Experimental Type 2 diabetes and lipotoxicity-associated neuroinflammation involve mitochondrial DNA-mediated cGAS/STING axis: implication of Type-1 interferon response in cognitive impairment.

Type-1 IFN (interferon)-associated innate immune response is increasingly getting attention in neurodegenerative and metabolic diseases like type 2 diabetes (T2DM). However, its significance in T2DM/lipotoxicity-induced neuroglia changes and cognitive impairment is missing. The present study aims to evaluate the involvement of cGAS (cyclic GMP-AMP synthase)-STING (stimulator of interferon gene), IRF3 (interferon regulatory factor-3), TBK (TANK binding kinase)-mediated Type-1 IFN response in the diabetic brain, and lipotoxicity (palmitate-bovine serum albumin conjugate/PA-BSA)-induced changes in cells (neuro2a and BV2). T2DM was induced in C57/BL6 mice by feeding on a high-fat diet (HFD, 60% Kcal) for 16 weeks and injecting a single dose of streptozotocin (100 mg/kg, i.p) in the 12th week. Plasma biochemical parameter analysis, neurobehavioral assessment, protein expression, and quantitative polymerase chain reaction study were carried out to decipher the hypothesis. T2DM-associated metabolic and lipotoxic stress led to mitochondrial impairment causing leakage of mtDNA to the cytoplasm further commencing cGAS activation and its downstream signaling. The diseased hippocampus and cortex showed decreased expression of synaptophysin (p < 0.01) and PSD-95 (p < 0.01, p < 0.05) with increased expression of cGAS (p < 0.001), p-STING (p < 0.001), p-STAT1 (signal transducer and activator of transcription) (p < 0.01), and IFN-ß (p < 0.001) compared to normal control. The IFN-ß/p-STAT1-mediated microglia activation was executed employing a conditioned media approach. C-176, a selective STING inhibitor, alleviated cGAS/p-STING/IFN-ß expression and proinflammatory microglia/M1-associated markers (CD16 expression, CXCL10, TNF-α, IL-1ß mRNA fold change) in the diabetic brain. The present study suggests Type-1IFN response may result in neuroglia dyshomeostasis affecting normal brain function. Alleviating STING signaling has the potential to protect T2DM-associated central ailment.

Combined Role of Interleukin-15 Stimulated Natural Killer Cell-Derived Extracellular Vesicles and Carboplatin in Osimertinib-Resistant H1975 Lung Cancer Cells with EGFR Mutations.

In this study, we evaluated IL-15 stimulated natural killer cell-derived EVs (NK-EVs) as therapeutic agents in vitro and in vivo in Osimertinib-resistant lung cancer (H1975R) with EGFR mutations (L858R) in combination with carboplatin (CBP). NK-EVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis, and atomic force microscopy imaging revealed vesicles with a spherical form and sizes meeting the criteria of exosomal EVs. Further, Western blot studies demonstrated the presence of regular EV markers along with specific NK markers (perforin and granzyme). EVs were also characterized by proteomic analysis, which demonstrated that EVs had proteins for natural killer cell-mediated cytotoxicity (Granzyme B) and T cell activation (perforin and plastin-2). Gene oncology analysis showed that these differentially expressed proteins are involved in programmed cell death and positive regulation of cell death. Further, isolated NK-EVs were cytotoxic to H1975R cells in vitro in 2D and 3D cell cultures. CBP's IC50 was reduced by approximately in 2D and 3D cell cultures when combined with NK-EVs. The EVs were then combined with CBP and administered by i.p. route to H1975R tumor xenografts, and a significant reduction in tumor volume in vivo was observed. Our findings show for the first time that NK-EVs target the PD-L1/PD-1 immunological checkpoint to induce apoptosis and anti-inflammatory response by downregulation of SOD2, PARP, BCL2, SET, NF-κB, and TGF-ß. The ability to isolate functional NK-EVs on a large scale and use them with platinum-based drugs may lead to new clinical applications. The results of the present study suggest the possibility of the combination of NK-cell-derived EVs and CBP as a viable immunochemotherapeutic strategy for resistant cancers.

Necrostatin-1S mitigates type-2 diabetes-associated cognitive decrement and lipotoxicity-induced neuro-microglia changes through p-RIPK-RIPK3-p-MLKL axis.

Type-2 diabetes mellitus (T2DM) is associated with neuroinflammation and cognitive decrement. Necroptosis programmed necrosis is emerging as the major contributing factor to central changes. It is best characterized by the upregulation of p-RIPK(Receptor Interacting Kinase), p-RIPK3, and the phosphorylated-MLKL (mixed-lineage kinase domain-like protein). The present study aims to evaluate the neuroprotective effect of Necrostatin (Nec-1S), a p-RIPK inhibitor, on cognitive changes in the experimental T2DM model in C57BL/6 mice and lipotoxicity-induced neuro-microglia changes in neuro2A and BV2 cells. Further, the study also explores whether Nec-1S would restore mitochondrial and autophago-lysosomal function.T2DM was developed in mice by feeding them a high-fat diet (HFD) for 16 weeks and injecting a single dose of streptozotocin (100 mg/kg, i.p) on the 12th week. Nec-1S was administered for 3 weeks at (10 mg/kg, i.p) once every 3 days. Lipotoxicity was induced in neuro2A, and BV2 cells using 200 µM palmitate/bovine serum albumin conjugate. Nec-1S (50 µM), and GSK-872(10 µM) were further used to explore their relative effect. The neurobehavioral performance was assessed using mazes and task-assisted performance tests. To decipher the hypothesis plasma parameters, western blot, immunofluorescence, microscopy, and quantitative reverse transcription-PCR studies were carried out. The Nec-1S treatment restored cognitive performance and reduced the p-RIPK-p-RIPK3-p-MLKL mediated neuro-microglia changes in the brain and in cells as well, under lipotoxic stress. Nec-1S reduced tau, and amyloid oligomer load. Moreover, Nec-1S restored mitochondrial function and autophago-lysosome clearance. The findings highlight the central impact of metabolic syndrome and how Nes-1S, by acting as a multifaceted agent, improved central functioning.

Piceatannol promotes neuroprotection by inducing mitophagy and mitobiogenesis in the experimental diabetic peripheral neuropathy and hyperglycemia-induced neurotoxicity.

Piceatannol (PCN), a SIRT1 activator, regulates multiple oxidative stress mechanism and has anti-inflammatory potential in various inflammatory conditions. However, its role in Diabetic insulted peripheral neuropathy (DN) remains unknown. Oxidative stress and mitochondrial dysfunction are major contributing factors to DN. Myriad studies have proven that sirtuin1 (SIRT1) stimulation convalesce nerve functions by activating mitochondrial functions like mitochondrial biogenesis and mitophagy. Diabetic neuropathy (DN) was provoked by injecting streptozotocin (STZ) at a dose of 55 mg/kg, i.p to male Sprague Dawley (SD) rats. Mechanical, thermal hyperalgesia was evaluated by using water immersion, Vonfrey Aesthesiometer, and Randall Sellito Calipers. Motor, sensory nerve conduction velocity was measured using Power Lab 4sp system whereas The Laser Doppler system was used to evaluate nerve blood flow. To induce hyperglycemia for the in vitro investigations, high glucose (HG) (30 mM) conditions were applied to Neuro2a cells. At doses of 5 and 10 µM, PCN was examined for its role in SIRT1 and Nrf2 activation. HG-induced N2A cells, reactive oxygen exposure, mitochondrial superoxides and mitochondrial membrane potentials were restored by PCN exposure, and their neurite outgrowth was enhanced. Peroxisome proliferator activated receptor-gamma coactivator-1α (PGC-1α) directed mitochondrial biogenesis was induced by increased SIRT1 activation by piceatannol. SIRT1 activation also enhanced Nrf2-mediated antioxidant signalling. Our study results inferred that PCN administration can counteract the decline in mitochondrial function and antioxidant activity in diabetic rats and HG-exposed N2A cells by increasing the SIRT1 and Nrf2 activities.

Activation of SIRT1 by silibinin improved mitochondrial health and alleviated the oxidative damage in experimental diabetic neuropathy and high glucose-mediated neurotoxicity.

BACKGROUND: Silibinin (SBN), a sirtuin 1 (SIRT1) activator, has been evaluated for its anti-inflammatory activity in many inflammatory diseases. However, its role in diabetes-induced peripheral neuropathy (DPN) remains unknown. The SIRT1 activation convalesces nerve functions by improving mitochondrial biogenesis and mitophagy. METHODS: DPN was induced by streptozotocin (STZ) at a dose of 55 mg/kg, i.p. in the male SD rats whereas neurotoxicity was induced in Neuro2A cells by 30 mM (high glucose) glucose. Neurobehavioural (nerve conduction velocity and nerve blood flow) western blot, immunohistochemistry, and immunocytochemistry were performed to evaluate the protein expression and their cellular localisation. RESULTS: Two-week SBN treatment improved neurobehavioural symptoms, SIRT1, PGC-1α, and TFAM expression in the sciatic nerve and HG insulted N2A cells. It has also maintained the mitophagy by up-regulating PARL, PINK1, PGAM5, LC3 level and provided antioxidant defence by upregulating Nrf2. CONCLUSION: SBN has shown neuroprotective potential in DPN through SIRT1 activation and antioxidant mechanism.

Role of MicroRNAs, Aptamers in Neuroinflammation and Neurodegenerative Disorders.

Exploring the microRNAs and aptamers for their therapeutic role as biological drugs has expanded the horizon of its applicability against various human diseases, explicitly targeting the genetic materials. RNA-based therapeutics are widely being explored for the treatment and diagnosis of multiple diseases, including neurodegenerative disorders (NDD). Latter includes microRNA, aptamers, ribozymes, and small interfering RNAs (siRNAs), which control the gene expression mainly at the transcriptional strata. One RNA transcript translates into different protein types; hence, therapies targeted at the transcriptional sphere may have prominent and more extensive effects than alternative therapeutics. Unlike conventional gene therapy, RNAs, upon delivery, can either altogether abolish or alter the synthesis of the protein of interest, therefore, regulating their activities in a controlled and diverse manner. NDDs like Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, Prion disease, and others are characterized by deposition of misfolded protein such as amyloid-ß, tau, α-synuclein, huntingtin and prion proteins. Neuroinflammation, one of the perquisites for neurodegeneration, is induced during neurodegenerative pathogenesis. In this review, we discuss microRNAs and aptamers' role as two different RNA-based approaches for their unique ability to regulate protein production at the transcription level, hence offering many advantages over other biologicals. The microRNA acts either by alleviating the malfunctioning RNA expression or by working as a replacement to lost microRNA. On the contrary, aptamer act as a chemical antibody and forms an aptamer-target complex.

PARP overactivation in neurological disorders.

Poly (ADP-ribose) polymerases (PARPs) constitute a family of enzymes associated with divergent cellular processes that are not limited to DNA repair, chromatin organization, genome integrity, and apoptosis but also found to play a crucial role in inflammation. PARPs mediate poly (ADP-ribosylation) of DNA binding proteins that is often responsible for chromatin remodeling thereby ensure effective repairing of DNA stand breaks although during the conditions of severe genotoxic stress PARPs direct the cell fate towards apoptotic events. Recent discoveries have pushed PARPs into the spotlight as targets for treating cancer, metabolic, inflammatory and neurological disorders. Of note, PARP-1 is the most abundant isoform of PARPs (18 member super family) which executes more than 90% of PARPs functions. Since oxidative/nitrosative stress actuated PARP-1 is linked to vigorous DNA damage and wide spread provocative inflammatory response that underlie the aetiopathogenesis of different neurological disorders, possibility of developing PARP-1 inhibitors as plausible neurotherapeutic agents attracts considerable research interest. This review outlines the recent advances in PARP-1 biology and examines the capability of PARP-1 inhibitors as treatment modalities in intense and interminable diseases of neuronal origin.

An Overview on ATP Dependent and Independent Proteases Including an Anterograde to Retrograde Control on Mitochondrial Function; Focus on Diabetes and Diabetic Complications.

Mitochondria are the central power stations of the cell involved with a myriad of cell signalling pathways that contribute for whole health status of the cell. It is a well known fact that not only mitochondrial genome encodes for mitochondrial proteins but there are several other mitochondrial specific proteins encoded by nuclear genome which regulate plethora of cell catabolic and anabolic process. Anterograde pathways include nuclear gene encoded proteins and their specific transport into the mitochondria and regulation of mitochondrial homeostasis. The retrograde pathways include crosstalk between the mitochondria and cytoplasmic proteins. Indeed, ATP dependent and independent proteases are identified to be very critical in balancing anterograde to retrograde signalling and vice versa to maintain the cell viability or cell death. Different experimental studies conducted on silencing the genes of these proteases have shown embryonic lethality, cancer cells death, increased hepatic glucose output, insulin tolerance, increased protein exclusion bodies, mitochondrial dysfunction, and defect in mitochondrial biogenesis, increased inflammation, Apoptosis etc. These experimental studies included from eubacteria to eukaryotes. Hence, many lines of theories proposed these proteases are conservative from eubacteria to eukaryotes. However, the regulation of these proteases at gene level is not clearly understood and still research is warranted. In this review, we articulated the origin and regulation of these proteases and the cross talk between the nucleus and mitochondria vice versa, and highlighted the role of these proteases in diabetes and diabetic complications in human diseases.