Mitochondrial calcium uptake capacity as a therapeutic target in the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) is an incurable autosomal-dominant neurodegenerative disorder initiated by an abnormally expanded polyglutamine domain in the huntingtin protein. It is proposed that abnormal mitochondrial Ca2+ capacity results in an increased susceptibility to mitochondrial permeability transition (MPT) induction that may contribute significantly to HD pathogenesis. The in vivo contribution of these hypothesized defects remains to be elucidated. In this proof-of-principle study, we examined whether increasing mitochondrial Ca2+ capacity could ameliorate the well-characterized phenotype of the R6/2 transgenic mouse model. Mouse models lacking cyclophilin D demonstrate convincingly that cyclophilin D is an essential component and a key regulator of MPT induction. Mitochondria of cyclophilin D knockout mice are particularly resistant to Ca2+ overload. We generated R6/2 mice with normal, reduced or absent cyclophilin D expression and examined the effect of increasing mitochondrial Ca2+ capacity on the behavioral and neuropathological features of the R6/2 model. A predicted outcome of this approach was the finding that cyclophilin D deletion enhanced the R6/2 brain mitochondria Ca2+ capacity significantly. Increased neuronal mitochondrial Ca2+ capacity failed to ameliorate either the behavioral and neuropathological features of R6/2 mice. We found no alterations in body weight changes, lifespan, RotaRod performances, grip strength, overall activity and no significant effect on the neuropathological features of R6/2 mice. The results of this study demonstrate that increasing neuronal mitochondrial Ca2+-buffering capacity is not beneficial in the R6/2 mouse model of HD.

Diminished expression of transcription factors nuclear factor kappaB and CCAAT/enhancer binding protein underlies a novel tumor evasion mechanism affecting macrophages of mammary tumor-bearing mice.

Interactions between malignant tumors and the host immune system shape the course of cancer progression. The molecular basis of such interactions is the subject of immense interest. Proinflammatory cytokines produced by macrophages are critical mediators of immune responses that contribute to the control of the advancement of neoplasia. We have shown that the expressions of interleukin 12 (IL-12) and inducible nitric oxide synthase (iNOS) are decreased in macrophages from mammary tumor-bearing mice. In this study, we investigated the causes of IL-12 dysregulation and found deficient nuclear factor kappaB (NFkappaB) and CCAAT/enhancer binding protein (C/EBP) expression and function in tumor bearers' peritoneal macrophages. The constitutive expressions of NFkappaB p50, c-rel, p65, and C/EBPalpha and beta, as well as the lipopolysaccharide-induced nuclear translocation and DNA binding of NFkappaB components and C/EBPalpha and beta, are profoundly impaired in macrophages from mice bearing D1-DMBA-3 tumors. Because similar findings occur with the iNOS gene, it seems that it represents a novel mechanism by which tumor-derived factors interfere with the host immune defenses.

Diminished PKC activity and decreased binding of transcription factors are involved in the impaired production of nitric oxide by macrophages from tumor-bearing mice.

In previous studies we have shown that peritoneal macrophages (PEM) from mammary tumor-bearing BALB/c mice (T-PEM) display a diminished ability to lyse tumor cells upon stimulation with LPS, a phenomenon that is associated to a lower production of nitric oxide, and that is reverted upon costimulation with IFN-gamma. The reduced lytic activity and NO production displayed by T-PEM upon LPS activation were earlier shown by us to be due to a diminished transcription of the inducible nitric oxide synthase (iNOS) gene. In the present study, we have investigated the participation of possible signaling molecules and transcription factors - PKC, NF-kappaB, C/EBP and IRF-1 - in the downregulation of NO production in LPS-activated T-PEM. It was found that PKC activity was greatly reduced in T-PEM as compared to normal macrophages, and did not respond to activation. Interestingly, the different PKC isozyme levels were not significantly altered in T-PEM, with the exception of PKC delta. Alterations in the binding activity of the transcription factors NF-kappaB and C/EBP appeared to be involved in the reduced transcription of iNOS previously observed in T-PEM after LPS activation. These results provide evidence that reductions in iNOS transcription secondary to alterations in cell signaling may be responsible for the diminished capacity of macrophages of LPS-activated tumor-bearers to produce NO and lyse tumor targets.

Murine epidermal cell antigen (Skn)-directed autoimmunity induced by transfer of CD4+ T cells.

While pathogenic T cells have been identified for several diseases with epithelial cell damage, an autoimmune T cell-mediated response targeted against a known keratinocyte antigen has not been reported. Previously we described an autoimmune response directed to the mouse epidermal cell antigens, Skn. For our murine model, primed Skn-immune lymphocytes are adoptively transferred to recipients, which develop lesions at the site of mild skin trauma. In this study we investigated the nature of the autoimmune component of the Skn response. A time-course study demonstrated a relationship between the number of primed Skn-immune cells injected and the severity of skin lesions in the recipients. Immunohistochemical staining revealed the presence of both CD4+ and CD8+ T cells in lesional skin, with a predominance of CD4+ T cells. To support a role for CD4+ T cells in the initiation of the autoimmune response, Skn-immune donor cells were either enriched or depleted of various subsets prior to transfer into recipients, which showed that CD4+, but not CD8+, T cells were essential for induction of lesions. Analysis of mRNA for T-helper (Th) cell cytokines in lesional skin displayed a Th 1 bias, and treatment with cyclosporin A (CsA) or anti-interleukin (IL)-2 antibody controlled the development of lesions. Overall the results clearly show an immunopathogenic profile consistent with a T cell-mediated mechanism.

Tissue transglutaminase overexpression does not modify the disease phenotype of the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) is a devastating autosomal-dominant neurodegenerative disorder initiated by an abnormally expanded polyglutamine in the huntingtin protein. Determining the contribution of specific factors to the pathogenesis of HD should provide rational targets for therapeutic intervention. One suggested contributor is the type 2 transglutaminase (TG2), a multifunctional calcium dependent enzyme. A role for TG2 in HD has been suggested because a polypeptide-bound glutamine is a rate-limiting factor for a TG2-catalyzed reaction, and TG2 can cross-link mutant huntingtin in vitro. Further, TG2 is up regulated in brain areas affected in HD. The objective of this study was to further examine the contribution of TG2 as a potential modifier of HD pathogenesis and its validity as a therapeutic target in HD. In particular our goal was to determine whether an increase in TG2 level, as documented in human HD brains, modulates the well-characterized phenotype of the R6/2 HD mouse model. To accomplish this objective a genetic cross was performed between R6/2 mice and an established transgenic mouse line that constitutively expresses human TG2 (hTG2) under control of the prion promoter. Constitutive expression of hTG2 did not affect the onset and progression of the behavioral and neuropathological HD phenotype of R6/2 mice. We found no alterations in body weight changes, rotarod performances, grip strength, overall activity, and no significant effect on the neuropathological features of R6/2 mice. Overall the results of this study suggest that an increase in hTG2 expression does not significantly modify the pathology of HD.

Immunophilin deficiency augments Ca2+-dependent glutamate release from mouse cortical astrocytes.

Immunophilins are receptors for immunosuppressive drugs such as the macrolides cyclosporin A (CsA) and FK506; correspondingly these immunophilins are referred to as cyclophilins and FK506-binding proteins (FKBPs). In particular, CsA targets cyclophilin D (CypD), which can modulate mitochondrial Ca(2+) dynamics. Since mitochondria have been implicated in the regulation of astrocytic cytosolic Ca(2+) (Ca(cyt)(2+)) dynamics and consequential Ca(2+)-dependent exocytotic release of glutamate, we investigated the role of CypD in this process. Cortical astrocytes isolated from CypD deficient mice Ppif(-/-) displayed reduced mechanically induced Ca(cyt)(2+) increases, even though these cells showed augmented exocytotic release of glutamate, when compared to responses obtained from astrocytes isolated from wild-type mice. Furthermore, acute treatment with CsA to inhibit CypD modulation of mitochondrial Ca(2+) buffering, or with FK506 to inhibit FKBP12 interaction with inositol-trisphosphate receptor of the endoplasmic reticulum, led to similar reductive effects on astrocytic Ca(cyt)(2+) dynamics, but also to an enhanced Ca(2+)-dependent exocytotic release of glutamate in wild-type astrocytes. These findings point to a possible role of immunophilin signal transduction pathways in astrocytic modulation of neuronal activity at the tripartite synapse.

Role of the proteasome in the downregulation of transcription factors NFkappaB and C/EBP in macrophages from tumor hosts.

Macrophages from mice bearing advanced mammary tumors are critically impaired in their immune functions, exhibiting reduced expression at the mRNA and protein levels of the crucial transcription factors, nuclear factor kappaB (NFkappaB) and CCAAT enhancer binding protein (C/EBP). We have previously shown that tumor-derived factors such as transforming growth factor beta (TGFbeta) and prostaglandin E2 (PGE2) modulate NFkappaB and C/EBP expression in macrophages. Transcriptional, post-transcriptional, translational and/or post-translational mechanisms may also play a role in altered levels of NFkappaB and C/EBP in macrophages from tumor hosts, contributing to impaired inflammatory response. One of the post-translational mechanisms that may tune down or recycle proteins in cells is the proteasomal pathway. Since upregulation of ubiquitin/proteasomal pathways has been described under cancer-induced cachexia, we examined the possible role of this proteolytic machinery in the decrease of NFkappaB and C/EBP proteins in macrophages from tumor hosts. Using MG-132 proteasome inhibitor to block the proteasome machinery in macrophages from normal and tumor-bearing animals we found that macrophages from tumor hosts display higher ubiquitination and proteolysis compared to those from normal mice and also that NFkappaB and C/EBP downregulation is reversed in these treated cells. Thus, proteasome degradation may contribute, at least in part, to NFkappaB and C/EBP impairment in macrophages from tumor-bearers.

Altered IL-12 Signaling Pathways Contribute to the Deficient IFN-γ Production by T Splenocytes from Tumor-bearing Mice.

IFN-γ is a crucial cytokine produced by T and NK cells. Previous work from our laboratory has reported that in T cells of BALB/c mice bearing the D1-DMBA-3 mammary tumor, IFN-γ production is down-regulated, due to decreased expression of IL-12 by macrophages of tumor bearers. IL-12 is the main inducer of IFN-γ production in T and NK cells. To exert its function, IL-12 interacts with its receptor (IL-12R), activating a JAK/STAT signaling pathway. Our investigation suggests that there is also a deficiency in the response to IL-12 by T cells from tumor hosts. The present work reports the results of RT-PCR experiments in the study of the IL-12R expression on T cells from normal and tumor bearers. Data showed a deficient expression of the IL-12Rß2 chain on T cells from tumor hosts. Gene expression arrays on IL-12-activated T cells from normal and tumor bearers confirmed the RT-PCR results, and also showed decreased expression of IL-18Rα in tumor bearers' T cells. Arrays also showed down-regulated expression of JAK2, STAT 1, 3, 4 and IRF-1. Finally, increased expression of SOCS 1,3,4,5 and 7, as well of Protein inhibitor of activated STATs (Pias) 1 and y was also observed in tumor bearers' T cells.