NEUROPEPTIDE LABSUMMER RESEARCH PROJECT 2014

Localized Expression of (b)Kalirin in Mouse BrainAlexandria Irace

BackgroundNeuron = electrically excitable cell in nervous system, communicate via electrochemical signals

• Dendrites receive signals• Axons transmit signals

Synapse = junction between neurons• Dendritic spines exhibit structural plasticity• Potentiation increase in spine volume

• How? Activation of various proteins and enzymes, such as Kalirin7

http://morphonix.com/software/education/science/brain/game/specimens/neuron_parts.html

• No Kal7 decreased spine density

• Kal7 is necessary for structural plasticity of dendritic spines as well as proper spine formation

Ma & Kiraly et al., J. Neurosci. 2008

Investigation

Alternative Promoter Usage • Structural significance – helical or unstructured• Functional significance – lipid binding• Developmental significance – B dominates in young

neurons, C begins to be used later in development

GE

NE

PR

OT

EIN

B Peptide

C PeptideMy project:Locational Significance – (b)KalirinDo certain neurons prefer to use the B promoter? Or is the B promoter expressed uniformly throughout the brain?

In Situ HybridizationRiboprobe Synthesis:1. (b)Kal RNA from adult wild type mouse brains DNA2. DNA put into plasmid vector3. Transcribe riboprobes

Antisense RNA – complementary to (b)Kal RNA in cells

Sense RNA (control) – (b)Kal sequence, cannot bindAntisense probe binds to (b)Kal mRNA in brain tissue:

Alkaline Phosphatase Staining

G U A C U G

C A U G A C

Antisense probe

mRNA in neurons

G U A C U G

C A U G A C1. Tagged uracils

Bind primary antibody conjugated to alkaline phosphatase2. Add substrate

Produce purple precipitate that stains cellsY

Antisense Sense

2x - Hippocampi 2x - Hippocampi

• Whole hippocampus is stained• Uniform (b)Kal expression

throughout hippocampus

Antisense - Cortex

2x – Cortex

IIIIIIIV

V

VI

• Uniform staining of cell bodies throughout cortex (besides layer I)• (b)Kalirin expression is not specific to a particular cortical layer

Antisense Sense

10x

- C

ort

ex10

x –

CA

310

x –

Den

tate

Gyr

us

Conclusions• The sense probe is an effective control to show that the

antisense binding signal is a real indication of (b)Kalirin expression (i.e. not random binding/staining)

• B promoter appears to be expressed uniformly in neurons of the hippocampus and cortex

Future Directions:• Look for C promoter expression

• Localized to same cells as B? Uniform expression?

Acknowledgements

• Thank you to Dr. Mains and Dr. Eipper for allowing me to work in their lab this summer and providing endless support and advice

• Thank you to Megan for being an incredible mentor and teacher throughout this whole process

• Thank you to everyone in the Eipper-Mains lab who made this experience so wonderful!