Axonal Growth Research Articles

Molecular signaling predicts corticospinal axon growth state and muscle response plasticity induced by neuromodulation

Electrical motor cortex stimulation can produce corticospinal system plasticity and enhance motor function after injury. We investigate molecular mechanisms of structural and physiological plasticity following electrical neuromodulation, focusing on identifying molecular predictors, or biomarkers, for durable plasticity. We used two neuromodulation protocols, repetitive multipulse stimulation (rMPS) and patterned intermittent theta burst stimulation (iTBS), incorporating different stimulation durations and follow-up periods. We compared neuromodulation efficacy in promoting corticospinal tract (CST) sprouting, motor cortex muscle evoked potential (MEP) LTP-like plasticity, and their associated molecular underpinnings. Only iTBS produced CST sprouting after short-term neuromodulation (1 d of stimulation; 9-d survival for sprouting expression); both iTBS and rMPS produced sprouting with long-term (10-d) neuromodulation. Significant mTOR signaling activation and phosphatase and tensin homolog (PTEN) protein deactivation predicted axon growth across all neuromodulation conditions that produced significant sprouting. Both neuromodulation protocols, regardless of duration, were effective in producing MEP enhancement. However, persistent LTP-like enhancement of MEPs at 30 d was only produced by long-term iTBS. Statistical modeling suggests that Stat3 signaling is the key mediator of MEP enhancement. Cervical spinal cord injury (SCI) alone did not affect baseline molecular signaling. Whereas iTBS and rMPS after SCI produced strong mTOR activation and PTEN deactivation, only iTBS produced Stat3 activation. Our findings support differential molecular biomarkers for neuromodulation-dependent structural and physiological plasticity and show that motor cortex epidural neuromodulation produces molecular changes in neurons that support axonal growth after SCI. iTBS may be more suitable for repair after SCI because it promotes molecular signaling for both CST growth and MEP plasticity.

Emerging role of microglia in the developing dopaminergic system: perturbation by early life stress

Early life stress correlates with a higher prevalence of neurological disorders, including autism, attention-deficit/hyperactivity disorder, schizophrenia, depression, and Parkinson's disease. These conditions, primarily involving abnormal development and damage of the dopaminergic system, pose significant public health challenges. Microglia, as the primary immune cells in the brain, are crucial in regulating neuronal circuit development and survival. From the embryonic stage to adulthood, microglia exhibit stage-specific gene expression profiles, transcriptome characteristics, and functional phenotypes, enhancing the susceptibility to early life stress. However, the role of microglia in mediating dopaminergic system disorders under early life stress conditions remains poorly understood. This review presents an up-to-date overview of preclinical studies elucidating the impact of early life stress on microglia, leading to dopaminergic system disorders, along with the underlying mechanisms and therapeutic potential for neurodegenerative and neurodevelopmental conditions. Impaired microglial activity damages dopaminergic neurons by diminishing neurotrophic support (e.g., insulin-like growth factor-1) and hinders dopaminergic axon growth through defective phagocytosis and synaptic pruning. Furthermore, blunted microglial immunoreactivity suppresses striatal dopaminergic circuit development and reduces neuronal transmission. Furthermore, inflammation and oxidative stress induced by activated microglia can directly damage dopaminergic neurons, inhibiting dopamine synthesis, reuptake, and receptor activity. Enhanced microglial phagocytosis inhibits dopamine axon extension. These long-lasting effects of microglial perturbations may be driven by early life stress–induced epigenetic reprogramming of microglia. Indirectly, early life stress may influence microglial function through various pathways, such as astrocytic activation, the hypothalamic–pituitary–adrenal axis, the gut–brain axis, and maternal immune signaling. Finally, various therapeutic strategies and molecular mechanisms for targeting microglia to restore the dopaminergic system were summarized and discussed. These strategies include classical antidepressants and antipsychotics, antibiotics and anti-inflammatory agents, and herbal-derived medicine. Further investigations combining pharmacological interventions and genetic strategies are essential to elucidate the causal role of microglial phenotypic and functional perturbations in the dopaminergic system disrupted by early life stress.

Unraveling the Nexus: The Role of Collapsin Response Mediator Protein 2 Phosphorylation in Neurodegeneration and Neuroregeneration

AbstractNeurodegenerative disease characterized by the progressive damage of the nervous system, and neuropathies caused by the neuronal injury are both led to substantial impairments in neural function and quality of life among geriatric populations. Recovery from nerve damage and neurodegenerative diseases present a significant challenge, as the central nervous system (CNS) has limited capacity for self-repair. Investigating mechanism of neurodegeneration and regeneration is essential for advancing our understanding and development of effective therapies for nerve damage and degenerative conditions, which can significantly enhance patient outcomes. Collapsin response mediator protein 2 (CRMP2) was first identified as a key mediator of axonal growth and guidance is essential for neurogenesis and neuroregeneration. Phosphorylation as a primary modification approach of CRMP2 facilitates its involvement in numerous physiological processes, including axonal guidance, neuroplasticity, and cytoskeleton dynamics. Prior research on CRMP2 phosphorylation has elucidated its involvement in the mechanisms of neurodegenerative diseases and nerve damage. Pharmacological and genetic interventions that alter CRMP2 phosphorylation have shown the potential to influence neurodegenerative diseases and promote nerve regeneration. Even with decades of research delving into the intricacies of CRMP2 phosphorylation, there remains a scarcity of comprehensive literature reviews addressing this topic. This absence of synthesis and integration of findings hampers the field’s progress by preventing a holistic understanding of CRMP2’s implications in neurobiology, thereby impeding potential advancements in clinical treatments and interventions. This review intends to compile investigations focused on the role of CRMP2 phosphorylation in both neurodegenerative disease models and injury models to summarizing impacts and offer novel insight for clinical therapies.

Bioactive Peptide Hydrogel Scaffold with High Fluidity, Thermosensitivity, and Neurotropism in 3D Spatial Structure for Promoted Repair of Spinal Cord Injury.

Spinal cord injury (SCI) has been considered a clinically challenging disease that is characterized by local disturbance of the microenvironment, which inhibits post-injury neural regeneration. The simulation of a microenvironment conducive to the regeneration of spinal cord is beneficial for SCI repair. In this study, bioactive composite hydrogels are developed that mimic the regenerative microenvironment of spinal cord for enhanced SCI repair. The fabricated composite hydrogels (CRP) based on chitosan (CS), RADA16 nanofibers, and nerve-promoted peptide (PPFLMLLKGSTR) exhibit excellent injectability, superior biodegradability and biocompatibility. In addition, the CRP hydrogels can formquickly (a few minutes) by mixing three components at human body temperature, showing high potential as a biomimetic matrix for in situ repair of SCI. The in vitro studies demonstrate that the CRP hydrogels can not only promote the proliferation and migration of bone marrow mesenchymal stem cells but also induce the proliferation and differentiation of neural stem cells (NSCs) into neurons. Meanwhile, the hydrogels reveal the efficiency of protecting neurons and promoting axonal growth. Furthermore, the in vivo tests prove that the CRP hydrogels can reduce post-SCI inflammatory responses, inhibit reactive astrocyte over-proliferation, and promote the migration, proliferation, and differentiation of endogenous NSCs, which agree well with the in vitro results. The pre-clinical test demonstrates that the CRP hydrogels restore the motor function in completely transected spinal cord rats, and the SCI repair mechanism may involve the activation of the PI3K/AKT/mTOR pathway. It is believed that the strategies shown in this work will be valuable for the design and synthesis of novel hydrogels for biomedical and tissue engineering applications.

The TOG5 domain of CKAP5 is required to interact with F-actin and promote microtubule advancement in neurons.

Microtubule (MT) and F-actin cytoskeletal crosstalk and organization are important aspects of axon guidance mechanisms, but how associated proteins facilitate this function remains largely unknown. While the MT-associated protein, CKAP5 (XMAP215/ch-TOG), has been best characterized as a MT polymerase, we have recently highlighted a novel role for CKAP5 in facilitating interactions between MT and F-actin in vitro and in embryonic Xenopus laevis neuronal growth cones. However, the mechanism by which it does so is unclear. Here, using in vitro reconstitution assays coupled with TIRF microscopy, we report that the TOG5 domain of CKAP5 is necessary for its ability to bind to and bundle actin filaments, as well as to crosslink MTs and F-actin in vitro. Additionally, we show that this novel MT/F-actin crosslinking function of CKAP5 is possible even in MT polymerase-incompetent mutants of CKAP5 in vivo. Indeed, CKAP5 requires both MT and F-actin binding, but not MT polymerization, to promote MT-F-actin alignment in growth cones and axon outgrowth. Taken together, our findings provide mechanistic insights into how MT populations penetrate the growth cone periphery through CKAP5-facilitated interaction with F-actin during axon outgrowth and guidance. [Media: see text] [Media: see text] [Media: see text].

TC10 differently controls the dynamics of Exo70 in growth cones of cortical and hippocampal neurons

The exocyst is an octameric protein complex that acts as a tether for GOLGI-derived vesicles at the plasma membrane during exocytosis. It is involved in membrane expansion during axonal outgrowth. Exo70 is a major subunit of the exocyst complex and is controlled by TC10, a Rho family GTPase. How TC10 affects the dynamics of Exo70 at the plasma membrane is not well understood. There is also evidence that TC10 controls Exo70 dynamics differently in nonpolar cells and axons. To address this, we used super-resolution microscopy to study the spatially resolved effects of TC10 on Exo70 dynamics in HeLa cells and the growth cone of cortical and hippocampal neurons. We generated single particle localization and trajectory maps and extracted mean square displacements, diffusion coefficients, and alpha coefficients to characterize Exo70 diffusion. We found that the diffusivity of Exo70 was different in nonpolar cells and in the growth cone of neurons. TC10 stimulated the mobility of Exo70 in HeLa cells, but decreased the diffusion of Exo70 in the growth cone of cortical neurons. In contrast to cortical neurons, TC10 overexpression did not affect the mobility of Exo70 in the axonal growth cone of hippocampal neurons. These data suggest that mainly exocyst tethering in cortical neurons was under the control of TC10.

Bioactive ECM-Mimicking Nerve Guidance Conduit for Enhancing Peripheral Nerve Repair

Extensive research efforts are being directed towards identifying alternatives to autografts for the treatment of peripheral nerve injuries (PNIs) with engineered nerve conduits (NGCs) identified as having potential for PNI patients. These NGCs, however, may not fulfill the necessary criteria for a successful transplant, such as sufficient mechanical structural support and functionalization. To address the aforementioned limitations of NGCs, the present investigation explored the development of double cross-linked hydrogels (o-CSMA-E) that integrate the biocompatibility of porcine tendon extracellular matrix (ECM) with the antimicrobial and conductive properties of methacrylated quaternary chitosan. The hydrogels had matrices that could promote the growth of axons and the transmission of neural signals. The hydrogels were subsequently incorporated into a nanofibrous PLLA-ZnO sheath scaffold (ZnO@PLLA) to emulate the natural nerve structure, guiding cell growth and facilitating nerve regeneration. The collaboration of core and sheath materials in ZnO@PLLA/o-CSMA-E nerve guidance conduits resulted in enhanced migration of Schwann cells, formation of myelin sheaths, and improved locomotion performance in rats with sciatic nerve defects when in vivo studies were undertaken. Notably, the in vivo studies demonstrated the similarity between the newly developed engineered NGCs and autologous transplants, with the newly engineered NGCs possessing the potential to promote functional recovery by mimicking the tubular structure and ECM of nerves.

Generation of a human induced pluripotent stem cell (iPSC) line from a patient with a CDKL5 gene mutation

CDKL5(Cyclin-dependent kinase-like 5) encodes a kinase that may influence components of molecular pathways associated with MeCP2 and regulates axon outgrowth, dendritic morphogenesis, and synapse formation early in life. Here, we report an induced pluripotent stem cell (iPSC) line generated from an epilepsy patient harboring the CDKL5 c.2684C > T (p.P895L) variant. Peripheral blood mononuclear cells from the patient were successfully reprogrammed into iPSCs that expressed pluripotency markers, a normal karyotype, and the capacity to differentiate into the three germ layers in vivo. This iPSC line is a potential resource for studying the pathogenic mechanisms of epilepsy.

Ex vivo magnetic resonance imaging of the human fetal brain.

The fetal brain undergoes a dynamic process of development during gestation, marked by well-orchestrated events such as neuronal proliferation, migration, axonal outgrowth, and dendritic arborization, mainly elucidated through histological studies. Ex vivo magnetic resonance imaging (MRI) has emerged as a useful tool for 3D visualization of the developing fetal brain, serving as a complementary tool to traditional histology. In this review, we summarized the commonly employed ex vivo MRI techniques and their advances in fetal brain imaging, as well as a standard protocol for postmortem fetal brain specimen collection and fixation. We then provided an overview of ex vivo MRI-based studies on the fetal brain. According to our review, ex vivo T1- or T2-weighted structural MRI has contributed to the characterization of the anatomy of transient neuronal proliferative zones, the basal ganglia, and the cortex. Diffusion MRI related techniques, such as diffusion tensor imaging and tractography, have helped to investigate the microstructural patterns of fetal brain tissue, as well as the early emergence and development of neuronal migration pathways and white matter bundles. Ex vivo MRI findings have shown strong histological correlations, supporting the potential of MRI in evaluating the developmental events in the fetal brain. Postmortem MRI examinations have also demonstrated comparable, and in certain cases, superior performance to traditional autopsy in revealing fetal brain abnormalities. In conclusion, ex vivo fetal brain MRI is an invaluable tool that provides unique insights into the early stages of brain development.

The Potential of Aloe Vera in Solution and in Blended Nanofibers Containing Poly (3-Hydroxybutyrate-Co-3-Hydroxyvalerate) as Substrates for Neurite Outgrowth.

This pilot study investigated the potential of aloe vera (AV) to promote neurite outgrowth in organotypic dorsal root ganglia (DRG) explants (n = 230) from neonatal rats (n = 15). Using this invitro model of acute axotomy, we assessed neurite outgrowth exceeding 1.5 times the explant diameter (viable explants) and measured the longest neurite length. These parameters were evaluated under control conditions and in cultures supplemented with commercial AV and four aligned scaffolds: poly-L-lactate (PLLA), polydioxanone (PDS), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and blended PHBV/AV. After 6 days of culture, explants were immunostained using neuron-specific marker Tuj1 and Schwann cell marker S100. Measurements were obtained with Image J software and analyzed using Jamovi 2.3. In control and AV dilution media, the study revealed radial tissue growth from the explant body with randomly oriented neurites, whereas in all scaffolds, bidirectional tissue growth occurred parallel to nanofibers. Binomial logistic regression analyses indicated that viable explants were more likely in the control group compared to PDS (p = 0.0042) and PHBV (p < 0.0001), with non-significant differences when compared to AV dilution, PLLA, and PHBV/AV. AV dilution showed a greater association with viable explants than PLLA (p = 0.0459), while non-significant difference was found between AV dilution and PHBV/AV. Additionally, the PHBV/AV scaffold predicted higher odds of viable explants than PLLA (p = 0.0479), PDS (p = 0.0001), and PHBV (p < 0.0001). Groups with similar probabilities of obtaining viable explants (control, AV dilution, and PHBV/AV) exhibited non-significant differences in their longest neurite lengths. In conclusion, control, AV dilution, and PHBV/AV yielded the highest probability of developing viable explants and the longest neurite lengths. This is the first study demonstrating the direct permissiveness of AV for axonal outgrowth. Furthermore, the blended PHBV/AV scaffold showed significant potential as a suitable scaffold for axonal regrowth and Schwann cell migration, ensuring controlled tissue formation for tissue engineering applications.

Chronic activation of corticospinal tract neurons after pyramidotomy injury enhances neither behavioral recovery nor axonal sprouting.

Modulation of neural activity is a promising strategy to influence the growth of axons and improve behavioral recovery after damage to the central nervous system. The benefits of neuromodulation likely depend on optimization across multiple input parameters. Here we used a chemogenetic approach to achieve continuous, long-term elevation of neural activity in murine corticospinal tract (CST) neurons. To specifically target CST neurons, AAV2-retro-DIO-hM3Dq-mCherry or matched mCherry control was injected to the cervical spinal cord of adult Emx1-Cre transgenic mice. Pilot studies verified efficient transgene expression in CST neurons and effective elevation of neural activity as assessed by cFos immunohistochemistry. In subsequent experiments mice were administered either DIO-hM3Dq-mCherry or control DIO-mCherry, were pre-trained on a pellet retrieval task, and then received unilateral pyramidotomy injury to selectively ablate the right CST. Mice then received continual clozapine via drinking water and weekly testing on the pellet retrieval task, followed by cortical injection of a viral tracer to assess cross-midline sprouting by the spared CST. After sacrifice at eight weeks post-injury immunohistochemistry for cFos verified elevated CST activity in hM3Dq-treated animals and immunohistochemistry for PKC-gamma verified unilateral ablation of the CST in all animals. Despite the chronic elevation of CST activity, however, both groups showed similar levels of cross-midline CST sprouting and similar success in the pellet retrieval task. These data indicate that continuous, long-term elevation of activity that is targeted specifically to CST neurons does not affect compensatory sprouting or directed forelimb movements.

Dual Role of Ibuprofen and Indomethacin in Promoting Peripheral Nerve Regeneration In Vitro.

Peripheral nerve injuries (PNI) can result in significant losses of motor and sensory function. Although peripheral nerves have an innate capacity for regeneration, restoration of function after severe injury remains suboptimal. The gold standard for peripheral nerve regeneration (PNR) is autologous nerve transplantation, but this method is limited by the generation of an additional surgical site, donor-site morbidity, and neuroma formation at the site of harvest. Although targeted drug compounds have the potential to influence axonal growth, there are no drugs currently approved to treat PNI. Therefore, we propose to repurpose commonly used nonsteroidal anti-inflammatory drugs (NSAIDs) to enhance PNR, facilitating easier clinical translation. Additionally, calcium signaling plays a crucial role in neuronal connectivity and regeneration, but how specific drugs modulate this process remains unclear. We developed an in vitro hollow channel collagen gel platform that successfully supports neuronal network formation. This study evaluated the effects of commonly used NSAIDs, namely ibuprofen and indomethacin, in our in vitro model of axonal growth, regeneration, and calcium signaling as potential treatments for PNI. Our results demonstrate enhanced axonal growth and regrowth with both ibuprofen and indomethacin, suggesting a positive influence on PNR. Further, these drugs showed enhanced calcium signaling dynamics, which we posit is a crucial aspect for nerve repair. Taken together, these findings highlight the potential of ibuprofen and indomethacin to be used as treatment options for PNI, given their dual capability to promote axonal growth and enhance calcium signaling.

Celsr3 drives development and connectivity of the acoustic startle hindbrain circuit.

In the developing brain, groups of neurons organize into functional circuits that direct diverse behaviors. One such behavior is the evolutionarily conserved acoustic startle response, which in zebrafish is mediated by a well-defined hindbrain circuit. While numerous molecular pathways that guide neurons to their synaptic partners have been identified, it is unclear if and to what extent distinct neuron populations in the startle circuit utilize shared molecular pathways to ensure coordinated development. Here, we show that the planar cell polarity (PCP)-associated atypical cadherins Celsr3 and Celsr2, as well as the Celsr binding partner Frizzled 3a/Fzd3a, are critical for axon guidance of two neuron types that form synapses with each other: the command-like neuron Mauthner cells that drive the acoustic startle escape response, and spiral fiber neurons which provide excitatory input to Mauthner cells. We find that Mauthner axon growth towards synaptic targets is vital for Mauthner survival. We also demonstrate that symmetric spiral fiber input to Mauthner cells is critical for escape direction, which is necessary to respond to directional threats. Moreover, we identify distinct roles for Celsr3 and Celsr2, as Celsr3 is required for startle circuit development while Celsr2 is dispensable, though Celsr2 can partially compensate for loss of Celsr3 in Mauthner cells. This contrasts with facial branchiomotor neuron migration in the hindbrain, which requires Celsr2 while we find that Celsr3 is dispensable. Combined, our data uncover critical and distinct roles for individual PCP components during assembly of the acoustic startle hindbrain circuit.

Complement C5a Implication in Axonal Growth After Injury.

Complement C5a protein has been shown to play a major role in tissue regeneration through interaction with its receptor (C5aR) on target cells. Expression of this receptor has been reported in the nervous system which, upon injury, has no treatment to restore the lost functions. This work aimed at investigating the Complement C5a effect on axonal growth after axotomy in vitro. Primary hippocampal neurons were isolated from embryonic Wistar rats. Cell expression of C5aR mRNA was verified by RT-PCR while its membrane expression, localization, and phosphorylation were investigated by immunofluorescence. Then, the effects of C5a on injured axonal growth were investigated using a 3D-printed microfluidic device. Immunofluorescence demonstrated that the primary cultures contained only mature neurons (93%) and astrocytes (7%), but no oligodendrocytes or immature neurons. Immunofluorescence revealed a co-localization of NF-L and C5aR only in the mature neurons where C5a induced the phosphorylation of its receptor. C5a application on injured axons in the microfluidic devices significantly increased both the axonal growth speed and length. Our findings highlight a new role of C5a in regeneration demonstrating an enhancement of axonal growth after axotomy. This may provide a future therapeutic tool in the treatment of central nervous system injury.

Glial cell derived pathway directs regenerating optic nerve axons toward the CNS midline.

Several RGC intrinsic signaling pathways have been shown to enhance RGC survival and RGC axonal growth after optic nerve injury. Yet an unresolved challenge for regenerating RGC axons is to properly navigate the optic chiasm located at the Central Nervous System midline. Here, we use live-cell imaging in larval zebrafish to show that regrowing RGC axons initiate growth toward the midline and extend along a trajectory similar to their original projection. From a candidate genetic screen, we identify the glycosyltransferase Lh3 to be required during the process of regeneration to direct regrowing RGC axons toward the midline. Moreover, we find that mutants in collagen 18a1 (col18a1), a putative Lh3 substrate, display RGC axonal misguidance phenotypes similar to those we observe in lh3 mutants, suggesting that lh3 may act through col18a1 during regeneration. Finally, we show that transgenic lh3 expression in sox10+ presumptive olig2+ oligodendrocytes located near the optic chiasm restores directed axonal growth. Combined these data identify lh3 and col18a1 as part of a glial derived molecular pathway critical for guiding in vivo regenerating RGC axons towards and across the optic chiasm.

MBL-1/Muscleblind regulates neuronal differentiation and controls the splicing of a terminal selector in Caenorhabditis elegans.

The muscleblind family of mRNA splicing regulators is conserved across species and regulates the development of muscles and the nervous system. However, how Muscleblind proteins regulate neuronal fate specification and neurite morphogenesis at the single-neuron level is not well understood. In this study, we found that the C. elegans Muscleblind/MBL-1 promotes axonal growth in the touch receptor neurons (TRNs) by regulating microtubule stability and polarity. Transcriptomic analysis identified dozens of MBL-1-controlled splicing events in genes related to neuronal differentiation or microtubule functions. Among the MBL-1 targets, the LIM-domain transcription factor mec-3 is the terminal selector for the TRN fate and induces the expression of many TRN terminal differentiation genes. MBL-1 promotes the splicing of the mec-3 long isoform, which is essential for TRN fate specification, and inhibits the short isoforms that have much weaker activities in activating downstream genes. MBL-1 promotes mec-3 splicing through three "YGCU(U/G)Y" motifs located in or downstream of the included exon, which is similar to the mechanisms used by mammalian Muscleblind and suggests a deeply conserved context-dependency of the splicing regulation. Interestingly, the expression of mbl-1 in the TRNs is dependent on the mec-3 long isoform, indicating a positive feedback loop between the splicing regulator and the terminal selector. Finally, through a forward genetic screen, we found that MBL-1 promotes neurite growth partly by inhibiting the DLK-1/p38 MAPK pathway. In summary, our study provides mechanistic understanding of the role of Muscleblind in regulating cell fate specification and neuronal morphogenesis.

Monoallelic de novo variants in DDX17 cause a neurodevelopmental disorder.

DDX17 is an RNA helicase shown to be involved in critical processes during the early phases of neuronal differentiation. Globally, we compiled a case-series of 11 patients with neurodevelopmental phenotypes harbouring de novo monoallelic variants in DDX17. All 11 patients in our case series had a neurodevelopmental phenotype, whereby intellectual disability, delayed speech and language, and motor delay predominated. We performed in utero cortical electroporation in the brain of developing mice, assessing axon complexity and outgrowth of electroporated neurons, comparing wild-type and Ddx17 knockdown. We then undertook ex vivo cortical electroporation on neuronal progenitors to quantitatively assess axonal development at a single cell resolution. Mosaic ddx17 crispants and heterozygous knockouts in Xenopus tropicalis were generated for assessment of morphology, behavioural assays, and neuronal outgrowth measurements. We further undertook transcriptomic analysis of neuroblastoma SH-SY5Y cells, to identify differentially expressed genes in DDX17-KD cells compared to controls. Knockdown of Ddx17 in electroporated mouse neurons in vivo showed delayed neuronal migration as well as decreased cortical axon complexity. Mouse primary cortical neurons revealed reduced axon outgrowth upon knockdown of Ddx17 in vitro. The axon outgrowth phenotype was replicated in crispant ddx17 tadpoles and in heterozygotes. Heterozygous tadpoles had clear neurodevelopmental defects and showed an impaired neurobehavioral phenotype. Transcriptomic analysis identified a statistically significant number of differentially expressed genes involved in neurodevelopmental processes in DDX17-KD cells compared to control cells. We have identified potential neurodevelopment disease-causing variants in a gene not previously associated with genetic disease, DDX17. We provide evidence for the role of the gene in neurodevelopment in both mammalian and non-mammalian species and in controlling the expression of key neurodevelopment genes.

Radixin: Roles in the Nervous System and Beyond.

Radixin is an ERM family protein that includes radixin, moesin, and ezrin. The importance of ERM family proteins has been attracting more attention, and studies on the roles of ERM in biological function and the pathogenesis of some diseases are accumulating. In particular, we have found that radixin is the most dramatically changed ERM protein in elevated glucose-treated Schwann cells. We systemically review the literature on ERM, radixin in focus, and update the roles of radixin in regulating cell morphology, interaction, and cell signaling pathways. The potential of radixin as a therapeutic target in neurodegenerative diseases and cancer was also discussed. Radixin research has focused on its cell functions, activation, and pathogenic roles in some diseases. Radixin and other ERM proteins maintain cell shape, growth, and motility. In the nervous system, radixin has been shown to prevent neurodegeneration and axonal growth. The activation of radixin is through phosphorylation of its conserved threonine residues. Radixin functions in cell signaling pathways by binding to membrane proteins and relaying the cell signals into the cells. Deficiency of radixin has been involved in the pathogenic process of diseases in the central nervous system and diabetic peripheral nerve injury. Moreover, radixin also plays a role in cell growth and drug resistance in multiple cancers. The trials of therapeutic potential through radixin modulation have been accumulating. However, the exact mechanisms underlying the roles of radixin are far from clarification. Radixin plays various roles in cells and is involved in developing neurodegenerative diseases and many types of cancers. Therefore, radixin may be considered a potential target for developing therapeutic strategies for its related diseases. Further elucidation of the function and the cell signaling pathways that are linked to radixin may open the avenue to finding novel therapeutic strategies for diseases in the nervous system and other body systems.

Rapid cyclic stretching induces a synthetic, proinflammatory phenotype in cultured human intestinal smooth muscle, with the potential to alter signaling to adjacent bowel cells.

Bowel smooth muscle experiences mechanical stress constantly during normal function, and pathologic mechanical stressors in disease states. We tested the hypothesis that pathologic mechanical stress could alter transcription to induce smooth muscle phenotypic class switching. Primary human intestinal smooth muscle cells (HISMCs), seeded on electrospun aligned poly-ε-caprolactone nano-fibrous scaffolds, were subjected to pathologic, high frequency (1 Hz) uniaxial 3% cyclic stretch (loaded) or kept unloaded in culture for 6 hours. Total RNA sequencing, qRT-PCR, and quantitative immunohistochemistry defined loading-induced changes in gene expression. NicheNet predicted how differentially expressed genes might impact HISMCs and other bowel cells. Loading induced differential expression of 4537 genes in HISMCs. Loaded HISMCs had a less contractile phenotype, with increased expression of synthetic SMC genes, proinflammatory cytokines, and altered expression of axon guidance molecules, growth factors and morphogens. Many differentially expressed genes encode secreted ligands that could act cell-autonomously on smooth muscle and on other cells in the bowel wall. HISMCs demonstrate remarkably rapid phenotypic plasticity in response to mechanical stress that may convert contractile HISMCs into proliferative, fibroblast-like cells or proinflammatory cells. These mechanical stress-induced changes in HISMC gene expression may be relevant for human bowel disease.

Netrin-1 stimulated axon growth requires the polyglutamylase TTLL1.

In the developing brain, neurons extend an axonal process through a complex and changing environment to form synaptic connections with the correct targets in response to extracellular cues. Microtubule and actin filaments provide mechanical support and drive axon growth in the correct direction. The axonal cytoskeleton responds to extracellular guidance cues. Netrin-1 is a multifunctional guidance cue that can induce alternate responses based on the bound receptor. The mechanism by which actin responds to Netrin-1 is well described. However, how Netrin-1 influences the microtubule cytoskeleton is less understood. Appropriate microtubule function is required for axon pathfinding, as mutations in tubulin phenocopy axon crossing defects of Netrin-1 and DCC mutants. Microtubule stabilization is required for attractive guidance cue response. The C-terminal tails of microtubules can be post-translationally modified. Post-translational modifications (PTMs) help control the microtubule cytoskeleton. We measured polyglutamylation in cultured primary mouse cortical neurons before and after Netrin-1 stimulation. We used immunohistochemistry to measure how Netrin-1 stimulation alters microtubule-associated protein localization. Next, we manipulated TTLL1 to determine if Netrin-1-induced axon growth and MAP localization depend on polyglutamylation levels. In this study, we investigated if Netrin-1 signaling alters microtubule PTMs in the axon. We found that microtubule polyglutamylation increases after Netrin-1 stimulation. This change in polyglutamylation is necessary for Netrin-1-induced axonal growth rate increases. We next determined that MAP1B and DCX localization changes in response to Netrin-1. These proteins can both stabilize the microtubule cytoskeleton and may be responsible for Netrin-1-induced growth response in neurons. The changes in DCX and MAP1B depend on TTLL1, a protein responsible for microtubule polyglutamylation.