Impact of chronic hyperglycemia on Small Heat Shock Proteins in diabetic rat brain.

Small heat shock proteins (sHsps) are a family of proteins. Some are induced in response to multiple stimuli and others are constitutively expressed. They are involved in fundamental cellular processes, including protein folding, apoptosis, and maintenance of cytoskeletal integrity. Hyperglycemia created during diabetes leads to neuronal derangements in the brain. In this study, we investigated the impact of chronic hyperglycemia on the expression of sHsps and heat shock transcription factors (HSFs), solubility and aggregation of sHsps and amyloidogenic proteins, and their role in neuronal apoptosis in a diabetic rat model. Diabetes was induced in Sprague-Dawley rats with streptozotocin and hyperglycemia was maintained for 16 weeks. Expressions of sHsps and HSFs were analyzed by qRT-PCR and immunoblotting in the cerebral cortex. Solubility of sHsps and amyloidogenic proteins, including α-synuclein and Tau, was analyzed by the detergent soluble assay. Neuronal cell death was analyzed by TUNEL staining and apoptotic markers. The interaction of sHsps with amyloidogenic proteins and Bax was assessed using co-immunoprecipitation. Hyperglycemia decreased Hsp27 and HSF1, and increased αBC, Hsp22, and HSF4 levels at transcript and protein levels. Diabetes induced the aggregation of αBC, Hsp22, α-synuclein, and pTau, as their levels were higher in the insoluble fraction. Additionally, diabetes impaired the interaction of αBC with α-synuclein and pTau. Furthermore, diabetes reduced the interaction of αBC with Bax, which may possibly contribute to neuronal apoptosis. Together, these results indicate that chronic hyperglycemia induces differential responses of sHsps by altering their expression, solubility, interaction, and roles in apoptosis.

Role of sorbitol-mediated cellular stress response in obesity-associated retinal degeneration.

PURPOSE: Obesity is a global health problem associated with several diseases including ocular complications. Earlier we reported progressive retinal degeneration because of obesity in a spontaneous obese rat (WNIN/Ob) model. In the current study, we examined the molecular mechanisms leading to retinal degeneration in WNIN/Ob rat. METHODS: Sorbitol was estimated by the fluorometric method in the retina of WNIN/Ob rats at different age (3-, 6- and 12- months), along with their respective lean rats. Immunoblotting was performed in the retina to assess the status of the insulin signaling pathway, ER stress and cellular stress (p38MAPK and ERK1/2). Human SK-N-SH cells were treated with 0.5 and 1.0 M sorbitol for 30 min to study insulin signaling, ER stress, and cellular stress. TUNEL assay was done to measure apoptosis. The retinal function in the rats was determined by electroretinogram. RESULTS: A gradual but significantly higher intracellular sorbitol accumulation was observed in the retina of obese rats from 3- to 12-months. The cellular osmotic stress has activated the insulin signaling mechanism without activating AKT and also triggered ER stress. Both the stresses activated the ERK and p38MAPK signaling causing apoptosis in the retina leading to retinal degeneration. Retinal dysfunction was confirmed by altered scotopic and photopic electroretinogram responses. These in vivo results were mimicked in SK-N-SH cells when exposed to sorbitol in vitro. CONCLUSIONS: These results suggest cellular stress due to sorbitol accumulation impairing the ER function, thereby leading to progressive retinal degeneration under obese conditions.

Nutrigenomics: Opportunities & challenges for public health nutrition.

The hierarchical information flow through DNA-RNA-protein-metabolite collectively referred to as 'molecular fingerprint' defines both health and disease. Environment and food (quality and quantity) are the key factors known to affect the health of an individual. The fundamental concepts are that the transition from a healthy condition to a disease phenotype must occur by concurrent alterations in the genome expression or by differences in protein synthesis, function and metabolites. In other words, the dietary components directly or indirectly modulate the molecular fingerprint and understanding of which is dealt with nutrigenomics. Although the fundamental principles of nutrigenomics remain similar to that of traditional research, a collection of comprehensive targeted/untargeted data sets in the context of nutrition offers the unique advantage of understanding complex metabolic networks to provide a mechanistic understanding of data from epidemiological and intervention studies. In this review the challenges and opportunities of nutrigenomic tools in addressing the nutritional problems of public health importance are discussed. The application of nutrigenomic tools provided numerous leads on biomarkers of nutrient intake, undernutrition, metabolic syndrome and its complications. Importantly, nutrigenomic studies also led to the discovery of the association of multiple genetic polymorphisms in relation to the variability of micronutrient absorption and metabolism, providing a potential opportunity for further research toward setting personalized dietary recommendations for individuals and population subgroups.

Attenuation of diabetic retinopathy in rats by ellagic acid through inhibition of AGE formation.

Diabetic retinopathy (DR) is a major concern for blindness all over the world. Diabetic retinopathy is associated with thickening of basement membrane, retinal thinning, retinal detachment, and pericyte death. Advanced glycation end products (AGEs) mediate the progression of DR by stimulating the expression of RAGE and VEGF which subsequently damages the blood-retinal barrier. Employing a set of in vitro protein glycation systems, earlier we demonstrated antiglycating potential of ellagic acid (EA). In this study, we evaluated the efficacy of EA to prevent in vivo accumulation of AGE and to ameliorate retinal changes in diabetic rats. Streptozotocin-induced diabetic rats were fed either with 0.2 or 2% EA in the diet for 12 weeks. Effect of EA on retinal function was assessed with electroretinogram (ERG). At the end of the experiment, rats were scarified and retina was collected. Histology was carried out with H&E staining and immunohistochemistry. Formation of AGE product (CML) and activation of RAGE was analyzed by immunoblotting and immunohistochemistry. Expression of GFAP, VEGF, Bax and HIF-1α was assessed by qRT-PCR and immunoblotting. Dietary supplementation of EA to diabetic rats resulted in: (1) inhibition of accumulation of CML and activation of RAGE in retina, (2) attenuation of expression of GFAP, VEGF, and HIF-1α in retina, (3) attenuation of cell death by reducing proapoptic mediator Bax and (4) amelioration of retinal thickness and function. In conclusion, EA attenuated the retinal abnormalities including angiogenesis, hypoxia and cell death by inhibiting AGE-RAGE mediated cellular events.

Altered ubiquitin-proteasome system leads to neuronal cell death in a spontaneous obese rat model.

BACKGROUND: Obesity is associated with various progressive age-related diseases, including neurological disorders. However, underlying molecular basis for increased risk of neurodegeneration in obesity is unknown. A suitable animal model would immensely help in understanding the obesity-linked neurological problems. METHODS: A spontaneously developed obese rat (WNIN/Ob) which is highly vulnerable for a variety of degenerative diseases was isolated from the existing WNIN stock rats. Ultrastructure of neurons in the cerebral cortex of 12-month old obese rats was evaluated by transmission electron microscopy. qRT-PCR and immunoblotting of ubiquitin C-terminal hydrolases (UCHs), ubiquitin, proteasomal sub-units, markers of ER stress and apoptosis were performed in the cerebral cortex. Proteasome activity was assayed by fluorometric method. Immunohistochemistry was performed for mediators of apoptosis, which was further confirmed by TUNEL assay. These investigations were also carried in high-fat diet-induced obese rat model. RESULTS: Neurons in the cerebral cortex of 12-month obese rats showed swollen mitochondria, disrupted ER and degenerating axons, nucleus and finally neurons. Results showed altered UPS, existence of ER stress, up-regulation of apoptotic markers and apoptosis in the cerebral cortex of obese rats. It appears that UCHL-1 mediated apoptosis through stabilizing p53 might play a role in neuronal cell death in obese rat. Similar changes were observed in the brain of diet-induced obese WNIN rats. CONCLUSION: Altered UPS could be one of the underlying mechanisms for the neuronal cell death in obese conditions. GENERAL SIGNIFICANCE: This is the first report to highlight the role of altered UPS in neurodegeneration due to obesity.

Anticancer effect of celastrol on human triple negative breast cancer: possible involvement of oxidative stress, mitochondrial dysfunction, apoptosis and PI3K/Akt pathways.

Signaling via the phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) is crucial for divergent physiological processes including transcription, translation, cell-cycle progression and apoptosis. The aim of work was to elucidate the anti-cancer effect of celastrol and the signal transduction pathways involved. Cytotoxic effect of celastrol was assessed by MTT assay on human triple negative breast cancer cells (TNBCs) and compared with that of MCF-7. Apoptosis induction was determined by AO/EtBr staining, mitochondrial membrane potential by JC-1, Annexin binding assays and modulation of apoptotic proteins and its effect on PI3K/Akt/mTOR pathway by western blotting. Celastrol induced apoptosis in TNBC cells, were supported by DNA fragmentation, caspase-3 activation and PARP cleavage. Meanwhile, celastrol triggered reactive oxygen species production with collapse of mitochondrial membrane potential, down-regulation of Bcl-2 and up-regulation of Bax expression. Celastrol effectively decreased PI3K 110α/85α enzyme activity, phosphorylation of Akt(ser473) and p70S6K1 and 4E-BP1. Although insulin treatment increased the phosphorylation of Akt(ser473), p70S6K1, 4E-BP1, celastrol abolished the insulin mediated phosphorylation. It clearly indicates that celastrol acts through PI3k/Akt/mTOR axis. We also found that celastrol inhibited the Akt/GSK3ß and Akt/NFkB survival pathway. PI3K/Akt/mTOR inhibitor, PF-04691502 and mTOR inhibitor rapamycin enhanced the apoptosis-inducing effect of celastrol. These data demonstrated that celastrol induces apoptosis in TNBC cells and indicated that apoptosis might be mediated through mitochondrial dysfunction and PI3K/Akt signaling pathway.

Expression and induction of small heat shock proteins in rat heart under chronic hyperglycemic conditions.

The induction of small heat shock proteins (sHsp) is observed under various stress conditions to protect the cells and organisms from adverse events including diabetes. Diabetic cardiomyopathy is a common complication of diabetes. Therefore, in this study, we investigated the expression of sHsp under chronic hyperglycemic conditions in rat heart. Hyperglycemia was induced in WNIN rats by intraperitoneal injection of streptozotocin and maintained for a period of 12weeks. Expression of sHsp, phosphorylation and translocation of phosphoforms of Hsp27 and αB-crystallin (αBC) from cytosolic fraction to cytoskeletal fraction was analyzed. While the expression of MKBP, HspB3, αBC was found to be increased in diabetic heart, expression of Hsp20 was decreased. Chronic hyperglycemia further induced phosphorylation of αBC at S59, S45, Hsp27 at S82, p38MAPK and p44/42MAPK. However, pS59-αBC and pS82-Hsp27 were translocated from detergent-soluble to detergent-insoluble fraction under hyperglycemic conditions. Furthermore, the interaction of pS82-Hsp27 and pS59-αBC with desmin was increased under hyperglycemia. However, the interaction of αBC and pS59-αBC with Bax was impaired by chronic hyperglycemia. These results suggest up regulation of sHsp (MKBP, HspB3 and αBC), phosphorylation and translocation of Hsp27 and αBC to striated sarcomeres and impaired interaction of αBC and pS59-αBC with Bax under chronic hyperglycemia.

Neuroprotective role of vitamin B12 in streptozotocin-induced type 1 diabetic rats.

Chronic hyperglycemia-induced neuropathological changes include neuronal apoptosis, astrogliosis, decrease in neurotrophic support, impaired synaptic plasticity, and impaired protein quality control (PQC) system. Vitamin B12 is indispensable for neuronal development and brain function. Several studies reported the neuroprotective effect of B12 supplementation in diabetic patients. However, the underlying molecular basis for the neuroprotective effect of B12 supplementation in diabetes needs to be thoroughly investigated. Two-month-old Sprague-Dawley rats were randomly assigned into three groups: Control (CN), diabetes (D; induced with streptozotocin; STZ), and diabetic rats supplemented with vitamin B12 (DBS; vitamin B12; 50 µg/kg) for four months. At the end of 4 months of experimentation, the brain was dissected to collect the cerebral cortex (CC). The morphology of CC was investigated with H&E and Nissl body staining. Neuronal apoptosis was determined with TUNEL assay. The components of neurotrophic support, astrogliosis, synaptic plasticity, and PQC processes were investigated by immunoblotting and immunostaining methods. H& E, Nissl body, and TUNEL staining revealed that diabetes-induced neuronal apoptosis and degeneration. However, B12 supplementation ameliorated the diabetes-induced neuronal apoptosis. Further, B12 supplementation restored the markers of neurotrophic support (BDNF, NGF, and GDNF), and synaptic plasticity (SYP, and PSD-95) in diabetic rats. Interestingly, B12 supplementation also attenuated astrogliosis, ER stress, and ameliorated autophagy-related proteins in diabetic rats. Overall, these findings suggest that B12 acts as a neuroprotective agent by inhibiting the neuropathological changes in STZ-induced type 1 diabetes. Thus, B12 supplementation could produce beneficial outcomes including neuroprotective effects in diabetic patients.

Emerging therapeutic roles of small heat shock protein-derived mini-chaperones and their delivery strategies.

The small heat shock protein (sHsp) family is a group of proteins in which some are induced in response to external stimuli, such as environmental and pathological stresses, while others are constitutively expressed. They show chaperone-like activity, protect cells from apoptosis, and maintain cytoskeletal architecture. Short sequences or fragments ranging from approximately 19-20 residues in sHsps were shown to display chaperone activity in vitro. These sequences are termed sHsp-derived mini-peptides/mini-chaperones. These peptides offer an advantage in providing protective and therapeutic effects over full-length proteins owing to their small molecular weight and easy uptake into the cells. Research on sHsp mini-chaperone therapy has recently received attention and advanced tremendously. sHsp mini-chaperones have shown a wide range of therapeutic effects, such as anti-aggregation of proteins, anti-apoptotic, anti-inflammatory, anti-oxidant, senolytic, and anti-platelet activity. The administration of mini-chaperones into the several disease animal models, including experimental autoimmune encephalomyelitis, cataract, age-related macular degeneration, glaucoma, and thrombosis through various routes reduced symptoms or prevented the progression of the disease. However, it was found that the therapeutic potential of sHsp mini-chaperones is limited by their short turnover and enzymatic degradation in circulation. Nonetheless, carrier molecules approach such as nanoparticles, cell penetration peptides, and extracellular vesicles increased their efficacy by enhancing the uptake, retention time, protection from enzymatic degradation, and site-specific delivery without altering their biological activity. In this context, this review highlights the recent advances in the therapeutic potential of sHsp-derived mini-chaperones, their effect in experimental animal models, and approaches for increasing their efficacy.

Implication of homocysteine in protein quality control processes.

Homocysteine (Hcy) is a key metabolite generated during methionine metabolism. The elevated levels of Hcy in the blood are reffered to as hyperhomocystenimeia (HHcy). The HHcy is caused by impaired metabolism/deficiency of either folate or B12 or defects in Hcy metabolism. Accumulating evidence suggests that HHcy is associated with cardiovascular and brain diseases including atherosclerosis, endothelial injury, and stroke etc. Vitamin B12 (cobalamin; B12) is a water-soluble vitamin essential for two metabolic reactions. It acts as a co-factor for methionine synthase and L-methylmalonyl-CoA mutase. Besides, it is also vital for DNA synthesis and maturation of RBC. Deficiency of B12 is associated with haematological and neurological disorders. Hyperhomocysteinemia (HHcy)-induced toxicity is thought to be mediated by the accumulation of Hcy and its metabolites, homocysteinylated proteins. Cellular protein quality control (PQC) is essential for the maintenance of proteome integrity, and cell viability and its failure contributes to the development of multiple diseases. Chaperones, unfolded protein response (UPR), ubiquitin-proteasome system (UPS), and autophagy are analogous strategies of PQC that maintain cellular proteome integrity. Recently, multiple studies reported that HHcy responsible for perturbation of PQC by reducing chaperone levels, activating UPR, and impairing autophagy. Besides, HHcy also induce cytotoxicity, inflammation, protein aggregation and apoptosis. It has been shown that some of the factors including altered SIRT1-HSF1 axis and irreversible homocysteinylation of proteins are responsible for folate and/or B12 deficiency or HHcy-induced impairment of PQC. Therefore, this review highlights the current understanding of HHcy in the context of cellular PQC and their pathophysiological and clinical consequences, epigenomic changes, therapeutic implications of B12, and chemical chaperones based on cell culture and experimental animal models.

Extracellular small heat shock proteins: exosomal biogenesis and function.

Small heat shock proteins (sHsps) belong to the family of heat shock proteins (Hsps): some are induced in response to multiple stressful events to protect the cells while others are constitutively expressed. Until now, it was believed that Hsps, including sHsps, are present inside the cells and perform intracellular functions. Interestingly, several groups recently reported the extracellular presence of Hsps, and sHsps have also been detected in sera/cerebrospinal fluids in various pathological conditions. Secretion into the extracellular milieu during many pathological conditions suggests additional or novel functions of sHsps in addition to their intracellular properties. Extracellular sHsps are implicated in cell-cell communication, activation of immune cells, and promoting anti-inflammatory and anti-platelet responses. Interestingly, exogenous administration of sHsps showed therapeutic effects in multiple disease models implying that extracellular sHsps are beneficial in pathological conditions. sHsps do not possess signal sequence and, hence, are not exported through the classical Endoplasmic reticulum-Golgi complex (ER-Golgi) secretory pathway. Further, export of sHsps is not inhibited by ER-Golgi secretory pathway inhibitors implying the involvement of a nonclassical secretory pathway in sHsp export. In lieu, lysoendosomal and exosomal pathways have been proposed for the export of sHsps. Heat shock protein 27 (Hsp27), αB-crystallin (αBC), and Hsp20 are shown to be exported by exosomes. Exosomes packaged with sHsps have beneficial effects in in vivo disease models. However, secretion mechanisms and therapeutic use of sHsps have not been elucidated in detail. Therefore, this review aimed at highlighting the current understanding of sHsps (Hsp27, αBC, and Hsp20) in the extracellular medium.

In Vivo Inhibition of Proteasome Activity and Tumour Growth by Murraya koenigii Leaf Extract in Breast Cancer Xenografts and by Its Active Flavonoids in Breast Cancer Cells.

Inhibition of the 26S proteasome is an attractive approach for anticancer therapy. Proteasome inhibitors are known to selectively target cancer cells and make them more sensitive to chemotherapeutic agents. Murraya koenigii is a medicinally important herb of Asian origin and a rich source of bioactive compounds such as flavonoids and alkaloids. In the present study, we investigated the proteasome inhibitory and apoptotic effect of M. koenigii leaf extract in vivo in a xenograft tumor mouse model, and also assessed the toxicity if any in normal mice. M. koenigii extract did not lead to any toxicity in mice. Analysis of extract revealed the presence of flavonoid compounds which act as proteasome inhibitors. Quercetin treatment led to the decrease in the cell viability and arrest of cells in G2/M phase. Quercetin, Apigenin, Kaempferol and Rutin; flavonoids present in the leaf extract, dose-dependently inhibited the endogenous 26S proteasome activity in MDA-MB-231 cells. Reduction in tumor growth was associated with a decrease in proteasomal enzyme activities in the treated groups. Increased caspase-3 activity and TUNEL-positive cells indicated enhanced apoptosis with Murraya leaf extract treatment. Decreased expression of angiogenic and anti-apoptotic gene markers is indicative of inhibition of angiogenesis and promotion of apoptosis in the leaf extract treated tumors.

Temperature-dependent structural and functional properties of a mutant (F71L) αA-crystallin: molecular basis for early onset of age-related cataract.

Previously we identified a novel mutation (F71L) in the αA-crystallin gene associated with early onset of age-related cataract. However, it is not known how the missense substitution translates into reduced chaperone-like activity (CLA), and how the structural and functional changes lead to early onset of the disease. Herein, we show that under native conditions the F71L-mutant is not significantly different from wild-type with regard to secondary and tertiary structural organization, hydrophobicity and the apparent molecular mass of oligomer but has substantial differences in structural and functional properties following a heat treatment. Wild-type αA-crystallin demonstrated increased CLA, whereas the F71L-mutant substantially lost its CLA upon heat treatment. Further, unlike the wild-type αA-subunit, F71L-subunit did not protect the αB-subunit in hetero-oligomeric complex from heat-induced aggregation. Moreover, hetero-oligomer containing F71L and αB in 3:1 ratio had significantly lower CLA upon thermal treatment compared to its unheated control. These results indicate that α-crystallin complexes containing F71L-αA subunits are less stable and have reduced CLA. Therefore, F71L may lead to earlier onset of cataract due to interaction with several environmental factors (e.g., temperature in this case) along with the aging process.