Zhen Fu

Nitric oxide (NO) has been implicated as a pathogenic mediator in a variety of central nervous system (CNS) disease states, including the animal model of multiple sclerosis (MS) and experimental allergic encephalomyelitis. We have examined post-mortem brain tissues collected from patients previously diagnosed with MS, as well as tissues collected from the brains of patients dying without neuropathies. Both Northern blot analysis and reverse transcriptase (RT)-driven in situ PCR (RT-in situ PCR) studies demonstrated that inducible NO synthase (iNOS) mRNA was present in the brain tissues from MS patients but was absent in equivalent tissues from normal controls. We have also performed experiments identifying the cell type responsible for iNOS expression by RT-in situ PCR in combination with immunohistochemistry. Concomitantly, we analyzed the tissues for the presence of the NO reaction product nitrotyrosine to demonstrate the presence of a protein nitrosylation adduct. We report here that iNOS mRNA was detectable in the brains of 100% of the CNS tissues from seven MS patients examined but in none of the three normal brains. RT-in situ PCR experiments also demonstrated the presence of iNOS mRNA in the cytoplasm of cells that also expressed the ligand recognized by the Ricinus communis agglutinin 1 (RCA-1), a monocyte/macrophage lineage marker. Additionally, specific labeling of cells was observed when brain tissues from MS patients were exposed to antisera reactive with nitrotyrosine residues but was significantly less plentiful in brain tissue from patients without CNS disease. These results demonstrate that iNOS, one of the enzymes responsible for the production of NO, is expressed at significant levels in the brains of patients with MS and may contribute to the pathology associated with the disease.

The putative role of nitric oxide in the neuropathogenesis of Borna disease was investigated by determining changes in the expression of inducible nitric oxide synthase (iNOS) mRNA and constitutively expressed NOS (cNOS) mRNA in brains of Borna disease virus (BDV)-infected rats. iNOS mRNA was not detected in normal rat brain but was identified in BDV-infected brain at 14 days postinfection (p.i.), reaching maximum levels at 21 days p.i., when neurological signs and inflammatory reactions in the brain were also at a peak. cNOS mRNA was expressed in both normal brain and infected brain, increasing markedly at 17 days p.i. and reaching a peak at 21 days p.i. In situ hybridization analysis revealed iNOS mRNA in some, but not all, BDV-infected regions of the brain, particularly in the basolateral cortex and the hippocampus. iNOS-positive cells, as identified immunohistologically, were preferentially localized in perivascular areas of the hippocampus and in outer cortical layers. These iNOS-positive cells resembled monocytes/macrophages in morphology and distribution pattern but were significantly fewer. The correlation of iNOS and cNOS mRNA expression with the development of neurological disease, as well as the enhanced expression of iNOS within brain regions with inflammatory lesions, strongly suggests that NO may contribute to pathogenesis of Borna disease.

Platelet-derived growth subunit A (PDGFA) plays critical roles in development of glioblastoma (GBM) with substantial evidence from TCGA database analyses and in vivo mouse models. So far, only platelet-derived growth receptor α (PDGFRA) has been identified as receptor for PDGFA. However, PDGFA and PDGFRA are categorized into different molecular subtypes of GBM in TCGA_GBM database. Our data herein further showed that activity or expression deficiency of PDGFRA did not effectively block PDGFA activity. Therefore, PDGFRA might be not necessary for PDGFA function.To profile proteins involved in PDGFA function, we performed co-immunoprecipitation (Co-IP) and Mass Spectrum (MS) and delineated the network of PDGFA-associated proteins for the first time. Unexpectedly, the data showed that EPHA2 could be temporally activated by PDGFA even without activation of PDGFRA and AKT. Furthermore, MS, Co-IP, in vitro binding thermodynamics, and proximity ligation assay consistently proved the interaction of EPHA2 and PDGFA. In addition, we observed that high expression of EPHA2 leaded to upregulation of PDGF signaling targets in TCGA_GBM database and clinical GBM samples. Co-upregulation of PDGFRA and EPHA2 leaded to worse patient prognosis and poorer therapeutic effects than other contexts, which might arise from expression elevation of genes related with malignant molecular subtypes and invasive growth. Due to PDGFA-induced EPHA2 activation, blocking PDGFRA by inhibitor could not effectively suppress proliferation of GBM cells, but simultaneous inhibition of both EPHA2 and PDGFRA showed synergetic inhibitory effects on GBM cells in vitro and in vivo. Taken together, our study provided new insights on PDGFA function and revealed EPHA2 as a potential receptor of PDGFA. EPHA2 might contribute to PDGFA signaling transduction in combination with PDGFRA and mediate the resistance of GBM cells to PDGFRA inhibitor. Therefore, combination of inhibitors targeting PDGFRA and EHA2 represented a promising therapeutic strategy for GBM treatment.