Amelioration of neuronal cell death in a spontaneous obese rat model by dietary restriction through modulation of ubiquitin proteasome system

Dietary restriction (DR) has been shown to increase longevity, delay onset of aging, reduce DNA damage and oxidative stress and prevent age-related decline of neuronal activity. We previously reported the role of altered ubiquitin proteasome system (UPS) in the neuronal cell death in a spontaneous obese rat model (WNIN/Ob rat). In this study, we investigated the effect of DR on obesity-induced neuronal cell death in a rat model. Two groups of 40-day-old WNIN/Ob rats were either fed ad libitum (Ob) or pair-fed with lean. The lean phenotype of WNIN/Ob rats served as ad libitum control. These animals were maintained for 6.5 months on their respective diet regime. At the end of the study, cerebral cortex was collected and markers of UPS, endoplasmic reticulum (ER) stress and autophagy were analyzed by quantitative real-time polymerase chain reaction, immunoblotting and immunohistochemistry. Chymotrypsin-like activity of proteasome was assayed by the fluorimetric method. Apoptotic cells were analyzed by TUNEL assay. DR improved metabolic abnormalities in obese rats. Alterations in UPS (up-regulation of UCHL1, down-regulation of UCHL5, declined proteasomal activity), increased ER stress, declined autophagy and increased expression of α-synuclein, p53 and BAX were observed in obese rats and DR alleviated these changes in obese rats. Further, DR decreased TUNEL-positive cells. In conclusion, DR in obese rats could not only restore the metabolic abnormalities but also preserved neuronal health in the cerebral cortex by preventing alterations in the UPS.     

Chlorella modulates insulin signaling pathway and prevents high-fat diet-induced insulin resistance in mice

Aims

The search for natural agents that minimize obesity-associated disorders is receiving special attention. In this regard, the present study aimed to evaluate the prophylactic effect of Chlorella vulgaris (CV) on body weight, lipid profile, blood glucose and insulin signaling in liver, skeletal muscle and adipose tissue of diet-induced obese mice.

Main methods

Balb/C mice were fed either with standard rodent chow diet or high-fat diet (HFD) and received concomitant treatment with CV for 12 consecutive weeks. Triglyceride, free fatty acid, total cholesterol and fractions of cholesterol were measured using commercial assay. Insulin and leptin levels were determined by enzyme-linked immunosorbent assay (ELISA). Insulin and glucose tolerance tests were performed. The expression and phosphorylation of IRβ, IRS-1 and Akt were determined by Western blot analyses.

Key findings

Herein we demonstrate for the first time in the literature that prevention by CV of high-fat diet-induced insulin resistance in obese mice, as shown by increased glucose and insulin tolerance, is in part due to the improvement in the insulin signaling pathway at its main target tissues, by increasing the phosphorylation levels of proteins such as IR, IRS-1 and Akt. In parallel, the lower phosphorylation levels of IRS-1^ser307 were observed in obese mice. We also found that CV administration prevents high-fat diet-induced dyslipidemia by reducing triglyceride, cholesterol and free fatty acid levels.

Significance

We propose that the modulatory effect of CV treatment preventing the deleterious effects induced by high-fat diet is a good indicator for its use as a prophylactic–therapeutic agent against obesity-related complications.     

Ubiquitin-proteasome system and ER stress in the brain of diabetic rats

The ubiquitin-proteasome system (UPS) has been implicated in the pathogenesis of many neurodegenerative diseases. Endoplasmic reticulum (ER) stress is shown to play a pathological role in the development of diabetes and its complications. Hence, the current study is aimed to investigate the role of UPS and ER stress in the cerebral cortex of diabetic rats and examine the therapeutic effect of 4-phenylbutyric acid (4-PBA), an ER stress inhibitor. Male Sprague-Dawley rats were divided into three groups: control, diabetes, and diabetes plus 4-PBA treatment group. Diabetes was induced by single intraperitoneal streptozotocin injection (37 mg/kg body weight [bw]), and 4-PBA was administered (40 mg/kg bw/d, intraperitoneal) for 2 months, starting from 2 months of diabetes induction. At the end of 4 months, cerebral cortex was collected for analysis. Declined proteasome activity and ubiquitin C-terminal hydrolase (UCH)-L1 expression, increased ubiquitinated proteins, and apoptosis were observed in the diabetic rats. The expression of the ubiquitin-activating enzyme E1, UCHL5, and ER stress markers (ATF6, pPERK, and CHOP) was markedly elevated, whereas the expression of ER-associated protein degradation (ERAD) components was downregulated in the diabetic rats. 4-PBA intervention attenuated ER stress, alterations in UPS, and ERAD components in diabetic rats. Importantly, neuronal apoptosis was lowered in 4-PBA-treated diabetic rats. Our observations demonstrate that altered UPS could be one of the underlying mechanisms of neuronal apoptosis in diabetes and chemical chaperones such as 4-PBA could be potential candidates for preventing these alterations under hyperglycemic conditions.     

Comparison between the effects of diallyl tetrasulfide on human retina pigment epithelial cells (ARPE-19) and HCT116 cells

Background

Diallyl mono- and polysulfanes from garlic are known to induce an adaptive cell response and the formation of antioxidants in cancer cells. In the case of a severe ER stress and a failure in the response, cancer cells eventually go into apoptosis. Only little is known about the response of normal cells upon treatment.

Methods

Normal ARPE-19 cells were treated with diallyl tetrasulfide to study their cellular response and the results were compared with those of HCT116 cancer cells. Cell viability was checked by an MTT assay and cytofluorimetry. The formation of superoxide radicals, H₂O₂ and thiols were determined and proteins involved in the ER stress response were also detected by Western blot analysis.

Results

We found that diallyl tetrasulfide induced reactive oxygen species (ROS) in normal cells similar to cancer cells in a time (0 to 60 min) and dose dependent manner (0 to 50 μM). The level of heme oxigenase-1 (HO-1) was up-regulated in both cell types. Initially, we found a decrease in the total thiol level in both cell types but in contrast to cancer cells, normal cells recovered from the decrease in the total thiol concentration within 60 min of treatment.

Conclusions

The recovery of the thiol concentration in normal cells treated with diallyl tetrasulfide seems to be responsible for the failure to induce the ER stress signalling pathway and finally apoptosis in normal cells.

General Significance

The difference in the recovery of the thiol status might be an explanation for the anti-carcinogenic effects of garlic compounds.     

Methyl pyruvate rescues mitochondrial damage caused by SIGMAR1 mutation related to amyotrophic lateral sclerosis

Background

Amyotrophic lateral sclerosis (ALS) is a disease caused by motor neuron degeneration. Recently, a novel SIGMAR1 gene variant (p.E102Q) was discovered in some familial ALS patients.

Methods

We address mechanisms underlying neurodegeneration caused by the mutation using Neuro2A cells overexpressing σ₁R^E102Q, a protein of a SIGMAR1 gene variant (p.E102Q) and evaluate potential amelioration by ATP production via methyl pyruvate (MP) treatment.

Results

σ₁R^E102Q overexpression promoted dissociation of the protein from the endoplasmic reticulum (ER) membrane and cytoplasmic aggregation, which in turn impaired mitochondrial ATP production and proteasome activity. Under ER stress conditions, overexpression of wild-type σ₁R suppressed ER stress-induced mitochondrial injury, whereas σ₁R^E102Q overexpression aggravated mitochondrial damage and induced autophagic cell death. Moreover, σ₁R^E102Q-overexpressing cells showed aberrant extra-nuclear localization of the TAR DNA-binding protein (TDP-43), a condition exacerbated by ER stress. Treatment of cells with the mitochondrial Ca^2 + transporter inhibitor Ru360 mimicked the effects of σ₁R^E102Q overexpression, indicating that aberrant σ₁R-mediated mitochondrial Ca^2 + transport likely underlies TDP-43 extra-nuclear localization, segregation in inclusion bodies, and ubiquitination. Finally, enhanced ATP production promoted by methyl pyruvate (MP) treatment rescued proteasome impairment and TDP-43 extra-nuclear localization caused by σ₁R^E102Q overexpression.

Conclusions

Our observations suggest that neurodegeneration seen in some forms of ALS are due in part to aberrant mitochondrial ATP production and proteasome activity as well as TDP-43 mislocalization resulting from the SIGMAR1 mutation.

General significance

ATP supplementation by MP represents a potential therapeutic strategy to treat ALS caused by SIGMAR1 mutation.     

Stimulation of cytosolic and mitochondrial calcium mobilization by indomethacin in Caco-2 cells: Modulation by the polyphenols quercetin,resveratrol and rutin

Background

The effect of indomethacin (INDO) on Ca^2 + mobilization, cytotoxicity, apoptosis and caspase activation and the potential protective effect of quercetin (QUE), resveratrol (RES) and rutin (RUT) were determined in Caco-2 cells.

Methods

Caco-2 cells were incubated with INDO in the presence or absence of QUE, RES or RUT. The concentrations of Ca^2 + in the cytosol (Fluo-3 AM) and mitochondria (Rhod-2 AM) were determined as well as the cytotoxicity (MTT reduction and LDH leakage), apoptosis (TUNEL) and caspase-3 and 9 activities.

Results

INDO promoted Ca^2 + efflux from the endoplasmic reticulum (ER), resulting in an early, but transient, increment of cytosolic Ca^2 + at 3.5 min, followed by a subsequent increment of intra-mitochondrial Ca^2 + at 24 min. INDO also induced cytotoxicity, apoptosis, and increased caspase activities and cytochrome c release. All these alterations were prevented by the inhibitors of the IP3R and RyR receptors, 2-Aminoethoxydiphenyl borate (2-APB) and dantrolene. QUE was the most efficient polyphenol in preventing Ca^2 + mobilization induced by INDO and all of its consequences including cytotoxicity and apoptosis.

Conclusions

In Caco-2 cells, INDO stimulates ER Ca^2 + mobilization, probably through the activation of IP3R and RyR receptors, and the subsequent entry of Ca^2 + into the mitochondria. Polyphenols protected the cells against the Ca^2 + mobilization induced by INDO and its consequences on cytotoxicity and apoptosis.

General significance

These results confirm the possibility of using polyphenols and particularly QUE for the protection of the gastroduodenal mucosa in subjects consuming NSAIDs.     

Age-dependent guanine oxidation in DNA of different brain regions of Wistar rats and prematurely aging OXYS rats

Background

Oxidative damage to the cell, including the formation of 8-oxoG, has been regarded as a significant factor in carcinogenesis and aging. An inbred prematurely aging rat strain (OXYS) is characterized by high sensitivity to oxidative stress, lipid peroxidation, protein oxidation, DNA rearrangements, and pathological conditions paralleling several human degenerative diseases including learning and memory deterioration.

Methods

We have used monoclonal antibodies against a common pre-mutagenic base lesion 8-oxoguanine (8-oxoG) and 8-oxoguanine DNA glycosylase (OGG1) in combination with indirect immunofluorescence microscopy and image analysis to follow the relative amounts and distribution of 8-oxoG and OGG1 in various cells of different brain regions from OXYS and control Wistar rats.

Results

It was shown that 8-oxoG increased with age in mature neurons, nestin- and glial fibrillary acidic protein (GFAP)-positive cells of hippocampus and frontal cortex in both strains of rats, with OXYS rats always displaying statistically significantly higher levels of oxidative DNA damage than Wistar rats. The relative content of 8-oxoG and OGG1 in nestin- and GFAP-positive cells was higher than in mature neurons in both Wistar and OXYS rats. However, there was no significant interstrain difference in the content of OGG1 for all types of cells and brain regions analyzed, and no difference in the relative content of 8-oxoG between different brain regions.

Conclusions

Oxidation of guanine may play an important role in the development of age-associated decrease in memory and learning capability of OXYS rats.

General significance

The findings are important for validation of the OXYS rat strain as a model of mammalian aging.     

Estrogen receptor alpha (ERα) promoter methylation status in tumor and serum DNA in Egyptian breast cancer patients

Background

Status of DNA methylation is one of the most common molecular alterations in human neoplasia. Because it is possible to detect these epigenetic alterations in the bloodstream of patients, we investigated the aberrant DNA methylation status of estrogen receptor alpha (ERα) in patient pretherapeutic sera and tissue.

Materials and methods

In this case control study the patient series consisted of 120 sporadic primary breast cancer cases and 100 patients with benign breast lesion. ER3, ER4, and ER5 primers were used for methylation-specific polymerase chain reaction (MSP) to analyze the CpG methylation of promoter region of ERα gene. Correlation between ER3, ER4, and ER5 methylation and clinicopathological characteristics of the patients was investigated.

Result

The methylation status of ER3, ER4 and ER5 was 65%, 26.7% and 61.7% in tissue respectively and 57.5%, 21.7% and 55.8% in serum respectively. The concordance between tumor and serum DNA methylation was 80%, 72% and 92% for ER3, ER4 and ER5 respectively.

Conclusions

This study demonstrated the potential utility of serum DNA methylation of ERα gene promoter as a non-invasive diagnostic and/or prognostic marker in patients with breast cancer.     

Cell death caused by quinazolinone HMJ-38 challenge in oral carcinoma CAL 27 cells: dissections of endoplasmic reticulum stress,mitochondrial dysfunction and tumor xenografts

Background

This investigation clearly clarified the synthesized and antimitotic compound, 2-(3′-methoxyphenyl)-6-pyrrolidinyl-4-quinazolinone (HMJ-38), addressing its target and precise mechanism of action. We hypothesized that HMJ-38 might sensitize apoptotic death of human oral carcinoma CAL 27 cells in vitro and inhibit xenograft tumor growth in vivo.

Methods

Cell viability was assessed utilizing MTT assay. HMJ-38-treated cells represented DNA fragmentation using agarose gel electrophoresis as further evidenced using TUNEL staining. Flow cytometric analyses, immunoblotting and quantitative RT-PCR were applied for protein and gene expression. Antitumor xenograft study was employed.

Results

HMJ-38 concentration- and time-dependently reduced viability of CAL 27 cells. The effect of intrinsic molecules was signalized during HMJ-38 exposure with disruption of ΔΨm, MPT pore opening and the release of various events from mitochondria undergoing cell apoptosis. HMJ-38 also markedly facilitated G₂/M phase arrest. HMJ-38 stimulated the activation of CDK1 activity that modulated phosphorylation on Ser70 of Bcl-2-mediated mitotic arrest and apoptosis. HMJ-38 triggered intracellular Ca^2 + release and activated related pivotal hallmarks of ER stress. HMJ-38 in nude mice bearing CAL 27 tumor xenografts decreased tumor growth. Furthermore, HMJ-38 enhanced caspase-3 gene expression and protein level in xenotransplanted tumors.

Conclusions

Early roles of mitotic arrest, unfolded protein response and mitochondria-dependent signaling contributed to apoptotic CAL 27 cell demise induced by HMJ-38. In in vivo experiments, HMJ-38 also efficaciously suppressed tumor volume in a xenotransplantation model.

General significance

This finding might fully support a critical event for HMJ-38 via induction of apoptotic machinery and ER stress against human oral cancer cells.     

Molecular and structural basis for N-glycan-dependent determination of glycoprotein fates in cells

Background

N-linked oligosaccharides operate as tags for protein quality control, consigning glycoproteins to different fates, i.e. folding in the endoplasmic reticulum (ER), vesicular transport between the ER and the Golgi complex, and ER-associated degradation of glycoproteins, by interacting with a panel of intracellular lectins in the early secretory pathway.

Scope of review

This review summarizes the current state of knowledge regarding the molecular and structural basis for glycoprotein-fate determination in cells that is achieved through the actions of the intracellular lectins and its partner proteins.

Major conclusions

Cumulative frontal affinity chromatography (FAC) data demonstrated that the intracellular lectins exhibit distinct sugar-binding specificity profiles. The glycotopes recognized by these lectins as fate determinants are embedded in the triantennary structures of the high-mannose-type oligosaccharides and are exposed upon trimming of the outer glucose and mannose residues during the N-glycan processing pathway. Furthermore, recently emerged 3D structural data offer mechanistic insights into functional interplay between an intracellular lectin and its binding partner in the early secretory pathway.

General significance

Structural biology approaches in conjunction with FAC methods provide atomic pictures of the mechanisms behind the glycoprotein-fate determination in cells. This article is a part of a Special issue entitled: Glycoproteomics.     

Blockage of TRPM7 channel induces hepatic stellate cell death through endoplasmic reticulum stress-mediated apoptosis

Aims

Proliferation is a ‘multiplier’ for extracellular matrix production and contraction of activated hepatic stellate cells (HSC) in fibrotic liver. Transient receptor potential melastatin-like 7 channels (TRPM7) are implicated in the survival and proliferation of several kinds of cells. This study was aimed to investigate the effect of TRPM7 blocker 2-APB on survival and proliferation of HSC and the underlying mechanisms.

Main methods

Rat HSC were stimulated by 2-APB for 24 h and then collected for further use. Cell viability was detected by MTT, and apoptosis was determined by AnnexinV/PI staining and TUNEL assay. Gene expressions of TRPM7, α-SMA, bcl-2, bax, and endoplasmic reticulum (ER) stress key members CHOP, caspase-12, ATF4, ATF6, Xbp1, GRP78 and calnexin were evaluated with quantitative RT-PCR. Quantifications of α-SMA, TRPM7, CHOP and GRP78 proteins were carried out by Western blot. Transmission electron microscopy and Xbp1 mRNA splicing analysis were also used for detection of ER stress.

Key findings

2-APB decreased TRPM7 and α-SMA expressions in primary HSC, and inhibited proliferation of activated HSC in a dose-dependent manner. 2-APB also decreased total count of activated HSC and increased the number of apoptotic cells. 2-APB increased expressions of bax and ER stress key factors CHOP, caspase-12, ATF4, ATF6, Xbp1, GRP78 and calnexin. Meanwhile, ultra-structural ER changes and spliced Xbp1 mRNA were also observed in 2-APB treated HSC.

Significance

Blockage of TRPM7 could inhibit activation and proliferation of primary HSC and induce apoptotic death of activated cells, in which ER stress was identified as one of possible underlying molecular bases.     

Omega-3 deficiency and neurodegeneration in the substantia nigra: Involvement of increased nitric oxide production and reduced BDNF expression

Background

Our previous study demonstrated that essential fatty acid (EFA) dietary restriction over two generations induced midbrain dopaminergic cell loss and oxidative stress in the substantia nigra (SN) but not in the striatum of young rats. In the present study we hypothesized that omega-3 deficiency until adulthood would reduce striatum's resilience, increase nitric oxide (NO) levels and the number of BDNF-expressing neurons, both potential mechanisms involved in SN neurodegeneration.

Methods

Second generation rats were raised from gestation on control or EFA-restricted diets until young or adulthood. Lipoperoxidation, NO content, total superoxide dismutase (t-SOD) and catalase enzymatic activities were assessed in the SN and striatum. The number of tyrosine hydroxylase (TH)- and BDNF-expressing neurons was analyzed in the SN.

Results

Increased NO levels were observed in the striatum of both young and adult EFA-deficient animals but not in the SN, despite a similar omega-3 depletion (~ 65%) in these regions. Increased lipoperoxidation and decreased catalase activity were found in both regions, while lower tSOD activity was observed only in the striatum. Fewer TH- (~ 40%) and BDNF-positive cells (~ 20%) were detected at the SN compared to the control.

Conclusion

The present findings demonstrate a differential effect of omega-3 deficiency on NO production in the rat's nigrostriatal system. Prolonging omega-3 depletion until adulthood impaired striatum's anti-oxidant resources and BDNF distribution in the SN, worsening dopaminergic cell degeneration.

General significance

Omega-3 deficiency can reduce the nigrostriatal system's ability to maintain homeostasis under oxidative conditions, which may enhance the risk of Parkinson's disease.     

Involvement of ASK1–p38 pathway in the pathogenesis of diabetes triggered by pancreatic ß cell exhaustion

Background

Diabetes mellitus is characterized by high blood glucose levels. Pancreatic ß cell death contributes to type 1 and type 2 diabetes. Akita mice, which harbor a human permanent neonatal diabetes-linked mutation (Cys96Tyr) in the insulin gene, are well established as an animal model of diabetes caused by pancreatic ß cell exhaustion. Mutant Insulin 2 protein (Ins2^C96Y) induces endoplasmic reticulum (ER) stress and pancreatic ß cell death in Akita mice, although the molecular mechanism of Ins^C96Y-induced cell death remains unclear.

Methods

We investigate the mechanisms of Ins2^C96Y-induced pancreatic ß cell death in vitro and in vivo, using p38 inhibitor (SB203580), MIN6 cell (pancreatic ß cell line), Akita mice and apoptosis signal-regulating kinase 1 (ASK1) knockout mice.

Results

The expression of Ins^C96Y activated the ASK1–p38 pathway. Deletion of ASK1 mitigated Ins^C96Y-induced pancreatic ß cell death and delayed the onset of diabetes in Akita mice. Moreover, p38 inhibitor suppressed Ins^C96Y-induced MIN6 cell death.

Conclusions

These findings suggest that ER stress-induced ASK1–p38 activation, which is triggered by the accumulation of Ins^C96Y, plays an important role in the pathogenesis of diabetes.

General significance

Pancreatic ß cell death caused by insulin overload appears to be involved in the pathogenesis of type 1 and type 2 diabetes. Inhibition of the ASK1–p38 pathway may be an effective therapy for various types of diabetes.     

Continuous oral administration of atorvastatin ameliorates brain damage after transient focal ischemia in rats

Aims

Pre-treatment with statins is known to ameliorate ischemic brain damage after experimental stroke, and is independent of cholesterol levels. We undertook pre- vs post-ischemic treatment with atorvastatin after focal cerebral ischemia in rats.

Main methods

Male Sprague–Dawley rats underwent transient 90-min middle cerebral artery occlusion (MCAO). Atorvastatin (20 mg/kg/day) or vehicle was administered orally. Rats were divided into vehicle-treated, atorvastatin pre-treatment, atorvastatin post-treatment, and atorvastatin continuous-treatment groups. In the pre-treatment, rats were given atorvastatin or vehicle for 7 days before MCAO. In the post-treatment, rats received atorvastatin or vehicle for 7 days after MCAO. Measurement of infarct volume, as well as neurological and immunohistochemical assessments, were done 24 h and 7 days after reperfusion.

Key findings

Each atorvastatin-treated group demonstrated significant reductions in infarct and edema volumes compared with the vehicle-treated group 24 h after reperfusion. Seven days after reperfusion, infarct volumes in the post-treatment group and continuous-treatment group (but not the pre-treatment group) were significantly smaller than in the vehicle-treated group. Only the continuous-treatment group had significantly improved neurological scores 7 days after reperfusion compared with the vehicle group. Post-treatment and continuous-treatment groups had significantly decreased lipid peroxidation, oxidative DNA damage, microglial activation, expression of tumor necrosis factor-alpha, and neuronal damage in the cortical ischemic boundary area after 7 days of reperfusion.

Significance

These results suggest that continuous oral administration (avoiding withdrawal) with statins after stroke may reduce the extent of post-ischemic brain damage and improve neurological outcome by inhibiting oxidative stress and inflammatory responses.     

Interplay between autophagy and apoptosis mediated by copper oxide nanoparticles in human breast cancer cells MCF7

Background

Metal oxide nanoparticles are well known to generate oxidative stress and deregulate normal cellular activities. Among these, transition metals copper oxide nanoparticles (CuO NPs) are more compelling than others and able to modulate different cellular responses.

Methods

In this work, we have synthesized and characterized CuO NPs by various biophysical methods. These CuO NPs (~ 30 nm) induce autophagy in human breast cancer cell line, MCF7 in a time- and dose-dependent manner. Cellular autophagy was tested by MDC staining, induction of green fluorescent protein-light chain 3 (GFP-LC3B) foci by confocal microscopy, transfection of pBABE-puro mCherry-EGFP-LC3B plasmid and Western blotting of autophagy marker proteins LC3B, beclin1 and ATG5. Further, inhibition of autophagy by 3-MA decreased LD₅₀ doses of CuO NPs. Such cell death was associated with the induction of apoptosis as revealed by FACS analysis, cleavage of PARP, de-phosphorylation of Bad and increased cleavage product of caspase 3. siRNA mediated inhibition of autophagy related gene beclin1 also demonstrated similar results. Finally induction of apoptosis by 3-MA in CuO NP treated cells was observed by TEM.

Results

This study indicates that CuO NPs are a potent inducer of autophagy which may be a cellular defense against the CuO NP mediated toxicity and inhibition of autophagy switches the cellular response into apoptosis.

Conclusions

A combination of CuO NPs with the autophagy inhibitor is essential to induce apoptosis in breast cancer cells.

General significance

CuO NP induced autophagy is a survival strategy of MCF7 cells and inhibition of autophagy renders cellular fate to apoptosis.