Genetic and phenotypic characterization of complex hereditary spastic paraplegia.

The hereditary spastic paraplegias are a heterogeneous group of degenerative disorders that are clinically classified as either pure with predominant lower limb spasticity, or complex where spastic paraplegia is complicated with additional neurological features, and are inherited in autosomal dominant, autosomal recessive or X-linked patterns. Genetic defects have been identified in over 40 different genes, with more than 70 loci in total. Complex recessive spastic paraplegias have in the past been frequently associated with mutations in SPG11 (spatacsin), ZFYVE26/SPG15, SPG7 (paraplegin) and a handful of other rare genes, but many cases remain genetically undefined. The overlap with other neurodegenerative disorders has been implied in a small number of reports, but not in larger disease series. This deficiency has been largely due to the lack of suitable high throughput techniques to investigate the genetic basis of disease, but the recent availability of next generation sequencing can facilitate the identification of disease-causing mutations even in extremely heterogeneous disorders. We investigated a series of 97 index cases with complex spastic paraplegia referred to a tertiary referral neurology centre in London for diagnosis or management. The mean age of onset was 16 years (range 3 to 39). The SPG11 gene was first analysed, revealing homozygous or compound heterozygous mutations in 30/97 (30.9%) of probands, the largest SPG11 series reported to date, and by far the most common cause of complex spastic paraplegia in the UK, with severe and progressive clinical features and other neurological manifestations, linked with magnetic resonance imaging defects. Given the high frequency of SPG11 mutations, we studied the autophagic response to starvation in eight affected SPG11 cases and control fibroblast cell lines, but in our restricted study we did not observe correlations between disease status and autophagic or lysosomal markers. In the remaining cases, next generation sequencing was carried out revealing variants in a number of other known complex spastic paraplegia genes, including five in SPG7 (5/97), four in FA2H (also known as SPG35) (4/97) and two in ZFYVE26/SPG15 Variants were identified in genes usually associated with pure spastic paraplegia and also in the Parkinson's disease-associated gene ATP13A2, neuronal ceroid lipofuscinosis gene TPP1 and the hereditary motor and sensory neuropathy DNMT1 gene, highlighting the genetic heterogeneity of spastic paraplegia. No plausible genetic cause was identified in 51% of probands, likely indicating the existence of as yet unidentified genes.

The blink reflex recovery cycle differs between essential and presumed psychogenic blepharospasm.

BACKGROUND: Psychogenic blepharospasm is difficult to distinguish clinically from benign essential blepharospasm (BEB). The blink reflex recovery cycle measures the excitability of human brainstem interneurons and is abnormal in BEB. We wished to study the blink reflex recovery cycle in patients with atypical (presumed psychogenic) blepharospasm (AB). METHODS: This was a prospective data collection study investigating the R2 blink reflex recovery cycle at interstimulus intervals (ISI) of 200, 300, 500, 1,000, and 3,000 msec in 10 patients with BEB, 9 patients with AB, and 9 healthy controls. All patients had spasm of the orbicularis oculi muscles. To compare individual patients, an R2 recovery index was calculated as average of the recovery values at ISIs of 200, 300, and 500 msec, with the upper limit of normal defined as mean (control group) + 2 SD. RESULTS: The R2 recovery cycle was significantly disinhibited in patients with BEB, whereas patients with AB did not differ from controls on a group level. The upper limit of normal for the R2 recovery index was 61%. The R2 index was abnormal in 9 out of 10 patients with BEB and in none of the patients with AB. CONCLUSIONS: A normal blink reflex recovery cycle indicates normal brainstem interneuron excitability. Assessment of the R2 recovery cycle may provide a useful diagnostic tool to distinguish patients with psychogenic blepharospasm from BEB and is worthy of further study.

Analysis of the factors influencing the cardiac phenotype in Friedreich's ataxia.

Friedreich's ataxia (FRDA) has been associated with both cardiac hypertrophy and to a lesser degree dilated cardiomyopathy. We have conducted a cross sectional magnetic resonance imaging (MRI) study of 25 patients with clinically and genetically confirmed FRDA and 24 healthy controls to analyse how disease parameters influence cardiac features in FRDA. MR cine imaging in the long and short axis planes was performed alongside clinical assessments. LV mass was most pronounced in FRDA patients with a larger genetic mutation (GAA1 repeats >600), earlier age of onset (<16years) and a shorter disease duration (<15 years). LV mass decreased with longer disease duration (>15 years), and independent of GAA1 repeat size and age of onset, suggesting cardiac thinning occurred with prolonged disease. Heart function was lower in patients with larger GAA1 repeat number and longer disease duration. Consequently, cardiac hypertrophy was more marked in FRDA patients with a larger GAA1 repeat number and younger age of onset, while prolonged disease duration was associated with lower LV mass and decreased heart function. It is important not only to understand the biochemical basis for these cardiac changes but also allow for these changes when assessing the effect of treatment of FRDA patients.

Protection against paraquat and A53T alpha-synuclein toxicity by cabergoline is partially mediated by dopamine receptors.

Both genetic and environmental factors are thought to be involved in the aetiology of Parkinson's disease (PD). Oxidative damage, mitochondrial and proteasomal dysfunction, and inflammatory change are considered to participate in PD pathogenesis. Dopamine agonists are used in the symptomatic treatment of PD but attention has recently also been focussed on their potential for use in slowing disease progression. We have studied the protective actions of the D2 dopamine agonist cabergoline in toxin (paraquat) and genetic (wild-type and mutant [A53T] alpha-synuclein) models of PD using SHSY-5Y cells. Cabergoline increased glutathione content, reduced free radical production and caspase-3 activation, increased mitochondrial membrane potential and ameliorated cell death in SHSY-5Y cells exposed to paraquat and this action was inhibited in part by D2 receptor blockade. Cabergoline also reduced the toxicity of wild-type and mutant alpha-synuclein expression following paraquat exposure by similar mechanisms. These results confirm the protective action of cabergoline in reducing cell death in two separate genetic and environmental model systems of PD.

Coenzyme Q10 and vitamin E deficiency in Friedreich's ataxia: predictor of efficacy of vitamin E and coenzyme Q10 therapy.

BACKGROUND AND PURPOSE: A pilot study of high dose coenzyme Q(10) (CoQ(10))/vitamin E therapy in Friedreich's ataxia (FRDA) patients resulted in significant clinical improvements in most patients. This study investigated the potential for this treatment to modify clinical progression in FRDA in a randomized double blind trial. METHODS: Fifty FRDA patients were randomly divided into high or low dose CoQ(10)/ vitamin E groups. The change in International Co-operative Ataxia Ratings Scale (ICARS) was assessed over 2 years as the primary end-point. A post hoc analysis was made using cross-sectional data. RESULTS: At baseline serum CoQ(10) and vitamin E levels were significantly decreased in the FRDA patients (P < 0.001). During the trial CoQ(10) and vitamin E levels significantly increased in both groups (P < 0.01). The primary and secondary end-points were not significantly different between the therapy groups. When compared to cross-sectional data 49% of all patients demonstrated improved ICARS scores. This responder group had significantly lower baseline serum CoQ(10) levels. CONCLUSIONS: A high proportion of FRDA patients have a decreased serum CoQ(10) level which was the best predictor of a positive clinical response to CoQ(10)/vitamin E therapy. Low and high dose CoQ(10)/vitamin E therapies were equally effective in improving ICARS scores.

International Cooperative Ataxia Rating Scale (ICARS): appropriate for studies of Friedreich's ataxia?

Clinicians require scientifically rigorous, clinically meaningful rating scales to evaluate the health impact of disease and treatment that cannot be measured using conventional laboratory instruments. This study evaluated the psychometric properties of the International Cooperative Ataxia Rating Scale (ICARS), a commonly used clinician-rated measure, in Friedreich's ataxia (FRDA). People with confirmed FRDA were assessed by using the ICARS. Two assumptions of its measurement model were tested: the legitimacy of reporting ICARS scores in FRDA, and the acceptability, reliability, and validity of total and subscale scores. Seventy-seven people with FRDA were assessed. The ICARS total score effectively satisfied all psychometric criteria tested. The posture and gait disturbances subscale also performed well. The other three subscales did not pass standard criteria for tests of scaling assumptions, reliability, and validity. This small study recommends only the use of the ICARS total score as a measure of FRDA. However, the extent to which this score quantifies the true extent of FRDA remains uncertain as our validity testing was limited, partly by the lack of appropriate validating measures. Further validity testing, and examination of responsiveness, is required before the ICARS can be recommended as an outcome measure for treatment trials of FDRA.

G209A mutant alpha synuclein expression specifically enhances dopamine induced oxidative damage.

Alpha synuclein protein may play an important role in familial and sporadic Parkinson's disease pathology. We have induced G209A mutant or wild-type alpha-synuclein expression in stable HEK293 cell models to determine if this influences markers of oxidative stress and damage under normal conditions or in the presence of dopamine or paraquat. Induced wild-type or mutant alpha-synuclein expression alone had no effect upon levels of oxidative stress or damage, as measured by glutathione levels or aconitase activity. Both wild-type and mutant alpha-synuclein expression decreased the oxidative damage induced by paraquat, although the protection was less marked with mutant alpha-synuclein expression. This suggests that alpha-synuclein expression may either have anti-oxidant properties or may upregulate cellular antioxidant levels, a function that was diminished by the G209A mutation. However, mutant but not wild-type alpha-synuclein expression specifically enhanced dopamine associated oxidative damage. Non-expressing cells treated with reserpine to inhibit the vesicular monoamine compartmentalisation produced similar results. However, consistent with the hypothesis that mutant alpha-synuclein disrupts vesicular dopamine compartmentalization, this effect was diminished in cells expressing mutant alpha-synuclein. This may result in increased dopamine metabolism and cause selective oxidative damage to dopaminergic cells.

Differences in toxicity of the catechol-O-methyl transferase inhibitors, tolcapone and entacapone to cultured human neuroblastoma cells.

Tolcapone and entacapone are catechol-O-methyltransferase (COMT) inhibitors used as adjuncts to levodopa in the treatment of Parkinson's disease (PD). The use of tolcapone has been limited by its hepatotoxicity, the cause of which remains uncertain. Tolcapone compound is an uncoupler of mitochondrial respiration in isolated mitochondria and this action may be relevant to its effect on liver function. We have examined the actions of COMT inhibitors on cultured cells, comparing them with those of the classical uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), in order to provide insight into their mechanism of potential toxicity. Tolcapone and FCCP were shown to be toxic to human neuroblastoma SH-SY5Y cells and caused a profound reduction in ATP synthesis. Entacapone was not toxic to SH-SY5Y. Tolcapone and FCCP were shown to be equally toxic to cells depleted of mtDNA and thus devoid of a functional respiratory chain. This study demonstrates that tolcapone markedly inhibits ATP synthesis in cultured cells mirroring the effects of a classical uncoupler. However its toxicity may also involve a mechanism independent of its effects upon oxidative phosphorylation.

Development of a flow cytometric assay to quantify lymphocyte adhesion to cytokine-stimulated human endothelial and biliary epithelial cells.

The adhesive interaction between T lymphocytes and parenchymal cells is of importance for many processes of the cellular immune response. This adhesion is regulated by the activation status of the T cell and by cytokines in the microenvironment which can alter adhesion molecule expression by endothelial and epithelial cells. In this study results from an isotopic adhesion assay were compared with those from a flow cytometric assay in order to determine which was most appropriate for the investigation of lymphocyte adhesion to human umbilical vein endothelial cells (HUVEC) and intrahepatic biliary epithelial cells (HIBEC). Treatment of both these cell types with the proinflammatory cytokines interferon-gamma (IFN-gamma) or tumour necrosis factor-alpha (TNF-alpha) significantly upregulated expression of intercellular adhesion molecule-1 (ICAM-1). Treatment with TNF-alpha also induced endothelial cells to express vascular cell adhesion molecule-1 (VCAM-1). The isotopic assay demonstrated increased adhesion of lymphoblasts to HUVEC which had been stimulated with cytokines for 15 h but failed to detect major changes in adhesion following 72 h of cytokine treatment of HUVEC or HIBEC. However, the flow cytometric assay reproducibly demonstrated increased adhesion following cytokine treatment for both these time periods; these increases corresponded with the changes in adhesion molecule expression by cytokine-stimulated HUVEC and HIBEC targets. The differences in apparent adhesion measured by the two assays after cytokine stimulation for 72 h may be explained by cytokine-induced changes in the morphology and confluency of cultured cells. Results of the isotopic assay are proportional to the total number of lymphoid cells bound by the cultured target cells and will be distorted by changes in effective target cell area. The flow cytometric assay measures the mean number of lymphoid cells bound by each target cell and is independent of the total binding area. It is concluded that the flow cytometric assay is more suitable than the isotopic technique for following time-dependent changes in the adhesion of leukocytes to cytokine-stimulated target cells.