Neuropathological features of genetically confirmed DYT1 dystonia: Investigating disease-specific inclusions Reema Paudel, UCL Institute of Neurology Aoife Kiely, UCL Institute of Neurology Abi Li, UCL Institute of Neurology Tammaryn Lashley, UCL Institute of Neurology Rina Bandopadhyay, UCL Institute of Neurology John Hardy, UCL Institute of Neurology Hyder Jinnah, Emory University Kailash Bhatia, UCL Institute of Neurology Henry Houlden, UCL Institute of Neurology Janice L. Holton, UCL Institute of Neurology Journal Title: Acta Neuropathologica Communications Volume: Volume 2, Number 1 Publisher: BioMed Central | 2014-01-27, Pages 159-159 Type of Work: Article | Final Publisher PDF Publisher DOI: 10.1186/s40478-014-0159-x Permanent URL: https://pid.emory.edu/ark:/25593/rz52g Final published version: http://dx.doi.org/10.1186/s40478-014-0159-x Copyright information: © 2014 Paudel et al.; licensee BioMed Central Ltd. This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/). Accessed September 25, 2021 4:11 AM EDT Paudel et al. Acta Neuropathologica Communications 2014, 2:159 http://www.actaneurocomms.org/content/2/1/159 RESEARCH Open Access Neuropathological features of genetically confirmed DYT1 dystonia: investigating disease- specific inclusions Reema Paudel1, Aoife Kiely2, Abi Li2, Tammaryn Lashley2, Rina Bandopadhyay2, John Hardy1, Hyder A Jinnah3, Kailash Bhatia1, Henry Houlden1 and Janice L Holton1,2* Abstract Introduction: Early onset isolated dystonia (DYT1) is linked to a three base pair deletion (ΔGAG) mutation in the TOR1A gene. Clinical manifestation includes intermittent muscle contraction leading to twisting movements or abnormal postures. Neuropathological studies on DYT1 cases are limited, most showing no significant abnormalities. In one study, brainstem intraneuronal inclusions immunoreactive for ubiquitin, torsinA and lamin A/C were described. Using the largest series reported to date comprising 7 DYT1 cases, we aimed to identify consistent neuropathological features in the disease and determine whether we would find the same intraneuronal inclusions as previously reported. Result: The pathological changes of brainstem inclusions reported in DYT1 dystonia were not replicated in our case series. Other anatomical regions implicated in dystonia showed no disease-specific pathological intracellular inclu- sionsorevidenceofmorethanmildneuronalloss. Conclusion: Our findings suggest that the intracellular inclusions described previously in DYT1 dystonia may not be a hallmark feature of the disorder. In isolated dystonia, DYT1 in particular, biochemical changes may be more relevant than the morphological changes. Keywords: DYT1, Neuropathology, Isolated dystonia, Inclusions Introduction [3]. Dystonia linked to a 3 base pair deletion (ΔGAG) mu- Dystonia is a common and heterogeneous neurological tation in exon 5 of the TOR1A gene on chromosome disorder. Recently, dystonia has been defined as a move- 9q34.11 is called DYT1 dystonia [4]. The new classifica- ment disorder characterized by sustained or intermittent tion system classified DYT1 dystonia as isolated dystonia muscle contractions causing abnormal, often repetitive, in which dystonia is the only clinical sign with the excep- movements, postures, or both with genetic heterogeneity tion of tremor [3]. The prevalence of isolated dystonia was reported. The term ‘dystonia musculorum deformans’ estimated at 330 per million in Rochester, Minnesota [5] was proposed in 1911 to describe a disorder in children and is about 152 per million in the European population with twisted postures, muscle spasms, gait abnormalities [6]. In DYT1 the inheritance is autosomal dominant with and clonic, rhythmic jerking movements [1]. In childhood the disease onset in childhood [7] and the most common onset cases dystonia tends to become generalized, while clinical features include muscle contractions affecting the remaining focal or segmental in adult onset cases [2]. A leg or arm, often progressing to generalized involvement newly proposed classification system classified dystonia with severe disability [8]. The mutation has been identified according to two axes: clinical characteristics and etiology in many diverse ethnic groups [9,10] and has a low pene- trance of 30–40% [10-12]. * Correspondence: [email protected] The TOR1A gene encodes a 332 amino acid protein, 1 Department of Molecular Neuroscience, UCL Institute of Neurology, London, torsinA, which has a cytoplasmic localization in cells. UK 2Queen Square Brain Bank, UCL Institute of Neurology, London, UK TorsinA is widely expressed throughout the central Full list of author information is available at the end of the article © 2014 Paudel et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Paudel et al. Acta Neuropathologica Communications 2014, 2:159 Page 2 of 8 http://www.actaneurocomms.org/content/2/1/159 nervous system in humans, but is found at particularly although systematic sampling of multiple regions for each high levels in dopaminergic neurons of the substantia case was not possible due to the retrospective nature of nigra, locus coeruleus, Purkinje cells, cerebellar dentate the study. Where possible the frontal and temporal cor- nucleus, basis pontis, thalamus, hippocampal formation, tices, striatum, globus pallidus, thalamus, subthalamic oculomotor nuclei and frontal cortex [13-16]. The exact nucleus, cerebellum, midbrain, pons, and medulla were function of torsinA remains unclear in humans. However, investigated. Brain regions available for study in each mutant torsinA protein has been shown to have aberrant case are indicated in Additional file 1: Table S1. The mid- cellular localization and impaired protein interactions and brain was absent in cases 2 and 3 and was only partially is associated with defective synaptic vesicle formation and represented in case 6. The pons including the reticular for- altered development of neuronal pathways [17]. mation was present in all cases except case 6. Much of the research in primary dystonia has focused on the role of the basal ganglia in disease [18-20]. How- Genetics ever, imaging studies of patients carrying a mutation in DNA was extracted from frozen brain tissue (cerebellum TOR1A causing primary dystonia revealed an increase in or frontal cortex) using DNeasy Blood and Tissue Kit metabolic brain activity not only in the basal ganglia but (Cat no 69504). Cases were sequenced for the recurring also in the cerebellum [21,22]. Neuropathological investiga- ΔGAG mutation in exon 5 of TOR1A using standard tion of clinically diagnosed primary dystonia cases has been Sanger polymerase chain reaction sequencing on an ABI generally disappointing with no specific abnormalities 3730XL machine (Applied Biosystems, Inc., Foster City, observed [23]. In genetically confirmed DYT1 cases, no CA, USA) and analyzed using Sequencher software. evidence of neuronal loss, inflammation or altered lo- calization of torsinA could be identified [15,23-26]. A Immunohistochemistry (IHC) possible reduction in striatal dopamine and the size of In brief, paraffin embedded tissue sections (7 μm) were cut pigmented neurons in the substantia nigra have been and stained using routine methods, including hematoxylin suggested [15,23,26]. The most interesting observation to and eosin, Gallyas silver and Luxol fast blue/cresyl violet. date has been the finding of perinuclear intraneuronal in- IHC staining was performed according to a standard clusions immunoreactive for ubiquitin, torsinA and lamin avidin-biotin complex protocol using the following anti- A/C in the periaqueductal grey matter, cuneiform and bodies: ubiquitin (1:200, Dako, Ely,UK), tau (1:600, AT8, pedunculopontine nuclei [27]. Similar inclusions were re- Autogen Bioclear, Calne, UK); AT100 (1:200, Innogenetics, ported in some of the DYT1 mouse models produced by Gent, Belgium), glial fibrillary acidic protein (GFAP; expression of transgenic human torsinA but this has not 1:1,000, Dako), Aβ (1:200, Dako), α-synuclein (1:50, been a consistent finding [28,29]. The intriguing observa- Novacastra, Newcastle, UK), α-synuclein (1:1000, BD tion of brainstem inclusions in DYT1 cases has not, so far, Transduction Biolabs, Oxford, UK), p62 (1:100, BD Bio- been replicated. science, Oxford, UK), CD68 (1:150, Dako), and TDP-43 The aims of this current study were: 1) to report the (1:2000, Protein Tech, Manchester, UK). Antibodies to neuropathological features of seven previously unreported ubiquitin, p62, and tau required pretreatment by pressure genetically proven DYT1 cases, 2) to determine whether cooking in sodium citrate buffer, pH 6.0, for 10 minutes any pathological features were consistently observed in all before IHC staining. Sections to be used for IHC staining cases and could be regarded as disease-related and 3) to with antibodies to α-synuclein and Aβ were treated with determine whether the perinuclear intracytoplasmic inclu- formic acid for 30 minutes before pressure cooking as sions previously