Scalable Hypothalamic Arcuate Neuron Differentiation from Human Pluripotent Stem Cells Suitable for Modeling Metabolic and Reproductive Disorders.

The hypothalamus, composed of several nuclei, is essential for maintaining our body's homeostasis. The arcuate nucleus (ARC), located in the mediobasal hypothalamus, contains neuronal populations with eminent roles in energy and glucose homeostasis as well as reproduction. These neuronal populations are of great interest for translational research. To fulfill this promise, we used a robotic cell culture platform to provide a scalable and chemically defined approach for differentiating human pluripotent stem cells (hPSCs) into pro-opiomelanocortin (POMC), somatostatin (SST), tyrosine hydroxylase (TH) and gonadotropin-releasing hormone (GnRH) neuronal subpopulations with an ARC-like signature. This robust approach is reproducible across several distinct hPSC lines and exhibits a stepwise induction of key ventral diencephalon and ARC markers in transcriptomic profiling experiments. This is further corroborated by direct comparison to human fetal hypothalamus, and the enriched expression of genes implicated in obesity and type 2 diabetes (T2D). Genome-wide chromatin accessibility profiling by ATAC-seq identified accessible regulatory regions that can be utilized to predict candidate enhancers related to metabolic disorders and hypothalamic development. In depth molecular, cellular, and functional experiments unveiled the responsiveness of the hPSC-derived hypothalamic neurons to hormonal stimuli, such as insulin, neuropeptides including kisspeptin, and incretin mimetic drugs such as Exendin-4, highlighting their potential utility as physiologically relevant cellular models for disease studies. In addition, differential glucose and insulin treatments uncovered adaptability within the generated ARC neurons in the dynamic regulation of POMC and insulin receptors. In summary, the establishment of this model represents a novel, chemically defined, and scalable platform for manufacturing large numbers of hypothalamic arcuate neurons and serves as a valuable resource for modeling metabolic and reproductive disorders.

High-throughput screening identifies cell cycle-associated signaling cascades that regulate a multienzyme glucosome assembly in human cells.

We have previously demonstrated that human liver-type phosphofructokinase 1 (PFK1) recruits other rate-determining enzymes in glucose metabolism to organize multienzyme metabolic assemblies, termed glucosomes, in human cells. However, it has remained largely elusive how glucosomes are reversibly assembled and disassembled to functionally regulate glucose metabolism and thus contribute to human cell biology. We developed a high-content quantitative high-throughput screening (qHTS) assay to identify regulatory mechanisms that control PFK1-mediated glucosome assemblies from stably transfected HeLa Tet-On cells. Initial qHTS with a library of pharmacologically active compounds directed following efforts to kinase-inhibitor enriched collections. Consequently, three compounds that were known to inhibit cyclin-dependent kinase 2, ribosomal protein S6 kinase and Aurora kinase A, respectively, were identified and further validated under high-resolution fluorescence single-cell microscopy. Subsequent knockdown studies using small-hairpin RNAs further confirmed an active role of Aurora kinase A on the formation of PFK1 assemblies in HeLa cells. Importantly, all the identified protein kinases here have been investigated as key signaling nodes of one specific cascade that controls cell cycle progression in human cells. Collectively, our qHTS approaches unravel a cell cycle-associated signaling network that regulates the formation of PFK1-mediated glucosome assembly in human cells.

Magnetic resonance measurements of cellular and sub-cellular membrane structures in live and fixed neural tissue.

We develop magnetic resonance (MR) methods for real-time measurement of tissue microstructure and membrane permeability of live and fixed excised neonatal mouse spinal cords. Diffusion and exchange MR measurements are performed using the strong static gradient produced by a single-sided permanent magnet. Using tissue delipidation methods, we show that water diffusion is restricted solely by lipid membranes. Most of the diffusion signal can be assigned to water in tissue which is far from membranes. The remaining 25% can be assigned to water restricted on length scales of roughly a micron or less, near or within membrane structures at the cellular, organelle, and vesicle levels. Diffusion exchange spectroscopy measures water exchanging between membrane structures and free environments at 100 s-1.

Dye-coupling between neonatal spinal motoneurons and interneurons revealed by prolonged back-filling of a ventral root with a low molecular weight tracer in the mouse.

We investigated dye-coupling between motoneurons in the L6 segment of the neonatal mouse spinal cord that contains limb-innervating motoneurons and sexually dimorphic motor nuclei. Using an isolated spinal cord preparation, we back-filled the cut, L6 ventral root with the small molecule Neurobiotin, or the much larger dextran-conjugated fluorophores for 16-24 hours. Motoneurons and parasympathetic preganglionic neurons were filled with both markers, but dye-coupling was only seen with Neurobiotin fills. Following a neurobiotin fill, fluorescence was observed in contralateral motoneurons, in motoneurons innervating adjacent ventral roots, and in ChAT-negative, putative interneurons outside of the motoneuron pools in addition to the directly back-labeled L6 motoneurons. It is known that the gap junction protein connexin-36 is widely expressed in the sexually dimorphic motoneurons of the L6 segment, suggesting that the dye-coupling is mediated by gap junctions. The technique has revealed previously unknown connections of motoneurons in the neonatal mouse cord that are likely to play important roles in the development and function of spinal circuits.

Identification of a novel spinal nociceptive-motor gate control for Aδ pain stimuli in rats.

Physiological responses to nociceptive stimuli are initiated within tens of milliseconds, but the corresponding sub-second behavioral responses have not been adequately explored in awake, unrestrained animals. A detailed understanding of these responses is crucial for progress in pain neurobiology. Here, high-speed videography during nociceptive Aδ fiber stimulation demonstrated engagement of a multi-segmental motor program coincident with, or even preceding, withdrawal of the stimulated paw. The motor program included early head orientation and adjustments of the torso and un-stimulated paws. Moreover, we observed a remarkably potent gating mechanism when the animal was standing on its hindlimbs and which was partially dependent on the endogenous opioid system. These data reveal a profound, immediate and precise integration of nociceptive inputs with ongoing motor activities leading to the initiation of complex, yet behaviorally appropriate, response patterns and the mobilization of a new type of analgesic mechanism within this early temporal nociceptive window.

Interactions between Dorsal and Ventral Root Stimulation on the Generation of Locomotor-Like Activity in the Neonatal Mouse Spinal Cord.

We investigated whether dorsal (DR) and ventral root (VR) stimulus trains engage common postsynaptic components to activate the central pattern generator (CPG) for locomotion in the neonatal mouse spinal cord. VR stimulation did not activate the first order interneurons mediating the activation of the locomotor CPG by sacrocaudal afferent stimulation. Simultaneous stimulation of adjacent dorsal or ventral root pairs, subthreshold for evoking locomotor-like activity, did not summate to activate the CPG. This suggests that locomotor-like activity is triggered when a critical class of efferent or afferent axons is stimulated and does not depend on the number of stimulated axons or activated postsynaptic neurons. DR- and VR-evoked episodes exhibited differences in the coupling between VR pairs. In DR-evoked episodes, the coupling between the ipsilateral and contralateral flexor/extensor roots was similar and stronger than the bilateral extensor roots. In VR-evoked episodes, ipsilateral flexor/extensor coupling was stronger than both the contralateral flexor/extensor and the bilateral extensor coupling. For both types of stimulation, the coupling was greatest between the bilateral L1/L2 flexor-dominated roots. This indicates that the recruitment and/or the firing pattern of motoneurons differed in DR and VR-evoked episodes. However, the DR and VR trains do not appear to activate distinct CPGs because trains of DR and VR stimuli at frequencies too low to evoke locomotor-like activity did so when they were interleaved. These results indicate that the excitatory actions of VR stimulation converge onto the CPG through an unknown pathway that is not captured by current models of the locomotor CPG.

Preservation of VGLUT1 synapses on ventral calbindin-immunoreactive interneurons and normal locomotor function in a mouse model of spinal muscular atrophy.

Dysfunction in sensorimotor synapses is one of the earliest pathological changes observed in a mouse model [spinal muscular atrophy (SMA)Δ7] of spinal muscular atrophy. Here, we examined the density of proprioceptive and cholinergic synapses on calbindin-immunoreactive interneurons ventral to the lateral motor column. This population includes inhibitory Renshaw interneurons that are known to receive synaptic input from muscle spindle afferents and from motoneurons. At postnatal day (P)13, near the end stage of the disease, the somatic area of calbindin(+) neurons in the L1/L2 and L5/L6 segments was reduced in SMAΔ7 mice compared with controls. In addition, the number and density of terminals expressing the glutamate vesicular transporter (VGLUT1) and the vesicular acetylcholine transporter (VAChT) were increased on calbindin(+) cells in the L1-L2 but not in the L5-L6 segments of SMAΔ7 mice. In addition, the isolated spinal cord of SMA mice was able to generate locomotor-like activity at P4-P6 in the presence of a drug cocktail or in response to dorsal root stimulation. These results argue against a generalized loss of proprioceptive input to spinal circuits in SMA and suggest that the loss of proprioceptive synapses on motoneurons may be secondary to motoneuron pathology. The increased number of VGLUT1(+) and VAChT(+) synapses on calbindin(+) neurons in the L1/L2 segments may be the result of homeostatic mechanisms. Finally, we have shown that abnormal locomotor network function is unlikely to account for the motor deficits observed in SMA mice at P4-6.

Retrograde loading of nerves, tracts, and spinal roots with fluorescent dyes.

Retrograde labeling of neurons is a standard anatomical method(1,2) that has also been used to load calcium and voltage-sensitive dyes into neurons(3-6). Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and low-cost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.

Early functional impairment of sensory-motor connectivity in a mouse model of spinal muscular atrophy.

To define alterations of neuronal connectivity that occur during motor neuron degeneration, we characterized the function and structure of spinal circuitry in spinal muscular atrophy (SMA) model mice. SMA motor neurons show reduced proprioceptive reflexes that correlate with decreased number and function of synapses on motor neuron somata and proximal dendrites. These abnormalities occur at an early stage of disease in motor neurons innervating proximal hindlimb muscles and medial motor neurons innervating axial muscles, but only at end-stage disease in motor neurons innervating distal hindlimb muscles. Motor neuron loss follows afferent synapse loss with the same temporal and topographical pattern. Trichostatin A, which improves motor behavior and survival of SMA mice, partially restores spinal reflexes, illustrating the reversibility of these synaptic defects. Deafferentation of motor neurons is an early event in SMA and may be a primary cause of motor dysfunction that is amenable to therapeutic intervention.

Long and short multifunicular projections of sacral neurons are activated by sensory input to produce locomotor activity in the absence of supraspinal control.

Afferent input from load and joint receptors has been shown to reactivate the central pattern generators for locomotion (CPGs) in spinal cord injury patients and thereby improve their motor function and mobility. Elucidation of the pathways interposed between the afferents and CPGs is critical for the determination of the capacity of sensory input to activate the CPGs when the continuity of the white matter tracts is impaired following spinal cord injury. Using electrophysiological recordings, confocal imaging studies of spinal neurons and surgical manipulations of the white matter, we show that the capacity of sacrocaudal afferent (SCA) input to produce locomotor activity in isolated rat spinal cords depends not only on long ascending pathways, but also on recruitment of sacral proprioneurons interposed between the second order neurons and the hindlimb CPGs. We argue that large heterogeneous populations of second-order and proprioneurons whose crossed and uncrossed axons project rostrally through the ventral, ventrolateral/lateral and dorsolateral white matter funiculi contribute to the generation of the rhythm by the stimulated sacrocaudal input. The complex organization and multiple projection patterns of these populations enable the sacrocaudal afferent input to activate the CPGs even if the white matter pathways are severely damaged. Further studies are required to clarify the mechanisms involved in SCA-induced locomotor activity and assess its potential use for the rescue of lost motor functions after spinal cord injury.