Articles in PresS. J Neurophysiol (August 30, 2017). doi:10.1152/jn.00945.2016
1
Spinal BDNF Induced Phrenic Motor Facilitation Requires PKCθ Activity
2 Ibis M. Agosto-Marlin1 and Gordon S. Mitchell 1,2
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1
Department of Comparative Biosciences University of Wisconsin Madison, WI 53706 and
2
Center for Respiratory Research and Rehabilitation Department of Physical Therapy and McKnight Brain Institute University of Florida, Gainesville, FL, 32610.
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*Corresponding author: Gordon S. Mitchell Department of Physical Therapy University of Florida 330 Center Drive Gainesville, FL, USA 32610 Phone: 352-273-6107 E-mail: [email protected]
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Contributions: I.M.A.M. collected all data. I.M.A.M. and G.S.M. contributed to experimental design, data analysis and manuscript development. Both authors approved the manuscript and do not have any conflicts of interest to declare. 1 Copyright © 2017 by the American Physiological Society.
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ABSTRACT
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Spinal brain derived neurotrophic factor (BDNF) is necessary and sufficient for certain
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forms of long-lasting phrenic motor facilitation (pMF). BDNF elicits pMF by binding to its
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high-affinity receptor, tropomyosin receptor kinase B (TrkB), on phrenic motor neurons,
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potentially activating multiple downstream signaling cascades. Canonical BDNF/TrkB
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signaling includes the: 1) Ras/RAF/MEK/ERK MAP Kinase; 2) phosphatidylinositol 3‐
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kinase/Akt; and 3) PLCγ/PKC pathways. Here, we demonstrate that spinal BDNF-
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induced pMF requires PLCγ/PKCθ in normal rats, but not MEK/ERK or PI3K/Akt
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signaling. Cervical intrathecal injections of MEK/ERK (U0126) or PI3K/Akt (PI-828; 100
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μM;12μL) inhibitors had no effect on BDNF-induced pMF (90min post-BDNF: U0126 +
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BDNF: 59±14%; PI-828 + BDNF: 59±8%; Inhibitor vehicle + BDNF: 56±7%; all p≥ 0.05).
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In contrast, PKCθ inhibition with TIP (0.86 mM; 12μL) prevented BDNF-induced pMF
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(90min post-BDNF: TIP + BDNF: -2±2%; p≤ 0.05 versus other groups). Thus, BDNF-
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induced pMF requires downstream PLCγ/PKCθ signaling contrary to initial expectations.
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2
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KEY WORDS
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respiratory
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PLC/PKCθ, motor neuron
plasticity,
phrenic
motor
facilitation,
BDNF,
MEK/ERK,
PI3K/AKT,
61 62
NEW AND NOTEWORTHY
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We demonstrate that BDNF induced pMF requires downstream signaling via PKCθ, but
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not MEK/ERK or PI3K/Akt signaling. These data are essential to understand the
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sequence of the cellular cascade leading to BDNF-dependent phrenic motor plasticity.
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INTRODUCTION
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Phrenic motor facilitation (pMF) can be elicited by acute intermittent hypoxia (AIH) or by
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drugs injected in the cervical spinal segments near the phrenic motor nucleus. When
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pMF is induced by AIH, it is known as phrenic long-term facilitation (pLTF; Devinney et
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al. 2013; Fuller et al. 2000; Hayashi et al. 1993; Mitchell et al. 2001; Mitchell and
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Johnson 2003; Powell et al. 1998); pMF is a more general term that includes AIH-
86
induced pLTF (Dale-Nagle EA et al. 2010; Mitchell and Johnson 2003). Multiple, distinct
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cellular cascades give rise to pMF.
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The Q-pathway to pMF is the predominant contributor to pLTF following moderate
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AIH; it is termed the Q-pathway since multiple Gq protein-coupled metabotropic
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receptors initiate a similar response (Dale-Nagle et al. 2010; Fuller et al. 2000). The
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prevailing cellular model of the Q-pathway involves (in sequence): 1) spinal serotonin
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type 2 receptor activation (5HT2; Fuller et al. 2001; MacFarlane et al. 2011), 2) protein
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kinase C-theta (PKCθ) activity (Devinney et al. 2015), 3) new synthesis and release of
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brain derived neurotrophic factor (BDNF; Baker-Herman et al. 2004), 4) activation of the
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high affinity BDNF receptor, tropomyosin receptor kinase B (TrkB; Baker-Herman et al.
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2004; Dale et al. 2017), and 5) MEK-ERK activation (Hoffman et al. 2012). Although the
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requirement for each molecule/step is necessary for pLTF, the specific sequence has
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been inferred, and not tested directly.
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New BDNF protein synthesis is necessary moderate AIH-induced pLTF, and
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BDNF/TrkB signaling alone elicits pMF (Baker-Herman et al. 2004). To establish the
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sequence of events downstream from BDNF/TrkB signaling, we studied intrathecal
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BDNF-induced pMF followed by selective inhibition of three potential TrkB signaling
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pathways. Canonical BDNF/TrkB signaling can involve any (or all) of the following: 1)
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Ras/Raf/MEK/ERK; 2) phosphatidylinositol 3‐ kinase (PI3K)/Akt; or 3) PLCγ/PKC
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signaling (Minichiello 2009). Based on several recent publications, we hypothesized that
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BDNF/TrkB signals via the MEK/ERK pathway with moderate AIH-induced pLTF
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(Hoffman et al., 2012; Devinney et al., 2013), whereas PKCθ signaling occurs upstream
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from new BDNF synthesis (Devinney et al., 2015). Thus, we hypothesized that
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MEK/ERK inhibition would abolish BDNF-induced pMF, with minimal impact from
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PI3K/Akt or PKCθ inhibition. To our surprise, BDNF-induced pMF requires PKCθ
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signaling, and is independent of MEK/ERK or PI3K/Akt signaling. Consequently,
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required MEK/ERK signaling in the Q-pathway to pMF must be upstream from
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BDNF/TrkB activation, at least in normal rats.
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MATERIALS AND METHODS
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Animals
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Adult male (3-4 months) Sprague-Dawley rats (Harlan, Colony 211 and 217) were used
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in all experiments. Rats were housed in a controlled environment (12h light/dark cycle;
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daily humidity and temperature monitoring), with food and water ad libitum. The
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University of Wisconsin Animal Care and Use committee approved all experimental
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protocols.
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Experimental Series
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We tested our hypothesis in three experimental series to investigate the role of three
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pathways associated with BDNF/TrkB signaling in other model systems. The first
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experimental series tested the hypothesis that: Spinal BDNF-induced phrenic motor
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facilitation is MEK/ERK dependent. In this series, each group received either: 1)
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intrathecal MEK/ERK inhibitor (U0126) prior to intrathecal BDNF (MEK/ERK inhibitor +
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BDNF; n=6); or 2) intrathecal MEK/ERK inhibitor (U0126) prior to intrathecal BDNF
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vehicle injections (aCSF+0.1%BSA) (MEK/ERK inhibitor + aCSF; n=6).
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The second experimental series tested the hypothesis that: BDNF-induced phrenic
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motor facilitation is AKT/PI3K independent. In this series, each group received
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either: 1) intrathecal AKT/PI3K inhibitor (PI-828) prior to intrathecal BDNF (AKT/PI3K
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inhibitor + BDNF; n=5); or 2) intrathecal AKT/PI3K inhibitor (PI-828) prior to intrathecal
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BDNF vehicle (aCSF+0.1%BSA) (AKT/PI3K inhibitor+ aCSF group; n=4).
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In the third experimental series, we tested the hypothesis that: BDNF-induced
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phrenic motor facilitation is PKCθ independent. In this series, groups received
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either: 1) intrathecal PKCθ inhibitor (TIP) before intrathecal BDNF (PKCθ inhibitor +
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BDNF; n=6); or 2) intrathecal PKCθ inhibitor (TIP) prior to intrathecal BDNF vehicle
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(aCSF+0.1%BSA) (PKCθ inhibitor + aCSF; n=5).
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Control Groups
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Controls groups included: 1) intrathecal 20%DMSO/80%Saline (Inhibitor vehicle for
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U0126 and PI-282) prior to intrathecal BDNF (n=5); 2) intrathecal aCSF (TIP Inhibitor
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vehicle) prior to intrathecal BDNF (n=5); 3) intrathecal 20%DMSO/80% Saline (Inhibitor
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vehicle for U0126 and PI-282) prior to intrathecal BDNF vehicle (aCSF+0.1%BSA)
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(n=3); and 4) intrathecal aCSF (TIP Inhibitor vehicle) prior to intrathecal BDNF vehicle
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(aCSF+0.1%BSA) (n=5).
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Since there were no statistically significant differences (two-way ANOVA, p=0.47)
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between 20%DMSO/80%Saline + BDNF (n=5) and 100% aCSF + BDNF (n=5), these
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groups were combined and renamed Inhibitor Vehicle + BDNF (n=10). Since there were
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no
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aCSF+0.1%BSA (n=3) and 100% aCSF + aCSF+0.1%BSA (n=5), these groups were
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also combined and renamed Time Controls (n=8).
significant
differences
(T-test,
p=0.30)
between
20%DMSO/80%Saline
+
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Surgical Protocol
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Rats were anesthetized using isoflurane in a closed chamber and placed on a
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temperature-regulated table. A nose cone was then used to continue isoflurane
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administration throughout the surgery (isoflurane, 3.5% in O2 50%, balance N2). Body
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temperature was assessed using a digital rectal probe and maintained between 36.5
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and 37.5°C. For intravenous infusions, a tail vein catheter (24G x 3⁄4 gauge in. iv
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catheter, Surflo) was placed (infusion rate: 0.5-1.2 ml·kg-1·h-1) throughout surgical
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preparations and the experimental protocol. Intravenous infusions were mixed to
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maintain fluid and acid-base balance (6:3:1 respectively) lactated Ringer’s solution,
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HesPan (6% Hetastarch in 0.9% sodium chloride) and bicarbonate solution (8.4%
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sodium bicarbonate solution). A tracheotomy was performed to enable artificial
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ventilation (Rodent Respirator, model 683, Harvard Apparatus, Holliston, MA; tidal
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volume 2.5 ml; frequency ~ 70-80). Before protocols began, the lungs were
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hyperinflated (2 breaths) every 1.5 hours to minimize alveolar collapse.
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A flow-through CO2 analyzer connected to the tracheal catheter was used to assess
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end-expired PCO2 levels (maintained between 40-45mmHg during surgical preparation;
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Capnogard, Novametrix, Wallingford, CT). To prevent entrainment of respiratory neural
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activity to the ventilator, rats were bilateraly vagotomized in the mid-cervical region. A
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catheter was placed in the right femoral artery (polyethylene catheter PE50, Intramedic)
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to monitor blood pressure and draw arterial blood samples for blood-gas and acid-base
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analysis (0.2–0.4 ml samples; ABL-800 Flex, Radiometer; Westlake, OH). Blood
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pressure was monitored continuously with a pressure transducer (Gould, P23ID).
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Measurements were made on blood samples drawn during baseline (BL), and at 15, 30,
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60 and 90 minutes post-treatment.
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The left phrenic nerve was isolated using a dorsal approach, cut distally,
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desheathed, and covered with a cotton ball soaked with saline until protocols began. A
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laminectomy (cervical-2) was performed in all rats and a small incision was made in the
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dura to place intrathecal catheters for drug delivery near the phrenic motor nucleus.
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Two soft silicone catheters (2 Fr; Access Technologies, Skokie, IL) were inserted 4 mm
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caudally from the C2 durotomy until the tip rested above the C4 segment. Intrathecal
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catheters were attached to 50-μl Hamilton syringes filled with appropriate solutions
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(inhibitors, BDNF or vehicles).
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After surgery, rats were converted to urethane anesthesia (1.85 g/kg, iv; delivered in
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multiple 0.2-0.4mL bolus injections over 15-20 minutes) while isoflurane was withdrawn.
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Once urethane anesthesia was established, anesthetic depth was confirmed via toe
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pinched with a hemostat, while monitoring changes in phrenic nerve activity, blood
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pressure and/or intentional movements. After conversion, a minimum of 1 h was
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allowed before initiating protocols. Rats were euthanized via urethane overdose at the
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end of experiments.
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Neurophysiological Measurements
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Pancuronium bromide (2.5 mg/kg iv) was used to paralyze rats during protocols. The
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phrenic nerve was covered in mineral oil, and placed on bipolar silver electrodes for
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nerve recordings. Phrenic nerve signals were amplified (gain 10,000X), band-pass
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filtered (100–10,000 Hz, model 1800, A-M Systems, Carlsborg, WA), rectified and
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integrated (Paynter filter, time constant 50 ms, MA-821, CWE, Ardmore, PA). Integrated
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phrenic nerve bursts were digitized (8kHz) and analyzed using a WINDAQ data-
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acquisition systems (DATAQ Instruments, Akron, OH).
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Before initiating protocols, the CO2 apneic threshold was determined by lowering
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end-tidal CO2 until phrenic nerve activity ceased for ~1 min. The recruitment threshold
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was then determined by slowly increasing end-tidal CO2 until nerve activity resumed.
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The end-tidal CO2 was raised ~2 mmHg above the recruitment threshold and ~15–20
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minutes were allowed to achieve a stable baseline.
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Drug treatments
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Brain derived neurotrophic factor (BDNF). Recombinant BDNF protein (Promega)
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was diluted in double distilled water to make a stock solution (5µg), aliquoted into
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multiple vials and stored at -20°C. The BDNF stock solution was diluted on the day of
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use to 100ng in aCSF+0.1%BSA solution. The intrathecal BDNF dose (100ng; 12uL)
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was the same as that used in a previous study from our group (Baker-Herman et al.
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2004). Control groups only received artificial cerebrospinal fluid (aCSF) (in mM: 120
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NaCl, 3 KCl,2 CaCl, 2 MgCl, 23 NaHCO3, 10 glucose, bubbled with 95%O2/5%CO2)
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with 0.1% BSA.
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MEK/ERK inhibitor (U0126) The membrane permeable MEK/ERK inhibitor (U0126
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or 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene, Promega) was
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dissolved in 100% DMSO, and then diluted with saline to create a 100 μM solution in
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20%DMSO/80%Saline. The inhibitor was diluted to its injected concentration on the day
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of use, and was administered (100 μM; 12μL) 20 minutes before BDNF (or BDNF
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vehicle) administration.
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Dose/response studies using intrathecal U0126 had been established in prior
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studies from our laboratory (Dale-Nagle et al. 2011; Dale et al. 2012; Hoffman et al.
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2012). We chose a dose shown to inhibit BDNF-dependent pLTF elicited by moderate
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AIH (Hoffman et al. 2012). Controls were 20%DMSO/80%Saline injections of the same
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volume. To demonstrate drug efficacy, we confirmed that this drug dose blocks
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moderate AIH-induced pLTF in two rats (data not shown).
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PI3K/Akt inhibitor (PI-828) The PI3K/Akt inhibitor (PI-828; 1,4-diamino-2,3-dicyano-
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1,4-bis(o-aminophenylmercapto) butadiene, Tocris) was dissolved in 100% DMSO and
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frozen in aliquots. The day of use, it was diluted with saline to a final concentration of
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100 μM in 20%DMSO/80%Saline. The inhibitor was administered (100 μM; 12μL) 20
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minutes before BDNF administration. Earlier studies from our laboratory used this same
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PI-828 dose, and confirmed efficacy (Dale-Nagle et al. 2011; Dale et al. 2012; Hoffman
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et al. 2012). For controls, we administered 20%DMSO/80%Saline at the same volume
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as the inhibitor. To confirm drug efficacy, we gave the same dose and concentration of
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the PI3K inhibitor prior to severe AIH since this form of pLTF is adenosine 2A receptor
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and PI3K/Akt dependent (Nichols et al., 2012); severe AIH-induced pLTF was
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successfully blocked in two rats (data not shown).
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PKCθ inhibitor (TIP). Theta inhibitory peptide (TIP; Calbiochem) is a myristoylated
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peptide mimicking the pseudosubstrate domain of PKCθ. TIP was dissolved in 100%
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aCSF and stored at -20°C (1mM). The day of use it was diluted with aCSF to a final
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concentration of 0.86 mM. The inhibitor was administered (0.86mM; 12 μL total, 1
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μL/15s) 15 minutes before BDNF administration. We previously demonstrated that this
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same dose and route of TIP administration blocks moderate AIH-induced pLTF
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(Devinney et al. 2015). For controls, we administered 100% aCSF at the same volume
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and rate of the inhibitor. To test that the drugs effect, we gave the same dose and
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concentration of TIP prior to delivering inactivity phrenic motor facilitation (iPMF). iPMF
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is a form of plasticity dependent on protein kinase C zeta (PKCζ), and independent from
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PKCθ (Baertsch and Baker-Herman 2015). As expected TIP failed to block iPMF (n = 2;
252
data not shown).
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Time Control
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Time control experiments controlled for time-dependent changes in phrenic nerve
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activity characteristic in anesthetized animals. Our time control experiments were the
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Inhibitor vehicle group (20%DMSO/80%Saline or aCSF) and the BDNF vehicle
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(aCSF+0.1% BSA). Inhibitor vehicle injections were done 15-20 minutes before BDNF
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vehicle, mimicking the timing of drug injections described above.
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Statistical analysis
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Integrated phrenic burst amplitude was normalized as a percent change from baseline.
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Respiratory frequency was also normalized as a change from baseline levels (% 11
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baseline), but was expressed as an absolute difference (bursts per minute). Statistical
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comparisons were made within and between treatment groups using a two-way ANOVA
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with a repeated measures design (Prism 6; GraphPad Software). The same statistical
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comparisons were done for mean arterial pressure (MAP), arterial blood gases, pH, and
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rectal temperature (BL and 90min time). To detect significant differences between
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experimental groups, a two-way repeated measures ANOVA was used (Prism 6;
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GraphPad Software); comparisons were made within and between treatment groups for
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phrenic nerve burst amplitude and frequency (BL, 15, 30, 60, and 90-min). Individual
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comparisons were made using Tukey’s post hoc test. To determine significance
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between control groups, two different tests were used. For the 20%DMSO/80%Saline +
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BDNF vs aCSF+ BDNF a two-way ANOVA was used and for the 20%DMSO/80%Saline
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+ aCSF+0.1%BSA vs 100% aCSF + aCSF+0.1%BSA, a T-test was performed. For all
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analyses, the significance level was set to 0.05; data are means ± SE.
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RESULTS
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Blood Gases and Arterial Pressure: Baseline and 90-min post-treatment
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Throughout protocols, PaCO2 was successfully regulated within ~2 mmHg from baseline
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value and PaO2 was kept above 150 mmHg. PaCO2 and PaO2 regulation during protocols
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was similar in all experimental groups (Table 1). Mean arterial pressure, temperature
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and pH were similar among groups at baseline and at 90 minutes post-hypoxia (Table
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1). Thus, differences in PaCO2, PaO2, MAP, temperature or pH regulation among groups
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cannot account for differential pMF responses observed in this study.
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BDNF induced pMF is MEK/ERK independent
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To test the hypothesis that BDNF-induced pMF requires MEK/ERK signaling, we
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delivered the MEK/ERK inhibitor U0126 (intrathecal delivery) 20 minutes before BDNF
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injections. Typical neurograms representing phrenic activity in Figure 2 (A-D) illustrate
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that MEK/ERK inhibition does not affect BDNF-induced pMF. Indeed, pMF magnitude
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was not significantly different when comparing the MEK/ERK inhibitor + BDNF group
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(60 minutes post-BDNF: 41±11%; 90 min: 59±14%; n=6) versus the Inhibitor vehicle +
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BDNF group (60 min: 49±7%; 90 min: 57±6%; n=10) (P≥0.05; Figure 1E).
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On the other hand, pMF was greater in both the MEK/ERK inhibitor + BDNF (60 min:
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41±11%; 90 min: 59±14%; n=6) and Inhibitor vehicle + BDNF (60 min: 49±7%; 90 min:
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57±6%; n=10) versus MEK/ERK inhibitor + aCSF (60 min: 15±2%; 90 min: 18±5%;
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n=6) and Time Control (60 min: 5±3%; 90 min: 4±3%; n=7) (P≤0.05; Figures 1E). The
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MEK/ERK inhibitor + aCSF and Time Control groups were not significantly different from
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one another at any time (P≥0.05; Figure 1E). There was no significant difference in
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frequency at any time in any group (P≥0.05; Figure 1F).
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BDNF induced pMF is PI3K/AKT independent
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To test the hypothesis that BDNF-induced pMF requires PI3K/Akt signaling, we
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delivered the PI3K/Akt inhibitor PI-828 (intrathecal delivery) 20 minutes before BDNF.
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Typical neurograms representing phrenic nerve output (Figures 2 A-D) illustrate that
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PI3K/Akt inhibitor does not affect BDNF-induced pMF. Indeed, pMF magnitude was not
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significantly different when comparing the PI3K/Akt inhibitor + BDNF group (60 minutes
13
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post-BDNF: 64±8%; 90 min: 59±8%; n=5) versus the Inhibitor vehicle + BDNF group (60
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min: 49±7%; 90 min: 57±6%; n=10) (P ≥ 0.05; Figure 2E).
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On the other hand, pMF was greater in both the PI3K/Akt inhibitor + BDNF (60 min:
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64±8%; 90 min: 59±8%; n=5) and Inhibitor vehicle + BDNF (60 min: 49±7%; 90 min:
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57±6%; n=10) versus PI3K/Akt inhibitor + aCSF (60 min: 8±9%; 90 min: 18±5%; n=4)
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and Time Control (60 min: 5±3%; 90 min: 4±3%; n=7) (P≤0.05; Figures 2E). The
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PI3K/Akt inhibitor + aCSF and Time Control groups were not significantly different from
316
one another at any time (P≥0.05; Figure 2E). There was no significant difference in
317
frequency at any time in any group (P≤0.05; Figure 2F).
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BDNF induced pMF is PKCθ dependent
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To test the hypothesis that BDNF-induced pMF is independent of PLC/PKCθ signaling,
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we delivered the PKCθ inhibitor TIP (intrathecal delivery) 15 minutes before BDNF
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administration. Typical neurograms representing phrenic nerve output are shown in
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Figure 3 (A-D), illustrating that, contrary to our hypothesis, PKCθ inhibition abolishes
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BDNF-induced pMF. In PKCθ inhibitor + BDNF treated rats (n=6), there was a
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significant decrease in the magnitude of pMF, starting at 30 minutes after BDNF
326
administration, relative to the Inhibitor vehicle + BDNF group (n=10) (30min PKCθ
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inhibitor+ BDNF 7±6%; versus Inhibitor vehicle + BDNF 26±6%; 60min PKCθ inhibitor +
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BDNF 12±5% versus Inhibitor vehicle + BDNF 49±7%; 90min PKCθ inhibitor + BDNF -
329
2±2% versus Inhibitor vehicle + BDNF: 57±6%; all P≤0.05; Figure 3E).
14
330
The PKCθ inhibitor + BDNF group was not significantly different at any time versus
331
PKCθ inhibitor + aCSF or Time Control groups (P≥0.05; Figure 3E). There were no
332
significant differences in frequency at any time in any group (P≥0.05; Figure 3F).
333 334
DISCUSSION
335
Contrary to our initial hypothesis, BDNF-induced pMF requires PKCθ activity, and is
336
independent of MEK/ERK or PI3K/Akt activity in normal rats (Figure 4). Doses and
337
selectivity of inhibitors were verified in other studies from our laboratory. Thus,
338
previously published working models of the Q-Pathway to pMF require revision (Dale-
339
Nagle et al. 2010; Dale-Nagle et al. 2010; Dale et al. 2014). We now suggest that the
340
correct protein activation sequence in moderate AIH-induced pLTF (ie. the Q-Pathway)
341
is: 1) MEK/ERK signaling upstream from new BDNF synthesis and release; and 2)
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BDNF/TrkB signaling via PLC/PKCθ activation. These findings elaborate our model and
343
increase understanding of mechanisms giving rise to phrenic motor plasticity.
344 345
BDNF induced pMF is MEK/ERK independent
346
We demonstrate that MEK/ERK signaling is not required for BDNF-induced pMF (Figure
347
1). These data are surprising since we initially hypothesized that BDNF-induced pMF
348
required MEK/ERK signaling based on the observation that MEK/ERK inhibition
349
abolishes moderate AIH-induced pLTF (Hoffman et al., 2012), and MEK/ERK is a
350
common TrkB signaling pathway (Minichiello 2009). Hoffman and colleagues (2012)
351
demonstrated that when the Q-pathway is elicited by moderate AIH, intrathecal U0126
352
blocks pLTF, demonstrating that MEK/ERK activity is necessary in the underlying
353
mechanism, but giving no real insight concerning where in the signaling cascade this 15
354
molecule was activated (Hoffman et al., 2012). When U0126 is administered < 5
355
minutes following moderate AIH, pLTF stalls, but does not return to baseline values,
356
thereby decreasing (but not reversing) pLTF (Hoffman et al., 2012). This observation is
357
consistent with the interpretation that ERK is upstream from BDNF synthesis versus
358
signaling downstream from TrkB activation in BDNF-dependent pMF.
359 360
BDNF induced pMF is PI3k/Akt independent
361
We confirmed that PI3K/Akt signaling is not necessary for BDNF-induced pMF, just as
362
with moderate AIH induced pLTF (Hoffman et al., 2012) (Figure 2). On the other hand,
363
the PI3K/Akt signaling pathway is necessary for the BDNF-independent, TrkB-
364
dependent S-pathway to pMF (Dale-Nagle et al. 2010; Golder et al. 2008; Hoffman et al.
365
2012; Nichols et al. 2012).
366 367
BDNF induced pMF is PKCθ dependent
368
Our finding that PKCθ signaling is necessary for BDNF-induced pMF (Figure 3) was
369
surprising, but is consistent with reports that TrkB can signal via PLC-γ/PKC in other
370
model systems (Minichiello 2009). We did not test the hypothesis that PLC-γ is
371
necessary for BDNF induced pMF since there are no selective PLC-γ inhibitors currently
372
available. On the other hand, Devinney and colleagues (2015) demonstrated that the
373
novel PKC isoform PKCθ is necessary for moderate AIH-induced pLTF, although it was
374
not determined if the necessary PKCθ activity is upstream versus downstream from
375
BDNF/TrkB signaling. Devinney et al (2015) also demonstrated PKCθ expression within
376
phrenic motor neurons, and that PKCθ knockdown via intrapleaural administration of
16
377
siRNAs targeting PKCθ mRNA abolishes pLTF. Here we used the peptide PKCθ
378
inhibitor TIP; it reliably reproduced the results of intrapleural siPKCθ injections, blocking
379
moderate AIH-induced pLTF (Devinney et al., 2015). Since TIP blocked BDNF induced
380
pMF, we suggest that the necessary PKCθ activity is downstream from BDNF/TrkB
381
activation in the Q pathway to pMF. Our findings do not rule out involvement of
382
additional PKC isoforms upstream from BDNF/TrkB signaling; for example, a currently
383
unknown conventional/novel PKC isoform is likely involved downstream from the Gq
384
protein-coupled receptors initiating the Q-pathway, but upstream from BDNF/TrkB
385
signaling. Investigating this possibility was beyond the scope of the present study.
386
Although we cannot assure that mechanisms of BDNF-induced pMF are identical to
387
those giving rise to moderate AIH-induced pLTF, the parsimonious explanation is that
388
PKCθ also acts downstream from BDNF/TrkB signaling following moderate AIH.
389 390
Significance and Conclusions
391
We conclude that BDNF-induced pMF requires PLC/PKCθ signaling, but is independent
392
of MEK/ERK or PI3K/Akt activation. On this basis, we suggest a new working model
393
with a revised sequence of protein activation in the Q-pathway to pMF (Figure 4C).
394
AIH induced pLTF is highly relevant as a model of motor neuron plasticity. In prior
395
studies, we demonstrated that moderate AIH (or repeated AIH) elicits plasticity in other
396
motor systems, including the hypoglossal motor nucleus (Bach and Mitchell 1996),
397
inspiratory intercostal motor neurons (Fregosi and Mitchell 1994; Navarrete-Opazo and
398
Mitchell 2014), recurrent laryngeal motor neurons (Xing et al. 2013) and cervical or
399
lumbar motor neurons that innervate the limbs (Lovett-Barr et al. 2012; Trumbower et
17
400
al. 2012), presumably via BDNF/TrkB-dependent mechanisms (Dale et al. 2014; Lovett-
401
Barr et al. 2012). Thus, cellular mechanisms revealed in the current study inform our
402
understanding of non-respiratory motor systems.
403
From another perspective, phrenic motor plasticity has already served as a guide to
404
clinical translation, inspiring the use of moderate AIH or repetitive AIH as a treatment to
405
restore breathing capacity and walking ability in neuromuscular disorders that
406
compromise movement, including cervical spinal injury and ALS (Dale et al. 2014;
407
Gonzalez-Rothi et al. 2015; Mahamed and Mitchell 2007). For example, AIH restores
408
breathing ability in rodent models of cervical spinal injury (Golder and Mitchell 2005;
409
Lovett-Barr et al. 2012; Navarrete-Opazo et al. 2017; Navarrete-Opazo et al. 2015) or
410
ALS (Nichols et al. 2013), and has already been demonstrated to induce functional
411
recovery of breathing (Tester et al. 2014) and walking ability (Hayes et al. 2014 ;
412
Navarrete-Opazo et al. 2017) in humans with chronic, incomplete spinal cord injury. By
413
greater understanding of cellular mechanisms giving rise to this plasticity, we can hope
414
to harness combinatorial strategies (including drugs) to enhance the functional benefits
415
of these promising therapeutic strategies. Alternately, specific drugs may be an
416
excellent alternative for patients that do not tolerate repeated hypoxic exposures by
417
inducing plasticity without systemic hypoxia (Almendros et al. 2014).
418 419 420 421 422
18
423 424 425
ACKNOWLEDGEMENTS We thank Bradley Wathen for expert technical assistance. Support provided by NIH Grant HL111598 and HL69064. IAM supported by NIH supplement to HL111598.
426
19
427
REFERENCES
428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472
Almendros I, Wang Y, and Gozal D. The polymorphic and contradictory aspects of intermittent hypoxia. Am J Physiol Lung Cell Mol Physiol 307: L129-140, 2014. Bach KB, and Mitchell GS. Hypoxia-induced long-term facilitation of respiratory activity is serotonin dependent. Respir Physiol 104: 251-260, 1996a. Baertsch NA, and Baker-Herman TL. Intermittent reductions in respiratory neural activity elicit spinal TNF-alpha-independent, atypical PKC-dependent inactivity-induced phrenic motor facilitation. American journal of physiology Regulatory, integrative and comparative physiology 308: R700-707, 2015. Baker-Herman T, Fuller D, Bavis R, Zabka A, Golder F, Doperalski N, Johnson R, Watters J, and Mitchell G. BDNF is necessary and sufficient for spinal respiratory plasticity following intermittent hypoxia. Nature neuroscience 7: 48-55, 2004. Dale-Nagle EA, Hoffman MS, MacFarlane PM, and Mitchell G. Multiple pathways to long-lasting phrenic motor facilitation. Advances in experimental medicine and biology 669: 225-230, 2010. Dale-Nagle EA, Hoffman MS, MacFarlane PM, Satriotomo I, Lovett-Barr MR, Vinit S, and Mitchell GS. Spinal plasticity following intermittent hypoxia: implications for spinal injury. Annals of the New York Academy of Sciences 1198: 252-259, 2010. Dale-Nagle EA, Satriotomo I, and Mitchell GS. Spinal vascular endothelial growth factor induces phrenic motor facilitation via extracellular signal-regulated kinase and Akt signaling. The Journal of neuroscience : the official journal of the Society for Neuroscience 31: 7682-7690, 2011. Dale EA, Ben Mabrouk F, and Mitchell GS. Unexpected benefits of intermittent hypoxia: enhanced respiratory and nonrespiratory motor function. Physiology 29: 39-48, 2014. Dale EA, Fields DP, Devinney MJ, and Mitchell GS. Phrenic motor neuron TrkB expression is necessary for acute intermittent hypoxia-induced phrenic long-term facilitation. Experimental neurology 287: 130-136, 2017. Dale EA, Satriotomo I, and Mitchell GS. Cervical spinal erythropoietin induces phrenic motor facilitation via extracellular signal-regulated protein kinase and Akt signaling. The Journal of neuroscience : the official journal of the Society for Neuroscience 32: 59735983, 2012. Devinney MJ, Huxtable AG, Nichols NL, and GS. M. Hypoxia-induced phrenic longterm facilitation: emergent properties. Annals of the New York Academy of Sciences 1279: 143-153, 2013. Devinney MJ, Fields DP, Huxtable AG, Peterson TJ, Dale EA, and Mitchell GS. Phrenic long-term facilitation requires PKCtheta activity within phrenic motor neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience 35: 8107-8117, 2015. Feldman JL, Mitchell GS, and Nattie EE. Breathing: rhythmicity, plasticity, chemosensitivity. Annual review of neuroscience 26: 239-266, 2003. Fregosi RF, and Mitchell GS. Long-term facilitation of inspiratory intercostal nerve activity following carotid sinus nerve stimulation in cats. The Journal of physiology 477 ( Pt 3): 469-479, 1994. Fuller D, Bach K, Baker T, Kinkead R, and Mitchell G. Long term facilitation of phrenic motor output. Respiration physiology 121: 135-146, 2000. 20
473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518
Fuller DD, Zabka AG, Baker TL, and Mitchell GS. Phrenic Long-term facilitation requires 5-HT receptor activation during but not following episodic hypoxia. Journal of applied physiology 90: 2001. Golder FJ, and Mitchell GS. Spinal synaptic enhancement with acute intermittent hypoxia improves respiratory function after chronic cervical spinal cord injury. The Journal of neuroscience : the official journal of the Society for Neuroscience 25: 29252932, 2005. Golder FJ, Ranganathan L, Satriotomo I, Hoffman M, Lovett-Barr MR, Watters JJ, Baker-Herman TL, and Mitchell GS. Spinal adenosine A2a receptor activation elicits long-lasting phrenic motor facilitation. The Journal of neuroscience : the official journal of the Society for Neuroscience 28: 2033-2042, 2008. Gonzalez-Rothi EJ, Lee KZ, Dale EA, Reier PJ, Mitchell GS, and Fuller DD. Intermittent Hypoxia and Neurorehabilitation. Journal of applied physiology jap 00235 02015, 2015. Hayashi F, Coles SK, Bach KB, Mitchell GS, and McCrimmon DR. Time-dependent phrenic nerve responses to carotid afferent activation: intact vs. decerebellate rats. Am J Physiol 265: R811-819, 1993. Hayes HB, Jayaraman A, Herrmann M, Mitchell GS, Rymer WZ, and Trumbower RD. Daily intermittent hypoxia enhances walking after chronic spinal cord injury: a randomized trial. Neurology 82: 104-113, 2014. Hoffman M, Nichols N, Macfarlane P, and Mitchell G. Phrenic long-term facilitation after acute intermittent hypoxia requires spinal ERK activation but not TrkB synthesis. Journal of applied physiology 113: 1184-1193, 2012. Lovett-Barr MR, Satriotomo I, Muir GD, Wilkerson JE, Hoffman MS, Vinit S, and Mitchell GS. Repetitive intermittent hypoxia induces respiratory and somatic motor recovery after chronic cervical spinal injury. The Journal of neuroscience : the official journal of the Society for Neuroscience 32: 3591-3600, 2012. MacFarlane PM, Vinit S, and Mitchell GS. Serotonin 2A and 2B receptor-induced phrenic motor facilitation: differential requirement for spinal NADPH oxidase activity. Neuroscience 178: 45-55, 2011. Mahamed S, and Mitchell GS. Is there a link between intermittent hypoxia-induced respiratory plasticity and obstructive sleep apnoea? Experimental physiology 92: 27-37, 2007. Minichiello L. TrkB signalling pathways in LTP and learning. Nature reviews Neuroscience 10: 850-860, 2009. Mitchell GS, Baker TL, Nanda SA, Fuller DD, Zabka AG, Hodgeman BA, Bavis RW, Mack KJ, and Olson EB, Jr. Invited review: Intermittent hypoxia and respiratory plasticity. Journal of applied physiology 90: 2466-2475, 2001. Mitchell GS, and Johnson SM. Neuroplasticity in respiratory motor control. Journal of applied physiology 94: 358-374, 2003. Navarrete-Opazo A, Dougherty BJ, and Mitchell GS. Enhanced recovery of breathing capacity from combined adenosine 2A receptor inhibition and daily acute intermittent hypoxia after chronic cervical spinal injury. Experimental neurology 287: 93-101, 2017. Navarrete-Opazo A, and Mitchell GS. Recruitment and plasticity in diaphragm, intercostal, and abdominal muscles in unanesthetized rats. Journal of applied physiology 117: 180-188, 2014. 21
519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537
Navarrete-Opazo A, Vinit S, Dougherty BJ, and Mitchell GS. Daily acute intermittent hypoxia elicits functional recovery of diaphragm and inspiratory intercostal muscle activity after acute cervical spinal injury. Experimental neurology 266: 1-10, 2015. Nichols NL, Dale EA, and Mitchell GS. Severe acute intermittent hypoxia elicits phrenic long-term facilitation by a novel adenosine-dependent mechanism. Journal of applied physiology 112: 1678-1688, 2012. Nichols NL, Van Dyke J, Nashold L, Satriotomo I, Suzuki M, and Mitchell GS. Ventilatory control in ALS. Respiratory physiology & neurobiology 189: 429-437, 2013. Powell FL, Milsom WK, and Mitchell GS. Time domains of the hypoxic ventilatory response. Respir Physiol 112: 123-134, 1998. Tester NJ, Fuller DD, Fromm JS, Spiess MR, Behrman AL, and Mateika JH. Longterm facilitation of ventilation in humans with chronic spinal cord injury. Am J Respir Crit Care Med 189: 57-65, 2014. Trumbower RD, Jayaraman A, Mitchell GS, and Rymer WZ. Exposure to acute intermittent hypoxia augments somatic motor function in humans with incomplete spinal cord injury. Neurorehabilitation and neural repair 26: 163-172, 2012. Xing T, Fong AY, Bautista TG, and Pilowsky PM. Acute intermittent hypoxia induced neural plasticity in respiratory motor control. Clinical and experimental pharmacology & physiology 40: 602-609, 2013.
538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 22
553
FIGURES AND TABLE
554
Figure 1. Representative traces of compressed integrated phrenic neurograms (A-D).
555
BDNF-induced pMF is MEK/ERK independent since intrathecal delivery of the
556
MEK/ERK inhibitor U0126 does not affect pMF. (A) Intrathecal MEK/ERK inhibitor
557
U0126 (100uM/12μL) administration 20 minutes before intrathecal BDNF administration
558
had no significant effect on pMF. (B) Intrathecal BDNF after the Inhibitor vehicle
559
(20%DMSO/80%Saline) elicits pMF. (C) MEK/ERK inhibitor + BDNF vehicle (aCSF)
560
does not exhibit pMF. (D) Intrathecal administration of Inhibitor vehicle + aCSF does not
561
exhibit pMF. (E) Group data for phrenic burst amplitude expressed as a percent change
562
from baseline. MEK/ERK inhibitor + BDNF (black circles, n=6), Inhibitor vehicle+ BDNF
563
(white triangles, n=10), MEK/ERK inhibitor + aCSF (white square, n=6), Time Control
564
(black triangle, n=8), were compared to determine significance between groups.
565
There were no significant differences at any time between MEK/ERK inhibitor +
566
BDNF versus Inhibitor vehicle + BDNF treated rats. There were no significant
567
differences at any time between MEK/ERK inhibitor+ aCSF versus Inhibitor vehicle+
568
aCSF. There were significant differences between the groups MEK/ERK inhibitor +
569
BDNF and Inhibitor vehicle+ BDNF versus MEK/ERK inhibitor + aCSF and Time Control
570
at 60 and 90 minutes post-BDNF or vehicle injection. Significance is P≤ 0.05; (#)-
571
indicates significance from MEK/ERK inhibitor + aCSF; (@)- indicates significance from
572
Inhibitor vehicle+ aCSF. (F) Group data for phrenic frequency expressed in
573
burst/minutes; there were no significant differences in frequency between any group at
574
any time.
575
23
576
Figure 2. Representative traces of compressed integrated phrenic neurograms (A-D).
577
BDNF- induced pMF is PI3K/Akt independent since the PI3K/Akt inhibitor PI-828 has no
578
effect. (A) Intrathecal PI3K/Akt inhibitor PI-828 (100uM/12uL) administration 20 minutes
579
before intrathecal BDNF does not affect pMF. (B) Intrathecal BDNF after the inhibitor
580
vehicle (20%DMSO/80% Saline) elicits pMF. (C) PI3K/Akt inhibitor + BDNF vehicle
581
(aCSF) does not exhibit pMF. (D) Intrathecal administration of Inhibitor vehicle + aCSF
582
does not exhibit pMF.
583
percent change from baseline. PI3K/Akt inhibitor + BDNF (black circles, n=5), Inhibitor
584
vehicle+ BDNF (white triangles, n=10), PI3K/Akt inhibitor +aCSF (white circle, n=5),
585
Time Control (black triangle, n=8), were compared to determine significance between
586
groups.
(E) Group data for phrenic burst amplitude expressed as a
587
There were no significant differences at any time between PI3K/Akt inhibitor + BDNF
588
and Inhibitor vehicle + BDNF treated rats. There were no significant differences at any
589
time between PI3K/Akt inhibitor + aCSF and Inhibitor vehicle+ aCSF. There were
590
significant differences between the groups PI3K/Akt inhibitor + BDNF and Inhibitor
591
vehicle+ BDNF versus PI3K/Akt inhibitor + aCSF and Time Control at 60 and 90
592
minutes post-BDNF. Significance is P≤ 0.05; (#)- indicates significance from PI3K/Akt
593
inhibitor PI-828 + aCSF; (@)- indicates significance from Inhibitor vehicle+ aCSF. (F)
594
Group data for phrenic frequency are expressed in burst/minutes; there were no
595
significant differences in frequency between any group at any time.
596 597
Figure 3. Representative traces of compressed integrated phrenic neurograms (A-D).
598
BDNF- induced pMF is PKCθ dependent since the PKCθ inhibitor TIP abolishes pMF.
24
599
(A) Intrathecal PKCθ inhibitor TIP (0.86mM/12uL) administration 15 minutes before
600
intrathecal BDNF abolishes pMF. (B) Intrathecal BDNF after the Inhibitor vehicle elicits
601
pMF. (C) PKCθ inhibitor + BDNF vehicle (aCSF) does not exhibit pMF. (D) Intrathecal
602
administration of Inhibitor vehicle + aCSF does not exhibit pMF. (E) Group data for
603
phrenic burst amplitude expressed as a percent change from baseline. PKCθ inhibitor +
604
BDNF (black circles, n=6), Inhibitor vehicle+ BDNF (white triangles, n=10), PKCθ
605
inhibitor + aCSF (white square, n=5), Time Control (black triangle, n=8), were compared
606
to determine significance between groups.
607
There were significant differences at 30, 60 and 90 minutes post-BDNF between
608
PKCθ inhibitor + BDNF versus Inhibitor vehicle+ BDNF, PKCθ inhibitor + aCSF and
609
Inhibitor vehicle+ aCSF.
610
Inhibitor vehicle+ BDNF, PKCθ inhibitor + aCSF and Inhibitor vehicle+ aCSF.
611
Significance is P≤ 0.05; (#)- indicates significance from PKCθ inhibitor + aCSF; (@)-
612
indicates significance from Time Control (%)- indicates significance from Inhibitor
613
vehicle+ BDNF. (F) Group data for phrenic frequency are expressed in burst/minutes;
614
there were no significant differences in frequency between any group at any time.
There was no significant difference at any time between
615 616
Figure 4. Summary of studies and new suggested working model of the Q-Pathway to
617
pMF (A-C) (A) Former working model of the Q-Pathway to pMF in which Gq coupled
618
receptors elicit complex downstream signaling. The initiating receptor was initially
619
postulated to signal via PKCθ, leading to new BDNF synthesis with subsequent TrkB
620
receptor activation. Downstream signaling was thought to be dependent on TrkB
621
induced MEK/ERK signaling, ultimately leading to pMF (Devinney et al. 2013; Baker-
25
622
Herman et al. 2004; Feldman et al. 2003; Mitchell et al. 2001). (B) We activated the Q-
623
Pathway by pharmacologically manipulating key proteins involved in the signaling
624
cascade leading to pMF (BDNF/TrkB). We administered inhibitors that blocked the three
625
canonical TrkB signaling pathways including: MEK/ERK, PI3K/Akt or PLC/PKC (PKCθ)
626
followed by intrathecal BDNF administration to elicit pMF. (C) The present study
627
demonstrates that PKCθ is the only pathway necessary for BDNF/TrkB induced pMF.
628
Thus, our working model of the Q pathway to pMF (A) requires revision; MEK/ERK must
629
be signaling upstream from BDNF/TrkB signaling, whereas PKCθ is a downstream
630
mediator (C).
631 632
26
Figure 1
Figure 2
Figure 3
Figure 4
Experimental Groups
PaCO2 (mmHg)
PaO2 (mmHg)
MAP (mmHg)
Temp (°C)
pH
Baseline
45.3±2.1
310.0±21.0
94.6±7.5
36.8±0.1
7.4±0
90 minutes
45.9±2.7
293.1±17.8
84.7.0±7.3
37.1±0.2
7.4±0
Baseline
47.0±1.7
317.1±11.8
101.3±0.6
37.1±0.2
7.4±0
90 minutes
47.5±1.7
276.5±25.9
91.2±8.0
37.2±0.2
7.4±0
Baseline
47.3±1.9
331.8±18.6
89.9±6.2
37.1±0.1
7.4±0
90 minutes
47.5±1.5
289.0±42.2
76.9±6.5
37.2±0.2
7.4±0
Baseline
49.5±1.6
308.5±24.2
114.9±2.1
37.1±0.2
7.4±0
90 minutes
49.5±1.7
283.2±36.0
97.1±1.4
37.2±0.1
7.4±0
Baseline
44.5±0.7
328.5±3.5
104.2±5.0
36.9±0.1
7.4±0
90 minutes
44.6±1.4
317.5±2.9
99.6±7.0
37.1±0.1
7.4±0
Baseline
43.6±0.9
323.25±4.7
109.6±6.4
37.5±0.1
7.4±0
90 minutes
42.7±0.9
303.0±11.4
92.8±5.7
37.1±0.1
7.4±0
Baseline
45.5±0.6
317.5±8.3
96.9±3.5
37.1±0.1
7.4±0
90 minutes
44.9±0.8
293.1±13.5
88.7±10.9
37.3±0.1
7.4±0
Baseline
45.5±3.9
294.7±39.8
100.4±6.1
37.2±0.2
7.4±0
90 minutes
44.8±3.6
295.2±33.3
88.7±10.9
37.1±0.2
7.4±0
MEK/ERK + BDNF
MEK/ERK + aCSF
PI3K/Akt + BDNF
PI3K/Akt + aCSF
PKCθ + BDNF
PKCθ + aCSF
Vehicle + BDNF
Vehicle + aCSF
Table 1. Arterial PaCO2, PaO2, mean arterial pressure (MAP), Temperature (°C) and pH during baseline and 90min of experimental groups. All values are expressed as means ± 1 SEM. Animals were treaed with the MEK/ERK inhibitor (U0126), PI3K/Akt inhibitor (PI-828), PKCθ inhibitor (TIP), inhibitor vehicle (20%
DMSO/80% saline (U0126 and PI-828) or 100% aCSF (TIP)) . The animals were either administered BDNF or aCSF (BDNF vehicle) post- inhibitor or post-inhibitor vehicle administration. There were no significant differences in any group at baseline versus 90 minutes.
1
Spinal BDNF Induced Phrenic Motor Facilitation Requires PKCθ Activity
2 Ibis M. Agosto-Marlin1 and Gordon S. Mitchell 1,2
3 4 5 6 7 8 9 10 11 12 13 14 15 16
1
Department of Comparative Biosciences University of Wisconsin Madison, WI 53706 and
2
Center for Respiratory Research and Rehabilitation Department of Physical Therapy and McKnight Brain Institute University of Florida, Gainesville, FL, 32610.
17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
*Corresponding author: Gordon S. Mitchell Department of Physical Therapy University of Florida 330 Center Drive Gainesville, FL, USA 32610 Phone: 352-273-6107 E-mail: [email protected]
32 33 34 35 36 37 38
Contributions: I.M.A.M. collected all data. I.M.A.M. and G.S.M. contributed to experimental design, data analysis and manuscript development. Both authors approved the manuscript and do not have any conflicts of interest to declare. 1 Copyright © 2017 by the American Physiological Society.
39
ABSTRACT
40
Spinal brain derived neurotrophic factor (BDNF) is necessary and sufficient for certain
41
forms of long-lasting phrenic motor facilitation (pMF). BDNF elicits pMF by binding to its
42
high-affinity receptor, tropomyosin receptor kinase B (TrkB), on phrenic motor neurons,
43
potentially activating multiple downstream signaling cascades. Canonical BDNF/TrkB
44
signaling includes the: 1) Ras/RAF/MEK/ERK MAP Kinase; 2) phosphatidylinositol 3‐
45
kinase/Akt; and 3) PLCγ/PKC pathways. Here, we demonstrate that spinal BDNF-
46
induced pMF requires PLCγ/PKCθ in normal rats, but not MEK/ERK or PI3K/Akt
47
signaling. Cervical intrathecal injections of MEK/ERK (U0126) or PI3K/Akt (PI-828; 100
48
μM;12μL) inhibitors had no effect on BDNF-induced pMF (90min post-BDNF: U0126 +
49
BDNF: 59±14%; PI-828 + BDNF: 59±8%; Inhibitor vehicle + BDNF: 56±7%; all p≥ 0.05).
50
In contrast, PKCθ inhibition with TIP (0.86 mM; 12μL) prevented BDNF-induced pMF
51
(90min post-BDNF: TIP + BDNF: -2±2%; p≤ 0.05 versus other groups). Thus, BDNF-
52
induced pMF requires downstream PLCγ/PKCθ signaling contrary to initial expectations.
53 54 55 56 57
2
58
KEY WORDS
59
respiratory
60
PLC/PKCθ, motor neuron
plasticity,
phrenic
motor
facilitation,
BDNF,
MEK/ERK,
PI3K/AKT,
61 62
NEW AND NOTEWORTHY
63
We demonstrate that BDNF induced pMF requires downstream signaling via PKCθ, but
64
not MEK/ERK or PI3K/Akt signaling. These data are essential to understand the
65
sequence of the cellular cascade leading to BDNF-dependent phrenic motor plasticity.
66 67 68 69 70 71 72 73 74 75 76 77 78 79
3
80
INTRODUCTION
81
Phrenic motor facilitation (pMF) can be elicited by acute intermittent hypoxia (AIH) or by
82
drugs injected in the cervical spinal segments near the phrenic motor nucleus. When
83
pMF is induced by AIH, it is known as phrenic long-term facilitation (pLTF; Devinney et
84
al. 2013; Fuller et al. 2000; Hayashi et al. 1993; Mitchell et al. 2001; Mitchell and
85
Johnson 2003; Powell et al. 1998); pMF is a more general term that includes AIH-
86
induced pLTF (Dale-Nagle EA et al. 2010; Mitchell and Johnson 2003). Multiple, distinct
87
cellular cascades give rise to pMF.
88
The Q-pathway to pMF is the predominant contributor to pLTF following moderate
89
AIH; it is termed the Q-pathway since multiple Gq protein-coupled metabotropic
90
receptors initiate a similar response (Dale-Nagle et al. 2010; Fuller et al. 2000). The
91
prevailing cellular model of the Q-pathway involves (in sequence): 1) spinal serotonin
92
type 2 receptor activation (5HT2; Fuller et al. 2001; MacFarlane et al. 2011), 2) protein
93
kinase C-theta (PKCθ) activity (Devinney et al. 2015), 3) new synthesis and release of
94
brain derived neurotrophic factor (BDNF; Baker-Herman et al. 2004), 4) activation of the
95
high affinity BDNF receptor, tropomyosin receptor kinase B (TrkB; Baker-Herman et al.
96
2004; Dale et al. 2017), and 5) MEK-ERK activation (Hoffman et al. 2012). Although the
97
requirement for each molecule/step is necessary for pLTF, the specific sequence has
98
been inferred, and not tested directly.
99
New BDNF protein synthesis is necessary moderate AIH-induced pLTF, and
100
BDNF/TrkB signaling alone elicits pMF (Baker-Herman et al. 2004). To establish the
101
sequence of events downstream from BDNF/TrkB signaling, we studied intrathecal
102
BDNF-induced pMF followed by selective inhibition of three potential TrkB signaling
4
103
pathways. Canonical BDNF/TrkB signaling can involve any (or all) of the following: 1)
104
Ras/Raf/MEK/ERK; 2) phosphatidylinositol 3‐ kinase (PI3K)/Akt; or 3) PLCγ/PKC
105
signaling (Minichiello 2009). Based on several recent publications, we hypothesized that
106
BDNF/TrkB signals via the MEK/ERK pathway with moderate AIH-induced pLTF
107
(Hoffman et al., 2012; Devinney et al., 2013), whereas PKCθ signaling occurs upstream
108
from new BDNF synthesis (Devinney et al., 2015). Thus, we hypothesized that
109
MEK/ERK inhibition would abolish BDNF-induced pMF, with minimal impact from
110
PI3K/Akt or PKCθ inhibition. To our surprise, BDNF-induced pMF requires PKCθ
111
signaling, and is independent of MEK/ERK or PI3K/Akt signaling. Consequently,
112
required MEK/ERK signaling in the Q-pathway to pMF must be upstream from
113
BDNF/TrkB activation, at least in normal rats.
114 115
MATERIALS AND METHODS
116
Animals
117
Adult male (3-4 months) Sprague-Dawley rats (Harlan, Colony 211 and 217) were used
118
in all experiments. Rats were housed in a controlled environment (12h light/dark cycle;
119
daily humidity and temperature monitoring), with food and water ad libitum. The
120
University of Wisconsin Animal Care and Use committee approved all experimental
121
protocols.
122 123
Experimental Series
124
We tested our hypothesis in three experimental series to investigate the role of three
125
pathways associated with BDNF/TrkB signaling in other model systems. The first
5
126
experimental series tested the hypothesis that: Spinal BDNF-induced phrenic motor
127
facilitation is MEK/ERK dependent. In this series, each group received either: 1)
128
intrathecal MEK/ERK inhibitor (U0126) prior to intrathecal BDNF (MEK/ERK inhibitor +
129
BDNF; n=6); or 2) intrathecal MEK/ERK inhibitor (U0126) prior to intrathecal BDNF
130
vehicle injections (aCSF+0.1%BSA) (MEK/ERK inhibitor + aCSF; n=6).
131
The second experimental series tested the hypothesis that: BDNF-induced phrenic
132
motor facilitation is AKT/PI3K independent. In this series, each group received
133
either: 1) intrathecal AKT/PI3K inhibitor (PI-828) prior to intrathecal BDNF (AKT/PI3K
134
inhibitor + BDNF; n=5); or 2) intrathecal AKT/PI3K inhibitor (PI-828) prior to intrathecal
135
BDNF vehicle (aCSF+0.1%BSA) (AKT/PI3K inhibitor+ aCSF group; n=4).
136
In the third experimental series, we tested the hypothesis that: BDNF-induced
137
phrenic motor facilitation is PKCθ independent. In this series, groups received
138
either: 1) intrathecal PKCθ inhibitor (TIP) before intrathecal BDNF (PKCθ inhibitor +
139
BDNF; n=6); or 2) intrathecal PKCθ inhibitor (TIP) prior to intrathecal BDNF vehicle
140
(aCSF+0.1%BSA) (PKCθ inhibitor + aCSF; n=5).
141 142
Control Groups
143
Controls groups included: 1) intrathecal 20%DMSO/80%Saline (Inhibitor vehicle for
144
U0126 and PI-282) prior to intrathecal BDNF (n=5); 2) intrathecal aCSF (TIP Inhibitor
145
vehicle) prior to intrathecal BDNF (n=5); 3) intrathecal 20%DMSO/80% Saline (Inhibitor
146
vehicle for U0126 and PI-282) prior to intrathecal BDNF vehicle (aCSF+0.1%BSA)
147
(n=3); and 4) intrathecal aCSF (TIP Inhibitor vehicle) prior to intrathecal BDNF vehicle
148
(aCSF+0.1%BSA) (n=5).
6
149
Since there were no statistically significant differences (two-way ANOVA, p=0.47)
150
between 20%DMSO/80%Saline + BDNF (n=5) and 100% aCSF + BDNF (n=5), these
151
groups were combined and renamed Inhibitor Vehicle + BDNF (n=10). Since there were
152
no
153
aCSF+0.1%BSA (n=3) and 100% aCSF + aCSF+0.1%BSA (n=5), these groups were
154
also combined and renamed Time Controls (n=8).
significant
differences
(T-test,
p=0.30)
between
20%DMSO/80%Saline
+
155 156
Surgical Protocol
157
Rats were anesthetized using isoflurane in a closed chamber and placed on a
158
temperature-regulated table. A nose cone was then used to continue isoflurane
159
administration throughout the surgery (isoflurane, 3.5% in O2 50%, balance N2). Body
160
temperature was assessed using a digital rectal probe and maintained between 36.5
161
and 37.5°C. For intravenous infusions, a tail vein catheter (24G x 3⁄4 gauge in. iv
162
catheter, Surflo) was placed (infusion rate: 0.5-1.2 ml·kg-1·h-1) throughout surgical
163
preparations and the experimental protocol. Intravenous infusions were mixed to
164
maintain fluid and acid-base balance (6:3:1 respectively) lactated Ringer’s solution,
165
HesPan (6% Hetastarch in 0.9% sodium chloride) and bicarbonate solution (8.4%
166
sodium bicarbonate solution). A tracheotomy was performed to enable artificial
167
ventilation (Rodent Respirator, model 683, Harvard Apparatus, Holliston, MA; tidal
168
volume 2.5 ml; frequency ~ 70-80). Before protocols began, the lungs were
169
hyperinflated (2 breaths) every 1.5 hours to minimize alveolar collapse.
170
A flow-through CO2 analyzer connected to the tracheal catheter was used to assess
171
end-expired PCO2 levels (maintained between 40-45mmHg during surgical preparation;
7
172
Capnogard, Novametrix, Wallingford, CT). To prevent entrainment of respiratory neural
173
activity to the ventilator, rats were bilateraly vagotomized in the mid-cervical region. A
174
catheter was placed in the right femoral artery (polyethylene catheter PE50, Intramedic)
175
to monitor blood pressure and draw arterial blood samples for blood-gas and acid-base
176
analysis (0.2–0.4 ml samples; ABL-800 Flex, Radiometer; Westlake, OH). Blood
177
pressure was monitored continuously with a pressure transducer (Gould, P23ID).
178
Measurements were made on blood samples drawn during baseline (BL), and at 15, 30,
179
60 and 90 minutes post-treatment.
180
The left phrenic nerve was isolated using a dorsal approach, cut distally,
181
desheathed, and covered with a cotton ball soaked with saline until protocols began. A
182
laminectomy (cervical-2) was performed in all rats and a small incision was made in the
183
dura to place intrathecal catheters for drug delivery near the phrenic motor nucleus.
184
Two soft silicone catheters (2 Fr; Access Technologies, Skokie, IL) were inserted 4 mm
185
caudally from the C2 durotomy until the tip rested above the C4 segment. Intrathecal
186
catheters were attached to 50-μl Hamilton syringes filled with appropriate solutions
187
(inhibitors, BDNF or vehicles).
188
After surgery, rats were converted to urethane anesthesia (1.85 g/kg, iv; delivered in
189
multiple 0.2-0.4mL bolus injections over 15-20 minutes) while isoflurane was withdrawn.
190
Once urethane anesthesia was established, anesthetic depth was confirmed via toe
191
pinched with a hemostat, while monitoring changes in phrenic nerve activity, blood
192
pressure and/or intentional movements. After conversion, a minimum of 1 h was
193
allowed before initiating protocols. Rats were euthanized via urethane overdose at the
194
end of experiments.
8
195
Neurophysiological Measurements
196
Pancuronium bromide (2.5 mg/kg iv) was used to paralyze rats during protocols. The
197
phrenic nerve was covered in mineral oil, and placed on bipolar silver electrodes for
198
nerve recordings. Phrenic nerve signals were amplified (gain 10,000X), band-pass
199
filtered (100–10,000 Hz, model 1800, A-M Systems, Carlsborg, WA), rectified and
200
integrated (Paynter filter, time constant 50 ms, MA-821, CWE, Ardmore, PA). Integrated
201
phrenic nerve bursts were digitized (8kHz) and analyzed using a WINDAQ data-
202
acquisition systems (DATAQ Instruments, Akron, OH).
203
Before initiating protocols, the CO2 apneic threshold was determined by lowering
204
end-tidal CO2 until phrenic nerve activity ceased for ~1 min. The recruitment threshold
205
was then determined by slowly increasing end-tidal CO2 until nerve activity resumed.
206
The end-tidal CO2 was raised ~2 mmHg above the recruitment threshold and ~15–20
207
minutes were allowed to achieve a stable baseline.
208 209
Drug treatments
210
Brain derived neurotrophic factor (BDNF). Recombinant BDNF protein (Promega)
211
was diluted in double distilled water to make a stock solution (5µg), aliquoted into
212
multiple vials and stored at -20°C. The BDNF stock solution was diluted on the day of
213
use to 100ng in aCSF+0.1%BSA solution. The intrathecal BDNF dose (100ng; 12uL)
214
was the same as that used in a previous study from our group (Baker-Herman et al.
215
2004). Control groups only received artificial cerebrospinal fluid (aCSF) (in mM: 120
216
NaCl, 3 KCl,2 CaCl, 2 MgCl, 23 NaHCO3, 10 glucose, bubbled with 95%O2/5%CO2)
217
with 0.1% BSA.
9
218
MEK/ERK inhibitor (U0126) The membrane permeable MEK/ERK inhibitor (U0126
219
or 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene, Promega) was
220
dissolved in 100% DMSO, and then diluted with saline to create a 100 μM solution in
221
20%DMSO/80%Saline. The inhibitor was diluted to its injected concentration on the day
222
of use, and was administered (100 μM; 12μL) 20 minutes before BDNF (or BDNF
223
vehicle) administration.
224
Dose/response studies using intrathecal U0126 had been established in prior
225
studies from our laboratory (Dale-Nagle et al. 2011; Dale et al. 2012; Hoffman et al.
226
2012). We chose a dose shown to inhibit BDNF-dependent pLTF elicited by moderate
227
AIH (Hoffman et al. 2012). Controls were 20%DMSO/80%Saline injections of the same
228
volume. To demonstrate drug efficacy, we confirmed that this drug dose blocks
229
moderate AIH-induced pLTF in two rats (data not shown).
230
PI3K/Akt inhibitor (PI-828) The PI3K/Akt inhibitor (PI-828; 1,4-diamino-2,3-dicyano-
231
1,4-bis(o-aminophenylmercapto) butadiene, Tocris) was dissolved in 100% DMSO and
232
frozen in aliquots. The day of use, it was diluted with saline to a final concentration of
233
100 μM in 20%DMSO/80%Saline. The inhibitor was administered (100 μM; 12μL) 20
234
minutes before BDNF administration. Earlier studies from our laboratory used this same
235
PI-828 dose, and confirmed efficacy (Dale-Nagle et al. 2011; Dale et al. 2012; Hoffman
236
et al. 2012). For controls, we administered 20%DMSO/80%Saline at the same volume
237
as the inhibitor. To confirm drug efficacy, we gave the same dose and concentration of
238
the PI3K inhibitor prior to severe AIH since this form of pLTF is adenosine 2A receptor
239
and PI3K/Akt dependent (Nichols et al., 2012); severe AIH-induced pLTF was
240
successfully blocked in two rats (data not shown).
10
241
PKCθ inhibitor (TIP). Theta inhibitory peptide (TIP; Calbiochem) is a myristoylated
242
peptide mimicking the pseudosubstrate domain of PKCθ. TIP was dissolved in 100%
243
aCSF and stored at -20°C (1mM). The day of use it was diluted with aCSF to a final
244
concentration of 0.86 mM. The inhibitor was administered (0.86mM; 12 μL total, 1
245
μL/15s) 15 minutes before BDNF administration. We previously demonstrated that this
246
same dose and route of TIP administration blocks moderate AIH-induced pLTF
247
(Devinney et al. 2015). For controls, we administered 100% aCSF at the same volume
248
and rate of the inhibitor. To test that the drugs effect, we gave the same dose and
249
concentration of TIP prior to delivering inactivity phrenic motor facilitation (iPMF). iPMF
250
is a form of plasticity dependent on protein kinase C zeta (PKCζ), and independent from
251
PKCθ (Baertsch and Baker-Herman 2015). As expected TIP failed to block iPMF (n = 2;
252
data not shown).
253 254
Time Control
255
Time control experiments controlled for time-dependent changes in phrenic nerve
256
activity characteristic in anesthetized animals. Our time control experiments were the
257
Inhibitor vehicle group (20%DMSO/80%Saline or aCSF) and the BDNF vehicle
258
(aCSF+0.1% BSA). Inhibitor vehicle injections were done 15-20 minutes before BDNF
259
vehicle, mimicking the timing of drug injections described above.
260 261
Statistical analysis
262
Integrated phrenic burst amplitude was normalized as a percent change from baseline.
263
Respiratory frequency was also normalized as a change from baseline levels (% 11
264
baseline), but was expressed as an absolute difference (bursts per minute). Statistical
265
comparisons were made within and between treatment groups using a two-way ANOVA
266
with a repeated measures design (Prism 6; GraphPad Software). The same statistical
267
comparisons were done for mean arterial pressure (MAP), arterial blood gases, pH, and
268
rectal temperature (BL and 90min time). To detect significant differences between
269
experimental groups, a two-way repeated measures ANOVA was used (Prism 6;
270
GraphPad Software); comparisons were made within and between treatment groups for
271
phrenic nerve burst amplitude and frequency (BL, 15, 30, 60, and 90-min). Individual
272
comparisons were made using Tukey’s post hoc test. To determine significance
273
between control groups, two different tests were used. For the 20%DMSO/80%Saline +
274
BDNF vs aCSF+ BDNF a two-way ANOVA was used and for the 20%DMSO/80%Saline
275
+ aCSF+0.1%BSA vs 100% aCSF + aCSF+0.1%BSA, a T-test was performed. For all
276
analyses, the significance level was set to 0.05; data are means ± SE.
277 278
RESULTS
279
Blood Gases and Arterial Pressure: Baseline and 90-min post-treatment
280
Throughout protocols, PaCO2 was successfully regulated within ~2 mmHg from baseline
281
value and PaO2 was kept above 150 mmHg. PaCO2 and PaO2 regulation during protocols
282
was similar in all experimental groups (Table 1). Mean arterial pressure, temperature
283
and pH were similar among groups at baseline and at 90 minutes post-hypoxia (Table
284
1). Thus, differences in PaCO2, PaO2, MAP, temperature or pH regulation among groups
285
cannot account for differential pMF responses observed in this study.
286 12
287
BDNF induced pMF is MEK/ERK independent
288
To test the hypothesis that BDNF-induced pMF requires MEK/ERK signaling, we
289
delivered the MEK/ERK inhibitor U0126 (intrathecal delivery) 20 minutes before BDNF
290
injections. Typical neurograms representing phrenic activity in Figure 2 (A-D) illustrate
291
that MEK/ERK inhibition does not affect BDNF-induced pMF. Indeed, pMF magnitude
292
was not significantly different when comparing the MEK/ERK inhibitor + BDNF group
293
(60 minutes post-BDNF: 41±11%; 90 min: 59±14%; n=6) versus the Inhibitor vehicle +
294
BDNF group (60 min: 49±7%; 90 min: 57±6%; n=10) (P≥0.05; Figure 1E).
295
On the other hand, pMF was greater in both the MEK/ERK inhibitor + BDNF (60 min:
296
41±11%; 90 min: 59±14%; n=6) and Inhibitor vehicle + BDNF (60 min: 49±7%; 90 min:
297
57±6%; n=10) versus MEK/ERK inhibitor + aCSF (60 min: 15±2%; 90 min: 18±5%;
298
n=6) and Time Control (60 min: 5±3%; 90 min: 4±3%; n=7) (P≤0.05; Figures 1E). The
299
MEK/ERK inhibitor + aCSF and Time Control groups were not significantly different from
300
one another at any time (P≥0.05; Figure 1E). There was no significant difference in
301
frequency at any time in any group (P≥0.05; Figure 1F).
302 303
BDNF induced pMF is PI3K/AKT independent
304
To test the hypothesis that BDNF-induced pMF requires PI3K/Akt signaling, we
305
delivered the PI3K/Akt inhibitor PI-828 (intrathecal delivery) 20 minutes before BDNF.
306
Typical neurograms representing phrenic nerve output (Figures 2 A-D) illustrate that
307
PI3K/Akt inhibitor does not affect BDNF-induced pMF. Indeed, pMF magnitude was not
308
significantly different when comparing the PI3K/Akt inhibitor + BDNF group (60 minutes
13
309
post-BDNF: 64±8%; 90 min: 59±8%; n=5) versus the Inhibitor vehicle + BDNF group (60
310
min: 49±7%; 90 min: 57±6%; n=10) (P ≥ 0.05; Figure 2E).
311
On the other hand, pMF was greater in both the PI3K/Akt inhibitor + BDNF (60 min:
312
64±8%; 90 min: 59±8%; n=5) and Inhibitor vehicle + BDNF (60 min: 49±7%; 90 min:
313
57±6%; n=10) versus PI3K/Akt inhibitor + aCSF (60 min: 8±9%; 90 min: 18±5%; n=4)
314
and Time Control (60 min: 5±3%; 90 min: 4±3%; n=7) (P≤0.05; Figures 2E). The
315
PI3K/Akt inhibitor + aCSF and Time Control groups were not significantly different from
316
one another at any time (P≥0.05; Figure 2E). There was no significant difference in
317
frequency at any time in any group (P≤0.05; Figure 2F).
318 319
BDNF induced pMF is PKCθ dependent
320
To test the hypothesis that BDNF-induced pMF is independent of PLC/PKCθ signaling,
321
we delivered the PKCθ inhibitor TIP (intrathecal delivery) 15 minutes before BDNF
322
administration. Typical neurograms representing phrenic nerve output are shown in
323
Figure 3 (A-D), illustrating that, contrary to our hypothesis, PKCθ inhibition abolishes
324
BDNF-induced pMF. In PKCθ inhibitor + BDNF treated rats (n=6), there was a
325
significant decrease in the magnitude of pMF, starting at 30 minutes after BDNF
326
administration, relative to the Inhibitor vehicle + BDNF group (n=10) (30min PKCθ
327
inhibitor+ BDNF 7±6%; versus Inhibitor vehicle + BDNF 26±6%; 60min PKCθ inhibitor +
328
BDNF 12±5% versus Inhibitor vehicle + BDNF 49±7%; 90min PKCθ inhibitor + BDNF -
329
2±2% versus Inhibitor vehicle + BDNF: 57±6%; all P≤0.05; Figure 3E).
14
330
The PKCθ inhibitor + BDNF group was not significantly different at any time versus
331
PKCθ inhibitor + aCSF or Time Control groups (P≥0.05; Figure 3E). There were no
332
significant differences in frequency at any time in any group (P≥0.05; Figure 3F).
333 334
DISCUSSION
335
Contrary to our initial hypothesis, BDNF-induced pMF requires PKCθ activity, and is
336
independent of MEK/ERK or PI3K/Akt activity in normal rats (Figure 4). Doses and
337
selectivity of inhibitors were verified in other studies from our laboratory. Thus,
338
previously published working models of the Q-Pathway to pMF require revision (Dale-
339
Nagle et al. 2010; Dale-Nagle et al. 2010; Dale et al. 2014). We now suggest that the
340
correct protein activation sequence in moderate AIH-induced pLTF (ie. the Q-Pathway)
341
is: 1) MEK/ERK signaling upstream from new BDNF synthesis and release; and 2)
342
BDNF/TrkB signaling via PLC/PKCθ activation. These findings elaborate our model and
343
increase understanding of mechanisms giving rise to phrenic motor plasticity.
344 345
BDNF induced pMF is MEK/ERK independent
346
We demonstrate that MEK/ERK signaling is not required for BDNF-induced pMF (Figure
347
1). These data are surprising since we initially hypothesized that BDNF-induced pMF
348
required MEK/ERK signaling based on the observation that MEK/ERK inhibition
349
abolishes moderate AIH-induced pLTF (Hoffman et al., 2012), and MEK/ERK is a
350
common TrkB signaling pathway (Minichiello 2009). Hoffman and colleagues (2012)
351
demonstrated that when the Q-pathway is elicited by moderate AIH, intrathecal U0126
352
blocks pLTF, demonstrating that MEK/ERK activity is necessary in the underlying
353
mechanism, but giving no real insight concerning where in the signaling cascade this 15
354
molecule was activated (Hoffman et al., 2012). When U0126 is administered < 5
355
minutes following moderate AIH, pLTF stalls, but does not return to baseline values,
356
thereby decreasing (but not reversing) pLTF (Hoffman et al., 2012). This observation is
357
consistent with the interpretation that ERK is upstream from BDNF synthesis versus
358
signaling downstream from TrkB activation in BDNF-dependent pMF.
359 360
BDNF induced pMF is PI3k/Akt independent
361
We confirmed that PI3K/Akt signaling is not necessary for BDNF-induced pMF, just as
362
with moderate AIH induced pLTF (Hoffman et al., 2012) (Figure 2). On the other hand,
363
the PI3K/Akt signaling pathway is necessary for the BDNF-independent, TrkB-
364
dependent S-pathway to pMF (Dale-Nagle et al. 2010; Golder et al. 2008; Hoffman et al.
365
2012; Nichols et al. 2012).
366 367
BDNF induced pMF is PKCθ dependent
368
Our finding that PKCθ signaling is necessary for BDNF-induced pMF (Figure 3) was
369
surprising, but is consistent with reports that TrkB can signal via PLC-γ/PKC in other
370
model systems (Minichiello 2009). We did not test the hypothesis that PLC-γ is
371
necessary for BDNF induced pMF since there are no selective PLC-γ inhibitors currently
372
available. On the other hand, Devinney and colleagues (2015) demonstrated that the
373
novel PKC isoform PKCθ is necessary for moderate AIH-induced pLTF, although it was
374
not determined if the necessary PKCθ activity is upstream versus downstream from
375
BDNF/TrkB signaling. Devinney et al (2015) also demonstrated PKCθ expression within
376
phrenic motor neurons, and that PKCθ knockdown via intrapleaural administration of
16
377
siRNAs targeting PKCθ mRNA abolishes pLTF. Here we used the peptide PKCθ
378
inhibitor TIP; it reliably reproduced the results of intrapleural siPKCθ injections, blocking
379
moderate AIH-induced pLTF (Devinney et al., 2015). Since TIP blocked BDNF induced
380
pMF, we suggest that the necessary PKCθ activity is downstream from BDNF/TrkB
381
activation in the Q pathway to pMF. Our findings do not rule out involvement of
382
additional PKC isoforms upstream from BDNF/TrkB signaling; for example, a currently
383
unknown conventional/novel PKC isoform is likely involved downstream from the Gq
384
protein-coupled receptors initiating the Q-pathway, but upstream from BDNF/TrkB
385
signaling. Investigating this possibility was beyond the scope of the present study.
386
Although we cannot assure that mechanisms of BDNF-induced pMF are identical to
387
those giving rise to moderate AIH-induced pLTF, the parsimonious explanation is that
388
PKCθ also acts downstream from BDNF/TrkB signaling following moderate AIH.
389 390
Significance and Conclusions
391
We conclude that BDNF-induced pMF requires PLC/PKCθ signaling, but is independent
392
of MEK/ERK or PI3K/Akt activation. On this basis, we suggest a new working model
393
with a revised sequence of protein activation in the Q-pathway to pMF (Figure 4C).
394
AIH induced pLTF is highly relevant as a model of motor neuron plasticity. In prior
395
studies, we demonstrated that moderate AIH (or repeated AIH) elicits plasticity in other
396
motor systems, including the hypoglossal motor nucleus (Bach and Mitchell 1996),
397
inspiratory intercostal motor neurons (Fregosi and Mitchell 1994; Navarrete-Opazo and
398
Mitchell 2014), recurrent laryngeal motor neurons (Xing et al. 2013) and cervical or
399
lumbar motor neurons that innervate the limbs (Lovett-Barr et al. 2012; Trumbower et
17
400
al. 2012), presumably via BDNF/TrkB-dependent mechanisms (Dale et al. 2014; Lovett-
401
Barr et al. 2012). Thus, cellular mechanisms revealed in the current study inform our
402
understanding of non-respiratory motor systems.
403
From another perspective, phrenic motor plasticity has already served as a guide to
404
clinical translation, inspiring the use of moderate AIH or repetitive AIH as a treatment to
405
restore breathing capacity and walking ability in neuromuscular disorders that
406
compromise movement, including cervical spinal injury and ALS (Dale et al. 2014;
407
Gonzalez-Rothi et al. 2015; Mahamed and Mitchell 2007). For example, AIH restores
408
breathing ability in rodent models of cervical spinal injury (Golder and Mitchell 2005;
409
Lovett-Barr et al. 2012; Navarrete-Opazo et al. 2017; Navarrete-Opazo et al. 2015) or
410
ALS (Nichols et al. 2013), and has already been demonstrated to induce functional
411
recovery of breathing (Tester et al. 2014) and walking ability (Hayes et al. 2014 ;
412
Navarrete-Opazo et al. 2017) in humans with chronic, incomplete spinal cord injury. By
413
greater understanding of cellular mechanisms giving rise to this plasticity, we can hope
414
to harness combinatorial strategies (including drugs) to enhance the functional benefits
415
of these promising therapeutic strategies. Alternately, specific drugs may be an
416
excellent alternative for patients that do not tolerate repeated hypoxic exposures by
417
inducing plasticity without systemic hypoxia (Almendros et al. 2014).
418 419 420 421 422
18
423 424 425
ACKNOWLEDGEMENTS We thank Bradley Wathen for expert technical assistance. Support provided by NIH Grant HL111598 and HL69064. IAM supported by NIH supplement to HL111598.
426
19
427
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Almendros I, Wang Y, and Gozal D. The polymorphic and contradictory aspects of intermittent hypoxia. Am J Physiol Lung Cell Mol Physiol 307: L129-140, 2014. Bach KB, and Mitchell GS. Hypoxia-induced long-term facilitation of respiratory activity is serotonin dependent. Respir Physiol 104: 251-260, 1996a. Baertsch NA, and Baker-Herman TL. Intermittent reductions in respiratory neural activity elicit spinal TNF-alpha-independent, atypical PKC-dependent inactivity-induced phrenic motor facilitation. American journal of physiology Regulatory, integrative and comparative physiology 308: R700-707, 2015. Baker-Herman T, Fuller D, Bavis R, Zabka A, Golder F, Doperalski N, Johnson R, Watters J, and Mitchell G. BDNF is necessary and sufficient for spinal respiratory plasticity following intermittent hypoxia. Nature neuroscience 7: 48-55, 2004. Dale-Nagle EA, Hoffman MS, MacFarlane PM, and Mitchell G. Multiple pathways to long-lasting phrenic motor facilitation. Advances in experimental medicine and biology 669: 225-230, 2010. Dale-Nagle EA, Hoffman MS, MacFarlane PM, Satriotomo I, Lovett-Barr MR, Vinit S, and Mitchell GS. Spinal plasticity following intermittent hypoxia: implications for spinal injury. Annals of the New York Academy of Sciences 1198: 252-259, 2010. Dale-Nagle EA, Satriotomo I, and Mitchell GS. Spinal vascular endothelial growth factor induces phrenic motor facilitation via extracellular signal-regulated kinase and Akt signaling. The Journal of neuroscience : the official journal of the Society for Neuroscience 31: 7682-7690, 2011. Dale EA, Ben Mabrouk F, and Mitchell GS. Unexpected benefits of intermittent hypoxia: enhanced respiratory and nonrespiratory motor function. Physiology 29: 39-48, 2014. Dale EA, Fields DP, Devinney MJ, and Mitchell GS. Phrenic motor neuron TrkB expression is necessary for acute intermittent hypoxia-induced phrenic long-term facilitation. Experimental neurology 287: 130-136, 2017. Dale EA, Satriotomo I, and Mitchell GS. Cervical spinal erythropoietin induces phrenic motor facilitation via extracellular signal-regulated protein kinase and Akt signaling. The Journal of neuroscience : the official journal of the Society for Neuroscience 32: 59735983, 2012. Devinney MJ, Huxtable AG, Nichols NL, and GS. M. Hypoxia-induced phrenic longterm facilitation: emergent properties. Annals of the New York Academy of Sciences 1279: 143-153, 2013. Devinney MJ, Fields DP, Huxtable AG, Peterson TJ, Dale EA, and Mitchell GS. Phrenic long-term facilitation requires PKCtheta activity within phrenic motor neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience 35: 8107-8117, 2015. Feldman JL, Mitchell GS, and Nattie EE. Breathing: rhythmicity, plasticity, chemosensitivity. Annual review of neuroscience 26: 239-266, 2003. Fregosi RF, and Mitchell GS. Long-term facilitation of inspiratory intercostal nerve activity following carotid sinus nerve stimulation in cats. The Journal of physiology 477 ( Pt 3): 469-479, 1994. Fuller D, Bach K, Baker T, Kinkead R, and Mitchell G. Long term facilitation of phrenic motor output. Respiration physiology 121: 135-146, 2000. 20
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Fuller DD, Zabka AG, Baker TL, and Mitchell GS. Phrenic Long-term facilitation requires 5-HT receptor activation during but not following episodic hypoxia. Journal of applied physiology 90: 2001. Golder FJ, and Mitchell GS. Spinal synaptic enhancement with acute intermittent hypoxia improves respiratory function after chronic cervical spinal cord injury. The Journal of neuroscience : the official journal of the Society for Neuroscience 25: 29252932, 2005. Golder FJ, Ranganathan L, Satriotomo I, Hoffman M, Lovett-Barr MR, Watters JJ, Baker-Herman TL, and Mitchell GS. Spinal adenosine A2a receptor activation elicits long-lasting phrenic motor facilitation. The Journal of neuroscience : the official journal of the Society for Neuroscience 28: 2033-2042, 2008. Gonzalez-Rothi EJ, Lee KZ, Dale EA, Reier PJ, Mitchell GS, and Fuller DD. Intermittent Hypoxia and Neurorehabilitation. Journal of applied physiology jap 00235 02015, 2015. Hayashi F, Coles SK, Bach KB, Mitchell GS, and McCrimmon DR. Time-dependent phrenic nerve responses to carotid afferent activation: intact vs. decerebellate rats. Am J Physiol 265: R811-819, 1993. Hayes HB, Jayaraman A, Herrmann M, Mitchell GS, Rymer WZ, and Trumbower RD. Daily intermittent hypoxia enhances walking after chronic spinal cord injury: a randomized trial. Neurology 82: 104-113, 2014. Hoffman M, Nichols N, Macfarlane P, and Mitchell G. Phrenic long-term facilitation after acute intermittent hypoxia requires spinal ERK activation but not TrkB synthesis. Journal of applied physiology 113: 1184-1193, 2012. Lovett-Barr MR, Satriotomo I, Muir GD, Wilkerson JE, Hoffman MS, Vinit S, and Mitchell GS. Repetitive intermittent hypoxia induces respiratory and somatic motor recovery after chronic cervical spinal injury. The Journal of neuroscience : the official journal of the Society for Neuroscience 32: 3591-3600, 2012. MacFarlane PM, Vinit S, and Mitchell GS. Serotonin 2A and 2B receptor-induced phrenic motor facilitation: differential requirement for spinal NADPH oxidase activity. Neuroscience 178: 45-55, 2011. Mahamed S, and Mitchell GS. Is there a link between intermittent hypoxia-induced respiratory plasticity and obstructive sleep apnoea? Experimental physiology 92: 27-37, 2007. Minichiello L. TrkB signalling pathways in LTP and learning. Nature reviews Neuroscience 10: 850-860, 2009. Mitchell GS, Baker TL, Nanda SA, Fuller DD, Zabka AG, Hodgeman BA, Bavis RW, Mack KJ, and Olson EB, Jr. Invited review: Intermittent hypoxia and respiratory plasticity. Journal of applied physiology 90: 2466-2475, 2001. Mitchell GS, and Johnson SM. Neuroplasticity in respiratory motor control. Journal of applied physiology 94: 358-374, 2003. Navarrete-Opazo A, Dougherty BJ, and Mitchell GS. Enhanced recovery of breathing capacity from combined adenosine 2A receptor inhibition and daily acute intermittent hypoxia after chronic cervical spinal injury. Experimental neurology 287: 93-101, 2017. Navarrete-Opazo A, and Mitchell GS. Recruitment and plasticity in diaphragm, intercostal, and abdominal muscles in unanesthetized rats. Journal of applied physiology 117: 180-188, 2014. 21
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Navarrete-Opazo A, Vinit S, Dougherty BJ, and Mitchell GS. Daily acute intermittent hypoxia elicits functional recovery of diaphragm and inspiratory intercostal muscle activity after acute cervical spinal injury. Experimental neurology 266: 1-10, 2015. Nichols NL, Dale EA, and Mitchell GS. Severe acute intermittent hypoxia elicits phrenic long-term facilitation by a novel adenosine-dependent mechanism. Journal of applied physiology 112: 1678-1688, 2012. Nichols NL, Van Dyke J, Nashold L, Satriotomo I, Suzuki M, and Mitchell GS. Ventilatory control in ALS. Respiratory physiology & neurobiology 189: 429-437, 2013. Powell FL, Milsom WK, and Mitchell GS. Time domains of the hypoxic ventilatory response. Respir Physiol 112: 123-134, 1998. Tester NJ, Fuller DD, Fromm JS, Spiess MR, Behrman AL, and Mateika JH. Longterm facilitation of ventilation in humans with chronic spinal cord injury. Am J Respir Crit Care Med 189: 57-65, 2014. Trumbower RD, Jayaraman A, Mitchell GS, and Rymer WZ. Exposure to acute intermittent hypoxia augments somatic motor function in humans with incomplete spinal cord injury. Neurorehabilitation and neural repair 26: 163-172, 2012. Xing T, Fong AY, Bautista TG, and Pilowsky PM. Acute intermittent hypoxia induced neural plasticity in respiratory motor control. Clinical and experimental pharmacology & physiology 40: 602-609, 2013.
538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 22
553
FIGURES AND TABLE
554
Figure 1. Representative traces of compressed integrated phrenic neurograms (A-D).
555
BDNF-induced pMF is MEK/ERK independent since intrathecal delivery of the
556
MEK/ERK inhibitor U0126 does not affect pMF. (A) Intrathecal MEK/ERK inhibitor
557
U0126 (100uM/12μL) administration 20 minutes before intrathecal BDNF administration
558
had no significant effect on pMF. (B) Intrathecal BDNF after the Inhibitor vehicle
559
(20%DMSO/80%Saline) elicits pMF. (C) MEK/ERK inhibitor + BDNF vehicle (aCSF)
560
does not exhibit pMF. (D) Intrathecal administration of Inhibitor vehicle + aCSF does not
561
exhibit pMF. (E) Group data for phrenic burst amplitude expressed as a percent change
562
from baseline. MEK/ERK inhibitor + BDNF (black circles, n=6), Inhibitor vehicle+ BDNF
563
(white triangles, n=10), MEK/ERK inhibitor + aCSF (white square, n=6), Time Control
564
(black triangle, n=8), were compared to determine significance between groups.
565
There were no significant differences at any time between MEK/ERK inhibitor +
566
BDNF versus Inhibitor vehicle + BDNF treated rats. There were no significant
567
differences at any time between MEK/ERK inhibitor+ aCSF versus Inhibitor vehicle+
568
aCSF. There were significant differences between the groups MEK/ERK inhibitor +
569
BDNF and Inhibitor vehicle+ BDNF versus MEK/ERK inhibitor + aCSF and Time Control
570
at 60 and 90 minutes post-BDNF or vehicle injection. Significance is P≤ 0.05; (#)-
571
indicates significance from MEK/ERK inhibitor + aCSF; (@)- indicates significance from
572
Inhibitor vehicle+ aCSF. (F) Group data for phrenic frequency expressed in
573
burst/minutes; there were no significant differences in frequency between any group at
574
any time.
575
23
576
Figure 2. Representative traces of compressed integrated phrenic neurograms (A-D).
577
BDNF- induced pMF is PI3K/Akt independent since the PI3K/Akt inhibitor PI-828 has no
578
effect. (A) Intrathecal PI3K/Akt inhibitor PI-828 (100uM/12uL) administration 20 minutes
579
before intrathecal BDNF does not affect pMF. (B) Intrathecal BDNF after the inhibitor
580
vehicle (20%DMSO/80% Saline) elicits pMF. (C) PI3K/Akt inhibitor + BDNF vehicle
581
(aCSF) does not exhibit pMF. (D) Intrathecal administration of Inhibitor vehicle + aCSF
582
does not exhibit pMF.
583
percent change from baseline. PI3K/Akt inhibitor + BDNF (black circles, n=5), Inhibitor
584
vehicle+ BDNF (white triangles, n=10), PI3K/Akt inhibitor +aCSF (white circle, n=5),
585
Time Control (black triangle, n=8), were compared to determine significance between
586
groups.
(E) Group data for phrenic burst amplitude expressed as a
587
There were no significant differences at any time between PI3K/Akt inhibitor + BDNF
588
and Inhibitor vehicle + BDNF treated rats. There were no significant differences at any
589
time between PI3K/Akt inhibitor + aCSF and Inhibitor vehicle+ aCSF. There were
590
significant differences between the groups PI3K/Akt inhibitor + BDNF and Inhibitor
591
vehicle+ BDNF versus PI3K/Akt inhibitor + aCSF and Time Control at 60 and 90
592
minutes post-BDNF. Significance is P≤ 0.05; (#)- indicates significance from PI3K/Akt
593
inhibitor PI-828 + aCSF; (@)- indicates significance from Inhibitor vehicle+ aCSF. (F)
594
Group data for phrenic frequency are expressed in burst/minutes; there were no
595
significant differences in frequency between any group at any time.
596 597
Figure 3. Representative traces of compressed integrated phrenic neurograms (A-D).
598
BDNF- induced pMF is PKCθ dependent since the PKCθ inhibitor TIP abolishes pMF.
24
599
(A) Intrathecal PKCθ inhibitor TIP (0.86mM/12uL) administration 15 minutes before
600
intrathecal BDNF abolishes pMF. (B) Intrathecal BDNF after the Inhibitor vehicle elicits
601
pMF. (C) PKCθ inhibitor + BDNF vehicle (aCSF) does not exhibit pMF. (D) Intrathecal
602
administration of Inhibitor vehicle + aCSF does not exhibit pMF. (E) Group data for
603
phrenic burst amplitude expressed as a percent change from baseline. PKCθ inhibitor +
604
BDNF (black circles, n=6), Inhibitor vehicle+ BDNF (white triangles, n=10), PKCθ
605
inhibitor + aCSF (white square, n=5), Time Control (black triangle, n=8), were compared
606
to determine significance between groups.
607
There were significant differences at 30, 60 and 90 minutes post-BDNF between
608
PKCθ inhibitor + BDNF versus Inhibitor vehicle+ BDNF, PKCθ inhibitor + aCSF and
609
Inhibitor vehicle+ aCSF.
610
Inhibitor vehicle+ BDNF, PKCθ inhibitor + aCSF and Inhibitor vehicle+ aCSF.
611
Significance is P≤ 0.05; (#)- indicates significance from PKCθ inhibitor + aCSF; (@)-
612
indicates significance from Time Control (%)- indicates significance from Inhibitor
613
vehicle+ BDNF. (F) Group data for phrenic frequency are expressed in burst/minutes;
614
there were no significant differences in frequency between any group at any time.
There was no significant difference at any time between
615 616
Figure 4. Summary of studies and new suggested working model of the Q-Pathway to
617
pMF (A-C) (A) Former working model of the Q-Pathway to pMF in which Gq coupled
618
receptors elicit complex downstream signaling. The initiating receptor was initially
619
postulated to signal via PKCθ, leading to new BDNF synthesis with subsequent TrkB
620
receptor activation. Downstream signaling was thought to be dependent on TrkB
621
induced MEK/ERK signaling, ultimately leading to pMF (Devinney et al. 2013; Baker-
25
622
Herman et al. 2004; Feldman et al. 2003; Mitchell et al. 2001). (B) We activated the Q-
623
Pathway by pharmacologically manipulating key proteins involved in the signaling
624
cascade leading to pMF (BDNF/TrkB). We administered inhibitors that blocked the three
625
canonical TrkB signaling pathways including: MEK/ERK, PI3K/Akt or PLC/PKC (PKCθ)
626
followed by intrathecal BDNF administration to elicit pMF. (C) The present study
627
demonstrates that PKCθ is the only pathway necessary for BDNF/TrkB induced pMF.
628
Thus, our working model of the Q pathway to pMF (A) requires revision; MEK/ERK must
629
be signaling upstream from BDNF/TrkB signaling, whereas PKCθ is a downstream
630
mediator (C).
631 632
26
Figure 1
Figure 2
Figure 3
Figure 4
Experimental Groups
PaCO2 (mmHg)
PaO2 (mmHg)
MAP (mmHg)
Temp (°C)
pH
Baseline
45.3±2.1
310.0±21.0
94.6±7.5
36.8±0.1
7.4±0
90 minutes
45.9±2.7
293.1±17.8
84.7.0±7.3
37.1±0.2
7.4±0
Baseline
47.0±1.7
317.1±11.8
101.3±0.6
37.1±0.2
7.4±0
90 minutes
47.5±1.7
276.5±25.9
91.2±8.0
37.2±0.2
7.4±0
Baseline
47.3±1.9
331.8±18.6
89.9±6.2
37.1±0.1
7.4±0
90 minutes
47.5±1.5
289.0±42.2
76.9±6.5
37.2±0.2
7.4±0
Baseline
49.5±1.6
308.5±24.2
114.9±2.1
37.1±0.2
7.4±0
90 minutes
49.5±1.7
283.2±36.0
97.1±1.4
37.2±0.1
7.4±0
Baseline
44.5±0.7
328.5±3.5
104.2±5.0
36.9±0.1
7.4±0
90 minutes
44.6±1.4
317.5±2.9
99.6±7.0
37.1±0.1
7.4±0
Baseline
43.6±0.9
323.25±4.7
109.6±6.4
37.5±0.1
7.4±0
90 minutes
42.7±0.9
303.0±11.4
92.8±5.7
37.1±0.1
7.4±0
Baseline
45.5±0.6
317.5±8.3
96.9±3.5
37.1±0.1
7.4±0
90 minutes
44.9±0.8
293.1±13.5
88.7±10.9
37.3±0.1
7.4±0
Baseline
45.5±3.9
294.7±39.8
100.4±6.1
37.2±0.2
7.4±0
90 minutes
44.8±3.6
295.2±33.3
88.7±10.9
37.1±0.2
7.4±0
MEK/ERK + BDNF
MEK/ERK + aCSF
PI3K/Akt + BDNF
PI3K/Akt + aCSF
PKCθ + BDNF
PKCθ + aCSF
Vehicle + BDNF
Vehicle + aCSF
Table 1. Arterial PaCO2, PaO2, mean arterial pressure (MAP), Temperature (°C) and pH during baseline and 90min of experimental groups. All values are expressed as means ± 1 SEM. Animals were treaed with the MEK/ERK inhibitor (U0126), PI3K/Akt inhibitor (PI-828), PKCθ inhibitor (TIP), inhibitor vehicle (20%
DMSO/80% saline (U0126 and PI-828) or 100% aCSF (TIP)) . The animals were either administered BDNF or aCSF (BDNF vehicle) post- inhibitor or post-inhibitor vehicle administration. There were no significant differences in any group at baseline versus 90 minutes.
