The Effects of BDNF on Nitric Oxide Synthase I Using Human SH-SY5Y Cells

The Effects of BDNF on Nitric Oxide Synthase I

Using Human SH-SY5Y Cells

By Amir Abyaneh, and Dr. Terrie Rife

Nitric Oxide Synthase I Nitric Oxide Synthase I (NOS I) is an

enzyme that catalyzes the reaction of a free radical, NO•.

Learning and Memory

Neurodegenerative Diseases/Problems

Why do we study it?

Nitric Oxide Synthase I

Human NOS I Structure

Note - Figure is not drawn to scale

Transcription

Translation

5’4 5’3 exon 3- 29

{ >20 kB }

exon 25’15’2

What We Know!Nerve Growth Factor

Trk A

p75

NOS 1 ↑

NEURON

Brain Derived Neurotrophic Factor

Neurotrophin ReceptorNGF – Nerve Growth Factor

P75 and TrkA

BDNF - Brain Derived Neurotrophic Factor

P75 and TrkB

Neurotrophin 3 P75 and Trk C

Neurotrophin 4/5 P75 and TrkB

Cell Lines human neuroblastoma cells SH-SY5Y A model for neuronal differentiation Responds to BDNF after being treated with Retinoic

Acid 5 days

Trk B

SH-SY5Y Cells

p75 RA

RA

RA

RA

RA

Establishing a Protocol

UntreatedRA w/ 10%FBS for 5 Days

BDNF for No Serum for 6 days

MethodsPretreatment with RA and 10% FBS for 5 DaysTreated with BDNF for 2, 4, 6, and 8 days, with a No Treatment controlRNA HarvestingReverse TranscriptionPCR Analysis

Primers – PCR analysis

433bp

380bp

No DNA

NT 2 RA 4 8

GADPH

nNOS

FUTURE PCR analysis of transcripts: 5’1 5’2 5’3 5’4

Note - Figure is not drawn to scale

Transcription

Translation

5’4 5’3 exon 3- 29{ >20 kB }

exon 25’15’2

Thanks