Inactivation of the peroxisomal ABCD2 transporter in the mouse leads to late-onset ataxia involving mitochondria, Golgi and endoplasmic reticulum damage

ATP-binding cassette (ABC) transporters facilitate unidirectional translocation of chemically diverse substances, ranging from peptides to lipids, across cell or organelle membranes. In peroxisomes, a subfamily of four ABC transporters (ABCD1 to ABCD4) has been related to fatty acid transport, because patients with mutations in ABCD1 (ALD gene) suffer from X-linked adrenoleukodystrophy (X-ALD), a disease characterized by an accumulation of very-long-chain fatty acids (VLCFAs). Inactivation in the mouse of the abcd1 gene leads to a late-onset neurodegenerative condition, comparable to the late-onset form of X-ALD [Pujol, A., Hindelang, C., Callizot, N., Bartsch, U., Schachner, M. and Mandel, J.L. (2002) Late onset neurological phenotype of the X-ALD gene inactivation in mice: a mouse model for adrenomyeloneuropathy. Hum. Mol. Genet., 11, 499-505.]. In the present work, we have generated and characterized a mouse deficient for abcd2, the closest paralog to abcd1. The main pathological feature in abcd2-/- mice is a late-onset cerebellar and sensory ataxia, with loss of cerebellar Purkinje cells and dorsal root ganglia cell degeneration, correlating with accumulation of VLCFAs in the latter cellular population. Axonal degeneration was present in dorsal and ventral columns in spinal cord. We have identified mitochondrial, Golgi and endoplasmic reticulum damage as the underlying pathological mechanism, thus providing evidence of a disturbed organelle cross-talk, which may be at the origin of the pathological cascade.     

Differential stability and fusion activity of Lyssavirus glycoprotein trimers

The oligomeric structure and the fusion activity of lyssavirus glycoprotein (G) was studied by comparing G from Mokola virus (GMok) and rabies virus (PV strain) (GPV), which are highly divergent lyssaviruses. G expressed at the surface of BSR cells upon either plasmid transfection or virus infection are shown to be mainly trimeric after cross-linking experiments. However, solubilization by a detergent (CHAPS) and analysis in sucrose sedimentation gradient evidenced that GMok trimer is less stable than GPV trimer. A chimeric glycoprotein (G Mok-PV) associating the N-terminal half of GMok to the C-terminal half part of GPV formed trimers with an intermediate stability, indicating that the G C-terminal domain is essential in trimer stability. A cell to cell fusion assay revealed that GMok (and not G Mok-PV) was able to induce fusion at a higher pH (0.5 pH unit) than GPV. Such differences in the oligomeric structure stability and in the fusion activity of lyssavirus glycoproteins may partly account for the previously reported differences of their immunogenic and pathogenic properties.     

Genetic analysis of the formation of the Ysc-Yop translocation pore in macrophages by Yersinia enterocolitica: role of LcrV, YscF and YopN

The Ysc-Yop type III secretion (TTS) system allows extracellular Yersinia bacteria, adhering to eukaryotic target cells, to inject Yop effector proteins in the cytosol of these cells. The secretion apparatus, called the injectisome, ends up with a needle-like structure made of YscF. YopN, one of the proteins secreted by the injectisome is thought to act as a plug. YopB, YopD and LcrV, three other proteins secreted by the injectisome and called 'translocators' form a pore allowing translocation of the Yop effectors across the target cell plasma membrane. Here, we tested the role of LcrV, YscF and YopN in the formation of this pore in macrophages by monitoring the release of the low-molecular-weight fluorescent dye BCECF (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, 623Da) and of the high-molecular-weight lactate dehydrogenase (LDH, 135 kDa). BCECF is released through the translocation pore itself provided no Yop effector is trafficking through the channel. In contrast, LDH is released by the osmotic lysis of the target cell that occurs after pore formation. This release is reduced by the GAP activity of YopE. In order to study the role of LcrV, one has to circumvent the regulatory effect of LcrV on the synthesis of YopB and YopD. We observed here that this regulatory role of LcrV is lost in a yopQ mutant and hence we studied the role of LcrV in a yopQ mutant background. A lcrV, yopQ double mutant was deficient in pore formation while able to produce YopB and YopD. Pore formation was restored by the introduction of lcrV(+) but not yopQ(+) confirming that LcrV itself is directly required for pore formation. Bacteria secreting only YopB, YopD and LcrV could form pores, showing that YopB, YopD and LcrV are sufficient for pore formation provided they are secreted by the same bacterium. LcrV is not involved in secretion of YopB and YopD as suggested previously. Bacteria producing normal Ysc injectisomes, including the YscF needle but no translocators did not form pores, indicating that the needle is not sufficient by itself for pore formation, as was also suggested. yopN mutant bacteria formed needles and released BCECF even if they secreted the effectors. This observation suggests that many translocation pores are not filled in the absence of YopN and thus that YopN might form a link between the needle and the pore, guiding the effectors.     

Origin and filiation of human plasmacytoid dendritic cells

Human plasmacytoid dendritic cells represent a rare population of leukocytes which produce high amounts of type I interferon in response to certain viruses. Although those cells were first described in 1958, there are still unsolved issues related to their origin and function. Recently, a leukemic counterpart of plasmacytoid dendritic cells was identified. Molecular approaches using either normal or leukemic plasmacytoid dendritic cells provide some new insights into the controversial lymphoid origin of those cells. The need for specific markers is still a critical aspect for the identification of plasmacytoid dendritic cells, whatever stage of differentiation, in normal as well as in pathological conditions. Hopefully, novel markers will allow delineation of the relationships between dendritic cells at different stages of differentiation/maturation along the myeloid and lymphoid lineages.     

Chronic exposure to superantigen induces regulatory CD4(+) T cells with IL-10-mediated suppressive activity

The repeated injection of bacterial superantigens (SAg), such as staphylococcus enterotoxin (SE) A or B, has been shown in mice to induce a state of unresponsiveness characterized by the lack of secretion of Th1 lymphokines, such as IL-2 and IFN-gamma, following subsequent SAg challenge. We made the observation, in vivo as well as in vitro, that unresponsiveness to SAg could be transferred from SEA- to SEB-reactive T cells (and reversibly from SEB- to SEA-specific T cells) in C57BL/6 mice but not in BALB/c mice. Since C57BL/6 mice, unlike BALB/c mice, possess TCR V(beta)3+ and V(beta)11+ T cells able to react with both SEA and SEB, we hypothesized that SAg-unresponsive V(beta)3(+) and V(beta)11+ T cells could mediate linked suppression of other SAg-reactive T cells. To analyze further this possibility, spleen cells from BALB/c mice made unresponsive to SEB were tested for their capacity to suppress the response of normal BALB/c cells to SEB. The production of both IFN-gamma and IL-2 following SEB stimulation was greatly impaired in co-cultures containing CD4(+) T cells, but not CD8(+) T cells, isolated from unresponsive animals. In vivo, the production of both IFN-gamma and IL-2 responses to SEB was dramatically reduced in animals adoptively transferred with unresponsive spleen cells. This suppression was abrogated in recipients injected with neutralizing anti-IL-10 antibodies. Moreover, in animals made unresponsive to SEB, SAg-reactive CD4(+) T cells were found to express high levels of CTLA-4, a molecule recently described to play an essential role in the suppressive function of regulatory T cells. Taken together these results demonstrate that the repetitive injection of SAg induces the differentiation of regulatory CD4(+) T cells capable of suppressing SAg-reactive naive T cells.     

Capacity of bottle-nosed dolphins for generalization based on a relative sign

Using a new modification of a previously proposed method in the bottle-nosed dolphin, a conditioned reflex was developed and consolidated to a relative spatial sign, namely the choice always of the left out of two identical objects under conditions of constant change of the position of this pair of stimuli against the background of one of the sides of the pen. The specialization was checked by means of the presentation of the pair of stimuli through various changes of the surrounding setting, including the discordance of external landmarks, as well as by the means of the presentation of the stimuli. Then the objects were replaced by others (12 pairs of stimuli) sharply differing from the initial stimuli in form, size, material, etc. The maintenance of appropriate choice, including with change in the medium of presentation of the stimuli) attested to the capacity of the dolphins to abstract and generalize on the basis of a relative sign, i. e., of abstraction and generalization in the to the left-to the right in general choice of two objects.Translated from Zhurnal Vysshei Nervnoi Deyatel'nosti imeni I. P. Pavlova, Vol. 40, No. 1, pp. 44–51, January–February, 1990.     

Increased resistance to anthelmintics of Haemonchus contortus eggs associated with changes in membrane fluidity of eggshells during embryonation

The embryonation of nematode eggs has been shown to increase their resistance to anthelmintics when parasites are submitted to egg hatch assays. Nevertheless, no mechanism has been suggested to explain this phenomenon. Earlier observations by other authors showed that the biochemical composition of eggshells is altered during the embryonation of eggs. The functional consequences of these changes have not been identified. We studied the changes in membrane environment (eggshells) of Haemonchus contortus eggs during the embryonation by fluidity measurements and their effects on nonspecific mechanisms of resistance to anthelmintics. We previously demonstrated that these mechanisms imply P-glycoproteins (Pgp) belonging to the multi-drug resistance (MDR) system and that the Pgp activity is very susceptible to their lipidic environment. The results obtained here show that the embryonation induced a significant and gradual increase in eggshell fluidity which was associated with an increased resistance to anthelmintics. Differences were observed between H. contortus isolates with various levels of resistance which might result from their specific biology and/or membrane composition. The membrane environment changes could act both on the solubilization of anthelmintics into the eggs and on the efflux of these lipophilic molecules by Pgp.     

Dysferlin mutations in LGMD2B, Miyoshi myopathy, and atypical dysferlinopathies

DYSF encoding dysferlin is mutated in Miyoshi myopathy and Limb-Girdle Muscular Dystrophy type 2B, the two main phenotypes recognized in dysferlinopathies. Dysferlin deficiency in muscle is the most relevant feature for the diagnosis of dysferlinopathy and prompts the search for mutations in DYSF. DYSF, located on chromosome 2p13, contains 55 coding exons and spans 150 kb of genomic DNA. We performed a genomic analysis of the DYSF coding sequence in 34 unrelated patients from various ethnic origins. All patients showed an absence or drastic decrease of dysferlin expression in muscle. A primary screening of DYSF using SSCP or dHPLC of PCR products of each of 55 exons of the gene was followed by sequencing whenever a sequence variation was detected. All together, 54 sequence variations were identified in DYSF, 50 of which predicting either a truncated protein or one amino-acid substitution and most of them (34 out of 54) being novel. In 23 patients, we identified two pathogenic mutations, while only one was identified in 11 patients. These mutations were widely spread in the coding sequence of the gene without any mutational "hotspot."