Sexual dimorphism in the dorsal root ganglia of neonatal mice identified by protein expression profiling with single-cell mass cytometry.

Development of neuronal and glial populations in the dorsal root ganglia (DRG) is required for detection of touch, body position, temperature, and noxious stimuli. While female-male differences in somatosensory perception have been previously reported, no study has examined global sex differences in the abundance of DRG cell types, and the developmental origin of these differences has not been characterized. To investigate whether sex-specific differences in neuronal and glial cell types arise in the DRG during development, we performed single-cell mass cytometry analysis on sex-separated DRGs from 4 separate litter replicates of postnatal day 0 (P0) C57/BL6 mouse pups. In this analysis, we observed that females had a higher abundance of total neurons (p = 0.0266), as well as an increased abundance of TrkB+ (p = 0.031) and TrkC+ (p = 0.04) neurons for mechanoreception and proprioception, while males had a higher abundance of TrkA+ (p = 0.025) neurons for thermoreception and nociception. Pseudotime comparison of the female and male datasets indicates that male neurons are more mature and differentiated than female neurons at P0. These findings warrant further studies to determine whether these differences are maintained across development, and their impact on somatosensory perception.

Global and Regional Damages in Retinal Ganglion Cell Axon Bundles Monitored Non-Invasively by Visible-Light Optical Coherence Tomography Fibergraphy.

Retinal ganglion cells (RGCs) exhibit compartmentalized organization, receiving synaptic inputs through their dendrites and transmitting visual information from the retina to the brain through the optic nerve. Little is known about the structure of RGC axon bundles extending from individual RGC somas to the optic nerve head (ONH) and how they respond to disease insults. We recently introduced visible-light optical coherence tomography fibergraphy (vis-OCTF), a technique for directly visualizing and analyzing mouse RGC axon bundles in vivo In this study, we validated vis-OCTF's ability to quantify RGC axon bundles with an increased number of RGCs using mice deficient in BCL2-associated X protein (BAX-/-). Next, we performed optic nerve crush (ONC) injury on wild-type (WT) mice and showed that the changes in RGC axon bundle width and thickness were location-dependent. Our work demonstrates the potential of vis-OCTF to longitudinally quantify and track RGC damage at single axon bundle level in optic neuropathies.SIGNIFICANCE STATEMENT Nearly all clinical and preclinical studies measure the retinal nerve fiber (RNFL) thickness as the sole indicator of retinal ganglion cell (RGC) damage without investigating RGC axon bundles directly. We demonstrated visible-light optical coherence tomography fibergraphy (vis-OCTF) to directly quantify global and regional RGC axon bundle organizations in vivo as a new biomarker for RGC health. We validated in vivo vis-OCTF measures using both confocal microscopy of the immunostained flat-mounted retina and numerical simulations. Vis-OCTF for monitoring RGC axon bundle organization has the potential to bring new insight into RGC damage in optic neuropathies.

Axonal spheroids in neurodegeneration.

Axonal spheroids are bubble-like biological features that form on most degenerating axons, yet little is known about their influence on degenerative processes. Their formation and growth has been observed in response to various degenerative triggers such as injury, oxidative stress, inflammatory factors, and neurotoxic molecules. They often contain cytoskeletal elements and organelles, and, depending on the pathological insult, can colocalize with disease-related proteins such as amyloid precursor protein (APP), ubiquitin, and motor proteins. Initial formation of axonal spheroids depends on the disruption of axonal and membrane tension governed by cytoskeleton structure and calcium levels. Shortly after spheroid formation, the engulfment signal phosphatidylserine (PS) is exposed on the outer leaflet of spheroid plasma membrane, suggesting an important role for axonal spheroids in phagocytosis and debris clearance during degeneration. Spheroids can grow until they rupture, allowing pro-degenerative factors to exit the axon into extracellular space and accelerating neurodegeneration. Though much remains to be discovered in this area, axonal spheroid research promises to lend insight into the etiologies of neurodegenerative disease, and may be an important target for therapeutic intervention. This review summarizes over 100 years of work, describing what is known about axonal spheroid structure, regulation and function.

p75NTR and DR6 Regulate Distinct Phases of Axon Degeneration Demarcated by Spheroid Rupture.

The regressive events associated with trophic deprivation are critical for sculpting a functional nervous system. After nerve growth factor withdrawal, sympathetic axons derived from male and female neonatal mice maintain their structural integrity for ∼18 h (latent phase) followed by a rapid and near unison disassembly of axons over the next 3 h (catastrophic phase). Here we examine the molecular basis by which axons transition from latent to catastrophic phases of degeneration following trophic withdrawal. Before catastrophic degeneration, we observed an increase in intra-axonal calcium. This calcium flux is accompanied by p75 neurotrophic factor receptor-Rho-actin-dependent expansion of calcium-rich axonal spheroids that eventually rupture, releasing their contents to the extracellular space. Conditioned media derived from degenerating axons are capable of hastening transition into the catastrophic phase of degeneration. We also found that death receptor 6, but not p75 neurotrophic factor receptor, is required for transition into the catastrophic phase in response to conditioned media but not for the intra-axonal calcium flux, spheroid formation, or rupture that occur toward the end of latency. Our results support the existence of an interaxonal degenerative signal that promotes catastrophic degeneration among trophically deprived axons.SIGNIFICANCE STATEMENT Developmental pruning shares several morphological similarities to both disease- and injury-induced degeneration, including spheroid formation. The function and underlying mechanisms governing axonal spheroid formation, however, remain unclear. In this study, we report that axons coordinate each other's degeneration during development via axonal spheroid rupture. Before irreversible breakdown of the axon in response to trophic withdrawal, p75 neurotrophic factor receptor-RhoA signaling governs the formation and growth of spheroids. These spheroids then rupture, allowing exchange of contents ≤10 kDa between the intracellular and extracellular space to drive death receptor 6 and calpain-dependent catastrophic degeneration. This finding informs not only our understanding of regressive events during development but may also provide a rationale for designing new treatments toward myriad neurodegenerative disorders.

TNFa/TNFR2 signaling is required for glial ensheathment at the dorsal root entry zone.

Somatosensory information from the periphery is routed to the spinal cord through centrally-projecting sensory axons that cross into the central nervous system (CNS) via the dorsal root entry zone (DREZ). The glial cells that ensheath these axons ensure rapid propagation of this information. Despite the importance of this glial-axon arrangement, how this afferent nerve is assembled during development is unknown. Using in vivo, time-lapse imaging we show that as centrally-projecting pioneer axons from dorsal root ganglia (DRG) enter the spinal cord, they initiate expression of the cytokine TNFalpha. This induction coincides with ensheathment of these axons by associated glia via a TNF receptor 2 (TNFR2)-mediated process. This work identifies a signaling cascade that mediates peripheral glial-axon interactions and it functions to ensure that DRG afferent projections are ensheathed after pioneer axons complete their navigation, which promotes efficient somatosensory neural function.

Replacing C189 in the bZIP domain of Zta with S, T, V, or A changes DNA binding specificity to four types of double-stranded DNA.

Zta is a bZIP transcription factor (TF) in the Epstein-Barr virus that binds unmethylated and methylated DNA sequences. Substitution of cysteine 189 of Zta to serine (Zta(C189S)) results in a virus that is unable to execute the lytic cycle, which was attributed to a change in binding to methylated DNA sequences. To learn more about the role of this position in defining sequence-specific DNA binding, we mutated cysteine 189 to four other amino acids, producing Zta(C189S), Zta(C189T), Zta(C189A), and Zta(C189V) mutants. Zta and mutants were used in protein binding microarray (PBM) experiments to evaluate sequence-specific DNA binding to four types of double-stranded DNA (dsDNA): 1) with cytosine in both strands (DNA(C|C)), 2) with 5-methylcytosine (5mC) in one strand and cytosine in the second strand (DNA(5mC|C)), 3) with 5-hydroxymethylcytosine (5hmC) in one strand and cytosine in the second strand (DNA(5hmC|C)), and 4) with both cytosines in all CG dinucleotides containing 5mC (DNA(5mCG)). Zta(C189S) and Zta(C189T) bound the TRE (AP-1) motif (TGAG/CTCA) more strongly than wild-type Zta, while binding to other sequences, including the C/EBP half site GCAA was reduced. Binding of Zta(C189S) and Zta(C189T) to DNA containing modified cytosines (DNA(5mC|C), DNA(5hmC|C), and DNA(5mCG)) was reduced compared to Zta. Zta(C189A) and Zta(C189V) had higher non-specific binding to all four types of DNA. Our data suggests that position C189 in Zta impacts sequence-specific binding to DNA containing modified and unmodified cytosine.

Delineating neurotrophin-3 dependent signaling pathways underlying sympathetic axon growth along intermediate targets.

Postganglionic sympathetic neurons detect vascular derived neurotrophin 3 (NT3) via the axonally expressed receptor tyrosine kinase, TrkA, to promote chemo-attraction along intermediate targets. Once axons arrive to their final target, a structurally related neurotrophic factor, nerve growth factor (NGF), also acts through TrkA to promote final target innervation. Does TrkA signal differently at these different locales? We previously found that Coronin-1 is upregulated in sympathetic neurons upon exposure to NGF, thereby endowing the NGF-TrkA complex with new signaling capabilities (i.e. calcium signaling), which dampens axon growth and branching. Based on the notion that axons do not express functional levels of Coronin-1 prior to final target innervation, we developed an in vitro model for axon growth and branching along intermediate targets using Coro1a-/- neurons grown in NT3. We found that, similar to NGF-TrkA, NT3-TrkA is capable of inducing MAPK and PI3K in the presence or absence of Coronin-1. However, unlike NGF, NT3 does not induce calcium release from intracellular stores. Using a combination of pharmacology, knockout neurons and in vitro functional assays, we suggest that the NT3-TrkA complex uses Ras/MAPK and/or PI3K-AKT signaling to induce axon growth and inhibit axon branching along intermediate targets. However, in the presence of Coronin-1, these signaling pathways lose their ability to impact NT3 dependent axon growth or branching. This is consistent with a role for Coronin-1 as a molecular switch for axon behavior and suggests that Coronin-1 suppresses NT3 dependent axon behavior.

Coronin-1 and calcium signaling governs sympathetic final target innervation.

Development of a functional peripheral nervous system requires axons to rapidly innervate and arborize into final target organs and then slow but not halt their growth to establish stable connections while keeping pace with organ growth. Here we examine the role of the NGF-TrkA effector protein, Coronin-1, on postganglionic sympathetic neuron final target innervation. In the absence of Coronin-1 we find that NGF-TrkA-PI3K signaling drives robust axon growth and branching in part by suppressing GSK3ß. In contrast, the presence of Coronin-1 (wild-type neurons) suppresses but does not halt NGF-TrkA-dependent growth and branching. This relative suppression in axon growth behaviors is due to Coronin-1-dependent calcium release via PLC-γ1 signaling, which releases PI3K-dependent suppression of GSK3ß. Finally, we demonstrate that Coro1a(-/-) mice display sympathetic axon overgrowth and overbranching phenotypes in the developing heart. Together with previous work demonstrating the Coronin-1 expression is NGF dependent, this work suggests that periods before and after NGF-TrkA-induced Coronin-1 expression (and likely other factors) defines two distinct axon growth states, which are critical for proper circuit formation in the sympathetic nervous system.

Emerging Roles of Neuronal Extracellular Vesicles at the Synapse.

Extracellular vesicles (EVs) are secreted from most, if not all, cell types and are implicated in short- and long-distance signaling throughout the body. EVs are also secreted from neurons and represent an emergent neuronal communication platform. Understanding the functional implications of EV signaling to recipient neurons and glia requires understanding the cell biology involved in EV biogenesis, cargo loading, secretion, uptake, and signal transduction in the recipient cell. Here we review these major questions of EV biology while highlighting recent new insights and examples within the nervous system, such as modulating synaptic function or morphogenesis in recipient neurons.

Trk activation of the ERK1/2 kinase pathway stimulates intermediate chain phosphorylation and recruits cytoplasmic dynein to signaling endosomes for retrograde axonal transport.

The retrograde transport of Trk-containing endosomes from the axon to the cell body by cytoplasmic dynein is necessary for axonal and neuronal survival. We investigated the recruitment of dynein to signaling endosomes in rat embryonic neurons and PC12 cells. We identified a novel phosphoserine on the dynein intermediate chains (ICs), and we observed a time-dependent neurotrophin-stimulated increase in intermediate chain phosphorylation on this site in both cell types. Pharmacological studies, overexpression of constitutively active MAP kinase kinase, and an in vitro assay with recombinant proteins demonstrated that the intermediate chains are phosphorylated by the MAP kinase ERK1/2, extracellular signal-regulated kinase, a major downstream effector of Trk. Live cell imaging with fluorescently tagged IC mutants demonstrated that the dephosphomimic mutants had significantly reduced colocalization with Trk and Rab7, but not a mitochondrial marker. The phosphorylated intermediate chains were enriched on immunoaffinity-purified Trk-containing organelles. Inhibition of ERK reduced the amount of phospho-IC and the total amount of dynein that copurified with the signaling endosomes. In addition, inhibition of ERK1/2 reduced the motility of Rab7- and TrkB-containing endosomes and the extent of their colocalization with dynein in axons. NGF-dependent survival of sympathetic neurons was significantly reduced by the overexpression of the dephosphomimic mutant IC-1B-S80A, but not WT IC-1B, further demonstrating the functional significance of phosphorylation on this site. These results demonstrate that neurotrophin binding to Trk initiates the recruitment of cytoplasmic dynein to signaling endosomes through ERK1/2 phosphorylation of intermediate chains for their subsequent retrograde transport in axons.

The good, the bald, and the hairy: A mechanosensor meets its fate at the target.

In this issue of Developmental Cell, Koutsioumpa et al. (2023) investigate the maturation of low-threshold mechanoreceptor nerve endings in both hairy and glabrous skin types and discover a critical role for target-derived BMP in the development of Meissner corpuscles in glabrous (i.e., hairless) skin.

Sympathetic neurons secrete retrogradely transported TrkA on extracellular vesicles.

Proper wiring of the peripheral nervous system relies on neurotrophic signaling via nerve growth factor (NGF). NGF secreted by target organs (i.e. eye) binds to the TrkA receptor expressed on the distal axons of postganglionic neurons. Upon binding, TrkA is internalized into a signaling endosome and retrogradely trafficked back to the soma and into the dendrites to promote cell survival and postsynaptic maturation, respectively. Much progress has been made in recent years to define the fate of the retrogradely trafficked TrkA signaling endosome, yet it has not been fully characterized. Here we investigate extracellular vesicles (EVs) as a novel route of neurotrophic signaling. Using the mouse superior cervical ganglion (SCG) as a model, we isolate EVs derived from sympathetic cultures and characterize them using immunoblot assays, nanoparticle tracking analysis, and cryo-electron microscopy. Furthermore, using a compartmentalized culture system, we find that TrkA derived from endosomes originating in the distal axon can be detected on EVs secreted from the somatodendritic domain. In addition, inhibition of classic TrkA downstream pathways, specifically in somatodendritic compartments, greatly decreases TrkA packaging into EVs. Our results suggest a novel trafficking route for TrkA: it can travel long distances to the cell body, be packaged into EVs, and be secreted. Secretion of TrkA via EVs appears to be regulated by its own downstream effector cascades, raising intriguing future questions about novel functionalities associated with TrkA+ EVs.

Role of the caspase-8/RIPK3 axis in Alzheimer's disease pathogenesis and Aß-induced NLRP3 inflammasome activation.

The molecular mediators of cell death and inflammation in Alzheimer's disease (AD) have yet to be fully elucidated. Caspase-8 is a critical regulator of several cell death and inflammatory pathways; however, its role in AD pathogenesis has not yet been examined in detail. In the absence of caspase-8, mice are embryonic lethal due to excessive receptor interacting protein kinase 3-dependent (RIPK3-dependent) necroptosis. Compound RIPK3 and caspase-8 mutants rescue embryonic lethality, which we leveraged to examine the roles of these pathways in an amyloid ß-mediated (Aß-mediated) mouse model of AD. We found that combined deletion of caspase-8 and RIPK3, but not RIPK3 alone, led to diminished Aß deposition and microgliosis in the mouse model of AD carrying human presenilin 1 and amyloid precursor protein with 5 familial AD mutations (5xFAD). Despite its well-known role in cell death, caspase-8 did not appear to affect cell loss in the 5xFAD model. In contrast, we found that caspase-8 was a critical regulator of Aß-driven inflammasome gene expression and IL-1ß release. Interestingly, loss of RIPK3 had only a modest effect on disease progression, suggesting that inhibition of necroptosis or RIPK3-mediated cytokine pathways is not critical during midstages of Aß amyloidosis. These findings suggest that therapeutics targeting caspase-8 may represent a novel strategy to limit Aß amyloidosis and neuroinflammation in AD.

A leptin-responsive hypothalamic circuit inputs to the circadian feeding network.

Salient cues, such as the rising sun or the availability of food, play a crucial role in entraining biological clocks, allowing for effective behavioral adaptation and ultimately, survival. While the light-dependent entrainment of the central circadian pacemaker (suprachiasmatic nucleus, SCN) is relatively well defined, the molecular and neural mechanisms underlying entrainment associated with food availability remains elusive. Using single nucleus RNA sequencing during scheduled feeding (SF), we identified a leptin receptor (LepR) expressing neuron population in the dorsomedial hypothalamus (DMH) that upregulates circadian entrainment genes and exhibits rhythmic calcium activity prior to an anticipated meal. We found that disrupting DMHLepR neuron activity had a profound impact on both molecular and behavioral food entrainment. Specifically, silencing DMHLepR neurons, mis-timed exogenous leptin administration, or mis-timed chemogenetic stimulation of these neurons all interfered with the development of food entrainment. In a state of energy abundance, repetitive activation of DMHLepR neurons led to the partitioning of a secondary bout of circadian locomotor activity that was in phase with the stimulation and dependent on an intact SCN. Lastly, we discovered that a subpopulation of DMHLepR neurons project to the SCN with the capacity to influence the phase of the circadian clock. This leptin regulated circuit serves as a point of integration between the metabolic and circadian systems, facilitating the anticipation of meal times.

Leptin receptor neurons in the dorsomedial hypothalamus input to the circadian feeding network.

Salient cues, such as the rising sun or availability of food, entrain biological clocks for behavioral adaptation. The mechanisms underlying entrainment to food availability remain elusive. Using single-nucleus RNA sequencing during scheduled feeding, we identified a dorsomedial hypothalamus leptin receptor-expressing (DMHLepR) neuron population that up-regulates circadian entrainment genes and exhibits calcium activity before an anticipated meal. Exogenous leptin, silencing, or chemogenetic stimulation of DMHLepR neurons disrupts the development of molecular and behavioral food entrainment. Repetitive DMHLepR neuron activation leads to the partitioning of a secondary bout of circadian locomotor activity that is in phase with the stimulation and dependent on an intact suprachiasmatic nucleus (SCN). Last, we found a DMHLepR neuron subpopulation that projects to the SCN with the capacity to influence the phase of the circadian clock. This direct DMHLepR-SCN connection is well situated to integrate the metabolic and circadian systems, facilitating mealtime anticipation.

A developmental atlas of somatosensory diversification and maturation in the dorsal root ganglia by single-cell mass cytometry.

Precisely controlled development of the somatosensory system is essential for detecting pain, itch, temperature, mechanical touch and body position. To investigate the protein-level changes that occur during somatosensory development, we performed single-cell mass cytometry on dorsal root ganglia from C57/BL6 mice of both sexes, with litter replicates collected daily from embryonic day 11.5 to postnatal day 4. Measuring nearly 3 million cells, we quantified 30 molecularly distinct somatosensory glial and 41 distinct neuronal states across all timepoints. Analysis of differentiation trajectories revealed rare cells that co-express two or more Trk receptors and over-express stem cell markers, suggesting that these neurotrophic factor receptors play a role in cell fate specification. Comparison to previous RNA-based studies identified substantial differences between many protein-mRNA pairs, demonstrating the importance of protein-level measurements to identify functional cell states. Overall, this study demonstrates that mass cytometry is a high-throughput, scalable platform to rapidly phenotype somatosensory tissues.

Metabolic homeostasis via BDNF and its receptors.

Metabolic disorders result from dysregulation of central nervous system and peripheral metabolic energy homeostatic pathways. To maintain normal energy balance, neural circuits must integrate feedforward and feedback signals from the internal metabolic environment to orchestrate proper food intake and energy expenditure. These signals include conserved meal and adipocyte cues such as glucose and leptin, respectively, in addition to more novel players including brain-derived neurotrophic factor (BDNF). In particular, BDNF's two receptors, tropomyosin related kinase B (TrkB) and p75 neurotrophin receptor (p75NTR), are increasingly appreciated to be involved in whole body energy homeostasis. At times, these two receptors even seem to functionally oppose one another's actions, providing the framework for a potential neurotrophin mediated energy regulatory axis, which we explore further here.

Long-distance regressive signaling in neural development and disease.

Nervous system development proceeds via well-orchestrated processes involving a balance between progressive and regressive events including stabilization or elimination of axons, synapses, and even entire neurons. These progressive and regressive events are driven by functionally antagonistic signaling pathways with the dominant pathway eventually determining whether a neural element is retained or removed. Many of these developmental sculpting events are triggered by final target innervation necessitating a long-distance mode of communication. While long-distance progressive signaling has been well characterized, particularly for neurotrophic factors, there remains relatively little known about how regressive events are triggered from a distance. Here we discuss the emergent phenomenon of long-distance regressive signaling pathways. In particular, we will cover (a) progressive and regressive cues known to be employed after target innervation, (b) the mechanisms of long-distance signaling from an endosomal platform, (c) recent evidence that long-distance regressive cues emanate from platforms like death receptors or repulsive axon guidance receptors, and (d) evidence that these pathways are exploited in pathological scenarios. This article is categorized under: Nervous System Development > Vertebrates: General Principles Signaling Pathways > Global Signaling Mechanisms Establishment of Spatial and Temporal Patterns > Cytoplasmic Localization.

A Microfluidic Culture Platform to Assess Axon Degeneration.

The field of microfluidics allows for the precise spatial manipulation of small amounts of fluids. Within microstructures, laminar flow of fluids can be exploited to control the diffusion of small molecules, creating desired microenvironments for cells. Cellular neuroscience has benefited greatly from devices designed to fluidically isolate cell bodies and axons. Microfluidic devices specialized for neuron compartmentalization are made of polydimethylsiloxane (PDMS) which is gas permeable, is compatible with fluorescence microscopy, and has low cost. These devices are commonly used to study signals initiated exclusively on axons, somatodendritic compartments, or even single synapses. We have also found that microfluidic devices allow for rapid, reproducible interrogation of axon degeneration. Here, we describe the methodology for assessing axonal degeneration in microfluidic devices. We describe several use cases, including enucleation (removal of cell bodies) and trophic deprivation to investigate axon degeneration in pathological and developmental scenarios, respectively.