Specification of dorsal root ganglia sensory neuron subpopulations derived from human pluripotent stem cells

The detection of sensations is essential for everyday functions and requires specialised dorsal root ganglia (DRG) sensory neurons to detect and transmit the stimuli to the central nervous system for processing. The DRG sensory neurons can be broadly classified as either (1) proprioceptors (that detect movement, muscle pressure, and tension), (2) low threshold mechanoreceptors (LTMRs) (that detect touch, hair deflection, and vibration) or (3) nociceptors (that detect pain arising from harmful thermal, mechanical, and chemical stimuli). Unfortunately, there are major challenges in studying sensory perception and disease, including the difficulty in acquiring human tissue samples and the limitations in the translatability of rodent models due to inherent differences between human and rodent sensory neurons. The use of human pluripotent stem cells (hPSCs) can circumvent these challenges by providing a constant source of human cells that can then be differentiated towards sensory neuron cultures. However, current protocols to generate sensory neuron cultures are often limited by low reproducibility, low neuronal yields, mixed populations of neurons, prevalence of nonneuronal cells within the cultures, as well as the requirement of long maturation stages to obtain functionally mature neurons. A promising approach to generate populations of functional sensory neurons is by mimicking sensory neurogenesis using a combined stepwise addition of extrinsic factors (small molecules and growth factors) to direct hPSCs towards progenitor states and neuronal types, combined with the induced expression of lineage-specifying transcription factors to drive the differentiation to a specific neuronal fate. Thus, the major aim of the work described in this thesis was to derive DRG sensory neurons using a combined extrinsic factor and induced transcription factor differentiation approach to generate cultures of sensory neurons and to then functionally characterise the sensory neurons. A key goal of this PhD thesis was to mimic sensory neurogenesis by inducing the expression of lineage specific transcription factors at a developmentally relevant progenitor cell type (i.e., enriched neural crest cells). The work presented in Chapter 3 describes the successful differentiation of hPSCs into caudal neural progenitors (CNPs), which were then further differentiated and enriched for neural crest cells. This protocol was then implemented in Chapters 4 and 5, which aimed to generate and functionally characterise hPSC-derived sensory neurons by inducing the expression of lineage specific transcription factors in the hPSC-derived neural crest cells. The work in Chapter 4 determined that the induced expression of the transcription factors, NEUROGENIN-1 (NGN1) or NEUROGENIN-2 (NGN2), in neural crest cells both significantly enhanced sensory neuron differentiation efficiency and generated a heterogeneous population of functional sensory neurons. The results presented in Chapter 5 demonstrated that the induced co-expression of the lineage specific transcription factors, NGN2 and RUNT RELATED TRANSCRIPTION FACTOR 3 (RUNX3) or NGN2 and SHORT STATURE HOMEOBOX 2 (SHOX2) in hPSC-derived neural crest cells generated enriched mature sensory neuron cultures that had expression and functional profiles consistent with proprioceptors or LTMRs, respectively. Additionally, the work described in Chapter 5 also aimed to investigate whether there are functional differences in the mechanosensory physiology between the two classes of hPSC-derived mechanosensory neurons and the molecular mechanisms by which the two classes of hPSC-derived mechanosensory neurons respond to stimuli. The mechanosensory neurons, denoted as induced-proprioceptor neurons (iPN) and induced-LTMR neurons (iLTMR) were exquisitely sensitive to mechanical stimuli and exhibited distinct mechanically sensitive responses to stretch and to submicrometer (0.1 μm) mechanical stimulation by probe indentation to the soma. Additionally, the iPN and iLTMR displayed different adaptation kinetics reflective of distinct sensory specialisations. Importantly, the iPN and iLTMR fired action potentials in response to \u3c 1.0 μm mechanical stimulation (probe indentation) and knockdown experiments demonstrated that these responses to mechanical stimulation were predominately mediated by PIEZO2. Taken together, the work described in this thesis demonstrates the successful generation of heterogenous and enriched populations of functional sensory neurons from hPSCs via the combination of extrinsic factors and induced expression of lineage specific transcription factors. The derived sensory neurons represent excellent models for the study of human sensory neuron development, peripheral neuropathies, mechanosensory physiology and for the development of directed therapies toward these neuronal populations that become compromised by trauma or neurodegenerative conditions

Computationally efficient modeling of proprioceptive signals in the upper limb for prostheses: a simulation study.

Accurate models of proprioceptive neural patterns could one day play an important role in the creation of an intuitive proprioceptive neural prosthesis for amputees. This paper looks at combining efficient implementations of biomechanical and proprioceptor models in order to generate signals that mimic human muscular proprioceptive patterns for future experimental work in prosthesis feedback. A neuro-musculoskeletal model of the upper limb with 7 degrees of freedom and 17 muscles is presented and generates real time estimates of muscle spindle and Golgi Tendon Organ neural firing patterns. Unlike previous neuro-musculoskeletal models, muscle activation and excitation levels are unknowns in this application and an inverse dynamics tool (static optimisation) is integrated to estimate these variables. A proprioceptive prosthesis will need to be portable and this is incompatible with the computationally demanding nature of standard biomechanical and proprioceptor modelling. This paper uses and proposes a number of approximations and optimisations to make real time operation on portable hardware feasible. Finally technical obstacles to mimicking natural feedback for an intuitive proprioceptive prosthesis, as well as issues and limitations with existing models, are identified and discussed

Stretch Activated Channels in Proprioceptive Organs of Crab and Crayfish Are Sensitive to Gadolinium but not Amiloride, Ruthenium Red or Low pH

The type of stretch activated receptors (SARs) in the chordotonal organs in the crab walking leg and of the muscle receptor organ (MRO) in the crayfish abdomen have not yet been classified as to their molecular or pharmacological profile. The purpose of this study is to examine the pharmacological profile of SARs in the proprioceptive neurons in the crab and crayfish models. Since many SARs share the pharmacological profile of displaying low pH or being proton sensitive (i.e. being more active) or blocked by the diuretic amiloride or ruthenium red as well as being blocked by the broad stretch activated channel blocker gadolinium (Gd3+), we used these agents to screen the receptors. Various displacement rates as well as static positions that activate the stretch activated receptors were used in examining their pharmacological profiles. Hour-long exposure to low pH decreased neural activity of the chordotonal organ of the crab more so than to amiloride or ruthenium red. The crayfish MRO did not show pH sensitivity or sensitivity to amiloride or ruthenium red. Gd3+ rapidly blocked neural activity in both the crab and crayfish. It appears these stretch activated receptors may not have a classification that is suited to the standard pharmacological profiles. The molecular makeup of the channels also awaits characterization. This could reveal a novel SAR subtype. Our neurophysiology course1 took this project on as a course-based undergraduate research experience (CURE) to address an authentic research question

The Effect of CO\u3csub\u3e2\u3c/sub\u3e, Intracellular pH and Extracellular pH on Mechanosensory Proprioceptor Responses in Crayfish and Crab

Proprioceptive neurons monitor the movements of limbs and joints to transduce the movements into electrical signals. These neurons function similarly in species from arthropods to humans. These neurons can be compromised in disease states and in adverse environmental conditions such as with changes in external and internal pH. We used two model preparations (the crayfish muscle receptor organ and a chordotonal organ in the limb of a crab) to characterize the responses of these proprioceptors to external and internal pH changes as well as raised CO2. The results demonstrate the proprioceptive organs are not highly sensitive to changes in extracellular pH, when reduced to 5.0 from 7.4. However, if intracellular pH is decreased by exposure to propionic acid or saline containing CO2, there is a rapid decrease in firing rate in response to joint movements. The responses recover quickly upon reintroduction of normal pH (7.4) or saline not tainted with CO2. These basic understandings may help to address the mechanistic properties of mechanosensitive receptors in other organisms, such as muscle spindles in skeletal muscles of mammals and tactile as well as pressure (i.e., blood pressure) sensory receptors

Proprioceptor subtype identity specified by limb-derived signals

The provision of proprioceptive feedback from limb muscle to spinal motor neuron is essential for the generation of coordinated movement. Proprioceptive sensory neurons form a precise matrix of connections with motor neurons and do so in the absence of patterned activity, implying the existence of proprioceptor subtype identities that mediate selective connectivity. The developing limb has been shown to influence the pattern of connections made by proprioceptors with motor neurons, suggesting that the patterning cues distributed along its cardinal axes are capable of influencing the molecular identities of proprioceptors. In this thesis, I describe efforts to characterize the molecular diversity of proprioceptors supplying distinct muscles located at different dorsoventral and proximodistal positions within the mouse hindlimb. I demonstrate the selective expression of several genes – cdh13, vstm2b, sema5a, and crtac1 – by proprioceptors supplying defined positional domains of the limb. I proceed to determine the limb tissue source of proprioceptor patterning information by examining the expression of these genes in mice in which one of three tissues encountered by proprioceptors – the motor axon, limb mesenchyme, and target muscle – has been genetically manipulated, revealing that both mesenchyme and muscle supply cues capable of directing proprioceptor gene expression. Finally, I show that one marker of proprioceptor muscle-type identity, cdh13, mediates the formation of selective connections between proprioceptors and motor neurons, thereby establishing a molecular link between proprioceptor subtype identity and patterned central connectivity

The role of the femoral chordotonal organ in motor control, interleg coordination, and leg kinematics in Drosophila melanogaster

Legged locomotion in terrestrial animals is often essential for mating and survival, and locomotor behavior must be robust and adaptable in order to be successful. The behavioral plasticity demonstrated by animals’ ability to locomote across diverse types of terrains and to change their locomotion in a task-dependent manner highlights the flexible and modular nature of locomotor networks. The six legs of insects are under the multi-level control of local networks for each limb and limb joint in addition to over-arching central control of the local networks. These networks, consisting of pattern-generating groups of interneurons, motor neurons, and muscles, receive modifying and reinforcing feedback from sensory structures that encode motor output. Proprioceptors in the limbs monitoring their position and movement provide information to these networks that is essential for the adaptability and robustness of locomotor behavior. In insects, proprioceptors are highly diverse, and the exact role of each type in motor control has yet to be determined. Chordotonal organs, analogous to vertebrate muscle spindles, are proprioceptive stretch receptors that span joints and encode specific parameters of relative movement between body segments. In insects, when leg chordotonal organs are disabled or manipulated, interleg coordination and walking are affected, but the simple behavior of straight walking on a flat surface can still be performed. The femoral chordotonal organ (fCO) is the largest leg proprioceptor and monitors the position and movements of the tibia relative to the femur. It has long been studied for its importance in locomotor and postural control. In Drosophila melanogaster, an ideal model organism due its genetic tractability, investigations into the composition, connectivity, and function of the fCO are still in their infancy. The fCO in Drosophila contains anatomical subgroups, and the neurons within a subgroup demonstrate similar responses to movements about the femur-tibia joint. Collectively, the experiments laid out in this dissertation provide a multi-faceted analysis of the anatomy, connectivity, and functional importance of subgroups of fCO neurons in D. melanogaster. The dissertation is divided into four chapters, representing different aspects of this complex and intriguing system. First, I present a detailed analysis of the composition of the fCO and its connectivity within the peripheral and central nervous systems. I demonstrate that the fCO is made up of anatomically distinct groups of neurons, each with their own unique features in the legs and ventral nerve cord. Second, I investigated the neuropeptide profile of the fCO and demonstrate that some fCO neurons express a susbtance that is known to act as a neuromodulator. Third, I demonstrate the sufficiency of subsets of fCO neurons to elicit reflex responses, highlighting the role of the Drosophila fCO in postural control. Lastly, I take this a step further and look into the functional necessity of these neuronal subsets for intra- and interleg coordination during walking. The importance of the fCO in motor control in D. melanogaster has been considered rather minor, though research into the topic is very limited. In the work laid out herein, I highlight the complexity of the Drosophila fCO and its role in the determination of locomotor behavior

Direction Selectivity in Drosophila Proprioceptors Requires the Mechanosensory Channel Tmc

Drosophila Transmembrane channel-like (Tmc) is a protein that functions in larval proprioception. The closely related TMC1 protein is required for mammalian hearing and is a pore-forming subunit of the hair cell mechanotransduction channel. In hair cells, TMC1 is gated by small deflections of microvilli that produce tension on extracellular tip-links that connect adjacent villi. How Tmc might be gated in larval proprioceptors, which are neurons having a morphology that is completely distinct from hair cells, is unknown. Here, we have used high-speed confocal microscopy both to measure displacements of proprioceptive sensory dendrites during larval movement and to optically measure neural activity of the moving proprioceptors. Unexpectedly, the pattern of dendrite deformation for distinct neurons was unique and differed depending on the direction of locomotion: ddaE neuron dendrites were strongly curved by forward locomotion, while the dendrites of ddaD were more strongly deformed by backward locomotion. Furthermore, GCaMP6f calcium signals recorded in the proprioceptive neurons during locomotion indicated tuning to the direction of movement. ddaE showed strong activation during forward locomotion, while ddaD showed responses that were strongest during backward locomotion. Peripheral proprioceptive neurons in animals mutant for Tmc showed a near-complete loss of movement related calcium signals. As the strength of the responses of wild-type animals was correlated with dendrite curvature, we propose that Tmc channels may be activated by membrane curvature in dendrites that are exposed to strain. Our findings begin to explain how distinct cellular systems rely on a common molecular pathway for mechanosensory responses.Peer ReviewedPostprint (published version

Understanding Hereditary Sensory and Autonomic Neuropathy type IV through a novel knock-in mouse model

in pain sensation. Indeed, a functional NGF-TrkA system is an essential requisite for the generation and maintenance of long-lasting thermal and mechanical hyperalgesia in adult mammals. Mutations in the gene encoding for TrkA are responsible for a rare condition, named Hereditary Sensory and Autonomic Neuropathy type IV (HSAN IV), characterized by the loss of response to noxious stimuli, sweating defects and cognitive impairment. However, to date, there is no available mouse model to properly understand how the NGF-TrkA system can lead to pathological phenotypes that are distinctive of HSAN IV. Since the diversity of HSAN IV TrkA-related mutations determines variable degrees of clinical phenotype and intellectual disabilities in affected individuals, we have decided to deeply investigate the missense Arg649Trp (R649W) mutation, located in the intracellular tyrosine kinase domain of TrkA receptor and known to induce a diminished kinase activity and reduced phosphorylation after NGF stimulation in transfected cells. First, by in vitro biochemical and biophysical analyses, I showed that the pathological R649W mutation leads to kinase-inactive TrkA, reducing the constitutive ubiquitination and also affecting the membrane dynamics and trafficking. Then, after the generation of the knock-in mouse line carrying the HSAN IV TrkAR649W mutation, I demonstrated that TrkAR649W/m mice displayed a lower response to thermal and chemical noxious stimuli, correlating with reduced skin innervation and altered expression of nociceptive markers in Dorsal Root Ganglia (DRGs). By performing a sweat assay, I also found that the pathological TrkAR649W mutation causes sweating deficits in HSAN IV TrkAR649W/m mice compared to TrkAh/m controls. Moreover, the R649W mutation decreased anxiety-like behavior and compromised cognitive abilities, by impairing spatial-working and social memory. In addition, the results obtained in this thesis uncovered unexplored roles of TrkA in thermoregulation and sociability. By exploiting suitable control animal models such as HSAN V NGFR100W/m and TrkA+/- mice, I demonstrated that HSAN IV TrkAR649W/m mice mimic the clinical phenotype of HSAN IV patients and they can be considered a suitable experimental platform to explain the clinical aspect of HSAN IV disease, also offering promising new routes for testing future therapies