NEUROPEPTIDE LAB SUMMER RESEARCH PROJECT 2014 Localized Expression of (b)Kalirin in Mouse Brain Alexandria Irace
Aug 08, 2015
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
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!