The neuroprotective WldS gene regulates expression of PTTG1 and erythroid differentiation regulator 1-like gene in mice and human cells

Abstract

Wallerian degeneration of injured neuronal axons and synapses is blocked in Wld(S) mutant mice by expression of an nicotinamide mononucleotide adenylyl transferase 1 (Nmnat-1)/truncated-Ube4b chimeric gene. The protein product of the Wld(S) gene localizes to neuronal nuclei. Here we show that Wld(S) protein expression selectively alters mRNA levels of other genes in Wld(S) mouse cerebellum in vivo and following transfection of human embryonic kidney (HEK293) cells in vitro. The largest changes, identified by microarray analysis and quantitative real-time polymerase chain reaction of cerebellar mRNA, were an approximate 10-fold down-regulation of pituitary tumour-transforming gene-1 (pttg1) and an approximate 5-fold up-regulation of a structural homologue of erythroid differentiation regulator-1 (edr1l-EST). Transfection of HEK293 cells with a Wld(S)-eGFP construct produced similar changes in mRNA levels for these and seven other genes, suggesting that regulation of gene expression by Wld(S) is conserved across different species, including humans. Similar modifications in mRNA levels were mimicked for some of the genes (including pttg1) by 1 mm nicotinamide adenine dinucleotide (NAD). However, expression levels of most other genes (including edr1l-EST) were insensitive to NAD. Pttg1(-/-) mutant mice showed no neuroprotective phenotype. Transfection of HEK293 cells with constructs comprising either full-length Nmnat-1 or the truncated Ube4b fragment (N70-Ube4b) demonstrated selective effects of Nmnat-1 (down-regulated pttg1) and N70-Ube4b (up-regulated edr1l-EST) on mRNA levels. Similar changes in pttg1 and edr1l-EST were observed in the mouse NSC34 motor neuron-like cell line following stable transfection with Wld(S). Together, the data suggest that the Wld(S) protein co-regulates expression of a consistent subset of genes in both mouse neurons and human cells. Targeting Wld(S)-induced gene expression may lead to novel therapies for neurodegeneration induced by trauma or by disease in humans.

Non-Nuclear WldSDetermines Its Neuroprotective Efficacy for Axons and SynapsesIn Vivo

“…We hypothesize now that this putative factor could be axonally transported Wld S itself. As rate of axonal transport declines with age (Cross et al, 2008) (Gillingwater et al, 2006;Simonin et al, 2007b) may be less linked to the high nuclear Wld S concentration than expected and feedback mechanisms from cytoplasmic Wld S could be one other explanation for the gene expression data. Previous data based on strong lentiviral overexpression of Nmnat1 in DRG neurons suggested efficacy was independent of subcellular targeting (Sasaki et al, 2006) although it is not clear whether this reflects the axon protection mechanism in vivo (Conforti et al, 2007).…”

“…A key question about the Wld S mechanism surrounds the subcellular site of its action (Fainzilber and Twiss, 2006). In vivo studies have consistently detected Wld S only in the nucleus (Mack et al, 2001;Samsam et al, 2003;Sajadi et al, 2004;Wilbrey et al, 2008), suggesting that Wld S confers its axonal effect indirectly by putative nuclear mechanisms (Araki et al, 2004;Gillingwater et al, 2006;Simonin et al, 2007a). However, the complete absence of Wld S in other cellular compartments could not be proven experimentally owing to detection limits and there are precedents for proteins acting in a subcellular compartment where they are barely detectable (Hamilton et al, 2001).…”

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

“…Second, expression of full-length Wld S protein alters the expression of a small subset of genes, but these changes are not fully reproduced when N-terminal sequences are deleted. 31 It is not yet known whether either of these effects is required for axon protection, or whether they are linked with one another. Importantly, however, both involve nuclear events, and the nucleus remains the only intracellular location in vivo where Wld S has been detected.…”

A Single Amino Acid Substitution (Cys249Trp) in Crb1 Causes Retinal Degeneration and Deregulates Expression of Pituitary Tumor Transforming GenePttg1

“…Pttg1 is a mitotic checkpoint protein that inhibits sister chromatid separation during mitosis. Several other functions have been attributed to this cytoplasmic and nuclear protein, such as regulation of gene transcription, cell proliferation, and apoptosis (Pei and Melmed, 1997;Heaney et al, 1999;Zhang et al, 1999;Yu et al, 2000;Bernal et al, 2002;Wang et al, 2003;Boelaert et al, 2004;Hamid and Kakar, 2004;Hamid et al, 2005;Gillingwater et al, 2006). Pttg1 Ϫ/Ϫ mice developed aberrations in cell cycle progression, hyperplasia in testis, and chromosomal breaks (Wang et al, 2001).…”