| dc.description.abstract | Neuron come in more than 500-fold range in cell size (include soma, dendrites, axons, and all arbors) in mammalian evolution and across structures (Keller et al., 2018; Mota & Herculano-Houzel, 2014a). This variability in neuronal cell size is also manifested within a structure. For example, in the rat cerebellar cortex, a typical Purkinje cell body is 10 to 20 µm in diameter, while a granule cell body is only 5 µm across. In comparison, the size of glial cells in the brain is largely invariable across species and structures (Dos Santos et al., 2020; Herculano-Houzel & Dos Santos, 2018; Mota & Herculano-Houzel, 2014a). Recent work from our lab also shows that average cell size in non-brain organs across mammalian species is also invariable (Gabi and Herculano-Houzel, in preparation), similar to glial cells in the brain. This is also true for cells of a same type within an organ. For instance, hepatocytes in the liver or beta cells in the pancreas are homogeneous in cell size (Ginzberg et al., 2015; Savage et al., 2007). The exception, other than neurons, are muscle cells, which are multi-nucleated (and thus have multiple genomes from which transcription and translation ensues, producing mRNAs and proteins), have elongated shape (which provides them with higher surface-to-volume ratio and access more capillary bed than oval-shaped cells), and are excitable (which makes rates of energy-use activity-dependent and variable).
How does neuronal cell size diversity come about across cells within a brain structure, across brain structures within a brain, and across brains of different species? How come the neuron is the only single-nucleated cell type in the body that is highly variable in cell size across species and across structures? And in all those cases, is it the same mechanism at play, such that variability in neuronal cell size across species is just a continuation of the variability in neuronal cell size across structures? This is the central question I addressed during my PhD.
Here I first formulate and test the hypothesis that evolutionary diversity in the size of neuronal but not non-neuronal cells is the result of evolutionary variation that is especially high in DNA sequences across species, whether protein-coding or regulatory, that are exclusively associated with neurons. To test this hypothesis, I calculate measurements of molecular evolution of protein-coding DNA sequence specifically expressed in different brain cell types as well as regulatory DNA sequences associated with different brain cell types across more than a hundred amniote species. I validate the results by repeating all measurements with four different expression datasets. Contrary to the prediction, I find that neuron-specific protein-coding genes and neuron-associated cis-regulatory DNA elements are very constrained in evolution, compared to their non-neuronal counterparts; in fact, my results show that neuron-specific sequences are as conserved as ATPase sequences, the benchmarks of extreme levels of negative selection. I also find that the most conserved neuron-specific genes are enriched in the gene ontology terms “transportation” and “signal-transduction”. I propose that a fair interpretation of these results is that “you don’t mess” with neuronal excitability: the fact that it cannot come to be without a large negative membrane potential presents both an opportunity and a challenge, for disturbances in the dynamic equilibrium of such a huge dam of charges has the potential to end in a dead neuron. I propose that once such a dam is built in a cell and the integrity of the latter depends on the continued dynamic equilibrium of the former, there is no going back. Similarly, I find that neuron-specific genes are more conserved than genes specifically expressed in other organs of the body, and only heart-specific and musculature-specific genes (which are thus also expressed in excitable cells) are peers to neurons in terms of the level of negative selection.
The finding that enormous evolutionary neuronal cell size diversity is simultaneous with highly conserved neuron-specific coding and regulatory sequences in these uniquely excitable cells led me to formulate a testable alternative hypothesis to explain such evolutionary diversity: That neuronal cell size is a self-regulated property that emerges during the development of each brain from the interplay between neuronal excitability (defined by gene expression patterns that are cell type-specific) and supply-limited energetic constraints to neuronal growth and activity, effectively imposing a trade-off between cell size and activity. As a result, and only because neurons do not increase in cell size isometrically (that is, they grow with marked changes in cell shape), I propose that larger neurons, but not cells of other types, are actually affordable, that is, viable.
In our proposed scenario of supply-limited energetic constraints to the combination of neuronal cell growth and activity, the two most energy-intensive processes in a neuron, I posit that increased neuronal cell size is possible as a trade-off with lower neuronal activity such that larger neuronal cell size should be associated with lower excitability, both within individual brains and across species. I then test that hypothesis using matching electrophysiology and morphology data of single cells from the human cortex, and find a strong predictability between lower firing rates and larger neuronal cell size, specifically of the total surface area of the dendritic surface.
Taken together, the research summarized in this dissertation strongly indicates that diversity in amniote neuronal cell size is not the result of evolutionary variation in neuron-specific DNA elements, which is the standard assumption made in the context of the Modern Synthesis of evolutionary theory to account for changes over evolutionary time in any characteristic of living beings. Rather, I argue that during development, neurons grow as much as they can, given (1) the rate of energy supplied to any volume of developing brain tissue and (2) depending on how many neurons share that same energy "cake". In this scenario, the key determining factor is the one true “requirement” for life, which is energy availability to power growth and sustain cell volume. For intrinsically excitable cells such as neurons, the allocation of the energy budget defined by (1) and (2) must in turn depend on intrinsic excitability (which is presumably determined by combinations of genes expressed in neurons of different types that determine the allocation of cell membranes to different neurite compartments and/or density of the specific types of membrane channels) on the one hand, and on how many neurons compete for a given rate of energy supply to the volume they jointly occupy. I propose that the rate of energy supply to any developing brain volume is determined by the density of capillaries and of supplying arteries to the brain, which in turn also controls the number of neurons that compete for that supply by imposing a limit to replication of neuronal progenitor cells at a pace that is clade-specific. This simple, testable, integrative theory can explain not only the large variation of neuronal cell size alone inside an individual body, but also the scaling of average neuronal cell size both within a clade and the difference in scaling rules across clades, and has wide application in medicine, as I outline in the conclusion of this dissertation. | |
