Study On The Neuroprotective Effects Of Val~8-GLP-1(7-36) Against Aβ1-40-Induced Neurotoxicity In Behavior, Electrophysiology And Calcium Imaging

Alzheimer disease (AD) is an age-related neurodegenerative disorder characterized by progressive learning impairment and memory loss followed with cognitive decline. The deposition of amyloidβ-protein (Aβ) in the brain, especially in the hippocampus and temporal cortex, is thought to be responsible for the deficit of learning and memory in AD patients. The neurotoxicity of Aβhas been widely reported in vivo and in vitro, including the impairment of spatial learning and memory and synaptic plasticity such as long-term potentiation (LTP). Calcium ion is one of the most important intracellular second messengers in the brain, being essential for a variety of neuronal functions such as neuronal development, synaptic transmission and plasticity, and the regulation of various metabolic pathways. Accumulating evidence suggested that the disruption of intracellular Ca²⁺ homeostasis is crucial to Aβinduced neurodegeneration. However, it is seriously short of effective neuroprotective strategies against Aβneurotoxicity up to now.Interestingly, it has been found that type 2 diabetes mellitus (T2DM), another degenerative disease, is a risk factor for developing AD in the elderly. One promising treatment for AD is using insulin-releasing gut hormone glucagon-like peptide-1 (GLP-1), a modulator used in T2DM therapy. GLP-1 can reduce the levels of Aβin the brain in vivo and reduce levels of amyloid precursor protein (APP) in cultured neuronal cells and possess neurotrophic properties to protect neurons against glutamate-induced apoptosis. Previous study has shown that the hippocampal LTP detrimental effect induced by Aβfragments was effectively prevented by GLP-1. More importantly, GLP-1 and GLP-1 receptors (GLP-1R) are expressed in the brain, including the hippocampus. However, whether GLP-1 can protect against Aβ-induced impairment of synaptic plasticity, especially the late phase of long-term potentiation (L-LTP) in hippocampus, is still an open question; the mechanism of neuroprotective role of GLP-1 in the brain has not yet been fully elucidated at the present time. The natural GLP-1 can be rapidly degraded by the enzyme dipeptidyl peptidase IV (DPP IV) and its half life is only 2-3 min in blood plasma. In contrast, Val⁸-GLP-1(7-36) is a GLP-1 analogue with profound resistance to DPP IV and greater biological activity.Therefore, Val⁸-GLP-1(7-36), not natural GLP-1, was used in the present study. By using the high-effective GLP-1 analogue, we investigated: (1) the effects of i.c.v. injection of Val⁸-GLP-1(7-36) on the Aβ1-40-induced impairment of spatial learning and memory of rats in a Morris water maze test; (2) the effects of i.c.v. injection of Val⁸-GLP-1(7-36) on the Aβ1-40-induced impairment of in vivo L-LTP in rat hippocampal CA1 region; (3) effects of Val⁸-GLP-1(7-36) on the Aβ1-40-induced changes in miniature postsynaptic currents (mEPSCs and mIPSCs) in hippocampal CA1 pyramidal neurons; (4) the effects of pretreatment with Val⁸-GLP-1(7-36) on Aβ1-40-induced elevation of [Ca²⁺]i in cultured primary rat cortical neurons.Partâ… Val⁸-GLP-1(7-36) Protects Against Aβ1-40-Induced Impairment of Spatial Learning and Memory in RatsTo characterize the neuroprotective role of Val⁸-GLP-1(7-36) in the brain, we investigated the effects of i.c.v. injection of Val⁸-GLP-1(7-36) on the Aβ1-40-induced impairment of spatial learning and memory of rats in a Morris water maze test and the alteration of CA1 neuronal morphology by HE staining after finishing the behavior experiment. The escape latency (s), distance traveled (cm) and swimming speed (cm/s) were recorded in hidden platform tests, and the percentage of the total time in the different quadrants was calculated in probe trials.The results showed that: (1) i.c.v. injection of 5 nmol Aβ1-40 impaired the spatial learning and memory of rats. In hidden platform test, the latencies and distances for searching for the platform were significantly larger compared with control from the 2nd to the 5th day (P<0.01). For example, the mean escape latencies and distances were 50±5.5 s and 1232.8±128.3 cm, significantly larger (P<0.01) than the values of 17.8±2.9 s as and 458.6±63.8 cm in the control group (P<0.01) at the 5th day of testing. In probe trial, the percentage of total time elapsed in the target quadrant in the Aβ1-40 group was only 28.4±3.8% in the Aβ1-40 group, significantly lower than 43.5±4.8% in control group (P<0.01). (2) Val⁸-GLP-1(7-36) alone improved spatial learning and memory of rats in a dose-dependent manner. In hidden platform test, 0.05 pmol Val⁸-GLP-1(7-36) had no effects on the escape latencies and distances (p>0.05); but 0.5 pmol and 5 pmol Val⁸-GLP-1(7-36) enhanced spatial learning ability of rats. In 5 consecutive days of testing, the latencies and distances were significant decreased in Val⁸-GLP-1(7-36) group compared with control (P<0.01), for example, on the 5th day of testing, the escape latencies in 0.05 pmol, 0.5 pmol and 5 pmol Val⁸-GLP-1(7-36) groups were 15.5±1.4 s, 11.1±1.1 s and 6.8±1.1 s (p<0.01), respectively, showing a dose-dependent decrease. In probe trial, the percentage of total time elapsed in the target quadrant in the 0.05 pmol, 0.5 pmol and 5 pmol Val⁸-GLP-1(7-36) groups were 44.9±4.4%, 48.5±4.9% and 51.4±3.8%, respectively, showing a significant increase with the increase of the concentration of Val⁸-GLP-1(7-36) (p<0.01). (3) Pretreatment of Val⁸-GLP-1(7-36) effectively protected spatial learning and memory against Aβ1-40-induced impairment in a dose-dependent manner. For example, on the 5th day of hidden platform test, the escape latencies in 0.05 pmol, 0.5 pmol and 5 pmol Val⁸-GLP-1(7-36) plus Aβ1-40 groups were 30.3±3.5 s, 28.4±2.5 s and 18.8±2.6 s, respectively, showing a dose-dependent decrease. In probe trial, the percentages of total time elapsed in the target quadrant were 40.2±4.7%, 46.4±4.6% and 48.3±4.7% for 0.05 pmol, 0.5 pmol and 5 pmol Val⁸-GLP-1(7-36) puls Aβ1-40 group, respectively, significantly higher than Aβ1-40 alone group (p<0.01), showing a significant dose-dependent increase (p<0.01). (4) Both Val⁸-GLP-1(7-36) and Aβ1-40 did not affect the vision and the swimming speeds of approximately 19 cm/s. (5) The most pronounced lesions were found in the CA1 sector in Aβ1-40 group, including reduced pyramidal neurons, diminished neuron density, partial neuronal degeneration and necrosis. However, pretreatment with Val⁸-GLP-1(7-36) obviously reduced Aβ1-40-induced damage of neuron, and the pathological changes were gradually alleviated with the increase of the concentration of Val⁸-GLP-1(7-36). These results indicated that i.c.v. administration of Aβ1-40 impaired spatial learning and memory of rats, while pretreatment with Val⁸-GLP-1(7-36) effectively reversed Aβ1-40-induced the impairment of cognitive function in a dose dependent manner, suggesting that natural GLP-1 in CNS may play an important positive role in maintaining normal cognitive function, and Val⁸-GLP-1(7-36) might be a promising strategy to ameliorate degenerative processes in AD.Partâ…¡Val⁸-GLP-1(7-36) Protects Against Aβ1-40-Induced Impairment of Hippocampal Late-Phase Long Term Potentiation in Rat Hippocampal CA1 Region in vivoThe effect of i.c.v. injection of Val⁸-GLP-1(7-36) on the Aβ1-40-induced impairment of in vivo L-LTP in rat hippocampal CA1 region was investigated in the present study. Hippocampal fEPSP were recorded in the CA1 region. Three sets of high frequency stimulation (HFS) with 5 min of interval were applied to produce a robust L-LTP.The results showed that: (1) Aβ1-40 did not affect the baseline fEPSP but significantly suppressed L-LTP. After application of 5 nmol Aβ1-40,the average fEPSP amplitudes before and 3 h after Aβ1-40 injection without HFSs were 99.5±1.4% and 100.2±1.11% (P>0.05), respectively. However, the average fEPSP amplitudes after HFSs were 129.6±3.3%, 108.1±5.4% and 101.1±7.2% in Aβ1-40 group at 1 h, 2 h and 3 h, respectively, significantly lower than the values of 153.0±4.5%, 140±4.8% and 139.38±5.5% in control group at the same time points (P<0.01). (2) Val⁸-GLP-1(7-36) alone did not affect the baseline fEPSP but enhanced the L-LTP in a dose-dependent manner. The baseline average fEPSP amplitudes induced by test stimulation before and 3 h after injection were 99.5±1.4% and 100.2±1.1% (P>0.05), 100.2±2.4% and 99.5±4.7% (P>0.05), 99.9±3.32% and 100.0±8.6% (P>0.05) in 0.05 pmol, 0.5 pmol and 5 pmol Val⁸-GLP-1(7-36) group (P>0.05), respectively. However, the averaged fEPSP amplitudes after HFSs were gradually increased with the increase of the concentration of Val⁸-GLP-1(7-36). For example, the averaged fEPSP amplitudes were 143.2±7.1%, 152.2±2.4% and 164.0±4.1% in the 0.05 pmol, 0.5 pmol and 5 pmol Val⁸-GLP-1(7-36) group at 3 h post-HFSs, respectively, significantly larger than the values in control group (143.2±1.5%, P<0.01) (3) Pretreatment with Val⁸-GLP-1(7-36) protected against Aβ1-40-induced impairment of L-LTP. The average fEPSP amplitudes in 0.05 pmol, 0.5 pmol and 5 pmol Val⁸-GLP-1(7-36) plus Aβ1-40 group were 130.3±6.0%, 138.9±6.0% and 139.0±5.3% at 3 h post-HFSs, respectively, significantly larger than the values in Aβ1-40 alone group (P<0.01).These results demonstrated that pretreatment with Val⁸-GLP-1(7-36) effectively reversed Aβ1-40-induced suppression of L-LTP in a dose-dependent manner in the rat hippocampal CA1 region in vivo. The results are well consistent with the results in the behavior experiment above and may partly explain the cellular mechanisms of GLP-1 in improving spatial learning and memory function.Partâ…¢Effects of Val⁸-GLP-1(7-36) on the Spontaneous Synaptic Activity in Hippocampal SlicesThe proper modulation of excitatory and inhibitory synaptic transmission is critical for brain function. To further reveal the cellular electrophysiological mechanism of neuroprotective role of Val⁸-GLP-1(7-36), we, in this part of experiment, examined spontaneous miniature postsynaptic currents (mEPSCs and mIPSCs) in hippocampal CA1 pyramidal neurons of rat brain slices by using whole-cell patch clamp technique.The results showed that: (1) Aβ1-40 significantly reduced the frequency of mEPSCs and mIPSCs and extended the decay time of mIPSCs. After application of 100 nM Aβ1-40 alone, the normalized frequency of mEPSCs reduced from 100.0±11.2% in control group to 37.9±8.8% (P<0.01); the normalized frequency of mIPSCs reduced from 100.0±5.1% in control group to 46.5±6.8% (P<0.01). However, the amplitude, 10-90% rise time and decay time of mEPSCs and the amplitude, 10-90% rise time of mIPSCs in Aβ1-40 group did not change (P>0.05). (2) Val⁸-GLP-1(7-36) (10 nM) did not change the amplitude, frequency, 10-90% rise time and decay time of mEPSCs and mIPSCs (P>0.05). (3) Pretreatment with Val⁸-GLP-1(7-36) improved Aβ1-40-induced change in frequency of mEPSCs and mIPSCs and decay time of mIPSCs. After pretreatment with Val⁸-GLP-1(7-36), the normalized frequency of mEPSCs significantly increased to 63.5±5.0% from 37.9±8.8% in Aβ1-40 alone group (P<0.01); the frequency of mIPSCs increased to 72.4±2.2% from 46.5±6.8% in Aβ1-40 alone group (P<0.01); the normalized decay time of mIPSCs decreased to 124.1±10.8% from 150.2±13.4% in Aβ1-40 group alone (P<0.01).These results clearly demonstrated that pretreatment of Val⁸-GLP-1(7-36) contributed to Aβ1-40 induce decrease of frequency of mEPSCs and mIPSCs and change of mIPSCs channel kinetics. These results may be helpful in explaining the mechanism of Aβ1-40 induced impairment in hippocampal synaptic plasticity. The decrease of the frequency of spontaneous mEPSCs will affect the presynaptic transmitter release, and the decrease of the frequency of spontaneous mIPSCs may cause the balance between excitation and inhibition of nervous system and enhance the cell excitotoxicity. Therefore, the effects of Val⁸-GLP-1(7-36) on the Aβ1-40-induced changes of mEPSCs and mIPSCs are positive in the regulation of neuronal activity, and GLP-1 and its analogue may represent an alternative and potentially valuable novel therapeutic intervention for reversing the neurodegenerative processes in AD.Partâ…£Effects of Val⁸-GLP-1(7-36) on Aβ1-40-Induced Calcium Influx in Cultured Primary Rat Cortical NeuronsBy utilizing calcium imaging via laser-scanning confocal fluorescent imaging technique, we investigated the effects of Aβ1-40 and Val⁸-GLP-1(7-36) on [Ca²⁺]i, especially the neuroprotective effects of Val⁸-GLP-1(7-36) against Aβ1-40-induced disruption of Ca²⁺ homeostasis in cultured primary rat cortical neurons.The results showed that: (1) Aβ1-40 alone significantly increased the intracellular calcium level. 18 min after application of 10μM Aβ1-40, the relative fluorescent intensity of cultured primary rat cortical neurons obviously increased from 100% in control to 191.1±21.6% (P<0.01). Pretreatment with 50μM AP-5, a specific inhibitor of NMDA receptor, or 5μM Nicardipine, a specific inhibitor of L-VDCC, suppressed Aβ1-40-induced [Ca²⁺]i elevations, with a relative fluorescent intensity of 142.9±5.3% and 152.1±1.7% (P<0.01), respectively. These results showed that Aβ1-40-induced [Ca²⁺]i elevations are mediated partly by L-type voltage-dependent calcium channels (L-VDCC) and N-methyl-D-aspartate (NMDA) receptor. (2) Val⁸-GLP-1(7-36) alone induced a transient elevation of [Ca²⁺]i in a dose-dependent manner. The fluorescent intensity of [Ca²⁺]i after application of different concentration of Val⁸-GLP-1(7-36) rapidly rised to peak values at 1 min after administration in a dose-dependent manner. The relative fluorescent intensity of [Ca²⁺]i were 102.6±1.1%, 126.5±4.5% (P<0.01) and 165.2±5.1% (P<0.01) in 10 nM, 100 nM and 1000 nM Val⁸-GLP-1(7-36) group, respectively, showing a dose-dependent increase (p<0.01). However, at the time point of 18 min after application, the relative fluorescent intensity of [Ca²⁺]i returned to baseline, being 100.1±0.54%, 100.3±1.0% and 99.6±1.6% in 10 nM, 100 nM and 1000 nM Val⁸-GLP-1(7-36) group, respectively. (3) Pretreatment with Val⁸-GLP-1(7-36) protected against Aβ1-40-induced elevation of [Ca²⁺]i in a dose-dependent manner. The relative fluorescent intensity of [Ca²⁺]i decreased to 165.6±3.4%, 141.3±3.3% and 123.6±3.5% in co-application of 10 nM, 100 nM and 1000 nM Val⁸-GLP-1(7-36) puls Aβ1-40 group, respectively, significantly lower than the value of 188.6±2.2% in Aβ1-40 alone group (P<0.01).These results demonstrated that Aβ1-40 alone significantly increase the intracellular calcium levels, which is closely related to the neurotoxicity of Aβ-induced calcium overload. The fact that pretreatment with Val⁸-GLP-1(7-36) effectively protected against Aβ1-40-induced elevation of [Ca²⁺]i in a dose-dependent manner suggested that the neuroprotective effects of Val⁸-GLP-1(7-36) in the behavioral and electrophysiological studies may result from the alleviation of Aβ-induced intracellular calcium overload.In conclusion, the present study, by using Morris water maze test, field potential recording, brain slice whole-cell patch clamp and confocal Ca²⁺ image technique, observed the effects of Val⁸-GLP-1(7-36) on the spatial learning and memory behavior, hippocampal L-LTP in vivo, spontaneous miniature postsynaptic currents (mEPSCs and mIPSCs) and [Ca²⁺]i of cultured cortical neurons of rats, and investigated the neuroprotective role of Val⁸-GLP-1(7-36) against Aβ1-40-induced neurotoxicity. The results indicated that Val⁸-GLP-1(7-36) can effectively protect against Aβ1-40-induced impairment of rat spatial learning and memory and hippocampal L-LTP in vivo; the neuroprotective effect of Val⁸-GLP-1(7-36) may be related to the modulation of synaptic transmission and intracellular calcium homeostasis. Therefore, the present study provides further behavioral and electrophysiological evidence for the neuroprotective effect of GLP-1 and its analogues Val⁸-GLP-1 (7-36), and reveals its possible cellular and channel mechanism, strongly suggesting that GLP-1 and its analogue might be one of the promising candidates for the treatment of AD in the future.