Transduction of E2F-1 TAT fusion proteins represses expression of hTERT in primary ductal breast carcinoma cell lines

<p>Abstract</p> <p>Background</p> <p>Telomerase expression is detectable in 81–95% of breast carcinomas and may serve as a therapeutic target. The objective of this study was to investigate repression of telomerase activity in primary ductal breast cancer cells through transcriptional regulation of the catalytic subunit hTERT. We hypothesized that inhibition of telomerase expression could be achieved via Tat mediated protein transduction of the repressor protein E2F-1.</p> <p>Methods</p> <p>Protein purification techniques were refined to yield biologically active Tat fusion proteins (TFPs) capable of transducing the breast cancer cell lines HCC1937 and HCC1599. Cell lines were treated with wildtype E2F-1 (E2F-1/TatHA), mutant E2F-1 (E132/TatHA) and a control Tat peptide (TatHA) for 24 hours. Total RNA was isolated from treated cells, reverse transcribed and fold changes in gene expression for hTERT determined via real-time RT-qPCR.</p> <p>Results</p> <p>Significant repression of the catalytic subunit of telomerase (hTERT) was present in both HCC1937 and HCC1599 cells following treatment with E2F-1/TatHA. In HCC1937 cells, hTERT was repressed 3.5-fold by E2F-1/TatHA in comparison to E132/TatHA (p < 0.0012) and the TatHA peptide controls (p < 0.0024). In HCC1599 cells, hTERT was also repressed with E2F-1/TatHA treatment by 4.0-fold when compared to the E132/TatHA control (p < 0.0001). A slightly lower hTERT repression of 3.3-fold was observed with E2F-1/TatHA in the HCC1599 cells when compared to the TatHA control (p < 0.0001).</p> <p>Conclusion</p> <p>These results suggest that transduction of E2F-1/TatHA fusion proteins in vitro is an effective repressor of hTERT expression in the primary ductal breast cancer cell lines HCC1937 and HCC1599.</p

In vivo impact of presynaptic calcium channel dysfunction on motor axons in episodic ataxia type 2

Ion channel dysfunction causes a range of neurological disorders by altering transmembrane ion fluxes, neuronal or muscle excitability, and neurotransmitter release. Genetic neuronal channelopathies affecting peripheral axons provide a unique opportunity to examine the impact of dysfunction of a single channel subtype in detail in vivo. Episodic ataxia type 2 is caused by mutations in CACNA1A, which encodes the pore-forming subunit of the neuronal voltage-gated calcium channel Cav2.1. In peripheral motor axons, this channel is highly expressed at the presynaptic neuromuscular junction where it contributes to action potential-evoked neurotransmitter release, but it is not expressed mid-axon or thought to contribute to action potential generation. Eight patients from five families with genetically confirmed episodic ataxia type 2 underwent neurophysiological assessment to determine whether axonal excitability was normal and, if not, whether changes could be explained by Cav2.1 dysfunction. New mutations in the CACNA1A gene were identified in two families. Nerve conduction studies were normal, but increased jitter in single-fibre EMG studies indicated unstable neuromuscular transmission in two patients. Excitability properties of median motor axons were compared with those in 30 age-matched healthy control subjects. All patients had similar excitability abnormalities, including a high electrical threshold and increased responses to hyperpolarizing (P < 0.00007) and depolarizing currents (P < 0.001) in threshold electrotonus. In the recovery cycle, refractoriness (P < 0.0002) and superexcitability (P < 0.006) were increased. Cav2.1 dysfunction in episodic ataxia type 2 thus has unexpected effects on axon excitability, which may reflect an indirect effect of abnormal calcium current fluxes during development

Mitochondrial DNA analysis from exome sequencing data improves the diagnostic yield in neurological diseases

A rapidly expanding catalogue of neurogenetic disorders has encouraged a diagnostic shift towards early clinical whole exome sequencing (WES). Adult primary mitochondrial diseases (PMDs) frequently exhibit neurological manifestations that overlap with other nervous system disorders. However, mitochondrial DNA (mtDNA) is not routinely analyzed in standard clinical WES bioinformatic pipelines. We reanalyzed 11,424 exomes, enriched with neurological diseases, for pathogenic mtDNA variants. Twenty‐four different mtDNA mutations were detected in 64 exomes, 11 of which were considered disease causing based on the associated clinical phenotypes. These findings highlight the diagnostic uplifts gained by analyzing mtDNA from WES data in neurological diseases

NfL and pNfH are increased in Friedreich's ataxia

Objective: To assess neurofilaments as neurodegenerative biomarkers in serum of patients with Friedreich’s ataxia. / Methods: Single molecule array measurements of neurofilament light (NfL) and heavy chain (pNfH) in 99 patients with genetically confirmed Friedreich’s ataxia. Correlation of NfL/pNfH serum levels with disease severity, disease duration, age, age at onset, and GAA repeat length. / Results: Median serum levels of NfL were 21.2 pg/ml (range 3.6–49.3) in controls and 26.1 pg/ml (0–78.1) in Friedreich’s ataxia (p = 0.002). pNfH levels were 23.5 pg/ml (13.3–43.3) in controls and 92 pg/ml (3.1–303) in Friedreich’s ataxia (p = 0.0004). NfL levels were significantly increased in younger patients (age 16–31 years, p < 0.001) and patients aged 32–47 years (p = 0.008), but not in patients of age 48 years and older (p = 0.41). In a longitudinal assessment, there was no difference in NfL levels in 14 patients with repeated sampling 2 years after baseline measurement. Levels of NfL correlated inversely with GAA1 repeat length (r = − 0.24, p = 0.02) but not with disease severity (r = − 0.13, p = 0.22), disease duration (r = − 0.06, p = 0.53), or age at onset (r = 0.05, p = 0.62). / Conclusion: Serum levels of NfL and pNfH are elevated in Friedreich’s ataxia, but differences to healthy controls decrease with increasing age. Long-term longitudinal data are required to explore whether this reflects a selection bias from early death of more severely affected individuals or a slowing down of the neurodegenerative process with age. In a pilot study over 2 years of follow-up—a period relevant for biomarkers indicating treatment effects—we found NfL levels to be stable

Toward allele-specific targeting therapy and pharmacodynamic marker for spinocerebellar ataxia type 3

Spinocerebellar ataxia type 3 (SCA3), caused by a CAG repeat expansion in the ataxin-3 gene (ATXN3), is characterized by neuronal polyglutamine (polyQ) ATXN3 protein aggregates. Although there is no cure for SCA3, gene-silencing approaches to reduce toxic polyQ ATXN3 showed promise in preclinical models. However, a major limitation in translating putative treatments for this rare disease to the clinic is the lack of pharmacodynamic markers for use in clinical trials. Here, we developed an immunoassay that readily detects polyQ ATXN3 proteins in human biological fluids and discriminates patients with SCA3 from healthy controls and individuals with other ataxias. We show that polyQ ATXN3 serves as a marker of target engagement in human fibroblasts, which may bode well for its use in clinical trials. Last, we identified a single-nucleotide polymorphism that strongly associates with the expanded allele, thus providing an exciting drug target to abrogate detrimental events initiated by mutant ATXN3. Gene-silencing strategies for several repeat diseases are well under way, and our results are expected to improve clinical trial preparedness for SCA3 therapies

SCAview: an Intuitive Visual Approach to the Integrative Analysis of Clinical Data in Spinocerebellar Ataxias

With SCAview, we present a prompt and comprehensive tool that enables scientists to browse large datasets of the most common spinocerebellar ataxias intuitively and without technical effort. Basic concept is a visualization of data, with a graphical handling and filtering to select and define subgroups and their comparison. Several plot types to visualize all data points resulting from the selected attributes are provided. The underlying synthetic cohort is based on clinical data from five different European and US longitudinal multicenter cohorts in spinocerebellar ataxia type 1, 2, 3, and 6 (SCA1, 2, 3, and 6) comprising > 1400 patients with overall > 5500 visits. First, we developed a common data model to integrate the clinical, demographic, and characterizing data of each source cohort. Second, the available datasets from each cohort were mapped onto the data model. Third, we created a synthetic cohort based on the cleaned dataset. With SCAview, we demonstrate the feasibility of mapping cohort data from different sources onto a common data model. The resulting browser-based visualization tool with a thoroughly graphical handling of the data offers researchers the unique possibility to visualize relationships and distributions of clinical data, to define subgroups and to further investigate them without any technical effort. Access to SCAview can be requested via the Ataxia Global Initiative and is free of charge

Survival in patients with spinocerebellar ataxia types 1, 2, 3, and 6 (EUROSCA): a longitudinal cohort study

BACKGROUND: Spinocerebellar ataxias are dominantly inherited progressive ataxia disorders that can lead to premature death. We aimed to study the overall survival of patients with the most common spinocerebellar ataxias (SCA1, SCA2, SCA3, and SCA6) and to identify the strongest contributing predictors that affect survival. METHODS: In this longitudinal cohort study (EUROSCA), we enrolled men and women, aged 18 years or older, from 17 ataxia referral centres in ten European countries; participants had positive genetic test results for SCA1, SCA2, SCA3, or SCA6 and progressive, otherwise unexplained, ataxias. Survival was defined as the time from enrolment to death for any reason. We used the Cox regression model adjusted for age at baseline to analyse survival. We used prognostic factors with a p value less than 0·05 from a multivariate model to build nomograms and assessed their performance based on discrimination and calibration. The EUROSCA study is registered with ClinicalTrials.gov, number NCT02440763. FINDINGS: Between July 1, 2005, and Aug 31, 2006, 525 patients with SCA1 (n=117), SCA2 (n=162), SCA3 (n=139), or SCA6 (n=107) were enrolled and followed up. The 10-year survival rate was 57% (95% CI 47–69) for SCA1, 74% (67–81) for SCA2, 73% (65–82) for SCA3, and 87% (80–94) for SCA6. Factors associated with shorter survival were: dysphagia (hazard ratio 4·52, 95% CI 1·83–11·15) and a higher value for the Scale for the Assessment and Rating of Ataxia (SARA) score (1·26, 1·19–1·33) for patients with SCA1; older age at inclusion (1·04, 1·01–1·08), longer CAG repeat length (1·16, 1·03–1·31), and higher SARA score (1·15, 1·10–1·20) for patients with SCA2; older age at inclusion (1·44, 1·20–1·74), dystonia (2·65, 1·21–5·53), higher SARA score (1·26, 1·17–1·35), and negative interaction between CAG and age at inclusion (0·994, 0·991–0·997) for patients with SCA3; and higher SARA score (1·17, 1·08–1·27) for patients with SCA6. The nomogram-predicted probability of 10-year survival showed good discrimination (c index 0·905 [SD 0·027] for SCA1, 0·822 [0·032] for SCA2, 0·891 [0·021] for SCA3, and 0·825 [0·054] for SCA6). INTERPRETATION: Our study provides quantitative data on the survival of patients with the most common spinocerebellar ataxias, based on a long follow-up period. These results have implications for the design of future interventional studies of spinocerebellar ataxias; for example, the prognostic survival nomogram could be useful for selection and stratification of patients. Our findings need validation in an external population before they can be used to counsel patients and their families. FUNDING: European Union 6th Framework programme, German Ministry of Education and Research, Polish Ministry of Scientific Research and Information Technology, European Union 7th Framework programme, and Fondation pour la Recherche Médicale

Mitochondrial DNA Analysis from Exome Sequencing Data Improves Diagnostic Yield in Neurological Diseases

A rapidly expanding catalog of neurogenetic disorders has encouraged a diagnostic shift towards early clinical whole exome sequencing (WES). Adult primary mitochondrial diseases (PMDs) frequently exhibit neurological manifestations that overlap with other nervous system disorders. However, mitochondrial DNA (mtDNA) is not routinely analyzed in standard clinical WES bioinformatic pipelines. We reanalyzed 11,424 exomes, enriched with neurological diseases, for pathogenic mtDNA variants. Twenty‐four different mtDNA mutations were detected in 64 exomes, 11 of which were considered disease causing based on the associated clinical phenotypes. These findings highlight the diagnostic uplifts gained by analyzing mtDNA from WES data in neurological diseases