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    • Recent studies revealed that synthetic oligomeric A decrease

    2021-09-02

    Recent studies revealed that synthetic oligomeric Aβ1-42 decreased GLT-1 expression and promoted mislocalization of GLT-1 from the cell surface of primary astrocytes, leading to glutamate dyshomeostasis in synapses (Abdul et al., 2009, Scimemi et al., 2013). To address the cause for reduction of astrocytic glutamate transport, we examined expression of EAATs. As expected, total and membrane protein expression of GLT-1 decreased in astrocytes after Aβ1-42 oligomers treatment. However, we found that astrocytic total and membrane protein expression of GLAST also decreased, which is not consistent with a previous study (Scimemi et al., 2013). This result implies that, in addition to GLT-1, dysfunction of astrocytic GLAST may participate in the pathogenesis of excitotoxicity in AD. Although GLT-1 is predominantly expressed by astrocytes, neurons also express GLT-1, which may contribute significantly to glutamate uptake into excitatory terminals (Chen et al., 2004, Chen et al., 2002, Petr et al., 2015). Therefore, we assessed the effect of Aβ1-42 oligomers on neuronal GLT-1 expression. However, no change was detected in our study. In addition, we also discovered that expression of GLAST and EAAC1 in neurons did not change. However, it is difficult to explain why neuronal glutamate uptake decreased in our study. A previous investigation demonstrated that accumulation of detergent-insoluble GLT-1 is related to cognitive impairment and neuropathological changes in the hippocampus and frontal cortex in AD patients (Woltjer et al., 2010). Thus, we hypothesize that the most likely reason for attenuation of neuronal glutamate transport by Aβ1-42 oligomers is that neuronal EAATs are transformed into detergent-insoluble forms with compromised function. Zumkehr et al. (2015) found that treatment with Aβ1-42 secreted into the CM from 7PA2-CHO dilution led to a significant decrease of GLT-1 mRNA and protein levels in a neuron-astrocyte co-culture system. However, in our study, we did not detect changes in GLT-1 mRNA and protein levels in co-cultures. This difference is possibly due to the different source of Aβ1-42 (We used synthetic Aβ1-42 oligomers; Zumkehr et al. used Aβ1-42 secreted from 7PA2-CHO cells). Previous studies of simple mixed neuron-glia cultures suggested that factors secreted by neurons could activate astroglial differentiation and expression of GLT-1 (Gegelashvili et al., 1997, Munir et al., 2000, Schlag et al., 1998). In addition, in a study by Yang et al. (2009), expression of GLT-1 in astrocytes combined with neuronal axons was significantly increased compared with that of astrocytes alone, indicating that axons can induce GLT-1 activation. We observed a reduction of GLAST protein level caused by Aβ1-42 oligomers, but this change did not affect glutamate uptake in co-cultures. All in all, little effect on glutamate uptake was observed in the co-cultures in our study. This could have been due to activation of GLT-1 in the co-culture system, which may have produced resistance to Aβ-induced excitotoxicity. To determine whether expression of GLT-1 and GLAST mRNA was regulated by Aβ1-42 oligomers, we employed qPCR and found that mRNA levels were not affected in astrocytes. These results agree with previous studies (Han et al., 2016, Li et al., 1997) and suggest that the Aβ1-42 oligomers-induced down-regulation of GLT-1 and GLAST protein expression in astrocytes is post-translational. Glutamate transporter functions can be regulated by transcriptional and post-translational mechanisms. The ubiquitin-proteasome system is a major non-lysosomal proteolytic pathway for selective degradation of cellular proteins and an important mechanism of post-translational regulation (Weng et al., 2006). In astrocytes, GLT-1 transporters are not static but rather highly mobile at the surface (Murphy-Royal et al., 2015). In several studies, ubiquitination has been shown to be involved in internalization of GLT-1 triggered by phorbol esters in a process dependent on the ubiquitin ligase Nedd4-2, an E3 ubiquitin ligase (Garcia-Tardon et al., 2012, Gonzalez-Gonzalez et al., 2008, Sheldon et al., 2008). Our results demonstrated that Aβ1-42 oligomers-induced reduction of astrocytic GLT-1 expression may be related to ubiquitination. Mechanisms underlying Aβ1-42 oligomers-induced endocytosis of glutamate transporters remain to be determined.