Korean J Physiol Pharmacol.  2013 Aug;17(4):331-338. 10.4196/kjpp.2013.17.4.331.

Neuroprotective Effects of AMP-Activated Protein Kinase on Scopolamine Induced Memory Impairment

• Kim SJ
• Lee JH
• Chung HS
• Song JH
• Ha J
• Bae H

• KMID: 2285468
• DOI: http://doi.org/10.4196/kjpp.2013.17.4.331

Abstract

AMP-activated protein kinase (AMPK), an important regulator of energy metabolism, is activated in response to cellular stress when intracellular levels of AMP increase. We investigated the neuroprotective effects of AMPK against scopolamine-induced memory impairment in vivo and glutamate-induced cytotoxicity in vitro. An adenovirus expressing AMPK wild type alpha subunit (WT) or a dominant negative form (DN) was injected into the hippocampus of rats using a stereotaxic apparatus. The AMPK WT-injected rats showed significant reversal of the scopolamine induced cognitive deficit as evaluated by escape latency in the Morris water maze. In addition, they showed enhanced acetylcholinesterase (AChE)-reactive neurons in the hippocampus, implying increased cholinergic activity in response to AMPK. We also studied the cellular mechanism by which AMPK protects against glutamate-induced cell death in primary cultured rat hippocampal neurons. We further demonstrated that AMPK WT-infected cells increased cell viability and reduced Annexin V positive hippocampal neurons. Western blot analysis indicated that AMPK WT-infected cells reduced the expression of Bax and had no effects on Bcl-2, which resulted in a decreased Bax/Bcl-2 ratio. These data suggest that AMPK is a useful cognitive impairment treatment target, and that its beneficial effects are mediated via the protective capacity of hippocampal neurons.

Keyword

Adenovirus; AMPK; Apoptosis; Learning and memory; Scopolamine

MeSH Terms

Acetylcholinesterase
Adenoviridae
AMP-Activated Protein Kinases
Animals
Annexin A5
Apoptosis
Blotting, Western
Cell Death
Cell Survival
Energy Metabolism
Hippocampus
Memory
Neurons
Neuroprotective Agents
Rats
Scopolamine Hydrobromide
United Nations
AMP-Activated Protein Kinases
Acetylcholinesterase
Annexin A5
Neuroprotective Agents
Scopolamine Hydrobromide

Figure

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  Fig. 1 Verification of the correct injection of the adenovirus into the hippocampal CA3. The correct injection of adenovirus was verified by Cresyl violet staining of the hippocampus CA3 prepared following microinjection of the viral suspension, and subsequent microscopic analysis (100×magnification). Arrow heads and numbers indicate hippocampal layers. Hippocampal layers are: subregion 1, CA3 stratum oriens; subregion 2, CA3 pyramidal cell layer; subregion 3, CA3 stratum radiatum; subregion 4, hippocampal fissure. Arrow indicates the lesion created by needle injection within the hippocampal CA3 regions.

• 

  Fig. 2 Alteration of escape latency and swimming distance during an acquisition test and time spent and distance around the platform during the retention test of the water maze test. Four trials per day for 4 consecutive days were conducted and the escape latency (A) and swimming distance (B) were measured. Four trials per day on the fifth day without the platform were conducted and the search latency (C) and distance (D) were measured. Data were analyzed using repeated measures ANOVA followed by Tukey's test. Each value represents the mean±SEM. *p<0.05, **p<0.01 and ***p<0.001 compared to AMPK-DN+scopolamine.

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  Fig. 3 Density of the AchE-reactive neurons in hippocampal CA1 and CA3 of the experimental groups. AchE positive nerve fibers in the hippocampal CA1 (A) and CA3 (B) molecular layer of experimental rats in AMPK-DN+saline group (a, d), AMPK-DN+scopolamine group (b, e), and AMPK-WT+scopolamine group (c, f). The brains were cut into 40 µm coronal sections and the scale bar represents 50 µm (100×magnification). The percentage of AMPK-DN+saline values of the density of acetylcholinesterase (AChE) stained nuclei in the hippocampal CA1 (C) and CA3 (D) areas after the water maze test. Data were analyzed using one-way ANOVA followed by the Tukey's test. Each value represents the mean±SEM. *p<0.05 compared to AMPK-DN+scopolamine.

• 

  Fig. 4 Phosphorylation of AMPK (p-AMPK) in CA3 of the hippocampus infected with Ad-AMPK-WT or Ad-AMPK-DN. Western blot analysis (A) of lysates obtained from normal hippocampus and those following Ad-AMPK-WT and -DN infection were performed using antibodies phosphorylated (P-AMPK) or total AMPK α (AMPK α). The intensities obtained from gel image of (A) were depicted as the ratio between total AMPK and P-AMPK (B).

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  Fig. 5 The effect of AMPK on glutamate-induced apopotosis of cells. Hippocampal neurons cultured in vitro for seven days were transfected with Ad-AMPK-WT and Ad-AMPK-DN in each group and then exposed to 100 µM glutamate with 10 µM of glycine in supplemented neuronal culture medium for 15 min at 37℃. Western blot assays were then conducted using anti-Bax and anti-Bcl-2. The experiment was repeated three times with similar results.

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  Fig. 6 The effect of AMPK on the apoptosis of primary cultured hippocampal neurons as determined by FITC-conjugated annexin V. Neonatal rat hippocampal neurons were cultured and infected with Ad-AMPK-WT or Ad-AMPK-DN, followed by exposure to glutamate and glycine. Anexin V positive cells were detected by flow cytometry (FACS Calibur, BD, San Diego, CA, USA) and analyzed using the CellQuest software.

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