Progranulin modulates zebrafish motoneuron development in vivoand rescues truncation defects associated with knockdown of Survival motor neuron 1

Babykumari P Chitramuthu,Denis G Kay,Andrew Bateman,David C Baranowski,Hugh PJ Bennett

doi:10.1186/1750-1326-5-41

Abstract

BackgroundProgranulin (PGRN) encoded by the GRN gene, is a secreted glycoprotein growth factor that has been implicated in many physiological and pathophysiological processes. PGRN haploinsufficiency caused by autosomal dominant mutations within the GRN gene leads to progressive neuronal atrophy in the form of frontotemporal lobar degeneration (FTLD). This form of the disease is associated with neuronal inclusions that bear the ubiquitinated TAR DNA Binding Protein-43 (TDP-43) molecular signature (FTLD-U). The neurotrophic properties of PGRN in vitro have recently been reported but the role of PGRN in neurons is not well understood. Here we document the neuronal expression and functions of PGRN in spinal cord motoneuron (MN) maturation and branching in vivo using zebrafish, a well established model of vertebrate embryonic development.ResultsWhole-mount in situ hybridization and immunohistochemical analyses of zebrafish embryos revealed that zfPGRN-A is expressed within the peripheral and central nervous systems including the caudal primary (CaP) MNs within the spinal cord. Knockdown of zfPGRN-A mRNA translation mediated by antisense morpholino oligonucleotides disrupted normal CaP MN development resulting in both truncated MNs and inappropriate early branching. Ectopic over-expression of zfPGRN-A mRNA resulted in increased MN branching and rescued the truncation defects brought about by knockdown of zfPGRN-A expression. The ability of PGRN to interact with established MN developmental pathways was tested. PGRN over-expression was found to reverse the truncation defect resulting from knockdown of Survival of motor neuron 1 (smn1). This is involved in small ribonucleoprotein biogenesis RNA processing, mutations of which cause Spinal Muscular Atrophy (SMA) in humans. It did not reverse the MN defects caused by interfering with the neuronal guidance pathway by knockdown of expression of NRP-1, a semaphorin co-receptor.ConclusionsExpression of PGRN within MNs and the observed phenotypes resulting from mRNA knockdown and over-expression are consistent with a role in the regulation of spinal cord MN development and branching. This study presents the first in vivo demonstration of the neurotrophic properties of PGRN and suggests possible future therapeutic applications in the treatment of neurodegenerative diseases.

Highlights

Progranulin (PGRN) encoded by the GRN gene, is a secreted glycoprotein growth factor that has been implicated in many physiological and pathophysiological processes
Zebrafish progranulin-A is expressed within the Central Nervous System (CNS) and Peripheral Nervous System (PNS) In our initial studies of zebrafish PGRN expression [32] we demonstrated that there is general zfPGRN-A knockdown results in disrupted development of multiple neural structures Antisense morpholino (MO) technology was employed to investigate the function of zfPGRN-A in neural development by knocking down gene expression by injection oligonucleotides into zebrafish embryos at the 1 to 2 cell stage
The efficacy of translation from injected mRNA was confirmed by injection of hPGRN mRNA or control green fluorescent protein mRNA which were shown by Western blot to be translated into protein products (Figure 3, Panel C)

Summary

Introduction

Progranulin (PGRN) encoded by the GRN gene, is a secreted glycoprotein growth factor that has been implicated in many physiological and pathophysiological processes. PGRN haploinsufficiency caused by autosomal dominant mutations within the GRN gene leads to progressive neuronal atrophy in the form of frontotemporal lobar degeneration (FTLD). This form of the disease is associated with neuronal inclusions that bear the ubiquitinated TAR DNA Binding Protein-43 (TDP-43) molecular signature (FTLD-U). Progranulin (PGRN) ( called granulin-epithelin precursor, PC-derived growth factor, transforming growth factor-e, acrogranin or proepithelin) is a secreted cause a progressive neurodegenerative disease, namely frontotemporal lobar degeneration associated with ubiquitin inclusions (FTLD-U). Familial FTLD is heterogeneous and in addition to GRN mutations, arise from mutations of MAPT, the gene encoding the Tau protein This variant differs histologically from FTLD-U in displaying Tau aggregation but lacking ubiquitinated TDP-43 inclusions [14]. FTLD-U is invariably fatal and no effective treatment exists at present [21]

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