Expanded Hexanucleotide GGGGCC Repeat Non-coding RNA Associated With ALS/FTD Cause Neurodegeneration

Amyotrophic lateral sclerosis (ALS) and Frontotemporal dementia (FTD) are two fatal neurodegenerative diseases without effective therapies. ALS has traditionally been considered a disorder in which degeneration of upper and lower motor neurons gives rise to progressive muscle weakness, spasticity and disturbances of speech, swallowing, or breathing. FTD is the second most common cause of presenile dementia in which degeneration of the frontal and temporal lobes of the brain results in progressive changes in personality, behavior, and language with relative preservation of perception and memory. ALS and FTD are two extremes of a spectrum of clinically, pathologically, and genetically overlapping disorders. ALS is increasingly recognized to be a multisystem disorder with impairment of frontotemporal functions such as cognition and behavior in up to50%of patients. Similarly, as many as half of FTD patients develop clinical symptoms of motor neuron dysfunction. Recently discovery showed that the transactive response DNA binding protein with Mr43kD (TDP-43) was mutant in the vast majority of ALS cases and in the most common pathological subtype of FTD, and TDP-43-positive inclusions within the CNS.A number of families have been reported with an autosomal dominant pattern of disease in which affected members may develop either FTD or ALS or both (FTD-ALS). Several of these families have shown genetic linkage to a region on chromosome9p21. The same chromosomal region has been identified in several large independent genome-wide association studies of both ALS and FTD, implicating the genetic defect at chromosome9p in sporadic forms of both diseases. Recently, two independent studies reported identification of the FTD/ALS gene defect on chromosome9p as being a massively expanded GGGGCC hexanucleotide repeat in a non-coding region of the chromosome9open reading frame72gene (C9ORF72). The mutation was found to result in loss of one alternatively spliced C9ORF72transcript and the formation of nuclear RNA foci composed of the hexanucleotide repeat. In these two studies, the C9ORF72mutation was found to be the most common genetic abnormality in familial and sporadic forms of both FTD and of ALS and was particularly frequent in patients and families with both conditions.Expansions of short tandem repeat of nucleotides in single genes cause hereditary neurological diseases in humans. The abnormally expanded microsatellites can lead to a variety of effects on genes, including the inhibition of transcription and the loss-of-function of protein products. In several cases, the repeat expansions confer toxicity to the mutant transcript RNAs, which colocalize with some host proteins and forms microscopic ribonuclear inclusions, which are capable of disrupting cell functions, leading to neurodegenerative diseases.However, the molecular pathogenesis underlying this expanded repeat remains unknown. There is no report show that whether and how expanded GGGGCC repeat non-coding RNA cause neurodegeneration. Here we developed both mammalian neuronal cell and Drosophila models of this expanded hexanucleotide repeat and showed that the expression of the expanded GGGGCC repeat RNA r(GGGGCC)30is sufficient to cause neurodegeneration. Furthermore, expanded GGGGCC repeat RNA could sequester specific RNA-binding protein Purine-rich single-stranded DNA-binding protein alpha (Pur alpha) from its normal functions, ultimately leading to neurodegeneration.Expanded GGGGCC repeats inhibit expression of gene downstream. To determine whether the expanded GGGGCC repeat could influent gene expression, we cloned both normal (3) and expanded (30) GGGGCC repeats into a mammalian expression vector pEFGP-N3. GGGGCC repeats were inserted into the5’-untranslated region (5’-UTR) between the transcriptional start site and translational start site of enhanced green fluorescent protein (EFGP) reporter gene. These constructs were transiently transfected into a mouse neuronal cell line, Neuro-2a cell, and EGFP expression was determined. At48hours post-transfection, cells transfected with the construct expressing the expanded GGGGCC repeats (GGGGCC)3o displayed significantly reduced EGFP mRNA and protein level compared to those transfected with either EGFP alone or the one expressing normal GGGGCC repeat (GGGGCC)3, suggesting that the expanded GGGGCC repeats can inhibit the transcription of gene downstream.Expanded GGGGCC repeats non-coding RNA cause neurodegeneration. To determine whether the expanded GGGGCC repeats RNA could cause neuronal toxicity, we cloned both normal (3) and expanded (30) GGGGCC repeats into a mammalian expression vector. These constructs were transiently transfected into Neuro-2a cells, and cell viability was determined with Cell-Titer Blue Assay. At48hours post-transfection, cells transfected with the construct expressing the expanded GGGGCC repeats (GGGGCC)30displayed significantly reduced viability compared to those transfected with either EGFP alone or the one expressing normal GGGGCC repeat (GGGGCC)3. Besides, by immunofluorescence staining we only found ubiquitin-positive inclusions in the cytoplasm expressing expanded GGGGCC repeats (GGGGCC)3o. These suggest that the expanded GGGGCC repeats RNA are sufficient to cause neuronal cell death.To further evaluate this in vivo, we expressed either normal (3) or expanded (30) GGGGCC repeats in Drosophila melanogaster to determine whether expanded GGGGCC repeats RNA also cause neuronal cell death. Both normal (3) and expanded (30) GGGGCC repeats were cloned into the Drosophila transformation vector pUAST-EGFP with a position similar to the mammalian vectors that we generated above. Control of transgene expression and tissue specificity was achieved by the GAL4/UAS (upstream activator sequence) system.Gmr-GAL4directed the expression of target gene specifically in retina of Drosophila. We saw that the expression of expanded repeats RNA (Gmr-GAL4/UAS-(GGGGCC)30-EGFP) could severely disrupt eye morphology including dramatic cell death, loss of pigmentation, and ommatidial disruption. We also examined phenotypes caused by the expression of rGGGGCC repeats in the eyes of different aged flies (Day1, Day7, Day14, Day21and Day28) and saw increased disruptions of eye morphology with the aged transgenic flies expressing the expanded GGGGCC repeats RNA, whereas expression of EGFP and normal repeats did not disrupt eye morphology up to28days.We took advantage of the UAS/GAL4system to drive the expression of our transgenes in motor neuron specifically using the motor neuron-specific driver, Ok371-GAL4. To determine the impact of the rGGGGCC repeat on motor neurons, we examined the locomotor activity of these flies using a Drosophila Activity Monitor (DAM) system. At Day7post-eclosion, we saw no difference in either the normal (OK371-GAL4/UAS-(GGGGCC)3-EGFP) or expanded (OK371-GAL4/UAS-(GGGGCC)30-EGFP) GGGGCC repeat flies compared to controls (OK371-GAL4/w1118); however, at Day28post-eclosion we observed a significant reduction of locomotor activity in the flies expressing the r(GGGGCC)3o repeat, but not the r(GGGGCC)3repeat compared to the control fly.These results suggest that the expression of expanded GGGGCC repeats non-coding RNA could induce progressive neurodegeneration in transgenic flies.Pur alpha binds to expanded GGGGCC repeat RNA in a sequence-specific fashion. One mechanism for expanded non-coding RNA mediated neuronal degeneration is sequestration of RBPs from their normal functions. Thus, we expect the expanded GGGGCC repeats non-coding RNA has same mechanism. Identifying any such specific RNA-binding protein(s) would be the key to understanding the molecular pathogenesis of neurodegeneration caused by the expanded GGGGCC repeats non-coding RNA. To explore this, we synthesized biotinylated r(GGGGCC)10probe, and incubated it with the whole cell lysate from mouse spinal cord. Streptavidin-coated magnetic beads were used to capture proteins that bound r(GGGGCC)10. We identified the specific RBPs binding to the rGGGGCC repeats by mass spectrometry. Most abundant protein sequenced was Pur alpha.To determine the specificity of these RBPs for the RNA-protein binding assay, we performed competition assays using unlabeled RNA oligos. The addition of excess unlabeled r(GGGGCC)10and r(CGG)10repeat abolished their ability to capture these protein(s). In the presence of unlabeled r(CGG)10, we found that the specific RBPs were partially reduced.To determine the specificity of Pur alpha for the rGGGGCC repeat, we expressed recombinant GST-tagged mouse and Drosophila Pur alpha. We performed RNA-protein binding assays by using different recombinant proteins and probe (32P-labeled r(GGGGCC)10). We found both mouse and Drosophila Pur alpha could bind to probe in a dose-dependent fashion, whereas essentially no binding was seen with the GST control or GST-hnRNP A2/B1. We performed Western blot using the RBPs pulled down by biotinylated r(GGGGCC)10. We found that both human and mouse Pur alpha, but not TDP-43, bind with rGGGGCC repeats. It suggests that the specific interaction between Pur alpha and rGGGGCC repeats is conserved among Drosophila, mouse and human.To confirm the interaction between Pur alpha and expanded GGGGCC repeats non-coding RNA in vivo, we performed RNA immunoprecipitation (RIP) using mouse Neuro-2a cells. In this experiment, we saw a significant enrichment of EGFP mRNA containing r(GGGGCC)30-EGFP, but not EGFP alone or r(GGGGCC)3-EGFP. These data demonstrate that Pur alpha is indeed associated with expanded rGGGGCC repeat-containing RNA in vivo.Pur alpha mitigates expanded GGGGCC repeat non-coding RNA-mediated neurodegeneration in mouse neuronal cells and Drosophila. To test the function of Pur alpha in the pathogenesis of expanded GGGGCC repeats non-coding RNA induced neuronal degeneration, we used Neuro-2a cells and transiently co-transfected the (GGGGCC)3o-EGFP construct along with the mammalian expression vectors that could express either Pur alpha or hnRNP A2/B1. At48hours post-transfection, we saw the cell death was attenuated in cells co-transfected with the (GGGGCC)3o-EGFP and Pur alpha. Furthermore, the knockdown of Pur alpha could significantly reduce the cell viability. We also examined the genetic interaction between Pur alpha and rGGGGCC repeat-mediated neurodegeneration in Drosophila. We crossed UAS-(GGGGCC)3o-EGFP transgenic flies with UAS-Pur alpha transgenic flies in the presence of the Gmr-GAL4driver. We found that overexpression of Pur alpha could suppress expanded GGGGCC repeats non-coding RNA mediated neurodegeneration.Taken together, our research suggests that expanded GGGGCC repeats non-coding RNA induce neurodegeneration by sequestration of normal RBPs from their normal function.