A missense point mutation in nerve growth factor (NGF^R100W) results in selective peripheral sensory neuropathy

Highlights

• A NGF^R100W knockin mouse model of Hereditary Sensory Autonomic Neuropathy V (HSAN V).

• Homozygous mutant mice showed severe,deficits in peripheral sensory structure and function.

• Heterozygous mutant mice developed progressive degeneration of small sensory fibers.

• Heterozygous mutant mice showed no apparent structural/functional changes in the brain.

• HSAN V is likely resulted from reduced secretion of NGF.

Abstract

The R100W mutation in nerve growth factor is associated with hereditary sensory autonomic neuropathy V in a Swedish family. These patients develop severe loss of perception to deep pain but with apparently normal cognitive functions. To better understand the disease mechanism, we examined a knockin mouse model of HSAN V. The homozygous mice showed significant structural deficits in intra-epidermal nerve fibers (IENFs) at birth. These mice had a total loss of pain perception at ∼2 months of age and often failed to survive to adulthood. Heterozygous mutant mice developed a progressive degeneration of small sensory fibers both behaviorally and functionally: they showed a progressive loss of IENFs starting at the age of 9 months accompanied with progressive loss of perception to painful stimuli such as noxious temperature. Quantitative analysis of lumbar 4/5 dorsal root ganglia revealed a significant reduction in small size neurons, while analysis of sciatic nerve fibers revealed the heterozygous mutant mice had no reduction in myelinated nerve fibers. Significantly, the amount of NGF secreted from mouse embryonic fibroblasts were reduced from both heterozygous and homozygous mice compared to their wild-type littermates. Interestingly, the heterozygous mice showed no apparent structural alteration in the brain: neither the anterior cingulate cortex nor the medial septum including NGF-dependent basal forebrain cholinergic neurons. Accordingly, these animals did not develop appreciable deficits in tests for brain function. Our study has thus demonstrated that the NGF^R100W mutation likely affects the structure and function of peripheral sensory neurons.

Introduction

Nerve growth factor (NGF) is a member of the neurotrophic factor family (NTF) that includes brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3) and neurotrophin 4 (NT-4) (Chao, 2003; Chao and Hempstead, 1995; Huang and Reichardt, 2001; Levi-Montalcini, 1987, 2004; Levi-Montalcini et al., 1995). These trophic factors act through two distinct receptors, Trk, the 140 kD tyrosine receptor kinase (TrkA for NGF; TrkB for BDNF, NT-3; TrkC for NT-4) and the 75 kD neurotrophin receptor (p75^NTR) to transmit signals in responsive neurons (Bothwell, 1995; Chao and Hempstead, 1995; Kaplan and Miller, 1997). The trophic function of NTFs is largely mediated by Trk, while p75^NTR has a more diverse effects on survival, differentiation and death of neurons (Casaccia-Bonnefil et al., 1998; Chao and Hempstead, 1995; Nykjaer et al., 2005).

NGF exerts potent trophic actions on sensory and sympathetic neurons of the peripheral nervous system (PNS)(Hamburger and Levi-Montalcini, 1949) and also regulates the trophic status of striatal and basal forebrain cholinergic neurons (BFCNs) of the central nervous system (CNS)(Conover and Yancopoulos, 1997; Kew et al., 1996; Lehmann et al., 1999; Levi-Montalcini and Hamburger, 1951; Li and Jope, 1995; Svendsen et al., 1994). Given its robust trophic effects, NGF has been investigated for therapeutic properties for treating both PNS and CNS diseases. For example, NGF was explored for treating/preventing degeneration of BFCNs in Alzheimer’s disease (AD) (Blesch and Tuszynski, 1995; Cuello et al., 2010; Eriksdotter Jonhagen et al., 1998; Hefti, 1994; Knusel and Gao, 1996; Koliatsos, 1996; Mufson et al., 2008; Olson, 1993; Rafii et al., 2014; Schindowski et al., 2008; Schulte-Herbruggen et al., 2008; Scott and Crutcher, 1994; Williams et al., 2006). However, some significant issues associated with administration of recombinant NGF such as back pain, injection site hyperalgesia, myalgia, weight loss led to the termination of these trials(Eriksdotter Jonhagen et al., 1998; Hefti, 1994; Knusel and Gao, 1996; Koliatsos, 1996; Olson, 1993; Scott and Crutcher, 1994). Other clinical efforts using NGF for treating diabetic neuropathies, HIV-induced peripheral neuropathies were also terminated due to the extreme side effects of pain(Apfel, 2002; Apfel et al., 1998; Hellweg and Hartung, 1990; Lein, 1995; McArthur et al., 2000; Pradat, 2003; Quasthoff and Hartung, 2001; Rask, 1999; Schifitto et al., 2001; Unger et al., 1998; Walwyn et al., 2006). These adverse effects such as severe pain associated with NGF administration has proven to be a significant roadblock for NGF-based therapies.

Indeed, NGF has also been recognized as a potent mediator of pain (Chuang et al., 2001; Lewin and Mendell, 1993; Lewin et al., 1993; Watanabe et al., 2008). This is further supported by genetic and clinical evidence demonstrating that both TrkA and p75^NTR-mediated signaling contributes to NGF-induced hyper-sensitization. For example, hereditary sensory and autonomic neuropathy type IV (HSAN IV) is resulted from recessive mutations in TrkA, these patients display pain insensitivity as well as mental retardation (Indo, 2001, 2002). Furthermore, many TrkA downstream effectors have also been implicated in NGF-mediated nociception: Pharmacological Inhibition of either the extracellular signal-regulated kinases (Erk) or phosphoinositide 3-kinase (PI3K) attenuates NGF-induced hyperalgesia (Zhang and Nicol, 2004). Activation of Phospholipase C (PLCγ) by NGF also potentiates nociceptive ion channels leading to hyperalgesia (Chuang et al., 2001). p75^NTR is also involved in NGF-induced hyperalgesia. For example, injecting a p75^NTR neutralizing antibody blocked NGF-induced hyperalgesia and NGF-mediated sensitization of action potentials in sensory neurons (Watanabe et al., 2008; Zhang and Nicol, 2004). Ceramide, a p75 downstream effector, is known to increase the number of action potentials in sensory neurons (Zhang et al., 2002, 2006). Nerve injury, axotomy, seizure, or ischemia can all cause an increase in both the expression and axonal transport of p75^NTR, thereby contributing to nociception (Zhou et al., 1996) (Roux et al., 1999). p75^NTR downstream signaling cascades were responsible for mechanical hyperalgesia following NGF injection (Khodorova et al., 2013). Therefore, both TrkA and p75^NTR receptor(s)-mediated signaling pathways play an important role in the pain signaling and function of NGF, although their relative contribution is yet to be defined.

The discovery of NGF mutations in human patients further highlights the importance of NGF in nociception. A homozygous mutation in the NGF gene [680C > A]+[681_682delGG] has been linked to hereditary sensory neuropathy in a consanguineous Arab family(Carvalho et al., 2011). Those affected individuals were completely unable to perceive pain, did not sweat, could not discriminate temperature (Carvalho et al., 2011). In addition, the patients had a chronic immunodeficiency and showed intellectual disabilities (Carvalho et al., 2011).

In a second case, a missense mutation in NGF (661C > T) was discovered in patients in consanguineous Swedish families who suffered from severe loss of deep pain, bone fractures and joint destruction(Einarsdottir et al., 2004). The disorder was classified as HSAN V (Online Mendelian Inheritance in Man (OMIM) # 608654). This particular mutation resulted in a substitution of tryptophan (W) for arginine (R) at Residue 211 in the proNGF polypeptide (pro-NGF^R221W that corresponds to Residue 100 in the mature protein: NGF^R100W) (Einarsdottir et al., 2004). The mutation is thus referred as NGF^R100W herein. Interestingly, unlike HSAN IV patients with recessive TrkA mutation(s) that develop pain insensitivity as well as mental retardation, HSAN V patients suffer from selective loss of pain sensation with normal cognitive function. This suggests that the NGF^R100W mutation causes selective loss of pain function, but likely retains intact trophic function (Einarsdottir et al., 2004; Minde et al., 2009, 2004; Minde, 2006; Perini et al., 2016; Sagafos et al., 2016). Therefore, NGF^R100W provides an important tool to uncouple the trophic function of NGF from its nociceptive actions (Capsoni, 2014; Capsoni et al., 2011; Cattaneo and Capsoni, 2019; Sung et al., 2018, 2019; Testa et al., 2019a; Yang et al., 2018).

Previous studies have revealed that the R100W mutation might disrupt the processing of proNGF to mature NGF (Larsson et al., 2009). We and others also examined the binding and signaling properties of the mature form of naturally occurring mutant NGF (NGF^R100W) and discovered that NGF^R100W retained its ability to bind to and signal through TrkA to induce trophic effects, but failed to bind or activate p75^NTR (Covaceuszach et al., 2010; Sung et al., 2018). Consistent with these studies, NGF^R100E or NGF^P61SR100E was also shown to be able to activate TrkA, but not p75^NTR signaling (Capsoni et al., 2011; Covaceuszach et al., 2010). Together, these studies have provided strong support for a role of p75^NTR in NGF-induced pain function. Failure to activate p75^NTR signaling is likely a major reason for loss of pain in HSAN V patients (Capsoni, 2014; Capsoni et al., 2011; Cattaneo and Capsoni, 2019; Sung et al., 2018, 2019; Testa et al., 2019a,b; Yang et al., 2018).

HSAN V is an extremely rare disease that makes it impossible to perform systematic study of signaling mechanism and function of NGF^R100W in HSAN V patients. Given that NGF is highly conserved between mice and human, mouse models of HSAN V harboring NGF^R100W have provided an important vehicle to carefully examine the effects of NGF^R100W on development/ maintenance of both the central nervous system (CNS) and the peripheral nervous system (PNS) in vivo (Testa et al., 2019a; Yang et al., 2018). Using the knock-in mouse model of HSAN V that harboring the mouse NGF^R100W allele, we previously reported that mice carrying homozygous NGF^R100W alleles (fln/fln) displayed normal embryonic development of major organs (heart, lung, liver, kidney, and spleen) showed normal gene expression of either TrkA or p75^NTR receptor (Yang et al., 2018). Interestingly, they exhibited extremely low counts of intra-epidermal nerve fibers (IENFs) at birth (Yang et al., 2018). In a recent study, Testa and colleagues have demonstrated that knocking in a human NGF allele carrying the R100W mutation resulted in a deficit in nociception but apparently with normal learning or memory (Testa et al., 2019b).

In the present study, we further characterized the impact of NGF^R100W on the structure and function of the brain and peripheral nervous system in the knock-in mouse model of HSAN V carrying mouse NGF^R100W mutation (Yang et al., 2018). We showed that unlike the homozygotes (fln/fln) that often failed to thrive to adulthood, development and survival of the heterozygotes (+/fln) showed no apparent difference from their wt (+/+) littermates. However, the +/fln mice developed a progressive degeneration of small sensory fibers, that were observed both behaviorally and functionally. Interestingly, the +/fln showed no obvious structural alterations in their brain and neuronal populations that included NGF-dependent basal forebrain cholinergic neurons (BFCNs). The +/fln mice did not develop appreciable deficits in learning and memory. Our results are in line with data from limited human studies of NGF^R100W carriers (Minde et al., 2009, 2004; Minde, 2006). Mechanistically, we showed the R100W mutation resulted in reduced secretion of mature NGF in primary mouse embryonic fibroblasts (MEFs) cultured from our mutant mice. These results are consistent with previous findings(Larsson et al., 2009; Testa et al., 2019b). We believe these in vivo findings will help to reignite efforts of using NGF as a therapeutic agent, that will lead to the development of ‘painless NGF’ therapies (Capsoni et al., 2011; Cattaneo and Capsoni, 2019; Sung et al., 2019; Testa et al., 2019b).