Differential Expression of Utrophin‐A and ‐B Promoters in the Central Nervous System (CNS) of Normal and Dystrophic mdx Mice

Abstract

Utrophin (Utrn) is the autosomal homolog of dystrophin, the Duchene Muscular Dystrophy (DMD) locus product and of therapeutic interest, as its overexpression can compensate dystrophin's absence. Utrn is transcribed by Utrn-A and -B promoters with mRNAs differing at their 5′ ends. However, previous central nervous system (CNS) studies used C-terminal antibodies recognizing both isoforms. As this distinction may impact upregulation strategies, we generated Utrn-A and -B promoter-specific antibodies, Taqman Polymerase chain reaction (PCR)-based absolute copy number assays, and luciferase-reporter constructs to study CNS of normal and dystrophic mdx mice. Differential expression of Utrn-A and -B was noted in microdissected and capillary-enriched fractions.At the protein level, Utrn-B was predominantly expressed in vasculature and ependymal lining, whereas Utrn-A was expressed in neurons, astrocytes, choroid plexus and pia mater. mRNA quantification demonstrated matching patterns of differential expression; however, transcription-translation mismatch was noted for Utrn-B in caudal brain regions. Utrn-A and Utrn-B proteins were significantly upregulated in olfactory bulb and cerebellum of mdx brain. Differential promoter activity, mRNA and protein expressions were studied in cultured C2C12, bEnd3, neurons and astrocytes. Promoter activity ranking for Utrn-A and -B was neurons > astrocytes > C2C12 > bEnd3 and bEnd3 > astrocytes > neurons > C2C12, respectively. Our results identify promoter usage patterns for therapeutic targeting and define promoter-specific differential distribution of Utrn isoforms in normal and dystrophic CNS.

Dystrophins, Utrophins, and Associated Scaffolding Complexes: Role in Mammalian Brain and Implications for Therapeutic Strategies

“…In neurons, utrophin labelling is localized along the somatic and dendritic membrane, which is at variance with the punctuate synaptic staining revealed by antidystrophin antibodies in synapses [39, 76]. The respective distributions of Utrn-A and B in the brain have been recently specified [78]: Utrn-A is mainly found in neuronal cells in various brain structures including olfactory bulb, cortex, medial septum, hypothalamus, hippocampus, cerebellum, and brainstem nuclei; it is also detected in pia mater of the meninge, choroid plexus, ependymal lining, and in some glial cells and vascular structures. Utrn-B is expressed to a lesser extent in the same brain regions and seems enriched in vascular elements.…”

Dystrophin–Glycoprotein Complex in Blood Cells

“…However, spontaneous upregulations also occur in nonmuscle tissues, such as in Dp71-deicient platelets [33] and, most importantly, in the brains of DMD mouse models [34]. However, their expression in distinct structures, as compared with dystrophin, may not relect functional compensation [24].…”

“…It has long been considered that utrophin and dystrophin share comparable functions during fetal development and adulthood, maintaining utrophin expression in adult dystrophic tissues, compensating for dystrophin loss, as has been observed in mdx skeletal and cardiac muscles [24,32]. However, spontaneous upregulations also occur in nonmuscle tissues, such as in Dp71-deicient platelets [33] and, most importantly, in the brains of DMD mouse models [34].…”

“…Transcription of full-length utrophin (Up395) is driven by two independent promoters: Utrn-A and Utrn-B. The Utrn-A protein is the main isoform in adult skeletal muscles, in contrast with Utrn-B, which is found in the vascular muscle endothelium [24]. G-utrophin, or Up113, was the irst short product identiied as a structural homolog of Dp116, while Up140 and Up71 are homologous to the short dystrophins Dp140 and Dp71, respectively; these short utrophins do not possess actin-binding sites in the N-terminal domain of the molecule [25].…”

A Method of Utrophin Up-Regulation through RNAi-Mediated Knockdown of the Transcription Factor EN1

“…24 The first promoter to be described (Promoter A) lies 5′ of exon 1 and the second promoter (Promoter B) lies 50 kb 3′ of exon 2 in the UTRN gene. 25 The complex transcriptional regulation of UTRN is not fully understood; however, facets of its transcriptional control have recently been identified. Both the ε-subunit promoter of the nicotinic acetylcholine receptor (nAChR) and promoter A in the UTRN gene contain an Nbox motif.…”