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A key to assembling materials on the surface of live neurons

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By Grace Huckins

When Anqi Zhang arrived at Stanford University as a postdoc, she had just spent six years learning to design and build brain implants: miniscule devices that could record the activity of neurons while causing minimal tissue damage. She became used to constructing devices herself, using metal films and precise beams of light. To begin this new phase of her career, however, Zhang decided to learn something completely new: genetic engineering. Instead of manufacturing devices herself, she would have to figure out how to leverage cellular machinery to do the assembling for her.

“It was very intimidating at first,” Zhang says. “I felt like I had to prove myself again, in this brand new field.”

For her postdoc, Zhang joined the Stanford University labs of Zhenan Bao, Professor of Chemical Engineering at, and Karl Deisseroth, Professor of Bioengineering and of Psychiatry and Behavioral Sciences. As part of a team of researchers, Zhang has designed a protein complex that partially lodges in the cell membranes of specific types of neurons, where it can spark substances injected into the brain to assemble cohesive materials. If those substances are conductive, they can change neurons’ sensitivity to stimuli, create electrical connections between neurons that previously functioned independently, or facilitate brain recording and stimulation. The team’s work was published in Science Advances this August.

polymer precursors and H2O2
Targeted cells express membrane-displayed horseradish peroxidase (HRP). Upon addition of small-molecule polymer precursors and H2O2, the membrane-displayed HRPs act as reaction centers, facilitating oxidative radical polymerization on targeted neurons. Image credit: Anqi Zhang, Bao Group, Stanford U.

The basic idea behind the project—building polymers from their component parts within biological tissue—isn’t a new one.  Karl Deisseroth and Zhenan Bao previously led a team that devised a way to deposit polymers on the surface of neurons in living animals. The trick was to genetically engineer the neurons so that they produced an enzyme that could catalyze polymerization reactions. Then, when the neurons were bathed in polymer components, the enzymes embedded in their membranes transformed the components into a cohesive material.

The approach worked, but was inefficient. While some of the enzyme did reach the surface of the neurons, where it could prompt the components to assemble into a functional polymer, plenty of it remained on the inside of the cell, floating around uselessly.  The challenge was to figure out how to put the enzyme in the right place. When Zhang worked with devices, she could easily redesign them, putting components precisely where she wished. To relocate the enzyme, however, Zhang had to use an indirect approach that harnessed the cell’s natural machinery.

Our cell membranes are swimming with proteins, each of which started life somewhere within the gooey cytoplasm that fills our cells. Membrane-bound proteins are efficiently sorted, packaged, and transported to their ultimate home at the cell’s margin. By binding her catalytic enzyme to one of these membrane proteins, Zhang hoped it would be carried to the same destination, a bit like a message tied to the leg of a homing pigeon.

But proteins can be finicky: Just because a protein winds up in the cell membrane under normal circumstances doesn’t mean it will get all the way there when weighed down by an extra enzyme. Finding the right protein carrier for her enzyme required persistence. “I was stuck for one and a half years,” Zhang says. “I made probably fifty constructs, and it was a lot of work.” Some protein carriers barely worked; others managed to haul some enzyme to the membrane but left plenty lingering in the cytoplasm.

Polyaniline on HRP (+) neurons
Bright-field image of live neuron with polymer deposition. Image credit: Anqi Zhang, Bao Group, Stanford U.

Finally, Zhang decided to test CD2, a protein that lives on the surface of the immune system’s T cells. It worked better than she could have hoped: Not only did it efficiently shuttle her enzyme to its target location on the cell’s surface, but it also successfully guided several other engineered proteins that she tested. CD2 was both effective and tolerant: a potential workhorse for carrying proteins to the surface of neurons.Already Zhang is testing the limits of what CD2 can do. In one experiment, she switched out her enzyme for one that will only create polymers if it’s stimulated by light. The genetic engineering approach already allows Zhang to target specific subclasses of neurons—only those that release the neurotransmitter dopamine, for example–—so adding a light-sensitive enzyme to the mix affords an impressive degree of finesse. Zhang’s hope is that she might one day be able to “draw” electrodes directly into the brain with a beam of light. That’s certainly not something she would have been able to do with her hand-built devices.

As much as Zhang is looking forward to leveraging CD2 to build minimally invasive brain interfaces, she is just as thrilled to see what CD2 might do in the hands of other scientists. If CD2 is as versatile as it seems, neuroscientists could use it to direct all sorts of engineered proteins to the cell membranes, for purposes far beyond what Zhang has yet imagined. “It’s the foundation of building all kinds of materials on the membrane,” she says.

Anqi Zhang is the lead author and a postdoctoral scholar in the Bao Lab and the Deisseroth lab at Stanford U.  Other Stanford co-authors include Kang Yong Loh, Ph.D. student in Chemistry; Chandan S. Kadur and Charu Ramakrishnan are Research Associates and Lab Managers for the Deisseroth Lab; Lukas Michalek is Postdoctoral Scholar in Chemical Engineering; Jiayi Dou is Postdoctoral Scholar in Bioengineering; Zhenan Bao is the K.K. Lee Professor of Chemical Engineering and also a member of Stanford Bio-X, the Stanford Cardiovascular Institute, the Maternal & Child Health Research Institute (MCHRI), the Precourt Institute for Energy, Sarafan ChEM-H, Stanford Woods Institute for the Environment, the Wu Tsai Human Performance Alliance, the Wu Tsai Neurosciences Institute, and an Investigator in CZ Biohub; Karl Deisseroth is the D.H. Chen Professor of Bioengineering and of Psychiatry and Behavioral Sciences, an Investigator at the Howard Hughes Medical Institute, and is also a member of Stanford Bio-X and the Wu Tsai Neurosciences Institute.

Zhenan Bao and Karl Deisseroth were supported by grants from the National Science Foundation and the W.M. Keck Foundation; Anqi Zhang was funded by the American Heart Association; Kang Yong Loh was supported by a Stanford ChEM-H Chemistry/Biology Interface Predoctoral Training Program grant and the Stanford Bio-X Bowes Fellowship; Lukas Michelak was funded by a Walter Benjamin Fellowship Program by the Deutsche Forschungsgemeinschaft grant. Part of this work was performed at Stanford University Cell Sciences Imaging Core Facility. Part of this work was performed at the Stanford Nano Shared Facilities (SNSF), supported by a National Science Foundation award.

The eWEAR-TCCI awards for science writing is a project commissioned by the Wearable Electronics Initiative (eWEAR) at Stanford University and made possible by funding through eWEAR industrial affiliates program member Shanda Group and the Tianqiao and Chrissy Chen Institute (TCCI®).