Posted on by Dr. Francis Collins
The spinal cord, as a key part of our body’s central nervous system, contains millions of neurons that actively convey sensory and motor (movement) information to and from the brain. Scientists have long sorted these spinal neurons into what they call “cardinal” classes, a classification system based primarily on the developmental origin of each nerve cell. Now, by taking advantage of the power of single-cell genetic analysis, they’re finding that spinal neurons are more diverse than once thought.
This image helps to visualize the story. Each dot represents the nucleus of a spinal neuron in a mouse; humans have a very similar arrangement. Most of these neurons are involved in the regulation of motor control, but they also differ in important ways. Some are involved in local connections (green), such as those that signal outward to a limb and prompt us to pull away reflexively when we touch painful stimuli, such as a hot frying pan. Others are involved in long-range connections (magenta), relaying commands across spinal segments and even upward to the brain. These enable us, for example, to swing our arms while running to help maintain balance.
It turns out that these two types of spinal neurons also have distinctive genetic signatures. That’s why researchers could label them here in different colors and tell them apart. Being able to distinguish more precisely among spinal neurons will prove useful in identifying precisely which ones are affected by a spinal cord injury or neurodegenerative disease, key information in learning to engineer new tissue to heal the damage.
This image comes from a study, published recently in the journal Science, conducted by an NIH-supported team led by Samuel Pfaff, Salk Institute for Biological Studies, La Jolla, CA. Pfaff and his colleagues, including Peter Osseward and Marito Hayashi, realized that the various classes and subtypes of neurons in our spines arose over the course of evolutionary time. They reasoned that the most-primitive original neurons would have gradually evolved subtypes with more specialized and diverse capabilities. They thought they could infer this evolutionary history by looking for conserved and then distinct, specialized gene-expression signatures in the different neural subtypes.
The researchers turned to single-cell RNA sequencing technologies to look for important similarities and differences in the genes expressed in nearly 7,000 mouse spinal neurons. They then used this vast collection of genomic data to group the neurons into closely related clusters, in much the same way that scientists might group related organisms into an evolutionary family tree based on careful study of their DNA.
The first major gene expression pattern they saw divided the spinal neurons into two types: sensory-related and motor-related. This suggested to them that one of the first steps in spinal cord evolution may have been a division of labor of spinal neurons into those two fundamentally important roles.
Further analyses divided the sensory-related neurons into excitatory neurons, which make neurons more likely to fire; and inhibitory neurons, which dampen neural firing. Then, the researchers zoomed in on motor-related neurons and found something unexpected. They discovered the cells fell into two distinct molecular groups based on whether they had long-range or short-range connections in the body. Researches were even more surprised when further study showed that those distinct connectivity signatures were shared across cardinal classes.
All of this means that, while previously scientists had to use many different genetic tags to narrow in on a particular type of neuron, they can now do it with just two: a previously known tag for cardinal class and the newly discovered genetic tag for long-range vs. short-range connections.
Not only is this newfound ability a great boon to basic neuroscientists, it also could prove useful for translational and clinical researchers trying to determine which specific neurons are affected by a spinal injury or disease. Eventually, it may even point the way to strategies for regrowing just the right set of neurons to repair serious neurologic problems. It’s a vivid reminder that fundamental discoveries, such as this one, often can lead to unexpected and important breakthroughs with potential to make a real difference in people’s lives.
 Conserved genetic signatures parcellate cardinal spinal neuron classes into local and projection subsets. Osseward PJ 2nd, Amin ND, Moore JD, Temple BA, Barriga BK, Bachmann LC, Beltran F Jr, Gullo M, Clark RC, Driscoll SP, Pfaff SL, Hayashi M. Science. 2021 Apr 23;372(6540):385-393.
What Are the Parts of the Nervous System? (Eunice Kennedy Shriver National Institute of Child Health and Human Development/NIH)
Spinal Cord Injury (National Institute of Neurological Disorders and Stroke/NIH)
Samuel Pfaff (Salk Institute, La Jolla, CA)
NIH Support: National Institute of Mental Health; National Institute of Neurological Disorders and Stroke; Eunice Kennedy Shriver National Institute of Child Health and Human Development
Posted on by Dr. Francis Collins
Gene editing has shown great promise as a non-heritable way to treat a wide range of conditions, including many genetic diseases and more recently, even COVID-19. But could a version of the CRISPR gene-editing tool also help deliver long-lasting pain relief without the risk of addiction associated with prescription opioid drugs?
In work recently published in the journal Science Translational Medicine, researchers demonstrated in mice that a modified version of the CRISPR system can be used to “turn off” a gene in critical neurons to block the transmission of pain signals . While much more study is needed and the approach is still far from being tested in people, the findings suggest that this new CRISPR-based strategy could form the basis for a whole new way to manage chronic pain.
This novel approach to treating chronic pain occurred to Ana Moreno, the study’s first author, when she was a Ph.D. student in the NIH-supported lab of Prashant Mali, University of California, San Diego. Mali had been studying a wide range of novel gene- and cell-based therapeutics. While reading up on both, Moreno landed on a paper about a mutation in a gene that encodes a pain-enhancing protein in spinal neurons called NaV1.7.
Moreno read that kids born with a loss-of-function mutation in this gene have a rare condition known as congenital insensitivity to pain (CIP). They literally don’t sense and respond to pain. Although these children often fail to recognize serious injuries because of the absence of pain to alert them, they have no other noticeable physical effects of the condition.
For Moreno, something clicked. What if it were possible to engineer a new kind of treatment—one designed to turn this gene down or fully off and stop people from feeling chronic pain?
Moreno also had an idea about how to do it. She’d been working on repressing or “turning off” genes using a version of CRISPR known as “dead” Cas9 . In CRISPR systems designed to edit DNA, the Cas9 enzyme is often likened to a pair of scissors. Its job is to cut DNA in just the right spot with the help of an RNA guide. However, CRISPR-dead Cas9 no longer has any ability to cut DNA. It simply sticks to its gene target and blocks its expression. Another advantage is that the system won’t lead to any permanent DNA changes, since any treatment based on CRISPR-dead Cas9 might be safely reversed.
After establishing that the technique worked in cells, Moreno and colleagues moved to studies of laboratory mice. They injected viral vectors carrying the CRISPR treatment into mice with different types of chronic pain, including inflammatory and chemotherapy-induced pain.
Moreno and colleagues determined that all the mice showed evidence of durable pain relief. Remarkably, the treatment also lasted for three months or more and, importantly, without any signs of side effects. The researchers are also exploring another approach to do the same thing using a different set of editing tools called zinc finger nucleases (ZFNs).
The researchers say that one of these approaches might one day work for people with a large number of chronic pain conditions that involve transmission of the pain signal through NaV1.7. That includes diabetic polyneuropathy, sciatica, and osteoarthritis. It also could provide relief for patients undergoing chemotherapy, along with those suffering from many other conditions. Moreno and Mali have co-founded the spinoff company Navega Therapeutics, San Diego, CA, to work on the preclinical steps necessary to help move their approach closer to the clinic.
Chronic pain is a devastating public health problem. While opioids are effective for acute pain, they can do more harm than good for many chronic pain conditions, and they are responsible for a nationwide crisis of addiction and drug overdose deaths . We cannot solve any of these problems without finding new ways to treat chronic pain. As we look to the future, it’s hopeful that innovative new therapeutics such as this gene-editing system could one day help to bring much needed relief.
 Long-lasting analgesia via targeted in situ repression of NaV1.7 in mice. Moreno AM, Alemán F, Catroli GF, Hunt M, Hu M, Dailamy A, Pla A, Woller SA, Palmer N, Parekh U, McDonald D, Roberts AJ, Goodwill V, Dryden I, Hevner RF, Delay L, Gonçalves Dos Santos G, Yaksh TL, Mali P. Sci Transl Med. 2021 Mar 10;13(584):eaay9056.
 Nuclease dead Cas9 is a programmable roadblock for DNA replication. Whinn KS, Kaur G, Lewis JS, Schauer GD, Mueller SH, Jergic S, Maynard H, Gan ZY, Naganbabu M, Bruchez MP, O’Donnell ME, Dixon NE, van Oijen AM, Ghodke H. Sci Rep. 2019 Sep 16;9(1):13292.
 Drug Overdose Deaths. Centers for Disease Control and Prevention.
Congenital insensitivity to pain (National Center for Advancing Translational Sciences/NIH)
Opioids (National Institute on Drug Abuse/NIH)
Mali Lab (University of California, San Diego)
Navega Therapeutics (San Diego, CA)
NIH Support: National Human Genome Research Institute; National Cancer Institute; National Institute of General Medical Sciences; National Institute of Neurological Disorders and Stroke