Elsevier

Experimental Cell Research

Volume 347, Issue 2, 1 October 2016, Pages 322-331
Experimental Cell Research

Research article
Metformin activation of AMPK-dependent pathways is neuroprotective in human neural stem cells against Amyloid-beta-induced mitochondrial dysfunction

https://doi.org/10.1016/j.yexcr.2016.08.013Get rights and content

Abstract

Alzheimer's disease (AD) is the general consequence of dementia and is diagnostic neuropathology by the cumulation of amyloid-beta (Aβ) protein aggregates, which are thought to promote mitochondrial dysfunction processes leading to neurodegeneration. AMP-activated protein kinase (AMPK), a critical regulator of energy homeostasis and a major player in lipid and glucose metabolism, is potentially implied in the mitochondrial deficiency of AD. Metformin, one of the widespread used anti- metabolic disease drugs, use its actions in part by stimulation of AMPK. While the mechanisms of AD are well established, the neuronal roles for AMPK in AD are still not well understood. In the present study, human neural stem cells (hNSCs) exposed to Aβ had significantly reduced cell viability, which correlated with decreased AMPK, neuroprotective genes (Bcl-2 and CREB) and mitochondria associated genes (PGC1α, NRF-1 and Tfam) expressions, as well as increased activation of caspase 3/9 activity and cytosolic cytochrome c. Co-treatment with metformin distinct abolished the Aβ-caused actions in hNSCs. Metformin also significantly rescued hNSCs from Aβ-mediated mitochondrial deficiency (lower D-loop level, mitochondrial mass, maximal respiratory function, COX activity, and mitochondrial membrane potential). Importantly, co-treatment with metformin significantly restored fragmented mitochondria to almost normal morphology in the hNSCs with Aβ. These findings extend our understanding of the central role of AMPK in Aβ-related neuronal impairment. Thus, a better understanding of AMPK might assist in both the recognition of its critical effects and the implementation of new therapeutic strategies in the treatment of AD.

Introduction

Alzheimer's disease (AD) is a serious neurodegenerative disorder of brain in the aged population. AD is the progressive neurodegenerative disease of aging and the general form of dementia [1], [2]. AD causes difficulty for patients, containing loss of independence, emotional suffering, cognitive impairment such as memory loss, and changes in behavioral symptoms [3], [4]. AD is correlated with numerous cellular alter in the brain, including the loss of synaptic structures, mitochondrial function, oxidative stress, inflammation, amyloid beta (Aβ) deposits, and neurofibrillary tangles [5], [6], [7], [8], [9], [10], [11], [12], [13], [14]. The gross pathology of AD described by Aβ peptide deposition into cerebral amyloid plaques [15]. Aβ peptides have a major role in the pathogenesis of the AD brains and illustrate the lesions invariably found in the hippocampus and cortex, and the subsequent neurodegeneration that follows [3], [15], [16], [17], [18], [19]. Aggregates of Aβ progress in extracellular plaques and are correlated with neurodegeneration in AD [20], [21], [22]. Several reports have indicated that Aβ is accountable for damaging synapses and mitochondria in neurons influenced by AD [23], [24], [25]. Aβ have been exhibited in mitochondrial membranes and to interact with mitochondrial proteins, change mitochondrial enzymes, disorganize electron transport chain, suppress ATP task, elicit free radical damage, and impair mitochondrial biogenesis [23], [24], [25], [26], [27].

AMP-activated protein kinase (AMPK) is a Ser/Thr kinase which shows a crucial role in the maintenance of energy balance at both the cellular and whole-body, senses degrees of adenosine triphosphate (ATP) [28], [29], [30], [31], [32]. When ATP levels decrease, AMPK is stimulated to provide the cells to modulate to the metabolic modify in the cell [33], [34], [35]. As a cellular energy sensor responding to low ATP levels, AMPK regulates several intracellular systems including the lipid metabolism, cellular glucose uptake by glucose transporter 4 (GLUT4), and mitochondria biogenesis [30], [36], [37], [38], [39], [40], [41]. AMPK is activated in response by consume cellular ATP, including low glucose, nutrient deprivation, oxidative stress, and hypoxia in effect to an increase in the AMP/ATP ratio [42], [43], [44], [45]. Raised Ca2+ levels and cytoplasmic AMP are the critical activators of neuronal AMPK signaling [46].

Research studies demonstrate that the AMPK are constantly showed in neurons and thus can be capability activated [46]. AMPK has demonstrated as a major sensor of cellular energy balance, and is potentially involved in a wide range of states, including AD [47], [48]. AMPK action has been shown to reduce with age, which may provide to decreased mitochondrial function with aging-related diabetes and AD [49], [50], [51], [52], [53], [54]. Diabetes major raise cognitive decline, indicating the association that cellular mechanisms of energy homeostasis are connect to AD pathogenesis [55]. Therefore, AMPK is a potential target to ameliorate disturbed brain energy biogenesis that is activated, might alter AD pathology and is a potential therapeutic target for AD [48], [56], [57], [58], [59]. Through activation of AMPK explained that it inhibits Aβ accumulation, meliorates mitochondrial dysfunction and suppress oxidative stress, and plays a beneficial effect in mouse brain [60]. Several studies exhibit that AMPK is a neuroprotective element against metabolic stress, involving a critical role in the prevention of AD pathogenesis [61], [62], [63]. Therefore, further studies required to explain the function of AMPK in either pathogenesis or treatment of AD.

Section snippets

Cell culture

GIBCO® human neural stem cells (hNSCs) were originally obtained from National Institutes of Health (NIH) approved H9 (WA09) human embryonic stem cells. Complete StemPro® NSC serum free medium (SFM) was used for optimal growth and expansion of GIBCO® hNSCs, and kept the hNSCs undifferentiated as described previously [64]. Complete StemPro® NSC SFM consists of KnockOut™ D-MEM/F-12 with 2% StemPro® Neural Supplement, 20 ng/ml of EGF, 20 ng/ml of bFGF, and 2 mM of GlutaMAX™-I.

Evaluation of cell growth

Cell viability was

Metformin rescued cell viability in hNSCs treated with Aβ via the AMPK pathway

The effects of AGEs on cell viability and caspase 3/9 (a marker of caspase cascade activation) activity in hNSCs were initially assessed. Compared to vehicle controls, hNSCs treated with Aβ for 72 h had significantly reduced cell viability (p<0.001) (Fig. 1A). In addition, hNSCs caspase 3 and 9 activities, detected after Aβ treatment for 72 h, were significantly increased 2-fold compared to their respective controls (p<0.001) (Fig. 1B, C). Furthermore, treatment with a AMPK agonist (metformin)

Discussion

Since, the identification of AD as mitochondrial diseases has been causing elevating attention [14], [72]. Moreover, experiments guided in animal as well as cellular models of AD demonstrate the benefit of mitochondrial protection for reduction neuronal degeneration [73]. AMPK activity has been shown to reduce with age, which may provide to decreased survivability and mitochondrial biogenesis with aging-correlated AD (Jornayvaz and Shulman, 2010). Here we exhibit our result of the central role

Conflict of interest

The authors have declared no conflict of interest.

Acknowledgments

The authors thank Chia-Nan Yen for proof-reading and editing the article. This work was supported by grants from the Ministry of Science and Technology, Taiwan (MOST 105–2314-B-030–005), Fu Jen Catholic University (A0104020 and A0204104), and Terry Whole Brain & Potential Development Center (Terry 105–11-01). The authors wish to thank for technical assistance of Electron Microscope Laboratory of Tzong Jwo Jang, collage of Medicine, Fu Jen Catholic University.

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