A method to study human synaptic protein-protein interactions by using flow cytometry coupled to proximity ligation assay (Syn-FlowPLA).

BACKGROUND: Synapses are highly specialized sites characterized by intricate networks of protein-protein interactions (PPIs) important to maintain healthy synapses. Therefore, mapping these networks could address unsolved questions about human cognition, synaptic plasticity, learning, and memory in physiological and pathological conditions. The limitation of analyzing synaptic interactions in living humans has led to the development of methods to isolate synaptic terminals (synaptosomes) from cryopreserved human brains. NEW METHOD: Here, we established a method to detect synaptic PPIs by applying flow cytometric proximity ligation assay (FlowPLA) to synaptosomes isolated from frozen human frontal cortex (FC) and hippocampus (HP) (Syn-FlowPLA). RESULTS: Applying this method in synaptosomes, we were able to detect the known post-synaptic interactions between distinct subtypes of N-methyl-D-aspartate glutamate receptors (NMDARs) and their anchoring postsynaptic density 95 protein (PSD95). Moreover, we detected the known pre-synaptic interactions between the SNARE complex proteins synaptosomal-associated protein of 25 kDa (SNAP25), synaptobrevin (VAMP2), and syntaxin 1a (STX1A). As a negative control, we analyzed the interaction between mitochondrial superoxide dismutase 2 (SOD2) and PSD95, which are not expected to be physically associated. COMPARISON WITH EXISTING METHODS: PPIs have been studied in vitro primarily by co-immunoprecipitation, affinity chromatography, protein-fragment complementation assays (PCAs), and flow cytometry. All these are valid approaches; however, they require more steps or combination with other techniques. PLA technology identifies PPIs with high specificity and sensitivity. CONCLUSIONS: The Syn-FlowPLA described here allows rapid analyses of PPIs, specifically within the synaptic compartment isolated from frozen autopsy specimens, achieving greater target sensitivity. Syn-FlowPLA, as presented here, is therefore a useful method to study human synaptic PPI in physiological and pathological conditions.

Tyrosine Kinase Inhibitors Reduce NMDA NR1 Subunit Expression, Nuclear Translocation, and Behavioral Pain Measures in Experimental Arthritis.

In the lumbar spinal cord dorsal horn, release of afferent nerve glutamate activates the neurons that relay information about injury pain. Here, we examined the effects of protein tyrosine kinase (PTK) inhibition on NMDA receptor NR1 subunit protein expression and subcellular localization in an acute experimental arthritis model. PTK inhibitors genistein and lavendustin A reduced cellular histological translocation of NMDA NR1 in the spinal cord occurring after the inflammatory insult and the nociceptive behavioral responses to heat. The PTK inhibitors were administered into lumbar spinal cord by microdialysis, and secondary heat hyperalgesia was determined using the Hargreaves test. NMDA NR1 cellular protein expression and nuclear translocation were determined by immunocytochemical localization with light and electron microscopy, as well as with Western blot analysis utilizing both C- and N-terminal antibodies. Genistein and lavendustin A (but not inactive lavendustin B or diadzein) effectively reduced (i) pain related behavior, (ii) NMDA NR1 subunit expression increases in spinal cord, and (iii) the shift of NR1 from a cell membrane to a nuclear localization. Genistein pre-treatment reduced these events that occur in vivo within 4 h after inflammatory insult to the knee joint with kaolin and carrageenan (k/c). Cycloheximide reduced glutamate activated upregulation of NR1 content confirming synthesis of new protein in response to the inflammatory insult. In addition to this in vivo data, genistein or staurosporin inhibited upregulation of NMDA NR1 protein and nuclear translocation in vitro after treatment of human neuroblastoma clonal cell cultures (SH-SY5Y) with glutamate or NMDA (4 h). These studies provide evidence that inflammatory activation of peripheral nerves initiates increase in NMDA NR1 in the spinal cord coincident with development of pain related behaviors through glutamate non-receptor, PTK dependent cascades.

Hippocampal stem cells promotes synaptic resistance to the dysfunctional impact of amyloid beta oligomers via secreted exosomes.

BACKGROUND: Adult hippocampal neurogenesis plays an important role in synaptic plasticity and cogntive function. We reported that higher numbers of neural stem cells (NSC) in the hippocampus of cognitively-intact individuals with high Alzheimer's disease (AD) pathology (plaques and tangles) is associated with decreased synaptic amyloid beta oligomers (Aßο), an event linked to onset of dementia in AD. While these findings suggest a link between NSC and synaptic resistance to Aßο, the involved mechanism remains to be determined. With this goal in mind, here we investigated the ability of exosomes secreted from hippocampal NSC to promote synaptic resilience to Aßo. METHODS: Exosomes isolated from media of hippocampus NSC (NSC-exo) or mature hippocampal neuronal (MN-exo) cultures were delivered intracerebroventricularly (ICV) to mice before assessment of Aßο-induced suppression of hippocampal long-term potentiation (LTP) and memory deficits. Aßο binding to synapses was assessed in cultured hippocampal neurons and on synaptosomes isolated from hippocampal slices from wild type mice and from an inducible mouse model of NSC ablation (Nestin-δ-HSV-TK mice) treated with exosomes. Expression of CaMKII and of AMPA and NMDA glutamate receptor subunits in synaptosomes was measured by western blot. Small RNA Deep sequencing was performed to identify microRNAs enriched in NSC-exo as compared to MN-exo. Mimics of select miRNAs were injected ICV. RESULTS: NSC-exo, but not MN-exo, abolished Aßo-induced suppression of LTP and subsequent memory deficits. Furthermore, in hippocampal slices and cultured neurons, NSC-exo significantly decreased Aßo binding to the synapse. Similarly, transgenic ablation of endogenous NSC increased synaptic Aßo binding, which was reversed by exogenous NSC-exo. Phosphorylation of synaptic CaMKII was increased by NSC-exo, while AMPA and NMDA receptors were not affected. Lastly, we identified a set of miRNAs enriched in NSC-exo that, when injected ICV, protected the synapses from Aßo-binding and Aßo-induced LTP inhibition. CONCLUSIONS: These results identify a novel mechanism linking NSC-exo and synaptic susceptibility to Aßo that may underscore cognitive resilience of certain individuals with increased neurogenesis in spite of AD neuropathology and unmask a novel target for the development of a new treatment concept for AD centered on promoting synaptic resilience to toxic amyloid proteins.

Peripheral adipose tissue insulin resistance alters lipid composition and function of hippocampal synapses.

Compelling evidence indicates that type 2 diabetes mellitus, insulin resistance (IR), and metabolic syndrome are often accompanied by cognitive impairment. However, the mechanistic link between these metabolic abnormalities and CNS dysfunction requires further investigations. Here, we evaluated whether adipose tissue IR and related metabolic alterations resulted in CNS changes by studying synapse lipid composition and function in the adipocyte-specific ecto-nucleotide pyrophosphate phosphodiesterase over-expressing transgenic (AtENPP1-Tg) mouse, a model characterized by white adipocyte IR, systemic IR, and ectopic fat deposition. When fed a high-fat diet, AtENPP1-Tg mice recapitulate essential features of the human metabolic syndrome, making them an ideal model to characterize peripherally induced CNS deficits. Using a combination of gas chromatography and western blot analysis, we found evidence of altered lipid composition, including decreased phospholipids and increased triglycerides (TG) and free fatty acid in hippocampal synaptosomes isolated from high-fat diet-fed AtENPP1-Tg mice. These changes were associated with impaired basal synaptic transmission at the Schaffer collaterals to hippocampal cornu ammonis 1 (CA1) synapses, decreased phosphorylation of the GluN1 glutamate receptor subunit, down-regulation of insulin receptor expression, and up-regulation of the free fatty acid receptor 1.

[Study of the effect of fluor protector on resistance to demineralization of milk beverages on enamel of primary teeth].

PURPOSE: To study the effect of fluor protector against demineralization of deciduous teeth enamel surface in milk beverages, to certify the mechanism of anti-demineralization of fluor protector, and provide laboratory basis for clinical practice of fluor protector and the prevention of infants and young children. METHODS: The enamel surfaces of 30 prepared deciduous teeth without caries were randomly divided into 3 groups. Group A was control group. Milk beverages were used in group B, and 0.1% fluor protector was used in group C. Each group included 10 specimens. All specimens were soaked in water tank at 37 degrees centigrade for 4 days, but specimens in groups B and C were soaked at intervals in 10mL yogurt drinks in water tank at 37 degrees centigrade, 9 minutes for one time and 3 times for one day. Scanning electron microscope (SEM) and spectrum analyzer were used to detect the changes in surface morphology of deciduous teeth enamel surface. The concentration values and the ratio of calcium/phosphorus were analyzed with SPSS11.5 software package. RESULTS: Enamel soaked in milk beverages had decreased Ca(2+) and P(3+) content significantly (P<0.01). Ca(2+) and P(3+) content in enamel in group C was significantly higher than in group B (P<0.01). Scanning electron microscopy showed enamel surface in group C had mild demineralization. CONCLUSIONS: Milk beverages can cause a strong acid demineralization in deciduous teeth enamel surface. The application of fluor protector in enamel surface can inhibit enamel demineralization in the milk beverages.

Percutaneous endoscopic gastrostomy/jejunostomy combined with percutaneous transhepatic biliary drainage in treating malignant biliary obstruction.

OBJECTIVE: To investigate the safety and efficacy of percutaneous endoscopic gastrostomy/jejunostomy (PEG/PEJ) combined with percutaneous transhepatic biliary drainage (PTCD) in treating malignant biliary obstruction. SUBJECTS AND METHODS: Nine patients (6 males and 3 females, average age 71.3 ± 5.5 years) with complete obstruction of the biliary tract were treated with PEG/PEJ after PTCD. The PEG/PEJ and PTCD tubes were linked outside of the abdominal wall to direct the externally drained bile back to the jejunum through the PEG/PEJ intestinal tube. Clinical symptoms and liver function were assessed following the treatment. RESULTS: The operations were successfully completed in the 9 patients within 40 min (average 35 ± 2.9 min). Clinical symptoms such as jaundice, abdominal distension, stomachache and diarrhea appeared but improved within 7 days of the operation. Serum levels of bilirubin, aspartate aminotransferase and alanine aminotransferase were reduced (p < 0.01) 4 weeks following the treatment. There were no procedural complications. CONCLUSIONS: Combined PEG/PEJ and PTCD appeared to be safe and effective in the management of malignant biliary obstruction. Further, larger-scale studies will be needed to verify findings of this report.

A peripheral neuroimmune link: glutamate agonists upregulate NMDA NR1 receptor mRNA and protein, vimentin, TNF-alpha, and RANTES in cultured human synoviocytes.

Human primary and clonal synovial cells were incubated with glutamate receptor agonists to assess their modulating influence on glutamate receptors N-methyl-d-aspartate (NMDA) NR1 and NR2 and inflammatory cytokines to determine potential for paracrine or autocrine (neurocrine) upregulation of glutamate receptors, as has been shown for bone and chondrocytes. Clonal SW982 synoviocytes constitutively express vimentin, smooth muscle actin (SMA), and NMDA NR1 and NR2. Coincubation (6 h) with glutamate agonists NMDA (5 microM), and the NMDA NR1 glycine site activator (+/-)1-aminocyclopentane-cis-1,3-dicarboxylic acid (5 muM), significantly increases cellular mRNA and protein levels of glutamate receptors, as well as increasing vimentin, SMA, tumor necrosis factor-alpha, and RANTES (regulated on activation, normal T-cell expressed and secreted), assessed qualitatively and quantitatively with nucleotide amplification, image analysis of immunocytochemical staining, fluorescein-activated cell sorting, Western blotting, and immunoassays. Human primary synovial cells harvested from patients with arthritic conditions also constitutively expressed NMDA NR1 with increases after agonist treatment. Glutamate receptor agonist-induced increases were blocked by the noncompetitive glutamate antagonist MK-801 (8 microg/ml) and NR1 blocking antibody. Coincubation with glutamate agonists and phorbol 12-myristate 13-acetate, a protein kinase C activator, significantly enhanced mean levels of TNF-alpha and RANTES in SW982 cell supernatants compared with incubation with either agent alone. Increases were diminished with protein kinase inhibitor and NR1 blocking antibody. The functional activation of glutamate receptors on human synoviocytes establishes a neurogenic cell signaling link between neurotransmitter glutamate released from nerve terminals and target cells in the joint capsule. The influence of glutamate on subsequent release of cellular proinflammatory mediators in non-neural tissue for activation of downstream immune events supports a peripheral neuroimmune link in arthritis.

Intermediate- and long-term recognition memory deficits in Tg2576 mice are reversed with acute calcineurin inhibition.

The Tg2576 transgenic mouse is an extensively characterized animal model for Alzheimer's disease (AD). Similar to AD, these mice suffer from progressive decline in several forms of declarative memory including contextual fear conditioning and novel object recognition (NOR). Recent work on this and other AD animal models suggests that initial cognitive deficits are due to synaptic dysfunction that, with the correct intervention, are fully treatable. We recently reported that acute calcineurin (CaN) inhibition with FK506 ameliorates one form of declarative memory (contextual fear conditioning) impairment in 5 months old Tg2576. This study tested whether acute CaN inhibition rescues deficits in an additional form of declarative memory, spontaneous object recognition, by employing the NOR paradigm. Furthermore, we determined whether FK506 rescue of NOR deficits depends on the retention interval employed and therefore is restricted to short-term, intermediate-term, or long-term memory (STM, ITM or LTM, respectively). In object recognition, Tg2576 are unimpaired when NOR is tested as a STM task and CaN inhibition with FK506 does not influence NOR STM performance in Tg2576 or WT mice. Tg2576 were impaired in NOR compared to WT mice when a 4 or 24h retention interval was employed to model ITM and LTM, respectively. Acute CaN inhibition prior to and during the training session reversed these deficits in Tg2576 mice with no effect on WT performance. Our findings demonstrate that aberrant CaN activity mediates object recognition deficits in 5 months old Tg2576 when NOR is employed as a test for ITM and LTM. In human AD, CaN inhibition may lead the way for therapeutics to improve declarative memory performance as demonstrated in a mouse model for AD.

Acute inhibition of calcineurin restores associative learning and memory in Tg2576 APP transgenic mice.

Misfolded amyloid beta peptide (Abeta) is a pathological hallmark of Alzheimer's disease (AD), a neurodegenerative illness characterized by cognitive deficits and neuronal loss. Transgenic mouse models of Abeta over-production indicate that Abeta-induced cognitive deficits occur in the absence of overt neuronal death, suggesting that while extensive neuronal death may be associated with later stages of the human disease, subtle physiological changes may underlie initial cognitive deficits. Therefore, identifying signaling elements involved in those Abeta-induced cognitive impairments that occur prior to loss of neurons may reveal new potential pharmacological targets. Here, we report that the enzymatic activity of calcineurin, a key protein phosphatase involved in phosphorylation-dependent kinase activity crucial for synaptic plasticity and memory function, is upregulated in the CNS of the Tg2576 animal model for Abeta over-production. Furthermore, acute treatment of Tg2576 mice with the calcineurin inhibitor FK506 (10mg/kg i.p.) improves memory function. These results indicate that calcineurin may mediate some of the cognitive effects of excess Abeta such that inhibition of calcineurin shall be further explored as a potential treatment to reverse cognitive impairments in AD.

[Ultrastructural changes of duodenal mucosas and their significance in patients with liver cirrhosis].

OBJECTIVE: To investigate the ultrastructural changes of duodenal mucosas and their significance in patients with liver cirrhosis (PLC). METHODS: Endoscopic biopsy duodenal mucosa specimens of 60 PLC and 18 healthy volunteers as controls were obtained. Ultrastructural changes of them were studied with transmission electron microscopy. These PLC were divided into groups A, B and C according to the Child-Pugh classification. The ultrastructural changes in the duodenal mucosas of each group were rated and compared with those of the other groups. PLC with and without ultrastructural changes of duodenal mucosas were divided into a positive group and a negative group. Levels of plasma Alb, TBil, PT, plasma endotoxin, and blood ammonia of the PLC were detected and compared. RESULTS: There were 20 PLC each in groups A, B, and C. Ultrastructural changes of duodenal mucosas were found in 5 PLC of group A, 9 in group B and 17 in group C. Among the 60 PLC, 52% had some changes in their duodenal mucosas. The changes included decrease and rupture of the microvilli; also karyopyknosis, karyorrhexis, widening of the gaps of the tight junction and tumefactions of mitochodrion of duodenal mucosa epithelial cells. No ultrastructural changes of duodenal mucosas were found in the control group. The rate of changes in the three Child-Pugh class groups and in the control group were 25%, 45%, 85%, 0% respectively (P < 0.01). The level of Alb of the positive group was significantly lower than that of the negative group (P < 0.01). Levels of plasma TBil, PT, endotoxin and blood ammonia of the positive group were significantly higher or longer than those of the negative group (P < 0.01). Levels of plasma Alb of the positive and negative groups were significantly lower than those of the control group (P < 0.01). Levels of TBil, PT, plasma endotoxin and blood ammonia of the positive and negative groups were significantly higher or longer than those of the control group (P < 0.01). CONCLUSION: There were ultrastructural changes of duodenal mucosas in PLC, especially in end-stage PLC. Ultrastructural changes of intestinal mucosas in the PLC may have important pathophysiological and clinical significance.