The role of mechanics in axonal stability and development.

Much of the focus of neuronal cell biology has been devoted to growth cone guidance, synaptogenesis, synaptic activity, plasticity, etc. The axonal shaft too has received much attention, mainly for its astounding ability to transmit action potentials and the transport of material over long distances. For these functions, the axonal cytoskeleton and membrane have been often assumed to play static structural roles. Recent experiments have changed this view by revealing an ultrastructure much richer in features than previously perceived and one that seems to be maintained at a dynamic steady state. The role of mechanics in this is only beginning to be broadly appreciated and appears to involve passive and active modes of coupling different biopolymer filaments, filament turnover dynamics and membrane biophysics. Axons, being unique cellular processes in terms of high aspect ratios and often extreme lengths, also exhibit unique passive mechanical properties that might have evolved to stabilize them under mechanical stress. In this review, we summarize the experiments that have exposed some of these features. It is our view that axonal mechanics deserves much more attention not only due to its significance in the development and maintenance of the nervous system but also due to the susceptibility of axons to injury and neurodegeneration.

Convergent Energy State-Dependent Antagonistic Signaling by Cocaine- and Amphetamine-Regulated Transcript (CART) and Neuropeptide Y (NPY) Modulates the Plasticity of Forebrain Neurons to Regulate Feeding in Zebrafish.

Dynamic reconfiguration of circuit function subserves the flexibility of innate behaviors tuned to physiological states. Internal energy stores adaptively regulate feeding-associated behaviors and integrate opposing hunger and satiety signals at the level of neural circuits. Across vertebrate lineages, the neuropeptides cocaine- and amphetamine-regulated transcript (CART) and neuropeptide Y (NPY) have potent anorexic and orexic functions, respectively, and show energy-state-dependent expression in interoceptive neurons. However, how the antagonistic activities of these peptides modulate circuit plasticity remains unclear. Using behavioral, neuroanatomical, and activity analysis in adult zebrafish of both sexes, along with pharmacological interventions, we show that CART and NPY activities converge on a population of neurons in the dorsomedial telencephalon (Dm). Although CART facilitates glutamatergic neurotransmission at the Dm, NPY dampens the response to glutamate. In energy-rich states, CART enhances NMDA receptor (NMDAR) function by protein kinase A/protein kinase C (PKA/PKC)-mediated phosphorylation of the NR1 subunit of the NMDAR complex. Conversely, starvation triggers NPY-mediated reduction in phosphorylated NR1 via calcineurin activation and inhibition of cAMP production leading to reduced responsiveness to glutamate. Our data identify convergent integration of CART and NPY inputs by the Dm neurons to generate nutritional state-dependent circuit plasticity that is correlated with the behavioral switch induced by the opposing actions of satiety and hunger signals.SIGNIFICANCE STATEMENT Internal energy needs reconfigure neuronal circuits to adaptively regulate feeding behavior. Energy-state-dependent neuropeptide release can signal energy status to feeding-associated circuits and modulate circuit function. CART and NPY are major anorexic and orexic factors, respectively, but the intracellular signaling pathways used by these peptides to alter circuit function remain uncharacterized. We show that CART and NPY-expressing neurons from energy-state interoceptive areas project to a novel telencephalic region, Dm, in adult zebrafish. CART increases the excitability of Dm neurons, whereas NPY opposes CART activity. Antagonistic signaling by CART and NPY converge onto NMDA-receptor function to modulate glutamatergic neurotransmission. Thus, opposing activities of anorexic CART and orexic NPY reconfigure circuit function to generate flexibility in feeding behavior.

Antagonistic Activities of Fmn2 and ADF Regulate Axonal F-Actin Patch Dynamics and the Initiation of Collateral Branching.

Interstitial collateral branching of axons is a critical component in the development of functional neural circuits. Axon collateral branches are established through a series of cellular processes initiated by the development of a specialized, focal F-actin network in axons. The formation, maintenance and remodeling of this F-actin patch is critical for the initiation of axonal protrusions that are subsequently consolidated to form a collateral branch. However, the mechanisms regulating F-actin patch dynamics are poorly understood. Fmn2 is a formin family member implicated in multiple neurodevelopmental disorders. We find that Fmn2 regulates the initiation of axon collateral protrusions in chick spinal neurons and in zebrafish motor neurons. Fmn2 localizes to the protrusion-initiating axonal F-actin patches and regulates the lifetime and size of these F-actin networks. The F-actin nucleation activity of Fmn2 is necessary for F-actin patch stability but not for initiating patch formation. We show that Fmn2 insulates the F-actin patches from disassembly by the actin-depolymerizing factor, ADF, and promotes long-lived, larger patches that are competent to initiate axonal protrusions. The regulation of axonal branching can contribute to the neurodevelopmental pathologies associated with Fmn2 and the dynamic antagonism between Fmn2 and ADF may represent a general mechanism of formin-dependent protection of Arp2/3-initiated F-actin networks from disassembly.SIGNIFICANCE STATEMENT Axonal branching is a key process in the development of functional circuits and neural plasticity. Axon collateral branching is initiated by the elaboration of F-actin filaments from discrete axonal F-actin networks. We show that the neurodevelopmental disorder-associated formin, Fmn2, is a critical regulator of axon collateral branching. Fmn2 localizes to the collateral branch-inducing F-actin patches in axons and regulates the stability of these actin networks. The F-actin nucleation activity of Fmn2 protects the patches from ADF-mediated disassembly. Opposing activities of Fmn2 and ADF exert a dynamic regulatory control on axon collateral branch initiation and may underly the neurodevelopmental defects associated with Fmn2.

Coupling of dynamic microtubules to F-actin by Fmn2 regulates chemotaxis of neuronal growth cones.

Dynamic co-regulation of the actin and microtubule subsystems enables the highly precise and adaptive remodelling of the cytoskeleton necessary for critical cellular processes, such as axonal pathfinding. The modes and mediators of this interpolymer crosstalk, however, are inadequately understood. We identify Fmn2, a non-diaphanous-related formin associated with cognitive disabilities, as a novel regulator of cooperative actin-microtubule remodelling in growth cones of both chick and zebrafish neurons. We show that Fmn2 stabilizes microtubules in the growth cones of cultured spinal neurons and in vivo. Super-resolution imaging revealed that Fmn2 facilitates guidance of exploratory microtubules along actin bundles into the chemosensory filopodia. Using live imaging, biochemistry and single-molecule assays, we show that a C-terminal domain in Fmn2 is necessary for the dynamic association between microtubules and actin filaments. In the absence of the cross-bridging function of Fmn2, filopodial capture of microtubules is compromised, resulting in destabilized filopodial protrusions and deficits in growth cone chemotaxis. Our results uncover a critical function for Fmn2 in actin-microtubule crosstalk in neurons and demonstrate that the modulation of microtubule dynamics via associations with F-actin is central to directional motility.

Synthesis of Phosphorodiamidate Morpholino Oligonucleotides Using Trityl and Fmoc Chemistry in an Automated Oligo Synthesizer.

Phosphorodiamidate morpholino oligonucleotides (PMOs) constitute 3 out of the 11 FDA-approved oligonucleotide-based drugs in the last 6 years. PMOs can effectively silence disease-causing genes and modify splicing. However, PMO synthesis has remained challenging for a variety of reasons: inefficient deprotection and coupling methods and instability of monomers. Here, we report the development of a suitable combination of resin supports, deblocking and coupling reagents for synthesizing PMOs using either trityl or Fmoc-protected chlorophosphoramidate monomers. The synthesized PMOs using both the methods on a solid support have been validated for gene silencing in a zebrafish model. The protocol was successfully transferred into an automated DNA synthesizer to make several sequences of PMOs, demonstrating for the first time the adaptation of regular PMOs in a commercial DNA synthesizer. Moreover, PMOs with longer than 20-mer sequences, including FDA-approved Eteplirsen (30-mer), were achieved in >20% overall yield that is superior to previous reports. Hybridization study shows that PMOs exhibit a higher binding affinity toward complementary DNA relative to the DNA/DNA duplex (>6 °C). Additionally, the introduction of Fmoc chemistry into PMOs opens up the possibility for PMO synthesis in commercial peptide synthesizers for future development.

Sensitivity of olfactory sensory neurons to food cues is tuned to nutritional states by Neuropeptide Y signaling.

Modulation of sensory perception by homeostatic feedback from physiological states is central to innate purposive behaviors. Olfaction is an important predictive modality for feeding-related behaviors and its modulation has been associated with hunger-satiety states. However, the mechanisms mapping internal states to chemosensory processing in order to modify behavior are poorly understood. In the zebrafish olfactory epithelium, a subset of olfactory sensory neurons (OSNs) and the terminal nerve projections express neuropeptide Y (NPY). Using a combination of neuronal activity and behavioral evaluation, we find that NPY signaling in the peripheral olfactory system of zebrafish is correlated with its nutritional state and is both necessary and sufficient for the olfactory perception of food-related odorants. NPY activity dynamically modulates the microvillar OSN activation thresholds and acts cooperatively with amino acid signaling resulting in a switch-like increase in OSN sensitivity in starved animals. We suggest that cooperative activation of phospholipase C by convergent signaling from NPY and amino acid receptors is central to this heightened sensitivity. This study provides ethologically relevant, physiological evidence for NPY signaling in the modulation of OSN sensitivity to food-associated amino acid cues. We demonstrate sensory gating directly at the level of OSNs and identify a novel mechanistic framework for tuning olfactory sensitivity to prevailing energy states. Cover Image for this issue: https://doi.org/10.1111/jnc.15091.

Formin 2 regulates the stabilization of filopodial tip adhesions in growth cones and affects neuronal outgrowth and pathfinding in vivo.

Growth cone filopodia are actin-based mechanosensory structures that are essential for chemoreception and the generation of contractile forces necessary for directional motility. However, little is known about the influence of filopodial actin structures on substrate adhesion and filopodial contractility. Formin 2 (Fmn2) localizes along filopodial actin bundles and its depletion does not affect filopodia initiation or elongation. However, Fmn2 activity is required for filopodial tip adhesion maturation and the ability of filopodia to generate traction forces. Dysregulation of filopodia in Fmn2-depleted neurons leads to compromised growth cone motility. Additionally, in mouse fibroblasts, Fmn2 regulates ventral stress fiber assembly and affects the stability of focal adhesions. In the developing chick spinal cord, Fmn2 activity is required cell-autonomously for the outgrowth and pathfinding of spinal commissural neurons. Our results reveal an unanticipated function for Fmn2 in neural development. Fmn2 regulates structurally diverse bundled actin structures, parallel filopodial bundles in growth cones and anti-parallel stress fibers in fibroblasts, in turn modulating the stability of substrate adhesions. We propose Fmn2 as a mediator of actin bundle integrity, enabling efficient force transmission to the adhesion sites.

Cytoskeletal Mechanisms of Axonal Contractility.

Mechanotransduction is likely to be an important mechanism of signaling in thin, elongated cells such as neurons. Maintenance of prestress or rest tension may facilitate mechanotransduction in these cells. In recent years, functional roles for mechanical tension in neuronal development and physiology are beginning to emerge, but the cellular mechanisms regulating neurite tension remain poorly understood. Active contraction of neurites is a potential mechanism of tension regulation. In this study, we have explored cytoskeletal mechanisms mediating active contractility of neuronal axons. We have developed a simple assay in which we evaluate contraction of curved axons upon trypsin-mediated detachment. We show that curved axons undergo contraction and straighten upon deadhesion. Axonal straightening was found to be actively driven by actomyosin contractility, whereas microtubules may subserve a secondary role. We find that although axons show a monotonous decrease in length upon contraction, subcellularly, the cytoskeleton shows a heterogeneous contractile response. Further, using an assay for spontaneous development of tension without trypsin-induced deadhesion, we show that axons are intrinsically contractile. These experiments, using novel experimental approaches, implicate the axonal cytoskeleton in tension homeostasis. Our data suggest that although globally, the axon behaves as a mechanical continuum, locally, the cytoskeleton is remodeled heterogeneously.

cAMP signaling mediates behavioral flexibility and consolidation of social status in Drosophila aggression.

Social rituals, such as male-male aggression in Drosophila, are often stereotyped and the component behavioral patterns modular. The likelihood of transition from one behavioral pattern to another is malleable by experience and confers flexibility to the behavioral repertoire. Experience-dependent modification of innate aggressive behavior in flies alters fighting strategies during fights and establishes dominant-subordinate relationships. Dominance hierarchies resulting from agonistic encounters are consolidated to longer-lasting, social-status-dependent behavioral modifications, resulting in a robust loser effect. We showed that cAMP dynamics regulated by the calcium-calmodulin-dependent adenylyl cyclase, Rut, and the cAMP phosphodiesterase, Dnc, but not the Amn gene product, in specific neuronal groups of the mushroom body and central complex, mediate behavioral plasticity necessary to establish dominant-subordinate relationships. rut and dnc mutant flies were unable to alter fighting strategies and establish dominance relationships during agonistic interactions. This real-time flexibility during a fight was independent of changes in aggression levels. Longer-term consolidation of social status in the form of a loser effect, however, required additional Amn-dependent inputs to cAMP signaling and involved a circuit-level association between the α/ß and γ neurons of the mushroom body. Our findings implicate cAMP signaling in mediating the plasticity of behavioral patterns in aggressive behavior and in the generation of a temporally stable memory trace that manifests as a loser effect.

Immunogenicity of SARS-CoV-2 vaccines BBV152 (COVAXIN®) and ChAdOx1 nCoV-19 (COVISHIELD™) in seronegative and seropositive individuals in India: a multicentre, nonrandomised observational study.

Background: There are limited global data on head-to-head comparisons of vaccine platforms assessing both humoral and cellular immune responses, stratified by pre-vaccination serostatus. The COVID-19 vaccination drive for the Indian population in the age group 18-45 years began in April 2021 when seropositivity rates in the general population were rising due to the delta wave of COVID-19 pandemic during April-May 2021. Methods: Between June 30, 2021, and Jan 28, 2022, we enrolled 691 participants in the age group 18-45 years across four clinical sites in India. In this non-randomised and laboratory blinded study, participants received either two doses of Covaxin® (4 weeks apart) or two doses of Covishield™ (12 weeks apart) as per the national vaccination policy. The primary outcome was the seroconversion rate and the geometric mean titre (GMT) of antibodies against the SARS-CoV-2 spike and nucleocapsid proteins post two doses. The secondary outcome was the frequency of cellular immune responses pre- and post-vaccination. Findings: When compared to pre-vaccination baseline, both vaccines elicited statistically significant seroconversion and binding antibody levels in both seronegative and seropositive individuals. In the per-protocol cohort, Covishield™ elicited higher antibody responses than Covaxin® as measured by seroconversion rate (98.3% vs 74.4%, p < 0.0001 in seronegative individuals; 91.7% vs 66.9%, p < 0.0001 in seropositive individuals) as well as by anti-spike antibody levels against the ancestral strain (GMT 1272.1 vs 75.4 binding antibody units/ml [BAU/ml], p < 0.0001 in seronegative individuals; 2089.07 vs 585.7 BAU/ml, p < 0.0001 in seropositive individuals). As participants at all clinical sites were not recruited at the same time, site-specific immunogenicity was impacted by the timing of vaccination relative to the delta and omicron waves. Surrogate neutralising antibody responses against variants-of-concern including delta and omicron was higher in Covishield™ recipients than in Covaxin® recipients; and in seropositive than in seronegative individuals after both vaccination and asymptomatic infection (omicron variant). T cell responses are reported from only one of the four site cohorts where the vaccination schedule preceded the omicron wave. In seronegative individuals, Covishield™ elicited both CD4+ and CD8+ spike-specific cytokine-producing T cells whereas Covaxin® elicited mainly CD4+ spike-specific T cells. Neither vaccine showed significant post-vaccination expansion of spike-specific T cells in seropositive individuals. Interpretation: Covishield™ elicited immune responses of higher magnitude and breadth than Covaxin® in both seronegative individuals and seropositive individuals, across cohorts representing the pre-vaccination immune history of most of the vaccinated Indian population. Funding: Corporate social responsibility (CSR) funding from Hindustan Unilever Limited (HUL) and Unilever India Pvt. Ltd. (UIPL).

A tale of two waves: Delineating diverse genomic and transmission landscapes driving the COVID-19 pandemic in Pune, India.

BACKGROUND: Modern response to pandemics, critical for effective public health measures, is shaped by the availability and integration of diverse epidemiological outbreak data. Tracking variants of concern (VOC) is integral to understanding the evolution of SARS-CoV-2 in space and time, both at the local level and global context. This potentially generates actionable information when integrated with epidemiological outbreak data. METHODS: A city-wide network of researchers, clinicians, and pathology diagnostic laboratories was formed for genome surveillance of COVID-19 in Pune, India. The genomic landscapes of 10,496 sequenced samples of SARS-CoV-2 driving peaks of infection in Pune between December-2020 to March-2022, were determined. As a modern response to the pandemic, a "band of five" outbreak data analytics approach was used. This integrated the genomic data (Band 1) of the virus through molecular phylogenetics with key outbreak data including sample collection dates and case numbers (Band 2), demographics like age and gender (Band 3-4), and geospatial mapping (Band 5). RESULTS: The transmission dynamics of VOCs in 10,496 sequenced samples identified B.1.617.2 (Delta) and BA(x) (Omicron formerly known as B.1.1.529) variants as drivers of the second and third peaks of infection in Pune. Spike Protein mutational profiling during pre and post-Omicron VOCs indicated differential rank ordering of high-frequency mutations in specific domains that increased the charge and binding properties of the protein. Time-resolved phylogenetic analysis of Omicron sub-lineages identified a highly divergent BA.1 from Pune in addition to recombinant X lineages, XZ, XQ, and XM. CONCLUSIONS: The band of five outbreak data analytics approach, which integrates five different types of data, highlights the importance of a strong surveillance system with high-quality meta-data for understanding the spatiotemporal evolution of the SARS-CoV-2 genome in Pune. These findings have important implications for pandemic preparedness and could be critical tools for understanding and responding to future outbreaks.

The Formin Fmn2b Is Required for the Development of an Excitatory Interneuron Module in the Zebrafish Acoustic Startle Circuit.

The formin family member Fmn2 is a neuronally enriched cytoskeletal remodeling protein conserved across vertebrates. Recent studies have implicated Fmn2 in neurodevelopmental disorders, including sensory processing dysfunction and intellectual disability in humans. Cellular characterization of Fmn2 in primary neuronal cultures has identified its function in the regulation of cell-substrate adhesion and consequently growth cone translocation. However, the role of Fmn2 in the development of neural circuits in vivo, and its impact on associated behaviors have not been tested. Using automated analysis of behavior and systematic investigation of the associated circuitry, we uncover the role of Fmn2b in zebrafish neural circuit development. As reported in other vertebrates, the zebrafish ortholog of Fmn2 is also enriched in the developing zebrafish nervous system. We find that Fmn2b is required for the development of an excitatory interneuron pathway, the spiral fiber neuron, which is an essential circuit component in the regulation of the Mauthner cell (M-cell)-mediated acoustic startle response. Consistent with the loss of the spiral fiber neurons tracts, high-speed video recording revealed a reduction in the short latency escape events while responsiveness to the stimuli was unaffected. Taken together, this study provides evidence for a circuit-specific requirement of Fmn2b in eliciting an essential behavior in zebrafish. Our findings underscore the importance of Fmn2 in neural development across vertebrate lineages and highlight zebrafish models in understanding neurodevelopmental disorders.

Transient systemic inflammation in adult male mice results in underweight progeny.

PROBLEM: While the testes represent an immune-privileged organ, there is evidence that systemic inflammation is accompanied by local inflammatory responses. We therefore examined whether transient systemic inflammation caused any inflammatory and functional consequences in murine testes. METHOD OF STUDY: Using a single systemic administration of Toll-like receptor (TLR) agonists [lipopolysaccharide (LPS) or peptidoglycan (PG) or polyinosinic-polycytidylic acid (polyIC)] in young adult male mice, we assessed testicular immune-inflammatory landscape and reproductive functionality. RESULTS: Our findings demonstrated a significant induction of testicular TNF-α, IL-1ß and IL-6 transcripts within 24 h of TLR agonist injection. By day 6, these cytokine levels returned to baseline. While there was no change in caudal sperm counts at early time points, eight weeks later, twofold decrease in sperm count and reduced testicular testosterone levels were evident. When these mice were subjected to mating studies, no differences in mating efficiencies or litter sizes were observed compared with controls. Nonetheless, the neonatal weights of progeny from LPS/PG/polyIC-treated sires were significantly lower than controls. Postnatal weight gain up to three weeks was also slower in the progeny of LPS/polyIC-treated sires. Placental weights at 17.5 days post-coitum were significantly lower in females mated to LPS- and polyIC-treated males. Given this likelihood of an epigenetic effect, we found lower testicular levels of histone methyltransferase enzyme, mixed-lineage leukaemia-1, in mice given LPS/PG/polyIC 8 weeks earlier. CONCLUSION: Exposure to transient systemic inflammation leads to transient local inflammation in the testes, with persistent sperm-mediated consequences for foetal development.

The HSPGs Syndecan and Dallylike bind the receptor phosphatase LAR and exert distinct effects on synaptic development.

The formation and plasticity of synaptic connections rely on regulatory interactions between pre- and postsynaptic cells. We show that the Drosophila heparan sulfate proteoglycans (HSPGs) Syndecan (Sdc) and Dallylike (Dlp) are synaptic proteins necessary to control distinct aspects of synaptic biology. Sdc promotes the growth of presynaptic terminals, whereas Dlp regulates active zone form and function. Both Sdc and Dlp bind at high affinity to the protein tyrosine phosphatase LAR, a conserved receptor that controls both NMJ growth and active zone morphogenesis. These data and double mutant assays showing a requirement of LAR for actions of both HSPGs lead to a model in which presynaptic LAR is under complex control, with Sdc promoting and Dlp inhibiting LAR in order to control synapse morphogenesis and function.

Complex interactions amongst N-cadherin, DLAR, and Liprin-alpha regulate Drosophila photoreceptor axon targeting.

The formation of stable adhesive contacts between pre- and post-synaptic neurons represents the initial step in synapse assembly. The cell adhesion molecule N-cadherin, the receptor tyrosine phosphatase DLAR, and the scaffolding molecule Liprin-alpha play critical, evolutionarily conserved roles in this process. However, how these proteins signal to the growth cone and are themselves regulated remains poorly understood. Using Drosophila photoreceptors (R cells) as a model, we evaluate genetic and physical interactions among these three proteins. We demonstrate that DLAR function in this context is independent of phosphatase activity but requires interactions mediated by its intracellular domain. Genetic studies reveal both positive and, surprisingly, inhibitory interactions amongst all three genes. These observations are corroborated by biochemical studies demonstrating that DLAR physically associates via its phosphatase domain with N-cadherin in Drosophila embryos. Together, these data demonstrate that N-cadherin, DLAR, and Liprin-alpha function in a complex to regulate adhesive interactions between pre- and post-synaptic cells and provide a novel mechanism for controlling the activity of Liprin-alpha in the developing growth cone.

Axonal cytomechanics in neuronal development.

For more than a century, mechanical forces have been predicted to govern many biological processes during development, both at the cellular level and in tissue homeostasis. The cytomechanics of the thin and highly extended neuronal axons have intrigued generations of biologists and biophysicists. However, our knowledge of the biophysics of neurite growth and development is far from complete. Due to its motile behavior and its importance in axonal pathfinding, the growth cone has received significant attention. A considerable amount of information is now available on the spatiotemporal regulation of biochemical signaling and remodeling of the growth cone cytoskeleton. However, the cytoskeletal organization and dynamics in the axonal shaft were poorly explored until recently. Driven by advances in microscopy, there has been a surge of interest in the axonal cytoskeleton in the last few years. A major emerging area of investigation is the relationship between the axonal cytoskeleton and the diverse mechanobiological responses of neurons. This review attempts to summarize our current understanding of the axonal cytoskeleton and its critical role in governing axonal mechanics in the context of neuronal development.

Fmn2 Regulates Growth Cone Motility by Mediating a Molecular Clutch to Generate Traction Forces.

Growth cone-mediated axonal outgrowth and accurate synaptic targeting are central to brain morphogenesis. Translocation of the growth cone necessitates mechanochemical regulation of cell-extracellular matrix interactions and the generation of propulsive traction forces onto the growth environment. However, the molecular mechanisms subserving force generation by growth cones remain poorly characterized. The formin family member, Fmn2, has been identified earlier as a regulator of growth cone motility. Here, we explore the mechanisms underlying Fmn2 function in the growth cone. Evaluation of multiple components of the adhesion complexes suggests that Fmn2 regulates point contact stability. Analysis of F-actin retrograde flow reveals that Fmn2 functions as a clutch molecule and mediates the coupling of the actin cytoskeleton to the growth substrate, via point contact adhesion complexes. Using traction force microscopy, we show that the Fmn2-mediated clutch function is necessary for the generation of traction stresses by neurons. Our findings suggest that Fmn2, a protein associated with neurodevelopmental and neurodegenerative disorders, is a key regulator of a molecular clutch activity and consequently motility of neuronal growth cones.

The axonal actin-spectrin lattice acts as a tension buffering shock absorber.

Axons span extreme distances and are subject to significant stretch deformations during limb movements or sudden head movements, especially during impacts. Yet, axon biomechanics, and its relation to the ultrastructure that allows axons to withstand mechanical stress, is poorly understood. Using a custom developed force apparatus, we demonstrate that chick dorsal root ganglion axons exhibit a tension buffering or strain-softening response, where its steady state elastic modulus decreases with increasing strain. We then explore the contributions from the various cytoskeletal components of the axon to show that the recently discovered membrane-associated actin-spectrin scaffold plays a prominent mechanical role. Finally, using a theoretical model, we argue that the actin-spectrin skeleton acts as an axonal tension buffer by reversibly unfolding repeat domains of the spectrin tetramers to release excess mechanical stress. Our results revise the current viewpoint that microtubules and their associated proteins are the only significant load-bearing elements in axons.

Axonal heparan sulfate proteoglycans regulate the distribution and efficiency of the repellent slit during midline axon guidance.

The presentation of secreted axon guidance factors plays a major role in shaping central nervous system (CNS) connectivity. Recent work suggests that heparan sulfate (HS) regulates guidance factor activity; however, the in vivo axon guidance roles of its carrier proteins (heparan sulfate proteoglycans, or HSPGs) are largely unknown. Here we demonstrate through genetic analysis in vivo that the HSPG Syndecan (Sdc) is critical for the fidelity of Slit repellent signaling at the midline of the Drosophila CNS, consistent with the localization of Sdc to CNS axons. sdc mutants exhibit consistent defects in midline axon guidance, plus potent and specific genetic interactions supporting a model in which HSPGs improve the efficiency of Slit localization and/or signaling. To test this hypothesis, we show that Slit distribution is altered in sdc mutants and that Slit and its receptor bind to Sdc. However, when we compare the function of the transmembrane Sdc to a different class of HSPG that localizes to CNS axons (Dallylike), we find functional redundancy, suggesting that these proteoglycans act as spatially specific carriers of common HS structures that enable growth cones to interact with and perceive Slit as it diffuses away from its source at the CNS midline.

CART neuropeptide modulates the extended amygdalar CeA-vBNST circuit to gate expression of innate fear.

Innate fear is critical for the survival of animals and is under tight homeostatic control. Deregulation of innate fear processing is thought to underlie pathological phenotypes including, phobias and panic disorders. Although central processing of conditioned fear has been extensively studied, the circuitry and regulatory mechanisms subserving innate fear remain relatively poorly defined. In this study, we identify cocaine- and amphetamine-regulated transcript (CART) neuropeptide signaling in the central amygdala (CeA) - ventral bed nucleus of stria terminalis (vBNST) axis as a key modulator of innate fear expression. 2,4,5-trimethyl-3-thiazoline (TMT), a component of fox faeces, induces a freezing response whose intensity is regulated by the extent of CART-signaling in the CeA neurons. Abrogation of CART activity in the CeA attenuates the freezing response and reduces activation of vBNST neurons. Conversely, ectopically elevated CART signaling in the CeA potentiates the fear response concomitant with enhanced vBNST activation. We show that local levels of CART signaling modulate the activation of CeA neurons by NMDA receptor-mediated glutamatergic inputs, in turn, regulating activity in the vBNST. This study identifies the extended amygdalar CeA-vBNST circuit as a CART modulated axis encoding innate fear. CART signaling regulates the glutamatergic excitatory drive in the CeA-vBNST circuit, in turn, gating the expression of the freezing response to TMT.