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We Still Don't Know How Zika Gets into the Brain

We investigated the role of AXL in Zika virus infection of neural progenitor cells. Here, I summarize our findings in laymen's terms.

Published onFeb 17, 2017
We Still Don't Know How Zika Gets into the Brain

A version of this article was published on on April 4, 2017 

I am not sure why, but I was never able to learn how to speak Spanish. Whenever I hear a wave of rolling Rs heading my way, I try to latch on to the few words I can understand before drowning. My conversation skills do not extend beyond being able to awkwardly ask where one can find the local library or inquire about your cousin’s ability to play the guitar. In the rare instance that a native speaker is able to understand my babbling, I can do little but smile and nod at their response. 

I am fully aware that many of my fellow Americans are not bilingual, but I have no excuse for my ignorance. My mother was born and raised in El Salvador, and despite her many attempts, I could never quite grasp her native language.  As a result, I am mute at large family gatherings, deaf to the heroic stories of my grandmother who raised three girls on her own during the Salvadoran civil war.

Though Mestizo blood nourishes the fingers that type these words, I often feel a deep disconnect from the place my ancestors called home.

Two separated spheres of brain cells in a dish forming a connection.
Two separated spheres of brain cells in a dish forming a connection.

In February 2016, I was given an opportunity to strengthen my Latin roots from the comfort of my laboratory bench in Massachusetts. The Zika virus was marching north from Brazil through Central America, disproportionately affecting the poor and brown. My boss, Dr. Kevin Eggan at Harvard University, came to me with a proposal. He suggested that I use the tools we have at our disposal to study the effects of the Zika virus on the brain. Given that El Salvador already had over 6,000 suspected cases of Zika at the time, I saw this as a chance to learn, contribute, and most importantly, fight for my family 3,000 miles away. We assembled a team at Harvard and Novartis and got to work.

Trying to understand an emerging threat

Before Zika started making international headlines, a group of scientists in France had already taken some of the first important steps towards understanding this mysterious virus. These researchers, who were investigating a strain of the virus that spread throughout the South Pacific in 2013, infected skin, lung, and kidney cells on a plastic dish to try to figure out how Zika was able to enter these cells. In October 2015, the paper describing their work was published and quickly became a must-read for Zika scientists everywhere. (In fact, my colleagues and I refer to this paper as the Wikipedia of Zika a.k.a. Zikapedia).

 One of the important things that these scientists discovered was that a protein (the building blocks of the cell) called AXL plays a critical role in Zika infection. We know from previous experiments that AXL can be found on the surface of cells and is important for cell growth and movement, as well as immune defense mechanisms.

(Disclaimer: To our knowledge, this protein was not named after Guns N’ Roses lead singer Axl Rose, though we hope we are wrong).

The Zikapedia group identified the relationship between AXL and Zika by performing simple, yet informative experiments. They added different compounds to the cells before and during infection, and found that when drugs that blocked or eliminated AXL were used, the virus was unable to enter and kill cells. Thus, they concluded that AXL participates in the Zika invasion of these types of cells.

At the time that Zikapedia was published, no one knew that this poorly understood virus was causing babies to be born with small brains, a condition known as microcephaly (micro meaning small, and ceph meaning head). Once the news began to spread that Zika infection is linked to microcephaly and other brain problems, scientists around the globe started trying to understand something that was not addressed in Zikapedia—How does the Zika virus get into brain cells?  

Turning off the brain cell assembly line

To answer this question, several research teams first determined that the virus prefers to infect a type of brain cell called neural progenitor cells. Early in the development of a fetus, these progenitors multiply in number and populate the brain with both neurons (the brain cells that communicate with each other through electrical and chemical signals) and glia cells (the brain cells that support and protect neurons). From this piece of information, it was obvious how Zika was causing microcephaly—by infecting and killing fetal progenitors, Zika shuts down the factory that produces neurons and glia. Hence, there are fewer brain cells and therefore smaller brains.

A sphere of dying Zika-infected brain cells in a dish.
A sphere of dying Zika-infected brain cells in a dish.

But why does Zika infect progenitor cells? Influenced by this early work, one group in California looked at many different cell types in the brain and found that the cells that are susceptible to Zika infection are coated with our old friend AXL. Based on this correlation, these researchers hypothesized that the Zika virus was entering the progenitors and other brain cells with the help of AXL. If this were true, it would mean that we could prevent or at least slow down Zika infections by interfering with the AXL protein, just like what was done in the Zikapedia paper in non-brain cells.

Disappointing, yet informative

Fueled by these findings, my team created human progenitor cells that were missing all traces of AXL. We cut AXL out of these cells using the CRISPR gene editing system, which essentially acts like microscopic scissors for DNA. (If you haven’t heard of CRISPR, you will soon. The rumor is that Jennifer Lopez is producing a show for NBC  that revolves around this gene editing technique. I can already hear myself yelling at the TV “That’s not how it works!”).

After removing AXL from the equation and checking to make sure that the loss of this protein did not cause any major health problems for these cells, we asked “Can the Zika virus still get in?” If the answer was no, it would suggest that Zika needs AXL in order to penetrate progenitor cells. If the answer was yes, it would imply that either AXL is not important at all or that there are other routes for viral entry.

Neural progenitor cells (blue/red) infected with Zika virus (yellow).
Neural progenitor cells (blue/red) infected with Zika virus (yellow).

To get an answer to this question, we added the virus to both the normal progenitors and the progenitors that were missing AXL. We then measured the levels of infection in the two groups side-by-side. Much to our surprise and disappointment, the normal cells and the AXL-lacking cells showed the same degree of infection and died at the same rates. The absence of AXL from the outer coat of the progenitor cells did not protect this particular brain cell type from Zika infection, a finding independently corroborated by another group using a slightly different method. Studies in mice missing AXL also showed this result, which is important because cells in a dish do not always behave the same way as cells in a living animal.

Even though we did not get the results that we had hoped for, we still revealed something important. Now we know that, unlike other cells in the brain, AXL is not the only “door” that Zika can use to enter progenitor cells. We now believe that progenitors are able to produce other access points for the virus, meaning that even though we closed the AXL entrance, we may have unknowingly left the back door open to Zika. 

Hopeful uncertainty

We may not have won this battle, but I am optimistic that we, or someone else, will. My team is planning the next series of experiments to try to answer our several remaining questions regarding this viral enigma. Should we succeed in figuring out how the virus gets into progenitor cells, then perhaps someday someone will figure out a way to safely disrupt this process in order to prevent future cases of Zika-related microcephaly.

I do not know if this will ever happen. I do not know if we will succeed. I do not know if we as a people will yet again lose yet another fight against yet another infectious disease. What I can tell you, though, is that we must keep trying, because if there is anything that we have learned from this global outbreak, it is that unlike me, Zika is multilingual.

Brian Wells: Does the brain produce AXL when it is infected with Zika and it helps it spread? Or does the body produce it normally for a function and it just also happens to help the Zika virus? When you wrote you edited out the AXL protein I thought "Does that affect anything else in the brain? If it does not then why is it in there?"
Michael Wells: This is a great question that I will fully address in the next draft. This is actually a question a reviewer asked when we submitted the paper to Cell Stem Cell and we had to experiments to show some of the effects (or lack thereof) on other proteins in these brain cells.
Gabriel Stein: Interesting. I thought from my first reading that 'express' referred to a cell's ability to create a given protein, and the rest of the paragraph made me think you were referring to a cell's ability to create any AXL, not how many copies it contains. So maybe it makes sense to talk about it in the context of production.
Gabriel Stein: What does it mean that cells "express" a protein? This feels like jargon that a general audience won't understand without an explanation. If possible, it would be best to find a different way to talk about it altogether.
+ 2 more...
Gabriel Stein: Lovely article, Mike! Great summary of past research and how yours fits in. Also, great headline. Heds are super important for getting people to read, so this is a very nice start.I think there's one missing piece here for general audiences: what about Zika's impact on the brain makes it so mysterious/weird/different from other viruses? My naive assumption is that most viruses can't enter the brain because of the blood/brain barrier? But I don't actually know, and I suspect most general audiences won't know either without a little context.
Elyse Timothy: You refer to neural progenitor cells as "progenitors" throughout, except In the first line of the fourth paragraph when you refer to them as "NPCs." Is this a typo?
Michael Wells: Ah good catch. I will make the change in the next draft. Thanks.