Ameliorative effects of omega-lycotoxin-Gsp2671e purified from the spider venom of Lycosa praegrandis on memory deficits of glutamate-induced excitotoxicity rat model

Abstract

Memory impairment is one of the main complications of Alzheimer's disease (AD). This condition can be induced by hyper-stimulation of N-Methyl-D-aspartate receptors (NMDARs) of glutamate in the hippocampus, which ends up to pyramidal neurons determination. The release of neurotransmitters relies on voltage-gated calcium channels (VGCCs) such as P/Q-types. Omega-lycotoxin-Gsp2671e (OLG1e) is a P/Q-type VGCC modulator with high affinity and selectivity. This bio-active small protein was purified and identified from the Lycosa praegrandis venom. The effect of this state-dependent low molecular weight P/Q-type calcium modulator on rats was investigated via glutamate-induced excitotoxicity by N-Methyl-D-aspartate. Also, Electrophysiological amplitude of field excitatory postsynaptic potentials (fEPSPs) in the input-output and Long-term potentiation (LTP) curves were recorded in mossy fiber and the amount of synaptophysin (SYN), synaptosomal-associated protein, 25 kDa (SNAP-25), and synaptotagmin 1(SYT1) genes expression were measured using Real-time PCR technique for synaptic quantification. The outcomes of the current study suggest that OLG1e as a P/Q-type VGCC modulator has an ameliorative effect on excitotoxicity-induced memory defects and prevents the impairment of pyramidal neurons in the rat hippocampus.

Keywords: animal venoms; calcium channel Cav2.1 (P/Q type); calcium channel modulators; memory; spider.

FIGURE 1

The Purification, and identification of OLG1e from the Lycosa praegrandis crude venom. (A) Gel-filtration chromatography of the crude venom performed on a GE Healthcare HiLoad 16/600 Superdex^® 75 pg prep grade column in 1 M PBS (pH 7.4), flow rate 0.7 ml/min. The fifth fraction is shown by black arrow. (B) Capillary electrophoresis of the sixth fraction was performed on a 50 μm uncoated silica column 1 M PBS (pH 4.7) for 5 min. The OLG1e fraction is shown by black arrow. (C) HPLC-ESI-MS of the OLG1e fraction was performed on a of Atlantis T3-C18 column. The spectrum results are shown in part (C).(D) N-terminal partial sequencing of the OLG1e protein. The singly charged ion of the N-terminal peptide (m/z, 1,406) was subjected to the fragmentation in the ion trap mass analyzer. Fragment ions observed are indicated above and below the peptide sequence.

FIGURE 2

The Morris water maze test (A) The effect of treatment with OLG1e on the average escape latency time during the training days. The data are shown as means ± SEM of 10 rats per group. (*p < 0.05 and **p < 0.01 and ***p < 0.001 and ****p < 0.0001) and (#p < 0.05). (B) The effect of treatment with OLG1e on time spent in the target quadrant, distance moved in the target quadrant, frequency of entry into the target quadrant from the test day. The data are shown as means ± SEM of 10 rats per group (*p < 0.05 and **p < 0.01 and ***p < 0.001 and ****p < 0.0001). (C) Scatter plot of part (B) (D) The swim path traces from the test day. The target zone is indicated with green star.

FIGURE 3

The Novel object recognition test (A) The effect of treatment with OLG1e on the d₂ index. The data are shown as means ± SEM of 10 rats per group. (*p < 0.05 and **p < 0.01 and ***p < 0.001 and ****p < 0.0001) (B) The effect of treatment with OLG1e on the R index. The data are shown as means ± SEM of 10 rats per group (*p < 0.05 and **p < 0.01 and ***p < 0.001 and ****p < 0.0001).

FIGURE 4

The Quantitative Real-Time PCR (A) The effect of treatment with OLG1e on the hippocampal SYN, SNAP-25, and SYT1 mRNA levels. The data are shown as means ± SEM of six rats per group. (*p < 0.05 and **p < 0.01 and ***p < 0.001 and ****p < 0.0001) (B) Heat map for SYN, SNAP-25, and SYT1 mRNA levels (Two-way ANOVA).

FIGURE 5

The Cresyl Violet Staining of rat hippocampus. (A) The healthy (Narrow red arrow) and dead (Narrow black arrow) pyramidal neurons are indicate in the CA₃ area. (B) The effect of treatment with OLG1e on the mean count of viable cells in CA3 of the hippocampus. The data are shown as means ± SEM of three rats per group (*p < 0.05 and **p < 0.01 and ***p < 0.001 and ****p < 0.0001). (C) The healthy (Narrow red arrow) and dead (Narrow black arrow) pyramidal neurons are indicate in the subiculum area. (D) The effect of treatment with OLG1e on the mean count of viable cells in subiculum of the hippocampus. The data are shown as means ± SEM of three rats per group (*p < 0.05 and **p < 0.01 and ***p < 0.001 and ****p < 0.0001).

FIGURE 6

Mossy Fiber circuit LTP (A) Long-term potentiation (LTP) curves of the field excitatory postsynaptic potential (fEPSP) amplitude in the hippocampal CA₃ for the all groups (n = 6). The data are shown as means ± SEM of six rats per group. (*p < 0.05 and **p < 0.01 and ***p < 0.001 and ****p < 0.0001), and (^#### p < 0.0001). (B) Sample traces of typical recorded fEPSPs in the hippocampal CA3 neurons before and after high-frequency stimulation (HFS) induction for the long-term potentiation (LTP) in experimental groups. (C) Experimental timeline. Timeline showing the sequence of events and behavioral testing the animals underwent.