Soft X-ray spectromicroscopy of human fibroblasts with impaired sialin function.

Salla disease (SD) is a lysosomal storage disease where free sialic acid (SA) accumulates in lysosomes due to the impaired function of a membrane protein, sialin. Synchrotron radiation-based scanning transmission soft X-ray spectromicroscopy (STXM) was used to analyze both SD patients' fibroblasts and normal human dermal fibroblasts (NHDF) from healthy controls. Both cell lines were also cultured with N-acetyl-d-mannosamine monohydrate (ManNAc) to see if it increased SA concentration in the cells. The STXM technique was chosen to simultaneously observe the morphological and chemical changes in cells. It was observed that free SA did not remain in the lysosomes during the sample processing, leaving empty vacuoles to the fibroblasts. The total cytosol and entire cell spectra, however, showed systematic differences between the SD and NHDF samples, indicating changes in the relative macromolecular concentrations of the cells. The NHDF cell lines contained a higher relative protein concentration compared to the SD cell lines, and the addition of ManNAc increased the relative protein concentration in both cell lines. In this study, two sample preparation methods were compared, resin-embedded thin sections and cells grown directly on sample analysis grids. While the samples grown on the grids exhibited clean, well-resolved spectra not masked by embedding resin, the low penetration depth of soft X-rays hindered the analysis to only the thin region of the microfilaments away from the thick nucleus.

Brain MRI findings in paediatric genetic disorders associated with white matter abnormalities.

AIM: To describe the specific brain magnetic resonance imaging (MRI) patterns of the paediatric genetic disorders associated with white matter abnormalities in Northern Finland. METHOD: In this retrospective population-based longitudinal study, brain MRI scans accumulated from 1990 to 2019 at Oulu University Hospital, Finland, were assessed. Inclusion criteria were defined as leukodystrophies or genetic diseases with significant white matter abnormalities that did not meet the criteria for leukodystrophy, at least one brain MRI, and age under 18 years at diagnosis. RESULTS: A total of 83 patients (48 males, 35 females) were found with 52 different diseases. The median age at the time of the brain MRI was 22 months (interquartile range [IQR] = 46 months). In 72 (87%) of the children, brain MRIs revealed abnormal findings, including cerebral white matter abnormalities (n = 49, 59%), brainstem signal abnormalities (n = 28, 34%), thinning of the corpus callosum (n = 30, 36%), delayed myelination (n = 11, 13%), and permanent hypomyelination (n = 9, 11%). INTERPRETATION: Symmetrical and bilateral white matter signal patterns of the brain MRI should raise suspicion of genetic disorders when the clinical symptoms are compatible. This study illustrates brain imaging patterns of childhood-onset genetic disorders in a population in Northern Finland and improves the diagnostic accuracy of rare genetic disorders.

Loss-of-function mutations of the TIE1 receptor tyrosine kinase cause late-onset primary lymphedema.

Primary lymphedema (PL), characterized by tissue swelling, fat accumulation, and fibrosis, results from defects in lymphatic vessels or valves caused by mutations in genes involved in development, maturation, and function of the lymphatic vascular system. Pathogenic variants in various genes have been identified in about 30% of PL cases. By screening of a cohort of 755 individuals with PL, we identified two TIE1 (tyrosine kinase with immunoglobulin- and epidermal growth factor-like domains 1) missense variants and one truncating variant, all predicted to be pathogenic by bioinformatic algorithms. The TIE1 receptor, in complex with TIE2, binds angiopoietins to regulate the formation and remodeling of blood and lymphatic vessels. The premature stop codon mutant encoded an inactive truncated extracellular TIE1 fragment with decreased mRNA stability, and the amino acid substitutions led to decreased TIE1 signaling activity. By reproducing the two missense variants in mouse Tie1 via CRISPR/Cas9, we showed that both cause edema and are lethal in homozygous mice. Thus, our results indicate that TIE1 loss-of-function variants can cause lymphatic dysfunction in patients. Together with our earlier demonstration that ANGPT2 loss-of-function mutations can also cause PL, our results emphasize the important role of the ANGPT2/TIE1 pathway in lymphatic function.

Hyperkinetic Movement Disorder Caused by the Recurrent c.892C>T NACC1 Variant.

BACKGROUND: Genetic syndromes of hyperkinetic movement disorders associated with epileptic encephalopathy and intellectual disability are becoming increasingly recognized. Recently, a de novo heterozygous NACC1 (nucleus accumbens-associated 1) missense variant was described in a patient cohort including one patient with a combined mitochondrial oxidative phosphorylation (OXPHOS) deficiency. OBJECTIVES: The objective is to characterize the movement disorder in affected patients with the recurrent c.892C>T NACC1 variant and study the NACC1 protein and mitochondrial function at the cellular level. METHODS: The movement disorder was analyzed on four patients with the NACC1 c.892C>T (p.Arg298Trp) variant. Studies on NACC1 protein and mitochondrial function were performed on patient-derived fibroblasts. RESULTS: All patients had a generalized hyperkinetic movement disorder with chorea and dystonia, which occurred cyclically and during sleep. Complex I was found altered, whereas the other OXPHOS enzymes and the mitochondria network seemed intact in one patient. CONCLUSIONS: The movement disorder is a prominent feature of NACC1-related disease.

A novel pathogenic SLC12A5 missense variant in epilepsy of infancy with migrating focal seizures causes impaired KCC2 chloride extrusion.

The potassium-chloride co-transporter 2, KCC2, is a neuron-specific ion transporter that plays a multifunctional role in neuronal development. In mature neurons, KCC2 maintains a low enough intracellular chloride concentration essential for inhibitory neurotransmission. During recent years, pathogenic variants in the KCC2 encoding gene SLC12A5 affecting the functionality or expression of the transporter protein have been described in several patients with epilepsy of infancy with migrating focal seizures (EIMFS), a devastating early-onset developmental and epileptic encephalopathy. In this study, we identified a novel recessively inherited SLC12A5 c.692G>A, p. (R231H) variant in a patient diagnosed with severe and drug-resistant EIMFS and profound intellectual disability. The functionality of the variant was assessed in vitro by means of gramicidin-perforated patch-clamp experiments and ammonium flux assay, both of which indicated a significant reduction in chloride extrusion. Based on surface immunolabeling, the variant showed a reduction in membrane expression. These findings implicate pathogenicity of the SLC12A5 variant that leads to impaired inhibitory neurotransmission, increasing probability for hyperexcitability and epileptogenesis.

ciRS-7 and miR-7 regulate ischemia-induced neuronal death via glutamatergic signaling.

Brain functionality relies on finely tuned regulation of gene expression by networks of non-coding RNAs (ncRNAs) such as the one composed by the circular RNA ciRS-7 (also known as CDR1as), the microRNA miR-7, and the long ncRNA Cyrano. We describe ischemia-induced alterations in the ncRNA network both in vitro and in vivo and in transgenic mice lacking ciRS-7 or miR-7. Our data show that cortical neurons downregulate ciRS-7 and Cyrano and upregulate miR-7 expression during ischemia. Mice lacking ciRS-7 exhibit reduced lesion size and motor impairment, while the absence of miR-7 alone results in increased ischemia-induced neuronal death. Moreover, miR-7 levels in pyramidal excitatory neurons regulate neurite morphology and glutamatergic signaling, suggesting a potential molecular link to the in vivo phenotype. Our data reveal the role of ciRS-7 and miR-7 in modulating ischemic stroke outcome, shedding light on the pathophysiological function of intracellular ncRNA networks in the brain.

Infantile onset encephalomyopathy, retinopathy, optic atrophy, and mitochondrial DNA depletion associated with a novel pathogenic DHX16 variant.

We studied a patient with mitochondrial DNA depletion in skeletal muscle and a multiorgan phenotype, including fatal encephalomyopathy, retinopathy, optic atrophy, and sensorineural hearing loss. Instead of pathogenic variants in the mitochondrial maintenance genes, we identified previously unpublished variant in DHX16 gene, a de novo heterozygous c.1360C>T (p. Arg454Trp). Variants in DHX16 encoding for DEAH-box RNA helicase have previously been reported only in five patients with a phenotype called as neuromuscular oculoauditory syndrome including developmental delay, neuromuscular symptoms, and ocular or auditory defects with or without seizures. We performed functional studies on patient-derived fibroblasts and skeletal muscle revealing, that the DHX16 expression was decreased. Clinical features together with functional data suggest, that our patient's disease is associated with a novel pathogenic DHX16 variant, and mtDNA depletion could be a secondary manifestation of the disease.

High NHLRC2 expression is associated with shortened survival in lung adenocarcinoma.

Background: Certain variants of NHL repeat (named after NCL-1, HT2A and LIN-41)-containing protein 2 (NHLRC2) gene have been linked to severe fibrotic interstitial lung disease in children. The aim of the current study was to evaluate the expression of NHLRC2 in lung cell and tissue samples from patients with lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC). Methods: The expression of NHLRC2 in lung tissue samples was studied by immunohistochemistry (102 ADC, 111 SCC), mRNA in situ hybridization (4 ADC, 3 SCC), and Western blot analysis (3 ADC, 2 SCC). The immunohistochemical NHLRC2 expression was measured by image analysis software and the percentage of NHLRC2-positive cancer cells was evaluated by semiquantitative analysis. The immunohistochemical results of NHLRC2 were compared with the clinical and histological characteristics of the patients. NHLRC2 protein levels in primary stromal and epithelial lung cancer cell lines were measured by Western blot analysis. Results: NHLRC2 was mainly expressed in cancer cells and inflammatory cells within the tumor. The NHLRC2 expression evaluated by image analysis method was significantly higher in ADC compared with that in SCC (P<0.001). High NHLRC2 expression was associated with reduced disease specific survival (P=0.002), overall survival (P=0.001), and high mitotic activity (P=0.042) in ADC. Additionally, the proportion of NHLRC2-positive cancer cells analyzed by the semiquantitative method was significantly higher in ADC than in SCC (P<0.001). Conclusions: NHLRC2 expression was higher in lung ADC than in SCC and its expression was associated with poor survival in ADC patients. Further studies are required to clarify the pathogenetic role of NHLRC2 in lung cancer.

DNA polymerase gamma variants and hepatotoxicity during maintenance therapy of childhood acute lymphoblastic leukemia: is there a causal relationship?

Hepatotoxicity is a frequent complication during maintenance therapy of acute lymphoblastic leukemia (ALL) with 6-mercaptopurine and methotrexate. Elevated levels of methylated 6-mercaptopurine metabolites (MeMP) are associated with hepatotoxicity. However, not all mechanisms are known that lead to liver failure in patients with ALL. Variants in the POLG gene, which encodes the catalytic subunit of mitochondrial DNA polymerase gamma (POLG1), have been related to drug-induced hepatotoxicity, for example, by sodium valproate. The association of common POLG variants with hepatotoxicity during maintenance therapy was studied in 34 patients with childhood ALL. Of the screened POLG variants, four different variants were detected in 12 patients. One patient developed severe hepatotoxicity without elevated MeMP levels and harbored a heterozygous POLG p.G517V variant, which was not found in the other patients.

Novel patients with NHLRC2 variants expand the phenotypic spectrum of FINCA disease.

Purpose: FINCA disease (Fibrosis, Neurodegeneration and Cerebral Angiomatosis, OMIM 618278) is an infantile-onset neurodevelopmental and multiorgan disease. Since our initial report in 2018, additional patients have been described. FINCA is the first human disease caused by recessive variants in the highly conserved NHLRC2 gene. Our previous studies have shown that Nhlrc2-null mouse embryos die during gastrulation, indicating the essential role of the protein in embryonic development. Defect in NHLRC2 leads to cerebral neurodegeneration and severe pulmonary, hepatic and cardiac fibrosis. Despite having a structure suggestive of an enzymatic role and the clinical importance of NHLRC2 in multiple organs, the specific physiological role of the protein is unknown. Methods: The clinical histories of five novel FINCA patients diagnosed with whole exome sequencing were reviewed. Segregation analysis of the biallelic, potentially pathogenic NHLRC2 variants was performed using Sanger sequencing. Studies on neuropathology and NHLRC2 expression in different brain regions were performed on autopsy samples of three previously described deceased FINCA patients. Results: One patient was homozygous for the pathogenic variant c.442G > T, while the other four were compound heterozygous for this variant and two other pathogenic NHLRC2 gene variants. All five patients presented with multiorgan dysfunction with neurodevelopmental delay, recurrent infections and macrocytic anemia as key features. Interstitial lung disease was pronounced in infancy but often stabilized. Autopsy samples revealed widespread, albeit at a lower intensity than the control, NHLRC2 expression in the brain. Conclusion: This report expands on the characteristic clinical features of FINCA disease. Presentation is typically in infancy, and although patients can live to late adulthood, the key clinical and histopathological features are fibrosis, infection susceptibility/immunodeficiency/intellectual disability, neurodevelopmental disorder/neurodegeneration and chronic anemia/cerebral angiomatosis (hence the acronym FINCA) that enable an early diagnosis confirmed by genetic investigations.

The Finnish genetic heritage in 2022 - from diagnosis to translational research.

Isolated populations have been valuable for the discovery of rare monogenic diseases and their causative genetic variants. Finnish disease heritage (FDH) is an example of a group of hereditary monogenic disorders caused by single major, usually autosomal-recessive, variants enriched in the population due to several past genetic drift events. Interestingly, distinct subpopulations have remained in Finland and have maintained their unique genetic repertoire. Thus, FDH diseases have persisted, facilitating vigorous research on the underlying molecular mechanisms and development of treatment options. This Review summarizes the current status of FDH, including the most recently discovered FDH disorders, and introduces a set of other recently identified diseases that share common features with the traditional FDH diseases. The Review also discusses a new era for population-based studies, which combine various forms of big data to identify novel genotype-phenotype associations behind more complex conditions, as exemplified here by the FinnGen project. In addition to the pathogenic variants with an unequivocal causative role in the disease phenotype, several risk alleles that correlate with certain phenotypic features have been identified among the Finns, further emphasizing the broad value of studying genetically isolated populations.

NHLRC2 expression is increased in idiopathic pulmonary fibrosis.

BACKGROUND: Variants of NHL repeat-containing protein 2 (NHLRC2) have been associated with severe fibrotic interstitial lung disease in early childhood and NHLRC2 has been listed as a differentially expressed gene between rapidly and slowly progressing idiopathic pulmonary fibrosis (IPF) patients. However, its cell type-specific localization in human lung tissue is unknown. The aim of this study was to evaluate NHLRC2 mRNA and protein expression in different cell types of lung tissue samples and to investigate the effect of transforming growth factor (TGF)-ß1 exposure on NHLRC2 expression in vitro. METHODS: The NHLRC2 expression in lung tissue samples was studied by immunohistochemistry (50 IPF, 10 controls) and mRNA in situ hybridization (8 IPF, 3 controls). The immunohistochemical NHLRC2 expression was quantified with image analysis software and associated with the clinical and smoking data of the patients. NHLRC2 expression levels in primary stromal and small airway epithelial cell lines after exposure to TGF-ß1 was measured by quantitative reverse transcription polymerase chain reaction and Western blot analysis. RESULTS: NHLRC2 expression was detected especially in bronchiolar epithelial cells, type II pneumocytes and macrophages in normal lung. In the lungs of IPF patients, NHLRC2 was mainly expressed in hyperplastic alveolar epithelial cells lining fibroblast foci and honeycombs. NHLRC2 expression assessed by image analysis was higher in IPF compared to controls (p < 0.001). Ever-smokers had more prominent NHLRC2 staining than non-smokers (p = 0.037) among IPF patients. TGF-ß1 exposure did not influence NHLRC2 levels in lung cell lines. CONCLUSIONS: NHLRC2 expression was higher in IPF compared to controls being widely expressed in type II pneumocytes, macrophages, bronchiolar epithelium, and hyperplastic alveolar epithelium. Additionally, its expression was not regulated by the exposure to TGF-ß1 in vitro. Further studies are needed to clarify the role of NHLRC2 in IPF.

Analysis of human brain tissue derived from DBS surgery.

BACKGROUND: Transcriptomic and proteomic profiling of human brain tissue is hindered by the availability of fresh samples from living patients. Postmortem samples usually represent the advanced disease stage of the patient. Furthermore, the postmortem interval can affect the transcriptomic and proteomic profiles. Therefore, fresh brain tissue samples from living patients represent a valuable resource of metabolically intact tissue. Implantation of deep brain stimulation (DBS) electrodes into the human brain is a neurosurgical treatment for, e.g., movement disorders. Here, we describe an improved approach to collecting brain tissues from surgical instruments used in implantation of DBS device for transcriptomics and proteomics analyses. METHODS: Samples were extracted from guide tubes and recording electrodes used in routine DBS implantation procedure to treat patients with Parkinson's disease, genetic dystonia and tremor. RNA sequencing was performed in tissues extracted from the recording microelectrodes and liquid chromatography-mass spectrometry (LC-MS) performed in tissues from guide tubes. To assess the performance of the current approach, the obtained datasets were compared with previously published datasets representing brain tissues. RESULTS: Altogether, 32,034 RNA transcripts representing the unique Ensembl gene identifiers were detected from eight samples representing both hemispheres of four patients. By using  LC-MS, we identified 734 unique proteins from 31 samples collected from 14 patients. The datasets are available in the BioStudies database (accession number S-BSST667). Our results indicate that surgical instruments used in DBS installation retain brain material sufficient for protein and gene expression studies. Comparison with previously published datasets obtained with similar approach proved the robustness and reproducibility of the protocol. CONCLUSIONS: The instruments used during routine DBS surgery are a useful source for obtaining fresh brain tissues from living patients. This approach overcomes the issues that arise from using postmortem tissues, such as the effect of postmortem interval on transcriptomic and proteomic landscape of the brain, and can be used for studying molecular aspects of DBS-treatable diseases.

Nhlrc2 is crucial during mouse gastrulation.

The loss of NHL repeat containing 2 (Nhlrc2) leads to early embryonic lethality in mice, but the exact timing is currently unknown. In this study, we determined the time of lethality for Nhlrc2 knockout (KO), C57BL/6NCrl-Nhlrc2tm1a(KOMP)Wtsi /Oulu, embryos and the in situ expression pattern of Nhlrc2 based on LacZ reporter gene expression during this period. Nhlrc2 KO preimplantation mouse embryos developed normally after in vitro fertilization. Embryonic stem (ES) cells established from KO blastocysts proliferated normally despite a complete loss of the NHLRC2 protein. Nhlrc2 KO embryos from timed matings implanted and were indistinguishable from their wildtype littermates on embryonic day (E) 6.5. On E7.5, Nhlrc2 KO embryo development was arrested, and on E8.5, only 6% of the genotyped embryos were homozygous for the Nhlrc2tm1a(KOMP)Wtsi allele. Nhlrc2 KO E8.5 embryos showed limited embryonic or extraembryonic tissue differentiation and remained at the cylinder stage. Nhlrc2 expression was ubiquitous but strongest in the epiblast/ectoderm and extraembryonic ectoderm on E6.5 and E7.5. NHLRC2 is essential for early postimplantation development, and its loss leads to failed gastrulation and amniotic folding in mice. Future studies on the evolutionarily conserved NHLRC2 will provide new insights into the molecular pathways involved in the early steps of postimplantation development.

INFRAFRONTIER quality principles in systemic phenotyping.

Improving reproducibility and replicability in preclinical research is a widely discussed and pertinent topic, especially regarding ethical responsibility in animal research. INFRAFRONTIER, the European Research Infrastructure for the generation, phenotyping, archiving, and distribution of model mammalian genomes, is addressing this issue by developing internal quality principles for its different service areas, that provides a quality framework for its operational activities. This article introduces the INFRAFRONTIER Quality Principles in Systemic Phenotyping of genetically altered mouse models. A total of 11 key principles are included, ranging from general requirements for compliance with guidelines on animal testing, to the need for well-trained personnel and more specific standards such as the exchange of reference lines. Recently established requirements such as the provision of FAIR (Findable, Accessible, Interoperable, Reusable) data are also addressed. For each quality principle, we have outlined the specific context, requirements, further recommendations, and key references.

Modeling Rare Human Disorders in Mice: The Finnish Disease Heritage.

The modification of genes in animal models has evidently and comprehensively improved our knowledge on proteins and signaling pathways in human physiology and pathology. In this review, we discuss almost 40 monogenic rare diseases that are enriched in the Finnish population and defined as the Finnish disease heritage (FDH). We will highlight how gene-modified mouse models have greatly facilitated the understanding of the pathological manifestations of these diseases and how some of the diseases still lack proper models. We urge the establishment of subsequent international consortiums to cooperatively plan and carry out future human disease modeling strategies. Detailed information on disease mechanisms brings along broader understanding of the molecular pathways they act along both parallel and transverse to the proteins affected in rare diseases, therefore also aiding understanding of common disease pathologies.

Epidemiological, clinical, and genetic characteristics of paediatric genetic white matter disorders in Northern Finland.

AIM: To examine the epidemiological, clinical, and genetic characteristics of paediatric patients with genetic white matter disorders (GWMDs) in Northern Finland. METHOD: A longitudinal population-based cohort study was conducted in the tertiary catchment area of Oulu University Hospital from 1990 to 2019. Patients were identified retrospectively by International Statistical Classification of Diseases and Related Health Problems codes in hospital records and prospectively by attending physicians. Inclusion criteria were children younger than 18 years with defined GWMDs or genetic disorders associated with white matter abnormalities (WMAs) on brain magnetic resonance imaging. RESULTS: Eighty patients (mean age [SD] at the end of the study 11y [8y 6mo], range 0-35y; 45 males, 35 females) were diagnosed with a defined GWMD. The cumulative childhood incidence was 30 per 100 000 live births. Regarding those patients with 49 distinct GWMDs, 20% had classic leukodystrophies and 80% had genetic leukoencephalopathies. The most common leukodystrophies were cerebral adrenoleukodystrophy, Krabbe disease, and Salla disease. Additionally, 29 patients (36%) had genetic aetiologies not previously associated with brain WMAs or they had recently characterised GWMDs, including SAMD9L- and NHLRC2-related neurological disorders. Aetiology was mitochondrial in 21% of patients. The most common clinical findings were motor developmental delay, intellectual disability, hypotonia, and spasticity. INTERPRETATION: The cumulative childhood incidence of childhood-onset GWMDs was higher than previously described. Comprehensive epidemiological and natural history data are needed before future clinical trials are undertaken. What this paper adds Forty-nine distinct genetic white matter disorders (GWMDs) were identified, with 20% of cases being classic leukodystrophies. The cumulative childhood incidence of GWMDs was higher than described previously. A considerable proportion (36%) of GWMDs were previously undefined or recently characterised GWMDs. Mitochondrial aetiology was more common (21%) than previously reported.

Loss of DIAPH1 causes SCBMS, combined immunodeficiency, and mitochondrial dysfunction.

BACKGROUND: Homozygous loss of DIAPH1 results in seizures, cortical blindness, and microcephaly syndrome (SCBMS). We studied 5 Finnish and 2 Omani patients with loss of DIAPH1 presenting with SCBMS, mitochondrial dysfunction, and immunodeficiency. OBJECTIVE: We sought to further characterize phenotypes and disease mechanisms associated with loss of DIAPH1. METHODS: Exome sequencing, genotyping and haplotype analysis, B- and T-cell phenotyping, in vitro lymphocyte stimulation assays, analyses of mitochondrial function, immunofluorescence staining for cytoskeletal proteins and mitochondria, and CRISPR-Cas9 DIAPH1 knockout in heathy donor PBMCs were used. RESULTS: Genetic analyses found all Finnish patients homozygous for a rare DIAPH1 splice-variant (NM_005219:c.684+1G>A) enriched in the Finnish population, and Omani patients homozygous for a previously described pathogenic DIAPH1 frameshift-variant (NM_005219:c.2769delT;p.F923fs). In addition to microcephaly, epilepsy, and cortical blindness characteristic to SCBMS, the patients presented with infection susceptibility due to defective lymphocyte maturation and 3 patients developed B-cell lymphoma. Patients' immunophenotype was characterized by poor lymphocyte activation and proliferation, defective B-cell maturation, and lack of naive T cells. CRISPR-Cas9 knockout of DIAPH1 in PBMCs from healthy donors replicated the T-cell activation defect. Patient-derived peripheral blood T cells exhibited impaired adhesion and inefficient microtubule-organizing center repositioning to the immunologic synapse. The clinical symptoms and laboratory tests also suggested mitochondrial dysfunction. Experiments with immortalized, patient-derived fibroblasts indicated that DIAPH1 affects the amount of complex IV of the mitochondrial respiratory chain. CONCLUSIONS: Our data demonstrate that individuals with SCBMS can have combined immune deficiency and implicate defective cytoskeletal organization and mitochondrial dysfunction in SCBMS pathogenesis.

TT2020 meeting report on the 16th Transgenic Technology Meeting.

The 16th transgenic technology (TT) meeting of the International Society of Transgenic technology (ISTT) took place on October 26-29th 2020 and was quite unique as it was the first-ever virtual meeting in the history of ISTT events. Dr. Rebecca Haffner-Krausz at Weizmann Institute of Science, Israel, was the local organizer of the meeting, which attracted 756 registered participants from 32 different countries.

Transmembrane Prolyl 4-Hydroxylase is a Novel Regulator of Calcium Signaling in Astrocytes.

Prolyl 4-hydroxylases (P4Hs) have vital roles in regulating collagen synthesis and hypoxia response. A transmembrane P4H (P4H-TM) is a recently identified member of the family. Biallelic loss of function P4H-TM mutations cause a severe autosomal recessive intellectual disability syndrome in humans, but functions of P4H-TM are essentially unknown at cellular level. Our microarray data on P4h-tm-/- mouse cortexes where P4H-TM is abundantly expressed indicated expression changes in genes involved in calcium signaling and expression of several calcium sequestering ATPases was upregulated in P4h-tm-/- primary mouse astrocytes. Cytosolic and intraorganellar calcium imaging of P4h-tm-/- cells revealed that receptor-operated calcium entry (ROCE) and store-operated calcium entry (SOCE) and calcium re-uptake by mitochondria were compromised. HIF1, but not HIF2, was found to be a key mediator of the P4H-TM effect on calcium signaling. Furthermore, total internal reflection fluorescence (TIRF) imaging showed that calcium agonist-induced gliotransmission was attenuated in P4h-tm-/- astrocytes. This phenotype was accompanied by redistribution of mitochondria from distal processes to central parts of the cell body and decreased intracellular ATP content. Our data show that P4H-TM is a novel regulator of calcium dynamics and gliotransmission.