C. difficile intoxicates neurons and pericytes to drive neurogenic inflammation.

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated gastrointestinal infections1,2. The exaggerated colonic inflammation caused by C. difficile toxins such as toxin B (TcdB) damages tissues and promotes C. difficile colonization3-6, but how TcdB causes inflammation is unclear. Here we report that TcdB induces neurogenic inflammation by targeting gut-innervating afferent neurons and pericytes through receptors, including the Frizzled receptors (FZD1, FZD2 and FZD7) in neurons and chondroitin sulfate proteoglycan 4 (CSPG4) in pericytes. TcdB stimulates the secretion of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from neurons and pro-inflammatory cytokines from pericytes. Targeted delivery of the TcdB enzymatic domain, through fusion with a detoxified diphtheria toxin, into peptidergic sensory neurons that express exogeneous diphtheria toxin receptor (an approach we term toxogenetics) is sufficient to induce neurogenic inflammation and recapitulates major colonic histopathology associated with CDI. Conversely, mice lacking SP, CGRP or the SP receptor (neurokinin 1 receptor) show reduced pathology in both models of caecal TcdB injection and CDI. Blocking SP or CGRP signalling reduces tissue damage and C. difficile burden in mice infected with a standard C. difficile strain or with hypervirulent strains expressing the TcdB2 variant. Thus, targeting neurogenic inflammation provides a host-oriented therapeutic approach for treating CDI.

PRDM12 Is Transcriptionally Active and Required for Nociceptor Function Throughout Life.

PR domain-containing member 12 (PRDM12) is a key developmental transcription factor in sensory neuronal specification and survival. Patients with rare deleterious variants in PRDM12 are born with congenital insensitivity to pain (CIP) due to the complete absence of a subtype of peripheral neurons that detect pain. In this paper, we report two additional CIP cases with a novel homozygous PRDM12 variant. To elucidate the function of PRDM12 during mammalian development and adulthood, we generated temporal and spatial conditional mouse models. We find that PRDM12 is expressed throughout the adult nervous system. We observed that loss of PRDM12 during mid-sensory neurogenesis but not in the adult leads to reduced survival. Comparing cellular biophysical nociceptive properties in developmental and adult-onset PRDM12 deletion mouse models, we find that PRDM12 is necessary for proper nociceptive responses throughout life. However, we find that PRDM12 regulates distinct age-dependent transcriptional programs. Together, our results implicate PRDM12 as a viable therapeutic target for specific pain therapies even in adults.

Human Pluripotent Stem Cells-Based Therapies for Neurodegenerative Diseases: Current Status and Challenges.

Neurodegenerative diseases are characterized by irreversible cell damage, loss of neuronal cells and limited regeneration potential of the adult nervous system. Pluripotent stem cells are capable of differentiating into the multitude of cell types that compose the central and peripheral nervous systems and so have become the major focus of cell replacement therapies for the treatment of neurological disorders. Human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC)-derived cells have both been extensively studied as cell therapies in a wide range of neurodegenerative disease models in rodents and non-human primates, including Parkinson's disease, stroke, epilepsy, spinal cord injury, Alzheimer's disease, multiple sclerosis and pain. In this review, we discuss the latest progress made with stem cell therapies targeting these pathologies. We also evaluate the challenges in clinical application of human pluripotent stem cell (hPSC)-based therapies including risk of oncogenesis and tumor formation, immune rejection and difficulty in regeneration of the heterogeneous cell types composing the central nervous system.

Human induced pluripotent stem cell-derived GABAergic interneuron transplants attenuate neuropathic pain.

Neuropathic pain causes severe suffering, and most patients are resistant to current therapies. A core element of neuropathic pain is the loss of inhibitory tone in the spinal cord. Previous studies have shown that foetal GABAergic neuron precursors can provide relief from pain. However, the source of these precursor cells and their multipotent status make them unsuitable for therapeutic use. Here, we extend these findings by showing, for the first time, that spinally transplanted, terminally differentiated human induced pluripotent stem cell-derived GABAergic (iGABAergic) neurons provide significant, long-term, and safe relief from neuropathic pain induced by peripheral nerve injury in mice. Furthermore, iGABAergic neuron transplants survive long term in the injured spinal cord and show evidence of synaptic integration. Together, this provides the proof in principle for the first viable GABAergic transplants to treat human neuropathic pain patients.

Peripheral straightjacket (α2δ Ca2+ channel subunit) expression is required for neuropathic sensitization in Drosophila.

Nerve injury leads to devastating and often untreatable neuropathic pain. While acute noxious sensation (nociception) is a crucial survival mechanism and is conserved across phyla, chronic neuropathic pain is considered a maladaptive response owing to its devastating impact on a patient's quality of life. We have recently shown that a neuropathic pain-like response occurs in adult Drosophila. However, the mechanisms underlying this phenomenon are largely unknown. Previous studies have shown that the α2δ peripheral calcium channel subunit straightjacket (stj) is a conserved factor required for thermal pain perception. We demonstrate here that stj is required in peripheral ppk+ sensory neurons for acute thermal responses and that it mediates nociceptive hypersensitivity in an adult Drosophila model of neuropathic pain-like disease. Given that calcium channels are the main targets of gabapentinoids (pregabalin and gabapentin), we assessed if these drugs can alleviate nociceptive hypersensitivity. Our findings suggest that gabapentinoids may prevent nociceptive hypersensitivity by preserving central inhibition after nerve injury. Together, our data further highlight the similarity of some mechanisms for pain-like conditions across phyla and validates the scientific use of Drosophila neuropathic sensitization models for analgesic drug discovery. This article is part of the Theo Murphy meeting issue 'Evolution of mechanisms and behaviour important for pain'.

Nerve injury drives a heightened state of vigilance and neuropathic sensitization in Drosophila.

Injury can lead to devastating and often untreatable chronic pain. While acute pain perception (nociception) evolved more than 500 million years ago, virtually nothing is known about the molecular origin of chronic pain. Here we provide the first evidence that nerve injury leads to chronic neuropathic sensitization in insects. Mechanistically, peripheral nerve injury triggers a loss of central inhibition that drives escape circuit plasticity and neuropathic allodynia. At the molecular level, excitotoxic signaling within GABAergic (γ-aminobutyric acid) neurons required the acetylcholine receptor nAChRα1 and led to caspase-dependent death of GABAergic neurons. Conversely, disruption of GABA signaling was sufficient to trigger allodynia without injury. Last, we identified the conserved transcription factor twist as a critical downstream regulator driving GABAergic cell death and neuropathic allodynia. Together, we define how injury leads to allodynia in insects, and describe a primordial precursor to neuropathic pain may have been advantageous, protecting animals after serious injury.

Molecular dissection of box jellyfish venom cytotoxicity highlights an effective venom antidote.

The box jellyfish Chironex fleckeri is extremely venomous, and envenoming causes tissue necrosis, extreme pain and death within minutes after severe exposure. Despite rapid and potent venom action, basic mechanistic insight is lacking. Here we perform molecular dissection of a jellyfish venom-induced cell death pathway by screening for host components required for venom exposure-induced cell death using genome-scale lenti-CRISPR mutagenesis. We identify the peripheral membrane protein ATP2B1, a calcium transporting ATPase, as one host factor required for venom cytotoxicity. Targeting ATP2B1 prevents venom action and confers long lasting protection. Informatics analysis of host genes required for venom cytotoxicity reveal pathways not previously implicated in cell death. We also discover a venom antidote that functions up to 15 minutes after exposure and suppresses tissue necrosis and pain in mice. These results highlight the power of whole genome CRISPR screening to investigate venom mechanisms of action and to rapidly identify new medicines.

Dissecting Motor Neuron Disease With Drosophila melanogaster.

Motor Neuron Disease (MND) typically affects patients during the later stages of life, and thus, MND is having an increasingly devastating impact on diagnosed individuals, their families and society. The umbrella term MND refers to diseases which cause the progressive loss of upper and/or lower motor neurons and a subsequent decrease in motor ability such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). The study of these diseases is complex and has recently involved the use of genome-wide association studies (GWAS). However, in the case of MND, it has been difficult to identify the complex genetics involved in subtypes, and functional investigation of new candidate disease genes is warranted. Drosophila is a powerful model for addressing these complex diseases. The UAS/Gal4/Gal80 system allows for the upregulation of Drosophila genes, the "knockdown" of genes and the ectopic expression of human genes or mutations in a tissue-specific manner; often resulting in Drosophila models which exhibit typical MND disease pathologies. These can then be further interrogated to identify disease-modifying genes or mutations and disease pathways. This review will discuss two common MNDs and the current Drosophila models which are being used to research their genetic basis and the different pathologies of MND.

Developing Modern Pain Therapies.

Chronic pain afflicts as much as 50% of the population at any given time but our methods to address pain remain limited, ineffective and addictive. In order to develop new therapies an understanding of the mechanisms of painful sensitization is essential. We discuss here recent progress in the understanding of mechanisms underlying pain, and how these mechanisms are being targeted to produce modern, specific therapies for pain. Finally, we make recommendations for the next generation of targeted, effective, and safe pain therapies.

Tauopathy induced by low level expression of a human brain-derived tau fragment in mice is rescued by phenylbutyrate.

Human neurodegenerative tauopathies exhibit pathological tau aggregates in the brain along with diverse clinical features including cognitive and motor dysfunction. Post-translational modifications including phosphorylation, ubiquitination and truncation, are characteristic features of tau present in the brain in human tauopathy. We have previously reported an N-terminally truncated form of tau in human brain that is associated with the development of tauopathy and is highly phosphorylated. We have generated a new mouse model of tauopathy in which this human brain-derived, 35 kDa tau fragment (Tau35) is expressed in the absence of any mutation and under the control of the human tau promoter. Most existing mouse models of tauopathy overexpress mutant tau at levels that do not occur in human neurodegenerative disease, whereas Tau35 transgene expression is equivalent to less than 10% of that of endogenous mouse tau. Tau35 mice recapitulate key features of human tauopathies, including aggregated and abnormally phosphorylated tau, progressive cognitive and motor deficits, autophagic/lysosomal dysfunction, loss of synaptic protein, and reduced life-span. Importantly, we found that sodium 4-phenylbutyrate (Buphenyl®), a drug used to treat urea cycle disorders and currently in clinical trials for a range of neurodegenerative diseases, reverses the observed abnormalities in tau and autophagy, behavioural deficits, and loss of synapsin 1 in Tau35 mice. Our results show for the first time that, unlike other tau transgenic mouse models, minimal expression of a human disease-associated tau fragment in Tau35 mice causes a profound and progressive tauopathy and cognitive changes, which are rescued by pharmacological intervention using a clinically approved drug. These novel Tau35 mice therefore represent a highly disease-relevant animal model in which to investigate molecular mechanisms and to develop novel treatments for human tauopathies.