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Astrocytic YAP protects the optic nerve and retina in an experimental autoimmune encephalomyelitis model through TGF-β signaling.

ABSTRACT: Rationale: Optic neuritis is one of main symptoms in multiple sclerosis (MS) that causes visual disability. Astrocytes are pivotal regulators of neuroinflammation in MS, and astrocytic yes-associated protein (YAP) plays a critical role in neuroinflammation. Meanwhile, YAP signaling is involved in visual impairment, including glaucoma, retinal choroidal atrophy and retinal detachment. However, the roles and underlying mechanisms of astrocytic YAP in neuroinflammation and demyelination of MS-related optic neuritis (MS-ON) remains unclear. Methods: To assess the functions of YAP in MS-ON, experimental autoimmune encephalomyelitis (EAE, a common model of MS) was established, and mice that conditional knockout (CKO) of YAP in astrocytes, YAP^GFAP-CKO mice, were successfully generated. Behavior tests, immunostaining, Nissl staining, Hematoxylin-Eosin (HE) staining, TUNEL staining, Luxol Fast Blue (LFB) staining, electron microscopy (EM), quantitative real-time PCR (qPCR), gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) by RNA sequencing were used to examine the function and mechanism of YAP signaling based on these YAP^GFAP-CKO mice and EAE model mice. To further explore the potential treatment of YAP signaling in EAE, EAE mice were treated with various drugs, including SRI-011381 that is an agonist of transforming growth factor-β (TGF-β) pathway, and XMU-MP-1 which inhibits Hippo kinase MST1/2 to activate YAP. Results: We found that YAP was significantly upregulated and activated in the astrocytes of optic nerve in EAE mice. Conditional knockout of YAP in astrocytes caused more severe inflammatory infiltration and demyelination in optic nerve, and damage of retinal ganglion cells (RGCs) in EAE mice. Moreover, YAP deletion in astrocytes promoted the activation of astrocytes and microglia, but inhibited the proliferation of astrocytes of optic nerve in EAE mice. Mechanically, TGF-β signaling pathway was significantly down-regulated after YAP deletion in astrocytes. Additionally, both qPCR and immunofluorescence assays confirmed the reduction of TGF-β signaling pathway in YAP^GFAP-CKO EAE mice. Interestingly, SRI-011381 partially rescued the deficits in optic nerve and retina of YAP^GFAP-CKO EAE mice. Finally, activation of YAP signaling by XMU-MP-1 relieved the neuroinflammation and demyelination in optic nerve of EAE mice. Conclusions: These results suggest astrocytic YAP may prevent the neuroinflammatory infiltration and demyelination through upregulation of TGF-β signaling and provide targets for the development of therapeutic strategies tailored for MS-ON.

SUBMITTER: Wu Q

PROVIDER: S-EPMC8344002 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe oral immunomodulatory agent laquinimod is currently evaluated for multiple sclerosis (MS) treatment. Phase II and III studies demonstrated a reduction of degenerative processes. In addition to anti-inflammatory effects, laquinimod might have neuroprotective properties, but its impact on the visual system, which is often affected by MS, is unknown. The aim of our study was to investigate potential protective effects of laquinimod on the optic nerve and retina in an experimental autoimmune encephalomyelitis (EAE) model.MethodsWe induced EAE in C57/BL6 mice via MOG35-55 immunization. Animals were divided into an untreated EAE group, three EAE groups receiving laquinimod (1, 5, or 25 mg/kg daily), starting the day post-immunization, and a non-immunized control group. Thirty days post-immunization, scotopic electroretinograms were carried out, and mice were sacrificed for histopathology (HE, LFB), immunohistochemistry (MBP, Iba1, Tmem119, F4/80, GFAP, vimentin, Brn-3a, cleaved caspase 3) of the optic nerve and retina, and retinal qRT-PCR analyses (Brn-3a, Iba1, Tmem119, AMWAP, CD68, GFAP). To evaluate the effect of a therapeutic approach, EAE animals were treated with 25 mg/kg laquinimod from day 16 when 60% of the animals had developed clinical signs of EAE.ResultsLaquinimod reduced neurological EAE symptoms and improved the neuronal electrical output of the inner nuclear layer compared to untreated EAE mice. Furthermore, cellular infiltration, especially recruited phagocytes, and demyelination in the optic nerve were reduced. Microglia were diminished in optic nerve and retina. Retinal macroglial signal was reduced under treatment, whereas in the optic nerve macroglia were not affected. Additionally, laquinimod preserved retinal ganglion cells and reduced apoptosis. A later treatment with laquinimod in a therapeutic approach led to a reduction of clinical signs and to an improved b-wave amplitude. However, no changes in cellular infiltration and demyelination of the optic nerves were observed. Also, the number of retinal ganglion cells remained unaltered.ConclusionFrom our study, we deduce neuroprotective and anti-inflammatory effects of laquinimod on the optic nerve and retina in EAE mice, when animals were treated before any clinical signs were noted. Given the fact that the visual system is frequently affected by MS, the agent might be an interesting subject of further neuro-ophthalmic investigations.

Project description:Glaucoma is a multifactorial disease and especially mechanisms occurring independently from an elevated intraocular pressure (IOP) are still unknown. Likely, the immune system contributes to the glaucoma pathogenesis. Previously, IgG antibody depositions and retinal ganglion cell (RGC) loss were found in an IOP-independent autoimmune glaucoma model. Therefore, we investigated the possible participation of the complement system in this model. Here, rats were immunized with bovine optic nerve homogenate antigen (ONA), while controls (Co) received sodium chloride (n = 5-6/group). After 14 days, RGC density was quantified on flatmounts. No changes in the number of RGCs could be observed at this point in time. Longitudinal optic nerve sections were stained against the myelin basic protein (MBP). We could note few signs of degeneration processes. In order to detect distinct complement components, retinas and optic nerves were labeled with complement markers at 3, 7, 14, and 28 days and analyzed. Significantly more C3 and MAC depositions were found in retinas and optic nerves of the ONA group. These were already present at day 7, before RGC loss and demyelination occurred. Additionally, an upregulation of C3 protein was noted via Western Blot at this time. After 14 days, quantitative real-time PCR revealed significantly more C3 mRNA in the ONA retinas. An upregulation of the lectin pathway-associated mannose-serine-protease-2 (MASP2) was observed in the retinas as well as in the optic nerves of the ONA group after 7 days. Significantly more MASP2 in retinas could also be observed via Western Blot analyses at this point in time. No effect was noted in regard to C1q. Therefore, we assume that the immunization led to an activation of the complement system via the lectin pathway in retinas and optic nerves at an early stage in this glaucoma model. This activation seems to be an early response, which then triggers degeneration. These findings can help to develop novel therapy strategies for glaucoma patients.

Project description:Background/Aims:Increasing evidence have associated mitochondrial dysfunction and reactive oxygen species (ROS) generation to neuron loss in multiple sclerosis (MS). PGC-1α regulates mitochondrial biogenesis and respiration and plays a pivotal role in modulating ROS levels. Here, we investigated the potential function of peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α) in the mammalian MS disease model, EAE experimental autoimmune encephalomyelitis (EAE). Methods:We used WT mice to evaluate the change of PGC during the course of EAE. The transgenic mice with neuron-specific overexpression of PGC-1α was applied to explore how PGC1 in neuron affects EAE outcome. A neuronal cell line of lentivirus transfection leading to PGC-1α markedly increased was applied to verify the protective effects of PGC1 in neurons. RNA-seq was employed to explore the potential target genes and signaling pathways. Results: We observed that PGC-1α were decreased at 13d, continuously decreased at 20d, then mildly increased at 30d in wild-type (WT) EAE mice, which is accompanied with alterations of mitochondrial antioxidants SOD2, Prx3, Trx2 and UCP4,5. Our study revealed that PGC-1αf/f Eno2-Cre mice improved EAE clinical symptoms, ameliorated inflammation and demyelination in spinal cords, increased mitochondrial antioxidants SOD2, Prx3, Trx2 and UCPs, reduced the production of ROS and inhibited the apoptotic pathways. In addition, PGC-1αf/f Eno2-Cre mice showed decreased apoptotic neurons compared with the WT EAE mice. In vitro, overexpressing PGC-1α by lentivirus transfection revealed similar results with that of in vivo by using NSC-34 cells treated with 1mg/ml lipopolysaccharide (LPS). Furthermore, RNA-seq analysis showed that apoptotic processes were significantly enriched in the top 10 significant GO terms of differentially expressed (DE) gene and apotosis pathway was significantly enriched in KEGG pathway analysis. Conclusion: Taken together, our findings indicate that upregulation of neuronal PGC-1α protected neuron apotosis in EAE and in vitro.

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Astrocytic YAP protects the optic nerve and retina in an experimental autoimmune encephalomyelitis model through TGF-β signaling.

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