川陈皮素改善衰老认知功能损伤的作用与机制

随着世界人口的老龄化,与年龄相关认知功能障碍的威胁越来越大.研究年龄相关认知功能损伤的发病机制及寻找有效的防治策略具有重要意义.我们之前的研究表明,衰老小鼠海马中S-亚硝基谷胱甘肽还原酶(S-nitrosoglutathione reductase,GSNOR)显著升高,神经元特异性高表达GSNOR转基因小鼠在行为学检测中表现出认知功能障碍.然而,其分子机制仍不清楚.在本研究中发现,CREB信号通路在GSNOR高表达转基因小鼠及原代培养小鼠海马神经元中均被GSNOR下调.在Y迷宫中检测表明,连续7 d腹腔注射CREB激活剂川陈皮素,能改善GSNOR过表达小鼠的认知损伤.进一步通过恐惧箱实验及Y迷宫测试研究川陈皮素对自然衰老小鼠认知功能的作用,发现川陈皮素能显著提高自然衰老小鼠在Y迷宫测试中的正确选择率以及在恐惧箱中的冻结时间,表明川陈皮素能显著改善衰老相关的认知功能.同样,川陈皮素上调了CREB磷酸化以及PSD95和Glu R1的水平,表明CREB信号上调在改善自然衰老认知功能损伤中发挥了重要作用.本研究为衰老认知功能损伤机制及改善方法提供了新的依据,GSNOR转基因小鼠也可能成为一种新的认知功能损伤模型.     

Optic, olfactory, and vestibular dysmorphogenesis in the homozygous mouse insertional mutant Tg9257

The transgene insertional mutation 9257 on mouse chromosome 18 was originally identified by the circling behavior caused by vestibular abnormalities in heterozygous mutants. To characterize the homozygous phenotype, we generated F2 offspring from the cross (C57BL/6J-tg/+ x DBA/2J). Eye defects ranging in severity from microphthalmia to anophthalmia were observed in the tg/tg offspring. Dysmorphic development of the lens was evident as early as E10.5 in homozygous transgenic mice. Apparent agenesis of the lateral semicircular canal was evident at E14.5. Anomalies of nasomaxillary structures and olfactory neuroepithelium were present in heterozygous and homozygous transgenic mice. The 9257 mutation provides a model for analysis of the morphogenesis of these three neurosensory systems and their associated bony structures.     

含P301L突变的Tau转基因小鼠纯合子品系的建立及鉴定

目的:建立含P301L突变的tau转基因小鼠的纯合子品系。方法:雄原核显微注射法获得含P301L突变的tau转基因阳性首建鼠,通过SYBR Green实时荧光定量PCR法和传统育种方式结合鉴定纯合子和杂合子。结果:共选育出95只纯合子,鉴定出的纯合子具有优于杂合子模拟老年痴呆生物学特性改变的优势。结论:外源性基因tau能稳定遗传,采用的SYBR Green实时荧光定量PCR和传统育种方式结合筛选鉴定纯合子和杂合子快速、经济、可靠。     

Mice deficient for the vesicular acetylcholine transporter are myasthenic and have deficits in object and social recognition

An important step for cholinergic transmission involves the vesicular storage of acetylcholine (ACh), a process mediated by the vesicular acetylcholine transporter (VAChT). In order to understand the physiological roles of the VAChT, we developed a genetically altered strain of mice with reduced expression of this transporter. Heterozygous and homozygous VAChT knockdown mice have a 45% and 65% decrease in VAChT protein expression, respectively. VAChT deficiency alters synaptic vesicle filling and affects ACh release. Whereas VAChT homozygous mutant mice demonstrate major neuromuscular deficits, VAChT heterozygous mice appear normal in that respect and could be used for analysis of central cholinergic function. Behavioral analyses revealed that aversive learning and memory are not altered in mutant mice; however, performance in cognitive tasks involving object and social recognition is severely impaired. These observations suggest a critical role of VAChT in the regulation of ACh release and physiological functions in the peripheral and central nervous system.     

Postweaning, forebrain-specific perturbation of the oxytocin system impairs fear conditioning

Oxytocin (Oxt) and vasopressin (Avp) are important for a wide variety of behaviors and the use of transgenic mice lacking the peptides or their receptors, particularly when their loss is spatially and temporally manipulated, offers an opportunity to closely examine their role in a particular behavior. We used a cued fear conditioning paradigm to examine associative learning in three lines of transgenic mice: mice that constitutively lack vasopressin 1a (Avpr1a(-/-)) or Oxt receptors (Oxtr(-/-)) and mice that have Oxt receptor loss restricted to the forebrain that begins postweaning (Oxtr(FB/FB)). Oxtr(-/-) and Avpr1a(-/-) mice have normal conditioned freezing. Oxtr(FB/FB) mice have a reduction in freezing behavior during acquisition, as well as during context and cue retention. In addition to reduction of Oxtr in the central nucleus of the amygdala, in vitro receptor autoradiography showed that the Oxtr(FB/FB) mice have significantly reduced levels of Avpr1a only in that structure. Our results show that postweaning alteration of the distribution of Oxtr receptors is critically important for fear behavior, an effect mirrored in the neural structures that mediate it. While constitutive knockouts of Oxtr and Avpr1a are useful for identifying the neural underpinnings of some behaviors, compensatory mechanisms within some circuits may obscure other behavioral roles.     

Enhanced neuronal plasticity and elevated endogenous sAPPα levels in mice over-expressing MMP9

Evidence accumulating during the past few years points to a significant role of matrix metalloproteinase 9 (MMP9) enzymatic activity in synaptic plasticity and cognitive processes. We have previously demonstrated that MMP9 is involved in receptor-mediated α-secretase-like cleavage of APP in vitro, resulting in increased secretion of sAPPα, the soluble N-terminal product of the non-amyloidogenic pathway known to be involved in neuronal plasticity and memory formation. To study the in vivo role of MMP9, we have generated transgenic mice over-expressing MMP9 in the brain. Herein, we demonstrate that MMP9 transgenic animals display enhanced performance in the non-spatial novel object recognition and the spatial water-maze task and that their enhanced performance was accompanied by increased dendritic spine density in the hippocampus and cortex following behavioural testing. Consistent with the above observations, the electrophysiological analysis revealed prolonged maintenance of long-term synaptic potentiation in hippocampal slices from MMP9 transgenic mice. Moreover, elevated sAPPα levels in the hippocampus and cortex of MPP9 transgenic animals were also observed. Overall, our results extend previous findings on the physiological role of MMP9 in neuronal plasticity and furthermore reveal that, APP may be one of the physiological proteolytic targets of MMP9 in vivo.     

A Comprehensive Behavioral Test Battery to Assess Learning and Memory in 129S6/Tg2576 Mice

Transgenic Tg2576 mice overexpressing human amyloid precursor protein (hAPP) are a widely used Alzheimer’s disease (AD) mouse model to evaluate treatment effects on amyloid beta (Aβ) pathology and cognition. Tg2576 mice on a B6;SJL background strain carry a recessive rd1 mutation that leads to early retinal degeneration and visual impairment in homozygous carriers. This can impair performance in behavioral tests that rely on visual cues, and thus, affect study results. Therefore, B6;SJL/Tg2576 mice were systematically backcrossed with 129S6/SvEvTac mice resulting in 129S6/Tg2576 mice that lack the rd1 mutation. 129S6/Tg2576 mice do not develop retinal degeneration but still show Aβ accumulation in the brain that is comparable to the original B6;SJL/Tg2576 mouse. However, comprehensive studies on cognitive decline in 129S6/Tg2576 mice are limited. In this study, we used two dementia mouse models on a 129S6 background—scopolamine-treated 129S6/SvEvTac mice (3–5 month-old) and transgenic 129S6/Tg2576 mice (11–13 month-old)–to establish a behavioral test battery for assessing learning and memory. The test battery consisted of five tests to evaluate different aspects of cognitive impairment: a Y-Maze forced alternation task, a novel object recognition test, the Morris water maze, the radial arm water maze, and a Y-maze spontaneous alternation task. We first established this behavioral test battery with the scopolamine-induced dementia model using 129S6/SvEvTac mice and then evaluated 129S6/Tg2576 mice using the same testing protocol. Both models showed distinctive patterns of cognitive impairment. Together, the non-invasive behavioral test battery presented here allows detecting cognitive impairment in scopolamine-treated 129S6/SvEvTac mice and in transgenic 129S6/Tg2576 mice. Due to the modular nature of this test battery, more behavioral tests, e.g. invasive assays to gain additional cognitive information, can easily be added.     

Orexin deficiency modulates cognitive flexibility in a sex-dependent manner

Cognitive flexibility is an important executive function and refers to the ability to adapt behaviors in response to changes in the environment. Of note, many brain disorders are associated with impairments in cognitive flexibility. Several classical neurotransmitter systems including dopamine, acetylcholine and noradrenaline are shown to be important for cognitive flexibility, however, there is not much known about the role of neuropeptides. The neuropeptide orexin, which is brain-widely released by neurons in the lateral hypothalamus, is a major player in maintaining sleep/wake cycle, feeding behavior, arousal, and motivational behavior. Recent studies showed a role of orexin in attention, cognition and stress-induced attenuation of cognitive flexibility by disrupting orexin signaling locally or systemically. However, it is not known so far whether brain-wide reduction or loss of orexin affects cognitive flexibility. We investigated this question by testing male and female orexin-deficient mice in the attentional set shifting task (ASST), an established paradigm of cognitive flexibility. We found that orexin deficiency impaired the intra-dimensional shift phase of the ASST selectively in female homozygous orexin-deficient mice and improved the first reversal learning phase selectively in male homozygous orexin-deficient mice. We also found that these orexin-mediated sex-based modulations of cognitive flexibility were not correlated with trait anxiety, narcoleptic episodes, and reward consumption. Our findings highlight a sexually dimorphic role of orexin in regulating cognitive flexibility and the need for further investigations of sex-specific functions of the orexin circuitry.     

外源性人TIMP-1基因在转基因小鼠染色体上的整合及定位

为探讨外源基因人基质金属蛋白酶组织抑制物-1（human tissue inhibitor of metalloproteinase-1, hTIMP-1）基因在转基因小鼠家系染色体上的整合和精确定位,应用Southrn印迹检测外源基因在染色体上整合的位点及拷贝数.结果表明,外源基因是以单拷贝、单位点形式整合;应用荧光原位杂交（fluorescence in situ hybridization, FISH）技术检测F4～F20代转基因小鼠中外源基因的整合.结果证明,该家系转基因小鼠自F4代起是纯合子,外源基因整合在17号染色体E区;反向PCR法（Inverse PCR, IPCR）克隆出约3.8 kb外源基因整合位点处的侧翼序列.分析表明,外源基因整合在17号染色体E1.3区,ALK（anaplastic lymphoma kinase, ALK）基因第23个内含子区域.结果提示,获得的转基因小鼠为纯系,外源基因hTIMP-1已稳定整合在转基因小鼠染色体上,并能遗传给后代.     

Effects of BRCA1 transgene expression on murine mammary gland development and mutagen-induced mammary neoplasia

To characterize the role of BRCA1 in mammary gland development and tumor suppression, a transgenic mouse model of BRCA1 overexpression was developed. Using the mouse mammary tumor virus (MMTV) promoter/enhancer, transgenic mice expressing human BRCA1 or select mutant controls were generated. Transgenic animals examined during adolescence were shown to express the human transgene in their mammary glands. The mammary glands of 13-week-old virgin homozygous MMTV-BRCA1 mice presented the morphology of moderately increased lobulo-alveolar development. The mammary ductal trees of both hemizygous and homozygous MMTV-BRCA1t340 were similar to those of control non-transgenic littermates. Interestingly, both hemi- and homozygous mice expressing a splice variant of BRCA1 lacking the N-terminal RING finger domain (MMTV-BRCA1sv) exhibited marked mammary lobulo-alveolar development, particularly terminal end bud proliferation. Morphometric analyses of mammary gland whole mount preparations were used to measure epithelial staining indices of ~35% for homozygous MMTV-BRCA1 mice and ~60% for both hemizygous and homozygous MMTV-BRCA1sv mice versus ~25% for non-transgenic mice. Homozygous MMTV-BRCA1 mice showed delayed development of tumors when challenged with 7,12 dimethylbenzanthracene (DMBA), relative to non-transgenic and homozygous BRCA1t340 expressing mice. In contrast, homozygous MMTV-BRCA1sv transgenic animals were sensitized to DMBA treatment and exhibited a very rapid onset of mammary tumor development and accelerated mortality. MMTV-BRCA1 effects on mortality were restricted to DMBA-induced tumors of the mammary gland. These results demonstrate in vivo roles for BRCA1 in both mammary gland development and in tumor suppression against mutagen-induced mammary gland neoplasia.     

BMP Signaling Mediates Effects of Exercise on Hippocampal Neurogenesis and Cognition in Mice

Exposure to exercise or to environmental enrichment increases the generation of new neurons in the adult hippocampus and promotes certain kinds of learning and memory. While the precise role of neurogenesis in cognition has been debated intensely, comparatively few studies have addressed the mechanisms linking environmental exposures to cellular and behavioral outcomes. Here we show that bone morphogenetic protein (BMP) signaling mediates the effects of exercise on neurogenesis and cognition in the adult hippocampus. Elective exercise reduces levels of hippocampal BMP signaling before and during its promotion of neurogenesis and learning. Transgenic mice with decreased BMP signaling or wild type mice infused with a BMP inhibitor both exhibit remarkable gains in hippocampal cognitive performance and neurogenesis, mirroring the effects of exercise. Conversely, transgenic mice with increased BMP signaling have diminished hippocampal neurogenesis and impaired cognition. Exercise exposure does not rescue these deficits, suggesting that reduced BMP signaling is required for environmental effects on neurogenesis and learning. Together, these observations show that BMP signaling is a fundamental mechanism linking environmental exposure with changes in cognitive function and cellular properties in the hippocampus.     

Pharmacokinetics of acute tryptophan depletion using a gelatin-based protein in male and female Wistar rats

In a previous publication we observed aberrant levels of the human reduced folate carrier (hRFC) in cortex from fetal Down syndrome (DS) subjects. Immunoreactivity for hRFC was increased as the only chromosome 21 gene product studied. We, therefore, analyzed mice transgenic for hRFC (TghRFC1) and wild-type (WT) mice for cognitive functions, behavior and in an observational neurological battery (FOB). Cognitive functions were evaluated by the Morris water maze (MWM), the open field (OF) was used for exploratory behavior, locomotor activity and anxiety-related behavior. The elevated plus maze (EPM) was used to confirm findings in the OF testing anxiety-related behavior and the rota rod (RR) to evaluate motor function. In the MWM TghRFC1 mice performed significantly worse (P < 0.0003) on the probe trial than WT mice. In the FOB visual placing was significantly reduced inTghRFC1 mice. In the OF TghRFC1 mice crossed twice as often (P < 0.029) and in the EPM individuals from this group showed a reduced number of exits from the closed arm (P < 0.044) compared to WT mice. TghRFC1 mice showed impaired performance on the RR, spending one-fourth of the time of WT on the revolving rod (P < 0.0003). Cognitive impairment is an obligatory symptom of DS and this deficiency corresponds to findings in the MWM of mice transgenic for hRFC. Findings of visual placing and failure on the RR may reflect impaired motor performance including muscular hypotonia in DS subjects. Increased crossings in the OF may indicate modulated anxiety-related behavior observed in patients with DS.     

Is It Possible to Improve Memory Function by Upregulation of the Cholesterol 24S-Hydroxylase (CYP46A1) in the Brain?

We previously described a heterozygous mouse model overexpressing human HA-tagged 24S-hydroxylase (CYP46A1) utilizing a ubiquitous expression vector. In this study, we generated homozygotes of these mice with circulating levels of 24OH 30–60% higher than the heterozygotes. Female homozygous CYP46A1 transgenic mice, aged 15 months, showed an improvement in spatial memory in the Morris water maze test as compared to the wild type mice. The levels of N-Methyl-D-Aspartate receptor 1, phosphorylated-N-Methyl-D-Aspartate receptor 2A, postsynaptic density 95, synapsin-1 and synapthophysin were significantly increased in the hippocampus of the CYP46A1 transgenic mice as compared to the controls. The levels of lanosterol in the brain of the CYP46A1 transgenic mice were significantly increased, consistent with a higher synthesis of cholesterol. Our results are discussed in relation to the hypothesis that the flux in the mevalonate pathway in the brain is of importance in cognitive functions.     

A partial reduction of VDAC1 enhances mitophagy,autophagy, synaptic activities in a transgenic Tau mouse model

Alzheimer''s disease (AD) is the most common cause of mental dementia in the aged population. AD is characterized by the progressive decline of memory and multiple cognitive functions, and changes in behavior and personality. Recent research has revealed age‐dependent increased levels of VDAC1 in postmortem AD brains and cerebral cortices of APP, APPxPS1, and 3xAD.Tg mice. Further, we found abnormal interaction between VDAC1 and P‐Tau in the AD brains, leading to mitochondrial structural and functional defects. Our current study aimed to understand the impact of a partial reduction of voltage‐dependent anion channel 1 (VDAC1) protein on mitophagy/autophagy, mitochondrial and synaptic activities, and behavior changes in transgenic TAU mice in Alzheimer''s disease. To determine if a partial reduction of VDAC1 reduces mitochondrial and synaptic toxicities in transgenic Tau (P301L) mice, we crossed heterozygote VDAC1 knockout (VDAC1^+/−) mice with TAU mice and generated double mutant (VDAC1^+/−/TAU) mice. We assessed phenotypic behavior, protein levels of mitophagy, autophagy, synaptic, other key proteins, mitochondrial morphology, and dendritic spines in TAU mice relative to double mutant mice. Partial reduction of VDAC1 rescued the TAU‐induced behavioral impairments such as motor coordination and exploratory behavioral changes, and learning and spatial memory impairments in VDAC1^+/−/TAU mice. Protein levels of mitophagy, autophagy, and synaptic proteins were significantly increased in double mutant mice compared with TAU mice. In addition, dendritic spines were significantly increased; the mitochondrial number was significantly reduced, and mitochondrial length was increased in double mutant mice. Based on these observations, we conclude that reduced VDAC1 is beneficial in symptomatic‐transgenic TAU mice.     

Behavioral assessment of Alzheimer's transgenic mice following long-term Abeta vaccination: task specificity and correlations between Abeta deposition and spatial memory.

Long-term vaccinations with human beta-amyloid peptide 1-42 (Abeta1-42) have recently been shown to prevent or markedly reduce Abeta deposition in the PDAPP transgenic model of Alzheimer's disease (AD). Using a similar protocol to vaccinate 7.5-month-old APP (Tg2576) and APP+PS1 transgenic mice over an 8-month period, we previously reported modest reductions in brain Abeta deposition at 16 months. In these same mice, Abeta vaccinations had no deleterious behavioral effects and, in fact, benefited the mice by providing partial protection from age-related deficits in spatial working memory in the radial arm water maze task (RAWM) at 15.5 months. By contrast, control-vaccinated transgenic mice exhibited impaired performance throughout the entire RAWM test period at 15.5 months. The present study expands on our initial report by presenting additional behavioral results following long-term Abeta vaccination, as well as correlational analyses between cognitive performance and Abeta deposition in vaccinated animals. We report that 8 months of Abeta vaccinations did not reverse an early-onset balance beam impairment in transgenic mice. Additionally, in Y-maze testing at 16 months, all mice showed comparable spontaneous alternation irrespective of genotype or vaccination status. Strong correlations were nonetheless present between RAWM performance and extent of "compact" Abeta deposition in both the hippocampus and the frontal cortex of vaccinated APP+PS1 mice. Our results suggest that the behavioral protection of long-term Abeta vaccinations is task specific, with preservation of hippocampal-associated working memory tasks most likely to occur. In view of the early short-term memory deficits exhibited by AD patients, Abeta vaccination of presymptomatic AD patients could be an effective therapeutic to protect against such cognitive impairments.     

ApoB100/LDLR-/- hypercholesterolaemic mice as a model for mild cognitive impairment and neuronal damage

Recent clinical findings support the notion that the progressive deterioration of cholesterol homeostasis is a central player in Alzheimer's disease (AD). Epidemiological studies suggest that high midlife plasma total cholesterol levels are associated with an increased risk of AD. This paper reports the plasma cholesterol concentrations, cognitive performance, locomotor activity and neuropathological signs in a murine model (transgenic mice expressing apoB100 but knockout for the LDL receptor [LDLR]) of human familial hypercholesterolaemia (FH). From birth, these animals have markedly elevated LDL-cholesterol and apolipoprotein B100 (apoB100) levels. These transgenic mice were confirmed to have higher plasma cholesterol concentrations than wild-type mice, an effect potentiated by aging. Further, 3-month-old transgenic mice showed cholesterol (total and fractions) concentrations considerably higher than those of 18-month-old wild-type mice. The hypercholesterolaemia of the transgenic mice was associated with a clear locomotor deficit (as determined by rotarod, grip strength and open field testing) and impairment of the episodic-like memory (determined by the integrated memory test). This decline in locomotor activity and cognitive status was associated with neuritic dystrophy and/or the disorganization of the neuronal microtubule network, plus an increase in astrogliosis and lipid peroxidation in the brain regions associated with AD, such as the motor and lateral entorhinal cortex, the amygdaloid basal nucleus, and the hippocampus. Aortic atherosclerotic lesions were positively correlated with age, although potentiated by the transgenic genotype, while cerebral β-amyloidosis was positively correlated with genetic background rather than with age. These findings confirm hypercholesterolaemia as a key biomarker for monitoring mild cognitive impairment, and shows these transgenic mice can be used as a model for cognitive and psycho-motor decline.     

Impotence induced by a single gene mutation

Inbred male mice homozygous for the stubby gene mutation were reported to be sterile nineteen years ago. However, the basis of the sterility has not been identified since testicular steroidogenesis and spermatogenesis are normal. In the present studies, the sexual behavior of stubby mice and their coisogenic, normal siblings was compared quantitatively. The experimental observations provided unequivocal evidence that the phenotypic basis of the sterility of stubby mice is impotency. Stubby mice represent the first animal model for the study of impotence and provide the first evidence of an autosomal gene mutation that has a primary effect on male sexual behavior.     

PNMT transgenic mice have an aggressive phenotype.

PNMT (phenylethanolamine-N-methyl-transferase) is the enzyme that catalyzes the formation of epinephrine from norepinephrine. In transgenic mice over-expressing PNMT, observations revealed a very high level of aggression compared to their background strain, C57BL/6J. To evaluate the influence of PNMT on aggression and emotionality in this transgenic line, single-sex male and female groups were independently established that consisted of either four wild-type mice or four transgenic mice overexpressing PNMT. The members of each group were littermates. Mixed single-sex groups consisting of two transgenic mice and two wild-type mice were also established. Almost no fights were observed within the female groups. In males, the transgenic line showed a significantly higher level of fighting than controls (p=0.007) and mixed male groups (p=0.02). Housing mice from the transgenic line in mixed groups with wild-type mice seems to decrease the level of aggression in the transgenic line. In conclusion, this is the first study to demonstrate a clear, significant increase in aggression arising from PNMT overexpression. This suggests an important role for central epinephrine levels in aggressive behavior.     

Enhanced learning and memory in GAT1 heterozygous mice

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. The termination of GABA transmission is through the action of a family of membrane proteins, called GABA transporters (GAT1-4). It is well established that GABA system is involved in the modulation of memory. Our previous study showed that homozygous GAT1(-/-) mice exhibited impaired hippocampus-dependent learning and memory. To evaluate the impact of endogenous reduced GABA reuptake on mice cognitive behaviors, the ability of learning and memory of heterozygous GAT1(+/-) mice was detected by the passive avoidance paradigm and Morris water maze. The hole board paradigm was also used to measure changes in anxiety-related behavior or exploratory behavior in such mice. As one form of synaptic plasticity, long-term potentiation was recorded in the mouse hippocampal CA1 area. We found that GAT1(+/-) mice displayed increased learning and memory, decreased anxiety-like behaviors, and highest synaptic plasticity compared with wild-type and homozygous GAT1(-/-) mice. Our results suggest that a moderate reduction in GAT1 activity causes the enhancement of learning and memory in mice.