Human TrkAR649W mutation impairs nociception, sweating and cognitive abilities: a mouse model of HSAN IV.

A functional nerve growth factor NGF-Tropomyosin Receptor kinase A (TrkA) system is an essential requisite for the generation and maintenance of long-lasting thermal and mechanical hyperalgesia in adult mammals. Indeed, mutations in the gene encoding for TrkA are responsible for a rare condition, named Hereditary Sensory and Autonomic Neuropathy type IV (HSAN IV), characterized by the loss of response to noxious stimuli, anhidrosis and cognitive impairment. However, to date, there is no available mouse model to properly understand how the NGF-TrkA system can lead to pathological phenotypes that are distinctive of HSAN IV. Here, we report the generation of a knock-in mouse line carrying the HSAN IV TrkAR649W mutation. First, by in vitro biochemical and biophysical analyses, we show that the pathological R649W mutation leads to kinase-inactive TrkA also affecting its membrane dynamics and trafficking. In agreement with the HSAN IV human phenotype, TrkAR649W/m mice display a lower response to thermal and chemical noxious stimuli, correlating with reduced skin innervation, in addition to decreased sweating in comparison to TrkAh/m controls. Moreover, the R649W mutation decreases anxiety-like behavior and compromises cognitive abilities, by impairing spatial-working and social memory. Our results further uncover unexplored roles of TrkA in thermoregulation and sociability. In addition to accurately recapitulating the clinical manifestations of HSAN IV patients, our findings contribute to clarifying the involvement of the NGF-TrkA system in pain sensation.

Structural analysis of TrkA mutations in patients with congenital insensitivity to pain reveals PLCγ as an analgesic drug target.

Chronic pain is a major health issue, and the search for new analgesics has become increasingly important because of the addictive properties and unwanted side effects of opioids. To explore potentially new drug targets, we investigated mutations in the NTRK1 gene found in individuals with congenital insensitivity to pain with anhidrosis (CIPA). NTRK1 encodes tropomyosin receptor kinase A (TrkA), the receptor for nerve growth factor (NGF) and that contributes to nociception. Molecular modeling and biochemical analysis identified mutations that decreased the interaction between TrkA and one of its substrates and signaling effectors, phospholipase Cγ (PLCγ). We developed a cell-permeable phosphopeptide derived from TrkA (TAT-pQYP) that bound the Src homology domain 2 (SH2) of PLCγ. In HEK-293T cells, TAT-pQYP inhibited the binding of heterologously expressed TrkA to PLCγ and decreased NGF-induced, TrkA-mediated PLCγ activation and signaling. In mice, intraplantar administration of TAT-pQYP decreased mechanical sensitivity in an inflammatory pain model, suggesting that targeting this interaction may be analgesic. The findings demonstrate a strategy to identify new targets for pain relief by analyzing the signaling pathways that are perturbed in CIPA.

Clinical, genomics and networking analyses of a high-altitude native American Ecuadorian patient with congenital insensitivity to pain with anhidrosis: a case report.

BACKGROUND: Congenital insensitivity to pain with anhidrosis (CIPA) is an extremely rare autosomal recessive disorder characterized by insensitivity to pain, inability to sweat and intellectual disability. CIPA is caused by mutations in the neurotrophic tyrosine kinase receptor type 1 gene (NTRK1) that encodes the high-affinity receptor of nerve growth factor (NGF). CASE PRESENTATION: Here, we present clinical and molecular findings in a 9-year-old girl with CIPA. The high-altitude indigenous Ecuadorian patient presented several health problems such as anhidrosis, bone fractures, self-mutilation, osteochondroma, intellectual disability and Riga-Fede disease. After the mutational analysis of NTRK1, the patient showed a clearly autosomal recessive inheritance pattern with the pathogenic mutation rs763758904 (Arg602*) and the second missense mutation rs80356677 (Asp674Tyr). Additionally, the genomic analysis showed 69 pathogenic and/or likely pathogenic variants in 46 genes possibly related to phenotypic heterogeneity, including the rs324420 variant in the FAAH gene. The gene ontology enrichment analysis showed 28 mutated genes involved in several biological processes. As a novel contribution, the protein-protein interaction network analysis showed that NTRK1, SPTBN2 and GRM6 interact with several proteins of the pain matrix involved in the response to stimulus and nervous system development. CONCLUSIONS: This is the first study that associates clinical, genomics and networking analyses in a Native American patient with consanguinity background in order to better understand CIPA pathogenesis.

Histamine Modulates Sweating and Affects Clinical Manifestations of Atopic Dermatitis.

Many factors such as food or environmental allergens, bacteria, fungi, and mental stress aggravate the condition of atopic dermatitis (AD) eczema. Sweating can also exacerbate AD, and patients are aware of that. In the past, it has been reported that contamination of skin surface antigens by sweat induces acute allergic reactions and that sweating functions of AD patients via axonal reflexes are decreased. Histamine demonstrably inhibits acetylcholine-induced sweating in both mice and humans via histamine H1 receptor-mediated signaling. In sweat glands, acetylcholine inactivates glycogen synthase kinase 3ß (GSK3ß), a kinase involved in endocytosis and secretion, whereas simultaneous stimulation with histamine activates GSK3ß and inhibits sweat secretion. Thus, histamine might be involved in the mechanism of abnormal skin dryness in patients with AD via decreasing sweat secretion. On another front, some patients secrete sweat normally. Patients with regular sweating are prone to develop skin disorders such as papules or erythema by residual sweat left on the skin surface. Patients with decreased sweating are prone to develop disorders characterized by xerosis, lichenoid changes, prurigo by elevated skin temperature, skin dryness, and compromised skin conditions. Careful inspection of skin manifestations provides a good indication of a patient's ability to sweat.

New Etiology of Cholinergic Urticaria.

Cholinergic urticaria (CholU) is characterized by pinpoint-sized, highly pruritic wheals occurring upon sweating. Both direct and indirect theories in the interaction of acetylcholine (ACh) with mast cells have been put forward in the sweating-associated histamine release from mast cells. In the mechanism of indirect involvement of ACh, patients are hypersensitive to sweat antigen(s) and develop wheals in response to sweat substances leaking from the syringeal ducts to the dermis, possibly by obstruction of the ducts. Some patients with CholU exhibit a positive reaction to intradermal injection of their own diluted sweat, representing 'sweat allergy (hypersensitivity)'. Regarding the direct interaction theory between ACh and mast cells, we found that CholU with anhidrosis and hypohidrosis lacks cholinergic receptor M3 (CHRM3) expression in eccrine sweat gland epithelial cells. The expression of CHRM3 is completely absent in the anhidrotic areas and lowly expressed in the hypohidrotic areas. In the hypohidrotic area, where CholU occurs, it is hypothesized that ACh released from nerves cannot be completely trapped by cholinergic receptors of eccrine glands and overflows to the adjacent mast cells, leading to wheals.

Sweat, the driving force behind normal skin: an emerging perspective on functional biology and regulatory mechanisms.

The various symptoms associated with excessive or insufficient perspiration can significantly reduce a patient's quality of life. If a versatile and minimally invasive method could be established for returning sweat activity to normalcy, there is no question that it could be used in the treatment of many diseases that are believed to involve perspiration. For this reason, based on an understanding of the sweat-gland control function and sweat activity, it was necessary to conduct a comprehensive search for the factors that control sweating, such as the central and peripheral nerves that control sweat-gland function, the microenvironment surrounding the sweat glands, and lifestyle. We focused on the mechanism by which atopic dermatitis leads to hypohidrosis and confirmed that histamine inhibits acetylcholinergic sweating. Acetylcholine promotes the phosphorylation of glycogen synthesis kinase 3ß (GSK3ß) in the sweat-gland secretory cells and leads to sensible perspiration. By suppressing the phosphorylation of GSK3ß, histamine inhibits the movement of sweat from the sweat-gland secretory cells through the sweat ducts, which could presumably be demonstrated by dynamic observations of the sweat glands using two-photon microscopy. It is expected that the discovery of new factors that control sweat-gland function can contribute to the treatment of diseases associated with dyshidrosis.

Commentary on ''dynamic analysis of histamine-mediated attenuation of acetylcholine-induced sweating via GSK3ß activation''.

Matsui et al. (2014) delineate a novel pathomechanism for attenuated sweating in atopic dermatitis (AD). They provide evidence that acetylcholine-induced sweating can be inhibited via H1-receptor activation. This work suggests that H1 blockers may be useful in patients with AD.

Dynamic analysis of histamine-mediated attenuation of acetylcholine-induced sweating via GSK3ß activation.

Sweating has been associated with the exacerbation of atopic dermatitis (AD) in diverse ways. Acetylcholine (ACh)-mediated sweating is known to be attenuated in AD, but its cause remains obscure. To address this issue, the impact of histamine on ACh-induced sweating was evaluated. Sweating was measured by counting the number of active sweat pores by the starch-iodine reaction and dynamic optical coherence tomography; sweat was visualized using two-photon excitation fluorescence microscopy in mice and the quantitative sudomotor axon reflex test in humans. Both histamine receptor antagonists and H1 receptor (H1R)-knockout (KO) mice were used to determine methodological specificity. Histamine demonstrably inhibited ACh-induced sweating in both mice and humans via H1R-mediated signaling. In sweat glands, ACh inactivated glycogen synthase kinase 3ß (GSK3ß), a kinase involved in endocytosis and secretion, whereas simultaneous stimulation with histamine activated GSK3ß. Results of two-photon excitation fluorescence microscopy confirmed the dynamic motion of sweat and sweat glands after ACh treatment, showing that simultaneous stimulation with histamine altered their dynamic properties. These results indicate that histamine inhibits sweat gland secretions by blocking ACh-induced inactivation of GSK3ß. Histamine-mediated hypohidrosis might be involved in the mechanism of abnormal skin dryness in patients with AD.

Comparison of the spread of three botulinum toxin type A preparations.

Botulinum toxins are frequently used in esthetics to improve the appearance of facial wrinkles. In this setting, precise localization of the neurotoxin is required to produce the desired clinical effects. Unwanted effects can occur if the neurotoxin diffuses into untargeted muscle. Therefore, a neurotoxin with low and predictable spread would be preferable for esthetic applications. The aim of this study was to investigate the spread of three approved botulinum toxin type A preparations, with and without complexing proteins, by measuring and comparing the size of the anhidrotic halos they produced following injection of equivalent doses in an identical volume into the forehead of patients. The results showed that incobotulinumtoxinA and onabotulinumtoxinA displayed comparable spread at 6 weeks (maximal area of anhidrosis within 6 weeks) and area under the effect curve (AUEC) over 6 months. However, abobotulinumtoxinA, when assuming a 1:2.5 injection volume ratio, produced a statistically significantly greater maximal area of anhidrosis within 6 weeks and AUEC over 6 months compared with incobotulinumtoxinA. All preparations were well tolerated. The results of this study demonstrate that incobotulinumtoxinA and onabotulinumtoxinA have comparable spread, while abobotulinumtoxinA has significantly greater spread than incobotulinumtoxinA.

Immunolocalization of aquaporin-5 in normal human skin and hypohidrotic skin diseases.

Aquaporin (AQP)-5 has been shown to be expressed in the secretory parts of mouse, rat and horse sweat glands. However, the precise localization of AQP-5 in normal and diseased human skin has not been fully determined. The aim of the present study was to further clarify the immunolocalization of AQP-5 in normal human skin and hypohidrotic skin diseases. Normal human scalp skin and biopsies from skin affected by hypohidrotic diseases were analyzed for AQP-5 and/or dermcidin expression by immunohistochemistry, immunofluorescence and/or immunoelectronmicroscopy. AQP-5 was expressed on the apical and basolateral plasma membranes of the clear cells in eccrine sweat coils, but not in ductal components or apocrine glands. Numbers of AQP-5-positive coils in the secretory part of eccrine sweat glands were decreased in Sjögren's syndrome, but not in skin affected by idiopathic segmental anhidrosis or idiopathic pure sudomotor failure. AQP-5 was mostly localized to the plasma membranes of clear cells in the secretory coils of eccrine sweat glands, suggesting that it plays a role in producing the primary sweat fluid.

Nerve growth factor, pain, itch and inflammation: lessons from congenital insensitivity to pain with anhidrosis.

NGF is a well-known neurotrophic factor essential for the survival and maintenance of primary afferent neurons and sympathetic neurons. NGF is also an inflammatory mediator associated with pain and itch. Congenital insensitivity to pain with anhidrosis is a genetic disorder due to loss-of-function mutations in the NTRK1 gene encoding TrkA, a receptor tyrosine kinase for NGF. Since patients with congenital insensitivity to pain with anhidrosis lack NGF-dependent unmyelinated (C-) and thinly myelinated (Aδ-) fibers, and their dermal sweat glands are without innervation, they exhibit no pain, itch, signs of neurogenic inflammation or sympathetic skin responses. Based on the pathophysiology of congenital insensitivity to pain with anhidrosis, this article indicates how NGF-dependent neurons are essential for the establishment of neural networks for interoception and homeostasis, and play crucial roles in brain-immune-endocrine interactions in pain, itch and inflammation. In addition, it refers to involvements of the NGF-TrkA system in various disease states, and potential pharmacological effects when this system is targeted.

Immunolocalization of aquaporin-5 expression in sweat gland cells from normal and anhidrotic horses.

Western blot analysis showed that sweat gland cells from freely sweating horses expressed the water channel aquaporin-5 (AQP-5). Immunohistochemistry revealed a strong AQP-5-like activity reaction at the apical membrane of the glandular secretory cells, which was absent from the surrounding myoepithelium and all other skin structures. In anhidrotic horses, AQP-5 was also found at the apical membrane of the luminal sweat gland cells, but the level of expression reduced with the length of time that the animal had displayed anhidrosis. The level of AQP-5 expression was substantially reduced in animals with long-term anhidrosis, hence implicating water channel impairment as a possible factor in the development of this disorder.

Reduced epidermal growth factor receptor expression in hypohidrotic ectodermal dysplasia and Tabby mice.

Patients with hypohidrotic ectodermal dysplasia (HED) and Tabby (Ta) mice lack sweat glands and there is compelling evidence that these phenotypes are caused by mutations in the same highly conserved but unidentified X-linked gene. Previous studies showed that exogenous epidermal growth factor (EGF) reversed the Ta phenotype but the EGF status in HED patients has not been studied at all. Studies reported herein investigated the hypothesis that the EGF signaling pathway is involved in HED/Ta. Fibroblasts from HED patients had a two- to eightfold decrease in binding capacity for (125)I-labeled EGF, a decreased expression of the immunoreactive 170-kD EGF receptor (EGFR) protein, and a corresponding reduction in EGFR mRNA. Reduced expression of the EGFR also was observed in Ta fibroblasts and liver membranes. Other aspects of the EGF signaling pathway, including EGF concentration in urine and plasma, were normal in both HED patients and Ta mice. We propose that a decreased expression of the EGFR plays a causal role in the HED/Ta phenotype.

Acquired idiopathic generalized anhidrosis: clinical manifestations and histochemical studies.

A 40-year-old male developed complete absence of sweating except for slight sweating in the axillar region. Histopathologic examination of the skin revealed lymphocytes infiltration around the sweat glands and coarse and irregular arrangement of the eccrine glands. Immunohistochemical staining using anti-CD3, CD4, and CD8 antibodies revealed that CD3 positive cells were dominant in the lesion. After intensive glucocorticoid treatment, generalized sweating was almost completely recovered.