RESUMEN

Migration of endothelial and many other cells requires spatiotemporal regulation of protrusive and contractile cytoskeletal rearrangements that drive local cell shape changes. Unexpectedly, the small GTPase Rho, a crucial regulator of cell movement, has been reported to be active in both local cell protrusions and retractions, raising the question of how Rho activity can coordinate cell migration. Here, we show that Rho activity is absent in local protrusions and active during retractions. During retractions, Rho rapidly activated ezrin-radixin-moesin proteins (ERMs) to increase actin-membrane attachment, and, with a delay, nonmuscle myosin 2 (NM2). Rho activity was excitable, with NM2 acting as a slow negative feedback regulator. Strikingly, inhibition of SLK/LOK kinases, through which Rho activates ERMs, caused elongated cell morphologies, impaired Rho-induced cell contractions, and reverted Rho-induced blebbing. Together, our study demonstrates that Rho activity drives retractions by sequentially enhancing ERM-mediated actin-membrane attachment for force transmission and NM2-dependent contractility.

Asunto(s)

Actomiosina , Movimiento Celular , Forma de la Célula , Proteínas del Citoesqueleto , Actomiosina/metabolismo , Humanos , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas de Unión al GTP rho/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de la Membrana/metabolismo , Actinas/metabolismo , Animales , Miosina Tipo II/metabolismo

RESUMEN

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.

Asunto(s)

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/metabolismo

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Proteínas con Dominio LIM , Neoplasias de la Boca , Transducción de Señal , Humanos , Transducción de Señal/efectos de los fármacos , Proteínas con Dominio LIM/metabolismo , Proteínas con Dominio LIM/antagonistas & inhibidores , Neoplasias de la Boca/tratamiento farmacológico , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/antagonistas & inhibidores , Antineoplásicos/farmacología , Antineoplásicos/química , Quinasas Janus/antagonistas & inhibidores , Quinasas Janus/metabolismo , Proliferación Celular/efectos de los fármacos , Factores de Transcripción STAT/metabolismo , Factores de Transcripción STAT/antagonistas & inhibidores , Progresión de la Enfermedad , Ensayos de Selección de Medicamentos Antitumorales

RESUMEN

Activity-regulated cytoskeleton-associated protein (Arc) plays a crucial role in synaptic plasticity, a process integral to learning and memory. Arc transcription is induced within a few minutes of stimulation, making it a useful marker for neuronal activity. However, the specific neuronal activity patterns that initiate Arc transcription have remained elusive due to the inability to observe mRNA transcription in live cells in real time. Using a genetically encoded RNA indicator (GERI) mouse model that expresses endogenous Arc mRNA tagged with multiple GFPs, we investigated Arc transcriptional activity in response to various electrical field stimulation patterns. The GERI mouse model was generated by crossing the Arc-PBS knock-in mouse, engineered with binding sites in the 3' untranslated region (UTR) of Arc mRNA, and the transgenic mouse expressing the cognate binding protein fused to GFP. In dissociated hippocampal neurons, we found that the pattern of stimulation significantly affects Arc transcription. Specifically, theta-burst stimulation consisting of high-frequency (100 Hz) bursts delivered at 10 Hz frequency induced the highest rate of Arc transcription. Concurrently, the amplitudes of nuclear calcium transients also reached their peak with 10 Hz burst stimulation, indicating a correlation between calcium concentration and transcription. However, our dual-color single-cell imaging revealed that there were no significant differences in calcium amplitudes between Arc-positive and Arc-negative neurons upon 10 Hz burst stimulation, suggesting the involvement of other factors in the induction of Arc transcription. Our live-cell RNA imaging provides a deeper insight into the complex regulation of transcription by activity patterns and calcium signaling pathways.

Asunto(s)

Proteínas del Citoesqueleto , Hipocampo , Ratones Transgénicos , Proteínas del Tejido Nervioso , Neuronas , Transcripción Genética , Animales , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Hipocampo/metabolismo , Hipocampo/fisiología , Neuronas/metabolismo , Neuronas/fisiología , Transcripción Genética/fisiología , Ratones , Estimulación Eléctrica , ARN Mensajero/metabolismo , Células Cultivadas , Calcio/metabolismo , Ratones Endogámicos C57BL

RESUMEN

The application of nanoscale scaffolds has become a promising strategy in vaccine design, with protein-based nanoparticles offering desirable avenues for the biocompatible and efficient delivery of antigens. Here, we presented a novel endogenous capsid-forming protein, activated-regulated cytoskeleton-associated protein (ARC), which could be engineered through the plug-and-play strategy (SpyCatcher3/SpyTag3) for multivalent display of antigens. Combined with the self-assembly capacity and flexible modularity of ARC, ARC-based vaccines elicited robust immune responses against Mpox or SARS-CoV-2, comparable to those induced by ferritin-based vaccines. Additionally, ARC-based nanoparticles functioned as immunostimulants, efficiently stimulating dendritic cells and facilitating germinal center responses. Even without adjuvants, ARC-based vaccines generated protective immune responses in a lethal challenge model. Hence, this study showed the feasibility of ARC as a novel protein-based nanocarrier for multivalent surface display of pathogenic antigens and demonstrated the potential of exploiting recombinant mammalian retrovirus-like protein as a delivery vehicle for bioactive molecules.

Asunto(s)

Vacunas contra la COVID-19 , COVID-19 , Nanopartículas , SARS-CoV-2 , Animales , Nanopartículas/química , Ratones , SARS-CoV-2/inmunología , COVID-19/prevención & control , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/química , Humanos , Ratones Endogámicos BALB C , Proteínas de la Cápside/química , Proteínas de la Cápside/inmunología , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Femenino , Células Dendríticas/inmunología , Nanovacunas , Proteínas del Tejido Nervioso

RESUMEN

Repeated cocaine use produces adaptations in brain function that contribute to long-lasting behaviors associated with cocaine use disorder (CUD). In rodents, the activity-regulated cytoskeleton-associated protein (Arc) can regulate glutamatergic synaptic transmission, and cocaine regulates Arc expression and subcellular localization in multiple brain regions, including the nucleus accumbens (NAc)-a brain region linked to CUD-related behavior. We show here that repeated, non-contingent cocaine administration in global Arc KO male mice produced a dramatic hypersensitization of cocaine locomotor responses and drug experience-dependent sensitization of conditioned place preference (CPP). In contrast to the global Arc KO mice, viral-mediated reduction of Arc in the adult male, but not female, NAc (shArcNAc) reduced both CPP and cocaine-induced locomotor activity, but without altering basal miniature or evoked glutamatergic synaptic transmission. Interestingly, cell type-specific knockdown of Arc in D1 dopamine receptor-expressing NAc neurons reduced cocaine-induced locomotor sensitization, but not cocaine CPP; whereas, Arc knockdown in D2 dopamine receptor-expressing NAc neurons reduced cocaine CPP, but not cocaine-induced locomotion. Taken together, our findings reveal that global, developmental loss of Arc produces hypersensitized cocaine responses; however, these effects cannot be explained by Arc's function in the adult mouse NAc since Arc is required in a cell type- and sex-specific manner to support cocaine-context associations and locomotor responses.

Asunto(s)

Cocaína , Proteínas del Citoesqueleto , Proteínas del Tejido Nervioso , Núcleo Accumbens , Animales , Núcleo Accumbens/metabolismo , Núcleo Accumbens/efectos de los fármacos , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Masculino , Ratones , Femenino , Cocaína/farmacología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D1/genética , Locomoción/efectos de los fármacos , Trastornos Relacionados con Cocaína/metabolismo , Trastornos Relacionados con Cocaína/genética , Trastornos Relacionados con Cocaína/fisiopatología , Ratones Endogámicos C57BL , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Transmisión Sináptica

RESUMEN

With the rapid progress in deciphering the pathogenesis of Alzheimer's disease (AD), it has been widely accepted that the accumulation of misfolded amyloid ß (Aß) in the brain could cause the neurodegeneration in AD. Although much evidence demonstrates the neurotoxicity of Aß, the role of Aß in the nervous system are complex. However, more comprehensive studies are needed to understand the physiological effect of Aß40 monomers in depth. To explore the physiological mechanism of Aß, we employed mass spectrometry to investigate the altered proteomic events induced by a lower submicromolar concentration of Aß. Human neuroblastoma SH-SY5Y cells were exposed to five different concentrations of Aß1-40 monomers and collected at four time points. The proteomic analysis revealed the time-course behavior of proteins involved in biological processes, such as RNA splicing, nuclear transport and protein localization. Further biological studies indicated that Aß40 monomers may activate PI3K/AKT signaling to regulate p-Tau, Ezrin and MAP2. These three proteins are associated with dendritic morphogenesis, neuronal polarity, synaptogenesis, axon establishment and axon elongation. Moreover, Aß40 monomers may regulate their physiological forms by inhibiting the expression of BACE1 and APP via activation of the ERK1/2 pathway. A comprehensive exploration of pathological and physiological mechanisms of Aß is beneficial for exploring novel treatment.

Asunto(s)

Enfermedad de Alzheimer , Péptidos beta-Amiloides , Proteómica , Humanos , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Proteómica/métodos , Línea Celular Tumoral , Ácido Aspártico Endopeptidasas/metabolismo , Ácido Aspártico Endopeptidasas/genética , Fragmentos de Péptidos/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas tau/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Proteínas Proto-Oncogénicas c-akt/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteoma/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Sistema de Señalización de MAP Quinasas

RESUMEN

The SP/KLF family of transcription factors harbour three C-terminal C2H2 zinc fingers interspersed by two linkers which confers DNA-binding to a 9-10 bp motif. Mutations in KLF1, the founding member of the family, are common. Missense mutations in linker two result in a mild phenotype. However, when co-inherited with loss-of-function mutations, they result in severe non-spherocytic hemolytic anemia. We generate a mouse model of this disease by crossing Klf1+/- mice with Klf1H350R/+ mice that harbour a missense mutation in linker-2. Klf1H350R/- mice exhibit severe hemolysis without thalassemia. RNA-seq demonstrate loss of expression of genes encoding transmembrane and cytoskeletal proteins, but not globins. ChIP-seq show no change in DNA-binding specificity, but a global reduction in affinity, which is confirmed using recombinant proteins and in vitro binding assays. This study provides new insights into how linker mutations in zinc finger transcription factors result in different phenotypes to those caused by loss-of-function mutations.

Asunto(s)

Hemólisis , Factores de Transcripción de Tipo Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Animales , Ratones , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Mutación Missense , Humanos , Anemia Hemolítica/genética , Anemia Hemolítica/metabolismo , Ratones Noqueados , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Masculino , Dedos de Zinc , Femenino , Mutación

RESUMEN

Individuals with the Autism Susceptibility Candidate 2 (AUTS2) gene disruptions exhibit symptoms such as intellectual disability, microcephaly, growth retardation, and distinct skeletal and facial differences. The role of AUTS2 in neurodevelopment has been investigated using animal and embryonic stem cell models. However, the precise molecular mechanisms of how AUTS2 influences neurodevelopment, particularly in humans, are not thoroughly understood. Our study employed a 3D human cerebral organoid culture system, in combination with genetic, genomic, cellular, and molecular approaches, to investigate how AUTS2 impacts neurodevelopment through cellular signaling pathways. We used CRISPR/Cas9 technology to create AUTS2-deficient human embryonic stem cells and then generated cerebral organoids with these cells. Our transcriptomic analyses revealed that the absence of AUTS2 in cerebral organoids reduces the populations of cells committed to the neuronal lineage, resulting in an overabundance of cells with a transcription profile resembling that of choroid plexus (ChP) cells. Intriguingly, we found that AUTS2 negatively regulates the WNT/ß-catenin signaling pathway, evidenced by its overactivation in AUTS2-deficient cerebral organoids and in luciferase reporter cells lacking AUTS2. Importantly, treating the AUTS2-deficient cerebral organoids with a WNT inhibitor reversed the overexpression of ChP genes and increased the downregulated neuronal gene expression. This study offers new insights into the role of AUTS2 in neurodevelopment and suggests potential targeted therapies for neurodevelopmental disorders.

Asunto(s)

Diferenciación Celular , Proteínas del Citoesqueleto , Neuronas , Organoides , Factores de Transcripción , Vía de Señalización Wnt , Humanos , Organoides/metabolismo , Neuronas/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Sistemas CRISPR-Cas , beta Catenina/metabolismo , beta Catenina/genética , Células Madre Embrionarias Humanas/metabolismo , Encéfalo/metabolismo , Encéfalo/patología

RESUMEN

Glutamate is involved in fundamental functions, including neuronal plasticity and memory. Astrocytes are integral elements involved in synaptic function, and the GLT-1 transporter possesses a critical role in glutamate uptake. Here, we study the role of GLT-1, specifically located in astrocytes, in the consolidation, expression, reconsolidation and persistence of spatial object recognition memory in rats. Administration of dihydrokainic acid (DHK), a selective GLT-1 inhibitor, into the dorsal hippocampus around a weak training which only induces short-term memory, promotes long-term memory formation. This promotion is prevented by hippocampal administration of protein-synthesis translation inhibitor, blockade of Activity-regulated cytoskeleton-associated protein (Arc) translation or Brain-Derived Neurotrophic Factor (BDNF) action, which are plasticity related proteins necessary for memory consolidation. However, DHK around a strong training, which induces long-term memory, does not affect memory consolidation. Administration of DHK before the test session impairs the expression of long-term memory, and this effect is dependent of Arc translation. Furthermore, DHK impairs reconsolidation if applied before a reactivation session, and this effect is independent of Arc translation. These findings reveal specific consequences on spatial memory stages developed under hippocampal GLT-1 blockade, shedding light on the intricate molecular mechanisms, governed in part for the action of glia.

Asunto(s)

Astrocitos , Factor Neurotrófico Derivado del Encéfalo , Proteínas del Citoesqueleto , Ácido Glutámico , Hipocampo , Memoria Espacial , Animales , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/fisiología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Memoria Espacial/efectos de los fármacos , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Masculino , Ratas , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Ácido Glutámico/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/antagonistas & inhibidores , Ratas Wistar , Ácido Kaínico/farmacología , Ácido Kaínico/análogos & derivados , Consolidación de la Memoria/efectos de los fármacos

RESUMEN

Membrane remodeling drives a broad spectrum of cellular functions, and it is regulated through mechanical forces exerted on the membrane by cytoplasmic complexes. Here, we investigate how actin filaments dynamically tune their structure to control the active transfer of membranes between cellular compartments with distinct compositions and biophysical properties. Using intravital subcellular microscopy in live rodents we show that a lattice composed of linear filaments stabilizes the granule membrane after fusion with the plasma membrane and a network of branched filaments linked to the membranes by Ezrin, a regulator of membrane tension, initiates and drives to completion the integration step. Our results highlight how the actin cytoskeleton tunes its structure to adapt to dynamic changes in the biophysical properties of membranes.

Asunto(s)

Citoesqueleto de Actina , Actinas , Membrana Celular , Animales , Citoesqueleto de Actina/metabolismo , Membrana Celular/metabolismo , Actinas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Ratas , Ratones , Fusión de Membrana

RESUMEN

INTRODUCTION: Traumatic brain injury (TBI) is a complex pathophysiological process, and increasing attention has been paid to the important role of post-synaptic density (PSD) proteins, such as glutamate receptors. Our previous study showed that a PSD protein Arc/Arg3.1 (Arc) regulates endoplasmic reticulum (ER) stress and neuronal necroptosis in traumatic injury in vitro. AIM: In this study, we investigated the expression, regulation and biological function of Arc in both in vivo and in vitro experimental TBI models. RESULTS: Traumatic neuronal injury (TNI) induced a temporal upregulation of Arc in cortical neurons, while TBI resulted in sustained increase in Arc expression up to 24 h in rats. The increased expression of Arc was mediated by the activity of metabotropic glutamate receptor 5 (mGluR5), but not dependent on the intracellular calcium (Ca2+) release. By using inhibitors and antagonists, we found that TNI regulates Arc expression via Gq protein and protein turnover. In addition, overexpression of Arc protects against TBI-induced neuronal injury and motor dysfunction both in vivo and in vitro, whereas the long-term cognitive function was not altered. To determine the role of Arc in mGluR5-induced protection, lentivirus-mediated short hairpin RNA (shRNA) transfection was performed to knockdown Arc expression. The mGluR5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG)-induced protection against TBI was partially prevented by Arc knockdown. Furthermore, the CHPG-induced attenuation of Ca2+ influx after TNI was dependent on Arc activation and followed regulation of AMPAR subunits. The results of Co-IP and Ca2+ imaging showed that the Arc-Homer1 interaction contributes to the CHPG-induced regulation of intracellular Ca2+ release. CONCLUSION: In summary, the present data indicate that the mGluR5-mediated Arc activation is a protective mechanism that attenuates neurotoxicity following TBI through the regulation of intracellular Ca2+ hemostasis. The AMPAR-associated Ca2+ influx and ER Ca2+ release induced by Homer1-IP3R pathway might be involved in this protection.

Asunto(s)

Lesiones Traumáticas del Encéfalo , Proteínas del Citoesqueleto , Proteínas de Andamiaje Homer , Proteínas del Tejido Nervioso , Neuronas , Ratas Sprague-Dawley , Receptor del Glutamato Metabotropico 5 , Animales , Lesiones Traumáticas del Encéfalo/metabolismo , Lesiones Traumáticas del Encéfalo/patología , Receptor del Glutamato Metabotropico 5/metabolismo , Receptor del Glutamato Metabotropico 5/antagonistas & inhibidores , Masculino , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/biosíntesis , Ratas , Proteínas de Andamiaje Homer/metabolismo , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Modelos Animales de Enfermedad , Células Cultivadas , Corteza Cerebral/metabolismo , Calcio/metabolismo , Glicina/análogos & derivados , Fenilacetatos

RESUMEN

2,5-hexanedione (HD) is the γ-diketone metabolite of industrial organic solvent n-hexane, primarily responsible for n-hexane neurotoxicity. Previous studies have shown that the formation of pyrrole adducts (PAs) is crucial for the toxic axonopathy induced by HD. However, the exact mechanism underlying PAs-induced axonal degeneration remains unclear. Recently, Sterile α and toll/interleukin 1 receptor motif-containing protein 1 (SARM1) has been identified as the central executor of axon degeneration. This study was designed to investigate the role of SARM1-mediated axon degeneration in rats exposed to HD. Furthermore, the causal relationship between PAs and SARM1-mediated axon degeneration was further explored using Sarm1 KO mice. Our findings suggest that HD causes axon degeneration and neuronal loss in animals. Mechanistic studies revealed that HD activates SARM1-dependent axonal degeneration machinery. In contrast, Sarm1 KO attenuates motor dysfunction and rescues neuron loss following HD exposure. Interestingly, the PAs formed by the binding of HD to proteins primarily accumulate on mitochondria, leading to mitochondrial dysfunction. This dysfunction serves as an upstream event in HD-induced nerve injuries. Our findings highlight the crucial role of PAs formation in the major pathological changes during n-hexane poisoning, providing a potential therapeutic target for n-hexane neuropathy.

Asunto(s)

Proteínas del Dominio Armadillo , Axones , Proteínas del Citoesqueleto , Hexanonas , Mitocondrias , Animales , Proteínas del Dominio Armadillo/metabolismo , Proteínas del Dominio Armadillo/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Hexanonas/toxicidad , Axones/efectos de los fármacos , Axones/metabolismo , Ratas , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Ratones , Ratones Noqueados , Masculino , Ratas Sprague-Dawley , Síndromes de Neurotoxicidad , Hexanos/toxicidad

RESUMEN

Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) is an immediate early gene that plays a vital role in learning and memory. Arc protein has structural and functional properties similar to viral Group-specific antigen (Gag) protein and mediates the intercellular RNA transfer through virus-like capsids. However, the regulators and secretion pathway through which Arc capsids maneuver cargos are unclear. Here, we identified that phosphatidylinositol-3-phosphate (PI3P) mediates Arc capsid assembly and secretion through the endosomal-multivesicular body (MVB) pathway. Indeed, reconstituted Arc protein preferably binds to PI3P. In HEK293T cells, Arc forms puncta that colocalize with FYVE, an endosomal PI3P marker, as well as Rab5 and CD63, early endosomal and MVB markers, respectively. Superresolution imaging resolves Arc accumulates within the intraluminal vesicles of MVB. CRISPR double knockout of RalA and RalB, crucial GTPases for MVB biogenesis and exocytosis, severely reduces the Arc-mediated RNA transfer efficiency. RalA/B double knockdown in cultured rat cortical neurons increases the percentage of mature dendritic spines. Intake of extracellular vesicles purified from Arc-expressing wild-type, but not RalA/B double knockdown, cells in mouse cortical neurons reduces their surface GlutA1 levels. These results suggest that unlike the HIV Gag, whose membrane targeting requires interaction with plasma-membrane-specific phosphatidyl inositol (4,5) bisphosphate (PI(4,5)P2), the assembly of Arc capsids is mediated by PI3P at endocytic membranes. Understanding Arc's secretion pathway helps gain insights into its role in intercellular cargo transfer and highlights the commonality and distinction of trafficking mechanisms between structurally resembled capsid proteins.

Asunto(s)

Proteínas del Citoesqueleto , Proteínas del Tejido Nervioso , Fosfatos de Fosfatidilinositol , Humanos , Animales , Fosfatos de Fosfatidilinositol/metabolismo , Células HEK293 , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Cuerpos Multivesiculares/metabolismo , Cápside/metabolismo , Ratones , Ratas , Endosomas/metabolismo

RESUMEN

The morphology of dendritic spines, the postsynaptic compartment of most excitatory synapses, decisively modulates the function of neuronal circuits as also evident from human brain disorders associated with altered spine density or morphology. Actin filaments (F-actin) form the backbone of spines, and a number of actin-binding proteins (ABP) have been implicated in shaping the cytoskeleton in mature spines. Instead, only little is known about the mechanisms that control the reorganization from unbranched F-actin of immature spines to the complex, highly branched cytoskeleton of mature spines. Here, we demonstrate impaired spine maturation in hippocampal neurons upon genetic inactivation of cyclase-associated protein 1 (CAP1) and CAP2, but not of CAP1 or CAP2 alone. We found a similar spine maturation defect upon overactivation of inverted formin 2 (INF2), a nucleator of unbranched F-actin with hitherto unknown synaptic function. While INF2 overactivation failed in altering spine density or morphology in CAP-deficient neurons, INF2 inactivation largely rescued their spine defects. From our data we conclude that CAPs inhibit INF2 to induce spine maturation. Since we previously showed that CAPs promote cofilin1-mediated cytoskeletal remodeling in mature spines, we identified them as a molecular switch that control transition from filopodia-like to mature spines.

Asunto(s)

Proteínas del Citoesqueleto , Espinas Dendríticas , Forminas , Hipocampo , Proteínas de Microfilamentos , Espinas Dendríticas/metabolismo , Animales , Ratones , Forminas/metabolismo , Forminas/genética , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Hipocampo/metabolismo , Hipocampo/citología , Células Cultivadas , Neuronas/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Ratones Noqueados , Humanos , Proteínas Portadoras

RESUMEN

Depression is a prevalent and intricate mental disorder. The involvement of small RNA molecules, such as microRNAs in the pathogenesis and neuronal mechanisms underlying the depression have been documented. Previous studies have demonstrated the involvement of microRNA-143-3p (miR-143-3p) in the process of fear memory and pathogenesis of ischemia; however, the relationship between miR-143-3p and depression remains poorly understood. Here we utilized two kinds of mouse models to investigate the role of miR-143-3p in the pathogenesis of depression. Our findings reveal that the expression of miR-143-3p is upregulated in the ventral hippocampus (VH) of mice subjected to chronic restraint stress (CRS) or acute Lipopolysaccharide (LPS) treatment. Inhibiting the expression of miR-143-3p in the VH effectively alleviates depressive-like behaviors in CRS and LPS-treated mice. Furthermore, we identify Lasp1 as one of the downstream target genes regulated by miR-143-3p. The miR-143-3p/Lasp1 axis primarily affects the occurrence of depressive-like behaviors in mice by modulating synapse numbers in the VH. Finally, miR-143-3p/Lasp1-induced F-actin change is responsible for the synaptic number variations in the VH. In conclusion, this study enhances our understanding of microRNA-mediated depression pathogenesis and provides novel prospects for developing therapeutic approaches for this intractable mood disorder.

Asunto(s)

Proteínas del Citoesqueleto , Depresión , Hipocampo , MicroARNs , Animales , MicroARNs/genética , MicroARNs/metabolismo , Hipocampo/metabolismo , Ratones , Depresión/metabolismo , Depresión/genética , Masculino , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Ratones Endogámicos C57BL , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/metabolismo , Conducta Animal , Modelos Animales de Enfermedad , Estrés Psicológico/metabolismo , Regulación de la Expresión Génica

RESUMEN

MICAL proteins represent a unique family of actin regulators crucial for synapse development, membrane trafficking, and cytokinesis. Unlike classical actin regulators, MICALs catalyze the oxidation of specific residues within actin filaments to induce robust filament disassembly. The potent activity of MICALs requires tight control to prevent extensive damage to actin cytoskeleton. However, the molecular mechanism governing MICALs' activity regulation remains elusive. Here, we report the cryo-EM structure of MICAL1 in the autoinhibited state, unveiling a head-to-tail interaction that allosterically blocks enzymatic activity. The structure also reveals the assembly of C-terminal domains via a tripartite interdomain interaction, stabilizing the inhibitory conformation of the RBD. Our structural, biochemical, and cellular analyses elucidate a multi-step mechanism to relieve MICAL1 autoinhibition in response to the dual-binding of two Rab effectors, revealing its intricate activity regulation mechanisms. Furthermore, our mutagenesis study of MICAL3 suggests the conserved autoinhibition and relief mechanisms among MICALs.

Asunto(s)

Actinas , Microscopía por Crioelectrón , Oxigenasas de Función Mixta , Humanos , Actinas/metabolismo , Oxigenasas de Función Mixta/metabolismo , Oxigenasas de Función Mixta/química , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/química , Unión Proteica , Citoesqueleto de Actina/metabolismo , Modelos Moleculares , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Dominios Proteicos , Calponinas

RESUMEN

BACKGROUND: Obesity is well-established as a significant contributor to the development of insulin resistance (IR) and diabetes, partially due to elevated plasma saturated free fatty acids like palmitic acid (PA). Grb10-interacting GYF Protein 2 (GIGYF2), an RNA-binding protein, is widely expressed in various tissues including the liver, and has been implicated in diabetes-induced cognitive impairment. Whereas, its role in obesity-related IR remains uninvestigated. METHODS: In this study, we employed palmitic acid (PA) exposure to establish an in vitro IR model in the human liver cancer cell line HepG2 with high-dose chronic PA treatment. The cells were stained with fluorescent dye 2-NBDG to evaluate cell glucose uptake. The mRNA expression levels of genes were determined by real-time qRT-PCR (RT-qPCR). Western blotting was employed to examine the protein expression levels. The RNA immunoprecipitation (RIP) was used to investigate the binding between protein and mRNA. Lentivirus-mediated gene knockdown and overexpression were employed for gene manipulation. In mice, an IR model induced by a high-fat diet (HFD) was established to validate the role and action mechanisms of GIGYF2 in the modulation of HFD-induced IR in vivo. RESULTS: In hepatocytes, high levels of PA exposure strongly trigger the occurrence of hepatic IR evidenced by reduced glucose uptake and elevated extracellular glucose content, which is remarkably accompanied by up-regulation of GIGYF2. Silencing GIGYF2 ameliorated PA-induced IR and enhanced glucose uptake. Conversely, GIGYF2 overexpression promoted IR, PTEN upregulation, and AKT inactivation. Additionally, PA-induced hepatic IR caused a notable increase in STAU1, which was prevented by depleting GIGYF2. Notably, silencing STAU1 prevented GIGYF2-induced PTEN upregulation, PI3K/AKT pathway inactivation, and IR. STAU1 was found to stabilize PTEN mRNA by binding to its 3'UTR. In liver cells, tocopherol treatment inhibits GIGYF2 expression and mitigates PA-induced IR. In the in vivo mice model, GIGYF2 knockdown and tocopherol administration alleviate high-fat diet (HFD)-induced glucose intolerance and IR, along with the suppression of STAU1/PTEN and restoration of PI3K/AKT signaling. CONCLUSIONS: Our study discloses that GIGYF2 mediates obesity-related IR by disrupting the PI3K/AKT signaling axis through the up-regulation of STAU1/PTEN. Targeting GIGYF2 may offer a potential strategy for treating obesity-related metabolic diseases, including type 2 diabetes.

Asunto(s)

Proteínas Portadoras , Resistencia a la Insulina , Hígado , Fosfohidrolasa PTEN , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Proteínas de Unión al ARN , Transducción de Señal , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Fosfohidrolasa PTEN/metabolismo , Fosfohidrolasa PTEN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Ratones , Hígado/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Células Hep G2 , Ácido Palmítico , Masculino , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Ratones Endogámicos C57BL , Dieta Alta en Grasa/efectos adversos

RESUMEN

Multiple studies have shown that astrocytes in the medullary dorsal horn (MDH) play an important role in the development of pathologic pain. However, little is known about the structural reorganization of the peripheral astrocytic processes (PAP), the main functional part of the astrocyte, in MDH in neuropathic state. For this, we investigated the structural relationship between PAP and their adjacent presynaptic axon terminals and postsynaptic dendrites in the superficial laminae of the MDH using electron microscopical immunohistochemistry for ezrin, a marker for PAP, and quantitative analysis in a rat model of neuropathic pain following chronic constriction injury of the infraorbital nerve (CCI-ION). We found that, compared to controls, in rats with CCI-ION, (1) the number, % area, surface density, and volume fraction of ezrin-positive (+) PAP, as well as the fraction of synaptic edge apposed by ezrin + PAP and the degree of its coverage of presynaptic axon terminals and postsynaptic dendrites increased significantly, (2) these effects were abolished by administration of the mGluR5 antagonist 2-methyl-6-(phenylethynyl) pyridine (MPEP). These findings indicate that PAP undergoes structural reorganization around the central synapses of sensory afferents following nerve injury, suggest that it may be mediated by mGluR5, and may represent the structural basis for enhancing astrocyte-neuron interaction in neuropathic pain.

Asunto(s)

Astrocitos , Modelos Animales de Enfermedad , Neuralgia , Ratas Sprague-Dawley , Asta Dorsal de la Médula Espinal , Animales , Astrocitos/metabolismo , Astrocitos/patología , Neuralgia/patología , Neuralgia/metabolismo , Masculino , Asta Dorsal de la Médula Espinal/metabolismo , Asta Dorsal de la Médula Espinal/patología , Ratas , Bulbo Raquídeo/metabolismo , Bulbo Raquídeo/patología , Receptor del Glutamato Metabotropico 5/metabolismo , Proteínas del Citoesqueleto/metabolismo , Dendritas/metabolismo , Dendritas/patología , Terminales Presinápticos/metabolismo , Terminales Presinápticos/patología , Terminales Presinápticos/ultraestructura

RESUMEN

Our investigation has unveiled a series of pyridine-based SARM1 inhibitors, with the lead compound TH-408 exhibiting remarkable potency, achieving an IC50 value of 0.46 µM. This exceptional inhibitory effect significantly curtailed SARM1-mediated cell death across diverse biological models. This finding highlights the promising therapeutic potential for neurodegenerative disorders by disrupting SARM1 activation and advances our understanding of molecular interventions in these complex disorders, including the regulation of NAD+ metabolism.

Asunto(s)

Proteínas del Dominio Armadillo , Muerte Celular , Proteínas del Citoesqueleto , Piridinas , Piridinas/farmacología , Piridinas/química , Piridinas/síntesis química , Humanos , Proteínas del Citoesqueleto/antagonistas & inhibidores , Proteínas del Citoesqueleto/metabolismo , Muerte Celular/efectos de los fármacos , Proteínas del Dominio Armadillo/metabolismo , Proteínas del Dominio Armadillo/antagonistas & inhibidores , NAD+ Nucleosidasa/metabolismo , NAD+ Nucleosidasa/antagonistas & inhibidores , NAD+ Nucleosidasa/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/síntesis química , Relación Estructura-Actividad , Estructura Molecular