Peripheral sensitization of nociceptors is believed to be a key driver of chronic pain states. Here, we sought to study the effects of a modified version of inflammatory soup on the excitability of human stem cell-derived sensory neurons. For this, we used a preexisting and a novel stem cell line, modified to stably express the calcium sensor GCamP6f. Upon treatment with inflammatory soup, we observed no changes in neuronal transcription or functional responses upon calcium imaging and only a very minor increase in resting membrane potential (RMP) via whole cell patch clamping: control RMP (-71.31 +/- 1.1 mV) vs inflammatory soup RMP (-67.74 +/- 1.29 mV), uncorrected 2-tailed independent samples t test, P = 0.0383. Similarly, small changes were observed when treating mouse primary sensory neurons with inflammatory soup. A semi-systematic reexamination of past literature further indicated that observed effects of inflammatory mediators on dissociated sensory neuron cultures are generally small. We conclude that modelling inflammation-induced peripheral sensitization in vitro is nontrivial and will require careful selection of mediators and/or more complex, longitudinal multicellular setups. Especially in the latter, our novel GCamP6f-induced pluripotent stem cell line may be of value.

JAK inhibitors (JAKi) are widely used antiinflammatory drugs. Recent data suggest that JAKi have superior effects on pain reduction in rheumatoid arthritis (RA). However, the underlying mechanisms for this observation are not fully understood. We investigated whether JAKi can act directly on human sensory neurons. We analyzed RNA-seq datasets of sensory neurons and found that they expressed JAK1 and STAT3. Addition of cell-free RA synovial fluid to human induced pluripotent stem cell-derived (iPSC-derived) sensory neurons led to phosphorylation of STAT3 (pSTAT3), which was completely blocked by the JAKi tofacitinib. Compared with paired serum, RA synovial fluid was enriched for the STAT3 signalling cytokines IL-6, IL-11, LIF, IFN-α, and IFN-β, with their requisite receptors present in peripheral nerves postmortem. Accordingly, these recombinant cytokines induced pSTAT3 in iPSC-derived sensory neurons. Furthermore, IL-6 + sIL-6R and LIF upregulated expression of pain-relevant genes with STAT3-binding sites, an effect that was blocked by tofacitinib. LIF also induced neuronal sensitization, highlighting this molecule as a putative pain mediator. Finally, over time, tofacitinib reduced the firing rate of sensory neurons stimulated with RA synovial fluid. Together, these data indicate that JAKi can act directly on human sensory neurons, providing a potential mechanistic explanation for their suggested superior analgesic properties.

Objective: Clinical studies suggest that compared to anti-TNF treatment, JAK inhibitors (JAKi) are superior in reducing pain in rheumatoid arthritis (RA). The underlying mechanisms for this observation are still unknown. Sensory neurons transmit noxious signals from inflamed joints to the central nervous system, where a pain percept is generated. We investigated whether JAKi exert direct effects on sensory neurons. Methods: In-house and public RNA sequencing datasets of sensory neurons were analysed for relevant JAK/STAT and cytokine-receptor gene expression. Human induced pluripotent stem cell (IPSC)-derived sensory neurons were stimulated with serum and synovial fluid (SF) from individuals with RA, or with selected cytokines that were found in RA SF by Luminex. Phosphorylation of STAT3 (pSTAT3) was assessed by Western blot. Sensory neuron activation was examined by recording neuronal firing using multi-electrode array and measuring expression levels of pain-relevant genes with STAT3-binding sites. Results: Cell-free RA synovial fluid induced pSTAT3 in IPSC-derived sensory neurons, an effect which was completely blocked by the JAKi tofacitinib. Compared to paired serum, RA SF was enriched for the JAK/STAT cytokines IL-6, IL-11, LIF, IFN-alpha and IFN-beta, with their requisite receptors present on sensory neurons. Stimulation of IPSC-derived sensory neurons with these recombinant cytokines recapitulated pSTAT3 induction in these cells. Furthermore, IL-6+sIL-6R or LIF upregulated expression of pain-relevant genes which was blocked by tofacitinib. Finally, we provided evidence that LIF can induce neuronal sensitisation. Conclusion: Our data indicate that JAKi can act directly on sensory neurons, providing a potential mechanistic explanation for their suggested superior analgesic properties. ### Competing Interest Statement LST and FD have received consultancy fees and/or research funding from AbbVie, CESAS Medical, GSK, Sanofi A/S, Ono Pharmaceuticals, UCB outside this work. OB is a cofounder, CEO and shareholder of LIFE & BRAIN GmbH. The remaining authors do not declare any conflicts of interest.

Human induced pluripotent stem cells (hiPSCs) are a promising approach to study neurological and neuropsychiatric diseases. Most methods to record the activity of these cells have major drawbacks as they are invasive or they do not allow single cell resolution. Genetically encoded voltage indicators (GEVIs) open the path to high throughput visualization of undisturbed neuronal activity. However, conventional GEVIs perturb membrane integrity through inserting multiple copies of transmembrane domains into the plasma membrane. To circumvent large add-ons to the plasma membrane, we used a minimally invasive novel hybrid dark quencher GEVI to record the physiological and pathological firing patterns of hiPSCs-derived sensory neurons from patients with inherited erythromelalgia, a chronic pain condition associated with recurrent attacks of redness and swelling in the distal extremities. We observed considerable differences in action potential firing patterns between patient and control neurons that were previously overlooked with other recording methods. Our system also performed well in hiPSC-derived forebrain neurons where it detected spontaneous synchronous bursting behavior, thus opening the path to future applications in other cell types and disease models including Parkinson’s disease, Alzheimer’s disease, epilepsy, and schizophrenia, conditions associated with disturbances of neuronal activity and synchrony.

ABSTRACT:Induced pluripotent stem cells (iPSCs) have enabled the generation of various difficult-to-access cell types such as human nociceptors. A key challenge associated with human iPSC-derived nociceptors (hiPSCdNs) is their prolonged functional maturation. While numerous studies have addressed the expression of classic neuronal markers and ion channels in hiPSCdNs, the temporal development of key signaling cascades regulating nociceptor activity has remained largely unexplored. In this study, we used an immunocytochemical high-content imaging approach alongside electrophysiological staging to assess metabotropic and ionotropic signaling of large scale-generated hiPSCdNs across 70 days of in vitro differentiation. During this period, the resting membrane potential became more hyperpolarized, while rheobase, action potential peak amplitude, and membrane capacitance increased. After 70 days, hiPSCdNs exhibited robust physiological responses induced by GABA, pH shift, ATP, and capsaicin. Direct activation of protein kinase A type II (PKA-II) through adenylyl cyclase stimulation with forskolin resulted in PKA-II activation at all time points. Depolarization-induced activation of PKA-II emerged after 35 days of differentiation. However, effective inhibition of forskolin-induced PKA-II activation by opioid receptor agonists required 70 days of in vitro differentiation. Our results identify a pronounced time difference between early expression of functionally important ion channels and emergence of regulatory metabotropic sensitizing and desensitizing signaling only at advanced stages of in vitro cultivation, suggesting an independent regulation of ionotropic and metabotropic signaling. These data are relevant for devising future studies into the development and regulation of human nociceptor function and for defining time windows suitable for hiPSCdN-based drug discovery.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease in humans and remains to have a fatal prognosis. Recent studies in animal models and human ALS patients indicate that increased reactive oxygen species (ROS) play an important role in the pathogenesis. Considering previous studies revealing the influence of ROS on mitochondrial physiology, our attention was focused on mitochondria in the murine ALS model, wobbler mouse. The aim of this study was to investigate morphological differences between wild-type and wobbler mitochondria with aid of superresolution structured illumination fluorescence microscopy, TEM, and TEM tomography. To get an insight into mitochondrial dynamics, expression studies of corresponding proteins were performed. Here, we found significantly smaller and degenerated mitochondria in wobbler motor neurons at a stable stage of the disease. Our data suggest a ROS-regulated, Ox-CaMKII-dependent Drp1 activation leading to disrupted fission-fusion balance, resulting in fragmented mitochondria. These changes are associated with numerous impairments, resulting in an overall self-reinforcing decline of motor neurons. In summary, our study provides common pathomechanisms with other ALS models and human ALS cases confirming mitochondria and related dysfunctions as a therapeutic target for the treatment of ALS.

Bacterial products can act on neurons to alter signaling and function. In the present study, we found that dorsal root ganglion (DRG) sensory neurons are enriched for ANTXR2, the high-affinity receptor for anthrax toxins. Anthrax toxins are composed of protective antigen (PA), which binds to ANTXR2, and the protein cargoes edema factor (EF) and lethal factor (LF). Intrathecal administration of edema toxin (ET (PA + EF)) targeted DRG neurons and induced analgesia in mice. ET inhibited mechanical and thermal sensation, and pain caused by formalin, carrageenan or nerve injury. Analgesia depended on ANTXR2 expressed by Nav1.8+ or Advillin+ neurons. ET modulated protein kinase A signaling in mouse sensory and human induced pluripotent stem cell-derived sensory neurons, and attenuated spinal cord neurotransmission. We further engineered anthrax toxins to introduce exogenous protein cargoes, including botulinum toxin, into DRG neurons to silence pain. Our study highlights interactions between a bacterial toxin and nociceptors, which may lead to the development of new pain therapeutics.

The controlled differentiation of pluripotent stem cells (PSCs) into neurons and glia offers a unique opportunity to study early stages of human central nervous system development under controlled conditions in vitro. With the advent of cell reprogramming and the possibility to generate induced pluripotent stem cells (iPSCs) from any individual in a scalable manner, these studies can be extended to a disease- and patient-specific level. Autism spectrum disorder (ASD) is considered a neurodevelopmental disorder, with substantial evidence pointing to early alterations in neurogenesis and network formation as key pathogenic drivers. For that reason, ASD represents an ideal candidate for stem cell-based disease modeling. Here, we provide a concise review on recent advances in the field of human iPSC-based modeling of syndromic and non-syndromic forms of ASD, with a particular focus on studies addressing neuronal dysfunction and altered connectivity. We further discuss recent efforts to translate stem cell-based disease modeling to 3D via brain organoid and cell transplantation approaches, which enable the investigation of disease mechanisms in a tissue-like context. Finally, we describe advanced tools facilitating the assessment of altered neuronal function, comment on the relevance of iPSC-based models for the assessment of pharmaceutical therapies and outline potential future routes in stem cell-based ASD research.

Bacterial toxins are able to act on neurons to modulate signaling and function. Here, we find that nociceptive sensory neurons that mediate pain are enriched in the receptor for anthrax toxins, ANTXR2. Anthrax Edema Toxin (ET) induced cAMP and PKA signaling in Nav1.8+ nociceptive neurons and modulated pain in vivo . Peripherally administered ET mediated mechanical allodynia in naïve mice and during B. anthracis infection. Intrathecally administered ET produced analgesic effects, potently blocking pain-like behaviors in multiple mouse models of inflammatory and chronic neuropathic pain. Nociceptor-specific ablation of ANTXR2 attenuated ET-induced signaling and analgesia. Modified anthrax toxin successfully delivered exogenous protein cargo into nociceptive neurons, illustrating utility of the anthrax toxin system as a molecular platform to target pain. ET further induced signaling in human iPSC-derived sensory neurons. Our findings highlight novel interactions between a bacterial toxin and nociceptors that may be utilized for developing new pain therapeutics.SUMMARY ANTXR2 expression on nociceptive neurons allows selective targeting and modulation of pain by native and engineered anthrax toxins.

Amyotrophic lateral sclerosis is a devastating motor neuron disease and to this day not curable. While 5–10% of patients inherit the disease (familiar ALS), up to 95% of patients are diagnosed with the sporadic form (sALS). ALS is characterized by the degeneration of upper motor neurons in the cerebral cortex and of lower motor neurons in the brainstem and spinal cord. The wobbler mouse resembles almost all phenotypical hallmarks of human sALS patients and is therefore an excellent motor neuron disease model. The motor neuron disease of the wobbler mouse develops over a time course of around 40 days and can be divided into three phases: p0, presymptomatic; p20, early clinical; and p40, stable clinical phase. Recent findings suggest an essential implication of the NAD+-producing enzyme Nmnat2 in neurodegeneration as well as maintenance of healthy axons. Here, we were able to show a significant downregulation of both gene and protein expression of Nmnat2 in the spinal cord of the wobbler mice at the stable clinical phase. The product of the enzyme NAD+ is also significantly reduced, and the values of the reactive oxygen species are significantly increased in the spinal cord of the wobbler mouse at p40. Thus, the deregulated expression of Nmnat2 appears to have a great influence on the cellular stress in the spinal cord of wobbler mice.