Bi-allelic loss-of-function variants in BCAS3 cause a syndromic neurodevelopmental disorder.

BCAS3 microtubule-associated cell migration factor (BCAS3) is a large, highly conserved cytoskeletal protein previously proposed to be critical in angiogenesis and implicated in human embryogenesis and tumorigenesis. Here, we established BCAS3 loss-of-function variants as causative for a neurodevelopmental disorder. We report 15 individuals from eight unrelated families with germline bi-allelic loss-of-function variants in BCAS3. All probands share a global developmental delay accompanied by pyramidal tract involvement, microcephaly, short stature, strabismus, dysmorphic facial features, and seizures. The human phenotype is less severe compared with the Bcas3 knockout mouse model and cannot be explained by angiogenic defects alone. Consistent with being loss-of-function alleles, we observed absence of BCAS3 in probands' primary fibroblasts. By comparing the transcriptomic and proteomic data based on probands' fibroblasts with those of the knockout mouse model, we identified similar dysregulated pathways resulting from over-representation analysis, while the dysregulation of some proposed key interactors could not be confirmed. Together with the results from a tissue-specific Drosophila loss-of-function model, we demonstrate a vital role for BCAS3 in neural tissue development.

Expression, purification and characterization of two Clostridium acetobutylicum flavodoxins: potential electron transfer partners for CYP152A2.

Two flavodoxin genes from Clostridium acetobutylicum, CacFld1 (CAC0587) and CacFld2 (CAC3417), were expressed in Escherichia coli and investigated for their ability to support activity of CYP152A2, a fatty acid hydroxylase from C. acetobutylicum. E. coli flavodoxin reductase (FdR) was used as a redox partner, since flavodoxin reductase CacFdR (CAC0196) from C. acetobutylicum could not be purified in a functional form. CacFld1 was shown to accept electrons from FdR and transfer them to CYP152A2. Since H2O2 was generated by uncoupling at different stages of the reconstituted electron transfer chain, catalase was used as H2O2 scavenger in order to exclude peroxygenation by CYP152A2. The reconstituted P450 system with CacFld1 and FdR oxidized myristic acid with a K(M) of 137 µM and a k(cat) of 36 min⁻¹. Furthermore, the hydroxylase activity of CYP152A2 towards myristic acid with CacFld1 was 17-fold higher than without CacFld1. Along with CYP152A2 and a physiological flavodoxin reductase, CacFld1 is therefore likely to be involved in oxygen detoxification in C. acetobutylicum. Flavodoxin CacFld2 did not accept electrons from NADPH-reduced FdR, though it cannot be excluded as a candidate redox partner for CYP152A2 in the presence of an appropriate physiological reductase.

Comparative Transcriptional Profiling of Motor Neuron Disorder-Associated Genes in Various Human Cell Culture Models.

Disease modeling requires appropriate cellular models that best mimic the underlying pathophysiology. Human origin and an adequate expression of the disease protein are pre-requisites that support information from a model to be meaningful. In this study we investigated expression profiles of (i) PBMCs and (ii) fibroblasts as patient derived cells as well as (iii) lymphoblasts and (iv) induced pluripotent stem cells (iPSC) as immortalized sources, and (v) iPSC-derived cortical neurons to assess their aptitude to model motor neuron diseases (MNDs) including hereditary spastic paraplegia (HSP), amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). We generated all five different cell types from two healthy donors and performed RNA sequencing to display expression patterns in MND-related genes. For the ten most common HSP genotypes we validated gene expression by qPCR. To verify the results on protein level, proteome analysis of fibroblasts, iPSCs and cortical neurons was performed. Depending on the specific MND gene we found largely different expression patterns. Out of 168 MND-related genes, 50 had their highest expression in iPSC-derived cortical neurons, 41 were most strongly expressed in fibroblasts, 26 in lymphoblasts, 22 in iPSCs, and 14 in PBMCs. Pathophysiologically related MNDs like HSPs associated with axonal transport deficits shared highest expression in cortical neurons. 15 MND-related genes were not detectable in any of the analyzed cell types. This may reflect the critical dependency of motor neurons on support of other cell types like oligodendrocytes which express myelin proteins like L1CAM (SPG1), PLP1 (SPG2) and MAG (SPG75) which are lacking in neurons but cause MNDs if mutated. This study provides comprehensive information on expression of genes associated with a large spectrum of MNDs. Expression profiles can be used to inform on appropriate cell models for genotype specific motor neuron research.

FIG4 mutations leading to parkinsonism and a phenotypical continuum between CMT4J and Yunis Varón syndrome.

BACKGROUND: Charcot-Marie-Tooth disease type 4J (CMT4J) originates from mutations in the FIG4 gene and leads to distal muscle weakness. Two null alleles of FIG4 cause Yunis Varón syndrome with severe central nervous system involvement, cleidocranial dysmorphism, absent thumbs and halluces and early death. OBJECTIVES: To analyse the phenotypic spectrum of FIG4-related disease and explore effects of residual FIG4 protein. METHODS: Phenotyping of five new patients with FIG4-related disease. Western Blot analyses of FIG4 from patient fibroblasts. RESULTS: Next generation sequencing revealed compound heterozygous variants in FIG4 in five patients. All five patients presented with peripheral neuropathy, various degree of dysmorphism and a central nervous system involvement comprising Parkinsonism in 3/5 patients, cerebellar ataxia (1/5), spasticity of lower limbs (1/5), epilepsy (1/5) and/or cognitive deficits (2/5). Onset varied between the first and the seventh decade. There was no residual FIG4 protein detectable in fibroblasts of the four analysed patients. CONCLUSION: This study extends the phenotypic spectrum of FIG4-related disease to Parkinsonism as a feature and demonstrates new phenotypes on a continuum between CMT4J and Yunis Varón syndrome.

Decreased Na+/K+ ATPase Expression and Depolarized Cell Membrane in Neurons Differentiated from Chorea-Acanthocytosis Patients.

Loss of function mutations of the chorein-encoding gene VPS13A lead to chorea-acanthocytosis (ChAc), a neurodegenerative disorder with accelerated suicidal neuronal cell death, which could be reversed by lithium. Chorein upregulates the serum and glucocorticoid inducible kinase SGK1. Targets of SGK1 include the Na+/K+-ATPase, a pump required for cell survival. To explore whether chorein-deficiency affects Na+/K+ pump capacity, cortical neurons were differentiated from iPSCs generated from fibroblasts of ChAc patients and healthy volunteers. Na+/K+ pump capacity was estimated from K+-induced whole cell outward current (pump capacity). As a result, the pump capacity was completely abolished in the presence of Na+/K+ pump-inhibitor ouabain (100 µM), was significantly smaller in ChAc neurons than in control neurons, and was significantly increased in ChAc neurons by lithium treatment (24 hours 2 mM). The effect of lithium was reversed by SGK1-inhibitor GSK650394 (24 h 10 µM). Transmembrane potential (Vm) was significantly less negative in ChAc neurons than in control neurons, and was significantly increased in ChAc neurons by lithium treatment (2 mM, 24 hours). The effect of lithium on Vm was virtually abrogated by ouabain. Na+/K+ α1-subunit transcript levels and protein abundance were significantly lower in ChAc neurons than in control neurons, an effect reversed by lithium treatment (2 mM, 24 hours). In conclusion, consequences of chorein deficiency in ChAc include impaired Na+/K+ pump capacity.

Generation of a homozygous CRISPR/Cas9-mediated knockout human iPSC line for the STUB1 locus.

STUB1/CHIP is a central component of cellular protein homeostasis and interacts with key proteins involved in the pathogenesis of many neurodegenerative diseases. Missense and truncating mutations in STUB1 lead to SCAR16. For ideal in vitro disease modelling with isogenic controls, we generated a CHIP knockout cell line from a healthy control with no CHIP functionality, but remaining genomic integrity and verified pluripotency.

mRNA as a Novel Treatment Strategy for Hereditary Spastic Paraplegia Type 5.

Hereditary spastic paraplegia type 5 is a neurodegenerative disease caused by loss-of-function mutations in the CYP7B1 gene encoding the oxysterol 7-α-hydroxylase involved in bile acid synthesis in the liver. Lack of CYP7B1 leads to an accumulation of its oxysterol substrates, in particular 25-hydroxycholesterol and 27-hydroxycholesterol that are able to cross the blood-brain barrier and have neurotoxic properties. A potential therapeutic strategy for SPG5 is the replacement of CYP7B1 by administration of mRNA. Here, we studied the intravenous application of formulated mouse and human CYP7B1 mRNA in mice lacking the endogenous Cyp7b1 gene. A single-dose injection of either mouse or human CYP7B1 mRNA led to a pronounced degradation of oxysterols in liver and serum within 2 days of treatment. Pharmacokinetics indicate a single injection of human CYP7B1 mRNA to be effective in reducing oxysterols for at least 5 days. Repetitive applications of mRNA were safe for at least 17 days and resulted in a significant reduction of neurotoxic oxysterols not only in liver and serum but also to some extent in the brain. Our study highlights the potential to use mRNA as a novel therapy to treat patients with SPG5 disease.

Natural History of Squamous Intraepithelial Lesions in Pregnancy and Mode of Delivery.

BACKGROUND: Numerous studies have addressed the impact of mode of delivery on the natural history of squamous intraepithelial lesions (SIL) in pregnant women. However, the literature is still contradictory. PATIENTS AND METHODS: In the course of a retrospective analysis, data of 63 pregnant women with abnormal cervical smears who were referred to our Outpatient Department for pre-invasive lesions of the cervix were analyzed. The study was conducted at the General Hospital in Vienna, Austria, between 2010 and 2015. Data collection included demographics, delivery route and diagnostic results of cervical lesions by cytology, colposcopy, human papilloma virus (HPV) testing, histological report of punch biopsy and, if applicable, cone biopsy. RESULTS: Among 63 women who met the inclusion criteria, 40 (63%) delivered vaginally and 23 (37%) underwent caesarean section. Postpartum regression of cervical dysplasia was documented in 15 women delivering vaginally and in 10 who had a caesarean section (p=0.641). Among those women who delivered vaginally, three had progression and in 22 women the lesions persisted postpartum. In the group of women with caesarean section, one had progression and the lesions of 12 women persisted after delivery. No woman had progression to invasive disease. CONCLUSION: The mode of delivery does not significantly influence the natural history of cervical dysplastic lesions in pregnant women. The numbers of spontaneous regressions to normal cervical cytology during pregnancy were similar in both groups.

Inactivation of the Mouse L-Proline Transporter PROT Alters Glutamatergic Synapse Biochemistry and Perturbs Behaviors Required to Respond to Environmental Changes.

The endogenous neutral amino acid L-proline exhibits a variety of physiological and behavioral actions in the nervous system, highlighting the importance of accurately regulating its extracellular abundance. The L-proline transporter PROT (Slc6A7) is believed to control the spatial and temporal distribution of L-proline at glutamatergic synapses by rapid uptake of this amino acid into presynaptic terminals. Despite the importance of members of the Slc6 transporter family regulating neurotransmitter signaling and homeostasis in brain, evidence that PROT dysfunction supports risk for mental illness is lacking. Here we report the disruption of the PROT gene by homologous recombination. Mice defective in PROT displayed altered expression of glutamate transmission-related synaptic proteins in cortex and thalamus. PROT deficiency perturbed mouse behavior, such as reduced locomotor activity, decreased approach motivation and impaired memory extinction. Thus, our study demonstrates that PROT regulates behaviors that are needed to respond to environmental changes in vivo and suggests that PROT dysfunctions might contribute to mental disorders showing altered response choice following task contingency changes.

Establishment of STUB1/CHIP mutant induced pluripotent stem cells (iPSCs) from a patient with Gordon Holmes syndrome/SCAR16.

STUB1/CHIP is a central component of cellular protein homeostasis and interacts with key proteins involved in the pathogenesis of many neurodegenerative diseases. Here, we reprogrammed human skin fibroblasts from a 12-year-old male patient with recessive spinocerebellar ataxia type 16 (OMIM #615768), carrying compound heterozygous mutations (c.355C>T, c.880A>T) in STUB1. Genomic integrity of the iPSC line HIHCNi001-A without transgene integration and genomic aberration but with maintained disease-relevant mutations was proven by SNP array analysis and Sanger sequencing while pluripotency was verified by the expression of important pluripotency markers and the capacity to differentiate into cells of all three germ layers.

GlyT1 determines the glycinergic phenotype of amacrine cells in the mouse retina.

The amino acid glycine acts as a neurotransmitter at both inhibitory glycinergic and excitatory glutamatergic synapses predominantly in caudal regions of the central nervous system but also in frontal brain regions and the retina. After its presynaptic release and binding to postsynaptic receptors at caudal glycinergic synapses, two high-affinity glycine transporters GlyT1 and GlyT2 remove glycine from the extracellular space. Glycinergic neurons express GlyT2, which is essential for the presynaptic replenishment of the transmitter, while glial-expressed GlyT1 was shown to control the extracellular glycine concentration. Here we show that GlyT1 expressed by glycinergic amacrine cells of the retina does not only contribute to the control of the extracellular glycine concentration in the retina but is also essential for the maintenance of the glycinergic transmitter phenotype of this cell population. Specifically, loss of GlyT1 from the glycinergic AII amacrine cells impairs AII-mediated glycinergic neurotransmission and alters regulation of the extracellular glycine concentration, without changes in the overall distribution and/or size of glycinergic synapses. Taken together, our results suggest that GlyT1 expressed by amacrine cells in the retina combines functions covered by neuronal GlyT2 and glial GlyT1 at caudal glycinergic synapses.

Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody.

The P2X7 channel is involved in the pathogenesis of various CNS diseases. An increasing number of studies suggest its presence in neurons where its putative functions remain controversial for more than a decade. To resolve this issue and to provide a model for analysis of P2X7 functions, we generated P2X7 BAC transgenic mice that allow visualization of functional EGFP-tagged P2X7 receptors in vivo. Extensive characterization of these mice revealed dominant P2X7-EGFP protein expression in microglia, Bergmann glia, and oligodendrocytes, but not in neurons. These findings were further validated by microglia- and oligodendrocyte-specific P2X7 deletion and a novel P2X7-specific nanobody. In addition to the first quantitative analysis of P2X7 protein expression in the CNS, we show potential consequences of its overexpression in ischemic retina and post-traumatic cerebral cortex grey matter. This novel mouse model overcomes previous limitations in P2X7 research and will help to determine its physiological roles and contribution to diseases.

Generation of optic atrophy 1 patient-derived induced pluripotent stem cells (iPS-OPA1-BEHR) for disease modeling of complex optic atrophy syndromes (Behr syndrome).

Human skin fibroblasts were isolated from a 48-year-old patient carrying compound heterozygous mutations (c.610+364G>A and c.1311A>G) in OPA1, responsible for early onset optic atrophy complicated by ataxia and pyramidal signs (Behr syndrome; OMIM #210000). Fibroblasts were reprogrammed using episomal plasmids carrying hOCT4, hSOX2, hKLF4, hL-MYC and hLIN28. The generated transgene-free line iPS-OPA1-BEHR showed no additional genomic aberrations, maintained the disease-relevant mutations, expressed important pluripotency markers and was capable to differentiate into cells of all three germ layers in vitro. The generated iPS-OPA1-BEHR line might be a useful platform to study the pathomechanism of early onset complicated optic atrophy syndromes.

Establishment of SPAST mutant induced pluripotent stem cells (iPSCs) from a hereditary spastic paraplegia (HSP) patient.

Human skin fibroblasts were isolated from a 40-year-old hereditary spastic paraplegia patient carrying an intronic splice site mutation (c.1687+2T>A) in SPAST, leading to hereditary spastic paraplegia type 4 (SPG4). Fibroblasts were reprogrammed using episomal plasmids carrying hOCT4, hSOX2, hKLF4, hL-MYC and hLIN28. The generated transgene-free line iPS-SPG4-splice retained the specific mutation with no additional genomic aberrations, expressed pluripotency markers and was able to differentiate into cells of all germ layers in vitro. The generated iPS-SPG4-splice line might be a useful platform to study the pathomechanism of SPG4.

[Achilles heel of COPD]. / Atemnot und gehunfähigkeit.

We report the case of a 73 year old patient with a COPD. A severe exazerbation was treated with glucocorticoids and Quinolones. A few days later both ankles were tender and painful. Ultrasound examination showed bilateral Achilles tendon ruptures. Diagnostics, etiology and therapeutic options are discussed.

Lectin binding patterns and carbohydrate mediation of sperm binding to llama oviductal cells in vitro.

Sperm binding to oviductal epithelium would be involved in sperm reservoir formation in the utero tubal junction (UTJ). Although in other mammals sperm-oviduct interaction has been proved to be mediated by carbohydrate-recognition mechanisms, the factors implicated in the sperm adhesion to oviductal epithelium of llama are still unknown. In order to assess the role of carbohydrates present in the mucosa surface, we examined the distribution of glycoconjugates in the llama oviduct by confocal lectin-histochemistry. Mannosyl, glucosyl, N-acetylglucosaminyl, galactosyl, N-acetylgalactosaminyl and sialic acid residues were detected in the oviductal mucose glycocalyx. By incubation of UTJ oviductal explants with LCA, DBA, UEA-1 or PNA lectin previous to co-culture with sperm, we observed a significant decrease in sperm binding only with LCA lectin. In the mucosa surface there were numerous d-glucosyl and D-manosyl residues, which were spotted by this lectin. Probably, this fact promotes the whole covering of the oviduct luminal surface by the sugar-lectin complex, preventing sperm access and adhesion of further residues. However, sperm incubation with mannose or glucose does not significantly prevent binding, which means that glucose and mannose would not be involved in a specific sperm-oviduct interaction. On the other hand, we observed a high reduction in sperm binding to UTJ explants with N-acetylgalactosamine and galactose (p<0.001). Coincidentally, binding sites for N-acetylgalactosamine-PAA-FITC conjugate were observed on the whole surface of the sperm, supporting the concept that llama sperm have lectin-like molecules in their surface, as is the case in other mammals. Probably, these lectin-like molecules, by means of N-acetylgalactosamine and galactose recognition, could link the sperm to the oviductal mucosa with the purpose of forming storing sites in the UTJ. Our results support the idea that more than one carbohydrate could participate in sperm reservoir formation in the llama UTJ oviductal segment.

Cytochrome P450 monooxygenase from Clostridium acetobutylicum: a new alpha-fatty acid hydroxylase.

Cytochrome P450 monooxygenase from the anaerobic microorganism Clostridium acetobutylicum (CYP152A2) has been produced in Escherichia coli. CYP152A2 was shown to bind a broad range of saturated and unsaturated fatty acids and corresponding methyl esters and demonstrated a high peroxygenase activity of up to 200min(-1) with myristic acid. Although a high concentration of hydrogen peroxide of 200microM was necessary for high activities of the enzyme, it led to a fast enzyme inactivation within 2-4min. This might reflect the natural function of CYP152A2 as a rapid hydrogen peroxide scavenging enzyme. In two different reconstituted systems with NADPH, CYP152A2 was able to convert 10 times more substrate, if provided with flavodoxin and flavodoxin reductase from E. coli and even 30-40 times more substrate with the CYP102A1-reductase from Bacillus megaterium. According to the clear preference for hydroxylation at alpha-position, CYP152A2 can be referred to as fatty acid alpha-hydroxylase.