The findings in our current study indicate a link between impaired AMPK signalling and mitochondrial respiratory chain dysfunction in human neuroblastoma SH-SY5Y cells. The SH-SY5Y cell line has been used extensively as an in vitro model system of peripheral sensory neurons as they exhibit traits of sensory neuron phenotype . Thus, the rationale for this approach is that the assay performed in this cell line can be a predictor of efficacy in human cells and will be useful for future drug screening endeavors . In addition, this work provides important background information that will underpin future molecular studies not feasible in primary neurons, e.g. proteomic studies to understand molecular pharmacology at the M1R (for example, see ). We observed MT7 and pirenzepine treatment enhanced AMPK phosphorylation, augmented mitochondrial complex protein expression as well as enhanced mitochondrial function in the SH-SY5Y cell line and these data support our previous report in DRG neurons [29, 30, 24, 23]. Importantly, siRNA targeting AMPK significantly blocked the drug-induced up-regulation of mitochondrial OXPHOS proteins resulting in a suppressed oxidative phosphorylation system.
Dynamic morphological changes in mitochondria are required to maintain a homogenous population of functional mitochondria to ensure continuous and optimal mitochondrial respiration. Optimal mitochondrial function is a key factor for axonal outgrowth and repair [37, 61]. Mitochondrial abnormalities have been proposed to mediate development of diabetic complications through cellular dysfunction in endothelial cells, skeletal muscle, cardiomyocytes and neurons [23, 26, 24, 62–64]. Mitochondrial biogenesis is triggered by the AMPK-PGC-1α-Nrf1 pathway which, in turn, regulates the expression of both mitochondrial and nuclear genes encoding respiratory chain subunits and other proteins that are required for mitochondrial function [65, 66]. Energy supplementation provided by this pathway is required for axonal outgrowth and neuronal growth . Previous studies have highlighted that activation of AMPK can elevate neurite outgrowth. For example, resveratrol, an activator of AMPK, drives axonal outgrowth and was protective against diabetic neuropathy in STZ-induced diabetic rats [24, 67]. Recent studies also report IGF-1-mediated up-regulation of mitochondrial respiration together with a dose-dependent stimulation of ATP production through AMPK in a type 1 model of diabetes . Other works have determined that certain mitochondrial complexes and mitochondrial membrane potential were impaired in cortical tissues and primary DRG neurons from diabetic rat but the cellular mechanisms are not completely understood [23, 68, 25].
The present study demonstrates for the first time that blockade of the M1R by the specific antagonist MT7 or the selective antagonist pirenzepine causes an augmentation of the mitochondrial membrane potential (MMP) in both cultured SH-SY5Y cells and DRG neurons. This stimulatory effect on MMP was time dependent and triggered within 1 h. MMP is a parameter for mitochondrial metabolic state and provides an estimate of the ATP production within individual mitochondria . The AMPK inhibitor, Compound C, abolished the pirenzepine and MT7-mediated up-regulation of mitochondrial MMP. SiRNA-based inhibition of endogenous AMPK exhibited a similar suppression of the pirenzepine enhancement of MMP. These novel observations in neurons provide functional evidence linking AMPK and alterations in mitochondrial performance, such as maintenance of MMP.
M1R activation inhibits voltage-gated Kv7 potassium channels that mediate the M-current in sympathetic neurons [43, 70]. M-current (IM) is a low-threshold, slowly activating potassium current in sympathetic neurons where it functions as a “brake” for neurons receiving persistent excitatory input . The M-current is strongly suppressed by M1R activation [42, 71, 45, 72] which is known to play an important role in modulating neuronal excitability and its suppression is predicted to increase input resistance in response to excitatory synaptic inputs [73–75, 70]. M-current inhibition via M1R activation by acetylcholine is phosphatidylinositol-4,5-bisphosphate (PIP2)-dependent with depletion of PIP2 dramatically decreasing Kv7 channel open probability [76, 77]. Acute ACh activation of M1R promotes PIP2 hydrolysis through phospholipase C activation, resulting in PKC phosphorylation and generation of inositol triphosphate, which induces endoplasmic reticulum Ca2+ release . Downstream Ca2+-dependent pathways drive closing of Kv7 channels, and the outcome is an enhanced propensity for depolarization of the plasma membrane. Interestingly, activated PKC may also contribute to the muscarinic inhibition of Kv7 channels . Activated PKC phosphorylates the C-terminus in the calmodulin (CaM) binding site of the Kv7.2 subunit assisted by A-kinase-anchoring protein AKAP79/150. The phosphorylated state of the channel destabilizes the Kv7 channel/PIP2 complex and consequently PIP2 hydrolysis suppresses the M-current [80–83].
Kv7/M-channel activity represents an integral regulator of PNS sensitivity downstream of multiple transduction mechanisms likely to contribute to dampening of peripheral pain pathways . They are densely expressed at the sites of spike generation e.g., axon initial segment of central neurons and terminals of peripheral nociceptive neurons [85, 86]. Previous investigations of the role of Kv7 in regulating neuronal excitability, pain pathways and nociceptive behaviors utilized pharmacological M-channel blockers or enhancers [41, 87–90]. M-current perturbations were strongly implicated in neuronal hyperexcitability underlying epilepsy and ALS [87, 91], neuroinflammation  and neuropathic pain [93, 94]. M-current “opener” compounds have been suggested to be efficacious in preventing brain damage after multiple types of insults/diseases, such as stroke, traumatic brain injury, drug addiction and mood disorders . However, sensory neurons express Kv7 channels and exhibit the M-current, activated at near resting potential such that at subthreshold potentials produce a prominent outward current [42, 41, 96] helping to keep the resting potential within a hyperpolarized range but an initiating role of M1R in this pathway has not been directly elucidated [41, 97]. In accordance with this concept, the present experiments revealed that antimuscarinic drugs pirenzepine or MT7 have a novel mechanism of action acting as putative positive modulators of Kv7 M-channels, i.e. Kv7 channel opener/enhancers in SH-SY5Y cells and sensory neurons. Consequently M1R antagonists help to establish the neuronal resting membrane potential by providing a continual hyperpolarizing influence and make the neurons less excitable. The effects of pirenzepine/MT7 on M-current activation were reversed by muscarinic agonist muscarine leading to increased responsiveness of neurons toward depolarizing stimuli. As such, the data presented here offer promising evidence for the pivotal role of Kv7 channel as a target of M1R antagonists to stabilize membrane potential as well as dampening deviations in depolarizion and, therefore, preventing ectopic firing and spontaneous pain. Importantly, upon axotomy, sensory neurons exhibit spontaneous electrical activity that consumes extensive ATP [98–101]. M1R antagonism enhances neurite outgrowth of axotomized adult sensory neurons in culture. Therefore blockade of the M-current would reduce possibility of depolarization, thus theoretically preserving ATP to support actin-treadmilling in the growth cone and enhancing axon outgrowth [102, 29].