Elsevier

Neurobiology of Aging

Volume 34, Issue 1, January 2013, Pages 248-262
Neurobiology of Aging

Regular article
Rho guanine nucleotide exchange factor is an NFL mRNA destabilizing factor that forms cytoplasmic inclusions in amyotrophic lateral sclerosis

https://doi.org/10.1016/j.neurobiolaging.2012.06.021Get rights and content

Abstract

Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive disorder of unknown etiology characterized by the selective degeneration of motor neurons. Recent evidence supports the hypothesis that alterations in RNA metabolism in motor neurons can explain the development of protein inclusions, including neurofilamentous aggregates, observed in this pathology. In mice, p190RhoGEF, a guanine nucleotide exchange factor, is involved in neurofilament protein aggregation in an RNA-triggered transgenic model of motor neuron disease. Here, we observed that rho guanine nucleotide exchange factor (RGNEF), the human homologue of p190RhoGEF, binds low molecular weight neurofilament mRNA and affects its stability via 3′ untranslated region destabilization. We observed that the overexpression of RGNEF in a stable cell line significantly decreased the level of low molecular weight neurofilament protein. Furthermore, we observed RGNEF cytoplasmic inclusions in ALS spinal motor neurons that colocalized with ubiquitin, p62/sequestosome-1, and TAR (trans-active regulatory) DNA-binding protein 43 (TDP-43). Our results provide further evidence that RNA metabolism pathways are integral to ALS pathology. This is also the first described link between ALS and an RNA binding protein with aggregate formation that is also a central cell signaling pathway molecule.

Introduction

Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive disorder characterized by the selective degeneration of motor neurons resulting in paralysis and death 3 to 5 years after onset in most patients (Strong et al., 2005). There are no treatments that will arrest disease progression and the cause remains elusive. Protein aggregate formation in motor neurons is a neuropathological hallmark of this disease. These aggregates contain proteins such as neurofilament (NF) (Kondo et al., 1986), peripherin (He and Hays, 2004), TAR DNA binding protein of 43 kDa (TDP-43) (Arai et al., 2006, Neumann et al., 2006), fused in sarcoma/translocated in liposarcoma (FUS/TLS) (Kwiatkowski et al., 2009, Vance et al., 2009), copper/zinc superoxide dismutase 1 (SOD1) (Shaw et al., 2008, Stieber et al., 2000), ubiquitin (Arai et al., 2006, Neumann et al., 2006), and 14-3-3 (Kawamoto et al., 2004). Additionally, motor neurons also show a selective decrease in the levels of polyadenylated mRNA, low molecular weight neurofilament (NFL) mRNA, α-internexin mRNA, and peripherin mRNA (Bergeron et al., 1994, Wong et al., 2000). These data, together with the participation of TDP-43 and FUS/TLS as RNA binding proteins (Buratti et al., 2001, Crozat et al., 1993), supports the hypothesis that alterations in RNA metabolism in motor neurons can lead to the development of protein aggregates in ALS (Strong, 2010). This hypothesis has been supported with a recent report showing a pathological association of an expanded hexanucleotide repeat in the noncoding region of C9ORF72 with ALS (Dejesus-Hernandez et al., 2011, Renton et al., 2011).

The NF proteins are a highly conserved family of neuronal intermediate filament proteins. There are 3 members of the NF family, a 68-kDa form (NFL), a 160-kDa form (middle molecular weight NF), and a 200-kDa form (high molecular weight NF). NF subunits assemble as heteropolymers in which the initial polymerization of NFL subunits is required for normal NF architecture formation (for review see Strong, 1999). In transgenic mouse models overexpressing peripherin or high molecular weight NF in which there is targeted disruption of the NFL gene, selective death of motor neurons is observed, along with motor dysfunction and the formation of intermediate filament aggregates, including intraneuronal NF aggregates (Beaulieu et al., 1999, Beaulieu et al., 2000). These results highlight the importance of NF stoichiometry in motor neurons and the importance of NFL mRNA stability as a possible cause of the protein aggregates observed in ALS (Szaro and Strong, 2010, Thyagarajan et al., 2007).

In mice, it has been observed that p190RhoGEF, a guanine nucleotide exchange factor (GEF) (van Horck et al., 2001) interacts with a destabilizing region of NFL mRNA providing stability to the transcript (Cañete-Soler et al., 2001). This protein is involved in NF protein aggregation observed in an RNA-triggered transgenic model of motor neuron disease (Lin et al., 2005, Nie et al., 2002). Previously, we showed that the RNA binding domain of the human homologue of p190RhoGEF, called rho guanine nucleotide exchange factor (RGNEF), can interact with NFL mRNA (Volkening et al., 2010). Here, we show that full length RGNEF is an RNA binding protein that acts as an NFL mRNA stability factor via 3′ untranslated region (UTR) destabilization and reduces NFL protein levels when overexpressed in HEK293T cells. Moreover, we show that RGNEF also presents a pathogenic phenotype including aggregate formation in motor neurons of ALS patients.

Section snippets

Cases

Postmortem frozen and formalin-fixed, paraffin-embedded tissues were collected as part of the ALS protocol at London Health Sciences Centre (London, Ontario, Canada). Six control cases (4 male, 2 female, age range 61–74 years), 13 sporadic ALS (sALS; 8 male, 5 female, age range 45–80 years) cases, and 3 familial ALS (fALS; 2 male, 1 female, age range 56–71 years) cases without known mutations in the coding sequence of SOD1, FUS/TLS, and TDP-43 and without C9ORF72 expanded repeats were used in

Human RGNEF shows a high similarity with murine p190RhoGEF

An amino acid (aa) sequence comparison analysis was performed to determine the similarity between the murine p190RhoGEF (GenBank U73199) and its human homolog, RGNEF (GenBank BC157846). These proteins share the same conserved domains (Fig. 1A). After Basic Local Alignment Search Tool analysis (BLAST utility; blast.ncbi.nlm.nih.gov), we observed an 80% identity between p190RhoGEF and RGNEF at the protein level. The GEF domain (mouse: aa 847–1039; human: aa 850–1042 according the conserved

Discussion

The major finding in this study is that RGNEF, the human homolog of the mouse protein p190RhoGEF, is a bifunctional protein that appears to be involved in the pathology of ALS. This manifests as aggregates and skeins that colocalize with other proteins that have been described as pathological in ALS (TDP-43, ubiquitin, p62/Sequestosome-1). We have shown that RGNEF is an RNA binding protein that acts as an RNA destabilizing factor for human NFL mRNA via its 3′ UTR, and can regulate NFL protein

Disclosure statement

The authors declare that they have no competing interests.

Acknowledgements

The authors thank Dr. Rosa Rademakers for the C9ORF72 expanded repeats genotyping of ALS patients and Dr. Wouter H. Moolenaar for kindly providing pcDNA-p190RhoGEF plasmid. This work was supported by the Canadian Institute of Health Research (CIHR), The McFeat Family Fund, and the ALS Society of Canada.

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