Fabrice P. Cordelières

BoneJ: Free and extensible bone image analysis in ImageJ

Bone geometry is commonly measured on computed tomographic (CT) and X-ray microtomographic (μCT) images. We obtained hundreds of CT, μCT and synchrotron μCT images of bones from diverse species that needed to be analysed remote from scanning hardware, but found that available software solutions were expensive, inflexible or methodologically opaque. We implemented standard bone measurements in a novel ImageJ plugin, BoneJ, with which we analysed trabecular bone, whole bones and osteocyte lacunae. BoneJ is open source and free for anyone to download, use, modify and distribute.

Histone Deacetylase 6 Inhibition Compensates for the Transport Deficit in Huntington's Disease by Increasing Tubulin Acetylation

A defect in microtubule (MT)-based transport contributes to the neuronal toxicity observed in Huntingtons disease (HD). Histone deacetylase (HDAC) inhibitors show neuroprotective effects in this devastating neurodegenerative disorder. We report here that HDAC inhibitors, including trichostatin A (TSA), increase vesicular transport of brain-derived neurotrophic factor (BDNF) by inhibiting HDAC6, thereby increasing acetylation at lysine 40 of α-tubulin. MT acetylation in vitro and in cells causes the recruitment of the molecular motors dynein and kinesin-1 to MTs. In neurons, acetylation at lysine 40 of α-tubulin increases the flux of vesicles and the subsequent release of BDNF. We show that tubulin acetylation is reduced in HD brains and that TSA compensates for the transport- and release-defect phenotypes that are observed in disease. Our findings reveal that HDAC6 inhibition and acetylation at lysine 40 of α-tubulin may be therapeutic targets of interest in disorders such as HD in which intracellular transport is altered.

The IGF-1/Akt Pathway Is Neuroprotective in Huntington's Disease and Involves Huntingtin Phosphorylation by Akt

In the search for neuroprotective factors in Huntingtons disease, we found that insulin growth factor 1 via activation of the serine/threonine kinase Akt/PKB is able to inhibit neuronal death specifically induced by mutant huntingtin containing an expanded polyglutamine stretch. The IGF-1/Akt pathway has a dual effect on huntingtin-induced toxicity, since activation of this pathway also results in a decrease in the formation of intranuclear inclusions of mutant huntingtin. We demonstrate that huntingtin is a substrate of Akt and that phosphorylation of huntingtin by Akt is crucial to mediate the neuroprotective effects of IGF-1. Finally, we show that Akt is altered in Huntingtons disease patients. Taken together, these results support a potential role of the Akt pathway in Huntingtons disease.

Vesicular Glycolysis Provides On-Board Energy for Fast Axonal Transport

Fast axonal transport (FAT) requires consistent energy over long distances to fuel the molecular motors that transport vesicles. We demonstrate that glycolysis provides ATP for the FAT of vesicles. Although inhibiting ATP production from mitochondria did not affect vesicles motility, pharmacological or genetic inhibition of the glycolytic enzyme GAPDH reduced transport in cultured neurons and in Drosophila larvae. GAPDH localizes on vesicles via a huntingtin-dependent mechanism and is transported on fast-moving vesicles within axons. Purified motile vesicles showed GAPDH enzymatic activity and produced ATP. Finally, we show that vesicular GAPDH is necessary and sufficient to provide on-board energy for fast vesicular transport. Although detaching GAPDH from vesicles reduced transport, targeting GAPDH to vesicles was sufficient to promote FAT in GAPDH deficient neurons. This specifically localized glycolytic machinery may supply constant energy, independent of mitochondria, for the processive movement of vesicles over long distances in axons.

Cystamine and cysteamine increase brain levels of BDNF in Huntington disease via HSJ1b and transglutaminase

There is no treatment for the neurodegenerative disorder Huntington disease (HD). Cystamine is a candidate drug; however, the mechanisms by which it operates remain unclear. We show here that cystamine increases levels of the heat shock DnaJ-containing protein 1b (HSJ1b) that are low in HD patients. HSJ1b inhibits polyQ-huntingtin-induced death of striatal neurons and neuronal dysfunction in Caenorhabditis elegans. This neuroprotective effect involves stimulation of the secretory pathway through formation of clathrin-coated vesicles containing brain-derived neurotrophic factor (BDNF). Cystamine increases BDNF secretion from the Golgi region that is blocked by reducing HSJ1b levels or by overexpressing transglutaminase. We demonstrate that cysteamine, the FDA-approved reduced form of cystamine, is neuroprotective in HD mice by increasing BDNF levels in brain. Finally, cysteamine increases serum levels of BDNF in mouse and primate models of HD. Therefore, cysteamine is a potential treatment for HD, and serum BDNF levels can be used as a biomarker for drug efficacy.

Ciliogenesis is regulated by a huntingtin-HAP1-PCM1 pathway and is altered in Huntington disease

Huntington disease (HD) is a devastating autosomal-dominant neurodegenerative disorder. It is caused by expansion of a CAG repeat in the first exon of the huntingtin (HTT) gene that encodes a mutant HTT protein with a polyglutamine (polyQ) expansion at the amino terminus. Here, we demonstrate that WT HTT regulates ciliogenesis by interacting through huntingtin-associated protein 1 (HAP1) with pericentriolar material 1 protein (PCM1). Loss of Htt in mouse cells impaired the retrograde trafficking of PCM1 and thereby reduced primary cilia formation. In mice, deletion of Htt in ependymal cells led to PCM1 mislocalization, alteration of the cilia layer, and hydrocephalus. Pathogenic polyQ expansion led to centrosomal accumulation of PCM1 and abnormally long primary cilia in mouse striatal cells. PCM1 accumulation in ependymal cells was associated with longer cilia and disorganized cilia layers in a mouse model of HD and in HD patients. Longer cilia resulted in alteration of the cerebrospinal fluid flow. Thus, our data indicate that WT HTT is essential for protein trafficking to the centrosome and normal ciliogenesis. In HD, hypermorphic ciliogenesis may affect signaling and neuroblast migration so as to dysregulate brain homeostasis and exacerbate disease progression.

Bloom’s Syndrome and PICH Helicases Cooperate with Topoisomerase IIα in Centromere Disjunction before Anaphase

Centromeres are specialized chromosome domains that control chromosome segregation during mitosis, but little is known about the mechanisms underlying the maintenance of their integrity. Centromeric ultrafine anaphase bridges are physiological DNA structures thought to contain unresolved DNA catenations between the centromeres separating during anaphase. BLM and PICH helicases colocalize at these ultrafine anaphase bridges and promote their resolution. As PICH is detectable at centromeres from prometaphase onwards, we hypothesized that BLM might also be located at centromeres and that the two proteins might cooperate to resolve DNA catenations before the onset of anaphase. Using immunofluorescence analyses, we demonstrated the recruitment of BLM to centromeres from G2 phase to mitosis. With a combination of fluorescence in situ hybridization, electron microscopy, RNA interference, chromosome spreads and chromatin immunoprecipitation, we showed that both BLM-deficient and PICH-deficient prometaphase cells displayed changes in centromere structure. These cells also had a higher frequency of centromeric non disjunction in the absence of cohesin, suggesting the persistence of catenations. Both proteins were required for the correct recruitment to the centromere of active topoisomerase IIα, an enzyme specialized in the catenation/decatenation process. These observations reveal the existence of a functional relationship between BLM, PICH and topoisomerase IIα in the centromere decatenation process. They indicate that the higher frequency of centromeric ultrafine anaphase bridges in BLM-deficient cells and in cells treated with topoisomerase IIα inhibitors is probably due not only to unresolved physiological ultrafine anaphase bridges, but also to newly formed ultrafine anaphase bridges. We suggest that BLM and PICH cooperate in rendering centromeric catenates accessible to topoisomerase IIα, thereby facilitating correct centromere disjunction and preventing the formation of supernumerary centromeric ultrafine anaphase bridges.

Automated Cell Tracking and Analysis in Phase-Contrast Videos (iTrack4U): Development of Java Software Based on Combined Mean-Shift Processes

Cell migration is a key biological process with a role in both physiological and pathological conditions. Locomotion of cells during embryonic development is essential for their correct positioning in the organism; immune cells have to migrate and circulate in response to injury. Failure of cells to migrate or an inappropriate acquisition of migratory capacities can result in severe defects such as altered pigmentation, skull and limb abnormalities during development, and defective wound repair, immunosuppression or tumor dissemination. The ability to accurately analyze and quantify cell migration is important for our understanding of development, homeostasis and disease. In vitro cell tracking experiments, using primary or established cell cultures, are often used to study migration as cells can quickly and easily be genetically or chemically manipulated. Images of the cells are acquired at regular time intervals over several hours using microscopes equipped with CCD camera. The locations (x,y,t) of each cell on the recorded sequence of frames then need to be tracked. Manual computer-assisted tracking is the traditional method for analyzing the migratory behavior of cells. However, this processing is extremely tedious and time-consuming. Most existing tracking algorithms require experience in programming languages that are unfamiliar to most biologists. We therefore developed an automated cell tracking program, written in Java, which uses a mean-shift algorithm and ImageJ as a library. iTrack4U is a user-friendly software. Compared to manual tracking, it saves considerable amount of time to generate and analyze the variables characterizing cell migration, since they are automatically computed with iTrack4U. Another major interest of iTrack4U is the standardization and the lack of inter-experimenter differences. Finally, iTrack4U is adapted for phase contrast and fluorescent cells.

The Bloom Syndrome Protein Limits the Lethality Associated with RAD51 Deficiency

Little is known about the functional interaction between the Blooms syndrome protein (BLM) and the recombinase RAD51 within cells. Using RNA interference technology, we provide the first demonstration that RAD51 acts upstream from BLM to prevent anaphase bridge formation. RAD51 downregulation was associated with an increase in the frequency of BLM-positive anaphase bridges, but not of BLM-associated ultrafine bridges. Time-lapse live microscopy analysis of anaphase bridge cells revealed that BLM promoted cell survival in the absence of Rad51. Our results directly implicate BLM in limiting the lethality associated with RAD51 deficiency through the processing of anaphase bridges resulting from the RAD51 defect. These findings provide insight into the molecular basis of some cancers possibly associated with variants of the RAD51 gene family. Mol Cancer Res; 8(3); 385–94

Inhibition of DNA damage repair by artificial activation of PARP with siDNA

One of the major early steps of repair is the recruitment of repair proteins at the damage site, and this is coordinated by a cascade of modifications controlled by phosphatidylinositol 3-kinase-related kinases and/or poly (ADP-ribose) polymerase (PARP). We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation. Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. Therefore, comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both recruit proteins involved in single-strand break repair (PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break repair (53BP1, NBS1, RAD51 and DNA-PK). By these ways, Pbait and Dbait disorganize DNA repair, thereby sensitizing cells to various treatments. Single-strand breaks repair inhibition depends on direct trapping of the main proteins on both molecules. Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations.