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Proceedings of the National Academy of... Jan 2020Tuberous Sclerosis Complex (TSC) is a rare genetic disease that manifests with early symptoms, including cortical malformations, childhood epilepsy, and TSC-associated...
Tuberous Sclerosis Complex (TSC) is a rare genetic disease that manifests with early symptoms, including cortical malformations, childhood epilepsy, and TSC-associated neuropsychiatric disorders (TANDs). Cortical malformations arise during embryonic development and have been linked to childhood epilepsy before, but the underlying mechanisms of this relationship remain insufficiently understood. Zebrafish have emerged as a convenient model to study elementary neurodevelopment; however, without in-depth functional analysis, the Tsc2-deficient zebrafish line cannot be used for studies of TANDs or new drug screening. In this study, we found that the lack of Tsc2 in zebrafish resulted in heterotopias and hyperactivation of the mTorC1 pathway in pallial regions, which are homologous to the mammalian cortex. We observed commissural thinning that was responsible for brain dysconnectivity, recapitulating TSC pathology in human patients. The lack of Tsc2 also delayed axonal development and caused aberrant tract fasciculation, corresponding to the abnormal expression of genes involved in axon navigation. The mutants underwent epileptogenesis that resulted in nonmotor seizures and exhibited increased anxiety-like behavior. We further mapped discrete parameters of locomotor activity to epilepsy-like and anxiety-like behaviors, which were rescued by reducing tyrosine receptor kinase B (TrkB) signaling. Moreover, in contrast to treatment with vigabatrin and rapamycin, TrkB inhibition rescued brain dysconnectivity and anxiety-like behavior. These data reveal that commissural thinning results in the aberrant regulation of anxiety, providing a mechanistic link between brain anatomy and human TANDs. Our findings also implicate TrkB signaling in the complex pathology of TSC and reveal a therapeutic target.
Topics: Animals; Anxiety; Disease Models, Animal; Epilepsy; Female; Humans; Intracellular Signaling Peptides and Proteins; Mechanistic Target of Rapamycin Complex 1; Receptor, trkB; Seizures; Tuberous Sclerosis; Zebrafish; Zebrafish Proteins
PubMed: 31932427
DOI: 10.1073/pnas.1910834117 -
International Journal of Molecular... Dec 2019The G protein-coupled cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and their endocannabinoid (eCBs) ligands, have been implicated in several aspects of brain...
The G protein-coupled cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and their endocannabinoid (eCBs) ligands, have been implicated in several aspects of brain wiring during development. Here we aim to assess whether interfering with CB1R affects development, neuritogenesis and pathfinding of GnRH and AgRP neurons, forebrain neurons that control respectively reproduction and appetite. We pharmacologically and genetically interfered with CB1R in zebrafish strains with fluorescently labeled GnRH3 and the AgRP1 neurons. By applying CB1R antagonists we observed a reduced number of GnRH3 neurons, fiber misrouting and altered fasciculation. Similar phenotypes were observed by CB1R knockdown. Interfering with CB1R also resulted in a reduced number, misrouting and poor fasciculation of the AgRP1 neuron's axonal projections. Using a bioinformatic approach followed by qPCR validation, we have attempted to link CB1R functions with known guidance and fasciculation proteins. The search identified stathmin-2, a protein controlling microtubule dynamics, previously demonstrated to be coexpressed with CB1R and now shown to be downregulated upon interference with CB1R in zebrafish. Together, these results raise the likely possibility that embryonic exposure to low doses of CB1R-interfering compounds could impact on the development of the neuroendocrine systems controlling sexual maturation, reproduction and food intake.
Topics: Agouti-Related Protein; Animals; Axons; Benzoxazines; Embryo, Nonmammalian; Embryonic Development; Gonadotropin-Releasing Hormone; Morpholines; Morpholinos; Naphthalenes; Neurons; Pyrrolidonecarboxylic Acid; Receptor, Cannabinoid, CB1; Zebrafish; Zebrafish Proteins
PubMed: 31881740
DOI: 10.3390/ijms21010168 -
Trends in Biochemical Sciences Jan 2020Netrin is a prototypical axon guidance cue. Structural studies have revealed how netrin interacts with the deleted in colorectal cancer (DCC) receptor, other receptors,... (Review)
Review
Netrin is a prototypical axon guidance cue. Structural studies have revealed how netrin interacts with the deleted in colorectal cancer (DCC) receptor, other receptors, and co-factors for signaling. Recently, genetic studies suggested that netrin is involved in neuronal haptotaxis, which requires a reversible adhesion process. Structural data indicate that netrin can also mediate trans-adhesion between apposing cells decorated with its receptors on the condition that the auxiliary guidance cue draxin is present. Here, we propose that netrin is involved in conditional adhesion, a reversible and localized process that can contribute to cell adhesion and migration. We suggest that netrin-mediated adhesion and signaling are linked, and that local environmental factors in the ventricular zone, the floor plate, or other tissues coordinate its function.
Topics: Animals; Cell Adhesion; DCC Receptor; Humans; Netrins; Signal Transduction
PubMed: 31704057
DOI: 10.1016/j.tibs.2019.10.005 -
Journal of Tissue Engineering and... Nov 2019The central feature of peripheral motor axons is their remarkable lengths as they project from a motor neuron residing in the spinal cord to distant target muscle....
The central feature of peripheral motor axons is their remarkable lengths as they project from a motor neuron residing in the spinal cord to distant target muscle. However, current in vitro models have not replicated this feature owing to challenges in generating motor axon tracts beyond a few millimeters in length. To address this, we have developed a novel combination of microtissue engineering and mechanically assisted growth techniques to create long-projecting centimeter-scale motor axon tracts. Here, primary motor neurons were isolated from rat spinal cords and induced to form engineered microspheres via forced aggregation in custom microwells. This technique yielded healthy motor neurons projecting dense, fasciculated axonal tracts. Within our custom-built mechanobioreactors, motor neuron culture conditions, neuronal/axonal architecture, and mechanical growth conditions were optimized to generate parameters for robust and efficient stretch growth of motor axons. We found that axons projecting from motor neuron aggregates were able to tolerate displacement rates at least 10 times greater than those by axons projecting from dissociated motor neurons. The growth and structural characteristics of these stretch-grown motor axons were compared with that of benchmark stretch-grown sensory axons, revealing increased motor axon fasciculation. Finally, motor axons were integrated with myocytes and stretch grown to create novel long-projecting axonal-myocyte constructs that recreate characteristic dimensions of native nerve-muscle anatomy. This is the first demonstration of mechanical elongation of spinal motor axons and may have applications as anatomically inspired in vitro testbeds or as tissue-engineered living scaffolds for targeted axon tract reconstruction following nervous system injury or disease.
Topics: Animals; Axons; Bioreactors; Motor Neurons; Rats; Rats, Sprague-Dawley
PubMed: 31469944
DOI: 10.1002/term.2955 -
Functional divergence of Plexin B structural motifs in distinct steps of olfactory circuit assembly.ELife Jun 2019Plexins exhibit multitudinous, evolutionarily conserved functions in neural development. How Plexins employ their diverse structural motifs in vivo to perform distinct...
Plexins exhibit multitudinous, evolutionarily conserved functions in neural development. How Plexins employ their diverse structural motifs in vivo to perform distinct roles is unclear. We previously reported that Plexin B (PlexB) controls multiple steps during the assembly of the olfactory circuit (Li et al., 2018b). Here, we systematically mutagenized structural motifs of PlexB and examined the function of these variants in these multiple steps: axon fasciculation, trajectory choice, and synaptic partner selection. We found that the extracellular Sema domain is essential for all three steps, the catalytic site of the intracellular RapGAP is engaged in none, and the intracellular GTPase-binding motifs are essential for trajectory choice and synaptic partner selection, but are dispensable for fasciculation. Moreover, extracellular PlexB cleavage serves as a regulatory mechanism of PlexB signaling. Thus, the divergent roles of PlexB motifs in distinct steps of neural development contribute to its functional versatility in neural circuit assembly.
Topics: Animals; Animals, Genetically Modified; Axons; Drosophila Proteins; Drosophila melanogaster; Nerve Tissue Proteins; Neurogenesis; Olfactory Bulb; Receptors, Cell Surface; Semaphorins; Signal Transduction; Smell
PubMed: 31225795
DOI: 10.7554/eLife.48594 -
Developmental Cell May 2019
PubMed: 31063761
DOI: 10.1016/j.devcel.2019.04.028 -
Scientific Reports Apr 2019Ceramide phosphoethanolamine (CPE), a major sphingolipid in invertebrates, is crucial for axonal ensheathment in Drosophila. Darkfield microscopy revealed that an...
Ceramide phosphoethanolamine (CPE), a major sphingolipid in invertebrates, is crucial for axonal ensheathment in Drosophila. Darkfield microscopy revealed that an equimolar mixture of bovine buttermilk CPE (milk CPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (diC18:1 PC) tends to form tubules and helical ribbons, while pure milk CPE mainly exhibits amorphous aggregates and, at low frequency, straight needles. Negative staining electron microscopy indicated that helices and tubules were composed of multilayered 5-10 nm thick slab-like structures. Using different molecular species of PC and CPE, we demonstrated that the acyl chain length of CPE but not of PC is crucial for the formation of tubules and helices in equimolar mixtures. Incubation of the lipid suspensions at the respective phase transition temperature of CPE facilitated the formation of both tubules and helices, suggesting a dynamic lipid rearrangement during formation. Substituting diC18:1 PC with diC18:1 PE or diC18:1 PS failed to form tubules and helices. As hydrated galactosylceramide (GalCer), a major lipid in mammalian myelin, has been reported to spontaneously form tubules and helices, it is believed that the ensheathment of axons in mammals and Drosophila is based on similar physical processes with different lipids.
Topics: Animals; Axon Fasciculation; Drosophila; Galactosylceramides; Lipid Bilayers; Membranes; Molecular Conformation; Nervous System; Phase Transition; Phosphatidylcholines; Sphingomyelins
PubMed: 30967612
DOI: 10.1038/s41598-019-42247-1 -
The Journal of Neuroscience : the... Jun 2019Selective cargo transport into axons and dendrites over the microtubule network is essential for neuron polarization. The axon initial segment (AIS) separates the axon...
Selective cargo transport into axons and dendrites over the microtubule network is essential for neuron polarization. The axon initial segment (AIS) separates the axon from the somatodendritic compartment and controls the microtubule-dependent transport into the axon. Interestingly, the AIS has a characteristic microtubule organization; it contains bundles of closely spaced microtubules with electron dense cross-bridges, referred to as microtubule fascicles. The microtubule binding protein TRIM46 localizes to the AIS and when overexpressed in non-neuronal cells forms microtubule arrays that closely resemble AIS fascicles in neurons. However, the precise role of TRIM46 in microtubule fasciculation in neurons has not been studied. Here we developed a novel correlative light and electron microscopy approach to study AIS microtubule organization. We show that in cultured rat hippocampal neurons of both sexes, TRIM46 levels steadily increase at the AIS during early neuronal differentiation and at the same time closely spaced microtubules form, whereas the fasciculated microtubules appear at later developmental stages. Moreover, we localized TRIM46 to the electron dense cross-bridges and show that depletion of TRIM46 causes loss of cross-bridges and increased microtubule spacing. These data indicate that TRIM46 has an essential role in organizing microtubule fascicles in the AIS. The axon initial segment (AIS) is a specialized region at the proximal axon where the action potential is initiated. In addition the AIS separates the axon from the somatodendritic compartment, where it controls protein transport to establish and maintain neuron polarity. Cargo vesicles destined for the axon recognize specialized microtubule tracks that enter the AIS. Interestingly the microtubules entering the AIS form crosslinked bundles, called microtubule fascicules. Recently we found that the microtubule-binding protein TRIM46 localizes to the AIS, where it may organize the AIS microtubules. In the present study we developed a novel correlative light and electron microscopy approach to study the AIS microtubules during neuron development and identified an essential role for TRIM46 in microtubule fasciculation.
Topics: Animals; Axon Fasciculation; Axon Initial Segment; Cell Polarity; Cells, Cultured; Cytoskeleton; Female; Hippocampus; Male; Microtubules; Neurons; Rats; Tripartite Motif Proteins
PubMed: 30967428
DOI: 10.1523/JNEUROSCI.3105-18.2019 -
Frontiers in Cell and Developmental... 2019Neuron-Glia related cell adhesion molecule (NrCAM) is a candidate autism risk factor that promotes axon guidance through cytoskeletal linkages in developing brain but...
Neuron-Glia related cell adhesion molecule (NrCAM) is a candidate autism risk factor that promotes axon guidance through cytoskeletal linkages in developing brain but its role in limbic circuitry has not been investigated. hybridization (ISH) and immunofluorescence staining showed that NrCAM is expressed in the developing amygdalar pathway of mouse embryos during outgrowth of projections in the stria terminalis, a major limbic tract that interconnects the central amygdala (CeA) with key targets in the bed nucleus of the stria terminalis (BNST). Analysis of fiber tracts in NrCAM mutant mice by Neurofilament protein immunohistochemistry showed pronounced defasciculation and misprojection of fibers in the ST. The defasciculation phenotype may result from impairment in NrCAM homophilic inter-axonal adhesion or axon repulsion from the secreted ligand Semaphorin 3F, which is expressed in limbic areas in proximity to the ST. Behavioral testing indicated that NrCAM null mice were impaired in context-dependent fear conditioning, in accord with altered amygdala-BNST connectivity, but displayed normal cued (tone-shock) conditioning. Results are consistent with the novel finding that NrCAM mediates fasciculation of axon fibers in the ST important for proper amygdalar-BNST circuitry and response to contextual fear conditioning.
PubMed: 30766872
DOI: 10.3389/fcell.2019.00009 -
Neuron Feb 2019In vertebrates, commissural axons extend ventrally toward the floor plate in the spinal cord and hindbrain. Netrin-1, secreted by floor plate cells, was proposed to...
In vertebrates, commissural axons extend ventrally toward the floor plate in the spinal cord and hindbrain. Netrin-1, secreted by floor plate cells, was proposed to attract commissural axons at a distance. However, recent genetic studies in mice have shown that netrin-1 is also produced by ventricular zone (VZ) progenitors and that in the hindbrain, it represents the main source of netrin-1 for commissural axons. Here, we show that genetically deleting netrin-1 either from the VZ or the floor plate does not prevent midline crossing in the spinal cord, although axon pathfinding and fasciculation are perturbed. Strikingly, the VZ and floor plate act synergistically, as the simultaneous ablation of netrin-1 from these two sources severely impedes crossing. These results suggest that floor-plate-derived netrin-1 has a distinct impact on commissural axons in the spinal cord and hindbrain.
Topics: Animals; Axon Guidance; Cerebral Ventricles; Female; Male; Mice; Netrin-1; Neurons; Rhombencephalon; Spinal Cord
PubMed: 30661739
DOI: 10.1016/j.neuron.2018.12.024