NNS 2015 Program & Abstract Book

University of Texas Medical Branch, Neuroscience & Cell Biology,

Galveston, USA

Tianjin Medical University, Department of Orthopedic Surgery,

Tianjin, China

Sun Yat-sen University, Department of Spine Surgery, Guanzhou,

China

Direct manipulation of endogenous neural stem cells (eNSCs) is an at-

tractive strategy to repair damage and replenish cells lost after spinal cord

injury (SCI). However, the gliogenic microenvironment drives differ-

entiation of eNSCs towards astrocytes. Previously we guided human

NSCs (hNSCs) to generate motor neurons (MNs) in vitro by regulating

the PI3K or STAT3 signaling pathway. Here we ask whether a combi-

nation of PI3K and STAT3 would enhance MN differentiation and de-

crease gliogenesis in vitro and in vivo. Both hNSCs and mouse NSCs

(mNSCs) generated more HB9

cells (a MN marker) after priming with

FGF2 and PI3K/STAT3 inhibitors, as compared to controls. To model

SCI in vitro, we performed a moderate stretch injury on hNSCs. The

combination treatment with FGF2 and PI3K/STAT3 inhibitors greatly

increased the number of HB9

cells, suggesting more MNs differentiated

from hNSCs after injury in vitro. For in vivo studies, FGF2, Heparin and

PI3K/ STAT3 inhibitors were intrathecally infused after contusion SCI.

Such treatments dramatically increased NeuN and ChAT (another MN

marker) expressing cells and decreased GFAP

cells 4-week post injury.

More excitingly, the locomotor functional score in the FGF2/Heparin/

inhibitor group was significantly higher than in the control groups, ac-

companied by improved rearing times. In summary, MN differentiation

from neural stem cells can be induced by FGF2 together with PI3K and

STAT3 inhibitors both in vitro and in vivo. Our novel findings suggest

that the gliogenic microenvironment after SCI can be manipulated to

allow endogenous spinal cord NSCs to generate neurons instead of as-

trocytes, and therefore, eNSCs can be attractive candidates as an alter-

native to cell transplantation to facilitate neural repair after SCI.

Keywords: neural stem cell, motor neuron, signaling pathway,

spinal cord injury

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INTERACTIVE ROLE OF MATRIX METALLOPROTEINASE

9 AND OSTEOPONTIN IN OLFACTORY BULB SYNAPTO-

GENESIS FOLLOWING TBI

Melissa Powell

, Linda Phillips

Virginia Commonwealth University, Anatomy and Neurobiology,

Richmond, USA

Traumatic brain injury (TBI) produces diffuse axotomy and synaptic

disruption, causing a variety of functional deficits. Axons of olfactory

receptor neurons (ORNs), which transmit sensory signals through the

cribriform plate, are particularly vulnerable to injury, resulting in deaf-

ferentation of olfactory bulb (OB) glomeruli. Normally, ORN turnover

produces continuous axon regeneration and reinnervation of the OB.

After trauma, this process becomes aberrant, often producing persistent

anosmia. Although the mechanism of OB synaptogenesis is not under-

stood, prior studies suggest matrix metalloproteinases (MMPs) regulate

OB synaptic repair after injury. MMP9 elevation/activity is documented

at sub-acute post-injury intervals, but its role in OB synaptogenesis re-

mains unclear. Recently, we posited that post-injury elevation of cyto-

kine osteopontin (OPN), an MMP substrate, represents a novel

mechanism for MMP9-mediated synaptic recovery. MMP cleaved OPN

binds integrin receptors, promoting glial proliferation and migration, as

well as cytokine and growth factor production for synaptogenesis. Here

we assessed MMP/OPN interaction in WT and MMP9 KO mouse OB

during acute (1d, 3d), degenerative (7d), and early regenerative (21d)

intervals. We hypothesized that, after central fluid percussion TBI, time-

dependent changes in MMP9 activity alter OPN fragment generation,

signaling neuroglial activation to promote OB synaptogenesis. Zymo-

graphic analysis showed

3 fold increase in MMP9 activity at 7d.

Western blot (WB) probe confirmed this increase was accompanied by

7d elevation of 47kD OPN integrin binding fragment. By 21d, MMP9

activity and OPN fragment production were below controls. With MMP9

KO, 47kD OPN expression at 7 and 21d was attenuated, supporting

MMP9 role in OB OPN processing after TBI. Further, OB ultrastructure

at 7d post-injury showed disrupted synaptic organization, an effect ex-

acerbated by MMP9 KO. Subsequent WB showed no OB change in the

common pre-synaptic marker Synapsin-I, however, ORN-specific ol-

factory marker protein (OMP) was reduced at 3d, returning to control

level by 21d. Notably, MMP9 KO prolonged OMP reduction beyond 7d,

likely interfering with ORN reinnervation. Collectively, these results

support MMP9/OPN interaction and OPN fragment signaling during OB

reactive synaptogenesis, particularly with regard to TBI-induced re-

innervation of deafferented glomeruli.

Support: NIH-NS056247, NS057758

Keywords: MMP9, Osteopontin, Synaptogenesis, Olfactory Bulb

A8-12

THE EFFECT OF MILD TRAUMATIC BRAIN INJURY

(MTBI) ON THE STRUCTURAL PLASTICITY OF THE AXON

INITIAL SEGMENT (AIS)

Michal Vascak

, Matthew L. Baer, John T. Povlishock

Virginia Commonwealth University School of Medicine, Department

of Anatomy & Neurobiology, Richmond, USA

The AIS is the site of action potential (AP) initiation, thereby a crucial

regulator of neural activity. Located on the proximal axon near the soma,

the precise position and length of the AIS varies with neuronal subtypes.

Ankyrin-G is the master structural protein regulating neuron excitability

via clustering voltage-gated sodium channels (NaV). In pyramidal neu-

rons, the high-density of NaV1.6 at the distal AIS sets the threshold for

AP generation. It has been shown that AIS modification alters neuronal

excitability following deafferentation of neural circuits. Recently, in

mTBI-mice, we have demonstrated dramatic alterations in the electro-

physiological status of intact neocortical pyramidal neurons, consistent

with AIS-specific changes, as well as the circuit disruption associated

with mTBI. Since AIS architecture modulates neuronal excitability, the

purpose of the current study was to determine if mTBI induces AIS

structural plasticity within a well-defined subset of intact neocortical

pyramidal neurons. Thy1-YFP mice exposed to either sham or mild

central fluid percussion injury were perfused after a 2-day recovery pe-

riod. Antibodies to ankyrin-G and NaV1.6 were used to fluorescently

label the AIS. Confocal microscopy was employed to identify intact

YFP

pyramidal neurons in layer 5 of S1 barrel field, whose axons were

continuous from the soma of origin to the subcortical white matter. Im-

munofluorescent profiles of ankyrin-G or NaV1.6 were then super-

imposed on the YFP

axonal traces to determine the start position with

respect to the somas of origin, and length. Alteration of these parameters

was interpreted as to reflect AIS structural plasticity. We found that while

mTBI had no effect on ankyrin-G start position, the length was signifi-

cantly reduced. This demonstrates a shortening of the AIS from the distal

end, where we also observed a peak in NaV1.6 immunofluorescent sig-

nal, consistent with the site of AP initiation. This change in AIS structure

most likely explains some of the electrophysiological abnormalities seen

within the intact neocortical pyramidal neuron population after mTBI.

Keywords: Mild Traumatic Brain Injury, Transgenic Mouse Model,

Plasticity, Axon Initial Segment, Ankyrin-G, Voltage-Gated Sodium

Channel

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