The goal of this project was to see if a hormone that prevents brain cells from dying could protect...

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Page 1: The goal of this project was to see if a hormone that prevents brain cells from dying could protect cells through a previously unknown pathway. We also.
Page 2: The goal of this project was to see if a hormone that prevents brain cells from dying could protect cells through a previously unknown pathway. We also.

The goal of this project was to see if a hormone that prevents brain cells from dying could protect cells through a previously unknown pathway.

We also aimed to develop a method to determine the concentration of a certain protein in cell samples using results of a normally qualitative analysis technique.

Page 3: The goal of this project was to see if a hormone that prevents brain cells from dying could protect cells through a previously unknown pathway. We also.

PACAP – pituitary adenlyate cyclase-activating polypeptide› Many protective functions in the central

nervous system PACAP binds to receptor receptor

activates G-protein G-protein activates AC AC produces cAMP› Known pathway: cAMP activates PKA› New pathway: cAMP activates MAPKs, which

activate ERK1/2

Genes are transcribed into proteins

Page 4: The goal of this project was to see if a hormone that prevents brain cells from dying could protect cells through a previously unknown pathway. We also.

Adapted from Ravni et al., 2008

Page 5: The goal of this project was to see if a hormone that prevents brain cells from dying could protect cells through a previously unknown pathway. We also.

Strokes trigger hypertoxicity› Elevated calcium and phosphate levels are

mediators of glutamatergic death PACAP regulates phosphate and

calcium homeostasis to prevent cell damage and death in vivo

Page 6: The goal of this project was to see if a hormone that prevents brain cells from dying could protect cells through a previously unknown pathway. We also.

NG108-15 and cortical cells Calibration cell samples:

Pharmacology cell samples: --- ddAd H89 U0126

PACAP (or forskolin)

PACAP + ddAd

PACAP + H89

PACAP + U0126

25μL PACAP 10% dilution 20% dilution 50% dilution

75% dilution 87.5% dilution

93.5% dilution

96.8% dilution

Page 7: The goal of this project was to see if a hormone that prevents brain cells from dying could protect cells through a previously unknown pathway. We also.
Page 8: The goal of this project was to see if a hormone that prevents brain cells from dying could protect cells through a previously unknown pathway. We also.

SDS-PAGE: Separated proteins by length Incubated in antibodies: phospho-ERK and

ERK Incubated in chemiluminescent substrate

Page 9: The goal of this project was to see if a hormone that prevents brain cells from dying could protect cells through a previously unknown pathway. We also.

Band Intensity vs. Protein Amount is not a linear relationship› Background deletion corrects

chemiluminescent substrate problems› Band intensity measured with ImageJ gel

analysis tool› Division by loading control corrects gel

loading variation› Calculated calibration equation via

hyperbolic regression script

Page 10: The goal of this project was to see if a hormone that prevents brain cells from dying could protect cells through a previously unknown pathway. We also.
Page 11: The goal of this project was to see if a hormone that prevents brain cells from dying could protect cells through a previously unknown pathway. We also.

NG108-15 cells

Cortical cells

Page 12: The goal of this project was to see if a hormone that prevents brain cells from dying could protect cells through a previously unknown pathway. We also.

Sum of errors:

Inaccurate for NG108-15 phospho-ERK blots and cortical ERK blots because the saturation point for band intensities was 12.5μL › curve was very sensitive to fluctuations at

smaller dilutions and flattened out

NG108-15 phospho-ERK 24,287,533

NG108-15 ERK 631,011

cortical phospho-ERK 160,739

cortical ERK 1,233,319

Page 13: The goal of this project was to see if a hormone that prevents brain cells from dying could protect cells through a previously unknown pathway. We also.

Non-canonical pathway via ERK rather than PKA activation exists in rat cortical cells

Analysis incomplete: did not have enough blots to correct curve due to chemiluminescent substrate difficulties› Background deletion problems› High blot-to-blot variation lead to high

standard deviations

Page 14: The goal of this project was to see if a hormone that prevents brain cells from dying could protect cells through a previously unknown pathway. We also.

Create calibration curve with more than two blots

Evaluate accuracy of method using known protein concentrations

Page 15: The goal of this project was to see if a hormone that prevents brain cells from dying could protect cells through a previously unknown pathway. We also.

Upregulation of other genes via ERK pathway Target gene discovered by microarray also

regulates calcium and phosphate concentrations in vitro

Pathway in other cells with PAC1 receptor Pathway could be targeted in drug

development if only exists in neuronal cells› prevent damage during neurodegenerative

disease progression or post ischemic insult

Page 16: The goal of this project was to see if a hormone that prevents brain cells from dying could protect cells through a previously unknown pathway. We also.

Montgomery Blair High School:

Susan Ragan Elizabeth Duval

National Institutes of Health:

Dr. Lee Eiden Yvonne Holighaus


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