The Neurotrophic Function of Glucagon-Like Peptide-1 Promotes Human Neuroblastoma Differentiation via the PI3K-AKT Axis

Abstract

Background: Neurons are terminally-differentiated cells that generally develop from neuronal stem cells stimulated by various neurotrophic factors such as NGF, BDNF, NT3, and NT-4. Neurotrophic factors have multiple functions for neurons, including enabling neuronal development, growth, and protection. Glucagon-like peptide-1 (GLP-1) is an intestinal-secreted incretin that enhances cellular glucose up-take to decrease blood sugar levels. However, many studies suggest that the function of GLP-1 is not limited to the regulation of blood sugar levels. Instead, it may also act as a neurotrophic factor with a role in ensuring neuronal survival and neurite outgrowth, as well as protecting synaptic plasticity and memory formation. Methods: The SH-SY5Y cells were differentiated by sequential treatments of retinoic acid and GLP-1 treatment within polyethylenimine-coated dishes under serum-free Neurobasal medium. PI3K inhibitor (LY294002) and MEK inhibitor (U0126) were used to determine the signaling pathway in regulation of neuronal differentiation. Neuronal marker (TUJ1) and synaptic markers (synapsin 1, synaptophysin, and PSD95) as well as single cell patch-clamp were applied to determine maturity of neurons. Antibodies of AMPA receptor, NMDA receptor subunit 2A, dopamine receptor D1, muscarinic acetylcholine receptor 2, and nicotinic acetylcholine receptor α4 were used to examine the types of differentiated neurons. Results: Our study’s results demonstrated that the treatment with GLP-1 of SH-SY5Y human neuroblastoma cells increased the expression of AMPA receptors, NMDA receptors, dopamine receptors, synaptic proteins-synapsin 1, synaptophysin, and postsynaptic density protein 95, but not muscular and nicotinic acetylcholine receptors. In addition, the biomarker of dividing neuronal cells, vimentin, was decreased after treatment with GLP-1. Tuj1 immunostaining images showed that GLP-1 induced neurite processes and the development of neuronal morphologies. The GLP-1-differentiated neurons were able to be induced to generate action potentials by single cell patch-clamp. Our study also suggested that the PI3K-AKT axis is the dominant signaling pathway promoting the differentiation of SH-SY5Y cells into mature and functional neurons in response to GLP-1 receptor activation. Conclusions: The sequential treatment of retinoic acid and GLP-1 within a serum-free medium is able to trigger the differentiation of SH-SY5Y cells into morphologically and physiologically mature glutamatergic and dopaminergic neurons.

Neurotoxic Effect of Flavonol Myricetin in the Presence of Excess Copper

“…Similar to our observations, PARP-1-mediated death of hippocampal neurons was associated with reduced ATP content and a lack of caspase-activation, as well as characterized by the pronounced deterioration of neuronal membrane properties due to the changed expression and function of AMPA receptors [ 52 ]. This mode of PARP-1-mediated death has been observed in neurodegenerative diseases, and the expression of AMPA receptors has been confirmed in undifferentiated SH-SY5Y cells [ 52 , 53 ].…”

The neuroprotective effects of glucagon-like peptide 1 in Alzheimer’s and Parkinson’s disease: An in-depth review

“…In PC12, SH-SY5Y and adult sensory neurons, GLP-1R activation was shown to stimulate the cytoskeletal actin/tubulin polymerisation to elicit neurite multiplication, branching and outgrowth in vitro ( Perry et al, 2002b ; Liu et al, 2006 ; Luciani et al, 2010 ; Kan et al, 2012 ), comparable to NGF ( Perry et al, 2002b ). A GLP-1 mimetic further promoted the expression of various synaptic proteins, including synapsin 1, synaptophysin and PSD-95, in SH-SY5Y cells ( Yang et al, 2020 ) and PSD-95 in the neocortex of adult mice ( Ohtake et al, 2014 ). As we describe elsewhere (section “GLP-1R agonists promote neurogenesis”), these pro-neuritic and pro-synaptic effects are the result of (cAMP/PKA-supported) CREB activation and brain derived neurotrophic factor (BDNF) expression by GLP-1 mimetics.…”

“…As indicated in another study using PC12 cells, GLP-1 seems to potentiate the differentiation-enhancing effect of other neurotrophins (NGF in this case) ( Perry et al, 2002b ). Indeed, when GLP-1 was applied to SH-SY5Y cells following initial retinoic acid treatment, the incretin hormone attenuated vimentin levels (a marker of proliferating neural progenitor cells) and induced AMPA, NMDA, and dopamine receptor and synaptic protein expression, neurite outgrowth, the development of neuron-like morphologies and the ability to generate action potentials ( Yang et al, 2020 ). Furthermore, amongst other effects, GLP-1R induction enhanced Na 2+ and L/T-type VDCC currents, paralleling those of mature neurons ( Luciani et al, 2010 ).…”

“…The use of pharmacological inhibitors demonstrated that the GLP-1-boosted growth, differentiation toward glutamatergic/dopaminergic neurons and survival were dependent on the PI3K/Akt/CREB axis and partially on MEK ( Yang et al, 2020 ). Other studies indicate that GLP-1R-induced Ras/Raf/MEK/ERK-signaling, as confirmed with MEK inhibitor treatment of PC12 cells, drive GAP-43 expression as well as GAP-43-associated neurite outgrowth and branching ( Liu et al, 2006 ).…”

Methods used to achieve different levels of the neuronal differentiation process in SH‐SY5Y and Neuro2a cell lines: An integrative review

“…PSD95, for example, is associated with synaptic maturation in synaptic regions, whereas SAP97 is associated with synapses and neuronal excitability. Synaptophysin, a membrane glycoprotein, promotes neurotransmission (Chaudhari & Ravanan, 2018; Tarsa & Balkowiec, 2009), whereas SV2 promotes neurotransmitter release (Ciruelas et al, 2019; Mahabadi & Taghibiglou, 2020; Yang et al, 2020).…”