[Introduction to Myelin Research].

Myelin is a multilamellar membrane structure formed by oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). It has been recognized as an insulator that is essential for the rapid and efficient propagation of action potentials by saltatory conduction. However, recently many studies have shown that myelin and myelin-forming cells interact with axons and regulate the nervous system far more actively than previously thought. For example, myelination changes axons dynamically and divides them into four distinct functional domains: node of Ranvier, paranode, juxtaparanode, and internode. Voltage-gated Na+ channels are clustered at the node, while K+ channels are at the juxtaparanode, and segregation of these channels by paranodal axoglial junction is necessary for proper axonal function. My research experience began at the neurology ward of the Niigata University Medical Hospital, where I saw a patient with peripheral neuropathy of unknown etiology more than 37 years ago. In the patient's serum, we found an autoantibody against a glycolipid enriched in the PNS. Since then, I have been interested in myelin because of its beautiful structure and unique roles in the nervous system. In this review, our recent studies related to CNS and PNS myelin are presented.

Robo2 Drives Target-Selective Peripheral Nerve Regeneration in Response to Glia-Derived Signals.

Peripheral nerves are divided into multiple branches leading to divergent synaptic targets. This poses a remarkable challenge for regenerating axons as they select their original trajectory at nerve branch-points. Despite implications for functional regeneration, the molecular mechanisms underlying target selectivity are not well characterized. Danio Rerio (zebrafish) motor nerves are composed of a ventral and a dorsal branch that diverge at a choice-point, and we have previously shown that regenerating axons faithfully select their original branch and targets. Here we identify robo2 as a key regulator of target-selective regeneration (sex of experimental subjects unknown). We demonstrate that robo2 function in regenerating axons is required and sufficient to drive target-selective regeneration, and that robo2 acts in response to glia located precisely where regenerating axons select the branch-specific trajectory to prevent and correct axonal errors. Combined, our results reveal a glia-derived mechanism that acts locally via axonal robo2 to promote target-selective regeneration.SIGNIFICANCE STATEMENT Despite its relevance for functional recovery, the molecular mechanisms that direct regenerating peripheral nerve axons toward their original targets are not well defined. Zebrafish spinal motor nerves are composed of a dorsal and a ventral branch that diverge at a stereotyped nerve branch-point, providing a unique opportunity to decipher the molecular mechanisms critical for target-selective regeneration. Using a combination of live cell imaging and molecular-genetic manipulations, we demonstrate that the robo2 guidance receptor is necessary and sufficient to promote target-selective regeneration. Moreover, we demonstrate that robo2 is part of a genetic pathway that generates transient, spatially restricted, and tightly coordinated signaling events that direct axons of the dorsal nerve branch toward their original, pre-injury targets.

Disturbance of phylogenetic layer-specific adaptation of human brain gene expression in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder with typical neuropathological hallmarks, such as neuritic plaques and neurofibrillary tangles, preferentially found at layers III and V. The distribution of both hallmarks provides the basis for the staging of AD, following a hierarchical pattern throughout the cerebral cortex. To unravel the background of this layer-specific vulnerability, we evaluated differential gene expression of supragranular and infragranular layers and subcortical white matter in both healthy controls and AD patients. We identified AD-associated layer-specific differences involving protein-coding and non-coding sequences, most of those present in the subcortical white matter, thus indicating a critical role for long axons and oligodendrocytes in AD pathomechanism. In addition, GO analysis identified networks containing synaptic vesicle transport, vesicle exocytosis and regulation of neurotransmitter levels. Numerous AD-associated layer-specifically expressed genes were previously reported to undergo layer-specific switches in recent hominid brain evolution between layers V and III, i.e., those layers that are most vulnerable to AD pathology. Against the background of our previous finding of accelerated evolution of AD-specific gene expression, here we suggest a critical role in AD pathomechanism for this phylogenetic layer-specific adaptation of gene expression, which is most prominently seen in the white matter compartment.

Direction selectivity in retinal bipolar cell axon terminals.

The ability to encode the direction of image motion is fundamental to our sense of vision. Direction selectivity along the four cardinal directions is thought to originate in direction-selective ganglion cells (DSGCs) because of directionally tuned GABAergic suppression by starburst cells. Here, by utilizing two-photon glutamate imaging to measure synaptic release, we reveal that direction selectivity along all four directions arises earlier than expected at bipolar cell outputs. Individual bipolar cells contained four distinct populations of axon terminal boutons with different preferred directions. We further show that this bouton-specific tuning relies on cholinergic excitation from starburst cells and GABAergic inhibition from wide-field amacrine cells. DSGCs received both tuned directionally aligned inputs and untuned inputs from among heterogeneously tuned glutamatergic bouton populations. Thus, directional tuning in the excitatory visual pathway is incrementally refined at the bipolar cell axon terminals and their recipient DSGC dendrites by two different neurotransmitters co-released from starburst cells.

Axonal CB1 Receptors Mediate Inhibitory Bouton Formation via cAMP Increase and PKA.

Experience-dependent formation and removal of inhibitory synapses are essential throughout life. For instance, GABAergic synapses are removed to facilitate learning, and strong excitatory activity is accompanied by the formation of inhibitory synapses to maintain coordination between excitation and inhibition. We recently discovered that active dendrites trigger the growth of inhibitory synapses via CB1 receptor-mediated endocannabinoid signaling, but the underlying mechanism remained unclear. Using two-photon microscopy to monitor the formation of individual inhibitory boutons in hippocampal organotypic slices from mice (both sexes), we found that CB1 receptor activation mediated the formation of inhibitory boutons and promoted their subsequent stabilization. Inhibitory bouton formation did not require neuronal activity and was independent of Gi/o-protein signaling, but was directly induced by elevating cAMP levels using forskolin and by activating Gs-proteins using DREADDs. Blocking PKA activity prevented CB1 receptor-mediated inhibitory bouton formation. Our findings reveal that axonal CB1 receptors signal via unconventional downstream pathways and that inhibitory bouton formation is triggered by an increase in axonal cAMP levels. Our results demonstrate an unexpected role for axonal CB1 receptors in axon-specific, and context-dependent, inhibitory synapse formation.SIGNIFICANCE STATEMENT Coordination between excitation and inhibition is required for proper brain function throughout life. It was previously shown that new inhibitory synapses can be formed in response to strong excitation to maintain this coordination, and this was mediated by endocannabinoid signaling via CB1 receptors. As activation of CB1 receptors generally results in the suppression of synaptic transmission, it remained unclear how CB1 receptors can mediate the formation of inhibitory synapses. Here we show that CB1 receptors on inhibitory axons signal via unconventional intracellular pathways and that inhibitory bouton formation is triggered by an increase in axonal cAMP levels and requires PKA activity. Our findings point to a central role for axonal cAMP signaling in activity-dependent inhibitory synapse formation.

White matter demyelination predates axonal injury after ischemic stroke in cynomolgus monkeys.

Unraveling the pathology of stroke is a prerequisite for designing therapeutic strategies. It was reported that myelin injury exceeded axonal loss in the peri-infarct region of rodent white matter stroke. An in-depth investigation of the post-stroke white matter damage in higher-order species might innovate stroke intervention. In this study, adult male cynomolgus monkeys received surgical middle cerebral artery occlusion (MCAO), and serial magnetic resonance scans to non-invasively assess brain damage. Spontaneous movements were recorded to evaluate post-stroke behavior. The axon and myelin loss, as well as immune cell infiltration were examined using immunohistochemistry. Magnetic resonance imaging revealed cerebral infarcts and white matter injury after MCAO in monkeys, which were confirmed by neurological deficits. Immunostaining of white matter fibers showed substantial demyelination whilst retention of axons in the infarcts 8 days post MCAO, while a progressive loss of myelin and axons was observed after one month. Gliosis, microglia activation, and leukocyte infiltration were identified in the lesions. These results demonstrate that demyelination predates axonal injury in non-human primate ischemic stroke, which provides a time window for stroke intervention focusing on prevention of progressive axonal loss through myelin regeneration.

Mossy Cells in the Dorsal and Ventral Dentate Gyrus Differ in Their Patterns of Axonal Projections.

Mossy cells (MCs) of the dentate gyrus (DG) are a major group of excitatory hilar neurons that are important for regulating activity of dentate granule cells. MCs are particularly intriguing because of their extensive longitudinal connections within the DG. It has generally been assumed that MCs in the dorsal and ventral DG have similar patterns of termination in the inner one-third of the dentate molecular layer. Here, we demonstrate that axonal projections of MCs in these two regions are considerably different. MCs in dorsal and ventral regions were labeled selectively with Cre-dependent eYFP or mCherry, using two transgenic mouse lines (including both sexes) that express Cre-recombinase in MCs. At four to six weeks following unilateral labeling of MCs in the ventral DG, a dense band of fibers was present in the inner one-fourth of the molecular layer and extended bilaterally throughout the rostral-caudal extent of the DG, replicating the expected distribution of MC axons. In contrast, following labeling of MCs in the dorsal DG, the projections were more diffusely distributed. At the level of transfection, fibers were present in the inner molecular layer, but they progressively expanded into the middle molecular layer and, most ventrally, formed a distinct band in this region. Optical stimulation of these caudal fibers expressing ChR2 demonstrated robust EPSCs in ipsilateral granule cells and enhanced the effects of perforant path stimulation in the ventral DG. These findings suggest that MCs in the dorsal and ventral DG differ in the distribution of their axonal projections and possibly their function.SIGNIFICANCE STATEMENT Mossy cells (MCs), a major cell type in the hilus of the dentate gyrus (DG), are unique in providing extensive longitudinal and commissural projections throughout the DG. Although it has been assumed that all MCs have similar patterns of termination in the inner molecular layer of the DG, we discovered that the axonal projections of dorsal and ventral MCs differ. While ventral MC projections exhibit the classical pattern, with dense innervation in the inner molecular layer, dorsal MCs have a more diffuse distribution and expand into the middle molecular layer where they overlap and interact with innervation from the perforant path. These distinct locations and patterns of axonal projections suggest that dorsal and ventral MCs may have different functional roles.

Expression of the type 1 cannabinoid receptor (CB1R) in CCK-immunoreactive axon terminals in the basolateral amygdala of the rhesus monkey (Macaca mulatta).

Studies in rodents have shown that interactions between cholecystokinin (CCK) and the endogenous cannabinoid system in the basolateral nuclear complex of the amygdala (BNC) modulate anxiety-like behavior and fear learning/expression. One of the main cell types implicated is a CCK-immunoreactive (CCK+) basket cell that innervates the somata of pyramidal projection neurons (PNs) and expresses the type 1 cannabinoid receptor (CB1R) in its axon terminals. Although numerous studies have elucidated the anatomy and physiology of these CCK+/CB1R + interneurons in rodents, it has not been determined if they exist in primates. The present investigation used immunohistochemical techniques in the monkey to answer this question. It was found that the monkey BNC, as in rodents, has a very high density of CB1R + axons, including CB1R + axon terminals that form basket-like plexuses contacting somata of PNs. These axons, as well as axons in the neuropil, exhibit extensive colocalization of CCK and CB1R. These findings suggest that the same synaptic mechanisms involved in CCK-CB1R interactions in rodents may also apply to primates, and that therapies that target the cannabinoid system in the BNC may be useful for treating fear and anxiety in human patients.

Axonal extension from dorsal root ganglia on fibrillar and highly aligned poly(lactic acid)-polypyrrole substrates obtained by two different techniques: Electrospun nanofibres and extruded microfibres.

The biological behaviour of Schwann cells (SCs) and dorsal root ganglia (DRG) on fibrillar, highly aligned and electroconductive substrates obtained by two different techniques is studied. Mats formed by nanometer-sized fibres of poly(lactic acid) (PLA) are obtained by the electrospinning technique, while bundles formed by micrometer-sized extruded PLA fibres are obtained by grouping microfibres together. Both types of substrates are coated with the electrically conductive polymer polypyrrole (PPy) and their morphological, physical and electrical characterization is carried out. SCs on micrometer-sized substrates show a higher motility and cell-cell interaction, while a higher cell-material interaction with a lower cell motility is observed for nanometer-sized substrates. This higher motility and cell-cell interaction of SCs on the micrometer-sized substrates entails a higher axonal growth from DRG, since the migration of SCs from the DRG body is accelerated and, therefore, the SCs tapestry needed for the axonal growth is formed earlier on the substrate. A higher length and area of the axons is observed for these micrometer-sized substrates, as well as a higher level of axonal sprouting when compared with the nanometer-sized ones. These substrates offer the possibility of being electrically stimulated in different tissue engineering applications of the nervous system.

Wrapping axons in mammals and Drosophila: Different lipids, same principle.

Plasma membranes of axon-wrapping glial cells develop specific cylindrical bilayer membranes that surround thin individual axons or axon bundles. Axons are wrapped with single layered glial cells in lower organisms whereas in the mammalian nervous system, axons are surrounded with a characteristic complex multilamellar myelin structure. The high content of lipids in myelin suggests that lipids play crucial roles in the structure and function of myelin. The most striking feature of myelin lipids is the high content of galactosylceramide (GalCer). Serological and genetic studies indicate that GalCer plays a key role in the formation and function of the myelin sheath in mammals. In contrast to mammals, Drosophila lacks GalCer. Instead of GalCer, ceramide phosphoethanolamine (CPE) has an important role to ensheath axons with glial cells in Drosophila. GalCer and CPE share similar physical properties: both lipids have a high phase transition temperature and high packing, are immiscible with cholesterol and form helical liposomes. These properties are caused by both the strong headgroup interactions and the tight packing resulting from the small size of the headgroup and the hydrogen bonds between lipid molecules. These results suggest that mammals and Drosophila wrap axons using different lipids but the same conserved principle.

Morphological and neurochemical characterisation of anterogradely labelled spinal sensory and autonomic nerve endings in the mouse bladder.

The bladder is innervated by axons of sympathetic and parasympathetic efferent nerves, and by spinal afferent neurons. The objective was to characterise anatomically and immunohistochemically the terminal endings of sensory and autonomic motor nerve endings in wholemount preparations of the mouse bladder. We used both anterograde labelling of pelvic and hypogastric nerves ex vivo and anterograde labelling from lumbosacral dorsal root ganglia (DRG) in vivo in male and female mice. These were combined with immunohistochemistry for major markers of sensory, sympathetic and parasympathetic nerves. Selective labelling of spinal afferent endings following dextran biotin-labelling from DRGs in vivo showed no co-localisation of VAChT or TH in sensory terminals in the detrusor and suburothelial plexus. Biotinamide was applied ex vivo to nerve trunks arising in the pelvic ganglion and running towards the bladder. Among the filled axons, 38% of detrusor fibres and 47% of suburothelial axons were immunoreactive for calcitonin-gene related peptide (CGRP). Vesicular acetylcholine transporter (VAChT) immunoreactivity was present in 26% of both detrusor and suburothelial axons. For tyrosine hydroxylase (TH), the proportions were 15% and 17%, respectively. Three major morphological types of CGRP-immunoreactive nerve endings were distinguished in the bladder wall: simple, branching and complex. VAChT-immunoreactive parasympathetic axons had simple and branching endings; TH immunoreactive axons all had simple morphologies. Our findings revealed that different subtypes of sensory and autonomic nerve endings can be reliably identified by combining anterograde labelling ex vivo with specific immunohistochemical markers, although morphologically some of these types of endings were indistinguishable.

In Vivo Calcium Imaging During Axon Degeneration in Zebrafish.

In vivo calcium imaging in zebrafish provides the ability to investigate calcium dynamics within neurons. Utilizing genetically encoded calcium sensors it is possible to monitor calcium signals within a single axon during axon injury and degeneration with high temporal and spatial resolution. Here we will describe in vivo, time-lapse confocal imaging methods of calcium imaging. Imaging of calcium dynamics with genetically encoded calcium sensors (GECS) within living axons can serve as a method to assess axonal physiology and effects of pharmacologic and genetic manipulation, as well as characterize responses to different injury models.

Oligodendrocyte pathology exceeds axonal pathology in white matter in human amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease. The majority of cases are sporadic (sALS), while the most common inherited form is due to C9orf72 mutation (C9ALS). A high burden of inclusion pathology is seen in glia (including oligodendrocytes) in ALS, especially in C9ALS. Myelin basic protein (MBP) messenger RNA (mRNA) must be transported to oligodendrocyte processes for myelination, a possible vulnerability for normal function. TDP43 is found in pathological inclusions in ALS and is a component of mRNA transport granules. Thus, TDP43 aggregation could lead to MBP loss. Additionally, the hexanucleotide expansion of mutant C9ALS binds hnRNPA2/B1, a protein essential for mRNA transport, causing potential further impairment of hnRNPA2/B1 function, and thus myelination. Using immunohistochemistry for p62 and TDP43 in human post-mortem tissue, we found a high burden of glial inclusions in the prefrontal cortex, precentral gyrus, and spinal cord in ALS, which was greater in C9ALS than in sALS cases. Double staining demonstrated that the majority of these inclusions were in oligodendrocytes. Using immunoblotting, we demonstrated reduced MBP protein levels relative to PLP (a myelin component that relies on protein not mRNA transport) and neurofilament protein (an axonal marker) in the spinal cord. This MBP loss was disproportionate to the level of PLP and axonal loss, suggesting that impaired mRNA transport may be partly responsible. Finally, we show that in C9ALS cases, the level of oligodendroglial inclusions correlates inversely with levels of hnRNPA2/B1 and the number of oligodendrocyte precursor cells. We conclude that there is considerable oligodendrocyte pathology in ALS, which at least partially reflects impairment of mRNA transport. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Morphogenesis Guided by 3D Patterning of Growth Factors in Biological Matrices.

Three-dimensional (3D) control over the placement of bioactive cues is fundamental to understand cell guidance and develop engineered tissues. Two-photon patterning (2PP) provides such placement at micro- to millimeter scale, but nonspecific interactions between proteins and functionalized extracellular matrices (ECMs) restrict its use. Here, a 2PP system based on nonfouling hydrophilic photocages and Sortase A (SA)-based enzymatic coupling is presented, which offers unprecedented orthogonality and signal-to-noise ratio in both inert hydrogels and complex mammalian matrices. Improved photocaged peptide synthesis and protein functionalization protocols with broad applicability are introduced. Importantly, the method enables 2PP in a single step in the presence of fragile biomolecules and cells, and is compatible with time-controlled growth factor presentation. As a corollary, the guidance of axons through 3D-patterned nerve growth factor (NGF) within brain-mimetic ECMs is demonstrated. The approach allows for the interrogation of the role of complex signaling molecules in 3D matrices, thus helping to better understand biological guidance in tissue development and regeneration.

Remyelination alters the pattern of myelin in the cerebral cortex.

Destruction of oligodendrocytes and myelin sheaths in cortical gray matter profoundly alters neural activity and is associated with cognitive disability in multiple sclerosis (MS). Myelin can be restored by regenerating oligodendrocytes from resident progenitors; however, it is not known whether regeneration restores the complex myelination patterns in cortical circuits. Here, we performed time lapse in vivo two photon imaging in somatosensory cortex of adult mice to define the kinetics and specificity of myelin regeneration after acute oligodendrocyte ablation. These longitudinal studies revealed that the pattern of myelination in cortex changed dramatically after regeneration, as new oligodendrocytes were formed in different locations and new sheaths were often established along axon segments previously lacking myelin. Despite the dramatic increase in axonal territory available, oligodendrogenesis was persistently impaired in deeper cortical layers that experienced higher gliosis. Repeated reorganization of myelin patterns in MS may alter circuit function and contribute to cognitive decline.

Spinal Interneurons With Dual Axon Projections to Knee-Extensor and Hip-Extensor Motor Pools.

The central nervous system (CNS) may simplify control of limb movements by activating certain combinations of muscles together, i.e., muscle synergies. Little is known, however, about the spinal cord interneurons that activate muscle synergies by exciting sets of motoneurons for different muscles. The turtle spinal cord, even without brain inputs and movement-related sensory feedback, can generate the patterns of motoneuron activity underlying forward swimming, three forms of scratching, and limb withdrawal. Spinal interneurons activated during scratching are typically activated during all three forms of scratching, to different degrees, even though each form of scratching has its own knee-hip synergy. Such spinal interneurons are also typically activated rhythmically during scratching motor patterns, with hip-related timing. We proposed a hypothesis that such interneurons that are most active during rostral scratch stimulation project their axons to both knee-extensor and hip-flexor motoneurons, thus generating the rostral scratch knee-hip synergy, while those interneurons most active during pocket scratch stimulation project their axons to both knee-extensor and hip-extensor motoneurons, thus generating the pocket scratch knee-hip synergy. The activity of the entire population would then generate the appropriate synergy, depending on the location of sensory stimulation. Mathematical modeling has demonstrated that this hypothesis is feasible. Here, we provide one test of this hypothesis by injecting two fluorescent retrograde tracers into the regions of knee-extensor motoneurons (more rostrally) and hip-extensor motoneurons (more caudally). We found that there were double-labeled interneurons, which projected their axons to both locations. The dual-projecting interneurons were widely distributed rostrocaudally, dorsoventrally, and mediolaterally within the hindlimb enlargement and pre-enlargement spinal segments examined. The existence of such dual-projecting interneurons is consistent with the hypothesis that they contribute to generating the knee-hip synergy for pocket scratching. The dual-projecting interneurons, however, were only about 1% of the total interneurons projecting to each location, which suggests that they might be one of several contributors to the appropriate knee-hip synergy. Indirect projections to both motor pools and/or knee extensor-dedicated interneurons might also contribute. There is evidence for dual-projecting spinal interneurons in frogs and mice as well, suggesting that they may contribute to limb motor control in a variety of vertebrates.

Cofilin dysregulation alters actin turnover in frataxin-deficient neurons.

Abnormalities in actin cytoskeleton have been linked to Friedreich's ataxia (FRDA), an inherited peripheral neuropathy characterised by an early loss of neurons in dorsal root ganglia (DRG) among other clinical symptoms. Despite all efforts to date, we still do not fully understand the molecular events that contribute to the lack of sensory neurons in FRDA. We studied the adult neuronal growth cone (GC) at the cellular and molecular level to decipher the connection between frataxin and actin cytoskeleton in DRG neurons of the well-characterised YG8R Friedreich's ataxia mouse model. Immunofluorescence studies in primary cultures of DRG from YG8R mice showed neurons with fewer and smaller GCs than controls, associated with an inhibition of neurite growth. In frataxin-deficient neurons, we also observed an increase in the filamentous (F)-actin/monomeric (G)-actin ratio (F/G-actin ratio) in axons and GCs linked to dysregulation of two crucial modulators of filamentous actin turnover, cofilin-1 and the actin-related protein (ARP) 2/3 complex. We show how the activation of cofilin is due to the increase in chronophin (CIN), a cofilin-activating phosphatase. Thus cofilin emerges, for the first time, as a link between frataxin deficiency and actin cytoskeleton alterations.

Immunogold labeling of synaptic vesicle proteins in developing hippocampal neurons.

Synaptic vesicles (SV) contain high concentrations of specific proteins. How these proteins are transported from soma to synapses, and how they become concentrated at SV clusters at presynaptic terminals were examined by immunogold electron microscopy in dissociated rat hippocampal neurons at 3-6 days in culture, a developmental stage when axonal transport of SV proteins is robust. In neuronal somas, labels for the SV integral membrane proteins (synaptophysin, SV2, VAMP/synaptobrevin, and synaptotagmin) were localized at Golgi complexes and other membranous structures that were dispersed in the cytoplasm as individual vesicle/vacuoles. These vesicles/vacuoles became aggregated in axons, with the size of aggregates ranging from 0.2 to 2 µm in length. Pleomorphic vesicle/vacuoles within the aggregate were typically larger (50-300 nm) than SVs, which were uniform in size at ~ 40 nm. These pleomorphic vesicles/vacuoles are probably transport cargos carrying SV integral membrane proteins from the soma, and then are preferentially sorted into axons at early developmental stages. Serial thin sections of young axons indicated that many labeled aggregates were not synaptic, and in fact, some of these axons were without dendritic contacts. In contrast, labels for two SV-associated proteins, synapsin I and α-synuclein, were not localized at the Golgi complexes or associated with membranous structures in the soma, but were dispersed in the cytoplasm. However, these SV-associated proteins became highly concentrated on clusters of SV-like vesicles in axons, and such clusters were already distinctive in axons as early as 3 days in culture. These clusters consisted of ~ 4-30 vesicles in single thin sections, and the vesicles were of a uniform size (~ 40 nm). Serial sectioning analysis showed that these clusters could be part of nascent synapses or exist in axons without any dendritic contact. Importantly, the vesicles were intensely labeled for SV integral membrane proteins as well as SV-associated proteins. Thus, these EM observations reveal that the two groups of proteins, SV integral membrane and SV-associated, proceed through different routes of biosynthesis and axon transport, and are only sorted into the same final compartment, SV clusters, when they are in the axons.

Axonal localization of the fragile X family of RNA binding proteins is conserved across mammals.

Spatial segregation of proteins to neuronal axons arises in part from local translation of mRNAs that are first transported into axons in ribonucleoprotein particles (RNPs), complexes containing mRNAs and RNA binding proteins. Understanding the importance of local translation for a particular circuit requires not only identifying axonal RNPs and their mRNA cargoes, but also whether these RNPs are broadly conserved or restricted to only a few species. Fragile X granules (FXGs) are axonal RNPs containing the fragile X related family of RNA binding proteins along with ribosomes and specific mRNAs. FXGs were previously identified in mouse, rat, and human brains in a conserved subset of neuronal circuits but with species-dependent developmental profiles. Here, we asked whether FXGs are a broadly conserved feature of the mammalian brain and sought to better understand the species-dependent developmental expression pattern. We found FXGs in a conserved subset of neurons and circuits in the brains of every examined species that together include mammalian taxa separated by up to 160 million years of divergent evolution. A developmental analysis of rodents revealed that FXG expression in frontal cortex and olfactory bulb followed consistent patterns in all species examined. In contrast, FXGs in hippocampal mossy fibers increased in abundance across development for most species but decreased across development in guinea pigs and members of the Mus genus, animals that navigate particularly small home ranges in the wild. The widespread conservation of FXGs suggests that axonal translation is an ancient, conserved mechanism for regulating the proteome of mammalian axons.

Linoleic acid-derived metabolites constitute the majority of oxylipins in the rat pup brain and stimulate axonal growth in primary rat cortical neuron-glia co-cultures in a sex-dependent manner.

In adult rats, omega-6 linoleic acid (LA, 18:2n-6) serves as a precursor to oxidized LA metabolites (OXLAMs) known to regulate multiple signaling processes in the brain. However, little is known regarding the levels or role(s) of LA and its metabolites during brain development. To address this gap, fatty acids within various brain lipid pools, and their oxidized metabolites (oxylipins) were quantified in brains from 1-day-old male and female pups using gas chromatography and liquid chromatography coupled to tandem mass spectrometry, respectively. Primary neuron-glia co-cultures derived from postnatal day 0-1 male and female rat neocortex were exposed to vehicle (0.1% ethanol), LA, the OXLAM 13-hydroxyoctadecadienoic acid (13-HODE), or prostaglandin E2 at 10-1000 nM for 48 h to test their effects on neuronal morphology. In both male and female pups, LA accounted for 1-3% of fatty acids detected in brain phospholipids and cholesteryl esters. It was not detected in triacylglycerols, and free fatty acids. Unesterified OXLAMs constituted 47-53% of measured unesterified oxylipins in males and females (vs. ~5-7% reported in adult rat brain). Of these, 13-HODE was the most abundant, accounting for 30-33% of measured OXLAMs. Brain fatty acid and OXLAM concentrations did not differ between sexes. LA and 13-HODE significantly increased axonal outgrowth. Separate analyses of cultures derived from male versus female pups revealed that LA at 1, 50, and 1000 nM, significantly increased axonal outgrowth in female but not male cortical neurons, whereas 13-HODE at 100 nM significantly increased axonal outgrowth in male but not female cortical neurons. prostaglandin E2 did not alter neuronal outgrowth in either sex. This study demonstrates that OXLAMs constitute the majority of unesterified oxylipins in the developing rat brain despite low relative abundance of their LA precursor, and highlights a novel role of LA and 13-HODE in differentially influencing neuronal morphogenesis in the developing male and female brain.