Fibulin-4 deficiency differentially affects cytoskeleton structure and dynamics as well as TGFß signaling.

Fibulin-4 is an extracellular matrix (ECM) protein essential for elastogenesis and mutations in this protein lead to aneurysm formation. In this study, we isolated vascular smooth muscle cells (VSMCs) from mice with reduced fibulin-4 protein expression (Fibulin-4R/R) and from mice with a smooth muscle cell specific deletion of the Fibulin-4 gene (Fibulin-4f/-/SM22Cre+). We subsequently analyzed and compared the molecular consequences of reduced Fibulin-4 expression versus total ablation of Fibulin-4 expression with regard to effects on the SMC specific contractile machinery, cellular migration and TGFß signaling. Analysis of the cytoskeleton showed that while Fibulin-4f/-/SM22Cre+ VSMCs lack smooth muscle actin (SMA) fibers, Fibulin-4R/R VSMCs were able to form SMA fibers. Furthermore, Fibulin-4f/-/SM22Cre+ VSMCs showed a decreased pCofilin to Cofilin ratio, suggesting increased actin depolymerization, while Fibulin-4R/R VSMCs did not display this decrease. Yet, both Fibulin-4 mutant VSMCs showed decreased migration. We found increased activation of TGFß signaling in Fibulin-4R/R VSMCs. However, TGFß signaling was not increased in Fibulin-4f/-/SM22Cre+ VSMCs. From these results we conclude that both reduction and absence of Fibulin-4 leads to structural and functional impairment of the SMA cytoskeleton. However, while reduced levels of Fibulin-4 result in increased TGFß activation, complete absence of Fibulin-4 does not result in increased TGFß activation. Since both mouse models show thoracic aortic aneurysm formation, we conclude that not only hampered TGFß signaling, but also SMA cytoskeleton dynamics play an important role in aortic aneurysmal disease.

Synaptic vesicle dynamic changes in a model of fragile X.

BACKGROUND: Fragile X syndrome (FXS) is a single-gene disorder that is the most common heritable cause of intellectual disability and the most frequent monogenic cause of autism spectrum disorders (ASD). FXS is caused by an expansion of trinucleotide repeats in the promoter region of the fragile X mental retardation gene (Fmr1). This leads to a lack of fragile X mental retardation protein (FMRP), which regulates translation of a wide range of messenger RNAs (mRNAs). The extent of expression level alterations of synaptic proteins affected by FMRP loss and their consequences on synaptic dynamics in FXS has not been fully investigated. METHODS: Here, we used an Fmr1 knockout (KO) mouse model to investigate the molecular mechanisms underlying FXS by monitoring protein expression changes using shotgun label-free liquid-chromatography mass spectrometry (LC-MS(E)) in brain tissue and synaptosome fractions. FXS-associated candidate proteins were validated using selected reaction monitoring (SRM) in synaptosome fractions for targeted protein quantification. Furthermore, functional alterations in synaptic release and dynamics were evaluated using live-cell imaging, and interpretation of synaptic dynamics differences was investigated using electron microscopy. RESULTS: Key findings relate to altered levels of proteins involved in GABA-signalling, especially in the cerebellum. Further exploration using microscopy studies found reduced synaptic vesicle unloading of hippocampal neurons and increased vesicle unloading in cerebellar neurons, which suggests a general decrease of synaptic transmission. CONCLUSIONS: Our findings suggest that FMRP is a regulator of synaptic vesicle dynamics, which supports the role of FMRP in presynaptic functions. Taken together, these studies provide novel insights into the molecular changes associated with FXS.

Mutations in a TGF-ß ligand, TGFB3, cause syndromic aortic aneurysms and dissections.

BACKGROUND: Aneurysms affecting the aorta are a common condition associated with high mortality as a result of aortic dissection or rupture. Investigations of the pathogenic mechanisms involved in syndromic types of thoracic aortic aneurysms, such as Marfan and Loeys-Dietz syndromes, have revealed an important contribution of disturbed transforming growth factor (TGF)-ß signaling. OBJECTIVES: This study sought to discover a novel gene causing syndromic aortic aneurysms in order to unravel the underlying pathogenesis. METHODS: We combined genome-wide linkage analysis, exome sequencing, and candidate gene Sanger sequencing in a total of 470 index cases with thoracic aortic aneurysms. Extensive cardiological examination, including physical examination, electrocardiography, and transthoracic echocardiography was performed. In adults, imaging of the entire aorta using computed tomography or magnetic resonance imaging was done. RESULTS: Here, we report on 43 patients from 11 families with syndromic presentations of aortic aneurysms caused by TGFB3 mutations. We demonstrate that TGFB3 mutations are associated with significant cardiovascular involvement, including thoracic/abdominal aortic aneurysm and dissection, and mitral valve disease. Other systemic features overlap clinically with Loeys-Dietz, Shprintzen-Goldberg, and Marfan syndromes, including cleft palate, bifid uvula, skeletal overgrowth, cervical spine instability and clubfoot deformity. In line with previous observations in aortic wall tissues of patients with mutations in effectors of TGF-ß signaling (TGFBR1/2, SMAD3, and TGFB2), we confirm a paradoxical up-regulation of both canonical and noncanonical TGF-ß signaling in association with up-regulation of the expression of TGF-ß ligands. CONCLUSIONS: Our findings emphasize the broad clinical variability associated with TGFB3 mutations and highlight the importance of early recognition of the disease because of high cardiovascular risk.

Transporters involved in the hepatic uptake of (99m)Tc-mebrofenin and indocyanine green.

BACKGROUND & AIMS: (99m)Tc-mebrofenin hepatobiliary scintigraphy (HBS) and the indocyanine green (ICG) clearance test are used for the assessment of hepatic function before and after liver surgery. The hepatic uptake of (99m)Tc-mebrofenin and ICG is considered similar to the uptake of organic anions such as bilirubin and bile acids. Little is known about hepatic uptake mechanisms of both compounds and recent evidence suggests that the hepatic transporters for ICG and (99m)Tc-mebrofenin are distinct. The aim of this study was to identify the specific human hepatic transporters of (99m)Tc-mebrofenin and ICG. METHODS: The uptake of (99m)Tc-mebrofenin was investigated in cRNA-injected Xenopus laevis oocytes expressing human OATP1B1, OATP1B3, OATP2B1, or NTCP. Chinese hamster ovary (CHO) cells stably expressing OATP1B1, OATP1B3, OATP2B1, or NTCP were used as a mammalian expression system. ICG transport into CHO cells was additionally imaged with confocal microscopy. RESULTS: We demonstrated that OATP1B1 and OATP1B3 are involved in the transport of (99m)Tc-mebrofenin. OATP1B1 showed an approximately 1.5-fold higher affinity for (99m)Tc-mebrofenin compared to OATP1B3. ICG is transported by OATP1B3 and NTCP. CONCLUSIONS: The transporter specificity of (99m)Tc-mebrofenin and ICG partially overlaps as both compounds are transported by OATP1B3. (99m)Tc-mebrofenin is also taken up by OATP1B1, whereas ICG is additionally transported by NTCP.

Shear stress affects the intracellular distribution of eNOS: direct demonstration by a novel in vivo technique.

The focal location of atherosclerosis in the vascular tree is correlated with local variations in shear stress. We developed a method to induce defined variations in shear stress in a straight vessel segment of a mouse. To this end, a cylinder with a tapered lumen was placed around the carotid artery, inducing a high shear stress field. Concomitantly, regions of low shear stress and oscillatory shear stress were created upstream and down-stream of the device, respectively. This device was used in mice transgenic for an eNOS3GFP fusion gene. We observed a strong induction of endothelial nitric oxide synthase-green fluorescent protein (eNOS-GFP) mRNA expression in the high shear stress region compared with the other regions (P < .05). Quantification of eNOS-GFP fluorescence or of immunoreactivity to the Golgi complex or to platelet endothelial cell adhesion molecule 1 (PECAM-1) showed an increase in the high shear stress region (P < .05) compared with nontreated carotid arteries. Colocalization of eNOS-GFP with either the Golgi complex or PECAM-1 also responded to alterations of shear stress. In conclusion, we showed a direct response of mRNA and protein expression in vivo to induced variations of shear stress. This model provides the opportunity to study the relationship between shear stress alterations, gene expression, and atherosclerosis.

Transport kinetics of FMRP containing the I304N mutation of severe fragile X syndrome in neurites of living rat PC12 cells.

Lack of fragile X mental retardation protein (FMRP) causes the fragile X syndrome, a common form of inherited mental retardation. The syndrome usually results from the expansion of a CGG repeat in the FMR1 gene with consequent transcriptional silencing of FMR1. However, one missense mutation (Ile304Asn) was reported in the second KH domain of the protein involved in RNA binding. The protein containing this mutation showed an impaired function, leading to an extremely severe phenotype. In the present report, we have studied the role of FMRP I304N in living PC12 cells to better understand the (dys) function of this mutant FMRP. We have generated an FMR1 I304N-EGFP stably transfected PC12 cell line with an inducible expression system (Tet-On) for regulated expression of the FMRP I304N-EGFP fusion protein. After Dox-induction, FMRP I304N-EGFP was localized in the neurites of PC12 cells; however, no granules were formed as has been recently demonstrated for the normal FMRP. Time-lapse microscopy in combination with bleaching technology illustrated that although FMRP I304N-EGFP does not form visible granules, the transport into the neurites is microtubule dependent. Immunoprecipitation with antibodies against GFP demonstrates that FMRP I304N-EGFP coprecipitate with both the 60S ribosomal protein P0 and FXR1P, suggesting that the mutant FMRP is still able to form complexes, however, with different characteristics compared to normal FMRP.

Centrosomes split in the presence of impaired DNA integrity during mitosis.

A well-established function of centrosomes is their role in accomplishing a successful mitosis that gives rise to a pair of identical daughter cells. We recently showed that DNA replication defects and DNA damage in Drosophila embryos trigger centrosomal changes, but it remained unclear whether comparable centrosomal responses can be provoked in somatic mammalian cells. To investigate the centrosomal organization in the presence of impaired DNA integrity, live and ultrastructural analysis was performed on gamma-tubulin-GFP and EGFP-alpha-tubulin-expressing Chinese hamster ovary cells. We have shown that during mitosis in the presence of incompletely replicated or damaged DNA, centrosomes split into fractions containing only one centriole. This results in the formation of multipolar spindles with extra centrosome-like structures. Despite the extra centrosomes and the multipolarity of the spindles, cells do exit from mitosis, resulting in severe division errors. Our data provide evidence of a novel mechanism showing how numerous centrosomes and spindle defects can arise and how this can lead to the formation of aneuploid cells.

Visualization of microtubule growth in cultured neurons via the use of EB3-GFP (end-binding protein 3-green fluorescent protein).

Several microtubule binding proteins, including CLIP-170 (cytoplasmic linker protein-170), CLIP-115, and EB1 (end-binding protein 1), have been shown to associate specifically with the ends of growing microtubules in non-neuronal cells, thereby regulating microtubule dynamics and the binding of microtubules to protein complexes, organelles, and membranes. When fused to GFP (green fluorescent protein), these proteins, which collectively are called +TIPs (plus end tracking proteins), also serve as powerful markers for visualizing microtubule growth events. Here we demonstrate that endogenous +TIPs are present at distal ends of microtubules in fixed neurons. Using EB3-GFP as a marker of microtubule growth in live cells, we subsequently analyze microtubule dynamics in neurons. Our results indicate that microtubules grow slower in neurons than in glia and COS-1 cells. The average speed and length of EB3-GFP movements are comparable in cell bodies, dendrites, axons, and growth cones. In the proximal region of differentiated dendrites approximately 65% of EB3-GFP movements are directed toward the distal end, whereas 35% are directed toward the cell body. In more distal dendritic regions and in axons most EB3-GFP dots move toward the growth cone. This difference in directionality of EB3-GFP movements in dendrites and axons reflects the highly specific microtubule organization in neurons. Together, these results suggest that local microtubule polymerization contributes to the formation of the microtubule network in all neuronal compartments. We propose that similar mechanisms underlie the specific association of CLIPs and EB1-related proteins with the ends of growing microtubules in non-neuronal and neuronal cells.

Loss of HR6B ubiquitin-conjugating activity results in damaged synaptonemal complex structure and increased crossing-over frequency during the male meiotic prophase.

The ubiquitin-conjugating enzymes HR6A and HR6B are the two mammalian homologs of Saccharomyces cerevisiae RAD6. In yeast, RAD6 plays an important role in postreplication DNA repair and in sporulation. HR6B knockout mice are viable, but spermatogenesis is markedly affected during postmeiotic steps, leading to male infertility. In the present study, increased apoptosis of HR6B knockout primary spermatocytes was detected during the first wave of spermatogenesis, indicating that HR6B performs a primary role during the meiotic prophase. Detailed analysis of HR6B knockout pachytene nuclei showed major changes in the synaptonemal complexes. These complexes were found to be longer. In addition, we often found depletion of synaptonemal complex proteins from near telomeric regions in the HR6B knockout pachytene nuclei. Finally, we detected an increased number of foci containing the mismatch DNA repair protein MLH1 in these nuclei, reflecting a remarkable and consistent increase (20 to 25%) in crossing-over frequency. The present findings reveal a specific requirement for the ubiquitin-conjugating activity of HR6B in relation to dynamic aspects of the synaptonemal complex and meiotic recombination in spermatocytes.

Transport of fragile X mental retardation protein via granules in neurites of PC12 cells.

Lack of fragile X mental retardation protein (FMRP) causes fragile X syndrome, a common form of inherited mental retardation. FMRP is an RNA binding protein thought to be involved in translation efficiency and/or trafficking of certain mRNAs. Recently, a subset of mRNAs to which FMRP binds with high affinity has been identified. These FMRP-associated mRNAs contain an intramolecular G-quartet structure. In neurons, dendritic mRNAs are involved in local synthesis of proteins in response to synaptic activity, and this represents a mechanism for synaptic plasticity. To determine the role of FMRP in dendritic mRNA transport, we have generated a stably FMR1-enhanced green fluorescent protein (EGFP)-transfected PC12 cell line with an inducible expression system (Tet-On) for regulated expression of the FMRP-GFP fusion protein. After doxycycline induction, FMRP-GFP was localized in granules in the neurites of PC12 cells. By using time-lapse microscopy, the trafficking of FMRP-GFP granules into the neurites of living PC12 cells was demonstrated. Motile FMRP-GFP granules displayed two types of movements: oscillatory (bidirectional) and unidirectional anterograde. The average velocity of the granules was 0.19 micro m/s with a maximum speed of 0.71 micro m/s. In addition, we showed that the movement of FMRP-GFP labeled granules into the neurites was microtubule dependent. Colocalization studies further showed that the FMRP-GFP labeled granules also contained RNA, ribosomal subunits, kinesin heavy chain, and FXR1P molecules. This report is the first example of trafficking of RNA-containing granules with FMRP as a core constituent in living PC12 cells.