SARA captures disparate progression and responsiveness in spinocerebellar ataxias.

BACKGROUND: The Scale for Assessment and Rating of Ataxia (SARA) is a widely used clinical scale to assess cerebellar ataxia but faces some criticisms about the relevancy of all its items. OBJECTIVES: To prepare for future clinical trials, we analyzed the progression of SARA and its items in several polyQ spinocerebellar ataxias (SCA) from various cohorts. METHODS: We included data from patients with SCA1, SCA2, SCA3, and SCA6 from four cohorts (EUROSCA, RISCA, CRC-SCA, and SPATAX) for a total of 850 carriers and 3431 observations. Longitudinal progression of the SARA and its items was measured. Cohort, stage and genetic effects were tested. We looked at the respective contribution of each item to the total scale. Sensitivity to change of the scale and the impact of item removal was evaluated by calculating sample sizes needed in various scenarios. RESULTS: Longitudinal progression was significantly different between cohorts in SCA1, SCA2 and SCA3, the EUROSCA cohort having the fastest progression. Advanced-stage patients were progressing slower in SCA2 and SCA6. Items were not contributing equally to the full scale through ataxia severity: gait, stance, hand movement, and heel-shin contributed the most in the early stage, and finger-chase, nose-finger, and sitting in later stages. Few items drove the sensitivity to the change of SARA, but changes in the scale structure could not improve its sensitivity in all populations. CONCLUSION: SARA and its item's progression pace showed high heterogeneity across cohorts and SCAs. However, no combinations of items improved the responsiveness in all SCAs or populations taken separately.

DNAzyme Cleavage of CAG Repeat RNA in Polyglutamine Diseases.

CAG repeat expansion is the genetic cause of nine incurable polyglutamine (polyQ) diseases with neurodegenerative features. Silencing repeat RNA holds great therapeutic value. Here, we developed a repeat-based RNA-cleaving DNAzyme that catalyzes the destruction of expanded CAG repeat RNA of six polyQ diseases with high potency. DNAzyme preferentially cleaved the expanded allele in spinocerebellar ataxia type 1 (SCA1) cells. While cleavage was non-allele-specific for spinocerebellar ataxia type 3 (SCA3) cells, treatment of DNAzyme leads to improved cell viability without affecting mitochondrial metabolism or p62-dependent aggresome formation. DNAzyme appears to be stable in mouse brain for at least 1 month, and an intermediate dosage of DNAzyme in a SCA3 mouse model leads to a significant reduction of high molecular weight ATXN3 proteins. Our data suggest that DNAzyme is an effective RNA silencing molecule for potential treatment of multiple polyQ diseases.

A CRISPR-Cas13a Based Strategy That Tracks and Degrades Toxic RNA in Myotonic Dystrophy Type 1.

Cas13a, an effector of type VI CRISPR-Cas systems, is an RNA guided RNase with multiplexing and therapeutic potential. This study employs the Leptotrichia shahii (Lsh) Cas13a and a repeat-based CRISPR RNA (crRNA) to track and eliminate toxic RNA aggregates in myotonic dystrophy type 1 (DM1) - a neuromuscular disease caused by CTG expansion in the DMPK gene. We demonstrate that LshCas13a cleaves CUG repeat RNA in biochemical assays and reduces toxic RNA load in patient-derived myoblasts. As a result, LshCas13a reverses the characteristic adult-to-embryonic missplicing events in several key genes that contribute to DM1 phenotype. The deactivated LshCas13a can further be repurposed to track RNA-rich organelles within cells. Our data highlights the reprogrammability of LshCas13a and the possible use of Cas13a to target expanded repeat sequences in microsatellite expansion diseases.

Dysphagia in spinocerebellar ataxias type 1, 2, 3 and 6.

BACKGROUND: Dysphagia is a common symptom and may be a cause of death in patients with spinocerebellar ataxias (SCAs). However, little is known about at which disease stage dysphagia becomes clinically relevant. Therefore, our study aims to investigate the prevalence of dysphagia in different disease stages of SCA 1, 2, 3 and 6. METHODS: We studied 237 genetically confirmed patients with SCA 1, 2, 3, 6 from the Clinical Research Consortium for SCAs and investigated the prevalence of self-reported dysphagia and the association between dysphagia and other clinical characteristics. We further stratified ataxia severity and studied the prevalence of dysphagia at each disease stage. RESULTS: Dysphagia was present in 59.9% of SCA patients. Patients with dysphagia had a longer disease duration and more severe ataxia than patients without dysphagia (patients with dysphagia vs. without dysphagia, disease duration (years): 14.51 ± 8.91 vs. 11.22 ± 7.82, p = .001, scale for the assessment and rating of ataxia [SARA]: 17.90 ± 7.74 vs. 13.04 ± 7.51, p = .000). Dysphagia was most common in SCA1, followed by SCA3, SCA 6, and SCA 2. Dysphagia in SCA1 and 3 was associated robustly with ataxia severity, whereas this association was less obvious in SCA2 and 6, demonstrating genotype-specific clinical variation. CONCLUSION: Dysphagia is a common clinical symptom in SCAs, especially in the severe disease stage. Understanding dysphagia in SCA patients can improve the care of these patients and advance knowledge on the roles of the cerebellum and brainstem control in swallowing.

The impact of ethnicity on the clinical presentations of spinocerebellar ataxia type 3.

BACKGROUND: For a variety of sporadic neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis, it is well-established that ethnicity does affect the disease phenotypes. However, how ethnicity contributes to the clinical symptoms and disease progressions in monogenetic disorders, such as spinocerebellar ataxia type 3 (SCA3), remains less studied. METHODS: We used multivariable linear and logistical regression models in 257 molecularly-confirmed SCA3 patients (66 Caucasians, 43 African Americans, and 148 Asians [composed of 131 Chinese and 17 Asian Americans]) to explore the influence of ethnicity on age at onset (AAO), ataxia severity, and non-ataxia symptoms (i.e. depression, tremor, and dystonia). RESULTS: We found that Asians had significantly later AAO, compared to Caucasians (ß = 4.75, p = 0.000) and to African Americans (ß = 6.64, p = 0.000) after adjusting for the pathological CAG repeat numbers in ATXN3. African Americans exhibited the most severe ataxia as compared to Caucasians (ß = 3.81, p = 0.004) and Asians (ß = 4.39, p = 0.001) after taking into consideration of the pathological CAG repeat numbers in ATXN3 and disease duration. Caucasians had a higher prevalence of depression than African Americans (ß = 1.23, p = 0.040). Ethnicity had no influence on tremor or dystonia. CONCLUSIONS: Ethnicity plays an important role in clinical presentations of SCA3 patients, which could merit further clinical studies and public health consideration. These results highlight the role of ethnicity in monogenetic, neurodegenerative disorders.

Tremor in the Degenerative Cerebellum: Towards the Understanding of Brain Circuitry for Tremor.

Cerebellar degenerative pathology has been identified in tremor patients; however, how the degenerative pathology could contribute to tremor remains unclear. If the cerebellar degenerative pathology can directly drive tremor, one would hypothesize that tremor is likely to occur in the diseases of cerebellar ataxia and follows the disease progression in such disorders. To further test this hypothesis, we studied the occurrence of tremor in different disease stages of classical cerebellar degenerative disorders: spinocerebellar ataxias (SCAs). We further separately analyzed postural tremor and rest tremor, two forms of tremor that both involve the cerebellum. We also explored tremor in different subtypes of SCAs. We found that 18.1% of SCA patients have tremor. Interestingly, SCA patients with tremor have worse ataxia than those without tremor. When stratifying patients into mild, moderate, and severe disease stages according to the severity of ataxia, moderate and severe SCA patients more commonly have tremor than those with mild ataxia, the effect most prominently observed in postural tremor of SCA3 and SCA6 patients. Finally, tremor can independently contribute to worse functional status in SCA2 patients, even after adjusting for ataxia severity. Tremor is more likely to occur in the severe stage of cerebellar degeneration when compared to mild stages. Our results partially support the cerebellar degenerative model of tremor.

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.

Human iPSC Models to Study Orphan Diseases: Muscular Dystrophies.

PURPOSE OF REVIEW: Muscular dystrophies (MDs) are a spectrum of muscle disorders, which are caused by a number of gene mutations. The studies of MDs are limited due to lack of appropriate models, except for Duchenne muscular dystrophy (DMD), myotonic dystrophy type 1 (DM1), facioscapulohumeral muscular dystrophy (FSHD), and certain type of limb-girdle muscular dystrophy (LGMD). Human induced pluripotent stem cell (iPSC) technologies are emerging to offer a useful model for mechanistic studies, drug discovery, and cell-based therapy to supplement in vivo animal models. This review will focus on current applications of iPSC as disease models of MDs for studies of pathogenic mechanisms and therapeutic development. RECENT FINDINGS: Many and more human disease-specific iPSCs have been or being established, which carry the natural mutation of MDs with human genomic background. These iPSCs can be differentiated into specific cell types affected in a particular MDs such as skeletal muscle progenitor cells, skeletal muscle fibers, and cardiomyocytes. Human iPSCs are particularly useful for studies of the pathogenicity at the early stage or developmental phase of MDs. High-throughput screening using disease-specific human iPSCs has become a powerful technology in drug discovery. While MD iPSCs have been generated for cell-based replacement therapy, recent advances in genome editing technologies enabled correction of genetic mutations in these cells in culture, raising hope for in vivo genome therapy, which offers a fundamental cure for these daunting inherited MDs. SUMMARY: Human disease-specific iPSC models for MDs are emerging as an additional tool to current disease models for elucidating disease mechanisms and developing therapeutic intervention.

Therapeutic Genome Editing for Myotonic Dystrophy Type 1 Using CRISPR/Cas9.

Myotonic dystrophy type 1 (DM1) is caused by a CTG nucleotide repeat expansion within the 3' UTR of the Dystrophia Myotonica protein kinase gene. In this study, we explored therapeutic genome editing using CRISPR/Cas9 via targeted deletion of expanded CTG repeats and targeted insertion of polyadenylation signals in the 3' UTR upstream of the CTG repeats to eliminate toxic RNA CUG repeats. We found paired SpCas9 or SaCas9 guide RNA induced deletion of expanded CTG repeats. However, this approach incurred frequent inversion in both the mutant and normal alleles. In contrast, the insertion of polyadenylation signals in the 3' UTR upstream of the CTG repeats eliminated toxic RNA CUG repeats, which led to phenotype reversal in differentiated neural stem cells, forebrain neurons, cardiomyocytes, and skeletal muscle myofibers. We concluded that targeted insertion of polyadenylation signals in the 3' UTR is a viable approach to develop therapeutic genome editing for DM1.

Furamidine Rescues Myotonic Dystrophy Type I Associated Mis-Splicing through Multiple Mechanisms.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant, CTG•CAG microsatellite expansion disease. Expanded CUG repeat RNA sequester the muscleblind-like (MBNL) family of RNA-binding proteins, thereby disrupting their normal cellular function which leads to global mis-regulation of RNA processing. Previously, the small molecule furamidine was shown to reduce CUG foci and rescue mis-splicing in a DM1 HeLa cell model and to rescue mis-splicing in the HSALR DM1 mouse model, but furamidine's mechanism of action was not explored. Here we use a combination of biochemical, cell toxicity, and genomic studies in DM1 patient-derived myotubes and the HSALR DM1 mouse model to investigate furamidine's mechanism of action. Mis-splicing rescue was observed in DM1 myotubes and the HSALR DM1 mouse with furamidine treatment. Interestingly, while furamidine was found to bind CTG•CAG repeat DNA with nanomolar affinity, a reduction in expanded CUG repeat transcript levels was observed in the HSALR DM1 mouse but not DM1 patient-derived myotubes. Further investigation in these cells revealed that furamidine treatment at nanomolar concentrations led to up-regulation of MBNL1 and MBNL2 protein levels and a reduction of ribonuclear foci. Additionally, furamidine was shown to bind CUG RNA with nanomolar affinity and disrupted the MBNL1 -CUG RNA complex in vitro at micromolar concentrations. Furamidine's likely promiscuous interactions in vitro and in vivo appear to affect multiple pathways in the DM1 mechanism to rescue mis-splicing, yet surprisingly furamidine was shown globally to rescue many mis-splicing events with only modest off-target effects on gene expression in the HSALR DM1 mouse model. Importantly, over 20% of the differentially expressed genes were shown to be returned, to varying degrees, to wild-type expression levels.

Elevated Peripheral Myelin Protein 22, Reduced Mitotic Potential, and Proteasome Impairment in Dermal Fibroblasts from Charcot-Marie-Tooth Disease Type 1A Patients.

A common form of hereditary autosomal dominant demyelinating neuropathy known as Charcot-Marie-Tooth disease type 1A (CMT1A) is linked with duplication of the peripheral myelin protein 22 (PMP22) gene. Although studies from animal models have led to better understanding of the pathobiology of these neuropathies, there continues to be a gap in the translation of findings from rodents to humans. Because PMP22 was originally identified in fibroblasts as growth arrest specific gene 3 (gas3) and is expressed broadly in the body, it was tested whether skin cells from neuropathic patients would display the cellular pathology observed in Schwann cells from rodent models. Dermal fibroblasts from two CMT1A pedigrees with confirmed PMP22 gene duplication were studied. Samples from age-matched non-neuropathic individuals were used as controls. CMT1A patient-derived cultures contain approximately 1.5-fold elevated levels of PMP22 mRNA, exhibit reduced mitotic potential, and display intracellular protein aggregates as compared to cells from unaffected individuals. The presence of cytosolic PMP22 coincides with a decrease in proteasome activity and an increase in autophagy-lysosomal proteins, including LC3-II and LAMP1. These results indicate that the abnormalities in the subcellular processing of excess PMP22 elicit a detectable response in human CMT1A fibroblasts, a phenotype that resembles Schwann cells from neuropathic mice. These findings support the use of human CMT1A fibroblasts as a platform for therapy testing.

Dystonia and ataxia progression in spinocerebellar ataxias.

BACKGROUND: Dystonia is a common feature in spinocerebellar ataxias (SCAs). Whether the presence of dystonia is associated with different rate of ataxia progression is not known. OBJECTIVES: To study clinical characteristics and ataxia progression in SCAs with and without dystonia. METHODS: We studied 334 participants with SCA 1, 2, 3 and 6 from the Clinical Research Consortium for Spinocerebellar Ataxias (CRC-SCA) and compared the clinical characteristics of SCAs with and without dystonia. We repeatedly measured ataxia progression by the Scale for Assessment and Rating of Ataxia every 6 months for 2 years. Regression models were employed to study the association between dystonia and ataxia progression after adjusting for age, sex and pathological CAG repeats. We used logistic regression to analyze the impact of different repeat expansion genes on dystonia in SCAs. RESULTS: Dystonia was most commonly observed in SCA3, followed by SCA2, SCA1, and SCA6. Dystonia was associated with longer CAG repeats in SCA3. The CAG repeat number in TBP normal alleles appeared to modify the presence of dystonia in SCA1. The presence of dystonia was associated with higher SARA scores in SCA1, 2, and 3. Although relatively rare in SCA6, the presence of dystonia was associated with slower progression of ataxia. CONCLUSIONS: The presence of dystonia is associated with greater severity of ataxia in SCA1, 2, and 3, but predictive of a slower progression in SCA6. Complex genetic interactions among repeat expansion genes can lead to diverse clinical symptoms and progression in SCAs.

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.

Postural Tremor and Ataxia Progression in Spinocerebellar Ataxias.

BACKGROUND: Postural tremor can sometimes occur in spinocerebellar ataxias (SCAs). However, the prevalence and clinical characteristics of postural tremor in SCAs are poorly understood, and whether SCA patients with postural tremor have different ataxia progression is not known. METHODS: We studied postural tremor in 315 patients with SCA1, 2, 3, and 6 recruited from the Clinical Research Consortium for Spinocerebellar Ataxias (CRC-SCA), which consists of 12 participating centers in the United States, and we evaluated ataxia progression in these patients from January 2010 to August 2012. RESULTS: Among 315 SCA patients, postural tremor was most common in SCA2 patients (SCA1, 5.8%; SCA2, 27.5%; SCA3, 12.4%; SCA6, 16.9%; p = 0.007). SCA3 patients with postural tremor had longer CAG repeat expansions than SCA3 patients without postural tremor (73.67 ± 3.12 vs. 70.42 ± 3.96, p = 0.003). Interestingly, SCA1 and SCA6 patients with postural tremor had a slower rate of ataxia progression (SCA1, ß = -0.91, p < 0.001; SCA6, ß = -1.28, p = 0.025), while SCA2 patients with postural tremor had a faster rate of ataxia progression (ß = 1.54, p = 0.034). We also found that the presence of postural tremor in SCA2 patients could be influenced by repeat expansions of ATXN1 (ß = -1.53, p = 0.037) and ATXN3 (ß = 0.57, p = 0.018), whereas postural tremor in SCA3 was associated with repeat lengths in TBP (ß = 0.63, p = 0.041) and PPP2R2B (ß = -0.40, p = 0.032). DISCUSSION: Postural tremor could be a clinical feature of SCAs, and the presence of postural tremor could be associated with different rates of ataxia progression. Genetic interactions between ataxia genes might influence the brain circuitry and thus affect the clinical presentation of postural tremor.

RAN Translation Regulated by Muscleblind Proteins in Myotonic Dystrophy Type 2.

Several microsatellite-expansion diseases are characterized by the accumulation of RNA foci and RAN proteins, raising the possibility of a mechanistic connection. We explored this question using myotonic dystrophy type 2, a multisystemic disease thought to be primarily caused by RNA gain-of-function effects. We demonstrate that the DM2 CCTG⋅CAGG expansion expresses sense and antisense tetrapeptide poly-(LPAC) and poly-(QAGR) RAN proteins, respectively. In DM2 autopsy brains, LPAC is found in neurons, astrocytes, and glia in gray matter, and antisense QAGR proteins accumulate within white matter. LPAC and QAGR proteins are toxic to cells independent of RNA gain of function. RNA foci and nuclear sequestration of CCUG transcripts by MBNL1 is inversely correlated with LPAC expression. These data suggest a model that involves nuclear retention of expansion RNAs by RNA-binding proteins (RBPs) and an acute phase in which expansion RNAs exceed RBP sequestration capacity, are exported to the cytoplasm, and undergo RAN translation. VIDEO ABSTRACT.

Elimination of Toxic Microsatellite Repeat Expansion RNA by RNA-Targeting Cas9.

Microsatellite repeat expansions in DNA produce pathogenic RNA species that cause dominantly inherited diseases such as myotonic dystrophy type 1 and 2 (DM1/2), Huntington's disease, and C9orf72-linked amyotrophic lateral sclerosis (C9-ALS). Means to target these repetitive RNAs are required for diagnostic and therapeutic purposes. Here, we describe the development of a programmable CRISPR system capable of specifically visualizing and eliminating these toxic RNAs. We observe specific targeting and efficient elimination of microsatellite repeat expansion RNAs both when exogenously expressed and in patient cells. Importantly, RNA-targeting Cas9 (RCas9) reverses hallmark features of disease including elimination of RNA foci among all conditions studied (DM1, DM2, C9-ALS, polyglutamine diseases), reduction of polyglutamine protein products, relocalization of repeat-bound proteins to resemble healthy controls, and efficient reversal of DM1-associated splicing abnormalities in patient myotubes. Finally, we report a truncated RCas9 system compatible with adeno-associated viral packaging. This effort highlights the potential of RCas9 for human therapeutics.

In Situ Printing-then-Mixing for Biological Structure Fabrication Using Intersecting Jets.

Although traditional three-dimensional bioprinting technology is suitable for many tissue engineering applications, various biomaterials and constructs call for bioprinting innovations. There is a need for the fabrication of complex structures from reactive biomaterials as well as heterogeneous structures with controlled material compositions. In particular, during reactive material printing, reactive solutions/suspensions that undergo changes in rheological properties or cytocompatibility are not printable using traditional bioprinting approaches that require all components of bioinks to be mixed before deposition. The objective of this study is to develop and implement an intersecting jets-based inkjet bioprinting approach, which enables voxel-resolution printing-then-mixing for the fabrication of biological structures using reactive materials as well as structures having a compositional gradient. Inkjetting is implemented herein as a versatile technique to simultaneously deposit droplets of disparate materials at controlled locations where active collision, mixing, and coalescence occur. For reactive material printing, neural stem cell (NSC) spheres are fabricated from reactive PuraMatrix hydrogel solution and physiological cell suspension, and cell-laden alginate structures are also printed in air directly from reactive sodium alginate and calcium chloride solutions. For heterogeneous structure printing, collagen sheets with a hydroxyapatite (HAP) content gradient are fabricated to demonstrate the unique online control of material composition throughout a structure. It is demonstrated that the proposed bioprinting approach is feasible for applications that utilize reactive materials or require heterogeneous compositions.

The Initial Symptom and Motor Progression in Spinocerebellar Ataxias.

The aim of this study is to determine whether the initial symptom associates with motor progression in spinocerebellar ataxias (SCAs). SCAs are clinically heterogeneous and the initial presentation may represent different subtypes of SCA with different motor progression. We studied 317 participants with SCAs1, 2, 3, and 6 from the Clinical Research Consortium for SCAs (CRC-SCA) and repeatedly measured the severity of ataxia for 2 years. SCA patients were divided into gait-onset and non-gait-onset (speech, vision, and hand dexterity) groups based on the initial presentation. In addition to demographic comparison, we employed regression models to study ataxia progression in these two groups after adjusting for age, sex, and pathological CAG repeats. The majority of SCA patients had gait abnormality as an initial presentation. The pathological CAG repeat expansions were similar between the gait-onset and non-gait-onset groups. In SCA1, gait-onset group progressed slower than non-gait-onset group, while gait-onset SCA6 group progressed faster than their counterpart. In addition, the disease presented 9 years later for SCA2 gait-onset group than non-gait-onset group. Initial symptoms of SCA3 did not influence age of onset or disease progression. The initial symptom in each SCA has a different influence on age of onset and motor progression. Therefore, gait and non-gait-onset groups of SCAs might represent different subtypes of the diseases.