RESUMEN
Major advances have been made in utilizing human-induced pluripotent stem cells (hiPSCs) for regenerative medicine. Nevertheless, the delivery and integration of hiPSCs into target tissues remain significant challenges, particularly in the context of retinal ganglion cell (RGC) restoration. In this study, we introduce a promising avenue for providing directional guidance to regenerated cells in the retina. First, we developed a technique for construction of gradient interfaces based on functionalized conductive polymers, which could be applied with various functionalized ehthylenedioxythiophene (EDOT) monomers. Using a tree-shaped channel encapsulated with a thin PDMS and a specially designed electrochemical chamber, gradient flow generation could be converted into a functionalized-PEDOT gradient film by cyclic voltammetry. The characteristics of the successfully fabricated gradient flow and surface were analyzed using fluorescent labels, time of flight secondary ion mass spectrometry (TOF-SIMS), and X-ray photoelectron spectroscopy (XPS). Remarkably, hiPSC-RGCs seeded on PEDOT exhibited improvements in neurite outgrowth, axon guidance and neuronal electrophysiology measurements. These results suggest that our novel gradient PEDOT may be used with hiPSC-based technologies as a potential biomedical engineering scaffold for functional restoration of RGCs in retinal degenerative diseases and optic neuropathies.
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Células Madre Pluripotentes Inducidas , Polímeros , Células Ganglionares de la Retina , Humanos , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/citología , Células Madre Pluripotentes Inducidas/citología , Polímeros/química , Orientación del Axón , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Propiedades de Superficie , Conductividad Eléctrica , Factores de Crecimiento Nervioso/metabolismo , Axones/metabolismo , Axones/fisiologíaRESUMEN
Neurons establish functional connections responsible for how we perceive and react to the world around us. Communication from a neuron to its target cell occurs through a long projection called an axon. Axon distances can exceed 1 m in length in humans and require a dynamic microtubule cytoskeleton for growth during development and maintenance in adulthood. Stathmins are microtubule-associated proteins that function as relays between kinase signaling and microtubule polymerization. In this review, we describe the prolific role of Stathmins in microtubule homeostasis with an emphasis on emerging roles for Stathmin-2 (Stmn2) in axon integrity and neurodegeneration. Stmn2 levels are altered in Amyotrophic Lateral Sclerosis and loss of Stmn2 provokes motor and sensory neuropathies. There is growing potential for employing Stmn2 as a disease biomarker or even a therapeutic target. Meeting this potential requires a mechanistic understanding of emerging complexity in Stmn2 function. In particular, Stmn2 palmitoylation has a surprising contribution to axon maintenance through undefined mechanisms linking membrane association, tubulin interaction, and axon transport. Exploring these connections will reveal new insight on neuronal cell biology and novel opportunities for disease intervention.
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Axones , Microtúbulos , Estatmina , Estatmina/metabolismo , Microtúbulos/metabolismo , Humanos , Axones/metabolismo , Axones/fisiología , Animales , Membrana Celular/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patologíaRESUMEN
The in vivo three-dimensional genomic architecture of adult mature neurons at homeostasis and after medically relevant perturbations such as axonal injury remains elusive. Here, we address this knowledge gap by mapping the three-dimensional chromatin architecture and gene expression program at homeostasis and after sciatic nerve injury in wild-type and cohesin-deficient mouse sensory dorsal root ganglia neurons via combinatorial Hi-C, promoter-capture Hi-C, CUT&Tag for H3K27ac and RNA-seq. We find that genes involved in axonal regeneration form long-range, complex chromatin loops, and that cohesin is required for the full induction of the regenerative transcriptional program. Importantly, loss of cohesin results in disruption of chromatin architecture and severely impaired nerve regeneration. Complex enhancer-promoter loops are also enriched in the human fetal cortical plate, where the axonal growth potential is highest, and are lost in mature adult neurons. Together, these data provide an original three-dimensional chromatin map of adult sensory neurons in vivo and demonstrate a role for cohesin-dependent long-range promoter interactions in nerve regeneration.
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Axones , Cromatina , Cohesinas , Regeneración Nerviosa , Regiones Promotoras Genéticas , Células Receptoras Sensoriales , Animales , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/fisiología , Ratones , Regiones Promotoras Genéticas/genética , Cromatina/metabolismo , Regeneración Nerviosa/genética , Regeneración Nerviosa/fisiología , Axones/metabolismo , Axones/fisiología , Humanos , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Elementos de Facilitación Genéticos/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Ganglios Espinales/metabolismo , Ganglios Espinales/citología , Nervio Ciático/metabolismoRESUMEN
Objective: To evaluate the safety of COVID-19 vaccines in patients with multiple sclerosis (MS) by assessing their impact on serum neurofilament light chain (sNfL) levels as a marker of neuroaxonal damage. Methods: Single-center observational longitudinal study including patients with MS who consecutively received their initial vaccination against SARS-CoV-2 at Hospital Universitario Ramón y Cajal, following the first national immunization program in Spain. Serum samples were collected at baseline and after receiving the second dose of the vaccine. sNfL levels were quantified using the single molecule array (SIMOA) technique. Adverse events, including clinical or radiological reactivation of the disease, were recorded. Results: Fifty-two patients were included (median age, 39.7 years [range, 22.5-63.3]; 71.2% female). After SARS-CoV-2 vaccination, no increased inflammatory activity, either determined by the presence of relapses and/or new MRI lesions and/or high sNfL levels, was detected. Accordingly, there was no difference between median sNfL levels before and after vaccination (5.39 vs. 5.76 pg/ml, p=0.6). Despite this, when looking at baseline patient characteristics before vaccination, younger age associated with disease activity after vaccination (OR 0.87, 95% CI: 0.77-0.98, p=0.022). Larger studies are needed to validate these results. Conclusion: COVID-19 vaccines did not cause reactivation of disease at a clinical, radiological or molecular level, thus suggesting that they are safe in MS patients.
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Vacunas contra la COVID-19 , COVID-19 , Esclerosis Múltiple , Proteínas de Neurofilamentos , SARS-CoV-2 , Humanos , Femenino , Masculino , Adulto , Persona de Mediana Edad , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/sangre , COVID-19/inmunología , COVID-19/prevención & control , COVID-19/sangre , Vacunas contra la COVID-19/efectos adversos , Vacunas contra la COVID-19/inmunología , Proteínas de Neurofilamentos/sangre , SARS-CoV-2/inmunología , Adulto Joven , Estudios Longitudinales , Biomarcadores/sangre , Axones/patología , España/epidemiología , Vacunación/efectos adversosRESUMEN
Objective: To explore the potential evidence of active peripheral nerve necrosis when n-hexane produces toxic effects on peripheral nerves. Methods: In May 2023, 36 SPF grade SD male rats with a body weight of 200-220 g were divided into 4 groups with 9 rats in each group and given normal saline and different doses of n-hexane (168, 675, 2 700 mg/kg) by gavage for 6 consecutive weeks (5 days/week). Three rats in each group were killed at the 2nd, 4th and 6th week, respectively. The spinal cord to sciatic nerve tissue was broken and the supernatant was extracted for SDS-PAGE protein isolation. The expression level of Sarm1 protein was analyzed with the ß-Actin color strip of internal reference protein by Western blot. The expression of Sarm1 protein was analyzed by the gray ratio of the two. At the 6th week, the sciatic nerve sections of the each group were observed by light microscope and electron microscope. Results: The number of axons was obviously reduced by light microscopy. According to electron microscope, myelin lesions were mainly local disintegration, deformation, and different thickness. The deformation of axonal surface became smaller. The axons in the nerve bundle membrane showed degeneration and reduction. The gray ratio of Sarm1 protein and internal reference protein bands in each group had no significant change at the second week of exposure, and the ratio of SARM1 protein to internal reference protein bands was 1.47 in the high dose group at the fourth week, and 1.51 and 1.89 in the middle and high dose group at the sixth week, respectively. Conclusion: Waller's degeneration was observed in sciatic neuropathologic manifestations of n-hexane-poisoned rats, and the expression level of Sarm1 protein increased.
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Hexanos , Nervio Ciático , Animales , Masculino , Ratas , Proteínas del Dominio Armadillo/metabolismo , Axones/metabolismo , Axones/patología , Proteínas del Citoesqueleto/metabolismo , Ratas Sprague-Dawley , Sarín/toxicidad , Sarín/envenenamiento , Nervio Ciático/metabolismoRESUMEN
Spatially and temporally accurate termination of axon outgrowth, a process called axon termination, is required for efficient, precise nervous system construction and wiring. The mechanosensory neurons that sense low-threshold mechanical stimulation or gentle touch have proven exceptionally valuable for studying axon termination over the past 40â years. In this Review, we discuss progress made in deciphering the molecular and genetic mechanisms that govern axon termination in touch receptor neurons. Findings across model organisms, including Caenorhabditis elegans, Drosophila, zebrafish and mice, have revealed that complex signaling is required for termination with conserved principles and players beginning to surface. A key emerging theme is that axon termination is mediated by complex signaling networks that include ubiquitin ligase signaling hubs, kinase cascades, transcription factors, guidance/adhesion receptors and growth factors. Here, we begin a discussion about how these signaling networks could represent termination codes that trigger cessation of axon outgrowth in different species and types of mechanosensory neurons.
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Axones , Transducción de Señal , Animales , Axones/metabolismo , Axones/fisiología , Mecanorreceptores/metabolismo , Caenorhabditis elegans/metabolismo , Drosophila/metabolismoRESUMEN
Recent studies highlighted the role of platelet-rich plasma (PRP) in progenitor cell homing, migration, and nerve cell regeneration while also inhibiting fibrosis and apoptosis in cavernous nerve injury (CNI). The aim of this study was to investigate the effect of PRP administration on axon and collagen regeneration in CNI. A true experimental study using a post-test-only control group design was conducted. Twenty-five male Wistar rats (Rattus norvegicus), weighing 200-300 grams, were divided into five groups: two control groups (sham procedure and negative control), and three experimental groups receiving local PRP, intraperitoneal PRP, and a combination of local and intraperitoneal PRP. The cavernous nerve was injured with a hemostasis clamp for one minute before 200 µL of 200 PRP was injected locally, intraperitoneally, or both, depending on the group. After four weeks, the rats were euthanized, tissue segments (2 mm) from each cavernous nerve and mid-penis were collected and analyzed for collagen density, axon diameter, and number of myelinated axons. Our study found that collagen growth was slower in CNI group without PRP (sham procedure) compared to all PRP groups (local, intraperitoneal, and combination). The intraperitoneal PRP group had the highest collagen density at 5.62 µm; however, no significant difference was observed in collagen density among all groups (p=0.056). Similar axon diameter was found across the groups, with no statistically significant difference observed (p=0.856). In the number of myelinated axons, a significant difference was found among all groups with significantly more axons in local PRP and combined local and intraperitoneal PRP groups compared to others (p=0.026). In conclusion, PRP administration improved the number of myelinated axons in CNI, suggesting PRP role in CNI regeneration and the potential for an innovative approach to treating erectile dysfunction associated with CNI.
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Axones , Colágeno , Disfunción Eréctil , Regeneración Nerviosa , Pene , Plasma Rico en Plaquetas , Ratas Wistar , Animales , Masculino , Colágeno/metabolismo , Ratas , Regeneración Nerviosa/efectos de los fármacos , Regeneración Nerviosa/fisiología , Axones/fisiología , Axones/patología , Axones/efectos de los fármacos , Pene/inervación , Pene/efectos de los fármacos , Disfunción Eréctil/terapia , Disfunción Eréctil/tratamiento farmacológico , Modelos Animales de Enfermedad , Traumatismos de los Nervios Periféricos/terapiaRESUMEN
Computer haptics (CH) is about integration of tactile sensation and rendering in Metaverse. However, unlike computer vision (CV) where both hardware infrastructure and software programs are well developed, a generic tactile data capturing device that serves the same role as what a camera does for CV, is missing. Bioinspired by electrophysiological processes in human tactile somatosensory nervous system, here we propose a tactile scanner along with a neuromorphically-engineered system, in which a closed-loop tactile acquisition and rendering (re-creation) are preliminarily achieved. Based on the architecture of afferent nerves and intelligent functions of mechano-gating and leaky integrate-and-fire models, such a tactile scanner is designed and developed by using piezoelectric transducers as axon neurons and thin film transistor (TFT)-based neuromorphic circuits to mimic synaptic behaviours and neural functions. As an example, the neuron-like tactile information of surface textures is captured and further used to render the texture friction of a virtual surface for "recreating" a "true" feeling of touch.
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Tacto , Humanos , Tacto/fisiología , Percepción del Tacto/fisiología , Neuronas/fisiología , Axones/fisiologíaRESUMEN
In this study, we develop an in silico model of a neuron's behaviour under demyelination caused by a cytokine storm to investigate the effects of viral infections in the brain. We use a comprehensive model to measure how cytokine-induced demyelination affects the propagation of action potential (AP) signals within a neuron. We analysed the effects of neuron-neuron communications by applying information and communication theory at different levels of demyelination. Our simulations demonstrate that virus-induced degeneration can play a role in the signal power and spiking rate, which compromise the propagation and processing of information between neurons. We propose a transfer function to model the weakening effects on the AP. Our results show that demyelination induced by a cytokine storm not only degrades the signal but also impairs its propagation within the axon. Our proposed in silico model can analyse virus-induced neurodegeneration and enhance our understanding of virus-induced demyelination.
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Simulación por Computador , Enfermedades Desmielinizantes , Neuronas , Enfermedades Desmielinizantes/patología , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/virología , Neuronas/metabolismo , Humanos , Modelos Neurológicos , Potenciales de Acción , Síndrome de Liberación de Citoquinas , Animales , Citocinas/metabolismo , Axones/metabolismo , Axones/patologíaRESUMEN
Traumatic brain injury (TBI) affects millions globally, with a majority of TBI cases being classified as mild, in which diffuse pathologies prevail. Two of the pathological hallmarks of TBI are diffuse axonal injury (DAI) and microglial activation. While progress has been made investigating the breadth of TBI-induced axonal injury and microglial changes in rodents, the neuroinflammatory progression and interaction between microglia and injured axons in humans is less well understood. Our group previously investigated microglial process convergence (MPC), in which processes of non-phagocytic microglia directly contact injured proximal axonal swellings, in rats and micropigs acutely following TBI. These studies demonstrated that MPC occurred on injured axons in the micropig, but not in the rat, following diffuse TBI. While it has been shown that microglia co-exist and interact with injured axons in humans post-TBI, the occurrence of MPC has not been quantitatively measured in the human brain. Therefore, in the current study we sought to validate our pig findings in human postmortem tissue. We investigated MPC onto injured axonal swellings and intact myelinated fibers in cases from individuals with confirmed DAI and control human brain tissue using multiplex immunofluorescent histochemistry. We found an increase in MPC onto injured axonal swellings, consistent with our previous findings in micropigs, indicating that MPC is a clinically relevant phenomenon that warrants further investigation.
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Axones , Lesión Axonal Difusa , Microglía , Humanos , Microglía/patología , Microglía/metabolismo , Axones/patología , Axones/metabolismo , Animales , Masculino , Porcinos , Lesión Axonal Difusa/patología , Lesión Axonal Difusa/metabolismo , Femenino , Encéfalo/patología , Encéfalo/metabolismo , Lesiones Traumáticas del Encéfalo/patología , Lesiones Traumáticas del Encéfalo/metabolismo , Persona de Mediana Edad , Autopsia , Adulto , Anciano , RatasRESUMEN
Glaucoma is a blinding disease where the retinal ganglion cells and their axons degenerate. Degradation of axonal microtubules is thought to play a critical role in the pathogenesis, but the mechanism is unknown. Here we investigate whether microtubule disruption in glaucoma can be alleviated by metabolic rescue. The integrity of axonal microtubules and the morphology of the retinal nerve fibers were evaluated by second-harmonic generation microscopy in a mouse model of glaucoma, DBA/2J, which received a dietary supplement of nicotinamide (NAM) for reducing metabolic stress. It was compared with control DBA/2J, which did not receive NAM, and non-glaucomatous DBA/2J-Gpnmb+. We found that the morphology of the retinal nerve fibers, but not axonal microtubules, are significantly protected by NAM. The decoupling is analogous to microtubule deficit, a glaucoma pathology in which axonal microtubules exhibit rapid degradation compared to the morphology of the retinal nerve fibers. Understanding microtubule deficit could provide insights into the divergent responses to NAM. From co-registered images of second-harmonic generation and immunofluorescence, it was determined that microtubule deficit was not due to a shortage of tubulins. Furthermore, microtubule deficit colocalized with the sectors in which the retinal ganglion cells were disconnected from the brain, suggesting that microtubule disruption is associated with axonal transport deficit in glaucoma. Together, our data suggests significant role axonal microtubules play in glaucomatous degeneration, offering a new opportunity for neuroprotection.
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Modelos Animales de Enfermedad , Glaucoma , Ratones Endogámicos DBA , Microtúbulos , Niacinamida , Células Ganglionares de la Retina , Animales , Glaucoma/patología , Glaucoma/metabolismo , Glaucoma/tratamiento farmacológico , Niacinamida/farmacología , Niacinamida/uso terapéutico , Ratones , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/patología , Células Ganglionares de la Retina/metabolismo , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Axones/efectos de los fármacos , Axones/metabolismo , Axones/patología , Microscopía/métodos , Fibras Nerviosas/efectos de los fármacos , Fibras Nerviosas/patología , Fibras Nerviosas/metabolismoRESUMEN
Understanding the sequence of cellular responses and their contributions to pathomorphogical changes in spinal white matter injuries is a prerequisite for developing efficient therapeutic strategies for spinal cord injury (SCI) as well as neurodegenerative and inflammatory diseases of the spinal cord such as amyotrophic lateral sclerosis and multiple sclerosis. We have developed several types of surgical procedures suitable for acute one-time and chronic recurrent in vivo multiphoton microscopy of spinal white matter [1]. Sophisticated surgical procedures were combined with transgenic mouse technology to image spinal tissue labeled with up to four fluorescent proteins (FPs) in axons, astrocytes, microglia, and blood vessels. To clearly separate the simultaneously excited FPs, spectral unmixing including iterative procedures was performed after imaging the diversely labeled spinal white matter with a custom-made 4-channel two-photon laser-scanning microscope. In our longitudinal multicellular studies of injured spinal white matter, we imaged axonal dynamics and invasion of microglia and astrocytes for a time course of over 200 days after SCI. Our methods offer ideal platforms for investigating acute and chronic cellular dynamics, cell-cell interactions, and metabolite fluctuations in health and disease as well as pharmacological manipulations in vivo.
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Axones , Ratones Transgénicos , Traumatismos de la Médula Espinal , Sustancia Blanca , Animales , Sustancia Blanca/patología , Sustancia Blanca/metabolismo , Sustancia Blanca/diagnóstico por imagen , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/diagnóstico por imagen , Axones/patología , Axones/metabolismo , Neuroglía/metabolismo , Neuroglía/patología , Ratones , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Médula Espinal/patología , Médula Espinal/metabolismo , Microglía/metabolismo , Microglía/patología , Astrocitos/metabolismo , Astrocitos/patologíaRESUMEN
BACKGROUND: Traumatic brain injury (TBI) causes axon tearing and synapse degradation, resulting in multiple neurological dysfunctions and exacerbation of early neurodegeneration; the repair of axonal and synaptic structures is critical for restoring neuronal function. C-C Motif Chemokine Ligand 5 (CCL5) shows many neuroprotective activities. METHOD: A close-head weight-drop system was used to induce mild brain trauma in C57BL/6 (wild-type, WT) and CCL5 knockout (CCL5-KO) mice. The mNSS score, rotarod, beam walking, and sticker removal tests were used to assay neurological function after mTBI in different groups of mice. The restoration of motor and sensory functions was impaired in CCL5-KO mice after one month of injury, with swelling of axons and synapses from Golgi staining and reduced synaptic proteins-synaptophysin and PSD95. Administration of recombinant CCL5 (Pre-treatment: 300 pg/g once before injury; or post-treatment: 30 pg/g every 2 days, since 3 days after injury for 1 month) through intranasal delivery into mouse brain improved the motor and sensory neurological dysfunctions in CCL5-KO TBI mice. RESULTS: Proteomic analysis using LC-MS/MS identified that the "Nervous system development and function"-related proteins, including axonogenesis, synaptogenesis, and myelination signaling pathways, were reduced in injured cortex of CCL5-KO mice; both pre-treatment and post-treatment with CCL5 augmented those pathways. Immunostaining and western blot analysis confirmed axonogenesis and synaptogenesis related Semaphorin, Ephrin, p70S6/mTOR signaling, and myelination-related Neuregulin/ErbB and FGF/FAK signaling pathways were up-regulated in the cortical tissue by CCL5 after brain injury. We also noticed cortex redevelopment after long-term administration of CCL5 after brain injury with increased Reelin positive Cajal-Rerzius Cells and CXCR4 expression. CCL5 enhanced the growth of cone filopodia in a primary neuron culture system; blocking CCL5's receptor CCR5 by Maraviroc reduced the intensity of filopodia in growth cone and also CCL5 mediated mTOR and Rho signalling activation. Inhibiting mTOR and Rho signaling abolished CCL5 induced growth cone formation. CONCLUSIONS: CCL5 plays a critical role in starting the intrinsic neuronal regeneration system following TBI, which includes growth cone formation, axonogenesis and synaptogensis, remyelination, and the subsequent proper wiring of cortical circuits. Our study underscores the potential of CCL5 as a robust therapeutic stratagem in treating axonal injury and degeneration during the chronic phase after mild brain injury.
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Axones , Quimiocina CCL5 , Ratones Endogámicos C57BL , Ratones Noqueados , Animales , Ratones , Quimiocina CCL5/metabolismo , Axones/metabolismo , Axones/fisiología , Lesiones Traumáticas del Encéfalo/metabolismo , Lesiones Traumáticas del Encéfalo/fisiopatología , Masculino , Neuronas/metabolismo , Lesiones Encefálicas/metabolismo , NeurogénesisRESUMEN
ER-phagy is a specialized form of autophagy, defined by the lysosomal degradation of ER subdomains. ER-phagy has been implicated in relieving the ER from misfolded proteins during ER stress upon activation of the unfolded protein response (UPR). Here, we identified an essential role for the ER chaperone calnexin in regulating ER-phagy and the UPR in neurons. We showed that chemical induction of ER stress triggers ER-phagy in the somata and axons of primary cultured motoneurons. Under basal conditions, the depletion of calnexin leads to an enhanced ER-phagy in axons. However, upon ER stress induction, ER-phagy did not further increase in calnexin-deficient motoneurons. In addition to increased ER-phagy under basal conditions, we also detected an elevated proteasomal turnover of insoluble proteins, suggesting enhanced protein degradation by default. Surprisingly, we detected a diminished UPR in calnexin-deficient early cortical neurons under ER stress conditions. In summary, our data suggest a central role for calnexin in orchestrating both ER-phagy and the UPR to maintain protein homeostasis within the ER.
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Calnexina , Estrés del Retículo Endoplásmico , Retículo Endoplásmico , Respuesta de Proteína Desplegada , Calnexina/metabolismo , Animales , Retículo Endoplásmico/metabolismo , Ratones , Autofagia , Neuronas Motoras/metabolismo , Axones/metabolismo , Células Cultivadas , Lisosomas/metabolismo , Neuronas/metabolismoRESUMEN
Traumatic spinal cord injury (tSCI) has complex pathophysiological events that begin after the initial trauma. One such event is fibroglial scar formation by fibroblasts and reactive astrocytes. A strong inhibition of axonal growth is caused by the activated astroglial cells as a component of fibroglial scarring through the production of inhibitory molecules, such as chondroitin sulfate proteoglycans or myelin-associated proteins. Here, we used neural precursor cells (aldynoglia) as promoters of axonal growth and a fibrin hydrogel gelled under alkaline conditions to support and guide neuronal cell growth, respectively. We added Tol-51 sulfoglycolipid as a synthetic inhibitor of astrocyte and microglia in order to test its effect on the axonal growth-promoting function of aldynoglia precursor cells. We obtained an increase in GFAP expression corresponding to the expected glial phenotype for aldynoglia cells cultured in alkaline fibrin. In co-cultures of dorsal root ganglia (DRG) and aldynoglia, the axonal growth promotion of DRG neurons by aldynoglia was not affected. We observed that the neural precursor cells first clustered together and then formed niches from which aldynoglia cells grew and connected to groups of adjacent cells. We conclude that the combination of alkaline fibrin with synthetic sulfoglycolipid Tol-51 increased cell adhesion, cell migration, fasciculation, and axonal growth capacity, promoted by aldynoglia cells. There was no negative effect on the behavior of aldynoglia cells after the addition of sulfoglycolipid Tol-51, suggesting that a combination of aldynoglia plus alkaline fibrin and Tol-51 compound could be useful as a therapeutic strategy for tSCI repair.
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Axones , Fibrina , Ganglios Espinales , Animales , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Ganglios Espinales/citología , Axones/metabolismo , Axones/efectos de los fármacos , Fibrina/metabolismo , Hidrogeles/química , Hidrogeles/farmacología , Ratas , Glucolípidos/farmacología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citología , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/patología , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/citología , Movimiento Celular/efectos de los fármacosRESUMEN
Investigating and understanding the biomechanical kinematics and kinetics of human brain axonal fibers during head impact process is crucial to study the mechanisms of Traumatic Axonal Injury (TAI). Such a study may require the explicit incorporation of brain fiber tracts into the host brain in order to distinguish the mechanical states of axonal fibers and brain tissue. Herein we extend our previously developed human head model by using an embedded element method to include fiber tracts reconstructed from diffusion tensor images in a host brain with the purpose of numerically tracking the deformation state of axonal fiber tracts during a head impact simulation. The updated model is validated by comparing its prediction of intracranial pressures with experimental data, followed by a thorough study of the effects of element types used for fiber tracts and the stiffness ratios of fiber to host brain. The validated model is also used to predict and visualize the damaged region of fiber tracts during the head impact process based on different injury criteria. The model is promising in tracking the state of fiber tracts and can add more objective functions such as axonal fiber deformation if used in the future design optimization of head protective equipment such as a football helmet.
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Axones , Encéfalo , Análisis de Elementos Finitos , Humanos , Axones/fisiología , Modelos Neurológicos , Fenómenos Biomecánicos/fisiología , Imagen de Difusión Tensora , Simulación por ComputadorRESUMEN
Motoneurons critically depend on precise spatial and temporal control of translation for axon growth and the establishment and maintenance of neuromuscular connections. While defects in local translation have been implicated in the pathogenesis of motoneuron disorders, little is known about the mechanisms regulating axonal protein synthesis. Here, we report that motoneurons derived from Hnrnpr knockout mice show reduced axon growth accompanied by lowered synthesis of cytoskeletal and synaptic components in axons. Mutant mice display denervated neuromuscular junctions and impaired motor behavior. In axons, hnRNP R is a component of translation initiation complexes and, through interaction with O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (Ogt), modulates O-GlcNAcylation of eIF4G. Restoring axonal O-GlcNAc levels rescued local protein synthesis and axon growth defects of hnRNP R knockout motoneurons. Together, these findings demonstrate a function of hnRNP R in controlling the local production of key factors required for axon growth and formation of neuromuscular innervations.
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Axones , Factor 4G Eucariótico de Iniciación , Ribonucleoproteínas Nucleares Heterogéneas , Ratones Noqueados , Neuronas Motoras , Biosíntesis de Proteínas , Animales , Ratones , Acetilglucosamina/metabolismo , Axones/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/genética , Neuronas Motoras/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , N-Acetilglucosaminiltransferasas/genética , Unión Neuromuscular/metabolismoRESUMEN
Ciliopathies are characterized by the absence or dysfunction of primary cilia. Despite the fact that cognitive impairments are a common feature of ciliopathies, how cilia dysfunction affects neuronal development has not been characterized in detail. Here, we show that primary cilium-mediated signaling is required cell-autonomously by neurons during neural circuit formation. In particular, a functional primary cilium is crucial during axonal pathfinding for the switch in responsiveness of axons at a choice point or intermediate target. Using different animal models and in vivo, ex vivo and in vitro experiments, we provide evidence for a crucial role of primary cilium-mediated signaling in long-range axon guidance. The primary cilium on the cell body of commissural neurons transduces long-range guidance signals sensed by growth cones navigating an intermediate target. In extension of our finding that Shh is required for the rostral turn of post-crossing commissural axons, we suggest a model implicating the primary cilium in Shh signaling upstream of a transcriptional change of axon guidance receptors, which in turn mediate the repulsive response to floorplate-derived Shh shown by post-crossing commissural axons.
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Orientación del Axón , Axones , Cilios , Proteínas Hedgehog , Transducción de Señal , Cilios/metabolismo , Animales , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Ratones , Axones/metabolismo , Conos de Crecimiento/metabolismo , Neuronas/metabolismoRESUMEN
BACKGROUND: Although WD repeat domain 45 (WDR45) mutations have been linked to ß -propeller protein-associated neurodegeneration (BPAN), the precise molecular and cellular mechanisms behind this disease remain elusive. This study aims to shed light on the impacts of WDR45-deficiency on neurodegeneration, specifically axonal degeneration, within the midbrain dopaminergic (DAergic) system. We hope to better understand the disease process by examining pathological and molecular alterations, especially within the DAergic system. METHODS: To investigate the impacts of WDR45 dysfunction on mouse behaviors and DAergic neurons, we developed a mouse model in which WDR45 was conditionally knocked out in the midbrain DAergic neurons (WDR45cKO). Through a longitudinal study, we assessed alterations in the mouse behaviors using open field, rotarod, Y-maze, and 3-chamber social approach tests. We utilized a combination of immunofluorescence staining and transmission electron microscopy to examine the pathological changes in DAergic neuron soma and axons. Additionally, we performed proteomic and lipidomic analyses of the striatum from young and aged mice to identify the molecules and processes potentially involved in the striatal pathology during aging. Further more, primary midbrain neuronal culture was employed to explore the molecular mechanisms leading to axonal degeneration. RESULTS: Our study of WDR45cKO mice revealed a range of deficits, including impaired motor function, emotional instability, and memory loss, coinciding with the profound reduction of midbrain DAergic neurons. The neuronal loss, we observed massive axonal enlargements in the dorsal and ventral striatum. These enlargements were characterized by the accumulation of extensively fragmented tubular endoplasmic reticulum (ER), a hallmark of axonal degeneration. Proteomic analysis of the striatum showed that the differentially expressed proteins were enriched in metabolic processes. The carbohydrate metabolic and protein catabolic processes appeared earlier, and amino acid, lipid, and tricarboxylic acid metabolisms were increased during aging. Of note, we observed a tremendous increase in the expression of lysophosphatidylcholine acyltransferase 1 (Lpcat1) that regulates phospholipid metabolism, specifically in the conversion of lysophosphatidylcholine (LPC) to phosphatidylcholine (PC) in the presence of acyl-CoA. The lipidomic results consistently suggested that differential lipids were concentrated on PC and LPC. Axonal degeneration was effectively ameliorated by interfering Lpcat1 expression in primary cultured WDR45-deficient DAergic neurons, proving that Lpcat1 and its regulated lipid metabolism, especially PC and LPC metabolism, participate in controlling the axonal degeneration induced by WDR45 deficits. CONCLUSIONS: In this study, we uncovered the molecular mechanisms underlying the contribution of WDR45 deficiency to axonal degeneration, which involves complex relationships between phospholipid metabolism, autophagy, and tubular ER. These findings greatly advance our understanding of the fundamental molecular mechanisms driving axonal degeneration and may provide a foundation for developing novel mechanistically based therapeutic interventions for BPAN and other neurodegenerative diseases.
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Axones , Neuronas Dopaminérgicas , Lipidómica , Mesencéfalo , Ratones Noqueados , Proteómica , Animales , Ratones , Axones/metabolismo , Axones/patología , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Mesencéfalo/metabolismo , Mesencéfalo/patología , Degeneración Nerviosa/patología , Degeneración Nerviosa/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patologíaRESUMEN
Clathrin-mediated endocytosis has characteristic features in neuronal dendrites and presynapses, but how membrane proteins are internalized along the axon shaft remains unclear. We focused on clathrin-coated structures and endocytosis along the axon initial segment (AIS) and their relationship to the periodic actin-spectrin scaffold that lines the axonal plasma membrane. A combination of super-resolution microscopy and platinum-replica electron microscopy on cultured neurons revealed that AIS clathrin-coated pits form within "clearings", circular areas devoid of actin-spectrin mesh. Actin-spectrin scaffold disorganization increased clathrin-coated pit formation. Cargo uptake and live-cell imaging showed that AIS clathrin-coated pits are particularly stable. Neuronal plasticity-inducing stimulation triggered internalization of the clathrin-coated pits through polymerization of branched actin around them. Thus, spectrin and actin regulate clathrin-coated pit formation and scission to control endocytosis at the AIS.