1N3R-tau induces S phase arrest in HEK293 cells.

<p><b>(A)</b> The distribution of HEK293 with six tau isoforms in sub G1 phase. <b>(B)</b> The distribution of HEK293 with six tau isoforms in G1 phase. *<i>P</i> < 0.05, compared with vector. <b>(C)</b> The distribution of HEK293 with six tau isoforms in S phase. **<i>P</i> < 0.01, compared with vector. <b>(D)</b> The distribution of HEK293 with six tau isoforms in G2/M phase. *<i>P</i> < 0.05, compared with vector.</p

1N3R-tau inhibits cell proliferation measured by BrdU incorporation.

<p>Fluorescent micrographs of eGFP and BrdU expression with six tau isoforms in N2a cells after 48 h transfection. GFP (Green), BrdU (Red). Quantification of BrdU-positive cells in eGFP expressing N2a cells with six tau isoforms after 48 h transfection. Scale bar = 50 μm. All values are standardized with vector. *<i>P</i> < 0.05, compared with vector.</p

1N3R tau induces translocation of cyclin E from nucleus to cytoplasm.

<p><b>(A)</b> N2a cells were transfected transiently with full length tau (2N4R), 1N3R tau and empty vector for 48 h, then cytoplasm and nuclear proteins were extracted and cyclin A, cyclin B1, cyclin D1, cyclin E, Cdk2, Cdk4 were detected by western blotting. DM1A and Lamin B1 represented loading and nuclear reference respectively. <b>(B)</b> Quantitative analysis of (A). All values are standardized with vector. *<i>P</i> < 0.05, compared with vector.</p

Magnesium reverses the LTP deficiency induced by ICV-STZ.

<p>Rats were divided into sham-operated control (Con), sham-operated plus 100 mg/kg magnesium control (Mg), and STZ ICV (STZ) or STZ ICV plus 100 mg/kg magnesium (STZ+Mg) groups. During the 3^rd week after ICV-STZ treatment, the hippocampal slices were prepared and an ideographic electrophysiology recording set-up with a stimulating electrode and recording electrode were placed in the CA3 and CA1 regions (A). The representative analog traces of evoked potentials before (solid line) and after (broken line) high-frequency stimulation (HFS) were recorded (B). Normalized field excitatory postsynaptic potential (fEPSP) slopes were measured in four groups (C), and the relative ratio of fEPSP increments after HFS (D) was calculated. Data were presented as means ± SD. *<i>P</i><0.05 versus the control group, <i>#P</i><0.05 versus the STZ group.</p

Intraperitoneal supplementation of magnesium rescues ICV-STZ-induced learning and memory deficits with elevation of brain magnesium level.

<p>The experiments were designed as shown in panel A. Rats were divided into six groups, i.e., sham-operated control (Con), sham-operated plus 100 mg/kg magnesium control (Mg), STZ ICV (STZ), STZ ICV plus 50 mg/kg magnesium (STZ+Mg50), STZ ICV plus 100 mg/kg magnesium (STZ+Mg100) and STZ ICV plus 200 mg/kg magnesium (STZ+Mg200) groups, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108645#s2" target="_blank">Methods</a>. During the 3^rd week after ICV-STZ treatment, the rats were trained in Morris watwe maze for six consecutive days to measure the learning capacity, and memory was tested on the 7^th day via removal of the hidden platform. The escape latencies to find the hidden platform were recorded daily (B). For the memory test, the time spent in the target quadrant (C), the swimming tracks (D) and numbers of crossings (E) in the target quadrant were calculated. The rats were sacrificed after the behavioral tests, and the magnesium levels in the cerebral cortex (F) and hippocampus (G) were measured. Data were presented as means ± SD. *<i>P</i><0.05 versus the control group, <i>#P</i><0.05 versus the STZ group.</p

Magnesium increases synaptic proteins in the hippocampus of ICV-STZ-treated rats.

<p>Rats were divided into sham operated control (Con), sham operated plus 100 mg/kg magnesium control (Mg), STZ ICV (STZ) or STZ ICV plus 100 mg/kg magnesium (STZ+Mg) groups and treated as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108645#pone-0108645-g001" target="_blank">Figure 1A</a>. The levels of presynaptic and postsynaptic proteins in whole hippocampal extracts were measured using Western blotting (A) and quantitative analysis (B). The expression of synapsin I, PSD95, PSD93, GluR1 and GluR2 was significantly reduced in the hippocampus of ICV-STZ-treated rats, while supplementation of magnesium restored the levels. Levels of synaptophysin, NR2A and NR2B were not significantly changed. Data were presented as means ± SD. *<i>P</i><0.05 versus the control group, <i>#P</i><0.05 versus the STZ group.</p

Magnesium Protects Cognitive Functions and Synaptic Plasticity in Streptozotocin-Induced Sporadic Alzheimer’s Model

<div><p>Alzheimer’s disease (AD) is characterized by profound synapse loss and impairments of learning and memory. Magnesium affects many biochemical mechanisms that are vital for neuronal properties and synaptic plasticity. Recent studies have demonstrated that the serum and brain magnesium levels are decreased in AD patients; however, the exact role of magnesium in AD pathogenesis remains unclear. Here, we found that the intraperitoneal administration of magnesium sulfate increased the brain magnesium levels and protected learning and memory capacities in streptozotocin-induced sporadic AD model rats. We also found that magnesium sulfate reversed impairments in long-term potentiation (LTP), dendritic abnormalities, and the impaired recruitment of synaptic proteins. Magnesium sulfate treatment also decreased tau hyperphosphorylation by increasing the inhibitory phosphorylation of GSK-3β at serine 9, thereby increasing the activity of Akt at Ser473 and PI3K at Tyr458/199, and improving insulin sensitivity. We conclude that magnesium treatment protects cognitive function and synaptic plasticity by inhibiting GSK-3β in sporadic AD model rats, which suggests a potential role for magnesium in AD therapy.</p></div

Magnesium suppresses GSK-3β with no effect on PP2A in the hippocampus of ICV-STZ-treated rats.

<p>Rats were divided into sham-operated control (Con), sham-operated plus 100 mg/kg magnesium control (Mg), STZ ICV (STZ) or STZ ICV plus 100 mg/kg magnesium (STZ+Mg) groups and treated as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108645#pone-0108645-g001" target="_blank">Figure 1A</a>. The total GSK-3β, GSK-3β (Ser9), GSK-3β (Tyr216), total PP2A and PP2A (pY307) levels in whole hippocampus extracts were measured using Western blotting (A, C) and quantitative analysis (B, D). The level of p-GSK-3β (Ser9) was significantly decreased in ICV-STZ-treated rats, and magnesium supplementation restored the levels. No alterations in total GSK-3β, p-GSK-3β (Y216), total PP2A and PP2A (pY307) levels were detected in these groups. Data were presented as means ± SD. *<i>P</i><0.05 versus the control group, <i>#P</i><0.05 versus the STZ group.</p

Magnesium reverses ICV-STZ-induced dendritic spines and synapse impairments.

<p>Rats were divided into sham-operated control (Con), sham-operated plus 100 mg/kg magnesium control (Mg), STZ ICV (STZ) or STZ ICV plus 100 mg/kg magnesium (STZ+Mg) groups and treated as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108645#pone-0108645-g001" target="_blank">Figure 1A</a>. The representative morphology alterations of neurons in the hippocampal CA1 regions were displayed by Golgi staining. Representative photomicrographs of primary dendrites in the hippocampal CA1 region were shown (A). Quantification of dendrite number (B) and mushroom-type dendrites (C) were calculated. The number of dendritic branches and mushroom percentage in the hippocampus of ICV-STZ-treated rats decreased markedly, and supplement of magnesium almost fully reversed the dendritic complexity. Data were presented as means ± SD. *<i>P</i><0.05 versus the control group, <i>#P</i><0.05 versus the STZ group.</p

Magnesium stimulates the activity of Akt and PI3K in the hippocampus of ICV-STZ-treated rats.

<p>Rats were divided into sham-operated control (Con), sham-operated plus 100 mg/kg magnesium control (Mg), STZ ICV (STZ) or STZ ICV plus 100 mg/kg magnesium (STZ+Mg) groups and treated as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108645#pone-0108645-g001" target="_blank">Figure 1A</a>. The total Akt, Akt (Thr308), Akt (Ser473), total PI3K and PI3K (Tyr458/199) levels in whole hippocampus extracts were measured using Western blotting (A, C) and quantitative analysis (B, D), respectively. The p-Akt (Ser473) and p-PI3K (Tyr458/199) levels decreased significantly in ICV-STZ-treated rats, and these changes were fully restored by magnesium supplement. There was no change in total Akt, Akt (Thr308) and PI3K. Data were presented as means ± SD. *<i>P</i><0.05 versus the control group, <i>#P</i><0.05 versus the STZ group.</p