Neanderthal introgression in SCN9A impacts mechanical pain sensitivity.

The Nav1.7 voltage-gated sodium channel plays a key role in nociception. Three functional variants in the SCN9A gene (encoding M932L, V991L, and D1908G in Nav1.7), have recently been identified as stemming from Neanderthal introgression and to associate with pain symptomatology in UK BioBank data. In 1000 genomes data, these variants are absent in Europeans but common in Latin Americans. Analysing high-density genotype data from 7594 Latin Americans, we characterized Neanderthal introgression in SCN9A. We find that tracts of introgression occur on a Native American genomic background, have an average length of ~123 kb and overlap the M932L, V991L, and D1908G coding positions. Furthermore, we measured experimentally six pain thresholds in 1623 healthy Colombians. We found that Neanderthal ancestry in SCN9A is significantly associated with a lower mechanical pain threshold after sensitization with mustard oil and evidence of additivity of effects across Nav1.7 variants. Our findings support the reported association of Neanderthal Nav1.7 variants with clinical pain, define a specific sensory modality affected by archaic introgression in SCN9A and are consistent with independent effects of the Neanderthal variants on Nav1.7 function.

Investigating genotype-phenotype relationship of extreme neuropathic pain disorders in a UK national cohort.

The aims of our study were to use whole genome sequencing in a cross-sectional cohort of patients to identify new variants in genes implicated in neuropathic pain, to determine the prevalence of known pathogenic variants and to understand the relationship between pathogenic variants and clinical presentation. Patients with extreme neuropathic pain phenotypes (both sensory loss and gain) were recruited from secondary care clinics in the UK and underwent whole genome sequencing as part of the National Institute for Health and Care Research Bioresource Rare Diseases project. A multidisciplinary team assessed the pathogenicity of rare variants in genes previously known to cause neuropathic pain disorders and exploratory analysis of research candidate genes was completed. Association testing for genes carrying rare variants was completed using the gene-wise approach of the combined burden and variance-component test SKAT-O. Patch clamp analysis was performed on transfected HEK293T cells for research candidate variants of genes encoding ion channels. The results include the following: (i) Medically actionable variants were found in 12% of study participants (205 recruited), including known pathogenic variants: SCN9A(ENST00000409672.1): c.2544T>C, p.Ile848Thr that causes inherited erythromelalgia, and SPTLC1(ENST00000262554.2):c.340T>G, p.Cys133Tr variant that causes hereditary sensory neuropathy type-1. (ii) Clinically relevant variants were most common in voltage-gated sodium channels (Nav). (iii) SCN9A(ENST00000409672.1):c.554G>A, pArg185His variant was more common in non-freezing cold injury participants than controls and causes a gain of function of NaV1.7 after cooling (the environmental trigger for non-freezing cold injury). (iv) Rare variant association testing showed a significant difference in distribution for genes NGF, KIF1A, SCN8A, TRPM8, KIF1A, TRPA1 and the regulatory regions of genes SCN11A, FLVCR1, KIF1A and SCN9A between European participants with neuropathic pain and controls. (v) The TRPA1(ENST00000262209.4):c.515C>T, p.Ala172Val variant identified in participants with episodic somatic pain disorder demonstrated gain-of-channel function to agonist stimulation. Whole genome sequencing identified clinically relevant variants in over 10% of participants with extreme neuropathic pain phenotypes. The majority of these variants were found in ion channels. Combining genetic analysis with functional validation can lead to a better understanding as to how rare variants in ion channels lead to sensory neuron hyper-excitability, and how cold, as an environmental trigger, interacts with the gain-of-function NaV1.7 p.Arg185His variant. Our findings highlight the role of ion channel variants in the pathogenesis of extreme neuropathic pain disorders, likely mediated through changes in sensory neuron excitability and interaction with environmental triggers.

CLC Anion/Proton Exchangers Regulate Secretory Vesicle Filling and Granule Exocytosis in Chromaffin Cells.

ClC-3, ClC-4, and ClC-5 are electrogenic chloride/proton exchangers that can be found in endosomal compartments of mammalian cells. Although the association with genetic diseases and the severe phenotype of knock-out animals illustrate their physiological importance, the cellular functions of these proteins have remained insufficiently understood. We here study the role of two Clcn3 splice variants, ClC-3b and ClC-3c, in granular exocytosis and catecholamine accumulation of adrenal chromaffin cells using a combination of high-resolution capacitance measurements, amperometry, protein expression/gene knock out/down, rescue experiments, and confocal microscopy. We demonstrate that ClC-3c resides in immature as well as in mature secretory granules, where it regulates catecholamine accumulation and contributes to the establishment of the readily releasable pool of secretory vesicles. The lysosomal splice variant ClC-3b contributes to vesicle priming only with low efficiency and leaves the vesicular catecholamine content unaltered. The related Cl-/H+ antiporter ClC-5 undergoes age-dependent downregulation in wild-type conditions. Its upregulation in Clcn3-/- cells partially rescues the exocytotic mutant defect. Our study demonstrates how different CLC transporters with similar transport functions, but distinct localizations can contribute to vesicle functions in the regulated secretory pathway of granule secretion in chromaffin cells.SIGNIFICANCE STATEMENT Cl-/H+ exchangers are expressed along the endosomal/lysosomal system of mammalian cells; however, their exact subcellular functions have remained insufficiently understood. We used chromaffin cells, a system extensively used to understand presynaptic mechanisms of synaptic transmission, to define the role of CLC exchangers in neurosecretion. Disruption of ClC-3 impairs catecholamine accumulation and secretory vesicle priming. There are multiple ClC-3 splice variants, and only expression of one, ClC-3c, in double Cl-/H+ exchanger-deficient cells fully rescues the WT phenotype. Another splice variant, ClC-3b, is present in lysosomes and is not necessary for catecholamine secretion. The distinct functions of ClC-3c and ClC-3b illustrate the impact of expressing multiple CLC transporters with similar transport functions and separate localizations in different endosomal compartments.

Studying human nociceptors: from fundamentals to clinic.

Chronic pain affects one in five of the general population and is the third most important cause of disability-adjusted life-years globally. Unfortunately, treatment remains inadequate due to poor efficacy and tolerability. There has been a failure in translating promising preclinical drug targets into clinic use. This reflects challenges across the whole drug development pathway, from preclinical models to trial design. Nociceptors remain an attractive therapeutic target: their sensitization makes an important contribution to many chronic pain states, they are located outside the blood-brain barrier, and they are relatively specific. The past decade has seen significant advances in the techniques available to study human nociceptors, including: the use of corneal confocal microscopy and biopsy samples to observe nociceptor morphology, the culture of human nociceptors (either from surgical or post-mortem tissue or using human induced pluripotent stem cell derived nociceptors), the application of high throughput technologies such as transcriptomics, the in vitro and in vivo electrophysiological characterization through microneurography, and the correlation with pain percepts provided by quantitative sensory testing. Genome editing in human induced pluripotent stem cell-derived nociceptors enables the interrogation of the causal role of genes in the regulation of nociceptor function. Both human and rodent nociceptors are more heterogeneous at a molecular level than previously appreciated, and while we find that there are broad similarities between human and rodent nociceptors there are also important differences involving ion channel function, expression, and cellular excitability. These technological advances have emphasized the maladaptive plastic changes occurring in human nociceptors following injury that contribute to chronic pain. Studying human nociceptors has revealed new therapeutic targets for the suppression of chronic pain and enhanced repair. Cellular models of human nociceptors have enabled the screening of small molecule and gene therapy approaches on nociceptor function, and in some cases have enabled correlation with clinical outcomes. Undoubtedly, challenges remain. Many of these techniques are difficult to implement at scale, current induced pluripotent stem cell differentiation protocols do not generate the full diversity of nociceptor populations, and we still have a relatively poor understanding of inter-individual variation in nociceptors due to factors such as age, sex, or ethnicity. We hope our ability to directly investigate human nociceptors will not only aid our understanding of the fundamental neurobiology underlying acute and chronic pain but also help bridge the translational gap.

Spatial filter and its application in three-dimensional single molecule localization microscopy.

Single molecule localization microscopy (SMLM) allows the imaging of cellular structures with resolutions five to ten times below the diffraction limit of optical microscopy. It was originally introduced as a two-dimensional technique based on the localization of single emitters as projection onto the x-y imaging plane. The determination of the axial position of a fluorescent emitter is only possible by additional information. Here we report a method (spatial filter SMLM (SFSMLM)) that allows to determine the axial positions of fluorescent molecules and nanoparticles on the nanometer scale by the usage of two spatial filters, which are placed in two otherwise identical emission detection channels. SFSMLM allows axial localization in a range of ca. 1.5 µm with a localization precision of 15 - 30 nm in axial direction. The technique was utilized for localizing and imaging small cellular structures - e.g. actin filaments, vesicles and mitochondria - in three dimensions.