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Subacute and chronic proteomic and phosphoproteomic analyses of a mouse model of traumatic brain injury at two timepoints and comparison with chronic traumatic encephalopathy in human samples


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Repetitive mild traumatic brain injury (r-mTBI) is the most widespread type of brain trauma worldwide. The cumulative injury effect triggers long-lasting pathological and molecular changes that may increase risk of chronic neurodegenerative diseases. R-mTBI is also characterized by changes in the brain proteome, where the majority of molecules altered early post-TBI are different from those altered at more chronic phases. This differentiation may contribute to the heterogeneity of available data on potential therapeutic targets and may present an obstacle in developing effective treatments. Here, we aimed to characterize a proteome profile of r-mTBI in a mouse model at two time points – 3 and 24 weeks post last TBI, as this may be a more relevant therapeutic window for individuals suffering negative consequences of r-mTBI. We identified a great number of proteins and phosphoproteins that remain continuously dysregulated from 3 to 24 weeks. These proteins may serve as effective therapeutic targets for sub-acute and chronic stages of post r-mTBI. We also compared canonical pathway activation associated with either total proteins or phosphoproteins and revealed that they both are upregulated at 24 weeks. However, at 3 weeks post-TBI, only pathways associated with total proteins are upregulated, while pathways driven by phosphoproteins are downregulated. Finally, to assess the translatability of our data, we compared proteomic changes in our mouse model with those reported in autopsied human samples of Chronic Traumatic Encephalopathy (CTE) patients compared to controls. We observed 39 common proteins that were upregulated in both species and 24 common pathways associated with these proteins. These findings support the translational relevance of our mouse model of r-mTBI for successful identification and translation of therapeutic targets.
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Morinetal. Molecular Brain (2022) 15:62
Subacute andchronic proteomic
andphosphoproteomic analyses ofamouse
model oftraumatic brain injury attwo
timepoints andcomparison withchronic
traumatic encephalopathy inhuman samples
Alexander Morin1,2* , Roderick Davis1, Teresa Darcey1, Michael Mullan1,2, Benoit Mouzon1,2,3 and
Fiona Crawford1,2,3
Repetitive mild traumatic brain injury (r-mTBI) is the most widespread type of brain trauma worldwide. The cumula-
tive injury effect triggers long-lasting pathological and molecular changes that may increase risk of chronic neurode-
generative diseases. R-mTBI is also characterized by changes in the brain proteome, where the majority of molecules
altered early post-TBI are different from those altered at more chronic phases. This differentiation may contribute to
the heterogeneity of available data on potential therapeutic targets and may present an obstacle in developing effec-
tive treatments. Here, we aimed to characterize a proteome profile of r-mTBI in a mouse model at two time points
– 3 and 24 weeks post last TBI, as this may be a more relevant therapeutic window for individuals suffering negative
consequences of r-mTBI. We identified a great number of proteins and phosphoproteins that remain continuously
dysregulated from 3 to 24 weeks. These proteins may serve as effective therapeutic targets for sub-acute and chronic
stages of post r-mTBI. We also compared canonical pathway activation associated with either total proteins or phos-
phoproteins and revealed that they both are upregulated at 24 weeks. However, at 3 weeks post-TBI, only pathways
associated with total proteins are upregulated, while pathways driven by phosphoproteins are downregulated. Finally,
to assess the translatability of our data, we compared proteomic changes in our mouse model with those reported in
autopsied human samples of Chronic Traumatic Encephalopathy (CTE) patients compared to controls. We observed
39 common proteins that were upregulated in both species and 24 common pathways associated with these pro-
teins. These findings support the translational relevance of our mouse model of r-mTBI for successful identification
and translation of therapeutic targets.
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Repetitive mild traumatic brain injury (mTBI) is a rec-
ognized risk for long term neurodegenerative disabilities
such as Alzheimer’s Disease (AD), Chronic Traumatic
Encephalopathy (CTE), and other forms of dementia
[13]. Numerous studies of repetitive mild TBI (r-mTBI),
especially in contact sport players, have revealed a
months-to-years-long period between the injury and the
onset of cognitive deficits [46]. During this time, the
brain undergoes a complex array of neuropathological
changes varying from axonal injury, vascular damage and
inflammation to neurodegeneration [4, 79]. Some of
Open Access
1 Roskamp Institute, Sarasota, USA
Full list of author information is available at the end of the article
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Morinetal. Molecular Brain (2022) 15:62
these processes are specific to only acute or chronic time
points while others are present in both. Understanding
such temporal heterogeneity may be crucial for therapeu-
tic target identification.
Mass-spectrometry (MS) based proteomics and phos-
phoproteomics allow for the unbiased investigation of
large numbers of proteins and associated biological pro-
cesses and comparison of datasets from TBI versus con-
trol samples could identify potential therapeutic targets.
However, to date, the number of published studies on
mTBI proteomics, and especially phosphoproteomics,
remains scarce. In their 2018 review, Sowers etal. out-
lined only ten TBI-related published proteomic studies,
five of which were conducted in human samples (TBI,
AD or CTE), two in in-vitro models and three in in-
vivo animal models [10]. Among them, only one study
has focused on mTBI in animal models [11] and none in
human samples. Between 2010 and 2017, an independ-
ent meta-analysis by Ganau etal. identified only 16 omic
papers on TBI, 8 clinical and 8 animal studies, ranging by
the type of analysis, injury severity, animal species, and
other variables [12]. Phosphoproteomics represents an
even less well investigated area with only a single study
conducted in an mTBI mouse model [13]. Combined,
the published data do not present a consistent message,
owing to the diverse models of TBI under investigation,
and differences in timing of assessment post-injury, spe-
cies, animal genotypes, and other factors. Altogether, the
heterogeneity of these variables plus the existing com-
plexity of mTBI pathology preclude clear comparison
between different omic datasets.
Here, we designed a study that addresses the temporal
changes of r-mTBI using proteomic- and phosphoprot-
eomic-based approaches within a single animal model.
Mice (hTau) received 5 closed-head injuries over 9days,
and cortical tissues were collected at either 3 or 24weeks
post last injury, representing subacute and chronic peri-
ods, respectively [14, 15]. In our previous studies, this
r-mTBI model results in sustained lifelong behavioral
deficits, glial activation, and tau phosphorylation up to
24months post last injury [16, 17]. e choice of hTau
mice, which express the six isoforms of human tau, is to
better mimic tauopathies involved in chronic forms of
neurodegeneration post TBI, as we have previously not
observed persistence of any TBI-dependent changes in
Tau in wild type mice [18]. To validate the clinical trans-
latability of our model, we compared the murine pro-
teome with previously acquired human CTE proteomic
data. ere is a lack of open-access raw datasets from
human TBI, but a publicly available dataset from corti-
ces of human CTE cases is available [19], and this raw
dataset of protein changes in human CTE samples (Pro-
teomeXchange) allowed us to analyze it in the same way
as we did with our own data. We hypothesize that cellular
mechanisms found to be similarly altered in human sam-
ples and in our mouse model may represent translational
therapeutic targets which can be further validated and
pursued in our model.
Commonly altered proteins andphosphoproteins post‑TBI
Mouse cortices were extracted at 3weeks (sub-acute—
SA) or 24weeks (chronic—CH) after the last injury or
sham procedure (Fig. 1A). Each group had n = 4 mice
(Fig. 1C). Proteomic and phosphoproteomic analyses
were conducted as shown in Fig.1B. Overall, 4511 total
proteins and 861 phosphoproteins were identified in both
cohorts. If a protein was not detected in at least one TBI
sample or one Sham sample, it was excluded from further
analysis. ose with abundance ratios (a.r.) > 1.2 or < 0.5
were selected for further analysis to represent upregu-
lation or downregulation, respectively [20, 21]. e SA
cohort was characterized by 881 up- and 15 down-regu-
lated proteins and 90 up- and 379 down-regulated phos-
phoproteins, while the CH mice had 1069 up- and 197
down-regulated proteins and 240 up- and 62 down-regu-
lated phosphoproteins (Fig.1D). Raw data is provided as
a supplement (Additional file1).
To demonstrate persistent changes from 3 to 24weeks
post-TBI, we identified proteins which were similarly
altered in response to r-mTBI in both cohorts of mice.
Overall, both SA and CH cohorts showed an upregula-
tion of 349 total and 57 phosphoproteins and a down-
regulation of 1 total and 21 phosphoproteins (Fig. 2).
Given the much higher number of significantly altered
proteins in the upregulated group, our analysis focused
primarily on the datasets of 349 total and 57 phospho-
proteins. Stratification of these proteins by their a.r.
identified PDE2A (phosphodiesterase 2A) as the total
protein most significantly altered in response to r-mTBI
(a.r.: SA-2.83, CH-2.29), followed by MRPL53 (mitochon-
drial ribosomal protein L53, a.r.: SA-2.78, CH-2.45), TTR
(transthyretin, a.r.: SA-2.65, CH-2.31), and TTBK1 (tau
tubulin kinase 1, a.r.: SA-2.59, CH-2.06) (Fig.2B). Phos-
phorylated proteins with the greatest change in expres-
sion in TBI vs sham which were common to both cohorts
included HSP90B1 (heat shock protein 90 beta family
member 1), PSMA5 (proteasome subunit alpha type-5),
PC (pyruvate carboxylase), MAP2K1 (mitogen-activated
protein kinase 1), FGF12 (fibroblast growth factor 12),
and PDXK (pyridoxal kinase) (Fig.2C).
As a proof of concept, we selected a protein showing
a significant level of change in both TBI cohorts com-
pared to shams, PDE2A, for the antibody-based valida-
tion. Quantitative analysis of immunofluorescent staining
of PDE2A (Fig.3A) was performed across somatomotor,
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Morinetal. Molecular Brain (2022) 15:62
Fig. 1 A Timeline of the study. Mice received 5 consecutive mTBI over 9 days (48 h interval between the injuries) at 3 months of age. They were
further divided into 2 cohorts: subacute (SA) and chronic (CH). SA mice were euthanized at 3 weeks after the last injury, CH mice at 24 weeks. B
Proteomic/phosphoproteomic design. Mouse cortices of both cohorts were processed using TMT labeling. Phospho-enrichment was performed
using TiO2 beads. C The number of mice (n = 4) in each group. D Total number of identified proteins. Detected proteins and phosphoproteins were
described as up- or down-regulated using thresholds for abundance ratio (a.r.) > 1.2 or < 0.5, respectively
Fig. 2 A Number of total proteins and phosphoproteins identified in SA and CH cohorts stratified by their a.r. as up- (> 1.2) or downregulated
(< 0.5). Venn diagrams show the overlapping proteins between two cohorts. B Heatmap of the top 10 commonly upregulated total proteins
stratified by the log10 values of a.r. C Heatmap of the top 10 commonly upregulated phosphoproteins stratified by the log10 values of a.r
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Morinetal. Molecular Brain (2022) 15:62
anterior cingulate, and retrosplenial areas corresponding
to the parts of cortex that were used for the proteomic
analyses. It demonstrated an increase in PDE2A-positive
cells in both SA (p < 0.05) and CH (p < 0.001) r-mTBI
cohorts compared to their respective shams (Fig.3B).
Commonly altered canonical pathways post‑TBI
In addition to the comparison of individual proteins, we
further decided to compare canonical pathways between
the cohorts due to the possibility that the same pathways
could be activated through TBI-dependent modulation
of different proteins. e activation state was calculated
using z-score where a positive value infers a pathway’s
upregulation, and a negative value infers downregula-
tion. A comparison analysis of responses in canoni-
cal pathways resulting from expression changes in total
proteins revealed a pattern of activation of many of the
same pathways in both SA and CH cohorts (Fig. 4A).
e top processes included Ephrin Receptor Signaling
(z-score: SA-3.32, CH-2.71), Integrin Signaling (z-score:
SA-3.87, CH-1.39), Leukocyte Extravasation Signal-
ing (z-score: SA-3.32, CH-1.89), EIF2 Signaling (z-score:
SA-2.00, CH-2.71) and others. From the whole list, only
two pathways were identified as being inhibited in both
two cohorts—RHOGDI Signaling (z-score: SA- 1.89,
CH- 1.67) and PTEN Signaling (z-score: SA- 2.53,
CH- 0.30). In contrast, the z-scores for canonical
pathways based on the phosphoprotein analyses showed
almost exclusively opposite direction activation in SA
versus CH cohorts, suggesting downregulation of those
pathways at the SA timepoint and upregulation at the
CH timepoint (Fig.4B). A complete list of molecules con-
stituting these pathways and of the molecules that were
significantly regulated in both the SA and CH cohorts is
provided in the Additional file2 and Additional file3: Fig.
Overlap ofmouse proteome withhuman CTE
In order to evaluate the relevance of our r-mTBI mouse
model to human clinical chronic outcomes, we accessed
a proteomic database of CTE cases (n = 11) with a his-
tory of concussions, which was available from the
ProteomeXchange dataset PXD007694 [19]. e total
proteome of both SA and CH cohorts was compared to
that of the CTE dataset, and the overlapping upregu-
lated proteins were identified (no overlap of downreg-
ulated proteins across all three groups was observed).
During the conversion of mouse protein accession
numbers to their respective proteins in the human pro-
teome, some molecules did not have a match and were
thus excluded from the comparison. Hence, the num-
ber of upregulated proteins utilized for the TBI-CTE
comparison is different from the originally identified
proteins as shown in Fig.1. From the proteins that were
Fig. 3 A Immunofluorescent staining for PDE2A in the cortex. B The ratio of the PDE2A positive cells to the total number of cells (DAPI) shows an
increase in TBI vs sham in both SA (p < 0.05) and CH cohorts (p < 0.001). Data were analyzed using two-way ANOVA
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Morinetal. Molecular Brain (2022) 15:62
successfully converted from mouse to human, we iden-
tified 39 proteins that were significantly upregulated in
all three cohorts compared to their respective controls
(Fig.5A, B). A comparison analysis of canonical path-
ways predicted to be responding in the mouse mod-
els of r-mTBI and human CTE (Fig. 5C) revealed 24
common pathways. Of these, 23 pathways were upregu-
lated in all cohorts and the remaining one was down-
regulated across all cohorts. e upregulated pathways
included Synaptogenesis Signaling Pathway, GNRH
signaling, Ephrin Signaling, Synaptic Long-Term Depres-
sion, Neurovascular Coupling and others. e single
Fig. 4 Bar graphs representing activation state (z-score) of canonical pathways by total (A) or phospho-proteome (B). Upregulation is described
with a positive z-score, downregulation with a negative z-score
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Morinetal. Molecular Brain (2022) 15:62
pathway downregulated in all cohorts was RHOGDI
Signaling (z-score: SA- 1.89, CH- 1.67, CTE- 2.00).
e goal of this study was to describe proteomic and
phosphoproteomic changes common to sub-acute (SA)
and chronic (CH) time points after r-mTBI that might
represent the best potential targets in a heterogene-
ous TBI population. Overall, we showed that both SA
and CH profiles share a large number of upregulated
proteins, while the phosphoproteome profiles were,
in general, more specific for each time-point. e SA
phosphoproteome was dominated by downregulated
proteins while at the CH timepoint the response to
r-mTBI was largely upregulation of phosphoproteins.
Only a few of the downregulated phosphoproteins in
SA mice remained below the sham expression levels by
the chronic time point, suggesting their specificity to
sub-acute post-TBI response. Such dynamics reflect the
transient nature of protein phosphorylation after r-mTBI
underscoring the importance of post-TBI timing of cer-
tain pathological responses and the limitations they
might place on therapeutic windows for potential inter-
ventions. However, the goal of this study was to evaluate
Fig. 4 continued
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Morinetal. Molecular Brain (2022) 15:62
proteins that kept consistent levels of regulation from 3
to 24weeks after TBI and hence our analyses was pri-
marily focused on the total proteome.
Studying proteins that are similarly regulated at dif-
ferent timepoints post-injury is an important undertak-
ing. Owing to the heterogeneity of human TBI itself, as
well as the potential timepoints post TBI at which inter-
vention may be initiated, a reasonable first approach to
determining therapeutic targets that could work in mul-
tiple scenarios is to identify TBI dependent changes that
persist over a period of weeks or months in a preclinical
model, as this could translate to clinically relevant time-
frames for therapeutic intervention in the human patient
population. Such an approach has been proposed as
the next logical move in TBI research by several scien-
tists [22, 23]. In their review, Kenzie etal. performed a
causal-loop analysis of post-mTBI mechanisms and dem-
onstrated a nonlinear pattern of mTBI recovery [22]. e
acute phase is often characterized by cell loss and axonal
damage, while at subacute and chronic phases, neuroin-
flammation and neuroplasticity mechanisms are preva-
lent [23]. Such heterogeneity is traced down to individual
proteins which comprise diverse proteomic profiles at
different timepoints post TBI. For example, in their study,
Lizhnyak and Ottens demonstrated that at 14days after
moderate TBI, 52% of dysregulated proteins were novel
compared to the proteins identified at the 2-day time-
point [23]. In our study, 72% of proteins dysregulated at
24weeks after r-mTBI, (both up- and down-regulated)
had not been identified as dysregulated at the 3-week
timepoint. For effective treatment approaches it will be
critical to understand the lifespan of any proposed thera-
peutic target. To date, many treatments have shown effi-
cacy in preclinical models but have not been successful
with clinical translation, and a contributor to this may
have been insufficient interrogation of the viable thera-
peutic window at the preclinical stage.
So far, only a few proteomic studies have proposed
potential targets in relation to timing post injury. Ojo and
colleagues measured altered proteins in the cortex and
hippocampus of C57BL/6J mice at 3, 6, 9 and 12months
after r-mTBI, using the same 5-hit model we utilized in
our current study [24]. Among the most dysregulated
processes common for all time-points were PI3K/AKT,
PKA, and PPARα/RXRα signaling in the hippocam-
pus, and PKA, GNRH, and B cell receptor signaling in
the cortex. To our knowledge, the only other proteomic
study that compares acute/sub-acute and chronic time
points after mild TBI was conducted by Song etal. [11].
ey measured temporal changes in the total proteome
of Sprague–Dawley rats at 1 day, 7days or 6 months
after either a single mTBI or a 3-hit r-mTBI. In addition
to demonstrating larger TBI-dependent fold-changes
in protein expression, they described two upregulated
proteins, PDE10A and GNAL, which did not return to
the sham levels even at 6months post-r-mTBI. PDE10A
belongs to the class of phosphodiesterases, as does
PDE2A, highlighted in our current study, which is a
group of enzymes that hydrolyze cAMP and cGMP, ter-
minating their signaling [25]. In the brain, they contrib-
ute to learning and memory, while in the periphery they
play an important role in macrophage differentiation [26].
Fig. 5 A An overlap of total upregulated proteins between the SA and CH cohorts in a mouse model of r-mTBI and the human CTE samples. B
Interaction map of the 39 identified overlapping proteins (STRING 11.5, confidence score > 0.7). C Overlapping canonical pathways stratified by the
activation z-score
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Morinetal. Molecular Brain (2022) 15:62
Inhibition of PDE2 prevents degradation of cGMP lead-
ing to enhanced long-term potentiation and improved
memory [27, 28]. Hence, prolonged increased levels of
PDE2 might be associated with chronic memory deficits.
e same mice as used in the current paper underwent
behavioral assessment at sub-acute and chronic time
points [29, 30] and demonstrated r-mTBI-dependent
impaired learning and memory. Using immunofluores-
cent imaging, we have confirmed an increased number
of PDE2A-expressing cells in the cortex after r-mTBI
at both 3- and 24-weeks post injury. Further validation
of PDE2A as a target, and the effects of its inhibition in
injured mice, will be a direction for future studies.
In addition to PDE2A, another protein that was
increased at 3 and 24 weeks post injury was tau tubu-
lin kinase 1 (TTBK1), which is known to be linked to
the development of several neurodegenerative diseases
such as ALS, FTLD, and AD [31]. In AD, TTBK1 phos-
phorylates tau at Ser422 contributing to the formation
of neurofibrillary tangles [32]. Animal models support
these observations showing that mice expressing human
TTBK1 develop tau phosphorylation, gliosis, and spa-
tial memory impairment [33]. In our previous study, we
demonstrated hyperphosphorylation of tau at r231 in
aged hTau at 3weeks after the same r-mTBI model, but
not in young mice [16, 29]. While no clear correlation
can be made between TTBK1 and tau in our SA and CH
cohorts, these independent findings highlight a possible
role of tau in the chronicity of TBI pathology. One of our
future goals is to investigate additional phospho-epitopes
of tau that can be linked to TTBK1 activity.
An intriguing trend was observed when we created an
activation profile of canonical pathways based on either
phospho- or total proteins. While canonical pathways
derived from the TBI-dependent changes in the total
proteome were consistently upregulated between SA
and CH cohorts, phospho-based pathways were largely
downregulated in the SA cohort but upregulated in the
CH cohort. Due to the lack of similar studies in the lit-
erature, it is hard to compare these findings with other
models. When we compared our total protein data with
CTE data, we found that both human and mouse data
demonstrated a great number of upregulated pathways
with only one downregulated process (RHOGDI). e
large number of pathways shown to be activated in our
mouse models which are also activated in CTE speaks to
the relevance of our model for investigations of human
TBI pathogenesis.
Our study has several limitations that are worth
acknowledging. First, the use of homogenized tissue
from the whole cortex may have masked detection of
molecular responses that are cell specific. Future studies
utilizing astroglial and microglial fractions instead of the
whole tissue may reveal more specialized mechanisms
involved in TBI pathogenesis, especially those related to
neuroinflammation. Secondly, common caveats of pro-
teomic studies are the biological and technical limita-
tions. ey may result in a decreased sensitivity for low
abundance proteins, inaccuracies in ratio calculations,
and false negative/positive results. To minimize the lat-
ter, we applied strict cutoff thresholds for identification
of significant changes in protein expression, and high-
stringency analysis of protein–protein interactions for
canonical pathways identification. Lastly, a sample size of
four mice per group presents statistical limitations, and a
higher-powered study would increase the accuracy of the
observed signal. However, we relied on our previous data
where a similar sample size of four was used in a r-mTBI
model and showed statistically significant changes in
TBI-induced pathology and behavior [14, 15, 29, 30, 34].
Moreover, tissue described in the current manuscript
was used in several other analyses where it demonstrated
increased neuroinflammation, tauopathy, and spatial
memory deficits [29, 30].
Overall, our data signify the importance of investigat-
ing not only the total proteome but also the phospho-
proteome and comparing animal data with human for
clinical relevance. is approach may help to identify
therapeutic targets for TBI but also provides a broad
timeline of the progression of TBI pathobiology. Future
studies will focus on validating candidate proteins and
pathways that exhibit both therapeutic and translational
Male and female hTau mice (n = 16) 12–14 weeks old
(weight 19-25g) were sourced from Jackson Laborato-
ries (Bar Harbor, ME). e animals were housed under
standard laboratory conditions (14-h light/10-h dark
cycle, 23 ± 1 °C, 50 ± 5% humidity) with free access to
food and water. All procedures were carried out under
Institutional Animal Care and Use Committee approval
(Roskamp Institute IACUC) and in accordance with the
National Institutes of Health Guide for the Care and Use
of Laboratory Animals.
Repetitive mild TBI
For sham/mTBI procedures, all animals underwent anes-
thesia with 1.5mL/min of oxygen and 3% isoflurane for
3 min on a heated platform to prevent hypothermia.
Mice assigned to TBI procedures were also maintained
on a heating pad during the injury procedure to pre-
vent hypothermia. e head of each animal was fixed in
a stereotaxic frame, and a 5 mm blunt metal impactor
was positioned midway on the sagittal suture. e injury
Page 9 of 12
Morinetal. Molecular Brain (2022) 15:62
was triggered at 5m/s velocity and 1.0mm depth, with
a dwell time of 200ms, using a myNeuroLab controller
device (Impact One Stereotaxic Impactor, Richmond,
IL). All TBI mice experienced short-term apnea (< 20s)
and showed no skull fractures. All animals (sham and
TBI) recovered from anesthesia on a heating pad and
were then returned to their cages with water and soft
food access. In total, mice received 5 hits (or in the case
of sham mice, 5 anesthesias of the same duration as TBI
mice), one every 48h for the duration of 9days as shown
in Fig.1A. After the final injury, mice were split into 2
cohorts: sub-acute (SA) and chronic (CH). A total of
four groups was formed—SA Sham, SA TBI, CH Sham,
CH TBI—with n = 4 mice in each group. SA mice were
euthanized at 3weeks after the last injury while CH were
euthanized at 24weeks post last injury, and their half-
cortices were extracted for proteomic analysis.
Tissue extraction andhomogenization
Cortical tissue was homogenized using 500 µL Mam-
malian Protein Extraction Reagent (MPER) per sam-
ple containing 1% EDTA and 1% protease/phosphatase
inhibitors cocktail. Samples were sonicated 3 times for
10min and centrifuged at 10,000rpm at 4°C for 10min.
Supernatants were separated and used for further
Sample preparation
Frozen tissue homogenates were thawed, and their pro-
tein content was determined using a bicinchoninic acid
(BCA) assay. Samples were immediately aliquoted to
avoid variation in the protein concentrations due to
freeze/thaw cycles. A control sample was created by
pooling an aliquot containing 60µg of protein from each
of the 48 samples into one Eppendorf tube. Next, six
150µg aliquots of this control sample were transferred
to separate 2mL Eppendorf tubes. A 10 µL aliquot of a
protein standard mixture was added to each control rep-
licate and 100mM triethylammonium bicarbonate buffer
(TEAB) was added appropriately to adjust the final vol-
ume to 100 µL. Samples were randomized and divided
into six batches with one overlapping repeat sample per
batch for between-batch quality control validation. At
this stage all batches were frozen at 20°C.
Further sample processing was performed in batches
and was guided by ermo Scientific’s instructions for
TMT 10-plex Mass Tag Labeling Kits and Reagents,
although not strictly followed. Each batch consisted of
one control replicate, eight true samples and one repeat
sample. Processing of one or two batches of samples was
started every 2days.
Protein reduction and alkylation were accomplished
by the addition of 5 µL of 200mM tris(2-carboxyethyl)
phosphine (TCEP) per sample, incubated for 1h at 55°C,
followed by 5 µL of 375mM iodoacetamide, incubated
for 30min at room temperature in the dark. e protein
was then precipitated with 600 µL of cold acetone, and
the process proceeded overnight at 20°C. e next day,
samples were pelleted by centrifugation at 8000×g for
15min at 4°C. e supernatant was aspirated, and pellets
were allowed to dry for approximately 20min. e pro-
tein was then resuspended in 100 µL of 50mM TEAB via
a 1-h incubation in a thermocycler at 37°C followed by a
1.5-h bath sonication with intermittent vortexing.
Protein digestion
Protein was digested first with Lys-C, incubated in a ther-
mocycler at 37°C for 1h, and then with Trypsin, incu-
bated in the thermocycler at 37 °C overnight. Samples
originally containing 150µg of protein received 3 µL of
(0.25µg/µL) Lys-C, and those originally containing only
50µg of protein received 1 µL. Trypsin (0.5µg/µL) was
added in the same manner. Samples were then stored at
20°C until ready for peptide labeling.
Peptide labeling
TMT10plex labeling reagents were brought to room tem-
perature and dissolved in anhydrous isopropanol. For
each batch, the combined sample was labeled with TMT-
126 and the repeat sample was labeled with TMT-131.
Labeling of true samples were randomized. Each sam-
ple was combined with one mass-tagging reagent and
incubated at room temperature for 1h. Subsequently, 8
µL of 5% hydroxylamine was added to each sample and
incubated for 15min to quench the reaction. e total
volume for each labeled sample was combined into one
new Eppendorf tube and stored at 20 °C. Once all
batches reached this stage, the six samples were desalted
using Pierce C18 spin columns per product instructions.
e instructions include an optional wash step for the
removal of excess TMT reagent. e samples were then
concentrated on a SpeedVac until sample volumes had
been reduced to approximately 25 µL and then stored at
Phosphopeptide enrichment was performed using TiO2
beads using steps modified from Huang etal. [35]. Vari-
ous acidic acetonitrile solutions were used for the sample
buffer, and for peptide binding and washing. Ammonia
hydrate solutions were used for the elution phase. Sam-
ple buffer was added to the samples and the resulting
solution was transfer pipetted into an Eppendorf tube
containing conditioned TiO2 beads. Sample tubes were
Page 10 of 12
Morinetal. Molecular Brain (2022) 15:62
placed on a shaker for 1 h to facilitate binding of the
phosphopeptides. TiO2 beads were collected after a brief
spin on a tabletop mini-centrifuge and the supernatant
representing the unbound peptide fraction was removed
and saved. e beads were washed with a three-step
procedure. e supernatants were discarded after these
steps. Bound analytes, enriched in phosphopeptides,
were eluted in two steps with the addition of ammonia
hydrate solutions. e supernatants were saved after
each elution step and combined. e phosphopeptide
enriched fractions were then concentrated on a Speed-
Vac until volumes had been reduced to 25–50 µL. Next,
an aliquot of 0.1% TFA (aq.) was added to each in prepa-
ration for clean-up on Pierce C18 spin columns per prod-
uct instructions.
LC/MS/MS analysis
Samples were analyzed on a LC/MS system comprised
of an Easy nLC 1000 (ermo) coupled to a Q Exactive
hybrid quadrupole-Orbitrap mass spectrometer with
a NanoFlex source (ermo Scientific). Peptides were
trapped on an Acclaim PepMap 100 (75µm × 20mm,
ermo Scientific) and desalted. Chromatography was
performed on an analytical column (75µm × 150mm,
ermo Scientific) packed with C18, 2µm particles using
a 115-min water/acetonitrile reversed-phase gradient
e Q Exactive was operated in data-dependent acqui-
sition (DDA) mode. Full scan MS spectra (m/z 400–1800)
were acquired in the Orbitrap analyzer with a resolving
power of 70,000 (at m/z 200). e fifteen most intense
multiply charged ions (z 2) were sequentially isolated
with a 1.0Da isolation width and fragmented in the colli-
sion cell by higher-energy collisional dissociation (HCD).
Fragment ions were mass analyzed to a 35,000 resolving
power (at m/z 200).
Peptide andprotein identication andquantication
Raw data were processed using Proteome Discoverer
software (version 2.1, ermo Scientific). e MS/MS
spectra were searched against a Uniprot mouse protein
database (downloaded February 2018) using a target-
decoy strategy. Reporter ion intensities were extracted
from nonredundant peptide spectral matches (PSMs)
and the ratios were determined relative to the combined
Tissue processing andimmunouorescence
At the time of euthanasia, one hemisphere per mouse
was post fixed in a solution of 4% paraformaldehyde at
4 °C for 48 h, dehydrated in graded ethanol solutions,
cleared in xylene, and embedded in paraffin. Serial
sections (6 µm thick) were cut onto positively charged
glass slides and rehydrated in ethanol solutions of
decreasing concentrations. Slides were boiled in citrate
buffer (pH 6.0) for 7min for antigen retrieval, transferred
to a Sudan Black solution for 15min to prevent autoflu-
orescence and blocked for 1h with UltraCruz Blocking
Reagent (Santa Cruz: sc-516214). Fluorescent staining
was performed with the antibodies for PDE2A (FabGen-
nix: PD2A-101AP) during the overnight incubation.
On the next day, secondary antibodies AlexaFluor555
(ermo Fisher Scientific: A31572) were applied. Slides
were mounted with ProLong Gold Antifade 4’,6-diami-
dino-2-phenylindole (DAPI) Mount. Imaging was per-
formed using a confocal microscope (LSM 800 Zeiss)
at 20× magnification. Quantification of the fluorescent
images was performed using the LSM 800 Zeiss and the
number of cells per selected region of interest (ROI) in
the cortex was measured.
Data analysis
e acquired abundance ratios for 5r-mTBI samples were
normalized to time-matched controls and used to calcu-
late the up- (> 1.2) or downregulation (< 0.5) of proteins/
phosphoproteins. ese cutoff levels were chosen based
on our previous practice and supported by independ-
ent publications [20, 21]. Functional analyses, canoni-
cal pathways and networks were generated through the
use of IPA (QIAGEN Inc., https:// www. qiage nbioi nform
atics. com/ produ cts/ ingen uity- pathw ay- analy sis) [36].
e interaction map was constructed using STRING
software (version 11.5). e parameters were set to show
the full network type indicating both physical and func-
tional associations between the proteins with high confi-
dence interaction score (> 0.7). Edges coloring represents
the type of interaction according to the default settings
(https:// string- db. org/).
r-mTBI: Repetitive mild traumatic brain injury; AD: Alzheimer’s disease; CTE:
Chronic traumatic encephalopathy; MS: Mass Spectrometry; SA: Sub-acute;
CH: Chronic; a.r.: Abundance ratio; DDA: Data dependent acquisition; HCD:
Higher-energy collisional dissociation; PSM: Peptide specific matches; ROI:
Region of interest; IPA: Ingenuity pathway analysis.
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s13041- 022- 00945-4.
Additional le1. Raw data of proteins and phosphoproteins.
Additional le2. Proteins and phosphoproteins in the overlapping
Additional le3. Molecules driving the overrepresented canonical
Page 11 of 12
Morinetal. Molecular Brain (2022) 15:62
We are thankful to the Department of Veterans Affairs for providing funding
for this study. We are also thankful to Eric Dammer and Chadwick Hales (Emory
University) who provided an open access data of the human CTE (PXD007694)
via the ProteomeXchange server.
Author contributions
AM performed animal injuries, sample collection, pathology, data analysis and
manuscript writing. RD performed LC/MS/MS analysis. TD performed sample
processing, protein digestion/labeling, phospho-enrichment. BM supervised
animal procedures, assisted with euthanasia. FC conceived and designed the
research and is a VA Research Career Scientist (Award # IK6BX005385). FC and
BM are CENC investigators. All authors reviewed the final manuscript and
approved its publication. All authors read and approved the final manuscript.
The study was supported by the Department of Veterans Affairs V Merit
[I01RX002334 (PI: F. Crawford)] and by the Roskamp Institute. The contents
do not represent the views of the Department of the Veterans Affairs or the
United States Government.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from
the corresponding author on reasonable request.
Ethics approval and consent to participate
All procedures were carried out under Institutional Animal Care and Use
Committee approval (Roskamp Institute IACUC) and in accordance with the
National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1 Roskamp Institute, Sarasota, USA. 2 The Open University, Milton Keynes, UK.
3 The James A Haley Veterans’ Administration, Tampa, USA.
Received: 19 April 2022 Accepted: 28 June 2022
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Full-text available
To date, an overwhelming number of preclinical studies have addressed acute treatment in mild TBI (mTBI) and repetitive mTBI (r-mTBI), whereas, in humans, there often exists a significant time gap between the injury and the first medical intervention. Our study focused on a delayed treatment with anatabine, an anti-inflammatory compound, in hTau mice using two different models of r-mTBI. The rationale for using two models of the same impact but different frequencies (5 hit mTBI over 9 days and 24 hit mTBI over 90 days) was chosen to address the heterogeneity of r-mTBI in clinical population. Following the last injury in each model, three months elapsed before the initiation of treatment. Anatabine was administered in drinking water for 3 months thereafter. Our data demonstrated that a 3-month delayed treatment with anatabine mitigated astrogliosis in both TBI paradigms but improved cognitive functions only in more-frequently-injured mice (24 hit mTBI). We also found that anatabine decreased the phosphorylation of tau protein and NFκB, which were increased after r-mTBI in both models. The ability of anatabine to suppress these mechanisms suggests that delayed treatment can be effective for clinical population of r-mTBI. The discrepancy between the two models with regard to changes in cognitive performance suggests that r-mTBI heterogeneity may influence treatment efficiency and should be considered in therapeutic development.
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Repeated exposure to mild TBI (mTBI) has been linked to an increased risk of Alzheimer’s disease (AD), chronic traumatic encephalopathy (CTE) and other neurodegenerative diseases. Some pathological features typically observed in AD have been found in postmortem brains of TBI and CTE, hence treatments tested for AD have a potential to be effective against r-mTBI outcomes. Neuroinflammation may present a possible answer due to its central role both in acute brain injury and in chronic degenerative-like disorders. Our previous studies have shown that drug nilvadipine, acting as an inhibitor of spleen tyrosine kinase (SYK), is effective at reducing inflammation, tau hyperphosphorylation and amyloid production in AD mouse models. To demonstrate the effect of nilvadipine in the absence of age-related variables, we introduced the same treatment to young r-mTBI mice. We further investigate therapeutic mechanisms of nilvadipine using its racemic properties. Both enantiomers, (+)-nilvadipine and (−)-nilvadipine, can lower SYK activity, whereas (+)-nilvadipine is also a potent L-type calcium channel blocker (CCB) and shown to be anti-hypertensive. All r-mTBI mice exhibited increased neuroinflammation and impaired cognitive performance and motor functions. Treatment with racemic nilvadipine mitigated the TBI-induced inflammatory response and significantly improved spatial memory, whereas (−)-enantiomer decreased microgliosis and improved spatial memory but failed to reduce the astroglial response to as much as the racemate. These results suggest the therapeutic potential of SYK inhibition that is enhanced when combined with the CCB effect, which indicate a therapeutic advantage of multi-action drugs for r-mTBI.
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Alzheimer’s disease (AD), the most prevalent form of dementia, is characterized by two pathological hallmarks: Tau-containing neurofibrillary tangles and amyloid-β protein (Aβ)-containing neuritic plaques. The goal of this study is to understand mild traumatic brain injury (mTBI)-related brain proteomic changes and tau-related biochemical adaptations that may contribute to AD-like neurodegeneration. We found that both phosphorylated tau (p-tau) and the ratio of p-tau/tau were significantly increased in brains of mice collected at 3 and 24 h after exposure to 82-kPa low-intensity open-field blast. Neurological deficits were observed in animals at 24 h and 7 days after the blast using Simple Neuroassessment of Asymmetric imPairment (SNAP) test, and axon/dendrite degeneration was revealed at 7 days by silver staining. Liquid chromatography-mass spectrometry (LC-MS/MS) was used to analyze brain tissue labeled with isobaric mass tags for relative protein quantification. The results from the proteomics and bioinformatic analysis illustrated the alterations of axonal and synaptic proteins in related pathways, including but not being limited to substantia nigra development, cortical cytoskeleton organization, and synaptic vesicle exocytosis, suggesting a potential axonal damage caused by blast-induced mTBI. Among altered proteins found in brains suffering blast, microtubule-associated protein 1B, stathmin, neurofilaments, actin binding proteins, myelin basic protein, calcium/calmodulin-dependent protein kinase, and synaptotagmin I were representative ones involved in altered pathways elicited by mTBI. Therefore, TBI induces elevated phospho-tau, a pathological feature found in brains of AD, and altered a number of neurophysiological processes, supporting the notion that blast-induced mTBI as a risk factor contributes to AD pathogenesis. LC/MS-based profiling has presented candidate target/pathways that could be explored for future therapeutic development.
Objective: Scleral remodeling plays a key role in axial elongation in myopia. The aim of the present study was to identify the proteomics changes and specific signaling networks to gain insight into the molecular basis of scleral remodeling in myopic eyes. Methods: Guinea pig form-deprivation myopia was induced with a translucent diffuser on a random eye for 4 weeks, while the other eye served as the contralateral control group. The axial length and refraction were measured at the beginning and end of the treatment. The proteins were extracted from the sclerae of each group and prepared for quantitative isobaric tags for relative and absolute quantification (iTRAQ) labeling combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The coexpression networks and protein functions were analyzed using Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA). Quantitative real-time PCR (qRT-PCR) and western blotting were performed to confirm the authenticity and accuracy of the iTRAQ results. Results: After 4 weeks, the form-deprivation eyes developed significant degrees of myopia, and the axial length increased statistically significantly (p<0.05). A total of 2,579 unique proteins with <1% false discovery rate (FDR) were identified. Furthermore, 56 proteins were found to be upregulated, and 84 proteins were found to be downregulated, with a threshold of a 1.2-fold change and p<0.05 in the myopia group, when compared to the control group. Further bioinformatics analysis indicated that 44 of 140 differentially expressed proteins were involved in cellular movement and cellular assembly and organization. The qRT-PCR or western blotting results confirmed that myosin IIB, ACTIN3, and cellular cytoskeletons were downregulated, while RhoA and RAP1A were upregulated in the sclera in myopic eyes. These results were consistent with the proteomics results. Conclusions: Proteomics and bioinformatics results can be helpful for identifying proteins and providing new insights for better understanding of the molecular mechanism underlying scleral remodeling. These results revealed that the proteins associated with cellular movement and cellular assembly and organization are altered during the development of myopia. Furthermore, RhoA plays a key role in the pathways involved in cellular movement and cellular assembly and organization.
Background: Neurodegenerative disorders have been reported in elite athletes who participated in contact sports. The incidence of neurodegenerative disease among former professional soccer players has not been well characterized. Methods: We conducted a retrospective cohort study to compare mortality from neurodegenerative disease among 7676 former professional soccer players (identified from databases of Scottish players) with that among 23,028 controls from the general population who were matched to the players on the basis of sex, age, and degree of social deprivation. Causes of death were determined from death certificates. Data on medications dispensed for the treatment of dementia in the two cohorts were also compared. Prescription information was obtained from the national Prescribing Information System. Results: Over a median of 18 years, 1180 former soccer players (15.4%) and 3807 controls (16.5%) died. All-cause mortality was lower among former players than among controls up to the age of 70 years and was higher thereafter. Mortality from ischemic heart disease was lower among former players than among controls (hazard ratio, 0.80; 95% confidence interval [CI], 0.66 to 0.97; P = 0.02), as was mortality from lung cancer (hazard ratio, 0.53; 95% CI, 0.40 to 0.70; P<0.001). Mortality with neurodegenerative disease listed as the primary cause was 1.7% among former soccer players and 0.5% among controls (subhazard ratio [the hazard ratio adjusted for competing risks of death from ischemic heart disease and death from any cancer], 3.45; 95% CI, 2.11 to 5.62; P<0.001). Among former players, mortality with neurodegenerative disease listed as the primary or a contributory cause on the death certificate varied according to disease subtype and was highest among those with Alzheimer's disease (hazard ratio [former players vs. controls], 5.07; 95% CI, 2.92 to 8.82; P<0.001) and lowest among those with Parkinson's disease (hazard ratio, 2.15; 95% CI, 1.17 to 3.96; P = 0.01). Dementia-related medications were prescribed more frequently to former players than to controls (odds ratio, 4.90; 95% CI, 3.81 to 6.31; P<0.001). Mortality with neurodegenerative disease listed as the primary or a contributory cause did not differ significantly between goalkeepers and outfield players (hazard ratio, 0.73; 95% CI, 0.43 to 1.24; P = 0.24), but dementia-related medications were prescribed less frequently to goalkeepers (odds ratio, 0.41; 95% CI, 0.19 to 0.89; P = 0.02). Conclusions: In this retrospective epidemiologic analysis, mortality from neurodegenerative disease was higher and mortality from other common diseases lower among former Scottish professional soccer players than among matched controls. Dementia-related medications were prescribed more frequently to former players than to controls. These observations need to be confirmed in prospective matched-cohort studies. (Funded by the Football Association and Professional Footballers' Association.).
Tau-tubuline kinases (TTBK) are a family of serine/threonine and tyrosine kinases recently discovered and implicated in the phosphorylation of important substrates such as tau, tubuline or TDP-43. Its two homologs, TTBK1 and TTBK2, show different expression patterns and different involvements in physiological mechanisms of great importance such as mitosis, ciliogenesis and neurotransmission. Their phosphorylation activity has also linked them to the development of neurodegenerative diseases like Alzheimer's disease, amyotrophic lateral sclerosis or spinocerebellar ataxia type 11. There are currently only three inhibitors of these kinases described in the literature. This review intends to give an overview of the structure, expression, physiological and pathological mechanisms of both kinases as well as an extended analysis on the molecules that can inhibit them. The final analysis of all this information led us to propose TTBK1 as a new target for the treatment of neurodegenerative diseases and its selective inhibitors as potential effective drugs for the treatment of these severe unmet disorders.