Ghanshyam Swarup

Purpose: Diabetic retinopathy (DR) is a neurovascular complication of diabetes (DM) causing the loss of neurons (ganglion cells) in the retina. The disease etiology and potential pathogenic mechanisms in this disease remains unclear. In the present study, we aimed to further understand the key and novel pathogenic mechanisms involved in DR pathogenesis by taking cues from our global proteomics data. Methodology: The study was approved by the institutional review board (IRB) of LVPEI, Hyderabad, India. Vitreous humour samples (PDR; n=3, DM; n=3, Control; n=3) were collected from patients undergoing vitrectomy and subjected to LC-MS-MS analysis. The acquired raw data were searched against the human vitreous proteome and was further analysed by various bioinformatic and proteomic tools. Western blotting and IHC was performed to validate crucial pathways. Blood samples from patients (DM, PDR & NPDR) and controls (n=50); cadaveric retinas from diabetic and non-diabetic donors (n=10) and epiretinal membranes (ERM, n=10) from DR cases and controls were collected and RNA was isolated. Quantitative expression of genes involved in autophagy were performed. ɅɅCT was compared across different categories and significance estimated using a student t-test. Result: A total of 1079 proteins were identified with 16 completely novel proteins in eye proteome. Top pathways identified were autophagy, inflammation, LXR/RXR activation (lipid metabolism), ROS generation by macrophages, apoptosis and protein degradation. Regulatory proteins identified were associated with cell death, phagocytic activation, angiogenesis and apoptosis. Autophagy inducers such as ROS was found to be accumulated in the DR vitreous. TREM2, microglial receptor was identified as a novel protein in PDR vitreous. The expression of TREM2, an autophagy-associated gene was significantly (p-value = 0.05) upregulated in all categories as compared to control (NDM and/or NDM/No-DR). TREM2 protein also seemed to colocalise with microglial marker F4/80 in retinal tissues and intense expression was observed near the blood vessels in case of PDR retina. Other autophagy-associated markers were also differentially regulated in DR as compared to controls. Conclusion: This study emphasises on the strong role of autophagy pathways and its associated genes in the development of DR.

Autophagy has emerged as a critical innate immune mechanism for host elimination of intracellular pathogens, however, the role of the autophagy receptor optineurin during mycobacterial infection is not fully understood. To address this lacuna, we infected bone marrow-derived macrophages (BMDMs) derived from Optn ^+/+ and Optn ^-/- mice with Mycobacterium smegmatis , and observed the infection outcome at sequential time points. While low multiplicity of infection (MOI) did not show any significant difference between BMDMs from the two groups, at high MOI Optn ^-/- mice-derived macrophages showed significantly lower colony forming unit counts, as well as lower cell counts at 12 h and 24 h post-infection. Quantification of cell numbers and nuclear morphologies at various time points post-infection indicated a markedly higher cell death in the optineurin-deficient macrophages. Optineurin-deficient macrophages showed significantly lower levels of the autophagosomal protein LC3-II upon infection, indicating a potential role for optineurin in regulating autophagy during mycobacterial infection. Moreover, when stimulated by bacterial LPS, optineurin deficient macrophages, showed altered levels of the inflammatory cytokine pro-IL-1β. These observations taken together suggest a novel regulatory role for optineurin during mycobacterial infection, with its deficiency leading to an impairment in macrophage responses.

Transferrin and its receptor play an important role in iron homeostasis. Binding of transferrin to its receptor (TFRC, transferrin receptor protein 1) initiates endocytic trafficking and subsequent recycling of TFRC to the plasma membrane. RAB8-positive tubules emanating from the endocytic recycling compartment play an important role in receptor recycling. However, the signaling pathways or mechanisms that mediate formation of RAB8-positive tubules are not clear. Here, we have investigated the role of transferrin-induced signaling in the regulation of RAB8- and MICAL-L1-positive tubules. Addition of transferrin to the serum starved HeLa cells resulted in enhanced recruitment of RAB8 as well as MICAL-L1 to the tubules, which was mediated by TFRC. Dynasore, an inhibitor of dynamin and endocytosis, completely blocked transferrin-induced formation of RAB8/MICAL-L1-positive tubules. RAB8 showed strong colocalization with MICAL-L1 on the tubules. Blocking of SRC or AKT kinase activity by specific inhibitors abolished transferrin-induced recruitment of RAB8 and MICAL-L1 to the tubules. Recycling of transferrin receptor was inhibited by blocking of AKT activity. TBC1D17, a GTPase activating protein for RAB8, inhibited RAB8/MICAL-L1-positive tubule formation. A phospho-mimicking mutant S366D of TBC1D17 did not inhibit formation of RAB8-positive tubules. Overall, these results show that transferrin induces TFRC mediated signaling dependent on endocytosis that is essential for the formation of RAB8- and MICAL-L1-positive tubules involved in recycling of transferrin receptor. Our results also show that AKT regulates transferrin-induced formation of RAB8- and MICAL-L1-positive tubules, which might be mediated by phosphorylation of TBC1D17.

Optineurin/OPTN polymorphism, M98K is associated with normal tension glaucoma in certain populations, and genetic evidence shows its interaction with tumour necrosis factor–alpha (TNFα) polymorphism in causing glaucoma. Endoplasmic reticulum (ER) stress is also associated with glaucoma. We hypothesized that M98K‐OPTN may sensitize retinal ganglion cells to various types of stress. To test this hypothesis, stable clones of a retinal cell line, 661W, expressing either wild‐type (WT)‐OPTN or M98K‐OPTN were generated and examined for their survival under various stress conditions. Compared with WT‐OPTN expressing cells, M98K‐OPTN expressing cells showed significantly lower cell survival and higher activation of caspase‐3 and caspase‐8 upon treatment with tunicamycin (an inducer of ER stress) or TNFα. Levels of ER stress sensors IRE1α, PERK and ATF6 were significantly higher in M98K‐OPTN expressing cells. Tunicamycin treatment resulted in significantly higher induction of ER stress marker CHOP and several other ER stress response genes regulated by IRE1α‐XBP1, PERK‐ATF4 and ATF6 pathways, in M98K‐OPTN expressing cells. Splicing of XBP1 and ATF6 activation was higher in tunicamycin‐treated M98K‐OPTN expressing cells. Increased levels of PERK and IRE1α proteins in M98K‐OPTN expressing cells were dependent on autophagy. Overall, our results show that M98K‐OPTN sensitizes retinal cells to TNFα and ER stress‐induced cell death. We also show that M98K‐OPTN alters ER stress response signalling, which possibly enhances the sensitivity of retinal cells to ER stress. Our results provide support to the hypothesis that M98K‐OPTN may cooperate with other genetic or environmental factors to cause retinal ganglion cell death associated with glaucoma.

NLRP3 is a cytoplasmic receptor protein, which initiates caspase-1 mediated inflammatory immune response upon detection of invading pathogen or a wide array of internal distress signals. Several gain-of function mutations of NLRP3 cause hereditary disorder of cold-induced hyper-inflammation known as familial cold autoinflammatory syndrome-1 (FCAS1). Although, caspase-1 activation and downstream interleukin-1β / interleukin-18 maturation are common effectors in pathophysiology of this disorder, molecular mechanisms of how exposure to subnormal temperature triggers mutant NLRP3-inflammsome activity is not understood. Here, we show that endogenous NLRP3 is in complex with HSC70 (HSPA8), and this interaction is reduced upon exposure to cold. FCAS-causing NLRP3-L353P and NLRP3-R260W mutants show enhanced interaction with HSC70. Upon exposure to subnormal temperature, NLRP3-L353P and NLRP3-R260W show enhanced inflammasome formation, increased caspase-1 activation and reduced interaction with HSC70. Knockdown of HSC70 results in increased inflammasome formation by L353P and R260W mutants of NLRP3. Our results suggest that interaction with HSC70 suppresses inflammasome formation by FCAS-causing NLRP3 mutants at physiological temperature, and loss of this inhibitory association at subnormal temperature causes aggravated inflammasome formation and caspase-1 activation leading to interleukin-1β maturation. These results provide evidence for HSC70 being a cold-sensor and a temperature-dependent regulator of inflammatory signaling by FCAS-causing NLRP3 mutants.