Synthesis and Degradation of Poly(ADP-ribose) in Zebrafish Brain Exposed to Aluminum

Abstract

Poly(ADPribosyl)ation is a post-translational protein modification, catalyzed by poly(ADP-ribose) polymerase (PARPs) enzymes, responsible for ADP-ribose polymer synthesis (PAR) from NAD⁺. PAR turnover is assured by poly(ADPR) glycohydrolase (PARGs) enzymes. In our previous study, the altered histology of zebrafish brain tissue, resulting in demyelination and neurodegeneration also with poly(ADPribosyl)ation hyperactivation, was demonstrated after aluminum (Al) exposure for 10 and 15 days. On the basis of this evidence, the aim of the present research was to study the synthesis and degradation of poly(ADP-ribose) in the brain of adult zebrafish exposed to 11 mg/L of Al for 10, 15, and 20 days. For this reason, PARP and PARG expression analyses were carried out, and ADPR polymers were synthesized and digested. The data showed the presence of different PARP isoforms, among which a human PARP1 counterpart was also expressed. Moreover, the highest PARP and PARG activity levels, responsible for the PAR production and its degradation, respectively, were measured after 10 and 15 days of exposure. We suppose that PARP activation is related to DNA damage induced by Al, while PARG activation is needed to avoid PAR accumulation, which is known to inhibit PARP and promote parthanatos. On the contrary, PARP activity decrease at longer exposure times suggests that neuronal cells could adopt the stratagem of reducing polymer synthesis to avoid energy expenditure and allow cell survival.

Keywords: PARG; PARP; aluminum; brain; neurodegeneration; poly(ADP-ribose); zebrafish.

Figure 1

12% SDS-PAGE (a) and immunoblotting with anti-PARP (b), anti-PARP1 N20 (c), anti-PAR (d), and anti-β-actin (e) on control and T10, T15, and T20 samples. Densitometric analysis (f). Bars represent mean ± SD. Similar letters indicate no significant differences between treated groups.

Figure 2

12% SDS-PAGE (a) and immunoblotting with anti-PARG antibodies (b) and anti-β-actin (c) on Ctrl and T10, T15, and T20 brain homogenates. Densitometric analysis (d). Bars represent mean ± SD. Similar letters indicate no significant differences in densitometry values between the treated groups for both immunopositive bands.

Figure 3

Isolation of protein-free [³²P]-labeled poly(ADP-ribose). [³²P]ADPR oligomers from Ctrl and T20 brain homogenates; [³²P]ADPR polymers from T10 and T15 brain homogenates; degradation of [³²P]poly(ADPR) produced in brain homogenates exposed to Al for 15 days (T15*). [³²P]NAD⁺ (200 cpm), bromophenol blue (BRΦ), and xylene cyanol (XC) migrating as a tetramer, an octamer, and a 20 mer of ADP-ribose (ADPR), respectively.

Figure 4

TLC of reaction products. Protein-free [³²P]PAR (300 cpm) purified from brain homogenate exposed to Al for 15 days (T15) and degraded by PARG activity (T15*). Nucleotide standards were [³²P]NAD⁺ (300 cpm), [³²P]PAR (300 cpm), and [³²P]ADP-ribose (300 cpm).