Journal of Neurotrauma Autophagy modulation by lanthionine ketimine ethyl ester improves long-term outcome following central fluid percussion injury in the mouse (doi: 10.1089/neu.2015.4196) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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Full Title: Autophagy modulation by Lanthionine ketimine ethyl ester improves long-term outcome following central fluid percussion injury in the mouse
Running Title: Autophagy modulation improves mouse brain injury Kenneth Hensleyab, PhD, Aleksandra Poteshkinad, BS, Ming F. Johnsond, MD, Pirooz Eslamid, MD, S. Prasad Gabbitac, PhD, Alexandar M. Hristova, PhD, Kalina M. Venkova-Hristovaa, PhD and Marni E. Harris-Whited+, PhD a
Department of Pathology and bDepartment of Neurosciences University of Toledo Health Science Campus 3000 Arlington Avenue, Toledo, OH USA 43614 Phone: 419-383-3442 Fax: 419-383-3066 c
P2D Bioscience, Inc., Suite 105, 10101 Alliance RoadCincinnati, OH 45242 Phone: 513-475-6618 Fax: 513-475-6618 d
Veterans Administration-Greater Los Angeles Healthcare System David Geffen School of Medicine at UCLA, Dept. of Medicine 11301 Wilshire Blvd., (151) Los Angeles, CA 90073 Phone: 310-478-3711 x49371 Fax: 310-268-4346 e
+Corresponding author: Marni E. Harris-White Address, phone and fax are the same for all authors within the same institution. Email addresses are as follows: (KH)
[email protected]; (AP)
[email protected]; (MFJ)
[email protected]; (PE)
[email protected]; (SPG)
[email protected]; (AMH)
[email protected]; (KMVH)
[email protected]; (MEHW)
[email protected]
Journal of Neurotrauma Autophagy modulation by lanthionine ketimine ethyl ester improves long-term outcome following central fluid percussion injury in the mouse (doi: 10.1089/neu.2015.4196) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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ABSTRACT Diffuse axonal injury is recognized as a progressive and long-term consequence of traumatic brain injury. Axonal injury can have sustained negative consequences on neuronal functions such as anterograde and retrograde transport and cellular processes such as autophagy that depend on cytoarchitecture and axon integrity. These changes can lead to somatic atrophy and an inability to repair and promote plasticity. Obstruction of the autophagic process has been noted following brain injury and rapamycin, a drug used to stimulate autophagy, has demonstrated positive effects in brain injury models. The optimization of drugs to promote beneficial autophagy without negative side effects could be used to attenuate traumatic brain injury and promote improved outcome. Lanthionine ketimine ethyl ester, a bioavailable derivative of a natural sulfur amino acid metabolite, has demonstrated effects on autophagy both in vitro and in vitro. Thirty minutes following a moderate central fluid percussion injury, and throughout the survival period, lanthionine ketimine ethyl ester was administered and mice subsequently evaluated for learning and memory impairments and biochemical and histological changes over a 5 week period. Lanthionine ketimine ethyl ester, which we have previously shown to modulate autophagy markers and alleviate pathology and slow cognitive decline in the 3xTgAD mouse model, spared cognition and pathology following central fluid percussion injury through a mechanism involving autophagy modulation.
Key Words: Diffuse axon injury, autophagy, behavior, pathology
Journal of Neurotrauma Autophagy modulation by lanthionine ketimine ethyl ester improves long-term outcome following central fluid percussion injury in the mouse (doi: 10.1089/neu.2015.4196) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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INTRODUCTION The discovery of therapeutics for traumatic brain injury (TBI) remains a formidable challenge. TBI begins with high-energy acceleration or deceleration of the brain within the skull or with penetration of the brain. Varying with the severity of the injury, primary cellular injury is followed by a secondary injury response. There are many aspects of brain injury to consider in both the acute and chronic phases of injury and a large number of pathophysiological events are triggered by TBI including, but not limited to, ischemia, changes in oxygen delivery and metabolism, excitotoxicity, apoptosis, central and peripheral inflammation, demyelination and associated white matter pathology, alterations in neurogenesis and seizures.
Management of acute
alterations such as regulation of blood circulation, intracranial pressure, oxygen levels and treatment of hematomas can be implemented for acute stabilization of the patient and to limit brain damage. However, an effective pharmacological treatment to limit injury and/or promote functional recovery evades the clinic. Neuronal perturbations and axonal changes have long been described as a feature of human head injury.1-8 It has been demonstrated that a limited number of axons undergo primary axotomy from impact forces that create shear, tensile and compressive strains and that a majority of injured axons degenerate via secondary axotomy.9,
10
derangements
Secondary axotomy may occur due to a combination of focal in
axolemmal
structure
and
function,
disruption
of
the
microtubule/neurofilament network, ionic dysregulation, proteolytic activity, and impaired axonal transport.8,
11-15
The demonstration of axonal swelling and disconnection
occurring adjacent to intact, unaltered axons is consistent with the phenomenon of
Journal of Neurotrauma Autophagy modulation by lanthionine ketimine ethyl ester improves long-term outcome following central fluid percussion injury in the mouse (doi: 10.1089/neu.2015.4196) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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4 diffuse axonal injury (DAI) described in both animals and humans.8,
16, 17
DAI is a
common pathology noted in all severities of TBI16, 18 and can occur over long periods of time.19 Evolving complications from TBI can be observed months to decades after the initial trauma
20
contributing to psychiatric disorders, cognitive dysfunction, brain
atrophy, chronic neuroinflammation and the development of neurodegenerative disorders such as Alzheimer’s Disease and chronic traumatic encephalopathy (CTE).21 The family of collapsin response mediator proteins (CRMPs) plays a significant physiological role in neuron cell body and axon stability within the central nervous system. CRMPs are a family of neuronal phosphoproteins that regulate microtubule assembly. All CRMPs are expressed intracellularly at high levels during development in the central nervous system, but it is CRMP2 that remains at high levels in the adult mammalian CNS within neurons and specific oligodendrocytes.22 CRMP2 is a microtubule-associated protein (MAP), phosphorylated by cyclin dependent kinase-5 (Cdk-5) and glycogen synthase kinase-
(GSK3β) enzymes.23 Normally, CRMP2
stabilizes microtubules by binding tubulin24 and facilitates protein trafficking by adapting kinesin-1 to protein cargo packages including neurotrophin receptors25 and actin nucleation factors important for maintenance of synapse stability.26 Studies have demonstrated that aberrant phosphorylation or cleavage of CRMP2 is detrimental to axon integrity and neuron survival.27,
28
Alterations in CRMP2 and CRMP4 protein
patterns have been shown to be associated with the process of ‘‘protrusion-like’’ bead formation, a sign of axonal and neuronal degeneration.29 Changes in CRMP2 expression, phosphorylation or cleavage may have an adverse effect on its ability to bind cytoskeletal proteins and degrade the integrity of the axon.
Journal of Neurotrauma Autophagy modulation by lanthionine ketimine ethyl ester improves long-term outcome following central fluid percussion injury in the mouse (doi: 10.1089/neu.2015.4196) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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5 Protein quality control and degradation play important roles in CNS homeostasis. The fidelity of the autophagic process is vital to CNS cells, and post-mitotic neurons in particular, that utilize autophagy to selectively dispose of misfolded or aggregated proteins and defective organelles.30 Autophagy is essential to neurons as a reduction in autophagy has been implicated in neuronal cell death31,
32
and neurodegenerative
diseases including Alzheimer’s33, Parkinson’s34-37, Huntington’s38, lateral sclerosis
(ALS).40,
41
Spatial and
39
, and amyotrophic
mechanical processes
ensure that
autophagosomes move processively along the axon and multiple scaffolding proteins likely cooperate in this efficient transport. In the axon, autophagosomes undergo longrange microtubule-based transport that is coupled to compartment maturation.33,
42, 43
The role of autophagy in TBI is clouded by conflicting reports of autophagy being both detrimental and beneficial.
44-46
Although there is substantial evidence that markers of
autophagy are increased in both human and animal brains following TBI, it is not clear whether those changes result in functional autophagy. We have recently demonstrated that the axonal scaffolding protein, CRMP2, is involved in autophagy as engineered knockdown of CRMP2 reduces autophagy flux.47 Further, Lanthionine ketimine ethyl ester (LKE), previously demonstrated to dramatically reduce the accumulation of aggregated amyloid and tau in an AD mouse model
48
, binds to the microtubule-
associated protein, CRMP249-51 and stimulates autophagy in mammalian CNS cells.47 In this study, we utilized the mouse central fluid percussion model, a model of diffuse axonal injury.52 The progressive nature of DAI suggests that there is a period of time in which a pharmacological treatment might be effective to stabilize neuronal architecture, stimulate productive autophagy and allow repair mechanisms to function.
Journal of Neurotrauma Autophagy modulation by lanthionine ketimine ethyl ester improves long-term outcome following central fluid percussion injury in the mouse (doi: 10.1089/neu.2015.4196) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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To this effect, 30 minutes following a moderate TBI, and throughout the survival period,
LKE was administered and mice evaluated for learning and memory impairments at 3
and 5 weeks. Biochemical and histological analyses were also performed at 3d and 5
weeks post TBI.
Journal of Neurotrauma Autophagy modulation by lanthionine ketimine ethyl ester improves long-term outcome following central fluid percussion injury in the mouse (doi: 10.1089/neu.2015.4196) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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MATERIALS AND METHODS Surgical preparation and injury induction The procedures by which mice were subjected to central fluid percussion injury (cFPI) were modified, with appropriate scaling, from those previously described using rats
53
. Briefly, C57BL6/129SVJ mice, 4 months of age and weighing 20–26g, were
surgically prepared for the induction of cFPI. Each animal was anesthetized in an anesthesia chamber with 3% isoflurane in O2. After induction, each animal’s thigh was shaved for intraoperative physiological monitoring and placed in a stereotactic frame (David Kopf Instruments, Tujunga, CA USA) fitted with a nose cone to maintain anesthesia with 1–2% isoflurane in O2. A thermostatically controlled heating pad (Harvard Apparatus, Holliston, MA USA) was then placed under the animal and set to maintain body temperature at 37°C during the surgery. A midline sagittal incision was made to expose the skull from bregma to lambda. The skull was cleaned and dried and a 3.0 mm circular craniotomy (using a trephine drill bit inserted into a drill mounted to the stereotaxic frame; Harvard Apparatus) was then made along the sagittal suture midway between bregma and lambda, leaving the underlying dura intact. Care was taken not to generate heat during the craniotomy procedure. A sterile Leur-Loc syringe hub was then cut away from a 20-gauge needle and affixed to the craniotomy site using cyanoacrylate. Upon confirming the integrity of the seal between the hub and the skull, dental acrylic was then applied around the hub to provide stability during the induction of injury. After the dental acrylic hardened, the scalp was sutured around the hub, topical bacitracin and marcaine were applied to the incision site, and the animal was removed from anesthesia and monitored in a warmed cage until fully ambulatory (~60–
Journal of Neurotrauma Autophagy modulation by lanthionine ketimine ethyl ester improves long-term outcome following central fluid percussion injury in the mouse (doi: 10.1089/neu.2015.4196) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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8 90 min). For the induction of injury, 2 hours following craniotomy surgery, each animal was reanesthetized with 3% isoflurane in 100% O2, and the male end of a spacing tube was inserted into the hub. The female end of the hub spacer assembly, filled with normal saline, was attached on to the male end of the fluid percussion apparatus (Custom Design and Fabrication; Virginia Commonwealth University; Richmond, VA). An injury of moderate severity (1.73 +/- 0.03 atmospheres) was administered by releasing a pendulum onto a fluid-filled piston to induce a brief fluid pressure pulse upon the intact dura. The pressure pulse measured by the transducer was displayed on a storage oscilloscope (Tektronix 5111), and the peak pressure was recorded. After injury, the animals were visually monitored for recovery of spontaneous respiration. The hub and dental acrylic were removed en bloc, and the incision closed with VetBond veterinary adhesive before recovery from anesthesia/ unconsciousness. The duration of transient unconsciousness was determined by measuring the time it took each animal to recover the following reflexes: toe pinch, tail pinch and righting. After recovery of the righting reflex, animals were placed in a warmed holding cage to ensure the maintenance of normothermia and monitored during recovery before being returned to the vivarium. For animals receiving a sham injury, all of the above steps were followed with the exception of the release of the pendulum to induce the injury. To verify a significant injury effect, righting reflex recovery times were analyzed by ANOVA and a Tukey’s post hoc analysis. All experiments were approved by the institutional animal care and use committee (protocol #2002-11) at the Veterans Administration-Greater Los Angeles Healthcare System (Animal welfare assurance #3002-01) in accordance with National Institutes of Health guidelines.
Journal of Neurotrauma Autophagy modulation by lanthionine ketimine ethyl ester improves long-term outcome following central fluid percussion injury in the mouse (doi: 10.1089/neu.2015.4196) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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9 Treatment R-LK-5-ethyl ester (LKE) was synthesized from 3-bromopyruvate (TCI America, Portland, OR USA) and L-cysteine-ethyl ester HCl (Alfa Aesar, Wood Hill MA USA).49, 54 LKE was formulated at 1000 ppm (100 mg/kg/mouse/day) into AIN93M rodent diets (Dyets, Inc, Bethlehem, PA USA) blind-coded “A” or “B”, and stored at -80oC until the week of use. To insure that mice received LKE in the first 3 days following surgery, 100 mg/kg/d LKE was delivered via intraperitoneal (ip) injection (in physiological saline) and then fed in the diet thereafter for longer survival time points.
Motor evaluation To determine if mice had motor impairments following injury, a battery of motor tasks was given to mice at 3 different time points. At 3 days following injury, a ledge test, hind limb clasping and gait analysis were performed.55 The ledge test is a direct measure of coordination performed by observing the mouse as it walks along the cage ledge and lowers itself into its cage. A normal mouse will typically walk along the ledge without losing its balance, and will lower itself back into the cage using its paws. This was assigned a score of 0. If the mouse lost it’s footing while walking along the ledge, but otherwise appears coordinated, it received a score of 1. If it did not effectively use its hind legs, or lands on its head rather than its paws when descending into the cage, it received a score of 2. If it fell off the ledge while walking or attempting to lower itself, or was shaky and refused to move, it receives a score of 3. For hind limb clasping analysis, the mouse was grasped by the tail near its base and lifted clear of all surrounding objects for 10 seconds. If the hind limbs were consistently splayed outward,
Journal of Neurotrauma Autophagy modulation by lanthionine ketimine ethyl ester improves long-term outcome following central fluid percussion injury in the mouse (doi: 10.1089/neu.2015.4196) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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10 away from the abdomen, it was assigned a score of 0. If one hind limb was retracted toward the abdomen for more than 50% of the time suspended, it received a score of 1 and if both hind limbs were partially retracted toward the abdomen for more than 50% of the time, it received a score of 2. If it’s hind limbs were entirely retracted and touching the abdomen for more than 50% of the time, it received a score of 3. Gait is a measure of coordination and muscle function. Gait analysis was performed by removing the mouse from its cage and placing it on a flat surface with its head facing away from the investigator and observing the mouse from behind as it walks. If the mouse moved normally, with its body weight supported on all limbs, with its abdomen not touching the ground, and with both hind limbs participating evenly, it received a score of 0. If it showed a tremor or appeared to limp while walking, it received a score of 1. If it showed a severe tremor, severe limp, lowered pelvis, or the feet point away from the body during locomotion (duck feet), it received a score of 2. If the mouse had difficulty moving forward and dragged its abdomen along the ground, it received a score of 3. At day 15, a visible platform swim evaluation was performed to assess visual and swimming impairments. Ambulatory velocities were evaluated during the acquisition phase of the Barnes maze testing (days 30-33). These are described below in the respective sections. Treatment groups were blinded to the operator.
Morris water maze To test hippocampal-dependent spatial cognition, mice were trained in the standard Morris water maze (MWM) with a hidden platform as previously described 56, 57 with minor modifications. The MWM consisted of a white, circular, water-filled pool
Journal of Neurotrauma Autophagy modulation by lanthionine ketimine ethyl ester improves long-term outcome following central fluid percussion injury in the mouse (doi: 10.1089/neu.2015.4196) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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11 (diameter, 1.20 m; height, 0.5 m at 24oC; maintained by a thermostat-driven heating system attached to the underside of the water tank) opacified by white powdered tempura paint. The tank was surrounded by solid white walls to eliminate distractions. Three solid black cues (different shapes) were affixed to the inside walls of the tank at three locations (N, E and W). A white escape platform (15 cm diameter, height 24 cm) was located 1 cm below the water surface in a fixed position (NE quadrant, 22 cm away from the wall). In each daily swim block (4 swim trials per block, 30 s inter-trial interval), mice were placed at one of the starting locations, facing the wall, in random order (N, S, E, W, including permutations of the four starting points per session) and were allowed to swim until they located the platform. Mice failing to find the platform within 60 s were placed on it for 15 s (the same period of time as the successful animals). Mice floating/not actively searching for the platform or demonstrating >80% thigmotaxing behavior were eliminated from the analysis. A probe trial was performed 24 hours after the final training block. The platform was removed from the pool and the mice performed a 60 second swim. At the end of every trial the mice were allowed to dry for 15 min in a heated enclosure and were returned to their home cage. The cued (visible platform) session was performed to test swimming speed and visual acuity. The visible platform was elevated 1 cm above the water and its position was clearly indicated by a visible cue (black tape around the elevated portion of the platform). Other than the black-taped platform, no other cues were. The cued test (4 trials) was performed three days prior to the hidden training protocol. Anymaze software (Stoelting Co., Wood Dale, IL USA) was used to capture and analyze the MWM data. The person performing behavioral tasks was blinded to treatment condition. MWM was evaluated from 15-21
Journal of Neurotrauma Autophagy modulation by lanthionine ketimine ethyl ester improves long-term outcome following central fluid percussion injury in the mouse (doi: 10.1089/neu.2015.4196) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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12 days. This time was chosen to allow for significant recovery post injury and to allow LKE enough time to impact long-term outcome. Barnes Maze The method used in our lab was adapted from a published protocol and review article.58 A 20-hole Barnes maze apparatus was used (Any Maze). White curtains were used around the maze to reduce room cues. Three cues were placed in close proximity to the maze for use in spatial navigation. In the pre-training trial, the mouse was placed in the middle of the maze under a dark colored box allowing the mouse to be in random orientation before each trial. After 10 s had elapsed, the chamber was lifted, and the mouse allowed to explore the maze for 3 min. Aversive stimuli (sounds, wind, harsh lighting) were not utilized. Errors and latency were recorded during acquisition and testing. Errors were defined as nose pokes and head deflections over any hole that does not have the target box. Latency was defined as the time it took to locate the target box. Mice were trained for four trials per day for 4 days with an inter-trial interval of at least 15 min. After each trial, the entire maze was cleaned with an unscented, mild, dilute soap solution. Mice not actively moving throughout the maze were eliminated from the analysis. 48 hrs after the last training trial, a probe trial was conducted to evaluate short-term memory retention. Any Maze software was used to capture and analyze the data. Barnes maze was performed at 30-35 days (called the 5 week time point).
Journal of Neurotrauma Autophagy modulation by lanthionine ketimine ethyl ester improves long-term outcome following central fluid percussion injury in the mouse (doi: 10.1089/neu.2015.4196) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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13 Tissue collection At the end of Barnes maze testing, animals were anesthetized with 100 mg/kg pentobarbital and a blood sample taken via cardiac puncture followed by cardiac perfusion with HEPES buffer [10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 137 mM NaCl, 4.6 mM NaCl, 4.6 mM KCl, 1.1 mM KH2PO4, 0.6 mM MgSO4 and 1.1 mM ethylenediamine tetraacetic acid (EDTA), 1 mM Na3VO4 and protease inhibitor cocktail (Roche complete ultra mini, Sigma Aldrich, St. Louis, MO USA)]. Hippocampus and cortex were dissected from one hemisphere, snap frozen in N2(l) and stored at -80oC. The contralateral hemisphere was immersion fixed in 10% neutral buffered formalin (Thermo Fisher Scientific, Waltham, MA USA) for 24 h followed by step-wise (10-20-30%) sucrose cryopreservation and embedding in preparation for cryostat sectioning.
Immunohistochemistry Formalin fixed, cryopreserved brains were sagitally sectioned on a Leica cryostat at 25 um thickness and stored at -20oC until immunolabeling. Immunohistochemical labeling was performed on free-floating sections using a VectaStain Elite ABC kit (Vector Laboratories) with diaminobenzadine chromagen. Antibodies were: phospho-cJun Ser63 (1:1000; Cell Signaling Technologies) and phosphorylated neurofilament H (SMI-31,1:500; EMD Millipore, Billerica, MA USA). Sections were mounted on glass slides and coverslipped using Permount® mounting media (Thermo Fisher Scientific). Specificity of antibody immunoreactivity was confirmed by elimination of the primary
Journal of Neurotrauma Autophagy modulation by lanthionine ketimine ethyl ester improves long-term outcome following central fluid percussion injury in the mouse (doi: 10.1089/neu.2015.4196) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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14 antibody during the staining procedure. This negative control staining resulted in the loss of staining for these antibodies as predicted. Western Blot Frozen tissue was homogenized in lysis buffer (10 mM Tris-HCl, 0.5 M NaCl, 0.3 M Sucrose, pH 7.4 and containing 1% Triton-X100, 5 mM EDTA, 10 mM dithiothreitol, 1 mM Na3VO4, 1 mM NaF, and protease inhibitor cocktail (Roche, Complete Ultra Mini and PhosphoSTOP) at a ratio of 1:20 (1 mg tissue = 20 μL of lysis buffer). The resulting homogenates were assayed for protein concentration by the DC Protein Assay (Biorad, Hercules, CA USA). Samples were adjusted to equal protein concentration, mixed 1:1 with loading dye containing 2% B-mercaptoethanol, boiled and frozen at -20oC until use.
For LC3 blot: 10ug of total lysate was resolved on 4-20% TGX gel (Biorad) at 100V for 100 min and transferred on a PVDF membrane for 2h at 60V. Chloroquinetreated HeLa cells were used as the positive autophagy control (Cell Signaling Technology #11972S). All other proteins (10-30ug) were resolved on 7.5% TGX gels (Biorad) and blotted onto a nitrocellulose membrane. Membranes were blocked in 5 % dry-milk, incubated in primary antibody overnight and developed using ECL chemiluminescent reagents (SuperSignal West Pico, Thermo Fisher Scientific; or ECL Prime, GE Healthcare, Pittsburgh, PA USA). Blots were imaged using a Syngene G:Box and automated GENEsys control software (Syngene, Frederick, MD USA). Blots were normalized by reprobing with Beta-actin.
All primary antibodies were purchased from Cell Signaling Technologies unless otherwise noted and used at a 1:1000 dilution:
Phospho-mTOR-Ser2448; mTOR
Journal of Neurotrauma Autophagy modulation by lanthionine ketimine ethyl ester improves long-term outcome following central fluid percussion injury in the mouse (doi: 10.1089/neu.2015.4196) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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15 (7C10); Phospho-ULK1 (S757); ULK1 (D8H5); Beclin1; Ubiquitin; LC3B/MAP1LC3B (Novus); CRMP2 (EMD Millipore); B-Actin (Pierce-Thermo Fisher Scientific).
Luxol Fast Blue Brain sections were removed from the -20oC freezer, thawed and rinsed 3 x 5 min in TBS followed by mounting on glass slides. Fat was removed from the sections by overnight incubation in a 1:1 ethanol/chloroform solution. Sections were rehydrated by successive 5 min incubations in 100/85/70% ethanol followed by H2O. Sections were then placed into a 1% Luxol fast blue solution (Sigma Aldrich, St. Louis, MO USA) for 2 hrs at 60oC. Staining was differentiated with 3 rounds of: 0.05% lithium carbonate for 30 s followed by 70% ethanol for 30 s and then H2O. Sections were dehydrated in 95% and 100% ethanol for 5 min each followed by 5 min in CitriSolve and then coverslipped with DPX (Thermo Fisher Scientific).
Cleaved tau ELISA Cleaved tau (C-tau) was measured using our previously characterized ELISA
59
and using three monoclonal antibodies (C-tau7, -8 and -12) that demonstrate noncompetitive binding to C-tau. Immulon 2 plates were coated with affinity-purified C-tau12 (1:200 in PBS; 100 uL/well) for 1 hr at r.t. and then blocked overnight with 5% nonfat dry milk and 0.5% gelatin in Tris-buffered saline (TBS). Plates were washed 4x with 0.1% Tween in TBS (TBST), and samples added in triplicate, incubated with rocking for 1h at r.t, and then washed 4x with TBST. HRP-conjugated C-tau7 and -8 (1:1000) were added for 1h with rocking at r.t. Plates were washed 4x with TBST and color developed using tetramethylbenzidine as substrate. The reaction was stopped after 25 minutes
Journal of Neurotrauma Autophagy modulation by lanthionine ketimine ethyl ester improves long-term outcome following central fluid percussion injury in the mouse (doi: 10.1089/neu.2015.4196) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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16 and the plate read at 450 nm. A standard curve was generated using affinity-purified human C-tau. Negative controls included exclusion of Ctau12, deletion of sample, or deletion of horseradish peroxidase (HRP)-conjugated C-tau7/C-tau8 from the assay. Experimental C-tau samples demonstrating optical density values less than the sensitivity of the assay were assigned a value of zero. The sensitivity of this ELISA is 30 pg per well and demonstrates an intra-assay variation of less than 7% and an interassay variation of less than 10%.
Plasma and Brain LKE Levels Male C57BL/6 adult mice were purchased from Jackson Laboratories at 19-21 g body weight and randomized to 3 groups (n=6 per group) fed AIN93M diet containing 0, 1 or 1,000 ppm LKE. This treatment regimen provides daily oral doses of approximately 15 mg/kg LKE for the 1 ppm-modified diet and 150 mg/kg LKE for the 1,000 ppmmodified diet, calculated from actual measured food consumption.
Fresh food and
water were provided ad libitum and food intake and body weight were measured daily for 15 days. At the end of the study (10-12 AM on day 15) the mice were euthanized by CO2 inhalation followed by immediate exsanguination via cardiac puncture and harvesting of tissue specimens (brain cortex). Blood samples (0.5-1 ml) were placed in heparinized eppendorf tubes on ice. Plasma was obtained by centrifugation and stored in sealed 1.5 ml tubes at -80oC until analysis. Plasma concentration of LKE was determined by LC-MS/MS analyses using tolbutamide as a standard for extraction efficiency.
Journal of Neurotrauma Autophagy modulation by lanthionine ketimine ethyl ester improves long-term outcome following central fluid percussion injury in the mouse (doi: 10.1089/neu.2015.4196) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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17 Brain samples were carefully dissected to remove cortices and meninges were discarded.
Cortices were weighed by subtraction in pre-weighed eppendorf
microcentrifuge tubes and frozen immediately then stored at -80oC until analysis. Tissue was lysed by addition of 2 volumes of phosphate-buffered saline, and sonication. A 50 L aliquot of brain lysate was mixed with 0.2 mL of extraction solvent and processed for LC-MS/MS.
Statistics All data are graphically presented as mean ± SEM unless otherwise specified. In the case of single mean comparisons, data were analyzed by two-tailed unpaired t-tests or Mann-Whitney tests appropriate to data distributions.
In case of multiple
comparisons, data were analyzed by one-or two-way ANOVA with post-hoc Bonferroni multiple comparisons using GraphPad Prism Software v 5.0a (GraphPad).
Journal of Neurotrauma Autophagy modulation by lanthionine ketimine ethyl ester improves long-term outcome following central fluid percussion injury in the mouse (doi: 10.1089/neu.2015.4196) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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18
RESULTS Moderate cFPI in mice induced a transient behavioral suppression of the righting reflex in all injured animals of 6.3 +/- 0.3 min that was significantly longer than that of 1.2 +/- 0.03 min for sham-injured animals (p