C9orf72-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility

Abstract

Hexanucleotide repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this mutation leads to these neurodegenerative diseases remains unclear. Here, we use human induced pluripotent stem cell-derived motor neurons to show that C9orf72 repeat expansions impair microtubule-based transport of mitochondria, a process critical for maintenance of neuronal function. Cargo transport defects are recapitulated by treating healthy neurons with the arginine-rich dipeptide repeat proteins (DPRs) that are produced by the hexanucleotide repeat expansions. Single-molecule imaging shows that these DPRs perturb motility of purified kinesin-1 and cytoplasmic dynein-1 motors along microtubules in vitro. Additional in vitro and in vivo data indicate that the DPRs impair transport by interacting with both microtubules and the motor complexes. We also show that kinesin-1 is enriched in DPR inclusions in patient brains and that increasing the level of this motor strongly suppresses the toxic effects of arginine-rich DPR expression in a Drosophila model. Collectively, our study implicates an inhibitory interaction of arginine-rich DPRs with the axonal transport machinery in C9orf72-associated ALS/FTD and thereby points to novel potential therapeutic strategies. INTRODUCTIONA GGGGCC (G4C2) repeat expansion in the C9orf72 gene is the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9-ALS/FTD) (1,2). Since the association between the hexanucleotide repeat expansions (HREs) and these neurodegenerative diseases was discovered, three non-mutually exclusive pathological mechanisms have been proposed. The first is a loss-of-function scenario due to decreased expression of C9orf72 transcript and protein observed in C9-ALS/FTD patients (1,3). The second is an RNA gain-of-function mechanism caused by the accumulation of expanded repeat transcripts that sequester numerous RNA-binding proteins (4-9). The third proposed mechanism is a protein gain-of-function via the generation of pathological dipeptide repeat proteins (DPRs) originating from non-ATG mediated translation of the expanded repeat transcripts (10-13).This repeat-associated non-ATG (RAN) translation occurs in all reading frames of sense and antisense transcripts resulting in five DPR proteins: poly-GR and poly-GA exclusively from the sense transcript, poly-PR and poly-PA exclusively from the antisense transcript, and poly-GP from both transcripts (10-13). DPRs are found in cytoplasmic inclusions in C9-ALS/FTD post-mortem brain and spinal cord tissue, and also have been detected in motor neurons differentiated from patient-derived induced pluripotent stem cells (iPSCs) (5,10,11,(14)(15)(16)(17)(18). The arginine-rich DPRs -poly-PR and poly-GR -are potently toxic in numerous disease models (14,(19)(20)(21)(22)(23)(24)(25)(26)(27), and have been shown to cause mitochondrial (28,29) and endoplasmic reticulum stress (26), as well as disturbances...

High content live profiling reveals concomitant gain and loss of function pathomechanisms in C9ORF72 amyotrophic lateral sclerosis

“…Consistently, treatment of iPSC-derived control MNs with recombinant DPRs of only 20 repeats were shown (currently under revision at Science translational medicine) to be sufficient to inhibit the microtubule-based motor proteins kinesin-1 and dynein in axons (48).…”

“…We DPRs provides already itself a sufficient explanation of the underlying mechanism as a pending report (under review at Science translational medicine) shows that DPRs can directly inhibit microtubule-based axonal transport of mitochondria and RNA granules by interfering with kinesin-1 and dynein in vivo as well as in vitro-recapitulated (i.e. cell-free) motility assays using recombinant DPRs (48). Moreover, we found augmented DSBs at D80 concurring with DPR foci within same MNs in C9 and C9-KO ( fig.…”

C9orf72 loss-of-function: a trivial, stand-alone or additive mechanism in C9 ALS/FTD?

“…supra), most C9 patient iPSC-derived neurons exhibit an overall reduction (50%) in C9orf72 transcripts (Table 2) [3,29,78,104,117]. On the contrary, a recent study did not observe these changes in individual and total transcript levels [40]. Also, fibroblasts and iPSCs do not show these consistent transcript alterations (Table 2) [3,29,40,98].…”

Targeting Axonal Transport: A New Therapeutic Avenue for ALS

“…While another study has shown that age-dependent organelle transport defects in iPSC-derived motor neurons from ALS patients carrying TARDBP mutations is independent from TDP-43 aggregation [31], thus, a clear interplay between TDP-43 aggregation and axonal transport still needs to be clarified. Recently, we found that arginine-rich dipeptide repeat proteins (DPRs), which are the pathological translational products from C9orf72 repeat expansion, can cause axonal transport defects in human stem cell-derived motor neurons [32]. We found that several components of the axonal transport machinery interact with arginine-rich DPRs both in vitro and in vivo.…”

“…We found that several components of the axonal transport machinery interact with arginine-rich DPRs both in vitro and in vivo. It has been proposed that argininerich DPRs might directly affect axonal transport through an inhibitory interaction with the microtubule-based transport machinery [32].…”