Georgios Baskozos

Excessive primary afferent activity sustains neuropathic pain, but safe and effective therapeutic inhibition has proved elusive. Realising the promise of gene therapies to meet this challenge requires strategies to identify and selectively target the neuronal populations responsible. Here, we use chemo- and optogenetic approaches to demonstrate that activity in injured afferents drives spontaneous pain but is dispensable for normal sensory function, thereby creating a therapeutic opportunity. Guided by chromatin accessibility profiling, we create an injury-responsive viral vector that confers >95% specificity for injured sensory neurons. Combined with the translationally relevant chemogenetic PSAM ⁴ -GlyR, this targeting module effectively silences hyperexcitable human sensory neurons and alleviates spontaneous neuropathic pain in mice. These findings establish precision cell - state targeting of sensory neurons as a promising therapeutic strategy for chronic pain.

Introduction: Whiplash-associated disorder grade II (WADII) is characterised by persistent pain in the absence of frank nerve injury, yet its molecular mechanisms remain unclear. Objectives: To identify molecular signatures of 2 recovery groups characterised by persistent moderate/severe and minimal symptoms in WADII using RNA-sequencing of blood samples 6 months postinjury. Methods: We performed bulk transcriptional analyses on blood samples collected from the primary cohort of individuals with WADII 6 months postinjury (n = 15/recovery group). Findings were replicated through meta-analyses using an independent cohort of chronic WAD. Blood cellular composition was estimated via deconvolution analyses across both cohorts. Results: Although there were no differentially expressed genes between recovery groups, we identified sex-specific gene expression signatures of recovery. In the primary WADII cohort, HLA-DQA1, HEBP1, and NECTIN2 showed lower expression in females with moderate/severe symptoms compared with minimal symptoms. SLC12A1 and MXRA7 showed lower expression in males with moderate/severe pain, whereas HLA-G was upregulated. Gene expression meta-analysis in females identified 6 differentially expressed genes, including replication of lower HLA-DQA1 expression from the primary cohort. There were no differentially expressed genes in the meta-analyses in males or between recovery groups. Deconvolution analyses revealed diverging trends between sexes for blood cell types and whiplash symptom severity scores. Ligand–receptor pair analyses further suggested distinct sex-specific recovery trajectories. Conclusion: Transcriptional profiling revealed sex-specific molecular blood signatures associated with persistent whiplash symptoms 6 months postinjury. Future research is needed to further characterise sex-specific mechanisms after whiplash injury to improve prognosis and targeted management strategies.

Human sensory neuron models are an important resource for studying pain mechanisms and axon injury and repair. Current systems are limited by accessibility, scalability, or incomplete functional maturation. The HD10.6 human dorsal root ganglion-derived immortalised cell line represents a promising alternative; however, its maturation trajectory and suitability for disease modelling remain incompletely defined. Here, we performed a longitudinal, multi-modal characterisation of HD10.6 cells during differentiation over 28 days. Bulk RNA sequencing revealed progressive transcriptional remodelling, with temporal up-regulation of neuronal and nociceptor-associated gene programmes, including ion channels implicated in pain signalling. Protein-level analyses confirmed increased expression of key nociceptor markers and neuropeptides, including TRPV1, Nav1.7, Nav1.8 and CGRP. Functional assays demonstrated the emergence of sensory neuron-like properties over time. Calcium imaging revealed increasing responsiveness to capsaicin, allyl isothiocyanate, α,β-MeATP, and prostaglandin E2, while patch-clamp electrophysiology at DIV 21 after maturation showed repetitive firing of action potential and, most importantly, exhibited TTX-Resistant sodium currents. These findings establish a temporal relationship between transcriptional changes and functional competence. Finally, we evaluated the utility of HD10.6 neurons for modelling axon degeneration. Treatment with vacor-induced robust neurite degeneration, which was attenuated by pharmacological inhibition of SARM1, demonstrating engagement of conserved axon degeneration pathways. Together, our findings define the progressive maturation of HD10.6 sensory neurons and establish this system as a scalable human platform for studying nociceptor biology and SARM1-dependent axon degeneration.

Nociceptors are primary afferent neurons that sense noxious stimuli. They can be activated by tissue injury as well as the accompanying local immune response. We have shown that following nerve injury in mice, cytotoxic Natural Killer (NK) cells infiltrate the peripheral nerve where they interact with stress-induced ligands of the activating receptor NKG2D ( Klrk1 ). However, the diversity and specificity of NKG2D receptor ligands among sensory neuron subtypes, and translation of this mechanism to human cells, remains unknown. We used dorsal root ganglion (DRG) neurons cultured from C57BL/6J mice of both sexes with fluorescently labelled sensory neuron lines ( Scn10a , Mrgprd , Calca , Trpv1 , Th , Thy1 ), as well as human induced pluripotent stem cell-derived (hiPSCd)-sensory neurons after laser ablation, as in vitro models of axonal injury. We assessed the expression of NKG2D ligands by quantitative polymerase chain reaction (PCR) corroborated by publicly available RNA sequencing datasets and validated with single-cell PCR. Recombinant NKG2D receptor proteins were used in live cell-based assays to reveal the subcellular membrane localisation of NKG2D ligands with quantification by semi-automated image analysis. Functional interactions between human NK cells and sensory neurons were confirmed with co-cultures in microfluidic devices. We show that NKG2D ligands are expressed exclusively in unmyelinated DRG neurons after injury. Soluble mouse NKG2D receptors bound to puncta along distal neurites of injured axons enriched predominantly in Mrgprd -expressing non-peptidergic nociceptors. We observed low-level binding of soluble human NKG2D receptors to neurites of hiPSCd sensory neurons that increased after axonal laser ablation. Degeneration of hiPSCd sensory neurites by interleukin (IL-2) primed human NK cells was prevented by an NKG2D blocking antibody. The induction and enrichment of functional NKG2D receptor ligands selectively on pathological nerve fibres could aid the diagnosis of peripheral neuropathy in chronic pain conditions, and sheds new light on the potential role of nociceptive neurons in regulating the local tissue immune microenvironment.

Polyamines are regulatory metabolites with key roles in transcription, translation, cell signalling and autophagy¹. They are implicated in multiple neurological disorders, including stroke, epilepsy and neurodegeneration, and can regulate neuronal excitability through interactions with ion channels². Polyamines have been linked to pain, showing altered levels in human persistent pain states and modulation of pain behaviour in animal models³. However, the systems governing polyamine transport within the nervous system remain unclear. Here, undertaking a genome-wide association study (GWAS) of chronic pain intensity in the UK Biobank (UKB), we found a significant association between pain intensity and variants mapping to the SLC45A4 gene locus. In the mouse nervous system, Slc45a4 expression is enriched in all sensory neuron subtypes within the dorsal root ganglion, including nociceptors. Cell-based assays show that SLC45A4 is a selective plasma membrane polyamine transporter, and the cryo-electron microscopy (cryo-EM) structure reveals a regulatory domain and basis for polyamine recognition. Mice lacking SLC45A4 show normal mechanosensitivity but reduced sensitivity to noxious heat- and algogen-induced tonic pain that is associated with reduced excitability of C-polymodal nociceptors. Our findings therefore establish a role for neuronal polyamine transport in pain perception and identify a target for therapeutic intervention in pain treatment.

Nociceptors are primary afferent neurons that sense noxious stimuli. They can be activated by tissue injury as well as the accompanying local immune response. We have shown that following nerve injury in mice cytotoxic Natural Killer (NK) cells infiltrate the peripheral nerve and interact with stress-induced ligands of the activating receptor NKG2D ( Klrk1 ). However, the diversity and specificity of NKG2D receptor ligands among sensory neuron subtypes, and translation of this mechanism to humans, remains unknown. We used dorsal root ganglion (DRG) neurons cultured from C57BL/6J mice of both sexes with fluorescently-labelled sensory neuron lineages ( Scn10a , Mrgprd, Calca, Trpv1 , Th , Thy1 ), as well as human induced pluripotent stem cell derived (hiPSCd)-sensory neurons after laser ablation, as in vitro models of axonal injury. We assessed expression of NKG2D ligands by quantitative polymerase chain reaction (PCR) corroborated by publicly available RNA sequencing datasets and validated with single-cell PCR. Recombinant NKG2D receptor proteins in live cell-based assays were used to reveal the subcellular membrane localisation of NKG2D ligands with quantification by a semi-automated image analysis. Functional interactions between human NK cells and sensory neurons were confirmed with co-cultures in microfluidic devices. We show that NKG2D ligands are expressed exclusively in unmyelinated DRG neurons after injury. NKG2D-receptors bound to puncta along distal neurites of injured axons enriched predominantly in Mrgprd -expressing non-peptidergic nociceptors. We observed low-level binding of human NKG2D-receptors to neurites of hiPSCd sensory neurons that increased after axonal laser ablation. Degeneration of hiPSCd sensory neurons neurites by interleukin (IL-2) primed human NK cells was prevented by an NKG2D blocking antibody. The induction and enrichment of functional NKG2D receptor ligands selectively on pathological nerve fibres could aid the diagnosis of peripheral neuropathy in chronic pain conditions, and sheds new light on the potential role of nociceptive neurons in regulating the local tissue immune microenvironment.

Assessing disease progression and informing clinical trials in peripheral neuropathy would benefit from objective and responsive fluid biomarkers closely linked to disease biology. This is particularly important in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and Guillain–Barré syndrome (GBS), the most common inflammatory neuropathies, where reliable biomarkers of peripheral demyelination would help identify, and potentially measure, active disease and responses to treatment. We postulated that periaxin, a protein exclusively expressed by myelinating Schwann cells, could serve as a fluid biomarker of demyelinating peripheral neuropathy. We developed a single molecule array (Simoa)-based immunoassay to measure plasma periaxin in patients with CIDP (n = 45, including longitudinal samples across a discovery cohort and a validation cohort, for a total of 77 time points), GBS (n = 30, 66 time points), Charcot–Marie–Tooth disease (CMT, n = 20), CNS disease controls with multiple sclerosis (n = 30) and healthy controls (n = 30). We also evaluated whether periaxin is released in myelinating co-cultures following immune-mediated demyelination and axonal damage, comparing results with uninjured cultures. Plasma periaxin effectively distinguishes peripheral from CNS diseases, with significantly elevated levels in CIDP, GBS and CMT, but not in CNS disease or healthy controls (all P < 0.01). In CIDP, periaxin discriminates patients with active disease from those with inactive disease (P < 0.0001), and plasma levels decrease following treatment with intravenous immunoglobulin (IVIg). Elevated periaxin strongly predicts clinical worsening at 1 year [sensitivity 99%, specificity 72%, area under the curve (AUC) 0.86 (95% confidence interval, CI: 0.67–1)]. In GBS, peak levels of plasma periaxin and the ratio of periaxin to axonal biomarkers [neurofilament light chain (NfL) and peripherin] discriminate most cases of acute inflammatory demyelinating polyradiculoneuropathy (AIDP) from acute motor axonal neuropathy (AMAN), as classified by electrophysiology (sensitivity 100%, specificity 86%, AUC = 0.94, 95% CI: 0.81–1). Serial measurements showed that plasma periaxin levels peak 2 to 3 weeks after GBS symptom onset, followed by a gradual decline in the weeks thereafter. In vitro, periaxin is higher following immune-mediated demyelination compared with axonal damage and control conditions. Plasma periaxin is a biomarker of peripheral nerve demyelination. Combined with axonal fluid biomarkers and existing clinical scales, periaxin has the potential to improve the clinical management of peripheral neuropathies, accelerating advances in care and experimental research.

Whiplash-associated disorders (WADs) affect 20–50 million individuals globally each year, with ≤50% developing persistent pain. WAD grade II (WADII) is the most common type and is characterized by neck symptoms and musculoskeletal signs without apparent nerve injury on routine diagnostic testing. However, emerging evidence suggests that nerve pathology might be present in some people with WADII. This longitudinal cohort study aimed to investigate comprehensively the presence, temporal patterns and prognostic value of nerve pathology and neuropathic pain in acute WADII. A prospective longitudinal cohort study was conducted with 129 acute participants with WADII (median age 36.0 years, 58% female) and 36 healthy controls (median age 39.0 years, 61% female). Participants with WADII were recruited within 4 weeks of injury from local emergency departments. Data collection included bedside neurological assessments, quantitative sensory testing, intraepidermal nerve fibre density and serum neurofilament light chain concentrations. Follow-up assessments were conducted 6 months after injury. Signs of neuropathic pain were present in 65% (84/129) acutely and persisted in 32% (21/66) 6 months post-injury. Bedside neurological assessment revealed that somatosensory loss-of-function was present in 54% (70/129) acutely, reducing to 25% (17/67) 6 months post-injury. Quantitative sensory testing demonstrated significantly reduced cold, warm, thermal sensory limen, mechanical and vibration detection thresholds in acute WADII compared with controls (d > 0.47). Acute loss-of-function in at least one quantitative sensory testing parameter was present in 67.6% (85/126) of WADII. At 6 months, participants with WADII showed persistent hypoaesthesia to warm, thermal sensory limen and mechanical detection thresholds, and decreased mechanical pain and pressure pain sensitivity compared with controls (d > 0.44). These functional neurological changes were accompanied by elevated serum neurofilament light chain levels in acute WADII compared with controls [d = −0.52 (95% confidence interval −0.94, −0.10)]. Intraepidermal nerve fibre densities at the index finger were not significantly different between groups. However, dermal myelin basic protein+/protein gene product+ myelinated nerve bundles at the index finger were reduced 6 months post-injury in WADII compared with controls [d = 0.69 (0.26, 1.11)]. Multivariable linear regression suggested that bedside tests for hypoaesthesia at the index finger were prognostic for whiplash-related upper quadrant pain 6 months post-injury (r2 = 0.13, P = 0.02). In conclusion, two-thirds of participants with acute WADII initially exhibited signs of neuropathic pain and nerve pathology. At the 6-month follow-up, neuropathic pain persisted in one-third of participants with WADII, and nerve pathology persisted in two-thirds. These findings challenge the traditional musculoskeletal classification of WADII and underscore the need for targeted neurological assessments and treatment.

Summary: Multi-electrode array (MEA) generate electrophysiological data that can be used to functionally characterize excitable cells. MEA data can be complex to analyse in a reproducible manner, with current data analysis tools often calculating parameters at the whole-well level. Here we present MEAanalysis, an open-source R package [GPL (≥2)] able to visualize burst parameters at the single electrode level downstream of AxIS Navigator software (Axion BioSystems) processing, thus increasing our understanding of an excitable cell network’s spatiotemporal variability. Availability and implementation: The package is hosted on and can be installed from the following GitHub repository: https://github.com/egordon2/MEA-analysis-package. User feedback provided via email or the GitHub issues tab will inform cycles of development.