Chronic Stress Contributes to Cognitive Dysfunction and Hippocampal Metabolic Abnormalities in APP/PS1 Mice.

BACKGROUND/AIMS: Stress response is determined by the brain, and the brain is a sensitive target for stress. Our previous experiments have confirmed that once the stress response is beyond the tolerable limit of the brain, particularly that of the hippocampus, it will have deleterious effects on hippocampal structure and function; however, the metabolic mechanisms for this are not well understood. METHODS: Here, we used morris water maze, elisa and gas chromatography-time of flight/mass spectrometry to observe the changes in cognition, neuropathology and metabolomics in the hippocampus of APP/PS1 mice and wild-type (C57) mice caused by chronic unpredictable mild stress (CUMS), we also further explored the correlation between cognition and metabolomics. RESULTS: We found that 4 weeks of CUMS aggravated cognitive impairment and increased amyloid-ß deposition in APP/PS1 mice, but did not affect C57 mice. Under non-stress conditions, compared with C57 mice, there were 8 different metabolites in APP/PS1 mice. However, following CUMS, 3 different metabolites were changed compared with untreated C57 mice. Compared to APP/PS1 mice, there were 7 different metabolites in APP/PS1+CUMS mice. Among these alterations, 3-hydroxybutyric acid, valine, serine, beta-alanine and o-phosphorylethanolamine, which are involved in sphingolipid metabolism, synthesis and degradation of ketone bodies, and amino acid metabolism. CONCLUSION: The results indicate that APP/PS1 mice are more vulnerable to stress than C57 mice, and the metabolic mechanisms of stress-related cognitive impairment in APP/PS1 mice are related to multiple pathways and networks, including sphingolipid metabolism, synthesis and degradation of ketone bodies, and amino acid metabolism.

Chronic high-frequency repetitive transcranial magnetic stimulation improves age-related cognitive impairment in parallel with alterations in neuronal excitability and the voltage-dependent Ca2+ current in female mice.

Chronic high-frequency repetitive transcranial magnetic stimulation (rTMS) is a noninvasive method to increase the excitability of neurons, and it induces long-term effects that can improve symptoms related to neurodegenerative diseases, including cognitive ability. The present study was undertaken to identify the mechanism by which rTMS improves cognitive impairments in mice. The novel object recognition test in vivo was used to evaluate the cognitive function of the mice. Whole-cell patch-clamp recordings were used to evaluate the neuronal excitability, including the resting membrane potential, the number of action potentials induced by depolarized current, after-hyperpolarization, and the voltage-dependent Ca(2+) current in hippocampal slices. We found that the aged mice showed impairments in cognitive function, and high-frequency (25Hz) rTMS for 14 consecutive-days ameliorated the impairments. Whole-cell patch-clamp recordings showed that, compared to matured mice, the hippocampal CA1 pyramidal neurons of aged mice showed significantly hyperpolarized resting membrane potential, significantly decreased numbers of action potentials after injection of depolarizing current, and significantly increased after-hyperpolarization after an action potential. The exposure to high-frequency rTMS significantly improved the above deficits in the neuronal excitability in the aged rTMS mice. Consistent with the above changes, the exposure to high-frequency rTMS also significantly decreased the voltage-dependent Ca(2+) current of the neurons compared with the aged sham mice. These data suggested that the rTMS could improve the age-related cognitive impairment in parallel with regulating the neuronal excitability and modifying the voltage-dependent Ca(2+) channels.

High Frequency Repetitive Transcranial Magnetic Stimulation Alleviates Cognitive Impairment and Modulates Hippocampal Synaptic Structural Plasticity in Aged Mice.

Normal aging is accompanied by hippocampus-dependent cognitive impairment, which is a risk factor of Alzheimer's disease. This study aims to investigate the effect of high frequency-repetitive transcranial magnetic stimulation (HF-rTMS) on hippocampus-dependent learning and memory in aged mice and explore its underlying mechanisms. Forty-five male Kunming mice (15 months old) were randomly divided into three groups: aged sham, 5 Hz rTMS, and 25 Hz rTMS. Two sessions of 5 Hz or 25 Hz rTMS comprising 1,000 pulses in 10 trains were delivered once a day for 14 consecutive days. The aged sham group was treated by the reverse side of the coil. In the adult sham group, 15 male Kunming mice (3 months old) were treated the same way as the aged sham group. A Morris water maze (MWM) was conducted following the stimulation, and synaptic ultrastructure was observed through a transmission electron microscope. HF-rTMS improved spatial learning and memory impairment in the aged mice, and 5 Hz was more significant than 25 Hz. Synaptic plasticity-associated gene profiles were modified by HF-rTMS, especially neurotrophin signaling pathways and cyclic adenosine monophosphate response element binding protein (CREB) cofactors. Compared to the aged sham group, synaptic plasticity-associated proteins, i.e., synaptophysin (SYN) and postsynaptic density (PSD)-95 were increased; brain-derived neurotrophic factor (BDNF) and phosphorylated CREB (pCREB) significantly increased after the 5 Hz HF-rTMS treatment. Collectively, our results suggest that HF-rTMS ameliorated cognitive deficits in naturally aged mice. The 5 Hz rTMS treatment significantly enhanced synaptic structural plasticity and activated the BDNF/CREB pathway in the hippocampus.

Damage to the nigrostriatal system in the MPTP-treated SAMP8 mouse.

Application of aged animals to studies of Parkinson's disease (PD) will be beneficial to improve the understanding of its pathogenesis. The senescence-accelerated mouse prone8 (SAMP8) mouse has an early onset of senility and a short life span, characterized by learning and memory impairment, and affective disturbance in the aging process. There is no animal currently being used as a PD model that exhibits these characteristics. Application of the SAMP8 mouse to PD research may have several merits. For the first time, we have investigated damage of the nigrostriatal system in the SAMP8 mouse induced by 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). Male SAMP8 mice (12 weeks) were treated with four subcutaneous injections of MPTP (20mg/kg at 2h intervals): spontaneous activity decreased significantly after the third injection, and recovered 48h after the first injection. In MPTP-SAMP8 mice, the tyrosine hydroxylase (TH)-positive neuronal loss at 6h (7.06%), 24h (12.79%), 3 days (22.49%), and 8 days (42.39%), while striatal dopamine (DA) levels decreased at 6h by 79.09%, at 24h by 80.33%, at 3 days by 83.86%, and at 8 days by 80.14%. These results indicated that there were marked decreases in striatal DA levels and a loss of dopaminergic neurons in the substantia nigra, with the behavior change following shortly thereafter, in MPTP-SAMP8 mice. On the basis of the current findings, the SAMP8 mouse is also vulnerable to neurotoxic effects of MPTP. These data suggest that the SAMP8 mouse may be utilized in PD research.

Enriched environment elevates expression of growth associated protein-43 in the substantia nigra of SAMP8 mice.

An enriched environment protects dopaminergic neurons from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neuronal injury, but the underlying mechanism for this is not clear. Growth associated protein-43 (GAP-43) is closely associated with neurite outgrowth and axon regeneration during neural development. We speculate that an enriched environment can reduce damage to dopaminergic neurons by affecting the expression of GAP-43. This study is designed to test this hypothesis. Three-month-old female senescence-accelerated mouse prone 8 (SAMP8) mice were housed for 3 months in an enriched environment or a standard environment. These mice were then subcutaneously injected in the abdomen with 14 mg/kg MPTP four times at 2-hour intervals. Morris water maze testing demonstrated that learning and memory abilities were better in the enriched environment group than in the standard environment group. Reverse-transcription polymerase chain reaction, immunohistochemistry and western blot assays showed that mRNA and protein levels of GAP-43 in the substantia nigra were higher after MPTP application in the enriched environment group compared with the standard environment group. These findings indicate that an enriched environment can increase GAP-43 expression in SAMP8 mice. The upregulation of GAP-43 may be a mechanism by which an enriched environment protects against MPTP-induced neuronal damage.

Cholesterol, 24-Hydroxycholesterol, and 27-Hydroxycholesterol as Surrogate Biomarkers in Cerebrospinal Fluid in Mild Cognitive Impairment and Alzheimer's Disease: A Meta-Analysis.

Abnormal cholesterol metabolism is an established feature of Alzheimer's disease (AD). Cerebrospinal fluid (CSF) is the fluid surrounding the central nervous system, and the protein and lipid content alterations in the CSF could be biomarkers for degenerative changes in the brain. The laboratory diagnosis of AD is limited to the analysis of three biomarkers in CSF: Aß42, total tau, and phospho-tau. The purpose of this analysis is to systematically analyze the available data describing the biomarkers of cholesterol and its metabolites in the CSF of subjects with AD. MEDLINE, EMBASE, and the Cochrane Central database were systematically queried to collect studies that have evaluated the markers of cholesterol and its metabolites in the CSF of subjects with mild cognitive impairment (MCI) or AD and age-matched controls. Analysis of the published data shows that the levels of cholesterol are increased in MCI subjects; 24-hydroxycholesterol and 27-hydroxycholesterol are elevated in AD and MCI subjects compared to controls. There is a significant dysfunction of cholesterol metabolism in the CSF of AD subjects. This analysis indicates that in addition to the available biomarkers in the CSF, such as Aß42, total tau, and phospho-tau, 24-hydroxycholesterol, 27-hydroxycholesterol, and cholesterol appear to be sensitive biomarkers for the evaluation of MCI and AD.

Low-frequency transcranial magnetic stimulation is beneficial for enhancing synaptic plasticity in the aging brain.

In the aging brain, cognitive function gradually declines and causes a progressive reduction in the structural and functional plasticity of the hippocampus. Transcranial magnetic stimulation is an emerging and novel neurological and psychiatric tool used to investigate the neurobiology of cognitive function. Recent studies have demonstrated that low-frequency transcranial magnetic stimulation (≤1 Hz) ameliorates synaptic plasticity and spatial cognitive deficits in learning-impaired mice. However, the mechanisms by which this treatment improves these deficits during normal aging are still unknown. Therefore, the current study investigated the effects of transcranial magnetic stimulation on the brain-derived neurotrophic factor signal pathway, synaptic protein markers, and spatial memory behavior in the hippocampus of normal aged mice. The study also investigated the downstream regulator, Fyn kinase, and the downstream effectors, synaptophysin and growth-associated protein 43 (both synaptic markers), to determine the possible mechanisms by which transcranial magnetic stimulation regulates cognitive capacity. Transcranial magnetic stimulation with low intensity (110% average resting motor threshold intensity, 1 Hz) increased mRNA and protein levels of brain-derived neurotrophic factor, tropomyosin receptor kinase B, and Fyn in the hippocampus of aged mice. The treatment also upregulated the mRNA and protein expression of synaptophysin and growth-associated protein 43 in the hippocampus of these mice. In conclusion, brain-derived neurotrophic factor signaling may play an important role in sustaining and regulating structural synaptic plasticity induced by transcranial magnetic stimulation in the hippocampus of aging mice, and Fyn may be critical during this regulation. These responses may change the structural plasticity of the aging hippocampus, thereby improving cognitive function.

Efficient differentiation of neural stem cells induced by the rat bone marrow stromal cells.

Neural stem cells (NSCs) are valuable self-renewing cells that can maintain the capacity to differentiate into specific brain cell types. NSCs may repair and even replace the brain tissue, and ultimatley promoting the central nervous system regeneration. Therefore, it is important, for scientists and pjysicians, to study the method for efficient culture and differentiation of NSCs. Our previous study demonstrated that Bone Marrow Stromal Cells (BMSCs) can directly regulate the differentiation of NSCs into neurons, and soluble molecules excreted by BMSCs played a key role in this process. Hereby, we further identified the BMSCs-induced neurons could form the synapses, convey dopamine and express voltage-depend and receptor-depend calcium channels. Moreover, the extracellular signal-regulated protein kinase ERK1/2 pathway was founded to be involved in the process of neuron differentiation and proliferation by the in vitro experiments. Finally, by using protein array, we, for the first time, found that the cytokine-induced neutrophil chemoattractant-3 (CINC-3, a small molecule cytokine) can promote the leukocytes invasion into the inflammation site, and have the ability to induce mesencephal NSCs into neurons. Consequently, these positive findings suggested that our BMSCs-induced culture system could provide a useful tool to investigate the molecular mechanisms of neural differentiation of NSCs, which may be benifical for neurodegenerative diseases in the near future.

[Changes of heme oxygenase-1 expression in the nigrostriatal system of MPTP-treated SAMP8 mouse].

AIM: To explore the relationship between the expression of hemeoxygenase-1 (HO-1) and the dopaminergic system impairment in MPTP-treated SAMP8 mice. METHODS: 6-month-old male SAMP8 mice received MPTP (20 mg/kg) subcutaneous injection at 2-h intervals for 4 times, and the control group was treated with an equal volume of normal saline. Mice were sacrificed at 6 h, 24 h, 3 d and 8 d after the first injection for the detection of the changes of tyrosine hydroxylase (TH) and HO-1 in the nigrostriatal system by immunohistochemistry and Western blotting. RESULTS: TH-positive neuronal loss was visible at 6 h (14.23%, P<0.05), 24 h (23.85%, P<0.01), 3 d (36.77%, P<0.001), and 8 d (45.90%, P<0.001), and the significant progression of dopaminergic neuronal loss occurred most prominently in the MPTP group from 24 h to 3 d (24 h vs 3 d, P<0.05). There was a significant decrease of striatal TH immunoreactive cells in the MPTP group (P<0.05). Additionally, HO-1 positive cells were detected in striatum just only at 3 d, with the increase of HO-1 protein expression in MPTP groups. Western blot analysis showed no change of HO-1 protein levels in the midbrain after MPTP treatment compared to those of the normal saline group. CONCLUSION: MPTP caused the loss of dopaminergic neuron number and the decrease of TH protein levels in SAMP8 mice. The up-regulation of HO-1 was ephemeral, and its effects related with Parkinson's disease was limited in this study.

[Neurotoxicity effect of MPP+ on primary cultured mesencephalon neurons of SAMP8 mouse].

AIM: To investigate the effect of MPP+ on the midbrain neurons of SAMP8 mouse in vitro, which provide the cell model for PD study. METHODS: SAMP8 mouse primary midbrain cell cultures were obtained from mice within 1 day after birth. On the sixth day after culturing, the cultures were treated with 100 micromol/L of MPP+ for 6 hours, 9 hours, 12 hours and 24 hours, then the cells were fixed and followed by immunofluorescence or Western blot analysis for the expressed location and the level of TH respectively. RESULTS: MPP+ led to the morphological changes of primary cultured midbrain neurons of SAMP8 mouse. The neurons had intact morphous with strong immunoreactivity, many and long dendrites in control group. Compared with the control group, the neurons had fewer and thinness dendrites with weak immunoreactivity in MPP+ group. MPP+ significantly decreased midbrain neurons numbers with marked decrease of TH protein levels after its treatment for 9 h. CONCLUSION: MPP+ had neurotoxicity effect on primary cultured midbrain neurons of SAMP8 mouse. Since MPP+ led to marked decrease of neuronal numbers and TH protein levels, it is suggested that MPP+ can cause the DA neuronal degeneration in primary midbrain cell cultures.

Microglial activation and age-related dopaminergic neurodegeneration in MPTP-treated SAMP8 mice.

Senescence-accelerated mouse prone 8 (SAMP8) has an early onset of senility and a shorter life span, providing with cognitive impairment. Contrasted with C57BL/6 mouse, which is most commonly used in the study of Parkinson's disease (PD), SAMP8 needs shorter period of breeding and might be good candidate for the investigation of cognitive impairment in PD. Studies had shown the increase of sensibility to 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) with aging in C57BL/6 mouse. However, the sensitivity of MPTP neurotoxicity depends on the strains of animal and the exact mechanisms of the progression of PD promoted by aging is lack of consensus. Here, we showed after MPTP injection, the spontaneous activity of both young (3-month-old) and old (6-month-old) SAMP8 decreased dramatically, and the old mice required longer recovery time. Immunohistochemical and immunoblot analysis revealed that old mice displayed significant reductions in the dopaminergic neuron numbers and tyrosine hydroxylase (TH) protein. Microglia protein (CD11b) in the striatum of old mice increased more pronouncedly than that in the young mice from 24 h to 3 days. Inducible nitric oxide synthase (iNOS) in the striatum remarkably increased, however, no discernible difference between the two groups was found. These results suggested that the sensibility to MPTP increased with aging in SAMP8. A greater increase of microglial activation in old mice may be a possible mechanism to explain how advancing age predisposes the dopamine system to parkinsonism. The MPTP-SAMP8 model will start a new consideration for the study of PD.

Neuroprotective effects of enriched environment in MPTP-treated SAMP8 mice.

Neuroprotective effects of enriched environment (EE) have been well established. Recent study suggests that exposure to EE can protect dopaminergic neurons against MPTP-induced Parkinsonism. After 64 female SAMP8 mice were reared in EE and standard environment (SE) for 3 months, the effects of EE and SE were compared on behavioural change, tyrosine hydroxylase (TH) immunoreaction positive neuron and dopaminetransporter (DAT) expression in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-treated SAMP8. EE mice showed decreased spontaneous activity compared with SE mice. But EE+MPTP mice showed less decreased spontaneous activity compared with SE+MPTP mice. Otherwise, EE mice showed increased percentage of entries into the open arms and percentage of time spent in the open arms. Furthermore, EE mice demonstrated reduced neurotoxicity, with less decreased TH mRNA and protein expression in Substantia Nigra (SN) after MPTP administration compared with SE mice. SE mice showed a 53.77% loss of TH-positive neurons, whereas EE mice only showed a 42.28% loss. Moreover, EE mice showed decreased DAT mRNA and protein expression compared with SE mice. These data demonstrate that EE can protect dopaminergic neurons against MPTP-induced neuronal damage, which suggest that the probability of developing Parkinson's disease (PD) may be related to life environment.