Low Dose Administration of Glutamate Triggers a Non-Apoptotic, Autophagic Response in PC12 Cells

Abstract

Background/Aims: Increasing amounts of the neurotransmitter glutamate are associated with excitotoxicity, a phenomenon related both to homeostatic processes and neurodegenerative diseases such as multiple sclerosis. Methods: PC12 cells (rat pheochromocytoma) were treated with various concentrations of the non-essential amino acid glutamate for 0.5-24 hours. The effect of glutamate on cell morphology was monitored with electron microscopy and haematoxylin-eosin staining. Cell survival was calculated with the MTT assay. Expression analysis of chaperones associated with the observed phenotype was performed using either Western Blotting at the protein level or qRT-PCR at the mRNA level. Results: Administration of glutamate in PC12 cells in doses as low as 10 μM causes an up-regulation of GRP78, GRP94 and HSC70 protein levels, while their mRNA levels show the opposite kinetics. At the same time, GAPDH and GRP75 show reduced protein levels, irrespective of their transcriptional rate. On a cellular level, low concentrations of glutamate induce an autophagy-mediated pro-survival phenotype, which is further supported by induction of the autophagic marker LC3. Conclusion: The findings in the present study underline a discrete effect of glutamate on neuronal cell fate depending on its concentration. It was also shown that a low dose of glutamate orchestrates a unique expression signature of various chaperones and induces cell autophagy, which acts in a neuroprotective fashion.

Extracellular HSP70 Activates ERK1/2, NF-kB and Pro-Inflammatory Gene Transcription Through Binding with RAGE in A549 Human Lung Cancer Cells

“…Mitogen-activated protein kinases (MAPKs) are a family of proteins involved in a wide range of cell responses mediated by inflammatory stimuli and previous works observed that RAGE activation leads to fast cell responses involving MAPK signaling cascade [28-32]. Thus, we analyzed the effect of eHSP70 on the activation state of the MAPKs ERK1/2, p38 and JNK.…”

Glutamate induces autophagy via the two-pore channels in neural cells

“…Because this concentration of glutamate does not induce toxicity and cell death, it is conceivable that the induced autophagy is related to the physiological function of this neurotransmitter [37]. …”

“…This indicates that physiological and stimulatory conditions lead to autophagy induction, which may act selectively to remove damaged organelles in a cytoprotective process [36, 37]. Glutamate is also a metabolic substrate, and particularly in astrocytes it exerts a critical role in energy production, by recycling the excessive glutamate released in synaptic cleft and converting to α-ketoglutarate and ammonia [38] therefore, in both neurons and glia, it is a source of energy, which is important for the storage of neurotransmitters as well as brain function [35].…”

Pharmacological modulation of autophagy: therapeutic potential and persisting obstacles

“…Cytosolic Ca 2+ is a potent activator of autophagy, and several excitotoxic neurotransmitters — including glutamate, N -methyl-D-aspartate (NMDA) and kainic acid — induce the expression of autophagy biomarkers in the rodent brain, possibly as a compensatory response to damage 62,63 . The depletion of BECN1 by siRNAs as well as pharmacological inhibitors of autophagy aggravated (whereas rapamycin and trehalose limited) the demise of primary rodent neurons and multiple neuronal cell lines of human origin that were exposed to glutamate, NMDA or kainic acid in vitro 64,65 , which suggests that autophagy supports neuronal viability in the course of excitotoxic challenges.…”