The antioxidants alpha-lipoic acid and N-acetylcysteine reverse memory impairment and brain oxidative stress in aged SAMP8 mice

Oxidative stress may play a crucial role in age-related neurodegenerative disorders. Here, we examined the ability of two antioxidants, alpha-lipoic acid (LA) and N-acetylcysteine (NAC), to reverse the cognitive deficits found in the SAMP8 mouse. By 12 months of age, this strain develops elevated levels of Abeta and severe deficits in learning and memory. We found that 12-month-old SAMP8 mice, in comparison with 4-month-old mice, had increased levels of protein carbonyls (an index of protein oxidation), increased TBARS (an index of lipid peroxidation) and a decrease in the weakly immobilized/strongly immobilized (W/S) ratio of the protein-specific spin label MAL-6 (an index of oxidation-induced conformational changes in synaptosomal membrane proteins). Chronic administration of either LA or NAC improved cognition of 12-month-old SAMP8 mice in both the T-maze footshock avoidance paradigm and the lever press appetitive task without inducing non-specific effects on motor activity, motivation to avoid shock, or body weight. These effects probably occurred directly within the brain, as NAC crossed the blood-brain barrier and accumulated in the brain. Furthermore, treatment of 12-month-old SAMP8 mice with LA reversed all three indexes of oxidative stress. These results support the hypothesis that oxidative stress can lead to cognitive dysfunction and provide evidence for a therapeutic role for antioxidants.     

Heat shock proteins in neurodegenerative disorders and aging

Many members of the heat shock protein family act in unison to refold or degrade misfolded proteins. Some heat shock proteins also directly interfere with apoptosis. These homeostatic functions are especially important in proteinopathic neurodegenerative diseases, in which specific proteins misfold, aggregate, and kill cells through proteotoxic stress. Heat shock protein levels may be increased or decreased in these disorders, with the direction of the response depending on the individual heat shock protein, the disease, cell type, and brain region. Aging is also associated with an accrual of proteotoxic stress and modulates expression of several heat shock proteins. We speculate that the increase in some heat shock proteins in neurodegenerative conditions may be partly responsible for the slow progression of these disorders, whereas the increase in some heat shock proteins with aging may help delay senescence. The protective nature of many heat shock proteins in experimental models of neurodegeneration supports these hypotheses. Furthermore, some heat shock proteins appear to be expressed at higher levels in longer-lived species. However, increases in heat shock proteins may be insufficient to override overwhelming proteotoxic stress or reverse the course of these conditions, because the expression of several other heat shock proteins and endogenous defense systems is lowered. In this review we describe a number of stress-induced changes in heat shock proteins as a function of age and neurodegenerative pathology, with an emphasis on the heat shock protein 70 (Hsp70) family and the two most common proteinopathic disorders of the brain, Alzheimer’s and Parkinson’s disease.     

DHEAS improves learning and memory in aged SAMP8 mice but not in diabetic mice

Dehydroepiandrosterone sulfate (DHEAS) has been reported to improve memory in aged animals and suggested as a treatment for age-related dementias. The SAMP8 mouse, a model of Alzheimer's disease, has an age-related impairment in learning and memory and an increase in brain levels of amyloid precursor protein (APP) and amyloid beta protein (Abeta). Male SAMP8 mice also have a decrease in testosterone, to which DHEA is a precursor. Diabetes has been suggested as a model of aging and to be linked to Alzheimer's disease. Diabetics can have memory deficits and lower DHEAS levels. Here, we examined the effects of chronic oral DHEAS on acquisition and retention for T-maze footshock avoidance in 12 mo male SAMP8 mice and in CD-1 mice with streptozocin-induced diabetes. Learning and memory were improved in aged SAMP8 mice, but not in CD-1 mice with streptozocin-induced diabetes. These findings suggest that DHEAS is more effective in reversing the cognitive impairments associated with overexpression of Abeta than with diabetes.     

Rapamycin activates autophagy in Hutchinson-Gilford progeria syndrome: implications for normal aging and age-dependent neurodegenerative disorders

While rapamycin has been in use for years in transplant patients as an antirejection drug, more recently it has shown promise in treating diseases of aging, such as neurodegenerative disorders and atherosclerosis. We recently reported that rapamycin reverses the cellular phenotype of fibroblasts from children with the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). We found that the causative aberrant protein, progerin, was cleared through autophagic mechanisms when the cells were treated with rapamycin, suggesting a new potential treatment for HGPS. Recent evidence shows that progerin is also present in aged tissues of healthy individuals, suggesting that progerin may contribute to physiological aging. While it is intriguing to speculate that rapamycin may affect normal aging in humans, as it does in lower organisms, it will be important to identify safer analogues of rapamycin for chronic treatments in humans in order to minimize toxicity. In addition to its role in HGPS and normal aging, we discuss the potential of rapamycin for the treatment of age-dependent neurodegenerative diseases.     

Regulation of aging and oxidative stress pathways in aged pancreatic islets using alpha-lipoic acid

Molecular and Cellular Biochemistry - Oxidative stress has been involved in the aging process and the pathogenesis of type-2 diabetes, which is a serious health problem worldwide. This study...     

Proteomic analysis of early response lymph node proteins in mice treated with titanium dioxide nanoparticles

Human exposure to nanoparticles is inevitable from natural and anthropogenic sources. Titanium dioxide (TiO2) nanoparticles are increasingly being used in pharmaceutical and cosmetic products. Previous studies revealed that TiO2 levels were significantly increased in tissues (e.g., lymph nodes) after mice were injected with nanosized TiO2. To identify early response lymph node proteins to TiO2 nanoparticles, groups of mice were intradermally injected with a low dose of DeGussa P25 TiO2 nanoparticles or vehicle alone. The proteomes of lymph nodes at 24 h were quantitatively analyzed using trypsin-catalyzed 16O/18O labeling in conjunction with two-dimensional liquid chromatography separation and tandem mass spectrometry (2DLC-MS/MS). A total of 33 proteins were significantly changed (over 1.3-fold, p<0.05) in the mice treated with TiO2 nanoparticles, which accounted for approximately 1% of the total proteins identified. The differentially expressed proteins mainly involve the immune response (e.g., inflammation), lipid and fatty acid metabolism, mRNA processing, and nucleosome assembly. Regulation of functionally distinct classes of proteins could be mediated by estrogen receptor (ESR1), PPARγ, and c-Myc signalings, etc. The differentially expressed proteins identified in this experiment could represent early response proteins to TiO2 nanoparticle treatment in mouse lymph nodes.     

Pathological implications of nucleic acid interactions with proteins associated with neurodegenerative diseases

Protein misfolding disorders (PMDs) refer to a group of diseases related to the misfolding of particular proteins that aggregate and deposit in the cells and tissues of humans and other mammals. The mechanisms that trigger protein misfolding and aggregation are still not fully understood. Increasing experimental evidence indicates that abnormal interactions between PMD-related proteins and nucleic acids (NAs) can induce conformational changes. Here, we discuss these protein–NA interactions and address the role of deoxyribonucleic (DNA) and ribonucleic (RNA) acid molecules in the conformational conversion of different proteins that aggregate in PMDs, such as Alzheimer’s, Parkinson’s, and prion diseases. Studies on the affinity, stability, and specificity of proteins involved in neurodegenerative diseases and NAs are specifically addressed. A landscape of reciprocal effects resulting from the binding of prion proteins, amyloid-β peptides, tau proteins, huntingtin, and α-synuclein are presented here to clarify the possible role of NAs, not only as encoders of genetic information but also in triggering PMDs.     

Mechanisms of DHA-enriched phospholipids in improving cognitive deficits in aged SAMP8 mice with high-fat diet

Recent studies have shown that a high-fat diet (HFD) is involved in both metabolic dysfunction and cognitive deficiency and that docosahexaenoic-acid-enriched phospholipids (DHA-PLs) have beneficial effects on obesity and cognitive impairment. However, there are only a few studies comparing differences between DHA-PC and DHA-PS in HFD-induced Alzheimer's disease (AD) models. After 8 weeks feeding with HFD, 10-month-old SAMP8 mice were fed with 1% (w/w) DHA-PC or 1% DHA-PS (biosynthesized from DHA-PC) for 8 weeks; we then tested the behavioral performances in the Barnes maze test and Morris maze test. The changes of the generation and accumulation of Aβ, oxidative stress, apoptosis, neuroinflammation and neurotrophic factors were also measured. The results indicated that both DHA-PC and DHA-PS significantly improved the metabolic disorders and cognitive deficits. Both DHA-PC and DHA-PS could ameliorate oxidative stress, and DHA-PS presented more notable benefits than DHA-PC on Aβ pathology, mitochondrial damage, neuroinflammation and neurotrophic factors; DHA-PS was for the first time found to increase the production of insoluble Aβ (less pathogenic) in this AD model. These data suggest that DHA-PLs can significantly improve cognitive deficiency, and the molecular mechanisms for this closely relate to the phospholipid polar groups.     

Proteomic identification of specific oxidized proteins in ApoE-knockout mice: relevance to Alzheimer's disease

We have examined oxidized proteins in the brain regions of wild-type (WT) and ApoE-knockout (KO) animals. Total protein oxidation in the hippocampus of young-KO (6 month) animals was approximately 2-fold greater than that of young-WT (6 month) animals and was similar to that of old-WT (18 month) and old-KO (18 month) animals. In the cortex of the same animals, the levels of total protein oxidation in all four groups were not significantly different. Two-dimensional electrophoresis (2-DE) coupled with immunostaining for protein carbonylation revealed six specific oxidation-sensitive proteins, the oxidation levels of which were increased in young-KO, old-WT, and old-KO mice compared with young-WT mice. These six oxidation-sensitive proteins were identified by mass spectrometry as glial fibrillary acidic protein, creatine kinase BB, disulfide isomerase, chaperonin subunit 5, dihydropyrimidase-related protein 2, and mortalin. These results indicate that the ApoE gene product offers protection against age-associated oxidative damage in the brain. Moreover, two of these proteins, creatine kinase and dihydropyrimidase-related protein 2, have recently been found to be oxidized in the brains of human subjects with Alzheimer's disease [Aksenov et al. J. Neurochem. 74: 2520-2527; 2000; Castegna et al. J. Neurochem. 82: 1524-1532; 2002]. These data suggest that the ApoE-knockout mouse serves as an appropriate model for studying pathogenic oxidative mechanisms influencing risk and progression of Alzheimer's disease.     

Proteomic identification of brain proteins that interact with dynein light chain LC8

Cytoplasmic dynein is a large minus end-directed microtubule motor that translocates cargos towards the minus end of microtubules. Light chain 8 of the dynein machinery (LC8) has been reported to interact with a large variety of proteins that possess K/RSTQT or GIQVD motifs in their sequence, hence permitting their transport in a retrograde manner. Yeast two-hybrid analysis has revealed that in brain, LC8 associates directly with several proteins such as neuronal nitric oxide synthase, guanylate kinase domain-associated protein and gephyrin. In this work, we report the identification of over 40 polypeptides, by means of a proteomic approach, that interact with LC8 either directly or indirectly. Many of the neuronal proteins that we identified cluster at the post-synaptic terminal, and some of them such as phosphofructokinase, lactate dehydrogenase or aldolase are directly involved in glutamate metabolism. Other pool of proteins identified displayed the LC8 consensus binding motif. Finally, recombinant LC8 was produced and a library of overlapping dodecapeptides (pepscan) was employed to map the LC8 binding site of some of the proteins that were previously identified using the proteomic approach, hence confirming binding to the consensus binding sites.     

Proteomic analysis of eccrine sweat: implications for the discovery of schizophrenia biomarker proteins

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and multiple reaction monitoring mass spectrometry (MRM-MS) proteomics analyses were performed on eccrine sweat of healthy controls, and the results were compared with those from individuals diagnosed with schizophrenia (SZ). This is the first large scale study of the sweat proteome. First, we performed LC-MS/MS on pooled SZ samples and pooled control samples for global proteomics analysis. Results revealed a high abundance of diverse proteins and peptides in eccrine sweat. Most of the proteins identified from sweat samples were found to be different than the most abundant proteins from serum, which indicates that eccrine sweat is not simply a plasma transudate and may thereby be a source of unique disease-associated biomolecules. A second independent set of patient and control sweat samples were analyzed by LC-MS/MS and spectral counting to determine qualitative protein differential abundances between the control and disease groups. Differential abundances of selected proteins, initially determined by spectral counting, were verified by MRM-MS analyses. Seventeen proteins showed a differential abundance of approximately 2-fold or greater between the SZ pooled sample and the control pooled sample. This study demonstrates the utility of LC-MS/MS and MRM-MS as a viable strategy for the discovery and verification of potential sweat protein disease biomarkers.     

Testosterone deficiency accelerates neuronal and vascular aging of SAMP8 mice: protective role of eNOS and SIRT1

Oxidative stress and atherosclerosis-related vascular disorders are risk factors for cognitive decline with aging. In a small clinical study in men, testosterone improved cognitive function; however, it is unknown how testosterone ameliorates the pathogenesis of cognitive decline with aging. Here, we investigated whether the cognitive decline in senescence-accelerated mouse prone 8 (SAMP8), which exhibits cognitive impairment and hypogonadism, could be reversed by testosterone, and the mechanism by which testosterone inhibits cognitive decline. We found that treatment with testosterone ameliorated cognitive function and inhibited senescence of hippocampal vascular endothelial cells of SAMP8. Notably, SAMP8 showed enhancement of oxidative stress in the hippocampus. We observed that an NAD(+)-dependent deacetylase, SIRT1, played an important role in the protective effect of testosterone against oxidative stress-induced endothelial senescence. Testosterone increased eNOS activity and subsequently induced SIRT1 expression. SIRT1 inhibited endothelial senescence via up-regulation of eNOS. Finally, we showed, using co-culture system, that senescent endothelial cells promoted neuronal senescence through humoral factors. Our results suggest a critical role of testosterone and SIRT1 in the prevention of vascular and neuronal aging.     

Proteomic analysis of cervical cancer cells treated with suberonylanilide hydroxamic acid

Suberonylanilide hydroxamic acid (SAHA) is an orally administered histone deacetylase inhibitor (HDACI) that has shown significant antitumour activity in a variety of tumour cells. To identify proteins involved in its antitumour activity, we utilized a proteomic approach to reveal protein expression changes in the human cervical cancer cell line HeLa following SAHA treatment. Protein expression profiles were analysed by 2-dimensional polyacrylamide gel electrophoresis (2-DE) and protein identification was performed on a MALDI-Q-TOF MS/MS instrument. As a result, a total of nine differentially expressed proteins were visualized by 2-DE and Coomassie brilliant blue (CBB) staining. Further, all the changed proteins were positively identified via mass spectrometry (MS)/MS analysis. Of these, PGAM1 was significantly downregulated in HeLa cells after treatment with SAHA. Moreover, PGAM1 has been proven to be downregulated in another cervical cancer cell line (CaSki) by western blot analysis. Together, using proteomic tools, we identified several differentially expressed proteins that underwent SAHA-induced apoptosis. These changed proteins may provide some clues to a better understanding of the molecular mechanisms underlying SAHA-induced apoptosis in cervical cancer.     

Proteomic analysis of amniotic membrane prepared for human transplantation: characterization of proteins and clinical implications

Amniotic membrane is commonly exploited in several surgical procedures. Despite a freeze preservation period, it is reported to retain wound healing, anti-angiogenic, antiinflammatory and anti-scarring properties; however, little is known about the active protein content. 2-DE analysis of transplant-ready amniotic membrane (TRAM) was performed. The effects of preservation and processing on amnion proteome were investigated, and the major proteins in the TRAM characterized using mass spectrometry and immunoblotting. This identified a spectrum of proteins including thrombospondin, mimecan, BIG-H3, and integrin alpha 6. Preservation compromises cellular viability resulting in selective elution of soluble cellular proteins, leaving behind extracellular matrix-associated and cell structural proteins. A number of key architectural proteins common to the architecture of the ocular surface were demonstrated in AM, which are involved in homeostasis and wound healing. Handling procedures alter the protein composition of amniotic membrane prepared for transplantation. Without standardization, there will be inter-membrane variation, which may compromise the desired therapeutic effect of transplant ready amniotic membrane.     

Proteomics for the identification of specifically oxidized proteins in brain: Technology and application to the study of neurodegenerative disorders

Summary. Proteomics offers the opportunity elucidate the complex protein interactions of cellular systems by studying the products of genes, i.e., proteins, and their structure, function and localization. The purpose of proteomics is to explain the information contained in the genome sequences in order to provide clues on cellular events, especially related to disease.Our proteomic approach has made possible the identification of specifically oxidized proteins in Alzheimers disease (AD) brain, providing for the first time evidence on how oxidative stress plays a crucial role in AD-related neurodegeneration. This represents an example of the use of proteomics to solve biological problems related to disease. The field, which is still in its infancy, represents a very promising way to elucidate mechanism of disease at a protein level. However, the techniques that support its development present several limitations and require introduction of new tools and innovation in order to achieve a fast, reliable and sensitive method to understand normal biological processes and their regulation as well as these cellular properties in disease.     

Effects of aging and dietary antler supplementation on the calcium-regulating hormones and bone status in ovariectomized SAMP8 mice

This study was conducted to investigate the effects of aging and long-term dietary antler supplementation on the calcium-regulating hormones and bone status in ovariectomized (Ovx) SAMP8 mice. The female SAMP8 mice were divided into four groups (in each group n = 6), Ovx or sham operated at the age of 2 months, and fed with 0.2% antler containing diet or control diet from the age of 2.5 months. The samples were collected at the age of 3, 6, 9, 12, and 15 months, respectively, for physicochemical analyses, biochemical analyses, and the determination of hormones by radioimmunoassay. The results showed that plasma calcium (Ca) concentrations were maintained in a narrow range in all groups throughout the whole experimental period. With aging and/or ovariectomy, plasma parathyroid hormone (PTH) and 1,25-dihydroxycholecalciferol (1,25-(OH)2-D3) levels increased, and plasma phosphorus (P) and calcitonin (CT) levels decreased, and the femoral bone densities and Ca contents increased during the earlier stage, and then decreased gradually in all groups. Plasma PTH and 1,25-(OH)2-D3 levels in the Ovx mice were significantly higher than those in the intact mice, and plasma P concentrations, plasma CT levels, femoral bone densities, and femoral Ca contents in the Ovx mice were significantly lower than those in the intact mice. In addition, the decreases of plasma P levels, plasma CT levels, femoral bone densities, and femoral Ca contents, and the increases of plasma PTH levels were moderated by antler administration in both Ovx and intact mice. However, there was no effect of the dietary antler supplementation on the plasma 1,25-(OH)2-D3 levels in the female mice. It is concluded that prolonged dietary antler supplementation has important positive effects on bone loss with age and/ or ovarian function deficiency.     

Neuronal expression of constitutive heat shock proteins: implications for neurodegenerative diseases

Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis have been termed "protein misfolding disorders." These diseases differ widely in frequency and impact different classes of neurons. Heat shock proteins provide a line of defense against misfolded, aggregation-prone proteins and are among the most potent suppressors of neurodegeneration in animal models. Analysis of constitutively expressed heat shock proteins revealed variable levels of Hsc70 and Hsp27 in different classes of neurons in the adult rat brain. The differing levels of these constitutively expressed heat shock proteins in neuronal cell populations correlated with the relative frequencies of the previously mentioned neurodegenerative diseases.     

Nitrite treatment rescues cardiac dysfunction in aged mice treated with conjugated linoleic acid

Conjugated linoleic acid (cLA) is a commercially available weight-loss supplement that is not currently regulated by the U.S. FDA. Numerous studies suggest that cLA mediates protection against diseases including cancer, diabetes, atherosclerosis, immune function, and obesity. Based upon these reports, it was hypothesized that supplementation with cLA would improve heart function in aged wild-type (WT) mice. At 10 months of age, mice were treated with cLA, nitrite, or the combination of the two. Echocardiograms revealed that cardiac function was decreased in aged compared to young WT mice, as determined by percentage of fractional shortening. Also, contrary to the hypothesis, mice that received cLA (6-week treatment) had significantly worse cardiac function compared to controls. This effect was attenuated when mice were cotreated with cLA and nitrite. Taken together, these results suggest that cLA-mediated cardiac injury can be circumvented by nitrite supplementation in a murine model of aging.