Promotion of retroviral entry in the absence of envelope protein by chlorpromazine

AbstractRetrovirus packaging cell lines that express the Moloney murine leukemia virus gag, pol, and env genes and a retroviral vector genome can produce virus particles that are capable of transducing cells. Normally if the packaging cell line does not produce a functional viral fusion glycoprotein, such as the retroviral envelope protein or a foreign viral glycoprotein, then the viruses will be incapable of transducing cells. We have found that incubating envelope protein-deficient virus particles bound to cells with chlorpromazine leads to transduction. Chlorpromazine (CPZ) is a membrane-active reagent that is commonly used to induce the hemifusion to fusion transition when membrane fusion is mediated by partially defective viral glycoproteins. The concentration and pH dependence of the promotion of transduction by CPZ is consistent with a role for CPZ micelle formation in viral entry. These data indicate that caution is warranted when experiments concerning membrane fusion completion promoted by CPZ are analyzed

BATF transgenic mice: Investigating the role of AP-1 in the development and function of natural killer T lymphocytes

BATF belongs to the AP-1 family of bZIP transcription factors and dimerizes with Jun proteins to negatively regulate the transcription of AP-1 target genes. BATF is expressed in hematopoietic cells and previous studies suggested a role for BATF in modulating the AP-1 activity of thymic and peripheral T cells. To examine this further, transgenic mice were generated in which HA epitope tagged BATF is expressed using the constitutive, T cell-specific p56lck or hCD2 promoters. While BATF transgenic mice exhibit a normal profile of the major T cell subsets, cytometric, molecular and functional analysis has revealed that BATF transgenic mice are specifically deficient in glycolipid reactive, Natural Killer T (NKT) lymphocytes. Analysis of the residual NKT cells in BATF transgenic mice with CD1d multimer technology has revealed that HA-BATF targets all classes of NKT cell regardless of Vβ usage or CD4 expression. Developmental tracking experiments have demonstrated that HA-BATF impacts the expansion, maturation, and survival of positively selected, yet immature, NKT cells within the thymus. However, transgenic NKT cells that manage to complete maturation can form reduced, but stable, peripheral NKT cell populations that appear tolerant of HA-BATF and are maintained throughout the life of the mice. The phenotype of BATF transgenic mice suggested for the first time that AP-1 is present in NKT cells and plays an important role in early NKT cell development. Purification of thymic NKT cells and analysis of AP-1 transcripts has revealed the full complement of AP-1 factors. In addition, AP-1 DNA binding activity has been detected in NKT cell hybridomas and primary cultures within six hours of treatment with plate bound CD1d and α-galactosylceramide. Analysis of antigen presentation has revealed that antigen presenting cells present glycolipid ligand and induce AP-1 activity in the same six-hour time frame. Lastly, transcription of the AP-1 target gene IL-4 in response to α-GalCer is a rapid event occurring within 3 hours of activation and appears sensitive to ERK inhibition. When coupled with the phenotype of BATF transgenic mice, the data presented begins to characterize AP-1 as an early response factor in NKT cells and a critical mediator of NKT cell survival and development in the thymus

Neural-specific deletion of Htra2 causes cerebellar neurodegeneration and defective processing of mitochondrial OPA1.

HTRA2, a serine protease in the intermembrane space, has important functions in mitochondrial stress signaling while its abnormal activity may contribute to the development of Parkinson's disease. Mice with a missense or null mutation of Htra2 fail to thrive, suffer striatal neuronal loss, and a parkinsonian phenotype that leads to death at 30-40 days of age. While informative, these mouse models cannot separate neural contributions from systemic effects due to the complex phenotypes of HTRA2 deficiency. Hence, we developed mice carrying a Htra2-floxed allele to query the consequences of tissue-specific HTRA2 deficiency. We found that mice with neural-specific deletion of Htra2 exhibited atrophy of the thymus and spleen, cessation to gain weight past postnatal (P) day 18, neurological symptoms including ataxia and complete penetrance of premature death by P40. Histologically, increased apoptosis was detected in the cerebellum, and to a lesser degree in the striatum and the entorhinal cortex, from P25. Even earlier at P20, mitochondria in the cerebella already exhibited abnormal morphology, including swelling, vesiculation, and fragmentation of the cristae. Furthermore, the onset of these structural anomalies was accompanied by defective processing of OPA1, a key molecule for mitochondrial fusion and cristae remodeling, leading to depletion of the L-isoform. Together, these findings suggest that HTRA2 is essential for maintenance of the mitochondrial integrity in neurons. Without functional HTRA2, a lifespan as short as 40 days accumulates a large quantity of dysfunctional mitochondria that contributes to the demise of mutant mice

Muscle strength and total activity are decreased in <i>Htra2</i>-deficient mice.

<p>(<i>A–D</i>) The hind limb suspension test was performed on HTRA2-deficient neonates. HTRA2 KO mice performed poorly compared to WT littermates in hang time (<i>A</i>) and number of pull attempts (<i>C</i>) from P7. NesKO neonates performed poorly compared to NesWT littermates in hang time (<i>B</i>) and number of pull attempts (<i>D</i>) from P8 (n = 28 (HTRA2 WT), 19 (HTRA2 KO), 10 (NesWT), 16 (NesKO)). (<i>E–F</i>) The weanling observation total activity score was reduced in P19–21 HTRA2 KO animals (<i>E</i>) compared to WT littermates, and NesKO animals (<i>F</i>) compared to NesWT littermates (n = 19 (HTRA2 WT), 12 (HTRA2 KO), 7 (NesWT), 4 (NesKO)). (<i>G–H</i>) Grip strength was reduced in HTRA2 KO (<i>G</i>) and NesKO (<i>H</i>) animals compared to respective WT littermates (n = 17 (HTRA2 WT), 9 (HTRA2 KO) 11 (NesWT), 5 (NesKO)). Data represents Mean ± SEM (#: 0.05≤p≤0.10, *: p≤0.05, **: p≤0.001, ***: p≤0.0001 by independent t-tests).</p

Neural deletion of <i>Htra2</i> is sufficient to generate neurological phenotypes.

<p>(<i>A</i>) Exons 2 to 4 of <i>Htra2</i> were flanked with loxP sites, with a FRT flanked neo cassette 3′ to exon 4. Expression of <i>FlpE</i> causes deletion of the selection cassette. Cre-mediated deletion causes excision of exons 2 to 4. Small arrows beneath the allele constructs denote the position of genotyping primers. (<i>B</i>) PCR from genomic DNA can distinguish WT (+, arrow, 279 bp), KO (–, filled arrowhead, 358 bp) and floxed (f, empty arrowhead, 313 bp) alleles of <i>Htra2</i>. (<i>C</i>) Western blot analysis confirmed loss of HTRA2 protein (arrow) in all tissues of HTRA2 KO mice and reduction in brain of NesKO mice (arrowheads denote non-specific bands). The levels of HTRA2 protein in NesKO spleen and thymus were comparable with NesWT. Cx: cortex, Mb: midbrain, Hb: hindbrain. PHB2 was used as a loading control. (<i>D</i>) HTRA2 KO mice and NesKO mice were smaller than WT littermates by comparison. The size of the thymus and spleen was reduced although brain was relatively normal in size (representative animals shown at P30, scale bar: 1 cm.). (<i>E</i>) Body weight of HTRA2 KO and NesKO mice did not increase beyond P18 (<i>n</i> = 56 (HTRA2 WT), 62 (HTRA2 KO), 35 (NesWT), 25 (NesKO), error bars indicate SEM).</p

Brain-specific disruption of OPA1 processing.

<p>(<i>A</i>) Levels of autophagy markers are unchanged in P33 HTRA2 KO brain. The LC3β-II to LC3β-I ratio is unchanged in P33 brain and there is no accumulation of P62 (n = 3). Actin is included as a loading control. (<i>B</i>) Processing of OPA1 was altered in HTRA2 KO brain at P25 but not in other tissues, with increased abundance of S-OPA1 (filled arrowhead) and decreased L-OPA1 (open arrowhead). MFN2 was not altered when compared to WT. VDAC was used as a loading control. Calculating the ratio of S-OPA1 to L-OPA1 for tissues from P25–P30 HTRA2 KO mice confirmed changes in OPA1 processing were present in the brain but not in other tissues when compared to WT (***: p<0.0001 by t-test, n≥3 per genotype). (<i>C</i>) Altered processing could be detected in HTRA2 KO brain from P9 onwards. PHB2 was used as a loading control. Calculating the ratio of S-OPA1 to L-OPA1 for P9–P10 brains confirmed the change in processing (***: p<0.0001 by t-test, n≥6 per genotype). (<i>D</i>) Processing changes were also detected in NesKO brain at P25. PHB2 was used as a loading control. Calculating the ratio of S-OPA1 to L-OPA1 for brain from NesKO and NesWT mice at P24–P26 confirmed OPA1 processing was significantly altered (**: p<0.01 by t-test, n≥3 per genotype). KO denotes HTRA2 KO, WT denotes HTRA2 WT, nKO denotes NesKO and nWT denotes NesWT.</p