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they themselves form synapses


Neural circuits in the mouse retina. Conical photoreceptors (red) allow color vision; bipolar neurons (magenta) relay information further down the circuit; and a bipolar neuron subtype (green) helps process signals detected by other photoreceptors in dim light

As we mammals age, many of us begin to lose our sight because the neurons in our retinas degenerate. Our retinal ganglion cells can be attacked by glaucoma, or our rods and cones (photoreceptors) can be eroded by macular degeneration or retinitis pigmentosa. Somewhere in evolution we lost our ability to regenerate these types of cells, just as we lost the ability to regenerate limbs. Once they’re gone, they’re gone.

Retinitis pigmentosa is caused by irreversible degeneration of rod and cone cells

But we humans have developed other things really well: the ability to use reason and the desire to support ourselves. And these attributes have brought us close to compensating for some of our evolutionary shortcomings.

It’s quite amazing that we can now turn human stem cells into retinal “organoids” — little balls that contain all the different cell types it takes to make a retina work, even organized in the right layers.

Retinal organs mimic the structure and function of the human retina to serve as a platform to study the underlying causes of retinal diseases, test new drug therapies, and provide a source of cells for transplantation.

But now we have learned that if we divide the organoid into individual cells, these cells are able to spontaneously form signal communication connections (synapses) with other retinal cells. This means that a patient could have their own stem cells grown in retinal cells and applied to their own retina, these new cells could functionally replace the old ones, and vision could be restored. No gene therapy required, thank you very much.

You can read all about this last hurdle being overcome in the University of Wisconsin laboratories of Drs. David Gamm and Xinyu Zhao in January 4 issue of Proceedings of the National Academy of Sciences.

Last year, Gamm’s lab had watch that rods and cones (photoreceptors) made from stem cells can react to light as healthy people do. It’s a great development for making single cells for therapy, but to be part of a functioning retina, these rods and cones must be able to transmit their signals to the rest of the retina. This happens through synapses, wafer-thin connections between neurons through which signaling molecules (mainly glutamate) have passed:

Schematic arrangement of retinal neurons. Synapses are marked with black arrows

Retinal organoids (ROs) gave Gamm and Zhao hope that defective parts of a retina could be reconstructed for real from stem cells, because not only do all RO cells form the layers they are supposed to train, but they also create links to each other inside the RO with synapses. You can see how similar the structure of an OR is to a real retina when it comes to cell types and synapses (shaded in green):

Green, antibody to Bassoon (synaptic marker); white, Hoechst (core marker). ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

So the question is, if we break those RO cells and apply the right ones to the patient’s retina, will they be able to remake those synapse connections? That’s what the Gamm and Zhao labs set out to test here.

They broke some RO with papain, which is an enzyme in papaya used as a meat tenderizer and aids in digestion but is also known to destroy synapses. (So ​​no injecting papain directly into your eyeballs, okay?)

If you score a papaya directly on the tree, the papain latex ooze out

After the papain treatment, they found that proteins important for synapse function were fortunately still there, but had somehow receded into the cells. So it looked like the cells would have a good chance of re-establishing synapses with each other if they could just find their bearings.

They cultured these RO cells together as individuals for 20 days on a plate, in a situation similar to what they would encounter when applied to a real retina. But how do you know if neurons have formed these tiny synapses and if these synapses are working?

Luckily, there’s a nifty way to do this called “synaptic tracing”. It turns out that the rabies virus can be transmitted between neurons alone thanks to functional synapses, so we can use it to find out not only if synapses are present, but also how well they are functioning. (Now seems like a good time to add the rabies virus to the very long, but ever-growing list of things not to inject into your eyeballs.)

The way it’s done is very cool, and stick with me here because you’ll get some colorful shots at the end that will make it pretty obvious what happened.

First we need to get the rabies virus to infect only a small percentage of our cells without trashing the whole culture, and we also need to mark those cells as “starters” somehow. another one. So we need to do a little configuration first.

We’re going to start with a different virus – the lentivirus – in which we put a gene for green fluorescent protein (GFP) which we directed to the nucleus. We will then be able to spot all the cells infected with our lentivirus, because they will have a big green dot in the center. We can do some trial and error with the amount of lentivirus we use to end up with about 5% of our cells infected.

We’re going to put two more genes in our lentivirus called TVA and Rgp, and we’ll see why they’re both important in a second.

Then we’re going to go ahead and infect our cells with the rabies virus, but we’re going to change the gene for its envelope protein. Usually it’s Rgp, but we’ll replace it with one called Env. Viruses that use Env as their envelope proteins can only infect cells that have TVA, and that’s exactly why we put TVA in our green-dotted cells. Now we can release the rabies virus on the culture, and it will only infect the green cells.

We will put a gene for mCherry (a red fluorescent protein) into our rabies virus, so that all the cells infected with it will have a red color throughout the cell, and it will be easy to spot the cells infected with the rage. So our green dot “starter” cells are all going to get infected with rabies because they all have TVA, and that will turn our “starter” cells festive red and green.

Remember that we also put the Rgp gene in our lentivirus, so our green dot cells also make the Rgp protein. Once the rabies virus infects our green dot cells, they will revert to their original coat protein, become themselves again, and… ohhhhhhh.

So now about 5% of our cells are red and green “starter” cells, and they can infect other cells in the culture with rabies (and give them a red color) only if they are connected to other cells by functional synapses! If this happens, we should see red blood cells without green dots, i.e. rabies-infected cells that were not starter cells. Bam! That’s your visualization, and now let’s go…

A good check to start with is the whole system we just talked about, but no Rgp in the lentivirus. This means that starter cells should not be able to infect other cells, because rabies will not have its normal coat protein. All we should see are starter cells, colored red and green.

Thus, the small graph on the left below shows red and green starter cells unable to infect other cells, even though there are active synapses. The bluer images on the left have an additional spot called DAPI, which detects DNA with a blue color, so each cell will appear blue. This way you can visualize the percentage of infected cells as starter cells. Then on the right side we get rid of the blue DAPI so you only see red and green. Notice that all those that are red also have a green dot.

Seed cells (red and green) that cannot infect other cells, even through active synapses

OK, now let’s do the real test, where Rgp is included in the lentivirus, so now the rabies virus can infect other cells, but only by active synapses. Same agreement on colors, and now we hope to see only red neurons:

Starter cells are able to infect other neurons if we have active synapses. Looks like we do!

We see a lot of rabies infections of non-starter cells, which means we have active synapses! And that means we are to clinical trials!

“We’ve quilted this story together in the lab, one piece at a time, to build confidence that we’re headed in the right direction,” says Gamm, who patented the organoids and co-founded Madison-based Opsis Therapeutics, which adapts technology to treat human eye disorders based on findings from UW-Madison. “All of this leads, ultimately, to human clinical trials, which are clearly the next step.”

After confirming the presence of synaptic connections, the researchers analyzed the cells involved and found that the most common types of retinal cells forming synapses were photoreceptors – rods and cones – which are lost in diseases like retinitis pigmentosa and age-related macular degeneration. .as in some eye lesions. The second most common cell type, retinal ganglion cells, are degenerated in optic nerve disorders like glaucoma.

“It was an important revelation for us,” says Gamm. “It really shows the potentially broad impact that these retinal organs could have.”

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