mtIF3 is locally translated in axons and regulates mitochondrial translation for axonal growth

Background The establishment and maintenance of functional neural connections relies on appropriate distribution and localization of mitochondria in neurites, as these organelles provide essential energy and metabolites. In particular, mitochondria are transported to axons and support local energy production to maintain energy-demanding neuronal processes including axon branching, growth, and regeneration. Additionally, local protein synthesis is required for structural and functional changes in axons, with nuclear-encoded mitochondrial mRNAs having been found localized in axons. However, it remains unclear whether these mRNAs are locally translated and whether the potential translated mitochondrial proteins are involved in the regulation of mitochondrial functions in axons. Here, we aim to further understand the purpose of such compartmentalization by focusing on the role of mitochondrial initiation factor 3 (mtIF3), whose nuclear-encoded transcripts have been shown to be present in axonal growth cones. Results We demonstrate that brain-derived neurotrophic factor (BDNF) induces local translation of mtIF3 mRNA in axonal growth cones. Subsequently, mtIF3 protein is translocated into axonal mitochondria and promotes mitochondrial translation as assessed by our newly developed bimolecular fluorescence complementation sensor for the assembly of mitochondrial ribosomes. We further show that BDNF-induced axonal growth requires mtIF3-dependent mitochondrial translation in distal axons. Conclusion We describe a previously unknown function of mitochondrial initiation factor 3 (mtIF3) in axonal protein synthesis and development. These findings provide insight into the way neurons adaptively control mitochondrial physiology and axonal development via local mtIF3 translation.

Loss of Neuropilin2a/b or Sema3fa alters olfactory sensory axon dynamics and protoglomerular targeting

Background Olfactory Sensory Neuron (OSN) axons project from the zebrafish olfactory epithelium to reproducible intermediate target locations in the olfactory bulb called protoglomeruli at early stages in development. Two classes of OSNs expressing either OMP or TRPC2 exclusively target distinct, complementary protoglomeruli. Using RNAseq, we identified axon guidance receptors nrp2a and nrp2b, and their ligand sema3fa, as potential guidance factors that are differentially expressed between these two classes of OSNs. Methods To investigate their role in OSN axon guidance, we assessed the protoglomerular targeting fidelity of OSNs labeled by OMP:RFP and TRPC2:Venus transgenes in nrp2a, nrp2b, or sema3fa mutants. We used double mutant and genetic interaction experiments to interrogate the relationship between the three genes. We used live time-lapse imaging to compare the dynamic behaviors of OSN growth cones during protoglomerular targeting in heterozygous and mutant larvae. Results The fidelity of protoglomerular targeting of TRPC2-class OSNs is degraded in nrp2a, nrp2b, or sema3fa mutants, as axons misproject into OMP-specific protoglomeruli and other ectopic locations in the bulb. These misprojections are further enhanced in nrp2a;nrp2b double mutants suggesting that nrp2s work at least partially in parallel in the same guidance process. Results from genetic interaction experiments are consistent with sema3fa acting in the same biological pathway as both nrp2a and nrp2b. Live time-lapse imaging was used to examine the dynamic behavior of TRPC2-class growth cones in nrp2a mutants compared to heterozygous siblings. Some TRPC2-class growth cones ectopically enter the dorsal-medial region of the bulb in both groups, but in fully mutant embryos, they are less likely to correct the error through retraction. The same result was observed when TRPC2-class growth cone behavior was compared between sema3fa heterozygous and sema3fa mutant larvae. Conclusions Our results suggest that nrp2a and nrp2b expressed in TRPC2-class OSNs help prevent their mixing with axon projections in OMP-specific protoglomeruli, and further, that sema3fa helps to exclude TRPC2-class axons by repulsion from the dorsal-medial bulb.

Analysis of growth cone extension in standardized coordinates highlights self-organization rules during wiring of the Drosophila visual system

A fascinating question in neuroscience is how ensembles of neurons, originating from different locations, extend to the proper place and by the right time to create precise circuits. Here, we investigate this question in the Drosophila visual system, where photoreceptors re-sort in the lamina to form the crystalline-like neural superposition circuit. The repeated nature of this circuit allowed us to establish a data-driven, standardized coordinate system for quantitative comparison of sparsely perturbed growth cones within and across specimens. Using this common frame of reference, we investigated the extension of the R3 and R4 photoreceptors, which is the only pair of symmetrically arranged photoreceptors with asymmetric target choices. Specifically, we found that extension speeds of the R3 and R4 growth cones are inherent to their cell identities. The ability to parameterize local regularity in tissue organization facilitated the characterization of ensemble cellular behaviors and dissection of mechanisms governing neural circuit formation.

Human pluripotent stem cell-derived photoreceptors switch from cell autonomous axon extension to non-cell autonomous process pulling during synaptic marker redistribution.

Photoreceptors (PRs) are the primary visual sensory cells, and their loss leads to blindness that is currently incurable. Cell replacement therapy holds promise as a therapeutic approach to restore vision to those who have lost PRs through damage or disease. While PR transplant research is ongoing in animal models, success is hindered by our limited understanding of PR axon growth during development and regeneration. Using a human pluripotent stem cell (hPSC) reporter line that labels PRs (WA09 CRX+/tdTomato), we generated retinal organoids in order to study mechanisms of PR process extension. We found that the earliest born PRs exhibit autonomous axon extension from dynamic terminals that appear similar to projection neuron growth cones. However, as hPSC-derived PRs age from 40 to 80 days of differentiation, they lose dynamic terminals in 2D plated cultures and within 3D retinal organoids, which does not correlate with cell birth date. Using a rod-specific hPSC reporter line (WA09 NRL+/eGFP), we further determined that rod PRs never form motile growth cones. Interestingly, PRs without motile terminals are still capable of extending axons, but neurites are generated from process stretching via their attachment to motile non-PR cells, which underlies the observed differences in PR neurite lengths on different substrata. While immobile PR terminals express actin, it is less polymerized and less organized than actin present in motile terminals. However, immobile PRs do localize synaptic proteins to their terminals, suggesting a normal developmental progression. These findings help inform the development of PR transplant therapies to treat blinding diseases and provide a platform to test treatments that restore autonomous PR axon extension.

An Emerging Role of PRRT2 in Regulating Growth Cone Morphology

Mutations in the PRRT2 gene are the main cause for a group of paroxysmal neurological diseases including paroxysmal kinesigenic dyskinesia, episodic ataxia, benign familial infantile seizures, and hemiplegic migraine. In the mature central nervous system, the protein has both a functional and a structural role at the synapse. Indeed, PRRT2 participates in the regulation of neurotransmitter release, as well as of actin cytoskeleton dynamics during synaptogenesis. Here, we show a role of the protein also during early stages of neuronal development. We found that PRRT2 accumulates at the growth cone in cultured hippocampal neurons. Overexpression of the protein causes an increase in the size and the morphological complexity of growth cones. In contrast, the growth cones of neurons derived from PRRT2 KO mice are smaller and less elaborated. Finally, we demonstrated that the aberrant shape of PRRT2 KO growth cones is associated with a selective alteration of the growth cone actin cytoskeleton. Our data support a key role of PRRT2 in the regulation of growth cone morphology during neuronal development.

Cofilin Regulates Filopodial Structure and Flexibility in Neuronal Growth Cones

Filopodia are actin-rich cytoskeletal protrusions at the leading edge of motile cells. In neuronal growth cones they function as antennae, guiding axonal growth toward the appropriate cellular targets. Proper brain development relies on robust axonal guidance mechanisms, so it is imperative to understand how the actin cytoskeleton functions in remodeling to meet the demands of growth cone exploration. Here we show by cryo-electron tomography and fluorescence imaging that filopodia in neuronal growth cones switch between fascin-linked and cofilin-decorated states, and that this transition regulates the exclusion of fascin from the cofilactin bundle at the filopodial base by hyper-twisting individual filaments and rearranging their packing. Additionally, we show that cofilactin bundles contribute to the flexibility of filopodial actin networks, thus, likely regulating the efficiency of targeted neurite outgrowth.

Time to stop; agent-based modelling of chemoaffinity with competition

In the classic Chemoaffinity theory, the retinotectal axon projection is thought to use pairs of orthogonal signalling gradients in the retina to specify the eventual location of synapses made on the surface of the tectum/superior colliculus. Similar orthogonal gradients in the tectum provide a coordinate system which allows the axons to match their prespecified destination with the correct location. Although the Ephrins have been shown to guide axons toward their destination, there has yet to emerge a complete account of the local interactions which halt the axonal growth cones in the correct locations to recreate the topography of the retinal cells. The model of Simpson and Goodhill (2011) provides an account of the basic topographic arrangement of cells on the tectum, as well as reproducing well known surgical and genetic manipulation experiments. However, it suffers from the absence of a local chemotactic guidance mechanism. Instead, each agent in their model is given instantaneous knowledge of the vector that would move it toward its pre specified destination. In addition to the globally supervised chemoaffinity term, Simpson and Goodhill (2011) introduced a competitive interaction for space between growth cone agents and a receptor-ligand axon-axon interaction in order to account for the full set of experimental manipulations. Here, we propose the replacement of the chemoaffinity term with a gradient following model consisting of axonal growth cone agents which carry receptor molecule expression determined by their soma's location of origin on the retina. Growth cones move on the simulated tectum guided by two pairs of opposing, orthogonal signalling molecules representing the Ephrin ligands. We show that with only the chemoaffinity term and a receptor-ligand based axon-axon interaction term (meaning that all growth cone interactions are by receptor-ligand signalling), a full range of experimental manipulations to the retinotectal system can be reproduced. Furthermore, we show that the observation that competition is not and essential requirement for axons to find their way (Gosse et al., 2008) is also accounted for by the model, due to the opposing influences of signalling gradient pairs. Finally, we demonstrate that, assuming exponentially varying receptor expression in the retina, ligand expression should either be exponential if the receptor-ligand signal induces repulsion (i.e. gradient descent) or logarithmic if the signal induces attraction (gradient ascent). Thus, we find that a model analogous to the one we presented in James et al. (2020) that accounts for murine barrel patterning is also a candidate mechanism for the arrangement of the more continuous retinotectal system.

The PH/MyTH4/FERMmolecule MAX-1 inhibits UNC-5 activity in regulation of VD growth cone protrusion in Caenorhabditis elegans

UNC-6/Netrin is a secreted conserved guidance cue that regulates dorsal-ventral axon guidance of C. elegans and in the vertebral spinal cord. In the polarity/protrusion model of VD growth cone guidance away from ventrally-expressed UNC-6 (repulsion), UNC-6 first polarizes the growth cone via the UNC-5 receptor such that filopodial protrusions are biased dorsally. UNC-6 then regulates a balance of protrusion in the growth cone based upon this polarity. UNC-5 inhibits protrusion ventrally, and the UNC-6 receptor UNC-40/DCC stimulates protrusion dorsally, resulting in net dorsal growth cone outgrowth. UNC-5 inhibits protrusion through the flavin monooxygenases FMO-1, 4, and 5 and possible actin destabilization, and inhibits pro-protrusive microtubule entry into the growth cone utilizing UNC-33/CRMP. The PH/MyTH4/FERM myosin-like protein was previously shown to act with UNC-5 in VD axon guidance utilizing axon guidance endpoint analysis. Here, we analyzed the effects of MAX-1 on VD growth cone morphology during outgrowth. We found that max-1 mutant growth cones were smaller and less protrusive than wild-type, the opposite of the unc-5 mutant phenotype. Furthermore, genetic interactions suggest that MAX-1 might normally inhibit UNC-5 activity, such that in a max-1 mutant growth cone, UNC-5 is overactive. Our results, combined with previous studies suggesting that MAX-1 might regulate UNC-5 levels in the cell or plasma membrane localization, suggest that MAX-1 attenuates UNC-5 signaling by regulating UNC-5 stability or trafficking. In summary, in the context of growth cone protrusion, MAX-1 inhibits UNC-5, demonstrating the mechanistic insight that can be gained by analyzing growth cones during outgrowth in addition to axon guidance endpoint analysis.

Visualising the cytoskeletal machinery in neuronal growth cones using cryo-electron tomography

Neurons navigate long distances and extend axons to form the complex circuitry of the mature brain. This depends on the coordinated response and continuous remodelling of the microtubule and F-actin networks in the axonal growth cone. Growth cone architecture remains poorly understood at nanoscales. We therefore investigated mouse hippocampal neuron growth cones using cryo-electron tomography to directly visualise their three-dimensional subcellular architecture with molecular detail. Our data show the hexagonal arrays of actin bundles that form filopodia penetrate the growth cone interior and terminate in the transition zone. We directly observe the modulation of these and other growth cone actin bundles by alteration of individual F-actin helical structures. Blunt-ended microtubules predominate in the growth cone, frequently contain lumenal particles and carry lattice defects. Investigation of the effect of absence of doublecortin, a neurodevelopmental cytoskeleton regulator, on growth cone cytoskeleton shows no major anomalies in overall growth cone organisation or in F-actin subpopulations. However, our data suggest that microtubules sustain more structural defects, highlighting the importance of microtubule integrity during growth cone migration.