Publications by authors named "Troels Tolstrup Nielsen"

17 Publications

  • Page 1 of 1

Novel Homozygous Truncating Variant Widens the Spectrum of Early-Onset Multisystemic SYNE1 Ataxia.

Cerebellum 2021 Jul 28. Epub 2021 Jul 28.

Department of Neurology, University of Copenhagen, Rigshospitalet, Inge Lehmanns Vej 8, 2100, Copenhagen, Denmark.

Pathogenic variants in the SYNE1 gene are associated with a phenotypic spectrum spanning from late-onset, slowly progressive, relatively pure ataxia to early-onset, fast progressive multisystemic disease. Since its first description in 2007 as an adult-onset ataxia in French Canadian families, subsequent identification of patients worldwide has widened the clinical spectrum and increased the number of identified pathogenic variants. We report a 20-year-old Faroese female with early-onset progressive gait problems, weakness, dysphagia, slurred speech, orthostatic dizziness, and urge incontinence. Neurological examination revealed mild cognitive deficits, dysarthria, broken slow pursuit, hypometric saccades, weakness with spasticity, hyperreflexia, absent ankle reflexes, ataxia, and wide-based, spastic gait. Magnetic resonance imaging displayed atrophy of the cerebellum, brainstem, and spinal cord. Severely prolonged central motor conduction time and lower motor neuron involvement was demonstrated electrophysiologically. Fluorodeoxyglucose-positron emission tomography (FDG-PET) scan showed hypometabolism of the cerebellum and right frontal lobe. Muscle biopsy revealed chronic neurogenic changes and near-absent immunostaining for Nesprin-1. Next-generation sequencing revealed a previously undescribed homozygous truncating, likely pathogenic variant in the SYNE1 gene. The patient's mother and paternal grandfather were heterozygous carriers of the variant. Her father's genotype was unobtainable. We expand the list of likely pathogenic variants in SYNE1 ataxia with a novel homozygous truncating variant with proximity to the C-terminus and relate it to a phenotype comprising early-onset cerebellar deficits, upper and lower motor neuron involvement and cognitive deficits. Also, we report novel findings of focally reduced frontal lobe FDG-PET uptake and motor evoked potential abnormalities suggestive of central demyelination.
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http://dx.doi.org/10.1007/s12311-021-01308-wDOI Listing
July 2021

Serum Neurofilament Light in Patients with Frontotemporal Dementia Caused by CHMP2B Mutation.

Dement Geriatr Cogn Disord 2020 24;49(6):533-538. Epub 2021 Feb 24.

Neurogenetics Clinic & Research Lab, Danish Dementia Research Centre, Copenhagen, Denmark.

Introduction: The potential of neurofilament light (NfL) as a blood-based biomarker is currently being investigated in autosomal dominant neurodegenerative disease. This study explores the clinical utility of serum-NfL in frontotemporal dementia due to CHMP2B mutation (FTD-3).

Methods: This cross-sectional study included serum and CSF data from 38 members of the Danish FTD-3 family: 12 affected CHMP2B mutation carriers, 10 presymptomatic carriers, and 16 noncarriers. Serum-NfL levels measured by single-molecule array (Simoa) technology were tested for associations with the clinical groups and clinical parameters. Serum and CSF data were compared, and CSF/serum-albumin ratio was included as a measure of blood-brain barrier (BBB) function.

Results: Serum-NfL concentrations were significantly increased in symptomatic CHMP2B mutation carriers compared to presymptomatic carriers and in both groups compared to healthy family controls. Serum-NfL levels appear to increase progressively with age in presymptomatic carriers, and this is perhaps followed by a change in trajectory when patients become symptomatic. Measurements of NfL in serum and CSF were highly correlated and fold-changes in serum and CSF between clinical groups were similar. Increase in serum-NFL levels was correlated with reduced ACE-score. Higher CSF/serum-albumin ratios were demonstrated in FTD-3 patients, but this did not affect the significant associations between serum-NfL and clinical groups.

Conclusion: Serum-NfL could be utilized as an accurate surrogate marker of CSF levels to segregate symptomatic CHMP2B carriers, presymptomatic carriers, and non-carriers. The observed indication of BBB dysfunction in FTD-3 patients did not confound this use of serum-NfL. The results support the occurrence of mutation-related differences in NfL dynamics in familial FTD.
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http://dx.doi.org/10.1159/000513877DOI Listing
February 2021

Compromised IGF signaling causes caspase-6 activation in Huntington disease.

Exp Neurol 2020 10 2;332:113396. Epub 2020 Jul 2.

Centre for Molecular Medicine and Therapeutics (CMMT), Department of Medical Genetics, University of British Columbia, 950 West 28th Avenue, Vancouver, BC V5Z 4H4, Canada. Electronic address:

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expansion of a polyglutamine repeat in the huntingtin (HTT) protein. Aberrant activation of caspase-6 and cleavage of mutant HTT generating the toxic N-terminal 586 HTT fragment are important steps in the pathogenesis of HD. Similarly, alterations in the insulin-like growth factor 1 (IGF-1) signaling pathway have been implicated in the disease as a result of decreased plasma IGF-1 levels in HD patients. In addition, two recent studies have demonstrated therapeutic benefit of IGF-1 treatment in mouse models of HD. Since IGF-1 promotes pro-survival pathways, we examined the relationship between IGF-1 signaling and aberrant caspase-6 activation in HD. Using immortalized mouse striatal cells expressing wild-type (STHdhQ7) or mutant HTT (STHdhQ111), we show that reduced levels of IGF-1 are associated with enhanced activation of caspase-6, increased cell death, and mutant HTT cleavage in a cellular stress paradigm. We demonstrate that IGF-1 supplementation reverses these effects and lowers the level of the toxic 586 HTT fragment. In addition, transcriptional analysis in the R6/2 HD transgenic mouse model demonstrated that the IGF-1 signaling system is dysregulated at multiple levels in several tissues including liver, muscle, and brain. Among these changes, we found increased expression of IGF-1 binding protein 3 (IGFBP-3), which may further reduce the bioavailability of IGF-1 as a consequence of increased IGF-1 binding. Our findings thus suggest that the therapeutic benefit of IGF-1 supplementation in HD may be significantly improved if other defects in the IGF-1 signaling pathway are corrected concurrently.
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http://dx.doi.org/10.1016/j.expneurol.2020.113396DOI Listing
October 2020

Enhancement of Autophagy and Solubilization of Ataxin-2 Alleviate Apoptosis in Spinocerebellar Ataxia Type 2 Patient Cells.

Cerebellum 2020 Apr;19(2):165-181

Neurogenetics Clinic & Research Laboratory, Danish Dementia Research Centre, Department of Neurology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark.

Spinocerebellar ataxia type 2 (SCA2), a rare polyglutamine neurodegenerative disorder caused by a CAG repeat expansion in the ataxin-2 gene, exhibits common cellular phenotypes with other neurodegenerative disorders, including oxidative stress and mitochondrial dysfunction. Here, we show that SCA2 patient cells exhibit higher levels of caspase-8- and caspase-9-mediated apoptotic activation than control cells, cellular phenotypes that we find to be exacerbated by reactive oxygen species (ROS) and inhibition of autophagy. We also suggest that oligomerization of mutant ataxin-2 protein is likely to be the cause of the observed cellular phenotypes by causing inhibition of autophagy and by inducing ROS generation. Finally, we show that removal of ataxin-2 oligomers, either by increasing autophagic clearance or by oligomer dissolution, appears to alleviate the cellular phenotypes. Our results suggest that oligomerized ataxin-2 and oxidative stress affect autophagic clearance in SCA2 cells, contributing to the pathophysiology, and that activation of autophagy or clearance of oligomers may prove to be effective therapeutic strategies.
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http://dx.doi.org/10.1007/s12311-019-01092-8DOI Listing
April 2020

Inflammatory markers of CHMP2B-mediated frontotemporal dementia.

J Neuroimmunol 2018 11 17;324:136-142. Epub 2018 Aug 17.

Danish Dementia Research Centre, Department of Neurology, Rigshospitalet, University of Copenhagen, Denmark.

Histopathological studies and animal models have suggested an inflammatory component in the pathomechanism of the CHMP2B associated frontotemporal dementia (FTD-3). In this cross-sectional study, serum and cerebrospinal fluid were analyzed for inflammatory markers in CHMP2B mutation carriers. Serum levels of CCL4 were increased throughout life. Serum levels of IL-15, CXCL10, CCL22 and TNF-α were significantly associated with cognitive decline, suggesting a peripheral inflammatory response to neurodegeneration. CSF levels of sTREM2 appeared to increase more rapidly with age in CHMP2B mutation carriers. The identification of a peripheral inflammatory response to disease progression supports the involvement of an inflammatory component in FTD-3.
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http://dx.doi.org/10.1016/j.jneuroim.2018.08.009DOI Listing
November 2018

Early microgliosis precedes neuronal loss and behavioural impairment in mice with a frontotemporal dementia-causing CHMP2B mutation.

Hum Mol Genet 2017 03;26(5):873-887

Department of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK.

Frontotemporal dementia (FTD)-causing mutations in the CHMP2B gene lead to the generation of mutant C-terminally truncated CHMP2B. We report that transgenic mice expressing endogenous levels of mutant CHMP2B developed late-onset brain volume loss associated with frank neuronal loss and FTD-like changes in social behaviour. These data are the first to show neurodegeneration in mice expressing mutant CHMP2B and indicate that our mouse model is able to recapitulate neurodegenerative changes observed in FTD. Neuroinflammation has been increasingly implicated in neurodegeneration, including FTD. Therefore, we investigated neuroinflammation in our CHMP2B mutant mice. We observed very early microglial proliferation that develops into a clear pro-inflammatory phenotype at late stages. Importantly, we also observed a similar inflammatory profile in CHMP2B patient frontal cortex. Aberrant microglial function has also been implicated in FTD caused by GRN, MAPT and C9orf72 mutations. The presence of early microglial changes in our CHMP2B mutant mice indicates neuroinflammation may be a contributing factor to the neurodegeneration observed in FTD.
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http://dx.doi.org/10.1093/hmg/ddx003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409096PMC
March 2017

SCA28: Novel Mutation in the AFG3L2 Proteolytic Domain Causes a Mild Cerebellar Syndrome with Selective Type-1 Muscle Fiber Atrophy.

Cerebellum 2017 02;16(1):62-67

Danish Dementia Research Centre, Neurogenetics Clinic, Department of Neurology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark.

The spinocerebellar ataxias (SCA) are a group of rare inherited neurodegenerative diseases characterized by slowly progressive cerebellar ataxia, resulting in unsteady gait, clumsiness, and dysarthria. The disorders are predominantly inherited in an autosomal dominant manner. Mutations in the gene AFG3L2 that encodes a subunit of the mitochondrial m-AAA protease have previously been shown to cause spinocerebellar ataxia type 28 (SCA28). Here, we present the clinical phenotypes of three patients from a family with autosomal dominant cerebellar ataxia and show by molecular genetics and in silico modelling that this is caused by a novel missense mutation in the AFG3L2 gene. Furthermore, we show, for the first time, fluorodeoxyglucose-positron emission tomography (FDG-PET) scans of the brain and selective type I fiber atrophy of skeletal muscle of SCA28 patients indicating non-nervous-system involvement in SCA28 as well.
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http://dx.doi.org/10.1007/s12311-016-0765-1DOI Listing
February 2017

Frontotemporal dementia caused by CHMP2B mutation is characterised by neuronal lysosomal storage pathology.

Acta Neuropathol 2015 Oct 10;130(4):511-23. Epub 2015 Sep 10.

Department of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK.

Mutations in the charged multivesicular body protein 2B (CHMP2B) cause frontotemporal dementia (FTD). We report that mice which express FTD-causative mutant CHMP2B at physiological levels develop a novel lysosomal storage pathology characterised by large neuronal autofluorescent aggregates. The aggregates are an early and progressive pathology that occur at 3 months of age and increase in both size and number over time. These autofluorescent aggregates are not observed in mice expressing wild-type CHMP2B, or in non-transgenic controls, indicating that they are a specific pathology caused by mutant CHMP2B. Ultrastructural analysis and immuno- gold labelling confirmed that they are derived from the endolysosomal system. Consistent with these findings, CHMP2B mutation patient brains contain morphologically similar autofluorescent aggregates. These aggregates occur significantly more frequently in human CHMP2B mutation brain than in neurodegenerative disease or age-matched control brains. These data suggest that lysosomal storage pathology is the major neuronal pathology in FTD caused by CHMP2B mutation. Recent evidence suggests that two other genes associated with FTD, GRN and TMEM106B are important for lysosomal function. Our identification of lysosomal storage pathology in FTD caused by CHMP2B mutation now provides evidence that endolysosomal dysfunction is a major degenerative pathway in FTD.
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http://dx.doi.org/10.1007/s00401-015-1475-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4575387PMC
October 2015

Tubulin polymerization-promoting protein (TPPP/p25α) promotes unconventional secretion of α-synuclein through exophagy by impairing autophagosome-lysosome fusion.

J Biol Chem 2013 Jun 29;288(24):17313-35. Epub 2013 Apr 29.

Department of Cellular and Molecular Medicine, The Panum Institute, University of Copenhagen, 2200 Copenhagen N, Denmark.

Aggregation of α-synuclein can be promoted by the tubulin polymerization-promoting protein/p25α, which we have used here as a tool to study the role of autophagy in the clearance of α-synuclein. In NGF-differentiated PC12 catecholaminergic nerve cells, we show that de novo expressed p25α co-localizes with α-synuclein and causes its aggregation and distribution into autophagosomes. However, p25α also lowered the mobility of autophagosomes and hindered the final maturation of autophagosomes by preventing their fusion with lysosomes for the final degradation of α-synuclein. Instead, p25α caused a 4-fold increase in the basal level of α-synuclein secreted into the medium. Secretion was strictly dependent on autophagy and could be up-regulated (trehalose and Rab1A) or down-regulated (3-methyladenine and ATG5 shRNA) by enhancers or inhibitors of autophagy or by modulating minus-end-directed (HDAC6 shRNA) or plus-end-directed (Rab8) trafficking of autophagosomes along microtubules. Finally, we show in the absence of tubulin polymerization-promoting protein/p25α that α-synuclein release was modulated by dominant mutants of Rab27A, known to regulate exocytosis of late endosomal (and amphisomal) elements, and that both lysosomal fusion block and secretion of α-synuclein could be replicated by knockdown of the p25α target, HDAC6, the predominant cytosolic deacetylase in neurons. Our data indicate that unconventional secretion of α-synuclein can be mediated through exophagy and that factors, which increase the pool of autophagosomes/amphisomes (e.g. lysosomal disturbance) or alter the polarity of vesicular transport of autophagosomes on microtubules, can result in an increased release of α-synuclein monomer and aggregates to the surroundings.
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http://dx.doi.org/10.1074/jbc.M112.401174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682534PMC
June 2013

Dysfunctional mitochondrial respiration in the striatum of the Huntington's disease transgenic R6/2 mouse model.

PLoS Curr 2013 Apr 2;5. Epub 2013 Apr 2.

Institute of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen; Section of Molecular Medicine, Department of Clinical Biochemistry, Genetics and Immunology, Statens Serum Institut, Copenhagen, Denmark.

Metabolic dysfunction and mitochondrial involvement are recognised as part of the pathology in Huntington's Disease (HD). Post-mortem examinations of the striatum from end-stage HD patients have shown a decrease in the in vitro activity of complexes II, III and IV of the electron transport system (ETS). In different models of HD, evidence of enzyme defects have been reported in complex II and complex IV using enzyme assays. However, such assays are highly variable and results have been inconsistent. We investigated the integrated ETS function ex vivo using a sensitive high-resolution respirometric (HRR) method. The O2 flux in a whole-cell sample combined with the addition of mitochondrial substrates, uncouplers and inhibitors enabled us to accurately quantitate the function of individual mitochondrial complexes in intact mitochondria, while retaining mitochondrial regulation and compensatory mechanisms. We used HRR to examine the mitochondrial function in striata from 12-week old R6/2 mice expressing exon 1 of human HTT with 130 CAG repeats. A significant reduction in complex II and complex IV flux control ratios was found in the R6/2 mouse striatum at 12 weeks of age compared to controls, confirming previous findings obtained with spectrophotometric enzyme assays.
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http://dx.doi.org/10.1371/currents.hd.d8917b4862929772c5a2f2a34ef1c201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3614423PMC
April 2013

Cognitive impairment in the preclinical stage of dementia in FTD-3 CHMP2B mutation carriers: a longitudinal prospective study.

J Neurol Neurosurg Psychiatry 2013 Feb 10;84(2):170-6. Epub 2012 Nov 10.

Memory Disorders Research Group, Department of Neurology, Rigshospitalet, Copenhagen University Hospital, N6702, 9 Blegdamsvej, Copenhagen 2100, Denmark.

Objective And Methods: A longitudinal study spanning over 8 years and including 17 asymptomatic individuals with CHMP2B mutations was conducted to assess the earliest neuropsychological changes in autosomal dominant neurodegenerative disease frontotemporal dementia (FTD) linked to chromosome 3 (FTD-3). Subjects were assessed with neuropsychological tests in 2002, 2005 and 2010.

Results: Cross-sectional analyses showed that the mutation carriers scored lower on tests of psychomotor speed, working memory, executive functions and verbal memory than a control group consisting of not-at-risk family members and spouses. Longitudinal analyses showed a gradual decline in psychomotor speed, working memory capacity and global executive measures in the group of non-demented mutation carriers that was not found in the control group. In contrast, there were no significant group differences in domain scores on memory or visuospatial functions. On an individual level the cognitive changes over time varied considerably.

Conclusion: Subjects with CHMP2B mutation show cognitive changes dominated by executive dysfunctions, years before they fulfil diagnostic criteria of FTD. However, there is great heterogeneity in the individual cognitive trajectories.
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http://dx.doi.org/10.1136/jnnp-2012-303813DOI Listing
February 2013

Germ-line CAG repeat instability causes extreme CAG repeat expansion with infantile-onset spinocerebellar ataxia type 2.

Eur J Hum Genet 2013 Jun 10;21(6):626-9. Epub 2012 Oct 10.

Memory Disorders Research Group, Neurogenetics Clinic, Department of Neurology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark.

The spinocerebellar ataxias (SCA) are a genetically and clinically heterogeneous group of diseases, characterized by dominant inheritance, progressive cerebellar ataxia and diverse extracerebellar symptoms. A subgroup of the ataxias is caused by unstable CAG-repeat expansions in their respective genes leading to pathogenic expansions of polyglutamine stretches in the encoded proteins. In general, unstable CAG repeats have an uninterrupted CAG repeat, whereas stable CAG repeats are either short or interrupted by CAA codons, which - like CAG codons - code for glutamine. Here we report on an infantile SCA2 patient who, due to germ-line CAG repeat instability in her father, inherited an extremely expanded CAG repeat in the SCA2 locus. Surprisingly, the expanded allele of the father was an interrupted CAG repeat sequence. Furthermore, analyses of single spermatozoa showed a high frequency of paternal germ-line repeat sequence instability of the expanded SCA2 locus.
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http://dx.doi.org/10.1038/ejhg.2012.231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3658194PMC
June 2013

Severe and rapidly progressing cognitive phenotype in a SCA17-family with only marginally expanded CAG/CAA repeats in the TATA-box binding protein gene: a case report.

BMC Neurol 2012 Aug 13;12:73. Epub 2012 Aug 13.

Memory Disorders Research Group, Neurogenetics Clinic, Department of Neurology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark.

Background: The autosomal dominant spinocerebellar ataxias (SCAs) confine a group of rare and heterogeneous disorders, which present with progressive ataxia and numerous other features e.g. peripheral neuropathy, macular degeneration and cognitive impairment, and a subset of these disorders is caused by CAG-repeat expansions in their respective genes. The diagnosing of the SCAs is often difficult due to the phenotypic overlap among several of the subtypes and with other neurodegenerative disorders e.g. Huntington's disease.

Case Presentation: We report a family in which the proband had rapidly progressing cognitive decline and only subtle cerebellar symptoms from age 42. Sequencing of the TATA-box binding protein gene revealed a modest elongation of the CAG/CAA-repeat of only two repeats above the non-pathogenic threshold of 41, confirming a diagnosis of SCA17. Normally, repeats within this range show reduced penetrance and result in a milder disease course with slower progression and later age of onset. Thus, this case presented with an unusual phenotype.

Conclusions: The current case highlights the diagnostic challenge of neurodegenerative disorders and the need for a thorough clinical and paraclinical examination of patients presenting with rapid cognitive decline to make a precise diagnosis on which further genetic counseling and initiation of treatment modalities can be based.
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http://dx.doi.org/10.1186/1471-2377-12-73DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3475097PMC
August 2012

ATXN2 with intermediate-length CAG/CAA repeats does not seem to be a risk factor in hereditary spastic paraplegia.

J Neurol Sci 2012 Oct 3;321(1-2):100-2. Epub 2012 Aug 3.

Danish Dementia Research Centre, Neurogenetics Clinic, Department of Neurology, Rigshospitalet, Copenhagen University Hospital, Denmark.

Hereditary spastic paraplegia (HSP) confines a group of heterogeneous neurodegenerative disorders characterized by progressive spasticity and lower limb weakness. Age of onset is highly variable even in familial cases with known mutations suggesting that the disease is modulated by other yet unknown parameters. Although progressive gait disturbances, lower limb spasticity and extensor plantar responses are hallmarks of HSP these characteristics are also found in other neurodegenerative disorders, e.g. amytrophic lateral sclerosis (ALS). HSP has been linked to ALS and frontotemporal degeneration with motor neuron disease (FTD-MND), since TDP-43 positive inclusions have recently been found in an HSP subtype, and TDP-43 are found in abundance in pathological inclusions of both ALS and FTD-MND. Furthermore, ataxin-2 (encoded by the gene ATXN2), a polyglutamine containing protein elongated in spinocerebellar ataxia type 2, has been shown to be a modulator of TDP-43 induced toxicity in ALS animal and cell models. Finally, it has been shown that ATXN2 with non-pathogenic intermediate-length CAG/CAA repeat elongations (encoding the polyglutamine tract) is a genetic risk factor of ALS. Considering the similarities in the disease phenotype and the neuropathological link between ALS and HSP we hypothesized that intermediate-length CAG/CAA repeats in ATXN2 could be a modulator of HSP. We show that in a cohort of 181 HSP patients 4.9 % of the patients had intermediate-length CAG/CAA repeats in ATXN2 which was not significantly different from the frequencies in a Danish control cohort or in American and European control populations. However, the mean age of onset was significantly lower in HSP patients with intermediate-length CAG/CAA repeats in ATXN2 compared to patients with normal length repeats. Based on these results we conclude that ATXN2 is most likely not a risk factor of HSP, whereas it might serve as a modulator of age of onset.
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http://dx.doi.org/10.1016/j.jns.2012.07.036DOI Listing
October 2012

Reversal of pathology in CHMP2B-mediated frontotemporal dementia patient cells using RNA interference.

J Gene Med 2012 Aug;14(8):521-9

Danish Dementia Research Centre, Department of Neurology, Rigshospitalet, Copenhagen University Hospital, Denmark.

Background: Frontotemporal dementia is the second most common form of young-onset dementia after Alzheimer's disease, and several genetic forms of frontotemporal dementia are known. A rare genetic variant is caused by a point mutation in the CHMP2B gene. CHMP2B is a component of the ESCRT-III complex, which is involved in endosomal trafficking of proteins targeted for degradation in lysosomes. Mutations in CHMP2B result in abnormal endosomal structures in patient fibroblasts and patient brains, probably through a gain-of-function mechanism, suggesting that the endosomal pathway plays a central role in the pathogenesis of the disease.

Methods: In the present study, we used lentiviral vectors to efficiently knockdown CHMP2B by delivering microRNA embedded small hairpin RNAs.

Results: We show that CHMP2B can be efficiently knocked down in patient fibroblasts using an RNA interference approach and that the knockdown causes reversal of the abnormal endosomal phenotype observed in patient fibroblasts.

Conclusions: This is the first description of a treatment that reverses the cellular pathology caused by mutant CHMP2B and suggests that RNA interference might be a feasible therapeutic strategy. Furthermore, it provides the first proof of a direct link between the disease-causing mutation and the cellular phenotype in cells originating from CHMP2B mutation patients.
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http://dx.doi.org/10.1002/jgm.2649DOI Listing
August 2012

Knockdown of GAD67 protein levels normalizes neuronal activity in a rat model of Parkinson's disease.

J Gene Med 2011 Mar;13(3):188-97

CNS Gene Therapy Unit, Wallenberg Neuroscience Center, Department of Experimental Medical Science, Lund University, Lund, Sweden.

Background: Dopamine depletion of the striatum is one of the hallmarks of Parkinson's disease. The loss of dopamine upregulates GAD67 expression in the striatal projection neurons and causes other changes in the activity of the basal ganglia circuit.

Methods: To normalize the GAD67 expression in the striatum after dopamine depletion, we developed several lentiviral vectors that express RNA interference (RNAi) directed against GAD67 mitochondrial RNA. The vectors were injected into the striatum of hemiparkinsonian rats and the level of GAD67 protein as well as a marker of neuronal activity, mtCO1, was analyzed using western blots.

Results: Unilateral lesions of the dopamine neurons in substantia nigra resulted in an increased level of GAD67 protein in the ipsilateral striatum. Furthermore, we detected significantly higher levels of mtCO1, after dopamine depletion in the striatum. Using a lentiviral vectors with a synthetic miRNA scaffold to deliver RNAi, we were able to normalize the GAD67 protein levels in the parkinsonian rat striatum. In addition, we were able to normalize the increased neural activity, which resulted from the loss of dopamine as measured by the marker mtCO1.

Conclusions: We conclude that RNAi directed against GAD67 may be a valid approach to correct the dysregulation of the basal ganglia circuit in a rat model of Parkinson's disease. The possibility to correct for a loss of dopamine using nondopamimetic tools is interesting because it may be more directed towards the casual mechanisms of the motor symptoms.
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http://dx.doi.org/10.1002/jgm.1555DOI Listing
March 2011

Neuron-specific RNA interference using lentiviral vectors.

J Gene Med 2009 Jul;11(7):559-69

Department of Cellular and Molecular Medicine, Panum Instituttet, University of Copenhagen, Denmark.

Background: Viral vectors have been used in several different settings for the delivery of small hairpin (sh) RNAs. However, most vectors have utilized ubiquitously-expressing polymerase (pol) III promoters to drive expression of the hairpin as a result of the strict requirement for precise transcriptional initiation and termination. Recently, pol II promoters have been used to construct vectors for RNA interference (RNAi). By embedding the shRNA into a micro RNA-context (miRNA) the endogenous miRNA processing machinery is exploited to achieve the mature synthetic miRNA (smiRNA), thereby expanding the possible promoter choices and eventually allowing cell type specific down-regulation of target genes.

Methods: In the present study, we constructed lentiviral vectors expressing smiRNAs under the control of pol II promoters to knockdown gene expression in cell culture and in the brain.

Results: We demonstrate robust knockdown of green fluorescent protein using lentiviral vectors driving RNAi from the ubiquitously-expressing promoter of the cytomegalovirus (CMV) and, in addition, we show for the first time neuron-specific knockdown in the brain using a neuron-specific promoter. Furthermore, we show that the expression pattern of the presumed ubiquitously-expressing CMV promoter changes over time from being expressed initially in neurons and glial cells to being expressed almost exclusively in neurons in later stages.

Conclusions: In the present study, we developed vectors for cell-specific RNAi for use in the brain. This offers the possibility of specifically targeting RNAi to a subset of cells in a complex tissue and may prove to be of great importance in the design of future gene therapeutic paradigms.
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http://dx.doi.org/10.1002/jgm.1333DOI Listing
July 2009
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