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.

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.

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.

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.

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.

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 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.

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.

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.

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.

Regulation of degenerative spheroids after injury.

Neuronal injury leads to rapid, programmed disintegration of axons distal to the site of lesion. Much like other forms of axon degeneration (e.g. developmental pruning, toxic insult from neurodegenerative disorder), Wallerian degeneration associated with injury is preceded by spheroid formation along axons. The mechanisms by which injury leads to formation of spheroids and whether these spheroids have a functional role in degeneration remain elusive. Here, using neonatal mouse primary sympathetic neurons, we investigate the roles of players previously implicated in the progression of Wallerian degeneration in injury-induced spheroid formation. We find that intra-axonal calcium flux is accompanied by actin-Rho dependent growth of calcium rich axonal spheroids that eventually rupture, releasing material to the extracellular space prior to catastrophic axon degeneration. Importantly, after injury, Sarm1-/- and DR6-/-, but not Wlds (excess NAD+) neurons, are capable of forming spheroids that eventually rupture, releasing their contents to the extracellular space to promote degeneration. Supplementation of exogenous NAD+ or expressing WLDs suppresses Rho-dependent spheroid formation and degeneration in response to injury. Moreover, injured or trophically deprived Sarm1-/- and DR6-/-, but not Wlds neurons, are resistant to degeneration induced by conditioned media collected from wild-type axons after spheroid rupture. Taken together, these findings place Rho-actin and NAD+ upstream of spheroid formation and may suggest that other mediators of degeneration, such as DR6 and SARM1, mediate post-spheroid rupture events that lead to catastrophic axon disassembly.

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.

Collapsin Response Mediator Protein 4 (CRMP4) Facilitates Wallerian Degeneration and Axon Regeneration following Sciatic Nerve Injury.

In contrast to neurons in the CNS, damaged neurons from the peripheral nervous system (PNS) regenerate, but this process can be slow and imperfect. Successful regeneration is orchestrated by cytoskeletal reorganization at the tip of the proximal axon segment and cytoskeletal disassembly of the distal segment. Collapsin response mediator protein 4 (CRMP4) is a cytosolic phospho-protein that regulates the actin and microtubule cytoskeleton. During development, CRMP4 promotes growth cone formation and dendrite development. Paradoxically, in the adult CNS, CRMP4 impedes axon regeneration. Here, we investigated the involvement of CRMP4 in peripheral nerve injury in male and female Crmp4-/- mice following sciatic nerve injury. We find that sensory axon regeneration and Wallerian degeneration are impaired in Crmp4-/- mice following sciatic nerve injury. In vitro analysis of dissociated dorsal root ganglion (DRG) neurons from Crmp4-/- mice revealed that CRMP4 functions in the proximal axon segment to promote the regrowth of severed DRG neurons and in the distal axon segment where it facilitates Wallerian degeneration through calpain-dependent formation of harmful CRMP4 fragments. These findings reveal an interesting dual role for CRMP4 in proximal and distal axon segments of injured sensory neurons that coordinately facilitate PNS axon regeneration.

The p75 neurotrophin receptor in AgRP neurons is necessary for homeostatic feeding and food anticipation.

Networks of neurons control feeding and activity patterns by integrating internal metabolic signals of energy balance with external environmental cues such as time-of-day. Proper circadian alignment of feeding behavior is necessary to prevent metabolic disease, and thus it is imperative that molecular players that maintain neuronal coordination of energy homeostasis are identified. Here, we demonstrate that mice lacking the p75 neurotrophin receptor, p75NTR, decrease their feeding and food anticipatory behavior (FAA) in response to daytime, but not nighttime, restricted feeding. These effects lead to increased weight loss, but do not require p75NTR during development. Instead, p75NTR is required for fasting-induced activation of neurons within the arcuate hypothalamus. Indeed, p75NTR specifically in AgRP neurons is required for FAA in response to daytime restricted feeding. These findings establish p75NTR as a novel regulator gating behavioral response to food scarcity and time-of-day dependence of circadian food anticipation.

In many animals, specific types of neurons, such as the hypothalamic hunger neurons, are tasked with regulating food consumption, integrating internal signals of hunger. In general, individuals eat if food becomes available when they are hungry; if food is absent, they will start moving to find new resources. Finally, if food always comes at the same time, animals will increase their activity just before it is delivered. Neurotrophins are a family of proteins that have many essential roles in the brain. In recent years, they have been shown to interact with the circadian clock, the built-in mechanism that helps animals stay synchronized with the cycle of day and night. A protein known as p75NTR is present in nerve cells, including hypothalamic hunger neurons: there, it helps to relay messages from a neurotrophin which, amongst other roles, controls food intake. However, it was unclear whether p75NTR played a role in regulating feeding behaviors, especially in a circadian manner. To investigate this question, Podyma et al. genetically engineered a group of mice lacking p75NTR, and a group missing the protein only in their hypothalamic hunger neurons. Both types of mutants had abnormal control of their feeding behavior: compared to normal mice, they fed less (and lost more weight) after they had been deprived of food overnight, or when they faced food shortage over multiple days. In addition, the mutants failed to move more before being fed. However, these feeding patterns were only affected during daytime, while they were preserved at night. These results reveal a new role for p75NTR in hypothalamic hunger neurons. Dissecting the biological processes that control food intake is key since obesity levels are increasing around the world. In particular, the relationship between food intake and the circadian clock is an important avenue of research as time-restricted diets (where food intake is only allowed during specific periods of the day) are growing in popularity.

Dopamine Signaling in the Suprachiasmatic Nucleus Enables Weight Gain Associated with Hedonic Feeding.

The widespread availability of energy-dense, rewarding foods is correlated with the increased incidence of obesity across the globe. Overeating during mealtimes and unscheduled snacking disrupts timed metabolic processes, which further contribute to weight gain. The neuronal mechanism by which the consumption of energy-dense food restructures the timing of feeding is poorly understood. Here, we demonstrate that dopaminergic signaling within the suprachiasmatic nucleus (SCN), the central circadian pacemaker, disrupts the timing of feeding, resulting in overconsumption of food. D1 dopamine receptor (Drd1)-null mice are resistant to diet-induced obesity, metabolic disease, and circadian disruption associated with energy-dense diets. Conversely, genetic rescue of Drd1 expression within the SCN restores diet-induced overconsumption, weight gain, and obesogenic symptoms. Access to rewarding food increases SCN dopamine turnover, and elevated Drd1-signaling decreases SCN neuronal activity, which we posit disinhibits downstream orexigenic responses. These findings define a connection between the reward and circadian pathways in the regulation of pathological calorie consumption.