Interleukin-1α links peripheral CaV2.2 channel activation to rapid adaptive increases in heat sensitivity in skin.

Neurons have the unique capacity to adapt output in response to changes in their environment. Within seconds, sensory nerve endings can become hypersensitive to stimuli in response to potentially damaging events. The underlying behavioral response is well studied, but several of the key signaling molecules that mediate sensory hypersensitivity remain unknown. We previously discovered that peripheral voltage-gated CaV2.2 channels in nerve endings in skin are essential for the rapid, transient increase in sensitivity to heat, but not to mechanical stimuli, that accompanies intradermal capsaicin. Here we report that the cytokine interleukin-1α (IL-1α), an alarmin, is necessary and sufficient to trigger rapid heat and mechanical hypersensitivity in skin. Of 20 cytokines screened, only IL-1α was consistently detected in hind paw interstitial fluid in response to intradermal capsaicin and, similar to behavioral sensitivity to heat, IL-1α levels were also dependent on peripheral CaV2.2 channel activity. Neutralizing IL-1α in skin significantly reduced capsaicin-induced changes in hind paw sensitivity to radiant heat and mechanical stimulation. Intradermal IL-1α enhances behavioral responses to stimuli and, in culture, IL-1α enhances the responsiveness of Trpv1-expressing sensory neurons. Together, our data suggest that IL-1α is the key cytokine that underlies rapid and reversible neuroinflammatory responses in skin.

Interleukin-1α links peripheral CaV2.2 channel activation to rapid adaptive increases in heat sensitivity in skin.

Neurons have the unique capacity to adapt output in response to changes in their environment. Within seconds, sensory nerve endings can become hypersensitive to stimuli in response to potentially damaging events. The underlying behavioral response is well studied, but several of the key signaling molecules that mediate sensory hypersensitivity remain unknown. We previously discovered that peripheral voltage-gated CaV2.2 channels in nerve endings in skin are essential for the rapid, transient increase in sensitivity to heat, but not to mechanical stimuli, that accompanies intradermal capsaicin. Here we report that the cytokine interleukin-1α (IL-1α), an alarmin, is necessary and sufficient to trigger rapid heat and mechanical hypersensitivity in skin. Of 20 cytokines screened, only IL-1α was consistently detected in hind paw interstitial fluid in response to intradermal capsaicin and, similar to behavioral sensitivity to heat, IL-1α levels were also dependent on peripheral CaV2.2 channel activity. Neutralizing IL-1α in skin significantly reduced capsaicin-induced changes in hind paw sensitivity to radiant heat and mechanical stimulation. Intradermal IL-1α enhances behavioral responses to stimuli and, in culture, IL-1α enhances the responsiveness of Trpv1-expressing sensory neurons. Together, our data suggest that IL-1α is the key cytokine that underlies rapid and reversible neuroinflammatory responses in skin.

Neuroma Analysis in Humans: Standardizing Sample Collection and Documentation.

INTRODUCTION: The biology of symptomatic neuromas is poorly understood, particularly the factors causing pain in human neuromas. Pain presence varies among and within individuals, with some having painful and nonpainful neuromas. To bridge these knowledge gaps, our group developed a protocol for assessing neuroma pain and collecting tissue for molecular analysis. This manuscript outlines our workflow and challenges and aims to inspire other centers to share their experiences with these tissues. METHODS: For every included patient and collected nerve or bone tissue specimens, we perform a detailed chart review and a multifaceted analysis of pain and pain perception immediately before surgery. We collect patient-reported outcome measures (PROMs) on pain, function, and mental well-being outcomes at preoperative assessment and at the 6-month follow-up postoperatively. Before surgery, the patient is assessed once again to obtain an immediate preoperative pain status and identify potential differences in pain intensity of different neuromas. Intraoperatively, specimens are obtained and their gross anatomical features are recorded, after which they are stored in paraformaldehyde or frozen for later sample analyses. Postoperatively, patients are contacted to obtain additional postoperative PROMs. RESULTS: A total of 220 specimens of nerve tissue have been successfully obtained from 83 limbs, comprising 95 specimens of neuromas and 125 specimens of nerves located proximal to the neuromas or from controls. CONCLUSIONS: Our approach outlines the methods combining specimen collection and examination, including both macroscopic and molecular biological features, with PROMs, encompassing physical and psychological aspects, along with clinical metadata obtained through clinical teams and chart review.

Neuronal STING activation in amyotrophic lateral sclerosis and frontotemporal dementia.

The stimulator of interferon genes (STING) pathway has been implicated in neurodegenerative diseases, including Parkinson's disease and amyotrophic lateral sclerosis (ALS). While prior studies have focused on STING within immune cells, little is known about STING within neurons. Here, we document neuronal activation of the STING pathway in human postmortem cortical and spinal motor neurons from individuals affected by familial or sporadic ALS. This process takes place selectively in the most vulnerable cortical and spinal motor neurons but not in neurons that are less affected by the disease. Concordant STING activation in layer V cortical motor neurons occurs in a mouse model of C9orf72 repeat-associated ALS and frontotemporal dementia (FTD). To establish that STING activation occurs in a neuron-autonomous manner, we demonstrate the integrity of the STING signaling pathway, including both upstream activators and downstream innate immune response effectors, in dissociated mouse cortical neurons and neurons derived from control human induced pluripotent stem cells (iPSCs). Human iPSC-derived neurons harboring different familial ALS-causing mutations exhibit increased STING signaling with DNA damage as a main driver. The elevated downstream inflammatory markers present in ALS iPSC-derived neurons can be suppressed with a STING inhibitor. Our results reveal an immunophenotype that consists of innate immune signaling driven by the STING pathway and occurs specifically within vulnerable neurons in ALS/FTD.

Biology and pathophysiology of symptomatic neuromas.

ABSTRACT: Neuromas are a substantial cause of morbidity and reduction in quality of life. This is not only caused by a disruption in motor and sensory function from the underlying nerve injury but also by the debilitating effects of neuropathic pain resulting from symptomatic neuromas. A wide range of surgical and therapeutic modalities have been introduced to mitigate this pain. Nevertheless, no single treatment option has been successful in completely resolving the associated constellation of symptoms. While certain novel surgical techniques have shown promising results in reducing neuroma-derived and phantom limb pain, their effectiveness and the exact mechanism behind their pain-relieving capacities have not yet been defined. Furthermore, surgery has inherent risks, may not be suitable for many patients, and may yet still fail to relieve pain. Therefore, there remains a great clinical need for additional therapeutic modalities to further improve treatment for patients with devastating injuries that lead to symptomatic neuromas. However, the molecular mechanisms and genetic contributions behind the regulatory programs that drive neuroma formation-as well as the resulting neuropathic pain-remain incompletely understood. Here, we review the histopathological features of symptomatic neuromas, our current understanding of the mechanisms that favor neuroma formation, and the putative contributory signals and regulatory programs that facilitate somatic pain, including neurotrophic factors, neuroinflammatory peptides, cytokines, along with transient receptor potential, and ionotropic channels that suggest possible approaches and innovations to identify novel clinical therapeutics.

S. aureus drives itch and scratch-induced skin damage through a V8 protease-PAR1 axis.

Itch is an unpleasant sensation that evokes a desire to scratch. The skin barrier is constantly exposed to microbes and their products. However, the role of microbes in itch generation is unknown. Here, we show that Staphylococcus aureus, a bacterial pathogen associated with itchy skin diseases, directly activates pruriceptor sensory neurons to drive itch. Epicutaneous S. aureus exposure causes robust itch and scratch-induced damage. By testing multiple isogenic bacterial mutants for virulence factors, we identify the S. aureus serine protease V8 as a critical mediator in evoking spontaneous itch and alloknesis. V8 cleaves proteinase-activated receptor 1 (PAR1) on mouse and human sensory neurons. Targeting PAR1 through genetic deficiency, small interfering RNA (siRNA) knockdown, or pharmacological blockade decreases itch and skin damage caused by V8 and S. aureus exposure. Thus, we identify a mechanism of action for a pruritogenic bacterial factor and demonstrate the potential of inhibiting V8-PAR1 signaling to treat itch.

iPSC motor neurons, but not other derived cell types, capture gene expression changes in postmortem sporadic ALS motor neurons.

Motor neuron degeneration, the defining feature of amyotrophic lateral sclerosis (ALS), is a primary example of cell-type specificity in neurodegenerative diseases. Using isogenic pairs of induced pluripotent stem cells (iPSCs) harboring different familial ALS mutations, we assess the capacity of iPSC-derived lower motor neurons, sensory neurons, astrocytes, and superficial cortical neurons to capture disease features including transcriptional and splicing dysregulation observed in human postmortem neurons. At early time points, differentially regulated genes in iPSC-derived lower motor neurons, but not other cell types, overlap with one-third of the differentially regulated genes in laser-dissected motor neurons from ALS compared with control postmortem spinal cords. For genes altered in both the iPSC model and bona fide human lower motor neurons, expression changes correlate between the two populations. In iPSC-derived lower motor neurons, but not other derived cell types, we detect the downregulation of genes affected by TDP-43-dependent splicing. This reduction takes place exclusively within genotypes known to involve TDP-43 pathology.

Multi-target modulation of ion channels underlying the analgesic effects of α-mangostin in dorsal root ganglion neurons.

BACKGROUND: α-Mangostin is a xanthone isolated from the pericarps of mangosteen fruit with, and has analgesic properties. Although the effects suggest an interaction of α-mangostin with ion channels in the nociceptive neurons, electrophysiological investigation of the underlying mechanism has not been performed. HYPOTHESIS: We hypothesized that α-Mangostin exerts its analgesic effects by modulating the activity of various ion channels in dorsal root ganglion (DRG) neurons. METHODS: We performed a whole-cell patch clamp study using mouse DRG neurons, HEK293T cells overexpressing targeted ion channels, and ND7/23 cells. Molecular docking (MD) and in silico absorption, distribution, metabolism, and excretion (ADME) analyses were conducted to obtain further insights into the binding sites and pharmacokinetics, respectively. RESULTS: Application of α-mangostin (1-3 µM) hyperpolarized the resting membrane potential (RMP) of small-sized DRG neurons by increasing background K+ conductance and thereby inhibited action potential generation. At micromolar levels, α-mangostin activates TREK-1, TREK-2, or TRAAK, members of the two-pore domain K+ channel (K2P) family known to be involved in RMP formation in DRG neurons. Furthermore, capsaicin-induced TRPV1 currents were potently inhibited by α-mangostin (0.43 ± 0.27 µM), and partly suppressed tetrodotoxin-sensitive voltage-gated Na+ channel (NaV) currents. MD simulation revealed that multiple oxygen atoms in α-mangostin may form stable hydrogen bonds with TREKs, TRAAK, TRPV1, and NaV channels. In silico ADME tests suggested that α-mangostin may satisfy the drug-likeness properties without penetrating the blood-brain barrier. CONCLUSION: The analgesic properties of α-mangostin might be mediated by the multi-target modulation of ion channels, including TREK/TRAAK activation, TRPV1 inhibition, and reduction of the tetrodotoxin-sensitive NaV current. The findings suggest that the phytochemical can be a multi-ion channel-targeting drug and an alternative drug for effective pain management.

Phenotypic screen identifies the natural product silymarin as a novel anti-inflammatory analgesic.

Sensory neuron hyperexcitability is a critical driver of pathological pain and can result from axon damage, inflammation, or neuronal stress. G-protein coupled receptor signaling can induce pain amplification by modulating the activation of Trp-family ionotropic receptors and voltage-gated ion channels. Here, we sought to use calcium imaging to identify novel inhibitors of the intracellular pathways that mediate sensory neuron sensitization and lead to hyperexcitability. We identified a novel stimulus cocktail, consisting of the SSTR2 agonist L-054,264 and the S1PR3 agonist CYM5541, that elicits calcium responses in mouse primary sensory neurons in vitro as well as pain and thermal hypersensitivity in mice in vivo. We screened a library of 906 bioactive compounds and identified 24 hits that reduced calcium flux elicited by L-054,264/CYM5541. Among these hits, silymarin, a natural product derived from milk thistle, strongly reduced activation by the stimulation cocktail, as well as by a distinct inflammatory cocktail containing bradykinin and prostaglandin E2. Silymarin had no effect on sensory neuron excitability at baseline, but reduced calcium flux via Orai channels and downstream mediators of phospholipase C signaling. In vivo, silymarin pretreatment blocked development of adjuvant-mediated thermal hypersensitivity, indicating potential use as an anti-inflammatory analgesic.

Comprehensive evaluation of human-derived anti-poly-GA antibodies in cellular and animal models of C9orf72 disease.

Hexanucleotide G4C2 repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Dipeptide repeat proteins (DPRs) generated by translation of repeat-containing RNAs show toxic effects in vivo as well as in vitro and are key targets for therapeutic intervention. We generated human antibodies that bind DPRs with high affinity and specificity. Anti-GA antibodies engaged extra- and intra-cellular poly-GA and reduced aggregate formation in a poly-GA overexpressing human cell line. However, antibody treatment in human neuronal cultures synthesizing exogenous poly-GA resulted in the formation of large extracellular immune complexes and did not affect accumulation of intracellular poly-GA aggregates. Treatment with antibodies was also shown to directly alter the morphological and biochemical properties of poly-GA and to shift poly-GA/antibody complexes to more rapidly sedimenting ones. These alterations were not observed with poly-GP and have important implications for accurate measurement of poly-GA levels including the need to evaluate all centrifugation fractions and disrupt the interaction between treatment antibodies and poly-GA by denaturation. Targeting poly-GA and poly-GP in two mouse models expressing G4C2 repeats by systemic antibody delivery for up to 16 mo was well-tolerated and led to measurable brain penetration of antibodies. Long-term treatment with anti-GA antibodies produced improvement in an open-field movement test in aged C9orf72450 mice. However, chronic administration of anti-GA antibodies in AAV-(G4C2)149 mice was associated with increased levels of poly-GA detected by immunoassay and did not significantly reduce poly-GA aggregates or alleviate disease progression in this model.

A reference human induced pluripotent stem cell line for large-scale collaborative studies.

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.

Endothelial cells regulate astrocyte to neural progenitor cell trans-differentiation in a mouse model of stroke.

The concept of the neurovascular unit emphasizes the importance of cell-cell signaling between neural, glial, and vascular compartments. In neurogenesis, for example, brain endothelial cells play a key role by supplying trophic support to neural progenitors. Here, we describe a surprising phenomenon where brain endothelial cells may release trans-differentiation signals that convert astrocytes into neural progenitor cells in male mice after stroke. After oxygen-glucose deprivation, brain endothelial cells release microvesicles containing pro-neural factor Ascl1 that enter into astrocytes to induce their trans-differentiation into neural progenitors. In mouse models of focal cerebral ischemia, Ascl1 is upregulated in endothelium prior to astrocytic conversion into neural progenitor cells. Injecting brain endothelial-derived microvesicles amplifies the process of astrocyte trans-differentiation. Endothelial-specific overexpression of Ascl1 increases the local conversion of astrocytes into neural progenitors and improves behavioral recovery. Our findings describe an unexpected vascular-regulated mechanism of neuroplasticity that may open up therapeutic opportunities for improving outcomes after stroke.

Spinal motor neuron transplantation to enhance nerve reconstruction strategies: Towards a cell therapy.

Nerve transfers have become a powerful intervention to restore function following devastating paralyzing injuries. A major limitation to peripheral nerve repair and reconstructive strategies is the progressive, fibrotic degeneration of the distal nerve and denervated muscle, eventually precluding recovery of these targets and thus defining a time window within which reinnervation must occur. One proven strategy in the clinic has been the sacrifice and transfer of an adjacent distal motor nerve to provide axons to occupy, and thus preserve (or "babysit"), the target muscle. However, available nearby nerves are limited in severe brachial plexus or spinal cord injury. An alternative and novel proposition is the transplantation of spinal motor neurons (SMNs) derived from human induced pluripotent stem cells (iPSCs) into the target nerve to extend their axons to occupy and preserve the targets. These cells could potentially be delivered through minimally invasive or percutaneous techniques. Several reports have demonstrated survival, functional innervation, and muscular preservation following transplantation of SMNs into rodent nerves. Advances in the generation, culture, and differentiation of human iPSCs now offer the possibility for an unlimited supply of clinical grade SMNs. This review will discuss the previous reports of peripheral SMN transplantation, outline key considerations, and propose next steps towards advancing this approach to clinic. Stem cells have garnered great enthusiasm for their potential to revolutionize medicine. However, this excitement has often led to premature clinical studies with ill-defined cell products and mechanisms of action, particularly in spinal cord injury. We believe the peripheral transplantation of a defined SMN population to address neuromuscular degeneration will be transformative in augmenting current reconstructive strategies. By thus removing the current barriers of time and distance, this strategy would dramatically enhance the potential for reconstruction and functional recovery in otherwise hopeless paralyzing injuries. Furthermore, this strategy may be used as a permanent axon replacement following destruction of lower motor neurons and would enable exogenous stimulation options, such as pacing of transplanted SMN axons in the phrenic nerve to avoid mechanical ventilation in high cervical cord injury or amyotrophic lateral sclerosis.

Human sensorimotor organoids derived from healthy and amyotrophic lateral sclerosis stem cells form neuromuscular junctions.

Human induced pluripotent stem cells (iPSC) hold promise for modeling diseases in individual human genetic backgrounds and thus for developing precision medicine. Here, we generate sensorimotor organoids containing physiologically functional neuromuscular junctions (NMJs) and apply the model to different subgroups of amyotrophic lateral sclerosis (ALS). Using a range of molecular, genomic, and physiological techniques, we identify and characterize motor neurons and skeletal muscle, along with sensory neurons, astrocytes, microglia, and vasculature. Organoid cultures derived from multiple human iPSC lines generated from individuals with ALS and isogenic lines edited to harbor familial ALS mutations show impairment at the level of the NMJ, as detected by both contraction and immunocytochemical measurements. The physiological resolution of the human NMJ synapse, combined with the generation of major cellular cohorts exerting autonomous and non-cell autonomous effects in motor and sensory diseases, may prove valuable to understand the pathophysiological mechanisms of ALS.

A high-content platform for physiological profiling and unbiased classification of individual neurons.

High-throughput physiological assays lose single-cell resolution, precluding subtype-specific analyses of activation mechanism and drug effects. We demonstrate APPOINT (automated physiological phenotyping of individual neuronal types), a physiological assay platform combining calcium imaging, robotic liquid handling, and automated analysis to generate physiological activation profiles of single neurons at large scale. Using unbiased techniques, we quantify responses to sequential stimuli, enabling subgroup identification by physiology and probing of distinct mechanisms of neuronal activation within subgroups. Using APPOINT, we quantify primary sensory neuron activation by metabotropic receptor agonists and identify potential contributors to pain signaling. We expand the role of neuroimmune interactions by showing that human serum directly activates sensory neurons, elucidating a new potential pain mechanism. Finally, we apply APPOINT to develop a high-throughput, all-optical approach for quantification of activation threshold and pharmacologically validate contributions of ion channel families to optical activation.

Effects of mexiletine on hyperexcitability in sporadic amyotrophic lateral sclerosis: Preliminary findings from a small phase II randomized controlled trial.

BACKGROUND: To collect preliminary data on the effects of mexiletine on cortical and axonal hyperexcitability in sporadic amyotrophic lateral sclerosis (ALS) in a phase 2 double-blind randomized controlled trial. METHODS: Twenty ALS subjects were randomized to placebo and mexiletine 300 or 600 mg daily for 4 wk and assessed by transcranial magnetic stimulation and axonal excitability studies. The primary endpoint was change in resting motor threshold (RMT). RESULTS: RMT was unchanged with 4 wk of mexiletine (combined active therapies) as compared to placebo, which showed a significant increase (P = .039). Reductions of motor evoked potential (MEP) amplitude (P = .013) and accommodation half-time (P = .002), secondary outcome measures of cortical and axonal excitability, respectively, were also evident at 4 wk on mexiletine. CONCLUSIONS: The relative stabilization of RMT in the treated subjects was unexpected and could be attributed to unaccounted sources of error or chance. However, a possible alternative cause is neuromodulation preventing an increase. The change in MEP amplitude and accommodation half-time supports the reduction of cortical and axonal hyperexcitability with mexiletine.

Effect of Ezogabine on Cortical and Spinal Motor Neuron Excitability in Amyotrophic Lateral Sclerosis: A Randomized Clinical Trial.

Importance: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of the motor nervous system. Clinical studies have demonstrated cortical and spinal motor neuron hyperexcitability using transcranial magnetic stimulation and threshold tracking nerve conduction studies, respectively, although metrics of excitability have not been used as pharmacodynamic biomarkers in multi-site clinical trials. Objective: To ascertain whether ezogabine decreases cortical and spinal motor neuron excitability in ALS. Design, Setting, and Participants: This double-blind, placebo-controlled phase 2 randomized clinical trial sought consent from eligible participants from November 3, 2015, to November 9, 2017, and was conducted at 12 US sites within the Northeast ALS Consortium. Participants were randomized in equal numbers to a higher or lower dose of ezogabine or to an identical matched placebo, and they completed in-person visits at screening, baseline, week 6, and week 8 for clinical assessment and neurophysiological measurements. Interventions: Participants were randomized to receive 600 mg/d or 900 mg/d of ezogabine or a matched placebo for 10 weeks. Main Outcomes and Measures: The primary outcome was change in short-interval intracortical inhibition (SICI; SICI-1 was used in analysis to reflect stronger inhibition from an increase in amplitude) from pretreatment mean at screening and baseline to the full-dose treatment mean at weeks 6 and 8. The secondary outcomes included levels of cortical motor neuron excitability (including resting motor threshold) measured by transcranial magnetic stimulation and spinal motor neuron excitability (including strength-duration time constant) measured by threshold tracking nerve conduction studies. Results: A total of 65 participants were randomized to placebo (23), 600 mg/d of ezogabine (23), and 900 mg/d of ezogabine (19 participants); 45 were men (69.2%) and the mean (SD) age was 58.3 (8.8) years. The SICI-1 increased by 53% (mean ratio, 1.53; 95% CI, 1.12-2.09; P = .009) in the 900-mg/d ezogabine group vs placebo group. The SICI-1 did not change in the 600-mg/d ezogabine group vs placebo group (mean ratio, 1.15; 95% CI, 0.87-1.52; P = .31). The resting motor threshold increased in the 600-mg/d ezogabine group vs placebo group (mean ratio, 4.61; 95% CI, 0.21-9.01; P = .04) but not in the 900-mg/d ezogabine group vs placebo group (mean ratio, 1.95; 95% CI, -2.64 to 6.54; P = .40). Ezogabine caused a dose-dependent decrease in excitability by several other metrics, including strength-duration time constant in the 900-mg/d ezogabine group vs placebo group (mean ratio, 0.73; 95% CI, 0.60 to 0.87; P < .001). Conclusions and Relevance: Ezogabine decreased cortical and spinal motor neuron excitability in participants with ALS, suggesting that such neurophysiological metrics may be used as pharmacodynamic biomarkers in multisite clinical trials. Trial Registration: ClinicalTrials.gov Identifier: NCT02450552.

Measurement of axonal excitability: Consensus guidelines.

Measurement of axonal excitability provides an in vivo indication of the properties of the nerve membrane and of the ion channels expressed on these axons. Axonal excitability techniques have been utilised to investigate the pathophysiological mechanisms underlying neurological diseases. This document presents guidelines derived for such studies, based on a consensus of international experts, and highlights the potential difficulties when interpreting abnormalities in diseased axons. The present manuscript provides a state-of-the-art review of the findings of axonal excitability studies and their interpretation, in addition to suggesting guidelines for the optimal performance of excitability studies.

Modeling cell-autonomous motor neuron phenotypes in ALS using iPSCs.

Amyotrophic lateral sclerosis (ALS) is an aggressive and uniformly fatal degenerative disease of the motor nervous system. In order to understand underlying disease mechanisms, researchers leverage a host of in vivo and in vitro models, including yeast, worms, flies, zebrafish, mice, and more recently, human induced pluripotent stem cells (iPSCs) derived from ALS patients. While mouse models have been the main workhorse of preclinical ALS research, the development of iPSCs provides a new opportunity to explore molecular phenotypes of ALS within human cells. Importantly, this technology enables modeling of both familial and sporadic ALS in the relevant human genetic backgrounds, as well as a personalized or targeted approach to therapy development. Harnessing these powerful tools requires addressing numerous challenges, including different variance components associated with iPSCs and motor neurons as well as concomitant limits of reductionist approaches. In order to overcome these obstacles, optimization of protocols and assays, confirmation of phenotype robustness at scale, and validation of findings in human tissue and genetics will cement the role for iPSC models as a valuable complement to animal models in ALS and more broadly among neurodegenerative diseases.