Gene expression in oligodendrocytes during remyelination reveals cholesterol homeostasis as a therapeutic target in multiple sclerosis.

Regional differences in neurons, astrocytes, oligodendrocytes, and microglia exist in the brain during health, and regional differences in the transcriptome may occur for each cell type during neurodegeneration. Multiple sclerosis (MS) is multifocal, and regional differences in the astrocyte transcriptome occur in experimental autoimmune encephalomyelitis (EAE), an MS model. MS and EAE are characterized by inflammation, demyelination, and axonal damage, with minimal remyelination. Here, RNA-sequencing analysis of MS tissues from six brain regions suggested a focus on oligodendrocyte lineage cells (OLCs) in corpus callosum. Olig1-RiboTag mice were used to determine the translatome of OLCs in vivo in corpus callosum during the remyelination phase of a chronic cuprizone model with axonal damage. Cholesterol-synthesis gene pathways dominated as the top up-regulated pathways in OLCs during remyelination. In EAE, remyelination was induced with estrogen receptor-ß (ERß) ligand treatment, and up-regulation of cholesterol-synthesis gene expression was again observed in OLCs. ERß-ligand treatment in the cuprizone model further increased cholesterol synthesis gene expression and enhanced remyelination. Conditional KOs of ERß in OLCs demonstrated that increased cholesterol-synthesis gene expression in OLCs was mediated by direct effects in both models. To address this direct effect, ChIP assays showed binding of ERß to the putative estrogen-response element of a key cholesterol-synthesis gene (Fdps). As fetal OLCs are exposed in utero to high levels of estrogens in maternal blood, we discuss how remyelinating properties of estrogen treatment in adults during injury may recapitulate normal developmental myelination through targeting cholesterol homeostasis in OLCs.

Cell-specific and region-specific transcriptomics in the multiple sclerosis model: Focus on astrocytes.

Changes in gene expression that occur across the central nervous system (CNS) during neurological diseases do not address the heterogeneity of cell types from one CNS region to another and are complicated by alterations in cellular composition during disease. Multiple sclerosis (MS) is multifocal by definition. Here, a cell-specific and region-specific transcriptomics approach was used to determine gene expression changes in astrocytes in the most widely used MS model, experimental autoimmune encephalomyelitis (EAE). Astrocyte-specific RNAs from various neuroanatomic regions were attained using RiboTag technology. Sequencing and bioinformatics analyses showed that EAE-induced gene expression changes differed between neuroanatomic regions when comparing astrocytes from spinal cord, cerebellum, cerebral cortex, and hippocampus. The top gene pathways that were changed in astrocytes from spinal cord during chronic EAE involved decreases in expression of cholesterol synthesis genes while immune pathway gene expression in astrocytes was increased. Optic nerve from EAE and optic chiasm from MS also showed decreased cholesterol synthesis gene expression. The potential role of cholesterol synthesized by astrocytes during EAE and MS is discussed. Together, this provides proof-of-concept that a cell-specific and region-specific gene expression approach can provide potential treatment targets in distinct neuroanatomic regions during multifocal neurological diseases.

Molecular Mechanisms Mediating Retinal Reactive Gliosis Following Bone Marrow Mesenchymal Stem Cell Transplantation.

A variety of diseases lead to degeneration of retinal ganglion cells (RGCs) and their axons within the optic nerve resulting in loss of visual function. Although current therapies may delay RGC loss, they do not restore visual function or completely halt disease progression. Regenerative medicine has recently focused on stem cell therapy for both neuroprotective and regenerative purposes. However, significant problems remain to be addressed, such as the long-term impact of reactive gliosis occurring in the host retina in response to transplanted stem cells. The aim of this work was to investigate retinal glial responses to intravitreally transplanted bone marrow mesenchymal stem cells (BM-MSCs) to help identify factors able to modulate graft-induced reactive gliosis. We found in vivo that intravitreal BM-MSC transplantation is associated with gliosis-mediated retinal folding, upregulation of intermediate filaments, and recruitment of macrophages. These responses were accompanied by significant JAK/STAT3 and MAPK (ERK1/2 and JNK) cascade activation in retinal Muller glia. Lipocalin-2 (Lcn-2) was identified as a potential new indicator of graft-induced reactive gliosis. Pharmacological inhibition of STAT3 in BM-MSC cocultured retinal explants successfully reduced glial fibrillary acidic protein expression in retinal Muller glia and increased BM-MSC retinal engraftment. Inhibition of stem cell-induced reactive gliosis is critical for successful transplantation-based strategies for neuroprotection, replacement, and regeneration of the optic nerve.

Identification of retinal ganglion cell neuroprotection conferred by platelet-derived growth factor through analysis of the mesenchymal stem cell secretome.

The development of neuroprotective strategies to attenuate retinal ganglion cell death could lead to novel therapies for chronic optic neuropathies such as glaucoma. Intravitreal transplantation of mesenchymal stem cells slows retinal ganglion cell death in models of optic nerve injury, but the mechanism of action remains unclear. Here we characterized the neuroprotective effects of mesenchymal stem cells and mesenchymal stem cell-derived factors in organotypic retinal explant culture and an in vivo model of ocular hypertensive glaucoma. Co-culture of rat and human bone marrow-derived mesenchymal stem cells with retinal explants increased retinal ganglion cell survival, after 7 days ex vivo, by ∼2-fold and was associated with reduced apoptosis and increased nerve fibre layer and inner plexiform layer thicknesses. These effects were not demonstrated by co-culture with human or mouse fibroblasts. Conditioned media from mesenchymal stem cells conferred neuroprotection, suggesting that the neuroprotection is mediated, at least partly, by secreted factors. We compared the concentrations of 29 factors in human mesenchymal stem cell and fibroblast conditioned media, and identified 11 enriched in the mesenchymal stem cell secretome. Treatment of retinal explants with a cocktail of these factors conferred retinal ganglion cell neuroprotection, with factors from the platelet-derived growth factor family being the most potent. Blockade of platelet-derived growth factor signalling with neutralizing antibody or with small molecule inhibitors of platelet-derived growth factor receptor kinase or downstream phosphatidylinositol 3 kinase eliminated retinal ganglion cell neuroprotection conferred by mesenchymal stem cell co-culture. Intravitreal injection of platelet-derived growth factor -AA or -AB led to profound optic nerve neuroprotection in vivo following experimental induction of elevated intraocular pressure. These data demonstrate that mesenchymal stem cells secrete a number of neuroprotective proteins and suggest that platelet-derived growth factor secretion in particular may play an important role in mesenchymal stem cell-mediated retinal ganglion cell neuroprotection. Furthermore, platelet-derived growth factor may represent an independent target for achieving retinal ganglion cell neuroprotection.

Retinal ganglion cell survival and axon regeneration in WldS transgenic rats after optic nerve crush and lens injury.

BACKGROUND: We have previously shown that the slow Wallerian degeneration mutation, whilst delaying axonal degeneration after optic nerve crush, does not protect retinal ganglion cell (RGC) bodies in adult rats. To test the effects of a combination approach protecting both axons and cell bodies we performed combined optic nerve crush and lens injury, which results in both enhanced RGC survival as well as axon regeneration past the lesion site in wildtype animals. RESULTS: As previously reported we found that the Wld(S) mutation does not protect RGC bodies after optic nerve crush alone. Surprisingly, we found that Wld(S) transgenic rats did not exhibit the enhanced RGC survival response after combined optic nerve crush and lens injury that was observed in wildtype rats. RGC axon regeneration past the optic nerve lesion site was, however, similar in Wld(S) and wildtypes. Furthermore, activation of retinal glia, previously shown to be associated with enhanced RGC survival and axon regeneration after optic nerve crush and lens injury, was unaffected in Wld(S) transgenic rats. CONCLUSIONS: RGC axon regeneration is similar between Wld(S) transgenic and wildtype rats, but Wld(S) transgenic rats do not exhibit enhanced RGC survival after combined optic nerve crush and lens injury suggesting that the neuroprotective effects of lens injury on RGC survival may be limited by the Wld(S) protein.

The astrocyte transcriptome in EAE optic neuritis shows complement activation and reveals a sex difference in astrocytic C3 expression.

Multiple sclerosis (MS) is a neuroinflammatory multifocal disorder. Optic neuritis is common in MS and leads to visual disability. No current treatments repair this damage. Discerning gene expression changes within specific cell types in optic nerve (ON) may suggest new treatment targets for visual disability in MS. Astrocytes are pivotal regulators of neuroinflammation, playing either detrimental or beneficial roles. Here, we used RiboTag technology to characterize the astrocyte-specific transcriptome in ON in the experimental autoimmune encephalomyelitis (EAE) model of MS. RNA sequencing analysis showed the Complement Cascade and Cholesterol Biosynthesis Pathways as the most enriched and de-enriched pathways, respectively, in ON astrocytes in EAE. Expression of complement component 3 (C3) was confirmed to be increased in ON astrocytes at the protein level during EAE. A bigger increase in C3 expressing ON astrocytes was found in EAE females versus healthy females, as compared to that in EAE males versus healthy males. Also, there was worse retinal ganglion cell (RGC) and axonal loss in EAE females. Regression analyses showed a negative correlation between C3 expressing astrocytes and RGC density. This cell-specific and sex-specific investigation of the optic nerve provides targets for the development of therapeutic strategies tailored for optic neuritis in MS.

Design of a Novel Gene Therapy Construct to Achieve Sustained Brain-Derived Neurotrophic Factor Signaling in Neurons.

Brain-derived neurotrophic factor (BDNF) acting through the tropomyosin-related receptor-B (TrkB) is an important signaling system for the maintenance and survival of neurons. Gene therapy using either recombinant adeno-associated virus (AAV) or lentiviral vectors can provide sustained delivery of BDNF to tissues where reduced BDNF signaling is hypothesized to contribute to disease pathophysiology. However, elevation in BDNF at target sites has been shown to lead to a downregulation of TrkB receptors, thereby reducing the effect of chronic BDNF delivery over time. A novel gene sequence has been designed coding both the ligand (BDNF) and the TrkB receptor in a single transgene separated by a short viral-2A sequence. The single transgene is efficiently processed intracellularly in vitro and in vivo to yield the two mature proteins, which are then independently transported to their final cellular locations: TrkB receptors to the cell surface, and BDNF contained within secretory vesicles. To accommodate the coding sequences of both BDNF and TrkB receptors within the narrow confines of the AAV vectors (4.7 kb pairs), the coding region for the pro-domain of BDNF was removed and the signal peptide sequence modified to improve production, intracellular transport, and secretion of mature BDNF (mBDNF). Intracellular processing and efficacy was shown in HEK293 cells and SH-SY5Y neuroblastoma cells using plasmid DNA and after incorporating the TrkB-2A-mBDNF into an AAV2 vector. Increased BDNF/TrkB-mediated intracellular signaling pathways were observed after AAV2 vector transfection while increased TrkB phosphorylation could be detected in combination with neuroprotection from hydrogen peroxide-induced oxidative stress. Correct processing was also shown in vivo in mouse retinal ganglion cells after AAV2 vector administration to the eye. This novel construct is currently being investigated for its efficacy in animal models to determine its potential to progress to human clinical studies in the future.

Retinal ganglion cell neuroprotection by an angiotensin II blocker in an ex vivo retinal explant model.

PURPOSE: An ex vivo organotypic retinal explant model was developed to examine retinal survival mechanisms relevant to glaucoma mediated by the renin angiotensin system in the rodent eye. METHODS: Eyes from adult Sprague Dawley rats were enucleated immediately post-mortem and used to make four retinal explants per eye. Explants were treated either with irbesartan (10 µM), vehicle or angiotensin II (2 µM) for four days. Retinal ganglion cell density was estimated by ßIII tubulin immunohistochemistry. Live imaging of superoxide formation with dihydroethidium (DHE) was performed. Protein expression was determined by Western blotting, and mRNA expression was determined by RT-PCR. RESULTS: Irbesartan (10 µM) almost doubled ganglion cell survival after four days. Angiotensin II (2 µM) reduced cell survival by 40%. Sholl analysis suggested that irbesartan improved ganglion cell dendritic arborisation compared to control and angiotensin II reduced it. Angiotensin-treated explants showed an intense DHE fluorescence not seen in irbesartan-treated explants. Analysis of protein and mRNA expression determined that the angiotensin II receptor At1R was implicated in modulation of the NADPH-dependent pathway of superoxide generation. CONCLUSION: Angiotensin II blockers protect retinal ganglion cells in this model and may be worth further investigation as a neuroprotective treatment in models of eye disease.

Release of vancomycin and gentamicin from a contact lens versus a fibrin coating applied to a contact lens.

PURPOSE: To develop a contact lens capable of releasing antibiotics for a minimum of 8 hours for the treatment of bacterial keratitis. METHODS: Fibrin gel was loaded with vancomycin or gentamicin and then shaped into a curved disc. The disc was then used to coat the surface of a commercial contact lens or was sealed between two lenses. Separate contact lenses were soaked in solutions of vancomycin or gentamicin. The in vitro release kinetics for each system was determined using PBS at 37°C and a particle-enhanced turbidimetric inhibition immunoassay. The bioactivity of the antibiotics released from the fibrin was confirmed by using a microbiological assay. RESULTS: Vancomycin and gentamicin were released at similar rates from soaked contact lenses and a coating of fibrin gel; however, the amounts of antibiotic delivered by the two systems differed considerably. The fibrin coating released over three times more gentamicin but less than one-fifth that of the lenses soaked in vancomycin. When fibrin was encapsulated between two contact lenses, significantly more controlled release was observed. For all systems, bactericidal amounts of vancomycin and gentamicin were released throughout the three-day testing period. CONCLUSIONS: As a delivery system, fibrin gel loaded with gentamicin performs better than contact lenses soaked in gentamicin. The opposite is true for vancomycin, where soaked lenses outperform fibrin gel. These systems could potentially be used as a treatment for bacterial keratitis.