Role of Sialidase in Long-Term Potentiation at Mossy Fiber-CA3 Synapses and Hippocampus-Dependent Spatial Memory.

Sialic acid bound to glycans in glycolipids and glycoproteins is essential for synaptic plasticity and memory. Sialidase (EC 3.2.1.18), which has 4 isozymes including Neu1, Neu2, Neu3 and Neu4, regulates the sialylation level of glycans by removing sialic acid from sialylglycoconjugate. In the present study, we investigated the distribution of sialidase activity in rat hippocampus and the role of sialidase in hippocampal memory processing. We previously developed a highly sensitive histochemical imaging probe for sialidase activity, BTP3-Neu5Ac. BTP3-Neu5Ac was cleaved efficiently by rat Neu2 and Neu4 at pH 7.3 and by Neu1 and Neu3 at pH 4.6. When a rat hippocampal acute slice was stained with BTP3-Neu5Ac at pH 7.3, mossy fiber terminal fields showed relatively intense sialidase activity. Thus, the role of sialidase in the synaptic plasticity was investigated at mossy fiber terminal fields. The long-term potentiation (LTP) at mossy fiber-CA3 pyramidal cell synapses was impaired by 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA), a sialidase inhibitor. DANA also failed to decrease paired-pulse facilitation after LTP induction. We also investigated the role of sialidase in hippocampus-dependent spatial memory by using the Morris water maze. The escape latency time to reach the platform was prolonged by DANA injection into the hippocampal CA3 region or by knockdown of Neu4 without affecting motility. The results show that the regulation of sialyl signaling by Neu4 is involved in hippocampal memory processing

Differences and relationships between weightbearing and non-weightbearing dorsiflexion range of motion in foot and ankle injuries

Abstract Background This study aimed to: (1) identify assessment methods that can detect greater ankle dorsiflexion range of motion (DROM) limitation in the injured limb; (2) determine whether differences in weightbearing measurements exist even in the absence of DROM limitations in the injured limb according to non-weightbearing measurements; and (3) examine associations between DROM in the weightbearing and non-weightbearing positions and compare those between a patient group with foot and ankle injuries and a healthy group. Methods Eighty-two patients with foot and ankle injuries (e.g., fractures, ligament and tendon injuries) and 49 healthy individuals participated in this study. Non-weightbearing DROM was measured under two different conditions: prone position with knee extended and prone position with knee flexed. Weightbearing DROM was measured as the tibia inclination angle (weightbearing angle) and distance between the big toe and wall (weightbearing distance) at maximum dorsiflexion. The effects of side (injured, uninjured) and measurement method on DROM in the patient groups were assessed using two-way repeated-measures ANOVA and t-tests. Pearson correlations between measurements were assessed. In addition, we analyzed whether patients without non-weightbearing DROM limitation (≤ 3 degrees) showed limitations in weightbearing DROM using t-tests with Bonferroni correction. Results DROM in patient groups differed significantly between legs with all measurement methods (all: P < 0.001), with the largest effect size for weightbearing angle (d = 0.95). Patients without non-weightbearing DROM limitation (n = 37) displayed significantly smaller weightbearing angle and weightbearing distance on the injured side than on the uninjured side (P < 0.001 each), with large effect sizes (d = 0.97–1.06). Correlation coefficients between DROM in non-weightbearing and weightbearing positions were very weak (R = 0.17, P = 0.123) to moderate (R = 0.26–0.49, P < 0.05) for the patient group, and moderate to strong for the healthy group (R = 0.51–0.69, P < 0.05). Conclusions DROM limitations due to foot and ankle injuries may be overlooked if measurements are only taken in the non-weightbearing position and should also be measured in the weightbearing position. Furthermore, DROM measurements in non-weightbearing and weightbearing positions may assess different characteristics, particularly in patient group. Level of evidence Level IV, cross-sectional study

Impairment of hippocampus-dependent memory by knockdown of Neu4.

<p>(A) <i>Neu4</i> mRNA levels in the hippocampus and cerebral cortex were measured after continuous injection of Neu4-targeting (<i>n</i> = 9) or non-targeting siRNA (<i>n</i> = 10) into the dorsal third ventricle for 7 days. *<i>P</i> < 0.05 vs. non-targeting siRNA (unpaired t<i>-test</i>). (B) Morris water maze test was performed under the effect of Neu4-targeting siRNA (<i>n</i> = 8), non-targeting (<i>n</i> = 10) or vehicle (<i>n</i> = 10). Latency time at the 1^st trial on day 2 was statistically analysed (one-way ANOVA with Newman-Keuls Multiple Comparison Test, <i>F</i>_2,25 = 4.77). (C) Motility was assessed in the first trial of the training session on day 1.</p

Difference in BTP3-Neu5Ac cleavage ability among sialidase isozymes.

<p>(A) Schematic staining mechanism of BTP3-Neu5Ac. (B and C) C-terminal Myc-tagged rat sialidase isozymes, Neu1, Neu2, Neu3 and Neu4, were expressed in C6 rat glioma cells. Lysate of sialidase isozyme-transfected cells (+) or mock-transfected cells (-, background level) was incubated in ACSF (pH 7.3, <i>n</i> = 4) (B) or sodium acetate buffer (pH 4.6, <i>n</i> = 4) (C) containing 200 μM BTP3-Neu5Ac. Then, fluorescence (green) was observed on a PVDF membrane under UV light (upper images). The amount of hydroryzed-BTP3 is shown as a bar graph after subtraction of each background level.</p

Impairment of mossy fiber-CA3 LTP by a sialidase inhibitor.

<p>(A) LTP was induced at 30 min by tetanic stimulation in the presence of 50 μM AP5. DANA (300 μM), 1 μM DCG-IV and 10 μM CNQX were applied at 10–70, 70–80 and 80–90 min, respectively. <i>n</i> = 7 each. The inset shows representative fEPSPs recorded at the times indicated by numbers in the graph. Scale bars, 0.1 mV and 5 ms. (B) The magnitudes of LTP were averaged at 60–65 min. *<i>P</i> < 0.05 (unpaired <i>t</i>-test with Welch's correction). (C) fEPSPs were averaged at 13–18 min (before LTP), 25–30 min (before LTP with AP5) and 60–65 min (during LTP) in the vehicle (ACSF)-treated group and at 5–10 min (before LTP), 13–18 min (before LTP with DANA), 25–30 min (before LTP with DANA+AP5) and 60–65 min (during LTP with DANA) in the DANA-treated group. **<i>P</i> < 0.01, ***<i>P</i> < 0.001 (repeated measures ANOVA with Bonferroni's multiple comparison test) (D) PPF was measured at the times indicated by squares (white: ACSF, orange: DANA) and roman numerals in panel (A). *<i>P</i> < 0.05 (paired <i>t</i>-test).</p

Sialidase activity imaging in rat hippocampus.

<p>Rat hippocampus and the surrounding region was stained with 100 μM BTP3-Neu5Ac (A) or with 100 μM BTP3-Neu5Ac +10 mM DANA (B) at pH 7.3. cc: corpus callosum, fi: hippocampal fimbria. Arrows, CA3 striatum lucidum; arrowheads, hilus. Scale bar, 0.5 mm.</p

Filopodium-derived vesicles produced by MIM enhance the migration of recipient cells

Extracellular vesicles (EVs) are classified as large EVs (l-EVs, or microvesicles) and small EVs (s-EVs, or exosomes). S-EVs are thought to be generated from endosomes through a process that mainly depends on the ESCRT protein complex, including ALG-2 interacting protein X (ALIX). However, the mechanisms of l-EV generation from the plasma membrane have not been identified. Membrane curvatures are generated by the bin-amphiphysin-rvs (BAR) family proteins, among which the inverse BAR (I-BAR) proteins are involved in filopodial protrusions. Here, we show that the I-BAR proteins, including missing in metastasis (MIM), generate l-EVs by scission of filopodia. Interestingly, MIM-containing l-EV production was promoted by in vivo equivalent external forces and by the suppression of ALIX, suggesting an alternative mechanism of vesicle formation to s-EVs. The MIM-dependent l-EVs contained lysophospholipids and proteins, including IRS4 and Rac1, which stimulated the migration of recipient cells through lamellipodia formation. Thus, these filopodia-dependent l-EVs, which we named as filopodia-derived vesicles (FDVs), modify cellular behavior