Quantifying Neurofilament and Tubulin Segregation in IDPN Axons

Quantifying Neurofilament and Tubulin Segregation in IDPN Axons Ellora Majumder

The Anthony Brown Lab • Studies cytoskeleton of nerve cells and the mechanism for axonal transport • Focus: The role of neurofilaments in health and disease • Expertise: light microscopy, protein biochemistry, and cell culture

Zoom in on the axon Brain Neuron neurofilament organelle Axon microtubule

Organelle Movement Along Microtubule Organelle Microtubule

Filaments moving in a cortical neuron Wang L. and Brown A. , 2010

Axon Importance = make contact with other cells Axon Transport = movement is crucial to cell survival Diseases: • Amyotrophic Lateral Sclerosis (ALS) • Charcot Marie Tooth Disease Normal motor neuron Degenerate cell

Clark, Arthur W. , John W. Griffin, and Donald L. Price. "The Axonal Pathology In Chronic Idpn Intoxication. " Journal of Neuropathology and Experimental Neurology 39. 1 (1980): 42 -55. Web.

The Big Question What is the mechanism by which this movement is disrupted in disease?

IDPN: Iminodipropionitrile • Originally used to study neurolathyrism (containing ODAP toxin) • Studied in rats, needs to be reproduced in mice • Can be used to model neurologic diseases • IDPN is cheaper, more readily available IDPN

Methods • Mice injected with IDPN and saline twice a week for 3 weeks • The nerve is cut out, fixed with formaldehyde, and baked into • resin 1 micron thick sections are then cut • The paper thin sections then undergo immunostaining for neurofilament and tubulin

Segregation Normal = neurofilament = microtubules IDPN

Before injection 3 weeks saline injections = Neurofilament = Tubulin = Overlap 3 weeks IDPN injections

Processing • Goal: To quantify thousands of axons in an unbiased way • Automated processing = less subjectivity, more reproducible outcome

Quantification: Segregation Index Jaccard Index = Overlap/Total Axon Area IDPN Index of 0 Contro l Index of 1

Control IDPN Neurofilament Tubulin Overlay

6 7 2 Axon Number 6 7 0. 38 8 9 Axon Number 2 2 2 1 1 6 6 1 2 3 4 4 3 3 5 5 1 7 7 4 77 4 8 5 6 6 9 1 9 0 10 3 0. 21 5 Average: 0. 35 0. 09 4 3 0. 53 0. 63 0. 74 0. 52 1 0. 16 5 0. 15 4 IDPN AXON INDEX 0. 09 Average: 0. 88 Index Value (from 0 -1) 0. 92 0. 89 3 0. 94 2 0. 95 0. 83 1 0. 79 0. 85 Index Value (from 0 -1) CONTROL AXON INDEX 10

Conclusion and Future Directions • Results support the claim of segregation between neurofilament and tubulin in IDPN mice • Goal: to understand neurofilament accumulation in disease • Understand the mechanism behind neurofilament transport • Segregation Swelling Disease

Acknowledgments The Brown Lab at The Ohio State University • • • Dr. Anthony Brown Paula Monsma J. Dan Fenn Elizabeth Stone Atsuko Uchida The Wellington School • • Dr. Brandon Sullivan Mr. Brautigam Mr. Frim Ms. Lin

Picture Sources • • Nerve images from J. Dan Fenn and Paula Monsma https: //upload. wikimedia. org/ http: //images. clipartpanda. com/muscle-clipart-nervous_muscle_innervation_web. jpg https: //i 2. wp. com/neurosciencenews. com/neuroscience_images/idpn-3 -3 Iminodipropionitrile. jpg http: //cen. acs. org/content/cen/articles/91/i 43/ http: //animaljusticeproject. com/AJP/extimages/p_20131204073947_6827. jpg https: //www. nature. com/articleassets/npg/nrm/journal/v 1/n 2/images/nrm 1100_153 a_f 3. gif www. wellington. org