In Vivo Brain Sodium Disequilibrium in ATP1A3‐Related Rapid‐Onset Dystonia‐Parkinsonism

dorsal midbrain, lending additional metabolic support to functional imaging data, suggesting changes in the SCol in dystonia. Various lines of evidence ranging from animal studies reporting improvements in lesions, gene expression experiments in monogenic forms, human structural and functional imaging, and eye-blink classical condition experiments point toward an involvement of efferent cerebellar structures in dystonia pathophysiology. Thus far, it proved difficult to conclude regarding how far cerebellar activity in dystonia is causal, contributory, or compensatory. In contrast to increased metabolic activity on cerebellar glucose-positron emission tomography imaging, which can be interpreted as both possibly causative and compensatory, our observation of decreased enzyme activity is compatible with a primary deficit within the cerebellar outflow tract. The b-GAL results overall argue against a purely GCasemediated effect but more likely general lysosomal activity changes in dystonia. Larger genetic studies are planned to elucidate whether lysosomal dysfunction in dystonia is associated with a specific gene, such as GBA, or broader mechanisms regulating lysosomal function. Mechanistically, endosomallysosomal deficiency has recently been reported to be implicated in dystonia due to mutations affecting the homotypic fusion and vacuole protein sorting complex, postulating disrupted cellular processes in motor control networks as a possible mechanism. Similarly, network signaling abnormalities and synaptic dysfunction have been described in the context of lysosomal storage disorders, and future studies should explore if they provide a possible mechanistic relation between lysosomal and network dysfunction in dystonia. Brain regions in this study were chosen based on their presumed role in dystonia pathophysiology and tissue availability and thus are not representative. We acknowledge the limitations regarding phenotypical information and statistical power due to the paucity of dystonia brain donors (Table S1). The presence of signs of pathological aging in some donors, reflecting the age at death, was balanced between groups and unlikely to have affected results. Medicationrelated bias seems equally unlikely, especially for botulinum toxin injections, the most frequently used medication in our sample. In summary, our observations provide preliminary evidence for a possible role of lysosomal dysfunction in isolated dystonia. Although the enzyme activity pattern identified points to a primary role of cerebellar output-/brainstem structures, the exact mechanism of how lysosomal dysfunction causes dystonia remains to be established.

document primarily affected cerebellar efferents, but also the dorsal midbrain, lending additional metabolic support to functional imaging data, suggesting changes in the SCol in dystonia. 2 Various lines of evidence ranging from animal studies reporting improvements in lesions, gene expression experiments in monogenic forms, human structural and functional imaging, and eye-blink classical condition experiments point toward an involvement of efferent cerebellar structures in dystonia pathophysiology. 1,3 Thus far, it proved difficult to conclude regarding how far cerebellar activity in dystonia is causal, contributory, or compensatory. 3 In contrast to increased metabolic activity on cerebellar glucose-positron emission tomography imaging, which can be interpreted as both possibly causative and compensatory, 1 our observation of decreased enzyme activity is compatible with a primary deficit within the cerebellar outflow tract.
The b-GAL results overall argue against a purely GCasemediated effect but more likely general lysosomal activity changes in dystonia. Larger genetic studies are planned to elucidate whether lysosomal dysfunction in dystonia is associated with a specific gene, such as GBA, or broader mechanisms regulating lysosomal function. Mechanistically, endosomallysosomal deficiency has recently been reported to be implicated in dystonia due to mutations affecting the homotypic fusion and vacuole protein sorting complex, postulating disrupted cellular processes in motor control networks as a possible mechanism. 4 Similarly, network signaling abnormalities 5 and synaptic dysfunction 6 have been described in the context of lysosomal storage disorders, and future studies should explore if they provide a possible mechanistic relation between lysosomal and network dysfunction in dystonia.
Brain regions in this study were chosen based on their presumed role in dystonia pathophysiology 1,2,7 and tissue availability and thus are not representative. We acknowledge the limitations regarding phenotypical information and statistical power due to the paucity of dystonia brain donors (Table  S1). The presence of signs of pathological aging in some donors, reflecting the age at death, was balanced between groups and unlikely to have affected results. Medicationrelated bias seems equally unlikely, especially for botulinum toxin injections, the most frequently used medication in our sample.
In summary, our observations provide preliminary evidence for a possible role of lysosomal dysfunction in isolated dystonia. Although the enzyme activity pattern identified points to a primary role of cerebellar output-/brainstem structures, the exact mechanism of how lysosomal dysfunction causes dystonia remains to be established.

Supporting Data
Additional Supporting Information may be found in the online version of this article at the publisher's web-site.

In Vivo Brain Sodium Disequilibrium in ATP1A3-Related
Rapid-Onset Dystonia-Parkinsonism ATP1A3-related neurological disorders display a broad clinical spectrum with three predominant phenotypes, including rapid-onset dystonia-parkinsonism (RDP). 1 The ATP1A3 gene encoding the α-subunit (subtype 3) of the Na + /K + -ATPase enzyme maintains the neuronal electrochemical gradient by removing intracellular sodium in exchange for extracellular potassium ions, which is essential for regulating the excitability of neurons, cell volume, and neurotransmission. 2,3 This study used 23 Na-MRI (magnetic resonance imaging) employing the nuclear magnetic resonance of sodium, with a combination of total sodium (tNa) and intracellular-weighted sodium imaging (inversion recovery 23 Na-MRI [IR-Na]). 4 The latter measurement is particularly interesting considering that decreased Na + /K + -ATPase activity is expected to lead to an accumulation of intracellular sodium, which might serve as a direct measure of the proposed disease mechanisms in ATP1A3-related disorders.
A 45-year-old male patient with RDP harboring a heterozygous missense mutation in the ATP1A3 gene [NM_152296.5 (ATP1A3):c.2788C>T(p.Arg930Trp)] and seven age/sexmatched (45.4 AE 2.4 years) control subjects were enrolled. Neuroimaging was performed on a 3T Siemens MAGNETOM Skyra MRI scanner using a 64-channel head/neck coil (Siemens) and a dual-tuned quadrature head coil 1 H/ 23 Na (RAPID Biomedical). Preprocessing of sodium images and voxel-based morphometry was done using the SPM12 software package and CAT12 toolbox. Volumes (T1) and the mean voxel intensities (tNa and IR-Na images) for caudate, putamen, pallidum, thalamus, supplementary motor area (SMA), precentral gyrus (PrecG), postcentral gyrus (PostcG), and the cerebellum were extracted using the Neuromorphometrics brain atlas. We selected the side-averaged regions of interest (ROIs) based on their involvement in the development of dystonia. 5 In addition, the occipital lobe (OccL) served as a control ROI because it is not implicated in the pathophysiology of dystonia. Extracted ROI values were scaled to the total intracranial volume (TIV) and z-transformed. More details on the methods and the case description can be found in the Supporting Information Methods and Video S1.
The volumetry values of our index patient were within the reference range of the control group (Supporting Information  Fig. S1D). The assessment of tNa content demonstrated marked differences for most of the earlier-mentioned ROIs contrary to the control ROI (Fig. 1C). The cerebellum showed the highest tNa z-score (6.29) and also the highest IR-Na zscore (2.79) (Fig. 1D).
Our study provides the first in-vivo evidence that sodium predominantly accumulates in the cerebellum of patients with RDP, which appears to be driven by intracellular accumulation. The measurement of a sodium disequilibrium is supported by previous reports showing pathophysiological involvement of the cerebellum in the development of ATP1A3-related disorders and other forms of dystonia. 5,6 In rodent models, cerebellar injection of ouabain (a pharmacological inhibitor of the Na + / K + -ATPase enzyme) or an ATP1A3-directed small hairpin RNA leads to the development of dystonia-like phenotypes. 7 In addition, cerebellar pathologies either caused by structural lesions or consequent to inherited ataxias can result in dystonia in humans. 5 Overall, these results indicate that tNa and IR-Na imaging may be suitable for studying ATP1A3-related disorders and should be applied once it becomes broadly available to future clinical trials. Future studies in patients with other forms of dystonia or parkinsonism are needed to evaluate the pathophysiological specificity of 23 Na-MRI in ATP1A3-related neurological disorders.

Supporting Data
Additional Supporting Information may be found in the online version of this article at the publisher's web-site.

Extreme Clinical Variability Among Carriers of Pathogenic Variant in SSBP1
Extreme Clinical Variability Among Carriers of Pathogenic Variant in SSBP1: Beyond Optic Atrophy Optic atrophy 13 with retinal and foveal abnormalities (OPA13, OMIM #165510) is an autosomal dominant optic atrophy caused by pathogenic variants in SSBP1. This is a nuclear gene that encodes tetrameric mitochondrial singlestranded DNA binding protein (mtSSB), an essential protein for mitochondrial replication and maintenance. 1 Mutations in nuclear genes associated with mitochondrial DNA (mtDNA) replisome can lead to damage to mtDNA and variable degrees of disturbances in oxidative phosphorylation and, as a