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Venous system pathologies have increasingly been linked to clinically relevant disorders of CSF circulation whereas the exact coupling mechanisms still remain unknown. In this work, flow dynamics of both systems were studied using real-time phase-contrast flow MRI in 16 healthy subjects during normal and forced breathing. Flow evaluations in the aqueduct, at cervical level C3 and lumbar level L3 for both the CSF and venous fluid systems reveal temporal modulations by forced respiration. During normal breathing cardiac-related flow modulations prevailed, while forced breathing shifted the dominant frequency of both CSF and venous flow spectra towards the respiratory component and prompted a correlation between CSF and venous flow in the large vessels. The average of flow magnitude of CSF was increased during forced breathing at all spinal and intracranial positions. Venous flow in the large vessels of the upper body decreased and in the lower body increased during forced breathing. Deep respiration couples interdependent venous and brain fluid flow—most likely mediated by intrathoracic and intraabdominal pressure changes. Further insights into the driving forces of CSF and venous circulation and their correlation will facilitate our understanding how the venous system links to intracranial pressure regulation and of related forms of hydrocephalus.

In 215 patients with a median age of 16 years, fingolimod was superior to interferon beta-1a in reducing relapses of multiple sclerosis and the accumulation of new lesions on MRI over a 2-year period. Seizures occurred in 5.6% of patients in the fingolimod group.

Translational readthrough gives rise to low abundance proteins with C-terminal extensions beyond the stop codon. To identify functional translational readthrough, we estimated the readthrough propensity (RTP) of all stop codon contexts of the human genome by a new regression model in silico, identified a nucleotide consensus motif for high RTP by using this model, and analyzed all readthrough extensions in silico with a new predictor for peroxisomal targeting signal type 1 (PTS1). Lactate dehydrogenase B (LDHB) showed the highest combined RTP and PTS1 probability. Experimentally we show that at least 1.6% of the total cellular LDHB is targeted to the peroxisome by a conserved hidden PTS1. The readthrough-extended lactate dehydrogenase subunit LDHBx can also co-import LDHA, the other LDH subunit, into peroxisomes. Peroxisomal LDH is conserved in mammals and likely contributes to redox equivalent regeneration in peroxisomes. Amino acids are the building blocks of proteins, and the order of the amino acids in a protein is determined by the order in which ‘codons’ appear in a messenger RNA molecule. Most codons represent a specific amino acid, but there are also three stop codons that are used to mark the end of a protein. When the cellular machinery that ‘translates’ the messenger RNA molecule into a protein encounters a stop codon, it stops and releases the completed protein. Sometimes, however, the stop codon is not interpreted as a stop signal, and the translation of the messenger RNA molecule continues until another stop codon is encountered. This process is known as readthrough. Some organisms, in particular viruses and fungi, use readthrough to produce a wider range of proteins than their genomes would otherwise allow. While readthrough also occurs in higher organisms such as mammals, it is not known if the resulting proteins perform extra functions that the original protein does not perform. A number of factors affect whether readthrough occurs when an mRNA template is being translated. For example, each of the three stop codons has a different likelihood of having its stop signal misinterpreted, and the mRNA sequence that surrounds the stop codon can also affect the likelihood of readthrough. Schueren et al. have developed a computational model that estimates how common this form of translational readthrough is in the human genome. The model was based on the identity of the stop codons themselves and the surrounding mRNA sequence. This model was then combined with another model that identifies proteins that are targeted to a structure inside a cell called the peroxisome, which is where a number of essential energy-releasing reactions take place. The combined model enabled Schueren et al. to identify proteins that both perform functions in the peroxisome and are likely to be formed by readthrough. The combined model suggested a protein that is a part of lactate dehydrogenase: an enzyme that speeds up chemical reactions that are important for the cell to produce energy. Low levels of lactate dehydrogenase had previously been found in the peroxisome, despite it apparently lacking a specific sequence of amino acids that proteins need to have to enter the peroxisome. However, Schueren et al. confirmed experimentally that readthrough does occur for the lactate dehydrogenase component identified by the model, revealing that it contains a ‘hidden’ peroxisome-targeting region. Furthermore, when more translational readthrough occurred, more lactate dehydrogenase was found in the peroxisomes. This unusual way that lactate dehydrogenase enters the peroxisome is an example of how the cell optimizes the used of the genetic information encoded in the genome and in messenger RNA. Translational readthrough always ensures that a certain proportion of lactate dehydrogenase will be brought to the peroxisome. The computational model developed here will be a valuable tool to identify other such proteins produced from genomes, including the human genome and those of other species.

Autosomal recessive (AR) gene defects are the leading genetic cause of intellectual disability (ID) in countries with frequent parental consanguinity, which account for about 1/7th of the world population. Yet, compared to autosomal dominant de novo mutations, which are the predominant cause of ID in Western countries, the identification of AR-ID genes has lagged behind. Here, we report on whole exome and whole genome sequencing in 404 consanguineous predominantly Iranian families with two or more affected offspring. In 219 of these, we found likely causative variants, involving 77 known and 77 novel AR-ID (candidate) genes, 21 X-linked genes, as well as 9 genes previously implicated in diseases other than ID. This study, the largest of its kind published to date, illustrates that high-throughput DNA sequencing in consanguineous families is a superior strategy for elucidating the thousands of hitherto unknown gene defects underlying AR-ID, and it sheds light on their prevalence.

Infantile-onset RNaseT2 deficient leukoencephalopathy is characterised by cystic brain lesions, multifocal white matter alterations, cerebral atrophy, and severe psychomotor impairment. The phenotype is similar to congenital cytomegalovirus brain infection and overlaps with type I interferonopathies, suggesting a role for innate immunity in its pathophysiology. To date, pathophysiological studies have been hindered by the lack of mouse models recapitulating the neuroinflammatory encephalopathy found in patients. In this study, we generated Rnaset2 −/− mice using CRISPR/Cas9-mediated genome editing. Rnaset2 −/− mice demonstrate upregulation of interferon-stimulated genes and concurrent IFNAR1-dependent neuroinflammation, with infiltration of CD8 + effector memory T cells and inflammatory monocytes into the grey and white matter. Single nuclei RNA sequencing reveals homeostatic dysfunctions in glial cells and neurons and provide important insights into the mechanisms of hippocampal-accentuated brain atrophy and cognitive impairment. The Rnaset2 −/− mice may allow the study of CNS damage associated with RNaseT2 deficiency and may be used for the investigation of potential therapies. Studies on interferon-driven brain pathology have so far been hampered by the lack of appropriate animal models. Here the authors characterize RNASET2-deficient mice and show that neuroinflammation and brain atrophy are IFNAR1-dependent.

RNaseT2-deficient cystic leukoencephalopathy (CLE) presents with severe psychomotor retardation, cystic brain lesions, white matter alterations, and cerebral atrophy. The Rnaset2 −/− mouse mirrors key features of this disease and represents the first murine model with a distinct neurological phenotype for type I interferonopathies. Rnaset2 −/− mice exhibit activated microglia, perivascular monocyte and CD8 + T cell infiltration, and hippocampal accentuated atrophy. However, the mechanisms linking interferon-driven neuroinflammation to neurodegeneration remain unclear, underscoring the need to clarify which molecular processes contribute to tissue injury in a time-dependent manner. We found a sustained upregulation of interferon-stimulated genes (IRF9, RIG-I) over three to 28 weeks of age in the brains of Rnaset2 −/− mice compared to controls. Expression of the chemokines Ccl2, Ccl5 , and Cxcl10 peaked early but declined thereafter. Pyroptosis-related markers (ASC, CASP1, GSDMD) were significantly increased already at three to 6 weeks of age and decreased thereafter, whereas apoptotic markers such as Bax, Bad, Bid, CASP3, CASP8, and PARP were not differentially expressed compared to controls. Finally, Cd3e as well as Tnf peaked later (at 17 weeks of age) and declined at 28 weeks. Interestingly, double IHC confirmed the co-localization of the pyroptosis-related marker ASC with the microglia marker IBA-1. Taken together, these findings support the notion that pyroptosis is an early, disease-associated event restricted to microglia that likely contributes to establishing a proinflammatory milieu prior to T cell infiltration and brain atrophy. Targeting pyroptosis could therefore represent a potential strategy to attenuate neurodegeneration in type I interferon–driven neuroinflammatory disorders.

Mitochondria contain their own DNA (mtDNA) and a dedicated gene expression machinery. As the mitochondrial dimensions are close to the diffraction limit of classical light microscopy, the spatial distribution of mitochondrial proteins and in particular of mitochondrial mRNAs remains underexplored. Here, we establish single-molecule fluorescence in situ hybridization (smFISH) combined with STED and MINFLUX super-resolution microscopy (nanoscopy) to visualize individual mitochondrial mRNA molecules and associated proteins. STED nanoscopy reveals the spatial relationships between distinct mRNA species and proteins such as the RNA granule marker GRSF1, demonstrating adaptive changes in mRNA distribution and quantity in challenged mammalian cells and patient-derived cell lines. Notably, STED-smFISH shows the release of mRNAs during apoptosis, while MINFLUX reveals the folding of the mRNAs into variable shapes, as well as their spatial proximity to mitochondrial ribosomes. These protocols are transferable to various cell types and open new avenues for understanding mitochondrial gene regulation in health and disease. Stoldt, Maass, and colleagues present a study where smFISH is combined with STED and MINFLUX microscopy to map mitochondrial mRNA at nanometre resolution, enabling the exploration of the structural folding and distribution of mRNAs within mitochondria.

Alternating hemiplegia of childhood (AHC) is a rare neurological disorder characterised by early-onset episodes of hemiplegia, dystonia, various paroxysmal symptoms, and developmental impairment. Almost all cases of AHC are sporadic but AHC concordance in monozygotic twins and dominant transmission in a family with a milder phenotype have been reported. Thus, we aimed to identify de-novo mutations associated with this disease. We recruited patients with clinically characterised AHC from paediatric neurology departments in Germany and with the aid of a parental support group between Sept, 2004, and May 18, 2012. We used whole-exome sequencing of three proband-parent trios to identify a disease-associated gene and then tested whether mutations in the gene were also present in the remaining patients and their healthy parents. We analysed genotypes and characterised their associations with the phenotypic spectrum of the disease. We studied 15 female and nine male patients with AHC who were aged 8–35 years. ATP1A3 emerged as the disease-associated gene in AHC. Whole-exome sequencing showed three heterozygous de-novo missense mutations. Sequencing of the 21 remaining affected individuals identified disease-associated mutations in ATP1A3 in all patients, including six de-novo missense mutations and one de-novo splice-site mutation. Because ATP1A3 is also the gene associated with rapid-onset dystonia-parkinsonism (DYT12, OMIM 128235) we compared the genotypes and phenotypes of patients with AHC in our cohort with those of patients with rapid-onset dystonia-parkinsonism reported in the scientific literature. We noted overlapping clinical features, such as abrupt onset of dystonic episodes often triggered by emotional stress, a rostrocaudal (face to arm to leg) gradient of involvement, and signs of brainstem dysfunction, as well as clearly differentiating clinical characteristics, such as episodic hemiplegia and quadriplegia. Mutation analysis of the ATP1A3 gene in patients who met clinical criteria for AHC allows for definite genetic diagnosis and sound genetic counselling. AHC and rapid-onset dystonia-parkinsonism are allelic diseases related to mutations in ATP1A3 and form a phenotypical continuum of a dystonic movement disorder. Eva Luise and Horst Köhler Foundation for Humans with Rare Diseases.

The dynamics of human CSF in brain and upper spinal canal are regulated by inspiration and connected to the venous system through associated pressure changes. Upward CSF flow into the head during inspiration counterbalances venous flow out of the brain. Here, we investigated CSF motion along the spinal canal by real-time phase-contrast flow MRI at high spatial and temporal resolution. Results reveal a watershed of spinal CSF dynamics which divides flow behavior at about the level of the heart. While forced inspiration prompts upward surge of CSF flow volumes in the entire spinal canal, ensuing expiration leads to pronounced downward CSF flow, but only in the lower canal. The resulting pattern of net flow volumes during forced respiration yields upward CSF motion in the upper and downward flow in the lower spinal canal. These observations most likely reflect closely coupled CSF and venous systems as both large caval veins and their anastomosing vertebral plexus react to respiration-induced pressure changes.

Transcription factor NRF2, encoded by NFE2L2 , is the master regulator of defense against stress in mammalian cells. Somatic mutations of NFE2L2 leading to NRF2 accumulation promote cell survival and drug resistance in cancer cells. Here we show that the same mutations as inborn de novo mutations cause an early onset multisystem disorder with failure to thrive, immunodeficiency and neurological symptoms. NRF2 accumulation leads to widespread misregulation of gene expression and an imbalance in cytosolic redox balance. The unique combination of white matter lesions, hypohomocysteinaemia and increased G-6-P-dehydrogenase activity will facilitate early diagnosis and therapeutic intervention of this novel disorder. The NRF2 transcription factor regulates the response to stress in mammalian cells. Here, the authors show that activating mutations in NRF2, commonly found in cancer cells, are found in four patients with a multisystem disorder characterized by immunodeficiency and neurological symptoms.