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1.
Neuropathol Appl Neurobiol ; 49(5): e12935, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-37705188

RESUMEN

AIMS: Fibroblast growth factor (FGF) signalling is dysregulated in multiple sclerosis (MS) and other neurological and psychiatric conditions, but there is little or no consensus as to how individual FGF family members contribute to disease pathogenesis. Lesion development in MS is associated with increased expression of FGF1, FGF2 and FGF9, all of which modulate remyelination in a variety of experimental settings. However, FGF9 is also selectively upregulated in major depressive disorder (MDD), prompting us to speculate it may also have a direct effect on neuronal function and survival. METHODS: Transcriptional profiling of myelinating cultures treated with FGF1, FGF2 or FGF9 was performed, and the effects of FGF9 on cortical neurons investigated using a combination of transcriptional, electrophysiological and immunofluorescence microscopic techniques. The in vivo effects of FGF9 were explored by stereotactic injection of adeno-associated viral (AAV) vectors encoding either FGF9 or EGFP into the rat motor cortex. RESULTS: Transcriptional profiling of myelinating cultures after FGF9 treatment revealed a distinct neuronal response with a pronounced downregulation of gene networks associated with axonal transport and synaptic function. In cortical neuronal cultures, FGF9 also rapidly downregulated expression of genes associated with synaptic function. This was associated with a complete block in the development of photo-inducible spiking activity, as demonstrated using multi-electrode recordings of channel rhodopsin-transfected rat cortical neurons in vitro and, ultimately, neuronal cell death. Overexpression of FGF9 in vivo resulted in rapid loss of neurons and subsequent development of chronic grey matter lesions with neuroaxonal reduction and ensuing myelin loss. CONCLUSIONS: These observations identify overexpression of FGF9 as a mechanism by which neuroaxonal pathology could develop independently of immune-mediated demyelination in MS. We suggest targeting neuronal FGF9-dependent pathways may provide a novel strategy to slow if not halt neuroaxonal atrophy and loss in MS, MDD and potentially other neurodegenerative diseases.


Asunto(s)
Trastorno Depresivo Mayor , Esclerosis Múltiple , Animales , Ratas , Factor 1 de Crecimiento de Fibroblastos , Factor 2 de Crecimiento de Fibroblastos , Factor 9 de Crecimiento de Fibroblastos
2.
J Vis Exp ; (196)2023 06 30.
Artículo en Inglés | MEDLINE | ID: mdl-37458443

RESUMEN

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of neurons, astrocytes, oligodendrocytes, and microglia. Due to increasing efforts to replace and reduce animal use, a variety of in vitro cell culture systems have been developed to explore innate cell properties, which allow the development of therapeutics for CNS infections and pathologies. Whilst certain research questions can be addressed by human-based cell culture systems, such as (induced) pluripotent stem cells, working with human cells has its own limitations with regard to availability, costs, and ethics. Here, we describe a unique protocol for isolating and culturing cells from embryonic mouse brains. The resulting mixed neural cell cultures mimic several cell populations and interactions found in the brain in vivo. Compared to current equivalent methods, this protocol more closely mimics the characteristics of the brain and also garners more cells, thus allowing for more experimental conditions to be investigated from one pregnant mouse. Further, the protocol is relatively easy and highly reproducible. These cultures have been optimized for use at various scales, including 96-well based high throughput screens, 24-well microscopy analysis, and 6-well cultures for flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. This culture method is a powerful tool to investigate infection and immunity within the context of some of the complexity of the CNS with the convenience of in vitro methods.


Asunto(s)
Astrocitos , Neuronas , Animales , Ratones , Humanos , Células Cultivadas , Neuronas/patología , Astrocitos/fisiología , Encéfalo , Técnicas de Cultivo de Célula , Inmunidad Innata
3.
Nat Neurosci ; 26(7): 1218-1228, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37386131

RESUMEN

Axonal degeneration determines the clinical outcome of multiple sclerosis and is thought to result from exposure of denuded axons to immune-mediated damage. Therefore, myelin is widely considered to be a protective structure for axons in multiple sclerosis. Myelinated axons also depend on oligodendrocytes, which provide metabolic and structural support to the axonal compartment. Given that axonal pathology in multiple sclerosis is already visible at early disease stages, before overt demyelination, we reasoned that autoimmune inflammation may disrupt oligodendroglial support mechanisms and hence primarily affect axons insulated by myelin. Here, we studied axonal pathology as a function of myelination in human multiple sclerosis and mouse models of autoimmune encephalomyelitis with genetically altered myelination. We demonstrate that myelin ensheathment itself becomes detrimental for axonal survival and increases the risk of axons degenerating in an autoimmune environment. This challenges the view of myelin as a solely protective structure and suggests that axonal dependence on oligodendroglial support can become fatal when myelin is under inflammatory attack.


Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Ratones , Animales , Humanos , Vaina de Mielina/metabolismo , Axones/metabolismo , Esclerosis Múltiple/patología , Encefalomielitis Autoinmune Experimental/patología , Factores de Riesgo
4.
Stem Cell Reports ; 18(2): 519-533, 2023 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-36608690

RESUMEN

Demyelinating disorders of the central nervous system (CNS) occur when myelin and oligodendrocytes are damaged or lost. Remyelination and regeneration of oligodendrocytes can be achieved from endogenous oligodendrocyte precursor cells (OPCs) that reside in the adult CNS tissue. Using a cuprizone mouse model of demyelination, we show that infusion of fractalkine (CX3CL1) into the demyelinated murine brain increases de novo oligodendrocyte formation and enhances remyelination in the corpus callosum and cortical gray matter. This is achieved by increased OPC proliferation in the cortical gray matter as well as OPC differentiation and attenuation of microglia/macrophage activation both in corpus callosum and cortical gray matter. Finally, we show that activated OPCs and microglia/macrophages express fractalkine receptor CX3CR1 in vivo, and that in OPC-microglia co-cultures fractalkine increases in vitro oligodendrocyte differentiation by modulating both OPC and microglia biology. Our results demonstrate a novel pro-regenerative role of fractalkine in a demyelinating mouse model.


Asunto(s)
Enfermedades Desmielinizantes , Remielinización , Ratones , Animales , Quimiocina CX3CL1 , Oligodendroglía/fisiología , Vaina de Mielina , Modelos Animales de Enfermedad , Diferenciación Celular/fisiología , Ratones Endogámicos C57BL
5.
Glia ; 71(3): 509-523, 2023 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-36354016

RESUMEN

Healthy myelin sheaths consist of multiple compacted membrane layers closely encasing the underlying axon. The ultrastructure of CNS myelin requires specialized structural myelin proteins, including the transmembrane-tetraspan proteolipid protein (PLP) and the Ig-CAM myelin-associated glycoprotein (MAG). To better understand their functional relevance, we asked to what extent the axon/myelin-units display similar morphological changes if PLP or MAG are lacking. We thus used focused ion beam-scanning electron microscopy (FIB-SEM) to re-investigate axon/myelin-units side-by-side in Plp- and Mag-null mutant mice. By three-dimensional reconstruction and morphometric analyses, pathological myelin outfoldings extend up to 10 µm longitudinally along myelinated axons in both models. More than half of all assessed outfoldings emerge from internodal myelin. Unexpectedly, three-dimensional reconstructions demonstrated that both models displayed complex axonal pathology underneath the myelin outfoldings, including axonal sprouting. Axonal anastomosing was additionally observed in Plp-null mutant mice. Importantly, normal-appearing axon/myelin-units displayed significantly increased axonal diameters in both models according to quantitative assessment of electron micrographs. These results imply that healthy CNS myelin sheaths facilitate normal axonal diameters and shape, a function that is impaired when structural myelin proteins PLP or MAG are lacking.


Asunto(s)
Sistema Nervioso Central , Proteína Proteolipídica de la Mielina , Vaina de Mielina , Glicoproteína Asociada a Mielina , Animales , Ratones , Axones/metabolismo , Sistema Nervioso Central/metabolismo , Ratones Noqueados , Microscopía Electrónica de Rastreo , Proteínas de la Mielina/metabolismo , Vaina de Mielina/metabolismo , Glicoproteína Asociada a Mielina/genética , Proteína Proteolipídica de la Mielina/genética
6.
Front Mol Neurosci ; 15: 860410, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35493328

RESUMEN

Zika virus (ZIKV) is a neurotropic flavivirus recently linked to congenital ZIKV syndrome in children and encephalitis and Guillain-Barré syndrome in adults. Neurotropic viruses often use axons to traffic to neuronal or glial cell somas where they either remain latent or replicate and proceed to infect new cells. Consequently, it has been suggested that axon degeneration could represent an evolutionarily conserved mechanism to limit viral spread. Whilst it is not known if ZIKV transits in axons, we previously reported that ZIKV infection of glial cells in a murine spinal cord-derived cell culture model of the CNS is associated with a profound loss of neuronal cell processes. This, despite that postmitotic neurons are relatively refractory to infection and death. Here, we tested the hypothesis that ZIKV-associated degeneration of neuronal processes is dependent on activation of Sterile alpha and armadillo motif-containing protein 1 (SARM1), an NADase that acts as a central executioner in a conserved axon degeneration pathway. To test this, we infected wild type and Sarm1 homozygous or heterozygous null cell cultures with ZIKV and examined NAD+ levels as well as the survival of neurons and their processes. Unexpectedly, ZIKV infection led to a rapid SARM1-independent reduction in NAD+. Nonetheless, the subsequent profound loss of neuronal cell processes was SARM1-dependent and was preceded by early changes in the appearance of ß-tubulin III staining. Together, these data identify a role for SARM1 in the pathogenesis of ZIKV infection, which may reflect SARM1's conserved prodegenerative function, independent of its NADase activity.

7.
Exp Neurol ; 354: 114113, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35569511

RESUMEN

Iron released from oligodendrocytes during demyelination or derived from haemoglobin breakdown products is believed to amplify oxidative tissue injury in multiple sclerosis (MS). However, the pathophysiological significance of iron-containing haemoglobin breakdown products themselves is rarely considered in the context of MS and their cellular specificity and mode of action remain unclear. Using myelinating cell cultures, we now report the cytotoxic potential of hemin (ferriprotoporphyrin IX chloride), a major degradation product of haemoglobin, is 25-fold greater than equimolar concentrations of free iron in myelinating cultures; a model that reproduces the complex multicellular environment of the CNS. At low micro molar concentrations (3.3 - 10 µM) we observed hemin preferentially binds to myelin and axons to initiate a complex detrimental response that results in targeted demyelination and axonal loss but spares neuronal cell bodies, astrocytes and the majority of oligodendroglia. Demyelination and axonal loss in this context are executed by a combination of mechanisms that include iron-dependent peroxidation by reactive oxygen species (ROS) and ferroptosis. These effects are microglial-independent, do not require any initiating inflammatory insult and represent a direct effect that compromises the structural integrity of myelinated axons in the CNS. Our data identify hemin-mediated demyelination and axonal loss as a novel mechanism by which intracerebral degradation of haemoglobin may contribute to lesion development in MS.


Asunto(s)
Hemina , Esclerosis Múltiple , Axones/patología , Sistema Nervioso Central/patología , Hemina/metabolismo , Hemina/farmacología , Humanos , Hierro/metabolismo , Esclerosis Múltiple/patología , Vaina de Mielina/patología , Oligodendroglía/metabolismo , Estrés Oxidativo
8.
Cell Rep ; 37(4): 109889, 2021 10 26.
Artículo en Inglés | MEDLINE | ID: mdl-34706227

RESUMEN

Astrocyte-derived cholesterol supports brain cells under physiological conditions. However, in demyelinating lesions, astrocytes downregulate cholesterol synthesis, and the cholesterol that is essential for remyelination has to originate from other cellular sources. Here, we show that repair following acute versus chronic demyelination involves distinct processes. In particular, in chronic myelin disease, when recycling of lipids is often defective, de novo neuronal cholesterol synthesis is critical for regeneration. By gene expression profiling, genetic loss-of-function experiments, and comprehensive phenotyping, we provide evidence that neurons increase cholesterol synthesis in chronic myelin disease models and in patients with multiple sclerosis (MS). In mouse models, neuronal cholesterol facilitates remyelination specifically by triggering oligodendrocyte precursor cell proliferation. Our data contribute to the understanding of disease progression and have implications for therapeutic strategies in patients with MS.


Asunto(s)
Colesterol , Esclerosis Múltiple , Vaina de Mielina , Células Precursoras de Oligodendrocitos/metabolismo , Remielinización/genética , Animales , Colesterol/biosíntesis , Colesterol/genética , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Noqueados , Esclerosis Múltiple/genética , Esclerosis Múltiple/metabolismo , Vaina de Mielina/genética , Vaina de Mielina/metabolismo
9.
J Anat ; 239(6): 1241-1255, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34713444

RESUMEN

A century ago this year, Pío del Río-Hortega (1921) coined the term 'oligodendroglia' for the 'interfascicular glia' with very few processes, launching an extensive discovery effort on his new cell type. One hundred years later, we review his original contributions to our understanding of the system of cytoplasmic channels within myelin in the context of what we observe today using light and electron microscopy of genetically encoded fluorescent reporters and immunostaining. We use the term myelinic channel system to describe the cytoplasm-delimited spaces associated with myelin; being the paranodal loops, inner and outer tongues, cytoplasm-filled spaces through compact myelin and further complex motifs associated to the sheath. Using a central nervous system myelinating cell culture model that contains all major neural cell types and produces compact myelin, we find that td-tomato fluorescent protein delineates the myelinic channel system in a manner reminiscent of the drawings of adult white matter by Río-Hortega, despite that he questioned whether some cytoplasmic figures he observed represented artefact. Together, these data lead us to propose a slightly revised model of the 'unrolled' sheath. Further, we show that the myelinic channel system, while relatively stable, can undergo subtle dynamic shape changes over days. Importantly, we capture an under-appreciated complexity of the myelinic channel system in mature myelin sheaths.


Asunto(s)
Sistema Nervioso Central , Vaina de Mielina , Citoplasma , Microscopía Electrónica , Oligodendroglía
10.
Glia ; 69(8): 2023-2036, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-33942402

RESUMEN

Some children with proven intrauterine Zika virus (ZIKV) infection who were born asymptomatic subsequently manifested neurodevelopmental delays, pointing to impairment of development perinatally and postnatally. To model this, we infected postnatal day (P) 5-6 (equivalent to the perinatal period in humans) susceptible mice with a mammalian cell-propagated ZIKV clinical isolate from the Brazilian outbreak in 2015. All infected mice appeared normal up to 4 days post-intraperitoneal inoculation (dpi), but rapidly developed severe clinical signs at 5-6 dpi. All nervous tissue examined at 5/6 dpi appeared grossly normal. However, anti-ZIKV positive cells were observed in the optic nerve, brain, and spinal cord; predominantly in white matter. Co-labeling with cell type specific markers demonstrated oligodendrocytes and astrocytes support productive infection. Rarely, ZIKV positive neurons were observed. In spinal cord white matter, which we examined in detail, apoptotic cells were evident; the density of oligodendrocytes was significantly reduced; and there was localized microglial reactivity including expression of the NLRP3 inflammasome. Together, our observations demonstrate that a clinically relevant ZIKV isolate can directly impact oligodendrocytes. As primary oligodendrocyte cell death can lead later to secondary autoimmune demyelination, our observations may help explain neurodevelopmental delays in infants appearing asymptomatic at birth and commend lifetime surveillance.


Asunto(s)
Infección por el Virus Zika , Virus Zika , Animales , Modelos Animales de Enfermedad , Femenino , Ratones , Neuronas , Oligodendroglía , Embarazo , Infección por el Virus Zika/complicaciones
11.
Viruses ; 13(1)2021 Jan 11.
Artículo en Inglés | MEDLINE | ID: mdl-33440758

RESUMEN

Understanding how Zika virus (Flaviviridae; ZIKV) affects neural cells is paramount in comprehending pathologies associated with infection. Whilst the effects of ZIKV in neural development are well documented, impact on the adult nervous system remains obscure. Here, we investigated the effects of ZIKV infection in established mature myelinated central nervous system (CNS) cultures. Infection incurred damage to myelinated fibers, with ZIKV-positive cells appearing when myelin damage was first detected as well as axonal pathology, suggesting the latter was a consequence of oligodendroglia infection. Transcriptome analysis revealed host factors that were upregulated during ZIKV infection. One such factor, CCL5, was validated in vitro as inhibiting myelination. Transferred UV-inactivated media from infected cultures did not damage myelin and axons, suggesting that viral replication is necessary to induce the observed effects. These data show that ZIKV infection affects CNS cells even after myelination-which is critical for saltatory conduction and neuronal function-has taken place. Understanding the targets of this virus across developmental stages including the mature CNS, and the subsequent effects of infection of cell types, is necessary to understand effective time frames for therapeutic intervention.


Asunto(s)
Axones/virología , Enfermedades Desmielinizantes/etiología , Infección por el Virus Zika/complicaciones , Infección por el Virus Zika/virología , Virus Zika/fisiología , Animales , Biomarcadores , Traumatismos del Nervio Craneal/etiología , Traumatismos del Nervio Craneal/metabolismo , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Humanos , Ratones , Ratas , Transcriptoma
12.
Nat Neurosci ; 24(1): 47-60, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33349711

RESUMEN

The repair of inflamed, demyelinated lesions as in multiple sclerosis (MS) necessitates the clearance of cholesterol-rich myelin debris by microglia/macrophages and the switch from a pro-inflammatory to an anti-inflammatory lesion environment. Subsequently, oligodendrocytes increase cholesterol levels as a prerequisite for synthesizing new myelin membranes. We hypothesized that lesion resolution is regulated by the fate of cholesterol from damaged myelin and oligodendroglial sterol synthesis. By integrating gene expression profiling, genetics and comprehensive phenotyping, we found that, paradoxically, sterol synthesis in myelin-phagocytosing microglia/macrophages determines the repair of acutely demyelinated lesions. Rather than producing cholesterol, microglia/macrophages synthesized desmosterol, the immediate cholesterol precursor. Desmosterol activated liver X receptor (LXR) signaling to resolve inflammation, creating a permissive environment for oligodendrocyte differentiation. Moreover, LXR target gene products facilitated the efflux of lipid and cholesterol from lipid-laden microglia/macrophages to support remyelination by oligodendrocytes. Consequently, pharmacological stimulation of sterol synthesis boosted the repair of demyelinated lesions, suggesting novel therapeutic strategies for myelin repair in MS.


Asunto(s)
Enfermedades Desmielinizantes/patología , Microglía/fisiología , Esteroles/biosíntesis , Animales , Colesterol/metabolismo , Desmosterol/metabolismo , Encefalomielitis Autoinmune Experimental , Femenino , Perfilación de la Expresión Génica , Humanos , Inflamación/metabolismo , Inflamación/patología , Metabolismo de los Lípidos , Receptores X del Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Esclerosis Múltiple , Oligodendroglía/metabolismo , Fagocitosis , Escualeno/metabolismo
13.
J Virol ; 95(1)2020 12 09.
Artículo en Inglés | MEDLINE | ID: mdl-33028720

RESUMEN

Zika virus (ZIKV) envelope (E) protein is the major target of neutralizing antibodies in infected hosts and thus represents a candidate of interest for vaccine design. However, a major concern in the development of vaccines against ZIKV and the related dengue virus is the induction of cross-reactive poorly neutralizing antibodies that can cause antibody-dependent enhancement (ADE) of infection. This risk necessitates particular care in vaccine design. Specifically, the engineered immunogens should have their cross-reactive epitopes masked, and they should be optimized for eliciting virus-specific strongly neutralizing antibodies upon vaccination. Here, we developed ZIKV subunit- and virus-like particle (VLP)-based vaccines displaying E in its wild-type form or E locked in a covalently linked dimeric (cvD) conformation to enhance the exposure of E dimers to the immune system. Compared with their wild-type derivatives, cvD immunogens elicited antibodies with a higher capacity to neutralize virus infection in cultured cells. More importantly, these immunogens protected animals from lethal challenge with both the African and Asian lineages of ZIKV, impairing virus dissemination to brain and sexual organs. Moreover, the locked conformation of E reduced the exposure of epitopes recognized by cross-reactive antibodies and therefore showed a lower potential to induce ADE in vitro Our data demonstrated a higher efficacy of the VLPs in comparison with that of the soluble dimer and support VLP-cvD as a promising ZIKV vaccine.IMPORTANCE Infection with Zika virus (ZIKV) leads to the production by the host of antibodies that target the viral surface envelope (E) protein. A subset of these antibodies can inhibit virus infection, thus making E a suitable candidate for the development of vaccine against the virus. However, the anti-ZIKV E antibodies can cross-react with the E protein of the related dengue virus on account of the high level of similarity exhibited by the two viral proteins. Such a scenario may lead to severe dengue disease. Therefore, the design of a ZIKV vaccine requires particular care. Here, we tested two candidate vaccines containing a recombinant form of the ZIKV E protein that is forced in a covalently stable dimeric conformation (cvD). They were generated with an explicit aim to reduce the exposure of the cross-reactive epitopes. One vaccine is composed of a soluble form of the E protein (sE-cvD), the other is a more complex virus-like particle (VLP-cvD). We used the two candidate vaccines to immunize mice and later infected them with ZIKV. The animals produced a high level of inhibitory antibodies and were protected from the infection. The VLP-cvD was the most effective, and we believe it represents a promising ZIKV vaccine candidate.


Asunto(s)
Vacunas de Partículas Similares a Virus/inmunología , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/inmunología , Infección por el Virus Zika/prevención & control , Virus Zika/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Acrecentamiento Dependiente de Anticuerpo , Protección Cruzada , Ratones , Conformación Proteica , Multimerización de Proteína , Vacunación , Proteínas del Envoltorio Viral/química , Virus Zika/clasificación
14.
Acta Neuropathol Commun ; 8(1): 135, 2020 08 13.
Artículo en Inglés | MEDLINE | ID: mdl-32792006

RESUMEN

Progressive multi-focal leukoencephalopathy (PML) is a potentially fatal encephalitis caused by JC polyomavirus (JCV). PML principally affects people with a compromised immune system, such as patients with multiple sclerosis (MS) receiving treatment with natalizumab. However, intrathecal synthesis of lipid-reactive IgM in MS patients is associated with a markedly lower incidence of natalizumab-associated PML compared to those without this antibody repertoire. Here we demonstrate that a subset of lipid-reactive human and murine IgMs induce a functional anti-viral response that inhibits replication of encephalitic Alpha and Orthobunyaviruses in multi-cellular central nervous system cultures. These lipid-specific IgMs trigger microglia to produce IFN-ß in a cGAS-STING-dependent manner, which induces an IFN-α/ß-receptor 1-dependent antiviral response in glia and neurons. These data identify lipid-reactive IgM as a mediator of anti-viral activity in the nervous system and provide a rational explanation why intrathecal synthesis of lipid-reactive IgM correlates with a reduced incidence of iatrogenic PML in MS.


Asunto(s)
Autoanticuerpos/líquido cefalorraquídeo , Inmunoglobulina M/líquido cefalorraquídeo , Leucoencefalopatía Multifocal Progresiva/inmunología , Lípidos/inmunología , Esclerosis Múltiple , Animales , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Humanos , Huésped Inmunocomprometido/inmunología , Inmunoglobulina M/inmunología , Factores Inmunológicos/efectos adversos , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/inmunología , Natalizumab/efectos adversos , Ratas , Ratas Sprague-Dawley
15.
Methods Mol Biol ; 2143: 233-261, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32524485

RESUMEN

Transmission electron microscopy of central nervous system white matter has provided unparalleled access to the ultrastructural features of axons, their myelin sheaths, and the major cells of white matter; namely, oligodendrocytes, oligodendrocyte precursors, astrocytes, and microglia. In particular, it has been invaluable in elucidating pathological changes in axons and myelin following experimentally induced injury or genetic alteration, in animal models. While also of value in the examination of human white matter, the tissue is rarely fixed adequately for the types of detailed analyses that can be performed on well-preserved samples from animal models, perfusion fixed at the time of death. In this chapter we describe methods for obtaining, processing, and visualizing white matter samples using transmission electron microscopy of perfusion fixed tissue and for unbiased morphometry of white matter, with particular emphasis on axon and myelin pathology. Several advanced electron microscopy techniques are now available, but this method remains the most expedient and accessible for routine ultrastructural examination and morphometry.


Asunto(s)
Microscopía Electrónica de Transmisión/métodos , Degeneración Walleriana/patología , Sustancia Blanca/ultraestructura , Animales , Axones/ultraestructura , Ácido Cacodílico , Disección/métodos , Resinas Epoxi , Formaldehído , Glutaral , Humanos , Microtomía/métodos , Vaina de Mielina/ultraestructura , Neuroglía/ultraestructura , Tetróxido de Osmio , Anhídridos Ftálicos , Polímeros , Coloración y Etiquetado/métodos , Adhesión del Tejido/métodos , Fijación del Tejido/métodos
16.
Brain ; 143(5): 1383-1399, 2020 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-32419025

RESUMEN

Pelizaeus-Merzbacher disease is a fatal X-linked leukodystrophy caused by mutations in the PLP1 gene, which is expressed in the CNS by oligodendrocytes. Disease onset, symptoms and mortality span a broad spectrum depending on the nature of the mutation and thus the degree of CNS hypomyelination. In the absence of an effective treatment, direct cell transplantation into the CNS to restore myelin has been tested in animal models of severe forms of the disease with failure of developmental myelination, and more recently, in severely affected patients with early disease onset due to point mutations in the PLP1 gene, and absence of myelin by MRI. In patients with a PLP1 duplication mutation, the most common cause of Pelizaeus-Merzbacher disease, the pathology is poorly defined because of a paucity of autopsy material. To address this, we examined two elderly patients with duplication of PLP1 in whom the overall syndrome, including end-stage pathology, indicated a complex disease involving dysmyelination, demyelination and axonal degeneration. Using the corresponding Plp1 transgenic mouse model, we then tested the capacity of transplanted neural stem cells to restore myelin in the context of PLP overexpression. Although developmental myelination and axonal coverage by endogenous oligodendrocytes was extensive, as assessed using electron microscopy (n = 3 at each of four end points) and immunostaining (n = 3 at each of four end points), wild-type neural precursors, transplanted into the brains of the newborn mutants, were able to effectively compete and replace the defective myelin (n = 2 at each of four end points). These data demonstrate the potential of neural stem cell therapies to restore normal myelination and protect axons in patients with PLP1 gene duplication mutation and further, provide proof of principle for the benefits of stem cell transplantation for other fatal leukodystrophies with 'normal' developmental myelination.


Asunto(s)
Encéfalo/patología , Modelos Animales de Enfermedad , Células-Madre Neurales/trasplante , Enfermedad de Pelizaeus-Merzbacher/patología , Animales , Humanos , Masculino , Ratones Transgénicos , Mutación , Proteína Proteolipídica de la Mielina/genética , Vaina de Mielina/patología , Enfermedad de Pelizaeus-Merzbacher/genética
17.
Sci Adv ; 6(11): eaax6328, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32195337

RESUMEN

Alterations to the gut microbiome are associated with various neurological diseases, yet evidence of causality and identity of microbiome-derived compounds that mediate gut-brain axis interaction remain elusive. Here, we identify two previously unknown bacterial metabolites 3-methyl-4-(trimethylammonio)butanoate and 4-(trimethylammonio)pentanoate, structural analogs of carnitine that are present in both gut and brain of specific pathogen-free mice but absent in germ-free mice. We demonstrate that these compounds are produced by anaerobic commensal bacteria from the family Lachnospiraceae (Clostridiales) family, colocalize with carnitine in brain white matter, and inhibit carnitine-mediated fatty acid oxidation in a murine cell culture model of central nervous system white matter. This is the first description of direct molecular inter-kingdom exchange between gut prokaryotes and mammalian brain cells, leading to inhibition of brain cell function.


Asunto(s)
Carnitina , Clostridiales/metabolismo , Microbioma Gastrointestinal , Mucosa Intestinal , Sustancia Blanca/metabolismo , Animales , Carnitina/análogos & derivados , Carnitina/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Masculino , Ratones
18.
Acta Neuropathol Commun ; 7(1): 212, 2019 12 19.
Artículo en Inglés | MEDLINE | ID: mdl-31856924

RESUMEN

Fibroblast growth factor (FGF) signaling contributes to failure of remyelination in multiple sclerosis, but targeting this therapeutically is complicated by its functional pleiotropy. We now identify FGF2 as a factor up-regulated by astrocytes in active inflammatory lesions that disrupts myelination via FGF receptor 2 (FGFR2) mediated activation of Wingless (Wnt) signaling; pharmacological inhibition of Wnt being sufficient to abrogate inhibition of myelination by FGF2 in tissue culture. Using a novel FGFR1-selective agonist (F2 V2) generated by deleting the N-terminal 26 amino acids of FGF2 we demonstrate polarizing signal transduction to favor FGFR1 abrogates FGF mediated inhibition of myelination but retains its ability to induce expression of pro-myelinating and immunomodulatory factors that include Cd93, Lif, Il11, Hbegf, Cxcl1 and Timp1. Our data provide new insights into the mechanistic basis of remyelination failure in MS and identify selective activation of FGFR1 as a novel strategy to induce a neuroprotective signaling environment in multiple sclerosis and other neurological diseases.


Asunto(s)
Astrocitos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/biosíntesis , Esclerosis Múltiple/metabolismo , Fibras Nerviosas Mielínicas/metabolismo , Neuroprotección/fisiología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/biosíntesis , Animales , Astrocitos/química , Astrocitos/patología , Factor 2 de Crecimiento de Fibroblastos/análisis , Factor 2 de Crecimiento de Fibroblastos/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/genética , Esclerosis Múltiple/patología , Fibras Nerviosas Mielínicas/patología , Ratas , Ratas Sprague-Dawley
19.
Immunology ; 158(4): 253-254, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31729761

RESUMEN

Regulatory T cells (Tregs) are important for limiting inflammation-dependent damage in neural tissue. However, Tregs have also been shown to inhibit neural repair associated with type 2 (anti-inflammatory/wound healing) immune responses. Recently, it was demonstrated that Sirtuins, a family of proteins that contribute to the control of cellular responses to metabolic stimuli, influence the functions of Tregs. Specifically, SIRT4 was found to suppress the anti-neuroinflammatory activity of Tregs infiltrating the spinal cord following injury; when SIRT4 expression was genetically suppressed, Tregs made more anti-inflammatory factors, IL-10, FoxP3, and transforming growth factor beta (TGFß). Thus, understanding how the SIRT4-Treg pathway can be manipulated could provide useful avenues to control both pathogenic and neuroprotective immune responses.


Asunto(s)
Neuronas/inmunología , Traumatismos de la Médula Espinal/inmunología , Médula Espinal/inmunología , Linfocitos T Reguladores/inmunología , Animales , Factores de Transcripción Forkhead/metabolismo , Humanos , Inmunidad , Interleucina-10/metabolismo , Proteínas Mitocondriales/metabolismo , Inflamación Neurogénica , Neuroprotección , Sirtuinas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Cicatrización de Heridas
20.
Glob Health Action ; 12(1): 1666566, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31640505

RESUMEN

Zika Preparedness Latin American Network (ZikaPLAN) is a research consortium funded by the European Commission to address the research gaps in combating Zika and to establish a sustainable network with research capacity building in the Americas. Here we present a report on ZikaPLAN`s mid-term achievements since its initiation in October 2016 to June 2019, illustrating the research objectives of the 15 work packages ranging from virology, diagnostics, entomology and vector control, modelling to clinical cohort studies in pregnant women and neonates, as well as studies on the neurological complications of Zika infections in adolescents and adults. For example, the Neuroviruses Emerging in the Americas Study (NEAS) has set up more than 10 clinical sites in Colombia. Through the Butantan Phase 3 dengue vaccine trial, we have access to samples of 17,000 subjects in 14 different geographic locations in Brazil. To address the lack of access to clinical samples for diagnostic evaluation, ZikaPLAN set up a network of quality sites with access to well-characterized clinical specimens and capacity for independent evaluations. The International Committee for Congenital Anomaly Surveillance Tools was formed with global representation from regional networks conducting birth defects surveillance. We have collated a comprehensive inventory of resources and tools for birth defects surveillance, and developed an App for low resource regions facilitating the coding and description of all major externally visible congenital anomalies including congenital Zika syndrome. Research Capacity Network (REDe) is a shared and open resource centre where researchers and health workers can access tools, resources and support, enabling better and more research in the region. Addressing the gap in research capacity in LMICs is pivotal in ensuring broad-based systems to be prepared for the next outbreak. Our shared and open research space through REDe will be used to maximize the transfer of research into practice by summarizing the research output and by hosting the tools, resources, guidance and recommendations generated by these studies. Leveraging on the research from this consortium, we are working towards a research preparedness network.


Asunto(s)
Brotes de Enfermedades/prevención & control , Infección por el Virus Zika/epidemiología , Infección por el Virus Zika/prevención & control , Américas , Brasil , Creación de Capacidad/organización & administración , Anomalías Congénitas/epidemiología , Anomalías Congénitas/prevención & control , Femenino , Accesibilidad a los Servicios de Salud/organización & administración , Humanos , Recién Nacido , Control de Mosquitos/organización & administración , Vigilancia de la Población , Embarazo , Virus Zika , Infección por el Virus Zika/diagnóstico
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