Amino acid analog toxicity in primary rat neuronal and astrocyte cultures: implications for protein misfolding and TDP-43 regulation

Abstract

Amino acid analogs promote translational errors that result in aberrant protein synthesis and have been used to understand the effects of protein misfolding in a variety of physiological and pathological settings. TDP-43 is a protein that is linked to protein aggregation and toxicity in a variety of neurodegenerative diseases. This study exposed primary rat neurons and astrocyte cultures to established amino acid analogs (canavanine and azetidine-2-carboxylic acid) and showed that both cell types undergo a dose-dependent increase in toxicity, with neurons exhibiting a greater degree of toxicity compared with astrocytes. Neurons and astrocytes exhibited similar increases in ubiquitinated and oxidized protein following analog treatment. Analog treatment increased heat shock protein (Hsp) levels in both neurons and astrocytes. In neurons, and to a lesser extent astrocytes, the levels of TDP-43 increased in response to analog treatment. Taken together, these data indicate that neurons exhibit preferential toxicity and alterations in TDP-43 in response to increased protein misfolding compared with astrocytes.

Figure 1. Amino acid analog treatment induces dose and time dependent cell death in primary rat neurons as compared to astrocytes

Cells were treated with increasing concentrations of the amino acid analog, AZC (A &B) for 3 hours or the cells were treated with 5mM AZC (C) for the indicated time intervals. Cell viability was analyzed after post treatment. Neurons were observed to have significantly higher levels of cell death in response to increasing concentrations of analog treatment as compared to astrocyte cultures. Cells treated with 5 mM of analog showed time dependent cell death with 24 hours of treatment (C), inducing more than 50% of cell death in neurons. Results using morphological criteria as well as nuclear condensation/fragmentation gave nearly identical results. Data (B&C) are presented as the mean and S.E.M. of results from three different sets of independent experiments (n=5). *p and **p < 0.05 compared to the untreated neurons and astrocytes respectively.

Figure 2. Amino acid analog treatment induces dose and time dependent cell death in primary rat neurons as compared to astrocytes

Cells were treated with increasing concentrations of the amino acid analog, Canavanine (A &B) for 3 hours or the cells were treated with 5mM Canavanine (C) for the indicated time intervals. Cell viability was analyzed after post treatment. Neurons were observed to have significantly higher levels of cell death in response to increasing concentrations of analog treatment as compared to astrocyte cultures. Cells treated with 5 mM of analog showed time dependent cell death with 24 hours of treatment (C), inducing more than 50% of cell death in neurons. Results using morphological criteria as well as nuclear condensation/fragmentation gave nearly identical results. Data (B&C) are presented as the mean and S.E.M. of results from 5 different sets of independent experiments (n= 5). *p and **p < 0.05 compared to the untreated neurons and astrocytes respectively.

Figure 3. The levels of ubiquitinated and oxidized proteins are increased in time dependent manner following analog treatment

Rat primary cortical neurons and astrocyte cultures were analyzed for the levels of ubiquitinated (A) and oxidized proteins (B) following treatment with 5mM AZC or Canavanine for 3 hours. Results showed an increase in ubiquitination and oxidation levels with the increase in time of treatment. Beta actin was used to show equal loading of protein lysates. Data are presented as the mean and S.E.M. of results from five independent experiments. *p and **p < 0.05 compared to the untreated neurons and astrocytes respectively.

Figure 4. Induction of heat shock proteins in neurons and astrocytes following time course treatment with amino acid analogs

Lysates from rat primary cortical neurons and astrocyte cultures were analyzed by Western blotting for the levels of heat shock proteins HSP70 (A& B) and HSP 40 (C & D) following amino acid analog treatment, with 5mM AZC or Canavanine, for different time intervals. Beta actin was used to show equal loading of protein lysates. Data represents results from five independent experiments done under similar conditions. See supplementary Figure 1 for quantification.

Figure 5. Levels of TDP43 in neurons and astrocytes following treatment with amino acid analogs

Whole cell lysates (A) from rat primary cortical neurons and astrocyte cultures were analyzed by western blotting for the levels of TDP 43 following treatment with different concentrations of amino acid analog, AZC or Canavanine for 3 hours. Beta actin was used to show equal loading of protein lysates. Insoluble fractions of (B) lysates from rat primary cortical neurons and astrocyte cultures were analyzed by western blotting for the levels of TDP 43 following treatment with 5mM amino acid analog, AZC or Canavanine for 3 hours. Data represents findings from five independent experiments done under similar conditions. See supplementary Figure 2 for quantification.