Generation of homozygous and heterozygous REEP1 knockout induced pluripotent stem cell lines by CRISPR/Cas9 gene editing.

REEP1 is a transmembrane protein in the endoplasmic reticulum (ER) membrane that is involved in shaping and remodeling of the ER. Mutations in REEP1 cause SPG31, an autosomal dominant form of hereditary spastic paraplegia (HSP). Here we show the generation of a homozygous and a heterozygous REEP1 knockout induced pluripotent stem cell line suitable for in vitro disease modelling using the CRISPR/Cas9 editing system.

Generation of two SPAST knockout human induced pluripotent stem cell lines to create a model for Hereditary Spastic Paraplegia type 4.

Spastin is a an ATPase that severs microtubules therby regulating amount and mobility of these structures. Mutations in the SPAST gene (SPG4) are the most common form of Hereditary Spastic Paraplegia (HSP). Here, we report the generation of a homozygous and a heterozygous SPAST knockout induced pluripotent stem cell (iPSC) line from a healthy control iPSC line using CRISPR/Cas9 technology.

CHIP mutations affect the heat shock response differently in human fibroblasts and iPSC-derived neurons.

C-terminus of HSC70-interacting protein (CHIP) encoded by the gene STUB1 is a co-chaperone and E3 ligase that acts as a key regulator of cellular protein homeostasis. Mutations in STUB1 cause autosomal recessive spinocerebellar ataxia type 16 (SCAR16) with widespread neurodegeneration manifesting as spastic-ataxic gait disorder, dementia and epilepsy. CHIP-/- mice display severe cerebellar atrophy, show high perinatal lethality and impaired heat stress tolerance. To decipher the pathomechanism underlying SCAR16, we investigated the heat shock response (HSR) in primary fibroblasts of three SCAR16 patients. We found impaired HSR induction and recovery compared to healthy controls. HSPA1A/B transcript levels (coding for HSP70) were reduced upon heat shock but HSP70 remained higher upon recovery in patient- compared to control-fibroblasts. As SCAR16 primarily affects the central nervous system we next investigated the HSR in cortical neurons (CNs) derived from induced pluripotent stem cells of SCAR16 patients. We found CNs of patients and controls to be surprisingly resistant to heat stress with high basal levels of HSP70 compared to fibroblasts. Although heat stress resulted in strong transcript level increases of many HSPs, this did not translate into higher HSP70 protein levels upon heat shock, independent of STUB1 mutations. Furthermore, STUB1(-/-) neurons generated by CRISPR/Cas9-mediated genome editing from an isogenic healthy control line showed a similar HSR to patients. Proteomic analysis of CNs showed dysfunctional protein (re)folding and higher basal oxidative stress levels in patients. Our results question the role of impaired HSR in SCAR16 neuropathology and highlight the need for careful selection of proper cell types for modeling human diseases.

GPT2 mutations cause developmental encephalopathy with microcephaly and features of complicated hereditary spastic paraplegia.

Various genetic defects can cause intellectual and developmental disabilities (IDDs). Often IDD is a symptom of a more complex neurodevelopmental or neurodegenerative syndrome. Identifying syndromic patterns is substantive for diagnostics and for understanding the pathomechanism of a disease. Recessive glutamate pyruvate transaminase (GPT2) mutations have recently been associated with IDD in 4 families. Here, we report a novel recessive GPT2 stop mutation p.Gln24* causing a complex IDD phenotype in a homozygous state in 5 patients from 2 consanguineous Arab families. By compiling clinical information of these individuals and previously described GPT2 patients a recognizable neurodevelopmental and potentially neurodegenerative phenotype can be assigned consisting of intellectual disability, pyramidal tract affection with spastic paraplegia, microcephaly and frequently epilepsy. Because of the consistent presence of pyramidal tract affection in GPT2 patients, we further suggest that GPT2 mutations should be considered in cases with complex hereditary spastic paraplegia.

Consensus Paper: Neurophysiological Assessments of Ataxias in Daily Practice.

The purpose of this consensus paper is to review electrophysiological abnormalities and to provide a guideline of neurophysiological assessments in cerebellar ataxias. All authors agree that standard electrophysiological methods should be systematically applied in all cases of ataxia to reveal accompanying peripheral neuropathy, the involvement of the dorsal columns, pyramidal tracts and the brainstem. Electroencephalography should also be considered, although findings are frequently non-specific. Electrophysiology helps define the neuronal systems affected by the disease in an individual patient and to understand the phenotypes of the different types of ataxia on a more general level. As yet, there is no established electrophysiological measure which is sensitive and specific of cerebellar dysfunction in ataxias. The authors agree that cerebellar brain inhibition (CBI), which is based on a paired-pulse transcranial magnetic stimulation (TMS) paradigm assessing cerebellar-cortical connectivity, is likely a useful measure of cerebellar function. Although its role in the investigation and diagnoses of different types of ataxias is unclear, it will be of interest to study its utility in this type of conditions. The authors agree that detailed clinical examination reveals core features of ataxia (i.e., dysarthria, truncal, gait and limb ataxia, oculomotor dysfunction) and is sufficient for formulating a differential diagnosis. Clinical assessment of oculomotor function, especially saccades and the vestibulo-ocular reflex (VOR) which are most easily examined both at the bedside and with quantitative testing techniques, is of particular help for differential diagnosis in many cases. Pure clinical measures, however, are not sensitive enough to reveal minute fluctuations or early treatment response as most relevant for pre-clinical stages of disease which might be amenable to study in future intervention trials. The authors agree that quantitative measures of ataxia are desirable as biomarkers. Methods are discussed that allow quantification of ataxia in laboratory as well as in clinical and real-life settings, for instance at the patients' home. Future studies are needed to demonstrate their usefulness as biomarkers in pharmaceutical or rehabilitation trials.

[Ataxias and hereditary spastic paraplegias]. / Ataxien und hereditäre spastische Spinalparalysen.

Hereditary ataxias and spastic paraplegias are genetic disorders with age-dependent nearly complete penetrance. The mostly monogenetic etiology allows one to establish the diagnosis, study pathogenesis and to develop new causative therapeutic approaches for these diseases. Both the causative genes as well as the clinical presentation overlap considerably between hereditary ataxias and spastic paraplegias. This strongly argues towards a united classification for these two groups of diseases. Next generation sequencing technologies have greatly expanded the number of genes known to be causative for hereditary ataxias and spastic paraplegias and allow simultaneous time- and cost-effective diagnostic testing of > 200 genes. However, repeat expansions and large genomic deletions must be considered separately. Here, we suggest a pragmatic algorithm for genetic testing in hereditary ataxias and spastic paraplegias that we have developed in our specialized outpatient clinics. Detailed phenotyping remains crucial to interpret the multitude of genetic variants discovered by high throughput sequencing techniques. Despite recent technical advances, a substantial proportion of ataxia and spastic paraplegia families are still without a molecular diagnosis. Beside new and so far undetected ataxia and spasticity genes, unusual mutation types including noncoding variants and polygenic inheritance patterns may contribute. Because of these clinical, genetic, and technological challenges, patients with hereditary ataxias and spastic paraplegias should be referred to specialized centers offering research and clinical studies. This will also help to recruit representative patient cohorts for upcoming interventional trials.

A cognitive brain-computer interface for patients with amyotrophic lateral sclerosis.

Brain-computer interfaces (BCIs) are often based on the control of sensorimotor processes, yet sensorimotor processes are impaired in patients suffering from amyotrophic lateral sclerosis (ALS). We devised a new paradigm that targets higher-level cognitive processes to transmit information from the user to the BCI. We instructed five ALS patients and twelve healthy subjects to either activate self-referential memories or to focus on a process without mnemonic content while recording a high-density electroencephalogram (EEG). Both tasks are designed to modulate activity in the default mode network (DMN) without involving sensorimotor pathways. We find that the two tasks can be distinguished after only one experimental session from the average of the combined bandpower modulations in the theta- (4-7Hz) and alpha-range (8-13Hz), with an average accuracy of 62.5% and 60.8% for healthy subjects and ALS patients, respectively. The spatial weights of the decoding algorithm show a preference for the parietal area, consistent with modulation of neural activity in primary nodes of the DMN.

CYP2U1 mutations in two Iranian patients with activity induced dystonia, motor regression and spastic paraplegia.

Hereditary spastic paraplegia (HSP) is a heterogeneous condition characterized by progressive spasticity and weakness in the lower limbs. It is divided into two major groups, complicated and uncomplicated, based on the presence of additional features such as intellectual disability, ataxia, seizures, peripheral neuropathy and visual problems. SPG56 is an autosomal recessive form of HSP with complicated and uncomplicated manifestations, complicated being more common. CYP2U1 gene mutations have been identified as responsible for SPG56. Intellectual disability, dystonia, subclinical sensory motor neuropathy, pigmentary degenerative maculopathy, thin corpus callosum and periventricular white-matter hyperintensities were additional features noted in previous cases of SPG56. Here we identified two novel mutations in CYP2U1 in two unrelated patients by whole exome sequencing. Both patients had complicated HSP with activity-induced dystonia, suggesting dystonia as an additional finding in SPG56. Two out of 14 previously reported patients had dystonia, and the addition of our patients suggests dystonia in a quarter of SPG56 patients. Developmental regression has not been reported in SPG56 patients so far but both of our patients developed motor regression in infancy.

Cerebellar pathology in Friedreich's ataxia: atrophied dentate nuclei with normal iron content.

BACKGROUND: In Friedreich's ataxia (FA) the genetically decreased expression of the mitochondrial protein frataxin leads to disturbance of the mitochondrial iron metabolism. Within the cerebellum the dentate nuclei (DN) are primarily affected. Histopathological studies show atrophy and accumulation of mitochondrial iron in DN. Dentate iron content has been suggested as a biomarker to measure the effects of siderophores/antioxidant treatment of FA. We assessed the iron content and the volume of DN in FA patients and controls based on ultra-high-field MRI (7 Tesla) images. METHODS: Fourteen FA patients (mean age 38.1 yrs) and 14 age- and gender-matched controls participated. Multi-echo gradient echo and susceptibility weighted imaging (SWI) sequences were acquired on a 7 T whole-body scanner. For comparison SWI images were acquired on a 1.5 T MR scanner. Volumes of the DN and cerebellum were assessed at 7 and 1.5 T, respectively. Parametric maps of T2 and T2* sequences were created and proton transverse relaxation rates were estimated as a measure of iron content. RESULTS: In FA, the DN and the cerebellum were significantly smaller compared to controls. However, proton transverse relaxation rates of the DN were not significantly different between both groups. CONCLUSIONS: Applying in vivo MRI methods we could demonstrate significant atrophy of the DN in the presence of normal iron content. The findings suggest that relaxation rates are not reliable biomarkers in clinical trials evaluating the potential effect of FA therapy.

Leukodystrophies underlying cryptic spastic paraparesis: frequency and phenotype in 76 patients.

BACKGROUND AND PURPOSE: In chronic progressive spasticity of the legs many rare causes have to be considered, including leukodystrophies due to neurometabolic disorders. To determine the frequency of leukodystrophies and the phenotypic spectrum patients with cryptic spasticity of the legs were screened for underlying neurometabolic abnormalities. METHODS: Seventy-six index patients presenting with adult-onset lower limb spasticity of unknown cause consistent with autosomal recessive inheritance were included in this study. Screening included serum levels of very long chain fatty acids for X-linked adrenoleukodystrophy/adrenomyeloneuropathy and lysosomal enzyme activities in leukocytes for metachromatic leukodystrophy, GM1-gangliosidosis, Tay-Sachs, Sandhoff and Krabbe disease. If clinical evidence was indicative of other types of leukodystrophies, additional genetic testing was conducted. Clinical characterization included neurological and psychiatric features and magnetic resonance imaging. RESULTS: Basic screening detected one index patient with metachromatic leukodystrophy, two patients with Krabbe disease and four patients with adrenoleukodystrophy/adrenomyeloneuropathy. Additional genetic testing revealed one patient with vanishing white matter disease. These patients accounted for an overall share of 11% of leukodystrophies. One patient with Krabbe disease and three patients with adrenoleukodystrophy/adrenomyeloneuropathy presented with pure spasticity of the lower limbs, whilst one patient each with Krabbe disease, metachromatic leukodystrophy and adrenoleukodystrophy/adrenomyeloneuropathy showed additional complicating symptoms. CONCLUSIONS: Adult patients presenting with cryptic spasticity of the legs should be screened for underlying X-linked adrenoleukodystrophy/adrenomyeloneuropathy and lysosomal disorders, irrespective of the presence of additional complicating symptoms. Leukodystrophies may manifest as late as the sixth decade and hyperintensity of cerebral white matter on magnetic resonance FLAIR images is not obligatory.

Consensus paper: management of degenerative cerebellar disorders.

Treatment of motor symptoms of degenerative cerebellar ataxia remains difficult. Yet there are recent developments that are likely to lead to significant improvements in the future. Most desirable would be a causative treatment of the underlying cerebellar disease. This is currently available only for a very small subset of cerebellar ataxias with known metabolic dysfunction. However, increasing knowledge of the pathophysiology of hereditary ataxia should lead to an increasing number of medically sensible drug trials. In this paper, data from recent drug trials in patients with recessive and dominant cerebellar ataxias will be summarized. There is consensus that up to date, no medication has been proven effective. Aminopyridines and acetazolamide are the only exception, which are beneficial in patients with episodic ataxia type 2. Aminopyridines are also effective in a subset of patients presenting with downbeat nystagmus. As such, all authors agreed that the mainstays of treatment of degenerative cerebellar ataxia are currently physiotherapy, occupational therapy, and speech therapy. For many years, well-controlled rehabilitation studies in patients with cerebellar ataxia were lacking. Data of recently published studies show that coordinative training improves motor function in both adult and juvenile patients with cerebellar degeneration. Given the well-known contribution of the cerebellum to motor learning, possible mechanisms underlying improvement will be outlined. There is consensus that evidence-based guidelines for the physiotherapy of degenerative cerebellar ataxia need to be developed. Future developments in physiotherapeutical interventions will be discussed including application of non-invasive brain stimulation.

Inventory of Non-Ataxia Signs (INAS): validation of a new clinical assessment instrument.

Although ataxia is by definition the prominent symptom of ataxia disorders, there are various neurological signs that may accompany ataxia in affected patients. Reliable and quantitative assessment of these signs is important because they contribute to disability, but may also interfere with ataxia. Therefore we devised the Inventory of Non-Ataxia Signs (INAS), a list of neurological signs that allows determining the presence and severity of non-ataxia signs in a standardized way. INAS underwent a rigorous validation procedure that involved a trial of 140 patients with spinocerebellar ataxia (SCA) for testing of inter-rater reliability and another trial of 28 SCA patients to assess short-term intra-rater reliability. In addition, data of the ongoing EUROSCA natural history study were used to determine the reproducibility, responsiveness and validity of INAS. Inter-rater reliability and short-term test-retest reliability was high, both for the total count and for most of the items. However, measures of responsiveness, such as the smallest detectable change and the clinically important change were not satisfactory. In addition, INAS did not differentiate between subjects that were subjectively stable and those that worsened in the 2-year observation period. In summary, INAS and INAS count showed good reproducibility, but unsatisfactory responsiveness. The present analysis and published data from the EUROSCA natural history study suggest that INAS is a valid measure of extracerebellar involvement in progressive ataxia disorders. As such, it is useful as a supplement to the measures of ataxia, but not as a primary outcome measure in future interventional trials.

Prospective analysis of falls in dominant ataxias.

In a previous retrospective study, we demonstrated that falls are common and often injurious in dominant spinocerebellar ataxias (SCAs) and that nonataxia features play an important role in these falls. Retrospective surveys are plagued by recall bias for the presence and details of prior falls. We therefore sought to corroborate and extend these retrospective findings by means of a prospective extension of this fall study. 113 patients with SCA1, SCA2, SCA3 or SCA6, recruited from the EuroSCA natural history study, were asked to keep a fall diary in between their annual visits to the participating centres. Additionally, patients completed a detailed questionnaire about the first three falls, to identify specific fall circumstances. Relevant disease characteristics were retrieved from the EuroSCA registry. 84.1% of patients reported at least one fall during a time period of 12 months. Fall-related injuries were common and their frequency increased with that of falls. The presence of nonataxia symptoms was associated with a higher fall frequency. This study confirms that falls are a frequent and serious complication of SCA, and that the presence of nonataxia symptoms is an important etiological factor in its occurrence.

Spinocerebellar ataxia type 1 (SCA1): new pathoanatomical and clinico-pathological insights.

AIMS: Spinocerebellar ataxia type 1 (SCA1) represents the first molecular genetically characterized autosomal dominantly inherited cerebellar ataxia and is assigned to the CAG-repeat or polyglutamine diseases. Owing to limited knowledge about SCA1 neuropathology, appropriate pathoanatomical correlates of a large variety of SCA1 disease symptoms are missing and the neuropathological basis for further morphological and experimental SCA1 studies is still fragmentary. METHODS: In the present study, we investigated for the first time serial tissue sections through the complete brains of clinically diagnosed and genetically confirmed SCA1 patients. RESULTS: Brain damage in the three SCA1 patients studied went beyond the well-known brain predilection sites of the underlying pathological process. Along with neuronal loss in the primary motor cortex, it included widespread degeneration of gray components of the basal forebrain, thalamus, brainstem and cerebellum, as well as of white matter components in the cerebellum and brainstem. It involved the motor cerebellothalamocortical and basal ganglia-thalamocortical circuits, the visual, auditory, somatosensory, oculomotor, vestibular, ingestion-related, precerebellar, basal forebrain cholinergic and midbrain dopaminergic systems. CONCLUSIONS: These findings show for the first time that the extent and severity of brain damage in SCA1 is very similar to that of clinically closely related spinocerebellar ataxias (that is, SCA2, SCA3 and SCA7). They offer suitable explanations for poorly understood SCA1 disease symptoms and will facilitate the interpretation of further morphological and experimental SCA1 studies.

Disease severity affects quality of life of hereditary spastic paraplegia patients.

BACKGROUND AND PURPOSE: Hereditary spastic paraplegia (HSP) causes progressive gait disturbance because of degeneration of the corticospinal tract. To assess its impact on Health-Related Quality of Life (HRQoL), we analyzed the correlation of HRQoL with disease severity and clinical symptoms in HSP. METHODS: HRQoL was assessed by the Short-Form 36 (SF-36) Mental and Physical Component summary scores (MCS and PCS) in 143 German patients with HSP. Disease severity was assessed by the Spastic Paraplegia Rating Scale (SPRS) and landmarks of walking ability. Patients with 'pure' or 'complicated' HSP were compared. RESULTS: Higher SPRS scores indicating higher disease severity correlated significantly with lower PCS (r = -0.63; P < 0.0005) and MCS (r = -0.38; P < 0.0005) scores. MCS and PCS were reduced in patients with 'complicated' forms compared to 'pure' HSP and with decreasing walking ability. CONCLUSION: HRQoL is substantially impaired in patients with HSP and decreases with disease severity and the presence of 'complicating' symptoms. Patients are most affected by the physical restraints of their disease, but mental health is impaired as well. HRQoL is a valid parameter in HSP that should be considered in upcoming therapeutical trials.

Pathoanatomy of cerebellar degeneration in spinocerebellar ataxia type 2 (SCA2) and type 3 (SCA3).

The cerebellum is one of the well-known targets of the pathological processes underlying spinocerebellar ataxia type 2 (SCA2) and type 3 (SCA3). Despite its pivotal role for the clinical pictures of these polyglutamine ataxias, no pathoanatomical studies of serial tissue sections through the cerebellum have been performed in SCA2 and SCA3 so far. Detailed pathoanatomical data are an important prerequisite for the identification of the initial events of the underlying disease processes of SCA2 and SCA3 and the reconstruction of its spread through the brain. In the present study, we performed a pathoanatomical investigation of serial thick tissue sections through the cerebellum of clinically diagnosed and genetically confirmed SCA2 and SCA3 patients. This study demonstrates that the cerebellar Purkinje cell layer and all four deep cerebellar nuclei consistently undergo considerable neuronal loss in SCA2 and SCA3. These cerebellar findings contribute substantially to the pathogenesis of clinical symptoms (i.e., dysarthria, intention tremor, oculomotor dysfunctions) of SCA2 and SCA3 patients and may facilitate the identification of the initial pathological alterations of the pathological processes of SCA2 and SCA3 and reconstruction of its spread through the brain.

Paroxysmal choreoathetosis/spasticity (DYT9) is caused by a GLUT1 defect.

OBJECTIVE: Mutations in SLC2A1, encoding the glucose transporter type 1 (GLUT1), cause a broad spectrum of neurologic disorders including classic GLUT1 deficiency syndrome, paroxysmal exercise-induced dyskinesia (PED, DYT18), and absence epilepsy. A large German/Dutch pedigree has formerly been described as paroxysmal choreoathetosis/spasticity (DYT9) and linked close to but not including the SLC2A1 locus on chromosome 1p. We tested whether 1) progressive spastic paraparesis, in addition to PED, as described in DYT9, and 2) autosomal dominant forms of hereditary spastic paraparesis (HSP) without PED are caused by SLC2A1 defects. METHODS: The German/Dutch family and an Australian monozygotic twin pair were clinically (re-)investigated, and 139 index cases with dominant or sporadic HSP in which relevant dominant genes were partially excluded were identified from databanks. SLC2A1 was sequenced in all cases in this observational study and the functional effects of identified sequence variations were tested in glucose uptake and protein expression assays. RESULTS: We identified causative mutations in SLC2A1 in both families, which were absent in 400 control chromosomes, cosegregated with the affection status, and decreased glucose uptake in functional assays. In the 139 index patients with HSP without paroxysmal dyskinesias, we only identified one sequence variation, which, however, neither decreased glucose uptake nor altered protein expression. CONCLUSIONS: This study shows that DYT9 and DYT18 are allelic disorders and enlarges the spectrum of GLUT1 phenotypes, now also including slowly progressive spastic paraparesis combined with PED. SLC2A1 mutations were excluded as a cause of HSP without PED in our cohort.

The natural history of spinocerebellar ataxia type 1, 2, 3, and 6: a 2-year follow-up study.

OBJECTIVE: To obtain quantitative data on the progression of the most common spinocerebellar ataxias (SCAs) and identify factors that influence their progression, we initiated the EUROSCA natural history study, a multicentric longitudinal cohort study of 526 patients with SCA1, SCA2, SCA3, or SCA6. We report the results of the 1- and 2-year follow-up visits. METHODS: As the primary outcome measure we used the Scale for the Assessment and Rating of Ataxia (SARA, 0-40), and as a secondary measure the Inventory of Non-Ataxia Symptoms (INAS, 0-16) count. RESULTS: The annual increase of the SARA score was greatest in SCA1 (2.18 ± 0.17, mean ± SE) followed by SCA3 (1.61 ± 0.12) and SCA2 (1.40 ± 0.11). SARA progression in SCA6 was slowest and nonlinear (first year: 0.35 ± 0.34, second year: 1.44 ± 0.34). Analysis of the INAS count yielded similar results. Larger expanded repeats and earlier age at onset were associated with faster SARA progression in SCA1 and SCA2. In SCA1, repeat length of the expanded allele had a similar effect on INAS progression. In SCA3, SARA progression was influenced by the disease duration at inclusion, and INAS progression was faster in females. CONCLUSIONS: Our study gives a comprehensive quantitative account of disease progression in SCA1, SCA2, SCA3, and SCA6 and identifies factors that specifically affect disease progression.