IFT74 variants cause skeletal ciliopathy and motile cilia defects in mice and humans.

Motile and non-motile cilia play critical roles in mammalian development and health. These organelles are composed of a 1000 or more unique proteins, but their assembly depends entirely on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). In mammals, malfunction of non-motile cilia due to IFT dysfunction results in complex developmental phenotypes that affect most organs. In contrast, disruption of motile cilia function causes subfertility, disruption of the left-right body axis, and recurrent airway infections with progressive lung damage. In this work, we characterize allele specific phenotypes resulting from IFT74 dysfunction in human and mice. We identified two families carrying a deletion encompassing IFT74 exon 2, the first coding exon, resulting in a protein lacking the first 40 amino acids and two individuals carrying biallelic splice site mutations. Homozygous exon 2 deletion cases presented a ciliary chondrodysplasia with narrow thorax and progressive growth retardation along with a mucociliary clearance disorder phenotype with severely shorted cilia. Splice site variants resulted in a lethal skeletal chondrodysplasia phenotype. In mice, removal of the first 40 amino acids likewise results in a motile cilia phenotype but with little effect on primary cilia structure. Mice carrying this allele are born alive but are growth restricted and developed hydrocephaly in the first month of life. In contrast, a strong, likely null, allele of Ift74 in mouse completely blocks ciliary assembly and causes severe heart defects and midgestational lethality. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia resulting from increased mechanical stress and repair needs could account for the motile cilia phenotype observed in human and mice.

Renewal of oligodendrocyte lineage reverses dysmyelination and CNS neurodegeneration through corrected N-acetylaspartate metabolism.

Myelinating oligodendrocytes are essential for neuronal communication and homeostasis of the central nervous system (CNS). One of the most abundant molecules in the mammalian CNS is N-acetylaspartate (NAA), which is catabolized into L-aspartate and acetate by the enzyme aspartoacylase (ASPA) in oligodendrocytes. The resulting acetate moiety is thought to contribute to myelin lipid synthesis. In addition, affected NAA metabolism has been implicated in several neurological disorders, including leukodystrophies and demyelinating diseases such as multiple sclerosis. Genetic disruption of ASPA function causes Canavan disease, which is hallmarked by increased NAA levels, myelin and neuronal loss, large vacuole formation in the CNS, and early death in childhood. Although NAA's direct role in the CNS is inconclusive, in peripheral adipose tissue, NAA-derived acetate has been found to modify histones, a mechanism known to be involved in epigenetic regulation of cell differentiation. We hypothesize that a lack of cellular differentiation in the brain contributes to the disruption of myelination and neurodegeneration in diseases with altered NAA metabolism, such as Canavan disease. Our study demonstrates that loss of functional Aspa in mice disrupts myelination and shifts the transcriptional expression of neuronal and oligodendrocyte markers towards less differentiated stages in a spatiotemporal manner. Upon re-expression of ASPA, these oligodendrocyte and neuronal lineage markers are either improved or normalized, suggesting that NAA breakdown by Aspa plays an essential role in the maturation of neurons and oligodendrocytes. Also, this effect of ASPA re-expression is blunted in old mice, potentially due to limited ability of neuronal, rather than oligodendrocyte, recovery.

IFT74 variants cause skeletal ciliopathy and motile cilia defects in mice and humans.

Motile and non-motile cilia are critical to mammalian development and health. Assembly of these organelles depends on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). A series of human and mouse IFT74 variants were studied to understand the function of this IFT subunit. Humans missing exon 2, which codes for the first 40 residues, presented an unusual combination of ciliary chondrodysplasia and mucociliary clearance disorders while individuals carrying biallelic splice site variants developed a lethal skeletal chondrodysplasia. In mice, variants thought to remove all Ift74 function, completely block ciliary assembly and result in midgestational lethality. A mouse allele that removes the first 40 amino acids, analogous to the human exon 2 deletion, results in a motile cilia phenotype with mild skeletal abnormalities. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia could account for the motile cilia phenotype observed in human and mice.

Deficient spermiogenesis in mice lacking Rlim.

The X-linked gene Rlim plays major roles in female mouse development and reproduction, where it is crucial for the maintenance of imprinted X chromosome inactivation in extraembryonic tissues of embryos. However, while females carrying a systemic Rlim knockout (KO) die around implantation, male Rlim KO mice appear healthy and are fertile. Here, we report an important role for Rlim in testis where it is highly expressed in post-meiotic round spermatids as well as in Sertoli cells. Systemic deletion of the Rlim gene results in lower numbers of mature sperm that contains excess cytoplasm, leading to decreased sperm motility and in vitro fertilization rates. Targeting the conditional Rlim cKO specifically to the spermatogenic cell lineage largely recapitulates this phenotype. These results reveal functions of Rlim in male reproduction specifically in round spermatids during spermiogenesis.

UPRmt scales mitochondrial network expansion with protein synthesis via mitochondrial import in Caenorhabditis elegans.

As organisms develop, individual cells generate mitochondria to fulfill physiological requirements. However, it remains unknown how mitochondrial network expansion is scaled to cell growth. The mitochondrial unfolded protein response (UPRmt) is a signaling pathway mediated by the transcription factor ATFS-1 which harbors a mitochondrial targeting sequence (MTS). Here, using the model organism Caenorhabditis elegans we demonstrate that ATFS-1 mediates an adaptable mitochondrial network expansion program that is active throughout normal development. Mitochondrial network expansion requires the relatively inefficient MTS in ATFS-1, which allows the transcription factor to be responsive to parameters that impact protein import capacity of the mitochondrial network. Increasing the strength of the ATFS-1 MTS impairs UPRmt activity by increasing accumulation within mitochondria. Manipulations of TORC1 activity increase or decrease ATFS-1 activity in a manner that correlates with protein synthesis. Lastly, expression of mitochondrial-targeted GFP is sufficient to expand the muscle cell mitochondrial network in an ATFS-1-dependent manner. We propose that mitochondrial network expansion during development is an emergent property of the synthesis of highly expressed mitochondrial proteins that exclude ATFS-1 from mitochondrial import, causing UPRmt activation.

Thermus thermophilus Argonaute Functions in the Completion of DNA Replication.

In many eukaryotes, Argonaute proteins, guided by short RNA sequences, defend cells against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. Here, we report that TtAgo also participates in DNA replication. In vivo, TtAgo binds 15- to 18-nt DNA guides derived from the chromosomal region where replication terminates and associates with proteins known to act in DNA replication. When gyrase, the sole T. thermophilus type II topoisomerase, is inhibited, TtAgo allows the bacterium to finish replicating its circular genome. In contrast, loss of gyrase and TtAgo activity slows growth and produces long sausage-like filaments in which the individual bacteria are linked by DNA. Finally, wild-type T. thermophilus outcompetes an otherwise isogenic strain lacking TtAgo. We propose that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes generated by DNA replication.

Loss of Arf4 causes severe degeneration of the exocrine pancreas but not cystic kidney disease or retinal degeneration.

Arf4 is proposed to be a critical regulator of membrane protein trafficking in early secretory pathway. More recently, Arf4 was also implicated in regulating ciliary trafficking, however, this has not been comprehensively tested in vivo. To directly address Arf4's role in ciliary transport, we deleted Arf4 specifically in either rod photoreceptor cells, kidney, or globally during the early postnatal period. Arf4 deletion in photoreceptors did not cause protein mislocalization or retinal degeneration, as expected if Arf4 played a role in protein transport to the ciliary outer segment. Likewise, Arf4 deletion in kidney did not cause cystic disease, as expected if Arf4 were involved in general ciliary trafficking. In contrast, global Arf4 deletion in the early postnatal period resulted in growth restriction, severe pancreatic degeneration and early death. These findings are consistent with Arf4 playing a critical role in endomembrane trafficking, particularly in the pancreas, but not in ciliary function.

In situ tissue engineering: endothelial growth patterns as a function of flow diverter design.

BACKGROUND: Vascular remodeling in response to implantation of a tissue engineering scaffold such as a flow diverter (FD) leads to the cure of intracranial aneurysms. We hypothesize that the vascular response is dependent on FD design, and CD34+ progenitor cells play an important role in the endothelialization of the implant. METHODS: Sixteen rabbit aneurysms were randomly treated with two different single-layer braided FDs made of cobalt-chrome alloys. The FD-48 and FD-72 devices had 48 and 72 wires, respectively. Aneurysm occlusion rate was assessed during the final digital subtraction angiogram at 10, 20, 30, and 60 days (n=2 per device per time point). Implanted vessels were analyzed with scanning electron microscopy for tissue coverage, endothelialization, and immuno-gold labeling for CD34+ cells. RESULTS: Complete aneurysm occlusion rates were similar between the devices; however, complete or near complete occlusion was more frequently observed in aneurysms with neck ≤4.2 mm (p=0.008). Total tissue coverage at 10 days over the surface of the FD-48 and FD-72 devices was 56.4±11.6% and 76.6±3.6%, respectively. Endothelial cell growth over the surface was time-dependent for the FD-72 device (Spearman's r=0.86, p=0.013) but not for the FD-48 device (Spearman's r=-0.59, p=0.094). The endothelialization score was marginally correlated with the distance from the aneurysm neck for the FD-48 device (Spearman's r=1, p=0.083) but not for the FD-72 device (Spearman's r=0.8, p=0.33). CD34+ cells were present along the entirety of both devices at all time points. CONCLUSIONS: This study gives preliminary evidence that temporal and spatial endothelialization is dependent on FD design. Circulating CD34+ progenitor cells contribute to endothelialization throughout the healing process.

Genetic link between renal birth defects and congenital heart disease.

Structural birth defects in the kidney and urinary tract are observed in 0.5% of live births and are a major cause of end-stage renal disease, but their genetic aetiology is not well understood. Here we analyse 135 lines of mice identified in large-scale mouse mutagenesis screen and show that 29% of mutations causing congenital heart disease (CHD) also cause renal anomalies. The renal anomalies included duplex and multiplex kidneys, renal agenesis, hydronephrosis and cystic kidney disease. To assess the clinical relevance of these findings, we examined patients with CHD and observed a 30% co-occurrence of renal anomalies of a similar spectrum. Together, these findings demonstrate a common shared genetic aetiology for CHD and renal anomalies, indicating that CHD patients are at increased risk for complications from renal anomalies. This collection of mutant mouse models provides a resource for further studies to elucidate the developmental link between renal anomalies and CHD.

CFAP54 is required for proper ciliary motility and assembly of the central pair apparatus in mice.

Motile cilia and flagella play critical roles in fluid clearance and cell motility, and dysfunction commonly results in the pediatric syndrome primary ciliary dyskinesia (PCD). CFAP221, also known as PCDP1, is required for ciliary and flagellar function in mice and Chlamydomonas reinhardtii, where it localizes to the C1d projection of the central microtubule apparatus and functions in a complex that regulates flagellar motility in a calcium-dependent manner. We demonstrate that the genes encoding the mouse homologues of the other C. reinhardtii C1d complex members are primarily expressed in motile ciliated tissues, suggesting a conserved function in mammalian motile cilia. The requirement for one of these C1d complex members, CFAP54, was identified in a mouse line with a gene-trapped allele. Homozygous mice have PCD characterized by hydrocephalus, male infertility, and mucus accumulation. The infertility results from defects in spermatogenesis. Motile cilia have a structural defect in the C1d projection, indicating that the C1d assembly mechanism requires CFAP54. This structural defect results in decreased ciliary beat frequency and perturbed cilia-driven flow. This study identifies a critical role for CFAP54 in proper assembly and function of mammalian cilia and flagella and establishes the gene-trapped allele as a new model of PCD.

Quantitative analysis of high-resolution, contrast-enhanced, cone-beam CT for the detection of intracranial in-stent hyperplasia.

BACKGROUND: Intracranial in-stent hyperplasia is a stroke-associated complication that requires routine surveillance. OBJECTIVE: To compare the results of in vivo experiments to determine the accuracy and precision of in-stent hyperplasia measurements obtained with modified C-arm contrast-enhanced, cone-beam CT (CE-CBCT) imaging with those obtained by 'gold standard' histomorphometry. Additionally, to carry out clinical analyses comparing this CE-CBCT protocol with digital subtraction angiography (DSA). METHODS: A non-binned CE-CBCT protocol (VasoCT) was used that acquires x-ray images with a small field-of-view and applies a full-scale reconstruction algorithm providing high-resolution three-dimensional (3D) imaging with 100 µm isotropic voxels. In an vivo porcine model, VasoCT cross-sectional area measurements were compared with gold standard vessel histology. VasoCT and DSA were used to calculate in-stent stenosis in 23 imaging studies. RESULTS: Porcine VasoCT cross-sectional stent, lumen, and in-stent hyperplasia areas strongly correlated with histological measurements (r(2)=0.97, 0.93, 0.90; slope=1.14, 1.07, and 0.76, respectively; p<0.0001). Clinical VasoCT percentage stenosis correlated well with DSA percentage stenosis (r(2)=0.84; slope=0.76), and the two techniques were free of consistent bias (Bland-Altman, bias=3.29%; 95% CI -14.75% to 21.33%). An illustrative clinical case demonstrated the advantages of VasoCT, including 3D capability and non-invasive IV contrast administration, for detection of in-stent hyperplasia. CONCLUSIONS: C-arm VasoCT is a high-resolution 3D capable imaging technique that has been validated in an animal model for measurement of in-stent tissue growth. Successful clinical implementation of the protocol was performed in a small case series.

Arabidopsis separase functions beyond the removal of sister chromatid cohesion during meiosis.

Separase is a capase family protease that is required for the release of sister chromatid cohesion during meiosis and mitosis. Proteolytic cleavage of the alpha-kleisin subunit of the cohesin complex at the metaphase-to-anaphase transition is essential for the proper segregation of chromosomes. In addition to its highly conserved role in cleaving the alpha-kleisin subunit, separase appears to have acquired additional diverse activities in some organisms, including involvement in mitotic and meiotic anaphase spindle assembly and elongation, interphase spindle pole body positioning, and epithelial cell reorganization. Results from the characterization of Arabidopsis (Arabidopsis thaliana) separase (ESP) demonstrated that meiotic expression of ESP RNA interference blocked the proper removal of cohesin from chromosomes and resulted in the presence of a mixture of fragmented chromosomes and intact bivalents. The presence of large numbers of intact bivalents raised the possibility that separase may also have multiple roles in Arabidopsis. In this report, we show that meiotic expression of ESP RNA interference blocks the removal of cohesin during both meiosis I and II, results in alterations in nonhomologous centromere association, disrupts the radial microtubule system after telophase II, and affects the proper establishment of nuclear cytoplasmic domains, resulting in the formation of multinucleate microspores.

The Arabidopsis cohesin protein SYN3 localizes to the nucleolus and is essential for gametogenesis.

Alpha-kleisins are core components of meiotic and mitotic cohesin complexes. Arabidopsis contains genes for four alpha-kleisin proteins encoded by SYN genes. SYN1, a REC8 ortholog, is essential for meiosis, while SYN2 and SYN4 appear to be SCC1 orthologs and function in mitosis. Our analysis of AtSYN3 shows that it localizes primarily in the nucleolus of both meiotic and mitotic cells. Furthermore, analysis of plants containing an AtSYN3 T-DNA knockout mutation demonstrated that it is essential for megagametogenesis and plays an important role in pollen. These results suggest that SYN3 may not function as part of a typical cohesin complex; rather it may have evolved a specialized role in controlling rDNA structure, transcription or rRNA processing.

Subdioecy in Consolea spinosissima (Cactaceae): breeding system and embryological studies.

The breeding system and the embryology of Consolea spinosissima, a tree-like opuntioid endemic to Jamaica, were investigated. Morphological and embryological studies revealed that the species is subdioecious, with three sexual morphs present in the 150 × 120 m plot studied at Hellshire Hills, Jamaica. The female morph has pistillate flowers with open stigma lobes, no pollen grains, and sets fruit. The male morph has cryptic staminate flowers with closed stigma lobes, viable pollen grains, and a nonfunctional gynoecium that does not set seed. The weak hermaphrodite morph has low fruit set and "perfect" flowers that superficially resemble the functionally staminate flowers of the male morph. These perfect flowers reach anthesis with viable pollen grains, with no or only a few functional ovules, and with the style supporting pollen tube growth. Embryological studies showed that the critical stage for sex determination occurs earlier in pistillate than in staminate and perfect flowers. Anthers of pistillate flowers abort prior to microspore tetrad formation, whereas ovules of the staminate and perfect flowers degenerate after the complete maturation of the embryo sac. Based on flower structure and embryological data, we hypothesize that the ancestor of C. spinosissima is/was hermaphroditic.