Ameliorative effect of SIRT1 in postpartum depression mediated by upregulation of the glucocorticoid receptor.

Recent evidence has confirmed the association of glucocorticoid receptor (GR) gene variants with the "stress" endocrine axis in postpartum depression (PPD). Sirtuin 1(SIRT1) is an NAD+-dependent histone deacetylase and transcriptional enhancer of GR. However, to date, the function of the SIRT1 gene in the regulation of GR expression in PPD remains to be fully determined. A hormone-stimulated pregnancy (HSP) and subsequent "postpartum" withdrawal of estrogen was employed to mimic the fluctuations in estradiol associated with pregnancy and postpartum. We confirmed that estradiol benzoate withdrawal (EW)-rats displayed depression- and anxiety-like behaviors. These behavioral dysfunctions are associated with attenuated expression of SIRT1 and GR in the hippocampus. To assess the role of SIRT1, as well as its regulatory target directly, a selective SIRT1 activator (SRT2104) was infused into the hippocampus of EW-rats. We found that pharmacological activation of hippocampal SIRT1 blocks the development of depression-related, but not anxiety-related, phenotypes of PPD. In addition, the activation of SIRT1 leads to an increase in hippocampal GR expression in EW-rats. We further confirmed that SIRT1 physically interacts with GR in a glucocorticoid-dependent manner. Taken together, our results suggest that neuropathology in PPD is caused, at least in part, by the inhibition of the SIRT1-GR signaling pathway. Elevating SIRT1 levels, either pharmacologically or through other means, could represent a therapeutic strategy for PPD.

PGC-1α reduces Amyloid-ß deposition in Alzheimer's disease: Effect of increased VDR expression.

Amyloid-ß (Aß) is the core component of amyloid plaques of Alzheimer's disease (AD). Recent evidence has confirmed that Aß triggers neurodegeneration by dramatically suppressing vitamin D receptor (VDR) expression. Thus far, the onset mechanisms and means of preventing AD are largely unknown. Perioxisome proliferator-activated receptor-γ coactivator (PGC-1α), as a transcriptional coactivator of VDR could protect cells against oxidative stress. Thus, upregulation of PGC-1α is a candidate therapeutic strategy for AD. To investigate the effect of PGC-1α in AD, and to illuminate the precise involvement of VDR in the neuroprotective strategy, the varies of molecular of PGC-1α and VDR were studied in APP/PS-1 double transgenic (2xTg-AD) mice at 6 months of age, significant reduction in the expression of PGC-1α and VDR was found in their hippocampus and the cortex. Besides, a specific mouse line, Dlx5/6-Cre:PGC-1αfl/fl in which the PGC-1α deficiency was limited to the hippocampus and the cortex, was used to study the target intervention of PGC-1α, decreased expression of VDR and increased oxidative damage were observed in AD-related brain regions by PGC-1α deficiency. To explore the function and therapeutic strategy of PGC-1α in AD, an adeno-associated virus (AAV) was used to induce PGC-1α overexpressed in the hippocampus of 2xTg-AD mice. Overexpressed PGC-1α results in a remarkable increase in the levels of VDR associated with a significant reduction in the expression of Aß plaques and of 8-oxo-dG in 2xTg-AD mice. These data may have ramifications for neuroprotective strategies targeting overexpression of PGC-1α in Alzheimer's disease.

Asiatic acid, a pentacyclic triterpene in Centella asiatica, attenuates glutamate-induced cognitive deficits in mice and apoptosis in SH-SY5Y cells.

AIM: To investigate whether asiatic acid (AA), a pentacyclic triterpene in Centella asiatica, exerted neuroprotective effects in vitro and in vivo, and to determine the underlying mechanisms. METHODS: Human neuroblastoma SH-SY5Y cells were used for in vitro study. Cell viability was determined with the MTT assay. Hoechst 33342 staining and flow cytometry were used to examine the apoptosis. The mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were measured using fluorescent dye. PGC-1α and Sirt1 levels were examined using Western blotting. Neonatal mice were given monosodium glutamate (2.5 mg/g) subcutaneously at the neck from postnatal day (PD) 7 to 13, and orally administered with AA on PD 14 daily for 30 d. The learning and memory of the mice were evaluated with the Morris water maze test. HE staining was used to analyze the pyramidal layer structure in the CA1 and CA3 regions. RESULTS: Pretreatment of SH-SY5Y cells with AA (0.1-100 nmol/L) attenuated toxicity induced by 10 mmol/L glutamate in a concentration-dependent manner. AA 10 nmol/L significantly decreased apoptotic cell death and reduced reactive oxygen species (ROS), stabilized the mitochondrial membrane potential (MMP), and promoted the expression of PGC-1α and Sirt1. In the mice models, oral administration of AA (100 mg/kg) significantly attenuated cognitive deficits in the Morris water maze test, and restored lipid peroxidation and glutathione and the activity of SOD in the hippocampus and cortex to the control levels. AA (50 and 100 mg/kg) also attenuated neuronal damage of the pyramidal layer in the CA1 and CA3 regions. CONCLUSION: AA attenuates glutamate-induced cognitive deficits of mice and protects SH-SY5Y cells against glutamate-induced apoptosis in vitro.

Differential effects of overexpression of wild-type and mutant human alpha-synuclein on MPP+-induced neurotoxicity in PC12 cells.

The genetic background and the pathogenesis of familial Parkinson's disease (PD) have not been fully elucidated. Two missense mutations in the alpha-synuclein gene, A30P and A53T, have been linked to familial PD. Increasing evidence suggests the involvement of alpha-synuclein, the dopamine transporter (DAT), and neurotoxins in the pathogenesis of PD, but their relationships to the death of nigral cells remains poorly understood. In the present study, we used the PC12 cell line, a well recognized model of the nigral cell, to investigate the effects of overexpression of wild-type (WT) and mutant human alpha-synuclein on MPP+-induced neurotoxicity. We found that overexpression of mutant alpha-synuclein enhanced the toxicity of MPP+ to PC12 cells and elevated intracellular reactive oxygen species (ROS) levels, whereas overexpression of WT alpha-synuclein protected PC12 cells against MPP+ toxicity and lowered ROS levels. Furthermore, assays of 131I-FP-beta-CIT binding with DAT membrane fractions showed that WT and mutant alpha-synuclein had different effects on the expression of DAT on the cell membrane following exposure to MPP+. WT alpha-synuclein reduced the toxic effect of MPP+ by facilitating DAT internalization, while both A30P and A53T alpha-synuclein aggravated the toxic effect of MPP+ by reducing DAT internalization. These data indicate that alpha-synuclein regulates ROS levels and DAT surface expression in dopaminergic neurons, and thus changes the response of these cells to MPP+.

[Construction of eukaryotic expression vector containing hSNCA gene and its pathogenic mutants and its expression in PC12 cells].

AIM: To construct recombinant eukaryotic expression vectors pEGFP-C3-SNCA containing human wild-type (WT) and pathogenic mutations A30P, A53T alpha-synuclein (SNCA), and to obtain monoclonal PC12 cell lines overexpressing human wild-type and pathogenic mutations A30P, A53T alpha-synuclein by stable transfection. METHODS: Human wild type SNCA gene was cloned by using RT-PCR. By T-A extension cloning, the gene was ligated with T-vector and sequenced. Based on it, the recombinant eukaryotic expression vectors containing wild type SNCA synonymous mutation or its G88C (Ala30Pro) and G209A (Ala53Thr) pathogenic mutation were constructed by site-directed mutagensis using primer variance in mononucleotide. And different recombinant plasmids pEGFP-C3-SNCA were identified with PCR, restriction enzyme digestion and DNA sequencing. PC12 cells were transfected with different recombinant plasmids pEGFP-C3-SNCA by liposome transfection method, screened with G418, and subcloned by limited dilution method to obtain different monoclonal PC12 cell lines stably over-expressing human wild-type, A30P or A53T alpha-synuclein, respectively, and PC12 cells stably transfected with pEGFP-C3 were used as control group. These monoclonal PC12 cell lines were identified with RT-PCR, Western blot and fluorescence microscopy. RESULTS: According to result of PCR, the double digestion and gene sequencing, it was comfirmed that recombinant eukaryotic expression vectors containing wild type SNCA synonymous mutation or its Ala30Pro and Ala53Thr pathogenic mutation had been constructed successfully. By means of RT-PCR, Western blot and fluorescence microscope, it was comfirmed that objective gene sequences in PC12 cells were stably over-expressed. CONCLUSION: The recombinant pEGFP-C3-SNCA vectors containing wild type SNCA synonymous mutation or its Ala30Pro and Ala53Thr pathogenic mutations had been constructed successfully. The transfected monoclonal PC12 cells are obtained in which three SNCA gene isoforms had stable expression.