Metformin Alleviates Neurocognitive Impairment in Aging Via Activation OfAMPK/ BDNF/PI3K Pathway PART 2

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Also, activation of AMPK signaling suppresses inflammatory reactions via activating SIRT129, stimulating FOXO proteins35 and inhibiting ER stress, and reducing oxidative stress. All these mechanisms will subsequently repress NF-κB signaling and neuroinflammation associated with aging36. In addition to its anti-inflammatory effects, AMPK is involved in various activities, including angiogenesis, autophagy enhancement, and mitochondrial protein induction16.

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Although the AMPK-dependent protective roles in different contexts have been reported, the AMPK-independent manners of metformin are less studied. The present study revealed that there was significant downregulation of the PI3K gene in the D-gal group when compared with the control group, while metformin coadministration led to the up-regulation of its level when compared with the D-gal group. PI3K/Akt is one of the important signaling pathways in cell apoptosis prevention. Activation of PI3K can be followed by Akt1 phosphorylation. Akt1 can inhibit the phosphorylation of JNK3 which promotes the activation of c-Jun. C-Jun is a protein that can induce the expression of apoptotic proteins37. Tus, Akt1 activation will result in the inactivation of JNK3 and c-Jun, and, then the proportion of cell survival is improved. Therefore, we explored the effects of metformin on cellular apoptosis by studying the immunohistochemical reaction of the apoptotic markers caspase-3 and Bcl-2. This study revealed an increment in the expression of the apoptotic marker caspase-3 with a reduction of the expression of the anti-apoptotic marker Bcl-2 in the hippocampus of the D-gal group when compared with the control group. Supporting our results, previous studies confirmed that mitochondrial ROS induces the activation of a large number of mitochondrial apoptotic proteins, leading to cellular apoptosis and organ damage38. Many apoptotic proteins are closely related to anti-apoptotic proteins in the aging induced by the injection of D-gal39. Caspase-3 is known to be a key factor of apoptosis in mammals40. Te Bcl-2 protein is a key player in the inhibition of apoptosis. It is a known factor in cell aging, and its overexpression can effectively prevent the apoptosis induced by free radicals41. It is commonly believed that Bcl-2 acts downstream of caspase-3 activation and, thus, apoptosis is inhibited42. As shown in our results, metformin administration significantly decreases the Caspase-3/Bcl-2 ratio. These results were by other studies that suggest that activation of AMPK by metformin up-regulates the Bcl-2 protein expression so it protects against apoptotic cell death induced by D-gal and increases neuronal viability43.

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BDNF is an interesting molecular candidate that could help establish a link between molecular and biochemical alterations and memory defects associated with aging. Optimal cognitive function is linked to efficient neuronal plasticity. Memory defects associated with aging might be coupled to alterations in the expression and regulation of plasticity-related proteins such as BDNF which is an important neurotrophic factor. It has been demonstrated that reduction of BDNF leads to neuronal atrophy and death44.

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The present study revealed that the BDNF level in the D-gal group was significantly lower when compared with the control group. A decrease in BDNF and/or its receptors in aging animals was evident in previous studies45. Mizisin et al. suggested that galactose metabolism by aldose reductase influenced axonal function and structure by altering the production of nerve and muscle BDNF46. On the contrary, treatment with metformin was associated with a significant increase in BDNF when compared with the D-gal group. Our results were in agreement with previously published reports which demonstrated that metformin up-regulates BDNF via AMPK activation47.

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Due to its critical role in LTP, BDNF has been postulated to be an essential part of the cellular mechanism supporting memory formation and maintenance by promoting synaptic consolidation. BDNF increases memory storage by favoring changes in spine morphology leading to the stabilization of LTP. BDNF can also increase the number, size, and complexity of dendritic spines. Furthermore, BDNF increases neurogenesis through changes in cell proliferation10. The binding of BDNF to TrkB receptors induces PI3K activity which inhibits apoptosis and promotes cell survival22.

Taken together, our study suggests that the anti-aging effects of metformin in improving neurocognitive impairment could be, at least in part, due to the activation of the AMPK/BDNF/PI3K pathway.

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The hippocampus plays an important role in learning and memory consolidation as well as in behaviors and mood regulation, and where both functional and structural plasticity occurs well into adulthood. Previous studies reported that the hippocampus undergoes several structural changes both grossly and at the cellular level with aging48. H & E study of the hippocampus in the D-gal group showed severe neuronal degeneration with multiple apoptotic bodies and gliosis. This result could be explained by the deleterious effects of d-galactose in the induction of oxidative stress, inflammation, and apoptosis. Additionally, we evaluated the expression levels of a synaptic marker protein (synaptophysin) in the hippocampus. Synaptophysin is a marker of synaptic plasticity. It is used as a specific marker for the presynaptic terminal, and its level is related to the synaptic density. Our results showed that the expression of synaptophysin was significantly lower in the D-gal group when compared with the control group. These results were in line with previously reported studies that demonstrated D-galinduced synaptogenesis impairment in the hippocampus3. However, metformin co-treatment with D-gal restored the synaptophysin expression and hippocampal tissue structure to levels close to their respective control levels. Evidence has shown that metformin promotes rodent and human neurogenesis and enhances spatial memory formation24,41. The increment in synaptic density and neurogenesis in the hippocampus goes hand in hand with the improvement in the neurobehavioral tests’ results in this group. This could be explained by the antioxidant, anti-inflammatory, and antiapoptotic effects of metformin, which are mediated by the activation of the AMPK/BDNF/ PI3K signaling pathway.

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Conclusions

Our findings support the use of D-gal in the rat model to carry out aging-related studies. We concluded that metformin could alleviate memory impairment and cognitive deficits caused by aging. The mechanisms likely involved are amelioration of neuro-inflammation, attenuation of oxidative stress, enhancement of the expression of the anti-apoptotic protein Bcl-2, as well as the promotion of neurogenesis and synaptic plasticity. We believed that these mechanisms could be mediated via activation of the AMPK/BDNF/PI3K pathway. To the best of our knowledge, this is the first study to demonstrate the action of metformin on improving cognitive impairment in aged rats via activation of this pathway. Therefore, our findings suggest that metformin is a useful anti-aging agent.

Data availability

All the data generated or analyzed during this study are included in this published manuscript.

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Received: 30 March 2022; Accepted: 21 September 2022

Published online: 12 October 2022

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Acknowledgments

The authors thank the Faculty of Medicine at Menoufa University for providing most of the required facilities.

Author contributions

O.A. designed the study and coordinated the laboratory work, performed the animal studies, carried out molecular biology experiments, analyzed the data, and drafted the manuscript. R.M.S. carried out the histopathology and immunohistochemistry studies and analyzed the data. R.A.A.A.E. participated in the study design, performed the animal studies, participated in the biochemical analysis, and helped in drafting the manuscript. All authors approved the final version of the manuscript. The authors declare that all data were generated in-house and that no paper mill was used.