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1.
Glia ; 71(7): 1683-1698, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-36945189

RESUMO

There is an urgent need for therapies that target the multicellular pathology of central nervous system (CNS) disease. Modified, nonanticoagulant heparins mimic the heparan sulfate glycan family and are known regulators of multiple cellular processes. In vitro studies have demonstrated that low sulfated modified heparin mimetics (LS-mHeps) drive repair after CNS demyelination. Herein, we test LS-mHep7 (an in vitro lead compound) in experimental autoimmune encephalomyelitis (EAE) and cuprizone-induced demyelination. In EAE, LS-mHep7 treatment resulted in faster recovery and rapidly reduced inflammation which was accompanied by restoration of animal weight. LS-mHep7 treatment had no effect on remyelination or on OLIG2 positive oligodendrocyte numbers within the corpus callosum in the cuprizone model. Further in vitro investigation confirmed that LS-mHep7 likely mediates its pro-repair effect in the EAE model by sequestering inflammatory cytokines, such as CCL5 which are upregulated during immune-mediated inflammatory attacks. These data support the future clinical translation of this next generation modified heparin as a treatment for CNS diseases with active immune system involvement.


Assuntos
Doenças do Sistema Nervoso Central , Encefalomielite Autoimune Experimental , Animais , Camundongos , Cuprizona/toxicidade , Sulfatos/efeitos adversos , Oligodendroglia/patologia , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Corpo Caloso/patologia , Doenças do Sistema Nervoso Central/patologia , Heparitina Sulfato/uso terapêutico , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Bainha de Mielina/patologia
2.
Bioinformatics ; 35(21): 4528-4530, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31095292

RESUMO

SUMMARY: MyelinJ is a free user friendly ImageJ macro for high throughput analysis of fluorescent micrographs such as 2D-myelinating cultures and statistical analysis using R. MyelinJ can analyse single images or complex experiments with multiple conditions, where the ggpubr package in R is automatically used for statistical analysis and the production of publication quality graphs. The main outputs are percentage (%) neurite density and % myelination. % neurite density is calculated using the normalize local contrast algorithm, followed by thresholding, to adjust for differences in intensity. For % myelination the myelin sheaths are selected using the Frangi vesselness algorithm, in conjunction with a grey scale morphology filter and the removal of cell bodies using a high intensity mask. MyelinJ uses a simple graphical user interface and user name system for reproducibility and sharing that will be useful to the wider scientific community that study 2D-myelination in vitro. AVAILABILITY AND IMPLEMENTATION: MyelinJ is freely available at https://github.com/BarnettLab/MyelinJ. For statistical analysis the freely available R and the ggpubr package are also required. MyelinJ has a user guide (Supplementary Material) and has been tested on both Windows (Windows 10) and Mac (High Sierra) operating systems. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Algoritmos , Software , Neuritos , Reprodutibilidade dos Testes
3.
Glia ; 67(4): 668-687, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30585359

RESUMO

The lack of endogenous repair following spinal cord injury (SCI) accounts for the frequent permanent deficits for which effective treatments are absent. Previously, we demonstrated that low sulfated modified heparin mimetics (LS-mHeps) attenuate astrocytosis, suggesting they may represent a novel therapeutic approach. mHeps are glycomolecules with structural similarities to resident heparan sulfates (HS), which modulate cell signaling by both sequestering ligands, and acting as cofactors in the formation of ligand-receptor complexes. To explore whether mHeps can affect the myelination and neurite outgrowth necessary for repair after SCI, we created lesioned or demyelinated neural cell co-cultures and exposed them with a panel of mHeps with varying degrees and positions of their sulfate moieties. LS-mHep7 enhanced neurite outgrowth and myelination, whereas highly sulfated mHeps (HS-mHeps) had attenuating effects. LS-mHeps had no effects on myelination or neurite extension in developing, uninjured myelinating cultures, suggesting they might exert their proregenerating effects by modulating or sequestering inhibitory factors secreted after injury. To investigate this, we examined conditioned media from cultures using chemokine arrays and conducted an unbiased proteomics approach by applying TMT-LC/MS to mHep7 affinity purified conditioned media from these cultures. Multiple protein factors reported to play a role in damage or repair mechanisms were identified, including amyloid betaA4. Amyloid beta peptide (1-42) was validated as an important candidate by treating myelination cultures and shown to inhibit myelination. Thus, we propose that LS-mHeps exert multiple beneficial effects on mechanisms supporting enhanced repair, and represent novel candidates as therapeutics for CNS damage.


Assuntos
Doenças do Sistema Nervoso Central/tratamento farmacológico , Doenças do Sistema Nervoso Central/metabolismo , Doenças Desmielinizantes/tratamento farmacológico , Heparitina Sulfato/uso terapêutico , Recuperação de Função Fisiológica/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Animais , Animais Recém-Nascidos , Antimetabólitos/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Desoxiuridina/farmacologia , Embrião de Mamíferos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas da Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito/metabolismo , Neuritos/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Medula Espinal/citologia
4.
Viruses ; 13(1)2021 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-33440758

RESUMO

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.


Assuntos
Axônios/virologia , Doenças Desmielinizantes/etiologia , Infecção por Zika virus/complicações , Infecção por Zika virus/virologia , Zika virus/fisiologia , Animais , Biomarcadores , Traumatismos dos Nervos Cranianos/etiologia , Traumatismos dos Nervos Cranianos/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Camundongos , Ratos , Transcriptoma
5.
Nat Rev Neurol ; 16(4): 229-240, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32099190

RESUMO

Spinal cord injury (SCI) remains one of the biggest challenges in the development of neuroregenerative therapeutics. Cell transplantation is one of numerous experimental strategies that have been identified and tested for efficacy at both preclinical and clinical levels in recent years. In this Review, we briefly discuss the state of human olfactory cell transplantation as a therapy, considering both its current clinical status and its limitations. Furthermore, we introduce a mesenchymal stromal cell derived from human olfactory tissue, which has the potential to induce multifaceted reparative effects in the environment within and surrounding the lesion. We argue that no single therapy will be sufficient to treat SCI effectively and that a combination of cell-based, rehabilitation and pharmaceutical interventions is the most promising approach to aid repair. For this reason, we also introduce a novel pharmaceutical strategy based on modifying the activity of heparan sulfate, an important regulator of a wide range of biological cell functions. The multi-target approach that is exemplified by these types of strategies will probably be necessary to optimize SCI treatment.


Assuntos
Heparitina Sulfato/uso terapêutico , Transplante de Células-Tronco Mesenquimais/métodos , Mucosa Olfatória/citologia , Traumatismos da Medula Espinal/terapia , Regeneração da Medula Espinal , Transplante de Células/métodos , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Heparina/uso terapêutico , Heparitina Sulfato/análogos & derivados , Humanos , Células-Tronco Mesenquimais/citologia , Regeneração Nervosa , Neuroglia , Mucosa Olfatória/fisiologia , Neurônios Receptores Olfatórios
6.
Biology (Basel) ; 8(3)2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31261710

RESUMO

In vitro cell-based assays have been fundamental in modern drug discovery and have led to the identification of novel therapeutics. We have developed complex mixed central nervous system (CNS) cultures, which recapitulate the normal process of myelination over time and allow the study of several parameters associated with CNS damage, both during development and after injury or disease. In particular, they have been used as a reliable screen to identify drug candidates that may promote (re)myelination and/or neurite outgrowth. Previously, using these cultures, we demonstrated that a panel of low sulphated heparin mimetics, with structures similar to heparan sulphates (HSs), can reduce astrogliosis, and promote myelination and neurite outgrowth. HSs reside in either the extracellular matrix or on the surface of cells and are thought to modulate cell signaling by both sequestering ligands, and acting as co-factors in the formation of ligand-receptor complexes. In this study, we have used these cultures as a screen to address the repair potential of numerous other commercially available sulphated glycomolecules, namely heparosans, ulvans, and fucoidans. These compounds are all known to have certain characteristics that mimic cellular glycosaminoglycans, similar to heparin mimetics. We show that the N-sulphated heparosans promoted myelination. However, O-sulphated heparosans did not affect myelination but promoted neurite outgrowth, indicating the importance of structure in HS function. Moreover, neither highly sulphated ulvans nor fucoidans had any effect on remyelination but CX-01, a low sulphated porcine intestinal heparin, promoted remyelination in vitro. These data illustrate the use of myelinating cultures as a screen and demonstrate the potential of heparin mimetics as CNS therapeutics.

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