Systematic and quantitative analysis of stop codon readthrough in Rett syndrome nonsense mutations.

Rett syndrome (RTT) is a neurodevelopmental disorder resulting from genetic mutations in the methyl CpG binding protein 2 (MeCP2) gene. Specifically, around 35% of RTT patients harbor premature termination codons (PTCs) within the MeCP2 gene due to nonsense mutations. A promising therapeutic avenue for these individuals involves the use of aminoglycosides, which stimulate translational readthrough (TR) by causing stop codons to be interpreted as sense codons. However, the effectiveness of this treatment depends on several factors, including the type of stop codon and the surrounding nucleotides, collectively referred to as the stop codon context (SCC). Here, we develop a high-content reporter system to precisely measure TR efficiency at different SCCs, assess the recovery of the full-length MeCP2 protein, and evaluate its subcellular localization. We have conducted a comprehensive investigation into the intricate relationship between SCC characteristics and TR induction, examining a total of 14 pathogenic MeCP2 nonsense mutations with the aim to advance the prospects of personalized therapy for individuals with RTT. Our results demonstrate that TR induction can successfully restore full-length MeCP2 protein, albeit to varying degrees, contingent upon the SCC and the specific position of the PTC within the MeCP2 mRNA. TR induction can lead to the re-establishment of nuclear localization of MeCP2, indicating the potential restoration of protein functionality. In summary, our findings underscore the significance of SCC-specific approaches in the development of tailored therapies for RTT. By unraveling the relationship between SCC and TR therapy, we pave the way for personalized, individualized treatment strategies that hold promise for improving the lives of individuals affected by this debilitating neurodevelopmental disorder. KEY MESSAGES: The efficiency of readthrough induction at MeCP2 premature termination codons strongly depends on the stop codon context. The position of the premature termination codon on the transcript influences the readthrough inducibility. A new high-content dual reporter assay facilitates the measurement and prediction of readthrough efficiency of specific nucleotide stop contexts. Readthrough induction results in the recovery of full-length MeCP2 and its re-localization to the nucleus. MeCP2 requires only one of its annotated nuclear localization signals.

Correction: Nonsense mutation suppression is enhanced by targeting different stages of the protein synthesis process.

[This corrects the article DOI: 10.1371/journal.pbio.3002355.].

Tissue-specific roles of peroxisomes revealed by expression meta-analysis.

Peroxisomes are primarily studied in the brain, kidney, and liver due to the conspicuous tissue-specific pathology of peroxisomal biogenesis disorders. In contrast, little is known about the role of peroxisomes in other tissues such as the heart. In this meta-analysis, we explore mitochondrial and peroxisomal gene expression on RNA and protein levels in the brain, heart, kidney, and liver, focusing on lipid metabolism. Further, we evaluate a potential developmental and heart region-dependent specificity of our gene set. We find marginal expression of the enzymes for peroxisomal fatty acid oxidation in cardiac tissue in comparison to the liver or cardiac mitochondrial ß-oxidation. However, the expression of peroxisome biogenesis proteins in the heart is similar to other tissues despite low levels of peroxisomal fatty acid oxidation. Strikingly, peroxisomal targeting signal type 2-containing factors and plasmalogen biosynthesis appear to play a fundamental role in explaining the essential protective and supporting functions of cardiac peroxisomes.

Nonsense mutation suppression is enhanced by targeting different stages of the protein synthesis process.

The introduction of premature termination codons (PTCs), as a result of splicing defects, insertions, deletions, or point mutations (also termed nonsense mutations), lead to numerous genetic diseases, ranging from rare neuro-metabolic disorders to relatively common inheritable cancer syndromes and muscular dystrophies. Over the years, a large number of studies have demonstrated that certain antibiotics and other synthetic molecules can act as PTC suppressors by inducing readthrough of nonsense mutations, thereby restoring the expression of full-length proteins. Unfortunately, most PTC readthrough-inducing agents are toxic, have limited effects, and cannot be used for therapeutic purposes. Thus, further efforts are required to improve the clinical outcome of nonsense mutation suppressors. Here, by focusing on enhancing readthrough of pathogenic nonsense mutations in the adenomatous polyposis coli (APC) tumor suppressor gene, we show that disturbing the protein translation initiation complex, as well as targeting other stages of the protein translation machinery, enhances both antibiotic and non-antibiotic-mediated readthrough of nonsense mutations. These findings strongly increase our understanding of the mechanisms involved in nonsense mutation readthrough and facilitate the development of novel therapeutic targets for nonsense suppression to restore protein expression from a large variety of disease-causing mutated transcripts.

Live-Cell Imaging of Peroxisomal Calcium Levels and Dynamics.

Calcium (Ca2+) is an intracellular messenger that plays an essential role in a variety of cellular processes ranging from early embryonic events to muscle contraction and neuron excitability. Measurement of cytosolic, endoplasmic reticulum (ER), and mitochondrial Ca2+ has contributed immensely to our understanding of cellular physiology. Here we describe the measurement of peroxisomal Ca2+ using ratiometric Ca2+ sensors, enabling measurement of absolute Ca2+ concentration and its dynamics in living cells.

Calcium in peroxisomes: An essential messenger in an essential cell organelle.

Calcium is a central signal transduction element in biology. Peroxisomes are essential cellular organelles, yet calcium handling in peroxisomes has been contentious. Recent advances show that peroxisomes are part of calcium homeostasis in cardiac myocytes and therefore may contribute to or even shape their calcium-dependent functionality. However, the mechanisms of calcium movement between peroxisomes and other cellular sites and their mediators remain elusive. Here, we review calcium handling in peroxisomes in concert with other organelles and summarize the most recent knowledge on peroxisomal involvement in calcium dynamics with a focus on mammalian cells.

Fluorescence lifetime DNA-PAINT for multiplexed super-resolution imaging of cells.

DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution technique highly suitable for multi-target (multiplexing) bio-imaging. However, multiplexed imaging of cells is still challenging due to the dense and sticky environment inside a cell. Here, we combine fluorescence lifetime imaging microscopy (FLIM) with DNA-PAINT and use the lifetime information as a multiplexing parameter for targets identification. In contrast to Exchange-PAINT, fluorescence lifetime PAINT (FL-PAINT) can image multiple targets simultaneously and does not require any fluid exchange, thus leaving the sample undisturbed and making the use of flow chambers/microfluidic systems unnecessary. We demonstrate the potential of FL-PAINT by simultaneous imaging of up to three targets in a cell using both wide-field FLIM and 3D time-resolved confocal laser scanning microscopy (CLSM). FL-PAINT can be readily combined with other existing techniques of multiplexed imaging and is therefore a perfect candidate for high-throughput multi-target bio-imaging.

How to Detect Isolated PEX10-Related Cerebellar Ataxia?

A 4-year-old boy presented with subacute onset of cerebellar ataxia. Neuroimaging revealed cerebellar atrophy. Metabolic screening tests aiming to detect potentially treatable ataxias showed an increased value (fourfold upper limit of normal) for phytanic acid and elevated very-long-chain fatty acid (VLCFA) ratios (C24:0/C22:0 and C26:0/C22:0), while absolute concentrations of VLCFA were normal. Genetic analysis identified biallelic variants in PEX10. Immunohistochemistry confirmed pathogenicity in the patients' cultured fibroblasts demonstrating peroxisomal mosaicism with a general catalase import deficiency as well as conspicuous peroxisome morphology as an expression of impaired peroxisomal function. We describe for the first time an elongated peroxisome morphology in a patient with PEX10-related cerebellar ataxia.A literature search yielded 14 similar patients from nine families with PEX10-related cerebellar ataxia, most of them presenting their first symptoms between 3 and 8 years of age. In 11/14 patients, the first and main symptom was cerebellar ataxia; in three patients, it was sensorineural hearing impairment. Finally, all 14 patients developed ataxia. Polyneuropathy (9/14) and cognitive impairment (9/14) were common associated findings. In 12/13 patients brain MRI showed cerebellar atrophy. Phytanic acid was elevated in 8/12 patients, while absolute concentrations of VLCFA levels were in normal limits in several patients. VLCFA ratios (C24:0/C22:0 and/or C26:0/C22:0), though, were elevated in 11/11 cases. We suggest including measurement of phytanic acid and VLCFA ratios in metabolic screening tests in unexplained autosomal recessive ataxias with cerebellar atrophy, especially when there is an early onset and symptoms are mild.

Stop Codon Context-Specific Induction of Translational Readthrough.

Premature termination codon (PTC) mutations account for approximately 10% of pathogenic variants in monogenic diseases. Stimulation of translational readthrough, also known as stop codon suppression, using translational readthrough-inducing drugs (TRIDs) may serve as a possible therapeutic strategy for the treatment of genetic PTC diseases. One important parameter governing readthrough is the stop codon context (SCC)-the stop codon itself and the nucleotides in the vicinity of the stop codon on the mRNA. However, the quantitative influence of the SCC on treatment outcome and on appropriate drug concentrations are largely unknown. Here, we analyze the readthrough-stimulatory effect of various readthrough-inducing drugs on the SCCs of five common premature termination codon mutations of PEX5 in a sensitive dual reporter system. Mutations in PEX5, encoding the peroxisomal targeting signal 1 receptor, can cause peroxisomal biogenesis disorders of the Zellweger spectrum. We show that the stop context has a strong influence on the levels of readthrough stimulation and impacts the choice of the most effective drug and its concentration. These results highlight potential advantages and the personalized medicine nature of an SCC-based strategy in the therapy of rare diseases.

Peroxisomes contribute to intracellular calcium dynamics in cardiomyocytes and non-excitable cells.

Peroxisomes communicate with other cellular compartments by transfer of various metabolites. However, whether peroxisomes are sites for calcium handling and exchange has remained contentious. Here we generated sensors for assessment of peroxisomal calcium and applied them for single cell-based calcium imaging in HeLa cells and cardiomyocytes. We found that peroxisomes in HeLa cells take up calcium upon depletion of intracellular calcium stores and upon calcium influx across the plasma membrane. Furthermore, we show that peroxisomes of neonatal rat cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes can take up calcium. Our results indicate that peroxisomal and cytosolic calcium signals are tightly interconnected both in HeLa cells and in cardiomyocytes. Cardiac peroxisomes take up calcium on beat-to-beat basis. Hence, peroxisomes may play an important role in shaping cellular calcium dynamics of cardiomyocytes.

Dysferlin links excitation-contraction coupling to structure and maintenance of the cardiac transverse-axial tubule system.

AIMS: The multi-C2 domain protein dysferlin localizes to the T-Tubule system of skeletal and heart muscles. In skeletal muscle, dysferlin is known to play a role in membrane repair and in T-tubule biogenesis and maintenance. Dysferlin deficiency manifests as muscular dystrophy of proximal and distal muscles. Cardiomyopathies have been also reported, and some dysferlinopathy mouse models develop cardiac dysfunction under stress. Generally, the role and functional relevance of dysferlin in the heart is not clear. The aim of this study was to analyse the effect of dysferlin deficiency on the transverse-axial tubule system (TATS) structure and on Ca2+ homeostasis in the heart. METHODS AND RESULTS: We studied dysferlin localization in rat and mouse cardiomyocytes by immunofluorescence microscopy. In dysferlin-deficient ventricular mouse cardiomyocytes, we analysed the TATS by live staining and assessed Ca2+ handling by patch-clamp experiments and measurement of Ca2+ transients and Ca2+ sparks. We found increasing co-localization of dysferlin with the L-type Ca2+-channel during TATS development and show that dysferlin deficiency leads to pathological loss of transversal and increase in longitudinal elements (axialization). We detected reduced L-type Ca2+-current (ICa,L) in cardiomyocytes from dysferlin-deficient mice and increased frequency of spontaneous sarcoplasmic reticulum Ca2+ release events resulting in pro-arrhythmic contractions. Moreover, cardiomyocytes from dysferlin-deficient mice showed an impaired response to ß-adrenergic receptor stimulation. CONCLUSIONS: Dysferlin is required for TATS biogenesis and maintenance in the heart by controlling the ratio of transversal and axial membrane elements. Absence of dysferlin leads to defects in Ca2+ homeostasis which may contribute to contractile heart dysfunction in dysferlinopathy patients.

Functions of Vertebrate Ferlins.

Ferlins are multiple-C2-domain proteins involved in Ca2+-triggered membrane dynamics within the secretory, endocytic and lysosomal pathways. In bony vertebrates there are six ferlin genes encoding, in humans, dysferlin, otoferlin, myoferlin, Fer1L5 and 6 and the long noncoding RNA Fer1L4. Mutations in DYSF (dysferlin) can cause a range of muscle diseases with various clinical manifestations collectively known as dysferlinopathies, including limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. A mutation in MYOF (myoferlin) was linked to a muscular dystrophy accompanied by cardiomyopathy. Mutations in OTOF (otoferlin) can be the cause of nonsyndromic deafness DFNB9. Dysregulated expression of any human ferlin may be associated with development of cancer. This review provides a detailed description of functions of the vertebrate ferlins with a focus on muscle ferlins and discusses the mechanisms leading to disease development.

Staying in Healthy Contact: How Peroxisomes Interact with Other Cell Organelles.

Peroxisomes share extensive metabolic connections with other cell organelles. Membrane contact sites (MCSs) establish and maintain such interactions, and they are vital for organelle positioning and motility. In the past few years peroxisome interactions and MCSs with other cellular organelles have been explored extensively, resulting in the identification of new MCSs, the tethering molecules involved, and their functional characterization. Defective tethering and compartmental communication can lead to pathological conditions that can be termed 'organelle interaction diseases'. We review peroxisome-organelle interactions in mammals and summarize the most recent knowledge of mammalian peroxisomal organelle contacts in health and disease.

Multiple Localization by Functional Translational Readthrough.

In a compartmentalized cell, correct protein localization is crucial for function of virtually all cellular processes. From the cytoplasm as a starting point, proteins are imported into organelles by specific targeting signals. Many proteins, however, act in more than one cellular compartment. In this chapter, we discuss mechanisms by which proteins can be targeted to multiple organelles with a focus on a novel gene regulatory mechanism, functional translational readthrough, that permits multiple targeting of proteins to the peroxisome and other organelles. In mammals, lactate and malate dehydrogenase are the best-characterized enzymes whose targeting is controlled by functional translational readthrough.

Super-resolution imaging reveals the sub-diffraction phenotype of Zellweger Syndrome ghosts and wild-type peroxisomes.

Peroxisomes are ubiquitous cell organelles involved in many metabolic and signaling functions. Their assembly requires peroxins, encoded by PEX genes. Mutations in PEX genes are the cause of Zellweger Syndrome spectrum (ZSS), a heterogeneous group of peroxisomal biogenesis disorders (PBD). The size and morphological features of peroxisomes are below the diffraction limit of light, which makes them attractive for super-resolution imaging. We applied Stimulated Emission Depletion (STED) microscopy to study the morphology of human peroxisomes and peroxisomal protein localization in human controls and ZSS patients. We defined the peroxisome morphology in healthy skin fibroblasts and the sub-diffraction phenotype of residual peroxisomal structures ('ghosts') in ZSS patients that revealed a relation between mutation severity and clinical phenotype. Further, we investigated the 70 kDa peroxisomal membrane protein (PMP70) abundance in relationship to the ZSS sub-diffraction phenotype. This work improves the morphological definition of peroxisomes. It expands current knowledge about peroxisome biogenesis and ZSS pathoethiology to the sub-diffraction phenotype including key peroxins and the characteristics of ghost peroxisomes.

Dual Reporter Systems for the Analysis of Translational Readthrough in Mammals.

Translational readthrough, the decoding of stop codons as sense codons, leads to C-terminal extension of proteins which may lead to the formation of protein isoforms with distinct properties from the original protein. Two proteins have recently been identified that are targeted to the peroxisome via hidden peroxisomal targeting signals in their readthrough extensions. This noninduced basal translational readthrough can be distinguished from pharmacological induction of readthrough by aminoglycosides or other small molecules, which can be used for the treatment of diseases caused by premature stop (termination) codons (PTCs). Readthrough of both, natural stop codons and PTCs, can be quantified in cell culture using reporter systems. In the present article, we describe two dual reporter systems, based on combined fluorescence/luminescence measurement and flow cytometric fluorescence measurement, respectively. Further, we provide a protocol for a fast and efficient cloning procedure of reporter constructs. The dual reporter systems described here help to analyze the peroxisome-specific isoforms of readthrough enzymes as well as potential readthrough therapeutics.

The Use of Glycosylation Tags as Reporters for Protein Entry into the Endoplasmic Reticulum in Yeast and Mammalian Cells.

N-glycosylation is a process occurring in the Endoplasmic Reticulum (ER) in nearly every organism. Proteins containing a glycosylation site are quickly glycosylated by oligosaccharyltransferases once the glycosylation site is exposed to the ER lumen. The oligosaccharide tree is then modified and proteins are targeted to specific organelles or subcompartments. For a long time peroxisomal membrane proteins (PMP) were thought to be targeted directly to the peroxisome. However, in the course of recent years, several PMPs were found to be targeted via the ER. Glycosylation increases the molecular weight of a protein, which is easily detected by Western blotting. Glycosylation tags like the opsin tag are therefore useful tools in the study of ER entry of peroxisomal proteins.

Synaptic UNC13A protein variant causes increased neurotransmission and dyskinetic movement disorder.

Munc13 proteins are essential regulators of neurotransmitter release at nerve cell synapses. They mediate the priming step that renders synaptic vesicles fusion-competent, and their genetic elimination causes a complete block of synaptic transmission. Here we have described a patient displaying a disorder characterized by a dyskinetic movement disorder, developmental delay, and autism. Using whole-exome sequencing, we have shown that this condition is associated with a rare, de novo Pro814Leu variant in the major human Munc13 paralog UNC13A (also known as Munc13-1). Electrophysiological studies in murine neuronal cultures and functional analyses in Caenorhabditis elegans revealed that the UNC13A variant causes a distinct dominant gain of function that is characterized by increased fusion propensity of synaptic vesicles, which leads to increased initial synaptic vesicle release probability and abnormal short-term synaptic plasticity. Our study underscores the critical importance of fine-tuned presynaptic control in normal brain function. Further, it adds the neuronal Munc13 proteins and the synaptic vesicle priming process that they control to the known etiological mechanisms of psychiatric and neurological synaptopathies.

Dysferlin mediates membrane tubulation and links T-tubule biogenesis to muscular dystrophy.

The multi-C2 domain protein dysferlin localizes to the plasma membrane and the T-tubule system in skeletal muscle; however, its physiological mode of action is unknown. Mutations in the DYSF gene lead to autosomal recessive limb-girdle muscular dystrophy type 2B and Miyoshi myopathy. Here, we show that dysferlin has membrane tubulating capacity and that it shapes the T-tubule system. Dysferlin tubulates liposomes, generates a T-tubule-like membrane system in non-muscle cells, and links the recruitment of phosphatidylinositol 4,5-bisphosphate to the biogenesis of the T-tubule system. Pathogenic mutant forms interfere with all of these functions, indicating that muscular wasting and dystrophy are caused by the dysferlin mutants' inability to form a functional T-tubule membrane system.

Pex35 is a regulator of peroxisome abundance.

Peroxisomes are cellular organelles with vital functions in lipid, amino acid and redox metabolism. The cellular formation and dynamics of peroxisomes are governed by PEX genes; however, the regulation of peroxisome abundance is still poorly understood. Here, we use a high-content microscopy screen in Saccharomyces cerevisiae to identify new regulators of peroxisome size and abundance. Our screen led to the identification of a previously uncharacterized gene, which we term PEX35, which affects peroxisome abundance. PEX35 encodes a peroxisomal membrane protein, a remote homolog to several curvature-generating human proteins. We systematically characterized the genetic and physical interactome as well as the metabolome of mutants in PEX35, and we found that Pex35 functionally interacts with the vesicle-budding-inducer Arf1. Our results highlight the functional interaction between peroxisomes and the secretory pathway.