Protective effect of xanthohumol against age-related brain damage

It has been recently shown that xanthohumol, a flavonoid present in hops, possesses antioxidant, anti-inflammatory and chemopreventive properties. However, its role in the aging brain has not been addressed so far. Therefore, this study aimed to investigate the possible neuroprotective activity of xanthohumol against age-related inflammatory and apoptotic brain damage in male senescence-accelerated prone mice (SAMP8). Animals were divided into 4 groups: Untreated young mice, untreated old mice and old mice treated either with 1 mg kg⁻¹ day⁻¹ or 5 mg kg⁻¹ day⁻¹ xanthohumol. Young and old senescence accelerated resistant mice (SAMR1) were used as controls. After 30 days of treatment, animals were sacrificed and their brains were collected and immediately frozen in liquid nitrogen. mRNA (GFAP, TNF-α, IL-1β, AIF, BAD, BAX, XIAP, NAIP and Bcl-2) and protein (GFAP, TNF-α, IL-1β, AIF, BAD, BAX, BDNF, synaptophysin and synapsin) expressions were measured by RT-PCR and Western blotting, respectively. Significant increased levels of pro-inflammatory (TNF-α, IL-1β) and pro-apoptotic (AIF, BAD, BAX) markers were observed in both SAMP8 and SAMR1 old mice compared to young animals (P<.05) and also in SAMP8 untreated old mice compared to SAMR1 (P<.05). These alterations were significantly less evident in animals treated with both doses of xanthohumol (P<.05). Also, a reduced expression of synaptic markers was observed in old mice compared to young ones (P<.05) but it significantly recovered with 5 mg kg⁻¹ day⁻¹ xanthohumol treatment (P<.05). In conclusion, xanthohumol treatment modulated the inflammation and apoptosis of aged brains, exerting a protective effect on damage induced by aging.     

Oxidative damage to RNA and expression patterns of MTH1 in the hippocampi of senescence-accelerated SAMP8 mice and Alzheimer's disease patients

Mammalian MTH1 protein, a MutT-related protein, catalyzes the hydrolysis of 8-oxo-7,8-dihydroguanosine triphosphate (8-oxoGTP) to monophosphate, thereby preventing incorporation of 8-oxo-7,8-dihydroguanine (8-oxoguanine) into RNA. In this study, we applied immunohistochemistry to follow the expression of MTH1 and the amount of 8-oxoguanine in RNA during aging. There were increased amounts of 8-oxoguanine in RNA in the CAl and CA3 subregions of hippocampi of 8- and 12-month-old SAMP8 mice, which exhibited early aging syndromes and declining learning and memory abilities compared to those of age-matched control SAMR1 mice. The expression levels of MTH1 in the hippocampi of 8- and 12-month-old SAMP8 mice were significantly lower than those of control mice. Therefore, in this mouse model, age-related accumulation of 8-oxoguanine in RNA is correlated with decreased expression of MTH1. Increased amounts of 8-oxoguanine in the RNA, and decreased expression of MTH1 were also observed in the hippocampi of patients suffering from Alzheimer’s disease. These results suggest that MTH1 deficiency might be a causative factor for aging and age-related disorders.     

Dysfunction of astrocytes in senescence-accelerated mice SAMP8 reduces their neuroprotective capacity

Early onset increases in oxidative stress and tau pathology are present in the brain of senescence-accelerated mice prone (SAMP8). Astrocytes play an essential role, both in determining the brain's susceptibility to oxidative damage and in protecting neurons. In this study, we examine changes in tau phosphorylation, oxidative stress and glutamate uptake in primary cultures of cortical astrocytes from neonatal SAMP8 mice and senescence-accelerated-resistant mice (SAMR1). We demonstrated an enhancement of abnormally phosphorylated tau in Ser(199) and Ser(396) in SAMP8 astrocytes compared with that of SAMR1 control mice. Gsk3beta and Cdk5 kinase activity, which regulate tau phosphorylation, was also increased in SAMP8 astrocytes. Inhibition of Gsk3beta by lithium or Cdk5 by roscovitine reduced tau phosphorylation at Ser(396). Moreover, we detected an increase in radical superoxide generation, which may be responsible for the corresponding increase in lipoperoxidation and protein oxidation. We also observed a reduced mitochondrial membrane potential in SAMP8 mouse astrocytes. Glutamate uptake in astrocytes is a critical neuroprotective mechanism. SAMP8 astrocytes showed a decreased glutamate uptake compared with those of SAMR1 controls. Interestingly, survival of SAMP8 or SAMR1 neurons cocultured with SAMP8 astrocytes was significantly reduced. Our results indicate that alterations in astrocyte cultures from SAMP8 mice are similar to those detected in whole brains of SAMP8 mice at 1-5 months. Moreover, our findings suggest that this in vitro preparation is suitable for studying the molecular and cellular processes underlying early aging in this murine model. In addition, our study supports the contention that astrocytes play a key role in neurodegeneration during the aging process.     

Differential expression of the liver proteome in senescence accelerated mice

The senescence-accelerated mouse (SAM) is a useful animal model to study aging or age-associated disorders due to its inherited aging phenotype. To investigate proteins involved in the aging process in liver, we compared the young (4- or 20-week old) and the aged group (50-week-old) of SAMP8 (short life span) and SAMR1 (control) mice, and identified 85 differentially expressed distinct proteins comprising antioxidation, glucose/amino acid metabolism, signal transduction and cell cycle systems using proteomics tools. For the antioxidation system, the aged SAMP8 mice showed a large increase in glutathione peroxidase and decreases in glutathione-S-transferase and peroxiredoxin, ranging from 2.5- to 5-fold, suggesting lower detoxification potentials for oxidants in the aged SAMP8 liver. Similarly, levels of key glycolytic enzymes decreased greatly in the aged SAMP8 compared to SAMR1, indicating a disturbance in glucose homeostasis that may be closely related to the typical deficits in learning and memory of the aged SAMP8. Protein profiles of amino acid metabolic enzymes suggest that accumulation of glutamine and glutamate in tissues of the aged SAMP8 may be due to hyperexpression of ornithine aminotransferase and/or glutamate dehydrogenase. Decreases in levels of proteins involved in signal transduction/apoptosis (e.g., cathepsin B) in the aged SAMP8 may support the previously proposed negative relationship between apoptosis and aging. However, the changes described above were not markedly seen in the young group of both strains. For cell cycle systems, levels of selenium binding protein increased about four-fold with age in SAMP8. Yet, almost no change occurred in either the young or the aged SAMR1, which may explain problems associated with cell proliferation and tissue regeneration in the aged SAMP8. In conclusion, composite profiles of key proteins involved in age-related processes enable assessment of accelerated senescence and the appearance of senescence-related pathologies in the aged SAMP8.     

Diabetes exacerbates amyloid and neurovascular pathology in aging‐accelerated mice

Mounting evidence supports a link between diabetes, cognitive dysfunction, and aging. However, the physiological mechanisms by which diabetes impacts brain function and cognition are not fully understood. To determine how diabetes contributes to cognitive dysfunction and age‐associated pathology, we used streptozotocin to induce type 1 diabetes (T1D) in senescence‐accelerated prone 8 (SAMP8) and senescence‐resistant 1 (SAMR1) mice. Contextual fear conditioning demonstrated that T1D resulted in the development of cognitive deficits in SAMR1 mice similar to those seen in age‐matched, nondiabetic SAMP8 mice. No further cognitive deficits were observed when the SAMP8 mice were made diabetic. T1D dramatically increased Aβ and glial fibrillary acidic protein immunoreactivity in the hippocampus of SAMP8 mice and to a lesser extent in age‐matched SAMR1 mice. Further analysis revealed aggregated Aβ within astrocyte processes surrounding vessels. Western blot analyses from T1D SAMP8 mice showed elevated amyloid precursor protein processing and protein glycation along with increased inflammation. T1D elevated tau phosphorylation in the SAMR1 mice but did not further increase it in the SAMP8 mice where it was already significantly higher. These data suggest that aberrant glucose metabolism potentiates the aging phenotype in old mice and contributes to early stage central nervous system pathology in younger animals.     

Acutely administered melatonin restores hepatic mitochondrial physiology in old mice

Damage to mitochondria as a result of the intrinsic generation of free radicals is theoretically involved in the processes of cellular aging. Herein, we investigated whether acutely administered melatonin, due to its free radical scavenging activity, would influence mitochondrial metabolism. Mitochondrial respiratory activity and respiratory chain complex I and IV activities in liver mitochondria from a strain of senescence-accelerated-prone mice (SAMP8) and a strain of senescence-accelerated-resistant mice (SAMR1) were measured when the animals were 12 months of age. Respiratory control index (RCI), ADP/O ratio, State 3 respiration and dinitrophenol (DNP)-dependent uncoupled respiration were significantly lower in SAMP8 than in SAMR1. In contrast, State 4 respiration was significantly higher in SAMP8 than in SAMR1. Activities of complexes I and IV in SAMP8 were significantly lower than in SAMR1. Melatonin administration (10mg/kg body weight, intraperitoneally) 1h prior to sacrifice significantly increased RCI, ADP/O ratio, State 3 respiration and DNP-induced uncoupled respiration in SAMP8 while also significantly reducing State 4 respiration in SAMP8. The injection of melatonin also significantly increased complex I activity in both mouse strains and complex IV activity in the liver of SAMP8 mice. These results document an age-related decrease in hepatic mitochondrial function in SAM which can be modified by an acute pharmacological injection of melatonin; the indole stimulated mitochondrial respiratory chain activity which would likely reduce deteriorative oxidative changes in mitochondria that normally occur in advanced age.     

Mitochondrial dysfunction in platelets and hippocampi of senescence-accelerated mice

Senescence-accelerated mice (SAM) strains are useful models to understand the mechanisms of age-dependent degeneration. In this study, measurements of the mitochondrial membrane potential (Δψ_m) of platelets and the Adenosine 5^′-triphosphate (ATP) content of hippocampi and platelets were made, and platelet mitochondria were observed in SAMP8 (faster aging mice) and SAMR1 (aging resistant control mice) at 2, 6 and 9 months of age. In addition, an Aβ-induced (Amyloid beta-protein) damage model of platelets was established. After the addition of Aβ, the Δψ_m of platelets of SAMP8 at 1and 6 months of age were measured. We found that platelet Δψ_m, and hippocampal and platelet ATP content of SAMP8 mice decreased at a relatively early age compared with SAMR1. The platelets of 6 month-old SAMP8 showed a tolerance to Aβ-induced damages. These results suggest that mitochondrial dysfunction might be one of the mechanisms leading to age-associated degeneration in SAMP mice at an early age and the platelets could serve as a biomarker for detection of mitochondrial function and age related disease.     

Effects of age on plasma levels of calcium-regulating hormones and bone status in male SAMP8 mice

This study was undertaken to examine whether the plasma levels of calcium-regulating hormones and bone status alter with age in male senescence accelerated mice (SAM), SAMP8. Age-matched senescence-resistant mice, SAMR1, were used as controls. The blood and femur samples were collected at 2.5 months of age (M) and then monthly from 3 to 12 M for physicochemical analyses, biochemical analyses, and the determination of hormones by radioimmunoassay. With advancing age, the plasma calcitonin (CT) levels decreased progressively, and the plasma parathyroid hormone (PTH) and 1,25-dihydroxycholecalciferol (1,25(OH)2D3) levels increased in both SAMR1 and SAMP8. The plasma calcium concentrations were maintained within a narrow range throughout the experimental period, while the plasma phosphorus (P) concentrations decreased with age in both strains. In contrast to SAMR1, the curves of age-related changes in the plasma CT levels and P concentrations were lower, and those in the plasma PTH levels were higher in SAMP8. The femoral bone densities and calcium contents increased gradually with age from the beginning of the experiment and peaked at 6 M in both strains, then declined. Those peaks were lower in SAMP8 than in SAMR1. These results indicate that the male SAMP8 develops osteoporotic signs earlier than SAMR1, and is proved to be a satisfactory animal model for longitudinal studies related to osteoporosis for men.     

Regional differences in PACAP transport across the blood-brain barrier in mice: a possible influence of strain,amyloid beta protein,and age

The blood–brain barrier (BBB) controls the exchange of peptides and regulatory proteins between the central nervous system (CNS) and the blood. Transport across the BBB of such regulatory substances is altered in animal models of Alzheimer’s disease. These alterations could lead to cognitive impairments or diminish their therapeutic potential. Here, we measured the transport rate of radioactively labeled pituitary adenylate cyclase-activating polypeptide (PACAP) from blood into whole brain and into 11 brain regions in three groups of mice: young (2 months old) ICR, young (2 months old) SAMP8, and aged (12 months old) SAMP8 mice. The SAMP8 is a strain which develops impaired learning and memory with aging that correlates with an age-related increase in brain levels of amyloid β protein (AβP). PACAP is a powerful neurotrophin that may have a therapeutic role in neurodegenerative diseases. We found that I-PACAP crossed the BBB fastest at the hypothalamus and the hippocampus in all three groups. Slower transport rates into the whole brain, the olfactory bulb, the hypothalamus, and the hippocampus for aged SAMP8 mice was likely related to differences both from strain and expression of AβP with aging.     

The level of butyrylcholinesterase activity increases and the content of the mRNA remains unaffected in brain of senescence-accelerated mouse SAMP8

Looking at cholinesterases (ChEs) changes in age-related mental impairment, the expression of ChEs in brain of senescence accelerated-resistant (SAMR1) and senescence accelerated-prone (SAMP8) mice was studied. Acetylcholinesterase (AChE) activity was unmodified and BuChE activity increased twofold in SAMP8 brain. SAMR1 brain contained many AChE-T mRNAs, less BuChE and PRiMA mRNAs and scant AChE-R and AChE-H mRNAs. Their content unchanged in SAMP8 brain. Amphiphilic (G(4)(A)) and hydrophilic (G(4)(H)) AChE and BuChE tetramers, besides amphiphilic dimers (G(2)(A)) and monomers (G(1)(A)) were identified in SAMR1 brain and their distribution was little modified in SAMP8 brain. Blood plasma does not seem to provide the excess of BuChE activity in SAMP8 brain; it probably arises from glial cell changes owing to astrocytosis.     

A Biological Model of Accelerated Senescence: II. Age-Related Changes in the Number of Developing Male Germ Cells and Sertoli Cells in the Gonads of Senescence-Accelerated Mice

It was shown that in immature three- to four-week-old mice prone to accelerated senescence (SAMP1 strain), the number of spermatogonia, pachytene spermatocytes, and circular spermatids exceeded that in mice resistant to accelerated senescence (SAMR1 strain) by more than two times. Differences were found in the pattern of age-related changes in the number of meiotic and postmeiotic cells in the sexually mature SAMP1 and SAMR1 mice. In the gonads of SAMP1 and SAMR1 mice, the number of Sertoli cells was unstable.     

The Hippocampal Proteomic Analysis of Senescence-Accelerated Mouse: Implications of Uchl3 and Mitofilin in Cognitive Disorder and Mitochondria Dysfunction in SAMP8

Senescence-accelerated mouse prone 8 (SAMP8) is considered as a useful animal model for age-related learning and memory impairments. Hippocampus, a critical brain region associated with cognitive decline during normal aging and various neurodegenerative diseases, appeared a series of abnormalities in SAMP8. To investigate the molecular mechanisms underlying age-related cognitive disorders, we used 2-DE coupled with MALDI TOF/TOF MS to analyze the differential protein expression of the hippocampus of SAMP8 at 6-month-old compared with the age-matched SAM/resistant 1 (SAMR1) which shows normal aging process. Two proteins were found to be markedly changed in SAMP8 as compared to SAMR1: ubiquitin carboxyl-terminal hydrolase L3 (Uchl3), implicating in cytosolic proteolysis of oxidatively damaged proteins, was down-regulated while mitofilin, a vital protein for normal mitochondria function, exhibited four isoforms with a consistent basic shift of isoelectric point among the soluble hippocampal proteins in SAMP8 compared with SAMR1. The alterations were confirmed by Western blotting analysis. The analysis of their expression changes may shed light on the mechanisms of learning and memory deficits and mitochondrial dysfunction as observed in SAMP8.     

Antibody to beta-amyloid protein increases acetylcholine in the hippocampus of 12 month SAMP8 male mice

Amyloid beta protein (Abeta) is the primary constituent of plaque seen in Alzheimer's disease. Abeta is proposed to play an etiological role in Alzheimer's disease and to be a cause of the decrease in the level of acetylcholine in the hippocampus. The SAMP8 strain of mouse develops age-related increases in Abeta and deficits in learning and memory by 12 months of age. We examined in 12 month old SAMP8 mice the effects of giving antibody to Abeta by septal or intracerebroventricular (ICV) injection on acetylcholine levels in the hippocampus. Antibody to Abeta increased acetylcholine in the hippocampus over 100% after ICV injection and over 200% after septal injection. Injection of rabbit serum, antibody directed towards mouse IgG, or a blocking antibody directed towards human interleukin-1beta were without effect. These results suggest that antagonism of Abeta increases acetylcholine concentrations in the hippocampus, an area important for learning and memory.     

Production of age-related DNA strand breakage in brain cells of senescence-accelerated prone (SAMP1) mouse.

Amounts of DNA strand breaks were estimated by the proportion of cells without tails (PCWT) and the average lengths of tail momentum (ALTM) in comet images of tissue cells of senescence-accelerated prone (SAMP1) mouse and senescence-accelerated resistant (SAMR1) mouse. The PCWT and ALTM of brain cells from SAMR1 were unchanged from 4 to 15 months of age. In the case of SAMP1 brain cells, the PCWT decreased and the ALTM increased in an age-related manner from 8 to 15 months of age. In the cases of liver and kidney, the PCWT and the ALTM of both SAMP1 and SAMR1 cells showed constant values from 4 to 15 months of ages.     

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.     

Age-related changes in blood pressure in the senescence-accelerated mouse (SAM): aged SAMP1 mice manifest hypertensive vascular disease

Age-related changes in systolic blood pressure were assessed, using the senescence-accelerated mouse (SAM) model for aging research with strains SAMR1, SAMP1, and SAMP8. Each of the strains manifested a characteristic change in blood pressure with age. The SAMR1 strain, with normal aging, did not have chronologic changes from 2 to 27 months of age. The SAMP1 strain, with accelerated senescence, had a significant increase in blood pressure with age, and some (8 of 39) mice manifested hypertensive vascular disease characterized by high blood pressure, cardiac hypertrophy, and arteriolar fibrinoid necrosis at 11 to 14 months of age. The gradual increase in blood pressure after 8 to 10 months was considered to be preceded by progressive renal changes, from glomerulonephritis to contraction of the kidney, suggesting that the high blood pressure in the SAMP1 strain was of renal origin. Blood pressure in the SAMP8 strain, with age-related deficits in learning and memory, gradually decreased after 5 to 7 months of age, and was suggested to be due to the astrogliotic changes in response to spongiform degeneration in the medulla oblongata at 11 to 14 and 15 to 18 months of age.     

Morphological changes of skeletal muscle, tendon and periosteum in the senescence-accelerated mouse (SAMP6): a murine model for senile osteoporosis

SAMP6, a substrain of senescence-accelerated mouse, was developed as an animal model for senile osteoporosis. Previously we observed age-related changes of the bone in SAMP6. In the present study, we investigated the morphology of the skeletal muscle, tendon and periosteum in SAMP6 and age-matched normal mouse SAMR1. We did not find any significant differences between SAMR1 and SAMP6 at 1 and 2 months of age. As compared with SAMR1, the cross-sectional area of type I and type II muscle fibers of the soleus muscle were significantly low in SAMP6 at 8 months of age. The projections in the interface of the muscle-tendon junctions were significantly decreased in SAMP6 at 8 months of age. The number of fibroblasts and the diameter of the tendon collagen fibers in Achilles fiber were significantly reduced in SAMP6 at 8 months of age. The diameter of Sharpey's fiber reduced in SAMP6 at 5 and 8 months of age. Some chondrocytes in the insertions of Achilles tendon and some osteogenic cells in the periosteum showed degenerative changes in SAMP6 at 5 and 8 months of age. The pronounced degenerative changes were detected in the skeletal muscle, muscle-tendon junction, tendon, tendon-bone interface and periosteum in SAMP6 with age. These findings indicated the atrophy of skeletal muscle, degeneration of tendon and periosteum in SAMP6, which may be involved in the bone loss for senile osteoporosis.     

Senescence-Accelerated Mouse (SAM) with Special References to Neurodegeneration Models,SAMP8 and SAMP10 Mice

The SAM strains, a group of related inbred strains consisting of senescence-prone inbred strains (SAMP) and senescence-resistant inbred strains (SAMR), have been successfully developed by selective inbreeding of the AKR/J strain of mice donated by the Jackson laboratory in 1968. The characteristic feature of aging common to the SAMP and SAMR is accelerated senescence and normal aging, respectively. Furthermore, SAMP and SAMR strains of mice manifest various pathobiological phenotypes spontaneously. Among SAMP strains, SAMP8 and SAMP10 mice show age-related behavioral deterioration such as deficits in learning and memory, emotional disorders (reduced anxiety-like behavior and depressive behavior) and altered circadian rhythm associated with certain pathological, biochemical and pharmacological changes. Here, the previous and recent literature on SAM mice are reviewed with an emphasis on SAMP8 and SAMP10 mice. A spontaneous model like SAM with distinct advantages over the gene-modified model is hoped by investigators to be used more widely as a biogerontological resource to explore the etiopathogenesis of accelerated senescence and neurodegenerative disorders.     

Curcumin prevents mitochondrial dysfunction in the brain of the senescence-accelerated mouse-prone 8

The aging brain suffers mitochondrial dysfunction and a reduced availability of energy in the form of ATP, which in turn may cause or promote the decline in cognitive, sensory, and motor function observed with advancing age. There is a need for animal models that display some of the pathological features of human brain aging in order to study their prevention by e.g. dietary factors. We thus investigated the suitability of the fast-aging senescence-accelerated mouse-prone 8 (SAMP8) strain and its normally aging control senescence-accelerated mouse-resistant 1 (SAMR1) as a model for the age-dependent changes in mitochondrial function in the brain. To this end, 2-months old male SAMR1 (n = 10) and SAMP8 mice (n = 7) were fed a Western type diet (control groups) for 5 months and one group of SAMP8 mice (n = 6) was fed an identical diet fortified with 500 mg curcumin per kg. Dissociated brain cells and brain tissue homogenates were analyzed for malondialdehyde, heme oxygenase-1 mRNA, mitochondrial membrane potential (MMP), ATP concentrations, protein levels of mitochondrial marker proteins for mitochondrial membranes (TIMM, TOMM), the mitochondrial permeability transition pore (ANT1, VDAC1, TSPO), respiration complexes, and fission and fusion (Fis, Opa1, Mfn1, Drp1). Dissociated brain cells isolated from SAMP8 mice showed significantly reduced MMP and ATP levels, probably due to significantly diminished complex V protein expression, and increased expression of TSPO. Fission and fusion marker proteins indicate enhanced mitochondrial fission in brains of SAMP8 mice. Treatment of SAMP8 mice with curcumin improved MMP and ATP and restored mitochondrial fusion, probably by up-regulating nuclear factor PGC1α protein expression. In conclusion, SAMP8 compared to SAMR1 mice are a suitable model to study age-dependent changes in mitochondrial function and curcumin emerges as a promising nutraceutical for the prevention of neurodegenerative diseases that are accompanied or caused by mitochondrial dysfunction.