ANK3, which encodes the scaffolding protein ankyrin-G, is a major risk gene for bipolar disorder and schizophrenia, yet what it does in adult neurons has been unclear. This study deletes Ank3 from mouse forebrain excitatory neurons either before birth or from adolescence and finds a convergent adult profile: hyperactivity and less anxiety-like and depression-like behavior, with social behavior intact. The neurons become less active, their membrane proteome shifts, and myelin basic protein falls, a protein made by oligodendrocytes rather than by the neurons that lost Ank3.
0:00Welcome to Base by Base, the paper cast that brings genomics to you wherever you are. Thanks for listening, and don't forget to follow and rate us in your podcast app. Base by Base is now on YouTube too, at Base by Base, where every episode gets a video with chapters and the full description.
0:14Come subscribe. Glad to be here for another deep dive. So, to kick things off today, I want you to imagine a really bizarre biological paradox. Picture an animal that's just exhibiting wildly hyperactive behavior.
0:28Like constantly moving around. Yeah, moving constantly, taking abnormal risks, showing almost 0 anxiety. And logically, you know, if you were to peer inside the animal's brain, you would assume the neural circuitry is just running hot.
0:40Oh, absolutely. You'd picture the brain cells, the neurons just firing out of control in this chaotic electrical storm. Right, exactly. But what if you look directly at those specific neurons and discover they are actually firing less?
0:54Like they are sluggish? They are dim. It's massive disconnect. It really is. We have this biological situation where a quiet underactive neuron is somehow driving an intensely noisy, hyperactive behavioral state.
1:08And, you know, that completely flips our standard assumptions about how the brain works. We have this natural tendency to think that loud behavior equals loud cells. Yeah, which makes intuitive sense. Right.
1:20But to figure out how a quiet, underactive neuron creates a maniclike state, we have to kind of pull the camera back. We have to look past the electrical firing of the neurons themselves, and look at the physical architecture of the brain.
1:33The structural stuff. Exactly. The scaffolding, the white matter insulation, and the literal cellular docking ports where these cells actually connect to each other. Well, today we celebrate the work of Se Yun Yun, Peter Penzas, and their team at Northwestern University, Feinbrook School of Medicine, who have advanced our understanding of the genetic risk factors for neuropsychiatric conditions.
1:53Their recent research tackles this exact paradox we're talking about. And, uh, it centers on a specific gene called ANK3. Okay, let's unpack this because Anne K3 is a pretty big deal, right? Oh, in the world of psychiatric genetics, ANK3 is a heavy hitter, like large scale global studies, genome wide association studies that look at hundreds of thousands of people, have repeatedly flagged ANK3 as a major genetic risk factor for bipolar disorder and schizophrenia.
2:22Wow. Yeah, and we'd known about this statistical link for years. But the barrier we keep running into is the mechanism. Because knowing a gene is associated with a condition tells you basically nothing about what that gene is actually doing on the factory floor of the brain.
2:37Right. It's just a correlation at that point. So if we look at that factory floor, the ANK3 gene codes for a protein called Enchyron G. Let's try to visualize what Enchyron G actually does. I like to think of it structurally.
2:50Yeah, if a neuron is like a skyscraper, and Kyron G is the critical scaffolding or like the rebar built into the foundation in the upper floors. That's a great analogy. It sits at the Axon initial segment, which is the exact spot where the neuron decides to fire its electrical signal.
3:07And it sits at the synapses, the specialized ports where it connects to other cells. It acts as a physical tether. Exactly. It holds the cells machinery, like the ion channels, locked into their proper places.
3:18So obviously, if you start ripping that structural rebar out of the building, the whole function of the skyscraper is going to warp. And you weren't just warping the one skyscraper. You might be altering the entire city blocks infrastructure.
3:32Right. So to test what happens when that rebar is removed, the Northwestern team used mouse models. But before we get into the mechanics of how they did this, we really need to set a firm boundary on what we are looking at here.
3:47Important disclaimer time. Yes. They studied male mice, and these mice are modeling specific bipolar like traits, specifically hyperactivity, and a lack of normal anxiety. So we aren't saying these mice have bipolar disorder.
4:02No, we definitely aren't. Human psychiatric disorders involve, you know, incredibly complex cognitive and emotional and social dimensions. Things you can't see in a mouse. Right. It simply cannot be perfectly replicated in an animal model.
4:16But what we can do is study the biological roots of specific behavioral traits that are tied to those genetic risk factors. Okay, that makes sense. So we have these male mice, and the researchers want to rip the rebar out of the skyscraper.
4:30They want to delete the ANK3 gene. Yes, but they didn't just knock it out globally across the whole body from conception. Right. They use something called Cree recombinase technology to be incredibly precise, right?
4:42Yeah, creeper combinates is essentially molecular wizardry. It's this genetic tool that acts like highly specific programmable molecular scissors. So the researchers can engineer the mouse so that the Ink 3 gene is only deleted in a very specific type of cell.
4:59Which, in this case, is the fore brain excitatory neurons. Exactly. Even more crucially, they can control when those scissors activate. So they set up 2 distinct timelines for the mice. Okay, what were the timelines?
5:13Well, in one group, they used a driver called EMX one Cree to delete the gene prenatally, like way before the mouse was even born. And in the second group, they used cam 2 alpha tree to wait and delete the gene much later when the mouse was in adolescence in adulthood.
5:29Wait, if the rebar's getting removed either way, why go through the immense effort of creating two completely different developmental timelines? Because the brain is not a static organ. I mean, building a house from scratch is very different from renovating a house that's already built and lived in.
5:43Oh I see. Yeah, in developmental biology, if a gene is missing during fetal development, the brain might wire itself entirely differently to compensate. Losing a gene early often has these cascading severe effects.
5:55So they wanted to see if adult loss was different. Right. They hypothesize that pulling the rebar out before the skyscraper is built would yield a vastly different behavioral and cellular outcome compared to pulling it out in an adult brain where all the wiring is already established.
6:10So they have these 2 timelines, early developmental loss versus adult loss. And they need to see how this changes the mice, starting with behavior. So they put them through several behavioral assays. The standard suite of tests, yeah.
6:22Like the open field test, the elevated 0 maze and the force swim tests. Let's break down what these actually measure for our listeners. Sure. So prey animals like mice have a natural instinct called thigmotaxis.
6:35They uh, they like to hug the walls. Because they don't want to be eaten. Exactly. They stay in dark, enclosed spaces because out in the open, they're vulnerable to predators. So in an elevated 0 maze, which is basically a raised circular track where 2 sections have high walls, and 2 sections are totally exposed ledges.
6:52A typical mouse would just cautiously stick to the waltz sections, right? Right. So if a mouse is spending abnormal amounts of time running around on the exposed ledges, It's displaying a significant reduction in anxiety like behavior.
7:04It's basically taking massive risks. And the open field test is pretty similar. Just tracking how far and fast they run around a big empty box to measure basil locomotion or hyperactivity. Exactly. And then the forced swim test evaluates how quickly they give up trying to escape water, which is, uh, it's a classic measure for depression like traits.
7:23So did the timeline actually matter? Did the mice who lost the gene before birth act differently than the mice who lost it as adults? Surprisingly, the timing didn't change the traits at all. Really? Yeah, we saw striking, convergent behavioral abnormalities in both groups.
7:38Whether the gene was deleted prenatally or in adulthood. The male mice became wildly hyperactive. So they were just running constantly? Yes, they traveled way further in the open field test. They showed severely reduced anxiety in the zero maze, marching right out onto the exposed ledges.
7:55And they showed decreased depression like responses in the swim test. So basically, just maintaining this scaffolding in an adult brain is every bit as critical for regulating behavior as it is during early fetal development.
8:07Yeah, you can't just build the brain properly and assume it will run itself. It requires constant structural maintenance. Wow. But this brings us back to the paradox we opened with. The mice are running around like crazy.
8:20The behavioral output is extremely loud. So they decide to look at the cellular output using calcium imaging. Right. Calcium imaging lists researchers watch living neurons fire in real time. They introduce this fluorescent indicator called GCamp into the neurons.
8:36So it's essentially like installing a microscopic dimmer switch inside the cell that disclose neon green every time electrical current passes through it. That's a good way to picture it. When a neuron fires an action potential.
8:48Calcium ions flood into the cell. This G camp sensor binds to that calcium, changes its shape and emits light. So using 2 photon laser scanning microscopes on brain slices, you can physically watch the neural circuits lighting up.
9:03Exactly. And you'd assume, given the manic-like behavior that these brain slices were just glowing like a Christmas tree. But they weren't. What's fascinating here is they were dim. They were very dim.
9:15The spontaneous neuronal activity and cultured neurons just dropped. And when the researchers looked at acute brain slices, and artificially stimulated the neurons to force them to fire. The peak calcium amplitude, the brightness and strength of that firing was significantly blunted.
9:33So the neurons missing the ink 3 gene became less active, not overactive. but less active. Yes, absolutely less active. We have to be very clear on that. The loss of that rebar, that Enchyron G scaffolding clearly depressed the neuron's ability to fire effectively.
9:52The cell is basically physically struggling to depolarize and send a STRING signal. Right, because the structural integrity at the Axon initial segment is fundamentally compromised, so the neuron goes quiet.
10:04But the behavior gets loud. It's so counterintuitive. So to figure out how a quiet cell causes this behavioral cascade, they had to look at what was happening to the actual molecular inventory of the brain.
10:18And to do that, they use a technique called TMTLCMS proteomics to analyze the brain tissue. Okay, let's translate that for everyone. Mass spectrometry is basically taking the brain tissue, separating the different cellular components in a centrifuge, chopping all the proteins up, tagging them with heavy molecular weights, and running them through a magnetic field, right?
10:37Yep. It identifies exactly what proteins are present and in what quantities. And they specifically looked at the P2 fraction. Let's explain what that is, because it becomes incredibly important later in the deep dive.
10:49So when you spin brain tissue in a centrifuge. It separates out based on density. The P2 fraction is the membrane enriched fraction. It's basically the outer envelope of the cells and the general surrounding machinery.
11:03Got it. And when they analyze this general P2 fraction and the brains of the mice, missing Ink 3. They found massive proteomic remodeling. 75 different proteins were significantly altered. So the brain is clearly trying to adapt to the missing scaffolding.
11:20And some of the proteins that were upregulated, meaning the brain started manufacturing more of them, are known risk genes for entirely different disorders. Yeah, that was surprising. Like a protein called Ryr2, which is linked to schizophrenia and tauc 2, which is linked to autism spectrum disorder.
11:37Both of those just spiked in concentration. It suggests the neural network is desperately trying to compensate for the missing Anchyron G, by leaning on these other structural and signaling pathways. But amidst all of this remodeling, there was one massive unexpected drop.
11:53And here's where it gets really interesting. A protein called myelin basic protein, or MVP, was severely down regulated. Severely. Now, let me make sure I'm not jumping to conclusions here because the name MVP is a dead giveaway.
12:08It's a critical structural component of myelin, which is the fatty insulation that wraps around nerve axons to help signals travel efficiently. Correct. But myelin isn't made by the excitatory neurons where they deleted the N3 gene.
12:22You've hit on the exact reason this was such a shock. MVP is produced by an entirely different class of support cells in the brain called oligodendrousytes. Wow. Yeah, the researchers deleted a gene inside a neuron, and a vital insulation protein produced by a completely different neighbor cell just crashed.
12:41So, wait, does that mean the insulation is literally melting away? Like, do these mice have less white matter? Are they experiencing active demylination? We have to be incredibly careful here, because it is totally natural to assume that less MVP means the myelin sheath is degrading.
12:58Right. But when the researchers use super resolution microscopy to physically look at the structure, the mileage heat diameter did not change significantly. Oh, really? Yeah. The physical insulation was still there.
13:10wrapping the nerves. What changed was the molecular composition. Specifically, a massive reduction in the expression of the MVP protein inside that insulation. And we still don't know the specific cell type behind that lower MVP signal.
13:25So we need to call it reduced MVP expression, not proven mylin loss. The pipe isn't broken, but the material the pipe is made of is chemically altered. Exactly. And it wasn't even a global issue across the whole brain.
13:40It was hyperlocalized, wasn't it? Yes. Using immunofluoresence to map the brain. They found the drop in MVP was highly specific to layers 2, 3, and 4 of the primary somaticensory cortex. Which are the specific upper layers of the cortex responsible for processing touch and sensory input from the outside world?
13:58Right. The deeper layers, like layer 5 and entirely different regions, like the hippocampus, were completely untouched. The MVP levels there were totally normal. So we have a highly localized, highly specific deficit in the molecular makeup of the brain's insulation.
14:15Now, knowing that deleting Ink 3 causes the specific drop in MVP alongside these hyperactive traits, the researchers introduced a final clinical variable, which was lithium. Right, lithium, which is one of the oldest, most foundational mood stabilizers used in human psychiatry to treat bipolar disorder.
14:35So, they fed the mice a diet enriched with lithium for three weeks. If they gave these hyperactive mice lithium, Did it calm them down? Well, this specific paper actually didn't retest their behavior. Wait, they didn't?
14:48No, because they didn't really need to. An earlier study using this exact same mouse model already proved that chronic lithium treatment rescues the behavioral deficits. It reverses the hyperactivity. Oh, I see.
15:00Yeah, the question this paper wanted to answer was, if we know lithium fixes the behavior, what is it doing to the proteins to achieve that? Is it going in and fixing all 75 of those altered proteins? Right.
15:13Does it act like a biological factory reset? Like, does it just push the big red button and force the entire P2 membrane fraction back to factory settings? It absolutely does not. The effects of lithium were surgically precise.
15:27When they analyze that broad P2 membrane fraction again, lithium didn't fix the MVP expression, it was still severely down regulated. Really? And it completely ignored those upregulated risk proteins we talked about.
15:40YR2 and Tawak 2, they were made totally elevated, untouched by the lithium. So we definitely shouldn't say lithium reversed the protium, but if it didn't fix the general cell membrane, how is it fixing the behavior?
15:54They had to dig deeper into a totally different cellular compartment. They looked at the P3 fraction. Okay, remind us how that's different. Remember how P2 is the general membrane envelope? Well, the P3 fraction is much denser.
16:08It is the postsynaptic density enriched fraction. synapsis. Right. It represents the highly specialized docking ports. The exact microscopic junctions where 2 neurons physically connect and communicate.
16:20The actual point of contact. Exactly. And in that highly specific P3 synaptic fraction. Chronic lithium treatment completely restored MVP expression back to healthy normal levels. That is wild. It selectively rescued the insulation protein strictly at the synaptic docking port, while completely ignoring the rest of the cellular membrane.
16:41It is wildly specific. But, you know, we still have a missing puzzle piece here. So what does this all mean? We have this cascade of events. You delete a gene in a neuron. The neuron gets quiet, and somehow a protein made by an oligodendrosite neighbor drops in a specific cortical layer.
16:59Right. How does a quiet neuron cause a drop in an insulation protein made by a completely different cell? Well, this is the frontier of the research. The exact biological mechanism remains unknown, but the researchers put forward a highly compelling hypothesis.
17:15Okay, let's hear it. It relies on axoglial signaling. Elegadendrocytes don't just blindly wrap nerves in mylan. They actually rely on constant electrical and chemical text messages from the neurons to know how to maintain that insulation.
17:30It is a bidirectional communication loop. Oh, wow. And because the neuron's missing Ink 3 are firing less. their peak calcium amplitudes are blunted. They simply aren't sending the text messages. That is the leading hypothesis.
17:44Because the excitatory neurons are sluggish and underactive. They failed to send the necessary signals that command the neighboring Oligadentrocytes to maintain MVP. So the neurons inability to fire properly creates a local communication breakdown in the sensory cortex.
17:59Exactly, which leads to this targeted drop in the insulation protein. If we connect this to the bigger picture, What does that lithium finding tell us about how nude stabilizers actually work? I mean, we tend to think of psychiatric drugs as just broadly calming the brain down, like turning down a global volume dial.
18:17Right, but this research challenges that global volume dial concept entirely. Lithium's highly selective compartment-specific restoration of MVP, specifically at the P3 synaptic fraction, suggest something profound.
18:31Which is? It suggests that mood stabilizers might exert their therapeutic effects, at least in part, by targeting the health of the white matter, and reinforcing the structural integrity of the synapse itself.
18:44It isn't just silencing noisy cells. It is physically repairing the axoglial communication infrastructure right at the site where neurons connect. It is repairing the city block, not just the skyscraper.
18:57That's a perfect way to put it. But as we navigate this, we definitely have to respect the boundaries of the data. For you listening, the limitations here are important markers for where the science goes next.
19:08First of all, they only studied male mice. Right. And as we noted earlier, we are looking at specific behavioral traits, not the full spectrum of a human psychiatric disorder. Absolutely. And while that axoglial communication breakdown is a beautiful hypothesis.
19:24The exact molecular signals between the underactive neuron and the oligodendracite remain an open question for future studies to one tangle. Those limitations are exactly what give the next generation of researchers their marching orders.
19:37Very true. But even with those boundaries firmly in place. I think the central insight we can extract from this deep dive is incredibly clear. Summarize it for us Deleting the N3 risk gene in adult excitatory neurons paradoxically reduces their cellular activity while driving a wildly hyperactive behavioral state.
19:58This loss triggers a surprising targeted drop in myelin basic protein expression across specific cortical layers. And that highly specific deficit in the brain's insulation network is something that chronic lithium treatment can partially restore right at the synapse.
20:14We spent so much time looking at the neurons, trying to figure out why they are misfiring. When the real culprit might be the physical environment they are sitting in. It changes the entire paradigm. Which raises an important question to think about.
20:26What does this mean for how we design future psychiatric treatments? Should we be looking past the neurons themselves and focusing on the insulation that connects them? This episode was based on an open access article under the CCBY4.0 license.
20:41You can find a direct link to the paper and the license in our episode description. If you enjoyed this, follow or subscribe in your podcast app and leave a 5 star rating. If you'd like to support our work, use the donation link in the description.
20:53Now, stay with us for an original track created especially for this episode and inspired by the article you've just heard about. Thanks for listening and join us next time as we explore more science base by base.
