Diabetic retinopathy (DR) is a significant cause of blindness in working-age adults worldwide. Lack of effective strategies to prevent or reduce vision loss is a major problem. Since the degeneration of retinal neurons is an early event in the diabetic retina, studies to characterize the molecular mechanisms of diabetes-induced retinal neuronal damage and dysfunction are of high significance. We have demonstrated that spermine oxidase (SMOX), a mediator of polyamine oxidation is critically involved in causing neurovascular damage in the retina. The involvement of SMOX in diabetes-induced retinal neuronal damage is completely unknown. Utilizing the streptozotocin-induced mouse model of diabetes, the impact of the SMOX inhibitor, MDL 72527, on neuronal damage and dysfunction in the diabetic retina was investigated. Retinal function was assessed by electroretinography (ERG) and retinal architecture was evaluated using spectral domain-optical coherence tomography. Retinal cryosections were prepared for immunolabeling of inner retinal neurons and retinal lysates were used for Western blotting. We observed a marked decrease in retinal function in diabetic mice compared to the non-diabetic controls. Treatment with MDL 72527 significantly improved the ERG responses in diabetic retinas. Diabetes-induced retinal thinning was also inhibited by the MDL 72527 treatment. Our analysis further showed that diabetes-induced retinal ganglion cell damage and neurodegeneration were markedly attenuated by MDL 72527 treatment. These results strongly implicate SMOX in diabetes-induced retinal neurodegeneration and visual dysfunction.

Polyamine oxidation plays a major role in neurodegenerative diseases. Previous studies from our laboratory demonstrated that spermine oxidase (SMOX, a member of the polyamine oxidase family) inhibition using MDL 72527 reduced neurodegeneration in models of retinal excitotoxicity and diabetic retinopathy. However, the mechanisms behind the neuroprotection offered by SMOX inhibition are not completely studied. Utilizing the experimental model of retinal excitotoxicity, the present study determined the impact of SMOX blockade in retinal neuroinflammation. Our results demonstrated upregulation in the number of cells positive for Iba-1 (ionized calcium-binding adaptor molecule 1), CD (Cluster Differentiation) 68, and CD16/32 in excitotoxicity-induced retinas, while MDL 72527 treatment reduced these changes, along with increases in the number of cells positive for Arginase1 and CD206. When retinal excitotoxicity upregulated several pro-inflammatory genes, MDL 72527 treatment reduced many of them and increased anti-inflammatory genes. Furthermore, SMOX inhibition upregulated antioxidant signaling (indicated by elevated Nrf2 and HO-1 levels) and reduced protein-conjugated acrolein in excitotoxic retinas. In vitro studies using C8-B4 cells showed changes in cellular morphology and increased reactive oxygen species formation in response to acrolein (a product of SMOX activity) treatment. Overall, our findings indicate that the inhibition SMOX pathway reduced neuroinflammation and upregulated antioxidant signaling in the retina.

Increased TERT mRNA is associated with disease relapse in favorable histology Wilms tumor (WT). This study sought to understand the mechanism of increased TERT expression by determining the association between TERT and WT1 and N-MYC, two proteins important in Wilms tumor pathogenesis that have been shown to regulate TERT expression. Three out of 45 (6.7%) WTs and the corresponding patient-derived xenografts harbored canonical gain-of-function mutations in the TERT promoter. This study identified near ubiquitous hypermethylation of the TERT promoter region in WT compared to normal kidney. WTs with biallelic inactivating mutations in WT1 (7/45, 15.6%) were found to have lower TERT expression by RNA-seq and qRT-PCR and lower telomerase activity determined by the telomerase repeat amplification protocol. Anaplastic histology and increased percentage of blastema were positively correlated with higher TERT expression and telomerase activity. In vitro shRNA knockdown of WT1 resulted in decreased expression of TERT, reduced colony formation, and decreased proliferation of WiT49, an anaplastic WT cell line with wild-type WT1. CRISPR-Cas9-mediated knockout of WT1 resulted in decreased expression of telomere-related gene pathways. However, an inducible Wt1-knockout mouse model showed no relationship between Wt1 knockout and Tert expression in normal murine nephrogenesis, suggesting that WT1 and TERT are coupled in transformed cells but not in normal kidney tissues. N-MYC overexpression resulted in increased TERT promoter activity and TERT transcription. Thus, multiple mechanisms of TERT activation are involved in WT and are associated with anaplastic histology and increased blastema. This study is novel because it identifies potential mechanisms of TERT activation in Wilms tumor that could be of therapeutic interests.