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    • br Materials and methods br

    2022-09-30


    Materials and methods
    Results Aberrant Aβ has been suggested to be an initiating factor for glutamate excitotoxicity in AD pathology. To investigate whether Aβ1-42 alters glutamate uptake in astrocytes, neurons, and astrocyte-neuron co-cultures, we used synthetic Aβ1-42 peptide. Aβ1-42 has fast aggregation kinetics (Bitan et al., 2003, Yan and Wang, 2006), and Aβ1-42 appears to be more pathogenic than Aβ1-40 (Benilova et al., 2012, Kuperstein et al., 2010, Matos et al., 2008). Despite the fact that these peptides tend to form amyloid fibrils and aggregates, it is widely accepted that soluble Aβ oligomers are more toxic than monomeric or fibrillar Aβ (Gerakis et al., 2016, Haass and Selkoe, 2007, Kayed and Lasagna-Reeves, 2013), and they are now considered the major cause of early synaptic loss in AD (Klein et al., 2004, Sultana and Butterfield, 2008). Aβ1-42 oligomers inhibited glutamate uptake in cultured astrocytes and neurons. Glutamate uptake after Aβ1-42 oligomers treatment for 24h was significantly reduced to 40.4±5.6% (p<0.05) in astrocytes compared with controls, and there was a slight reduction (to about 91.4±5.0%, p>0.05) after Aβ1-42 oligomers treatment for 48h (Fig. 1A). Similarly, glutamate uptake in neurons decreased to 87.6±4.1% (0.05) after Aβ1-42 oligomers treatment for 24h and to 84.9±3.0% (0.5) after treatment for 48h (Fig. 1B). However, there was no significant effect on glutamate uptake in astrocyte-neuron co-cultures (Fig. 1C). Aβ1-42 oligomers decreased total protein levels of GLAST and GLT-1 in astrocytes and GLAST in co-cultures. During neuronal activity, glutamate is released into the synaptic cleft and rapidly taken up by excitatory amino caspofungin transporters. To determine whether inhibition of glutamate uptake induced by Aβ1-42 oligomers was due to reduction in glutamate transporter levels, we measured the total level of EAATs by western blot. In astrocytes, we found that GLAST expression decreased to 76.5±6.1% after 24h of Aβ1-42 oligomers treatment, but no significant change was observed caspofungin after 48h of Aβ1-42 oligomers treatment (Fig. 2A), which was further confirmed by immunocytofluorescence (Fig. 2B, C). In addition, Aβ1-42 oligomers significantly decreased total levels of GLT-1 compared with controls after 24h of treatment (63.8±8.5%), and a slight decrease was observed after 48h of treatment (79.2±2.5%) (Fig. 2D). Consistent with these results, Aβ1-42 oligomers significantly decreased total levels of GLT-1 in 24h as detected by immunocytofluorescence (Fig. 2E, F). In neurons, no changes in total expression levels of EAATs were observed after Aβ1-42 oligomers treatment for 24h and 48h (Fig. 3A–D). In astrocyte-neuron co-cultures, total GLAST protein levels decreased to 63.6±2.2% after 24h of Aβ1-42 oligomers treatment and to 58.9±15.8% after 48h of treatment (Fig. 3E, F), but no changes in GLT-1 and EAAC1 protein levels were observed (Fig. 3G, H). Aβ1-42 oligomers decreased membrane levels of GLT-1 and GLAST in astrocytes but not in neurons or co-cultures. In general, only EAATs anchored to the plasma membrane are capable of transporting glutamate. Therefore, we further investigated whether EAATs protein expression at the membrane level was altered by Aβ1-42 oligomers. Unexpectedly, the result differed from the total level of EAATs in astrocytes. After Aβ1-42 oligomers treatment for 24h, the membrane protein level of GLAST in astrocytes decreased to 68.8±4.2% compared with controls (Fig. 4A). Similarly, compared with controls, the membrane level of GLT-1 in astrocytes decreased to 65.9±11.1% after 24h of Aβ1-42 oligomers treatment and to 72.1±2.3% after 48h of treatment (Fig. 4B), which was consistent with the results from immunocytofluorescence (Fig. 4C–F). In contrast, there was no effect on membrane expression of EAATs in neurons (Fig. S2A–D) and co-cultures (Fig. S2E–H). Furthermore, there was little change in EAATs mRNA levels in astrocytes (Fig. S3A, B) and neurons (Fig. S3C–E). In co-cultures, no change was observed in GLAST and GLT-1 mRNA levels after Aβ1-42 oligomers treatment (Fig. S3F, G), but there was a decrease in EAAC1 mRNA levels (Fig. S3H).