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

A hexanucleotide repeat expansion in the C9orf72 gene is 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 show using patient stem cell–derived motor neurons that the repeat expansion impairs microtubule-based transport, a process critical for neuronal survival. Cargo transport defects are recapitulated by treating neurons from healthy individuals with proline-arginine and glycine-arginine dipeptide repeats (DPRs) produced from the repeat expansion. Both arginine-rich DPRs similarly inhibit axonal trafficking in adult Drosophila neurons in vivo. Physical interaction studies demonstrate that arginine-rich DPRs associate with motor complexes and the unstructured tubulin tails of microtubules. Single-molecule imaging reveals that microtubule-bound arginine-rich DPRs directly impede translocation of purified dynein and kinesin-1 motor complexes. Collectively, our study implicates inhibitory interactions of arginine-rich DPRs with axonal transport machinery in C9orf72-associated ALS/FTD and thereby points to potential therapeutic strategies.

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

10.1101/835082 ◽

2019 ◽

Cited By ~ 4

Author(s):

Laura Fumagalli ◽

Florence L. Young ◽

Steven Boeynaems ◽

Mathias De Decker ◽

Arpan R. Mehta ◽

...

Keyword(s):

Axonal Transport ◽

Motor Neurons ◽

Single Molecule ◽

Induced Pluripotent Stem Cell ◽

Cytoplasmic Dynein ◽

Inhibitory Interaction ◽

Hexanucleotide Repeat ◽

Repeat Expansions

ABSTRACTHexanucleotide 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.

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Effect of G4C2-Repeat Expansions on the Motion of Lysosomes Inside Neurites.

10.1101/2021.10.29.466472 ◽

2021 ◽

Author(s):

Maria Mytiliniou ◽

Joeri A.J. Wondergem ◽

Marleen Feliksik ◽

Thomas Schmidt ◽

Doris Heinrich

Keyword(s):

Neuronal Cell ◽

Repeat Expansion ◽

Time Resolved ◽

Lower Velocity ◽

Hexanucleotide Repeat ◽

Repeat Expansions ◽

Local Mean ◽

Hexanucleotide Repeat Expansion ◽

Axonal Trafficking ◽

Lateral Sclerosis

The G4C2 hexanucleotide repeat expansion in the c9orf72 locus is one among a plethora of mutations associated with amyotrophic lateral sclerosis. It accounts for the majority of disease cases. The exact processes underlying the pathology of this mutation remain elusive, yet recent evidence suggests a mechanism that disrupts axonal trafficking. Here, we used a neuronal cell line with and without the G4C2 repeats, and implemented time-resolved local mean squared displacement analysis to characterize the motion of lysosomes inside neurites. Neurites were either aligned along chemically patterned lines, or oriented randomly on the substrate. We confirmed that in the presence of the G4C2 repeats, lysosome motion was affected. Lysosomes had a smaller reach exhibited lower velocity, especially inside aligned neurites. At the same time they became more active with increasing length of the G4C2 repeats when the neurites were randomly oriented. The duration of diffusive and super-diffusive lysosome transport remained unaffected for both neurite geometries and for all lengths of the repeats, but the displacement and velocity was decreased on varying the repeat number and neurite geometry. Lastly, the ratio of anterograde/retrograde/neutral trajectories was affected disparately for the two neurite geometries. Our observations support the hypothesis that impaired axonal trafficking emerges in the presence of the G4C2 hexanucleotide repeat expansion.

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Nucleocytoplasmic Proteomic Analysis Uncovers eRF1 and Nonsense Mediated Decay as Modifiers of ALS C9orf72 Toxicity

10.1101/677419 ◽

2019 ◽

Author(s):

Juan A. Ortega ◽

Elizabeth L. Daley ◽

Sukhleen Kour ◽

Marisa Samani ◽

Liana Tellez ◽

...

Keyword(s):

Translation Termination ◽

Protein Translation ◽

Subcellular Fractionation ◽

Nucleocytoplasmic Transport ◽

Repeat Expansion ◽

Nonsense Mediated Decay ◽

Hexanucleotide Repeat ◽

Hexanucleotide Repeat Expansion ◽

Lateral Sclerosis

SUMMARYThe most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a hexanucleotide repeat expansion in C9orf72 (C9-HRE). While RNA and dipeptide repeats produced by the C9-HRE disrupt nucleocytoplasmic transport, the proteins that become redistributed remain unknown. Here, we utilized subcellular fractionation coupled with tandem mass spectrometry and identified 126 proteins, enriched for protein translation and RNA metabolism pathways, which collectively drive a shift towards a more cytosolic proteome in C9-HRE cells. Amongst these was eRF1, which regulates translation termination and nonsense-mediated decay (NMD). eRF1 accumulates within elaborate nuclear envelope invaginations in patient iPSC-neurons and postmortem tissue and mediates a protective shift from protein translation to NMD-dependent mRNA degradation. Overexpression of eRF1 and the NMD-driver UPF1 ameliorate C9-HRE toxicity in vivo. Our findings provide a resource for proteome-wide nucleocytoplasmic alterations across neurodegeneration-associated repeat expansion mutations and highlight eRF1 and NMD as therapeutic targets in C9orf72-associated ALS/FTD.

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Knocking out C9ORF72 Exacerbates Axonal Trafficking Defects Associated with Hexanucleotide Repeat Expansion and Reduces Levels of Heat Shock Proteins

Stem Cell Reports ◽

10.1016/j.stemcr.2020.01.010 ◽

2020 ◽

Vol 14 (3) ◽

pp. 390-405 ◽

Cited By ~ 12

Author(s):

Masin Abo-Rady ◽

Norman Kalmbach ◽

Arun Pal ◽

Carina Schludi ◽

Antje Janosch ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Proteins ◽

Repeat Expansion ◽

Hexanucleotide Repeat ◽

Hexanucleotide Repeat Expansion ◽

Trafficking Defects ◽

Shock Proteins ◽

Axonal Trafficking

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Receptor-Dependent and -Independent Axonal Retrograde Transport of Poliovirus in Motor Neurons

Journal of Virology ◽

10.1128/jvi.02225-08 ◽

2009 ◽

Vol 83 (10) ◽

pp. 4995-5004 ◽

Cited By ~ 39

Author(s):

Seii Ohka ◽

Mai Sakai ◽

Stephanie Bohnert ◽

Hiroko Igarashi ◽

Katrin Deinhardt ◽

...

Keyword(s):

Sciatic Nerve ◽

Axonal Transport ◽

Motor Neurons ◽

Retrograde Transport ◽

Cytoplasmic Dynein ◽

Dependent Manner ◽

Fast Transport ◽

Axonal Trafficking

ABSTRACT Poliovirus (PV), when injected intramuscularly into the calf, is incorporated into the sciatic nerve and causes an initial paralysis of the inoculated limb in transgenic (Tg) mice carrying the human PV receptor (hPVR/CD155) gene. We have previously demonstrated that a fast retrograde axonal transport process is required for PV dissemination through the sciatic nerves of hPVR-Tg mice and that intramuscularly inoculated PV causes paralytic disease in an hPVR-dependent manner. Here we showed that hPVR-independent axonal transport of PV was observed in hPVR-Tg and non-Tg mice, indicating that several different pathways for PV axonal transport exist in these mice. Using primary motor neurons (MNs) isolated from these mice or rats, we demonstrated that the axonal transport of PV requires several kinetically different motor machineries and that fast transport relies on a system involving cytoplasmic dynein. Unexpectedly, the hPVR-independent axonal transport of PV was not observed in cultured MNs. Thus, PV transport machineries in cultured MNs and in vivo differ in their hPVR requirements. These results suggest that the axonal trafficking of PV is carried out by several distinct pathways and that MNs in culture and in the sciatic nerve in situ are intrinsically different in the uptake and axonal transport of PV.

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