Parkinson's disease: Alterations in iron and redox biology as a key to unlock therapeutic strategies.

A plethora of studies indicate that iron metabolism is dysregulated in Parkinson's disease (PD). The literature reveals well-documented alterations consistent with established dogma, but also intriguing paradoxical observations requiring mechanistic dissection. An important fact is the iron loading in dopaminergic neurons of the substantia nigra pars compacta (SNpc), which are the cells primarily affected in PD. Assessment of these changes reveal increased expression of proteins critical for iron uptake, namely transferrin receptor 1 and the divalent metal transporter 1 (DMT1), and decreased expression of the iron exporter, ferroportin-1 (FPN1). Consistent with this is the activation of iron regulator protein (IRP) RNA-binding activity, which is an important regulator of iron homeostasis, with its activation indicating cytosolic iron deficiency. In fact, IRPs bind to iron-responsive elements (IREs) in the 3ꞌ untranslated region (UTR) of certain mRNAs to stabilize their half-life, while binding to the 5ꞌ UTR prevents translation. Iron loading of dopaminergic neurons in PD may occur through these mechanisms, leading to increased neuronal iron and iron-mediated reactive oxygen species (ROS) generation. The "gold standard" histological marker of PD, Lewy bodies, are mainly composed of α-synuclein, the expression of which is markedly increased in PD. Of note, an atypical IRE exists in the α-synuclein 5ꞌ UTR that may explain its up-regulation by increased iron. This dysregulation could be impacted by the unique autonomous pacemaking of dopaminergic neurons of the SNpc that engages L-type Ca+2 channels, which imparts a bioenergetic energy deficit and mitochondrial redox stress. This dysfunction could then drive alterations in iron trafficking that attempt to rescue energy deficits such as the increased iron uptake to provide iron for key electron transport proteins. Considering the increased iron-loading in PD brains, therapies utilizing limited iron chelation have shown success. Greater therapeutic advancements should be possible once the exact molecular pathways of iron processing are dissected.

Hox transcription factors and their elusive mammalian gene targets.

The Hox family of homeodomain transcription factors regulate numerous pathways during developmental and normal cellular processes. All Hox proteins recognise similar sequences in vitro yet display functional diversity in an in vivo environment. This review focuses on the transcriptional and functional specificity elicited by Hox proteins, giving an overview of homeodomain-DNA interactions and the gain of binding specificity through cooperative binding with cofactors. Furthermore, currently identified mammalian Hox target genes are presented, of which the most striking feature is that very few direct Hox targets have been identified. The direct targets participate in an array of cellular functions including organogenesis and cellular differentiation, cell adhesion and migration and cell cycle and apoptotic pathways. A further assessment of identified mammalian promoter targets and the contribution of bases outside the canonical recognition motif is given, highlighting roles they may play in either trans-activation or repression by Hox proteins.

Genomic organisation and alternative splicing of mouse and human thioredoxin reductase 1 genes.

BACKGROUND: Thioredoxin reductase (TR) is a redox active protein involved in many cellular processes as part of the thioredoxin system. Presently there are three recognised forms of mammalian thioredoxin reductase designated as TR1, TR3 and TGR, that represent the cytosolic, mitochondrial and novel forms respectively. In this study we elucidated the genomic organisation of the mouse (Txnrd1) and human thioredoxin reductase 1 genes (TXNRD1) through library screening, restriction mapping and database mining. RESULTS: The human TXNRD1 gene spans 100 kb of genomic DNA organised into 16 exons and the mouse Txnrd1 gene has a similar exon/intron arrangement. We also analysed the alternative splicing patterns displayed by the mouse and human thioredoxin reductase 1 genes and mapped the different mRNA isoforms with respect to genomic organisation. These isoforms differ at the 5' end and encode putative proteins of different molecular mass. Genomic DNA sequences upstream of mouse exon 1 were compared to the human promoter to identify conserved elements. CONCLUSIONS: The human and mouse thioredoxin reductase 1 gene organisation is highly conserved and both genes exhibit alternative splicing at the 5' end. The mouse and human promoters share some conserved sequences.

Expression and localization of thioredoxin during early implantation in the marmoset monkey.

Thioredoxin is a powerful redox protein expressed in invasive cytotrophoblasts and essential for blastocyst implantation in mice. Isolated marmoset thioredoxin cDNA showed that the deduced amino acid sequence differed from the human sequence by four amino acids. The close homology of thioredoxin in the two species enabled us to use monoclonal antibodies against human thioredoxin to detect marmoset thioredoxin in implantation sites, blastocysts and culture medium. Immunocytochemistry on marmoset implantation sites, on pregnancy days 12 and 15, showed that thioredoxin is highly expressed in uterine luminal epithelium, glands and in some endometrial stromal cells. In attached blastocysts, thioredoxin staining was detected in mural and polar trophoblast cells and both visceral and parietal endoderm, whereas no staining was present in the inner cell mass. A similar pattern of thioredoxin expression was detected in hatched blastocysts attached to Matrigel in tissue culture. Trophoblastic vesicles derived from blastocysts expressed thioredoxin in inner endoderm-like cells and outer trophoblast-like cells and secreted thioredoxin into the culture medium. These experiments have demonstrated thioredoxin expression during early stages of embryo-maternal interaction. We propose that thioredoxin protects the early placenta from oxidative damage and that the marmoset is a valuable model for studying thioredoxin regulation and function during implantation and blastocyst differentiation.

Expression and localisation of thioredoxin in mouse reproductive tissues during the oestrous cycle.

Thioredoxin expression within the reproductive tissues of the female mouse was analysed during the oestrous cycle stages of dioestrus, oestrus and metoestrus by Western blot analyses and immunocytochemistry. From Western blot analyses the expression of thioredoxin was found to be increased in oestrus compared to dioestrus and metoestrus. Localisation of thioredoxin within the reproductive organs of the mouse during the oestrous cycle has shown that the expression of thioredoxin is specific for distinct areas within the reproductive organs. These areas are the stratified squamous epithelium of the vagina, the simple columnar epithelium and the uterine glands of the uterus, the ciliated columnar epithelium of the oviduct, the corpus lutea, the interstitial cells and the secondary follicles of the ovary. The discrete cellular localisation and oestrous dependence of thioredoxin expression are suggestive of specific roles in various reproductive processes.

Neovascularization of the Xenopus embryo.

The receptor tyrosine kinase, Flk-1 or VEGFR-2, and its ligand, vascular endothelial growth factor (VEGF) are required for the development of the embryonic vasculature. Targeted disruption of either gene in mice results in the failure of vascular system formation. The Xenopus homologues of flk-1 and VEGF have been cloned and their expression has been examined throughout early embryonic development. These studies indicate that flk-1 is expressed in groups of endothelial precursor cells which will form the major blood vessels of the embryo, including the posterior cardinal veins, the dorsal aorta, the vitelline veins, and the endocardium. VEGF expression is found in tissues adjacent to the mesenchyme containing the flk-1-expressing endothelial precursors. Expression of both flk-1 and VEGF is transient, appearing as the primary vascular plexus is forming and declining steadily after the onset of functional embryonic circulation. After establishment of the primary vascular structures, flk-1 expression is also observed in the intersegmental veins which form by an angiogenic mechanism. Overall, these results support a role for VEGF/flk-1 signaling in both vasculogenesis and angiogenesis in the Xenopus embryo. When VEGF is expressed ectopically in Xenopus embryos by microinjection of either plasmid DNA or synthetic mRNA, large, disorganized vascular structures are produced. This result indicates that ectopic VEGF is capable of altering the architecture of the developing vascular network.

Cardiac troponin I is a heart-specific marker in the Xenopus embryo: expression during abnormal heart morphogenesis.

Cardiac troponin I (troponin Ic) expression is restricted to the heart at all stages of Xenopus development. Whole-mount in situ hybridization and Northern blot analysis indicates that troponin Ic is first expressed in tailbud embryos (stage 28) about the time of the first cytological heart differentiation and about 24 hr before beating tissue is observed. We have used this marker to examine abnormal heart morphogenesis in embryos treated with retinoic acid and lithium. When retinoic acid is administered to embryos prior to heart specification, heart tissue is reduced and often completely ablated. When embryos are treated after heart specification, but before the heart primordium migrates to the ventral midline, the migration is unaffected but smaller, abnormal hearts result. Lithium treatment of cleavage stage embryos causes an increase in heart tissue. In severely dorsalized embryos, heart tissue can be found around the entire embryo with the exception of a small gap at the most dorsal point. This gap indicates that migration of the heart to the ventral midline does not occur in these embryos. Later in development, a centrally located, beating heart is observed in dorsalized embryos. The timing of its appearance suggests that it is formed by movements normally associated with heart morphogenesis rather than migration.

XNkx-2.5, a Xenopus gene related to Nkx-2.5 and tinman: evidence for a conserved role in cardiac development.

We have isolated a Xenopus homeodomain sequence, XNkx-2.5, which shows significant similarity to mouse Nkx-2.5 and to the Drosophila tinman gene product. In Drosophila, tinman is required for formation of the heart and visceral mesoderm structures. In situ hybridization studies show that XNkx-2.5 is expressed in the heart region during early Xenopus development and later is also expressed in gut tissue. The observed similarity of sequences and expression patterns suggests that the regulatory mechanisms underlying heart formation may be conserved between distant species.

Analysis of a variant Max sequence expressed in Xenopus laevis.

Max is a small helix-loop-helix protein which forms heterodimers with members of the Myc protein family. Myc/Max heterodimers exhibit sequence-specific DNA binding with much greater affinity than Myc homodimers. The Xenopus laevis homologue of Max, XMax, is shorter than the equivalent mammalian protein. This difference results from the deletion of a 24 amino acid sequence located near the C-terminus of the protein. Xenopus max transcripts undergo alternative splicing. In addition to the 27 base alternatively spliced sequence (exon A) previously detected in mice and humans, some Xmax transcripts also contain an 81 base sequence (exon B) at a second site within the coding sequence. Although exon B insertion alters part of the leucine zipper protein/protein interaction domain, the resulting XMax protein retains the ability to form stable heterodimers with both c-Myc and N-Myc. Xmax mRNA is present at approximately constant levels during early development. This contrasts with the rapidly changing levels of c-myc and N-myc mRNA in the embryo and has implications for regulation of gene expression during differentiation. All four alternatively spliced forms of Xmax mRNA are present during development and in all adult tissue examined.

Two neural-cell adhesion molecule (NCAM)-encoding genes in Xenopus laevis are expressed during development and in adult tissues.

Expression of genes encoding neural-cell adhesion molecules (NCAM) is regulated to a large extent at the level of alternative splicing. Xenopus laevis contains two functional copies of the NCAM gene and comparison of the amino acid sequences of NCAM proteins derived from both genes shows that they differ at approximately 8% of positions. Differential expression of these proteins during development or in adult tissues could provide an additional level of regulation above that obtained by alternative splicing. PCR analysis however, shows that both NCAM genes are expressed at similar levels throughout embryonic development and in all adult tissues examined.

Identification of molecules involved in the 'early pregnancy factor' phenomenon.

An isolated preparation from ovine placental extracts which was active in the rosette inhibition assay mimicking the activity of the so-called 'early pregnancy factor' (EPF) has been shown to contain a 12 kDa polypeptide which could be partially resolved from low-molecular-weight active moieties. N-terminal amino acid sequence analysis of the polypeptide indicated that it was ovine thioredoxin, an identification confirmed by isolation and complete sequence analysis of the corresponding cDNA. The cDNA for human thioredoxin was expressed in Escherichia coli and the recombinant protein isolated and purified. Pure recombinant thioredoxin alone did not induce the expression of increased rosette inhibition titres (RITs) when tested in the rosette inhibition assay; but, when tested in combination with cell stimuli such as platelet-activating factor (PAF) or serum, it allowed the expression of increased RITs where none was achieved in its absence. Thioredoxin acted in the assay to reverse a refractory state normally induced by these stimuli, allowing lipoxygenase-dependent moieties also induced by the stimuli to exert their effects, resulting in the expression of increased RITs. Antibodies to recombinant thioredoxin removed from pregnancy sera the capacity to induce increased RITs, i.e. to express EPF activity, thus establishing a role for thioredoxin or thioredoxin-like proteins and associated molecules in the mechanisms which allow pregnancy sera to induce increased RITs. Based on a consideration of these and other results, a new model for the study of the EPF phenomenon is presented and discussed.

Isolation and characterization of human thioredoxin-encoding genes.

Thioredoxin (Trx) has recently been demonstrated to be an essential component of the early pregnancy factor activity of pregnancy serum. Here, we report the structure and sequence of human Trx-encoding genes (Trx) by analysis of genomic clones. The Trx gene extends over 13 kb and consists of five exons encoding a 12-kDa protein. A 700-bp fragment upstream from the start codon functions as a promoter when inserted in front of a human growth hormone-encoding reporter gene in tissue-culture cells. This promoter region is very G + C rich and does not contain a classical TATA or CCAAT box, but has three consensus sequences for high-affinity Sp1 binding. Southern analysis demonstrated the presence of several Trx genes in the human genome. The number includes at least one inactive copy as shown by the isolation and sequencing of an inactive pseudogene.