While Wild-type Research Articles

Dissecting a Role for Melanopsin in Behavioural Light Aversion Reveals a Response Independent of Conventional Photoreception

Melanopsin photoreception plays a vital role in irradiance detection for non-image forming responses to light. However, little is known about the involvement of melanopsin in emotional processing of luminance. When confronted with a gradient in light, organisms exhibit spatial movements relative to this stimulus. In rodents, behavioural light aversion (BLA) is a well-documented but poorly understood phenomenon during which animals attribute salience to light and remove themselves from it. Here, using genetically modified mice and an open field behavioural paradigm, we investigate the role of melanopsin in BLA. While wildtype (WT), melanopsin knockout (Opn4−/−) and rd/rd cl (melanopsin only (MO)) mice all exhibit BLA, our novel methodology reveals that isolated melanopsin photoreception produces a slow, potentiating response to light. In order to control for the involvement of pupillary constriction in BLA we eliminated this variable with topical atropine application. This manipulation enhanced BLA in WT and MO mice, but most remarkably, revealed light aversion in triple knockout (TKO) mice, lacking three elements deemed essential for conventional photoreception (Opn4−/− Gnat1−/− Cnga3−/−). Using a number of complementary strategies, we determined this response to be generated at the level of the retina. Our findings have significant implications for the understanding of how melanopsin signalling may modulate aversive responses to light in mice and humans. In addition, we also reveal a clear potential for light perception in TKO mice.

Reduced anticipatory locomotor responses to scheduled meals in ghrelin receptor deficient mice

Ghrelin, an orexigenic hormone produced by the stomach, is secreted in anticipation of scheduled meals and in correlation with anticipatory locomotor activity. We hypothesized that ghrelin is directly implicated in stimulating locomotor activity in anticipation of scheduled meals. To test this hypothesis, we observed 24 h patterns of locomotor activity in mice with targeted mutations of the ghrelin receptor gene (GHSR KO) and wild-type littermates, all given access to food for 4 h daily for 14 days. While wild type (WT) and GHSR KO mice produced increases in anticipatory locomotor activity, anticipatory locomotor activity in GHSR KO mice was attenuated ( P<0.05). These behavioral measures correlated with attenuated levels of Fos immunoreactivity in a number of hypothalamic nuclei from GHSR KO placed on the same restricted feeding schedule for 7 days and sacrificed at ZT4. Interestingly, seven daily i.p. ghrelin injections mimicked hypothalamic Fos expression patterns to those seen in mice under restricted feeding schedules. These data suggest that ghrelin acts in the hypothalamus to augment locomotor activity in anticipation of scheduled meals.

Estrogen deficiency results in apoptosis in the frontal cortex of adult female aromatase knockout mice

The aromatase knockout (ArKO) mouse is completely estrogen deficient. We previously detected apoptosis in the hypothalamus of 1 year-old male ArKO mice. This study shows that 12 week-old female ArKO mice display spontaneous apoptosis of pyramidal neurons in the frontal cortex while wild-type (WT) littermates show no signs of apoptosis. Concomitantly, bcl-2 related anti-apoptotic genes are down-regulated whereas the pro-apoptotic gene TRADD is up-regulated in the female ArKO frontal cortex. This phenotype can be rescued by 3-week replacement of 17β-estradiol. Furthermore, the apoptosis phenotype is exacerbated in 12–15 month-old female ArKO mice, which have 30% less neurons in the frontal cortex and lower brain weights than WT counterparts. These data show that estrogens are essential for the survival of female cortical neurons even in the absence of pathological conditions or external assaults. Our observations also demonstrate the sexually dimorphic susceptibility of neurons to estrogen deficiency.

ZEB1 regulates the latent-lytic switch in infection by Epstein-Barr virus.

The immediate-early (IE) BZLF1 gene of Epstein-Barr virus (EBV) regulates the switch between latent and lytic infection by EBV. We previously showed that the cellular transcription factor ZEB1 binds to a sequence element, ZV, located at nt −17 to −12 relative to the transcription initiation site of the BZLF1 promoter, Zp, repressing transcription from Zp in a transient transfection assay. Here, we report the phenotype in the context of a whole EBV genome of a variant of EBV strain B95.8 containing a 2-bp substitution mutation in the ZV element of Zp that reduced, but did not eliminate, ZEB1 binding to Zp. Strikingly, epithelial 293 cells latently infected with the EBV ZV mutant spontaneously produced IE-, early-, and late-gene products and infectious virus, while wild-type (WT)-infected 293 cells did not and have never been reported to do so. Furthermore, treatment with the chemical inducers sodium butyrate and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) led to an additional order-of-magnitude production of infectious virus in the ZV mutant–infected 293 cells, but still no virus in the WT-infected 293 cells. Similarly, ZV mutant–infected Burkitt's lymphoma BJAB cells accumulated at least 10-fold more EBV IE mRNAs than did WT-infected BJAB cells, with TPA or sodium butyrate treatment leading to an additional 5- to 10-fold accumulation of EBV IE mRNAs in the ZV mutant–infected cells. Thus, we conclude that ZEB1 binding to Zp plays a central role in regulating the latent-lytic switch in EBV-infected epithelial and B cells, suggesting ZEB1 as a target for lytic-induction therapies in EBV-associated malignancies.

Early epidermal destruction with subsequent epidermal hyperplasia is a unique feature of the papilloma-independent squamous cell carcinoma phenotype in PKCepsilon overexpressing transgenic mice.

Protein kinase C epsilon (PKCepsilon) overexpressing transgenic (PKCepsilon Tg) mice develop papilloma-independent squamous cell carcinomas (SCC) elicited by 7,12-dimethylbenz[a]anthracene (DMBA) tumor initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA) tumor promotion. We examined whether epidermal cell turnover kinetics was altered during the development of SCC in PKCepsilon Tg mice. Dorsal skin samples were fixed for histological examination. A single application of TPA resulted in extensive infiltration of polymorphonuclear neutrophils (PMNs) into the epidermis at 24 h after TPA treatment in PKCepsilon Tg mice while wild-type (WT) mouse skin showed focal infiltration by PMNs. Complete epidermal necrosis was observed at 48 h in PKCepsilon Tg mice only; at 72 h, epidermal cell regeneration beginning from hair follicles was observed in PKCepsilon Tg mice. Since the first TPA treatment to DMBA-initiated PKCepsilon Tg mouse skin led to epidermal destruction analogous to skin abrasion, we propose the papilloma-independent phenotype may be explained by death of initiated interfollicular cells originally destined to become papillomas. Epidermal destruction did not occur after multiple doses of TPA, presumably reflecting adaptation of epidermis to chronic TPA treatment. Prolonged hyperplasia in the hair follicle may result in the early neoplastic lesions originally described by Jansen et al. (2001) by expanding initiated cells in the hair follicles resulting in the subsequent development of SCC.

Kinetic Evidence for Interaction of Human Immunodeficiency Virus Type 1 Reverse Transcriptase with the 3‘-OH of the Incoming dTTP Substrate

Two previously identified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants, Q151N and V148I, are known to have reduced dNTP binding affinity but possess wild-type chemical catalysis rates. Structural modeling based on the crystal structure of the HIV-1 RT ternary complex with dTTP proposes that Q151N loses the interaction with the 3'-OH of the incoming dTTP and that V148I disrupts positioning of Q151 for this interaction. On the basis of this, we predicted that while wild-type (WT) HIV-1 RT would have decreased binding affinity to dTTP analogues lacking 3'-OH, compared to dTTP, the Q151N and V148I RT mutants should have decreased but similar affinity to both dTTP and dTTP analogues. Pre-steady-state kinetics on WT RT showed 14- and 53-fold higher K(d) values for the 3'-OH lacking ddTTP and acyTTP, compared to dTTP. In contrast, the Q151N and V148I mutants, which were predicted to have lost H-bonding interaction with the 3'-OH of dTTP, showed higher but similar K(d) values for dTTP, ddTTP, and acyTTP. Interestingly, the Q151N and V148I RTs bound to AZTTP approximately 12 and 18 times more tightly than to dTTP, respectively. Our structure modeling suggests that these RT mutants can interact with the azido moiety of AZTTP, which is 1.4 A longer than the 3'-OH of dTTP. The kinetic data presented in this report demonstrate the functional role of the Q151 residue in HIV-1 RT interaction with dTTP and its analogues containing chemical modifications at the 3'-C of the sugar moiety.

Mutant alpha-actinin-4 promotes tumorigenicity and regulates cell motility of a human lung carcinoma.

The precise role of alpha-actinin-4 encoding gene (ACTN4) is not very well understood. It has been reported to elicit tumor suppressor activity and to regulate cellular motility. To further assess the function of human ACTN4, we studied a lung carcinoma cell line expressing a mutated alpha-actinin-4, which is recognized as a tumor antigen by autologous CD8(+) cytotoxic T lymphocytes (CTL). Confocal immunofluorescence microscopy indicated that, while wild-type (WT) alpha-actinin-4 stains into actin cytoskeleton and cell surface ruffles, the mutated protein is only dispersed in the cytoplasm of the lung carcinoma cells. This loss of association with the cell surface did not appear to correlate with a decrease in in vitro alpha-actinin-4 crosslinking to filamentous (F)-actin. Interestingly, experiments using cell lines stably expressing ACTN4 demonstrated that as opposed to WT gene, mutant ACTN4 was unable to inhibit tumor cell growth in vitro and in vivo. Moreover, the expression of mutant alpha-actinin-4 resulted in the loss of tumor cell capacity to migrate. The identification of an inactivating mutation in ACTN4 emphasizes its role as a tumor suppressor gene and underlines the involvement of cytoskeleton alteration in tumor development and metastasis.