Molecular mechanisms underlying nucleotide repeat expansion disorders.

The human genome contains over one million short tandem repeats. Expansion of a subset of these repeat tracts underlies over fifty human disorders, including common genetic causes of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (C9orf72), polyglutamine-associated ataxias and Huntington disease, myotonic dystrophy, and intellectual disability disorders such as Fragile X syndrome. In this Review, we discuss the four major mechanisms by which expansion of short tandem repeats causes disease: loss of function through transcription repression, RNA-mediated gain of function through gelation and sequestration of RNA-binding proteins, gain of function of canonically translated repeat-harbouring proteins, and repeat-associated non-AUG translation of toxic repeat peptides. Somatic repeat instability amplifies these mechanisms and influences both disease age of onset and tissue specificity of pathogenic features. We focus on the crosstalk between these disease mechanisms, and argue that they often synergize to drive pathogenesis. We also discuss the emerging native functions of repeat elements and how their dynamics might contribute to disease at a larger scale than currently appreciated. Lastly, we propose that lynchpins tying these disease mechanisms and native functions together offer promising therapeutic targets with potential shared applications across this class of human disorders.

What repeat expansion disorders can teach us about the Central Dogma.

Pathogenic repeat sequences underlie several human disorders, including amyotrophic lateral sclerosis, Huntington's disease, and myotonic dystrophy. Here, we speak to several researchers about how repeat sequences have been implicated in affecting all aspects of the Central Dogma of molecular biology through their effects on DNA, RNA, and protein.

Ribosome queuing enables non-AUG translation to be resistant to multiple protein synthesis inhibitors.

Aberrant translation initiation at non-AUG start codons is associated with multiple cancers and neurodegenerative diseases. Nevertheless, how non-AUG translation may be regulated differently from canonical translation is poorly understood. Here, we used start codon-specific reporters and ribosome profiling to characterize how translation from non-AUG start codons responds to protein synthesis inhibitors in human cells. These analyses surprisingly revealed that translation of multiple non-AUG-encoded reporters and the endogenous GUG-encoded DAP5 (eIF4G2/p97) mRNA is resistant to cycloheximide (CHX), a translation inhibitor that severely slows but does not completely abrogate elongation. Our data suggest that slowly elongating ribosomes can lead to queuing/stacking of scanning preinitiation complexes (PICs), preferentially enhancing recognition of weak non-AUG start codons. Consistent with this model, limiting PIC formation or scanning sensitizes non-AUG translation to CHX. We further found that non-AUG translation is resistant to other inhibitors that target ribosomes within the coding sequence but not those targeting newly initiated ribosomes. Together, these data indicate that ribosome queuing enables mRNAs with poor initiation context-namely, those with non-AUG start codons-to be resistant to pharmacological translation inhibitors at concentrations that robustly inhibit global translation.

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 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.

Ribosomal quality control factors inhibit repeat-associated non-AUG translation from GC-rich repeats.

A GGGGCC (G4C2) hexanucleotide repeat expansion in C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), while a CGG trinucleotide repeat expansion in FMR1 leads to the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). These GC-rich repeats form RNA secondary structures that support repeat-associated non-AUG (RAN) translation of toxic proteins that contribute to disease pathogenesis. Here we assessed whether these same repeats might trigger stalling and interfere with translational elongation. We find that depletion of ribosome-associated quality control (RQC) factors NEMF, LTN1 and ANKZF1 markedly boost RAN translation product accumulation from both G4C2 and CGG repeats while overexpression of these factors reduces RAN production in both reporter assays and C9ALS/FTD patient iPSC-derived neurons. We also detected partially made products from both G4C2 and CGG repeats whose abundance increased with RQC factor depletion. Repeat RNA sequence, rather than amino acid content, is central to the impact of RQC factor depletion on RAN translation-suggesting a role for RNA secondary structure in these processes. Together, these findings suggest that ribosomal stalling and RQC pathway activation during RAN translation inhibits the generation of toxic RAN products. We propose augmenting RQC activity as a therapeutic strategy in GC-rich repeat expansion disorders.

Neural evidence of switch processes during semantic and phonetic foraging in human memory.

Humans may retrieve words from memory by exploring and exploiting in "semantic space" similar to how nonhuman animals forage for resources in physical space. This has been studied using the verbal fluency test (VFT), in which participants generate words belonging to a semantic or phonetic category in a limited time. People produce bursts of related items during VFT, referred to as "clustering" and "switching." The strategic foraging model posits that cognitive search behavior is guided by a monitoring process which detects relevant declines in performance and then triggers the searcher to seek a new patch or cluster in memory after the current patch has been depleted. An alternative body of research proposes that this behavior can be explained by an undirected rather than strategic search process, such as random walks with or without random jumps to new parts of semantic space. This study contributes to this theoretical debate by testing for neural evidence of strategically timed switches during memory search. Thirty participants performed category and letter VFT during functional MRI. Responses were classified as cluster or switch events based on computational metrics of similarity and participant evaluations. Results showed greater hippocampal and posterior cerebellar activation during switching than clustering, even while controlling for interresponse times and linguistic distance. Furthermore, these regions exhibited ramping activity which increased during within-patch search leading up to switches. Findings support the strategic foraging model, clarifying how neural switch processes may guide memory search in a manner akin to foraging in patchy spatial environments.

Identification of PSMB5 as a genetic modifier of fragile X-associated tremor/ataxia syndrome.

Fragile X­associated tremor/ataxia syndrome (FXTAS) is a debilitating late-onset neurodegenerative disease in premutation carriers of the expanded CGG repeat in FMR1 that presents with a spectrum of neurological manifestations, such as gait ataxia, intention tremor, and parkinsonism [P. J. Hagerman, R. J. Hagerman, Ann. N. Y. Acad. Sci. 1338, 58­70 (2015); S. Jacquemont et al., JAMA 291, 460­469 (2004)]. Here, we performed whole-genome sequencing (WGS) on male premutation carriers (CGG55­200) and prioritized candidate variants to screen for candidate genetic modifiers using a Drosophila model of FXTAS. We found 18 genes that genetically modulate CGG-associated neurotoxicity in Drosophila, such as Prosbeta5 (PSMB5), pAbp (PABPC1L), e(y)1 (TAF9), and CG14231 (OSGEPL1). Among them, knockdown of Prosbeta5 (PSMB5) suppressed CGG-associated neurodegeneration in the fly as well as in N2A cells. Interestingly, an expression quantitative trait locus variant in PSMB5, PSMB5rs11543947-A, was found to be associated with decreased expression of PSMB5 and delayed onset of FXTAS in human FMR1 premutation carriers. Finally, we demonstrate evidence that PSMB5 knockdown results in suppression of CGG neurotoxicity via both the RAN translation and RNA-mediated toxicity mechanisms, thereby presenting a therapeutic strategy for FXTAS.

Non-canonical initiation factors modulate repeat-associated non-AUG translation.

Repeat-associated non-AUG (RAN) translation of expanded repeat-mutation mRNA 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, DENR and 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 the survival of Drosophila expressing expanded GGGGCC repeats. Taken together, these data support a role for alternative initiation factors in RAN translation and suggest these may serve as novel therapeutic targets in neurodegenerative disease.

Mechanistic convergence across initiation sites for RAN translation in fragile X associated tremor ataxia syndrome.

Repeat associated non-AUG (RAN) translation of CGG repeats in the 5'UTR of FMR1 produces toxic proteins that contribute to fragile X-associated tremor/ataxia syndrome (FXTAS) pathogenesis. The most abundant RAN product, FMRpolyG, initiates predominantly at an ACG upstream of the repeat. Accurate FMRpolyG measurements in FXTAS patients are lacking. We used data-dependent acquisition and parallel reaction monitoring (PRM) mass spectrometry coupled with stable isotope labeled standard peptides to identify signature FMRpolyG fragments in patient samples. Following immunoprecipitation, PRM detected FMRpolyG signature peptides in transfected cells, and FXTAS tissues and cells, but not in controls. We identified two amino-terminal peptides: an ACG-initiated Ac-MEAPLPGGVR and a GUG-initiated Ac-TEAPLPGGVR, as well as evidence for RAN translation initiation within the CGG repeat itself in two reading frames. Initiation at all sites increased following cellular stress, decreased following eIF1 overexpression and was eIF4A and M7G cap-dependent. These data demonstrate that FMRpolyG is quantifiable in human samples and FMR1 RAN translation initiates via similar mechanisms for near-cognate codons and within the repeat through processes dependent on available initiation factors and cellular environment.

CGG Repeat-Associated Non-AUG Translation Utilizes a Cap-Dependent Scanning Mechanism of Initiation to Produce Toxic Proteins.

Repeat-associated non-AUG (RAN) translation produces toxic polypeptides from nucleotide repeat expansions in the absence of an AUG start codon and contributes to neurodegenerative disorders such as ALS and fragile X-associated tremor/ataxia syndrome. How RAN translation occurs is unknown. Here we define the critical sequence and initiation factors that mediate CGG repeat RAN translation in the 5' leader of fragile X mRNA, FMR1. Our results reveal that CGG RAN translation is 30%-40% as efficient as AUG-initiated translation, is m(7)G cap and eIF4E dependent, requires the eIF4A helicase, and is strongly influenced by repeat length. However, it displays a dichotomous requirement for initiation site selection between reading frames, with initiation in the +1 frame, but not the +2 frame, occurring at near-cognate start codons upstream of the repeat. These data support a model in which RAN translation at CGG repeats uses cap-dependent ribosomal scanning, yet bypasses normal requirements for start codon selection.

CGG repeats trigger translational frameshifts that generate aggregation-prone chimeric proteins.

CGG repeat expansions in the FMR1 5'UTR cause the neurodegenerative disease Fragile X-associated tremor/ataxia syndrome (FXTAS). These repeats form stable RNA secondary structures that support aberrant translation in the absence of an AUG start codon (RAN translation), producing aggregate-prone peptides that accumulate within intranuclear neuronal inclusions and contribute to neurotoxicity. Here, we show that the most abundant RAN translation product, FMRpolyG, is markedly less toxic when generated from a construct with a non-repetitive alternating codon sequence in place of the CGG repeat. While exploring the mechanism of this differential toxicity, we observed a +1 translational frameshift within the CGG repeat from the arginine to glycine reading frame. Frameshifts occurred within the first few translated repeats and were triggered predominantly by RNA sequence and structural features. Short chimeric R/G peptides form aggregates distinct from those formed by either pure arginine or glycine, and these chimeras induce toxicity in cultured rodent neurons. Together, this work suggests that CGG repeats support translational frameshifting and that chimeric RAN translated peptides may contribute to CGG repeat-associated toxicity in FXTAS and related disorders.

Dissecting the roles of EIF4G homologs reveals DAP5 as a modifier of CGG repeat-associated toxicity in a Drosophila model of FXTAS.

Neurodegeneration in Fragile X-associated tremor/ataxia syndrome (FXTAS) is caused by a CGG trinucleotide repeat expansion in the 5' UTR of FMR1. Expanded CGG repeat RNAs form stable secondary structures, which in turn support repeat-associated non-AUG (RAN) translation to produce toxic peptides. The parameters that impact RAN translation initiation efficiency are not well understood. Here we used a Drosophila melanogaster model of FXTAS to evaluate the role of the eIF4G family of eukaryotic translation initiation factors (EIF4G1, EIF4GII and EIF4G2/DAP5) in modulating RAN translation and CGG repeat-associated toxicity. DAP5 knockdown robustly suppressed CGG repeat-associated toxicity and inhibited RAN translation. Furthermore, knockdown of initiation factors that preferentially associate with DAP5 (such as EIF2ß, EIF3F and EIF3G) also selectively suppressed CGG repeat-induced eye degeneration. In mammalian cellular reporter assays, DAP5 knockdown exhibited modest and cell-type specific effects on RAN translation. Taken together, these data support a role for DAP5 in CGG repeat associated toxicity possibly through modulation of RAN translation.

A pharmacophore-based approach to demonstrating the scope of alcohol dehydrogenases.

Barriers to the ready adoption of biocatalysis into asymmetric synthesis for early stage medicinal chemistry are addressed, using ketone reduction by alcohol dehydrogenase as a model reaction. An efficient substrate screening approach is used to show the wide substrate scope of commercial alcohol dehydrogenase enzymes, with a high tolerance to chemical groups employed in drug discovery (heterocycle, trifluoromethyl and nitrile/nitro groups) observed. We use our screening data to build a preliminary predictive pharmacophore-based screening tool using Forge software, with a precision of 0.67/1, demonstrating the potential for developing substrate screening tools for commercially available enzymes without publicly available structures. We hope that this work will facilitate a culture shift towards adopting biocatalysis alongside traditional chemical catalytic methods in early stage drug discovery.

Social Search and Resource Clustering as Emergent Stable States.

Social search has stably evolved across various species and is often used by humans to search for resources (such as food, information, social partners). In turn, these resources frequently come distributed in patches or clusters. In the current work, we use an ecologically inspired agent-based model to investigate whether social search and clustering are stable outcomes of the dynamical mutual interactions between the two. While previous research has studied unidirectional influences of social search on resource clustering and vice versa, the current work investigates the consequential patterns emerging from their two-way interactions over time. In our model, consumers evolved search strategies (ranging from competitive to social) as adaptations to their environmental resource structures, and resources varied in distributions (ranging from random to clustered) that were shaped by agents' consumption patterns. Across four experiments, we systematically analyzed the patterns of influence that search strategies and environment structure have on each other to identify stable attractor states of both. In Experiment 1, we fixed resource clustering at various levels and observed its influence on social search, and in Experiment 2, we observed the influence of social search on resource distribution. In both these experiments we found that increasing levels of one variable produced increases in the other; however, at very high levels of the manipulated variable, the dependent variable tended to fall. Finally in Experiments 3 and 4, we studied the dynamics that arose when resource clustering and social search could both change and mutually influence each other, finding that low levels of social search and clustering were stable attractor states. Our simple 2D model yielded results that qualitatively resemble those across a wide range of search domains (from physical search for food to abstract search for information), highlighting some stable outcomes of mutually interacting consumer/resource systems.

The International Fragile X Premutation Registry: building a resource for research and clinical trial readiness.

FMR1 premutation cytosine-guanine-guanine repeat expansion alleles are relatively common mutations in the general population that are associated with a neurodegenerative disease (fragile X-associated tremor/ataxia syndrome), reproductive health problems and potentially a wide range of additional mental and general health conditions that are not yet well-characterised. The International Fragile X Premutation Registry (IFXPR) was developed to facilitate and encourage research to better understand the FMR1 premutation and its impact on human health, to facilitate clinical trial readiness by identifying and characterising diverse cohorts of individuals interested in study participation, and to build community and collaboration among carriers, family members, researchers and clinicians around the world. Here, we describe the development and content of the IFXPR, characterise its first 747 registrants from 32 countries and invite investigators to apply for recruitment support for their project(s). With larger numbers, increased diversity and potentially the future clinical characterisation of registrants, the IFXPR will contribute to a more comprehensive and accurate understanding of the fragile X premutation in human health and support treatment studies.

Eating sustainably: Conviction or convenience?

Environmentally sustainable food consumption is one component of addressing climate change. Previous research has largely approached sustainable food consumption by investigating individual behaviors, without a broader conceptualization of what motivates food consumers to act sustainably. Using a representative sample of Indiana consumers, we explore sustainability across a range of food behaviors through latent class analysis, controlling for environmental attitudes, spatial access to food, and consumer demographics. This approach allows us to go beyond consumer segmentation analysis to explore how consumers conceptualize sustainable food behavior. The largest class of consumers (44% of the sample) appear either unwilling or unable to pay more for sustainability but are more likely to engage in sustainable behaviors that intersect with self-oriented attributes such as health benefits and lower cost. A second class (34%) consists of consumers who seem to be primarily motivated by the single issue of buying organic, are on average higher income, more educated, have better access to food, and are not opposed to paying for sustainability. Consumers in the smallest and most highly motivated group (9%) in terms of sustainability attitudes and self-perceived sustainability focus on local food production and are generally rural dwelling with less income. Only 13% of consumers engage in few to no sustainable behaviors, and these people notably exhibit the least sustainable attitudes. These findings illustrate the ways in which food sustainability is more nuanced than often characterized-much of it is driven by convenience and self-interest rather than reputation with respect to sustainability or conviction about environmental outcomes. This work also highlights how a combination of social, psychological, and spatial barriers exists and shape how different consumer groups conceptualize sustainable food consumption.

COVID-19 and friendships: Agreeableness and neuroticism are associated with more concern about COVID-19 and friends' risky behaviors.

Given the importance of friendships during challenging times and the mixed associations between personality traits and disease-related behaviors, we investigated the correlations between personality traits and perceptions of friendships during the COVID-19 pandemic. Data were collected as part of a longitudinal investigation of the correlations between the pandemic and various cooperative relationships. In this investigation, we found that agreeableness and neuroticism predicted participants being more concerned about COVID-19 and bothered by friends' risky behavior, and extraversion predicted enjoying helping friends during the pandemic. Our results suggest that personality differences are associated with how individuals cope with friends' risky behaviors during the COVID-19 pandemic.

Quantifying changes in societal optimism from online sentiment.

Individuals can hold contrasting views about distinct times: for example, dread over tomorrow's appointment and excitement about next summer's vacation. Yet, psychological measures of optimism often assess only one time point or ask participants to generalize about their future. Here, we address these limitations by developing the optimism curve, a measure of societal optimism that compares positivity toward different future times that was inspired by the Treasury bond yield curve. By performing sentiment analysis on over 3.5 million tweets that reference 23 future time points (2 days to 30 years), we measured how positivity differs across short-, medium-, and longer-term future references. We found a consistent negative association between positivity and the distance into the future referenced: From August 2017 to February 2020, the long-term future was discussed less positively than the short-term future. During the COVID-19 pandemic, this relationship inverted, indicating declining near-future- but stable distant-future-optimism. Our results demonstrate that individuals hold differentiated attitudes toward the near and distant future that shift in aggregate over time in response to external events. The optimism curve uniquely captures these shifting attitudes and may serve as a useful tool that can expand existing psychometric measures of optimism.

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

GGGGCC (G4C2) hexanucleotide repeat expansions in the endosomal trafficking gene C9orf72 are the most common genetic cause of ALS and frontotemporal dementia. Repeat-associated non-AUG (RAN) translation of this expansion through near-cognate initiation codon usage and internal ribosomal entry generates toxic proteins that accumulate in patients' brains and contribute to disease pathogenesis. The helicase protein DEAH-box helicase 36 (DHX36-G4R1) plays active roles in RNA and DNA G-quadruplex (G4) resolution in cells. As G4C2 repeats are known to form G4 structures in vitro, we sought to determine the impact of manipulating DHX36 expression on repeat transcription and RAN translation. Using a series of luciferase reporter assays both in cells and in vitro, we found that DHX36 depletion suppresses RAN translation in a repeat length-dependent manner, whereas overexpression of DHX36 enhances RAN translation from G4C2 reporter RNAs. Moreover, upregulation of RAN translation that is typically triggered by integrated stress response activation is prevented by loss of DHX36. These results suggest that DHX36 is active in regulating G4C2 repeat translation, providing potential implications for therapeutic development in nucleotide repeat expansion disorders.