PIEZO2 and perineal mechanosensation are essential for sexual function.

Despite the potential importance of genital mechanosensation for sexual reproduction, little is known about how perineal touch influences mating. We explored how mechanosensation affords exquisite awareness of the genitals and controls reproduction in mice and humans. Using genetic strategies and in vivo functional imaging, we demonstrated that the mechanosensitive ion channel PIEZO2 (piezo-type mechanosensitive ion channel component 2) is necessary for behavioral sensitivity to perineal touch. PIEZO2 function is needed for triggering a touch-evoked erection reflex and successful mating in both male and female mice. Humans with complete loss of PIEZO2 function have genital hyposensitivity and experience no direct pleasure from gentle touch or vibration. Together, our results help explain how perineal mechanoreceptors detect the gentlest of stimuli and trigger physiologically important sexual responses, thus providing a platform for exploring the sensory basis of sexual pleasure and its relationship to affective touch.

PIEZO2 in somatosensory neurons controls gastrointestinal transit.

The gastrointestinal tract is in a state of constant motion. These movements are tightly regulated by the presence of food and help digestion by mechanically breaking down and propelling gut content. Mechanical sensing in the gut is thought to be essential for regulating motility; however, the identity of the neuronal populations, the molecules involved, and the functional consequences of this sensation are unknown. Here, we show that humans lacking PIEZO2 exhibit impaired bowel sensation and motility. Piezo2 in mouse dorsal root, but not nodose ganglia is required to sense gut content, and this activity slows down food transit rates in the stomach, small intestine, and colon. Indeed, Piezo2 is directly required to detect colon distension in vivo. Our study unveils the mechanosensory mechanisms that regulate the transit of luminal contents throughout the gut, which is a critical process to ensure proper digestion, nutrient absorption, and waste removal.

Human Stem Cell-Derived TRPV1-Positive Sensory Neurons: A New Tool to Study Mechanisms of Sensitization.

Somatosensation, the detection and transduction of external and internal stimuli such as temperature or mechanical force, is vital to sustaining our bodily integrity. But still, some of the mechanisms of distinct stimuli detection and transduction are not entirely understood, especially when noxious perception turns into chronic pain. Over the past decade major progress has increased our understanding in areas such as mechanotransduction or sensory neuron classification. However, it is in particular the access to human pluripotent stem cells and the possibility of generating and studying human sensory neurons that has enriched the somatosensory research field. Based on our previous work, we describe here the generation of human stem cell-derived nociceptor-like cells. We show that by varying the differentiation strategy, we can produce different nociceptive subpopulations with different responsiveness to nociceptive stimuli such as capsaicin. Functional as well as deep sequencing analysis demonstrated that one protocol in particular allowed the generation of a mechano-nociceptive sensory neuron population, homogeneously expressing TRPV1. Accordingly, we find the cells to homogenously respond to capsaicin, to become sensitized upon inflammatory stimuli, and to respond to temperature stimulation. The efficient and homogenous generation of these neurons make them an ideal translational tool to study mechanisms of sensitization, also in the context of chronic pain.

The Form and Function of PIEZO2.

Mechanosensation is the ability to detect dynamic mechanical stimuli (e.g., pressure, stretch, and shear stress) and is essential for a wide variety of processes, including our sense of touch on the skin. How touch is detected and transduced at the molecular level has proved to be one of the great mysteries of sensory biology. A major breakthrough occurred in 2010 with the discovery of a family of mechanically gated ion channels that were coined PIEZOs. The last 10 years of investigation have provided a wealth of information about the functional roles and mechanisms of these molecules. Here we focus on PIEZO2, one of the two PIEZO proteins found in humans and other mammals. We review how work at the molecular, cellular, and systems levels over the past decade has transformed our understanding of touch and led to unexpected insights into other types of mechanosensation beyond the skin.

Decoding Cellular Mechanisms for Mechanosensory Discrimination.

Single-cell RNA-sequencing and in vivo functional imaging provide expansive but disconnected views of neuronal diversity. Here, we developed a strategy for linking these modes of classification to explore molecular and cellular mechanisms responsible for detecting and encoding touch. By broadly mapping function to neuronal class, we uncovered a clear transcriptomic logic responsible for the sensitivity and selectivity of mammalian mechanosensory neurons. Notably, cell types with divergent gene-expression profiles often shared very similar properties, but we also discovered transcriptomically related neurons with specialized and divergent functions. Applying our approach to knockout mice revealed that Piezo2 differentially tunes all types of mechanosensory neurons with marked cell-class dependence. Together, our data demonstrate how mechanical stimuli recruit characteristic ensembles of transcriptomically defined neurons, providing rules to help explain the discriminatory power of touch. We anticipate a similar approach could expose fundamental principles governing representation of information throughout the nervous system.

Assigning transcriptomic class in the trigeminal ganglion using multiplex in situ hybridization and machine learning.

ABSTRACT: Single cell sequencing has provided unprecedented information about the transcriptomic diversity of somatosensory systems. Here, we describe a simple and versatile in situ hybridization (ISH)-based approach for mapping this information back to the tissue. We illustrate the power of this approach by demonstrating that ISH localization with just 8 probes is sufficient to distinguish all major classes of neurons in sections of the trigeminal ganglion. To further simplify the approach and make transcriptomic class assignment and cell segmentation automatic, we developed a machine learning approach for analyzing images from multiprobe ISH experiments. We demonstrate the power of in situ class assignment by examining the expression patterns of voltage-gated sodium channels that play roles in distinct somatosensory processes and pain. Specifically, this analysis resolves intrinsic problems with single cell sequencing related to the sparseness of data leading to ambiguity about gene expression patterns. We also used the multiplex in situ approach to study the projection fields of the different neuronal classes. Our results demonstrate that the surface of the eye and meninges are targeted by broad arrays of neural classes despite their very different sensory properties but exhibit idiotypic patterns of innervation at a quantitative level. Very surprisingly, itch-related neurons extensively innervated the meninges, indicating that these transcriptomic cell classes are not simply labeled lines for triggering itch. Together, these results substantiate the importance of a sensory neuron's peripheral and central connections as well as its transcriptomic class in determining its role in sensation.

Transcriptional Programming of Human Mechanosensory Neuron Subtypes from Pluripotent Stem Cells.

Efficient and homogeneous in vitro generation of peripheral sensory neurons may provide a framework for novel drug screening platforms and disease models of touch and pain. We discover that, by overexpressing NGN2 and BRN3A, human pluripotent stem cells can be transcriptionally programmed to differentiate into a surprisingly uniform culture of cold- and mechano-sensing neurons. Although such a neuronal subtype is not found in mice, we identify molecular evidence for its existence in human sensory ganglia. Combining NGN2 and BRN3A programming with neural crest patterning, we produce two additional populations of sensory neurons, including a specialized touch receptor neuron subtype. Finally, we apply this system to model a rare inherited sensory disorder of touch and proprioception caused by inactivating mutations in PIEZO2. Together, these findings establish an approach to specify distinct sensory neuron subtypes in vitro, underscoring the utility of stem cell technology to capture human-specific features of physiology and disease.

PIEZO2 mediates injury-induced tactile pain in mice and humans.

Tissue injury and inflammation markedly alter touch perception, making normally innocuous sensations become intensely painful. Although this sensory distortion, known as tactile allodynia, is one of the most common types of pain, the mechanism by which gentle mechanical stimulation becomes unpleasant remains enigmatic. The stretch-gated ion channel PIEZO2 has been shown to mediate light touch, vibration detection, and proprioception. However, the role of this ion channel in nociception and pain has not been resolved. Here, we examined the importance of Piezo2 in the cellular representation of mechanosensation using in vivo imaging in mice. Piezo2-knockout neurons were completely insensitive to gentle dynamic touch but still responded robustly to noxious pinch. During inflammation and after injury, Piezo2 remained essential for detection of gentle mechanical stimuli. We hypothesized that loss of PIEZO2 might eliminate tactile allodynia in humans. Our results show that individuals with loss-of-function mutations in PIEZO2 completely failed to develop sensitization and painful reactions to touch after skin inflammation. These findings provide insight into the basis for tactile allodynia, identify the PIEZO2 mechanoreceptor as an essential mediator of touch under inflammatory conditions, and suggest that this ion channel might be targeted for treating tactile allodynia.

Effects of topographical and mechanical property alterations induced by oxygen plasma modification on stem cell behavior.

Polymeric substrates intended for cell culture and tissue engineering are often surface-modified to facilitate cell attachment of most anchorage-dependent cell types. The modification alters the surface chemistry and possibly topography. However, scant attention has been paid to other surface property alterations. In studying oxygen plasma treatment of polydimethylsiloxane (PDMS), we show that oxygen plasma treatment alters the surface chemistry and, consequently, the topography and elasticity of PDMS at the nanoscale level. The elasticity factor has the predominant effect, compared with the chemical and topographical factors, on cell adhesions of human mesenchymal stem cells (hMSCs). The enhanced focal adhesions favor cell spreading and osteogenesis of hMSCs. Given the prevalent use of PDMS in biomedical device construction and cell culture experiments, this study highlights the importance of understanding how oxygen plasma treatment would impact subsequent cell-substrate interactions. It helps explain inconsistency in the literature and guides preparation of PDMS-based biomedical devices in the future.

Targeted delivery to single cells in precisely controlled microenvironments.

The heterogeneous nature of cells can be an issue for in vitro analysis of cell function due to cell type differences within a population. Observations are most often averaged and dependent on the homogeneity or lack thereof for most cell types. Patterning of features at the sub-cellular scale (< 10 µm) allows for single cell manipulation. Additionally, the ability to pattern multiple materials simultaneously with nanoscale precision enables facile fabrication of multiplexed cellular microenvironment arrays. Here we use this ability to deliver different materials to single or few cells within hundreds of microns of each other on the same substrate. Calcein AM, Calcein Red AM and quantum dots are delivered to live single or few cells. This allows for exposing limited cell numbers to many well defined conditions, thus opening the possibility of single cell based assays.

Development of universal antidotes to control aptamer activity.

With an ever increasing number of people taking numerous medications, the need to safely administer drugs and limit unintended side effects has never been greater. Antidote control remains the most direct means to counteract acute side effects of drugs, but, unfortunately, it has been challenging and cost prohibitive to generate antidotes for most therapeutic agents. Here we describe the development of a set of antidote molecules that are capable of counteracting the effects of an entire class of therapeutic agents based upon aptamers. These universal antidotes exploit the fact that, when systemically administered, aptamers are the only free extracellular oligonucleotides found in circulation. We show that protein- and polymer-based molecules that capture oligonucleotides can reverse the activity of several aptamers in vitro and counteract aptamer activity in vivo. The availability of universal antidotes to control the activity of any aptamer suggests that aptamers may be a particularly safe class of therapeutics.

Effect of treatment, during primary infection, on establishment and clearance of cellular reservoirs of HIV-1.

Patients in whom virologic suppression is achieved with highly active antiretroviral therapy (HAART) retain long-lived cellular reservoirs of human immunodeficiency virus type 1 (HIV-1); this retention is an obstacle to sustained control of infection. To assess the impact that initiating treatment during primary HIV-1 infection has on this cell population, we analyzed the decay kinetics of HIV-1 DNA and of infectivity associated with cells activated ex vivo in 27 patients who initiated therapy before or <6 months after seroconversion and in whom viremia was suppressed to <50 copies/mL. The clearance rates of cellular reservoirs could not be distinguished by these techniques (median half-life, 20 weeks) during the first year of HAART. The clearance of HIV-1 DNA slowed significantly during the subsequent 3 years of treatment (median half-life, 70 weeks), consistent with heterogeneous cellular reservoirs being present. Total cell-associated infectivity (CAI) after 1 year of treatment was undetectable (<0.07 infectious units/million cells [IUPM]) in most patients initiating treatment during primary infection either before (9/9) or <6 months after (6/8) seroconversion. In contrast, all 17 control patients who initiated HAART during chronic infection retained detectable CAI after 3-6 years of treatment (median reservoir size, 1.1 IUPM; P<.0005). These results suggest that treatment <6 months after seroconversion may facilitate long-term control of cellular reservoirs that maintain HIV-1 infection during treatment.

Amplification of acetylcholine-binding catenanes from dynamic combinatorial libraries.

Directed chemical synthesis can produce a vast range of molecular structures, but the intended product must be known at the outset. In contrast, evolution in nature can lead to efficient receptors and catalysts whose structures defy prediction. To access such unpredictable structures, we prepared dynamic combinatorial libraries in which reversibly binding building blocks assemble around a receptor target. We selected for an acetylcholine receptor by adding the neurotransmitter to solutions of dipeptide hydrazones [proline-phenylalanine or proline-(cyclohexyl)alanine], which reversibly combine through hydrazone linkages. At thermodynamic equilibrium, the dominant receptor structure was an elaborate [2]-catenane consisting of two interlocked macrocyclic trimers. This complex receptor with a 100 nM affinity for acetylcholine could be isolated on a preparative scale in 67% yield.