A key protein expressed in epithelial “skin” cells plays a critical role in protecting the structure and function of cells in the nervous system of a model nematode, a type of worm, according to new research published today by Fred Hutch Cancer Center scientists in the journal Developmental Cell.
The new study examines the relationship between two known cell types of the nervous system, glial cells and neurons, and the epithelial cells surrounding them at the apical tip (the nose) of the powerful model organism Caenorhabditis elegans. This tiny worm species (about one millimeter in length) has gained much attention in the last decade and a half within the scientific community for its remarkable ability to model critical aspects of nervous system pathways that have been conserved throughout evolution.
Principal investigator Aakanksha Singhvi, PhD, of Fred Hutch’s Basic Sciences Division directed the new study, building on her previous work elucidating the importance of glial cells in working alongside neurons to pick up accurate sensory messages from the environment. It’s an area of research that has fascinated her since her first forays into academic neuroscience over a quarter-century ago.
“In sensory biology we ask about different sensory systems and how they integrate into the brain,” Singhvi said. “How is the mapping and organization happening in the brain? Problems in the brain’s ability to receive and process information from the outside world properly is linked to aging, and a lot of neurological diseases.”
Old findings lead to new questions — and new discoveries
Singhvi’s previous work found that glial cells play an essential role in maintaining the shape of the neurons around them, which affects how the worm senses its external world.
In the new study, Singhvi’s lab uncovered a role for a third cell type found outside the nervous system in preserving the structural integrity of the glial-neuron unit throughout the life of the adult animal. A protein called UNC-23 that is expressed in these neighboring cells, called epithelial (skin) cells, was found to be a key player in protecting multiple sensory glia-neuron units in the head of the animal from mechanical stress.
“That a third cell was involved was an absolute surprise for us as a finding,” Singhvi stressed.
Neuron receptive endings: Receiving critical communication from the environment
Neurons collect information directly from the outside environment at specialized structures: think of the hairs inside the inner ear that receive information from passing sound waves, or the stalks of microvilli emerging from taste bud receptors that collect information from food particles in the mouth. These structures then transmit the information by secreting molecules known as neurotransmitters through gaps between neuron cells known as synaptic clefts.
These neurotransmitters are then picked up by neighbor neurons at their specialized structures in a messaging cascade that eventually reaches the brain. In this way, our circuits communicate signals that help regulate a myriad of cellular processes, from metabolism and muscle contraction, to how we respond to information we receive from the outside world. The shape of these special “receiver” structures, or neuron receptive endings (NREs), play a critical role in receiving external environmental signals. In short, the shape of the neuron-ending itself determines its ability to properly perform the task of accurate sensory perception.
In previous work, Singhvi and her colleagues had pointed to the importance of neighboring glial cells in maintaining the shape of the NREs of many neurons. Singhvi and colleagues had further shown that a specific type of glial cell, AMsh glia, controls the shape of the NRE of sensory neurons including AFD (termed AFD-NRE), the primary thermosensory neuron in C. elegans. Defects in the shape of AFD-NRE lead to the misfunctioning of the neuron: A potentially lethal threat for an animal that must sense heat danger or risk wiggling into a too-hot environment.
UNC-23: A new player in the glial-neuron relationship
To dig further into how animals maintain their NRE shapes, Singhvi turned to the genetic method of forward screening: a classic technique used to create many different mutations in a model organism. By exposing the animals to a known mutagen (a chemical with the ability to intercalate, or weave itself into, the DNA of the organism), genes can be disrupted and kept from functioning properly. Mutants are then chosen for study based on their resulting phenotypes (that is, observable traits and/or behaviors, NRE shape in this case). This technique allows researchers to investigate mutants with specific defects to determine the genetic source of the defect, and from there to ask, where, how, and when the implicated gene works during the life of the animal.
Singhvi’s lab identified that when the C. elegans gene unc-23 was mutated, the size of the NRE on the AFD neruon was altered. (The standard naming convention for C. elegans proteins utilizes all capital letters, while the corresponding gene that encodes the protein is italicized and written in lowercase letters. Thus unc-23 is the gene that, when transcribed and translated by the cell’s machinery, yields the functional protein UNC-23.) As a coincidence, it was the exact same DNA change as unc-23(e25), a mutation that had been identified 50 years ago in the first genetic screen ever performed on this model organism. The mutants demonstrated significant overgrowth of AFD-NRE, a phenotype that they dubbed “meander.”
“It was very fascinating, because [unlike other mutants], the AFD-NRE in unc-23 mutant animals was actually becoming super-extra-long,” Singhvi said. “So, it was a very intriguing mutant for me. But what was initially confusing was that try as we could, we just couldn’t get a clear answer if the gene was working in the glia or the neuron.”
Singhvi enlisted Cecilia G. Martin, then a research technician in her lab and currently a trainee in the University of Washington’s Medical Scientist Training Program (MD/PhD), to work on this project. They worked together to brainstorm different potential mechanisms, which led them to look outside the box and consider if other nearby cell types in the C. elegans head, the epithelial or muscles, could be contributing to the shape of a neuron.
A series of experiments unearthed a fascinating link between epithelial cells, glial cells and the shape of the AFD-NRE. Singhvi’s lab determined that UNC-23, expressed in the epithelial cells (the animal’s skin), is a critical protein for maintaining the shape of the glia and neurons of the sensory nerves in the animal’s head.
When the UNC-23 protein is altered, as in the mutant unc-23(e25), both the epithelial cell shape and its apical polarity (essentially, how it is arranged in the organism relative to the tip of the nose) stretch due to mechanical stress brought on by the animal’s movement. That stretching causes a loss in a key component of the cytoskeleton, an internal structure that helps cells maintain their shape, in the adjacent AMsh glial cells (SMA-1, a protein with the human ortholog ßH-Spectrin). The loss of UNC-23 also causes disorganization in filamentous actin protein in the AMsh glia, another key component maintaining cellular shape.
Singhvi’s team discovered that the ability of epithelial UNC-23 to maintain the glial-neuron shape was specific to a defined part of the animal’s anatomy (the head). Even more surprisingly, they found that it acts only at a specific developmentally critical period (at the juvenile-adult transition stage) in its lifespan. When the protein does not work properly, the animal’s sensory neuron NREs and glia shapes get progressively worse in the later-stage adult animal (similar to premature aging), and the animal dies much earlier than it otherwise would.
UNC-23 is an “Hsp co-chaperone protein.” The “Hs” in the name stands for “heat shock”: one of several cellular stresses that can result in misfolded (and therefore misfunctioning) proteins. Misfolded proteins are either degraded by the cell’s protective machinery, or they accumulate or can be mistakenly trafficked to wrong cellular locations. Humans have an "ortholog" of UNC-23, known as BAG2 (for Bcl-2-associated athanogene), which descended from the same protein in a distant ancestor of worms and humans. BAG2 helps cells deal with stress and fold proteins properly. It is involved in many cell processes related to cell survival and is implicated in a number of human diseases, including cancer and neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease.
To develop a deeper understanding of the mechanisms by which UNC-23 functions in the tricellular epithelial cell/AMsh glial cell/AFD neuron coupling, Singhvi’s team, with research technician James Bent later joining Martin, conducted several additional experiments. Using techniques as varied as fluorescent-tag visualization of specific proteins and cellular structures, advanced genetic tricks of double/triple gene manipulations, live imaging of neuron activity, and drug treatment assays, Singhvi’s lab determined that UNC-23 works in concert with another protein, HSP-1/Hsp70, to regulate AFD-NREs. The study also determined the specifics of how intracellular junctions between epithelial cells are affected in unc-23 mutants, and how the epithelial cell transmits information about mechanical stress to glial cells, which in turn accordingly affect neuron shape.
Together, this group of experiments represents about six years of work for the Singhvi Lab — a fast pace for this type of research, which, Singhvi noted, would take much longer if the C. elegans model organisms had a more complicated nervous system or a longer lifespan.
