CRISPR/Cas13d targeting suppresses repeat-associated non-AUG translation of C9orf72 hexanucleotide repeat RNA.

A hexanucleotide GGGGCC repeat expansion in the non-coding region of the C9orf72 gene is the most common genetic mutation identified in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The resulting repeat RNA and dipeptide repeat proteins from non-conventional repeat translation have been recognized as important markers associated with the diseases. CRISPR/Cas13d, a powerful RNA-targeting tool, has faced challenges in effectively targeting RNA with stable secondary structures.

Multivalent GU-rich oligonucleotides sequester TDP-43 in the nucleus by inducing high molecular weight RNP complexes.

TDP-43 nuclear clearance and cytoplasmic aggregation are hallmarks of TDP-43 proteinopathies. We recently demonstrated that binding to endogenous nuclear GU-rich RNAs sequesters TDP-43 in the nucleus by restricting its passive nuclear export. Here, we tested the feasibility of synthetic RNA oligonucleotide-mediated augmentation of TDP-43 nuclear localization.

Elevated nuclear TDP-43 induces constitutive exon skipping.

Background: Cytoplasmic inclusions and loss of nuclear TDP-43 are key pathological features found in several neurodegenerative disorders, suggesting both gain- and loss-of-function mechanisms of disease. To study gain-of-function, TDP-43 overexpression has been used to generate in vitro and in vivo model systems.

Methods: We analyzed RNA-seq datasets from mouse and human neurons overexpressing TDP-43 to explore species specific splicing patterns.

DDX3X overexpression decreases dipeptide repeat proteins in a mouse model of C9ORF72-ALS/FTD.

Hexanucleotide repeat expansion in C9ORF72 (C9) is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One of the proposed pathogenic mechanisms is the neurotoxicity arising from dipeptide repeat (DPR) proteins produced by repeat-associated non-AUG (RAN) translation. Therefore, reducing DPR levels emerges as a potential therapeutic strategy for C9ORF72-ALS/FTD.

RNA-mediated ribonucleoprotein assembly controls TDP-43 nuclear retention.

TDP-43 is an essential RNA-binding protein strongly implicated in the pathogenesis of neurodegenerative disorders characterized by cytoplasmic aggregates and loss of nuclear TDP-43. The protein shuttles between nucleus and cytoplasm, yet maintaining predominantly nuclear TDP-43 localization is important for TDP-43 function and for inhibiting cytoplasmic aggregation. We previously demonstrated that specific RNA binding mediates TDP-43 self-assembly and biomolecular condensation, requiring multivalent interactions via N- and C-terminal domains.

RNA-mediated ribonucleoprotein assembly controls TDP-43 nuclear retention.

TDP-43 is an essential RNA-binding protein strongly implicated in the pathogenesis of neurodegenerative disorders characterized by cytoplasmic aggregates and loss of nuclear TDP-43. The protein shuttles between nucleus and cytoplasm, yet maintaining predominantly nuclear TDP-43 localization is important for TDP-43 function and for inhibiting cytoplasmic aggregation. We previously demonstrated that specific RNA binding mediates TDP-43 self-assembly and biomolecular condensation, requiring multivalent interactions via N- and C-terminal domains.

Multivalent GU-rich oligonucleotides sequester TDP-43 in the nucleus by inducing high molecular weight RNP complexes.

TDP-43 nuclear clearance and cytoplasmic aggregation are hallmarks of TDP-43 proteinopathies. We recently demonstrated that binding to endogenous nuclear GU-rich RNAs sequesters TDP-43 in the nucleus by restricting its passive nuclear export. Here, we tested the feasibility of synthetic RNA oligonucleotide-mediated augmentation of TDP-43 nuclear localization.

Elevated nuclear TDP-43 induces constitutive exon skipping.

Cytoplasmic inclusions and loss of nuclear TDP-43 are key pathological features found in several neurodegenerative disorders, suggesting both gain- and loss-of-function mechanisms of disease. To study gain-of-function, TDP-43 overexpression has been used to generate and model systems. Our study shows that excessive levels of nuclear TDP-43 protein lead to constitutive exon skipping that is largely species-specific.

Nuclear RNA binding regulates TDP-43 nuclear localization and passive nuclear export.

Nuclear clearance of the RNA-binding protein TDP-43 is a hallmark of neurodegeneration and an important therapeutic target. Our current understanding of TDP-43 nucleocytoplasmic transport does not fully explain its predominantly nuclear localization or mislocalization in disease. Here, we show that TDP-43 exits nuclei by passive diffusion, independent of facilitated mRNA export.

Emerging Therapies and Novel Targets for TDP-43 Proteinopathy in ALS/FTD.

Nuclear clearance and cytoplasmic mislocalization of the essential RNA binding protein, TDP-43, is a pathologic hallmark of amyotrophic lateral sclerosis, frontotemporal dementia, and related neurodegenerative disorders collectively termed "TDP-43 proteinopathies." TDP-43 mislocalization causes neurodegeneration through both loss and gain of function mechanisms. Loss of TDP-43 nuclear RNA processing function destabilizes the transcriptome by multiple mechanisms including disruption of pre-mRNA splicing, the failure of repression of cryptic exons, and retrotransposon activation.

Nuclear export and translation of circular repeat-containing intronic RNA in C9ORF72-ALS/FTD.

C9ORF72 hexanucleotide GGGGCC repeat expansion is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat-containing RNA mediates toxicity through nuclear granules and dipeptide repeat (DPR) proteins produced by repeat-associated non-AUG translation. However, it remains unclear how the intron-localized repeats are exported and translated in the cytoplasm.

Nuclear Transport Assays in Permeabilized Mouse Cortical Neurons.

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases. Moreover, there is a growing recognition of cell-specific differences in nuclear pore complex structure, prompting a need to adapt nuclear transport methods for use in neurons. Permeabilized cell assays, in which the plasma membrane is selectively perforated by digitonin, are widely used to study passive and active nuclear transport in immortalized cell lines but have not been applied to neuronal cultures.

A Helicase Unwinds Hexanucleotide Repeat RNA G-Quadruplexes and Facilitates Repeat-Associated Non-AUG Translation.

The expansion of a hexanucleotide repeat GGGGCC (G4C2) in the gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The G4C2 expansion leads to repeat-associated non-AUG (RAN) translation and the production of toxic dipeptide repeat (DPR) proteins, but the mechanisms of RAN translation remain enigmatic. Here, we report that the RNA helicase DHX36 is a robust positive regulator of RAN translation.

UPF1 reduces C9orf72 HRE-induced neurotoxicity in the absence of nonsense-mediated decay dysfunction.

Multiple cellular pathways have been suggested to be altered by the C9orf72 GGGGCC (GC) hexanucleotide repeat expansion (HRE), including aspects of RNA regulation such as nonsense-mediated decay (NMD). Here, we investigate the role that overexpression of UPF1, a protein involved in NMD, plays in mitigating neurotoxicity in multiple models of C9orf72 ALS/FTD. First, we show that NMD is not altered in our endogenous induced pluripotent stem cell (iPSC)-derived spinal neuron (iPSN) model of C9orf72 ALS (C9-ALS) or postmortem motor cortex tissue from C9-ALS patients.

arginine-rich dipeptide repeat proteins disrupt karyopherin-mediated nuclear import.

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases, including ALS caused by a hexanucleotide repeat expansion. However, the mechanism(s) remain unclear. Karyopherins, including importin β and its cargo adaptors, have been shown to co-precipitate with the arginine-containing dipeptide repeat proteins (R-DPRs), poly-glycine arginine (GR) and poly-proline arginine (PR), and are protective in genetic modifier screens.

CRISPR-Cas9 Screens Identify the RNA Helicase DDX3X as a Repressor of C9ORF72 (GGGGCC)n Repeat-Associated Non-AUG Translation.

Hexanucleotide GGGGCC repeat expansion in C9ORF72 is the most prevalent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One pathogenic mechanism is the aberrant accumulation of dipeptide repeat (DPR) proteins produced by the unconventional translation of expanded RNA repeats. Here, we performed genome-wide CRISPR-Cas9 screens for modifiers of DPR protein production in human cells.

Distal denervation in the SOD1 knockout mouse correlates with loss of mitochondria at the motor nerve terminal.

Impairment of mitochondrial transport has long been implicated in the pathogenesis of neuropathy and neurodegeneration. However, the role of mitochondria in stabilizing motor nerve terminals at neuromuscular junction (NMJ) remains unclear. We previously demonstrated that mice lacking the antioxidant enzyme, superoxide dismutase-1 (Sod1-/-), develop progressive NMJ denervation.

C9ORF72 GGGGCC repeat-associated non-AUG translation is upregulated by stress through eIF2α phosphorylation.

Hexanucleotide repeat expansion in C9ORF72 is the most frequent cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we demonstrate that the repeat-associated non-AUG (RAN) translation of (GGGGCC) -containing RNAs into poly-dipeptides can initiate in vivo without a 5'-cap. The primary RNA substrate for RAN translation of C9ORF72 sense repeats is shown to be the spliced first intron, following its excision from the initial pre-mRNA and transport to the cytoplasm.

Poly(GP) proteins are a useful pharmacodynamic marker for -associated amyotrophic lateral sclerosis.

There is no effective treatment for amyotrophic lateral sclerosis (ALS), a devastating motor neuron disease. However, discovery of a GC repeat expansion in the gene as the most common genetic cause of ALS has opened up new avenues for therapeutic intervention for this form of ALS. GC repeat expansion RNAs and proteins of repeating dipeptides synthesized from these transcripts are believed to play a key role in -associated ALS (c9ALS).

C9ORF72-ALS/FTD: Transgenic Mice Make a Come-BAC.

For five years, since the landmark discovery of the C9ORF72 hexanucleotide repeat expansion in ALS/FTD, a transgenic mouse model has remained elusive. Now, two laboratories (Liu et al., 2016; Jiang et al.