Biology subjects

Todd, P. K.

Publications and source records attributed to Todd, P. K..

10 recordsLinked to original sources

bioRxiv2026

Structural basis for repeat-associated non-AUG translation initiation on C9orf72 mRNA

A GGGGCC (G4C2) hexanucleotide repeat expansion in the first intron of C9orf72 is the most common monogenic cause of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). This element serves as a template for repeat-associated non-AUG (RAN) translation, which generates neurotoxic dipeptide repeat (DPR) proteins that aggregate in patient brains and contribute to neurodegeneration in model systems. To understand the mechanisms underlying RAN translation initiation, we used cryo-electron microscopy to determine the structure of an in vitro assembled human late-stage C9orf72 translation initiation complex at 3.2 A resolution and a near-native complex purified from human cell lysate at 3.5 A resolution. RAN translation on C9orf72 mRNA initiates predominantly at a near-cognate CUG codon that is located 24 nucleotides upstream of the G4C2 repeat and is flanked by a strong endogenous Kozak sequence. The structures further reveal a direct interaction between the G4C2 repeat and 18S ribosomal RNA expansion segment 9 (ES9S) on the 40S small ribosomal subunit through a kissing-loop interaction. Preventing this RNA-RNA interaction using complementary antisense oligonucleotides significantly reduces RAN translation in vitro and in patient-derived iNeurons, indicating that this structural interaction is functionally important. These findings provide mechanistic insights into RAN translation initiation and highlight a novel potential therapeutic approach to mitigate toxic DPR production in C9orf72-associated neurodegenerative disease.

molecular biology↗

bioRxiv2023

AAGGG repeat expansions trigger RFC1-independent synaptic dysregulation in human CANVAS Neurons

Cerebellar ataxia with neuropathy and vestibular areflexia syndrome (CANVAS) is a late onset, recessively inherited neurodegenerative disorder caused by biallelic, non-reference pentameric AAGGG(CCCTT) repeat expansions within the second intron of replication factor complex subunit 1 (RFC1). To investigate how these repeats cause disease, we generated CANVAS patient induced pluripotent stem cell (iPSC) derived neurons (iNeurons) and utilized calcium imaging and transcriptomic analysis to define repeat-elicited gain-of-function and loss-of-function contributions to neuronal toxicity. AAGGG repeat expansions do not alter neuronal RFC1 splicing, expression, or DNA repair pathway functions. In reporter assays, AAGGG repeats are translated into pentapeptide repeat proteins that selectively accumulate in CANVAS patient brains. However, neither these proteins nor repeat RNA foci were detected in iNeurons, and overexpression of these repeats in isolation did not induce neuronal toxicity. CANVAS iNeurons exhibit defects in neuronal development and diminished synaptic connectivity that is rescued by CRISPR deletion of a single expanded allele. These phenotypic deficits were not replicated by knockdown of RFC1 in control neurons and were not rescued by ectopic expression of RFC1. These findings support a repeat-dependent but RFC1-independent cause of neuronal dysfunction in CANVAS, with important implications for therapeutic development in this currently untreatable condition. SummaryHuman CANVAS neurons exhibit transcriptional and functional synaptic defects that are corrected by heterozygous repeat deletion but are independent of the gene within which they reside--RFC1.

neuroscience↗

bioRxiv2023

Stress granule formation helps to mitigate neurodegeneration

Cellular stress pathways that inhibit translation initiation lead to transient formation of cytoplasmic RNA/protein complexes known as stress granules. Many of the proteins found within stress granules and the dynamics of stress granule formation and dissolution are implicated in neurodegenerative disease. Whether stress granule formation is protective or harmful in neurodegenerative conditions is not known. To address this, we took advantage of the alphavirus protein nsP3, which selectively binds dimers of the central stress granule nucleator protein G3BP (rin in Drosophila) and markedly reduces stress granule formation without directly impacting the protein translational inhibitory pathways that trigger stress granule formation. In Drosophila and rodent neurons, reducing stress granule formation with nsP3 had modest impacts on lifespan even in the setting of serial stress pathway induction. In contrast, reducing stress granule formation in models of ataxia, amyotrophic lateral sclerosis and frontotemporal dementia largely exacerbated disease phenotypes. These data support a model whereby stress granules mitigate, rather than promote, neurodegenerative cascades.

neuroscience↗

bioRxiv2021

DENR/MCTS1 knockdown modulates repeat-associated non-AUG translation

Repeat associated non-AUG (RAN) translation of mRNAs containing repeat-expansion mutations produces toxic peptides in neurons of patients suffering from neurodegenerative diseases. Recent findings indicate that RAN translation in diverse model systems is not inhibited by cellular stressors that impair global translation through phosphorylation of the alpha subunit of eIF2, the essential eukaryotic translation initiation factor that brings the initiator tRNA to the 40S ribosome. Using in vitro, cell-based, and Drosophila models, we examined the role of alternative ternary complex factors that may function in place of eIF2, including eIF2A, eIF2D, and DENR/MCTS1. Among these factors, DENR knockdown had the greatest inhibitory effect on RAN translation of expanded GGGGCC and CGG repeat reporters, and its reduction improved survival of Drosophila expressing expanded GGGGCC repeats. Taken together, these data support a role for alternative initiation factors in RAN translation and suggest they may serve as novel therapeutic targets in neurodegenerative disease.

neuroscience↗

bioRxiv2021

The RNA helicase DHX36/G4R1 modulates C9orf72 GGGGCC repeat-associated translation

GGGGCC (G4C2) hexanucleotide repeat expansions (HRE) in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat-associated non-AUG (RAN) translation of this expansion generates toxic proteins that accumulate in patient brains and contribute to disease pathogenesis. The DEAH-Box Helicase 36 (DHX36/G4R1) plays active roles in RNA and DNA G-quadruplex (G4) resolution in cells. As G4C2 repeats form G4 structures in vitro, we sought to determine the impact of manipulating DHX36 expression on repeat transcription and RAN translation. We found that DHX36 depletion suppresses RAN translation from reporter constructs in a repeat length dependent manner while overexpression of DHX36 enhances RAN translation from G4C2 reporter RNAs. Taken together, these results suggest that DHX36 is active in regulating G4C2 repeat translation, providing potential implications for therapeutic development in nucleotide repeats expansion disorders.

molecular biology↗

bioRxiv2021

In vivo CGG repeat RNA binding protein capture identifies RAN translation modifiers and suppressors of repeat toxicity.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by a transcribed CGG repeat expansion in the 5 UTR of FMR1. Expanded CGG repeat RNAs both sequester RNA-binding proteins (RBPs) into nuclear foci and undergo repeat-associated non-AUG (RAN) translation into toxic homopolymeric peptides. RBPs that interact with CGG repeats may play a pivotal role in foci formation and/or RAN translation. Here we employed a CGG repeat RNA-tagging system to capture and identify CGG repeat binding RBPs in vivo under different cellular conditions. We found that several SR (serine/arginine-rich domain) proteins interact with CGG repeat RNAs basally and under cellular stress. These same proteins strongly modify toxicity in a Drosophila model of FXTAS, improving eye degeneration and survival. Furthermore, genetic or pharmacological targeting of the serine/arginine protein kinases (SRPKs) suppresses RAN translation in cellular reporters and toxicity in fly models of FXTAS and C9orf72 ALS/FTD. Finally, pharmacological targeting of SRPK1 supressed CGG repeat toxicity and enhanced survival in rodent neurons. Taken together, these findings demonstrate roles for CGG repeat RNA binding proteins in both RAN translation and repeat toxicity and suggest SRPK inhibition may serve as a possible therapeutic strategy in repeat expansion disorders.

neuroscience↗

bioRxiv2020

Near-cognate initiation generates FMRpolyG from CGG repeats in Fragile X associated Tremor Ataxia Syndrome

Repeat associated non-AUG (RAN) translation of FMR1 5 UTR CGG repeats produces toxic homo-polymeric proteins that accumulate within ubiquitinated inclusions in Fragile X-associated tremor/ataxia syndrome (FXTAS) patient brains and model systems. The most abundant RAN product, FMRpolyG, initiates predominantly at an ACG codon located just 5 to the repeat. Methods to accurately measure FMRpolyG in FXTAS patients are lacking. Here we used data dependent acquisition (DDA) and parallel reaction monitoring (PRM) mass spectrometry coupled with stable isotope labeled standard peptides (SIS) to identify potential signature FMRpolyG fragments in patient cells and tissues. Following immunoprecipitation (IP) enrichment, we detected FMRpolyG signature peptides by PRM in transfected cells, FXTAS human samples and patient derived stem cells, but not in controls. Surprisingly, we identified two amino-terminal peptides: one beginning with methionine (Ac-MEAPLPGGVR) initiating at an ACG, and a second beginning with threonine (Ac-TEAPLPGGVR), initiating at a GUG. Abundance of the threonine peptide was enhanced relative to the methionine peptide upon activation of the integrated stress response. In addition, loss of the eIF2 alternative factor, eIF2A, or enhanced expression of initiation factor eIF1, preferentially suppressed GUG initiated FMRpolyG synthesis. These data demonstrate that FMRpolyG is quantifiable in human samples and that RAN translation on FMR1 initiates at specific near cognate codons dependent on available initiation factors and cellular environment.

neuroscience↗

bioRxiv2020

The carboxyl-terminus of RAN translated GGGGCC nucleotide repeat expansions modulates toxicity in models of ALS/FTD

An intronic hexanucleotide repeat expansion in C9ORF72 causes familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This repeat is thought to elicit toxicity through RNA mediated protein sequestration and repeat-associated non-AUG (RAN) translation of dipeptide repeat proteins (DPRs). We generated a series of transgenic Drosophila models expressing GGGGCC (G4C2) repeats either inside of an artificial intron within a GFP reporter or within the 5 UTR of GFP placed in different downstream reading frames. Expression of 484 intronic repeats elicited minimal alterations in eye morphology, viability, longevity, or larval crawling but did trigger RNA foci formation, consistent with prior reports. In contrast, insertion of repeats into the 5 UTR elicited differential toxicity that was dependent on the reading frame of GFP relative to the repeat. Greater toxicity correlated with a short and unstructured carboxyl terminus in the glycine-arginine (GR) RAN protein reading frame. This change in C-terminal sequence triggered nuclear accumulation of all three RAN DPRs. A similar differential toxicity and dependence on the GR carboxyl terminus was observed when repeats were expressed in rodent neurons. The presence of the native carboxyl-termini across all three reading frames was partly protective. Taken together, these findings suggest that carboxyl terminal sequences outside of the repeat region may alter the behavior and toxicity of dipeptide repeat proteins derived from GGGGCC repeats.

genetics↗

bioRxiv2018

Translation of upstream open reading frames in a model of neuronal differentiation

Upstream open reading frames (uORFs) initiate translation within mRNA 5 leaders, and have the potential to alter main coding sequence (CDS) translation on transcripts in which they reside. Ribosome profiling (RP) studies suggest that translating ribosomes are pervasive within 5 leaders across model systems. However, the significance of this observation remains unclear. To explore a role for uORF usage in neuronal differentiation, we performed RP on undifferentiated and differentiated human neuroblastoma cells. Using a spectral coherence algorithm (SPECtre), we identify 4,954 uORFs across 31% of all neuroblastoma transcripts. These uORFs predominantly utilize non-AUG initiation codons and exhibit translational efficiencies (TE) comparable to annotated coding regions. Usage of both AUG initiated uORFs and a conserved and consistently translated subset of non-AUG initiated uORFs correlates with repressed CDS translation. Ribosomal protein transcripts are enriched in uORFs, and select uORFs on such transcripts were validated for expression. With neuronal differentiation, we observed an overall positive correlation between translational shifts in uORF/CDS pairs. However, a subset of transcripts exhibit inverse shifts in translation of uORF/CDS pairs. These uORFs are enriched in AUG initiation sites, non-overlapping, and shorter in length.\n\nCumulatively, CDSs downstream of uORFs characterized by persistent translation show smaller shifts in TE with neuronal differentiation relative to CDSs without a predicted uORF, suggesting that fluctuations in CDS translation are buffered by uORF translation. In sum, this work provides insights into the dynamic relationships and potential regulatory functions of uORF/CDS pairs in a model of neuronal differentiation.

molecular biology↗

bioRxiv2018

Targeted Reactivation of FMR1 Transcription in Fragile X Syndrome Embryonic Stem Cells

Fragile X Syndrome (FXS) is the most common inherited cause of intellectual disability and autism. It results from expansion of a CGG nucleotide repeat in the 5 untranslated region of FMR1. Large expansions elicit repeat and promoter hyper-methylation, heterochromatin formation, FMR1 transcriptional silencing, and loss of the Fragile X protein, FMRP. Efforts aimed at correcting the sequelae resultant from FMRP loss have thus far proven insufficient, perhaps because of FMRPs pleiotropic functions. As the repeats do not disrupt the FMRP coding sequence, reactivation of endogenous FMR1 gene expression could correct the proximal event in FXS pathogenesis. Here we utilize the CRISPR/dCAS9 system to selectively re-activate transcription from the silenced FMR1 locus. Fusion of the transcriptional activator VP192 to dCAS9 robustly enhances FMR1 transcription and increases FMRP levels when targeted directly to the CGG repeat in human cells. Using a previously uncharacterized FXS human embryonic stem cell (hESC) line which acquires transcriptional silencing with serial passaging, we achieved locus-specific transcriptional re-activation of FMR1 mRNA expression despite promoter and repeat methylation. These studies demonstrate that FMR1 mRNA expression can be selectively reactivated in human patient cells, creating a pathway forward for therapeutic development in Fragile X Syndrome.

neuroscience↗