Introduction

Progesterone Modulates the Phosphorylation of Akt in a Closed Skull Traumatic Brain Injury Model

Justin Garling, MSII, Lora Watts, PhD, Shane Sprague, BS, David F. Jimenez, MD, FACS, Murat Digicaylioglu, MD, PhD

The University of Texas Health Science Center at San Antonio

Conclusions

• Traumatic Brain Injury (TBI) affects nearly 1.7 million people in the United States each year, but not all head injuries result in TBI.2

• There are currently no FDA approved drugs on the market to treat TBI

• Progesterone is a hydrophobic steroid hormone that has been shown in recent studies to exhibit neuroprotective effects.

• This study aims to determine if progesterone is involved in the regulation of Akt via the Serine 473 and Threonine 308 phosphorylation sites.

References

Progesterone (100 nM) treated astrocyte culture:

• Increased phosphorylation of Akt at Thr308• Decreased phosphorylation of Akt at

Ser473• Small decrease in total Akt

TBI mice treated with Progesterone:

• Significantly increased phosphorylation of Akt at Thr308 site in the hippocampus compared to sham (72 hours post-TBI)

Significance of Researching Progesterone:

• To identify the active groups on the progesterone molecule.

• Progesterone is a large molecule with solubility and blood brain barrier penetration problems.

• To find a progesterone mimetic

Progesterone Treatment:

Mice were treated with progesterone by injection post-TBI at 1hr (i.p.), 6hr (s.c.), 24hr (s.c.), 48hr (s.c.), and then sacrificed at 72hrs. (Note: i.p.=intraperitoneal s.c.= subcutaneous)

Methods

Mouse Traumatic Brain Injury (TBI) model:

A 5mm disc is placed over the right side of the exposed skull between the bregma and lambda sutures. Mice were impacted using a pneumatic impactor which fires at a rate of 4.5m/s striking the disc depressing the skull 2mm.

Future Directions

• Time-response curves at the progesterone concentrations which produced the most significant changes in the dose-response curves (100nM)

• Oxygen Glucose Deprivation (OGD) in astrocyte and cortical neuron cultures

• P-mTOR pathway relation to progesterone and Akt

• Intranasal progesterone treatment

Progesterone

DAPI

MergedPGR

GFAP

DAPI PGR Merged

Results

Progesterone Treated Astrocyte Culture(Dose response curves)

Progesterone treated 72hr TBI Mice

P-Akt (T308)

Actin

Total Akt

P-Akt (S473)

P-Akt (T308)

Total Akt

1nM 10nMVC 20nM 40nM 100nM 1μM 20μMProgesterone

concentrations:

HCtx Ctx Ctx CtxH H H

TBI w/P4 TBI w/P4 Sham w/P4 Sham w/V

Figure 1: The progesterone treated astrocyte cultures were incubated for 30min. The vehicle (V) used in all experiments was 2-hydroxypropyl-β-cyclodextrin. The right cortex (Ctx) and hippocampus (H) were isolated in the progesterone treated 72hr TBI mice

Figure 3: Nissl Staining. Nissl stains rough endoplasmic reticulum. All Nissl stainings show the hippocampus of the impacted side of C57Black/6 mice. None of the mice shown were treated with progesterone.

Sham 24hr TBI 72hr TBI

100X

4

0X

2

0X

This work was supported by the Department of Neurosurgery

Expression of Progesterone Receptor

Figure 2: Progesterone receptor in Astrocyte culture and mouse cortex. DAPI=blue, nuclei. PGR (progesterone receptor)= green. GFAP (Glial Fibrillary Acidic Protein)= red, and is a structural protein of astrocytes.

Hippocampus of TBI mice

1. Brinton, R., Thompson, R. & Foy, M. Progesterone Receptors: Form and Function in Brain. Neuroendocrinology 29, 313-339 (2008).

2. Control, C.f.D. CDC - Injury - Traumatic Brain Injury(Atlanta, 2010).

3. Cutler, S., Cekic, M., Miller, D. & Stein, D. Progesterone Improves Acute Recovery after Traumatic Brain Injury in the Aged Rat. Journal of Neurotrauma, 1475-1486 (2007).

4. CW, G., SW, H. & DG, S. Behavioral effects and anatomic correlates after brain injury: a progesterone dose-response study. Pharmacology, Biochemistry, and Behavior, 231-242 (2003).

5. Wright, D., Bauer, M., Hoffman, S. & Stein, D. Serum Progesterone Levels Correlate with Decreased Cerebral Edema after Traumatic Brain Injury in Male Rats. Journal of Neurotrauma 18 (2001).

ProgesteroneProgesterone

AktAktPP

PPS473S473

T308T308

InflammationInflammationApoptosisApoptosisEdemaEdemaGrowthGrowth

Cell Viability