Kainic Acid-Induced Excitotoxicity Leads to the Activation of Heat Shock Response.

Heat shock response (HSR) which is regulated by heat shock factor 1 (HSF1) is the most important mechanism and the major regulator that prevents protein aggregation in neurodegenerative diseases. Excitotoxicity, which is the accumulation of excess glutamate in synaptic cleft, is observed in age-dependent neurodegenerative diseases and also in stroke, epilepsy, and brain trauma. Only a few studies in the literature show the link between excitotoxicity and HSR. In this study, we aimed to show the molecular mechanism underlying this link. We applied heat shock (HS) treatment and induced excitotoxicity with kainic acid (KA) in neuroblastoma (SHSY-5Y) and glia (immortalized human astrocytes (IHA)) cells. We observed that, only in SHSY-5Y cells, heat shock preconditioning increases cell survival after KA treatment. GLT-1 mRNA expression is increased as a result of KA treatment and HS due to the elevation of HSF1 binding to GLT-1 promoter which was induced by HSF1 phosphorylation and sumolation in SHSY-5Y cells. Additionally, glutamine synthetase and glutaminase expressions are increased after HS preconditioning in SHSY-5Y cells indicating that HS activates glutamate metabolism modulators and accelerates the glutamate cycle. In glia cells, we did not observe the effect of HS preconditioning. In summary, heat shock preconditioning might be protective against excitotoxicity-related cell death and degeneration.

The Regulation of GLT-1 Degradation Pathway by SIRT4.

Glial cells give rise to glioblastoma multiform as a primary brain tumor. In glioblastomas, neurons are destroyed via excitotoxicity which is the accumulation of excess glutamate in synaptic cavity. Glutamate Transporter 1 (GLT-1) is the main transporter that absorbs the excessive glutamate. Sirtuin 4 (SIRT4) was shown to have a potential protective role against excitotoxicity in previous studies. In this study, the regulation of dynamic GLT-1 expression by SIRT4 was analyzed in glia (immortalized human astrocytes) and glioblastoma (U87) cells. The expression of GLT-1 dimers and trimers were reduced and the ubiquitination of GLT-1 was increased in glioblastoma cells when SIRT4 was silenced; however GLT-1 monomer was not affected. In glia cells, SIRT4 reduction did not affect GLT-1 monomer, dimer, trimer expression or the ubiquitination of GLT-1. The phosphorylation of Nedd4-2 and the expression of PKC did not change in glioblastoma cells when SIRT4 was silenced but increased in glia cells. We also showed that SIRT4 deacetylates PKC in glia cells. In addition, GLT-1 was shown to be deacetylated by SIRT4 which might be a priority for ubiquitination. Therefore, we conclude that GLT-1 expression is regulated differently in glia and glioblastoma cells. SIRT4 activators or inhibitors of ubiquitination may be used to prevent excitotoxicity in glioblastomas.