Nascent Neurons in Early Animals

Some of the most primitive animals have no nerves or neurons... how do they know what is going on?

We often think of our brains as computers, but while human-made computers are (so far) strictly electrical, our brains have a significantly different basis. The electrical component is comparatively slow, and confined to conduction along the membranes of single cells. Each of these neurons communicate with others using chemicals, mostly at specialized synapses, but also via other small compounds, neuropeptides, and hormones. That is why drugs have so many interesting effects, from anesthesia to anti-depression and hallucination. These properties suggest that the brain and its neurons began, evolutionarily speaking, as chemically excitable cells, before they became somewhat reluctant electrical conductors.

Thankfully, a few examples of early stages of animal evolution still exist. The main branches of the early divergence of animals are sponges (porifera), jellies and corals (ctenophora, cnidiaria), bilaterians (us), and an extremely small family of placozoa. Neural-type functions appear to have evolved independently in each of these lineages, from origins that are clearest in what appears to be the most primitive of them, the placozoa. These are pancake-like organisms of three cell layers, hardly more complex than a single-celled paramecium. They have about six cell types in all, and glide around using cilia, engulfing edible detritus. They have no neurons, let alone synaptic connections between them, yet they have excitable cells that secrete what we would call neuropeptides, that tell nearby cells what to do. Substrances like enkephalins, vasopressin, neurotensin, and the famous glucagon-like peptide are part of the managerie of neuropeptides at work in our own brains and bodies.

A placozoan, about a millimeter wide. They are sort of a super-amoeba, attaching to and gliding over surfaces underwater and eating detritus. They are heavily ciliated, with only a few cell types divided in top, middle, and bottom cell layers. The proto-neural peptidergic cells make up ~13% of cells in this body.

The fact is that excitable cells long predate neurons. Even bacteria can sense things from outside, orient, and respond to them. As eukaryotes, placozoans inherited a complex repertoire of sense and response systems, such as G-protein coupled receptors (GPCRs) that link sensation of external chemicals with cascades of internal signaling. GPCRs are the dominant signaling platforms, along with activatable ion channels, in our nervous systems. So a natural hypothesis for the origin of nervous systems is that they began with chemical sensing and inter-cell chemical signaling systems that later gained electrical characteristics to speed things up, especially as more cells were added, body size increased, and local signaling could not keep up. Jellies, for instance, have neural nets that are quite unlike, and evolutionarily distinct from, the centralized systems of animals, yet use a similar molecular palette of signaling molecules, receptors, and excitation pathways. 

Placozoans, which date to maybe 800 million years ago, don't even have neurons, let alone neural nets or nervous systems. A recent paper labored to catalog what they do have, however, finding a number of pre-neural characteristics. For example, the peptidergic cell type, which secretes peptides that signal to neighboring cells, expresses 25 or more GPCRs, receptors for those same peptides and other environmental chemicals. They state that these GPCRs are not detectably related to those of animals, so placozoans underwent their own radiation, evolving/diversifying a primordial receptor into hundreds that exist in its genome today. The researchers even go so far as to employ the AI program Alpha Fold to model which GPCRs bind to which endogenously produced peptides, in an attempt to figure out the circuitry that these organisms employ.

This peptidergic cell type also expresses other neuron-like proteins, like neuropeptide processing enzymes, transcription regulators Sox, Pax, Jun, and Fos, a neural-specific RNA polyadenylation enzyme, a suite of calcium sensitive channels and signaling components, and many components of the presynaptic scaffold, which organizes the secretion of neuropeptides and other transmitters in neurons, and in placozoa presumably organizes its secretion of its quasi-neuropeptides. So of the six cell types, the peptidergic cell appears to be specialized for signaling, is present in low abundance, and expresses a bunch of proteins that in other lineages became far more elaborated into the neural system. Peptidergic cells do not make synapses or extended cell processes, for example. What they do is to offer this millimeter-sized organism a primitive signaling and response capacity that, in response to environmental cues, prompts it to alter its shape and movement by distributing neuropeptides to nearby effector cells that do the gliding and eating that the peptidergic cells can't do.

A schematic of neural-like proteins expressed in placozoa, characteristic of more advanced presynaptic secretory neural systems. These involve both secretion of neuropeptides (bottom left and middle), the expression of key ion channels used for cell activation (Ca++ channels), and the expression of cell-cell adhesion and signaling molecules (top right).

Why peptides? The workhorse of our brain synapses are simpler chemicals like serotonin, glutamate, and norepinephrine. Yet the chemical palette of such simple compounds is limited, and each one requires its own enzymatic machinery for synthesis. Neuropeptides, in contrast, are typically generated by cleavage of larger proteins encoded from the genome. Thus the same mechanism (translation and cleavage) can generate a virtually infinite variety of short and medium sized peptide sequences, each of which can have its own meaning, and have a GPCR or other receptor tailored to detecting it. The scope of experimentation is much greater, given normal mutation and duplication events through evolutionary time, and the synthetic pipeline much easier to manage. Our nervous systems use a wide variety of neuropeptides, as noted above, and our immune system uses an even larger palette of cytokines and chemokines, upwards of a hundred, each of which have particular regulatory meanings.

An evolutionary scheme describing the neural and proto-neural systems observed among primitive animals.

The placozoan relic lineages show that nervous systems arose in gradual fashion from already-complex systems of cell-cell signaling that focused on chemical rather than electrical signaling. But very quickly, with the advent of only slighly larger and more complex body plans, like those of hydra or jellies, the need for speed forced an additional mode of signaling- the propagation of electrical activity within cells, (the proto-neurons), and their physical extension to capitalize on that new mode of rapid conduction. But never did nervous systems leave behind their chemical roots, as the neurons in our brains still laboriously conduct signals from one neuron to the next via the chemical synapse, secreting a packet of chemicals from one side, and receiving that signal across the gap on the other side.

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Emergency- Call UCP!

Uncoupling proteins in mitochondria provide a paradoxical safety valve.

One of the great insights of biochemistry in the last century was the chemiosmotic theory, which finally described the nature of power flows in the mitochondrion. Everyone knew that energetic electrons were spun off the metabolism (burning) of food via the electron transport chain, ending up re-united with oxygen (creating the CO2 we breathe out). But how was that power transmitted to ATP? The key turned out to be a battery-like state across the mitochondrial membrane, where protons are pumped out by the electron transport chain, and then come back in while turning the motor of the ATP synthase to phosphorylate ADP into ATP. It is the (proton) concentration and charge difference (that is, the chemiosmotic gradient) across the inner mitochondrial membrane that stores and transmits this power- a clever and flexible system for energizing the mitochondrion and, indirectly, the rest of the cell.

Schematic view of the electron transport chain proteins, as well as the consumer of its energy, the ATP synthase. The inside of the mitochondrial matrix is at top, where core metabolism takes place to generate electrons, resulting in protons pumped out towards the bottom. Protons return through the ATP synthase (right) to power the phosphorylation (so-called oxidative phosphorylation) of ADP to ATP.

Chemiosmotic theory taught us that mitochondria are always charged up, keeping a balance of metabolism and ATP production going, all dependent on the tightness of the inner mitochondrial membrane, which was the "plate" that keeps the protons and other ions sealed apart. But over the years, leaks kept cropping up. In the human genome, there are at least six uncoupling proteins, or UCPs, which let protons through this membrane, on purpose. What is the deal with that?

One use of these proteins is easy enough to understand- the generation of heat in brown fat. Brown fat is brown because it has a lot of mitochondria, which are brown because of the many metal- and iron-hosting enzymes that operate at the core of metabolism. UCP1 is present in brown fat to generate heat by letting the engine run free, as it were. It is as simple as that. But most of the time, inefficiency is not really the point. The other UCP proteins have very different roles. On the whole, however, it is estimated that proton leaks from all sources eat up about a fourth of our metabolic energy, and thus evidently play a role in making us warm blooded, even apart from specialized brown fat.

A more general schematic that adds UCP proteins to the view above. Leaks also happen through other channels, such as the membrane itself, and also the ANT protein, at low and non-regulated rates..

One big problem of mitochondria is that they are doing some quite dangerous chemistry. The electrons liberated from metabolism of food have a lot of energy, and the electron transport chain is really more like a high voltage power station. The proteins in this chain are all structured to squeeze all the power they can out of the electrons and into the proton gradient. But that runs the risk of squeezing too hard. If there is a holdup anywhere, things can back up and electrons leak out. If that happens, they are likely to combine with oxygen in an uncontrolled way that generates compounds like peroxide, superoxide, and hydroxy radicals. These are highly reactive (customarily termed ROS, for reactive oxygen species) and can do a great deal of damage in the cell. ROS is used in some signaling systems, such as the pathway by which glucose stimulates insulin secretion in the pancreas, but generally, ROS is very bad for the cell and rises exponentially with the severity of blockages in the electron transport chain. Many theories relating to aging and how to address it revolve around the ongoing damage from ROS.

Thus the more important role for the other UCP proteins is to function as a safety valve for overall power flow through mitochondrial metabolism- a metaphorical steam valve. UCP proteins are known to be inducible by ROS, and when activated, allow protons to run back into the matrix, which relieves the pressure upstream on all the electron transport chain proteins, which are furiously pumping out protons in response to the overall metabolic rate of fat/sugar usage. While metabolism is regulated at innumerable points, it is evident that, on a moment-to-moment basis, an extra level of regulation, i.e. relief, is needed at this UCP level to keep the system humming with minimal chemical damage to the rest of the cell.

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The Lucky Country

The story of California, the story of the US, and optimism about free frontiers.

I am reading "California, the great exception". This classic from 1949 by Cary McWilliams is stoutly jingoistic and pro-California. But it also provides a deeper analysis of the many things that made California such an optimistic and happy place. Mainly, it boils down to free land and rapid settlement by ambitious working people. The Native Californians were so weak, and so ruthlessly extirpated, that they did not present the irritating conflict that happened elsewhere in the US. California's gold was so widely and thinly distributed (as placer in streams) that mining was a matter of small partnerships, not huge businesses, as it became elsewhere in the West, in the deep hard rock silver and later copper mines of Nevada (Carson city and the Comstock lode) and Montana (Butte). The immigrants were of working age and enthusiastic to work, dismissing slavery and corporatism in favor of a rapacious entrepreneurialism. 

California never had a paternal territorial government, but transitioned directly from self-rule to statehood, its riches speaking volumes to the national government in Washington. And the national government was anxious lest secessionist sentiment spread to the still far-distant west, so it funded the building of a transcontinental railway, during the civil war when money must have been extremely tight. That feared secession was not to join the South, but rather to found a new and prosperous nation on the West Coast. San Francisco went on to serve as the financial capital of the West, particularly of western mining, creating almost overnight a collusus to rival the centers of the East. In due time, gushers of oil also appeared on the California landscape. It is no wonder that Californians became fundamentally optimistic, ready to take on huge challenges such as water management, building a great education system, and the entertainment of the world.

California was also blessed by weak neighbors on all sides. There were no foreign policy predicaments or military threats. It could nurse its riches in peace. It was, in concentrated form, the story of America- of a new continent limited more by its ability to attract and grow its population than by its land and the riches that land held. An isolated continent that wrote its society almost on a blank slate- a new government and a melting pot of people from many places. 

Bound for California, around 1850.

How stark is the contrast to a country like Ukraine, neighbor of imperialist Russia and before that host to the Scythians, Goths, and Huns. A flat land exposed on all sides, that has been overrun countless times. A fertile land, but always contested. The idea that history would stop, that Ukraine could join the West, and enjoy its riches in peace and security- that turns out to have been a dream that bullies in the neighborhood have a different view on. Better to beat up on the little "brother" than to build up both nations and economies through beneficial exchange and prosperity. Better for both to go down in flames than that the little "brother" escapes the bully's clutches into a more humane world.

But the happy place of the US and Calfornia has hit some rough patches too. It turns out that our resource riches are not endless after all. The foundation of material wealth- the agricultural land, the mines, the lumber- underwrote social and technological innovation. No wonder the US was first in flight, and led the way in electricity, automobiles, the internet, the cell phone. Now we have an innovation economy, and get much of our materials and lower-grade goods from far-off places. The people we have attracted and continue to attract are the new wealth, but therein lies a conflict. Places like California have huge homeless populations because we have ceased to grow, ceased to embody the hope and optimism of our lucky past. Conflict has raised its head. There is no more free land, or gold in the streams. Now, with the land all parcelled up and the forests mowed down, everyone wants to hold on to what they have, and damn those who come after. Prop 13 was the perfect expression of this sour and conservative mood- let the newcomers pay for public services, not us.

California is transitioning from a visionary frontier into a cramped, normal, and not especially lucky place. The fabulous climate is suffering under fire and drought. The population is growing significantly older, while next generation is educated less well then their parents. The app innovation economy has fostered a nightmare of surveillance and social dysfunction. The pull of a new frontier is so strong, however, that some of our richest people now imagine it on other planets. The irony of sending rockets, fueled by vast amounts of fossil carbon and compressed oxygen, to other worlds where there isn't even air to breathe, let alone plants to cut down, begs belief. It is the final gasp of a dream that somewhere, out there, is another lucky country.

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Neutrophils Ask: How Did I Get Here?

With apologies to the Talking Heads... how the amoeboid cells of our immune system travel around in response to outside cues like cytokines.

Amoeboid cells seem so alive and even conscious. They seek out prey, engulf, and kill it. How is that done, and what are they thinking? Molecular biologists naturally come at this from a molecular perspective, asking what the signals are, how are they received, what pathways relay them to the cytoskeleton, and so forth. No soul is assumed, and none has been found, despite the great complexity of these cells and their activities.

The story starts with receptors at the surface, which can sense many of the cytokines of the immune system, of which there are roughly a hundred. These have many roles, including pro-inflammatory and anti-inflammatory effects. Neutrophils, which are the subject of today's paper, also have receptors that directly sense pathogens, like bacterial cell coats, viral double stranded RNA, and also broken cells, like DNA out in the environment where it shouldn't be. One question is how these cells sense shallow gradients- they can orient properly with as little as two percent difference in concentration between back and front. This is thought to involve pretty strong feedback systems that accentuate the stronger signal and then keep strengthening it in concert with the cytoskeleton that the receptors ultimately organize and orient. But that then leads to the next question of what turns this feedback process off, preventing locking on one target, so that neutrophils can turn on a dime and pursue a new target, if needed?

The molecular basics of cell orientation in eukaryotes have taken a long time to establish. The cell surface receptors typically activate G proteins, specifically the beta/gamma subunit, which can activate an enzyme called PI3 kinase (PI3K). This enzyme puts a phosphate group on the membrane lipid inositol, generating inositol triphosphate, or IP3. This lipid is a sort of beacon, which attracts a variety of other proteins to come to the membrane, among which is DOCK2, and other members of its family of guanine exchange proteins, which in turn activate RAC, by encouraging it to release GDP and bind GTP. RAC is a key node here that is active with GTP. RAC then activates other proteins like WAVE and PAK1, which go on to activate ARP2 and its family members, which are, finally, the proteins which nucleate extension and branching of actin in filaments, which provide the actual power behind cell protrusions and movement.

A sketch of the signaling cascade from outside the cell to cytoskeletal re-orientation. R stands for receptor. One form of feedback is shown, which is positive reinforcement from locally active Rac and actin, back to PI3K. This helps the local front stay coherent in pursuit of prey or gradients of signals.

It has also been found that both RAC and actin have some kind of local positive feedback effect on neutrophils, allowing migrating cells to establish stable fronts that respond to gradients of stimulating molecules. At the same time, there is a global negative regulation system, mostly due to the tension from actin and on the cell membrane, which encourages retraction of cellular fronts that are not experiencing stimulating signals. All this obviously contributes to the ability of cells to go one way, and have their back ends follow. 

The current paper asked in a little more fine grained detail how the front mechanism works- how does it avoid locking up from positive feedback, and how does it allow other areas of the cell to take over if they see stimulation on their sides? They set up a remarkable system of light-activated PI3 kinase, where they could shine blue light on one side of the engineered cells and see them move in that direction, from the excess PI3K activity. This system derives from an obscure bacterial protein that rearranges a flavin cofactor under blue light, in a way that can allow binding surfaces to be hidden or revealed. 

In the key experiement, they shined light on one side of their cells, then turned it off for a bit, and the shined light on the entire cell. This tests whether there is a residual effect from the prior stimulation. Would the cells be entrained to keep going where they were going before? Or would they not care, or would they try something new? The answer clearly (and reproducibly) was that they struck off in a new direction. This shows that there is a habituation or inhibition mechanism at work, over some slow time period, which acts in activated regions. 

 The source for this video is the main paper behind this post. The dashed circle indicates where the researchers shined their blue light which induces local PI3K activity. Note how at first, they are leading the cell by just the front. When this cell gets to the midline, they switch to illuminating the whole cell, to ask whether there is residual activation or inhibition from the earlier illumination. The observation that the cell then veers off opposite to the original stimulation indicates that inhibition is the residual effect from the former activation.

 

Such habituation is a critical piece of behavior that follows gradients. It gets used to what it just saw, and if the next unit is the same intensity, it doesn't care that much (though probably will keep going). If the next unit of stimulation is increased, then it will keep going. But if it is decreased, then the inhibition kicks in and the front slows down, allowing other areas of the cell to expand if they are seeing increased gradients. Thus temporal and spatial gradients can both be negotiated, using a finely tuned mix of positive and negative feedbacks.

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