Abstract
PARP6, a member of a family of enzymes (17 in humans) known as poly-ADP-ribose polymerases (PARPs), is a neuronally enriched PARP. While previous studies from our group show that Parp6 is a regulator of dendrite morphogenesis in rat hippocampal neurons, its function in the nervous system in vivo is poorly understood. Here, we describe the generation of a Parp6 loss-of-function mouse model for examining the function of Parp6 during neurodevelopment in vivo. Using CRISPR-Cas9 mutagenesis, we generated a mouse line that expressed a Parp6 truncated variant (Parp6TR) in place of Parp6WT. Unlike Parp6WT, Parp6TR is devoid of catalytic activity. Homozygous Parp6TR do not exhibit obvious neuromorphological defects during development, but nevertheless die perinatally. This suggests that Parp6 catalytic activity is important for postnatal survival. We also report PARP6 mutations in six patients with several neurodevelopmental disorders, including microencephaly, intellectual disabilities, and epilepsy. The most severe mutation in PARP6 (C563R) results in the loss of catalytic activity. Expression of Parp6C563R in hippocampal neurons decreases dendrite morphogenesis. To gain further insight into PARP6 function in neurons we also performed a BioID proximity labeling experiment in hippocampal neurons and identified several microtubule-binding proteins (e.g., MAP-2) using proteomics. Taken together, our results suggest that PARP6 is an essential microtubule-regulatory gene in mice, and that the loss of PARP6 catalytic activity has detrimental effects on neuronal function in humans.
Highlights
ADP-ribosylation (ADPRylation) is a reversible regulatory post-translational modification that results in the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD+) to amino acids on proteins
To further study the role of Parp6 in the nervous system in vivo, we generated a Parp6 knockout mouse line using the CRISPR-Cas9 mutagenesis system, using a single-guide RNA sequence corresponding to exon 18 in the C-terminal catalytic domain of Parp6 (Figure 1A)
The mutation resulted in a frameshift mutation 91 residues into the catalytic domain, and a subsequent early termination codon at amino acid 507, which resulted in a truncated Parp6 protein (Parp6TR) that was devoid of the catalytic domain (Figure 1B, Table S4)
Summary
ADP-ribosylation (ADPRylation) is a reversible regulatory post-translational modification that results in the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD+) to amino acids on proteins. The enzymes that catalyze ADPRylation belong to a 17-member family known as the poly-(ADP-ribose) polymerases (PARPs), named after PARP1, the first and best characterized member of the PARP family [1,2]. The protein expression of Parp peaks during a critical period of dendritic growth and branching in primary rat hippocampal neurons [7]. Knockdown of Parp in embryonic rat hippocampal neurons decreases dendritic complexity in vitro and in vivo [7]. Overexpression of Parp increases dendritic complexity, and this effect depends on the catalytic activity of Parp. Overexpression of Parp increases dendritic complexity, and this effect depends on the catalytic activity of Parp6 Together, these results show that Parp catalytic activity plays a critical role in regulating dendrite morphogenesis during development
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