Understanding pain perception through genetic painlessness diseases: The role of NGF and proNGF.

Nerve growth factor (NGF), by binding to TrkA and p75(NTR) receptors, regulates the survival and differentiation of sensory neurons during development and mediates pain transmission and perception during adulthood, by acting at different levels of the nervous system. Key to understanding the role of NGF as a pain mediator is the finding that mutations (namely, R121W, V232fs and R221W) in the NGF gene cause painlessness disease Hereditary Sensory and Autonomic Neuropathy type V (HSAN V). Here we shall review the consequences of these NGF mutations, each of which results in specific clinical signs: R221W determines congenital pain insensitivity with no overt cognitive disabilities, whereas V232fs and R121W also result in intellectual disability, thus showing similarities to HSAN IV, which is caused by mutations in TrkA, rather than to HSAN V. Comparing the cellular, biochemical and clinical findings of these mutations could help in better understanding not only the possible mechanisms underlying HSAN V, but also mechanisms of NGF signalling and roles. These mutations alter the balance between NGF and proNGF in favour of an accumulation of the latter, suggesting a possible role of proNGF as a molecule with an analgesic role. Furthermore, the neurotrophic and pronociceptive functions of NGF are split by the R221W mutation, making NGF variants based on this mutation interesting for designing therapeutic applications for many diseases. This review emphasizes the possibility of using the mutations involved in "painlessness" clinical disorders as an innovative approach to identify new proteins and pathways involved in pain transmission and perception.Outstanding questions: Why do homozygous HSAN V die postnatally? What is the cause of this early postnatal lethality?Is the development of a mouse or a human feeling less pain affecting higher cognitive and perceptual functions?What is the consequence of the HSAN V mutation on the development of joints and bones? Are the multiple fractures observed in HSAN V patients due exclusively to the carelessness consequent to not feeling pain, or also to an intrinsic frailty of their bones?Are heterodimers of NGF(WT) and NGF(R221W) in the heterozygote state formed? And if so, what are the properties of these heterodimeric proteins?How is the processing of proNGF(R221W) to NGF(R221W) affected by the mutation

Spectroscopy of very hot plasma in non-flaring parts of a solar limb active region: spatial and temporal properties

In this work we investigate the thermal structure of an off-limb active region in various non-flaring areas, as it provides key information on the way these structures are heated. In particular, we concentrate in the very hot component (>3 MK) as it is a crucial element to discriminate between different heating mechanisms. We present an analysis using Fe and Ca emission lines from both SOHO/SUMER and HINODE/EIS. A dataset covering all ionization stages from Fe X to Fe XIX has been used for the thermal analysis (both DEM and EM). Ca XIV is used for the SUMER-EIS radiometric cross-calibration. We show how the very hot plasma is present and persistent almost everywhere in the core of the limb AR. The off-limb AR is clearly structured in Fe XVIII. Almost everywhere, the EM analysis reveals plasma at 10 MK (visible in Fe XIX emission) which is down to 0.1% of EM of the main 3 MK plasma. We estimate the power law index of the hot tail of the EM to be between -8.5 and -4.4. However, we leave an open question on the possible existence of a small minor peak at around 10 MK. The absence in some part of the AR of Fe XIX and Fe XXIII lines (which fall into our spectral range) enables us to determine an upper limit on the EM at such temperatures. Our results include a new Ca XIV 943.59 \AA~ atomic model

Reconnection nanojets in the solar corona

P.A. acknowledges STFC support from grant numbers ST/R004285/2 and ST/T000384/1 and support from the International Space Science Institute, Bern, Switzerland to the International Teams on ‘Implications for coronal heating and magnetic fields from coronal rain observations and modeling’ and ‘Observed Multi-Scale Variability of Coronal Loops as a Probe of Coronal Heating’. This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 647214). P.T. was also supported by contracts 8100002705 and SP02H1701R from Lockheed-Martin to the Smithsonian Astrophysical Observatory (SAO), and NASA contract NNM07AB07C to the SAO.The solar corona is shaped and mysteriously heated to millions of degrees by the Sun’s magnetic field. It has long been hypothesized that the heating results from a myriad of tiny magnetic energy outbursts called nanoflares, driven by the fundamental process of magnetic reconnection. Misaligned magnetic field lines can break and reconnect, producing nanoflares in avalanche-like processes. However, no direct and unique observations of such nanoflares exist to date, and the lack of a smoking gun has cast doubt on the possibility of solving the coronal heating problem. From coordinated multi-band high-resolution observations, we report on the discovery of very fast and bursty nanojets, the telltale signature of reconnection-based nanoflares resulting in coronal heating. Using state-of-the-art numerical simulations, we demonstrate that the nanojet is a consequence of the slingshot effect from the magnetically tensed, curved magnetic field lines reconnecting at small angles. Nanojets are therefore the key signature of reconnection-based coronal heating in action.PostprintPeer reviewe

First Report of Phytophthora Insolita and P. Inflata on Rhododendron in Ohio

During August 2003, we conducted a statewide survey of rhododendrons to determine if Phytophthora ramorum was present in Ohio ornamental nurseries. In total, 240 samples were randomly collected in 12 nurseries throughout Ohio from rhododendrons showing foliar necrotic lesions and twig dieback symptoms

Disentangling the signaling complexity of nerve growth factor receptors by CRISPR/Cas9

The binding of nerve growth factor (NGF) to the tropomyosin-related kinase A (TrkA) and p75(NTR) receptors activates a large variety of pathways regulating critical processes as diverse as proliferation, differentiation, membrane potential, synaptic plasticity, and pain. To ascertain the details of TrkA-p75(NTR) interaction and cooperation, a plethora of experiments, mostly based on receptor overexpression or downregulation, have been performed. Among the heterogeneous cellular systems used for studying NGF signaling, the PC12 pheochromocytoma-derived cell line is a widely used model. By means of CRISPR/Cas9 genome editing, we created PC12 cells lacking TrkA, p75(NTR), or both. We found that TrkA-null cells become unresponsive to NGF. Conversely, the absence of p75(NTR) enhances the phosphoiylation of TrkA and its effectors. Using a patch-clamp, we demonstrated that the individual activation of TrkA and p75(NTR) by NGF results in antagonizing effects on the membrane potential. These newly developed PC12 cell lines can be used to investigate the specific roles of TrkA and p75(NTR) in a genetically defined cellular model, thus providing a useful platform for future studies and further gene editing

A triheptanoin-supplemented diet rescues hippocampal hyperexcitability and seizure susceptibility in FoxG1+/- mice

The Forkhead Box G1 (FOXG1) gene encodes a transcription factor with an essential role in the mammalian telencephalon development. FOXG1-related disorders, caused by deletions, intragenic mutations or duplications, are usually associated with severe intellectual disability, autistic features, and, in 87% of subjects, epileptiform manifestations. In at least a subset of the patients with FoxG1 mutations, seizures remain intractable, prompting the need for novel therapeutic options. To address this issue, we took advantage of a haploinsufficient animal model, the FoxG1+/- mouse. In vivo electrophysiological analyses of FoxG1+/- mice detected hippocampal hyperexcitability, which turned into overt seizures upon delivery of the proconvulsant kainic acid, as confirmed by behavioral observations. These alterations were associated with decreased expression of the chloride transporter KCC2. Next, we tested whether a triheptanoin-based anaplerotic diet could have an impact on the pathological phenotype of FoxG1+/- mice. This manipulation abated altered neural activity and normalized the enhanced susceptibility to proconvulsant-induced seizures, in addition to rescuing the altered expression of KCC2 and increasing the levels of the GABA transporter vGAT. In conclusion, our data show that FoxG1 haploinsufficiency causes dysfunction of hippocampal circuits and increases the susceptibility to a proconvulsant insult, and that these alterations are rescued by triheptanoin dietary treatment

Reversal of neurological deficits by painless nerve growth factor in a mouse model of Rett syndrome

: Rett syndrome is a rare genetic neurodevelopmental disease, affecting 1 in over 10,000 females born worldwide, caused by de novo mutations in the X-chromosome-located methyl-CpG-binding protein 2 (MeCP2) gene. Despite the great effort put forth by the scientific community, a therapy for this devastating disease is still needed. Here, we tested the therapeutic effects of a painless mutein of the Nerve Growth Factor, called human NGF painless (hNGFp), via a non-invasive intranasal delivery in female MeCP2+/- mice. Of note, previous work had demonstrated a broad biodistribution of hNGFp in the mouse brain by the nasal delivery route. We report that (1) the long-term lifelong treatment of MeCP2+/- mice with hNGFp, starting at 2 months of age, increased the chance of survival while also greatly improving behavioral parameters. Furthermore, when we assessed the phenotypic changes brought forth by (2) a short-term 1-month-long hNGFp-treatment, starting at 3 months of age (right after the initial presentation of symptoms), we observed the rescue of a well-known neuronal target population of NGF, cholinergic neurons in the medial septum. Moreover, we reveal a deficit in microglial morphology in MeCP2+/- mice, completely reversed in treated animals. This effect on microglia is in line with reports showing microglia to be a TrkA-dependent non-neuronal target cell population of NGF in the brain. To understand the immunomodulatory activity of hNGFp, we analyzed the cytokine profile after hNGFp treatment in MeCP2+/- mice, to discover that the treatment recovered the altered expression of key neuroimmune-communication molecules, such as fractalkine. The overall conclusion is that hNGFp delivered intranasally can ameliorate symptoms in the MeCP2+/- model of Rett syndrome, by exerting strong neuroprotection with a dual mechanism of action: directly on target neurons and indirectly via microglia

Changes in pharyngeal aerobic microflora in oral breathers after palatal rapid expansion

BACKGROUND: The purpose of this study was to investigate in oral breathing children the qualitative and quantitative effects on aerobic and facultatively anaerobic oropharyngeal microflora of respiratory function improved by rapid palatal expansion (RPE). METHODS: In an open clinical trial, we studied 50 oral breathers, aged 8 to 14 years and suffering from both maxillary constriction and posterior cross-bite. At baseline, patients were examined by a single otorhinolaryngologist (ENT), confirming nasal obstruction in all subjects by posterior rhino-manometric test. Patients were evaluated three times by oropharyngeal swabs:1) at baseline (T = 0); 2) after palatal spreading out (T = 1); and 3) at the end of RPE treatment (T = 2). With regard to the microbiological aspect, the most common and potentially pathogenic oral microrganisms (i.e. Streptococcus pyogenes, Diplococcus pneumoniae, Staphylococcus aureus, Haemophilus spp, Branhamella catarrhalis, Klebsiella pneumoniae, Candida albicans) were specifically detected in proper culture plates, isolated colonies were identified by means of biochemical tests and counted by calibrated loop. The data were analyzed by means of the following tests: Chi-square test, Fisher's exact test and Wilcoxon's test. RESULTS: After the use of RME there was a statistically significant decrease of Staphylococcus aureus stock at CFU/mLat T1(P = 0.0005; Z = -3,455 by Wilcoxon Rank test) and T2 (P < 0.0001; Z = -4,512 by Wilcoxon Rank test) vs T0. No significant changes were found for the other examined microrganisms. CONCLUSION: Our data suggest that RPE therapy in oral breathers may strongly reduce the pathogenic aerobic and facultatively anaerobic microflora in the oral pharynx after a normalization of the upper airways function, and may reduce the risk of respiratory infections

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

A functional nerve growth factor (NGF)-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 clarify the involvement of the NGF-TrkA system in pain sensation