A Ufd2/D4Cole1e chimeric protein and overexpression of Rbp7 in the slow Wallerian degeneration ( Wld ^S ) mouse

Abstract

Exons of three genes were identified within the 85-kilobase tandem triplication unit of the slow Wallerian degeneration mutant mouse, C57BL͞Wld S . Ubiquitin fusion degradation protein 2 (Ufd2) and a previously undescribed gene, D4Cole1e, span the proximal and distal boundaries of the repeat unit, respectively. They have the same chromosomal orientation and form a chimeric gene when brought together at the boundaries between adjacent repeat units in Wld S . The chimeric mRNA is abundantly expressed in the nervous system and encodes an in-frame fusion protein consisting of the N-terminal 70 amino acids of Ufd2, the C-terminal 302 amino acids of D4Cole1e, and an aspartic acid formed at the junction. Antisera raised against synthetic peptides detect the expected 43-kDa protein specifically in Wld S brain. This expression pattern, together with the previously established role of ubiquitination in axon degeneration, makes the chimeric gene a promising candidate for Wld. The third gene altered by the triplication, Rbp7, is a novel member of the cellular retinoid-binding protein family and is highly expressed in white adipose tissue and mammary gland. The whole gene lies within the repeat unit leading to overexpression of the normal transcript in Wld S mice. However, it is undetectable on Northern blots of Wld S brain and seems unlikely to be the Wld gene. These data reveal both a candidate gene for Wld and the potential of the Wld S mutant for studies of ubiquitin and retinoid metabolism.

NAD+ and axon degeneration revisited: Nmnat1 cannot substitute for WldS to delay Wallerian degeneration

“…Nmnat1 and Wallerian degeneration L Conforti et al as described previously, 14 and mouse monoclonal anti-b-actin (Abcam) was used as a loading control.…”

“…Two antibodies raised against the Wld S protein, 183 and Wld-18, were used to demonstrate expression of Ube4b/Nmnat1 and W258A, as described previously. 8,14 Expression of Nmnat1 was demonstrated with a polyclonal antibody to Nmnat1 (D20 from Santa Cruz Biotech, 1 : 100 concentration).…”

“…6 Our groups showed that the gene identified by positional cloning [12][13][14] delays Wallerian degeneration in transgenic mice and virally transduced dorsal root ganglion (DRG) explant cultures. 15,16 Wld S encodes a fusion protein containing 70 N-terminal amino acids of ubiquitination factor Ube4b, full-length nicotinamide adenine dinucleotide (NAD þ )-synthesising enzyme nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1), and a unique 18-amino-acid joining sequence.…”

WldS, Nmnats and axon degeneration-progress in the past two decades

“…Genetic studies showed that the slow Wallerian degeneration phenotype in the Wld S mice is due to the over-expression of an fusion protein Wld S that consists of the amino-terminal 70 amino acids of UFD2, which is an evolutionally conserved protein involved in protein poly-ubiquitination (Koegl et al, 1999), and the nicotinamide mononucleotide adenylyltransferase (Nmnat1) (Conforti et al, 2000). Initially, there was some controversy regarding whether individual fragments of the Wld S gene (UFD2 or Nmnat1), or the entire chimeric gene, are responsible for the phenotypes (Araki et al, 2004;Wang et al, 2005;Zhai et al, 2006;Conforti et al, 2007).…”

“…Perhaps the Nmnat1 expressed in transgenic mice generated by Conforti (Conforti et al, 2007) did not reach a high level and still primarily localized in the nucleus; therefore, insufficient amounts of Nmnat1 were available in the axons for the protective effects. In addition, although many reports suggest that Wld S is localized to the nucleus of neurons in the Wld S mice (Coleman, 2005), all of these studies relied on an antibody prepared against an 18-amino acid peptide in Wld S (between UFD2 and Nmnat1) (Conforti et al, 2000;Fang et al, 2005). UFD2 itself localizes to both the nucleus and axons (Fang et al, 2005), and thus the fusion protein Wld S might be brought to axons by the UFD2 fragment.…”