Sub-diffraction error mapping for localisation microscopy images

Assessing the quality of localisation microscopy images is highly challenging due to the difficulty in reliably detecting errors in experimental data. The most common failure modes are the biases and errors produced by the localisation algorithm when there is emitter overlap. Also known as the high density or crowded field condition, significant emitter overlap is normally unavoidable in live cell imaging. Here we use Haar wavelet kernel analysis (HAWK), a localisation microscopy data analysis method which is known to produce results without bias, to generate a reference image. This enables mapping and quantification of reconstruction bias and artefacts common in all but low emitter density data. By avoiding comparisons involving intensity information, we can map structural artefacts in a way that is not adversely influenced by nonlinearity in the localisation algorithm. The HAWK Method for the Assessment of Nanoscopy (HAWKMAN) is a general approach which allows for the reliability of localisation information to be assessed

The spectrum of neurodevelopmental, neuromuscular and neurodegenerative disorders due to defective autophagy

Primary dysfunction of autophagy due to Mendelian defects affecting core components of the autophagy machinery or closely related proteins have recently emerged as an important cause of genetic disease. This novel group of human disorders may present throughout life and comprises severe early-onset neurodevelopmental and more common adult-onset neurodegenerative disorders. Early-onset (or congenital) disorders of autophagy often share a recognizable "clinical signature," including variable combinations of neurological, neuromuscular and multisystem manifestations. Structural CNS abnormalities, cerebellar involvement, spasticity and peripheral nerve pathology are prominent neurological features, indicating a specific vulnerability of certain neuronal populations to autophagic disturbance. A typically biphasic disease course of late-onset neurodegeneration occurring on the background of a neurodevelopmental disorder further supports a role of autophagy in both neuronal development and maintenance. Additionally, an associated myopathy has been characterized in several conditions. The differential diagnosis comprises a wide range of other multisystem disorders, including mitochondrial, glycogen and lysosomal storage disorders, as well as ciliopathies, glycosylation and vesicular trafficking defects. The clinical overlap between the congenital disorders of autophagy and these conditions reflects the multiple roles of the proteins and/or emerging molecular connections between the pathways implicated and suggests an exciting area for future research. Therapy development for congenital disorders of autophagy is still in its infancy but may result in the identification of molecules that target autophagy more specifically than currently available compounds. The close connection with adult-onset neurodegenerative disorders highlights the relevance of research into rare early-onset neurodevelopmental conditions for much more common, age-related human diseases.Peer reviewe

Out-of-Frame Mutations in ACTN2 Last Exon Cause a Dominant Distal Myopathy With Facial Weakness

Background and Objectives To clinically, genetically, and histopathologically characterize patients presenting with an unusual combination of distal myopathy and facial weakness, without involvement of upper limb or shoulder girdle muscles. Methods Two families with a novel form of actininopathy were identified. Patients had been followed up over 10 years. Their molecular genetic diagnosis was not clear after extensive investigations, including analysis of candidate genes and FSHD1-related D4Z4 repeats. Results Patients shared a similar clinical phenotype and a common pattern of muscle involvement. They presented with a very slowly progressive myopathy involving anterior lower leg and facial muscles. Muscle MRI finding showed complete fat replacement of anterolateral compartment muscles of the lower legs with variable involvement of soleus and gastrocnemius but sparing thigh muscles. Muscle biopsy showed internalized nuclei, myofibrillar disorganization, and rimmed vacuoles. High-throughput sequencing identified in each proband a heterozygous single nucleotide deletion (c.2558del and c.2567del) in the last exon of the ACTN2 gene. The deletions are predicted to lead to a novel but unstructured slightly extended C-terminal amino acid sequence. Discussion Our findings indicate an unusual form of actininopathy with specific molecular and clinical features. Actininopathy should be considered in the differential diagnosis of distal myopathy combined with facial weakness.Peer reviewe

Epigenetic changes as a common trigger of muscle weakness in congenital myopathies

Congenital myopathies are genetically and clinically heterogeneous conditions causing severe muscle weakness, and mutations in the ryanodine receptor gene (RYR1) represent the most frequent cause of these conditions. A common feature of diseases caused by recessive RYR1 mutations is a decrease of ryanodine receptor 1 protein content in muscle. The aim of the present investigation was to gain mechanistic insight into the causes of this reduced ryanodine receptor 1. We found that muscle biopsies of patients with recessive RYR1 mutations exhibit decreased expression of muscle-specific microRNAs, increased DNA methylation and increased expression of class II histone deacetylases. Transgenic mouse muscle fibres over-expressing HDAC-4/HDAC-5 exhibited decreased expression of RYR1 and of muscle-specific miRNAs, whereas acute knock-down of RYR1 in mouse muscle fibres by siRNA caused up-regulation of HDAC-4/HDAC-5. Intriguingly, increased class II HDAC expression and decreased ryanodine receptor protein and miRNAs expression were also observed in muscles of patients with nemaline myopathy, another congenital neuromuscular disorder. Our results indicate that a common pathophysiological pathway caused by epigenetic changes is activated in some forms of congenital neuromuscular disorder

Insights into the Role of a Cardiomyopathy-Causing Genetic Variant in ACTN2

Pathogenic variants in ACTN2, coding for alpha-actinin 2, are known to be rare causes of Hyper-trophic Cardiomyopathy. However, little is known about the underlying disease mechanisms. Adult heterozygous mice carrying the Actn2 p.Met228Thr variant were phenotyped by echocar-diography. For homozygous mice, viable E15.5 embryonic hearts were analysed by High Reso-lution Episcopic Microscopy and wholemount staining, complemented by unbiased proteomics, qPCR and Western blotting. Heterozygous Actn2 p.Met228Thr mice have no overt phenotype. Only mature males show molecular parameters indicative of cardiomyopathy. By contrast, the variant is embryonically lethal in the homozygous setting and E15.5 hearts show multiple morphological abnormalities. Molecular analyses, including unbiased proteomics, identified quantitative abnormalities in sarcomeric parameters, cell cycle defects and mitochondrial dys-function. The mutant alpha-actinin protein is found to be destabilised, associated with increased activity of the ubiquitin-proteosomal system. This missense variant in alpha-actinin renders the protein less stable. In response, the ubiquitin-proteosomal system is activated; a mechanism which has been implicated in cardiomyopathies previously. In parallel, lack of functional al-pha-actinin is thought to cause energetic defects through mitochondrial dysfunction. This seems, together with cell cycle defects, the likely cause of death of the embryos. The defects also have wide-ranging morphological consequences

The spectrum of neurodevelopmental, neuromuscular and neurodegenerative disorders due to defective autophagy

Primary dysfunction of autophagy due to Mendelian defects affecting core components of the autophagy machinery or closely related proteins have recently emerged as an important cause of genetic disease. This novel group of human disorders may present throughout life and comprises severe early-onset neurodevelopmental and more common adult-onset neurodegenerative disorders. Early-onset (or congenital) disorders of autophagy often share a recognizable "clinical signature," including variable combinations of neurological, neuromuscular and multisystem manifestations. Structural CNS abnormalities, cerebellar involvement, spasticity and peripheral nerve pathology are prominent neurological features, indicating a specific vulnerability of certain neuronal populations to autophagic disturbance. A typically biphasic disease course of late-onset neurodegeneration occurring on the background of a neurodevelopmental disorder further supports a role of autophagy in both neuronal development and maintenance. In addition, an associated myopathy has been characterized in several conditions. The differential diagnosis comprises a wide range of other multisystem disorders, including mitochondrial, glycogen and lysosomal storage disorders, as well as ciliopathies, glycosylation and vesicular trafficking defects. The clinical overlap between the congenital disorders of autophagy and these conditions reflects the multiple roles of the proteins and/or emerging molecular connections between the pathways implicated and suggests an exciting area for future research. Therapy development for congenital disorders of autophagy is still in its infancy but may result in the identification of molecules that target autophagy more specifically than currently available compounds. The close connection with adult-onset neurodegenerative disorders highlights the relevance of research into rare early-onset neurodevelopmental conditions for much more common, age-related human diseases

Phosphoregulation of the Titin-cap Protein Telethonin in Cardiac Myocytes

Telethonin (also known as titin-cap or t-cap) is a muscle-specific protein whose mutation is associated with cardiac and skeletal myopathies through unknown mechanisms. Our previous work identified cardiac telethonin as an interaction partner for the protein kinase D catalytic domain. In this study, kinase assays used in conjunction with MS and site-directed mutagenesis confirmed telethonin as a substrate for protein kinase D and Ca(2+)/calmodulin-dependent kinase II in vitro and identified Ser-157 and Ser-161 as the phosphorylation sites. Phosphate affinity electrophoresis and MS revealed endogenous telethonin to exist in a constitutively bis-phosphorylated form in isolated adult rat ventricular myocytes and in mouse and rat ventricular myocardium. Following heterologous expression in myocytes by adenoviral gene transfer, wild-type telethonin became bis-phosphorylated, whereas S157A/S161A telethonin remained non-phosphorylated. Nevertheless, both proteins localized predominantly to the sarcomeric Z-disc, where they partially replaced endogenous telethonin. Such partial replacement with S157A/S161A telethonin disrupted transverse tubule organization and prolonged the time to peak of the intracellular Ca(2+) transient and increased its variance. These data reveal, for the first time, that cardiac telethonin is constitutively bis-phosphorylated and suggest that such phosphorylation is critical for normal telethonin function, which may include maintenance of transverse tubule organization and intracellular Ca(2+) transients