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Our group has
been systematically experimenting with the permanent middle cerebral artery
occlusion (PMCAO) mouse model revealing large number of genes differentially
expressed in the ischemic brain, hemispheres and targeting specific tissues in
the ischemic core and penumbra brain regions after pituitary adenylate-cyclase
activating polypeptide (PACAP) intervention. Utilizing a high throughput DNA
microarray-based whole mouse genome analysis on the Agilent platform by
two-color/dye-swap approach for genome-wide transcriptome datasets, the
neuroprotective effects of the PACAP38 (1 pmol) treatment is being revealed
step by step in the PMCAO model mouse brain. This report revisits the large
volume of data (dye-swap based gene inventory) specifically obtained from the
ischemic core and penumbra regions of the ischemic brain by filtering it over
the brain hemispheres data and healthy control brain in conjunction with
Ingenuity Pathway Analysis (IPA) analysis. In conjunction with targeted Western
analysis of a potential key player in PACAP-mediated neuroprotection, namely
CRMP2, previously identified by us using 2-DGE analysis and other researchers,
these combined results suggest a role for PACAP in regulating CRMP2 function
through GSK3β and upstream signaling networks as revealed by the bioinformatics
analysis of specific genome data at 6 h post-treatment with PACAP38. A
schematic and hypothetical model shows genes potentially involved behind
neuroprotective function of PACAP38 in the ischemic brain early on is presented.
Keywords: PACAP38, Ischemic
core and penumbra, Mice, DNA microarray, CRMP2
INTRODUCTION
Pituitary adenylate-cyclase activating
polypeptide (PACAP) [1-3] has neuroprotective functions especially under the
condition of brain ischemia [4-18]. Based on these evidences, some from our own
group using the permanent middle cerebral artery occlusion (hereafter referred
to as PMCAO) mouse model [6,7,11-16] and that from other researchers in stroke
models [17-22] this neuropeptide was suggested to be a potential therapy in
counteracting effects of brain ischemia or stroke.
It is to be noted that our group’s research uses the intraluminal filament technique-based PMCAO model over the transient MCAO, which results in reperfusion injury, and is avoided in our research model. To address a question on whether PACAP38 treatment has indeed an effect on the brain infarction size (in mice), previous researches have quantitatively/qualitatively (by 2, 3, 5-triphenyltetrazolium chloride, TTC, staining) demonstrated that PACAP38 treatment improved brain infarction compared to a control 0.9% saline [6,19,12; and references therein]. Thus, previous results demonstrated PACAP38 efficacy for neuroprotection of the brain following an ischemic insult in mice.
A unique aspect of our group’s
PACAP38 research is the strategy involving a large-scale DNA microarray-based
[23] high-throughput omics analysis on the PMCAO model brain hemispheres and
regions, over a period of time to step by step create an inventory of genes
(transcriptome database, freely accessible on the NCBI GEO database -
http://www.ncbi.nlm.nih.gov/geo/info/linking.html, under accession numbers -
GSE28201, GSE37565, GSE62884, GSE 67421) [12-16]. These gene lists and
expression profiles have revealed their modulation in the ischemic brain with
or without PACAP38 treatment at two different time points of 6 and 24 h
post-treatment, providing the first inventory of the mouse ischemic brain
transcriptome [12-14]. All the above researches resulted in a vast gene
database resource on novel molecular factors and potential mechanisms that
might underlie the neuroprotective effects of PACAP using animal models, and
that may be used to give new insight into the potential therapeutic use of
PACAP38 and/or its analogs [6, 7, 11-17,19-22]. For example, Tamas and
colleagues have indicated in their review that the role of PACAP as a
neurotrophic factor has now been validated and the data suggesting an important
function of PACAP in neuronal regeneration promises its use as a therapeutic
agent in injuries to the nervous system [22]. Rivnyak and co-workers have
recently reviewed the effects of PACAP on the transcriptome and proteome in the
brain [18].
PACAP38 effect comprises of numerous and interlinked molecular genetic factors and/or pathways as revealed from all these studies. As a next step we have narrowed down to specifically dissected brain regions in the ischemic brain, namely the ischemic/infract core (hereafter abbreviated IC) and the penumbra (hereafter abbreviated P or ischemic P, IP, when looking at the ischemic region) to analyze these gene inventories and get new insight into PACAP38 effect (Figure 1).
Our recent hypothesis is that targeting the P
and IC regions in the ischemic brain would give information on the unique
expressions of genes involved in modulating the neuroprotective effect of
PACAP38 early on (i.e., 6 h post ischemia) in the ischemic insult. The 6 h time
period has been specifically examined in our current analysis of gene
expressions in order to know what signaling process occur at this time point,
which is the acute phase following PMCAO. As generally believed, during the
stroke onset (acute phase) ischemic injury causes a blockage of nutrients via
deprivation of blood and there is irreversible damage to the ischemic core; and
compared to the 24 h chronic phase where there may be some remodeling signaling
in the penumbra (IP). Therefore, our experiment examining the effect of PACAP38
intervention at the 6 h time point is critical to see and seek out the
signaling factors working in the IC and P(IP), and how the remodeling via
PACAP38 is occurring to cause improvements in the brain in both the IC and the
P(IP). Therefore, at present the 24 h time point (chronic phase) has not been
examined in the present analysis.
Thus, this current report, first, puts
forward a bioinformatics-based evidence to explain the nature of the genes
expressed in specifically dissected P(IP) and IC regions in relation to
recovery effect of PACAP38 treatment early on (6 h post ischemic insult), in
PMCAO mouse model. Second, based on the bioinformatics analyses and gene
functions and pathways unravelled during this study, targeted protein analysis
was performed using immunoblotting with specific antibodies to further support
the role of the previously identified collapsing response mediator protein 2
(CRMP2) [24], and other related up-stream potential target molecules for the
proposed neuroprotective mechanisms behind PACAP38 action in mice brain
ischemia.
MATERIALS
& METHODS
DNA microarray data usage and ingenuity pathway
analysis (IPA)
The present study uses raw whole gene
expression data from previously conducted experiments in our laboratory and
unpublished data (but deposited in the NCBI GEO database: GSE28201, GSE37565,
GSE62884, GSE 67421; Figure 1) [12-16]. All the animal research (animal care
and experimental procedures) were previously performed with the relevant
guidelines and regulations which were approved by the Institutional Animal Care
and Use Committee of Showa University (School of Medicine), Tokyo, Japan. PMCAO
model mice were generated and brain regions dissected and processed for DNA
microarray analysis as described previously, including PACAP38
injection/treatment [6,12-14]. Utilizing these gene expression datasets, the
biological function and network analyses were performed using IPA (Ingenuity®
Systems, www.ingenuity.com). Concurrent to the previous experiments, a new
analysis was performed using the IPA tool [Content version: 23814503 (Release
Date: 2015-03-22), QIAGEN]. The dataset from the DNA microarray which includes
the differentially expressed (≧/≦ 1.5/0.75-fold) genes
and their corresponding fold change values, was uploaded as an Excel spread
sheet into the IPA tool. To create gene data (bio-functions, canonical pathways
and upstream regulators), the genes were overlaid onto a global molecular
network that was developed from information that was contained in the ingenuity
knowledge base. The functional analysis identified the biological functions
that were most significant to the dataset (p-value <0.05) according to a
right-tailed Fisher’s exact test.
Extraction of total
soluble protein
Total
protein was extracted from approximately 50 mg sample powders (each P/IP and IC brain
region; n=6 each, and sample powders were prepared from pooled brain regions
that were extremely small in size and quantity due to the microdissection form
the mentioned sampling regions of the brain) using a lysis buffer that
contained Thiourea and Tris (LB-TT) for extraction of brain proteins [13, 25]. Composition of slightly modified LB-TT
was as follows: 7 M (w/v) urea, 42 g; 2 M (w/v) Thiourea, 15.2 g; 4 % (w/v) CHAPS, 4.0 g; 18 mM (w/v) Tris-HCl (pH
8.0), 1.8 mL; 14 mM (w/v) Trizma base, 169.5 mg; 0.2 % (v/v) Triton X-100, 0.2 mL; 50 mM (w/v) DTT, 771.5 mg; 1 % (v/v) pH 3-10 Ampholyte, 1 mL; and two EDTA-free
proteinase inhibitor (Roche) tablets in a total volume of 100 mL at room temperature (RT). Protein extraction was initiated by
quickly adding 1 mL of LB-TT to the 2 mL microfuge tube containing sample powder following removal from
the -80°C deep freezer, and immediately mixed by vortexing (at full speed using a
Lab mixer, Iwaki, Tokyo, Japan) for 1 min at RT. Protein solution in LB-TT was
incubated at RT for 30 min with mixing by vortexing (30 s) and sonication (30 s
in a water-bath type sonicator) for a total of 5 times. Insoluble protein
pellet and/or debris were pelleted by centrifugation at 18,500 g for 15 min at 20ºC using a high-speed refrigerated micro centrifuge (MX-150, TOMY, Tokyo,
Japan). Clear supernatant (around 900 mL) was transferred to a new 1.5 mL microfuge tube and stored at -80°C as the total soluble protein. Protein
concentration was determined with a Coomassie PlusTM Protein Assay Kit (PIERCE, Rockford, IL, USA) using bovine serum albumin (BSA) as a standard and a
NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA).
Western blot analysis
SDS-PAGE (mini-gel) was carried out on the separated
proteins as per standard protocols proteins [13, 25]. The well separated proteins were electro transferred onto a
polyvinyldifluoride [PVDF (Trans-Blot Turbo Midi PVDF, 0.2 mM, Transfer Packs kit; Cat. no. 170-4157)]
using the Trans-Blot Turbo Transfer System (Bio-Rad; StandardSD protocol; 25V,
1.0 A, 30 min). Following the transfer of proteins on the PVDF membrane
(confirmed by visualizing all the 10 colored molecular mass standards), the
membrane was incubated in 25 mL of 5 % blocking solution (Block-Ace powder,
Cat. no. UK-B80, DS Pharma, Osaka, Japan; Yukizirushi, Sapporo, Hokkaido,
Japan) for 1 h under constant slow shaking at RT. Blocking solution was
prepared by dissolving 4 g powder in 80 mL 1X TTBS [10X TTBS: NaCl, 80 g; 1 M
Tris-HCl, pH 7.5, 200 mL; Tween-20, 5 mL]. Western blotting and detection was
carried out using the Immun-Star WesternC Chemiluminescent Kit (Cat. no.
170-5070, Bio-Rad) following the manufacturer’s instructions. Blocking solution
was decanted and the membrane was washed once in 1X TTBS (5 min), followed by
incubation in 25 mL of primary antibody solution [PAS; 1 mL rabbit anti-CRMP2 phophoT514 protein
antibody (Cat. no. ab62478; 100 mg, 2 mg/mL; Abcam, www.abcam.co.jp;
anti-CRMP2 phophoSer522 and anti-CRMP2 phophoThr555] for 1 h, as above. The
membrane was then washed with 25 mL of 1X TTBS for five times. The last TTBS
wash was decanted and the membrane was incubated in 25 mL of secondary antibody
solution [SAS; 0.5 mL of Amersham, ECL anti-rabbit IgG, HRP linked species-specific whole
antibody (from Donkey); Cat. no. NA 934; GE Healthcare] for 1 h with slow
shaking at RT. The 1X TTBS wash step was repeated five times. For blot
development, the luminol/enhancer and peroxide buffer solutions were mixed in a
1:1 ratio (1 mL: 1 mL; one membrane volume) and spread over the membrane and incubated
at RT for 5 min. Excess solution was drained by touching one end of the
membrane on a KimWipe paper towel and the signal (cross-reacting protein bands)
was visualized using the ChemiDoc XRS+imaging system (Bio-Rad). The Western
blot analysis was repeated at least three times, and representative data from
one blot is shown.
RESULTS AND DISCUSSION
Specifically up- and down-regulated genes in ischemic
core and penumbra following PACAP38 treatment and their IPA
IPA-based bioinformatics analysis provided a number of
IC-specific genes: 241 (up-regulated)/229 (down-regulated) and also
P(IP)-specific genes: 181 (up-regulated)/236 (down-regulated) at 6 h
post-ischemia following the injection of PACAP38 (Figure 1). The 6 h
early time point was targeted because of early visualization of the brain
regions and ischemia (also by TTC
staining which shows the ischemic region in the brain) in the brain along with numerous gene expression changes [12-16] and the very early treatments for stroke
given within 4 to 6 h of the stroke. Keeping in mind the hypothesis that
PACAP38 should be effective by its nature as a neuroprotective peptide/agent on
the cells (or neurons) by direct action or indirect action through modulating
the cells through activating the release of neuroprotective factors. Thus the
authors wished to see if the narrowed down microarray-based transcriptome
contains these hints and whether they can be delineated in the IC and P(IP)
regions, on the very early time point (acute phase) of 6 h post-ischemia.
Remarkable differences were seen upon performing IPA and generating the
bio-functions, canonical pathways and upstream regulators for both IC (Figures
2, 3 and 4) and P(IP) (Figures 5, 6 and 7).
Both IC and
the P(IP) regions are affected by the PACAP38 injection in various gene
bio-functions and pathways and upstream targets; a greater number of gene
categories are being affected in the IC by PACAP38. In the IC gene categories
of bio function: lipid metabolism, small molecule biochemistry, gene
expression, cell to cell signaling and interaction, others (down-regulated) to
up-regulated genes in cellular development to embryonic development and cell
morphology are seen (Figure 2); In the IC, gene functions can be sub-divided
mainly into antithrombotic, ant-inflammation and which are expected if PACAP38
is acting to suppress the spread of the ischemia. This is not the case in the
P(IP) region. In the case of bio-functions, PACAP38 appears to show a general
decrease in the categories of the nervous system development and function
(sensation and nociception functions), cell cycle, tissue development and
cellular growth and proliferation; all these are related to reduction in
cellular proliferation function in the P(IP) region (Figure 5). This can
be expected as the penumbra (IP) is supposed to be the site or boundary between
active recovery (IC, under PACAP38) and the presumably healthy regions.
However, we find that an increase in the gene category of endocrine system disorders
with benign thyroid disease as the annotated function is seen in the P(IP) (Figure
5). For example, one of the prominent genes is CTLA4 which suppresses the
T-cells sending them turn-off signals in case of chronic infections [26]. This
anti-inflammation function of CTLA4 would be required for the P(IP) region and
which should not be actively producing a defensive response by the T cells.
Similarly, the TTR gene is being up-regulated and which along with BDNF has
been shown to have neuroprotective effects [15, 27-29]. However, it is to be noted that TTR and especially BNDF may have
different functions depending on the cells and conditions and that is why we
see it (BDNF) being reduced in the annotated function of sensation.
Second, in the canonical pathways, IL1 signaling, dopamine receptor signaling, acute phase response signaling, CDK5 signaling, and cAMP-mediated signaling show a decrease in the P(IP) region (Figure 6) compared to the opposite gene functions in the IC (Figure 3). Anti-inflammation and blood coagulation are the main events occurring in the P(IP) region following PACAP38 treatment, especially with the cAMP-mediated pathway having an effect on the blood coagulation. Considering the fact that the region around the core is healthy, it would not be desirable to have a higher level of blood flow in response to the ischemic insult. In the case of the IC region, we see that Ga12/13 signaling function,leukocyte extravasation signaling, LXR/AXR signaling is being decreased. These decreased functions can be sub-divided into anticoagulation, anti-inflammation. The G12/13 family is well known for its involvement in cell proliferation and morphology, and PACAP38 might be function to prevent cell proliferation till the recovery process is best suited for new cell/neuron growth. Interestingly, the anti-inflammation response function might require a reduction in the fatty acid synthesis, and this is what we find (IL1RN) in the IC region. On the other hand, Wnt/β-catenin signaling may be more pronounced in the P(IP) region as compared to the IC region where it may both be induced and suppressed.
Thirdly, in the case of identified upstream regulators
(Figures 4 and 7), we can see that proliferation (STAT5B) is reduced in
the P(IP) region compared to the growth factor (WISP2) which is activated.
These play active roles in the central nervous system and their modulation by
PACAP38 suggests PACAP38 recruitment of these molecular factors and pathways
for aiding the recovery process in the P(IP) region. Taken together, these
different gene functions being differentially expressed under PACAP38 injection
in the IC and P(IP) regions of the ischemic brain are evidences of a specific
and different PACAP38 action in the IC and P(IP), in light of its
neuroprotective role in the ischemic brain. Further, in the IC region, as
upstream regulators, MKL1 and MKL2 can be seen along with their target
molecules, and where both are inhibited (Figure 4). The
MKL/myocardin-like protein 1 or myocardin-related transcription factor A
(MRTF-A) produces a protein which is a key regulator of smooth muscle cell
differentiation. Interestingly, the protein has other functions, including in
the intracerebral hemorrhagic stroke and a role in maintaining cerebral small
vessel (blood) integrity [30]. This makes for an important finding for inhibited MLK1/MRTF-A function also
in neuroprotective functions under PACAP38 treatment. In the P(IP), two
upstream regulators, STAT5B (signal transducer and activator of
transcription-5, inhibited) and WISP2 (Wnt-1 inducible
signaling pathway protein-2, activated) were identified (Figure
7). Both these are now know to be involved in the ischemic insult signaling
cascades. For example, the STAT5B has been shown to contribute to the
erythropoietin-mediated neuroprotection against hippocampal neuronal death
after transient global cerebral ischemia [31], whereas, the WISP2, a member of the connective tissue growth factor and
nephroblastoma overexpressed gene family of matricellular proteins is critical
in growth factor mediated cell proliferation [32,33]. In summary, the identification of these three (MKL1, STAT5B and WISP2)
factors in the ischemic brain regions supports the proposed PACAP-mediated
neuroprotective function.
Six-hour post ischemic response in the penumbra might
involve cell cycle suppression in conjunction with CRMP2 function
Previously, we had shown that the CRPM2 [24] protein
is accumulated during the ischemia following PACAP38 treatment, especially at 6
h post-ischemia, and we also showed that the additional band observed in our
experiments might be not a degradative product but having some role in
neuroprotection following PACAP38 treatment [14,15]. Recently, using brain-specific Crmp2 knockout (cKO) mice it was shown that these
mice not only display molecular, cellular, structural and behavioral deficit
but also that a loss of Crmp2 in the hippocampus leads to reduced
long-term potentiation, abnormal NMDA receptor composition, aberrant dendrite
development and defective synapse formation in CA1 neurons, and a knockdown of crmp2 specifically in newborn neurons results in
stage-dependent defects in their development during adult hippocampal
neurogenesis [34]. This recent paper [34] provides the strongest evidence to date
on the importance of CRMP2 in axonal growth/neurogenesis, and we attempted to
link our gene data with CRMP2 action, and which is presented as a model in Figure 8 (upper panel). The identified
(DNA microarray-based gene expressions) up-stream up-regulated (in red
arrowheads) and down-regulated (in green arrowheads) molecules are marked in
the diagram, which is linked to the phosphorylation-dephosphorylation of the
CRMP2 protein, in our model (Figure 8,
lower panel). PACAP38 influences the regulation of other gene functions as
indicated, especially those acting to suppress the GSK3β-pathway; it may be
possible that CRMP2 action is enhanced in the penumbra (IP).
Our combined data also focuses on the short form of CRMP2 which could play an important role in nerve regeneration upon treatment by PACAP38. A study by Rogemond et al. [35] looked at a new short isoform of 58 kDa, expressed during brain development, and derived from C-terminal processing of the CRMP2B subtype. Those authors demonstrated its presence in the nuclear fraction of brain extract and showed that this short CRMP2 induces neurite outgrowth inhibition in neuroblastoma cells and suppressed axonal growth in cultured cortical neurons [35]. They went on to suggest that the post-transcriptionally processed and nuclear localized CRMP2 may be key in the regulation of neurite outgrowth in brain development. Rogemond et al. [35] study shows a link with our finding of CRMP2 involvement and its post-translational modified state among the signaling pathways identified via the IPA analysis in IC and IP regions. Dephosphorylation of CRMP2 by PACAP38 was confirmed by Western blotting. As a result of using the CRMP2 antibodies of Ser522, Thr555 and Thr514, it was confirmed that the addition of PACAP38 promoted the dephosphorylation (Figure 8, upper panel-A, B, C). RhoA signaling, CDK5 signaling pathways are involved in CRMP2 dephosphorylation (Ser522, Thr555, Thr514), and gsk3β [36]. The phosphorylation of Gsk3β by PACAP was confirmed by Western blotting. Gsk3β is known to be inactivated by phosphorylation (GSK3β-Ser9). As a result of Western blotting, it was confirmed that the expression level of Gsk3β was reduced by PACAP38, but it was not confirmed that phosphorylation (GSK3β-Ser9) was promoted. As a result of the IPA pathway analysis, PACAP38 suppressed RhoA Signaling and its upstream Gα12/13 Signaling in the 6 h IC, and suppressed CDK5 Signaling in 6h IP, as well as CXCR4 signaling, ILK signaling, ERK/MAPK signaling, Wnt/β-activation of catenin signaling was predicted. As shown in Figure 8 (lower panel) from these results, in the IC 6 h scenario RhoA signaling and its upstream Gα12/13 signaling dephosphorylates CRMP2-Thr555 and suppresses GSK3β, and in the IP 6 h scenario the CDK5 signaling dephosphorylated CRMP2-Ser522. It was suggested that CXCR4 Signaling, ILK Signaling, ERK/MAPK Signaling, and Wnt/βcatenin signaling are involved in GSK3β suppression and CRMP2-Thr514 dephosphorylation.
CONCLUSION
Using
the unique datasets of specifically up and down-regulated genes in the IC and P(IP)
regions post PACAP38 treatment, we have provided new insight into how PACAP38
might act in the ischemic brain of mice. The datasets have been obtained from
the highly credible and reproducible Agilent Gene Expression Microarray
platform wherein we utilized the two-color and dye-swap approach (Agilent
Technologies). Our results suggest how the PACAP38 might be affecting the
recovery of the ischemic region by targeting the adjacent P(IP) region
signalling pathways and this neurogenesis or new nerve growth would be critical
for the recovery from the ischemic insult and therein to recovery. However, the above discussion based on the obtained
bioinformatics gene analysis (IPA) will have to be proved further in wet-lab
experiments and downstream analyses in new studies. It is our view that a
bioinformatics strategy to dissect out genes specifically to the ischemic and
adjoining regions will be most helpful in not only pinpointing gene candidates
potentially involved in mediating the neuroprotective effect of PACAP38 but
also support the reasons behind the current therapy for stroke in human
patients. A recently published paper utilized
metabolomics, the third high-throughput technique in the omics arsenal, to
suggest the involvement of small molecule metabolites in brain injury and
functional recovery. Their study [37] combined with transcriptomic and
proteomic data (recently reviewed by [18]) imply that continued research and
interlinking big data (omics) will be critical to truly understand the brain
stroke and mechanisms therein, which would then lead to not only diagnostic
markers but also therapeutic drugs.
ACKNOWLEDGMENT
1. Kimura C, Ohkubo S, Ogi K,
Hosoya M, Itoh Y, et al. (1990) A novel peptide which stimulates adenylate
cyclase: Molecular cloning and characterization of the ovine and human cDNAs.
Biochem Biophys Res Commun 166: 81-89.
2. Arimura A (1998) Perspectives
on pituitary adenylate cyclase activating polypeptide (PACAP) in the
neuroendocrine, endocrine, and nervous systems. J Physiol 48: 301-331.
3. Shioda S, Ozawa H, Dohi K,
Mizushima H, Matsumoto K, et al. (1998) PACAP protects hippocampal neurons
against apoptosis: Involvement of JNK/SAPK signaling pathway. Ann N Y Acad Sci
865: 111-117.
4. Gusev EI, Skvortsova VI (2003)
Brain Ischemia. Kluwer Academic/Plenum Publishers, New York.
5. Ohtaki H, Funahashi H, Dohi K,
Oguro T, Horai R, et al. (2003) Suppression of oxidative neuronal damage after
transient middle cerebral artery occlusion in mice lacking interleukin-1.
Neurosci Res 45: 313-324.
6. Ohtaki H, Nakamachi T, Dohi K,
Aizawa Y, Takaki A, et al. (2006) Pituitary adenylate cyclase-activating
polypeptide (PACAP) decreases ischemic neuronal cell death in association with
IL-6. Proc Natl Acad Sci USA 103: 7488-7493.
7. Ohtaki H, Nakamachi T, Dohi K,
Shioda S (2007) Role of PACAP in ischemic neural death. J Mol Neurosci 36:
16-25.
8. Ohtaki H, Takeda T, Dohi K,
Yofu S, Nakamachi T, et al. (2008) Increased mitochondrial DNA oxidative damage
after transient middle cerebral artery occlusion in mice. Neurosci Res 58:
349-355.
9. Ohtaki H, Ylostalo JH, Foraker
JE, Robinson AP, Reger RL, et al. (2008) Stem/progenitor cells from bone marrow
decrease neuronal death in global ischemia by modulation of inflammatory/immune
responses. Proc Natl Acad Sci USA 105: 14638-14643.
10. Nakamachi T, Endo S, Ohtaki H,
Yin L, Kenji D, et al. (2005) Orexin-1 receptor expression after global
ischemia in mice. Regul Pep 126: 49-54.
11. Nakamachi T, Ohtaki H, Yofu S,
Dohi K, Watanabe J, et al. (2010) Endogenous pituitary adenylate cyclase
activating polypeptide is involved in suppression of edema in the ischemic
brain. Acta Neurochir Suppl 106: 43-46.
12. Hori M, Nakamachi T, Rakwal R,
Shibato J, Nakamura K, et al. (2012) Unraveling the ischemic brain transcriptome
in a permanent middle cerebral artery occlusion mouse model by DNA microarray
analysis. Dis Model Mech 5: 270-283.
13. Hori M, Nakamachi T, Rakwal R,
Shibato J, Ogawa T, et al. (2012) Transcriptomics and proteomics analyses of
the PACAP38 influenced ischemic brain in permanent middle cerebral artery
occlusion model mice. J Neuroinflammation 9: 256.
14. Hori M, Nakamachi T, Shibato
J, Rakwal R, Tsuchida M, et al. (2014) PACAP38 differentially effects genes and
CRMP2 protein expression in ischemic core and penumbra regions of permanent
middle cerebral artery occlusion model mice brain. Int J Mol Sci 15:
17014-17034.
15. Hori M, Nakamachi T, Shibato
J, Rakwal R, Shioda S, et al. (2015) Unraveling the specific ischemic core and
penumbra transcriptome in the permanent middle cerebral artery occlusion model
mice brain treated with the neuropeptide PACAP38. Microarrays 4: 2-24.
16. Hori M, Nakamachi T, Shibato
J, Rakwal R, Shioda S, et al. (2015) Unraveling the progression of ischemic
core genome-wide by bioinformatics analysis of permanent middle cerebral artery
occlusion (PMCAO) mouse model brain regions genes. Transcriptomics 3: 112.
17. Tamas A, Reglodi D, Szanto Z,
Borsiczky B, Nemeth J, Lengvari I (2002) Comparative neuroprotective effects of
preischemic PACAP and VIP administration in permanent occlusion of the middle
cerebral artery in rats. Neuroendocrinol
Letts 23: 249-254.
18. Rivnyak A, Kiss P, Tamas A,
Balogh D, Reglodi D (2018) Review on PACAP-Induced transcriptomic and proteomic
changes in neuronal development and repair. Int J Mol Sci 19: 1020.
19. Chen Y, Samal B, Hamelink CR,
Xiang CC, Chen Y, et al. (2006) Neuroprotection by endogenous and exogenous
PACAP following stroke. Regul Pept 137: 4-19.
20. Dejda A, Seaborn T, Bourgault
S, Touzani O, Fournier A, et al. (2011) PACAP and a novel stable analog protect
rat brain from ischemia: Insight into the mechanisms of action. Peptides 32:
1207-1216.
21. Reglodi D, Kiss P, Szabadfi K,
Atlasz T, Gabriel R, et al. (2012) PACAP is an endogenous protective
factor-insights from PACAP-deficient mice. J Mol Neurosci 48: 482-492.
22. Tamas A, Reglodi D, Farkas O,
Kovesdi E, Pal J, et al. (2012) Effect of PACAP in central and peripheral nerve
injuries. Int J Mol Sci 13: 8430-8448.
23. DeRisi JL, Iyer VR, Brown PO
(1997) Exploring the metabolic and genetic control of gene expression on a
genomic scale. Science 278: 680-686.
24. Goshima I, Nakamura F,
Strittmatter P, Strittmatter SM (1995) Collapsin-induced growth cone collapse
mediated by an intracellular protein related to UNC-33. Nature 376: 509-514.
25. Masuo Y, Hirano M, Shibato J,
Nam HW, Fournier I, et al. (2011) Sample Preparation in Biological Mass
Spectrometry. Springer.
26. Walunas TL, Lenschow DJ,
Bakker CY, Linsley P (1994) CTLA-4 can function as a negative regulator of T
cell activation. Immunity 1: 405-413.
27. Pettersson LM, Geremia NM,
Ying Z, Verge VM (2014) Injury-associated PACAP expression in rat sensory and
motor neurons is induced by endogenous BDNF. PLoS One 9: e100730.
28. Vieira M, Gomes JR, Saraiva MJ
(2014) Transthyretin induces insulin-like growth factor 1nuclear translocation
regulating its levels in the Hippocampus. Mol Neurobiol.
29. Lazarovici P, Cohen G,
Arien-Zakay H, Chen J, Zhang C, et al. (2012) Multimodal neuroprotection
induced by PACAP38 in oxygen-glucose deprivation and middle cerebral artery
occlusion stroke models. J Mol Neurosci 48: 526-540.
30. Weinl C, Vega SC, Riehle H,
Stritt C, Calaminus C, et al. (2015) Endothelial depletion of murine SRF/MRTF
provokes intracerebral hemorrhagic stroke. Proc Natl Acad Sci USA 112:
9914-9919.
31. Zhang F, Wang S, Cao G, Gao Y,
Chen J (2007) Signal transducers and activators of transcription 5 contributes
to erythropoietin-mediated neuroprotection against hippocampal neuronal death
after transient global cerebral ischemia. Neurobiol Dis 25: 45-53.
32. Holboum KP, Acharya KR, Perbal
B (2008) The CCN family of proteins: Structure-function relationships. Trends
Biochemical Science 33: 461-473.
33. Xiao G, Tang Z, Yuan X, Yuan
J, Zhao J, et al. (2015) The expression of Wnt-1 inducible signaling pathway
protein-2 in astrocytoma: Correlation between pathological grade and clinical
outcome. Oncol Lett 9: 235-240.
34. Zhang H, Kang E, Wang Y, Yang
C, Yu H, et al. (2016) Brain-specific Crmp2 deletion leads to neuronal
development deficits and behavioral impairments in mice. Nature Communications
7: 11773.
35. Rogemond V, Auger C, Giraudon
P, Becchi M, Auvergnon N, et al. (2008) Processing and nuclear localization of
CRMP2 during brain development induce neurite outgrowth inhibition. J Biol Chem
283: 14751-14761.
36. Arimura N, Ménager C, Kawano
Y, Yoshimura T, Kawabata S, et al. (2005) Phosphorylation by Rho kinase
regulates CRMP-2 activity in growth cones. Mol Cell Biol 25: 9973-9984.
37. Cheng X, Yang YL, Li WH, Liu
M, Zhnag SS, et al. (2020) Dynamic alterations of brain injury, functional
recovery and metabolites profile after cerebral ischemia/reperfusion in rats
contributes to potential biomarkers. J Mol Neurosci (in press).
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