Widespread alternative splicing dysregulation occurs presymptomatically in CAG expansion spinocerebellar ataxias.

The spinocerebellar ataxias (SCAs) are a group of dominantly inherited neurodegenerative diseases, several of which are caused by CAG expansion mutations (SCAs 1, 2, 3, 6, 7 and 12) and more broadly belong to the large family of over 40 microsatellite expansion diseases. While dysregulation of alternative splicing is a well defined driver of disease pathogenesis across several microsatellite diseases, the contribution of alternative splicing in CAG expansion SCAs is poorly understood. Furthermore, despite extensive studies on differential gene expression, there remains a gap in our understanding of presymptomatic transcriptomic drivers of disease. We sought to address these knowledge gaps through a comprehensive study of 29 publicly available RNA-sequencing datasets. We identified that dysregulation of alternative splicing is widespread across CAG expansion mouse models of SCAs 1, 3 and 7. These changes were detected presymptomatically, persisted throughout disease progression, were repeat length-dependent, and were present in brain regions implicated in SCA pathogenesis including the cerebellum, pons and medulla. Across disease progression, changes in alternative splicing occurred in genes that function in pathways and processes known to be impaired in SCAs, such as ion channels, synaptic signalling, transcriptional regulation and the cytoskeleton. We validated several key alternative splicing events with known functional consequences, including Trpc3 exon 9 and Kcnma1 exon 23b, in the Atxn1154Q/2Q mouse model. Finally, we demonstrated that alternative splicing dysregulation is responsive to therapeutic intervention in CAG expansion SCAs with Atxn1 targeting antisense oligonucleotide rescuing key splicing events. Taken together, these data demonstrate that widespread presymptomatic dysregulation of alternative splicing in CAG expansion SCAs may contribute to disease onset, early neuronal dysfunction and may represent novel biomarkers across this devastating group of neurodegenerative disorders.

Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9.

Transcription of expanded microsatellite repeats is associated with multiple human diseases, including myotonic dystrophy, Fuchs endothelial corneal dystrophy, and C9orf72-ALS/FTD. Reducing production of RNA and proteins arising from these expanded loci holds therapeutic benefit. Here, we tested the hypothesis that deactivated Cas9 enzyme impedes transcription across expanded microsatellites. We observed a repeat length-, PAM-, and strand-dependent reduction of repeat-containing RNAs upon targeting dCas9 directly to repeat sequences; targeting the non-template strand was more effective. Aberrant splicing patterns were rescued in DM1 cells, and production of RAN peptides characteristic of DM1, DM2, and C9orf72-ALS/FTD cells was drastically decreased. Systemic delivery of dCas9/gRNA by adeno-associated virus led to reductions in pathological RNA foci, rescue of chloride channel 1 protein expression, and decreased myotonia. These observations suggest that transcription of microsatellite repeat-containing RNAs is more sensitive to perturbation than transcription of other RNAs, indicating potentially viable strategies for therapeutic intervention.

The alternative initiation factor eIF2A plays key role in RAN translation of myotonic dystrophy type 2 CCUG•CAGG repeats.

Repeat-associated non-ATG (RAN) proteins have been reported in 11 microsatellite expansion disorders but the factors that allow RAN translation to occur and the effects of different repeat motifs and alternative AUG-like initiation codons are unclear. We studied the mechanisms of RAN translation across myotonic dystrophy type 2 (DM2) expansion transcripts with (CCUG) or without (CAGG) efficient alternative AUG-like codons. To better understand how DM2 LPAC and QAGR RAN proteins are expressed, we generated a series of CRISPR/Cas9-edited HEK293T cell lines. We show that LPAC and QAGR RAN protein levels are reduced in protein kinase R (PKR)-/- and PKR-like endoplasmic reticulum kinase (PERK)-/- cells, with more substantial reductions of CAGG-encoded QAGR in PKR-/- cells. Experiments using mutant eIF2α-S51A HEK293T cells show that p-eIF2α is required for QAGR production. In contrast, LPAC levels were only partially reduced in these cells, suggesting that both non-AUG and close-cognate initiation occur across CCUG RNAs. Overexpression of the alternative initiation factor eIF2A increases LPAC and QAGR protein levels but, notably, has a much larger effect on QAGR expressed from CAGG-expansion RNAs that lack efficient close-cognate codons. The effects of eIF2A on increasing LPAC are consistent with previous reports that eIF2A affects CUG-initiation translation. The observation that eIF2A also increases QAGR proteins is novel because CAGG expansion transcripts do not contain CUG or similarly efficient close-cognate AUG-like codons. For QAGR but not LPAC, the eIF2A-dependent increases are not seen when p-eIF2α is blocked. These data highlight the differential regulation of DM2 RAN proteins and eIF2A as a potential therapeutic target for DM2 and other RAN diseases.

Repeat instability as the basis for human diseases and as a potential target for therapy.

Expansions of repetitive DNA sequences cause numerous human neurological and neuromuscular diseases. Ongoing repeat expansions in patients can exacerbate disease progression and severity. As pathogenesis is connected to repeat length, a potential therapeutic avenue is to modulate disease by manipulating repeat expansion size--targeting DNA, the root-cause of symptoms. How repeat instability is mediated by DNA replication, repair, recombination, transcription and epigenetics may explain its contribution to pathogenesis and give insights into therapeutic strategies to block expansions or induce contractions.

A CTG repeat-selective chemical screen identifies microtubule inhibitors as selective modulators of toxic CUG RNA levels.

A CTG repeat expansion in the DMPK gene is the causative mutation of myotonic dystrophy type 1 (DM1). Transcription of the expanded CTG repeat produces toxic gain-of-function CUG RNA, leading to disease symptoms. A screening platform that targets production or stability of the toxic CUG RNA in a selective manner has the potential to provide new biological and therapeutic insights. A DM1 HeLa cell model was generated that stably expresses a toxic r(CUG)480 and an analogous r(CUG)0 control from DMPK and was used to measure the ratio-metric level of r(CUG)480 versus r(CUG)0. This DM1 HeLa model recapitulates pathogenic hallmarks of DM1, including CUG ribonuclear foci and missplicing of pre-mRNA targets of the muscleblind (MBNL) alternative splicing factors. Repeat-selective screening using this cell line led to the unexpected identification of multiple microtubule inhibitors as hits that selectively reduce r(CUG)480 levels and partially rescue MBNL-dependent missplicing. These results were validated by using the Food and Drug Administration-approved clinical microtubule inhibitor colchicine in DM1 mouse and primary patient cell models. The mechanism of action was found to involve selective reduced transcription of the CTG expansion that we hypothesize to involve the LINC (linker of nucleoskeleton and cytoskeleton) complex. The unanticipated identification of microtubule inhibitors as selective modulators of toxic CUG RNA opens research directions for this form of muscular dystrophy and may shed light on the biology of CTG repeat expansion and inform therapeutic avenues. This approach has the potential to identify modulators of expanded repeat-containing gene expression for over 30 microsatellite expansion disorders.

Therapeutic strategies for C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia.

PURPOSE OF REVIEW: An intronic G4C2 expansion mutation in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). Although there are currently no treatments for this insidious, fatal disease, intense research has led to promising therapeutic strategies, which will be discussed here. RECENT FINDINGS: Therapeutic strategies for C9-ALS/FTD have primarily focused on reducing the toxic effects of mutant expansion RNAs or the dipeptide repeat proteins (DPRs). The pathogenic effects of G4C2 expansion transcripts have been targeted using approaches aimed at promoting their degradation, inhibiting nuclear export or silencing transcription. Other promising strategies include immunotherapy to reduce the DPRs themselves, reducing RAN translation, removing the repeats using DNA or RNA editing and manipulation of downstream disease-altered stress granule pathways. Finally, understanding the molecular triggers that lead to pheno-conversion may lead to opportunities that can delay symptomatic disease onset. SUMMARY: A large body of evidence implicates RAN-translated DPRs as a main driver of C9-ALS/FTD. Promising therapeutic strategies for these devastating diseases are being rapidly developed with several approaches already in or approaching clinical trials.

Intron retention induced by microsatellite expansions as a disease biomarker.

Expansions of simple sequence repeats, or microsatellites, have been linked to ∼30 neurological-neuromuscular diseases. While these expansions occur in coding and noncoding regions, microsatellite sequence and repeat length diversity is more prominent in introns with eight different trinucleotide to hexanucleotide repeats, causing hereditary diseases such as myotonic dystrophy type 2 (DM2), Fuchs endothelial corneal dystrophy (FECD), and C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). Here, we test the hypothesis that these GC-rich intronic microsatellite expansions selectively trigger host intron retention (IR). Using DM2, FECD, and C9-ALS/FTD as examples, we demonstrate that retention is readily detectable in affected tissues and peripheral blood lymphocytes and conclude that IR screening constitutes a rapid and inexpensive biomarker for intronic repeat expansion disease.

Abnormal nuclear aggregation and myotube degeneration in myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is caused by CTG nucleotide repeat expansions in the 3'-untranslated region (3'-UTR) of the dystrophia myotonica protein kinase (DMPK) gene. The expanded CTG repeats encode toxic CUG RNAs that cause disease, largely through RNA gain-of-function. DM1 is a fatal disease characterized by progressive muscle wasting, which has no cure. Regenerative medicine has emerged as a promising therapeutic modality for DM1, especially with the advancement of induced pluripotent stem (iPS) cell technology and therapeutic genome editing. However, there is an unmet need to identify in vitro outcome measures to demonstrate the therapeutic effects prior to in vivo clinical trials. In this study, we examined the muscle regeneration (myotube formation) in normal and DM1 myoblasts in vitro to establish outcome measures for therapeutic monitoring. We found normal proliferation of DM1 myoblasts, but abnormal nuclear aggregation during the early stage myotube formation, as well as myotube degeneration during the late stage of myotube formation. We concluded that early abnormal nuclear aggregation and late myotube degeneration offer easy and sensitive outcome measures to monitor therapeutic effects in vitro.

Mitigating RNA Toxicity in Myotonic Dystrophy using Small Molecules.

This review, one in a series on myotonic dystrophy (DM), is focused on the development and potential use of small molecules as therapeutics for DM. The complex mechanisms and pathogenesis of DM are covered in the associated reviews. Here, we examine the various small molecule approaches taken to target the DNA, RNA, and proteins that contribute to disease onset and progression in myotonic dystrophy type 1 (DM1) and 2 (DM2).

Altered levels of the splicing factor muscleblind modifies cerebral cortical function in mouse models of myotonic dystrophy.

Myotonic dystrophy (DM) is a progressive, multisystem disorder affecting skeletal muscle, heart, and central nervous system. In both DM1 and DM2, microsatellite expansions of CUG and CCUG RNA repeats, respectively, accumulate and disrupt functions of alternative splicing factors, including muscleblind (MBNL) proteins. Grey matter loss and white matter changes, including the corpus callosum, likely underlie cognitive and executive function deficits in DM patients. However, little is known how cerebral cortical circuitry changes in DM. Here, flavoprotein optical imaging was used to assess local and contralateral responses to intracortical motor cortex stimulation in DM-related mouse models. In control mice, brief train stimulation generated ipsilateral and contralateral homotopic fluorescence increases, the latter mediated by the corpus callosum. Single pulse stimulation produced an excitatory response with an inhibitory-like surround response mediated by GABAA receptors. In a mouse model of DM2 (Mbnl2 KO), we observed prolonged and increased responsiveness to train stimulation and loss of the inhibition from single pulse stimulation. Conversely, mice overexpressing human MBNL1 (MBNL1-OE) exhibited decreased contralateral response to train stimulation and reduction of inhibitory-like surround to single pulse stimulation. Therefore, altering levels of two key DM-associated splicing factors modifies functions of local cortical circuits and contralateral responses mediated through the corpus callosum.

Systematic review of professional liability when prescribing ß-lactams for patients with a known penicillin allergy.

OBJECTIVE: To describe medical negligence and malpractice cases in which a patient with a known penicillin allergy received a ß-lactam and experienced an adverse reaction related to the ß-lactam. DATA SOURCES: Lexis-Nexus, Westlaw, and Google Scholar were searched. STUDY SELECTIONS: Medical negligence and malpractice cases were eligible for inclusion if they met the following criteria: the plaintiff had a known penicillin allergy, received a ß-lactam, and experienced an adverse event. All United States federal and state cases were eligible. RESULTS: Twenty-seven unique cases met the inclusion criteria. Eighteen cases involved the receipt of a penicillin-based antibiotic; of these cases with a known legal outcome, the plaintiff (patient or representative) prevailed or settled in 3 cases and defendants (providers) prevailed in 7 cases. Seven cases involved the receipt of a cephalosporin; of these cases with a known legal outcome, the plaintiff settled with physicians before trial in 1 case and defendants prevailed in 3 cases. Two cases involved the receipt of a carbapenem. Defendants prevailed in one case and the legal outcome of the other case is unknown. In cases in which the defense successfully moved for summary judgment, judges cited a lack of scientific evidence demonstrating a cephalosporin or carbapenem was contraindicated for a patient with a penicillin allergy. CONCLUSION: The cases with published legal outcomes found limited professional liability for clinicians who prescribed cephalosporins or carbapenems to a patient with a known penicillin allergy. These results may decrease the litigation fears of practitioners and risk managers within health care systems.

Antifungal-Associated Drug-Induced Cardiac Disease.

The etiology of cardiomyopathies are classified into 4 main groupings (dilated, hypertrophic, restrictive, and idiopathic) and can be mechanistically caused by myocarditis, conduction abnormalities, focal direct injury, or nutritional deficiency. Based on our review of this topic, evidence suggests that echinocandin-related cardiac dysfunction is a mitochondrial drug-induced disease caused by focal direct myocyte injury. With caspofungin or anidulafungin administration into the heart via central line, exposure is likely extreme enough to induce the acute toxicity. Chronic or low-dose exposure may lead to hypertrophic cardiomyopathy; however, only acute exposures have been explored to date.

Repeat-associated non-ATG (RAN) translation in neurological disease.

Well-established rules of translational initiation have been used as a cornerstone in molecular biology to understand gene expression and to frame fundamental questions on what proteins a cell synthesizes, how proteins work and to predict the consequences of mutations. For a group of neurological diseases caused by the abnormal expansion of short segments of DNA (e.g. CAG•CTG repeats), mutations within or outside of predicted coding and non-coding regions are thought to cause disease by protein gain- or loss-of-function or RNA gain-of-function mechanisms. In contrast to these predictions, the recent discovery of repeat-associated non-ATG (RAN) translation showed expansion mutations can express homopolymeric expansion proteins in all three reading frames without an AUG start codon. This unanticipated, non-canonical type of protein translation is length-and hairpin-dependent, takes place without frameshifting or RNA editing and occurs across a variety of repeat motifs. To date, RAN proteins have been reported in spinocerebellar ataxia type 8 (SCA8), myotonic dystrophy type 1 (DM1), fragile X tremor ataxia syndrome (FXTAS) and C9ORF72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). In this article, we review what is currently known about RAN translation and recent progress toward understanding its contribution to disease.

Counting the Cost of Daptomycin Versus Vancomycin in Hospitalized Patients: A Cost Minimization Analysis.

Daptomycin use for gram-positive infections has increased. This cost minimization analysis aimed to determine cost and/or time savings of daptomycin over vancomycin. The estimated hospital cost savings was US$166.41 per patient, and pharmacist time saved of almost 20 minutes per patient. Daptomycin has the potential to save both time and money.

Evidence of cis-acting factors in replication-mediated trinucleotide repeat instability in primate cells.

The mechanism of disease-associated trinucleotide repeat instability involves cis-acting factors (cis-elements) in the vicinity of the repeat, but the nature of these elements is unknown. One cis-element may be the location of the replication origin relative to the repeat. We have used an SV40 DNA replication system to investigate the effect of the location of replication initiation on (CTG)(n)*(CAG)(n) stability in primate cells. Depending on the distance between the SV40 replication origin and the repeat tract, templates with 79 repeats yield predominantly expansions or predominantly deletions or remain intact. All templates with 17 repeats are stable. Thus, cis-elements that affect the sites of Okazaki fragment initiation relative to the repeat are crucial determinants of instability. This model system recapitulates the bias for expansions observed in many of the diseases associated with trinucleotide repeats. Our results might explain the variable amounts of CTG/CAG instability that are observed in different chromosomal contexts.

Alternative Splicing Outcomes Across an RNA-Binding Protein Concentration Gradient.

Alternative splicing (AS) is a dynamic RNA processing step that produces multiple RNA isoforms from a single pre-mRNA transcript and contributes to the complexity of the cellular transcriptome and proteome. This process is regulated through a network of cis-regulatory sequence elements and trans-acting factors, most-notably RNA binding proteins (RBPs). The muscleblind-like (MBNL) and RNA binding fox-1 homolog (RBFOX) are two well characterized families of RBPs that regulate fetal to adult AS transitions critical for proper muscle, heart, and central nervous system development. To better understand how the concentration of these RBPs influences AS transcriptome wide, we engineered a MBNL1 and RBFOX1 inducible HEK-293 cell line. Modest induction of exogenous RBFOX1 in this cell line modulated MBNL1-dependent AS outcomes in 3 skipped exon events, despite significant levels of endogenous RBFOX1 and RBFOX2. Due to background RBFOX levels, we conducted a focused analysis of dose-dependent MBNL1 skipped exon AS outcomes and generated transcriptome wide dose-response curves. Analysis of this data demonstrates that MBNL1-regulated exclusion events may require higher concentrations of MBNL1 protein to properly regulate AS outcomes compared to inclusion events and that multiple arrangements of YGCY motifs can produce similar splicing outcomes. These results suggest that rather than a simple relationship between the organization of RBP binding sites and a specific splicing outcome, that complex interaction networks govern both AS inclusion and exclusion events across a RBP gradient.

Quercetin selectively reduces expanded repeat RNA levels in models of myotonic dystrophy.

Myotonic dystrophy is a multisystemic neuromuscular disease caused by either a CTG repeat expansion in DMPK (DM1) or a CCTG repeat expansion in CNBP (DM2). Transcription of the expanded alleles produces toxic gain-of-function RNA that sequester the MBNL family of alternative splicing regulators into ribonuclear foci, leading to pathogenic mis-splicing. There are currently no approved treatments that target the root cause of disease which is the production of the toxic expansion RNA molecules. In this study, using our previously established HeLa DM1 repeat selective screening platform, we identified the natural product quercetin as a selective modulator of toxic RNA levels. Quercetin treatment selectively reduced toxic RNA levels and rescued MBNL dependent mis-splicing in DM1 and DM2 patient derived cell lines and in the HSALR transgenic DM1 mouse model where rescue of myotonia was also observed. Based on our data and its safety profile for use in humans, we have identified quercetin as a priority disease-targeting therapeutic lead for clinical evaluation for the treatment of DM1 and DM2.

The contribution of Raman scattering to the fluorescence of the polyene antibiotic amphotericin B.

Fluorescence has recently been applied to the analysis of the molecular organization state of the polyene antibiotic amphotericin B (AmB) in solution or in lipid membranes. The polyene chain of AmB monomer gives rise to two fluorescence emissions; S(1)(2(1)A(g)) → S(0)(1(1)A(g)) between 500 and 700 nm, S(2)(1(1)B(u)) → S(0)(1(1)A(g)) between 400 and 500 nm. However, Raman scattering might interfere with the S(2) → S(0) emission fluorescence due to the weak fluorescence quantum yield and close proximity to the exciting lines. In fact, we show here that a change in the excitation wavelength results in a shift of three emission bands, an effect which excludes their assignment to fluorescence. These bands originate from the water Raman at 3382 cm(-1)and AmB resonance Raman at 1556 and 1153 cm(-1). As a consequence, some former conclusions on the molecular organization state of AmB should be reconsidered.

CTCF cis-regulates trinucleotide repeat instability in an epigenetic manner: a novel basis for mutational hot spot determination.

At least 25 inherited disorders in humans result from microsatellite repeat expansion. Dramatic variation in repeat instability occurs at different disease loci and between different tissues; however, cis-elements and trans-factors regulating the instability process remain undefined. Genomic fragments from the human spinocerebellar ataxia type 7 (SCA7) locus, containing a highly unstable CAG tract, were previously introduced into mice to localize cis-acting "instability elements," and revealed that genomic context is required for repeat instability. The critical instability-inducing region contained binding sites for CTCF -- a regulatory factor implicated in genomic imprinting, chromatin remodeling, and DNA conformation change. To evaluate the role of CTCF in repeat instability, we derived transgenic mice carrying SCA7 genomic fragments with CTCF binding-site mutations. We found that CTCF binding-site mutation promotes triplet repeat instability both in the germ line and in somatic tissues, and that CpG methylation of CTCF binding sites can further destabilize triplet repeat expansions. As CTCF binding sites are associated with a number of highly unstable repeat loci, our findings suggest a novel basis for demarcation and regulation of mutational hot spots and implicate CTCF in the modulation of genetic repeat instability.

T. asahii pulmonary infection as a complication of TNF-inhibitor and steroids: posaconazole pharmacotherapy and risk analysis.

We report the first documented Trichosporon asahii infection in a patient with connective tissue disease treated with a Tumor Necrosis Factor (TNF) inhibitor and describe an institutional root cause analysis for TNF inhibitor-associated infections. Fourteen patients with incident fungal infections during TNF inhibitor treatment were identified. They were matched with uncomplicated patients receiving TNF inhibitors or with rheumatoid arthritis (RA) patients managed without TNF inhibitors. We found that patients acquiring fungal infections were more likely to have graft versus host disease (GVHD) (p<0.05). Furthermore, infected patients were more likely (OR=24.4) to have multiple immunosuppressive therapies over the controls as well as several risk factors identified by the Infectious Disease Society ofAmerica (IDSA). The 3 patient deaths in our study were associated with GVHD and infliximab. Trichosporon was isolated in 1 patient receiving adalimumab. Our results suggest that these high risk patients be monitored closely for fungal infection.