In Huntington disease, cellular toxicity is particularly caused by toxic protein fragments generated from the mutant huntingtin (HTT) protein. By modifying the HTT protein, we aim to reduce... Show moreIn Huntington disease, cellular toxicity is particularly caused by toxic protein fragments generated from the mutant huntingtin (HTT) protein. By modifying the HTT protein, we aim to reduce proteolytic cleavage and ameliorate the consequences of mutant HTT without lowering total HTT levels. To that end, we use an antisense oligonucleotide (AON) that targets HTT pre-mRNA and induces partial skipping of exon 12, which contains the critical caspase-6 cleavage site. Here, we show that AON-treatment can partially restore the phenotype of YAC128 mice, a mouse model expressing the full-length human HTT gene including 128 CAG-repeats. Wild-type and YAC128 mice were treated intracerebroventricularly with AON12.1, scrambled AON or vehicle starting at 6 months of age and followed up to 12 months of age, when MRI was performed and mice were sacrificed. AON12.1 treatment induced around 40% exon skip and protein modification. The phenotype on body weight and activity, but not rotarod, was restored by AON treatment. Genes differentially expressed in YAC128 striatum changed toward wild-type levels and striatal volume was preserved upon AON12.1 treatment. However, scrambled AON also showed a restorative effect on gene expression and appeared to generally increase brain volume. Show less
Velde, N.M. van de; Koeks, Z.; Signorelli, M.; Verwey, N.; Overzier, M.; Bakker, J.A.; ... ; Niks, E.H. 2023
Background and Objectives: The slow and variable disease progression of Becker muscular dystrophy (BMD) urges the development of biomarkers to facilitate clinical trials. We explored changes in 3... Show moreBackground and Objectives: The slow and variable disease progression of Becker muscular dystrophy (BMD) urges the development of biomarkers to facilitate clinical trials. We explored changes in 3 muscle-enriched biomarkers in serum of patients with BMD over 4-year time and studied associations with disease severity, disease progression, and dystrophin levels in BMD. Methods: We quantitatively measured creatine kinase (CK) using the International Federation of Clinical Chemistry reference method, creatine/creatinine(ratio) (Cr/Crn) using liquid chromatography-tandem mass spectrometry, and myostatin with ELISA in serum and assessed functional performance using the North Star Ambulatory Assessment (NSAA), 10-meter run velocity (TMRv), 6-Minute Walking Test (6MWT), and forced vital capacity in a 4-year prospective natural history study. Dystrophin levels were quantified in the tibialis anterior muscle using capillary Western immunoassay. The correlation between biomarkers, age, functional performance, mean annual change, and prediction of concurrent functional performance was analyzed using linear mixed models. Results: Thirty-four patients with 106 visits were included. Eight patients were nonambulant at baseline. Cr/Crn and myostatin were highly patient specific (intraclass correlation coefficient for both = 0.960). Cr/Crn was strongly negatively correlated, whereas myostatin was strongly positively correlated with the NSAA, TMRv, and 6MWT (Cr/Crn rho = -0.869 to -0.801 and myostatin rho = 0.792 to 0.842, all p < 0.001). CK showed a negative association with age (p = 0.0002) but was not associated with patients' performance. Cr/Crn and myostatin correlated moderately with the average annual change of the 6MWT (rho = -0.532 and 0.555, p = 0.02). Dystrophin levels did not correlate with the selected biomarkers nor with performance. Cr/Crn, myostatin, and age could explain up to 75% of the variance of concurrent functional performance of the NSAA, TMRv, and 6MWT. Discussion: Both Cr/Crn and myostatin could potentially serve as monitoring biomarkers in BMD, as higher Cr/Crn and lower myostatin were associated with lower motor performance and predictive of concurrent functional performance when combined with age. Future studies are needed to more precisely determine the context of use of these biomarkers. Show less
Velde, N.M. van de; Koeks, Z.; Signorelli, M.; Verwey, N.; Overzier, M.; Bakker, J.A.; ... ; Niks, E.H. 2023
Background and ObjectivesThe slow and variable disease progression of Becker muscular dystrophy (BMD) urges the development of biomarkers to facilitate clinical trials. We explored changes in 3... Show moreBackground and ObjectivesThe slow and variable disease progression of Becker muscular dystrophy (BMD) urges the development of biomarkers to facilitate clinical trials. We explored changes in 3 muscle-enriched biomarkers in serum of patients with BMD over 4-year time and studied associations with disease severity, disease progression, and dystrophin levels in BMD.MethodsWe quantitatively measured creatine kinase (CK) using the International Federation of Clinical Chemistry reference method, creatine/creatinineratio (Cr/Crn) using liquid chromatography–tandem mass spectrometry, and myostatin with ELISA in serum and assessed functional performance using the North Star Ambulatory Assessment (NSAA), 10-meter run velocity (TMRv), 6-Minute Walking Test (6MWT), and forced vital capacity in a 4-year prospective natural history study. Dystrophin levels were quantified in the tibialis anterior muscle using capillary Western immunoassay. The correlation between biomarkers, age, functional performance, mean annual change, and prediction of concurrent functional performance was analyzed using linear mixed models.ResultsThirty-four patients with 106 visits were included. Eight patients were nonambulant at baseline. Cr/Crn and myostatin were highly patient specific (intraclass correlation coefficient for both = 0.960). Cr/Crn was strongly negatively correlated, whereas myostatin was strongly positively correlated with the NSAA, TMRv, and 6MWT (Cr/Crn rho = −0.869 to −0.801 and myostatin rho = 0.792 to 0.842, all p < 0.001). CK showed a negative association with age (p = 0.0002) but was not associated with patients' performance. Cr/Crn and myostatin correlated moderately with the average annual change of the 6MWT (rho = −0.532 and 0.555, p = 0.02). Dystrophin levels did not correlate with the selected biomarkers nor with performance. Cr/Crn, myostatin, and age could explain up to 75% of the variance of concurrent functional performance of the NSAA, TMRv, and 6MWT.DiscussionBoth Cr/Crn and myostatin could potentially serve as monitoring biomarkers in BMD, as higher Cr/Crn and lower myostatin were associated with lower motor performance and predictive of concurrent functional performance when combined with age. Future studies are needed to more precisely determine the context of use of these biomarkers. Show less
Velde, N.M. van de; Koeks, Z.; Signorelli, M.; Verwey, N.; Overzier, M.; Bakker, J.A.; ... ; Niks, E.H. 2023
Background and Objectives The slow and variable disease progression of Becker muscular dystrophy (BMD) urges the development of biomarkers to facilitate clinical trials. We explored changes in 3... Show moreBackground and Objectives The slow and variable disease progression of Becker muscular dystrophy (BMD) urges the development of biomarkers to facilitate clinical trials. We explored changes in 3 muscle-enriched biomarkers in serum of patients with BMD over 4-year time and studied associations with disease severity, disease progression, and dystrophin levels in BMD.Methods We quantitatively measured creatine kinase (CK) using the International Federation of Clinical Chemistry reference method, creatine/creatinineratio (Cr/Crn) using liquid chromatography–tandem mass spectrometry, and myostatin with ELISA in serum and assessed functional performance using the North Star Ambulatory Assessment (NSAA), 10-meter run velocity (TMRv), 6-Minute Walking Test (6MWT), and forced vital capacity in a 4-year prospective natural history study. Dystrophin levels were quantified in the tibialis anterior muscle using capillary Western immunoassay. The correlation between biomarkers, age, functional performance, mean annual change, and prediction of concurrent functional performance was analyzed using linear mixed models.Results Thirty-four patients with 106 visits were included. Eight patients were nonambulant at baseline. Cr/Crn and myostatin were highly patient specific (intraclass correlation coefficient for both = 0.960). Cr/Crn was strongly negatively correlated, whereas myostatin was strongly positively correlated with the NSAA, TMRv, and 6MWT (Cr/Crn rho = −0.869 to −0.801 and myostatin rho = 0.792 to 0.842, all p < 0.001). CK showed a negative association with age (p = 0.0002) but was not associated with patients' performance. Cr/Crn and myostatin correlated moderately with the average annual change of the 6MWT (rho = −0.532 and 0.555, p = 0.02). Dystrophin levels did not correlate with the selected biomarkers nor with performance. Cr/Crn, myostatin, and age could explain up to 75% of the variance of concurrent functional performance of the NSAA, TMRv, and 6MWT.Discussion Both Cr/Crn and myostatin could potentially serve as monitoring biomarkers in BMD, as higher Cr/Crn and lower myostatin were associated with lower motor performance and predictive of concurrent functional performance when combined with age. Future studies are needed to more precisely determine the context of use of these biomarkers. Show less
Velde, N.M. van de; Koeks, Z.; Signorelli, M.; Verwey, N.; Overzier, M.; Bakker, J.A.; ... ; Niks, E.H. 2022
While the genetic cause of Huntington disease (HD) is known since 1993, still no cure exists. Therapeutic development would benefit from a method to monitor disease progression and treatment... Show moreWhile the genetic cause of Huntington disease (HD) is known since 1993, still no cure exists. Therapeutic development would benefit from a method to monitor disease progression and treatment efficacy, ideally using blood biomarkers. Previously, HD-specific signatures were identified in human blood representing signatures in human brain, showing biomarker potential. Since drug candidates are generally first screened in rodent models, we aimed to identify HD signatures in blood and brain of YAC128 HD mice and compare these with previously identified human signatures. RNA sequencing was performed on blood withdrawn at two time points and four brain regions from YAC128 and control mice. Weighted gene co-expression network analysis was used to identify clusters of co-expressed genes (modules) associated with the HD genotype. These HD-associated modules were annotated via text-mining to determine the biological processes they represented. Subsequently, the processes from mouse blood were compared with mouse brain, showing substantial overlap, including protein modification, cell cycle, RNA splicing, nuclear transport, and vesicle-mediated transport. Moreover, the disease-associated processes shared between mouse blood and brain were highly comparable to those previously identified in human blood and brain. In addition, we identified HD blood-specific pathology, confirming previous findings for peripheral pathology in blood. Finally, we identified hub genes for HD-associated blood modules and proposed a strategy for gene selection for development of a disease progression monitoring panel. Show less
Rutten, J.W.; Hack, R.J.; Duering, M.; Gravesteijn, G.; Dauwerse, J.G.; Overzier, M.; ... ; Oberstein, S.A.J.L. 2020
Objective To determine the small vessel disease spectrum associated with cysteine-alteringNOTCH3variants in community-dwelling individuals by analyzing the clinical and neuroimaging features of UK... Show moreObjective To determine the small vessel disease spectrum associated with cysteine-alteringNOTCH3variants in community-dwelling individuals by analyzing the clinical and neuroimaging features of UK Biobank participants harboring such variants. Methods The exome and genome sequencing datasets of the UK Biobank (n = 50,000) and cohorts of cognitively healthy elderly (n = 751) were queried for cysteine-alteringNOTCH3variants. Brain MRIs of individuals harboring such variants were scored according to Standards for Reporting Vascular Changes on Neuroimaging criteria, and clinical information was extracted with ICD-10 codes. Clinical and neuroimaging data were compared to age- and sex-matched UK Biobank controls and clinically diagnosed patients from the Dutch cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) registry. Results We identified 108 individuals harboring a cysteine-alteringNOTCH3variant (2.2 of 1,000), of whom 75% have a variant that has previously been reported in CADASIL pedigrees. Almost all variants were located in 1 of the NOTCH3 protein epidermal growth factor-like repeat domains 7 to 34. White matter hyperintensity lesion load was higher in individuals withNOTCH3variants than in controls (p= 0.006) but lower than in patients with CADASIL with the same variants (p< 0.001). Almost half of the 24 individuals with brain MRI had a Fazekas score of 0 or 1 up to age 70 years. There was no increased risk of stroke. Conclusions Although community-dwelling individuals harboring a cysteine-alteringNOTCH3variant have a higher small vessel disease MRI burden than controls, almost half have no MRI abnormalities up to age 70 years. This shows thatNOTCH3cysteine altering variants are associated with an extremely broad phenotypic spectrum, ranging from CADASIL to nonpenetrance. Show less
CADASIL is a vascular protein aggregation disorder caused by cysteine-altering NOTCH3 variants, leading to mid-adult-onset stroke and dementia. Here, we report individuals with a cysteine-altering... Show moreCADASIL is a vascular protein aggregation disorder caused by cysteine-altering NOTCH3 variants, leading to mid-adult-onset stroke and dementia. Here, we report individuals with a cysteine-altering NOTCH3 variant that induces exon 9 skipping, mimicking therapeutic NOTCH3 cysteine correction. The index came to our attention after a coincidental finding on a commercial screening MRI, revealing white matter hyperintensities. A heterozygous NOTCH3 c.1492G>T, p.Gly498Cys variant, was identified using a gene panel, which was also present in four first- and second-degree relatives. Although some degree of white matter hyperintensities was present on MRI in all family members with the NOTCH3 variant, the CADASIL phenotype was mild, as none had lacunes on MRI and there was no disability or cognitive impairment above the age of 60 years. RT-PCR and Sanger sequencing analysis on patient fibroblast RNA revealed that exon 9 was absent from the majority of NOTCH3 transcripts of the mutant allele, effectively excluding the mutation. NOTCH3 aggregation was assessed in skin biopsies using electron microscopy and immunohistochemistry and did not show granular osmiophilic material and only very mild NOTCH3 staining. For purposes of therapeutic translatability, we show that, in cell models, exon 9 exclusion can be obtained using antisense-mediated exon skipping and CRISPR/Cas9-mediated genome editing. In conclusion, this study provides the first in-human evidence that cysteine corrective NOTCH3 exon skipping is associated with less NOTCH3 aggregation and an attenuated phenotype, justifying further therapeutic development of NOTCH3 cysteine correction for CADASIL. Show less
This study investigates changes with respect to increasing protein levels in dystrophic nerves of two mdx mouse models of Duchenne muscular dystrophy (DMD). We propose that these nerve changes... Show moreThis study investigates changes with respect to increasing protein levels in dystrophic nerves of two mdx mouse models of Duchenne muscular dystrophy (DMD). We propose that these nerve changes result from progressive ongoing damage to neuromuscular junctions (NMJs) due to repeated intrinsic bouts of necrosis in dystrophic muscles. We compared sciatic nerves from classic mdx mice aged 13, 15 and 18 months (M), with D2.mdx mice (on DBA2 background) aged 9 and 13 M, using immunoblotting to quantify levels of 7 proteins. The neuronal proteins S100 beta and Tau5 were increased by 13 M in mdx nerves (compared with WT), indicating ongoing myonecrosis in this strain. In striking contrast there was no difference in levels of these neuronal proteins for D2.mdx and D2.WT sciatic nerves at 13 M, indicating reduced myonecrosis over this time in D2.mdx mice compared with mdx. These novel changes in mdx sciatic nerves by 13 M, suggest early denervation or neurodegeneration of dystrophic nerves that is likely irreversible and progressive. This neuronal readout of persistent myonecrosis may provide a useful new long-term biomarker for preclinical studies that aim to reduce myonecrosis, plus such neuronal changes present potential new drug targets to help maintain the function of DMD muscles. Show less
Gravesteijn, G.; Munting, L.P.; Overzier, M.; Mulder, A.A.; Hegeman, I.; Derieppe, M.; ... ; Oberstein, S.A.J.L. 2019
CADASIL is a NOTCH3-associated cerebral small vessel disease. A pathological ultrastructural disease hallmark is the presence of NOTCH3-protein containing deposits called granular osmiophilic... Show moreCADASIL is a NOTCH3-associated cerebral small vessel disease. A pathological ultrastructural disease hallmark is the presence of NOTCH3-protein containing deposits called granular osmiophilic material (GOM), in small arteries. How these GOM deposits develop over time and what their role is in disease progression is largely unknown. Here, we studied the progression of GOM deposits in humanized transgenic NOTCH3(Arg182Cys) mice, compared them to GOM deposits in patient material, and determined whether GOM deposits in mice are associated with a functional CADASIL phenotype. We found that GOM deposits are not static, but rather progress in ageing mice, both in terms of size and aspect. We devised a GOM classification system, reflecting size, morphology and electron density. Six-month-old mice showed mostly early stage GOM, whereas older mice and patient vessels showed predominantly advanced stage GOM, but also early stage GOM. Mutant mice did not develop the most severe GOM stage seen in patient material. This absence of end-stage GOM in mice was associated with an overall lack of histological vascular pathology, which may explain why the mice did not reveal functional deficits in cerebral blood flow, cognition and motor function. Taken together, our data indicate that GOM progress over time, and that new GOM deposits are continuously being formed. The GOM staging system we introduce here allows for uniform GOM deposit classification in future mouse and human studies, which may lead to more insight into a potential association between GOM stage and CADASIL disease severity, and the role of GOM in disease progression. Show less
Putten, M. van; Putker, K.; Overzier, M.; Adamzek, W.A.; Pasteuning-Vuhman, S.; Plomp, J.J.; Aartsma-Rus, A. 2019
Background: Spinocerebellar ataxia type 3 (SCA3) is a progressive neurodegenerative disorder caused by expansion of the polyglutamine repeat in the ataxin-3 protein. Expression of mutant ataxin-3... Show moreBackground: Spinocerebellar ataxia type 3 (SCA3) is a progressive neurodegenerative disorder caused by expansion of the polyglutamine repeat in the ataxin-3 protein. Expression of mutant ataxin-3 is known to result in transcriptional dysregulation, which can contribute to the cellular toxicity and neurodegeneration. Since the exact causative mechanisms underlying this process have not been fully elucidated, gene expression analyses in brains of transgenic SCA3 mouse models may provide useful insights.Methods: Here we characterised the MJD84.2 SCA3 mouse model expressing the mutant human ataxin-3 gene using a multi-omics approach on brain and blood. Gene expression changes in brainstem, cerebellum, striatum and cortex were used to study pathological changes in brain, while blood gene expression and metabolites/lipids levels were examined as potential biomarkers for disease.Results: Despite normal motor performance at 17.5 months of age, transcriptional changes in brain tissue of the SCA3 mice were observed. Most transcriptional changes occurred in brainstem and striatum, whilst cerebellum and cortex were only modestly affected. The most significantly altered genes in SCA3 mouse brain were Tmc3, Zfp488, Cart, and Chdh. Based on the transcriptional changes, a-adrenergic and CREB pathways were most consistently altered for combined analysis of the four brain regions. When examining individual brain regions, axon guidance and synaptic transmission pathways were most strongly altered in striatum, whilst brainstem presented with strongest alterations in the pi-3 k cascade and cholesterol biosynthesis pathways. Similar to other neurodegenerative diseases, reduced levels of tryptophan and increased levels of ceramides, di- and triglycerides were observed in SCA3 mouse blood.Conclusions: The observed transcriptional changes in SCA3 mouse brain reveal parallels with previous reported neuropathology in patients, but also shows brain region specific effects as well as involvement of adrenergic signalling and CREB pathway changes in SCA3. Importantly, the transcriptional changes occur prior to onset of motor- and coordination deficits. Show less
Toonen, L.J.A.; Overzier, M.; Evers, M.M.; Leon, L.G.; Zeeuw, S.A.J. van der; Mei, H.L.; ... ; Roon-Mom, W.M.C. van 2018
Limb-girdle muscular dystrophy types 2D and 2F (LGMD 2D and 2F) are autosomal recessive disorders caused by mutations in the alpha- and delta sarcoglycan genes, respectively, leading to severe... Show moreLimb-girdle muscular dystrophy types 2D and 2F (LGMD 2D and 2F) are autosomal recessive disorders caused by mutations in the alpha- and delta sarcoglycan genes, respectively, leading to severe muscle weakness and degeneration. The cause of the disease has been well characterized and a number of animal models are available for pre-clinical studies to test potential therapeutic interventions. To facilitate transition from drug discovery to clinical trials, standardized procedures and natural disease history data were collected for these mouse models. Implementing the TREAD-NMD standardized operating procedures, we here subjected LGMD2D (SGCA-null), LGMD2F (SGCD-null) and wild type (C57BL/6J) mice to five functional tests from the age of 4 to 32 weeks. To assess whether the functional test regime interfered with disease pathology, sedentary groups were taken along. Muscle physiology testing of tibialis anterior muscle was performed at the age of 34 weeks. Muscle histopathology and gene expression was analysed in skeletal muscles and heart. Muscle histopathology and gene expression was analysed in skeletal muscles and heart. Mice successfully accomplished the functional tests, which did not interfere with disease pathology. Muscle function of SGCA- and SGCD-null mice was impaired and declined over time. Interestingly, female SGCD-null mice outperformed males in the two and four limb hanging tests, which proved the most suitable non-invasive tests to assess muscle function. Muscle physiology testing of tibialis anterior muscle revealed lower specific force and higher susceptibility to eccentric-induced damage in LGMD mice. Analyzing muscle histopathology and gene expression, we identified the diaphragm as the most affected muscle in LGMD strains. Cardiac fibrosis was found in SGCD-null mice, being more severe in males than in females. Our study offers a comprehensive natural history dataset which will be useful to design standardized tests and future pre-clinical studies in LGMD2D and 2F mice Show less
Pasteuning-Vuhman, S.; Boertje-van der Meulen, J.W.; Putten, M.M. van; Overzier, M.; Dijke, P. ten; Kielbasa, S.M.; ... ; Loomans, C.J.M. 2017