William D. Willis, Jr, MD, PhD Memorial Lecture: The evolutionary history of nerve growth factor and nociception

Introduction Rare pathological mutations in a small group of genes in humans can lead to pain insensitivity,4,19 and such cases have been instrumental in driving drug discovery programs for new analgesics.75 A less frequently trodden track is the examination of why some animals have evolved specific types of pain insensitivity. In this review, I will focus on insights that the biology of African mole-rats has given us in understanding the molecular basis of naturally occurring pain insensitivity.27,89,91,114 Many millions of years ago, evolution selected for changes in African mole-rat genes producing specific types of pain insensitivity.27 It is striking that evolution selected for changes in genes and signal transduction pathways that were subsequently identified as being critical for human nociception, which is a major theme of this short review. It has long been known that naturally occurring chemical substances, called algogens, evoke painful sensations or avoidance in animals and humans. One of the best characterized algogens is capsaicin, a natural substance and the active ingredient of chili peppers, and the substance that confers the burn of hot peppers.11,12 There is a huge literature on capsaicin, characterizing its neurotoxic effects in neonatal animals, as well as its use as a substance to produce acute experimental pain in humans.41,124 Capsaicin causes burning sensations in humans and is thought to induce a similar sensation in most vertebrates, one major exception being all birds, which have evolved insensitivity to capsaicin likely because they are seed carriers for capsicum plants.49 It is not clear when capsaicin insensitivity evolved in the bird lineage, but it is possible that the ancestors of birds, ie, nonavian dinosaurs, were already insensitive to capsaicin. In 2008, we discovered that capsaicin was not a universal irritant in mammals because our studies on the nociceptive system of the naked mole-rat (Heterocephalus glaber) revealed that this unusual subterranean mammal was completely insensitive to capsaicin.91 Further experiments also revealed that the naked mole-rat displays no nerve growth factor (NGF)-induced thermal hyperalgesia,66,91 a phenomenon that is dependent on the presence of the capsaicin-activated ion channel TRPV1.17 Naked mole-rats have fascinated biologists for several decades because this underground-dwelling species native to East Africa has a unique biology. Naked mole-rats are 1 of only 2 eusocial mammals that live in very large colonies (up to 300 individuals) with a single breeding female who is the queen.42,43 In the late 1970s, Dr Jenny Jarvis from the University of Cape Town was the first to show that these animals could be kept and bred in the laboratory, and today naked mole-rat colonies are maintained in many laboratories worldwide. Naked mole-rats have so many extreme physiological features that we would need an entire review to detail them all. Some of their most striking physiological adaptations include an extraordinarily long maximum life span of at least 36 years,100 apparent resistance to cancer, and resistance to extreme hypercapnia and hypoxia.92 In this review, I will examine the questions of how and why naked mole-rats, and more recently other African mole-rat species, lost certain types of pain sensitivity. 2. Nerve growth factor: recent history The naked mole-rat evolved changes in the NGF signaling that probably underlies pain insensitivity in this species.89,91 Nerve growth factor is the founding member of the neurotrophin family. In the past 20 years, the link between the biology of NGF and pain has been well established.35,60,73,95 There are at least 5 major pharmaceutical companies running clinical trials of humanized antibodies designed to sequester NGF for the treatment of pain in conditions as varied as osteoarthritis, lower back pain, and interstitial cystitis.8,14,28,52,57,106 The first example of an NGF-sequestering drug is tanezumab, a humanized monoclonal antibody that potently binds NGF developed by Rinat/Pfizer.57 At the time of writing, it was not clear whether such drugs will gain regulatory approval because a very small percentage of treated patients had experienced severe joint damage as a side effect of treatment.106 Because an NGF signaling axis is undoubtedly important in the etiology of pain, it is important to understand how NGF functions in the context of nociception and above all in the context of inflammatory hyperalgesia. Here, I will briefly review the mechanistic basis of how NGF functions in nociception and chronic pain. The availability of antibody tools to manipulate the endogenous levels of NGF allowed researchers to address the functional consequences of NGF sequestration. Initially, efforts focused on identifying precisely which neuronal populations depend on NGF for survival and when. It is through such experiments and later genetics that we got to know that NGF is required for the survival of sympathetic ganglion neurons and a large proportion of embryonic sensory neurons that are destined to become nociceptors.23,101,102 It seems that both sympathetic and sensory neurons largely lose their absolute dependency on NGF for survival in the postnatal period.101,102 In the 1980s and 1990s, developmental biologists asked which neurons required the neurotrophins for survival during development.59 For example, it was known that all sensory neurons that express TrkA (tropomyosin receptor kinase A, also known as NTRK1), the main receptor of NGF during embryonic development, require NGF to survive, but it was also clear that this population is not phenotypically homogenous.23,74 Nerve growth factor continues to be synthesized in the peripheral targets into adulthood and it has long been noted that the levels of NGF in the target correlate very well with the density of sympathetic and sensory innervation.54,59,109 Studies in the 1980s already showed that it is primarily neuropeptide-containing nociceptive sensory neurons in the adults that respond to NGF.59 Thus the neuropeptide content, primarily substance P (SP) and calcitonin gene-related peptide (CGRP), of sensory neurons innervating tissues high in NGF, such as the skin, was observed to be high compared to tissues low in NGF.78 Lindsay and Harmar had already demonstrated that NGF directly upregulates the SP content of adult sensory neurons.69 TrkA receptor expression is a feature of all developing nociceptors in the embryo, but its expression is extinguished in postnatal, small-diameter, nonpeptidergic nociceptors.83 The high-affinity TrkA receptor, which is the primary NGF-signaling receptor, is coexpressed in neuropeptide-positive nociceptors in adults. In mature animals, the peripheral tissue could be shown to influence the chemical composition of sensory afferents. Thus, in experiments where a cutaneous nerve was rerouted to the NGF-poor skeletal muscle and a muscle nerve was rerouted to NGF-rich skin, the SP content changed to match that characteristic of the new target, eg, muscle nerve innervating skin now had a high SP content.77,78 What was even more striking was the fact that the central connectivity of muscle afferents that had been redirected to skin now resembled that of normal skin afferents.62 These results led us to conduct the first serious test of the idea that a neurotrophic factor could regulate synaptic strength in the nervous system. We decided to artificially raise the levels of NGF in the skeletal muscle, in this case the gastrocnemius muscle, by chronically pumping NGF into the muscle. By making extracellular recordings from spinal dorsal horn neurons, we knew that only very few of these neurons receive strong synaptic drive from afferents innervating skeletal muscle. However, after exposure to NGF, skeletal muscle afferents showed a huge increase in their ability to excite dorsal horn neurons and this increase was very large when compared to effects of muscle afferents innervating the contralateral, untreated muscle.68 This was the very first demonstration that a neurotrophic factor can modulate synaptic strength. Shortly afterwards, an and more of this idea from the of which showed that both and neurotrophic factor can increase the strength of in with a time in the of these studies the of a huge and important of how neurotrophins regulate and synaptic strength in the nervous system. Nerve growth factor and the is as an increase in the of a after an or an inflammatory is the of an that is not to peripheral of the primary because the afferents that the be directly by the This of is thought to be to of central to from the the it was clear that the basis of was to a large dependent on a phenomenon central Thus, strong of nociceptors to a and at between primary sensory neurons and dorsal horn neurons, and this in synaptic strength can hyperalgesia. is after or but can also be after skin of that or A example of and is that after of capsaicin to the on the of NGF in the of nociceptors in and noted that that had been to of NGF were more to and untreated These led us to a more of the effects of NGF on nociceptive in the our a single of NGF and which for several and to be because first apparent after the and at The fact that a single NGF, can into a of functional changes with all the of after the of whether NGF was for inflammatory hyperalgesia. This was in of that NGF was in the nerve after of the It to use antibodies in to show whether an in NGF was a first in producing hyperalgesia. a new of inflammatory was where NGF a that the link between and the nociceptive sensory neurons that and and The features of this are shown in the of that have been the discovery that NGF is for inflammatory hyperalgesia. after we that NGF could induce we as well as other whether antibodies could or and The first 2 showed that both the and that a could be by the of of TrkA to the human of the that endogenous NGF were also shown to be of and with a in The that of NGF-signaling in inflammatory conditions has a major effect has now been in many It is important to that human NGF has also been to humans with effects to in of peripheral and central from and or from expression and the of ion in the of the peripheral nerve Nerve growth factor signaling the of substance and from the central of sensory neurons, which in neurons in the of synaptic in the spinal dorsal central neurotrophic calcitonin gene-related a mechanistic the in which NGF could produce and after One feature of this was the idea that the that the are from that the We that an important was that NGF is of changes in the peripheral of that them to to require the of changes in expression that to central that In the past 20 years, has been in the molecular that peripheral hyperalgesia. has also been in understanding and new that both central and peripheral be the molecular basis of which is to be in One that seems to be important for the of central is the increase in of from the central of nociceptors of from the central of nociceptors the receptor on spinal and this is thought to lead to through of the receptor For a more review on the of central The nociceptive system in naked mole-rats The first examination of sensory in the naked mole-rat demonstrated that the skin of this largely species is by both and In most can be into and nonpeptidergic their expression of neuropeptide such as and However, in the naked in the skin were observed to be of SP and The of neuropeptide-positive sensory neurons in the naked mole-rat the of whether this species had lost this sensory population in the of One to examine such a is to the of in peripheral which is with Many have the of and in cutaneous and observed that are to as many as A in these However, in naked mole-rats, this is and the low is to an absolute of nociceptors and not to of Thus, naked mole-rats have cutaneous nociceptors other which us to subterranean mole-rat species that are to the naked In a of from species to the the naked mole-rat was the only species with low cutaneous to of the and to that this was for by a of an of It is likely that mammals have evolved cutaneous as the skin is to with this that muscle and not high of to A as is in the skin and this is of all mammals all the naked and a of mammals a to in muscle A for the cutaneous in naked mole-rats is that they largely sensory are to most other humans are naked and human cutaneous also show a to In normal it has been observed that is naturally occurring of sensory neurons with in the postnatal This of is in a in the to from in to in adults. We recently observed a but more in naked mole-rats, such that postnatal naked mole-rat have a to of that to in The that are in to the biology of NGF and its receptor and in mole-rats that and SP show pain and the of in naked mole-rat nociceptors the of whether naked mole-rats pain insensitivity to The of nociceptors is completely in the and even the with 300 neurons, has a of the of nociceptors is demonstrated by humans with insensitivity to pain a that can to a of nociceptive and The of nociceptors observed in some with is to mutations in NGF or its receptor in which the the NGF receptor TrkA was also insensitivity to pain and is also with mutations in other for example, and this seems to be for normal developmental expression of the NGF receptor the fact that cutaneous in naked mole-rats have a of our physiological studies have shown that the receptor of these neurons not from in This was the case for nerve with nociceptive as well as for nociceptors as an skin nerve The major population is as both to and thermal and they the of The population are which are only by and which the functional population to have compared to these 2 populations were in naked mole-rats with receptor that were to of other Thus, the of in the naked mole-rat not to be to of one functional and the of in these seems to be to their The in between other and the naked mole-rat were more when we their to are that directly produce pain sensation in humans and animals and by directly specific ion or by potently sensory that pain. Capsaicin is a and when into a which is to its specific of the receptor 1 channel a and ion channel by Capsaicin naked mole-rat as by recordings with the and recordings from dorsal However, the fact that naked mole-rat nociceptors are by capsaicin, it to evoke in naked In of capsaicin causes of the to this phenomenon also not in naked is it that capsaicin of nociceptors not lead to pain The of nociceptive to the spinal in the dorsal and because neurons are that are at the peripheral are also at the in the dorsal one can recordings from spinal neurons and capsaicin to of which an in the neurons with which nociceptors are In the of neurons that are by nociceptors are in the dorsal but in the naked mole-rat as many dorsal horn neurons are to nociceptors as are neurons in This connectivity of nociceptors why capsaicin a in but in the naked Thus, of nociceptors in the naked mole-rat both and dorsal horn We that between in the and dorsal horn could such a pain when SP was into the naked for example through capsaicin into the was to evoke a and thermal The receptor for SP is only in the dorsal horn of the naked mole-rat spinal and of nociceptors by capsaicin in the presence of spinal SP to a of that of a of SP was also shown to in naked and evoke a thermal that is both and pain in mole-rats In to changes in acute nociception, a of inflammatory have been to examine the of in naked is a substance that acute and late of which occurring in the naked mole-rat are compared to capsaicin to evoke thermal in the naked mole-rat and a similar of thermal was observed after of and NGF, was by Nerve growth thermal is a but naked mole-rat sensory neurons respond to capsaicin, which that of in naked mole-rat is to the of thermal hyperalgesia. of naked mole-rat demonstrated that it has normal and capsaicin Naked mole-rat sensory neurons also express the NGF receptor but even in neurons, NGF to induce of However, NGF naked mole-rat when in sensory neurons from that and when coexpressed with TrkA in naked mole-rat results in naked mole-rat and the naked mole-rat experiments naked mole-rat TrkA and a TrkA naked showed that of the naked mole-rat TrkA receptor is less at producing that the in the demonstrated that NGF of TrkA is less in signaling as shown by of compared to Thus, the of thermal in naked mole-rats is primarily to TrkA of the naked mole-rat TrkA that between 1 and that are unique to the naked mole-rat in the kinase of TrkA are likely for the signaling the of changes in the TrkA kinase the observed in the naked one would a naked mole-rat with the TrkA but this is not However, we have with the critical from the naked mole-rat into the results with these that they show inflammatory pain Thus, the naked mole-rat more years ago, the mole-rat had already discovered the of NGF The molecular tools to such as are in the of and However, the can also be to such as the naked We recently and a very large that us to directly the expression levels of from the and spinal not only between the naked mole-rat and but also other African mole-rat We have all these so that species as well as expression levels of can be and Here, I these to examine the idea that the mutations in which can in show expression or unique in the naked mole-rat It is clear that 2 and show expression in the naked mole-rat compared to the in the also expression The has been shown to be a developmental of and is important for the of is a which has a at the the of which has been directly in as well as from our we the naked mole-rat and 2 African species to for that be unique that the naked mole-rat has a compared to other mammals and 2 unique in the of the There is a that this be in the of the unusual nociceptive system in the naked The is in adult sensory neurons but it is this has a in in the of levels for and in the of and naked from P P P A of the of human and from with mole-rat East African and mole-rat from and from a The naked mole-rat was also from that the and be as an of the where human mutations were 2 where a unique seems to be in the naked mole-rat the that is as long in the mole-rat as it is in the human of pain in naked mole-rats In to naked mole-rats also show no pain in to of similar to pain in to have been observed the which the of observed in naked mole-rats unique capsaicin naked mole-rat no of naked mole-rat was in all are also capsaicin or more can sensory neurons by the or of several of ion of ion and and of certain studies have shown that naked mole-rat neurons to both and have been and expression of seems to be between and naked mole-rat sensory the of naked mole-rat and have a similar to their the is insensitive and seems to the when in which sensory to some It was that naked mole-rat sensory neurons can be by but we not in nociceptors pain The first to this with the that can potently channel are for and as demonstrated by the fact that pathological mutations in some genes that of mutations in the to changes in which be the primary for The is important for and and the naked mole-rat that when into the human of at certain that excite We also showed that the low is a more of in naked mole-rat nociceptors in the Thus, our that at least one major by which naked mole-rats have evolved insensitivity is through specific changes in that of the a The sensory of the naked mole-rat recently led us to whether pain insensitivity is a occurring African underground-dwelling The naked mole-rat to the a very group of mole-rats that have most of We in mole-rat species and one species from Africa We capsaicin, and the is the active ingredient of and and is a that is a specific of ion not mammals and but also and this We also showed that naked mole-rats after of Thus, naked mole-rats can the as all other so We discovered that one mole-rat species was completely insensitive to capsaicin mole-rat and 2 species were insensitive to mole-rat and East African The that the of and pain in African are most we also identified the first case of insensitivity to the chemical in the The mole-rat is a African mole-rat that to the and other of the and all these animals were to in the channel that the channel less to the However, the mole-rat was even to and displays no or in the first of the The mole-rat was also unique so that it no in the It is to in this context that a in the human is with pain in a large We from sensory tissues from the species and changes in expression the This revealed a single for in the mole-rat that a This channel has been in a in We that in sensory neurons, the is to of nociceptors that for this idea was by mole-rats with an of the channel with the that this would sensitivity. We which has been shown to and after the mole-rat to both and the effects of that the mole-rat has evolved an unusual and molecular to become to pain and in such a could be to new We also that the mole-rat lost to because it has to its with the the of which is a in African of the species, as on the basis of were on the basis of and an insensitivity to the years to of a large of African mole-rat species for we discovered 2 new of in the Cape mole-rat and the East African The Cape mole-rat and East African are subterranean but are both animals, they not be to the of probably experienced by the naked both the East African and the Cape mole-rat have in the of the channel that a of in and single into or both in all subterranean African It be noted that has been shown to increase the of the of the channel the revealed of evolution in the of the channel so for example, many species the naked but species the or human These that the of the channel is with of that could produce tissue is with In our recent we that the Cape mole-rat was the only species that has the in of the channel as the naked mole-rat and not the more This is only the of such a which is so the only that has been shown to increase the of the also revealed that a of the channel in has that are unique to the African mole-rats of the The functional of such to be At least in the case of the naked it seems to that exposure to high levels of as of in colonies could be one factor driving insensitivity. Naked mole-rats show an resistance to which not produce in this We have also shown that naked mole-rats to that when to extreme hypoxia.92 It seems that insensitivity evolved as a to pain in the naked mole-rats to have and a It was recently that levels are in naked mole-rat but that the is less that with changes in However, the levels of in where the of the to be We that naked mole-rats in of animals to and it is very likely that animals at the of such will be to very low and high Because the species observed to types of it was all the more that we could also genes that were in the in the of all We could show that all species showed levels of ion would be by of such genes and However, we also discovered new genes in that also all species with pain It is to that of physiological in the of being to capsaicin, or a to the species in In some we can the that the of the species, eg, insensitivity in the naked mole-rat this species with tissue with hypercapnia with it is also possible that some have evolved for that are not primarily with pain For example, the specific of nociceptors that was observed only in the most mole-rat species, the naked be an to a need for an to it sensory The naked mole-rat a very which at a Thus, it has been that other in this species, such as of and of are to where it is not of but not all that also be a that the of the In studies on African mole-rats have so revealed molecular to pain. the molecular changes by mole-rats to pain have to be with the causes of human the main genes with in 2 and are directly with pain insensitivity in naked a to how evolution can produce that will in the us how to best for a of human as well as pain. of The has no of to