Kif3a interacts with Dynactin subunit p150Glued to organize centriole subdistal appendages

Formation of cilia, microtubule-based structures that function in propulsion and sensation, requires Kif3a, a subunit of Kinesin II essential for intraflagellar transport (IFT). We have found that, Kif3a is also required to organize centrioles. In the absence of Kif3a, the subdistal appendages of centrioles are disorganized and lack p150(Glued) and Ninein. Consequently, microtubule anchoring, centriole cohesion and basal foot formation are abrogated by loss of Kif3a. Kif3a localizes to the mother centriole and interacts with the Dynactin subunit p150(Glued). Depletion of p150(Glued) phenocopies the effects of loss of Kif3a, indicating that Kif3a recruitment of p150(Glued) is critical for subdistal appendage formation. The transport functions of Kif3a are dispensable for subdistal appendage organization as mutant forms of Kif3a lacking motor activity or the motor domain can restore p150(Glued) localization. Comparison to cells lacking Ift88 reveals that the centriolar functions of Kif3a are independent of IFT. Thus, in addition to its ciliogenic roles, Kif3a recruits p150(Glued) to the subdistal appendages of mother centrioles, critical for centrosomes to function as microtubule-organizing centres

Kif3a interacts with Dynactin subunit p150Glued to organize centriole subdistal appendages

Formation of cilia, microtubule-based structures that function in propulsion and sensation, requires Kif3a, a subunit of Kinesin II essential for intraflagellar transport (IFT). We have found that, Kif3a is also required to organize centrioles. In the absence of Kif3a, the subdistal appendages of centrioles are disorganized and lack p150(Glued) and Ninein. Consequently, microtubule anchoring, centriole cohesion and basal foot formation are abrogated by loss of Kif3a. Kif3a localizes to the mother centriole and interacts with the Dynactin subunit p150(Glued). Depletion of p150(Glued) phenocopies the effects of loss of Kif3a, indicating that Kif3a recruitment of p150(Glued) is critical for subdistal appendage formation. The transport functions of Kif3a are dispensable for subdistal appendage organization as mutant forms of Kif3a lacking motor activity or the motor domain can restore p150(Glued) localization. Comparison to cells lacking Ift88 reveals that the centriolar functions of Kif3a are independent of IFT. Thus, in addition to its ciliogenic roles, Kif3a recruits p150(Glued) to the subdistal appendages of mother centrioles, critical for centrosomes to function as microtubule-organizing centres

Alexander Disease Mutations Produce Cells with Coexpression of Glial Fibrillary Acidic Protein and NG2 in Neurosphere Cultures and Inhibit Differentiation into Mature Oligodendrocytes

BackgroundAlexander disease (AxD) is a rare disease caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). The disease is characterized by presence of GFAP aggregates in the cytoplasm of astrocytes and loss of myelin.ObjectivesDetermine the effect of AxD-related mutations on adult neurogenesis.MethodsWe transfected different types of mutant GFAP into neurospheres using the nucleofection technique.ResultsWe find that mutations may cause coexpression of GFAP and NG2 in neurosphere cultures, which would inhibit the differentiation of precursors into oligodendrocytes and thus explain the myelin loss occurring in the disease. Transfection produces cells that differentiate into new cells marked simultaneously by GFAP and NG2 and whose percentage increased over days of differentiation. Increased expression of GFAP is due to a protein with an anomalous structure that forms aggregates throughout the cytoplasm of new cells. These cells display down-expression of vimentin and nestin. Up-expression of cathepsin D and caspase-3 in the first days of differentiation suggest that apoptosis as a lysosomal response may be at work. HSP27, a protein found in Rosenthal bodies, is expressed less at the beginning of the process although its presence increases in later stages.ConclusionOur findings seem to suggest that the mechanism of development of AxD may not be due to a function gain due to increase of GFAP, but to failure in the differentiation process may occur at the stage in which precursor cells transform into oligodendrocytes, and that possibility may provide the best explanation for the clinical and radiological images described in AxD

Mesenchymal stem cells improve motor functions and decrease neurodegeneration in ataxic mice

The main objective of this work is to demonstrate the feasibility of using bone marrow-derived stem cells in treating a neurodegenerative disorder such as Friedreich's ataxia. In this disease, the dorsal root ganglia of the spinal cord are the first to degenerate. Two groups of mice were injected intrathecally with mesenchymal stem cells isolated from either wild-type or Fxntm1Mkn/Tg(FXN)YG8Pook (YG8) mice. As a result, both groups presented improved motor skills compared to nontreated mice. Also, frataxin expression was increased in the dorsal root ganglia of the treated groups, along with lower expression of the apoptotic markers analyzed. Furthermore, the injected stem cells expressed the trophic factors NT3, NT4, and BDNF, which bind to sensory neurons of the dorsal root ganglia and increase their survival. The expression of antioxidant enzymes indicated that the stem cell-treated mice presented higher levels of catalase and GPX-1, which are downregulated in the YG8 mice. There were no significant differences in the use of stem cells isolated from wild-type and YG8 mice. In conclusion, bone marrow mesenchymal stem cell transplantation, both autologous and allogeneic, is a feasible therapeutic option to consider in delaying the neurodegeneration observed in the dorsal root ganglia of Friedreich's ataxia patients.This work was supported by FARA/FARA Ireland (Friedreich's Ataxia Research Alliance), ASOGAF (Friedreich's ataxia association of Granada), Science and Innovation Ministry (MICINN BFU2010-27326), GVA Prometeo grant 2009/028, PROMETEOII GRANT 2014/014, Tercel (RD06/0010/0023 & RD06/0010/24), MEC-CONSOLIDER CSD2007-00023, Cinco P menos Foundation, EUCOMM, Fundacion Diogenes-Elche city government, Walk on Project, and MAPFRE Foundation.Peer reviewe

Meox2/Tcf15 Heterodimers Program the Heart Capillary Endothelium for Cardiac Fatty Acid Uptake

BACKGROUND: Microvascular endothelium in different organs is specialized to fulfill the particular needs of parenchymal cells. However, specific information about heart capillary endothelial cells (ECs) is lacking. METHODS AND RESULTS: Using microarray profiling on freshly isolated ECs from heart, brain, and liver, we revealed a genetic signature for microvascular heart ECs and identified Meox2/Tcf15 heterodimers as novel transcriptional determinants. This signature was largely shared with skeletal muscle and adipose tissue endothelium and was enriched in genes encoding fatty acid (FA) transport-related proteins. Using gain- and loss-of-function approaches, we showed that Meox2/Tcf15 mediate FA uptake in heart ECs, in part, by driving endothelial CD36 and lipoprotein lipase expression and facilitate FA transport across heart ECs. Combined Meox2 and Tcf15 haplodeficiency impaired FA uptake in heart ECs and reduced FA transfer to cardiomyocytes. In the long term, this combined haplodeficiency resulted in impaired cardiac contractility. CONCLUSIONS: Our findings highlight a regulatory role for ECs in FA transfer to the heart parenchyma and unveil 2 of its intrinsic regulators. Our insights could be used to develop new strategies based on endothelial Meox2/Tcf15 targeting to modulate FA transfer to the heart and remedy cardiac dysfunction resulting from altered energy substrate usage.status: publishe

Transplantation of hMSCs and hMAPCs induced an increase in angiogenesis.

<p>A) CD31 immunostaining in the ischemic boundary zone at different time points after cell transplantation (2 days, 4 days, 7 days and 28 days) (Scale bar = 100 µm). B) Quantitative analysis of angiogenesis after cell transplantation (n = 4). *<i>p</i><0.05 and ** <i>p</i><0.001 by Bonferroni test.</p

Glial-scar inhibitory effect after hMSCs and hMAPCs transplantation.

<p>Immunoflurescence analysis of fibronectin (A) and NG2-CSP (B) 4 days after hMSCs and hMAPCs transplantation. The intensity of the staining varied according to the color scale shown in the left corner in each panel. C) Quantitative analysis of NG2-CSP immunostaining. *<i>P</i><0.01 in PBS-treated group vs cell transplanted groups. Scale bar = 100 µm.</p

Effect of transplantation of hMSCs and hMAPCs in tissue loss.

<p>A) Nissl-staining photomicrographs of coronal sections that show the loss of tissue at 28 days after transplantation. The dashed line marks tissue loss. Scale bar = 1000 µm; B) Quantification of tissue loss 28 days after cell transplantation n = 4 *<i>p</i><0.05 PBS-treated group vs cell transplanted groups by Bonferroni test.</p