Splice modulators target PMS1 to reduce somatic expansion of the Huntington's disease-associated CAG repeat.

Huntington's disease (HD) is a dominant neurological disorder caused by an expanded HTT exon 1 CAG repeat that lengthens huntingtin's polyglutamine tract. Lowering mutant huntingtin has been proposed for treating HD, but genetic modifiers implicate somatic CAG repeat expansion as the driver of onset. We find that branaplam and risdiplam, small molecule splice modulators that lower huntingtin by promoting HTT pseudoexon inclusion, also decrease expansion of an unstable HTT exon 1 CAG repeat in an engineered cell model. Targeted CRISPR-Cas9 editing shows this effect is not due to huntingtin lowering, pointing instead to pseudoexon inclusion in PMS1. Homozygous but not heterozygous inactivation of PMS1 also reduces CAG repeat expansion, supporting PMS1 as a genetic modifier of HD and a potential target for therapeutic intervention. Although splice modulation provides one strategy, genome-wide transcriptomics also emphasize consideration of cell-type specific effects and polymorphic variation at both target and off-target sites.

Somatic CAG Repeat Stability in a Transgenic Sheep Model of Huntington's Disease.

Somatic instability of the huntingtin (HTT) CAG repeat mutation modifies age-at-onset of Huntington's disease (HD). Understanding the mechanism and pathogenic consequences of instability may reveal therapeutic targets. Using small-pool PCR we analyzed CAG instability in the OVT73 sheep model which expresses a full-length human cDNA HTT transgene. Analyses of five- and ten-year old sheep revealed the transgene (CAG)69 repeat was remarkably stable in liver, striatum, and other brain tissues. As OVT73 sheep at ten years old have minimal cell death and behavioral changes, our findings support instability of the HTT expanded-CAG repeat as being required for the progression of HD.

PMS1 as a target for splice modulation to prevent somatic CAG repeat expansion in Huntington's disease.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder whose motor, cognitive, and behavioral manifestations are caused by an expanded, somatically unstable CAG repeat in the first exon of HTT that lengthens a polyglutamine tract in huntingtin. Genome-wide association studies (GWAS) have revealed DNA repair genes that influence the age-at-onset of HD and implicate somatic CAG repeat expansion as the primary driver of disease timing. To prevent the consequent neuronal damage, small molecule splice modulators (e.g., branaplam) that target HTT to reduce the levels of huntingtin are being investigated as potential HD therapeutics. We found that the effectiveness of the splice modulators can be influenced by genetic variants, both at HTT and other genes where they promote pseudoexon inclusion. Surprisingly, in a novel hTERT-immortalized retinal pigment epithelial cell (RPE1) model for assessing CAG repeat instability, these drugs also reduced the rate of HTT CAG expansion. We determined that the splice modulators also affect the expression of the mismatch repair gene PMS1, a known modifier of HD age-at-onset. Genome editing at specific HTT and PMS1 sequences using CRISPR-Cas9 nuclease confirmed that branaplam suppresses CAG expansion by promoting the inclusion of a pseudoexon in PMS1, making splice modulation of PMS1 a potential strategy for delaying HD onset. Comparison with another splice modulator, risdiplam, suggests that other genes affected by these splice modulators also influence CAG instability and might provide additional therapeutic targets.

Multiple nutrient transporters enable cells to mitigate a rate-affinity tradeoff.

Eukaryotic genomes often encode multiple transporters for the same nutrient. For example, budding yeast has 17 hexose transporters (HXTs), all of which potentially transport glucose. Using mathematical modelling, we show that transporters that use either facilitated diffusion or symport can have a rate-affinity tradeoff, where an increase in the maximal rate of transport decreases the transporter's apparent affinity. These changes affect the import flux non-monotonically, and for a given concentration of extracellular nutrient there is one transporter, characterised by its affinity, that has a higher import flux than any other. Through encoding multiple transporters, cells can therefore mitigate the tradeoff by expressing those transporters with higher affinities in lower concentrations of nutrients. We verify our predictions using fluorescent tagging of seven HXT genes in budding yeast and follow their expression over time in batch culture. Using the known affinities of the corresponding transporters, we show that their regulation in glucose is broadly consistent with a rate-affinity tradeoff: as glucose falls, the levels of the different transporters peak in an order that mostly follows their affinity for glucose. More generally, evolution is constrained by tradeoffs. Our findings indicate that one such tradeoff often occurs in the cellular transport of nutrients.

Tissue-specific and repeat length-dependent somatic instability of the X-linked dystonia parkinsonism-associated CCCTCT repeat.

X-linked dystonia-parkinsonism (XDP) is a progressive adult-onset neurodegenerative disorder caused by insertion of a SINE-VNTR-Alu (SVA) retrotransposon in the TAF1 gene. The SVA retrotransposon contains a CCCTCT hexameric repeat tract of variable length, whose length is inversely correlated with age at onset. This places XDP in a broader class of repeat expansion diseases, characterized by the instability of their causative repeat mutations. Here, we observe similar inverse correlations between CCCTCT repeat length with age at onset and age at death and no obvious correlation with disease duration. To gain insight into repeat instability in XDP we performed comprehensive quantitative analyses of somatic instability of the XDP CCCTCT repeat in blood and in seventeen brain regions from affected males. Our findings reveal repeat length-dependent and expansion-based instability of the XDP CCCTCT repeat, with greater levels of expansion in brain than in blood. The brain exhibits regional-specific patterns of instability that are broadly similar across individuals, with cerebellum exhibiting low instability and cortical regions exhibiting relatively high instability. The spectrum of somatic instability in the brain includes a high proportion of moderate repeat length changes of up to 5 repeats, as well as expansions of ~ 20- > 100 repeats and contractions of ~ 20-40 repeats at lower frequencies. Comparison with HTT CAG repeat instability in postmortem Huntington's disease brains reveals similar brain region-specific profiles, indicating common trans-acting factors that contribute to the instability of both repeats. Analyses in XDP brains of expansion of a different SVA-associated CCCTCT located in the LIPG gene, and not known to be disease-associated, reveals repeat length-dependent expansion at overall lower levels relative to the XDP CCCTCT repeat, suggesting that expansion propensity may be modified by local chromatin structure. Together, the data support a role for repeat length-dependent somatic expansion in the process(es) driving the onset of XDP and prompt further investigation into repeat dynamics and the relationship to disease.

Mutations causing Lopes-Maciel-Rodan syndrome are huntingtin hypomorphs.

Huntington's disease pathogenesis involves a genetic gain-of-function toxicity mechanism triggered by the expanded HTT CAG repeat. Current therapeutic efforts aim to suppress expression of total or mutant huntingtin, though the relationship of huntingtin's normal activities to the gain-of-function mechanism and what the effects of huntingtin-lowering might be are unclear. Here, we have re-investigated a rare family segregating two presumed HTT loss-of-function (LoF) variants associated with the developmental disorder, Lopes-Maciel-Rodan syndrome (LOMARS), using whole-genome sequencing of DNA from cell lines, in conjunction with analysis of mRNA and protein expression. Our findings correct the muddled annotation of these HTT variants, reaffirm they are the genetic cause of the LOMARS phenotype and demonstrate that each variant is a huntingtin hypomorphic mutation. The NM_002111.8: c.4469+1G>A splice donor variant results in aberrant (exon 34) splicing and severely reduced mRNA, whereas, surprisingly, the NM_002111.8: c.8157T>A NP_002102.4: Phe2719Leu missense variant results in abnormally rapid turnover of the Leu2719 huntingtin protein. Thus, although rare and subject to an as yet unknown LoF intolerance at the population level, bona fide HTT LoF variants can be transmitted by normal individuals leading to severe consequences in compound heterozygotes due to huntingtin deficiency.

Pan-Genome-Scale Network Reconstruction: Harnessing Phylogenomics Increases the Quantity and Quality of Metabolic Models.

A genome-scale network reconstruction (GENRE) is a knowledgebase for an organism and has various applications. Available genome sequences have risen in recent years, but the number of curated GENREs has not kept pace. Existing yeast GENREs contain significant commission and omission errors. Current practices limit the quantity and quality of GENREs. An open and transparent phylogenomic-driven framework is outlined to address these issues. The method is demonstrated with 33 yeasts and fungi in Dikarya. A pan-fungal metabolic network called FYRMENT (Fungal and Yeast Metabolic Network) (https://github.com/LMSE/FYRMENT) is created, and annotated with ortholog groups from AYbRAH (https://github.com/LMSE/AYbRAH). Metabolic models for lower-level taxons are compiled. The fungal pan-GENRE contains 1553 orthologs, 2759 reactions, 2251 metabolites. The GENREs have higher genomic and metabolic coverage than existing yeast and fungal GENREs created with other methods. Metabolic simulations show the maximum amino acid yields from glucose differs between yeast lineages, indicating metabolic networks have evolved. Curating genomes and reactions at higher taxonomic-levels increases the quantity and quality of GENREs than conventional approaches. This approach can scale to other branches in the tree of life.

Patterns of CAG repeat instability in the central nervous system and periphery in Huntington's disease and in spinocerebellar ataxia type 1.

The expanded HTT CAG repeat causing Huntington's disease (HD) exhibits somatic expansion proposed to drive the rate of disease onset by eliciting a pathological process that ultimately claims vulnerable cells. To gain insight into somatic expansion in humans, we performed comprehensive quantitative analyses of CAG expansion in ~50 central nervous system (CNS) and peripheral postmortem tissues from seven adult-onset and one juvenile-onset HD individual. We also assessed ATXN1 CAG repeat expansion in brain regions of an individual with a neurologically and pathologically distinct repeat expansion disorder, spinocerebellar ataxia type 1 (SCA1). Our findings reveal similar profiles of tissue instability in all HD individuals, which, notably, were also apparent in the SCA1 individual. CAG expansion was observed in all tissues, but to different degrees, with multiple cortical regions and neostriatum tending to have the greatest instability in the CNS, and liver in the periphery. These patterns indicate different propensities for CAG expansion contributed by disease locus-independent trans-factors and demonstrate that expansion per se is not sufficient to cause cell type or disease-specific pathology. Rather, pathology may reflect distinct toxic processes triggered by different repeat lengths across cell types and diseases. We also find that the HTT CAG length-dependent expansion propensity of an individual is reflected in all tissues and in cerebrospinal fluid. Our data indicate that peripheral cells may be a useful source to measure CAG expansion in biomarker assays for therapeutic efforts, prompting efforts to dissect underlying mechanisms of expansion that may differ between the brain and periphery.

A novel C-terminal degron identified in bacterial aldehyde decarbonylases using directed evolution.

BACKGROUND: Aldehyde decarbonylases (ADs), which convert acyl aldehydes into alkanes, supply promising solution for producing alkanes from renewable feedstock. However the instability of ADs impedes their further application. Therefore, the current study aimed to investigate the degradation mechanism of ADs and engineer it towards high stability. RESULTS: Here, we describe the discovery of a degradation tag (degron) in the AD from marine cyanobacterium Prochlorococcus marinus using error-prone PCR-based directed evolution system. Bioinformatic analysis revealed that this C-terminal degron is common in bacterial ADs and identified a conserved C-terminal motif, RMSAYGLAAA, representing the AD degron (ADcon). Furthermore, we demonstrated that the ATP-dependent proteases ClpAP and Lon are involved in the degradation of AD-tagged proteins in E. coli, thereby limiting alkane production. Deletion or modification of the degron motif increased alkane production in vivo. CONCLUSION: This work revealed the presence of a novel degron in bacterial ADs responsible for its instability. The in vivo experiments proved eliminating or modifying the degron could stabilize AD, thereby producing higher titers of alkanes.

Publisher Correction: MEMOTE for standardized genome-scale metabolic model testing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genetic Risk Underlying Psychiatric and Cognitive Symptoms in Huntington's Disease.

BACKGROUND: Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded CAG repeat in the HTT gene. It is diagnosed following a standardized examination of motor control and often presents with cognitive decline and psychiatric symptoms. Recent studies have detected genetic loci modifying the age at onset of motor symptoms in HD, but genetic factors influencing cognitive and psychiatric presentations are unknown. METHODS: We tested the hypothesis that psychiatric and cognitive symptoms in HD are influenced by the same common genetic variation as in the general population by 1) constructing polygenic risk scores from large genome-wide association studies of psychiatric and neurodegenerative disorders and of intelligence and 2) testing for correlation with the presence of psychiatric and cognitive symptoms in a large sample (n = 5160) of patients with HD. RESULTS: Polygenic risk score for major depression was associated specifically with increased risk of depression in HD, as was schizophrenia risk score with psychosis and irritability. Cognitive impairment and apathy were associated with reduced polygenic risk score for intelligence. CONCLUSIONS: Polygenic risk scores for psychiatric disorders, particularly depression and schizophrenia, are associated with increased risk of the corresponding psychiatric symptoms in HD, suggesting a common genetic liability. However, the genetic liability to cognitive impairment and apathy appears to be distinct from other psychiatric symptoms in HD. No associations were observed between HD symptoms and risk scores for other neurodegenerative disorders. These data provide a rationale for treatments effective in depression and schizophrenia to be used to treat depression and psychotic symptoms in HD.

AYbRAH: a curated ortholog database for yeasts and fungi spanning 600 million years of evolution.

Budding yeasts inhabit a range of environments by exploiting various metabolic traits. The genetic bases for these traits are mostly unknown, preventing their addition or removal in a chassis organism for metabolic engineering. Insight into the evolution of orthologs, paralogs and xenologs in the yeast pan-genome can help bridge these genotypes; however, existing phylogenomic databases do not span diverse yeasts, and sometimes cannot distinguish between these homologs. To help understand the molecular evolution of these traits in yeasts, we created Analyzing Yeasts by Reconstructing Ancestry of Homologs (AYbRAH), an open-source database of predicted and manually curated ortholog groups for 33 diverse fungi and yeasts in Dikarya, spanning 600 million years of evolution. OrthoMCL and OrthoDB were used to cluster protein sequence into ortholog and homolog groups, respectively; MAFFT and PhyML reconstructed the phylogeny of all homolog groups. Ortholog assignments for enzymes and small metabolite transporters were compared to their phylogenetic reconstruction, and curated to resolve any discrepancies. Information on homolog and ortholog groups can be viewed in the AYbRAH web portal (https://lmse.github.io/aybrah/), including functional annotations, predictions for mitochondrial localization and transmembrane domains, literature references and phylogenetic reconstructions. Ortholog assignments in AYbRAH were compared to HOGENOM, KEGG Orthology, OMA, eggNOG and PANTHER. PANTHER and OMA had the most congruent ortholog groups with AYbRAH, while the other phylogenomic databases had greater amounts of under-clustering, over-clustering or no ortholog annotations for proteins. Future plans are discussed for AYbRAH, and recommendations are made for other research communities seeking to create curated ortholog databases.

An interspecies malate-pyruvate shuttle reconciles redox imbalance in an anaerobic microbial community.

Microbes in ecosystems often develop coordinated metabolic interactions. Therefore, understanding metabolic interdependencies between microbes is critical to deciphering ecosystem function. In this study, we sought to deconstruct metabolic interdependencies in organohalide-respiring consortium ACT-3 containing Dehalobacter restrictus using a combination of metabolic modeling and experimental validation. D. restrictus possesses a complete set of genes for amino acid biosynthesis yet when grown in isolation requires amino acid supplementation. We reconciled this discrepancy using flux balance analysis considering cofactor availability, enzyme promiscuity, and shared protein expression patterns for several D. restrictus strains. Experimentally, 13C incorporation assays, growth assays, and metabolite analysis of D. restrictus strain PER-K23 cultures were performed to validate the model predictions. The model resolved that the amino acid dependency of D. restrictus resulted from restricted NADPH regeneration and predicted that malate supplementation would replenish intracellular NADPH. Interestingly, we observed unexpected export of pyruvate and glutamate in parallel to malate consumption in strain PER-K23 cultures. Further experimental analysis using the ACT-3 transfer cultures suggested the occurrence of an interspecies malate-pyruvate shuttle reconciling a redox imbalance, reminiscent of the mitochondrial malate shunt pathway in eukaryotic cells. Altogether, this study suggests that redox imbalance and metabolic complementarity are important driving forces for metabolite exchange in anaerobic microbial communities.

Inactivation of the transcription factor mig1 (YGL035C) in Saccharomyces cerevisiae improves tolerance towards monocarboxylic weak acids: acetic, formic and levulinic acid.

Toxic concentrations of monocarboxylic weak acids present in lignocellulosic hydrolyzates affect cell integrity and fermentative performance of Saccharomyces cerevisiae. In this work, we report the deletion of the general catabolite repressor Mig1p as a strategy to improve the tolerance of S. cerevisiae towards inhibitory concentrations of acetic, formic or levulinic acid. In contrast with the wt yeast, where the growth and ethanol production were ceased in presence of acetic acid 5 g/L or formic acid 1.75 g/L (initial pH not adjusted), the m9 strain (Δmig1::kan) produced 4.06 ± 0.14 and 3.87 ± 0.06 g/L of ethanol, respectively. Also, m9 strain tolerated a higher concentration of 12.5 g/L acetic acid (initial pH adjusted to 4.5) without affecting its fermentative performance. Moreover, m9 strain produced 33% less acetic acid and 50-70% less glycerol in presence of weak acids, and consumed acetate and formate as carbon sources under aerobic conditions. Our results show that the deletion of Mig1p provides a single gene deletion target for improving the acid tolerance of yeast strains significantly.

Biocatalytic production of adipic acid from glucose using engineered Saccharomyces cerevisiae.

Adipic acid is an important industrial chemical used in the synthesis of nylon-6,6. The commercial synthesis of adipic acid uses petroleum-derived benzene and releases significant quantities of greenhouse gases. Biocatalytic production of adipic acid from renewable feedstocks could potentially reduce the environmental damage and eliminate the need for fossil fuel precursors. Recently, we have demonstrated the first enzymatic hydrogenation of muconic acid to adipic acid using microbial enoate reductases (ERs) - complex iron-sulfur and flavin containing enzymes. In this work, we successfully expressed the Bacillus coagulans ER in a Saccharomyces cerevisiae strain producing muconic acid and developed a three-stage fermentation process enabling the synthesis of adipic acid from glucose. The ability to express active ERs and significant acid tolerance of S. cerevisiae highlight the applicability of the developed yeast strain for the biocatalytic production of adipic acid from renewable feedstocks.

Exploring Bacterial Carboxylate Reductases for the Reduction of Bifunctional Carboxylic Acids.

Carboxylic acid reductases (CARs) selectively reduce carboxylic acids to aldehydes using ATP and NADPH as cofactors under mild conditions. Although CARs attracts significant interest, only a few enzymes have been characterized to date, whereas the vast majority of CARs have yet to be examined. Herein the authors report that 12 bacterial CARs reduces a broad range of bifunctional carboxylic acids containing oxo-, hydroxy-, amino-, or second carboxyl groups with several enzymes showing activity toward 4-hydroxybutanoic (4-HB) and adipic acids. These CARs exhibits significant reductase activity against substrates whose second functional group is separated from the carboxylate by at least three carbons with both carboxylate groups being reduced in dicarboxylic acids. Purified CARs supplemented with cofactor regenerating systems (for ATP and NADPH), an inorganic pyrophosphatase, and an aldo-keto reductase catalyzes a high conversion (50-76%) of 4-HB to 1,4-butanediol (1,4-BDO) and adipic acid to 1,6-hexanediol (1,6-HDO). Likewise, Escherichia coli strains expressing eight different CARs efficiently reduces 4-HB to 1,4-BDO with 50-95% conversion, whereas adipic acid is reduced to a mixture of 6-hydroxyhexanoic acid (6-HHA) and 1,6-HDO. Thus, our results illustrate the broad biochemical diversity of bacterial CARs and their compatibility with other enzymes for applications in biocatalysis.

The Genetic Modifiers of Motor OnsetAge (GeM MOA) Website: Genome-wide Association Analysis for Genetic Modifiers of Huntington's Disease.

BACKGROUND: Huntington's disease (HD) is a dominantly inherited disease caused by a CAG expansion mutation in HTT. The age at onset of clinical symptoms is determined primarily by the length of this CAG expansion but is also influenced by other genetic and/or environmental factors. OBJECTIVE: Recently, through genome-wide association studies (GWAS) aimed at discovering genetic modifiers, we identified loci associated with age at onset of motor signs that are significant at the genome-wide level. However, many additional HD modifiers may exist but may not have achieved statistical significance due to limited power. METHODS: In order to disseminate broadly the entire GWAS results and make them available to complement alternative approaches, we have developed the internet website "GeM MOA" where genetic association results can be searched by gene name, SNP ID, or genomic coordinates of a region of interest. RESULTS: Users of the Genetic Modifiers of Motor Onset Age (GeM MOA) site can therefore examine support for association between any gene region and age at onset of HD motor signs. GeM MOA's interactive interface also allows users to navigate the surrounding region and to obtain association p-values for individual SNPs. CONCLUSIONS: Our website conveys a comprehensive view of the genetic landscape of modifiers of HD from the existing GWAS, and will provide the means to evaluate the potential influence of genes of interest on the onset of HD. GeM MOA is freely available at https://www.hdinhd.org/.