加速衰老小鼠脑组织中的衰老相关基因的表达

从分子水平上研究衰老对大脑的影响有助于揭示机体衰老的分子机理 ,也有助于揭示衰老相关性脑功能异常的发生过程。本研究应用DDRT PCR方法研究衰老相关基因在SAM (Senescence acceleratedmouse)小鼠脑组织中表达的变化情况。在SAMR1TA、SAMP8/Ta、SAMP1 0 /Ta三个品系中 ,通过比较不同鼠龄SAMP1 0 /Ta (2、 4、 1 2、 1 8月龄 )的基因表达情况 ,发现在 4月龄和 1 2月龄分别有一个差异表达片段 ;对不同鼠龄的SAMP8/Ta (2、 4、 1 1月龄 )经差显比较 ,发现在 2月龄和 1 1月龄各有一差异表达片段。在不同品系的比较中发现了 1 6个差异性片段 ,分别属于SAMP1 0 /Ta (3个 )、SAMP8/Ta (6个 )和SAMR1TA (7个 )。测序结果经检索显示 ,它们分别与下列基因转录产物同源 :热休克识别蛋白 70、ATP依赖性线粒体RNA螺旋酶、DleumRNA、小鼠X染色体RP2 3 334C4克隆DNA序列、还原型辅酶Q 细胞色素c还原酶复合物 7 2kD亚单位、 6 0S核糖体蛋白L2 1、FIS、苯基烷基胺钙离子拮抗物结合蛋白、岩藻糖基转移酶 9、胶质细胞源性神经营养因子家族受体α1、内切核酸酶 /逆转录酶、PER1蛋白相关超级融原核蛋白、中心体蛋白CG NAP、转铁蛋白重链基因、巢蛋白 2基因、DNA依赖性蛋白激酶催化亚单位基因 prkdc     

快速老化模型小鼠海马正反向抑制消减cDNA文库的构建

目的:构建快速老化模型小鼠(SAM)海马正反向抑制消减cDNA文库,以揭示SAMP8学习记忆脑老化的机制,同时为研究阿尔茨海默病(AD)的发病机制提供线索。方法:以快速老化模型小鼠SAMP8和SAMR1海马的总RNA为材料,采用抑制消减杂交方法和蓝白斑筛选克隆构建文库,并用PCR鉴定了文库的质量。结果:成功构建了12月龄雄性SAMP8和SAMR1海马的正反向抑制消减cDNA文库,其中正向文库包含864个克隆,反向文库包含960个克隆,阳性克隆率为96.16%,插入片段范围为250～2000bp。结论:SAMP8和SAMR1海马的正反向抑制消减cDNA文库的构建,为进一步筛选鉴定SAMR1和SAMP8海马差异表达基因提供了丰富的实验材料。     

The effect of aging on the mineral status of female SAMP1 and SAMR1

The effect of aging on the status of macrominerals and trace elements in tissues was studied using two strains (SAMP1 and SAMR1) of senescence accelerated mouse. Two-month-old, 6-mo-old, and 10-mo-old female SAMP1 and SAMR1 mice were fed a commercial diet. Iron, zinc, copper, calcium, magnesium, phosphorus, sulfur, sodium, and potassium concentrations in blood, liver, kidney, brain, and tibia of the mice were determined. The copper concentration in the brain was significantly increased with age in SAMP1 and SAMR1. In addition, the brain copper levels in SAMP1 were significantly higher than that in SAMR1 at respective ages. The calcium concentration in the kidney was significantly increased with age, but the copper and phosphorus concentrations significantly decreased with age in SAMP1 and SAMR1. In the liver of SAMR1, all minerals measured in this study except for sodium and potassium were significantly decreased with age. In addition, all mineral concentrations in the liver of 2-mo-old mice in SAMR1 except for copper and sodium were markedly higher than those in SAMP1 of the same age. These results suggest that the genetic factor is related to the age-associated mineral changes in tissues.     

Characterization of Senescence-Accelerated Mouse Prone 6 (SAMP6) as an Animal Model for Brain Research

The senescence-accelerated mouse (SAM) was developed by selective breeding of the AKR/J strain, based on a graded score for senescence, which led to the development of both senescence-accelerated prone (SAMP), and senescence-accelerated resistant (SAMR) strains. Among the SAMP strains, SAMP6 is well characterized as a model of senile osteoporosis, but its brain and neuronal functions have not been well studied. We therefore decided to characterize the central nervous system of SAMP6, in combination with different behavioral tests and analysis of its biochemical and pharmacological properties. Multiple behavioral tests revealed higher motor activity, reduced anxiety, anti-depressant activity, motor coordination deficits, and enhanced learning and memory in SAMP6 compared with SAMR1. Biochemical and pharmacological analyses revealed several alterations in the dopamine and serotonin systems, and in long-term potentiation (LTP)-related molecules. In this review, we discuss the possibility of using SAMP6 as a model of brain function.     

Viral complementation allows HIV-1 replication without integration

Previous studies have reported that various inbred SAM mouse strains differ markedly with regard to a variety of parameters, such as capacity for learning and memory, life spans and brain histopathology. A potential cause of differences seen in these strains may be based on the fact that some strains have a high concentration of infectious murine leukemia virus (MuLV) in the brain, whereas other strains have little or no virus. To elucidate the effect of a higher titer of endogenous retrovirus in astroglial cells of the brain, we established astroglial cell lines from SAMR1 and SAMP8 mice, which are, respectively, resistant and prone to deficit in learning and memory and shortened life span. MuLV-negative astroglial cell lines established from ICR mice served as controls. Comparison of these cell lines showed differences in: 1) levels of the capsid antigen CAgag in both cell lysates and culture media, 2) expression of genomic retroelements, 3) the number of virus particles, 4) titer of infectious virus, 5) morphology, 6) replication rate of cells in culture and final cell concentrations, 7) expression pattern of proinflammatory cytokine genes. The results show that the expression of MuLV is much higher in SAMP8 than SAMR1 astrocyte cultures and that there are physiological differences in astroglia from the 2 strains. These results raise the possibility that the distinct physiological differences between SAMP8 and SAMR1 are a function of activation of endogenous retrovirus.     

Adiposity-Related Biochemical Phenotype in Senescence-Accelerated Mouse Prone 6 (SAMP6)

Senescence-accelerated mouse prone 6 (SAMP6) is a model of senile osteoporosis. From 10 to 22 wk of age, SAMP6 mice were heavier than age-matched AKR/J and SAMR1 mice. Body mass indices of 10- and 25-wk-old SAMP6 mice were higher than those of age-matched AKR/J and SAMR1 mice, indicating obesity in the SAMP6 animals. We compared the blood biochemical values among SAMP6, SAMR1, and AKR/J mice to assess whether the SAMP6 strain has abnormal obesity-related parameters. Plasma glucose, triglyceride, insulin, and leptin levels were higher in 10-wk-old SAMP6 mice than in age-matched SAMR1 and AKR/J mice, whereas plasma glucagon and adiponectin levels in 25-wk-old SAMP6 were lower compared with those in age-matched SAMR1 and AKR/J. Total cholesterol levels in SAMR1 and SAMP6 mice at 10 and 25 wk of age were higher than those in AKR/J mice. Hepatic lipid levels were higher in 10- and 25-wk-old SAMP6 mice compared with age-matched AKR/J and SAMR1 animals. These results indicate that SAMP6 mice exhibit obesity and hyperlipidemia, suggesting that the SAMP6 strain is a potential tool for the study of hyperlipidemia.Abbreviations: BMI, body mass indexThe senescence-accelerated mouse strains were developed through selective breeding of AKR/J mice based on graded scores for senescence and pathologic phenotypes.⁴⁴ The 9 senescence-prone (SAMP) strains all have a shortened lifespan and display an early onset of senescence after normal development and maturation, whereas the 3 senescence-resistant (SAMR) strains are resistant to early senescence and serve as controls. Among the SAMP strains, SAMP8 and SAMP10 exhibit deficits in learning and memory at a relatively early stage in their lifespan.^6,30 In contrast, SAMP6 mice are considered to be a model of senile osteoporosis, with their low bone mass and slow bone loss;²⁴ the bone mineral density of SAMP6 mice decreases after 4 mo of age.^14,17Our regular measurement of body weight revealed that SAMP6 mice were significantly higher between 10 and 22 wk of age than were age-matched SAMR1 and AKR/J. Based on this observation, we decided to compare body mass indices (BMIs), blood biochemical values, and liver sections among mice of these strains at 10 and 25 wk of age, which respectively correspond to the beginning and end of a period of significant body weight gain in SAMP6 mice compared with age-matched SAMR1 and AKR/J. Increased BMIs of SAMP6 mice at 10- and 25 wk compared with those of age-matched AKR/J and SAMR1 animals would indicate obesity in the SAMP6. In addition, because osteoblasts and adipocytes are thought to share a common precursor cell, osteoporosis and enhanced adipogenesis may be related. For example, adipogenesis in the bone marrow increases with aging and during osteoporosis,^15,33,34 and increased bone turnover occurs in hypercholesterolemic or dyslipidemic patients.²² Therefore obesity in SAMP6 mice might be due at least in part to enhanced adipogenesis. We measured and compared blood biochemical values among SAMP6, SAMR1, and AKR/J (the founder for the SAM strains) mice to assess whether the SAMP6 strain has abnormalities in blood biochemical markers, such as triglycerides or cholesterol.     

Trimethylamine‐N‐oxide promotes brain aging and cognitive impairment in mice

Gut microbiota can influence the aging process and may modulate aging‐related changes in cognitive function. Trimethylamine‐N‐oxide (TMAO), a metabolite of intestinal flora, has been shown to be closely associated with cardiovascular disease and other diseases. However, the relationship between TMAO and aging, especially brain aging, has not been fully elucidated. To explore the relationship between TMAO and brain aging, we analysed the plasma levels of TMAO in both humans and mice and administered exogenous TMAO to 24‐week‐old senescence‐accelerated prone mouse strain 8 (SAMP8) and age‐matched senescence‐accelerated mouse resistant 1 (SAMR1) mice for 16 weeks. We found that the plasma levels of TMAO increased in both the elderly and the aged mice. Compared with SAMR1‐control mice, SAMP8‐control mice exhibited a brain aging phenotype characterized by more senescent cells in the hippocampal CA3 region and cognitive dysfunction. Surprisingly, TMAO treatment increased the number of senescent cells, which were primarily neurons, and enhanced the mitochondrial impairments and superoxide production. Moreover, we observed that TMAO treatment increased synaptic damage and reduced the expression levels of synaptic plasticity‐related proteins by inhibiting the mTOR signalling pathway, which induces and aggravates aging‐related cognitive dysfunction in SAMR1 and SAMP8 mice, respectively. Our findings suggested that TMAO could induce brain aging and age‐related cognitive dysfunction in SAMR1 mice and aggravate the cerebral aging process of SAMP8 mice, which might provide new insight into the effects of intestinal microbiota on the brain aging process and help to delay senescence by regulating intestinal flora metabolites.     

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.     

Superoxide dismutase in senescence-accelerated mouse retina

To examine the relationship between retinal ageing and superoxide dismutase, we studied the dismutase, with immunohistochemistry and immunoquantitative analysis, in the retina of senescence-accelerated mice P8/Ta (SAMP8/Ta) 3 and 12 months after birth. Accelerated senescence-resistant mice R1TA (SAMR1TA), which show no acceleration of senescence, were used as controls. In SAMP8/Ta, copper-zinc superoxide dismutase and manganese superoxide dismutase immunoreactivity in the photoreceptor inner segments, the outer nuclear layer and the inner nuclear layer increased earlier than in the controls. The increase in both superoxide dismutases with age occurred not only in SAMP8/Ta retinas but also in the controls. In conclusion, we propose the possibility that SAMP8/Ta undergo deterioration not only of learning and memory but also acceleration of senescence in the retina. The dismutases also appear to increase with normal ageing in the retina.     

Novel frame-shift mutation in Slc5a2 encoding SGLT2 in a strain of senescence-accelerated mouse SAMP10

The senescence-accelerated mouse prone10 (SAMP10) strain, a model of aging, exhibits cognitive impairments and cerebral atrophy. We noticed that SAMP10/TaSlc mice, a SAMP10 substrain, have developed persistent glucosuria over the past few years. In the present study, we characterized SAMP10/TaSlc mice and further identified a spontaneous mutation in the Slc5a2 gene encoding sodium-glucose co-transporter (SGLT) 2. The mean concentration of urine glucose was high in SAMP10/TaSlc mice and increased further with advancing age, whereas other strains of senescence-accelerated mice, including SAMP1/SkuSlc, SAMP6/TaSlc and SAMP8/TaSlc or normal aging control SAMR1/TaSlc mice, exhibited no detectable glucose in urine. SAMP10/TaSlc mice consumed increasing amounts of food and water compared to SAMR1/TaSlc mice, suggesting the compensation of polyuria and the loss of glucose. Oral glucose tolerance tests showed decreased glucose reabsorption in the kidney of SAMP10/TaSlc mice. In addition, blood glucose levels decreased in an age-dependent fashion. The kidney was innately larger than that of control mice with no histological alterations. We examined the expression levels of glucose transporters in the kidney. Among SGLT1, SGLT2, glucose transporter (GLUT) 1 and GLUT2, we found a significant decrease only in the level of SGLT2. DNA sequencing of SGLT2 in SAMP10/TaSlc mice revealed a single nucleotide deletion of guanine at 1236, which resulted in a frameshift mutation that produced a truncated protein. We designate this strain as SAMP10/TaSlc-Slc5a2^slc (SAMP10-ΔSglt2). Recently, SGLT2 inhibitors have been demonstrated to be effective for the treatment of patients with type 2 diabetes (T2D). SAMP10-ΔSglt2 mice may serve as a unique preclinical model to study the link between aging-related neurodegenerative disorders and T2D.     

Frontiers of Model Animals for Neuroscience:Two Prosperous Aging Model Animals forPromoting Neuroscience Research

A model animal showing spontaneous onset is a useful tool for investigating the mechanism of disease. Here, I would like to introduce two aging model animals expected to be useful for neuroscience research: the senescence-accelerated mouse (SAM) and the klotho mouse. The SAM was developed as a mouse showing a senescence-related phenotype such as a short lifespan or rapid advancement of senescence. In particular, SAMP8 and SAMP10 show age-related impairment of learning and memory. SAMP8 has spontaneous spongy degeneration in the brain stem and spinal cord with aging, and immunohistochemical studies reveal excess protein expression of amyloid precursor protein and amyloid β in the brain, indicating that SAMP8 is a model for Alzheimer’s disease. SAMP10 also shows age-related impairment of learning and memory, but it does not seem to correspond to Alzheimer’s disease because senile plaques primarily composed of amyloid β or neurofibrillary tangles primarily composed of phosphorylated tau were not observed. However, severe atrophy in the frontal cortex, entorhinal cortex, amygdala, and nucleus accumbens can be seen in this strain in an age-dependent manner, indicating that SAMP10 is a model for normal aging. The klotho mouse shows a phenotype, regulated by only one gene named α-klotho, similar to human progeria. The α-klotho gene is mainly expressed in the kidney and brain, and oxidative stress is involved in the deterioration of cognitive function of the klotho mouse. These animal models are potentially useful for neuroscience research now and in the near future.     

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.     

Ultrastructure of the water-clear cell in the parathyroid gland of SAMP6 mice

Although the parathyroid water-clear cell is very rare, it has clinical significance because of its association with parathyroid hyperplasia or adenoma. SAMP6, a substrain of senescence-accelerated mouse, was developed as an animal model for senile osteoporosis. We investigated the morphology of the parathyroid glands in SAMP6 and age-matched normal mouse SAMR1. The parathyroid water-clear cells, which contained numerous vacuoles and the crystalloid inclusions, were found in SAMP6 mice at 5, 8 and 12 months of age. It was noted that the number of water-clear cells increased with aging, which are fairly consistent with the change of the serum parathyroid hormone (PTH) level. We did not find any water-clear cells in the parathyroid glands of SAMR1 mice. The existence of water-clear cells may represent hyperfunction of the parathyroid glands in SAMP6.     

Identification of differentially expressed genes in senescence-accelerated mouse testes by suppression subtractive hybridization analysis

Senescence-accelerated mouse (SAM) strains constitute a model of accelerated senescence coupled with a short lifespan and the early development of various age-related disorders. To identify differential gene expression in testes between senescence-accelerated SAMP1 and control SAMR1 mice, we performed suppression subtractive hybridization. We observed that the expression of three genes related to cell proliferation (myosin regulatory light chain B, aldolase 1A isoform, and cytochrome c oxidase subunit VIc) were upregulated and four genes implicated in spermatogenesis were downregulated in SAMP1 mice. Asb-8, a member of ankyrin repeat-containing proteins, was abundantly expressed in the testes and downregulated in SAMP1. The other three downregulated genes (germ cell-specific gene 1, T-complex polypeptide 1b, and activator of cAMP responsive element modulator in testis) have been reported to regulate late-stage spermatogenesis. These gene expression profiles might explain the findings of early testicular maturation and rapid decline in the ability to produce spermatozoa with advancing age in SAMP1 mice.     

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.     

Senescence-Accelerated Mouse (SAM) strains have a spontaneous mutation in the Abcb1a gene.

Senescence-Accelerated Mouse (SAM) strains are used as animal models for gerontological research. Here, we report that the SAMR1 strain, which shows a high sensitivity to toxicity of the parasiticide ivermectin, has a spontaneous retroviral insertional mutation in the ATP-binding cassette, sub-family B (MDR/TAP), member 1A (Abcb1a) gene. This mutation is identical to that found in Crl:CF1-Abcb1a mice, which are also highly sensitive to ivermectin due to the mutation. The mutant Abcb1a allele was found in SAMR4, SAMR5, SAMP1, SAMP6, SAMP7, and SAMP9, but not in SAMP3, SAMP8, SAMP10, SAMP11, and other outbred and inbred strains, including 129/SvJ strains. These results impart both caution and promise in the use of SAM strains in studies of biological processes in which P-glycoprotein participates.     

快速老化小鼠海马差异表达cDNA芯片的制作

老年性痴呆(Alzheimer’s disease,AD)是老年人群中最普遍的痴呆类型,是一种神经退行性紊乱疾病,目前临床上还没有有效的治疗方法。快速老化小鼠亚系P8(senescence-accelerated mouse prone8,SAMP8)是研究增龄相关性认知缺陷机制以及研究脑老化机制的良好动物,同时也是研究AD较为理想的实验动物模型之一。cDNA芯片技术可以同时规模研究成千上万个基因的表达,尤其适于AD这种多机制、多靶标、多途径的复杂疾病的研究,为了揭示AD的发病机制,发现用于治疗AD的药物靶标,以SAMP8和SAMR1海马抑制消减cDNA文库中的cDNA片段为材料,以β-actin和G3PDH为内参,设计了16×(1×14)点阵方案,并点制了含有3136个点的SAM海马差异表达cDNA芯片。芯片背景均匀一致,点的大小均一,排列规则整齐。在靶分子与探针杂交过程中,进行了杂交条件和洗涤芯片的优化。将杂交结果进行统计分析,选择差异表达的cDNA进行测序并进行生物信息学分析,用实时定量RT-PCR对部分基因的表达进行了验证,检测了芯片筛选结果的可靠性。该芯片的成功制备为进一步进行差异表达基因的筛选和研究提供了良好的手段,并将成为揭示SAMP8脑老化和AD发病机制的有力手段。     

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.     

P518/Qrfp sequence polymorphisms in SAMP6 osteopenic mouse

Mice lacking GPR103A expression display osteopenia. Analysis of mouse quantitative trait loci literature associated with bone mineral density suggested GPR103A ligand P518/Qrfp (chromosome 2qB) as a candidate osteoporosis gene. Promoter and coding regions of mouse P518/Qrfp were sequenced from genomic DNA obtained from the osteoporosis-prone strain SAMP6 and control strains SAMR1, A/J, AKR/J, BALB/c, C3H/HeJ, C57BL/6J, and DBA/2J. Four single-nucleotide polymorphisms (SNPs) were identified in only SAMP6 genomic DNA, g.-1773 T-->C, g.110 A-->G (N37S), g.188 G-->A (R63K), and g.135 T-->C (H45H). The promoter SNP generated a novel neuron-restrictive silencing factor binding site, a repressor that decreases gene expression in nonneuronal tissues. TaqMan analysis demonstrated fivefold lower P518/Qrfp liver expression in SAMP6 versus SAMR1 or C57BL/6J control strains. Tissue distribution of human, mouse, and rat P518/Qrfp and its receptors showed expression in bone and spinal cord. A direct role for P518/Qrfp function in maintaining bone mineral density is suggested.