RESUMEN
EXPRESSION OF CONCERN: J.-Z. Yu, J. Kuret, and M. M. Rasenick, "Transient Expression of Fluorescent Tau Proteins Promotes Process Formation in PC12 Cells: Contributions of the Tau C-terminus to This Process," Journal of Neuroscience Research 67, no. 5 (2002): 625-633, https://doi.org/10.1002/jnr.10152. This Expression of Concern for the above article published online on 16 January 2002, in Wiley Online Library (wileyonlinelibrary.com), has been published by agreement between the journal Editors-in-Chief, Cristina A. Ghiani and J. Paula Warrington; and Wiley Periodicals LLC. The Expression of Concern has been agreed following concerns raised regarding suspected duplication between the two images, Tau23-GFP (72 hours) presented in Figure 4a and Tau 24 (174-383)-GFP (24 hours) presented in Figure 5a. The authors acknowledge the duplication but due to the length of time that has elapsed since the study was conducted and published, they were unable to provide an explanation or the original data. The journal has decided to issue an Expression of Concern to alert the readers.
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Proteínas tau , Proteínas tau/metabolismo , Proteínas tau/genética , Células PC12 , Animales , Ratas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Neuritas/metabolismoRESUMEN
Sustained compressive injury (SCI) in the brain is observed in numerous injury and pathological scenarios, including tumors, ischemic stroke, and traumatic brain injury-related tissue swelling. Sustained compressive injury is characterized by tissue loading over time, and currently, there are few in vitro models suitable to study neural cell responses to strain-dependent sustained compressive injury. Here, we present an in vitro model of sustained compressive neural injury via centrifugation. Spheroids were made from neonatal rat cortical cells seeded at 4000 cells/spheroid and cultured for 14 days in vitro. A subset of spheroids was centrifuged at 104, 209, 313 or 419 rads/s for 2 minutes. Modeling the physical deformation of the spheroids via finite element analyses, we found that spheroids centrifuged at the aforementioned angular velocities experienced pressures of 10, 38, 84 and 149 kPa, respectively, and compressive (resp. tensile) strains of 10% (5%), 18% (9%), 27% (14%) and 35% (18%), respectively. Quantification of LIVE-DEAD assay and Hoechst 33342 nuclear staining showed that centrifuged spheroids subjected to pressures above 10 kPa exhibited significantly higher DNA damage than control spheroids at 2, 8, and 24 hours post-injury. Immunohistochemistry of ß3-tubulin networks at 2, 8, and 24 hours post-centrifugation injury showed increasing degradation of microtubules over time with increasing strain. Our findings show that cellular injuries occur as a result of specific levels and timings of sustained tissue strains. This experimental SCI model provides a high throughput in vitro platform to examine cellular injury, to gain insights into brain injury that could be targeted with therapeutic strategies.
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Supervivencia Celular , Neuritas , Esferoides Celulares , Animales , Esferoides Celulares/patología , Ratas , Neuritas/metabolismo , Neuritas/patología , Estrés Mecánico , Corteza Cerebral/patología , Células Cultivadas , Ratas Sprague-Dawley , Daño del ADN , CentrifugaciónRESUMEN
Primary neuronal cultures are commonly used to study genetic and exogenous factors influencing neuronal development and maturation. During development, neurons undergo robust morphological changes involving expansion of dendritic arbor, formation of dendritic spines, and expression of synaptic proteins. In this chapter, we will cover methodological approaches allowing quantitative assessment of in vitro cultured neurons. Various quantitative characteristics of dendritic arbor can be derived based on immunostaining against anti-microtubule-associated protein 2 followed by dendrite tracing with the SNT plug-in of the FIJI software package. The number and subtypes of dendritic spines can be assessed by double labeling with DiI and Phalloidin iFluor448 followed by laser scanning confocal microscopy analysis. Finally, expression of presynaptic and postsynaptic proteins can be determined by immunohistochemistry and quantification using several available software packages including FIJI and Imaris, which also allows for 3D rendering and statistical displaying of the expression level of synaptic proteins.
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Espinas Dendríticas , Neuritas , Neuronas , Animales , Espinas Dendríticas/metabolismo , Neuronas/metabolismo , Neuronas/citología , Neuritas/metabolismo , Microscopía Confocal , Células Cultivadas , Ratones , Programas Informáticos , Inmunohistoquímica/métodos , Neurogénesis , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Sinapsis/metabolismoRESUMEN
Dendrite morphology and dendritic spines are key features of the neuronal networks in the brain. Abnormalities in these features have been observed in patients with psychiatric disorders and mouse models of these diseases. In utero electroporation is an easy and efficient gene transfer system for developing mouse embryos in the uterus. By combining with the Cre-loxP system, the morphology of individual neurons can be clearly and sparsely visualized. Here, we describe how this labeling system can be applied to visualize and evaluate the dendrites and dendritic spines of cortical neurons.
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Espinas Dendríticas , Electroporación , Neuritas , Animales , Electroporación/métodos , Ratones , Femenino , Neuritas/metabolismo , Espinas Dendríticas/metabolismo , Embarazo , Útero/citología , Técnicas de Transferencia de Gen , Neuronas/citología , Neuronas/metabolismoRESUMEN
The molecular mechanisms underlying neurite formation include multiple crosstalk between pathways such as membrane trafficking, intracellular signaling, and actin cytoskeletal rearrangement. To study the proteins involved in such complex pathways, we present a detailed workflow of the sample preparation for mass spectrometry-based proteomics and data analysis. We have also included steps to perform label-free quantification of proteins that will help researchers quantify changes in the expression levels of key regulators of neuronal morphogenesis on a global scale.
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Neuritas , Proteómica , Proteómica/métodos , Neuritas/metabolismo , Animales , Humanos , Espectrometría de Masas/métodos , Proteoma/metabolismo , Proteoma/análisis , Cromatografía Liquida/métodosRESUMEN
The use of time-lapse live imaging enables us to track the dynamic changes in neurites during their formation. Ex vivo live imaging with acute brain slices provides a more physiological environment than cultured cells. To accomplish this, a certain method of labeling is necessary to visualize and identify neurite morphology. To understand the dynamics of neurite structure at early stages of neurite formation, we describe in this chapter ex vivo live imaging using a confocal microscope at P0 in combination with in utero electroporation (IUE).
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Encéfalo , Electroporación , Neuritas , Animales , Electroporación/métodos , Neuritas/metabolismo , Encéfalo/citología , Encéfalo/embriología , Encéfalo/diagnóstico por imagen , Ratones , Femenino , Microscopía Confocal/métodos , Imagen de Lapso de Tiempo/métodos , Embarazo , NeurogénesisRESUMEN
The therapy of large defects in peripheral nerve injury (PNI) suffers from several drawbacks, especially the lack of autologous nerve donors. Nerve conduits are considered as a solution for nerve injury treatment, but biocompatibility improvements is still required for conduits prepared with synthetic materials. Cell-derived extracellular matrix (ECM) has drawn attention due to its lower risk of immunogenic response and independence from donor availability. The goal of this study is to coat bone mesenchymal stem cell-derived ECMs on poly(lactic-co-glycolic) acid (PLGA) conduits to enhance their ability to support neural growth and neurite extensions. The ECM-coated conduits have better hydrophilic properties than the pure PLGA conduits. A marked increase on PC12 and RSC96 cells' viability, proliferation and dorsal root ganglion neurite extension was observed. Quantitative PCR analysis exhibited a significant increase in markers for cell proliferation (GAP43), neurite extension (NF-H, MAP2, andßIII-tubulin) and neural function (TREK-1). These results show the potential of ECM-coated PLGA conduits in PNI therapy.
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Proliferación Celular , Supervivencia Celular , Matriz Extracelular , Células Madre Mesenquimatosas , Regeneración Nerviosa , Neuritas , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Animales , Ratas , Neuritas/metabolismo , Células PC12 , Matriz Extracelular/metabolismo , Células Madre Mesenquimatosas/citología , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Regeneración Nerviosa/efectos de los fármacos , Andamios del Tejido/química , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/farmacología , Ganglios Espinales , Traumatismos de los Nervios Periféricos/terapia , Ingeniería de Tejidos/métodos , Polímeros/química , Ensayo de MaterialesRESUMEN
Spinal and bulbar muscular atrophy (SBMA) is a slowly progressing neuromuscular disease caused by a polyglutamine (polyQ)-encoding CAG trinucleotide repeat expansion in the androgen receptor (AR) gene, leading to AR aggregation, lower motor neuron death, and muscle atrophy. AR is a ligand-activated transcription factor that regulates neuronal architecture and promotes axon regeneration; however, whether AR transcriptional functions contribute to disease pathogenesis is not fully understood. Using a differentiated PC12 cell model of SBMA, we identified dysfunction of polyQ-expanded AR in its regulation of neurite growth and maintenance. Specifically, we found that in the presence of androgens, polyQ-expanded AR inhibited neurite outgrowth, induced neurite retraction, and inhibited neurite regrowth. This dysfunction was independent of polyQ-expanded AR transcriptional activity at androgen response elements (ARE). We further showed that the formation of polyQ-expanded AR intranuclear inclusions promoted neurite retraction, which coincided with reduced expression of the neuronal differentiation marker ß-III-Tubulin. Finally, we revealed that cell death is not the primary outcome for cells undergoing neurite retraction; rather, these cells become senescent. Our findings reveal that mechanisms independent of AR canonical transcriptional activity underly neurite defects in a cell model of SBMA and identify senescence as a pathway implicated in this pathology. These findings suggest that in the absence of a role for AR canonical transcriptional activity in the SBMA pathologies described here, the development of SBMA therapeutics that preserve this activity may be desirable. This approach may be broadly applicable to other polyglutamine diseases such as Huntington's disease and spinocerebellar ataxias.
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Neuritas , Receptores Androgénicos , Receptores Androgénicos/metabolismo , Receptores Androgénicos/genética , Animales , Neuritas/metabolismo , Ratas , Células PC12 , Senescencia Celular , Péptidos/metabolismo , Humanos , Trastornos Musculares Atróficos/metabolismo , Trastornos Musculares Atróficos/genética , Trastornos Musculares Atróficos/patología , Mutación , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologíaRESUMEN
In the developing human brain, only 53 stochastically expressed clustered protocadherin (cPcdh) isoforms enable neurites from individual neurons to recognize and self-avoid while simultaneously maintaining contact with neurites from other neurons. Cell assays have demonstrated that self-recognition occurs only when all cPcdh isoforms perfectly match across the cell boundary, with a single mismatch in the cPcdh expression profile interfering with recognition. It remains unclear, however, how a single mismatched isoform between neighboring cells is sufficient to block erroneous recognitions. Using systematic cell aggregation experiments, we show that abolishing cPcdh interactions on the same membrane (cis) results in a complete loss of specific combinatorial binding between cells (trans). Our computer simulations demonstrate that the organization of cPcdh in linear array oligomers, composed of cis and trans interactions, enhances self-recognition by increasing the concentration and stability of cPcdh trans complexes between the homotypic membranes. Importantly, we show that the presence of mismatched isoforms between cells drastically diminishes the concentration and stability of the trans complexes. Overall, we provide an explanation for the role of the cPcdh assembly arrangements in neuronal self/non-self-discrimination underlying neuronal self-avoidance.
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Cadherinas , Neuronas , Isoformas de Proteínas , Humanos , Neuronas/metabolismo , Cadherinas/metabolismo , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Comunicación Celular , Simulación por Computador , Neuritas/metabolismo , Membrana Celular/metabolismoRESUMEN
The neurological effects and underlying pathophysiological mechanisms of sports-related concussion (SRC) in active young boxers remain poorly understood. This study aims to investigate the impairment of white matter microstructure and assess changes in glymphatic function following SRC by utilizing neurite orientation dispersion and density imaging (NODDI) on young boxers who have sustained SRC. A total of 60 young participants were recruited, including 30 boxers diagnosed with SRC and 30 healthy individuals engaging in regular exercise. The assessment of whole-brain white matter damage was conducted using diffusion metrics, while the evaluation of glymphatic function was performed through diffusion tensor imaging (DTI) analysis along the perivascular space (DTI-ALPS) index. A two-sample t-test was utilized to examine group differences in DTI and NODDI metrics. Spearman correlation and generalized linear mixed models were employed to investigate the relationship between clinical assessments of SRC and NODDI measurements. Significant alterations were observed in DTI and NODDI metrics among young boxers with SRC. Additionally, the DTI-ALPS index in the SRC group exhibited a significantly higher value than that of the control group (left side: 1.58 vs. 1.48, PFDR = 0.009; right side: 1.61 vs. 1.51, PFDR = 0.02). Moreover, it was observed that the DTI-ALPS index correlated with poorer cognitive test results among boxers in this study population. Repetitive SRC in active young boxers is associated with diffuse white matter injury and glymphatic dysfunction, highlighting the detrimental impact on brain health. These findings highlight the importance of long-term monitoring of the neurological health of boxers.
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Boxeo , Conmoción Encefálica , Imagen de Difusión Tensora , Sistema Glinfático , Neuritas , Sustancia Blanca , Humanos , Sustancia Blanca/diagnóstico por imagen , Sustancia Blanca/patología , Sistema Glinfático/diagnóstico por imagen , Masculino , Conmoción Encefálica/diagnóstico por imagen , Conmoción Encefálica/fisiopatología , Adolescente , Neuritas/fisiología , Boxeo/lesiones , Boxeo/fisiología , Femenino , Estudios de Casos y Controles , Adulto Joven , Traumatismos en Atletas/diagnóstico por imagen , Traumatismos en Atletas/fisiopatologíaRESUMEN
BACKGROUND: New daily persistent headache (NDPH) is a rare primary headache with unclear pathogenesis. Neuroimaging studies of NDPH are limited, and controversy still exists. Diffusion tensor imaging (DTI) is commonly used to study the white matter. However, lacking specificity, the potential pathological mechanisms of white matter microstructural changes remain poorly understood. In addition, the intricacy of gray matter structures impedes the application of the DTI model. Here, we applied an advanced diffusion model of neurite orientation dispersion and density imaging (NODDI) to study the white matter and cortical gray matter microstructure in patients with NDPH. METHODS: This study assessed brain microstructure, including 27 patients with NDPH, and matched 28 healthy controls (HCs) by NODDI. The differences between the two groups were assessed by tract-based spatial statistics (TBSS) and surface-based analysis (SBA), focusing on the NODDI metrics (neurite density index (NDI), orientation dispersion index (ODI), and isotropic volume fraction (ISOVF)). Furthermore, we performed Pearson's correlation analysis between the NODDI indicators and clinical characteristics. RESULTS: Compared to HCs, patients with NDPH had a reduction of density and complexity in several fiber tracts. For robust results, the fiber tracts were defined as comprising more than 100 voxels, including bilateral inferior fronto-occipital fasciculus (IFOF), left superior longitudinal fasciculus (SLF) and inferior longitudinal fasciculus (ILF), as well as right corticospinal tract (CST). Moreover, the reduction of neurite density was uncovered in the left superior and middle frontal cortex, left precentral cortex, and right lateral orbitofrontal cortex and insula. There was no correlation between the NODDI metrics of these brain regions and clinical variables or scales of relevance after the Bonferroni correction. CONCLUSIONS: Our research indicated that neurite loss was detected in both white matter and cortical gray matter of patients with NDPH.
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Imagen de Difusión Tensora , Sustancia Gris , Sustancia Blanca , Humanos , Sustancia Blanca/diagnóstico por imagen , Sustancia Blanca/patología , Femenino , Sustancia Gris/diagnóstico por imagen , Sustancia Gris/patología , Masculino , Adulto , Persona de Mediana Edad , Corteza Cerebral/diagnóstico por imagen , Corteza Cerebral/patología , Trastornos de Cefalalgia/diagnóstico por imagen , Trastornos de Cefalalgia/patología , Neuritas/patologíaRESUMEN
Cell functions rely on intracellular transport systems distributing bioactive molecules with high spatiotemporal accuracy. The endoplasmic reticulum (ER) tubular network constitutes a system for delivering luminal solutes, including Ca2+, across the cell periphery. How the ER structure enables this nanofluidic transport system is unclear. Here, we show that ER membrane-localized reticulon 4 (RTN4/Nogo) is sufficient to impose neurite outgrowth inhibition in human cortical neurons while acting as an ER morphoregulator. Improving ER transport visualization methodologies combined with optogenetic Ca2+ dynamics imaging and in silico modeling, we observed that ER luminal transport is modulated by ER tubule narrowing and dilation, proportional to the amount of RTN4. Excess RTN4 limited ER luminal transport and Ca2+ release, while RTN4 elimination reversed the effects. The described morphoregulatory effect of RTN4 defines the capacity of the ER for peripheral Ca2+ delivery for physiological releases and thus may constitute a mechanism for controlling the (re)generation of neurites.
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Calcio , Retículo Endoplásmico , Neuronas , Proteínas Nogo , Retículo Endoplásmico/metabolismo , Proteínas Nogo/metabolismo , Humanos , Calcio/metabolismo , Neuronas/metabolismo , Neuritas/metabolismo , Transporte Biológico , Proyección Neuronal/efectos de los fármacosRESUMEN
The gene product of ocular albinism 1 (OA1)/G-protein-coupled receptor (GPR)143 is a receptor for L-3,4-dihydroxyphenylanine (l-DOPA), the most effective agent for Parkinson's disease. When overexpressed, human wild-type GPR143, but not its mutants, inhibits neurite outgrowth in PC12 cells. We investigated the downstream signaling pathway for GPR143-induced inhibition of neurite outgrowth. Nifedipine restored GPR143-induced neurite outgrowth inhibition to the level of control transfectant but did not affect outgrowth in GPR143-knockdown cells. Cilnidipine and flunarizine also suppressed the GPR143-induced inhibition, but their effects at higher concentrations still occurred even in GPR143-knockdown cells. These results suggest that GPR143 regulates neurite outgrowth via L-type calcium channel(s).
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Canales de Calcio Tipo L , Proyección Neuronal , Nifedipino , Receptores Acoplados a Proteínas G , Células PC12 , Animales , Ratas , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/genética , Nifedipino/farmacología , Proyección Neuronal/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiología , Humanos , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Proteínas del Ojo/farmacología , Flunarizina/farmacología , Transducción de Señal/efectos de los fármacos , Levodopa/farmacología , Técnicas de Silenciamiento del Gen , Neuritas/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Glicoproteínas de MembranaRESUMEN
The cell-adhesion molecule NEPH1 is required for maintaining the structural integrity and function of the glomerulus in the kidneys. In the nervous system of Drosophila and C. elegans, it is involved in synaptogenesis and axon branching, which are essential for establishing functional circuits. In the mammalian nervous system, the expression regulation and function of Neph1 has barely been explored. In this study, we provide a spatiotemporal characterization of Neph1 expression in mouse dorsal root ganglia (DRGs) and spinal cord. After the neurogenic phase, Neph1 is broadly expressed in the DRGs and in their putative targets at the dorsal horn of the spinal cord, comprising both GABAergic and glutamatergic neurons. Interestingly, we found that PRRXL1, a homeodomain transcription factor that is required for proper establishment of the DRG-spinal cord circuit, prevents a premature expression of Neph1 in the superficial laminae of the dorsal spinal cord at E14.5, but has no regulatory effect on the DRGs or on either structure at E16.5. By chromatin immunoprecipitation analysis of the dorsal spinal cord, we identified four PRRXL1-bound regions within the Neph1 introns, suggesting that PRRXL1 directly regulates Neph1 transcription. We also showed that Neph1 is required for branching, especially at distal neurites. Together, our work showed that Prrxl1 prevents the early expression of Neph1 in the superficial dorsal horn, suggesting that Neph1 might function as a downstream effector gene for proper assembly of the DRG-spinal nociceptive circuit.
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Ganglios Espinales , Proteínas de Homeodominio , Neuritas , Asta Dorsal de la Médula Espinal , Factores de Transcripción , Animales , Ratones , Asta Dorsal de la Médula Espinal/metabolismo , Asta Dorsal de la Médula Espinal/citología , Neuritas/metabolismo , Neuritas/fisiología , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Ganglios Espinales/metabolismo , Ganglios Espinales/citología , Ganglios Espinales/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas del Tejido NerviosoRESUMEN
Air pollution is an environmental factor associated with an increased risk of neurodegenerative diseases, such as Alzheimer's and Parkinson's, characterized by decreased cognitive abilities and memory. The limited models of sporadic Alzheimer's disease fail to replicate all pathological hallmarks of the disease, making it challenging to uncover potential environmental causes. Environmentally driven models of Alzheimer's disease are thus timely and necessary. We used live-cell confocal fluorescent imaging combined with high-resolution stimulated emission depletion (STED) microscopy to follow the response of retinoic acid-differentiated human neuroblastoma SH-SY5Y cells to nanomaterial exposure. Here, we report that exposure of the cells to some particulate matter constituents reproduces a neurodegenerative phenotype, including extracellular amyloid beta-containing plaques and decreased neurite length. Consistent with the existing in vivo research, we observed detrimental effects, specifically a substantial reduction in neurite length and formation of amyloid beta plaques, after exposure to iron oxide and diesel exhaust particles. Conversely, after exposure to engineered cerium oxide nanoparticles, the lengths of neurites were maintained, and almost no extracellular amyloid beta plaques were formed. Although the exact mechanism behind this effect remains to be explained, the retinoic acid differentiated SH-SY5Y cell in vitro model could serve as an alternative, environmentally driven model of neurodegenerative diseases, including Alzheimer's disease.
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Péptidos beta-Amiloides , Neuritas , Material Particulado , Proteínas tau , Humanos , Material Particulado/toxicidad , Neuritas/efectos de los fármacos , Péptidos beta-Amiloides/metabolismo , Línea Celular Tumoral , Proteínas tau/metabolismo , Placa Amiloide , Enfermedad de Alzheimer/inducido químicamente , Tretinoina/farmacología , Nanopartículas/química , Nanopartículas/toxicidadRESUMEN
Spinal cord injury induces significant cellular stress responses. The Heat Shock Protein 90 (HSP90) plays a pivotal role as a molecular chaperone and is crucial for protein folding, stabilization, and cellular signaling pathways. Despite its important function in stress adaptation, the specific expression patterns and functional roles of HSP90 after nerve injury remain unclear. This study aimed to elucidate the expression dynamics and functional implications of HSP90 following central nervous system (CNS) injury. Using western blotting and immunohistochemical analyses, we observed upregulation of HSP90 expression in spinal cord tissues and within injured neurons in a spinal cord contusion injury model. Additionally, HSP90 was found to enhance neurite outgrowth in primary cortical neurons cultured in vitro. Furthermore, in a glutamate-induced neuronal injury model, the expression of HSP90 was up-regulated, and overexpression of HSP90 promoted neurite re-growth in damaged neurons. Overall, our findings highlight the critical involvement of HSP90 in the neural response to injury and offer valuable insights into potential therapeutic strategies for CNS repair.
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Proteínas HSP90 de Choque Térmico , Traumatismos de la Médula Espinal , Proteínas HSP90 de Choque Térmico/metabolismo , Animales , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Neuronas/metabolismo , Células Cultivadas , Ratas Sprague-Dawley , Proyección Neuronal/fisiología , Regulación hacia Arriba , Médula Espinal/metabolismo , Neuritas/metabolismo , Masculino , RatasRESUMEN
Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) is a pleiotropic peptide known to promote many beneficial processes following neural damage and cell death after stroke. Despite PACAP's known neurotrophic and anti-inflammatory properties, it has not realized its translational potential due to a poor pharmacokinetic profile (non-linear PK/PD), and limited Blood-Brain Barrier Penetration (BBB) permeability. We have previously shown that glycosylation of PACAP increases stability and enhances BBB penetration. In addition, our prior studies showed reduced neuronal cell death and neuroinflammation in models of Parkinson's disease and Traumatic Brain Injury (TBI). In this study we show that a PACAP(1-27) glucoside retains the known neurotrophic activity of native PACAP(1-27)in vitro and a 5-day daily treatment regimen (100 nM) leads to neurite-like extensions in PC12 cells. In addition, we show that intraperitoneal injection of a PACAP(1-27) lactoside (10 mg/kg) with improved BBB-penetration, given 1-hour after reperfusion in a Transient Middle Cerebral Artery Occlusion (tMCAO) mouse model, reduces the infarct size after the ischemic injury in males significantly by â¼ 36 %, and the data suggest a dose-dependency. In conclusion, our data support further development of PACAP glycopeptides as promising novel drug candidates for the treatment of stroke, an area with an urgent clinical need.
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Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Animales , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/administración & dosificación , Masculino , Ratas , Ratones , Células PC12 , Ratones Endogámicos C57BL , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/patología , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/patología , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/uso terapéutico , Modelos Animales de Enfermedad , Glicósidos/farmacología , Glicósidos/uso terapéutico , Glicósidos/administración & dosificación , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Neuritas/efectos de los fármacos , Neuritas/patologíaRESUMEN
The accumulation of amyloid-ß (Aß) in the brain is the first pathological mechanism to initiate Alzheimer's disease (AD) pathogenesis. However, the precise role of Aß in the disease progression remains unclear. Through decades of research, prolonged inflammation has emerged as an important core pathology in AD. Previously, a study has demonstrated the neurotoxic effect of Aß-induced neuroinflammation in neuron-glia co-culture at 72 h. Here, we hypothesise that initial stage Aß may trigger microglial inflammation, synergistically contributing to the progression of neurite lesions relevant to AD progression. In the present study, we aimed to determine whether olanzapine, an antipsychotic drug possessing anti-inflammatory properties, can ameliorate Aß-induced progressive neurite lesions. Our study reports that Aß induces neurite lesions with or without inflammatory microglial cells in vitro. More intriguingly, the present study revealed that Aß exacerbates neurite lesions in synergy with microglia. Moreover, the time course study revealed that Aß promotes microglia-mediated neurite lesions by eliciting the secretion of pro-inflammatory cytokines. Furthermore, our study shows that olanzapine at lower doses prevents Aß-induced microglia-mediated progressive neurite lesions. The increase in pro-inflammatory cytokines induced by Aß is attenuated by olanzapine administration, associated with a reduction in microglial inflammation. Finally, this study reports that microglial senescence induced by Aß was rescued by olanzapine. Thus, our study provides the first evidence that 1 µM to 5 µM of olanzapine can effectively prevent Aß-induced microglia-mediated progressive neurite lesions by modulating microglial inflammation. These observations reinforce the potential of targeting microglial remodelling to slow disease progression in AD.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Microglía , Neuritas , Olanzapina , Olanzapina/farmacología , Microglía/efectos de los fármacos , Microglía/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Citocinas/metabolismo , Antiinflamatorios/farmacología , Ratones , Células Cultivadas , Antipsicóticos/farmacología , Técnicas de Cocultivo , Humanos , Ratones Endogámicos C57BLRESUMEN
Purpose: Retinal ganglion cells (RGCs) connect the retina to the brain. Proper development of the axons and dendrites of RGCs is the basis for these cells to function as projection neurons to deliver visual information to the brain. The purpose of this study was to investigate the function of Shtn1 (which encodes shootin1) in RGC neurite development. Methods: Immunofluorescence (IF) was used to characterize the expression pattern of marker genes. An in vitro direct somatic cell reprogramming system was used to generate RGC-like neurons (iRGCs), which was subsequently used to study the function of Shtn1. Short-hairpin RNAs (shRNAs) were used to knock down Shtn1, and the coding sequence (CDS) of Shtn1 was used to overexpress the gene. Lentiviruses were used to deliver shRNAs or CDSs into iRGCs. The patch clamp technique was used to measure the electrophysiological properties of the iRGCs. RNA sequencing (RNA-seq) was used to examine transcriptome expression. Results: Using IF, we demonstrated that shootin1 is distinctively expressed in RGCs during the period in which RGCs actively develop and adjust the connections of their neurites with upstream and downstream neurons. Using the iRGC system, we demonstrated that Shtn1 promotes the growth and complexity of neurites and thus the electrophysiological maturation, of iRGCs. RNA-seq analyses showed that Shtn1 may also regulate gene expression and neurogenesis in iRGCs. Conclusions: Shtn1 promotes RGC neurite development. These findings improve our understanding of the molecular machinery governing RGC neurite development and may help to optimize future RGC regeneration methods.
Asunto(s)
Proteínas del Tejido Nervioso , Neuritas , Células Ganglionares de la Retina , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/citología , Animales , Neuritas/fisiología , Neuritas/metabolismo , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Reprogramación Celular/fisiología , Células Cultivadas , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp , Neurogénesis/fisiología , Neurogénesis/genéticaRESUMEN
Fluorescence imaging is widely used for the mesoscopic mapping of neuronal connectivity. However, neurite reconstruction is challenging, especially when neurons are densely labelled. Here, we report a strategy for the fully automated reconstruction of densely labelled neuronal circuits. Firstly, we establish stochastic super-multicolour labelling with up to seven different fluorescent proteins using the Tetbow method. With this method, each neuron is labelled with a unique combination of fluorescent proteins, which are then imaged and separated by linear unmixing. We also establish an automated neurite reconstruction pipeline based on the quantitative analysis of multiple dyes (QDyeFinder), which identifies neurite fragments with similar colour combinations. To classify colour combinations, we develop unsupervised clustering algorithm, dCrawler, in which data points in multi-dimensional space are clustered based on a given threshold distance. Our strategy allows the reconstruction of neurites for up to hundreds of neurons at the millimetre scale without using their physical continuity.