Redox manipulation alters hippocampal neuron response to semaphorin 3A

The function of the brain necessarily rests upon the proper cumulative functioning of billions of neurons. This accumulation of neuronal function requires a sophisticated network between neurons with neuronal axons sending signals and neuronal dendrites typically receiving them. The plastic nature of the brain requires this network to be first formed and then remodeled on a continuous basis. This sophisticated and flexible network is guided by a variety of navigational cues. These cues include the secreted protein known as semaphorin 3a (Sema3A) and various redox reactions such as the oxidizing function of Mical proteins on f-actin that function downstream of Sema3A. However, what is less understood is how various redox conditions alter these guiding cues. This work investigates the subcellular redox state of hippocampal neurons and the effects redox environments have on the navigational cue Sema3A. I first investigated the unperturbed cytosolic subcellular glutathione redox state of rat hippocampal neurons. I monitored this redox state in live cells using a redox sensitive green fluorescent protein (roGFP) specific to glutathione (GSH). Using this roGFP at three different time points on primary cultured hippocampal rat neurons, I found that axons and dendrites do not uniformly maintain distinct glutathione redox states. While axons and dendrites react in a near opposite manner to each other in response to Sema3A, this does not necessitate the maintenance of a distinct subcellular cytosolic redox niche. Next, I tested the hypothesis that oxidizing conditions would increase the f-actin collapsing effect of Sema3A on axons and increased growth on dendrites. To accomplish this task, I bathed cultured rat hippocampal neurons for 24 hours with or without Sema3A and with or without oxidizing (hydrogen peroxide) or reducing (GSH) conditions and measured the lengths of axons and dendrites. I discovered that reducing conditions with Sema3A produced longer dendrites as predicted but that oxidizing conditions with Sema3A produced no change in axon length compared to the control group despite Sema3A and oxidizing conditions alone producing shorter length axons. I then repeated 24-hour bath treatments with gradients of either hydrogen peroxide (H2O2) or diamide (DIA) with/without Sema3A after first monitoring the initial effects of the treatments using spatial light interference microscopy (SLIM) in real time. The effects of reducing conditions on dendritic growth was consistent with the initial hypothesis but the effects of oxidizing conditions revealed a more complex relationship between Sema3A and redox states. Finally, I wanted to see if the results from 24-hour baths with different conditions were replicated in real time in isolated individual axon tips. I used SLIM to monitor dry mass changes and filopodial dynamics in rat hippocampal neuronal axons exposed to different conditions within microfluidic devices (µFDs). I found there to be significant diversity in both filopodial dynamics and dry mass change in the axonal tips of hippocampal rat neurons exposed to various combinations of Sema3A and redox states. These diverse reactions included differences between Sem3a exposure alone versus Sema3A and hydrogen peroxide combined. I then subjected these SLIM images to automated image analysis and discovered multiple interactions between Sema3A and GSH, H2O2, as well as diamide (DIA). The results of these investigations further untangle the behavior of hippocampal neurons that maintain a homogenous cytosolic redox state while also being sensitive to their redox environment. The redox environment does indeed alter hippocampal neuronal response to Sema3A with dendrites increasing in length as predicted but with axons behaving in an unexpected manner. Interestingly, H2O2 and DIA do not interact with Sema3A in the same way despite both chemicals being oxidizers. These results improve the field’s knowledge about Sema3A and redox signaling in the brain and will aid further research in the mechanisms of axonal/dendritic navigation.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Cell and Developmental Biology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2024

Author dc:creator

• Norsworthy, Miles David

Contributors dc:contributor

• Gillette, Martha U
• Brieher, William
• Smith, Andrew
• Li, Xin

• Hydrogen Peroxide
• Diamide
• Glutathione
• Cell
• Development
• Slim
• Real Time Imaging
• Microfluidic
• Microenvironment
• Hippocampus
• Dendrite
• Neuron
• Brain
• Redox
• Oxidation
• Sema
• Sema3a
• Semaphorin
• Axon

Statement dc:rights

• Copyright 2024 Miles Norsworthy

Language dc:language

eng, en

Handle dc:identifier

https://hdl.handle.net/2142/127324

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/127324