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Evidence for a non-canonical JAK/STAT signaling pathway in the synthesis of the brain’s major ion channels and neurotransmitter receptors

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Background: Brain-derived neurotrophic factor (BDNF) is a major signaling molecule that the brain uses to control a vast network of intracellular cascades fundamental to properties of learning and memory, and cognition. While much is known about BDNF signaling in the healthy nervous system where it controls the mitogen activated protein kinase (MAPK) and cyclic-AMP pathways, less is known about its role in multiple brain disorders where it contributes to the dysregulated neuroplasticity seen in epilepsy and traumatic brain injury (TBI). We previously found that neurons respond to prolonged BDNF exposure (both in vivo (in models of epilepsy and TBI) and in vitro (in BDNF treated primary neuronal cultures)) by activating the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway. This pathway is best known for its association with inflammatory cytokines in non-neuronal cells. Results: Here, using deep RNA-sequencing of neurons exposed to BDNF in the presence and absence of well characterized JAK/STAT inhibitors, and without non-neuronal cells, we determine the BDNF transcriptome that is specifically regulated by agents that inhibit JAK/STAT signaling. Surprisingly, the BDNF-induced JAK/STAT transcriptome contains ion channels and neurotransmitter receptors coming from all the major classes expressed in the brain, along with key modulators of synaptic plasticity, neurogenesis, and axonal remodeling. Analysis of this dataset has revealed a unique non-canonical mechanism of JAK/STATs in neurons as differential gene expression mediated by STAT3 is not solely dependent upon phosphorylation at residue 705 and may involve a BDNF-induced interaction of STAT3 with Heterochromatin Protein 1 alpha (HP1α). Conclusions: These findings suggest that the neuronal BDNF-induced JAK/STAT pathway involves more than STAT3 phosphorylation at 705, providing the first evidence for a non-canonical mechanism that may involve HP1α. Our analysis reveals that JAK/STAT signaling regulates many of the genes associated with epilepsy syndromes where BDNF levels are markedly elevated. Uncovering the mechanism of this novel form of BDNF signaling in the brain may provide a new direction for epilepsy therapeutics and open a window into the complex mechanisms of STAT3 transcriptional regulation in neurological disease.
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R E S E A R C H A R T I C L E Open Access
Evidence for a non-canonical JAK/STAT
signaling pathway in the synthesis of the
brains major ion channels and
neurotransmitter receptors
Kathryn M. Hixson
1,2
, Meaghan Cogswell
1
, Amy R. Brooks-Kayal
3
and Shelley J. Russek
1,2,4*
Abstract
Background: Brain-derived neurotrophic factor (BDNF) is a major signaling molecule that the brain uses to control
a vast network of intracellular cascades fundamental to properties of learning and memory, and cognition. While
much is known about BDNF signaling in the healthy nervous system where it controls the mitogen activated
protein kinase (MAPK) and cyclic-AMP pathways, less is known about its role in multiple brain disorders where it
contributes to the dysregulated neuroplasticity seen in epilepsy and traumatic brain injury (TBI). We previously
found that neurons respond to prolonged BDNF exposure (both in vivo (in models of epilepsy and TBI) and in vitro
(in BDNF treated primary neuronal cultures)) by activating the Janus Kinase/Signal Transducer and Activator of
Transcription (JAK/STAT) signaling pathway. This pathway is best known for its association with inflammatory
cytokines in non-neuronal cells.
Results: Here, using deep RNA-sequencing of neurons exposed to BDNF in the presence and absence of well
characterized JAK/STAT inhibitors, and without non-neuronal cells, we determine the BDNF transcriptome that is
specifically regulated by agents that inhibit JAK/STAT signaling. Surprisingly, the BDNF-induced JAK/STAT
transcriptome contains ion channels and neurotransmitter receptors coming from all the major classes expressed in
the brain, along with key modulators of synaptic plasticity, neurogenesis, and axonal remodeling. Analysis of this
dataset has revealed a unique non-canonical mechanism of JAK/STATs in neurons as differential gene expression
mediated by STAT3 is not solely dependent upon phosphorylation at residue 705 and may involve a BDNF-induced
interaction of STAT3 with Heterochromatin Protein 1 alpha (HP1α).
Conclusions: These findings suggest that the neuronal BDNF-induced JAK/STAT pathway involves more than
STAT3 phosphorylation at 705, providing the first evidence for a non-canonical mechanism that may involve HP1α.
Our analysis reveals that JAK/STAT signaling regulates many of the genes associated with epilepsy syndromes
where BDNF levels are markedly elevated. Uncovering the mechanism of this novel form of BDNF signaling in the
brain may provide a new direction for epilepsy therapeutics and open a window into the complex mechanisms of
STAT3 transcriptional regulation in neurological disease.
Keywords: JAK/STAT, Neurons, RNAseq, BDNF, HP1α, Epilepsy
© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
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* Correspondence: srussek@bu.edu
1
Laboratory of Translational Epilepsy, Department of Pharmacology &
Experimental Therapeutics, Boston University School of Medicine (BUSM),
Boston, USA
2
Graduate Program for Neuroscience (GPN), Boston University (BU), Boston,
USA
Full list of author information is available at the end of the article
Hixson et al. BMC Genomics (2019) 20:677
https://doi.org/10.1186/s12864-019-6033-2
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Background
In the injured brain, it has been suggested that neuro-
genesis [1], mossy fiber sprouting [2], and hippocampal
cell death [3] contribute to aberrant neuronal circuit
reorganization. One manifestation of this reorganization
is thought to be altered long-term potentiation (LTP)-in-
duced synaptic plasticity [4], which is associated with al-
tered levels of particular neurotransmitter receptors and
ion channels [5,6]. As the functional properties of neu-
rons change, a state of unchecked overexcitation may
occur and is accompanied by decreased GABA-mediated
inhibition [7]. Many believe that a major mechanism for
this altered brain plasticity is the increased synthesis and
release of Brain-derived Neurotrophic Factor (BDNF), a
major brain signaling molecule that plays an important
role throughout life. BDNF is a neurotrophin ubiqui-
tously expressed in the whole brain with a major role in
activity-dependent alterations in neuronal morphology
and synaptogenesis. Protein and mRNA levels of BDNF
are markedly increased after seizures in brain regions in-
volved in epileptogenesis [8,9] and mutations in BDNF
can reduce the development of spontaneous seizures in
animal models [10]. Moreover, a conditional deletion of
the high-affinity BDNF receptor TrkB in a subset of neu-
rons, is sufficient to completely eliminate all behavioral
evidence of the progression of epilepsy in the kindling
model [11] and it has been shown that enhanced TrkB
signaling can exacerbate epileptogenesis [12].
In addition to the TrkB-mediated BDNF response, the
p75 Neurotrophin Receptor (p75NTR, also referred to as
NGFR) is a low-affinity receptor for the cleaved mature
form of BDNF while a high-affinity receptor for the
uncleaved form, proBDNF. p75NTR levels are elevated
after Status Epilepticus (SE) in animals and increased ac-
tivation by proBDNF, like mature BDNF, increases sus-
ceptibility to seizures [13,14]. Interestingly, our labs
have shown that increased synthesis of proBDNF, and
not mature BDNF, is the first response of the injured
brain in the pilocarpine (PILO) model of epilepsy prior
to SE-induced increases of p75NTR [15]. This finding
suggests that high levels of proBDNF may exert their ef-
fects through TrkB rather than p75NTR.
In the normal brain, BDNF activation of its recep-
tors regulates multiple signaling pathways: mitogen-
activated protein kinase/extracellular signal-regulated
protein kinase (MAPK/ERK), phospholipase Cγ
(PLCγ), phosphoinositide 3-kinase (PI3K), c-Jun N-
terminal kinase (JNK), and NFkB [16]. It is well
established that BDNF modulates long-term potenti-
ation (LTP) at Schaffer collateral-CA1 and mossy
fiber-CA3 hippocampal synapses by acting through
TrkB [1721]. Although p75NTR is not implicated in
LTP, it plays a significant role in learning and mem-
ory through its modulation of hippocampal long-term
depression (LTD) by altering AMPA receptor expres-
sion [22].
Studies from our laboratories demonstrate that in
addition to the signaling pathways described above, in-
creased levels of BDNF activate the Janus kinase/signal
transducer and activator of transcription (JAK/STAT)
pathway both in vivo, in the rat PILO model of epilepsy,
and in vitro, in BDNF-treated primary cultured neurons
[23]. The JAK/STAT pathway is a signaling cascade that
has a prominent role in immune function and cancer de-
velopment. The canonical pathway involves the binding
of JAK to its target (such as a cytokine or hormone re-
ceptor), subsequent JAK phosphorylation that stimulates
the recruitment and activation of STAT proteins (phos-
pho-STAT (p-STAT)) and their movement as dimers
into the nucleus where they function as transcriptional
activators. Although JAK/STATs regulate transcription
through this canonical pathway, more recently, non-ca-
nonical JAK/STAT signaling has been discovered where
JAKs can act independently of STATs to regulate tran-
scription in the nucleus, and STATs can function inde-
pendently of their phosphorylation state to maintain
genome stability via interactions with heterochromatin
protein 1 (HP1) [24]. Increasing evidence also suggests
that HP1 may be present at euchromatin as well as het-
erochromatin and involved in gene transcription [25,
26]. While the role, and mechanism, of JAK/STAT sig-
naling in the brain is still not completely understood, it
is clear that it is an important part of how the brain reg-
ulates its synaptic connections [27].
Research from our laboratory has demonstrated that
activation of the JAK/STAT pathway results in the ex-
pression of the inducible cAMP early repressor (ICER)
via STAT3-mediated gene regulation. Moreover, we
show that ICER represses the gene (Gabra1/GABRA1)
coding for the α1 subunit of the type A γ-aminobutyric
acid (GABA) receptor (GABAR), the major inhibitory
neurotransmitter receptor in the brain, and that such re-
pression reduces the number of α1 containing GABARs
in neurons [28]. Most importantly, reduced α1 subunit
gene expression also occurs in epilepsy patients [5]as
well as in other disorders of the nervous system [2931].
In addition, use of the JAK/STAT inhibitor, WP1066, in
the PILO rat model of epilepsy reduces the number of
spontaneous seizures after the latent period [32]. We
now hypothesize that in addition to controlling the gene
regulation of Gabra1/GABRA1, the BDNF-induced JAK/
STAT pathway controls the expression of diverse gene
products that are involved in synaptic plasticity as well
as in the epileptogenic process that follows brain injury.
We also hypothesize that attenuating this pathway may
provide a gateway to new treatments for refractory epi-
lepsy and other brain disorders that display dysregulated
neuroplasticity.
Hixson et al. BMC Genomics (2019) 20:677 Page 2 of 16
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To test these hypotheses, and to gain a comprehen-
sive understanding of the genome impacted by BDNF-
induced JAK/STAT activation, we exposed primary cul-
tured neurons to BDNF with and without JAK/STAT
inhibitors and performed deep RNA-sequencing (RNA-
seq) in order to determine the gene set within the
BDNF response that could be ascribed to JAK/STAT
activation (as defined by WP1066 and Ruxolitinib
(Ruxo), a potent inhibitor of JAK1/2 activation [33]).
We now report evidence that suggests BDNF acts
through the JAK/STAT pathway to execute changes in:
the expression of ion channels, neurotransmitter and
GPCR receptors, and the expression of key modulators
of synaptic plasticity, neurogenesis, and axonal remod-
eling. Many of these functions were previously ascribed
to BDNF, however, the role of the JAK/STAT pathway
in their manifestation has never been previously ex-
plored. Our studies also reveal the presence of many
new BDNF target genes providing a broader landscape
for BDNF mediated gene regulation of the healthy and
impaired nervous system.
Results
BDNF signaling and the identification of epilepsy-
associated gene networks
Primary cultured neurons were treated with BDNF (0.7
nM, a physiologically relevant level) or vehicle control
(water) to determine the genome response to prolonged
receptor activation (4 h) (Fig. 1). Using RNA-sequencing
for open discovery, we identified a total of 2869 differen-
tially expressed genes (DEGs) whose levels change (in-
crease or decrease) in response to BDNF signaling
(Fig. 2a) (CTRL vs V + B gene list, Additional file 5). In-
spection of the gene set using Ingenuity Pathway Ana-
lysis (IPA) [34] revealed that only 83 of these genes have
been previously associated with BDNF (as determined
using the Ingenuity Knowledge Base, a curated database
of all historical scientific information in IPA databases),
suggesting that the majority of genes identified in this
study are novel in their association with the BDNF sig-
naling pathway. A subset of 194 potential BDNF regu-
lated genes are associated with seizure disorders
(BDNF and epilepsy, Additional file 5), an umbrella
A
C
B
Fig. 1 Schematic of experimental design and analysis.aTimeline representation of the drug treatment protocol, including the 1 h drug or DMSO
(vehicle, V) pretreatment (initiated at 1 h), administration of aqueous BDNF (B) or water (vehicle) treatment (Time = 0 h) and time of cell
collection and RNA extraction (4 h). b6-well plate showing each one of the 5 treatment groups and the abbreviations by which they will be
referred to in the text: DMSO+Water (CTRL), DMSO+BDNF (V + B), 100 nM Ruxolitinib+BDNF (RX1 + B), 10 μM Ruxolitinib+BDNF (RX2 + B), 10 μM
WP1066 + BDNF (WP + B). cDiagram represents the approach taken to identify differential gene expression (DEG) in response to JAK/STAT
pathway inhibition. BDNF through its receptors activates multiple signaling pathways (represented by blue arrows) that impact transcription.
Differential expression analysis comparing V + B vs. CTRL treatment groups (green box) will reveal the total set of genes that are regulated by
BDNF-induced intracellular signaling pathways. To identify BDNF DEGs that are specific to JAK/STAT signaling, comparisons are made between
the groups pretreated with JAK inhibitors (RX1 + B, RX2 + B, WP + B) and the group pretreated with vehicle (V + B) (purple box); DEGs from this
comparison (where JAK/STAT inhibition reverses BDNF stimulation or inhibition) are thought to be associated with the JAK/STAT pathway
Hixson et al. BMC Genomics (2019) 20:677 Page 3 of 16
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category in IPA that includes all epilepsy classes as rep-
resented in the curated knowledge base. Moreover, seiz-
ure disorder is the primary most significant neurological
disorder identified by enrichment analysis of the gene
pool (p= 3.29E-27).
In addition to the 194 epilepsy-associated genes, path-
way analysis suggests that increased levels of BDNF al-
ters the expression of essential neurobiological gene sets
that are required for brain development and that are
dysregulated during epileptogenesis in animal models
and human patients. For instance, the differentially
expressed set of transcripts from BDNF-treated primary
neurons is highly enriched for genes essential to axonal
guidance (p= 1.09E-11); further, there is a striking en-
richment that is specific to all major classes of ion chan-
nels and neurotransmitter/neuropeptide receptors, with
133 of 599 total genes related to one of these categories
regulated by prolonged BDNF exposure (4 h) (p= 2.66E-
11) (Fig. 2b). Specifically, the differentially expressed
transcripts for receptors include those activated by
GABA, glutamate, acetylcholine, dopamine, opioids,
serotonin, galanin, and neuropeptide Y, covering both
metabotropic and ionotropic receptor subtypes. Most
classes of ion channel genes are also represented, includ-
ing those for potassium, sodium, and calcium, where
mutations have been shown to underlie specific genetic
epilepsies [3537]. There is also a specific decrease (1.73
fold) in the expression of Gabra1 that is a member of
the GABAR gene cluster which we have previously dem-
onstrated to be regulated by BDNF using qRT-PCR in
extracts of BDNF-treated primary neurons and hippo-
campal tissue extracted from the in vivo PILO model of
temporal lobe epilepsy [23]. As expected, the neuronal
RNA-seq dataset revealed that prolonged exposure to
BDNF increases BDNF transcript levels, a finding that is
well established in the literature [38]. Given our previous
identification of a novel JAK/STAT pathway in neurons
that is regulated by BDNF, we asked whether any
A
C
B
Fig. 2 JAK/STAT inhibitors reverse BDNF-induced gene expression. aTOP: Venn diagram representation of all protein-coding genes in the rat
genome (in accordance with Strand NGS notation) with all differentially expressed genes (DEGs) between primary neurons treated with
DMSO+BDNF vs. DMSO+Water. BOTTOM: Venn diagram representation of all DEGs in DMSO+Water vs. DMSO+BDNF (BDNF, Red, 2869),
WP1066 + BDNF vs. DMSO+BDNF (WP1066,Orange, 5170 total) and 10 μM Ruxo+BDNF vs. DMSO+BDNF (RX2, Blue, 5393 total genes). bReceptor
and Ion Channel expression is altered by BDNF and rescued by JAK inhibition. TOP: List of Ion Channels and Receptor Subunits whose expression
is altered by 4-h BDNF treatment. Color represents direction and degree of fold change (Red: up, Green: down) sorted by receptor or channel
type. BOTTOM: Receptor and Ion Channel reversal in expression by addition of WP or RX2 (white receptors are not affected by JAK/STAT
inhibitors). Response to BDNF in presence of WP was used as the basis for coloring receptors depicted in the diagram. Red: Upregulated, Green:
Downregulated. cHeatmap of all DEGs (columns) associated with Epilepsy (IPA) that are affected by exposure to BDNF. DW: DMSO+Water, DB:
DMSO+BDNF, RX1: 100 nM Ruxo+BDNF, RX2:10 μM Ruxo+BDNF, WP: 10 μM WP1066 + BDNF. Green: low expression, red: high expression
Hixson et al. BMC Genomics (2019) 20:677 Page 4 of 16
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members of the JAK/STAT pathway might be regulated
at a transcriptional level in response to BDNF receptor
activation. Our dataset indicates significant enrichment
for transcripts linked to JAK/STAT signaling (p= 6.03E-
5) and in particular, an enrichment of Jak1 (1.6-fold en-
richment) and Jak2 (1.8-fold enrichment).
Neuropharmacological dissection of BDNF-induced JAK/
STAT signaling reveals a non-canonical pathway in
neurons
Primary cultured neurons were pre-treated with agents
known to inhibit the JAK/STAT pathway (Fig. 1)by
interfering with JAK kinase activity, either directly by
inhibiting JAK phosphorylation or stimulating JAK deg-
radation. Two different antagonists were employed in
these studies: the small molecule WP1066 that has been
shown to inhibit JAK2/STAT3 signaling and degrade
total JAK2 [39] and Ruxolitinib (Ruxo), currently in use
as a cancer therapeutic, which acts on JAK1 and JAK2
by forming two hydrogen bonds with the hinge region of
the JAK protein (the segment that connects the N-lobe
to the C-lobe of the kinase domain) and reduces phos-
phorylation of the JAK2 protein at Tyr1007/1008 [33].
Based on our earlier discovery that siRNA knockdown of
STAT3 reverses BDNF-induced downregulation in the
levels of Gabra1 subunit mRNAs [23], as does WP1066,
we used RNA-seq to identify the full set of genes re-
sponsive to JAK inhibition. This strategy enables us to
unmask those genes in the BDNF transcriptome that
may be JAK/STAT dependent.
Comparing the transcriptomes BDNF (V + B) or 100
nM Ruxo + BDNF (RX1 + B), we were surprised to find
that there was no difference between these two tran-
scriptomes, as statistically assessed using DSEQ2 (0
DEGs) (FDR = 0.05, Wald test). To make sure that Ruxo
treatment at 100 nM was active in the primary cultures
of our studies, Western blot was performed using the
same drug stock as used in cultures that generated the
RNA-seq libraries. In this control experiment, Ruxo
(100 nM) potently inhibited STAT3 phosphorylation at
A
C
B
Fig. 3 RX1 and RX2 reduce levels of phospho-STAT3 but only RX2 blocks ICER induction.aRepresentative Western Blot analysis of whole-cell
protein extracts from primary neurons 9 DIV pretreated with100nM Ruxolitinib (RX1), 10uM Ruxo (RX2), or DMSO for 1 h before the addition of
BDNF. Cells were collected 30 min after BDNF administration and probed with anti pSTAT3, STAT3 and β-Actin. bQuantitation of signals from
densitometry is displayed as mean percent change (±SEM) relative to the DMSO+Water control group (**p < 0.01). Ruxolitinib significantly
reduces the levels of P-STAT3 with no change in total STAT3. cGraphical representation of results from real-time PCR analysis using specific
Taqman probe and primers. RNA was extracted from cells collected 4 h after BDNF administration. Transcript levels are shown as the mean values
(±SEM) of the ratio relative to the DMSO+Water control group for Icer n = 6. Note, that while RX1 blocks pSTAT3, it does not block the effect of
BDNF treatment on ICER induction, while WP and RX2 do
Hixson et al. BMC Genomics (2019) 20:677 Page 5 of 16
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30 min (80% + 3.07 reduction of BDNF signal) and at
4 h (73% reduction of BDNF signal, Fig. 3). Therefore,
despite reducing STAT3 phosphorylation at Tyr 705,
our data taken together demonstrate that 100 nM Ruxo
does not alter BDNF-induced gene regulation, including
changes in ICER expression (Fig. 3c). This finding sug-
gests that the BDNF-induced JAK/STAT pathway is not
STAT3 phosphorylation-dependent even though knock-
down of STAT3 with siRNAs prevents ICER induction
and Gabra1 downregulation [23]. Note that in our
current studies BDNF does not significantly increase
STAT3 phosphorylation but Ruxo still inhibits the basal
state of its activation. These observations favor the
hypothesis that neurons use a non-canonical mechanism
of JAK/STAT signaling which may be relevant to epi-
lepsy, and potentially to learning and memory where
BDNF is a critical signaling molecule, but require further
experimentation.
Using pharmacology to reduce the dimensionality of the
dataset and identify potential BDNF-induced JAK/STAT
target genes
While we discovered that co-treatment of neurons with
BDNF (V + B) vs. RX1 + B produced similar transcrip-
tomes, we found that a marked increase in the
concentration of Ruxo (10 μM) plus BDNF (RX2 + B) at-
tenuated the expression of multiple genes that were al-
tered in response to BDNF, and most importantly, those
that we previously identified in the PILO model of epi-
lepsy. We next compared the RX2 DEG as determined
by DESEQ2 (FDR = 0.05, Wald test) to those generated
from cultures treated with 10 μM WP1066 plus BDNF
(WP + B) and generated a subset of genes whose BDNF-
induced expression was reversed back to vehicle control
by both drug treatments. Using these JAK/STAT inhibi-
tors as probes for unmasking potential genes that are
regulated by the BDNF-induced JAK/STAT pathway, we
identified 1559 of the 2659 BDNF target genes that may
be most associated with JAK/STAT signaling in neurons.
Interrogation of IPA networks specific to this shared
gene set revealed that 131 genes are epilepsy-related and
seizure disorder is still the top most significant neuro-
logical disease (P= 7.31E-22). Considering that 131 of
the 194 BDNF-regulated epilepsy-linked genes are re-
versed by JAK/STAT inhibitors (68%), it suggests that
JAK/STAT signaling may be a large component of
BDNF-related epileptogenesis. This can be best appreci-
ated by looking at the heat map displayed in Fig. 2c
which depicts the expression levels of all 194 epilepsy-
linked genes that are changed by BDNF treatment. The
results clearly show that WP + B (10 μM) and Ruxo
(RX2 + B, 10 μM) block BDNF-induced changes in neur-
onal gene expression for epilepsy-linked genes and both
appear to have a similar gene expression signature to
that of DMSO+Water control. Note that Ruxo at its
lower concentration (RX1 + B, 100 nM) looks similar to
BDNF alone.
Gene ontology and pathway analysis
Gene Ontology (GO) term analysis of the WP + B/RX2 +
B dataset, performed using EnrichR [40,41], reveals that
the 1559 overlapping genes are significantly associated
with molecular functions related to receptor and channel
activity (found in 8/10 top molecular functions) (Fig. 4a,
top panel). All of the top 10 biological process GO terms
include the words synaptic transmission(Fig. 4a, mid-
dle panel). These consistent findings strongly suggest
that both WP and Ruxo have the capacity to regulate
synaptic transmission at the gene expression level in the
brain. When compared with the total list of receptor/ion
channel related genes that were regulated by BDNF (as
depicted in Fig. 2b, top panel), 107/133 genes (80%) were
reversed by treatment with JAK/STAT inhibitors (Fig. 2b,
bottom panel). This is best seen by the color reversal in
the receptors for all but a few that were not significantly
affected by pre-treatment with the inhibitor prior to
BDNF activation (displayed in white).
In addition, neurotransmitter receptors, axonal guid-
ance, GPCR and c-AMP signaling were found to be
amongst the top KEGG pathways associated with the
shared gene set of WP + B and RX2 + B (Fig. 4a, bottom
panel). Members of the JAK/STAT signaling pathway
were also identified as significant gene targets (P=
1.044E-3) suggesting that BDNF may also participate in
the feedback regulation of the pathway. Figure 4bisa
schematic representation showing many of the compo-
nent molecules that make up the JAK/STAT pathway,
with expression of all the colored genes being affected
by BDNF treatments and all the ones with a purple
border reversed by JAK/STAT inhibitor treatments
WP + B and RX2 + B.
In addition to the significant relationship to epilepsy
identified by IPA (see Additional file 1: Table S1), the
overlapping WP/RX2 gene set had a significant link to
neuroinflammation despite the neuronal cultures being
largely devoid of any glial cells, 41 genes of the BDNF-
induced set of 56 neuroinflammation-linked genes were
reversed by WP + B or RX2 + B treatment (73% of the
gene set) (Fig. 4c).
Functional relationships between genes as described in
the literature
Pathway and GO analysis identified the following epi-
leptogenic processes, in addition to inflammation, as
being enriched in the WP + B/RX2 + B overlapping data
set: synaptic plasticity, receptors and ion channels, epi-
lepsy, transcription factors, proliferation, and neurogen-
esis. In order to visualize the functional connectivity of
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the genes involved in epileptogenesis, we created a func-
tional network of the most significant genes as deter-
mined by a p-value of < 1 × 10^10 and a fold-change > 2
(Additional file 3: Figure S1). Genes that met these con-
ditions were separated into the lists of the epileptogenic
processes, as described above. In order to relate these
genes to one another and their epileptogenic functions,
we created a network that portrays their function, ex-
pression fold change, direction, and functional protein
relationships in Cytoscape [42](Fig. 5a). Using GENEMa-
nia, a Cytoscape tool containing a database of experi-
mental data showing protein-protein interactions [43],
we determined potential functional relationships. Each
node in the network represents a gene while the edges
represents a protein interaction. Node color is deter-
mined by the epileptogenic category. Due to the inter-
play between these functions, many genes within one
category overlap with another category. For instance,
Egr3 plays a role in 5 of the 6 categories: synaptic
plasticity, epilepsy, transcription factors, proliferation,
and neurogenesis. The color in this case is determined
by whichever category comes first in this list: synaptic
plasticity, receptors and ion channels, epilepsy, transcrip-
tion factors, proliferation, and neurogenesis, where the
order is determined by the number of genes in that cat-
egory from smallest to largest. This was done to avoid
having the majority of the nodes colored for the largest
category (neurogenesis). For Egr3, it is yellow represent-
ing synaptic plasticity, however, its location in the net-
work is near the center because the proximity of the
node to other categories represents its link to those sec-
ondary categories. In addition, the Venn diagram in 5B
also shows the degree of overlap for each category. For
instance, there are 108 total genes related to receptors
and ion channels. In the list, 61 of those did not belong
to any other category, 11 were also linked to neurogen-
esis, 5 to receptors, neurogenesis and proliferation,14 to
receptors and epilepsy, 6 to receptors, epilepsy and
AB
C
Fig. 4 Pathway Enrichment and Gene Ontology analysis of the overlapping RX2/WP dataset. aPathways and functions enriched in the list of
1559 genes altered by WP and 10 μM Ruxo (RX2). Gene Ontology and KEGG enrichment analysis performed using EnrichR. bGenes involved in
the canonical JAK/STAT signaling pathway with functional representation. Genes are colored by degree of fold change 9 with BDNF alone (see
panel legend). Purple border signifies that WP + B and RX2 + B reverse effects of BDNF on gene expression. cList of DEGs involved in
neuroinflammatory signaling and degree of fold change of WP + B vs V + B. Listed in order of FC most negative to most positive
Hixson et al. BMC Genomics (2019) 20:677 Page 7 of 16
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neurogenesis, and 2 to receptors, neurogenesis, epilepsy
and proliferation.
Differentially expressed genes exclusive to BDNF/RX2
contain a rich set of epilepsy-associated targets not
present in the WP1066 exclusive list
To further dissect the regulatory contribution of the
two JAK/STAT inhibitors, we examined the list of
DEGs (as determined by DESEQ2, FDR = 0.05, Wald
test) that differed between WP + B and RX2 + B treat-
ments. Four hundred seventeen DEGs are reversed by
RX2 + B treatment that are absent with WP + B and
610 DEGs are reversed by WP + B that are not by
RX2 + B (Fig. 2a, bottom panel). Interestingly, the
genes whose expression is exclusively reversed by
RX2 + B are significantly related to neurodegeneration,
and brain-related movement disorders (i.e., Hunting-
tons disease) as well as to epilepsy. On the other
hand, those exclusively regulated by WP + B were not
highly associated with epilepsy or other brain disor-
ders. Additional file 4: Figure S2 contains the EnrichR
generated KEGG pathway analysis for this gene set (Add-
itional file 4: Figure S2A). Nine of the 10 top related ca-
nonical pathways differ from the KEGG list of overlapping
WP/RX2 DEGs (Fig. 4b). Top genes involved in the KEGG
pathways listed are also shown in a clustergram (Add-
itional file 4: Figure S2B). The list of genes exclusively
regulated by RX2+ B that are linked to epilepsy, Hunting-
tons disease, and neurodegeneration (E) are also pre-
sented (Additional file 4: Figure S2C-E).
qRT-PCR gene validation of datasets
Many of the genes identified in these RNA-seq studies
were originally reported by us in the literature (ICER,
Gabra1,Egr3), using a variety of molecular approaches
[23,32,44]. To further validate the new datasets, we gen-
erated an additional set of cultures, originating from the
embryos of multiple pregnant rats, and chose the follow-
ing genes for qRT-PCR because of their interest in the
field of epilepsy: Dopamine Receptor D5 (Drd5), Galinin
Receptor 1 (Galr1), γ2 subunit of the GABAR (Gabrg2),
Glutamate metabotropic receptor 1 (Grm1), and their re-
lationship to the JAK/STAT pathway: Inducible cAMP
early repressor (Icer), myeloid leukemia cell differentiation
protein (Mcl1), Suppressor Of Cytokine Signaling 3
(Socs3), Cyclin D1 (Ccnd1), and C-X-C Motif Chemokine
Receptor 4 (Cxcr4) (Fig. 6). In most cases, findings from
qRT-PCR were consistent with those of RNA-seq. Quanti-
tated results from select validations are shown (Fig. 6a-d).
A results table for all candidates where validation was per-
formed is included in Fig. 6e. All significance was deter-
mined using One-Way ANOVA followed by Tukeystest
for multiple comparisons.
Fig. 5 Functional Network of DEGs involved in epileptogenic-related processes.aNode color represents primary category, determined by order
indicated in panel legend. Node size indicates degree of fold change (FC). Border color indicates direction of FC. Gray lines show functional
connectivity determined by GeneMania in Cytoscape. Location in network roughly determined by category and multiple associations with
neighboring and other categories. (Genes located near the center are involved in multiple categories). bVenn diagram of genes contained in
network diagram in A demonstrating overlap of the genes in each category, neurogenesis (blue), receptors (red), epilepsy (gray), synaptic
plasticity (yellow), and proliferation (green). Gene associations provided by IPA
Hixson et al. BMC Genomics (2019) 20:677 Page 8 of 16
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Comparison of DEGs with previously published ChIP-seq
datasets
Using a published set of validated STAT3 targets from
chromatin immunoprecipitation (ChIP)-sequencing (ChIP-
seq) assays, performed with glioblastoma cells [45], we
generated a list of 308 genes that were also present in
our WP + B/RX2 + B datasets (Fig. 7). Of particular
interest are BDNF, Calcium Voltage-Gated Channel
Auxiliary Subunit Beta 4 (Cacnb4),Gabrg2,Grm1,
Jak1/2, and Sodium Voltage-Gated Channel Alpha Sub-
unit 1(Scn1a). These genes have all been associated
with epilepsy models and with human mutations in epi-
lepsy patients [35,4648].
HP1αassociation with STAT3 in neurons
As a first step to uncover the non-canonical mechanism
underlying BDNF-induced JAK/STAT signaling in neu-
rons, we asked whether BDNF treatment alters the asso-
ciation of STAT3 with Heterochromatin Protein 1 Alpha
(HP1α), a mediator of gene silencing that is found in
heterochromatin. An association of STAT3 with HP1α
has been previously reported in the literature [49] and
we expected to find that BDNF might decrease
association thereby releasing the DNA for the subse-
quent binding of transcription factors and transcrip-
tional elongation. To our surprise, however, BDNF
increased association of the two proteins which was
blocked by the JAK/STAT inhibitors WP and RX2
(Fig. 8).
Discussion
There is a strong body of evidence suggesting that BDNF
signaling through its TrkB receptor contributes in a
major way to the development of TLE, as suggested by
animal models of epilepsy [11,50] and their potential
relationship to human epilepsy brain pathology [51].
BDNF plays a critical role in the cell biological processes
that regulate the structural plasticity of dentate granule
cells and the enhanced excitation associated with
changes in LTP and compromised GABA-mediated in-
hibition [17,46,50,52]. Although the role BDNF plays
in brain disorders is better understood now than at the
time of its discovery, many key aspects of its complex
regulatory programs remain a mystery. We designed our
study to gather a comprehensive list of genes whose
expression is either upregulated or downregulated by
Fig. 6 qRT-PCR validation of BDNF regulated genes in presence of JAK/STAT inhibitors.Primary neurons 9 DIV were pretreated with100nM Ruxolitinib
(RX1), 10uM Ruxo (RX2), 10uM WP1066 (WP) or DMSO for 1 h before the addition of BDNF. a-dGraphical representation of selected results of real time
PCR analysis using Taqman probe and primers. RNA was extracted from cells collected 4 h after BDNF administration. mRNA levels are shown as the
mean values (±SEM) of the ratio relative to DMSO+Water control group for aGalr1 n=3,bDrd5 n=3,cGrm1 n=4, dGabrg2 n=4. eCompilation
of all genes tested for validation with qRT-PCR represented alphabetically. Values are the ratio relative to DMSO+Water for RNAseq (top row) and qRT-
PCR (2nd row) for comparison. Indication of a validating result (3rd row) is marked in green. Results marked with Yes(Y) followed by the level of
significance indicate a significant result consistent with RNA-seq. Yes- Not significant (Y-NS) seen in all the RX1 is the expected result consistent with
our RNA-seq findings. Measurements that did not reach significance are marked in gray as Not Significant (NS). Significance was tested using One-Way
ANOVA followed by Tukeys test for multiple comparisons. (*p< 0.05, **p< 0.01, ***p< 0.0005, ** **p<.0001)
Hixson et al. BMC Genomics (2019) 20:677 Page 9 of 16
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increased exposure to BDNF, as is believed to occur in
response to seizures and other brain insults such as trau-
matic brain injury [53].
The use of primary rat neurons treated with recombin-
ant BDNF allows us to pinpoint the effects of BDNF
without the background of additional signaling pathways
that may be activated in TLE models. Our study provides
a comprehensive list of the changes in the transcriptome
that occur in response to a relatively short but continuous
exposure to BDNF (4 h) at a concentration of 0.7 nM that
Fig. 7 The WP + B/RX2 + B overlapping datasets contain previously validated STAT3 ChIP-sequencing target genes. List of all WP + B/RX2 + B
genes (308) that are validated STAT3 ChIP-seq targets (as determined by Zhang, et.al, 2013) in alphabetical order. Highlighted in yellow are genes
of special interest
Fig. 8 Binding of HP1 to STAT3 is enhanced by BDNF signaling in neurons. aRepresentative Western Blot image of coimmunoprecipitation
assays using whole-cell extracts of 9-DIV primary cortical neurons treated (30 min) with Water or 0.7 nM BDNF. Protein was precipitated with a
specific HP1 antibody (Cell Signaling Technologies #2616) and the elution analyzed by SDS-PAGE. Membrane was immunoblotted with an anti-
STAT3 antibody (Cell Signaling Technologies #4904) to detect co-association of HP1 with STAT3, (n= 4). bQuantitation of STAT3 association
with HP1 from dataset in A.cRepresentative Western Blot image: Inhibitors of JAK/STAT signaling (WP1066 (WP) and Ruxolitinib 2 (RX2))
reduce HP1αantibody precipitation of STAT3. Data was normalized to βactin levels which did not change upon BDNF treatment, * = p< 0.05
Hixson et al. BMC Genomics (2019) 20:677 Page 10 of 16
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is relevant to endogenous levels in neurons that activate
TrKB receptors and not p75NTR (unpublished data from
our laboratory). A collection of the BDNF regulated genes
within the list have been previously identified both in
treated cultures and in vivo by our laboratory and others.
They include Gabra1, the inducible cAMP early repressor
(ICER) (which negatively regulates Gabra1) [23], BDNF it-
self [38], and multiple ion channel genes, as well as genes
that code for important markers of neurogenesis and cell
proliferation [54,55].
In addition to being consistent with the findings in the
literature, our results reveal new BDNF-induced gene
targets that are linked to epilepsy. Continuous exposure
to BDNF alters the expression of 194 epilepsy-linked
genes identified by IPA. The following genes are of par-
ticular interest to us because of their marked level of
transcriptional alteration and their involvement in
epilepsy:
Dopamine receptor D5 (Drd5)- A D1-type dopamine
receptor whose knockdown has been shown to be
protective against seizures [56]. In our dataset, the
neuronal expression of Drd5 increases nearly 15-fold
in response to BDNF treatment.
Galinin receptor 1 (Galr1)- Galanin is a
neuropeptide in the brain that inhibits glutamate
release and galanin agonists delivered to the brain
can inhibit seizures [57]. Our dataset reveals a 35-
fold increase in Galr1 expression, demonstrating the
complex medley of both protective and pathogenic
changes in genome expression that are invoked by
increasing levels of neuronal BDNF.
Glutamate metabotropic receptor 1 (Grm1)- Grm1
has been associated with the alterations in synaptic
plasticity and glutamate disturbances that result in
neuronal overexcitation, a hallmark of epilepsy [48,
58]. Grm1 mutations can also cause spinocerebellar
ataxia type 44, a genetic disease that can present
with seizures. In our dataset, BDNF treatment led to
a 2.45-fold increase in the expression of Grm1.
γ2 subunit of the GABAR (Gabrg2)- the Gabrg2
subunit of GABAR is important for benzodiazepine
action and mutations lead to epilepsy [5962]. In
this study, RNA-seq revealed a 2.38-fold decrease in
the expression of Gabrg2 in response to BDNF.
Our new results suggest that sustained levels of BDNF
at endogenous concentrations (0.7 nM) can dysregulate
a major segment of the epilepsy-associated transcrip-
tome. Recent discovery of the role of BDNF in cancer
suggests that understanding how BDNF orchestrates its
diverse effects on genome dysregulation will also provide
a new window on cancer chemotherapies [6365]. Sur-
prisingly, beyond its activation of downstream targets
like the cAMP regulatory element binding (CREB) pro-
tein, whose relationship to BDNF has been rigorously
described [66,67], we still know little about the role of
other BDNF controlled transcriptional regulators and
their intracellular signaling partners that contribute to
disease.
Using the TLE model of epilepsy, we previously showed
that during the latent period after pilocarpine-induced SE,
there is a marked upregulation in activated CREB and an
induction in the expression of the inducible cAMP early
repressor (ICER) [23]. We also reported that activated
CREB interacts with ICER to downregulate the levels of
Gabra1 transcripts leading to a decrease in α1-containing
GABARs in hippocampal neurons. In our studies, we dis-
covered that BDNF was linked to the JAK/STAT pathway
as 1) STAT3 knockdown blocked ICER induction in re-
sponse to BDNF, 2) STAT3 was found on the ICER pro-
moter in response to BDNF treatment (as measured by
chromatin immunoprecipitation (ChIP), 3) JAK/STAT
inhibitors (pyridone 6 and WP1066) blocked ICER induc-
tion and Gabra1 downregulation, and 4) in vivo applica-
tion of WP1066 at the time of pilocarpine-induced SE
reduced the frequency of subsequent spontaneous seizures
in these animals [32].
Based on these findings, we asked whether the BDNF
transcriptome, as identified in our prior studies, could
be separated into those where the BDNF response was
blocked by exposure to JAK/STAT inhibitors (Fig. 1)
and those that were regulated by other pathways. We
chose two inhibitors, WP1066 (a JAK2 inhibitor with di-
verse mechanisms of inhibition [39]), that we showed
was active in primary cultures and in vivo, and Ruxoliti-
nib (Ruxo), a high-affinity JAK1/2 inhibitor that blocks
JAK activation and is currently used in the treatment of
patients with high-risk myelofibrosis. To our surprise,
when we compared the transcriptome of cells exposed
to BDNF, and to low dose Ruxo plus BDNF (RX1,100
nM + B), we did not see a significant difference between
the two datasets. As we show in Fig. 3, this was not due
to any issue with the ability of the stock drug to block
STAT3 phosphorylation in sister dishes. Phosphoryl-
ation-independent STAT3 activity, however, has been
described in multiple contexts [49,68] suggesting that
JAK/STAT signaling in neurons may act in a non-ca-
nonical fashion, at least when activated by prolonged
BDNF signaling as studied in our research. A role for
unphosphorylated STATs in genome stability and for
nuclear JAK2 in the regulation of histone 3 Y41 phos-
phorylation has been described [69,70] and is the sub-
ject of our future studies.
Despite the lack of response of the transcriptome to
BDNF in the presence of the high affinity concentration
of Ruxo (RX1, 100 nM), which inhibits STAT3 phos-
phorylation, WP1066 and Ruxo at 10 μM (RX2)
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produced substantial changes in gene expression
(Fig. 2a). By comparing the two datasets (WP1066 + B
and RX2 + B) we were able to decrease the dimensional-
ity of the BDNF transcriptome into those most relevant
by their association with epilepsy. In fact, using enrich-
ment analysis, we discovered an association with
epilepsy that is more pronounced than any other neuro-
logical disease, due to the large proportion of differen-
tially expressed epilepsy-associated genes present within
the WP + B and RX2 + B subset (68%) (see Additional
file 2: Table S2).
Pathway analysis also revealed that JAK/STAT inhib-
ition had a marked effect on reversing the BDNF-in-
duced transcriptional response for genes organized in
pathways that control neurogenesis, neuroinflammation,
synaptic plasticity, receptor expression, and synaptic
transmission. Alterations in these functions exist in the
epileptogenic brain, which is known to have altered
neurogenesis, mossy-fiber sprouting, inflammation and
neuronal pruning leading to increased plasticity, and al-
tered excitation and inhibition at the synapse [2,3,71].
They also expand upon the recent findings that JAK/
STAT signaling plays an important role in learning and
memory [27], suggesting that impairment in such signal-
ing may be implicated in the cognitive comorbidities of
the condition. In the future, it will be important to study
these changes in the context of an epileptogenic animal
model in vivo where the STAT3 neuronal response can
be separated from its nonneuronal counterparts in astro-
cytes and microglia.
Given that multiple epilepsy-associated genes have
been shown to have functional binding sites for STAT3,
including BDNF itself (Fig. 7), and considering the sub-
stantial relationship between prolonged exposure to
BDNF and the ability of JAK/STAT inhibitors to reverse
the differential expression of epilepsy associated genes in
our studies, we hypothesize that identifying the mechan-
ism that connects BDNF signaling to this novel non-ca-
nonical role of JAK/STATs in neurons has the potential
to reveal new strategies in the treatment of intractable
epilepsy, or its comorbidities, and provide a new window
on how neuropharmacology can be used to identify the
complex cross-talk between different intracellular signal-
ing pathways in different cell types.
As a first step towards identifying the novel mecha-
nism(s) that drive differential gene expression in response
to dysregulated BDNF, we discovered that, contrary to our
expectation, BDNF increases the association of HP1αwith
STAT3 (see Fig. 8). Perhaps this is not so surprising as
HP1αhas also been associated with euchromatin and is
particularly important in gene induction [25,26]. Future
studies will be aimed at learning more about the func-
tional consequences of such protein/protein interactions
and their potential role in disease.
Conclusion
We have employed deep RNA-seq to determine the re-
sponse of BDNF on the transcriptome of neurons and
have employed a neuropharmacological approach to re-
duce the dimensionality of the datasets to identify BDNF
responsive genes whose transcriptional polarity is re-
versed by two well characterized JAK/STAT inhibitors.
Our results show that this subset of genes contains many
with previous association to epilepsy in both animal
models and human patients. The BDNF-induced JAK/
STAT gene set is highly enriched for genes involved in
synaptic neurotransmission, and contains targets from
all the major classes of ion channels and neurotransmit-
ter receptors of the brain. In particular, the gene set in-
cludes those known to play a role in epileptogenesis
through the regulation of synaptic plasticity, neurogen-
esis, transcriptional regulation, neuroinflammation and
proliferation. Some of the genes are known to contain
functional STAT recognition sites as verified by EN-
CODE, while others remain to be interrogated. Pharma-
cological analysis also revealed that phosphorylation of
STAT3 at Tyr705 most likely does not control BDNF-in-
duced JAK/STAT regulation of genome expression in
neurons, suggesting that the mechanism is non-canon-
ical. This conclusion is consistent with the additional ob-
servation that BDNF regulates the association of HP1α
with STAT3, suggesting that it either regulates hetero-
chromatin structure or plays a novel role in euchroma-
tin. Most importantly, the RNA-seq datasets highlight
the importance of the JAK/STAT pathway in neurons
and stimulate further discovery in this area based on its
potential relevance to brain function and the need to
identify new therapeutic strategies for the treatment of
intractable neurological disorders.
Methods
Cell culture and treatments
Primary neocortical neurons were dissected from neocor-
tex of 3 E18 Sprague-Dawley rat embryos (Charles River
Laboratories). Pregnant dams were euthanized by CO 2
according to approved Boston University Institutional ani-
mal care and use (IACUC) protocol (AN14327). Embry-
onic brains were removed and placed in ice-cold Ca 2+
/Mg 2+ free (CMF) media [Ca 2+ /Mg 2+ free Hanks BSS,
4.2 mM sodium bicarbonate,1 mM pyruvate, 20 mM
HEPES, 3 mg/mL BSA, pH 7.257.3]. Cortices were dis-
sected under a dissection microscope, trypsinized, cen-
trifuged, and triturated in plating media [Neurobasal
media (Invitrogen), 10% fetal bovine serum (Gibco),
100 U/mL penicillin, 100 μg/mL streptomycin, 200 mM
glutamax (Gibco)]. Dissociated neurons were plated on
Poly-L-lysine coated 6-well plates (1x10
6
cells/well) and
placed in an incubator (37 °C/5% CO 2) for attachment
to plate. 1 h later, plating media was removed and
Hixson et al. BMC Genomics (2019) 20:677 Page 12 of 16
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replaced with 2 mL of defined medium [Neurobasal
media (Invitrogen), B27 serum-free supplement
(Gibco), 100 U/mL penicillin, 100 μg/mL streptomycin,
200 mM glutamax (Gibco)]. Neurons were cultured in
the incubator for 9 days until use (9 days in vitro
[DIV]). Treatments: RNA-seq and P-STAT3 Western
Blot- At 9DIV, conditioned media was removed from
wells so each well had an equal volume of 1.5 ml. Cells
were pretreated for 1-h with vehicle dimethyl sulfoxide
(DMSO) at 0.1%, Ruxolitinib (Sellckchem S1378; 100
nM or 10uM), or WP1066 (EMD Millipore 573,097;
10uM). 1.5 μL of nuclease-free water (vehicle) or aque-
ous Brain-derived neurotrophic factor (EMD Millipore
GF029; BDNF final concentration 0.7 nM) was then
added to the media (total volume 1.5 ml) for 4 h before
collection. Due to the small volume of the BDNF or
water vehicle addition, effect on media salt concentra-
tion was negligible. N= 3, each N represents cells col-
lected from one pregnant dam. Enough primary cells
were collected from each N sufficient for 1 biological
replicate of each treatment. HP1αPulldown: At 9-DIV
cells were pretreated for 1-h with dimethyl sulfoxide
(DMSO) at 0.1%, Ruxolitinib (Sellckchem S1378;
10uM), or WP1066 (EMD Millipore 573,097; 10uM).
Nuclease-free water vehicle or Brain-derived neuro-
trophic factor was then added (EMD Millipore GF029;
0.7 nM) for 30 min before collection, N=4.
RNA extraction and library preparation
At the end of the 4-h treatment period, cells were collected
and RNA extracted using the Qiagen RNeasy Micro Kit
with DNAase treatment (Cat. 74,004). RNA was run in the
Agilent Bioanalyzer to determine RNA integrity number
(RIN). RIN of all samples was 9.0. mRNA was selected by
using the NEBNext® Poly(A) mRNA Magnetic Isolation
Module (E7490S) before using the NEBNext® UltraDirec-
tional RNA Library Prep Kit for Illumina® (E7420S) with
Agencourt AMPure XP Clean up beads (A63881) to make
RNA into strand specific cDNA libraries with multiplexing
barcodes from the NEBNext® Multiplex Oligos for Illu-
mina® (Index Primers Set 1) kit (E7335S).
Illumina sequencing
Sequencing was performed at the Boston University
Microarray Core and University of Chicago Genome
Center. Fragmentation and concentration were analyzed
on the Agilent Bioanalyzer and Fragment analyzer prior
to sample pooling. Runs were either sequenced at 75 bp
single-end reads on the Illumina NextSeq sequencer to a
depth of 100120 million reads per sample or at 50 bp
single-end reads on the Illumina HiSeq sequencer to a
depth of 90100 reads per sample. All samples were
multiplexed and pooled using barcodes to separate bio-
logical replicates.
RNA-seq bioinformatics
Data analysis was performed using Strand NGS software,
Version 3.2, Build 237,248.© Strand Life Sciences,
Bangalore, India. Proprietary Strand NGS algorithms
were used to conduct read alignment to the rat genome
build rn6 RefSeq Genes (2016.05.11). Reads were filtered
based on the following parameters: Max number of
novel splices: 1, Min alignment score: 90, Number of
gaps allowed: 5, number of matches to be output for
each read: 1, exclude reads with alignment length less
than: 25, ignore reads with matches more than: 5, Trim
3end with average base quality less than: 10. RNA
quantification was performed by the Strand NGS soft-
ware, which counts the total number of reads that map
to each gene and exon in the genome, and is reported as
raw counts. Normalization of the raw counts was
achieved using the DESeq method, which accounts for
difference in the total number of reads between samples
and log transforms the data [72]. R version 3.1.2 was
used with DEseq2 version 1.6.1 to determine differential
expression FDR = 0.05, Wald test type. The list of signifi-
cant genes was then used in subsequent analyses. Dis-
ease involvement data was analyzed through the use of
IPA with proprietary statistical algorithms (QIAGEN
Inc., https://www.qiagenbioinformatics.com /products/
ingenuitypathway-analysis). Cytoscape 3.5.1 software was
used with GeneMANIA for functional network gener-
ation with [42,43]. EnrichR was used for GO-term ana-
lysis and KEGG pathway analysis [40,41].
Real-time quantitative reverse transcriptase PCR (qRT-PCR)
Primary neurons were prepared, treated, and RNA ex-
tracted, as described above for RNA-seq. The Applied
Biosystems 1-step RNA to Ct (cat. 4,392,938) kit was
used and protocol was followed as directed: 50 ng of
RNA template, 10 μL of 2x mastermix, 0.5 μLofeach
40x primer/probe kit (Ppia and the gene of interest),
0.5 μL of the enzyme mix, and nuclease free water was
added to each 20 μL reaction. Each reaction was per-
formed with 2 technical replicates in a 384-well plate.
All taqman primer/probe kits were ordered from
Thermo Fisher Scientific. Assay ID: Ppia (housekeeping):
Rn00690933_m1 VIC-MGB; Galr1: Rn04932425_m1
FAM-MGB; Drd5: Rn00562768_s1 FAM-MGB; Jak2:
Rn00676341_m1 FAM-MGB; Gabrg2: Rn01464079_m1
FAM-MGB; Grm1: Rn00566625_m1 FAM-MGB; Icer:
Rn00569145_m1 FAM-MGB; Ccnd1: Rn00432359_m1
FAM-MGB; Socs3: Rn00585674_s1 FAM-MGB; Mcl1:
Rn00821024_g1 FAM-MGB; Cxcr4: Rn00573522_s1.
The Applied Biosystems 7900 HT Real-Time PCR sys-
tem was used to run with the following cycling parame-
ters: 48 °C 15 min. Hold (Reverse Transcription), 95 °C
10 min. Hold, 40 cycles: 95 °C 15 s, 60 °C 1 min. After
Hixson et al. BMC Genomics (2019) 20:677 Page 13 of 16
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
run completion, the ΔΔCT method was used to calculate
the relative values of the transcripts.
Western blot and immunoprecipitation
Primary cortical cultures used for Western Blot were pre-
pared and treated with drugs the same as described for
RNA-seq. After the 4-h period of BDNF treatment, cells
were washed once with ice-cold PBS solution (PBS +
EDTA, 1x PhosStop (Roche) and 1x Protease Inhibitor
cocktail (Roche cOmplete protease inhibitor)) and centri-
fugedin1.5mLtubesat5000RPMfor4minat4°C.PBS
was removed and cell pellet was resuspended in 1x RIPA
buffer (with 1x Protease and Phosphatase inhibitors). Ly-
sates were incubated on a rotator at 4 °C for 15 min, then
centrifuged at 13000 RPM for 10 min. Supernatant was
transferred to a fresh tube and aliquoted for Western Blot
or Immunoprecipitation. For non-immunoprecipitated
samples, 35 μg of sample was diluted to 10 μLinRIPAbuf-
fer and 10 μL of 2x SDS Tris-Glycine loading buffer
(Invitrogen LC2676) with 50 mM DTT. For immunopre-
cipitation, Active Motif Universal Magnetic Co-IP kit (Cat.
#54002) was used. Three hundred microgram of protein
was incubated overnight at 4 °C with 15 μLHP1αantibody
(Cell Signaling Technology #2616). Samples were then in-
cubated in magnetic bead solution from the kit for 1 h be-
fore elution into 20uL SDS Tris-Glycine loading buffer
(Invitrogen LC2676) with 50 mM DTT. All samples were
boiled at 95 °C for 5 min, centrifuged and loaded into a
Novex 10% Wedgewell gel (Invitrogen XP00100BOX). The
gel was run at 220 V for 40 min and wet transfer was
performed at 33 V for 2 h to a nitrocellulose membrane.
Membrane was quickly washed in TBST then blocked in
5% milk in TBST for 1 h at room temperature. Three 5 min
washes in TBST were performed. Membrane was incubated
overnight at 4 °C in primary antibody then washed 3 × 10
minutes in TBST. HRP-conjugated secondary was added
for 1 h at room temperature, then 3 washes in 1% milk
were performed prior to imaging the blot with ECL (BioRad
Clarity #1705060). Membrane was stripped using the GM
Biosciences One Minute Advance Stripping buffer
#GM6031. Antibodies were used as follows: 1:1000 pSTAT3
(CST #9131S) in 5% BSA, 1:5000 anti-rabbit HRP-conju-
gated secondary antibody (EMD Millipore AP132P) in 1%
milk in TBST. 1:2000 total STAT3 (CST #4904S) in 5%
milk in TBST. 1:6000 β-actin (Sigma A5441) in 1% milk in
TBST, 1:2500 Anti-mouse HRP-conjugated secondary anti-
body (Vector Labs PI2000) in 1% milk in TBST.
Additional files
Additional file 1: Table S1. Genes of interest in the dataset and their
relationship to epilepsy.Several genes of particular interest to the field of
epilepsy are listed with the degree of FC from DMSO+Water vs. DMSO+BDNF
and the FC of DMSO+BDNF vs. WP1066 + BDNF. Many have not been
associated with the JAK/STAT pathway or BDNF regulation. The name of the
gene, the related function, and a reference to support the potential
relationship with epilepsy is listed. (PDF 196 kb)
Additional file 2: Table S2. Top Neurological disease relationships
determined by IPA. (A) Top 10 significantly enriched neurological diseases in
the BDNF transcriptome with their p-value (IPA). (B) Top 10 significantly
enriched neurological diseases in the WP/RX2 vs. BDNF set of differentially
expressed genes listed with their p-value from IPA. (PDF 180 kb)
Additional file 3: Figure S1. Volcano Plot of BDNF regulated genes
whose response is reversed by WP + B/RX2 + B. Representation of degree
of fold change and significance used to narrow down the list of top
genes to include in the Fig. 3network. Teal-genes upregulated by both
WP + B and RX2 + B; Blue: genes upregulated by WP + B only; Red: genes
downregulated by both WP +B and RX2 +B; Pink: genes downregulated
only by WP + B; Yellow: genes that did not meet cutoffs of > 2 FC, p<
1e-10. Teal and Red gene list used for Fig. 3network. Maximum log10 p
value detected was 45, p values more significant than 45 are represented
as log10 of 45. (PDF 103 kb)
Additional file 4: Figure S2. Enrichment Analysis of BDNF regulated
genes whose expression is reversed exclusively by 10uM Ruxo.
Enrichment analysis was performed on the set of 417 DEGs between
RX2 + B vs. V + B that were not overlapping with WP +B. (A) KEGG
pathway analysis as generated in EnrichR ranked and colored by p value.
(B) Top gene clusters involved in the KEGG canonical pathways from A.
(C-E) Neurological diseases significantly associated with the RX2 exclusive
gene set for (C) Epilepsy, (D) Huntingtons disease, (E)
Neurodegeneration. (PDF 279 kb)
Additional file 5: List of significant genes and FC values for all relevant
comparisons. Comprehensive list of genes that were significantly different
when comparing various treatments (PDF 493 kb)
Abbreviations
AMPA: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; BDNF: Brain-
derived neurotrophic factor; ChIP: Chromatin Immunoprecipitation;
DEG: Differentially expressed genes; Drd5: Dopamine receptor D5;
ERK: Extracellular-signal-regulated kinase; GABA: Gamma-Aminobutyric Acid;
Gabra1: Gaba A receptor alpha 1 subunit; Gabrg2: Gaba A receptor gamma 2
subunit; Galr1: Galinin receptor 1; GO: Gene ontology; GPCR: G-protein-
coupled receptor; Grm1: Glutamate metabatropic receptor 1;
HP1α: Heterochromatin Protein 1 alpha; IACUC: Institutional animal care and
use; Icer: Inducible cyclic-AMP early repressor; IPA: Ingenuity pathway
analysis; JAK/STAT: Janus kinases (JAK); JNK: c-Jun N-terminal kinase;
LTD: Long-term depression; LTP: Long-term potentiation; MAPK: Mitogen-
activated protein kinase; mRNA: Messenger ribonucleic acid; NF-κB: Nuclear
factor kappa-light-chain-enhancer of activated B cells; NGFR: Nerve growth
factor receptor; P75NTR: p75 Neurotrophin receptor; PI3K: Phosphoinositide
3-kinase; PILO: Pilocarpine; PLCγ: Phospholipase C gamma; qRT-
PCR: Quantitative real-time polymerase chain reaction; RUXO: Ruxolitinib;
SE: Status Epilepticus; STAT: Signal Transducer and Activator of Transcription;
TBI: Traumatic brain injury; TLE: Temporal lobe epilepsy; TrkB: Tropomyosin
receptor kinase B
Acknowledgements
We would like to acknowledge the work of previous members of the Russek
laboratory that have provided the creative environment needed to pursue
the mechanism of this novel signaling pathway in the brain, Drs. Rebecca
Benham and Kristen Hokenson, as well as our colleagues in the Department
of Pharmacology and the Graduate Program for Neuroscience. We also thank
the members of the Brooks-Kayal laboratory for providing a wonderful colle-
gial environment for translational research efforts.
Authorscontributions
KH performed the experiments, analysis, and wrote the manuscript with
input from all authors. MC assisted with study design and data analysis. ABK
assisted with study design and results interpretation. SR oversaw study
design, experimental work, data analysis and read, edited and wrote the final
manuscript. All authors read and approved the final manuscript.
Hixson et al. BMC Genomics (2019) 20:677 Page 14 of 16
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Funding
Funding was generously provided by the following grants:
NIGMS T32GM008541; stipend support and partial graduate training for K.
Hixson.
NINDS R01NS051710; supported data collection, design, analysis,
interpretation, and writing. PIs A.R. Brooks-Kayal and S.J. Russek.
NINDS R21 NS083057; enabled the initial discovery of JAK/STAT inhibitor
selection and use in vivo. PIs A.R. Brooks-Kayal and S.J. Russek.
CURE Epilepsy Multidisciplinary Award; funding brought together research
laboratories and helped build hypotheses for R01 and R21 applications. PIs
A.R. Brooks-Kayal and S.J. Russek.
Availability of data and materials
All data generated and analyzed during our study which support the
conclusions of the article are included in the Additional files. All aligned
sequencing files (.bam) are publicly available in the NCBI SRA database (SRA
accession: PRJNA554446) at this link:
http://www.ncbi.nlm.nih.gov/bioproject/554446
Ethics approval and consent to participate
Rats were sacrificed in accordance with the Boston University practice for
laboratory animal procedures. The protocol (AN14327) was approved by the
Boston University Institutional Animal Care and Use committee (IACUC).
Consent for publication
Not Applicable.
Competing interests
There are no conflicts of interest for any of the authors on this manuscript.
Author details
1
Laboratory of Translational Epilepsy, Department of Pharmacology &
Experimental Therapeutics, Boston University School of Medicine (BUSM),
Boston, USA.
2
Graduate Program for Neuroscience (GPN), Boston University
(BU), Boston, USA.
3
Department of Pediatric Neurology, University of
Colorado Anschutz Medical Campus, Aurora, USA.
4
Department of Biology,
Boston University (BU), Boston, USA.
Received: 23 January 2019 Accepted: 15 August 2019
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... STAT3 was identified as an important factor in emotional responses, related to depression, schizophrenia, and bipolar disorder, and found to be a key coordinator of cytokine activation in cellular immune responses, and in the "immune hypothesis," it has a close link to psychopathology [21][22][23]. In addition to cytokines, the activation of STAT3 can also reflect the role of upstream regulators related to neurological function, including growth factors, hormones, and endocannabinoids [24,25]. Kwon et al. revealed that STAT3 regulates depression-related behaviors via neuronal M-CSF-mediated synaptic activity [26]. ...
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... Using a full transcriptome sequencing approach on BDNF-treated neurons with and without JAK/STAT inhibitors, Hixson et al. [126] have determined the complete pool of genes that undergo BDNF-dependent JAK/STAT-mediated regulation in cultured cortical neurons. Their analyses revealed 2869 differentially regulated genes whose expression changes after BDNF application. ...
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... Using a full transcriptome sequencing approach on BDNF-treated neurons with and without JAK/STAT inhibitors, Hixson et al. [125] have determined the complete pool of genes that undergo BDNF-dependent JAK/STAT-mediated regulation in cultured cortical neurons. ...
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... In a similar vein, uSTAT3 mediates induction of ion channels and neurotransmitter receptors in the brain [85] and can also augment the expression of select STAT1-and STAT2-responsive genes by increasing promoter accessibility [86]. However, in several cases 'canonical' signaling can also increase the transcription of the respective STAT gene, thereby elevating uSTAT levels that leads to sustained basal transcriptional activation over a longer time frame. ...
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... Atypical activation of JAK-STAT signaling is mainly related to the neuroinflammatory processes aggravating neuronal impairment in Epilepsy. Diverse stimuli of cytotoxic substances released by activated glial cells in response to prolonged seizure-induced brain damage like axon sprouting represent neuroinflammation in epilepsy [60]. The elevated levels of cytokines (TNF-α, IL-6, IL-1β) cause secondary development of seizures that are adversely associated with neuronal apoptosis by activating JAK-STAT in glial cells favoring neuroinflammation in epilepsy [32,61]. ...
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The brain-derived neurotrophic factor (BDNF) is synthesized as a precursor, namely proBDNF, which can be processed into mature BDNF (mBDNF). Evidences suggest that proBDNF signaling through p75(NTR) may account for the emergence of neurological disorders. These findings support the view that the relative availability of mBDNF and proBDNF forms is an important mechanism underlying brain circuit formation and cognitive functions. Here we describe novel insights into the proBDNF/p75(NTR) mechanisms and function in vivo in modulating neuronal circuit and synaptic plasticity during the first postnatal weeks in rats. Our results showed that increased proBDNF/p75(NTR) signaling during development maintains a depolarizing γ-aminobutyric acid (GABA) response in a KCC2-dependent manner in mature neuronal cells. This resulted in altered excitation/inhibition balance and enhanced neuronal network activity. The enhanced proBDNF/p75(NTR) signaling ultimately led to increased seizure susceptibility that was abolished by in vivo injection of function blocking p75(NTR) antibody. Altogether, our study shed new light on how proBDNF/p75(NTR) signaling can orchestrate the GABA excitatory/inhibitory developmental sequence leading to depolarizing and excitatory actions of GABA in adulthood and subsequent epileptic disorders.
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