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Stress is an important risk factor of Alzheimer's disease (AD). It has been evidenced that stress could induce tau phosphorylation and increase tau insolubility in brain; however, little is known about the interactional effect of stress with aging on tauopathy. Therefore, we explored the effects of aging on stress-induced tauopathy and the potential mechanism in mouse model of chronic restraint stress (CRS). Here we found that in general, the level of phosphorylated tau (P-tau) was higher in brain of middle-aged mice than that in adult mice under physiological conditions. CRS-induced tau phosphorylation and its insolubility were more prominent in middle-aged mice. The increase of AT8-labeled insoluble P-tau was dramatic in middle-aged mice, which was highly ubiquitinated but did not form PHF structures. The levels of chaperones were relatively lower in middle-aged mice brain; CRS further reduced the expression, especially for HDJ2/HSP40. CRS also suppressed the expression of Pin1, the peptidylprolyl cis/trans isomerase, in middle-aged mice but not in adult mice. Downregulation of HSP40 or Pin1 caused an increase of transfected extraneous tau in 293 cells. Rosmarinic acid (RA) could effectively suppress the elevation of P-tau and insoluble P-tau formation induced by CRS, and reversed the abnormal changes of chaperones and Pin1 particularly in middle-aged mice. Taken together, our findings provided evidence that aging could be a promoting factor in stress-induced tauopathy, which was relevant with malregulation of chaperones and Pin1, and RA might be a promising beneficial agent for stress-induced tauopathy. [ABSTRACT FROM AUTHOR]

Abstract: Huntington’s disease (HD) and other polyglutamine (polyQ) neurodegenerative diseases are characterized by neuronal accumulation of the disease protein, suggesting that the cellular ability to handle abnormal proteins is compromised. As a multi-subunit protein localized in the mitochondria of eukaryotic cells, the F0F1-ATP synthase α belongs to the family of stress proteins HSP60. Currently, mounting evidences indicate F0F1-ATP synthase α may play a role in neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). Recently, ATP synthase α was reported to have protective and therapeutic roles in primary cardiacmyocytes of iron-overloaded rats by lowering ROS production. However, little is understood about the role of ATP synthase α in cell death and neurodegeneration. Here, we demonstrate that overexpression of ATP synthase α suppresses huntingtin (htt) polyQ aggregation and toxicity in transfected SH-SY5Y cell lines. Overexpression of ATP synthase α is able to protect cell death caused by polyglutamine-expanded htt. Transient overexpression of ATP synthase α suppresses the aggregate formation by estimation of polyQ aggregation, Western blot analysis, and filter trap assay (FTA) in transfected SH-SY5Y cells. These results indicated that ATP synthase α has a strong inhibitory effect on polyglutamine aggregate formation and toxicity in vitro, and suggest a novel neuroprotective role of ATP synthase α. [Copyright &y& Elsevier]

Stress is an important risk factor of Alzheimer's disease (AD). It has been evidenced that stress could induce tau phosphorylation and increase tau insolubility in brain; however, little is known about the interactional effect of stress with aging on tauopathy. Therefore, we explored the effects of aging on stress-induced tauopathy and the potential mechanism in mouse model of chronic restraint stress (CRS). Here we found that in general, the level of phosphorylated tau (P-tau) was higher in brain of middle-aged mice than that in adult mice under physiological conditions. CRS-induced tau phosphorylation and its insolubility were more prominent in middle-aged mice. The increase of AT8-labeled insoluble P-tau was dramatic in middle-aged mice, which was highly ubiquitinated but did not form PHF structures. The levels of chaperones were relatively lower in middle-aged mice brain; CRS further reduced the expression, especially for HDJ2/HSP40. CRS also suppressed the expression of Pin1, the peptidylprolyl cis/trans isomerase, in middle-aged mice but not in adult mice. Downregulation of HSP40 or Pin1 caused an increase of transfected extraneous tau in 293 cells. Rosmarinic acid (RA) could effectively suppress the elevation of P-tau and insoluble P-tau formation induced by CRS, and reversed the abnormal changes of chaperones and Pin1 particularly in middle-aged mice. Taken together, our findings provided evidence that aging could be a promoting factor in stress-induced tauopathy, which was relevant with malregulation of chaperones and Pin1, and RA might be a promising beneficial agent for stress-induced tauopathy.

To investigate the murine double minute 2 (MDM2) localization and expression pattern in brain, immunohistochemistry, immunofluorescent staining and immunoblotting methods were used to analyze it in brains of Kunming mice during postnatal development, in brains of adult SD rats and in primarily cultured neurons. The distribution of MDM2 and markers of axon initial segment (AIS) was analyzed by double immunolabeling. In addition, Nutlin-3, a MDM2 antagonist, was injected into hippocampus to analyze the effect on the distribution of MDM2 and AIS protein Nav1.6 in AIS. The results showed that the dynamic expression patterns of MDM2 protein in cerebral cortex and hippocampus of Kunming mice after birth were different. However, it was similar that MDM2 was gradually enriched to AIS during postnatal development, especially after postnatal day 7. The MDM2 in AIS was also observed in different brain regions of adult SD rat brain and in primarily cultured neurons, where MDM2 was colocalized with AIS markers such as AnkG and Nav1.6. In addition, hippocampal injection of Nutlin-3 could induce the loss of the characteristic distribution of MDM2 in AIS. Moreover, Nutlin-3 not only caused a decrease of Nav1.6 distributing in AIS, but also disrupted the polarized distribution of MAP2 in neurons. These results indicate that MDM2 can be enriched at the AIS of adult rodent brain, which might play a role in regulation of the maintenance of AIS function and neuronal polarity.

Small ubiquitin-related modifiers (SUMOs) belong to an important class of ubiquitin like proteins. SUMOylation is a post-translational modification process that regulates the functional properties of many proteins, among which are several proteins implicated in neurodegenerative diseases. This study was aimed to investigate the changes of SUMO-1 expression and modification, and the relationship between SUMO-1 and Alzheimer's disease (AD) pathology in APP/PS1 transgenic AD mice. Using Western blot, co-immunoprecipitation and immunofluorescent staining methods, the SUMO-1 expression and modification and its relation to tau, amyloid precursor protein (APP) and β-amyloid protein (Aβ) in the 12-month-old APP/PS1 transgenic AD mice were analyzed. The results showed that: (1) Compared with the normal wild-type mice, the expression and modification of SUMO-1 increased in brain of AD mice, which was accompanied by an increase of ubiquitination; (2) In RIPA soluble protein fraction of cerebral cortex, co-immunoprecipitation analysis showed tau SUMOylated by SUMO-1 increased in AD mice, however, AT8 antibody labeled phosphorylated tau was less SUMOylated whereas PS422 antibody labeled phosphorylated tau was similar to control mice; (3) Double immunofluorescent staining showed that SUMO-1 could distributed in amyloid plaques, appearing that some of SUMO-1 diffused in centre of some plaques and some of SUMO-1 co-localized with AT8 labeled phosphorylated tau forming punctate aggregates around amyloid plaques which was concerned as dystrophic neurites, however, less Aβ, APP and PS422 labeled phosphorylated tau were found co-localized with SUMO-1. These results suggest that SUMO-1 expression and modification increase abnormally in transgenic AD mice, which may participate in modulation of the formation of senile plaques and dystrophic neurites.

The ATP synthase is known to play important roles in ATP generation and proton translocation within mitochondria. Here, we now provide evidence showing the presence of functional ecto-ATP synthase on the neuronal surface. Immunoblotting revealed that the α, β subunits of ATP synthase F1 portion are present in isolated fractions of plasma membrane and biotin-labelled surface protein from primary cultured neurons; the surface distribution of α, β subunits was also confirmed by immunofluorescence staining. Moreover, α and β subunits were also found in fractions of plasma membrane and lipid rafts isolated from rat brain, and flow cytometry analysis showed α subunits on the surface of acutely isolated brain cells. Activity assays showed that the extracellular ATP generation of cultured neurons could be compromised by α, β subunit antibodies and ATP synthase inhibitors. pH(i) (intracellular pH) analysis demonstrated that at low extracellular pH, α or β subunit antibodies decreased pHi of primary cultured neurons. Therefore, ATP synthase on the surface of neurons may be involved in the machineries of extracellular ATP generation and pH(i) homoeostasis.

Huntington's disease (HD) and other polyglutamine (polyQ) neurodegenerative diseases are characterized by neuronal accumulation of the disease protein, suggesting that the cellular ability to handle abnormal proteins is compromised. As a multi-subunit protein localized in the mitochondria of eukaryotic cells, the F(0)F(1)-ATP synthase alpha belongs to the family of stress proteins HSP60. Currently, mounting evidences indicate F(0)F(1)-ATP synthase alpha may play a role in neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD). Recently, ATP synthase alpha was reported to have protective and therapeutic roles in primary cardiacmyocytes of iron-overloaded rats by lowering ROS production. However, little is understood about the role of ATP synthase alpha in cell death and neurodegeneration. Here, we demonstrate that overexpression of ATP synthase alpha suppresses huntingtin (htt) polyQ aggregation and toxicity in transfected SH-SY5Y cell lines. Overexpression of ATP synthase alpha is able to protect cell death caused by polyglutamine-expanded htt. Transient overexpression of ATP synthase alpha suppresses the aggregate formation by estimation of polyQ aggregation, Western blot analysis, and filter trap assay (FTA) in transfected SH-SY5Y cells. These results indicated that ATP synthase alpha has a strong inhibitory effect on polyglutamine aggregate formation and toxicity in vitro, and suggest a novel neuroprotective role of ATP synthase alpha.

It is recognized in recent years that activation of delta-opioid receptor (DOR) elicits neuroprotection against hypoxia and ischemia. However, the underlying mechanisms are not well understood yet. Mitochondrial dysfunction plays a key role in hypoxic neuronal injury, but the effect of DOR activation on neurons with a mitochondrial respiratory chain deficiency is poorly elucidated. In this study we tested the effects of DOR activation and inhibition on cultured cortical neurons after inhibiting mitochondrial respiratory chain with sodium azide (NaN(3)) in days 8 cultures. Neuronal injury was assessed by lactate dehydrogenase release. Changes in DOR proteins were investigated using an antibody against the N-terminus of the DOR, which recognizes the 60, 48, and 32 kDa proteins. Our main findings are that 1) delta- but not mu-opioid receptor activation reduces NaN(3)-induced neuronal damage, and this neuroprotective effect is abolished by DOR antagonist (naltrindole, NTI); 2) prolonged DOR inhibition with NTI further increases NaN(3)-induced neuronal damage; 3) NaN(3) treatment down-regulates DOR protein levels in neurons, and the 60 and 32 kDa proteins are particularly sensitive; 4) DADLE, besides activating DOR directly, also reverses the decrease of neuronal DOR protein levels induced by NaN(3), which may contribute greatly to its neuroprotective effect; 5) NTI reverses NaN(3)-induced down-regulation of DOR proteins as well, the effect of NTI amplifying NaN(3)-induced neuronal damage therefore is probably due to its inhibition on DOR activity only. In conclusion, these data suggest that DOR activation plays an important role in neuroprotection against mitochondrial respiratory chain injury.

Objective: To explore whether there are beta-amyloid protein (Abeta) binding elements in heart-beneficial recipe (HBR, a compound traditional Chinese herbal medicine), which can ameliorate the cytotoxicity of Abeta., Methods: The extract of HBR and Abeta(1-40) were co-precipitated, and the Abeta(1-40) in pellets was detected by immunoblotting. Affi-gel-Abeta(1-40) was constructed, and Affi-gel-Abeta(1-40) affinity elements from the extract of HBR were analyzed by high-performance liquid chromatography (HPLC). The assay of lactic dehydrogenase (LDH) release from the primary cultured rat cortex neurons was used to evaluate the cytotoxicity of Abeta(1-42), and the protection effects of the HBR serum and the Affi-gel-Abeta(1-40) treated HBR serum., Results: Immunoblotting examination showed Abeta(1-40) could be co-precipitated with components of HBR following co-incubation, and the amount of Abeta(1-40) within pellets decreased when the HBR extract was diluted. Abeta(1-40) affinity elements from the extract of HBR, eluted from Affi-gel-Abeta(1-40) by glycine solution (pH=2.5), could be detected by HPLC-fluorescent detector system. The analysis of LDH release showed that exposure of neurons to 5 micromol/L Abeta(1-42) for 48 h caused a significant increase of LDH release in either a serum free or 10% serum contained culture condition (P<0.01). The rat HBR serum was able to suppress Abeta(1-42) induced LDH release (P<0.05), whereas Affi-gel-Abeta(1-40) treated HBR serum still maintained the ability to attenuate Abeta(1-42) induced LDH release although the effect was somewhat decreased compared with Affi-gel treated HBR serum., Conclusion: There are Abeta affinity components in HBR, which could not increase the Abeta cytotoxicity, but might be able to inhibit the cytotoxicity of Abeta. The results implied that the exploration of Abeta affinity elements from Chinese medicinal recipe which is effective for Alzheimer disease, might be an important direction in Alzheimer disease therapeutic research area.

Recent evidence indicates that the aberrant neuronal expression of mitotic proteins in Alzheimer's disease (AD) brain may be related to AD pathological changes. To investigate whether the toxicity of beta-amyloid protein (Abeta) induces mitotic proteins expression in adult rat brain, we used immunohistochemical and integral optical density analytic method to analyze the adult rat brains, which had been injected with Abeta(25-35) into unilateral amygdala. Results showed that the levels of neurofibrillary tangle (NFT) related phosphorylated tau protein and apoptosis related protein Bax were increased in Abeta(25-35) injected rat brains, meanwhile the aberrantly expression of mitotic protein cyclin A and cyclin B1 was also detected at 7 d after operation, but the level of cyclin A decreased and cyclin B1 disappeared at 21 d. Immunofluorescence double labeling presented that cyclin B1 was partially co-localized with Bax or phosphorylated tau protein, whereas Bax and phosphorylated tau protein seldom co-localized. These results suggest that Abeta causes mitotic protein expression in adult brain neurons, which may die through apoptosis or may be affected by AD NFT-related tau phosphorylation.

To study the relationship between tau hyperphosphorylation and the function of glutamate transporter okadaic acid (OA), a protein phosphatase inhibitor, 20 ng in a 0.5 microl volume, was injected into the frontal cortex of rat brain and immunostaining was used to observe the phosphorylation of tau protein and the expression of excitatory amino acid transporter 1 (EAAT1) in the brain following the injection. The results showed that (1) the neurons in the center of the injection region displayed cytoplasmic shrinkage, swelling, nuclear pyknosis, and dislocation at the early stage, and necrosis appeared 3 d after the injection. However, most neurons in the peri-injected areas showed normal morphological characters with immuno positive reaction for AT8, a tau phosphorylated marker; (2) morphological analysis showed that tau hyperphosphorylation caused by OA treatment was mainly observed in the axons and dendrites of neuronal cells at 6 h in the cell body at 1 d, which brought about dystrophic neurites and neurofibrillary tangle (NFT)-like pathological changes; (3) the induction of glutamate transporter EAAT1 was observed in the involved areas corresponding to that with AT8 immunopositive staining, and the number of EAAT1-positive staining cells markedly increased at 12 h (P<0.01), peaked at 1 d (P<0.001), then decreased at 3 d following the injection. Combined with a confocal laser scanning microscopic analysis, double fluorescent immunostaining showed that EAAT1 positive staining appeared in neurons as well as astrocytes in the peri-injected areas of the frontal cortex. These results demonstrate that OA increases glutamate transporter EAAT1 expression in neurons while it induces tau hyperphosphorylation. However, the mechanism and significance of the induction of glutamate transporter EAAT1 expression remain to be further elucidated.