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

2019 ◽  
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.

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

2021 ◽  
Vol 7 (15) ◽  
pp. eabg3013
Author(s):  
Laura Fumagalli ◽  
Florence L. Young ◽  
Steven Boeynaems ◽  
Mathias De Decker ◽  
Arpan R. Mehta ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Motor Neurons ◽  
Single Molecule ◽  
Repeat Expansion ◽  
Physical Interaction ◽  
Hexanucleotide Repeat ◽  
Hexanucleotide Repeat Expansion ◽  
Axonal Trafficking ◽  
Inhibitory Interactions

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.

scholarly journals A cell-penetrant peptide blocking C9ORF72-repeat RNA nuclear export suppresses neurodegeneration

2021 ◽  
Author(s):  
Lydia M Castelli ◽  
Alvaro Sanchez-Martinez ◽  
Ya-Hui Lin ◽  
Santosh Kumar Upadhyay ◽  
Adrian Higginbottom ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Nuclear Export ◽  
Promising Alternative ◽  
Hexanucleotide Repeat ◽  
Repeat Expansions ◽  
A Cell ◽  
Models Of Disease ◽  
Lateral Sclerosis

Hexanucleotide repeat expansions in C9ORF72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), a spectrum of incurable debilitating neurodegenerative diseases. Here, we report a novel ALS/FTD drug concept with in vivo and in vitro therapeutic activity in preclinical models of C9ORF72-ALS/FTD. Our data demonstrate that supplementation or oral administration of a cell-penetrant peptide, which competes with the SRSF1:NXF1 interaction, confers neuroprotection by inhibiting the nuclear export of pathological C9ORF72-repeat transcripts in various models of disease including primary neurons, patient-derived motor neurons and Drosophila. Our drug-like rationale for disrupting the nuclear export of microsatellite repeat transcripts in neurological disorders provides a promising alternative to conventional small molecule inhibitors often limited by poor blood-brain barrier penetrance.

scholarly journals Smcr8 deficiency disrupts axonal transport-dependent lysosomal function and promotes axonal swellings and gain of toxicity in C9ALS/FTD mouse models

2019 ◽  
Vol 28 (23) ◽  
pp. 3940-3953 ◽  
Author(s):  
Chen Liang ◽  
Qiang Shao ◽  
Wei Zhang ◽  
Mei Yang ◽  
Qing Chang ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Mouse Models ◽  
Motor Neurons ◽  
Neuromuscular Junctions ◽  
Motor Behavior ◽  
In Vivo Studies ◽  
Lysosomal Function ◽  
Repeat Expansions ◽  
Axonal Swellings

Abstract G4C2 repeat expansions in an intron of C9ORF72 cause the most common familial amyotrophic lateral sclerosis and frontotemporal dementia (collectively, C9ALS/FTD). Mechanisms and mediators of C9ALS/FTD pathogenesis remain poorly understood. C9orf72 and Smcr8 form a protein complex. Here, we show that expression of Smcr8, like C9orf72, is reduced in C9ALS/FTD mouse models and patient tissues. Since Smcr8 is highly conserved between human and mouse, we evaluated the effects of Smcr8 downregulation in mice. Smcr8 knockout (KO) mice exhibited motor behavior deficits, which resemble those of C9ALS/FTD mouse models, and displayed axonal swellings in their spinal cords and neuromuscular junctions. These deficits are caused by impaired autophagy-lysosomal functions due to disrupted axonal transport in mutant motor neurons. Consistent with its interaction with C9orf72 and their downregulation in patient tissues, Smcr8 deficiency exacerbated autophagy-lysosomal impairment in C9orf72 KO mice. The disease relevance of Smcr8 downregulation was reflected by exacerbated axonal swellings and gain of toxicity pathology arising from Smcr8 haploinsufficiency in a mouse model of C9ALS/FTD. Thus, our in vivo studies suggested that Smcr8 deficiency impairs axonal transport dependent autophagy-lysosomal function and exacerbates axonal degeneration and gain of toxicity in C9ALS/FTD mouse models.

scholarly journals DYNC1H1 mutations associated with neurological diseases compromise processivity of dynein–dynactin–cargo adaptor complexes

2017 ◽  
Vol 114 (9) ◽  
pp. E1597-E1606 ◽  
Author(s):  
Ha Thi Hoang ◽  
Max A. Schlager ◽  
Andrew P. Carter ◽  
Simon L. Bullock
Keyword(s):  
Single Molecule ◽  
Heavy Chain ◽  
Muscular Atrophy ◽  
Recombinant Expression ◽  
Neurological Diseases ◽  
Expression System ◽  
Cytoplasmic Dynein ◽  
In Vitro Motility

Mutations in the human DYNC1H1 gene are associated with neurological diseases. DYNC1H1 encodes the heavy chain of cytoplasmic dynein-1, a 1.4-MDa motor complex that traffics organelles, vesicles, and macromolecules toward microtubule minus ends. The effects of the DYNC1H1 mutations on dynein motility, and consequently their links to neuropathology, are not understood. Here, we address this issue using a recombinant expression system for human dynein coupled to single-molecule resolution in vitro motility assays. We functionally characterize 14 DYNC1H1 mutations identified in humans diagnosed with malformations in cortical development (MCD) or spinal muscular atrophy with lower extremity predominance (SMALED), as well as three mutations that cause motor and sensory defects in mice. Two of the human mutations, R1962C and H3822P, strongly interfere with dynein’s core mechanochemical properties. The remaining mutations selectively compromise the processive mode of dynein movement that is activated by binding to the accessory complex dynactin and the cargo adaptor Bicaudal-D2 (BICD2). Mutations with the strongest effects on dynein motility in vitro are associated with MCD. The vast majority of mutations do not affect binding of dynein to dynactin and BICD2 and are therefore expected to result in linkage of cargos to dynein–dynactin complexes that have defective long-range motility. This observation offers an explanation for the dominant effects of DYNC1H1 mutations in vivo. Collectively, our results suggest that compromised processivity of cargo–motor assemblies contributes to human neurological disease and provide insight into the influence of different regions of the heavy chain on dynein motility.

scholarly journals DYNC1H1 mutations associated with neurological diseases compromise processivity of dynein-dynactin-cargo adaptor complexes

2016 ◽  
Author(s):  
Ha Thi Hoang ◽  
Max A. Schlager ◽  
Andrew P. Carter ◽  
Simon L Bullock
Keyword(s):  
Single Molecule ◽  
Heavy Chain ◽  
Muscular Atrophy ◽  
Recombinant Expression ◽  
Neurological Diseases ◽  
Expression System ◽  
Cytoplasmic Dynein ◽  
Recombinant Expression System

Mutations in the human DYNC1H1 gene are associated with neurological diseases. DYNC1H1 encodes the heavy chain of cytoplasmic dynein-1, a 1.4 MDa motor complex that traffics organelles, vesicles and macromolecules towards microtubule minus ends. The effects of the DYNC1H1 mutations on dynein motility, and consequently their links to neuropathology, are not understood. Here, we address this issue using a recombinant expression system for human dynein coupled to single-molecule resolution in vitro motility assays. We functionally characterise 14 DYNC1H1 mutations identified in humans diagnosed with malformations in cortical development (MCD) or spinal muscular atrophy with lower extremity predominance (SMALED), as well as three mutations that cause motor and sensory defects in mice. Two of the human mutations, R1962C and H3822P, strongly interfere with dynein’s core mechanochemical properties. The remaining mutations selectively compromise the processive mode of dynein movement that is activated by binding to the accessory complex dynactin and the cargo adaptor BICD2. Mutations with the strongest effects on dynein motility in vitro are associated with MCD. The vast majority of mutations do not affect binding of dynein to dynactin and BICD2, and are therefore expected to result in linkage of cargoes to dynein-dynactin complexes that have defective long-range motility. This observation offers an explanation for the dominant effects of DYNC1H1 mutations in vivo. Collectively, our results suggest that compromised processivity of cargo-motor assemblies contributes to human neurological disease and provide insight into the influence of different regions of the heavy chain on dynein motility.

scholarly journals A motor neuron disease–associated mutation in p150Glued perturbs dynactin function and induces protein aggregation

2006 ◽  
Vol 172 (5) ◽  
pp. 733-745 ◽  
Author(s):  
Jennifer R. Levy ◽  
Charlotte J. Sumner ◽  
Juliane P. Caviston ◽  
Mariko K. Tokito ◽  
Srikanth Ranganathan ◽  
...  
Keyword(s):  
Cell Death ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Mitotic Spindle ◽  
Cytoplasmic Dynein ◽  
Aggregate Formation ◽  
Microtubule Motor ◽  
Delayed Recovery

The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. Dynactin is ubiquitously expressed in eukaryotes, but a G59S mutation in the p150Glued subunit of dynactin results in the specific degeneration of motor neurons. This mutation in the conserved cytoskeleton-associated protein, glycine-rich (CAP-Gly) domain lowers the affinity of p150Glued for microtubules and EB1. Cell lines from patients are morphologically normal but show delayed recovery after nocodazole treatment, consistent with a subtle disruption of dynein/dynactin function. The G59S mutation disrupts the folding of the CAP-Gly domain, resulting in aggregation of the p150Glued protein both in vitro and in vivo, which is accompanied by an increase in cell death in a motor neuron cell line. Overexpression of the chaperone Hsp70 inhibits aggregate formation and prevents cell death. These data support a model in which a point mutation in p150Glued causes both loss of dynein/dynactin function and gain of toxic function, which together lead to motor neuron cell death.

scholarly journals Lis1 has two opposing modes of regulating cytoplasmic dynein

2017 ◽  
Author(s):  
Morgan E. DeSantis ◽  
Michael A. Cianfrocco ◽  
Zaw Min Htet ◽  
Phuoc Tien Tran ◽  
Samara L. Reck-Peterson ◽  
...  
Keyword(s):  
Single Molecule ◽  
Intracellular Transport ◽  
Cytoplasmic Dynein ◽  
The Novel ◽  
Microtubule Binding ◽  
In Vivo Experiments ◽  
Cryo Electron Microscopy ◽  
Functional Versatility

SummaryRegulation is central to the functional versatility of cytoplasmic dynein, a motor involved in intracellular transport, cell division, and neurodevelopment. Previous work established that Lis1, a conserved and ubiquitous regulator of dynein, binds to its motor domain and induces a tight microtubule-binding state in dynein. The work we present here—a combination of biochemistry, single-molecule assays, cryo-electron microscopy and in vivo experiments—led to the surprising discovery that Lis1 has two opposing modes of regulating dynein, being capable of inducing both low and high affinity for the microtubule. We show that these opposing modes depend on the stoichiometry of Lis1 binding to dynein and that this stoichiometry is regulated by the nucleotide state of dynein’s AAA3 domain. We present data on the in vitro and in vivo consequences of abolishing the novel Lis1-induced weak microtubule-binding state in dynein and propose a new model for the regulation of dynein by Lis1.

scholarly journals A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity

2014 ◽  
Vol 25 (5) ◽  
pp. 669-678 ◽  
Author(s):  
Kaeling Tan ◽  
Anthony J. Roberts ◽  
Mark Chonofsky ◽  
Martin J. Egan ◽  
Samara L. Reck-Peterson
Keyword(s):  
Genome Sequencing ◽  
Single Molecule ◽  
Intracellular Transport ◽  
Screening Method ◽  
Cytoplasmic Dynein ◽  
Catalytic Core ◽  
Single Molecule Studies ◽  
Novel Alleles

The timely delivery of membranous organelles and macromolecules to specific locations within the majority of eukaryotic cells depends on microtubule-based transport. Here we describe a screening method to identify mutations that have a critical effect on intracellular transport and its regulation using mutagenesis, multicolor-fluorescence microscopy, and multiplex genome sequencing. This screen exploits the filamentous fungus Aspergillus nidulans, which has many of the advantages of yeast molecular genetics but uses long-range microtubule-based transport in a manner more similar to metazoan cells. Using this method, we identified seven mutants that represent novel alleles of components of the intracellular transport machinery: specifically, kinesin-1, cytoplasmic dynein, and the dynein regulators Lis1 and dynactin. The two dynein mutations identified in our screen map to dynein's AAA+ catalytic core. Single-molecule studies reveal that both mutations reduce dynein's velocity in vitro. In vivo these mutants severely impair the distribution and velocity of endosomes, a known dynein cargo. In contrast, another dynein cargo, the nucleus, is positioned normally in these mutants. These results reveal that different dynein functions have distinct stringencies for motor performance.

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

2009 ◽  
Vol 83 (10) ◽  
pp. 4995-5004 ◽  
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.

scholarly journals Glycine-alanine dipeptide repeats spread rapidly in a repeat length- and age-dependent manner in the fly brain

2019 ◽  
Vol 7 (1) ◽  
Author(s):  
Javier Morón-Oset ◽  
Tessa Supèr ◽  
Jacqueline Esser ◽  
Adrian M. Isaacs ◽  
Sebastian Grönke ◽  
...  
Keyword(s):  
Dependent Manner ◽  
Genetic Screens ◽  
Hexanucleotide Repeat ◽  
Repeat Expansions ◽  
Alanine Dipeptide ◽  
The Brain ◽  
Lateral Sclerosis ◽  
Dipeptide Repeat

AbstractHexanucleotide repeat expansions of variable size in C9orf72 are the most prevalent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Sense and antisense transcripts of the expansions are translated by repeat-associated non-AUG translation into five dipeptide repeat proteins (DPRs). Of these, the polyGR, polyPR and, to a lesser extent, polyGA DPRs are neurotoxic, with polyGA the most abundantly detected DPR in patient tissue. Trans-cellular transmission of protein aggregates has recently emerged as a major driver of toxicity in various neurodegenerative diseases. In vitro evidence suggests that the C9 DPRs can spread. However, whether this phenomenon occurs under more complex in vivo conditions remains unexplored. Here, we used the adult fly brain to investigate whether the C9 DPRs can spread in vivo upon expression in a subset of neurons. We found that only polyGA can progressively spread throughout the brain, which accumulates in the shape of aggregate-like puncta inside recipient cells. Interestingly, GA transmission occurred as early as 3 days after expression induction. By comparing the spread of 36, 100 and 200 polyGA repeats, we found that polyGA spread is enhanced upon expression of longer GA DPRs. Transmission of polyGA is greater in older flies, indicating that age-associated factors exacerbate the spread. These data highlight a unique propensity of polyGA to spread throughout the brain, which could contribute to the greater abundance of polyGA in patient tissue. In addition, we present a model of early GA transmission that is suitable for genetic screens to identify mechanisms of spread and its consequences in vivo.

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