Advances and challenges in modeling inherited peripheral neuropathies using iPSCs.

Inherited peripheral neuropathies (IPNs) are a group of diseases associated with mutations in various genes with fundamental roles in the development and function of peripheral nerves. Over the past 10 years, significant advances in identifying molecular disease mechanisms underlying axonal and myelin degeneration, acquired from cellular biology studies and transgenic fly and rodent models, have facilitated the development of promising treatment strategies. However, no clinical treatment has emerged to date. This lack of treatment highlights the urgent need for more biologically and clinically relevant models recapitulating IPNs. For both neurodevelopmental and neurodegenerative diseases, patient-specific induced pluripotent stem cells (iPSCs) are a particularly powerful platform for disease modeling and preclinical studies. In this review, we provide an update on different in vitro human cellular IPN models, including traditional two-dimensional monoculture iPSC derivatives, and recent advances in more complex human iPSC-based systems using microfluidic chips, organoids, and assembloids.

A humanized chemogenetic system inhibits murine pain-related behavior and hyperactivity in human sensory neurons.

Hyperexcitability in sensory neurons is known to underlie many of the maladaptive changes associated with persistent pain. Chemogenetics has shown promise as a means to suppress such excitability, yet chemogenetic approaches suitable for human applications are needed. PSAM4-GlyR is a modular system based on the human α7 nicotinic acetylcholine and glycine receptors, which responds to inert chemical ligands and the clinically approved drug varenicline. Here, we demonstrated the efficacy of this channel in silencing both mouse and human sensory neurons by the activation of large shunting conductances after agonist administration. Virally mediated expression of PSAM4-GlyR in mouse sensory neurons produced behavioral hyposensitivity upon agonist administration, which was recovered upon agonist washout. Stable expression of the channel led to similar reversible suppression of pain-related behavior even after 10 months of viral delivery. Mechanical and spontaneous pain readouts were also ameliorated by PSAM4-GlyR activation in acute and joint pain inflammation mouse models. Furthermore, suppression of mechanical hypersensitivity generated by a spared nerve injury model of neuropathic pain was also observed upon activation of the channel. Effective silencing of behavioral hypersensitivity was reproduced in a human model of hyperexcitability and clinical pain: PSAM4-GlyR activation decreased the excitability of human-induced pluripotent stem cell-derived sensory neurons and spontaneous activity due to a gain-of-function NaV1.7 mutation causing inherited erythromelalgia. Our results demonstrate the contribution of sensory neuron hyperexcitability to neuropathic pain and the translational potential of an effective, stable, and reversible humanized chemogenetic system for the treatment of pain.

An iPSC model of hereditary sensory neuropathy-1 reveals L-serine-responsive deficits in neuronal ganglioside composition and axoglial interactions.

Hereditary sensory neuropathy type 1 (HSN1) is caused by mutations in the SPTLC1 or SPTLC2 sub-units of the enzyme serine palmitoyltransferase, resulting in the production of toxic 1-deoxysphingolipid bases (DSBs). We used induced pluripotent stem cells (iPSCs) from patients with HSN1 to determine whether endogenous DSBs are neurotoxic, patho-mechanisms of toxicity and response to therapy. HSN1 iPSC-derived sensory neurons (iPSCdSNs) endogenously produce neurotoxic DSBs. Complex gangliosides, which are essential for membrane micro-domains and signaling, are reduced, and neurotrophin signaling is impaired, resulting in reduced neurite outgrowth. In HSN1 myelinating cocultures, we find a major disruption of nodal complex proteins after 8 weeks, which leads to complete myelin breakdown after 6 months. HSN1 iPSC models have, therefore, revealed that SPTLC1 mutation alters lipid metabolism, impairs the formation of complex gangliosides, and reduces axon and myelin stability. Many of these changes are prevented by l-serine supplementation, supporting its use as a rational therapy.

Molecular and cellular correlates of human nerve regeneration: ADCYAP1/PACAP enhance nerve outgrowth.

We only have a rudimentary understanding of the molecular and cellular determinants of nerve regeneration and neuropathic pain in humans. This cohort study uses the most common entrapment neuropathy (carpal tunnel syndrome) as a human model system to prospectively evaluate the cellular and molecular correlates of neural regeneration and its relationship with clinical recovery. In 60 patients undergoing carpal tunnel surgery [36 female, mean age 62.5 (standard deviation 12.2) years], we used quantitative sensory testing and nerve conduction studies to evaluate the function of large and small fibres before and 6 months after surgery. Clinical recovery was assessed with the global rating of change scale and Boston Carpal Tunnel Questionnaire. Twenty healthy participants provided normative data [14 female, mean age 58.0 (standard deviation 12.9) years]. At 6 months post-surgery, we noted significant recovery of median nerve neurophysiological parameters (P < 0.0001) and improvements in quantitative sensory testing measures of both small and large nerve fibre function (P < 0.002). Serial biopsies revealed a partial recovery of intraepidermal nerve fibre density [fibres/mm epidermis pre: 4.20 (2.83), post: 5.35 (3.34), P = 0.001], whose extent correlated with symptom improvement (r = 0.389, P = 0.001). In myelinated afferents, nodal length increased postoperatively [pre: 2.03 (0.82), post: 3.03 (1.23), P < 0.0001] suggesting that this is an adaptive phenomenon. Transcriptional profiling of the skin revealed 31 differentially expressed genes following decompression, with ADCYAP1 (encoding pituitary adenylate cyclase activating peptide, PACAP) being the most strongly upregulated (log2 fold-change 1.87, P = 0.0001) and its expression was associated with recovery of intraepidermal nerve fibres. We found that human induced pluripotent stem cell-derived sensory neurons expressed the receptor for PACAP and that this peptide could significantly enhance axon outgrowth in a dose-dependent manner in vitro [neurite length PACAP 1065.0 µm (285.5), vehicle 570.9 µm (181.8), P = 0.003]. In conclusion, carpal tunnel release is associated with significant cutaneous reinnervation, which correlates with the degree of functional improvement and is associated with a transcriptional programme relating to morphogenesis and inflammatory processes. The most highly dysregulated gene ADCYAP1 (encoding PACAP) was associated with reinnervation and, given that this peptide signals through G-protein coupled receptors, this signalling pathway provides an interesting therapeutic target for human sensory nerve regeneration.

Establishing Myelinating Cocultures Using Human iPSC-Derived Sensory Neurons to Investigate Axonal Degeneration and Demyelination.

Complex signaling between Schwann cells and axons are vital for peripheral neuron development, myelination, and repair. The interaction between these two cell types can be modeled in vitro by coculturing rodent Schwann cells and neurons together. These have in the past been used with great success to help unravel the bidirectional signaling mechanisms that lead to Schwann cell proliferation and myelination. To provide more translatable potential, we have developed myelinating cocultures using human, induced pluripotent stem cell (iPSC)-derived neurons. Under the right conditions, the human neurons are efficiently myelinated by rat Schwann cells, demonstrating successful cross-species signaling. This chapter describes all the necessary steps to generate these myelinating cocultures and methods to investigate and quantify various aspects of myelination. The myelinating cocultures can be maintained in excellent health for over 1 year, facilitating their use to study developmental or chronic disease processes. With this in mind, we have used the cocultures to model a sensory neuropathy which displays clinically with both axonal and demyelinating features. In the cocultures, we found evidence of extensive axonal degeneration and demyelination demonstrated by axonal swelling and fragmentation, and myelin disintegration. The myelinating cocultures can therefore be used to study complex, human disease processes that result in both axonal and myelin-associated degenerative processes.

Defining the Functional Role of NaV1.7 in Human Nociception.

Loss-of-function mutations in NaV1.7 cause congenital insensitivity to pain (CIP); this voltage-gated sodium channel is therefore a key target for analgesic drug development. Utilizing a multi-modal approach, we investigated how NaV1.7 mutations lead to human pain insensitivity. Skin biopsy and microneurography revealed an absence of C-fiber nociceptors in CIP patients, reflected in a reduced cortical response to capsaicin on fMRI. Epitope tagging of endogenous NaV1.7 revealed the channel to be localized at the soma membrane, axon, axon terminals, and the nodes of Ranvier of induced pluripotent stem cell (iPSC) nociceptors. CIP patient-derived iPSC nociceptors exhibited an inability to properly respond to depolarizing stimuli, demonstrating that NaV1.7 is a key regulator of excitability. Using this iPSC nociceptor platform, we found that some NaV1.7 blockers undergoing clinical trials lack specificity. CIP, therefore, arises due to a profound loss of functional nociceptors, which is more pronounced than that reported in rodent models, or likely achievable following acute pharmacological blockade. VIDEO ABSTRACT.

The Role of Voltage-Gated Sodium Channels in Pain Signaling.

Acute pain signaling has a key protective role and is highly evolutionarily conserved. Chronic pain, however, is maladaptive, occurring as a consequence of injury and disease, and is associated with sensitization of the somatosensory nervous system. Primary sensory neurons are involved in both of these processes, and the recent advances in understanding sensory transduction and human genetics are the focus of this review. Voltage-gated sodium channels (VGSCs) are important determinants of sensory neuron excitability: they are essential for the initial transduction of sensory stimuli, the electrogenesis of the action potential, and neurotransmitter release from sensory neuron terminals. Nav1.1, Nav1.6, Nav1.7, Nav1.8, and Nav1.9 are all expressed by adult sensory neurons. The biophysical characteristics of these channels, as well as their unique expression patterns within subtypes of sensory neurons, define their functional role in pain signaling. Changes in the expression of VGSCs, as well as posttranslational modifications, contribute to the sensitization of sensory neurons in chronic pain states. Furthermore, gene variants in Nav1.7, Nav1.8, and Nav1.9 have now been linked to human Mendelian pain disorders and more recently to common pain disorders such as small-fiber neuropathy. Chronic pain affects one in five of the general population. Given the poor efficacy of current analgesics, the selective expression of particular VGSCs in sensory neurons makes these attractive targets for drug discovery. The increasing availability of gene sequencing, combined with structural modeling and electrophysiological analysis of gene variants, also provides the opportunity to better target existing therapies in a personalized manner.

Comprehensive analysis of long noncoding RNA expression in dorsal root ganglion reveals cell-type specificity and dysregulation after nerve injury.

Dorsal root ganglion (DRG) neurons provide connectivity between peripheral tissues and the spinal cord. Transcriptional plasticity within DRG sensory neurons after peripheral nerve injury contributes to nerve repair but also leads to maladaptive plasticity, including the development of neuropathic pain. This study presents tissue and neuron-specific expression profiling of both known and novel long noncoding RNAs (LncRNAs) in the rodent DRG after nerve injury. We have identified a large number of novel LncRNAs expressed within the rodent DRG, a minority of which were syntenically conserved between the mouse, rat, and human, and including, both intergenic and antisense LncRNAs. We have also identified neuron type-specific LncRNAs in the mouse DRG and LncRNAs that are expressed in human IPS cell-derived sensory neurons. We show significant plasticity in LncRNA expression after nerve injury, which in mice is strain and gender dependent. This resource is publicly available and will aid future studies of DRG neuron identity and the transcriptional landscape in both the naive and injured DRG. We present our work regarding novel antisense and intergenic LncRNAs as an online searchable database, accessible from PainNetworks (http://www.painnetworks.org/). We have also integrated all annotated gene expression data in PainNetworks, so they can be examined in the context of their protein interactions.

Hereditary sensory neuropathy type 1-associated deoxysphingolipids cause neurotoxicity, acute calcium handling abnormalities and mitochondrial dysfunction in vitro.

Hereditary sensory neuropathy type 1 (HSN-1) is a peripheral neuropathy most frequently caused by mutations in the SPTLC1 or SPTLC2 genes, which code for two subunits of the enzyme serine palmitoyltransferase (SPT). SPT catalyzes the first step of de novo sphingolipid synthesis. Mutations in SPT result in a change in enzyme substrate specificity, which causes the production of atypical deoxysphinganine and deoxymethylsphinganine, rather than the normal enzyme product, sphinganine. Levels of these abnormal compounds are elevated in blood of HSN-1 patients and this is thought to cause the peripheral motor and sensory nerve damage that is characteristic of the disease, by a largely unresolved mechanism. In this study, we show that exogenous application of these deoxysphingoid bases causes dose- and time-dependent neurotoxicity in primary mammalian neurons, as determined by analysis of cell survival and neurite length. Acutely, deoxysphingoid base neurotoxicity manifests in abnormal Ca2+ handling by the endoplasmic reticulum (ER) and mitochondria as well as dysregulation of cell membrane store-operated Ca2+ channels. The changes in intracellular Ca2+ handling are accompanied by an early loss of mitochondrial membrane potential in deoxysphingoid base-treated motor and sensory neurons. Thus, these results suggest that exogenous deoxysphingoid base application causes neuronal mitochondrial dysfunction and Ca2+ handling deficits, which may play a critical role in the pathogenesis of HSN-1.

Immune or Genetic-Mediated Disruption of CASPR2 Causes Pain Hypersensitivity Due to Enhanced Primary Afferent Excitability.

Human autoantibodies to contactin-associated protein-like 2 (CASPR2) are often associated with neuropathic pain, and CASPR2 mutations have been linked to autism spectrum disorders, in which sensory dysfunction is increasingly recognized. Human CASPR2 autoantibodies, when injected into mice, were peripherally restricted and resulted in mechanical pain-related hypersensitivity in the absence of neural injury. We therefore investigated the mechanism by which CASPR2 modulates nociceptive function. Mice lacking CASPR2 (Cntnap2-/-) demonstrated enhanced pain-related hypersensitivity to noxious mechanical stimuli, heat, and algogens. Both primary afferent excitability and subsequent nociceptive transmission within the dorsal horn were increased in Cntnap2-/- mice. Either immune or genetic-mediated ablation of CASPR2 enhanced the excitability of DRG neurons in a cell-autonomous fashion through regulation of Kv1 channel expression at the soma membrane. This is the first example of passive transfer of an autoimmune peripheral neuropathic pain disorder and demonstrates that CASPR2 has a key role in regulating cell-intrinsic dorsal root ganglion (DRG) neuron excitability.

Functional imaging in microfluidic chambers reveals sensory neuron sensitivity is differentially regulated between neuronal regions.

Primary afferent sensory neurons are incredibly long cells, often traversing distances of over 1 m in humans. Cutaneous sensory stimuli are transduced in the periphery by specialised end organs or free nerve endings, which code the stimulus into electrical action potentials that propagate towards the central nervous system. Despite significant advances in our knowledge of sensory neuron physiology and ion channel expression, many commonly used techniques fail to accurately model the primary afferent neuron in its entirety. In vitro experiments often focus on the cell somata and neglect the fundamental processes of peripheral stimulus transduction and action potential propagation. Despite this, these experiments are commonly used as a model for cellular investigations of the receptive terminals. We demonstrate that ratiometric calcium imaging performed in compartmentalised sensory neuron cultures can be used to directly and accurately compare the sensitivity and functional protein expression of isolated neuronal regions in vitro. Using microfluidic chambers, we demonstrate that the nerve terminals of cultured dorsal root ganglion neurons can be depolarised to induce action potential propagation, which has both tetrodotoxin-resistant and tetrodotoxin-sensitive components. Furthermore, we show that there is a differential regulation of proton sensitivity between the sensory terminals and somata in cultured sensory neurons. We also demonstrate that capsaicin sensitivity is highly dependent on embryonic dissection age. This approach enables a comprehensive method to study the excitability and regional sensitivity of cultured sensory neurons on a single-cell level. Examination of the sensory terminals is crucial to further understand the properties and diversity of dorsal root ganglion sensory neurons.

Using an engineered glutamate-gated chloride channel to silence sensory neurons and treat neuropathic pain at the source.

See Basbaum (doi:10.1093/brain/awx227) for a scientific commentary on this article. Peripheral neuropathic pain arises as a consequence of injury to sensory neurons; the development of ectopic activity in these neurons is thought to be critical for the induction and maintenance of such pain. Local anaesthetics and anti-epileptic drugs can suppress hyperexcitability; however, these drugs are complicated by unwanted effects on motor, central nervous system and cardiac function, and alternative more selective treatments to suppress hyperexcitability are therefore required. Here we show that a glutamate-gated chloride channel modified to be activated by low doses of ivermectin (but not glutamate) is highly effective in silencing sensory neurons and reversing neuropathic pain-related hypersensitivity. Activation of the glutamate-gated chloride channel expressed in either rodent or human induced pluripotent stem cell-derived sensory neurons in vitro potently inhibited their response to both electrical and algogenic stimuli. We have shown that silencing is achieved both at nerve terminals and the soma and is independent of membrane hyperpolarization and instead likely mediated by lowering of the membrane resistance. Using intrathecal adeno-associated virus serotype 9-based delivery, the glutamate-gated chloride channel was successfully targeted to mouse sensory neurons in vivo, resulting in high level and long-lasting expression of the channel selectively in sensory neurons. This enabled reproducible and reversible modulation of thermal and mechanical pain thresholds in vivo; analgesia was observed for 3 days after a single systemic dose of ivermectin. We did not observe any motor or proprioceptive deficits and noted no reduction in cutaneous afferent innervation or upregulation of the injury marker ATF3 following prolonged glutamate-gated chloride channel expression. Established mechanical and cold pain-related hypersensitivity generated by the spared nerve injury model of neuropathic pain was reversed by ivermectin treatment. The efficacy of ivermectin in ameliorating behavioural hypersensitivity was mirrored at the cellular level by a cessation of ectopic activity in sensory neurons. These findings demonstrate the importance of aberrant afferent input in the maintenance of neuropathic pain and the potential for targeted chemogenetic silencing as a new treatment modality in neuropathic pain.

Co-cultures with stem cell-derived human sensory neurons reveal regulators of peripheral myelination.

See Saporta and Shy (doi:10.1093/awx048) for a scientific commentary on this article.Effective bidirectional signalling between axons and Schwann cells is essential for both the development and maintenance of peripheral nerve function. We have established conditions by which human induced pluripotent stem cell-derived sensory neurons can be cultured with rat Schwann cells, and have produced for the first time long-term and stable myelinating co-cultures with human neurons. These cultures contain the specialized domains formed by axonal interaction with myelinating Schwann cells, such as clustered voltage-gated sodium channels at the node of Ranvier and Shaker-type potassium channel (Kv1.2) at the juxtaparanode. Expression of type III neuregulin-1 (TIIINRG1) in induced pluripotent stem cell-derived sensory neurons strongly enhances myelination, while conversely pharmacological blockade of the NRG1-ErbB pathway prevents myelination, providing direct evidence for the ability of this pathway to promote the myelination of human sensory axons. The ß-secretase, BACE1 is a protease needed to generate active NRG1 from the full-length form. Due to the fact that it also cleaves amyloid precursor protein, BACE1 is a therapeutic target in Alzheimer's disease, however, consistent with its role in NRG1 processing we find that BACE1 inhibition significantly impairs myelination in our co-culture system. In order to exploit co-cultures to address other clinically relevant problems, they were exposed to anti-disialosyl ganglioside antibodies, including those derived from a patient with a sensory predominant, inflammatory neuropathy with mixed axonal and demyelinating electrophysiology. The co-cultures reveal that both mouse and human disialosyl antibodies target the nodal axolemma, induce acute axonal degeneration in the presence of complement, and impair myelination. The human, neuropathy-associated IgM antibody is also shown to induce complement-independent demyelination. Myelinating co-cultures using human induced pluripotent stem cell-derived sensory neurons thus provide insights into the cellular and molecular specialization of axoglial signalling, how pharmacological agents may promote or impede such signalling and the pathogenic effects of ganglioside antibodies.awx012media15372351982001.