmed et al., 2011;
Vigneswara et al., 2014). The siCASP2 is currently in
Phase III clinical trials for the treatment of non-arteritic
ischaemic optic neuropathy as a result of our preclinical
work (Ahmed et al., 2011); http://www.eyeactnow.com).
Attenuation of the DNA damage response after optic
nerve crush injury in vivo using Mre11 or ATM inhibitors significantly neuroprotected RGC from death. RGC
neuroprotection was >90% after inhibition of mirin and
KU-60019, which is on a par with the inhibition of caspase-2 using an siRNA (siCASP2) (Ahmed et al., 2011;
Vigneswara and Ahmed, 2016). Out of all of the strategies to promote RGC neuroprotection, siCASP2 treatment appears to be the most effective (Thomas et al.,
2017). Other treatments such as lens injury and cAMP þ
oncomudulin only protect to a maximum of 24% of
RGC at 21 days after ONC (Leon et al., 2000; Yin et al.,
2006). Even combinatorial treatments such as phosphatase and tensin homologue deletion þ zymosan þ cAMP
only protected a maximum of 36% of RGC at 10–
12 weeks after ONC (de Lima et al., 2012). The delivery
of CNTF, a well-studied neurotrophic factor for RGC,
promoted <60% RGC survival at 2 weeks (Pernet et al.,
2013), declining to 31% at 5 weeks (Leaver et al., 2006)
and 20% at 8 weeks (Pernet et al., 2013) after ONC. In
contrast, twice weekly injections of mirin and KU-60019
neuroprotected >90% of RGC at 24 days after ONC.
Although we have not studied time points beyond
| 17
24 days, we would expect that this level of neuroprotection would continue as we have observed for siCASP2
for 12 weeks after ONC (Vigneswara and Ahmed, 2016).
Therefore, inhibition of Mre11 and ATM to our knowledge is on par with the current best treatment for RGC
neuroprotection, namely siCASP2.
Importantly, although siCASP2 offers >90% RGC neuroprotection, it does not promote RGC axon regeneration
(Vigneswara et al., 2014). In contrast, Mre11 and ATM
inhibitors not only promoted significant RGC survival,
but also promoted significant RGC axon regeneration.
Other treatments such lens injury and cAMP þ oncomodulin only promote the regeneration of <1000 RGC
axons at 1 mm from the lesion at 21 days after ONC
(Leon et al., 2000; Yin et al., 2006). ZymosanþcAMP
and phosphatase and tensin homologue deletion þ
Zymosan þ cAMP only promote the regeneration of
<100 axons at 3 mm from the lesion site at 2 weeks after
ONC (Kurimoto et al., 2010; de Lima et al., 2012).
Meanwhile, CNTF only promotes the regeneration of
<20 axons at 1.5 mm beyond the lesion site after
2 weeks and <50 axons at 4 mm beyond the lesion site
after 8 weeks (Leaver et al., 2006; Pernet et al., 2013).
Our results showing robust RGC axon regeneration of
>1000 axons at 4 mm beyond the lesion site suggest that
inhibition of Mre11 and ATM supersedes some of the
best treatments that have been reported to date to promote RGC axon regeneration. Moreover, our results
demonstrate that mirin or KU-60019 on their own cannot only protect RGC from death but also promote their
axons to regenerate, making translation to the clinical
scenario easier due to the requirement of delivering a single molecule to activate both neuroprotective and axon
regenerative pathways.
Attenuating the DNA damage
response in SCI
Attenuation of the DNA damage response after DC injury using Mre11 or ATM inhibitors promoted significant
DC axon regeneration and electrophysiological, sensory
and locomotor improvements. This is despite the presence
of spinal cord cavities that are characteristic of DC crush
injury in the rat. However, after DC injury, DRGN do
not die and hence neuroprotection in vivo could not be
assessed. Nonetheless, in vitro experiments with both
Mre11 and ATM inhibitors demonstrated significant
DRGN neuroprotection and hence, we can surmise that
the inhibitors are DRGN neuroprotective. The recovery
of function after SCI in adult rats administered Mre11 or
ATM inhibitors and confirmed using shMre11 and
shATM, is striking and, to the best of our knowledge,
unprecedented. The improvements in sensory and locomotor function that we achieved are perhaps more remarkable given that DC injury in adult rats results in
spinal cord cavities but cavitation is absent after DC injury in the mouse (Surey et al., 2014). Instead in the
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However, ATM inhibitors have not yet been tested in trials for neurodegeneration. In addition to double-strand
breaks, ATM is activated by multiple inputs, including reactive oxygen species, has a reported 700 potential downstream phosphorylation targets in multiple pathways. In
contrast, the MRN complex has no reported functions
other than in DNA damage repair, which potentially
makes it a simpler target for therapy.
Would long-term targeting of the MRN complex as a
therapeutic strategy for neurodegeneration cause cancer?
Potentially, yes. However, heterozygous carriers of mutations in Mre11, Rad50, Nbs1/Nbn have only a small increase in cancer incidence over the lifecourse (Bartkova
et al., 2008; Kuusisto et al., 2011; Damiola et al., 2014).
This suggests that targeting the MRN complex may a
feasible strategy, particular for more rapidly progressing
neurodegenerative disorders such as ALS. Potentially, a
genetic approach to partially knockdown MRN complex
levels via shRNA would be more effective and could be
largely restricted to the CNS using viral delivery.
BRAIN COMMUNICATIONS 2019: Page 17 of 21
18
| BRAIN COMMUNICATIONS 2019: Page 18 of 21
pharmacotherapy that is currently approved for use in
SCI, but this has failed to show clinically significant
effects and is now often used off-label (Varma et al.,
2013). Several other experimental therapies are currently
in the early phases of development, including the Rho antagonist, Cethrin; anti-Nogo antibodies and autologous
mesenchymal stem cell transplants. However, completed
clinical trials of these reagents have either not been
reported, abandoned or concluded with no significant
improvements reported. Consequently, there is a clear unmet need for a therapy in SCI that would not only promote
neuroprotection
but
also
promote
axon
regeneration. It is clear from our previous work that neuroprotection and axon regeneration are signalled by different signalling pathways (Ahmed et al., 2010) and our
study is the first to report single inhibitors capable of
stimulating both processes. A ‘single hit’ molecule that
can modify both neuroprotective and axon regenerative
pathways would be an additional advantage for a potential therapy to treat the devastating loss of function that
ensues after SCI in humans. Here, we report that DNA
damage response inhibitors are good candidates.
In conclusion, our article highlights a novel therapeutic
strategy by targeting the response of neurons to doublestrand breaks to protect neurons from death and promote
their axon regeneration. This strategy prevents loss of
function in multiple neuropathological paradigms and
suggests new treatment possibilities for neurological
conditions.
Supplementary material
Supplementary
material
Communications online.
is
available
at
Brain
Acknowledgements
We thank Emma Richards for help with the initial stages of
this project, Drs Damien Crowther and Mel Feany for providing Drosophila stocks and Professors Martin Berry and
Malcolm Taylor for helpful comments on the manuscript.
Funding
This work was supported by a UK Biotechnology and
Biological Sciences Research Council New Investigator
award BB/N008472/1 (R.I.T.), AMA was supported by
Marie-Curie ITN grant INsecTIME PITN-GA-2012-316790
(C.P.K), Saudi Education Ministry PhD Studentship (S.A.
and Z.A.), University of Birmingham Bryant Bequest PhD
Studentship (A.M.T. and Z.A.) and a UK Medical Research
Council Confidence in Concept award (B.K).
Downloaded from https://academic.oup.com/braincomms/article/1/1/fcz005/5526876 by guest on 13 July 2022
mouse, the lesion sites fill with dense fibrous connective
tissues which allow some regeneration of axons (Bilgen
et al., 2007). In contrast, cavitation in the rat results in
worsening of the initial injury, leading to disconnection
of more axons over a period of several months (Tang
et al., 2003; Byrnes et al., 2010).
Blunt injury, such as compression or contusion, account
for more than >80% of all SCI in humans (Taoka and
Okajima, 1998; Metz et al., 2000; Brechtel et al., 2006).
Our rat DC crush model replicates many aspects of the
human pathology well, including evolution of the injury
over time and morphological aspects, such as spinal cord
atrophy, myelomalacia, cavity, cyst and syrinx formation
and cord disruption (Gruner, 1992; Taoka and Okajima,
1998; Kwon et al., 2002). Importantly, cavity and cysts
form and increase over time, thus transecting more axons
and causing ongoing damage to the spinal cord architecture. Our study, therefore, addresses axon regeneration
and functional recovery in a model that cavitates, as
opposed to mouse models where cavitation is absent.
Taken together, these clinically relevant features suggest
our approach could be a very effective therapy for
patients.
The Mre11 and ATM inhibitors used in this study are
potent, small-molecule inhibitors that are specific for their
targets and have a low IC50. These inhibitors were developed has chemotherapy agents for cancer. DNA damage-induced tumour cell killing has been demonstrated in
preclinical models with mirin and KU-60019 (Hosoya
and Miyagawa, 2014) and other ATM inhibitors are currently in trials that potentially could be re-purposed for
SCI. Importantly, our method of administering the compounds—by intrathecal injection—can be translated
directly to human therapy. This method gives direct access of compounds to the injury site, which often requires
lower drug doses with greater efficiency than other routes
of administration (Bottros and Christo, 2014), limiting
the potential unwanted side effects on other cells in the
CNS such as glia.
In our experiments, the mirin and KU-60019 compounds were delivered directly after injury but this could
also happen in human patients since most cases attend
emergency care immediately. For neurodegeneration, a
viral-mediated genetic knockdown approach may prove
to be more effective by effecting a partial knockdown in
MRN complex levels over a longer period; this may also
prove to be the case in SCI treatments. However, there
are caveats to this approach. Inhibiting the DNA damage
response may have negative consequences to cells and
system functions if applied systemically. Even if used
intrathecally as suggested by us, there may be negative
consequences on dividing cells such as glia in the CNS.
Hence, caution must be used when using these compounds in treating human conditions.
Current therapies for SCI are limited and only offer
palliative relief with none reversing the underlying damage to axons. Methylprednisolone is the only
R. I. Tuxworth et al.
Attenuating DNA damage is neuroregenerative
Competing interests
The authors report no competing interests.
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