Biology subjects

Woolf, C. J.

Publications and source records attributed to Woolf, C. J..

32 records · Page 2Linked to original sources

bioRxiv2022

Scalable Generation of Pseudo-Unipolar Sensory Neurons from Human Pluripotent Stem Cells

Development of new non-addictive analgesics requires advanced strategies to differentiate human pluripotent stem cells (hPSCs) into relevant cell types amenable for translational research. Here, we developed a highly efficient and reproducible method that differentiates hPSCs into peptidergic and non-peptidergic nociceptors. By modulating specific cell signaling pathways, hPSCs were first converted into SOX10+ neural crest cells, followed by differentiation into sensory neurons with an in vivo-like pseudo-unipolar morphology. Detailed characterization confirmed that the hPSC-derived nociceptors displayed molecular and cellular features comparable to native dorsal root ganglion (DRG) neurons, and expressed high-threshold primary sensory neuron markers, transcription factors, neuropeptides, and over 150 ion channels and receptors, including critical pain-relevant drug targets (e.g., TRPV1, TAC1, CALCA, NAV1.7, NAV1.8). Moreover, after confirming robust functional activities and differential response to noxious stimuli and specific drugs, a robotic cell culture system was employed to produce large quantities of human sensory neurons, which can be used to develop nociceptor-selective analgesics.

developmental biology↗

bioRxiv2022

Development of a PET radioligand for α2δ-1 subunit of calcium channels for imaging neuropathic pain

Neuropathic pain affects 7-10% of the adult population. Being able to accurately monitor biological changes underlying neuropathic pain will improve our understanding of neuropathic pain mechanisms and facilitate the development of novel therapeutics. Positron emission tomography (PET) is a noninvasive molecular imaging technique that can provide quantitative information of biochemical changes at the whole-body level by using radiolabeled ligands. One important biological change underlying the development of neuropathic pain is the overexpression of 2{delta}-1 subunit of voltage-dependent calcium channels (the target of gabapentin). Thus, we hypothesized that a radiolabeled form of gabapentin may allow imaging changes in 2{delta}-1 for monitoring the underlying pathophysiology of neuropathic pain. Here, we report the development of two 18F-labeled derivatives of gabapentin (trans-4-[18F]fluorogabapentin and cis-4-[18F]fluorogabapentin) and their evaluation in healthy rats and a rat model of neuropathic pain (spinal nerve ligation model). Both isomers were found to selectively bind to the 2{delta}-1 receptor with trans-4-[18F]fluorogabapentin having a higher affinity. Both tracers displayed around 1.5- to 2-fold increased uptake in injured nerves over the contralateral uninjured nerves when measured by gamma counting ex vivo. Although the small size of the nerves and the signal from surrounding muscle prevented visualizing these changes using PET, this work demonstrates that fluorinated derivatives of gabapentin retain binding to 2{delta}-1 and that their radiolabeled forms can be used to detect pathological changes in vitro and ex vivo. Furthermore, this work confirms that 2{delta}-1 is a promising target for imaging specific features of neuropathic pain.

neuroscience↗

bioRxiv2022

Core Transcription Programs Controlling Injury-Induced Neurodegeneration of Retinal Ganglion Cells

Neurodegenerative diseases are characterized by neuronal death and regenerative failure. However, gene regulatory programs governing how initial neuronal injuries lead to neuronal death remain poorly understood. In adult mice, optic nerve crush (ONC) injury, which severs all axons of retinal ganglion cells (RGCs), results in massive death of axotomized RGCs and regenerative failure of survivors. We performed an in vivo CRISPR/Cas9-based genome-wide screen of 1893 transcription factors (TFs) to seek repressors of RGC survival and axon regeneration following ONC. In parallel, we profiled the epigenetic and transcriptional landscapes of injured RGCs by ATAC-seq and RNA-seq to identify critical injury responsive TFs and their targets. Remarkably, these independent analyses converged on a set of four ATF/CEBP transcription factors - ATF3, ATF4, C/EBP{gamma} and CHOP (Ddit3) - as critical regulators of survival. Further studies indicate that these TFs contribute to two pro-death transcriptional programs: ATF3/CHOP preferentially regulate pathways activated by cytokines and innate immunity, whereas ATF4/C/EBP{gamma} regulate pathways engaged by intrinsic neuronal stressors. Manipulation of these TFs also protects RGCs in an experimental model of glaucoma, a prevalent disease in which RGCs die. Together, our results reveal core transcription programs that transform an initial axonal insult into a degenerative result and suggest novel strategies for treating neurodegenerative diseases.

neuroscience↗

bioRxiv2021

Integrin-driven Axon Regeneration in the Spinal Cord Activates a Distinctive CNS Regeneration Program

The peripheral branch of sensory dorsal root ganglion (DRG) neurons regenerates readily after injury unlike their central branch in the spinal cord. However extensive regeneration and reconnection of sensory axons in the spinal cord can be driven by the expression of 9 integrin and its activator kindlin- 1(9k1), which enable axons to interact with tenascin-C. To elucidate the mechanisms and downstream pathways affected by activated integrin expression and central regeneration, we conducted transcriptomic analyses of DRG sensory neurons transduced with 9k1, and controls, with and without axotomy of the central branch. Expression of 9k1 without the central axotomy led to upregulation of a known PNS regeneration program, including many genes associated with peripheral nerve regeneration. Coupling 9k1 treatment with dorsal root axotomy led to extensive central axonal regeneration and caused expression of a distinctive CNS regeneration program, including genes associated with ubiquitination, autophagy, endoplasmic reticulum, trafficking, and signalling. Pharmacological inhibition of these processes blocked the regeneration of axons from DRGs and human iPS-derived sensory neurons, validating their causal contributions. This CNS regeneration- associated program showed little correlation with either embryonic development or PNS regeneration programs. Potential transcriptional drivers of this CNS program coupled to regeneration include Mef2a, Runx3, E2f4, Tfeb, Yy1. Signalling from integrins primes sensory neurons for regeneration, but their axon growth in the CNS is associated with a distinctive program that differs from that involved in PNS regeneration.

neuroscience↗

bioRxiv2021

Demyelination induces transcriptional reprogramming in proprioceptive and Aβ rapidly adapting low-threshold-mechanoreceptor neurons.

Schwann cells, the main glial cell in the peripheral nervous system (PNS), ensheath bundles of small unmyelinated axons or form myelin on larger axons. PNS injuries initiate transcriptional reprograming in both Schwann cells and sensory neurons that promotes regeneration. While the factors that initiate the transcriptional reprograming in Schwann cells are well characterized, the full range of stimuli that initiate this reprograming in sensory neurons remain elusive. Here, using a genetic model of Schwann cell ablation, we find that Schwann cell loss results in transient PNS demyelination without overt axonal loss. By profiling sensory ganglia at single-cell resolution we show that this demyelination induces transcriptional reprogramming preferably in proprioceptive and A{beta} RA-LTMR neurons. Transcriptional reprograming is assumed to be a cell autonomous response of sensory neurons to mechanical axonal injury. By identifying similar reprograming in non-injured, demyelinated neurons, our study suggests that this reprograming represents a non-cell autonomous transcriptional response of sensory neurons to the loss of axon-Schwann cell interactions. HighlightsO_LIAblation of Schwann cells results in transient PNS demyelination, without overt axonal loss. C_LIO_LISchwann cell loss results in transcriptional reprograming in specific sensory neurons. C_LIO_LISpinal nerve transection (mechanical injury of axons) and demyelination (intact axons) induces similar transcriptional responses in DRG neurons. C_LIO_LIThe transcriptional response to demyelination among DRG neurons is specific to the large myelinated proprioceptive and A{beta} RA-LTMR neurons. C_LI

neuroscience↗

bioRxiv2021

Screening for axon regeneration promoting compounds with human iPSC-derived motor neurons

CNS neurons do not regenerate after injury, leading to permanent functional deficits. Although sensory and motor neuron axons do regrow after peripheral nerve injury, functional outcome is limited due to the incomplete and slow regrowth. The lack of human-relevant assays suitable for large-scale drug screens has limited neuro-repair therapy discovery. To address this we developed a phenotypic screening strategy using human induced pluripotent stem cell-derived motor neurons to identify axon-regeneration promoting compounds and targets. The screens involve both re-plating human motor neurons on chondroitin sulfate proteoglycans and measuring regeneration responses to laser axotomy in spot cultures, and from them we identified multiple hits that promote injured axon regrowth. The top hit blebbistatin, a non-muscle myosin II inhibitor, accelerated axon regeneration and functional recovery after sciatic nerve injury in vivo. Human "injury in a dish" assays are suitable, therefore, to screen for therapeutic interventions that can induce or accelerate axon regeneration.

neuroscience↗

bioRxiv2021

Cannabidiol activates neuronal Kv7 channels

Cannabidiol (CBD), a chemical found in the Cannabis sativa plant, is a clinically effective antiepileptic drug whose mechanism of action is unknown. Using a fluorescence-based thallium flux assay, we performed a large-scale screen and found enhancement of flux through heterologously-expressed human Kv7.2/7.3 channels by CBD. Using patch clamp recordings, we found that CBD at low concentrations activates Kv7.2/7.3 channels at subthreshold voltages, with 100 nM CBD producing a doubling of current at -50 mV. CBD shifted the voltage-dependence of channels in the hyperpolarizing direction, producing a shift in the midpoint of activation by [~]-14 mV at 300 nM. CBD also effectively enhanced native M-current in both mouse superior cervical ganglion neurons and rat hippocampal neurons. The potent enhancement of Kv2/7.3 channels by CBD seems likely to contribute to its effectiveness as an antiepileptic drug by reducing neuronal hyperexcitability.

neuroscience↗

bioRxiv2021

A Multiparametric Activity Profiling Platform for Neuron Disease Phenotyping and Drug Screening

Patient stem cell-derived models enable imaging of complex disease phenotypes and the development of scalable drug discovery platforms. Current preclinical methods for assessing cellular activity do not, however, capture the full intricacies of disease-induced disturbances, and instead typically focus on a single parameter, which impairs both the understanding of disease and the discovery of effective therapeutics. Here, we describe a cloud-based image processing and analysis platform that captures the intricate activity profile revealed by GCaMP fluorescent recordings of intracellular calcium changes and enables discovery of molecules that correct 153 parameters that define the amyotrophic lateral sclerosis motor neuron disease phenotype. In a high-throughput screen we identified compounds that revert the multiparametric disease profile to that found in healthy cells, a novel and robust measure of therapeutic potential quite distinct from unidimensional screening. This platform can guide the development of therapeutics that counteract the multifaceted pathological features of diseased cellular activity.

neuroscience↗

bioRxiv2020

Transcription factor network analysis identifies REST/NRSF as an intrinsic regulator of CNS regeneration

The inability of neurons to regenerate long axons within the CNS is a major impediment to improving outcome after spinal cord injury, stroke, and other CNS insults. Recent advances have uncovered an intrinsic program that involves coordinate regulation by multiple transcription factors that can be manipulated to enhance growth in the peripheral nervous system. Here, we used a system-genomics approach to characterize regulatory relationships of regeneration-associated transcription factors, identifying RE1-Silencing Transcription Factor (REST; Neuron-Restrictive Silencer Factor, NRSF) as a predicted upstream suppressor of a pro-regenerative gene program associated with axon regeneration in the CNS. We validate our predictions using multiple paradigms, showing that mature mice bearing cell type-specific deletions of REST or expressing dominant-negative mutant REST showed improved regeneration of the corticospinal tract and optic nerve, accompanied by upregulation of regeneration-associated genes in cortical motor neurons and retinal ganglion cells, respectively. These analyses identify a novel role for REST as an upstream suppressor of the intrinsic regenerative program in the CNS and demonstrate the power of a systems biology approach involving integrative genomics and bio-informatics to predict key regulators of CNS repair.

neuroscience↗

bioRxiv2020

DeepEthogram: a machine learning pipeline for supervised behavior classification from raw pixels

Researchers commonly acquire videos of animal behavior and quantify the prevalence of behaviors of interest to study nervous system function, the effects of gene mutations, and the efficacy of pharmacological therapies. This analysis is typically performed manually and is therefore immensely time consuming, often limited to a small number of behaviors, and variable across researchers. Here, we created DeepEthogram: software that takes raw pixel values of videos as input and uses machine learning to output an ethogram, the set of user-defined behaviors of interest present in each frame of a video. We used convolutional neural network models that compute motion in a video, extract features from motion and single frames, and classify these features into behaviors. These models classified behaviors with greater than 90% accuracy on single frames in videos of flies and mice, matching expert-level human performance. The models accurately predicted even extremely rare behaviors, required little training data, and generalized to new videos and subjects. DeepEthogram runs rapidly on common scientific computer hardware and has a graphical user interface that does not require programming by the end-user. We anticipate DeepEthogram will enable the rapid, automated, and reproducible assignment of behavior labels to every frame of a video, thus accelerating all those studies that quantify behaviors of interest. Code is available at: https://github.com/jbohnslav/deepethogram

neuroscience↗

bioRxiv2019

Transcriptional reprogramming of distinct peripheral sensory neuron subtypes after axonal injury

Primary somatosensory neurons are specialized to transmit specific types of sensory information through differences in cell size, myelination, and the expression of distinct receptors and ion channels, which together define their transcriptional and functional identity. By transcriptionally profiling sensory ganglia at single-cell resolution, we find that different somatosensory neuronal subtypes undergo a remarkably consistent and dramatic transcriptional response to peripheral nerve injury that both promotes axonal regeneration and suppresses cell identity. Successful axonal regeneration leads to a restoration of neuronal cell identity and the deactivation of the growth program. This injury-induced transcriptional reprogramming requires Atf3, a transcription factor which is induced rapidly after injury and is necessary for axonal regeneration and functional recovery. While Atf3 and other injury-induced transcription factors are known for their role in reprogramming cell fate, their function in mature neurons is likely to facilitate major adaptive changes in cell function in response to damaging environmental stimuli.

neuroscience↗

bioRxiv2019

Convergent neural representations of acute nociceptive pain in healthy volunteers: A large-scale fMRI meta-analysis

Characterizing a reliable, pain-related neural signature is critical for translational applications. Many prior fMRI studies have examined acute pain-related brain activation in healthy participants. However, synthesizing these data to identify convergent patterns of activation can be challenging due to the heterogeneity of experimental designs and samples. To address this challenge, we conducted a comprehensive meta-analysis of fMRI studies of stimulus-induced pain in healthy participants. Following pre-registration, two independent reviewers evaluated 4,927 abstracts returned from a search of 8 databases, with 222 fMRI experiments meeting inclusion criteria. We analyzed these experiments using Activation Likelihood Estimation with rigorous type I error control (voxel height p < 0.001, cluster p < 0.05 FWE-corrected) and found a convergent, largely bilateral pattern of pain-related activation in the secondary somatosensory cortex, insula, midcingulate cortex, and thalamus. Notably, these regions were consistently recruited regardless of stimulation technique, location of induction, and participant sex. These findings suggest a highly-conserved core set of pain-related brain areas, encouraging applications as a biomarker for novel therapeutics targeting acute pain.\n\nHIGHLIGHTSO_LIPain stimulation recruits a core set of pain-related brain regions.\nC_LIO_LIThis core set includes thalamus, SII, insula and mid-cingulate cortex.\nC_LIO_LIThese regions were recruited regardless of stimulus modality and stimulus location.\nC_LI

neuroscience↗

bioRxiv2017

Enhanced neuronal regeneration in the CAST/Ei mouse strain is linked to expression of differentiation markers after injury

Peripheral nerve regeneration after injury requires a broad program of transcriptional changes. We investigated the basis for the enhanced nerve regenerative capacity of the CAST/Ei mouse strain relative to C57BL/6 mice. RNA sequencing of dorsal root ganglia (DRG) showed a CAST/Ei specific upregulation of Ascl1 after injury. Ascl1 overexpression in C57BL/6 mice DRG neurons enhanced their neurite outgrowth. Ascl1 is regulated by miR-7048-3p, which is down-regulated in CAST/Ei mice. Inhibition of miR-7048-3p enhances neurite outgrowth. Following injury, CAST/Ei neurons largely retained their mature neuronal profile as determined by single cell RNAseq, whereas the C57BL/6 neurons acquired an immature profile. These findings suggest that one facet of the enhanced regenerative phenotype is preservation of neuronal identity in response to injury.

molecular biology↗

bioRxiv2017

Mouse embryonic stem cells can differentiate via multiple paths to the same state

In embryonic development, cells must differentiate through stereotypical sequences of intermediate states to generate mature states of a particular fate. By contrast, direct programming can generate similar fates through alternative routes, by directly expressing terminal transcription factors. Yet the cell state transitions defining these new routes are unclear. We applied single-cell RNA sequencing to compare two mouse motor neuron differentiation protocols: a standard protocol approximating the embryonic lineage, and a direct programming method. Both undergo similar early neural commitment. Then, rather than transitioning through spinal intermediates like the standard protocol, the direct programming path diverges into a novel transitional state. This state has specific and abnormal gene expression. It opens a loop or worm hole in gene expression that converges separately onto the final motor neuron state of the standard path. Despite their different developmental histories, motor neurons from both protocols structurally, functionally, and transcriptionally resemble motor neurons from embryos.

developmental biology↗