Low sulfated heparan sulfate mimetic differentially affects repair in immune-mediated and toxin-induced experimental models of demyelination.

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.

MyelinJ: an ImageJ macro for high throughput analysis of myelinating cultures.

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.

Low sulfated heparins target multiple proteins for central nervous system repair.

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.

Zika Virus Infection Leads to Demyelination and Axonal Injury in Mature CNS Cultures.

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.

Multi-target approaches to CNS repair: olfactory mucosa-derived cells and heparan sulfates.

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.

The Use of Myelinating Cultures as a Screen of Glycomolecules for CNS Repair.

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.