Enantioselective Fluorocyclizations Mediated by Amino-Acid-Derived Phthalazine

Article abstract of DOI:10.1002/adsc.201900814

Novel amino-acid-derived phthalazine reagents have been designed and synthesized for the enantioselective fluorocyclizations of prochiral indoles. The scope of reaction is evidenced by 28 examples of fluorinated furoindole and pyrroloindole heterocycles b

Full text of DOI:10.1002/adsc.201900814

NeuroscienceLetters699(2019)199–205
Contents lists available at ScienceDirect
Neuroscience Letters
journal homepage: www.elsevier.com/locate/neulet
Research article
Proinflammatory and amyloidogenic S100A9 induced by traumatic brain
injury in mouse model
Chao Wanga,1, Igor A. Iashchishyna,1, John Karaa, Vito Foderàb, Valeria Vetric,
⁎
Giuseppe Sancataldoc, Niklas Marklundd, Ludmilla A. Morozova-Rochea,
a Department of Medical Biochemistry and Biophysics, Umeå University, 90187 Umeå, Sweden
b Department of Pharmacy, University of Copenhagen, 2100 Copenhagen, Denmark
c Dipartimento di Fisica e Chimica e Aten Center Universitá di Palermo, 90128 Palermo, Italy
d Department of Neurosurgery, Uppsala University Hospital, 751 85 Uppsala, Sweden
G R A P H I C A L A B S T R A C T
A R T I C L E I N F O
A B S T R A C T
Keywords:
Traumatic brain injury (TBI) represents a significant risk factor for development of neurodegenerative diseases
such as Alzheimer’s and Parkinson’s. The S100A9-driven amyloid-neuroinflammatory cascade occurring during
primary and secondary TBI events can serve as a mechanistic link between TBI and Alzheimer’s as demonstrated
recently in the human brain tissues. Here by using immunohistochemistry in the controlled cortical impact TBI
mouse model we have found pro-inflammatory S100A9 in the brain tissues of all mice on the first and third post-
TBI days, while 70% of mice did not show any S100A9 presence on seventh post-TBI day similar to controls. This
indicates that defensive mechanisms effectively cleared S100A9 in these mouse brain tissues during post-TBI
recovery. By using sequential immunohistochemistry we have shown that S100A9 was produced by both neu-
ronal and microglial cells. However, Aβ peptide deposits characteristic for Alzheimer’s disease were not detected
in any post-TBI animals. On the first and third post-TBI days S100A9 was found to aggregate intracellularly into
amyloid oligomers, similar to what was previously observed in human TBI tissues. Complementary, by using
Rayleigh scatting, intrinsic fluorescence and atomic force microscopy we demonstrated that in vitro S100A9 self-
assembles into amyloid oligomers within minutes. Its amyloid aggregation is highly dependent on changes of
environmental conditions such as variation of calcium levels, pH, temperature and reduction/oxidation, which
might be relevant to perturbation of cellular and tissues homeostasis under TBI. Present results demonstrate that
Alzheimer’s disease
Amyloid
Neuroinflammation
Oligomerization
S100A9
Traumatic brain injury
⁎ Corresponding author.
E-mail address: ludmilla.morozova-roche@umu.se (L.A. Morozova-Roche).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.neulet.2019.02.012
Received 20 January 2019; Received in revised form 7 February 2019; Accepted 8 February 2019
Availableonline10February2019
0304-3940/©2019ElsevierB.V.Allrightsreserved.

C. Wang, et al.
NeuroscienceLetters699(2019)199–205
S100A9 induction mechanisms in TBI are similar in mice and humans, emphasizing that S100A9 is an important
marker of brain injury and therefore can be a potential therapeutic target.
1. Introduction
according to previously published methods [12,17,32]. Totally 27 adult
male C57BL/6 mice with an initial weight of 25–32 g were used
Traumatic brain injury (TBI) is one of the leading causes of death
and disability worldwide, including the developing world, due to road
accidents, falls, collision sports and military incidents [22,28]. TBI is
considered also a significant risk factor for neurodegenerative ailments
such as Alzheimer’s (AD) and Parkinson’s (PD) diseases [11,14,31]. The
pathophysiology of TBI consists of two main phases, a primary phase of
damage (mechanical impact) and secondary or delayed damage. The
primary damage occurs at the moment of insult and includes contusion
and laceration, diffuse axonal injury and intracranial hemorrhage
[21,25]. By contrast, secondary damage may manifest clinically in
hours, days and even years after injury. Ischemia, brain swelling,
neurochemical alterations, neurodegeneration and amyloid disposition
are all well-recognized secondary injury mechanisms. However, the
tissue and cell damages after TBI are complex and still only partially
understood.
(Scanbur). Each batch of animals, selected for one, three or seven days
of post-TBI survival, were randomized into sham injury or CCI groups.
TBI mice brain tissue with post-TBI periods of one, three and seven days
(six animals per group) were compared with control sham-operated
mice including four, one and four animals, corresponding to each post-
TBI group, respectively [13]. Following induction with 3.5% isoflurane
in air for up to two minutes, the mice were moved to a stereotaxic
frame. Anesthesia was maintained using 1.4% isoflurane in a mixture of
nitrous oxide and oxygen (70/30%) delivered through a nose cone.
Body temperature was controlled and maintained at 37 °C throughout
surgery using a thermistor-controlled heating pad. The scalp was
opened by a midline incision after local anesthesia of bupivacaine
®
(Marcaine , AstraZeneca). Using a dental drill, a 4-mm diameter cra-
niotomy was created over the left parietal cortex between the bregma
and lambda sutures and TBI was induced using a 2.7 mm diameter
piston set to compress the brain for 0.5 mm at a speed of 3.1 m/s,
producing a large focal injury [12]. Sham-injured (control) animals
received identical anesthesia and were subjected to scalp incision,
drilling of the bone and craniectomy exposing the intact dura, although
brain injury was not induced. After the injury, the bone flap was put
back in both sham and brain injured animals, the wound was closed and
the mouse was placed in an awakening cage under a heat lamp. The
frozen brain tissue was sectioned to 14 μm thick slices.
Recently we have demonstrated in human brain tissues that the
S100A9-driven amyloid-neuroinflammatory cascade was triggered at
very early post-TBI time and on the longer time scale it may serve as a
mechanistic link between TBI and AD [37,38]. It was manifested in
abundant production and deposition of highly amyloidogenic and
proinflammatory S100A9 protein in human TBI and, if S100A9 level
was sustained during prolonged secondary inflammation, it may lead to
the AD development characterized by Aβ and S100A9 amyloid oli-
gomer, fibril and plaque formation and deposition [37,38]. S100A9
have been also implicated in other neurological diseases such as PD [9]
and cerebral ischemia [27], as well as many other inflammation-related
conditions including cardiovascular disease [19], malaria [30] and
numerous cancers [35]. Moreover intranasal injection of S100A9
amyloids in aging mouse model induced wide-spread tissue stress re-
sponses and invoked an AD-like behavioral impairment in the passive
avoidance test [10]. By contrast S100A9 knockdown attenuated
memory impairment and reduced amyloid plaque burden in the AD
transgenic mouse model [7], suggesting that S100A9 can be a pro-
spective target for therapeutic interventions.
2.2. Immunohistochemistry
Single and sequential immunohistochemistry on the same tissue
sections was performed as described previously [38]. Mouse S100A9
(sc-8115) and CD68 (sc-70761) antibodies were purchased from Santa
Cruz Biotechnology. Mouse Aβ (ab11132) and NeuN (ab104225) anti-
bodies were purchased from Abcam. Anti-rabbit (MP-7401) and anti-
mouse (MP-7402) secondary antibodies were purchased from Vector
Laboratories. Anti-goat (ab6885) was purchased from Abcam. A11 an-
tibodies were generated by R. Kayed [15].
Here we examined S100A9 responses in the TBI brain tissues during
the first post-injury week of wild-type mice subjected to controlled
cortical impact (CCI). These in vivo findings were compared to inherent
aggregation properties of S100A9 observed in vitro and to the previous
observations of S100A9 excessive production in the human TBI tissues
[37]. Increasing evidence demonstrates that TBI profoundly alters the
cerebral acid base homeostasis, leading to the brain tissue acidosis,
which consequently causes neuronal damage and poor survival out-
come [4,6,29,33]. Therefore, here we have conducted in vitro experi-
ments under acidic conditions, examining their effect on S100A9
amyloid formation.
2.3. AFM
In vitro produced amyloid samples were imaged by using a Bruker
Bioscope Catalyst microscope operated in the peak force mode at 1 kHz
frequency with 0.6 N/m stiff cantilevers. Amyloid samples were de-
posited on the surface of freshly cleaved mica (Ted Pella) for 15 min,
washed 3 × 100 μl by deionized water and dried at room temperature.
The dimensions of amyloid aggregates were measured in the AFM cross-
sections and the probability density function (PDF) was reconstructed
defined as the probability of finding aggregate with certain height
normalized on the whole sample set.
2. Materials and methods
2.4. Rayleigh scattering and intrinsic fluorescence
2.1. Animals and CCI surgery
Rayleigh scattering and intrinsic fluorescence signals were recorded
simultaneously during S100A9 amyloid aggregation in 1 cm path quartz
cuvette by using a Jasco FP-8500 equipped with a Peltier-thermostat.
After thermal equilibration, the emission spectra were recorded with
0.5 nm wavelength intervals, emission and excitation bandwidth of
2.5 nm, 100 nm/min scan speed and 1 s integration time. Emission
spectra in the range of 275–450 nm were obtained under excitation at
280 nm. Light scattering at 90° was also measured as the maximum of
the elastic peaks of excitation light. Excitation spectra were measured
All experimental procedures with TBI mice treatment and handling
their tissue samples were approved by the medical ethics committees on
animal experiments of the Uppsala University, followed the rules of the
Swedish Animal Welfare Agency and EC Directive 86/609/EEC for
animal experiments. TBI was induced by CCI. All animals were housed
in an environmentally controlled room in a 12 h light-dark cycle and
were allowed a minimum of one week acclimatization prior to the
surgical procedure. A CCI brain injury or sham injury was performed
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after each kinetic experiment to check if any variations of the excitation
band profile has happened. All experiments were performed in tripli-
cate and the average intensity was used for fitting and calculation of
3. Results
3.1. S100A9 in ex vivo TBI mouse brain tissues
keff
.
The presence of S100A9 was evaluated in the TBI mouse brain tis-
sues on the first, third and seventh days (six animals per group) after
CCI and compared with control animals (nine animals in the group). In
TBI mouse brain tissues S100A9 immunopositive staining was observed
in the parietal cortex in proximity to the cortical region of impact; the
corresponding area is encircled by red line in Fig. 1A and C. The
2.5. Kinetics data fit
S100A9 amyloid kinetics were described by nucleated polymeriza-
tion model with some modification as described previously [37].
Fig. 1. Post-TBI S100A9 induction in
mouse brain tissues. (A, C) Overall
view of immunohistochemistry with
S100A9 antibodies of mouse brain
tissue; the area affected by focal TBI is
encircled by red line. Magnified area
affected by TBI is presented in insertion
in (C); S100A9 immunopositive cells
and blood vessels are shown as red
spots. (B) Overall view of control
mouse brain tissue showing the lack of
S100A9 immunostaining. Scale bars
are 2000 μm in (A, B), 1000 μm (C) and
50 μm in (C, insertion). (D) Number of
S100A9 immunonegative (red bars)
and immunopositive (blue bars) mouse
brain tissues in control and post-TBI
groups. Post-TBI time in days is shown
along x-axis. (E‒G) Representative
images
of
sequential
im-
munohistochemistry of microglial cells
in post-TBI tissues with pair of (E)
S100A9 and (F) CD68 antibodies, re-
spectively, and (G) the superposition of
the corresponding images in pseudo-
colors, i.e. S100A9 staining is shown in
green and CD68 – in red. Scale bars are
50 μm. (H-J) Representative images of
sequential immunohistochemistry of
neuronal cells in post-TBI tissues with
(H) S100A9 and (I) NeuN antibodies,
respectively, and (J) the superposition
of the corresponding images in pseudo-
colors, i.e. S100A9 staining is shown in
green and NeuN – in purple. Scale bars
are 50 μm.
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enlarged view of S100A9 immunopositive blood vessels and individual
cells are shown in an insertion to Fig. 1C. No immunostaining with
S100A9 antibodies was detected in the contralateral brain tissues and in
the brain tissues of control animals (Fig. 1A–C). If all animals on the
first and third post-TBI days were characterized by the S100A9 positive
immunostaining in the area of impact, on the seventh post-TBI days
only 30% of animals showed S100A9-immunopositive staining
(Fig. 1D). Since S100A9 is a proinflammatory or damage-associated
pattern molecule [34], this indicates that the level of neuroinflamma-
tion induced by trauma in the mouse brain tissues subsided with in-
creasing post-TBI period. In order to identify in which cells S100A9 was
induced after trauma, the mouse brain tissues of all animals on the day
one and three after TBI were subjected to sequential im-
munohistochemistry with a pair of S100A9 and CD68 antibodies, the
latter were specific to microglia cells (Fig. 1E–G). The overlap of
S100A9 and CD68 immunostaining patterns, especially clearly pre-
sented in the superimposed image (Fig. 1G), indicates that S100A9 was
produced by microglial cells following the trauma. Even more so, the
sequential immunohistochemistry with the pair of S100A9 and NeuN
antibodies (Fig. 1H–J), the latter were specific to neuronal cells, re-
vealed that neurons also produced S100A9. Importantly, in all TBI
mouse groups no S100A9 precursor-plaques were noticed (Fig. 1A–C).
Immunohistochemistry with Aβ antibodies also did not reveal any re-
activity either in TBI affected or control tissues (data not shown). Se-
quential immunohistochemistry with S100A9 and amyloid oligomer
specific A11 antibodies of the representative TBI tissues collected on the
first and third post-TBI days revealed that some cells were co-stained
with both antibodies as shown by arrows in Fig. 2A and B, respectively,
thus suggesting that S100A9 was prompted to aggregate into amyloid
oligomers intracellularly after TBI. By contrast, in the contralateral to
TBI areas no immunopositive staining were observed either with A11 or
S100A9 antibodies (Fig. 2C and D).
oligomers (Fig. 3C). Already after 10 min of incubation the whole array
of the self-assembled structures have emerged including the chains of
round-shaped aggregates, curved protofilaments and larger grained
aggregates, which spanned the wide distribution of heights form 1 to
15 nm (Fig. 3D and E). They grow further and clumped into large
amyloid aggregates after 24 h incubation, corresponding the plateau
region (Fig. 3E). S100A9 incubated under the same pH conditions but
lower temperature 42 °C formed numerous small and flexible fibrils
with an average heights in AFM cross-sections of 2.5 nm (Fig. 3H),
which showed less tendency to clump.
4. Discussion
Recently we have demonstrated in human TBI and AD tissues that
S100A9 is involved both in the amyloid and neuroinflammatory pa-
thological cascades and can play a critical role paving the way from TBI
to AD [37,38]. Inflammation is a complex process including multiple
inter-linked responses such as extensive microglial activation, infiltra-
tion of blood-derived mononuclear phagocytes and lymphocytes and
significant rise of pro-inflammatory cytokines and alarmins– all da-
maging reactions, which can sustain inflammation and exacerbate
amyloid formation and neurodegeneration [3,18,24,26]. Since S100A9
was found to be a part of these complex responses in humans, it was
important to examine the involvement of S100A9 in TBI conditions in
wild-type mouse model. Inflammation occurs early in the CCI model
used in the present report [16], and when inflammation is attenuated,
behavioral and histological outcome is commonly improved [2]. Si-
milar underlying molecular and cellular mechanisms involved in both
mammalian species will lead to better understanding of this process and
enable to develop new therapeutic targets and diagnostic markers. Here
by using single and sequential immunohistochemistry we have shown
that S100A9 production was induced immediately after the trauma and
spread in the area affected by the impact. It was produced by both
neuronal and microglial cells immediately after trauma in all studied
animals and was cleared by the body clearance mechanisms in 70% of
3.2. S1009 amyloid formation in vitro modified by calcium-binding and
reducing agent
We have examined the effect of calcium-binding (addition of 1 mM
CaCl2 or 1 mM EDTA) and reducing conditions (0.5 mM DTT) on
S100A9 amyloid formation, since the significant perturbation of tissue
and cell homoeostasis usually occurring under TBI [6,29] may affect the
S100A9 calcium-binding sites and Cys 3 residue and consequently its
amyloid self-assembly (Fig. 3). The amyloid formation kinetics of
S100A9 without any added compounds were monitored by Rayleigh
scattering, sensitive to the size and shape of amyloids, and intrinsic
fluorescence (Fig. 3A). Both assays resulted in similar kinetics, char-
acterized by significant growth of the corresponding scattering or in-
trinsic fluorescence signals right from the beginning of incubation and
leading to plateau regions after ca. 15 h (Fig. 3A). Intrinsic fluorescence
spectra of S100A9 followed in the course of amyloid formation were
also characterized by blue-shift of the spectral maxima from 346 nm to
336 nm (by ca. 10 nm), indicating that single Trp 88 of S100A9 became
more buried within the amyloid interior (Fig. 3A, insertion). The
amyloid kinetics in the presence of CaCl2, EDTA and reducing agent
DTT were monitored by Rayleigh scattering (Fig. 3B). Under all these
conditions the kinetic curves were fitted by using the nucleation de-
pendent polymerization model [23]. Since the addition of 1 mM CaCl2
to S100A9 saturates both calcium-binding sites and increases protein
stability [36], these resulted in slower amyloid formation, with the
effective keff rate constant twice lower than in the presence of EDTA.
Interestingly, addition of reducing agent DTT also significantly in-
creased the rate of the S100A9 holo-form amyloid assembly (by ca. 1.5
times), but did not produced any substantial effect on the self-assembly
of the S100A9 apo-form (Fig. 3B). The amyloid self-assemblies of
S100A9 were monitored by AFM and representative images are shown
in Fig. 3C–H. Already after a few minutes of incubation at 57 °C S100A9
formed round-shaped structures, some of which represent amyloid
Fig. 2. Post-TBI amyloid oligomerization observed in mouse brain tissues.
Representative images of sequential immunostaining with pair of (A) S100A9
and (B) amyloid oligomeric A11 antibodies, respectively, in post-TBI area of
mouse brain tissues. The cells co-immunostained with both antibodies are
shown by red and black arrows in (A) and (B) figures, respectively. Lack of
immunostaining with (C) S100A9 and (D) amyloid oligomeric A11 antibodies
applied sequentially to the contralateral brain area. Magnified insertions are
shown in the upper right corners. Scale bars are 50 μm in (A and B), 500 μm in
(C and D) and 100 μm in (C and D insertions).
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Fig. 3. S100A9 amyloid self-assembly monitored in vitro. (A) Normalized kinetics of S100A9 amyloid formation monitored by intrinsic fluorescence (shown by
blue squares) and Rayleigh scattering (red circles). Blue shift of S100A9 intrinsic fluorescence spectrum maxima in the course of amyloid formation is shown in
insertion. (B) Kinetics of S100A9 amyloid formation monitored by Rayleigh scattering in the presence of 1 mM CaCl2 (shown in red), 1 mM CaCl2 and 0.5 mM DDT
(blue), 1 mM EDTA (orange), 1 mM EDTA and 0.5 mM DDT [5] and without additives (purple). Effective rate constants Keff are shown in insertion. 20 μM S100A9 was
incubated in 20 mM Na acetate, pH 4.5, 57 °C and without agitation (unless specified). (C) AFM images of S100A9 amyloid oligomers formed after 2 min of
incubation at 57 °C without additives; (D) –amyloid protofilaments formed after 10 min; (E) Distribution of the heights of amyloid protofilaments shown in (D) as
measured in AFM cross-sections. PDF is presented along y-axis and heights of protofilaments in nm – along x-axis. (F) AFM images of amyloid clumps formed after
24 h of incubation at pH 4.5 and 57 °C and (G) – S100A9 fibrils formed after 4 h of incubation at pH 4.5 and 42 °C. Insertion: 4× magnified image of amyloid fibrils.
(H) Distribution of the heights of amyloid fibrils shown in (G) as measured in AFM cross-sections. PDF is presented along y-axis and heights of protofilaments in nm –
along x-axis.
animals on seventh post-TBI day. Moreover S100A9 self-assembled in
some cells into amyloid oligomers as evident in the sequential im-
munohistochemistry with the pair of S100A9 and A11 antibodies. We
have shown in human TBI that amyloid oligomerization of S100A9 and
in lesser extend Aβ peptide was correlated with the activation of
apoptotic makers such as Bax and activated caspase-3 [37]. Therefore,
amyloid oligomerization could be a critical step leading to pathological
consequences also in the mouse model, since the amyloid oligomers are
viewed as the most toxic species compared to relatively inert amyloid
fibrils [1]. Our in vitro studies of S100A9 amyloid self-assembly under
the acidic conditions, which are often implicated in TBI [4,6,20,29],
have revealed that S100A9 forms amyloid oligomers and protofibrils
already after minutes of its incubation and develops mature fibrils and
larger clumps after four and twenty four hours, respectively. Since
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primary and secondary post-TBI events may perturb cellular and tissue
homeostasis, abundantly produced S100A9 will be exposed to changing
conditions, including pH, temperature, oxidation/reduction and cal-
cium concentration, some of which may promote its self-assembly into
amyloids. Previously we have shown that S100A9 amyloid oligomers
and protofibrils self-assembled under both neutral and acidic pH 4.5
were the most toxic to neuronal cell culture, causing cell apoptosis. The
cytotoxicity of the fibrillar species were significantly reduced, though
still detectable [9,37,38]. In addition, in vivo studies in mouse model,
when S100A9 amyloids were administered intranasally, the oligomers
caused the most pronounced neuronal stress responses in cerebral tis-
sues and behavioral impairment in the passive avoidance test of all
studied animals [10]. Thus, if amyloid oligomers and fibrils are not
cleared by cellular defense mechanisms these may trigger the amyloid-
neuroinflammatory cascade [37,38]. Recent findings that S100A9 in
human CSF can serve as a biomarker not only AD but already mild
cognitive impairment [8] indicate that S100A9 is involved in the early
AD pathology and can be viewed as a therapeutic target already at early
stages of neurodegeneration.
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Declarations of interest
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Availability of data and materials
The data and materials are available from the corresponding author
upon the first request.
Acknowledgements
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We acknowledge Fredrik Clausen for performing the experimental
TBI model. We acknowledge the Biochemical Imaging Center at Umeå
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