NNS 2015 Program & Abstract Book

A1-06

DISRUPTED AUTOPHAGY AFTER SPINAL CORD INJURY

IS ASSOCIATED WITH ER STRESS AND NEURONAL CELL

DEATH

Shuo Liu

, Chinmoy Sarkar

, Alan Faden

, Eugene Koh

, Junfang

Marta Lipinski

University of Maryland, Baltimore, Anesthesiology, Baltimore, USA

University of Maryland, Baltimore, Orthopaedics, Baltimore, USA

Autophagy is a catabolic mechanism facilitating degradation of cy-

toplasmic proteins and organelles in a lysosome-dependent manner.

Autophagy flux is necessary for normal neuronal homeostasis and its

dysfunction contributes to neuronal cell death in several neurode-

generative diseases. Although signs of elevated autophagy have been

reported after SCI, its mechanism, cell type specificity, and relation-

ship with cell death remain unknown.

In a rat model of contusive SCI, we observed accumulation of LC3-II

positive autophagosomes starting at post-trauma day 1. This was ac-

companied by a pronounced accumulation of autophagy substrate pro-

tein p62/SQSTM1, indicating that early elevation of autophagy markers

reflects disrupted autophagosome degradation. Levels of lysosomal

protease cathepsin D (CTSD) and numbers of CTSD positive lysosomes

were also decreased at this time, suggesting that lysosomal damage may

contribute to the observed defect in autophagy flux. Normalization of

SQSTM1 levels started by day 7 after SCI, and was associated with

increased CTSD levels. Therefore, increase in the size and activity of

the lysosomal compartment may eventually help restore autophagy flux.

At day 1 after SCI accumulation of autophagosomes was most

pronounced in ventral horn motor neurons and dorsal column oligo-

dendrocytes and microglia. In motor neurons disruption of autophagy

strongly correlated with evidence of endoplasmic reticulum (ER)

stress. As autophagy is thought to protect against ER stress, its dis-

ruption after SCI could contribute to ER stress-induced neuronal

apoptosis. Consistently, motor neurons showing disrupted autophagy

co-expressed ER-stress associated initiator caspase 12 and cleaved

executioner caspase 3. Together these findings indicate that SCI

causes lysosomal dysfunction that contributes to disruption of au-

tophagy flux and associated ER stress-induced neuronal apoptosis.

Keywords: autophagy, ER stress, apoptosis, lysosome, rat contusive

SCI

A1-07

LYSOSOMAL DAMAGE LEADS TO INHIBITION OF AU-

TOPHAGY AND CONTRIBUTES TO NEURONAL CELL

DEATH AFTER TBI

Chinmoy Sarkar, Zaorui Zhao, Stephanie Aungst, Boris Sabirzhanov,

Alan Faden,

Marta Lipinski

University of Maryland, Baltimore, Anesthesiology, Baltimore, USA

Disruption of autophagy, a lysosome-dependent intracellular degra-

dation process, has been implicated in both acute and chronic neu-

rodegenerative diseases. Although increase in markers of autophagy

has been reported in the brain after traumatic brain injury (TBI), its

cell type specificity, mechanisms and function remain unknown.

Following controlled cortical impact (CCI) brain injury in

GFP-

LC3

transgenic mice, we observed accumulation of autophagosomes

in the ipsilateral cortex and hippocampus starting by 24 hours after

injury. This accumulation was not due to increased initiation of au-

tophagy but rather to decrease in clearance of autophagosomes, as

reflected by accumulation of SQSTM1/p62. This was confirmed by

ex vivo

studies demonstrating impaired autophagy flux in brain slices

from injured as compared to control animals. The impairment of au-

tophagy was at least in part caused by TBI-induced decrease in ly-

sosomal function, evidenced by lower protein levels and enzymatic

activity of cathepsin D (CTSD) in the injured cortex. CTSD was also

abnormally localized to the cytosol after TBI, suggesting that lyso-

somal dysfunction was caused by injury-induced lysosomal mem-

brane permeabilization.

At 1 day after TBI autophagy flux was inhibited predominantly in

neurons. At that time we observed co-localization of GFP-LC3

signal with markers of caspase dependent (cleaved caspase 3, cas-

pase 12) and caspase-independent (AIF) cell death, indicating that

inhibition of the autophagy-lysosomal pathway contributes to neu-

ronal cell death. Taken together, our data demonstrate that autop-

hagic clearance is compromised after TBI due to injury-induced

lysosomal damage and likely contributes to neuronal cell death.

Autophagic flux was restored by day 7, at which point autophagy

could become neuroprotective. We propose that restoration of ly-

sosomal function and autophagy flux may represent novel thera-

peutic strategies to limit neuronal loss after TBI.

Keywords: autophagy, lysosome, transgenic mouse, CCI, injury

mechanisms

A1-08

SILICONE PAD DEMONSTRATES SIMILAR LEVEL OF EF-

FECTIVENESS AS NEOPRENE PAD ON FLUID PERCUS-

SION INJURY DEVICE

Maggie Parsley

, Bridget Hawkins, Ian Bolding, Donald Prough,

Douglas DeWitt

University of Texas Medical Branch, Anesthesiology, Galveston, USA

Introduction:

Traumatic brain injury (TBI) affects millions of

American civilians and military service personnel. One of the models

used to study TBI is the fluid percussion injury device (FPI). Tradi-

tionally, a neoprene pad has been used on the end of the plunger of the

FPI device. We tested a silicone-based pad to determine if it would

produce injury levels similar to that of the neoprene pad.

Methods:

Male Sprague Dawley rats were anesthetized, intubated,

mechanically ventilated and prepared for FPI. Animals were randomly

assigned to receive sham, moderate FPI with neoprene pad (nFPI) or

moderate FPI with silicone pad (sFPI). Anesthesia was discontinued

and, upon return of a withdrawal reflex in response to paw pinch,

animals received FPI. Return of righting reflex (RR) time measurements

commenced immediately upon injury. Animals survived for 24 hours

prior to being euthanized and their brains were collected and fresh-

frozen. Ten coronal (10mM thickness) sections were collected every

section throughout the injury site. Sections were stained with

.001% Fluorojade-C (FJ), and positive cells were counted in the CA1/2

and CA3 regions of the hippocampus by two blinded investigators.

Results:

Overall, there were no statistically significant differences

between the two pad materials used on the FPI device when delivering

moderate (2.0 atm) severity levels. There was a significant correlation

between the RR measurements and the numbers of FJ-positive (in-

jured) neurons in the rat hippocampus. We conclude from these data

that the silicone and neoprene pads deliver a similar level of injury

and either can be used on the FPI device.

These studies were completed as part of an interdisciplinary re-

search team funded by The Moody Project for Translational Trau-

matic Brain Injury Research.

Keywords: fluid percussion, model comparison, righting reflex, cell

death

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