DTYMK is essential for genome integrity and neuronal survival.

Nucleotide metabolism is a complex pathway regulating crucial cellular processes such as nucleic acid synthesis, DNA repair and proliferation. This study shows that impairment of the biosynthesis of one of the building blocks of DNA, dTTP, causes a severe, early-onset neurodegenerative disease. Here, we describe two unrelated children with bi-allelic variants in DTYMK, encoding dTMPK, which catalyzes the penultimate step in dTTP biosynthesis. The affected children show severe microcephaly and growth retardation with minimal neurodevelopment. Brain imaging revealed severe cerebral atrophy and disappearance of the basal ganglia. In cells of affected individuals, dTMPK enzyme activity was minimal, along with impaired DNA replication. In addition, we generated dtymk mutant zebrafish that replicate this phenotype of microcephaly, neuronal cell death and early lethality. An increase of ribonucleotide incorporation in the genome as well as impaired responses to DNA damage were observed in dtymk mutant zebrafish, providing novel pathophysiological insights. It is highly remarkable that this deficiency is viable as an essential component for DNA cannot be generated, since the metabolic pathway for dTTP synthesis is completely blocked. In summary, by combining genetic and biochemical approaches in multiple models we identified loss-of-function of DTYMK as the cause of a severe postnatal neurodegenerative disease and highlight the essential nature of dTTP synthesis in the maintenance of genome stability and neuronal survival.

Pathophysiology and targets for treatment in hereditary galactosemia: A systematic review of animal and cellular models.

Since the first description of galactosemia in 1908 and despite decades of research, the pathophysiology is complex and not yet fully elucidated. Galactosemia is an inborn error of carbohydrate metabolism caused by deficient activity of any of the galactose metabolising enzymes. The current standard of care, a galactose-restricted diet, fails to prevent long-term complications. Studies in cellular and animal models in the past decades have led to an enormous progress and advancement of knowledge. Summarising current evidence in the pathophysiology underlying hereditary galactosemia may contribute to the identification of treatment targets for alternative therapies that may successfully prevent long-term complications. A systematic review of cellular and animal studies reporting on disease complications (clinical signs and/or biochemical findings) and/or treatment targets in hereditary galactosemia was performed. PubMed/MEDLINE, EMBASE, and Web of Science were searched, 46 original articles were included. Results revealed that Gal-1-P is not the sole pathophysiological agent responsible for the phenotype observed in galactosemia. Other currently described contributing factors include accumulation of galactose metabolites, uridine diphosphate (UDP)-hexose alterations and subsequent impaired glycosylation, endoplasmic reticulum (ER) stress, altered signalling pathways, and oxidative stress. galactokinase (GALK) inhibitors, UDP-glucose pyrophosphorylase (UGP) up-regulation, uridine supplementation, ER stress reducers, antioxidants and pharmacological chaperones have been studied, showing rescue of biochemical and/or clinical symptoms in galactosemia. Promising co-adjuvant therapies include antioxidant therapy and UGP up-regulation. This systematic review provides an overview of the scattered information resulting from animal and cellular studies performed in the past decades, summarising the complex pathophysiological mechanisms underlying hereditary galactosemia and providing insights on potential treatment targets.

Nucleotide sugar profiles throughout development in wildtype and galt knockout zebrafish.

Nucleotide sugars (NS) are fundamental molecules in life and play a key role in glycosylation reactions and signal conduction. Several pathways are involved in the synthesis of NS. The Leloir pathway, the main pathway for galactose metabolism, is crucial for production of uridine diphosphate (UDP)-glucose and UDP-galactose. The most common metabolic disease affecting this pathway is galactose-1-phosphate uridylyltransferase (GALT) deficiency, that despite a lifelong galactose-restricted diet, often results in chronically debilitating complications. Alterations in the levels of UDP-sugars leading to galactosylation abnormalities have been hypothesized as a key pathogenic factor. However, UDP-sugar levels measured in patient cell lines have shown contradictory results. Other NS that might be affected, differences throughout development, as well as tissue specific profiles have not been investigated. Using recently established UHPLC-MS/MS technology, we studied the complete NS profiles in wildtype and galt knockout zebrafish (Danio rerio). Analyses of UDP-hexoses, UDP-hexosamines, CMP-sialic acids, GDP-fucose, UDP-glucuronic acid, UDP-xylose, CDP-ribitol, and ADP-ribose profiles at four developmental stages and in tissues (brain and gonads) in wildtype zebrafish revealed variation in NS levels throughout development and differences between examined tissues. More specifically, we found higher levels of CMP-N-acetylneuraminic acid, GDP-fucose, UDP-glucuronic acid, and UDP-xylose in brain and of CMP-N-glycolylneuraminic acid in gonads. Analysis of the same NS profiles in galt knockout zebrafish revealed no significant differences from wildtype. Our findings in galt knockout zebrafish, even when challenged with galactose, do not support a role for abnormalities in UDP-glucose or UDP-galactose as a key pathogenic factor in GALT deficiency, under the tested conditions.

Δ9-tetrahydrocannabivarin impairs epithelial calcium transport through inhibition of TRPV5 and TRPV6.

Compounds extracted from the cannabis plant, including the psychoactive Δ9-tetrahydrocannabinol (THC) and related phytocannabinoids, evoke multiple diverse biological actions as ligands of the G protein-coupled cannabinoid receptors CB1 and CB2. In addition, there is increasing evidence that phytocannabinoids also have non-CB targets, including several ion channels of the transient receptor potential superfamily. We investigated the effects of six non-THC phytocannabinoids on the epithelial calcium channels TRPV5 and TRPV6, and found that one of them, Δ9-tetrahydrocannabivarin (THCV), exerted a strong and concentration-dependent inhibitory effect on mammalian TRPV5 and TRPV6 and on the single zebrafish orthologue drTRPV5/6. Moreover, THCV attenuated the drTRPV5/6-dependent ossification in zebrafish embryos in vivo. Oppositely, 11-hydroxy-THCV (THCV-OH), a product of THCV metabolism in mammals, stimulated drTRPV5/6-mediated Ca2+ uptake and ossification. These results identify the epithelial calcium channels TRPV5 and TRPV6 as novel targets of phytocannabinoids, and suggest that THCV-containing products may modulate TRPV5- and TRPV6-dependent epithelial calcium transport.

Impaired fertility and motor function in a zebrafish model for classic galactosemia.

Classic galactosemia is a genetic disorder of galactose metabolism, caused by severe deficiency of galactose-1-phosphate uridylyltransferase (GALT) enzyme activity due to mutations of the GALT gene. Its pathogenesis is still not fully elucidated, and a therapy that prevents chronic impairments is lacking. In order to move research forward, there is a high need for a novel animal model, which allows organ studies throughout development and high-throughput screening of pharmacologic compounds. Here, we describe the generation of a galt knockout zebrafish model and present its phenotypical characterization. Using a TALEN approach, a galt knockout line was successfully created. Accordingly, biochemical assays confirm essentially undetectable galt enzyme activity in homozygotes. Analogous to humans, galt knockout fish accumulate galactose-1-phosphate upon exposure to exogenous galactose. Furthermore, without prior exposure to exogenous galactose, they exhibit reduced motor activity and impaired fertility (lower egg quantity per mating, higher number of unsuccessful crossings), resembling the human phenotype(s) of neurological sequelae and subfertility. In conclusion, our galt knockout zebrafish model for classic galactosemia mimics the human phenotype(s) at biochemical and clinical levels. Future studies in our model will contribute to improved understanding and management of this disorder.

Biochemical Characterizations of Human TMPK Mutations Identified in Patients with Severe Microcephaly: Single Amino Acid Substitutions Impair Dimerization and Abolish Their Catalytic Activity.

Deoxythymidylate kinase (TMPK) is a key enzyme in the synthesis of deoxythymidine triphosphate (dTTP). Four TMPK variants (P81L, A99T, D128N, and a frameshift) have been identified in human patients who suffered from severe neurodegenerative diseases. However, the impact of these mutations on TMPK function has not been clarified. Here we show that in fibroblasts derived from a patient, the P81L and D128N mutations led to a complete loss of TMPK activity in mitochondria and extremely low and unstable TMPK activity in cytosol. Despite the lack of TMPK activity, the patient-derived fibroblasts apparently grew normal. To investigate the impact of the mutations on the enzyme function, the mutant TMPKs were expressed, purified, and characterized. The wild-type TMPK mainly exists as a dimer with high substrate binding affinity, that is, low K M value and high catalytic efficiency, that is, k cat/K M. In contrast, all mutants were present as monomers with dramatically reduced substrate binding affinity and catalytic efficiencies. Based on the human TMPK structure, none of the mutated amino acids interacted directly with the substrates. By structural analysis, we could explain why the respective amino acid substitutions could drastically alter the enzyme structure and catalytic function. In conclusion, TMPK mutations identified in patients represent loss of function mutations but surprisingly the proliferation rate of the patient-derived fibroblasts was normal, suggesting the existence of an alternative and hitherto unknown compensatory TMPK-like enzyme for dTTP synthesis. Further studies of the TMPK enzymes will help to elucidate the role of TMPK in neuropathology.

Multiview deconvolution approximation multiphoton microscopy of tissues and zebrafish larvae.

Imaging in three dimensions is necessary for thick tissues and small organisms. This is possible with tomographic optical microscopy techniques such as confocal, multiphoton and light sheet microscopy. All these techniques suffer from anisotropic resolution and limited penetration depth. In the past, Multiview microscopy-imaging the sample from different angles followed by 3D image reconstruction-was developed to address this issue for light sheet microscopy based on fluorescence signal. In this study we applied this methodology to accomplish Multiview imaging with multiphoton microscopy based on fluorescence and additionally second harmonic signal from myosin and collagen. It was shown that isotropic resolution was achieved, the entirety of the sample was visualized, and interference artifacts were suppressed allowing clear visualization of collagen fibrils and myofibrils. This method can be applied to any scanning microscopy technique without microscope modifications. It can be used for imaging tissue and whole mount small organisms such as heart tissue, and zebrafish larva in 3D, label-free or stained, with at least threefold axial resolution improvement which can be significant for the accurate quantification of small 3D structures.

Dopamine-neurotransmission and nociception in zebrafish: An anti-nociceptive role of dopamine receptor drd2a.

Dopamine (DA) is an important modulator in nociception and analgesia. Spinal DA receptors are involved in descending modulation of the nociceptive transmission. Genetic variations within DA neurotransmission have been associated with altered pain sensitivity and development of chronic pain syndromes. The variant rs6277 in dopamine receptor 2 a (drd2a) has been associated with a decreased D2 receptor availability and increased nociception. The aim of this study is to further characterize the role of DA neurotransmission in nociception and the anti-nociceptive function of drd2a. The phenotype caused by rs6277 was modelled in zebrafish larvae using morpholino's and the effect on nociception was tested using a validated behavioural assay. The anti-nociceptive role of drd2a was tested using pharmacological intervention of D2 agonist Quinpirole. The experiments demonstrate that a decrease in drd2a expression results in a pro-nociceptive behavioural phenotype (P = 0.016) after a heat stimulus. Furthermore, agonism of drd2a with agonist Quinpirole (0.2 µM) results in dose-dependent anti-nociception (P = 0.035) after a heat stimulus. From these results it is concluded that the dopamine receptor drd2a is involved in anti-nociceptive behaviour in zebrafish. The model allows further screening and testing of genetic variation and treatment involved in nociception.

A zebrafish model to study small-fiber neuropathy reveals a potential role for GDAP1.

Mutations in genes involved in mitochondrial dynamics (fusion and fission) have been implicated in many peripheral neuropathies. We hypothesized that defects in these genes could result in a phenotype resembling features of small-fiber neuropathy (SFN). This was investigated in zebrafish by knocking down two genes involved in mitochondrial dynamics gdap1 (possibly fission and motility) and opa1 (fusion) using established morpholinos. Our read-outs were nerve density in the caudal fin and a behavioral response to temperature changes, both based on comparable hallmarks of SFN in patients. Knockdown of gdap1 resulted in zebrafish embryos with a reduced density of sensory neurites compared to control morpholino-injected embryos. Furthermore, these embryos demonstrated a decreased temperature-related activity. In contrast, a knockdown of opa1 did not affect the density of sensory neurites nor the temperature-related activity. However, only the opa1 morphants had an effect on mitochondrial network morphology. As we were not able to visualize the mitochondria in the neurons, it could well be that changes in the mitochondrial network remained undetected. Our data indicate that GDAP1 knockdown affects sensory neurite development, however, it is unclear if a problem in mitochondrial fission and network formation is the pathophysiological mechanism. Although we did not observe an effect of inhibiting mitochondrial fusion during development, we still propose that genes involved in mitochondrial dynamics should be screened for mutations in patients with SFN.

Expression of pathogenic SCN9A mutations in the zebrafish: A model to study small-fiber neuropathy.

Small-fiber neuropathy (SFN) patients experience a spectrum of sensory abnormalities, including attenuated responses to non-noxious temperatures in combination with a decreased density of the small-nerve fibers. Gain-of-function mutations in the voltage-gated sodium channels SCN9A, SCN10A and SCN11A have been identified as an underlying genetic cause in a subpopulation of patients with SFN. Based on clinical-diagnostic tests for SFN, we have set up a panel of two read-outs reflecting SFN in zebrafish, being nerve density and behavioral responses. Nerve density was studied using a transgenic line in which the sensory neurons are GFP-labelled. For the behavioral experiments, a temperature-controlled water compartment was developed. This device allowed quantification of the behavioral response to temperature changes. By using these read-outs we demonstrated that zebrafish embryos transiently overexpressing the pathogenic human SCN9A p.(I228M) or p.(G856D) mutations both have a significantly decreased density of the small-nerve fibers. Additionally, larvae overexpressing the p.(I228M) mutation displayed a significant increase in activity induced by temperature change. As these features closely resemble the clinical hallmarks of SFN, our data suggest that transient overexpression of mutant human mRNA provides a model for SFN in zebrafish. This disease model may provide a basis for testing the pathogenicity of novel genetic variants identified in SFN patients. Furthermore, this model could be used for studying SFN pathophysiology in an in vivo model and for testing therapeutic interventions.

Genetic defects in mtDNA-encoded protein translation cause pediatric, mitochondrial cardiomyopathy with early-onset brain disease.

This study aims to identify gene defects in pediatric cardiomyopathy and early-onset brain disease with oxidative phosphorylation (OXPHOS) deficiencies. We applied whole-exome sequencing in three patients with pediatric cardiomyopathy and early-onset brain disease with OXPHOS deficiencies. The brain pathology was studied by MRI analysis. In consanguineous patient 1, we identified a homozygous intronic variant (c.850-3A > G) in the QRSL1 gene, which was predicted to cause abnormal splicing. The variant segregated with the disease and affected the protein function, which was confirmed by complementation studies, restoring OXPHOS function only with wild-type QRSL1. Patient 2 was compound heterozygous for two novel affected and disease-causing variants (c.[253G > A];[938G > A]) in the MTO1 gene. In patient 3, we detected one unknown affected and disease-causing variants (c.2872C > T) and one known disease-causing variant (c.1774C > T) in the AARS2 gene. The c.1774C > T variant was present in the paternal copy of the AARS2 gene, the c.2872C > T in the maternal copy. All genes were involved in translation of mtDNA-encoded proteins. Defects in mtDNA-encoded protein translation lead to severe pediatric cardiomyopathy and brain disease with OXPHOS abnormalities. This suggests that the heart and brain are particularly sensitive to defects in mitochondrial protein synthesis during late embryonic or early postnatal development, probably due to the massive mitochondrial biogenesis occurring at that stage. If both the heart and brain are involved, the prognosis is poor with a likely fatal outcome at young age.

Replication Errors Made During Oogenesis Lead to Detectable De Novo mtDNA Mutations in Zebrafish Oocytes with a Low mtDNA Copy Number.

Of all pathogenic mitochondrial DNA (mtDNA) mutations in humans, ∼25% is de novo, although the occurrence in oocytes has never been directly assessed. We used next-generation sequencing to detect point mutations directly in the mtDNA of 3-15 individual mature oocytes and three somatic tissues from eight zebrafish females. Various statistical and biological filters allowed reliable detection of de novo variants with heteroplasmy ≥1.5%. In total, we detected 38 de novo base substitutions, but no insertions or deletions. These 38 de novo mutations were present in 19 of 103 mature oocytes, indicating that ∼20% of the mature oocytes carry at least one de novo mutation with heteroplasmy ≥1.5%. This frequency of de novo mutations is close to that deducted from the reported error rate of polymerase gamma, the mitochondrial replication enzyme, implying that mtDNA replication errors made during oogenesis are a likely explanation. Substantial variation in the mutation prevalence among mature oocytes can be explained by the highly variable mtDNA copy number, since we previously reported that ∼20% of the primordial germ cells have a mtDNA copy number of ≤73 and would lead to detectable mutation loads. In conclusion, replication errors made during oogenesis are an important source of de novo mtDNA base substitutions and their location and heteroplasmy level determine their significance.

Differences in Strength and Timing of the mtDNA Bottleneck between Zebrafish Germline and Non-germline Cells.

We studied the mtDNA bottleneck in zebrafish to elucidate size, timing, and variation in germline and non-germline cells. Mature zebrafish oocytes contain, on average, 19.0 × 10(6) mtDNA molecules with high variation between oocytes. During embryogenesis, the mtDNA copy number decreases to ∼170 mtDNA molecules per primordial germ cell (PGC), a number similar to that in mammals, and to ∼50 per non-PGC. These occur at the same developmental stage, implying considerable variation in mtDNA copy number in (non-)PGCs of the same female, dictated by variation in the mature oocyte. The presence of oocytes with low mtDNA numbers, if similar in humans, could explain how (de novo) mutations can reach high mutation loads within a single generation. High mtDNA copy numbers in mature oocytes are established by mtDNA replication during oocyte development. Bottleneck differences between germline and non-germline cells, due to early differentiation of PGCs, may account for different distribution patterns of familial mutations.